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Assessment of surgical site infection risk factors at Imam Reza hospital, Mashhad, Iran between 2006 and 2011
<p> <bold>Background:</bold> The present study was conducted to establish the patterns and risk factors of surgical site infections in our institution between 2006 and 2011. </p><p> <bold>Methods:</bold> This was a retrospective cross-sectional study. The surgical site infection (SSI) was identified based on the presence of ICD-10-CM diagnostic code in hospital discharge records. By using a standardized data collection form predictor variables including patient characteristics, preoperative, intra-operative and postoperative data were obtained. </p><p> <bold>Results:</bold> Ninety five patients fulfilled the inclusion criteria. The patients were admitted for various procedures including both elective (62.1%) and emergency (37.9%) operations. Colectomy (13.7%) was the leading procedure followed by umbilical herniation (12.6) and appendix perforation (12.6%). The mean age was 47.13 years with standard deviation of 19.60 years. Twenty percent were addicted to opium. Midline incision above and below the umbilicus (40%) had the highest prevalence of infection. Most patients (46.3%) had cleancontaminated wounds and 30.5% had contaminated one. The quantitative variables which were also measured include duration of surgery, pre-operative and post-operative hospital stay with the mean of 2.9&#x000b1;1.45 hours, 1.02&#x000b1;1.42 and 7.75&#x000b1;6.75 days respectively. The most antibiotics prescribed post-operatively were the combination of ceftriaxone and metronidazole (51.6%). </p><p> <bold>Conclusion:</bold> The contaminated and clean-contaminated wounds are associated with higher rate of SSIs. Also, there was a converse relation between length of surgical incision and rate of SSIs. In overall, we found type of surgery as the main risk factor in developing the SSIs. </p>
<contrib contrib-type="author"><name><surname>Motie</surname><given-names>Mohammad Reza</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Ansari</surname><given-names>Majid</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Nasrollahi</surname><given-names>Hamid Reza</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Surgical site infections (SSIs) as one of the most common causes of nosocomial infections is accounted for 20 to 25% of all nosocomial infections worldwide (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>). The SSIs are the most common complication following surgery, with reported rates up to 30% (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R3" ref-type="bibr">3</xref>). These infections place a substantial burden on healthcare cost as a result of increased post-operative morbidity and mortality (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R4" ref-type="bibr">4</xref>-<xref rid="R7" ref-type="bibr">7</xref>). They are responsible for 30 to 40% of the deaths in the postoperative period (<xref rid="R8" ref-type="bibr">8</xref>). </p><p> With regard to the multifactorial condition of SSI, it is important to detect these factors, to investigate the procedures that bear the highest risk and, if possible, define suitable indices that can predict the risk of SSI (<xref rid="R9" ref-type="bibr">9</xref>). </p><p> The present study was conducted to establish the patterns and risk factors of surgical site infections at Imam Reza hospital in Iran between 2006 and 2011. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> This is a retrospective cross-sectional study. The SSI was identified based on the presence of ICD-10-CM diagnosis code in hospital discharge records of patients admitted to general surgery ward of Imam Reza hospital, Mashhad, Iran between 2006 and 2011. </p><p> Surgical site infections were defined according to the CDC (The Centers for Disease Control and Prevention) criteria. All patients who met inclusion criteria were enrolled into the study. By using a standardized data collection form predictor variables including patient characteristics, preoperative, intra-operative and postoperative data were obtained. Study data included type of surgery, wound class, infection degree, incision site, type and duration of operation, type of prophylaxis and duration of antimicrobial therapy, use of drain, preoperative and postoperative hospital stay. Data were analyzed using The SPSS software </p><p> Patients diagnosed with surgical site infections who were identifiable based on the presence of ICD-10-CM diagnosis code in hospital discharge records were included. </p><p> Patients with more than one surgery during hospitalization and those who underwent minor surgery (surgeries that doesn&#x02019;t require anesthesia or respiratory aid) were excluded from the study. </p><sec id="s2-1"><title>Statistical analysis</title><p> Descriptive statistics such as frequency table is derived for categorical variables and mean and standard deviation (SD) for numerical variables. All statistical analysis was done using SPSS statistical software. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> Retrospective review of patients&#x02019; medical records showed that 95 patients fulfilled the inclusion criteria. Study population included 51 (53.7%) males and 44 (46.3%) females with age ranged 12-88 years. The mean age was 47.13 (SD: 19.60) years. The patients were admitted for various procedures of both elective (62.1%) and emergency (37.9%) operations as shown in <xref ref-type="table" rid="T1">table 1</xref>. Colectomy (13.7%) was the leading procedure followed by umbilical herniation (12.6) and appendix perforation (12.6%) (<xref ref-type="table" rid="T1">Table 1</xref>). </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Various surgical procedures and their frequency. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Surgical Category</td><td align="center" rowspan="1" colspan="1">No.</td><td align="center" rowspan="1" colspan="1">%</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Colectomy</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">13.7</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Umbilical Herniation</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">12.6</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Appendix Perforation</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">12.6</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Colon Cancer</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8.4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Cholecystectomy</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">6.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Appendectomy</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">4.2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Bowel Obstruction</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">3.2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Mesenteric Ischemia</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Bowel Fistula</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Volvulus</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Aortic Aneurysm</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Iliac Aneurysm</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">3.2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gastric Perforation</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Hydatid Cyst</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">5.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Limb Amputation</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">4.2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Bowel Perforation</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Mastectomy</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Inguinal Herniation</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">4.2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Pilonidal Sinus</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">5.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gastric Banding</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Bronchobiliary Sinus</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gastrointestinal Bleeding</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Trauma</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2.1</td></tr></tbody></table></table-wrap><p> Patient history of smoking, addiction, past history of diseases (including hypertension, diabetes and autoimmune diseases) and medications (corticosteroid and antibiotic therapy) were assessed. Nineteen patients (19.7%) were addicted to opium 8 (8.3%) with diabetes, other factors ratio was less than 8% for each. </p><p> Except one case (laparoscopic gastric binding), all patients had undergone open surgery (96.8%). </p><p> The highest incidence of infection were identified in patients with midline incision above and below the umbilicus (40%) followed by midline incision below the umbilicus (8.4%) and above the umbilicus (4.7%). </p><p> The organ involvements were also recorded based on the information contained in the operation description sheet. In most cases (26%) all abdominal viscera such as appendix, colon, small intestine, ovaries and fallopian tubes have been involved during operations. The small intestine and the colon involvement alone occurred in 21.1% of cases (<xref ref-type="table" rid="T2">Table 2</xref>). </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Organ involvements during operation </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Organ involvements</td><td align="center" rowspan="1" colspan="1">No.</td><td align="center" rowspan="1" colspan="1">%</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Abdominal Viscera</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">26.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Colon and Small Intestine</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">21.1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Colon</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">7.4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Liver</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">5.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Limbs</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">5.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Genital Organs</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">5.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gall Bladder</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">4.2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Breast</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Stomach</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr></tbody></table></table-wrap><p> The highest incidence of wound infections was observed in 44 patients (46.3%) with clean-contaminated wounds. Ninety nine patients (30.5%) had contaminated and 20 patients (21.1%) had clean wounds, and 2 cases identified as dirty wounds. The highest degree of infection (65.3%) was serous secretion without wound dehiscence (<xref ref-type="table" rid="T3">Table 3</xref>). </p><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> Wound infection degree in patients </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Wound infection degree</td><td align="center" rowspan="1" colspan="1">No.</td><td align="center" rowspan="1" colspan="1">%</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Inflammation and redness</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">8.4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Serous secretion without wound dehiscence</td><td align="center" rowspan="1" colspan="1">62</td><td align="center" rowspan="1" colspan="1">65.3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Superficial wound dehiscence</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">17.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Deep wound dehiscence</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">4.2</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Abscess unassociated with the operated organ</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2.1</td></tr></tbody></table></table-wrap><p> The mean duration of surgery, pre-operative and post-operative hospital stays was 2.9&#x000b1;1.45 hours, 1.02&#x000b1;1.42 and 7.75&#x000b1;6.75 days respectively. The missing data included duration of surgery and pre-operative and post-operative hospital stay in 40% and 33% of patients, respectively. </p><p> Surgical drains were used in 44.2% of cases and in 82.1% of cases, the wound was closed initially. </p><p> Only for 4.2% of patients, preoperative antimicrobial prophylaxes were recorded. Ceftriaxone and metronidazole were administered for 2.1% of patients; and equal proportions of patients received metronidazole and erythromycin or ceftriaxone, metronidazole and ciprofloxacine (1.1%). </p><p> The most antibiotics prescribed post-operatively were the combination of ceftriaxone and metronidazole (51.6%). </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> In this study, the mean age was 47.13 years with standard deviation of 19.60 years. There were 51 (53.7%) males and 44 (46.3%) females in age range of 12-88 years. Similar demographic observation was reported by other studies. The rate of SSI was higher in males than in females. This could be explained by multiple risk factors in males such as addiction to opium. In accordance with our study, previous studies have shown that patients suffering from pre-morbid diseases, such as diabetes mellitus and hypertension are at high risk of developing SSI (<xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R8" ref-type="bibr">8</xref>, <xref rid="R10" ref-type="bibr">10</xref>, <xref rid="R11" ref-type="bibr">11</xref>). Cigarette smoking was significantly found to be associated with SSI in other studies (<xref rid="R5" ref-type="bibr">5</xref>, <xref rid="R10" ref-type="bibr">10</xref>, <xref rid="R2" ref-type="bibr">2</xref>). In contrast, in our study only 3.2% had a history of cigarette smoking; on the contrary 20% were addicted to drugs, which showed significant association with development of SSI. </p><p> In the present study the patients were admitted for various procedures including both elective (62.1%) and emergency (37.9%) operations. The higher rate of SSI in elective surgeries can be explained due to the higher rate of contaminated and clean contaminated wounds in elective surgeries of our institution. Colectomy (13.7%) was the leading procedure followed by umbilical herniation (12.6%), and appendix perforation (12.6%) (<xref ref-type="table" rid="T1">Table 1</xref>). The highest incidence of infection were identified in patients with midline incision above and below the umbilicus (40%) followed by midline incision below the umbilicus (8.4%) and above the umbilicus (4.7%). </p><p> Fiorio et al meta-analysis on 3066 surgical procedures demonstrated highest incidence of SSI in small bowel (16.3%) and colon surgery (12.5%) (<xref rid="R7" ref-type="bibr">7</xref>). Our findings confirmed previous knowledge that surgeries with an increased microbial load in the operative field are associated with higher risk of SSI (<xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R13" ref-type="bibr">13</xref>). </p><p> For a long time, surgical wound classification has been recognized as an important predictive&#x000a0;factor in developing surgical site infections after surgery (<xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R14" ref-type="bibr">14</xref>-<xref rid="R16" ref-type="bibr">16</xref>).In our study, as well as previous studies, the incidence of SSI was statistically higher in clean contaminated (46.3%) and contaminated (30.5%) wounds. </p><p> A prolonged pre-operative hospital stay has been reported to increase the rate of surgical site infection (<xref rid="R17" ref-type="bibr">17</xref>). A length of operation of more than 3 hours leads to 4 times higher risk of SSI (<xref rid="R9" ref-type="bibr">9</xref>). In present study, the means for duration of surgery, pre-operative and post-operative hospital stay were 2.91.45 hours, 1.021.42 and 7.756.75 days, respectively. This is not in accordance with the literature regarding the risk of SSI determined by the duration of the surgery and pre-operative hospital stay (<xref rid="R5" ref-type="bibr">5</xref>-<xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R9" ref-type="bibr">9</xref>). In addition, the use of surgical drains has been reported to be associated with an increased risk of SSI which was confirmed in this study (28-30). </p><p> Preoperative antimicrobial prophylaxis was only administered in 4.2% of patients. This can be explained by using routine antibiotics for every procedure, and also it necessitates the use of antibiotic policy regarding different therapeutic procedures for these patients. The most prescribed antibiotics used post-operatively were ceftriaxone -metronidazole (51.6%) and metronidazole (8.4%). Other studies however reported using different antibiotics (<xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R9" ref-type="bibr">9</xref>). </p><p> This investigation had limitations because of missing data including prophylactic antibiotics and ICD-10-CM diagnostic code in hospital discharge records. </p><p> The comparison of SSI incidence between hospitals from various locations and countries must always be attentive, due to specific characteristics for each place and patient population that make it difficult to reach valid conclusions. The ideal situation would be for each hospital to critically analyze its own data, preferably focusing on the historical series and then particularizing it for various types of surgery. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Surgical site infection is highly related to the type of wound, namely contaminated and clean contaminated wounds and associated with higher rate of SSI. Also, it seems that there is a converse relation between length of surgical incision and the rate of SSIs. In short, we found that the type of surgery considered to be the main risk factor in developing SSI. </p></sec>
Supporting-emotional needs of Iranian parents with premature infants admitted to Neonatal Intensive Care Units
<p> <bold>Background:</bold> Having an infant in the neonatal intensive care unit (NICU) is a stressful and painful experience. Unlike to normal births, this birth is associated with admission and separation of infant from parents. The aim of this study was to compile the supporting-emotional needs of Iranian parents who have a premature infant admitted in (NICU). </p><p> <bold>Methods:</bold> This study was performed using qualitative research approach. Twelve participants including 9 parents whose infant had been hospitalized in NICU; two nurses and one physician were also selected for sampling purposes. Data were gathered using semi-structured interview. Data were analyzed by inductive content analysis approach. </p><p> <bold>Results:</bold> Four subcategories emerged from data analysis expressed supporting-emotional needs of parents of premature infants admitted in NICU. These subcategories were: Need for interaction with infant, Need to medical team&#x02019;s empathy, need to exchange support with spouse, and Need to get help from others. </p><p> <bold>Conclusion:</bold> In order to develop mutual bonding with infant and attain parental roles, parents need to be close their neonate, also receive empathy and support to find a way to meet their needs. Participants in this study announced that resolving these needs can help parents to feel more confidence in infant's care and reduce their negative feelings. </p>
<contrib contrib-type="author"><name><surname>Aliabadi</surname><given-names>Faranak</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Kamali</surname><given-names>Mohammad</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Borimnejad</surname><given-names>Leili</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rassafiani</surname><given-names>Mehdi</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rasti</surname><given-names>Mehdi</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Shafaroodi</surname><given-names>Narges</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rafii</surname><given-names>Foroogh</given-names></name><xref ref-type="aff" rid="A07"> <sup>7</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Askary Kachoosangy</surname><given-names>Reihaneh</given-names></name><xref ref-type="aff" rid="A08"> <sup>8</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> The birth of a neonate usually makes the family happy. However, the early birth of a neonate can lead to different vague emotions in parents (<xref rid="R1" ref-type="bibr">1</xref>). When a preterm neonate is born, parents do not feel enough psychological, emotional and physical readiness and would undergo too much stress (<xref rid="R2" ref-type="bibr">2</xref>). In this situation, parents are confused and concern about survival of their neonate and their long term complications (<xref rid="R3" ref-type="bibr">3</xref>). Premature neonates are very fragile and need to receive special care in a Neonatal Intensive Care Unit (NICU) (<xref rid="R4" ref-type="bibr">4</xref>). Length of hospitalization depends on medical condition and may vary from a few days to several weeks or even months 5). Early separation from the neonate increases parents&#x02019; NICU-related stress. Furthermore, prolonged separation between mother and her neonate causes maternal stress, anxiety, depression and psychological reactions and feelings such as frustration, guilt, hostility, irritability, fear, grief, and feeling of failure (<xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R7" ref-type="bibr">7</xref>). These parents lose their self-confidence and feel incompetency in playing their parental roles. They also alienate from their neonate which may lead to problems in parent-baby attachment (<xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R7" ref-type="bibr">7</xref>). In general, it can be said that in this situation, the natural parental process may be disrupted. Difficulties in communicating with neonate may cause developmental delay in infant (<xref rid="R6" ref-type="bibr">6</xref>). On the other hand, ignoring the parental emotions and their psychological stress will lead to trouble in bonding with infant, especially at the time of discharge or transition to home which causes increased vulnerability (<xref rid="R6" ref-type="bibr">6</xref>). According to family-centered interventions, health system should pay attention to the important parental roles. In family-centered approach, parents participate in decision making about their neonate and have the opportunity of choosing a therapeutic method which is proper for their conditions (<xref rid="R8" ref-type="bibr">8</xref>-<xref rid="R9" ref-type="bibr">9</xref>). </p><p> Occupational therapy is one of the members of rehabilitation team which has an important role in providing services for preterm neonates and their parents. Although occupational therapists have their own frameworks and principles in their specialty, they strictly recommended to present family-centered services, so should consider parents&#x02019; needs and priorities to deliver more effective and helpful therapeutic interventions (<xref rid="R8" ref-type="bibr">8</xref>-<xref rid="R10" ref-type="bibr">10</xref>).Also, family-centered intervention is widely suggested to other professions who work with children. As understanding of the parents&#x02019; experience of having an infant admitted to the NICU increases, professionals will be better prepared to meet parental needs and alleviate parental suffering (<xref rid="R11" ref-type="bibr">11</xref>). Therefore, there is an increasing need to improve knowledge about parents&#x02019; needs, feelings, and stresses. Considering the limited literature which exclusively can describe the needs of Iranian parents of preterm infants admitted in NICU, and this fact that existing researches are not completely generalizable to Iran&#x02019;s context, running further researches to obtain better perspective in this field is strongly needed. Such information can be attained mainly by qualitative researches. The aim of this qualitative content analysis study was to compile the supporting-emotional needs of Iranian parents with premature infants, admitted in NICU. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> A qualitative design and a thematic content analysis were chosen to achieve the aim of the study. Analyzing the content of narrative data helps identifying prominent themes and patterns among the themes and is a method that is useful for studying phenomena about which little is known (<xref rid="R12" ref-type="bibr">12</xref>). </p><sec id="s2-1"><title> Sample and data collection method </title><p> In this study, parents, nurses, and a physician at the hospitals affiliated to Iran University of Medical Sciences (IUMS) in Tehran, Iran contributed. </p><p> Each participant was interviewed using a semi-structured format with open-ended questions. Interviews audio taped. The interviewer was a female occupational therapist, expert in neonatology and with a 16 year experience in the field of pediatric family-centered occupational therapy and 10 years of experience in early intervention. She has not been involved directly in the care of these infants. Interviews were conducted at a time and place of convenience to the participants. One participant was interviewed in his own home and others were interviewed in a quiet private room within the hospital. Prior to the beginning of each interview, researcher informed the participant the purpose of study and permission was sought to tape record. The participants were also guaranteed confidentiality and anonymity in the presentation of the results. </p><p> The interview began with questions about their infant&#x02019;s current condition and the parents&#x02019; social and demographic information. Then, parents were asked: &#x0201c;Please tell me what you do in NICU? The interview was supported by questions about their feelings and needs in this situation. They were encouraged to express themselves freely in narrative form. Each interview lasted approximately 20 to 60 minutes. The interviews took the form of a conversation, and the interviewer used active listening skills in this process. </p><p> Inclusion criteria considered for parents were: being Persian-speaker; having a singleton, low birth weight premature infant with gestational age of 28 to 37 weeks in NICU for at least 7 days; having no genetic disorder, no grade 3 and 4 intra ventricular hemorrhage, no Apgar score under 7, no cardiopulmonary arrest in their infants; and no history of having another infant in NICU. The mean length of hospitalization in NICU was 34.5 days. Exclusion criterion was the participant&#x02019;s dissuasion in any step of the study. </p></sec><sec id="s2-2"><title> Data analysis </title><p> The interviews were tape recorded, transcribed and then, the transcriptions were reviewed for accuracy by some other writers of this article which were expert in fields of occupational therapy and/or qualitative researches. Each transcription was coded by hand, using the qualitative content analysis method (<xref rid="R13" ref-type="bibr">13</xref>). Using this method, each interview was coded line by line by interviewer (first author) who was qualified in qualitative research methods and a research assistant (fifth author) who was trained in this field. These primary codes were meaningful statements, which lengths from a word to one or more complete sentence(s), in participant&#x02019;s own words. In the next step, these primary codes were reworded into shorter phrases. After that, similar phrases were collected and subcategories developed. Finally, the main category was emerged through more abstraction of subcategories. This process is shown in <xref ref-type="boxed-text" rid="B1">Box 1</xref>. </p><boxed-text position="float" id="B1" orientation="portrait"><sec sec-type="Box 1"><title>Box 1.Data analysis process</title><p> -First phase. Line by line coding </p><p> <italic>&#x0201c;I just want him to be around me, I want to hold him, I stand beside him and I start playing with him when His smile makes me calm and love to be around him and hug him.&#x0201d;</italic> </p><p> &#x0201c;It would be good if parents could see their child whenever they want. For example, when my husband comes to see our child, our baby is slept. Or my husband loves to come back from work at night to see our baby, which is impossible. He loves to see our baby opens his eyes and smiles or start laughing. We want to speak with him and hear his voice. It makes us calm.&#x0201d; </p><p> -Second phase. Phrasing shorter codes </p><p> Want to have baby around herself to have an opportunity to see, hold, play with, and hug him whenever they want </p><p> love to speak with baby and hear his voice </p><p> -Third phase. Developing final codes </p><p> Need to embrace and hug the neonate </p><p> Need to speak with neonate and hear her/his voice </p><p> -Fourth phase. Making and naming </p><p> Need to interact with the neonate </p></sec></boxed-text><p> Credibility of data was provided through member checking, peer checking, prolonged engagement with participants and data and, maximum variation of sampling. Member checks confirmed whether or not interpretations of participants' statements were accurate. Ten of twelve participants had the opportunity to review their full transcript of their coded interview at the next visit, while two of them (both were parents) did not check their statements due to discharge from hospital and their reluctance with being followed. The use of peer checking process helped decreasing possible biases. For this purpose, 2 investigators (sixth and seventh authors) independently reviewed each interview to code them and reached an acceptable agreement on them with main research team. Prolonged engagement with participants and data helped researcher to achieve a better communication with participants and have a deep understanding of context. </p></sec><sec id="s2-3"><title> Ethical considerations </title><p> Verbal and written informed consent was acquired from each participant according to a protocol approved by the research ethics committee of Iran University of Medical Sciences. Participants were informed that participation in this study is voluntarily and they could refuse to enter or withdraw from the study at any time. Interviews were conducted with the presence of only the interviewer and the interviewee and participants were identified by an identification code to protect their privacy and confidentiality. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> Participant&#x02019;s characteristics: A purposive sample of 12 participants (5 mothers, 4 fathers, 2 nurses, and 1 physician) participated in this study. </p><p> Nurses had master&#x02019;s degree in nursing and 5 to 7 years of experience working in NICU, and physician also was a subspecialty in neonatology with 18 years of experience. Parents&#x02019; age ranged between 20 and 42 years. Six of parents were first-time parents, two were second-time parents, and only one of them was third-time parents. The neonates were born with a gestational age between 28 and 36 weeks. The mean length of hospitalization in NICU was 34.5 days (<xref ref-type="table" rid="T1">Table 1</xref>). </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table1 </label><caption><title> Participants / infant demographics </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Gender</td><td align="center" rowspan="1" colspan="1">Role</td><td align="center" rowspan="1" colspan="1">Age</td><td align="center" rowspan="1" colspan="1">Education</td><td align="center" rowspan="1" colspan="1"> Infant Gestational<break/>Age at Birth (wk) </td><td align="center" rowspan="1" colspan="1"> Length of hospitalization<break/>(day) </td><td align="center" rowspan="1" colspan="1">Birth Order</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">mother</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">diploma</td><td align="center" rowspan="1" colspan="1">32</td><td align="center" rowspan="1" colspan="1">29</td><td align="center" rowspan="1" colspan="1">Third</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">mother</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">diploma</td><td align="center" rowspan="1" colspan="1">36</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">first</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">father</td><td align="center" rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">Scholastic degree</td><td align="center" rowspan="1" colspan="1">31</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">first</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">mother</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">Pre university</td><td align="center" rowspan="1" colspan="1">34</td><td align="center" rowspan="1" colspan="1">28</td><td align="center" rowspan="1" colspan="1">first</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">father</td><td align="center" rowspan="1" colspan="1">28</td><td align="center" rowspan="1" colspan="1">diploma</td><td align="center" rowspan="1" colspan="1">28</td><td align="center" rowspan="1" colspan="1">36</td><td align="center" rowspan="1" colspan="1">second</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">mother</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">diploma</td><td align="center" rowspan="1" colspan="1">31</td><td align="center" rowspan="1" colspan="1">29</td><td align="center" rowspan="1" colspan="1">first</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">father</td><td align="center" rowspan="1" colspan="1">28</td><td align="center" rowspan="1" colspan="1">Bachelor</td><td align="center" rowspan="1" colspan="1">34</td><td align="center" rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">first</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">mother</td><td align="center" rowspan="1" colspan="1">29</td><td align="center" rowspan="1" colspan="1">Associate Degree</td><td align="center" rowspan="1" colspan="1">34</td><td align="center" rowspan="1" colspan="1">49</td><td align="center" rowspan="1" colspan="1">second</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">father</td><td align="center" rowspan="1" colspan="1">36</td><td align="center" rowspan="1" colspan="1">master</td><td align="center" rowspan="1" colspan="1">33</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">first</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">nurse</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">master</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">nurse</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">master</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">physician</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">subspecialist</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr></tbody></table></table-wrap><p> Analyzing the data and interviews of present study in order to compiling and identifying the supporting-emotional needs of parents with premature infants resulted in four subcategories. </p><p> The supporting-emotional needs category and its subcategories and codes are illustrated in <xref ref-type="table" rid="T2">Table 2</xref>. </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Supporting-emotional needs of parents of premature infants, admitted in NICU </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Category</td><td rowspan="1" colspan="1">Subcategory</td><td rowspan="1" colspan="1">Open codes</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="9" colspan="1"> Supporting-emotional needs </td><td rowspan="1" colspan="1">Need to interact with the neonate</td><td rowspan="1" colspan="1">Need to speak with neonate and hear her/his voice</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to embrace and hug the neonate</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to medical team&#x02019;s empathy</td><td rowspan="1" colspan="1">Need to have a respectful and empathic relationship established by the medical staff</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to be ensured and satisfied of proper care providing for their neonate by staff</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to receive appropriate answers by staff</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to exchange support with spouse</td><td rowspan="1" colspan="1">Need to be understood by the spouse</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to interact with spouse</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to get help from others</td><td rowspan="1" colspan="1">Need to get help from family</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Need to get help from significant others</td></tr></tbody></table></table-wrap><sec id="s3-1"><title> Need to interact with neonate </title><p> One of subcategories of the supporting-emotional needs which parents and other participants reported was &#x0201c;Need to interact with neonate&#x0201d;. This subcategory contains two codes: "Need to speak with neonate and hear her/his voice" and "Need to embrace and hug the neonate". Hospitalizing a baby in NICU is a critical situation for many parents which may cause arousing negative feelings in them. Separation of parent and neonate has been recognized as a major source of stress for parents. Accordingly, being near the neonate and touching her/his up may reduce parents&#x02019; stress and develop emotional support. The parents explained the need to have closeness and proximity and belonging to their infant. One of the participants expressed this need to: </p><p> <italic>&#x0201c;I just want him to be around me, I want to hold him, I stand beside him and I start playing with him when he open his eyes and smiles or start laughing. His smile makes me calm and love to be around him and hug him.&#x0201d;</italic> </p><p> Parents also tended to spend more time and to be near their infant whenever they want. For example: </p><p> <italic>&#x0201c;It would be good if parents could see their child whenever they want. For example, when my husband comes to see our child, our baby is slept. Or my husband loves to come back from work at night to see our baby, which is impossible. He loves to see our baby opens his eyes and smiles or start laughing. We want to speak with him and hear his voice. It makes us calm.&#x0201d; </italic> </p></sec><sec id="s3-3"><title> Need to medical team&#x02019;s empathy </title><p> Another subcategory of supporting-emotional needs is &#x0201c;Need to medical team&#x02019;s empathy&#x0201d; which includes three codes: "Need to have a respectful and empathic relationship established by the medical staff", "Need to be ensured and satisfied of proper care providing for their neonate by staff", and "Need to receive appropriate answers from staff". Parents need to have a respectful and empathic relationship with medical team and wish to be understood by them. They believe that healthcare professionals as a team can play a big role in reducing their stress. As one participant said: </p><p> <italic>&#x0201c;Personnel should be changed with someone who shows their heart and great to us. Some of them have bad attitude toward you and they don&#x02019;t care about you when you need to talk to them.&#x0201d;</italic> </p><p> Participants stated that relying on the quality of care delivered by staff may reduce their stress and negative feelings, even when they are not present in the neonate&#x02019;s bedside. One participant said: </p><p> <italic>&#x0201c;My baby needs me to be here all day long, because personnel don&#x02019;t do their job right, they really don&#x02019;t take care of us thoroughly and I have to be here all the time.&#x0201d;</italic> </p><p> Participants stated that the lack of staff responding to their questions about the neonate can increase anxiety and fear, also lead to inappropriate communication with the staff. For example: </p><p> <italic>&#x0201c;To get a chance to ask my question about my baby, I pass by twenty of them but it makes me feel more anxious when they don&#x02019;t answer my questions. I think there is something wrong with my baby which is scary.&#x0201d;</italic> </p></sec><sec id="s3-4"><title> Need to exchange support with spouse </title><p> This subcategory includes two codes: "Need to be understood by spouse" and "Need to interact with spouse". Most parents are eager to exchange supportive roles with their mate. This concept is expressed in the words of one of participants as: </p><p> <italic>&#x0201c;The first thing I do is to take care of my wife because she becomes very weak after her delivery. I have heard this is because a mild depression that she might have gotten after her delivery and she needs me to talk to her and understand her. I do my best to take care of her.&#x0201d;</italic> </p><p> Or: </p><p> <italic>&#x0201c;They need their husband&#x02019;s emotional supports. For example, bad attitudes irritate them really bad which is very common in our busy hospitals. Nurses and physicians don&#x02019;t have enough time to listen to mothers as much so spouse should take care of themselves.&#x0201d;</italic> </p></sec><sec id="s3-5"><title> Need to get help from others </title><p> In this study need to get help from others was considered as a subcategory. This subcategory includes two codes: " Need to get help from family" and "Need to get help from significant others". Most parents, especially mothers, could not do all things related to themselves or neonate due to critical situation of infant, fatigue and need recovering from labor. So they need to get help from other person and kin to perform the duties and parents&#x02019; roles. One participant said: </p><p> &#x0201c;The only person who is allowed to take care of the baby is his mother, but the mother herself needs someone else to take care of her. It would be great if someone else like my baby&#x02019;s grandmother or aunt could help me to take care of him.&#x0201d; </p></sec></sec><sec id="s5"><title> Discussion </title><p> The present study is one of the limited qualitative studies that compile the needs of parents of premature infants. According to the results of this study, supporting-emotional needs of parents of premature infants admitted in NICU were categorized in 4 categories. The results indicated that all parents in this study wanted to be close to the neonate as much as possible. Separation from their baby is experienced as the most stressful part of having a preterm birth. </p><p> Some studies (<xref rid="R14" ref-type="bibr">14</xref>-<xref rid="R15" ref-type="bibr">15</xref>) show that mothers experience frustration when separated from their neonate. When separation occurs, mothers and their neonate have no opportunity to experience mutual bonding. The results of this study are in consistent with the study of Valizadeh et al (2007). Valizadeh et al pointed out that restricted contact with neonate is one of the resources of stress and influence on infant-parent interaction (<xref rid="R16" ref-type="bibr">16</xref>). Therefore, there might be a delay in the process of attaining maternal role identity, while this process must be initiated as soon as possible (<xref rid="R14" ref-type="bibr">14</xref>). Participants in this study pointed out that they want to be close to their neonates and have the possibility of caring directly and contact with baby at any time. Neu et al stated that kangaroo-care, as a way of establishing close relationship and contact with infant, was perceived by mothers as a positive and pleasant experience (<xref rid="R17" ref-type="bibr">17</xref>). </p><p> Participants noted the importance of receiving empathy from medical staff and establishing a respectful and empathic relationship with them. Reviewing previous studies showed that parents of premature infants emphasized the importance of proper communication with the medical team (<xref rid="R18" ref-type="bibr">18</xref>). Valizadeh et al pointed out that giving hope and support by nurses can cause development and promotion in the mother-infant attachment and relationship. Also, mothers use it to cope with the event of sudden preterm birth as a preventive strategy to solve this crisis (<xref rid="R16" ref-type="bibr">16</xref>).When parents feel to be supported by the staff, their concerns will be reduced. Being understood by staff is also very important which causes parents feel that their neonate receive good and appropriate care (<xref rid="R14" ref-type="bibr">14</xref>),also their negative feelings may be controlled. Arockiasamy et al pointed out that giving information to parents can cause increasing their understanding and control over the situation. Furthermore, he emphasized on consistency of information content which can be provided by the staff (<xref rid="R18" ref-type="bibr">18</xref>). According to Van Rooyen, providing accurate and understandable information to parents is necessary to overcome negative emotions of parents (<xref rid="R19" ref-type="bibr">19</xref>). </p><p> Parents devote a plenty of time to thinking about their neonate and are interested in receiving support to find a way to meet their needs (<xref rid="R20" ref-type="bibr">20</xref>). Parents stated that although they do not receive enough support from staff in some cases, it may be provided to them in other ways. Since attaining support is one of their fundamental needs, parents seek for alternatives to meet it. They look for it in all available resources in their context to feel being protected. It seems that most available resources of support for Iranian parents are: their spouse, other mothers who are in the same situation, therapeutic staff, family members, and God. </p><p> Parents said that whenever they have an encouraging and loving conversation with their spouse, they feel better. In addition, when they see that their mate, especially husband, is happy and have a good relationship with the neonate, they feel supported. Moreover, talking with other mothers in NICU or receiving help from them make mothers more powerful to face problems. They announced the same experience with their family member. According to Lindberg&#x02019;s study, mothers stated that support can be obtained from infant&#x02019;s father and/or from speaking with other mothers who were in unit (<xref rid="R14" ref-type="bibr">14</xref>). Short-daily conversations and talks can be an important tool for the support of parents, especially mothers. These talks will help them feel confident in the care of the infant and give better care to their infant (<xref rid="R21" ref-type="bibr">21</xref>). Valizadeh et al pointed out to the role of husband and family supports on attachment development, also its influence on improving mother&#x02019;s morale and empowering her on facing the crisis and coping with it (<xref rid="R16" ref-type="bibr">16</xref>). The relationship between family members and support for the family is one of the main sources of adaptability (<xref rid="R16" ref-type="bibr">16</xref>). In addition, in the Lindberg&#x02019;s study, fathers noted to importance of relationship with their wives and conversation with other parents who were in unit (<xref rid="R1" ref-type="bibr">1</xref>).Hughes et al (1994) reported that fathers often cope with this situation by interaction with others and seeking social supports but mothers cope with it by talking to their husbands as an emotional and psychological support (<xref rid="R15" ref-type="bibr">15</xref>). Mothers use some strategies to decrease risk and increase safety of their newborn in the NICU. These strategies include negotiating with the medical team and creating a strong relationship with the mothers, their husband, family, and friends (<xref rid="R22" ref-type="bibr">22</xref>-<xref rid="R23" ref-type="bibr">23</xref>). Receiving support from staff, husband, other family member, and significant others are factors which enable mothers to manage the critical situation they are exposed to it (<xref rid="R11" ref-type="bibr">11</xref>).If parents receive incorrect or incomplete information about their infant, they will feel stress and anxiety (<xref rid="R23" ref-type="bibr">23</xref>).Since traditional resources of support including family and other nonprofessional persons may present incomplete or even wrong information to parents, medical team&#x02019;s role in this area seems to be very important. </p></sec><sec sec-type="conclusion" id="s4"><title>Conclusion</title><p> Participants in this study announced that resolving these needs can help parents to feel more confidence in infant's care and reduce their negative feelings such as anxiety, depression, inefficacy, and guilt feeling. </p></sec>
Pseudoachalasia: a diagnostic challenge
<p>Here, we present a case of a 78-year-old man that underwent gastrointestinal endoscopy because of one- month history of dysphagia to liquids and solid foods with accompanying weight loss. On endoscopy, there was distal esophageal stenosis. Multiple biopsies were obtained. Histologic examination of the samples revealed normal tissue. The stenosis was treated by dilatation and abdomino pelvic computed tomography scanning was performed to search for an underlying malignant lesion that showed a mass adjacent to distal esophagus. We did endosonography- guided fine needle aspiration of the mass. It was a squamous cell carcinoma (SCC). Malignancy is a challenging diagnosis in patients with dysphagia and near normal endoscopy. To our knowledge, there are a few reports of SCC to cause it.</p>
<contrib contrib-type="author"><name><surname>Roushan</surname><given-names>Nader</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Zolfaghari</surname><given-names>Abolfazl</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Asadi</surname><given-names>Mehrnaz</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Taslimi</surname><given-names>Reza</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Achalasia is a primary esophageal motility disorder presenting with dysphagia to liqiuds and solid foods. It is usually diagnosed using barium swallow study and manometry. Barium study shows narrowing of esophagogastric junction (EGJ) while manometry shows decreased or absent distal esophageal peristalsis with increased lower esophageal sphincter (LES) pressure and impaired LES relaxation with deglutition. Esophagus appears to be normal on endoscopic examination (<xref rid="R1" ref-type="bibr">1</xref>). </p><p> Pseudoachalasia is characterised by achalasia-like symptoms caused by secondary etiologies. Clinical, radiologic and endoscopic findings resemble those of achalasia but treatment and prognosis are different in these conditions. Therefore, discriminating between these two disorders is important (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R5" ref-type="bibr">5</xref>). </p></sec><sec id="s2"><title>Case report</title><p> 78-years-old man was admitted to our hospital due to 10-days history of intractable postprandial vomiting. He complained of progressive dysphagia presented initially to solid foods and then to liquids for a month. During this time, he had 15 kilogram weight loss. </p><p> He smoked for decades and noticed occasional productive cough with no relevant diagnostic work-up. He had history of hypothyroidism after being treated by radio &#x02013;iodine for the primary hyperthyroidism when he was 60-years-old. However, at the time of presentation, he was in euthyroid state. </p><p> On clinical examination, he appeared ill and dehydrated. Except for bilateral temporal wasting, the whole clinical examination was normal. Tests of blood and electrolytes showed white blood cell (WBC): 9,700 /&#x000b5;l, Hb: 11.2 g/dl, MCV: 86/fl: Platelet count; 283,000 /&#x000b5;l: ESR; 125 mm/ hour, Na: 137 and K: 3.6 meq/ l. Renal and liver function assays were normal as serum protein electrophoresis study. </p><p> There was no evidence of hepatobiliary or splenic abnormality on abdominal sonography. </p><p> On upper endoscopy, esophagus appeared to be diffusely erythematous with multiple white plaques compatible with candidiasis; due to tight LES stenosis, attempts to pass the endoscope into stomach was futile. After dilatation with the Savary dilators, all parts of stomach were visualized that seemed to have normal mucosa. Multiple biopsies were obtained from distal esophagus and proximal stomach (both side of LES); but histologic examination revealed normal mucosa. </p><p> Abdominal and chest CT scan showed an abnormal soft tissue mass around distal esophagus with a solitary 2 cm pulmonary nodule (SPN) near right lung hilum. His medical records were unavailable to evaluate the growth rate of SPN. </p><p> On endosonography (EUS), a 42 mm hypoechoic mass was seen at LES extending beneath the mucosa ,involving all layers of esophageal wall albeit without any regional lymph node abnormality. Fine needle aspiration (FNA) guided by EUS was done (<xref ref-type="fig" rid="F1">Fig.1</xref>). Histological study of samples showed a well differentiated squamous cell carcinoma (<xref ref-type="fig" rid="F2">Fig. 2</xref>). We did not evaluate the SPN as he refused to undergo further diagnostic work-up. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-54-g001"/><statement><p>Fine needle aspiration of the mass during endosonography.</p></statement></fig><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-54-g002"/><statement><p>Squamous cell carcinoma pathologic preparation obtained during endosonography by Fine needle aspiration.</p></statement></fig><p> The tumor stage was T3N0M0. Therefore, he received chemo radiotherapy with platinium-based regimen. Thereafter, dysphagia improved significantly. Unfortunately, he died six months after treatment due to a heart attack. </p></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Motility type dysphagia defined by difficult passage of both liquids and solid foods to stomach is a diagnostic challenge to physicians. The differential diagnosis includes a wide spectrum of various disorders. At one end are benign motility disorders such as diffuse esophageal spasm, hypertensive LES and achalasia, and at the other extreme, are ominous malignant cancers mimicking achalasia. Dysphagia to both liquid and solid foods that is not due to primary neuronal disease and instead is due to secondary malignant or non-malignant causes is called pseudoachalasia; nonmalignant diseases include pancreatic pseudocyst, amyloidosis, sarcoidosis, neurofibromatosis, aortic aneurysm, juvenile Sjogren syndrome, chronic idiopathic intestinal pseudo obstruction, familial glucocorticoid deficiency syndrome, and a few surgeries (vagotomy, bariatric, Nissen fundoplication) (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R5" ref-type="bibr">5</xref>-<xref rid="R12" ref-type="bibr">12</xref>). Esophagogastric junction adenocarcinoma comprises the most common malignant cause of pseudoachalasia (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R3" ref-type="bibr">3</xref>, <xref rid="R13" ref-type="bibr">13</xref>); the others include cancers of pancreas, esophagus, lung, kidney, hepatobililary, lymphoma and mesothelioma (<xref rid="R2" ref-type="bibr">2</xref>). Malignant neoplasms comprise 4% of achalasia-like syndromes (<xref rid="R3" ref-type="bibr">3</xref>, <xref rid="R13" ref-type="bibr">13</xref>). Dysmotility in achalasia is caused by unrecognized causes that destroy distal esophageal and LES inhibitory neurons; these cause LES muscle contraction with no relaxation in deglutition. Suggested mechanisms for pseudoachalasia include encircling and compressing LES by tumor, infiltration of myentric plexus branches or vagus nerves, and paraneoplastic neuropathy without direct infiltration of nerves by malignant cells (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R5" ref-type="bibr">5</xref>). </p><p> Standard treatment of achalasia, including balloon dilatation and surgical myotomy are ineffective and even dangerous for pseudoachalasia and delays timely diagnosis of malignant neoplasm (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R3" ref-type="bibr">3</xref>, <xref rid="R5" ref-type="bibr">5</xref>). So every attempts should be done to reach correct diagnosis. </p><p> Endoscopy is the first diagnostic modality in a patient with new onset dysphagia (<xref rid="R14" ref-type="bibr">14</xref>); it detects structural esophageal and gastric abnormalities; especially tumors. In achalasia-like syndrome, it may show mass, nodularity, irregular mucosa or ulcer, pointing to pseudoachalasia rather than achalasia (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R15" ref-type="bibr">15</xref>). Even in normal appearing mucosa, difficult passage of endoscope through LES suggests peudoachalasia in contrast to achalasia that endoscope can be passed with subtle pressure through LES (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R2" ref-type="bibr">2</xref>). </p><p> In the case of normal appearing esophagus on endoscopy and new onset dysphagia, the physician must be vigilant to the presence of an underlying malignant lesion, especially when the patient is old, and there is significant weight loss (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R13" ref-type="bibr">13</xref>, <xref rid="R16" ref-type="bibr">16</xref>). In such circumstances, CT scan and EUS are helpful diagnostic tools. CT scan may show asymmetric wall thickening of distal esophagus and even a well-defined mass (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R5" ref-type="bibr">5</xref>). EUS would reveal more information as it precisely defines esophageal wall layers and any abnormal tissue; EUS also can be used for tissue acquisition by FNA, when endoscopic biopsies are inconclusive (<xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R17" ref-type="bibr">17</xref>, <xref rid="R18" ref-type="bibr">18</xref>). </p><p> In the literature, squamous cell carcinoma as a cause of pseudoachalasia has been shown to be derived from esophagus, liver, lung and uterine cervix (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R5" ref-type="bibr">5</xref>, <xref rid="R13" ref-type="bibr">13</xref>). Our case was probably due to a primary lung squamous cell cancer with metastasis to distal esophagus because he was smoker and had a SPN in right lung. </p></sec><sec sec-type="conclusion" id="s4"><title>Conclusion</title><p> Pseudoachalasia should be in mind when approaching to every patient with suspected achalasia and especially alarm signs should be questioned; on the other hand, it should be kept in mind that simple mucosal biopsy do not exclude pseudoachalasia and in suspected patients further work up such as CT scan and EUS may be warranted. </p></sec>
Solitary fibrous tumor of the intrathoracic goiter
<p>Solitary Fibrous Tumors (SFTs) are rare primary pleural neoplasms which have recently been reported in extra-thoracic sites. In this report, solitary fibrous tumor arising in an intra-thoracic goiter with no evidence of cervical mass in a 74-year-old obese man who was found to have a large superior mediastinal mass with tracheal deviation on Chest X-Ray is presented.</p>
<contrib contrib-type="author"><name><surname>Vaziri</surname><given-names>Mohammad</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Molanaei</surname><given-names>Saadat</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Tamannaei</surname><given-names>Zeinab</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Solitary Fibrous Tumors (SFTs) are rare primary pleural neoplasms which have recently been reported in extra-thoracic sites such as the meninges, nasal cavity, oral cavity, pharynx, epiglottis, salivary glands, thyroid, breast, kidney, bladder and spinal cord (<xref rid="R1" ref-type="bibr">1</xref>). In this report, solitary fibrous tumor arising in an intra-thoracic goiter with no evidence of cervical mass is presented. </p></sec><sec id="s2"><title>Case Report</title><p> A 74-year-old obese man primarily admitted for prostatectomy, was found to have a large superior mediastinal mass with tracheal deviation on Chest X-Ray (<xref ref-type="fig" rid="F1">Fig. 1</xref>). The patient had a previous history of cough and dyspnea with no personal attention and clinical evaluation. The physical examination was unremarkable with no cervical mass or adenopathy. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-51-g001"/><statement><p>Chest X-Ray and CT scan: Chest X-Ray shows a mass to the left of the trachea in the antero-superior portion of the visceral compartment with a characteristic tracheal deviation beginning in the cervical portion of the trachea which is typical of an intra-thoracic goiter. CT Scan reveals a large retrosternal mass with well-defined borders and non-homogeneity with discrete non-enhancing low-density areas located anteriorly in the visceral compartment of mediastinum. There is right-sided tracheal deviation and compression with no invasion to surrounding structures.</p></statement></fig><p> Computerized Tomography (CT) scan revealed a large retrosternal mass with right-sided tracheal deviation and compression extending from thyroid tissue with no invasion to surrounding structures and no apparent cervical tumor (<xref ref-type="fig" rid="F1">Fig. 1</xref>). No Ultrasonographic examination or fine-needle aspiration was carried out and in the context of normal laboratory tests (including thyroid function tests) and acceptable cardio-pulmonary evaluation, surgery via a cervical incision with no extension to sternum or thoracic cavity was performed. </p><p> In cases of suspected intra thoracic goiters (as in this case with a characteristic CT Scan) needle biopsy is unnecessary and is not recommended as a part of the diagnostic evaluation because an occult tumor would be inaccessible to random biopsy. The indications of trans-thoracic needle biopsy include any symptoms or signs of malignancy such as hoarseness (vocal cord paralysis), Horner&#x02019;s syndrome, Superior Vena Cava Syndrome, tracheal invasion and when a dominant cold nodule is clearly present. The clinical presentation and CT Scan findings in this patient did not suggest any malignant tumor. </p><p> Intra-operative findings included a well-circumscribed and brown-yellow mass with no involvement of surrounding structures which following total resection was 12*7*5 cm in size and weighing 114 grams. No intra-operative frozen section assessment was required because preoperative diagnosis based on clinical and imaging evaluation, was an intra-thoracic nonmalignant goiter and there was no intra-operative findings to justify this procedure. </p><p> Complete (near total) thyroidectomy was performed in order to significantly reduce the recurrence rate and patient is maintained on thyroid hormone replacement therapy with normal thyroid function tests. </p><p> Microscopic examination showed goiterous thyroid containing a neoplasm composed of spindle cells in a patternless growth intermingled with collagen bundles (<xref ref-type="fig" rid="F2">Fig. 2</xref>). The tumor had high cellularity with no atypia, rich vascularization, rare mitotic figures and no necrosis. </p><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-51-g002"/><statement><p>Histopathologic view: The pathologic picture clearly shows simultaneous presence of thyroid tissue and solitary fibrous tumor which contains goiterous thyroid containing a neoplasm composed of spindle cells in a patternless growth intermingled with collagen bundles .The right portion of the picture shows IHC staining of the tumor with Immunohistochemistry positive reaction to CD 99.</p></statement></fig><p> According to the pathologist opinion, no high power field of histopathology finding was necessary because the reported diagnosis (thyroid SFT) was readily made by the depicted magnification (x4) and characteristic IHC evaluation. No Ki67 labeling index and TTF-1 or thyroglobulin immunohistochemistry assessments were required because the pathologist was certain of the diagnosis of a solitary fibrous tumor. </p><p> Immunohistochemistry (<xref ref-type="fig" rid="F2">Fig. 2</xref>) revealed positive reaction for Vimentin, CD 34 and CD 99 in addition to negative reaction to Cytokeratin, Desmin and S100. After two years of follow-up, no evidence of local recurrence or distant metastasis is recorded. </p></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Most patients with substernal goiters which are considered masquerading lesion of a mediastinal tumor are thick-necked women in the seventh or eighth decade of life with a long-standing benign lesion. It is very unusual to encounter a patient with an intra-thoracic goiter containing a tumor such as Solitary Fibrous Tumor (SFT). </p><p> SFT is a rare primary localized pleural tumor which has been known by a variety of names that reflect its clinical course and controversies surrounding its histogenesis (<xref rid="R2" ref-type="bibr">2</xref>). Occurrence of this tumor in various reported extra-thoracic sites including thyroid (<xref rid="R1" ref-type="bibr">1</xref>) may demonstrate a mesenchymal origin of this mysterious neoplasm. </p><p> The distinction of SFT from other spindle cell malignancies may be difficult especially in the thyroid gland which may be the site of metastatic spread from other organs (mostly lung, kidney and larynx). Primary thyroid spindle cell lesions can be derived from follicular, C-cell or mesenchymal components and may be the result of neoplastic processes including Riedel thyroiditis, solitary fibrous tumor, leiomyoma, medullary carcinoma, anaplastic carcinoma, sarcoma and squamous cell carcinoma (<xref rid="R3" ref-type="bibr">3</xref>). </p><p> SFTs are usually encapsulated well-circumscribed masses with smooth external surfaces which on cut section are grey-white to tan with possible areas of hemorrhage or necrosis. Histologically, localized fibrous tumors appear as low-grade neoplasms of variable cellularity which is inversely related to the collagen content with minimal nuclear pleomorphism and rare mitoses. The most frequent microscopic pattern is the &#x0201c;patternless pattern&#x0201d; in which there is intermingling of tumor cells and collagen in a random fashion and the second most common pattern is hemangiopericytoma-like appearance. </p><p> The cornerstone diagnostic tool for SFT is immunohistochemistry and the findings of CD34, vimentin positive and keratin negative tumor are so characteristic that make the exclusion of other tumors relatively straightforward. In this regard it has been suggested that mesenchymal tumors of the thyroid, reported in previous studies as Leiomyoma, Neurilemmoma and Hemangiopericytoma should probably classified as SFT (<xref rid="R3" ref-type="bibr">3</xref>). </p><p> The diagnosis of a thyroid SFT is rarely reached before surgical excision and pathological examination of the mass and because of the diversity of histologic patterns, percutaneous biopsy samples are insufficient for diagnosis. Accordingly we think that radiological tools like ultrasonography and percutaneous techniques such as FNA are not useful and indicated in a probable thyroid SFT as well as in any intrathoracic goiter. </p><p> Due to rarity of thyroid SFT in general and SFT arising in an intrathoracic goiter in particular, sound prediction and recommendation regarding the clinical behavior of the tumor or necessity of adjuvant therapy, respectively can not be made but cumulative data from previous reports suggest a benign nature and similar clinical-histological characteristics of its pleural counterpart (<xref rid="R4" ref-type="bibr">4</xref>). However, malignant solitary fibrous tumor of the thyroid with local recurrence and pulmonary metastasis has been reported (<xref rid="R5" ref-type="bibr">5</xref>). </p><p> We conclude that careful attention should be paid to the morphological and histological characteristics of thyroid SFT as the most important indicators of the outcome and all SFTs need long-term follow-up with aggressive surgical resection as the treatment of choice for the recurrence. </p></sec>
An unusual, giant and benign condyloma acuminatum lesion on the genital area for more than a decade
<p>Condyloma acuminatum, is a rare sexually transmitted disease. The virus responsible for condyloma is human papillomavirus.HPV-6 and HPV-11 are the most commonly detected HPV genotypes, but at least 20 other HPV genotypes have occasionally been found in genital wart tissue specimens. In fact genital HPV infection is common among sexually active populations. This disease may occur at any age after puberty and always seen in the mucosal area. It is characterized by slow growth. We reported here a case of a 42-year-old female patient with a 12 year history of a condyloma acuminatum lesion in her genitalia area without any medical treatment.</p>
<contrib contrib-type="author"><name><surname>Aminimoghaddam</surname><given-names>Soheila</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Badakhsh</surname><given-names>Mohammad Hossein</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Taftachi</surname><given-names>Farrokh</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Nejadisalami</surname><given-names>Forough</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mahmoudzadeh</surname><given-names>Fatemeh</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Genital warts or Condylomata acuminatum is a highly contagious sexually transmitted disease caused by wide variety of sub-types of human papillomavirus (HPV) (i.e. types 6 and 11 are responsible for 90% of genital warts) (<xref rid="R1" ref-type="bibr">1</xref>). It spreads through direct skin-to-skin contact during oral, genital, or anal sex with an infected partner (<xref rid="R2" ref-type="bibr">2</xref>). The prevalence of HPV has been observed at 27% of all sexually active people, with an increase of up to 45% between the ages of 14 and 33 but in overall 1% of the adult population are affected by condyloma acuminatum (<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R4" ref-type="bibr">4</xref>). Condyloma acuminatum lesions characterized as cauliflowerlike masses of tissues that can be found on the urethra, penis, female genitalia, perianal area, or rectum (<xref rid="R5" ref-type="bibr">5</xref>). The lesions are typically limited to a few centimeters in diameter at the time of presentation. We report the case of a 42-year-old female with a 12 year history of a rather large wart. </p></sec><sec id="s2"><title>Case Report</title><p> A 42-year-old woman came to oncology outpatient department complaining of extensive lesion in the genital part of the body for more than 12 years ago. She has 3 sons which all born by vaginal delivery. She married at age of twenty-four years and then divorced 12 years later and she did not have a history of extramarital contact. Also she gave a history of smoking for more than 20 years, one pack each day. Her last mense occurred two weeks ago, with the clinical and past medical history within normal limits. The Condyloma size was 25 to 5 cm (<xref ref-type="fig" rid="F1">Fig.1</xref>) and grown for 12 years ago with pain that healed spontaneously without any treatment. During this she had itching and pain in her genitalia region and no treatment was applied. No other lesions were seen in other part of the body. Pathologic examination of lesions showed koilocytic changes that is pathognomic of HPV infection in specimen. The detection of epithelial acanthosis with atypical koilocytosis on histological examination, as well as the finding of human papillomavirus genotype 6 by in situ DNA hybridization, confirmed the diagnosis of condyloma acuminatum. Routine serological testing for other sexually transmitted infections, including human immunodeficiency virus, yielded negative results. After obtaining signed informed consent, wide local excision was done (<xref ref-type="fig" rid="F2">Fig.2</xref>). Follow-up visits occurred 15 days and 1, 3, 6, and 12 months after treatment; no relapses were registered. </p><fig id="F1" orientation="portrait" position="float"><label> Fig.1 </label><graphic xlink:href="MJIRI-28-55-g001"/><statement><p>Condyloma acuminatum lesion on the genital area</p></statement></fig><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-55-g002"/><statement><p> Clinical resolution after surgical incision</p></statement></fig></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Condylomata acuminatum are caused by infection with the human papilloma virus. Most warts are caused by human papilloma virus genotype 6 or 11 (<xref rid="R2" ref-type="bibr">2</xref>). Genital condylomata are commonly transmitted by sexual contact. Risk factors for HPV transmission are: multiple sexual partners, prostitution, homosexuality, lack of hygiene, and chronic genital infections. It seems the most important risk factors in this patient are lack of hygiene and heavy smoking. </p><p> Condylomata acuminatum lesions may sometimes be localized on extragenital mucous membranes, such as lingual, anus, axilla and some other mucous membranes (<xref rid="R6" ref-type="bibr">6</xref>), but this case had no lesions in the other parts of her body. </p><p> We describe here the first case, to our knowledge, of condyloma acuminatum with giant lesions with no sign of neoplasia or dysplasia in the tissue for more than 12-year without any treatment. Lesions were treated by local excision. It seems that condyloma accuminatum might growth with no dysplasia and neoplasia if they were not treated. </p></sec>
Retroperitoneal bronchogenic cyst: a case report
<p> Bronchogenic cysts are among developmental disorders of the primitive foregut which are typically found above the diaphragm. Bronchial cysts discovered in the abdominal cavity or retroperitoneum are extremely rare. We present a rare case of a retroperitoneal bronchogenic cyst which was incidentally detected after a wrestling injury in a 23-year-old man who had a negative medical history. Although initial imaging studies suggested an adrenal tumor, histopathological analysis provided a definite diagnosis of bronchogenic cyst. Though rare, bronchogenic cysts must be considered in the differential diagnosis of retroperitoneal cystic lesions. This is the first case of a retroperitoneal bronchogenic cyst reported in Iran. </p>
<contrib contrib-type="author"><name><surname>Mirsadeghi</surname><given-names>Ali</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Farrokhi</surname><given-names>Farid</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Fazli-Shahri</surname><given-names>Azadeh</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Gholipour</surname><given-names>Bahareh</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Bronchogenic cysts are benign cystic congenital aberrations resulting from an abnormal budding of the tracheobronchial tree between 26 and 40 days of embryogenesis. They are usually discovered in the thorax, especially in the mediastinum. Rarely, they can develop below the diaphragm, and a retroperitoneal position is exceptionally unusual (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R3" ref-type="bibr">3</xref>). To our knowledge, only 66 cases have been indexed in the Pubmed among the English literature (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R8" ref-type="bibr">8</xref>), and there has been no report in Iran. </p><p> Herein, we present a caseof bronchogenic cyst occurred in the retroperitoneum in a 23-year-old man with a review of relevant literature. </p></sec><sec id="s2"><title>Case report</title><p> A 23-year-old man, who had been injured during a wrestling competition, was referred to our hospital with an abdominal pain. He had no significant past medical history. On physical examination, he was a thin young patient with stable vital signs. He had pain and mild tenderness in left upper quadrant region. Laboratory tests including complete blood counts, liver function test (LFT) and blood chemistry were normal. Anti-hydatic cyst antibody test was negative. Erythrocyte sedimentation rate (ESR) was high (62 mm/h) and culture for enterococcus was positive. Abdominal computed tomography scan revealed a complicated cystic lesion with dimensions of 118 x 130 mm between spleen and the left kidney and a compression effect into the kidney. The mass had septa and calcification in its inferior part. Using contrast enhancement, no abnormal vessel or enhancement was noted (<xref ref-type="fig" rid="F1">Fig.1</xref>). Thus the patient underwent a semi-elective operation. During the operation, a huge cystic structure was discovered filled with thick, brownish secretions in retroperitoneal area. The cyst was about 20 x 20 x 20 cm in diameter and adjacent to spleen, adrenal gland and kidney. Postoperative course was uneventful; the patient was discharged on day 3, and had remained asymptomatic during the follow-up period of 4 years. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-56-g001"/><statement><p> Computed tomographic scan demonstrating a sharply marginated mass with septa and calcification between spleen and the left kidney with compression effect into the kidney. </p></statement></fig><p> On gross examination, the specimen consisted of an opened creamy brown ovoid cystic lesion, measuring 10 x 8 x 7 cm with irregular surface. On opening internal surface was brownish and irregular, wall thickness from 0.2 cm to 1.5 cm. The cyst was submitted for microscopic examination. </p><p> Histological sections showed fragments of cystic wall, lined by bronchial type pseudostratified cylindrical epithelium resting on fibrovascular connective tissue containing extensive foci of mononuclear inflammatory cells aggregates and presence of smooth muscle fascicles. There were foci of non-neoplastic adrenal tissue attached to external surface of the cystic lesion. A subsequent cytological examination were negative for malignancy and acute inflammatory fluid. These findings were consistent with a bronchogenic cyst (<xref ref-type="fig" rid="F2">Fig. 2</xref>). </p><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-56-g002"/><statement><p>Microscopic section of the bronchogenic cyst showing cystic cavities lined by pseudostratified cylindrical epithelium on a thick fibrous wall containing cartilage, seromucous glands, smooth muscle fascicles and extensive foci of mononuclear inflammatory cells aggregates (H&#x00026;E, x 40).</p></statement></fig></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Bronchogenic cysts are rare primitive foregut-derived developmental anomalies with bronchial type pseudostratified cylindrical epithelium which are usually discovered above the diaphragm. Subdiaphragmatic cysts are extremely rare. Studies have shown that retroperitoneal bronchogenic cysts usually occur in both sexes in equal ratio and a wide age range (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R3" ref-type="bibr">3</xref>, <xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R8" ref-type="bibr">8</xref>). </p><p> Although the exact pathogenesis is still unknown, in 1985, this hypothesis was put forward by Sumiyoshi et al. (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R3" ref-type="bibr">3</xref>, <xref rid="R8" ref-type="bibr">8</xref>) that a retroperitoneal bronchial cyst can be resulted from pinching off and trapping of the abnormal buds of the tracheobronchial tree after migration in to the abdomen before fusion of the diaphragm components. There is another alternative theory which seems less reliable and described intraabdominal aberrant budding from the primitive foregut (<xref rid="R4" ref-type="bibr">4</xref>). </p><p> In most instances, retroperitoneal bronchogenic cysts have occurred in the left adrenal gland or the superior body of the pancreas region; and they were asymptomatic and were discovered incidentally during an imaging of the chest (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R3" ref-type="bibr">3</xref>, <xref rid="R8" ref-type="bibr">8</xref>). There is an increasing tendency between size of the cyst and age of the patient (<xref rid="R5" ref-type="bibr">5</xref>). </p><p> There are some differential diagnosis for a retroperitoneal cyst lined by pseudostratified ciliated cylindrical epithelium, such as intra-abdominal cystic teratoma, bronchopulmonary sequestration, cyst of urogenital and mullerian origin, and other foregut cysts, in addition to a bronchogenic cyst. Present of secretory respiratory lining epithelium (cuboid or cylindrical ciliated epithelium) surrounding by smooth muscle similar to those in normal bronchi, as in this case, along with presence of cartilage, elastic tissues and seromucous glands, clarifies a definite pathological diagnosis of bronchogenic cyst (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R8" ref-type="bibr">8</xref>). </p><p> Regardless of being disputable, surgical resectioneven in asymptomatic cases is recommended to obtain definitive histological diagnosis and avoid future probable development of symptoms and complications, such as infection,hemorrhage, or neoplasia within the cyst (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R8" ref-type="bibr">8</xref>). </p><p> Despite the fact that the occurrence of bronchogenic cysts in the retroperitoneum is extremely rare, and also their preoperative diagnosis is so difficult, they must be considered in the differential diagnosis of retroperitoneal cystic lesions. </p></sec>
Combined therapeutic effects of low power laser (980nm) and CoQ10 on Neuropathic Pain in adult male rat
<p> <bold>Background:</bold> Neuropathic pain (NP) is one of the most suffering medical conditions that often fail to respond to certain pain therapy. Although its exact etiology is still unknown the role of reactive oxygen species (ROS) and oxidative stress were explored by many researchers. Neuropathies either central or peripheral lead to painful condition as well as social and economic isolation, thus various therapies were used to treat or reduce the pain. Laser therapy and antioxidant drugs have separately considered as treatment for NP, but the combination of them have not been used yet. In order to study the combination effects of Low Level Laser Therapy (LLLT) and Coenzyme Q<sub>10</sub> (CoQ<sub>10</sub>) the present study was designed. </p><p> <bold>Methods:</bold> Sixty adult male rats (230-320g) were used in this experimental study that divided into six groups (n=10). Chronic constriction injury (CCI) was used to induce neuropathic pain. The CoQ10 or vehicle, a low level laser of 980nm was used for two consecutive weeks. Thermal and mechanical paw withdrawal thresholds were assessed before and after surgery on 7<sup>th</sup> and 14<sup>th</sup> days. </p><p> <bold>Results:</bold> As we expected CCI decreased the pain threshold, whereas CoQ<sub>10</sub> administration for two weeks increased mechanical and thermal threshold. The same results obtained for laser therapy using the CCI animals. Combination of laser 980nm with CoQ<sub>10</sub> also showed significant differences in CCI animals. </p><p> <bold>Conclusion:</bold> Based on our findings the combination of CoQ10 with LLLT showed better effects than each one alone. In this regard we believe that there might be cellular and molecular synergism in simultaneous use of CoQ<sub>10</sub> and LLLT on pain relief. </p>
<contrib contrib-type="author"><name><surname>Jameie</surname><given-names>Seyed Behnamedin</given-names></name></contrib><contrib contrib-type="author"><name><surname>Masoumipoor</surname><given-names>Masoumeh</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Janzadeh</surname><given-names>Atousa</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Nasirinezhad</surname><given-names>Farinaz</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Kerdari</surname><given-names>Mahdieh</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Soleimani</surname><given-names>Maryam</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Various agents including direct nerve and spinal cord trauma; viral infections and metabolic diseases such as diabetes may trigger neuropathic pain (NP). As clinical laboratory examination shows that the neuropathic pain is often independent of any obvious signs of inflammation; it is sometimes described as &#x02018;non-inflammatory pain&#x02019;. The International Association for the Study of Pain (IASP) de&#x0fb01;nes NP as pain &#x02018;initiated or caused by a primary lesion or dysfunction in the nervous system&#x02019; (<xref rid="R1" ref-type="bibr">1</xref>). Despite of this definition that refers to mainly the cause, the exact mechanism of the events following the injury or trauma remain poorly understood. There are certain evidences that confirm cellular events following injury that mainly emphasize on the role of cellular organelles such as mitochondria (<xref rid="R2" ref-type="bibr">2</xref>). The first mitochondrial dysfunction described in the 1960s and during the last two decades the role that it plays in health, disease, and aging have been reported by others. Several studies showed that free radical; oxidative stress and inflammation have major role in the pathogenesis of neurodegenerative diseases, such as amyotrophic lateral sclerosis, epilepsy, migraine headaches, strokes, Alzheimer and Parkinson&#x02019;s diseases and NP resulting in mitochondrial dysfunction (<xref rid="R3" ref-type="bibr">3</xref>-<xref rid="R5" ref-type="bibr">5</xref>). Mitochondrial dysfunction has been shown in rats with painful peripheral neuropathies (<xref rid="R6" ref-type="bibr">6</xref>). It is reported that the major reason for mitochondrial dysfunction is reactive oxygen species (ROS) production thus antioxidant could be a good candidate and therapeutic strategy for decreasing the severity of the damage to mitochondria. Ubiquinone CoQ<sub>10</sub> is a vital cofactor in complexes I to III of the mitochondrial electron transport chain, which acts as an electron acceptor and also a key component of the mitochondrial respiratory chain for adenosine triphosphate synthesis (<xref rid="R7" ref-type="bibr">7</xref>-<xref rid="R9" ref-type="bibr">9</xref>). In addition to its unique role in mitochondria, CoQ<sub>10</sub> is a potent antioxidant and scavenging free radicals and inhibiting lipid per oxidation (<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R11" ref-type="bibr">11</xref>). The CoQ<sub>10</sub> has bioenergetics and anti-inflammatory effects that protect against apoptosis of neurons and cells from oxidative stress in vivo (<xref rid="R12" ref-type="bibr">12</xref>&#x02013;<xref rid="R15" ref-type="bibr">15</xref>). The previous studies carried out by Kohli et al. and El-Abhar et al. showed that CoQ<sub>10</sub> possesses anti-ulcer potential as well (<xref rid="R16" ref-type="bibr">16</xref>,<xref rid="R17" ref-type="bibr">17</xref>). Ghule et al. have documented that CoQ<sub>10</sub> provides protection against isoproterenol-induced cardiotoxicity and cardiac hypertrophy preclinically, and Burke et al. showed that it could be used as a treatment for systolic hypertensive patient (<xref rid="R18" ref-type="bibr">18</xref>,<xref rid="R19" ref-type="bibr">19</xref>). CoQ<sub>10</sub> treatment improves endothelial function and blood flow; thus, long-term treatment may be effective by improving oxygenation of the peripheral nerves (<xref rid="R20" ref-type="bibr">20</xref>). Hernandez-Ojeda et al. reported that a 12-wk treatment with ubiquinone significantly improves diabetic polyneuropathy in patients with type II diabetes (<xref rid="R21" ref-type="bibr">21</xref>). </p><p> As drug therapy it may lead to unwanted and undesirable side effects hence other therapeutic procedures including physical methods have been improved due to the absence of side effects (<xref rid="R22" ref-type="bibr">22</xref>). Low level laser therapy (LLLT) is well known for its anti-inflammatory, analgesic and tissue repair effects (<xref rid="R23" ref-type="bibr">23</xref>-<xref rid="R28" ref-type="bibr">28</xref>). It seems that some mechanisms of low level laser involved in mitochondrial respiratory chain and oxidative stress biomarkers (<xref rid="R29" ref-type="bibr">29</xref>). According to Karu et al. laser exposure can lead to an increase in mitochondrial electrochemical activity and ATP synthesis (<xref rid="R30" ref-type="bibr">30</xref>). Eells et al. showed that cytochrome c oxidase is the main photo acceptor of laser light (<xref rid="R31" ref-type="bibr">31</xref>). Other researchers postulated that laser therapy influences oxidative stress parameters such as changes in antioxidant enzyme activity and the production of ROS (<xref rid="R32" ref-type="bibr">32</xref>-<xref rid="R35" ref-type="bibr">35</xref>). The absorption of laser light have shown to accelerate the transfer of electrons (respiratory chain) and induces an initial ROS production, specifically increasing the production of superoxide anion (<xref rid="R32" ref-type="bibr">32</xref>). Despite the known clinical effects of LLLT and CoQ<sub>10</sub>, to our knowledge there are no study on combined effects on NP. Thus the present study designed first to compare the effects of CoQ<sub>10</sub> and LLLT on neuropathic pain model and explore the combination of these two procedures on same model. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><sec id="s2-1"><title>Animals</title><p> Sixty adult male Wistar rats (250&#x02013;320 g) were used in this study with food and water ad libitum. The animals were divided into six groups (n=10) as follows: </p><list list-type="bullet"><list-item><p> CCI group: animals that were subjected to surgical procedure, without undergoing treatment&#x0200f;. </p></list-item><list-item><p> Coenzyme Q<sub>10</sub> group: received 200mg /kg/day intraperitoneal (i.p) injection of Co Q<sub>10</sub> (Tishcon, NY,USA&#x0200f;) </p></list-item><list-item><p> Vehicle group: received 200mg /kg/day (i.p) injection vehicle of CoQ<sub>10</sub> </p></list-item><list-item><p> Laser therapy group (980nm): laser irradiation with energy density of 4 J/cm<sup>2</sup> and intensity of 0.248 (W/cm<sup>2</sup>&#x0200f;) </p></list-item><list-item><p> Laser 980nm+CoQ<sub>10</sub> group: received 200mg /kg/day (i.p) injection of CoQ<sub>10</sub> and laser 980nm&#x0200f;. </p></list-item><list-item><p> Laser 980nm+Vehicle group: received 200mg /kg/day (i.p) injection vehicle of CoQ<sub>10</sub> and laser 980nm&#x0200f;. </p></list-item></list><p> All animals were subjected to the behavioral evaluation before surgery. To induce NP, the sciatic nerve injury model described by Bennett and Xie was used (<xref rid="R36" ref-type="bibr">36</xref>). </p><sec id="s2-2"><title>Treatment</title><p> The day after surgery, vehicle and CoQ<sub>10</sub> groups received 200 mg/kg (i.p) injection of CoQ<sub>10</sub> (CoQ<sub>10</sub> is in LiQsorb form) for 14 days. </p><p> A CW diode laser emitter with following specification was used in this study. A laser with wavelength of 980 nm, power of 70mW (Aixiz; model: AH980-6015AC), the energy density of 4 J/cm<sup>2</sup>, power density of 0.248 W/cm<sup>2</sup> and beam area ~ 0.238 cm<sup>2</sup>was used. The irradiation was as follows: two points on two ends of surgical incision and another at the mn time 11.3s for visible wavelength and 16.13s for NIR one. </p><p> Laser calibration was done prior to use. Three points of the surgical incision were irradiated transcutaneously with no direct skin conidpoint. Treatment was started on the first day after the surgery and was continued for two weeks daily at the same time between 10-12 AM. </p></sec><sec id="s2-3"><title>Functional analysis</title><p> Behavioral study was carried out before and after surgery on 14<sup>th</sup> day. </p></sec><sec id="s2-4"><title> Thermal withdrawal threshold </title><p>Using a Plantar Test apparatus (UgoBasile, Italy) thermal hyperalgesia, the latency to withdrawal of the hind paws from a focused beam of radiant heat applied to the plantar surface. The animals were placed in an acrylic box with glass floor and the plantar surface of their hind paw exposed to a beam of infrared radiant heat. The paw withdrawal latencies were recorded at infrared intensity of 50 and three trials for the right hind paws were performed and for each reading, the apparatus was set at a cut-off time of 25s. Each trial separated by an interval time of 5 minutes.</p></sec><sec id="s2-5"><title> Mechanical withdrawal threshold </title><p>Mechanical paw withdrawal thresholds were assessed with the Randall&#x02013;Selitto method using an Analgesy-meter apparatus (UgoBasile, Italy). This instrument exerts a force that was increased at a constant rate. The force was applied to the hind paw of the rat, which was placed on a small plinth under a cone shaped pusher with a rounded tip (1.5 mm in diameter).The rat was held upright with the head and limb to be tested free, but most of its body cradled in the hands of the experimenter. The paw was then put under the pusher until the rat withdrew the hind paw. Each hind paw was tested twice, with a 10 min interval between the measurements and mechanical paw withdrawal thresholds were calculated as the average of two consecutive measurements.</p></sec><sec id="s2-6"><title> Statistical analysis </title><p>Using SPSS 19.0 statistical analysis was done, and the results presented as means &#x000b1; SD, and p-value less than 0.05 was considered to be significant.</p></sec></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> For functional evaluation of gait we used the Plantar Test and Randall&#x02013;Selitto method preoperatively and those recorded on the 14<sup>th</sup> day after surgery. Our results were as follow: </p><sec id="s3-1"><title>Plantar Test</title><p> The thermal withdrawal threshold of the control group was, on average, 18.91&#x000b1;4.08 sec of the data recorded prior to the injury. For the CCI group 12.42&#x000b1;4.82 sec and10.70&#x000b1;5.02 sec on the 7<sup>th</sup> day and the 14<sup>th</sup> day respectively after surgery. </p><p> For the CoQ<sub>10</sub> group, the mean values were 18.91&#x000b1;4.09 sec and 16.15&#x000b1;4.52 sec on the 7<sup>th</sup> and the 14<sup>th</sup> day respectively after surgery. For the Vehicle group, the mean values were 14.67&#x000b1;5.64 sec and 13.86&#x000b1;4.91 sec on the 7<sup>th</sup> and the 14<sup>th</sup> respectively day after surgery. For the LLLT 980nm group, the mean values were 16.13&#x000b1;4.11 sec and 14.18&#x000b1;3.35 sec on the 7<sup>th</sup> and the 14<sup>th</sup> day respectively after surgery. For the LLLT 980nm+CoQ<sub>10</sub> group, the mean values were 19.02&#x000b1;3.02 sec and 19.11&#x000b1;4.61 sec on the 7<sup>th</sup> and the 14<sup>th</sup> day after surgery. For the LLLT 980nm+Vehicle group, the mean values were 17.64&#x000b1;5.22 sec and 17.54&#x000b1;4.96 sec on the 7<sup>th</sup> and the 14<sup>th</sup> day respectively after surgery. By using ANOVA, the results among the CCI and treatment groups considered significant. However, there were no significant difference between the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days of the CoQ<sub>10</sub> group and the control group; but there was significant difference between the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days in the LLLT 980nm group and the control group (p&#x0003c; 0.01, p&#x0003c; 0.001) respectively; and there were no significant difference between the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days in the LLLT 980nm+CoQ<sub>10</sub> group and the Control group; also between LLLT 980nm+vehicle group and the control group. The comparison between the LLLT 980nm+CoQ<sub>10</sub> group and LLLT 980nm+Vehicle group showed there was no significant difference in both values. There was no significant difference between the 7<sup>th</sup> post-surgery days in the LLLT 980nm+CoQ<sub>10</sub> group and the CoQ<sub>10</sub> group and there was significant difference between the 7<sup>th</sup> post-surgery day of the LLLT 980nm+Q<sub>10</sub> group and the LLLT 980nm group at (p&#x0003c; 0.01). Comparison of the results among the LLLT 980nm+CoQ<sub>10</sub> group and treatment groups (CoQ<sub>10</sub> and LLLT 980nm) illustrated significant difference (p&#x0003c; 0.05, p&#x0003c; 0.001) on the 14<sup>th</sup> day after surgery (<xref ref-type="fig" rid="F1">Fig 1</xref>&#x00026;<xref ref-type="fig" rid="F2">Fig 2</xref>). </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-58-g001"/><statement><p> Mean values of the Thermal Paw Withdrawal Threshold obtained from the groups during the study period (before surgery (control), the 7<sup>th</sup> day after surgery). Asterisks represent significant differences from CCI group (*** p&#x0003c; 0.001) and (### p&#x0003c; 0.001, ## p&#x0003c; 0.01) represent significant differences from control group. </p></statement></fig><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-58-g002"/><statement><p> Mean values of the Thermal Paw Withdrawal Threshold obtained from the groups during the study period (before surgery (control), the 14<sup>th</sup> day after surgery). Asterisks represent significant differences from CCI group (*** p&#x0003c; 0.001, **p&#x0003c; 0.01, *P&#x0003c; 0.05) and (### p&#x0003c; 0.001, ## p&#x0003c; 0.01) represent significant differences from control group. </p></statement></fig></sec><sec id="s3-2"><title>Randall&#x02013;Selitto method</title><p> The mean of mechanical withdrawal threshold of the control group was 19.18&#x000b1;4.66 g before surgery. The means for the CCI group were 10.17&#x000b1;4.18 g and 9.15&#x000b1;4.20 g on the 7<sup>th</sup> and the 14<sup>th</sup> day respectively after surgery. For the CoQ<sub>10</sub> group, the mean values were 15.29&#x000b1;5.50 g and 13.19&#x000b1;4.12 g on the 7<sup>th</sup> and the 14<sup>th</sup> day after surgery. For the Vehicle group, the mean values were 12.09&#x000b1;3.92 sec and 9.75&#x000b1;3.15 sec on the 7<sup>th</sup> and the 14<sup>th</sup> day respectively after surgery. For the LLLT 980nm group, the mean values were 14.15&#x000b1;4.25 g and 12.35&#x000b1;5.28 g on the 7<sup>th</sup> day and the 14<sup>th</sup> day respectively after surgery. For the LLLT 980nm+CoQ<sub>10</sub> group, the mean values were 14.65&#x000b1;5.77 sec and 12.72&#x000b1;4.63 sec on the 7<sup>th</sup> and the 14<sup>th</sup> day respectively after surgery. For the LLLT 980nm+Vehicle group, the mean values were 13.6&#x000b1;3.50 sec and 10.98&#x000b1;2.99 sec on the 7<sup>th</sup> and the 14<sup>th</sup> day respectively after surgery. Statistical analysis indicated that the difference between control group and the CCI group on the 7th, 14th post-surgery days, was significant at (p&#x0003c; 0.001); also difference between CoQ<sub>10</sub> group and the CCI group on the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days, was significant (p&#x0003c; 0.001, p&#x0003c; 0.01); and difference between LLLT 980nm group and the CCI group on the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days, was also significant (p&#x0003c;0.01, p&#x0003c;0.05). Moreover, difference between LLLT 980nm+CoQ<sub>10</sub> group and the CCI group on the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days, was significant (p&#x0003c; 0.001, p&#x0003c; 0.05). Difference between LLLT 980nm+Vehicle group and the CCI group on the 7<sup>th</sup>post-surgery day was significant (p&#x0003c; 0.01) but there was no significant difference on 14<sup>th</sup> day post-surgery. Also, there was significant difference between the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days of the treatment groups and the control group (p&#x0003c; 0.001). In a comparison between the LLLT 980nm+CoQ10 and LLLT 980nm+Vehicle groups, there were no significant differences in the values on the 7<sup>th</sup>, 14<sup>th</sup> post-surgery days and comparison between the LLLT 980nm+CoQ<sub>10</sub> group and treatment groups (CoQ<sub>10</sub> and LLLT 980nm), showed no significant difference on the 7<sup>th</sup>, 14<sup>th</sup> days (<xref ref-type="fig" rid="F3">Fig 3</xref> &#x00026; <xref ref-type="fig" rid="F4">Fig 4</xref>). </p><fig id="F3" orientation="portrait" position="float"><label> Fig. 3 </label><graphic xlink:href="MJIRI-28-58-g003"/><statement><p> Mean values of the Mechanical Paw Withdrawal Threshold obtained from the groups during the study period (before surgery (control), and 7<sup>th</sup> day after surgery).Asterisks represent significant differences from CCI group (*** p&#x0003c;0.001, ** p&#x0003c; 0.01) and (### p&#x0003c; 0.001, ## p&#x0003c; 0.01) represent significant differences from control group. </p></statement></fig><fig id="F4" orientation="portrait" position="float"><label> Fig. 4 </label><graphic xlink:href="MJIRI-28-58-g004"/><statement><p> Mean values of the Mechanical Paw Withdrawal Threshold obtained from the groups during the study period (before surgery (control), and 14<sup>th</sup> day after surgery).Asterisks represent significant differences from CCI group (*** p&#x0003c;0.001, ** p&#x0003c; 0.01, * p&#x0003c; 0.05) and (### p&#x0003c; 0.001) represent significant differences from control group. </p></statement></fig></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Our finding showed that LLLT and CoQ10 alone and combined after 1 week increase thermal and mechanical sense thresholds compared to the CCI animals. The results were the same for two weeks of intervention. </p><p> To explain the cellular mechanisms that led to these findings, the nature and pathology of NP should be considered. It is generally accepted that neuropathic pain hyperalgesia depends on an increase in proinflammatory cytokines (<xref rid="R37" ref-type="bibr">37</xref>). In addition, NO a diffusible multifunctional transcellular messenger also contribute to hyperalgesia. It acts directly by sensitizing peripheral nerve or indirectly by influencing the local inflammatory process. NO is also involved in the transmission and modulation of nociceptive information at the periphery, spinal cord and supraspinal level (<xref rid="R38" ref-type="bibr">38</xref>). Other factors including cytokines, such as tumor necrosis factor alpha (TNF-&#x003b1;) and interleukin-10 (IL-10) are important for pain behavior following nerve injury. It is shown that these factors are associated with mitochondrial dysfunction and lead to increasing ROS production and oxidative stress generation (<xref rid="R39" ref-type="bibr">39</xref>). In addition to their pronociceptive action, TNF-&#x003b1; and NO act as proapoptotic messengers (<xref rid="R43" ref-type="bibr">43</xref>,<xref rid="R44" ref-type="bibr">44</xref>). TNF-&#x003b1; is responsible for a cascade of cellular events that result in mitochondrial dysfunction. It acts via reducing complex III activity, increasing ROS production and causing damage to mtDNA (<xref rid="R40" ref-type="bibr">40</xref>-<xref rid="R41" ref-type="bibr">41</xref>). </p><p> The LLLT is widely used for its cellular therapeutic effects, which lead to wound healing, pain relief, reduction of edema and inflammation. During the period of LLLT, absorption of red or near-infrared photons by cytochrome c oxidase in the mitochondrial respiratory chain causes an increase in cellular respiration (<xref rid="R42" ref-type="bibr">42</xref>). Silveira et al. evaluated the effects of low level laser therapy (904 nm) with varied irradiation intensity on mitochondrial respiratory chain activity and some oxidative stress markers. They showed LLLT reduces the complex II activity of the mitochondrial respiratory chain and they concluded that LLLT could protect the cell against oxidative damage to membrane lipids, due to the decreases in both superoxide anion production and oxidative stress. (<xref rid="R43" ref-type="bibr">43</xref>). The reduction of oxidative damages has been postulated as one of the main mechanisms following using LLLT which induces an increase in SOD activity, thus lead to decrease in tissue damages and the maximization of the healing process (<xref rid="R44" ref-type="bibr">44</xref>-<xref rid="R46" ref-type="bibr">46</xref>). Various mechanisms for therapeutic efficacy of low level laser irradiation have been proposed, including increases in mitochondrial activity and ATP levels, production of low levels of reactive oxygen species, induction of transcription factors NF-&#x003ba;B, and inhibition of apoptosis (<xref rid="R47" ref-type="bibr">47</xref>). Khalil et al. have shown that N-type Ca<sup>2+</sup> channel activation plays a role in nerve repair (<xref rid="R48" ref-type="bibr">48</xref>). Following increased ATP and protein synthesis after LLLT, the expressions of growth factors and cytokines increase and activation of calcium channels resulting in increased intracellular calcium concentration, ultimately lead to cell survival (<xref rid="R49" ref-type="bibr">49</xref>-<xref rid="R55" ref-type="bibr">55</xref>). </p><p> Increases in pain relief factor such as beta-endorphins, blocked depolarization of C-fiber afferent nerves (<xref rid="R56" ref-type="bibr">56</xref>), axonal sprouting and nerve cell regeneration (<xref rid="R57" ref-type="bibr">57</xref>), decreased bradikynin levels, ion channel normalization (<xref rid="R58" ref-type="bibr">58</xref>), stabilization of the cell membrane (<xref rid="R59" ref-type="bibr">59</xref>), enhancement of ATP synthesis (<xref rid="R60" ref-type="bibr">60</xref>), stimulated vasodilation along with release histamine, NO and serotonin (<xref rid="R61" ref-type="bibr">61</xref>), reduction in interleukin-1&#x003b2; levels (<xref rid="R62" ref-type="bibr">62</xref>), increasing angiogenesis (<xref rid="R63" ref-type="bibr">63</xref>), enhancing superoxide dismutase (<xref rid="R64" ref-type="bibr">64</xref>), decreasing C-reactive protein and neopterin levels (<xref rid="R65" ref-type="bibr">65</xref>) are other reported mechanisms for reducing pain by red and near infrared light . </p><p> Regarding CoQ10 it is reported that it can decrease neurological symptoms in patients with Parkinson and Huntington diseases. It is also shown that CoQ<sub>10</sub> may play an important role in neuroprotection against diabetic neuropathy and other neurodegenerative disorders (<xref rid="R66" ref-type="bibr">66</xref>, <xref rid="R67" ref-type="bibr">67</xref>). Zhang et al. demonstrated the potential benefits of CoQ<sub>10</sub> as a potent antioxidant and its ability to relieve neuropathic pain in the type I diabetic mouse model (<xref rid="R68" ref-type="bibr">68</xref>). Also, Shi et al. reported that the CoQ<sub>10</sub> may represent a promising therapeutic strategy for type II diabetic neuropathy (<xref rid="R69" ref-type="bibr">69</xref>). The CoQ<sub>10</sub> neuroprotection may also leads to functional improvement of respiratory chain activity and prevention of neuronal apoptosis (<xref rid="R68" ref-type="bibr">68</xref>). It also acts throughout inhibiting oxidative stress and reducing inflammation by down-regulating of proinflammatory factors (<xref rid="R11" ref-type="bibr">11</xref>). The CoQ<sub>10</sub> intensely reduced apoptotic cell death, attenuated ATP decrease, and hindered DNA fragmentation elicited by all apoptotic stimuli that is accompanied by inhibition of mitochondrial depolarization, cytochrome c release (<xref rid="R70" ref-type="bibr">70</xref>). Tsai et al. showed that CoQ<sub>10</sub> significantly reduced the activation of NF-&#x003ba;B, suppressed the expression of P<sub>53</sub> and the expression of Bax and led to a significant increase in expression of the antiapoptotic protein Bcl-2 and suppressing oxidative stress-related responses by modulating NO-related signaling (<xref rid="R71" ref-type="bibr">71</xref>). The role of CoQ<sub>10</sub> is to reduce hypertension-mediated oxidative damage (<xref rid="R72" ref-type="bibr">72</xref>), increases the antioxidant capacity of glutathione reductase and superoxide dismutase (SOD) also reported (<xref rid="R73" ref-type="bibr">73</xref>). Nonetheless the CoQ<sub>10</sub> treatment improves endothelial function and blood flow; thus, long-term treatment may be effective by improving oxygenation of the peripheral nerves (<xref rid="R74" ref-type="bibr">74</xref>). An increase in the concentration of CoQ<sub>10</sub> might affect mitochondrial respiratory function and early supplementation should be administrated in cases of deficiency (<xref rid="R77" ref-type="bibr">77</xref>). Since these events are due to mitochondrial PTP opening, Papucci et al. suggested the antiapoptotic activity of CoQ<sub>10</sub> could be related to its ability to prevent PTP opening and thus apoptosis (<xref rid="R70" ref-type="bibr">70</xref>). It is reported by Singh et al. that CoQ<sub>10</sub> supplements could increase the levels of vitamins A, C, and E (<xref rid="R75" ref-type="bibr">75</xref>, <xref rid="R76" ref-type="bibr">76</xref>), hence some of its effects might be related to this function. </p><p> Since better results was obtained in combined therapy (CoQ<sub>10</sub>+LLLT 980nm) and based on our knowledge from literature we believe that there might be separated or synergetic mechanisms for this phenomenon. It is possible that each of these modalities that we used acts in its own way to reduce pain, prevents apoptosis or inhibits the inflammation process. There are numerous evidences presented that support this possibility. It is also possible that they acted together with same or other mechanisms. From this point of view it is shown that LLLT and CoQ<sub>10</sub> under different or same pathway are simultaneously able to inhibit proinflamatory process (<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R23" ref-type="bibr">23</xref>, <xref rid="R25" ref-type="bibr">25</xref>), enhancing mitochondrial respiratory chain (<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R42" ref-type="bibr">42</xref>) inhibiting or down regulating the apoptotic cascade (<xref rid="R43" ref-type="bibr">43</xref>, <xref rid="R68" ref-type="bibr">68</xref>, <xref rid="R71" ref-type="bibr">71</xref>) and decreasing the effects of oxidative stress (<xref rid="R72" ref-type="bibr">72</xref>-<xref rid="R74" ref-type="bibr">74</xref>, <xref rid="R43" ref-type="bibr">43</xref>-<xref rid="R46" ref-type="bibr">46</xref>). The specific mechanisms for these events are still unknown and more studies needed to explain them, and the possibility of adverse effects of LLLT and CoQ<sub>10</sub> should be considered in future. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> The CoQ<sub>10</sub> can prevent deleterious effects of nerve injury and laser application at 980nm was also effective in promoting early functional recovery. The combination of CoQ<sub>10</sub> with LLLT showed better effects than each one alone. There might be cellular and molecular synergism in simultaneous use of CoQ<sub>10</sub> and LLLT. Under careful guide clinical application of these modalities may be used in treatment of the NP. </p></sec>
Causality relationships between coagulation factors in type 2 diabetes mellitus: path analysis approach
<p> <bold>Background:</bold> Type 2 diabetic mellitus patients are amongst the most susceptible groups to vascular abnormalities, which predominantly lead to myocardial disease. The hypercoagulable state has been widely studied by researchers as being the major suspicious mechanism facilitating the consecutive chain of molecular events leading to these complications. However, there is no consensus on the definition of the hypercoagulable state with respect to coagulation quantities, their interrelations and basic factor(s) initiating this pathogenic event, by which the prognosis of myocardial complications could be determined. </p><p> <bold>Methods:</bold> Path analysis was used to study the interactions between coagulation factors as well as other factors beyond coagulation factors in relation with pathogenic events in both diabetics and healthy subjects. In the present work, coagulation factors of 40 healthy and 40 type 2 diabetics were determined experimentally. The data were then analyzed using SPSS and AMOS software. Multivariate regression analysis was done to draw path diagrams. </p><p> <bold>Results:</bold> Our results show that FII, as the main cause for hypercoagulable state, is directly induced by FX and FVIII in normal individuals and by FX, FXI, FV and VWF cofactors in diabetic patients. </p><p> <bold>Conclusion:</bold> In general, our findings showed complicated relationship between coagulation factors and their effects either separately or combined. </p>
<contrib contrib-type="author"><name><surname>Dayer</surname><given-names>Mohammad Reza</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mard-Soltani</surname><given-names>Maysam</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Dayer</surname><given-names>Mohammad Saaid</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Alavi</surname><given-names>Sayed Mohammad Reza</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Type 2 diabetes mellitus is one of the mostly incriminated conditions for the onset of atherosclerosis, endothelial, vascular and thrombotic complications (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R5" ref-type="bibr">5</xref>). Increased concentration of coagulation factors is widely reported in type 2 diabetes mellitus (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R8" ref-type="bibr">8</xref>). Fibrinogen coagulation factor with plasma concentration of 200-400mg/dL and half-life of 4-6 days is converted to insoluble protein fibrin, by FIIa (<xref rid="R9" ref-type="bibr">9</xref>-<xref rid="R10" ref-type="bibr">10</xref>). In type 2 diabetic patients with or without vascular complications, fibrinogen concentrations do not indicate remarkable difference; hence fibrinogen may not be an important causal factor for vascular complications (<xref rid="R11" ref-type="bibr">11</xref>-<xref rid="R12" ref-type="bibr">12</xref>). </p><p> Thrombin or FIIa is a serine protease enzyme produced by proteolytic cleavage of its precursor FII (prothrombin) by FXa with aim of FV and phospholipid membrane (<xref rid="R13" ref-type="bibr">13</xref>-<xref rid="R15" ref-type="bibr">15</xref>). In diabetics, FII has been reported neither to increase in concentration (more than 10mg/dL) nor to be a risk factor for vascular events (<xref rid="R15" ref-type="bibr">15</xref>). However FX is a plasma glycoprotein with mean concentration of 1mg/dL and a half-life of 2-2.5 days. The activation pathway of FX to FXa is the committed step stands at the crossroad between intrinsic and extrinsic pathways of coagulation. Coagulation FV with 0.4-1.4 mg/dL and 0.5-1.5 days of half-life in turn is activated by FXa and FIIa. There are many reports which show no significant increase in FV in diabetic patients without prognostic importance (<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R16" ref-type="bibr">16</xref>-<xref rid="R17" ref-type="bibr">17</xref>). </p><p> In the intrinsic pathway FX is activated by FIXa via formation of TENASE complex with coagulation factor VIII and calcium ions on phospholipid membranes. The plasma concentration of FIX showed no remarkable difference between normal and diabetics either with or without artery complications, although elevated FIX was reported in patients with myocardial infarction (<xref rid="R16" ref-type="bibr">16</xref>, <xref rid="R18" ref-type="bibr">18</xref>-<xref rid="R19" ref-type="bibr">19</xref>). Coagulation FVIII which is activated by FIIa, tends to increase in diabetes mellitus but with no correlation with vascular complications (<xref rid="R19" ref-type="bibr">19</xref>-<xref rid="R20" ref-type="bibr">20</xref>). Some researchers hold that increased concentrations of both FVIII and its carrier vWF (von-Willebrand factor) in diabetes mellitus render them susceptible to heart attack (<xref rid="R21" ref-type="bibr">21</xref>-<xref rid="R22" ref-type="bibr">22</xref>). It has been shown that the activation of FXI and FIX is required for continued formation of FIIa (<xref rid="R23" ref-type="bibr">23</xref>). In diabetic patients, there are elevated concentrations of plasma FXI and FXII of which the former factor (FXI) has been shown to be involved in myocardial infarction (<xref rid="R16" ref-type="bibr">16</xref>, <xref rid="R20" ref-type="bibr">20</xref>, <xref rid="R24" ref-type="bibr">24</xref>-<xref rid="R27" ref-type="bibr">27</xref>). This coagulation factor exerts its pathogenic effect via FIX and FX activation (<xref rid="R23" ref-type="bibr">23</xref>, <xref rid="R28" ref-type="bibr">28</xref>). Multivariate regression analysis showed that FXI is an independent factor for thrombotic complications (<xref rid="R29" ref-type="bibr">29</xref>-<xref rid="R32" ref-type="bibr">32</xref>). Coagulation FXII seems of no prognostic importance in vascular complication. </p><p> In extrinsic pathway, FX is activated by FVII/TF complex on phospholipid membrane in the presence of calcium ions. Coagulation FVII circulates in plasma as a zymogen of a short half-life (5-8 hours) at 0.05mg/dL concentration (<xref rid="R33" ref-type="bibr">33</xref>-<xref rid="R34" ref-type="bibr">34</xref>). High concentration of FVII acts as independent variable that correlates with ischemic heart disease (<xref rid="R10" ref-type="bibr">10</xref>-<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R13" ref-type="bibr">13</xref>, <xref rid="R35" ref-type="bibr">35</xref>). In contrast to FVIIa, inactivated FVII shows partial enzymatic activity at high concentration in the presence of TF, so that it may activate FIX; promote FXa formation and leads to artery problems (<xref rid="R36" ref-type="bibr">36</xref>-<xref rid="R37" ref-type="bibr">37</xref>). Tissue factor is necessary for the initiation of extrinsic coagulation pathway through FVII activation. (<xref rid="R3" ref-type="bibr">3</xref>, <xref rid="R38" ref-type="bibr">38</xref>-<xref rid="R39" ref-type="bibr">39</xref>). Raised blood glucose and hyperinsulinemia in T2DM induces a marked increase in TF activity (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R20" ref-type="bibr">20</xref>, <xref rid="R40" ref-type="bibr">40</xref>-<xref rid="R42" ref-type="bibr">42</xref>). There are reports showing that TF is increased in coronary complications (<xref rid="R43" ref-type="bibr">43</xref>-<xref rid="R44" ref-type="bibr">44</xref>) </p><p> Since FII is the last target of coagulation cascade either from intrinsic or extrinsic origin play a determinant role in initiation of vascular complication, this study has chosen it as a most important dependent variable in a hypothetical model or path resemble coagulation cascade to edit and verify it as a causality model for coagulation factors. The main purpose of the present study is to extract a path model using our hypothetical model and experimental data obtained for diabetics and normal individuals via multivariate regression analysis (path analysis) (<xref rid="R45" ref-type="bibr">45</xref>-<xref rid="R46" ref-type="bibr">46</xref>). The obtained model expected to be important model that gives a prospect on the path relations between coagulation factors e.g. the extent to which one factor affects other coagulation factors. This effect may be positive effect (threatening factors) or negative effect (safety factors) against vascular complications. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> <italic>Subjects Selection:</italic> Forty patients with type 2 diabetes mellitus were selected based on their medical history as well as forty normal volunteers, all with no history of micro/macro vascular complications, neuropathy, nephropathy, insulin therapy, psychiatric illness or smoking. Subjects with backgrounds of any acute or chronic diseases were excluded. Chronic diseases of retinopathy were followed by regular checking of patients' visual acuity, nephropathy (rise in serum creatinine concentration and microalbuminurea) and foot sensation. All subjects were nominated by Dezfoul Ganjavian Hospital, Khuzestan Province, IRAN. The body mass index, BMI, was calculated to be 18 to 25 kg/m<sup>2</sup>. The only medication used by diabetics was anti-diabetic metformin pill having no insulin. </p><p> <italic>Sample Preparation:</italic> Blood samples were collected from 12-h fasted subjects. Plasma was prepared by twice centrifugation at 1500&#x000d7;g for 10 min each at 15&#x02013;18 &#x000b0;C and was then stored in polypropylene tubes at &#x02212;70 &#x000b0;C until used for measurement. All samples were assayed in duplicate. Plasma samples showing evidence of hemolysis were discarded. </p><p> <italic>Analytical method:</italic> Fasting glucose of subjects was measured by PAD/GOD Method (CinnaGen Incorporation, Glucose Diagnostic Kit, Shahrak Ekbatan, Tehran, Iran). Duplicated glucose level of fasting blood plasma glucose of &#x02265;126 mg/dL was used as criteria for type 2 diabetes mellitus in patients with at least 8h fasting with no caloric intake. The patients were selected based on their medical history with no signs of insulin resistance and no history of insulin treatment. The coagulation assay method used for the determination of vWF, Fibrinogen, II, V, VII, VIII, IX, X, XI in plasma was performed as reported previously (<xref rid="R46" ref-type="bibr">46</xref>). All experimental procedures involving human participants were conducted with due attention to the guidelines approved by the Research Ethical Committee of Shahid Chamran University (Ahwaz, Iran). </p><sec id="s2-1"><title>Statistical analysis</title><p> <italic>Coagulation Factor Correlation:</italic> The correlation between coagulation factors results were analyzed using the Statistical Package for the Social Science (SPSS-PC, version 15. SPSS, Inc., Chicago, IL). The significance level of 0.05 was used for correlation tests. </p><p> <italic>Path analysis:</italic> Path analysis was used to investigate the relation between coagulation factors in normal and diabetic individuals and to survey probable causal effects of some coagulation factors on a certain coagulation factor. Path analysis made it possible to describe and interpret the correlation between some coagulation factors and their effects on coagulation factor II of 40 diabetics and 40 normal voluntrees in a path model using <bold>Amos16</bold> software (Arbuckle, J. L. (2006). Amos (Version 16.0) (Computer Program). Chicago: SPSS). The obtaind structural equation models were tested and confimed at the significance level of 0.05. The causality model defines some hierarchical regression models between coagulation factors and FII variable (the effect). Paths between variables are drawn from independent to dependent variables with directional arrow for every regression model (arrowhead on one end only). A two-way arrow between two variables indicates that the correlation between these two variables will remain unanalyzed. For every regression the total variance in dependent variable is theorized to be caused either by independent variables of the model or by extraneous variables (<bold>e</bold>) not considered in the model. An arrow from <bold>e</bold> indicates the influence of these extraneous variables. Hence, the total variances of dependent variable become the sum of variance caused by both independent variables and extraneous variables. Each independent variable may affect the dependent variable directly and/or indirectly, i.e. via mediation factors. Accordingly, the total effect of an independent variable on a certain dependent variable is the sum of its direct and indirect effects. Each path has a coefficient showing the standardized coefficient of regressing independent variable on dependent variable of the relevant path. A coefficient may be positive (increase of independent variable elevates dependent variable when all other independent variables are held constant) or negative (increase of independent variable decreases dependent variable). The indirect effect was determined by multiplying the path coefficients of intervening variables (<xref rid="R47" ref-type="bibr">47</xref>). </p></sec></sec><sec sec-type="results" id="s4"><title>Results</title><p> In order to construct path analysis model between coagulation factors we made a pair wise Pearson test between coagulation factors and only the factors showing significant 2-tailed correlations were selected as suitable for model construction. These data are shown in <xref ref-type="table" rid="T1">table 1(a</xref>-<xref ref-type="table" rid="T2">b)</xref>. The best refined model for our hypothetical model obtained by path analysis calculated for healthy subjects is presented in <xref ref-type="fig" rid="F1">figure 1</xref>. In this model, FX and FVIII are the only independent variables which showed direct effects on FII. FX seemed to be the determinant factor for FII and the major risk factor for vascular complications. <xref ref-type="table" rid="T3">Table 2</xref> summarizes path coefficients of total, direct and indirect effects of coagulation factors of the model on FII. As represented in <xref ref-type="table" rid="T3">table 2</xref>, the maximum total effect exerted by FX on FII is as large as 0.642. This is followed by FVIII, FVII, VWF and FXI which show positive effects of 0.514, 0.444, 0.299 and 0.211 respectively. Whereas FV shows negative effect of 0.170 on FII. However only FX and FVIII show direct effects on FII while, other factors show indirect effects. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Pearson correlation of coagulation factors with 2-tailed significance in parenthesis for a, 40 normal and b, 40 diabetic groups. <xref ref-type="table" rid="T1">Table 1-a</xref> </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td colspan="6" rowspan="1">VWF</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VII</td><td align="center" rowspan="1" colspan="1"> -0.226<break/>(0.399) </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">VII</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">II</td><td align="center" rowspan="1" colspan="1"> -0.156<break/>(0.536) </td><td align="center" rowspan="1" colspan="1"> 0.480<break/>(0.038) </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">II</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">X</td><td align="center" rowspan="1" colspan="1"> -0.456<break/>(0.066) </td><td align="center" rowspan="1" colspan="1"> 0.736<break/>(0.001) </td><td align="center" rowspan="1" colspan="1"> 0.637<break/>(0.003) </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">X</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1"> 0.712<break/>(0.003) </td><td align="center" rowspan="1" colspan="1"> -0.285<break/>(0.285) </td><td align="center" rowspan="1" colspan="1"> 0.329<break/>(0.182) </td><td align="center" rowspan="1" colspan="1"> -0.352<break/>(0.166) </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">XI</td><td align="center" rowspan="1" colspan="1"> -0.205<break/>(0.447) </td><td align="center" rowspan="1" colspan="1"> -0.163<break/>(0.532) </td><td align="center" rowspan="1" colspan="1"> 0.258<break/>(0.285) </td><td align="center" rowspan="1" colspan="1"> -0.050<break/>(0.839) </td><td align="center" rowspan="1" colspan="1"> 0.320<break/>(0.211) </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">XI</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">V</td><td align="center" rowspan="1" colspan="1"> -0.624<break/>(0.006) </td><td align="center" rowspan="1" colspan="1"> -0.015<break/>(0.952) </td><td align="center" rowspan="1" colspan="1"> -0.008<break/>(0.973) </td><td align="center" rowspan="1" colspan="1"> 0.334<break/>(0.151) </td><td align="center" rowspan="1" colspan="1"> -0.613<break/>(0.007) </td><td align="center" rowspan="1" colspan="1"> 0.038<break/>(0.879) </td></tr></tbody></table></table-wrap><table-wrap id="T2" orientation="portrait" position="float"><label> Table 1-b </label><caption><title> Table 1-b </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td colspan="9" rowspan="1">VWF</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VII</td><td align="center" rowspan="1" colspan="1"> 0.034<break/>(0.839) </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">VII</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">II</td><td align="center" rowspan="1" colspan="1"> -0.178<break/>(0.286) </td><td align="center" rowspan="1" colspan="1"> 0.694<break/>(&#x0003c;0.001) </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">II</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">X</td><td align="center" rowspan="1" colspan="1"> -0.043<break/>(0.797) </td><td align="center" rowspan="1" colspan="1"> 0.586<break/>(&#x0003c;0.001) </td><td align="center" rowspan="1" colspan="1"> 0.868<break/>(&#x0003c;0.001) </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">X</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1"> 0.144<break/>(0.387) </td><td align="center" rowspan="1" colspan="1"> -0.006<break/>(0.971) </td><td align="center" rowspan="1" colspan="1"> -0.046<break/>(0.779) </td><td align="center" rowspan="1" colspan="1"> 0.076<break/>(0.640) </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">FIB</td><td align="center" rowspan="1" colspan="1"> 0.256<break/>(0.121) </td><td align="center" rowspan="1" colspan="1"> 0.251<break/>(0.119) </td><td align="center" rowspan="1" colspan="1"> 0.200<break/>(0.215) </td><td align="center" rowspan="1" colspan="1"> 0.279<break/>(0.081) </td><td align="center" rowspan="1" colspan="1"> 0.377<break/>(0.016) </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">FIB</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">XI</td><td align="center" rowspan="1" colspan="1"> -0.008<break/>(0.9620 </td><td align="center" rowspan="1" colspan="1"> 0.336<break/>(0.036) </td><td align="center" rowspan="1" colspan="1"> 0.218<break/>(0.183) </td><td align="center" rowspan="1" colspan="1"> 0.359<break/>(0.025) </td><td align="center" rowspan="1" colspan="1"> 0.525<break/>(0.001) </td><td align="center" rowspan="1" colspan="1"> 0.377<break/>(0.018) </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">XI</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">V</td><td align="center" rowspan="1" colspan="1"> 0.303<break/>(0.064) </td><td align="center" rowspan="1" colspan="1"> 0.522<break/>(0.001) </td><td align="center" rowspan="1" colspan="1"> 0.485<break/>(0.002) </td><td align="center" rowspan="1" colspan="1"> 0.398<break/>(0.011) </td><td align="center" rowspan="1" colspan="1"> 0.206<break/>(0.201) </td><td align="center" rowspan="1" colspan="1"> 0.256<break/>(0.110) </td><td align="center" rowspan="1" colspan="1"> 0.428<break/>(0.007) </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">V</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">IX</td><td align="center" rowspan="1" colspan="1"> 0.070<break/>(0.679) </td><td align="center" rowspan="1" colspan="1"> 0.546<break/>(&#x0003c;0.001) </td><td align="center" rowspan="1" colspan="1"> 0.553<break/>(&#x0003c;0.001) </td><td align="center" rowspan="1" colspan="1"> 0.642<break/>(&#x0003c;0.001) </td><td align="center" rowspan="1" colspan="1"> 0.190<break/>(0.247) </td><td align="center" rowspan="1" colspan="1"> 0.186<break/>(0.257) </td><td align="center" rowspan="1" colspan="1"> 0.581<break/>(&#x0003c;0.001) </td><td align="center" rowspan="1" colspan="1"> 0.341<break/>(0.034) </td></tr></tbody></table></table-wrap><table-wrap id="T3" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Total, direct and indirect effects of coagulation factors on FII in the best path diagram obtained from path analysis shown in <xref ref-type="fig" rid="F1">figure1</xref>. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> Total <break/>effect </td><td align="center" rowspan="1" colspan="1"> Direct<break/> effect </td><td align="center" rowspan="1" colspan="1"> Indirect<break/> effect </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">V</td><td align="center" rowspan="1" colspan="1">-.170</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">-.170</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">XI</td><td align="center" rowspan="1" colspan="1">.211</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">.211</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VWF</td><td align="center" rowspan="1" colspan="1">.299</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">.299</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VII</td><td align="center" rowspan="1" colspan="1">.444</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">.444</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1">.514</td><td align="center" rowspan="1" colspan="1">.514</td><td align="center" rowspan="1" colspan="1">.000</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">X</td><td align="center" rowspan="1" colspan="1">.642</td><td align="center" rowspan="1" colspan="1">.698</td><td align="center" rowspan="1" colspan="1">-.056</td></tr></tbody></table></table-wrap><p> <xref ref-type="fig" rid="F2">Figure 2</xref> show the best refined model for our hypothetical model obtained by path analysis calculated for diabetic patients. In this model, FX, FV, VWF and FXI show direct effects. <xref ref-type="table" rid="T4">Table 3</xref> summarizes the total, direct and indirect effects of coagulation factors on FII. As depicted in <xref ref-type="table" rid="T4">Table 3</xref>, FX and FV have positive effects of 0.837 and 0.340 respectively on FII but VWF and FXI have negative effects of 0.254 and 0.222 respectively. In path model for diabetic patients, as in <xref ref-type="fig" rid="F2">Figure 2</xref>, all variables except FX, FXI, FV and VWF have only indirect effects on FII. The unique coagulation factor that affects FII by dual action of direct effects of 0.340 and indirect effects of 0.334, is FXI. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-59-g001"/><statement><p>The best path diagram obtained from multivariate done for 40 normal individuals. All variable are putted in a rectangle, a single headed arrow from independent variable shows a path relation. The standardized path coefficients are calculated for direct relations between independent and dependent variables and putted over on headed arrows. The error terms that are shown by e are uncorrelated with variables of the model and with each other.</p></statement></fig><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-59-g002"/><statement><p>The best path diagram obtained from multivariate done for 40 type2 diabetics. All variable are putted in a rectangle, a single headed arrow from independent variable shows a path relation. A double-headed arrow is connecting two variables that are merely correlated with our obvious path relations. The standardized path coefficients are calculated for direct relations between independent and dependent variables and putted over on headed arrows. The error terms that are shown by e are uncorrelated with variables of the model and with each other.</p></statement></fig><table-wrap id="T4" orientation="portrait" position="float"><label> Table 3 </label><caption><title> The total, direct and indirect coefficients obtained in casualty model in diabetic patient extracted from multivariate regression done for 40 patients with type 2 diabetes mellitus. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td style="border-width: 0px 1px 0pt 0px ; border-style: none solid none none;border-color:#fff8e8" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Total effect</td><td align="center" rowspan="1" colspan="1">Direct effect</td><td align="center" rowspan="1" colspan="1">Indirect effect</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VWF</td><td align="center" rowspan="1" colspan="1">-.254</td><td align="center" rowspan="1" colspan="1">-.254</td><td align="center" rowspan="1" colspan="1">.000</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Glc</td><td align="center" rowspan="1" colspan="1">-.101</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">-.101</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1">.038</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">.038</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">XI</td><td align="center" rowspan="1" colspan="1">.112</td><td align="center" rowspan="1" colspan="1">-.222</td><td align="center" rowspan="1" colspan="1">.334</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VII</td><td align="center" rowspan="1" colspan="1">.232</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">.232</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">V</td><td align="center" rowspan="1" colspan="1">.340</td><td align="center" rowspan="1" colspan="1">.340</td><td align="center" rowspan="1" colspan="1">.000</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">IX</td><td align="center" rowspan="1" colspan="1">.518</td><td align="center" rowspan="1" colspan="1">.000</td><td align="center" rowspan="1" colspan="1">.518</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">X</td><td align="center" rowspan="1" colspan="1">.837</td><td align="center" rowspan="1" colspan="1">.837</td><td align="center" rowspan="1" colspan="1">.000</td></tr></tbody></table></table-wrap><p> Next to coagulation factor X, Factor IX is an important factor which imposes variance on FII with positive effect of about 0.52, while, FV, FVII, FXI and FVIII are ranked at lower causative situations in terms of inducing variances on FII. This finding implies that increase in concentration of factors X, IX, V, VII, XI and VIII induce increase in FII variance and could be considered as threatening factors for cardiovascular problems given their total effects. This model shows that glucose and VWF exert negative effects on FII, counteracting the effects of other factors on vascular disease. The effect of VWF, as FVIII carrier, could be easily interpreted, since it takes up the free FVIII from plasma decreasing its active form involved in coagulation phenomenon. However, the negative effects of glucose on FII could not be so easily interpreted. </p></sec><sec sec-type="discussion" id="s5"><title>Discussion</title><p> As shown in <xref ref-type="fig" rid="F1">Figures 1</xref> and <xref ref-type="fig" rid="F2">2</xref>, the extent of correlation between coagulation factors in diabetics is more evident than in normal group i.e. in the case of diabetics, most of coagulation factors are correlated and hence the numbers of variables included in the model become higher than in the case of normal group. This may be the reason for the more hypercoagulable conditions stated in diabetic condition (<xref rid="R46" ref-type="bibr">46</xref>-<xref rid="R48" ref-type="bibr">48</xref>) .It is of great importance to remember that the main goal of the current research is to find out an acceptable model by which the biological events that lead to micro or macro vascular complication could be elucidated. </p><p> <italic>Model Fit Assessment:</italic> In order to assess how our model fits the data, we used the model to check the data fit degree. In this connection, we used some qualification parameters including (<xref ref-type="table" rid="T5">Table 4</xref>): </p><table-wrap id="T5" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Model fit parameter extracted using AMOS 16 software for two model obtained for normal and diabetic groups. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Chi-square</td><td align="center" rowspan="1" colspan="1">DF</td><td align="center" rowspan="1" colspan="1">P-value</td><td align="center" rowspan="1" colspan="1">GFI</td><td align="center" rowspan="1" colspan="1">RMSEA</td><td align="center" rowspan="1" colspan="1">CFI</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Diabetics</td><td align="center" rowspan="1" colspan="1">28.697</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">0.094</td><td align="center" rowspan="1" colspan="1">0.893</td><td align="center" rowspan="1" colspan="1">0.106</td><td align="center" rowspan="1" colspan="1">0.949</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Normal</td><td align="center" rowspan="1" colspan="1">12.302</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">0.582</td><td align="center" rowspan="1" colspan="1">0.898</td><td align="center" rowspan="1" colspan="1">0.000</td><td align="center" rowspan="1" colspan="1">0.901</td></tr></tbody></table></table-wrap><list list-type="order"><list-item><p> Chi-square statistic, as the first index used. This test is a single number that adds up all the differences between our actual data and the data expected if there is no difference. If the actual data and expected data (if no difference) are identical, the Chi-square value is zero. A bigger difference will give a bigger Chi-square value. The most important characteristic of this statistic is its p-value criterion. As shown, the calculated p-value exceeds 0.05. Therefore, both models (healthy normal and diabetic models) are consistent with the null hypothesis and hence according to this criterion, both models are acceptable models. </p></list-item><list-item><p> The second index used was GFI or the goodness of fit index. GFI says what proportion of the variance in the sample variance-covariance matrix is accounted for the model. This should exceed 0.9 for a good model. According to this index, both models are acceptable models but they are not good fit. </p></list-item><list-item><p> The Root Mean Square Error of Approximation or RMSEA is used as a measure of total error and estimates the lack of fitted model compared to the saturated model with all correlated factors linked to each other. RMSEA value of 0.05 or less indicates good fit, and 0.08 or less indicates adequate fit. Based on this index the model extracted here for normal individuals show a good model while the model of diabetic is a poor model. </p></list-item><list-item><p> Comparative Fit Index (CFI) is an index well performs well even with small size sample. This statistics ranges from 0.0 to 1.0 with values closer to 1.0 indicating good fit. Accordingly our models for healthy and diabetics show good fits (<xref rid="R47" ref-type="bibr">47</xref>). </p></list-item></list><p> However there are limitations for path models including the necessity of adequate sample size, normal distribution of the data and the presence of linear relation between studied variable which all match our data. </p><p> <italic>Bidirectional relations:</italic> <xref ref-type="table" rid="T6">Table 5</xref> shows simple correlations between the coagulation factors due to unanalyzed relation represented by two-headed arrows which seems to add values to the extracted models. Taking into account such correlations will enable including, within the model, certain invisible relations between coagulation factors which may not be perceived immediately. As mentioned earlier, there may be one- way relations between each pair of these coagulation factors or otherwise spurious associations caused by a third factor as if it be biological one. In this context, FV tends to correlate with VWF, and glucose with FVIII in a way to construct a plausible model as in <xref ref-type="table" rid="T6">Table 5</xref>. This finding may open a new horizon of research for studying the mechanisms behind such causality relationship and beyond. The correlation which exists between FV and either VWF or glucose in diabetic model is absent in normal model. So, what is the factor behind this discrepancy between diabetics and normal individuals? The other bidirectional relations are formed with glucose in diabetic patients. Glucose tends to have bidirectional association with each of FV, FVII and FVIII. This may lead to hypercoagulable states and hence life-threatening cardiovascular events in diabetic patients. </p><table-wrap id="T6" orientation="portrait" position="float"><label> Table 5 </label><caption><title> simple correlation for coagulation factors that connected with two headed arrows in diabetic model shown in <xref ref-type="fig" rid="F2">figure 2</xref>. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">VII</td><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1">V</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">vWF</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.23</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">GLC</td><td align="center" rowspan="1" colspan="1">0.30</td><td align="center" rowspan="1" colspan="1">0.38</td><td align="center" rowspan="1" colspan="1">0.36</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">VIII</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.18</td></tr></tbody></table></table-wrap></sec><sec sec-type="conclusion" id="s6"><title>Conclusion</title><p> Path analysis findings indicate that the analysis is a powerful method that helps to discover invisible relations between coagulation factors. Our results gave two acceptable models, which adequate fit the data for normal and diabetic patients. According to the first model for normal individuals, as shown in<xref ref-type="fig" rid="F1">Figure 1</xref> and <xref ref-type="table" rid="T3">Table 2</xref>, FX and FVIII induce the largest direct effects on FII with total effects of .642 and .514 respectively. On the other hand, FV, FXI, VWF and FVII show indirect effects on FII. While in the second model for diabetic group, as shown in <xref ref-type="fig" rid="F2">Figure 2</xref> and <xref ref-type="table" rid="T4">Table 3</xref>, the most direct effects are exerted by coagulation factors VWF, FX, FV and FXI on FII. As per this model, in addition to Glucose (Glc), FVIII and FV, factor XI also exerts indirect effect on FII simultaneously. </p><p> Our findings show that the obtained path model or diagram for coagulation factors are more complicated in diabetic patients than in normal individuals and convey that a sudden increase in the synthesis of each coagulation factors or their activation may trigger the initiation of coagulation cascade, leading to vascular clot formation with myocardial consequences. </p></sec>
Photographic facial soft tissue analysis of healthy Iranian young adults: anthropometric and angular measurements
<p> <bold>Background:</bold> Soft tissue profile can be widely different in various populations. Furthermore, this profile can be also continues to change throughout life. However, there are few studies that quantitatively evaluate the soft tissue profile in Iranian population. In order to determine normal reference values of facial parts in our populations, we aimed to measure standards for facial soft tissue parameters in Iranian young population. </p><p> <bold>Methods:</bold> The study samples included 155 medical students at the Firouzgar hospital in winter 2011. The soft tissue facial profiles were digitally analyzed using linear measurements and angles made with standardized photographic records, taken in a natural head position, to determine the average soft tissue facial profile for males and females. </p><p> <bold>Results:</bold> There was a statistically significant difference between males and females in 21 of our 26 measurements. The most prominent differences between the genders were observed in the measurements taken from the face region. Minimum frontal breadth and supraorbital breadth were larger in males than in females. Except for middle face height measurement, other horizontal and vertical measurements for the face were larger in males than in females, indicating wider and higher faces in men than in women. Some measurements of facial angles are discrepant between the two genders. </p><p> <bold>Conclusion:</bold> Due to the specific features of Iranian facial soft tissue values and also observable differences in facial measurements and angles between men and women, the Iranian standard values on facial measurements and angles should be given more attention, especially by plastic and cosmetic surgeons. </p>
<contrib contrib-type="author"><name><surname>Asghari</surname><given-names>Alimohamad</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rajaeih</surname><given-names>Shahin</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Hassannia</surname><given-names>Fatemeh</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Tavakolifard</surname><given-names>Negah</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Fattahi Neisyani</surname><given-names>Hamed</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Kamrava</surname><given-names>Seyed Kamran</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Jalessi</surname><given-names>Maryam</given-names></name><xref ref-type="aff" rid="A07"> <sup>7</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Omidian</surname><given-names>Parisa</given-names></name><xref ref-type="aff" rid="A08"> <sup>8</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> The analysis of the human face is a science and an art, utilizing both aesthetic and anthropologic tools. The appearance of the face is influenced by age, sex, race, and ethnicity (<xref rid="R1" ref-type="bibr">1</xref>). In addition, the quantitative measurements of face can be widely changes following growth as well as after surgical procedures (<xref rid="R2" ref-type="bibr">2</xref>). Furthermore, facial measurements are also an integral part of the evaluation of dimorphism. Therefore, planning a surgery on the face with the aim of restoration of facial components or maintaining the beauty should be performed by considering each of these factors (<xref rid="R3" ref-type="bibr">3</xref>). On the other hand, due to the increasing scrutiny placed upon the facial appearance at the completion of maxillofacial and cosmetic surgeries, a great deal of research has been conducted to determine what factors contribute to facial esthetics naturally through growth as well as through treatment (<xref rid="R4" ref-type="bibr">4</xref>). Consequently, the assessment of the soft tissue profile is an important part of diagnosis and treatment plan those in need of these types of surgeries (<xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R6" ref-type="bibr">6</xref>). Photogrammetry has been introduced as an alternative to direct measurements to obtain angles and distances between facial landmarks. Obtaining measurements from photographs is less intrusive to the patient and more cost-effective, it provides a permanent record of the face that can be accessed at a later time (<xref rid="R6" ref-type="bibr">6</xref>,<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R8" ref-type="bibr">8</xref>). </p><p> There are numerous studies which demonstrated that the soft tissue profile can be widely different in various populations. Furthermore, this profile can also be continued to change throughout life from childhood into adulthood. However, there are few studies that quantitatively evaluate the soft tissue profile in Iranian population. In order to determine normal reference values of facial parts in our populations, we aimed to measure standards for facial soft tissue parameters in Iranian young population. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> This analytical cross sectional study samples included 155 medical students at the Firouzgar hospital who had an appropriate health condition and enrolled in the hospital in winter 2011. All participants were explained how to implement the project and then they all had written consent to participate in the study. A brief questionnaire for all baseline characteristics including age, gender, and race was completed and inclusion criteria were checked by an ENT resident. Dental class I occlusion (the mesiobuccal groove of the maxillary first molar sitting within the mesiobuccal groove of the mandibular first molar (<xref rid="R9" ref-type="bibr">9</xref>)) was performed for all subjects that the normal state considered as inclusion criterion. History of orthodontic treatment was also considered as exclusion criterion. Other exclusion criteria were history of trauma to the face and facial fractures, facial cosmetic surgery, or any significant deformity in the nose or face. </p><p> Photography devices included a camera (canon D 5 35mm) and a tripod that used to prevent vibration and considering the height of subject for setting up horizontal axis of the camera lens. To avoid red eye in the picture, the camera flash the mode set for red eye effect. The primary flash was attached to the tripod by a lateral arm, at a distance of 27 cm from the optic axis to avoid the &#x0201c;red-eye effect&#x0201d; on the records. A secondary flash was placed behind the subject to enlight the background and eliminate undesirable shadows from the contours of the facial profile. The primary and secondary flashes were synchronized to improve the image. Distance between the camera and subject fixed at 2m and the visual axis was parallel to the floor. To get the actual size of the frontal and lateral views, a one-centimeter benchmark in the middle of the irforehead and in the cheek was considered respectively. Before taking the picture if the person had to wear glasses, the glass was removed. Standardized facial photographs were obtained: with a fully opened eye, no smile, and gently closed lips, and with visible forehead and neck. For each subject, a single operator located the standard anthropometric landmarks on digital photographic images. All data were obtained from standardized digital photographic images using the standard anthropometric measuring method. After locating a total of 19 soft tissue facial landmarks on frontal view (<xref ref-type="fig" rid="F1">Fig. 1</xref>) and 15 landmarks on lateral view (<xref ref-type="fig" rid="F2">Fig. 2</xref>), angles (26 measurements on frontal view and 9 angles on lateral view) were measured using AutoCAD 2008 software and recorded in the checklists. Anthropometric landmarks used in this study are presented in <xref ref-type="table" rid="T1">Table 1</xref> and <xref ref-type="fig" rid="F1">Figures 1</xref> &#x00026; <xref ref-type="fig" rid="F2">2</xref>, and 26 standard anthropometric measurements on right and left side of the face are shown in <xref ref-type="table" rid="T2">Table 2</xref>. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-49-g001"/><statement><p> Facial landmarks in frontal view (tr-trichion, g-glabella, n-nasion, mf-maxillofrontale, prpronasale, al-alare, sbal-subalare, sn-subnasale, cph-crista philter, ls-labialesuperius, ch-cheilion, sto-stomion, lilabialeinferius, sl-sublabiale, Me-menton, zy-zygion, gogonion, ft-frontotemporale, fz-frontozygomaticus) </p></statement></fig><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-49-g002"/><statement><p>2. Facial landmarks in lateral view (G-glabella, N- nasion, Mn- mid nasal, Prn- pronasal, Cm, Sn-subnasal, Ls-labial superior, Li-labial inferior, SL- sub labal, Pg-pogonion, Me- menton, C- cervical, Trg- tragus, Ort point- junction of true vertical and true horizontal. Reference lines)</p></statement></fig><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Anthropometric landmarks used in the study </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Landmarks</td><td rowspan="1" colspan="1">Region</td><td rowspan="1" colspan="1">Definition</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">tr-trichion</td><td rowspan="1" colspan="1">Cranial</td><td rowspan="1" colspan="1">Midpoint of the hairline</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">g-glabella</td><td rowspan="1" colspan="1">Cranial</td><td rowspan="1" colspan="1">The most prominent point in the median sagital plane between the supraorbital ridges</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">n-nasion</td><td rowspan="1" colspan="1">Face</td><td rowspan="1" colspan="1">The midpoint of the nasofrontal suture</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">mf-maxillofrontale</td><td rowspan="1" colspan="1">Nose</td><td rowspan="1" colspan="1">The anterior lacrimal crest of the maxilla at the frontomaxillary suture</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">pr-pronasale</td><td rowspan="1" colspan="1">Nose</td><td rowspan="1" colspan="1">The most protruded point of the nasal tip</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">al-alare</td><td rowspan="1" colspan="1">Nose</td><td rowspan="1" colspan="1">The most lateral point on the nasal ala</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">sbal-subalare</td><td rowspan="1" colspan="1">Nose</td><td rowspan="1" colspan="1"> The point on the lower margin of the base of the nasal ala where the<break/>ala disappears into the upper lip skin </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">sn-subnasale</td><td rowspan="1" colspan="1">Face</td><td rowspan="1" colspan="1">The junction between the lower border of the nasal septum, the partition that divides the nostrils, and the cutaneous portion of the upper lip in the midline</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">cph-crista philter</td><td rowspan="1" colspan="1">Orolabial</td><td rowspan="1" colspan="1"> The point on the crest of the philtrum, the vertical groove in the<break/>median portion of the upper lip, just above the vermilion border </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">ls-labialesuperius</td><td rowspan="1" colspan="1">Orolabial</td><td rowspan="1" colspan="1">The midpoint of the vermilion border of the upper lip</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">ch-cheilion</td><td rowspan="1" colspan="1">Orolabial</td><td rowspan="1" colspan="1">The outer corner of the mouth where the outer edges of the upper and lower vermilions meet</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">sto-stomion</td><td rowspan="1" colspan="1">Face</td><td rowspan="1" colspan="1">The midpoint of the labial fissure when the lips are closed naturally</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">li-labialeinferius</td><td rowspan="1" colspan="1">Orolabial</td><td rowspan="1" colspan="1">The midpoint of the vermilion border of the lower lip</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">sl-sublabiale</td><td rowspan="1" colspan="1">Face</td><td rowspan="1" colspan="1">The midpoint of the labiomental sulcus</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Me-menton</td><td rowspan="1" colspan="1">Face</td><td rowspan="1" colspan="1">The lowest point in the midline on the lower border of the chin</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">zy-zygion</td><td rowspan="1" colspan="1">Face</td><td rowspan="1" colspan="1">The most lateral point on the zygomatic arch</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">go-gonion</td><td rowspan="1" colspan="1">Face</td><td rowspan="1" colspan="1">The most lateral point at the angle of the mandible</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">ft-frontotemporale</td><td rowspan="1" colspan="1">Cranial</td><td rowspan="1" colspan="1">The most medial point on the temporal crest of the frontal bone</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">fz-frontozygomaticus</td><td rowspan="1" colspan="1">Cranial</td><td rowspan="1" colspan="1">The most lateral point on the frontozygomatic suture</td></tr></tbody></table></table-wrap><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Standard anthropometric measurements </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Region</td><td rowspan="1" colspan="1">Measurement name</td><td rowspan="1" colspan="1">Plane</td><td rowspan="1" colspan="1">Landmarks</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Cranial</td><td rowspan="1" colspan="1">Minimum frontal breadth</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">frontotemporale&#x02013;frontotemporale</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Supraorbital breadth</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">frontozygomaticus-frontozygomaticus</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Forehead height I</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">trichion&#x02013;glabella</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Forehead height II</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">trichion-nasion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Facial</td><td rowspan="1" colspan="1">Middle face height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">glabella&#x02013;subnasale</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Maximum facial breadth</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">zygion-zygion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Bigonial breadth</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">gonion&#x02013;gonion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Physiognomic face height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">trichion&#x02013;Me(menton)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Morphologic face height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">nasiongnathion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Upper face height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">nasion&#x02013;stomion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Anterior mandibular height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">stomion&#x02013;Me(menton)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Chin height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">sublabiale-gnathion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Lower face height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">subnasale-gnathion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nasal</td><td rowspan="1" colspan="1">Nose height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">nasion-subnasale</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Nasal bridge length</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">nasion-pronasale</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Nose width</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">alare-alare</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Nasal root width</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">maxillofrontale-maxillofrontale</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Nostril floor width</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">subalare-subnasale</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Oral-labial</td><td rowspan="1" colspan="1">Philtrum width</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">crista philtre-crista philtre</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Labial fissure width</td><td rowspan="1" colspan="1">Horizontal line</td><td rowspan="1" colspan="1">cheilion-cheilion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Philtrum length</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">subnasale-labialesuperius</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Upper vermilion height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">labialesuperius-stomion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Upper lip height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">subnasalestomion</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Lower lip height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">stomion-sublabiale</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Cutaneous lower lip height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">labialeinferius-sublabiale</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Lower vermilion height</td><td rowspan="1" colspan="1">Vertical line</td><td rowspan="1" colspan="1">labialeinferius-stomion</td></tr></tbody></table></table-wrap><p> Results were presented as mean/SD for quantitative variables and compared using t test or Mann-Whitney U test if required. Statistical significance was determined at a p value of &#x02264; 0.05. All statistical analysis was performed using SPSS software (version 16.0, SPSS Inc., Chicago, Illinois). </p></sec><sec sec-type="results" id="s3"><title>Results</title><p> The results of the craniofacial anthropometric measurements for all 155 subjects (72 men with the mean age of 22.19/2.42 years and 83 women with the mean age of 23.38/3.24 years) were summarized by gender in <xref ref-type="table" rid="T3">Table 3</xref>. All measurements were given in millimeters. Craniofacial measurements were compared between males and females. A statistically significant difference existed between males and females in 21 of our 26 measurements. The most prominent differences between the genders were observed in the measurements taken from the face region. In this study, 19 facial landmarks were marked by the same investigator. After one month, this same investigator marked the landmarks on the 40frontal and lateral images (10 male, 10 female) that were selected randomly from the study population. Analysis was performed to obtain a G reliability coefficient. As a result, the analysis of the rate indicated good repeatability for both female and male subjects (G = 0.91). </p><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> Standard craniofacial anthropometric measurements in Iranian population in men and women </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td style="border-width: 0px 1px 0pt 0px ; border-style: none solid none none;border-color:#fff8e8" rowspan="1" colspan="1">Facial measurements</td><td align="center" rowspan="1" colspan="1"> Male<break/>(mean) </td><td align="center" rowspan="1" colspan="1">SD (SEM)</td><td align="center" rowspan="1" colspan="1"> Female<break/>(mean) </td><td align="center" rowspan="1" colspan="1">SD (SEM)</td><td align="center" rowspan="1" colspan="1">Total (mean)</td><td align="center" rowspan="1" colspan="1">SD (SEM)</td><td align="center" rowspan="1" colspan="1">p-value</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Minimum frontal breadth</td><td align="center" rowspan="1" colspan="1">107.433</td><td align="center" rowspan="1" colspan="1">7.5873</td><td align="center" rowspan="1" colspan="1">94.564</td><td align="center" rowspan="1" colspan="1">7.1996</td><td align="center" rowspan="1" colspan="1">100.542</td><td align="center" rowspan="1" colspan="1">9.7778</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Supra-orbital breadth</td><td align="center" rowspan="1" colspan="1">129.108</td><td align="center" rowspan="1" colspan="1">6.7111</td><td align="center" rowspan="1" colspan="1">115.102</td><td align="center" rowspan="1" colspan="1">5.6665</td><td align="center" rowspan="1" colspan="1">121.608</td><td align="center" rowspan="1" colspan="1">9.3260</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nasal root width</td><td align="center" rowspan="1" colspan="1">21.383</td><td align="center" rowspan="1" colspan="1">1.3058</td><td align="center" rowspan="1" colspan="1">20.054</td><td align="center" rowspan="1" colspan="1">1.7883</td><td align="center" rowspan="1" colspan="1">20.672</td><td align="center" rowspan="1" colspan="1">1.7121</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Maximum facial breadth</td><td align="center" rowspan="1" colspan="1">138.050</td><td align="center" rowspan="1" colspan="1">9.6521</td><td align="center" rowspan="1" colspan="1">124.698</td><td align="center" rowspan="1" colspan="1">6.6860</td><td align="center" rowspan="1" colspan="1">130.900</td><td align="center" rowspan="1" colspan="1">10.5541</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nose width</td><td align="center" rowspan="1" colspan="1">39.771</td><td align="center" rowspan="1" colspan="1">2.6555</td><td align="center" rowspan="1" colspan="1">35.022</td><td align="center" rowspan="1" colspan="1">2.2258</td><td align="center" rowspan="1" colspan="1">37.228</td><td align="center" rowspan="1" colspan="1">3.3964</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nostril floor width</td><td align="center" rowspan="1" colspan="1">14.183</td><td align="center" rowspan="1" colspan="1">2.3627</td><td align="center" rowspan="1" colspan="1">12.167</td><td align="center" rowspan="1" colspan="1">1.6870</td><td align="center" rowspan="1" colspan="1">13.104</td><td align="center" rowspan="1" colspan="1">2.2597</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Philtrum width</td><td align="center" rowspan="1" colspan="1">15.737</td><td align="center" rowspan="1" colspan="1">2.7116</td><td align="center" rowspan="1" colspan="1">12.790</td><td align="center" rowspan="1" colspan="1">2.1680</td><td align="center" rowspan="1" colspan="1">14.159</td><td align="center" rowspan="1" colspan="1">2.8403</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Labial fissure width</td><td align="center" rowspan="1" colspan="1">53.133</td><td align="center" rowspan="1" colspan="1">4.7927</td><td align="center" rowspan="1" colspan="1">48.057</td><td align="center" rowspan="1" colspan="1">6.3020</td><td align="center" rowspan="1" colspan="1">50.415</td><td align="center" rowspan="1" colspan="1">6.1798</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Bigonial breadth</td><td align="center" rowspan="1" colspan="1">127.229</td><td align="center" rowspan="1" colspan="1">10.9665</td><td align="center" rowspan="1" colspan="1">105.960</td><td align="center" rowspan="1" colspan="1">7.4428</td><td align="center" rowspan="1" colspan="1">115.840</td><td align="center" rowspan="1" colspan="1">14.0782</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Forehead height I</td><td align="center" rowspan="1" colspan="1">58.933</td><td align="center" rowspan="1" colspan="1">6.1150</td><td align="center" rowspan="1" colspan="1">55.751</td><td align="center" rowspan="1" colspan="1">6.9009</td><td align="center" rowspan="1" colspan="1">57.229</td><td align="center" rowspan="1" colspan="1">6.7181</td><td align="center" rowspan="1" colspan="1">0.003</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Forehead height II</td><td align="center" rowspan="1" colspan="1">68.500</td><td align="center" rowspan="1" colspan="1">5.7529</td><td align="center" rowspan="1" colspan="1">65.113</td><td align="center" rowspan="1" colspan="1">7.2194</td><td align="center" rowspan="1" colspan="1">66.686</td><td align="center" rowspan="1" colspan="1">6.7736</td><td align="center" rowspan="1" colspan="1">0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nasal bridge length</td><td align="center" rowspan="1" colspan="1">40.350</td><td align="center" rowspan="1" colspan="1">6.7173</td><td align="center" rowspan="1" colspan="1">41.498</td><td align="center" rowspan="1" colspan="1">4.6480</td><td align="center" rowspan="1" colspan="1">40.965</td><td align="center" rowspan="1" colspan="1">5.7128</td><td align="center" rowspan="1" colspan="1">0.213</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Philtrum length</td><td align="center" rowspan="1" colspan="1">17.567</td><td align="center" rowspan="1" colspan="1">2.5546</td><td align="center" rowspan="1" colspan="1">14.243</td><td align="center" rowspan="1" colspan="1">3.9960</td><td align="center" rowspan="1" colspan="1">15.787</td><td align="center" rowspan="1" colspan="1">3.7784</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Upper vermilion height</td><td align="center" rowspan="1" colspan="1">6.025</td><td align="center" rowspan="1" colspan="1">1.6215</td><td align="center" rowspan="1" colspan="1">6.312</td><td align="center" rowspan="1" colspan="1">1.1496</td><td align="center" rowspan="1" colspan="1">6.179</td><td align="center" rowspan="1" colspan="1">1.3916</td><td align="center" rowspan="1" colspan="1">0.212</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Lower vermilion height</td><td align="center" rowspan="1" colspan="1">9.600</td><td align="center" rowspan="1" colspan="1">2.6691</td><td align="center" rowspan="1" colspan="1">9.820</td><td align="center" rowspan="1" colspan="1">1.6889</td><td align="center" rowspan="1" colspan="1">9.718</td><td align="center" rowspan="1" colspan="1">2.1944</td><td align="center" rowspan="1" colspan="1">0.534</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Cutaneous lower lip height</td><td align="center" rowspan="1" colspan="1">10.967</td><td align="center" rowspan="1" colspan="1">3.3117</td><td align="center" rowspan="1" colspan="1">8.025</td><td align="center" rowspan="1" colspan="1">2.0247</td><td align="center" rowspan="1" colspan="1">9.392</td><td align="center" rowspan="1" colspan="1">3.0667</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Chin height</td><td align="center" rowspan="1" colspan="1">31.754</td><td align="center" rowspan="1" colspan="1">3.6766</td><td align="center" rowspan="1" colspan="1">25.302</td><td align="center" rowspan="1" colspan="1">2.4955</td><td align="center" rowspan="1" colspan="1">28.299</td><td align="center" rowspan="1" colspan="1">4.4687</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Lower lip height</td><td align="center" rowspan="1" colspan="1">20.5667</td><td align="center" rowspan="1" colspan="1">3.02115</td><td align="center" rowspan="1" colspan="1">17.8458</td><td align="center" rowspan="1" colspan="1">2.28331</td><td align="center" rowspan="1" colspan="1">19.1097</td><td align="center" rowspan="1" colspan="1">2.97279</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Anterior mandibular height</td><td align="center" rowspan="1" colspan="1">52.3208</td><td align="center" rowspan="1" colspan="1">4.63711</td><td align="center" rowspan="1" colspan="1">43.1482</td><td align="center" rowspan="1" colspan="1">3.62503</td><td align="center" rowspan="1" colspan="1">47.4090</td><td align="center" rowspan="1" colspan="1">6.16238</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Midface height</td><td align="center" rowspan="1" colspan="1">76.029</td><td align="center" rowspan="1" colspan="1">5.9859</td><td align="center" rowspan="1" colspan="1">73.770</td><td align="center" rowspan="1" colspan="1">5.3207</td><td align="center" rowspan="1" colspan="1">74.819</td><td align="center" rowspan="1" colspan="1">5.7334</td><td align="center" rowspan="1" colspan="1">0.015</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Morphological face height</td><td align="center" rowspan="1" colspan="1">128.350</td><td align="center" rowspan="1" colspan="1">8.9877</td><td align="center" rowspan="1" colspan="1">116.918</td><td align="center" rowspan="1" colspan="1">7.4709</td><td align="center" rowspan="1" colspan="1">122.228</td><td align="center" rowspan="1" colspan="1">9.9840</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Physiognomical face height</td><td align="center" rowspan="1" colspan="1">196.850</td><td align="center" rowspan="1" colspan="1">12.8803</td><td align="center" rowspan="1" colspan="1">182.031</td><td align="center" rowspan="1" colspan="1">11.5016</td><td align="center" rowspan="1" colspan="1">188.915</td><td align="center" rowspan="1" colspan="1">14.2092</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Middle face height</td><td align="center" rowspan="1" colspan="1">62.004</td><td align="center" rowspan="1" colspan="1">5.1380</td><td align="center" rowspan="1" colspan="1">62.577</td><td align="center" rowspan="1" colspan="1">4.2623</td><td align="center" rowspan="1" colspan="1">62.311</td><td align="center" rowspan="1" colspan="1">4.6826</td><td align="center" rowspan="1" colspan="1">0.455</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Upper lip height</td><td align="center" rowspan="1" colspan="1">23.592</td><td align="center" rowspan="1" colspan="1">1.8256</td><td align="center" rowspan="1" colspan="1">20.555</td><td align="center" rowspan="1" colspan="1">3.9837</td><td align="center" rowspan="1" colspan="1">21.966</td><td align="center" rowspan="1" colspan="1">3.5064</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Lower face height</td><td align="center" rowspan="1" colspan="1">75.912</td><td align="center" rowspan="1" colspan="1">5.4588</td><td align="center" rowspan="1" colspan="1">63.704</td><td align="center" rowspan="1" colspan="1">6.1785</td><td align="center" rowspan="1" colspan="1">69.375</td><td align="center" rowspan="1" colspan="1">8.4488</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nose height</td><td align="center" rowspan="1" colspan="1">52.437</td><td align="center" rowspan="1" colspan="1">5.0226</td><td align="center" rowspan="1" colspan="1">53.214</td><td align="center" rowspan="1" colspan="1">3.5721</td><td align="center" rowspan="1" colspan="1">52.854</td><td align="center" rowspan="1" colspan="1">4.3100</td><td align="center" rowspan="1" colspan="1">0.276</td></tr></tbody></table></table-wrap><p> Cranial: Four measurements were performed, two vertical (tr-n, tr-g) and two horizontal (ft-ft, fz-fz). Comparison of the measurement of cranial region showed that minimum frontal breadth and supraorbital breadth were larger in males than in females. Also, vertical measurements of forehead height (tr - g, tr &#x02013; n) were discrepant between the two genders. </p><p> Face: Seven vertical (g-sn, tr-gn, n-gn, n-sto, sto-gn, sl-gn, sn-gn) and two horizontal (zy-zy, go-go) measurements were performed. Except for middle face height measurement, other measurements (including maximum facial breadth, bigonial breadth, physiognomic face height, morphologic face height, upper face height, anterior mandibular height, chin height, lower face height) were larger in males than females. Measurements of the face showed that males had wider and higher faces. </p><p> Nasal: Five measurements were made for the nasal region. Although nose width and nostril floor width show statistically significant differences between males and females, nose height, nasal bridge length, and nasal root width were not different. </p><p> Orolabial: Comparing anthropometric measurements from the orolabial region, we found that all measurements were larger in males than in females, except for the upper and lower vermilion heights. </p><p> Angles: Descriptive measurements of angles are summarized in <xref ref-type="table" rid="T4">Table 4</xref> and compared between the two genders. </p><table-wrap id="T4" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Standard craniofacial angle measurements in Iranian population in men and women </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Facial angles</td><td align="center" rowspan="1" colspan="1"> Male<break/>(mean) </td><td align="center" rowspan="1" colspan="1">SD (SEM)</td><td align="center" rowspan="1" colspan="1"> Female<break/>(mean) </td><td align="center" rowspan="1" colspan="1">SD (SEM)</td><td align="center" rowspan="1" colspan="1">Total (mean)</td><td align="center" rowspan="1" colspan="1">SD (SEM)</td><td align="center" rowspan="1" colspan="1">p-value</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">G&#x02013;N &#x02013;Prn, nasofrontal</td><td align="center" rowspan="1" colspan="1">132.917</td><td align="center" rowspan="1" colspan="1">6.9402</td><td align="center" rowspan="1" colspan="1">141.161</td><td align="center" rowspan="1" colspan="1">5.9041</td><td align="center" rowspan="1" colspan="1">138.860</td><td align="center" rowspan="1" colspan="1">7.2032</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">N&#x02013;Prn/N&#x02013;Ort, vertical nasal</td><td align="center" rowspan="1" colspan="1">32.125</td><td align="center" rowspan="1" colspan="1">4.6374</td><td align="center" rowspan="1" colspan="1">30.242</td><td align="center" rowspan="1" colspan="1">5.6559</td><td align="center" rowspan="1" colspan="1">30.767</td><td align="center" rowspan="1" colspan="1">5.4312</td><td align="center" rowspan="1" colspan="1">0.119</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Prn&#x02013;Sn&#x02013;Ls, nasolabial</td><td align="center" rowspan="1" colspan="1">106.625</td><td align="center" rowspan="1" colspan="1">11.9011</td><td align="center" rowspan="1" colspan="1">103.581</td><td align="center" rowspan="1" colspan="1">14.5766</td><td align="center" rowspan="1" colspan="1">104.430</td><td align="center" rowspan="1" colspan="1">13.8815</td><td align="center" rowspan="1" colspan="1">0.324</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Li&#x02013;Sl&#x02013;Pg, mentolabial</td><td align="center" rowspan="1" colspan="1">122.000</td><td align="center" rowspan="1" colspan="1">11.8211</td><td align="center" rowspan="1" colspan="1">124.258</td><td align="center" rowspan="1" colspan="1">10.0506</td><td align="center" rowspan="1" colspan="1">123.628</td><td align="center" rowspan="1" colspan="1">10.5519</td><td align="center" rowspan="1" colspan="1">0.413</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Sn&#x02013;Prn/N- Prn, nasal</td><td align="center" rowspan="1" colspan="1">75.500</td><td align="center" rowspan="1" colspan="1">8.7377</td><td align="center" rowspan="1" colspan="1">79.887</td><td align="center" rowspan="1" colspan="1">7.6074</td><td align="center" rowspan="1" colspan="1">78.663</td><td align="center" rowspan="1" colspan="1">8.1307</td><td align="center" rowspan="1" colspan="1">0.037</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">N-Mn-Prn, nasal dorsum</td><td align="center" rowspan="1" colspan="1">174.167</td><td align="center" rowspan="1" colspan="1">4.2290</td><td align="center" rowspan="1" colspan="1">178.032</td><td align="center" rowspan="1" colspan="1">2.5797</td><td align="center" rowspan="1" colspan="1">176.953</td><td align="center" rowspan="1" colspan="1">3.5576</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">G-Pg/C-Me, cervicomental</td><td align="center" rowspan="1" colspan="1">101.417</td><td align="center" rowspan="1" colspan="1">6.9590</td><td align="center" rowspan="1" colspan="1">91.952</td><td align="center" rowspan="1" colspan="1">7.2302</td><td align="center" rowspan="1" colspan="1">94.593</td><td align="center" rowspan="1" colspan="1">8.2979</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">N-Trg-Sn, med facial</td><td align="center" rowspan="1" colspan="1">27.625</td><td align="center" rowspan="1" colspan="1">1.7399</td><td align="center" rowspan="1" colspan="1">30.581</td><td align="center" rowspan="1" colspan="1">2.8718</td><td align="center" rowspan="1" colspan="1">29.756</td><td align="center" rowspan="1" colspan="1">2.9182</td><td align="center" rowspan="1" colspan="1">&#x0003c; 0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Sn-Trg-Me inf facial third</td><td align="center" rowspan="1" colspan="1">34.458</td><td align="center" rowspan="1" colspan="1">2.2838</td><td align="center" rowspan="1" colspan="1">34.903</td><td align="center" rowspan="1" colspan="1">3.2629</td><td align="center" rowspan="1" colspan="1">34.779</td><td align="center" rowspan="1" colspan="1">3.0153</td><td align="center" rowspan="1" colspan="1">0.478</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">G-Sn-Pg, facial convexity</td><td align="center" rowspan="1" colspan="1">167.417</td><td align="center" rowspan="1" colspan="1">3.5743</td><td align="center" rowspan="1" colspan="1">167.774</td><td align="center" rowspan="1" colspan="1">4.4332</td><td align="center" rowspan="1" colspan="1">167.674</td><td align="center" rowspan="1" colspan="1">4.1937</td><td align="center" rowspan="1" colspan="1">0.700</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">G-Prn-Pg, total facial</td><td align="center" rowspan="1" colspan="1">137.875</td><td align="center" rowspan="1" colspan="1">3.7103</td><td align="center" rowspan="1" colspan="1">138.145</td><td align="center" rowspan="1" colspan="1">4.1125</td><td align="center" rowspan="1" colspan="1">138.070</td><td align="center" rowspan="1" colspan="1">3.9846</td><td align="center" rowspan="1" colspan="1">0.770</td></tr></tbody></table></table-wrap><p> Nose: There was significant difference in the angles of nasal (N-Prn-Sn), nasal dorsum, (N-Mn-Prn), and nasofrontal (G&#x02013;N &#x02013;Prn) between men and women, while no differences was detected in vertical nasal (N&#x02013;Prn/N&#x02013;Ort) angle between them. </p><p> Nasal Base and upper lip: This region was assessed by measuring Nasolabial (Prn&#x02013;Sn&#x02013;Ls) angle that ranged 85 to 123 degree in men and 79 to 120 degree in women. Mentolabial (Li&#x02013;Sl&#x02013;Pg) angle were not different between men and women. The average of cervicomental (G-Pg/C-Me) angle was 101.41/6.95 degree in men and 91.94/7.23 degree in women with a significant difference. The measurements of other angles were not meaningful between the two genders. </p><p> Facial convexity was assessed using facial convexity (G-Sn-Pg (angle with the mean of 167.67/4.19. Also, total facial convexity was measured by Total Facial (G &#x02013; Prn &#x02013; Pg) angle with the average of 138.86/7.20 degree. </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Many studies have been performed for better understanding of how growth and development could affects the skeleton, dentition, and the soft tissue profile in children and adolescence (<xref rid="R10" ref-type="bibr">10</xref>). It has been recently presented statistical significance in the interethnic variability of the neoclassical facial measurements with a 95 percent confidence level, allowing classification of the facial measurements into five distinct levels of variability including least variable, less variable intermediate, intermediate, more variable intermediate, and most variable (<xref rid="R11" ref-type="bibr">11</xref>). </p><p> Comparison of our facial measurements with the findings of other studies among different populations especially among Asians showed major similarities in different landmarks and facial angles. Similar to studies on Turkish people, in our observation, the most prominent differences between the sexes were observed in the measurements taken from the face region (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R12" ref-type="bibr">12</xref>). In our study, all four measurements of skull including two vertical and two horizontal were discrepant between men and women. Also, in facial measurement, except for middle face height measurement, other measurements including maximum facial breadth, bigonial breadth, physiognomic face height, morphologic face height, upper face height, anterior mandibular height, chin height, lower face height) for the face were larger in males than females. Gender differences were also present in the mentolabial and cervicomental angles. However, when this was compared to Chinese adults, most of the measured angles in our analysis were similar in the two genders, while all the linear measurements in Chinese peoples were larger in men than women, but all the angular measurements were smaller in men than women (<xref rid="R13" ref-type="bibr">13</xref>). Also, in some studies on both eastern and western European nations, most of the facial angles including nasofrontal, nasolabial, mentolabial, and nasal tip angle were larger in women than men (<xref rid="R14" ref-type="bibr">14</xref>,<xref rid="R15" ref-type="bibr">15</xref>). In Croatian people, almost all vertical variables were larger in the males, except the length of the nasal tip, which was larger in females (<xref rid="R16" ref-type="bibr">16</xref>). </p><p> With respect to the results of anthropometric and angular measurements, we decided to propound schematic figures of young Iranian adults in soft facial tissue (<xref ref-type="fig" rid="F3">Fig. 3</xref>). </p><fig id="F3" orientation="portrait" position="float"><label> Fig. 3 </label><graphic xlink:href="MJIRI-28-49-g003"/><statement><p> Schematic figures of young Iranian adults of soft facial tissue (With respect to the results of anthropometric and angular measurements) </p></statement></fig></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Our study revealed a highly significant sex dimorphism in the soft tissue profile, presenting that form for both size and shape differe between male and female soft tissue profiles that was similarly shown in some other populations especially neighboring countries of Iran. Due to the specific features of Iranian facial soft tissue values and also observable differences in facial measurements and angles between men and women, the Iranian standard values on facial measurements and angles should be given more attention, especially by plastic and cosmetic surgeons. </p></sec>
Assessing validity and reliability of Dundee ready educational environment measure (DREEM) in Iran
<p> <bold>Background:</bold> If an institute is looking for improvement of its learning environment, a reliable and valid assessment tool is needed for measurement of the educational environment .The Dundee Ready Educational Environment Measure (DREEM) has been used in various studies to evaluate the educational environment. However, psychometric evaluations of the instrument seem necessary, for all known versions of the instrument. The aim of this study was to investigate the reliability and validity of Persian version of the DREEM in the major clinical wards in teaching hospitals affiliated to Iran University of Medical Sciences. </p><p> <bold>Methods:</bold> This descriptive - analytical study, involved medical students (clinical stagers and interns) in 4 major clinical wards. In this study, DREEM questionnaire was reviewed in content, face validity and construct validity through confirmatory factor analysis. The reliability was calculated according to test - retest and the internal consistency was measured using Cronbach's alpha coefficient. </p><p> <bold>Results:</bold> A total number of 267 questionnaires were completed by medical stagers (60%) and interns (40%) including 181 females and 82 males. The mean age of stagers and interns were 23.60 &#x000b1; 1.27 and 25.45 &#x000b1; 1.22 years, respectively. The total mean of the questionnaire was calculated as 96.15 (93.5375, 98.7547) out of 176, with 95% confidence interval. The face validity of the questionnaire was confirmed. The mean of content validity ratio (CVR) was calculated as 0.35, and 6 questions were omitted in this step. The content validity index (CVI) was 0.39. The reliability coefficient mean was 0.71. In confirmatory factor analysis five factors were confirmed that changed the orientation of some questions. The Cronbach's alpha coefficient of the whole questionnaire was obtained as 0.914. </p><p> <bold>Conclusion:</bold> The modified and validates DREEM questionnaire in Persian language with 44 items and appropriate psychometric attributes is capable of being used in assessment of clinical education environments in Iran. </p>
<contrib contrib-type="author"><name><surname>Koohpayehzadeh</surname><given-names>Jalil</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Hashemi</surname><given-names>Akram</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Soltani Arabshahi</surname><given-names>Kamran</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Bigdeli</surname><given-names>Shoaleh</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Moosavi</surname><given-names>Maryam</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Hatami</surname><given-names>Kamran</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Baradaran</surname><given-names>Hamid Reza</given-names></name><xref ref-type="aff" rid="A07"> <sup>7</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> In 1998, the World Federation for Medical Education emphasized on the role of learning environment as one of the&#x000a0;evaluation goals of medical education programs (<xref rid="R1" ref-type="bibr">1</xref>). Medical education environment is one of the extraordinary complexities sharing on complexities with working,&#x000a0;specialized and training environments, however, with unique intellectual intricacies. This environment includes a host of areas that are specified by specific tasks assumed at specific times for specific objectives (<xref rid="R2" ref-type="bibr">2</xref>). Evaluating educational environments (both academic and clinical) is the key to achieve high-quality, student-centered curriculums (<xref rid="R3" ref-type="bibr">3</xref>). In other global studies, the need for measuring educational atmosphere has been noted as an important indicator in educational settings, and the necessity of its quantification has been emphasized (<xref rid="R4" ref-type="bibr">4</xref>). If we can determine the operational components in the educational environment, the institution atmosphere or in the programs and evaluate the attitudes of students and teachers toward them, thus, we would have a basis for their modification in order to facilitate the learning experience in relation to the educational objectives (<xref rid="R5" ref-type="bibr">5</xref>). The measurement action is an essential component for scientific research, whether in natural sciences, social sciences or in health sciences and it certainly plays a critical role in the health sciences (<xref rid="R6" ref-type="bibr">6</xref>). Quantitative measurement of learning environment requires a tool or a questionnaire. Choosing such an instrument should be based on the quality of measurement process suggesting the use of a special tool or instrument fitness for measuring educational environment. Quality or such psychometric characteristics are generally performed entitled as validity and reliability. A valid and reliable measure of learning environment leads to meaningful measurement of educational environment of an institution, and thus, a perfect tool to improve the learning environment is achieved (<xref rid="R7" ref-type="bibr">7</xref>). Since 1970s, some tool-oriented studies have been carried out to measure students' attitudes towards learning experiences and educational environments. Differences between educational environments have led to developing of various educational questionnaires. The first tool was Medical School Environment or MSLES, which was developed in 1970. Thence, a path to develop further tools in medical education appeared that according to a recent systematic review in 2010, its number has been reported as 19 cases (<xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R8" ref-type="bibr">8</xref>). </p><p> One of the benchmarks for measuring the environment and the atmosphere of education is Dundee Ready Educational Environment Measure (DREEM), which was developed in 1997 by Susan Roff et al. in the University of Dundee in Scotland using standard Grounded theory and Delphi procedures methods on nearly 100 educators on health specialty from around the world, and examining on over 1,000 students in different countries, it was validated in order to measure and assess the atmosphere of medical education. Employing a combination of qualitative and quantitative methods led to develop a multi-dimensional and an independent of particular culture tool (<xref rid="R9" ref-type="bibr">9</xref>). It was demonstrated that the DREEM have been accepted as a useful tool for gathering the feedback of strengths and weaknesses of educational environment in the educational institutions (<xref rid="R10" ref-type="bibr">10</xref>). Any measurement tool must have some properties to be useful for the purpose it has been built for. Specialists in measurement and evaluation for tests have considered a lot of features. The most important and emphasized ones by the professionals in examination survey include validity and reliability (<xref rid="R11" ref-type="bibr">11</xref>). </p><p> In Iran, the DREEM questionnaire has been frequently used to evaluate the clinical learning environment. Since the study of validity and reliability of the aforementioned questionnaire in Persian language has not done so far in the form of a specific project, we decided to examine the reliability and validity of the DREEM questionnaire in evaluation of educational environment of major clinical wards (Internal Medicine, Obstetrics &#x00026; Gynecology, General Surgery and Pediatric) from the perspective of medical students (trainees and interns) in a few teaching hospital affiliated to Iran University of Medical Sciences. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> This is a descriptive - analytical study. The study population was medical students (interns and stagers) from major clinical wards (Internal Medicine - Gynecology and Obstetrics - Pediatrics and General Surgery) of the Iran University of Medical Sciences. They were&#x000a0;under education in teaching hospitals affiliated to the University (including HazratRasoolAkram, Firoozgar, Hazrat Ali Asghar and ShahidAkbarabadi hospitals). The total number in academic year of 2012-2013 has been estimated as follows: 122 students in Pediatrics; 155 students in Internal Medicine, 98 students in General Surgery and 90 students in Obstetrics and Gynecology. Data sampling was done using stratified random sampling method. Sample size was estimated according to the structure validity review using factor analysis method 5 times of the number of questions in the instrument (<xref rid="R12" ref-type="bibr">12</xref>), which was equivalent to 250 subjects. The number was selected randomly and proportional to the size of each groupin each stager and internship grades. First, the required permission was obtained in order to use the DREEM questionnaire developed by Susan Roff et al. at Dundee University, Scotland in 1997 and revised in 2005. Then, the questionnaire, which is originally in English, was translated into Farsi under the supervision of&#x000a0;faculty advisors. Then, it was back translated by someone fluent in English and adapted to the original questionnaire, and the gap in compliance was corrected. </p><sec id="s2-1"><title>Study methodology in content and face validity</title><p> The questionnaire was given to 20 experts familiar with clinical education. </p><p> In the review of face validity, which is the questionnaire appearance and logical sequence of questions in the questionnaire and their legibility (<xref rid="R11" ref-type="bibr">11</xref>), a number of questions were rewritten. </p><p> The questionnaire content validity was investigated regarding two aspects of ratio (CVR) and index (CVI). </p><p> The content validity ratio (CVR) is direct linear conversion of panel members&#x02019; comments that have chosen the necessary option (<xref rid="R13" ref-type="bibr">13</xref>). </p><p> Two methods were used to determine the content validity: </p><list list-type="order"><list-item><p> Using the CVR formula (<xref rid="R14" ref-type="bibr">14</xref>) Thus, for each question was a CVR. </p></list-item><list-item><p> From the average of experts&#x02019; judgments </p></list-item></list><p> Given that each question was ranked on a 3-degree scale of Essential, Useful but not essential and Not necessary: </p><list list-type="bullet"><list-item><p> Essential: 2 Points </p></list-item><list-item><p> Useful but not essential: 0 Point </p></list-item><list-item><p> Not necessary: 1 Point </p></list-item></list><p> For each question, a mean score between 0 and 2 was obtained (<xref rid="R13" ref-type="bibr">13</xref>). </p><p> Determining a criterion for acceptance or rejection of the questions was as follows: </p><list list-type="bullet"><list-item><p> Acceptance of the question </p></list-item></list><p> 1. If the CVR calculated in the formula was equal to 0.42 or higher, this number would be 16 based on Lawshe Table CVR values (<xref rid="R14" ref-type="bibr">14</xref>). </p><p> 2. If the CVR calculated by the formula was between zero and 0.42 and the mean of judgments was equal to 1.1 or greater (In different studies, this range is different). </p><list list-type="bullet"><list-item><p> Rejecting the question, if the CVR value was less than zero and the mean of judgments was less than 1.1. </p></list-item></list><p> According to the results of the two methods, a number of questions were rejected;&#x000a0;some were modified and the rest were accepted. </p><p> Also, reviewing the content validity index (CVI), which indicates the universality of judgments related to validity or applicability of the model, test or the final instrument, the overall CVI was calculated using the formula (<xref rid="R13" ref-type="bibr">13</xref>, <xref rid="R15" ref-type="bibr">15</xref>). </p></sec><sec id="s2-2"><title>Study of approach of consistency reliability</title><p> After conducting a pilot study, the correlation coefficients between test and retest questions were investigated using Spearman's correlation coefficient, and the questions with correlation coefficient tending toward zero were revised. </p></sec><sec id="s2-3"><title>Evaluation method of internal consistency reliability</title><p> To evaluate the reliability of the internal consistency, the Cronbach's alpha coefficient calculation was used. </p></sec><sec id="s2-4"><title>Study method of construct validity</title><p> The confirmatory factor analysis was used to examine construct validity during the following steps: </p><list list-type="bullet"><list-item><p> First step: Examining the data number proportionality using Kaiser - Meyer - Olkin (KMO) test and their homogeneity for factor analysis using Bartlett's Test of Sphericity </p></list-item><list-item><p> Second step: Extraction of the factors using&#x000a0;principal components analysis method (<xref rid="R16" ref-type="bibr">16</xref>). </p></list-item></list></sec><sec id="s2-5"><title> Statistical analysis </title><p> Data analysis was performed using SPSS software version 16. The required statistical methods include confirmatory factor analysis methods and Pearson correlation tests and Cronbach's alpha coefficient determination. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> 267 questionnaires were completed by stagers and interns. </p><p> The questionnaire overall mean was obtained as 96.15(95% CI: 93.5375, 98.7547) from 176 that according to the interpretation of modified DREEM scores, positives points are more than negatives ones (<xref rid="R17" ref-type="bibr">17</xref>). </p><p> Interpretations of the factors are as follows: </p><list list-type="bullet"><list-item><p> Students' perception of learning: A more positive perception </p></list-item><list-item><p> Students' perception of teachers: Moving in the right direction </p></list-item><list-item><p> Students' academic self-perceptions: Feeling more on the positive side </p></list-item><list-item><p> Students' perceptions of atmosphere: A more positive atmosphere </p></list-item><list-item><p> Students' social self-perception: Not too bad (<xref rid="R17" ref-type="bibr">17</xref>). </p></list-item></list><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Demographic data </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Educational Hospital(n)</td><td rowspan="1" colspan="1"> Ali Asghar<break/>RasouleAkram<break/>Firouzgar<break/>AkbarAbadi </td><td align="center" rowspan="1" colspan="1"> 53<break/>93<break/>74<break/>47 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Ward(n)</td><td rowspan="1" colspan="1"> internal medicine<break/>pediatrics<break/>surgery<break/>gynecology </td><td align="center" rowspan="1" colspan="1"> 69<break/>88<break/>38<break/>72 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age(year)</td><td rowspan="1" colspan="1"> 24.30&#x000b1;1.24<break/><break/>Lower bound<break/>Upper bound </td><td align="center" rowspan="1" colspan="1"> <break/> <break/>24.1149<break/>24.4945 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gender (F / M)(n)</td><td rowspan="1" colspan="1">181 / 82</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Stage (Stager / Intern) (n, %)</td><td rowspan="1" colspan="1">(161,60% )/ (106, 40%)</td></tr></tbody></table></table-wrap><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Modified DREEM scores </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Subscale</td><td align="center" rowspan="1" colspan="1">Mean score</td><td align="center" rowspan="1" colspan="1">95% Confidence Interval</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' perception of learning(44 points)</td><td align="center" rowspan="1" colspan="1">21.80&#x000b1;6</td><td align="center" rowspan="1" colspan="1">(21.0788,22.5242)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' perception of teachers(36 points)</td><td align="center" rowspan="1" colspan="1">21.66&#x000b1;5.24</td><td align="center" rowspan="1" colspan="1">(21.0318,22.2941)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' academic self-perceptions(28 points)</td><td align="center" rowspan="1" colspan="1">14.90&#x000b1;4.55</td><td align="center" rowspan="1" colspan="1">(14.3509,15.4468)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' perceptions of atmosphere(40 points)</td><td align="center" rowspan="1" colspan="1">21.97&#x000b1;6.56</td><td align="center" rowspan="1" colspan="1">(21.1836,22.7640)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' social self-perception(28 points)</td><td align="center" rowspan="1" colspan="1">15.81&#x000b1;3.72</td><td align="center" rowspan="1" colspan="1">(15.3607,16.2572)</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total(176 points)</td><td align="center" rowspan="1" colspan="1">96.15&#x000b1;21.64</td><td align="center" rowspan="1" colspan="1">(93.5375,98.7547)</td></tr></tbody></table></table-wrap><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> CVR values Mean of judgment And the Accept or Remove results of each item </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Items</td><td align="center" rowspan="1" colspan="1">CVR</td><td align="center" rowspan="1" colspan="1">Mean of judgment</td><td align="center" rowspan="1" colspan="1">Accept or Remove</td><td align="center" rowspan="1" colspan="1">Items</td><td align="center" rowspan="1" colspan="1">CVR</td><td align="center" rowspan="1" colspan="1">Mean of judgment</td><td align="center" rowspan="1" colspan="1">Accept or Remove</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">26</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">1.33</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">1.17</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">1.83</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">28</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">29</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">1.33</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">1.17</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">31</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">1.33</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">32</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">33</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">-0.5</td><td align="center" rowspan="1" colspan="1">0.50</td><td align="center" rowspan="1" colspan="1">R</td><td align="center" rowspan="1" colspan="1">34</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">1.33</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">-0.17</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">R</td><td align="center" rowspan="1" colspan="1">35</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">1.17</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">36</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">37</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">1.17</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">38</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">39</td><td align="center" rowspan="1" colspan="1">-0.17</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">R</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">1.17</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">40</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">1.33</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">1.83</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">-0.33</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">R</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">-0.17</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">R</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">1.83</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">43</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">1.83</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">1.33</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">0.83</td><td align="center" rowspan="1" colspan="1">1.83</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">45</td><td align="center" rowspan="1" colspan="1">0.67</td><td align="center" rowspan="1" colspan="1">1.67</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">1.33</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">46</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">1.17</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1">-0.67</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">R</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">48</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">49</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">A</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.50</td><td align="center" rowspan="1" colspan="1">A</td><td align="center" rowspan="1" colspan="1">50</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">A</td></tr></tbody></table></table-wrap><table-wrap id="T4" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Internal consistency reliability of DREEM </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Subscale</td><td align="center" rowspan="1" colspan="1">Number of items</td><td align="center" rowspan="1" colspan="1">Cronbach's Alpha</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' perception of learning</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0.722</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' perception of teachers</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">0.739</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' academic self-perceptions</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">0.759</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' perceptions of atmosphere</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.771</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Students' social self-perception</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">0.446</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">0.914</td></tr></tbody></table></table-wrap><sec id="s3-1"><title>Validity</title></sec><sec id="s3-2"><title>Face validity</title><p> After the study of face validity of the questionnaire, the questions 7, 11, 12, 25, 27, 35, 42, 47 and 50 were amended again in writing. </p></sec><sec id="s3-3"><title>Content Validity</title><p> The sum of the Content Validity Ratios (&#x02211; CVR) was found 17.33 and Mean of CVR was computed 0.35. </p><p> The questions 9, 10, 17, 22, 39 and 42 were removed from the questionnaire. </p><p> The Content Validity Index (CVI) was found 0.39. </p></sec><sec id="s3-4"><title> Reliability </title><p> The Mean of Test-Retest reliability of DREEM was 0.71 and The Consistency reliability was in an acceptable range. </p><p> Considering that the Cronbach'salpha coefficient has become more than 0.7, we can say the instrument has reliability regarding internal consistency. Meanwhile, the Cronbach'salpha coefficient of the factors was obtained in the range of 0.446 to 0.771. </p></sec><sec id="s3-5"><title> Factor analysis </title><p> The KMO value was calculated as 0.892, which shows the proportion of samples number for factor analysis and the Bartlett's sphericity test became significant at significance error of 0.05 (p&#x0003c;0.001), indicating the homogeneity of the data for factor analysis testing. </p><p> After factors extraction, 5 factors with Eigen values higher than 1 were obtained, which covered the variance of 43.471%. </p></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> After evaluation of the reliability and validity of the Persian version DREEM, the face validity of the questionnaire was confirmed, and the content validity index and the average of content validity ratio were obtained as 0.39 and 0.35, respectively. The mean of the CVR and CVI were low level totally. Perhaps we can say in review of content validity that the CVR value and the low average of judgments in some questions leading to their omission from the questionnaire were due to differences in the cultural context of the countries (e.g. questions 17 and 39). The overall content validity will be higher if the value of the CVI is closer to 0.99 and vice versa. Among the studies conducted in various countries, one study has been also done in Greece in 2010 by IDK Dimoliatis et al. entitles as validating the Greek translation of DREEM, in which the face and content validity of the questionnaire (qualitatively) has been studied. The face and content validity were optimized; however, due to no limitation of international results, no question was removed or added (<xref rid="R18" ref-type="bibr">18</xref>). </p><p> The instrument consistency reliability was better in interns than to trainees; but, both were in an acceptable range. The instrument consistency reliability was reported as average in Getulio R, De Oliveira Filho et al. study in Brazil in 2005 (<xref rid="R19" ref-type="bibr">19</xref>); also in Greece, the test and retest reliabilities were both obtained as 0.9. Examining the validity and reliability of Pakistani version the instrument conducted by JunaidSarfraz Khan et al.&#x000a0;in 2011, the Spearman - Brown correlation coefficients (0.868) indicated the reliability of the analysis (<xref rid="R20" ref-type="bibr">20</xref>). </p><p> Review results of the internal consistency of the Persian version of the tool along with the results obtained in countries, including Brazil, Greece, Malaysia (<xref rid="R21" ref-type="bibr">21</xref>), China (<xref rid="R22" ref-type="bibr">22</xref>), Spain (<xref rid="R23" ref-type="bibr">23</xref>), Germany (<xref rid="R3" ref-type="bibr">3</xref>), New Zealand (<xref rid="R8" ref-type="bibr">8</xref>) and Pakistan with similar studies are given in the following table. It can be said that in all the translated versions of the instrument, the reliability of parallelism had a reasonable value <xref ref-type="table" rid="T6">Table 6</xref>. </p><table-wrap id="T5" orientation="portrait" position="float"><label> Table 5 </label><caption><title> Item factor loading </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Scale in original Questionnaire</td><td align="center" rowspan="1" colspan="1">Suggested scale</td><td align="center" rowspan="1" colspan="1">The most factor loading in proportion to the 5 &#x02013;scale domain</td><td align="center" rowspan="1" colspan="1">Scale 1</td><td align="center" rowspan="1" colspan="1">Scale 2</td><td align="center" rowspan="1" colspan="1">Scale 3</td><td align="center" rowspan="1" colspan="1">Scale 4</td><td align="center" rowspan="1" colspan="1">Scale 5</td><td align="center" rowspan="1" colspan="1">Item</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.552</td><td align="center" rowspan="1" colspan="1">0.552</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.472</td><td align="center" rowspan="1" colspan="1">0.472</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.498</td><td align="center" rowspan="1" colspan="1">0.498</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">-0.307</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">3</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">0.356</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.356</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">0.377</td><td align="center" rowspan="1" colspan="1">0.350</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.377</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">5</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.507</td><td align="center" rowspan="1" colspan="1">0.507</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.401</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">6</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.515</td><td align="center" rowspan="1" colspan="1">0.515</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">7</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.492</td><td align="center" rowspan="1" colspan="1">0.492</td><td align="center" rowspan="1" colspan="1">0.309</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">8</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.596</td><td align="center" rowspan="1" colspan="1">0.596</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.575</td><td align="center" rowspan="1" colspan="1">0.575</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">10</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.533</td><td align="center" rowspan="1" colspan="1">0.533</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">11</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">0.315</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.315</td><td align="center" rowspan="1" colspan="1">12</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">13</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.616</td><td align="center" rowspan="1" colspan="1">0.616</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">14</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.547</td><td align="center" rowspan="1" colspan="1">0.547</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">15</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.438</td><td align="center" rowspan="1" colspan="1">0.427</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.438</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">16</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.629</td><td align="center" rowspan="1" colspan="1">0.629</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">17</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.652</td><td align="center" rowspan="1" colspan="1">0.652</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">18</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.613</td><td align="center" rowspan="1" colspan="1">0.613</td><td align="center" rowspan="1" colspan="1">0.391</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">19</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.666</td><td align="center" rowspan="1" colspan="1">0.666</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">20</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.326</td><td align="center" rowspan="1" colspan="1">-0.515</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.326</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">21</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.445</td><td align="center" rowspan="1" colspan="1">0.445</td><td align="center" rowspan="1" colspan="1">-0.401</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">22</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.441</td><td align="center" rowspan="1" colspan="1">0.441</td><td align="center" rowspan="1" colspan="1">-0.415</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">23</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">0.323</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.323</td><td align="center" rowspan="1" colspan="1">24</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.683</td><td align="center" rowspan="1" colspan="1">0.683</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">25</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.499</td><td align="center" rowspan="1" colspan="1">0.499</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">-0.441</td><td align="center" rowspan="1" colspan="1">26</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.451</td><td align="center" rowspan="1" colspan="1">0.451</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">-0.423</td><td align="center" rowspan="1" colspan="1">27</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.476</td><td align="center" rowspan="1" colspan="1">0.476</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">28</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.485</td><td align="center" rowspan="1" colspan="1">0.485</td><td align="center" rowspan="1" colspan="1">0.301</td><td align="center" rowspan="1" colspan="1">0.429</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">29</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.577</td><td align="center" rowspan="1" colspan="1">0.577</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">30</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.542</td><td align="center" rowspan="1" colspan="1">0.542</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">31</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.318</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">-0.413</td><td align="center" rowspan="1" colspan="1">0.318</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">32</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.622</td><td align="center" rowspan="1" colspan="1">0.622</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">33</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.664</td><td align="center" rowspan="1" colspan="1">0.664</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">34</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.637</td><td align="center" rowspan="1" colspan="1">0.637</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">35</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.660</td><td align="center" rowspan="1" colspan="1">0.660</td><td align="center" rowspan="1" colspan="1">-0.303</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">36</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.655</td><td align="center" rowspan="1" colspan="1">0.655</td><td align="center" rowspan="1" colspan="1">-0.348</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">37</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.730</td><td align="center" rowspan="1" colspan="1">0.730</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">38</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.546</td><td align="center" rowspan="1" colspan="1">0.546</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">39</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.479</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.479</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">-0.322</td><td align="center" rowspan="1" colspan="1">40</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.519</td><td align="center" rowspan="1" colspan="1">0.519</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">41</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">0.484</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.484</td><td align="center" rowspan="1" colspan="1">-0.307</td><td align="center" rowspan="1" colspan="1">42</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.489</td><td align="center" rowspan="1" colspan="1">0.489</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">43</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">0.303</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">0.303</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">44</td></tr></tbody></table></table-wrap><table-wrap id="T6" orientation="portrait" position="float"><label> Table 6 </label><caption><title> Comparison of Internal consistency of different versions among countries </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Country</td><td align="center" rowspan="1" colspan="1">Total Cronbach&#x02019;s Alfa</td><td align="center" rowspan="1" colspan="1">Subscales Cronbach's Alfa</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Iran</td><td align="center" rowspan="1" colspan="1">0.914</td><td align="center" rowspan="1" colspan="1">0.446-0.771</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Brazil</td><td align="center" rowspan="1" colspan="1">0.93</td><td align="center" rowspan="1" colspan="1">0.58-0.93</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Greek</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.48-0.79</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Malaysia</td><td align="center" rowspan="1" colspan="1">0.936</td><td align="center" rowspan="1" colspan="1">0.58 &#x02013; 0.82</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">China</td><td align="center" rowspan="1" colspan="1">0.949</td><td align="center" rowspan="1" colspan="1">0.623-0.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Spain</td><td align="center" rowspan="1" colspan="1">0.91</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Germany</td><td align="center" rowspan="1" colspan="1">0.92 &#x00026;amp; 0.94</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">New Zealand</td><td align="center" rowspan="1" colspan="1">0.90 &#x00026;amp; 0.92</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Pakistan</td><td align="center" rowspan="1" colspan="1">0.91</td><td align="center" rowspan="1" colspan="1"> </td></tr></tbody></table></table-wrap><p> Reviewing the structure validity, the KMO test and Bartlett's Test of Sphericity provided&#x000a0;acceptable values for performing factor analysis; in factor extraction using analysis of&#x000a0;principal components, 5 factors were obtained. The model without rotation compared to the rotating models, indicated a better fitness with the core composition of the questionnaire based on our data. According to the questionnaire items content, it is recommended: </p><p> The questions 3-5-6-8-9-10-15-16-18-19-21-22-23-25-26-28-29-30-31-32-33-35-36-37-39-40-43 remain at the core axis and the 4-27-42 questions will be put at suggesting topics. (It should be noted that the number of items is provided after 6 questions deleting in the content validity review). </p><p> Structure validity has been also discussed in other studies. For example, in a study conducted in Brazil using the DREEM questionnaire, the construct validity was demonstrated. The factor analysis found 5 factors that explained the variance of 52% (<xref rid="R19" ref-type="bibr">19</xref>). </p><p> In a study in Malaysia in 2012 carried out by MuhamadSaifulBahriYusoff, the confirmatory factor analysis was used in examining the structure validity, which rejected the suggesting 5-factor structure fitness (<xref rid="R21" ref-type="bibr">21</xref>). </p><p> In a study conducted in 2009 in China by Jian Wang et al., 5 factors were found by analysis of principal components through Oblimin and Kaiser normalizing rotation method in performing factor analysis. The main factors names were kept, but some items in each area were changed. The five factors all had factor loadings greater than 1, and totally explained the variance of 52.186% (<xref rid="R22" ref-type="bibr">22</xref>). </p><p> In the Greek version of the instrument, factor analysis created significant areas; however, did not match everywhere with the original version (<xref rid="R18" ref-type="bibr">18</xref>). </p><p> In the Swedish version of DREEM, the exploratory factor analysis was also used due to poor fitness of the confirmatory factor analysis, which proposed 5 new factors for the tool (<xref rid="R24" ref-type="bibr">24</xref>). </p><p> In the Pakistani version of the tool, both exploratory and confirmatory factor analysis of areas were created;&#x000a0;however, mismatch with the original version was high due to English to Pakistani cultural differences (<xref rid="R20" ref-type="bibr">20</xref>). </p><p> The German version of factor analysis showed that 5 dimensions compared to 5 areas considered by DREEM authors as necessary conditions are slightly divergent. Finally, the DREEM fitness was shown only just for the students but also for teachers in measuring learning environment (<xref rid="R3" ref-type="bibr">3</xref>). </p><p> According to the results of this study, the Persian modified version of DREEM consisting of 44 questions in 5 axes provided with the introduced combination in results and appropriate psychometric properties, has the capabilities of being used in assessment of our country&#x02019;s medical and clinical training system, and can be used to assess the clinical training provided for medical students. The researchers are able to use the modified questionnaire for employing the questionnaire within domestic clinical environments. They can also use the original DREEM questionnaire translation to achieve the comparison with the original DREEM questionnaire in an international approach. </p></sec><sec sec-type="conclusion" id="s6"><title>Conclusion</title><p> In some studies it is suggested that a few of items be deleted or edited due to those countries cultural issues or students conceptions, but in order to maintain international results no question is deleted. </p><p> Results of this study and similar studies in Sweden, Pakistan, China and Greece in oppose to previous claims shows DREEM questionnaire is not a questionnaire independent to culture. </p><p> According to the results of this study, Persian module of the DREEM including 44 questions in 5 domains with suitable valid specifications can be used in assessment of clinical educational environments in our country. </p><p> Researchers can refer to each of the questionnaire: </p><list list-type="simple"><list-item><p> - The modified questionnaire for clinical educational environment surveys in our country. </p></list-item><list-item><p> - The translated original questionnaire in comparison with original DREEM in international approaches. </p></list-item></list></sec>
Emergency medicine specialty may improve patient satisfaction
Could not extract abstract
<contrib contrib-type="author"><name><surname>Abbasi</surname><given-names>Saeed</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Farsi</surname><given-names>Davood</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Bahrani</surname><given-names>Maryam</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Davari</surname><given-names>Saeed</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Pishbin</surname><given-names>Elham</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Kianmehr</surname><given-names>Nahid</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rezai</surname><given-names>Mahdi</given-names></name><xref ref-type="aff" rid="A07"> <sup>7</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Yazdanpanah</surname><given-names>Reza</given-names></name><xref ref-type="aff" rid="A08"> <sup>8</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mofidi</surname><given-names>Mani</given-names></name><xref ref-type="aff" rid="A09"> <sup>9</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec id="s1"><title>Brief Communication</title><p> Emergency Department (ED) is a setting where patients usually present with acute and severe illnesses (<xref rid="R1" ref-type="bibr">1</xref>). Nowadays, patient satisfaction has become an important outcome of health care services and shows quality of care that provides valuable information about the care delivered by an ED system (<xref rid="R2" ref-type="bibr">2</xref>-<xref rid="R4" ref-type="bibr">4</xref>). A patient&#x02019;s experience at an ED can influence his or her choice of hospital when seeking future care and may generate either positive or negative comments among their social sphere (<xref rid="R5" ref-type="bibr">5</xref>). Patient satisfaction depends on many factors such as length of stay at ED, severity of illness, demographic characteristics of patients, patient&#x02019;s origin and language barriers (<xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R7" ref-type="bibr">7</xref>). We conducted the current study to find out whether the presence of Emergency Medicine (EM) system is effective in patient satisfaction in the EDs. </p><p> This was a cross sectional study on patient satisfaction in EDs of two tertiary centers over a period of 12 weeks. EM residents with supervision of EM faculties provided care at the first center (academic ED) and general practitioners provided care for patients at the second hospital (non academic ED). Both hospitals had an annual number of ED visitors of approximately 40,000 patients with medical and surgical complaints. 3 EM residents and 3 general practitioners were present in each hospital respectively. EM faculties did not visit the patients alone; also there were no interns or residents (other than EM residents in academic ED) in both hospitals. After approval by the Local Ethics Committee (LEC), 12 days were randomly chosen from the 12 weeks of the study period. During a 24-hour period, 40 patients (about 30% of patients presented at the EDs) were randomly chosen to complete the questionnaire. </p><p> During 12 weeks period, 716 patients enrolled in the study. The study population consisted of 355 patients (49.6%) from the academic hospital and 361 (50.4%) patients from the non-academic hospital. </p><p> There was no difference between two hospitals regarding sex, mean of age and educational levels of patients. Patients&#x02019; opinion about quality of care in EDs including staff performance, facilities, and physical environment are shown in (<xref ref-type="table" rid="T1">Table 1</xref>). We found that all the items (except secretor) received significantly higher score in academic section of ED (p&#x0003c;0.001). </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Patients&#x02019; Opinions on each Component of the Emergency Department in both Hospitals </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="2" colspan="1">Variable (%)</td><td align="center" colspan="2" rowspan="1">Excellent</td><td align="center" colspan="2" rowspan="1">Good</td><td align="center" colspan="2" rowspan="1">Fair</td><td align="center" colspan="2" rowspan="1">Poor</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">EM</td><td align="center" rowspan="1" colspan="1">GP</td><td align="center" rowspan="1" colspan="1">EM</td><td align="center" rowspan="1" colspan="1">GP</td><td align="center" rowspan="1" colspan="1">EM</td><td align="center" rowspan="1" colspan="1">GP</td><td align="center" rowspan="1" colspan="1">EM</td><td align="center" rowspan="1" colspan="1">GP</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Physicians&#x02019; attitude</td><td align="center" rowspan="1" colspan="1">31.3</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">57.7</td><td align="center" rowspan="1" colspan="1">49.6</td><td align="center" rowspan="1" colspan="1">9.9</td><td align="center" rowspan="1" colspan="1">32.7</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">7.8</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Physicians&#x02019; competency</td><td align="center" rowspan="1" colspan="1">29.9</td><td align="center" rowspan="1" colspan="1">6.9</td><td align="center" rowspan="1" colspan="1">51</td><td align="center" rowspan="1" colspan="1">36</td><td align="center" rowspan="1" colspan="1">17.7</td><td align="center" rowspan="1" colspan="1">41.8</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">15.2</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nurses&#x02019; attitude</td><td align="center" rowspan="1" colspan="1">27.6</td><td align="center" rowspan="1" colspan="1">8.9</td><td align="center" rowspan="1" colspan="1">57.2</td><td align="center" rowspan="1" colspan="1">50.7</td><td align="center" rowspan="1" colspan="1">13.8</td><td align="center" rowspan="1" colspan="1">32.7</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">7.8</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Secretary</td><td align="center" rowspan="1" colspan="1">30.1</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">64.8</td><td align="center" rowspan="1" colspan="1">78.9</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">9.1</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Treatment strategies</td><td align="center" rowspan="1" colspan="1">21.1</td><td align="center" rowspan="1" colspan="1">6.4</td><td align="center" rowspan="1" colspan="1">50.1</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1">26.5</td><td align="center" rowspan="1" colspan="1">38.5</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">14.1</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Equipments</td><td align="center" rowspan="1" colspan="1">15.8</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">43.1</td><td align="center" rowspan="1" colspan="1">39.6</td><td align="center" rowspan="1" colspan="1">36.6</td><td align="center" rowspan="1" colspan="1">46.3</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">10.2</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Waiting time for visiting by physician</td><td align="center" rowspan="1" colspan="1">29.6</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">63.7</td><td align="center" rowspan="1" colspan="1">55.4</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">21.3</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">13.3</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Physical environment</td><td align="center" rowspan="1" colspan="1">11.8</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">49.3</td><td align="center" rowspan="1" colspan="1">42.4</td><td align="center" rowspan="1" colspan="1">35.8</td><td align="center" rowspan="1" colspan="1">43.8</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">8.9</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Laboratory</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">69.9</td><td align="center" rowspan="1" colspan="1">65.7</td><td align="center" rowspan="1" colspan="1">15.2</td><td align="center" rowspan="1" colspan="1">24.4</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3.9</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Radiology</td><td align="center" rowspan="1" colspan="1">24.2</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">62.5</td><td align="center" rowspan="1" colspan="1">60.7</td><td align="center" rowspan="1" colspan="1">11.3</td><td align="center" rowspan="1" colspan="1">23.5</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">5</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Turnaround Time</td><td align="center" rowspan="1" colspan="1">27.6</td><td align="center" rowspan="1" colspan="1">6.6</td><td align="center" rowspan="1" colspan="1">62.5</td><td align="center" rowspan="1" colspan="1">56</td><td align="center" rowspan="1" colspan="1">7.3</td><td align="center" rowspan="1" colspan="1">24.4</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">13</td></tr><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Overall satisfaction (after one week)</td><td align="center" rowspan="1" colspan="1">28.2</td><td align="center" rowspan="1" colspan="1">4.4</td><td align="center" rowspan="1" colspan="1">57.7</td><td align="center" rowspan="1" colspan="1">38</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">16.6</td></tr></tbody></table><table-wrap-foot><fn><p> EM: Emergency Medicine, GP: General Practitioner </p></fn></table-wrap-foot></table-wrap><p> One week after discharge from the hospital, we called patients and asked them about the reasons for satisfaction and dissatisfaction. The details are shown in (<xref ref-type="table" rid="T2">Table 2</xref>). The overall satisfaction was significantly higher in academic hospitals. (Chi<sup>2</sup>, p&#x0003c;0.001) </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Patients&#x02019; satisfaction on each Component of the Emergency Department in both Hospitals after One Week from Discharge </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="2" colspan="1">Variable (%)</td><td align="center" colspan="2" rowspan="1">Satisfying</td><td align="center" colspan="2" rowspan="1">Dissatisfying</td><td rowspan="2" colspan="1">p value</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">EM</td><td align="center" rowspan="1" colspan="1">GP</td><td align="center" rowspan="1" colspan="1">EM</td><td align="center" rowspan="1" colspan="1">GP</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Admission Time</td><td align="center" rowspan="1" colspan="1">98.9</td><td align="center" rowspan="1" colspan="1">84.2</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">15.8</td><td align="center" rowspan="1" colspan="1">Fisher&#x02019;s Exact, &#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Physicians</td><td align="center" rowspan="1" colspan="1">96.9</td><td align="center" rowspan="1" colspan="1">60.9</td><td align="center" rowspan="1" colspan="1">3.1</td><td align="center" rowspan="1" colspan="1">39.1</td><td align="center" rowspan="1" colspan="1"> Chi<sup>2</sup>, &#x0003c;0.001 </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Nurses</td><td align="center" rowspan="1" colspan="1">96.6</td><td align="center" rowspan="1" colspan="1">87.8</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">12.2</td><td align="center" rowspan="1" colspan="1"> Chi<sup>2</sup>, &#x0003c;0.001 </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Secretors</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">99.4</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">Fisher&#x02019;s Exact, 0.499</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Physical Place</td><td align="center" rowspan="1" colspan="1">95.2</td><td align="center" rowspan="1" colspan="1">84.5</td><td align="center" rowspan="1" colspan="1">4.8</td><td align="center" rowspan="1" colspan="1">15.5</td><td align="center" rowspan="1" colspan="1"> Chi<sup>2</sup>, &#x0003c;0.001 </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Laboratory</td><td align="center" rowspan="1" colspan="1">99.4</td><td align="center" rowspan="1" colspan="1">97.2</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">Fisher&#x02019;s Exact, 0.037</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Radiology</td><td align="center" rowspan="1" colspan="1">99.2</td><td align="center" rowspan="1" colspan="1">96.1</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">Fisher&#x02019;s Exact, 0.012</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Discharge Time</td><td align="center" rowspan="1" colspan="1">98.6</td><td align="center" rowspan="1" colspan="1">79.2</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">20.8</td><td align="center" rowspan="1" colspan="1"> Chi<sup>2</sup>, &#x0003c;0.001 </td></tr></tbody></table></table-wrap><p> 83 percent of patients who admitted at the academic hospital responded that they may recommend the hospital to their relatives or friends. One explanation about the effect of EM specialists in various aspects of satication was that in academic hospital, all process of patient care were supervised by EM faculties and each staff or worker took into account a better care for patients. </p><p> We found that in the academic hospital, the majority of patients were discharged by EM system. In contrast to other hospital, most patients were either admitted or left against medical advice. Unnecessary admission result in unfruitful outcomes such as less available empty beds for actual necessary hospitalization, overcrowding of ED, more waiting time for admission, more discharge by written consent and higher dissatisfaction rate. The number of leave against medical advice was significantly lower in academic hospital. These events probably related to better decision making and better disposition which was done by EM system. Blanco-Abril et al showed that overall satisfaction was higher among those ED patients who returned home than those who were admitted to the hospital (<xref rid="R8" ref-type="bibr">8</xref>). The most important reason for patient dissatisfaction in non academic hospital was related to the physicians' performance (39.1%; 95% CI, 34.3-44.3). But at the academic hospital, only 3.1% (95% CI, 1.4-5.1) of patients were dissatisfied with the physicians. </p><p> In our study, waiting time for the first visit was an important factor for dissatisfaction in non-academic hospital (15.8%; 95% CI, 11.9-19.7). This value was 1.1% (95% CI, 0.3-2.3) in academic hospital. The results from two studies conducted in 2 academic centers of Turkey and Pakistan have shown that prolongation of waiting time was the major concern for patients' dissatisfaction (<xref rid="R6" ref-type="bibr">6</xref>,<xref rid="R9" ref-type="bibr">9</xref>). Some studies revealed that clear expression of real situation to the patients had a significant influence in decreasing the sense of elongated waiting time and leads to an increase in the general satisfaction with the ED experience (<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R11" ref-type="bibr">11</xref>). </p><p> In conclusion, this study showed that the presence of EM specialist in EDs could be one of the important factors that decreases patient complaints and increases patient satisfaction. </p></sec>
Effect of university policies on research productions: a scientometric study
<p> <bold>Background:</bold> This study aimed to assess the effect of new package of interventions on scientific productions rate in Shahid Beheshti University of Medical Sciences. </p><p> <bold>Methods:</bold> Through a health system research, we extracted policies from the strategic plan of the university and 10 interventions were developed to increase the scientific productions in terms of quality, quantity and commercialization and to develop infrastructure for research in health service provision and education. For evaluating the effectiveness of interventions, citation and publication indicators for individuals and schools were analyzed using descriptive statistics and t-test. They were extracted from Scopus and ISI web of knowledge during period of 1/1/2009 to 30/5/2012. </p><p> <bold>Results:</bold> There was an increasing trend in scientific productions from 2009 to mid-2012. We found 60 percent of total scientific productions of the university were published during last 3.5 years. During this 3.5 years, 10 more percentile of faculty members involved in research. Schools of pharmacy, Medicine and Health had the highest scientific products. Mean for h-index was 1.5 (SD=2.49) in ISI and 1.9 (SD=2.89) in Scopus database (p&#x0003c;0.001). </p><p> <bold>Conclusion:</bold> Effective policies and interventions lead to 46% increase in scientific productions from 2009 to 2010 and 56% increase from 2010 to 2011. </p>
<contrib contrib-type="author"><name><surname>Sohrabi</surname><given-names>Mohammad-Reza</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rahmati-Roodsari</surname><given-names>Mohammad</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rahmdar</surname><given-names>Saeid-Reza</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> As the era of sciences extended, research organizations increasingly perceived the need for scientometrics indices for comparison of researchers and also trend analysis. Hirsch introduced h-index as one of the most acceptable indices in 2005 (<xref rid="R1" ref-type="bibr">1</xref>). It is not limited only to the researchers; Braun et al introduced a Hirsch-type index for journals which was defined as &#x0201c;h published papers, each of them has at least h citations&#x0201d;; they suggested it as a supplement for impact factor (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R3" ref-type="bibr">3</xref>). Recently researchers recommended to use h-index to distinguish &#x0201c;hot topics&#x0201d; from &#x0201c;old topics&#x0201d; (<xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R5" ref-type="bibr">5</xref>). </p><p> Iranian Ministry of Health and Medical Education (MOHME) developed a comprehensive scientific national map (including values, goals, priorities, policies and strategies). In research era, sub-secretary for research and technology of Iranian MOHME developed a plan based on this map and national development plans in 2007; in this plan duties of each 42 universities of medical sciences have been clearly defined to attain the goals. They had to train human resources, prepare suitable setting and equipment, and facilitated research process. These universities also developed their strategic plan based on these duties. Although there have had strategic plans and related interventions in research system of the universities, but their effect on performance and scientific production of the universities remained unclear. As we did not find any published evidence on effect of research system interventions on scientific productions of medical universities, managers can use this information for evidence based policy making and management in the university (<xref rid="R6" ref-type="bibr">6</xref>). </p><p> We conducted this study in order to assess the effect of these new policies and related interventions on scientometrics indices of Shahid Beheshti University of Medical Sciences (SBUMS) and its faculties during period of 1/1/2009 to 30/5/2012. This university is the second ranked university in research among 42 universities of medical sciences in Iran and we assessed all the faculty members in this study (<xref rid="R7" ref-type="bibr">7</xref>). </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> SBUMS has more than 1200 faculty members, 10 schools, and 48 research centers. It prepares health services for more than 80% of citizens of metropolitan of Tehran, the capital of Iran by this time. We designed a package of interventions based on the policies in sub-secretary for research and technology of SBUMS; then assessed its effect on scientometrics indices. </p><sec id="s2-1"><title>Policies</title><p> Strategic plan of SBUMS contains policies like: to expand applied and fundamental Health system researches; to extend national and international relationship in research; to develop context for scientific production using new technologies; to support researchers; to improve efficacy and effectiveness of research; to absorb and extend resources; to improve research products in terms of quality, quantity and commercialization; to develop infrastructure for research in health service provision and education. </p></sec><sec id="s2-2"><title>Interventions</title><p> Based on strategic planning, following interventions were implemented: </p><p> 1) There was a gap in the process of conducting scientific productions from theories or questions of faculty members. We needed some intermediate personnel to do the administrative process of research; they should facilitate the process of developing proposals from an idea. In order to train human resources, we designed an educational program for research development and management and presented it for researchers and research centers. Duration of this course was 300 hours in 12 educational blocks. This course empowered the manpower to design, manage and finalize a research proposal, manage its accomplishment and even wiring articles drafts. We named these personnel as research workers. 2) Access to full text articles of scientific journals was a major concern; Sub-secretary for research subscribed to major sources of scientific product like ISI web of knowledge, Scopus, Oxford, ProQuest, and Ovid. To be more enriched, back files of 800 journals were bought too. All the faculty members and researchers could use this database at the university website and through other related IPs. 3) In order to develop research setting and facilities we established a comprehensive central laboratory. New technologies like advanced PCR were employed in this laboratory. 4) Other intervention for setting a baseline for research was developing a central animal lab. In this lab new technologies like MRI (Magnetic Resonance Imaging) for animals were used. 5) First incubator for pharmaceutical sciences established to communicate university to industry. 6) We facilitate administrative processes of research using web-based software; Submitting proposals, progress reports, result of ethics committee and other administrative affairs were done through this software. 7) To conduct researches toward goals of organization, research priorities for schools and sub-secretaries of the university in two separate projects were defined (<xref rid="R8" ref-type="bibr">8</xref>, <xref rid="R9" ref-type="bibr">9</xref>). In these research projects the recommended method of council on health research for development was used (<xref rid="R10" ref-type="bibr">10</xref>); all the stakeholders were defined in different relevant organization and also in community level. In next step and stakeholder analysis was done. The priorities extracted through brain storming technique and then finalized using Delphi technique. These projects revealed the fields of research priorities in the university in different categories of basic sciences, clinical sciences and health system research. The University encouraged researchers to use these priorities by allocating resources only for them. 8) A supportive funding organization (Alborz Persian-Drug Fund) established to connect non-academic to academic organizations. This funding agency supports the researches that lead to a considerable product for health market. On the other hand a &#x0201c;clear room&#x0201d; established to attract financial resources for research from MOHME. 9) Extending the research centers from 20 to 48 research centers and organize them in context of 15 research school, each of them contain at least three subject related research center. These research schools were included in five relevant research institutes, each of them cover at least three research schools. This process is ongoing and new research centers are adding every year. It decreased the financial burden of over-head costs of staffing and maintenances.10) The 10<sup>th</sup> intervention defined as special financial support for faculties that disseminated the results of their projects as articles. These bonuses were defined based on journals&#x02019; scientific credit, Impact Factor and indexing. More bonuses were paid for PubMed and ISI web of knowledge indexed journals as the first degree journals; they paid more for journals with higher impact factors. This bonus was besides the grunts they received for the projects. On the other side faculty members&#x02019; articles- at least one English language article in PubMed or ISI journals- may facilitate annual promotional benefit. Qualified faculty members in Iran receive an employment benefit each year by which their salary increases about five percent. This benefit was only based on their educational activities before that. By this new rule they encouraged to be more active in research fields. </p></sec><sec id="s2-3"><title>Outcomes assessment</title><p> We considered two years as latent period, from 2007 to 2009, for interventions to take effect and outcomes revealed. For evaluating the effectiveness of interventions, research team considered descriptive statistics of publications in each year, total publications during last three years, row number of citations, total number of citations during last three years, h-index for individuals and means of publications in each year and last three years, number of citations, total number of citations during last three years for schools. They were extracted from Scopus and ISI web of knowledge during period of 1/1/2009 to 30/5/2012. Final list of faculty members based on their schools, scientific rank and employment situation requested from human resource office of the university. In order to prevent biases one expert faculty member in search who had history of working in two sub-secretaries for education and for research and technology of the university extracted these indices. He Searched data bases considering that Iranian names and family names may be written in different formats in English and sometimes one person has different profiles in these databases. Using specific protocols of Scopus and ISI web of knowledge and general rules of search such as using asterisk &#x0201c;*&#x0201d;, conjunctions -&#x0201c;OR&#x0201d;, &#x0201c;AND&#x0201d;, NOT&#x0201d;-, quotations &#x0201c;&#x0201d;,dashes and other relevant techniques for covering different writing formats all the faculty members of the university were assessed on scientometrics indices. </p><p> These indices were summarized based on schools. The trend of row number of scientific production extracted and presented in table 4. The indices were presented as descriptive statistics and were compared using t-student test when appropriate. Statistical analyses were performed using SPSS software, version 16.0 (SPSS Inc, Chicago, USA). </p><p> This study was approved by the Research Ethics Committee of SBUMS and was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association. </p></sec></sec><sec sec-type="results" id="s6"><title>Results</title><p> Totally 1203 faculty members were evaluated. Among these faculty members 128 (10.7%) were professors, 286 (23.8%) were associate professors, 626 (52.2%) were assistant professor, 156 (13.1%) were instructors and for the other 7, there was no scientific rank available in database. </p><p> There was totally 4605 article in Scopus since 1985 submitted by affiliation of Shahid Beheshti University of Medical Sciences, among them almost 60% of scientific productions were published from 2009 to mid-2012. The trend was increasing as about 420 (10%) of them were published in 2009, 613 (13%) in 2010, 957 (21%) in 2011 and 792 (17%) in first half of 2012. </p><p> Total articles and citations, their means based on years and also databases were presented in <xref ref-type="table" rid="T1">Table 1</xref>. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> means for scientometrics indices of faculty members of Shahid Beheshti University of Medical Sciences in Scopus and ISI during 2009-mid 2012 </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" colspan="2" rowspan="1">Scopus</td><td align="center" colspan="2" rowspan="1">ISI</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Articles</td><td align="center" rowspan="1" colspan="1">Citations</td><td align="center" rowspan="1" colspan="1">Articles</td><td align="center" rowspan="1" colspan="1">Citations</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Mid 2012</td><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">844</td><td align="center" rowspan="1" colspan="1">6486</td><td align="center" rowspan="1" colspan="1">635</td><td align="center" rowspan="1" colspan="1">4119</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Mean (SD)</td><td align="center" rowspan="1" colspan="1">0.7 (2.08)</td><td align="center" rowspan="1" colspan="1">5.4 (19.80)</td><td align="center" rowspan="1" colspan="1">0.5 (1.49)</td><td align="center" rowspan="1" colspan="1">3.4 (13.04)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">2011</td><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">1924</td><td align="center" rowspan="1" colspan="1">13283</td><td align="center" rowspan="1" colspan="1">1556</td><td align="center" rowspan="1" colspan="1">9018</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Mean (SD)</td><td align="center" rowspan="1" colspan="1">1.6 (4.27)</td><td align="center" rowspan="1" colspan="1">11.1 (37.65)</td><td align="center" rowspan="1" colspan="1">1.3 (3.42)</td><td align="center" rowspan="1" colspan="1">7.5 (26.84)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">2010</td><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">1717</td><td align="center" rowspan="1" colspan="1">10077</td><td align="center" rowspan="1" colspan="1">1279</td><td align="center" rowspan="1" colspan="1">6857</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Mean (SD)</td><td align="center" rowspan="1" colspan="1">1.4 (3.62)</td><td align="center" rowspan="1" colspan="1">8.4 (30.52)</td><td align="center" rowspan="1" colspan="1">1.1 (2.72)</td><td align="center" rowspan="1" colspan="1">5.7 (21.98)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">2009</td><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">1494</td><td align="center" rowspan="1" colspan="1">7222</td><td align="center" rowspan="1" colspan="1">1154</td><td align="center" rowspan="1" colspan="1">5050</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Mean (SD)</td><td align="center" rowspan="1" colspan="1">1.2 (3.15)</td><td align="center" rowspan="1" colspan="1">6 (23.34)</td><td align="center" rowspan="1" colspan="1">1 (2.63)</td><td align="center" rowspan="1" colspan="1">4.2 (18.68)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">2009-mid2012</td><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">5135</td><td align="center" rowspan="1" colspan="1">30582</td><td align="center" rowspan="1" colspan="1">3986</td><td align="center" rowspan="1" colspan="1">30925</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Mean (SD)</td><td align="center" rowspan="1" colspan="1">4.3 (10.40)</td><td align="center" rowspan="1" colspan="1">25.4 (90.56)</td><td align="center" rowspan="1" colspan="1">3.3 (8.18)</td><td align="center" rowspan="1" colspan="1">17.4 (66.62)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">10698</td><td align="center" rowspan="1" colspan="1">54300</td><td align="center" rowspan="1" colspan="1">8295</td><td align="center" rowspan="1" colspan="1">35909</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Mean (SD)</td><td align="center" rowspan="1" colspan="1">8.81 (22)</td><td align="center" rowspan="1" colspan="1">45.1 (176.44)</td><td align="center" rowspan="1" colspan="1">6.9 (18.07)</td><td align="center" rowspan="1" colspan="1">29.9 (124.25)</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">h-index : Mean (SD)</td><td align="center" rowspan="1" colspan="1">1.9 (2.89)</td><td align="center" rowspan="1" colspan="1">1.5 (2.49)</td></tr></tbody></table></table-wrap><p> In Scopus database, 80% of faculty members had at least one publication. After 2009, one more percentile involved in research and had publication in this database. H-index for 60% of them was one or more. In ISI web of knowledge 70% of faculty members had at least one indexed publication. After 2010, ten more percent of faculty members involved in research and had articles indexed in this database. There was the same situation for citations in both databases. Scientometrics indices are presented as percentile groups of faculty members for Scopus in <xref ref-type="table" rid="T2">Table 2</xref> and for ISI in <xref ref-type="table" rid="T3">Table 3</xref>. </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Percentiles for scientometrics indices of faculty members of Shahid Beheshti University of Medical Sciences in Scopus during 2009-mid 2012 </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" colspan="11" rowspan="1">Percentiles</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">40</td><td align="center" rowspan="1" colspan="1">50</td><td align="center" rowspan="1" colspan="1">60</td><td align="center" rowspan="1" colspan="1">70</td><td align="center" rowspan="1" colspan="1">80</td><td align="center" rowspan="1" colspan="1">90</td><td align="center" rowspan="1" colspan="1">95</td><td align="center" rowspan="1" colspan="1">97</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total articles</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1">34</td><td align="center" rowspan="1" colspan="1">45</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total citations</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">38</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">222</td><td align="center" rowspan="1" colspan="1">335</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles mid-2012</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles 2011</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles 2010</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">6.9</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles 2009</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">5.8</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations mid-2012</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">36.8</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations 2011</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">26</td><td align="center" rowspan="1" colspan="1">54.6</td><td align="center" rowspan="1" colspan="1">78.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations 2010</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">64.6</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations 2009</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">32</td><td align="center" rowspan="1" colspan="1">48.9</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">H-index</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">9</td></tr></tbody></table></table-wrap><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> Percentiles for scientometrics indices of faculty members of Shahid Beheshti University of Medical Sciences in ISI during 2009-mid 2012 </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" colspan="11" rowspan="1">Percentiles</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">40</td><td align="center" rowspan="1" colspan="1">50</td><td align="center" rowspan="1" colspan="1">60</td><td align="center" rowspan="1" colspan="1">70</td><td align="center" rowspan="1" colspan="1">80</td><td align="center" rowspan="1" colspan="1">90</td><td align="center" rowspan="1" colspan="1">95</td><td align="center" rowspan="1" colspan="1">97</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total articles</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">28.9</td><td align="center" rowspan="1" colspan="1">38.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total citations</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">64</td><td align="center" rowspan="1" colspan="1">152.6</td><td align="center" rowspan="1" colspan="1">242.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles mid-2012</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles 2011</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles 2010</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">7.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Articles 2009</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">6</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations mid-2012</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">16.9</td><td align="center" rowspan="1" colspan="1">24.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations 2011</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">39.9</td><td align="center" rowspan="1" colspan="1">57.9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations 2010</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">32</td><td align="center" rowspan="1" colspan="1">45</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Citations 2009</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">34</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">H-index</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">8.9</td></tr></tbody></table></table-wrap><p> Comparing the databases showed that means for h-index is statistically significant different in ISI and Scopus (p&#x0003c;0.001) (<xref ref-type="table" rid="T1">Table 1</xref>). </p><p> <xref ref-type="table" rid="T4">Table 4</xref> shows the scientific publications during 2009-2011 in Scopus and ISI data bases divided by schools. Comparing means based on year showed that there was no statistically significant difference between means in all schools in all selected years (p&#x0003e;0.05 for all) except for clinical departments of school of medicine in which in year 2010 (p=0.015) and 2009 (p=0.001) means for articles in Scopus was statistically significant higher than ISI; but in 2011 the means had no difference (p=0.098). </p><table-wrap id="T4" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Mean (Standard Deviation) for Articles of Schools of Shahid Beheshti university of Medical Sciences in Scopus and ISI databases during 2009-2011 </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="2" colspan="1">Number of faculty members</td><td align="center" colspan="2" rowspan="1">2009</td><td align="center" colspan="2" rowspan="1">2010</td><td align="center" colspan="2" rowspan="1">2011</td><td align="center" colspan="2" rowspan="1">h-index</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Schools</td><td align="center" rowspan="1" colspan="1">Scopus</td><td align="center" rowspan="1" colspan="1">ISI</td><td align="center" rowspan="1" colspan="1">Scopus</td><td align="center" rowspan="1" colspan="1">ISI</td><td align="center" rowspan="1" colspan="1">Scopus</td><td align="center" rowspan="1" colspan="1">ISI</td><td align="center" rowspan="1" colspan="1">Scopus</td><td align="center" rowspan="1" colspan="1">ISI</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Medicine (Faculties of clinical disciplines )</td><td align="center" rowspan="1" colspan="1">569</td><td align="center" rowspan="1" colspan="1"> 1.1<break/> (1.83) </td><td align="center" rowspan="1" colspan="1"> 0.7<break/> (1.46) </td><td align="center" rowspan="1" colspan="1"> 1.3<break/> (2.29) </td><td align="center" rowspan="1" colspan="1"> 0.95 <break/>(1.86) </td><td align="center" rowspan="1" colspan="1"> 1.3 <break/>(2.47) </td><td align="center" rowspan="1" colspan="1"> 1<break/> (2.02) </td><td align="center" rowspan="1" colspan="1">1.64 (2.29)</td><td align="center" rowspan="1" colspan="1"> 1.3<break/> (2.08) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Medicine (Faculties of basic Sciences disciplines)</td><td align="center" rowspan="1" colspan="1">103</td><td align="center" rowspan="1" colspan="1"> 1.8 <break/>(3.35) </td><td align="center" rowspan="1" colspan="1"> 1.62 <break/>(3.30) </td><td align="center" rowspan="1" colspan="1"> 1.7<break/> (2.69) </td><td align="center" rowspan="1" colspan="1"> 1.54<break/> (2.60) </td><td align="center" rowspan="1" colspan="1"> 2.2<break/> (3.29) </td><td align="center" rowspan="1" colspan="1"> 2 <break/>(3.34) </td><td align="center" rowspan="1" colspan="1">2.9 (3.26)</td><td align="center" rowspan="1" colspan="1"> 2.4<break/> (2.89) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Dentistry</td><td align="center" rowspan="1" colspan="1">122</td><td align="center" rowspan="1" colspan="1"> 0.42<break/> (0.88) </td><td align="center" rowspan="1" colspan="1"> 0.36<break/>(0.87) </td><td align="center" rowspan="1" colspan="1"> 0.45 <break/>(1.13) </td><td align="center" rowspan="1" colspan="1"> 0.38 <break/>(0.99) </td><td align="center" rowspan="1" colspan="1"> 0.62<break/> (1.33) </td><td align="center" rowspan="1" colspan="1">0.55 (1.23)</td><td align="center" rowspan="1" colspan="1"> 1 <break/>(2.02) </td><td align="center" rowspan="1" colspan="1"> 0.89<break/> (1.66) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Pharmacy</td><td align="center" rowspan="1" colspan="1">40</td><td align="center" rowspan="1" colspan="1"> 2.1<break/> (2.44) </td><td align="center" rowspan="1" colspan="1"> 1.9<break/> (2.30) </td><td align="center" rowspan="1" colspan="1"> 2 <break/>(2.46) </td><td align="center" rowspan="1" colspan="1"> 1.8 <break/>(2.29) </td><td align="center" rowspan="1" colspan="1"> 2.2 <break/>(2.33) </td><td align="center" rowspan="1" colspan="1">1.7 (1.83)</td><td align="center" rowspan="1" colspan="1"> 5<break/> (3.73) </td><td align="center" rowspan="1" colspan="1"> 3.9<break/> (3.53) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Rehabilitation</td><td align="center" rowspan="1" colspan="1">38</td><td align="center" rowspan="1" colspan="1"> 0.13<break/> (0.41) </td><td align="center" rowspan="1" colspan="1"> 0.13<break/> (0.34) </td><td align="center" rowspan="1" colspan="1"> 0.32<break/> (0.62) </td><td align="center" rowspan="1" colspan="1"> 0.29 <break/>(0.70) </td><td align="center" rowspan="1" colspan="1"> 0.42<break/> (0.79) </td><td align="center" rowspan="1" colspan="1">0.26 (0.50)</td><td align="center" rowspan="1" colspan="1">0.42 (1.20)</td><td align="center" rowspan="1" colspan="1"> 0.37<break/> (1.10) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Paramedical Sciences</td><td align="center" rowspan="1" colspan="1">62</td><td align="center" rowspan="1" colspan="1"> 0.35 <break/>(0.93) </td><td align="center" rowspan="1" colspan="1"> 0.23<break/> (96) </td><td align="center" rowspan="1" colspan="1"> 0.60 <break/>(1.34) </td><td align="center" rowspan="1" colspan="1"> 0.39<break/> (1.22) </td><td align="center" rowspan="1" colspan="1"> 0.68 <break/>(1.59) </td><td align="center" rowspan="1" colspan="1">0.48 (1.21)</td><td align="center" rowspan="1" colspan="1">0.90 (1.54)</td><td align="center" rowspan="1" colspan="1"> 0.66 <break/>(1.28) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Public Health</td><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1"> 1.1<break/> (1.71) </td><td align="center" rowspan="1" colspan="1"> 0.6 <break/>(1.18) </td><td align="center" rowspan="1" colspan="1"> 1.2 <break/>(2.89) </td><td align="center" rowspan="1" colspan="1"> 0.9 <break/>(1.91) </td><td align="center" rowspan="1" colspan="1"> 1.7 <break/>(2.97) </td><td align="center" rowspan="1" colspan="1">1.4 (3.26)</td><td align="center" rowspan="1" colspan="1">2.1 (3.21)</td><td align="center" rowspan="1" colspan="1"> 1.2<break/> (2.46) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Health, safety &#x00026; environment</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1"> 0.1<break/> (0.48) </td><td align="center" rowspan="1" colspan="1"> 0.1 <break/>(0.44) </td><td align="center" rowspan="1" colspan="1"> 0.1 <break/>(0.30) </td><td align="center" rowspan="1" colspan="1"> 0.1<break/> (0.22) </td><td align="center" rowspan="1" colspan="1"> 0.4<break/> (0.74) </td><td align="center" rowspan="1" colspan="1">0.2 (0.54)</td><td align="center" rowspan="1" colspan="1"> 0.6<break/> (1.63) </td><td align="center" rowspan="1" colspan="1"> 0.5<break/>(1.54) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Nutrition</td><td align="center" rowspan="1" colspan="1">35</td><td align="center" rowspan="1" colspan="1"> 0.7 <break/>(1.38) </td><td align="center" rowspan="1" colspan="1"> 0.9<break/> (2.13) </td><td align="center" rowspan="1" colspan="1"> 2.3 <break/>(4.1) </td><td align="center" rowspan="1" colspan="1"> 1.6<break/> (3.13) </td><td align="center" rowspan="1" colspan="1"> 2.6<break/> (4.69) </td><td align="center" rowspan="1" colspan="1">2.2 (3.86)</td><td align="center" rowspan="1" colspan="1"> 2.5<break/> (3.88) </td><td align="center" rowspan="1" colspan="1"> 1.8 <break/>(3.45) </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">School of Nursery and Midwifery</td><td align="center" rowspan="1" colspan="1">54</td><td align="center" rowspan="1" colspan="1"> 0.1<break/> (0.53) </td><td align="center" rowspan="1" colspan="1"> 0.1<break/> (0.52) </td><td align="center" rowspan="1" colspan="1"> 0.2 <break/>( 0.53) </td><td align="center" rowspan="1" colspan="1"> 0.1<break/> (0.33) </td><td align="center" rowspan="1" colspan="1"> 0.4<break/> (1.23) </td><td align="center" rowspan="1" colspan="1">0.2 (0.82)</td><td align="center" rowspan="1" colspan="1"> 0.4<break/> (1.08) </td><td align="center" rowspan="1" colspan="1"> 0.2 <break/>(0.71) </td></tr></tbody></table></table-wrap></sec><sec sec-type="discussion" id="s7"><title>Discussion</title><p> In this study we found scientific productions of faculty members in SBMUS have increased following new policies and interventions. Scientific productions during last 3.5 years exceed those in 24 years before the study. There was continuous growth during few years of the study. Considering that indexing articles in Scopus and ISI took mean time of 2-3 months, we expect another rapid growth in 2012 as we had 792 articles only in first half of 2012. </p><p> There is an increasing trend of published articles in the country. In order to control the probable bias and showing the effect of our interventions the national research data considered as control group. If we compare these two trends. Country publication trend, as Saboury reported, showed 12% increase from 2009 to 2010 and 32% from 2010 to 2011 in ISI database. Our study showed 46% increase from 2009 to 2010 and 56% from 2010 to 2011.This high differences support the effectiveness of our interventions (<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R12" ref-type="bibr">12</xref>). This finding supports the causal effect of our interventions on increasing trend of scientific productions. </p><p> H-index used for assessment of articles citations in this study. This index is best known index for evaluating researchers function but there are some weak points. As Bornmann discussed h-index is suitable for young researchers because old researchers if even had no new publication their h-index will increase by the time (<xref rid="R13" ref-type="bibr">13</xref>); on the other side, if they had high h-index, new publication can&#x02019;t increase their h-index in short time. To overcome this issue we also assessed row number of publications during last 3.5 years divided by year. Batista et al found that the percent of researcher with h-index&#x0003e;10 defers between disciplines; they conclude it is difficult to compare scientist of deferent disciplines (<xref rid="R14" ref-type="bibr">14</xref>). H-index is insensitive to un-cited or less-cited articles; on the other hand if a paper has higher citations than h (even double or triple of h-index) the h-index remains constant. They proposed a new g-index to cover this weak point but it is not used generally yet (<xref rid="R15" ref-type="bibr">15</xref>). Sangwal also noted this weak point and focused on &#x0201c;conceptualization of tapered Hirsch index h<sub>T</sub>, circular citation area radius R and citation acceleration a&#x0201d; (<xref rid="R16" ref-type="bibr">16</xref>). </p><p> Braun mentioned lack of standards for different disciplines and sub-disciplines and number of authors in each article are main concern in evaluating h-index. Faculties in more specialized disciplines have lower h-index in general and others in general or interdisciplinary eras like cancer, Diabetes or nutrition have higher h-index. We also assessed the h-index and scientific productions in different schools and found that their values were different. Schools of Medicine, Pharmacy, and Nutrition had the higher means compare to the other Schools; Post-graduate disciplines in master or PhD degrees and residency programs present in these schools more than the others and this may explain the differences. School of Health also had higher mean score; department of epidemiology may affect the scientometrics indices of this school. All the authors had the same weight in h-index; this was another challenging point of h-index in ours and also Braun study (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> Analysis of data showed that there were some other points for intervention. One of them is considerable number of faculty members that had no publication indexed in these two databases. In next Step University authorities should develop policies and plans to involve them in qualified researches. Before that the cause of their disaffiliation should be cleared. Next studies could be focused on the effect of each intervention separately. </p><p> The means of articles in ISI and Scopus were statistically different. H-index can be variable based on data base as Bar-ilain compared three main databases of ISI, Scopus and Google-Scholar (<xref rid="R17" ref-type="bibr">17</xref>). We found in our study that we assessed only articles indexed in Scopus and ISI; but many faculties have articles in Persian -or in English- and most of them did not indexed in these two databases: so this doesn&#x02019;t show all research efforts of faculty members. Rip also noted that scientometrics is limited to a main data base of ISI and the other data bases have not the same characteristic (<xref rid="R18" ref-type="bibr">18</xref>). It might be difficult for them to write in English as Van leeuwen et al, mentioned (<xref rid="R19" ref-type="bibr">19</xref>). On the other hand, editing the articles and the higher fees for the publication process in some journals may push them to publish their articles in journals of other databases which are free and open access. To overcome this concern university has had some interventions like financial supports but they can be more focused and facilitated. </p><p> Although there was a clear methodology but we had some limitations in practice. We could not assess the pure effect of every intervention as they were used as a package. The other important limitation of this study was different spelling of Persian names in English. For Example family name of &#x0201d;mousavi&#x0201d; which is a common in Iran can be written by 27 another spellings of &#x0201c;moosavi, mosavi, musavi, mousavie, moosavie, mosavie, musavie, mousavei, moosavei, mosavei, musavei, mousavii, moosavii, mosavii, musavii, mousavy, moosavy, mosavy, musavy, mousavey, moosavey, mosavey, musave, mousavee, moosavee, mosavee, musavee&#x0201d;. When we searched this name using asterisk, there were many more names with additional suffixes especially in ISI, and make it difficult for judgment. Considering this point there may be some under-estimation in counting publications and H-index. Van Raan also confirmed this under-estimation in non-English countries and in articles with high number of authors (<xref rid="R20" ref-type="bibr">20</xref>). </p><p> Scientific writing should prepare solutions for health problems. Writing for writing is not the case. The main concern in research is paying attention to applied researches (<xref rid="R21" ref-type="bibr">21</xref>). Researches should be ordered by industry or other users or by a third party for them. Unfortunately this process is not matured in developing countries. A considerable portion of their researches are copies of developing countries researches without any native modification. New policies should consider whether studies are designed based on appropriate questions and focus on quality of researches in next studies. </p></sec><sec sec-type="conclusion" id="s8"><title>Conclusion</title><p> There is increasing trend in scientific production of SBMUS in both Scopus and ISI data bases. It could be mostly due to the effect interventions in research system during last five years. As Iran is one of the third high ranked in Middle East in terms of scientometrics (<xref rid="R22" ref-type="bibr">22</xref>), it is important to monitor progress of scientific production by yearly interval. </p></sec>
A pilot randomized controlled trial on the effectiveness of inclusion of a distant learning component into empathy training
<p> <bold>Background:</bold> Studies have shown a gradual decline in empathy of medical trainees with increasing years of education. Methods to augment empathy show some promise, but the most effective methods are both expensive and time consuming. To assess effectiveness of communication skills training program as a distant learning method in improving empathy. </p><p> <bold>Methods:</bold> Fourteen first year residents of psychiatry were randomly allocated to either participate in a two day workshop on communications skills (attending group) or to watch the videotape of the first day and participate in the second day (distance learning group). Assessments included Jefferson Scale of Empathy (JSE) and objective assessment of empathy (OAE) during a simulated interview, before and 3 months after the training. </p><p> <bold>Results:</bold> The empathy was significantly increased in the attending group as measured by OAE. The score of JSE also increased in the attending group but did not reach a significance level. No increase in empathy was seen in the distance learning group. </p><p> <bold>Conclusion:</bold> Watching the videotape of the workshop is not effective in improving empathy of residents. More interactive methods should be sought if we plan to use distance learning methods in enhancement of empathy. </p>
<contrib contrib-type="author"><name><surname>Nasr Esfahani</surname><given-names>Mehdi</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Behzadipour</surname><given-names>Mojgan</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Jalali Nadoushan</surname><given-names>Amirhossein</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Shariat</surname><given-names>Seyed Vahid</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Empathy is an important part of every relationship and especially patient-physician relationship. Nonetheless, there is a considerable uncertainty on the definition of empathy in the literature (<xref rid="R1" ref-type="bibr">1</xref>). Recently, Hojat has tried to decrease the ambiguity around the concept of physician empathy by defining it as &#x0201c;a predominantly cognitive (rather than emotional) attribute that involves an understanding (rather than feeling) of experiences, concerns and perspectives of the patient, combined with a capacity to communicate this understanding&#x0201d; (<xref rid="R2" ref-type="bibr">2</xref>). However, empathy has also been defined as a concept that includes emotive, moral, cognitive, and behavioral dimensions (<xref rid="R3" ref-type="bibr">3</xref>). In this view, emotional engagement, as well as the cognitive understanding, is crucial for effective empathy. </p><p> Despite the vagueness of its definition, there is a consensus on the beneficial effects of empathy on both treatment outcomes and patient satisfaction (<xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R5" ref-type="bibr">5</xref>). But the disappointing issue is that, several studies have shown a decreasing trend of empathy scores in medical students and residents with increasing years of education (<xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R9" ref-type="bibr">9</xref>). Therefore, it is an important concern of medical educators to have effective and less complicated training methods for enhancement of empathy. </p><p> In a review, Hojat et al has mentioned 10 approaches that has been used for enhancement of empathy in health professionals (<xref rid="R10" ref-type="bibr">10</xref>), including improving interpersonal skill, exposure to role models, shadowing a patient, hospitalization experiences etc. In another study, Stepien and Baernstein reviewed strategies for teaching empathy to medical students (<xref rid="R3" ref-type="bibr">3</xref>). Both reviews have mentioned that empathy could be improved via different strategies. However, the studies on enhancement of empathy have limitations that make further researches necessary, including small sample size, lack of appropriate control group, lack of specificity of the instrument for physician empathy, and not assessing the durability of change. </p><p> Communication skill training is one of the more frequently studied methods, which has been shown to be effective in improving empathy both in short and long term (<xref rid="R11" ref-type="bibr">11</xref>-<xref rid="R13" ref-type="bibr">13</xref>). However, the method is time consuming and a limited number of participants can take part in each workshop. As a result, these programs should be repeated for several groups of target populations. An alternative cheaper method with comparable effectiveness would be of great interest. This pilot study was designed to assess if participating in a part of the program and watching the videotape of the other part could be as effective as participating in the workshop in improving empathy after 3 months or not. </p><p> Furthermore, we would like to see if the score of the scale that is frequently used for assessment of empathy (Jefferson Scale of Empathy) is correlated with objective assessment of empathy performed by specialists and with the evaluation of a standardized patient from empathy of physician. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><sec id="s2-5"><title>Participants</title><p> Subjects of the study included 14 first year residents of psychiatry of the Tehran Institute of Psychiatry and Department of Psychiatry of Iran University of Medical Sciences during the study. All of the approached residents accepted to participate in the study. Subjects were randomly allocated to two groups of 7 residents, considering gender as a stratum to have two male residents in each group. All of the participants were between 30 to 40 years old. </p></sec><sec id="s2-1"><title>Intervention and study groups</title><p> Our intervention included a training workshop of communication skills, which was taught by an experienced academic psychiatrist on two days with a one week interval. Each day of the workshop lasted 6 hours. First day of the workshop included a 2 hour lecture on basic communication skills and one role playing on &#x0201c;clinical setting and basic communication skills in therapeutic relationship&#x0201d;. The second day of the workshop included two different role plays on &#x0201c;getting information from patient&#x0201d; and &#x0201c;giving information to patient&#x0201d;. All of the role plays of the two day workshop were performed twice; first, with some mistakes and negative points in the performance of the &#x0201c;physician&#x0201d;, and then in a professionally correct manner. After the &#x0201c;flawed&#x0201d; performance, residents discussed the negative points with the instructor. Furthermore, each session ended with a group discussion on the topics of that session. </p><p> One group of the subjects was planned to attend the two days of the workshop (attending group) and the other group to view the videotape of the first day and to receive a text on the topic of communication skills and to attend the second session of the workshop (distance learning group). Only one of the subjects of the attending group declared that she cannot participate in the first day of the workshop and was then replaced with another subject from the other group. </p></sec><sec id="s2-2"><title>Ethical considerations</title><p> All of the participants could freely choose to participate in the study or not and all of them signed a written form of informed consent. The study was approved by the ethics committee of Tehran University of Medical Sciences. All of the data were considered as confidential and not disclosed to those not involved in the study. </p></sec><sec id="s2-3"><title>Assessments</title><p> Jefferson Scale of Empathy (JSE) is a self-report scale specifically designed to assess patient-physician empathy that is currently translated to more than 38 languages including Persian. Its Persian version has been shown to have acceptable validity and reliability (<xref rid="R8" ref-type="bibr">8</xref>, <xref rid="R14" ref-type="bibr">14</xref>). JSE has 20 items that each item is scored in a 7 point Likert type scale from strongly disagree to strongly agree. All of the subjects completed JSE before and three months after the intervention. Both exploratory and confirmatory factor analysis have shown that JSE item could be grouped into three factors; namely, perspective taking, compassionate care, and standing in patient&#x02019;s shoes (<xref rid="R8" ref-type="bibr">8</xref>, <xref rid="R15" ref-type="bibr">15</xref>). </p><p> <italic>Objective assessment of empathy (OAE) during interview:</italic> We tried to assess empathy of the subjects objectively during their interview with standardized patients (SP) both before and 3 months after the intervention. For pre-training objective assessment of empathy we used the scores of the subjects in the routine OSCE that was performed one month before the intervention. This exam included 4 stations that in each of these stations two board certified psychiatrists independently rated the performance of the residents. We only extracted scores of the two items that were dedicated to assessment of empathy and communication skills of the subjects in these stations and added them up to a single score. For post-training objective assessment of empathy we held an interview with an SP, during which two board-certified psychiatrists rated the empathy and communication skills of the subjects in two items that were again summed up to a single score. In order to make the pre- and post-training scores comparable, we divided the obtained scores by the maximum possible score. The raters and the SP were blind to the group status of the subjects. </p><p> Jefferson Scale of Patient&#x02019;s Perception of Physician Empathy (JSPPPE) is a 5 item scale developed to assess the perception of the patient from the empathy of his/her physician (<xref rid="R16" ref-type="bibr">16</xref>). Each item is scored in a 5 point Likert type scale from strongly disagree to strongly agree. The scale has been translated to Persian and used in a study (<xref rid="R17" ref-type="bibr">17</xref>). In the objective evaluation of the subjects 3 months after the intervention, the SP was asked to rate all of the subjects using this scale, immediately after each interview. The SP was a board certified psychiatrist and did not know any of the subjects and was also blind to the group status of the subjects. </p></sec><sec id="s2-4"><title>Statistical analyses</title><p> All of the data were entered to SPSS software version 16 and analyzed using descriptive statistics including mean and standard deviation as well as Mann-Whitney <bold>U</bold> test to compare the groups before training, Wilcoxon signed ranks to compare each group before and after the training, Pearson correlation to assess the correlation of the scores of JSE, JSPPPE, and OAE. We also subtracted the mean scores of JSE before and after training and compared this score between the two groups using Mann Whitney <bold>U</bold> test. The significance level of less than 0.05 was considered for all of the analytical tests. </p><p> The study is registered as a clinical trial in the Iranian Registry for Clinical Trials website with the IRCT ID of IRCT2012090510749N1. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> The two groups did not differ in terms of gender, marital status, and age. Each group included 2 male and 3 married residents. Mean age of the attending group was 31.3 (SD=3.9, median=30, IQR=7) and 29.4 in the distance learning group (SD=1.5, median=30, IQR=3). However, the attending group had a non-significant higher baseline score of JSE (m=122.7, SD=4.8) and OAE (m=0.73, SD=0.13) compared to the distance learning group (JSE: m=116.3, SD=11.2, U=12.5, p=0.124; OAE: m=0.6, SD=0.16, U=11.5, p=0.174) (<xref ref-type="table" rid="T1">Table 1</xref>). </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Scores of OAE and JSE and its three factors in the two groups of study (attending group and distance learning group) before and three months after the training </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="2" colspan="1">Outcome</td><td align="center" colspan="2" rowspan="1">Attending group</td><td align="center" colspan="2" rowspan="2">Statistical sig.</td><td align="center" colspan="2" rowspan="1">Distance learning group</td><td align="center" colspan="2" rowspan="2">Statistical sig.</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> Pre training<break/>Mean (SD) </td><td align="center" rowspan="1" colspan="1"> Post training<break/>Mean (SD) </td><td align="center" rowspan="1" colspan="1"> Pre training<break/>Mean (SD) </td><td align="center" rowspan="1" colspan="1"> Post training<break/>Mean (SD) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Z</td><td align="center" rowspan="1" colspan="1">p</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Z</td><td align="center" rowspan="1" colspan="1">p</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">JSE</td><td align="center" rowspan="1" colspan="1">122.7 (4.8)</td><td align="center" rowspan="1" colspan="1">125 (7.9)</td><td align="center" rowspan="1" colspan="1">-0.76</td><td align="center" rowspan="1" colspan="1">0.44</td><td align="center" rowspan="1" colspan="1">116.3 (11.2)</td><td align="center" rowspan="1" colspan="1">114.3 (14.1)</td><td align="center" rowspan="1" colspan="1">-0.33</td><td align="center" rowspan="1" colspan="1">0.73</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Factor I</td><td align="center" rowspan="1" colspan="1">59.7 (3.8)</td><td align="center" rowspan="1" colspan="1"> 60.3 (<xref rid="R6" ref-type="bibr">6</xref>) </td><td align="center" rowspan="1" colspan="1">-0.105</td><td align="center" rowspan="1" colspan="1">0.91</td><td align="center" rowspan="1" colspan="1">54.3 (7.1)</td><td align="center" rowspan="1" colspan="1">52.6 (7.6)</td><td align="center" rowspan="1" colspan="1">-0.76</td><td align="center" rowspan="1" colspan="1">0.44</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Factor II</td><td align="center" rowspan="1" colspan="1">52.4 (2.6)</td><td align="center" rowspan="1" colspan="1">53.6 (1.8)</td><td align="center" rowspan="1" colspan="1">-0.93</td><td align="center" rowspan="1" colspan="1">0.35</td><td align="center" rowspan="1" colspan="1">51.1 (3.8)</td><td align="center" rowspan="1" colspan="1">49.9 (5.1)</td><td align="center" rowspan="1" colspan="1">-0.94</td><td align="center" rowspan="1" colspan="1">0.34</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Factor III</td><td align="center" rowspan="1" colspan="1">10.6 (1.1)</td><td align="center" rowspan="1" colspan="1">11.1 (1.1)</td><td align="center" rowspan="1" colspan="1">-1.63</td><td align="center" rowspan="1" colspan="1">0.102</td><td align="center" rowspan="1" colspan="1">10.9 (1.6)</td><td align="center" rowspan="1" colspan="1">10.9 (2.5)</td><td align="center" rowspan="1" colspan="1">-1.18</td><td align="center" rowspan="1" colspan="1">0.23</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">OAE</td><td align="center" rowspan="1" colspan="1">0.73 (0.13)</td><td align="center" rowspan="1" colspan="1">0.79 (0.15)</td><td align="center" rowspan="1" colspan="1">-2.02</td><td align="center" rowspan="1" colspan="1">0.043*</td><td align="center" rowspan="1" colspan="1">0.6 (0.16)</td><td align="center" rowspan="1" colspan="1">0.6 (0.1)</td><td align="center" rowspan="1" colspan="1">-0.31</td><td align="center" rowspan="1" colspan="1">0.75</td></tr></tbody></table></table-wrap><sec id="s3-1"><title>Change in empathy</title><p> Three months after the training, mean JSE score increased by more than 2 units in the attending group, but this change was not significant, probably due to our small sample size. Regarding the factors of JSE, factor II and III that are negatively worded items tended to increase, but factor I did not show a difference (<xref ref-type="table" rid="T1">Table 1</xref>). The difference made in each of the two groups before and after training was also compared with the other group, but did not reach significance level (U=19.5, p=0.52). The score of OAE significantly increased in the attending group, and showed no change in distance learning group. </p></sec><sec id="s3-2"><title>Correlation of empathy measures</title><p> JSE had a moderate correlation with OAE (0.39) and JSPPPE (0.37). There was a strong correlation between JSPPPE and OAE (0.85) (<xref ref-type="table" rid="T2">Table 2</xref>). </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Correlation of the scores of JSE, with OAE and JSPPPE </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Variables</td><td align="center" colspan="2" rowspan="1">1</td><td align="center" colspan="2" rowspan="1">2</td><td align="center" colspan="2" rowspan="1">3</td><td align="center" colspan="2" rowspan="1">4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">r</td><td align="center" rowspan="1" colspan="1">p </td><td align="center" rowspan="1" colspan="1">r</td><td align="center" rowspan="1" colspan="1">p </td><td align="center" rowspan="1" colspan="1">r</td><td align="center" rowspan="1" colspan="1">p </td><td align="center" rowspan="1" colspan="1">r</td><td align="center" rowspan="1" colspan="1">p </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> 1.JSE posttest </td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> 2. JSE pretest </td><td align="center" rowspan="1" colspan="1">0.59</td><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> 3. OAE </td><td align="center" rowspan="1" colspan="1">0.39</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.32</td><td align="center" rowspan="1" colspan="1">0.31</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> 4. JSPPPE </td><td align="center" rowspan="1" colspan="1">0.37</td><td align="center" rowspan="1" colspan="1">0.23</td><td align="center" rowspan="1" colspan="1">0.42</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">0.85</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.001</td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr></tbody></table></table-wrap></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> This pilot study suggests that a two day communication skills workshop can be effective in improving empathy of the first year residents of psychiatry after three months. However, participating in one of the two days of this workshop and watching the videotape of the other day is not effective in improving empathy. Both OAE and JSE showed the improvement of empathy in the attending group after the intervention. However, the difference was not significant in the latter. All of the three factors of JSE shared a part in this improvement. This suggests that the beneficial effect of intervention has not been limited to a single factor. Interestingly, none of the factors of JSE showed an improvement in the distant learning group. This pattern of findings suggests that the difference in JSE could have reached significance level if our sample size was larger. Each group of our study only included seven participants, which limited the power of the study. </p><p> There are several studies that have assessed the effectiveness of communication skills workshops on empathy, but this is the first study on improvement of empathy with a distance learning component. Therefore, it is not possible to compare this finding with the previous studies. The reason for the lack of effectiveness of training in the distance learning group could be related to possible noncooperative participants that might not have seen the videotape and not have read the text provided to them. After the study we enquired about these issues and two of the residents admitted that they had not watched the videotape or read the texts before the second session. It is well known that success of students in distance learning is directly related to their motivation (<xref rid="R18" ref-type="bibr">18</xref>, <xref rid="R19" ref-type="bibr">19</xref>); and it is not an easy task to keep the students motivated out of the class. Another possible reason for non-effectiveness of our intervention in distance learning group to improve empathy could be the fact that, face to face encounter is a necessary component in training empathy. Because empathy is a quality that largely depends on interpersonal engagement. </p><p> Another point that we should consider in discussing the results is that, despite random allocation of the subjects to the study groups, the two groups did not have equal empathy levels before the intervention. Although the difference did not reach significance level, this should be considered as a limitation of our study. It is possible that the group with higher empathy has been more motivation to learn about empathy and communication skills than the other group. </p><p> Our study also showed that JSE has a moderate correlation with JSPPPE and OAE. The correlation of JSE and JSPPPE is consistent with the findings of two other studies that have reported comparable correlation coefficients (<xref rid="R16" ref-type="bibr">16</xref>, <xref rid="R20" ref-type="bibr">20</xref>). The observed correlations are actually indicators of concurrent validity of JSE. It should be emphasized that in our study the SP that completed JSPPPE was a psychiatrist, which could more carefully evaluate the professional behavior of the residents. </p><p> In this study we also assessed the correlation of JSPPPE with OAE, which showed a very high correlation. This is in support of the validity of JSPPPE that has not been as widely studied and validated as JSE. We suggest that JSPPPE is a concise and valid tool to assess physician empathy from a third person perspective. </p><p> This study had some limitation that should be considered. First, as a pilot study, we had a small sample size, which limits the power of the study. Some of the comparisons could have become significant, if we had a larger sample. Second, the two groups of the study were not totally equal at the baseline, as mentioned above. Third, we did not assess the subjects immediately after the intervention, so we could not assess the early effectiveness in this study. However, we know that the long-term effectiveness is more important and is the desirable effect that is expected from the intervention. </p><p> Further research on adding distance learning components to empathy training could focus on other more interactive methods or some aspects of virtual reality to the program. Moreover, it is not known if adding components of distance learning to education of empathy (instead of replacing it with some parts of the educational program) would have an additive beneficial effect or not. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Inclusion of distance learning methods in empathy training is a new area of research, which merits further study. Face to face interaction seems to be an essential component of empathy training, and finding an alternative for it in distance learning seems to be the major challenge. Balancing our expectations, as well as developing more interactive methods might be helpful in advancing this important and interesting area of research. </p></sec>
Severe neonatal hyperbilirubinemia leading to exchange transfusion
<p> <bold>Background:</bold> Severe neonatal hyperbilirubinemia is associated with significant morbidity and mortality. This study was conducted to investigate the causes of severe hyperbilirubinemia leading to Exchange Transfusion (ET) from March 2009 to March 2011 in Bahrami children hospital, Tehran, Iran in order to establish guidelines to prevent profound jaundice &#x00026; ET. </p><p> <bold>Methods:</bold> 94 neonates underwent ET for severe hyperbilirubinemia data for demographic data, and onset of jaundice, history of severe hyperbilirubinemia in siblings, blood group of both mother and neonate, G6PD activity, hemoglobin, hematocrite, reticulocyte count, peripheral blood smear, total and direct bilirubin before and after ET, direct and indirect Coombs, times of transfusion and the cause of hyperbilirubinemia were all recorded for analysis. </p><p> <bold>Results:</bold> Ninety four neonates (56.4% boys and 43.6% girls) underwent ET with a mean birth weight of 1950&#x000b1;40 g and a mean gestational age of 35.2&#x000b1;1.4 weeks. Premature labor, breastfeeding jaundice, ABO incompatibility and G6PDD with the frequency of 59(63%), 33(35%), 25(24/5%) and 12(12.8%) were of major causes of ET. </p><p> <bold>Conclusions:</bold> Predisposing factors for severe hyperbilirubinemia in this study were premature labor, breastfeeding jaundice, ABO incompatibility and G6PDD. The authors recommend prevention of premature labor, reevaluation of successful breastfeeding education for mothers and screening infants for blood group and G6PD In the first of life. Arranging earlier and continuous visits in neonates with these risk factors during the first four days of life is also recommended. </p>
<contrib contrib-type="author"><name><surname>Alizadeh Taheri</surname><given-names>Peymaneh</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Sadeghi</surname><given-names>Mandana</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Sajjadian</surname><given-names>Negar</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Pathologic neonatal hyperbilirubinemia is associated with significant morbidity and mortality. It is estimated to be the most common cause of neonatal hospital readmission in North America (<xref rid="R1" ref-type="bibr">1</xref>). It may lead to bilirubin accumulation in basal ganglia and brain stem nuclei and lead to kernicterus. If the infants survive the acute phase, which is marked by lethargy, hypotonia, poor feeding and opisthotonus, they may develop chronic encephalopathy. This condition is manifested by cerebral palsy,sensory neural hearing loss, dental dysplasia, upward gaze paralysis and mental retardation (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> Causes of severe neonatal hyperbilirubinemia are categorized either as hemolytic (blood group mismatch, sepsis, G6PD deficiency) or non-hemolytic (breast feeding jaundice, internal hemorrhage, gestational diabetes, pyloric stenosis, hypothyroidism and some mutations in hepatic enzymes). Prematurity, jaundice in the first 24 hours of life, jaundice noted before discharge from hospital, a history of jaundice treated with phototherapy in siblings and Asian race are other predisposing factors for severe hyperbilirubinemia noted by various studies (<xref rid="R3" ref-type="bibr">3</xref>-<xref rid="R5" ref-type="bibr">5</xref>). </p><p> Phototherapy is the main method of treatment for neonatal jaundice. However, severe hyperbilirubinemia is mainly treated by Exchange Transfusion (ET).According to American Academy of Pediatrics (AAP), neonates with weight &#x02265; 2500 grams in healthy term, ET is indicated when indirect bilirubin level reaches&#x02265; 25 mg/dl and &#x02265;20 mg/dl and in cases with risk factors or gestational age of35-37 wk and well despite 6 hr of intensive phototherapy. In newborns of 35-37 wk and risk factors, ET is indicated when the indirect bilirubin level reaches &#x02265; 18 mg/dl despite 6 hr of intensive phototherapy (<xref rid="R2" ref-type="bibr">2</xref>). ET is associated with many complications such as hypersensitivity reactions, sepsis, catheter-induced vascular damage, hypotension, necrotizing enterocolitis, etc (<xref rid="R5" ref-type="bibr">5</xref>). </p><p> The present study has been conducted to investigate the causes of severe hyperbilirubinemia leading to exchange transfusion from March 2009 to March 2011 in Bahrami children hospital, Tehran, Iran. The aim was to identify neonatal predisposing factors that can be prevented by screening or other measures, thus reducing the incidence of kernicterus, which can be prevented and also to avoid ET and its side effects. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> All infants below 30 days who underwent ET for severe hyperbilirubinemia in Bahrami Children Hospital during 2009-2011 were enrolled into this cross sectional study. A questionnaire prepared consisted of neonates&#x02019; gender, gestational age, age at the time of admission, birth weight, time of jaundice appearance, history of severe hyperbilirubinemia in siblings, blood group and Rh of both mother and neonate, G6PD activity, complete blood count, reticulocyte count, peripheral blood smear, total and direct bilirubin before and after exchange transfusion, direct Coombs in mother and indirect Coombs in neonates, times of transfusion, any other diagnostic laboratory data based on attending neonatologist's decision and the cause of hyperbilirubinemia stated by the attending neonatologist. Criteria for diagnosis of ABO incompatibility were type O maternal blood group and A, B or AB neonatal blood group associated with neonatal Hb drop. Positive coombs, peripheral blood spherocytosis and high corrected reticulocyte count were other diagnostic criteria. Icteric breastfed newborns with the beginning of jaundice in the second or third day of life and 2% or more weight loss per day of life with or without uremia or hypernatremia were considered breastfeeding jaundice. </p><p> The data were analyzed with SPSS software, version 16. </p></sec><sec sec-type="results" id="s3"><title>Results</title><p> During the study period, 94 neonates (56.4% boys and 43.6% girls) underwent exchange transfusion due to severe hyperbilirubinemia. The infants had a mean birth weight of 1950&#x000b1;40 g and a mean gestational age of 35.2&#x000b1;1.4 weeks. Fifty nine (63%) of neonates were preterm, and history of neonatal jaundice in siblings was reported in 50% of cases. However, only 18(19%) had a history of severe hyperbilirubinemia leading to ET in their siblings. </p><p> Most cases (40.5%) of severe hyperbilirubinmia started to become icteric on the second day after birth. </p><p> First day jaundice was observed in 10 neonates, all of them due to hemolysis. <xref ref-type="table" rid="T1">Table 1</xref> shows a summary of underlying factors for severe hyperbilirubinemia according to the onset of jaundice. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Underlying causes of severe hyperbilirubinemia leading to ET according to the date of jaundice appearance </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Start date of jaundice</td><td rowspan="1" colspan="1">Underlying causes</td><td rowspan="1" colspan="1">N (%)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">First day after birth</td><td rowspan="1" colspan="1"> ABO mismatch<break/>Rh mismatch<break/>G6PD deficiency </td><td rowspan="1" colspan="1"> 7(7.4%)<break/>2(2.1%)<break/>1(1.06%) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Second day after birth</td><td rowspan="1" colspan="1"> ABO mismatch<break/>Rh mismatch<break/>G6PD deficiency<break/>Infection<break/>Breast feeding jaundice </td><td rowspan="1" colspan="1"> 13(13.82%)<break/>2(2.1%)<break/>6(6.3%)<break/>8(8.5%)<break/>9(9.6%) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Third day after birth</td><td rowspan="1" colspan="1"> ABO mismatch<break/>Rh mismatch<break/>G6PD deficiency<break/>Infection<break/>Breast feeding jaundice </td><td rowspan="1" colspan="1"> 3(3.2%)<break/>2(2.1%)<break/>2(2.1%)<break/>3(3.2%)<break/>14(14.9%) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Forth day after birth</td><td rowspan="1" colspan="1"> Rh mismatch<break/>G6PD deficiency<break/>Breast feeding jaundice </td><td rowspan="1" colspan="1"> 2(2.1%)<break/>2(2.1%)<break/>8(8.5%) </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Fifth day after birth and later<break/> </td><td rowspan="1" colspan="1"> G6PD deficiency<break/>Breast feeding jaundice </td><td rowspan="1" colspan="1"> 1(1.6%)<break/>2(2.1%) </td></tr></tbody></table></table-wrap><p> The majority of infants (91.5%) had only one episode of ET, and hemolysis was the sole underlying reason in the remaining who had 2 or 3 episodes of ET. Totally, after prematurity, the second most prevalent underlying factor for severe hyperbilirubinemia was breastfeeding jaundice that included 33(35%) of neonates. We found that the next most prevalent causes were ABO mismatch and G6PD deficiency (<xref ref-type="table" rid="T2">Table 2</xref>). No cases of cephalhematoma, hypothyroidism or metabolic cases led to blood exchange. There was no kriglernajar cases in our study because no significant hyperbilirubinemia was found after discharging and fallow up. </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Prevalence of predisposing factors of severe hyperbilirubinemia leading to ET </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Predisposing factor</td><td align="center" rowspan="1" colspan="1">Numbers (%)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Hemolytic<break/>ABO mismatch<break/>Rh mismatch<break/>G6PD deficiency<break/>Sepsis </td><td align="center" rowspan="1" colspan="1"> <break/>23(24.5%)<break/>8(8.5%)<break/>12(12.8%)<break/>11(11.7%) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Non-hemolytic<break/>Breast feeding jaundice<break/>prematurity </td><td align="center" rowspan="1" colspan="1"> <break/>33(35%)<break/>59(63%) </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Idiopathic</td><td align="center" rowspan="1" colspan="1">7(7.5%)</td></tr></tbody></table></table-wrap></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> According to epidemiological studies, some risk factors are associated with severe hyperbilirubinemia in neonates. The risk factors are male gender, jaundice presenting in the first 24 hours after birth, jaundice noted at discharge from the hospital, previous sibling with jaundice, preterm labor, breast feeding, Rh and ABO incompatibility, G6PD deficiency and sepsis (<xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R6" ref-type="bibr">6</xref>). </p><p> According to the Canadian Pediatric Society, gestational age of 35-36 weeks is an important risk factor for hyperbilirubinemia (<xref rid="R7" ref-type="bibr">7</xref>). Kuzniewicz et al also reported gestational age to be the main predictor of severe hyperbilirubinemia (<xref rid="R8" ref-type="bibr">8</xref>). In our series, infants had a mean gestational age of 35.2 weeks and fifty nine (63%) of neonates were preterm. </p><p> Most cases (40.5%) of severe hyperbilirubinmia started on the second day after birth. This corresponds with views of American Academy of Pediatrics and Canadian Pediatric Society, which recommend clinical assessment of infants for jaundice within the first 48 hours of birth (<xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R7" ref-type="bibr">7</xref>). However, clinical assessment of newborns is not an accurate means of assessing the severity of hyperbilirubinemia (<xref rid="R1" ref-type="bibr">1</xref>). </p><p> In the present study, breastfeeding jaundice, as the most important factor associated with severe hyperbilirubinemia and according to Salas and Mazzi (<xref rid="R9" ref-type="bibr">9</xref>), is encountered in 33(35%) of cases. The data also corresponds with findings of Huang et al (<xref rid="R6" ref-type="bibr">6</xref>), who reported an odds ratio of 3.2 for severe hyperbilirubinemia in neonates who were breastfed (<xref rid="R10" ref-type="bibr">10</xref>). Inadequate intake may lead to dehydration, increased enterohepetic cycle and development of hyperbilirubinemia. Guidelines advise mothers to nurse their infants at least 8 to 12 times per day for the first several days, rooming in with night feeding, discouraging 5% dextrose or water supplementation and ongoing lactation support for reducing the incidence of breast feeding jaundice (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> In this study, ABO mismatch was the third most common cause of severe hyperbilirubinemia and accounted for 24.5% of cases. This is similar to the reports of Badee and Sanpavat who reported ABO mismatch in 22% and 21.3% of cases, respectively (<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R12" ref-type="bibr">12</xref>). However, according to Canadian surveillance program, the most common causes of severe hyperbilirubinemia were incompatibility ABO blood group and G6PD deficiency. </p><p> The G6PD deficiency was the fourth most prevalent risk factor for severe hyperbilirubinemia leading to ET and accounted for 12(12.8%) of cases. Badee also reported G6PD Deficiency in 19.1% of neonates who underwent ET (<xref rid="R11" ref-type="bibr">11</xref>). According to Johnson et al, G6PD deficiency was considered to be the main cause of hyperbilirubinemia in 19 of 61 (31.5%) infants who developed kernicterus (<xref rid="R14" ref-type="bibr">14</xref>). </p><p> Sepsis accounted for 11(11.7%) cases of severe hyperbilirubinemia. On the other hand, sepsis was the most common underlying cause for ET in the study performed by Koosha and Rafizadeh (<xref rid="R15" ref-type="bibr">15</xref>). </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Prematurity was the most prevalent risk factor for severe hyperbilirubinemia. Thus following protocols for prevention of premature labor is recommended. Educating mothers for breast feeding in regard to the right times and method of feeding are important issue that should be considered. Since blood group mismatch and G6PD deficiency were the third and fourth most prevalent underlying factors, screening infants for blood group and if proven to be cost effective, G6PD activity in the first day of life are also recommended. Arranging earlier and continuous visits in neonates with these risk factors during the first four days of life are also mandatory. </p></sec>
Perioperative antibiotic prophylaxis in elective surgeries in Iran
<p> <bold>Background:</bold> The use of antibiotic prior to surgery is widely accepted. The WHO has recommended the use of ATC/DDD (Anatomical Therapeutic Chemical / Defined Daily Dose) for the analysis of drug utilization. The aims of the present study are 1) to analyze the assessment of prophylactic antibiotic usage prior to surgery, 2) to assess the drug administration based on antibiograms and 3) to compare the results with the national and international standards. </p><p> <bold>Methods:</bold> The present study used ATC/DDD, in a retrospective manner. Cefazolin, ceftazidime, gentamicin, ciprofloxacin, metronidazole, vancomycin, imipenem and penicillin G from 21st March to 21st June 2011 were analyzed in a hospital. Out of 516 medical records, 384 patients had received prophylactic antibiotics. </p><p> <bold>Results:</bold> In comparison, the orthopaedic ward had used more antibiotics. The results showed that antibiotics were not selected based on the antibiogram antibiotic programs. Patients in the age range of 20-30 years were the most recipients of the antibiotics. Men had received more antibiotic in comparison with women. About 75% (384 out of 516) of patients in the study received antibiotics as prophylaxis. Cefazolin was the most frequently prescribed antibiotic. </p><p> <bold>Conclusion:</bold> Our findings showed differences in comparison with national and international studies, but insignificant differences. Data on in-hospital antibiotic usage are varying widely not only due to different antibiotic policies but also due to different methods of mesurement. These differences make the comparison difficult. </p>
<contrib contrib-type="author"><name><surname>Foroutan</surname><given-names>Behzad</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Foroutan</surname><given-names>Reza</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> The principles of antibiotic prophylaxis against postsurgical infection were established in laboratory studies in the early 1960s (<xref rid="R1" ref-type="bibr">1</xref>). Later this strategy has been applied to many areas of clinical surgery (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R3" ref-type="bibr">3</xref>). Prophylaxis is desirable and is based on a combination of perioperative preparation, surgical techniques, perioperative antibiotic prophylaxis and postoperative wound care. According to the Infectious Diseases Society of America, rational use of antibiotics requires the use of antibiotics with the appropriate medication that could influence the clinical needs of patients in certain geographic areas with the lowest side effects and cost of medications to the patient may be imposed (<xref rid="R4" ref-type="bibr">4</xref>). Despite the advances in surgical techniques there are still a significant number of postsurgical complications, the most common being, surgical wound infections, sepsis, respiratory and cardiovascular complications and thromboembolic events (<xref rid="R5" ref-type="bibr">5</xref>). Antibiotic prophylaxis has been routinely used to prevent such complications (<xref rid="R6" ref-type="bibr">6</xref>). The quantity of antibiotic use in hospitals and the community has been shown to correlate with antimicrobial resistance, resulting in increased morbidity, mortality, and cost of health care (<xref rid="R6" ref-type="bibr">6</xref>). Some evidences have shown that 30 &#x02013; 60 % of cases of mismanagement and poor prescription or administration that cause these errors came by physicians, distributors or self-medication resulting inappropriate prescribing and unnecessary use of antibiotics raise antibiotic resistance as well as adverse drug events and expenditures (<xref rid="R7" ref-type="bibr">7</xref>). </p><p> The WHO recommended the Anatomical Therapeutic Chemical (ATC) methodology and Defined Daily Dose (DDD) as a measuring unit. This style facilitate to design and perform a standardized and repeatable drug consumption studies (<xref rid="R8" ref-type="bibr">8</xref>). The DDD is suitable as a statistical measure of drug consumption (<xref rid="R9" ref-type="bibr">9</xref>). It is used to standardize the comparison of drug usage between different drugs or different health care environments. The DDD is not to be confused with the therapeutic dose or with the dose actually prescribed by a physician for an individual patient. The system is used internationally and the number of users is increasing. Prescribed antibiotics were classified by generic names and according to the WHO/ATC .The purpose of the ATC/DDD system is to serve as a tool for drug utilization research in order to improve quality of drug use (<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R11" ref-type="bibr">11</xref>). One component of this is the presentation and comparison of drug consumption statistics at international and other levels. </p><p> In the present study antibiotic prophylactic regimens used before elective surgeries procedures were evaluated in a university-affiliated multidisciplinary hospital in Iran. </p><sec id="s1-1"><title>Experimental Procedure </title><p> <italic>The ATC (Anatomical Therapeutic Chemical) classification:</italic> The Norwegian Medicinal Depot set off the ATC system in the 1970s. In 1982 the WHO Collaborating Centre for Drug Statisics Methodology in Oslo synchronized it. The center modified the ATC codes as necessary and maintained an online database and published index. Drugs were divided into special groups according to the organ or system on which they perform and/or their therapeutic and chemical characteristics. At least one ATC code was assigned for each drug. Afterward drugs classified into five different groups. See <xref ref-type="table" rid="T1">Table 1</xref> for ATC antibiotics codes, J01 class, which was analyzed in the present study (<xref ref-type="table" rid="T1">Table 1</xref>). </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Cumulative overview of some DDD (Defined Daily Dose) performed in the period 1982-2011(Last updated: 2011-12-19, WHO). Table shows only some of the J01 class of ATC drugs classification. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">ATC Code</td><td align="center" rowspan="1" colspan="1"> ATC Drug Name</td><td align="center" rowspan="1" colspan="1"> DDD</td><td align="center" rowspan="1" colspan="1"> U </td><td align="center" rowspan="1" colspan="1">Administration Rote</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">J01DB04</td><td align="center" rowspan="1" colspan="1"> Cefazolin</td><td align="center" rowspan="1" colspan="1"> 3</td><td align="center" rowspan="1" colspan="1"> g</td><td align="center" rowspan="1" colspan="1"> P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">J01DD02</td><td align="center" rowspan="1" colspan="1"> Ceftazidime </td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1"> g</td><td align="center" rowspan="1" colspan="1"> P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> J01DD04</td><td align="center" rowspan="1" colspan="1"> Cefrtiaxone </td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1"> g</td><td align="center" rowspan="1" colspan="1"> P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">J01MA02 </td><td align="center" rowspan="1" colspan="1">Ciprofloxacin </td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1"> g </td><td align="center" rowspan="1" colspan="1">P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> J01XD01</td><td align="center" rowspan="1" colspan="1"> Metronidazol </td><td align="center" rowspan="1" colspan="1">1.5 </td><td align="center" rowspan="1" colspan="1">g </td><td align="center" rowspan="1" colspan="1">P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> J01GB03 </td><td align="center" rowspan="1" colspan="1">Gentamycin </td><td align="center" rowspan="1" colspan="1">0.24 </td><td align="center" rowspan="1" colspan="1">g </td><td align="center" rowspan="1" colspan="1">P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">J01GB06</td><td align="center" rowspan="1" colspan="1"> Amikacin</td><td align="center" rowspan="1" colspan="1"> 1 </td><td align="center" rowspan="1" colspan="1">g</td><td align="center" rowspan="1" colspan="1"> P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> J01XA01</td><td align="center" rowspan="1" colspan="1"> Vancomycin </td><td align="center" rowspan="1" colspan="1">2 </td><td align="center" rowspan="1" colspan="1">g </td><td align="center" rowspan="1" colspan="1">P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> J01FF01 </td><td align="center" rowspan="1" colspan="1">Clindamycin</td><td align="center" rowspan="1" colspan="1"> 1.8</td><td align="center" rowspan="1" colspan="1"> g </td><td align="center" rowspan="1" colspan="1">P</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">J01DH51</td><td align="center" rowspan="1" colspan="1"> Imipenem &#x00026; enzyme inhibitor (refer to Imipenem)</td><td align="center" rowspan="1" colspan="1"> 2 </td><td align="center" rowspan="1" colspan="1">g</td><td align="center" rowspan="1" colspan="1">P</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> J01CE01</td><td align="center" rowspan="1" colspan="1"> Penicillin G Na 5000000u (is equivalent to 312 mg)</td><td align="center" rowspan="1" colspan="1">3.6 </td><td align="center" rowspan="1" colspan="1">g </td><td align="center" rowspan="1" colspan="1">P</td></tr></tbody></table></table-wrap></sec><sec id="s1-2"><title>Setting and study period</title><p> A hospital-based study was conducted in Imam Hossein Hospital, Shahrood, Iran. The hospital is a university-affiliated multidisciplinary with 313 beds. Now the center of ear, nose and throat, surgery, ophthalmology, internal medicine, general surgery, neurology, cardiology, obstetrics and gynecology, pediatrics, urology, orthopedics, neurology, psychiatry, infectious disease, infants, dialysis, ICU, CCU, NICU and is part of the Clinical and Laboratory medicine. This study was carried out for a period of ninety following days from 21<sup>st</sup> March to 21<sup>st</sup> June 2011 in the orthopedic and general surgery wards (<xref ref-type="table" rid="T2">Tables 2</xref> and <xref ref-type="table" rid="T3">3</xref> ). </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Type of surgeries performed during the 90 days of the study (n = 516, 357 male plus 159 female). It would be mentioned that 129 patients had surgeries without antibiotics prophylaxis in the period of the study. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1"> Type of Surgery </td><td align="center" rowspan="1" colspan="1">Total Number of patients in month 1</td><td align="center" rowspan="1" colspan="1"> Total Number of patients in month 2 </td><td align="center" rowspan="1" colspan="1"> Total Number of patients in month 3 </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> General </td><td align="center" rowspan="1" colspan="1"> 79 </td><td align="center" rowspan="1" colspan="1">56</td><td align="center" rowspan="1" colspan="1"> 58 </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Orthopedic </td><td align="center" rowspan="1" colspan="1">159</td><td align="center" rowspan="1" colspan="1"> 80</td><td align="center" rowspan="1" colspan="1"> 84</td></tr></tbody></table></table-wrap><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> Patients&#x02019; age (years) in each month with and without antibiotic prophylaxis. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Range of Age/ Period of Study</td><td align="center" rowspan="1" colspan="1"> &#x0003c; 10 </td><td align="center" rowspan="1" colspan="1"> 11-20 </td><td align="center" rowspan="1" colspan="1"> 21-30 </td><td align="center" rowspan="1" colspan="1"> 31-40 </td><td align="center" rowspan="1" colspan="1"> 41-50 </td><td align="center" rowspan="1" colspan="1"> 51-60 </td><td align="center" rowspan="1" colspan="1"> 61-70 </td><td align="center" rowspan="1" colspan="1"> 71-80 </td><td align="center" rowspan="1" colspan="1"> &#x0003e; 80 </td><td align="center" rowspan="1" colspan="1"> Patients with antibiotic prophylaxis </td><td align="center" rowspan="1" colspan="1"> Patients without antibiotic prophylaxis </td><td align="center" rowspan="1" colspan="1"> Total </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Month 1</td><td align="center" rowspan="1" colspan="1"> 33</td><td align="center" rowspan="1" colspan="1"> 51</td><td align="center" rowspan="1" colspan="1"> 65 </td><td align="center" rowspan="1" colspan="1">32</td><td align="center" rowspan="1" colspan="1"> 24 </td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1"> 10</td><td align="center" rowspan="1" colspan="1"> 5 </td><td align="center" rowspan="1" colspan="1">2 </td><td align="center" rowspan="1" colspan="1">147</td><td align="center" rowspan="1" colspan="1"> 91</td><td align="center" rowspan="1" colspan="1"> 238</td><td align="center" rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Month 2 </td><td align="center" rowspan="1" colspan="1">16 </td><td align="center" rowspan="1" colspan="1">26 </td><td align="center" rowspan="1" colspan="1">46</td><td align="center" rowspan="1" colspan="1"> 17</td><td align="center" rowspan="1" colspan="1"> 15 </td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1"> 3 </td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2 </td><td align="center" rowspan="1" colspan="1">120</td><td align="center" rowspan="1" colspan="1"> 16</td><td align="center" rowspan="1" colspan="1"> 136</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Month 3</td><td align="center" rowspan="1" colspan="1"> 19</td><td align="center" rowspan="1" colspan="1"> 25</td><td align="center" rowspan="1" colspan="1"> 45 </td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1"> 22 </td><td align="center" rowspan="1" colspan="1">9 </td><td align="center" rowspan="1" colspan="1">4 </td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"> 1</td><td align="center" rowspan="1" colspan="1"> 117</td><td align="center" rowspan="1" colspan="1"> 25 </td><td align="center" rowspan="1" colspan="1">142</td></tr></tbody></table></table-wrap></sec><sec id="s1-3"><title>Study population and operation characteristics</title><p> From a total of 516 patients, 384 cases (men = 265 and women = 119) were prescribed prophylactic antibiotics before their surgical operations. The median age of the patients was 26 years. Most of them were residents of Shahrood, however, some of them came from near cities and villages. The median operation duration was 58 minutes (interquartile range 42 to 83). </p></sec><sec id="s1-4"><title>Data gathering procedure</title><p> Data was collected from elective surgeries from general surgery and orthopedic wards. The general surgery had 37 and orthopedic ward had 26 beds. The other wards of the hospital were excluded to prevent data accumulation. This would help the research to focus on the wards which were used more antibiotics. </p><p> Orthopedic and general surgery bed occupancy for the first six months were 82.7% and 83.4% respectively. Therefore, the occupancy indexes for the orthopedic and general surgery were 0.827 and 0.834 respectively. The average occupancy index for both wards was 0.83. </p><p> A data collection form was designed by the authors. The pre-designed form included items regarding patient demographics, type of surgical procedure, drug history and allergy, choice of antibiotic regimen, time of administration, dose and repeated doses and duration of operation. To assess whether the data collection form was filled correctly and accurately, the author checked the items that must be measured. If it was comprehensive enough to collect all the information needed to address the purpose and goals of the study then a pilot test for 30 patients was set up. The form was filled out by 5 paramedic students. The contribution of students was just filling the forms based on patients&#x02019; medical profiles and records. </p><p> Retrospective follow-up was performed to the day of discharge, using patients&#x02019; profiles and records. Data collection was made based on the officially authorized agreement between research committee of the university and the hospital to access to the patients&#x02019; profiles and records. </p><p> On completion, the data was reviewed, organized, tabulated and analyzed. To perform a standardized and reproducible study, the ATC/DDD methodology was used. The quantity of systemic antibiotics prescribed for in-patients over a period of 90 days was converted to DDDs which is then calculated as DDD per 100 bed-days. The following equation is used to calculate and compare antibiotic usage in hospitals. </p></sec><sec id="s1-5"><title>Definition of DDD</title><p> In the present study, DDD was used as a unit to calculate the total antibiotics prescribed. The DDD is the assumed average preservation dose per day for a drug used for its main indication in adults. A DDD will only be assigned for drugs that already have an ATC code. The defined daily dose is a unit of measurement and does not necessarily reflect the recommended or Prescribed Daily Dose. Doses for individual patients and patient groups will often differ from the DDD and will necessarily have to be based on individual characteristics (e.g. age and weight) and pharmacokinetic concerns. The DDDs per 100 bed-days is a useful aspect when in-hospital drug consumption is considered. For example 100 DDD per 100 bed days specifies that for instance 20 indivuals get a certain treatment for 5 days. </p></sec><sec id="s1-6"><title>DDD/ 100 bed-days</title><p> To calculate the DDD per 100 bed days the number of units administrated in a given period was multiplied by 100 and the divided by: the multiplication of "DDD", "number of days in the period", "number of beds" and the "occupancy index". In the present study the occupancy index was 0.83 for the wards. The number of days in the study was 90 and the total numbers of beds in the general surgery and orthopedic wards were 63. The DDD value for each antibiotic is given by the DDD/ATC WHO system. </p><p> <disp-formula id="m1"><mml:math id="n1"><mml:mrow><mml:mi>D</mml:mi><mml:mi>D</mml:mi><mml:mi>D</mml:mi><mml:mo>/</mml:mo><mml:mn>100</mml:mn><mml:mtext>&#x02009;</mml:mtext><mml:mi>b</mml:mi><mml:mi>e</mml:mi><mml:mi>d</mml:mi><mml:mtext>&#x02009;</mml:mtext><mml:mi>d</mml:mi><mml:mi>a</mml:mi><mml:mi>y</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:mi>A</mml:mi><mml:mi>n</mml:mi><mml:mi>t</mml:mi><mml:mi>i</mml:mi><mml:mi>b</mml:mi><mml:mi>i</mml:mi><mml:mi>o</mml:mi><mml:mi>t</mml:mi><mml:mi>i</mml:mi><mml:mi>c</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>c</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mi>s</mml:mi><mml:mi>u</mml:mi><mml:mi>m</mml:mi><mml:mi>p</mml:mi><mml:mi>t</mml:mi><mml:mi>i</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>i</mml:mi><mml:mi>n</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>g</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>m</mml:mi><mml:mi>s</mml:mi><mml:mo stretchy="false">)</mml:mo><mml:mo>&#x000d7;</mml:mo><mml:mn>100</mml:mn></mml:mrow><mml:mrow><mml:mi>D</mml:mi><mml:mi>D</mml:mi><mml:mi>D</mml:mi><mml:mo>&#x000d7;</mml:mo><mml:mi>n</mml:mi><mml:mi>u</mml:mi><mml:mi>m</mml:mi><mml:mi>b</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>o</mml:mi><mml:mi>f</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>d</mml:mi><mml:mi>a</mml:mi><mml:mi>y</mml:mi><mml:mi>s</mml:mi><mml:mo>&#x000d7;</mml:mo><mml:mi>n</mml:mi><mml:mi>u</mml:mi><mml:mi>m</mml:mi><mml:mi>b</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>o</mml:mi><mml:mi>f</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>b</mml:mi><mml:mi>e</mml:mi><mml:mi>d</mml:mi><mml:mi>s</mml:mi><mml:mo>&#x000d7;</mml:mo><mml:mi>o</mml:mi><mml:mi>c</mml:mi><mml:mi>c</mml:mi><mml:mi>u</mml:mi><mml:mi>p</mml:mi><mml:mi>a</mml:mi><mml:mi>n</mml:mi><mml:mi>c</mml:mi><mml:mi>y</mml:mi><mml:mtext>&#x02003;</mml:mtext><mml:mi>i</mml:mi><mml:mi>n</mml:mi><mml:mi>d</mml:mi><mml:mi>e</mml:mi><mml:mi>x</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula> </p><p> The occupancy index was calculated every month and was derived by dividing the number of occupied beds by the total number of beds in the wards. </p></sec><sec id="s1-7"><title>Statistical analysis</title><p> All data were analyzed using SPSS, version 21. Frequencies and percentages were calculated. </p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><p> The results revealed that cephalosporins are used in highly variable dosages for different indications, which should be reflected in the assigned DDDs. Indications for cephalosporins prescription (i.e. the severity of the infections) vary rather extensive from one country to another. The assigned DDDs are placed in the upper area of the dose range for moderate to severe infections. J01DB First-generation cephalosporins have relatively narrow spectrum of activity focused primarily on the gram-positive cocci. </p><p> The percentage of patients receiving perioperative antibiotic was 74.41% (<xref ref-type="table" rid="T4">Table 4</xref>). </p><table-wrap id="T4" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Type of parenteral administrated antibiotic prior to surgery (n = 384). The numbers in brackets show the number of patients. The numbers aside brackets show the total dosage form which were applied. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1"> Period of Study/ Antibiotics Used </td><td rowspan="1" colspan="1"> Month 1 </td><td rowspan="1" colspan="1">Month 2</td><td rowspan="1" colspan="1"> Month 3</td><td rowspan="1" colspan="1"> Total </td><td rowspan="1" colspan="1"> WHO/DDD</td><td rowspan="1" colspan="1"> DDD/ 100 bed days </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Cephazoline 500mg </td><td rowspan="1" colspan="1"> 98 (29)</td><td rowspan="1" colspan="1"> 15 (3)</td><td rowspan="1" colspan="1"> 17 (3)</td><td rowspan="1" colspan="1"> 130</td><td rowspan="1" colspan="1"> 3 </td><td rowspan="1" colspan="1">26.43</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Cephazoline 1g </td><td rowspan="1" colspan="1"> 1267 (76)</td><td rowspan="1" colspan="1"> 1237 (73)</td><td rowspan="1" colspan="1"> 1003 (68)</td><td rowspan="1" colspan="1"> 3667 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Ceftazidime 500mg </td><td rowspan="1" colspan="1"> 1 (1)</td><td rowspan="1" colspan="1"> 4 (1)</td><td rowspan="1" colspan="1"> 5 (2)</td><td rowspan="1" colspan="1"> 10</td><td rowspan="1" colspan="1"> 4 </td><td rowspan="1" colspan="1"> 0.02 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Cefrtiaxone 1g </td><td rowspan="1" colspan="1"> 70 (5)</td><td rowspan="1" colspan="1"> 72 (6)</td><td rowspan="1" colspan="1"> 18 (2)</td><td rowspan="1" colspan="1"> 160</td><td rowspan="1" colspan="1"> 2 </td><td rowspan="1" colspan="1"> 1.7 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Ciprofluxacin 200mg </td><td rowspan="1" colspan="1"> 62 (5)</td><td rowspan="1" colspan="1"> 40 (1)</td><td rowspan="1" colspan="1"> 57 (4)</td><td rowspan="1" colspan="1"> 159 </td><td rowspan="1" colspan="1">0.5 </td><td rowspan="1" colspan="1"> 1.73 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Ciprofluxacin 500mg </td><td rowspan="1" colspan="1">4 (1)</td><td rowspan="1" colspan="1"> 14 (3)</td><td rowspan="1" colspan="1"> - (-) </td><td rowspan="1" colspan="1"> 18 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Metronidazol 500mg </td><td rowspan="1" colspan="1"> 12 (2)</td><td rowspan="1" colspan="1"> - (-)</td><td rowspan="1" colspan="1"> - (-)</td><td rowspan="1" colspan="1"> 12</td><td rowspan="1" colspan="1">1.5 </td><td rowspan="1" colspan="1"> 0.08 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Gentamycin 80mg </td><td rowspan="1" colspan="1">136 (21)</td><td rowspan="1" colspan="1"> 96 (15)</td><td rowspan="1" colspan="1"> 122 (17)</td><td rowspan="1" colspan="1"> 354 </td><td rowspan="1" colspan="1">0.24 </td><td rowspan="1" colspan="1"> 2.5 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Amikacin 500mg </td><td rowspan="1" colspan="1"> 16 (3)</td><td rowspan="1" colspan="1">94 (11)</td><td rowspan="1" colspan="1"> 66 (7)</td><td rowspan="1" colspan="1"> 176</td><td rowspan="1" colspan="1"> 1 </td><td rowspan="1" colspan="1"> 1.87 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Vancomycin 500mg </td><td rowspan="1" colspan="1"> 45 (3)</td><td rowspan="1" colspan="1"> -(-) </td><td rowspan="1" colspan="1">65 (5)</td><td rowspan="1" colspan="1"> 110 </td><td rowspan="1" colspan="1">2 </td><td rowspan="1" colspan="1"> 0.58 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Clindamycin 300mg </td><td rowspan="1" colspan="1"> 3 (1)</td><td rowspan="1" colspan="1"> 237 (5)</td><td rowspan="1" colspan="1"> 88 (6)</td><td rowspan="1" colspan="1"> 328</td><td rowspan="1" colspan="1"> 1.8</td><td rowspan="1" colspan="1"> 1.36 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Clindamycin 600mg </td><td rowspan="1" colspan="1">- (-)</td><td rowspan="1" colspan="1"> (-) </td><td rowspan="1" colspan="1">28 (2) </td><td rowspan="1" colspan="1"> 28 </td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Imipenem 500mg </td><td rowspan="1" colspan="1"> - (-)</td><td rowspan="1" colspan="1"> 33 (1)</td><td rowspan="1" colspan="1"> - (-)</td><td rowspan="1" colspan="1"> 33 </td><td rowspan="1" colspan="1">2 </td><td rowspan="1" colspan="1"> 0.17 </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Penicillin G Na 5000000u </td><td rowspan="1" colspan="1"> - (-)</td><td rowspan="1" colspan="1">12 (1)</td><td rowspan="1" colspan="1"> 4 (1)</td><td rowspan="1" colspan="1"> 16</td><td rowspan="1" colspan="1"> 3.6</td><td rowspan="1" colspan="1"> 0 </td></tr></tbody></table></table-wrap><p> There was not any evidence in patients&#x02019; files related to selecting the antibiotics by antibiograms. Host factors such as immunity contribute to the selective process. Antibiotics themselves may support bacterial diversity, either mediated by the random drift effect or triggering the increase of mutational proceedings under bacterial stress. Analysis of selective environment-related antibiotic-host-bacteria interactions is essential to reach better outcomes. </p></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Based on the main goal of the present study which was the usage pattern of prophylactic antibiotics in elective surgeries the utilization of antibacterial agents, J01 class, was evaluated and compared between the surgical wards of the hospital using the WHO ATC/DDD procedure. Most commonly used agent in the wards, according to the DDD was cefazoline followed by gentamicin and amikacin. Perioperative antimicrobial prophylaxis was used in 384 elective surgeries (74.4%). The DDD/100 bed-days for the perioperative antimicrobial prophylaxis for the most frequently used antimicrobials, cefazolin, in the period of the study was 26.43 (<xref ref-type="table" rid="T4">Table 4</xref>). No statistically significant difference in antibiotic utilization was found between the wards when measured by DDD/100 bed days. However, the total DDD of antibacterial agents utilized was less than 100 DDD/100 bed days in both units indicating reasonably acceptable use of antibiotics as prophylaxis. Typically, prophylactic antimicrobials are not indicated for clean surgeries (<xref rid="R12" ref-type="bibr">12</xref>). They are particularly beneficial in surgical procedures associated with a high rate of infection and the agent chosen should have activity against the most common surgical wound pathogens. Cephalosporins are appropriate first-line antimicrobials for most surgical procedures, targeting the most likely organisms (<xref rid="R12" ref-type="bibr">12</xref>,<xref rid="R13" ref-type="bibr">13</xref>). It is advisable to avoid broad-spectrum antimicrobial therapy that may lead to the development of antimicrobial resistance (<xref rid="R14" ref-type="bibr">14</xref>). In addition the results of the present study showed lesser than those reported from surgical departments in hospitals in Spain, Estonia and Sweden (<xref rid="R15" ref-type="bibr">15</xref>). </p><p> It should be mentioned that our results is not in parallel with other studies which have been previousy published such as Mahdaviazad et al, 2011 (<xref rid="R16" ref-type="bibr">16</xref>) and Hatam et al, 2011 (<xref rid="R17" ref-type="bibr">17</xref>) because our results did not indicate that over- and misuse of prophylactic antibiotics in Imam Hossein Hospital. Therefore, the novelty of the present research communication was that antibiotics consumption in the surgical wards was acceptable in Imam Hossein hospital. This would mark this hospital as a clean health care environment with research, education and treatment missions. </p><p> A study at Al-Watani governmental hospital in Nablus, Palestine revealed that the use of antibacterial agents was less than optimal and reached a total of 39 DDD/ 100 bed-days (<xref rid="R18" ref-type="bibr">18</xref>). In contrast in a study carried out at Emam hospital in Sari, Iran, the total DDD/100 bed-days in general surgery was 121 in 2000 and this declined to 107 in 2005 (<xref rid="R19" ref-type="bibr">19</xref>). </p><p> The difference between the results of our study and those published from the other countries may be attributed to the infection control and prevention programs, as well as special guidelines for antibiotic use in surgical wards. Antimicrobial administration is not recommended for all surgical procedures. The present study revealed that antibiotics&#x02019; consumption in the orthopedic and general surgery wards was acceptable. However it is difficult to compare the results with the other studies. For example a study done by Akalin et al revealed that the most frequently used antimicrobials were cefazolin with 117.9 DDD/100-operation (<xref rid="R20" ref-type="bibr">20</xref>). Thus reports on antibiotic use, often lack complete definitions of the units of measurement, hampering the comparison of data between hospitals around the world (<xref rid="R21" ref-type="bibr">21</xref>-<xref rid="R23" ref-type="bibr">23</xref>). </p><p> It is generally accepted that the use of perioperative antibiotics prophylaxis reduces the risk of postoperative infection sites (<xref rid="R24" ref-type="bibr">24</xref>-<xref rid="R28" ref-type="bibr">28</xref>); yet few studies have described the association between perioperative antibiotics usages and the risk of surgical complication (<xref rid="R29" ref-type="bibr">29</xref>). In addition there are a few published descriptions or comparisons of antibiotic consumption. This lack of information has hindered rational discussions about desirable levels of consumption (<xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R15" ref-type="bibr">15</xref>). </p><p> Another noticeable concern is this fact that the use of perioperative antibiotics might be standard practice in hospitals that provide generally better care, so perhaps this better care, not antibiotics, caused the better outcomes of patients receiving antibiotics prophylaxis (<xref rid="R14" ref-type="bibr">14</xref>,<xref rid="R30" ref-type="bibr">30</xref>-<xref rid="R34" ref-type="bibr">34</xref>). There is a possibility that physicians who do not use prophylactic antibiotics have higher complications rates because the care they deliver is inadequate in other ways (<xref rid="R35" ref-type="bibr">35</xref>). Since we might expect patients operated between 6 PM to 6 AM to be at increased risk either because inadequate staffing or urgent conditions (<xref rid="R36" ref-type="bibr">36</xref>). These are the concerns that would influence the patients&#x02019; outcomes without any relations with antibiotics as prophylaxis (<xref rid="R37" ref-type="bibr">37</xref>). </p><p> As well as antibiotic prophylaxis being a generally effective intervention for preventing postoperative site infection, the level of this effectiveness would appear to be reasonably independent of what type of surgery is being considered. Therefore, the general prevailing attitude that antibiotic prophylaxis should be assumed to be ineffective unless its effectiveness has been experimentally proven beyond doubt for the specific type of surgery being considered, perhaps should be revised. In particular, a sensible philosophy would be to assume that antibiotic prophylaxis is effective in reducing the risk of wound infection for all types of surgery, even ones where no clinical trial data exists and make exceptions to this rule if, for certain types of surgery, it can be proved to the contrary. </p></sec><sec sec-type="conclusion" id="s4"><title>Conclusion</title><p> The findings showed differences when compared with national and international studies although the difference was not dramatic. Data on in-hospital antibiotic usage are varying widely not only due to different antibiotic policies but also due to different methods of measurement. These differences make the comparison difficult. </p></sec><sec id="s5"><title>Limitation</title><p> Our study had some limitations, including the retrospective design and lack of randomizationd. Moreover, a small number of patients were analyzed from a single center. Furthermore, we could not judge the appropriateness of the empirical antibiotic treatment in this study. Thus further studies will need to assess this valuable and important issue. </p></sec>
Oral magnesium supplementation in type II diabetic patients
<p> <bold>Background:</bold> Magnesium is the second most abundant intracellular cation. It plays an important role in insulin homeostasis and glucose metabolism through multiple enzymatic reactions. With increasing data on magnesium deficiency in diabetic patients and epidemiological studies demonstrating magnesium deficiency as a risk factor for diabetes, it is logical to search for its possible beneficial effects on diabetes control and prevention. We aimed to determine whether oral magnesium supplementation improves metabolic control, lipid profile and blood pressure in patients with type II diabetes. </p><p> <bold>Methods:</bold> Fifty four patients with type II diabetes were included in a randomized double blind placebocontrolled clinical trial.Patients received either placebo or 300 mg elemental magnesium (as magnesium sulfate -MgSo4-) daily, for 3 months. Metabolic control, lipid profile, blood pressure, magnesium status, hepatic enzymes, hemoglobin concentration, and anthropometric indices were determined in the beginning and at the end of the study. </p><p> <bold>Results:</bold> Daily administration of 300 mg elemental magnesium for 3 months, significantly improved fasting blood glucose (183.9&#x000b1;15.43 to 125.8&#x000b1;6.52 vs. 196.5&#x000b1;28.12 to 136.5&#x000b1;7.94, p&#x0003c; 0.0001), 2-hour post prandial glucose (239.1&#x000b1;74.75 to 189.1&#x000b1;60mg/dl vs. 246.4&#x000b1;97.37 to 247.8&#x000b1;86.74mg/dl, p&#x0003c; 0.01), lipid profile, blood pressure and hepatic enzymes. </p><p> <bold>Conclusion:</bold> Oral magnesium supplementation with proper dosage has beneficial effects on blood glucose, lipid profile, and blood pressure in patients with type II diabetes. </p>
<contrib contrib-type="author"><name><surname>Solati</surname><given-names>Mehrdad</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Ouspid</surname><given-names>Elham</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Hosseini</surname><given-names>Saeedeh</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Soltani</surname><given-names>Nepton</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Keshavarz</surname><given-names>Mansoor</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Dehghani</surname><given-names>Mohsen</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> &#x0200f;Magnesium (Mg) is the second most abundant intracellular cation (<xref rid="R1" ref-type="bibr">1</xref>). Mg homeostasis and metabolism is affected by multiple factors but it is in part, a determinant of cellular ionic environment and cellular responsiveness to external stimuli such as insulin and glucose (<xref rid="R2" ref-type="bibr">2</xref>). Mg is a cofactor in the cell membrane glucose-transporting mechanism, as well as in various enzymes in carbohydrate oxidation. It is also involved in insulin secretion, binding, and activity at various levels (<xref rid="R3" ref-type="bibr">3</xref>). It is suggested that Mg depletion contributes to states of insulin resistance such as hypertension, metabolic syndrome and type II diabetes (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> &#x0200f;Some studies have shown that hypomagnesaemia, which is a frequent condition in patients with diabetes, could be involved in the development of complications related to diabetes (<xref rid="R4" ref-type="bibr">4</xref>). However total serum Mg concentrations do not reflect the Mg status or intracellular pool, and intracellular or serum ionized Mg. Mg depletion can be seen with normal total serum Mg concentration (<xref rid="R2" ref-type="bibr">2</xref>). Previous studies have shown different results for oral Mg administration in patients with diabetes. Purvis and colleagues found that the administration of 386 mg magnesium chloride for 6 weeks could significantly decrease systolic blood pressure, without any effect on metabolic indices or lipid profile (<xref rid="R5" ref-type="bibr">5</xref>). Other studies have shown different effects of Mg supplementation on blood pressure (<xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R7" ref-type="bibr">7</xref>). Paolisso and co-workers found that the administration of 15.8mmol magnesium pidolate for 4 weeks improved insulin stimulated glucose uptake and glucose oxidation (<xref rid="R8" ref-type="bibr">8</xref>). Lima and colleagues compared supplementation with 20.7 or 41.4mmol magnesium oxide and placebo for 30 days; they found that supplementation with 20.7mmol Mg did not show any beneficial effects, but a dosage of 41.4 mmol significantly reduced fructosamine (<xref rid="R9" ref-type="bibr">9</xref>). Klitgard and colleagues used 500 mg of magnesium oxide daily for 24 weeks and reported a significant reduction in total cholesterol (TC), low density cholesterol (LDL C.), and Apo lipoprotein &#x000df;, but no effect on glycosylated hemoglobin (HbA1c) or fasting blood glucose (FBG) (<xref rid="R10" ref-type="bibr">10</xref>). Yokota and co-workers stated that Mg supplementation with a natural preparation from a water lake had reduced homeostasis model assessment index (HOMA-IR), but not FBG or HbA1c (<xref rid="R11" ref-type="bibr">11</xref>). </p><p> &#x0200f;In previous studies we found that oral MgSO4 administration in diabetic animal models could decrease blood glucose and blood pressure (<xref rid="R12" ref-type="bibr">12</xref>). We also reported that MgSo4 had a beneficial effect on animal model diabetic vessel complications (<xref rid="R13" ref-type="bibr">13</xref>). Based on our previous studies and increasing data on Mg deficiency in patients with diabetes (<xref rid="R12" ref-type="bibr">12</xref>-<xref rid="R17" ref-type="bibr">17</xref>), as well as epidemiological studies reporting Mg deficiency as a risk factor for diabetes (<xref rid="R18" ref-type="bibr">18</xref>-<xref rid="R22" ref-type="bibr">22</xref>), it is logical to search for its possible beneficial effects on diabetes control and prevention. </p><p> &#x0200f;We aimed to determine whether oral Mg supplementation improves blood glucose, lipid profile, blood pressure, hepatic enzymes, hemoglobin concentration, and anthropometric indices, as control parameters for patients with type II diabetes. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><sec id="s2-1"><title> Patients </title><p> &#x0200f;In this randomized double-blind controlled trial we included patients with type II diabetes who did not have obvious disturbances in Mg metabolism, with any level of diabetes control, and were available and willing to participate. Therefore, patients with type II diabetes aged 20&#x02013;60 years were recruited from an out-patient primary level medical care office, specific for diabetic patients in Bandar-Abbas, southern Iran. </p><p> &#x0200f;Before the study all patients completed a questionnaire. Also, they were clinically evaluated and blood samples were withdrawn in order to exclude patients who were using insulin, calcium channel blockers, and Mg or calcium (Ca) containing supplements. Moreover, patients who had experienced renal insufficiency (serum creatinine levels more than 1.3 mg/dl in women and more than 1.5 mg/dl in men), elevated hepatic enzymes (more than 3 folds over normal values), recent infections (less than one month prior to study) and chronic inflammatory diseases, cerebrovascular accident (CVA) and acute coronary syndrome (ACS) less than one month prior to the study and were alcoholic, pregnant, or drug abusers were excluded. </p><p> &#x0200f;After obtaining informed consent, 54 patients with type II diabetes were ultimately enrolled in the study and randomly allocated to receive either Mg supplementation or placebo for 3 moths using the simple randomization method. The study flow diagram is shown in <xref ref-type="fig" rid="F1">figure 1</xref>. The patients and personnel were blind to group assignment. Only the study analyst saw unblended data, but was not in contact with the participants. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-67-g001"/><statement><p>Study felow diagram.</p></statement></fig><p> &#x0200f;The present study was a randomized double-blind placebo-controlled clinical trial. All patients and researcher except the one who was responsible for randomization, were blind to the study protocol. </p><p> &#x0200f;The study was approved by the Ethics Committee of the Faculty of Medicine of Bandar Abbas University of Medical Sciences, and registered in the Iranian registry of clinical trials (IRCT), with a membership number of 1707. </p><p> &#x0200f;Initially, 10ml blood was withdrawn from the antecubital vein of all participants to measure variables including FBG as the primary outcome, 2-hour post prandial blood glucose (2hr PP BS), HbA1C, fasting plasma insulin (FPI) as indicators of metabolic control, hemoglobin concentration (Hb), triglycerides (TG), TC, LDL C., high density cholesterol (HDL C.) as indicators of diabetes complications, serum Ca and Mg levels, urine Mg level, and Ca /Mg ratio as indicators of diabetes-related vascular complications (<xref rid="R23" ref-type="bibr">23</xref>). Hepatic enzymes (Alanine transferase [ALT] and Aspartate transferase [AST]) were also measured as indicators of hepatic side effects of Mg. Blood pressure was measured at baseline using the standard method (<xref rid="R24" ref-type="bibr">24</xref>). HOMA-IR index (<xref rid="R25" ref-type="bibr">25</xref>) was calculated at the baseline and at the end of the study using FBG and FPI; it was then used as an indicator for insulin resistance and metabolic control. </p><p> &#x0200f;All patients were visited by an endocrinologist, who was blind to the grouping, and their medications were adjusted. Glibenclamide, metformin, atorvastatin, and enalapril were used for treatment. After the initiation of the intervention, the patients were visited every month to be evaluated for adherence to pharmacological treatment. Blood pressure (Microlife BPAG1-20, Microlife, Swiss) and FBG (glucometer, EasyGluco, Infopia CO., Korea) were measured monthly during the study period and all laboratory measurements were done completely. All participants were visited by the same endocrinologist every month and their drugs were readjusted. At the end of the intervention the changes in glucose lowering drug dosage were recorded and used as indicators of metabolic control. </p></sec><sec id="s2-2"><title> &#x0200f;Drugs </title><p> &#x0200f;Capsules containing MgSo4 were used as magnesium supplement. Each capsule contained 100 mg elemental Mg. Each patient used 3 capsules per day; one with each meal adding up to a daily supplementation of 300 mg elemental Mg, according to our pilot study. The placebo capsules were similar to the Mg capsules and were used 3 times a day. Mg and placebo capsules were prepared at the Herbal Drugs Research Center of Tehran University of Medical Sciences, Tehran, Iran. </p></sec><sec id="s2-3"><title> Measurements </title><p> &#x0200f;Height and weight were measured using standard protocols when the patients were wearing light clothing without shoes. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (<xref rid="R24" ref-type="bibr">24</xref>). </p><p> &#x0200f;FBG was measured every three weeks using the glucose&#x02013;oxidase method (Glucose - monoreagent kit, Kimia Pajhouhan, Iran) at baseline and every month thereafter during the study period. Two-hr PP BS was also measured using the glucose&#x02013;oxidase method. Enzymatic methods were used for the measurement of TC (CHOD-PAP cholesterol kit, Pars Azmun, Iran), LDL C. (LDL- C kit, Pars Azmun, Iran), HDL C. (CHOD-PAP cholesterol kit, Pars Azmun, Iran), and TG (GPO-PAP triglycerides kit, Pars Azmun, Iran). FPI levels were measured using Elisa test (Monobind insulin microplate Elisa test, Monobind Inc., USA). Serum Mg concentrations (Colorimetric Magnesium kit, Pars Azmun, Iran) and Hb concentration (Colorimetric Hb kit, Pars Azmun, Iran) were measured with the colorimetric method. Nycocard HbA1c kit (Axis-shild, UK) was used for HbA1C measurement. Cresol phethalein complex method (Pars Azmun, Iran) was used to measure serum Ca and monoreagent method (Pars Azmun, Iran) was used for the measurement of AST and ALT. </p><p> &#x0200f;Insulin resistance was determined by the homeostasis model assessment index (defined as HOMA&#x02013;IR= FBG(mg/dl)&#x000d7;FPI (&#x000b5;u/ml) &#x02215;405) (<xref rid="R25" ref-type="bibr">25</xref>). </p></sec><sec id="s2-4"><title> &#x0200f;Statistical analyses </title><p> &#x0200f;Data were expressed as mean &#x000b1; SD. The normal distribution of our populations (Gaussian distributions) was tested using the Kolmogorov and Smirnov method. Comparisons between groups were analyzed using the T test. Paired-T test was used to analyze difference between means of each group before and after the intervention and different multiple statistical tests such as Bonferroni and Newman-Keuls tests were used to adjust for the effect of other medications changes. SPSS software, version 13, was used for the analyses. p&#x0003c;0.05 was considered as statistically significant. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> &#x0200f;Fifty four patients were enrolled in our study, and were randomly allocated into Mg (n=26) and placebo (n=28) groups. The Mg group received 300 mg elemental Mg daily and the placebo group received placebo capsules. One woman in the placebo group developed pyelonephritis during the study and was excluded from the study to receive appropriate treatment. Five other participants in the placebo group were lost to follow-up, because they moved from Bandar Abbas. In the Mg group, one patient was excluded because of becoming pregnant. Twenty five patients in the Mg group and twenty two patients in the placebo group completed the three-month intervention successfully (<xref ref-type="fig" rid="F1">Fig. 1</xref>). Mg supplement was well tolerated and did not cause any adverse effects in the participants apart from slight diarrhea in two patients without the need for terminating the treatment. </p><p> &#x0200f;The results of this study before the intervention are shown in <xref ref-type="table" rid="T1">Table 1</xref>. <xref ref-type="table" rid="T2">Table 2</xref> demonstrates the study results in both groups after the intervention. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Study variables in Mg treated and placebo group before intervention. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> <bold>MgSO</bold> <sub>4</sub> <bold> group (n =25)</bold> </td><td align="center" rowspan="1" colspan="1">Placebo group (n = 22)</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Baseline</td><td align="center" rowspan="1" colspan="1">Baseline</td><td align="center" rowspan="1" colspan="1">p</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age (years)</td><td align="center" rowspan="1" colspan="1">46.76 &#x000b1; 9</td><td align="center" rowspan="1" colspan="1">50.15 &#x000b1; 6</td><td align="center" rowspan="1" colspan="1"> <sup>Not significant</sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Sex (male/female)</td><td align="center" rowspan="1" colspan="1"> 7/18 </td><td align="center" rowspan="1" colspan="1"> 6/16 </td><td align="center" rowspan="1" colspan="1"> <sup>Not significant</sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Duration of diabetes (years)</td><td align="center" rowspan="1" colspan="1"> 4.11 &#x000b1; 4.23 </td><td align="center" rowspan="1" colspan="1"> 5.35 &#x000b1; 4.01 </td><td align="center" rowspan="1" colspan="1"> <sup>Not significant</sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> BMI (kg/m<sup>2</sup>) </td><td align="center" rowspan="1" colspan="1"> 26.19 &#x000b1; 2.86 </td><td align="center" rowspan="1" colspan="1"> 26.89 &#x000b1; 5.23 </td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Systolic blood pressure ( mm Hg)</td><td align="center" rowspan="1" colspan="1">117.9 &#x000b1; 14.99</td><td align="center" rowspan="1" colspan="1">120.8 &#x000b1; 18.31</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Diastolic blood pressure ( mm Hg)</td><td align="center" rowspan="1" colspan="1">72.91 &#x000b1; 8.10</td><td align="center" rowspan="1" colspan="1">73.75 &#x000b1; 8.82</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Serum Mg (mg/dl)</td><td align="center" rowspan="1" colspan="1">2.15 &#x000b1; 0.33</td><td align="center" rowspan="1" colspan="1">2.03 &#x000b1; 0.58</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Urine Mg (mg/dl)</td><td align="center" rowspan="1" colspan="1">5.67 &#x000b1; 1.77</td><td align="center" rowspan="1" colspan="1">5.54 &#x000b1; 1.56</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Calcium to magnesium ratio</td><td align="center" rowspan="1" colspan="1">4.58 &#x000b1; 0.68</td><td align="center" rowspan="1" colspan="1">4.79 &#x000b1; 1.</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Fasting blood glucose (mg/dl)</td><td align="center" rowspan="1" colspan="1">183.9 &#x000b1; 15.43</td><td align="center" rowspan="1" colspan="1">196.5 &#x000b1; 28.12</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">2hr PP blood glucose (mg/dl)</td><td align="center" rowspan="1" colspan="1">239.1 &#x000b1; 74.75</td><td align="center" rowspan="1" colspan="1">246.4 &#x000b1; 97.37</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">HbA1C (%)</td><td align="center" rowspan="1" colspan="1">8.33 &#x000b1; 1.47</td><td align="center" rowspan="1" colspan="1">8.30 &#x000b1; 1.99</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">HOMA-IR index</td><td align="center" rowspan="1" colspan="1">2.47 &#x000b1; 2.71</td><td align="center" rowspan="1" colspan="1">2.37 &#x000b1; 3.02</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Fasting plasma insulin (qIU/ml)</td><td align="center" rowspan="1" colspan="1">7.12 &#x000b1; 8.02</td><td align="center" rowspan="1" colspan="1">6.82 &#x000b1; 8.60</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Triglycerides (mg/dl)</td><td align="center" rowspan="1" colspan="1"> 162.6 &#x000b1; 68.92 </td><td align="center" rowspan="1" colspan="1">188 &#x000b1; 69.90</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">217.7&#x000b1;106.49</td><td align="center" rowspan="1" colspan="1">201&#x000b1;54.07</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">LDL cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">118.3 &#x000b1; 32.55</td><td align="center" rowspan="1" colspan="1">130.65 &#x000b1; 33.52</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">HDL cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">44.30 &#x000b1; 10.20</td><td align="center" rowspan="1" colspan="1">47.45 &#x000b1; 10.50</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Non HDL cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">149.35 &#x000b1; 35.07</td><td align="center" rowspan="1" colspan="1">158.38 &#x000b1; 26.92</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Hemoglobin concentration (gr/dl)</td><td align="center" rowspan="1" colspan="1">13.45 &#x000b1; 1.26</td><td align="center" rowspan="1" colspan="1">13.32 &#x000b1; 1.29</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Aspartate transfrase (IU/ml)</td><td align="center" rowspan="1" colspan="1">18 &#x000b1; 5.40</td><td align="center" rowspan="1" colspan="1">21 &#x000b1; 6.50</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Alanin transfrase (IU/ml)</td><td align="center" rowspan="1" colspan="1">22.80 &#x000b1; 2.22</td><td align="center" rowspan="1" colspan="1">23.81 &#x000b1; 1.89</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Metformin dosage ( mg )</td><td align="center" rowspan="1" colspan="1">1045.45 &#x000b1; 598.60</td><td align="center" rowspan="1" colspan="1">1136.36 &#x000b1; 551.85</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Glibenclamid dosage ( mg )</td><td align="center" rowspan="1" colspan="1">11.36 &#x000b1; 5.04</td><td align="center" rowspan="1" colspan="1">11.75 &#x000b1; 6.66</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Enalapril dosage (mg)</td><td align="center" rowspan="1" colspan="1">15 &#x000b1; 4.56</td><td align="center" rowspan="1" colspan="1">15 &#x000b1; 2.88</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Atorvastatin dosage (mg)</td><td align="center" rowspan="1" colspan="1">22.5 &#x000b1; 2.5</td><td align="center" rowspan="1" colspan="1">23.33 &#x000b1; 6.6</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr></tbody></table><table-wrap-foot><fn><p> Data are means &#x000b1; SD. </p></fn></table-wrap-foot></table-wrap><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Study variables in Mg treated and placebo group after 3 months intervention. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> <bold>MgSO</bold> <sub>4</sub> <bold> group (n =25)</bold> </td><td align="center" rowspan="1" colspan="1">Placebo group (n = 22)</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">End</td><td align="center" rowspan="1" colspan="1">End</td><td align="center" rowspan="1" colspan="1">p</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age (years)</td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Sex (male/female)</td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Duration of diabetes (years)</td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td><td align="center" rowspan="1" colspan="1"> <sup>________</sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> BMI (kg/m<sup>2</sup>) </td><td align="center" rowspan="1" colspan="1">26.41 &#x000b1; 3.29</td><td align="center" rowspan="1" colspan="1">27.28 &#x000b1; 5.13</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Systolic blood pressure ( mm Hg)</td><td align="center" rowspan="1" colspan="1">102.8 &#x000b1; 9.51</td><td align="center" rowspan="1" colspan="1">117.2 &#x000b1; 3.48</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Diastolic blood pressure ( mm Hg)</td><td align="center" rowspan="1" colspan="1">62.85 &#x000b1; 7.55</td><td align="center" rowspan="1" colspan="1">70 &#x000b1; 7.07</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.05</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Serum Mg (mg/dl)</td><td align="center" rowspan="1" colspan="1">2.16 &#x000b1; 0.36</td><td align="center" rowspan="1" colspan="1">2.23 &#x000b1; 0.50</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Urine Mg (mg/dl)</td><td align="center" rowspan="1" colspan="1">5.99 &#x000b1; 2</td><td align="center" rowspan="1" colspan="1">4.56 &#x000b1; 1.72</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.05</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Calcium to magnesium ratio</td><td align="center" rowspan="1" colspan="1">4.64 &#x000b1; 1.46</td><td align="center" rowspan="1" colspan="1">5.21 &#x000b1; 1.65</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Fasting blood glucose (mg/dl)</td><td align="center" rowspan="1" colspan="1">125.8 &#x000b1; 6.52</td><td align="center" rowspan="1" colspan="1">136.5 &#x000b1; 7.94</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.0001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">2hr PP blood glucose (mg/dl)</td><td align="center" rowspan="1" colspan="1">189.1 &#x000b1; 60.05</td><td align="center" rowspan="1" colspan="1">247.8 &#x000b1; 86.74</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.01</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">HbA1C (%)</td><td align="center" rowspan="1" colspan="1">7.90 &#x000b1; 1.68</td><td align="center" rowspan="1" colspan="1">7.61 &#x000b1; 1.59</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">HOMA-IR index</td><td align="center" rowspan="1" colspan="1">5.01 &#x000b1; 2.47</td><td align="center" rowspan="1" colspan="1">4.65 &#x000b1; 3.48</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Fasting plasma insulin (qIU/ml)</td><td align="center" rowspan="1" colspan="1">15.90 &#x000b1; 8.02</td><td align="center" rowspan="1" colspan="1">13.35 &#x000b1; 11.53</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Triglycerides (mg/dl)</td><td align="center" rowspan="1" colspan="1">158.7 &#x000b1; 77.16</td><td align="center" rowspan="1" colspan="1">175.5 &#x000b1; 107.5</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Total cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">176.2 &#x000b1; 48.33</td><td align="center" rowspan="1" colspan="1">182.7 &#x000b1; 56.23</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">LDL cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">93.63 &#x000b1; 24.58</td><td align="center" rowspan="1" colspan="1">120.4 &#x000b1; 34.86</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.01</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">HDL cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">41.7 &#x000b1; 8.79</td><td align="center" rowspan="1" colspan="1">43.65 &#x000b1; 11.20</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Non HDL cholesterol (mg/dl)</td><td align="center" rowspan="1" colspan="1">125.30 &#x000b1; 23.19</td><td align="center" rowspan="1" colspan="1">152.16 &#x000b1; 37.05</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Hemoglobin concentration (gr/dl)</td><td align="center" rowspan="1" colspan="1">13.46 &#x000b1; 1.51</td><td align="center" rowspan="1" colspan="1">13.18 &#x000b1; 1.71</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Aspartate transfrase (IU/ml)</td><td align="center" rowspan="1" colspan="1">18.15 &#x000b1; 10.40</td><td align="center" rowspan="1" colspan="1">24.92 &#x000b1; 12.12</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Alanin transfrase (IU/ml)</td><td align="center" rowspan="1" colspan="1">22.30 &#x000b1; 2.83</td><td align="center" rowspan="1" colspan="1">33.93 &#x000b1; 5.70</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.05</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Metformin dosage ( mg )</td><td align="center" rowspan="1" colspan="1">1371.42 &#x000b1; 625.21</td><td align="center" rowspan="1" colspan="1">1750 &#x000b1; 379.77</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.01</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Glibenclamid dosage ( mg )</td><td align="center" rowspan="1" colspan="1">10.76 &#x000b1; 7.10</td><td align="center" rowspan="1" colspan="1">13.31 &#x000b1; 6.18</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Enalapril dosage (mg)</td><td align="center" rowspan="1" colspan="1">13 &#x000b1; 4.6</td><td align="center" rowspan="1" colspan="1">23 &#x000b1; 6.6</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.01</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Atorvastatin dosage (mg)</td><td align="center" rowspan="1" colspan="1">15 &#x000b1; 1.88</td><td align="center" rowspan="1" colspan="1">17.5 &#x000b1; 3.65</td><td align="center" rowspan="1" colspan="1">Not significant</td></tr></tbody></table><table-wrap-foot><fn><p> Data are means &#x000b1; SD. </p></fn></table-wrap-foot></table-wrap><p> &#x0200f;We found that Mg supplementation could significantly decrease both systolic (p&#x0003c; 0.001) and diastolic (p&#x0003c; 0.05) blood pressure (<xref ref-type="table" rid="T2">Table 2</xref>). Differences in the blood pressure were significant, even when antihypertensive drugs were taken into account. </p><p> &#x0200f;Urine Mg concentration was also measured, showing a significant increase in the Mg group compared with the placebo group (p&#x0003c;0.05). We measured the Ca/Mg ratio as an indicator for atherosclerosis; and observed that this ratio was higher in the placebo group at the end of the study but the difference between the two groups was not significant. </p><p> &#x0200f;Blood glucose measurements showed reduced FBG and 2hr PP BS levels in the participants of both groups which was significantly lower in the Mg group compared with the placebo group (p&#x0003c;0.0001 for percentage of FBG reduction and p&#x0003c;0.01 for decrease in 2hr PP BS). At the end of the study, the dosage of metformin used in the placebo group was significantly higher than the group treated with Mg (p&#x0003c;0.01). As shown in <xref ref-type="table" rid="T2">table 2</xref>, we also found a significant decrease in plasma LDL C. levels in the Mg treated group compared with the placebo group (p&#x0003c;0.01). After oral Mg supplementation non-HDL cholesterol level showed a significant reduction in the Mg treated group compared with the placebo group (p&#x0003c;0.001). </p><p> &#x0200f;AST levels showed no significant difference, but the interesting finding was related to the ALT levels. In the placebo group ALT levels were rising during the study, but in the Mg treated group these levels remained unchanged (p&#x0003c;0.05). </p></sec><sec sec-type="discussion" id="s5"><title>Discussion</title><p> &#x0200f;We found that oral Mg supplementation could improve glycemic control, blood pressure, and lipid profile in patients with type II diabetes without having any important complications. </p><p> &#x0200f;Since we had previously found beneficial effects in supplementation with oral Mg (as MgSo4) on streptozotocin induced diabetic rats (<xref rid="R12" ref-type="bibr">12</xref>, <xref rid="R13" ref-type="bibr">13</xref>), we designed a double-blind placebo-controlled clinical trial with the best preparation and dosage of Mg according to our previous studies and other investigations. The purpose of this study was to determine whether oral Mg supplementation improves metabolic control, lipid profile, blood pressure, hepatic enzymes, hemoglobin concentration and anthropometric indices, as parameters of diabetes control in patients with type II diabetes. </p><p> &#x0200f;Considering the increasing prevalence of diabetes and its complications throughout the world, researchers are trying hard to discover new prevention and treatment methods for diabetes (<xref rid="R26" ref-type="bibr">26</xref>, <xref rid="R27" ref-type="bibr">27</xref>). Mg supplementation had proven to be effective in controlling type II diabetes (<xref rid="R2" ref-type="bibr">2</xref>), and numerous studies on animal models have found consistent results (<xref rid="R12" ref-type="bibr">12</xref>, <xref rid="R13" ref-type="bibr">13</xref>). Recently, some clinical trials have studied the effectiveness of oral Mg supplementation in controlling type II diabetic patients and found favorable results; although there is not enough data to confirm these benefits yet (<xref rid="R4" ref-type="bibr">4</xref>-<xref rid="R11" ref-type="bibr">11</xref>). </p><p> &#x0200f;Blood glucose measurements in this study indicated that the participants of both groups had reduced FBG and 2hr PP BS levels. This reduction is mainly due to the glucose lowering agents prescribed by the endocrinologist in monthly visits. Although the endocrinologist was blind to the group assignment, reduction of FBG and 2hrPP BS was significantly higher in the Mg supplemented group. To the best of our knowledge, almost all previous clinical trials have failed to show any changes in FBG (<xref rid="R5" ref-type="bibr">5</xref>-<xref rid="R11" ref-type="bibr">11</xref>). This failure in showing beneficial effects of Mg on FBG can be explained by the differences in Mg dosage and duration of supplementation. As in our previous studies on diabetic rat models we found that supplementation with just a dosage of 10 g/L MgSO4 in drinking water could improve glycemic control and the histological structure of the pancreas and aorta (<xref rid="R12" ref-type="bibr">12</xref>). Dosages less than 10 g/L, did not show beneficial effects and dosage more than 10 g/L, could reduce blood glucose but had deleterious effects on the histological structure of the pancreas (<xref rid="R12" ref-type="bibr">12</xref>). </p><p> &#x0200f;Of course, the beneficial effects of Mg on FBG and 2hr PP BS in our study can be explained by their critical role in carbohydrate metabolism and post receptor reactions in insulin receptors (<xref rid="R2" ref-type="bibr">2</xref>). However, the important point is that although glycemic control (FBG &#x00026; 2hr PP BS) was improved, we found no significant difference in HOMA-IR index and FPI levels between the two groups. This finding implies that the beneficial effects of Mg in diabetes control cannot be only due to improvement of insulin sensitivity or insulin secretion. Therefore we think that, one of the main sites of Mg action can be the muscles. It is possible that Mg can help, translocation of glucose transporter number 4 (GLUT 4) to the cell membrane, to take place. </p><p> &#x0200f;On the other hand, it should be kept in mind that in our study both groups were blindly treated by oral hypoglycemic agents. The main used oral hypoglycemic agent was metformin in both groups. Metformin dosage was significantly lower in the Mg treated patients after three months of Mg supplementation compared with the placebo group. </p><p> &#x0200f;The significant decrease in LDL cholesterol in the Mg treated patients in this study infers the beneficial effects of Mg on atherogenic lipid profile, as shown in our previous study in an animal model (<xref rid="R13" ref-type="bibr">13</xref>) and other similar studies (<xref rid="R9" ref-type="bibr">9</xref>, <xref rid="R10" ref-type="bibr">10</xref>). This effect can be explained by the role of Mg in the activity of lipoprotein lipase enzyme as described by Rayssiguire and colleagues (<xref rid="R28" ref-type="bibr">28</xref>). Mg deficiency enhances catecholamine secretion which results in increased lipolysis. Enhancement of lipolysis and subsequent elevation of plasma free fatty acids may lead to an increase in VLDL and TG synthesis and secretion and elevated plasma TG concentration (<xref rid="R28" ref-type="bibr">28</xref>) </p><p> &#x0200f;Our results were in agreement with our previous study, showing significant decrease in systolic, diastolic, and mean arterial blood pressure (<xref rid="R13" ref-type="bibr">13</xref>). These effects result from changes in intracellular ionic environment (<xref rid="R2" ref-type="bibr">2</xref>). When the intracellular Ca/Mg ratio increases as a result of Mg deficiency, vascular smooth muscle tone and smooth muscle cell response to external constrictor stimuli increases, which leads to vasoconstriction and as a result elevation of blood pressure (<xref rid="R2" ref-type="bibr">2</xref>). In our previous studies (<xref rid="R13" ref-type="bibr">13</xref>) we showed that the administration of magnesium can decrease mesenteric vascular bed sensitivity to phenylephrine and decrease Ca/Mg ratio. We also showed that magnesium decrease collagen thickness, intima/media thickness and the lumen/ media ratio in aorta (<xref rid="R13" ref-type="bibr">13</xref>). So it seems that administration of magnesium can decrease blood pressure due to prevent vascular morphological changes and decrease vascular sensitivity to neurotransmitter. ALT levels significantly increases in the placebo group during the study while there was no significant increase in ALT levels in the Mg group. The elevation of hepatic enzymes in the placebo group could be a sign of fatty liver disease. The higher increase in metformin dosage in the placebo group might be responsible for the significant rise of ALT levels in this group compared with the Mg supplemented group. </p><p> &#x0200f;In patients supplemented with Mg, urinary Mg increased significantly compared with the placebo group. Increased urinary Mg without plasma Mg changes may be due to an increase in ionized or intracellular Mg in these patients. </p></sec><sec sec-type="conclusion" id="s6"><title>Conclusion</title><p> &#x0200f;According to the results of our study and the previous ones, we can conclude that not only Mg supplementation can be helpful in diabetes control, but also the effective dosage and duration of supplementation, and the patients who need the supplementation should be considered. Therefore, Mg is also recommended as an inexpensive, easy to use, natural adjuvant therapy for patients with type II diabetic. </p></sec>
Hospitalizations and its related factors in HIV/AIDS patients in Tehran, Iran
<p> <bold>Background:</bold> HIV/AIDS patients are mainly hospitalized for HIV-related diseases and opportunistic infections.ions. This study was performed to determine the causes of hospitalization and its related factors in HIV/AIDS patients in Tehran&#x02019;s Imam Khomeini Hospital during 2009-2012. </p><p> <bold>Methods:</bold> This study was a descriptive cross-sectional study. HIV patients admitted to the Imam Khomeini Hospital were included in the study through census method, during the study. Demographic variables, hepatitis co-infection, CD4 count, history of receiving anti- retroviral therapy (ART), cause of admission, length of hospitalization and patient's outcome were recorded. Data were analyzed by SPSS software and by means of Chisquare and Mann Whitney U tests. </p><p> <bold>Results:</bold> During the study, 555 HIV patients were included in, 84.9% of whom were male, with the mean age of 36.59&#x000b1;8.51 years and the average length of hospitalization for 16.04&#x000b1;18.82 days. Opportunistic infections were the most common cause of hospitalization (46.5%) with prevalent of which was pulmonary tuberculosis being the most prevalent (37.6%). Patients suffering from opportunistic infections had significantly lower CD4 count and longer hospitalization than the other diseases. A significant difference was detected between patients outcome and the history of ART. </p><p> <bold>Conclusion:</bold> Low CD4 count may contribute to an increase in number and length of hospitalization in HIV/AIDS patients. Accordingly, it appears to affect outcome of their treatment and ART was accompanied by a drop in the death rate of hospitalized patients. </p>
<contrib contrib-type="author"><name><surname>Hajiabdolbaghi</surname><given-names>Mahboobeh</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Jafari</surname><given-names>Sirous</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mansouri</surname><given-names>Sedighe</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Hedayat Yaghoobi</surname><given-names>Mojtaba</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Human Immunodeficiency Virus/ Acquired Immunodeficiency Syndrome (HIV/AIDS) is a spreading epidemic that devours more victims day in day out and imposes a lot of cost on national health systems of several countries. According to the latest statistics publishe d by the ministry of Health and Medical Education of Iran, by 2012 a total of 24290 people infected with HIV/AIDS had been identified in the country (<xref rid="R1" ref-type="bibr">1</xref>), and the estimated number of infected people is much higher than this figure. Generally, the rate and length of hospitalization in HIV/AIDS patients is more than other patients. Each hospitalization is costly for patients as well as the health care systems <sup> ( <xref rid="R2" ref-type="bibr">2</xref>) </sup>. Several studies have evaluated the rate and causes of hospitalization among HIV/AIDS patients <sup> (<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R4" ref-type="bibr">4</xref>) </sup>. Causes of hospitalization in this population are mainly due to HIV/AIDS related diseases and or opportunistic infections. With the introduction of Anti-Retroviral Therapy (ART) since 1996 and a combined antiretroviral therapy, the treatment of this disease has been revolutionized (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> A recent studies carried out in the industrialized countries, indicated a decreasing trend in the rate of hospitalization and the causes of hospitalization in HIV patients are showing a changing trend away from opportunistic infections and towards other diseases with the widespread use of antiretroviral therapy (<xref rid="R4" ref-type="bibr">4</xref>-<xref rid="R8" ref-type="bibr">8</xref>). However, increasing side effects in patients must be considered (<xref rid="R7" ref-type="bibr">7</xref>). But it seems that opportunistic infections are the leading cause of hospitalization in the poor and developing countries yet (<xref rid="R9" ref-type="bibr">9</xref>-<xref rid="R15" ref-type="bibr">15</xref>). Age, gender, CD4 count, underlying diseases and prolonged infections were amongst factors leading to the hospitalization of patients (<xref rid="R6" ref-type="bibr">6</xref>,<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R11" ref-type="bibr">11</xref>&#x00026;<xref rid="R16" ref-type="bibr">16</xref>). </p><p> So far, only one study has conducted the causes in hospitalization in HIV/AIDS patients conducted in Iran, retrospectively on 52 patients during 2000 to 2005 (<xref rid="R15" ref-type="bibr">15</xref>). </p><p> This study was performed to determine the causes of hospitalization in HIV/AIDS patients and its related factors in Tehran&#x02019;s Imam Khomeini Hospital during 2009-2012. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> This study was a descriptive cross-sectional in one population which included all hospitalized patients in Imam Khomeini hospital in a four-year period. The sample size of this study embraced through census method in which all HIV/AIDS patients admitted in this Hospital were included in this study. </p><p> The variables in this study included age, gender, underlying diseases, IDU (injection drug user) status, HCV and HBV co-infection, CD4 count, ART history, anemia, thrombocytopenia, cause of admission (based on ICD-10), length of hospitalization and the final outcome of the patients which were obtained from hospital patients records and these information were recorded in the questionnaire prepared beforehand. </p><p> At all stages of the study, confidentiality of information obtained from the hospital records was observed. </p><p> Data were analyzed and classified by SPSS software through which the frequency, mean and standard deviation were calculated. Kolmogorov Smirnov test was used to determine the normal distribution of the quantitative variables and Chi-square, Mann Whitney U tests were applied to analyze the data and compare the studied variables as well. </p></sec><sec sec-type="results" id="s3"><title>Results</title><p> According to the findings of this study of 555 cases of hospitalized HIV/AIDS patients studied. 471 (84.9%) were men and 84 (15.1%) women (male to female ratio 6.5 to 1). Furthermore, 417 cases (75.1%) had a history of IDU. </p><p> The HIV infection had been diagnosed for the first time in 138 cases (24.9%) in their first admissions. </p><p> The mean age of the samples was 36.59&#x000b1;8.51 years (range 16-67 years), and the mean duration of hospitalization was 16.04&#x000b1;18.82 days (range 1-322 days). </p><p> Of all patients 498 (89.7%) were hospitalized in the infectious diseases ward, and 15 (2.7%), 13 (2.3%) and 29 (5.2%) cases were hospitalized in internal medicine, surgery and other wards respectively. </p><p> The frequency of hospitalization of patients was 140 (25.2%), 136 (24.5%), 131 (23.6%), and 148 cases (26.7%) in the years 2009, 2010, 2011, and 2012 respectively. </p><p> The overall mortality rate in HIV/AIDS patients admitted to the hospital in four years course of the study was 101 cases (18.2%), in which 31 (30.7%) were diagnosed with HIV in their recent hospitalization. </p><p> The frequency of mortality in patients was 25 (24.8%), 33 (32.7%), 17 (16.8%), and 26 cases (25.7%) in the years 2009, 2010, 2011, and 2012 respectively. </p><p> Of all the HIV patients in the hospital during the study period, 258 (46.5%) were admitted due to opportunistic infections and 297 cases (53.5%) hospitalized for other illness such as skin and soft tissue diseases in 37 (12.5%), and liver and bile ducts diseases in 35 (11.8%). 15 (2.7%) of all admissions were because of malignancy and 6 cases (1.1%) were hospitalized due to drug reactions. </p><p> The most common opportunistic infections in the samples were pulmonary tuberculosis, brain toxoplasmosis, and Pneumocystis jirovecii pneumonia in 97 (37.6%), 47 (18.2%), and 34 (13.2%) respectively. </p><p> Among the HIV patients 138 (24.9%) were hospitalized due to tuberculosis infection from which, pulmonary TB, miliary TB, TB lymphadenitis, pleural TB, TB meningitis, pericardial TB, TB of larynx, peritonitis TB, genitalia TB, and abdominal TB were seen in 97 (70.3%), 18 (13.0%), 8 (5.8%), 5 (3.6%), 4 (2.9%), 1 (0.72%), 1 (0.72%), 2 (1.44%), 1 (0.72%) and 1 cases (0.72%), respectively. </p><p> Co-infection with hepatitis C and B, was observed in 331 (59.6%) and 74 patients (13.3%) respectively and the mean of CD4+ T lymphocyte was 144.12&#x000b1;181.73 (range 5-1400) on admission. The CD4 count was less than 200 in 386 (75.8%), between200-349 in 58 (11.4%), 350-499 in 33 (5/6%) and equal to or greater than 500 cells per micro liter in 32 (6.3%). The CD4 counts in 46 (8.3% of all cases) were also uncertain. </p><p> 139 patients (30.0%) were treated with ART and the mean of time spent in the receiving ART was 24.49&#x000b1;27.08 months (range 1-108 months). </p><p> The mean platelet count in samples was 183000&#x000b1;102154 (range 240 to 568000) and 226 cases (40.7%) had thrombocytopenia (less than 150,000) on admission. Mean of hemoglobin in samples was 10.48&#x000b1;2.31 gr/dl (range 3-18.5 gr/dl) and 494 cases (89%) were anemic (<xref ref-type="table" rid="T1">Table 1</xref>). </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Demographic and laboratory characteristics of patients admitted with opportunistic infections and other patients enrolled in the study. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Characteristics</td><td align="center" colspan="2" rowspan="1">Cause of admission</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">p </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Opportunistic Infection</td><td align="center" rowspan="1" colspan="1">Other</td><td align="center" rowspan="1" colspan="1">total</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age (year)</td><td align="center" rowspan="1" colspan="1">36.36&#x000b1;7.74</td><td align="center" rowspan="1" colspan="1">36.77&#x000b1;9.21</td><td align="center" rowspan="1" colspan="1">36.59&#x000b1;8.51</td><td align="center" rowspan="1" colspan="1">0.94</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Disease duration (month)</td><td align="center" rowspan="1" colspan="1">31.21&#x000b1;33.71</td><td align="center" rowspan="1" colspan="1">32.47&#x000b1;37.46</td><td align="center" rowspan="1" colspan="1">32.86&#x000b1;34.48</td><td align="center" rowspan="1" colspan="1">0.73</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Admission duration (day)</td><td align="center" rowspan="1" colspan="1">17.77&#x000b1;14.84</td><td align="center" rowspan="1" colspan="1">14.77&#x000b1;21.20</td><td align="center" rowspan="1" colspan="1">16.04&#x000b1;18.82</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.0001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Hb (gr/dl)</td><td align="center" rowspan="1" colspan="1">10.14&#x000b1;2.18</td><td align="center" rowspan="1" colspan="1">10.73&#x000b1;2.38</td><td align="center" rowspan="1" colspan="1">10.48&#x000b1;2.31</td><td align="center" rowspan="1" colspan="1">0.004</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Platelet count (count/&#x000b5;l)</td><td align="center" rowspan="1" colspan="1">184000&#x000b1;101929</td><td align="center" rowspan="1" colspan="1">183000&#x000b1;102477</td><td align="center" rowspan="1" colspan="1">183000&#x000b1;102154</td><td align="center" rowspan="1" colspan="1">0.75</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">CD4 count (cell/&#x000b5;l)</td><td align="center" rowspan="1" colspan="1">82.64&#x000b1;117.35</td><td align="center" rowspan="1" colspan="1">192.83&#x000b1;207.36</td><td align="center" rowspan="1" colspan="1">144.12&#x000b1;181.73</td><td align="center" rowspan="1" colspan="1">&#x0003c;0.0001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">ART history [n (%)]</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">73 (28.3)</td><td align="center" rowspan="1" colspan="1">66 (22.2)</td><td align="center" rowspan="1" colspan="1">139 (30.0)</td><td align="center" rowspan="1" colspan="1"> <break/>0.73 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">85 (71.7)</td><td align="center" rowspan="1" colspan="1">231 (77.8)</td><td align="center" rowspan="1" colspan="1">316(70.0)</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gender [n (%)]</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> Male</td><td align="center" rowspan="1" colspan="1">217 (84.1)</td><td align="center" rowspan="1" colspan="1">250 (84.2)</td><td align="center" rowspan="1" colspan="1">467 (84.1)</td><td align="center" rowspan="1" colspan="1"> <break/>0.96 </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> Female</td><td align="center" rowspan="1" colspan="1">41 (15.9)</td><td align="center" rowspan="1" colspan="1">47 (15.8)</td><td align="center" rowspan="1" colspan="1">88 (15.9)</td><td align="center" rowspan="1" colspan="1"> </td></tr></tbody></table></table-wrap><p> Patients admitted due to the opportunistic infections had significantly lower CD4 counts, longer hospital stay and lower hemoglobin levels in comparison with other causes (p &#x0003c;0.005) (<xref ref-type="table" rid="T1">Table 1</xref>). </p><p> Of 101 deceased HIV/AIDS patients, only 13 (12.9%) had received ART (p=0.006). In 54.4% (55 cases) of the deceased patients, opportunistic infection was the cause of admission (p=0.128) and the most common cause of hospital death in 25 (24.7%) was pulmonary tuberculosis. </p><p> History of IDU, hepatitis C and B co-infection were seen in 79.2%, 49.5% and 16.8% of all HIV/AIDS deaths respectively (p&#x0003e; 0.05). In 74.3% (75 cases) of deceased patients, CD4 count was less than 200 (p=0.01). </p><p> Patients who died had lower CD4 count, hemoglobin levels and platelet counts (p&#x0003c;0.05) (<xref ref-type="table" rid="T2">Table 2</xref>). </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Demographic and laboratory characteristics of patients who died and other patients enrolled in the study. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1"> Characteristics </td><td colspan="2" align="center" rowspan="1">Outcome</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"> p </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"> Death </td><td align="center" rowspan="1" colspan="1">Other</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Age (year) </td><td align="center" rowspan="1" colspan="1"> 37.23&#x000b1;8.40</td><td align="center" rowspan="1" colspan="1"> 35.94&#x000b1;8.12</td><td align="center" rowspan="1" colspan="1"> 0.17</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Disease duration (month) </td><td align="center" rowspan="1" colspan="1"> 27.73&#x000b1;29.98</td><td align="center" rowspan="1" colspan="1"> 31.78&#x000b1;36.96</td><td align="center" rowspan="1" colspan="1"> 0.49 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Admission duration (day) </td><td align="center" rowspan="1" colspan="1"> 12.75&#x000b1;13.64</td><td align="center" rowspan="1" colspan="1"> 17.57&#x000b1;20.44</td><td align="center" rowspan="1" colspan="1"> &#x0003c;0.0001 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Hb (gr/dl) </td><td align="center" rowspan="1" colspan="1"> 9.9&#x000b1;2.21</td><td align="center" rowspan="1" colspan="1"> 10.5&#x000b1;2.26</td><td align="center" rowspan="1" colspan="1"> 0.02 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Platelet count (count/&#x003bc;l) </td><td align="center" rowspan="1" colspan="1">130000&#x000b1;81259 </td><td align="center" rowspan="1" colspan="1">190000&#x000b1;103331</td><td align="center" rowspan="1" colspan="1"> &#x0003c;0.0001 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> CD4 count (cell/&#x003bc;l) </td><td align="center" rowspan="1" colspan="1"> 94.94&#x000b1;122.306</td><td align="center" rowspan="1" colspan="1"> 144.33&#x000b1;186.927</td><td align="center" rowspan="1" colspan="1"> 0.02 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> HAART history [n (%)] </td><td rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> Yes </td><td align="center" rowspan="1" colspan="1"> 13 (12.9) </td><td align="center" rowspan="1" colspan="1">126 (27.8)</td><td align="center" rowspan="1" colspan="1"> 0.006 </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1"> 88 (87.1)</td><td align="center" rowspan="1" colspan="1"> 328 (72.2)</td></tr></tbody></table></table-wrap><p> A significant difference was not found between the duration mean of hospital stay in patients who received ART (15.19&#x000b1;12.50 days) and those without ART (16.32&#x000b1;20.50 days), and also patients with hepatitis C co-infection (17.25&#x000b1;22.19 days) and without hepatitis C co-infection (14.25 &#x000b1; 12.06 days) (p&#x0003e; 0.05). Regression analysis showed no significant relation between hospitalization and risk factors (ART and hepatitis C) (p=0.54 and p=0.6 respectively). </p><p> There was significant difference between the mean duration of hospital stay in patients with CD4 count of less than 200 in comparison with other cases (17.73&#x000b1;20.91 days versus 13.54&#x000b1;13.03 days) (p=0.001). </p><p> 82.6%, 51.4%, and 20.3% of patients in the current admission had CD4 count less than 200, were infected with opportunistic infections and died in the same admission respectively and 93.5% of those patients who received ART had less than 350 CD4 count. </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Our findings showed that the most hospitalized HIV/AIDS patients in the study period were men with a history of IDU and CD4 counts less than 200 (in the advanced stage of AIDS) and the most prevalent cause of admission was opportunistic infections of pulmonary TB as the most common cause. In addition, about a quarter of the patients had a history of receiving ART. </p><p> HIV infection leads to progressive deficiency in cellular immunity and increases risk of opportunistic infections and mortality in these patients (<xref rid="R17" ref-type="bibr">17</xref>). By increasing viral load and reducing CD4 count, HIV-positive individuals are increasingly susceptible to opportunistic infections that may lead to hospitalization (<xref rid="R18" ref-type="bibr">18</xref>). A clear reduction in mortality and rates of hospitalization of HIV/AIDS patients have occurred with the availability of antiretroviral therapy in the industrialized countries since 1996. The majority of former studies have revealed the relationship between applying ART and the reduction of mortality in these patients (<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R9" ref-type="bibr">9</xref>,<xref rid="R16" ref-type="bibr">16</xref>&#x00026;<xref rid="R19" ref-type="bibr">19</xref>). Currently the leading cause of death in the industrialized countries has shifted from opportunistic infections to chronic diseases in HIV/AIDS patients (<xref rid="R6" ref-type="bibr">6</xref>,<xref rid="R20" ref-type="bibr">20</xref>-<xref rid="R24" ref-type="bibr">24</xref>). Meanwhile, due to less availability of ART in poor and developing countries, it seems that opportunistic infections remain the major cause of hospitalization and death in these patients (<xref rid="R9" ref-type="bibr">9</xref>,<xref rid="R10" ref-type="bibr">10</xref> &#x00026;<xref rid="R15" ref-type="bibr">15</xref>). </p><p> In our study only quarter of patients had history of ART due to the fact that a high percentage of them were in advanced stage of AIDS (CD4 count at 75.8% of samples were less than 200), this would reveal the lack of convenient access to ART and also show delayed presentation or late diagnosis in patients in Iran. However in some previous studies, a quarter of patients were under ART too (<xref rid="R16" ref-type="bibr">16</xref>). In our study 24.9% of patients were diagnosed with HIV/AIDS in current admission, this figure in the conducted study in England was 17% (<xref rid="R25" ref-type="bibr">25</xref>). Some previous studies also have reported late presentation and late diagnosis in people with HIV/AIDS in different populations (<xref rid="R13" ref-type="bibr">13</xref>,<xref rid="R16" ref-type="bibr">16</xref>,<xref rid="R26" ref-type="bibr">26</xref>-<xref rid="R28" ref-type="bibr">28</xref>). The interesting point in our study was that CD4 counts were less than 200 in 82.6% of patients who diagnosed with HIV/AIDS in current hospitalization. This means that a majority of our patients were diagnosed in the advanced stages of AIDS, whereas in the conducted study in the UK this figure was 56.3% (<xref rid="R25" ref-type="bibr">25</xref>). It seems that the disease screening is still a very important topic in our country and especial consideration and wide educational services are necessary to promote community awareness about this disease and its transmission. Nonetheless, two points are worth mentioning; first, since Imam hospital is a sub-specialty and a reference hospital in the country, a high percentage of admitted patients in advanced stage of AIDS may be justified; the next point is the high figure of IDU among hospitalized patients (75.1%) which can justify their being more in advanced disease stages and more hospitalization rate in these patients. Previous studies also have shown a meaningful relationship between unemployment, poor hygienic, and IDU with hospitalization rate in patients with HIV/AIDS (<xref rid="R18" ref-type="bibr">18</xref>). </p><p> Tuberculosis is the most common opportunistic infection in HIV/AIDS patients in a formerly published study in Iran, but CD4 count was not regarded in that study (<xref rid="R15" ref-type="bibr">15</xref>). </p><p> In the present study, the mean hospital stay was 16.04 days which is partly in consistent with the mean of hospital stay in the carried out study in Cameroon (14 days) (<xref rid="R11" ref-type="bibr">11</xref>) and is less than the reported mean hospital stay in Ethiopia (<xref rid="R10" ref-type="bibr">10</xref>), it is more than the reported mean hospital stay in studies conducted in industrialized countries (<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R14" ref-type="bibr">14</xref>,<xref rid="R16" ref-type="bibr">16</xref>&#x00026; <xref rid="R18" ref-type="bibr">18</xref>). It seems that high percentage of advanced AIDS cases in our study may help explain the longer duration of their hospital stay .There might be other factors influencing the length of hospitalization for which further studies have to be taken into consideration. </p><p> There were not any significant differences between the mean duration of hospital stay in HIV/AIDS patients receiving ART (15.19 days) and those who did not (16.32 days) (p=0.7). While in the Vidigal et al study (<xref rid="R5" ref-type="bibr">5</xref>) the mean duration of hospital stay in patients not receiving ART was reported 17.5 days in comparison with 7.7 days in the group receiving ART. </p><p> The mean age of our samples was 36.59 years which was less than reported figures in the United states (<xref rid="R6" ref-type="bibr">6</xref>)and Canada (<xref rid="R16" ref-type="bibr">16</xref>) (40 and 38 years respectively), but it was higher than the reported mean age (32.8 years) in Ethiopia (<xref rid="R16" ref-type="bibr">16</xref>). </p><p> In Vidigal et al study the mean age of the patients who received ART was 34 years versus 30.6 years in those who did not (<xref rid="R5" ref-type="bibr">5</xref>). </p><p> It appears that the application of ART as well as the reduction of mortality has boosted life expectancy resulting in higher mean age in patients in countries such as United States and Canada. </p><p> The majority of the study subjects were males (84.9%). This figure is in consistent with the majority of formerly conducted studies (6,12&#x00026; 18), particularly the one in Iran (<xref rid="R15" ref-type="bibr">15</xref>). </p><p> The most prevalent cause of hospitalization in our samples was opportunistic infections (46.5%), and with pulmonary tuberculosis as the highest rate (37.6%), also patients with opportunistic infections significantly had lower CD4 count and longer duration of hospital stay (p=0.000). These findings are in completing consistent with the results of a former study conducted in our country (<xref rid="R15" ref-type="bibr">15</xref>). In studied conducted in Ethiopia (<xref rid="R10" ref-type="bibr">10</xref>) and India (<xref rid="R13" ref-type="bibr">13</xref>) tuberculosis was also the most prevalent cause of hospitalization in patients, while in performed studies in the industrialized countries, causes of admission showed a trend away from AIDS-related diseases (<xref rid="R6" ref-type="bibr">6</xref>,<xref rid="R16" ref-type="bibr">16</xref>). Non AIDS-related diseases have risen at 42% in the study performed in Canada (<xref rid="R16" ref-type="bibr">16</xref>) and the rate of hospitalization due to opportunistic infections was reduced from 31% in the years 1994-1996 to 9.5% in the years 2003-2005 in a study conducted in the United States (<xref rid="R6" ref-type="bibr">6</xref>). Based on our findings in comparison with previous studies in other countries, widespread application of ART and the use of new drugs with greater efficacy as well as identification patterns of drug resistance in our country are absolutely necessary and tangible. </p><p> In our study 18.2% of patients who were admitted with HIV/AIDS died and the most prevalent cause of death was opportunistic infections (54.4%), and only 12.9% of deceased cases were receiving ART (p=0.006). This figure in comparison to 31% and 27% of reported mortality in studies conducted in Ethiopia (<xref rid="R10" ref-type="bibr">10</xref>) and Cameron (<xref rid="R11" ref-type="bibr">11</xref>) is much less, but our findings were much higher than 7% overall mortality rate which reported by Floris-Moore et al (<xref rid="R8" ref-type="bibr">8</xref>) during the years 1996 to 2000 in the United States. Meanwhile, the number of the deceased patients due to opportunistic infections during the Floris-Moore et al study period was reduced from 40 to 27 percent (<xref rid="R8" ref-type="bibr">8</xref>) and their results showed that patients who received ART had a lower risk of hospitalization. Thus, once again, this puts emphasize on the development of the use of ART in Iran. </p><p> Hepatitis C co-infection was seen in 59.6% of our samples, although the mean of hospital stay was higher in patients with HCV co-infection compared to the group without (17.25 versus 14.25 days), but there was no significant difference between the two groups (p&#x0003e;0.05). Falster et al (<xref rid="R7" ref-type="bibr">7</xref>) and Vidigal et al (<xref rid="R5" ref-type="bibr">5</xref>) noted the co-infection of hepatitis C as an independent factor which increases hospitalization and length of hospital stay in HIV/AIDS patients. </p><p> Our limitations include, lack of data on patients who were admitted because of mental illness since they referred to other centers, and unavailability CD4 count in some patients. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Based on our findings, the majority of admitted patients to Imam Khomeini hospital were male with IDU history in that the opportunistic infections were the most prevalent cause of admission. </p><p> Low CD4 count may contribute to an increase in number and length of hospitalization in HIV/AIDS patients. Accordingly, it appears to affect outcome of these patients. ART was accompanied by a drop in the death rate of hospitalized patients; however, a further study should be carried out into an intermediate variable which contributes to this fact. </p><p> Also, in further studies, various anti-retroviral therapies along with their effects should be compared. Moreover, the impact of the new generation drugs on the cause, and length of hospitalization, as well as the outcome of the patients is capable of being taken into account. Furthermore, we could conduct a research on educating, and promoting the public awareness about AIDS as well as the dire role of controlling the IDU, which are the leading means of transmission of HIV in the country, to decline the incidence of opportunistic infections. </p></sec>
Association of Dowling-Degos disease and multiple seborrheic keratosis in a "Christmas tree pattern"
<p>Dowling-Degos disease is a rare sporadic or autosomal dominant pigmentary entity, in which clusters of papules and reticulate macules slowly develop with predominance in flexural regions. This entity is due to mutations in the keratin 5 gene, and is related with other cutaneous disorders. We report the sporadic form of Dowling-Degos disease in an elderly man with multiple seborrheic keratosis in a "Christmas tree" pattern. Worthy of note in this case study is the lesions evolved for over than 30 years. The aim is to describe the association of these keratoses with Dowling-Degos disease in a healthy man.</p>
<contrib contrib-type="author"><name><surname>dos Santos</surname><given-names>Vitorino Modesto</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Pereira</surname><given-names>Nayanne Lays dos Santos</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Silva</surname><given-names>Renata Faria</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Silva</surname><given-names>Fabio Henrique de Oliveira</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Garcia</surname><given-names>Cacilda Joyce Ferreira da Silva</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Sousa</surname><given-names>Maria Aparecida Alves de Figueiredo</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Dowling-Degos disease or reticular pigment anomaly of flexures is a rare autosomal dominant genodermatosis clinically characterized by the gradually progressive development of symmetrical reticulate pigmentation of the flexures, perioral comedone-like lesions and pitted scars (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R7" ref-type="bibr">7</xref>). Classical changes can appear in childhood, but adult onset is more common, usually in the third or fourth decade of life, affecting both genders with predominance of 2:1 in females (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R7" ref-type="bibr">7</xref>). The &#x0201c;Christmas tree&#x0201d; pattern may be found in skin lesions of exanthematic psoriasis, Kaposi sarcoma, mycosis fungoides, pityriasisrosea, and secondary syphilis (<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R9" ref-type="bibr">9</xref>). The aim is to describe multiple seborrheickeratosis in a &#x0201c;Christmas tree&#x0201d; pattern affecting a healthy man with Dowling-Degos disease. </p></sec><sec id="s2"><title>Case report</title><p> A 75-year-old afro descendent man was hospitalized with transient ischemic attack (TIA). Because of diabetes mellitus, arterial hypertension, and antecedent ischemic cerebrovascular events, he was taking captopril, metformin, indapamide, and simvastatin. Physical examination, showed perioral acneiform pits and comedones, and symmetric bilateral pigmented papules in the flexures (neck, axilla, and groin) and around the ears. There were thoracodorsalmacules with a &#x0201c;Christmas tree&#x0201d; pattern, with no remarkable skin changes in extremities (<xref ref-type="fig" rid="F1">Fig. 1</xref>). He was 45-years-old at the onset of lesions, which slowly appeared in the groins and axillae and after affected ears and neck. Over three decades, the pigmented changes had progressed and evolved asymptomatic, except for rare pruritus. He wondered that the skin papules were associated with his natural ageing; and the pigmented macules over the trunk as a consequence of his afro descendent origin. Otherwise, he observed that the main development of hyperkeratotic papules occurred in areas of friction. He denied family history of similar lesions. During the last two years, he had been under clinical outpatient surveillance. Laboratory tests and imaging studies were done to discard the hypothesis of eventual unsuspected malignancy. Routine controls, tumor markers, gastrointestinal endoscopy, and chest images were normal. The histopathology study of flexural lesions showed keratin plugs within thedilated follicularostium of pseudocysts from seborrheic papules, increased melanin in the basal membrane, melanophages in the superficial dermis, melanocytes in the granular layer, and thin branches of epidermal downgrowths in the upper dermis (<xref ref-type="fig" rid="F2">Fig. 2</xref>). The clinical features and microscopic findings were consistent with the diagnosis of Dowling-Degos disease coexistent with seborrheic keratosis. Taking in account the absence of any consistent finding of internal malignancy or HIV infection, he was discharged to home after improvement of the neurological manifestations related to TIA. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-68-g001"/><statement><p>Bilateral perioral acneiformcomedones and pits, symmetric pigmented papules in the flexural regions (neck, axillae, and groins) and around the ears. There were thoracodorsal dark macules presenting a &#x0201c;Christmas tree&#x0201d; pattern, and no remarkable skin changes in extremities.</p></statement></fig><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-68-g002"/><statement><p> (Histopathology features of specimens from the flexural lesions). A: Seborrheic keratosis associated with epidermal downward filiform elongations (arrows) to the upper dermis (HE, x20); B: Keratin plugs within dilated follicules (arrowheads) appearing like horny pseudocysts (HE, x20); C: Conspicuous deposits of melanin (arrows) in the cells of basal layer (HE, x40); and D: Increased number of melanophages (arrows) in the upper dermis (HE, x40).</p></statement></fig></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Dowling (1938) and Degos (1954) were the first to describe the type of reticular pigmentation called dark dot disease, Dowling-Degos disease, or reticular pigment anomaly of flexures (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R5" ref-type="bibr">5</xref>-<xref rid="R7" ref-type="bibr">7</xref>). This entity is due to mutations in the keratin 5 gene, and is related with other cutaneous disorders. Acropigmentation of Dohi, Dowling-Degos disease, Galli-Galli disease, and reticulate acropigmentation of Kitamura, can be overlapped and may be variants of the same entity (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R3" ref-type="bibr">3</xref>-<xref rid="R6" ref-type="bibr">6</xref>). Associated conditions include epidermal cysts, hidradenitissuppurative, keratoacanthoma, pilonidal cysts, seborrheic keratosis and squamous cell carcinoma (<xref rid="R2" ref-type="bibr">2</xref>-<xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R6" ref-type="bibr">6</xref>). These phenomena are indicative of the role played by simultaneous underlying defects of follicular proliferation (<xref rid="R5" ref-type="bibr">5</xref>). The pigmented changes typically follow a slow and usually asymptomatic course (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>), with symmetrical growth of the lesions over several years, and worsening after sunlight exposition (<xref rid="R2" ref-type="bibr">2</xref>). Confluence of papules in areas of friction, and episodes of mild pruritus may be observed (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R4" ref-type="bibr">4</xref>). The changes can appear with diverse tones of brown, black or blue discoloration, and in addition to flexures, the round to oval lesions may affect the face, chest, perineum, or extremities (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R4" ref-type="bibr">4</xref>). Histopathology of Dowling-Degos disease include adenoid or reticulated type of seborrheic keratosis with epidermal thickening of basaloid cells, increased deposits of melanin in the basal layer, numerous melanophages in the papillary dermis, epidermal rete ridges with downward filiform elongation (&#x0201c;antler-like&#x0201d;), and dilated follicles appearing as keratin-filled cysts (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R7" ref-type="bibr">7</xref>). This old Brazilian man presented a longstanding development of a reticular pigment anomaly of flexures coexistent with seborrheic <underline>keratoses</underline>, in addition to numerous hyperchromic macules on his back mimicking the &#x0201c;Christmas tree&#x0201d; pattern. An initial concern should be about the eventual paraneoplastic significance of this finding. Progressive seborrheic keratosis are common findings among patients of elderly groups, and may be found in individuals with the diagnosis of Dowling-Degos disease (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R6" ref-type="bibr">6</xref>). Because of the esthetically consequences, patients frequently search for interventional strategies (<xref rid="R5" ref-type="bibr">5</xref>); although medical treatments are not effective to control their cutaneous changes, ablative laser therapy constitutes a successful option (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R6" ref-type="bibr">6</xref>,<xref rid="R7" ref-type="bibr">7</xref>). The thoracodorsal changes with &#x0201c;Christmas tree&#x0201d; distribution could be due to a Wolf isotopic response, for example associated with an undiagnosed episode of juvenile or early-adult pityriasis rosea (10). In conclusion, the elderly man herein reported had characteristic changes of sporadic Dowling-Degos disease, which coexisted for over than 30 years with hyperpigmented thoracodorsal lesions presenting a &#x0201c;Christmas tree&#x0201d; pattern. Although with inherent weaknesses of single case studies, the present report of concomitant uncommon conditions may contribute to better knowledge about their pathogeneses still unclear. </p></sec>
Rare variants of cutaneous leishmaniasis presenting as eczematous lesions
<p>Cutaneous Leishmaniasis may present with clinical presentation such as zosteriform, sporotrichoid and erysipeloid. The eczema variant has rarely been reported. We report a 27- year- old patient with atypical cutaneous leishmaniasis resembling eczema on the hand of a man in Yazd province in the central of Iran.</p>
<contrib contrib-type="author"><name><surname>Ayatollahi</surname><given-names>Jamshid</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Fattahi Bafghi</surname><given-names>Ali</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Shahcheraghi</surname><given-names>Seyed Hossein</given-names></name></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Cutaneous Leishmaniasis is a common protozoan disease, caused by Leishmania; and it is an important public &#x02013; health problem in Iran (<xref rid="R1" ref-type="bibr">1</xref>). In its most common clinical picture it presents as nodules, papules or nodoloulcerative lesions. Unusual clinical presentations have been reported occasionally and include annular, sporotrichoid, palmoplantar, erysipeloid, whitlow, paronychial and impetigo- form (<xref rid="R2" ref-type="bibr">2</xref>-<xref rid="R4" ref-type="bibr">4</xref>). We present a patient with eczema form, a very rare and chronic variant of cutaneous leishmaniasis. </p></sec><sec id="s2"><title>Case Report</title><p> A 27- year &#x02013; old man was referred to our clinic with a 3- month history of an exudatng lesion on the hand (<xref ref-type="fig" rid="F1">Fig. 1</xref>). It had started as a small insect-bite-like lesion and progressed slowly. He denied any history of burns, trauma, drug intake or allergic disorder. </p><fig id="F1" orientation="portrait" position="float"><label> Fig.1 </label><graphic xlink:href="MJIRI-28-71-g001"/><statement><p>Lesion on the posterior of hand</p></statement></fig><p> The patient was an army soldier. There was no history of a similar disease in the patient and his family. He alsohad notany history oftuberculosisorcontact withtubercular patients. The patient denied risk factors associated with HIV and also reported no chills, fever, pain or constitutional symptoms. </p><p> The total blood count, CRP, Erythrocyte sedimentation rate (ESR), FBS, and intradermal purified protein derivative (PPD) skin test and HIV serology were all normal. </p><p> On examination, there was a crusted plaque on the posterior surface of the hand. The plaque had dirty- brown crust and multiple papulopustules. There was no lymph node or palpable lymphatic cord. The clinical picture was consistent with eczema. Previous treatments icluding steroid, antihistamine and antibiotics failed to heal the lesion and its slow progression. </p><p> Special stains and cultures were negative for acid- fast bacteria, fungi, and other bacteria. Because thepatientwas residingin an endemic area ofdisease, wasaskedtoperform cutaneous leishmaniasis test and touch preparations stained with Wright &#x02013; Giemsa preparation were positive for leishmania. </p><p> The patient was treated with meglumineantimoniate (Glucantime), pentavalent antimony, at a dosage of 20 mg/kg per day intra-muscularly for 20 days (treatment administered by center for control diseases of Iran). After completion of therapy, the lesion had partially healed, and after 3 months, the ulcer healed completely. The side effect of meglumineantimoniate was mild arthralgias, myalgias, and pain at the injection site but otherwise tolerated the medication well. </p></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Cutaneous Leishmaniasis is caused by obligate intracellular protozoa of the genus leishmania. Rodents and canids are as common reservoir hosts and humans as incidental hosts (<xref rid="R1" ref-type="bibr">1</xref>). The vectors are sandflies of the genus phlebotomus in the old world. The incubation period ranges from a week to many months. Lesions typically appear on exposed areas of the body. The first manifestation is usually a papule at the site of the sandfly bite, which progressively increase in size and eventually ulcerate. Multiple primary lesions, regional adenopathy, sporotrichoid form, Zoster- form, impetigo &#x02013; form, erysipeloid, and whitlow- form are variably present (<xref rid="R4" ref-type="bibr">4</xref>-<xref rid="R7" ref-type="bibr">7</xref>). Here, we described a case of cutaneous leishmaniasis showing unusual presentation, resembling eczema on the hand with a response to treatment with meglumineantimoniate (<xref rid="R8" ref-type="bibr">8</xref>). The precise pathogenesis of the eczema form of the cutaneous leishmaniasis has been poorly documented. The clinical manifestation in cutaneous leishmaniasis depends on the infecting Leishmania species and host immune response, which is largely mediated through cellular immunity. Other factors include the site of infection, the number of parasites inoculated and nutritional status of the host. However, in the eczematous form of the cutaneous leishmaniasis, one factor could be the epidermal invasion by Leishmania causing an intense cell- mediated immune response leading to severe inflammatory and eczematous changes (<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R9" ref-type="bibr">9</xref>). </p><p> In our report the large size and the eczematous appearance of the lesion was in itself very rare, because there was no primary nodule or plaque. Our patient was an otherwise healthy, young adult with no history of other skin or systemic disease or atopy. Whether the eczematous appearance resulted from an atypical Leishmania strain or from lack of response or a specific immune response is not clear (<xref rid="R10" ref-type="bibr">10</xref>). </p><p> In another report a very rare case of bilateral and symmetrical cutaneous leishmaniasis was presented as eczema-like eruptions with localization exclusively on dorsal aspect of both hands (<xref rid="R11" ref-type="bibr">11</xref>). In another study, a 60-year-old man was presented with ulcerated infiltrative plaques over his face. The diagnosis was confirmed to be cutaneous leishmaniasis as eczema-like eruptions by histological examination and polymerase chain reaction assay of the skin biopsy. In our study the eruptions were on the hand (<xref rid="R12" ref-type="bibr">12</xref>). </p></sec><sec sec-type="conclusion" id="s4"><title>Conclusion</title><p> In endemic areas or in cases with recenet travel to endemic areas, it is necessary for the physician to be aware of atypical skin lesion and it should be investigated for cutaneous leishmaniasis. </p></sec>
Evaluation of the relationship between Helicobacter Pylori infection and Hyperemesis Gravidarum
<p> <bold>Background:</bold> Hyperemesis gravidarum (HEG) is one of the many problems during pregnancy; its etiology has not been clearly understood. Inflammatory factors like helicobacter pylori infection has been considered as a risk factor in some studies. The purpose of the present study is to find a relationship between Helicobacter Pylori (H.P) infection and hyperemesis gravidarum (HEG). </p><p> <bold>Methods:</bold> A case control study was performed on two groups of pregnant women who were in the first trimester of their pregnancies. Case group were pregnant women with hyperemesis gravidarum and control group were pregnant women with the same gestational age but without hyperemesis gravidarum. In both groups, IgG for H.P was measured and compared between the 2 groups. </p><p> <bold>Results:</bold> Totally, 175 pregnant women were evaluated; 78 women with HEG and 97 without. Both groups had no statistically significant difference according to age, gestational age, gravidity, and body mass index (BMI). 51 women out of 78 (65.4%) in HEG group and 43 women (44.3%) in the control group were IgG positive for HP, which showed a significant difference (p=0.005); OR= 2.37, CI 95%= 1.28-4.38. Also, mean serum level of IgG was higher in the HEG group (42.1 &#x000b1; 3.75 VS 32.6 &#x000b1; 3.65, p= 0.05). Between the different variables of age, gestational age, gravidity and HP infection, only HP infection was found as a risk factor for HEG using logistic regression model (p=0.011); OR= 2.522, CI 95%= 1.23-5.14. </p><p> <bold>Conclusion:</bold> HP infection is higher in HEG cases and may be considered as its risk factor. </p>
<contrib contrib-type="author"><name><surname>Kazemzadeh</surname><given-names>Mashaallah</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Kashanian</surname><given-names>Maryam</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Baha</surname><given-names>Bita</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Sheikhansari</surname><given-names>Narges</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Nausea and vomiting is a common and unpleasant problem during pregnancy with a frequency of 75% to 80% of pregnancies (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R2" ref-type="bibr">2</xref>). </p><p> It usually starts between first and second missed menstrual period and can continue up to 14-16 weeks of pregnancy (<xref rid="R1" ref-type="bibr">1</xref>). </p><p> In some women nausea and vomiting is very severe and does not respond to simple diet manipulation and antiemetic agents. This culminates in dehydration, electrolytic imbalance and starvation ketosis and is called as hyper emesis gravid arum (HEG). </p><p> The frequency of HEG is about one in 200 to 1.5% of pregnancies (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R3" ref-type="bibr">3</xref>) and although its definition has not yet been standardized, the accepted clinical pattern includes persistent vomiting, dehydration, ketosis, electrolyte imbalance and weight loss (more than 5% of body weight) (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> A scoring system of Pregnancy- Unique Quantification of Emesis and Nausea (PUQE) has been used for its evaluation (<xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R5" ref-type="bibr">5</xref>). </p><p> The exact etiology of HEG is not clearly defined, but it can be considered as a multi factorial problem (<xref rid="R6" ref-type="bibr">6</xref>), which is under investigation (<xref rid="R7" ref-type="bibr">7</xref>). A relationship between HP and HEG has recently been reported (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R9" ref-type="bibr">9</xref>). Chronic infection with HP has been reported to have a role in producing HEG in a study (<xref rid="R8" ref-type="bibr">8</xref>). In this study 61.8% of pregnant women with HEG had positive HP genome, while 27.6% of pregnant women without HEG had this genome. The researchers concluded that chronic infection with HP should be considered as an important factor in the pathogenesis of HEG, even if it is not the sole factor (<xref rid="R8" ref-type="bibr">8</xref>). </p><p> Usual nausea and vomiting of pregnancy is normally accompanied with improvement in the pregnancy outcome including less abortion, preterm labor and still birth, and also, less instance of low birth weight, intrauterine growth restriction (IUGR) and fetal mortality (<xref rid="R2" ref-type="bibr">2</xref>). In contrast HEG is accompanied with more maternal complications including splenic avulsion, esophageal rupture, Mallory - Weiss tears, pneumo thorax, peripheral neuropathy, and preeclampsia, and also more IUGR and fetal mortality. </p><p> HP is a gram-negative bacterium, which is colonized in the gastric mucosa and causes an increased production of reactive oxygen species (ROS), and decreased plasma antioxidant like ascorbic acid (<xref rid="R9" ref-type="bibr">9</xref>). HEG may be considered as an oxidative stress state, which is determined, by high ROS activity and low antioxidant state. </p><p> Goldberg et al (<xref rid="R10" ref-type="bibr">10</xref>) performed a systematic review on 14 case- control studies about the relationship between HP and HEG in 2007. They concluded that the study does show a relationship, however, heterogeneity between studies has limited the results. In contrast in a study by Jacobson et al (<xref rid="R11" ref-type="bibr">11</xref>), a relationship between HP and HEG was not reported and therefore, there is no consensus on the role of HP on the genesis of HEG. </p><p> The purpose of the present study is to evaluate a relationship between HP and HEG. Regarding to the fact that in different studies, having employment, age, maternal BMI, psychogenic factors and race have been found as confounding factors (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R11" ref-type="bibr">11</xref>), in the present study, the women were considered to be homogenous and confounding factors has been omitted using logistic regression model. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> The study was conducted as a case control study on pregnant women who referred to the prenatal clinic of Akbarabadi Teaching Hospital in Tehran, Iran, between October 2009- March 2010. </p><p> Women with HEG were the case group and the control group was women without. Sampling was performed as a convenient and non-random sequential sampling. Inclusion criteria were gestational age of 6-16 weeks of pregnancy (according to a reliable LMP and ultrasound confirmation), singleton, wanted pregnancy, being housewife, and BMI between18.5-24.9. </p><p> Exclusion criteria included molar pregnancy, history of any systemic disorder or drug use except ordinary supplementation (i.e. folic Acid), history of any gastro intestinal (GI) disorder or GI problems, smoking or drug abuse, hyperthyroidism, and known psychological problems. </p><p> All women had a low socioeconomic condition and the study was performed in a public hospital in downtown Teheran with mostly low income habitants. </p><p> A sample size of 90 in each group was considered sufficient in order to obtain a power of 90% (&#x003b1;= 0.05,1-&#x003b2;= 0.085) with a significance level of 5%. </p><p> <disp-formula id="m1"><mml:math id="n1"><mml:mrow><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn><mml:mo>&#x000d7;</mml:mo><mml:msup><mml:mrow><mml:mo stretchy="false">[</mml:mo><mml:msub><mml:mi>z</mml:mi><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:mn>1</mml:mn><mml:mo>&#x02212;</mml:mo><mml:mi>a</mml:mi><mml:mo>/</mml:mo><mml:mn>2</mml:mn><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>z</mml:mi><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:mn>1</mml:mn><mml:mo>&#x02212;</mml:mo><mml:mi>&#x003b2;</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:msub><mml:mo stretchy="false">]</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:msup><mml:mi>s</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:mrow><mml:mrow><mml:msup><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:msub><mml:mi>&#x003bc;</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>&#x02212;</mml:mo><mml:msub><mml:mi>&#x003bc;</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mo stretchy="false">)</mml:mo></mml:mrow><mml:mn>2</mml:mn></mml:msup></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula> </p><p> Written informed consent was obtained from all participants who were fully informed about the study and advised that the study had no extra expense for participants. </p><p> HEG was defined as vomiting of more than 3 times per 24 hours plus weight loss of more than 3 Kg and keton in the urine, and the score of PUQE of more than 13. PUQE is a scoring system for quantifying of the severity of nausea and vomiting which is based on 3 physical signs. These include nausea, vomiting and retching and its validity has been confirmed (<xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R5" ref-type="bibr">5</xref>, <xref rid="R12" ref-type="bibr">12</xref>, <xref rid="R13" ref-type="bibr">13</xref>). Duration of nausea) hour), the number of the episodes of retching and vomiting, during 24 hours, would be evaluated (<xref rid="R5" ref-type="bibr">5</xref>, <xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R12" ref-type="bibr">12</xref>, <xref rid="R13" ref-type="bibr">13</xref>). Total score is between 3-15, in which score of 3-6 is mild, 7-12 is moderate and 13-15 is considered as severe (<xref rid="R6" ref-type="bibr">6</xref>). </p><p> Twomili liter bloods was obtained from all eligible participants for measurement of HP antibody at the time of the entrance the study and thenIgG was determined by ELIZA (Radin, K5HPG, Italy). </p><p> Serum level of IgG of more than 20 Iu/ml was considered as positive and serum level of less than 15 Iu/ml was considered negative. The serum level between15-20 was considered suspicious and repeated 2-4 weeks later. If the second titer of IgG was less than 15 Iu/ml, it was considered as negative, and if it was higher than 20 Iu/ml, it was considered positive and the samples, which were between 15-20 Iu/ml, were removed from the study (excluded from the study). </p><p> The serum level of IgG, and also, positive or negative results of the tests were recorded in order to compare. The obtained data were analyzed using SPSS 17. Chi-square test, student t- test in the case with normal distribution and non-parametric tests for the cases without normal distribution were used for analysis. Odd ratio was calculated to determine the relationship between HP and HEG. In order to omit the effects of confounding factors, logistic regression model was used. P value of less than 0.05 was considered significant. </p></sec><sec sec-type="results" id="s3"><title>Results</title><p> 175 pregnant women entered the study of whom, 78 were in the HEG group and 97 women were in the control group (without HEG). </p><p> The groups showed no significant difference according to age, gestational age, gravidity and BMI (<xref ref-type="table" rid="T1">Table 1</xref>). </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> The characteristics of women of both groups. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td colspan="2" rowspan="1">Characteristics</td><td align="center" rowspan="1" colspan="1">HEG group n=78</td><td align="center" rowspan="1" colspan="1">No HEG group n=97</td><td align="center" rowspan="1" colspan="1">p</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age (year)M&#x000b1; SD</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">28.02 &#x000b1; 5.8</td><td align="center" rowspan="1" colspan="1">27.3 &#x000b1; 5.4</td><td align="center" rowspan="1" colspan="1">0.423</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gravidityn (%)</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">30 (38.5%)</td><td align="center" rowspan="1" colspan="1">41 (42.3%)</td><td align="center" rowspan="1" colspan="1">0.852</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">28 (35.9%)</td><td align="center" rowspan="1" colspan="1">34 (35.1%)</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x02265;3</td><td align="center" rowspan="1" colspan="1">20 (25.6%)</td><td align="center" rowspan="1" colspan="1">22 (22.7%))</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Gestational age (weeks) M&#x000b1; SD</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">11.5 &#x000b1;2.3</td><td align="center" rowspan="1" colspan="1">11.6 &#x000b1;2.1</td><td align="center" rowspan="1" colspan="1">0.870</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> BMI (kg/m<sup>2</sup>)M&#x000b1; SD </td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">22.4 &#x000b1;5.3</td><td align="center" rowspan="1" colspan="1">23.1&#x000b1; 4.7</td><td align="center" rowspan="1" colspan="1">0.606</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Positive IgGn (%)</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">51 (65.4%)</td><td align="center" rowspan="1" colspan="1">43 (44.3%)</td><td align="center" rowspan="1" colspan="1">0.005</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Serum level of IgG M&#x000b1; SD</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">42.1 &#x000b1;3.75</td><td align="center" rowspan="1" colspan="1">32.6&#x000b1; 3.65</td><td align="center" rowspan="1" colspan="1">0.05</td></tr></tbody></table></table-wrap><p> In the HEG group, 51 cases (65.4%) were positive for HP IgG, against 43 cases (44.3%) in the control group which showed a significant difference (<xref ref-type="table" rid="T1">Table 1</xref>) (OR= 2.27, CI95%= 1.28-4.38). Also, serum IgG level was significantly higher in the HEG group (<xref ref-type="table" rid="T1">Table 1</xref>). </p><p> Using logistic regression stepwise model, age (OR= 0.982, CI95%= 0.917-1.0450, p=0.591), gestational age (p=0.951, OR= 1.005, CI 95%= 0.852-1.186), gravidity 1 (p=0.430, OR= 0.569, CI 95%=0.0141-2.306), gravidity 2 (p=0.359, OR= 0.527, CI 95%= 0.234- 2.069), gravidity 3 or more (p=0.220, OR= 0.298, CI 95%= 0.091-1.334), did not show any relationship with HEG and only HP had correlation with HEG (p=0.011, OR= 2.522, CI 95%=1.236-5.143). </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> In the present study, positive tests of HP IgG in the cases of HEG was higher and confounding factors like age, gestational age, BMI, number of previous pregnancies did not show correlation with HEG, and only HP showed a significant correlation. </p><p> In a study by Jacobson et al (<xref rid="R11" ref-type="bibr">11</xref>), correlation between sero positive cases of HP and HEG has not been shown, while, age, and race showed significant correlation with HEG. </p><p> The results of this study are not in agreement with the present study. At the same time, study (<xref rid="R14" ref-type="bibr">14</xref>) reported no correlation between less severe nausea and vomiting of pregnancy and HP, but, they found more cases of HP in the more severe cases of pregnancy vomiting. </p><p> The researchers concluded that HP probably potentiates the progression of mild nausea and vomiting to more severe one. </p><p> Also study (<xref rid="R15" ref-type="bibr">15</xref>), reported that although the cases of HEG were moresero- positive for HP than the cases without HEG, they couldn&#x02019;t show any correlation between HP sero- positivity and the duration of HEG or the time of its beginning. Therefore, the researchers concluded that HP probably increases the severity of HEG, but is not a sole factor. </p><p> The other study, which was performed on Hispanic pregnant women (<xref rid="R16" ref-type="bibr">16</xref>), could not show more HP sero-positive cases in the pregnant women with HEG than no HEG cases. </p><p> The result of the above mentioned studies are in agreement with the other studies (<xref rid="R17" ref-type="bibr">17</xref>-<xref rid="R19" ref-type="bibr">19</xref>). </p><p> In contrast, correlation between seropositivity for HP and HEG has been confirmed in studies (<xref rid="R20" ref-type="bibr">20</xref>-<xref rid="R23" ref-type="bibr">23</xref>). In a systematic review and met analysis which was performed on different case- control studies (<xref rid="R24" ref-type="bibr">24</xref>), the researchers showed that HP exposure has been accompanied with a greater risk of HEG. However, there was heterogeneity between studies, with variable reasons. This study stated that regarding the high prevalence of HP, public health consequence of HP with respect to HEG, should be considered significantly. Even the other study (<xref rid="R25" ref-type="bibr">25</xref>), suggested that screening of HP should be added to the HEG diagnostic tests. </p><p> HP infection was accompanied with more cases of HEG in a dose response manner and also stronger in Africans than non- Africans (<xref rid="R26" ref-type="bibr">26</xref>). Different etiologic factors have been suggested for HEG, of which the most recent is the correlation between HP infection and HEG and also cell free fetal DNA (<xref rid="R27" ref-type="bibr">27</xref>). </p><p> Pregnancy dependent hormones like HCG, progestin and estrogen have been studied a lot and other hormones like leptin, placental growth factor, prolactin, thyroid and adrenal hormones have been considered in the genesis of HEG. In addition, infections, immunologic, psycho logic, metabolic and anatomic factors have been reported, and it seems reasonable to performed more studies for finding the etiologic factors and pathogenesis of HEG (<xref rid="R1" ref-type="bibr">1</xref>, <xref rid="R28" ref-type="bibr">28</xref>). </p><p> Specific IgG tests and stool antigen for diagnosis of HP both have been suggested as good screening tests for HP in the cases of HEG (<xref rid="R29" ref-type="bibr">29</xref>) in early pregnancy. The other study (<xref rid="R30" ref-type="bibr">30</xref>), suggested the correlation between HP infection and HEG using stool antigen test, and reported that this test is better than serologic tests. </p><p> Endoscopic evaluation and biopsy for finding HP has been performed in one study in the case of HEG (<xref rid="R31" ref-type="bibr">31</xref>) and showed a significant difference with the cases without HEG. The study concluded that HP infection should be considered in the cases of refractory HEG (<xref rid="R31" ref-type="bibr">31</xref>). However, in study (<xref rid="R32" ref-type="bibr">32</xref>), stool antigen test for HP showed no significant difference in the cases with and without HEG. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> HP infection was higher in HEG cases in this study, and may be considered as its risk factor, however, regarding the above -mentioned studies and controversies between the studies, the role of HP infection in the pathogenesis of HEG should be investigated more fully to consider the different confounding factors, and routine serologic analysis for HP infection is not suggested in all cases of HEG (<xref rid="R17" ref-type="bibr">17</xref>). </p></sec>
Quality of sleep for hospitalized patients in Rasoul-Akram hospital
<p> <bold>Background:</bold> Sleep disturbances have negative effects on medical conditions, mental health and cognitive performance. It was shown that about 60% of inpatients suffer from sleep problems. The aim of this study was to assess the correlation between sleep quality and other factors in the inpatients of Rasoul-e-Akram hospital. </p><p> <bold>Methods:</bold> In this cross-sectional study, all the hospitalized patients in twelve wards of Rasoul-e-Akram hospital during September 2012, were examined. Sleeping habits of 209 inpatients of different wards were assessed through the Persian version of Pittsburgh Sleep Questionnaire (PSQI). A self-designed 18- question questionnaire was conducted for all patients in order to assess their attitude to interior and atmosphere of wards. Content validity and test retest reliability were evaluated. The pain level was also measured by the visual analog scale (VAS) and scores analyzed by the statistical methods of frequency, percentage, chi-square and logistic regression. </p><p> <bold>Results:</bold> The mean of the total scores in PSQI was 8.8&#x000b1;4.8 and 70.8% of the patients were 'poor sleepers' (global PSQI&#x0003e; 5). Age and gender had no effect on the PSQI total score, but the number of roommates, type of the ward, hospitalization period, presence and severity of pain, taking sleep medication and attitude toward the overall atmosphere and interior of wards have caused deviation in scores. </p><p> <bold>Conclusion:</bold> Sleep problems are quite frequent in medical inpatients. Pain management and modification of the ward interior and atmosphere can impact inpatients sleep quality. </p>
<contrib contrib-type="author"><name><surname>Ghanbari Jolfaei</surname><given-names>Atefeh</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Makvandi</surname><given-names>Alena</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Pazouki</surname><given-names>Abdolreza</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Proper sleep is essential for a person&#x02019;s health and wellbeing. Generally less than 6 hours sleep is considered to be insomnia and associated with increasing risk of accidents, fallings, irritability and fatigability, cognitive decline, psychomotor disturbances and morbidity and mortality. Moreover insomnia can lead to high blood pressure and greater C-reactive protein (CRP) concentration, which are inductive of coronary artery disease (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R8" ref-type="bibr">8</xref>). </p><p> According to the National Sleep Foundation (NSF), sixty percent of American adults suffer from sleep disorders (<xref rid="R1" ref-type="bibr">1</xref>). Pourafkary et al. showed that prevalence of sleep disorders in general population of Iran was 35.22%. Moreover daytime sleepiness (64%), difficulty in falling sleep (41.3%), nightmare (16.9%), sleep talking (26.4%), sleep paralysis (16.5%), sleep terror (9.6%) and sleep walking (7.1%) were the frequent sleep disturbances in their study (<xref rid="R9" ref-type="bibr">9</xref>). </p><p> Sleep disturbances were also frequently reported by hospitalized patients. The majority of patients suffer from insomnia in the first three days of hospitalization. (<xref rid="R10" ref-type="bibr">10</xref>) There were various precipitants for sleep disturbances in inpatients, which include: psychiatric disorders, anticipatory anxiety for surgical or diagnostic procedures, and type of medical and surgical problems (<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R11" ref-type="bibr">11</xref>). </p><p> For instance, Pimental et.al study, done in two hospitals in Belo Horizonte, showed that the main causes of sleep disturbances were environmental factors including noise (45.7%), temperature (22.9%) and number of roommates (20.0%). Furthermore, 29% of the inpatients could not sleep, because of their medical condition and 17% became insomniac because of psychological problems (<xref rid="R12" ref-type="bibr">12</xref>). </p><p> Sleep quality is particularly important in inpatients of a general hospital. </p><p> It was shown that the rate of hospitalization increased among inpatients suffering from sleep disturbances (<xref rid="R13" ref-type="bibr">13</xref>-<xref rid="R14" ref-type="bibr">14</xref>). </p><p> Accordingly, it is valuable to determine the risk factors for sleep disturbances in inpatients of general hospitals. Unfortunately, there are few studies of sleep problems among Iranian inpatients of general hospitals. This study assessed the sleep quality and its correlation with the aforementioned subjects. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><sec id="s2-1"><title>Participants and setting</title><p> In this cross-sectional study, all the inpatients of Rasoul-e-Akram hospital-a training hospital affiliated to Iran University of medical sciences who were hospitalized during September 2012, included in this study. The patients were enrolled to the study through available non-probability sampling. </p><p> Inclusion criteria were: being hospitalized for at least seven days, 18 years old or above, being conscious, literate and able to communicate in Persian. </p><p> All the patients filled the informed consent and were aware that refusing to participate in the study would not influence their treatment process. </p><p> At the end of September, 209 inpatients of twelve wards including Internal Medicine, Orthopedics, Obstetrics-Gynecology, Neurology-neurosurgery, Surgery, Post-CCU, Pain, Dermatology-Oncology, ENT, Nephrology, Psychiatry and Ophthalmology were asked about their quality of sleep, severity of pain and attitude towards wards atmosphere. </p></sec><sec id="s2-2"><title> Procedure </title><p> The quality of sleep was assessed through the Persian version of Pittsburgh Sleep Quality Index (PSQI). </p><p> The PSQI is a self-rated questionnaire designed by Buysse et al. for assessing subjective sleep quality over the past month. In this study we changed the past month to past week. </p><p> The psychometric properties in insomniac patients and excessive daytime sleepiness were confirmed to be fair (<xref rid="R15" ref-type="bibr">15</xref>). It consists of seven &#x02018;component&#x02019; scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction, all of which are summed to a single global score. In a previous study, the Persian version of PSQI was validated and its reliability were also assessed (Chronbach&#x02019;sa=0.83). Total score more than 5 is defined as sleep disturbance (<xref rid="R16" ref-type="bibr">16</xref>). </p><p> The pain level was measured by the visual analog scale (VAS).Patients were asked to assess the level of their average pain by placing a mark on a horizontal line that was 10 cm long. </p><p> Inspired by Vartiainen and Hakola (<xref rid="R17" ref-type="bibr">17</xref>) a questionnaire with 18 questions was designed to assess patients&#x02019; satisfaction of the ward interior and the general ward atmosphere. Each question had five grades (totally disagree, slightly disagree, hard to say, slightly agree, totally agree). And the total score was 90. To evaluate thecontent validityofthisquestionnaire delphi method was used. Six experts (five associative professor of psychiatry and one assistant professor of psychiatry) revised the questions in three rounds. Test retest reliability was evaluated within 5 days for 42 inpatients(r=0. 74). </p><p> The Data were analyzed using correlation, chi-square,ANOVA, t-test and logistic regression. The level of significance was set up at p&#x0003c;0.01. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> Subjects&#x02019; demographic characteristics are presented in <xref ref-type="table" rid="T1">Table 1</xref>. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Demographic and other characteristics of subjects (n=209) </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Variable</td><td align="center" rowspan="1" colspan="1">No (%)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Gender<break/>Female<break/>male </td><td align="center" rowspan="1" colspan="1"> <break/> 106(50.7) <break/> 103(49.3) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age</td><td align="center" rowspan="1" colspan="1">38+_12</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Mean duration of hospitalization(MIN-MAX)</td><td align="center" rowspan="1" colspan="1">9.1+_2.1(7-32)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Mean numbers of roommates(MIN-MAX)</td><td align="center" rowspan="1" colspan="1">1.8+_1.2(0-8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> types of sleeping medication<break/>clonazepam<break/>lorazepam<break/>diazepam<break/>oxazepam<break/>alprazolam<break/>zolpidem </td><td align="center" rowspan="1" colspan="1"> <break/>34<break/>1<break/>22<break/>11<break/>1<break/>4 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Pain<break/>No<break/>Mild<break/>Mod<break/>Severe<break/>Extremely severe </td><td align="center" rowspan="1" colspan="1"> <break/>119(56.9)<break/>28(13.4)<break/>34(16.3)<break/>21(10)<break/>7(3.3) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> PSQI mean component scores<break/>subjective sleep quality<break/>sleep latency<break/>sleep duration<break/>habitual sleep efficiency<break/>sleep disturbances<break/>use of sleeping medication<break/>daytime dysfunction<break/>total </td><td align="center" rowspan="1" colspan="1"> <break/>1.29&#x000b1;0.8<break/><italic>1.38</italic>&#x000b1;1<break/>1.89&#x000b1;0.8<break/>1.22&#x000b1;1.1<break/>1.22&#x000b1;0.5<break/>1.2&#x000b1;0.8<break/>0.9&#x000b1;0.8<break/><italic>8.8</italic>&#x000b1;4.8 </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Ward<break/>medicine<break/>Orthopedics<break/>Obstetrics-Gynecology<break/>Neurology-neurosurgery<break/>Surgery<break/>Post-CCU<break/>pain<break/>Dermatology-Oncology<break/>ENT<break/>Nephrology<break/>Psychiatry<break/>Ophthalmology </td><td align="center" rowspan="1" colspan="1"> <break/>40(19.1)<break/>30(14.4)<break/>23(11)<break/>20(9.6)<break/>29(13.9)<break/>14(6.7)<break/>14(6.7)<break/>14(6.7)<break/>10(4.8)<break/>10(4.8)<break/>10(4.8)<break/>9(4.3) </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Satisfaction of wards atmosphere and interior</td><td align="center" rowspan="1" colspan="1">69.48+-9.13</td></tr></tbody></table></table-wrap><p> 70.8% of the samples had PSQI score more than 5.The mean score of The PSQI was 8.8&#x000b1;4.82. </p><p> The results showed that the frequency of a total score of greater than 5 in the Pittsburgh questionnaire did not differ regarding to gender (p=0.36) and age (p=0.28). </p><p> In contrast, using correlation test total score of the Pittsburgh questionnaire was significantly different based on number of room-mates (p=0.01, r=0.164), duration of hospitalization (p=0.002, r=0.218), presence and severity of pain (p=0.001, r=0.501), taking sleep medications (p=0.0001, r=0.247) and satisfaction of the ward. (p=0.001,r=-0.263) </p><p> In summary, higher number of roommates, longer duration of hospitalization, presence and severity of pain, sleep medications and negative attitude towards ward atmosphere were accompanied with poor sleep. </p><p> Out of the patients hospitalized in different wards, mean score of subjective sleep quality and mean of sleep duration were the highest in neurologic patients and the lowest in psychiatric and orthopedic patients. Nevertheless the rate of taking sleep medicines and the mean scores of daytime dysfunction were the lowest in neurologic patients and the highest in psychiatric and orthopedic patients. Using ANOVA, it was revealed that the total score of PSQI was significantly different between inpatients of different wards (F=3.671, dftotal=207, p=0.0001). </p><p> Using logistic regression total score of PSQI was not significantly different based on pain (p=0.037), type of ward (p=0.471), satisfaction of ward (p=0.605), gender (p=0.278), age (p=0.161), number of roommates (p=0.144) and duration of hospitalization (p=0.152) and the only variant that significantly affected sleep quality was taking sleep medicine (p=0.001). </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> In this study, the mean of total score of PSQI was 8.8&#x000b1;4.8 and 70.8%of the samples gained atotal score greater than 5. </p><p> The mean of total score of PSQI in advanced cancer patients in Mistakidou&#x02019;s study was 11.72&#x000b1;4.4 (<xref rid="R18" ref-type="bibr">18</xref>). The different results could be due to the differences in sampling methods, because sleep disturbance has been one of the most frequent side effects experienced by patients with cancer (<xref rid="R19" ref-type="bibr">19</xref>,<xref rid="R20" ref-type="bibr">20</xref>) and it could be the reason for higher global score of PSQI in that study. </p><p> A study carried out by Dogan et al. (2005) showed that the PSQI mean score in 150 hospitalized patients of a general hospital was 7.9 (<xref rid="R11" ref-type="bibr">11</xref>). Their sampling method was more similar to ours. Similarly, they pointed out that patients in psychiatric ward experienced more sleep problem than the other wards. </p><p> We did not find a significant relationship between demographic factors and sleep, conversely some of the studies demonstrated that the quality of sleep was influenced by demographic factors such as age and gender. For example in the study by Dogan et al. showed that female patients had worse sleep quality than male patients. And in Sendir&#x02019;s study, aging was a risk factor of poor sleeping (<xref rid="R21" ref-type="bibr">21</xref>). In a study, which was conducted in a general hospital in Zahedan, Iran, it was shown that 62% of elderly inpatients had sleep disorders, although they used a self-designed questionnaire for assessing the quality of sleep and they did not compare the sleep of elderly inpatients with other ages (<xref rid="R22" ref-type="bibr">22</xref>). </p><p> We found out that patients dissatisfaction from the ward, higher number of roommates and severity of pain were associated with poor sleep. Lane et al. and Sendir et al. also reported that compared to demographic factors, environmental factors such as noise and factors, such as pain and tension had a greater effect on sleep quality (<xref rid="R21" ref-type="bibr">21</xref>,<xref rid="R23" ref-type="bibr">23</xref>)and these findings indicate the importance of management of pain and modification of the ward atmosphere and interior in improving sleep patterns in hospitalized patients. </p><p> Lei et al. also carried out a study on 397 patients in a general hospital. They indicated that 56.7% of patients suffered from sleep disturbances; and the mean score of PSQIafter hospitalization was 7.34 and the most precipitants of poor sleep had other concerns, such as the need to use the toilet at nights and environmental factors (<xref rid="R24" ref-type="bibr">24</xref>). </p><p> In a study conducted on 200 inpatients of university-affiliated hospitals in Babol, Iran, was shown that 63.6% of patients had sleep problems; the mean score of PSQI as 5.97&#x000b1;3.02 and consistent with our study, a negative correlation was found between sleep quality and the duration of hospitalization. The reason might be that the longer hospitalization is associated with more severe medical conditions, demoralization and adjustment disorder (<xref rid="R25" ref-type="bibr">25</xref>). </p><p> Our study indicated that sleep problems were more in patients of psychiatric and orthopedic wards, although after using multiple regressions and adjusting for other factors, the type of the ward was no longer associated with poor sleep. Similarly other studies reported the most sleep problems in patients of psychiatric and orthopedic wards (<xref rid="R11" ref-type="bibr">11</xref>,<xref rid="R25" ref-type="bibr">25</xref>). The high rates of sleep disturbances in orthopedic patients can be explained by the high prevalence of pain in them. Other studies also demonstrated that secondary and primary sleep disorders are more common in patients with psychiatric disorders (<xref rid="R26" ref-type="bibr">26</xref>). In Pourafkary&#x02019;s study, psychiatric patients compared with general population used more hypnotic agents and the prevalence of sleep disorders in them was significantly higher than general population (<xref rid="R9" ref-type="bibr">9</xref>).And it might be the cause of high proportion of sleep disturbances in inpatients of psychiatric wards. </p><p> As it was expected, taking benzodiazepines was associated with higher scores of PSQI (<xref rid="R26" ref-type="bibr">26</xref>-<xref rid="R27" ref-type="bibr">27</xref>). </p><p> The present findings should be settled considering the limitations. The data were gatheredthrough a cross-sectional study. Furthermore, there were plenty of factors which influence the quality of sleep; factors such as psychiatric disorders, type of medical and surgical illness, concerns about diagnostic procedures, prognosis, outcome and treatment, health anxiety and other factors which we did not control. </p><p> Additional research is necessary to resolve these limitations. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Sleep problems are quite frequent in medical inpatients. With regard to the complications of sleep disorders and their important role on the course of diseases and quality of life, it is crucial to assess sleep disorders among hospitalized patients, and educate them about sleep hygiene. Furthermore, strategies like pain management and modification of the ward interior and atmosphere could be taken for prevention and management of sleep problems in these patients. </p></sec>
Ergonomic intervention, workplace exercises and musculoskeletal complaints: a comparative study
<p> <bold>Background:</bold> Musculoskeletal disorders are among the most prevalent occupational disorders in different jobs such as office work. Some interventions such as ergonomic modifications and workplace exercises are introduced as the methods for alleviating these disorders. In this study we compared the effect of ergonomic modifications and workplace exercises on musculoskeletal pain and discomfort in a group of office workers. </p><p> <bold>Methods:</bold> In an interventional study on office workers, the effect of two interventions was compared. Ergonomic modification consisted of correcting the arrangement of workstation and changing some equipment; workplace exercises included stretching exercises focusing on neck, shoulders, low back, and hand and wrist. Musculoskeletal complaints were assessed and compared before and after 1 month interventions. </p><p> <bold>Results:</bold> The frequency of musculoskeletal complaints was high before the study. Both interventions significantly reduced complaints in a similar manner except for low back pain which was reduced in exercise group more than the other group. </p><p> <bold>Conclusion:</bold> In this study we found a beneficial short-term effect for both ergonomic modifications and stretching work-place exercises on reducing musculoskeletal pain in office workers. </p>
<contrib contrib-type="author"><name><surname>Mehrparvar</surname><given-names>Amir Houshang</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Heydari</surname><given-names>Mohammad</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mirmohammadi</surname><given-names>Seyyed Jalil</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mostaghaci</surname><given-names>Mehrdad</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Davari</surname><given-names>Mohammad Hossein</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Taheri</surname><given-names>Mahmoud</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Musculoskeletal disorders are among the most prevalent occupational disorders in different jobs. There is substantial evidence that such ergonomic risk factors as repetition, awkward posture, contact stress and force if overcome worker's biomechanical capabilities may lead to work-related musculoskeletal disorders (WMSDs) (<xref rid="R1" ref-type="bibr">1</xref>). It is said that WMSDs are leading causes of absenteeism and disability (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R3" ref-type="bibr">3</xref>). </p><p> Office work due to computer use is now a job with a high prevalence of WMSDs (<xref rid="R4" ref-type="bibr">4</xref>). Most office workers now routinely use a computer and its accessories as a part of their equipment in the workplace and this equipment creates many ergonomic risk factors, especially awkward postures (<xref rid="R5" ref-type="bibr">5</xref>). Therefore, musculoskeletal complaints in different parts of the body, especially, neck, shoulder, wrist and hand are common in this occupational group (<xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R7" ref-type="bibr">7</xref>). Some studies have shown an incidence of about 50% for WMSDs among video display terminal (VDT) users in different parts of the body (<xref rid="R5" ref-type="bibr">5</xref>, <xref rid="R8" ref-type="bibr">8</xref>). </p><p> In order to overcome musculoskeletal disorders or symptoms, some interventions are used such as training, ergonomic modifications, rest breaks, and workplace exercises with various effects (<xref rid="R9" ref-type="bibr">9</xref>-<xref rid="R16" ref-type="bibr">16</xref>). </p><p> It is obviously proved in several studies that ergonomic modifications can decrease the frequency of musculoskeletal pain or discomfort among office workers. Amick et al. assessed this effect after changing the chairs in an office environment and found considerable results (<xref rid="R9" ref-type="bibr">9</xref>), they also found that training alone can also reduce the frequency of MSDs although to a level lower than ergonomic change; this result was also observed in the authors' previous study on office workers (<xref rid="R14" ref-type="bibr">14</xref>). Arnetz et al. found that workplace ergonomic intervention can decrease absenteeism among office workers (<xref rid="R17" ref-type="bibr">17</xref>). </p><p> Although it has been shown in some studies that ergonomic modifications are significantly effective for alleviating MSDs, they are costly, which is an important issue especially in developing countries. Thus considering other interventions such as training, rest breaks, or workplace exercises are probably more practical in these countries. </p><p> Some studies have shown that installing ergonomic programs based on training, or workplace exercises are also effective in reducing of WMSDs. Authors in their previous study found a beneficial effect for training in reduction of awkward postures (<xref rid="R14" ref-type="bibr">14</xref>). Tsauo et al. found a beneficial effect for an intensive team-exercise program in reducing neck and shoulder symptoms in sedentary workers (<xref rid="R18" ref-type="bibr">18</xref>). Coury et al. in a systematic review found that workplace exercises can significantly reduce neck pain among office workers, although this effect was not significant for other parts of the body (<xref rid="R19" ref-type="bibr">19</xref>). </p><p> Although studies have shown a beneficial effect for ergonomic modifications and exercise, compliance especially over time is a concern (<xref rid="R20" ref-type="bibr">20</xref>). In another hand, some studies have failed to show a significant effect for ergonomic modifications or exercise. (<xref rid="R13" ref-type="bibr">13</xref>). A systematic review conducted by Verhagen et al. failed to show a clear effect of some ergonomic modifications and exercises on work-related complaints of the arm, neck and shoulder (<xref rid="R16" ref-type="bibr">16</xref>). </p><p> In this study, we aimed to evaluate and compare the effect of ergonomic modifications and workplace exercises on musculoskeletal pain and discomfort in a group of office workers. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> This was an interventional study conducted on 184 office workers. According to previous studies sample size was calculated to be at least 80 subjects for each group (<xref rid="R13" ref-type="bibr">13</xref>). </p><sec id="s2-1"><title>Subjects</title><p> Two departments in the central office of the university were selected to join the study and subjects in each department were assigned in the same intervention program to avoid contamination between groups. Two groups were working in different buildings of an office with similar jobs and similar environmental conditions (lighting, temperature and dampness). All subjects in both departments were office workers who worked on a VDT most of their working time. Their job was sedentary in a 7-hour morning shift (from 8 AM to 3 PM). They had half an hour rest period at 1:30 PM for praying. They worked at least 3 hours on a computer workstation and were sitting on a chair more than 5 hours during their work shift. </p><p> The intervention in two groups included the following activities: One group received ergonomic modifications in their workstation and equipment and the other received training to exercise regularly in the workplace. Those with previous known musculoskeletal diseases and those with second jobs containing ergonomic hazards were excluded from the study. Eight subjects in the first group (3 due to job change, 3 due to previous diseases and 2 due to second job) and 12 subjects in the second group (1 due to job change, 4 due to previous diseases and 7 due to second job) were excluded from the study at the beginning or during the study. </p><p> Musculoskeletal complaints were assessed by Nordic questionnaire (<xref rid="R25" ref-type="bibr">25</xref>) before and after intervention and the change after intervention was compared between two groups. An informed consent was obtained from each participant. </p></sec><sec id="s2-2"><title>Intervention</title><p> All workstations in the first group (ergonomic modification), were surveyed by two occupational medicine specialists and one industrial hygienist in order to find non-ergonomic conditions and equipment. Then the arrangement of the equipment was corrected according to ergonomic rules (change in desk placement, seat height, position of keyboard, mouse and monitor) using OSHA VDT workstation checklist (<xref rid="R21" ref-type="bibr">21</xref>, <xref rid="R22" ref-type="bibr">22</xref>), and non-ergonomic equipment (including mouse, keyboard, and mouse pad) were changed and copy holder and foot rest were added to the workstation when needed. No change was performed in the chair and desk due to monetary limitations of the study. </p><p> In the second group (exercise) a workplace exercise program was planned for all participants. The program consisted of two 1-quarter periods of office exercises including stretching exercises focusing on neck, shoulder, wrist, back and low back at 10 AM and 12. After assessment of musculoskeletal complaints, all participants in the second group participated in a training session (1 hour in 20-person groups) about exercises and all planned exercises were explained to them by an occupational medicine specialist through an oral lecture. In these training sessions, exercises were presented using pictures. After the training session a poster containing the picture of exercises and a brief explanation sent to their e-mail and they were asked to do exercises regularly at the planned times. An occupational medicine resident supervised the exercises every other day. </p><p> One month after the interventions, participants were assessed again by the same questionnaire. </p><p> The study was sponsored by Shahid Sadoughi University of Medical Sciences and approved by ethics committee of the university. Data was analyzed by SPSS software (ver. 19) using independent samples or paired samples T-test and chi square or Mc Nemar test. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> In total 181 subjects were selected, and after considering exclusion criteria, 164 individuals enrolled the study (83 subjects in the ergonomic modification and 81 in the exercise group). <xref ref-type="fig" rid="F1">Diagram 1</xref> shows subjects&#x02019; allocation in two groups. Gender distribution was similar in both groups (49% males and 51% females). <xref ref-type="table" rid="T1">Table 1</xref> compares the demographic characteristics of two groups. </p><fig id="F1" orientation="portrait" position="float"><label> Diagram 1 </label><graphic xlink:href="MJIRI-28-69-g001"/><statement><p>The allocation of two groups</p></statement></fig><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Demographic variables in the study groups (ergonomic modification and exercise). </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Variables</td><td align="center" rowspan="1" colspan="1">Study groups**</td><td align="center" rowspan="1" colspan="1">Mean</td><td align="center" rowspan="1" colspan="1">SD*</td><td align="center" rowspan="1" colspan="1">P value</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">38.95</td><td align="center" rowspan="1" colspan="1">7.48</td><td align="center" rowspan="1" colspan="1">.650</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">38.40</td><td align="center" rowspan="1" colspan="1">8.00</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Work experience</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">15.09</td><td align="center" rowspan="1" colspan="1">7.30</td><td align="center" rowspan="1" colspan="1">.077</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">14.07</td><td align="center" rowspan="1" colspan="1">7.14</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">BMI</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">24.86</td><td align="center" rowspan="1" colspan="1">3.30</td><td align="center" rowspan="1" colspan="1">.29</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">26.04</td><td align="center" rowspan="1" colspan="1">3.48</td><td align="center" rowspan="1" colspan="1"> </td></tr></tbody></table><table-wrap-foot><fn><p> * SD: standard deviation, ** 1: Ergonomic modification group, 2: Exercise group </p></fn></table-wrap-foot></table-wrap><p> Body part with the most frequent complaints was low back. There was no difference between two groups regarding the frequency of musculoskeletal complaints in different parts of the body before intervention except for elbow. The ergonomic modification group complained of more symptoms in the elbow than exercise group.<xref ref-type="fig" rid="F2">Figure 1</xref> shows this comparison. </p><fig id="F2" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-69-g002"/><statement><p>Frequency of musculoskeletal complaints in two groups before intervention.</p></statement></fig><p> There was a significant reduction (partial or complete improvement) in musculoskeletal complaints after intervention in both groups. We could not find any significant difference between two groups in neck, shoulder, hand and wrist after the interventions (p= 0.508, 0.243, and 0.575, respectively for each area). <xref ref-type="table" rid="T2">Table 2</xref> shows the frequency of musculoskeletal complaints in different body parts.Although change in the frequency of musculoskeletal symptoms in low back area was significantly higher in the exercise group (p= 0.03). <xref ref-type="fig" rid="F3">Figures 2</xref> through <xref ref-type="fig" rid="F6">5</xref> show the changes in the frequency of musculoskeletal complaints in different parts of the body. </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Characteristics of musculoskeletal complaints in different parts of body in both groups before intervention. </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="4" colspan="1">Complaints</td><td rowspan="4" colspan="1"/><td align="center" colspan="8" rowspan="1">Frequency in different parts of body, N (%)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" colspan="8" rowspan="1">Group</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" colspan="3" rowspan="1">Job modification</td><td align="center" colspan="5" rowspan="1">Exercise</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Neck</td><td align="center" rowspan="1" colspan="1">Shoulder</td><td align="center" rowspan="1" colspan="1">Low back</td><td align="center" rowspan="1" colspan="1">Wrist and hand</td><td align="center" rowspan="1" colspan="1">Neck</td><td align="center" rowspan="1" colspan="1">Shoulder</td><td align="center" rowspan="1" colspan="1">Low back</td><td align="center" rowspan="1" colspan="1">Wrist and hand</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Pain severity</td><td align="center" rowspan="1" colspan="1">Mild</td><td align="center" rowspan="1" colspan="1"> 12 <break/>(14.45) </td><td align="center" rowspan="1" colspan="1">20 (24.09)</td><td align="center" rowspan="1" colspan="1"> 13<break/>(15.66) </td><td align="center" rowspan="1" colspan="1"> 12<break/>(14.45) </td><td align="center" rowspan="1" colspan="1"> 14 <break/>(17.28) </td><td align="center" rowspan="1" colspan="1"> 18<break/>(22.22) </td><td align="center" rowspan="1" colspan="1"> 19<break/>(23.46) </td><td align="center" rowspan="1" colspan="1"> 16<break/>(19.75) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Moderate</td><td align="center" rowspan="1" colspan="1"> 38 <break/>(45.78) </td><td align="center" rowspan="1" colspan="1">27 (32.53)</td><td align="center" rowspan="1" colspan="1"> 33<break/>(39.75) </td><td align="center" rowspan="1" colspan="1"> 26<break/>(31.32) </td><td align="center" rowspan="1" colspan="1"> 35 <break/>(43.21) </td><td align="center" rowspan="1" colspan="1"> 27<break/>(32.23) </td><td align="center" rowspan="1" colspan="1"> 26<break/>(32.09) </td><td align="center" rowspan="1" colspan="1"> 35<break/>(43.21) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Severe</td><td align="center" rowspan="1" colspan="1"> 8<break/> (9.64) </td><td align="center" rowspan="1" colspan="1"> 2<break/> (2.41) </td><td align="center" rowspan="1" colspan="1"> 18<break/>(21.68) </td><td align="center" rowspan="1" colspan="1"> 14<break/>(16.86) </td><td align="center" rowspan="1" colspan="1"> 11 <break/>(13.58) </td><td align="center" rowspan="1" colspan="1"> 10<break/>(12.34) </td><td align="center" rowspan="1" colspan="1"> 15<break/>(18.52) </td><td align="center" rowspan="1" colspan="1"> 17<break/>(20.98) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Pain frequency</td><td align="center" rowspan="1" colspan="1">Only once</td><td align="center" rowspan="1" colspan="1"> 0 <break/>(0) </td><td align="center" rowspan="1" colspan="1"> 0 <break/>(0) </td><td align="center" rowspan="1" colspan="1"> 0<break/>(0) </td><td align="center" rowspan="1" colspan="1"> 0<break/>(0) </td><td align="center" rowspan="1" colspan="1"> 1 <break/>(1.23) </td><td align="center" rowspan="1" colspan="1"> 1<break/>(1.23) </td><td align="center" rowspan="1" colspan="1"> 2<break/>(2.47) </td><td align="center" rowspan="1" colspan="1"> 2<break/>(2.47) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">More than once, not every day</td><td align="center" rowspan="1" colspan="1"> 36<break/> (43.37) </td><td align="center" rowspan="1" colspan="1">34 (40.97)</td><td align="center" rowspan="1" colspan="1">41 (49.38)</td><td align="center" rowspan="1" colspan="1"> 29 <break/>(34.93) </td><td align="center" rowspan="1" colspan="1"> 50 <break/>(61.5) </td><td align="center" rowspan="1" colspan="1">43 (53.07)</td><td align="center" rowspan="1" colspan="1">44 (56.78)</td><td align="center" rowspan="1" colspan="1"> 51 <break/>(62.95) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Every day</td><td align="center" rowspan="1" colspan="1"> 21 <break/>(25.30) </td><td align="center" rowspan="1" colspan="1">14 (16.87)</td><td align="center" rowspan="1" colspan="1"> 22<break/>(26.50) </td><td align="center" rowspan="1" colspan="1"> 23<break/>(27.71) </td><td align="center" rowspan="1" colspan="1"> 9 <break/>(11.11) </td><td align="center" rowspan="1" colspan="1"> 10<break/>(12.34) </td><td align="center" rowspan="1" colspan="1"> 12<break/>(14.81) </td><td align="center" rowspan="1" colspan="1"> 4<break/>(4.94) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Pain duration</td><td align="center" rowspan="1" colspan="1">Less than 1 day</td><td align="center" rowspan="1" colspan="1"> 19 <break/>(22.89) </td><td align="center" rowspan="1" colspan="1">25 (30.12)</td><td align="center" rowspan="1" colspan="1"> 14<break/>(16.86) </td><td align="center" rowspan="1" colspan="1"> 22<break/>(26.51) </td><td align="center" rowspan="1" colspan="1"> 25 <break/>(30.84) </td><td align="center" rowspan="1" colspan="1"> 25<break/>(30.86) </td><td align="center" rowspan="1" colspan="1"> 28<break/>(34.57) </td><td align="center" rowspan="1" colspan="1"> 26<break/>(32.09) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">1-7 days</td><td align="center" rowspan="1" colspan="1"> 23 <break/>(27.71) </td><td align="center" rowspan="1" colspan="1">23 (27.71)</td><td align="center" rowspan="1" colspan="1"> 28<break/>(33.73) </td><td align="center" rowspan="1" colspan="1"> 19<break/>(22.89) </td><td align="center" rowspan="1" colspan="1"> 39 <break/>(48.15) </td><td align="center" rowspan="1" colspan="1"> 35<break/>(43.21) </td><td align="center" rowspan="1" colspan="1"> 34<break/>(41.97) </td><td align="center" rowspan="1" colspan="1"> 36<break/>(44.44) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x0003e; 7 days</td><td align="center" rowspan="1" colspan="1"> 32 <break/>(38.55) </td><td align="center" rowspan="1" colspan="1"> 21 <break/>(25.3) </td><td align="center" rowspan="1" colspan="1"> 34<break/>(40.95) </td><td align="center" rowspan="1" colspan="1"> 30<break/>(36.13) </td><td align="center" rowspan="1" colspan="1"> 14<break/> (17.28) </td><td align="center" rowspan="1" colspan="1"> 18<break/>(22.22) </td><td align="center" rowspan="1" colspan="1"> 18<break/>(22.22) </td><td align="center" rowspan="1" colspan="1"> 16<break/>(19.75) </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td colspan="2" rowspan="1">Absenteeism due to pain</td><td align="center" rowspan="1" colspan="1"> 13 <break/>(15.66) </td><td align="center" rowspan="1" colspan="1"> 4<break/> (4.82) </td><td align="center" rowspan="1" colspan="1">17 (20.38)</td><td align="center" rowspan="1" colspan="1"> 7<break/>(8.43) </td><td align="center" rowspan="1" colspan="1"> 7<break/>(8.43) </td><td align="center" rowspan="1" colspan="1">11 (13.58)</td><td align="center" rowspan="1" colspan="1"> 22<break/>(27.16) </td><td align="center" rowspan="1" colspan="1"> 8<break/>(9.87) </td></tr></tbody></table></table-wrap><fig id="F3" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-69-g003"/><statement><p>Reduction of the musculoskeletal complaints in neck.</p></statement></fig><fig id="F4" orientation="portrait" position="float"><label> Fig. 3 </label><graphic xlink:href="MJIRI-28-69-g004"/><statement><p> Reduction of the musculoskeletal complaints in shoulder.</p></statement></fig><fig id="F5" orientation="portrait" position="float"><label> Fig. 4 </label><graphic xlink:href="MJIRI-28-69-g005"/><statement><p>Reduction of the musculoskeletal complaints in wrist/hand.</p></statement></fig><fig id="F6" orientation="portrait" position="float"><label> Fig. 5 </label><graphic xlink:href="MJIRI-28-69-g006"/><statement><p>Reduction of the musculoskeletal complaints in low back area.</p></statement></fig></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Computer work is one of the main jobs which may lead to several kinds of musculoskeletal symptoms and discomfort. Ergonomic interventions by modifications in the equipment or arrangement of the workstation, training, rest breaks and workplace exercises have been proved to reduce musculoskeletal symptoms in many workplaces, especially office work. The strongest improvements have been observed by ergonomic modifications, but some of these modifications are costly and difficult to recommend in many workplaces especially in developing countries. </p><p> In this study, we compared the effect of workplace exercises, as a more practical intervention, and ergonomic modifications in reducing the frequency of musculoskeletal complaints among office workers. </p><p> The frequency of musculoskeletal complaints was high in this study. We found a significant reduction in musculoskeletal complaints one month after both interventions which was consistent with some previous studies, the efficacy of both ergonomic modifications and workplace exercises (<xref rid="R9" ref-type="bibr">9</xref>, <xref rid="R17" ref-type="bibr">17</xref>, <xref rid="R23" ref-type="bibr">23</xref>-<xref rid="R25" ref-type="bibr">25</xref>). This change was observed in the most important at risk areas of body (i.e. neck, shoulder, hand and wrist and low back). </p><p> Regarding ergonomic modifications, we could change the arrangement of workstation and some non-ergonomic equipment. We were unable to modify non-ergonomic chairs or desks due to monetary limitations, thus our focus was on neck and hand/wrist more than other parts of the body. We found a significant reduction of musculoskeletal complaints in neck, shoulder, hand/wrist and low back. This result was also observed in the exercise group. The improvement in musculoskeletal complaints was similar in both groups except for low back region in which the effect of workplace exercises was more than ergonomic interventions. This was probably due to our limitation for changing chair or desk, but our exercise program included exercises for back and low back regions as well. In this study we assessed only short-term effects of the interventions after one month. More studies are required to assess long-term effect and durability of the interventions. </p><p> Anderson et al., consistent with the results of the current study, showed the positive effect of exercise on neck, although they used specific strengthening but we used some stretching exercises. They also assessed the effect of general fitness training which showed a small acute pain reduction (<xref rid="R23" ref-type="bibr">23</xref>). Alexandre et al. found a positive effect of exercise on low back pain among nursing personnel, although their exercise program was different from ours (<xref rid="R24" ref-type="bibr">24</xref>). </p><p> Amick et al. found a clear effect after using ergonomic chair with training (<xref rid="R9" ref-type="bibr">9</xref>). Sjogren et al. in a cluster randomized controlled trial evaluated the effects of a workplace physical exercise intervention on the intensity of symptoms in the neck and shoulders and found that physical exercise intervention resulted in a slight, but statistically significant decrease in the intensity of neck symptoms. The intervention had no effect on the intensity of shoulder symptoms (<xref rid="R25" ref-type="bibr">25</xref>). </p><p> Sihawong et al. in a literature review assessed the effectiveness of various types of exercise for prevention and treatment of nonspecific neck pain in office workers and found a positive effect for muscle strengthening or endurance exercises (<xref rid="R26" ref-type="bibr">26</xref>). </p><p> Boocock et al. in a systematic review found that the use of some mechanical and modifier </p><p> interventions were effective in preventing and managing neck/upper extremity musculoskeletal conditions and fibromyalgia (<xref rid="R27" ref-type="bibr">27</xref>). Tsaou et al. found that an intensive team-exercise program was beneficial for alleviating neck and shoulder symptoms in sedentary workers which is in agreement with the results of the current study (<xref rid="R18" ref-type="bibr">18</xref>). </p><p> Maher et al. in a systematic review of randomized controlled trials showed that workplace exercise was effective, but education ineffective, and workplace modification plus education was of unknown value in preventing low back pain (<xref rid="R13" ref-type="bibr">13</xref>) which is in contrast to the results we found in this study, although ergonomic modifications and workplace exercise programs were various in different studies. </p><p> Maul et al. found that supervised physical training could effectively improve functional capacity and reduced low back pain. This study showed a long-term benefit for training as well (<xref rid="R28" ref-type="bibr">28</xref>). In the current study we assessed only the short-term effect of exercise. </p><p> Van Poppel et al. could not find a beneficial effect for lumbar supports, education, or exercise in the primary prevention of low back pain at the workplace which was contrary to the results of the current study (<xref rid="R29" ref-type="bibr">29</xref>). </p><p> This study had some limitations: Our study suffered from monetary deficiencies, hence our ergonomic modification was not complete and we could not change non-ergonomic chairs or desks. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> There is inconsistency between the results of different studies about the effect of exercise or ergonomic modifications on alleviating musculoskeletal complaints or disorders. One of the principal explanations for this inconsistency could be different methods of the interventions. In this study we found a beneficial short-term effect for both ergonomic modifications and stretching work-place exercises on reducing musculoskeletal complaints in office workers. </p></sec>
The comparison of spinal curves and hip and ankle range of motions between old and young persons
<p> <bold>Background:</bold> Falls have been strongly associated with decreased physical activity and impaired mobility. Reduced range of motion, as a consequence of muscle stiffness, has been indicated to assume a positive relationship to fall incidence. Also clinical observations suggest that maintaining the normal spinal curves is associated with the prevention of spinal, knee and hip disorders. Thus, the aim of this study was to compare hip and ankle range of motions and thoracic and lumbar curves between young and old persons </p><p> <bold>Methods:</bold> Using a nonprobability sampling 30 elderly persons at average of 68.14 &#x000b1;4.03 years of age and 30 young people (age 23.37 &#x000b1; 2.31 years) through a case &#x02013; control design participated in the study. Maximal hip extension and ankle dorsiflexion range of motions were measured by a standard goniometer. Thoracic and lumbar curvatures were measured by a flexible ruler in both groups. Independent t test were used to statistically analyze differences between groups. </p><p> <bold>Results:</bold> Compared with the young group, the elderly group had decreased hip extension and ankle dorsiflexion motions (p&#x0003c;0.01). The result of independent t test showed that the mean of lumbar curve was higher in young group (31.29&#x000b1; 6.37) than elderly subjects (27.93&#x000b1;8.11) ,however, no significant difference was found between two groups (p=0.08). The result also showed increasing thoracic curvature with aging (young group=34.43&#x000b1;13.27, old group= 36.19&#x000b1;8.97), however, no significant difference was found between two groups </p><p> <bold>Conclusion:</bold> Findings suggest decreased ankle and hip joint range of motions should be considered in rehabilitation of elderly people. </p>
<contrib contrib-type="author"><name><surname>Nodehi-Moghadam</surname><given-names>Afsun</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Taghipour</surname><given-names>Morteza</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Goghatin Alibazi</surname><given-names>Razie</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Baharlouei</surname><given-names>Hamzeh</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Many investigators have found an association between spinal alignment and low back pain. Low back pain and hip and knee joints pain are major concerns in the elderly, whose population is increasing (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R3" ref-type="bibr">3</xref>). Some clinical studies have found an alteration in spinal curves with increasing age. Altered spinal curves may cause disk degeneration, radicular pain and vertebral fractures in the elderly (<xref rid="R4" ref-type="bibr">4</xref>,<xref rid="R5" ref-type="bibr">5</xref>). Sagittal spinal curvatures act as a shock absorber. A flattened spine exerts one-fifth the structural strength compared to a spine with thoraco-lumbar curves (<xref rid="R4" ref-type="bibr">4</xref>). In addition, spinal mobility was proven as an important factor relating quality of life (<xref rid="R6" ref-type="bibr">6</xref>,<xref rid="R7" ref-type="bibr">7</xref>). Moreover, studies have shown hyperkyphosis is associated with decreased mobility, poor balance and greater body sway, which in turn is correlated with increased risk of falls, vertebral fractures, chronic back pain and functional decline (<xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R7" ref-type="bibr">7</xref>). </p><p> Reduced range of motion, as a natural consequence of the muscle-tendon unit and surrounding connective tissue stiffness with aging has been considered as a major cause of falls. Some studies have indicated that reduced range of motion, especially in hip and ankle joints may increase the risk of falls (<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R9" ref-type="bibr">9</xref>). Gait studies of healthy elderly have shown decreased walking speed, less range of motion of the hip, knee, and ankle joints, and increased anterior pelvis tilt (<xref rid="R9" ref-type="bibr">9</xref>,<xref rid="R10" ref-type="bibr">10</xref>). </p><p> Muscle &#x02013;tendon tightness that associated with aging process especially affect the bi-articular muscles. Kerrigan et al (<xref rid="R8" ref-type="bibr">8</xref>) showed that elderly fallers are characterized by marked hip tightness. They also reported an increase in peak hip extension as a result of 10 week stretching exercises (<xref rid="R11" ref-type="bibr">11</xref>). In addition, studies with older persons have shown an association between improved flexibility and increases in activities of daily living and quality of life (<xref rid="R6" ref-type="bibr">6</xref>, <xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R12" ref-type="bibr">12</xref>) </p><p> It seems that contractures of bi-articular muscles of hip, knee and ankle hip flexion contractures to occur with reduced of physical activity in elderly (<xref rid="R8" ref-type="bibr">8</xref>). Also, the standing posture depends not only on spinal alignment but also on hip, knee and ankle joints alignments (<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R3" ref-type="bibr">3</xref>). Therefore, it is important to explore this issue in the Iranian elderly. The present study aimed to compare the thoracic and lumbar curves and hip extension and ankle dorsiflexion range of motions between old and young persons </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> 30 healthy old persons over 60 years old (age range, 63-78 yrs, 16 female, 14 male) were recruited to participate in this study. The participants had been living in the facility care unit and were medically stable with no evidence of vestibular or neuromuscular conditions. All the subjects were independent in daily living activities no walking aids. The control group (young group, n=30) with an average age of 23.37 &#x000b1; 2.31 years was height, weight and sex matched, with same exclusion criteria. Before participating in the study, all subjects signed an informed consent form approved by the human subjects committee of University of Social Welfare &#x00026; Rehabilitation Sciences. </p><sec id="s2-1"><title>Test procedures</title><p> A standard goniometer was used for measuring passive range of motion of hip and ankle joints. Each measurement was repeated twice to allow evaluation of intratester reliability. The right and left lower extremities were assessed for each participant .The mean of the two measurements was considered as the joint motion value. For the measurement of passive hip extension the subject lies prone. Relaxed hip extension angle was measured using a goniometer and defined as the angle between the horizontal and a line from the greater trochanter to the lateral femoral condyle (<xref rid="R8" ref-type="bibr">8</xref>). Length of plantar flexors was determined by measuring the amount of ankle joint dorsiflexion with the knee extended. The subject was positioned in the prone position with the foot hanging off the table and the subtalar joint maintained in the neutral position. Dorsiflexion was measured with a standard goniometer as the angle formed by the lateral midline of the leg on a line from the head of the fibula to the tip of the lateral malleolus and the lateral midline of the foot in line with the border of calcaneus (<xref rid="R13" ref-type="bibr">13</xref>). The average measurement of two trials with 5-second pause between trials was recorded. </p><p> Subject&#x02019;s spinal curves were assessed using flexible ruler and the measurement performed with the subject standing in relaxed posture. For the measurement of spinal curves, the flexible ruler is placed between two marked points (T1, T12) vertebrae for measurement of kyphosis and T12, S2 vertebrae for measurement of lordosis) and shaped as spinal curves. The flexible ruler was removed and placed on a piece of white paper so the spinal curves could be copied by a pencil along the flexible ruler. A vertical line was drawn to connect the T1 to T12 and T12 to S2 landmarks (total length of curve/L line) and the maximal width of spinal curvature measured in centimeter (H line). Then using the equation &#x004e8;=4&#x000d7; [arc tang (2H/L)] the degree of spinal curves were calculated (<xref rid="R14" ref-type="bibr">14</xref>). </p><p> The data was analyzed using the SPSS statistical software version 19. Means and standard deviation were calculated. The intra-class correlation coefficient (ICC), two way mixed effect model, and standard error of measurement (SEM values) were used to assess intra-tester reliability of the measurement. We calculated the ICC (<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R1" ref-type="bibr">1</xref>) as described by Shrout and Fleiss (<xref rid="R15" ref-type="bibr">15</xref>), because only one judge evaluated the same population of subjects. </p><p> Independent t tests were performed to compare hip extension and ankle dorsiflexion range of motions and thoracic and lumbar curves between the young and elderly persons. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> <xref ref-type="table" rid="T1">Table 1</xref> presents the ICC and SEM values for test- retest reliability of the each measurement taken in the pilot study. ICC values for the all measurements were greater than 0.90. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Intraclass correlation coefficient (ICC) and standard error of measurement (SEM) values for intratester reliability. (N=10 subjects) </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td style="border-width: 0px 1px 0pt 0px ; border-style: none solid none none;border-color:#fff8e8" rowspan="1" colspan="1">Variables</td><td align="center" style="border-width: 0px 1px 0pt 0px ; border-style: none solid none none;border-color:#fff8e8" rowspan="1" colspan="1">ICC</td><td align="center" rowspan="1" colspan="1">SEM</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Lumbar curve</td><td align="center" rowspan="1" colspan="1">0.92</td><td align="center" rowspan="1" colspan="1">0.87</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Thoracic curve</td><td align="center" rowspan="1" colspan="1">0.90</td><td align="center" rowspan="1" colspan="1">0.82</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Hip extension range</td><td align="center" rowspan="1" colspan="1">0.93</td><td align="center" rowspan="1" colspan="1">1.10</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Ankle dorsiflexion range</td><td align="center" rowspan="1" colspan="1">0.97</td><td align="center" rowspan="1" colspan="1">1.56</td></tr></tbody></table></table-wrap><p> Descriptive statistics for the measurement scores in two groups presented in <xref ref-type="table" rid="T2">Table 2</xref>. </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Results of independent t test comparing of hip extension and ankle dorsiflexion(plantar flexors length) ranges and spinal curves between the young and old groups </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="2" colspan="1">Variables (measurement unit)</td><td align="center" colspan="2" rowspan="1">Group(Mean&#x000b1;SD)</td><td align="center" rowspan="2" colspan="1">Mean differences</td><td align="center" rowspan="2" colspan="1">p</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">young</td><td align="center" rowspan="1" colspan="1">old</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Lumbar curve (degree)</td><td align="center" rowspan="1" colspan="1">31.29&#x000b1;6.37</td><td align="center" rowspan="1" colspan="1">27.93&#x000b1;8.11</td><td align="center" rowspan="1" colspan="1">3.36</td><td align="center" rowspan="1" colspan="1">0.08</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Thoracic curve (degree)</td><td align="center" rowspan="1" colspan="1">34.43&#x000b1;13.27</td><td align="center" rowspan="1" colspan="1">36.19&#x000b1;8.97</td><td align="center" rowspan="1" colspan="1">1.76</td><td align="center" rowspan="1" colspan="1">0.55</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Right Hip extension range (degree)</td><td align="center" rowspan="1" colspan="1">24&#x000b1;4.70</td><td align="center" rowspan="1" colspan="1">13.6&#x000b1;5.99</td><td align="center" rowspan="1" colspan="1">3.40</td><td align="center" rowspan="1" colspan="1">p&#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Left Hip extension range (degree)</td><td align="center" rowspan="1" colspan="1">16.37&#x000b1;3.08</td><td align="center" rowspan="1" colspan="1">8.72&#x000b1;4.56</td><td align="center" rowspan="1" colspan="1">9.97</td><td align="center" rowspan="1" colspan="1">p&#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Right Ankle dorsiflexion range (degree)</td><td align="center" rowspan="1" colspan="1">17.230&#x000b1;3.19</td><td align="center" rowspan="1" colspan="1">6.57&#x000b1;8.30</td><td align="center" rowspan="1" colspan="1">10.66</td><td align="center" rowspan="1" colspan="1">p&#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Left Ankle dorsiflexion range (degree)</td><td align="center" rowspan="1" colspan="1">16.83&#x000b1;3.10</td><td align="center" rowspan="1" colspan="1">6.86&#x000b1;8.40</td><td align="center" rowspan="1" colspan="1">10.94</td><td align="center" rowspan="1" colspan="1">p&#x0003c;0.001</td></tr></tbody></table></table-wrap><p> The result of independent t test showed that the mean of lumbar curve was higher in young group (31.29&#x000b1; 6.37) than elderly ones (27.93&#x000b1;8.11), however, no significant difference was found between two groups (p=0.08) (<xref ref-type="table" rid="T2">Table 2</xref>). The result also showed increased thoracic curvature with aging process (young group=34.43&#x000b1;13.27, old group=36.19&#x000b1;8.97), however, no significant difference was found between two groups(p=0.55). </p><p> The result of independent t test also showed that young person&#x02019;s demonstrated a significantly higher hip extension (hip flexors length) with ankle dorsiflexion (plantar flexors length) range of motions than elderly group (p&#x0003c;0.01)(<xref ref-type="table" rid="T2">Table 2</xref>). </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Due to increasing elderly population is worldwide, concern over health problems and medical and social costs of the elderly are growing. The data from this study demonstrated that reduced hip extension and ankle dorsiflexion occur in elderly (p&#x0003c;0.01). In agreement with previous studies (<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R11" ref-type="bibr">11</xref>,<xref rid="R16" ref-type="bibr">16</xref>) the present study showed that reduced hip extensors or hip flexion contractures occur in Iranian elderly. There is a tendency with age to develop contractures of the hips, because the hip joint is less often fully stretched due to reduction in physical activity (<xref rid="R10" ref-type="bibr">10</xref>). On the other hand, the only regular daily activity that extends the hip to its maximum and thereby stretching the hip flexors are walking and running (<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R15" ref-type="bibr">15</xref>). Thus a decline in these activities along with aging will contribute to hip flexors tightness and contracture (<xref rid="R10" ref-type="bibr">10</xref>). It also suggested that hip flexion contractures or reduced hip extension may lead to an increase in anterior pelvic tilt and decrease in stride length and walking speed in elderly people (<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R11" ref-type="bibr">11</xref>). A reduction in hip extension range and stride length may be particularly important in situations requiring rapid changes in stride length such as, attempting to change walking speeds rapidly, or when faced with uneven surfaces or obstacles (<xref rid="R8" ref-type="bibr">8</xref>,<xref rid="R16" ref-type="bibr">16</xref>). </p><p> The sagittal motion around the hip joint is achieved by the hip joint itself and by the support of the lumbar spine (<xref rid="R17" ref-type="bibr">17</xref>). In our measurement of lumbar lordosis, the mean angle in the old and young group were 27.93&#x000b1;8.11 and 31.29&#x000b1;6.37respectively and the thoracic mean angle in the old and young group 36.19&#x000b1;8.97 and 34.43&#x000b1;13.27, respectively, however these differences were not significant. </p><p> Some studies showed that degenerative changes of the lumbar spine in middle-aged and elderly people can lead to decrease in lumbar lordosis and thoracic kyphosis (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R18" ref-type="bibr">18</xref>). Berlemann et al (<xref rid="R19" ref-type="bibr">19</xref>) and Tsuji et al (<xref rid="R2" ref-type="bibr">2</xref>) demonstrated that in elderly people, decreasing lumbar lordosis lead to increasing thigh muscle function and knee flexion while standing .Offierski and Macnab (<xref rid="R3" ref-type="bibr">3</xref>) and Tsuji et al (<xref rid="R2" ref-type="bibr">2</xref>) reported a correlation between spine and hip joint pain and lumbar kyphosis and knee flexion position, respectively. They reported that concurrent diseases at hip-spine and knee-spine are common clinical problems in elderly (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R3" ref-type="bibr">3</xref>). It also suggested that kyphotic condition alters balance and increases the incidence of falling (<xref rid="R17" ref-type="bibr">17</xref>,<xref rid="R20" ref-type="bibr">20</xref>). The flattening of the lumbar spine thrusts the old person forward. It increases the load at anterior part of vertebral body and increases fall risk (<xref rid="R4" ref-type="bibr">4</xref>). </p><p> Our finding also showed that old people have decreased length of the calf muscles (ankle dorsiflexion range) than young people. According to previous studies, the old people had changes in the ankle joint capsule, associated ligaments fascia, the skin and decreased calf muscles length (<xref rid="R21" ref-type="bibr">21</xref>,<xref rid="R22" ref-type="bibr">22</xref>). Aging is known to bring about a loss of functional motor units, a decrease in the number and the size of both muscle fibers (type I, II), with the possibility of selective atrophy of type II fibers. The reduction in the number of functional motor units and muscle fibers may account for the decreased muscle mass, strength and extensibility observed for the older people (<xref rid="R21" ref-type="bibr">21</xref>). Gajdosik also showed that the older women had decreased maximal isometric strength of the calf muscles (<xref rid="R21" ref-type="bibr">21</xref>). Changing calf muscle function due to decreased length and strength may have some impact on ambulation and static and dynamic standing balance that warrant further investigation (<xref rid="R21" ref-type="bibr">21</xref>,<xref rid="R22" ref-type="bibr">22</xref>). </p></sec>
The effect of preterm birth on vestibular evoked myogenic potentials in children
<p> <bold>Background:</bold> Preterm birth is a significant global health problem with serious short- and long-term consequences. This study examined the long term effects of preterm birth on vestibular evoked myogenic potentials (VEMPs) among preschool-aged children. </p><p> <bold>Methods:</bold> Thirty-one children with preterm and 20 children with term birth histories aged 5.5 to 6.5 years were studied. Each child underwent VEMPs testing using a 500 Hz tone-burst stimulus with a 95 dB nHL (normal hearing level) intensity level. </p><p> <bold>Results:</bold> The mean peak latencies of the p13 and n23 waves in the very preterm group were significantly longer than for the full-term group (p&#x02264; 0.041). There was a significant difference between very and mildly preterm children in the latency of peak p13 (p= 0.003). No significant differences existed between groups for p13-n23 amplitude and the interaural amplitude difference ratio. The tested ear and gender did not affect the results of the test. </p><p> <bold>Conclusion:</bold> Prolonged VEMPs in very preterm children may reflect neurodevelopmental impairment and incomplete maturity of the vestibulospinal tract (sacculocollic reflex pathway), especially myelination. VEMPs is a non-invasive technique for investigating the vestibular function in young children, and considered to be an appropriate tool for evaluating vestibular impairments at the low brainstem level. It can be used in follow-ups of the long-term effects of preterm birth on the vestibular system. </p>
<contrib contrib-type="author"><name><surname>Eshaghi</surname><given-names>Zahra</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Jafari</surname><given-names>Zahra</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Shaibanizadeh</surname><given-names>Abdolreza</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Jalaie</surname><given-names>Shohreh</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Ghaseminejad</surname><given-names>Azizeh</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Early interaction with a marginally inappropriate extra-uterine environment in preterm infants could play a crucial role in short- and long-term sequelae. Results from previous studies confirm the presence of high rates of neurodevelopmental impairment among preschool children with preterm birth history that span neuromotor functioning, cognition, language, and emotional/behavioral adjustment (<xref rid="R1" ref-type="bibr">1</xref>). The average rate of preterm birth is 9.6% Worldwide (<xref rid="R2" ref-type="bibr">2</xref>). In the last two decades, the rate of survival after preterm birth, especially in socio-economically developed countries, has significantly increased (<xref rid="R3" ref-type="bibr">3</xref>). This is mainly the result of improvements in neonatal intensive care. Studies investigating brain structural development in prematurely born group consistently show structural alterations in both cortical and sub-cortical structures at birth and, considerably, throughout childhood and adolescence (<xref rid="R4" ref-type="bibr">4</xref>). </p><p> Vestibular evoked myogenic potential (VEMP) is a short-latency response of the sternocleidomastoid muscle following high level of acoustic stimulation. It is a manifestation of the vestibulocollic re&#x0fb02;ex, originated in the saccular macula, which moves through the vestibular nerve and nucleus, the vestibulospinal tract, on to the motor neurons of the sternocleidomastoid muscle (<xref rid="R5" ref-type="bibr">5</xref>). VEMPs are used for diagnoses ranging from otologic disorders to neuropathies and brainstem lesions (<xref rid="R6" ref-type="bibr">6</xref>). Delayed reflex is typically seen in central pathology. If there is no conductive hearing loss, the lesions of the end-organs, primary afferents, and the nerve root entry zone lead to absence of response (<xref rid="R5" ref-type="bibr">5</xref>). </p><p> Although studies on VEMPs in preterm groups are rare in the literature, the available results of studies on preterm infants consistently have shown that they are at risk of disability related to maturation of the vestibulocollic reflex (<xref rid="R7" ref-type="bibr">7</xref>-<xref rid="R9" ref-type="bibr">9</xref>). The vestibular system is important to motor development; vestibular dysfunction may lead to delayed postural control and locomotor development at later ages (<xref rid="R10" ref-type="bibr">10</xref>). Children with histories of preterm birth are at greater risk for motor disabilities and poor postural control than those with normal deliveries (<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R12" ref-type="bibr">12</xref>). This group should be followed closely for vestibular deficiency-related complications. The present study investigated the long term effects of preterm birth on the vestibulocollic reflex pathway. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> This comparative-analytic study was performed on 31 children with histories of preterm birth (18 males, 13 females) and 20 with term births as controls (9 males, 11 females). The subjects were 5.5 to 6.5 yr of age and enrolled in the audiology clinic at school of rehabilitation between February and July of 2013. Children with preterm history were divided into very preterm (VP; less than 32 wk) and mildly preterm (MP; 32 to 37 wk) groups. The VP group comprised 10 children (6 males, 4 females) and the MP group 21 children (12 males, 9 females). The mean gestational age for the VP, MP, and control groups were 30.6 &#x000b1; 0.94 (28-31 wk), 34.9 &#x000b1; 1.28 (33-37 wk), and 39.6 &#x000b1; 0.74 (38-41 wk), respectively. The exclusion criteria for all groups were a history of congenital abnormalities, ototoxic drug use, recurrent ear infections, previous ear surgery, high fever, head trauma, mental retardation, and psychological problems (based on parental report). Parental approval, good general health of the child, absence of abnormalities of the external and middle ear, normal peripheral hearing (tested by conventional pure-tone audiometry and normal results for tympanometry) were considered as inclusion criteria for all children. The subjects were selected through a review of their medical records for the past 5.5 to 6.5 yr. </p><p> An ICS Charter EP device was used to record surface electromyographic activity of the SCM muscle. The active electrodes were attached to the upper half of the bilateral SCM muscles; a reference electrode was attached to the suprasternal notch and a ground electrode to the forehead. To elicit the desired response from each ear, the child was trained to rotate his or her head toward the contralateral side and keep his or her head in this position for several seconds (<xref rid="R13" ref-type="bibr">13</xref>). The children were awake and none sedated during the course of the test. Short tone bursts (95 dB nHL, 500 Hz each, rise-and-fall time = 2 ms, plateau time = 0 ms) were delivered monaurally using an ER-3A insert receiver. The stimulation rate was 5.1 Hz with the analysis time of 100 ms. The 150 responses to stimuli were then averaged, and their band-pass filtered (10-1500 Hz) and amplified (5000&#x000d7;) (<xref rid="R14" ref-type="bibr">14</xref>). Measurements were repeated twice to check test wave reproducibility. </p><p> Normal distribution of data was assessed by the One-Sample Kolmogorov-Smirnovtest. The One-way ANOVA Post-Hoc LSD ctest was used to compare the results. For statistical analysis, the data were presented as a mean &#x000b1; (standard deviation (SD)). All statistical analyses were done using SPSS Statistics 18.0 at significance level of 0.05. This study was approved by the ethics committee of Tehran University of Medical Sciences. </p></sec><sec sec-type="results" id="s3"><title>Results</title><p> No statistically significant differences were observed between results of the both ears (p&#x02265; 0.215). Biphasic waveforms for VEMPs were obtained for all children in the three groups. The mean latencies of the p13 and n23 waves for all groups are compared in <xref ref-type="fig" rid="F1">Fig. 1</xref>. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-75-g001"/><statement><p>Comparison of the mean p13 and n23 latency between three groups.</p></statement></fig><p> According to the one-way ANOVA statistical test the difference between three groups was significant in p13 latency (F=5.68, p=0.005), but not significant for n23 latency (F=2.33, p=0.10), p13-n23 amplitude (F=0.12, p=0.88) and inter-aural amplitude difference ratios (F=0.49, p=0.60). Results from precise analysis by ANOVA Post-Hoc LSD test are shown in <xref ref-type="table" rid="T1">Table 1</xref>. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Comparison of the VEMPs in three groups </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Normal Children (N)</td><td align="center" rowspan="1" colspan="1"> Mildly preterm<break/>(MP) </td><td align="center" rowspan="1" colspan="1">Very preterm(VP)</td><td align="center" rowspan="1" colspan="1">Groups</td><td align="center" rowspan="1" colspan="1">p</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">N (ears)</td><td align="center" rowspan="1" colspan="1">40</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">N,MP</td><td align="center" rowspan="1" colspan="1">0.906</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">P13 Latency (ms)</td><td align="center" rowspan="1" colspan="1">14.89 (0.69)</td><td align="center" rowspan="1" colspan="1">14.92 (1.19)</td><td align="center" rowspan="1" colspan="1">15.73 (1.02)</td><td align="center" rowspan="1" colspan="1">N,VP</td><td align="center" rowspan="1" colspan="1">0.002</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">MP,VP</td><td align="center" rowspan="1" colspan="1">0.003</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">N,MP</td><td align="center" rowspan="1" colspan="1">0.773</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">n23 Latency (ms)</td><td align="center" rowspan="1" colspan="1">21.65 (1.22)</td><td align="center" rowspan="1" colspan="1">21.75 (1.79)</td><td align="center" rowspan="1" colspan="1">22.51 (1.41)</td><td align="center" rowspan="1" colspan="1">N,VP</td><td align="center" rowspan="1" colspan="1">0.041</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">MP,VP</td><td align="center" rowspan="1" colspan="1">0.068</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">N,MP</td><td align="center" rowspan="1" colspan="1">0.924</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">p13-n23 amplitude (&#x003bc;V)</td><td align="center" rowspan="1" colspan="1">162.13 (122.9)</td><td align="center" rowspan="1" colspan="1">160.00 (84.46)</td><td align="center" rowspan="1" colspan="1">139.77 (79.57)</td><td align="center" rowspan="1" colspan="1">N,VP</td><td align="center" rowspan="1" colspan="1">0.627</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">MP,VP</td><td align="center" rowspan="1" colspan="1">0.681</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">N,MP</td><td align="center" rowspan="1" colspan="1">0.943</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Interaural amplitude difference ratio</td><td align="center" rowspan="1" colspan="1">18.82 (9.69)</td><td align="center" rowspan="1" colspan="1">19.05 (17.24)</td><td align="center" rowspan="1" colspan="1">24.59 (13.49)</td><td align="center" rowspan="1" colspan="1">N,VP</td><td align="center" rowspan="1" colspan="1">0.348</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">MP,VP</td><td align="center" rowspan="1" colspan="1">0.377</td></tr></tbody></table><table-wrap-foot><fn><p> Data are expressed as mean (SD). </p></fn></table-wrap-foot></table-wrap><p> Significant differences were observed in the latency of the p13 (p= 0.002) and n23 (p= 0.041) waves between the VP group and control group. <xref ref-type="fig" rid="F2">Fig. 2</xref> shows sample responses recorded from the right ear in one VP and one normal child. A significantly prolonged p13 latency was observed in the VP group over the MP group (p= 0.003). Moreover there was no significant difference between the MP and control groups for all test parameters (p&#x02265; 0.773). </p><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-75-g002"/><statement><p> VEMPs result of very preterm child (A) verses normal child (B). </p></statement></fig><p> There was no significant difference between the three groups in p13-n23 amplitude (p&#x02265; 0.627) and interaural amplitude difference ratios (p&#x02265; 0.348). Sex had no effect on the results (p&#x02265; 0.103). No significant differences were observed between the left and right ears for each group. </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Although, no similar study has been published on preschool aged children with preterm birth history, previous studies on term children reported non-significant differences between the right and left ears (<xref rid="R15" ref-type="bibr">15</xref>-<xref rid="R17" ref-type="bibr">17</xref>). It appears that maturation occurs similarly for ears on both sides. This reflective response generates from the lower brainstem and laterality has little effect on this potential. </p><p> The VP group showed significantly longer p13 and n23 latencies than the control group. Studies on preterm infants have also shown abnormal VEMPs results. Ecevit et al. (<xref rid="R7" ref-type="bibr">7</xref>) studied 17 late preterm infants (mean gestational age: 35.11 wk &#x000b1; 0.78) at 8 wk postnatal and found that the latencies of p13 and n23 in preterm infants were significantly longer than for term infants. Erbek et al. (<xref rid="R8" ref-type="bibr">8</xref>) studied 50 newborns with a mean gestational age of 31.7 &#x000b1; 2.7 wk (range= 26-36 wk). The results showed a significant difference between abnormal VEMPs rates for preterm and full term infants. Wang et al. (<xref rid="R9" ref-type="bibr">9</xref>) studied 27 low-risk preterm neonates and found that mean latency of peaks p13 and n23 in the preterm group were significantly longer than those in the full-term group. It has been reported that conditions affecting the vestibulospinal tract result VEMPs delay and that vestibule disorders frequently led to lack of response (<xref rid="R18" ref-type="bibr">18</xref>).Imaging studies confirmed structural alterations in cortical and sub-cortical structures at birth, and in childhood and adolescence in the preterm group (<xref rid="R4" ref-type="bibr">4</xref>). In other hand, previous studies have shown that the auditory brainstem response (ABR) was affected by preterm birth in infancy and at preschool age. For instance, Hasani et al. (<xref rid="R19" ref-type="bibr">19</xref>) studied 30 children with histories of preterm birth with 4-6 yr of age. Significant differences in the ABR test results were observed for the inter-peak intervals of the I-III and III-V waves and the absolute latency of the III wave. Overall, prolonged VEMPs in VP children may reflect incomplete maturation (especially myelination) and deficits in their vestibulospinal tract. </p><p> In the present study, no significant difference was found between the MP and control groups for wave latency of VEMPs. In contrast, Ecevit et al. (<xref rid="R7" ref-type="bibr">7</xref>) reported that wave latency for VEMPs in late preterm infants (34-36 wk) was significantly longer than those with full term. This condition may relate to the delay in maturation of the sacculocollic pathways in the MP group in infancy. In other hand, the VP was significantly different from the MP group for p13 latency. Previous studies have shown that the risk of mortality, morbidity, and neurodevelopmental impairment was tightly linked to gestational age and reached the highest rate for infants born at less than 32 wk (very preterm) (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R20" ref-type="bibr">20</xref>). Consequently, increased gestational age (GA) in children born at GA &#x0003e; 32 wk gestation showed less long-term sequelae than children with GA &#x0003c; 32 wk. </p><p> The p13-n23 amplitude did not differ between the three groups of children in this study. This is consistent with the findings of Wang et al. (<xref rid="R9" ref-type="bibr">9</xref>) on preterm and term infants; these groups showed no significant differences in the raw and corrected p13-n23 amplitudes. VEMPs amplitude is dependent upon sound intensity and constriction of the SCM muscle (<xref rid="R21" ref-type="bibr">21</xref>). Accordingly, since there are large variability in response amplitude, especially for the very young, fewer studies have considered it. The interaural amplitude difference ratio is the more informative parameter for investigating unilateral vestibular disorders (<xref rid="R14" ref-type="bibr">14</xref>). In the present study, the interaural amplitude difference ratio showed no significant difference between groups. These findings support the possibility of a symmetrical function of the vestibular system for both ears. </p><p> No significant difference was observed between the two genders for the test parameters. Previous studies have also reported no effect for gender on VEMPs responses (<xref rid="R15" ref-type="bibr">15</xref>, <xref rid="R22" ref-type="bibr">22</xref>, <xref rid="R23" ref-type="bibr">23</xref>). These findings could be a consequence of the elicitation of these reflecting responses from the lower brainstem (<xref rid="R24" ref-type="bibr">24</xref>). </p><p> Findings of the present study revealed the positive effect of augmented in age on the improvement of VEMPs results. Thus, in comparison with Wang et al. (<xref rid="R9" ref-type="bibr">9</xref>) study which VEMPs was not appeared in 74% of preterm neonates, in the present study, VEMPs was obtained in all children of the studied groups. Although, in the present study, abnormal VEMPs results were still observed, especially in the VP group.The early exposure to an extra-uterine environment for the VP group could exposed the neurodevelopmental process of these infants to physiological, psychological, and environmental hazards (<xref rid="R25" ref-type="bibr">25</xref>). It can be concluded that deficits in the central vestibular pathway could be related to incomplete myelinization of the neural pathways and the consequent delay in nerve conduction can leads to abnormalities such as delayed p13 and n23 waves in VEMPs. </p><p> Improvements in hygiene and reductions in the birth rate have decreased the incidence of preterm birth in Iran and, consequently, decreased the pool of these children with a history of preterm birth (especially very preterm). Future studies should expand access to the population sample of preterm children in different regions so that longitudinal studies can be designed to obtain more precise information about the effect of preterm birth on vestibular evoked myogenic potentials and the neurodevelopmental process on improvements in this response. Moreover, behavioral assessments of the vestibular system, especially on static balance skills, and electrophysiological tests can clarify the role of structural disabilities in balance and provide subtle information for counseling and planning useful treatment programs. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> The significant finding of the current study is the prolonged latency of p13 and n23 waves in the VEMPs in children with very preterm birth histories compared with those with histories of mildly preterm and full term births. Possibly, the delay in and deficit of the neural pathway of the vestibulocollic reflex and inadequate neural synchronization were the main causes for the difference. The results of this study provide information about effects of preterm birth for both specialists and parents. Since the VEMPs test evaluates the function of a part of the vestibular system, the probable effects of preterm birth on other aspects of the vestibular and balance systems should be studied. Other area of future research should explore the usage of VEMPs testing on performance of vestibulocollic reflex among children with preterm birth at the preschool age. </p></sec><sec id="s6"><title>Acknowledgments</title><p> This study was approved by the Tehran University of Medical Sciences, grant number 91/D/260/4955. We are grateful to the Mofid, AkbarabadiShaheedand Comprehensive Women hospitals for their assistance in the implementation of this project. We also extend our appreciation to the parents of our subjects for their help in this research. </p></sec><sec id="s7"><title>Conflict of interest</title><p> The authors declare that they have no competing interests. </p></sec>
Anti-cyclic citrullinated peptide antibodies in ulcerative colitis, and its relation with disease activity
<p> <bold>Background:</bold> Ulcerative colitis an inflammatory bowel disease (IBD) and chronically idiopathic immune related that associates with extraintestinal manifestations such as arthritis. Despite of the highly specificity of anticyclic citrullinated peptide (CCP) antibodies for rheumatoid arthritis, their role in IBD remains unclear. There are only a few studies on the prevalence of anti-CCP antibodies in patients with IBD. This study aimed to assess the prevalence of anti- CCP antibodies in ulcerative colitis and to investigate possible associations with their clinical and laboratory characteristics </p><p> <bold>Methods:</bold> In this cross-sectional study, 93 consecutive patients with ulcerative colitis referred to gastroenterology clinics in Razi referral hospital of Rasht, Iran, from September 2010 to September 2011. Rheumatologic examination, demographic data and clinical presentation of patients were recorded on specially prepared data sheets. Blood sample was collected for assessment of anti-CCP and other laboratory tests. Data were analyzed by the Chi square test, Fisher Exact test and student t test, using the SPSS 20 software for Windows, and P value less than 0.05 was considered significant. </p><p> <bold>Results:</bold> Of 93 patients, anti-CCP antibodies detected in 10.8% of cases (CI 95%: 4.5-17.1%). There were a significant relation between the prevalence of anti CCP positivity and aphthous ulcers and ocular manifestations whereas other parameters were not significantly related. </p><p> <bold>Conclusion:</bold> Anti CCP may have a possible role in some ulcerative colitis manifestations but there was no association between the presence of these antibodies and activity or extension of inflammatory colitis. We suggest other studies especially molecular studies to investigate other aspects of these antibodies in IBD patients </p>
<contrib contrib-type="author"><name><surname>Shafaghi</surname><given-names>Afshin</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mansour-ghanaei</surname><given-names>Fariborz</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Rostamnejad</surname><given-names>Maryam</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Amir Maafi</surname><given-names>Alireza</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Haji-abbasi</surname><given-names>Asghar</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Froutan</surname><given-names>Hossein</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> The inflammatory bowel diseases (IBD), Crohn&#x02019;s disease (CD) and ulcerative colitis (UC), are chronic disorders of immune system that affect gastrointestinal tract in genetically susceptible patients (<xref rid="R1" ref-type="bibr">1</xref>). </p><p> Twenty five percent of patients with this condition also develop several extraintestinal manifestations and 6&#x02013;46% of these, complicate with musculoskeletal involvements as the most common extraintestinal manifestations including articular, periarticular, and muscular involvement, osteoporosis and fibromyalgia. IBD related arthropathy, develop joint destruction that classified as an inflammatory arthritis (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R3" ref-type="bibr">3</xref>). </p><p> On the other hand, historically we know that anti-CCP antibody titers, first introduced in 1998, have a prognostic value in destruction of joint in RA (<xref rid="R4" ref-type="bibr">4</xref>-<xref rid="R5" ref-type="bibr">5</xref>). (With sensitivity of 80% and a specificity of 98%)and studies show that radiographic damages in rheumatoidarthritis (RA) patients with positiveanti-CCP antibodies are severe than other anti-CCP-negative ones. Moreover recently several studies demonstrated the association between this autoantibodis and arthropathies in other inflammatory conditions such as psoriatic arthritis (PsA), juvenile idiopathic arthritis and palindromic rheumatism (<xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R8" ref-type="bibr">8</xref>). </p><p> Martinez et al showed that anti-CCP antibodies are associated with erosive arthritis in SLE (<xref rid="R9" ref-type="bibr">9</xref>)and Gottenberg and colleagues found that 7.5% of patients with primary Sj&#x000f6;gren syndrome were positive for anti-CCP antibodies. (<xref rid="R10" ref-type="bibr">10</xref>) </p><p> Prediction of which IBD patient will develop the arthritis and discrimination of IBD-related arthritis from other anti-CCP positive arthritides could be an important point in treatment and prevention from late musculoskeletal sequels. Nevertheless there are only a few studies on the prevalence of anti-CCP antibodies in patients with IBD. </p><p> The aims of this study were to assess the prevalence of anti- CCP antibodies in ulcerative colitis cases and to investigate any associations with their clinical and laboratory characteristics. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> In this cross sectional study, 93 consecutive patients with UC referred to gastroenterology clinics in Razi referral hospital of Rasht, Iran, from September 2010 to September 2011, were enrolled. The diagnosis was established by classic criteria at least 6 months before study entry (<xref rid="R11" ref-type="bibr">11</xref>-<xref rid="R12" ref-type="bibr">12</xref>). </p><p> Patients with established or suspected diagnosis of RA and other rheumatologic diseases were excluded. Disease activity was classified based on Truelove &#x00026;Witts Criteria in these patients. Colonoscopy was done at this time for endoscopic and pathologic findings. </p><p> Disease extension was classified as ulcerative proctitis if&#x000a0;inflammation&#x000a0;limited to the rectum, ulcerative proctosigmoiditis refers to disease limited to the rectum and sigmoid colon, and not involving the descending colon. Left-sided or distal ulcerative colitis is defined as disease that extends beyond the rectum and as far proximally as the splenic flexure. Extensive colitis&#x000a0;refers to&#x000a0;disease extends beyond the splenic flexure but not as far as the cecum and ultimately pancolitis,&#x000a0;when the inflammatory process extends beyond the splenic flexure&#x000a0;involving the cecum. </p><p> All patients had a complete rheumatologic examination by an expert rheumatologist at the time of IBD diagnosis. At this stage, past and current occurrence of peripheral arthritis was evaluated in all cases. Arthritis was defined as at least one anamnestic or current episode of pain, swelling, and increased skin temperature in one or more joints. Peripheral arthritis diagnosed by clinical examination and peripheral joint disease, diagnosed by disease history, were recorded separately. X-rays performed when clinical findings were suggestive of erosive arthritis. Peripheral arthritis associated with UC was considered when other causes of joint diseases were ruled out. </p><p> Demographic data such as age, sex, and clinical presentation including articular manifestations (erythema, warmness and motion limitation), ocular manifestations(episcleritis, uveitis), dermatologic manifestations (erythema nodosum, pyodermagangrenosum) of patients were recorded on specially prepared data sheet. </p><p> Blood sample was collected during routine venepuncture .Laboratory tests including complete blood count, erythrocyte sedimentation rate, C-reactive protein and anti-CCP. </p><p> Anti-CCP reactivity was determined using a commercially available citrullinated protein antibodies ELISA kit of first generation (Genesis Diagnostics, UK), in which citrullinated recombinant rat filagrin is used as the antigen for the detection of anticitrullinated protein antibodies. The assay was performed according to the manufacturer&#x02019;s instructions. Anti-CCP antibodies were measured in U/mL and were considered to be positive at a cut-off value of&#x02265; 6.25 units/mL.This study was approved by the local ethical committee and informed consent for drawing extra blood at the time of routine venepuncture was obtained from subjects. </p><sec id="s2-1"><title>Statistical analysis</title><p> Results were expressed as mean &#x000b1; standard deviation (SD) or as number (percentage). Normality of the variables&#x02019; distribution was tested by using the one sample Kolmogorov-Smirnov test. Analysis was performed by using Chi square test, Fisher Exact test and student T test. All statistical analyses were done by the SPSS 20 software for Windows and p values less than 0.05 considered significant. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><p> Patients included in this study, 63(67.7%) male and 30(32.3%) female, with 15&#x02013;79 years old and the mean age of 35.23&#x000b1;13.68 years. </p><p> Demographic, clinical and laboratory characteristics of patients with and without anti-CCP were listed and summarized separately in <xref ref-type="table" rid="T1">Table 1</xref>. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Demographic, clinical and laboratory characteristics of patients with ulcerative colitis </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">Anti-CCP positive(n=10)</td><td align="center" rowspan="1" colspan="1"> Anti-CCP negative<break/>(n=83) </td><td align="center" rowspan="1" colspan="1"> <bold>p</bold> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Male/female</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">6/4</td><td align="center" rowspan="1" colspan="1">57/26</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age, mean &#x000b1; SD (years)</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">34.55&#x000b1;10.33</td><td align="center" rowspan="1" colspan="1">35.36&#x000b1; 12.65</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Duration of disease(years, mean &#x000b1; SD)</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">4.2 &#x000b1;1.3</td><td align="center" rowspan="1" colspan="1">4.4 &#x000b1;1.5</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Smokers</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3(30%)</td><td align="center" rowspan="1" colspan="1">23(27.7%)</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Treatment modality (Surgical/Medical )</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0/10</td><td align="center" rowspan="1" colspan="1">0/83</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Treatment with AZA,6MP</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">8/10(80%)</td><td align="center" rowspan="1" colspan="1">54/83(65%)</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Disease Extension</td><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Ulcerative proctitis</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Proctosigmoiditis</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Left-sided colitis</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Extensive colitis</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Disease Presentation</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Bowel movements (per day)</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x02264;4</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">66</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">4-6</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x02265;6</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Blood in stool</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">small</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">64</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Moderate</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">severe</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Arthralgia</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Arthritis</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Erythema nodosum</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Uveitis</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0.001*</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Aphthous ulcer</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">0.014*</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Fever</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x0003c;37.5&#x000b0;C mean (&#x0003c;99.5&#x000b0;F)</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">82</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x0003e;37.5&#x000b0;C mean (&#x0003e;99.5&#x000b0;F)</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Pulse rate(mean pulse)</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x0003c;60</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">60-90</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">70</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">&#x0003e;90</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Endoscopic appearance</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Erythema, decreased vascular pattern, fine granularity</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">29</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Marked erythema, coarse granularity, absent vascular markings, contact bleeding, no ulcerations</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">43</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Spontaneous bleeding, ulcerations</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Laboratory findings</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Hemoglobin (g/dl)</td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Normal or Mild (Hb&#x0003e;11)</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">88</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Moderate (Hb&#x0003e;10.5-11)</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Severe(Hb&#x0003c;10.5)</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">CRP(+)</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">76</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">ESR(&#x0003e;30 mm)</td><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">74</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><fn><p> * p values less than 0.05 were considered significant- NS= not significant </p></fn></table-wrap-foot></table-wrap><p> Anti-CCP antibodies were detected in 10.8% (95% CI: 4.5-17.1%) of patients. Only one anti-CCP positive patients had ocular manifestations (uveitis) compared to 0 out of 83 anti-CCP negative UC patients. </p><p> There were ten patients with aphthous ulcers, in which 30% of them (95% CI: 2-58%) were anti-CCP positive (p=0.014). Other parameters were not significantly different between two groups. </p><p> It is considered that the majority (93.5 %) of patient with UC classified as mild. There was no relation between disease activity or disease extension of UC and anti-CCP positivity. </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Anti-CCP antibodies are important diagnostic and prognostic markers for patient with RA. </p><p> These antibodies are directed toward epitopes that pass citrullination process; peptidyl arginine deiminase (PAD) enzymes modify structure and function of proteins by converting arginine residues to citrulline in post translational stage. This modification participates in regulation of immune system by converting the chemokines (<xref rid="R13" ref-type="bibr">13</xref>-<xref rid="R14" ref-type="bibr">14</xref>). </p><p> Inflammation of the joint, subsequent event that leads to citrulination of synovial proteins and developing anti-CCP are known mechanisms occurred in RA (<xref rid="R15" ref-type="bibr">15</xref>-<xref rid="R16" ref-type="bibr">16</xref>). Unlike RA, IBD does not include a homogenous complications and the role of antibodies against the citrullinated peptide is also not well understood in this condition. </p><p> Not many studies have evaluated anti-CCP in IBD despite of relatively high prevalence of musculoskeletal involvement in this disease. </p><p> Previous studies have shown that anti-CCP antibodies had a low prevalence in patients with IBD. Papamichael et al found that anti-CCP was positive in only 1 out of 84 patients (1.1%) with CD and 0 out of 50 case of UC. (<xref rid="R17" ref-type="bibr">17</xref>) This prevalence was reported 1.8% by Koutroubakis et al and 1.2% for IgA subclass of anti CCP by Haga et al. (<xref rid="R18" ref-type="bibr">18</xref>-<xref rid="R19" ref-type="bibr">19</xref>). </p><p> In the present study, it was found that anti-CCP was detected in 10/93 (10.7%) patients with UC. 13.9% of patients had peripheral arthritis that in 23.07% of them anti CCP detected but in Papamichaels study no clinical evidence of arthritis was seen in anti-CCP-positive patient. </p><p> Despite of the higher rate of anti-CCP antibody that was seen in our study, no significant association between the prevalence of anti-CCP positivity and IBD related arthritis was found. This result is similar to studies of Papamichaels and Koutroubakis but in earlier study by Haga et al, significant association between IgA class antibodies and arthritis in patients with IBD was shown (<xref rid="R17" ref-type="bibr">17</xref>-<xref rid="R19" ref-type="bibr">19</xref>). </p><p> Moreover, previous studies suggested highly specificity of these markers for RA, recent literature also have shown that anti-CCP antibodies may also be developed in the other inflammatory conditions such as PsA, juvenile idiopathic arthritis and palindromic rheumatism (<xref rid="R6" ref-type="bibr">6</xref>-<xref rid="R8" ref-type="bibr">8</xref>). </p><p> For example Gupta reported that anti-CCP antibodies were more frequently detectable in the sera of JIA patients with severe manifestations like erosions and deformity and concluded that anti-CCP antibodies could be important markers to predict severe complications of this disease (<xref rid="R20" ref-type="bibr">20</xref>) but, in our study no significant relation was found. </p><p> This differences suggested that pathogenesis of arthritis and other extraintestinal manifestations in UC may differ from mechanisms occurred in RA. According to the significant association that was seen in recent study by Huga, et al, relation between pathological changes in the intestinal mucosa of IBD patients with arthritis, could be considered. (<xref rid="R18" ref-type="bibr">18</xref>) We suggest further studies are needed to confirm this relation. </p><p> In present study there were significant differences in oral and ocular manifestation between positive and negative anti-CCP IBD patients. The probable reason for association between aphthous ulcers and anti-CCP positivity may be similarity of mouth antigens as a part of gastrointestinal tracts and joint antigens that confirmed by Huga, et al study. </p><p> In conclusion, we found a low prevalence of anti-CCP positivity in UC patients but it was greater than previous reports. This study had some limitations including patient&#x02019;s participation for colonoscopic evaluation, poor preparation of colon, overall diagnostic performance of antibody assay especially false positive and negative results, and also treatment effects on antibody positivity and its changes with drugs treatment (especially corticosteroids and immunosuppressive drugs). </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Our study has shown that there was no significant relation between UC activities, extension and presence of UC arthritis with anti-CCP positivity .We suggest that more studies are required on IBD patients especially on crohns disease patients and its clinical significance and also molecular studies &#x000a0;&#x000a0;to investigate other aspects of these antibodies in groups of patients. </p></sec>
Comparison of the efficacy of methotrexate and actinomycin D in the treatment of patients with stage I low risk gestational trophoblastic neoplasia (GTN)
<p> <bold>Background:</bold> Gestational trophoblastic neoplasia (GTN) refers to malignant lesions that arise from abnormal proliferation of placental trophoblast. Even in its metastatic forms GTN is curable with a cure rate of 90-100 %. Currently, methotrexate with or without folic acid, andactinomycin D is recommended for low risk GTN. The aim of this study is to compare the efficacy of methotrexate and actinomycin D as the first-line single chemotherapeutic agents for women with low-risk gestational trophoblastic neoplasia (LR-GTN). </p><p> <bold>Methods:</bold> A total of 30 women with LR-GTN were randomized to receive a weekly pulsed dose of 40 mg/m (2) of methotrexate intramuscularly (n=15) or a pulsed intravenous bolus of 1.25 mg/m (2) of actinomycin D every 2 weeks (n=15). An additional cycle was administered as consolidation treatment following normalization of the serum level of beta-human chorionic gonadotropin (&#x002c2;10 IU/L). </p><p> <bold>Results:</bold> Complete remission was achieved in 53.3% of patients in the methotrexate group and 86.7% in the actinomycin D group (p&#x002c2;0.04). The mean number of treatment cycles needed to achieve response was lower in the actinomycin D group (4.3 vs. 6.5). The mean duration from beginning of treatment till achieving complete remission was 9.6 weeks for the Act group and 13 weeks for the MTX group. </p><p> <bold>Conclusion:</bold> Actinomycin D may be a better option than methotrexate as a first-line chemotherapy agent for patients with LR-GTN but larger multicenter randomized controlled trials should be conducted to establish the most appropriate regimen for these patients. </p>
<contrib contrib-type="author"><name><surname>Shahbazian</surname><given-names>Nahid</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Razi</surname><given-names>Taghi</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Razi</surname><given-names>Shima</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Yazdanpanah</surname><given-names>Leila</given-names></name></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Gestational trophoblastic neoplasia (GTN) refers to malignant lesions tha arise from abnormal proliferation of placental trophoblast. These groups of diseases have a wide variety and can be divided into 4 groups: 1.Invasive mole, 2. Choriocarcinoma, 3. Placenta Site Trophoblastic Tumor, 4. Epitheloid Trophoblastic Tumor (ETT) (<xref rid="R1" ref-type="bibr">1</xref>). </p><p> In general, about 15% of patients with complete molar pregnancies develop GTN. The International Federation of Gynecology and Obstetrics (FIGO) reported the hCG criteria used for the diagnosis of post-molar GTN to include: 1) an hCG level plateau, plus or minus 10 % of baseline recorded over a 3-week duration (days 1,7,14,21); 2) an hCG level rise greater than 10% above baseline recorded over a 2-week duration (days 1,7,14); and 3) persistence of hCG for morethan 6 months after molar evacuation (<xref rid="R2" ref-type="bibr">2</xref>). The most common symptoms are: irregular vaginal bleeding, uterine sub involution or asymmetric enlargement, and theca lutein cysts (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> In the modified World Health Organization (WHO) prognostic scoring system as adapted by FIGO, each stage is divided into high risk and low risk groups. Prognostic factors including (age at diagnosis, preceding pregnancy, interval between preceding and index pregnancy, pretreatment serum level of &#x003b2;-hCG, size of largest tumor whether uterine or metastatic, site of metastases, number of metastases identified, and number of drugs included in previous failed chemotherapy) are scored as 0-4 and then calculated as final score. Patient's final score equal or less than 6 is low risk and a score higher than 6 is high risk. Low risk GTN (LR-GTN) is defined as molar pregnancy with WHO score lower than six. This categorization can be used to predict the patient's response to treatment with a single drug because most of the time low risks cases are treated with only one drug (<xref rid="R3" ref-type="bibr">3</xref>). </p><p> Despite the inevitability of death in untreated GTN, its treatment with chemotherapy is extremely effective. Therefore, it is the main modality of treatment in patient with GTN. </p><p> Even in its metastatic forms, GTN is curable with cure rate of 90-100 % (<xref rid="R4" ref-type="bibr">4</xref>). </p><p> The first successful treated GTN with Methotrexate (MTX) was reported in 1956. Currently, MTX with or without folic acid, Actinomycin D (Act) recommended for low risk GTN. MTX drug regimens are the most commonly used treatments. This treatment was associated with a 49% to 92.3% improvement (<xref rid="R5" ref-type="bibr">5</xref>). </p><p> The aim of this study was comparing the therapeutic effects of MTX and Act to find the best available treatment for these patients in south western region of Iran. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> In this clinical trial the participants included were all patients diagnosed with Stage I Low Risk GTN, referred to a gynecology clinic in Ahvaz, Iran from 2009 to 2011. Each patient was scored according to the WHO system; and low risk group was also determined (<xref ref-type="table" rid="T1">Table 1</xref>). Written consent was completed by all patients and the study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Science. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Characteristics of Methotrexate and Actinomycin D group </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Group 1(ACT-D)</td><td align="center" rowspan="1" colspan="1">Group 2(MTX)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Age (year)</td><td align="center" rowspan="1" colspan="1">26.47&#x000b1;6.9</td><td align="center" rowspan="1" colspan="1">26.87&#x000b1;9.6</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">score</td><td align="center" rowspan="1" colspan="1">2.33&#x000b1;1.17</td><td align="center" rowspan="1" colspan="1">2.33&#x000b1;1.34</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Number of Gravidity</td><td align="center" rowspan="1" colspan="1">2.8&#x000b1;2.59</td><td align="center" rowspan="1" colspan="1">2.6&#x000b1;2.16</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">&#x003b2;-hCG level before treatment</td><td align="center" rowspan="1" colspan="1">33591.7&#x000b1;47853</td><td align="center" rowspan="1" colspan="1">19650.53&#x000b1;39280.2</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Chemotherapy cycles for complete remission</td><td align="center" rowspan="1" colspan="1">4.33&#x000b1;2.55</td><td align="center" rowspan="1" colspan="1">6.53&#x000b1;3.29</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Duration of Treatment (day)</td><td align="center" rowspan="1" colspan="1">67.2&#x000b1;29.2</td><td align="center" rowspan="1" colspan="1">91.67&#x000b1;54.697</td></tr></tbody></table></table-wrap><p> Thirty participants randomly divided into two groups (each including 15 participants). This study's exclusion criteria were: </p><list list-type="order"><list-item><p> Previous cancer patients for whom chemotherapy is not possible (with the exception of non-melanoma skin cancer&#x0200f;) </p></list-item><list-item><p> Patients who had previously received chemotherapy due to GTN&#x0200f;. </p></list-item><list-item><p> Patients after developing a malignancy were free of disease for less than 5 years&#x0200f;. </p></list-item><list-item><p> Patients with abnormal liver, renal or hematologic function&#x0200f;. </p></list-item></list><p> Before starting chemotherapy in each group, in addition to collecting history and physical examination, tests such as: CBC, &#x003b2;-hCG, LDH, SGOT, SGPT, Total bilirubin, Alkaline-Phosphatase, BUN, Creatinine, Thyroid function tests ( TFT), chest x-ray (CXR), CT scan or Abdominopelvic sonography were done. Patients were randomly divided into ACT and MTX groups. In the ACT group patients received a dose of 1.25 mg/m<sup>2</sup>, intravenously, every two weeks and &#x003b2;-hCG levels were checked each week until a negative level was achieved. In the MTX group patients received a dose of 40 mg/m<sup>2</sup>, intramuscular, each week and &#x003b2;-hCG levels were checked each week until a negative level was achieved. Response to treatment is defined as achieving &#x003b2;-hCG levels less than 6mlU/mL over three consecutive weeks and no response to treatment is defined as less than 10% decrease in &#x003b2;-hCG values in three consecutive weeks or more than 20% rise in &#x003b2;-hCG values over two consecutive weeks. An extra course of chemotherapy as consolidation therapy was considered for each group after achievement of negative &#x003b2;-hCG levels. Failure of response to treatment leads to a change of drug and transfer to the opposite group. </p><p> After the first negative &#x003b2;-hCG serum level, it was checked weekly until negative &#x003b2;-hCG levels were maintained for three consecutive weeks and after that it was checked monthly. Complication rates in each group were assessed by a questionnaire at weekly visits. In this questionnaire factors such as mucosal ulcers, inhibition of bone marrow, hepatotoxicity, nausea, vomiting and skin necrosis were assessed and the prevalence of these complications were compared in both groups. </p><p> Data analyzed was done using SPSS version 19. Statistical comparison was performed using Chi-square and independent t-tests. p&#x0003c;0.05 was considered as statistically significant. </p></sec><sec sec-type="results" id="s3"><title>Results</title><p> Of 30 participants, one group (15 persons) was treated with Actinomycin D and another group (15 persons) was treated with Methotrexate. Characteristics of two groups are shown in <xref ref-type="table" rid="T1">Table 1</xref>. </p><p> There was no statistical difference between the two groups in terms of age (p=1). The disease score in both groups were compared and did not have significant difference (p=0.7). There was no statistical difference between the two groups in terms of gravidity (p=0.82). There was no statistical difference between the two groups in terms of &#x003b2;-hCG levels prior to treatment (p=0.39). </p><p> The mean number of courses of chemotherapy to achieve complete remission in the Actinomycin D group was 4.33 courses compared to 6.53 courses in the Methotrexate group. These numbers are statistically different (p=0.04) and Actinomycin D group received a less number of courses of chemotherapy. </p><p> Average duration of the course of treatment in Actinomycin D group was 67.2 days (9.5 weeks) and 91.67 days (13 weeks) in Methotrexate group. However, there was no statistical difference in the duration of treatment between the two groups (p=0.13). </p><p> In terms of response to treatment, in Actinomycin D group 13 participants responded to treatment, two did not respond and then were treated with Methotrexate. One patient responded to Methotrexate, the other did not respond and a multi-drug treatment (including etoposide, methotrexate, Actinomycin D, EMA/CO, cyclophosphamide, vincristine) was used and the patient was treated. </p><p> In the Methotrexate group, 8 patients responded to treatment, 7 patients did not respond and were referred for treatment with Actinomycin D, 4 patients responded to treatment but 3 of the 7 patients did not respond to Actinomycin D and were treated with a multi-drug regimen of EMA/CO. </p><p> The difference between the responses to treatment was statistically significant (p=0.04) and Actinomycin D was more effective. Comparison of response to treatment in two groups is shown in <xref ref-type="fig" rid="F1">Fig. 1</xref>. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-78-g001"/><statement><p>Comparison of mean duration of treatment in Methotrexate and Actinomycin D group</p></statement></fig><p> Regarding the side effects of these two drugs the following findings were seen: </p><list list-type="order"><list-item><p> Two participants in the Methotrexate group experienced nausea and vomiting during treatment&#x0200f;. </p></list-item><list-item><p> In the Actinomycin D group no cases of mucosal ulcers were seen but one case was seen in the Methotrexate group&#x0200f;. </p></list-item><list-item><p> One case of elevated liver enzymes was seen in the Methotrexate group&#x0200f;. </p></list-item></list><p> The rate of complications including mucosal lesions, increased liver enzymes, vomiting and other issues did not have statistically significant differences between the two groups (p=1,0.86,0.46 respectively). </p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> In comparison of the effects of Methotrexate and Actinomycin D to find the best treatment for patients with GTN, the Actinomycin D group received fewer chemotherapy courses to achieve remission and in general it was more effective. </p><p> So far different regimes and protocols have been reported for the treatment of low-risk GTD. But the best and most cost efficient protocol with minimal toxicity has not been achieved yet. However, each institution and country, based on their circumstances and their patient's conditions, select the appropriate treatment for their patients. For example, in Brazil the recommended treatment in deprived areas is Methotrexate despite the fact that the efficacy did not differ between Methotrexate and Actinomycin D groups. Additionally, patient satisfaction was higher in the Actinomycin group (<xref rid="R15" ref-type="bibr">15</xref>). </p><p> In our study, complication rates were not different between the two groups; but in practice, were higher in the Methotrexate group. In three clinical trials, complete remission with MTX pulse therapy was 49-53%, markedly lower than ACT-D pulse treatment which was 69-90% (<xref rid="R18" ref-type="bibr">18</xref>-<xref rid="R20" ref-type="bibr">20</xref>). In one study, a 5-day regimen of Actinomycin was compared to an 8-day regimen of methotrexate and Folinic acid (MTX-FA) to treat GTD without metastases; complete remission was 100% in the Actinomycin group and 74% in the Methotrexate group (<xref rid="R17" ref-type="bibr">17</xref>). In a retrospective study by Abrao et al, low-risk GTD patients treated with 5-day regimen of ACT-D or MTX and Folinic acid (MTX-FA) or a combination of ACT-D and MTX were analyzed; no significant difference in the recovery rate of the three groups was seen (61.4%, 69% and 79.1% respectively). The side effects in the combination therapy group were 62.5%, in the 5 day ACT-D regimen group were 19% and in the 5 day MTX regimen group were 28.6% (p=0.0003) (<xref rid="R15" ref-type="bibr">15</xref>). </p><p> In a study by Yarndy et al., the mean number of chemotherapy courses to achieve complete remission was 4.8 in ACT-D group and 6.8 in the MTX group and the duration of treatment in the ACT-D group was longer (<xref rid="R18" ref-type="bibr">18</xref>). In our study the number of courses of chemotherapy in patients treated with Actinomycin D was also less than the other group (4.33 versus 6.53). With this we can conclude that patients are more likely to be satisfied with Actinomycin D. But it should be noted that the duration of the course of treatment in this study was not statistically different between the two groups. In other studies, mean number of chemotherapy courses to achieve complete remission was 4 courses with Actinomycin D (<xref rid="R17" ref-type="bibr">17</xref>). In a prospective study by the Gynecology Oncology Group (GOG), ACT-D pulse regime once every two weeks with a remission rate of 73% was compared with MTX regime once every week with a remission rate of 58%. ACT-D pulse regime once every two weeks intravenously was more effective than intramuscular MTX regime once every week (<xref rid="R20" ref-type="bibr">20</xref>). In our study the response rate was higher in ACT-D group compared to MTX group (86.6% compared to53.3%). In one study in Thailand, drugs were given as 5 day doses and again the effectiveness of ACT-D was higher than MTX (<xref rid="R17" ref-type="bibr">17</xref>) .In Baptista et al. study MTX, ACT-D and etoposide were compared. Remission rate with etoposide was 100%, with ACT-D was 90% and with MTX was 50%. Similar to this study ACT-D was more effective than MTX. They found mean time intervals between beginning treatment and remission similar in both groups (<xref rid="R21" ref-type="bibr">21</xref>). </p><p> In a study by Yarandi et al., the most important predictor of treatment response was &#x003b2;-hCG levels before treatment. However, in our study despite equal serum &#x003b2;-hCG levels in both groups, response to treatment was more in the ACT-D group (<xref rid="R18" ref-type="bibr">18</xref>). </p><p> Finally, given the findings of this study and previous studies, Actinomycin D is a more effective treatment for low-risk GTD. Moreover, because ACT-D has the lowest toxicity levels, it may offer the best option to treat patients with low-risk GTD. More research is needed to determine the most ideal one-drug regimen. Limitation of this study was its sample size. We recommend larger population for future studies. </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Actinomycin D may be a better option than methotrexate as a first-line chemotherapy agent for patients with LR-GTN but larger multicenter randomized controlled trials should be conducted to establish the most appropriate regimen for these patients. </p></sec>
Extra-axial medulloblastoma in cerebello-pontine angle: A report of a rare case with literature review
<p>Medulloblastoma is quite uncommon in the adult population and even rarer in extra-axial site in cerebello-pontine (CP) angle. In this report, a 23-year-old male patient with a two month history of deafness, nausea, vomiting and ataxia is presented. Clinical and radiological findings demonstrated a heterogeneously enhanced extra-axial lesion in the right CP angle. Total excision was performed and the histopathological features of medulloblastoma were confirmed. After surgery, the patient had no neurological deficit and the audiometric findings were improved. In addition, he underwent adjuant radiotherapy and no sign of metastatic mass was observed in follow-up spinal cord MRI. Although extremely rare, medulloblastoma must be considered in the differential diagnosis of extra-axial CP angle lesions.</p>
<contrib contrib-type="author"><name><surname>Bahrami</surname><given-names>Eshagh</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Bakhti</surname><given-names>Sahar</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Fereshtehnejad</surname><given-names>Seyed-Mohammad</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Parvaresh</surname><given-names>Mansour</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Khani</surname><given-names>Mohammad Reza</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Medulloblastoma is a common childhood tumor of the posterior fossa, which accounts for 21.8% of all pediatric primary malignant central nervous system tumors in Iran (<xref rid="R1" ref-type="bibr">1</xref>). However, this malignancy is quite rare in the adult population with the incidence rate of 0.4% to 1% among all primary adult brain tumors (<xref rid="R2" ref-type="bibr">2</xref>). Most of the cases with cerebello-pontine (CP) angle medulloblastomas are intra-axial, while only a few of them represents extra-axially that makes this site of tumor even extremely rare in adults (<xref rid="R3" ref-type="bibr">3</xref>). In the current report, we outline the clinical features, imaging results and surgical treatment of an extra-axial medulloblastoma in CP angle of a young man. Thereafter, relevant literature and the few similar cases are discussed in order to review the available evidences to manage this rare site of tumor. </p></sec><sec id="s2"><title>Case Report</title><p> A 23 year old man presented with a two month history of deafness of the right ear followed by nausea, vomiting and ataxia. Neurological examination yielded normal findings, except for audiometric examination that showed hearing loss and ataxic gait. In particular, audiometric assessment demonstrated a discrete sensorineural hearing loss of 50 dB at 1 KHz to 8 KHz. Magnetic resonance imaging (MRI) revealed a cystic and necrotic lesion in the right CP angle. The lesion was heterogenously more hypointense on T1 and hyperintense on T2 (<xref ref-type="fig" rid="F1">Fig. 1</xref>). The lesion enhanced on T1 weighted MRI images after injection of gadolinium (<xref ref-type="fig" rid="F2">Fig. 2</xref>). Spinal cord MRI did not reveal any evidence of metastasis. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-57-g001"/><statement><p>Axial MRI shows the heterogeneous extra-axial lesion with cystic component on T1 (A) and T2 (B) in the right CP angle.</p></statement></fig><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-57-g002"/><statement><p> Brain MRI in coronal (A), axial (B) and sagittal (C); Gadolinium injection shows a right CP angle lesion with heterogeneous enhancement of solid component and compression effect on the 4<sup>th</sup> ventricle </p></statement></fig><p> He underwent right retromastoid craniectomy and gross total excision of the lesion with facial nerve monitoring. There was a clear plane between the tumor and cerebellum, whereas it was adherent to tent laterally. Histopathologic evaluation showed &#x0201c;pale islands&#x0201d; consisting of micronodular, reticulin-free zones with a low magnification appearance similar to follicular lymphoid hyperplasia. The lesion was characterized by reduced cellularity, a rarefied fibrillar matrix and compact undifferentiated area (<xref ref-type="fig" rid="F3">Fig. 3</xref>). After surgery, the patient had no neurological deficit and his audiometric findings were improved. The postoperative MRI showed total extirpation of the tumor (<xref ref-type="fig" rid="F4">Fi. 4-A</xref>). In addition, he underwent radiotherapy as adjuvant treatment. The latest follow-up was performed one-year post-operation by means of brain MRI, which shows no lesion in the right CP angle (<xref ref-type="fig" rid="F4">Fig. 4-B</xref>). </p><fig id="F3" orientation="portrait" position="float"><label> Fig. 3 </label><graphic xlink:href="MJIRI-28-57-g003"/><statement><p> Desmoplastic/nodular medulloblastoma. Microphotograph of histopathology slide (H and E staining). Micronodular zones of reduced cellularity (pale islands) are a striking feature of this medulloblastoma variant. </p></statement></fig><fig id="F4" orientation="portrait" position="float"><label> Fig. 4 </label><graphic xlink:href="MJIRI-28-57-g004"/><statement><p> Post-operative MRI shows total resection of tumor with no detectable enhancement in the right CP angle promptly after surgery (A) and one-year post-operation follow-up (B) </p></statement></fig></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p> Medulloblastoma is a predominantly pediatric tumor commonly occurring intra-axially in the cerebellar vermis as the most common site of origin (<xref rid="R4" ref-type="bibr">4</xref>). Origin of medulloblastoma may be either from germinal cells or their remnants situated at the end of inferior medullary velum or from remnants of the external granular layer, however, the exact origin is not certainly known yet (<xref rid="R5" ref-type="bibr">5</xref>-<xref rid="R6" ref-type="bibr">6</xref>). Adult medulloblastomas usually arise from the surface of the cerebellum or pons and 50% of these are laterally located (<xref rid="R5" ref-type="bibr">5</xref>,<xref rid="R7" ref-type="bibr">7</xref>). Extra-axially, the tumors have been localized in the tentorial region or the CP angle (<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R8" ref-type="bibr">8</xref>). In another point of view, there are only a few cases of CP angle medulloblastomas and most of them are intra-axial, which makes the extra-axial site of this tumor extremely rare. Recently, two misdiagnosed adult cases of medulloblastoma of the CP angle are reported, which were wrongly marked as vestibular schwannoma and petrosal meningioma during the preoperative radiological assessment (<xref rid="R9" ref-type="bibr">9</xref>). </p><p> Development of this tumor in the CP angle may be from the remnants of the external granular layer in the cerebellar hemisphere, including the flocculus which faces the CP angle (<xref rid="R10" ref-type="bibr">10</xref>-<xref rid="R11" ref-type="bibr">11</xref>). It may grow to occupy the CPA through two ways including lateral extension from the 4<sup>th</sup> ventricle through the foramen of Lushka, or direct exophytic growth from the site of origin at the surface of the cerebellum or pons (<xref rid="R6" ref-type="bibr">6</xref>). The lack of association with any cerebellar tissue and extra-axial location in the region of CP angle is an extremely rare phenomenon. Based on the latest literature review, less than 10 cases of extra-axial adulthood medulloblastoma have been reported in the CP angle. </p><p> <xref ref-type="table" rid="T1">Table 1</xref> summarizes the characteristics, clinical manifestation and treatment work-up for these reported cases. During the adulthood, patient age at diagnosis ranges from 21 to 52 years, mostly often occurring in the late 20s and early 30s (<xref rid="R12" ref-type="bibr">12</xref>) including the current case in our report with 23 years of age. It seems that a male preponderance is also seen for the reported cases. Regarding the clinical manifestations, no specific features have been ascribed to CP angle medulloblastomas. However, there are some characteristics that may help distinguish them from other CP angle lesions, mainly the most common tumor, acoustic neuroma followed by meningiomas, primary cholesteatomas and epidermoid tumours (<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R13" ref-type="bibr">13</xref>). Involvement of the V<sup>th</sup>, VI<sup>th</sup>, VII<sup>th</sup>, VIII<sup>th</sup> and lower cranial nerve and signs of cerebellar dysfunction are commonly noted in CP angle lesions (<xref rid="R14" ref-type="bibr">14</xref>). Early onset of progressive cerebellar signs and gait ataxia may indicate an axial origin of tumor, whereas positional nystagmus may be an early sign suggestive of an acoustic schwannoma (<xref rid="R11" ref-type="bibr">11</xref>). Hearing impairment and VII<sup>th</sup> nerve involvement is usually less common and a late feature of a CP angle medulloblastoma; however, this feature has been even reported as initial symptom in a few cases (<xref rid="R11" ref-type="bibr">11</xref>) including ours that also complained from diminution of hearing as initial symptom. As shown in <xref ref-type="table" rid="T1">Table 1</xref> , headache, nausea, vomiting and ataxia are among the most common symptoms reported in these cases. Related findings could be found in neurological examination such as papilloedema, hemiparesis, gait disturbances, facial paresis and etc. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Characteristics and work-up of published adult cases with extra-axial medulloblastoma in cerebello-pontine angle </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Author</td><td align="center" rowspan="1" colspan="1">Year</td><td align="center" rowspan="1" colspan="1">Patient&#x02019;s Age</td><td align="center" rowspan="1" colspan="1">Patient&#x02019;s Sex</td><td align="center" rowspan="1" colspan="1">Presentation</td><td align="center" rowspan="1" colspan="1">Neurological Examination</td><td align="center" rowspan="1" colspan="1">Treatment</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Becker et al. <sup> (<xref rid="R8" ref-type="bibr">8</xref>) </sup> </td><td align="center" rowspan="1" colspan="1">1995</td><td align="center" rowspan="1" colspan="1"> 32 yr<break/>52 yr </td><td align="center" rowspan="1" colspan="1"> Female<break/>Female </td><td align="center" rowspan="1" colspan="1"> Headache, <break/>vomiting </td><td align="center" rowspan="1" colspan="1">-</td><td align="center" rowspan="1" colspan="1">-</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Akay et al. <sup> (<xref rid="R6" ref-type="bibr">6</xref>) </sup> </td><td align="center" rowspan="1" colspan="1">2003</td><td align="center" rowspan="1" colspan="1">21 yr</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">Headache, nausea, vomiting, ataxia</td><td align="center" rowspan="1" colspan="1">Bilateral papilloedema, hemiparesis, hemihypesthesia</td><td align="center" rowspan="1" colspan="1">Partial excision, radiotherapy, chemotherapy</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Gil-Sal&#x000fa; et al. <sup> (<xref rid="R12" ref-type="bibr">12</xref>) </sup> </td><td align="center" rowspan="1" colspan="1">2004</td><td align="center" rowspan="1" colspan="1">40 yr</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">Headache, vomiting, hearing difficulties</td><td align="center" rowspan="1" colspan="1">One-side trigeminal 1st and 2nd nerves deficit</td><td align="center" rowspan="1" colspan="1">Total excision, adjuvant therapy</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Fallah et al. <sup> (<xref rid="R3" ref-type="bibr">3</xref>) </sup> </td><td align="center" rowspan="1" colspan="1">2009</td><td align="center" rowspan="1" colspan="1">47 yr</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">Headache, nausea, vomiting</td><td align="center" rowspan="1" colspan="1">Normal</td><td align="center" rowspan="1" colspan="1">Tumor resection, radiotherapy</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Furtado et al. <sup> (<xref rid="R5" ref-type="bibr">5</xref>) </sup> </td><td align="center" rowspan="1" colspan="1">2009</td><td align="center" rowspan="1" colspan="1">32 yr</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1"> Headache,<break/>Vomiting, gait unsteadiness </td><td align="center" rowspan="1" colspan="1">Papilloedema, right-sided dymetria, dysdiadokokinasia, gait ataxia</td><td align="center" rowspan="1" colspan="1">Total excision, referred to radiation oncologist for further management</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Singh et al. <sup> (<xref rid="R18" ref-type="bibr">18</xref>) </sup> </td><td align="center" rowspan="1" colspan="1">2011</td><td align="center" rowspan="1" colspan="1">21 yr</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">Headache, vomiting, ataxia, left facial weakness</td><td align="center" rowspan="1" colspan="1">Papilloedema, left lower motor neuron facial paresis, left IX and X cranial nerve paresis, left cerebellar signs</td><td align="center" rowspan="1" colspan="1">Total excision, failed to receive radiotherapy (recurrent lesions and metastasis after 15 months)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Spina et al. <sup> (<xref rid="R9" ref-type="bibr">9</xref>) </sup> </td><td align="center" rowspan="1" colspan="1">2013</td><td align="center" rowspan="1" colspan="1"> 22 yr<break/>26 yr </td><td align="center" rowspan="1" colspan="1"> Male<break/>Female </td><td align="center" rowspan="1" colspan="1"> Headache,<break/>hearing loss, tinnitus, dizziness and ataxia </td><td align="center" rowspan="1" colspan="1"> Weakness of the right arm, slight<break/>left nystagmus and a mild peripheral deficit of the left facial<break/>nerve </td><td align="center" rowspan="1" colspan="1">Total tumor resection, radiotherapy</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1">Present Study</td><td align="center" rowspan="1" colspan="1">2013</td><td align="center" rowspan="1" colspan="1">23 yr</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">Deafness, nausea, vomiting, ataxia</td><td align="center" rowspan="1" colspan="1">Hearing loss in audiometry, ataxic gait</td><td align="center" rowspan="1" colspan="1">Total excision, radiotherapy</td></tr></tbody></table></table-wrap><p> As we also found in our reported case, MRI assessment often shows heterogeneously gadolinium-enhancement lesions (<xref rid="R8" ref-type="bibr">8</xref>). However, there are some previous reports where the tumor has demonstrated a homogenous enhancement pattern, which may lead to misdiagnosis (<xref rid="R3" ref-type="bibr">3</xref>,<xref rid="R12" ref-type="bibr">12</xref>). </p><p> Histopathollogicaly, desmoplastic variant of medulloblastoma is more common in adults than the classical type (<xref rid="R5" ref-type="bibr">5</xref>). This variant is hemispheric in location and is more often associated with cysts and necrosis in comparison with classical medulloblastomas; however, there are no pathognomonic MRI criteria to differentiate between these two types (<xref rid="R15" ref-type="bibr">15</xref>-<xref rid="R16" ref-type="bibr">16</xref>). Medulloblastomas are known to metastasize through cerebrospinal fluid into the spinal canal, leptomeninges, and supratentorial regions. Metastasis in medulloblastomas varies between 38 and 60% in various series, with the spinal canal being the commonest site with approximately 58% (<xref rid="R17" ref-type="bibr">17</xref>). Spinal metastasis from CP angle medulloblastoma is very rare and until today just one case was reported with involvement of spine where the patients failed to receive post-surgical adjunctive radiotherapy (<xref rid="R18" ref-type="bibr">18</xref>). We followed the patient for probable metastasis by spinal cord MRI and no metastasis is reported yet. </p><p> Adult medulloblastomas are treated similar to pediatric medulloblastoma (<xref rid="R14" ref-type="bibr">14</xref>). A highly suspicious medulloblastoma should be purposed in atypical posterior fossa lesions in adults (<xref rid="R11" ref-type="bibr">11</xref>). A pre-operative diagnosis based on clinical assessment and radiological findings supports complete resection followed by beneficial adjuvant therapy. </p></sec><sec sec-type="conclusion" id="s4"><title>Conclusion</title><p> Conclusively, although extra-axial site of adulthood medulloblastoma is extremely rare, this tumor must be considered in the differential diagnosis of extra-axial CP angle lesions. Appropriate diagnostic and surgical work-up should be performed including total resection and adjuant therapy. Any neurological deterioration seen in followed-up patient must be evaluated for metastasis. </p></sec>
Quality of published Iranian medical education research studies: a systematic review
<p> <bold>Background:</bold> Research in medical education has been paid more attention than before; however the quality of research reporting has not been comprehensively appraised. To evaluate the methodological and reporting quality of Iranian published medical education articles. </p><p> <bold>Methods:</bold> Articles describing medical students, residents, fellows or program evaluation were included. Articles related to continuing medical education or faculty development, review articles and reports, and studies considering both medical and nonmedical students were excluded. We searched MEDLINE through PubMed in addition to major Iranian medical education search engines and databases including Scientific Information Database (SID) from March 2003 to March 2008. The Medical Education Research Quality Index (MERSQI) scale and the Consolidated Standards of Reporting Trials (CONSORT 2001) were used for experimental studies and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) was utilized for observational studies. </p><p> <bold>Results:</bold> Ninety five articles were found to be related to the medical education research in Iran including 16 (16.8%) experimental studies. Total MERSQI scores ranged between 3.82 and 13.09 with the mean of 8.39 points. Mean domain scores were highest for data analysis (1.85) and lowest for validity (0.61). The most frequently reported item was background (96%) and the least reported was the study limitations (16%). </p><p> <bold>Conclusion:</bold> The quality of published medical education research in Iran seems to be suboptimal. </p>
<contrib contrib-type="author"><name><surname>Golnari</surname><given-names>Pedram</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Sodagari</surname><given-names>Faezeh</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Baradaran</surname><given-names>Hamid Reza</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Medical education research has been improved extensively during recent years (<xref rid="R1" ref-type="bibr">1</xref>-<xref rid="R2" ref-type="bibr">2</xref>) as evidenced by several national and international journals and the increasing number of education reports published in scientific journals (<xref rid="R3" ref-type="bibr">3</xref>-<xref rid="R4" ref-type="bibr">4</xref>). Medical education research is frequently criticized for lack of generalizability and rigor by stakeholders such as professional organizations, journal editors, universities, teaching institutions and education researchers, maintaining that the quality of medical education research is inadequate and greater methodological rigor is needed (<xref rid="R5" ref-type="bibr">5</xref>-<xref rid="R9" ref-type="bibr">9</xref>). </p><p> Evaluating and improving methodological quality of the medical education research should be a central consideration to ensure that current educational efforts for training future physicians can improve patient care (<xref rid="R10" ref-type="bibr">10</xref>). A few methods and scales have been proposed for quality assessment in medical education. Among these, Medical Education Research Study Quality Instrument (MERSQI) (<xref rid="R11" ref-type="bibr">11</xref>) has been used with strong content, criterion, and predictive validity as well as inter-rater and intra-rater reliability. This metric has been shown to be a useful tool for educators, reviewers, and journal editors to assess the quality of medical education research (<xref rid="R11" ref-type="bibr">11</xref>-<xref rid="R13" ref-type="bibr">13</xref>). As for evaluating the quality of reporting, Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) and Consolidated Standards of Reporting Trials (CONSORT) statements are used as the most comprehensive guides and standards for reporting observational and interventional studies, respectively (<xref rid="R14" ref-type="bibr">14</xref>-<xref rid="R15" ref-type="bibr">15</xref>). </p><p> In Iran, medical students, residents, and fellows are now being trained in more than 45 medical universities and affiliated teaching hospitals (<xref rid="R16" ref-type="bibr">16</xref>-<xref rid="R17" ref-type="bibr">17</xref>). Following establishment of medical educational development centers in these universities, medical education research has been improved quantitatively during recent years (<xref rid="R18" ref-type="bibr">18</xref>). However, the quality of these published articles and reports have not been appraised and evaluated from methodological point of view. </p><p> Therefore, we conducted this systematic review with two aims: a) to explore the quality of reporting of experimental and not experimental research studies in medical education; b) to evaluate the methodological quality of medical education research studies. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> The study protocol was approved by the research council of the Medical Education and Development Center of Iran University of Medical Sciences. </p><sec id="s2-1"><title>Design</title><p> This study is a systematic review of the methodological and reporting quality of the published articles in the field of medical education in Iran. </p></sec><sec id="s2-2"><title>Search strategy</title><p> Published articles related to medical education research conducted in Iran were included in this review. Studies published between March 2003 and March 2008 were searched by two of authors (PG and FS) in MEDLINE through PubMed (accessed 15 October 2009). A search on the Scientific Information Database (SID) in which all Iranian journals are indexed was also conducted (accessed 15 October 2009). Combinations of the words related to medical education (medical education, teaching, and learning) and learners&#x02019; level (student, intern, resident and fellow) were used. Three major Iranian medical education journals (Strides in Development of Medical Education, Journal of Medical Education, and Iranian Journal of Medical Education) were hand-searched to identify additional studies published during the time interval. For the studies published in 2 different journals or in different languages, only the first publication was included regardless of the language or the journal. </p></sec><sec id="s2-3"><title>Study selection</title><p> Two reviewers (PG and FS) independently reviewed the titles and abstracts of the 762 retrieved articles and selected the eligible studies for inclusion. Medical education research was defined as any research study pertaining to medical students, residents, fellows, faculty members, or program evaluation. Studies with cross-sectional, case-control, cohort, and post-test only designs, as well as uncontrolled trials, non-randomized trials, and randomized-controlled trials which had been conducted in Iran were included. Articles related to continuing medical education or faculty development, as well as review articles and reports were excluded. Studies considering both medical and nonmedical students (nursing, allied medicine, dentistry, etc.) were also excluded. When titles and abstracts were not sufficient for determining the eligibility, the full articles were identified and reviewed. Any disagreements between the reviewers were resolved by consensus. </p></sec><sec id="s2-4"><title>Study review and data extraction</title><p> Full-texts of relevant studies were obtained and reviewed by reviewers (PG and FS). The viewers were not blind to the study location, authors affiliation or citation information. A standardized form was designed to extract the data from included studies. The following information was extracted from each article: location of the study, the highest academic degree of the authors, source of funding, study design, study population, sample size, outcomes, and the axis of the study. We categorized the studies into 3 axes of teaching, learning, and evaluation. </p></sec><sec id="s2-5"><title>Methodological quality assessment</title><p> Medical Education Research Study Quality Instrument (MERSQI) (<xref rid="R11" ref-type="bibr">11</xref>) was used for assessing the methodological quality of included studies. This instrument can assess the methodological quality of medical education studies in 6 domains including study design, sampling, type of data, validation of evaluation instrument, data analysis, and outcomes measured. By scoring in these domains, each medical education research can be scored between 6 and 18; 6 showing the lowest quality and 18 for the highest quality. Strong content, criterion, and predictive validity as well as inter-rater and intra-rater reliability have been reported previously for MERSQI scores (<xref rid="R11" ref-type="bibr">11</xref>-<xref rid="R13" ref-type="bibr">13</xref>). </p></sec><sec id="s2-6"><title>Reporting quality assessment</title><p> Quality of reporting was assessed using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist (<xref rid="R14" ref-type="bibr">14</xref>) and the 2001 revision of the Consolidated Standards of Reporting Trials (CONSORT) statement (<xref rid="R15" ref-type="bibr">15</xref>) for observational and experimental studies, respectively. </p></sec><sec id="s2-7"><title>Data analysis</title><p> Quantitative synthesis of data and meta-analysis was not possible to perform due to the heterogeneity of aims, scopes, designs, and population studied. All statistical analyses were performed using the Statistical Package for Social Sciences, version 15 for Windows&#x02122; (SPSS&#x000ae; Inc., Chicago, IL). Data were summarized as mean &#x000b1;SD and count (percent) for continuous and categorical variables, respectively. The normality of data was assessed using One-Sample Kolmogorov-Smirnov test. For non-normal data, median and range was used. MERSQI score calculation was used based on the reported method by Reed et al (<xref rid="R11" ref-type="bibr">11</xref>). Student's t-test and one-way analysis of variances (ANOVA) was used when comparing continuous variables (i.e. MERSQI scores) between two and more than two subgroups, respectively. All tests of significance were two-tailed and considered to be significant at P value less than 0.05. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><sec id="s3-1"><title>Study search and selection results</title><p> Overall, a total of 856 articles were retrieved from MEDLINE, SID and hand search which is illustrated in <xref ref-type="fig" rid="F1">figure 1</xref>. Screening of the titles and abstracts reduced this number to 174 articles. For these articles, full-texts were obtained and reviewed. Finally, 95 articles were found to be eligible for inclusion. Using time limits were not possible in SID search engine, thus we manually excluded the articles published out of the selected time period. </p><fig id="F1" orientation="portrait" position="float"><label> Fig.1 </label><graphic xlink:href="MJIRI-28-79-g001"/><statement><p> Literature search and study selection process for identifying Iranian medical education articles published between 2003 and 2008.</p></statement></fig></sec><sec id="s3-2"><title>Characteristics of included studies</title><p> <xref ref-type="table" rid="T1">Table 1</xref> summarizes the characteristics of the included studies. The articles were published in 6 international journals indexed in MEDLINE, 3 exclusive Iranian medical education journals, and 19 Iranian non-medical education journals. Sample size of included studies ranged from 11 to 1370 with a median of 75 participants. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Characteristics of included studies </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Characteristics</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Number (%)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Source of article</td><td rowspan="1" colspan="1">MEDLINE</td><td rowspan="1" colspan="1">7 (7.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Iranian medical education journals</td><td rowspan="1" colspan="1">60 (63.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Iranian general journals</td><td rowspan="1" colspan="1">28 (29.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Study design</td><td rowspan="1" colspan="1">Observational</td><td rowspan="1" colspan="1">79 (83.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Experimental</td><td rowspan="1" colspan="1">16 (16.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Centers</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">87 (91.6)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">3 (3.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">&#x0003e;2</td><td rowspan="1" colspan="1">5 (5.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Axis</td><td rowspan="1" colspan="1">Teaching</td><td rowspan="1" colspan="1">44 (46.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Learning</td><td rowspan="1" colspan="1">30 (42.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Assessment/evaluation</td><td rowspan="1" colspan="1">11 (11.6)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Highest academic rank of authors</td><td rowspan="1" colspan="1">Professor</td><td rowspan="1" colspan="1">17 (17.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Associate professor</td><td rowspan="1" colspan="1">24 (25.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Assistant professor</td><td rowspan="1" colspan="1">36 (37.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Instructor</td><td rowspan="1" colspan="1">2 (2.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Student</td><td rowspan="1" colspan="1"> 1 (<xref rid="R1" ref-type="bibr">1</xref>) </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Not mentioned</td><td rowspan="1" colspan="1">15 (15.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Funding source</td><td rowspan="1" colspan="1">Ministry of health and medical education</td><td rowspan="1" colspan="1">3 (3.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Academic</td><td rowspan="1" colspan="1">17 (17.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Research center</td><td rowspan="1" colspan="1">2 (2.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Not mentioned</td><td rowspan="1" colspan="1">73 (76.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Number of study participants</td><td rowspan="1" colspan="1">&#x0003c;50</td><td rowspan="1" colspan="1">29 (30.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">50-100</td><td rowspan="1" colspan="1">27 (28.4)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">&#x0003e;100</td><td rowspan="1" colspan="1">39 (41.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Population studied *</td><td rowspan="1" colspan="1">Basic science students</td><td rowspan="1" colspan="1">18 (18.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Physiopathology students</td><td rowspan="1" colspan="1">8 (8.4)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Clinical clerkship students</td><td rowspan="1" colspan="1">36 (37.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Interns</td><td rowspan="1" colspan="1">42 (44.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">residents</td><td rowspan="1" colspan="1">16 (16.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Fellowship</td><td rowspan="1" colspan="1">0</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Faculty members</td><td rowspan="1" colspan="1">13 (13.7)</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Other **</td><td rowspan="1" colspan="1">3 (3.3)</td></tr></tbody></table><table-wrap-foot><fn><p> * Percents do not add up 100 due to multiple target populations in some studies </p><p> ** Including morning reports, medical faculties, exams or multiple choice questions each with one study. </p></fn></table-wrap-foot></table-wrap><p> Teaching with 44 (46.3%) studies was the most common axis of the medical education articles and assessment/evaluation was the least with 11 (11.6%). The majority of studies evaluated interns (42 studies) and clerkship students (36 studies) as the main target population. None of the studies assessed fellowship training. The highest academic rank of the authors was assistant professor in 36 (37.9%) articles and associate professor and professor were next with 24 (25.3%) and 10 (10.5%), respectively. In 23 articles, the academic rank for authors was not mentioned. Financial support was reported only in 22 articles in which 17 (17.9%) had university support, 3 (3.2%) were sponsored by the Iranian Ministry of Health and Medical Education and for the other 2 articles a research center support was provided. </p></sec><sec id="s3-3"><title>Methodological quality</title><p> The majority of studies (79, 83.2%) had a single-group cross-sectional or single group post-test only design and only 5 studies (5.3%) were designed as a randomized-controlled trial. Almost all studies had been conducted in one center (87, 91.5%) and only 5 studies were multi-institutional. Among 56 studies with applicable response rate, 18 (32.2%) failed to report or had less than 50% response rate. In 62 studies (65.3%), outcome assessments were based on the study participants&#x02019; assessment while objective measurements were performed only in 33 (37.4%) studies. Only 15, (15.8%) studies reported the content validity evidence for the evaluation instrument they used for outcome assessment. Almost one third of the studies (32, 33.7%) had an inappropriate data analysis and among 63 studies with appropriate analysis, 13 studies (21%) used descriptive analysis only. </p><p> More than two thirds of studies (67, 70.5%) assessed satisfaction, attitude, opinion, perception, or general facts as an outcome. Twenty five (26.3%) assessed the knowledge or skills of the participants and in the remaining 3, participants behavior was assessed. None of the studies assessed any patients or healthcare system outcomes (<xref ref-type="table" rid="T2">Table 2</xref>). </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Methodological quality of Iranian medical education published articles between 2003 and 2008 based on the MERSQI Scale (N=95) </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Domain</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"> MERSQI item</td><td rowspan="1" colspan="1"> Studies Number (%)</td><td rowspan="1" colspan="1"> Item Mean &#x000b1;SD</td><td rowspan="1" colspan="1"> Domain Mean &#x000b1;SD</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Study design</td><td rowspan="1" colspan="1"> Design </td><td rowspan="1" colspan="1">Single group cross-sectional or single group post-test only </td><td rowspan="1" colspan="1">79 (83.2)</td><td rowspan="1" colspan="1"> 1.18 &#x000b1;0.49</td><td rowspan="1" colspan="1"> 1.18 &#x000b1;0.49</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"> Single group pre-test &#x00026; post-test </td><td rowspan="1" colspan="1"> 6 (6.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Nonrandomized, 2group</td><td rowspan="1" colspan="1"> 5 (5.3)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Randomized controlled trial</td><td rowspan="1" colspan="1"> 5 (5.3)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Sampling </td><td rowspan="1" colspan="1"> No. of institutions studied</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1"> 87 (91.5)</td><td rowspan="1" colspan="1"> 0.56 &#x000b1;0.23 </td><td rowspan="1" colspan="1">1.20 &#x000b1;0.72</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1"> 3 (3.2)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">&#x0003e;2 </td><td rowspan="1" colspan="1"> 5 (5.3)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Response rate, % </td><td rowspan="1" colspan="1">Not applicable </td><td rowspan="1" colspan="1"> 41 (43.2)</td><td rowspan="1" colspan="1">1.12 &#x000b1;0.46</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">&#x0003c;50 or not reported </td><td rowspan="1" colspan="1"> 18 (18.9)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">50-74 </td><td rowspan="1" colspan="1"> 5 (5.3)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">&#x02265;75 </td><td rowspan="1" colspan="1">31 (32.6) </td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Type of data</td><td rowspan="1" colspan="1">Type of data </td><td rowspan="1" colspan="1">Assessment by study participant</td><td rowspan="1" colspan="1"> 62 (65.3)</td><td rowspan="1" colspan="1">1.69 &#x000b1; 0.95</td><td rowspan="1" colspan="1">1.69 &#x000b1; 0.95</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Objective measurement </td><td rowspan="1" colspan="1">33 (37.4)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Validity of evaluation instrument</td><td rowspan="1" colspan="1">Internal structure</td><td rowspan="1" colspan="1">Not applicable </td><td rowspan="1" colspan="1">20 (31.1)</td><td rowspan="1" colspan="1">0.52 &#x000b1;0.50</td><td rowspan="1" colspan="1">0.61 &#x000b1;0.70</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Reported</td><td rowspan="1" colspan="1"> 39 (41.1)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Not reported </td><td rowspan="1" colspan="1">36 (37.8)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Content </td><td rowspan="1" colspan="1">Not applicable </td><td rowspan="1" colspan="1">20 (21.1)</td><td rowspan="1" colspan="1"> 5 (5.3)</td><td rowspan="1" colspan="1">0.20 &#x000b1;0.4</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Reported </td><td rowspan="1" colspan="1"> 15 (15.8)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Not reported </td><td rowspan="1" colspan="1">60 (63.2)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Relationships to other variables </td><td rowspan="1" colspan="1">Not applicable </td><td rowspan="1" colspan="1">20 (21.1)</td><td rowspan="1" colspan="1">0.05 &#x000b1;0.22</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Reported </td><td rowspan="1" colspan="1"> 4 (4.2)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Not reported </td><td rowspan="1" colspan="1">71 (74.7)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Data Analysis</td><td rowspan="1" colspan="1">Appropriateness of analysis</td><td rowspan="1" colspan="1"> Data analysis inappropriate for study design or type of data </td><td rowspan="1" colspan="1"> 32 (33.7) </td><td rowspan="1" colspan="1"> 0.66 &#x000b1; 0.47</td><td rowspan="1" colspan="1">1.85 &#x000b1;1.36</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Data analysis appropriate for study design or type of data</td><td rowspan="1" colspan="1"> 63 (66.3)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Complexity of analysis</td><td rowspan="1" colspan="1">Inappropriate </td><td rowspan="1" colspan="1"> 32 (33.7)</td><td rowspan="1" colspan="1">1.18 &#x000b1;0.91</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Descriptive analysis only </td><td rowspan="1" colspan="1">13 (13.7)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Beyond descriptive analysis </td><td rowspan="1" colspan="1">50 (52.6)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Outcomes</td><td rowspan="1" colspan="1">Outcomes</td><td rowspan="1" colspan="1"> Satisfaction, attitudes, perceptions,opinions, general facts</td><td rowspan="1" colspan="1">67 (70.5)</td><td rowspan="1" colspan="1"> 1.16 &#x000b1;0.26 </td><td rowspan="1" colspan="1">1.16 &#x000b1;0.26</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Knowledge, skills </td><td rowspan="1" colspan="1">25 (26.3)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Behaviours </td><td rowspan="1" colspan="1">3 (3.2)</td><td rowspan="1" colspan="1"/></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Patient/health care outcome </td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1"/></tr></tbody></table></table-wrap><p> Total MERSQI scores of the 95 included studies ranged between 3.82 and 13.09 with the mean &#x000b1;SD of 8.39 &#x000b1;2.28 points. The highest mean domain score was in data analysis domain with 1.85 &#x000b1;1.36 while the validity of evaluation instrument domain showed the lowest MERSQI score (0.61 &#x000b1;0.70). Although not statistically significant, the total MERSQI score was higher for experimental studies comparing to the observational studies (9.07 &#x000b1;2.79 versus 8.26 &#x000b1;2.16; p=0.019). The total MERSQI score was not significantly different between the teaching (8.19 &#x000b1;2.42), learning (8.66 &#x000b1;2.11), and assessment/evaluation (8.22 &#x000b1;2.41) axes (p=0.63). </p></sec><sec id="s3-4"><title>Reporting quality</title><p> <xref ref-type="table" rid="T3">Tables 3</xref> and <xref ref-type="table" rid="T4">4</xref> summarize the frequency of each item in observational and experimental studies. Among 79 observational studies, 23 (29.1%) studies did not report their study design in the abstract and in 10 studies (12.7%), the reported design was incorrect according to the method section. In method section, participant eligibility was the most reported item (71 articles, 82.9%) and the sample size calculation method was the least (2 articles, 2.5%). In results section, slightly more than half of the studies (46, 58.2%) reported the descriptive data. Key results, limitations of the study and generalizability of the results were discussed in the discussion section of 72 (91.1%), 11 (13.9%) and 12 (15.2%) studies, respectively. </p><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> Reporting quality of 79 observational Iranian medical education published articles between 2003 and 2008 based on the modified STROBE checklist </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Paper Section</td><td rowspan="1" colspan="1">Item</td><td align="center" rowspan="1" colspan="1">Descriptor</td><td align="center" rowspan="1" colspan="1">Frequency (%)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Title and abstract</td><td rowspan="1" colspan="1">Study design</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">46 (58.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">23 (29.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Incorrect*</td><td align="center" rowspan="1" colspan="1">10 (12.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Introduction</td><td rowspan="1" colspan="1">Background/rationale</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">75 (94.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">4 (5.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Objectives</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">74 (93.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">5 (6.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Methods</td><td rowspan="1" colspan="1">Study design</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">50 (63.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">23 (29.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Incorrect*</td><td align="center" rowspan="1" colspan="1">6 (7.6)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Setting</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">66 (83.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">13 (16.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Participants eligibility</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">71 (89.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">8 (10.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Data sources/ measurement</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">68 (86.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">11 (13.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Sampling method</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">51 (64.6)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">28 (35.4)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Study size calculation</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">2 (2.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">40 (50.6)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">37 (46.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Statistical methods</td><td rowspan="1" colspan="1">Appropriate</td><td align="center" rowspan="1" colspan="1">53 (67.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Inappropriate**</td><td align="center" rowspan="1" colspan="1">12 (15.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">14 (17.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Results</td><td rowspan="1" colspan="1">Participants flow</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">1 (1.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">78 (98.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Descriptive data</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">46 (58.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">33 (41.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Other analyses</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">2 (2.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">77 (97.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Discussion</td><td rowspan="1" colspan="1">Key results</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">72 (91.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">7 (8.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Limitations</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">11 (13.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">68 (86.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Interpretation</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">68 (86.1)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">11 (13.9)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Generalizability</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">12 (15.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">67 (84.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Other Information</td><td rowspan="1" colspan="1">Funding</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">21 (22.6)</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not mentioned</td><td align="center" rowspan="1" colspan="1">63 (79.7)</td></tr></tbody></table><table-wrap-foot><fn><p> * Inappropriateness of the reported study design with method section or ambiguous designs reported (e.g. descriptive-analytic study) </p><p> ** Inappropriateness of the statistical tests with variables, not using the statistical tests reported in method section, or not reporting the p values </p></fn></table-wrap-foot></table-wrap><table-wrap id="T4" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Reporting quality of 16 experimental Iranian medical education published articles between 2003 and 2008 based on the modified CONSORT 2001 checklist </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Paper Section</td><td rowspan="1" colspan="1">Item</td><td align="center" rowspan="1" colspan="1">Descriptor</td><td align="center" rowspan="1" colspan="1">Frequency (%)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Title and abstract</td><td rowspan="1" colspan="1">Random allocation</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">4 (25)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">1 (6.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">11 (68.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Introduction</td><td rowspan="1" colspan="1">Background</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">16 (100)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Methods</td><td rowspan="1" colspan="1">Participants (Inclusion/Exclusion)</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">16 (100)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Setting</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">10 (62.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">6 (37.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Interventions</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">13 (81.2)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">3 (18.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Objectives</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">15 (93.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">1 (6.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Sampling method</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">8 (50)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">8 (50)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Sample size calculation</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">2 (12.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">11 (68.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">3 (18.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Randomization-Sequence generation</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">5 (31.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">5 (31.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">6 (37.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Randomization-Allocation concealment</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">5 (31.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">11 (68.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Randomization-Implementation</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">5 (31.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">11 (68.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Blinding (masking)</td><td align="center" rowspan="1" colspan="1">Participant</td><td align="center" rowspan="1" colspan="1">1 (6.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Outcome assessor</td><td align="center" rowspan="1" colspan="1">2 (12.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Outcome assessor &#x00026; analyst</td><td align="center" rowspan="1" colspan="1">1 (6.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">1 (6.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">11 (68.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Statistical methods</td><td align="center" rowspan="1" colspan="1">Appropriate</td><td align="center" rowspan="1" colspan="1">10 (62.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Inappropriate*</td><td align="center" rowspan="1" colspan="1">4 (25)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">2 (12.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Results</td><td rowspan="1" colspan="1">Participants flow</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">2 (12.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">4 (25)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">10 (62.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Recruitment/Follow-up</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">8 (50)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">6 (37.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">Not applicable</td><td align="center" rowspan="1" colspan="1">2 (12.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Baseline data</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">9 (56.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">7 (43.7)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Numbers analyzed</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">8 (50)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">8 (50)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Ancillary analyses</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">16 (100)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Discussion</td><td rowspan="1" colspan="1">Interpretation</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">14 (87.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">2 (12.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Generalizability</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">2 (12.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">14 (87.5)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Overall evidence</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">11 (68.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">5 (31.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">limitations</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">5 (31.3)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">11 (68.8)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Other Information</td><td rowspan="1" colspan="1">Funding</td><td align="center" rowspan="1" colspan="1">Yes</td><td align="center" rowspan="1" colspan="1">6 (37.5)</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1"> </td><td align="center" rowspan="1" colspan="1">No</td><td align="center" rowspan="1" colspan="1">10 (62.5)</td></tr></tbody></table><table-wrap-foot><fn><p> * Inappropriateness of the statistical tests with variables, not using the statistical tests reported in method section, or not reporting the p values </p></fn></table-wrap-foot></table-wrap><p> As for experimental studies, all 16 studies mentioned the eligibility criteria. Intervention description was not provided in 3 (18.8%) studies and sampling method was reported in only half of the studies. Randomization-sequence generation was reported only in half of the applicable articles and none of the studies reported the allocation concealment method. In results, baseline data was not provided in 7 (43.7%) of the experiments and half of the studies did not report the number analyzed in statistical analyses. Generalizability was the least reported item in the discussion section with only 2 (12.5%) studies reporting it. </p></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Although medical education research had been known as an important field of research in medical universities in Iran, to our best of knowledge, this systematic review is the first study aimed to assess the methodological quality of these reports. Our results revealed that the overall methodological quality is suboptimal and some important elements are not routinely reported in published papers. These findings are in line with other internationally reports. </p><p> Other studies in medical education have described suboptimal reporting methodological quality (<xref rid="R11" ref-type="bibr">11</xref>-<xref rid="R13" ref-type="bibr">13</xref>). For example Cook et al (<xref rid="R19" ref-type="bibr">19</xref>-<xref rid="R20" ref-type="bibr">20</xref>) in two systematic reviews showed that many essential elements of scientific reporting were frequently missing from articles describing medical education experiments, including a critical literature review, study design statement, definition of the comparison or control group. </p><p> The lowest MERSQI score was observed in the validity of evaluation instruments domain in our systematic review which is consistent with other studies (<xref rid="R11" ref-type="bibr">11</xref>-<xref rid="R13" ref-type="bibr">13</xref>) and confirms previous reports that studies rarely report validity assessments for their evaluation instruments (<xref rid="R21" ref-type="bibr">21</xref>-<xref rid="R22" ref-type="bibr">22</xref>). The most reported measure of validity observed in our review was internal structure while in other studies content validity was more frequently reported (<xref rid="R19" ref-type="bibr">19</xref>-<xref rid="R20" ref-type="bibr">20</xref>). One explanation for this difference is that authors and journal reviewers in Iran might pay more attention to the measures of internal validity (e.g. Cronbach&#x02019;s alpha) than the description of how adequately items represent the content of the items. </p><p> Our systematic review demonstrated that only 17% of studies in this review used an interventional design, that only 8.5% have been conducted in more than one center, and that only 3.2% assessed the learner behaviors and none of them assessed the patient/health care outcomes which highlights the need to increase methodological rigor in medical education research. More interventional studies with control groups are needed to make comparisons meaningful and applicable in the field of medical education. Such interventional studies can produce high level of evidence for educators and help them select the best methods and approaches for training medical students. Multi-institutional research is essential in order to test the effects of educational interventions across educational sites and environments. What works in one university or faculty may not work in another depending on the culture and processes conducted in each site. In medical education research, a significant amount of studies are measuring trainee satisfaction and performance and relatively little attention is paid to the effect of medical education on patient or healthcare outcomes which are fundamental goals of training physicians (<xref rid="R23" ref-type="bibr">23</xref>). </p><p> The majority of studies evaluated interns (44.2%) and clerkship students (37.9%) as the main target population. Few studies assessed the residency training (16.8%) and none of them assessed the fellowship training, highlighting the gap in this field. </p><p> Financial support was reported only in 23.2% of the studies, showing the need for greater funding for medical education research. Reed et al (<xref rid="R11" ref-type="bibr">11</xref>) showed a significant association between funding and study quality (as measured by the MERSQI), providing evidence to support the call to increase funding for medical education research in the USA. Policy reform that increases funding support may promote high-quality medical education research (<xref rid="R11" ref-type="bibr">11</xref>, <xref rid="R24" ref-type="bibr">24</xref>). </p><p> Evaluation of the reporting quality of the reviewed articles revealed that although peer-review process is performed in medical education journals, several essential elements of scientific reporting were still missing. This is consistent with previous studies (<xref rid="R19" ref-type="bibr">19</xref>) and highlights another weakness in medical education articles and their reviewing process for publication in journals. We noticed that in 13% of the studies, the implied study design was different from what the authors stated in their articles; this finding is confirmed by another study in the USA stating that statement of the study design is missing in almost 80% of abstracts of published medical education articles (<xref rid="R20" ref-type="bibr">20</xref>). </p></sec><sec id="s5"><title>Limitations</title><p> This study faced some limitations. Firstly, some medical education articles published in Iranian non-medical education journals may be missing from this review due to the lack of a reliable search engine in the SID, the main electronic database of scientific journals in Iran. Secondly, MERSQI is developed to assess the methodological quality of the studies rather than their reporting quality. However, low reporting quality has influence on the MERSQI score. It is probable that authors conducted better studies but simply did not report in the manuscript all the items that MERSQI needs. </p></sec><sec id="s6"><title>Implications for future research</title><p> This suboptimal quality status may reflect the need for using statistical consultants in research teams for improving the quality of inferences and also using more methodological and analytical oriented reviewers which could correct analytic flaws before publication or reject studies with fatal flaws. </p><p> Authors and journal reviewers in Iran should pay more attention to the measures of internal and external validities for medical education research design or report before publishing. Comparing to medicine, research in education may be more complex, confounding factors may be more apparent, content may be more implicit and controlled trials may be difficult. Thus, higher quality of research is mandated and correct designing research studies in this field is of paramount importance. </p></sec><sec sec-type="conclusion" id="s7"><title>Conclusion</title><p> It could be concluded that a) majority of published medical education research have been observational studies rather than experimental; b) the reliability and validity of assessment tools used in the Iranian educational studies to assess the success of their educational efforts have not been properly reported and indicated; c) overall, the quality of the reporting of research studies in medical education in Iran seems not strong; and d) more robust and rigorous research studies are needed in the future. </p></sec><sec id="s8"><title>Acknowledgements</title><p> The authors would like to thank Dr. Kamran Soltani-Arabshahi and Dr. Mohammad E Khamseh from Iran University of Medical Sciences and also Dr. David Cook from Mayo Clinic for their expert advice. This project has been granted funding (No. M/396) by deputy of research of Iran University of Medical Sciences. Ethical approval is not applicable. </p></sec><sec id="s9"><title>Conflict of Interest</title><p> The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. </p></sec>
A meta-synthesis study of literature review and systematic review published in nurse prescribing
<p> <bold>Background:</bold> Prescribing represents a new aspect of practice for nurses. To make qualitative results more accessible to clinicians, researchers, and policy makers, individuals are urged to synthesize findings from related studies. Therefore this study aimed to aggregate and interpret existing literature review and systematic studies to obtain new insights on nurse prescription. </p><p> <bold>Methods:</bold> This was a qualitative meta synthesis study using Walsh and Downe process. In order to obtain data all Digital National Library of Medicine's databases, search engines and several related sites were used. Full texts with "review and nurs* prescri* " words in the title or abstract in English language and published without any time limitation were considered. After eliminating duplicate and irrelevant studies, 11 texts were selected. Data analysis was conducted using qualitative content analysis. Multiple codes were compared based on the differences and similarities and divided to the categories and themes. </p><p> <bold>Results:</bold> The results from the meta synthesis of the 11 studies revealed 8 themes namely: leading countries in prescribing, views, features, infrastructures, benefits, disadvantages, facilitators and barriers of nursing prescription that are discussed in this article. The results led to a schematic model. </p><p> <bold>Conclusion:</bold> Despite the positive view on nurse prescribing, there are still issues such as legal, administrative, weak research and educational deficiencies in academic preparation of nurses that needs more effort in these areas and requires further research. </p>
<contrib contrib-type="author"><name><surname>Darvishpour</surname><given-names>Azar</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Joolaee</surname><given-names>Soodabeh</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Cheraghi</surname><given-names>Mohammad Ali</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> Many countries have introduced non-medical prescription to improve efficiency and access to medication, particularly where access to doctors can be difficult (<xref rid="R1" ref-type="bibr">1</xref>). The number of countries where nurses are legally permitted to prescribe medication has grown considerably over the last two decades (<xref rid="R2" ref-type="bibr">2</xref>). Now in many countries (Australia, USA, UK, Canada, Ireland, etc) specific groups of nurses are allowed to prescribe various drugs (<xref rid="R3" ref-type="bibr">3</xref>). </p><p> In fact, in countries with a well-developed education system for nurses, opportunities exist for nurses to obtain advanced practice training and graduate education; in the US, Canada and the United Kingdom. For example, a nurse practitioner typically defined as a registered nurse who is educated to function in an advanced clinical role and prescribing is one aspect of this advanced clinical role (<xref rid="R4" ref-type="bibr">4</xref>). </p><p> The benefits of non-medical prescribing have been consistently reported in the literature (<xref rid="R1" ref-type="bibr">1</xref>). Improved speed and convenience of access to medicines have been consistently reported as key benefits of non-medical prescription by patients and health professionals (<xref rid="R1" ref-type="bibr">1</xref>). </p><p> According to this, the World Health Organization (WHO) in the fifth meeting of the regional advisory panel in the Eastern Mediterranean nursing convention (Islamabad, Pakistan, 2001) recommended that it required nurses as first-line health care providers to be empowered and prepared to prescribe properly (<xref rid="R5" ref-type="bibr">5</xref>). However, nurses in many countries still do not have the authority to prescribe medication. The introduction of major policy initiatives, such as nurse prescribing, requires high level of discussion and policy development to ensure successful implementation (<xref rid="R6" ref-type="bibr">6</xref>). Accordingly, each country can utilize international experiences to minimize their inevitable losses and damages. It can give new insight regarding the different aspects and necessity of implementation of nurse drug prescribing in leading countries. A significant number of review studies have been conducted related to nurse prescribing (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref>-<xref rid="R16" ref-type="bibr">16</xref>). Each of these studies has contributed to our understanding of nurse prescribing, but to both advance knowledge and policy making for its implementation, knowledge synthesis across multiple studies is required. To make qualitative results more accessible to clinicians, researchers, and policymakers, individuals are urged to synthesize findings from related studies (<xref rid="R17" ref-type="bibr">17</xref>). Thus the purpose of this study was to aggregate and interpret existing literature review and systematic review studies to obtain new insights on nurse prescribing drugs, and to present a schematic model of nurse prescribing that can be a useful framework for its implementation. According to the purpose of the study, the following questions were considered in literature reviews: </p><list list-type="simple"><list-item><p> - What is the overall view on nurse prescribing? </p></list-item><list-item><p>- What are the positive and negative outcomes of nurse prescribing? </p></list-item><list-item><p>- What are the barriers and facilitators for its implementation?</p></list-item></list></sec><sec sec-type="methods" id="s2"><title>Methods</title><p> Qualitative Meta synthesis method was applied to conduct this study. The purpose of the Meta synthesis is to dig below the surface of what is currently understood regarding a phenomenon, to emerge with the kernel of a new truth, a better kind of understanding of something (<xref rid="R18" ref-type="bibr">18</xref>). Metasynthesis, a systematic synthesis of findings across qualitative studies, seeks to generate new interpretations for which there is a consensus within a particular field of study (<xref rid="R19" ref-type="bibr">19</xref>). A qualitative meta synthesis integrates individual qualitative studies by bringing together and breaking down the findings (themes, metaphors, or categorizations) of individual studies, elucidating the key features, and combining these findings into a transformed whole &#x02013;a single description of the findings that authentically represents all of the cases (<xref rid="R20" ref-type="bibr">20</xref>). </p><p> The meta synthesis process employed in this study followed that done by Walsh and Downe (2005) (<xref rid="R21" ref-type="bibr">21</xref>). Walsh and Downe developed and proposed a seven-step approach for the qualitative meta-synthesis: (<xref rid="R1" ref-type="bibr">1</xref>) framing a meta-synthesis exercise, (<xref rid="R2" ref-type="bibr">2</xref>) locating relevant papers, (<xref rid="R3" ref-type="bibr">3</xref>) deciding what to include, (<xref rid="R4" ref-type="bibr">4</xref>) appraising studies, (<xref rid="R5" ref-type="bibr">5</xref>) comparing and contrasting exercise, (<xref rid="R6" ref-type="bibr">6</xref>) reciprocating translation, and (<xref rid="R7" ref-type="bibr">7</xref>) synthesizing translation (<xref rid="R21" ref-type="bibr">21</xref>). </p><p> Search strategy included all of the following databases, search interfaces and journals of INLM (Integrated Digital National Library of Medicine) from August 14, 2012 to December week 4 2012: EBSCO host (CINAHL), EBSCO host (Medline), Ovid SP (MEDLINE(R)), Cochran Library, Scopus, Web of science, Elsevier, Emelard, JAMA Journals, Wiley, Oxford Journals, Springer and Thieme Journals and few related sites (World Health Organization (www.who.int), websites for health professionals (<uri xlink:href="http://www.nurse-prescriber.co">http://www.nurse-prescriber.co</uri>. uk), Google Scholar (scholar.google.com) and <uri xlink:href="http://journals.cambridge.org/action/">http://journals.cambridge.org/action/</uri> search). </p><p> The inclusion, exclusion criteria and appropriate data sources were identified. The inclusion criteria included: all studies without time restriction that contain search terms (review and nurs* prescri*) in the title or abstract, with published full text in English. The review studies that had a search strategy were included but review studies of experts' opinion and without a search strategy were excluded from the meta synthesis. </p><p> One researcher screened all titles and abstracts and two other researchers during supervising the process of study, screened full texts as relevant to the purpose of study. Quality assessment and data extraction was carried out by three researchers. </p><p> The qualities of included papers were appraised using AMSTAR tool (Assessment of Multiple Systematic Reviews consists of 11 items) (<xref rid="R22" ref-type="bibr">22</xref>) and CASP tool (Critical Appraisal Skills Program for Review articles with 10 items) (<xref rid="R23" ref-type="bibr">23</xref>). Studies were awarded a score between 0 and 11 for AMSTAR and 0-10 for CASP tool. Studies rated as low, medium, and high quality. </p></sec><sec sec-type="results" id="s3"><title>Results</title><p> The electronic search yielded 5829 results in which. 49 were duplicated and 5739 had no relevant to the research objectives and therefore excluded. This reduced the number of texts to 41. The full texts of the remaining 41articles were reviewed and a further 30 put aside and not reviewed (i.e. book review). Finally, 11 studies were selected, reviewed and included in this study (<xref rid="R2" ref-type="bibr">2</xref>,<xref rid="R7" ref-type="bibr">7</xref>-<xref rid="R16" ref-type="bibr">16</xref>).<xref ref-type="fig" rid="F1">Figure 1</xref> shows summary of the selection process for articles used in the study. <xref ref-type="table" rid="T1">Table 1</xref> illustrates search strategy of Ovid SP (MEDLINE(R)) </p><fig id="F1" orientation="portrait" position="float"><label> Fig.1 </label><graphic xlink:href="MJIRI-28-77-g001"/><statement><p>The flowchart for the search and selection process</p></statement></fig><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Search Strategy of Ovid SP (MEDLINE(R)) </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Search history</td><td rowspan="1" colspan="1">Search Terms</td><td align="center" rowspan="1" colspan="1">Studies Returned, n</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">nurs* prescri*.m_titl.</td><td align="center" rowspan="1" colspan="1">338</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td rowspan="1" colspan="1">Limit 1 to (english language and yr=''1946-2012'')</td><td align="center" rowspan="1" colspan="1">324</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td rowspan="1" colspan="1">Limit 1 to (english language and full text and yr=''1946-2012'')</td><td align="center" rowspan="1" colspan="1">61</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td rowspan="1" colspan="1">nurs* prescri* and review.m_titl.</td><td align="center" rowspan="1" colspan="1">9</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td rowspan="1" colspan="1">nurs* and prescri* and review.ti.</td><td align="center" rowspan="1" colspan="1">20</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">6</td><td rowspan="1" colspan="1">Limit 5 to (english language and full text)</td><td align="center" rowspan="1" colspan="1">5</td></tr></tbody></table></table-wrap><p> 4 of these 11 review articles were considered as systematic review and 7 literature review. Systematic review articles were evaluated by means of AMSTAR in which three were strong (score of 0.8) and one moderate (score of 0.51 to 0.79). Review articles with CASP (Critical Appraisal Skills Program) tool were tested that three were evaluated as "strong" (score more than 0.8) and 4 "moderate" articles (score of 0.51 to 0.79). </p><p> All 11 studies that met the inclusion criteria were read line by line several times in order to get the intended message of the content as well as an accurate impression of the context. The results and conclusions of studies were considered as &#x0201c;meaning units&#x0201d; and coded, integrated, synthesized and categorized. Multiple codes based on the differences and similarities compared and divided into category and themes. The results of meta synthesis revealed 8 themes namely: Leading countries in prescribing, Views, Features, Infrastructures, Benefits, Disadvantages, Facilitators and Barriers. Examples illustrating these themes can be found in <xref ref-type="table" rid="T1">Tables 1</xref>, <xref ref-type="table" rid="T2">2</xref> and <xref ref-type="table" rid="T3">3</xref>. These themes are described bellow. </p><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Cods, Categories and Illustrations of Features of nurse prescribing identified by the meta- synthesis </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1"> <bold>Categories</bold> </td><td rowspan="1" colspan="1">Cods</td><td rowspan="1" colspan="1">Illustrations</td><td rowspan="1" colspan="1">Authors</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Quality and safety of practice</td><td rowspan="1" colspan="1">-No differences in the health status of patients treated with Physicians and nurses</td><td rowspan="1" colspan="1">"No differences in health status were found"</td><td rowspan="1" colspan="1"> Horrocks, et al., 2002 <sup> (<xref rid="R7" ref-type="bibr">7</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">-Long consultation nurse practitioners than to physicians</td><td rowspan="1" colspan="1">"Nurse practitioners had longer consultations and made more investigations than did doctors"</td><td rowspan="1" colspan="1"> Horrocks, et al., 2002<sup> (<xref rid="R7" ref-type="bibr">7</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">- No differences in prescriptions, return consultations, or referrals</td><td rowspan="1" colspan="1">"No differences were found in prescriptions, return consultations, or referrals."</td><td rowspan="1" colspan="1"> Horrocks, et al., 2002<sup> (<xref rid="R7" ref-type="bibr">7</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">-Better quality of care Quality of care in some ways for nurse practitioner consultations</td><td rowspan="1" colspan="1">"Quality of care was in some ways better for nurse practitioner consultations"</td><td rowspan="1" colspan="1"> Horrocks, et al., 2002<sup> (<xref rid="R7" ref-type="bibr">7</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">some studies in primary and secondary care found that nurses had longer consultation times than GPs, although does not report the statistical significance of this finding.</td><td rowspan="1" colspan="1"> Van Ruth, et al., 2008 <sup> (<xref rid="R12" ref-type="bibr">12</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">-No security problem</td><td rowspan="1" colspan="1">"The synthesis revealed no major safety concerns as a result of the implementation of PGDs."</td><td rowspan="1" colspan="1"> Price, et al., 2012<sup> (<xref rid="R16" ref-type="bibr">16</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">similar or better therapeutic Clinical parameters of nursing</td><td rowspan="1" colspan="1">"Clinical parameters were the same or better for treatment by nurses"</td><td rowspan="1" colspan="1"> Van Ruth, et al., 2008<sup> (<xref rid="R12" ref-type="bibr">12</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">-Similar or higher patient satisfaction with nurse prescribing</td><td rowspan="1" colspan="1">"Eight studies all found that patients being treated by nurses were just as satisfied or more satisfied than patients being treated by physicians"</td><td rowspan="1" colspan="1"> Van Ruth, et al., 2008<sup> (<xref rid="R12" ref-type="bibr">12</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">-Same or better quality of perceived nurses care</td><td rowspan="1" colspan="1">"perceived quality of care by nurses is similar or better."</td><td rowspan="1" colspan="1"> Van Ruth, et al., 2008<sup> (<xref rid="R12" ref-type="bibr">12</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Confidence in prescribing</td><td rowspan="1" colspan="1">- Felt confident</td><td rowspan="1" colspan="1">"All studies reported that the majority of respondents felt confident in their prescribing."</td><td rowspan="1" colspan="1"> Creedon , et al., 2009<sup> (<xref rid="R13" ref-type="bibr">13</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">- moderately confident</td><td rowspan="1" colspan="1">"Most respondents that prescribed less than three times per week were moderately confident and felt somewhat limited by the nurse prescribers&#x02019; formulary"</td><td rowspan="1" colspan="1"> O' Connell, et al., 2009<sup> (<xref rid="R14" ref-type="bibr">14</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">-Feeling of Limited prescription</td><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Areas of nurse prescribing</td><td rowspan="1" colspan="1">nurses prescribing practices</td><td rowspan="1" colspan="1">Nine studies investigated nurses prescribing practices consisted of antibiotics, anti hypertensives, cardiovascular drugs, dermatological and skin conditions, analgesics, diabetic medications and controlled drugs.</td><td rowspan="1" colspan="1"> O' Connell, et al., 2009<sup> (<xref rid="R14" ref-type="bibr">14</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Prescribing patterns</td><td rowspan="1" colspan="1">diversity of forces leding to nurse prescribing</td><td rowspan="1" colspan="1">"A diversity of external and internal forces has led to the introduction of nurse prescribing internationally."</td><td rowspan="1" colspan="1"> Kroezen, et al., 2011<sup> (<xref rid="R2" ref-type="bibr">2</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">"The legal, educational and organizational conditions under which nurses prescribe medicines vary considerably between countries; from situations where nurses prescribe independently to situations in which prescribing by nurses is only allowed under strict conditions and supervision of physicians."</td><td rowspan="1" colspan="1"> Kroezen, et al., 2011<sup> (<xref rid="R2" ref-type="bibr">2</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">variation in prescribing patterns</td><td rowspan="1" colspan="1">"There is some variation in the prescribing patterns of district nurses&#x02019;, health visitors&#x02019; and practice nurses"</td><td rowspan="1" colspan="1"> Latter and Courtenay, 2004<sup> (<xref rid="R10" ref-type="bibr">10</xref>) </sup> </td></tr></tbody></table></table-wrap><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> Summary of barriers of nurse prescribing identified by the meta-synthesis </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1"> <bold>Categories</bold> </td><td rowspan="1" colspan="1">Cods</td><td rowspan="1" colspan="1">Illustrations</td><td rowspan="1" colspan="1">Authors</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Legal limitations</td><td rowspan="1" colspan="1">limitations of Nurse Prescribers&#x02019; Formulary</td><td rowspan="1" colspan="1">There is some variation in the prescribing patterns of district nurses&#x02019;, health visitors&#x02019; and practice nurses, and the limitations of the original Nurse Prescribers&#x02019; Formulary (NPF) have been highlighted</td><td rowspan="1" colspan="1"> Latter and Courtenay, 2004<sup> (<xref rid="R10" ref-type="bibr">10</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">legal restrictions</td><td rowspan="1" colspan="1">All Western-European and Anglo-Saxon countries that have realised or initiated nurse prescribing have imposed legal restrictions on which categories of nurses can prescribe medicines, what, how much and to whom they can prescribe, and whether they are allowed to do so on an independent basis or under the supervision of a physician.</td><td rowspan="1" colspan="1"> Kroezen, et al., 2011 <sup> (<xref rid="R2" ref-type="bibr">2</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Executive Factors</td><td rowspan="1" colspan="1">implementation barriers</td><td rowspan="1" colspan="1">implementation barriers emerged from the empirical and anecdotal literature, including funding problems, delays in practicing and obtaining prescription pads, encumbering clinical management plans and access to records.</td><td rowspan="1" colspan="1"> Cooper, et al., 2008 <sup> (<xref rid="R11" ref-type="bibr">11</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">safety concerns</td><td rowspan="1" colspan="1">There were a number of safety concerns identified including: nurses using their professional judgment to deliberately work outside the parameters of PGDs (Miles et al, 2001), poor record keeping (Brooks et al, 2003; Deave et al, 2003; Baileff, 2007) and the development of PGDs that failed to comply with legal requirements (Deave et al, 2003).</td><td rowspan="1" colspan="1"> Price, et al., 2012 <sup> (<xref rid="R16" ref-type="bibr">16</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Humanistic Factors</td><td rowspan="1" colspan="1">Lack of confidence in applied pharmacology and therapeutics among nurses</td><td rowspan="1" colspan="1">although patients were consent with nurses prescribing medication, nurses lacked confidence in applied pharmacology and therapeutics and hence, required additional scientific education</td><td rowspan="1" colspan="1"> Banning, 2004<sup> (<xref rid="R8" ref-type="bibr">8</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">nurses&#x02019; fears of becoming overconfident</td><td rowspan="1" colspan="1">Bradley et al (2007) reported nurses&#x02019; fears of becoming overconfident and prescribing outside their competency area.</td><td rowspan="1" colspan="1"> Creedon , et al., 2009<sup> (<xref rid="R13" ref-type="bibr">13</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">medical apathy</td><td rowspan="1" colspan="1">medical apathy and independent prescribing potentially undermine the success of SP</td><td rowspan="1" colspan="1"> Cooper, et al., 2008<sup> (<xref rid="R11" ref-type="bibr">11</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Educational deficiencies</td><td rowspan="1" colspan="1">Lack of doctors knowledge on the training of nurse rescribers</td><td rowspan="1" colspan="1">The Swedish GPs interviewed in Wilhelmsson and Foldeive&#x02019;s (2003) study lacked knowledge on the training of nurse prescribers, which could account for their negative views on nurse prescribing</td><td rowspan="1" colspan="1"> Creedon , et al., 2009 <sup> (<xref rid="R13" ref-type="bibr">13</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">substantial gaps in the knowledge base</td><td rowspan="1" colspan="1">Our review suggests that there are substantial gaps in the knowledge base to help evidence based policy making in this arena.</td><td rowspan="1" colspan="1"> Bhanbhro, et al., 2011 <sup> (<xref rid="R15" ref-type="bibr">15</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">deficits in the scientific preparation of nurses</td><td rowspan="1" colspan="1">This review has drawn attention to the deficits in the scientific preparation of nurses in applied pharmacology and therapeutics.</td><td rowspan="1" colspan="1"> Banning, 2004<sup> (<xref rid="R8" ref-type="bibr">8</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Research weaknesses</td><td rowspan="1" colspan="1">methodological weaknesses</td><td rowspan="1" colspan="1">there are both methodological weaknesses and under-researched issues that point to the need for further research into this important policy initiative.</td><td rowspan="1" colspan="1"> Latter and Courtenay, 2004 <sup> (<xref rid="R10" ref-type="bibr">10</xref>) </sup> </td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1"> </td><td rowspan="1" colspan="1">Empirical studies were often methodological weaknesses and under-evaluation of safety, economic analysis and patients&#x02019; experiences were identified in empirical studies</td><td rowspan="1" colspan="1"> Cooper, et al., 2008 <sup> (<xref rid="R11" ref-type="bibr">11</xref>) </sup> </td></tr></tbody></table></table-wrap><p> Further research is needed to provide a picture for investigated concept. The results were lead to a schematic model (<xref ref-type="fig" rid="F2">Fig. 2</xref>). This model of nurse prescribing can help policymakers to identify areas for attention. </p><fig id="F2" orientation="portrait" position="float"><label> Fig. 2 </label><graphic xlink:href="MJIRI-28-77-g002"/><statement><p>Schematic model of nurse prescribing medicine</p></statement></fig><sec id="s3-1"><title>Leading countries in prescription drug</title><p> The results showed that four studies in this meta synthesis focused on the leading countries in prescription drug (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R13" ref-type="bibr">13</xref>-<xref rid="R15" ref-type="bibr">15</xref>). </p><p> Bhanbhro et al (2011) found that the majority of studies about nurse prescribing were undertaken in the UK with only one from USA, Canada, Botswana and Zimbabwe each (<xref rid="R15" ref-type="bibr">15</xref>). In addition, Kroezen et al (2011) showed that seven Western European and Anglo-Saxon countries (Australia, Canada, Ireland, New Zealand, Sweden, the UK and the USA) had implemented nurse prescribing medicines. Netherlands and Spain were in the process of introducing nurse prescribing drug policies (<xref rid="R2" ref-type="bibr">2</xref>). </p></sec><sec id="s3-2"><title>Views relating to nurse prescribing</title><p> Nine studies indicated the views (doctors, nurses, and patients) related to nurse prescriber (<xref rid="R8" ref-type="bibr">8</xref>-<xref rid="R16" ref-type="bibr">16</xref>). All of these studies have reported positive views on nurse. For example Latter and Courtenay (2004) stated: &#x0201c;The review highlights that nurse prescribing has generally been evaluated positively to date&#x0201d; (<xref rid="R10" ref-type="bibr">10</xref>). </p><p> Also Van Ruth et al (2008) mentioned "The effects of nurse prescribing seem positive, although the high risk of bias in the studies means they must be regarded with caution" (<xref rid="R12" ref-type="bibr">12</xref>). </p><p> In addition, Bhanbhro et al. (2011) reported that non-medical prescribing was widely accepted and viewed positively by patients and professionals (<xref rid="R15" ref-type="bibr">15</xref>). </p><p> Only two studies noted indirectly negative views of physicians on the nurse practitioners prescribing medicine as follows: </p><p> "Nurses and pharmacists were positive about supplementary prescribing (SP) but the medical profession were more critical and lacked awareness/ understanding, according to the identified literature" (<xref rid="R11" ref-type="bibr">11</xref>). </p><p> Also Creedon, et al (2009) quoted Wilhelmsson and Foldeivi (2003) that the Swedish GPs had lacked knowledge on the training of nurse prescribers, which could account for their negative views on nurse prescribing (<xref rid="R13" ref-type="bibr">13</xref>). </p><p> However, these negative views can be a barrier that should be given particular importance to the implementation of nurse prescriber. </p></sec><sec id="s3-3"><title>Features</title><p> Results showed that seven authors came up with the issue of Features of nurse prescribing (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R10" ref-type="bibr">10</xref>, <xref rid="R12" ref-type="bibr">12</xref>-<xref rid="R14" ref-type="bibr">14</xref>,<xref rid="R16" ref-type="bibr">16</xref>).This issue consists of 4 category (Prescribing patterns, Areas of nurse prescribing, Confidence in prescribing and Quality and safety of practice) that emerged in this study. <xref ref-type="table" rid="T2">Table 2</xref> shows summary of emerged cods and categories of this issue. </p><p> The findings showed the most important feature of nurse prescribing was long consultation results of Horrocks et al (2002) and Van Ruth et al (2008) studies. These studies noted that longer consultations lead to better quality of care and increase patient satisfaction and improve patient-centered care as the main goal of health care system (<xref rid="R7" ref-type="bibr">7</xref>, <xref rid="R12" ref-type="bibr">12</xref>). </p></sec><sec id="s3-4"><title>Benefits</title><p> Eight studies (<xref rid="R7" ref-type="bibr">7</xref>-<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R12" ref-type="bibr">12</xref>-<xref rid="R14" ref-type="bibr">14</xref>,<xref rid="R16" ref-type="bibr">16</xref>) investigated benefits of nurse prescribing. These benefits were categorized as: benefits for health system, patients, and nurses. Benefits for health system consists of better care delivery, acquiring patients satisfaction, reduction in number of required personnel, effectiveness and potentially reduction in costs. </p><p> There were some benefits for patients including convenience, easier access to drugs and care, receive better care, health promotion, earlier treatment, reduce in waiting time for medication, safety, satisfaction of care, improvement in compliance with medications, prevention of relapses, closeness and proximity of patients to health care providers, make sense of the nurses concerns expressed by patients and reduction in the number of journeys some carers had to make. And benefits for nurses included reduction in workload, time saving, professional autonomy, professional development, satisfaction of role, increasing clinical competence, better use of nurses' skills and experience, recognition abilities, capabilities and expertise of nurses, better accountability, enhance the effective monitoring and reporting of adverse drug reactions, improve communication between the professional health care team. In addition, the evidence showed that nurse prescribing was also useful for physicians and other health care professionals. Jabareen (2008) quoted Crossman (2006) that Nurse prescribing allows GPs to take on more complex cases (<xref rid="R27" ref-type="bibr">27</xref>). Also Bowskill (2009) quoted Jordan, Knight and Pointon (2004) and Jones et al (2007) that nurse prescribing was a useful means for reducing professional distance, enabling nurse prescribers to work more closely with medical colleagues (<xref rid="R28" ref-type="bibr">28</xref>). </p></sec><sec id="s3-5"><title>Disadvantages</title><p> Two studies (<xref rid="R9" ref-type="bibr">9</xref>, <xref rid="R16" ref-type="bibr">16</xref>) mentioned disadvantages of nurse prescribing. Harris (2004) stated this Disadvantage as follows: "Most of nurses felt that there was additional work associated with record keeping and administration" (<xref rid="R9" ref-type="bibr">9</xref>). Price et al (2012) has quoted the disadvantage as "safety concerns" including: working outside the parameters of Patient Group Directions (PGD), poor record keeping and the development of PGD that failed to comply with legal requirements (<xref rid="R16" ref-type="bibr">16</xref>). </p></sec><sec id="s3-6"><title>Infrastructures</title><p> In total, the meta synthesis showed that three authors investigated the infrastructures of nurse prescribing (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R10" ref-type="bibr">10</xref>, <xref rid="R15" ref-type="bibr">15</xref>). Most other studies were not relevant to the main study question. This issue covered three categories (legal status, educational and organizational conditions). </p><p> Only one study stated the legal restrictions as follows: "All Western-European and Anglo-Saxon countries that have realized or initiated nurse prescribing have imposed legal restrictions on which categories of nurses can prescribe medicines" (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> Two studies (<xref rid="R2" ref-type="bibr">2</xref>, <xref rid="R15" ref-type="bibr">15</xref>) addressed organizational conditions as illustrated in the following comment: </p><p> "The gradual growth over time of legislative authority and in the numbers of non-medical prescribers, particularly nurses, in some countries suggests that the acceptability of nonmedical prescribing is based on the perceived value to the health care system as a whole (<xref rid="R15" ref-type="bibr">15</xref>) and "It is nonetheless clear that most countries operate some sort of mandatory registration system in which nurse prescribers have to be registered before they are allowed to prescribe" (<xref rid="R2" ref-type="bibr">2</xref>). </p><p> One study (<xref rid="R2" ref-type="bibr">2</xref>) which focused on educational conditions stated: "nurses are required to successfully complete a prescribing course before they are allowed to start prescribing and one of the most important requirements for nurses internationally to enter prescribing programs is sufficient clinical experience" and also: "nurses must have three - five years of clinical experience in their own field of practice, before they are eligible for endorsements as a nurse practitioner and hence for prescribing medicines". </p></sec><sec id="s3-7"><title>Barriers</title><p> The results indicated barriers to nurse prescribing categories included: legal limitations, executive factors, educational deficiencies, research weaknesses, humanistic factors such as concerns about the adequacy of pharmacologic knowledge and unaware of physicians about nurse prescribing education (<xref ref-type="table" rid="T3">Table 3</xref>). </p></sec><sec id="s3-8"><title>Facilitators</title><p> The results of the meta synthesis indicated the facilitators of nurse prescribing such as training and educational preparations, managerial factor for support mechanisms and competency assessment, organizational factors to aware physicians and other staff about nurse prescribing. <xref ref-type="table" rid="T4">Table 4</xref> shows summary of facilitators in nurse prescribing implementation identified by the meta-synthesis. </p><table-wrap id="T4" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Summary of <underline>facilitators</underline> of nurse prescribing identified by the meta-synthesis </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1"> <bold>Categories</bold> </td><td rowspan="1" colspan="1">Cods</td><td rowspan="1" colspan="1">Illustrations</td><td rowspan="1" colspan="1">Authors</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">Educational factors</td><td rowspan="2" colspan="1"> Appropriate education and training to safe and effective prescribing need for further training </td><td rowspan="1" colspan="1">appropriate education and training were essential not only for safe and effective prescribing but also for a wider role in medicines management </td><td rowspan="1" colspan="1"> Harris, 2004<sup> (<xref rid="R9" ref-type="bibr">9</xref>) </sup> </td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"> Both Luker et al. (1997) and Brooks et al. (2001) also comment on nurses&#x02019; need for further training as the expansions to the formulary are introduced. </td><td rowspan="1" colspan="1"> Latter and Courtenay, 2004<sup> (<xref rid="R10" ref-type="bibr">10</xref>) </sup> </td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">Educational needs</td><td rowspan="1" colspan="1"> Tyler &#x00026; Hicks (2001) survey of family planning nurses&#x02019;training needs for prescribing identified nurses&#x02019; views on the top 15 training needs, that included research, advanced clinical activities, applied pharmacology, administration and technical activities. </td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1"/><td rowspan="2" colspan="1">The need for scientific education in applied pharmacology</td><td rowspan="1" colspan="1"> although patients were consent with nurses prescribing medication, nurses lacked confidence in applied pharmacology and therapeutics and hence, required additional scientific education </td><td rowspan="1" colspan="1"> Banning, 2004<sup> (<xref rid="R8" ref-type="bibr">8</xref>) </sup> </td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"> One can suggest that pre-registration nurses should receive a comprehensive scientific foundation in applied pharmacology and therapeutics and professional knowledge in order to prepare them for post graduate education and training in medication management </td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">Managerial factors</td><td rowspan="1" colspan="1">support mechanisms</td><td rowspan="1" colspan="1"> Two studies looked specifically at the support mechanisms nurses require in practice to enable good prescribing. </td><td rowspan="1" colspan="1"> Latter and Courtenay, 2004 <sup> (<xref rid="R10" ref-type="bibr">10</xref>) </sup> </td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">nurses&#x02019; confidence with Supplementary prescribing to prescribe independently.</td><td rowspan="1" colspan="1">Supplementary prescribing was found to be useful in the initial stages as it builds nurses&#x02019; confidence to prescribe independently.</td><td rowspan="1" colspan="1"> Creedon , et al.,2009<sup> (<xref rid="R13" ref-type="bibr">13</xref>) </sup> </td></tr><tr><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">competency assessment</td><td rowspan="1" colspan="1">In order to improve safety, increased competency assessment and training was recommended (Baxter et al, 2002; Jones, 2002b; Larsen, 2004; Baileff, 2007), in conjunction with the development of national PGDs by the DH (Baxter et al, 2002). Methods of competency assessment found to be effective included knowledge assessment via questionnaire (Brooks et al, 2003) and role play (Bacon et al, 2003).</td><td rowspan="1" colspan="1"> Price, et al., 2012<sup> (<xref rid="R16" ref-type="bibr">16</xref>) </sup> </td></tr><tr><td rowspan="1" colspan="1">Organizational factors</td><td rowspan="1" colspan="1">awareness of physicians and other staff</td><td rowspan="1" colspan="1">There was a perception that nurse and pharmacist independent prescribing may supersede supplementary prescribing</td><td rowspan="1" colspan="1"> Cooper, et al., 2008<sup> (<xref rid="R11" ref-type="bibr">11</xref>) </sup> </td></tr></tbody></table></table-wrap><p> As the results show in Schematic model (<xref ref-type="fig" rid="F2">Fig. 2</xref>), barriers and facilitators are directly in contact with infrastructures. In fact, with structural reforming, the barriers will be eliminated and the implementation of the nurse prescribing will be facilitated. </p></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> Our analysis showed different aspects of nurse prescribing that can help other countries act on its implementation with greater recognition. </p><p> The first theme "the leading countries in prescribing" depicted that the implementation of nurse prescribing in UK was higher than other countries. Nurse drug prescribing in the UK has grown significantly over the last decade and it is well established as a mainstream qualification (<xref rid="R29" ref-type="bibr">29</xref>). </p><p> Despite this fact that nurse prescribing has had well growth in European and American countries, but unfortunately, there is no evidence to indicate its implementation in Asian countries. This may be attributed due to diverse policy of health care system, cultural, economical, political and social conditions dominated in these countries that the nurses. Hence, despite the useful information derived from leading countries, the health policy makers should consider their context for nurse prescribing implementation. </p><p> This issue can also be looked at the other side that may be nurses are prescribing in these countries now, but it has not been published in online articles. As the Miles et al (2006) mentioned detailed information about the specific nature of nurse prescribing in low-resource settings was generally lacking in the literature (<xref rid="R30" ref-type="bibr">30</xref>). </p><p> The theme "Views"(second theme) indicated the views of different stakeholders (doctors, nurses, and patients) on nurse prescribing. Although all reviews studies reported positive views regarding nurse prescribing but negative views of physicians was reported which may be due to lack of knowledge on the training of nurse prescribers. Health professions have often specific knowledge in their field and do not have sufficient awareness about other professions. This may cause resistance to implementation of such activities that have overlap with their areas of functions. However, the health policy makers should pay special attention to this issue in their strategic planning and provide proper context for professional's familiarity with other fields. For instance it is recommended some of nursing courses be taught by physicians. In this way, doctors may become more familiar with the content of nursing courses and also nurses learn better interaction with physicians. </p><p> Another theme in our findings was" features of nurse prescribing". One of the features of nurse prescribing noted in studies was longer consultations of nurse prescribers than physicians that can lead patient-centered care as a main goal of health care system. This feature is a unique characteristic of nursing that is affected by holistic perspective toward patient. Based on the Irajpour et al (2012) study, more problems raise when some professionals treat patients as medical cases only worth studying rather than a human who needs health services (<xref rid="R31" ref-type="bibr">31</xref>). Thus nurse prescribing with their holistic view may probably face fewer problems. </p><p> "Benefits" and "disadvantages" were the two other themes emerged in our study. </p><p> These results showed in contrast to the considerable benefits of nurse prescribing, there are a few disadvantages that can be corrected by managerial planning. Each of identified benefits is enough important to support the development of nurse prescribing in all countries. These benefits are likely to be extended if nurse practitioners are able to prescribe (<xref rid="R32" ref-type="bibr">32</xref>). Here's a point worth mentioning that these reviews despite the many benefits of nurse prescribing have not addressed cost-benefit and cost- effectiveness of nurse prescribing. In other words, there are very few published evaluations about the clinical outcomes and cost effectiveness of nurse prescribing, particularly in direct comparison with doctors, the traditional prescribers of medication (<xref rid="R30" ref-type="bibr">30</xref>). </p><p> For example it is mentioned in Venning et al (2000) study that clinical care and health service costs of nurse practitioners and general practitioners were similar. However, they concluded if nurse practitioners were able to maintain the benefits while reducing their return consultation rate or shortening consultation times, they could be more cost effective than general practitioners (<xref rid="R33" ref-type="bibr">33</xref>). Another possible explanation for cost- effectiveness of nurse prescribing may be the view of hospitals in the past which considered nurses as cheap labor and believed they &#x0201c;owned&#x0201d; nurses. Today, many hospitals hold the same view (<xref rid="R34" ref-type="bibr">34</xref>). Thus with a simple count it may be concluded that the nurse prescribing is both cost-benefit and cost-effectiveness. However more information is needed about the cost-benefit and cost-effectiveness of nurse prescribing that point to the need for further research. </p><p> Another theme in this meta-synthesis was infrastructures. Although the main issues for implementation of nurse prescribing (legal status, educational and organizational conditions) have been expressed in studies, but there is no available evidence regarding the cultural and political context that strongly affect on the entire topic of nurse prescribing. Importance of this issue is stated in Mills et al study (2oo6) who mentioned some of issues may be somewhat irrelevant when it comes to politically and economically unstable environments (<xref rid="R30" ref-type="bibr">30</xref>). </p><p> Thus it seems that one of the priorities of nurse prescribing implementation is providing cultural and political context. However these factors can affect as the barriers in nurse prescribing implementation because they can influence on views of policy makers and managers. Then their views can impact on strategic planning, implementation, and supportive structure or even on interaction of medical team colleagues. </p><p> The results identified a broad range of barriers and facilitators (Theme 7 and 8). These factors should be inferred as guides for policymakers to implementation of the nurse prescribing. Also the results revealed there are still issues such as legal, administrative, weak research and educational deficiencies in leading countries which needs for more effort to be made in these areas. Based on this review lack of confidence in applied pharmacology and therapeutics is an issue categorized in human factors as the barrier of nurse drug prescription. Conversely, self-confidence is a facilitator in nurse prescribing. On the other hand, delegating authority and enhancing self-confidence of nurses can help them to apply their knowledge in practice (<xref rid="R35" ref-type="bibr">35</xref>). </p><p> Miles et al (2oo6) expressed there are key elements of success in developing mechanisms for nurse prescribing such as strong political, support of nursing and academic programs to educate. (<xref rid="R30" ref-type="bibr">30</xref>). However, one of the problems highlighted with training programs is that many academic nursing education programs are not always feasible, affordable or attainable, particularly in rural areas (<xref rid="R30" ref-type="bibr">30</xref>). Support mechanisms are other important issues that are addressed in this meta synthesis. For providing this the nurse managers can play an important role. This support can undertake any facilitating activity, such as providing facilities, means and equipment, information, education, rewards and even some symbolic behaviors that nurses perceive to be facilitating (<xref rid="R35" ref-type="bibr">35</xref>). Also the support that physicians give in terms of ongoing training and supervision is important key in nurse prescribing (<xref rid="R36" ref-type="bibr">36</xref>). </p></sec><sec sec-type="conclusion" id="s5"><title>Conclusion</title><p> Considering the findings of this study, it seems that nursing prescribing is positive experience and other countries can introduce its implementation without serious concern. In this metasynthesis, the most important factors identified including providing valuable information for nurse managers and policy makers. However, the results revealed there are still issues such as legal, administrative, weak research and educational deficiencies in leading countries that needs more effort in these areas. Appropriate training and support mechanisms are needed in order to facilitate implementation of nurse prescribing. In spite of the useful information that has been obtained from the study of the leading countries, cultural and social factors of each country are also of great importance. Hence health policy makers should identify barriers and facilitators within their own context and use this information to promote health system. </p></sec><sec id="s8"><title>Ethical Considerations</title><p> Although the ethical issues were minimal in this study since no human subjects were involved, the researchers still had an ethical responsibility arising from this study. In order to fulfill this, the researchers considered the ethical responsibility as follows: </p><list list-type="bullet"><list-item><p> Commitment to respecting moral rights of authors and no forging parts or all of the results (data making&#x0200f;) </p></list-item><list-item><p> Intentional manipulation of data or analyzes (falsification), or reporting methods not used by the researchers </p></list-item></list><sec id="s8-1"><title>Recommendations for future research</title><p> There are little evidences reporting the economic and financial outcomes of non-medical prescribing. It is therefore recommended that future studies explore cost-benefit and cost-effectiveness of nurse prescribing. </p><p> Considering the importance of the cultural context of health systems in implementation of programs, policymakers must recognize this within their own context. It is suggested that next research should be on understanding of infrastructures and existing context of nurse prescribing in each country. </p><p> There is also a need for more theory-based research on nurse prescribing. Future research should address this. </p></sec></sec><sec id="s6"><title>Limitations</title><p> Limitations of this synthesis include the exclusion of unpublished and in progress reviews and systematic reviews. </p></sec><sec id="s7"><title>Acknowledgements</title><p> This study is part of a PhD thesis financially supported by Tehran University of Medical Sciences (Grant Number: 1929). </p></sec><sec id="s9"><title>Conflict of Interest</title><p> No conflict of interest associated with this work. </p></sec>
How within-city socioeconomic disparities affect life expectancy? Results of Urban HEART in Tehran, Iran
<p> <bold>Background:</bold> There is substantial lack of knowledge about the role of socioeconomic status (SES) indicators on life expectancy (LE) within-cities, especially within mega-cities. We aimed to investigate the disparities of LE within city districts of Tehran, Iran, and specify how SES inequalities play role on LE. </p><p> <bold>Methods:</bold> The death and population data for 2010 by different age, gender, and residency district were obtained from the main cemetery of Tehran and statistical centre of Iran, respectively. Age-specific mortality rates and consequently LE were calculated for all 22 districts by different genders. Finally, based on the results of first Tehran's Urban Health Equity Assessment and Response Tool (Urban HEART) project in 2008, the influence of social classes (SCs), total costs, and education indicators were analyzed on LE at birth (e0). </p><p> <bold>Results:</bold> The e0 for total males and females in Tehran were calculated as 74.6 and 78.4 years for 2010, respectively. The maximum LE of 80 years was observed in females of northern part with higher SES, and the minimum e0 of 72.7 years observed in males of southern part with lower SES. The e0 gender gap among districts was 5.5 years for females and 3.7 years for males. The highest and lowest mean of e0 observed in SC1 (highest class) and SC5 (lowest class), were 77.6 and 76.0 years, respectively. The lowest mean of e0 observed in the first group of total costs indicator and was 76.2 years. In addition, the lowest observed mean of e0 was in the first category of education indicator (illiterate) and was 76.0 years. </p><p> <bold>Conclusion:</bold> Results indicate substantial disparities in LE within city districts. This confirms that SES disparities within-cities would have direct influences on LE. </p>
<contrib contrib-type="author"><name><surname>Mokhayeri</surname><given-names>Yaser</given-names></name><xref ref-type="aff" rid="A01"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Mahmoudi</surname><given-names>Mahmood</given-names></name><xref ref-type="aff" rid="A02"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Haghdoost</surname><given-names>Ali Akbar</given-names></name><xref ref-type="aff" rid="A03"> <sup>3</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Amini</surname><given-names>Hassan</given-names></name><xref ref-type="aff" rid="A04"> <sup>4</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Asadi-Lari</surname><given-names>Mohsen</given-names></name><xref ref-type="aff" rid="A05"> <sup>5</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Holakouie Naieni</surname><given-names>Kourosh</given-names></name><xref ref-type="aff" rid="A06"> <sup>6</sup> </xref></contrib>
Medical Journal of the Islamic Republic of Iran
<sec sec-type="introduction" id="s1"><title>Introduction</title><p> According to world health report 2000, disability-adjusted life expectancy (LE) at birth in Iran for 1999 was 60.5 years, and Iran had a rank of 96 among 191 member states of World Health Organization (WHO) (<xref rid="R1" ref-type="bibr">1</xref>). This was upgraded, moreover, to 71.56 years in 2003 (<xref rid="R2" ref-type="bibr">2</xref>), and in the most recent report, life expectancy at birth increased to 73 years in 2009 (<xref rid="R3" ref-type="bibr">3</xref>). </p><p> Generally, there are considerable credible literatures that report influence of socioeconomic measures on LE (<xref rid="R4" ref-type="bibr">4</xref>-<xref rid="R8" ref-type="bibr">8</xref>). In brief, little association has been reported between LE and gross national product (GNP) among rich countries of the world (<xref rid="R4" ref-type="bibr">4</xref>). However, a potent relationship between socioeconomic status (SES) of the households and children mortality rates, exist within the country in which mortality rates increase gradually in households with lower SES (<xref rid="R4" ref-type="bibr">4</xref>, <xref rid="R9" ref-type="bibr">9</xref>). In addition, education level has an inverse relation with mortality rates. For example, mortality rates have been reported to be less in well educated people of every parts of the world either developing or developed countries (<xref rid="R9" ref-type="bibr">9</xref>-<xref rid="R13" ref-type="bibr">13</xref>), where indicates the LE gap within educational groups is still rising to the extent that educational differentials may depict 30% of changes in LE (<xref rid="R12" ref-type="bibr">12</xref>). Nevertheless, albeit there is large body of literature in global- and national-scale on the influence of SES disparities on LE, considerable lack of knowledge exist on how SES factors play role on LE within-cities, especially within mega-cities. </p><p> In this study, the authors aimed a) to investigate the disparities of LE within 22 city-districts of Tehran, Iran, and b) to analyze the influence of SES disparities on LE within city-districts. </p></sec><sec sec-type="methods" id="s2"><title>Methods</title><sec id="s2-1"><title> Study area </title><p> Tehran, the capital of Iran, is the largest and most populated city of the country. The population at the time of study was about 8.2 million people (<xref rid="R14" ref-type="bibr">14</xref>). The city has a large area of about 613 km2, and divided administratively into 22 districts (<xref rid="R15" ref-type="bibr">15</xref>). The structure of the city is diverse in which socioeconomic situation of northern households are higher than southern parts. </p></sec><sec id="s2-2"><title> LE calculations </title><p> In this analytic-descriptive study, we used census method to estimate LE within city districts. The main cemetery of Tehran, Behesht-e-Zahra which provides necessary service for family of deceased people is officially under subsidiary of Tehran Municipality where all death data are recorded. Thus, the number of deaths in 2010 obtained from this cemetery was based on age, gender, and residency district (<xref rid="R16" ref-type="bibr">16</xref>). The population data were also derived from the statistical centre of Iran by the same catagories (<xref rid="R14" ref-type="bibr">14</xref>). Age-specific mortality rates, thereafter, were calculated for all 22 districts of Tehran by different genders. Based on the age-specific mortality rates, the estimated LE for all Tehran's districts through various gender and age groups, were at birth or &#x0003c;1 year-old, 1-4, 5-9,&#x02026;, 75-79, and &#x0003e;80 years-old. To estimate life tables we used MORTPAK 4 software&#x02014;The United Nations Software Package for Mortality Measurements&#x02014;with 17 different applications, using "COPMAR" and "MATCH" applications to estimate the LE (<xref rid="R17" ref-type="bibr">17</xref>). After determining the LE for all 22 districts, we plotted the results using geographic information system (GIS) to visualize the distribution of LE over the city districts. </p></sec><sec id="s2-3"><title> Socioeconomic measures </title><p> In order to determine the influence of socioeconomic measures on LE within city districts, results of the first Tehran's Urban Health Equity Assessment and Response Tool (Urban HEART-1) project in 2008 were extracted (<xref rid="R18" ref-type="bibr">18</xref>). Briefly, the Tehran's Urban HEART project evaluated 42 indicators in six principal policy domains including &#x0201c;Physical and Infra-structure,&#x0201d; &#x0201c;Human and Social Development,&#x0201d; &#x0201c;Economic,&#x0201d; &#x0201c;Governance,&#x0201d; &#x0201c;Health&#x0201d; and &#x0201c;Nutrition&#x0201d; to quantify socioeconomic inequalities in a large population-based survey within Tehran megacity. In the current study, three indicators, namely &#x0201c;Socioeconomic Status (SES)&#x0201d; according to job, &#x0201c;Total Costs&#x0201d; (non-food), and &#x0201c;Education&#x0201d; were extracted and analyzed to explore the relationship with LE (<xref rid="R18" ref-type="bibr">18</xref>). </p><p> Social classes were determined according to the household head&#x02019;s occupation, and are summarized into six social classes (SCs). </p><p> Total Costs was a single quantitative variable, derived from a series of detailed questions regarding every aspect of household expenditures reported by the family head, which were finally categorized into five quintiles that represent 20 percent of total data for each category. </p><p> Education was categorized into five groups including &#x0201c;Illiterate,&#x0201d; &#x0201c;Primary,&#x0201d; &#x0201c;High-school and Diploma,&#x0201d; &#x0201c;Bachelor of Science (BSc) and under&#x0201d; and &#x0201c;Master of Science (MSc) and Higher.&#x0201d; </p></sec><sec id="s2-4"><title> Statistical analyses </title><p> Kolmogorov-Smirnov test was used to test the normality of LE. Descriptive statistics were applied to SES, total costs, and education indicators. Levene's test was used to investigate the homogeneity (equality) of variances between sub-groups. Since the variances among the sub-groups were not equal, hence non-parametric tests were used. Moreover Kruskal-Wallis test was used to test differences between sub-groups and post-hoc test used for pairwise multiple comparisons between sub-groups. </p></sec></sec><sec sec-type="results" id="s3"><title>Results</title><sec id="s3-1"><title> LE </title><p> The LE for total males and females in Tehran were calculated as 74.6 and 78.4 years for 2010, respectively (p&#x0003c; 0.001). The maximum LE of 80 years was observed in females of northern part (1, 4, and 5 districts) with higher socioeconomic status, and the minimum LE of 72.7 years observed in males of southern part (district 9) with lower socioeconomic status, and with a gap of 7.3 years (<xref ref-type="fig" rid="F1">Fig. 1</xref>). The maximum and minimum observed LE for males in districts of northern (district 1) and southern part (district 9), were 78.2 and 72.7 years, respectively. The maximum LE for females estimated as 80 years in districts of northern part (districts of 1, 4, and 5), while the minimum was 76.3 years in district 9 in southern part. The LE gender gap among districts was 5.5 years for females and 3.7 for males. Regardless of gender difference, the maximum and minimum LEs observed in districts of northern (district 1) and southern part (district 9), were 79.1 and 74.5, respectively. </p><fig id="F1" orientation="portrait" position="float"><label> Fig. 1 </label><graphic xlink:href="MJIRI-28-80-g001"/><statement><p>Estimated distribution of life expectancy at birth for a) males, b) females and c) both genders in 22 districts of Tehran, Iran, 2010.</p></statement></fig><p> <xref ref-type="table" rid="T1">Table 1</xref> shows LE at birth in all 22 districts of Tehran in both genders. Also, <xref ref-type="table" rid="T2">Tables 2</xref> and <xref ref-type="table" rid="T3">3</xref> show LE of aforementioned age groups by different genders in all 22 districts of Tehran. Moreover,<xref ref-type="fig" rid="F1">Fig. 1</xref> illustrates the geographic distribution of LE in all 22 districts of Tehran megacity by various genders. Descriptive statistics of SES, education, and total costs indicators, also, are shown in <xref ref-type="table" rid="T4">Table 4</xref>. </p><table-wrap id="T1" orientation="portrait" position="float"><label> Table 1 </label><caption><title> Life expectancy at birth within Tehran megacity by different districts, genders and geographic location </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">District</td><td align="center" rowspan="1" colspan="1">Males</td><td align="center" rowspan="1" colspan="1">Females</td><td align="center" rowspan="1" colspan="1"> Both <break/>genders </td><td align="center" rowspan="1" colspan="1">Geographical location</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">78.2</td><td align="center" rowspan="1" colspan="1">80.0</td><td align="center" rowspan="1" colspan="1">79.1</td><td align="center" rowspan="1" colspan="1">North</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">75.9</td><td align="center" rowspan="1" colspan="1">79.6</td><td align="center" rowspan="1" colspan="1">77.7</td><td align="center" rowspan="1" colspan="1">North</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">75.9</td><td align="center" rowspan="1" colspan="1">79.5</td><td align="center" rowspan="1" colspan="1">77.7</td><td align="center" rowspan="1" colspan="1">North</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">78.0</td><td align="center" rowspan="1" colspan="1">80.0</td><td align="center" rowspan="1" colspan="1">79.0</td><td align="center" rowspan="1" colspan="1">North</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">77.8</td><td align="center" rowspan="1" colspan="1">80.0</td><td align="center" rowspan="1" colspan="1">78.9</td><td align="center" rowspan="1" colspan="1">North</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">74.7</td><td align="center" rowspan="1" colspan="1">78.4</td><td align="center" rowspan="1" colspan="1">76.5</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">74.2</td><td align="center" rowspan="1" colspan="1">77.7</td><td align="center" rowspan="1" colspan="1">75.9</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">74.0</td><td align="center" rowspan="1" colspan="1">77.3</td><td align="center" rowspan="1" colspan="1">75.6</td><td align="center" rowspan="1" colspan="1">West</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">72.7</td><td align="center" rowspan="1" colspan="1">76.3</td><td align="center" rowspan="1" colspan="1">74.5</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">73.1</td><td align="center" rowspan="1" colspan="1">77.1</td><td align="center" rowspan="1" colspan="1">75.1</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">73.7</td><td align="center" rowspan="1" colspan="1">77.0</td><td align="center" rowspan="1" colspan="1">75.3</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">72.8</td><td align="center" rowspan="1" colspan="1">76.7</td><td align="center" rowspan="1" colspan="1">74.7</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">73.1</td><td align="center" rowspan="1" colspan="1">77.7</td><td align="center" rowspan="1" colspan="1">75.4</td><td align="center" rowspan="1" colspan="1">West</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">74.3</td><td align="center" rowspan="1" colspan="1">77.6</td><td align="center" rowspan="1" colspan="1">75.9</td><td align="center" rowspan="1" colspan="1">West</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">74.7</td><td align="center" rowspan="1" colspan="1">78.6</td><td align="center" rowspan="1" colspan="1">76.6</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">72.9</td><td align="center" rowspan="1" colspan="1">76.4</td><td align="center" rowspan="1" colspan="1">74.6</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">72.8</td><td align="center" rowspan="1" colspan="1">77.2</td><td align="center" rowspan="1" colspan="1">75.0</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">74.0</td><td align="center" rowspan="1" colspan="1">77.5</td><td align="center" rowspan="1" colspan="1">75.7</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">73.0</td><td align="center" rowspan="1" colspan="1">76.8</td><td align="center" rowspan="1" colspan="1">74.9</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">74.0</td><td align="center" rowspan="1" colspan="1">79.9</td><td align="center" rowspan="1" colspan="1">76.9</td><td align="center" rowspan="1" colspan="1">South</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">74.0</td><td align="center" rowspan="1" colspan="1">79.1</td><td align="center" rowspan="1" colspan="1">76.5</td><td align="center" rowspan="1" colspan="1">East</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1">75.6</td><td align="center" rowspan="1" colspan="1">79.7</td><td align="center" rowspan="1" colspan="1">77.6</td><td align="center" rowspan="1" colspan="1">East</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">74.6</td><td align="center" rowspan="1" colspan="1">78.4</td><td align="center" rowspan="1" colspan="1">76.5</td><td align="center" rowspan="1" colspan="1">-</td></tr></tbody></table></table-wrap><table-wrap id="T2" orientation="portrait" position="float"><label> Table 2 </label><caption><title> Life expectancy of males in Tehran megacity by different districts and age-groups in 2010 </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Age group / District</td><td align="center" rowspan="1" colspan="1">1_4</td><td align="center" rowspan="1" colspan="1">5_9</td><td align="center" rowspan="1" colspan="1">10_14</td><td align="center" rowspan="1" colspan="1">15_19</td><td align="center" rowspan="1" colspan="1">20_24</td><td align="center" rowspan="1" colspan="1">25_29</td><td align="center" rowspan="1" colspan="1">30_34</td><td align="center" rowspan="1" colspan="1">35_39</td><td align="center" rowspan="1" colspan="1">40_44</td><td align="center" rowspan="1" colspan="1">45_49</td><td align="center" rowspan="1" colspan="1">50_54</td><td align="center" rowspan="1" colspan="1">55_59</td><td align="center" rowspan="1" colspan="1">60_64</td><td align="center" rowspan="1" colspan="1">65_69</td><td align="center" rowspan="1" colspan="1">70_74</td><td align="center" rowspan="1" colspan="1">75_79</td><td align="center" rowspan="1" colspan="1">80&#x0003c;</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">77.54</td><td align="center" rowspan="1" colspan="1">73.55</td><td align="center" rowspan="1" colspan="1">68.57</td><td align="center" rowspan="1" colspan="1">63.59</td><td align="center" rowspan="1" colspan="1">58.64</td><td align="center" rowspan="1" colspan="1">53.70</td><td align="center" rowspan="1" colspan="1">48.76</td><td align="center" rowspan="1" colspan="1">43.82</td><td align="center" rowspan="1" colspan="1">38.89</td><td align="center" rowspan="1" colspan="1">34.00</td><td align="center" rowspan="1" colspan="1">29.22</td><td align="center" rowspan="1" colspan="1">24.65</td><td align="center" rowspan="1" colspan="1">20.38</td><td align="center" rowspan="1" colspan="1">16.41</td><td align="center" rowspan="1" colspan="1">12.77</td><td align="center" rowspan="1" colspan="1">9.64</td><td align="center" rowspan="1" colspan="1">7.11</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">75.58</td><td align="center" rowspan="1" colspan="1">71.62</td><td align="center" rowspan="1" colspan="1">66.65</td><td align="center" rowspan="1" colspan="1">61.69</td><td align="center" rowspan="1" colspan="1">56.77</td><td align="center" rowspan="1" colspan="1">51.89</td><td align="center" rowspan="1" colspan="1">47.00</td><td align="center" rowspan="1" colspan="1">42.10</td><td align="center" rowspan="1" colspan="1">37.23</td><td align="center" rowspan="1" colspan="1">32.40</td><td align="center" rowspan="1" colspan="1">27.72</td><td align="center" rowspan="1" colspan="1">23.29</td><td align="center" rowspan="1" colspan="1">19.17</td><td align="center" rowspan="1" colspan="1">15.37</td><td align="center" rowspan="1" colspan="1">11.92</td><td align="center" rowspan="1" colspan="1">8.99</td><td align="center" rowspan="1" colspan="1">6.65</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">75.58</td><td align="center" rowspan="1" colspan="1">71.62</td><td align="center" rowspan="1" colspan="1">66.65</td><td align="center" rowspan="1" colspan="1">61.69</td><td align="center" rowspan="1" colspan="1">56.77</td><td align="center" rowspan="1" colspan="1">51.89</td><td align="center" rowspan="1" colspan="1">47.00</td><td align="center" rowspan="1" colspan="1">42.10</td><td align="center" rowspan="1" colspan="1">37.23</td><td align="center" rowspan="1" colspan="1">32.40</td><td align="center" rowspan="1" colspan="1">27.72</td><td align="center" rowspan="1" colspan="1">23.29</td><td align="center" rowspan="1" colspan="1">19.17</td><td align="center" rowspan="1" colspan="1">15.37</td><td align="center" rowspan="1" colspan="1">11.92</td><td align="center" rowspan="1" colspan="1">8.99</td><td align="center" rowspan="1" colspan="1">6.65</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">77.37</td><td align="center" rowspan="1" colspan="1">73.38</td><td align="center" rowspan="1" colspan="1">68.39</td><td align="center" rowspan="1" colspan="1">63.41</td><td align="center" rowspan="1" colspan="1">58.47</td><td align="center" rowspan="1" colspan="1">53.54</td><td align="center" rowspan="1" colspan="1">48.60</td><td align="center" rowspan="1" colspan="1">43.67</td><td align="center" rowspan="1" colspan="1">38.74</td><td align="center" rowspan="1" colspan="1">33.86</td><td align="center" rowspan="1" colspan="1">29.08</td><td align="center" rowspan="1" colspan="1">24.52</td><td align="center" rowspan="1" colspan="1">20.27</td><td align="center" rowspan="1" colspan="1">16.31</td><td align="center" rowspan="1" colspan="1">12.69</td><td align="center" rowspan="1" colspan="1">9.58</td><td align="center" rowspan="1" colspan="1">7.07</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">77.19</td><td align="center" rowspan="1" colspan="1">73.21</td><td align="center" rowspan="1" colspan="1">68.22</td><td align="center" rowspan="1" colspan="1">63.24</td><td align="center" rowspan="1" colspan="1">58.30</td><td align="center" rowspan="1" colspan="1">53.37</td><td align="center" rowspan="1" colspan="1">48.44</td><td align="center" rowspan="1" colspan="1">43.51</td><td align="center" rowspan="1" colspan="1">38.59</td><td align="center" rowspan="1" colspan="1">33.71</td><td align="center" rowspan="1" colspan="1">28.94</td><td align="center" rowspan="1" colspan="1">24.40</td><td align="center" rowspan="1" colspan="1">20.15</td><td align="center" rowspan="1" colspan="1">16.21</td><td align="center" rowspan="1" colspan="1">12.61</td><td align="center" rowspan="1" colspan="1">9.51</td><td align="center" rowspan="1" colspan="1">7.02</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">74.61</td><td align="center" rowspan="1" colspan="1">70.67</td><td align="center" rowspan="1" colspan="1">65.71</td><td align="center" rowspan="1" colspan="1">60.76</td><td align="center" rowspan="1" colspan="1">55.88</td><td align="center" rowspan="1" colspan="1">51.02</td><td align="center" rowspan="1" colspan="1">46.16</td><td align="center" rowspan="1" colspan="1">41.30</td><td align="center" rowspan="1" colspan="1">36.45</td><td align="center" rowspan="1" colspan="1">31.67</td><td align="center" rowspan="1" colspan="1">27.04</td><td align="center" rowspan="1" colspan="1">22.67</td><td align="center" rowspan="1" colspan="1">18.63</td><td align="center" rowspan="1" colspan="1">14.92</td><td align="center" rowspan="1" colspan="1">11.55</td><td align="center" rowspan="1" colspan="1">8.70</td><td align="center" rowspan="1" colspan="1">6.44</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">74.21</td><td align="center" rowspan="1" colspan="1">70.29</td><td align="center" rowspan="1" colspan="1">65.33</td><td align="center" rowspan="1" colspan="1">60.39</td><td align="center" rowspan="1" colspan="1">55.52</td><td align="center" rowspan="1" colspan="1">50.68</td><td align="center" rowspan="1" colspan="1">45.83</td><td align="center" rowspan="1" colspan="1">40.98</td><td align="center" rowspan="1" colspan="1">36.14</td><td align="center" rowspan="1" colspan="1">31.38</td><td align="center" rowspan="1" colspan="1">26.77</td><td align="center" rowspan="1" colspan="1">22.43</td><td align="center" rowspan="1" colspan="1">18.42</td><td align="center" rowspan="1" colspan="1">14.74</td><td align="center" rowspan="1" colspan="1">11.41</td><td align="center" rowspan="1" colspan="1">8.60</td><td align="center" rowspan="1" colspan="1">6.36</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">74.06</td><td align="center" rowspan="1" colspan="1">70.14</td><td align="center" rowspan="1" colspan="1">65.19</td><td align="center" rowspan="1" colspan="1">60.25</td><td align="center" rowspan="1" colspan="1">55.38</td><td align="center" rowspan="1" colspan="1">50.54</td><td align="center" rowspan="1" colspan="1">45.70</td><td align="center" rowspan="1" colspan="1">40.85</td><td align="center" rowspan="1" colspan="1">36.02</td><td align="center" rowspan="1" colspan="1">31.27</td><td align="center" rowspan="1" colspan="1">26.67</td><td align="center" rowspan="1" colspan="1">22.34</td><td align="center" rowspan="1" colspan="1">18.34</td><td align="center" rowspan="1" colspan="1">14.67</td><td align="center" rowspan="1" colspan="1">11.36</td><td align="center" rowspan="1" colspan="1">8.55</td><td align="center" rowspan="1" colspan="1">6.33</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">73.06</td><td align="center" rowspan="1" colspan="1">69.18</td><td align="center" rowspan="1" colspan="1">64.25</td><td align="center" rowspan="1" colspan="1">59.33</td><td align="center" rowspan="1" colspan="1">54.49</td><td align="center" rowspan="1" colspan="1">49.69</td><td align="center" rowspan="1" colspan="1">44.88</td><td align="center" rowspan="1" colspan="1">40.07</td><td align="center" rowspan="1" colspan="1">35.29</td><td align="center" rowspan="1" colspan="1">30.58</td><td align="center" rowspan="1" colspan="1">26.04</td><td align="center" rowspan="1" colspan="1">21.78</td><td align="center" rowspan="1" colspan="1">17.86</td><td align="center" rowspan="1" colspan="1">14.26</td><td align="center" rowspan="1" colspan="1">11.03</td><td align="center" rowspan="1" colspan="1">8.30</td><td align="center" rowspan="1" colspan="1">6.15</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">73.37</td><td align="center" rowspan="1" colspan="1">69.47</td><td align="center" rowspan="1" colspan="1">64.53</td><td align="center" rowspan="1" colspan="1">59.61</td><td align="center" rowspan="1" colspan="1">54.75</td><td align="center" rowspan="1" colspan="1">49.95</td><td align="center" rowspan="1" colspan="1">45.13</td><td align="center" rowspan="1" colspan="1">40.31</td><td align="center" rowspan="1" colspan="1">35.51</td><td align="center" rowspan="1" colspan="1">30.78</td><td align="center" rowspan="1" colspan="1">26.22</td><td align="center" rowspan="1" colspan="1">21.95</td><td align="center" rowspan="1" colspan="1">18.00</td><td align="center" rowspan="1" colspan="1">14.38</td><td align="center" rowspan="1" colspan="1">11.12</td><td align="center" rowspan="1" colspan="1">8.37</td><td align="center" rowspan="1" colspan="1">6.21</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">73.83</td><td align="center" rowspan="1" colspan="1">69.91</td><td align="center" rowspan="1" colspan="1">64.97</td><td align="center" rowspan="1" colspan="1">60.03</td><td align="center" rowspan="1" colspan="1">55.17</td><td align="center" rowspan="1" colspan="1">50.34</td><td align="center" rowspan="1" colspan="1">45.51</td><td align="center" rowspan="1" colspan="1">40.67</td><td align="center" rowspan="1" colspan="1">35.85</td><td align="center" rowspan="1" colspan="1">31.10</td><td align="center" rowspan="1" colspan="1">26.52</td><td align="center" rowspan="1" colspan="1">22.21</td><td align="center" rowspan="1" colspan="1">18.23</td><td align="center" rowspan="1" colspan="1">14.57</td><td align="center" rowspan="1" colspan="1">11.28</td><td align="center" rowspan="1" colspan="1">8.49</td><td align="center" rowspan="1" colspan="1">6.29</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">73.14</td><td align="center" rowspan="1" colspan="1">69.25</td><td align="center" rowspan="1" colspan="1">64.32</td><td align="center" rowspan="1" colspan="1">59.40</td><td align="center" rowspan="1" colspan="1">54.55</td><td align="center" rowspan="1" colspan="1">49.76</td><td align="center" rowspan="1" colspan="1">44.95</td><td align="center" rowspan="1" colspan="1">40.13</td><td align="center" rowspan="1" colspan="1">35.34</td><td align="center" rowspan="1" colspan="1">30.63</td><td align="center" rowspan="1" colspan="1">26.08</td><td align="center" rowspan="1" colspan="1">21.82</td><td align="center" rowspan="1" colspan="1">17.89</td><td align="center" rowspan="1" colspan="1">14.29</td><td align="center" rowspan="1" colspan="1">11.05</td><td align="center" rowspan="1" colspan="1">8.32</td><td align="center" rowspan="1" colspan="1">6.17</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">73.37</td><td align="center" rowspan="1" colspan="1">69.47</td><td align="center" rowspan="1" colspan="1">64.53</td><td align="center" rowspan="1" colspan="1">59.61</td><td align="center" rowspan="1" colspan="1">54.75</td><td align="center" rowspan="1" colspan="1">49.95</td><td align="center" rowspan="1" colspan="1">45.13</td><td align="center" rowspan="1" colspan="1">40.31</td><td align="center" rowspan="1" colspan="1">35.51</td><td align="center" rowspan="1" colspan="1">30.78</td><td align="center" rowspan="1" colspan="1">26.22</td><td align="center" rowspan="1" colspan="1">21.95</td><td align="center" rowspan="1" colspan="1">18.00</td><td align="center" rowspan="1" colspan="1">14.38</td><td align="center" rowspan="1" colspan="1">11.12</td><td align="center" rowspan="1" colspan="1">8.37</td><td align="center" rowspan="1" colspan="1">6.21</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">74.29</td><td align="center" rowspan="1" colspan="1">70.36</td><td align="center" rowspan="1" colspan="1">65.41</td><td align="center" rowspan="1" colspan="1">60.47</td><td align="center" rowspan="1" colspan="1">55.59</td><td align="center" rowspan="1" colspan="1">50.75</td><td align="center" rowspan="1" colspan="1">45.89</td><td align="center" rowspan="1" colspan="1">41.04</td><td align="center" rowspan="1" colspan="1">36.20</td><td align="center" rowspan="1" colspan="1">31.44</td><td align="center" rowspan="1" colspan="1">26.82</td><td align="center" rowspan="1" colspan="1">22.48</td><td align="center" rowspan="1" colspan="1">18.47</td><td align="center" rowspan="1" colspan="1">14.78</td><td align="center" rowspan="1" colspan="1">11.44</td><td align="center" rowspan="1" colspan="1">8.62</td><td align="center" rowspan="1" colspan="1">6.38</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">74.61</td><td align="center" rowspan="1" colspan="1">70.67</td><td align="center" rowspan="1" colspan="1">65.71</td><td align="center" rowspan="1" colspan="1">60.76</td><td align="center" rowspan="1" colspan="1">55.88</td><td align="center" rowspan="1" colspan="1">51.02</td><td align="center" rowspan="1" colspan="1">46.16</td><td align="center" rowspan="1" colspan="1">41.30</td><td align="center" rowspan="1" colspan="1">36.45</td><td align="center" rowspan="1" colspan="1">31.67</td><td align="center" rowspan="1" colspan="1">27.04</td><td align="center" rowspan="1" colspan="1">22.67</td><td align="center" rowspan="1" colspan="1">18.63</td><td align="center" rowspan="1" colspan="1">14.92</td><td align="center" rowspan="1" colspan="1">11.55</td><td align="center" rowspan="1" colspan="1">8.70</td><td align="center" rowspan="1" colspan="1">6.44</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">73.21</td><td align="center" rowspan="1" colspan="1">69.33</td><td align="center" rowspan="1" colspan="1">64.39</td><td align="center" rowspan="1" colspan="1">59.47</td><td align="center" rowspan="1" colspan="1">54.62</td><td align="center" rowspan="1" colspan="1">49.82</td><td align="center" rowspan="1" colspan="1">45.01</td><td align="center" rowspan="1" colspan="1">40.19</td><td align="center" rowspan="1" colspan="1">35.40</td><td align="center" rowspan="1" colspan="1">30.68</td><td align="center" rowspan="1" colspan="1">26.13</td><td align="center" rowspan="1" colspan="1">21.86</td><td align="center" rowspan="1" colspan="1">17.93</td><td align="center" rowspan="1" colspan="1">14.32</td><td align="center" rowspan="1" colspan="1">11.07</td><td align="center" rowspan="1" colspan="1">8.34</td><td align="center" rowspan="1" colspan="1">6.18</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">73.14</td><td align="center" rowspan="1" colspan="1">69.25</td><td align="center" rowspan="1" colspan="1">64.32</td><td align="center" rowspan="1" colspan="1">59.40</td><td align="center" rowspan="1" colspan="1">54.55</td><td align="center" rowspan="1" colspan="1">49.76</td><td align="center" rowspan="1" colspan="1">44.95</td><td align="center" rowspan="1" colspan="1">40.13</td><td align="center" rowspan="1" colspan="1">35.34</td><td align="center" rowspan="1" colspan="1">30.63</td><td align="center" rowspan="1" colspan="1">26.08</td><td align="center" rowspan="1" colspan="1">21.82</td><td align="center" rowspan="1" colspan="1">17.89</td><td align="center" rowspan="1" colspan="1">14.29</td><td align="center" rowspan="1" colspan="1">11.05</td><td align="center" rowspan="1" colspan="1">8.32</td><td align="center" rowspan="1" colspan="1">6.17</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">74.06</td><td align="center" rowspan="1" colspan="1">70.14</td><td align="center" rowspan="1" colspan="1">65.19</td><td align="center" rowspan="1" colspan="1">60.25</td><td align="center" rowspan="1" colspan="1">55.38</td><td align="center" rowspan="1" colspan="1">50.54</td><td align="center" rowspan="1" colspan="1">45.70</td><td align="center" rowspan="1" colspan="1">40.85</td><td align="center" rowspan="1" colspan="1">36.02</td><td align="center" rowspan="1" colspan="1">31.27</td><td align="center" rowspan="1" colspan="1">26.67</td><td align="center" rowspan="1" colspan="1">22.34</td><td align="center" rowspan="1" colspan="1">18.34</td><td align="center" rowspan="1" colspan="1">14.67</td><td align="center" rowspan="1" colspan="1">11.36</td><td align="center" rowspan="1" colspan="1">8.55</td><td align="center" rowspan="1" colspan="1">6.33</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">73.29</td><td align="center" rowspan="1" colspan="1">69.40</td><td align="center" rowspan="1" colspan="1">64.46</td><td align="center" rowspan="1" colspan="1">59.54</td><td align="center" rowspan="1" colspan="1">54.69</td><td align="center" rowspan="1" colspan="1">49.88</td><td align="center" rowspan="1" colspan="1">45.07</td><td align="center" rowspan="1" colspan="1">40.25</td><td align="center" rowspan="1" colspan="1">35.45</td><td align="center" rowspan="1" colspan="1">30.73</td><td align="center" rowspan="1" colspan="1">26.18</td><td align="center" rowspan="1" colspan="1">21.90</td><td align="center" rowspan="1" colspan="1">17.96</td><td align="center" rowspan="1" colspan="1">14.35</td><td align="center" rowspan="1" colspan="1">11.10</td><td align="center" rowspan="1" colspan="1">8.36</td><td align="center" rowspan="1" colspan="1">6.19</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">74.06</td><td align="center" rowspan="1" colspan="1">70.14</td><td align="center" rowspan="1" colspan="1">65.19</td><td align="center" rowspan="1" colspan="1">60.25</td><td align="center" rowspan="1" colspan="1">55.38</td><td align="center" rowspan="1" colspan="1">50.54</td><td align="center" rowspan="1" colspan="1">45.70</td><td align="center" rowspan="1" colspan="1">40.85</td><td align="center" rowspan="1" colspan="1">36.02</td><td align="center" rowspan="1" colspan="1">31.27</td><td align="center" rowspan="1" colspan="1">26.67</td><td align="center" rowspan="1" colspan="1">22.34</td><td align="center" rowspan="1" colspan="1">18.34</td><td align="center" rowspan="1" colspan="1">14.67</td><td align="center" rowspan="1" colspan="1">11.36</td><td align="center" rowspan="1" colspan="1">8.55</td><td align="center" rowspan="1" colspan="1">6.33</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">74.06</td><td align="center" rowspan="1" colspan="1">70.14</td><td align="center" rowspan="1" colspan="1">65.19</td><td align="center" rowspan="1" colspan="1">60.25</td><td align="center" rowspan="1" colspan="1">55.38</td><td align="center" rowspan="1" colspan="1">50.54</td><td align="center" rowspan="1" colspan="1">45.70</td><td align="center" rowspan="1" colspan="1">40.85</td><td align="center" rowspan="1" colspan="1">36.02</td><td align="center" rowspan="1" colspan="1">31.27</td><td align="center" rowspan="1" colspan="1">26.67</td><td align="center" rowspan="1" colspan="1">22.34</td><td align="center" rowspan="1" colspan="1">18.34</td><td align="center" rowspan="1" colspan="1">14.67</td><td align="center" rowspan="1" colspan="1">11.36</td><td align="center" rowspan="1" colspan="1">8.55</td><td align="center" rowspan="1" colspan="1">6.33</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1">75.33</td><td align="center" rowspan="1" colspan="1">71.38</td><td align="center" rowspan="1" colspan="1">66.41</td><td align="center" rowspan="1" colspan="1">61.45</td><td align="center" rowspan="1" colspan="1">56.55</td><td align="center" rowspan="1" colspan="1">51.67</td><td align="center" rowspan="1" colspan="1">46.78</td><td align="center" rowspan="1" colspan="1">41.90</td><td align="center" rowspan="1" colspan="1">37.03</td><td align="center" rowspan="1" colspan="1">32.21</td><td align="center" rowspan="1" colspan="1">27.54</td><td align="center" rowspan="1" colspan="1">23.13</td><td align="center" rowspan="1" colspan="1">19.03</td><td align="center" rowspan="1" colspan="1">15.26</td><td align="center" rowspan="1" colspan="1">11.83</td><td align="center" rowspan="1" colspan="1">8.91</td><td align="center" rowspan="1" colspan="1">6.59</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">74.53</td><td align="center" rowspan="1" colspan="1">70.59</td><td align="center" rowspan="1" colspan="1">65.63</td><td align="center" rowspan="1" colspan="1">60.69</td><td align="center" rowspan="1" colspan="1">55.80</td><td align="center" rowspan="1" colspan="1">50.95</td><td align="center" rowspan="1" colspan="1">46.09</td><td align="center" rowspan="1" colspan="1">41.23</td><td align="center" rowspan="1" colspan="1">36.39</td><td align="center" rowspan="1" colspan="1">31.61</td><td align="center" rowspan="1" colspan="1">26.98</td><td align="center" rowspan="1" colspan="1">22.62</td><td align="center" rowspan="1" colspan="1">18.59</td><td align="center" rowspan="1" colspan="1">14.88</td><td align="center" rowspan="1" colspan="1">11.53</td><td align="center" rowspan="1" colspan="1">8.68</td><td align="center" rowspan="1" colspan="1">6.43</td></tr></tbody></table></table-wrap><table-wrap id="T3" orientation="portrait" position="float"><label> Table 3 </label><caption><title> Life expectancy of females in Tehran megacity by different districts and age-groups in 2010 </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td align="center" rowspan="1" colspan="1">Age group / District</td><td align="center" rowspan="1" colspan="1">1_4</td><td align="center" rowspan="1" colspan="1">5_9</td><td align="center" rowspan="1" colspan="1">10_14</td><td align="center" rowspan="1" colspan="1">15_19</td><td align="center" rowspan="1" colspan="1">20_24</td><td align="center" rowspan="1" colspan="1">25_29</td><td align="center" rowspan="1" colspan="1">30_34</td><td align="center" rowspan="1" colspan="1">35_39</td><td align="center" rowspan="1" colspan="1">40_44</td><td align="center" rowspan="1" colspan="1">45_49</td><td align="center" rowspan="1" colspan="1">50_54</td><td align="center" rowspan="1" colspan="1">55_59</td><td align="center" rowspan="1" colspan="1">60_64</td><td align="center" rowspan="1" colspan="1">65_69</td><td align="center" rowspan="1" colspan="1">70_74</td><td align="center" rowspan="1" colspan="1">75_79</td><td align="center" rowspan="1" colspan="1">80&#x0003c;</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">79.57</td><td align="center" rowspan="1" colspan="1">75.60</td><td align="center" rowspan="1" colspan="1">70.62</td><td align="center" rowspan="1" colspan="1">65.63</td><td align="center" rowspan="1" colspan="1">60.66</td><td align="center" rowspan="1" colspan="1">55.69</td><td align="center" rowspan="1" colspan="1">50.73</td><td align="center" rowspan="1" colspan="1">45.78</td><td align="center" rowspan="1" colspan="1">40.86</td><td align="center" rowspan="1" colspan="1">35.97</td><td align="center" rowspan="1" colspan="1">31.18</td><td align="center" rowspan="1" colspan="1">26.49</td><td align="center" rowspan="1" colspan="1">21.90</td><td align="center" rowspan="1" colspan="1">17.50</td><td align="center" rowspan="1" colspan="1">13.42</td><td align="center" rowspan="1" colspan="1">9.87</td><td align="center" rowspan="1" colspan="1">6.98</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">79.23</td><td align="center" rowspan="1" colspan="1">75.27</td><td align="center" rowspan="1" colspan="1">70.29</td><td align="center" rowspan="1" colspan="1">65.31</td><td align="center" rowspan="1" colspan="1">60.34</td><td align="center" rowspan="1" colspan="1">55.38</td><td align="center" rowspan="1" colspan="1">50.42</td><td align="center" rowspan="1" colspan="1">45.48</td><td align="center" rowspan="1" colspan="1">40.56</td><td align="center" rowspan="1" colspan="1">35.69</td><td align="center" rowspan="1" colspan="1">30.91</td><td align="center" rowspan="1" colspan="1">26.24</td><td align="center" rowspan="1" colspan="1">21.67</td><td align="center" rowspan="1" colspan="1">17.30</td><td align="center" rowspan="1" colspan="1">13.26</td><td align="center" rowspan="1" colspan="1">9.76</td><td align="center" rowspan="1" colspan="1">6.91</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">79.15</td><td align="center" rowspan="1" colspan="1">75.19</td><td align="center" rowspan="1" colspan="1">70.21</td><td align="center" rowspan="1" colspan="1">65.23</td><td align="center" rowspan="1" colspan="1">60.26</td><td align="center" rowspan="1" colspan="1">55.30</td><td align="center" rowspan="1" colspan="1">50.35</td><td align="center" rowspan="1" colspan="1">45.41</td><td align="center" rowspan="1" colspan="1">40.49</td><td align="center" rowspan="1" colspan="1">35.62</td><td align="center" rowspan="1" colspan="1">30.85</td><td align="center" rowspan="1" colspan="1">26.18</td><td align="center" rowspan="1" colspan="1">21.62</td><td align="center" rowspan="1" colspan="1">17.25</td><td align="center" rowspan="1" colspan="1">13.22</td><td align="center" rowspan="1" colspan="1">9.73</td><td align="center" rowspan="1" colspan="1">6.89</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">79.57</td><td align="center" rowspan="1" colspan="1">75.60</td><td align="center" rowspan="1" colspan="1">70.62</td><td align="center" rowspan="1" colspan="1">65.63</td><td align="center" rowspan="1" colspan="1">60.66</td><td align="center" rowspan="1" colspan="1">55.69</td><td align="center" rowspan="1" colspan="1">50.73</td><td align="center" rowspan="1" colspan="1">45.78</td><td align="center" rowspan="1" colspan="1">40.86</td><td align="center" rowspan="1" colspan="1">35.97</td><td align="center" rowspan="1" colspan="1">31.18</td><td align="center" rowspan="1" colspan="1">26.49</td><td align="center" rowspan="1" colspan="1">21.90</td><td align="center" rowspan="1" colspan="1">17.50</td><td align="center" rowspan="1" colspan="1">13.42</td><td align="center" rowspan="1" colspan="1">9.87</td><td align="center" rowspan="1" colspan="1">6.98</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">79.57</td><td align="center" rowspan="1" colspan="1">75.60</td><td align="center" rowspan="1" colspan="1">70.62</td><td align="center" rowspan="1" colspan="1">65.63</td><td align="center" rowspan="1" colspan="1">60.66</td><td align="center" rowspan="1" colspan="1">55.69</td><td align="center" rowspan="1" colspan="1">50.73</td><td align="center" rowspan="1" colspan="1">45.78</td><td align="center" rowspan="1" colspan="1">40.86</td><td align="center" rowspan="1" colspan="1">35.97</td><td align="center" rowspan="1" colspan="1">31.18</td><td align="center" rowspan="1" colspan="1">26.49</td><td align="center" rowspan="1" colspan="1">21.90</td><td align="center" rowspan="1" colspan="1">17.50</td><td align="center" rowspan="1" colspan="1">13.42</td><td align="center" rowspan="1" colspan="1">9.87</td><td align="center" rowspan="1" colspan="1">6.98</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">78.26</td><td align="center" rowspan="1" colspan="1">74.32</td><td align="center" rowspan="1" colspan="1">69.35</td><td align="center" rowspan="1" colspan="1">64.38</td><td align="center" rowspan="1" colspan="1">59.42</td><td align="center" rowspan="1" colspan="1">54.48</td><td align="center" rowspan="1" colspan="1">49.54</td><td align="center" rowspan="1" colspan="1">44.62</td><td align="center" rowspan="1" colspan="1">39.73</td><td align="center" rowspan="1" colspan="1">34.89</td><td align="center" rowspan="1" colspan="1">30.16</td><td align="center" rowspan="1" colspan="1">25.54</td><td align="center" rowspan="1" colspan="1">21.04</td><td align="center" rowspan="1" colspan="1">16.75</td><td align="center" rowspan="1" colspan="1">12.82</td><td align="center" rowspan="1" colspan="1">9.43</td><td align="center" rowspan="1" colspan="1">6.70</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">77.70</td><td align="center" rowspan="1" colspan="1">73.78</td><td align="center" rowspan="1" colspan="1">68.82</td><td align="center" rowspan="1" colspan="1">63.85</td><td align="center" rowspan="1" colspan="1">58.90</td><td align="center" rowspan="1" colspan="1">53.97</td><td align="center" rowspan="1" colspan="1">49.05</td><td align="center" rowspan="1" colspan="1">44.14</td><td align="center" rowspan="1" colspan="1">39.27</td><td align="center" rowspan="1" colspan="1">34.46</td><td align="center" rowspan="1" colspan="1">29.75</td><td align="center" rowspan="1" colspan="1">25.16</td><td align="center" rowspan="1" colspan="1">20.70</td><td align="center" rowspan="1" colspan="1">16.46</td><td align="center" rowspan="1" colspan="1">12.58</td><td align="center" rowspan="1" colspan="1">9.26</td><td align="center" rowspan="1" colspan="1">6.59</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">77.38</td><td align="center" rowspan="1" colspan="1">73.48</td><td align="center" rowspan="1" colspan="1">68.52</td><td align="center" rowspan="1" colspan="1">63.56</td><td align="center" rowspan="1" colspan="1">58.61</td><td align="center" rowspan="1" colspan="1">53.69</td><td align="center" rowspan="1" colspan="1">48.77</td><td align="center" rowspan="1" colspan="1">43.88</td><td align="center" rowspan="1" colspan="1">39.02</td><td align="center" rowspan="1" colspan="1">34.21</td><td align="center" rowspan="1" colspan="1">29.52</td><td align="center" rowspan="1" colspan="1">24.95</td><td align="center" rowspan="1" colspan="1">20.51</td><td align="center" rowspan="1" colspan="1">16.30</td><td align="center" rowspan="1" colspan="1">12.45</td><td align="center" rowspan="1" colspan="1">9.17</td><td align="center" rowspan="1" colspan="1">6.53</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">76.61</td><td align="center" rowspan="1" colspan="1">72.74</td><td align="center" rowspan="1" colspan="1">67.80</td><td align="center" rowspan="1" colspan="1">62.84</td><td align="center" rowspan="1" colspan="1">57.92</td><td align="center" rowspan="1" colspan="1">53.01</td><td align="center" rowspan="1" colspan="1">48.12</td><td align="center" rowspan="1" colspan="1">43.24</td><td align="center" rowspan="1" colspan="1">38.41</td><td align="center" rowspan="1" colspan="1">33.64</td><td align="center" rowspan="1" colspan="1">28.98</td><td align="center" rowspan="1" colspan="1">24.46</td><td align="center" rowspan="1" colspan="1">20.07</td><td align="center" rowspan="1" colspan="1">15.92</td><td align="center" rowspan="1" colspan="1">12.15</td><td align="center" rowspan="1" colspan="1">8.95</td><td align="center" rowspan="1" colspan="1">6.39</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">77.23</td><td align="center" rowspan="1" colspan="1">73.33</td><td align="center" rowspan="1" colspan="1">68.38</td><td align="center" rowspan="1" colspan="1">63.41</td><td align="center" rowspan="1" colspan="1">58.47</td><td align="center" rowspan="1" colspan="1">53.55</td><td align="center" rowspan="1" colspan="1">48.64</td><td align="center" rowspan="1" colspan="1">43.75</td><td align="center" rowspan="1" colspan="1">38.89</td><td align="center" rowspan="1" colspan="1">34.10</td><td align="center" rowspan="1" colspan="1">29.41</td><td align="center" rowspan="1" colspan="1">24.85</td><td align="center" rowspan="1" colspan="1">20.42</td><td align="center" rowspan="1" colspan="1">16.22</td><td align="center" rowspan="1" colspan="1">12.39</td><td align="center" rowspan="1" colspan="1">9.12</td><td align="center" rowspan="1" colspan="1">6.50</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">77.15</td><td align="center" rowspan="1" colspan="1">73.26</td><td align="center" rowspan="1" colspan="1">68.30</td><td align="center" rowspan="1" colspan="1">63.34</td><td align="center" rowspan="1" colspan="1">58.40</td><td align="center" rowspan="1" colspan="1">53.48</td><td align="center" rowspan="1" colspan="1">48.57</td><td align="center" rowspan="1" colspan="1">43.68</td><td align="center" rowspan="1" colspan="1">38.83</td><td align="center" rowspan="1" colspan="1">34.04</td><td align="center" rowspan="1" colspan="1">29.35</td><td align="center" rowspan="1" colspan="1">24.80</td><td align="center" rowspan="1" colspan="1">20.38</td><td align="center" rowspan="1" colspan="1">16.18</td><td align="center" rowspan="1" colspan="1">12.36</td><td align="center" rowspan="1" colspan="1">9.10</td><td align="center" rowspan="1" colspan="1">6.48</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">76.92</td><td align="center" rowspan="1" colspan="1">73.04</td><td align="center" rowspan="1" colspan="1">68.08</td><td align="center" rowspan="1" colspan="1">63.13</td><td align="center" rowspan="1" colspan="1">58.19</td><td align="center" rowspan="1" colspan="1">53.28</td><td align="center" rowspan="1" colspan="1">48.38</td><td align="center" rowspan="1" colspan="1">43.49</td><td align="center" rowspan="1" colspan="1">38.65</td><td align="center" rowspan="1" colspan="1">33.86</td><td align="center" rowspan="1" colspan="1">29.19</td><td align="center" rowspan="1" colspan="1">24.65</td><td align="center" rowspan="1" colspan="1">20.24</td><td align="center" rowspan="1" colspan="1">16.07</td><td align="center" rowspan="1" colspan="1">12.27</td><td align="center" rowspan="1" colspan="1">9.03</td><td align="center" rowspan="1" colspan="1">6.44</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">77.70</td><td align="center" rowspan="1" colspan="1">73.78</td><td align="center" rowspan="1" colspan="1">68.82</td><td align="center" rowspan="1" colspan="1">63.85</td><td align="center" rowspan="1" colspan="1">58.90</td><td align="center" rowspan="1" colspan="1">53.97</td><td align="center" rowspan="1" colspan="1">49.05</td><td align="center" rowspan="1" colspan="1">44.14</td><td align="center" rowspan="1" colspan="1">39.27</td><td align="center" rowspan="1" colspan="1">34.46</td><td align="center" rowspan="1" colspan="1">29.75</td><td align="center" rowspan="1" colspan="1">25.16</td><td align="center" rowspan="1" colspan="1">20.70</td><td align="center" rowspan="1" colspan="1">16.46</td><td align="center" rowspan="1" colspan="1">12.58</td><td align="center" rowspan="1" colspan="1">9.26</td><td align="center" rowspan="1" colspan="1">6.59</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">77.62</td><td align="center" rowspan="1" colspan="1">73.71</td><td align="center" rowspan="1" colspan="1">68.74</td><td align="center" rowspan="1" colspan="1">63.78</td><td align="center" rowspan="1" colspan="1">58.83</td><td align="center" rowspan="1" colspan="1">53.90</td><td align="center" rowspan="1" colspan="1">48.98</td><td align="center" rowspan="1" colspan="1">44.07</td><td align="center" rowspan="1" colspan="1">39.21</td><td align="center" rowspan="1" colspan="1">34.39</td><td align="center" rowspan="1" colspan="1">29.69</td><td align="center" rowspan="1" colspan="1">25.11</td><td align="center" rowspan="1" colspan="1">20.65</td><td align="center" rowspan="1" colspan="1">16.42</td><td align="center" rowspan="1" colspan="1">12.55</td><td align="center" rowspan="1" colspan="1">9.24</td><td align="center" rowspan="1" colspan="1">6.57</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">78.42</td><td align="center" rowspan="1" colspan="1">74.48</td><td align="center" rowspan="1" colspan="1">69.50</td><td align="center" rowspan="1" colspan="1">64.53</td><td align="center" rowspan="1" colspan="1">59.57</td><td align="center" rowspan="1" colspan="1">54.62</td><td align="center" rowspan="1" colspan="1">49.68</td><td align="center" rowspan="1" colspan="1">44.76</td><td align="center" rowspan="1" colspan="1">39.86</td><td align="center" rowspan="1" colspan="1">35.02</td><td align="center" rowspan="1" colspan="1">30.28</td><td align="center" rowspan="1" colspan="1">25.65</td><td align="center" rowspan="1" colspan="1">21.14</td><td align="center" rowspan="1" colspan="1">16.84</td><td align="center" rowspan="1" colspan="1">12.89</td><td align="center" rowspan="1" colspan="1">9.48</td><td align="center" rowspan="1" colspan="1">6.73</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">76.69</td><td align="center" rowspan="1" colspan="1">72.82</td><td align="center" rowspan="1" colspan="1">67.87</td><td align="center" rowspan="1" colspan="1">62.91</td><td align="center" rowspan="1" colspan="1">57.98</td><td align="center" rowspan="1" colspan="1">53.08</td><td align="center" rowspan="1" colspan="1">48.18</td><td align="center" rowspan="1" colspan="1">43.30</td><td align="center" rowspan="1" colspan="1">38.47</td><td align="center" rowspan="1" colspan="1">33.69</td><td align="center" rowspan="1" colspan="1">29.03</td><td align="center" rowspan="1" colspan="1">24.50</td><td align="center" rowspan="1" colspan="1">20.11</td><td align="center" rowspan="1" colspan="1">15.96</td><td align="center" rowspan="1" colspan="1">12.18</td><td align="center" rowspan="1" colspan="1">8.97</td><td align="center" rowspan="1" colspan="1">6.40</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">77.31</td><td align="center" rowspan="1" colspan="1">73.41</td><td align="center" rowspan="1" colspan="1">68.45</td><td align="center" rowspan="1" colspan="1">63.49</td><td align="center" rowspan="1" colspan="1">58.54</td><td align="center" rowspan="1" colspan="1">53.62</td><td align="center" rowspan="1" colspan="1">48.71</td><td align="center" rowspan="1" colspan="1">43.81</td><td align="center" rowspan="1" colspan="1">38.95</td><td align="center" rowspan="1" colspan="1">34.15</td><td align="center" rowspan="1" colspan="1">29.46</td><td align="center" rowspan="1" colspan="1">24.90</td><td align="center" rowspan="1" colspan="1">20.47</td><td align="center" rowspan="1" colspan="1">16.26</td><td align="center" rowspan="1" colspan="1">12.42</td><td align="center" rowspan="1" colspan="1">9.14</td><td align="center" rowspan="1" colspan="1">6.51</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">77.54</td><td align="center" rowspan="1" colspan="1">73.63</td><td align="center" rowspan="1" colspan="1">68.67</td><td align="center" rowspan="1" colspan="1">63.70</td><td align="center" rowspan="1" colspan="1">58.76</td><td align="center" rowspan="1" colspan="1">53.83</td><td align="center" rowspan="1" colspan="1">48.91</td><td align="center" rowspan="1" colspan="1">44.01</td><td align="center" rowspan="1" colspan="1">39.14</td><td align="center" rowspan="1" colspan="1">34.33</td><td align="center" rowspan="1" colspan="1">29.63</td><td align="center" rowspan="1" colspan="1">25.05</td><td align="center" rowspan="1" colspan="1">20.60</td><td align="center" rowspan="1" colspan="1">16.38</td><td align="center" rowspan="1" colspan="1">12.52</td><td align="center" rowspan="1" colspan="1">9.21</td><td align="center" rowspan="1" colspan="1">6.56</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">76.99</td><td align="center" rowspan="1" colspan="1">73.11</td><td align="center" rowspan="1" colspan="1">68.16</td><td align="center" rowspan="1" colspan="1">63.20</td><td align="center" rowspan="1" colspan="1">58.26</td><td align="center" rowspan="1" colspan="1">53.35</td><td align="center" rowspan="1" colspan="1">48.44</td><td align="center" rowspan="1" colspan="1">43.56</td><td align="center" rowspan="1" colspan="1">38.71</td><td align="center" rowspan="1" colspan="1">33.92</td><td align="center" rowspan="1" colspan="1">29.25</td><td align="center" rowspan="1" colspan="1">24.70</td><td align="center" rowspan="1" colspan="1">20.29</td><td align="center" rowspan="1" colspan="1">16.11</td><td align="center" rowspan="1" colspan="1">12.30</td><td align="center" rowspan="1" colspan="1">9.05</td><td align="center" rowspan="1" colspan="1">6.46</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">79.48</td><td align="center" rowspan="1" colspan="1">75.52</td><td align="center" rowspan="1" colspan="1">70.53</td><td align="center" rowspan="1" colspan="1">65.55</td><td align="center" rowspan="1" colspan="1">60.58</td><td align="center" rowspan="1" colspan="1">55.61</td><td align="center" rowspan="1" colspan="1">50.65</td><td align="center" rowspan="1" colspan="1">45.71</td><td align="center" rowspan="1" colspan="1">40.78</td><td align="center" rowspan="1" colspan="1">35.90</td><td align="center" rowspan="1" colspan="1">31.11</td><td align="center" rowspan="1" colspan="1">26.42</td><td align="center" rowspan="1" colspan="1">21.84</td><td align="center" rowspan="1" colspan="1">17.45</td><td align="center" rowspan="1" colspan="1">13.38</td><td align="center" rowspan="1" colspan="1">9.84</td><td align="center" rowspan="1" colspan="1">6.97</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">78.82</td><td align="center" rowspan="1" colspan="1">74.87</td><td align="center" rowspan="1" colspan="1">69.89</td><td align="center" rowspan="1" colspan="1">64.92</td><td align="center" rowspan="1" colspan="1">59.95</td><td align="center" rowspan="1" colspan="1">55.00</td><td align="center" rowspan="1" colspan="1">50.05</td><td align="center" rowspan="1" colspan="1">45.11</td><td align="center" rowspan="1" colspan="1">40.21</td><td align="center" rowspan="1" colspan="1">35.35</td><td align="center" rowspan="1" colspan="1">30.59</td><td align="center" rowspan="1" colspan="1">25.94</td><td align="center" rowspan="1" colspan="1">21.40</td><td align="center" rowspan="1" colspan="1">17.06</td><td align="center" rowspan="1" colspan="1">13.07</td><td align="center" rowspan="1" colspan="1">9.62</td><td align="center" rowspan="1" colspan="1">6.82</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1">79.32</td><td align="center" rowspan="1" colspan="1">75.35</td><td align="center" rowspan="1" colspan="1">70.37</td><td align="center" rowspan="1" colspan="1">65.39</td><td align="center" rowspan="1" colspan="1">60.42</td><td align="center" rowspan="1" colspan="1">55.46</td><td align="center" rowspan="1" colspan="1">50.50</td><td align="center" rowspan="1" colspan="1">45.56</td><td align="center" rowspan="1" colspan="1">40.64</td><td align="center" rowspan="1" colspan="1">35.76</td><td align="center" rowspan="1" colspan="1">30.98</td><td align="center" rowspan="1" colspan="1">26.30</td><td align="center" rowspan="1" colspan="1">21.73</td><td align="center" rowspan="1" colspan="1">17.35</td><td align="center" rowspan="1" colspan="1">13.30</td><td align="center" rowspan="1" colspan="1">9.79</td><td align="center" rowspan="1" colspan="1">6.93</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td align="center" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">78.26</td><td align="center" rowspan="1" colspan="1">74.32</td><td align="center" rowspan="1" colspan="1">69.35</td><td align="center" rowspan="1" colspan="1">64.38</td><td align="center" rowspan="1" colspan="1">59.42</td><td align="center" rowspan="1" colspan="1">54.48</td><td align="center" rowspan="1" colspan="1">49.54</td><td align="center" rowspan="1" colspan="1">44.62</td><td align="center" rowspan="1" colspan="1">39.73</td><td align="center" rowspan="1" colspan="1">34.89</td><td align="center" rowspan="1" colspan="1">30.16</td><td align="center" rowspan="1" colspan="1">25.54</td><td align="center" rowspan="1" colspan="1">21.04</td><td align="center" rowspan="1" colspan="1">16.75</td><td align="center" rowspan="1" colspan="1">12.82</td><td align="center" rowspan="1" colspan="1">9.43</td><td align="center" rowspan="1" colspan="1">6.70</td></tr></tbody></table></table-wrap><table-wrap id="T4" orientation="portrait" position="float"><label> Table 4 </label><caption><title> Ecological relationship between life expectancy and social classes, level of education, and family expenditures </title></caption><table frame="box" rules="none"><tbody><tr style="border-width: 1px 1px 1pt 1px ; border-style: solid solid solid solid;border-color:#49abc4;background-color:#49abc4;color:#fff8e8"><td rowspan="1" colspan="1">Main variable</td><td rowspan="1" colspan="1"> Subgroups</td><td rowspan="1" colspan="1"> Mean (SD)</td><td rowspan="1" colspan="1"> p-value</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="6" colspan="1"> Social class</td><td rowspan="1" colspan="1"> SC-1</td><td rowspan="1" colspan="1"> 77.19 (1.42)</td><td rowspan="6" colspan="1"> &#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> SC-2</td><td rowspan="1" colspan="1"> 76.74 (1.50)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">SC-3a</td><td rowspan="1" colspan="1"> 76.51 (1.50)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">SC-3b </td><td rowspan="1" colspan="1"> 76.28 (1.44)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">SC-4 </td><td rowspan="1" colspan="1"> 76.32 (1.45)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1">SC-5</td><td rowspan="1" colspan="1"> 76.05 (1.36)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"> Total </td><td rowspan="1" colspan="1"> 76.48 (1.47)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="6" colspan="1"> Education </td><td rowspan="1" colspan="1"> Illiterate</td><td rowspan="1" colspan="1"> 76.00 (1.34)</td><td rowspan="6" colspan="1">&#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Primary </td><td rowspan="1" colspan="1"> 76.17 (1.39)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> High-school and Diploma</td><td rowspan="1" colspan="1"> 76.46 (1.48)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Bachelor of Science and Under</td><td rowspan="1" colspan="1"> 76.98 (1.49)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Master of Science and Higher</td><td rowspan="1" colspan="1"> 77.38 (1.39)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Total </td><td rowspan="1" colspan="1"> 76.47 (1.49)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="6" colspan="1"> Household costs</td><td rowspan="1" colspan="1"> First quintile (20%) </td><td rowspan="1" colspan="1"> 76.20 (1.39)</td><td rowspan="6" colspan="1">&#x0003c;0.001</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Second quintile (40%)</td><td rowspan="1" colspan="1"> 76.30 (1.44)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Third quintile (60%) </td><td rowspan="1" colspan="1"> 76.45 (1.48)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Fourth quintile (80%)</td><td rowspan="1" colspan="1"> 76.61 (1.53)</td></tr><tr style="border-width: 0px 1px 0pt 1px ; border-style: none solid none solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Fifth quintile (100%)</td><td rowspan="1" colspan="1"> 76.91 (1.55)</td></tr><tr style="border-width: 0px 1px 1pt 1px ; border-style: none solid solid solid;border-color:#49abc4"><td rowspan="1" colspan="1"> Total </td><td rowspan="1" colspan="1"> 76.50 (1.50)</td></tr></tbody></table></table-wrap></sec><sec id="s3-2"><title> LE and SES </title><p> The highest and lowest mean of LE observed in SC1 and SC5, were 77.19 and 76.05 years, respectively. The variances between sub-groups of SES indicator were not equal (p &#x0003c; 0.001). Meanwhile, the mean of LE was significantly different in SES classes (p &#x0003c; 0.001). Notably, the mean of LE within the SC3b and SC4 was not different (p &#x0003e; 0.1). </p></sec><sec id="s3-3"><title> LE and Total Costs </title><p> The lowest mean of LE was observed in the first group (percentile 20), with the lowest non-food costs, and calculated 76.20 years. The variances between sub-groups of total costs indicator were not equal (p&#x0003c;0.001). Meanwhile, the mean of LE was significantly different in total costs groups (p&#x0003c; 0.001). </p></sec><sec id="s3-4"><title> LE and Education </title><p> The lowest observed mean of LE in the first category (illiterate), was 76 years. The variances between sub-groups of education indicator were not equal (p&#x0003c;0.001). Meanwhile, the mean of LE was significantly different in education groups (p&#x0003c; 0.001). </p></sec></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p> In this research, we studied intra-city differences for LE in Tehran mega-city. We further tried to visualize distribution of LE across 22 districts of Tehran for better understanding of associated changes in LE of general population. In addition, using results of Tehran&#x02019;s Urban HEART-1 project (<xref rid="R18" ref-type="bibr">18</xref>), the role of SES, total costs, and education disparities were characterized on LE within various districts. As a main result, substantial disparities were observed among LE of Tehran&#x02019;s 22 districts. As illustrated in <xref ref-type="fig" rid="F1">Figure 1</xref>, the populations of the northern part districts, are most advantaged districts with higher SES, benefited with more LE compared to southern districts. Meanwhile, the maximum LE was 80 years in females living in north and minimum 72.7 years in males of southern parts which indicate that socioeconomic inequalities within cities could have direct influence on LE within Tehran population. </p><p> This is the first report of direct calculation of life expectancy using registered data in Tehran, which inturn correlated with contextual ecologic data of households living in different districts. In previous study, Fereshtehnejad et al. (2010) reported an estimation of LE using a large population sample, which indicated that LE was largely influenced by contextual variables, such as level of education, family size, household expenditures, and social class (<xref rid="R19" ref-type="bibr">19</xref>). </p><p> Although the influences of various variables of socioeconomic position (<xref rid="R20" ref-type="bibr">20</xref>-<xref rid="R24" ref-type="bibr">24</xref>), GNP (<xref rid="R25" ref-type="bibr">25</xref>), and education level (<xref rid="R12" ref-type="bibr">12</xref>-<xref rid="R13" ref-type="bibr">13</xref>, <xref rid="R26" ref-type="bibr">26</xref>), on LE have been studied in global and national-scales among populations of countries and states, there are few reports that study these relations within city populations (<xref rid="R9" ref-type="bibr">9</xref>). For example, a study in Philadelphia demonstrated that child mortality rate (CMR) in 1971 was 22-per-thousands while at the same time the gap for CMR in two different districts was 18-per-thousands (<xref rid="R27" ref-type="bibr">27</xref>). Haan et al., (1987), also, studied white and non-white residents of two federally specified impoverished and advantaged areas of Oakland, Alameda, California for nine-years from 1965 to 1974, and reported that both resident groups of poor areas experienced significantly higher mortalities versus well-off areas (<xref rid="R28" ref-type="bibr">28</xref>). McCord and Freeman (1990) studied male residents of Central Harlem health district in New York City, where 41 percent were below the poverty line and predominantly black. They reported an extremely high mortality for them identical or less to rural males of less developed countries (<xref rid="R29" ref-type="bibr">29</xref>). Geronimus et al., (1996) studied residents of sixteen persistent poor and affluent areas of different cities in the United States, where the mortality rates of black males (i.e., in Harlem) were escalated compared to advantaged residential places (<xref rid="R30" ref-type="bibr">30</xref>). Guest et al., (1998) studied 308 populations of Chicago (77 community areas by gender and by two ethnicities) and estimated their mortality rates. In fact, they have found consistent results with McCord and Freeman (1990) in which mortality rates of black males were three-fold more than non-black males and six-fold more than non-black females (<xref rid="R31" ref-type="bibr">31</xref>). Thus, mortality rates and LE have been reported to be quite different within city areas. </p><p> In essence, extensive national- and regional-scale studies have studied the role of different socioeconomic and sociodemographic status measures on longevity and life expectancy (<xref rid="R9" ref-type="bibr">9</xref>, <xref rid="R24" ref-type="bibr">24</xref>, <xref rid="R32" ref-type="bibr">32</xref>-<xref rid="R35" ref-type="bibr">35</xref>). Kulkarni et al., (2011) studied LE disparities across United States counties and found a considerable gap of 15.2 and 12.5 years for men and women, respectively (<xref rid="R24" ref-type="bibr">24</xref>). Clarke et al., (2010) reported a substantial impact of neighborhood SES among different age, genders, and ethnicities on LE. They have found that those males of California, which are in the poorest 20% neighborhoods, have the same LE as males of those developing countries that low LEs have been reported for them (<xref rid="R32" ref-type="bibr">32</xref>). In another report among general population by Tarkiainen et al., (2012), the role of Finnish people&#x02019;s income was studied on LE over 20 years. Noteworthy, a gap of LE by 5.1&#x02005;years among males and 2.9&#x02005;years among females was observed within maximum and minimum income quintiles (<xref rid="R34" ref-type="bibr">34</xref>). Tobias and Cheung (2003), also, studied three ethnicities (i.e., M&#x000e4;ori, Pacific, and European) of New Zealand within four periods from 1995 to 2000, and analyzed LE with increasing/decreasing of deprivation in small areas. Thus, they found a strong association between LE and deprivation, in which longevity of males living in rich areas was nine-years more than their counterparts in deprived areas (<xref rid="R36" ref-type="bibr">36</xref>). Besides, a negative correlation between education level of the people and life expectancy has been reported in Bangladesh and Norwegian territories in which educated people have higher longevity (<xref rid="R9" ref-type="bibr">9</xref>, <xref rid="R33" ref-type="bibr">33</xref>). Identically, an inverse relationship between education level of the people and longevity has been reported from the United States in which higher mortality rates have been reported for poorly educated people compared with those benefited from better education (<xref rid="R37" ref-type="bibr">37</xref>). </p></sec><sec id="s5"><title>Limitations</title><p> The first limitation of this study was due to high and unpredictable rate of migration and relocation among the districts. This might have changed somewhat the amount of calculated LEs. The second limitation was ecologic design of the study, which harbored some inherent weaknesses. In fact, ecologic studies suffer from effect of a variety of confounding factors. The third limitation which highlighted specifically in developing countries as well as Iran was mortality registration. The mortality system of record in developing countries suffers from some weaknesses, especially in lower age groups like those with fewer than one year old. This problem might also affect the amount of calculated LEs. However, although the resulted were affected by these limitations, we believe there are considerable validities within these data. </p></sec><sec sec-type="conclusion" id="s6"><title>Conclusion</title><p> Our findings highlight the inequalities in life expectancy across districts of a megacity, in particular the SES differentials of LE. This study demonstrated that LE had important different within city districts, especially within mega-cities. These inequalities, in fact, underline the importance of resources management within specific regions of the cities&#x02014;to enhance the quality of life as well as the LE of populations. Meanwhile, LE may be influenced by different factors, such as socioeconomic position, and variations of LE within city districts can be a mirror of inequalities. Nonetheless the results of this study may pave the way for health policy makers, urban planners and health professionals to think more about socioeconomic determinants of this important outcome of health status. </p></sec>
Steroid hormone synthetic pathways in prostate cancer
<p>While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa) since the seminal recognition of the disease as androgen-dependent by Huggins and Hodges in 1941, therapy is uniformly marked by progression to castration-resistant prostate cancer (CRPC) over a period of about 18 months, with an ensuing median survival of 1 to 2 years. Importantly, castration does not eliminate androgens from the prostate tumor microenvironment. Castration resistant tumors are characterized by elevated tumor androgens that are well within the range capable of activating the AR and AR-mediated gene expression, and by steroid enzyme alterations which may potentiate <italic>de novo</italic> androgen synthesis or utilization of circulating adrenal androgens. The dependence of CRPC on intratumoral androgen metabolism has been modeled <italic>in vitro</italic> and <italic>in vivo</italic>, and residual intratumoral androgens are implicated in nearly every mechanism by which AR-mediated signaling promotes castration-resistant disease.</p><p>These observations suggest that tissue based alterations in steroid metabolism contribute to the development of CRPC and underscore these metabolic pathways as critical targets of therapy. Herein, we review the accumulated body of evidence which strongly supports intracrine (tumoral) androgen synthesis as an important mechanism underlying PCa progression. We first discuss the presence and significance of residual prostate tumor androgens in the progression of CRPC. We review the classical and non-classical pathways of androgen metabolism, and how dysregulated expression of these enzymes is likely to potentiate tumor androgen production in the progression to CRPC. Next we review the <italic>in vitro</italic> and <italic>in vivo</italic> data in human tumors, xenografts, and cell line models which demonstrate the capacity of prostate tumors to utilize cholesterol and adrenal androgens in the production of testosterone (T) and dihydrotestosterone (DHT), and briefly review the potential role of exogenous influences on this process. Finally, we discuss the emerging data regarding mechanisms of response and resistance to potent ligand synthesis inhibitors entering clinical practice, and conclude by discussing the implications of these findings for future therapy.</p>
<contrib contrib-type="author" corresp="yes"><name><surname>Mostaghel</surname><given-names>Elahe A.</given-names></name></contrib><aff id="aff1">Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle WA, <country>USA</country></aff>
Translational Andrology and Urology
<sec><title>Presence of intratumoral androgens despite castration</title><p>The efficacy of androgen deprivation therapy (ADT) is routinely based on achieving castrate levels of serum T, defined as &#x0003c;20 ng/dL. However, tissue androgen levels in the setting of benign prostatic hyperplasia (BPH), primary prostate tumors, locally recurrent prostate cancer (PCa), or metastatic castration-resistant prostate cancer (CRPC) have consistently demonstrated that castration does not eliminate androgens from the prostate tumor microenvironment.</p><p>Geller <italic>et al</italic>. examined prostatic DHT levels by radioimmunoassay (RIA) and demonstrated that castration by orchiectomy (or megace plus DES) reduced prostatic DHT levels by 75-80% to 1 ng/g in some but not all patients. Epithelial and stromal cell protein synthesis was strongly correlated with tissue DHT levels, and prostatic DHT levels were further reduced when castration was combined with adrenal androgen blockade by ketoconazole (<xref rid="r1" ref-type="bibr">1</xref>-<xref rid="r6" ref-type="bibr">6</xref>), suggesting the goal of therapy should be to decrease prostatic DHT to as low as possible, a concept similarly framed in early studies by Labrie (<xref rid="r7" ref-type="bibr">7</xref>).</p><p>Incomplete suppression of prostate tissue androgens by castration has been subsequently confirmed in numerous studies of short and long term castration therapy (<xref rid="r8" ref-type="bibr">8</xref>). Treatment of BPH patients for 3 months with an LHRH agonist decreased intraprostatic T levels by 75%, to about 0.1 ng/g, and DHT levels by 90%, to 0.48 ng/g (<xref rid="r9" ref-type="bibr">9</xref>). In men with PCa 6 months of neoadjuvant ADT with castration and flutamide reduced prostatic DHT levels by 75% to about 1.35 ng/g (<xref rid="r10" ref-type="bibr">10</xref>). Notably, tumor differentiation based on Gleason grading has been correlated with change in tissue DHT, with an 85% decrease measured in Gleason 6 cancers, but only a 60% decrement in Gleason 7-10 tumors (<xref rid="r11" ref-type="bibr">11</xref>). This finding indicates that tumor type-specific changes in androgen metabolism may impact responses to systemic T suppression.</p><p>Residual androgens have also been demonstrated in both locally recurrent and metastatic castration resistant tumors. Testosterone levels in locally recurrent tumors from castrate patients were <italic>equivalent</italic> to those of BPH patients, and DHT levels were only reduced 80%, to about 0.4 ng/g (<xref rid="r12" ref-type="bibr">12</xref>). Compared to primary prostate tumors from untreated patients (T 0.25 ng/g, DHT 2.75 ng/g) androgen levels in metastatic CRPC tumors obtained via rapid autopsy showed 3-fold higher T levels and an inverted ratio of T to DHT (T 0.74 ng/g; DHT 0.25 ng/g) (<xref rid="r13" ref-type="bibr">13</xref>). Adrenal androgens have also been detected at significant levels in prostate tissue of castrate men. Prostatic levels of dihydroepiandrosterone (DHEA), DHEA-sulfate (DHEA-S), and androstenedione (AED) were decreasedby about 50% in castrate patients and far exceeded values of T and DHT in recurrent tumors (<xref rid="r12" ref-type="bibr">12</xref>). No decrease in prostatic levels of 5-androstenediol were found after castration (<xref rid="r14" ref-type="bibr">14</xref>), which is of particular significance as this androgen has been shown to bind wild type AR without being inhibited by flutamide or bicalutamide (<xref rid="r15" ref-type="bibr">15</xref>).</p><p>Two recently reported studies demonstrate that the addition of androgen synthesis inhibitors to castration therapy can lower prostate androgens below that achieved with standard androgen blockade. The addition of dutasteride and ketoconazole to combined androgen blockade (CAB) for 3 months prior to prostatectomy lowered prostate DHT from 0.92 ng/g (in the CAB arm) to 0.03 ng/g (<xref rid="r16" ref-type="bibr">16</xref>). In a second study, the potent CYP17A inhibitor abiraterone was added to LHRH agonist therapy for 3 or 6 months prior to prostatectomy. Abiraterone decreased prostate tissue DHT from 1.3 ng/g (in men treated with LHRH agonist therapy alone) to 0.18 ng/g and also decreased prostate levels of AED and DHEA (<xref rid="r17" ref-type="bibr">17</xref>).</p></sec><sec><title>Significance of intratumoral androgens in progression of CRPC</title><p>These findings clearly demonstrate that achieving castrate levels of circulating T does not eliminate androgens from the prostate tumor microenvironment. The ability of DHT in the range observed in castrate tumors (~1 nm, 0.5 to 1.0 ng/g) to activate the AR, stimulate expression of AR-regulated genes, and promote androgen mediated tumor growth has been convincingly demonstrated in both <italic>in vitro</italic> and <italic>in vivo</italic> studies (<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r18" ref-type="bibr">18</xref>-<xref rid="r21" ref-type="bibr">21</xref>), and is evidenced by the nearly universal rise in serum PSA that accompanies CRPC progression.</p><p>Residual tissue androgens are implicated in driving the majority of mechanisms whereby persistent AR-mediated signaling drives castration resistant disease. These mechanisms include AR overexpression, AR mutations that broaden ligand specificity and/or confer sensitivity to adrenal androgens, alterations in AR coactivators and/or corepressors that modulate AR stability and ligand sensitivity, and activation of the AR or downstream regulatory molecules by &#x0201c;cross talk&#x0201d; with other signaling pathways. Restoration of AR expression and signaling in a xenograft model was both necessary and sufficient to drive progression from androgen-dependent to castration resistant growth, allowing tumor cell proliferation in 80% lower androgen concentrations (<xref rid="r22" ref-type="bibr">22</xref>). Importantly, ligand binding was required for hormone refractory growth, and modest increases in AR expression were sufficient to support signaling in a low androgen environment.</p><p>The clinical relevance of intratumoral androgens in promoting CRPC tumor growth is confirmed by the clinical responses to agents targeting residual androgen pathway activity. These include historical responses described in response to adrenalectomy and/or hypophysectomy (<xref rid="r23" ref-type="bibr">23</xref>,<xref rid="r24" ref-type="bibr">24</xref>); the limited but consistent ~5% overall survival benefit seen in meta-analyses of CAB (<xref rid="r25" ref-type="bibr">25</xref>-<xref rid="r27" ref-type="bibr">27</xref>); the observation that nearly 30% of recurrent prostate tumors demonstrate at least transient clinical responses to secondary or tertiary hormonal manipulation (<xref rid="r28" ref-type="bibr">28</xref>); and most recently, the striking clinical response observed with novel ligand synthesis inhibitors such as abiraterone, and potent AR inhibitors such as enzalutamide (<xref rid="r29" ref-type="bibr">29</xref>,<xref rid="r30" ref-type="bibr">30</xref>). Perhaps most importantly, emerging studies suggest that response and resistance to abiraterone is associated with tumoral evidence of upregulated androgen synthesis, clearly demonstrating the importance of intratumoral androgen metabolism in CRPC tumor survival (<xref rid="r31" ref-type="bibr">31</xref>-<xref rid="r33" ref-type="bibr">33</xref>).</p></sec><sec><title>Pathways of androgen metabolism</title><p>The source of residual androgens within prostate tumors of castrate men has not been fully elucidated, but is generally attributed to the uptake and conversion of circulating adrenal androgens (<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r34" ref-type="bibr">34</xref>), and somewhat more controversially, to <italic>de novo</italic> biosynthesis of androgens from progesterone or cholesterol precursors (<xref rid="r35" ref-type="bibr">35</xref>). Here we review the classical pathways of <italic>de novo</italic> androgen synthesis in adrenal and peripheral tissues [<xref ref-type="fig" rid="f1"><italic>Figure 1</italic></xref>, reviewed in (<xref rid="r36" ref-type="bibr">36</xref>)], the enzymatic pathways mediating prostate androgen metabolism, and the so called &#x02018;back-door&#x02019; pathway of androgen synthesis. A general outline of the classical and non-classical steroidogenic pathways is provided in <xref ref-type="fig" rid="f2"><italic>Figure 2</italic></xref>.</p><fig id="f1" fig-type="figure" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Steroid hormone synthesis pathways in the adrenal gland and testis. A. Steroid synthesis in the adrenal gland occurs in three zones, each with a specific complement of enzymes. The zona glomerulosa contains the enzymes necessary to produce aldosterone. The zona fasciculata and reticularis additionally express CYP17A. The hydroxylase activity of CYP17A is active in the zona fasiculata resulting in the production of cortiso. Due to tissue-specific expression of the cytochrome b5 coregulator, the lyase activity of CYP17A is only present in the zona reticularis and drives efficient production of DHEA which is then sulfated to DHEA-S. 17&#x003b1;-OH progesterone is a poor substrate for CYP17A lyase (dotted arrow) and thus androstenedione is formed at lower levels. The zona fasciculate and zona reticularis are sensitive to the ACTH feedback stimulation that occurs when cortisol production is suppressed by inhibition of CYP17A. Agents specifically targeting the lyase but not hydroxylase activity of CYP17A would not inhibit cortisol synthesis and are anticipated to induce less ACTH feedback stimulation; B. Testicular androgen synthesis follows a similar pathway to DHEA formation as that in the zona reticularis. Due to the absence of SULT2A1, and the presence of HSD3B2 and HSD17B3, DHEA is efficiently converted to testosterone.</p></caption><graphic xlink:href="tau-02-03-212-f1"/></fig><fig id="f2" fig-type="figure" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Classical and non-classical pathways of androgen biosynthesis. Cholesterol is converted to C21 precursors (pregnenolone and progesterone) by the action of STAR and CYP11A. In the classical pathway (light gray arrows) C21 steroids are converted to the C19 adrenal androgens DHEA and androstenedione (AED) by the sequential hydroxylase and lyase activity of CYP17A. Due to the substrate preference the lyase activity of CYP17 (which requires the cytochrome b5 cofactor) favors production of DHEA. DHEA (from intrinsic or circulating sources depending on the tissue) is subsequently acted on by HSD3B and HSD17B3 (or AKR1C3) to form testosterone (T) which is converted to DHT via SRD5A. In the backdoor pathway (hatched arrows) the progestin intermediates are acted on first by the activity of SRD5A and the reductive activity of AKR1C2 prior to the lyase activity of CYP17A. Androsterone is then acted on by HSD17B3 (or AKR1C3) and must undergo an oxidative step mediated by RL-HSD (or others) to generate DHT. In a third pathway, termed the 5&#x003b1;-Androstanedione pathway (dark gray arrows) DHEA and AED are produced as in the classical pathway. However, instead of conversion of AED to T followed by the activity of SRD5A to produce DHT, the enzymatic sequence is reversed such that AED is converted first by SRD5A to 5&#x003b1;-Androstanedione and then by HSD17B3 (or AKR1C3) to DHT.</p></caption><graphic xlink:href="tau-02-03-212-f2"/></fig><sec><title>Androgen synthesis in the adrenal gland and peripheral tissues</title><p>Steroid hormone synthesis begins with transfer of a 27-carbon (C-27) cholesterol molecule from the outer mitochondrial membrane to the inner membrane by steroidogenic acute regulatory protein (STAR), followed by its conversion to the C-19 steroid, pregnenolone via CYP11A (side change cleavage enzyme). Subsequent metabolism to progesterone, mineralocorticoids, glucocorticoids (all C-21steroids), androgens (C-19) or estrogens (C-18) is dictated in a tissue-specific manner, driven by the expression of specific enzymes and catalytic cofactors.</p><p>CYP17A, expressed in the adrenal gland, testis and ovary, is a single enzyme with one active site which catalyzes sequential but independent hydroxylase and lyase reactions, both of which are required for converting C21 progestins to androgens and estrogens, either along the delta-5 pathway from pregnenolone or the delta-4 pathway from progesterone. The hydroxylase activity of CYP17A for pregnenolone and progesterone is similar, but its lyase activity for delta-5 and -4 substrates requires the activity of the cytochrome <italic>b5</italic> cofactor, and is approximately 50 times more efficient for converting the delta-5 substrate 17-OH pregnenolone to DHEA than the delta-4 substrate 17-OH progesterone to AED (<xref rid="r36" ref-type="bibr">36</xref>). HSD3B enzymes catalyze the conversion of delta-5 to -4 steroids. Whereas HSD3B2 is the primary isoform expressed in adrenal, testis and ovary, HSD3B1 (10 fold more efficient) is the isoform expressed in peripheral tissues such as skin, breast, prostate, placenta and brain (<xref rid="r36" ref-type="bibr">36</xref>).</p><p>In adrenal steroidogenesis [<xref ref-type="fig" rid="f1"><italic>Figure 1A</italic></xref>, reviewed in (<xref rid="r37" ref-type="bibr">37</xref>)], the zona glomerulosa lacks CYP17A activity and produces aldosterone via the sequential activity of HSD3B2, CYP21A1, and CYP11B on pregnenolone. Both the zona fasiculata and zona reticularis express CYP17A, but the zona fasiculata does not express the cytochrome <italic>b5</italic> cofactor required to catalyze the lyase activity of CYP17A, channeling precursors to production of glucocorticoids. The differential expression of cytochrome <italic>b5</italic> in the zona reticularis catalyzes the lyase activity of CYP17 10-fold, leading to robust production of DHEA, followed by conversion to DHEA-S via the sulfotransferase activity of SULT2A1. The zona reticularis is characterized by low expression of HSD3B2, favoring conversion of pregnenolone to DHEA and DHEA-S, although small amounts are converted to AED (<xref rid="r38" ref-type="bibr">38</xref>).</p><p>Less recognized is that the human zona reticularis also expresses AKR1C3, which mediates the final step in T synthesis from AED. Notably, in a small study of 8 women, adrenal vein T levels increased 6-fold (18.5 to 116 ng/dL) before and after ACTH stimulation (<xref rid="r39" ref-type="bibr">39</xref>). In a much earlier study, selective adrenal vein catheterization in men also demonstrated adrenal to peripheral venous T gradients, although a compensatory increase in adrenal production of T was not observed in castrate <italic>vs.</italic> intact men (<xref rid="r40" ref-type="bibr">40</xref>).</p><p>Leydig cells of the testis (<xref ref-type="fig" rid="f1"><italic>Figure 1B</italic></xref>) express similar metabolic machineries, including STAR, CYP11A, and the preference of CYP17A for delta-4 substrates, allowing them to produce DHEA from cholesterol, but with several key differences, including absence of SULT2A1, preventing conversion to DHEA-S, and abundant expression of HSD3B2, which mediates the delta-4 to -5 conversion required to generate T. The final steps in T biosynthesis are catalyzed by HSD17B3 and/or HSD17B5 (called AKR1C3). HSD17B3 is primarily expressed in testicular Leydig cells, while AKR1C3 mediates production of T and DHT in peripheral tissues. The activity of HSD3B2 sand HSD17B3 thus drives the stepwise conversion of DHEA to T, via either AED or androst-5-ene-3, 17-diol (5-androstenediol or A5-diol).</p></sec><sec><title>Androgen synthesis in the prostate and pre-receptor control of DHT metabolism</title><p>The uptake of circulating androgens and the local synthesis of active steroids in peripheral target tissues such as breast, prostate and skin has been termed intracrinology and involves the paracrine diffusion and conversion of steroid substrates among neighboring cell types with different enzyme capacities (<xref rid="r41" ref-type="bibr">41</xref>). In the prostate, circulating T from the Leydig cells is converted to DHT by SRD5A2 present in both basal and luminal epithelial cells. Circulating DHEA-S can be converted to AED, T and DHT via the activity of HSD3B1, AKR1C3 and SRD5A2 present in basal epithelial cells (<xref rid="r42" ref-type="bibr">42</xref>,<xref rid="r43" ref-type="bibr">43</xref>). Circulating T or that produced in the basal cells diffuses to the AR positive luminal cells where it is then converted to DHT by SRD5A2 (<xref rid="r41" ref-type="bibr">41</xref>).</p><p>Prostate tissue also demonstrates epithelial cell expression of phase I (reducing) and phase II (conjugating) DHT catabolizing enzymes that act in concert to regulate access of DHT to the AR. AKR1C1 is the primary enzyme responsible for the irreversible reduction of DHT to the weak metabolite, 5&#x003b1;-androstane-3,17-diol (3&#x003b1;-androstanediol or 3&#x003b1;-diol, a low affinity AR ligand), whereas AKR1C2 catalyzes the reversible conversion of DHT to 5&#x003b1;-androstane-3,17-diol (3&#x003b2;-diol, a pro-apoptotic ligand of estrogen receptor beta, ER) (<xref rid="r44" ref-type="bibr">44</xref>). The reductase activity of AKR1C2, coupled with the reverse oxidative activity of specific 3&#x003b1;-HSD enzymes is a critical molecular switch regulating access of DHT to the AR (<xref rid="r44" ref-type="bibr">44</xref>-<xref rid="r47" ref-type="bibr">47</xref>).</p><p>Candidate enzymes mediating the reversible conversion of 3&#x003b2;-diol to DHT include RL-HSD (17BHSD6), 17BHSD10, RODH4, RDH5, and DHRS9. Transcripts of RL-HSD and 17BHSD10 are highly expressed in the prostate, however several studies suggest RL-HSD is more active in converting 3&#x003b2;-diol to DHT in prostate cells (<xref rid="r48" ref-type="bibr">48</xref>,<xref rid="r49" ref-type="bibr">49</xref>). Basal epithelial cell expression of RL-HSD is present at the protein level, while transcript profiling of cultured epithelial and stromal cells detects stromal expression as well (<xref rid="r48" ref-type="bibr">48</xref>,<xref rid="r50" ref-type="bibr">50</xref>). RL-HSD also acts as an epimerase to convert 3&#x003b2;-diol to 3&#x003b1;-diol, although at much higher substrate concentrations (<xref rid="r51" ref-type="bibr">51</xref>). Recently, RL-HSD was also shown to directly catalyze conversion of physiologic levels of DHT to 3&#x003b1;-diol, suggesting RL-HSD is involved in maintaining the intraprostatic balance of DHT, 3&#x003b1;-diol and 3&#x003b2;-diol (<xref rid="r50" ref-type="bibr">50</xref>).</p><p>The glucuronidating enzymes UGT2B15 and UTG2B17 located in prostate luminal and basal epithelial cells, respectively, irreversibly terminate the androgen signal by glucuronidation of 3&#x003b2;-diol (as well as T, DHT and other metabolites), and are major determinants of the androgen signal in PCa cell lines (<xref rid="r52" ref-type="bibr">52</xref>-<xref rid="r54" ref-type="bibr">54</xref>). UGDH is required to generate the substrate for glucuronide conjugation (UDP-glucuronate), and over-expression of UGDH increases the generation of glucuronidated androgens (<xref rid="r55" ref-type="bibr">55</xref>). Thus, the relative activity of AKR1C2 in converting DHT to 3&#x003b2;-diol, and of RL-HSD and UGT2B17 in competing for conversion of 3&#x003b2;-diol back to DHT or to 3&#x003b2;-diol-G, respectively, will collectively determine the amount of active steroid available for AR ligand occupancy.</p></sec><sec><title>Classical and backdoor pathways of androgen metabolism</title><p>In the classical pathway of androgen synthesis discussed above (<xref ref-type="fig" rid="f2"><italic>Figure 2</italic></xref>, light gray arrows), C21 steroids generated from cholesterol such as pregnenolone and progesterone are first converted to the C19 steroids DHEA and AED via sequential hydroxylase and lyase activity of CYP17A and are then acted on by HSD17B3 to generate T, with peripheral conversion of T to DHT carried out by SRD5A2 in target tissues. However, in steroidogenic tissues in which both CYP17A and SRD5A are co-expressed, an alternate route to DHT, called the &#x02018;back-door&#x02019; pathway (<xref ref-type="fig" rid="f2"><italic>Figure 2</italic></xref>, hatched arrows) is possible wherein C21 steroids undergo 5&#x003b1;-reduction by SRD5A <italic>prior</italic> to being acted upon by the lyase activity of CYP17A (<xref rid="r56" ref-type="bibr">56</xref>). In fact, 17&#x003b1;-OH progesterone is a better substrate for SRD5A (especially SRD5A1) than either AED or T (<xref rid="r57" ref-type="bibr">57</xref>). Since 17&#x003b1;-OH dihydroprogesterone (the 5&#x003b1;-reduced product of 17&#x003b1;-OH progesterone) is a poor substrate for the lyase activity of CYP17A, synthesis proceeds via the 3&#x003b1;-reduction of 17&#x003b1;-OH dihydroprogesterone by ARK1C2, which yields 17&#x003b1;-OH allo-pregnanolone, an excellent substrate for CYP17A lyase activity that is minimally dependent on cytochrome <italic>b5</italic> (<xref rid="r58" ref-type="bibr">58</xref>). Androsterone generated by the lyase activity of CYP17A is then acted upon by HSD17B3 or AKR1C3 to generate 3&#x003b2;-diol. In this case, a reverse oxidative step catalyzed by RL-HSD (not required in the classical pathway) is required to generate DHT (<xref rid="r36" ref-type="bibr">36</xref>). This pathway, e.g., 5&#x003b1;-reduction of C21 steroids prior to the action of CYP17A lyase, occurs in the testis of the immature mouse and the tammar wallaby, is also hypothesized to occur in ovarian hyperandrogenism and polycystic ovarian syndromes, as the human ovary expresses both CYP17A and SRD5A (<xref rid="r56" ref-type="bibr">56</xref>).</p><p>Interestingly, production of DHT in mouse testis via this mechanism is specifically mediated by type 1 and not the type 2 isoform of SRD5A (<xref rid="r59" ref-type="bibr">59</xref>). This observation is of relevance to prostatic androgen metabolism in that a clear shift from SRD5A2 to SRD5A1 expression occurs in the transition from benign to neoplastic prostate tissue (discussed below). Moreover, human CYP17A displays markedly more robust lyase activity for the 5&#x003b1;-reduced progesterone intermediate 17&#x003b1;-OH allo-pregnanolone than for the classical substrates 17&#x003b1;-OH pregnenolone or 17&#x003b1;-OH progesterone, such that the combination of increased SRD5A1 activity in conjunction with expression of CYP17A in PCa tissue may favor <italic>de novo</italic> synthesis via the backdoor pathway over the classical pathway (<xref rid="r60" ref-type="bibr">60</xref>). Importantly, while these &#x02018;backdoor&#x02019; pathways to DHT bypass conventional intermediates of AED and T, it is worth emphasizing that <italic>the backdoor pathway requires the same enzymatic conversions which produce DHT via the conventional pathway</italic>; all that differs is the order in which the enzymes mediate the reactions.</p></sec></sec><sec><title>Altered expression of steroidogenic enzymes in progression to CRPC</title><p>Primary PCa and castration resistant tumors are characterized by a number of changes in steroidogenic gene expression that are consistent with either promoting conversion of adrenal androgens to DHT, inhibiting conversion of DHT to inactive metabolites, or in case of CRPC tumors, mediating <italic>de novo</italic> synthesis of androgens from cholesterol and/or progestin precursors. Here we review the alterations observed in prostate tumors during the progression to CRPC and discuss implications of these changes for determining intra-tumor androgen levels.</p><sec><title>Altered expression of steroidogenic genes in primary PCa</title><p>Perhaps the most consistently observed alteration in prostate tumors is a subtotal loss of tumoral SRD5A2, the principle isoform of this enzyme expressed in benign prostate tissue (<xref rid="r61" ref-type="bibr">61</xref>), and a relative shift in primary and recurrent prostate tumors to expression of SRD5A1 (<xref rid="r34" ref-type="bibr">34</xref>,<xref rid="r62" ref-type="bibr">62</xref>,<xref rid="r63" ref-type="bibr">63</xref>) [although some studies have shown Gleason grade-related increases in both SRD5A1 and SRD5A2 (<xref rid="r64" ref-type="bibr">64</xref>)]. The significance of this shift was recently elucidated by the group of Sharifi who demonstrated that (I) the 5&#x003b1;-reduction of AED to 5&#x003b1;-androstanedione is a required step for DHT synthesis in PCa cells (rather than direct 5&#x003b1;-reduction of T to DHT); (II) this conversion is specifically mediated by SRD5A1; and (III) that in PCa cells T and AED are actually negligible substrates for SRD5A2 (<xref rid="r60" ref-type="bibr">60</xref>) (possibly related to the altered redox environment of tumor cells as SRD5A1 and 2 have different pH optima). These data support previous findings that SRD5A activity in PCa cells has a preference for AED rather than T (<xref rid="r65" ref-type="bibr">65</xref>), as well as initial studies of SRD5A1 which found AED to be a better substrate for 5&#x003b1;-reduction than T (<xref rid="r66" ref-type="bibr">66</xref>,<xref rid="r67" ref-type="bibr">67</xref>).</p><p>Sharifi <italic>et al.</italic> call this the 5&#x003b1;-androstandione pathway (<xref ref-type="fig" rid="f2"><italic>Figure 2</italic></xref>, dark arrow) and suggest that the upregulation of SRD5A1 observed in the transition to CRPC reflects selection of tumors cells capable of efficiently synthesizing DHT via this pathway. Interestingly, a recent report demonstrated that progression to CRPC was correlated with a higher pre-treatment ratio of T to DHT in prostate biopsies taken before the start of ADT [T<sup>:</sup>DHT ratio 0.19 pg/mg (0.98 to 4.92 pg/mg) <italic>vs.</italic> 0.05 pg/mg (0.45 to 16.89 pg/mg) in patients who did not develop CRPC] (<xref rid="r68" ref-type="bibr">68</xref>). It is tempting to speculate that this elevated ratio of T to DHT may reflect tumor cells with pre-treatment loss of SRD5A2 activity, followed by induction of SRD5A1-mediated DHT production via 5&#x003b1;-androstanedione under the selection pressure of ADT. Altered expression of a third SRD5A isozyme, SRD5A3, has also been reported, with increased expression observed in primary and castration recurrent prostate tumors (<xref rid="r69" ref-type="bibr">69</xref>). The importance and/or activity of this enzyme in PCa progression awaits further evaluation (<xref rid="r70" ref-type="bibr">70</xref>).</p><p>Differential changes in the expression of reductive and oxidative enzyme pairs favoring the conversion of inactive diones to active androgens (e.g., AED to T, or androstanedione to DHT) have been observed in primary prostate tumors, including increased tumor expression of the reductive enzymes HSD17B3 (<xref rid="r71" ref-type="bibr">71</xref>) and AKR1C3 (<xref rid="r34" ref-type="bibr">34</xref>,<xref rid="r43" ref-type="bibr">43</xref>,<xref rid="r72" ref-type="bibr">72</xref>), and decreased expression of the oxidative enzyme catalyzing the reverse reaction, HSD17B2 (<xref rid="r71" ref-type="bibr">71</xref>,<xref rid="r73" ref-type="bibr">73</xref>), suggesting a shift in tumoral androgen metabolism to the formation of T and DHT. While increased prostate tumor expression of HSD17B4, which has unidirectional oxidative activity, has been observed, this isoform (also known as D-bifunctional protein or DBP) has a unique peroxisomal targeting sequence and acts primarily in peroxisomal -chain oxidation of fatty acids (<xref rid="r74" ref-type="bibr">74</xref>).</p><p>Similarly, primary PCa demonstrates a selective loss of both AKR1C2 and AKR1C1 versus paired benign tissues, accompanied by a reduced capacity to metabolize DHT to 3&#x003b2;-diol, resulting in increased tumoral DHT levels (<xref rid="r47" ref-type="bibr">47</xref>). Increased expression of HSD17B10, one of the oxidative enzyme capable of mediating the back conversion of 3&#x003b2;-diol to DHT, has also been observed in malignant epithelial cells compared to normal, similarly consistent with an increased capacity to generate DHT in tumor tissue (<xref rid="r75" ref-type="bibr">75</xref>). In contrast, epithelial expression of RL-HSD (which can mediate either conversion of 3&#x003b2;-diol to DHT or of DHT to 3&#x003b1;-diol) is lost in primary PCa, which is hypothesized to reflect loss of the 3&#x003b2;-diol/ER mediated growth inhibition pathway during malignant transformation (<xref rid="r50" ref-type="bibr">50</xref>).</p><p>Another enzyme which may modulate prostate tissue androgen levels is SULT2B1, which shows selective loss of expression in tumor <italic>vs.</italic> benign prostate epithelial cells (<xref rid="r76" ref-type="bibr">76</xref>). While SULT2A1 is the primary phase II enzyme responsible for sulfonation in the adrenal gland, SULT2B1b is highly expressed in the prostate and may limit the pool of unconjugated DHEA available for conversion to AED. This is consistent with a report demonstrating increased DHEA-stimulated LNCaP proliferation in cells with knockdown of SULT2B1b (<xref rid="r77" ref-type="bibr">77</xref>).</p></sec><sec><title>Altered expression of steroidogenic genes in castration resistant prostate tumors</title><p>CRPC tumors demonstrate altered expression of numerous genes in the steroid synthesis pathway, including genes involved in cholesterol metabolism, <italic>de novo</italic> steroidogenesis, as well as utilization of adrenal androgens, suggesting that castration resistant tumors may have the ability to utilize cholesterol, progesterone and/or adrenal precursors for conversion to T and DHT (<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r34" ref-type="bibr">34</xref>). Changes related to cholesterol metabolism include increased expression of squalene epoxidase (SQLE), the rate-limiting enzyme in cholesterol synthesis, as well other genes in this pathway such as HMG-CoA synthase, squalene synthetase and lanosterol synthase (<xref rid="r35" ref-type="bibr">35</xref>). In a study comparing CRPC with primary tumors, the relative expression of numerous transcripts involved in <italic>de novo</italic> androgen biosynthesis and adrenal androgen utilization were altered, including increased expression of HSD3B2 (1.8), AKR1C3 (5.3), SRD5A1 (2.1), SRD5A2 (0.54), AKR1C2 (3.4), AKR1C1 (3.1) and UGT2B15 (3.5). Another study of CRPC metastases in which elevated levels of tumor T and DHT were also measured (T 0.74 ng/g, DHT 0.25 ng/g), showed elevated expression of STAR, CYP17A, HSD3B1/2, HSD17B3, AKR1C3, SRD5A, UGT2B15/17, CYP19A and decreased SRD5A2 (<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r78" ref-type="bibr">78</xref>,<xref rid="r79" ref-type="bibr">79</xref>). Interestingly, CYP17A has also been demonstrated to have squalene epoxidase activity in assays using recombinant CYP17A and in a mouse Leydig tumor cell line (<xref rid="r80" ref-type="bibr">80</xref>), suggesting it may have a dual role in CRPC steroid metabolism. Other studies have not specifically found increased expression of CYP17A in CRPC tumors, but have demonstrated similar findings suggestive of intracrine utilization of adrenal androgens, including increased expression of HSD17B3 and AKR1C3 (<xref rid="r34" ref-type="bibr">34</xref>,<xref rid="r81" ref-type="bibr">81</xref>,<xref rid="r82" ref-type="bibr">82</xref>). Also of note, AKR1C3 has recently been identified as an AR coactivators and thus may play dual roles in promoting ligand synthesis and AR activation (<xref rid="r83" ref-type="bibr">83</xref>).</p><p>A gain of function SNP in HSD3B1 (1245C; N367T, population frequency 22%) has recently been identified as a somatic mutation in CRPC tumors (<xref rid="r84" ref-type="bibr">84</xref>). Three of 25 CRPC tumors with wild type germline DNA at this site had acquired the gain of function mutation in the tumor, and 3 of 11 CRPC tumors with heterozygous germline DNA, showed loss of heterozygosity of the wild type allele. Expression of this variant resulted in increased protein levels of HSD3B1, rendered the protein resistant to ubiquitination and degradation, and lead to increased levels of intratumoral DHT production. Compared to the poor conversion of DHEA to AED in LAPC4 cells which do not have this mutation, the mutant allele was shown to account for the efficient flux of DHEA to AED in LNCaP cells, and was also detected in the VCaP cell line.</p><p>Notably, the expression of enzymes involved in <italic>de novo</italic> steroidogenesis, including MLN64 (homolog of STAR), CYP11A, CYP17A and HSD3B has also been demonstrated in primary prostate tissues (<xref rid="r78" ref-type="bibr">78</xref>,<xref rid="r85" ref-type="bibr">85</xref>). While a role for <italic>de novo</italic> steroidogenesis <italic>per se</italic> in primary prostate tumors is less likely, these observations suggest that the selection pressure of ADT may lead to upregulated expression of these enzymes and reconstitution of tumor androgen levels in CRPC.</p></sec></sec><sec><title>Intracrine steroidogenesis in the continuum from normal prostate to CRPC</title><p>The ability of prostate tissue and prostate tumors to mediate the intracrine conversion of adrenally derived androgens or cholesterol to downstream androgens of T and DHT has been evaluated in normal rat and human prostate, in primary prostate tumors, in CRPC tumors, and <italic>in vitro</italic> and <italic>in vivo</italic> models of CRPC. Here we review the evidence in each of these setting that demonstrate the activity of steroidogenic pathways in the continuum from normal prostate to CRPC.</p><sec><title>Evaluation of steroidogenesis in normal prostate and PCa tissue</title><p>A number of early studies attempted to directly examine the steroidogenic ability of rat and human prostate tissue by evaluating the conversion of exogenously administered radiolabeled-adrenal androgens to T or DHT. Bruchovsky administered radioactively labeled androgens including T, DHT, and the adrenal androgens DHEA and AED to castrated male rats and evaluated prostatic metabolites at 60 minutes after injection (<xref rid="r86" ref-type="bibr">86</xref>). Following administration of DHEA approximately 1% and 8% of the recovered radioactivity was found in T and DHT respectively, with AED it was 2% and 12%, respectively, compared to 37% conversion of exogenously administered T into DHT. Labrie <italic>et al.</italic> demonstrated that administering DHEA or AED to castrate adult rats at levels found in the serum of adult men led to increased prostatic DHT levels and increases in ventral prostate weight (<xref rid="r87" ref-type="bibr">87</xref>). In the Dunning R3327 prostate carcinoma model, administration of adrenal androgens to castrate male rats increased tumor DHT levels and stimulated tumor growth to the level of intact controls (<xref rid="r88" ref-type="bibr">88</xref>).</p><p>In studies of human prostate tissue, Harper <italic>et al.</italic> evaluated prostate androgen metabolism by infusing eugonadal men with <sup>3</sup>H-T, <sup>3</sup>H-AED or <sup>3</sup>H-DHEA-sulfate (DHEA-S) thirty minutes prior to performing radical prostatectomy for BPH (<xref rid="r89" ref-type="bibr">89</xref>). The major metabolite present in prostate tissue after <sup>3</sup>H-T infusion was DHT (about 65% conversion). Infusion of <sup>3</sup>H-AED resulted in approximately 7-10% radioactivity associated with either T or DHT. <sup>3</sup>H-DHEA-S was primarily converted to DHEA (70-90%), with 1-3% conversion to T, DHT and AED. Consistent with these observations, a more recent study using mass spectrometry to identify metabolites formed from <italic>ex-vivo</italic> incubation of human prostate homogenate with DHEA demonstrated production of 5-androstenediol, T, DHT and androsterone (<xref rid="r90" ref-type="bibr">90</xref>). Together, these studies in rat and human prostate tissues suggest that while the most efficient substrate for DHT production in non-tumor prostate tissue is T, a limited amount of DHT is also formed from exogenous DHEA or AED, consistent with intracrine steroidogenesis.</p><p>In PCa tissues Acevedo <italic>et al.</italic> investigated the metabolism of C14 progesterone in primary cancer, but did not observe significant metabolic conversion beyond the formation of immediate progesterone derivatives (<xref rid="r91" ref-type="bibr">91</xref>). This finding is not necessarily unexpected, as studies have now clearly demonstrated that it is CRPC tumors in which steroidogenic genes capable of <italic>de novo</italic> synthesis are upregulated. Klein <italic>et al.</italic> evaluated the presence of adrenal androgens and steroid metabolizing activity (including SRD5A, HSD3B, and HSD17B) <italic>ex vivo</italic> in hormone naive tumors and lymph node metastases. Although malignant tissue had a sub-total loss of SRD5A activity, they found that primary tumors and metastases possessed the capacity to metabolize adrenal androgen precursors along the pathway to DHT (<xref rid="r61" ref-type="bibr">61</xref>). Di Silverio <italic>et al.</italic> demonstrated the conversion of DHEA-S to DHEA within PCa tissue extracts from both eugonadal and castrate men (<xref rid="r92" ref-type="bibr">92</xref>), and Klein <italic>et al.</italic> subsequently confirmed the presence of the steroid sulfatase required for conversion of DHEA-S to DHEA within prostate epithelial tissue (<xref rid="r93" ref-type="bibr">93</xref>). Consistent with their observation that the primary route to DHT in PCa cells is from AED to androstenedione rather than from AED to T, Sharifi&#x02019;s group demonstrated robust conversion of AED to androstenedione and limited to no metabolism of AED to T in biopsy tissue from two patients with CRPC (<xref rid="r60" ref-type="bibr">60</xref>).</p></sec><sec><title>Experimental models of de novo steroidogenesis in CRPC</title><p>Studies using <italic>in vitro</italic> and <italic>in vivo</italic> models of CRPC support the concept of intratumoral androgen synthesis, including both adrenal androgen utilization and <italic>de novo</italic> androgen synthesis (<xref rid="r94" ref-type="bibr">94</xref>). Numerous studies using CRPC xenograft models in castrate mice have demonstrated measurement of substantial intratumor androgen levels (<xref rid="r31" ref-type="bibr">31</xref>,<xref rid="r32" ref-type="bibr">32</xref>,<xref rid="r95" ref-type="bibr">95</xref>-<xref rid="r99" ref-type="bibr">99</xref>). As rodent adrenal glands do not synthesize significant amounts of adrenal androgens, these findings are suggestive of <italic>de novo</italic> steroidogenesis from cholesterol or progesterone precursors. Notably, circulating levels of exogenously administered cholesterol were associated with tumor size (R=0.3957, P=0.0049) and intratumoral T levels (R=0.41, P=0.0023) in subcutaneous LNCaP tumors grown in hormonally intact mice, and were directly correlated with tumoral expression of CYP17A (R=0.4073, P=0.025). Since the hypercholesterolemia did not raise circulating androgen levels, these data suggest the administered cholesterol led to increased intratumoral androgens via <italic>de novo</italic> steroidogenesis.</p><p>More directly addressing this question, a number of groups have carried out <italic>in vitro</italic> studies with radiolabeled cholesterol precursors to demonstrate intratumoral conversion to downstream metabolites. The androgen-independent LNCaP derivative (C81) showed higher expression of STAR, CYP11A and CYP17A compared to its androgen-dependent counterpart (C33) and was shown to directly convert radioactive cholesterol into T (<xref rid="r100" ref-type="bibr">100</xref>). Increases in expression of genes responsible for accumulation of free cholesterol and cholesterol synthesis including LDLR, SRB1, ABCA1, STAR, ACAT, HMG-CoA and CYP11A were demonstrated in a xenograft LNCaP model (<xref rid="r97" ref-type="bibr">97</xref>,<xref rid="r101" ref-type="bibr">101</xref>,<xref rid="r102" ref-type="bibr">102</xref>) (as well as increases in transcripts encoding CYP17A, AKR1C1, AKR1C2, AKR1C3, HSD17B2, and SRD5A1) (<xref rid="r101" ref-type="bibr">101</xref>). Conversion of C14-acetic acid to DHT was observed in these xenografts, and tumors were shown to metabolize H3-progesterone to six different intermediates upstream of 5&#x003b1;-DHT, suggesting occurrence of steroidogenesis via both classic and &#x0201c;backdoor&#x0201d; pathways (<xref rid="r101" ref-type="bibr">101</xref>). In a study of six prostate cell lines (LnCaP, 22Rv1, DU145, RWPE1, PC3 and ALVA4), expression of CYP11A, CYP17A, HSD3B2, 17BSHD3 was detected in all, with conversion of C14-labled cholesterol to T and DHT demonstrated in each cell line, albeit with different efficiencies (<xref rid="r78" ref-type="bibr">78</xref>). It should be noted that other studies have not detected expression of CYP17A or evidence for <italic>de novo</italic> steroidogenesis in PCa cell lines (<xref rid="r103" ref-type="bibr">103</xref>,<xref rid="r104" ref-type="bibr">104</xref>).</p></sec><sec><title>Exogenous influences on intratumoral androgen biosynthesis</title><p>A number of exogenous factors including cytokines, growth factors and paracrine cellular interactions have been found to promote steroid production in PCa cell lines. IL-6 is implicated in cross-talk and regulation of AR activity and PCa growth, but may also play a role in modulating androgen synthesis. Treatment of LNCaP cells with IL-6 induced the expression of steroidogenic enzymes including CYP11A, HSD3B2, AKR1C3 and HSD17B3, and increased levels of T in lysates of cells grown in serum free media by 2 fold (<xref rid="r105" ref-type="bibr">105</xref>).</p><p>In a study designed to evaluate the effects of insulin on steroidogenesis, exposure of LNCaP cells to insulin caused an increase in transcript levels of cholesterol and steroid synthesizing genes, including SREBP1, STAR, CYP11A, CYP17A, HSD3B2, HSD17B3, and SRD5A1, which were confirmed at the protein level for a number of genes including CYP11A1 and CYP17A1. In parallel, insulin increased intracellular levels of pregnenolone, 17&#x003b1;-OH progesterone, DHEA and T, and incubation of insulin-treated LNCaP and VCaP cells with C14-acetate resulted in detection of radiolabeled pregnan-3,20-dione, AED, T and androsterone (<xref rid="r99" ref-type="bibr">99</xref>). In similar studies evaluating the effect of IGF2 on steroidogenesis, these authors demonstrated increased conversion of C14-acetate to pregnan-3,20-dione, pregnan-3,17-diol-20-one, androsterone, AED, and T (<xref rid="r106" ref-type="bibr">106</xref>).</p><p>Receptors for luteinizing hormone (LH), the target of LH releasing hormone (LHRH) agonist therapy in the brain, have also been demonstrated in PCa specimens and may play a role in steroidogenesis (<xref rid="r107" ref-type="bibr">107</xref>). Exposure of both androgen-sensitive (LNCaP) and androgen-independent (22RV1 and C4-2B) PCa cell lines to LH increased the protein expression of steroidogenic enzymes including STAR, CYB5B, CYP11A, and 3BHSD, and a 2.5 fold increase in progesterone synthesis was observed in LH treated C4-2B cells compared to controls (<xref rid="r108" ref-type="bibr">108</xref>). These data suggest that LH may have a role in the regulation of steroid biosynthesis in PCa cells and identify the LH receptor as a potential therapeutic target.</p><p>Several studies have indicated that bone-marrow and PCa-derived stromal cells may play an important role in facilitating androgen biosynthesis in PCa cells. Whereas DHEA induced little or no PSA expression in monocultures of LAPC-4 PCa cells, co-culture with PCa-associated stromal cells resulted in marked stimulation of PSA expression, likely mediated by stromal cell generation of T from DHEA (as T was detected in a time and dose-dependent manner in PCa-stromal cell monocultures treated with DHEA) (<xref rid="r109" ref-type="bibr">109</xref>). Similarly, the impact of DHEA on PSA promoter activity in LNCaP cells was markedly enhanced in the presence of PCa-derived stromal cells (<xref rid="r110" ref-type="bibr">110</xref>). Knockdown of AR in the LNCaP cells abrogated this effect, while coculture with PCa-stromal cells transfected with AR shRNA did not, suggesting paracrine factors secreted by the stromal cells act on the LNCaP AR. Furthermore, following DHEA treatment, T and DHT concentrations were ~5-fold higher in the PCa-stromal/LNCaP coculture <italic>vs.</italic> the LNCaP monoculture. Interestingly, normal-prostate stroma, bone-marrow stroma, lung stroma and bone-derived stromal cells also induced an increase in PSA expression, although the strongest effects were noted with PCa-stromal cells. In a separate study of bone-marrow stromal cells, resting mesenchymal cells were found to express HSD3B and SRD5A protein, while incubation with DHEA resulted in the additional expression of HSD17B5 (<xref rid="r111" ref-type="bibr">111</xref>). These findings indicate that metabolism of androgen precursors in PCa-associated stromal cells may facilitate and/or potentiate the maintenance of intratumoral androgen levels in CRPC tumors.</p><p>Together these studies provide evidence supporting the role of steroidogenesis in reactivating AR signaling in CRPC, and highlight the interplay between cytokines, growth factors, and paracrine stromal and epithelial cell interactions in this mechanism.</p></sec></sec><sec><title>Response and resistance to potent steroidogenesis inhibition in CRPC</title><p>Collectively, these studies demonstrate the capacity of primary and castration resistant prostate tumors to carry out the intracrine conversion of adrenal androgens to DHT, while the <italic>in vitro</italic> and <italic>in vivo</italic> experimental models clearly show that PCa cells are capable of <italic>de novo</italic> steroidogenesis starting from cholesterol and/or progesterone precursors. These findings cannot address the efficiency with which these pathways are active in human CRPC tumors <italic>in situ</italic>, but they strongly support the premise that the residual androgens measured in CRPC tumors reflect the increased expression and activity of enzymes mediating <italic>de novo</italic> steroidogenesis and adrenal androgen utilization. These data provide mechanistic support for the role of intracrine androgen production in maintaining the tumor androgen microenvironment in CRPC and underscore these metabolic pathways as critical therapeutic targets.</p><p>Given its central role in the production of either adrenal or tumor-derived androgens, CYP17A has emerged as a primary target of novel therapeutics. Abiraterone, a pregnenolone derivative that acts as a selective irreversible inhibitor of both the hydroxylase and lyase activity CYP17A, is the first of these agents to enter clinical practice. While clinical responses have been impressive, not all patients respond, the duration of response is variable, and a majority of men eventually progress with a rising PSA. Although the mechanisms determining response and mediating resistance to CYP17A inhibition have not been fully elucidated, emerging clinical and pre-clinical data suggest several possibilities.</p><p>Perhaps most importantly, pre-clinical studies provided the first <italic>in vivo</italic> confirmation that the clinical effect of abiraterone was associated with suppression of tumor androgen levels. Clinical studies have clearly demonstrated abiraterone-mediated suppression of serum androgens, including suppression of DHEA by approximately 75% and of DHEA-S, AED, and T to essentially undetectable (<xref rid="r112" ref-type="bibr">112</xref>,<xref rid="r113" ref-type="bibr">113</xref>). As well, higher levels of AR and CYP17A staining in pre-treatment tumor-infiltrated bone marrow biopsies from men with CRPC were associated with longer responses to abiraterone treatment, supporting CYP17A mediated androgen production as the target of abiraterone activity (<xref rid="r41" ref-type="bibr">41</xref>). However, the efficacy of abiraterone in suppressing tumor androgens in men with CRPC remains to be demonstrated.</p><p>In this regard, treatment of castration resistant LuCaP35 and LuCaP23 xenografts significantly inhibited tumor growth, serum PSA, and intratumoral androgen levels, supporting the hypothesis that abiraterone&#x02019;s primary mechanism of action is through effects on tissue androgens (<xref rid="r31" ref-type="bibr">31</xref>). Seven days after starting treatment levels of T and DHT decreased from 0.49 to 0.03 pg/mg and 2.65 to 0.23 pg/mg, respectively in LuCaP23, and from 0.69 to 0.02 pg/mg and 3.5 to 0.24 pg/mg in LuCaP35. A similar impact of abiraterone on T and DHT levels was observed in separate study of castration resistant VCaP tumors (<xref rid="r32" ref-type="bibr">32</xref>). Notably, while androgen levels remained suppressed in LuCaP23 tumors recurring after therapy, increasing levels of T and DHT were observed in LuCaP35 tumors recurring on abiraterone.</p><p>Further evaluation demonstrated that these CRPC models responded to CYP17A inhibition with multiple mechanisms directed at maintaining AR signaling. This included upregulated expression of full length AR and ligand independent AR variants, as well as induction of steroidogenic genes (including the target gene, CYP17A), several of which showed strong correlations with DHT levels in recurrent tumors. Moreover, tumor biopsies from patients treated with the CYP17A inhibitor ketoconazole also demonstrated increased expression of transcripts encoding CYP17A compared to biopsies from CRPC patients not treated with ketoconazole (<xref rid="r114" ref-type="bibr">114</xref>).</p><p>These findings are consistent with clinical observations that patients progressing on abiraterone have a rise in PSA, suggesting reactivation of AR signaling. Development of resistance to abiraterone has not been associated with a rise in serum androgen levels or in bone marrow aspirate T levels (although 5&#x003b1;-androstanedione may be more appropriate to assess if the route to DHT bypasses T). However, numerous studies (reviewed above) show that circulating androgen levels do not reflect tumor cell androgen concentrations. Thus, in the setting of tumor progression on abiraterone, the rationale for focusing further therapeutic efforts on more potent AR antagonists and agents suppressing AR ligands remains strong.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>Data regarding the molecular response of PCa to hormone therapy continues to emerge, providing critical insight into cellular growth and signaling pathways that may be exploited as therapeutic targets. The presence of residual androgens and persistent activation of the AR signaling axis in CRPC suggest that a multi-targeted treatment approach to ablate all contributions to AR signaling within the prostate tumor will be required for optimal anti-tumor efficacy.</p><p>The molecular alterations occurring in CRPC tumors following abiraterone treatment suggest tumor-specific methods of addressing resistance, either through optimizing steroidogenic blockade or by inhibiting AR signaling. Importantly, a 2 to 3 fold increase in AR expression can render low androgen levels (in the range detected in the abiraterone-treated tumors) physiologically relevant in promoting AR driven growth (<xref rid="r22" ref-type="bibr">22</xref>). Combining CYP17A blockade with inhibitors of other critical components of the pathway such as HSD3B1 or SRD5A2 or with AR inhibitors could offset adaptive upregulation of CYP17A (<xref rid="r115" ref-type="bibr">115</xref>). Abiraterone at higher (but clinically achievable) concentrations can strongly inhibit HSD3B1 and 2 (<xref rid="r116" ref-type="bibr">116</xref>), and can antagonize the promiscuous T877A mutant AR (<xref rid="r117" ref-type="bibr">117</xref>), providing a rationale for dose-escalation of abiraterone at time of progression. However, data demonstrating the induction of full length and ligand-independent AR splice variants in abiraterone-treated tumors suggests combined strategies directed at targeting ligand synthesis with AR inhibitors may have the greatest efficacy.</p><p>The introduction of potent steroidogenic inhibitors such as abiraterone and novel AR inhibitors such as MDV3100 holds significant promise for improving the treatment of men with CRPC. However, the optimal timing, sequence, and potential combinatorial strategies using new AR pathway inhibitors are critical unanswered questions. Delineating mechanisms and biomarkers of resistance will be critical for rational trial design and for the stratification of men to treatment strategies with the highest likelihood of durable efficacy.</p></sec>
Multi-region hemispheric specialization differentiates human from nonhuman primate brain function
Could not extract abstract
<contrib contrib-type="author"><name><surname>Wey</surname><given-names>Hsiao-Ying</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Phillips</surname><given-names>Kimberley A.</given-names></name><address><phone>+1-210-9997102</phone><fax>+1-210-9998386</fax><email>[email protected]</email></address><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff4">4</xref><xref ref-type="aff" rid="Aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>McKay</surname><given-names>D. Reese</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff8">8</xref><xref ref-type="aff" rid="Aff9">9</xref></contrib><contrib contrib-type="author"><name><surname>Laird</surname><given-names>Angela R.</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Kochunov</surname><given-names>Peter</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff5">5</xref><xref ref-type="aff" rid="Aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>Davis</surname><given-names>M. Duff</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff5">5</xref><xref ref-type="aff" rid="Aff10">10</xref></contrib><contrib contrib-type="author"><name><surname>Glahn</surname><given-names>David C.</given-names></name><xref ref-type="aff" rid="Aff8">8</xref><xref ref-type="aff" rid="Aff9">9</xref></contrib><contrib contrib-type="author"><name><surname>Duong</surname><given-names>Timothy Q.</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Fox</surname><given-names>Peter T.</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff5">5</xref></contrib><aff id="Aff1"><label>1</label>Research Imaging Institute, University of Texas Health Science Center, San Antonio, TX 78229 USA </aff><aff id="Aff2"><label>2</label>Department of Radiology, University of Texas Health Science Center, San Antonio, TX 78229 USA </aff><aff id="Aff3"><label>3</label>Athinoula A Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Boston, MA 02129 USA </aff><aff id="Aff4"><label>4</label>Department of Psychology, Trinity University, 1 Trinity Place, San Antonio, TX 78212 USA </aff><aff id="Aff5"><label>5</label>Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX USA </aff><aff id="Aff6"><label>6</label>Department of Physics, Florida International University, Miami, FL USA </aff><aff id="Aff7"><label>7</label>Maryland Psychiatric Research Center, University of Maryland, Baltimore, MD 21201 USA </aff><aff id="Aff8"><label>8</label>Olin Neuropsychiatry Research Center, Institute of Living, Yale University, New Haven, CT USA </aff><aff id="Aff9"><label>9</label>Department of Psychiatry, Yale University, New Haven, CT 06511 USA </aff><aff id="Aff10"><label>10</label>Department of Pharmacology, University of Texas Health Science Center, San Antonio, TX 78229 USA </aff>
Brain Structure &#x00026; Function
<sec id="Sec1" sec-type="introduction"><title>Introduction</title><p>Hemispheric specialization refers to the differential functions of the left and right cerebral hemispheres. One of the most pronounced behavioral asymmetries in humans is hand dominance, with a majority of individuals expressing right-handedness. Though the neuroanatomical differences underlying this major behavioral specialization are minor (Amunts et al. <xref ref-type="bibr" rid="CR1">1996</xref>), motor-task functional activation studies using fMRI readily illustrate this behavior neurophysiologically (Biswal et al. <xref ref-type="bibr" rid="CR3">1995</xref>). Similarly, left-lateralized language dominance is the rule in humans, to the degree that left hemisphere lesions routinely yield aphasia, while right hemisphere lesions do so only rarely. As in the manual motor system, lateralized neuroanatomical correlates that subserve these functional differences are relatively modest (Geschwind and Levitsky <xref ref-type="bibr" rid="CR12">1968</xref>; Steinmetz <xref ref-type="bibr" rid="CR37">1996</xref>) while neurophysiological metrics acquired during speech paradigms mirror the behavioral asymmetry (Petersen et al. <xref ref-type="bibr" rid="CR27">1988</xref>; Fox et al. <xref ref-type="bibr" rid="CR8">2000</xref>). Finally, attentional dominance is a strongly lateralized function in humans, with hemi-spatial neglect occurring with right hemisphere lesions but not with left hemisphere lesions (Mesulam <xref ref-type="bibr" rid="CR25">1981</xref>). To our knowledge, there are no such anatomical asymmetries yet discovered that underlie this behavioral specialization, but functional imaging studies show this effect quite readily (Fox et al. <xref ref-type="bibr" rid="CR9">2006</xref>). Collectively, these observations illustrate that humans show extreme behavioral lateralization, which parallels task activation as imaged by fMRI. Neurobiological explanations of these marked brain-behavioral asymmetries should be sought with functional, rather than structural, imaging methods.</p><p>Nonhuman primates (NHP) also demonstrate hemispheric specialization. As in humans, the most marked expression of this is seen in hand dominance. Chimpanzees, baboons, and capuchin monkeys display hand dominance for various skilled motor tasks, though at levels of lateralization that are less pronounced than humans (Hopkins <xref ref-type="bibr" rid="CR16">2007</xref>). Anatomical asymmetries related to hand dominance have been reported in the motor cortices of great apes (Hopkins and Cantalupo <xref ref-type="bibr" rid="CR17">2004</xref>; Gannon et al. <xref ref-type="bibr" rid="CR11">1998</xref>; Hopkins et al. <xref ref-type="bibr" rid="CR18">2010</xref>) and monkeys (Phillips and Sherwood <xref ref-type="bibr" rid="CR28">2005</xref>; Phillips and Thompson <xref ref-type="bibr" rid="CR29">2013</xref>). Behavior and lesion studies indicate that monkeys, like humans, preferentially use the auditory system in the left hemisphere to process vocalizations (Heffner and Heffner <xref ref-type="bibr" rid="CR15">1984</xref>; Poremba et al. <xref ref-type="bibr" rid="CR30">2004</xref>), though again at levels less lateralized than humans. In addition, some indications suggest functional lateralization for the production of attention-getting vocalizations in chimpanzees which is associated with asymmetry of language area homologs (Taglialatela et al. <xref ref-type="bibr" rid="CR38">2008</xref>). To our knowledge there are no data on lateralization of attention in NHP. Thus, there appears to have been positive selection within the primate order for increasing complexity of hemispheric specialization.</p><p>Task-activation networks in humans correspond to intrinsic functional connectivity networks (ICNs) (Biswal et al. <xref ref-type="bibr" rid="CR3">1995</xref>; Fox and Raichle <xref ref-type="bibr" rid="CR7">2007</xref>; Smith et al. <xref ref-type="bibr" rid="CR35">2009</xref>). Functional connectivity can be inferred from spontaneous BOLD signal fluctuations arising from low frequency (&#x0003c;0.1&#x000a0;Hz) brain activity (Biswal et al. <xref ref-type="bibr" rid="CR3">1995</xref>; Damoiseaux et al. <xref ref-type="bibr" rid="CR6">2006</xref>). These ICNs provide a means of identifying the neurophysiological underpinnings of the brain&#x02019;s functional architecture, which in some cases reflects the underlying structural connectivity of the brain (van den Heuvel et al. <xref ref-type="bibr" rid="CR40">2009</xref>) without task-engagement. Furthermore, resting-state connectivity networks are robust in sleeping infants and anesthesia, hence this connectivity is intrinsic and can be examined in anesthetized NHP (Fransson et al. <xref ref-type="bibr" rid="CR10">2007</xref>; Vincent et al. <xref ref-type="bibr" rid="CR41">2007</xref>).</p><p>In the present study, we used resting-state functional imaging to examine the evolution of lateralized ICNs in representative primate species: humans, chimpanzees (a Great Ape), baboons (an Old World Primate), and capuchin monkeys (a New World Primate). We expected to find evidence for continuity of lateralized ICNs with increasing complexity within the primate order. However, if hemispheric specialization is unique to humans, as has been proposed (Crow <xref ref-type="bibr" rid="CR5">1998</xref>), then lateralized ICNs associated with motor function, attention, and especially language should only appear in humans.</p></sec><sec id="Sec2" sec-type="materials|methods"><title>Methods</title><sec id="Sec3"><title>Subjects</title><p>We acquired 100 resting-state fMRI scans from human participants (45 males, 55 females; age&#x000a0;=&#x000a0;43.2&#x000a0;&#x000b1;&#x000a0;12.1&#x000a0;years), five resting-state fMRI scans from chimpanzees (<italic>Pan troglodytes</italic>, 1 male, 4 females; age&#x000a0;=&#x000a0;24.8&#x000a0;&#x000b1;&#x000a0;12.5&#x000a0;years), 24 resting-state fMRI scans from baboons (<italic>Papio hamadryas</italic> spp., 14 females; age&#x000a0;=&#x000a0;12.7&#x000a0;&#x000b1;&#x000a0;4.5&#x000a0;years) and 25 resting-state fMRI scans from capuchin monkeys (<italic>Cebus apella</italic>, 3 males, 5 females; age&#x000a0;=&#x000a0;9.2&#x000a0;&#x000b1;&#x000a0;7.9&#x000a0;years). Human imaging data were provided by the Genetics of Brain Structure and Function study (structural MRI&#x02014;Kochunov and Davis <xref ref-type="bibr" rid="CR22">2010</xref>; resting-state fMRI&#x02014;Glahn et al. <xref ref-type="bibr" rid="CR13">2010</xref>). Humans were instructed to relax with eyes open during scans. Nonhuman primates were anesthetized with isoflurane (1&#x02013;2&#x000a0;%) for the purpose of restraint and to keep the subjects immobilized during the collection of the brain images. Subjects remained anesthetized throughout the MRI procedure while a veterinarian continually monitored respiration rate, heart rate, and oxygen consumption. The Institutional Review Board and Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio and/or the Texas Biomedical Research Institute (San Antonio, Texas) approved the research.</p></sec><sec id="Sec4"><title>Image acquisition</title><p>All MRI studies were performed on a 3T Siemens TIM TRIO. Gradient echo EPI was used for BOLD resting-state images with the following parameters: TR/TE&#x000a0;=&#x000a0;3000/30&#x000a0;ms. In NHPs, images were acquired with matrix&#x000a0;=&#x000a0;124&#x000a0;&#x000d7;&#x000a0;124, field of view (FOV)&#x000a0;=&#x000a0;12.4&#x000a0;&#x000d7;&#x000a0;12.4&#x000a0;cm (1&#x000a0;&#x000d7;&#x000a0;1&#x000a0;&#x000d7;&#x000a0;1.9&#x000a0;mm resolution), and 27 slices for 30&#x000a0;min; in human studies, images were acquired with matrix&#x000a0;=&#x000a0;128&#x000a0;&#x000d7;&#x000a0;128, FOV&#x000a0;=&#x000a0;22&#x000a0;&#x000d7;&#x000a0;22&#x000a0;cm (1.7&#x000a0;&#x000d7;&#x000a0;1.7&#x000a0;&#x000d7;&#x000a0;3&#x000a0;mm resolution), and 43 slices for 8.5&#x000a0;min. Each subject underwent high-resolution T1-weighted 3D Turbo-flash imaging with an adiabatic inversion contrast pulse and the following parameters: TE/TR/TI&#x000a0;=&#x000a0;3.04/2100/785&#x000a0;ms, flip angle&#x000a0;=&#x000a0;13&#x000b0;, and 800 micrometer isotropic voxel resolution. These images were subjected to retrospective motion correction to achieve optimal gray/white matter contrast (Kochunov and Davis <xref ref-type="bibr" rid="CR22">2010</xref>).</p></sec><sec id="Sec5"><title>Image pre-processing</title><p>Analyses were performed using The Oxford Center for Functional Magnetic Resonance Imaging (FMRIB) software, FSL (<ext-link ext-link-type="uri" xlink:href="http://www.fmrin.ox.ac.uk/fsl">www.fmrin.ox.ac.uk/fsl</ext-link>). Standard image pre-processing for functional MRI data was employed. Time-series data were skull-stripped using automated brain extraction software (Smith <xref ref-type="bibr" rid="CR34">2002</xref>) and motion corrected (Jenkinson et al. <xref ref-type="bibr" rid="CR20">2002</xref>). Images were then band-passed temporal filtered at 0.01&#x02013;0.08&#x000a0;Hz (fslmaths). The resulting data were spatial smoothed with either a 6&#x000a0;mm FWHM (capuchin monkeys and baboons) or an 8&#x000a0;mm FWHM (chimpanzees and humans) Gaussian kernel. Individual time-series data were then registered to its own high-resolution T1-weighting anatomical image and further registered to a standard template brain. Both co-registration steps use affine linear registration with 12 degrees-of-freedom. Human data were co-registered to an MNI-152 atlas brain, and data of NHPs were co-registered to brain study specific templates created by averaging anatomic images of each species.</p></sec><sec id="Sec6"><title>Independent component analysis</title><p>Intrinsic connectivity networks (ICNs) were derived using temporal-concatenation independent component analysis (Beckmann and Smith <xref ref-type="bibr" rid="CR2">2004</xref>), which is a well-established and robust data-driven group-level functional connectivity approach (Smith et al. <xref ref-type="bibr" rid="CR35">2009</xref>). To facilitate comparison of results herein with previous publications and to compare ICNs across species, the dimensionality of all ICA analyses was 20. The identification of ICNs was based on visual inspection of spatial similarity. In addition, to confirm the results were not biased due to difference in sample sizes (one hundred (8.5&#x000a0;min) datasets in human vs. five to twenty-five (30&#x000a0;min) datasets in NHPs), the same functional connectivity analysis using TC-ICA was applied for a subset of human subjects (<italic>N</italic>&#x000a0;=&#x000a0;18, 18 (8.5&#x000a0;min) data were chose to resemble 5 (30&#x000a0;min) worth of data in NHPs). Spatially similar ICNs were found in this subset of data.</p></sec><sec id="Sec7"><title>Lateralization index</title><p>Lateralization index (LI) was calculated using equation: <italic>LI</italic>&#x000a0;=&#x000a0;<italic>(Left</italic> &#x02013; <italic>Right)/(Left</italic>&#x000a0;+&#x000a0;<italic>Right),</italic> where <italic>Left</italic> and <italic>Right</italic> represents voxel counts from the region-of-interests (ROIs) defined within the left and right hemisphere of each ICN (<italic>Z</italic>&#x000a0;&#x0003e;&#x000a0;3), respectively. LI was derived from all primate species and plotted in Fig.&#x000a0;<xref rid="Fig4" ref-type="fig">4</xref>. A LI larger than 0 is considered left-lateralized, while a LI &#x0003c;0 is considered right-lateralized. A |LI|&#x000a0;&#x02265;&#x000a0;0.2 is typically considered as strongly lateralized.</p></sec><sec id="Sec8"><title>Homotopic connectivity analysis</title><p>Studies have proposed that the strength of homotopic connectivity as an index for the tendency of hemispherical asymmetry (Zuo et al. <xref ref-type="bibr" rid="CR42">2010</xref>). Homotopic connectivity (termed voxel-mirrored homotopic connectivity in early studies) was calculated between every pair of symmetric inter-hemispheric voxels. Specifically, we calculated the Pearson&#x02019;s correlation coefficient between the time series of each voxel and its symmetric inter-hemispheric voxel. The resulting correlation coefficients were then Fisher z-transformed. Individual z-maps representing the strength of homotopic connectivity were averaged by subject within each group and normalized by dividing each voxel by the average hemisphere value.</p></sec></sec><sec id="Sec9" sec-type="results"><title>Results</title><p>We computed the homotopic functional coherences and demonstrated similar spatial patterns among humans, chimpanzees, baboons, and capuchin monkeys (Fig.&#x000a0;<xref rid="Fig1" ref-type="fig">1</xref>). This suggests that the overall distributions of functional connectivity are similar across some primate species (Stark et al. <xref ref-type="bibr" rid="CR36">2008</xref>). Furthermore, resting-state based functional connectivity in humans and anesthetized NHP is broadly consistent and appropriate for finer grained analyses of specific networks (Rilling et al. <xref ref-type="bibr" rid="CR31">2007</xref>; Hutchison et al. <xref ref-type="bibr" rid="CR19">2011</xref>).<fig id="Fig1"><label>Fig.&#x000a0;1</label><caption><p>Inter-hemispherical synchrony of spontaneous brain activity. The strength of inter-hemispherical coherences is shown. The higher degree of coherence indicates a higher inter-hemispherical connectivity and coordination. The correspondence of the maps is proof-of-concept that the resting-state data contributing to the findings herein is broadly similar appropriate for cross-species comparison</p></caption><graphic xlink:href="429_2013_620_Fig1_HTML" id="MO1"/></fig> </p><p>Most of the ICNs, in both humans and NHP, were not lateralized. Several spatially similar ICNs, albeit with minor differences, were found across primate species and pertained to basic sensory-motor functions (Fig.&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>). Visual and somatosensory ICNs, including the occipital cortex and pre- and post-central sulcus, respectively, were robust in all primate species. In addition, the default mode network (DMN)&#x02014;which encompasses the anterior and posterior cingulate cortex, the medial and lateral superior parietal lobe, and the medial prefrontal cortex&#x02014;was spatially similar in all species. Furthermore, each group demonstrated a strikingly similar split of the DMN into two separate anterior and posterior components (Fig.&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref>).<fig id="Fig2"><label>Fig.&#x000a0;2</label><caption><p>Bilateral intrinsic connectivity networks across primate species. ICNs from the four primate species that correspond to basic bottom-up processing. The visual, sensory-motor, auditory, and cerebellar networks incorporate the input of information from the surround and the output of motor plans. The default mode network is associated with self-referential, non-directed processing. These ICNs were symmetric and most similar across species</p></caption><graphic xlink:href="429_2013_620_Fig2_HTML" id="MO2"/></fig> </p><p>We found the left- and right- asymmetric ICNs generally reported in humans to be present across species. Contrary to expectation, these ICNs were strikingly similar in all species, suggesting that functional laterality emerges early in the primate lineage (Fig.&#x000a0;<xref rid="Fig3" ref-type="fig">3</xref>). Laterality indices indicated significant lateralization of these ICNs in all species (Fig.&#x000a0;<xref rid="Fig4" ref-type="fig">4</xref>). This strongly argues against hemispheric specialization as a unique feature of humans. However, an unexpected and notable difference existed in humans, where two strongly left-lateralized and one right-lateralized ICN were consistently shown to include frontal and parietal components (Damoiseaux et al. <xref ref-type="bibr" rid="CR6">2006</xref>; Smith et al. <xref ref-type="bibr" rid="CR35">2009</xref>). In NHP, the frontal and parietal components were split into separate lateralized ICNs.<fig id="Fig3"><label>Fig.&#x000a0;3</label><caption><p>Unilateral intrinsic connectivity networks across primate species. Left- and right-lateralized ICNs that correspond top-down cognitive functionality. In humans, the left lateralized fronto-parietal network is associated with speech and language processing, while the right lateralized fronto-parietal network is associated with reasoning, attention, inhibition and working memory. These networks are confined to a single frontal node in non-human primates</p></caption><graphic xlink:href="429_2013_620_Fig3_HTML" id="MO3"/></fig> <fig id="Fig4"><label>Fig.&#x000a0;4</label><caption><p>Lateralization Indices (LI). Lateralization indices (LI) for ICNs illustrated in Figs.&#x000a0;<xref rid="Fig2" ref-type="fig">2</xref> and <xref rid="Fig3" ref-type="fig">3</xref>. ICNs are considered lateralized when the LI is larger than 0.2 or lower than &#x02212;0.2. While separate frontal and parietal networks were lateralized in all four groups (not shown), the fronto-parietal human networks were the only multi-region lateralized ICNs in any of the species</p></caption><graphic xlink:href="429_2013_620_Fig4_HTML" id="MO4"/></fig> </p></sec><sec id="Sec10" sec-type="discussion"><title>Discussion</title><p>Our results provide compelling evidence that functional laterality is phylogenetically conserved in primates. As in humans, functional laterality is far more profound than anatomical asymmetry. Furthermore, the notable difference in functional laterality between humans and NHP is fronto-parietal connectivity, where multi-region (inter-lobar) networks existed only in humans. While functional laterality alone is not a distinguishing characteristic of human brains, we suggest the observed pattern of multi-region functional connectivity is. Passingham (<xref ref-type="bibr" rid="CR26">2008</xref>) postulated that it is the connections of neurons, particularly those in association cortices, that account for the distinguishing mental characteristics of humans.</p><p>When not engaged in goal-directed behavior, spontaneous fluctuations in brain activity give rise to coherent and structured ICNs that are nearly identical to networks engaged during cognitive or behavioral tasks (Smith et al. <xref ref-type="bibr" rid="CR35">2009</xref>; Biswal et al. <xref ref-type="bibr" rid="CR4">2010</xref>). Given this high degree of correspondence between rest and task, the behavioral correlates of human networks are reliable and have been assessed using previously published ICA of task-based data (See Laird et al. <xref ref-type="bibr" rid="CR23">2011</xref> for detailed descriptions of each human network). In contrast, the default mode putatively supports self-referential or non-directed cognitive processing (Gusnard et al. <xref ref-type="bibr" rid="CR14">2001</xref>) and served as a benchmark in two regards for the current report. First, NHP subjects were anesthetized and humans were not. It is possible that the lateralized multi-region hemispheric, inter-lobar connectivity is present in NHPs yet masked by anesthesia. However, as the DMN&#x02014;which has inter-lobar connectivity&#x02014;was readily and uniformly detectable in all species, it was deemed unlikely that a lateralized functional network with inter-lobar connectivity would be masked by anesthesia. Second, consistency of default mode networks across species was used to affirm the validity of making group comparisons in groups of varying size. Because these default mode based benchmarks were met, implications of task-associated networks were assessed. </p><p>ICNs underlying basic input of sensory information from the surround and output of simple motor plans were extremely homogenous across humans and NHPs. The visual ICN was strongly linked to simple visual stimuli such as flashing checkerboards. This sensorimotor ICN was associated with action and somesthesis corresponding to hand movements, including tasks such as finger tapping, grasping, pointing, and electrical and vibrotactile stimulation. The auditory ICN was related to audition, including tone and pitch discrimination tasks. The cerebellar ICN was associated with action and somesthesis, including both overt and covert object recognition even though no other language or speech tasks were associated with this region. Indeed, the cross-species congruence of ICNs and behavioral repertoire in bottom-up processing is clear.</p><p>In contrast, human ICNs responsible for higher aspects of cognition (attention and language) required inter-lobar fronto-parietal connectivity, while NHP ICNs included only the frontal subcomponent. The left-lateralized fronto-parietal network strongly mapped to a host of semantic, phonologic, and orthographic language tasks such as word generation and covert reading, as well as working and explicit memory tasks, such as paired associate recall, cued encoding and recognition. The right-lateralized fronto-parietal ICN was associated with multiple cognitive processes, such as reasoning, inhibition, and memory. Moreover, top-down processing that is thought to be exclusive to humans evidently requires intrinsic connectivity of multiple brain regions.</p><p>Because our findings ultimately reflect connectivity, white matter tracts are surely involved. The superior longitudinal fasciculus, a group of white matter fibers located in the frontal, parietal and temporal regions provides connectivity within the fronto-parietal networks. Recent diffusion tensor imaging (DTI) and dissection studies revealed three different components of this perisylvian tract, which connect to specific cortical areas within the frontal, parietal and temporal lobes (Martino et al. <xref ref-type="bibr" rid="CR24">2012</xref>). In humans, this tract and working memory performance share genetic influence (Karlsgodt et al. <xref ref-type="bibr" rid="CR21">2010</xref>). Thiebaut de Schotten et al. (<xref ref-type="bibr" rid="CR39">2012</xref>) reported major differences in the arcuate fasciculus, the inferior fronto-occipital fasciculus, and the inferior aspect of the superior longitudinal fasciculus. Another DTI study revealed differences across humans, chimpanzees, and macaques in the arcuate fasciculus, the white matter tract connecting the frontal and temporal cortices, which is associated with language in humans (Rilling et al. <xref ref-type="bibr" rid="CR32">2008</xref>). Our results are in strong support of their overall premise, that similarities suggest preserved functions across anthropoids while connectivity differences indicate human specific functional specialization. We build upon these findings by demonstrating that the observed structural connectivity has multi-regional, lateralized physiological properties and is correlated with human specific behavior.</p></sec>
Type 1 Diabetes and Autoimmunity
<title>Abstract.</title><p>Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by the autoimmune response against pancreatic &#x003b2; cells. T1D is often complicated with other autoimmune diseases, and anti-islet autoantibodies precede the clinical onset of disease. The most common coexisting organ-specific autoimmune disease in patients with T1D is autoimmune thyroid disease, and its frequency is estimated at &#x0003e; 90% among patients with T1D and autoimmune diseases. The prevalence of anti-thyroid antibodies in children with T1D at disease onset is about 20% and is particularly common in girls. Furthermore, patients with anti-thyroid antibodies are 18 times more likely to develop thyroid disease than patients without anti-thyroid antibodies. Therefore, for early detection of autoimmune thyroid disease in children with T1D, measurement of anti-thyroid antibodies and TSH at T1D onset and in yearly intervals after the age of 12 yr is recommended. Anti-islet autoantibodies are predictive and diagnostic markers for T1D. The most frequently detected autoantibodies in Japanese patients are GAD autoantibodies (~80%) followed by IA-2 autoantibodies (~60%), insulin autoantibodies (~55%) and ZnT8 autoantibodies (~50%). In a combined analysis, 94% of Japanese patients with T1D can be defined as having type 1A diabetes. Furthermore, autoantibodies to ZnT8 and IA-2 are associated with childhood-onset and acute-onset patients. Thus, it is important to develop a diagnostic strategy for patients with type 1A diabetes in consideration of the age or mode of disease onset.</p>
<contrib contrib-type="author"><name><surname>Kawasaki</surname><given-names>Eiji</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1</label> Department of Diabetes and Metabolism, Nagasaki Harbor Medical Center City Hospital, Nagasaki, Japan</aff>
Clinical Pediatric Endocrinology
<sec sec-type="intro" id="s1"><title>Introduction</title><p>Type 1 diabetes (T1D) is an organ-specific autoimmune disease characterized by the selective destruction of pancreatic &#x003b2;-cells. The histopathology of T1D is defined by a decreased &#x003b2;-cell mass with infiltration of mononuclear cells into the islets of Langerhans, which was described in 1901 by Opie (<xref rid="r1" ref-type="bibr">1</xref>). This lesion was later called &#x02018;insulitis&#x02019;, and it is the hallmark of T1D. In 1965, Gepts reported that insulitis was observed in 70% of patients with acute-onset T1D and concluded that this disease was caused by a &#x003b2;-cell-specific autoimmune process (<xref rid="r2" ref-type="bibr">2</xref>). Furthermore, in the 1970s, Nerup demonstrated cellular autoimmunity in patients with T1D using the leukocyte migration test and speculated that cellular hypersensitivity was the counterpart of lymphocytic infiltration in islets (<xref rid="r3" ref-type="bibr">3</xref>). Therefore, he speculated that cell-mediated immunity could play an important part in the pathogenesis of T1D. As a view suggesting that T1D is an autoimmune disease, there is some evidence that T1D is often complicated with other autoimmune diseases or that anti-islet autoantibodies precede the clinical onset of the disease. In this article, I focus on these two points and review the recent knowledge.</p></sec><sec id="s2"><title>Type 1 Diabetes and Autoimmune Thyroid Disease</title><p>It is well known that T1D is frequently associated with other organ-specific autoimmune diseases, including autoimmune thyroid disease (AITD), pernicious anemia, and idiopathic Addison&#x02019;s disease (<xref rid="r4" ref-type="bibr">4</xref>). <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1</label><caption><title>The prevalence of autoimmune disease complicating type 1 diabetes</title></caption><graphic xlink:href="cpe-23-099-t001"/></table-wrap> summarizes the prevalence of organ-specific autoimmune disease complicating T1D in Japanese and Caucasoid patients (<xref rid="r5" ref-type="bibr">5</xref>). In Japanese patients with T1D, the most common coexisting organ-specific autoimmune disease is AITD (&#x0003e; 90%). The prevalence of anti-thyroid autoantibodies in children with T1D at disease onset is about 20%, and anti-thyroid autoantibodies are particularly common in girls. Furthermore, it is reported that the prevalence of anti-thyroid antibodies increases with increasing age and that the presence of anti-thyroid antibodies at diagnosis of T1D predicts the development of future thyroid disease (<xref rid="r6" ref-type="bibr">6</xref>). Patients with anti-thyroid antibodies are 18 times more likely to develop thyroid disease than patients without anti-thyroid antibodies (<xref rid="r7" ref-type="bibr">7</xref>) (<xref ref-type="fig" rid="fig_001">Fig.1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p>Risk for the development of autoimmune thyroid disease in children with type 1 diabetes. TPO, thyroid peroxidase antibodies. (Material from this publication has been used with the permission of American Diabetes Association from Glastras SJ, <italic>et al</italic>.: The role of autoimmunity at diagnosis of type 1 diabetes in the development of thyroid and celiac disease and microvascular complications. Diabetes Care 2005; 28(9): 2170-2175. Copyright and all rights reserved.)</p></caption><graphic xlink:href="cpe-23-099-g001"/></fig>). Therefore, for early detection of AITD in children with T1D, Glastras <italic>et al</italic>. suggested measurement of anti-thyroid antibodies and TSH at T1D onset and in yearly intervals after the age of 12 yr. Furthermore, the International Society for Pediatric and Adolescent Diabetes (ISPAD) Consensus Clinical Guidelines recommend the screening of thyroid function by analyzing circulating TSH at the diagnosis of diabetes and, thereafter, every 2nd yr in asymptomatic individuals without goiter and more frequent if goiter is present.</p><p>To characterize the T1D patients complicated with AITD (autoimmune polyendocrine syndrome type 3 variant, APS3v), we have analyzed the clinical characteristics of patients with APS3v who were consecutively diagnosed at Nagasaki University Hospital (<xref rid="r8" ref-type="bibr">8</xref>). A remarkable female predominance (M:F=1:4.4), a slow and older age of onset of T1D and a higher prevalence of GAD autoantibodies were observed in APS3v patients compared with T1D patients without AITD. Furthermore, among the patients with T1D and Graves&#x02019; disease, 60% of patients developed Graves&#x02019; disease preceding the onset of T1D, and 30% developed Graves&#x02019; disease after the onset of T1D; there were also a few patients who developed T1D and Graves&#x02019; disease simultaneously (10%). The interval between the onsets of T1D and Graves&#x02019; disease was less than 10 yr in most cases but was close to 20 yr or more than 20 yr in some cases (<xref ref-type="fig" rid="fig_002">Fig. 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p>The interval from the onset of type 1 diabetes to the onset of Graves&#x02019; disease in APS3v patients. T1D, type 1 diabetes; GD, Graves&#x02019; disease; APS3v, autoimmune polyendocrine syndrome type 3 variant.</p></caption><graphic xlink:href="cpe-23-099-g002"/></fig>).</p></sec><sec id="s3"><title>Anti-islet Autoantibodies in Type 1 Diabetes</title><p>Japanese T1D can be divided into three subtypes, i.e., the fulminant form, acute-onset form and slow-onset form (slowly-progressive form) (<xref rid="r9" ref-type="bibr">9</xref>). Among patients with them, those with slowly-progressive T1D are generally indistinguishable from type 2 diabetes if anti-islet autoantibodies are not examined.</p><p>In 1974, Bottazzo and MacCuish firstly described the presence of anti-islet autoantibodies (islet cell antibodies, ICA) in patients with autoimmune polyendocrine syndrome by an indirect immunofluorescence technique (<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r11" ref-type="bibr">11</xref>). In the 1990s, many investigators tried to find target autoantigens against ICA, and glutamic acid decarboxylase (GAD), insulinoma-associated antigen-2 (IA-2) and, more recently, zinc transporter 8 (ZnT8) were identified (<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>). Previous studies have reported that anti-islet autoantibodies were detected in &#x0003e; 90% of Caucasian patients with T1D (<xref rid="r14" ref-type="bibr">14</xref>, <xref rid="r15" ref-type="bibr">15</xref>). In a radioligand binding assay using an <italic>in vitro</italic> transcribed/translated <sup>35</sup>S-labeled protein, we identified GAD autoantibodies in 82% patients with Japanese T1D at disease onset (<xref rid="r16" ref-type="bibr">16</xref>). The next most frequently identified anti-islet autoantibodies in Japanese T1D were IA-2 autoantibodies (58%) followed by insulin autoantibodies (IAA) (55%) and ZnT8 autoantibodies (50%) (<xref ref-type="fig" rid="fig_003">Fig. 3</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p>Combined analysis of anti-islet autoantibodies in Japanese patients with type 1 diabetes at disease onset. GADA, GAD autoantibodies; IAA, insulin autoantibodies; ZnT8A, ZnT8 autoantibodies; IA-2A, IA-2 autoantibodies.</p></caption><graphic xlink:href="cpe-23-099-g003"/></fig>). Furthermore, the prevalence of autoantibodies to ZnT8 and IA-2 was inversely related to the onset age and significantly higher in childhood-onset patients compared with adult-onset patients (<xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2</label><caption><title>Combined analysis of anti-islet autoantibodies in childhood- and adult-onset patients with type 1 diabetes</title></caption><graphic xlink:href="cpe-23-099-t002"/></table-wrap>). Thus, autoantibodies to ZnT8 and IA-2 identify heterogeneity in the age of diabetes onset and are good markers of childhood-onset T1D.</p><p>Measurement of a combination of autoantibody markers has been suggested as a useful tool for determining type 1A diabetes. In a combined analysis, 94% of Japanese patients have at least one of these autoantibodies and are defined as having type 1A (autoimmune-mediated) diabetes (<xref rid="r16" ref-type="bibr">16</xref>) (<xref ref-type="fig" rid="fig_003">Fig. 3</xref>). However, the clinical utility of ZnT8 autoantibodies is limited over testing autoantibodies to GAD, IA-2 and insulin in childhood-onset patients. In our cohort, 90% of the childhood-onset patients had autoantibodies to GAD and/or IA-2, but inclusion of autoantibodies to insulin and/or ZnT8 did not increase the sensitivity for identifying type 1A diabetes. In contrast, inclusion of the ZnT8 autoantibodies reduced the number of autoantibody-negative subjects in the adult-onset patients from 8% to 5%, and 40% of patients who were negative for autoantibodies to GAD, IA-2, and insulin were positive for ZnT8 autoantibodies. Such a broader autoantibody response in adult-onset patients suggests that different pathogenic mechanisms may be involved between adult-onset and childhood-onset T1D.</p></sec><sec id="s4"><title>Anti-islet Autoantibodies and Specificity of &#x003b2; Cell Destruction</title><p>It is generally accepted that T1D is a T cell-mediated autoimmune disease and that circulating autoantibodies to various islet cell antigens are induced following the destruction of pancreatic &#x003b2; cells. Therefore, anti-islet autoantibodies are used as a predictive marker for the development of T1D. However, associations between the autoantibody positivity and the specificity of &#x003b2; cell destruction are variable depending on the target autoantigens. <xref rid="tbl_003" ref-type="table">Table 3</xref><table-wrap id="tbl_003" orientation="portrait" position="float"><label>Table 3</label><caption><title>Disease specificity of GAD autoantibodies</title></caption><graphic xlink:href="cpe-23-099-t003"/></table-wrap> summarizes the disease specificity of GAD autoantibodies. GAD autoantibodies were originally identified in patients with stiff-person syndrome regardless of the coexistence of T1D (<xref rid="r17" ref-type="bibr">17</xref>). Furthermore, GAD autoantibodies can be detected in other diseases such as APS1, AITD, or type 2 diabetes. We and others have previously reported the association between anti-thyroid autoimmunity and anti-islet autoantibodies, especially autoantibodies to GAD. Patients with T1D and AITD (i.e., APS3) show higher levels of GAD autoantibodies compared with patients with T1D alone in both cross-sectional and longitudinal observations (<xref rid="r18" ref-type="bibr">18</xref>). Because high levels of GAD autoantibodies are observed in insulin-deficient patients as in our case, production of GAD autoantibodies may not associated with the residual &#x003b2; cell antigens. Furthermore, it has been reported that GAD is not only expressed in &#x003b2; cells but also in the thyroid gland. In contrast, it is suggested that autoantibodies to IA-2 and ZnT8 are more specific markers of autoimmune-mediated &#x003b2; cell destruction.</p></sec><sec sec-type="conclusions" id="s5"><title>Conclusion</title><p>In this article, I reviewed the recent knowledge regarding the autoimmune diseases associated with T1D and anti-islet autoantibodies. Although the underlying mechanisms with respect to the development of multiple autoimmune diseases within the same person are largely unknown, recent progress including the identification of several loci with associations to more than one autoimmune disease (<xref rid="r19" ref-type="bibr">19</xref>) suggests that common genetic factors or immunological processes are present among the different autoimmune diseases. As the most common coexisting organ-specific autoimmune disease associated with Japanese T1D is autoimmune thyroid disease, children with T1D, or with a family history of T1D, should be aware of the tendency to develop additional autoimmune disorders, especially autoimmune thyroid disease.</p><p>The clinical utilities of anti-islet autoantibodies in patients with diabetes include diagnosis (type 1A or type 1B), prediction (progressor or non-progressor) and understanding of pathophysiology (insulitis-specific or nonspecific phenomenon) (<xref ref-type="fig" rid="fig_004">Fig. 4</xref><fig orientation="portrait" fig-type="figure" id="fig_004" position="float"><label>Fig. 4.</label><caption><p>Clinical utilities of anti-islet autoantibodies in patients with diabetes.</p></caption><graphic xlink:href="cpe-23-099-g004"/></fig>). It is especially necessary to pay attention to the interpretation of GAD autoantibodies. The development of a high-throughput assay to detect epitope-specific or immunoglobulin isotype-specific autoantibodies should warrant accurate diagnosis and prediction of autoimmune disorders.</p></sec>
Increased Secretion of Endogenous GH after Treatment with an Intranasal GH-releasing Peptide-2 Spray Does Not Promote Growth in Short Children with GH Deficiency
<title>Abstract.</title><p>We investigated whether treatment with an intranasal GH-releasing peptide (GHRP)-2 spray, which acts as a potent GH secretagogue that stimulates endogenous GH secretion, promotes growth in patients with GH deficiency (GHD). This study involved 126 prepubertal short children (81 males, 45 females) with a height SD score of &#x02013;2 SD or less, who had been diagnosed as having GHD based on GH stimulation tests, and in whom the serum GH concentrations increased up to 9 ng/ml after preliminary administration of an intranasal GHRP-2 spray. The subjects included in this study were divided into 3 groups by use of a double-blind method; that is 44 were placed into the placebo group (P group: 30 males, 14 females), 41 were placed into the GHRP-2 low dose group (L group: 25 males, 16 females), and 41 were placed into the GHRP-2 high dose group (H group: 26 males, 15 females). Those with a body wt of less than 20 kg were administered a placebo (P group), 50 &#x003bc;g of GHRP-2 (L group) or 100 &#x003bc;g of GHRP-2 (H group), and those with a body wt of 20 kg or more were administered a placebo (P group), 100 &#x000b5;g of GHRP-2 (L group) or 200 &#x000b5;g of GHRP-2 (H group) twice daily (morning and evening) for 48 continuous wk. Age and height SD scores at baseline were not significantly different among the three groups: 7.5 yr old and &#x02013;2.26 SD in the P group, 7.3 yr old and &#x02013;2.38 SD in the L group, and 7.5 yr old and &#x02013;2.27 SD in the H group. Of the 126 subjects, 44, 40 and 40 subjects in the P, L and H groups, respectively, completed the 48 continuous wk of treatment. The changes in the mean height SD scores (mean growth rate) after 48 wk of treatment in the P, L and H groups were 0.07 SD, 0.03 SD, and 0.02 SD, respectively, and thus no significant differences was observed among the 3 groups. Also no significant changes in blood IGF-I levels at baseline or after 48 wk of treatment were observed among the 3 groups. This study revealed that in patients with GHD, an increase in endogenous GH secretion as a result of treatment with GHRP-2 does not promote growth. It is speculated that the area under the curve of serum GH concentration by GHRP-2 spray is too small to produce biological effects. In conclusion, it was demonstrated that growth cannot be promoted by a transient increase in endogenous GH secretion.</p>
<contrib contrib-type="author"><name><surname>Tanaka</surname><given-names>Toshiaki</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hasegawa</surname><given-names>Yukihiro</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Yokoya</surname><given-names>Susumu</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Nishi</surname><given-names>Yoshikazu</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><aff id="aff1"><label>1</label> Tanaka Growth Clinic, Tokyo, Japan</aff><aff id="aff2"><label>2</label>Department of Endocrinology and Metabolism, Tokyo Metropolitan Children&#x02019;s Medical Center, Tokyo, Japan</aff><aff id="aff3"><label>3</label>Department of Medical Specialties, National Center for Child Health and Development, Tokyo, Japan</aff><aff id="aff4"><label>4</label>Department of Pediatrics, Hiroshima Red Cross Hospital &#x00026; Atomic-bomb Survivors Hospital, Hiroshima, Japan</aff>
Clinical Pediatric Endocrinology
<sec sec-type="intro" id="s1"><title>Introduction</title><p>In the late 1970s, Bowers <italic>et al.</italic> developed an artificial peptide that consists of 6 amino acids derived from methionine-enkephalin, acts as a GH secretagogue (GHS) and promotes the secretion of GH. Since then, various GHS have been synthesized, and in 1992, Bowers <italic>et al.</italic> prepared GH releasing peptide-2 (GHRP-2, KP-102: generic name pralmorelin hydrochloride), which is a potent peptide compound (<xref rid="r1" ref-type="bibr">1</xref>).</p><p>GHRP-2 effectively promotes GH secretion and is also approved in Japan as a diagnostic agent for severe adult GH deficiency (AGHD) and GH deficiency (GHD) in short children. Its cut-off value is 9 ng/ml for AGHD and 16 ng/ml for short children with GHD and thus is higher than that of the agents used for the GH secretion stimulation test for AGHD (1.8 ng/ml) (<xref rid="r2" ref-type="bibr">2</xref>) and for short children with GHD (6 ng/ml).</p><p>However, only a poor increase in the serum GH concentration is observed when GHRP-2 is administered orally due to the effects of food intake, and therefore, an intranasal GHRP-2 spray was developed. In 1997 Pihoker <italic>et al</italic>. (<xref rid="r3" ref-type="bibr">3</xref>) reported that, in 15 children with short stature (7 with idiopathic short stature, 8 with GHD) who showed peak GH levels &#x0003e;20 ng/ml after intravenous administration of GHRP-2 (1 &#x003bc;g/kg) and peak GH levels &#x0003e;10 ng/ml after administration of an intranasal spray preparation (5 to 15 &#x003bc;g/kg), a therapeutic effect was observed after 18 to 24 mo when the nasal spray was administered twice daily (5 to 15 &#x003bc;g/kg/dose) for a period of 3 mo and thereafter 3 times a d. As a result of the treatment with intranasal GHRP-2 spray, no changes in the serum IGF-I and IGF-BP-3 levels were observed, and the growth rate increased significantly from 3.7 &#x000b1; 0.2 cm/yr at baseline to 6.1 &#x000b1; 0.3 cm/yr after 6 mo of treatment; the authors concluded that the intranasal GHRP-2 spray is a promising agent for treatment of GHD. However, we could not find any other reports on its clinical usefulness that were published after this report.</p><p>Here we report the results of a double-blind clinical study that involved 126 children with GHD and was conducted for a period of 1 yr to investigate the growth promotion effect of an intranasal GHRP-2 spray.</p></sec><sec sec-type="methods" id="s2"><title>Subjects and Methods</title><p>This study included 84 facilities throughout Japan and involved 126 children (81 males, 45 females) who fulfilled the following criteria: 1) prepubertal boys aged &#x02265;4 to &#x0003c;10 yr old, and prepubertal girls aged &#x02265;4 to &#x0003c;9 yr old; 2) a height SD score of &#x02013;2 SD or less and peak GH levels of 6 ng/ml or less in at least 2 GH stimulation tests using insulin, glucagon, arginine, clonidine or L-DOPA (<xref rid="r4" ref-type="bibr">4</xref>); 3) an increased in the serum GH level to at least 9 ng/ml more than 30 or 45 min after administration in a preliminary test in which the subjects were administered an intranasal GHRP-2 spray (50 &#x003bc;g in those with a body wt of less than 20 kg, and 100 &#x003bc;g in those with a body wt of 20 kg or more).</p><p>All subjects had been diagnosed as having idiopathic GHD. According to the maximum peak GH value in the GH stimulation tests (mpGH), the subjects were classified into three types of GHD as follows: 15 patients with severe GHD (mpGH &#x02264; 5 ng/ml), 111 patients with moderate GHD (5 ng/ml &#x0003c; mpGH &#x02264; 10 ng/ml) and 46 patients with mild GHD (mpGH &#x0003e; 10 ng/ml). However, there were no difference in height SDS, serum IGF-I concentrations and growth velocity among the three types of GHD.</p><p>Subjects were enrolled by a double-blind method into 3 groups; that is 44 were included in the placebo group (group P: 30 males, 14 females), 41 were included in the GHRP-2 low dose group (group L: 25 males, 16 females), and 41 were included in the GHRP-2 high dose group (group H: 26 males, 15 females). Background and clinical factors of the subjects at baseline are presented for each group in <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1</label><caption><title>Clinical characteristics among three groups of GHRP-2 doses</title></caption><graphic xlink:href="cpe-23-107-t001"/></table-wrap>. No significant differences in conditions at birth, height of parents, mpGH, age, height, body wt, height SD scores, annual growth rate or serum IGF-I concentrations were observed among the 3 groups.</p><p>The peak GH values obtained in the groups P, L and H after preliminary treatment with the intranasal GHRP-2 spray were 16.2 &#x000b1; 7.5 ng/ml, 14.6 &#x000b1; 5.3 ng/ml and 16.7 &#x000b1; 5.8 ng/ml, respectively, and thus there were no significant differences among the 3 groups. The GHRP-2 spray was obtained from Kaken Pharmaceutical Co., Ltd. (Bunkyo-ku, Tokyo, Japan).</p><p>The subjects in group P received a placebo, while those in the other two groups were administered the intranasal GHRP-2 spray twice a day, in the morning before meals and in the evening before bedtime; for these two groups, subjects with a body wt of less than 20 kg were administered 50 &#x003bc;g (group L) or 100 &#x003bc;g (group H), and those with a body wt of 20 kg or more were administered 100 &#x003bc;g (group L) or 200 &#x003bc;g (group H). At the start of treatment, and after 24 and 48 wk of treatment with the intranasal GHRP-2 spray, blood was collected before administration and at 30, 45 and 60 min after administration to determine serum GH concentrations. The GH concentration was plotted against time, and the area under the curve (AUC) of the GH concentration was calculated as the sum of trapezia of the area under the GH concentration line.</p><p>Serum IGF-I concentrations were determined before initiation of treatment and after 24 and 48 wk of treatment. Height and body wt was measured at the start of treatment and after 4, 12, 24, 36 and 48 wk of treatment.</p><p>For comparisons between the groups, the Games-Howell test was performed.</p><p>This clinical study was conducted at the request of Kaken Pharmaceutical Co., Ltd., after obtaining approval from the ethics committees of each participating facility and written consent from a parent or guardian of each subject in compliance with the GCP guidelines.</p></sec><sec sec-type="results" id="s3"><title>Results</title><p>Of the 126 subjects, 44, 40 and 40 subjects in groups P, L and H, respectively, completed the 48-wk treatment period. None of the subjects reached puberty during the treatment period. The values that were determined are not shown separately for boys and girls, since no differences in measured values were observed between them.</p><p>The mean peak serum GH values at baseline and after 24 and 48 wk of treatment with the intranasal GHRP-2 spray (group P received a placebo) are shown for each group in <xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p>Mean peak serum GH concentrations after administration of the GHRP-2 nasal spray at the start of treatment and at 24 wk and 48 wk of treatment in three groups of GHRP-2 doses. Numbers of subjects are shown in parentheses. *p &#x0003c; 0.05.</p></caption><graphic xlink:href="cpe-23-107-g001"/></fig>. At baseline, the mean peak serum GH values were 4.5 ng/ml in group P, 13.2 ng/ml in group L, and 26.4 ng/ml in group H, and thus significant differences were observed among the 3 groups. After 24 wk of treatment, the mean peak serum GH values were 3.4 ng/ml in group P, 10.7 ng/ml in group L, and 17.8 ng/ml in group H, and thus significant differences were also observed among 3 groups at this time point. However, the mean peak GH values of groups L and H gradually decreased, and after 48 wk of treatment, the mean peak serum GH values were 4.0 ng/ml in group P, 12.0 ng/ml in group L, and 14.6 ng/ml in group H; thus no significant difference was observed between groups L and H.</p><p>At baseline, the mean AUC values were 1.94 ng/ml&#x030fb;hr in group P, 8.3 ng/ml&#x030fb;h in group L and 16.3 ng/ml&#x030fb;h in group H.</p><p>The changes in the serum IGF-I levels are shown in <xref ref-type="fig" rid="fig_002">Fig. 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p>Mean serum IGF-I concentrations before the GHRP-2 treatment and 48 wk after treatment in three groups of GHRP-2 doses. Vertical bars indicate the SD. Numbers of subjects are shown in parentheses.</p></caption><graphic xlink:href="cpe-23-107-g002"/></fig>. No significant differences were observed among the 3 groups, and no significant changes were observed in any of the 3 groups during the 48-wk treatment period. Growth rates (cm/yr) during the 1 yr before the start of treatment and during the 48-wk treatment period are shown in <xref ref-type="fig" rid="fig_003">Fig. 3</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p>Growth velocity before and during the 48 wk of treatment. Vertical bars indicate the SD. Numbers of subjects are shown in parentheses.</p></caption><graphic xlink:href="cpe-23-107-g003"/></fig>. During treatment, the mean growth rates in groups P, L and H were 5.4 cm/yr, 5.2 cm/yr and 5.1 cm/yr, respectively, and thus no significant differences were observed; no promotion of the growth rate was observed as result of treatment.</p><p>After 48 wk of treatment, the height SD scores in groups P, L and H were &#x02013;2.18 SD, &#x02013;2.35 SD, and &#x02013;2.24 SD, respectively, and thus no significant differences were observed among 3 groups: also no significant change in height SD score was observed in any of the 3 groups as result of treatment.</p><p>Adverse events were reported for 76 subjects (60.3%), and most of the adverse events were accidental such as acute upper respiratory tract inflammation, tonsillitis and otitis media; adverse events that were judged by the investigator as &#x0201c;possibly related&#x0201d; or &#x0201c;probably related&#x0201d; included borborygmus (in 3 subjects) and epistaxis, decreased blood pressure and eosinophilia (in the same subject).</p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p>This study involved subjects with idiopathic GHD, in whom decreased GH secretion had been confirmed by commonly used GH stimulation tests. However, since treatment with the potent intranasal GHRP-2 spray was confirmed to substantially increase GH secretion, we believe that this was a population of children with short stature in whom the ability to secrete GH was not severely impaired. Since there were no significant differences in clinical characteristics among the subjects with three types of GHD, it was suggested that their GH secretion capacities were not severely impaired, even in those with severe GHD. Classically, severely short children who were born by breech presentation and with asphyxia were diagnosed as having severe GHD with other pituitary hormone deficiencies and showed a decreased growth velocity and low IGF-I levels. However, nowadays babies with a breech presentation are born by Cesarean section. We classified short children as having severe GHD based on mpGH. But severe GHD did not show a decreased growth velocity and low IGF-I levels in this study, and therefore the subjects with severe GHD were clinically different from those with classical severe GHD. This fact demonstrated the uncertainty of GH stimulation tests in the diagnosis and classification of GHD as previously pointed out (<xref rid="r5" ref-type="bibr">5</xref>). Patients with brain tumors, such as craniopharyngioma, can be diagnosed as having severe GHD and usually show a very low mpGH. It might be better to revise the cutoff point for severe GHD to be a lower concentration of mpGH. The data in the present study were presented for boys and girls together, since no differences were observed between them. Furthermore, all subjects were prepubertal throughout the study. During the prepubertal period, the differences between boys and girls are minimal in terms of growth velocity, IGF-I levels and response to GH treatment in GHD.</p><p>Although the peak GH values after treatment with the intranasal GHRP-2 spray tended to decline slightly in group H and did not change much in group L during this clinical study, the GH values in groups H and L were significantly higher than in group P. However, even in groups H and L, no significant increase in IGF-I value was observed, and no improvement in the growth rate was observed after 1 yr of treatment.</p><p><xref ref-type="fig" rid="fig_004">Figure 4</xref><fig orientation="portrait" fig-type="figure" id="fig_004" position="float"><label>Fig. 4.</label><caption><p>Mean serum concentration after administration of nasal spray and subcutaneous injection of GH.</p></caption><graphic xlink:href="cpe-23-107-g004"/></fig> shows the changes in the mean GH concentrations after treatment by subcutaneous GH injection (25 &#x003bc;g/kg) (<xref rid="r6" ref-type="bibr">6</xref>) and the intranasal GHRP-2 spray in group H at the time when this clinical study was initiated. GH was subcutaneously injected into 12 short children (9 boys, 3 girls) aged from 6 yr to 12 yr (<xref rid="r6" ref-type="bibr">6</xref>). With these administration methods, high peak GH concentrations of 25.8 &#x000b1; 3.1 ng/ml and 25.0 &#x000b1; 15.8 ng/ml, respectively, were achieved, and there were no differences in the mean peak serum GH concentrations. However, the AUC was found to be much smaller during treatment with the intranasal GHRP-2 spray in group H (16.3 &#x000b1; 10.0 ng/ml&#x02022;h) than that during treatment with subcutaneous GH injection (230 &#x000b1; 7.0 ng/ml&#x02022;hr) (<xref rid="r6" ref-type="bibr">6</xref>), although concentrations were measured at different time points. When treatment is conducted by subcutaneous GH injection, the exogenous GH inhibits endogenous GH (<xref rid="r7" ref-type="bibr">7</xref>), and therefore, the dose that is injected should be an amount that is sufficient to maintain the serum GH concentration over a prolonged period of time and to promote growth by stimulating the production of IGF-I. The intranasal GHRP-2 spray stimulated a prompt GH discharge from the pituitary, but GH secretion did not continue. It is speculated that the effect of the GHRP-2 spray on the AUC of the serum concentration is too small to produce biological effects.</p><p>One could argue that twice a d administration of the spray is not enough to mimic the physiological GH secretion and that more frequent administration may be effective in increasing the AUC and serum IGF-I and promoting improvement of the growth velocity. When the AUC is taken into account, more than 10 times as much nasal spray is necessary to increase the AUC amount to be close to that caused by subcutaneous injection. Although physiological GH secretion is mainly observed during sleep in prepubertal children, frequent intranasal GHRP-2 administration during sleep is a clinically impossible means of mimicking physiological GH secretion. Moreover, it seems doubtful that the serum GH level would increase up to 25 ng/ml after every administration of the GHRP-2 spray.</p><p>Although we had expected that treatment with GHRP-2 would sufficiently stimulate the secretion of endogenous GH and promote growth because of the strong GH secretion-inducing properties of GHRP-2, no growth promoting effect was observed. We may have obtained different results from those reported by Pinhoker <italic>et al.</italic> (<xref rid="r3" ref-type="bibr">3</xref>) because the subjects in their study (<xref rid="r3" ref-type="bibr">3</xref>) had a lower growth rate before the start of treatment and the proportion of subjects with severe GHD was higher. Furthermore, 5 of the subjects were children with short stature who were 10 yr of age or older, and thus it is possible that the growth observed in those children during the GHRP-2 treatment overlapped with the growth spurt of puberty. Pinhoker <italic>et al</italic>. (<xref rid="r3" ref-type="bibr">3</xref>) also were not sure why the growth rates increased in their subjects without an increase in the IGF-I level. It is possible that no other clinical trials have been performed using GHRP-2 for growth promotion for the following reasons. Even if the growth rate was improved with this treatment, at the utmost, a mean growth rate of 6.1 cm/yr was achieved (<xref rid="r3" ref-type="bibr">3</xref>). This growth rate is comparable to the prepubertal growth rate of healthy children, and this improvement is considered poor compared with the mean growth response of 8 cm/yr in children with GHD treated with GH in the first yr.</p><p>Among the various attempts to promote the growth of children with short stature by increasing endogenous GH secretion, in addition to GHRP-2 (<xref rid="r3" ref-type="bibr">3</xref>), treatment with intravenous injection of GH-releasing hormone (GHRH) (<xref rid="r8" ref-type="bibr">8</xref>, <xref rid="r9" ref-type="bibr">9</xref>) has been reported, but due to difficulties in administration, it has not been possible to apply this method in a clinical setting. Furthermore, an oral treatment with clonidine was reported to be effective in one study (<xref rid="r10" ref-type="bibr">10</xref>); however, this treatment was found to be inefficient in subsequent studies (<xref rid="r11" ref-type="bibr">11</xref>, <xref rid="r12" ref-type="bibr">12</xref>).</p><p>At present, medications and supplements that may increase endogenous GH secretion and promote growth can be found in abundance on the Internet. A typical example is arginine. Arginine is used for the GH stimulation test, in which 0.5 g/kg of arginine is administered by intravenous drip infusion for 30 min to increase the blood arginine concentrations and stimulate GH secretion. Therefore, in the case of children weighing 30 kg, 15 g of arginine is infused. It is unlikely that the blood arginine concentration after oral administration of 2 to 3 g of arginine is capable of increasing GH secretion and promoting growth. Even though endogenous GH was sufficiently stimulated in the present study, no growth promotion effect was observed.</p><p>Therefore, it is necessary to issue a warning about the use of medications and supplements that can be found in abundance on the Internet and claim to promote GH secretion. The Japanese Society for Pediatric Endocrinology has set up a homepage for this purpose titled &#x0201c;Opinion of the Japanese Society for Pediatric Endocrinology with regard to warning patients about the use of supplements, etc., that claim to increase height.&#x0201d;</p><p>In conclusion, it was demonstrated that growth cannot be promoted by a transient increase in endogenous GH secretion.</p></sec>
A Novel Deletion Mutation of the Arginine Vasopressin Receptor 2 Gene in a Japanese Infant with Nephrogenic Diabetes Insipidus
Could not extract abstract
<contrib contrib-type="author"><name><surname>Daitsu</surname><given-names>Takashi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Igaki</surname><given-names>Junko</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Goto</surname><given-names>Masahiro</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hasegawa</surname><given-names>Yukihiro</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><aff id="aff1"><label>1</label> Department of Pediatrics, Yamagata City Hospital Saiseikan, Yamagata, Japan</aff><aff id="aff2"><label>2</label> Division of Endocrinology and Metabolism, Tokyo Metropolitan Children&#x02019;s Medical Center, Tokyo, Japan</aff><aff id="aff3"><label>3</label> Division of Genetic Research, Tokyo Metropolitan Children&#x02019;s Medical Center, Tokyo, Japan</aff>
Clinical Pediatric Endocrinology
<sec sec-type="intro" id="s1"><title>Introduction</title><p>Arginine vasopressin (AVP) is released from the posterior pituitary. It controls water balance homeostasis. It binds to arginine vasopressin receptor 2 (AVPR2) on the basolateral membrane of the kidney collecting duct and triggers activation of Gs proteins, which leads to increases in intracellular cAMP and the activity of protein kinase A. These increases cause trafficking of aquaporin-2 (AQP2) water channels to the apical membrane of collecting duct cells, resulting in increased water permeability and antidiuresis. The AVPR2 gene encodes a 7-transmembrane-spanning G protein-coupled receptor, which is located on chromosome Xq28. AVPR2 mutations, which are typically loss-of-function mutations, explain approximately 90% of cases of hereditary nephrogenic diabetes insipidus (NDI) (OMIM 304800). NDI is characterized by an inability to concentrate urine, resulting in excessive urine production, dehydration and thirst. Administration of exogenous AVP cannot restore the normal balance of water in most patients with AVPR2 mutations.</p><p>We describe a Japanese infant with NDI who has a novel deletion mutation of the AVPR2 gene.</p></sec><sec id="s2"><title>Case Report</title><p>A 4-mo-old boy was admitted to our hospital presenting with vomiting, failure to thrive, and hypernatremia. He was born at 40 wks&#x02019; gestation, weighing 2,887 g, and there were no problems in the perinatal period. He had no family history of NDI. At 3 mo of age, vomiting and failure to thrive began.</p><p>On admission, his body length was 59.5 cm (&#x02013;0.5 SD) and body weight was 5,464 g (&#x02013;1.2 SD). His skin turgor was normal and anterior fontanelle was flat. The results of laboratory findings on admission are summarized in <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1</label><caption><title>Laboratory data on admission</title></caption><graphic xlink:href="cpe-23-115-t001"/></table-wrap>. His serum sodium level was 150 mEq/L, and his chloride level was 110 mEq/L. His venous gas values were within normal limits. His urine volume was 1,085 mL/d (3,616 mL/m<sup>2</sup>/d), and his urine osmolality was low, in spite of high serum osmolality and an elevated AVP level.</p><p>He was diagnosed as having congenital NDI. Management was started by intravenous and oral fluid administration, followed by sodium restriction (1.0 mEq/kg/d) and administration of oral hydrochlorothiazide (2 mg/kg/d). Water intake and urinary volume before the therapies were about 1,400 mL/d and about 1,000 mL/d, respectively. After therapy, they were about 1,100 mL/d and about 800 mL/d, respectively. Two weeks after admission, his levels of serum electrolytes were normal.</p></sec><sec id="s3"><title>Mutational Analysis</title><p>Informed consent, based on the Helsinki Final Act of 1975, for a mutational analysis was obtained from the patient&#x02019;s parents. Genomic DNA was extracted from white blood cells. PCR and direct sequencing were conventionally performed. Analysis of the AVPR2 gene revealed a novel one-nucleotide deletion at position 368 (c.368delT) (<xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p>Sequence of the AVPR2 gene in the patient. The patient had a one-nucleotide deletion at position 368 (c.368delT). An arrow indicates the delation site.</p></caption><graphic xlink:href="cpe-23-115-g001"/></fig>). This mutation resulted in a frameshift and premature stop codon (M129*) in the third transmembrane domain. The analysis of the AQP2 gene was negative. Specimens from the parents were not available for analysis.</p></sec><sec sec-type="discussion" id="s4"><title>Discussion</title><p>At present, according to the Human Gene Mutation Database (HGMD; www.hgmd.cf.ac.uk), more than two hundred mutations in the AVPR2 gene have been detected, including nonsense mutations and frameshift mutations leading to stop codons. Although a correlation between genotype and phenotype has not been previously reported, partial correlation is thought to be present. For example, a large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface (<xref rid="r1" ref-type="bibr">1</xref>), resulting in a complete inability to concentrate urine.</p><p>The present patient had a novel one-nucleotide deletion at position 368 (c.368delT). This mutation resulted in a frameshift and premature stop codon (M129*) in the third transmembrane domain.</p><p>We consider that the M129* mutation is a causative mutation of NDI. There are at least 18 nonsense mutations beyond this codon (M129*), namely, W156*, W164*, S167*, Q180*, W193*, W200*, Q225*, E231*, E242*, W284*, W293*, W296*, L312*, W323* and R337*, and at least 28 types of deletion mutation introducing a stop codon beyond this codon (M129*). A nonsense mutation of the C-terminal intracellular tail (R337*) is a cause of NDI (<xref rid="r2" ref-type="bibr">2</xref>) and is predicted to produce a truncated mutant protein, which was reported not to be transported to the plasma membrane (<xref rid="r3" ref-type="bibr">3</xref>). The mutation in our case is also predicted to produce a truncated protein that is shorter than the R337* mutant protein.</p></sec>
Pilot Study on Interferon-&#x003b3;-producing T Cell Subsets after the Protective Vaccination with Radiation-attenuated Cercaria of <italic>Schistosoma japonicum</italic> in the Miniature Pig Model
<p>CLAWN miniature pig has been shown to serve as a suitable host for the experimental infection of <italic>Schistosoma japonicum</italic>. In this study, we found that radiation-attenuated cercaria (RAC) vaccine gave CLAWN miniature pigs protective immunity against subsequent challenge infection with <italic>S. japonicum</italic> cercaria. To characterize the protective immune response of the pig model vaccinated by attenuated cercaria, flow cytometric analysis of the reactive T cell subsets was performed. The intracellular interferon (IFN)-&#x003b3; and the cell surface markers revealed the peripheral blood CD3+ T-lymphocytes produced significant amounts of IFN-&#x003b3; during the immunization period and after the challenge infection. CD4+ &#x003b1;&#x003b2;-T cells as well as CD4+/CD8&#x003b1;<sup>mid</sup> double positive and/or CD8&#x003b1;<sup>high</sup> &#x003b1;&#x003b2;-T cells were the major IFN-&#x003b3;-producing CD3+ T cells. On the contrary, &#x003b3;&#x003b4; T cells did not produce intracellular IFN-&#x003b3;. Our results suggested that RAC-vaccinated miniature pigs showed effective protective immunity through the activation of &#x003b1;&#x003b2; T cells bearing antigen specific T-cell receptors but not through the activation of &#x003b3;&#x003b4; T cells.</p>
<contrib contrib-type="author"><name><surname>Abdel-Hafeez</surname><given-names>Ekhlas Hamed</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Watanabe</surname><given-names>Kanji</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kamei</surname><given-names>Kaori</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kikuchi</surname><given-names>Mihoko</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Honggen</given-names></name><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Daniel</surname><given-names>Boamah</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Chuanxin</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Hirayama</surname><given-names>Kenji</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><aff id="aff1"><label>1</label><institution>Department of Immunogenetics, Institute of Tropical Medicine (NEKKEN), Nagasaki University</institution>, <addr-line>1-14-2 Sakamoto, Nagasaki 852-8523, Japan</addr-line></aff><aff id="aff2"><label>2</label><institution>Department of Parasitology, Faculty of Medicine, Minia University</institution>, <addr-line>Minia, 61519, Egypt</addr-line></aff><aff id="aff3"><label>3</label><institution>Department of Parasitology, Institute of Tropical Medicine (NEKKEN), Nagasaki University</institution>, <addr-line>1-14-2 Sakamoto, Nagasaki 852-8523, Japan</addr-line></aff><aff id="aff4"><label>4</label><institution>Jiangxi Provintial Institute of Parasitic Diseases</institution>, <addr-line>Nanchang 330046, P.R. China</addr-line></aff><aff id="aff5"><label>5</label><institution>Jiangsu Institute of Parasitic Diseases</institution>, <addr-line>Wuxi, Jiangsu 214064, P.R. China</addr-line></aff>
Tropical Medicine and Health
<sec sec-type="introduction"><title>Introduction</title><p id="p001">Schistosomiasis is one of the neglected tropical diseases [<xref rid="B1" ref-type="bibr">1</xref>]. An estimated 200 million people suffer from schistosomiasis and 600 million people are at risk of contracting this disease. Recent re-evaluation of the disease burden has revealed that schistosomiasis exerts a far greater impact than was originally thought [<xref rid="B2" ref-type="bibr">2</xref>, <xref rid="B3" ref-type="bibr">3</xref>]. Among the several schistosoma species identified to date, <italic>Schistosoma japonicum</italic> is known to cause endemic diseases in Asia, especially in China and the Philippines [<xref rid="B4" ref-type="bibr">4</xref>&#x02013;<xref rid="B6" ref-type="bibr">6</xref>]. Despite the implementation of an extensive control program over the past 20 years, infection remains high in these endemic areas [<xref rid="B5" ref-type="bibr">5</xref>, <xref rid="B7" ref-type="bibr">7</xref>, <xref rid="B8" ref-type="bibr">8</xref>]. In contrast to other schistosoma species including <italic>S. mansoni</italic> and <italic>S. haematobium</italic>, <italic>S. japonicum</italic> is mainly harbored by animal species such as water buffalo, cattle and pigs [<xref rid="B9" ref-type="bibr">9</xref>]. Such a variety of hosts makes the control of <italic>S. japonicum</italic> difficult. Current control programs rely mainly on the annual administration of Praziquantel (PZQ) to residents of the endemic areas [<xref rid="B10" ref-type="bibr">10</xref>&#x02013;<xref rid="B12" ref-type="bibr">12</xref>]. PZQ is very effective at treating the disease but it does not protect against re-infection [<xref rid="B13" ref-type="bibr">13</xref>]. Subsequent <italic>S. japonicum</italic> infections are thought to contribute to both the transmission and persistence of schistosoma infection in the prevalent areas.</p><p id="p002">As the areas that require coverage by the annual PZQ treatment are very large [<xref rid="B13" ref-type="bibr">13</xref>], additional therapies are urgently required. The development of effective vaccines against <italic>S. japonicum</italic> infection is currently underway [<xref rid="B10" ref-type="bibr">10</xref>]. Two major prerequisites need to be met in developing the vaccine. First, the animal model should be able to tolerate <italic>S. japonicum</italic> infection over a long period and should be chosen from a natural reservoir host. In this context, we previously reported that the CLAWN miniature pig was a unique and appropriate experimental model of <italic>S. japonicum</italic> infection [<xref rid="B14" ref-type="bibr">14</xref>]. In addition, CLAWN miniature pigs are easy to handle due to their small size in comparison to domestic pigs. <italic>S. japonicum</italic> can infect and establish infection in this species of pig. The evaluation of long-term infection is also feasible in this pig within animal facilities. The second prerequisite to consider during vaccine study in animal models is that the animal should show an immune response against the parasite and subsequently be immunized against such parasites or parasite antigens [<xref rid="B10" ref-type="bibr">10</xref>]. Radiation-attenuated cercaria (RAC) may serve as a positive control, indicating that the host may potentially recognize the parasite and develop protective immunity. During <italic>S. mansoni</italic> infection in mice, RAC inoculation confers host protective immunity against subsequent infection [<xref rid="B2" ref-type="bibr">2</xref>]. However, unlike <italic>S. mansoni</italic> infection in mice, effective, protective immunity against subsequent infection with <italic>S. japonicum</italic> in mice conferred by RAC was minimal [<xref rid="B15" ref-type="bibr">15</xref>, <xref rid="B16" ref-type="bibr">16</xref>]. In our previous study, we reported that the CLAWN miniature pig showed protective immunity following RAC inoculation [<xref rid="B17" ref-type="bibr">17</xref>]. In order to assess the ability of the CLAWN miniature pig to mount an immune response that leads to immunization, we evaluated the effects of RAC inoculation on subsequent S. japonicum cercaria infection and analyzed the immune response elicited by RAC inoculation.</p></sec><sec sec-type="methods"><title>Methods</title><sec sec-type="animals"><title>Animals</title><p id="p003">Four CLAWN miniature pigs were purchased from the Japan Clawn Farm Institute (Kagoshima, Japan). The body weight of the animals used in this study varied from 2.5&#x02013;3.0&#x000a0;kg and the pigs were 5&#x02013;6 weeks of age. The pigs were separated into two groups based on their swine leukocyte antigen (SLA), fed standard nutrient chow based on their body weight and given water ad libitum. The experimental protocol was pre-approved by the Animal Ethics Committee of Nagasaki University (No. 071207-1).</p></sec><sec sec-type="2"><title>Parasite, parasitological technique and blood collection</title><p id="p004">One group of miniature pigs was pericutaneously inoculated twice with 400 RAC (total 20&#x000a0;krad, 33&#x000a0;Gy/min, using 6012 Co irradiator: Pony Industry Co., Ltd., Osaka, Japan) with a 3-week interval between inoculations. As a control, the other group was injected with PBS at the same time as the inoculated group. Four weeks after the irradiated cercaria inoculation, the miniature pigs were further challenged with 200 cercariae. The cercariae were shed from Oncomelania hupensis snails infected with a Chinese strain of <italic>S. japonicum</italic> and maintained at the Jiangsu Provincial Institute of Parasitic Disease Control (Wuxi, Jiangsu Province, China). <italic>S. japonicum</italic> adult worms were recovered from the liver and mesenteric veins using the perfusion method [<xref rid="B14" ref-type="bibr">14</xref>]. A portion of the left hepatic lobe (1&#x02013;2 cubic cm) was digested in 3% KOH at 37&#x000b0;C for 24&#x000a0;h after recording the weight. The egg number counted in one tenth of the digested fluid was evaluated to determine the total number of eggs per gram of tissue.</p></sec><sec sec-type="2"><title>Analysis of peripheral blood lymphocytes</title><p id="p005">Blood samples were collected from the auricular vein every 2 weeks. Whole peripheral blood was then lysed with ACK lysis buffer and stained with monoclonal antibodies. The antibodies used were as follows: anti-swine FITC-CD3 antibody (clone: BB23-8E68C8), Biotin-CD16 (FcG7), PE-CD4 (74-12-4), FITC-CD8 (76-2-11) and APC-gamma-delta T cell receptor (&#x003b3;&#x003b4; TCR) (clone: MAC320). All antibodies were purchased from Becton Dickinson (Tokyo, Japan). To analyze the source of IFN-&#x003b3;, peripheral blood lymphocytes (PBL) were cultured in RPMI-1640 medium supplemented with 10% FBS and 50&#x000a0;mM 2-mercaptoethanol. The cells were cultured at a density of 3 &#x000d7; 10<sup>6</sup>/ml in 48-well flat bottom culture plates (Corning, Inc., NY, USA) for 3 days with schistosoma adult worm antigen (SWA, 50&#x000a0;&#x003bc;g/ml). The cells were then cultured for 4&#x000a0;h with brefeldin A (10&#x000a0;&#x003bc;g/ml), PMA (10&#x000a0;ng/ml, Sigma-Aldrich, Tokyo Japan) and ionomycin (1&#x000a0;&#x003bc;g/ml, Sigma-Aldrich), harvested and stained with the fluorochrome-conjugated monoclonal antibodies (mAb) listed above for the analysis of cell surface markers. For the intracellular cytokine staining, following incubation with antibody, the cells were permeabilized using the Cytofix/Cytoperm Fixation Permeabilization Kit (Becton Dickinson) and stained with PE-(phycoerythrin) conjugated anti IFN-&#x003b3; mAb (clone: P2G10, Becton Dickinson). The stained samples were then applied to a FACS calibur (Becton Dickinson) and analyzed using the CellQuest program (Becton Dickinson) and FlowJo (Tree Star, OR, USA).</p></sec><sec sec-type="2"><title>Cytokine measurement</title><p id="p006">In a separate experiment, cells were stimulated with PHA (phytohemagglutinin) and cultured for 2 (IFN-&#x003b3;) to 4 (IL-4, IL-10) days. The IL-4, IL-10, IFN-&#x003b3; concentrations in the culture supernatant were measured using ELISA sandwich assay according to the manufacturer&#x02019;s instructions (R&#x00026;D Systems, MN, USA).</p></sec></sec><sec sec-type="results"><title>Results</title><sec sec-type="RAC inoculation confers"><title>RAC inoculation confers protective immunity to subsequent <italic>S. japonicum</italic> infection in CLAWN miniature pigs</title><p id="p007">In this study, CLAWN miniature pigs were immunized twice with RAC at 3-week intervals. Four weeks after the second immunization, CLAWN miniature pigs were inoculated with <italic>S. japonicum</italic> cercaria. The efficacy of immunization was examined by calculating the number of worms recovered from CLAWN miniature pigs 6 weeks after infection (Table <xref ref-type="table" rid="T1">1</xref>). In the immunized miniature pigs, the worm reduction rate was found to be greater than 80%. We also noticed a marginal number of deposited eggs in the liver in both vaccinated and control pigs. There was no difference between the two groups. The paucity of eggs in the liver was observed previously, but no clear reason was disclosed. This result confirmed the previous findings that RAC inoculation effectively conferred protective immunity against <italic>S. japonicum</italic> infection in miniature pigs [<xref rid="B17" ref-type="bibr">17</xref>].</p></sec><sec sec-type="2"><title>Analysis of the immune response</title><sec sec-type="3"><title>Cellular composition</title><p id="p008">In order to evaluate the immunity elicited by RAC inoculation, the composition of peripheral blood mononuclear cells (PBMC) was sequentially analyzed (<xref ref-type="fig" rid="F1">Fig. 1</xref>). We did not identify any significant differences between the CD3+ cell (T cells) and CD21+ cell (B cells) ratio between the immunized and control miniature pigs (data not shown). The ratio of T cell populations&#x02014;for example, the CD4+ cells, CD8&#x003b1;<sup>high</sup> cells and CD4+/CD8&#x003b1;<sup>mid</sup> cells&#x02014;in the immunized pigs also showed no difference from that in the control pigs (<xref ref-type="fig" rid="F1">Fig. 1</xref>a&#x02013;c). CD4+ T cells were increased 1 week after infection. In contrast, the CD8&#x003b1;<sup>high</sup> T cells were decreased at 1 week after infection in both groups of miniature pigs and then increased 3 weeks after infection (<xref ref-type="fig" rid="F1">Fig.&#x000a0;1</xref>a, b). CD4+/CD8&#x003b1;<sup>mid</sup> cells were increased at 3 weeks after infection (<xref ref-type="fig" rid="F1">Fig. 1</xref>c). One of the features of the pig PBMC is that it contains a large proportion of T cells that express &#x003b3;&#x003b4; TCR [<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B19" ref-type="bibr">19</xref>]. In this study, almost 50% of lymphocytes comprised T cells that expressed &#x003b3;&#x003b4; TCR prior to immunization. However, during the course of immunization and infection, the percentage of this population of cells in both the immunized and control pigs showed a remarkable decrease (<xref ref-type="fig" rid="F1">Fig. 1</xref>d). This finding suggests that &#x003b3;&#x003b4; TCR+ T cells play a minor role in the protective immunity elicited by RAC inoculation. Granulocytes, especially eosinophils, have been shown to play an important role in the development of protective immunity in schistosoma infections [<xref rid="B20" ref-type="bibr">20</xref>, <xref rid="B21" ref-type="bibr">21</xref>]. In our study, the eosinophil number was not increased following immunization, but increased 6 weeks following infection in both groups (data not shown).</p></sec></sec><sec sec-type="2"><title>Cytokine production</title><p id="p009">We also evaluated cytokine response using ELISA (<xref ref-type="fig" rid="F2">Fig. 2</xref>). In order to achieve this, peripheral blood was collected and the PBMC were isolated. PHA was added to PBMC for 2 days to stimulate IL-4 levels and 4 days to stimulate IFN-&#x003b3; and IL-10. Cytokines in the culture supernatant were then measured by ELISA. The RAC-immunized miniature pigs produced more IFN-&#x003b3; and IL-4 than the control miniature pigs during the immunization period, although the differences were not statistically significant due to the small number of pigs. IFN-&#x003b3; production was found to peak 3 weeks after infection. The amount of IFN-&#x003b3; was greater than that of IL-4 produced in the RAC-immunized miniature pig PBMC. Unlike IFN-&#x003b3; production, IL-4 production peaked at 1 week after infection. Throughout the course of immunization and infection, the immunized miniature pig PBMC produced significantly higher levels of IL-10 than the control miniature pigs.</p></sec><sec sec-type="2"><title>Cellular source of IFN-&#x003b3;</title><p id="p010">Our observation of cytokine response suggested that RAC immunization in miniature pigs elicits an IFN-&#x003b3;-mediated immune response, similar to the findings reported for <italic>S. mansoni</italic> infection in mice [<xref rid="B22" ref-type="bibr">22</xref>, <xref rid="B23" ref-type="bibr">23</xref>]. Therefore, we examined the cellular source of IFN-&#x003b3;, as few papers have reported the source of IFN-&#x003b3; during <italic>S. japonicum</italic> infection in pigs. In order to examine the cellular source of IFN-&#x003b3;, immunized miniature pig PBMC were cultured in the presence of SWA and then stimulated with PMA and ionomycin for 4&#x000a0;h. The PBMC were then stained for intracellular IFN-&#x003b3; and analyzed using flow cytometry (<xref ref-type="fig" rid="F3">Fig. 3</xref>). Based on forward and side scatter, IFN-&#x003b3;-positive cells were lymphocytes (data not shown). Among the lymphocytes observed, almost all of the IFN-&#x003b3;-positive cells were also positive for CD3 and negative for &#x003b3;&#x003b4; TCR (<xref ref-type="fig" rid="F3">Fig. 3</xref>a). Thus, we suggest that the conventional T cells expressing &#x003b1;&#x003b2; TCR are the most likely major source of IFN-&#x003b3; in the immunized miniature pigs. We also examined the natural killer (NK) cells, which are strong producers of IFN-&#x003b3; [<xref rid="B24" ref-type="bibr">24</xref>], using CD16 as a marker of NK cells among other lymphocytes. CD16 is a low affinity receptor for IgG and is expressed on monocytes and a population of NK cells [<xref rid="B25" ref-type="bibr">25</xref>]. Additional specific markers of porcine NK cells have yet to be clearly established [<xref rid="B18" ref-type="bibr">18</xref>].</p><p id="p011">We found a number of CD16+ lymphocytes, initially thought to be NK cells, that were positive for IFN-&#x003b3; (<xref ref-type="fig" rid="F3">Fig.&#x000a0;3</xref>b). However, this population only represented 2.3% of the total lymphocytes. In contrast, 15.9% of the lymphocyte population was positive for IFN-&#x003b3;, but not for CD16. This result suggests that only a small proportion of NK cells was able to produce IFN-&#x003b3;. The lymphocyte subpopulations were further examined to determine their CD4 and CD8&#x003b1;<sup>mid</sup> expression (<xref ref-type="fig" rid="F3">Fig. 3</xref>c). We found that the CD8&#x003b1;<sup>mid</sup> and CD8&#x003b1;<sup>high</sup> cells were positive for IFN-&#x003b3;, and that a portion of the CD4+ cells were also positive for IFN-&#x003b3;. In the pig lymphocyte population, it has also been shown that the CD8&#x003b1;<sup>mid</sup> cells express CD4 and that these cells were considered to form a part of the CD4+ T cell group [<xref rid="B26" ref-type="bibr">26</xref>]. Therefore, both CD8+ and CD4+ T cells represented the main producers of IFN-&#x003b3;.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p id="p012">This study revealed that CLAWN miniature pigs could be successfully immunized using RAC inoculation and that the specifically generated &#x003b1;&#x003b2; but not &#x003b3;&#x003b4; T cells produced high levels of IFN-&#x003b3; in response to antigens. IFN-&#x003b3; was found to be mainly produced by the CD4+/CD8&#x003b1;<sup>mid</sup> T cells and CD8+ T cells. As noted in previous reports including ours [<xref rid="B14" ref-type="bibr">14</xref>&#x02013;<xref rid="B16" ref-type="bibr">16</xref>], in both groups of pigs, CD4+ T cell number was increased 1 week after infection. At the same time, the CD8+ T cell number was decreased. In addition, RAC immunization itself reduced the CD8+ T cell number after the second immunization. Thus, RAC immunization appeared to increase the CD4+/CD8&#x003b1;<sup>mid</sup> T cell ratio. &#x003b3;&#x003b4; TCR+ T cells comprise one of the major components of the porcine PBMC. During infancy, these cells comprise 50% of the lymphocyte population [<xref rid="B18" ref-type="bibr">18</xref>, <xref rid="B19" ref-type="bibr">19</xref>]. As the pigs age, this ratio gradually decreases [<xref rid="B4" ref-type="bibr">4</xref>, <xref rid="B18" ref-type="bibr">18</xref>]. Since no remarkable differences were observed between the immunized and control groups, the observed decrease in &#x003b3;&#x003b4; TCR+ T cells may be attributable to the physiological changes caused by growth.</p><p id="p013">We also examined cytokine production using ELISA. In the immunized pig group, we found a higher level of IFN-&#x003b3; production than in the control group. This increased IFN-&#x003b3; production was even observed during the immunization period of -7 to -3 weeks of infection. In addition, the immunized group produced more IL-4, and RAC immunization activated production of both Th1 and Th2 cytokines in the lymphocytes. Since IFN-&#x003b3; production predominated over IL-4 production, RAC immunization induced IFN-&#x003b3;-based protective immunity, a finding also reported in a mouse model of <italic>S. mansoni</italic> infection [<xref rid="B22" ref-type="bibr">22</xref>, <xref rid="B23" ref-type="bibr">23</xref>]. IFN-&#x003b3; production peaked at 3 weeks after infection, a result that was consistent with the increase in CD8+ T cell and CD4+/CD8&#x003b1;<sup>mid</sup> T cell numbers in both groups. This observation was further supported by the results presented in <xref ref-type="fig" rid="F3">Fig. 3</xref> showing that IFN-&#x003b3; was mainly produced by these two types of T cells during the infection period. We suggest that CD8 T and CD4+/CD8&#x003b1;<sup>mid</sup> T cells, as well as the corresponding IFN-&#x003b3; production, increased during this period because these lymphocytes might accumulate in the lung 1&#x02013;2 weeks after infection. This accumulation is thought to have been caused by the migration of schistosomula into the lung tissue [<xref rid="B27" ref-type="bibr">27</xref>]. After the exit of schistosomula from the lung, the accumulated lymphocytes are probably dispersed into the peripheral blood before migrating further. We also found that IL-4 production reached a peak at 1 week after infection, earlier than IFN-&#x003b3; production. CD4+ cell number was also increased during this period, a finding suggesting that IL-4 was produced in CD4+ T cells during the early stages of infection. IL-10 was also found to be significantly produced in the immunized, but not control, group. Given that IL-10 is thought to function as a regulatory cytokine that reduces the inflammatory response, the synthesized IL-10 might be produced in response to inflammation induced by immunization. In order to determine the cellular source of IFN-&#x003b3;, we undertook intracellular cytokine staining. Since activation by schistosomal antigen alone was insufficient to detect intracellular cytokine signals, we also used PMA and ionomycin stimulation. As expected, CD3+ cells were shown to be the main source of IFN-&#x003b3; synthesis. We found that approximately half of the CD16+ cells demonstrated IFN-&#x003b3; production (2.3% of lymphocyte population); however, this level was less than that of the CD3+ population. According to Gerner W et al. [<xref rid="B18" ref-type="bibr">18</xref>], NK cells of porcine present CD3-negative and CD8a-positive. But, CD3-negative cells produced very little IFN-&#x003b3;. Therefore, CD16-positive cells producing IFN-&#x003b3; may be NKT cells (<xref ref-type="fig" rid="F3">Fig. 3</xref>). Furthermore, lymphocytes expressing &#x003b3;&#x003b4; TCR were not positive for IFN-&#x003b3;. This finding is consistent with the result in <xref ref-type="fig" rid="F1">Fig. 1</xref> demonstrating that this type of T cell was decreased over time. Thus, we suggest that &#x003b3;&#x003b4; TCR+ T cells play a minor role in RAC-induced IFN-&#x003b3; production.</p><p id="p014">In this study, we used CD16 as a marker of NK cells among the lymphocyte population. Although a CD16-population existed [<xref rid="B25" ref-type="bibr">25</xref>], our observations showed that almost all of the IFN-&#x003b3; producing cells were positive for CD3. Therefore, NK cells appeared to play a minor role in IFN-&#x003b3; synthesis.</p><p id="p015">Among the IFN-&#x003b3;+ cells, which are known to mainly comprise T cells expressing the &#x003b1;&#x003b2; chain, CD8&#x003b1;<sup>high</sup> and CD8&#x003b1;<sup>mid</sup> cells were positive for IFN-&#x003b3;. As shown in <xref ref-type="fig" rid="F3">Fig. 3</xref> (c), CD8&#x003b1;<sup>mid</sup> cells also expressed CD4. Thus, CD8&#x003b1;<sup>mid</sup> cells positive for CD4 and CD8&#x003b1;<sup>high</sup> cells are the source of RAC-induced IFN-&#x003b3; production. Considering the fact that the percentage of IFN-&#x003b3;+/CD4+/CD8&#x003b1;<sup>mid</sup> cells was almost twice that of IFN-&#x003b3;+/CD8&#x003b1;<sup>high</sup> cells, CD4+/CD8&#x003b1;<sup>mid</sup> cells may be the main producer of IFN-&#x003b3; in the miniature pig.</p><p id="p016">The fact that CD8&#x003b1;<sup>high</sup> cells also produce IFN-&#x003b3; may be attributable to the PMA and ionomycin used to activate PBL. PBMC were cultured with SWA for 3 days and then stimulated with PMA and ionomycin to preferentially simulate the schistosoma-specific cells. We expected that 3 days of culture with SWA would be sufficient to expand the specific cells. However, it may be possible that the PMA and ionomycin non-specifically simulated the cells. In our previous study, we reported that two proteins synthesized by eight candidate genes showed strong reactivity to the serum of RAC-immunized miniature pigs [<xref rid="B17" ref-type="bibr">17</xref>]. Given that RAC induced IFN-&#x003b3;-based immunity, it is expected that these two vaccine candidates may induce Th1 immunity against infection. Despite numerous reports emphasizing the role of IFN-&#x003b3; in RAC immunization, Th2 immunity has been shown to be important for protective immunity [<xref rid="B28" ref-type="bibr">28</xref>, <xref rid="B29" ref-type="bibr">29</xref>]. As shown in <xref ref-type="fig" rid="F2">Fig. 2</xref>b, the RAC-immunized group demonstrated a higher IL-4 production. In a human study, Th2 immunity was shown to be related to resistance against re-infection with <italic>S. mansoni</italic> and <italic>S. japonicum</italic> [<xref rid="B30" ref-type="bibr">30</xref>, <xref rid="B31" ref-type="bibr">31</xref>]. Thus, it may also be important to study the immunity governed by Th2 and elicited by immunization with adjuvant of cholera toxin or alum. In this study, RAC-induced IFN-&#x003b3;-based immunity and IFN-&#x003b3; were mainly produced by CD4+/CD8&#x003b1; <sup>mid</sup> and CD8&#x003b1; <sup>high</sup> cells.</p></sec>
Adrenal and thyroid function in the fetus and preterm infant
<p>Adrenal and thyroid hormones are essential for the regulation of intrauterine homeostasis, and for the timely differentiation and maturation of fetal organs. These hormones play complex roles during fetal life, and are believed to underlie the cellular communication that coordinates maternal-fetal interactions. They serve to modulate the functional adaptation for extrauterine life during the perinatal period. The pathophysiology of systemic vasopressor-resistant hypotension is associated with low levels of circulating cortisol, a result of immaturity of hypothalamic-pituitary-adrenal axis in preterm infants under stress. Over the past few decades, studies in preterm infants have shown abnormal clinical findings that suggest adrenal or thyroid dysfunction, yet the criteria used to diagnose adrenal insufficiency in preterm infants continue to be arbitrary. In addition, although hypothyroidism is frequently observed in extremely low gestational age infants, the benefits of thyroid hormone replacement therapy remain controversial. Screening methods for congenital hypothyroidism or congenital adrenal hyperplasia in the preterm neonate are inconclusive. Thus, further understanding of fetal and perinatal adrenal and thyroid function will provide an insight into the management of adrenal and thyroid function in the preterm infant.</p>
<contrib contrib-type="author" corresp="yes"><name><surname>Chung</surname><given-names>Hye Rim</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib>
Korean Journal of Pediatrics
<sec sec-type="intro"><title>Introduction</title><p>Adrenal and thyroid gland hormones are essential for the regulation of intrauterine homeostasis and timely differentiation and maturation of fetal organs; these hormones provide the cellular communications that coordinate maternal-fetal interactions<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3)</xref>. During the perinatal period, they serve to modulate functional adaptations for extrauterine life<xref rid="B4" ref-type="bibr">4)</xref>.</p><p>Over recent decades, the survival rate of preterm infants has improved, yet preterm infants continue to present with abnormal thyroid and cortisol axes. Infants with endocrine abnormalities are at an increased risk of abnormal development and morbidity<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6)</xref>. As the physiology of preterm infants differs from that of term infants and older children, and normal physiological hormone levels of preterm infants at different gestational ages (GAs) remain unclear, no definitive management of endocrine problems in preterm infants has been determined as yet<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11)</xref>.</p><p>This article reviews current understanding of the maturation of fetal adrenal and thyroid glands, and the roles of the adrenal and thyroid hormones during the infant's adaptation to extrauterine life; interpretation of clinical findings associated with these hormones in preterm infants is also discussed.</p></sec><sec><title>Fetal adrenal gland</title><p>The fetal adrenal gland exhibits a remarkable transformation in size, morphology, and function during the fetal and perinatal period. In contrast to the adrenal medulla, which is derived from the neuroectoderm, the adrenal cortex is of mesodermal origin. The primitive adrenal glands can be recognized by 3 to 4 weeks of gestation<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B13" ref-type="bibr">13)</xref>. The fetal adrenal gland is composed of three functional zones: a fetal zone (FZ), a transitional zone, and an outer definitive zone. The FZ mainly produces androgens, the transitional zone contains enzymes for cortisol production, and the definitive zone produces mineralocorticoids. The FZs become well differentiated by 9 to 12 weeks of gestation, and are capable of active steroidogenesis<xref rid="B12" ref-type="bibr">12)</xref>. The fetal adrenal gland grows rapidly; the combined glandular weight is approximately 8 g at term, at which time the FZ makes up about 80% of the mass of the gland, with a relative size that is 10 to 20 folds that of the adult adrenal gland<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B14" ref-type="bibr">14)</xref>. Soon after birth, the fetal adrenal gland undergoes rapid involution due to the rapid disappearance of the FZ; in contrast, the definitive zone, which comprises an inner zona fasciculata and an outer zona glomerulosa, proliferates<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B14" ref-type="bibr">14)</xref>.</p><p>The fetal adrenal gland expresses five steroidogenic enzymes: 17-hydroxylase and 17, 20-desmolase (CYP17 or P450c17), 21-hydroxylase (CYP21A2 or P450c21), cholesterol side-chain cleavage (CYP11A1 or P450scc), aldosterone synthase (CYP11B2 or P450c11), and 3&#x003b2;-hydroxysteroid dehydrogenase (3&#x003b2;HSD)<xref rid="B12" ref-type="bibr">12)</xref>. Since the FZ has relatively high steroid sulfotransferase activity and low 3&#x003b2;HSD activity, the major steroid products of the fetal adrenal gland are dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S)<xref rid="B14" ref-type="bibr">14)</xref>, and there is a limited amount of cortisol and aldosterone (<xref ref-type="fig" rid="F1">Fig. 1</xref>). Fetal steroidogenesis is largely programmed to produce inactive products, and provide DHEA substrates for placental estrone and estradiol production<xref rid="B14" ref-type="bibr">14)</xref>. There is complementary activity between the enzymes involved in steroid formation and transformation between the placental and fetal compartments<xref rid="B15" ref-type="bibr">15)</xref> (<xref ref-type="fig" rid="F1">Figs. 1</xref>, <xref ref-type="fig" rid="F2">2</xref>).</p><p>Prior to 23 weeks gestation, the human fetal adrenal cortex is unable to produce cortisol de novo and normally does not do so until as late as 30 weeks gestation<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B16" ref-type="bibr">16)</xref>. Near term, the fetal cortisol production rate in the blood per unit body weight is similar to that in the adult<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B14" ref-type="bibr">14)</xref>. About two thirds of fetal cortisol is derived from the fetal adrenal glands, and one third is derived from placental transfer. Fetal cortisol is converted to cortisone through an 11&#x003b2; hydroxysteroid dehydrogenase (11&#x003b2;HSD) in fetal tissues, and by midgestation, levels of circulating cortisone are 4 to 5 folds higher than cortisol concentrations<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18)</xref>.</p><p>Fetal steroidogenesis is regulated by the hypothalamic-pituitary-adrenal (HPA) axis. The adrenocorticotropic hormone (ACTH) feedback control system matures progressively during the second half of gestation and early neonatal period<xref rid="B12" ref-type="bibr">12)</xref>.</p><p>The steroid hormones produced by the fetal adrenal gland play key roles in the maintenance of pregnancy, intrauterine homeostasis, fetal maturation, and the initiation of parturition<xref rid="B14" ref-type="bibr">14)</xref>.</p></sec><sec><title>Fetal thyroid gland</title><p>The primordium of the human thyroid, which is derived from the epithelium of the pharyngeal floor, is initially recognizable at 16 to 17 days of gestation<xref rid="B12" ref-type="bibr">12)</xref>. The primitive stalk connecting the primordium with the pharyngeal floor elongates into the thyroglossal duct; cells from the lower portion of thyroglossal duct differentiate into thyroid tissue<xref rid="B12" ref-type="bibr">12)</xref>.</p><p>Embryogenesis of the human thyroid gland is largely completed by 10 to 12 weeks gestation, at which point tiny follicle precursors are visible<xref rid="B19" ref-type="bibr">19)</xref>. Thyroid hormones are detectable in fetal serum by 12 weeks gestation; at that point, both thyroxine (T4) and triiodothyronine (T3) are measurable; however, a large proportion of detectable hormones derive from the mother through placental transfer<xref rid="B20" ref-type="bibr">20)</xref>. During gestation, there is a gradual increase in the levels of thyroid hormones. While thyroglobulin (TG) can be identified in the fetal thyroid gland as early as the 5th week, maturation of TG secretion takes much longer<xref rid="B21" ref-type="bibr">21</xref>,<xref rid="B22" ref-type="bibr">22)</xref>. While iodide concentrating capacity can be detected in the thyroid of the 10- to 11-week fetus, the capacity of the fetal thyroid gland to reduce iodide trapping in response to excess iodide does not appear until 36- to 40-week gestation<xref rid="B23" ref-type="bibr">23)</xref>.</p><p>The fetus has detectable levels of thyroid-stimulating hormone (TSH) at GA 12 weeks. There is a moderate increase in TSH over the last two trimesters to levels of 6 to 8 mU/L at the time of delivery<xref rid="B24" ref-type="bibr">24)</xref>. The fetal thyrotroph responds to thyrotropin-releasing hormone (TRH) as early as 25 weeks gestation<xref rid="B24" ref-type="bibr">24)</xref>. The maturation of the negative feedback control of thyroid hormone synthesis occurs around midgestation<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24)</xref> (<xref ref-type="fig" rid="F3">Fig. 3</xref>).</p><p>During gestation, circulating concentrations of T4 and the active metabolite T3 are low, while the inactive metabolites, reverse T3 (rT3) and T3 sulfate, are high. This pattern is a consequence of both immaturity of the hypothalamic-pituitary-thyroid axis, and coordinated adjustments in the deiodinase system. The level of type 1 iodothyronine deiodinase (D1), which catalyzes T4 to T3 conversion, is low throughout gestation. Levels of type 2 deiodinase (D2), which converts T4 to T3, and type 3 deiodinase (D3), an inactivating deiodinase that converts T4 to rT3, are high<xref rid="B25" ref-type="bibr">25)</xref>. Despite low concentrations of circulating T3, by 20- to 26-week gestation T3 levels in the fetal brain are approaching 60%-80% of adult values. While the physiological significance of low circulating T3 concentrations throughout gestation is unknown, it has been suggested that its function may be to avoid tissue thermogenesis and potentiate the anabolic state of the rapidly growing fetus<xref rid="B26" ref-type="bibr">26)</xref>.</p><p>The placenta produces various hormones that can influence the fetal thyroid gland. The most important role of the placenta, however, is in regulating the passage of hormones and drugs from the mother to the embryo, a process that influences the fetal thyroid gland (<xref ref-type="fig" rid="F2">Fig. 2</xref>).</p></sec><sec><title>Fetal anterior pituitary</title><p>Rathke's pouch separates from the primitive pharyngeal stomodeum by 5-week gestation<xref rid="B27" ref-type="bibr">27)</xref>. The bony floor of the sella turcica is present by 7 weeks of gestation. Intact hypothalamic-pituitary portal vessels are present by 12 to 17 weeks of gestation. Maturation of the pituitary portal vascular system continues, extending to 30- to 35-weeks of gestation<xref rid="B12" ref-type="bibr">12)</xref>.</p></sec><sec><title>Role of endocrine system in transition to extrauterine life</title><p>After delivery, the neonate must initiate breathing and defend against hypothermia, hypoglycemia, and hypocalcemia, as the placental supply of energy and nutrients are abruptly removed. Fetal hormones, especially from the adrenal cortex and thyroid gland, rapidly respond to these changes.</p></sec><sec><title>Cortisol surge</title><p>Human fetal cortisol levels tend to be as low as 5-10 &#x000b5;g/mL until about 30-week gestation. Cortisol levels increase progressively, reaching ~20 &#x000b5;g/mL by 36 weeks of gestation, and 45 &#x000b5;g/mL at term. Cortisol increases further during labor, peaking to levels of ~200 &#x000b5;g/mL several hours after term delivery<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B28" ref-type="bibr">28)</xref>. This cortisol surge is mediated by a decrease in the conversion of cortisol to cortisone, with a simultaneous increase in cortisol production by the fetal adrenal gland. Cesarean section of the unlabored fetus blunts the postnatal rise in cortisol<xref rid="B29" ref-type="bibr">29)</xref>; also, during preterm birth, the cortisol responses are attenuated because of the immaturity and unresponsiveness of the adrenal gland<xref rid="B29" ref-type="bibr">29)</xref>.</p><p>The cortisol surge augments surfactant synthesis in lung tissue, increases reabsorption of liquid in the lung, increases methylation of norepinephrine to epinephrine, increases conversion of T4 to T3, facilitates ductus closure, induces maturation of several enzymes and transport processes of the small intestine, and stimulates maturation of hepatic enzymes<xref rid="B12" ref-type="bibr">12)</xref>. Prenatal inflammation, as observed in chorioamnionitis, leads to adrenal stimulation, which results in increased cortisol secretion<xref rid="B30" ref-type="bibr">30</xref>,<xref rid="B31" ref-type="bibr">31)</xref>.</p></sec><sec><title>Extrauterine thyroid adaptation</title><p>During parturition, the neonate must rapidly convert from the fetal state of predominant thyroid hormone inactivation to a state of relative thyroid hyperactivity; this is initiated by an abrupt increase in hypothalamic TRH and pituitary TSH secretion. The cold-stimulated TRH-TSH surge is short-lived and peaks at 30 minutes, with peak concentrations as high as 60 to 70 &#x000b5;U/L<xref rid="B32" ref-type="bibr">32)</xref>; thereafter, serum TSH concentrations progressively decrease to normal infant levels by 3 to 5 days, while serum-free T4 levels remain elevated for several weeks<xref rid="B33" ref-type="bibr">33)</xref>.</p><p>While acute ablation of thyroid function at birth has not been shown to greatly alter thermogenesis or cardiovascular adaptation, chronic inhibition of thyroid function prior to birth has been shown to interfere with postnatal cardiovascular adaptation and thermogenesis in newborn lambs<xref rid="B34" ref-type="bibr">34)</xref>. These results show that thyroid hormones play an important role in the preparation for birth, rather than in modulating endocrine adaptation to birth. Preterm infants have a blunted TSH surge, with very low levels of plasma T3 and T4, relative to term infants.</p></sec><sec><title>Adrenal gland of preterm infants</title><p>The main functions of the postnatal adrenal gland are to regulate protein, carbohydrate, lipid, and nucleic acid metabolism; maintain vascular responsiveness to circulating vasoconstrictors; oppose the increase in capillary permeability during acute inflammation; regulate extracellular water by reducing movement of water into cells and promoting water excretion; suppress the inflammatory response; and modulate central nervous system processing and behavior<xref rid="B5" ref-type="bibr">5)</xref>.</p><p>Activation of the HPA axis is crucial in maintaining homeostasis in response to stress; otherwise, the preterm infant would have limited ability to maintain postpartum homeostasis. Developmental immaturity and illness-induced adrenal insufficiency may contribute to inadequate adrenal function. In preterm infants, adrenal cortex function is closely related to the duration of gestation<xref rid="B35" ref-type="bibr">35)</xref>. However, in preterm infants of less than 30-week GAs, the cortisol production rate, assessed by urinary cortisol metabolites, approaches the cortisol production rate of older children and adults. The surge in cortisol production is absent in preterm infants during clinical illness<xref rid="B36" ref-type="bibr">36)</xref>. Although the human fetal adrenal cortex does not express the 3&#x003b2;HSD enzyme prior to 23-week gestation, there is no evidence of significant immaturity in adrenal 3&#x003b2;HSD activity in preterm infants born between 24-28 weeks of gestation<xref rid="B37" ref-type="bibr">37)</xref>. Blood concentrations of cortisol and other steroid hormones are no lower in preterm infants with late onset adrenal insufficiency than in control preterm infants<xref rid="B38" ref-type="bibr">38)</xref>. These findings suggest that while preterm infants might not have an absolute deficiency of cortisol production, their ability to synthesize sufficient cortisol for the corresponding degree of clinical stress may be limited.</p><sec><title>1. Adrenal insufficiency in preterm infant</title><p>Activation of the HPA axis is crucial in maintaining homeostasis in response to stress. While there is no evidence of clinical adrenocortical insufficiency in term infants, clinically ill and preterm infants may have limited ability to produce adequate amounts of glucocorticoids.</p><p>Systemic hypotension is a common complication in sick preterm infants. While the cause of hypotension in the preterm infant is multifactorial, multiple studies on extremely low birth weight infants have demonstrated that hypotension responds to glucocorticoids, while being refractory to volume expanders and vasopressors<xref rid="B39" ref-type="bibr">39</xref>,<xref rid="B40" ref-type="bibr">40)</xref>. Recent studies have demonstrated low levels of circulating cortisol in preterm infants under stress, suggesting that the pathophysiology of systemic hypotension is associated with the immaturity of the HPA axis<xref rid="B36" ref-type="bibr">36</xref>,<xref rid="B38" ref-type="bibr">38)</xref>.</p><p>Transient adrenocortical insufficiency of prematurity (TAP) is the term used to describe the clinical scenario wherein preterm newborns in the immediate postnatal period have normal or enhanced pituitary response; however, their adrenal glands have a transient inability to maintain cortisol homoeostasis<xref rid="B41" ref-type="bibr">41</xref>,<xref rid="B42" ref-type="bibr">42)</xref>. TAP is frequently associated with systemic hypotension and results from an immature HPA axis, and reduced ability of the adrenal glands to produce cortisol in response to deficiencies of intermediate enzymes in the synthesis pathway, such as 11&#x003b2;-hydroxylase<xref rid="B41" ref-type="bibr">41</xref>,<xref rid="B42" ref-type="bibr">42)</xref>. TAP is typically transient, and adrenal function tends to return to normal by 2-week postpartum. Therefore, glucocorticoid-responsive hypotension is not considered a common phenomenon in this population beyond 2-week postpartum. However, preterm infants sometimes develop late-onset glucocorticoid-responsive circulatory collapse<xref rid="B38" ref-type="bibr">38)</xref>. The pathophysiology of late-onset adrenal insufficiency in preterm infants (AIP) is not due to an absolute deficiency of cortisol production; instead, it may be due to a limited ability to synthesize sufficient cortisol for the corresponding degree of clinical stress<xref rid="B38" ref-type="bibr">38)</xref>. Clinical predictors of AIP include hypotension, oliguria, hyponatremia, lung edema, increased demand for oxygen in the absence of infection, hypovolemia, anemia, and the reopening of a patent ductus arteriosus.</p><p>There are no definitive diagnostic criteria for AIP. A presumptive diagnosis can be made in case the clinical picture indicates adrenal insufficiency, inappropriately low serum cortisol levels, and rapid recovery from signs of adrenal insufficiency following cortisol replacement. A serum cortisol level of &#x0003c;15 &#x000b5;g/dL is frequently used for diagnosis of AIP. This level was based on relative adrenal insufficiency in critically ill adults and a study of critically ill term neonates that demonstrated improvement in hemodynamic parameters with hydrocortisone therapy, selectively in patients with initial cortisol concentrations of &#x0003c;15 &#x000b5;g/dL<xref rid="B43" ref-type="bibr">43</xref>,<xref rid="B44" ref-type="bibr">44)</xref>. A cortisol increase of &#x0003c;9 &#x000b5;g/dL in response to low dose adrenocorticotropin (ACTH) stimulation (1 &#x000b5;g/kg of synthetic ACTH) is also used for the diagnosis of AIP. However, in preterm infants, neither baseline cortisol &#x0003c;15 &#x000b5;g/dL nor &#x00394;-cortisol &#x0003c;9 &#x000b5;g/dL were associated with the presence of relative adrenal insufficiency between days 5 and 7 postpartum<xref rid="B45" ref-type="bibr">45)</xref>. Some authors have recommended measuring the cortisol levels in serum or saliva in response to a corticotropin releasing hormone (CRH) test (1 &#x000b5;g/kg of hCRH) as a reliable method to evaluate the HPA axis in the preterm infant<xref rid="B46" ref-type="bibr">46</xref>,<xref rid="B47" ref-type="bibr">47)</xref>.</p><p>Hydrocortisone is greatly preferred over dexamethasone for treatment of AIP, because it has a limited effect on suppression of growth, and influences both glucocorticoids and mineralocorticoids. Various dosages and durations of hydrocortisone therapy have been used for replacement of AIP (<xref ref-type="table" rid="T1">Table 1</xref>). Further studies are warranted in order to verify the diagnostic criteria and optimal treatment of AIP.</p></sec><sec><title>2. Screening for congenital adrenal hyperplasia</title><p>Congenital adrenal hyperplasia (CAH), which is caused by 21-hydroxylase deficiency, is an inherited metabolic disorder that affects 1 per 16,000 neonates<xref rid="B48" ref-type="bibr">48</xref>,<xref rid="B49" ref-type="bibr">49)</xref>. Mass screening of neonates for 21-hydroxylase deficiency identifies both male and female infants, prevents incorrect sex assignment, and decreases mortality and morbidity due to salt-wasting crisis. Most newborn screening programs measure 17-hydroxyprogesterone (17-OHP) from dried blood spots on filter paper; however, 17-OHP measurement has a high false positive rate in preterm infants<xref rid="B50" ref-type="bibr">50)</xref>.</p><p>The mechanisms underlying high 17-OHP concentrations in preterm infants are unclear, because 21-hydroxylase is actively expressed during early midgestation and 3&#x003b2;HSD is expressed during late midgestation<xref rid="B1" ref-type="bibr">1)</xref>. Possible explanations for the increased levels of 17-OHP in preterm infants include an increase in the conversion of cholesterol to pregnenolone due to increased ACTH from postnatal stress<xref rid="B51" ref-type="bibr">51)</xref>; decrease in conversion of 11-deoxycortisol to cortisol due to delayed expression of 11&#x003b2;-hydroxylase<xref rid="B52" ref-type="bibr">52)</xref>; and decrease in the excretion of steroid metabolites in the kidney<xref rid="B50" ref-type="bibr">50)</xref>. Another probable explanation is that there is cross-reactivity while measuring 17-OHP, with other steroid metabolites such as 17-hydroxypregnenolone and its sulfated metabolites<xref rid="B53" ref-type="bibr">53</xref>,<xref rid="B54" ref-type="bibr">54)</xref>.</p><p>Conversely, antenatal corticosteroid administration can interfere with CAH screening programs, because corticosteroids are known to suppress the HPA axis<xref rid="B55" ref-type="bibr">55</xref>,<xref rid="B56" ref-type="bibr">56)</xref>. Since betamethasone and dexamethasone are similar in their ability to cross the placenta and suppress the fetal pituitary-adrenal axis, the use of antenatal corticosteroids may increase the risk of lowering 17-OHP levels in the blood spot, thus leading to false-negative results.</p><p>While screening preterm infants for CAH, the rates for false positive and false negative results are high; however, there is a low risk of missing a case of CAH that might lead to a salt-wasting crisis in the neonatal intensive care unit. Rescreening of preterm infants with elevated 17-OHP levels, with careful monitoring of the clinical status during intervals, is recommended<xref rid="B57" ref-type="bibr">57)</xref>.</p></sec></sec><sec><title>Thyroid function of preterm neonate</title><p>Postnatal thyroid function of preterm infants differs from that of term infants. Blunted postnatal TSH surges and low serum T4 levels are frequently observed in preterm neonates; this is generally referred to as hypothyroxinemia of prematurity<xref rid="B58" ref-type="bibr">58)</xref>. In contrast to typical congenital hypothyroidism, initial screening indicates a normal TSH level, followed by delayed TSH elevation in some preterm infants<xref rid="B59" ref-type="bibr">59)</xref>.</p><p>The main factors that influence thyroid function in preterm infants are immaturity of the hypothalamic-pituitary-thyroid axis, immature thyroid hormone synthesis, immature thyroid hormone metabolism, and systemic diseases. Insufficient or excessive iodine intake also influence preterm thyroid function<xref rid="B60" ref-type="bibr">60)</xref>.</p><sec><title>1. Hypothyroxinemia and delay in TSH elevation</title><p>Transient hypothyroxinemia of prematurity (THOP) is a condition that primarily affects preterm infants born at less than 30 weeks of gestation, and is characterized by low levels of circulating thyroid hormones despite normal levels of TSH<xref rid="B58" ref-type="bibr">58)</xref>.</p><p>A blunted TSH surge after birth is one of the reasons for low T4 levels in the preterm infant<xref rid="B61" ref-type="bibr">61)</xref>. The other reason is reduced storage of iodine, which can exist due to prematurity<xref rid="B20" ref-type="bibr">20</xref>,<xref rid="B62" ref-type="bibr">62)</xref>. In addition, very low birth weight infants usually have various systemic diseases and are given drugs such as dopamine, dobutamine, and morphine that affect the hypothalamic-pituitary-thyroidal axis. Thus, TSH levels are not representative of overall thyroid function in preterm infants.</p><p>The depth of the nadir and length of time before THOP resolves is related to GA. This condition usually resolves within 2 to 3 weeks, with progressive maturation of the hypothalamic-pituitary-thyroid axis<xref rid="B63" ref-type="bibr">63)</xref>. Although no consensus exists for THOP reference ranges, prevalence rates have been reported to be 35%-85% in very preterm infants<xref rid="B64" ref-type="bibr">64)</xref>.</p><p>Although transiently low levels of thyroid hormones are associated with higher rates of cerebral palsy and cognitive impairment in preterm infants, studies have not demonstrated the benefits of thyroid hormone replacement (<xref ref-type="table" rid="T2">Table 2</xref>). In a meta-analysis, prophylactic thyroid hormone replacement in preterm infants was not shown to be beneficial in reducing neonatal mortality or morbidity, or in improving neurodevelopmental outcomes<xref rid="B65" ref-type="bibr">65)</xref>.</p><p>The incidence of persistent hypothyroidism does not differ among preterm and term newborns; however, transient hypothyroidism is considerably more prevalent<xref rid="B59" ref-type="bibr">59)</xref>. The estimated incidence of delayed TSH elevation is up to 12% in preterm infants<xref rid="B63" ref-type="bibr">63</xref>,<xref rid="B64" ref-type="bibr">64)</xref>. Although the timing of this elevation varies, it usually develops between 2 and 6 weeks of age in most cases. Although the reasons for delayed TSH elevation in the preterm infant may be complex, iodine deficiency or excess are the likely reasons for transient hypothyroidism in the preterm infant. The daily iodine requirement of preterm infants is more than twice that of term infants<xref rid="B66" ref-type="bibr">66)</xref>, and studies conducted in Europe have demonstrated that most preterm infants have iodine deficiency<xref rid="B67" ref-type="bibr">67</xref>,<xref rid="B68" ref-type="bibr">68</xref>,<xref rid="B69" ref-type="bibr">69)</xref>. On the other hand, iodine excess is associated with delayed TSH elevation in the preterm infant<xref rid="B70" ref-type="bibr">70</xref>,<xref rid="B71" ref-type="bibr">71)</xref>. Since sodium/iodide symporters are expressed in the mammary gland, excessive iodine in the lactating mother can be directly transferred to the infant<xref rid="B72" ref-type="bibr">72)</xref>. The skin of preterm infants is thin and may absorb iodine easily, and preterm infants have many opportunities of exposure to iodine-containing disinfectants<xref rid="B73" ref-type="bibr">73)</xref>. Since downregulation of the sodium/iodide symporter (i.e., escape from the Wolff-Chaikoff effect) does not occur in the fetus until the third trimester and seems to appear at &#x0003e;35-week GA in preterm infants<xref rid="B12" ref-type="bibr">12)</xref>, thyroid function in preterm infants is vulnerable to excessive iodine intake. Dopamine is known to suppress thyrotropin release, and transfusion may affect thyroid function test results<xref rid="B74" ref-type="bibr">74)</xref>.</p></sec><sec><title>2. Screening for congenital hypothyroidism in preterm infant</title><p>As routine neonatal screening for congenital hypothyroidism may fail to detect the atypical form of hypothyroidism with delayed TSH elevation, recent screening guidelines recommend repeated screening in the preterm infant<xref rid="B75" ref-type="bibr">75</xref>,<xref rid="B76" ref-type="bibr">76)</xref>. The repeat specimen should be collected at either 2 weeks of age, or 2 weeks after the first screening test was carried out. However, repeat screening has not been adopted by all screening programs, because elevated TSH is mostly a transient problem<xref rid="B77" ref-type="bibr">77)</xref>. Further studies on the etiology and developmental outcomes of delayed TSH elevation are needed for better clinical practice.</p></sec></sec><sec sec-type="conclusions"><title>Conclusions</title><p>Adrenal and thyroid hormones play various roles in somatic development and maintenance of homeostasis throughout the fetal and neonatal periods. Whereas abnormal clinical findings associated with adrenal or thyroid dysfunction are not rare in preterm infants, the diagnostic criteria and optimal management have not been determined yet. Further understanding of fetal and perinatal adrenal and thyroid function will enhance clinician insight into the management of adrenal and thyroid dysfunction in the preterm infant. Further research is required to improve the understanding of the pathophysiology and management of adrenal and thyroid dysfunction in the preterm infant.</p></sec>
Advancements in the treatment of pediatric acute leukemia and brain tumor - continuous efforts for 100% cure
<p>Treatment outcomes of pediatric cancers have improved greatly with the development of improved treatment protocols, new drugs, and better supportive measures, resulting in overall survival rates greater than 70%. Survival rates are highest in acute lymphoblastic leukemia, reaching more than 90%, owing to risk-based treatment through multicenter clinical trials and protocols developed to prevent central nervous system relapse and testicular relapse in boys. New drugs including clofarabine and nelarabine are currently being evaluated in clinical trials, and other targeted agents are continuously being developed. Chimeric antigen receptor-modified T cells are now attracting interest for the treatment of recurrent or refractory disease. Stem cell transplantation is still the most effective treatment for pediatric acute myeloid leukemia (AML). However, in order to reduce treatment-related death after stem cell transplantation, there is need for improved treatments. New drugs and targeted agents are also needed for improved outcome of AML. Surgery and radiation therapy have been the mainstay for brain tumor treatment. However, chemotherapy is becoming more important for patients who are not eligible for radiotherapy owing to age. Stem cell transplant as a means of high dose chemotherapy and stem cell rescue is a new treatment modality and is often repeated for improved survival. Drugs such as temozolomide are new chemotherapeutic options. In order to achieve 100% cure in children with pediatric cancer, every possible treatment modality and effort should be considered.</p>
<contrib contrib-type="author"><name><surname>Ju</surname><given-names>Hee Young</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Hong</surname><given-names>Che Ry</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Shin</surname><given-names>Hee Young</given-names></name><degrees>MD</degrees><degrees>PhD</degrees><xref ref-type="aff" rid="A1"/></contrib>
Korean Journal of Pediatrics
<sec sec-type="intro"><title>Introduction</title><p>Since the 1960s, there have been incremental improvements in treatment options for pediatric cancers. These improvements were fundamental for the advancement of the field of medical oncology, and led to the development of the following important concepts for cancer treatment; combination chemotherapy, multimodal treatment, risk-based treatment, and neo-adjuvant chemotherapy. Based on these treatment concepts, the 5-year survival rate of pediatric cancer has increased to up to 85% (<xref ref-type="table" rid="T1">Table 1</xref>)<xref rid="B1" ref-type="bibr">1)</xref>. This advancement was mediated by improvements in prophylaxis and treatment of infection and bleeding, which had been the main cause of death in pediatric cancer patients in the early 1970s. Nevertheless, some patients remain refractory to treatment, and successful treatment after relapse is still difficult to achieve. This review focuses on the recent advancements to overcome refractory or relapsed disease, as well as current advances in supportive care.</p></sec><sec><title>Acute lymphoblastic leuke</title><p>The current 5-year survival rate of childhood acute lymphoblastic leukemia (ALL) exceeds 85% in developed countries. This improvement has been achieved by the optimal use of antileukemic agents, dose modification, improved drug combinations, optimized administration schedules, and better supportive care. As most patients with newly diagnosed ALL are cured, current studies are focused on curing relapsed or refractory patients, as well as the reduction of long-term treatment-related morbidity for survivors.</p><p>New agents are gradually applied in relapsed pediatric ALL patients (<xref ref-type="table" rid="T2">Table 2</xref>). Clofarabine is a drug approved by the United States Food and Drug Administration for pediatric patients prior to adult patients. It has shown promising results for relapsed pediatric patients when used as a single agent or as combination therapy along with standard chemotherapy agents as cyclophosphamide or etoposide<xref rid="B2" ref-type="bibr">2)</xref>. Clofarabine is currently being investigated in a clinical trial by the Children's Oncology Group (COG) in combination with cyclophosphamide and etoposide as front-line therapy for very high-risk pediatric ALL patients (clinical trial number NCT00408005). A multicenter clinical trial examining clofarabine in relapsed ALL children was initiated this year in Korea with cooperation of the Health Insurance Review &#x00026; Assessment Service and the Korean Society of Pediatric Hematology Oncology. If this clinical trial achieves greater than a 30% 3-year survival rate, then clofarabine will be covered by medical insurance. This is the first risk-sharing model for new drugs in Korea. Nelarabine is a particularly active nucleoside analogue for T-cell disease. A phase I study of nelarabine as a single agent for pediatric refractory malignancies demonstrated an overall response rate of 42%<xref rid="B3" ref-type="bibr">3)</xref>. Nelarabine is currently under investigation in a COG phase III trial for newly diagnosed T-cell ALL and lymphoblastic lymphoma (NCT00408005).</p><p>Tyrosine kinases are constitutively activated in a subgroup of leukemia and are therefore attractive targets for therapeutic intervention. The tyrosine kinase inhibitor imatinib has been shown to greatly improve survival when added to chemotherapy for children with Philadelphia chromosome-positive ALL, resulting in a 3-year event-free survival (EFS) of 80%<xref rid="B4" ref-type="bibr">4)</xref>. Following this promising result, hematopoietic stem cell transplantation (HSCT) was not performed for first complete remission (CR) in Philadelphia chromosome-positive ALL when minimal residual disease (MRD) was negative in a study performed at St. Jude hospital<xref rid="B5" ref-type="bibr">5)</xref>. Dasatinib, a second generation tyrosine kinase inhibitor is 325 times more potent than imatinib for inhibiting the BCR-ABL<xref rid="B5" ref-type="bibr">5)</xref>, and is able to cross the blood-brain barrier<xref rid="B6" ref-type="bibr">6)</xref>. Continuous use of dasatinib in combination with standard chemotherapy is currently being investigated in a clinical trial for children newly diagnosed with Philadelphia-chromosome positive ALL (NCT01460160).</p><p>Another actively studied field in pediatric cancer is cellular therapy. Multiple studies of cellular therapy for pediatric cancers are currently being performed with promising results<xref rid="B6" ref-type="bibr">6)</xref>. Chimeric antigen receptor (CAR)-modified T cells with specificity for CD19 have shown promising results in the treatment of pediatric ALL<xref rid="B6" ref-type="bibr">6)</xref>. Natural killer cells are lymphocytes of the innate immune system that can kill target cancer cells by ligand-mediated interactions with various killer inhibitor receptors (KIRs). Practical application of cellular therapy will greatly expand treatment options for pediatric ALL<xref rid="B7" ref-type="bibr">7)</xref>.</p></sec><sec><title>Acute myeloid leukemia</title><p>Acute myeloid leukemia (AML) accounts for 15%-20% of pediatric leukemia, and the overall survival (OS) of pediatric AML is approximately 60%-70%. The treatment outcomes of pediatric AML have improved with effective use of chemotherapeutics, improvement of supportive care, and risk-group stratification. However, this progress has plateaued, with survival rates substantially lower than those for ALL, where OS exceeds 80%. Current steps to further improve AML treatment include monitoring of MRD, and tailoring treatment with novel therapeutics.</p><p>In ALL, measurement of MRD by leukemia-specific polymerase chain reaction (PCR)-based quantitative techniques has emerged as a very powerful prognostic factor, and risk-directed therapy has resulted in the improvement of ALL EFS approaching 90%. The heterogeneity of AML has made MRD application difficult, but the use of quantitative PCR-technology has eliminated these difficulties. The AML02 multicenter clinical trial adopted MRD to establish the final risk assignment and to determine treatment for pediatric AML<xref rid="B8" ref-type="bibr">8)</xref>. According to this study, the use of chemotherapy and HSCT according to MRD-based risk grouping could improve outcomes in patients with childhood AML. Furthermore, the authors suggested that more sensitive flow-cytometric methods based on more than four-color analysis should improve the sensitivity of MRD testing and may improve the identification of patients who are likely to have poor early treatment response.</p><p>Novel therapeutic agents are emerging as new options for pediatric AML. Novel cytarabine derivatives (cladribine, fludarabine) have shown promising results. Unlike cytarabine, cladribine and fludarabine inhibit both DNA polymerase and ribonucleotide reductase, thereby depleting deoxynucleotide pools. Cladribine has shown encouraging results in combination with cytarabine<xref rid="B9" ref-type="bibr">9)</xref>, topotecan<xref rid="B10" ref-type="bibr">10)</xref>, and idarubicin<xref rid="B11" ref-type="bibr">11)</xref>. In particular, acute monoblastic leukemia (FAB M5) was shown to be most sensitive to cladribine. Fludarabine in combination with cytarabine with or without anthracycline is widely used as a reinduction regimen for children with relapsed AML<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B13" ref-type="bibr">13)</xref>. Clofarabine is a structural hybrid of cladribine and fludarabine, designed to have better efficacy and improved stability. For childhood AML, an ongoing randomized phase II study is comparing end-of-induction MRD for children treated with clofarabine and cytarabine versus conventional ADE therapy (cytarabine, daunorubicin, etoposide) (NCT 00703820).</p><p>Gemtuzumab ozogamicin (GO), a humanized anti-CD33 monoclonal antibody conjugated to the cytotoxic compound N-acetyl-gamma-calicheamicin dimethyl hydrazide, has shown improved response when added to cytarabine in relapsed/refractory AML<xref rid="B14" ref-type="bibr">14)</xref>. The AML02 clinical trial showed tolerability and efficacy of GO when incorporated in the induction chemotherapy treatment protocol.</p><p>FMS-like tyrosine kinase 3 (FLT3) inhibitors are emerging as a new targeted therapeutics in pediatric AML. The first generation FLT3 inhibitors such as CEP-701 (lestaurtinib) and PKC412 (midostaurin) are nonselective compounds with potent anti-FLT3 activity. Second generation FLT3 inhibitors such as AC220 (quizartinib), designed with increased potency and selectivity for FLT3 are currently being evaluated in early-phase clinical trials.</p><p>A key issue for pediatric cancer patients has been reducing exposure to cardiotoxic drugs. Liposomal daunorubicin causes less cardiotoxicity than the nonliposomal daunorubicin formulation by preferential release of daunorubicin in tumor cells<xref rid="B15" ref-type="bibr">15)</xref>. The liposomal daunorubicin option can decrease late events related to chemotherapy. Cardioprotective drugs such as cardioxane (dexrazoxane) help to greatly reduce the cardiac toxicity from anthracycline in long-term survivors<xref rid="B16" ref-type="bibr">16)</xref>. Reducing the mortality and morbidity of chemotherapy-associated complications such as cardiac toxicity is becoming increasingly important when treating childhood malignancies.</p><p>Allogeneic stem cell transplantation (SCT) is no longer considered the first choice of treatment for first CR in low risk AML. In patients with low-risk or intermediate-risk AML without a matched-related donor, treatment-related mortality and morbidity may exceed the benefit. However, most oncologists consider allogeneic SCT in relapsed AML. More studies are needed to refine the indications of allogeneic SCT in pediatric AML.</p><p>For patients who undergo allogeneic SCT, prophylaxis of complications is important for improving the OS. One example is the prevention of veno-occlusive disease (VOD) with lipo-prostaglandin E1 (PGE1). Incidence of VOD ranges from 11% to 31% of pediatric SCT, but the mortality rate has been reported to be up to 50%<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20)</xref>. Because of the high mortality rate, the prevention of VOD is critical. The effectiveness of PGE1 has been reported for prevention of VOD<xref rid="B21" ref-type="bibr">21)</xref>, and lipo-PGE1 was suggested as a more potent drug with fewer side effects when compared to PGE1<xref rid="B22" ref-type="bibr">22)</xref>. Recent studies have shown the effectiveness of lipo-PGE1 for prevention of VOD in pediatric SCT<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24)</xref>. Future efforts should be focused on reducing the transplantation-related mortality.</p></sec><sec><title>Brain tumor</title><p>Medulloblastoma is the most common pediatric malignant brain tumor, accounting for 20% of all cases<xref rid="B25" ref-type="bibr">25)</xref>. Medulloblastoma can be classified into the following subtypes: classic, desmoplastic, large cell, anaplastic, medullomyoblastoma, and melanotic medulloblastoma. Recent data suggest that the desmoplastic subtype has improved prognosis in young infants<xref rid="B26" ref-type="bibr">26)</xref>, but the large cell and anaplastic types have poor prognosis<xref rid="B27" ref-type="bibr">27)</xref>. The mainstay treatment of medulloblastoma/primitive neuroectodermal tumor (PNET) is surgical resection; craniospinal radiotherapy is also required for improved outcome. Recent medulloblastoma trials focus on reduced-dose craniospinal radiation and adjuvant chemotherapy for average-risk patients. Good survival rates were reported using sequential high-dose chemotherapy with autologous stem cell rescue following craniospinal radiation<xref rid="B28" ref-type="bibr">28)</xref>. The survival of children under 3 years of age with PNET remains poor. For children younger than 3 years of age who cannot receive radiotherapy because of possible cognitive dysfunction, high-dose chemotherapy with autologous stem cell rescue may be the optimal treatment. Recent genomic studies have identified distinct subtypes of medulloblastoma, which are defined by sonic hedgehog (SHH), Wnt, or other pathway activation. SHH antagonists and targeted agents for ErbB family receptors (erlotinib, lapatinib) are being explored. Development of targeted therapy is expected to expand treatment options for medulloblastoma.</p><p>High-grade glioma (HGG), including anaplastic astrocytoma and glioblastoma multiforme, together with diffuse intrinsic pontine glioma (DIPG), constitute approximately 25% of pediatric brain tumors. HGGs and DIPGs have poor survival outcomes with fewer than 10% of children with GBM or DIPG surviving 2 years after diagnosis<xref rid="B29" ref-type="bibr">29)</xref>. Treatment of HGG consists of surgery followed by radiation therapy combined with chemotherapy. Studies examining higher doses and different schedules of radiation fractionation to brainstem gliomas have shown no improvement in responses; standard radiation is still recommended because clinical improvement and tumor response is observed in about 30% of patients. In adult glioblastoma multiforme, adjuvant temozolomide has provided significant improvements in survival. However, in the 2011 COG study, results with temozolomide for HGGs and DIPG were similar to those obtained in the CCG-945 phase III trial with lomustine and vincristine following radiotherapy<xref rid="B30" ref-type="bibr">30)</xref>. A phase I trial with lomustine and temozolomide showed improved EFS, but no difference in OS<xref rid="B31" ref-type="bibr">31)</xref>. Multicenter prospective trials are needed to further improve the survival rates of HGG patients.</p></sec><sec><title>Advancements in supportive care</title><p>Current guidelines for the treatment of infection include empirical antibiotic therapy to cover the most likely pathogens such as gram-negative bacilli including Pseudomonas aeruginosa and viridans group streptococci in high-risk febrile neutropenia patients<xref rid="B32" ref-type="bibr">32)</xref>. Monotherapy with an antipseudomonal beta-lactam or a carbapenem is recommended as the initial treatment, and prompt administration of antibiotics has been shown to improve outcome of febrile neutropenia<xref rid="B33" ref-type="bibr">33)</xref>. Antifungal prophylaxis in cancer patients has been shown to lower the rate of invasive fungal infections. Patients at high risk of invasive fungal infection include those who need transplantation from an unrelated donor or a partially matched related donor, for the treatment of malignancy, bone marrow failure syndrome, or congenital immunodeficiency<xref rid="B34" ref-type="bibr">34</xref>,<xref rid="B35" ref-type="bibr">35</xref>,<xref rid="B36" ref-type="bibr">36)</xref>. Children with AML have a high incidence of invasive fungal infection, both in newly diagnosed and relapsed patients<xref rid="B37" ref-type="bibr">37</xref>,<xref rid="B38" ref-type="bibr">38)</xref>. In contrast, ALL patients are considered to be at high risk for invasive fungal infection only when they relapse<xref rid="B39" ref-type="bibr">39)</xref>. For these high-risk patients, agents used for antifungal prophylaxis include fluconazole. Fluconazole prevents candida infection but lacks activity against Aspergillus spp, which is the second-most common cause of invasive fungal infection in pediatric cancer patients. Thus, echinocandins such as caspofungin could be utilized to cover both molds and candida; however, further study is needed to evaluate the efficacy in children. The usage of colony-stimulating factors contributes to the control of infection in cancer patients. Routine use of granulocyte colony-stimulation factors decreased the incidence of febrile neutropenia and duration of hospitalization and may decrease delays in subsequent chemotherapy, but it does not lead to improvement in OS<xref rid="B40" ref-type="bibr">40</xref>,<xref rid="B41" ref-type="bibr">41</xref>,<xref rid="B42" ref-type="bibr">42</xref>,<xref rid="B43" ref-type="bibr">43)</xref>.</p><p>Prophylactic platelet transfusions have contributed to lowering the risk of bleeding in pediatric cancer patients. Prophylactic platelet transfusions are the standard of care for adult patients with hypoplastic thrombocytopenia after receiving chemotherapy or radiation<xref rid="B44" ref-type="bibr">44)</xref>. A randomized controlled study of pediatric cancer patients showed that patients who received prophylactic platelet transfusions had shorter number of days with bleeding<xref rid="B45" ref-type="bibr">45)</xref>, and another study revealed that prophylactic platelet transfusion resulted in fewer bleeding episodes<xref rid="B46" ref-type="bibr">46)</xref>. Another study showed that platelet counts of &#x0003c;10<sup>5</sup>/&#x000b5;L was independently associated with an increased probability of traumatic bloody lumbar puncture<xref rid="B47" ref-type="bibr">47)</xref>, which was associated with increased risk of relapse<xref rid="B48" ref-type="bibr">48)</xref>. Platelet transfusion had been in the form of platelet concentrate before the use of platelet pheresis; small children could not receive more than 2 units of platelet concentrate owing to the problem of volume overloading and bleeding could not be sufficiently controlled. Since the development of pheresis machine, small children can now receive more than 6 units of platelet at once without the risk of volume overloading so that bleeding can be controlled. Patients 0-5 years of age have been shown to have higher clinically significant bleeding incidence at PLADO (prophylactic platelet dose trial) analysis when compared to older patients<xref rid="B49" ref-type="bibr">49)</xref>; however, there was insufficient power to determine differences of bleeding outcomes. Future randomized controlled trials should be designed to compare platelet transfusion regimens.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>Survival rates of childhood cancers have continued to improve over the past decade, particularly for leukemia patients. Future efforts at improving survival in pediatric cancer patients should focus on relapsed or refractory disease. In particular, the application of targeted agents or cell therapies should be the subject of future studies of relapsed leukemia. Some pediatric and adolescent solid tumors have shown no definite advancement in survival over the past 10 to 20 years; these include Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Similarly, survival improvements have been modest for pediatric brain tumors over the past 10 to 15 years for most age groups1). There is a need for increased focus on developing novel therapies that are based on understanding the cellular pathways promoting tumor growth and survival.</p></sec>
Serum interleukin-1beta and tumor necrosis factor-alpha in febrile seizures: is there a link?
<sec><title>Purpose</title><p>Febrile seizures are induced by fever and are the most common type of seizures in children. Although numerous studies have been performed on febrile seizures, their pathophysiology remains unclear. Recent studies have shown that cytokines may play a role in the pathogenesis of febrile seizures. The present study was conducted to identify potential links between serum interleukin-1beta (IL-1&#x003b2;), tumor necrosis factor-alpha (TNF-&#x003b1;), and febrile seizures.</p></sec><sec><title>Methods</title><p>Ninety-two patients with simple or complex febrile seizures (46 patients per seizure type), and 46 controls with comparable age, sex, and severity of temperature were enrolled.</p></sec><sec><title>Results</title><p>The median concentrations of serum IL-1&#x003b2; in the simple, complex febrile seizure, and control groups were 0.05, 0.1, and 0.67 pg/mL, respectively (<italic>P</italic>=0.001). Moreover, the median concentrations of TNF-&#x003b1; in the simple, complex febrile seizure, and control groups were 2.5, 1, and 61.5 pg/mL, respectively (<italic>P</italic>=0.001). Furthermore, there were significant differences between the case groups in serum IL-1&#x003b2; and TNF-&#x003b1; levels (<italic>P</italic>&#x0003c;0.05).</p></sec><sec><title>Conclusion</title><p>Unlike previous studies, our study does not support the hypothesis that increased IL-1&#x003b2; and TNF-&#x003b1; production is involved in the pathogenesis of febrile seizures.</p></sec>
<contrib contrib-type="author" corresp="yes"><name><surname>Mahyar</surname><given-names>Abolfazl</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Ayazi</surname><given-names>Parviz</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Orangpour</surname><given-names>Reza</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Daneshi-Kohan</surname><given-names>Mohammad Mahdi</given-names></name><degrees>LD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Sarokhani</surname><given-names>Mohammad Reza</given-names></name><degrees>LD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Javadi</surname><given-names>Amir</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Habibi</surname><given-names>Morteza</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Talebi-Bakhshayesh</surname><given-names>Mousa</given-names></name><degrees>MSc</degrees><xref ref-type="aff" rid="A1"/></contrib>
Korean Journal of Pediatrics
<sec sec-type="intro"><title>Introduction</title><p>Febrile seizures are seizures induced by fever (temperature&#x02265;38&#x02103;). Febrile seizures usually occur in young children between the age of 6 months and 5 years<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2)</xref>. These patients do not have any central nervous infection, electrolyte imbalance, metabolic disorders, or a history of febrile seizures<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4)</xref>. Febrile seizures are classified into three types: simple, complex, and febrile status epilepticus. Simple febrile seizures are generalized, last for &#x0003c;15 minutes, and do not recur within 24 hours. Complex febrile seizures are more prolonged (&#x0003e;15 minutes), focal, and recur within 24 hours. Febrile status epilepticus seizures last for &#x0003e;30 minutes<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8)</xref>. This type of febrile seizure occurs in 2%-9% of children<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10)</xref>. While several cohort studies suggest that prognosis of febrile seizures is often good, epilepsy is observed in 5.4% of these patients<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12)</xref>. Although numerous studies have been performed on febrile seizures, their exact pathophysiology remains unknown<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14)</xref>. Cytokines may be one of the factors involved in the pathogenesis of febrile seizures<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18)</xref>. Cytokines are hormonal mediators involved in several infectious and immunological phenomena. Interleukin-1beta (IL-1&#x003b2;) and tumor necrosis factor-alpha (TNF-&#x003b1;) are the major cytokines<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16)</xref>. Tutuncuoglu et al.<xref rid="B17" ref-type="bibr">17)</xref> reported that increased production of IL-1&#x003b2; is involved in the pathogenesis of febrile seizures. In contrast, Tomoum et al.<xref rid="B18" ref-type="bibr">18)</xref> suggested that IL-1&#x003b2; do not play any roles in the pathogenesis of febrile seizures. Because of these controversial opinions, the present study was conducted to investigate the relationship between serum IL-1&#x003b2; and TNF-&#x003b1; with febrile seizures in children.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><p>This case-control study was performed at Qazvin Children Hospital affiliated to the Qazvin University of Medical Sciences in Qazvin, Iran in 2012. Case groups (92 patients) were consecutively selected from children admitted to the hospital after simple and complex febrile seizures. Control group comprised 46 febrile children without seizures. The ages of all patients were between 6 and 60 months. Sample size was calculated to determine 95% confidence coefficient and 80% power for statistical analysis<xref rid="B16" ref-type="bibr">16)</xref>. Consecutive sampling continued until the desired sample size was reached. Inclusion criteria for the febrile seizure groups were: (1) fever&#x02265;38&#x02103;, (2) presence of simple febrile seizure (generalized seizure lasting for &#x0003c;15 minutes), and (3) presence of complex febrile seizure (focal seizure lasting for &#x0003e;15 minutes that recurred within 24 hours)<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3)</xref>. Patients with central nervous system infections, electrolyte imbalance, metabolic disorders, neurological deficits, and a history of febrile seizures were excluded. The control group included febrile children without seizures who visited the hospital clinic because of common febrile diseases such as upper respiratory tract infection. Febrile diseases (viral or bacterial) were diagnosed according to clinical findings. Body temperature (axillary) was measured using a standard method<xref rid="B3" ref-type="bibr">3)</xref>. The ethics committee of the Research Department in the Qazvin University of Medical Sciences (project no. 284) approved the study. All parents were provided information regarding the research method in simple language. The children were included in the study after their parents agreed and signed the informed consent form. Four milliliters of blood was taken from the peripheral vessels of children in all the groups, and serum was obtained by centrifugation at 3,000 rpm for 5 minutes at 4&#x02103;. The serum was then poured into acid-washed tubes and stored in a refrigerator at -20&#x02103; until IL-1&#x003b2; and TNF-&#x003b1; assay. In the febrile seizure groups, blood samples were collected within 5 hours after the occurrence of a seizure episode<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B19" ref-type="bibr">19)</xref>. Concentrations of serum IL-1&#x003b2; and TNF-&#x003b1; were measured using enzyme-linked immunosorbent assay and commercially available kits (Cat. No. BE51011 and Cat.No.BE58351; IBL Co., Hamburg, Germany). All the samples were measured in duplicates to improve accuracy. Results were analyzed by chi-square test, analysis of variance, and nonparametric tests (Mann-Whitney <italic>U</italic> test and Kruskal-Wallis test). Cytokine levels were expressed as median and range, whereas variable values except age were expressed as mean&#x000b1; standard deviation. Statistical calculations were performed using SPSS ver. 15 (SPSS Inc., Chicago, IL, USA). <italic>P</italic>&#x0003c;0.05 was considered statistically significant.</p></sec><sec sec-type="results"><title>Results</title><p>The simple febrile seizures group included 25 male and 21 female patients. These values in complex febrile seizures and control groups were 26, 20 and 26, 20 patients, respectively (<italic>P</italic>= 0.971). The minimum and maximum ages of the children in the cases and control groups were 6 and 60 months, respectively. There were no statistically significant differences between the groups with respect to age, severity of temperature, duration of fever and type of febrile disease (viral or bacterial) (<italic>P</italic>&#x0003e;0.05) (<xref ref-type="table" rid="T1">Table 1</xref>). The median concentrations of serum IL-1&#x003b2; in simple, complex febrile seizures and control groups were 0.05, 0.1, and 0.67 pg/mL, respectively (<italic>P</italic>&#x0003c;0.0001). Also, the median concentrations of TNF-&#x003b1; in simple, complex febrile seizures and control groups were 2.5, 1, and 61.5 pg/mL, respectively (<italic>P</italic>&#x0003c;0.0001). Serum IL-1&#x003b2; and TNF-&#x003b1; concentrations were significantly different between the febrile seizures and control groups (<italic>P</italic>&#x0003c;0.05) (<xref ref-type="table" rid="T2">Table 2</xref>). The simple and complex febrile seizures groups also showed significant differences regarding serum IL-1&#x003b2; and TNF-&#x003b1; levels (<italic>P</italic>&#x0003c;0.05) (<xref ref-type="table" rid="T3">Tables 3</xref>, <xref ref-type="table" rid="T4">4</xref>; <xref ref-type="fig" rid="F1">Figs 1</xref>, <xref ref-type="fig" rid="F2">2</xref>). No significant differences were observed between the cases and control groups with respect to the correlation between degree of fever and serum IL-1&#x003b2; (r=0.081, <italic>P</italic>=0.442) and TNF-&#x003b1; levels (r=-0.059, <italic>P</italic>=0.574).</p></sec><sec sec-type="discussion"><title>Discussion</title><p>Although numerous studies have been performed on the identification of factors causing febrile seizures in children, the actual cause of the disease has not yet been detected<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20)</xref>. Few studies highlight the role of cytokines in febrile seizures<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>,<xref rid="B21" ref-type="bibr">21</xref>,<xref rid="B22" ref-type="bibr">22</xref>,<xref rid="B23" ref-type="bibr">23)</xref>. Cytokines are essentially protein or glycoprotein hormones, mostly between 8 and 30 kDa that can be produced in all tissues and by most cells. Cytokines are hormonal mediators produced in body in response to defensive and growth phenomena. The role of these mediators in infectious, immunological, and inflammatory phenomena is of special interest. Cytokines include ILs, chemokines, TNFs, interferons, etc. Of these, IL-1&#x003b2; and TNF-&#x003b1; are the important cytokines<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B21" ref-type="bibr">21)</xref>. One of the important role of IL-1&#x003b2; and TNF-&#x003b1; is direct and indirect modulating effects on neurons and neurotoxic neurotransmitters released during excitation or inflammation<xref rid="B18" ref-type="bibr">18)</xref>. So, this question was raised to us; what is the role of these cytokines in febrile seizures? Previous studies have shown contradictory results regarding the role of these two cytokines in the development of febrile seizures<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20)</xref>. Tutuncuoglu et al.<xref rid="B17" ref-type="bibr">17)</xref> reported that plasma IL-1&#x003b2; and cerebrospinal fluid TNF-&#x003b1; level in febrile seizure patients during the acute phase of the disease were significantly higher than those in controls were. However, plasma TNF-&#x003b1; levels and cerebrospinal fluid (CSF) IL-1&#x003b2; levels were not significantly different between the case and control groups. This study concluded that IL-1&#x003b2; is the likely factor influencing the pathogenesis of febrile seizures. Another study showed that induction of leukocytes by double-stranded RNA resulted in a large-scale production of IL-1&#x003b2; in febrile seizure patients as compared to that in controls; however, the levels of TNF-&#x003b1; did not change significantly between the two groups. This study concluded that IL-1&#x003b2; was a likely factor influencing the pathogenesis of febrile seizures<xref rid="B22" ref-type="bibr">22)</xref>. Helminen et al.<xref rid="B24" ref-type="bibr">24)</xref> showed that stimulation of peripheral blood mononuclear cells by liposaccharide in children with febrile seizures led to an increased production of IL-1&#x003b2; in these children as compared to that in control. Another study showed a significant correlation between IL-1&#x003b2; allele 2 and febrile seizures<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B25" ref-type="bibr">25)</xref>. Kanemoto et al.<xref rid="B25" ref-type="bibr">25)</xref> suggested that IL-1&#x003b2;-511T allele is a determining factor in the development of febrile seizures. A study by Dube et al.<xref rid="B26" ref-type="bibr">26)</xref> on an animal model showed that IL-1&#x003b2; plays a role in the incidence of febrile seizures by increasing N-methyl-D-aspartate function. Studies supporting the hypothesis that cytokines play an important role in the incidence of febrile seizures indicate that during infections, especially viral infections, immune cells such as macrophages, T cells, and B cells are stimulated and consequently secrete proinflammatory cytokines such as IL-1&#x003b2;, TNF-&#x003b1;, and IL-6<xref rid="B27" ref-type="bibr">27</xref>,<xref rid="B28" ref-type="bibr">28</xref>,<xref rid="B29" ref-type="bibr">29)</xref>. In addition to studies that support the role of IL-1&#x003b2; in the incidence of febrile seizures, there are studies that do not support the above hypothesis and suggest that no correlation exists between febrile seizures and IL-1&#x003b2; and TNF-&#x003b1; level. Lahat et al.<xref rid="B20" ref-type="bibr">20)</xref> reported that there was no significant difference in plasma IL-1&#x003b2; and cerebrospinal fluid TNF-&#x003b1; level between the febrile seizures children and control group. Haspolat et al.<xref rid="B16" ref-type="bibr">16)</xref> showed that cerebrospinal fluid IL-1&#x003b2; levels increased significantly in patients with febrile seizures as compared to those in controls; however, serum IL-1&#x003b2; and TNF-&#x003b1; level in the two groups were not significantly different. These results have also been confirmed by Virta et al.<xref rid="B15" ref-type="bibr">15)</xref> and Tomoum et al.<xref rid="B18" ref-type="bibr">18)</xref>. Another study also showed no significant difference between children with febrile seizures and those with meningitis and encephalitis with respect to plasma concentration of IL-1&#x003b2;<xref rid="B30" ref-type="bibr">30)</xref>. In the present study, serum IL-1&#x003b2; and TNF-&#x003b1; level in the simple and complex febrile seizure groups were lower than those in the control group. Moreover, no significant correlation was observed between the intensity of body temperature and the levels of IL-1&#x003b2; and TNF-&#x003b1; in the case and control groups. Lower serum levels of these cytokines in the febrile seizure groups than those in the control group indicate that production of cytokines does not increase during febrile seizures. Therefore, unlike previous studies the hypothesis that increased production of cytokines influences the incidence of febrile seizures is not convincing. Contradictory results of various studies may be due to the interference of confounding variables such as, time of sampling, severity of temperature, duration of fever, difficulty in measuring cytokines, type of infection, and sample size. Roth et al.<xref rid="B19" ref-type="bibr">19)</xref> showed that in guinea pigs, serum TNF-&#x003b1; level reached its maximum within 1 hour after injecting a bacterial endotoxin and decreased by 15%-20% after 2 hours to an extent that it was no longer measurable. The author of the above study recommended that TNF-&#x003b1; levels should be measured within 5 hours after the incidence of febrile seizure. Time of sampling is very important for measuring IL-1&#x003b2; levels. IL-1&#x003b2; increases within 1 hour after seizure, reaches its maximum level within 4-12 hours after seizure, and returns to its normal level after 24 hours<xref rid="B16" ref-type="bibr">16)</xref>. Thus, IL-1&#x003b2; is best measured within 12 hours after the incidence of seizure. In the present study, samples were prepared within 5 hours after the incidence of seizure, a period in which actual levels of the studied cytokines can be measured. Therefore, in this study, low levels of IL-1&#x003b2; and TNF-&#x003b1; in patient with febrile seizures did not correlate with the time of sampling. Another factor that may influences cytokines level is fever. Tournieretal reported that fever increases some interleukins and decreases others<xref rid="B31" ref-type="bibr">31)</xref>. Because, in the present study, there was no significant differences between 3 groups regarding severity of temperature (<italic>P</italic>=0.45) and duration of fever (<italic>P</italic>=0.916), thus fever cannot be a confounding variable. Another problem is measuring IL-1&#x003b2; level. Dinarello<xref rid="B32" ref-type="bibr">32)</xref> reported that measurement of IL-1&#x003b2; is very difficult because IL-1&#x003b2; binds to large proteins such as &#x003b1;2-macroglobulin and complements. In our study, method of measuring of interleukins was similar in all groups. It has been shown that proinflammatory cytokines increase after viral and bacterial infections<xref rid="B21" ref-type="bibr">21)</xref>. In the present study, types of diseases (viral and bacterial) were not significantly different between the case and control groups. Most of the studied children had three types of infections: upper respiratory tract infections, viral diarrhea, and viral pneumonia. Therefore, the type of infection in the groups could not interfere with the results of this study. Sample size is another confounding factor that must be considered for evaluating the results of various studies. We calculated sample size regularly according to biostatics formula. In addition, one more explanation for low level of IL-1&#x003b2; and TNF-&#x003b1; is suppuration effect of IL-10. There are some reports that IL-10 is increased in response to lipopolysaccharide in patients with febrile convulsion<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B33" ref-type="bibr">33)</xref>. Since, the etiology of disease in present study comparing the previous one is completely different, so it is not applicable in our study. Despite above discussion, still this question remains unanswered for us, whether low levels of these cytokines are related to the effect of seizure attack during infection? Responding to this question is very difficult. According to my knowledge and literature review there is no document to have satisfactory answer for this question. Donnelly et al.<xref rid="B34" ref-type="bibr">34)</xref> reported that IL-1&#x003b2; has an inhibitory effect on cell excitability and a neuroprotective role for seizure-induced hippocampal. Our study has one limitation, in that CSF IL-1&#x003b2; and TNF-&#x003b1; levels were not measured in patients with febrile seizures. The studied patients did not show any indications for undergoing lumbar puncture procedure, which is morally objectionable. As a result, unlike previous studies, our study does not support the hypothesis that increased IL-1&#x003b2; and TNF-&#x003b1; production is involved in the pathogenesis of febrile seizures.</p></sec>
The large-conductance calcium-activated potassium channel holds the key to the conundrum of familial hypokalemic periodic paralysis
<sec><title>Purpose</title><p>Familial hypokalemic periodic paralysis (HOKPP) is an autosomal dominant channelopathy characterized by episodic attacks of muscle weakness and hypokalemia. Mutations in the calcium channel gene, <italic>CACNA1S</italic>, or the sodium channel gene, <italic>SCN4A</italic>, have been found to be responsible for HOKPP; however, the mechanism that causes hypokalemia remains to be determined. The aim of this study was to improve the understanding of this mechanism by investigating the expression of calcium-activated potassium (K<sub>Ca</sub>) channel genes in HOKPP patients.</p></sec><sec><title>Methods</title><p>We measured the intracellular calcium concentration with fura-2-acetoxymethyl ester in skeletal muscle cells of HOKPP patients and healthy individuals. We examined the mRNA and protein expression of KCa channel genes (<italic>KCNMA1</italic>, <italic>KCNN1</italic>, <italic>KCNN2</italic>, <italic>KCNN3</italic>, and <italic>KCNN4</italic>) in both cell types.</p></sec><sec><title>Results</title><p>Patient cells exhibited higher cytosolic calcium levels than normal cells. Quantitative reverse transcription polymerase chain reaction analysis showed that the mRNA levels of the K<sub>Ca</sub> channel genes did not significantly differ between patient and normal cells. However, western blot analysis showed that protein levels of the <italic>KCNMA1</italic> gene, which encodes K<sub>Ca</sub>1.1 channels (also called big potassium channels), were significantly lower in the membrane fraction and higher in the cytosolic fraction of patient cells than normal cells. When patient cells were exposed to 50 mM potassium buffer, which was used to induce depolarization, the altered subcellular distribution of BK channels remained unchanged.</p></sec><sec><title>Conclusion</title><p>These findings suggest a novel mechanism for the development of hypokalemia and paralysis in HOKPP and demonstrate a connection between disease-associated mutations in calcium/sodium channels and pathogenic changes in nonmutant potassium channels.</p></sec>
<contrib contrib-type="author" corresp="yes"><name><surname>Kim</surname><given-names>June-Bum</given-names></name><degrees>MD</degrees><degrees>PhD</degrees><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Sung-Jo</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kang</surname><given-names>Sun-Yang</given-names></name><degrees>MS</degrees><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Yi</surname><given-names>Jin Woong</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Seung-Min</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="A4">4</xref></contrib>
Korean Journal of Pediatrics
<sec sec-type="intro"><title>Introduction</title><p>Familial hypokalemic periodic paralysis (HOKPP, OMIM no. 170400) is an autosomal dominant disorder that features reversible attacks of flaccid paralysis with concomitant hypokalemia<xref rid="B1" ref-type="bibr">1)</xref>. The first attack can occur any time between infancy and puberty, with the majority occurring around puberty. Paralytic attacks usually occur at night or early in the morning and last for hours and sometimes days. They can be induced by carbohydrate- or sodium-rich meals, strenuous exercise, emotional stress, and exposure to cold<xref rid="B2" ref-type="bibr">2)</xref>.</p><p>The majority of HOKPP cases are caused by mutations in the skeletal muscle voltage-gated calcium channel gene, <italic>CACNA1S</italic>, or in the sodium channel gene, <italic>SCN4A</italic><xref rid="B1" ref-type="bibr">1)</xref>. These mutations have been shown to contribute to an inward cation leakage current (referred to as the gating-pore current), which makes the muscle fibers of patients susceptible to abnormal depolarization in response to low extracellular potassium concentration<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4)</xref>. Alterations in the expression, subcellular localization, and/or kinetics of nonmutated potassium channels, which reduce outward potassium currents, have been implicated in the development of hypokalemia as well as pathological depolarization<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8)</xref>. However, the reason why potassium channels are affected by mutations in the <italic>CACNA1S</italic> or <italic>SCN4A</italic> gene is not yet clear.</p><p>A previous study demonstrated that skeletal muscle fibers from HOKPP patients exhibited increased intracellular calcium levels compared with normal cells<xref rid="B6" ref-type="bibr">6)</xref>. An increase in cytoplasmic calcium levels via its entry and/or release from intracellular stores triggers a variety of physiological processes, such as muscle contraction, neuronal excitability, vasoregulation, hormone secretion, and immune responses<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12)</xref>. The elevated intracellular calcium ions, however, also stimulate the activity of calcium-activated potassium (KCa) channels, of which the large-conductance KCa channels (also termed big potassium [BK] channels) primarily serve as negative feedback regulators that repolarize the cell by increasing potassium efflux. This can lead to membrane hyperpolarization and a consequent decrease in cell excitability<xref rid="B13" ref-type="bibr">13)</xref>.</p><p>We conducted this study to test the hypothesis that the expression patterns of the K<sub>Ca</sub> channel genes (<italic>KCNMA1</italic>, <italic>KCNN1</italic>, <italic>KCNN2</italic>, <italic>KCNN3</italic>, and <italic>KCNN4</italic>) in the skeletal muscle cells of HOKPP patients differ from those in normal cells and to find a mechanistic link between HOKPP mutant ion channels and pathogenic alterations to non-mutant potassium channels.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>1. Subjects</title><p>We reviewed the records of 185 patients who were being treated for HOKPP from March 2008 to April 2013 in the clinics of Seoul Children's Hospital and Konyang University Hospital. Each patient underwent genetic testing. All the patients fulfilled the diagnostic criteria for HOKPP, including (1) episodic attacks of muscle weakness with hypokalemia (&#x0003c;3.5 mEq/L); (2) positive family history or genetically confirmed skeletal calcium or sodium channel mutation; and (3) exclusion of secondary causes of hypokalemia, such as thyroid, adrenal, and renal disorders, and drug abuse<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B14" ref-type="bibr">14)</xref>. Mutation screening was performed by sequencing the entire coding region of the CACNA1S, SCN4A, and <italic>KCNJ2</italic> genes, as described elsewhere<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16)</xref>. The clinical details of some patients have been reported previously<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18)</xref>. Of these, we selected the three who presented with the most obvious symptoms of HOKPP in terms of both severity and frequency of paralytic attacks. These patients had the Arg1239Gly mutation in the <italic>CACNA1S</italic> gene. Three healthy volunteers acted as controls. All participants provided written informed consent, and the study was conducted in compliance with the guidelines of the Institutional Review Board of Konyang University Hospital.</p></sec><sec><title>2. Methods</title><sec><title>1) Skeletal muscle tissue sampling and preparation</title><p>Skeletal muscle biopsy samples were obtained as described previously7). The muscle biopsy samples were rinsed in phosphate buffered saline (PBS) with Ca<sup>2+</sup>-Mg<sup>2+</sup> and dissected into small pieces (diameter, 1-2 mm) with a sterilized scalpel. The tissue fragments were cultured in Dulbecco's modified Eagle's Medium (DMEM; Thermo Scientific, Marietta, OH, USA) supplemented with 5% fetal bovine serum (FBS; Thermo Scientific), 2&#x000b5;M glutamine (Sigma-Aldrich, St. Louis, MO, USA), 1% nonessential amino acids (Thermo Scientific), 0.1mM &#x003b2;-mercaptoethanol (Sigma-Aldrich), 5-ng/mL fibroblast growth factor-basic recombinant human protein (Invitrogen, Carlsbad, CA, USA), and 1% penicillin-streptomycin (Caisson, Logan, UT, USA) for 2 weeks at 37&#x02103; in an incubator containing 95% air and 5% CO<sub>2</sub> (Thermo Scientific). Isolation of skeletal muscle cells from the original culture was performed according to a method described previously<xref rid="B19" ref-type="bibr">19)</xref>.</p></sec><sec><title>2) Preparation of potassium buffers</title><p>Potassium buffer (4mM) at pH 7.35 (4mM KCl, 145mM NaCl, 1mM MgCl<sub>2</sub>, 0.5mM CaCl<sub>2</sub>, 5mM glucose, and 10mM 3-(N-morpholino) propanesulfonic acid [MOPS]) was prepared and used for exposing cells to a normal physiological concentration of extracellular potassium. To induce depolarization of skeletal muscle cells via a high concentration of extracellular potassium, 50 mM potassium buffer (50 mM KCl, 145 mM NaCl, 1 mM MgCl2, 0.5 mM CaCl<sub>2</sub>, 5 mM glucose, and 10 mM MOPS; pH 7.35) was prepared. The buffers were sterilized before use.</p></sec><sec><title>3) Culturing of skeletal muscle cells and treatment with potassium buffer solutions</title><p>Skeletal muscle cells obtained from HOKPP patients (patient cells) and healthy controls (normal cells) were cultured in DMEM containing 20% FBS (Thermo Scientific) and 1% penicillin-streptomycin at 37&#x02103; in an incubator containing 95% air and 5% CO<sub>2</sub>. To induce differentiation, skeletal muscle cells were incubated in DMEM supplemented with 2% horse serum (Thermo Scientific) and 1% penicillin-streptomycin for up to 5 days. The differentiation medium was replaced every 48 hours. Both normal and patient cells were collected for analysis during the 12th passage. The mRNA and protein levels of the KCa channel genes (<italic>KCNMA1</italic>, <italic>KCNN1</italic>, <italic>KCNN2</italic>, <italic>KCNN3</italic>, and <italic>KCNN4</italic>) were analyzed in both types of cells, prior to and at 1 hour after exposure to the 4 and 50 mM potassium buffers.</p></sec><sec><title>4) Measurement of cytosolic calcium levels</title><p>Cytosolic calcium levels were analyzed using fura-2-acetoxymethyl ester (Fura-2AM), a membrane-permeable, calcium-sensitive fluorescent dye (Sigma-Aldrich). Both normal and patient cells were loaded with 1&#x000b5;M Fura-2AM diluted in 4mM potassium buffer for 30 minutes in a CO<sub>2</sub> incubator. After washing with potassium buffer to remove residual dye, the cells were treated with trypsin-ethylenediaminetetraacetic acid (Caisson). They were then harvested, washed with Ca<sup>2+</sup>-free PBS, and analyzed by flow cytometry (Millipore, Billerica, MA, USA). All the experiment protocols were done in triplicate.</p></sec><sec><title>5) Quantitative reverse transcription polymerase chain reaction analysis</title><p>Total RNA from the skeletal muscle cells was extracted using TRIzol reagent (Invitrogen), and 100 ng was converted to cDNA by using SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's instructions. AccuPower PCR PreMix (Bioneer, Daejun, Korea) was added to the reaction mix, and quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using primers for KCNMA1, <italic>KCNN1</italic>, <italic>KCNN2</italic>, <italic>KCNN3</italic>, and <italic>KCNN4</italic>. Primers were designed with the VectorNTI10 (Invitrogen) and Primer3 software (<ext-link ext-link-type="uri" xlink:href="http://sourceforge.net/projects/primer3/">http://sourceforge.net/projects/primer3/</ext-link>). The sequences of these primers are available on request. The PCR conditions consisted of initial denaturation at 95&#x02103; for 5 minutes, followed by 30 cycles at 95&#x02103; for 30 seconds, 55&#x02103; for 30 seconds, and 72&#x02103; for 1 minute, with final extension at 72&#x02103; for 5 minutes. All the reactions were performed in triplicate. The mRNA expression levels for each gene were normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).</p></sec><sec><title>6) Western blot analysis</title><p>Prior to and after exposure to the two different concentrations of potassium buffers for 1 hour, the cytosolic and membranous protein fractions of normal and patient cells were separated and analyzed by western blotting. Detailed methods for subcellular fractionation and western blot analysis have been previously described8). To detect the channel proteins, primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for KCa1.1 (MaxiK&#x003b1; [B-1]), K<sub>Ca</sub>2.1 (SK1 [A-13]), K<sub>Ca</sub>2.2 (KCNN2 [U-24]), K<sub>Ca</sub>2.3 (SK3 [H-45]), K<sub>Ca</sub>3.1 (IK1 [D-5]), and &#x003b2;-actin (&#x003b2;-actin [C4]) were used in combination with antigoat, antimouse, or antirabbit secondary antibodies. The proteins were then quantified using the TINA 2.0 densitometric analytical system (Raytest Isotopenme&#x003b2;ger&#x000e4;te GmbH, Straubenhardt, Germany) according to the manufacturer's instructions. All the experiments were performed independently at least three times.</p></sec><sec><title>7) Statistical analysis</title><p>The IBM SPSS Statistics ver. 19.0 (IBM Co., Armonk, NY, USA) was used for statistical analysis. Data are presented as mean&#x000b1;standard deviation. Comparisons were made using a two-way analysis of variance test with <italic>P</italic>&#x0003c;0.05 indicating statistical significance.</p></sec></sec></sec><sec sec-type="results"><title>Results</title><sec><title>1. Cytosolic calcium levels in skeletal muscle cells</title><p>We examined cytosolic calcium levels in both patient and normal cells in 4mM potassium buffer using the calcium-sensitive dye Fura-2AM. The percentage of fluorescence was measured by flow cytometry. At least 5,000 cells were analyzed for each sample by using InCite software ver. 2.2.2 (Millipore), and the threshold was matched at SSC 291. Patient cells exhibited significantly higher levels of cytosolic calcium ions than normal cells (37.91%&#x000b1;5.33% vs. 27.32%&#x000b1;3.08%, <italic>P</italic>&#x0003c;0.05) (<xref ref-type="fig" rid="F1">Fig. 1</xref>).</p></sec><sec><title>2. KCa channel mRNA expression</title><p>We performed quantitative RT-PCR to compare the mRNA levels for the K<sub>Ca</sub> channel genes (<italic>KCNMA1</italic>, <italic>KCNN1</italic>, <italic>KCNN2</italic>, <italic>KCNN3</italic>, and <italic>KCNN4</italic>) between patient and normal (control) cells in both 4mM and 50mM potassium buffers. No significant differences were found at either potassium concentration (data not shown).</p></sec><sec><title>3. Western blot analysis to evaluate the expression patterns of KCa channel proteins</title><p>We compared the protein expression profiles of the KCa channels in patient versus normal cells, following exposure to 4 and 50mM potassium. Membranous and cytosolic fractions of the cells were separated, and the protein expression levels of the KCa channel genes were evaluated by western blot as mentioned above. No significant difference was observed for K<sub>Ca</sub>2.1, K<sub>Ca</sub>2.2, K<sub>Ca</sub>2.3, and K<sub>Ca</sub>3.1 (data not shown); however, the expression of K<sub>Ca</sub>1.1 (encoded by <italic>KCNMA1</italic>) differed significantly in patient cells: compared with control levels, expression was lower in the membrane fraction (<italic>P</italic>&#x0003c;0.05) and higher in the cytosolic fraction (<italic>P</italic>&#x0003c;0.05) for both 4 and 50 mM potassium buffers (<xref ref-type="fig" rid="F2">Figs. 2</xref>, <xref ref-type="fig" rid="F3">3</xref>).</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>Previous studies have reported alterations in the expression, subcellular localization, and/or kinetics of nonmutant potassium channels (but not BK channels) in HOKPP<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8)</xref>. However, the relationship between mutations in the <italic>CACNA1S</italic> or <italic>SCN4A</italic> gene and the pathological operation of nonmutant potassium channels has not yet been elucidated.</p><p>The mutant channels responsible for HOKPP have been shown to generate a nonselective inward cation leakage current through an aberrant gating pore that is open at the resting membrane potential<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4)</xref>. In agreement with the results of Puwanant and Ruff<xref rid="B6" ref-type="bibr">6)</xref>, we observed an increase in the cytoplasmic calcium levels of HOKPP patient muscle cells at a normal physiological concentration of extracellular potassium. Although the precise mechanism responsible for this increase is not known, from the findings of the above studies<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4)</xref>, we can reasonably infer that it occurs via inward calcium leakage through mutant channels and/or calcium release from intracellular stores in response to an inward cation current through the mutant channels at resting potential.</p><p>The BK channel exists as a tetrameric structure, composed of four &#x003b1;-subunits, either alone or associated with &#x003b2;-subunits. The &#x003b1;-subunit, encoded by <italic>KCNMA1</italic>, is the pore-forming unit, and the &#x003b2;-subunit, encoded by <italic>KCNMB1</italic>, <italic>KCNMB2</italic>, <italic>KCNMB3</italic>, or <italic>KCNMB4</italic>, is the modulatory unit. The different subunit compositions of BK channels give rise to its functional diversity in various tissues; for example, skeletal muscle BK is assembled from &#x003b1;-subunits alone, whereas vascular BK is composed of &#x003b1;+&#x003b2;1 and neuronal types are composed of &#x003b1;+&#x003b2;4 or &#x003b1;+&#x003b2;2<xref rid="B20" ref-type="bibr">20</xref>,<xref rid="B21" ref-type="bibr">21</xref>,<xref rid="B22" ref-type="bibr">22)</xref>. Additional mechanisms involving multiple promoters, alternative splicing, and metabolic regulation also modify BK functional properties, thus producing the diverse range of phenotypes necessary for the normal functioning of many tissues<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24</xref>,<xref rid="B25" ref-type="bibr">25</xref>,<xref rid="B26" ref-type="bibr">26)</xref>. Consistent with the wide distribution of BK channels throughout the human body, malfunction of BK channels has been implicated in various medical conditions, such as epilepsy, psychiatric disorders, deafness, hypertension, asthma, and urinary incontinence<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B27" ref-type="bibr">27</xref>,<xref rid="B28" ref-type="bibr">28</xref>,<xref rid="B29" ref-type="bibr">29</xref>,<xref rid="B30" ref-type="bibr">30</xref>,<xref rid="B31" ref-type="bibr">31)</xref>. BK channels play an essential role in regulating both the firing frequency and the action potential repolarization phase of muscle cells. BK channels also serve as molecular targets for drugs used in the treatment of HOKPP: acetazolamide and dichlorphenamide, which act by opening skeletal muscle BK channels<xref rid="B32" ref-type="bibr">32)</xref>.</p><p>This is the first study to demonstrate altered subcellular distribution of BK channels in the skeletal muscle cells of patients with HOKPP. Paralysis in HOKPP results from persistent membrane depolarization, and skeletal muscle fibers from patients with HOKPP are extremely susceptible to depolarization-induced inactivation<xref rid="B33" ref-type="bibr">33)</xref>. Given the pivotal role played by BK channels in action potential repolarization by increasing potassium efflux out of cells, the reduced membrane expression of BK channels in patient cells, observed in this study, correlates well with the characteristic pathological conditions of HOKPP, such as hypokalemia and paralysis.</p><p>The altered subcellular distribution of BK channels in patient cells in the 4 mM potassium buffer may represent a physiological mechanism developed to compensate for the chronic abnormal increase in cytoplasmic calcium at resting potential. Indeed, several posttranslational modifications have been reported to affect the membrane expression of BK channels<xref rid="B25" ref-type="bibr">25</xref>,<xref rid="B26" ref-type="bibr">26)</xref>. On the other hand, it is currently unclear how the altered subcellular distribution of BK channels is maintained in patient cells in the depolarizing 50 mM potassium buffer. Whether alternative splicing of the <italic>KCNMA1</italic> gene or certain posttranslational mechanisms modify the subcellular distribution of BK channels in patient cells remains to be determined.</p><p>In conclusion, we observed an abnormal increase in cytoplasmic calcium levels and altered subcellular distribution of BK channels in the skeletal muscle cells of patients with HOKPP. The altered distribution of BK channels represents a novel mechanism linking elevated intracellular calcium, induced by HOKPP-associated mutations, to the hypokalemia and paralysis that is symptomatic of the disease and also demonstrates a connection between HOKPP mutant ion channels and pathogenic changes in nonmutant potassium channels.</p></sec>
Serum procalcitonin as a diagnostic marker of neonatal sepsis
<sec><title>Purpose</title><p>We evaluated serum procalcitonin (PCT) as a diagnostic marker of neonatal sepsis, and compared PCT levels with C-reactive protein (CRP) levels.</p></sec><sec><title>Methods</title><p>We retrospectively reviewed the medical records of 269 neonates with a suspected infection, admitted to Wonkwang University School of Medicine &#x00026; Hospital between January 2011 and December 2012, for whom PCT and CRP values had been obtained. Neonates were categorized into 4 groups according to infection severity. CRP and PCT values were analyzed and compared, and their effectiveness as diagnostic markers was determined by using receiver operating characteristic (ROC) curve analysis. We also calculated the sensitivity, specificity, and positive, and negative predictive values.</p></sec><sec><title>Results</title><p>The mean PCT and CRP concentrations were respectively 56.27&#x000b1;81.89 and 71.14&#x000b1;37.17 mg/L in the "confirmed sepsis" group; 15.64&#x000b1;32.64 and 39.23&#x000b1;41.41 mg/L in the "suspected sepsis" group; 9.49&#x000b1;4.30 and 0.97&#x000b1;1.16 mg/L in the "mild infection" group; and 0.21&#x000b1;0.12 and 0.72&#x000b1;0.7 mg/L in the control group. High concentrations indicated greater severity of infection (P&#x0003c;0.001). Five of 18 patients with confirmed sepsis had low PCT levels (&#x0003c;1.0 mg/L) despite high CRP levels. In the ROC analysis, the area under the curve was 0.951 for CRP and 0.803 for PCT. The cutoff concentrations of 0.5 mg/L for PCT and 1.0 mg/L for CRP were optimal for diagnosing neonatal sepsis (sensitivity, 88.29% vs. 100%; specificity, 58.17% vs. 85.66%; positive predictive value, 13.2% vs. 33.3%; negative predictive value, 98.6% vs. 100%, respectively).</p></sec><sec><title>Conclusion</title><p>PCT is a highly effective early diagnostic marker of neonatal infection. However, it may not be as reliable as CRP.</p></sec>
<contrib contrib-type="author"><name><surname>Park</surname><given-names>In Ho</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Seung Hyun</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Seung Taek</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Oh</surname><given-names>Yeon Kyun</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib>
Korean Journal of Pediatrics
<sec sec-type="intro"><title>Introduction</title><p>Despite improvement in neonatal survival rates due to advanced neonatal treatment, neonatal infection-associated sepsis still has an important effect on neonatal morbidity and survival, as it affects 1-4 in 1,000 babies in developed countries<xref rid="B1" ref-type="bibr">1)</xref>. Diagnosing sepsis is very difficult because its symptoms are vague and nonspecific. Therefore, antibiotics are administered before culture results are obtained to reduce morbidity and mortality rates-increasing antibiotic resistance and hospital charges. Thus, an accurate and rapid diagnostic test is necessary.</p><p>The most reliable test for diagnosing sepsis is a blood culture; however, false-positive results often occur due to contamination or no culture growth. Although neutrophil, total white blood cell (WBC), absolute neutrophil count (ANC), and platelet counts and blood culture are ordered to screen for suspected sepsis, these values are ineligible as infection markers due to insufficient sensitivity and specificity<xref rid="B1" ref-type="bibr">1)</xref>. Thus, most hospitals commonly use C-reactive protein (CRP) levels as markers.</p><p>Recently, the effectiveness of procalcitonin (PCT) as an early diagnostic tool for neonatal sepsis has been reported. Research studies reported that PCT is more effective than CRP at follow-up, as PCT levels rise earlier and return to normal levels more rapidly than CRP levels<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9)</xref>. However, some studies have shown a decrease in PCT specificity associated with an increase in uninfected preterm babies, hypoxia, neonatal respiratory distress syndrome (RDS), and hematologic failure<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12)</xref>. Furthermore, one report suggested that PCT is only significant when used in combination with other tests, and it is not appropriate for diagnosing sepsis<xref rid="B13" ref-type="bibr">13)</xref>. Here, we compared PCT with CRP levels for diagnosing neonatal sepsis.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>1. Subject and group classification</title><p>We targeted &#x0003e;4-day-old and &#x0003c;30-day-old patients with suspected bacterial infection who had undergone antibiotic injection after CRT and PCT tests and were hospitalized in the neonatal intensive care unit, nursery, pediatric wards from January 2011 to December 2012; we excluded patients who had major congenital anomalies and liver diseases.</p><p>We included 269 patients in the study and divided them into 4 groups based on the severity of infection<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15)</xref>.</p><sec><title>1) Group I: confirmed sepsis group</title><p>&#x0003e;3 positive sepsis-related clinical symptoms; &#x0003e;2 positive sepsis-related blood test results; CRP levels &#x0003e;6 mg/L; and positive blood culture test results</p></sec><sec><title>2) Group II: suspected sepsis group</title><p>&#x0003e;3 positive sepsis-related clinical symptoms; &#x0003e;2 positive sepsis-related blood test results; CRP levels &#x0003e;6 mg/L; negative blood culture test</p></sec><sec><title>3) Group III: mild infection group</title><p>&#x0003e;2 positive sepsis-related clinical symptoms; &#x0003c;2 positive sepsis-related blood tests; CRP levels &#x0003c; 6 mg/L; negative blood culture test results</p></sec><sec><title>4) Group IV: no infection group (control group)</title><p>All babies, excluding groups I, II, and III.</p><p>The criteria for sepsis-related clinical symptoms1) were divided into the following groups: (1) General: fever, temperature instability, and poor feeding; (2) gastrointestinal system: abdominal distension and hepatomegaly; (3) respiratory system: apnea, dyspnea, retraction, and cyanosis; (4) cardiovascular system: pallor, mottling, tachycardia, and bradycardia; (5) central nervous system: irritability, lethargy, abnormal Moro reflex, fontanelle bulging, seizure, and hypotonia; (6) hematologic system: jaundice, splenomegaly, and petechiae.</p><p>Sepsis-related blood tests included total WBC, ANC, CRP levels, and platelet counts. Total WBC and ANC were analyzed based on the criteria described by Manroe et al.<xref rid="B16" ref-type="bibr">16)</xref> and Rodwell et al.<xref rid="B17" ref-type="bibr">17)</xref> scoring system. Leukopenia was defined as &#x0003c;5,000/mm<sup>3</sup>, leukocytosis &#x0003e;21,000/mm<sup>3</sup>, normal ANC 1,800-5,400/mm<sup>3</sup>, thrombocytopenia<xref rid="B18" ref-type="bibr">18)</xref> &#x0003c;150,000/mm<sup>3</sup>, and CRP levels were &#x0003e;6 mg/L (which was our hospital's criterion for infection), and these were analyzed retrospectively.</p></sec></sec><sec><title>2. Methods</title><p>We analyzed the results and divided patients into groups based on infection severity after investigating gestational age (GA), birth weight (BW), gender, CRP and PCT levels, blood culture, and clinical symptoms of the neonates with suspected bacterial infection.</p><p>Neonates with suspected bacterial infection were given antibiotics after blood sampling for total WBC, ANC, platelet count, CRP, PCT, and blood culture results.</p><p>Blood sampling was performed when bacterial infection was suspected. PCT was measured by enzyme-linked fluorescent assay using BRAHMS PCT (BioMerieux SA, Marcy l'Etoile, France) kit in the VIDAS equipment, and CRP levels were measured by immunoturbidimetric analysis using a CRP reagent kit (Sekisui Medical Co., Tokyo, Japan). To determine the diagnostic usefulness of PCT and CRP levels, we analyzed the receiver operating characteristic (ROC) curve and calculated the sensitivities, specificities, and positive and negative predictive values.</p></sec><sec><title>3. Statistics</title><p>For statistical analysis, characteristics of 4 patients group was assessed using analysis of variance and X<sup>2</sup> test; PCT and CRP values using the Kruskal-Wallis test; and multiple comparison between groups using Mann-Whitney <italic>U</italic> test. Significance was defined as a <italic>P</italic> value &#x0003c;0.01.</p></sec></sec><sec sec-type="results"><title>Results</title><sec><title>1. Patients' characteristics in each group</title><p>Of the 269 babies, 18 were included in the confirmed sepsis group, 56 in the suspected sepsis group, 81 in the mild infection group, and 114 in the control group. Patients' BWs were 2,370&#x000b1;1,090, 2,440&#x000b1;1,110, 2,430&#x000b1;3,840, and 2,140&#x000b1;960 g, respectively, and GAs were 34.8&#x000b1;6.92, 34.8&#x000b1;4.45, 32.8&#x000b1;5.07, and 33.4&#x000b1;4.64 weeks, respectively. BW and GA were not significantly different between the groups. There were more male (n=164) than female babies (n=105); however, the difference was not significant (<xref ref-type="table" rid="T1">Table 1</xref>).</p></sec><sec><title>2. PCT and CRP levels in each group</title><p>PCT levels were significantly high according to infection severity: 56.27&#x000b1;81.89 mg/L, confirmed sepsis group; 15.64&#x000b1;32.64 mg/L, suspected sepsis group; 9.49&#x000b1;4.30 mg/L, mild infection group; and 0.21&#x000b1;0.12 mg/L, control group (<italic>P</italic>&#x0003c;0.001). CRP values showed similar results (71.14&#x000b1;37.17, 39.23&#x000b1;41.41, 0.97&#x000b1;1.16, and 0.72&#x000b1; 0.7 mg/L, respectively, in each group, <italic>P</italic>&#x0003c;0.001) (<xref ref-type="table" rid="T2">Table 2</xref>).</p></sec><sec><title>3. Causative microorganisms in the confirmed sepsis group</title><p>Bacteria were cultured in 18 cases. Enterococcus faecium and Streptococcus were the most common microbes on blood culture (4 cases, 22%) (<xref ref-type="table" rid="T3">Table 3</xref>).</p><p>Of the 18 patients with confirmed sepsis, 2 patients (patient 8, 18) had slightly increased CRP values to 14.93 and 18.2 mg/L, respectively; however, their PCT values increased considerably to 200 and 141.18 mg/L, respectively. Therefore, PCT seems to be more reliable for diagnosing neonatal sepsis than CRP in 2 cases. However, despite increased CRP values in 5 cases, their PCT values remained very low, and were &#x0003c;0.5 mg/L (as the cutoff value) in 2 cases (patient 3 and 4) and &#x0003c;1 mg/L (as the cutoff value) in 3 cases (patient 2, 13, and 17), for detecting neonatal sepsis. Therefore, PCT may have a lower accuracy as a diagnostic marker of neonatal sepsis than CRP (<xref ref-type="table" rid="T3">Table 3</xref>).</p></sec><sec><title>4. ROC curve</title><p>ROC curve analysis was performed to determine the diagnostic usefulness of PCT compared with CRP for detecting neonatal sepsis. In the ROC curve, area under the curve (AUC) was higher for CRP (0.951, 95% confidence interval [CI], 0.918-0.973) than for PCT (0.803, 95% CI, 0.751-0.849), showing that CRP is more useful; however, PCT levels also showed a high result (<italic>P</italic>&#x0003c;0.0015) (<xref ref-type="fig" rid="F1">Fig. 1</xref>). PCT sensitivity and specificity were 88.89% and 58.17%, respectively, with a diagnostic threshold of 0.5 mg/L, and 72.22% and 69.32%, respectively with a threshold of 1 mg/L. The sensitivity and specificity of CRP were 100% and 78.09%, respectively, with a diagnostic threshold of 6 ng/L, and 100% and 85.66% with a threshold of 10 mg/L (<xref ref-type="table" rid="T4">Table 4</xref>). Therefore, PCT seems very useful for diagnosing neonatal sepsis with high sensitivity and specificity; however, the sensitivity and specificity of CRP were much higher. Moreover, the most sensitive diagnostic cutoff values were 0.5 mg/L for PCT and 10 mg/L for CRP. The positive predictive rate (0.5 mg/L, 13.2%; 1 mg/L, 14.4%) of PCT was very low, whereas the negative predictive rate (0.5 mg/L, 98.6%; 1 mg/L, 97.2%) showed a similar high result to CRP (6 and 10 mg/L, 100%) (<xref ref-type="table" rid="T4">Table 4</xref>).</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>Although blood culture results are important for diagnosing neonatal sepsis, it has a low rate of culture, making it difficult to diagnose neonatal sepsis early and resulting in unnecessary or delayed treatment. Therefore, a rapid test with the best degree of sensitivity, reliability, and predictability is required for the early diagnosis and treatment of neonatal sepsis.</p><p>The diagnostic markers of neonatal sepsis include total WBC and differential counts; an immature-to-total neutrophil ratio, &#x02265;0.2; neutropenia; thrombocytopenia; and levels of CRP, PCT, haptoglobin, fibrinogen, and cytokines (interleukin [IL] 6, IL-8, and tumor necrosis factor-&#x003b1;), etc., with the bacterial culture providing a definitive diagnosis<xref rid="B1" ref-type="bibr">1)</xref>. Among these markers, CRP is most commonly used in all hospitals during follow-up and diagnosis. Although CRP levels can be obtained easily and rapidly through an automatic method, and has a high sensitivity, its specificity is low, making it difficult to diagnose sepsis<xref rid="B19" ref-type="bibr">19)</xref>.</p><p>Comparatively, PCT is more specific than other markers and is detected faster than CRP. Therefore, PCT is believed to be a new marker of infection; however, its use as the only marker for early diagnosis of sepsis is controversial<xref rid="B19" ref-type="bibr">19)</xref>.</p><p>PCT, a precursor of calcitonin, is a peptide composed of 116 amino acids and a glycoprotein with a molecular weight of 14.5 KDa. In ordinary times, it is synthesized in the C cells of the thyroid gland and becomes the mature hormone calcitonin formed by 32 amino acids through proteolysis<xref rid="B20" ref-type="bibr">20)</xref>. Although it exists in extremely low concentrations in healthy persons, its levels increase in the presence of bacterial infections, such as sepsis, meningitis, and urethritis, and it rises rapidly in severe sepsis or septic shock<xref rid="B21" ref-type="bibr">21)</xref>. At that time, high serum PCT levels are found because macrophages and monocytes, and the C cells of the thyroid produce it through induction by bacterial endotoxin. Dandona et al.<xref rid="B22" ref-type="bibr">22)</xref> found that PCT is first detected 4 hours after injection of a small quantity of endotoxin in healthy people due to inflammation; its levels increase rapidly after 6-8 hours, reach a plateau, and then return to normal levels after 24 hours.</p><p>PCT levels show a physiologic increase 24-48 hours after birth, and it is normalized after 3 days <xref rid="B12" ref-type="bibr">12)</xref>. Other than infection, PCT levels increase in premature infants, hypoxia, RDS, and hemodynamic instability, decreasing its specificity in early-onset sepsis<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12)</xref>.</p><p>In 1993, Assicot et al.<xref rid="B23" ref-type="bibr">23)</xref> were the first to report increased PCT levels after severe bacterial infection, using a monoclonal immunoradiometric assay. Thereafter, many PCT studies have been reported, but few have found meaningful results for the early diagnosis of bacterial infection. Although CRP level is widely used as an indicator of acute infection, increased CRP levels after the beginning of inflammation is slower than that in PCT levels. This difference can be attributed to CRP starting to increase 4-6 hours later than PCT, after the beginning of inflammation, and reaching its peak about 36 hours later<xref rid="B21" ref-type="bibr">21)</xref>. Further, although PCT decreases 24-48 hours later after antibiotics are initiated and its level returns to normal 5 days after, CRP levels remains high for at least 24-48 hours, and then decline 5 days later despite treatment with antibiotics<xref rid="B24" ref-type="bibr">24)</xref>. Therefore, PCT is known as a more useful indicator for diagnosing early-onset sepsis in newborns because it increases earlier within 12 hours of life than either CRP or IL-6 levels, and it may also be useful for follow-up examination.</p><p>However, Blommendahl et al.<xref rid="B19" ref-type="bibr">19)</xref> suggested that PCT was not a better marker than CRP levels because PCT was affected by perinatal factors within 48 h of birth. A study by Chiesa et al. 24) on the perinatal influence on PCT suggests that the prenatal antibiotic therapy may be associated with false-negative PCT results in those with early neonatal sepsis. Furthermore, Martin-Denavit et al.<xref rid="B25" ref-type="bibr">25)</xref> mentioned that prenatal antibiotics administered to neonates 1 day after birth increases PCT level to a slightly higher concentration than normal.</p><p>We divided patients into 4 groups, who were suspected bacterial infection after 4 days to below 30 days of birth, according to their clinical states. PCT levels were higher in patients with more severe clinical symptoms such as that observed for CRP. Additionally, we performed ROC curve analysis to determine the diagnostic usefulness of PCT compared with CRP for detecting neonatal sepsis. In the ROC curve, the AUC for CRP was 0.951 and that for PCT was 0.803; thus, both markers had a great diagnostic value; however, CRP levels showed more usefulness. To check diagnosis accuracy, we used the following guidelines based on the AUC level: noninformative (0.5), less accurate (0.5&#x0003c;AUC&#x02264;0.7), moderately accurate (0.7&#x0003c;AUC&#x02264;0.9), and highly accurate (0.9&#x0003c;AUC&#x0003c;1)<xref rid="B26" ref-type="bibr">26)</xref>. Thus, based on our results, CRP is a highly accurate marker, whereas PCT is moderately accurate for the diagnosis of neonatal sepsis. Moreover, PCT levels remained normal to very low in 5 patients, while CRP levels increased (<xref ref-type="table" rid="T3">Table 3</xref>). Of these 5 cases, PCT values were &#x0003c;0.5 mg/L of the cutoff value for neonatal sepsis in 2 patients and &#x0003c;1 mg/L of cutoff value (mildly increased) in 3 patients. Therefore, PCT showed lower accuracy than CRP.</p><p>Our results suggest that the best cutoff value for diagnosing neonatal bacterial infection is 0.5 mg/L for PCT and 10 mg/L for CRP. PCT had a sensitivity of 88.79% and specificity of 58.17%, with a cutoff value 0.5 mg/L, while CRP had a sensitivity of 100% and specificity of 52.66%. Although the sensitivity and specificity was slightly lower for PCT, they are considered sufficiently high for a diagnostic marker. Previous studies have reported the sensitivity and specificity of PCT as follows: Sakha et al.<xref rid="B27" ref-type="bibr">27)</xref>, 66.7% and 50%; Boo et al.<xref rid="B28" ref-type="bibr">28)</xref>, 88.9% and 65.2%; Chiesa et al.<xref rid="B24" ref-type="bibr">24)</xref>, 92.6% and 97.5%; and Hatherill et al.<xref rid="B3" ref-type="bibr">3)</xref>, 92.6% and 97.5%, respectively. In a domestic study, the sensitivity, specificity, positive, and negative predictive values for PCT were 66.7%, 94.4%, 88.9%, and 81.0%, respectively, suggesting that PCT is a better clinical marker than CRP-although it is associated with a more expensive cost<xref rid="B29" ref-type="bibr">29)</xref>.</p><p>In our study, negative predictive values of PCT were 98.6% with a 0.5 mg/L cutoff value and 97.2% with a 1 mg/L cutoff value. Without sepsis, normal cases from sepsis cases could be distinguished and the results were the same as those with CRP. Ballot et al.<xref rid="B13" ref-type="bibr">13)</xref> reported a high negative predictability of 95% and Guibourdenche et al.<xref rid="B30" ref-type="bibr">30)</xref> emphasized its significance in PCT tests.</p><p>As stated above, although many reports have suggested that PCT is a more useful marker than CRP, our study highlights the risk of evaluating PCT levels as the only test for neonatal sepsis. This is because the sensitivity and specificity of PCT are slightly lower than those of CRP. Moreover, 5 of 18 patients with confirmed sepsis had PCT levels within the normal to very low level despite having high CRP levels.</p><p>Ballot et al.<xref rid="B13" ref-type="bibr">13)</xref> suggested that the PCT test alone was not sufficient to confirm neonatal sepsis, because of its slightly lower sensitivity, specificity, positive predictability, and AUC. Lapillonne et al.<xref rid="B10" ref-type="bibr">10)</xref> discovered that premature birth increased PCT concentrations without any bacterial infection; thus, it had no specificity despite of its diagnostic value. Furthermore, Monneret et al.<xref rid="B12" ref-type="bibr">12)</xref> found that high PCT levels observed on the second day after birth (enough to suspect bacterial infection) return to the physiologically normal range (&#x0003c;0.5 mg/L) on the fourth day, similar to other healthy babies. Their conclusion was as follows: "It is common in RDS and condition related to temporal hypoxia during labor so high PCT level doesn't mean the bacterial infection all the time."</p><p>Based on our analysis, PCT may not be a sufficiently reliable diagnostic marker of neonatal sepsis compared with CRP. Therefore, we conclude that PCT can be used as a diagnostic marker in combination with other tests for the diagnosis of neonatal sepsis.</p></sec>
Overlapping Guillain-Barr&#x000e9; syndrome and Bickerstaff's brainstem encephalitis associated with Epstein Barr virus
<p>A flaccid tetraparesis in Bickerstaff's brainstem encephalitis (BBE) is presumed to be a sign of overlapping Guillain-Barr&#x000e9; syndrome (GBS). In addition, BBE and Fisher syndrome, which are clinically similar and are both associated with the presence of the immunoglobulin G anti-GQ1b antibody, represent a specific autoimmune disease with a wide spectrum of symptoms that include ophthalmoplegia and ataxia. A 2-year-old boy presented with rapidly progressive ophthalmoplegia, ataxia, hyporeflexia, weakness of the lower extremities, and, subsequently, disturbance of consciousness. He experienced bronchitis with watery diarrhea and had laboratory evidence of recent infection with Epstein-Barr virus (EBV). He was diagnosed as having overlapping GBS and BBE associated with EBV and received treatment with a combination of immunoglobulin and methylprednisolone, as well as acyclovir, and had recovered completely after 3 months. In addition, he has not experienced any relapse over the past year. We suggest that combinations of symptoms and signs of central lesions (disturbance of consciousness) and peripheral lesions (ophthalmoplegia, facial weakness, limb weakness, and areflexia) are supportive of a diagnosis of overlapping GBS and BBE and can be helpful in achieving an early diagnosis, as well as for the administration of appropriate treatments.</p>
<contrib contrib-type="author" corresp="yes"><name><surname>Rho</surname><given-names>Young Il</given-names></name><degrees>MD</degrees><degrees>PhD</degrees><xref ref-type="aff" rid="A1"/></contrib>
Korean Journal of Pediatrics
<sec sec-type="intro"><title>Introduction</title><p>Bickerstaff's brainstem encephalitis (BBE) is a rare immune disorder in children. BBE is characterized by progressive ophthalmoplegia, ataxia, disturbance of consciousness, pyramidal signs, extensor plantar responses, and long track sensory disturbance<xref rid="B1" ref-type="bibr">1)</xref>. Limb weakness in patients with BBE was considered the result of overlapping with the axonal subtype of Guillain-Barr&#x000e9; syndrome (GBS)<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2)</xref>. Although the etiology of BBE is unknown, it has been linked to various antecedent infections, such as <italic>Campylobacter jejuni</italic>, Cytomegalovirus (CMV), <italic>Varicellar zoster</italic> virus, Epstein Barr virus (EBV), and <italic>Mycoplasma pneumoniae</italic><xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6)</xref>.</p><p>In overlapping cases, such as Miller Fisher syndrome (MFS) and GBS, the clinical features may cause confusion so that a correct diagnosis of BBE is difficult. Presence of antiganglioside antibodies (GQ, GM, and GD) in serum or CSF is informative for the diagnosis of BBE. Here, we describe a patient with overlapping GBS and BBE associated with EBV who recovered completely without relapse by treatment with a combination of immunoglobulin (Ig) and methylprednisolone as well as acyclovir. This case supports the hypothesized continuity between GBS and BBE.</p></sec><sec><title>Case report</title><p>A 2-year-old boy was referred to Chosun University Hospital with symptoms of ophthamoplegia, walking disturbance, and irritability. He had suffered from acute bronchitis and enteritis three weeks before admission.</p><p>On admission, the vital signs were body temperature of 36.5&#x02103;, pulse rate of 121 beats/min, and blood pressure of 100/60 mmHg. Mild abdominal distention was observed. The pupils were 4 mm bilaterally and light reflexes were intact. He was alert, with no other abnormal neurological findings except for ptosis and ophthalmoplegia (restriction of lateral gaze of both eyes). The muscle strength of his lower extremities was grade 2. Deep tendon reflexes of his lower extremities were absent, but sensation was intact. No signs of meningeal irritation were observed during admission. By the following day, he had become confused, had dysphasia, and had bilateral facial palsy. Treatment with intravenous immunoglobulin (IVIG) (0.4 g/kg/day for five days) was started. On the third day of admission, he developed difficult respiration. Muscle strength of his upper extremities was decreased to grade 2, and that of his lower extremities was decreased to grade 0. Treatment with a high dose of methylprednisolone (30 mg/kg for three days) was started. On the fourth day of admission, self respiration was absent. The patient's consciousness level worsened to a coma state. Pupil size decreased (2 mm/2 mm), and there were no light reflexes. His blood pressure and pulse rate had increased (120/80 mmHg, 200 beats/min, respectively). An angiotensin converting enzyme inhibitor was started for management of hypertension. He required assisted ventilation until day 19 due to respiratory failure.</p><p>White blood count (13,500/&#x000b5;L) mildly increased. Hemoglobin, platelet count, routine chemistries, liver function profile, ammonia, electrolytes, erythrocyte sedimentation rate, and cerebrospinal fluid (CSF) examination were within reference ranges. Blood tests for recent infection by CMV and the influenza virus were negative. The results of serum antibody testing for <italic>Mycoplasma</italic>, <italic>C. jejuni</italic> were negative. The result of a repeat CSF examination on day 3 was normal: pressure, 12 cmH<sub>2</sub>O; leukocytes, 4/&#x000b5;L; red blood cell, 1/&#x000b5;L; protein, 33.5 mg/dL; glucose, 65.3 mg/dL. The myelin basic protein and oligoclonal bands were normal. A specific polymerase chain reaction (PCR) examination of the CSF was negative for the Herpes virus group or for an Enterovirus infection. Serum and CSF antiganglioside antibodies, which were measured using an enzyme-linked immunoadsorbent assay, showed an absence of anti-GQ1b IgG and IgM antibodies, anti-GM1 IgG and IgM antibodies, and anti-GD1 IgG and IgM antibodies. A CSF analysis on day 5 showed a protein concentration of 54 mg/dL without leukocyte. A positive result was obtained for EBV viral capsid and antigen-IgG and IgM antibodies and EBV confirmed seroconversion of the nuclear antigen antibody. EBV-DNA was detected in the CSF by PCR. These findings indicate prior infection by EBV.</p><p>Brain and spine magnetic resonance images (MRIs) performed on the fourth and 13th days detected no abnormal signals in either the cerebrum or brainstem (<xref ref-type="fig" rid="F1">Fig. 1</xref>). An electroencephalogram (EEG) without sedative drugs showed slow wave bursts at both occipital lobes (<xref ref-type="fig" rid="F2">Fig. 2</xref>). Nerve conduction and electromyogram studies were not performed. Acyclovir was started for more effective treatment of EBV infection.</p><p>By the final day of treatment, the patient's mental status, blood pressure, and pulse rate were normal. Facial palsy, ptosis, ophthalmoplegia, dysarthria, and deep tendon reflex showed improvement. Muscle strength remained impaired and he exhibited a slight stagger while walking. Three months after the beginning of his neurological disease, he had almost completely recovered. One year after treatment, he had fully recovered without relapse.</p></sec><sec sec-type="discussion"><title>Discussion</title><p>BBE is a rare immune disorder in children, which was first described by Bickerstaff and Cloake<xref rid="B7" ref-type="bibr">7)</xref> in 1951. The clinical features and course of the condition, the associated auto-antibodies against relevant antigens, and the response to treatment all suggest that Bickerstaff's encephalitis is an autoimmune disease.</p><p>Like some other autoimmune diseases, the condition usually follows a minor infection, such as a respiratory tract infection or gastroenteritis. An immunological mechanism induced by infection could potentially play a pathogenic role in BBE<xref rid="B3" ref-type="bibr">3)</xref>. A previous infectious disease is frequently reported before the appearance of neurological symptoms, which might be suggestive of an infectious etiology<xref rid="B1" ref-type="bibr">1)</xref>. Our case was associated with EBV infection.</p><p>Major manifestations of BBE associated with facial and bulbar weakness, and a flaccid tetraparesis include acute ophthalmoplegia, ataxia, and disturbance of consciousness. After opthalmoplegia, facial weakness is more common than bulbar palsy. Involvement of respiratory muscles is rare and recovery is usually complete. Therefore, it is important to provide ventilation for all patients. BBE with limb weakness was considered the result of overlapping with the axonal subtype of GBS<xref rid="B1" ref-type="bibr">1)</xref>. BBE and MFS are similar clinically and are associated with presence of the IgG anti-GQ1b antibody, representing a specific autoimmune disease with a wide spectrum of symptoms, including ophthalmoplegia and ataxia<xref rid="B8" ref-type="bibr">8)</xref>. There is certainly an overlap between GBS, MFS, and BBE, as well as other conditions associated with antiganglioside antibodies, such as chronic ophthalmoplegia with the anti-GQ1b antibody<xref rid="B9" ref-type="bibr">9)</xref>.</p><p>A variety of CSF abnormalities with albumino-cytologic dissociation occur late in the illness in one third or more patients. Cumulative evidence has suggested a causal relationship between BBE, GBS, and Fisher Syndrome, and CSF albumino-cytologic dissociations are often being detected in all of them<xref rid="B4" ref-type="bibr">4)</xref>. CSF albumino-cytologic dissociation was found in 19% of cases of BBE during the first week after the beginning of the illness<xref rid="B1" ref-type="bibr">1)</xref>. The CSF protein concentration showed a progressive increase after the fourth week. In our case, CSF was normal during the six days after the beginning of illness, after that, CSF albumino-cytologic dissociation was detected.</p><p>The EEG is often abnormal but shows only slow wave activity, which also occurs in many other conditions, and is therefore of limited value in diagnosis. High intensity T2-weighted abnormalities of the brainstem or other parts of the central nervous system are observed on brain MRI in 30% of patients<xref rid="B1" ref-type="bibr">1)</xref>. Cerebral imaging is rarely useful for the diagnosis of BBE. Abnormal findings on MRI brain scans were reported in 39% of cases of BBE without limb weakness and in 23% of cases of BBE with limb weakness<xref rid="B10" ref-type="bibr">10)</xref>.</p><p>In patients with BBE with coexisting limb weakness, decreased motor nerve conduction velocities, prolonged distal latency, reduced compound muscle action potential amplitude, disappearance of F-wave or its prolonged latency, which is indicative of motor nerve demyelination and axon degeneration can be observed on an electromyogram. The somatosensory evoked potentials for early diagnosis are useful and may be more severely impaired than auditory brainstem responses in BBE<xref rid="B11" ref-type="bibr">11)</xref>.</p><p>There is no single diagnostic test which can diagnose BBE. Due to its diverse symptoms, early diagnosis is often difficult. Early diagnosis is important not only for the performance of effective immunotherapies but also for the avoidance of unnecessary evaluation testing and treatment, such as antiviral or antithrombotic agents. However, the diagnosis of BBE is still based on clinical criteria. The presence of anti-GQ1b antibodies and an abnormal brain MRI can help to support its diagnosis. However, the absence of anti-GQ1b antibodies and a normal brain MRI do not exclude the diagnosis.</p><p>Our patient was initially diagnosed with MFS and then diagnosed with overlapping GBS and BBE with specific clinical symptoms, including impaired consciousness and limb weakness, and CSF findings, although other tests which might be helpful for the diagnosis of BBE, such as anti-GQ1b antibodies and a brain MRI were normal.</p><p>IVIG is effective in immune-mediated disorders such as MFS and GBS<xref rid="B12" ref-type="bibr">12)</xref>. IVIG is also effective in treatment of BBE with hypothesized immune-mediated etiology, which is similar to the clinical and immunologic features of MFS or GBS, which suggests an overlapping spectrum of MFS and GBS. The therapeutic effect of IVIG (total dose of 2 g/kg given over two days) is related to the neutralization of pathogenic autoantibodies such as anti-GQ1b antibodies<xref rid="B13" ref-type="bibr">13)</xref>. BBE and GBS are so closely related that combined therapy of IVIG and high-dose methylprednisolone is a more efficacious therapy than IVIG alone. The prognosis was good, with most patients making a full clinical recovery over six months. Cases of recurrent BBE have been reported. IVIG was initially chosen for our patient because of similar symptoms of MFS or GBS. On the third hospital day, methylprednisolone was started because IVIG was ineffective and his mental state showed greater impairment. On the fifth hospital day, acyclovir was started because of a positive result for EBV in serum and CSF which is based on a previous report<xref rid="B3" ref-type="bibr">3)</xref>. Clinical responses to treatment were observed on the 23rd day, similar to a previous report<xref rid="B14" ref-type="bibr">14)</xref>, and he recovered completely without recurrence.</p><p>We think that our patient was overlapping GBS and BBE following EBV with specific clinical symptoms: progressive opthalmoplegia; ataxia; disturbance of consciousness; progressive bilateral limb weakness; facial weakness; urinary retention and hypertension. We suggest that combinations of symptoms and signs of central lesions (disturbance of consciousness) and peripheral lesions (ophthalmoplegia, facial weakness, limb weakness, and areflexia) are supportive of a diagnosis of overlapping GBS and BBE and can be helpful in achievement of an early diagnosis and for administration of appropriate treatment.</p></sec>
Inhaled iloprost for the treatment of patient with Fontan circulation
<p>Decreased exercise capacity after Fontan surgery is relatively common and the failure of the Fontan state gradually increases with age. However, there is no further treatment for patients with Fontan circulation. Pulmonary vasodilation therapy is an effective method to solve this problem because pulmonary vascular resistance is a major factor of the Fontan problem. Inhaled iloprost is a chemically stable prostacyclin analogue and a potent pulmonary vasodilator. We experienced two cases of Fontan patients treated with inhaled iloprost for 12 weeks. The first patient was an 18-year-old female with pulmonary atresia with an intact ventricular septum, and the second patient was a 22-year-old male with a double outlet right ventricle. Fifteen years have passed since both patients received Fontan surgery. While the pulmonary pressure was not decreased significantly, improved exercise capacity and cardiac output were observed without any major side effects in both patients. The iloprost inhalation therapy was well tolerated and effective for the symptomatic treatment of Fontan patients.</p>
<contrib contrib-type="author"><name><surname>Kim</surname><given-names>Yong Hyun</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Chae</surname><given-names>Moon Hee</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Choi</surname><given-names>Deok Young</given-names></name><degrees>MD</degrees><degrees>PhD</degrees><xref ref-type="aff" rid="A1"/></contrib>
Korean Journal of Pediatrics
<sec sec-type="intro"><title>Introduction</title><p>Since the first Fontan procedure was introduced by Fontan in 1971, the surgical technique has evolved and the survival rate has shown dramatic improvement. Recently, Fontan surgery has been performed as an operation of the final stage for congenital heart disease with single ventricle physiology<xref rid="B1" ref-type="bibr">1)</xref>.</p><p>In Fontan circulation the systemic venous pressure is the driving force of pulmonary circulation. Therefore, the pulmonary vascular resistance (PVR) must be lower than the antegrade transpulmonary flow resistance<xref rid="B2" ref-type="bibr">2)</xref>. Thus, even a mild increase in the PVR may interfere with pulmonary circulation which in turn may result in decreased preload of ventricle, diminished cardiac output, and declined exercise tolerance<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4)</xref>.</p><p>Loss of pulsatility of pulmonary flow, reduced growth of the pulmonary vasculature, micro and macro thromboembolism in pulmonary circulation and reduced ventricular function are underlying mechanisms that induce the high PVR in Fontan circulation<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7)</xref>.</p><p>Several studies<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12)</xref> reported the pulmonary vasodilating therapy had an effective influence on Fontan patients so we want to determine whether inhaled iloprost as a pulmonary vasodilator might have a role in the clinical symptoms, hemodynamics and exercise capacity of Fontan patients.</p><p>Ethical approval for this study and permission for acquiring, analyzing and reporting of patient's data were obtained in accordance with the guidelines of the Institutional Review Board of Gachon University Gil Medical Center (No. GIRBA 2476).</p></sec><sec><title>Case reports</title><sec><title>1. Case 1</title><p>An 18-year-old female with pulmonary atresia with intact ventricular septum status post Fontan surgery at age of three years complained of aggravated dyspnea on exertion (DOE). A physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area, liver and spleen were not palpable. Recently, she felt more difficult to climb the stairs due to shortness of breath. New York Heart Association (NYHA) class was III. She had been taking the digoxin, enalapril, and aspirin. The inhaled iloprost was administered at 60 microgram/day divided by six times for 12 weeks. Pre and post medication, laboratory test-including pro brain natriuretic peptide, cardiac catheterization, bicycle ergometer test, NYHA class, and 6-minute walking test-were performed. The DOE was improved about 4 weeks after the medication. After 12 weeks, the patient's systemic blood pressure and O2 saturation were increased. Six-minute walking distance was increased from 380 to 395 m and NYHA class changed from III to II. Maximal oxygen consumption was increased. In cardiac catheterization, the cardiac index and stroke volume were increased without significant change of pulmonary pressure and PVR. The results are shown in <xref ref-type="table" rid="T1">Table 1</xref>.</p></sec><sec><title>2. Case 2</title><p>A 22-year-old male with double outlet right ventricle status post Fontan surgery at age of five years complained of mild DOE only. Physical examination showed no specific findings. NYHA class was II. He volunteered for an attempt. He had been taking the enalapril and asprin. He also received the same medication (inhaled iloprost) and underwent several tests. After 12 weeks, the patient's systemic blood pressure, O2 saturation, and NYHA class were not changed significantly. N-terminal pro brain natriuretic peptide was decreased. The maximal oxygen consumption was increased. In cardiac catheterization, the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and PVR. The results are shown in <xref ref-type="table" rid="T2">Table 2</xref>.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>Despite the increased survival rate of patients who has congenital heart disease with single ventricular physiology due to advanced surgical technique, the Fontan surgery is still a palliative procedure. Development of the Fontan failure state might occur gradually over time; ventricular dysfunction, aggravated cyanosis, hepatic dysfunction, coagulopathy, protein loosing enteropathy, plastic bronchitis, arrhythmia etc.<xref rid="B1" ref-type="bibr">1)</xref>. Progressive decreasing cardiac output, and increasing central venous pressure can be also observed in Fontan physiology with age<xref rid="B1" ref-type="bibr">1)</xref>.</p><p>Most patients who underwent cardiac transplant long after Fontan surgery had elevated PVR before and immediately after the operation<xref rid="B13" ref-type="bibr">13)</xref>. Indeed, PVR is an important factor in the progressive decline in efficiency of the Fontan physiology. The loss of pulsatile blood flow of pulmonary circulation of Fontan patients is positively correlated with high PVR. The lowering of PVR may increase pulmonary blood flow and preload reserve in Fontan physiology, and for this reason the selective pulmonary vasodilating therapy is interesting as a means to improve the Fontan circulation<xref rid="B8" ref-type="bibr">8)</xref>. In addition, nonpulsatile pulmonary blood flow may reduce capillary recruitment and endothelial function. As a result of endothelial dysfunction, impairment in production of nitric oxide, prostacyclin and prolonged overexpression of endothelin-1 level can be observed<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B14" ref-type="bibr">14)</xref>.</p><p>Several authors have reported on the effects of Bosentan on Fontan patients<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10)</xref>. Most studies were based on high level of endothelin-1 in Fontan patients<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10)</xref>, the endothelin receptor antagonist induced improvement of clinical, hemodynamic parameters and exercise capacity. Experiences with sildenafil therapy on Fontan patients have also been reported<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12)</xref>. In a recent study, Rhodes et al.<xref rid="B15" ref-type="bibr">15)</xref> reported on the effect of inhaled iloprost on exercise function of Fontan patients. In this article the authors administered a single treatment with iloprost nebulizer just before exercise and concluded that iloprost improves the peak oxygen pulse and peak VO2 of patients with Fontan physiology.</p><p>In our two case studies, we found that 12 weeks of iloprost therapy showed an association with improvement of clinical, hemodynamic parameters and exercise capacity in patients with Fontan physiology. First case was considered Fontan failure because of aggravated DOE, functional class and pitting edema. The other case was voluntary. Unlike previous study using iloprost once, we used iloprost for three month period and positive results were achieved without major side effects. In addition, we found that iloprost was effective for Fontan patients with or without symptoms.</p><p>Although more advanced curable treatments of Fontan physiology have not been developed, pulmonary vasodilator therapy on Fontan circulation is one of good suggestion for symptomatic treatment of Fontan failure and improvement of the cardiac output. Conduct of further studies including a larger number of patients is necessary.</p></sec>
Role of modern 3D echocardiography in valvular heart disease
<p>Three-dimensional (3D) echocardiography has been conceived as one of the most promising methods for the diagnosis of valvular heart disease, and recently has become an integral clinical tool thanks to the development of high quality real-time transesophageal echocardiography (TEE). In particular, for mitral valve diseases, this new approach has proven to be the most unique, powerful, and convincing method for understanding the complicated anatomy of the mitral valve and its dynamism. The method has been useful for surgical management, including robotic mitral valve repair. Moreover, this method has become indispensable for nonsurgical mitral procedures such as edge to edge mitral repair and transcatheter closure of paravaluvular leaks. In addition, color Doppler 3D echo has been valuable to identify the location of the regurgitant orifice and the severity of the mitral regurgitation. For aortic and tricuspid valve diseases, this method may not be quite as valuable as for the mitral valve. However, the necessity of 3D echo is recognized for certain situations even for these valves, such as for evaluating the aortic annulus for transcatheter aortic valve implantation. It is now clear that this method, especially with the continued development of real-time 3D TEE technology, will enhance the diagnosis and management of patients with these valvular heart diseases.</p>
<contrib contrib-type="author" corresp="yes"><name><surname>Shiota</surname><given-names>Takahiro</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Echocardiography has been used to evaluate and diagnose patients with valvular heart disease for many years. New echocardiography methods with improved diagnostic accuracy have been proposed for over 40 years. In particular, 3-dimensional (D) echocardiography is now used clinically due to the development of the high-quality real-time transesophageal echocardiography (TEE). In general, 3D echocardiography allows visualization of cardiac structures such as the mitral valve (MV) from any spatial point of view. However, there are limitations to the currently available 3D ultrasound methods, especially the transthoracic version, due to its relatively low image quality and low time resolution. However, this method is widely used to evaluate valvular heart disease.</p><p>Herein, the author will discuss the clinical applications of 3D echocardiography for the mitral, aortic, and tricuspid valves (TVs) individually.</p></sec><sec><title>THE MITRAL VALVE</title><p>Among the four heart valves, 3D echocardiography, especially real-time 3D TEE, is most useful for the diagnosis and management of MV conditions.</p></sec><sec><title>MITRAL REGURGITATION</title><p>Mitral regurgitation (MR) is fundamentally classified as either organic or functional in etiology. Organic MR is usually caused by degenerative abnormalities, including valve prolapse and/or flail. Locating the prolapse and/or flail of the mitral leaflet (medial, central, and lateral) and its geometry is essential for selecting a surgical and/or transcatheter correction technique. However, conventional 2D echocardiography requires multiple views of the MV and mental reconstruction of the 3D image of the diseased structure. Many investigators have reported the usefulness of 3D echocardiography for visualizing, localizing, and quantifying MV abnormalities in patients with MR [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>]. The superiority of transthoracic real-time 3D echocardiography over conventional 2D echo methods in analyzing the anatomy of MV in patients with MR has been reported multiple times since the introduction of transthoracic real-time 3D echocardiography [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B13" ref-type="bibr">13</xref>].</p><p>The use of TEE has also been repeatedly reported to evaluate MV anatomy in patients with MR [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B11" ref-type="bibr">11</xref>]. However, 3D TEE was not clinically accepted until user-friendly real-time 3D TEE was introduced circa 2007. In 2008, Sugeng et al. [<xref rid="B11" ref-type="bibr">11</xref>] reported clinical use of real-time 3D TEE in 211 patients. Excellent visualization of the MV (85% to 91% for all scallops of both MV leaflets, the interatrial septum 84%, left atrial appendage 86%, and left ventricle 77%) was observed. This real-time 3D TEE yields high-quality images of the MV (<xref ref-type="fig" rid="F1">Fig. 1</xref>). Since these initial publications, many reports have described the use of real-time 3D TEE for imaging MV pathology [<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>,<xref rid="B21" ref-type="bibr">21</xref>,<xref rid="B22" ref-type="bibr">22</xref>,<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24</xref>,<xref rid="B25" ref-type="bibr">25</xref>,<xref rid="B26" ref-type="bibr">26</xref>,<xref rid="B27" ref-type="bibr">27</xref>,<xref rid="B28" ref-type="bibr">28</xref>,<xref rid="B29" ref-type="bibr">29</xref>,<xref rid="B30" ref-type="bibr">30</xref>,<xref rid="B31" ref-type="bibr">31</xref>,<xref rid="B32" ref-type="bibr">32</xref>,<xref rid="B33" ref-type="bibr">33</xref>,<xref rid="B34" ref-type="bibr">34</xref>,<xref rid="B35" ref-type="bibr">35</xref>,<xref rid="B36" ref-type="bibr">36</xref>,<xref rid="B37" ref-type="bibr">37</xref>,<xref rid="B38" ref-type="bibr">38</xref>,<xref rid="B39" ref-type="bibr">39</xref>]. For example, in 2013, we published a study on the superiority of real-time 3D TEE over 2D TEE for measuring the gap and width of a MV prolapse and flail [<xref rid="B22" ref-type="bibr">22</xref>]. As seen in this paper and many others, real-time 3D TEE provides a better overall perspective of the MV than 2D TEE, including the shape of the prolapse and the exact size and location of the MV, with the use of the so-called surgical view (<xref ref-type="fig" rid="F2">Fig. 2</xref>). This specific view facilitates communication between echocardiologists and surgeons and interventionists.</p><p>In patients with MR, color Doppler capability, which was initially introduced in reconstruction 3D systems, then later in real-time 3D transthoracic and real-time 3D TEE, can provide 3D images of regurgitant flow jets (<xref ref-type="fig" rid="F3">Fig. 3</xref>) and flow convergence [<xref rid="B40" ref-type="bibr">40</xref>,<xref rid="B41" ref-type="bibr">41</xref>,<xref rid="B42" ref-type="bibr">42</xref>,<xref rid="B43" ref-type="bibr">43</xref>,<xref rid="B44" ref-type="bibr">44</xref>,<xref rid="B45" ref-type="bibr">45</xref>,<xref rid="B46" ref-type="bibr">46</xref>,<xref rid="B47" ref-type="bibr">47</xref>,<xref rid="B48" ref-type="bibr">48</xref>]. The location and size of the flow convergence zone or proximal isovelocity surface area (PISA) can determine the location of the regurgitant orifice and severity of MR [<xref rid="B48" ref-type="bibr">48</xref>]. Such information, especially on the location of the regurgitant orifice, is critical for selection of an appropriate treatment protocol; i.e., either surgery or the edge-to-edge clip procedure [<xref rid="B49" ref-type="bibr">49</xref>]. For instance, for the edge-to-edge clip procedure, A2/P2 MR is preferred for the commissural origin of MR in the ongoing clinical mitral clip trial, Clinical Outcomes Assessment of Percutaneous Treatment, in the United States.</p><p>Also, color Doppler 3D echocardiography has demonstrated that in many conditions the flow convergence zone is not hemispherical, such as for irregular or asymmetrical orifices, and in patients with functional or ischemic MR [<xref rid="B47" ref-type="bibr">47</xref>,<xref rid="B50" ref-type="bibr">50</xref>,<xref rid="B51" ref-type="bibr">51</xref>,<xref rid="B52" ref-type="bibr">52</xref>,<xref rid="B53" ref-type="bibr">53</xref>]. Multiple investigators have proposed more realistic geometric shapes for flow convergence zones, such as a hemiellipse or hemiellipsoid, to obtain more accurate regurgitant volumes [<xref rid="B46" ref-type="bibr">46</xref>,<xref rid="B53" ref-type="bibr">53</xref>,<xref rid="B54" ref-type="bibr">54</xref>].</p><p>The vena contracta (VC) area determined by color Doppler 3D echocardiography has been repeatedly found useful for quantitatively defining the MR severity [<xref rid="B55" ref-type="bibr">55</xref>,<xref rid="B56" ref-type="bibr">56</xref>,<xref rid="B57" ref-type="bibr">57</xref>,<xref rid="B58" ref-type="bibr">58</xref>,<xref rid="B59" ref-type="bibr">59</xref>]. These studies also noted a variety of VC shapes, including a curved VC, in functional MR patients (<xref ref-type="fig" rid="F4">Fig. 4</xref>) [<xref rid="B57" ref-type="bibr">57</xref>]. Another recent study using 3D TEE demonstrated multiple VC shapes in a patient and added them together to determine MR severity [<xref rid="B58" ref-type="bibr">58</xref>]. The idea of 3D VC seems attractive because it is independent of geometric assumptions. However, the location and size of VC from the PISA to the distal jet may vary depending on the operator and cutting the VC in the exact plane in 3D space is difficult, especially when the jet is quite eccentric. In addition, the cutoff value of the VC area for severe MR has not been firmly established. Practically, therefore, one may prefer the size of the PISA when it is appropriately imaged with proper Nyquist limits to the 3D VC for determining MR severity. Both methods can be used together with classic regurgitant jet imaging to increase accuracy.</p><p>As mentioned above, edge-to-edge clip repair was approved for high-risk surgical patients with severe degenerative MR in the United States in October 2013. In the catheterization laboratory, 3D TEE can assist the positioning of the clip on the MV orifice, grasping of both MV leaflets, and evaluating the result, including visualization of the residual MR (<xref ref-type="fig" rid="F5">Fig. 5</xref>) [<xref rid="B60" ref-type="bibr">60</xref>,<xref rid="B61" ref-type="bibr">61</xref>,<xref rid="B62" ref-type="bibr">62</xref>,<xref rid="B63" ref-type="bibr">63</xref>]. At the author's institution, 3D TEE is indispensable for the success of this procedure. We reported previously the value of real-time 3D TEE for determining the unique shape, size and location of the atrial septal defect created by septal puncture with the large catheter and its sheath (24 F) used for the clip procedure [<xref rid="B64" ref-type="bibr">64</xref>].</p><p>Regarding postoperative evaluation of MV repair or replacement, 3D TEE has been shown to facilitate visualization of the entire structure of the new artificial valve [<xref rid="B65" ref-type="bibr">65</xref>]. In addition, color Doppler 3D TEE can delineate the location of the paravalvular MR, especially useful for transcatheter closure of the leak [<xref rid="B65" ref-type="bibr">65</xref>,<xref rid="B66" ref-type="bibr">66</xref>,<xref rid="B67" ref-type="bibr">67</xref>,<xref rid="B68" ref-type="bibr">68</xref>]. In our study, color Doppler 3D TEE showed the exact location of the circumferential orifice of paravalvular regurgitation around the artificial MV, thus assisting the transcatheter device closure procedure [<xref rid="B66" ref-type="bibr">66</xref>]. <xref ref-type="fig" rid="F6">Fig. 6</xref> shows a case of postoperative residual paravalvular MR. Color Doppler 3D TEE showed the exact location of the residual MR, which allowed immediate successful surgical correction (<xref ref-type="fig" rid="F6">Fig. 6</xref>).</p></sec><sec><title>MITRAL STENOSIS</title><p>Mitral stenosis (MS) is usually caused by rheumatic MV disease. Fusion of the commissure is the major cause of the stenosis. Conventional 2D echocardiography has been widely used to determine the smallest valve area. However, 2D echocardiography can only minimally visualize the entire MV and the subvalvular apparatus, resulting in erroneous measurement of the smallest valve area. Three-dimensional echocardiography has been reported to be superior to conventional 2D echocardiography for determining the smallest area and visualizing morphological abnormalities [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B29" ref-type="bibr">29</xref>,<xref rid="B32" ref-type="bibr">32</xref>,<xref rid="B69" ref-type="bibr">69</xref>,<xref rid="B70" ref-type="bibr">70</xref>,<xref rid="B71" ref-type="bibr">71</xref>,<xref rid="B72" ref-type="bibr">72</xref>,<xref rid="B73" ref-type="bibr">73</xref>,<xref rid="B74" ref-type="bibr">74</xref>,<xref rid="B75" ref-type="bibr">75</xref>,<xref rid="B76" ref-type="bibr">76</xref>,<xref rid="B77" ref-type="bibr">77</xref>,<xref rid="B78" ref-type="bibr">78</xref>]. In an early study, 3D echocardiography provided accurate and highly reproducible measurements of the mitral valve area (MVA) and was easily performed via an apical approach [<xref rid="B70" ref-type="bibr">70</xref>]. In another study, a real-time 3D echocardiographic system was used for MV planimetry [<xref rid="B72" ref-type="bibr">72</xref>]. This was reportedly more accurate than the Gorlin method for measurement of the valve area. The authors concluded that 3D echo planimetry may be a better reference method than the Gorlin method in terms of assessing the severity of rheumatic MS [<xref rid="B72" ref-type="bibr">72</xref>]. The recently introduced real-time 3D TEE yields striking images of MS in patients (<xref ref-type="fig" rid="F7">Fig. 7</xref>) [<xref rid="B79" ref-type="bibr">79</xref>]. Not only the stenosis but also the shape, location, and anatomical abnormalities of the MV leaflets, such as heavy calcification, are visualized intuitively. In a clinical study of 43 patients with rheumatic MV stenosis, 3D TEE allowed excellent assessment of commissural fusion and MVA planimetry (<xref ref-type="fig" rid="F7">Fig. 7</xref>) [<xref rid="B75" ref-type="bibr">75</xref>]. Also, a recent Korean study showed a tendency of overestimation of MVA by 2D planimetry and concluded that 3D TEE should be considered for accurate MVA assessment, especially in patients with a large left atrium and large angle between the lines of the true MV tip and the echo beam-to-the tip [<xref rid="B38" ref-type="bibr">38</xref>].</p><p>Additionally, in 63 consecutive patients with rheumatic MS, valve area assessment using the flow convergence (or PISA) method with a newly developed single-beat real-time 3D color Doppler echocardiography was reportedly feasible in a clinical setting and more accurate than the conventional 2D PISA method [<xref rid="B79" ref-type="bibr">79</xref>]. This new type of real-time 3D TEE seems clinically feasible and useful with and without color Doppler.</p></sec><sec><title>APPLICATION OF 3D ECHOCARDIOGRAPHY FOR BALLOON MITRAL VALVULOPLASTY</title><p>Application of 3D echocardiography for mitral valvuloplasty has also been reported [<xref rid="B80" ref-type="bibr">80</xref>,<xref rid="B81" ref-type="bibr">81</xref>,<xref rid="B82" ref-type="bibr">82</xref>,<xref rid="B83" ref-type="bibr">83</xref>]. In one of these studies, an old type of reconstruction 3D TEE enabled visualization of the MV, allowing visualization of commissural splitting and leaflet tears not seen on 2D echocardiography [<xref rid="B80" ref-type="bibr">80</xref>].</p><p>Thanks to the recent development of relatively high quality transthoracic real-time 3D echocardiography, improvement of valve area and changes in valve geometry after balloon valvuloplasty were reported in a clinical study [<xref rid="B81" ref-type="bibr">81</xref>]. In this study, transthoracic real-time 3D echo, instead of multiplane TEE 3D reconstruction, was employed to measure the valve area in 29 patients with rheumatic MS who underwent balloon valvuloplasty [<xref rid="B81" ref-type="bibr">81</xref>]. The authors concluded that transthoracic real-time 3D echocardiography is a feasible and accurate technique for measuring the MVA in patients with rheumatic MV stenosis [<xref rid="B81" ref-type="bibr">81</xref>]. In another study, real-time 3D echocardiography improved visualization of valvular anatomy and provided a unique assessment of the extent of commissural splitting [<xref rid="B84" ref-type="bibr">84</xref>].</p><p>Anwar et al. [<xref rid="B85" ref-type="bibr">85</xref>] proposed a new score system based on real-time transthoracic 3D echocardiography that was feasible and highly reproducible for the assessment of MV morphology in patients with MS. According to this study, the score system can provide incremental prognostic information in addition to the Wilkins score [<xref rid="B85" ref-type="bibr">85</xref>].</p><p>More recently, real-time 3D TEE showed its superiority for evaluating the efficacy of mitral valvuloplasty due to its higher-resolution MV images compared to the old-type reconstruction 3D TEE and transthoracic real-time 3D echocardiography [<xref rid="B86" ref-type="bibr">86</xref>].</p></sec><sec><title>THE MITRAL ANNULUS</title><p>Real-time 3D echocardiographic methods have been used to evaluate non-planarity and area changes in the mitral annulus in animals and humans [<xref rid="B87" ref-type="bibr">87</xref>,<xref rid="B88" ref-type="bibr">88</xref>,<xref rid="B89" ref-type="bibr">89</xref>,<xref rid="B90" ref-type="bibr">90</xref>,<xref rid="B91" ref-type="bibr">91</xref>,<xref rid="B92" ref-type="bibr">92</xref>,<xref rid="B93" ref-type="bibr">93</xref>,<xref rid="B94" ref-type="bibr">94</xref>,<xref rid="B95" ref-type="bibr">95</xref>,<xref rid="B96" ref-type="bibr">96</xref>,<xref rid="B97" ref-type="bibr">97</xref>,<xref rid="B98" ref-type="bibr">98</xref>]. Extracted 3D images obtained with multiplane TEE can also be used to evaluate non-planarity and area changes of the mitral annulus in patients with an annuloplasty ring [<xref rid="B99" ref-type="bibr">99</xref>,<xref rid="B100" ref-type="bibr">100</xref>]. The saddle-shaped geometry of the mitral annulus has been repeatedly reported and confirmed by 3D echocardiography, and its assessment of mitral annular size and function in control subjects and patients with cardiomyopathy was reportedly accurate and well correlated with magnetic resonance imaging (MRI) findings [<xref rid="B101" ref-type="bibr">101</xref>]. Three-dimensional echocardiography allowed quantitative analysis of not only the annulus geometry but also the valve tethering or tenting in ischemic cardiomyopathy and idiopathic cardiomyopathy [<xref rid="B91" ref-type="bibr">91</xref>,<xref rid="B94" ref-type="bibr">94</xref>,<xref rid="B102" ref-type="bibr">102</xref>,<xref rid="B103" ref-type="bibr">103</xref>]. In one recent study of real-time 3D TEE, the mitral annulus in functional MR was significantly larger, rounder, and flatter, and dilated further and became more flattened at late systole, compared to controls [<xref rid="B95" ref-type="bibr">95</xref>]. Considering the clinical importance of annuloplasty for managing such patients with MR, detailed geometric evaluation should be performed to improve surgical results. Real-time 3D TEE showed that in 35 patients undergoing elective surgical aortic valve replacement, the mitral annulus underwent significant geometric changes immediately postoperatively. A 16.3% reduction in the mitral annular area was observed. The anterior annulus underwent a greater reduction in length compared to the posterior annulus, which suggested mechanical compression by the prosthetic valve [<xref rid="B98" ref-type="bibr">98</xref>].</p></sec><sec><title>THE AORTIC VALVE</title><p>Considering its 3D structure, the aortic valve may prove to be one of the most important applications of 3D echocardiography [<xref rid="B104" ref-type="bibr">104</xref>,<xref rid="B105" ref-type="bibr">105</xref>,<xref rid="B106" ref-type="bibr">106</xref>,<xref rid="B107" ref-type="bibr">107</xref>].</p></sec><sec><title>AORTIC VALVE REGURGITATION</title><p>Aortic regurgitation (AR) is caused by valvular abnormalities such as a bicuspid aortic valve, senile degenerative (calcification) valvular disease, and rheumatic valve disease and also by aortic annular dilations such as Marfan syndrome and annular ectasia. In addition, another new type of AR has recently drawn the attention of cardiologists, interventionists, and surgeons due to the development of transaortic valve replacement (TAVR). AR post-TAVR is paravalvular in nature, and its severity is often difficult to determine. Three-dimensional echocardiography, especially real-time 3D TEE, is particularly important for the prevention and diagnosis of this type of AR [<xref rid="B108" ref-type="bibr">108</xref>,<xref rid="B109" ref-type="bibr">109</xref>]. In general, the role of 3D echocardiography in AR evaluation, including this post-TAVR AR, is probably twofold: providing detailed anatomical assessment of the valve and leakage location and size, and quantitative evaluation of the severity of AR [<xref rid="B110" ref-type="bibr">110</xref>].</p><p>Three-dimensional echocardiography, especially real-time 3D TEE, has an advantage over 2D echo methods for visualizing the aortic valve anatomy in depth (<xref ref-type="fig" rid="F8">Figs. 8</xref> and <xref ref-type="fig" rid="F9">9</xref>) [<xref rid="B111" ref-type="bibr">111</xref>]. We reported that real-time 3D TEE could reveal characteristic anatomical differences between type I (annular dilation) and type II (prolapsed) AR [<xref rid="B111" ref-type="bibr">111</xref>].</p><p>As for AR severity, quantitative assessment of AR with 2D echocardiography remains challenging. A recent clinical study in 32 patients with AR reported the accuracy of 2D and 3D transthoracic echocardiography (TTE) for AR quantification, using 3D three-directional velocity-encoded (VE)-MRI as the reference method. With color Doppler TTE, the 2D area was calculated using PISA. From the 3D TTE multiplanar reformation data, the 3D area was calculated using planimetry of the VC. Regurgitant volumes were obtained by multiplying the 2D and 3D area by the velocity-time integral of the AR jet, then compared with those obtained using VEMRI. For the entire population, the 3D TTE-derived regurgitant volume was highly correlated to the VEMRI-derived regurgitant volume (<italic>r</italic> = 0.94 and -13.6 to 15.6 mL per beat, respectively). In contrast, the 2D TTE-derived regurgitant volume showed a modest correlation and large limits of agreement with the VEMRI (<italic>r</italic> = 0.70 and -22.2 to 32.8 mL per beat, respectively). The investigators concluded that AR regurgitant volume quantification using 3D TTE is accurate, and is particularly advantageous over 2D TTE in patients with eccentric jets [<xref rid="B112" ref-type="bibr">112</xref>]. However, AR volume quantification is not often required in a clinical setting. Thus, 3D echo quantification as reported above may be impractical in many cases.</p></sec><sec><title>AORTIC VALVE STENOSIS</title><p>Aortic stenosis (AS) is either congenital (usually bicuspid) or acquired (degenerative calcific valve). The normal aortic valve area is ~3 to 4 cm<sup>2</sup>. In this current aging population, degenerative calcific aortic valve stenosis is the most commonly detected by conventional 2D echocardiography. In one clinical study on AS, 3D echocardiographic methods for planimetry of the aortic valve area showed good agreement with the standard TEE technique in patients with AS [<xref rid="B106" ref-type="bibr">106</xref>]. Also, 3D planimetry methods were at least as good as standard TEE and had better reproducibility [<xref rid="B106" ref-type="bibr">106</xref>]. The authors concluded that 3D aortic valve planimetry is a novel non-invasive technique that provides an accurate and reliable quantitative assessment of AS [<xref rid="B106" ref-type="bibr">106</xref>]. However, the image quality of the aortic valve with TTE is often suboptimal, which may hinder measurement of the smallest valve area. TEE is certainly superior to TTE in this regard. Multiple publications have reported the usefulness of 3D TEE, especially real-time 3D TEE for this purpose [<xref rid="B113" ref-type="bibr">113</xref>,<xref rid="B114" ref-type="bibr">114</xref>]. We reported better agreement between the continuity aortic valve area and planimetry area with the use of real-time 3D TEE than with conventional 2D echocardiography [<xref rid="B114" ref-type="bibr">114</xref>]. The advantage of 3D over 2D echocardiography is evidenced by the ability of the 2D plane to search for the smallest valve area in the 3D space (<xref ref-type="fig" rid="F10">Fig. 10</xref>) [<xref rid="B114" ref-type="bibr">114</xref>].</p></sec><sec><title>THE AORTIC ANNULUS</title><p>The geometry and size of the left ventricular outflow tract (LVOT) or the aortic annulus has become the focus of 3D computed tomography (CT) and echo research because of the development of TAVR and its residual AR. Greater than mild paravalvular AR after TAVR is reportedly a poor prognostic indicator. Thus, proper sizing of the aortic annulus is necessary for the success of TAVR. CT and 3D echocardiography, especially real-time 3D TEE, have contributed to the analysis of the shape and size of the aortic annulus or LVOT where the new aortic valve will be placed. Three-dimensional imaging techniques, including 3D echocardiography, can demonstrate that the shape of the annulus is not circular, but oval. Thus, 3D methods, including CT and 3D TEE, should be used to evaluate the aortic annulus area because 2D imaging techniques provide only a sagittal view, which may underestimate it [<xref rid="B109" ref-type="bibr">109</xref>,<xref rid="B114" ref-type="bibr">114</xref>,<xref rid="B115" ref-type="bibr">115</xref>,<xref rid="B116" ref-type="bibr">116</xref>,<xref rid="B117" ref-type="bibr">117</xref>]. In conjunction with this, assessment of LVOT stroke volume with 3D echocardiography is more accurate than that by the conventional 2D continuity method [<xref rid="B107" ref-type="bibr">107</xref>,<xref rid="B114" ref-type="bibr">114</xref>]. Thus, 3D echocardiography is highly recommended over 2D echo for determining the LVOT area [<xref rid="B114" ref-type="bibr">114</xref>].</p><p>Regarding the subaortic membrane, multiplane analysis of 3D datasets is reportedly a sensitive and accurate method for delineation of morphological details of discrete sub-AS, adding to information gained from 2D echocardiography [<xref rid="B118" ref-type="bibr">118</xref>]. Recently, we reported the value of real-time 3D TEE for evaluating dynamic changes in the LVOT in both the subaortic membrane and in obstructive hypertrophic cardiomyopathy (<xref ref-type="fig" rid="F11">Fig. 11</xref>) [<xref rid="B119" ref-type="bibr">119</xref>].</p></sec><sec><title>THE TRICUSPID VALVE</title><p>Assessment of TV size and function plays an important role in a number of disease states. However, all three TV leaflets (septal, anterior, and posterior) cannot be visualized in one cross-sectional view using either transthoracic or transesophageal 2D echocardiography. In contrast, 3D echocardiography allows visualization of the entire TV from any perspective (<xref ref-type="fig" rid="F12">Fig. 12</xref>). This capability significantly improves our understanding of the pathophysiological mechanism underlying various TV diseases.</p></sec><sec><title>TRICUSPID VALVE REGURGITATION</title><p>Causes of tricuspid regurgitation (TR) may be classified into two major categories, primary and secondary, as in MR. The former is caused by an anatomical abnormality of the TV itself while the latter is caused not by the valve itself, but by abnormalities of the surrounding or supporting structures, such as tricuspid annular dilation and/or RV dilation and dysfunction and pulmonary hypertension. Two-dimensional echocardiography is widely used to evaluate the cause and severity of TR. However, its clinical utility is far from perfect.</p><p>Three-dimensional echocardiography has been reported to be advantageous over 2D echocardiography for evaluation of the anatomical abnormalities of the TV and the location of the TR orifice. Primary TR is caused by a variety of anatomical abnormalities that can be better visualized using 3D than 2D echocardiography, including an apically located leaflet in Epstein disease, or a thickened and restricted leaflet in carcinoid syndrome [<xref rid="B120" ref-type="bibr">120</xref>,<xref rid="B121" ref-type="bibr">121</xref>,<xref rid="B122" ref-type="bibr">122</xref>,<xref rid="B123" ref-type="bibr">123</xref>,<xref rid="B124" ref-type="bibr">124</xref>,<xref rid="B125" ref-type="bibr">125</xref>,<xref rid="B126" ref-type="bibr">126</xref>]. One important finding with 3D echocardiography is lead-derived TR related to a pacer/device. Multiple publications have reported the usefulness of 3D echocardiography for detecting the location of the lead and its relationship to significant TR [<xref rid="B127" ref-type="bibr">127</xref>,<xref rid="B128" ref-type="bibr">128</xref>,<xref rid="B129" ref-type="bibr">129</xref>]. In one of these studies, 45 of 100 patients showed device-lead TV leaflet interference. The septal leaf let was the most commonly affected (n = 23). On multivariate analysis, the preimplantation VC width and the presence of an interfering lead were independently associated with postdevice TR. Additionally, the presence of an interfering lead was the only factor associated with TR worsening, increasing the likelihood of developing moderate or severe TR. The authors concluded that lead-leaflet interference as seen on 3D echocardiography is associated with TR after device lead placement, suggesting that 3D echocardiography should be used to assess lead interference in patients with significant TR [<xref rid="B127" ref-type="bibr">127</xref>].</p><p>One of the causes of secondary or functional TR is reportedly dilation of the tricuspid annulus, which can be determined using 3D echocardiography [<xref rid="B130" ref-type="bibr">130</xref>]. In another clinical 3D echocardiographic study of 54 patients with various degrees of functional TR, its severity was determined based mainly on septal leaf let tethering, septal-lateral annular dilatation, and the severity of pulmonary hypertension [<xref rid="B131" ref-type="bibr">131</xref>].</p></sec><sec><title>THE TRICUSPID ANNULUS</title><p>The geometry and size of the tricuspid annulus have been investigated using 3D echocardiography [<xref rid="B101" ref-type="bibr">101</xref>,<xref rid="B102" ref-type="bibr">102</xref>,<xref rid="B130" ref-type="bibr">130</xref>,<xref rid="B131" ref-type="bibr">131</xref>,<xref rid="B132" ref-type="bibr">132</xref>,<xref rid="B133" ref-type="bibr">133</xref>]. We found that a normal tricuspid annulus has a unique 3D geometry (<xref ref-type="fig" rid="F13">Fig. 13</xref>) [<xref rid="B130" ref-type="bibr">130</xref>]. With the 3D geometric concept, a new annuloplasty ring for a tricuspid annulus was developed and used in patients with severe TR (<xref ref-type="fig" rid="F12">Fig. 12</xref>) [<xref rid="B134" ref-type="bibr">134</xref>]. Short term results with this new annuloplasty ring appear to be satisfactory [<xref rid="B135" ref-type="bibr">135</xref>]. Three-dimensional echocardiography has been valuable not only for understanding complicated cardiac structures but also for developing new strategies, such as this new annuloplasty ring, for treatment of patients.</p></sec><sec><title>INFECTIVE ENDOCARDITIS</title><p>The current diagnostic protocol for endocarditis does not include 3D echocardiography, mainly because 2D echocardiography, especially 2D TEE, is fully capable in this regard. The author admits that 3D TEE is less sensitive than 2D TEE for detecting small vegetations due to its lower image quality. However, the shape, location, and extension of the endocarditis findings, including vegetations, perforations, and abscesses, are evaluated with greater accuracy and in more detail than with conventional 2D echocardiography (<xref ref-type="fig" rid="F14">Figs. 14</xref> and <xref ref-type="fig" rid="F15">15</xref>) [<xref rid="B20" ref-type="bibr">20</xref>,<xref rid="B65" ref-type="bibr">65</xref>,<xref rid="B136" ref-type="bibr">136</xref>,<xref rid="B137" ref-type="bibr">137</xref>,<xref rid="B138" ref-type="bibr">138</xref>]. It is almost impossible to image the entirety of a complicated mobile vegetation with 2D echocardiography. In contrast, 3D echocardiography, especially real-time 3D TEE, facilitates visualization of the full extension and motion of the complicated vegetation in one view from any desired angle (<xref ref-type="fig" rid="F15">Fig. 15</xref>). As a result, the maximum size of the vegetation was underestimated by 2D TEE as compared to 3D TEE (a mean difference of 3.2 mm) in our recent study [<xref rid="B138" ref-type="bibr">138</xref>].</p><p>In summary, 3D echocardiography has been shown to be useful for clarifying complicated valvular anatomy. In particular, real-time 3D TEE has reduced the technical and quality problems of previous 3D echocardiography and has resulted in widespread use of 3D echocardiography in patients with valvular heart disease.</p></sec>
The scientific achievements of the decades in Korean Acute Myocardial Infarction Registry
<p>The Korea Acute Myocardial Infarction Registry (KAMIR) was the first nationwide registry data collection designed to track outcomes of patients with acute myocardial infarction (AMI). These studies reflect the current therapeutic approaches and management for AMI in Korea. The results of KAMIR could help clinicians to predict the prognosis of their patients and identify better diagnostic and treatment tools to improve the quality of care. The KAMIR score was proposed to be a predictor of the prognosis of AMI patients. Triple antiplatelet therapy, consisting of aspirin, clopidogrel and cilostazol, was effective at preventing major adverse clinical outcomes. Drug-eluting stents were effective and safe in AMI patients with no increased risk of stent thrombosis. Statin therapy was effective in Korean AMI patients, including those with very low levels of low density cholesterol. The present review summarizes the 10-year scientific achievements of KAMIR from admission to outpatient care during long-term clinical follow-up.</p>
<contrib contrib-type="author"><name><surname>Kim</surname><given-names>Hyun Kuk</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Jeong</surname><given-names>Myung Ho</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Seung Hun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sim</surname><given-names>Doo Sun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Hong</surname><given-names>Young Joon</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ahn</surname><given-names>Youngkeun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Chong Jin</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Cho</surname><given-names>Myeong Chan</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Young Jo</given-names></name><xref ref-type="aff" rid="A4">4</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>The Korea Acute Myocardial Infarction Registry (KAMIR) was the first nationwide, population-based, multicenter registry, and was launched in November 2005. The ethics committee at each participating institution approved the study protocol. Informed consent was obtained from each patient. Trained study coordinators at each participating institution collected the data using a standardized format. Standardized definitions of all variables were determined by the steering committee board of KAMIR. Data were submitted via password-protected web-based electronic case report forms.</p><p>KAMIR is an ongoing registry that covers the complete spectrum of acute myocardial infarction (AMI), including long-term clinical follow-up. The registry included 55 representative centers, and contained data from ~52,000 patients over a 10-year period. A total of 122 publications have used KAMIR data, including 103 articles in Science Citation Index journals. The most important aspect of this great accomplishment is that the clinical data have been made available to all participating centers without limitations. The present review focuses on the results of publications regarding AMI, from admission to the outpatient department, over a 1-year follow-up. The publications were divided into three categories based on predictors of prognosis, treatment, and special patient groups.</p></sec><sec><title>PREDICTORS OF PROGNOSIS</title><sec><title>Risk stratification</title><p>Risk assessment at the time of admission and discharge is important for deciding treatment strategy and postdischarge management. Thrombolysis in myocardial infarction (TIMI) risk score and the Global Registry of Acute Coronary Event (GRACE) risk models are examples of tools for risk stratification. However, these models were developed and validated from the late 1990s to the early 2000s. The rate of percutaneous coronary intervention (PCI) and use of clopidogrel was only 30% during that period, whereas these treatments are used in approximately 90% of patients currently. Furthermore, previous models did not consider risk factors such as: admission hyperglycemia, the presence of stroke or peripheral artery disease, and left ventricular systolic function. Most importantly, TIMI and GRACE risk scores focused predominantly on western AMI patients. KAMIR score was developed for admission and postdischarge risk prediction over a 1-year period [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>]. The KAMIR score is composed of six independent variables related to 1-year mortality: age, Killip class, serum creatinine, no in-hospital PCI, left ventricular ejection fraction, and admission glucose (<xref ref-type="fig" rid="F1">Fig. 1</xref>). The KAMIR score exhibited significant differences in prediction accuracy compared with the GRACE model in a validation cohort (<xref ref-type="table" rid="T1">Table 1</xref>). The CHA2DS2-VASc scoring system was also useful for risk stratification in patients with AMI, irrespective of atrial fibrillation [<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>].</p></sec><sec><title>Prognostic factors in the admission period</title><sec><title>Coronary risk factors</title><p>Hypertensive AMI patients had worse outcomes, but antecedent hypertension was not associated with 1-year mortality [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>]. Patients with prior cerebrovascular disease (CVD) presented more frequently with non-ST segment elevation myocardial infarction (NSTEMI), were less likely to receive reperfusion therapy, and had worse clinical outcomes compared with patients without a history of CVD [<xref rid="B7" ref-type="bibr">7</xref>]. A family history of coronary artery disease was an independent prognostic factor, particularly in patients with low risk profiles and female gender [<xref rid="B8" ref-type="bibr">8</xref>]. Surprisingly, both smoking and obesity were risk factors for AMI development, but were associated with decreased mortality rates after AMI. These phenomena were also observed in KAMIR [<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>].</p></sec><sec><title>Arrival timing</title><p>A disparity between ST-segment elevation myocardial infarction (STEMI) and NSTEMI was observed [<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>]. Patients admitted during off-hours had a longer waiting time until their procedure, and received less PCI compared with on-hours visitors. In contrast, there was no clinical impact in patients with NSTEMI [<xref rid="B15" ref-type="bibr">15</xref>]. However, in-hospital mortality was increased in patients with STEMI [<xref rid="B16" ref-type="bibr">16</xref>]. An additional report showed that the time delay in STEMI was not associated with in-hospital mortality [<xref rid="B17" ref-type="bibr">17</xref>]. In addition to treatment delay, the incidence of AMI varied according to the season and month. Meteorological parameters, including air temperature, humidity, and sunshine duration, had a significant influence on the occurrence of AMI, particularly in young and female patients [<xref rid="B18" ref-type="bibr">18</xref>].</p></sec><sec><title>Clinical findings</title><p>Painless AMI occurred in individuals that were older, female or non-smokers, or had diabetes mellitus (DM) or renal failure; these patients had worse outcomes [<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>]. N-terminal pro-B-type natriuretic peptide levels were associated with short-term mortality in patients with primary PCI [<xref rid="B21" ref-type="bibr">21</xref>]. Renal dysfunction and C-reactive protein also had prognostic effects for AMI [<xref rid="B22" ref-type="bibr">22</xref>,<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24</xref>].</p></sec><sec><title>Coronary artery lesions</title><p>Unprotected left main artery-related AMI resulted in fatal in-hospital outcome, even in cases with non-culprit lesions [<xref rid="B25" ref-type="bibr">25</xref>,<xref rid="B26" ref-type="bibr">26</xref>]. However, no significant differences were detected in hospital survivors during long-term follow-up [<xref rid="B27" ref-type="bibr">27</xref>,<xref rid="B28" ref-type="bibr">28</xref>]. Patients with NSTEMI and total occluded infarct-related artery had a worse outcome than did those with non-occluded NSTEMI [<xref rid="B29" ref-type="bibr">29</xref>]. Left circumflex artery-related AMI was associated with reduced ST-segment elevation and treatment-delay [<xref rid="B30" ref-type="bibr">30</xref>]. Interestingly, AMI patients with non-significant stenosis in a coronary angiogram had a similar outcome as patients with one- or two-vessel disease [<xref rid="B31" ref-type="bibr">31</xref>].</p></sec></sec></sec><sec><title>TREATMENT OF AMI</title><sec><title>Pharmaceutical management</title><p>Medical treatment is an essential option and interventional management in patients with AMI. Intensive medical treatment might improve the in-hospital outcome of patients who did not undergo PCI [<xref rid="B32" ref-type="bibr">32</xref>]. &#x003b2;-Blocker treatment reduced the long-term mortality in patients with MI and preserved systolic function during PCI [<xref rid="B33" ref-type="bibr">33</xref>]. Insurmountable angiotensin II receptor blockers (ARB) were more effective than ARB in AMI [<xref rid="B34" ref-type="bibr">34</xref>]. Dual blockage of the renin-angiotensin system using angiotensin-converting enzyme inhibitor and ARB could increase the risk of adverse outcomes in severe renal dysfunction patients, without eliciting any clinical benefits [<xref rid="B35" ref-type="bibr">35</xref>]. Trimetazidine and nicorandil were not class I drugs in AMI. Additive effects of the use of these drugs have been reported; however, additional studies are needed [<xref rid="B36" ref-type="bibr">36</xref>,<xref rid="B37" ref-type="bibr">37</xref>].</p><p>The optimal loading dose of clopidogrel in Asian patients has not been investigated fully. Data in the KAMIR revealed that the standard loading dose clopidogrel group had similar clinical results compared with the double loading dose group before PCI [<xref rid="B38" ref-type="bibr">38</xref>,<xref rid="B39" ref-type="bibr">39</xref>]. Low-molecular-weight heparin was associated with a better clinical outcome than unfractionated heparin in patients with AMI [<xref rid="B40" ref-type="bibr">40</xref>,<xref rid="B41" ref-type="bibr">41</xref>,<xref rid="B42" ref-type="bibr">42</xref>].</p></sec><sec><title>Interventional treatment</title><sec><title>Strategy establishment</title><p>Early elective PCI after successful thrombolytic therapy for STEMI remains a controversial recommendation. Patients who underwent PCI within 48 hours of thrombolytic therapy had a long-term clinical benefit compared with those who underwent PCI later [<xref rid="B43" ref-type="bibr">43</xref>]. In early latecomer AMI (12 to 72 hours after symptom onset), PCI was associated with a significant improvement in the 12-month clinical outcome [<xref rid="B44" ref-type="bibr">44</xref>]. The optimal revascularization strategy in AMI patients, culprit-only or total revascularization, remains unclear. KAMIR reported different results between patients with STEMI or NSTEMI. In NSTEMI, multivessel PCI yielded a better clinical outcome without increasing in-stent restenosis and the progression of diseased vessels (hazard ratio [HR], 0.658; 95% confidence interval [CI], 0.45 to 0.96; <italic>p</italic> = 0.031) [<xref rid="B45" ref-type="bibr">45</xref>]. However, multivessel PCI increased target lesion revascularization without any clinical benefit in STEMI [<xref rid="B46" ref-type="bibr">46</xref>].</p></sec><sec><title>Stent implantation in AMI</title><p>Drug-eluting stents (DESs) were implanted in &#x0003e; 90% of AMI patients in Korea (<xref ref-type="table" rid="T2">Table 2</xref>) [<xref rid="B47" ref-type="bibr">47</xref>]. DESs were superior in terms of promoting target lesion revascularization and had similar safety outcomes compared with bare metal stents (BMSs) in patients with large coronary artery and renal insufficiency [<xref rid="B48" ref-type="bibr">48</xref>,<xref rid="B49" ref-type="bibr">49</xref>]. Among the DESs, sirolimus-eluting stents (SESs) and everolimus-eluting stents were similar or better than paclitaxel-eluting stents and early-type zotarolimus-eluting stents in terms of target lesion failure [<xref rid="B50" ref-type="bibr">50</xref>,<xref rid="B51" ref-type="bibr">51</xref>,<xref rid="B52" ref-type="bibr">52</xref>,<xref rid="B53" ref-type="bibr">53</xref>,<xref rid="B54" ref-type="bibr">54</xref>,<xref rid="B55" ref-type="bibr">55</xref>]. Intravascular ultrasound-guided PCI did not reduce restenosis and thrombosis in AMI patients [<xref rid="B56" ref-type="bibr">56</xref>]. Thrombus aspiration did not improve the clinical outcome of overall patients who underwent primary PCI. In a subgroup analysis, left anterior descending artery occlusion patients had a better prognosis when thrombus aspiration was performed. In addition, the use of a glycoprotein IIb/IIIa inhibitor had a synergistic effect with thrombus aspiration [<xref rid="B57" ref-type="bibr">57</xref>].</p></sec></sec><sec><title>Secondary prevention</title><sec><title>Statin after AMI</title><p>The optimal medical therapy after discharge was prescribed at suboptimal rates, particularly in high-risk AMI patients [<xref rid="B58" ref-type="bibr">58</xref>,<xref rid="B59" ref-type="bibr">59</xref>]. Early and continuous statin therapy improved the prognosis after AMI [<xref rid="B60" ref-type="bibr">60</xref>]. Lifesaving medications, including lipid-lowering drugs, were underused in patients with lower low-density lipoprotein cholesterol (LDL-C) levels. More intensive treatments, including lipid-lowering therapy, for AMI in patients with lower LDL-C level might improve clinical outcomes [<xref rid="B61" ref-type="bibr">61</xref>]. Statin therapy in patients with LDL-C levels &#x0003c; 70 mg/dL was also associated with improved clinical outcomes (<xref ref-type="fig" rid="F2">Fig. 2</xref>) [<xref rid="B62" ref-type="bibr">62</xref>]. Stent thrombosis could be reduced by statins in AMI patients with levels of high C-reactive protein [<xref rid="B63" ref-type="bibr">63</xref>]. Statins were also effective in patients with renal dysfunction [<xref rid="B64" ref-type="bibr">64</xref>].</p></sec><sec><title>Antiplatelet treatment</title><p>Dual antiplatelet therapy (DAPT) using aspirin combined with thienopyridine agents such as clopidogrel has become a mainstay of secondary prevention strategies after AMI. Clopidogrel has complimentary effects with aspirin, and reduces adverse cardiac events, including stent thrombosis, compared with aspirin plus warfarin or aspirin alone. However, clopidogrel is a prodrug that requires activation by specific hepatic cytochrome p450 enzymes. This two-step activation process is susceptible to interference by genetic polymorphisms, which can lead to the phenomenon of clopidogrel "nonresponders." Use of an increased dose, addition of other drugs, and development of more potent drugs have been attempted to overcome these shortcomings. In KAMIR, triple antiantiplatelet therapy (TAPT; DAPT + cilostazol) resulted in significantly lower incidences of cardiac death (HR, 0.52; 95% CI, 0.32 to 0.84; <italic>p</italic> = 0.007), total death (HR, 0.60; 95% CI, 0.41 to 0.89; <italic>p</italic> = 0.010), and total major adverse cardiac events (HR, 0.74; 95% CI, 0.58 to 0.95; <italic>p</italic> = 0.019) compared with DAPT in patients with STEMI at the 8-month follow-up [<xref rid="B65" ref-type="bibr">65</xref>]. However, the no-reflow phenomenon is a poor prognostic factor for in-hospital mortality after primary PCI [<xref rid="B66" ref-type="bibr">66</xref>]. Nevertheless, TAPT was superior to DAPT in patients with AMI who had no-reflow during PCI [<xref rid="B67" ref-type="bibr">67</xref>].</p></sec></sec></sec><sec><title>INDIVIDUAL PATIENT SUBGROUPS IN AMI</title><sec><title>Female gender</title><p>There were no gender differences in the initial treatment of AMI. However, the first KAMIR study showed that the success rate of PCI in NSTEMI was lower and the incidence of adverse events after 1 month was higher in females [<xref rid="B68" ref-type="bibr">68</xref>]. However, subsequent studies revealed that female gender itself was not an independent risk factor for in-hospital mortality in a multivariate analysis [<xref rid="B69" ref-type="bibr">69</xref>,<xref rid="B70" ref-type="bibr">70</xref>]. The gender effects might be age-dependent, or due to the presence of more comorbidities and severe hemodynamic conditions at presentation [<xref rid="B71" ref-type="bibr">71</xref>].</p></sec><sec><title>DM and metabolic syndrome</title><p>Generally, DM was a poor prognostic factor for mid-term (1 month to 1 year) clinical outcomes, but not for the short-term prognosis (~1 month) in AMI patients [<xref rid="B72" ref-type="bibr">72</xref>,<xref rid="B73" ref-type="bibr">73</xref>]. Glucose levels at the time of admission were a prognostic factor for short-term clinical outcomes in the KAMIR. Hyperglycemia at admission was an independent predictor of 1-month mortality in non-DM patients, but not in DM patients [<xref rid="B74" ref-type="bibr">74</xref>]. Hyperglycemia at admission was also associated with worse clinical outcomes in DM patients with poorly controlled blood sugar (hemoglobin A1c &#x0003e; 8.0%) [<xref rid="B75" ref-type="bibr">75</xref>]. In addition, metabolic syndrome was an important predictor of in-hospital mortality in STEMI patients, and the prognosis was worsen if DM or renal dysfunction was combined [<xref rid="B76" ref-type="bibr">76</xref>,<xref rid="B77" ref-type="bibr">77</xref>,<xref rid="B78" ref-type="bibr">78</xref>]. In metabolic syndrome patients with multi-vessel disease, coronary artery bypass graft had a favorable outcome in terms of revascularization compared with DES (4.8% vs. 12.2%, <italic>p</italic> = 0.014) [<xref rid="B79" ref-type="bibr">79</xref>]. However, there were no significant differences among different types of DES [<xref rid="B53" ref-type="bibr">53</xref>].</p></sec><sec><title>Renal dysfunction</title><p>The estimated glomerular filtration rate (eGFR) was associated with both short- and long-term mortality rates in AMI patients [<xref rid="B80" ref-type="bibr">80</xref>,<xref rid="B81" ref-type="bibr">81</xref>], and the association between lower eGFR and mortality weakened with increasing age [<xref rid="B82" ref-type="bibr">82</xref>]. However, patients with renal dysfunction had a lower likelihood of receiving optimal medical care [<xref rid="B83" ref-type="bibr">83</xref>]. Early invasive strategies improved the prognosis in patients with renal dysfunction, with the exception of those with severe chronic kidney disease [<xref rid="B84" ref-type="bibr">84</xref>].</p></sec><sec><title>Old age</title><p>PCI for AMI yielded favorable outcomes, even in elderly patients [<xref rid="B85" ref-type="bibr">85</xref>,<xref rid="B86" ref-type="bibr">86</xref>,<xref rid="B87" ref-type="bibr">87</xref>]. Generally, the clinical status of octogenarians was significantly inferior to that of nonoctogenarians in terms of Killip class &#x02265; II (34.8% vs. 22.5%, <italic>p</italic> &#x0003c; 0.001), multivessel disease (65.8% vs. 53.7%, <italic>p</italic> &#x0003c; 0.001). The rate of 1-year all-cause death was significantly higher in octogenarians compared with nonoctogenarians (22.3% vs. 6.5%, <italic>p</italic> &#x0003c; 0.001) [<xref rid="B88" ref-type="bibr">88</xref>]. However, elderly (&#x0003e; 75 years) AMI patients with cardiogenic shock had a similar outcome after PCI as younger patients [<xref rid="B89" ref-type="bibr">89</xref>]. Nonagenarian AMI patients had similar outcomes as octogenarians, despite the higher in-hospital and 1-year mortality rates [<xref rid="B87" ref-type="bibr">87</xref>].</p></sec></sec><sec sec-type="conclusions"><title>CONCLUSIONS</title><p>The KAMIR has generated a multitude of publications. In addition, the KAMIR score provides a simple and accurate assessment tool for improving risk stratification in contemporary clinical practice. The prognosis of AMI patients differs according to the presence of coronary risk factors, the time of arrival at the hospital, and clinical findings. Before the surgical procedure, a standard loading dose of clopidogrel was sufficient for most Korean patients. Low-molecular-weight heparin had a better clinical outcome than did unfractionated heparin. DES implantation in AMI patients reduced target lesion failure compared with BMS, and had similar safety profiles. Among DESs, everolimus-eluting and SESs showed better outcomes. Statins reduced adverse cardiac events, including stent thrombosis, irrespective of cholesterol levels. The addition of cilostazol to a dual antiplatelet agent improved clinical outcomes in patients with STEMI that experienced the no-reflow phenomenon during intervention. Individual patient factors, such as female gender, very old age, and chronic kidney disease, were associated with a poor clinical status, and such patients received guideline-directed treatments less frequently. However, thorough and appropriate management could improve the prognosis in these groups.</p></sec>
Recent advances in treatment of aplastic anemia
<p>Recent advances in the treatment of aplastic anemia (AA) made most of patients to expect to achieve a long-term survival. Allogeneic stem cell transplantation (SCT) from HLA-matched sibling donor (MSD-SCT) is a preferred first-line treatment option for younger patients with severe or very severe AA, whereas immunosuppressive treatment (IST) is an alternative option for others. Horse anti-thymocyte globuline (ATG) with cyclosporin A (CsA) had been a standard IST regimen with acceptable response rate. Recently, horse ATG had been not available and replaced with rabbit ATG in most countries. Subsequently, recent comparative studies showed that the outcomes of patients who received rabbit ATG/CsA were similar or inferior compared to those who received horse ATG/CsA. Therefore, further studies to improve the outcomes of IST, including additional eltrombopag, are necessary. On the other hand, the upper age limit of patients who are able to receive MSD-SCT as first-line treatment is a current issue because of favorable outcomes of MSD-SCT of older patients using fludarabine-based conditioning. In addition, further studies to improve the outcomes of patients who receive allogeneic SCT from alternative donors are needed. In this review, current issues and the newly emerging trends that may improve their outcomes in near futures will be discussed focusing the management of patients with AA.</p>
<contrib contrib-type="author"><name><surname>Shin</surname><given-names>Seung Hwan</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="author-notes" rid="FN1">*</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Sung Eun</given-names></name><xref ref-type="aff" rid="A2">2</xref><xref ref-type="author-notes" rid="FN1">*</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Lee</surname><given-names>Jong Wook</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Aplastic anemia (AA) is an clinical syndrome characterized by fatty replacement and decreased hematopoietic precursors of the bone marrow (BM), which results in peripheral blood (PB) pancytopenia [<xref rid="B1" ref-type="bibr">1</xref>]. It has been suggested that immune-mediated destruction of hematopoietic stem cells by activated cytotoxic T-cells expressing inhibitory cytokines such as interferon-&#x003b3; and tumor necrosis factor-&#x003b1; contribute to its occurrence [<xref rid="B2" ref-type="bibr">2</xref>]. These cytokines induces death of hematopoietic stem cells, at least partially through the Fas-dependent pathway of apoptosis [<xref rid="B3" ref-type="bibr">3</xref>]. The mechanism of activation of cytotoxic T-cells is unclear, but several potential factors which are associated with antigen recognition, susceptibility of immune response, and secretion of cytokines are found [<xref rid="B4" ref-type="bibr">4</xref>]. The annual incidence of AA is estimated to be 2 per 1,000,000 persons per year in Western countries [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>], but it is relatively higher in the Far East with 4 to 7 per 1,000,000 persons per year [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>]. When patients are diagnosed with AA, careful investigations to exclude other possible cause of pancytopenia with hypocellular BM, including hypocellular myelodysplastic syndrome, paroxysmal nocturnal hemoglobinuria, and late onset inherited BM failure disorders, are needed [<xref rid="B10" ref-type="bibr">10</xref>]. For their therapeutic decision making, disease has been stratified to moderate, severe, and very severe AA based on the degree of pancytopenia and BM hypocellularity [<xref rid="B11" ref-type="bibr">11</xref>].</p><p>Four decades ago, when there were only a few effective treatment options, patients with severe or very severe aplastic anemia (SAA) died 1 to 2 years after diagnosis due to fatal infections and/or hemorrhagic complications. However, recent treatment options including allogeneic stem cell transplantation (SCT) and immunosuppressive treatment (IST), mainly anti-thymocyte globuline (ATG) with cyclosporin A (CsA), have helped them to expect a long-term survival [<xref rid="B12" ref-type="bibr">12</xref>]. Nevertheless, there are still many challenges in management of patients with AA; approximately half of patients fail to achieve hematologic response and experience relapse or clonal evolution associated with myelodysplasia and leukemia after IST. On the other hand, a substantial proportion of patients who need MSD-SCT, especially those who experienced failure to a first-line IST, are not able to receive transplantation due to donor unavailability or significant comorbidities. In addition, graft failure and serious complications including graft-vs-host disease (GVHD) and fatal infections after allogeneic SCT limit its more relevant applications [<xref rid="B12" ref-type="bibr">12</xref>]. In this review, current issues and the newly emerging trends that may improve their outcomes in near futures will be discussed focusing the management of patients with AA.</p></sec><sec><title>CURRENT TREATMENT SCHEME IN PATIENTS WITH SAA</title><p>Based on recent reports for the management of patients with SAA, a treatment algorithm is represented with <xref ref-type="fig" rid="F1">Fig. 1</xref>. Allogeneic SCT from HLA-matched sibling donor (MSD-SCT) is the most preferred first-line treatment option for younger (&#x02264; 50 years) SAA patients with a suitable donor and no significant comorbidities. Other patients can receive IST with ATG/CsA as their initial treatment. Allogeneic SCT from available HLA-matched donor should be considered as a salvage treatment in patients who fail to achieve response to first-line IST. If they cannot receive it, appropriate alternative treatment options, including novel IST regimen, allogeneic SCT from familial mismatched donor (FMD-SCT), androgen, and supportive care, should be offered according to their individual circumstances.</p></sec><sec><title>IMMUNOSUPPRESSIVE TREAMTENT</title><sec><title>Historical aspect of IST for patients with AA: horse ATG/CsA as a standard IST regimen</title><p>Before allogeneic SCT and IST were relevantly used to the treatment of the patients with AA, high-dose androgen and/or supportive care including blood transfusion were the only available treatments, but their efficacy was observed in only a small proportion of patients [<xref rid="B12" ref-type="bibr">12</xref>]. Based on the recovery of autologous BM function after receiving horse ATG or allogeneic BM infusion with horse ATG conditioning [<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>], many investigators evaluated the efficacy of horse ATG alone for patients with AA and reported that a substantial proportion of patients could achieve hematologic response [<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>]. After Stryckmans et al. [<xref rid="B19" ref-type="bibr">19</xref>] reported the ability to induce hematological improvement of CsA, a phase III prospective study conducted by Frickhofen et al. [<xref rid="B20" ref-type="bibr">20</xref>] showed that patients who received IST using a combination of horse ATG and CsA achieved a higher overall response rate compared to those using horse ATG alone (70% vs. 46% at 6 months, <italic>p</italic> &#x0003c; 0.050). In addition, a subsequent report for long-term outcomes of this study showed a higher failure-free survival rate (39% vs. 24% at 11 years, <italic>p</italic> = 0.040) in the horse ATG/CsA group, but similar overall survival rate (58% vs. 54% at 11 years, <italic>p</italic> = 0.600) between the two groups because most nonresponders received salvage therapy [<xref rid="B21" ref-type="bibr">21</xref>]. Thereafter, other studies validated the efficacy horse ATG/CsA, which showed satisfactory results with overall response and overall survival rates of approximately 60% to 70% and 60 to 90%, respectively. Based on these reports, horse ATG/CsA has been accepted as a standard IST regimen for patients with AA [<xref rid="B22" ref-type="bibr">22</xref>,<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24</xref>].</p></sec><sec><title>Pretreatment factors predicting response and relapse rates of ATG/CsA for patients with AA</title><p>Although several studies showed acceptable outcomes of ATG/CsA, there were only limited reports for pretreatment factors predicting response and relapse rates of patients with AA who received it. A large retrospective study by the US National Institutes of Health (NIH) group to determine whether pretreatment blood counts can predict response rate for patients receiving horse ATG/CsA showed that younger age, higher absolute reticulocyte count (ARC) and absolute lymphocyte count (ALC) were associated with increased response rate. In this study, patients with higher ARC and ALC (&#x02265; 25 &#x000d7; 10<sup>9</sup>/L and &#x02265; 1 &#x000d7; 10<sup>9</sup>/L, respectively) achieved a higher overall response rate compared to those with lower ARC and ALC (83% vs. 41%, <italic>p</italic> &#x0003c; 0.001) [<xref rid="B25" ref-type="bibr">25</xref>]. In other studies, patients with HLA-DR15, normal cytogenetics, and paroxysmal nocturnal hemoglobinuria clone had higher overall response rate and quality of response compared to those without [<xref rid="B26" ref-type="bibr">26</xref>,<xref rid="B27" ref-type="bibr">27</xref>,<xref rid="B28" ref-type="bibr">28</xref>].</p><p>On the other hand, Saracco et al. [<xref rid="B29" ref-type="bibr">29</xref>] compared the relapse rate between those who responded to horse ATG/CsA that received rapid (0.8 mg/kg/month) and slow CsA (&#x02264; 0.7 mg/kg/month) tapering after maintaining stable blood counts lasting 3 or more months, and reported that was the relapse rate was higher in the rapid tapering group (60% vs. 8% at 10 years, <italic>p</italic> = 0.001). This study suggests that tapering CsA should be initiated after at least 1 year from treatment and taper carefully by 10% every month. Late relapse also can be observed in several long-term responders, which may have resulted from suboptimal therapeutic range of CsA by intolerability or poor compliance [<xref rid="B30" ref-type="bibr">30</xref>]. In addition, the NIH group reported that shorter telomere length of PB leukocytes at diagnosis was associated with higher relapse (odds ratio [OR] 0.16; 95% confidence interval [CI], 0.03 to 0.69; <italic>p</italic> = 0.010) and clonal evolution (OR, 0.29; 95% CI, 0.11 to 0.76; <italic>p</italic> = 0.010) rates with a lower overall survival (OR, 0.35; 95% CI, 0.17 to 0.73; <italic>p</italic> = 0.005) rate, but was not associated with overall response rate [<xref rid="B31" ref-type="bibr">31</xref>]. Conversely, Sakaguchi et al. [<xref rid="B32" ref-type="bibr">32</xref>] reported that pediatric patients with shorter telomere length of PB lymphocytes at diagnosis was associated with a lower overall response rate (OR, 22.0; 95% CI, 4.19 to 115; <italic>p</italic> &#x0003c; 0.001), but not relapse, clonal evolution, and overall survival rates. Considering these conflicting results, further large prospective studies to validate the relation between the outcomes of ATG/CsA and telomere length at diagnosis are needed.</p></sec><sec><title>The comparison of efficacy between patients with AA who received horse ATG/CsA and rabbit ATG/CsA as a first-line IST</title><p>Horse ATG/CsA had been used as a regimen of choice for first-line IST, whereas rabbit ATG/CsA has been used as a second-line regimen for patients with refractory or relapsed AA [<xref rid="B30" ref-type="bibr">30</xref>]. However, Lymphoglobulin (Sangstat, Lyon, France), which had once been the most widely used preparation of horse ATG, was withdrawn from the market in 2007 due to manufacturing difficulties. Thereafter, horse ATG has become unavailable and replaced with rabbit ATG in most countries.</p><p>Until recently, there have been several studies comparing the outcomes of AA patients who received horse ATG/CsA and rabbit ATG/CsA as a first-line IST (<xref ref-type="table" rid="T1">Table 1</xref>). A phase III randomized prospective study conducted by the NIH group showed that the horse ATG/CsA group showed higher overall response (68% vs. 37% at 6 months, <italic>p</italic> &#x0003c; 0.010) and overall survival (96% vs. 76% at 3 years, <italic>p</italic> = 0.040) rates compared to the rabbit ATG/CsA group [<xref rid="B33" ref-type="bibr">33</xref>]. A retrospective study by Brazilian group also reported that horse ATG/CsA group showed higher overall response (60% vs. 35% at 6 months, <italic>p</italic> &#x0003c; 0.050) and overall survival (78% vs. 55% at 2 years, <italic>p</italic> = 0.030) rates [<xref rid="B34" ref-type="bibr">34</xref>]. However, a phase II prospective study comparing the historical horse ATG/CsA control group conducted by the Cleveland Clinic and a retrospective study by Korean group showed similar overall response (58% vs. 50% at 1 year, <italic>p</italic> = 0.610; and 46% vs. 49% at 1 year, <italic>p</italic> = 0.740, respectively) and overall survival (64% vs. 65% at 5 years, <italic>p</italic> = 0.540; and 84% vs. 83% at 5 year, <italic>p</italic> = 0.460, respectively) rates were observed between two groups [<xref rid="B35" ref-type="bibr">35</xref>,<xref rid="B36" ref-type="bibr">36</xref>]. On the other hand, another phase II prospective study comparing the historical horse ATG/CsA control group conducted by the European Group for Blood and Marrow Transplantation (EBMT) as well as a recent large pediatric retrospective study by East Asian Group, showed similar overall response (67% vs. 60%, <italic>p</italic> &#x0003e; 0.050; and 60% vs. 55% at 6 months, <italic>p</italic> = 1.000) rates, but the horse ATG/CsA group showed higher overall survival (86% vs. 68% at 2 years, <italic>p</italic> = 0.010; and 92% vs. 87%, <italic>p</italic> &#x0003c; 0.010, respectively) rates compared to the rabbit ATG/CsA group [<xref rid="B37" ref-type="bibr">37</xref>,<xref rid="B38" ref-type="bibr">38</xref>].</p><p>Compared to horse ATG, rabbit ATG evokes more prolonged and profound lymphocytopenia, which leads to a lower absolute number of CD4<sup>+</sup> regulatory T-cells [<xref rid="B33" ref-type="bibr">33</xref>,<xref rid="B36" ref-type="bibr">36</xref>]. These findings can explain lower response and survival rates of the rabbit ATG/CsA group in several studies. However, it is suggested that discrepancies of other factors including the dose and duration of CsA administration and ethnic factors may result in conflicting results [<xref rid="B1" ref-type="bibr">1</xref>]. Notably, there has not been any study that showed superior outcomes of rabbit ATG/CsA over horse ATG/CsA. Therefore, horse ATG/CsA should be used preferentially as a first-line IST regimen and rabbit ATG/CsA should be considered only if horse ATG is unavailable.</p></sec><sec><title>Efforts to improve the outcomes of ATG/CsA by the addition of other agents</title><p>Because half of patients with AA experience failure to IST, there have been many efforts to improve its efficacy by adding other agents. However, prospective studies conducted by the NIH group failed to show that the addition of mycophenolate mofetil or silrolimus to horse ATG/CsA results in improved overall response rate and reduced relapse rate [<xref rid="B39" ref-type="bibr">39</xref>,<xref rid="B40" ref-type="bibr">40</xref>]. Meanwhile, a prospective study conducted by Teramura et al. [<xref rid="B41" ref-type="bibr">41</xref>] comparing the outcomes of patients who received horse ATG/CsA with or without granulocyte colony-stimulating factor (G-CSF) showed that patients who received G-CSF achieved higher overall response (77% vs. 57% at 4 years, <italic>p</italic> = 0.030) and lower relapse (15% vs. 42% at 4 years, <italic>p</italic> = 0.010) rates compared to those who did not, but there were no significant difference in overall survival rate (88% vs. 94% at 4 years, <italic>p</italic> = 0.440) and incidence of infectious complications (59% vs. 40%, <italic>p</italic> = 0.070). Conversely, similar overall response rate (73% vs. 66% at 6 years, <italic>p</italic> = 0.540) between the patients who received horse ATG/CsA with and without G-CSF was observed in another randomized study conducted by Tichelli et al. [<xref rid="B42" ref-type="bibr">42</xref>]. Because of conflicting results and insufficient data, many clinical centers have not included routine G-CSF administration with their IST protocol.</p><p>Eltrombopag, an oral form of nonpeptide thrombopoietin mimetic, was developed to induce platelet maturation and release by binding to thrombopoietin receptor (c-MPL) on megakaryocytes in patients with immune thrombocytopenic purpura [<xref rid="B43" ref-type="bibr">43</xref>]. However, a previous report, which showed that patients who lack c-MPL develop a form of congenital BM failure, suggested that thrombopoietin is critical to hematopoietic stem cell development and differentiation [<xref rid="B44" ref-type="bibr">44</xref>]. Based on this observation, a recent phase II prospective study was conducted to determine the eltrombopag can be effective for nonresponders to previous IST. In this study, 11 of 25 enrolled patients (44%) achieved hematological response in at least one-lineage and the normalization of BM tri-lineage hematopoiesis [<xref rid="B45" ref-type="bibr">45</xref>]. In its subsequent study, hematological response in at least one-lineage was observed in 17 of 43 enrolled patients (40%), including seven (16.3%) tri-lineage response. In addition, five patients (11.6%) who achieved sustained response maintained stable blood counts at a median 13 months (range, 1 to 15) after drug discontinuation [<xref rid="B46" ref-type="bibr">46</xref>]. These reports suggest that adding eltrombopag might improve the efficacy of horse ATG/CsA. A phase III randomized study to validate this hypothesis is currently ongoing (<ext-link ext-link-type="uri" xlink:href="www.clinicaltrials.gov">www.clinicaltrials.gov</ext-link> as #NCT01623167).</p></sec><sec><title>Salvage treatment for patients with AA refractory or relapsed to a first-line IST</title><p>Not only MSD-SCT but also allogeneic SCT from HLA-matched unrelated donor (URD-SCT) can be a considerable treatment option for SAA patients who failed to first-line IST. It is supported by a recent phase III prospective study conducted by Kosaka et al. [<xref rid="B47" ref-type="bibr">47</xref>], which showed a higher failure-free survival rate of pediatric patients who received URD-SCT compared to those who received horse ATG/CsA (84% vs. 10% at 5 years, <italic>p</italic> &#x0003c; 0.010) after experiencing failure to first-line IST. However, alternative treatment options, including an additional course of ATG/CsA or other IST regimens, also can be considered for patients who are ineligible for allogeneic SCT.</p><p>Studies for second course of ATG/CsA were not relatively abundant and reported a wide range of response rates. A phase II prospective multicenter study conducted by Di Bona et al. [<xref rid="B48" ref-type="bibr">48</xref>] reported that nonresponders to first-line horse ATG/CsA who received a second course rabbit ATG/CsA showed acceptable overall and complete response rates (70% and 30%, respectively). In addition, a retrospective study by the NIH group showed that overall response rates of a second course rabbit ATG/CsA at 6 months in patients who failed to achieve response or experienced relapse to first-line horse ATG/CsA were 30% and 65%, respectively [<xref rid="B49" ref-type="bibr">49</xref>]. However, a retrospective study by the NIH group showed that non-responders to first-line rabbit ATG/CsA who received a second course horse ATG/CsA achieved relatively poor overall response rate (21% at 3 months) [<xref rid="B50" ref-type="bibr">50</xref>]. Based on this result, It is not recommended second course of ATG/CSA in patients who failed achieve response to first-line rabbit ATG/CsA.</p><p>Alemtuzumab, which is a humanized anti-CD52 IgG1 monoclonal antibody with lymphocytopenic activity, or high-dose cyclophosphamide (CY) has been also used in patients with refractory or relapsed AA. A pilot study conducted by Risitano et al. [<xref rid="B51" ref-type="bibr">51</xref>] for 19 patients with AA (including 13 refractory) showed an overall response rate of 58% and complete response rate of 26% after receiving alemtuzumab (a total dose of 103 mg subcutaneously) and low-dose CsA. In addition, the NIH group investigated the role of alemtuzumab monotherapy (test dose of 1 mg followed by 10 mg/kg/day for 10 days) for patients with refractory or relapsed SAA, which showed overall response rates at 6 months of 37% and 56% with overall survival rates at 3 years of 83% and 86%, respectively [<xref rid="B52" ref-type="bibr">52</xref>]. In these studies, clinically significant infectious complications including cytomegalovirus reactivation and Epstein-Barr virus-related disease was not problematic. On the other hand, a recent report for long-term outcomes of CY (50 mg/kg for 4 days) in 23 patients with refractory AA showed feasible overall and complete response rates (47.8% and 21.7% at 10 years, respectively) with overall survival rate (61.8% at 10 years) [<xref rid="B53" ref-type="bibr">53</xref>]. However, CY for patients with AA should be used with caution because frequent fatal infectious complications followed by delayed neutrophil recovery in patients who received it as a first-line treatment were observed in a previous study [<xref rid="B54" ref-type="bibr">54</xref>].</p></sec></sec><sec><title>ALLOGENEIC SCT FOR PATIENTS WITH SAA</title><sec><title>Allogeneic MSD-SCT</title><p>MSD-SCT is the most preferred treatment option for younger SAA patients with a suitable donor and no significant comorbidities. Until recently, many investigators have explored optimal conditioning regimen for SAA patients who receive MSD-SCT to achieve sustained engraftment with minimal fatal complications (<xref ref-type="table" rid="T2">Table 2</xref>). In the initial experiences of MSD-SCT using CY alone conditioning, high incidence of graft failure was a major challenge, particularly in previous heavily transfused patients [<xref rid="B55" ref-type="bibr">55</xref>]. Subsequently, MSD-SCT using radiation, including local-field and total-body irradiation (TBI), plus CY conditioning was attempted and resulted in a lower incidence of graft failure, but was associated with relatively higher incidences of long-term regimen-related morbidities and mortality [<xref rid="B56" ref-type="bibr">56</xref>,<xref rid="B57" ref-type="bibr">57</xref>,<xref rid="B58" ref-type="bibr">58</xref>,<xref rid="B59" ref-type="bibr">59</xref>]. Thereafter, several investigators attempted MSD-SCT using CY plus ATG conditioning, which might induce both effective immunoablation and lymphoablation [<xref rid="B60" ref-type="bibr">60</xref>,<xref rid="B61" ref-type="bibr">61</xref>,<xref rid="B62" ref-type="bibr">62</xref>,<xref rid="B63" ref-type="bibr">63</xref>]. In a phase II prospective study conducted by the Seattle group, the incidence of neutrophil engraftment, acute and chronic GVHD for patients who received MSD-SCT using CY (200 mg/kg) plus horse ATG (90 mg/kg) conditioning were 95%, 15%, and 34%, respectively. Their overall survival rate was significantly higher compared to that of historical control group who received CY alone conditioning (92% vs. 72% at 3 years, <italic>p</italic> = 0.043) [<xref rid="B60" ref-type="bibr">60</xref>]. In addition, a retrospective study by Ades et al. [<xref rid="B62" ref-type="bibr">62</xref>] comparing the outcomes of patients who received MSD-SCT using CY (200 mg/kg) plus rabbit ATG (10 mg/kg) conditioning and thoraco-abdominal irradiation (TAI, 600 cGy) plus CY (150 mg/kg) conditioning showed that the CY/ATG group was associated with a lower incidence of acute (0% vs. 42%, <italic>p</italic> &#x0003c; 0.001) and chronic GVHD (42% vs. 64%, <italic>p</italic> = 0.025) with higher overall survival rate (OR, 6.20; 95% CI, 1.50 to 25.90; <italic>p</italic> &#x0003c; 0.001) compared to the TAI/CY group.</p><p>On the other hand, minimizing the incidence of GVHD is essential for SAA patients who receive allogeneic SCT, considering that they do not need its graft-versus-leukemia effect. A large registry-based study conducted by EBMT/Center for International Blood and Marrow Transplant Research (CIBMTR) comparing the outcomes of patients who received MSD-SCT using peripheral blood stem cells (PBSCs) and BM as a graft source showed that higher incidence of chronic GVHD (27% vs. 12%, <italic>p</italic> = 0.002) and lower overall survival rate (73% vs. 85% at 5 years, <italic>p</italic> = 0.024) were observed in the PBSC group compared to the BM group, especially in patients younger than 20 [<xref rid="B64" ref-type="bibr">64</xref>]. In addition, a phase III randomized study conducted by Gruppo Trapianto di Midollo Osseo/EBMT comparing the outcomes of patients who received MSD-SCT using CsA plus methotrexate (MTX) and CsA alone GVHD prophylaxis showed that the incidence of transplant-related mortality (3% vs. 15% at 1 year, <italic>p</italic> = 0.070) and overall survival rate (94% vs. 78% at 5 years, <italic>p</italic> = 0.050) were significantly superior in the CsA/MTX group compared to the CsA alone group [<xref rid="B65" ref-type="bibr">65</xref>]. Therefore, BM and CsA/MTX are widely used as the preferable stem cell source and GVHD prophylactic regimen for SAA patients who receive MSD-SCT.</p><p>On the other hand, the upper age limit of SAA patients who are able to receive MSD-SCT remains an unsolved challenge. In a large registry analysis conducted by CIBMTR, the impact of patients' age on transplant outcomes was analyzed. The incidence of neutrophil engraftment was not significantly different in all age groups, whereas patients older than 40 showed a significantly lower incidence of platelet engraftment compared to those younger than 20 (OR, 0.45; 95% CI, 0.25 to 0.83; <italic>p</italic> = 0.010), whereas not compared to those aged between 20 and 40 (OR, 0.60; 95% CI, 0.33 to 1.10; <italic>p</italic> = 0.098). The incidence of mortality for patients older than 40 was significantly higher compared to those of patients younger than 20 (OR, 2.70; 95% CI, 1.89 to 3.87; <italic>p</italic> &#x0003c; 0.001) and aged between 20 and 40 (OR, 1.60; 95% CI, 1.13 to 2.26; <italic>p</italic> = 0.008) [<xref rid="B66" ref-type="bibr">66</xref>]. This study showed that the incidence of mortality increased with age for patients who received MSD-SCT although it should be interpreted with caution because patients in this study cohort received heterogeneous conditioning regimens with mostly receiving CY (200 mg/kg) with or without ATG (71.4%). However, the difficulties of determining the most appropriate first-line treatment for older patients still remain because lower overall response and survival rates were also observed when they received IST [<xref rid="B25" ref-type="bibr">25</xref>].</p><p>On the other hand, a recent phase II prospective study conducted by EBMT which analyzed the outcomes of patients who received MSD-SCT using fludarabine (FLU, 120 mg/m<sup>2</sup>) plus CY (1,200 mg/m<sup>2</sup>) with rabbit ATG (15 mg/kg) showed that no significant difference in overall survival rate was observed between patients older than 40 and those aged between 30 and 40 [<xref rid="B67" ref-type="bibr">67</xref>]. We also reported the outcomes of 82 SAA patients who received MSD-SCT using FLU (180 mg/kg) plus CY (100 mg/kg) with rabbit ATG (10 mg/kg) conditioning. In this study, there was no significant difference in overall survival rate among patients with age lesser than 20, 20 to 39, 40 to 49 and 50 to 59 (88%, 97%, 92%, and 86% at 3 years, respectively, <italic>p</italic> = 0.426) [<xref rid="B68" ref-type="bibr">68</xref>]. Consequently, these reports suggest that FLU-based conditioning may be a promising option to overcome relatively poor outcomes of MSD-SCT in older patients. Therefore, MSD-SCT using FLU-based conditioning should be considered as a feasible first-line treatment for older patients.</p></sec><sec><title>Allogeneic URD-SCT</title><p>URD-SCT can be considered as a treatment option for SAA patients who failed first-line IST or require emergent allogeneic SCT if they have no suitable MSD. Two decades ago, only about one-third of patients who received URD-SCT experienced long-term survival [<xref rid="B69" ref-type="bibr">69</xref>]. Recently, however, relevant applications of high-resolution HLA typing for donor selection and more optimized conditioning regimen improved overall survival rate to approximately 70 to 80% [<xref rid="B70" ref-type="bibr">70</xref>,<xref rid="B71" ref-type="bibr">71</xref>,<xref rid="B72" ref-type="bibr">72</xref>,<xref rid="B73" ref-type="bibr">73</xref>,<xref rid="B74" ref-type="bibr">74</xref>].</p><p>The conditioning regimen for URD-SCT for SAA patients that could minimize the incidence of graft failure and regimen-related toxicities still remains uncertain. An initial phase II prospective study conducted by EBMT which analyzed the outcomes of patients who received URD-SCT using FLU (120 mg/m<sup>2</sup>) plus CY (1,200 mg/m<sup>2</sup>) with rabbit ATG (7.5 mg/kg) conditioning showed that the incidence of graft failure was 18% with an overall survival rate of 73% at 2 years. In this study, patients older than 14 showed a significantly higher incidence of graft failure (32% vs. 5%, <italic>p</italic> = 0.030) with a lower trend of overall survival rate (61% vs. 84% at 2 years, <italic>p</italic> = 0.200) [<xref rid="B70" ref-type="bibr">70</xref>], which suggested that the modification of conditioning was required. In addition, an EBMT registry-based analysis for the outcomes of patients who received URD-SCT using FLU (120 mg/m<sup>2</sup>) plus CY (1,200 mg/m<sup>2</sup>) with rabbit ATG (7.5 mg/kg) &#x000b1; low-dose TBI (200 cGY) conditioning showed that there was no significant difference in overall survival rate between the patients older and younger than 27 when they received low-dose TBI containing regimen (78% vs. 79% at 5 years, <italic>p</italic> &#x0003e; 0.050). However, relatively high incidence of mortalities by graft failure (7%), posttransplant lymphoproliferative disease (4%) and GVHD (4%) of this study remained to be a challenge to overcome [<xref rid="B71" ref-type="bibr">71</xref>]. On the other hand, the Seattle group conducted a dose-escalating study to find the most appropriate TBI dose, when used with CY (200 mg/kg) plus horse ATG (120 mg/kg) conditioning. After escalating or de-escalating 200 cGy based on the development of graft failure or regimen-related toxicities from staring 600 cGy, they found that 200 cGy was the most effective dose in minimizing regimen-related toxicities without sacrificing sustained engraftment [<xref rid="B72" ref-type="bibr">72</xref>]. In addition, we conducted a prospective study of de-escalating TBI to find optimal dose, when used with CY (120 mg/kg), which showed higher overall survival rate in the 800 cGy group was observed compared to the 1,000 and 1,200 cGy groups (92% vs. 40% vs. 44% at 3 years, <italic>p</italic> &#x0003c; 0.001) [<xref rid="B75" ref-type="bibr">75</xref>]. In a subsequent report of the long-term results for patients who received URD-SCT using TBI (800 cGy) plus CY (120 mg/kg) conditioning, all enrolled patients achieved sustained myeloid engraftment after a median of 13 days (range, 8 to 30) with acceptable incidences of acute and chronic GVHD (46% and 50%, respectively) and overall survival rate (88% at 5 years) [<xref rid="B76" ref-type="bibr">76</xref>]. The current available reports of outcomes for SAA patients who received URD-SCT are summarized in <xref ref-type="table" rid="T3">Table 3</xref>.</p><p>Because patients who received URD-SCT experience more frequent and severe GVHD compared to those who received MSD-SCT [<xref rid="B77" ref-type="bibr">77</xref>], more effective GVHD prophylaxis, especially for SAA patients, is needed. A matched-pair analysis based on the Japan Marrow Donor Program registry comparing the outcomes of patients who received URD-SCT using GVHD prophylaxis with FK506 and CsA along with MTX showed that the FK506/MTX group showed a higher overall survival rate (83% vs. 50% at 5 years, <italic>p</italic> = 0.012) with a lower trend for incidence of chronic GVHD (13% vs. 36%, <italic>p</italic> = 0.104) [<xref rid="B78" ref-type="bibr">78</xref>]. On the other hand, several patients unavoidably receive URD-SCT using PBSC as stem cells source and/or from partially HLA-URD. Because they are associated with a higher incidence of GVHD or lower survival rate [<xref rid="B76" ref-type="bibr">76</xref>,<xref rid="B79" ref-type="bibr">79</xref>], we conducted a phase II prospective study of adding low-dose rabbit ATG (2.5 mg/kg) to TBI (800 cGy) plus CY (120 mg/kg) conditioning in patients who received URD-SCT from these donors and compared the historical control group who did not receive additional low-dose rabbit ATG. In this study, the incidence of acute GVHD was significantly decreased (21% vs. 64%, <italic>p</italic> = 0.028), but not chronic GVHD (31% vs. 57%, <italic>p</italic> = 0.247) in patients who received low-dose rabbit ATG compared to the historical control group [<xref rid="B80" ref-type="bibr">80</xref>]. However, further studies are needed to determine whether this strategy is effective for SAA patients.</p></sec><sec><title>Allogeneic FMD-SCT</title><p>FMD-SCT had been considered a treatment option for patients with aggressive hematologic malignancies and lacking HLA-matched donors. Although there are several advantages including universal donor availability without delay, high incidences of graft failure and GVHD due to bidirectional barrier of HLA incompatibilities have been considered as a great concern [<xref rid="B81" ref-type="bibr">81</xref>,<xref rid="B82" ref-type="bibr">82</xref>]. However, recent advances in controlling these challenges extended its application to patients with less aggressive diseases, including SAA.</p><p>Until recently, several investigators have attempted to search optimal conditioning regimen and strategy of graft manipulations for SAA patients who receive FMD-SCT. An initial retrospective study by the Seattle group showed that patients who received unmanipulated FMD-SCT using more intensified conditioning consisted of TBI (1,200 cGy) and CY (120 mg/kg) achieved a higher incidence of sustained engraftment (83% vs. 29%, <italic>p</italic> &#x0003c; 0.050) and overall survival rate (50% vs. 0%, <italic>p</italic> &#x0003c; 0.050) compared to those using CY alone (200 mg/kg) conditioning [<xref rid="B83" ref-type="bibr">83</xref>]. Another retrospective study by Tzeng et al. [<xref rid="B84" ref-type="bibr">84</xref>] showed that no graft failure was observed in six patients who received unmanipulated FMD-SCT using TBI (800 cGY) plus CY (200 mg/kg) conditioning. In a recent retrospective study by the Peking group for patients who received FMD-SCT, all patients achieved sustained engraftment by using intensified conditioning that consisted of CY (200 mg/kg) plus rabbit (2.5 mg/kg) or horse ATG (20 mg/kg) with intravenous busufulan (6.4 mg/kg) following G-CSF primed BM and mobilized PBSC infusion, with acceptable overall survival rate (65% at 2 years) and incidences of acute and chronic GVHD (42% and 56%, respectively) [<xref rid="B85" ref-type="bibr">85</xref>]. In addition, various novel strategies including using posttransplant CY and selective CD3<sup>+</sup> T-cell depleted grafts have been also attempted to improve the outcomes of patients who receive FMD-SCT [<xref rid="B86" ref-type="bibr">86</xref>,<xref rid="B87" ref-type="bibr">87</xref>].</p></sec></sec><sec><title>IRON CHELATION THEARPY FOR PATIENTS WITH AA</title><p>A substantial proportion of patients with AA unavoidably receive blood transfusions as supportive care after receiving available treatments. Long-term regular transfusion may lead to iron overload, which is associated with significant morbidities and mortality [<xref rid="B88" ref-type="bibr">88</xref>,<xref rid="B89" ref-type="bibr">89</xref>]. Deferasirox is an oral iron chelator, whose efficiency has been proven for patients with various transfusion-dependent hematological diseases [<xref rid="B90" ref-type="bibr">90</xref>,<xref rid="B91" ref-type="bibr">91</xref>]. A recent sub-group analysis for 116 patients with AA, conducted by Lee et al. [<xref rid="B92" ref-type="bibr">92</xref>], of a large phase II prospective study which evaluated its efficacy and safety showed that their serum ferritin level significantly decreased (3,254 to 1,854 ng/mL, <italic>p</italic> &#x0003c; 0.001) at 1 year after deferasirox without significant toxicities except mild renal toxicities. A subsequent <italic>post hoc</italic> analysis of this study showed hematologic improvement was observed in a subset of patients (46%) who received deferasirox without concomitant IST with a significant difference in serum ferritin level change from baseline between those who achieved response and those who did not (-46% vs. -28%, <italic>p</italic> = 0.017) [<xref rid="B93" ref-type="bibr">93</xref>]. Other investigators also reported that a substantial proportion of patients who received deferasirox achieved hematological improvement [<xref rid="B94" ref-type="bibr">94</xref>,<xref rid="B95" ref-type="bibr">95</xref>,<xref rid="B96" ref-type="bibr">96</xref>], but further investigations are required to determine which mechanism was involved.</p></sec><sec sec-type="conclusions"><title>CONCLUSIONS</title><p>Although recent advances have helped most AA patients to achieve long-term survival, there are many unsolved challenges in their appropriate management. About half of patients who received IST as a first-line treatment still experience failure. Therefore, further studies are needed to improve the response rate and reduce the incidence of relapse after IST, especially considering the unavailability of horse ATG in many countries. Eltrombopag may be a promising additional agent to improve the efficacy of IST, but further validations for its beneficial effects are needed. In addition, more effective treatment for patients who are ineligible for allogeneic SCT after experiencing failure to first-line IST should be explored.</p><p>On the other hand, the upper age limit of SAA patients who are able to receive MSD-SCT as a first-line treatment should be re-determined, considering the favorable outcomes of older patients who received MSD-SCT using FLU-based conditioning. It may contribute to determine the most appropriate treatment option for them along with further studies to investigate pretreatment factors predicting response and relapse after fist-line IST. In addition, more optimized conditioning and effective GVHD prophylaxis should be also developed to improve the outcomes of allogeneic SCT from alternative donors.</p></sec>
The long-term outcome of balloon dilation versus botulinum toxin injection in patients with primary achalasia
Could not extract abstract
<contrib contrib-type="author"><name><surname>Kim</surname><given-names>Do Hoon</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Jung</surname><given-names>Hwoon-Yong</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<p>See Article on Page <related-article related-article-type="commentary-article" id="d35e100" vol="29" page="738" ext-link-type="pmc">738-745</related-article></p><p>Balloon dilation (BD) is currently the most commonly performed treatment for achalasia. Intrasphincteric botulinum toxin injection (BTI) is also used as an alternative to BD or laparoscopic Heller's myotomy with partial fundoplication. These treatments reduce lower esophageal sphincter (LES) pressure, resulting in improved esophageal emptying by gravity and in improved symptoms, such as dysphagia, regurgitation, chest pain, and weight loss; however, few studies have identified predictors of the long-term outcomes for BD versus BTI in patients with primary achalasia.</p><p>In the current issue of <italic>The Korean Journal of Internal Medicine</italic>, the long-term outcomes of BD versus BTI in patients with primary achalasia from a single institution were compared and the predictors of remission identified [<xref rid="B1" ref-type="bibr">1</xref>]. At a median follow-up of 61 months, BD appeared to be more effica cious than BTI in terms of long-term remission in the enrolled patients with achalasia. Independent factors predicting long-term remission included the treatment type and the difference in LES pressure.</p><p>Botulinum toxin (BT) can impede the release of acetylcholine from cholinergic neurons. Chemical denervation after BTI is intended to lower both basal and residual LES pressure, thereby reducing bolus obstruction [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>]. Commonly, 70% to 80% of referred patients show relieved or improved symptoms within 30 days after the procedure. According to a literature review performed by Bassotti and Annese [<xref rid="B4" ref-type="bibr">4</xref>], a single injection of BT is effective in approximately 85% of patients with achalasia, but its effect diminishes over time to 50% by 6 months and to 30% by 1 year. According to a review by Vaezi and Richter [<xref rid="B5" ref-type="bibr">5</xref>], 26% of patients are resistant to BT and show no clinical response, which is thought to be due to antibodies against the protein [<xref rid="B6" ref-type="bibr">6</xref>].</p><p>Although BTI is safe and easy to perform, it was found to be effective only in short-term evaluations, with reduced benefit within 2 years after injection and eventually no benefit with repeated injections [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>]. Because of these limitations, BTI is best reserved for patients who are too ill to undergo surgery, such as those who are elderly, those whose disease is complicated by overlapping diseases, or those who decline surgery or BD [<xref rid="B9" ref-type="bibr">9</xref>]. BTI is also suitable as a transition during periods in which more invasive treatments are not possible, for example, during pregnancy or temporal use of double or triple antiplatelet therapy. In addition, BTI has been used as a rescue treatment after unsuccessful BD or surgical myotomy [<xref rid="B10" ref-type="bibr">10</xref>]. However, there is increased difficulty with performing esophagomyotomy after BTI [<xref rid="B11" ref-type="bibr">11</xref>].</p><p>BD is the most cost-effective treatment for achalasia over a 5- to 10-year postprocedure period [<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B13" ref-type="bibr">13</xref>]. BD aims to fracture the muscularis propria forcibly, decreasing LES pressure and thereby improving bolus transit through the cardia. According to a review of 1,144 patients across 24 studies with an average follow-up of 37 months, BD showed good to excellent symptom relief in a graded manner in 74%, 86%, and 90% of patients treated with 30-, 35-, and 40-mm balloons, respectively [<xref rid="B8" ref-type="bibr">8</xref>]. Irrespectively of the protocol used, a large portion of patients will relapse, mainly during the first year after treatment [<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>]. After 4 to 6 years, nearly one-third of patients experience symptom relapse [<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>]. However, long-term remission, based on symptom recurrence, can be achieved in almost all patients by repeated BD [<xref rid="B17" ref-type="bibr">17</xref>]. Those patients with the best outcomes following BD tend to be older (&#x0003e; 40 years), female, and to present with type II patterns on high resolution manometry [<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>,<xref rid="B21" ref-type="bibr">21</xref>]. Several studies using long-term follow-up periods are available currently. Eckardt et al. [<xref rid="B22" ref-type="bibr">22</xref>] showed a 5-year follow-up response rate of 40% among patients with unique BD, and patients experiencing a relief in symptoms after 5 years were more likely to continue in this way. Zerbib et al. [<xref rid="B17" ref-type="bibr">17</xref>] reported estimated efficacies of 97% and 93% after 5 and 10 years, respectively, but most frequently in cases of repeated BD. In a study comprising 209 patients with a mean follow-up of 70 months, a 72% success rate with BD was observed [<xref rid="B16" ref-type="bibr">16</xref>]. However, in these studies, BD was not repeated routinely, rather performed on demand only for patients were are still symptomatic. In a meta-analysis performed by Weber et al. [<xref rid="B23" ref-type="bibr">23</xref>], the 10-year remission rate for BD was 47.9%, while the perforation rate was 2.4%. When performed by experienced operators, BD can achieve good to excellent outcomes (defined as an improved swallowing ability and an improved quality of life); however, only a few patients can be definitively treated by a single dilation, with most needing repeated dilations over a long-term follow-up [<xref rid="B24" ref-type="bibr">24</xref>].</p><p>A recent Cochrane Review compared 178 patients from six randomized, controlled trials after esophageal BD versus endoscopic BTI. At the 1-year follow-up, up to 74% of patients who underwent BTI experienced treatment failure, compared with 30% of patients who underwent BD [<xref rid="B25" ref-type="bibr">25</xref>]. In addition, Campos et al. [<xref rid="B26" ref-type="bibr">26</xref>] performed a systematic review and a meta-analysis on 7,855 achalasia patients and found better symptomatic relief in patients treated by BD compared with BTI.</p><p>Perforation is the most serious complication of BD, with an overall rate of 1.9% [<xref rid="B24" ref-type="bibr">24</xref>,<xref rid="B27" ref-type="bibr">27</xref>]. Most such cases should be managed by surgical correction, such as simple closure, second-look operation after simple drainage, or esophagectomy.</p><p>Recently, peroral endoscopic myotomy (POEM) has been introduced as a promising alternative to the current treatments. However, the POEM technique is difficult and requires extensive experience with therapeutic endoscopy. POEM is an elegant treatment resulting in excellent short-term results and is considered an alternative for achalasia. BD and laparoscopic Heller's myotomy have shortcomings, suggesting a need for a better treatment option. A recent POEM survey showed an overall clinical success rate of 98% after a mean follow-up of 9.3 months [<xref rid="B28" ref-type="bibr">28</xref>].</p><p>Therefore, BD should be used as the first-line treatment in Korean patients with achalasia due to its superior long-term clinical success rate. BTI is best reserved for patients who are too ill to undergo surgery and as a suitable transition during periods in which more invasive treatments are not possible. BTI can also be used as rescue treatment after unsuccessful BD or surgical myotomy. However, the number of effective treatments available for achalasia, such as POEM, is increasing. Short-term follow-up data for POEM are promising; however, more long-term follow-up data and prospective randomized trials comparing POEM with BD or surgical myotomy are needed to determine the potential of POEM as a new treatment option for achalasia.</p>
Appropriate candidates for statin use in heart failure
Could not extract abstract
<contrib contrib-type="author" corresp="yes"><name><surname>Seo</surname><given-names>Hong Seog</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<p>See Article on Page <related-article related-article-type="commentary-article" id="d35e93" vol="29" page="754" ext-link-type="pmc">754-763</related-article></p><p>Heart failure (HF) is a clinical syndrome resulting from structural and functional impairments of the heart associated with various cardiovascular diseases and is characterized by impaired cardiac performance, neurohormone imbalance, endothelial dysfunction, and inflammation [<xref rid="B1" ref-type="bibr">1</xref>]. Current guidelines for treating congestive heart failure (CHF) focus on improving cardiac performance and correcting the neurohormone changes [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>] Recently, significant advances have been made in the treatment of HF with renin-angiotensin-aldosterone system blockers, &#x003b2;-blockers, devices, diuretics, and digitalis [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>]. There has been a 40% reduction in the age-standardized death rate due to HF over the past two decades and a concomitant increase in the age of death from HF [<xref rid="B4" ref-type="bibr">4</xref>]. Despite advances in therapy, however, the 5-year mortality of HF is still approximately 50%, which is worse than that of many cancers [<xref rid="B5" ref-type="bibr">5</xref>]. Therefore, further strategies are needed to improve these poor outcomes.</p><p>Cardiac tissue inflammation plays a major role in the initiation and progression of HF [<xref rid="B6" ref-type="bibr">6</xref>]. Endothelial dysfunction is common in patients with CHF and is thought to be related to the clinical complications of CHF [<xref rid="B7" ref-type="bibr">7</xref>]. Endothelial dysfunction in CHF patients is often associated with increased inflammatory cytokines, reactive oxygen species, endothelial cell apoptosis, decreased endothelial nitric oxide synthase expression, reduced blood flow, and shear stress [<xref rid="B8" ref-type="bibr">8</xref>]. Following the initial insult of a cardiac event, increased production of proinflammatory cytokines, including tumor necrosis factor-&#x003b1; (TNF-&#x003b1;), interleukin (IL) 6, IL-1, and IL-18, compromises the cardiac tissue via the increased inflammatory response and through direct effects that alter cardiomyocyte structure and function. Cardiac myocyte hypertrophy, contractile dysfunction, cardiac myocyte apoptosis, and extracellular matrix remodeling are the major mechanisms by which CHF develops and progresses [<xref rid="B9" ref-type="bibr">9</xref>]. Although many of the deleterious effects of inflammatory mediators are potentially reversible once the inflammation subsides, HF remains a progressive process despite optimal therapy [<xref rid="B10" ref-type="bibr">10</xref>]. Consequently, anti-inflammatory strategies can be rationalized in patients with HF.</p><p>Some of these anti-inflammatory strategies include use of anti-TNF-&#x003b1; therapy with monoclonal antibodies [<xref rid="B11" ref-type="bibr">11</xref>] or soluble TNF receptor fusion proteins [<xref rid="B12" ref-type="bibr">12</xref>], &#x003b2;-adrenergic agonists [<xref rid="B13" ref-type="bibr">13</xref>], adenosine [<xref rid="B14" ref-type="bibr">14</xref>], phosphodiesterase inhibitors [<xref rid="B15" ref-type="bibr">15</xref>], amiodarone [<xref rid="B16" ref-type="bibr">16</xref>], ouabain [<xref rid="B17" ref-type="bibr">17</xref>], and estrogen [<xref rid="B18" ref-type="bibr">18</xref>]. In clinical trials, however, the use of either a soluble TNF receptor or an anti-TNF antibody did not benefit patients with HF [<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>], which contrasts with the results of experimental studies. The clinical benefits of other anticytokines in CHF are also not reassuring and patients with severe HF should not be treated with anticytokines [<xref rid="B10" ref-type="bibr">10</xref>].</p><p>The 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, commonly known as statins, have lipid-lowering, and pleiotropic effects, which include anti-inflammatory and plaque-stabilizing actions. Statins are used routinely in patients with coronary artery disease for secondary prevention [<xref rid="B19" ref-type="bibr">19</xref>]. In patients with HF, statins might directly improve the microvascular circulation and endothelial function by stimulating angiogenesis and modulating the synthesis and activity of endothelial nitric oxide synthase and endothelin-1 [<xref rid="B20" ref-type="bibr">20</xref>]. They also have anti-inflammatory and antioxidant effects and reduce the levels of inflammatory biomarkers and cytokines, irrespective of cholesterol levels [<xref rid="B21" ref-type="bibr">21</xref>]. In animal models, statins reduced angiotensin II receptor expression and matrix metalloproteinase secretion [<xref rid="B22" ref-type="bibr">22</xref>], resulting in cardiac remodeling [<xref rid="B23" ref-type="bibr">23</xref>]. All of these effects can play a critical role in HF progression and prognosis. Statins appear to have many pleiotropic effects believed to influence the pathophysiology of HF progression. However, the results of large randomized clinical trials conflict with other clinical studies [<xref rid="B24" ref-type="bibr">24</xref>]. Small prospective trials have suggested that statin treatment in patients with HF has a positive impact that is pleiotropic and independent of any underlying atherosclerotic disease [<xref rid="B25" ref-type="bibr">25</xref>]. Moreover, observational studies and post hoc analyses of randomized trials suggest that statin therapy more strongly influences the prognosis of patients with HF [<xref rid="B26" ref-type="bibr">26</xref>]. Based on these promising findings, large randomized trials of rosuvastatin were performed: the Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardio-Heart Failure (GISSI-HF) trial [<xref rid="B27" ref-type="bibr">27</xref>] and the Controlled Rosuvastatin Multinational Trial in Heart Failure (CORONA) [<xref rid="B28" ref-type="bibr">28</xref>]. The CORONA study was a large, randomized, placebo-controlled trial of rosuvastatin 10 mg versus a placebo in patients with chronic systolic HF of ischemic etiology [<xref rid="B28" ref-type="bibr">28</xref>]. The study enrolled 5,011 patients older than 60 years with New York Heart Association (NYHA) class II symptoms and an ejection fraction of less than 35%, or NYHA class III to IV symptoms and an ejection fraction of less than 40% with an average 3 years of follow-up. Rosuvastatin did not have survival benefits, but did reduce the number of HF hospitalizations in older patients with ischischemic systolic HF. The GISSI-HF trial was a multicenter, randomized, double-blind study that assessed the effect of N-3 polyunsaturated fatty acids and rosuvastatin 10 mg versus placebo on cardiovascular morbidity and mortality of patients with chronic symptomatic HF [<xref rid="B27" ref-type="bibr">27</xref>]. This study enrolled 4,574 HF patients and had broad criteria requiring NYHA class II to IV symptoms of any etiology. There were no exclusions based on ejection fraction or baseline cholesterol levels. Like the CORONA study, the GISSI-HF trial also did not show any significant effect of rosuvastatin on the clinical outcomes of patients with CHF of both ischemic and nonischemic etiologies after 3 years of follow-up. The results of these trials were contrary to what was expected, in that rosuvastatin did not reduce the number of deaths in patients with HF [<xref rid="B27" ref-type="bibr">27</xref>,<xref rid="B28" ref-type="bibr">28</xref>].</p><p>However, several issues should have been considered regarding the assessment of statin benefits in patients with ischemic HF. The first issue is related to the target disease status and the benefit of statin therapy being dependent on heart failure severity. Although many trials have demonstrated that statins reduce cardiovascular morbidity and mortality in patients with ischemic heart disease, this does not seem to hold true in patients with ischemic HF, as the CORONA trial showed [<xref rid="B28" ref-type="bibr">28</xref>]. According to the results of the JUPITER, CORONA, and AURORA trials, it is critical to start statin treatment as early as possible in the cardiovascular continuum [<xref rid="B29" ref-type="bibr">29</xref>]. Initiation of statin therapy when a patient is already at end-stage disease is most likely too late. Although it is vital to control all cardiovascular risk factors along the continuum, the beneficial effects of therapy differ depending on the stage at which treatment is started. Therefore, if statins are prescribed at earlier stages, the improvement in cardiovascular prognosis will be markedly greater than if therapy was initiated during later stages [<xref rid="B30" ref-type="bibr">30</xref>]. In the CORONA trial, the lowest N-terminal pro-B-type natriuretic peptide tertile (&#x0003c; 868 pg/mL) did benefit from rosuvastatin, showing a significant improvement in the primary end point [<xref rid="B31" ref-type="bibr">31</xref>]. It has been suggested that in milder HF, statins can modify coronary events, whereas in severe HF, statin use does not improve the progressive loss of pump function [<xref rid="B32" ref-type="bibr">32</xref>]. Another issue is related to the dose and characteristics of statins. Specifically, trials have shown increasingly beneficial results with higher doses [<xref rid="B33" ref-type="bibr">33</xref>]. Notably, both the CORONA and GISSI-HF trials, which had negative results, were conducted using low-dose rosuvastatin (10 mg). Different dose-dependent effects of atorvastatin treatment on the arterial wall properties and on indices of left ventricular remodeling have also been reported in ischemic HF [<xref rid="B34" ref-type="bibr">34</xref>]. Short-term treatment with atorvastatin 40 mg has beneficial effects on the arterial wall properties and improves the indices of left ventricle remodeling in HF patients, while no significant changes in these parameters occur after treatment with atorvastatin 10 mg/day. Therefore, high-dose statins are recommended for the treatment of HF.</p><p>Regarding the pharmacodynamic properties of statins, hydrophilic statins such as rosuvastatin rely on active transport into hepatocytes to exert their effect and have poor penetration into extrahepatic tissues. Consequently, they have a lower risk of adverse effects, but also have very low uptake by cardiac muscle. However, lipophilic statins including simvastatin tend to achieve higher levels of exposure in nonhepatic tissues and have very high cardiac muscle uptake [<xref rid="B35" ref-type="bibr">35</xref>]. A recent study of a real-life cohort followed for a maximum of 9.1 years showed that lipophilic statins were independently and significantly associated with a lower mortality risk, including patients with nonischemic HF etiology, which contrasts the results of large randomized trials with hydrophilic statins [<xref rid="B24" ref-type="bibr">24</xref>]. Another possible issue is the presence of comorbidities with HF. The results of the CORONA trial might have been influenced by the enrollment of elderly patients, since this age group might have comorbidities that could attenuate the potential benefits of treatment [<xref rid="B36" ref-type="bibr">36</xref>]. Furthermore, the drug could interact with the complex medical therapy typical in older patients with HF.</p><p>In this issue of <italic>The Korean Journal of Internal Medicine</italic>, Lee et al. [<xref rid="B37" ref-type="bibr">37</xref>] report the effects of intense versus mild lipid-lowering programs with statins by analyzing 69 ischemic CHF patients receiving pravastatin 10 mg or pitavastatin 4 mg daily. They found that in CHF patients with ischemic origin, both very-low-dose/low potency pravastatin and high-dose/high-potency pitavastatin had beneficial effects on cardiac remodeling and systolic function. According to the demographic characteristics, patients receiving low-dose pravastatin were younger and had lower levels of serum B-type natriuretic peptide than those receiving high-dose pitavastatin. These are intrinsic group-specific caveats that must be considered when assessing the beneficial effects of statins in patients with ischemic HF. Despite achieving higher low density lipoprotein cholesterol and lower high density lipoprotein cholesterol levels with low-dose pravastatin treatment, the patients in this group exhibited significant improvement only in exercise capacity. These results suggest the importance of patient characteristics when evaluating the effects of statin therapy in ischemic HF, which is consistent with the CORONA trial [<xref rid="B31" ref-type="bibr">31</xref>]. The authors also suggest that lowering cholesterol too aggressively might not be beneficial to CHF patients. They explained the lack of improvement in functional capacity and the possible side effect of fatigue, likely due to the different doses and lipophilicity of statins, and other possible toxic effects of statins, which include those described by the endotoxin lipoprotein, coenzyme Q10 (ubiquinone), and selenoprotein hypotheses. Despite these concerns, statin trials, systematic reviews, and meta-analyses of statin treatment in HF have revealed no detrimental effects and actually suggest favorable effects in HF populations [<xref rid="B38" ref-type="bibr">38</xref>]. In addition, some evidence from recent nonrandomized studies complements the findings of the small randomized trials, but suggests that lipophilic statins provide better outcomes than hydrophilic statins in patients with HF [<xref rid="B24" ref-type="bibr">24</xref>].</p><p>While large randomized trials of statins in HF reported negative results, statins are still expected to have beneficial effects in certain groups of patients with HF because their robust beneficial effects and minimal harmful effects are well documented in atherosclerotic disease, which has an underlying pathophysiology identical to that of HF. To conclude, the effect of statin therapy in ischemic HF suggests that the age of the study population, severity of HF, drug dosage, and pharmacokinetic character of statins must be considered more meticulously when designing clinical trials.</p>
Changes in the epidemiology and burden of community-acquired pneumonia in Korea
Could not extract abstract
<contrib contrib-type="author" corresp="yes"><name><surname>Yoon</surname><given-names>Hyoung Kyu</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<p>See Article on Page <related-article related-article-type="commentary-article" id="d35e93" vol="29" page="764" ext-link-type="pmc">764-773</related-article></p><p>Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Lower respiratory tract infections, including CAP, were the fourth leading cause of death worldwide, with an estimated 2.81 million deaths, and were the leading cause of years of life lost in 1990, and second in 2010 [<xref rid="B1" ref-type="bibr">1</xref>]. CAP is also the disease with the largest socio-economic burden worldwide, as measured using disability-adjusted life years (DALY), accounting for 94.5 million DALYs in 2004 [<xref rid="B2" ref-type="bibr">2</xref>]. Traditionally, death caused by CAP is regarded as the result of an unhygienic environment and inadequate nutrition, which occur mainly in undeveloped countries. CAP is still the leading cause of childhood mortality in developing countries and the most common cause of adult hospitalization. In 2011, 1.3 million children died from pneumonia, most in poor countries.</p><p>Despite the marked improvement in socioeconomic status in Korea, CAP remains a major public health problem. According to Statistics Korea data for 2013, pneumonia skyrocketed from 12th place to 6th place as a cause of death in Korea over the past 10 years, and the pneumonia death rate has increased from 5.7 per 100,000 in 2003 to 21.4 per 100,000 in 2013 [<xref rid="B3" ref-type="bibr">3</xref>]. Attention should be focused on discovering why the incidence of death due to pneumonia, which is considered a disease of unsanitary environments, is increasing in Korea. South Korea is a dramatic success story in terms of the economic growth that has taken place over the past three decades, and the quality of life in Korea equals that in developed countries. Therefore, the old concept of pneumonia death can no longer explain the change in pneumonia death in Korea. According to Statistics Korea for 2013, in 1983, the death rate due to pneumonia was 18.3 per 100,000 in patients younger than 15 years and 53.3 per 100,000 in those older than 65 years. In comparison, in 2013, the death rate in patients younger than 15 years had decreased to 0.2 per 100,000, while it more than tripled in patients older than 65 years, to 166.6 per 100,000. In addition, the death rate of people older than 80 years almost quadrupled from 162.8 per 100,000 in 1983 to 622.1 per 100,000 in 2013 [<xref rid="B3" ref-type="bibr">3</xref>]. Therefore, the issue of pneumonia at younger ages, which causes most of the socioeconomic loss in undeveloped countries, is not a problem in South Korea. The current problem in South Korea is pneumonia at older ages, and the increased death rate in older age groups caused by pneumonia comprises most pneumonia deaths.</p><p>There are many reasons for the changes in death due to pneumonia in Korea. The increased elderly population is a major factor. Korea is rapidly becoming an extremely aged society as it has the world's lowest birth rate. More interestingly, this change is occurring much more rapidly than in other countries with the same experiences. Generally, pneumonia occurs most frequently in the elderly. In addition, advanced age is a risk factor for CAP according to research on the Korean population [<xref rid="B4" ref-type="bibr">4</xref>]. Since the respiratory symptoms of pneumonia are less obvious in the elderly, an early diagnosis is difficult. Consequently, when patients present, they are severely ill and are more likely to be hospitalized than younger pneumonia patients, which results in higher medical costs for caregiver assistance [<xref rid="B5" ref-type="bibr">5</xref>]. Pneumonia patients older than 65 years cost 6.7-fold more than pneumonia patients 15 to 44 years old, and 75% of these medical costs are for hospitalization. Therefore, the prevention of pneumonia in the elderly and an early diagnosis are vital in terms of the effective utilization of healthcare resources.</p><p>The increasing elderly population could explain the increase in the total number of pneumonia deaths, but it cannot entirely explain the increase in the death rate per 100,000 people. This might be explained by the increase in the population with underlying diseases that might function as pneumonia risk factors. The risk factors for pneumonia include chronic obstructive lung disease, cardiovascular disease, diabetes mellitus, and smoking. Compared to individuals with no risk factors, the risk of pneumonia death is increased 4.2-fold in patients with asthma, 3.0-fold with lung disease, and 1.9-fold with cardiovascular disease. These risk factors are very common in old age and are often concomitant. The frequencies of the underlying conditions are factors that affect the clinical outcome of CAP. About half of the deaths in patients with CAP were attributable to the worsening of underlying disease [<xref rid="B6" ref-type="bibr">6</xref>].</p><p>In this issue of <italic>The Korean Journal of Internal Medicine</italic>, Lee and colleague [<xref rid="B7" ref-type="bibr">7</xref>] report the results of a retrospective, observational study of the disease burden of pneumonia in Korean adults older than 50 years. They observed that the elderly and those with underlying disease had a high disease burden related to treatment when hospitalized with CAP. This study has some limitations; e.g., its small sample size and the fact that it was conducted in university hospital. Despite these limitations, this study enhances our understanding of the importance of age and comorbidities in the disease burden of CAP in Korea. Their result also suggests that efforts at improving the control of underlying disease and adequate pneumonia prevention will decrease the burden of CAP.</p><p>The prolonged life expectancy and medical advances in Korea have increased the proportions of patients with CAP at advanced ages and with multiple comorbidities. These improvements have also had side effects: an increase in the number of elderly people at higher risk of pneumonia and death from pneumonia. Another concern is that the numbers of elderly patients in nursing homes and hospitals are increasing. These patients are likely to develop severe pneumonia caused by resistant pathogens, and the probability of death is expected to be higher; this is another critical cause of the increasing number of deaths from pneumonia.</p><p>In Korea, rapid social changes have occurred over the past 40 years with the extremely rapid economic growth, and changes that likely happened in other countries over 100 years have occurred in Korea within a generation. Similarly, the disease trends have changed from those of an undeveloped country to an advanced country over the past 40 years. However, few studies have analyzed these changes and identified the associated problems, including economic issues. Health spending is associated mainly with the proportion of elderly individuals in the population. Especially, the retirement of the 7 million baby boomers born between 1955 and 1963 will be a major challenge for the Korean health care system. In 2010, Korean baby boomers comprised about 15% of the total population. It is estimated that 3.1 million baby boomers are set to retire over the next 10 years. As baby boomers enter retirement, the proportion of the population 65 years and older will continue to increase, accelerating the inflation of health care expenditures and precipitating a crisis in the health insurance system [<xref rid="B8" ref-type="bibr">8</xref>]. These changes might result in increased morbidity and mortality from pneumonia in the near future. Therefore, it is crucial for policy makers and health care providers to provide comprehensive health services to prevent pneumonia. Vaccines targeting pneumococcal disease and influenza remain the mainstay for preventing CAP. Smoking cessation and respiratory hygiene measures, including the use of hand hygiene and masks, should be used to reduce respiratory infections [<xref rid="B9" ref-type="bibr">9</xref>]. Preventive strategies that identify and act on modifiable risk factors are paramount for reducing CAP-related death in the elderly.</p><p>In conclusion, it is mandatory to understand the recent changes in the epidemiology and socioeconomic burden of pneumonia in our country. Establishing a prevention system for this disease should be our next objective. Since the elderly population experiences the greatest number of deaths from pneumonia, an effective prevention system is desperately needed. Cost effectiveness, of course, is a factor, and active vaccination for pneumonia and influenza and other preventive activities for the elderly and higher-risk groups for pneumonia are required.</p>
Long-term outcomes of balloon dilation versus botulinum toxin injection in patients with primary achalasia
<sec><title>Background/Aims</title><p>We compared the long-term outcomes of balloon dilation versus botulinum toxin injection in Korean patients with primary achalasia and identified factors predicting remission.</p></sec><sec><title>Methods</title><p>We included 73 patients with achalasia newly diagnosed between January 1988 and January 2011. We ultimately enrolled 37 of 55 patients with primary achalasia through telephone interviews, who were observed for over 1 year. Short-term outcomes were evaluated from the medical records based on symptom relief after 1 month of treatment. Long-term outcomes were evaluated in a telephone interview using a questionnaire.</p></sec><sec><title>Results</title><p>Twenty-five patients were administered a botulinum toxin injection and 12 underwent balloon dilation. One month after the botulinum toxin injection, improvements were seen in chest pain (14 [56.0%] to 4 patients [16.0%]), regurgitation (16 [64.0%] to 4 [16.0%]), and dysphagia (25 [100.0%] to 5 [20.0%]). In the balloon dilation group, chest pain (8 [66.7%] to 1 [8.3%]), regurgitation (11 [91.7%] to 1 [8.3%]), and dysphagia (12 [100.0%] to 1 [8.3%]) had improved. A significant difference was observed in the mean remission duration between the botulinum toxin injection and balloon dilation groups (13 months [range, 1 to 70] vs. 29 months [range, 6 to 72], respectively; <italic>p</italic> = 0.036). Independent factors predicting long-term remission included treatment type (odds ratio [OR], 6.982; <italic>p</italic> = 0.036) and the difference in the lower esophageal sphincter pressure (OR, 7.198; <italic>p</italic> = 0.012).</p></sec><sec><title>Conclusions</title><p>Balloon dilation may be more efficacious than botulinum toxin for providing long-term remission in Korean patients with achalasia. Follow-up manometry may predict the long-term outcome.</p></sec>
<contrib contrib-type="author"><name><surname>Jung</surname><given-names>Ho Eun</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Lee</surname><given-names>Joon Seong</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Tae Hee</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Jin Nyoung</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Hong</surname><given-names>Su Jin</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Jin Oh</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Hyeon Geon</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Jeon</surname><given-names>Seong Ran</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Cho</surname><given-names>Joo Young</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Achalasia is a primary esophageal motor disorder characterized by incomplete relaxation of the lower esophageal sphincter (LES) and the absence of esophageal peristalsis caused by degeneration of Auerbach's plexus [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>]. The goal of therapy is to reduce LES pressure (LESP), resulting in improved esophageal emptying by gravity and improved symptoms, such as dysphagia, regurgitation, chest pain, and weight loss [<xref rid="B4" ref-type="bibr">4</xref>]. Pharmacological therapy, pneumatic balloon dilation (BD), and intrasphincteric botulinum toxin (Botox, Allergan, Irvine, CA, USA) injection are the medical treatment modalities used most commonly. Pharmacological therapy is ineffective. Pneumatic BD is used commonly and provides good symptomatic relief in 86% to 100% of cases [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>].</p><p>Intrasphincteric Botox injection has been used as an alternative to pneumatic BD or laparoscopic Heller's myotomy with partial fundoplication. Botox, a potent inhibitor of acetylcholine release from nerve endings, relaxes the LES by decreasing unopposed cholinergic stimulation of the LES. Some studies have shown that 65% to 100% of patients respond to a single injection within 1 month, but the effect is sustained for 12 months in only 15% to 75% [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>]. The treatment is very safe and effective in the short term for relieving symptoms and has few complications [<xref rid="B4" ref-type="bibr">4</xref>].</p><p>A few studies have compared the short-term outcomes of BD versus Botox injection in patients with primary achalasia. In a prospective study, Mikaeli et al. [<xref rid="B6" ref-type="bibr">6</xref>] reported that the 12-month remission rate was significantly higher after pneumatic dilation (53%) than after Botox injection (15%). Vaezi et al. [<xref rid="B8" ref-type="bibr">8</xref>] reported that 14/20 (70%) pneumatic dilation- and 7/22 (32%) Botox-treated patients were in symptomatic remission at 12 months.</p><p>LESP is measured after treatment to evaluate the efficacy of BD or intrasphincteric Botox injection by performing esophageal emptying scintigraphy to determine the barium height on an esophagram [<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>]. However, few studies have identified the predictors of long-term outcomes. Therefore, we compared the long-term outcomes for BD versus Botox injection in patients with primary achalasia and identified predictors of remission.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Patients</title><p>Diagnoses were established using clinical, radiological, and manometric criteria. The clinical criteria included dysphagia, regurgitation, chest pain, and weight loss, and the radiological criteria included bird's beak appearance of the LES, decreased esophageal peristalsis, and delayed esophageal emptying. The manometric diagnostic criteria consisted of aperistalsis of the esophageal body, increased LESP, and incomplete relaxation on swallowing. Patients with secondary achalasia were excluded based on esophagogastroduodenoscopy (EGD). This study was approved by the Institutional Review Board of Soonchunhyang University Hospital, and informed consent was obtained from all patients.</p><p>We enrolled 73 patients with achalasia newly diagnosed between January 1988 and January 2011. Exclusion criteria included gastric cancer (n = 2), postvagotomy (n = 2), no treatment (n = 12), and myotomy treatment (n = 2). With the exceptions of telephone interview failure (n = 18), contact failure due to the loss of a telephone number or address (n = 12), or refusal to interview (n = 6), we ultimately enrolled 37 of 55 patients with primary achalasia who were observed for 1 year: 25 underwent intrasphincteric Botox injection (Botox group) and 12 underwent BD group (<xref ref-type="fig" rid="F1">Fig. 1</xref>). The median patient age was 56 years (range, 19 to 89). Twenty-two males and 15 females were enrolled. The median ages of the Botox and BD groups were 58 years (range, 39 to 89) and 38 years (range, 19 to 75), respectively. Thirty-seven patients (100%) had dysphagia, 22 (59.5%) had chest pain, and 27 (73.0%) had regurgitation. The median LESP values were 35.5 mmHg (range, 5.1 to 98.0) and 27.1 mmHg (range, 13.7 to 63.5) in the Botox and BD groups, respectively (<xref ref-type="table" rid="T1">Table 1</xref>).</p></sec><sec><title>Methods</title><p>To evaluate the long-term treatment effect, we determined the patient's current symptoms through telephone interviews after reviewing their medical records. To obtain objective information, we conducted the interview using a predetermined questionnaire that included the status of symptom improvement after treatment, time of recurrence in case of recurrence, and information pertaining to the frequency and degree of dysphagia, regurgitation, and chest pain. These were classified using the Eckardt score [<xref rid="B18" ref-type="bibr">18</xref>]. The points for each clinical parameter were summed, and level 0 indicated a score &#x0003c; 2, level 1 a score of 2 to 4, level 2 a score of 5 to 6, and level 3 a score of &#x02265; 7. Eckardt levels of 0 to 1 indicated remission, whereas levels 2 to 3 indicated treatment failure. The rate of symptom improvement according to treatment method was based on the questionnaire, and we evaluated the short-term symptom improvement status compared with symptoms prior to the first treatment. Long-term symptom improvement was evaluated from the clinical medical records and a telephone interview.</p></sec><sec><title>Esophageal manometry</title><p>Esophageal manometry is perfusion manometry involving constant perfusion of water and measuring pressure through an EMC8 (Synthetics Medical, Stockholm, Sweden) measuring tube. This procedure was performed after we anesthetized the nasal cavity with lidocaine while the patient was sitting for 5 to 10 minutes to stabilize the reading. Using the station pull-through technique, we located the side pore in the stomach and measured LESP by subtracting the measurements at 1-cm intervals.</p><p>We performed high-resolution manometry (HRM) in eight patients who underwent BD using an HRM catheter (Sierra Scientific Instruments, Los Angeles, CA, USA), in which 36 columnar pressure sensors were located at 1-cm intervals. Each pressure sensor had a 2.5-mm-long detection device that detected pressure using pressure transmission technology (TactArray, Pressure Profile Systems, Los Angeles, CA, USA). The data measured by the pressure gauge were analyzed using ManoView software (Sierra Scientific Instruments).</p></sec><sec><title>Esophagogastroduodenoscopy</title><p>The EGD observations included the dilation status of the esophageal body, stasis of food in the esophagus, resistance of the LES, and contraction status of the LES with breathing when it was reversed inside the stomach. We also looked for tumors to distinguish secondary achalasia.</p></sec><sec><title>Balloon dilation</title><p>We performed EGD with sedation and the patients lying on their left sides. A guidewire was located in the stomach, and we inserted a balloon dilator (Rigiflex, Boston Scientific, Natick, MA, USA) into the stomach along the guidewire. We checked the location of the balloon using fluoroscopy or EGD. We then dilated the balloon using contrast medium until the curve in the unrelaxed LES was obvious. We attempted dilation for 15 to 60 seconds with 7 to 15 per square inch pressure using balloons with external diameters of 30, 35, and 40 mm. We performed EGD and esophageal manometry within 3 days after the operation as a tracking examination.</p></sec><sec><title>Intrasphincteric botulinum toxin injection</title><p>We inserted the EGD to the level of the LES after a 12-hour fast. We mixed 100 units Botox with 4 to 5 mL normal saline and injected 1 mL at four points around the LES using a syringe. We kept the patient <italic>nil per os</italic> the next day and looked for evidence of perforation on a chest radiograph. Afterwards, the patients were started on clear fluids and finally underwent follow-up EGD and esophageal manometry before discharge.</p></sec><sec><title>Statistical analysis</title><p>SPSS version 18.0 (IBM Co., Armonk, NY, USA) was used for the statistical analysis. Quantitative variables are presented as medians and ranges. The Kaplan-Meier method was used to assess symptomatic recovery. Fisher exact test was used to compare the remission rate of each symptom between the two treatment methods. The Kaplan-Meier method and log-rank test were used for analysis of the treatment predictors in the univariate analyses, and the Cox regression method was used for the multivariate analysis. <italic>p</italic> values &#x0003c; 0.05 were considered significant.</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Clinical progress</title><p>Thirty-seven of the 55 patients were contacted for telephone interviews; 25 had intrasphincteric Botox injections and 12 had undergone BD. Thirty-five patients showed symptom improvement after treatment, whereas two did not. Three of 24 in the Botox group who showed symptom improvement remained in remission, whereas 21 developed recurrent symptoms. Of these 21, 11 received an intrasphincteric Botox injection, and five underwent BD. Five of the 11 patients who showed improvement after BD as the first treatment continued in remission, and six had recurrent symptoms. One of the recurrent patients underwent two more rounds of BD (<xref ref-type="fig" rid="F2">Fig. 2</xref>).</p><p>No complications, such as esophageal perforation or bleeding requiring treatment, were observed. We performed a barium esophagogram, esophageal manometry, and EGD before and after BD or intrasphincteric Botox injection. If the patient had worsened subjective symptoms, the reoperation status was determined from these tests.</p><p>The median LESP was 32.2 mmHg (range, 5.1 to 98.0) before treatment and 22.5 mmHg (range, 0.8 to 48.0) after treatment. The difference was 18.4 mmHg (range, 0.0 to 65.0). During the median 61-month (range, 12 to 184) follow-up period, the median remission duration was 20 months (range, 1 to 72). The follow-up period was 56 months (range, 15 to 184) for the intrasphincteric Botox injection group and 33 months (range, 12 to 74) for the BD group.</p></sec><sec><title>Short-term symptom relief after the first treatment according to treatment method</title><p>The number of patients with chest pain in the Botox group decreased from 14 (56.0%) to 4 (16.0%), those with regurgitation decreased from 16 (64.0%) to 4 (16.0%), and those with dysphagia decreased from 25 (100.0%) to 5 (20.0%). The number of patients with chest pain in the BD group decreased from 8 (66.7%) to 1 (8.3%), regurgitation from 11 (91.7%) to 1 (8.3%), and dysphagia from 12 (100.0%) to 1 (8.3%) (<xref ref-type="table" rid="T2">Table 2</xref>).</p></sec><sec><title>The symptom-free period after the first treatment</title><p>The median time to the symptom-free period in the 35 patients who showed symptom improvement after the first treatment was 20 months. The symptom-free period was 13 months (range, 1 to 70) in the Botox group and 29 months (range, 6 to 72) in the BD group. The symptom-free period was significantly longer in the BD group (<italic>p</italic> = 0.036) than then Botox group (<xref ref-type="fig" rid="F3">Fig. 3</xref>).</p></sec><sec><title>Predictors of long-term outcome</title><p>Univariate analyses of age, sex, reflux, chest pain, and LESP after treatment were conducted, and the differences in LESP values before and after treatment were used to evaluate the effect of each factor on long-term outcome. The symptom-free period was significantly longer (<italic>p</italic> = 0.015) when the difference in the LESP before and after treatment was &#x0003e; 19 mmHg. The treatment method was also a significant predictor. Patients undergoing BD had a significantly longer symptom-free period (<italic>p</italic> = 0.036) than those who received Botox (<xref ref-type="table" rid="T3">Table 3</xref>).</p><p>The symptom-free period was significantly longer when the difference in LESP before and after treatment was &#x0003e; 19 mmHg, according to the Cox multivariate regression analysis for age, sex, difference in LESP, and treatment type (<italic>p</italic> = 0.012). BD also resulted in a significantly longer symptom-free period than that of intrasphincteric Botox injection after correcting for other variables, such as age and sex (<italic>p</italic> = 0.036) (<xref ref-type="table" rid="T4">Table 4</xref>).</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>The rates of therapeutic success from intrasphincteric Botox injection were 48%, 24%, and 8.7% after 1, 2, and 5 years, respectively, whereas the rates for BD were 66.7%, 58.3%, and 25%. Bansal et al. [<xref rid="B19" ref-type="bibr">19</xref>] also compared BD and intrasphincteric Botox injection and found respective success rates of 89% and 38% after 1 year. Gutschow et al. [<xref rid="B20" ref-type="bibr">20</xref>] found that symptoms recurred or continued in 6.3% of patients after 35 months of observations following BD, whereas they recurred or continued in 71.4% 9 months after intrasphincteric Botox injection. Other studies have shown that 65% to 100% of patients respond to a single injection within 1 month, and on further follow-up, this effect was sustained for 12 months in 15% to 75% [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>]. The results of our study were similar.</p><p>Perforation is a complication of BD and intrasphincteric Botox injection in 1% to 4% of cases [<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B21" ref-type="bibr">21</xref>]. However, no perforation occurred in our series.</p><p>Many studies have examined predictors of primary achalasia treatment. Alderliesten et al. [<xref rid="B13" ref-type="bibr">13</xref>] reported that the rate of recurrence is higher in younger patients and in those with a LESP &#x0003e; 10 mmHg 3 months after treatment. Nam et al. [<xref rid="B10" ref-type="bibr">10</xref>] reported that the remission rate was high if the difference in the LESP between before and after BD was &#x0003e; 13 mmHg. In addition, useful predictors have included sex, esophageal emptying scintigraphy, esophageal diameter on esophagography, length of retained barium, normalization of the reversed stomach-esophagus pressure, and occurrence of different simultaneous contraction configurations [<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B22" ref-type="bibr">22</xref>,<xref rid="B23" ref-type="bibr">23</xref>].</p><p>In this study, the remission duration was significantly longer if the difference in the LESP between before and after treatment was &#x0003e; 19 mmHg. BD was a more useful treatment than intrasphincteric Botox injection.</p><p>Selection bias was a potential limitation of this study due to its nonrandomized nature. The group undergoing intrasphincteric Botox injection showed selection bias due to age. We depended on the patients' memory of subjective symptoms and the recurrence period, which is a limitation of retrospective studies. The number of patients included was small, and 18 patients were excluded because they were unavailable for telephone interviews. Therefore, the study was impractical for a statistical interpretation. In addition, eight patients undergoing BD recently underwent HRM. The pressure on HRM appeared to be lower than that using conventional manometry. Hence, the examination we used could have affected the numerical pressure value. It was possible that the LESP appeared low in the group of patients who underwent BD.</p><p>Many patients remained symptomatic &#x0003e; 5 years after treatment. BD and intrasphincteric Botox injection do not restore destroyed nerves but alter the LES. Laparoscopic Heller's myotomy with partial fundoplication is another treatment option. A recent 2-year randomized prospective study showed that the efficacy of BD is similar to that of Heller's myotomy and Dor's fundoplication for treating achalasia [<xref rid="B24" ref-type="bibr">24</xref>]. Japanese and European researchers successfully conducted a peroral endoscopic myotomy (POEM), which is a submucosal myotomy using an endoscope, without complications [<xref rid="B25" ref-type="bibr">25</xref>,<xref rid="B26" ref-type="bibr">26</xref>]. A successful case of POEM was reported here. The number of effective treatments available for achalasia is increasing. Therefore, an evaluation of POEM is needed. However, this method does not treat the root problem; i.e., destruction of the LES, and it is also necessary to treat reflux. The root solution would be to restore the destroyed splanchnic nerve plexus. Neural stem cell transplantation is a future alternative treatment [<xref rid="B27" ref-type="bibr">27</xref>]. Many studies currently in progress are evaluating cerebral cortex neural stem cells and should assess the survival and side effects of transplanted stem cells.</p><p>In conclusion, BD had a better long-term outcome than that of intrasphincteric Botox injection. The symptom-free period was significantly longer if the difference in LESP was &#x0003e; 19 mmHg, suggesting the usefulness of manometric follow-up after treatment. A future prospective randomized study is needed to identify the predictors of long-term remission in patients with primary achalasia.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>In Korean patients with achalasia, the short-term symptom relief after intrasphincteric botulinum toxin injection and balloon dilation treatment are similar.</p></list-item><list-item><p>Ballon dilatation had a better long-term outcome than that of intrasphincteric Botox injection in the treatment of achalasia.</p></list-item><list-item><p>Independent factors predicting long-term remission included treatment type and the difference in the lower esophageal sphincter pressure. The symptom-free period was significantly longer if the difference in LESP was &#x0003e; 19 mmHg, suggesting the usefulness of manometric follow-up after treatment.</p></list-item></list> </p></boxed-text></sec>
The diagnostic utility of chest computed tomography scoring for the assessment of amiodarone-induced pulmonary toxicity
<sec><title>Background/Aims</title><p>Amiodarone is one of the most widely used antiarrhythmic agents; however, amiodarone-induced pulmonary toxicity (APT) can be irreversible and sometimes fatal. The aim of this study was to evaluate the feasibility of chest computed tomography (CT) as a diagnostic tool for APT and to assess the utility of the CT APT score as an index for predicting the severity of APT.</p></sec><sec><title>Methods</title><p>Patients underwent amiodarone treatment for various reasons, most often atrial fibrillation, for more than 2 years, and those that received a cumulative dose &#x0003e; 100 g were enrolled. A total of 34 patients who underwent chest CT between December 2011 and June 2012 were enrolled, whether or not they had clinical symptoms. The APT CT score was defined as the number of involved regions in the lung, which was divided into 18 regions (right and left, upper, middle, and lower, and central, middle, and peripheral). The CT findings were evaluated according to the total dose and duration of amiodarone treatment and the results of a pulmonary function test. Clinical symptoms and outcomes were also evaluated according to APT CT scores.</p></sec><sec><title>Results</title><p>Seven patients had positive APT CT scores (interstitial fibrosis in five, organizing pneumonia in one, and mixed interstitial fibrosis and organizing pneumonia in one), and these patients exhibited significantly lower diffusion capacity for carbon monoxide in the lungs compared with patients without an increased APT CT score (70.2% &#x000b1; 6.9% vs. 89.7% &#x000b1; 19.4%; <italic>p</italic> = 0.011). Three of the seven patients experienced overt APT that required hospital admission.</p></sec><sec><title>Conclusions</title><p>Chest CT is a useful diagnostic tool for APT, and the APT CT score might be a useful index for assessing the severity of APT.</p></sec>
<contrib contrib-type="author"><name><surname>Kang</surname><given-names>In Sook</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Kyung Jin</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Yookyung</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Park</surname><given-names>Seong-Hoon</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Amiodarone is one of the most widely used antiarrhythmic agents; however, amiodarone-induced pulmonary toxicity (APT) can be irreversible and sometimes fatal [<xref rid="B1" ref-type="bibr">1</xref>]. Furthermore, the long half-life and lipophilic nature of amiodarone, as well as its tendency to accumulate in various tissues, make it challenging to predict toxicity [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>].</p><p>The intracellular accumulation of phospholipids and T cell-mediated hypersensitivity are potential mechanisms of APT [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>]. Chest computed tomography (CT) findings related to amiodarone exposure are well defined and include lung parenchymal changes, septal thickening, interstitial fibrosis, high attenuation pleuroparenchymal opacities, and interstitial or alveolar opacities [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>]. These findings, unlike other drug-induced pulmonary changes, often present with asymmetric patterns, whether diffuse or patchy infiltration [<xref rid="B6" ref-type="bibr">6</xref>]. Although APT-related CT findings have been described, the relationship between the extent of these abnormal findings and the diagnosis or severity of APT remain unclear.</p><p>Several studies have assessed the effects of amiodarone cumulative total doses [<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>]. The long-term use of low maintenance doses of amiodarone can reach a high total cumulative dose as high as 101 to 151 g, which was associated with a significantly increased risk of APT (odds ratio [OR], 10.29); however, this risk reached a plateau at cumulative doses &#x0003e; 150 mg (OR, 9.5) [<xref rid="B9" ref-type="bibr">9</xref>].</p><p>The aim of the current study was to evaluate the utility of chest CT as a diagnostic tool for APT in chronic amiodarone users, particularly in individuals prescribed lower maintenance doses that result in a high total cumulative dose. In addition, we also assessed the usefulness of the CT APT score, a surrogate for the extent of APT, as an index for predicting the severity of APT.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Study population</title><p>This was a cross-sectional cohort study with a 1-year prospective follow-up. Patient medical records were reviewed retrospectively, and 1-year outcomes were observed prospectively after discontinuing amiodarone therapy. Patients who had been treated with amiodarone for &#x0003e; 2 years with a total cumulative dose &#x0003e; 100 g were enrolled based on information in the medical database between January 2000 to October 2011. A total of 65 patients were eligible for inclusion in the study. Patients who had no available medical records within the last 2 years (due to transfer to another hospital or lack of follow-up, n = 11), who could not discontinue amiodarone treatment (due to aggravation of underlying heart disease, n = 1), and who did not agree to inclusion in the study (n = 9) were excluded.</p><p>A total of 44 patients provided oral informed consent and were recruited into the study. They underwent chest CT between December 2011 and June 2012, whether they were symptomatic or asymptomatic. Patients with a medical history of chronic obstructive lung disease (COPD), interstitial lung disease, preexisting lung disease, or those with no known medical history of bronchiectasis but in whom lesions were evident on chest X-ray were excluded. The total cumulative amiodarone doses were calculated from the medical records. Seven of the 44 patients were excluded because their actual cumulative dose or the period of amiodarone treatment did not satisfy the enrollment criteria, and three patients were excluded due to preexisting lung disease (bronchiectasis, emphysema, and chronic bronchitis, respectively). Therefore, a total 34 patients were entered into the final analysis. All individuals discontinued amiodarone within 1 week of the chest CT and had been followed up for 1 year without taking amiodarone. This study was approved by the Institutional Review Board of Ewha Medical Center (IRB No. ECT 12-17A-22). Patients were enrolled after providing oral informed consent.</p></sec><sec><title>Computed tomography</title><p>All CT scans were assessed for the presence of APT, and an experienced radiologist (YK, 17 years of experience in chest CT) analyzed the CT patterns of APT. The CT findings were pulmonary lesions defined as follows: (1) pulmonary interstitial fibrosis besides bronchiectasis, pleural thickening, stable pulmonary tuberculosis, and linear atelectasis related to other lung problems; (2) pulmonary consolidation or ground-glass opacity that was not related to any etiology other than a medical history of amiodarone use; (3) pulmonary lesions with a high CT-attenuation not related to the calcification of another pulmonary disease.</p><p>The APT CT score was calculated for each patient by counting the number of involved regions in the lung, which was divided into 18 regions (right and left, upper, middle, and lower, and central, middle, and peripheral). Both lungs were divided into three regions in the craniocaudal direction: upper, above the carina; middle, between the carina and the inferior pulmonary vein; lower, below the inferior pulmonary vein. Each region was then divided into central, middle, and peripheral thirds. Therefore, the maximum APT CT score was 18, and the minimum APT CT score was 0. When different types of pulmonary lesions existed in the same region, that region was counted as "1," regardless of the number of types of pulmonary lesions (more detailed examples are presented in <xref ref-type="fig" rid="F1">Figs. 1</xref> and <xref ref-type="fig" rid="F2">2</xref>). For example, the presence of both pulmonary interstitial fibrosis and pulmonary consolidation in the right middle central region would be counted as 1.</p><p>Overt APT was defined when the individuals had a positive APT CT score and required hospital treatment due to clinical symptoms and/or respiratory failure related to amiodarone use. Clinical symptoms included cough, dyspnea, and/or fever.</p></sec><sec><title>Statistical analysis</title><p>The baseline characteristics of the subjects were compared using chi-square tests for categorical variables and Student t tests for continuous variables. Continuous variables were expressed as means &#x000b1; SD. Spearman's correlation analyses were performed to assess the correlation between variables. In addition, a forward stepwise logistic regression model was applied to identify variables related to the APT CT score. A value of <italic>p</italic> &#x0003c; 0.05 was used to define statistical significance, and all statistical analyses were performed using SPSS version 19.0 (IBM Co., Armonk, NY, USA).</p></sec></sec><sec sec-type="results"><title>RESULTS</title><p>The most common reason for amiodarone treatment in the study population was atrial fibrillation (AF): (1) AF alone (n = 21); (2) AF with valvular heart disease (n = 10); (3) AF and a medical history of aborted sudden cardiac death related to acute myocardial infarction (n = 2); (4) AF with ischemic heart disease (n = 1); and (5) supraventricular tachycardia with ischemic heart disease (n = 1). The mean age of the 34 subjects was 67.3 &#x000b1; 10.5 years, and 64.2% were female. The mean starting dose of amiodarone was 314.7 &#x000b1; 135.1 mg, and the mean total cumulative dose was 408.4 &#x000b1; 221.1 g over ~5.87 years (<xref ref-type="table" rid="T1">Table 1</xref>). Eleven patients (32.4%) had clinical symptoms such as cough (14.7%), fever (5.9%), and dyspnea (26.5%), and 11.8% of patients had more than two symptoms.</p><p>Seven patients had positive APT findings on CT scans. The CT pattern was a pulmonary interstitial fibrosis pattern of interlobular and intralobular septal thickenings predominantly involving the lower lungs in five patients (<xref ref-type="fig" rid="F1">Fig. 1</xref>), an organizing pneumonia pattern of consolidation and ground-glass opacity in one patient (<xref ref-type="fig" rid="F2">Fig. 2</xref>), and a mixed pattern of pulmonary interstitial fibrosis and organizing pneumonia in one patient (<xref ref-type="fig" rid="F3">Fig. 3</xref>).</p><p>The APT CT scores, clinical manifestations, chest radiographical, and CT findings of all patients are summarized in <xref ref-type="table" rid="T2">Table 2</xref>. Cases 1 to 3 had APT CT scores &#x0003c; 5, no symptoms, and no overt APT during the 1-year follow-up period. Case 3 had suspected APT, both on a chest X-ray and chest CT, but had no symptoms and did not experience APT. These patients were treated for septic shock that was unrelated to amiodarone. An APT CT score of 9 to 16 was observed in four patients, three of whom were admitted to the hospital for treatment of overt APT. Cases six (<xref ref-type="fig" rid="F3">Fig. 3</xref>) and seven (<xref ref-type="fig" rid="F2">Fig. 2</xref>), who showed an organizing pneumonia pattern, experienced acute respiratory failure that required ventilator care. Two of the three patients recovered completely after the discontinuation of amiodarone and medical treatment, but one patient died.</p><p>Overt APT was not evident in patients with negative APT CT scores. During follow-up, three minor events of supraventricular tachycardia, one morbidity of nonwitnessed outside hospital sudden cardiovascular collapse, and one unrelated minor event of community-acquired pneumonia occurred in the patients with negative APT CT scores. After the discontinuation of amiodarone, there were no newly developed instances of APT or APT relapse in any of the 34 patients during the 1-year follow-up.</p><p>The subjects were further divided into two groups according to positive and negative APT CT scores (PCT and NCT, respectively). There were no differences in age, gender, and medical history between the groups. Amiodarone-related variables, such as initial dose, total cumulative dose, and the duration of administration, were also comparable between groups (<xref ref-type="table" rid="T3">Table 3</xref>). The PCT group had a lower diffusion capacity for carbon monoxide in the lungs (DLCO) and less body surface area (BSA) than did the NCT group. In addition, the APT CT score was correlated negatively with BSA according to a forward stepwise logistic regression model (95% confidence interval, -2.075 to -0.263; <italic>p</italic> = 0.013).</p></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>The prevalence of APT is ~5% [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B10" ref-type="bibr">10</xref>], with an annual incidence of ~2% [<xref rid="B11" ref-type="bibr">11</xref>]. The current study showed that the incidence of overt APT was 8.8%; however, this might be exaggerated due to the excluded patients. A Japanese study reported that the 1-, 3-, and 5-year cumulative incidences of APT in amiodarone users on a 141-mg maintenance dose were 4.2%, 7.8%, and 10.6%, respectively [<xref rid="B12" ref-type="bibr">12</xref>]. This is consistent with the current observations.</p><p>APT is diagnosed by exclusion, and diagnoses may be based on existing findings or clinical symptoms (cough or dyspnea), particularly by the presence of local or diffuse opacities on chest CT and a decrease in DLCO from baseline [<xref rid="B1" ref-type="bibr">1</xref>]. However, there is no consensus on whether APT can be diagnosed only using symptoms, pulmonary function tests (PFT), chest CT, or pathology. Therefore, there are no pathognomonic findings regarding chest CT and APT.</p><p>The general guidelines for APT recommend performing baseline and surveillance of PFT, as well as chest X-rays [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B11" ref-type="bibr">11</xref>]. However, these recommendations are not always followed in real-world practice. The results of a cross-sectional retrospective study in a large tertiary-care hospital revealed that only 52% of patients underwent baseline PFT, and 60% underwent both chest X-ray and PFT surveillance [<xref rid="B11" ref-type="bibr">11</xref>]. The current results showed that PCT subjects also had a decreased DLCO compared with NCT subjects, although baseline DLCO data were not available.</p><p>Chest CT could be useful for differential diagnosis when no baseline data for PFT and/or chest X-ray are available, or in the presence of underlying pulmonary diseases such as COPD. The clinical symptoms for APT are nonspecific [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B10" ref-type="bibr">10</xref>]. Our data also revealed no difference in clinical symptoms between the two groups (<italic>p</italic> = 0.66, data not shown), and clinical symptoms presented regardless of APT. However, all three patients who were diagnosed with overt APT had clinical symptoms. Therefore, a positive APT CT score and the presence of clinical symptoms might be helpful for the diagnosis of APT in long-term amiodarone users. In particular, a high APT CT score such as &#x02265; 9 could be related to severe APT that requires hospital admission. Other patients with positive ATP CT scores but no symptoms or clinical events were not diagnosed with APT; therefore, no treatment was considered.</p><p>There are two major hypotheses regarding APT: immune-mediated hypersensitivity and the direct cytotoxicity of drug-induced phospholipidosis [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B13" ref-type="bibr">13</xref>]. Moreover, amiodarone has several pharmacological properties that contribute to its toxicities [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B14" ref-type="bibr">14</xref>], including the presence of two iodine atoms that result in the release of 7 mg iodine per 200 mg amiodarone (normal iodine intake, 150 to 200 &#x000b5;g/day), its lipophilic nature and high affinity for tissue accumulation, long half-life, and a large distribution volume. Consequently, amiodarone accumulates in fatty tissue, the liver, and lungs in chronic users, which leads to drug-induced direct organ toxicities. Iodine is highly attenuated on CT; therefore, the high attenuation of these organs on nonenhanced CT might represent iodine accumulation in the lungs and/or liver of chronic amiodarone users [<xref rid="B6" ref-type="bibr">6</xref>]. Increased lung attenuation seems to present in more advanced APT cases [<xref rid="B7" ref-type="bibr">7</xref>]. Case six in the current study showed characteristic high attenuation on nonenhanced CT in both the liver and lungs, which suggested iodine accumulation related to chronic amiodarone use (<xref ref-type="fig" rid="F3">Fig. 3A and 3C</xref>).</p><p>When a long-term amiodarone user with a cumulative dose &#x0003e; 100 g presents with newly developed symptoms, it is necessary to evaluate not only chest X-ray and PFT, but also chest CT. Cases six and seven experienced acute respiratory failure that required ventilator support. Both patients exhibited organized pneumonia patterns on chest CT, and their APT CT scores were 12 and 16, respectively. If the APT CT score is &#x02265; 9, the patient should be assessed and managed according to the possibility of severe APT and respiratory failure.</p><p>There are several known risk factors for APT, including old age, duration of amiodarone therapy and a high cumulative dose, pre-existing lung disease, and high oxygen therapy [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>]. In the current study, the two groups were similar in age, which might be due to the small number of patients, or age might not be a significant risk factor in long-term amiodarone users who have reached a high cumulative dose. However, there are no published data available regarding this specific situation.</p><p>In addition, the current study showed no significant differences according to the duration of treatment and cumulative dose between the two groups. This might be because our study population included only patients with a cumulative dose &#x0003e; 100 g and total treatment periods &#x0003e; 2 years. The total cumulative doses in the PCT and NCT groups were 449.6 and 398.7 g, respectively; the mean dose was higher in the PCT group, but not significantly (<italic>p</italic> = 0.59). BSA showed an inverse relationship with a positive APT CT score (<italic>p</italic> = 0.013), and patients in the PCT group had a higher total dose/BSA compared with the NCT group (288.7 g/m<sup>2</sup> vs. 229.4 g/m<sup>2</sup>), but not significantly. Therefore, this should be investigated further. In addition, a previous report [<xref rid="B9" ref-type="bibr">9</xref>] suggested that the risk of APT might plateau at very high cumulative doses &#x0003e; 150 g. However, another study revealed a significantly increased risk of ATP when the cumulative dose reached 140 to 230 g [<xref rid="B8" ref-type="bibr">8</xref>].</p><p>All patients had discontinued amiodarone ~1 week before the chest CT, and some were prescribed other antiarrhythmic agents as a substitute. All subjects were followed for at least 1 year after discontinuation of amiodarone. Some studies reported that the recurrence of APT after discontinuing amiodarone was related to the rapid tapering of steroids [<xref rid="B6" ref-type="bibr">6</xref>]. Nevertheless, our data did not reveal newly developed or recurrent APT. Two patients who received hospital treatment due to overt APT also did not show recurrent APT or additional lung problems during the follow-up period.</p><p>There are some limitations to our study. It had a small subject number and was a single-center study. Therefore, the results cannot be generalized. In addition, baseline PFT data were not available. We performed 1-year follow-up by prospective observation, and chest CTs were not repeated routinely. Therefore, we could not evaluate the reversibility of chest CT lesions in patients with positive APT CT scores without overt APT after discontinuing amiodarone. Consequently, the meaning of APT CT scores &#x0003c; 5 not associated with overt APT could not be assessed.</p><p>In conclusion, the APT CT score might be helpful for the assessment and management of APT in symptomatic long-term amiodarone users. Chest CT has value for differential diagnosis, and a high APT CT score might be related to pulmonary insufficiency and rapid progression of respiratory failure. There was little evidence to suggest new development or recurrence of APT after discontinuing amiodarone.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Computed tomography (CT) score is helpful in assessment and management of amiodarone-induced pulmonary toxicity (APT) for symptomatic long term amiodarone user.</p></list-item><list-item><p>Especially, high APT CT score may be related with pulmonary insufficiency and rapid progress of respiratory failure.</p></list-item><list-item><p>There was little evidence of new development or recurrence of APT after discontinuing amiodarone.</p></list-item></list> </p></boxed-text></sec>
Effects of intensive versus mild lipid lowering by statins in patients with ischemic congestive heart failure: Korean Pitavastatin Heart Failure (SAPHIRE) study
<sec><title>Background/Aims</title><p>This study was designed to evaluate the dose-effect relationship of statins in patients with ischemic congestive heart failure (CHF), since the role of statins in CHF remains unclear.</p></sec><sec><title>Methods</title><p>The South koreAn Pitavastatin Heart FaIluRE (SAPHIRE) study was designed to randomize patients with ischemic CHF into daily treatments of 10 mg pravastatin or 4 mg pitavastatin.</p></sec><sec><title>Results</title><p>The low density lipoprotein cholesterol level decreased by 30% in the pitavastatin group compared with 12% in the pravastatin (<italic>p</italic> &#x0003c; 0.05) group. Left ventricular systolic dimensions decreased significantly by 9% in the pitavastatin group and by 5% in the pravastatin group. Left ventricular ejection fraction (EF) improved significantly from 37% to 42% in the pitavastatin group and from 35% to 39% in the pravastatin group. Although the extent of the EF change was greater in the pitavastatin group (16% vs. 11%) than that in the pravastatin group, no significant difference was observed between the groups (<italic>p</italic> = 0.386). Exercise capacity, evaluated by the 6-min walking test, improved significantly in the pravastatin group (<italic>p</italic> &#x0003c; 0.001), but no change was observed in the pitavastatin group (<italic>p</italic> = 0.371).</p></sec><sec><title>Conclusions</title><p>Very low dose/low potency pravastatin and high dose/high potency pitavastatin had a beneficial effect on cardiac reverse remodeling and improved systolic function in patients with ischemic CHF. However, only pravastatin significantly improved exercise capacity. These findings suggest that lowering cholesterol too much may not be beneficial for patients with CHF.</p></sec>
<contrib contrib-type="author"><name><surname>Lee</surname><given-names>Hae-Young</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Cho</surname><given-names>Hyun-Jai</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Hee-Yul</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Jeon</surname><given-names>Hee-Kyung</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Shin</surname><given-names>Joon Han</given-names></name><xref ref-type="aff" rid="A4">4</xref></contrib><contrib contrib-type="author"><name><surname>Kang</surname><given-names>Suk-Min</given-names></name><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Baek</surname><given-names>Sang Hong</given-names></name><xref ref-type="aff" rid="A6">6</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Congestive heart failure (CHF) is becoming a major worldwide public health problem that requires a global response and enhanced survival after coronary artery disease, congenital or valvular heart disease, and societal aging. CHF is a complex clinical syndrome in which the heart cannot pump sufficient blood to the body. Various diseases or noxious stimuli, such as myocardial ischemia, high blood pressure, tachycardia, inflammation, or excess neurohormonal stimulation comprise the pathophysiological mechanisms of CHF. Significant strides have been made in the treatment of CHF by neurohormonal modulation using angiotensin-converting enzyme inhibitors, angiotensin-II receptor blockers, &#x003b2;-blockers, and aldosterone antagonists [<xref rid="B1" ref-type="bibr">1</xref>]. However, 1-year mortality in clinical trials remains high and exceeds 6% in patients with CHF and mild to moderate symptoms [<xref rid="B2" ref-type="bibr">2</xref>] and 12.8% in patients with CHF and moderate to severe symptoms [<xref rid="B3" ref-type="bibr">3</xref>], warranting further strategies to reduce adverse outcomes. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or statins are candidate drugs that have been investigated and used in patients with CHF.</p><p>It is well-established that statins reduce morbidity and mortality in patients with coronary artery disease, thus preventing its progression to CHF [<xref rid="B4" ref-type="bibr">4</xref>], and have cholesterol-independent pleiotropic effects by inhibiting mevalonate. Statins decrease isoprenoid production and consequently downregulate the inflammatory pathways mediated by Rho proteins [<xref rid="B5" ref-type="bibr">5</xref>] and reactive oxygen species generated by Rac proteins [<xref rid="B6" ref-type="bibr">6</xref>]. Additionally, many observational studies and retrospective and post hoc analyses of data from randomized trials of patients with various cardiovascular conditions suggest the survival benefit of statins in patients with CHF [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>]. However, two large randomized controlled trials suggested that statins do not benefit patients with heart failure. The Controlled Rosuvastatin Multinational Trial in Heart Failure (CORONA) [<xref rid="B10" ref-type="bibr">10</xref>] and Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardio-Heart Failure (GISSI-HF) [<xref rid="B11" ref-type="bibr">11</xref>] randomized patients to rosuvastatin or a matching placebo and evaluated long-term outcomes in patients with CHF. Regrettably, neither study showed improved survival in patients with CHF. However, recent studies have not confirmed the detrimental effects of statins in CHF patients reported in the CORONA and GISSI-HF trials [<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B13" ref-type="bibr">13</xref>]. Therefore, the effect of statins on patients with CHF is not clear, raising important clinical questions. In particular, concerns have been raised about potential adverse effects (AEs) of high-dose statin therapy [<xref rid="B14" ref-type="bibr">14</xref>], because observational studies have detected an association between low cholesterol levels and adverse outcomes in CHF patients [<xref rid="B15" ref-type="bibr">15</xref>]. A hypothesis was proposed that the decrease in ubiquinone levels caused by statin therapy may have deleterious consequences, because ubiquinone is a potential antioxidant and a key molecule in mitochondrial function and, thus, muscle strength [<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>].</p><p>Pitavastatin is a lipophilic statin with longer-acting cholesterol lowering effects than that of other statins. Pitavastatin also has high bioavailability and is minimally metabolized by the cytochrome P450 system [<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>]. The South koreAn Pitavastatin Heart FaIluRE (SAPHIRE) study was designed to randomize patients with CHF due to New York Heart Association functional class II to III ischemic heart disease into low potency, hydrophilic, 10 mg pravastatin or high potency, lipophilic, 4 mg pitavastatin daily groups and to evaluate systolic function and exercise capacity changes after 1 year.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Study population</title><p>This was a prospective, randomized, open label, active drug-controlled, and two parallel group-comparison study to evaluate the efficacy and safety of pitavastatin compared with pravastatin in patients with chronic ischemic heart failure. Clinically stable (NYHA class II to III) male and female patients with heart failure (age, 30 to 80 years), with a left ventricular ejection fraction (EF) &#x02264; 45% and serum low density lipoprotein cholesterol (LDL-C) &#x02265; 70 mg/dL at screening, were enrolled. Patients were excluded if they had any of the following conditions: decompensated heart failure (NYHA class IV), acute myocardial infarction, unstable angina, coronary artery bypass graft, percutaneous coronary intervention or valve surgery within 3 months, uncontrolled heart failure due to hypertrophic obstructive cardiomyopathy, mild-to-severe valvular stenosis, or severe (grade III/IV) valvular regurgitation. Female patients with child-bearing potential and pregnant and nursing women were excluded. This study was approved by the Institutional Review Board and the Korea Food and Drug Administration and was conducted in accordance with Korean Good Clinical Practice and the Declaration of Helsinki. This study has been registered at Clinical-Trials.gov (ID; NCT00701285). All patients signed written informed consent prior to participation.</p></sec><sec><title>Study design</title><p>The clinical trial was conducted at five clinical research centers and hospitals. All patients provided written informed consent prior to participation. Subjects who satisfied all inclusion/exclusion criteria were randomized either to the pravastatin group or the pitavastatin group. The pravastatin group was administered 10 mg pravastatin once daily and the pitavastatin group 2 mg pitavastatin once daily. If no AEs were determined by the investigators after 4 weeks, the pitavastatin dose was doubled to 4 mg once daily regardless of the serum cholesterol level, and the patients were treated for 52 weeks. The study design is summarized in <xref ref-type="fig" rid="F1">Fig. 1</xref>.</p><p>Patient compliance and the presence of AEs, regardless of an association with the study drugs, were evaluated at each visit by direct interview using a prespecified script and pill counts. Blood samples were collected to evaluate any changes during the administration of the study medications.</p><p>All laboratories in the hospitals that participated in this study used procedures that were in accordance with the guidelines of the Clinical and Laboratory Standards Institute for sample collection and handling. They regularly participated in proficiency tests and were accredited by the Korean Association of Quality Assurance for Clinical Pathology.</p></sec><sec><title>Clinical follow-up</title><p>The duration of the study was 52 weeks. Left ventricular dimensions and ejection fraction (EF) were measured at baseline and 52 weeks after initiating statin therapy using the modified Simpson's method. The patients also underwent the 6-minute walk test at baseline and 6 months after initiating statin therapy. B-natriuretic peptide (BNP) levels were compared at baseline and 6 months after initiating statin therapy by one central laboratory.</p></sec><sec><title>Outcome</title><p>The primary endpoint was the composite outcome of heart failure aggravation including hospitalization due to heart failure and total deaths. Hospitalization due to heart failure was determined by the investigators. The secondary outcome was the change in echocardiographic EF after statin treatment. Secondary endpoints included functional assessments using the 6-minute walk test and NYHA class, left ventricular end-diastolic pressure evaluated by the E/e' ratio [<xref rid="B20" ref-type="bibr">20</xref>], and BNP levels [<xref rid="B21" ref-type="bibr">21</xref>]. Safety endpoints included an evaluation of AEs, vital signs, and clinically significant abnormal values among the laboratory test results throughout the study.</p></sec><sec><title>Statistical analysis</title><p>Since no previous reports have compared the effect of statins in patients with heart failure, a sample size of 35 patients per group was needed to detect a 20.0% difference in LDL reduction between the two groups with a standard deviation of 27.0%, 80% power to reach a <italic>p</italic> value of 0.05, and a drop-out rate of 20%. The statistical analysis was performed using SAS version 9.2 (SAS Institute, Cary, NC, USA). Results are expressed as means, standard deviations, and ranges for baseline data, including demographics, medical history, physical examinations, and drug history. All data were compared by an independent biostatistician using one-way analysis of variance (continuous variables) or the chi-square test (discrete variables). The change between baseline and the last observation carried forward value was compared with the paired <italic>t</italic> test. No adjustment was made for baseline covariates.</p><p>The overall rate and frequency of AE occurrence and the rate and frequency of AE occurrence that met the following categories were summarized by treatment group and compared between the two treatment groups using Fisher exact test: AEs whose causality to the study drug could not be ruled out, severely intense AEs, serious AEs, and AEs that led to discontinuing the study drug. The t test was used with a two-sided significance level of 5% to determine if there was any difference between the two groups with respect to the change from baseline to the end of the study.</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Study population and baseline characteristics</title><p>Of the 85 screened patients from six study institutions, 16 were excluded during screening, and 69 were randomized (34 to the pitavastatin group and 35 to the pravastatin group). Three subjects randomized to the pitavastatin group and four subjects randomized to the pravastatin group were excluded before administration of the study drugs, as they violated the inclusion/exclusion criteria and withdrew their consent to participate in this study. After administrating the study drug, six subjects in the pitavastatin group and six subjects in the pravastatin group withdrew; thus, 50 subjects (25 in the pitavastatin group and 25 in the pravastatin group) completed the study (<xref ref-type="fig" rid="F2">Fig. 2</xref>). No significant difference was observed between the two groups for withdrawal rate (<italic>p</italic> = 0.833). Primary efficacy was analyzed in the intention-to-treat population (31 in the pitavastatin group and 31 in the pravastatin group), which included all subjects who received at least one dose of the study drug.</p><p>The majority of the patients were male (74%) with a mean age of 64.4 &#x000b1; 11.5 years. Although the prevalence of males was higher in the pravastatin group (84%) compared with the pitavastatin group (65%), no statistical difference was detected (<italic>p</italic> = 0.082). The NYHA class distribution was 95% in class II and 5% in class III. The mean duration of CHF was 2.7 &#x000b1; 3.7 years, and 48% of patients had a history of admission due to CHF; 24% of patients had diabetes mellitus, 64% had a history of myocardial infarction, and 57% had undergone coronary revascularization (percutaneous intervention 53%, coronary artery bypass graft 7%). Baseline patient characteristics did not differ between the groups (<xref ref-type="table" rid="T1">Table 1</xref>).</p></sec><sec><title>Change in lipoprotein values</title><p>LDL-C decreased significantly by 30% in the 4 mg pitavastatin group, from a mean of 114.58 to 79.45 mg/dL (<italic>p</italic> &#x0003c; 0.001). The reduction in LDL-C in the pitavastatin group was significantly greater than the 12% reduction in the 10 mg pravastatin group (<italic>p</italic> = 0.003).</p><p>Total cholesterol levels decreased significantly by 16% in the 4 mg pitavastatin group (<italic>p</italic> &#x0003c; 0.001) and by 19% in the 10 mg pravastatin group (<italic>p</italic> = 0.016). Although the extent of cholesterol reduction was greater in the pitavastatin group, no statistical difference was observed.</p><p>The HDL cholesterol level was increased by 9% in the 4 mg pitavastatin group with borderline significance (<italic>p</italic> = 0.052); however, no change was observed in the pravastatin group (<italic>p</italic> = 0.635). Triglyceride levels remained unchanged in both groups. The changes in cholesterol and lipoprotein levels are summarized in <xref ref-type="table" rid="T2">Table 2</xref>.</p></sec><sec><title>Left ventricular remodeling following statin treatment</title><p>The left ventricular systolic dimensions decreased significantly by 9% in the pitavastatin group (<italic>p</italic> = 0.011) and by 5% in the pravastatin group (<italic>p</italic> = 0.026). The difference between the two groups was not significant (<italic>p</italic> = 0.444) (<xref ref-type="fig" rid="F3">Fig. 3A</xref>).</p><p>EF improved significantly from 37% to 42% in the pitavastatin group (<italic>p</italic> = 0.003) and from 35% to 39% in the pravastatin group (<italic>p</italic> = 0.002). Although the extent of the EF change was greater in the pitavastatin group (16% vs. 11%), no statistical difference was observed between the groups (<italic>p</italic> = 0.386) (<xref ref-type="fig" rid="F3">Fig. 3B</xref>).</p><p>In contrast, left ventricular diastolic function, evaluated by the E/A ratio, remained unchanged in both groups (<italic>p</italic> = 0.88) (<xref ref-type="fig" rid="F3">Fig. 3C</xref>). The left atrial diameter also remained unchanged (data not shown). The changes in left ventricular dimensions and systolic and diastolic function evaluated by echocardiography are summarized in <xref ref-type="table" rid="T3">Table 3</xref>.</p></sec><sec><title>Exercise capacity, BNP changes, and outcomes following statin treatment</title><p>Exercise capacity evaluated by the 6-minute walk test improved significantly in the 10 mg pravastatin group (<italic>p</italic> &#x0003c; 0.001), however, no change was observed in the pitavastatin group (<italic>p</italic> = 0.371) (<xref ref-type="table" rid="T4">Table 4</xref>, <xref ref-type="fig" rid="F4">Fig. 4</xref>). More patients (n = 4) in the pravastatin group tended to show an improved NYHA class than those in the pitavastatin group (n = 2). BNP levels did not change significantly in either group.</p><p>No mortalities were observed during the 1-year treatment period. One patient in the pitavastatin group was admitted due to aggravated heart failure after 207 days of treatment. Two patients in the pravastatin group were admitted due to heart failure: one patient after 157 days and the other after the treatment phase ended (455 days).</p></sec><sec><title>Tolerability</title><p>No AE was reported before administration of the investigated products. Thirteen subjects (40%) in the pitavastatin group and 12 subjects (36%) in the pravastatin group experienced an AE; however, no treatment-related AEs were found in either group by the investigators. None of the laboratory parameters differed between the groups (data not shown).</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>The SAPHIRE study investigated the dose-effect relationship of statins in patients with ischemic CHF. Since no previous data on the dose-effect relationship among the extremely different doses/potencies of statins in CHF patients are available, this exploratory study was initiated with 35 patients enrolled per group as a baseline LDL-C-lowering efficacy trial. Due to the small sample size, our findings suggested two hypotheses regarding the effect of statins in patients with CHF. First, low dose, low potency pravastatin lowered LDL-C by 12%, and high dose, high potency pitavastatin lowered LDL-C by 30%. Both treatments improved left ventricular systolic function after 1 year, suggesting that these statins might have beneficial cardiac remodeling effects in patients with ischemic CHF. Second, in contrast to improved cardiac systolic function, high dose, high potency pitavastatin did not improve exercise capacity or functional capacity in patients with CHF, although low dose, low potency pravastatin showed some beneficial effects on exercise capacity, suggesting that lowering cholesterol too much may not be beneficial for muscle strength.</p><p>Despite the molecular biological activities of statins, which clearly show anti-inflammatory and antioxidant effects, the results of two randomized trials evaluating rosuvastatin and one evaluating pitavastatin failed to show a reduction in cardiovascular events [<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B22" ref-type="bibr">22</xref>]. The CORONA study randomized 5,011 patients with ischemic CHF to 10 mg rosuvastatin or placebo [<xref rid="B10" ref-type="bibr">10</xref>]. The GISSI-HF trial randomized 4,631 patients with CHF of all etiologies to 10 mg rosuvastatin or placebo [<xref rid="B11" ref-type="bibr">11</xref>]. The rosuvastatin treatment did not affect the primary endpoints in the two studies and only showed a benefit for the number of CHF hospitalizations in the CORONA trial. However, it was unclear whether the results of the CORONA study and the GISSI-HF trial can be generalized to all patients with CHF or to the different kinds of statins. A meta-analysis reported that the effects of statins on CHF are not class effects, and a significant benefit was found using lipophilic atorvastatin but not hydrophilic rosuvastatin [<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24</xref>]. However, a third randomized trial on lipophilic pitavastatin, the PEARL study, also failed to show benefits [<xref rid="B22" ref-type="bibr">22</xref>], which strongly suggests that the effects of statins on CHF are class effects.</p><p>Although it is still debated whether statins cause fatigue, thus, limiting exercise capacity, one study showed that even statins with modest potency, such as simvastatin and pravastatin, contribute to significant AEs on energy and fatigue with exertion [<xref rid="B25" ref-type="bibr">25</xref>]. A post hoc analysis of the CORONA study also reported a small but significant worsening of fatigue in older patients with systolic heart failure [<xref rid="B26" ref-type="bibr">26</xref>]. Myopathy, a rare but well-reported AE of statins, clearly increases with high-dose statin use compared with lower dose, low potency statin use [<xref rid="B25" ref-type="bibr">25</xref>,<xref rid="B27" ref-type="bibr">27</xref>], which may explain our findings. Another explanation regarding statin-related fatigue is that statins may increase the perception of fatigue through a central nervous system effect, and lipophilic statins increase this perception more than that of hydrophilic agents such as rosuvastatin [<xref rid="B28" ref-type="bibr">28</xref>].</p><p>Other hypotheses arguing against statin treatment in CHF patients are the endotoxin lipoprotein hypothesis, the coenzyme Q10 (ubiquinone) hypothesis, and the selenoprotein hypothesis [<xref rid="B29" ref-type="bibr">29</xref>]. In particular, the endotoxin lipoprotein hypothesis is related to lower cholesterol levels. Several studies have addressed the relationship and relevance between serum cholesterol level and outcomes in patients with CHF and consistently suggested that too low of cholesterol levels are associated with increased mortality [<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B30" ref-type="bibr">30</xref>].</p><p>The findings of the SAPHIRE study are quite interesting, showing that pravastatin and pitavastatin may have a beneficial cardiac reverse remodeling effect and may improve systolic function regardless of potency and lipophilicity/hydrophilicity. The absolute extent of the benefit was greater with the more potent pitavastatin, although no statistical significance was observed, mainly due to the small sample size. In contrast, high dose, high potency pitavastatin failed to improve exercise capacity or functional capacity in patients with CHF, although low dose, low potency pravastatin showed some beneficial exercise capacity effects. These findings may offer a new hypothesis that statin therapy is beneficial for cardiac reverse remodeling, at least in patients with ischemic CHF; however, the adequate dose and potency to maximize the effect must be determined.</p><p>Our findings should be considered in the context of several limitations. This study was designed as an exploratory study and enrolled a small number of patients. Therefore, a larger and longer duration study is warranted to confirm our findings. Second, we tried to narrow the scope to patients with ischemic CHF and to maximize the potency, dose, and characteristics of the statins due to the small sample size. Therefore, our findings cannot be generalized to all patients with CHF unless supported by further studies. Last, although low potency pravastatin may improve exercise capacity, we did not observe significant hemodynamic, echocardiographic, or BNP changes associated with CHF symptoms. Therefore, uncertainty remains as to whether a low dose statin is beneficial for exercise capacity.</p><p>In conclusion, both low dose pravastatin and high dose pitavastatin had a beneficial cardiac reverse remodeling effect and improved systolic function in patients with ischemic CHF. However, only low dose pravastatin improved exercise capacity significantly. These findings suggest that "too much" lowering of cholesterol might not be beneficial for functional capacity in patients with CHF.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>In heart failure patients with ischemic origin, statin treatment is required in order to prevent second ischemic attack.</p></list-item><list-item><p>However, 'too much' lowering of cholesterol might not be beneficial in heart failure patients especially in functional capacity recovery.</p></list-item></list> </p></boxed-text></sec>
Disease burden of pneumonia in Korean adults aged over 50 years stratified by age and underlying diseases
<sec><title>Background/Aims</title><p>This study was conducted to assess the disease burden of pneumonia according to age and presence of underlying diseases in patients admitted with community-acquired pneumonia (CAP).</p></sec><sec><title>Methods</title><p>We performed a retrospective, observational study and collected data targeting patients with CAP (&#x02265; 50 years) from 11 hospitals. Disease burden was defined as total per-capita medical fee, severity (CURB-65), hospital length of stay (LOS), and mortality.</p></sec><sec><title>Results</title><p>Of the 693 enrolled subjects, elderly subjects (age, &#x02265; 65 years) had a higher mean CURB-65 score (1.56 vs. 0.25; <italic>p</italic> &#x0003c; 0.01) and higher mortality than nonelderly subjects (4.4% [n = 21] vs. 0.5% [n = 1]; <italic>p</italic> = 0.00). In addition, the total cost of pneumonia treatment was higher in elderly patients compared to in nonelderly patients (KRW 2,088,190 vs. US $1,701,386; <italic>p</italic> &#x0003c; 0.01). Those with an underlying disease had a higher CURB-65 score (1.26 vs. 0.68; <italic>p</italic> &#x0003c; 0.01), were much older (mean age, 71.24 years vs. 64.24 years; <italic>p</italic> &#x0003c; 0.01), and had a higher mortality rate than those without an underlying disease (3.5% [n = 20] vs. 1.7% [n = 2]; <italic>p</italic> = 0.56). Total per-capita medical fees were higher (KRW 2,074,520 vs. US $1,440,471; <italic>p</italic> &#x0003c; 0.01) and hospital LOS was longer (mean, 8.38 days vs. 6.42 days; <italic>p</italic> &#x0003c; 0.01) in patients with underlying diseases compared to those without.</p></sec><sec><title>Conclusions</title><p>Due to the relatively high disease burden in Korea, particularly in the elderly and in those with an underlying disease, closer and more careful observation is needed to improve the outcomes of patients with CAP.</p></sec>
<contrib contrib-type="author"><name><surname>Lee</surname><given-names>Jung Yeon</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yoo</surname><given-names>Chul Gyu</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Hyo-Jin</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Jung</surname><given-names>Ki Suck</given-names></name><xref ref-type="aff" rid="A4">4</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Yoo</surname><given-names>Kwang Ha</given-names></name><xref ref-type="aff" rid="A5">5</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Community-acquired pneumonia (CAP) is a common and potentially serious illness. It is associated with considerable morbidity and mortality, particularly in elderly patients and those with significant underlying diseases [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>]. Some studies have indicated that the incidence of CAP increases substantially with age, and patients &#x0003e; 60 years account for 81.2% of all cases [<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>]. Furthermore, the hospitalization rate due to CAP increases with every decade of life until the eighth decade [<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>]. The overall annual incidence of CAP in adults also increases with age (14 per 1,000 person-year in adults age &#x02265; 65 years) [<xref rid="B6" ref-type="bibr">6</xref>]. Additionally, age is an independent risk factor for pneumonia, after controlling for confounding variables, such as underlying disease conditions, immobility, and use of tranquilizers [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>]. Moreover, the elderly are more susceptible to pneumonia due to the anatomical and physiological changes that occur in the lungs with age [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>].</p><p>A study conducted in Denmark demonstrated that the incidence of pneumonia requiring hospitalization increased by 50% from 1994 to 2004, and reported a persistently high mortality rate [<xref rid="B9" ref-type="bibr">9</xref>]. A Korean study of the pneumonia burden in 764 inpatients with CAP (age, &#x02265; 50 years) revealed that 3.2% patients died during treatment [<xref rid="B10" ref-type="bibr">10</xref>]. Mortality occurs primarily in the elderly and is frequently associated with underlying diseases. Moreover, CAP is not only an increasing mortality factor in these patients but is associated with a significant economic burden in the Asia-Pacific region [<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>].</p><p>Although many Korean studies have reported the incidence of CAP and its associated risk factors, there is a lack of information regarding the elderly, the group that most frequently experiences severe illness and underlying disease [<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>]. Furthermore, no comparative information is available on disease burden in those patients. To better understand the influence of age and underlying disease, we focused on the disease burden of inpatients with CAP according to aging factors and the presence of underlying diseases. Furthermore, we analyzed the variables associated with disease severity, cost, and outcome.</p></sec><sec sec-type="methods"><title>METHODS</title><p>The study setting and population were described previously by Yoo et al. [<xref rid="B10" ref-type="bibr">10</xref>]. This was a retrospective observational study conducted from January 1, 2008 to December 31, 2010. Subjects aged &#x02265; 50 years were recruited from 11 university hospitals and were enrolled in reverse temporal order from the end of treatment. Of the 764 cases of confirmed pneumonia, 56 subjects with malignant cancer and 15 cases with only a fungal pathogen were excluded from analyses.</p><sec><title>Data collection</title><p>We reported the methodological details of data collection previously [<xref rid="B10" ref-type="bibr">10</xref>]. Briefly, general and clinical characteristics included sex, age, underlying disease, comorbid conditions (solid organ transplant, neutropenia, use of immunosuppressant within 3 months, use of corticosteroid) and identified pathogen severity using the CURB-65 (confusion, urea &#x0003e; 7 mM/L [19 mg/dL], respiratory rate &#x02265; 30/min, systolic blood pressure &#x0003c; 90 mmHg or diastolic blood pressure &#x02264; 60 mmHg, and age &#x02265; 65 years) score, hospital length of stay (LOS), and survival status were collected.</p></sec><sec><title>Resource utilization</title><p>All resource utilization and economic data were derived from the patient's detailed direct medical costs (insured and uninsured charges). Insured or uninsured charges for medical resource items were calculated to reflect the actual status at each relevant institution. The 2010 health insurance fees were consistently applied as insured charges. Each cost was separated by the following pertinent charge departments: hospitalization, medication, diagnostic testing, laboratory testing, imaging testing, and procedures or surgery. If patient admission days were 2 or more, the transfer and discharge day were excluded from the cost analysis, and only medical resource utilization was applied. Site and frequency of admission were evaluated for hospitalization. Antibiotics and other pneumonia-related drugs were included as medications. Procedures for identifying bacteria (bronchoscopy, thoracentesis, histology, etc.) and sputum, blood, urine, and pleural fluid examinations were included as diagnostic tests. Hematology or urinalysis; names and frequency of imaging tests (simple chest radiography, chest computed tomography, magnetic resonance imaging, ultrasound, and echocardiography); and for procedures or surgery, any procedure to resolve pneumonia-associated complications were performed as laboratory tests. More detailed resource utilization and calculation methods for the cost analysis were described in our previous report [<xref rid="B10" ref-type="bibr">10</xref>]. The costs only for evaluating and managing CAP were included.</p></sec><sec><title>Statistical analysis</title><p>The SPSS version 20.0 (IBM Co., Armonk, NY, USA) was used for the analysis. Descriptive statistics are presented as results for the general and clinical characteristics of the elderly and nonelderly patients as well as those with or without an underlying disease. Categorical variables are described with counts and percentages. Means &#x000b1; SD for continuous variables are presented. The <italic>t</italic> test or Mann-Whitney test and chi-square test were used to determine the significance of differences. A multivariate analysis of risk factors that affected hospital LOS and costs of patients with pneumonia was conducted, and log-transformation was applied. A two-tailed <italic>p</italic> &#x0003c; 0.05 was considered to indicate significance. The average per-capita direct medical cost and average per-capita daily direct medical cost for all patients with pneumonia and the subgroups of patients (elderly or 50 to 64 years and those with or without underlying diseases) were calculated using the direct medical cost results, which were estimated from medical resources.</p></sec><sec><title>Ethics statement</title><p>The Institutional Review Boards at each site approved this study, and written informed consent was obtained from all patients. The approval number for Hallym University Sacred Heart Hospital was 2011-S016.</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Baseline characteristics</title><p>After excluding 71 (56 patients with active malignancy and 15 with a single fungal pathogen) of 764 patients, the results for 693 patients were analyzed. We separated all patients with pneumonia into groups according to age (elderly [age &#x02265; 65 years] or nonelderly [age, 50 to 64 years]), and presence of underlying disease (with or without underlying disease), and compared the baseline characteristics. Males comprised the majority of elderly patients (62.1% [298 patients] vs. 37.9% [182 patients], <italic>p</italic> &#x0003c; 0.01) (<xref ref-type="table" rid="T1">Table 1</xref>). We also separated all patients with pneumonia into those with or without an underlying disease (<xref ref-type="table" rid="T2">Table 2</xref>). Of all patients with pneumonia, 83.4% (578) had an underlying disease. Mean ages of patients with and without an underlying disease were 71.24 &#x000b1; 9.97 and 64.24 &#x000b1; 11.16 years (<italic>p</italic> &#x0003c; 0.01), respectively, and that for males was 60% (n = 347) versus 40% (n = 231), respectively (<italic>p</italic> &#x0003c; 0.01). A greater proportion of males had an underlying disease, and the mean age of males was greater than that of females.</p><p>A pathogen was identified in 32.9% of cases (228 patients). <italic>Streptococcus pneumoniae</italic> was detected in 26 (5.4%) of the nonelderly patients and 12 cases (5.6%) of the elderly had a pathogen (<xref ref-type="table" rid="T1">Table 1</xref>). <italic>S. pneumoniae</italic> was isolated from 32 cases (5.5%) with an underlying disease and six (5.2%) without (<xref ref-type="table" rid="T2">Table 2</xref>). Mixed infection with <italic>S. pneumoniae</italic> was noted in nine elderly patients (1.9 %; <italic>p</italic> = 0.10, data not shown) and 12 patients (2.1%) with an underlying disease (<italic>p</italic> = 0.71, data not shown). The distribution of pneumonia pathogens did not differ between the elderly and nonelderly groups and those with or without underlying diseases. Furthermore, gram-positive and -negative bacteria were isolated at a similar rate in elderly and nonelderly groups, same as the with underlying disease and without underlying diseases (<xref ref-type="table" rid="T1">Tables 1</xref> and <xref ref-type="table" rid="T2">2</xref>).</p></sec><sec><title>Disease severity, treatment duration, and treatment outcomes</title><p>The mean CURB-65 score in all patients with pneumonia was 1.16 points, and was higher in the elderly (1.56 &#x000b1; 0.78 vs. 0.25 &#x000b1; 0.52, <italic>p</italic> &#x0003c; 0.01) and those with an underlying disease than the nonenderly group without underlying disease (1.26 &#x000b1; 0.85 vs. 0.68 &#x000b1; 0.85, <italic>p</italic> &#x0003c; 0.01). Patients who scored &#x02265; 3 points comprised 10.2% of the elderly, which was significantly higher than that in the 50- to 64-year age group (10.2% [n = 49] vs. 0.5 % [n = 1], <italic>p</italic> &#x0003c; 0.01). Moreover, subjects with an underlying disease scored &#x02265; 3 points significantly more frequently than those without (7.6% [n = 44] vs. 5.2% [n = 6], <italic>p</italic> &#x0003c; 0.01) (<xref ref-type="table" rid="T3">Table 3</xref>).</p><p>LOS was longer in elderly patients (8.45 days vs. 7.16 days, <italic>p</italic> = 0.00), and in those with than without an underlying disease (8.38 days vs. 6.42 days, <italic>p</italic> &#x0003c; 0.01) (<xref ref-type="table" rid="T3">Table 3</xref>). A total of 3.2% of the 693 patients with pneumonia died. The mortality rate was 4.4% (21 patients) in the elderly and 0.5% (one patient) in the nonelderly group (<italic>p</italic> = 0.00). Similarly, the mortality rate was 3.5% (20 patients) in those with an underlying disease and 1.7% (two patients) in those without (<italic>p</italic> = 0.56) (<xref ref-type="table" rid="T3">Table 3</xref>).</p></sec><sec><title>Cost analysis</title><p>Total per-capita medical fees for all patients with pneumonia were KRW 1,969,303 (US $1,969; US $1 = 1,000 KRW); they were 2,088,190 KRW in the elderly group and 1,701,386 KRW in the nonelderly group (<italic>p</italic> &#x0003c; 0.01). Furthermore, the medical fees summed to 2,074,520 KRW in patients with an underlying disease and 1,440,471 KRW in those without (<italic>p</italic> &#x0003c; 0.01) (<xref ref-type="table" rid="T4">Table 4</xref>). The costs for hospitalization, medication, diagnostic testing, laboratory testing, imaging, and a procedure or surgery in those with an underlying disease were higher than in those without an underlying disease (<italic>p</italic> &#x0003c; 0.01) (<xref ref-type="table" rid="T4">Table 4</xref>). Elderly patients had a higher cost for each item compared to nonelderly patients, with the exception of diagnostic tests (<xref ref-type="table" rid="T4">Table 4</xref>). The percentage breakdown of costs for each item in the elderly was 28.2% for hospitalization, 22.3% for medication, 14.5% for diagnostic testing, 14.8% for laboratory testing, 10.4% for imaging, and 9.8% for a procedure or surgery. The underlying disease group had higher cost percentages for hospitalization (27.9%), medication (22.1%), diagnostic testing (15.2%), laboratory testing (14.7%), imaging (10.8%), and a procedure or surgery (9.3%). Subjects aged 50 to 64 years and those without an underlying disease showed similar proportions.</p></sec><sec><title>Associations among disease severity, hospital LOS, and cost</title><p>A univariate analysis presumed that the following factors were important for predicting patient outcome: age &#x0003e; 65 years, chronic cardiovascular disorder, chronic lung disease, CNS disorder, and chronic renal disorder (data not shown). Using these variables, we analyzed the factors associated with hospital LOS, cost, and severity (CURB-65 score). Hospital LOS was increased in the elderly (<italic>p</italic> &#x0003c; 0.033) and those with chronic lung disease (<italic>p</italic> = 0.035). Cost was increased in the elderly (<italic>p</italic> = 0.005), and those with a chronic cardiovascular disorder (<italic>p</italic> = 0.028) or chronic lung disease (<italic>p</italic> = 0.006) (<xref ref-type="table" rid="T5">Table 5</xref>). The propensity to score low on the CURB-65 was increased in the elderly (<italic>p</italic> &#x0003c; 0.001), in those with chronic renal disease (<italic>p</italic> &#x0003c; 0.001), and in those with a CNS disorder (<italic>p</italic> = 0.024). Moreover, a high CURB-65 score was more likely in patients with chronic lung disease (<italic>p</italic> = 0.014).</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>This study involved a subgroup analysis, which was first conducted by Yoo et al. [<xref rid="B10" ref-type="bibr">10</xref>]. In the current study, we focused on disease burden measured by total per-capita medical fees, severity (CURB-65), hospital LOS, and mortality, according to age and underlying diseases. Our findings demonstrated that disease burden was higher in the elderly (&#x02265; 65 years) and in patients with an underlying disease compared to those aged 50 to 64 years or without an underlying disease.</p><p>A cost analysis for pneumonia across studies is challenging due to differences in coding and data sources for the definition of pneumonia as well as the analytical methods used to calculate costs [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>]. According to a US pneumonia burden study, the mean LOS per hospital admission was 7.6 days, with a mean cost of US $6,949 per person (1997 value) [<xref rid="B5" ref-type="bibr">5</xref>]. This is consistent with the results of the retrospective analysis by Niederman et al. [<xref rid="B16" ref-type="bibr">16</xref>], who reported that mean hospital LOS and cost were 7.8 days and US $7,166 per person (1995 value) in patients &#x0003e; 65 years of age [<xref rid="B16" ref-type="bibr">16</xref>]. The overall annual hospital cost for CAP in elderly patients in the US is $4.4 billion to $4.8 billion [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B16" ref-type="bibr">16</xref>]. A US pneumonia cost analysis of hospital-treated pneumonia as a primary diagnosis in the elderly population in 2010, reported a conservative estimate of &#x0003e; US $7 billion, or US $9,749 per person (2005 to 2007 value); this imposes a tremendous burden on the US healthcare system [<xref rid="B14" ref-type="bibr">14</xref>]. However, those studies relied on Medicare administrative claims for the cost analysis, and the subjects selected may not have provided the actual costs of care. Additionally, limited clinical details of each individual were provided. This suggests the costs may have been overestimated as a result of inclusion of treatments unrelated to pneumonia or unobserved morbidities. In contrast, the costs in our study included both insured and uninsured charges for each medical resource item. Furthermore, we consistently applied the actual cost case by case when a difference existed between hospitals, particularly for uninsured charges. In contrast to these previous studies, which used only the International Classification of Diseases code to select subjects, the definition of pneumonia in our study was clear due to the diagnosis being confirmed by pulmonary specialists in tertiary teaching hospitals. In support of these results, another US study reported that the mortality rate can change according to the pneumonia disease coding used [<xref rid="B17" ref-type="bibr">17</xref>]. Few studies in Asia have determined whether the CAP economic burden can be compared with that of other regions. In Taiwan, the cost of one hospital admission and the total cost for CAP in the elderly were approximately US $3,221 and US $1,897,137, respectively [<xref rid="B18" ref-type="bibr">18</xref>]. Among all age groups in rural Thailand, the cost of hospitalization for an episode of pneumonia ranges from US $490.80 to US $628.60 [<xref rid="B19" ref-type="bibr">19</xref>]. However, the Thai study used national health insurance claims data only; therefore, selection bias may have existed. Furthermore, the case definition for the inclusion criteria and the cost analysis method were unclear.</p><p>In addition to the cost analysis method and disease coding, the population examined is an important factor for a cost analysis study. A multicenter, hospital-based study of the clinical and economic burden of invasive pneumococcal disease (IPD) reported mean direct medical costs of US $7,452 (mean, US $5,404 or US $8,756/case), and no difference between age and risk groups [<xref rid="B20" ref-type="bibr">20</xref>]. Similarly, Weycker et al. [<xref rid="B13" ref-type="bibr">13</xref>] reported that the direct medical cost per case did not differ significantly between risk and age groups (US $15,402 to US $31,849/case) [<xref rid="B13" ref-type="bibr">13</xref>]. The former calculated direct medical costs by adding only IPD-related costs for each subject, whereas the latter involved data from the 2004 Healthcare Cost and Utilization Project Nationwide Inpatient Sample using mean age- and risk-specific charges [<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B20" ref-type="bibr">20</xref>]. Those results are in agreement with our findings. Total costs for CAP in our elderly and underlying disease groups were significantly higher than in the nonelderly and without underlying disease groups, suggesting that the initial disease severity is an important factor for the cost analysis, as are the cost analysis and disease coding methods. As only subjects with severe disease were included, selection bias could have existed in those studies. Moreover, those studies may have reported considerably higher costs because they did not include actual costs, such as uninsured charges, as in our study. Therefore, our data are more accurate and can be generalized to an inpatient CAP population.</p><p>CAP is one of the most common causes of death in the infectious diseases category in Korea, and mortality rates reach 12% to 14%, even with antibiotic use [<xref rid="B21" ref-type="bibr">21</xref>]. A prospective surveillance study conducted in 14 tertiary hospitals in Asian countries (South Korea, China, Taiwan, Hong Kong, India, Singapore, Vietnam, and Philippines; Asian Network for Surveillance of Resistant Pathogens), showed that old age was a risk factor for mortality [<xref rid="B11" ref-type="bibr">11</xref>]. In fact, when patients were stratified by age, the mean CURB-65 severity score increased gradually with age (50 to 54 years, 0.16; 55 to 59 years, 0.30; 60 to 64 years, 0.29; 65 to 70 years, 1.40; data not shown) as in our study. Furthermore, the percentage of patients with a high CURB-65 score also increased markedly starting at age 65 yr (50 to 54 years, n = 0 [0%]; 55 to 59 years, n = 0 [0%]; 60 to 64 years, n = 1 [1.3%]; 65 to 70 years, n = 7 [6.9%]; data not shown). This is possibly because the CURB-65 score includes age &#x02265; 65 years as a factor in the severity scoring. We divided the patients into elderly (&#x02265; 65 years) or nonelderly (50 to 64 years) groups. The elderly group developed a higher disease burden. However, age is itself an important risk or prognostic factor for pneumonia. Age was not a predictive factor for mortality in a Korean study conducted to determine predictors of in-hospital mortality in patients with severe CAP who required mechanical ventilation [<xref rid="B22" ref-type="bibr">22</xref>]. Therefore, severity is more important than age in terms of mortality.</p><p>Because we defined the costs for disease burden, severity (CURB-65), hospital LOS, and mortality, we identified the variables associated with these factors. Cost and hospital LOS were significantly higher in the elderly and in patients with an underlying disease; however, no significant association with any variable was found in the multivariate analysis (data not shown). This might have been caused by a lower or slightly higher mortality rate in those with an underlying disease or the elderly. Although we cannot explain the low mortality, several factors are likely involved, such as the modest effect of higher pneumococcal vaccination rates on pneumonia risk; reduced smoking; and better treatment for chronic underlying disease; these are risk factors for pneumonia. Pooled data from observational studies have demonstrated the overall burden of CAP in patients with other medical conditions, such as chronic respiratory diseases, cardiovascular diseases, cerebrovascular diseases, dementia, and diabetes mellitus, which were the most frequent underlying diseases [<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B23" ref-type="bibr">23</xref>]. Up to two-thirds of patients had a chronic respiratory disease and almost half had a chronic cardiovascular disease [<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B23" ref-type="bibr">23</xref>]. Our results also indicate that ~90% of those with an underlying disease had chronic cardiovascular disease (52.4%, n = 363) or chronic lung disease (37.4%, n = 259). Disease severity and frequency of an underlying condition affected the outcomes. Mortensen et al. reported that about half of deaths in patients with CAP were attributable to worsening of a pre-existing condition [<xref rid="B24" ref-type="bibr">24</xref>]. Although we cannot explain these factors separately, we understand that age and underlying disease are important factors for predicting disease burden. An analysis of each individual patient with an underlying disease and a longitudinal assessment of outcomes are needed to better understand the associations between age and underlying disease, cost, disease severity, and hospital LOS. Some limitations of this investigation should be mentioned. As our study was performed in a tertiary referral center and did not include an outpatient population, in which CAP is treated more commonly, it is difficult to generalize our results. Another limitation of our study is that only subjects age &#x02265; 50 years were included; thus, our results may not be representative of the entire population of pneumonia patients in Korea.</p><p>The strengths of this study are the accuracy of the cost analysis for inpatient CAP. Due to the lack of information about the most frequent independent risk factors (elderly, patients with underlying disease) in Korea, we analyzed these patients using accurate medical resources, including uninsured items, which had not been applied in previous studies. Insured items were adjusted for annual inflation by consistently applying the 2010 insurance fees for each insurance medical resources. Furthermore, the nursing (or intensive care unit nursing) personnel category was applied by identifying the actual status in each institution. An accurate economic burden for uninsured items was calculated by focusing on publicly available uninsured data, as disclosed on the website of each institution. Although this was a retrospective study, patients with pneumonia at each hospital were identified in reverse temporal order from the treatment completion date (December 31, 2010) and were extracted relatively randomly; hence, little bias existed according to disease severity, age, sex, or the presence of underlying disease. Third, the study was conducted mostly in the pulmonary divisions of university hospitals; thus, the diagnosis of pneumonia and assessment of underlying diseases were accurate. We identified pathogens more aggressively using every possible diagnostic tools such as bronchoscopy, thoracentesis, histology, etc., and assessed treatment outcomes in &#x0003e; 90% of all cases.</p><p>In summary, we report herein the characteristics and medical costs of Korean pneumonia patients aged &#x02265; 50 years who received inpatient care. Patients were divided into elderly and non-elderly and those with and without an underlying disease. Total costs, hospital LOS, and disease severity were higher in the elderly and in those with an underlying disease than in the non-elderly and those without an underlying disease. Therefore, this disease imposes a considerable on both the individual, particularly the elderly and those with an underlying disease, and society in general. Furthermore, an actual cost analysis regarding all medical resources, including noninsurance charges, is essential; this should be based on an accurate disease diagnosis as well as on the presence of underlying disease, as discussed above. Given that being elderly and having chronic lung disease were associated with a higher disease burden, closer and more careful observations in these populations are essential to improve the outcomes of inpatient treatment of CAP.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Among patients hospitalized for community-acquired pneumonia (CAP), the elderly and those with an underlying disease had higher disease burdens.</p></list-item><list-item><p>A cost analysis that considers all medical resources based on accurate diagnoses is essential to assess more accurately the disease burden associated with CAP.</p></list-item></list> </p></boxed-text></sec>
The impact of high-flux dialysis on mortality rates in incident and prevalent hemodialysis patients
<sec><title>Background/Aims</title><p>The effect of high-flux (HF) dialysis on mortality rates could vary with the duration of dialysis. We evaluated the effects of HF dialysis on mortality rates in incident and prevalent hemodialysis (HD) patients.</p></sec><sec><title>Methods</title><p>Incident and prevalent HD patients were selected from the Clinical Research Center registry for end-stage renal disease (ESRD), a Korean prospective observational cohort study. Incident HD patients were defined as newly diagnosed ESRD patients initiating HD. Prevalent HD patients were defined as patients who had been receiving HD for &#x0003e; 3 months. The primary outcome measure was all-cause mortality.</p></sec><sec><title>Results</title><p>This study included 1,165 incident and 1,641 prevalent HD patients. Following a median 24 months of follow-up, the mortality rates of the HF and low-flux (LF) groups did not significantly differ in the incident patients (hazard ratio [HR], 1.046; 95% confidence interval [CI], 0.592 to 1.847; <italic>p</italic> = 0.878). In the prevalent patients, HF dialysis was associated with decreased mortality compared with LF dialysis (HR, 0.606; 95% CI, 0.416 to 0.885; <italic>p</italic> = 0.009).</p></sec><sec><title>Conclusions</title><p>HF dialysis was associated with a decreased mortality rate in prevalent HD patients, but not in incident HD patients.</p></sec>
<contrib contrib-type="author"><name><surname>Kim</surname><given-names>Hyung Wook</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Su-Hyun</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Young Ok</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Jin</surname><given-names>Dong Chan</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Ho Chul</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Choi</surname><given-names>Euy Jin</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Yong-Lim</given-names></name><xref ref-type="aff" rid="A4">4</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Yon-Su</given-names></name><xref ref-type="aff" rid="A5">5</xref></contrib><contrib contrib-type="author"><name><surname>Kang</surname><given-names>Shin-Wook</given-names></name><xref ref-type="aff" rid="A6">6</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Nam-Ho</given-names></name><xref ref-type="aff" rid="A7">7</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Chul Woo</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kim</surname><given-names>Yong Kyun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>The mortality rate of end-stage renal disease (ESRD) patients undergoing maintenance hemodialysis (HD) is higher compared with that of the general population [<xref rid="B1" ref-type="bibr">1</xref>]. More effective and well-tolerated HD treatment could improve the clinical outcomes of ESRD patients undergoing HD [<xref rid="B2" ref-type="bibr">2</xref>]. Dialysis duration and frequency, the nature of vascular access, and the quality of the dialysate influence the rates of short- and long-term complications in HD, which can affect the overall outcome. In particular, selection of the most effective dialyzer is an important determinant of HD treatment effectiveness. High-flux (HF) dialyzers, characterized by a higher degree of porosity compared with low-flux (LF) dialyzers, can clear a greater amount of medium-molecular-weight uremic toxins, such as &#x003b2;2-microglobulin, parathyroid hormone, advanced glycation products, and leptin [<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>].</p><p>Despite its superior ability to remove medium-molecular-weight toxins, the clinical benefit of HF dialysis remains controversial. Several observational studies indicated that HF dialysis confers a survival benefit [<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>]. However, two large, randomized clinical trials, the HEMO study and the European Membrane Permeability Outcome (MPO) study, reported no significant difference in the mortality rates associated with HF- and LF-dialysis [<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>].</p><p>This discrepancy could be due to differences in study design or population characteristics; we presently focus on the latter possibility. HF dialysis decreased the mortality rate in patients receiving &#x0003e; 3.7 years of dialysis prior to enrolment in the subgroup analysis of the HEMO study [<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>]. In contrast, there was no significant difference in the survival rates of HF versus LF dialysis patients receiving &#x0003c; 3.7 years of dialysis treatment prior to enrolment. This was also the case in the incident HD patients of the MPO study [<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>]. Furthermore, incident and prevalent HD patients differ in terms of the causes of, and risk factors for, mortality [<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>].</p><p>Therefore, we hypothesized that the impact on mortality rate of HF dialysis might differ between incident and prevalent HD patients, and might also vary in accordance with the duration of prior dialysis treatment in prevalent HD patients. This prospective cohort study, which included incident and prevalent HD patients, evaluated the impact of HF dialysis on mortality rates in incident and prevalent HD patients.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Study population</title><p>Each patient enrolled to the study was on the Clinical Research Center registry for ESRD. An ongoing, observational prospective cohort design was employed, using patients with ESRD drawn from 31 centers in Korea. Enrolment commenced in April 2009, and included adult (&#x0003e; 18 years of age) incident and prevalent dialysis patients. Incident HD patients were defined as newly diagnosed ESRD patients initiating HD. Prevalent HD patients were defined as patients who had been receiving HD for &#x0003e; 3 months. Totals of 1,283 incident patients and 1,784 prevalent patients were included. Patients undergoing hemodiafiltration (n = 104) were excluded, as were patients for whom information regarding the type of dialysis membrane used was not available (n = 157). Totals of 1,165 incident and 1,641 prevalent patients were included in the final analysis.</p><p>Demographic and clinical data were collected at enrolment. Assessment of dialysis characteristics and measurements of health were conducted every 6 months, until the termination of the follow-up period. Dates and causes of death were immediately reported during the follow-up period. The study was approved by the Medical Ethics Committees of the participating hospitals. Informed consent was provided by all patients prior to their inclusion.</p></sec><sec><title>Clinical and dialysis parameters</title><p>Baseline demographic and clinical data, including age, sex, body mass index (BMI), type of dialysis membrane used, cause of ESRD, comorbidities, laboratory investigation results, and therapeutic characteristics, were recorded. Cardiovascular disease was defined as the presence of coronary heart disease, peripheral vascular disease, or cerebrovascular disease. Serum hemoglobin, total cholesterol, albumin, creatinine, and urea levels were determined from blood samples. To assess the effect of dialysis membrane type on mortality rates, patients were divided into HF and LF dialysis groups in accordance with the type of dialysis membrane used during their treatment. HF dialysis was defined as an ultrafiltration coefficient of &#x02265; 20 mL/mmHg per hour, and a sieving coefficient, for &#x003b2;2-microglobulin, of &#x0003e; 0.6. LF dialysis was defined as an ultrafiltration coefficient of &#x0003c; 20 mL/mmHg per hour, with a sieving coefficient for &#x003b2;2-microglobulin of 0.</p><p>A total of 26 types of LF dialyzer, and 31 types of HF dialyzer, were observed in this study. In incident HD patients, the most frequently used LF dialyzer was the Gambro polyflux 14L (36.9% of cases; Gambro, Lund, Sweden); the most frequently used used HF dialyzer was the Fresenius Medical Care F60S (26.9% of cases; Fresenius Medical Care, Bad Homburg, Germany). In prevalent HD patients, the most frequently used LF dialyzer was the Gambro polyflux 14L (30.7% of cases); the most frequently used HF dialyzer was the Fresenius Medical Care FX 60 (21.3% of cases). The types of dialysis membrane materials used were also determined. In incident HD patients, all of the dialysis membranes were synthetic in the HF dialyzer group; in the LF dialyzer group, 99.7% of the membranes were synthetic, and 0.3% contained substituted cellulose. In prevalent HD patients, all dialysis membranes were synthetic in the HF dialyzer group; in the LF dialyzer group, 98.4% of membranes were synthetic and 1.6% contained substituted cellulose. All dialysis sessions were conducted without reusing dialyzers, and all dialysate solutions were bicarbonate-based. The single-pool Kt/V (spKt/V: K, dialyzer clearance; t, time; V, volume of water contained within the body) was determined using two-point urea modeling on the basis of the intradialytic reduction in blood urea and intradialytic weight loss [<xref rid="B21" ref-type="bibr">21</xref>].</p></sec><sec><title>Outcomes</title><p>The primary clinical outcome measure was all-cause mortality. For each death, the clinical center principal investigator completed a form documenting cause of death according to the study's classification system.</p></sec><sec><title>Statistical analyses</title><p>Continuous variables with normal distributions are presented as means &#x000b1; SD. Variables that are not normally distributed are presented as medians with ranges. Student t test, Mann-Whitney test, one-way analysis of variance and Kruskal-Wallis test were used, as appropriate, to assess differences in continuous variables. Categorical variables are presented as percentages. The Pearson's chi-square test or Fisher exact test was used to determine differences in categorical variables.</p><p>Absolute mortality rates were calculated per 100 person-years of follow-up. The primary outcome measure was mortality rate. All patients were followed until death (event), or until the study terminated, with censoring of data occurring either when patients underwent renal transplantation or were lost to follow-up following withdrawal of participation or transfer to a non-participating hospital. Survival curves for the HF and LF dialysis groups were estimated using the Kaplan-Meier method and the log-rank test. The Cox proportional hazard regression model was used to calculate the hazard ratio (HR), with 95% confidence intervals (CI), for all-cause mortality. For categorical variables, the assumption of proportional hazards over time was assessed by visual inspection of the log-minus-log survival plot. Analyses were adjusted for the following potential confounders: age, gender, BMI, diabetes mellitus, cardiovascular disease, causes of ESRD, duration of dialysis therapy, systolic blood pressure (BP), hemoglobin, serum albumin, serum total cholesterol, number of dialysis sessions per week, type of vascular access, and HD blood flow rates. To determine the interaction between the effect of HF dialysis on mortality and the duration of dialysis therapy in prevalent HD patients, we categorized the duration of prior dialysis therapy using quartiles, as follows (m): quartile 1, &#x0003c; 13.5; quartile 2, 13.5 to 33.0; quartile 3, 33.1 to 68.5; and quartile 4, &#x0003e; 68.5. Multivariate Cox regression analysis was performed for the different quartiles. A value of <italic>p</italic> &#x0003c; 0.05 was taken to indicate statistical significance. All analyses were performed using the SPSS version 11.5 (SPSS Inc., Chicago, IL, USA).</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Patient characteristics</title><p>Totals of 1,165 incident and 1,641 prevalent patients were included in this study. Of the incident HD patients, 71.3% (831 of 1,165) were dialyzed using LF dialysis membranes; 28.7% (334 of 1,165) were dialyzed using HF dialysis membranes. The baseline characteristics of incident HD patients are displayed in <xref ref-type="table" rid="T1">Table 1</xref>. The HF group was characterized by lower BMI, higher systolic BP, and lower serum albumin levels compared with the LF group. There were no significant group differences in age, gender, prevalence of diabetes mellitus, prevalence of cardiovascular disease, causes of ESRD, diastolic BP, serum hemoglobin levels, serum total cholesterol levels or spKt/V. There was no significant group difference in the number of arteriovenous grafts used for vascular access. Significantly more tunneled cuffed catheters were used in the LF group compared with the HF group; but arteriovenous fistula and uncuffed catheter use was significantly more prevalent in the HF group (at the point of enrolment). The LF group was characterized by lower HD blood flow rates and fewer weekly dialysis sessions.</p><p>In 50.8% (834 of 1,641) of cases, prevalent HD patients were dialyzed using LF dialysis membranes, with 49.2% (807 of 1,641) of patients dialyzed using HF dialysis membranes (at the point of enrolment). The baseline characteristics of prevalent HD patients are shown in <xref ref-type="table" rid="T2">Table 2</xref>. The HF group was characterized by a lower prevalence of diabetes mellitus and longer durations of dialysis therapy prior to enrolment compared with the LF group. The duration of dialysis therapy in the LF group ranged between 3 and 360 months (median, 28; interquartile range, 11 to 57), compared with a range of 3 and 328 months (median, 39; interquartile range, 16 to 78) in the HF group. Diabetes mellitus, as a primary cause of ESRD, was more prevalent in the LF group. Systolic BP was higher, and serum total cholesterol levels were lower, in the HF group. There were no significant group differences in age, gender, BMI, prevalence of cardiovascular diseases, diastolic BP, serum hemoglobin levels, serum albumin levels or spKt/V; or in the frequency of use of tunneled cuffed catheters and uncuffed catheters (for vascular access). The frequency with which an arteriovenous fistula was used for vascular access was higher in the HF group, but arteriovenous grafts were used more frequently in the LF group (at the point of enrolment). The LF group was characterized by lower HD blood flow rates and fewer dialysis sessions per week.</p></sec><sec><title>Effect of membrane flux on all-cause mortality</title><p>The median follow-up period was 24 months (interquartile range, 12 to 39). In total, 297 incident HD patients withdrew from the study for reasons other than death, comprising 43 patients who received kidney transplantation, 155 who were transferred to a nonparticipating hospital, 53 who withdrew their participation during treatment, and 46 who withdrew during the follow-up. Seventy-eight deaths occurred during the follow-up. The leading causes of death were cardiovascular events (30.6% of all deaths) and infectious diseases (26.4% of all deaths). The absolute mortality rate during the follow-up period was 4.4 deaths per 100 person-years. In univariate Cox regression analysis, HF dialysis was not associated with mortality (HR, 1.242; 95% CI, 0.761 to 2.029; <italic>p</italic> = 0.386). <xref ref-type="fig" rid="F1">Fig. 1</xref> depicts the Kaplan-Meier plot for all-cause mortality in incident HD patients according to HF and LF membrane use. There was no significant group difference (HF vs. LF) in survival rate (<italic>p</italic> = 0.384; log-rank test). Following adjustment for demographic variables, comorbidities, and laboratory data results, the adjusted HR for mortality in the HF group was 1.046 (95% CI, 0.592 to 1.847; <italic>p</italic> = 0.878), indicating that mortality rates for the incident HD patients did not differ significantly between the HF and LF groups (<xref ref-type="table" rid="T3">Table 3</xref>).</p><p>A total of 246 prevalent HD patients withdrew from the study for reasons other than death, comprising 71 patients who received kidney transplantations, 103 who transferred to a nonparticipating hospital, 38 who withdrew their participation during the treatment period, and 34 others who withdrew during the follow-up. One hundred and thirty-nine deaths occurred during the follow-up period. The leading causes of death were cardiovascular events (33.8% of all deaths) and infectious diseases (25.9% of all deaths). The absolute mortality rate during the follow-up period was 3.6 deaths per 100 person-years. In univariate Cox regression analysis, HF dialysis was associated with significantly reduced mortality rates (HR, 0.641; 95% CI, 0.454 to 0.906; <italic>p</italic> = 0.012). <xref ref-type="fig" rid="F2">Fig. 2</xref> depicts the Kaplan-Meier plot for all-cause mortality in prevalent HD patients according to HF or LF membrane use. Mortality was lower in the HF group compared with the LF group (<italic>p</italic> = 0.011; log-rank test). Even after adjusting for demographic variables, comorbid conditions and laboratory data, the adjusted HR for mortality was 0.606 (95% CI, 0.416 to 0.885; <italic>p</italic> = 0.009) in prevalent HD patients of the HF group: the HF group had a 39.4% lower risk of death compared with the LF group (<xref ref-type="table" rid="T4">Table 4</xref>).</p></sec><sec><title>Interaction between membrane flux and the duration of prior dialysis in prevalent HD patients</title><p>To determine the interaction between the effects of HF dialysis on mortality rate and the duration of dialysis therapy, in prevalent HD patients, we categorized the duration of prior dialysis therapy according to quartiles, as follows (m): quartile 1, &#x0003c; 13.5; quartile 2, 13.5 to 33.0; quartile 3, 33.1 to 68.5; and quartile 4, &#x0003e; 68.5. Multivariate Cox regression analysis indicated that HF dialysis was associated with a lower mortality rate in quartiles 1 (HR, 0.127; 95% CI, 0.032 to 0.512; <italic>p</italic> = 0.004) and 2 (HR, 0.355; 95% CI, 0.136 to 0.929; <italic>p</italic> = 0.035) compared with LF dialysis, with no significant group difference in mortality in quartiles 3 (HR, 0.853; 95% CI, 0.412 to 1.766; <italic>p</italic> = 0.668) or 4 (HR, 0.917; 95% CI, 0.442 to 1.901; <italic>p</italic> = 0.816) following adjustment for age, gender, use of an HF membrane, BMI, diabetes mellitus, cardiovascular disease, causes of ESRD, duration of dialysis therapy, systolic BP, diastolic BP, serum hemoglobin, albumin, or total cholesterol levels, total number of dialysis sessions per week, type of vascular access, and HD blood flow rate (<xref ref-type="table" rid="T5">Table 5</xref>).</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>In this prospective study, we demonstrated an association between HF dialysis and a decreased mortality rate in prevalent HD patients compared with LF dialysis. There was no significant difference between the HF and LF dialysis groups' mortality rates in incident HD patients. Our findings suggest that the impact of HF dialysis on mortality rates differs between incident and prevalent HD patients.</p><p>Our results accord with previous reports of a difference between incident and prevalent HD patients in the impact of HF dialysis on mortality rates. The survival benefit conferred by HF dialysis has also been described in several previous observational studies [<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>,<xref rid="B12" ref-type="bibr">12</xref>]. However, two large, randomized clinical trials, the MPO study (which included only incident HD patients) and the HEMO study (which included both incident and prevalent patients), revealed no significant difference in mortality rates between HF and LF dialysis groups [<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>]. The subgroup analysis of the HEMO study indicated that HF dialysis was associated with decreased all-cause mortality in patients receiving dialysis for &#x0003e; 3.7 years, prior to the commencement of the study, but not in the subgroup receiving dialysis for &#x02264; 3.7 years [<xref rid="B14" ref-type="bibr">14</xref>]. In our study, HF dialysis was associated with decreased all-cause mortality in a group of prevalent HD patients receiving dialysis for &#x02265; 3 months prior to study enrolment, but not in a group of incident HD patients.</p><p>The characteristics of the incident and prevalent patients in our study differed with respect to risk factors for mortality [<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>]. The mortality rate was highest in the first 3 months following the initiation of HD, and was also higher in incident compared with prevalent patients [<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>]. The most common risk factors for mortality in incident patients were the use of central venous catheters and inadequate predialysis nephrology care [<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B18" ref-type="bibr">18</xref>]. Because risk factors for mortality differ between incident and prevalent patients, this could confound any analysis of the effect of HF dialysis on the mortality rate in the whole cohort; therefore, we assessed the incident and prevalent patients separately.</p><p>Although there is no immediately obvious explanation for the differential impact of HF dialysis on mortality rates between incident and prevalent HD patients, several possible mechanisms can be proposed. First, central venous catheter access was employed more frequently in incident than in prevalent HD patients [<xref rid="B22" ref-type="bibr">22</xref>]. A central venous catheter was used in 71.5% of the incident HD patients (tunneled cuffed catheter, 55.7%; uncuffed catheter, 15.8%), compared with only 6.9% of the prevalent HD patients (tunneled cuffed catheter, 6.1%; uncuffed catheter, 0.8%). The low rate of venous catheter use in prevalent patients could have attenuated the survival benefit conferred by HF dialysis. Second, the mortality rate associated with withdrawal from dialysis was higher in the incident versus prevalent patients [<xref rid="B17" ref-type="bibr">17</xref>]. Bradbury et al. [<xref rid="B17" ref-type="bibr">17</xref>] reported that withdrawal accounted for 20% of deaths during the first 4 months following HD initiation, with 15% of deaths occurring in the subsequent 4 to 12 months. Higher death rates due to withdrawal in incident patients might overshadow the effects of HF dialysis on mortality. Third, previous exposure to LF or HF dialysis membranes could have affected the relationship between membrane flux type and mortality rate in the prevalent patients.</p><p>The interaction observed herein between the duration of prior dialysis, dialysis membrane flux type, and the mortality rate in prevalent HD patients is noteworthy. HF dialysis conferred a survival benefit in quartiles 1 and 2 of the duration of prior dialysis, but not quartiles 3 and 4. This suggests that, in prevalent HD patients, HF dialysis should be applied within 33 months following the initiation of dialysis, especially in prevalent patients who have received &#x0003c; 33 months of prior dialysis. The reason that HF dialysis conferred a survival benefit to prevalent patients who had received &#x0003c; 33 months of dialysis prior to entering the study is unclear. It is possible that longer durations of dialysis are associated with a greater accumulation of medium-molecular-weight toxins, such as &#x003b2;2-microglobulin [<xref rid="B23" ref-type="bibr">23</xref>]. The application of HF dialysis prior to the accumulation of significant amounts of such toxins could therefore be beneficial. However, our results are somewhat different to those of the HEMO study, in terms of the interaction between the duration of prior dialysis, the type of dialyzer membrane flux used and the mortality rate in prevalent HD patients. In the HEMO study, HF dialysis was associated with a lower risk of all-cause mortality, but only in quintiles in which patients had received &#x0003e; 6.09 years of prior dialysis [<xref rid="B14" ref-type="bibr">14</xref>]. Further studies are required to corroborate the relationships among HF dialysis, mortality rates and the duration of prior dialysis [<xref rid="B24" ref-type="bibr">24</xref>].</p><p>Our study had several limitations. First, we employed a prospective observational design. Accordingly, certain baseline characteristics differed between the HF and LF dialysis groups, especially in the context of the prevalent HD patients, thereby raising the possibility of selection bias. Randomized controlled trials comparing patients differing in terms of dialysis duration prior to enrolment are required to confirm the survival benefit conferred by HF dialysis according to the duration of prior dialysis. Second, the median follow-up period of 24 months was relatively short. Third, despite the multicenter nature of the study, the cohort consisted only of Korean patients. Thus, our results may not be generalizable to other, non-Asian ethnic groups receiving HD treatment.</p><p>In conclusion, HF dialysis was independently associated with decreased mortality compared with LF dialysis among prevalent HD patients, especially those who had received &#x0003c; 33 months of prior dialysis. However, HF dialysis conferred no significant survival benefit to incident HD patients. These findings suggest that HF dialysis differentially impacts upon mortality rates in incident versus prevalent HD patients, and further that the use of HF dialysis membranes should be recommended for prevalent patients within the 33 months following initiation of dialysis.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>High-flux (HF) dialysis was associated with a decreased mortality rate in prevalent hemodialysis (HD) patients, but not in incident HD patients.</p></list-item><list-item><p>The use of HF dialysis membranes should be recommended for prevalent patients.</p></list-item></list> </p></boxed-text></sec>
Clinical significance of nuclear factor &#x003ba;B and chemokine receptor CXCR4 expression in patients with diffuse large B-cell lymphoma who received rituximab-based therapy
<sec><title>Background/Aims</title><p>This study investigated the expression of nuclear factor &#x003ba;B (NF-&#x003ba;B) and the chemokine receptor (CXCR4) in patients with diffuse large B-cell lymphoma (DLBCL) who received rituximab-based therapy.</p></sec><sec><title>Methods</title><p>Seventy patients with DLBCL and treated with rituximab-CHOP (R-CHOP) were included, and immunohistochemistry was performed to determine the expression of NF-&#x003ba;B (I&#x003ba;B kinase &#x003b1;, p50, and p100/p52) and CXCR4. To classify DLBCL cases as germinal center B-cell-like (GCB) and non-GCB, additional immunohistochemical expression of CD10, bcl-6, or MUM1 was used in this study. The expression was divided into two groups according to the intensity score (negative, 0 or 1+; positive, 2+ or 3+).</p></sec><sec><title>Results</title><p>The median age of the patients was 66 years (range, 17 to 87), and 58.6% were male. Twenty-seven patients (38.6%) had stage III or IV disease at diagnosis. Twenty-three patients (32.9%) were categorized as high or high-intermediate risk according to their International Prognostic Indexs (IPIs). The overall incidence of bone marrow involvement was 5.7%. Rates of positive NF-&#x003ba;B and CXCR4 expression were 84.2% and 88.6%, respectively. High NF-&#x003ba;B expression was associated with CXCR4 expression (<italic>p</italic> = 0.002), and 56 patients (80.0%) showed coexpression. However, the expression of NF-&#x003ba;B or CXCR4 was not associated with overall survival and EFS. On multivariate analysis that included age, gender, performance status, stage, and the IPI, no significant association between the grade of NF-&#x003ba;B or CXCR4 expression and survival was observed.</p></sec><sec><title>Conclusions</title><p>The current study suggests that the tissue expression of NF-&#x003ba;B and CXCR4 may not be an independent prognostic marker in DLBCL patients treated with R-CHOP.</p></sec>
<contrib contrib-type="author"><name><surname>Shin</surname><given-names>Ho Cheol</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Seo</surname><given-names>Jongwon</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kang</surname><given-names>Byung Woog</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Moon</surname><given-names>Joon Ho</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chae</surname><given-names>Yee Soo</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Soo Jung</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Yoo Jin</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Han</surname><given-names>Seoae</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Seo</surname><given-names>Sang Kyung</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Jong Gwang</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sohn</surname><given-names>Sang Kyun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Tae-In</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Despite the benefits of adding rituximab (a chimeric immunoglobulin G1 monoclonal antibody targeting CD20) to cyclophosphamide, vincristine, adriamycin, and prednisolone (CHOP) therapy, the relapse rate is relatively high, and survival after relapse remains low, in patients with diffuse large B-cell lymphoma (DLBCL) [<xref rid="B1" ref-type="bibr">1</xref>]. The prognosis of patients with DLBCL has been shown to be influenced by several established surgical-pathological features. In particular, because DLBCL is considered a heterogeneous disease based on its development, growth, and progression depending on the deregulation of various cellular pathways, it can be subdivided into several subtypes according to its immunochemical features [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>]. Two representative examples are nuclear factor &#x003ba;B (NF-&#x003ba;B) and the chemokine receptor CXCR4.</p><p>The NF-&#x003ba;B family comprises five members that form homodimers or heterodimers, and can be activated through the classical or alternative pathway. Previous studies have demonstrated that abnormal activation of NF-&#x003ba;B contributes to lymphoma development and progression, as well as resistance of malignant cells to chemotherapy and radiation [<xref rid="B5" ref-type="bibr">5</xref>]. Therefore, NF-&#x003ba;B may be a predictor of prognosis in lymphoma patients or a beneficial target for lymphoma treatment, although the underlying mechanism in lymphoma remains unknown [<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>]. For example, Bavi et al. [<xref rid="B8" ref-type="bibr">8</xref>] reported a significant correlation between high expression of NF-&#x003ba;B and a poor prognosis in patients with DLBCL.</p><p>CXCR4 is an &#x003b1;-chemokine receptor specific for stromal derived factor-1 (SDF-1), which has potent chemotactic activity for lymphocytes. CXCR4 expression has been demonstrated in various tumors and was linked to metastasis to tissues containing a high concentration of SDF-1 [<xref rid="B9" ref-type="bibr">9</xref>]. In recent years, CXCR4 expression was also found in malignant lymphoma cell lines, and its inhibition by a monoclonal antibody enhanced apoptosis, decreased proliferation, and inhibited migration [<xref rid="B10" ref-type="bibr">10</xref>].</p><p>Interestingly, Okera et al. [<xref rid="B11" ref-type="bibr">11</xref>] reported that NF-&#x003ba;B expression was positively correlated with CXCR4 expression in prostate cancer. Furthermore, NF-&#x003ba;B has been suggested to regulate migration and metastasis by up-regulating the expression of CXCR4 in breast cancer and leukemia [<xref rid="B12" ref-type="bibr">12</xref>,<xref rid="B13" ref-type="bibr">13</xref>].</p><p>Accordingly, the present study analyzed the expression of these molecules and their impact on the survival of patients with DLBCL who received rituximab-based therapy.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Patients and treatment</title><p>We retrospectively reviewed the medical records of 70 patients with newly diagnosed DLBCL who received R-CHOP at Kyungpook National University Hospital (KNUH) between August 2003 and December 2009. All of the tissues investigated in the present study were obtained from excisional biopsy material. All patients were evaluated using standard laboratory tests, computed tomography (CT), and a unilateral bone marrow (BM) aspirate and biopsy at the time of diagnosis. Additional information was also recorded, including age, gender, performance status, the presence of B symptoms (fever, night sweats, weight loss), the presence of bulky disease (defined as tumor size &#x0003e; 10 cm), the presence of extranodal disease, the presence of BM involvement, the International Prognostic Index (IPI) [<xref rid="B14" ref-type="bibr">14</xref>], serum lactate dehydrogenase levels, hemoglobin levels, white blood cell counts, and platelet levels. All of the patients were staged according to the Ann Arbor Staging classification [<xref rid="B15" ref-type="bibr">15</xref>] using CT. All of the patients were treated with six cycles of rituximab plus CHOP (R-CHOP), while the patients with bulky disease received six cycles of R-CHOP with radiotherapy.</p></sec><sec><title>Immunohistochemistry and scoring of NF-&#x003ba;B and CXCR4 expression</title><p>Written informed consent for immunohistochemistry (IHC) was received from the patients, and the study was approved by the Institutional Review Board at KNUH. The tissue sections were deparaffinized in xylene and rehydrated using a graded series of ethanol solutions. A rabbit polyclonal anti-NF-&#x003ba;B p100/p52 antibody (dilution, 1:300; ab31409, Abcam, Cambridge, UK), NF-&#x003ba;B p50 antibody (1:150; sc-114, Santa Cruz Biotechnology, Santa Cruz, CA, USA), I&#x003ba;B kinase &#x003b1; (Ikk&#x003b1;) antibody (1:40; sc-7183, Santa Cruz Biotechnology), and rabbit anti-human CXCR4 polyclonal antibody (1:100; E18444, Spring Bioscience, Pleasanton, CA, USA) were used as primary antibodies. Immunohistochemical staining was carried out according to the manufacturer's protocol with the Ventana BenchMark XT autoimmunostainer (Ventana Medical Systems, Tucson, AZ, USA) using the ultraView kit. Primary antibodies were incubated at 37&#x02103; for 32 minutes, followed by standard Ventana signal amplification, counterstaining with hematoxylin for 4 minutes and staining with a bluing reagent for 4 minutes. Slides were then removed from the immunostainer, mounted and examined by light microscopy.</p></sec><sec><title>Interpretation of immunohistochemical analysis of NF-&#x003ba;B and CXCR4 expression</title><p>Based on prior studies, breast carcinoma tissue was used as the positive control for NF-&#x003ba;B expression. The tumors were classified into four grades: grade 0, no expression; grade 1, less than 10% expression; grade 2, between 10% and 50% expression; grade 3, more than 50% expression. For statistical reasons, the expression of each of NF-&#x003ba;B p100/p52, NF-&#x003ba;B p50, and Ikk&#x003b1; was analyzed as a dichotomous covariate-namely, NF-&#x003ba;B p100/p52-low (grade 0 and 1) versus NF-&#x003ba;B p100/52-high (grade 2 and 3), NF-&#x003ba;B p50-low (grade 0 and 1) versus NF-&#x003ba;B p50-high (grade 2 and 3), and NF-&#x003ba;B/Ikk&#x003b1;-low (grade 0 and 1) versus NF-&#x003ba;B/Ikk&#x003b1;-high (grade 2 and 3). We defined positive expression for NF-&#x003ba;B when the expression of p100/52, p50, and Ikk&#x003b1; was high. Placenta was used as the positive control for CXCR4. Staining was scored as follows: grade 0, no expression; grade 1, less than 10% expression; grade 2, between 10% and 50% expression; grade 3, more than 50% expression. CXCR4 overexpression (positive) was defined as grade 2 or 3. Two pathologists who were blinded to the patients' characteristics examined all the slides. The average percentage concordance for slides was 90.0%. In discordant cases, each pathologist retested and reviewed the slides again by capturing 10 pieces within a same sample.</p></sec><sec><title>Statistical analysis</title><p>Descriptive statistics are reported as proportions and medians. The baseline characteristics were compared using the chi-square and Fisher exact tests. A chi-square test was also used to assess the correlation between the levels of the two proteins. Overall survival (OS) was defined as the time from diagnosis to death from any cause. Event-free survival (EFS) was defined as the time from diagnosis to relapse or death from any cause [<xref rid="B16" ref-type="bibr">16</xref>]. The OS and EFS were calculated using the Kaplan-Meier test and compared using the log-rank test. Cox's proportional hazard regression model was used for the survival analyses. The analyses were adjusted for age, gender, performance status, stage, and IPI. The hazard ratio and 95% confidence interval were also estimated. A cutoff <italic>p</italic> value of 0.05 was adopted for all of the statistical analyses. All of the analyses were performed using SPSS version 14 (SPSS Inc., Chicago, IL, USA).</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Patients' characteristics</title><p>The patients' characteristics are summarized in <xref ref-type="table" rid="T1">Table 1</xref>. The total cohort included 70 patients. The median age was 60.7 years (range, 17 to 87), and 58.6% were male. Twenty-seven patients (38.6%) had stage III or IV disease at diagnosis. Twenty-three patients (32.9%) were categorized as high or high-intermediate risk according to their IPI. The overall incidence of BM involvement was 5.7%. Seven patients (10.0%) had bulky disease. The characteristics of the germinal center B-cell-like (GCB) and non-GCB groups were similar.</p></sec><sec><title>IHC analysis of NF-&#x003ba;B and CXCR4 expression</title><p>The NF-&#x003ba;B and CXCR4 expression results are shown in <xref ref-type="fig" rid="F1">Fig. 1</xref>. Positive NF-&#x003ba;B expression was found in 59 patients (84.3%) and CXCR4 in 62 patients (88.6%). Regarding the NF-&#x003ba;B and CXCR4 statuses, no significant difference was found between the GCB and non-GCB groups. High NF-&#x003ba;B expression was associated with CXCR4 expression (<italic>p</italic> = 0.002), and 56 patients (80.0%) showed coexpression (<xref ref-type="table" rid="T2">Table 2</xref>). With a median follow-up duration of 24.7 months (range, 0.3 to 66.4) among the patients alive at the last follow up, the 5-year OS and EFS rates were 80.0% and 73.7%, respectively. The expression of NF-&#x003ba;B or CXCR4 was not associated with OS or EFS (<xref ref-type="fig" rid="F2">Fig. 2</xref>). On multivariate analysis that included age, gender, performance status, stage, and IPI, no significant association was observed between the grade of NF-&#x003ba;B or CXCR4 expression and survival. The IPI and older age were independent prognostic factors of OS for patients with DLBCL (<xref ref-type="table" rid="T3">Table 3</xref>).</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>We investigated the relationship between the immunohistochemical expression of NF-&#x003ba;B and CXCR4 with the clinical outcome in patients with DLBCL. The results indicate that NF-&#x003ba;B and CXCR4 are abnormally expressed in DLBCL and coexpressed in 80% of these patients. Although the expression of NF-&#x003ba;B or CXCR4 was not associated with survival for DLBCL patients, it is important to understand the biological and molecular processes of DLBCL.</p><p>Because NF-&#x003ba;B is known to play major roles in the growth and metastasis of malignant tumors, many studies have focused on the clinical significance of its expression in tumors to predict the prognosis of solid tumors, including lymphoma [<xref rid="B17" ref-type="bibr">17</xref>]. The expression of NF-&#x003ba;B has been reported to be a significant marker of tumor recurrence or reduced survival, and this expression was independent of the conventional clinicopathological factors for DLBCL [<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B18" ref-type="bibr">18</xref>]. Furthermore, the activated B-cell subtype of DLBCL, characterized by its pattern of gene expression, shows constitutive activity of Ikk with accumulation of NF-&#x003ba;B proteins in the nucleus [<xref rid="B19" ref-type="bibr">19</xref>]. However, the relationship between NF-&#x003ba;B expression and prognosis remains unclear. For example, NF-&#x003ba;B activation in DLBCL is caused by genetic lesions affecting multiple other genes [<xref rid="B20" ref-type="bibr">20</xref>]. Additionally, treatment might affect the NF-&#x003ba;B signaling pathway in lymphoma, leading to altered survival in these patients [<xref rid="B6" ref-type="bibr">6</xref>]. Saito et al. [<xref rid="B21" ref-type="bibr">21</xref>] also reported that rituximab plus chemotherapy improved the survival of nongerminal-center-type untreated DLBCL, leading to increased sensitivity to chemotherapy by inhibiting the NF-&#x003ba;B signaling pathway. Thus, when taken together, the results remain inconsistent, although the findings of the present study differ in that all of the patients received rituximab-based chemotherapy.</p><p>The chemokine receptor CXCR4 is thought to be a metastatic indicator and poor prognostic factor in several hematologic and nonhematologic malignancies. Ahn et al. [<xref rid="B22" ref-type="bibr">22</xref>] reported that high CXCR4 expression is associated with a poor prognosis in patients with acute myeloid leukemia. In addition, increased CXCR4 levels have been found in several solid tumor types and may portend a worse prognosis [<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24</xref>,<xref rid="B25" ref-type="bibr">25</xref>]. In particular, CXCR4 has been implicated in regulating the metastasis of breast and prostate cancer and is under the control of NF-&#x003ba;B.</p><p>A recent <italic>in vitro</italic> study demonstrated that NF-&#x003ba;B regulates the motility of breast cancer cells by directly up-regulating the expression of CXCR4. Okera et al. [<xref rid="B11" ref-type="bibr">11</xref>] also suggested that CXCR4 expression is associated with the activities of interleukin 8, matrix metalloproteinase 9, and vascular endothelial growth factor, and these factors are often regulated by NF-&#x003ba;B. In the present study, DLBCL patients showed increased levels of NF-&#x003ba;B and CXCR4 coexpression, a finding that is consistent with a previous report. This finding also suggests that clinical studies of DLBCL with coexpression of these molecules should be considered separately from other lymphomas. In the present study, although positive expression of NF-&#x003ba;B seemed to be associated with slightly better survival than negative expression of NF-&#x003ba;B, the overall prognostic impact was not statistically significant. There are several possible explanations for this result. First, the sample size of 70 patients was too small to draw statistically significant conclusions. Second, although IHC is considered a good method for NF-&#x003ba;B testing, various ranges of positivity rates have been reported among studies. In fact, there is no consensus regarding the interpretation of NF-&#x003ba;B expression. Finally, the effect of chemotherapy may also have contributed to the survival outcomes, although no data regarding the relationship between NF-&#x003ba;B expression and the use of chemotherapy have been reported.</p><p>In conclusion, NF-&#x003ba;B and CXCR4 were abnormally expressed in DLBCL and were coexpressed in 80% of these patients. In the present study, both NF-&#x003ba;B and CXCR4 were found to be associated with the outcomes of Korean patients with DLBCL. However, further, larger-scale studies to clarify the role of NF-&#x003ba;B and CXCR4 as biomarkers for these patients are warranted.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Diffuse large B-cell lymphoma (DLBCL) can be subdivided into several subtypes according to the immunochemical features.</p></list-item><list-item><p>Nuclear factor &#x003ba;B (NF-&#x003ba;B) and CXCR4 were coexpressed in 80% of the patients.</p></list-item><list-item><p>The expression of NF-&#x003ba;B and CXCR4 may not be an independent prognostic marker for DLBCL patients treated with rituximab-CHOP.</p></list-item></list> </p></boxed-text></sec>
Kinetics of T-cell-based assays on cerebrospinal fluid and peripheral blood mononuclear cells in patients with tuberculous meningitis
<sec><title>Background/Aims</title><p>The goal of this study was to monitor tuberculosis (TB)-specific T-cell responses in cerebrospinal fluid-mononuclear cells (CSF-MCs) and peripheral blood mononuclear cells (PBMCs) in patients with tuberculous meningitis (TBM) over the course of anti-TB therapy.</p></sec><sec><title>Methods</title><p>Adult patients (&#x02265; 16 years) with TBM admitted to Asan Medical Center, Seoul, South Korea, were prospectively enrolled between April 2008 and April 2011. Serial blood or CSF samples were collected over the course of the anti-TB therapy, and analyzed using an enzyme-linked immunosorbent spot (ELISPOT) assay.</p></sec><sec><title>Results</title><p>Serial ELISPOT assays were performed on PBMCs from 17 patients (seven definite, four probable, and six possible TBM) and CSF-MC from nine patients (all definite TBM). The median number of interferon-gamma (IFN-&#x003b3;)-producing T-cells steadily increased during the first 6 months after commencement of anti-TB therapy in PBMCs. Serial CSF-MC ELISPOT assays revealed significant variability in immune responses during the first 6 weeks of anti-TB therapy, though early increases in CSF-MC ELISPOT results were associated with treatment failure or paradoxical response.</p></sec><sec><title>Conclusions</title><p>Serial analysis of PBMCs by ELISPOT during the course of treatment was ineffective for predicting clinical response. However, increases in TB-specific IFN-&#x003b3;-producing T-cells in CSF-MC during the early phase of anti-TB therapy may be predictive of clinical failure.</p></sec>
<contrib contrib-type="author"><name><surname>Park</surname><given-names>Ki-Ho</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Mi Suk</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Sang-Oh</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Choi</surname><given-names>Sang-Ho</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Yang Soo</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Woo</surname><given-names>Jun Hee</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kang</surname><given-names>Joong Koo</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Sang-Ahm</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kim</surname><given-names>Sung-Han</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p><italic>Mycobacterium tuberculosis</italic>-specific interferon-&#x003b3; (IFN-&#x003b3;) releasing assays (IGRAs) are an effective method for diagnosing tuberculous meningitis (TBM) in both peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid mononuclear cells (CSF-MC) [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>]. Serial analysis of tuberculosis (TB)-specific IFN-&#x003b3;-producing T-cell responses by IGRA over the course of anti-TB therapy may be useful in predicting clinical response [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>]; however, it is uncertain whether such an analysis would be beneficial in patients with TBM. Here, we evaluated TB-specific T-cell responses in CSF-MC and PBMCs using an enzyme-linked immunosorbent spot (ELISPOT) assay in patients with TBM during the course of anti-TB therapy.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Study design and patients</title><p>Adult patients (&#x02265; 16 years) with suspected TBM admitted to Asan Medical Center, a 2,700-bed tertiary hospital in Seoul, South Korea, were prospectively enrolled between April 2008 and April 2011. Patients with suspected TBM were categorized as definite, probable, or possible TBM, according to a recently proposed uniform case definition, with some modifications [<xref rid="B10" ref-type="bibr">10</xref>]. In brief, patients were classified as having "definite TBM" if clinical specimens were found to be positive for <italic>M. tuberculosis</italic> either by culture or through the use of an <italic>M. tuberculosis</italic> polymerase chain reaction assay. Patients were classified as having "probable TBM" or "possible TBM" according to the diagnostic scoring system [<xref rid="B10" ref-type="bibr">10</xref>]. All patients received standard TB therapy in accordance with the recommendations of the American Thoracic Society [<xref rid="B11" ref-type="bibr">11</xref>]. Only patients willing to provide serial blood or CSF samples over the course of anti-TB therapy were included in the final analysis. The study protocol was approved by the Institutional Review Board of Asan Medical Center.</p></sec><sec><title>ELISPOT assay</title><p>PBMCs and CSF-MCs were isolated from peripheral venous blood (~8 mL) and CSF (~4 mL), respectively, within 30 minutes of collection. Cells were then resuspended in appropriate medium, and analyzed using an ELISPOT assay (T-SPOT.TB, Oxford Immunotec, Oxford, UK) as described previously [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B12" ref-type="bibr">12</xref>]. The minimum threshold for a positive response was defined as &#x02265; 6 spot-forming cells per well (SFCs) after subtraction of the negative control. Indeterminate responses were defined as &#x0003c; 20 spots in the positive control well, or &#x0003e; 10 spots in the negative control well [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B12" ref-type="bibr">12</xref>].</p></sec></sec><sec sec-type="results"><title>RESULTS</title><p>Forty-six patients with TBM were prospectively enrolled during the study period. Of these 46 patients, 17 (37%), 8 (17%), and 21 (46%) were classified as having definite, probable, and possible TBM, respectively. Baseline ELISPOT assays were performed on PBMCs and CSF-MCs from all 46 patients; however, only 22 of these patients were willing to provide serial samples over the course of their treatment. Baseline clinical characteristics were similar between patients providing serial samples and those providing only pretreatment samples (<xref ref-type="table" rid="T1">Table 1</xref>).</p><p>Serial PBMC ELISPOT assays were performed on 17 patients (seven definite, four probable, and six possible TBM). Among the 15 patients exhibiting positive ELISPOT results at baseline, no negative reversions (&#x0003c; 6 spots) were observed during the course of treatment; positive conversion (&#x02265; 6 spots) occurred in one of two patients initially diagnosed as negative by ELISPOT. Median IFN-&#x003b3;-producing T-cell numbers steadily increased during the first 6 months after commencement of anti-TB therapy in these patients (<xref ref-type="fig" rid="F1">Fig. 1</xref>).</p><p>Serial CSF-MC ELISPOT assays were performed on nine patients with TBM (all definite TBM). Serial changes in the number of IFN-&#x003b3;-producing T-cells were associated with both white blood cell (WBC) counts and clinical responses (<xref ref-type="fig" rid="F2">Fig. 2</xref>). IFN-&#x003b3;-producing T-cell counts decreased in four patients with decreasing CSF WBC counts (patients 1 to 4), but increased in four others exhibiting increased CSF WBC counts (patients 6 to 9); one patient (patient 5) exhibited no change in IFN-&#x003b3;-producing T-cell numbers despite a decrease in CSF WBC counts. None of the five patients, in whom IFN-&#x003b3;-producing T-cells decreased or remained constant during the early phase of anti-TB therapy, experienced a worsening of clinical symptoms; however, all four patients with definite TBM, in whom IFN-&#x003b3;-producing T-cells increased, exhibited clear signs of clinical deterioration.</p></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>Analyses of serial TB-specific IFN-&#x003b3;-producing T-cell responses in CSF-MCs and PBMCs of patients with TBM remain limited. Here, we showed that TB-specific IFN-&#x003b3;-producing T-cell responses in PBMC increased steadily over the course of 6 months after initiation of anti-TB therapy. This finding is consistent with a previous ELISPOT analysis performed by our laboratory in which median TB-specific T-cell responses were shown to increase steadily for up to 6 months in 52 patients with extrapulmonary TB, followed by a decrease thereafter [<xref rid="B13" ref-type="bibr">13</xref>]. A more complete comparison was not possible however, as long-term follow-up (&#x0003e; 6 months) in patients with TBM was not performed.</p><p>While some studies have reported increased T-cell responses during or after anti-TB therapy [<xref rid="B13" ref-type="bibr">13</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>], consistent associations between anti-TB therapy and an attenuated T-cell response in PBMCs have also been observed [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B18" ref-type="bibr">18</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>]. The reasons underlying this discrepancy, along with the small number of negative reversions with increased T-cell responses described in this study are not clear. One possible explanation may be that extrapulmonary TB is more likely to exhibit a paradoxical response to anti-TB therapy [<xref rid="B21" ref-type="bibr">21</xref>]. Most studies examining IGRA kinetics during anti-TB therapy have focused exclusively on pulmonary TB [<xref rid="B5" ref-type="bibr">5</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B8" ref-type="bibr">8</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>,<xref rid="B17" ref-type="bibr">17</xref>,<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>], which may explain some of the different response kinetics observed in our studies. Further studies will be necessary to evaluate differences in immune response kinetics between pulmonary and extrapulmonary TB.</p><p>In addition to PBMC-related effects, we also observed increased TB-specific T-cell responses in CSF-MC during the early phase of anti-TB treatment, which were associated with clinical deterioration in patients with definite TBM. Previously, we had reported a case of TBM in which a therapy-induced increase in TB-specific T-cell responses in CSF-MC and PBMCs occurred [<xref rid="B22" ref-type="bibr">22</xref>]. Taken together, these studies suggest that the clinical deterioration seen during the early phase of TB therapy may be the result of TB-specific T-cell responses. Interestingly, these heightened T-cell responses occurred not only in patients who died as a result of TBM but also in those who experienced clinical deterioration despite CSF sterilization, but ultimately improved without modification to their anti-TB regimen (i.e., paradoxical response). These observations are consistent with our earlier work showing that an "immunologic paradox" often preceded a "therapeutic paradox," which is strongly predictive of poor outcomes [<xref rid="B22" ref-type="bibr">22</xref>]. Further studies will be needed to evaluate the prognostic value of increased TB-specific T-cell responses during anti-TB therapy, and the clinical utility of this phenomenon in patients with TBM.</p><p>Like all studies, this work is not without limitations. Ten of the seventeen patients (59%) in whom serial PBMC ELISPOT assays were performed were classified as having probable or possible TBM on the basis of clinical features and image findings alone, in the absence of microbiological confirmation. However, as existing bacteriological methods are not sufficiently sensitive to be used for diagnosing TBM alone, the majority of studies include patients based upon a combination of microbiological, clinical, and radiological criteria [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B23" ref-type="bibr">23</xref>,<xref rid="B24" ref-type="bibr">24</xref>]. These patients were subsequently classified using a recently proposed uniform case definition as a way to minimize selection bias [<xref rid="B10" ref-type="bibr">10</xref>]. Second, serial ELISPOT assays were performed in only a small number of patients with TBM. Although a clear trend towards increased T-cell responses in PBMCs was observed during the anti-TB therapy, the small sample size was insufficient for most statistical analyses. Finally, CSF-MC ELISPOT assays were not performed at regular intervals. Further studies should include a larger number of patients with serial measurement performed at regular intervals.</p><p>Despite these limitations, this study may be useful for understanding the immunologic response to anti-TB therapy in patients with TBM. Serial analysis of PBMCs by ELISPOT during the course of treatment was found to be ineffective for predicting clinical response; however, increases in TB-specific IFN-&#x003b3;-producing T-cells in CSF-MC during the early phase of anti-TB therapy may be predictive of clinical failure.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Serial peripheral blood mononuclear cell enzyme-linked immunosorbent spot assays during the course of treatment were found to be ineffective for predicting clinical response in patients with tuberculous meningitis (TBM).</p></list-item><list-item><p>Increases in tuberculosis (TB)-specific interferon-gamma producing T-cell response on cerebrospinal fluid-mononuclear cells during the early phase of anti-TB therapy may be predictive of clinical failure in patients with TBM.</p></list-item></list> </p></boxed-text></sec>
Clinical features of elderly chronic urticaria
<sec><title>Background/Aims</title><p>Chronic urticaria (CU) is defined as itchy wheals lasting 6 weeks or more. As the aged population increases worldwide, it is essential to identify the specific features of this disease in the elderly population.</p></sec><sec><title>Methods</title><p>We investigated the prevalence and clinical features of CU in elderly patients. Medical records of 837 CU patients from the outpatient Allergy Clinic of Ajou University Hospital, Korea were analyzed retrospectively. Patients with chronic spontaneous urticaria according to the EAACI/GA2LEN/EDF/WAO guidelines were included. Patients older than 60 years were defined as elderly.</p></sec><sec><title>Results</title><p>Of the 837 patients, 37 (4.5%) were elderly. In elderly versus nonelderly CU patients, the prevalence of atopic dermatitis (AD) was significantly higher (37.8% vs. 21.7%, respectively; <italic>p</italic> = 0.022), while that of aspirin intolerance was lower (18.9% vs. 43.6%, respectively; <italic>p</italic> = 0.003) in terms of comorbid conditions. The prevalences of serum specific immunoglobulin E antibodies to staphylococcal enterotoxin A and staphylococcal enterotoxin B were considerably higher in elderly CU patients with AD than in those without AD (37.5% vs. 0%, respectively).</p></sec><sec><title>Conclusions</title><p>Elderly patients with CU had a higher prevalence of AD. Therefore, there is a need to recognize the existence of AD in elderly CU patients.</p></sec>
<contrib contrib-type="author"><name><surname>Ban</surname><given-names>Ga-Young</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Mi-Yea</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Yoo</surname><given-names>Hye-Soo</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Nahm</surname><given-names>Dong-Ho</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Ye</surname><given-names>Young-Min</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Shin</surname><given-names>Yoo-Seob</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0003-2614-0303</contrib-id><name><surname>Park</surname><given-names>Hae-Sim</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Chronic urticaria (CU) is a common allergic skin disease. It is defined as itchy wheals with or without angioedema that usually persists for less more than 24 hours. According to current EAACI/GA<sup>2</sup>LEN/EDF/WAO guidelines [<xref rid="B1" ref-type="bibr">1</xref>], CU can be classified as spontaneous, physical, or other. Chronic spontaneous urticaria (CSU), usually called CU, is the most common subtype of all forms of nonacute urticaria, and is characterized by wheals that develop independently of external stimuli and last for a minimum of 6 weeks. The underlying causes of CU are difficult to identify in most patients [<xref rid="B2" ref-type="bibr">2</xref>].</p><p>The pathogenic mechanisms of CU are unclear. Evidence of an autoimmune etiology is reported in about 45% of CU patients, while in others the underlying cause remains unknown. Circulating autoantibodies specific to high-affinity immunoglobulin E (IgE) receptors or dermal mast cell bound IgE activate mast cells and induce degranulation with cytokine release [<xref rid="B3" ref-type="bibr">3</xref>]. Atopy is proposed to play a role in the pathogenesis of CU, especially in the aspirin-intolerant CU phenotype [<xref rid="B4" ref-type="bibr">4</xref>]. Several hypotheses exist regarding the relationship of atopy and aspirin intolerance; however, the exact association remains unclear [<xref rid="B5" ref-type="bibr">5</xref>]. One report has suggested that a personal or familial history of allergic disease is predictive of a response to anti-IgE monoclonal antibody (Omalizumab) therapy. This report also indicated that a personal or familial history of allergic disease might contribute to the development of CU [<xref rid="B6" ref-type="bibr">6</xref>].</p><p>Superantigens are virulent polypeptides produced by a variety of infectious organisms; those produced by Staphylococcus aureus are particularly important in allergic diseases such as asthma [<xref rid="B7" ref-type="bibr">7</xref>], aspirin exacerbated respiratory disease [<xref rid="B8" ref-type="bibr">8</xref>], chronic rhinosinusitis [<xref rid="B9" ref-type="bibr">9</xref>], atopic dermatitis (AD) [<xref rid="B10" ref-type="bibr">10</xref>], and CU [<xref rid="B11" ref-type="bibr">11</xref>]. The levels of specific IgE antibodies to superantigens correlate to disease severity in upper and lower airway disease [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B10" ref-type="bibr">10</xref>]. In addition, specific IgE antibodies to staphylococcal superantigens are thought to stimulate mast cells, basophils, and eosinophils, causing release of various mediators [<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B12" ref-type="bibr">12</xref>].</p><p>The aged population is increasing worldwide. Forty years ago, life expectancy in Korea was 60 years, with only 3% of the Korean population reaching age 65 years or more [<xref rid="B13" ref-type="bibr">13</xref><xref rid="B14" ref-type="bibr">,14</xref>]. In 2012, by contrast, those over the age of 65 years represented 12% of the Korean population. It is predicted that the population over age 65 years in Korea will increase to ~40% by 2060 [<xref rid="B14" ref-type="bibr">14</xref>]. All organ systems are affected by aging, and geriatric patients respond differently to environmental stimuli. Therefore, it is essential to identify specific features of chronic diseases in aged populations. Several studies have investigated the clinical presentation and etiopathogenesis of urticaria in the elderly population [<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>]; however, few have compared the clinical features of CU in the elderly to those of the nonelderly [<xref rid="B17" ref-type="bibr">17</xref>]. We investigated the prevalence and clinical features of CU in elderly versus nonelderly patients in Korea. The findings of this study reveal factors that play a significant role in elderly CU.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Subjects</title><p>We retrospectively analyzed the medical records of 837 CU patients (age range, 9 to 84 years) who were followed at the outpatient Allergy Clinic of Ajou University Hospital in South Korea. According to EAACI/GA2LEN/EDF/WAO guidelines [<xref rid="B1" ref-type="bibr">1</xref>], patients with physical or other secondary types of urticaria were excluded, while those with CSU were included. AD was diagnosed by allergy specialists when patients demonstrated skin itching, dryness, and recurrent typical eczema with a high eosinophil cataionic protein level, along with concomitant allergic diseases or atopy (as defined by a positive skin prick test or high serum-specific IgE antibodies to common inhalant allergens). Subjects were divided into two groups according to the their age at presentation: those younger than 60 years were classified as non-elderly, while those older than 60 years were classified as elderly [<xref rid="B18" ref-type="bibr">18</xref>]. This study was approved by the Institutional Review Board of Ajou University Hospital.</p></sec><sec><title>Urticaria activity scores and urticaria severity</title><p>The urticaria activity score (UAS) was used to assess disease severity. UAS was calculated using the quantity (0, no wheals; 1, &#x0003c; 10 wheals; 2, 10 to 50 wheals; 3, &#x0003e; 50 wheals), distribution range (0, none; 1, &#x0003c; 25% of the body surface area [BSA]; 2, 25% to 50% of BSA; 3, &#x0003e; 50% of BSA), mean diameter (0, no wheals; 1, &#x0003c; 1 cm; 2, 1 to 3 cm; 3, &#x0003e; 3 cm), and duration (0, no wheals; 1, &#x0003c; 4 hours; 2, 4 to 12 hours; 3, &#x0003e; 12 hours) of wheals, along with pruritus according to intensity (0, no pruritus; 1, mild; 2, moderate; 3, severe) within the last week, yielding a total score of 0 to 15 [<xref rid="B19" ref-type="bibr">19</xref>]. Severe CU was defined as a UAS &#x02265;13 at the initial visit [<xref rid="B20" ref-type="bibr">20</xref>].</p></sec><sec><title>Atopic status</title><p>Atopy was defined by a positive response to at least one common inhaled allergen, detected by either a skin prick test or serum-specific IgE test. Skin prick tests were performed with 55 common aeroallergens (Bencard, Brentford, UK) and the responses were considered positive if they produced a wheal of more than 3 mm in diameter, or if the ratio of the mean wheal diameter to that of the histamine control was &#x0003e; 1. Specific IgE antibodies were measured using the ImmunoCAP system (Thermo Fisher Scientific, Uppsala, Sweden); a level greater than or equal to 0.35 kU/L was considered a positive response [<xref rid="B18" ref-type="bibr">18</xref>].</p></sec><sec><title>Immune-related laboratory findings</title><p>Total IgE levels and specific IgE antibody levels for staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), and toxic shock syndrome toxin (TSST)-1 were obtained using the ImmunoCAP system (Thermo Fisher Scientific). Anti-thyroglobulin (TG) and anti-microsomal (MC) antibodies were detected by radioimmunoassay (BRAHMS Aktiengesellschaft, Hennigsdorf, Germany).</p></sec><sec><title>Oral provocation test with aspirin</title><p>An oral provocation test with aspirin was conducted on all study patients [<xref rid="B4" ref-type="bibr">4</xref>]. All medications, including anti-histamines, steroids, and leukotriene receptor antagonists, were discontinued 72 hours prior to the procedure. After a placebo challenge, 500 mg of aspirin (Rhonal, KunWha Pharmaceutical Co., Seoul, Korea) in tablet form were administered orally. Patients were observed every 30 minutes for 4 hours following aspirin administration for urticaria, angioedema, or changes in lung function. The appearance of urticaria within 4 hours without any change in forced expiratory volume in 1 second was considered a positive result.</p></sec><sec><title>Statistical analysis</title><p>Data for continuous variables were compared via Student <italic>t</italic> tests; Pearson chi-square or Fisher exact tests were used for categorical variables. All computations were performed using the SPSS version 16.0 (SPSS Inc., Chicago, IL, USA).</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Clinical characteristics and comorbidities</title><p><xref ref-type="table" rid="T1">Table 1</xref> summarizes the clinical characteristics of the subjects. Of 837 patients with CU, 37 (4.4%) were elderly. Both the nonelderly and elderly CU groups showed a female predominance (58.2% vs. 51.4%, respectively; <italic>p</italic> = 0.406). There were no significant differences between the two groups in mean UAS (<italic>p</italic> = 0.666) or the prevalence of severe CU (32.5% in nonelderly vs. 40% in elderly, <italic>p</italic> = 0.663). The atopy rate was significantly lower in the elderly CU group than in the non-elderly CU group (30.3% vs. 51.2%, respectively; <italic>p</italic> = 0.019). The prevalence of AD was considerably higher in the elderly CU group than the non-elderly CU group (37.8% vs. 21.7%, respectively; <italic>p</italic> = 0.022); however, there were no significant differences in the prevalence of other allergic diseases such as asthma or rhinitis. The prevalence of aspirin intolerance was significantly lower in the elderly CU group than the non-elderly CU group (18.9% vs. 43.6%, respectively; <italic>p</italic> = 0.003).</p></sec><sec><title>Laboratory findings in elderly and nonelderly CU patients</title><p>Levels of total IgE and the prevalence of serum-specific IgE antibodies to SEA, SEB, and TSST-1 exhibited no significant differences between the two age groups. No significant differences were noted in the prevalence of serum anti-TG and anti-MC antibodies between the two groups (<xref ref-type="table" rid="T2">Table 2</xref>).</p><p><xref ref-type="table" rid="T3">Table 3</xref> shows laboratory findings according to the coexistence of AD in elderly patients with CU. Elderly CU patients with AD showed no differences in total IgE levels or the prevalence of serum anti-TG or anti-MC antibodies compared to those without AD. The prevalence of serum-specific IgE to SEA/SEB was significantly higher in elderly CU patients with AD compared to those without AD (37.5% vs. 0%, respectively, <italic>p</italic> = 0.042); however, no significant difference was noted in the prevalence of serum-specific IgE to TSST-1 between the two groups.</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>It is estimated that about 0.5% to 1% of the population suffers from CU at any given time, and that about one quarter of the total population has experienced urticaria at some point during their lives. Both sexes can be affected, but in general, females suffer from urticaria nearly twice as frequently as males [<xref rid="B21" ref-type="bibr">21</xref>]. In our study, we found a lack of female predominance in the elderly CU group, despite a 57.9% proportion of females in the total study population. This finding is in concordance with a previous report of an almost equal sex distribution in an elderly CU group [<xref rid="B17" ref-type="bibr">17</xref>]. All age groups can be affected by CU, but the peak age is between 20 and 40 years [<xref rid="B21" ref-type="bibr">21</xref>]; therefore, few studies on CU in the aged population have been performed. In this study, despite a less stringent definition of elderly as those over 60 years (versus over 65 years used in previous studies), only 4.4% of the subjects were elderly, versus 9.4% in a previous study [<xref rid="B17" ref-type="bibr">17</xref>]. Despite the small percentage of CU patients defined as elderly, it is important to identify the specific clinical features of this group due to the aging of the population.</p><p>Structural and physiological changes occur in the skin with age. A reduction in dermal thickness and decrease in cutaneous vascularity and cellularity are associated with aging. The number of cutaneous mast cells also declines [<xref rid="B22" ref-type="bibr">22</xref>]. The major characteristic of CU pathogenesis is mast cell degranulation, which results in release of histamine and other chemical mediators. Cutaneous mast cell numbers are not increased in CU; however, an increased releasability of histamine (related to the histamine content or activation state of mast cells) may play a role in CU pathogenesis [<xref rid="B23" ref-type="bibr">23</xref>]. The features of elderly CU are not due to structural and physiological changes in aging skin; therefore, our results showing no significant differences between the two groups in UAS or in the prevalence of severe CU exist despite changes in dermal thickness and the number of mast cells. This similarity in UAS for both groups is in agreement with a previous report [<xref rid="B17" ref-type="bibr">17</xref>]. To estimate urticarial activity accurately, recent trials have used UAS 7, which averages the daily UAS over 1 week [<xref rid="B24" ref-type="bibr">24</xref>]. Because of the limitations of the UAS 7 in real practice, we applied a UAS including five components, as described previously [<xref rid="B19" ref-type="bibr">19</xref>,<xref rid="B20" ref-type="bibr">20</xref>]. Therefore, we speculate that CU disease activity and severity are not significantly different between the elderly and nonelderly.</p><p>Superantigens (derived mainly from <italic>S. aureus</italic>) can activate T cells, induce IgE synthesis by B cells, and stimulate inflammatory leukocytes (such as mast cells and eosinophils). CU patients were reported to have high levels of serum-specific IgE against SEA, SEB, and TSST-1 [<xref rid="B25" ref-type="bibr">25</xref>]. The prevalence of specific IgE antibodies against SEA, SEB, and TSST-1 were noted to be significantly higher in aspirin intolerant chronic urticaria (AICU) patients than in normal controls. Compared to aspirin tolerant chronic urticaria patients, levels of serum specific IgE to TSST-1 were significantly higher in AICU patients [<xref rid="B12" ref-type="bibr">12</xref>]. With regard to AD, one report described higher levels of specific IgE antibodies to staphylococcal superantigens in younger subjects [<xref rid="B26" ref-type="bibr">26</xref>]. However, another study found that 57% of adult AD patients had high levels of serum-specific IgE to staphylococcal superantigens, compared to only 34% of pediatric AD patients [<xref rid="B27" ref-type="bibr">27</xref>]. To our knowledge, no study has investigated differences in the prevalence of specific IgE antibodies to staphylococcal superantigens according to age within the adult population, especially in the case of CU. We identified no difference in the prevalence of specific IgE to staphylococcal superantigens between the elderly and nonelderly CU groups; however, the prevalences of specific IgE antibodies to SEA and SEB were significantly higher in elderly CU patients with AD than in those without. The rate of AD in the general population is 2% to 5% (~15% in children and young adults), and is believed to be lower in the elderly than young adults (~5.6%) [<xref rid="B27" ref-type="bibr">27</xref>,<xref rid="B28" ref-type="bibr">28</xref>]. Studies of the prevalence of AD accompanied by CU in the elderly are limited; however, one population-based cross-sectional survey reported higher rates of eczema and urticaria in adults [<xref rid="B28" ref-type="bibr">28</xref>]. The higher prevalence of AD in elderly CU patients in our study may be due to presumptive diagnosis of AD based on clinical signs [<xref rid="B17" ref-type="bibr">17</xref>]. In patients with AD, impaired skin barrier function facilitates the colonization of <italic>S. aureus</italic>; ceramidases from this organism degrade ceramide (a component in the lipid matrix of the skin barrier). By this mechanism, <italic>S. aureus</italic> enables continuous penetration of the skin barrier, leading to IgE sensitization [<xref rid="B29" ref-type="bibr">29</xref>]. Even normal-appearing aged skin has increased ceramidase activity, resulting in degradation of the lamellar structure of stratum corneum lipids [<xref rid="B30" ref-type="bibr">30</xref>]. Therefore, production of specific IgE antibodies to staphylococcal superantigens may play a synergistic role in the pathogenesis of elderly AD.</p><p>Aspirin intolerance and atopy were found more frequently in the nonelderly than the elderly CU group; however, it has already been noted that a personal or family history of atopy is a risk factor for aspirin intolerance in CU patients [<xref rid="B5" ref-type="bibr">5</xref>]. Most studies indicate a lower prevalence of atopy in the elderly population [<xref rid="B31" ref-type="bibr">31</xref>]; therefore, we tested a bivariate predictive model of nonelderly CU. The multiple logistic regression analysis included aspirin intolerance and atopy variables. However, aspirin intolerance was significantly related to age (<italic>p</italic> = 0.031), even after adjustment for the confounding effect of atopic status. It is widely known that aspirin aggravates CU in 20% to 30% of patients [<xref rid="B32" ref-type="bibr">32</xref>]. However, 42.5% of CU patients in our study were found to have aspirin intolerance, a considerably larger proportion than reported previously [<xref rid="B33" ref-type="bibr">33</xref>,<xref rid="B34" ref-type="bibr">34</xref>]. This is most likely due to recruitment of study subjects from a tertiary care university hospital. Nevertheless, the high prevalence of aspirin intolerance in nonelderly CU patients indicates a need to carefully question and educate CU patients about the possibility of aspirin allergy.</p><p>In conclusion, this study indicates that 4.5% of CU patients are elderly. There is a need to further investigate the relationship of AD and CU in elderly patients, due to the higher prevalence of AD in elderly versus nonelderly CU patients.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Clinical features of elderly chronic urticaria (CU) patients were different from those in younger population.</p></list-item><list-item><p>Atopic dermatitis is a common comorbid condition of elderly CU.</p></list-item><list-item><p>Evaluation of comorbid condition is needed for the management of elderly CU.</p></list-item></list> </p></boxed-text></sec>
Early effects of tumor necrosis factor inhibition on bone homeostasis after soluble tumor necrosis factor receptor use
<sec><title>Background/Aims</title><p>Our aim was to assess whether short-term treatment with soluble tumor necrosis factor (TNF) receptor affects circulating markers of bone metabolism in rheumatoid arthritis (RA) patients.</p></sec><sec><title>Methods</title><p>Thirty-three active RA patients, treated with oral disease-modifying antirheumatic drugs (DMARDs) and glucocorticoids for &#x0003e; 6 months, were administered etanercept for 12 weeks. Serum levels of bone metabolism markers were compared among patients treated with DMARDs at baseline and after etanercept treatment, normal controls and naive RA patients not previously treated with DMARDs (both age- and gender-matched).</p></sec><sec><title>Results</title><p>Bone-specific alkaline phosphatase (BSALP) and serum c-telopeptide (CTX)-1 levels were lower in RA patients treated with DMARDs than in DMARD-naive RA patients. After 12 weeks of etanercept treatment, serum CTX-1 and sclerostin levels increased. In patients whose DAS28 improved, the sclerostin level increased from 1.67 &#x000b1; 2.12 pg/mL at baseline to 2.51 &#x000b1; 3.03 pg/mL, which was statistically significant (<italic>p</italic> = 0.021). Increases in sclerostin levels after etanercept treatment were positively correlated with those of serum CTX-1 (<italic>r</italic> = 0.775), as were those of BSALP (<italic>r</italic> = 0.755).</p></sec><sec><title>Conclusions</title><p>RA patients treated with DMARDs showed depressed bone metabolism compared to naive RA patients. Increases in serum CTX-1 and sclerostin levels after short-term etanercept treatment suggest reconstitution of bone metabolism homeostasis.</p></sec>
<contrib contrib-type="author"><name><surname>Lim</surname><given-names>Mie Jin</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kwon</surname><given-names>Seong Ryul</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Joo</surname><given-names>Kowoon</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Son</surname><given-names>Min Jung</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Shin-Goo</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Park</surname><given-names>Won</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Rheumatoid arthritis (RA) is a chronic disease characterized by joint inflammation and local bone erosion. Progressive bone erosion and joint destruction results in progressive joint deformity, which is the hallmark of RA [<xref rid="B1" ref-type="bibr">1</xref>]. Tumor necrosis factor (TNF)-&#x003b1; is a key player in the pathogenesis of RA, and is associated with both inflammation and bone metabolism. TNF-&#x003b1; induces the secretion of multiple proinflammatory cytokines (e.g., interleukin [IL]-1, IL-6, IL-8, and granulocyte macrophage colony-stimulating factor), as well as dickkopf (DKK)-1, a regulator in the Wnt pathway, which is associated with bone formation. Blockade of TNF-&#x003b1; is known to arrest bone loss [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>], partly by stimulating bone formation [<xref rid="B4" ref-type="bibr">4</xref>]. Diarra et al. [<xref rid="B4" ref-type="bibr">4</xref>] showed that treatment with an anti-TNF blocker decreases circulating DKK-1 levels in RA patients, and a previous study suggested DKK-1 levels are associated with radiographic progression in RA patients [<xref rid="B5" ref-type="bibr">5</xref>]. Therefore, the role of the Wnt pathway, especially regulatory molecules such as DKK-1 and sclerostin, is a recent focus of RA research. However, data on the effects of <italic>in vivo</italic> TNF-&#x003b1; inhibition on general bone loss in RA patients are limited [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>]. Furthermore, the effects of etanercept, a soluble TNF receptor, on Wnt pathway antagonists have not been determined. Therefore, our aim was to assess whether short-term treatment with a soluble TNF receptor in RA patients affects circulating markers of bone metabolism, including sclerostin.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Patients</title><p>We enrolled 33 RA patients who had been treated with disease-modifying antirheumatic drugs (DMARDs) and glucocorticoids for &#x0003e; 6 months. Twelve patients were postmenopausal females, 15 were premenopausal females, and six were males. All patients fulfilled at least four of the 1987 revised American College of Rheumatology criteria for RA [<xref rid="B8" ref-type="bibr">8</xref>]. All patients had active RA with a 28 joint count disease activity score (DAS28) &#x0003e; 3.2 at inclusion [<xref rid="B9" ref-type="bibr">9</xref>], despite chronic use of DMARDs and glucocorticoids. Thirty of the 33 patients received combination DMARD treatment including methotrexate (MTX) at a dose of &#x0003e; 12.5 mg/week; DMARDs other than MTX were discontinued after starting etanercept therapy. The three patients who did not receive MTX prior to the study due to contraindications were treated with combination DMARDs, including hydroxychloroquine, azathioprine, sulfasalazine, and bucillamine; either hydroxychloroquine or azathioprine was continued with etanercept throughout the study period. Twenty-seven patients were treated with calcium and vitamin D supplements and two patients were taking osteoporosis medications (raloxifene and risedronate for each). All patients in this group were injected with etanercept at 25 mg twice per week for 12 weeks.</p><p>An equal number of age- and gender-matched healthy individuals who visited Inha University Hospital for health screening were recruited as normal controls. RA patients who were just diagnosed as seropositive RA and were not yet receiving any DMARD treatment were also included as naive RA.</p><p>Blood samples were collected from RA patients previously treated with DMARDs at baseline and after 12 weeks of etanercept treatment, normal controls, and naive RA patients. The demographic characteristics of patients are summarized in <xref ref-type="table" rid="T1">Table 1</xref>. This study was approved by the Ethics Committee of Inha University Hospital (No. 2010-0001) in January 2010 and informed consent was obtained from all participants.</p></sec><sec><title>Clinical assessments</title><p>Overall disease activity was evaluated by the DAS28, which includes three variables of 28 joint counts of tenderness and swelling and erythrocyte sedimentation rate (ESR). DAS28 was calculated using a calculator (online at <ext-link ext-link-type="uri" xlink:href="http://www.das-score.nl">www.das-score.nl</ext-link>). Acute-phase reactants, including ESR and C-reactive protein (CRP), were measured before and after 12 weeks of etanercept treatment. The European League Against Rheumatism (EULAR) response criteria were used to assess response to therapy after 12 weeks of treatment [<xref rid="B9" ref-type="bibr">9</xref>].</p></sec><sec><title>Biochemical analysis</title><p>Serum samples were stored immediately at -70&#x02103; until analysis. Serum levels of IL-6, receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), c-telopeptide (CTX)-1, sclerostin, bone-specific alkaline phosphatase (BSALP), DKK-1, and procollagen type 1 amino-terminal propeptide (P1NP) were measured using commercially available enzyme-linked immunosorbent assay kits (IL-6, R&#x00026;D Systems, Minneapolis, MN, USA: interassay coefficient of variation [CV] 6.9% to 7.8%; RANKL and OPG, Biomedica, Wien, Austria: interassay CV of RANKL 3% to 5%, interassay CV of OPG 4% to 10%; CTX-1, Nordic Bioscience Diagnostics A/S, Herlev, Denmark: interassay CV 2.5% to 10.9%; sclerostin, USCN, Wuhan, China: interassay CV 4% to 6%; BSALP, Quidel, San Diego, CA, USA: interassay CV 5% to 8%; DKK-1, Assay Designs, Michigan, USA: interassay CV 7.9% to 13.3%; P1NP, Cusabio, Wuhan, China).</p></sec><sec><title>Bone mineral density measurement</title><p>The bone mineral density (BMD) in RA patients was measured by dual-energy X-ray absorptiometry (QDR-4500A, Hologic Inc., Bedford, MA, USA). BMD was measured at baseline and 1 year after treatment in the lumbar spine, femoral neck, and total hip.</p></sec><sec><title>Statistical analysis</title><p>Data are presented as means &#x000b1; standard deviation. Results were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Data prior to and after etanercept treatment were compared using the paired samples <italic>t</italic> test. Comparisons between two independent groups were performed by independent samples <italic>t</italic> test. Correlations between parameters were assessed using Pearson's test. A value of <italic>p</italic> &#x02264; 0.05 was regarded as significant in all analyses.</p></sec></sec><sec sec-type="results"><title>RESULTS</title><p>RA patients previously treated with DMARDs received etanercept treatment for 12 weeks and reported no side effects throughout the study period.</p><sec><title>Effects of etanercept on systemic inflammation and RA disease activity</title><p>Etanercept treatment caused notable reductions in markers of systemic inflammation, including ESR and CRP (<xref ref-type="table" rid="T2">Table 2</xref>). The serum IL-6 level also declined significantly upon treatment (<italic>p</italic> &#x0003c; 0.001). RA disease activity was also improved (<italic>p</italic> &#x0003c; 0.001).</p></sec><sec><title>Effects of etanercept on bone metabolism</title><p>In RA patients treated chronically with DMARDs and glucocorticoids prior to etanercept, both BSALP and CTX-1 levels were significantly lower than in RA patients not yet treated with any DMARD or glucocorticoids (<italic>p</italic> = 0.004, <italic>p</italic> = 0.024, respectively). After 12 weeks of etanercept treatment, serum BSALP, sclerostin, and CTX-1 levels increased, the latter significantly (<xref ref-type="fig" rid="F1">Fig. 1</xref>). Increases in sclerostin levels after etanercept treatment correlated positively with those of serum CTX-1 (<italic>r</italic> = 0.775, <italic>p</italic> &#x0003c; 0.001). Examination of serum levels of bone metabolism markers revealed that changes in BSALP were correlated with those in CTX-1 (<italic>r</italic> = 0.755, <italic>p</italic> &#x0003c; 0.001) (<xref ref-type="fig" rid="F2">Fig. 2</xref>). Levels of other bone metabolism markers including, OPG/RANKL, P1NP, DKK-1, were not affected significantly by etanercept treatment (<xref ref-type="table" rid="T2">Table 2</xref>).</p><p>By the DAS28 EULAR response criteria, there were 27 responders and six nonresponders. In the responders, of the bone metabolism markers only serum sclerostin levels increased significantly, from 1.67 &#x000b1; 2.12 to 2.51 &#x000b1; 3.03 pg/mL (<italic>p</italic> = 0.021). Bone metabolism marker levels did not change in the nonresponder group.</p><p>Lumbar spinal and femoral neck BMD (g/cm<sup>2</sup>) greatly improved at approximately 1 year of treatment with etanercept (<italic>p</italic> = 0.050, <italic>p</italic> = 0.021, respectively). Total hip BMD also exhibited a trend towards recovery after treatment (<italic>p</italic> = 0.066) (<xref ref-type="table" rid="T3">Table 3</xref>).</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>The main finding of this study was that serum levels of the bone metabolism markers CTX-1 and BSALP were lower in RA patients treated chronically with DMARDs and glucocorticoids than in DMARD-naive RA patients. Etanercept treatment for 12 weeks improved RA disease activity and systemic inflammation, but serum levels of the bone metabolism markers CTX-1 and sclerostin remained elevated after treatment.</p><p>Our finding that chronic treatment with DMARDs and glucocorticoids reduces serum CTX-1 and BSALP levels is consistent with earlier reports that these drugs affect bone formation and resorption. MTX, which was taken by 91% of the RA patients in this study treated chronically with DMARDs, suppresses bone formation by inhibiting the differentiation of early osteoblastic cells [<xref rid="B10" ref-type="bibr">10</xref>]. MTX is also known to decrease deoxypyridinoline (DPD) and N-telopeptide of type 1 collagen levels in patients with RA, thereby ameliorating bone resorption [<xref rid="B11" ref-type="bibr">11</xref>]. Glucocorticoids, taken by 97% of the patients chronically treated with DMARDs, also affect bone formation and resorption. Chronic prednisolone at 5 mg daily for 8 weeks was found in a double-blinded randomized trial to significantly decrease serum P1NP, osteocalcin, and free urinary DPD levels [<xref rid="B12" ref-type="bibr">12</xref>].</p><p>Our finding of increased serum CTX-1 and sclerostin after 12 weeks of etanercept treatment has several implications for managing RA patients' bone density and disease. Serum CTX-1 is known to be more specific to bone resorption than other measurements, as these peptide fragments are generated by collagen degradation [<xref rid="B13" ref-type="bibr">13</xref>]. In contrast, sclerostin is a secreted Wnt antagonist that binds to low density lipoprotein receptor-related protein 5 (LRP5) and LRP6 to inhibit Wnt/&#x003b2;-catenin signaling [<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>,<xref rid="B16" ref-type="bibr">16</xref>], suggesting that it would inhibit bone formation [<xref rid="B17" ref-type="bibr">17</xref>].</p><p>The present finding that etanercept stimulates bone resorption and increases serum levels of a bone formation antagonist is inconsistent with previous studies showing improved bone metabolism after 12 weeks of etanercept therapy [<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B18" ref-type="bibr">18</xref>]. However, whether this indicates worsening of bone metabolism after short-term etanercept therapy is unclear. Polyzos et al. [<xref rid="B19" ref-type="bibr">19</xref>] showed that risedronate for 6 months in postmenopausal females significantly increased sclerostin levels; further, sclerostin levels were positively correlated with lumbar spinal BMD. Similarly, Terpos et al. [<xref rid="B20" ref-type="bibr">20</xref>] found that tocilizumab, an IL-6 blocker, increases the sclerostin level and increases the ratio of OPG to RANKL after 2 months of treatment. Thus, increases in sclerostin after etanercept use suggest not only decreased bone formation but also a therapeutic effect on RA-induced bone degradation. In addition, Polyzos et al. [<xref rid="B19" ref-type="bibr">19</xref>] reported that levels of bone metabolism markers such as CTX, P1NP, and total serum ALP were increased after 6 months of teriparatide treatment. In our study, the OPG/RANKL ratio, P1NP and BSALP were not altered significantly by etanercept therapy. However, its effects on serum CTX-1 and sclerostin and the correlation of serum CTX-1 with BSALP suggest that bone resorption and formation might be coupled and that etanercept might induce recovery of bone metabolism from DMARD- and glucocorticoid-induced depression.</p><p>The correlation of changes in sclerostin with changes in serum CTX-1 are consistent with the study of Modder et al. [<xref rid="B21" ref-type="bibr">21</xref>], who reported the same correlation after estrogen treatment. Although the underlying mechanism is unknown, sclerostin seems to affect bone resorption in addition to bone formation [<xref rid="B21" ref-type="bibr">21</xref>].</p><p>The fact that increases in the sclerostin level after etanercept treatment were significant only in DAS28 responders suggests that the effects of etanercept on bone metabolism might result from the reduction in systemic inflammation. Similarly, Sennels et al. [<xref rid="B22" ref-type="bibr">22</xref>] reported a significant increase in total serum RANKL in tandem with major decreases in serum CRP after 12 weeks of etanercept.</p><p>The main limitation of our study was that it was not randomized. Secondly, although comparisons between baseline and 12 weeks after etanercept therapy were performed in individual patients, comparisons between drug-naive patients and those treated chronically with DMARDs were conducted across groups. Though a longitudinal study would reduce the probability of individual variation, Korean treatment guidelines mean that such a study would take years; nonresponse after at least 6 months of oral DMARDs is required for treatment with a TNF inhibitor at insurance-covered costs. Individual variations such as differences in sex hormone levels could also have affected our results.</p><p>In conclusion, 12 weeks of etanercept therapy appears to improve systemic inflammation, but not bone turnover. However, it may begin to restore bone metabolism at this time point, which is depressed due in part to chronic use of MTX and glucocorticoids.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Twelve weeks of etanercept treatment dramatically reduces systemic inflammation.</p></list-item><list-item><p>Levels of circulating bone metabolism markers-including c-telopeptide-1 and sclerostin-are elevated after etanercept treatment.</p></list-item><list-item><p>Short-term etanercept therapy appears to restore homeostasis in bone metabolism following chronic use of methotrexate and glucocorticoids.</p></list-item></list> </p></boxed-text></sec>
Complex repetitive discharge on electromyography as a risk factor for malignancy in idiopathic inflammatory myopathy
<sec><title>Background/Aims</title><p>We investigated the electromyography (EMG) findings and demographic, clinical, and laboratory features that may predict the development of malignancy in patients with idiopathic inflammatory myopathy (IIM).</p></sec><sec><title>Methods</title><p>In total, 61 patients, 36 with dermatomyositis and 25 with polymyositis, were included. Patients were divided into those with and without malignancies, and comparisons were made between the groups in terms of their demographic, clinical, laboratory, and EMG findings.</p></sec><sec><title>Results</title><p>The frequencies of malignancies associated with dermatomyositis and polymyositis were 22% and 8%, respectively. Patients with malignancies showed a significantly higher incidence of dysphagia (odds ratio [OR], 21.50; 95% confidence interval [CI], 3.84 to 120.49), absence of interstitial lung disease (ILD; OR, 0.12; 95% CI, 0.01 to 0.98), and complex repetitive discharge (CRD) on the EMG (OR, 26.25; 95% CI, 2.67 to 258.52), versus those without. After adjustment for age, dysphagia and CRD remained significant, while ILD showed a trend for a difference but was not statistically significant. Multivariate analysis revealed that the CRD conferred an OR of 25.99 (95% CI, 1.27 to 531.86) for malignancy. When the frequency of malignancy was analyzed according to the number of risk factors, patients with three risk factors showed a significantly higher incidence of malignancy, versus those with fewer than two (<italic>p</italic> = 0.014).</p></sec><sec><title>Conclusions</title><p>We demonstrated for the first time that CRD on the EMG was an additional independent risk factor for malignancy in IIM. Further studies on a larger scale are needed to confirm the importance of CRD as a risk factor for malignancy in IIM.</p></sec>
<contrib contrib-type="author"><name><surname>Kim</surname><given-names>Na Ri</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Nam</surname><given-names>Eon Jeong</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kang</surname><given-names>Jong Wan</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Hyun Seok</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Im</surname><given-names>Churl Hyun</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kang</surname><given-names>Young Mo</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib>
The Korean Journal of Internal Medicine
<sec sec-type="intro"><title>INTRODUCTION</title><p>Idiopathic inflammatory myositis (IIMs), including dermatomyositis and polymyositis, is a group of chronic autoimmune conditions characterized by proximal muscle weakness and cutaneous lesions in dermatomyositis [<xref rid="B1" ref-type="bibr">1</xref>]. The risk of malignancy in patients with IIM, especially dermatomyositis, has been reported to be increased [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>]. Risk factors associated with concomitant malignancies in IIM include older age at onset, cutaneous necrosis, increased erythrocyte sedimentation rate, and presence of cutaneous leukoclastic vasculitis [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B8" ref-type="bibr">8</xref>]. The comorbidity of interstitial lung disease (ILD) and the absence of dysphagia are significantly preventative against malignancy [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>]. However, previous studies have focused on demographic, clinical, and laboratory markers associated with malignancy in patients with IIM.</p><p>Uchino et al. [<xref rid="B9" ref-type="bibr">9</xref>] found that the incidence of rare infiltrative-type muscle pathology may be a predictive marker of dermatomyositis or malignancy in dermatomyositis. The results indicated that a subset of muscle inflammation might be specifically associated with malignancy. On electromyography (EMG), IIM is characterized by the presence of prominent muscle membrane irritability [<xref rid="B10" ref-type="bibr">10</xref>]. The degree of abnormal muscle membrane irritability is believed to reflect ongoing disease activity [<xref rid="B10" ref-type="bibr">10</xref>]. However, to date, no association between EMG findings and malignancies in IIM has been established.</p><p>Because malignancy is one of the most important complications affecting the prognosis of patients with IIM, an extensive evaluation for malignancy in patients with IIM has been recommended [<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B6" ref-type="bibr">6</xref>]. In this study, we investigated the EMG findings as well as demographic, clinical, and laboratory features that may predict the development of malignancy in patients with IIM.</p></sec><sec sec-type="methods"><title>METHODS</title><sec><title>Patient enrollment</title><p>We conducted a retrospective study in patients with dermatomyositis and polymyositis admitted to Kyungpook National University Hospital between 1999 and 2013. The diagnoses of polymyositis and dermatomyositis were based on the criteria of Bohan and Peter [<xref rid="B11" ref-type="bibr">11</xref>]: (1) symmetric muscle weakness, (2) increased serum muscle enzymes, (3) myopathic changes on EMG, (4) typical histological findings on a muscle biopsy, and (5) characteristic dermatological manifestations. Two patients were diagnosed with amyopathic dermatomyositis (ADM) according to the criteria of Sontheimer [<xref rid="B12" ref-type="bibr">12</xref>]. Patients were divided into those with and without malignancies, and comparisons were made between the two groups in terms of their demographic factors, clinical findings, laboratory results, and EMG findings.</p></sec><sec><title>Patient demographics and clinical features</title><p>In this study, patients with hallmark cutaneous manifestations of dermatomyositis but for whom muscle testing did not reveal any abnormality were classified as having ADM, according to Sontheimer's criteria [<xref rid="B12" ref-type="bibr">12</xref>]. We included a total of 61 patients with dermatomyositis/polymyositis after excluding four patients with juvenile dermatomyositis, and two with ADM. We reviewed the medical records of these patients. The diagnosis of malignancy-associated IIM was retained if IIM occurred in the context of a recently diagnosed malignancy, or if a malignancy was diagnosed during the 2 years following the diagnosis of IIM (three patients were followed for less than 2 years after the diagnosis of IIM: two for 6 months and one for 9 months). Malignancy screening was performed in all patients, based on our institution's guidelines. All patients were screened for malignancy with laboratory tests including carcinoembryonic antigen, &#x003b1;-fetoprotein, carbohydrate antigen (CA 19-9), cancer antigen (CA-125), neuron-specific enolase, and &#x003b2;-human chorionic gonadotropin for females or prostate-specific antigen for males, radiographic imaging, including abdominal and chest computed tomographies (CTs), upper and lower endoscopic examinations, and positron emission tomography-CT if available. A muscle biopsy was performed in most cases. A nerve conduction study and needle EMGs were performed according to basic principles by one of two experienced electromyographers (JKS and HSS, Neurologist) with Medelec Synergy (VIASYS, London, UK). The classic triad of EMG findings of IIM (1) polyphasic, short duration motor unit potentials, (2) fibrillation, positive sharp waves, increased insertional activity, and (3) complex repetitive discharge (CRD) were evaluated [<xref rid="B10" ref-type="bibr">10</xref>,<xref rid="B11" ref-type="bibr">11</xref>].</p></sec><sec><title>Statistical analyses</title><p>Baseline characteristics were compared between groups using Student <italic>t</italic> test and the chi-square test, or Fisher exact test for categorical variables. An age-adjusted analysis was performed to compare malignancy-associated and nonmalignancy-associated IIM, using univariate logistic regression analysis. We calculated the odds ratios (ORs) and 95% confidence intervals (CIs) of the factors associated with malignancies in patients with IIM. The factors with a <italic>p</italic> value &#x0003c; 0.05 in the univariate analysis were subjected to a multivariate analysis. Multivariate logistic regression analysis was used to identify the relationship between the malignancy and predictor variables. The multivariate analysis used a step-wise backward regression model. The relative excess risk due to interaction (RERI) that was attributable to the interaction between the two variables [<xref rid="B13" ref-type="bibr">13</xref>] was calculated to estimate the excess risk. All statistical calculations were performed using the SPSS version 20 (IBM Co., Armonk, NY, USA). <italic>p</italic> values &#x0003c; 0.05 were considered to indicate statistical significance.</p></sec></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Demographic findings</title><p>Each patient was followed from the time of the initial diagnosis at our institute to either the date of death or the date of the latest follow-up. We excluded patients with a preceding malignancy who had been in remission for more than 3 years at the time of IIM diagnosis. Among 61 the patients, dermatomyositis was the most common type (n = 30, 49%), followed by polymyositis (n = 25, 41%), juvenile dermatomyositis (n = 4, 7%), and ADM (n = 2, 3%). Demographic and clinical data on these patients are presented in <xref ref-type="table" rid="T1">Table 1</xref>. The mean age at diagnosis was 45.3 years for dermatomyositis (including ADM/juvenile dermatomyositis) and 49.3 years for polymyositis. Patients with IIM were predominantly women (60.7%). Distributions of age at diagnosis, gender, and frequency of ILD were not significantly different between the two groups.</p></sec><sec><title>Clinical characteristics of malignancy in IIM patients</title><p>Malignancies were present in 16.4% of all patients (10/61). These included eight of the 36 patients with dermatomyositis (including ADM and juvenile dermatomyositis) (22.2%) and two of the 25 with polymyositis (8.0%). No patient with ADM or juvenile dermatomyositis had a malignancy. There was no significant difference in the risk of malignancy between dermatomyositis and polymyositis patients. Gastric cancer was the most frequent malignancy observed (two patients), followed by bladder cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, thyroid cancer, lung cancer, gallbladder cancer, and invasive thymoma (one patient each). Adenocarcinoma was the predominant histological type (50%). A comparison of the clinical findings of the 10 patients with malignancies and 51 without is presented in <xref ref-type="table" rid="T2">Table 2</xref>.</p><p>Patients with malignancies showed a significantly higher incidence of dysphagia (OR, 21.50; 95% CI, 3.84 to 120.49; <italic>p</italic> &#x0003c; 0.001) and a lower incidence of ILD (OR, 0.12; 95% CI, 0.01 to 0.98; <italic>p</italic> = 0.034), compared with those without. After adjustment for age, dysphagia remained significant, while ILD showed a trend for a difference but was not statistically significant. Serum concentrations of muscle enzymes, such as creatine phosphokinase, aldolase, and myoglobin, and the frequency of autoantibodies, including antinuclear, and anti-Jo-1, -Ro, and -La antibodies, showed no significant difference between IIM patients with and without malignancies.</p><p>EMG was performed in 42 patients, including five with malignancy. The number of abnormal EMG findings was significantly higher in patients with malignancies (OR, 9.05; 95% CI, 1.08 to 75.57; <italic>p</italic> = 0.044). Patients with malignancies showed an increased incidence of CRD (OR, 26.25; 95% CI, 2.67 to 258.52; <italic>p</italic> = 0.008), versus those without, which remained significant after adjustment for age.</p></sec><sec><title>Factors associated with underlying malignancies</title><p>In the next step, interactions among the three predictive factors that were statistically significantly associated with a higher risk of malignancy in patients with IIM were investigated. We chose these significant factors for multivariate logistic regression to determine the independent predictive factors for malignancies (<xref ref-type="table" rid="T3">Table 3</xref>). The presence of CRD on the EMG conferred an OR of 25.99 (95% CI, 1.27 to 531.86; <italic>p</italic> = 0.034) for developing a malignancy. We found that CRD was another independent factor associated with malignancy in IIM, while dysphagia showed marginal statistical significance (<italic>p</italic> &#x0003c; 0.1). Furthermore, we found that an increase in the number of these risk factors was associated with a higher malignancy incidence. Patients with three risk factors showed a significantly higher incidence of malignancies, compared with those with zero (<italic>p</italic> = 0.007) or one (<italic>p</italic> = 0.014) (<xref ref-type="fig" rid="F1">Fig. 1</xref>). RERI was calculated to determine the synergistic interaction between two variables among the three risk factors. There was no statistically significant excess risk attributable to an interaction between any two variables.</p></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><p>Risk factors associated with malignancies in diagnostic tests, such as EMG, or pathology besides the clinical and laboratory features have rarely been defined in patients with IIM. In this study, we found that patients with malignancies had a higher incidence of CRDs on EMG as well as dysphagia and absence of ILD, versus those without malignancies. Moreover, multivariate logistic regression analysis revealed that the CRD stood out as another independent risk factor for malignancy in IIM. When the frequency of malignancy was analyzed according to the number of risk factors, patients with three factors had a significantly increased risk of malignancies, compared with those with fewer than two.</p><p>The standardized incidence ratios for malignancy in patients with dermatomyositis have been reported to be up to 14.2% [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>]. The frequency of malignancies in patients with dermatomyositis and polymyositis has been reported to range from 15% to 35.7% and from 3.1% to 14.3%, respectively [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>,<xref rid="B14" ref-type="bibr">14</xref>,<xref rid="B15" ref-type="bibr">15</xref>]. Our study showed that the frequencies of malignancies associated with dermatomyositis and polymyositis were 22% and 8%, respectively, within the range reported in previous studies, and there was no significant difference in the risk of malignancy between dermatomyositis and polymyositis patients.</p><p>Ovarian, lung, pancreatic, stomach, and colorectal cancers, and lymphomas have been reported as the most commonly associated malignancies in dermatomyositis [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B4" ref-type="bibr">4</xref>]. In Asia, nasopharyngeal carcinoma is also reported frequently in IIM [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B7" ref-type="bibr">7</xref>]. Among the various histological types of malignancy associated with IIM, adenocarcinoma is reported most frequently [<xref rid="B4" ref-type="bibr">4</xref>]. We also found that the most common histological type of the malignancies was adenocarcinoma (50%), although the origin of the malignancies varied.</p><p>Risk factors associated with concomitant malignancies in IIM include older age, dysphagia, absence of ILD, males, muscle enzyme levels, rapid onset of skin and/or muscular symptoms, skin necrosis, and periungal erythema, which have been reported in different combinations in various studies [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B3" ref-type="bibr">3</xref>,<xref rid="B6" ref-type="bibr">6</xref>,<xref rid="B7" ref-type="bibr">7</xref>]. Of these factors, older age, dysphagia, and absence of ILD have been reported repeatedly as risk factors for malignancies. In this study, we also found that patients with malignancies had a higher frequency of dysphagia and absence of ILD, versus those without.</p><p>While the mechanism(s) behind the association between malignancy and dysphagia or absence of ILD have not been established, several hypotheses have been proposed. The association of dysphagia with malignancies might be explained by a mechanical component of gastric cancer or laryngeal muscle weakness due to cancer [<xref rid="B2" ref-type="bibr">2</xref>,<xref rid="B7" ref-type="bibr">7</xref>]. However, only two patients with gastric cancer and one with nasopharyngeal cancer were included in this study and that the significant difference between patients with and without malignancy was maintained after excluding these patients (85.7% vs. 15.7%, <italic>p</italic> &#x0003c; 0.001).</p><p>In the case of the negative association between ILD and malignancy, one possibility suggested in a previous study was that patients with ILD had a poor prognosis [<xref rid="B16" ref-type="bibr">16</xref>], indicating that they might not live long enough to develop malignancies [<xref rid="B7" ref-type="bibr">7</xref>]. However, the mean follow-up duration of patients with and without ILD was not significantly different in the present study, consistent with the findings of So et al. [<xref rid="B2" ref-type="bibr">2</xref>]. Another possibility is that the involvement of the muscular system in paraneoplastic syndrome may have a different mechanism, compared with that of IIM, which is associated with sparing of inflammatory cell infiltration in the lung.</p><p>Few studies have addressed the pathological characteristics of muscle in IIM with malignancy. In these studies, dermatomyositis patients with malignancies had less inflammatory cell infiltration and regeneration, versus those without [<xref rid="B9" ref-type="bibr">9</xref>,<xref rid="B14" ref-type="bibr">14</xref>]. Sampson et al. [<xref rid="B17" ref-type="bibr">17</xref>] reported that paraneoplastic necrotizing myopathy showed extensive necrosis but little or no lymphocytic infiltrate, possibly due to cytokine-mediated mechanisms of myocyte injury [<xref rid="B18" ref-type="bibr">18</xref>]. Consequently, humoral myocyte injury in malignancies associated with IIM, which shows the paucity of inflammatory cell infiltration, may induce chronic inflammation in the muscular system, thereby leading to muscular membrane irritability and a higher frequency of CRD on EMG.</p><p>CRD comprises trains of complex polyphasic potentials that repeat at a regular frequency (range, 5 to 100 Hz) and that characteristically begin and terminate abruptly [<xref rid="B19" ref-type="bibr">19</xref>]. The complex configuration of CRD is explained by the fact that it consists of action potentials generated by individual muscle fibers forming a closed circuit, activated through ephaptic transmission [<xref rid="B20" ref-type="bibr">20</xref>]. CRD is present in chronic diseases, such as inflammatory myositis, muscular dystrophies, and spinal muscular atrophy [<xref rid="B20" ref-type="bibr">20</xref>], while no reports on the association of CRD as a risk factor malignancy related with IIM, have to our knowledge been published. In the present study, we investigated additional risk factors and found that CRD was more frequently detected in patients with malignancies, versus those without. CRD on EMG together with dysphagia and the absence of ILD was analyzed by multivariate logistic regression, which identified CRD as another independent risk factor for malignancies.</p><p>We also evaluated the association between the frequency of malignancies and numbers of risk factors: CRD, dysphagia, and absence of ILD. Patients with all three factors had a significantly increased risk of malignancies, compared with those with fewer than two. However, there was no synergistic interaction between any two of the three risk factors. These data suggest that a higher number of risk factors may be a warning sign for hidden malignancies in IIM, thus suggesting the need for a thorough evaluation for malignancies. A limitation of our study was that it was a retrospective study at a single tertiary referral center, so some data were missing especially the results of EMG studies. Moreover, relatively few patients were enrolled.</p><p>In conclusion, we demonstrated for the first time that CRD on EMG was an independent risk factor for malignancies in IIM. Further studies on a larger scale are needed to confirm the importance of CRD as a risk factor for malignancies in IIM and to investigate the mechanism(s) underlying the association between CRD and the characteristic muscle pathology findings in IIM.</p></sec><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Patients with malignancies related to idiopathic inflammatory myopathy (IIM) had a higher incidence of complex repetitive discharge (CRD) on electromyography (<italic>p</italic> = 0.008) as well as dysphagia (<italic>p</italic> &#x0003c; 0.001) and the absence of interstitial lung disease (<italic>p</italic> = 0.034) versus those without malignancies.</p></list-item><list-item><p>Multivariate logistic regression analysis revealed that the CRD conferred an odds ratio of 25.99 (95% confidence interval, 1.27 to 531.86; <italic>p</italic> = 0.034) for malignancy.</p></list-item><list-item><p>A higher number of risk factors may be a warning sign for hidden malignancies in IIM, suggesting the need for a thorough evaluation for malignancies.</p></list-item></list> </p></boxed-text></sec>
Funtional significance of the intermediate lesion in a single coronary artery assessed by fractional flow reserve
Could not extract abstract
<contrib contrib-type="author"><name><surname>Ha</surname><given-names>Sang Jin</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kwon</surname><given-names>Se Hwan</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kim</surname><given-names>Soo-Joong</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib>
The Korean Journal of Internal Medicine
<p>To the Editor,</p><p>Congenital coronary artery anomalies are infrequently seen during coronary angiographic study, reportedly occurring in 0.64% to 1.3% of patients [<xref rid="B1" ref-type="bibr">1</xref>]. Most patients with a congenital coronary artery anomaly are asymptomatic; in rare cases, they may present with chest pain and have myocardial ischemia or other life-threatening conditions. We present a patient with the right coronary artery (RCA) originating from the terminal branch of the left circumflex (LCx) artery who presented with chest pain and was found to have a moderate stenotic lesion in the distal LCx artery. Fractional flow reserve (FFR) allowed us to evaluate this moderate stenotic lesion in a single coronary artery, and to treat with medical therapy only.</p><p>A 63-year-old man was admitted to the cardiology unit with atypical chest pain. He had hypertension as coronary heart disease risk factor. His vital signs were stable, and physical examination showed no abnormalities. A resting electrocardiogram demonstrated a convex-shaped ST-segment elevation in the precordial lead without reciprocal change. Laboratory findings including cardiac markers showed no abnormalities. Echocardiography revealed normal left ventricular morphology, no regional wall motion abnormality, and a diastolic dysfunction of relaxation abnormality. The patient underwent coronary computed tomographic (CT) angiography. It demonstrated the presence of a single coronary artery with the LCx artery continuing in the course of the RCA and minimal possibility of chronic total occlusion (CTO) of the RCA ostium with a collateral supply from the LCx artery (<xref ref-type="fig" rid="F1">Fig. 1</xref>). It also showed 60% diameter stenosis in the distal LCx artery (<xref ref-type="fig" rid="F1">Fig. 1</xref>).</p><p>To differentiate single coronary artery from CTO of the RCA ostium and confirm the degree of stenosis in the distal LCx artery, we performed coronary angiography. Selective left coronary angiography displayed normal origin and course of the left main, LCx, and left anterior descending arteries. The LCx artery did not terminate after reaching the crux; giving rise to the posterior descending branch, it coursed in the right atrioventricular groove as if it were the RCA and ended when it reached the right sinus of Valsalva (<xref ref-type="fig" rid="F2">Fig. 2A</xref>). There was a stenosis of 60% diameter in the distal part of the LCx proper where the RCA arose (<xref ref-type="fig" rid="F2">Fig. 2B</xref>). Attempts to engage the right coronary catheter into the RCA ostium were futile, and aortography obtained in the left anterior-oblique projection displayed the absence of the ostium of the RCA in the right sinus of Valsalva (<xref ref-type="fig" rid="F2">Fig. 2C</xref>). To decide the functional significance of the stenosis in the distal LCx artery, we performed an FFR study with a pressure wire and found that there was no critical functional stenosis (FFR, 0.9) (<xref ref-type="fig" rid="F2">Fig. 2D</xref>). The patient was discharged with medications of aspirin, statin, nitrate, and &#x003b2; blocker and free from chest pain.</p><p>A single coronary artery is an extremely rare congenital anomaly characterized by a single coronary artery ostium from an aortic sinus, and is seen in only 0.024% to 0.066% of patients who undergo conventional coronary angiography [<xref rid="B1" ref-type="bibr">1</xref>]. A single coronary artery is usually asymptomatic and has a benign course, and these patients have a normal life expectancy. The presence of a single coronary artery may bring about myocardial ischemia because of the inability to adequately sustain normal coronary circulation [<xref rid="B2" ref-type="bibr">2</xref>].</p><p>FFR is a physiologic parameter that can be readily measured during the invasive procedure and can evaluate the functional significance of coronary stenosis. In particular, FFR plays an important role when making a decision for percutaneous coronary intervention (PCI) of angiographically moderate stenosis and assessing accurately the functional consequences of a given coronary stenosis with unclear hemodynamic significance [<xref rid="B3" ref-type="bibr">3</xref>]. In addition, the clinical outcome of patients in whom PCI is deferred because FFR indicated no hemodynamically significant stenosis was very favorable [<xref rid="B4" ref-type="bibr">4</xref>]. In this case, FFR was a good assessment modality because the lesion of interest in the LCx artery was a part of a single coronary artery that supplied both the LCx artery and RCA territories. With FFR, we could make a decision for deferred PCI and choose medical treatment, even though coronary CT angiography and coronary angiography showed moderate stenosis in the LCx artery.</p><p>In conclusion, FFR could successfully evaluate the functional significance of a moderate stenotic lesion in a single coronary artery where the RCA originated from the terminal portion of the LCx artery.</p>
Graves' disease presenting with acute renal infarction
Could not extract abstract
<contrib contrib-type="author"><name><surname>Baek</surname><given-names>Cho-Ok</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Kyung Ae</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Tae Sun</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Jin</surname><given-names>Heung Yong</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<p>To the Editor,</p><p>Hyperthyroidism is associated with increased plasma levels of procoagulants and antifibrinolytic clotting factors such as fibrinogen and plasminogen activator inhibitor-1 [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B2" ref-type="bibr">2</xref>]. Similarly, thyrotoxicosis is associated with the occurrence of arterial thrombosis that is primarily due to atrial arrhythmias; the overall incidence of thromboembolism varies from 8% to 40% [<xref rid="B3" ref-type="bibr">3</xref>]. Thrombosis is often observed in patients with thyrotoxicosis but there are few reports of arterial thrombosis in conjunction with thyrotoxicosis that are independent of atrial arrhythmias. The present study is a case report of a patient with Graves' disease who presented with a renal infarction without atrial fibrillation.</p><p>A 54-year-old man visited our emergency department due to the sudden onset of left flank pain for 30 minutes that was accompanied by dysuria in the absence of other symptoms of pyelonephritis in conjunction with neck enlargement and eyeball protrusion. The patient did not have a medical history of diabetes, hypertension, or cardiovascular disease but he had a 10 pack-per-year history of smoking. An electrocardiogram (ECG) revealed sinus tachycardia but his chest X-ray was unremarkable. His vital signs were as follows: blood pressure of 140/90 mmHg, heart rate of 108 beats per minute, respiration rate of 22 breaths per minute, and body temperature of 36.5&#x02103;. His laboratory findings were as follows: urinary analysis of 5 to 9 red blood cells per high power field, +1 proteinuria, a thyroid stimulating hormone (TSH) level of 0.01 &#x000b5;U/mL (normal range, 0.55 to 4.78), a free thyroxine level of 28.33 pmol/L (normal range, 11.5 to 22.7), and a TSH-binding inhibitory immunoglobulin level of 40.00 IU/L (normal level, 1.75 or less). Coagulation tests, including prothrombin time and activated partial thromboplastin time, were performed while the patient was in the emergency room and were within normal ranges. In addition, the patient's levels of Factor VIII, von Willebrand factor, antithrombin III, fibrinogen, and plasminogen activator inhibitor-1 were also assessed to screen for coagulation and fibrinolytic system disorders. All levels were within normal ranges and further tests for antiphospholipid antibodies, cytoplasmic antineutrophil cytoplasmic antibodies, and perinuclear antineutrophil cytoplasmic antibodies, which are associated with vasculitis, were negative.</p><p>Initial Holter monitoring (24 hours) revealed sinus tachycardia but the patient's echocardiogram findings were normal. An enhanced abdominal computed tomography (CT) scan revealed a hypoechoic lesion with a clear wedge-shaped margin on the left kidney and a thrombus in the left renal artery (<xref ref-type="fig" rid="F1">Fig. 1A</xref>). The renal infarction was diagnosed based on the symptoms of the patient and typical CT findings and he was treated with intravenous unfractionated heparin infusions. A thyroid ultrasonography revealed increases in the size and vascularity of both thyroid glands (<xref ref-type="fig" rid="F1">Fig. 1B</xref>) and a technetium-99m thyroid scan was compatible with a diagnosis of Graves' disease (<xref ref-type="fig" rid="F1">Fig. 1C</xref>). The patient was initially treated with methimazole and was switched to warfarin therapy after 5 days of heparin infusions. A second round of Holter monitoring (24 hours) after 1 week showed nonspecific findings and a subsequent ECG showed a normal sinus rhythm.</p><p>Hyperthyroidism is associated with a hypercoagulable state and is thought to increase the risk of venous thrombosis [<xref rid="B2" ref-type="bibr">2</xref>]. However, the relationship between thyroid function and the risk of venous thrombosis has yet to be fully explored. Thyrotoxicosis shifts the haemostatic balance of an individual towards a hypercoagulable and hypofibrinolytic state [<xref rid="B2" ref-type="bibr">2</xref>], but unlike venous thrombosis, the arterial embolisms that are associated with thyrotoxicosis typically result from atrial arrhythmias. Patients with hyperthyroidism frequently present with atrial fibrillation, and its incidence is approximately 10% to 25% for all patients with thyrotoxicosis [<xref rid="B3" ref-type="bibr">3</xref>]. The overall rate of systemic embolisms is 8% to 40% in patients with thyrotoxic atrial fibrillation [<xref rid="B3" ref-type="bibr">3</xref>], but as in the present case, the presence of arterial thromboembolism in thyrotoxic patients without cardiac arrhythmia is very rare and not fully characterized. Only seven cases of acute cerebrovascular ischemic disease have been reported [<xref rid="B1" ref-type="bibr">1</xref>] and in some of these instances, including the present case, paroxysmal atrial fibrillation or vasculitis that may contribute to embolic phenomena could not be entirely excluded.</p><p>In the present case, the patient presented with a renal infarction during the course of thyrotoxicosis that was caused by hyperthyroidism induced by Graves' disease. Although the relationship that likely exists between arterial embolisms, particularly renal infarctions, and hyperthyroidism needs to be clarified, the present case suggests that uncontrolled thyrotoxicosis is associated with a risk of renal infarction.</p>
Persistent suboptimal molecular response in a patient with chronic myelogenous leukemia and Klinefelter syndrome
Could not extract abstract
<contrib contrib-type="author" corresp="yes"><name><surname>Chakraborty</surname><given-names>Rajshekhar</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Mukkamalla</surname><given-names>Shiva Kumar Reddy</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Singam</surname><given-names>Kranthi</given-names></name><xref ref-type="aff" rid="A1"/></contrib><contrib contrib-type="author"><name><surname>Calderon</surname><given-names>Natalia</given-names></name><xref ref-type="aff" rid="A1"/></contrib>
The Korean Journal of Internal Medicine
<p>To the Editor,</p><p>Klinefelter syndrome (KS) is the most prevalent sex chromosomal anomaly in males [<xref rid="B1" ref-type="bibr">1</xref>]. To date, no significant association between KS and chronic myelogenous leukemia (CML) has been documented [<xref rid="B2" ref-type="bibr">2</xref>].</p><p>We present a case of CML in a patient with KS who demonstrated a persistent, suboptimal molecular response despite therapy with second-generation tyrosine kinase inhibitors (TKIs). Furthermore, he had a deletion of the argininosuccinate synthetase (ASS) gene at the 9q34 locus. He had no clinically significant comorbidity related to KS, which was diagnosed incidentally while performing bone marrow cytogenetics. It is unclear whether the presence of the derivative chromosome 9 deletion and sex chromosome anomaly affects the outcome in these patients with CML.</p><p>A 35-year-old Hispanic male was diagnosed with CML (in the chronic phase) in July 2009. He had a history of tuberculosis treated for 9 months in 1996 in Colombia. He initially presented with generalized fatigue, splenomegaly, and a high white blood cell count of 133,000/&#x000b5;L. Initial bone marrow biopsy revealed markedly hypercellular marrow with increased granulocytes at all stages of maturation and micromegakaryocytes. Few immature cells stained positive for CD34 and CD117. Initial cytogenetic studies showed 94% of cells with Breakpoint Cluster Region-Abelson (BCR-ABL) rearrangement on fluorescence <italic>in situ</italic> hybridization, 9q34.1 deletion and an extra X chromosome (<xref ref-type="fig" rid="F1">Fig. 1</xref>). All of the 20 metaphase cells analyzed in cytogenetic studies showed an extracopy of an X chromosome and deletion of the ASS gene at 9q34 locus in bone marrow cells positive for BCR-ABL rearrangement.</p><p>He was started on imatinib mesylate 400 mg daily. Subsequently, he achieved a complete hematologic response and a major molecular response in 3 months with 3-log reduction in BCR-ABL transcripts but only a minor cytogenetic response at 6 months. His medication history was further investigated, and it was found that he was taking rifabutin as prescribed by his primary care physician for a positive skin purified protein derivative test. Due to the drug interaction between imatinib mesylate and rifabutin, the dose of imatinib was increased to 800 mg daily in March 2010. Despite increasing the imatinib dose, reverse transcription followed by real-time polymerase chain reaction (PCR) of peripheral blood showed a persistent increase in BCR-ABL1 transcripts (<xref ref-type="fig" rid="F2">Fig. 2</xref>). Dasatinib was subsequently started on June 2010 due to the persistent partial molecular response. In the interim, ABL kinase mutation analysis was performed and found to be negative. The patient was then evaluated for allotransplantation, but no related or unrelated matches were found. He was continued on dasatinib 100 mg daily. A complete cytogenetic response was finally achieved in April 2013. However, he continues to have a suboptimal molecular response with fluctuating levels of BCR-ABL1 transcripts in peripheral blood PCR studies.</p><p>Several case reports and cohort studies have suggested an increased risk for malignancies in males with KS. A recent British cohort study found a significantly increased risk and mortality from breast cancer, lung cancer, and non-Hodgkin lymphoma in males cytogenetically diagnosed with KS [<xref rid="B2" ref-type="bibr">2</xref>]. In the same study, an increased incidence of leukemia was also noted, but the difference was not statistically significant.</p><p>In the past, few cases of Philadelphia-chromosome-positive CML have been reported in males with KS [<xref rid="B1" ref-type="bibr">1</xref>,<xref rid="B3" ref-type="bibr">3</xref>]. In a study by Alimena et al. [<xref rid="B3" ref-type="bibr">3</xref>] on the cytogenetics of leukemic cells in patients with constitutional chromosomal anomalies, it was observed that acute leukemia occurred predominantly in patients with trisomy 21, whereas chronic myeloproliferative disorders were dominant in those with balanced translocations and sex chromosome anomalies, including KS. All of the patients presented with CML in the chronic phase, with the exception of one case reported by Toubai et al. [<xref rid="B1" ref-type="bibr">1</xref>] in which the patient presented in blast crisis and subsequently underwent allogeneic bone marrow transplantation from an unrelated donor. In our case, the patient presented in the chronic phase with bone marrow cytogenetics showing 47, XXY, t(9;22)(q34;q11) with deletion of ASS at 9q34.1.</p><p>It is unclear whether the presence of KS has any prognostic significance in patients with CML. The patient presenting with blast crisis reported by Toubai et al. [<xref rid="B1" ref-type="bibr">1</xref>] relapsed after bone marrow transplantation and subsequently died due to disease progression. Our patient has a persistent suboptimal molecular response despite therapy with second-generation TKIs.</p><p>Some controversy exists regarding the prognostic significance of large deletions at the t(9; 22) breakpoint in patients with CML. Quintas-Cardama et al. [<xref rid="B4" ref-type="bibr">4</xref>] reported a study of 352 patients with CML and found similar rates of cytogenetic response, overall survival and response duration with imatinib mesylate in those with or without derivative chromosome 9 deletion. Huntly et al. [<xref rid="B5" ref-type="bibr">5</xref>] reported lower rates of hematologic and cytogenetic responses in patients with deletions when treated with imatinib mesylate. To date, no study of the rates of molecular remission in patients with derivative chromosome 9 deletions and the effect of second-generation TKIs, if any, on survival and rates of remission (hematologic, cytogenetic, and molecular) in this subgroup of patients has been conducted.</p><p>To our knowledge, this is the first case report of CML in a patient with KS to address the outcome and response to TKIs. Our patient had a persistent suboptimal molecular response with fluctuating BCR-ABL activity in peripheral blood detected by PCR, despite therapy with second-generation TKIs. Further studies are required to identify the prognostic significance of KS and derivative chromosome 9 deletions in patients with CML.</p><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Leukemia, myelogenous, chronic, BCR-ABL positive</p></list-item><list-item><p>Klinefelter syndrome</p></list-item><list-item><p>Molecular remission</p></list-item></list> </p></boxed-text></sec>
Transformation into large-cell neuroendocrine carcinoma associated with acquired resistance to erlotinib in nonsmall cell lung cancer
Could not extract abstract
<contrib contrib-type="author"><name><surname>Lim</surname><given-names>Jeong Uk</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Woo</surname><given-names>In Sook</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Jung</surname><given-names>Yun Hwa</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Byeon</surname><given-names>Jae Ho</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Chan Kwon</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Tae Jung</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Hyo Rim</given-names></name><xref ref-type="aff" rid="A4">4</xref></contrib>
The Korean Journal of Internal Medicine
<p>To the Editor,</p><p>The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib is active in patients with metastatic lung adenocarcinoma carrying mutations in the EGFR gene. Although erlotinib has been found to improve markedly progression-free survival and quality of life, most patients who show an initial response eventually develop disease progression. Possible mechanisms of acquired or secondary resistance to erlotinib include second mutations in the EGFR gene such as T790M, activation of an alternative pathway including Met or HER2 amplification, histological transformation to small cell lung cancer (SCLC) and epithelial to mesenchymal transition [<xref rid="B1" ref-type="bibr">1</xref>]. However, acquired resistance due to histological transformation into a large cell neuroendocrine carcinoma (LCNEC) is uncommon. Herein we describe a patient who developed a LCNEC at the time of acquired resistance to erlotinib during treatment for primary lung adenocarcinoma.</p><p>A 33-year-old male was admitted to the Department of Neurosurgery because of headache and diplopia. Brain magnetic resonance imaging (MRI) revealed multiple metastatic masses in the left frontal and right temporal lobes and the left cerebellum of the brain. The largest mass in the left frontal lobe measured 3.4 cm and caused midline shifting of the brain (<xref ref-type="fig" rid="F1">Fig. 1</xref>).</p><p>A left frontal craniotomy was performed to remove the mass in the left frontal lobe. The specimen was positive for cytokeratin-7 (CK-7), thyroid transcription factor-1 (TTF-1), leukocyte common antigen, CK-20, and vimentin, indicating a well-differentiated adenocarcinoma (<xref ref-type="fig" rid="F2">Fig. 2</xref>). The patient was subsequently referred to the Department of Medical Oncology.</p><p>Chest computed tomography (CT) showed multiple nodules in both lung parenchyma and enlarged lymph nodes in both mediastinal and right hilar and right supraclavicular areas (<xref ref-type="fig" rid="F3">Fig. 3</xref>). Abdominal CT showed a heterogeneously enhanced mass, measuring 3.6 &#x000d7; 4.1 cm in the lower portion of the left kidney, along with multiple enlarged lymph nodes near the left renal vein and a low density mass 1.4 cm in size near the right kidney. A bone scan showed sacral metastasis.</p><p>The patient was diagnosed with metastatic lung adenocarcinoma. Analysis of the EGFR gene showed a deletion in exon 19.</p><p>The patient underwent CyberKnife surgery treatment of the left cerebellar hemisphere, right temporal lobe and left precentral gyrus, followed by two cycles of pemetrexed-cisplatin as first-line chemotherapy. However, follow-up CT showed progressive disease (<xref ref-type="fig" rid="F3">Fig. 3</xref>).</p><p>Second-line treatment with erlotinib for 2 months showed a partial response in the lung nodules, mediastinal lymph nodes, and kidney masses. Four months after erlotinib was started, the patient experienced pain in the hip and right leg. MRI of the L-spine and sacrum revealed a 3.8 &#x000d7; 7.8 &#x000d7; 4.4 cm lobular mass involving the right sacral body crossing the midline with extraosseous extension into the spinal canal and obliteration of the right side S1 nerve root. The patient was treated with palliative radiotherapy to the sacrum (total, 4,200 cGy in 14 fractions). Erlotinib was continued during palliative radiotherapy. After 10 months of treatment with erlotinib, the right lower paratracheal lymph node increased in size from 1 to 3 cm (<xref ref-type="fig" rid="F3">Fig. 3</xref>).</p><p>A rebiopsy of the right paratracheal lymph node using mediastinoscopy showed two different types of cells: adenocarcinoma cells, as shown on initial pathology, and LCNEC cells, comprising 50% of the microscopic field. The LCNEC cells were positive for chromogranin, CK-7, neuron specific enolase, and TTF-1 and negative for CD56a (<xref ref-type="fig" rid="F2">Fig. 2</xref>). The EGFR gene in the second biopsy specimen was not analyzed due to patient refusal. Despite receiving three cycles of etoposide-cisplatin chemotherapy, along with maintenance erlotinib to prevent disease flare up from erlotinib discontinuation, the disease progressed (<xref ref-type="fig" rid="F3">Fig. 3</xref>).</p><p>After 1 month observation period, the patient visited our emergency room with fever and dyspnea. Chest CT showed enlarged mediastinal lymph nodes invading the trachea and superior vena cava (SVC), consistent with SVC syndrome. Palliative emergency radiation treatment of the mediastinal mass (3,000 cGy in 10 fractions) was started immediately. Follow-up chest CT showed that several parenchymal nodules in both lungs had increased in size, while others had decreased and the size of the mediastinal lymph nodes had decreased. However, the SVC remained obstructed and salvage chemotherapy with topotecan was started. Topotecan chemotherapy was discontinued after one cycle due to worsening of the patient's general condition and tumor lysis syndrome. After 5 days of intensive care unit care, the patient died.</p><p>Acquired or secondary resistance to EGFR TKIs is associated with histological transformation into SCLC and changes in the molecular genetics of lung adenocarcinoma [<xref rid="B2" ref-type="bibr">2</xref>]. Therefore, a rebiopsy is warranted when disease progression is observed during treatment with an EGFR TKI. The mechanism of histological transformation remains unclear. A female patient diagnosed initially with adenocarcinoma subsequently developed SCLC upon disease progression, with the SCLC cells harboring an 18 bp deletion in exon 19 of the EGFR gene [<xref rid="B3" ref-type="bibr">3</xref>]. An analysis of the molecular genetic and histological features of 37 patients with progressive disease after durable response to EGFR TKI showed the EGFR T790M mutation in 18 patients (49%) and Met amplification and PIK3CA mutations in two patients (5%) each [<xref rid="B2" ref-type="bibr">2</xref>]. In five patients (14%), histological transformation to SCLC was confirmed based on expression of neuroendocrine markers, with the original EGFR mutation maintained. One of the five patients with SCLC transformation also had a mutation in PIK3CA, with four showing resistance during treatment with an EGFR TKI.</p><p>Two cases of histological transformation to neuroendocrine carcinoma during or after treatment with an EGFR TKI were reported recently [<xref rid="B4" ref-type="bibr">4</xref>,<xref rid="B5" ref-type="bibr">5</xref>]. For example, one patient with an adenocarcinoma showed transformation to combined SCLC and NSCLC with neuroendocrine morphology after 12 months of gefitinib treatment [<xref rid="B4" ref-type="bibr">4</xref>]. The patient showed disease progression after four cycles of pemetrexed-cisplatin combination chemotherapy and erlotinib treatment for 12 months, with an EGFR exon 19 deletion observed in a tissue specimen obtained at the time of progression. Therefore, whether histological transformation reflects acquired resistance to EGFR TKI remains unclear. Another patient showed transformation from an adenocarcinoma to EGFR-positive LCNEC after treatment with an EGFR TKI. Following surgery, this patient received radiation treatment concurrent with combination chemotherapy of vinorelbine and cisplatin for disease progression and adjuvant chemotherapy for stage IIIA lung adenocarcinoma with an EGFR mutation. This patient was treated with gefitinib for 24 months and then changed to erlotinib. Since the rebiopsy was obtained when the patient developed resistance to erlotinib, the time of phenotypic switch to LCNEC could not be determined [<xref rid="B5" ref-type="bibr">5</xref>].</p><p>The timing of histological transformation may require obtaining biopsy samples when the patient shows resistance to each type of systemic treatment. However, repeated biopsies are difficult in clinical practice.</p><p>Our patient presented with a combined tumor, consisting of adenocarcinoma and LCNEC components when he developed resistance to erlotinib. The pathological results were confirmed by the histological features of the tumor and with immunohistochemistry of neuroendocrine markers. Molecular genetic analysis of the rebiopsied tissue was not performed and changes in molecular genetic and epigenetic profiles occurring at the time of transformation into LCNEC were not determined because the patient refused further study.</p><p>Differential diagnosis of SCLC and LCNEC is difficult in some cases because they have different morphologies, such as organoid nesting, trabecular, rosette-like, and palisading patterns, with neuroendocrine differentiation confirmed immunohistochemically. LCNEC cells are larger than small resting lymphocytes. Compared with SCLC cells, the nucleoli in LCNEC cells are more prominent and their nuclear-cytoplasmic ratio is lower.</p><p>Our findings suggest that, during treatment with the EGFR TKI erlotinib, the adenocarcinoma had partially transformed to LCNEC or was undergoing histological transformation. Initial pathological diagnosis was confirmed since the tissue obtained with craniotomy showed no evidence of the LCNEC component. Moreover, rebiopsy of the paratracheal lymph node showed the tumor had a marked initial response to erlotinib but became enlarged again at the time of resistance to EGFR TKI.</p><p>The mechanisms of the LCNEC transformation during treatment with EGFR TKI and the development of acquired resistance require further investigations.</p><p>In conclusion, we described a patient with metastatic lung adenocarcinoma who experienced a histological transformation to combined LCNEC and adenocarcinoma of the lung during treatment with erlotinib.</p>
<italic>Parvimonas micra</italic> chest wall abscess following transthoracic lung needle biopsy
Could not extract abstract
<contrib contrib-type="author" corresp="yes"><name><surname>Gorospe</surname><given-names>Luis</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Bermudez-Coronel-Prats</surname><given-names>Isabel</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Gomez-Barbosa</surname><given-names>Carol F.</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Olmedo-Garcia</surname><given-names>Maria E.</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Ruedas-Lopez</surname><given-names>Angel</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Gomez del Olmo</surname><given-names>Vicente</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib>
The Korean Journal of Internal Medicine
<p>To the Editor,</p><p>Percutaneous transthoracic computed tomography (CT)-guided needle biopsy is a well-established, simple, reliable diagnostic interventional procedure for definitive pathologic diagnosis of thoracic neoplasms because of its high diagnostic yield and relatively low morbidity [<xref rid="B1" ref-type="bibr">1</xref>]. However, one of the most serious complications of CT-guided biopsy, although extremely rare, is implantation of carcinoma along the biopsy route.</p><p><italic>Parvimonas micra</italic> (formerly known as <italic>Peptostreptococcus micros</italic>) is a nonspore-forming anaerobic gram-positive coccus widely distributed as commensal flora in the oral cavity that, under immunosuppressed or traumatic conditions, can become pathogenic and cause brain, liver, and thoracic infections, as well as generalized necrotizing soft tissue infections. Within the thorax, <italic>P. micra</italic> may cause aspiration pneumonia, lung abscesses, empyema, and mediastinitis. Rarely, <italic>P. micra</italic> infections can also occur in patients with no recognized predisposing conditions [<xref rid="B2" ref-type="bibr">2</xref>].</p><p>We present a case of a patient suspected to have lung cancer who underwent two CT-guided transthoracic needle biopsies and developed a chest wall mass 10 days after the second percutaneous biopsy. This mass was ultimately confirmed to be a <italic>P. micra</italic> chest wall abscess.</p><p>A 67-year-old male ex-smoker with cough and constitutional syndrome (loss of appetite, fatigue, and progressive weight loss over 6 weeks) and no fever was referred to our institution after chest radiographs revealed a nonresolving opacity in the right lung base. His medical history was otherwise nonsignificant. Physical examination showed decreased breath sounds on auscultation at the right hemithorax. The patient had been treated with levofloxacin (500 mg/12 hours) for 7 days, but was not taking corticosteroids or other immunosuppressants. Tests for human immunodeficiency, hepatitis B, and hepatitis C viruses were negative. Thoracic CT showed a heterogeneous consolidation in the right lung involving both the right upper lobe and the right middle lobe (<xref ref-type="fig" rid="F1">Fig. 1</xref>). Large mediastinal adenopathies were also present. Lung carcinoma was suspected, but two bronchoscopic transbronchial biopsies failed to identify cancerous cells, so a CT-guided biopsy of the right lung lesion was performed.</p><p>The patient was placed in the supine position on the CT table and an 18 G semi-automated coaxial core biopsy needle (Bard Magnum, Bard, Tempe, AZ, USA) was inserted into the patient's thorax through the right pectoral muscle (<xref ref-type="fig" rid="F2">Fig. 2</xref>). Pathologic analysis of the obtained specimens also failed to reveal cancerous cells. The right lung opacity was punctured again 5 days later with the same coaxial system. Surprisingly, pathologic analysis was again nondiagnostic and only showed lymphocyte-rich inflammatory changes and nonspecific areas of organizing pneumonia. No immediate complications (i.e., pneumothorax or hemorrhage) were observed during these percutaneous procedures.</p><p>Ten days after the second transthoracic biopsy, the patient developed a painful mass on the right anterior chest wall. Since this mass was on the puncture site of the previous biopsy, needle track dissemination of carcinoma was suspected. A new thoracic CT revealed a low density well-defined lesion in the right pectoral muscle (<xref ref-type="fig" rid="F3">Fig. 3</xref>). This lesion was punctured and pus was aspirated. Microbiological culture of the aspirated pus identified clindamycin-susceptible <italic>P. micra</italic>.</p><p>The patient was started on clindamycin (600 mg/8 hours intravenous) and the anterior chest wall abscess was drained. After 8 weeks of treatment, the right lung consolidation had decreased in size, the chest wall abscess had resolved, and the patient's symptoms had improved. The patient's mouth was explored and was found to have suboptimal oral hygiene and periodontal disease. We believe that the lung consolidation was also caused by <italic>P. micra</italic> infection and that dissemination to the chest wall was a complication of percutaneous transthoracic needle biopsy.</p><p>Percutaneous transthoracic CT-guided needle biopsy is a well-established, simple, reliable diagnostic interventional procedure for definitive pathologic diagnosis of thoracic neoplasms since it has a high diagnostic yield and a relatively low morbidity [<xref rid="B1" ref-type="bibr">1</xref>]. However, immediate and delayed complications may occur. Among the common immediate complications are pneumothorax, hemoptysis/pulmonary haemorrhage, and hemothorax. Most of these complications are managed conservatively, although pneumothorax may require tube drainage. Air embolism is the most serious immediate complication and has a high mortality rate, although it is very rare. Delayed complications include infection and dissemination of cancer cells along the biopsy track. The latter complication is also exceedingly rare, with a reported frequency less than 0.04%, but the consequences can be devastating [<xref rid="B3" ref-type="bibr">3</xref>].</p><p>Factors involved in the likelihood of dissemination include thickness of the biopsy needle, number of punctures, distance between the tumor and chest wall, operator skill, tumor growth rate, and the patient's immune status [<xref rid="B4" ref-type="bibr">4</xref>]. Dissemination risk is also minimized by a coaxial technique that prevents contact between the biopsy needle and the chest wall.</p><p>In our case, an 18-gauge semi-automated biopsy needle was used twice (5 days apart). Dissemination occurred despite our use of a coaxial technique, which may be explained in our patient by the extensive contact of the lung consolidation with the anterior pleural surface of the right hemithorax, which could allow and facilitate direct dissemination of the infected tissue from the peripheral lung parenchyma to the chest wall through the small pleural defect created by the outer needle of the coaxial system. Since the pleural surface was punctured twice, this may have also increased the probability of chest pleural dissemination in our immunocompetent patient.</p><p><italic>P. micra</italic> is a nonspore-forming anaerobic gram-positive coccus widely distributed as normal flora in the oral mucosa. Predisposing conditions for this infection include immunodeficiency, diabetes, steroid treatment, previous surgery, and neoplasia. In these conditions, this bacteria can cause opportunistic infections in the central nervous system, thorax, abdomen, and pelvis, as well as generalized necrotizing soft tissue infections. Opportunistic infections caused by <italic>P. micra</italic> more commonly cause brain abscesses and endocarditis following dental manipulations. Within the thorax, <italic>P. micra</italic> can cause aspiration pneumonia, lung abscesses, empyema, and mediastinitis. Rarely, <italic>P. micra</italic> infections can also occur in patients with no recognized predisposing factors. According to various practice guidelines, such as those of the Infectious Diseases Society of America, anaerobic infections should be treated with either clindamycin or a combination of penicillin and a &#x003b2;-lactamase inhibitor for 2 to 4 weeks depending on response. In the case of an associated lung abscess, treatment is recommended for up to 3 months or until the chest radiograph clears, though treatment can be shortened with proper surgical drainage, as in our case [<xref rid="B2" ref-type="bibr">2</xref>].</p><p>The imaging findings of anerobic thoracic infections are not specific and may sometimes resemble lung carcinoma, as in our case. Common CT findings include lung consolidation, necrotic nodules and masses, and cavitation [<xref rid="B5" ref-type="bibr">5</xref>]. The chest wall is involved in a small number of patients. To the best of our knowledge, there are no previous reports describing chest wall dissemination of <italic>P. micra</italic> pulmonary infection following transthoracic lung biopsy.</p><p>In conclusion, we present the case of an immunocompetent patient who underwent two nondiagnostic transbronchial and CT-guided transthoracic needle biopsies due to suspicion of lung cancer. Ten days after the second percutaneous biopsy, the patient developed a chest wall mass that was ultimately confirmed to be a <italic>P. micra</italic> chest wall abscess.</p><sec><title>KEY MESSAGE</title><boxed-text position="float" orientation="portrait"><p> <list list-type="order"><list-item><p>Percutaneous lung biopsy</p></list-item><list-item><p>Computed tomography</p></list-item><list-item><p>Chest wall infection</p></list-item></list> </p></boxed-text></sec>
Crystals in a patient with asymptomatic proteinuria
Could not extract abstract
<contrib contrib-type="author"><name><surname>Lim</surname><given-names>Jin Han</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yhim</surname><given-names>Ho-Young</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Cha</surname><given-names>Eun Jung</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Moon Hyang</given-names></name><xref ref-type="aff" rid="A3">3</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kang</surname><given-names>Kyung Pyo</given-names></name><xref ref-type="aff" rid="A1">1</xref><xref ref-type="aff" rid="A2">2</xref></contrib>
The Korean Journal of Internal Medicine
<p>A 64-year-old male was referred to our hospital for evaluation of proteinuria. His medical history and physical examination were unremarkable. Laboratory findings revealed a white blood cell count of 4,900/mm<sup>3</sup>, hemoglobin level of 12.9 g/dL, platelet count of 157,000/mm<sup>3</sup>, blood urea nitrogen level of 18 mg/dL, and serum creatinine level of 0.77 mg/dL. Urinalysis showed 2+ proteinuria without hematuria. His 24-hour urine protein level was 920 mg/day. Serum protein electrophoresis showed a small M peak, and serum protein immunofixation showed monoclonal gammopathy with an immunoglobulin G (IgG) &#x003ba;-light chain. A bone marrow biopsy revealed normocellular marrow with minor monoclonal plasma cell infiltration (&#x0003c; 5%). The patient subsequently underwent a renal biopsy. Histologically, the size and cellularity of the glomeruli were normal with patent capillary lumina (<xref ref-type="fig" rid="F1">Fig. 1A</xref>). Immunofluorescence staining was positive for &#x003ba;-light chains in the tubular cytoplasm, but there was no staining for IgG, IgM, IgA, &#x003bb;-light chain, C3, or C1q (<xref ref-type="fig" rid="F1">Fig. 1B</xref>). Under electron microscopic examination, the proximal tubules contained sparsely scattered or abundantly packed rhomboid and rod-shaped medium electron-dense crystals in the cytoplasm with segmental loss of microvillus surfaces (<xref ref-type="fig" rid="F1">Fig. 1C</xref>). These findings are consistent with light chain proximal tubulopathy (LCPT) with crystal formation. Despite significant crystalline deposition in the proximal tubules, there was no evidence of Fanconi syndrome, and the patient had no metabolic or bone abnormalities. One year after diagnosis, the patient's hematologic and renal functions were stable without treatment.</p><p>Diagnosis of light chain crystal deposition may be challenging because the clinical and pathological manifestations are uncommon and poorly characterized, sometimes resulting in an incorrect or missed diagnosis. Although this patient presented with clinically asymptomatic proteinuria, a high index of suspicion was necessary to achieve an accurate diagnosis of LCPT without Fanconi syndrome. Diagnostic work-up for asymptomatic proteinuria should include protein electrophoresis and ultrastructural examination of renal biopsy specimens.</p>
Fibromuscular dysplasia: a cause of secondary hypertension
Could not extract abstract
<contrib contrib-type="author" corresp="yes"><name><surname>Shejul</surname><given-names>Yogesh Kashiram</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Viswanathan</surname><given-names>Muthu Krishnan</given-names></name><xref ref-type="aff" rid="A2">2</xref></contrib><contrib contrib-type="author"><name><surname>Jangale</surname><given-names>Prakash</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kulkarni</surname><given-names>Anjali</given-names></name><xref ref-type="aff" rid="A1">1</xref></contrib>
The Korean Journal of Internal Medicine
<p>A 38-year-old female presented to us with a history of fluctuating blood pressure. On physical examination, her blood pressure in the right arm was 180/110 mmHg. Physical examination revealed no other abnormalities. Her laboratory parameters were within normal limits. Renal artery duplex ultrasound was suggestive of significant stenosis in the left renal artery.</p><p>Subsequent renal angiography demonstrated a classical "string of beads" appearance of both the renal arteries (saccular dilatations and constrictions) that spared the ostium; this was suggestive of fibromuscular dysplasia. The left renal artery also exhibited short-segment web-like stenosis (70% to 80%) in the mid-segment with a significant gradient across it (<xref ref-type="fig" rid="F1">Fig. 1</xref>). Angioplasty of the stenotic segment was performed using an angioplasty balloon at a pressure of 10 atmospheres with good results (<xref ref-type="fig" rid="F2">Fig. 2</xref>). After angioplasty, the patient's blood pressure normalized and she was discharged without any antihypertensive medications. At the 6-month follow-up, her blood pressure remained normal and she required no antihypertensive medications.</p><p>Fibromuscular dysplasia is a noninflammatory, nonatherosclerotic vascular disease that commonly involves the renal and internal carotid arteries; however, it can involve any arterial bed. Renal fibromuscular dysplasia usually affects females from 15 to 50 years of age and accounts for around 10% of cases of renal artery stenosis. Renal fibromuscular dysplasia is classified based on the predominant arterial layer involved: intima, media, or adventitia. Involvement of the media is further subdivided into medial, perimedial, and hyperplastic medial fibromuscular dysplasia. The three most common and classically described subtypes include medial (70% of cases), perimedial (15% to 25%), and intimal fibrodysplasias (1% to 2%). The classical angiographic "string of beads" appearance is seen in medial and perimedial fibromuscular dysplasias. It is characterized by alternating stenoses and aneurysms and frequently involves the bilateral renal arteries.</p><p>The most accurate method for diagnosis of fibromuscular dysplasia remains catheter-based angiography.</p>
Comparison of the Growth of <italic>Lactobacillus delbrueckii</italic>, <italic>L. paracasei</italic> and <italic>L. plantarum</italic> on Inulin in Co-culture Systems
<p><italic>Lactobacillus delbrueckii</italic> TU-1, which apparently takes intact inulin into its cells and then degrades it intracellularly, was co-cultured <italic>in vitro</italic> with <italic>L.&#x000a0;paracasei</italic> KTN-5, an extracellular inulin degrader; or <italic>L.&#x000a0;plantarum</italic> 22A-3, a strain that is able to utilize fructose but not inulin; or both in order to prequalify inulin as a prebiotic agent <italic>in vivo</italic>. When <italic>L.&#x000a0;delbrueckii</italic> TU-1 was co-cultured with <italic>L.&#x000a0;paracasei</italic> KTN-5 on fructose or inulin, the growth of <italic>L.&#x000a0;delbrueckii</italic> TU-1 on inulin was markedly higher than that of <italic>L.&#x000a0;paracasei</italic> KTN-5, whereas the growth of <italic>L.&#x000a0;delbrueckii</italic> TU-1 on fructose was much lower than that of <italic>L.&#x000a0;paracasei</italic> KTN-5. These results suggest that <italic>L.&#x000a0;delbrueckii</italic> TU-1 and <italic>L.&#x000a0;paracasei</italic> KTN-5 were efficient at utilizing inulin and fructose, respectively. When <italic>L.&#x000a0;plantarum</italic> 22A-3 was co-cultured with <italic>L.&#x000a0;delbrueckii</italic> TU-1 on inulin, the growth of <italic>L.&#x000a0;plantarum</italic> 22A-3 was enhanced by <italic>L.&#x000a0;paracasei</italic> KTN-5 but not by <italic>L.&#x000a0;delbrueckii</italic> TU-1, suggesting that the fructose moiety that <italic>L.&#x000a0;paracasei</italic> KTN-5 released temporarily into the medium was &#x0201c;scavenged&#x0201d; by <italic>L.&#x000a0;plantarum</italic> 22A-3. Thus, <italic>L.&#x000a0;delbrueckii</italic> TU-1, <italic>L.&#x000a0;paracasei</italic> KTN-5, and <italic>L.&#x000a0;plantarum</italic> 22A-3 were then cultured altogether on inulin. The growth of <italic>L.&#x000a0;delbrueckii</italic> TU-1 was unaffected but that of <italic>L.&#x000a0;paracasei</italic> KTN-5 was markedly suppressed. This evidence suggests that prebiotic use of inulin supported the selective growth of intracellular inulin degraders such as <italic>L.&#x000a0;delbrueckii</italic> rather than extracellular inulin degraders such as <italic>L.&#x000a0;paracasei</italic> in the host microbiota.</p>
<contrib contrib-type="author"><name><surname>TAKAGI</surname><given-names>Risa</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>TSUJIKAWA</surname><given-names>Yuji</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>NOMOTO</surname><given-names>Ryohei</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>OSAWA</surname><given-names>Ro</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref rid="cor1" ref-type="corresp"><sup>*</sup></xref></contrib><aff id="aff1"><label>1</label>Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, 1&#x02013;1 Rokko-dai, Nada-ku, Kobe 657-8501, Japan</aff>
Bioscience of Microbiota, Food and Health
<sec sec-type="intro" id="s1"><title>INTRODUCTION</title><p>The <italic>Lactobacillus</italic> species are among the industrially important lactic acid bacteria, which are found in fermented food products and can provide many beneficial effects to their hosts as probiotic bacteria [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>]. For example, strains of <italic>L.&#x000a0;delbrueckii</italic> that are generally found in dairy products such as yogurt have been shown to have probiotic effects including antioxidative effects, immunostimulatory activity, and antitumor activity in both <italic>in vitro</italic> and <italic>in vivo</italic> studies [<xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r5" ref-type="bibr">5</xref>]. However, these beneficial effects may be transient because up to 100 trillion intestinal commensal microbes usually prevent probiotic bacteria from establishing themselves in the intestinal environment [<xref rid="r6" ref-type="bibr">6</xref>, <xref rid="r7" ref-type="bibr">7</xref>]. To overcome this limitation, the intake of prebiotics in addition to probiotic bacteria has been reported to be effective [<xref rid="r8" ref-type="bibr">8</xref>].</p><p>Prebiotics are nondigestible food ingredients that are not hydrolyzed by human digestive enzymes but are utilized by a limited number of bacteria in the colon and beneficially affect the host by improving the consistency of gastrointestinal microbiota [<xref rid="r9" ref-type="bibr">9</xref>, <xref rid="r10" ref-type="bibr">10</xref>]. The majority of candidate prebiotics are nondigestible oligosaccharides such as lactulose, raffinose, fructo-oligosaccharides, lactosucrose, xylo-oligosaccharides, and isomalto-oligosaccharides, but they also include highly polymerized saccharides such as resistant starch, pectin, and inulin [<xref rid="r10" ref-type="bibr">10</xref>]. In particular, the effects of prebiotics on inulin have been well studied. Inulin is a linear D-fructose polymer linked by beta-(2-1)-glycosidic bonds, with a terminal glucose moiety that is linked by an alpha-(1-2)-glycosidic bond [<xref rid="r11" ref-type="bibr">11</xref>]. The beta-(2-1)-glycosidic bond of inulin, including the first glucose-fructose bond, is not hydrolyzed to a great extent by any mammalian digestive enzymes [<xref rid="r12" ref-type="bibr">12</xref>]. Inulin extracted from chicory, which is a root vegetable, varies in the number of D-fructose units from a minimum of 2 to a maximum of 60. This relatively unprocessed inulin has a degree of polymerization (DP) ranging from 2 to approximately 60, and the average DP is approximately 12 [<xref rid="r11" ref-type="bibr">11</xref>].</p><p>Meanwhile, certain <italic>Lactobacillus</italic> strains, including strains of <italic>L.&#x000a0;paracasei</italic> and <italic>L.&#x000a0;delbrueckii</italic>, are able to grow in <italic>in vitro</italic> inulin monoculture systems [<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r15" ref-type="bibr">15</xref>]. Recently, Tsujikawa et al.&#x000a0;[<xref rid="r16" ref-type="bibr">16</xref>] provided evidence to suggest that <italic>L.&#x000a0;paracasei</italic> strains degrade inulin extracellularly to release free molecules of fructose and sucrose that can be subsequently transported into cells to be metabolized, whereas <italic>L.&#x000a0;delbrueckii</italic> strains take up intact inulin into cells and then degrade it intracellularly. If the above hypothesis is accurate, it would be interesting to see which species has an ecological advantage when both species are grown on inulin together. Furthermore, the growth of <italic>L.&#x000a0;paracasei</italic> rather than <italic>L.&#x000a0;delbrueckii</italic> may be suppressed by the presence of any fructose-utilizing concomitant bacteria. Therefore, in this study, we examined the ability of <italic>L.&#x000a0;delbrueckii</italic> to compete for inulin with <italic>L.&#x000a0;paracasei,</italic> an avid fructose-utilizing but not inulin-utilizing strain of <italic>L.&#x000a0;plantarum</italic> or both in co-culture systems.</p></sec><sec sec-type="materials|methods" id="s2"><title>MATERIALS AND METHODS</title><sec><title>Bacterial strains used</title><p>The bacterial strains used in this study were <italic>L.&#x000a0;delbrueckii</italic> TU-1, <italic>L.&#x000a0;paracasei</italic> KTN-5, and <italic>L.&#x000a0;plantarum</italic> 22A-3 (<xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Bacterial strains used in this study</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Strain</td><td align="center" rowspan="1" colspan="1">Origin</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>Lactobacillus delbrueckii</italic> TU-1</td><td align="left" rowspan="1" colspan="1">Yogurt commercially available in Japan</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Lactobacillus paracasei</italic> KTN-5</td><td align="left" rowspan="1" colspan="1">Fermented milk product commercially available in Japan</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Lactobacillus plantarum</italic> 22A-3</td><td align="left" rowspan="1" colspan="1">Nukaduke (rice bran pickling) of eggplants</td></tr></tbody></table></table-wrap>). It should be noted that these 3 <italic>Lactobacillus</italic> strains were isolated in our laboratory from dietary fermented food products that were commercially available in Japan and that their taxonomic identities were confirmed by our species-specific quantitative PCR assay, which will be described later. All bacterial isolates were stored in de Man-Rogosa-Sharpe (MRS) broth (Oxoid, Basingstoke, United Kingdom) at &#x02212;80&#x000b0;C until used.</p></sec><sec><title>Media and substrates used</title><p>Modified MRS medium [<xref rid="r17" ref-type="bibr">17</xref>], which was without glucose and supplemented with 0.5&#x02005;g/L L-cysteine hydrochloride (Wako Pure Chemical Industries, Osaka, Japan), hereafter referred to as the&#x02005;mMRS medium, was used as the basal fermentation medium throughout this study. The pH of the medium was adjusted to 6.5 before sterilization (121&#x000b0;C for 15&#x02005;min).</p><p>Glucose, fructose, or inulin was added to&#x02005;mMRS as the sole carbon source (2%, wt/vol). In all cases, these sugars were sterilized through membrane &#x0fb01;ltration using Millex<sup>&#x000ae;</sup> syringe filter units (pore size, 0.45 &#x003bc;m; Merck Millipore, Darmstadt, Germany) and were added aseptically to the sterile&#x02005;mMRS medium. All of these sugars were purchased from Wako (Osaka, Japan). The DP of inulin (Wako) is reported to vary between 3 and 60 according to the information supplied by the manufacturer.</p></sec><sec><title>Fermentation experiments</title><p>To perform mono- and co-culture fermentations, the strains were anaerobically subcultured twice in MRS broth, and the cultures obtained after 6 hr of anaerobic growth at 37&#x000b0;C were centrifuged at 4,000&#x02005;rpm for 15&#x02005;min. After centrifugation, the bacterial pellet was washed once with phosphate-buffered saline (PBS) [0.8% NaCl, 0.02% KH<sub>2</sub>PO<sub>4</sub>, 0.115% Na<sub>2</sub>HPO<sub>4</sub> (pH 7.4)] and resuspended in PBS until its copy number of the 16S rRNA gene in the bacterial genome reached 10<sup>8</sup> copies/ml, which was followed by the spotting of 50&#x02005;&#x000b5;L of this suspension on 5&#x02005;mL of MRS or&#x02005;mMRS containing glucose, fructose, or inulin (2%, wt/vol at final concentration). Incubations of the media were performed anaerobically at 37&#x000b0;C in an anaerobic jar (Mitsubishi Gas Chemical, Tokyo, Japan) for up to 48 hr during which approximately 500&#x02005;&#x000b5;L of the culture was withdrawn at 0, 3, 6, 12, 18, 24, and 48 hr after incubation. It should be noted that it was difficult to adjust the cell numbers of the initial inoculum of the <italic>L.&#x000a0;delbrueckii</italic> strain by the plate count method because its cells were tightly bound in a chain formation and could not be separated to single cells even by a rigorous vortexing to be spread on a plate. We thus adjusted the initial cell numbers of the stains to be equivalent to each other in terms of the copy number of the 16S rRNA genes.</p></sec><sec><title>DNA preparations</title><p>Whole genomic DNA from each culture was prepared essentially following the method of Marmur et al. [<xref rid="r18" ref-type="bibr">18</xref>]. Briefly, a 200 &#x003bc;l aliquot of each culture was transferred to sterile bead-beating tubes containing 300&#x02005;mg of glass beads (0.1&#x02005;mm). This was added to approximately 500&#x02005;&#x000b5;L of TE-saturated phenol, 250&#x02005;&#x000b5;L of lysis buffer, and 50&#x02005;&#x000b5;L of 10% sodium dodecyl sulfate. After centrifugation at 15,000&#x02005;rpm for 5&#x02005;min, the upper layer was removed to a new tube. Four hundred microliters of phenol:chloroform:isoamyl alcohol (25:24:1) was then added to the tube and centrifuged at 15,000&#x02005;rpm for 5&#x02005;min. The upper aqueous layer was carefully collected in a new tube. This sample was shaken in a FastPrep-24 Instrument (MP Biomedicals SARL, Illkirch, France) for 30&#x02005;s at maximum speed. DNA was precipitated by adding 275&#x02005;&#x000b5;L of isopropyl alcohol and a 1/10 volume of 3&#x02005;M sodium acetate, which had been placed in the tube at &#x02212;20&#x000b0;C for 10&#x02013;15&#x02005;min. The pellet was washed with 70% ice-cold ethanol by centrifugation at 15,000&#x02005;rpm for 5&#x02005;min, and DNA was dried under vacuum. DNA was subsequently dissolved again in TE buffer (10&#x02005;mM Tris-HCl, 1&#x02005;mM EDTA, pH 8.0).</p></sec><sec><title>Quantitative PCR (qPCR)</title><p>In order to quantify the growth of each strain in the co-cultures, we initially used the plate count method on our co-cultures but we found it extremely difficult to assign any colonies formed on the plates of mixed species co-cultures to the species by their appearance. Quantification of the <italic>L.&#x000a0;delbrueckii</italic>, <italic>L.&#x000a0;paracasei</italic>, and <italic>L.&#x000a0;plantarum</italic> 16S rRNA genes was therefore performed by quantitative PCR using a TP700 Thermal Cycler Dice Real Time System Lite (Takara, Ohtsu, Japan). Primer pairs targeting a partial 16S rRNA gene for <italic>L.&#x000a0;delbrueckii</italic>, <italic>L.&#x000a0;paracasei</italic>[<xref rid="r19" ref-type="bibr">19</xref>] and <italic>L.&#x000a0;plantarum</italic> were designed in this study and used for the qPCR assay (<xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title> Primers, amplicon sizes and standard bacterium used in quantitative PCR detection of target bacteria</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Target bacteria</td><td align="center" rowspan="1" colspan="1">Primer</td><td align="center" rowspan="1" colspan="1">Amplicon<break/>size (bp)</td><td align="center" rowspan="1" colspan="1">Standard bacterium</td><td align="center" rowspan="1" colspan="1">Reference</td></tr></thead><tbody><tr><td align="left" valign="top" rowspan="2" colspan="1"><italic>Lactobacillus delbrueckii</italic></td><td align="left" valign="top" rowspan="1" colspan="1">F: 5&#x02032;-GGRTGATTTGTTGGACGCTAG-3&#x02032;</td><td align="center" valign="top" rowspan="2" colspan="1">138</td><td align="center" valign="top" rowspan="2" colspan="1"><italic>Lactobacillus delbrueckii</italic> TU-1</td><td align="center" valign="top" rowspan="2" colspan="1">[<xref rid="r19" ref-type="bibr">19</xref>]</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">R: 5&#x02032;-GCCGCCTTTCAAACTTGAATC-3&#x02032;</td></tr><tr><td align="left" valign="top" rowspan="2" colspan="1"><italic>Lactobacillus paracasei</italic></td><td align="left" valign="top" rowspan="1" colspan="1">F: 5&#x02032;-GCACCGAGATTCAACATGG-3&#x02032;</td><td align="center" valign="top" rowspan="2" colspan="1">117</td><td align="center" valign="top" rowspan="2" colspan="1"><italic>Lactobacillus paracasei</italic> KTN-5</td><td align="center" valign="top" rowspan="2" colspan="1">[<xref rid="r19" ref-type="bibr">19</xref>]</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">R: 5&#x02032;-GGTTCTTGGATYTATGCGGTATTAG-3&#x02032;</td></tr><tr><td align="left" valign="top" rowspan="2" colspan="1"><italic>Lactobacillus plantarum</italic></td><td align="left" valign="top" rowspan="1" colspan="1">F: 5&#x02032;-TTTGAGTGAGTGGCGAACTG-3&#x02032;</td><td align="center" valign="top" rowspan="2" colspan="1">145</td><td align="center" valign="top" rowspan="2" colspan="1"><italic>Lactobacillus plantarum</italic> 22A-3</td><td align="center" valign="top" rowspan="2" colspan="1">This study</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">R: 5&#x02032;-CCAAAAGTGATAGCCGAAGC-3&#x02032;</td></tr></tbody></table></table-wrap>). The PCR conditions for <italic>L.&#x000a0;delbrueckii</italic> and <italic>L.&#x000a0;paracasei</italic> were as follows: 95&#x000b0;C for 10&#x02005;min and 40 cycles of 95&#x000b0;C for 15 sec and 60&#x000b0;C for 1&#x02005;min; those for <italic>L.&#x000a0;plantarum</italic> were as follows: 95&#x000b0;C for 1&#x02005;min and 40 cycles of 95&#x000b0;C for 5 sec and 60&#x000b0;C for 30 sec. To check the specificity of the amplifications, a melting curve was obtained by performing the following cycle: a denaturation step at 95&#x000b0;C for 15 sec, a 1&#x000b0;C increase in temperature every 20 sec starting at 60&#x000b0;C and ending at 95&#x000b0;C and a final step at 95&#x000b0;C for 15 sec. Standard curves for absolute quantification in the cultures were prepared using 10<sup>2</sup>&#x02013;10<sup>6</sup> copies of the PCR fragment of the 16S rRNA gene. The correlation coefficients for all the standard curves were above 0.99. For each assay, 1&#x02005;&#x000b5;L of DNA solution was added to 9&#x02005;&#x000b5;L of a PCR mixture containing 5 &#x003bc;lL of THUNDERBIRD<sup>TM</sup> SYBR<sup>&#x000ae;</sup> qPCR Mix (Toyobo, Osaka, Japan), 3.6&#x02005;&#x000b5;L of distilled water, and 200 nM of each primer.</p></sec><sec><title>Sugar degradation analysis by thin-layer chromatography (TLC)</title><p>To substantiate the observed difference in fructose and inulin utilization by the strains, TLC (Merck silica gel 60 plate) was performed on the spent cultures (approximately 2&#x02005;&#x000b5;L each) of <italic>L.&#x000a0;delbrueckii</italic> TU-1, <italic>L.&#x000a0;paracasei</italic> KTN-5, and <italic>L.&#x000a0;plantarum</italic> 22A-3 grown in&#x02005;mMRS containing 2% fructose or inulin for 24 hr, which were spotted along with solutions of fructose and inulin standard (2%, wt/vol) onto different lanes of a TLC plate. The plates were developed in 1-butanol:2-propanol:ethanol:water (3:2:3:4) solvent. The spots were visualized by spraying the plates with <italic>p</italic>-anisaldehyde (containing acetic acid and H<sub>2</sub>SO<sub>4</sub>) ethanol solution (Tokyo Chemical Industry, Tokyo, Japan) and heating them at 160&#x000b0;C for several minutes.</p></sec></sec><sec sec-type="results" id="s3"><title>RESULTS</title><sec><title>Monocultures of L.&#x000a0;delbrueckii TU-1, L.&#x000a0;paracasei KTN-5, and L.&#x000a0;plantarum 22A-3 in&#x02005;mMRS supplemented with glucose, fructose, or inulin</title><p>All 3 strains grew well on glucose or fructose (<xref ref-type="fig" rid="fig_001">Fig. 1A, 1B</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> Growth curves of the&#x02005;mMRS medium supplemented with a 2% (wt/vol) concentration of glucose (A), fructose (B), or inulin (C) and without the sugars (D).</p><p>&#x025cf;, Growth of <italic>L. delbrueckii</italic> TU-1; &#x025a1;, growth of <italic>L. paracasei</italic> KTN-5; &#x025b2;, growth of <italic>L. plantarum</italic> 22A-3. Each experiment was performed in triplicate.</p></caption><graphic xlink:href="bmfh-33-139-g001"/></fig>); in particular, the growth levels of <italic>L.&#x000a0;plantarum</italic> 22A-3 on both the sugars were higher than those of the other 2 strains. In addition, <italic>L.&#x000a0;paracasei</italic> KTN-5 and <italic>L.&#x000a0;plantarum</italic> 22A-3 reached a stationary phase in approximately 12 hr, whereas <italic>L.&#x000a0;delbrueckii</italic> TU-1 reached a stationary phase in approximately 24 hr (<xref ref-type="fig" rid="fig_001">Fig. 1A, 1B</xref>). <italic>L.&#x000a0;delbrueckii</italic> TU-1 grew faster on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>) than on glucose or fructose (<xref ref-type="fig" rid="fig_001">Fig. 1A, 1B</xref>), and the growth reached a stationary phase in approximately 6 hr; on the other hand, <italic>L.&#x000a0;paracasei</italic> KTN-5 (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>) showed a comparable growth pattern on glucose or fructose (<xref ref-type="fig" rid="fig_001">Fig. 1A, 1B</xref>). The growth level of <italic>L.&#x000a0;plantarum</italic> 22A-3 on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>) decreased to less than 1/10-fold of its level of growth on glucose or fructose (<xref ref-type="fig" rid="fig_001">Fig. 1A, 1B</xref>) and was comparable to that of its growth without the sugars (<xref ref-type="fig" rid="fig_001">Fig. 1D</xref>).</p><p>In the TLC analysis, a dark oval-shaped fructose spot was seen in the upper part of the chromatograph in&#x02005;mMRS with fructose that was not incubated with any strain (<xref ref-type="fig" rid="fig_002">Fig. 2A and 2B</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Thin-layer chromatography analysis of the sugar compositions of the spent cultures of the <italic>L. delbrueckii</italic> TU-1, <italic>L. paracasei</italic> KTN-5, and <italic>L. plantarum</italic> 22A-3 in the&#x02005;mMRS medium supplemented with a 2% (wt/vol) concentration of fructose (A) or inulin (B) after 24 hr of incubation.</p><p>Strains were grown and harvested as follows: (A) Lane 1, fructose (2%, wt/vol); lane 2, <italic>L. delbrueckii</italic> TU-1; lane 3, <italic>L. paracasei</italic> KTN-5; and lane 4, <italic>L. plantarum</italic> 22A-3. (B) Lane 1, fructose (2%, wt/vol); lane 2, <italic>L. delbrueckii</italic> TU-1; lane 3, <italic>L. paracasei</italic> KTN-5; lane 4, <italic>L. plantarum</italic> 22A-3; and lane 5, inulin (2%, wt/vol).</p></caption><graphic xlink:href="bmfh-33-139-g002"/></fig>, lane 1). The spent culture of each of the 3 strains grown in&#x02005;mMRS containing 2% fructose for 24 hr (<xref ref-type="fig" rid="fig_002">Fig. 2A</xref>, lane 2) showed fructose spots. However, the darkness intensity of <italic>L.&#x000a0;delbrueckii</italic> TU-1 was slightly less than that of the reference fructose spot, whereas those of <italic>L.&#x000a0;paracasei</italic> KTN-5 and <italic>L.&#x000a0;plantarum</italic> 22A-3 (<xref ref-type="fig" rid="fig_002">Fig. 2A</xref>, lanes 3 and 4) were much less than that of the reference fructose spot (<xref ref-type="fig" rid="fig_002">Fig. 2A</xref>, lane 1).&#x02005;mMRS with inulin that was not incubated with any strain (<xref ref-type="fig" rid="fig_002">Fig. 2B</xref>, lane 5) showed a markedly extended spot covering a wide area and a dark oval-shaped non-migrating inulin spot far below the fructose spot. The spent culture of <italic>L.&#x000a0;delbrueckii</italic> TU-1 grown in&#x02005;mMRS containing 2% inulin for 24 hr (<xref ref-type="fig" rid="fig_002">Fig. 2B</xref>, lanes 2) showed an inulin spot, but the darkness intensity decreased. The spent culture of <italic>L.&#x000a0;paracasei</italic> KTN-5 grown in&#x02005;mMRS containing 2% inulin for 24 hr (<xref ref-type="fig" rid="fig_002">Fig. 2B</xref>, lane 3) showed an inulin spot with a decreased darkness intensity as well as a dark oval-shaped spot in the upper part of the chromatograph. However, the spent culture of <italic>L.&#x000a0;plantarum</italic> 22A-3 inoculated in&#x02005;mMRS containing 2% inulin for 24 hr (<xref ref-type="fig" rid="fig_002">Fig. 2B</xref>, lane 4) showed an intact inulin spot.</p></sec><sec><title>Co-culture of L.&#x000a0;delbrueckii TU-1 and L.&#x000a0;paracasei KTN-5 in&#x02005;mMRS supplemented with fructose or inulin</title><p>In the co-culture of <italic>L.&#x000a0;delbrueckii</italic> TU-1 and <italic>L.&#x000a0;paracasei</italic> KTN-5 on fructose (<xref ref-type="fig" rid="fig_003">Fig. 3A</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p> Growth curves of a co-culture of <italic>L. delbrueckii</italic> TU-1 and <italic>L. paracasei</italic> KTN-5 in the&#x02005;mMRS medium with a 2% (wt/vol) concentration of fructose (A) or inulin (B).</p><p>&#x025cf;, Growth of <italic>L. delbrueckii</italic> TU-1; &#x025a1;, growth of <italic>L. paracasei</italic> KTN-5. Each experiment was performed in triplicate.</p></caption><graphic xlink:href="bmfh-33-139-g003"/></fig>), <italic>L.&#x000a0;delbrueckii</italic> TU-1 did not grow well, and the growth was suppressed compared with that in the monoculture on fructose (<xref ref-type="fig" rid="fig_001">Fig. 1B</xref>) but was comparable with that without the sugars (<xref ref-type="fig" rid="fig_001">Fig. 1D</xref>); on the other hand, <italic>L.&#x000a0;paracasei</italic> KTN-5 showed a growth pattern comparable with that in the monoculture on fructose (<xref ref-type="fig" rid="fig_001">Fig. 1B</xref>). In contrast, in the co-culture on inulin (<xref ref-type="fig" rid="fig_003">Fig. 3B</xref>), <italic>L.&#x000a0;delbrueckii</italic> TU-1 showed a comparable growth pattern to that of its monoculture on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>), whereas <italic>L.&#x000a0;paracasei</italic> KTN-5 did not grow well; its growth was slower than that in monoculture on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>).</p></sec><sec><title>Co-cultures of L.&#x000a0;plantarum 22A-3 with L.&#x000a0;delbrueckii TU-1 or with L.&#x000a0;paracasei KTN-5 in&#x02005;mMRS supplemented with inulin</title><p>In the co-culture of <italic>L.&#x000a0;plantarum</italic> 22A-3 and <italic>L.&#x000a0;delbrueckii</italic> TU-1 on inulin (<xref ref-type="fig" rid="fig_004">Fig. 4A</xref><fig orientation="portrait" fig-type="figure" id="fig_004" position="float"><label>Fig. 4.</label><caption><p> Growth curves of a co-culture of <italic>L. plantarum</italic> 22A-3 with <italic>L. delbrueckii</italic> TU-1 (A) or with <italic>L. paracasei</italic> KTN-5 (B) in the&#x02005;mMRS medium with a 2% (wt/vol) concentration of inulin.</p><p>&#x025cf;, Growth of <italic>L. delbrueckii</italic> TU-1; &#x025a1;, growth of <italic>L. paracasei</italic> KTN-5; &#x025b2;, growth of <italic>L. plantarum</italic> 22A-3. Each experiment was performed in triplicate.</p></caption><graphic xlink:href="bmfh-33-139-g004"/></fig>), <italic>L.&#x000a0;plantarum</italic> 22A-3 did not grow well, and the growth level was comparable with that of monoculture on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>) as well as without the sugars (<xref ref-type="fig" rid="fig_001">Fig. 1D</xref>). <italic>L.&#x000a0;delbrueckii</italic> TU-1 grew well and showed a growth almost comparable with that in monoculture on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>). In the co-culture of <italic>L.&#x000a0;plantarum</italic> 22A-3 and <italic>L.&#x000a0;paracasei</italic> KTN-5 (<xref ref-type="fig" rid="fig_004">Fig. 4B</xref>), the growth level of <italic>L.&#x000a0;plantarum</italic> 22A-3 was much higher than in monoculture on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>) and was comparable with that of monoculture on fructose (<xref ref-type="fig" rid="fig_001">Fig. 1B</xref>); on the other hand, the growth level of <italic>L.&#x000a0;paracasei</italic> KTN-5 decreased to less than 1/10-fold its growth when monocultured on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>).</p></sec><sec><title>Co-culture of L.&#x000a0;delbrueckii TU-1, L.&#x000a0;paracasei KTN-5, and L.&#x000a0;plantarum 22A-3 in&#x02005;mMRS supplemented with inulin</title><p>In the triple co-culture of <italic>L.&#x000a0;delbrueckii</italic> TU-1, <italic>L.&#x000a0;paracasei</italic> KTN-5, and <italic>L.&#x000a0;plantarum</italic> 22A-3 on inulin (<xref ref-type="fig" rid="fig_005">Fig. 5</xref><fig orientation="portrait" fig-type="figure" id="fig_005" position="float"><label>Fig. 5.</label><caption><p> Growth curve of a co-culture of <italic>L. delbrueckii</italic> TU-1, <italic>L. paracasei</italic> KTN-5, and <italic>L. plantarum</italic> 22A-3 in the&#x02005;mMRS medium with a 2% (wt/vol) concentration of inulin.</p><p>&#x025cf;, Growth of <italic>L. delbrueckii</italic> TU-1; &#x025a1;, growth of <italic>L. paracasei</italic> KTN-5; &#x025b2;, growth of <italic>L. plantarum</italic> 22A-3. Each experiment was performed in triplicate.</p></caption><graphic xlink:href="bmfh-33-139-g005"/></fig>), <italic>L.&#x000a0;delbrueckii</italic> TU-1 grew well, but the growth level was slightly decreased compared with its growth when monocultured on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>) as well as when co-cultured with <italic>L.&#x000a0;paracasei</italic> KTN-5 or <italic>L.&#x000a0;plantarum</italic> 22A-3 on inulin (<xref ref-type="fig" rid="fig_003">Fig. 3B, 4A</xref>). The growth of <italic>L.&#x000a0;paracasei</italic> KTN-5 (<xref ref-type="fig" rid="fig_005">Fig. 5</xref>) decreased markedly to less than 1/10-fold of its growth in the monoculture on inulin (<xref ref-type="fig" rid="fig_001">Fig. 1C</xref>), and it showed a growth pattern comparable with that when co-cultured with <italic>L.&#x000a0;plantarum</italic> 22A-3 on inulin (<xref ref-type="fig" rid="fig_004">Fig. 4B</xref>).</p></sec></sec><sec sec-type="discussion" id="s4"><title>DISCUSSION</title><p>Our TLC analysis confirmed that <italic>L.&#x000a0;paracasei</italic> KTN-5 degraded inulin to release free molecules of fructose extracellularly as reported by Tsujikawa et al. [<xref rid="r16" ref-type="bibr">16</xref>]. By contrast, <italic>L.&#x000a0;delbrueckii</italic> TU-1 did not release any fructose molecules in the culture medium, suggesting that the strain is capable of taking up intact inulin into its cells and subsequently degrading it intracellularly. Therefore, we speculated that <italic>L.&#x000a0;paracasei</italic> had to take 2 steps to utilize inulin, an initial step of degrading inulin to fructose and a subsequent step of transporting the released fructose molecules inside of its cells, while <italic>L.&#x000a0;delbrueckii</italic> only took one step to transport the entire inulin molecule into its cells. This in turn suggested that <italic>L.&#x000a0;delbrueckii</italic> could utilize inulin more readily than <italic>L.&#x000a0;paracasei.</italic> This was realized in our co-culture experiments of <italic>L.&#x000a0;delbrueckii</italic> TU-1 and <italic>L.&#x000a0;paracasei</italic> KTN-5, in which the growth of <italic>L.&#x000a0;delbrueckii</italic> TU-1 on inulin was markedly higher than that of <italic>L.&#x000a0;paracasei</italic> KTN-5, whereas that on fructose was much lower than that of <italic>L.&#x000a0;paracasei</italic> KTN-5, suggesting that <italic>L.&#x000a0;delbrueckii</italic> TU-1 was more efficient at utilizing inulin and that <italic>L.&#x000a0;paracasei</italic> KTN-5 was more efficient at utilizing fructose. This was also confirmed by our TLC analysis on fructose and inulin. Meanwhile, our TLC analysis also indicated that <italic>L.&#x000a0;plantarum</italic> 22A-3 could utilize fructose but not inulin.</p><p>To investigate the influence of <italic>L.&#x000a0;plantarum</italic> 22A-3 on the growth of <italic>L.&#x000a0;paracasei</italic> KTN-5 or <italic>L.&#x000a0;delbrueckii</italic> TU-1, we co-cultured <italic>L.&#x000a0;plantarum</italic> 22A-3 with <italic>L.&#x000a0;paracasei</italic> KTN-5 or <italic>L.&#x000a0;delbrueckii</italic> TU-1. In the co-culture experiment of <italic>L.&#x000a0;plantarum</italic> 22A-3 with <italic>L.&#x000a0;paracasei</italic> KTN-5 on inulin, <italic>L.&#x000a0;plantarum</italic> 22A-3 grew as well as in monoculture on fructose. However, <italic>L.&#x000a0;plantarum</italic> 22A-3 was unable to utilize inulin, and the growth of <italic>L.&#x000a0;paracasei</italic> KTN-5 greatly decreased when compared with monoculture on inulin or co-culture with <italic>L.&#x000a0;delbrueckii</italic> TU-1 on inulin. This suggests that <italic>L.&#x000a0;paracasei</italic> KTN-5 released fructose into the medium following extracellular inulin degradation and that <italic>L.&#x000a0;plantarum</italic> 22A-3 then used the released fructose; <italic>L.&#x000a0;plantarum</italic> 22A-3 was &#x0201c;scavenging&#x0201d; the free fructose molecules that were produced by <italic>L.&#x000a0;paracasei</italic> KTN-5, which were accumulating temporarily outside its cells. A similar finding was reported in a co-culture study in which <italic>Bacteroides thetaiotaomicron</italic> degraded inulin and released free fructose into the medium, thereby promoting concomitant bifidobacteria that were not able to degrade inulin themselves [<xref rid="r20" ref-type="bibr">20</xref>]. Meanwhile, in the co-culture experiment of <italic>L.&#x000a0;plantarum</italic> 22A-3 with <italic>L.&#x000a0;delbrueckii</italic> TU-1 on inulin, the growth of <italic>L.&#x000a0;delbrueckii</italic> TU-1 was comparable to its growth when monocultured on inulin, and that of <italic>L.&#x000a0;plantarum</italic> 22A-3 was comparable to its growth when monocultured without the sugars. This evidence suggests that <italic>L.&#x000a0;delbrueckii</italic> TU-1 did not produce extracellular fructose from inulin; thus, <italic>L.&#x000a0;plantarum</italic> 22A-3 could not &#x0201c;exploit&#x0201d; <italic>L.&#x000a0;delbrueckii</italic> TU-1 for free fructose. A similar case was observed in a co-culture study of <italic>Roseburia inulinivorans</italic> and <italic>Bifidobacterium longum</italic> on inulin in which an inulin-degrading <italic>R. inulinivorans</italic> strain did not promote the growth of a non-inulin-degrading <italic>B. longum</italic> strain [<xref rid="r21" ref-type="bibr">21</xref>].</p><p>In the triple co-culture experiment of <italic>L.&#x000a0;delbrueckii</italic> TU-1, <italic>L.&#x000a0;paracasei</italic> KTN-5, and <italic>L.&#x000a0;plantarum</italic> 22A-3 on inulin, the growth of <italic>L.&#x000a0;delbrueckii</italic> TU-1 was relatively unaffected by the presence of <italic>L.&#x000a0;plantarum</italic> 22A-3, but that of <italic>L.&#x000a0;paracasei</italic> KTN-5 was markedly suppressed. These results suggest that even in the presence of fructose-utilizing concomitant bacteria, <italic>L.&#x000a0;delbrueckii</italic> TU-1 had an ecological advantage as compared with <italic>L.&#x000a0;paracasei</italic> KTN-5 on inulin. In an actual intestinal environment, monosaccharides such as glucose and fructose are subject to fermentation by a large number of intestinal bacteria [<xref rid="r22" ref-type="bibr">22</xref>], and the competition with concomitant intestinal bacteria for such sugars would be therefore more pronounced than what we observed in the co-culture experiment. Based on this, we can speculate that the prebiotic use of inulin supports the selective growth of intracellular inulin degraders such as <italic>L.&#x000a0;delbrueckii</italic> but not that of extracellular inulin degraders such as <italic>L.&#x000a0;paracasei</italic>.</p><p>In various studies that have used <italic>in vitro</italic> gut simulation models and clinical <italic>in vivo</italic> trials with human volunteers, it has been demonstrated that inulin has the ability to selectively increase the number of <italic>Bifidobacterium</italic> spp. and <italic>Lactobacillus</italic> spp. [<xref rid="r23" ref-type="bibr">23</xref>,<xref rid="r24" ref-type="bibr">24</xref>,<xref rid="r25" ref-type="bibr">25</xref>], including species of <italic>B. adolescentis</italic> and <italic>L.&#x000a0;gasseri</italic>[<xref rid="r26" ref-type="bibr">26</xref>, <xref rid="r27" ref-type="bibr">27</xref>]. In addition, it is well known that inulin utilization by lactic acid bacteria is species- and even strain-dependent [<xref rid="r28" ref-type="bibr">28</xref>]. One of the explanations for this species specificity may be that these strains of the species, the growth of which is promoted by inulin have an ability to degrade inulin intracellularly rather than extracellularly. To evaluate the possibility that the growth of intracellular inulin-degrading bacteria such as <italic>L.&#x000a0;delbrueckii</italic> rather than extracellular inulin-degrading bacteria such as <italic>L.&#x000a0;paracasei</italic> is promoted when inulin is administered as a prebiotic supplement, further studies that use <italic>in vitro</italic> gut simulation models and <italic>in vivo</italic> trials are needed.</p></sec>
Beneficial Effects of Citrus Juice Fermented with <italic>Lactobacillus plantarum</italic> YIT 0132 on Japanese Cedar Pollinosis
<p>Recently, the prevalence of allergies in Japan has been increasing. Certain types of fruit juice and lactic acid bacteria are known to alleviate allergic symptoms. Therefore, we examined whether citrus juice fermented by a specific lactic acid bacteria can improve the symptoms of Japanese cedar pollinosis (JCPsis). <italic>Lactobacillus plantarum</italic> YIT 0132 (LP0132) was selected based on its high proliferative activity in citrus juice and anti-inflammatory interleukin-10-inducing activity. Dietary administration of heat-killed LP0132 cells or citrus juice fermented with LP0132 was found to significantly suppress nasal rubbing in a JCPsis mouse model, indicating relief of allergy symptoms. To evaluate the effects of LP0132-fermented citrus juice on pollinosis symptoms and quality of life (QOL) in humans with JCPsis, a single-blind, placebo-controlled, parallel-group clinical trial was conducted. The participants were 42 adults with JCPsis. They ingested 100&#x02005;mL of sterilized LP0132-fermented citrus juice (active group) or unfermented citrus juice (placebo group) once daily for 8 weeks. Immediately after the pollen peak when allergy symptoms and QOL loss were most severe, itchy eyes, itchy skin, and QOL loss by JCPsis were alleviated in the active group compared with the placebo group. At 10 weeks after starting the intervention, increased the levels of blood eosinophils were significantly suppressed in the active group compared with the placebo group. We conclude that continuous ingestion of citrus juice fermented with LP0132 may help alleviate the allergy symptoms and impaired QOL caused by JCPsis.</p>
<contrib contrib-type="author"><name><surname>HARIMA-MIZUSAWA</surname><given-names>Naomi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref rid="cor1" ref-type="corresp"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>IINO</surname><given-names>Tohru</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>ONODERA-MASUOKA</surname><given-names>Norie</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>KATO-NAGAOKA</surname><given-names>Noriko</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>KIYOSHIMA-SHIBATA</surname><given-names>Junko</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>GOMI</surname><given-names>Atsushi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>SHIBAHARA-SONE</surname><given-names>Harue</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>KANO</surname><given-names>Mitsuyoshi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>SHIDA</surname><given-names>Kan</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>SAKAI</surname><given-names>Masashi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>MIYAZAKI</surname><given-names>Kouji</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>ISHIKAWA</surname><given-names>Fumiyasu</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1</label>Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan</aff>
Bioscience of Microbiota, Food and Health
<sec sec-type="intro" id="s1"><title>INTRODUCTION</title><p>Japanese cedar pollinosis (JCPsis) is an allergic reaction to the pollen of the Japanese cedar (<italic>Cryptomeria japonica</italic>) and is the most common type of allergic rhinitis in Japan. Individuals with pollinosis experience sneezing, runny nose, blocked nose, and itchy eyes every spring. Those with severe symptoms frequently develop asthma or bronchitis as complications. According to epidemiological studies, the number of people in Japan with JCPsis was estimated to be about 30 million (26.5% of the Japanese population) in 2008 [<xref rid="r1" ref-type="bibr">1</xref>]. JCPsis has therefore been called the national disease of Japan. JCPsis reduces quality of life (QOL), and the cost of treatment with antihistamines, steroids, and other drugs is high. Furthermore, long-term administration of these drugs frequently causes side effects. Thus, ways to prevent or treat pollen allergies without drugs are urgently required.</p><p>The hygiene hypothesis states that the lack of exposure to infectious agents, symbiotic microorganisms, or parasites in early childhood increases susceptibility to allergic diseases [<xref rid="r2" ref-type="bibr">2</xref>]. According to this theory, the immunomodulatory effects of microbial contact in the intestine are important. By this reasoning, the involvement of lactic acid bacteria (LAB) in allergic diseases has recently received considerable interest. In fact, many clinical trials have been conducted to examine the effects of LAB on allergic diseases, and evidence for the health benefits of fermented milk and bacterial cells with respect to JCPsis has been accumulating [<xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r6" ref-type="bibr">6</xref>,<xref rid="r7" ref-type="bibr">7</xref>,<xref rid="r8" ref-type="bibr">8</xref>,<xref rid="r9" ref-type="bibr">9</xref>]. LAB with anti-allergy effects often induce the release of cytokines from immunocytes. For example, a strain of <italic>Lactobacillus pentosus</italic> has been reported to be a highly potent interleukin (IL)-10 inducer that produces an anti-allergic effect <italic>in vivo</italic>[<xref rid="r10" ref-type="bibr">10</xref>].</p><p>Some types of herbs and natural herbal products have been demonstrated to have anti-allergic potential. An example of this is hesperidin, a flavanone glycoside found abundantly in citrus fruits and known for beneficial effects such as anti-inflammatory activity, inhibition of degranulation of mast cells, relief of edema, and prevention of anaphylaxis [<xref rid="r11" ref-type="bibr">11</xref>,<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>]. In this study, we selected a strain of LAB showing both high proliferative activity in citrus juice and anti-allergic activity in an animal experiment, and conducted a clinical trial to examine the beneficial effects of citrus juice fermented with the selected LAB on allergy symptoms in individuals with JCPsis.</p></sec><sec sec-type="materials|methods" id="s2"><title>MATERIALS AND METHODS</title><sec><title>Bacterial culture</title><p>About 100 strains of LAB isolated from various traditional fermented foods or the environment were obtained from the culture collection of Yakult Central Institute for Microbiological Research (YIT; Tokyo, Japan).</p></sec><sec><title>Proliferative activity in citrus juice</title><p>LAB were pre-cultured in <italic>Lactobacilli</italic> MRS Broth (Difco<sup>TM</sup>, Becton Dickinson and Company, Detroit, MI, USA) at 37&#x000b0;C for 16 hr. Then, 40&#x02005;&#x000b5;L of the pre-culture containing almost 4&#x000d7;10<sup>5</sup> viable cells was inoculated into 10&#x02005;mL of 100% reconstituted citrus juice (Yakult Honsha Co., Ltd., Tokyo, Japan) and incubated at 37&#x000b0;C for 48 hr. Viable cells were counted by the agar plate method using <italic>Lactobacilli</italic> MRS Broth and expressed in colony-forming units (CFU)/ml.</p></sec><sec><title>Heat-killed LAB cells</title><p>LAB were cultured at 37&#x000b0;C for 48 hr in <italic>Lactobacilli</italic> MRS Broth, washed with sterilized water, killed by heating at 100&#x000b0;C for 30&#x02005;min and then lyophilized to prepare a powder of heat-killed cells.</p></sec><sec><title>IL-10- and IL-12-inducing activities of LAB in peritoneal macrophage culture</title><p>IL-10- and IL-12-inducing activities of LAB in peritoneal macrophages were evaluated according to a previously reported method [<xref rid="r14" ref-type="bibr">14</xref>]. Briefly, peritoneal macrophages were prepared from female BALB/c mice (8&#x02013;12 weeks old, Japan SLC, Shizuoka, Japan) 4 days after intraperitoneal injection of 4% thioglycollate broth (Difco<sup>TM</sup>, Becton Dickinson and Company, Detroit, MI, USA). Mouse peritoneal macrophages (2&#x000d7;10<sup>5</sup> cells/200&#x02005;&#x000b5;L/well) were incubated at 37&#x000b0;C for 24 hr in RPMI#1640 medium (Gibco<sup>&#x000ae;</sup>, Life Technologies, Carlsbad, CA, USA) containing 10% fetal calf serum together with heat-killed LAB cells (10 &#x000b5;g/ml). The concentrations of IL-10 and IL-12p70 in the co-culture supernatant were determined by enzyme-linked immunosorbent assay according to the method of Kaji et&#x000a0;al [<xref rid="r15" ref-type="bibr">15</xref>].</p></sec><sec><title>LP0132-fermented citrus juice</title><p>Valencia orange juice (Cutrale Co., Ltd., Araraquara, Brazil) was fermented with LP0132 at 37&#x000b0;C for 48 hr. This LP0132-fermented citrus juice was lyophilized for the animal experiment or sterilized for the clinical trial by heating at 90&#x000b0;C for 5&#x02005;min. The sterilized LP0132-fermented citrus juice was found to contain LP0132 cells at a concentration of approximately 8&#x000d7;10<sup>10</sup> cells/100&#x02005;mL using the DAPI counting method [<xref rid="r16" ref-type="bibr">16</xref>].</p></sec><sec><title>Anti-allergic effect in JCPsis mouse model</title><p>Anti-allergy effects were examined in a JCPsis mouse model according to the method of Yamamoto et al [<xref rid="r17" ref-type="bibr">17</xref>]. The experimental protocol is shown in <xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> Protocol for the animal experiment to evaluate anti-allergic effect of fermented citrus juice.</p></caption><graphic xlink:href="bmfh-33-147-g001"/></fig>. Female B6D2F1 mice (4 weeks old) were purchased from Charles River Laboratories Japan (Kanagawa, Japan) and fed a commercial non-purified diet (Oriental MF; Oriental Yeast Co., Ltd., Tokyo, Japan) during the habituation period for 1 week. Then the mice were assigned to 6 groups (blank, control, anti-histamine, unfermented juice, fermented juice, and killed cells) so that average body weight was similar between the groups. Mice were housed (4 mice per cage) in an air-conditioned room (at 25 &#x000b1; 1&#x000b0;C, with 12-hr light-dark cycle: lights on from 08:30 to 20:30, humidity 60 &#x000b1; 5%) with access to food and water <italic>ad libitum</italic>. Mice in the blank, control, and anti-histamine groups were fed a basal diet (AIN-76 containing 50&#x02005;g sucrose/100&#x02005;g; Oriental Yeast Co., Ltd), and those in the unfermented juice, fermented juice, and killed cells groups were fed a test diet for 45 days. The test diet for the unfermented and fermented juice groups was prepared by replacing 3.75&#x02005;g of sucrose in 100&#x02005;g of AIN-76 diet with the same amount of the lyophilized powder of raw Valencia orange juice or LP0132-fermented citrus juice, respectively. The diet for the fermented juice group contained approximately 5.3&#x000d7;10<sup>8</sup> CFU/kg of LP0132. The test diet for the killed cells group was prepared by mixing an AIN-76 diet with the heat-killed LP0132 cells at a concentration of 0.3&#x02005;mg powder/g, corresponding to about 6.6&#x000d7;10<sup>8</sup> cells/g, before heat killing.</p><p>As shown in <xref ref-type="fig" rid="fig_001">Fig. 1</xref> and <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Treatment conditions for the animal experiment to evaluate anti-allergy effects of fermented citrus juice</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Group</td><td align="center" rowspan="1" colspan="1">Sensitization <sup>a)</sup></td><td align="center" rowspan="1" colspan="1">Nasal injection</td><td align="center" rowspan="1" colspan="1">Loratadine administration <sup>d)</sup></td><td align="center" rowspan="1" colspan="1">Number of mice</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Blank</td><td align="center" rowspan="1" colspan="1">Done</td><td align="center" rowspan="1" colspan="1">Saline <sup>b)</sup></td><td align="center" rowspan="1" colspan="1">Not done</td><td align="center" rowspan="1" colspan="1">4</td></tr><tr><td align="left" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">Done</td><td align="center" rowspan="1" colspan="1">JCP extract <sup>c)</sup></td><td align="center" rowspan="1" colspan="1">Not done</td><td align="center" rowspan="1" colspan="1">12</td></tr><tr><td align="left" rowspan="1" colspan="1">Anti-histamine</td><td align="center" rowspan="1" colspan="1">Done</td><td align="center" rowspan="1" colspan="1">JCP extract <sup>c)</sup></td><td align="center" rowspan="1" colspan="1">Done</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td align="left" rowspan="1" colspan="1">Unfermented juice</td><td align="center" rowspan="1" colspan="1">Done</td><td align="center" rowspan="1" colspan="1">JCP extract <sup>c)</sup></td><td align="center" rowspan="1" colspan="1">Not done</td><td align="center" rowspan="1" colspan="1">12</td></tr><tr><td align="left" rowspan="1" colspan="1">Fermented juice</td><td align="center" rowspan="1" colspan="1">Done</td><td align="center" rowspan="1" colspan="1">JCP extract <sup>c)</sup></td><td align="center" rowspan="1" colspan="1">Not done</td><td align="center" rowspan="1" colspan="1">12</td></tr><tr><td align="left" rowspan="1" colspan="1">Killed cells</td><td align="center" rowspan="1" colspan="1">Done</td><td align="center" rowspan="1" colspan="1">JCP extract <sup>c)</sup></td><td align="center" rowspan="1" colspan="1">Not done</td><td align="center" rowspan="1" colspan="1">8</td></tr></tbody></table><table-wrap-foot><p><sup>a)</sup> Sensitized once a week for 3 weeks by intraperitoneal injection of Japanese cedar pollen (JCP) extract (<italic>Cryptomeria japonica</italic>) with adjuvant.</p><p><sup>b)</sup> Intranasally injected with saline once daily for 3 days.</p><p><sup>c)</sup> Intranasally challenged with a solution of Japanese cedar pollen extract once daily for 3 days.</p><p><sup>d)</sup> Oral administration of 0.2&#x02005;mg of loratadine 1 hr before intranasal challenge.</p></table-wrap-foot></table-wrap>, all mice were sensitized once a week for 3 weeks by intraperitoneal injection of 10 &#x000b5;g of Japanese cedar pollen (JCP) extract-Cj (LsL Co., Ltd., Osaka, Japan) with 2.0&#x02005;mg of alum adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Two weeks after the third sensitization, all mice except those in the blank group were intranasally challenged with 10&#x02005;&#x000b5;L of 0.1% solution of JCP extract; those in the blank group were injected with the equivalent amount of saline. The intranasal challenge (both nasal cavities) was performed once daily for 3 consecutive days, and the number of nasal rubbing events during 10&#x02005;min was counted immediately after the last challenge. In the anti-histamine group, loratadine (Claritin<sup>TM</sup>; Merck &#x00026; Co., Inc., Whitehouse Station, NJ, USA), a second-generation H<sub>1</sub> histamine antagonist drug, was administered at a dose of 0.2&#x02005;mg/mouse 1 hr before each challenge.</p><p>All animal experiments were conducted in accordance with the guidelines of the Animal Studies Committee of Yakult Central Institute for Microbiological Research (Tokyo, Japan).</p></sec><sec><title>Clinical trial</title><p>The participants enrolled in the clinical trial were 42 adults with JCPsis (average age, 38.2 &#x000b1; 10.2&#x02005;years) living in or near Tachikawa City, Tokyo, Japan. The exclusion criteria were food allergy, pregnant or lactating, and planning to become pregnant. Prior to enrollment, the purpose and protocol of the trial were fully explained to all the participants, after which signed informed consent was obtained. Throughout the test period, intake of beverages containing LAB or bifidobacteria; food containing LAB such as kimchi, cheese, and pickles; all citrus fruits except for grapefruit; and foods with anti-pollinosis potential were restricted. The use of eye drops, nose drops, and anti-pollinosis drugs and hospital visits were not restricted but were recorded in a diary. The protocol was approved by the Human Studies Committee of Yakult Central Institute for Microbiological Research (Tokyo, Japan), in accordance with the Declaration of Helsinki and the Committee&#x02019;s own guidelines.</p><p>A single-blind, placebo-controlled, parallel-group trial was conducted in the spring of 2010. Participants were divided into a placebo group and an active group according to a completely randomized design, so that there would be no bias in terms of age, sex ratio, body mass index, symptom score of pollen-induced disease during the last pollen season and pre-observation period, co-medications, and number of hospital visits. Once daily after lunch for 8 weeks, participants in the active group ingested 100&#x02005;mL of the sterilized LP0132-fermented citrus juice, and those in the placebo group consumed the equivalent amount of Valencia orange juice flavored to taste the same as the drink for the active group.</p><p>Throughout the 12-week test period (2 weeks pre-observation, 8 weeks sample intake, and 2 weeks post-observation), questionnaires were completed to assess symptoms of pollinosis (runny nose, sneezing, blocked nose, itchy nose, itchy eyes, and watery eyes) and QOL loss, in accordance with the Japan Rhinoconjunctivitis Quality of Life Questionnaire [<xref rid="r18" ref-type="bibr">18</xref>]. The degree of skin itchiness was evaluated using a similar questionnaire about symptoms of pollinosis. Blood samples were collected from a brachial vein just before completing the questionnaire and at 4 and 8 weeks after initiation of sample intake. The serum concentrations of immunoglobulin E (IgE) (total, specific for JCP, and specific for Japanese cypress pollen) were determined by fluorescent enzyme immunoassay. Blood eosinophil counts were determined by flow cytometry using an auto-analyzer (Sysmex XE-2100, Sysmex Co., Ltd., Hyogo, Japan).</p></sec><sec><title>Statistical analysis</title><p>In the animal experiment, data were compared between the groups by Tukey&#x02019;s test. In the clinical trial, questionnaire data were compared between the groups by Wilcoxon&#x02019;s rank-sum test, and blood data were compared between the groups by Welch&#x02019;s t-test. All statistical analyses were performed using the SAS Preclinical Package software for Windows (ver. 5.0, SAS Institute Japan Ltd., Tokyo, Japan). Two-sided p-values less than 0.05 were considered statistically significant.</p></sec></sec><sec sec-type="results" id="s3"><title>RESULTS</title><sec><title>Primary screening of LAB with high proliferative activity in citrus juice</title><p>The proliferative activities in citrus juice of about 100 LAB strains were compared. <xref ref-type="fig" rid="fig_002">Figure 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Proliferative activity of lactic acid bacteria in citrus juice.</p></caption><graphic xlink:href="bmfh-33-147-g002"/></fig> shows the results of 29 representative strains tested. Most <italic>Lactobacillus plantarum</italic> strains showed good proliferative activity, which was defined as more than 2&#x000d7;10<sup>8</sup> CFU/ml after 48 hr of culture in citrus juice, in contrast to <italic>Lactococcus lactis</italic> and <italic>Lactobacillus casei</italic> strains. Seven strains of <italic>L. plantarum</italic> (YIT 0132, YIT 0148, YIT 10015, YIT 10021, YIT 0181, YIT 0182, and YIT 0190) were selected for secondary screening.</p></sec><sec><title>Secondary screening of LAB with IL-10- and IL-12-inducing activities</title><p><xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title> Interleukin (IL)-10- and IL-12-inducing activities of the heat-killed cell powders of <italic>L. plantarum</italic> strains in mouse intraperitoneal macrophage culture</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Strain</td><td align="center" rowspan="1" colspan="1">IL-10<break/>(ng/ml)</td><td align="center" valign="middle" rowspan="1" colspan="1">IL-12p70<break/>(ng/ml)</td><td align="center" rowspan="1" colspan="1">IL-10/IL-12 ratio</td></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">YIT 0132</td><td align="center" valign="top" rowspan="1" colspan="1">2.54 &#x000b1; 0.28<sup>a)</sup></td><td align="center" valign="top" rowspan="1" colspan="1">7.92 &#x000b1; 0.93</td><td align="center" valign="top" rowspan="1" colspan="1">0.321 &#x000b1; 0.024</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">YIT 0148</td><td align="center" valign="top" rowspan="1" colspan="1">2.20 &#x000b1; 0.28</td><td align="center" valign="top" rowspan="1" colspan="1">9.98 &#x000b1; 2.22</td><td align="center" valign="top" rowspan="1" colspan="1">0.225 &#x000b1; 0.031</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">YIT 10015</td><td align="center" valign="top" rowspan="1" colspan="1">1.02 &#x000b1; 0.12</td><td align="center" valign="top" rowspan="1" colspan="1">12.09 &#x000b1; 2.04</td><td align="center" valign="top" rowspan="1" colspan="1">0.086 &#x000b1; 0.019</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">YIT 10021</td><td align="center" valign="top" rowspan="1" colspan="1">1.75 &#x000b1; 0.26</td><td align="center" valign="top" rowspan="1" colspan="1">18.33 &#x000b1; 1.93</td><td align="center" valign="top" rowspan="1" colspan="1">0.095 &#x000b1; 0.004</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">YIT 10181</td><td align="center" valign="top" rowspan="1" colspan="1">1.35 &#x000b1; 0.16</td><td align="center" valign="top" rowspan="1" colspan="1">19.30 &#x000b1; 0.75</td><td align="center" valign="top" rowspan="1" colspan="1">0.070 &#x000b1; 0.006</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">YIT 10182</td><td align="center" valign="top" rowspan="1" colspan="1">2.10 &#x000b1; 0.10</td><td align="center" valign="top" rowspan="1" colspan="1">8.86 &#x000b1; 0.61</td><td align="center" valign="top" rowspan="1" colspan="1">0.238 &#x000b1; 0.008</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">YIT 10190</td><td align="center" valign="top" rowspan="1" colspan="1">1.90 &#x000b1; 0.02</td><td align="center" valign="top" rowspan="1" colspan="1">9.78 &#x000b1; 0.88</td><td align="center" valign="top" rowspan="1" colspan="1">0.195 &#x000b1; 0.016</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Medium only</td><td align="center" valign="top" rowspan="1" colspan="1">0.56 &#x000b1; 0.11</td><td align="center" valign="top" rowspan="1" colspan="1">0.19 &#x000b1; 0.13</td><td align="center" valign="top" rowspan="1" colspan="1">&#x02013;</td></tr></tbody></table><table-wrap-foot><p><sup>a)</sup> Values are expressed as the mean &#x000b1; SD.</p></table-wrap-foot></table-wrap> shows the IL-10- and IL-12-inducing activities of heat-killed cells of LAB. All strains potently induced the production of both IL-12 and IL-10. Finally, LP0132 was selected on the basis of it having high IL-10-inducing activity and the highest ratio of IL-10/IL-12.</p></sec><sec><title>Anti-allergic effects of LP0132 in JCPsis mouse model</title><p><xref ref-type="fig" rid="fig_003">Figure 3</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p> Effect of <italic>Lactobacillus plantarum</italic> YIT 0132 on the allergenic reaction after intranasal challenge in the mouse model of Japanese cedar pollinosis. Numbers of nasal rubbing events after intranasal challenge are expressed as the mean &#x000b1; standard deviation. ***p&#x0003c;0.001 versus the control group.</p></caption><graphic xlink:href="bmfh-33-147-g003"/></fig> shows the number of nasal rubbing events after intranasal challenge in the mouse model of JCPsis. The number was significantly higher (p&#x0003c;0.001) in the control group than in the blank group that received the same amount of saline instead of intranasal challenge. Significant reductions in nasal rubbing were observed in both the fermented juice and killed cells groups compared with the control group, and these effects were comparable to those of the anti-histamine group (p&#x0003c;0.001). The unfermented juice group had a tendency toward a reduction in nasal rubbing (p=0.089) compared with the control group. No differences in body weight gain or food consumption were observed between the experimental groups.</p></sec><sec><title>Anti-allergic effects of sterilized LP0132-fermented citrus juice in individuals with JCPsis</title><p>With respect to the symptoms of pollinosis, the total score of all questionnaire items fluctuated over time, showing a pattern corresponding to the dispersion of JCP in Tachikawa City (<xref ref-type="fig" rid="fig_004">Fig. 4</xref><fig orientation="portrait" fig-type="figure" id="fig_004" position="float"><label>Fig. 4.</label><caption><p> Pollen dispersion in Tachikawa City in the spring of 2010 and the clinical trial schedule. The test samples were ingested from February 8 to April 5 (light gray). The questionnaires regarding pollen disease symptoms and quality of life were completed at 1-week intervals (white arrowheads), and blood samples were collected between February 8 and April 19 (black arrowheads).</p></caption><graphic xlink:href="bmfh-33-147-g004"/></fig>). In other words, there was a peak in JCP dispersion in Tachikawa City on March 11, and the highest scores for each questionnaire item were observed on March 15 (5 weeks after initiation of sample intake).</p><p><xref ref-type="fig" rid="fig_005">Figure 5A</xref><fig orientation="portrait" fig-type="figure" id="fig_005" position="float"><label>Fig. 5.</label><caption><p> Scores for nasal and eye symptoms (A) and quality of life (B) at 5 weeks after starting sample intake (on March 15). Values are expressed as mean scores of questionnaire items. *p&#x0003c;0.05; &#x02020;p&#x0003c;0.1 versus the placebo group.</p></caption><graphic xlink:href="bmfh-33-147-g005"/></fig> shows the average scores of questionnaire items when symptoms were worst. Most of the scores except for runny nose were lower in the active group than in the placebo group. The average score for itchy eyes in the active group was significantly lower (p&#x0003c;0.05) than that in the placebo group. With regard to QOL loss, each score for the 17 questionnaire items was highest on March 15 (data not shown). As shown in <xref ref-type="fig" rid="fig_005">Fig. 5B</xref>, all QOL loss scores were lower in the active group than in the placebo group. In the active group, the scores for &#x0201c;deterioration of memory&#x0201d; (p&#x0003c;0.05), &#x0201c;nervous in the company of others&#x0201d; (p&#x0003c;0.05), and &#x0201c;indisposed&#x0201d; (p&#x0003c;0.05) were significantly lower, and that for &#x0201c;decline in cognitive power&#x0201d; showed a weaker tendency toward a reduction compared with the placebo group (p=0.094).</p><p><xref ref-type="fig" rid="fig_006">Figure 6A to C</xref><fig orientation="portrait" fig-type="figure" id="fig_006" position="float"><label>Fig. 6.</label><caption><p> Time-dependent changes in scores for pollinosis symptoms. (A) Total score, (B) itchy skin score, (C) average itchiness score (skin, eyes and nose). Values are expressed as mean scores of questionnaire items. **p&#x0003c;0.01; *p&#x0003c;0.05; &#x02020;p&#x0003c;0.1 versus the placebo group. Squares, placebo group; circles, LP0132.</p></caption><graphic xlink:href="bmfh-33-147-g006"/></fig> shows the time-dependent changes in the total scores of all questionnaire items for pollinosis symptoms, scores for itching of skin, and the average score for itching (skin, eyes, and nose), respectively. As shown in <xref ref-type="fig" rid="fig_006">Fig. 6A</xref>, the total scores for all questionnaire items were highest a few days after the peak in JCP dispersion. As for nasal and eye symptoms, both scores showed a pattern corresponding to the dispersion of JCP, and were highest on March 15. Further, they were lower in the active group than in the placebo group at every time point. Significant difference in itchy skin score were observed on March 15 and April 5 (the end of sample intake), and a significant difference in total itchiness was observed on March 15.</p><p>As shown in <xref ref-type="fig" rid="fig_007">Fig. 7</xref><fig orientation="portrait" fig-type="figure" id="fig_007" position="float"><label>Fig. 7.</label><caption><p> Time-dependent changes in the proportion of blood eosinophils. Values are expressed as the mean &#x000b1; standard deviation. *p&#x0003c;0.05; &#x02020;p&#x0003c;0.1 versus the placebo group.</p></caption><graphic xlink:href="bmfh-33-147-g007"/></fig>, the percentage of the initial value of blood eosinophils in the active group was significantly lower on April 19 (p&#x0003c;0.05) and tended to be lower on April 5 compared with the placebo group (p=0.082). Significant between-group differences in serum IgE (total, specific for JCP, and specific for Japanese cypress pollen) were not observed at any time point (data not shown).</p><p>In the clinical trial, compliance was almost 100%, and there were no dropouts or side effects, such as diarrhea or abdominal pain. Also, there were no differences in the frequencies of drug use and hospital visits between the groups.</p></sec></sec><sec sec-type="discussion" id="s4"><title>DISCUSSION</title><p>The aim of this study was to find LAB with anti-allergy activity and to examine the effects of citrus juice fermented with the selected LAB on allergy symptoms of JCPsis. We demonstrated that most strains belonging to <italic>L. plantarum</italic> could proliferate well in citrus juice with a pH of 3.5, whereas other species of LAB tested could not (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). Since this pH is outside the range that general LAB can tolerate and proliferate in [<xref rid="r19" ref-type="bibr">19</xref>], the strains belonging to <italic>L. plantarum</italic> are thought to utilize special metabolic systems. One such system could be malolactic fermentation, a metabolic pathway for energy production that uses malic acid and plays an important role in wine production from acidic grape juice by <italic>L. plantarum</italic> and other LAB [<xref rid="r20" ref-type="bibr">20</xref>]. Malic acid was detected at a level of 0.03% in the citrus juice we used but not after fermentation (data not shown), suggesting that LP0132 utilized the malolactic fermentation pathway to proliferate in acidic citrus juice.</p><p>Previous studies have suggested that some strains of immunoregulatory probiotics that predominantly induce IL-10 can promote the development of regulatory T cells, thereby improving the symptoms of inflammatory diseases such as allergies, inflammatory bowel diseases and autoimmune diseases [<xref rid="r21" ref-type="bibr">21</xref>, <xref rid="r22" ref-type="bibr">22</xref>]. It has also been reported that LAB that induce a higher ratio of IL-10/IL-12 production have a potent immunoregulatory potential [<xref rid="r23" ref-type="bibr">23</xref>]. For these reasons, we used IL-10-inducing activity and the ratio of IL-10/IL-12 as markers of anti-allergic potential. Following the secondary screening, LP0132 was selected as a strain with both high IL-10-inducing activity and a high ratio of IL-10/IL-12 in macrophages (<xref rid="tbl_002" ref-type="table">Table 2</xref>).</p><p>The anti-allergy activity of the fermented citrus juice containing live LP0132 was demonstrated in the mouse model of JCPsis. For animals in the fermented juice group, nasal rubbing decreased to a level similar to that in the anti-histamine group (positive control), whereas the effect of unfermented citrus juice was lower (<xref ref-type="fig" rid="fig_003">Fig. 3</xref>). Some kinds of citrus juice contain hesperidin, which reduces the inflammatory reaction, inhibits the degranulation of mast cells [<xref rid="r13" ref-type="bibr">13</xref>], relieves edema, and prevents anaphylaxis. Therefore, we posit that this weak anti-allergic activity in the unfermented juice group is probably due to the action of hesperidin. No significant difference was detected between the fermented and unfermented juice groups, but on average, the number of nasal rubbing events was 15% lower in the fermented juice group than in the unfermented juice group. Kim et al. [<xref rid="r24" ref-type="bibr">24</xref>] reported that hesperidin may act as a potential IL-5 antagonist and have a therapeutic effect on allergic asthma. However, there is less information available about the anti-allergy mechanism of hesperidin, and we have not yet examined the involvement of IL-5 in the anti-allergic mechanism of LP0132. So at this point, it is difficult to estimate if hesperidin and LP0132 exert anti-allergic effects through the same or different pathways. The reasons why a significant additive effect was not observed is unknown, but we think the large standard deviation in each group was one of the reasons.</p><p>Since the powder from heat-killed LP0132 cells was also effective in the animal experiment, it was considered that viable cells are not always necessary for anti-allergy effects. Therefore, we conducted a clinical trial using sterile fermented citrus juice containing heat-killed LP0132.</p><p>The clinical trial was conducted in 2010. In the spring of 2010, the amount of JCP dispersed in the participants&#x02019; area of residence was lower than usual [<xref rid="r25" ref-type="bibr">25</xref>]. However, the symptomatic states of pollinosis and QOL were worsening in conjunction with the amount of JCP dispersed. Therefore, we consider that the clinical trial was conducted in a suitable setting for evaluating anti-pollinosis effects.</p><p>Our data demonstrate that the symptomatic states of pollinosis and impairments of QOL were alleviated in the active group as compared with the placebo group; that is, the average scores of itchy eyes, itchy skin (<xref ref-type="fig" rid="fig_005">Fig. 5A</xref>), and some QOL questionnaire items were significantly attenuated (<xref ref-type="fig" rid="fig_005">Fig. 5B</xref>).</p><p>It is interesting that, of the symptoms of pollinosis, the active drink particularly alleviated itching (of the nose, eyes, and skin) in this clinical trial (<xref ref-type="fig" rid="fig_006">Fig. 6B, C</xref>), indicating that it may help to improve QOL. Itching is thought to occur via stimulation of C-fibers by mediators released from mast cells, including histamine and tryptase [<xref rid="r26" ref-type="bibr">26</xref>]. Based on the remarkable effect on itching observed in our trial, a possible effect of LP0132-fermented citrus juice on skin itchiness in atopic dermatitis is also suggested. Jang et al. reported that oral administration of <italic>L. plantarum</italic> PM008 isolated from kimchi (Korean pickles) reduced nasal rubbing induced by subcutaneous administration of histamine in mice [<xref rid="r27" ref-type="bibr">27</xref>]. This suggests that the <italic>L. plantarum</italic> strain may alleviate itching by affecting a mechanism other than histamine release. LP0132 may also effectively suppress the processes after degranulation of mast cells, since no significant decrease in serum levels of IgE (total, specific for Japanese cedar pollen, or specific for cypress pollen) was observed.</p><p>The percentage of blood eosinophils gradually increased during the later phase of the trial in the placebo group, but this increase was not observed in the active group. A significant difference in the percentage of the initial value of blood eosinophils between the placebo and active groups was observed at 3 weeks after the pollen peak (<xref ref-type="fig" rid="fig_007">Fig. 7</xref>), whereas the improvement in clinical scores was observed immediately after the pollen peak. It is known that the ratio of eosinophils to total leukocytes increases after the onset of allergy, and so we speculate the difference between groups become more clear in the late stage due to accumulation of suppressive effects on the delayed reaction to JCPsis arising as a result of the continuous inhalation of antigen. Eosinophil cationic protein released during degranulation of eosinophils is closely associated with chronic inflammation or asthma [<xref rid="r28" ref-type="bibr">28</xref>]. Therefore, suppression of the level of eosinophils increase by LP0132-fermented citrus juice may also lead to benefits for individuals with other chronic allergies.</p><p>Although our animal experiment and clinical trial clearly showed that LP0132-fermented citrus juice improved the symptoms of JCPsis, the mechanism of action was not examined. Since the probiotic strain has a potent ability to induce IL-10, the immunoregulatory actions of IL-10, including downregulation of inflammatory responses and induction of Treg cells, might be responsible [<xref rid="r29" ref-type="bibr">29</xref>]. In a future study, we intend to clarify whether the fermented juice can promote IL-10 production and Treg cell development.</p><p>The present results show that it is not necessary for LP0132 to be alive to exert its anti-allergic activity, suggesting a mode of action other than the ecological competition of intestinal microbiota. This property provides advantages such as a long shelf life and reduced distribution costs of the product, which can be marketed as an anti-allergy drink. The fermented citrus juice also has some advantages with respect to the costs of manufacture and disposal compared with mixing raw citrus juice with heat-killed bacteria cultured separately. In conclusion, citrus juice fermented with the selected strain of <italic>L. plantarum</italic>, LP0132, showed anti-allergic potential. Continuous consumption of this juice may help in the relief of allergy symptoms and impaired QOL caused by JCPsis.</p></sec>
<italic>Lactobacillus acidophilus</italic> L-92 Cells Activate Expression of Immunomodulatory Genes in THP-1 Cells
<p>To understand the immunomodulatory effects of <italic>Lactobacillus acidophilus</italic> L-92 cells suggested from our previous study of <italic>in vivo</italic> anti-allergy and anti-virus effects, host immune responses in macrophage-like THP-1 cells after 4&#x02005;h (the early phase) and 24&#x02005;h (the late phase) of cocultivation with L-92 cells were investigated by transcriptome analysis. In the early phase of L-92 treatment, various transcription regulator genes, such as, <italic>NFkB1, NFkB2, JUN, HIVEP2</italic> and <italic>RELB,</italic> and genes encoding chemokines and cytokines, such as <italic>CCL4, CXCL11, CCL3</italic> and <italic>TNF,</italic> were upregulated. Two transmembrane receptor genes, <italic>TLR7</italic> and <italic>ICAM1</italic>, were also upregulated in the early phase of treatment. In contrast, many transmembrane receptor genes, such as <italic>IL7R, CD80, CRLF2, CD86, CD5, HLA-DQA1, IL2RA, IL15RA</italic> and <italic>CSF2RA</italic>, and some cytokine genes, including <italic>IL6, IL23A</italic> and <italic>CCL22</italic>, were significantly upregulated in the late phase after L-92 exposure. Some genes encoding cytokines, such as <italic>IL1A, IL1B</italic> and <italic>IL8,</italic> and the enzyme <italic>IDO1</italic> were upregulated at both the early and the late phases of treatment. These results suggest that probiotic L-92 might promote Th1 and regulatory T-cell responses by activation of the MAPK signaling pathway, followed by the NOD-like receptor signaling pathway in THP-1 cells.</p>
<contrib contrib-type="author"><name><surname>YANAGIHARA</surname><given-names>Sae</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>GOTO</surname><given-names>Hiroaki</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>HIROTA</surname><given-names>Tatsuhiko</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>FUKUDA</surname><given-names>Shinji</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>OHNO</surname><given-names>Hiroshi</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>YAMAMOTO</surname><given-names>Naoyuki</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref><xref rid="cor1" ref-type="corresp"><sup>*</sup></xref></contrib><aff id="aff1"><label>1</label>Microbiology and Fermentation Laboratory, Calpis Co. Ltd., 5&#x02013;11&#x02013;10 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa 252-0206, Japan</aff><aff id="aff2"><label>2</label>Intestinal Microbe Symbiosis Laboratory, RIKEN, Wako, Saitama 351-0198, Japan</aff><aff id="aff3"><label>3</label>Laboratory for Intestinal Ecosystem, RCAI, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan</aff><aff id="aff4"><label>4</label>Immunobiology Laboratory, Graduate School of Medical Life Science, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan</aff><aff id="aff5"><label>5</label>Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0052, Japan</aff><aff id="aff6"><label>6</label>Research and Development Planning Department, Calpis Co. Ltd., 5&#x02013;11&#x02013;10 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa 252-0206, Japan</aff>
Bioscience of Microbiota, Food and Health
<sec sec-type="intro" id="s1"><title>INTRODUCTION</title><p>A variety of lactic acid bacteria (LAB) have been isolated and used in mainly dairy foods and supplements for their health benefits in humans. One of the most important probiotic effects of LAB in a host would be its action on the gastrointestinal immune system, which may be beneficial in the prevention of gastrointestinal infections [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>], and inflammatory bowel diseases [<xref rid="r3" ref-type="bibr">3</xref>] and the alleviation of allergic diseases [<xref rid="r4" ref-type="bibr">4</xref>]. Among many probiotic strains that have been developed, <italic>Lactobacillus</italic> strains have been characterized as strong inducers of pro-inflammatory cytokines, such as IL-12 and tumor necrosis factor (TNF-&#x003b1;) [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r6" ref-type="bibr">6</xref>], and they seem to be different from the weak IL-12 inducer bifidobacteria.</p><p><italic>Lactobacillus acidophilus</italic> L-92 has been developed as a strain with anti-allergy effects for pollen allergy [<xref rid="r7" ref-type="bibr">7</xref>], perennial allergy [<xref rid="r8" ref-type="bibr">8</xref>], and atopic dermatitis [<xref rid="r9" ref-type="bibr">9</xref>, <xref rid="r10" ref-type="bibr">10</xref>], and it also has the ability to control gastrointestinal disorders [<xref rid="r11" ref-type="bibr">11</xref>]. To explain these <italic>in vivo</italic> anti-allergy effects, various mechanisms have been proposed, including an improvement in the balance of T helper 1 (Th1) cells and T helper 2 (Th2) cells, induction of apoptosis, induction or activation of regulatory T cells, and activation of the innate immune system [<xref rid="r9" ref-type="bibr">9</xref>, <xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>]. Our recent study demonstrated that surface layer protein A (SlpA) on the cell wall of the <italic>L. acidophilus</italic> L-92 strain is important for enhancement of the release of IL-12, known as a Th1-type cytokine, from splenocytes [<xref rid="r15" ref-type="bibr">15</xref>]. However, the detailed mechanisms of such immune modulation events are unknown.</p><p>The Peyer&#x02019;s patch, lamina propria and mesenteric lymph nodes of the gastrointestinal tract play a crucial role in controlling the gastrointestinal immune system. In the gastrointestinal immune system, contact between LAB and antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, is likely to be a particularly important process for initiation of host immune responses by priming innate and adaptive immunity [<xref rid="r16" ref-type="bibr">16</xref>]. Probiotic microbial signals are thought to be transmitted to DCs via Toll-like receptors [<xref rid="r17" ref-type="bibr">17</xref>]; however, few reports have addressed the action of probiotics on cells of the immune system. To improve understanding of the events in DCs, transcriptome analyses have been conducted both in animal studies and in human trials [<xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref>,<xref rid="r20" ref-type="bibr">20</xref>]. Macrophage-like THP-1 cells represent a well responding and physiologically meaningful model system for investigation of the context of innate immunity.</p><p>We herein report a detailed analysis of cell metabolism of macrophages in response to L-92 by using transcriptomic analysis on THP-1 cells, which show a response to L-92 cells similar to that of splenocytes by releasing IL-12. The present study suggests that probiotic L-92 might activate Th1 and regulatory T-cell responses by engaging the MAPK and NOD-like receptor signaling pathways.</p></sec><sec sec-type="materials|methods" id="s2"><title>MATERIALS AND METHODS</title><sec><title>THP-1 cell culture</title><p>The human monocyte cell line THP-1 [<xref rid="r21" ref-type="bibr">21</xref>, <xref rid="r22" ref-type="bibr">22</xref>] was obtained from the Health Science Research Resources Bank (Tokyo, Japan) and maintained in RPMI-1640 (Sigma, St. Louis, MO, USA) containing 10% (V/V) of heat-inactivated fetal bovine serum (FBS; Hana-Nesco Bio, Tokyo, Japan), streptomycin (100&#x02005;&#x000b5;g/ml) and penicillin (100 U/ml) (GIBCO, Grand Island, NY, USA) at 37&#x000b0;C in 5% CO<sub>2</sub>/95% air.</p></sec><sec><title>Preparation of heat-killed L-92 cells</title><p><italic>Lactobacillus acidophilus</italic> L-92 from our stock culture collection was cultured anaerobically at 37&#x000b0;C for 20 hr in de Man, Rogosa and Sharpe (MRS) broth (Difco Laboratories, Detroit, MI, USA), and washed with phosphate-buffered saline (PBS). Bacteria were suspended in PBS and killed by heating at 100&#x000b0;C for 10&#x02005;min.</p></sec><sec><title>L-92 treatment of THP-1 cells</title><p>For transcriptome analysis of THP-1 metabolism, THP-1 cells were grown in RPMI-1640 containing 10% (V/V) of heat-inactivated FBS, streptomycin (100&#x02005;&#x000b5;g/ml) and penicillin (100 U/ml) in a 250&#x02005;mL flask (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) at 37&#x000b0;C in 5% CO<sub>2</sub>/95% air and stimulated to macrophage-like cells by a method described previously, with some modifications [<xref rid="r23" ref-type="bibr">23</xref>]. Briefly, THP-1 cells were suspended in 2&#x02005;mL of RPMI-1640 at a cell density of 5 &#x000d7; 10<sup>6</sup> cells/ml and cultivated in a 6-well plate (Asahi Glass Co., Ltd., Tokyo, Japan). The cells were differentiated by cultivation in 1.2% (V/V) Me<sub>2</sub>SO containing RPMI-1640 for 24 hr and stimulated with IFN-&#x003b3; (5000 U/ml) for 16 hr. Unstained cells were counted as viable cell after treatment with Trypan Blue Solution (0.4%) (Sigma, St. Louis, MO, USA). Next, 1 &#x000d7; 10<sup>9</sup> cells of heat-killed L-92, suspended in 0.2&#x02005;mL of culture buffer, were added to stimulate the THP-1 cells. The THP-1 cells were then harvested after 4 and 24 hr of cocultivation with L-92 in the culture medium. The THP-1 cells were collected by centrifugation at 4&#x000b0;C for 10&#x02005;min at 430 &#x000d7; <italic>g</italic>, and cell pellets were washed twice with cold D-PBS (Sigma) for total RNA extraction. Cells untreated with L-92 cells and stimulated with IFN-&#x003b3; (5000 U/ml) for 16 hr were used as controls. Three samples were prepared from three independent experiments for each group.</p></sec><sec><title>Microarray analysis</title><p>Total RNA was extracted by using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Double-stranded cDNA was synthesized from 5&#x02005;&#x000b5;g of total RNA, and the cDNA was subjected to <italic>in vitro</italic> transcription in the presence of biotinylated nucleotide triphosphates. Human genome-wide gene expression was examined by using the GeneChip<sup>&#x000ae;</sup> Human Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA), which contains oligonucleotide probe sets for approximately 28,869 full-length genes and expressed sequence tags. The biotinylated cDNA was hybridized with a probe array for 16 hr at 45&#x000b0;C. The hybridized products were stained with streptavidin-phycoerythrin, and then scanned with a Hewlett-Packard Gene Array Scanner (Palo Alto, CA, USA). The fluorescence intensity of each probe was quantified by using the GeneChip Analysis Suite 5.0 software (Affymetrix). The level of gene expression was determined as the average difference by using the GeneChip software.</p></sec><sec><title>Statistical and functional analysis of microarray data</title><p>Data analysis was performed with GeneSpring software version 6.1 (Silicon Genetics, San Carlos, CA, USA). Expression data were considered significant when they differed at least twofold between L-92-treated and PBS-treated cells, and were statistically analyzed by the unpaired t-test with the Benjamini-Hochberg post test. P-values of less than 0.05 were considered significant. Biological interaction networks among regulated genes activated in response to L-92 were identified using Ingenuity Pathways Analysis (IPA). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used for deeper understanding of the metabolism of the altered gene expression.</p></sec><sec><title>Quantitative real-time (qRT)-PCR</title><p>A two-step RT-PCR reaction was employed. cDNA was synthesized according to the protocol of the Superscript III First-Strand cDNA Synthesis System (Invitrogen, Carlsbad, CA, USA) using 1&#x02005;&#x000b5;g of total RNA. cDNA was diluted, and frozen aliquots were stored at &#x02212;20&#x000b0;C. The cDNA generated was amplified using SYBR<sup>&#x000ae;</sup> Premix Ex Taq<sup>TM</sup> II (Tli RNaseH Plus) (Takara Bio, Inc., Shiga, Japan). All assays were performed on a TP800 Thermal Cycler Dice<sup>&#x000ae;</sup> Real Time System (Takara Bio, Inc.). Primers were prepared for amplification of specific genes, TNF, CRLF2, GAPDH, CD86, STAT4 and IL-8 genes. The sequences of the primers (forward and reverse primers) used in PCR reactions were 5&#x02032;-AGATGATCTGACTGCCTGGG-3&#x02032; and 5&#x02032;-CTGCTGCACTTTGGAGTGAT-3&#x02032; for TNF, 5&#x02032;-CTGATGCCACGAAAATCTCA-3&#x02032; and 5&#x02032;-TTCTCCATCAGGAATGGGAC-3&#x02032; for CRLF25&#x02019;, 5&#x02032;-GCACCGTCAAGGCTGAGAAC-3&#x02032; and 5&#x02032;-TGGTGAAGACGCCAGTGGA -3&#x02032; for GAPDH, 5&#x02032;-AGAGGAGCAGCACCAGAGAG-3&#x02032; and 5&#x02032;-CAGAAGCAGCCAAAATGGAT-3&#x02032; for CD86, 5&#x02032;-CACAGCTACATGCATTGGATT-3&#x02032;and 5&#x02032;-CGTGTTTCCAAAGAGAAAAACC-3&#x02032; for STAT4 and 5&#x02032;-CTGGCCGTGGCTCTCTTG-3&#x02032; and 5&#x02032;-CCTTGGCAAAACTGCACCTT-3&#x02032; for and IL8. A cycle threshold (Ct) was assigned at the beginning of the logarithmic phase of PCR amplification. Data were analyzed by ABI software, and the gene expression was quantified using the 2<sup>&#x02212;&#x00394;&#x00394;CT</sup> method and normalized to the constitutively expressed housekeeping gene <italic>GAPDH</italic>.</p></sec></sec><sec sec-type="results" id="s3"><title>RESULTS</title><sec><title>Altered gene pathways in THP-1 cells</title><p>To understand the host immunomodulatory effects of <italic>L. acidophilus</italic> L-92 cells suggested from previous studies of <italic>in vivo</italic> anti-allergy and anti-virus effects, responses in THP-1 cells after 4 hr (the early phase) and 24 hr (the late phase) of cocultivation with L-92 cells were investigated by transcriptome analysis. For cultivation of THP-1 cells in RPMI-1640 medium, a high concentration of IFN-&#x003b3; was added to enhance IL-12 release for the Th1 type cell response based on our previous study. Viability of the THP-1 cells after inoculation in RPMI-1640 medium with IFN-&#x003b3; at 16 hr was 96.3%. Differential expression analysis showed a significant (more than 2-fold) up- or downregulation of 282 genes in the early phase and 2,411 genes in the late phase in THP-1 cells caused by treatment with L-92 (data not shown).</p><p>Next, IPA was used to integrate the transcriptional networks for a more detailed understanding of the main networks linked to immunomodulatory responses in THP-1 cells, as described in the Materials and Methods. In the IPA analysis, the most significant differences in biological functions, referred to as &#x0201c;hematological process&#x0201d;, were found after 4 hr (66 molecules; p=1.34 &#x000d7; 10<sup>&#x02212;28</sup>) and 24 hr (253 molecules; p=1.42 &#x000d7; 10<sup>&#x02212;37</sup>) of treatment with L-92 cells. At the early phase of the treatment (4 hr), transcription regulators, cytokines and transmembrane receptors in particular showed highly altered expression (<xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Ingenuity pathway analysis for altered gene expression in THP-1 cells treated with heat-killed <italic>Lactobacillus acidophilus</italic> L-92 cells</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Category</td><td align="center" rowspan="1" colspan="1">4 hr</td><td align="center" rowspan="1" colspan="1">24 hr</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Transcription regulator</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">53</td></tr><tr><td align="left" rowspan="1" colspan="1">Enzyme</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">31</td></tr><tr><td align="left" rowspan="1" colspan="1">Transmembrane receptor</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">28</td></tr><tr><td align="left" rowspan="1" colspan="1">Cytokine</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">24</td></tr><tr><td align="left" rowspan="1" colspan="1">Kinase</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">22</td></tr><tr><td align="left" rowspan="1" colspan="1">G-protein coupled receptor</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">10</td></tr><tr><td align="left" rowspan="1" colspan="1">Phosphatase</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">7</td></tr><tr><td align="left" rowspan="1" colspan="1">Growth factor</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">5</td></tr><tr><td align="left" rowspan="1" colspan="1">Peptidase</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">4</td></tr><tr><td align="left" rowspan="1" colspan="1">Ion channel</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">3</td></tr><tr><td align="left" rowspan="1" colspan="1">Ligand-dependent nuclear receptor</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">3</td></tr><tr><td align="left" rowspan="1" colspan="1">Transporter</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">Other</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">62</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">66</td><td align="center" rowspan="1" colspan="1">253</td></tr></tbody></table></table-wrap>). In the late phase of the treatment with L-92, transcription regulators, enzymes, transmembrane receptors and cytokines were mostly upregulated (<xref rid="tbl_001" ref-type="table">Table 1</xref>). Over the course of the treatment, the majority of genes with altered expression were transcription regulators, enzymes, cytokines and transmembrane receptors (<xref rid="tbl_001" ref-type="table">Table 1</xref>). These results revealed that the genes showing changes in expression in THP-1 cells caused by L-92 treatment were mainly associated with immune response and transcriptional regulation.</p></sec><sec><title>Altered genes in THP-1 cells</title><p>For a deeper understanding of each cellular response in THP-1 cells caused by L-92 treatment, genes that showed significant changes, especially those in the transcription regulator, enzyme, transmembrane receptor and cytokine categories that showed over a 4-fold change after 4 hr of treatment and less than a 4-fold change from 4 hr to 24 hr of L-92 treatment, are listed in <xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title> Major altered genes categorized as transcription regulators, enzymes, transmembrane receptors and cytokines in THP-1 cells after L-92 treatment for 4 hr and 24 hr</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Category</td><td align="center" rowspan="2" colspan="1">Entrez Gene Name</td><td align="center" rowspan="2" colspan="1">Symbol</td><td align="center" colspan="2" rowspan="1">Fold change<hr/></td></tr><tr><td align="center" rowspan="1" colspan="1">0&#x02013;4 hr</td><td align="center" rowspan="1" colspan="1">4&#x02013;24 hr</td></tr></thead><tbody><tr><td align="left" colspan="3" rowspan="1">Early responding genes</td><td align="center" rowspan="1" colspan="1">&#x0003e; 4.0</td><td align="center" rowspan="1" colspan="1">4.0 &#x0003e;</td></tr><tr><td align="left" colspan="5" rowspan="1"> Cytokine</td></tr><tr><td align="left" rowspan="5" colspan="1"/><td align="left" rowspan="1" colspan="1">chemokine (C-C motif) ligand 4</td><td align="left" rowspan="1" colspan="1"><italic>CCL4</italic></td><td align="center" rowspan="1" colspan="1">58.29 </td><td align="center" rowspan="1" colspan="1">2.02 </td></tr><tr><td align="left" rowspan="1" colspan="1">chemokine (C-X-C motif) ligand 11</td><td align="left" rowspan="1" colspan="1"><italic>CXCL11</italic></td><td align="center" rowspan="1" colspan="1">27.97 </td><td align="center" rowspan="1" colspan="1">2.28 </td></tr><tr><td align="left" rowspan="1" colspan="1">chemokine (C-C motif) ligand 3</td><td align="left" rowspan="1" colspan="1"><italic>CCL3</italic></td><td align="center" rowspan="1" colspan="1">20.04 </td><td align="center" rowspan="1" colspan="1">2.77 </td></tr><tr><td align="left" rowspan="1" colspan="1">tumor necrosis factor</td><td align="left" rowspan="1" colspan="1"><italic>TNF</italic></td><td align="center" rowspan="1" colspan="1">14.57 </td><td align="center" rowspan="1" colspan="1">1.15 </td></tr><tr><td align="left" rowspan="1" colspan="1">interleukin 1 receptor antagonist</td><td align="left" rowspan="1" colspan="1"><italic>IL1RN</italic></td><td align="center" rowspan="1" colspan="1">5.11 </td><td align="center" rowspan="1" colspan="1">1.95 </td></tr><tr><td align="left" colspan="5" rowspan="1"> Transmembrane receptor</td></tr><tr><td align="left" rowspan="2" colspan="1"/><td align="left" rowspan="1" colspan="1">intercellular adhesion molecule 1</td><td align="left" rowspan="1" colspan="1"><italic>ICAM1</italic></td><td align="center" rowspan="1" colspan="1">10.01 </td><td align="center" rowspan="1" colspan="1">2.33 </td></tr><tr><td align="left" rowspan="1" colspan="1">toll-like receptor 7</td><td align="left" rowspan="1" colspan="1"><italic>TLR7</italic></td><td align="center" rowspan="1" colspan="1">6.63 </td><td align="center" rowspan="1" colspan="1">0.32 </td></tr><tr><td align="left" colspan="5" rowspan="1"> Enzyme</td></tr><tr><td align="left" rowspan="2" colspan="1"/><td align="left" rowspan="1" colspan="1">superoxide dismutase 2, mitochondrial</td><td align="left" rowspan="1" colspan="1"><italic>SOD2</italic></td><td align="center" rowspan="1" colspan="1">10.04 </td><td align="center" rowspan="1" colspan="1">2.50 </td></tr><tr><td align="left" rowspan="1" colspan="1">heme oxygenase (decycling) 1</td><td align="left" rowspan="1" colspan="1"><italic>HMOX1</italic></td><td align="center" rowspan="1" colspan="1">4.26 </td><td align="center" rowspan="1" colspan="1">2.21 </td></tr><tr><td align="left" colspan="5" rowspan="1"> Transcription regulator</td></tr><tr><td align="left" rowspan="5" colspan="1"/><td align="left" rowspan="1" colspan="1">jun proto-oncogene</td><td align="left" rowspan="1" colspan="1"><italic>JUN</italic></td><td align="center" rowspan="1" colspan="1">4.99 </td><td align="center" rowspan="1" colspan="1">2.72 </td></tr><tr><td align="left" rowspan="1" colspan="1">nuclear factor of kappa light polypeptide gene enhancer in B-cells 1</td><td align="left" rowspan="1" colspan="1"><italic>NFKB1</italic></td><td align="center" rowspan="1" colspan="1">5.18 </td><td align="center" rowspan="1" colspan="1">2.21 </td></tr><tr><td align="left" rowspan="1" colspan="1">nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100)</td><td align="left" rowspan="1" colspan="1"><italic>NFKB2</italic></td><td align="center" rowspan="1" colspan="1">6.10 </td><td align="center" rowspan="1" colspan="1">1.85 </td></tr><tr><td align="left" rowspan="1" colspan="1">human immunodeficiency virus type I enhancer binding protein 2</td><td align="left" rowspan="1" colspan="1"><italic>HIVEP2</italic></td><td align="center" rowspan="1" colspan="1">4.18 </td><td align="center" rowspan="1" colspan="1">2.29 </td></tr><tr><td align="left" rowspan="1" colspan="1">v-rel reticuloendotheliosis viral oncogene homolog B</td><td align="left" rowspan="1" colspan="1"><italic>RELB</italic></td><td align="center" rowspan="1" colspan="1">4.34 </td><td align="center" rowspan="1" colspan="1">1.04 </td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" colspan="3" rowspan="1">Slowly responding genes</td><td align="center" rowspan="1" colspan="1">0&#x02013;4 hr</td><td align="center" rowspan="1" colspan="1">4&#x02013;24 hr</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" colspan="3" rowspan="1"> Transmembrane receptor</td><td align="center" rowspan="1" colspan="1">4.0 &#x0003e;</td><td align="center" rowspan="1" colspan="1">&#x0003e; 4.0</td></tr><tr><td align="left" rowspan="9" colspan="1"/><td align="left" rowspan="1" colspan="1">interleukin 7 receptor</td><td align="left" rowspan="1" colspan="1"><italic>IL7R</italic></td><td align="center" rowspan="1" colspan="1">1.83 </td><td align="center" rowspan="1" colspan="1">63.99 </td></tr><tr><td align="left" rowspan="1" colspan="1">CD80 molecule</td><td align="left" rowspan="1" colspan="1"><italic>CD80</italic></td><td align="center" rowspan="1" colspan="1">1.74 </td><td align="center" rowspan="1" colspan="1">61.40 </td></tr><tr><td align="left" rowspan="1" colspan="1">cytokine receptor-like factor 2</td><td align="left" rowspan="1" colspan="1"><italic>CRLF2</italic></td><td align="center" rowspan="1" colspan="1">2.02 </td><td align="center" rowspan="1" colspan="1">52.00 </td></tr><tr><td align="left" rowspan="1" colspan="1">CD86 molecule</td><td align="left" rowspan="1" colspan="1"><italic>CD86</italic></td><td align="center" rowspan="1" colspan="1">0.86 </td><td align="center" rowspan="1" colspan="1">30.10 </td></tr><tr><td align="left" rowspan="1" colspan="1">CD5 molecule</td><td align="left" rowspan="1" colspan="1"><italic>CD5</italic></td><td align="center" rowspan="1" colspan="1">1.10 </td><td align="center" rowspan="1" colspan="1">16.20 </td></tr><tr><td align="left" rowspan="1" colspan="1">interleukin 15 receptor, alpha</td><td align="left" rowspan="1" colspan="1"><italic>IL15RA</italic></td><td align="center" rowspan="1" colspan="1">2.06 </td><td align="center" rowspan="1" colspan="1">4.37 </td></tr><tr><td align="left" rowspan="1" colspan="1">major histocompatibility complex, class II, DQ alpha 1</td><td align="left" rowspan="1" colspan="1"><italic>HLA-DQA1</italic></td><td align="center" rowspan="1" colspan="1">0.94 </td><td align="center" rowspan="1" colspan="1">5.93 </td></tr><tr><td align="left" rowspan="1" colspan="1">interleukin 2 receptor, alpha</td><td align="left" rowspan="1" colspan="1"><italic>IL2RA</italic></td><td align="center" rowspan="1" colspan="1">1.00 </td><td align="center" rowspan="1" colspan="1">4.98 </td></tr><tr><td align="left" rowspan="1" colspan="1">colony stimulating factor 2 receptor, alpha, low-affinity (granulocyte-macrophage)</td><td align="left" rowspan="1" colspan="1"><italic>CSF2RA</italic></td><td align="center" rowspan="1" colspan="1">0.85 </td><td align="center" rowspan="1" colspan="1">4.15 </td></tr><tr><td align="left" colspan="5" rowspan="1"> Cytokine</td></tr><tr><td align="left" rowspan="6" colspan="1"/><td align="left" rowspan="1" colspan="1">interleukin 12B (natural killer cell stimulatory factor 2, p40)</td><td align="left" rowspan="1" colspan="1"><italic>IL12B</italic></td><td align="center" rowspan="1" colspan="1">3.39 </td><td align="center" rowspan="1" colspan="1">18.10 </td></tr><tr><td align="left" rowspan="1" colspan="1">interleukin 6 (interferon, beta 2)</td><td align="left" rowspan="1" colspan="1"><italic>IL6</italic></td><td align="center" rowspan="1" colspan="1">1.08 </td><td align="center" rowspan="1" colspan="1">54.45 </td></tr><tr><td align="left" rowspan="1" colspan="1">interleukin 23, alpha subunit p19</td><td align="left" rowspan="1" colspan="1"><italic>IL23A</italic></td><td align="center" rowspan="1" colspan="1">1.29 </td><td align="center" rowspan="1" colspan="1">28.01 </td></tr><tr><td align="left" rowspan="1" colspan="1">secreted phosphoprotein 1</td><td align="left" rowspan="1" colspan="1"><italic>SPP1 </italic></td><td align="center" rowspan="1" colspan="1">2.73 </td><td align="center" rowspan="1" colspan="1">11.06 </td></tr><tr><td align="left" rowspan="1" colspan="1">chemokine (C-C motif) ligand 22</td><td align="left" rowspan="1" colspan="1"><italic>CCL22</italic></td><td align="center" rowspan="1" colspan="1">1.07 </td><td align="center" rowspan="1" colspan="1">17.16 </td></tr><tr><td align="left" rowspan="1" colspan="1">chemokine (C-C motif) ligand 19</td><td align="left" rowspan="1" colspan="1"><italic>CCL19</italic></td><td align="center" rowspan="1" colspan="1">1.13 </td><td align="center" rowspan="1" colspan="1">9.65 </td></tr><tr><td align="left" colspan="5" rowspan="1"> Transcription regulator</td></tr><tr><td align="left" rowspan="6" colspan="1"/><td align="left" rowspan="1" colspan="1">early B-cell factor 1</td><td align="left" rowspan="1" colspan="1"><italic>EBF1</italic></td><td align="center" rowspan="1" colspan="1">1.12 </td><td align="center" rowspan="1" colspan="1">41.89 </td></tr><tr><td align="left" rowspan="1" colspan="1">signal transducer and activator of transcription 4</td><td align="left" rowspan="1" colspan="1"><italic>STAT4</italic></td><td align="center" rowspan="1" colspan="1">1.42 </td><td align="center" rowspan="1" colspan="1">23.28 </td></tr><tr><td align="left" rowspan="1" colspan="1">runt-related transcription factor 1; translocated to, 1 (cyclin D-related)</td><td align="left" rowspan="1" colspan="1"><italic>RUNX1T1</italic></td><td align="center" rowspan="1" colspan="1">0.97 </td><td align="center" rowspan="1" colspan="1">8.18 </td></tr><tr><td align="left" rowspan="1" colspan="1">zinc finger protein, multitype 2</td><td align="left" rowspan="1" colspan="1"><italic>ZFPM2</italic></td><td align="center" rowspan="1" colspan="1">1.05 </td><td align="center" rowspan="1" colspan="1">5.65 </td></tr><tr><td align="left" rowspan="1" colspan="1">B-cell CLL/lymphoma 11A (zinc finger protein)</td><td align="left" rowspan="1" colspan="1"><italic>BCL11A</italic></td><td align="center" rowspan="1" colspan="1">0.85 </td><td align="center" rowspan="1" colspan="1">6.92 </td></tr><tr><td align="left" rowspan="1" colspan="1">growth factor independent 1 transcription repressor</td><td align="left" rowspan="1" colspan="1"><italic>GFI1</italic></td><td align="center" rowspan="1" colspan="1">&#x02013;2.21 </td><td align="center" rowspan="1" colspan="1">&#x02013;5.00 </td></tr><tr><td align="left" colspan="5" rowspan="1"> Enzyme</td></tr><tr><td align="left" rowspan="6" colspan="1"/><td align="left" rowspan="1" colspan="1">prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)</td><td align="left" rowspan="1" colspan="1"><italic>PTGS2</italic></td><td align="center" rowspan="1" colspan="1">2.58 </td><td align="center" rowspan="1" colspan="1">14.29 </td></tr><tr><td align="left" rowspan="1" colspan="1">adenosine deaminase</td><td align="left" rowspan="1" colspan="1"><italic>ADA</italic></td><td align="center" rowspan="1" colspan="1">1.07 </td><td align="center" rowspan="1" colspan="1">9.62 </td></tr><tr><td align="left" rowspan="1" colspan="1">sphingosine-1-phosphate lyase 1</td><td align="left" rowspan="1" colspan="1"><italic>SGPL1</italic></td><td align="center" rowspan="1" colspan="1">1.06 </td><td align="center" rowspan="1" colspan="1">5.90 </td></tr><tr><td align="left" rowspan="1" colspan="1">3&#x02019;-phosphoadenosine 5&#x02019;-phosphosulfate synthase 2</td><td align="left" rowspan="1" colspan="1"><italic>PAPSS2</italic></td><td align="center" rowspan="1" colspan="1">1.05 </td><td align="center" rowspan="1" colspan="1">5.47 </td></tr><tr><td align="left" rowspan="1" colspan="1">fibronectin 1</td><td align="left" rowspan="1" colspan="1"><italic>FN1</italic></td><td align="center" rowspan="1" colspan="1">0.95 </td><td align="center" rowspan="1" colspan="1">5.84 </td></tr><tr><td align="left" rowspan="1" colspan="1">coagulation factor XIII, A1 polypeptide</td><td align="left" rowspan="1" colspan="1"><italic>F13A1</italic></td><td align="center" rowspan="1" colspan="1">&#x02013;1.33 </td><td align="center" rowspan="1" colspan="1">&#x02013;12.62 </td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" colspan="3" rowspan="1">Constitutively responding genes</td><td align="center" rowspan="1" colspan="1">0&#x02013;4 hr</td><td align="center" rowspan="1" colspan="1">4&#x02013;24 hr</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" colspan="3" rowspan="1"> Cytokine</td><td align="center" rowspan="1" colspan="1">&#x0003e; 4.0</td><td align="center" rowspan="1" colspan="1">&#x0003e; 4.0</td></tr><tr><td align="left" rowspan="4" colspan="1"/><td align="left" rowspan="1" colspan="1">interleukin 1, beta</td><td align="left" rowspan="1" colspan="1"><italic>IL1B</italic></td><td align="center" rowspan="1" colspan="1">26.00 </td><td align="center" rowspan="1" colspan="1">4.09 </td></tr><tr><td align="left" rowspan="1" colspan="1">interleukin 8</td><td align="left" rowspan="1" colspan="1"><italic>IL8</italic></td><td align="center" rowspan="1" colspan="1">16.28 </td><td align="center" rowspan="1" colspan="1">4.84 </td></tr><tr><td align="left" rowspan="1" colspan="1">interleukin 1, alpha</td><td align="left" rowspan="1" colspan="1"><italic>IL1A</italic></td><td align="center" rowspan="1" colspan="1">4.32 </td><td align="center" rowspan="1" colspan="1">16.48 </td></tr><tr><td align="left" rowspan="1" colspan="1">Epstein-Barr virus induced 3</td><td align="left" rowspan="1" colspan="1"><italic>EBI3</italic></td><td align="center" rowspan="1" colspan="1">6.64 </td><td align="center" rowspan="1" colspan="1">7.44 </td></tr><tr><td align="left" colspan="5" rowspan="1"> Enzyme</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">indoleamine 2,3-dioxygenase 1</td><td align="left" rowspan="1" colspan="1"><italic>IDO1</italic></td><td align="center" rowspan="1" colspan="1">5.67 </td><td align="center" rowspan="1" colspan="1">30.67 </td></tr></tbody></table><table-wrap-foot><p>Early responding genes were those altered at 4 hr; slowly responding genes were those altered at 24 hr.</p></table-wrap-foot></table-wrap> as &#x0201c;early responding genes&#x0201d;. Among these genes, 6 categorized as &#x0201c;cytokine&#x0201d; were markedly upregulated after 4 hr of treatment but were moderately upregulated after 24 hr of treatment. Moreover, 2 genes in the &#x0201c;transmembrane receptor&#x0201d; category, 2, 2 genes in the &#x0201c;enzyme&#x0201d; category and 5 genes in the &#x0201c;transcription regulator&#x0201d; category were upregulated in THP-1 cells by L-92 treatment at 4 hr of treatment but showed moderate upregulation at 24 hr of treatment (<xref rid="tbl_002" ref-type="table">Table 2</xref>). In contrast, genes that showed less than a 4-fold change after 4 hr, but over a 4-fold increase in expression after 24 hr of treatment compared with that after 4 hr were categorized as &#x0201c;slowly responding genes&#x0201d; (<xref rid="tbl_002" ref-type="table">Table 2</xref>). Among these genes, 9 categorized as &#x0201c;transmembranes&#x0201d; were markedly upregulated after 24 hr but showed mild changes after 4 hr of treatment. Moreover, 6 genes in the &#x0201c;cytokine&#x0201d; category and 6 genes in &#x0201c;transcription regulator&#x0201d; category also showed a significant increase after 24 hr but only mild changes after 4 hr of treatment with L-92 (<xref rid="tbl_002" ref-type="table">Table 2</xref>). Finally, four genes categorized as &#x0201c;cytokine&#x0201d; and 1 gene categorized as an &#x0201c;enzyme&#x0201d; were constitutively expressed after 4 and 24 hr of treatment (&#x0201c;constitutively responding genes&#x0201d; in <xref rid="tbl_002" ref-type="table">Table 2</xref>). These results suggest that L-92 treatment in THP-1 cells might cause a rapid change in expression among genes in the &#x0201c;cytokine&#x0201d; category after 4 hr of treatment and subsequently among genes involved in cell signaling categorized here as &#x0201c;transmembrane receptors&#x0201d; and &#x0201c;cytokine&#x0201d; (<xref rid="tbl_002" ref-type="table">Table 2</xref>). For these cell responses, the activation of &#x0201c;transcription regulator&#x0201d; genes observed in the early and late phases of responses seems to be important for subsequent gene expression.</p></sec><sec><title>Quantitative analysis of the gene expressions in THP-1 cells</title><p>To validate the gene expression changes observed in THP-1 cells in response to L-92 cell treatment (<xref rid="tbl_002" ref-type="table">Table 2</xref>), qRT-PCR analysis was performed for 6 randomly selected genes with specific primer pairs: <italic>TNF</italic> (early responding genes), <italic>CRLF2, CD86 and STAT4</italic> (slowly responding genes); and <italic>IL8</italic> and <italic>IDO1 </italic>(constitutively responding genes). As shown in <xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> Changes in gene expressions from 0 to 4 hr (black bar) and 4 to 24 hr (white bar) observed in THP-1 cells after treatment with L-92 cells as shown by qRT-PCR analysis. The dotted line represents a 4-fold change in expression. The mean &#x000b1; SD levels of expression of differentially expressed genes and statistical significance of each gene&#x02019;s expression between 0 and 4 hr (black) and 4 and 24 hr (white) are shown. *p&#x0003c;0.05; **p&#x0003c;0.01 (determined by Student&#x02019;s t-test).</p></caption><graphic xlink:href="bmfh-33-157-g001"/></fig>, statistical changes were confirmed for most of the quantified gene expressions in L-92-treated THP-1 cells. The <italic>TNF</italic> gene categorized as an early responding gene was significantly upregulated at 4 hr after treatment (black) but not at 24&#x02005;hr after treatment (white) (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>). In contrast, in the genes categorized as slowly responding genes, <italic>CRLF2, CD86</italic> and <italic>STAT4</italic>, the change in expression of each upregulated gene was bigger at 24 hr (white) than that at 4 hr (black) after treatment (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>). Genes categorized as constitutively responding genes, <italic>IL8</italic> and <italic>IDO1,</italic> were upregulated both at 4 hr and 24hr (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>). As shown in <xref ref-type="fig" rid="fig_001">Fig. 1</xref>, the gene expressions randomly quantified by qRT-PCR in THP-1 cells after L-92 treatment showed changes similar to those quantified by microarray analysis (<xref rid="tbl_002" ref-type="table">Table 2</xref>).</p></sec><sec><title>Highlighted responses in THP-1 cells</title><p>These cell responses at 4 hr and 24 hr are illustrated in <xref ref-type="fig" rid="fig_002">Fig. 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Changes in gene expression in THP-1 cells after 4 hr of treatment with <italic>Lactobacillus acidophilus</italic> L-92 cells. Genes that were significantly upregulated by L-92 treatment were categorized by the IPA method as described in the Materials and Methods. Upregulated genes are indicated by red arrows. Black thin arrows represent the predicted flow of the pathway. Genes grouped by KEGG analysis are indicated by the red dotted line.</p></caption><graphic xlink:href="bmfh-33-157-g002"/></fig> as the early response and in <xref ref-type="fig" rid="fig_003">Fig. 3</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p> Changes in gene expression in THP-1 cells after 24 hr of treatment with <italic>Lactobacillus acidophilus</italic> L-92 cells. Genes that were significantly upregulated genes by L-92 treatment were categorized by the IPA method as described in the Materials and Methods. Upregulated genes are indicated by red arrows. Black thin arrows represent the predicted flow of the pathway. Genes grouped by KEGG analysis are indicated by the red dotted line.</p></caption><graphic xlink:href="bmfh-33-157-g003"/></fig> as the slow response. As summarized in <xref ref-type="fig" rid="fig_002">Fig. 2</xref>, in the early phase of the cellular response (4 hr of treatment), various transcription regulator genes, such as <italic>JUN</italic>, <italic>NFkB1</italic>, <italic>NFkB2</italic>, <italic>HIVEP2</italic> and <italic>RELB,</italic> as well as genes encoding chemokines and cytokines, such as <italic>CXCL11, CCL4,, CCL3, IL1B, IL8</italic> and <italic>TNF,</italic> were upregulated by L-92 treatment. Two transmembrane receptor genes, <italic>TLR7</italic> and <italic>ICAM1</italic>, were also upregulated at the early phase of the treatment (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). Moreover, the <italic>NOD2</italic> and <italic>MyD88</italic> genes, which were not involved in the hematological processes in the shown in <xref rid="tbl_002" ref-type="table">Table 2</xref>, were selected as important genes in the MAPK signal pathway and NOD-like receptor signal pathway by KEGG pathway analysis, with 2.99- fold and 1.25-fold upregulated gene expressions at 4 hr (p&#x0003c;0.05 and p&#x0003c;0.05, respectively) in response to L-92 treatment (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). During the slow response of the THP-1 cells (24 hr of treatment), mainly transmembrane receptors, such as <italic>IL2RA, IL7R, CD80, CRLF2, CD86, CD5, HLA-DQA1, IL15RA</italic> and <italic>CSF2RA</italic>, were activated (<xref ref-type="fig" rid="fig_003">Fig. 3</xref>). Some cytokine genes, namely <italic>IL6, IL23A</italic> and <italic>CCL22</italic>, were also significantly upregulated.</p><p>As listed in <xref rid="tbl_002" ref-type="table">Table 2</xref> and <xref ref-type="fig" rid="fig_002">Figs. 2</xref> and<xref ref-type="fig" rid="fig_003"> 3</xref>, in the early phase of the response in THP-1 cells, mainly transcription regulator genes and cytokines/chemokines were activated. Subsequently, many transmembrane receptor genes were mainly upregulated in THP-1 cells in the late phase of treatment.</p></sec></sec><sec sec-type="discussion" id="s4"><title>DISCUSSION</title><p>Increases in some Th1-type cytokines and chemokines have been observed in previous <italic>in vitro</italic> and <italic>in vivo</italic> studies with L-92 [<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r13" ref-type="bibr">13</xref>], although those studies did not include detailed analyses of the underlying mechanisms. In the present study, we first investigated the immunomodulatory responses that occurred in THP-1 cells as a model of APCs in response to probiotic L-92 cell exposure by using microarray analysis for a detailed understanding of the host gastrointestinal immune response. As expected from a previous study showing the probiotic anti-allergy effects of L-92 <italic>in vivo</italic>, many Th1-type cytokines and chemokines that would be expected to increase during anti-allergic and anti-inflammatory events were elevated in THP-1 cells treated with L-92 in the present study, as summarized in <xref ref-type="fig" rid="fig_002">Figs. 2</xref> and<xref ref-type="fig" rid="fig_003"> 3</xref>. The THP-1 response in the early phase involved mainly the activation of transcription regulator genes and cytokine/chemokine genes categorized as the MAPK signaling pathway and the NOD-like receptor signaling pathway (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). Subsequently, many transmembrane receptor and transcription regulator genes in the Toll-like receptor signaling pathway were upregulated in the late phase of the treatment (<xref ref-type="fig" rid="fig_003">Fig. 3</xref>). Taking into account the above results, the transcriptional upregulation observed in THP-1 cells co-cultured with L-92 seems to well explain the previous <italic>in vivo</italic> immunomodulatory effects of L-92 treatment [<xref rid="r7" ref-type="bibr">7</xref>,<xref rid="r8" ref-type="bibr">8</xref>,<xref rid="r9" ref-type="bibr">9</xref>,<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r13" ref-type="bibr">13</xref>, <xref rid="r15" ref-type="bibr">15</xref>].</p><p>Regarding the activation of the Th1-type immune response by L-92 in THP-1 cells, one of the first events would be the induction of transcription regulator genes involved in TLR-7 signaling; this would trigger the release of some of Th1-type cytokines and inflammatory cytokines in the MAPK signaling pathway (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). These molecules, together with the upregulation of surface markers involved in the maturation of APCs such as <italic>CD86, CD80</italic> and <italic>CD40</italic>, stimulate the activation of Th1 cells [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r23" ref-type="bibr">23</xref>]. These changes in gene expression observed in THP-1 cells strongly suggest that the <italic>in vivo</italic> anti-allergy effect of L-92 might be linked to a Th1-type host response in a phase-dependent manner in the gastrointestinal immune system.</p><p>Considering the constitutive upregulation of the indoleamine 2,3-dioxygenase (<italic>IDO</italic>) gene observed in the present study (<xref rid="tbl_002" ref-type="table">Table 2</xref>), other upregulated genes encoding cytokines, such as <italic>IL8, TNF, IL1B</italic> and <italic>IL12</italic>, seem to be closely linked to IDO enhancement. IDO has been identified as a key enzyme with immunomodulatory effects, resulting from its enzymatic activity that leads to catabolism of the essential amino acid L-tryptophane [<xref rid="r24" ref-type="bibr">24</xref>, <xref rid="r25" ref-type="bibr">25</xref>]. This enzyme is expressed in epithelial cells, macrophages, and DCs induced by proinflammatory cytokines, including type I and type II IFN [<xref rid="r26" ref-type="bibr">26</xref>]. Recent studies suggested the involvement of IL-1&#x003b2;, TNF-&#x003b1; and IL-12 in the induction of IDO in DCs [<xref rid="r27" ref-type="bibr">27</xref>, <xref rid="r28" ref-type="bibr">28</xref>] and engagement of CTLA-4 with CD80/CD86 on the membrane of DCs in the stimulation of IDO transcriptional expression and activity [<xref rid="r29" ref-type="bibr">29</xref>, <xref rid="r30" ref-type="bibr">30</xref>]. These observations suggest that the upregulation of CD80 and CD86 on the membrane of APCs caused by treatment with L-92 might be closely linked to activation of the <italic>IDO</italic> gene. IDO1 is known to induce regulatory T cells (Tregs) [<xref rid="r31" ref-type="bibr">31</xref>, <xref rid="r32" ref-type="bibr">32</xref>] and to inhibit T-cell proliferation and promote T-cell apoptosis by degradation of L-tryptophan to convert into toxic metabolites such as quinolinic acid and 3-hydroxy-anthranilic acid [<xref rid="r31" ref-type="bibr">31</xref>, <xref rid="r33" ref-type="bibr">33</xref>].</p><p>In conclusion, key molecules are induced in cultured THP-1 cells by L-92 stimulation that are suggested to play a central role in skewing toward Th1 cells and also in inducing T cell suppression networks. The present study on cultured THP-1 cell opens the way for understanding the immunomodulatory networks modulated by L-92 in the gastrointestinal immune system <italic>in vivo</italic>. The primary information on both gene profiling and the key genes altered in THP-1 cells after L-92 exposure obtained in the present study will be very important for a detailed understanding of the strain-dependent immune responses to other <italic>Lactobacillus</italic> species in future studies.</p></sec>
<italic>Mycobacterium tuberculosis</italic> PE25/PPE41 protein complex induces necrosis in macrophages: Role in virulence and disease reactivation?
<p>Necrotic cell death during TB infection is an important prerequisite for bacterial dissemination and virulence. The underlying mechanisms and the bacterial factors involved therein are not well understood. The <italic>Mycobacterium tuberculosis</italic> (<italic>M. tuberculosis</italic>) co-operonic PE25/PPE41 protein complex, similar to ESAT-6/CFP-10, belonging to the PE/PPE and ESAT-6 families of genes has co-expanded and co-evolved in the genomes of pathogenic mycobacteria. We report a novel role of this highly immunogenic PE25/PPE41 protein complex in inducing necrosis, but not apoptosis, in macrophages. We propose that these protein complexes of <italic>M. tuberculosis</italic>, secreted by similar/unique transport system (Type VII), have an important role in <italic>M. tuberculosis</italic> virulence and disease reactivation.</p>
<contrib contrib-type="author"><name><surname>Tundup</surname><given-names>Smanla</given-names></name><xref rid="af005" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">&#x0204e;</xref></contrib><contrib contrib-type="author"><name><surname>Mohareer</surname><given-names>Krishnaveni</given-names></name><xref rid="af010" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Hasnain</surname><given-names>Seyed E.</given-names></name><email>[email protected]</email><xref rid="af015" ref-type="aff">c</xref><xref rid="af020" ref-type="aff">d</xref><xref rid="cor1" ref-type="corresp">,&#x0204e;</xref></contrib>
FEBS Open Bio
<sec sec-type="intro" id="s0005"><label>1</label><title>Introduction</title><p>Cell death either due to apoptosis or necrosis is very common during infection with <italic>Mycobacterium tuberculosis</italic> (<italic>M. tuberculosis</italic>), the causative agent of tuberculosis (TB). Apoptosis of antigen presenting cells (APCs) is known to be an innate mechanism of the host to fight infections. <italic>M. tuberculosis</italic> induces apoptosis in APCs through TNF-&#x003b1;, toll like receptors (TLRs), Fas or by altering the expression of Bax/Bcl-xL <italic>via</italic> an oxygen dependent pathway <xref rid="b0005 b0010 b0015 b0020" ref-type="bibr">[1&#x02013;4]</xref>. Virulent strains of <italic>M. tuberculosis</italic> have been known to develop mechanisms that resist host apoptotic cell death <xref rid="b0025 b0030 b0035 b0040" ref-type="bibr">[5&#x02013;8]</xref>, however they are capable of inducing necrotic cell death, which helps in bacterial multiplication and dissemination <xref rid="b0230 b0235 b0055 b0060" ref-type="bibr">[9&#x02013;12]</xref>. On the other hand non-virulent strains like <italic>Mycobacterium bovis</italic> BCG and <italic>Mycobacterium smegmatis</italic> fail to induce necrosis of the host cells <xref rid="b0235 b0055" ref-type="bibr">[10,11]</xref>, suggesting that the factors released by the virulent strains interfere with the host cell death machinery for their release and subsequent infection of the neighboring (fresh) cells <xref rid="b0040" ref-type="bibr">[8]</xref>. Although necrotic cell death during <italic>M. tuberculosis</italic> infection is a common observation, very little is known about the mechanisms. A unique property of H<sub>37</sub>Rv strain in preventing apoptotic envelope formation leading to necrotic cell death of macrophages has been recently reported <xref rid="b0065" ref-type="bibr">[13]</xref>. Even though it is clear that host-pathogen interactions at the interface of cell death are a key to <italic>M. tuberculosis</italic> dissemination and virulence, the underlying mechanism(s) and the <italic>M. tuberculosis</italic> factors involved in the processes are very poorly understood.</p><p>RD1 locus, present in the genome of only the virulent mycobacterial strains, is believed to encode virulent factors ESAT-6 and CFP-10 complex secreted <italic>via</italic> a secretory locus called <italic>esx-1</italic> <xref rid="b0070" ref-type="bibr">[14]</xref>. ESX-1 locus also contains a pair of genes encoding PE/PPE proteins. PE/PPE proteins are coded by a family of genes that include PE, PPE and PGRS (or PE/PPE/PGRS) and these represent 10% of the coding capacity of the mycobacterial genome Although the exact roles of PE/PPE proteins in virulence and regulation of their expression in mycobacteria are not completely understood, it is believed that these have important function in mycobacterial virulence and pathogenesis <xref rid="b0075 b0080 b0085" ref-type="bibr">[15&#x02013;17]</xref>. The findings that PE/PPE genes are duplicated and expanded in the genomes of pathogenic mycobacteria during the course of evolution lends further support to their role in virulence <xref rid="b0090" ref-type="bibr">[18]</xref>. We earlier reported that the PE/PPE genes in the genome of <italic>M. tuberculosis</italic> are organized in operons with the members of the same family as well as those belonging to ESAT-6 like family <xref rid="b0095" ref-type="bibr">[19]</xref>. Proteins belonging to these families are secreted by pathogenic mycobacteria using specialized (Type VII) secretion systems. For example, ESAT-6 protein of RD-1 locus and PE25/PPE41 protein complex are secreted by pathogenic mycobacteria <italic>via</italic> Esx-1 and Esx-5 loci, respectively <xref rid="b0100 b0240 b0110" ref-type="bibr">[20&#x02013;22]</xref>. This supports the observation that the PE/PPE proteins and the ESAT-6 family of genes are co-evolved and co-expanded in the genome of pathogenic mycobacteria <xref rid="b0090 b0095" ref-type="bibr">[18,19]</xref>. This is also evident from the observations that the pathogenic mycobacteria harbor multiple <italic>esx</italic> secretory system containing genes encoding both PE/PPE and ESAT-6 family of proteins <xref rid="b0075 b0090 b0100" ref-type="bibr">[15,18,20]</xref>. Functionally, the <italic>esx-5</italic> locus induces bacterial dissemination by cell lysis during mycobacterial infection, suggesting that the proteins secreted through this locus play an important role in virulence <xref rid="b0110" ref-type="bibr">[22]</xref>. We previously reported that the PPE41 and PE25 proteins interact with each other and form complex in a fashion similar to ESAT-6 and CFP-10 <xref rid="b0095" ref-type="bibr">[19]</xref>. To form soluble complex, PE25 and PPE41 interact at the translational or translocation levels since PE25 or PPE41, when expressed individually in <italic>Escherichia coli</italic> cells, remain insoluble as opposed to when co-expressed as an operon <xref rid="b0095" ref-type="bibr">[19]</xref>. That this is indeed the case is evident from recent observation that co-expression of PE25 and PPE41, as well as the C-terminal of PE25 is indispensable for the secretion of PPE41 protein in pathogenic mycobacteria <xref rid="b0100" ref-type="bibr">[20]</xref>. We also reported that the PE25/PPE41 complex elicited stronger immune responses in TB patients as well as in a mouse model, compared to individually expressed PE25 or PPE41 proteins <xref rid="b0115 b0120" ref-type="bibr">[23,24]</xref>, thereby suggesting that PE25/PPE41 complex is recognized by the host immune system. The <italic>esx</italic> secretory systems, including <italic>esx-1</italic> and <italic>esx-5</italic>, are also involved in secretion of other PE/PPE and ESAT-6 proteins, which potentially form a part of the virulence component factors that help in reactivation and dissemination of the pathogenic mycobacteria. Based on the evidences that the PE25/PPE41 complex and ESAT-6 family of proteins are exported out of mycobacteria (<italic>via</italic> special secretory loci, <italic>esx-1</italic> and <italic>esx-5</italic>) and induce necrosis or host cell lysis, but not apoptosis, we hypothesize that the genes belonging to PE/PPE and ESAT-6 family facilitate bacterial dissemination, multiplication and infection of fresh/neighboring cells leading to increased virulence, pathogenicity and disease reactivation.</p></sec><sec sec-type="materials|methods" id="s0010"><label>2</label><title>Materials and methods</title><sec id="s0015"><label>2.1</label><title>Protein expression and purification</title><p>The purification of the recombinant protein was carried out as described previously <xref rid="b0095" ref-type="bibr">[19]</xref>. Briefly, <italic>E. coli</italic> BL-21 cells were transformed with pETDuet vector carrying both the <italic>pe25</italic> and <italic>ppe41</italic> genes for the expression of recombinant proteins. Soluble fraction of the co-expressed protein was loaded onto a cobalt affinity column and washed with 1X PBS containing 25&#x000a0;mM Imidazole. The bound proteins were eluted with 200&#x000a0;mM imidazole in 1X PBS and dialyzed against 1X PBS. To obtain the protein complex, the co-purified proteins were loaded onto Superose 6 column and the fractions corresponding to the sharp (&#x0223c;70&#x000a0;kDa) peak containing both PE25 and PPE41 proteins were collected, as described <xref rid="b0095" ref-type="bibr">[19]</xref>. The concentration of the recombinant proteins was measured using BCA protein assay kit (Pierce). The purified recombinant protein was treated with Polymyxin B sepharose and the endotoxin level was measured with LAL (Limulus Amebocyte Lysates) method, which was &#x0003c;10&#x000a0;pg/ml.</p></sec><sec id="s0020"><label>2.2.</label><title>MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay</title><p>RAW264.7 macrophages were seeded and stimulated with different concentrations of the recombinant protein in a final volume of 250&#x000a0;&#x003bc;l of RPMI per well. The cell culture was incubated at 37&#x000a0;&#x000b0;C under 5% CO<sub>2</sub> and 70% relative humidity. Cell cytotoxicity was tested by MTT assay by adding 10&#x000a0;&#x003bc;l of 5&#x000a0;mg/ml MTT. Cells were then incubated at 37&#x000a0;&#x000b0;C for 3&#x02013;4&#x000a0;h and then 100&#x000a0;&#x003bc;l of solubilization buffer (20% SDS, 50% N-Methyl Formamide) was added and absorbance was measured at 570&#x000a0;nm.</p></sec><sec id="s0025"><label>2.3</label><title>Enzyme linked immunosorbent assay (ELISA)</title><p>The levels of cytokines present in the culture supernatants were quantified by commercially available two-site sandwich enzyme-linked immunosorbent assay kit (BD OptEIA&#x02122; set Mouse, IL-12, IL-10 and TNF-&#x003b1;) according to the manufacturer&#x02019;s instructions. Briefly, 96 well plates were coated with coating antibody, incubated at 4&#x000a0;&#x000b0;C overnight, blocked with 2% BSA (blocking buffer) and incubated for 2&#x000a0;h at 37&#x000a0;&#x000b0;C. The culture supernatant was then added followed by detection antibody for 1&#x000a0;h at RT. The plates were then incubated with HRP conjugated secondary antibody for 1&#x000a0;h at 37&#x000a0;&#x000b0;C and the HRP activity was determined by using the substrate, o-phenylenediamine tetra-hydrochloride and H<sub>2</sub>O<sub>2</sub>. The plates were washed at every step of the incubation with wash buffer (1XPBS-Tween-20) for 5&#x02013;7 times. The reaction was terminated using 2&#x000a0;N H<sub>2</sub>SO<sub>4</sub> and the absorbance was measured at 492&#x000a0;nm in an ELISA reader.</p></sec><sec id="s0030"><label>2.4</label><title>TUNEL assay</title><p>Apoptosis was assayed using TUNEL assay kit (Promega), according to the manufacturer&#x02019;s instructions, in a 4 well chamber slide (BD, Bioscience). In brief, cells were washed with PBS, fixed with 3% paraformaldehyde and incubated for 30&#x000a0;min at RT. Cells were permeabilized with 0.1% Triton X-100 and incubated with reaction mixture containing fluorescein labeled dTTP and rdTTP transferase and allowed for nick end labeling for one hour at 37&#x000a0;&#x000b0;C. Reaction was stopped with 2X SSC. The slides were then rinsed with PBS and analyzed using confocal microscope.</p></sec><sec id="s0035"><label>2.5</label><title>Flow cytometry</title><p>Cell death assay was performed using Annexin V and propidium iodide (PI) staining kit (BD) according to the manufacturer&#x02019;s instructions. In brief, 0.2&#x000a0;&#x000d7;&#x000a0;10<sup>6</sup>&#x000a0;cells were stimulated with different concentrations of recombinant protein and incubated for different intervals of time. Cells were then harvested and washed in PBS twice and resuspended in 1X Annexin V binding buffer. Cells were stained with Annexin V and PI dyes, incubated for 15&#x000a0;min at RT and processed for flow cytometric analysis (BD, Vantage SE).</p></sec><sec id="s0040"><label>2.6</label><title>LDH assay</title><p>LDH release assay was carried out with the culture supernatant of RAW 264.7 macrophages stimulated with different concentration of recombinant protein and LPS (5&#x000a0;&#x003bc;g/ml). The reaction was carried out in a buffer containing 80&#x000a0;mM Tris&#x02013;HCl (pH 7.2), 200&#x000a0;mM NaCl, 1.6&#x000a0;mM pyruvate, 0.2&#x000a0;mM NADH and 50&#x000a0;&#x003bc;l of culture supernatant. Optical density at 340&#x000a0;nm was recorded for 30&#x000a0;min using spectrophotometer. LDH activity was measured by calculating the rate of oxidation of NADH per unit time per mg of protein.</p></sec><sec sec-type="results" id="s0045"><label>2.7</label><title>Statistical analyses</title><p>The experiments were carried out in triplicate and repeated 4 or more independent times. Statistical significance was determined using student&#x02019;s <italic>t</italic> test.</p></sec></sec><sec sec-type="results" id="s0050"><label>3</label><title>Results</title><sec id="s0055"><label>3.1</label><title>The PE25/PPE41 complex drives TNF-&#x003b1; production in macrophages</title><p>We used the polymyxin B treated recombinant PE25/PPE41 complex protein to stimulate <italic>in vitro</italic> cultured RAW264.7 macrophages and scored for inflammatory cytokines TNF-&#x003b1;, IL-10 and IL-12. Strong induction of TNF-&#x003b1;, indicative of pro-inflammatory response, could be seen when RAW264.7 macrophages were stimulated with the PE25/PPE41 protein complex. The up regulation of TNF-&#x003b1; by recombinant PE25/PPE41 complex was observed in a dose and time dependent manner (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). The production of TNF-&#x003b1; was observed as early as 2&#x000a0;h of incubation (&#x0223c;400&#x000a0;pg/ml). Recombinant PE or PPE protein when expressed individually could also induce TNF-&#x003b1; expression (data not shown). Control experiments with heat or protease treated protein failed to stimulate TNF-&#x003b1; secretion (<xref rid="f0005" ref-type="fig">Fig. 1</xref>), clearly demonstrating that the increase in TNF-&#x003b1; secretion is not due to endotoxin contamination but is due to the PE/PPE complex. The PE/PPE complex failed to drive production of IL-12 and IL-10 after incubation with the macrophages even up to 48&#x000a0;h (data not shown). These results demonstrate that the PE25/PPE41 protein complex is able to mount a strong pro-inflammatory response in the form of TNF-&#x003b1; production.</p></sec><sec id="s0060"><label>3.2</label><title>PE25/PPE41 complex induced cell death is not due to TNF-&#x003b1;, NO or NF&#x003ba;B mediated signaling</title><p><italic>In-vitro</italic> cultured murine macrophage RAW264.7 cells were treated with recombinant PE25/PPE41 protein complex and cell death was measured using MTT assay. It could be seen that PE25/PPE41 complex induces cell death in a concentration dependent manner, causing approximately 26%, 42% and 60% cell death at a concentration of 0.3, 3 and 10&#x000a0;&#x003bc;g/ml, respectively after 12&#x000a0;h of incubation (<xref rid="f0010" ref-type="fig">Fig. 2</xref>).</p><p>A member of the PE/PPE family &#x02013; PE_PGRS33 protein &#x02013; has been reported to induce TNF-&#x003b1; secretion in RAW264.7 macrophages leading to apoptotic cell death <xref rid="b0125" ref-type="bibr">[25]</xref>. Therefore, experiments were designed to investigate the role of TNF-&#x003b1;, nitric oxide (NO) or nuclear factor kappa B (NF&#x003ba;-B) mediated signaling in PE25/PPE41 protein complex mediated cell death. MTT assay was carried out after pre-incubation of macrophage cells with anti-TNF-&#x003b1; antibody, amino guanidine (AG, inhibitor of nitric oxide synthesis), or PDTC (inhibitor of nuclear factor kappa B), followed by incubation with varying concentrations (0.3&#x02013;10&#x000a0;&#x003bc;g/ml) of the PE/PPE complex. Expectedly, while PE/PPE protein complex caused cell death as a direct function of protein concentration, cell death remains unchanged in the absence of TNF&#x003b1;, NO or NF&#x003ba;B (<xref rid="f0015" ref-type="fig">Fig. 3</xref>). These results suggest that cell death induced by the PE/PPE protein complex is independent of NF&#x003ba;B, NO or TNF-&#x003b1; mediated signaling.</p></sec><sec id="s0065"><label>3.3</label><title>The PE25/PPE41 complex induces necrosis, but not apoptosis of mouse macrophages</title><p>In order to differentiate necrotic cell death from apoptosis, propidium iodide (PI)/Annexin V staining was carried out using macrophages stimulated with PE25/PPE41 complex (<xref rid="f0020" ref-type="fig">Fig. 4</xref>A and B). Propidium iodide easily passes through the ruptured membrane of dead cells and stains nucleic acids, but live cells or cells in early apoptotic phase are impermeable to PI dye. Unlike PI, Annexin V binds to phosphatidylserine with high affinity, which is externalized on the surface of apoptosed or dead cells, thus recognizing cells undergoing apoptosis even at early stage of cell death. Using flow cytometry staining, it could be seen that while PI uptake by the macrophages was a direct function of increasing concentration of the PE/PPE complex (<xref rid="f0020" ref-type="fig">Fig. 4</xref>A), the necrotic population of macrophages (PI<sup>+</sup>/AnnexinV<sup>+</sup>) increased at 2, 8 and 12&#x000a0;h of incubation, respectively (<xref rid="f0020" ref-type="fig">Fig. 4</xref>B). Absence of apoptotic population (PI<sup>&#x02212;</sup>/AnnexinV<sup>+</sup>) suggests that PE/PPE protein complex induces necrosis of macrophages but not apoptosis (<xref rid="f0020" ref-type="fig">Fig. 4</xref>B). These results clearly suggest that the PE25/PPE41 protein complex induces necrosis in macrophages in a time dependent manner. Apoptotic cell death was also ruled out by TUNNEL staining. Macrophages incubated with PE25/PPE41 complex at a concentration as high as 10&#x000a0;&#x003bc;g/ml were negative for TUNEL staining (<xref rid="f0020" ref-type="fig">Fig. 4</xref>C). Necrotic cell death was further validated by lactate dehydrogenase (LDH) release assay. LDH assay involves measurement of NADH oxidation to NAD by LDH (released in to the medium from ruptured cells) in presence of pyruvic acid. RAW264.7 macrophages were stimulated with PE25/PPE41 at two different concentrations of PE25/PPE41 protein complex and LDH released was measured in the culture supernatant. It could be seen that the PE25/PPE41 protein complex induced release of LDH into the supernatant of RAW264.7 macrophages in a concentration dependent manner (<xref rid="f0025" ref-type="fig">Fig. 5</xref>). Very low oxidation of NADH was seen in the supernatant of cells left unstimulated (<xref rid="f0025" ref-type="fig">Fig. 5</xref>) or stimulated with concentration as high as 5&#x000a0;&#x003bc;g/ml of LPS (<xref rid="f0025" ref-type="fig">Fig. 5</xref>). Taken together these results demonstrate that the PE25/PPE41 protein complex induces necrosis, but not apoptosis, in RAW 264.7 macrophage cells.</p></sec></sec><sec sec-type="discussion" id="s0070"><label>4</label><title>Discussion</title><p>Macrophages are the cellular host of mycobacterial infection and constitute the first line of defense without which the host is unable to provide complete protection <xref rid="b0130" ref-type="bibr">[26]</xref>. <italic>M. tuberculosis</italic> employs various strategies to counteract macrophage protective responses <xref rid="b0135" ref-type="bibr">[27]</xref>. Induction of necrotic cell death in macrophages is one such strategy that helps in bacterial dissemination and nutrients uptake inside the granulomas, while preventing apoptotic cell death, that acts as an innate defense against <italic>M. tuberculosis</italic> infection <xref rid="b0140" ref-type="bibr">[28]</xref>. It is unclear how <italic>M. tuberculosis</italic> induces host cell necrosis and regulates its growth and multiplication especially during the latent phase of the growth. Here we report that a PE/PPE complex induces macrophage cell death by necrosis. Based on the current findings and previous reports we suggest that one of the important virulent features of the secretory PE/PPE proteins is to induce necrotic cell death of macrophages that helps the bacterium to survive and multiply in the host.</p><p>Necrotic cell death commonly observed in mycobacterial lesion and granulomas is driven by bacterial factors but not due to secretion of TNF-&#x003b1; during the infection <xref rid="b0145 b0150" ref-type="bibr">[29,30]</xref>. Production of TNF-&#x003b1; in response to the PE25/PPE41 complex is suggestive of a host response elicited against the protein since it has been reported that TNF-&#x003b1; provides protection against <italic>M. tuberculosis</italic> infection perhaps through the induction of host cell apoptosis <xref rid="b0140 b0155 b0160" ref-type="bibr">[28,31,32]</xref>. While various pathways are known that mediate apoptotic cells death <xref rid="b0005 b0010 b0015 b0020" ref-type="bibr">[1&#x02013;4]</xref>, not much is known about the mechanisms driving necrotic cell death during mycobacterial infection. Our observations that necrotic cell death, in response to PE25/PPE41 complex, is independent of TNF-&#x003b1;, nitric oxide or NF-&#x003ba;B signaling suggests that PE25/PPE41 utilizes an unknown mechanism(s) to drive macrophage cell death that may ultimately contribute to virulence. Secretory apparatus, <italic>esx-5</italic>, has been known to be involved in caspase independent, cathepsin-B dependent cell death of macrophages during mycobacterial infection. Since PPE41 protein secretion is mediated by <italic>esx-5</italic> apparatus, the role of cathepsin B in induction of cell death in response to PE25/PPE41 protein complex cannot be ruled out <xref rid="b0240" ref-type="bibr">[21]</xref>. This ability of PE25/PPE41 protein complex to induce necrosis, supports the observation that necrotic cell death during mycobacterial infection is not driven by host response but by bacterial factor(s).</p><p>The association of PE/PPE and ESAT-6 gene families with virulence is evident from the observations that the duplication and expansion of the PE/PPE and ESAT-6 genes during the course of evolution occurred only in pathogenic mycobacteria <xref rid="b0090 b0095" ref-type="bibr">[18,19]</xref>. These genes are very uniquely organized in the genome with <italic>pe</italic> and <italic>ppe</italic> genes in operon with another member of <italic>pe/ppe</italic> or <italic>esat-6</italic> family of genes <xref rid="b0095" ref-type="bibr">[19]</xref> and evidently both ESAT-6 and PE/PPE family of proteins are secreted by Type VII/ESX secretory apparatus <xref rid="b0075" ref-type="bibr">[15]</xref>. This also suggests that PE/PPE and ESAT-6 family of genes may have played important role in adaptation and host specificity, as these were earlier shown to be differentially expressed in different species of pathogenic mycobacteria <xref rid="b0165 b0170 b0175" ref-type="bibr">[33&#x02013;35]</xref>. Although exact function of PE/PPE proteins remain unknown, reports suggest that these are surface localized or secreted outside by mycobacteria and may have direct role in virulence and pathogenesis <xref rid="b0080 b0110 b0180 b0185 b0190 b0195 b0245 b0205 b0210 b0250" ref-type="bibr">[16,22,36&#x02013;43]</xref>. One of the important virulence mechanisms of pathogenic mycobacteria appears to be their ability to induce necrosis of host immune cells, as not only PE25/PPE41 complex, ESAT-6 protein has also been shown to induce necrosis of immune cells <italic>via</italic> an unknown mechanism <xref rid="b0220" ref-type="bibr">[44]</xref>. The fact that the PE/PPE proteins mount both B- and T-cell immune responses <xref rid="b0115 b0120 b0225" ref-type="bibr">[23,24,45]</xref> provokes us to also suggest a novel role for these protein families, present exclusively in the genus Mycobacterium, in &#x02018;immune quorum sensing&#x02019; (<xref rid="f0030" ref-type="fig">Fig. 6</xref>) wherein these proteins are secreted to sense the immune status of the host after which through the possible involvement of cell surface constituents that influence interactions with other cells <xref rid="b0190" ref-type="bibr">[38]</xref>, convey a signal to stay dormant or undergo necrotic cell death. Based on our present and earlier findings, and existing evidences, we believe that the co-evolved and co-expanded PE/PPE and ESAT-6 family of proteins are secreted out, using sophisticated bacterial Type VII secretory systems, interact with host immune cells (<xref rid="f0030" ref-type="fig">Fig. 6</xref>), and facilitate mycobacterial dissemination and multiplication, which may ultimately lead to disease reactivation.</p></sec>
A new <italic>pma1</italic> mutation identified in a chronologically long-lived fission yeast mutant
<p>We isolated a chronologically long-lived mutant of <italic>Schizosaccharomyces pombe</italic> and found a new mutation in <italic>pma1</italic><sup>+</sup> that encoded for an essential P-type proton ATPase. An Asp-138 to Asn mutation resulted in reduced Pma1 activity, concomitant with an increase in the chronological lifespan of this fission yeast. This study corroborates our previous report indicating Pma1 activity is crucial for the determination of life span of fission yeast, and offers information for better understanding of the enzyme, Pma1.</p>
<contrib contrib-type="author"><name><surname>Naito</surname><given-names>Chikako</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Ito</surname><given-names>Hirokazu</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Oshiro</surname><given-names>Tomoko</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Ohtsuka</surname><given-names>Hokuto</given-names></name><xref rid="af010" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Murakami</surname><given-names>Hiroshi</given-names></name><xref rid="af015" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Aiba</surname><given-names>Hirofumi</given-names></name><email>[email protected]</email><xref rid="af010" ref-type="aff">b</xref><xref rid="cor1" ref-type="corresp">&#x0204e;</xref></contrib>
FEBS Open Bio
<sec sec-type="intro" id="s0005"><label>1</label><title>Introduction</title><p>The chronological lifespan of yeast cells is defined as the period during which cells can survive in a nondividing state and is determined by their viability after entry into the stationary phase <xref rid="b0005" ref-type="bibr">[1]</xref>. Studies on <italic>Saccharomyces cerevisiae</italic> have identified several novel longevity factors, including Ras2, Tor1, and Sch9 <xref rid="b0010" ref-type="bibr">[2]</xref>. In the fission yeast <italic>Schizosaccharomyces pombe</italic>, disruptions of <italic>pka1</italic><sup>+</sup> and <italic>sck2</italic><sup>+</sup> reportedly increased its chronological lifespan <xref rid="b0015" ref-type="bibr">[3]</xref>. The lifespan of <italic>S. pombe</italic> can also be extended by calorie restriction (CR), which is known to extend the life spans of various organisms from yeast to mammals. In <italic>S. pombe</italic>, this process relies on the Sty1 MAP kinase <xref rid="b0020" ref-type="bibr">[4]</xref>.</p><p>Because lifespan is a complex phenomenon, identifying new factors involved in regulating the chronological lifespan is essential for understanding lifespan as a whole. To this end, we have screened for <italic>S. pombe</italic> short-lived and long-lived mutants and identified <italic>lcf1</italic> and <italic>pma1</italic> mutants, respectively. <italic>lcf1</italic><sup>+</sup> encodes for a long-chain fatty acyl-CoA synthetase that is involved in fatty acid utilization and/or metabolism <xref rid="b0025" ref-type="bibr">[5]</xref>. <italic>pma1</italic><sup>+</sup> encodes for an essential P-type proton ATPase <xref rid="b0030" ref-type="bibr">[6]</xref>. We have also determined that deleting <italic>php2</italic>, which encodes for a subunit of the CCAAT-binding factor complex, results in extending the chronological lifespan of this fission yeast <xref rid="b0035" ref-type="bibr">[7]</xref>.</p><p>In this study we identified and characterized another allele of the <italic>pma1</italic> mutation that prolonged the chronological lifespan of <italic>S. pombe</italic>.</p></sec><sec sec-type="materials|methods" id="s0010"><label>2</label><title>Materials and methods</title><sec id="s0015"><label>2.1</label><title>Strains and media</title><p><italic>S. pombe</italic> strain JY333 (h<sup>-</sup> leu1-32 ade6-M216) was used for mutant screening. Strains were grown in SD medium [0.67% yeast nitrogen base without amino acids (Difco), and 2% glucose] supplemented with necessary growth requirements in standard amounts at 30&#x000a0;&#x000b0;C. Chronological lifespan analysis was done as described previously <xref rid="b0040" ref-type="bibr">[8]</xref>.</p></sec><sec id="s0020"><label>2.2</label><title>Linkage analysis</title><p>To conduct linkage analysis, a Km-resistant gene was inserted at 1824&#x02013;1835&#x000a0;bp downstream of <italic>pma1</italic><sup>+</sup> termination codon using previously described methods <xref rid="b0045" ref-type="bibr">[9]</xref>. Both the upstream and downstream regions of the desired insertion region were PCR-amplified using F1 and F2 primers and R1 and R2 primers, respectively. After mixing both DNA fragments with pFA6a-kanMX6, a PCR was performed using the F1 and R1 primers. JY336 (h<sup>+</sup> leu1-32 ade6-M210) was transformed with the amplified DNA fragment, and stable G418-resisitant transformants were selected. The chromosome construct was then confirmed by PCR using appropriate primers. The primers used were: F1, AGAAGTTATCGTGAGCTACG; F2, TTAATTAACCCGGGGATCCGGAAATCATTGATTTATCTATATAC; R1, GTCTTGGTCTGGTATCAACG; and R2, GTTTAAACGAGCTCGAATTCCATGGATAAGCTGCTAATCCATAAT.</p></sec><sec id="s0025"><label>2.3</label><title>Preparation of a Pma1 antibody</title><p>An antibody directed against Pma1 was prepared by immunizing a rabbit with the peptide MMNGKPKESRNQRSIEDL (Sigma&#x02013;Aldrich), which corresponded to amino acids 886&#x02013;903 of the Pma1 protein.</p></sec><sec id="s0030"><label>2.4</label><title>Pma1 ATPase assay</title><p>Pma1 ATPase activity was determined using previously described methods <xref rid="b0050" ref-type="bibr">[10]</xref>. Total cell lysates were used as the enzyme sources. This assay was conducted with or without 0.1&#x000a0;mM sodium vanadate, and released inorganic phosphate was determined using a Phospha C-Test (Wako Co., Japan). Vanadate-sensitive ATPase activity was determined and expressed as Pma1 activity.</p></sec><sec id="s0035"><label>2.5</label><title>Assay for glucose concentrations</title><p>Cells were grown in SD medium. In addition to monitoring cell growth, 20&#x000a0;&#x003bc;l of culture was sampled to determine the remaining glucose concentrations in medium using a Glucose CII-test kit (Wako Co., Ltd).</p></sec></sec><sec id="s0040"><label>3</label><title>Results and discussion</title><sec id="s0045"><label>3.1</label><title>L16 mutant phenotypic characterization</title><p>We previously screened for some long-lived mutant <italic>S. pombe</italic> candidates <xref rid="b0030" ref-type="bibr">[6]</xref>. In this study, we analyzed one uncharacterized mutant, designated L16. We first analyzed the long-lived phenotype of L16 that was grown in SD medium (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). As expected, L16 mutant cells&#x02019; viability was maintained for a long period after their entry into the stationary phase as compared with wild type cells (<xref rid="f0005" ref-type="fig">Fig. 1</xref>B). Cell morphology was also monitored along with cell growth in SD medium. As shown in <xref rid="f0005" ref-type="fig">Fig. 1</xref>C, there were no differences in cell morphology between wild type and L16 mutant cells during the logarithmic growth phase (sample point &#x0201c;a&#x0201d; shown in <xref rid="f0005" ref-type="fig">Fig. 1</xref>A). However, after entry into the stationary phase (sample points &#x0201c;b&#x0201d; and &#x0201c;c&#x0201d; in <xref rid="f0005" ref-type="fig">Fig. 1</xref>A), wild type cells exhibited abnormal morphologies, such as broken or shrunken figures. By comparison, many of the L16 mutant cells had normal morphologies. This phenotypic difference in morphology may have been due to the differences in viability after entry into the stationary phase.</p></sec><sec id="s0050"><label>3.2</label><title>Identifying a mutation site in the L16 mutant</title><p>We suspected a mutation in <italic>pma1</italic><sup>+</sup> that encodes for a plasma membrane P-type ATPase, as we previously isolated a long-lived mutant and identified its mutation in <italic>pma1</italic><sup>+</sup> <xref rid="b0030" ref-type="bibr">[6]</xref>. Thus, we sequenced the ORF region of <italic>pma1</italic><sup>+</sup> in the L16 mutant and identified one missense (G to A) mutation that caused an Asp-138 to Asn change in the predicted first extracellular domain of the Pma1 protein (<xref rid="f0010" ref-type="fig">Fig. 2</xref>). Next, we confirmed that the identified mutation (designated, <italic>pma1-L16</italic> allele) was the causative mutation for the long-lived phenotype of the L16 mutant.</p><p>First, a Km-resistant cassette was inserted in the downstream region of <italic>pma1</italic><sup>+</sup>, after which the Km-resistant strain was mated with the L16 mutant to assess the linkage between the Km-resistant phenotype and the chronologically long-lived phenotype. After crossing, phenotype analysis of the progeny revealed that 90% of chronologically long-lived cells (<italic>n</italic>&#x000a0;=&#x000a0;20) were Km-sensitive, which indicated that the <italic>pma1-L16</italic> mutation was located close to the site at which the Km-resistant cassette was inserted on the chromosome. Next, after crossing the L16 mutant with a wild type strain, we randomly isolated both long-lived cells (<italic>n</italic>&#x000a0;=&#x000a0;4) and non-long-lived cells (<italic>n</italic>&#x000a0;=&#x000a0;4), and sequenced their chromosome regions corresponding to the <italic>pma1-L16</italic> mutation. We confirmed that all long-lived cells and non-long-lived cells had a <italic>pma1-L16</italic> mutation and a wild type <italic>pma1</italic><sup>+</sup> allele, respectively. On the basis of these results, we concluded that the <italic>pma1-L16</italic> mutation was the causative mutation that conferred the long-lived phenotype.</p></sec><sec id="s0055"><label>3.3</label><title>L16 mutant ATPase activity</title><p>Pma1 is a P-type plasma membrane ATPase that is a hydrogen ion pump <xref rid="b0055 b0060" ref-type="bibr">[11,12]</xref>. H<sup>+</sup>-ATPase activity of the L16 mutant was analyzed to characterize the effect of the <italic>pma1-L16</italic> mutation. Cell lysates were prepared from cells after growth to the mid-logarithmic phase (OD<sub>600</sub>&#x000a0;=&#x000a0;1) and to the stationary phase (OD<sub>600</sub>&#x000a0;=&#x000a0;2), after which their H<sup>+</sup>-ATPase activities were determined. As shown in <xref rid="f0015" ref-type="fig">Fig. 3</xref>A, the ATPase activity of the L16 mutant was lower than that of wild type cells at the stationary phase. In <italic>S. cerevisiae</italic>, it is known that Pma1 H<sup>+</sup>-ATPase activity is positively regulated by the glucose concentration in the medium; when glucose is added to carbon-starved cells, their ATPase activity increases <xref rid="b0065 b0070" ref-type="bibr">[13,14]</xref>. Thus, we assessed <italic>S. pombe</italic> Pma1 activity in response to the glucose concentration.</p><p>Wild type and L16 mutant cells were grown in SD medium until the logarithmic growth phase was achieved, and were then transferred to SD media that contained different glucose concentrations. Cells were grown in each medium for 90&#x000a0;min, after which H<sup>+</sup>-ATPase activity was determined. As shown in <xref rid="f0015" ref-type="fig">Fig. 3</xref>B, wild type cells&#x02019; H<sup>+</sup>-ATPase activity was regulated by glucose, as their ATPase activity was low in medium without glucose and increased in response to the glucose concentration. For the L16 mutant, ATPase activity increased in response to glucose. However, the activity was lower than that of wild type cells in medium that contained 0.5&#x02013;2% glucose. These results suggested that the Pma1-L16 protein had defective ATPase activity but that its regulation by glucose was not affected. Taken together, we concluded that the L16 mutant had low H<sup>+</sup>-ATPase activity.</p></sec><sec id="s0060"><label>3.4</label><title>Expression profiles for <italic>pma1<sup>+</sup></italic> mRNA and Pma1 protein</title><p>To determine the expression profiles associated with <italic>pma1</italic><sup>+</sup>, wild-type and L16 mutant cells were grown in SD medium, after which <italic>pma1<sup>+</sup></italic> mRNA levels were determined by Northern hybridization. As shown in <xref rid="f0020" ref-type="fig">Fig. 4</xref>A, similar <italic>pma1<sup>+</sup></italic> mRNA levels were expressed in both L16 mutant and wild type cells. Next, we determined the amounts of Pma1 protein by Western blotting under the same growth conditions. As shown in <xref rid="f0020" ref-type="fig">Fig. 4</xref>B, similar amounts of Pma1 protein were expressed in both cell types. On the basis of these results, we concluded that there were no differences in the amounts and stabilities of the Pma1 protein expressed in L16 and wild type cells. This indicated that the specific H<sup>+</sup>-ATPase activity of the Pma1-L16 protein was lower than that of the wild type Pma1 protein.</p></sec><sec id="s0065"><label>3.5</label><title>The <italic>pma1-L16</italic> mutant consumes less glucose</title><p>Pma1 H<sup>+</sup>-ATPase is involved in H<sup>+</sup>-dependent nutrient uptake <xref rid="b0070" ref-type="bibr">[14]</xref>. Pma1 ATPase functions physiologically to pump protons out of a cell, thus generating an electrochemical gradient that drives solute uptake by an array of H<sup>+</sup>-coupled co-transporters. In <italic>S. pombe</italic>, glucose uptake is energy dependent and is driven by the plasma membrane ATPase-generated electrochemical gradient <xref rid="b0075" ref-type="bibr">[15]</xref>. We compared glucose consumption during growth between wild-type and L16 mutant cells (<xref rid="f0025" ref-type="fig">Fig. 5</xref>). This revealed that the L16 mutant consumed less glucose compared with wild type cells. This difference in glucose consumption was likely due to the differences in Pma1 activity that provides the proton gradient for glucose uptake.</p><p>On the basis of these findings, we propose the following scenario to explain the long-lived phenotype of the L16 mutant. In the L16 mutant, reduced Pma1 activity causes some defect in glucose uptake. This might cause physiological changes that are equivalent to changes caused by calorie restriction.</p><p>In summary, we propose that Pma1 ATPase activity is crucial for determining the chronological lifespan of <italic>S. pombe</italic>. Because Pma1 is conserved among many organisms, their chronological life spans might be manipulated by modulating Pma1 activity. Verification of this possibly novel means to regulate lifespan awaits further experimentation.</p></sec></sec>
An alternative allosteric regulation mechanism of an acidophilic <sc>l</sc>-lactate dehydrogenase from <italic>Enterococcus mundtii</italic> 15-1A
<p>A plant-derived <italic>Enterococcus mundtii</italic> 15-1A, that has been previously isolated from <italic>Brassica rapa</italic> L. subsp. <italic>nipposinica</italic> (L.H. Bailey) Hanelt var. <italic>linearifolia</italic> by our group, possesses two kinds of <sc>l</sc>-lactate dehydrogenase (<sc>l</sc>-LDH): LDH-1 and LDH-2. LDH-1 was activated under low concentration of fluctose-1,6-bisphosphate (FBP) at both pH 5.5 and 7.5. Although LDH-2 was also activated under the low concentration of FBP at pH 5.5, a high concentration of FBP is necessary to activate it at pH 7.5. The present study shows the crystal structures of the acidophilic LDH-2 in a complex with and without FBP and NADH. Although the tertiary structure of the ligands-bound LDH-2 is similar to that of the active form of other bacterial <sc>l</sc>-LDHs, the structure without the ligands is different from that of any other previously determined <sc>l</sc>-LDHs. Major structural alterations between the two structures of LDH-2 were observed at two regions in one subunit. At the <italic>N</italic>-terminal parts of the two regions, the ligands-bound form takes an &#x003b1;-helical structure, while the form without ligands displays more disordered and extended structures. A vacuum-ultraviolet circular dichroism analysis showed that the &#x003b1;-helix content of LDH-2 in solution is approximately 30% at pH 7.5, which is close to that in the crystal structure of the form without ligands. A D241N mutant of LDH-2, which was created by us to easily form an &#x003b1;-helix at one of the two parts, exhibited catalytic activity even in the absence of FBP at both pH 5.5 and 7.5.</p>
<contrib contrib-type="author"><name><surname>Matoba</surname><given-names>Yasuyuki</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Miyasako</surname><given-names>Masashi</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Matsuo</surname><given-names>Koichi</given-names></name><xref rid="af010" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Oda</surname><given-names>Kosuke</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Noda</surname><given-names>Masafumi</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Higashikawa</surname><given-names>Fumiko</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Kumagai</surname><given-names>Takanori</given-names></name><xref rid="af005" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Sugiyama</surname><given-names>Masanori</given-names></name><email>[email protected]</email><xref rid="af005" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">&#x0204e;</xref></contrib>
FEBS Open Bio
<sec sec-type="intro" id="s0005"><label>1</label><title>Introduction</title><p>Lactic acid bacteria (LAB) have their natural habitats in rather different environments and interact differently with human beings. In spite of having their different lifestyles, LAB predominantly obtain energy by the lactic acid fermentation. In addition to providing a characteristic flavor, lactic acid confers important preservative properties to fermented products <xref rid="b0005" ref-type="bibr">[1]</xref>. Lactic acid produced by LAB is also known to be a material essential to generate polylactic acid. The free energy generated during homolactic acid fermentation is 2&#x000a0;mol of ATP per 1&#x000a0;mol of glucose. The crucial enzyme in this pathway is lactate dehydrogenase (LDH), which is responsible for catalyzing the reversible reduction of pyruvate to <sc>l</sc>- or <sc>d</sc>-lactate <xref rid="b0010" ref-type="bibr">[2]</xref>. <sc>d</sc>- and <sc>l</sc>-LDHs stereospecifically convert pyruvate to <sc>d</sc>- and <sc>l</sc>-lactate, respectively. This reaction serves to balance the redox potential by oxidation of NADH to NAD.</p><p><sc>l</sc>-LDH (EC 1.1.1.27), which is widely distributed in nature, is a tetrameric enzyme composed of four subunits. The arrangement of the subunits displays 222 symmetry where three 2-fold axes, which we defined as <italic>X</italic>-, <italic>Y</italic>-, and <italic>Z</italic>-axes, are present. Each monomer has one active site, and the tetramer has two allosteric sites, each of which is situated at the <italic>Y</italic>-axis interface between two monomers. Bacterial <sc>l</sc>-LDHs are homotetrameric and usually allosteric enzymes, which require fructose-1,6-bisphosphate (FBP), an intermediate in the glycolysis pathway, for catalytic activities. On the other hand, vertebrate cells contain non-allosteric <sc>l</sc>-LDH isozymes.</p><p>The mechanism for the allosteric regulation of the bacterial <sc>l</sc>-LDHs was first elucidated from the structural studies of the enzyme from <italic>Bacillus</italic> (<italic>B</italic>.) <italic>stearothermophilus</italic> <xref rid="b0015 b0020 b0025" ref-type="bibr">[3&#x02013;5]</xref>. In the FBP-bound tetrameric structure, each FBP molecule interacts with four positively charged residues (Arg173 and His188 residues in each of two juxtaposed subunits). The tetramer of the <italic>B. stearothermophilus</italic> <sc>l</sc>-LDH dissociates into two inactive <italic>X</italic>-axis-related dimers in the absence of FBP <xref rid="b0030 b0035" ref-type="bibr">[6,7]</xref>. FBP may play a role in stabilizing and maintaining the tetrameric structure.</p><p>Another mechanism for allosteric regulation was proposed by the structural studies of the <italic>Bifidobacterium</italic> (<italic>Bf</italic>.) <italic>longum</italic> <sc>l</sc>-LDH <xref rid="b0040 b0045" ref-type="bibr">[8,9]</xref>. In this case, the inactive tetrameric structures without and with FBP and NADH were determined <xref rid="b0040 b0045" ref-type="bibr">[8,9]</xref>, as well as the active tetrameric structure with FBP, NADH, and oxamate, a pyruvate analog <xref rid="b0045" ref-type="bibr">[9]</xref>. The quaternary structure of the enzyme is significantly different between the inactive and active states, in which two <italic>Y</italic>-axis-related dimers in a tetramer take open and closed conformations, respectively. Furthermore, the structure of the inactive dimer of the <italic>B. stearothermophilus</italic> <sc>l</sc>-LDH <xref rid="b0025" ref-type="bibr">[5]</xref> resembles the structure of the <italic>X</italic>-axis-related dimer in the inactive tetramer of the <italic>Bf. longum</italic> <sc>l</sc>-LDH in many features. Particularly, Arg171 residue, which is essential for the binding of pyruvate, does not point toward the pyruvate-binding site in both inactive structures. Therefore, it was concluded that FBP plays an important role in inducing the inter-subunit rearrangement of the <sc>l</sc>-LDH tetramer from an inactive to an active state, which is accompanied by local intra-subunit conformational changes. However, the fact that the crystal structure of the FBP-bound inactive state is present indicates that FBP merely increases the ratio of the active state to the inactive one. The binding of pyruvate to the active site may complete the structural change from the inactive to the active state.</p><p>However, it is unclear what key structural feature is responsible for the distinct behavior of the allosteric and non-allosteric <sc>l</sc>-LDHs, since bacterial <sc>l</sc>-LDHs are highly divergent and evolutionarily distant from the vertebrate non-allosteric enzymes. Bacterial <sc>l</sc>-LDHs exhibit wide variation in their regulation properties, as well as in their primary structures <xref rid="b0010" ref-type="bibr">[2]</xref>. Some of them absolutely require FBP for their catalytic activities, whereas some enzymes can exhibit their activities even without FBP.</p><p>Among the allosteric bacterial <sc>l</sc>-LDHs, the enzyme from <italic>Lactobacillus</italic> (<italic>Lb</italic>.) <italic>casei</italic> shows unique properties, exhibiting a great pH-dependence in the enzyme activation <xref rid="b0050 b0055 b0060" ref-type="bibr">[10&#x02013;12]</xref>. The enzyme absolutely requires a high concentration of FBP (about 10<sup>&#x02212;1</sup>&#x000a0;M) for its maximum catalytic activity under the neutral pH condition, where it exhibits low affinity for the substrate pyruvate. In contrast, under a weak acidic condition, the enzyme exhibits activity even in the absence of FBP, despite the low affinity for pyruvate, while the addition of FBP to the enzyme increases the affinity for pyruvate. Structural and mutational analyses of the <italic>Lb. casei</italic> <sc>l</sc>-LDH indicated that two inter-subunit salt bridges formed between His20 and Asp264 and between His205 and Glu211 are important to form the active state <xref rid="b0070" ref-type="bibr">[14]</xref>. The salt bridges seem to be efficiently formed under the acidic condition, since the His residues are sufficiently protonated.</p><p>On the other hand, the <sc>l</sc>-LDH from <italic>Lb. pentosus</italic> is known to be an exceptionally non-allosteric enzyme <xref rid="b0075" ref-type="bibr">[15]</xref>, which constitutively exhibits high catalytic activity independently of FBP, similar to the vertebrate enzymes. The <italic>Lb. pentosus</italic> <sc>l</sc>-LDH constitutionally takes an active state independently of the binding of FBP. Structural analysis of the <italic>Lb. pentosus</italic> <sc>l</sc>-LDH revealed that the subunit interfaces of this enzyme are specifically stabilized to adopt the active state by increased numbers of inter-subunit salt bridges and hydrogen bonds and high geometrical complementarity <xref rid="b0080" ref-type="bibr">[16]</xref>.</p><p>Some strains of LAB produce antibacterial peptides, called bacteriocins, which are ribosomally synthesized and inhibitory to closely related Gram-positive bacteria <xref rid="b0085 b0090" ref-type="bibr">[17,18]</xref>. Our group has recently isolated a bacteriocin-producing LAB from <italic>Brassica rapa</italic> L. subsp. <italic>nipposinica</italic> (L.H. Bailey) Hanelt var. <italic>linearifolia</italic> in Japan. The isolated strain, designated 15-1A, was identified as <italic>Enterococcus</italic> (<italic>Ec</italic>.) <italic>mundtii</italic> <xref rid="b0095" ref-type="bibr">[19]</xref>. Whole genome sequence analysis of the 15-1A strain, now in progress in our laboratory, indicates that the strain contains at least two <sc>l</sc>-LDH-like proteins, designated LDH-1 and LDH-2. Another strain classified as <italic>Ec. mundtii</italic> was demonstrated to produce high yield of <sc>l</sc>-lactic acid with high optical purity from cellobiose or xylose <xref rid="b0100 b0105" ref-type="bibr">[20,21]</xref>. Therefore, elucidation of the regulation mechanism of the <italic>Ec. mundtii</italic> <sc>l</sc>-LDHs at the molecular level is important to know why this bacterium can efficiently produce <sc>l</sc>-lactic acid.</p><p>In the present study, we performed an enzyme kinetic analysis of the recombinant <sc>l</sc>-LDHs from <italic>Ec. mundtii</italic>. As a result, it was found that the <italic>Ec. mundtii</italic> LDH-2 exhibits acidophilic kinetic properties. Understanding the allosteric regulation mechanism of the <italic>Ec. mundtii</italic> LDH-2 will be useful for creating an artificial enzyme with an acidophilic feature in future work. To clarify the allosteric regulation mechanism of the acidophilic LDH-2, we performed crystallographic, vacuum-ultraviolet circular dichroism (VUVCD) and site-directed mutation analyses.</p></sec><sec sec-type="results" id="s0010"><label>2</label><title>Results</title><sec id="s0015"><label>2.1</label><title>Amino acid sequence of LDH-1 and LDH-2 from <italic>Ec. mundtii</italic> 15-1A</title><p><italic>Ec. mundtii</italic> 15-1A has two genes encoding <sc>l</sc>-LDH (LDH-1 and LDH-2). Redundancy of the gene encoding <sc>l</sc>-LDH was also found in many LAB including <italic>Lactococcus</italic> (<italic>Lc.</italic>) <italic>lactis</italic> <xref rid="b0110" ref-type="bibr">[22]</xref>, <italic>Lb. plantarum</italic> <xref rid="b0115" ref-type="bibr">[23]</xref>, and <italic>Ec</italic>. <italic>faecalis</italic> <xref rid="b0120" ref-type="bibr">[24]</xref>. There are two reports about the kinetic characterizations of two <sc>l</sc>-LDHs contained in one bacterium.</p><p><italic>Lc. lactis</italic> FI9078, which is a derivative of the MG1363 strain carrying a disruption of the <italic>ldh</italic> gene, can convert glucose to <sc>l</sc>-lactate, since the strain overexpresses the <italic>ldhB</italic> gene <xref rid="b0125" ref-type="bibr">[25]</xref>. The <italic>K</italic><sub>act</sub> value of LDHB, which is defined as the concentration of FBP necessary for activation at 50% of the maximum, was found to be strongly dependent on pH (700-fold increase in the pH range from 6.0 to 7.0), in contrast to LDH. That is, the <italic>Lc. lactis</italic> LDHB is active under the neutral pH condition only when the FBP concentration is high enough, as with the <italic>Lb. casei</italic> <sc>l</sc>-LDH. However, the <italic>Lc. lactis</italic> LDHB is different from the <italic>Lb. casei</italic> <sc>l</sc>-LDH, since the affinity of the former enzyme for NADH, but not for pyruvate, is lowered with the increase of pH even in the presence of a high concentration of FBP.</p><p><italic>Ec. faecalis</italic> V583 also contains two <sc>l</sc>-LDH-encoding genes (<italic>ldh-</italic>1 and <italic>ldh-</italic>2). The roles of the two genes were studied using knockout mutants <xref rid="b0130" ref-type="bibr">[26]</xref>. Deletion of <italic>ldh-</italic>1 caused a metabolic shift from homolactic fermentation to ethanol, formate, and acetoin production, with a high level of formate production even under aerobic conditions. On the other hand, deletion of <italic>ldh-</italic>2 kept the homolactic fermentation, suggesting that LDH-2 plays only a minor role in the lactate production. Kinetic study showed that the <italic>K<sub>m</sub></italic> values of LDH-2 for both pyruvate and NADH are larger than those of LDH-1, indicating the minor role in lactate production <xref rid="b0135" ref-type="bibr">[27]</xref>.</p><p>The sequence identity between the <italic>Ec. mundtii</italic> LDH-1 and LDH-2 is 44.9% (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). However, the <italic>Ec. mundtii</italic> LDH-1 shows a high sequence identity with the <italic>Ec. faecalis</italic> LDH-1 (86.2%) and the <italic>Lc. lactis</italic> LDH (68.7%), whereas the <italic>Ec. mundtii</italic> LDH-2 shows a high sequence identity with the <italic>Ec. faecalis</italic> LDH-2 (65.0%) and the <italic>Lc. lactis</italic> LDHB (71.0%). These results indicate that LDH-1 mainly plays a role in <sc>l</sc>-lactate production in <italic>Ec. mundtii</italic>, while LDH-2 plays an additional role.</p></sec><sec id="s0020"><label>2.2</label><title>Kinetic properties of LDH-1 and LDH-2 from <italic>Ec. mundtii</italic> 15-1A</title><p>To characterize the <italic>Ec. mundtii</italic> LDH-1 and LDH-2, we performed enzyme kinetic analysis using each recombinant enzyme, which was overexpressed in <italic>Escherichia</italic> (<italic>E.</italic>) <italic>coli</italic> and purified almost to homogeneity. At first, we investigated the effect of pH on the catalytic activities of LDH-1 and LDH-2. The pH profiles of the activities of LDH-1 and LDH-2 in the presence of 20&#x000a0;mM pyruvate, 1.5&#x000a0;mM NADH, and 0.1&#x000a0;mM FBP are depicted in <xref rid="f0010" ref-type="fig">Fig. 2</xref>. LDH-1 maintains a high level of activity between pH 4.0 and pH 7.5. On the other hand, the activity of LDH-2 was maximal at pH 5.5 and then gradually decreased with the increase of pH.</p><p>The results of the detailed kinetic analysis, which was done at pH 5.5 and pH 7.5, are summarized in <xref rid="t0005" ref-type="table">Table 1</xref>. In the presence of 3&#x000a0;mM FBP, both LDH-1 and LDH-2 showed hyperbolic kinetic responses to increasing concentrations of pyruvate or NADH independently of pH. The <italic>K<sub>m</sub></italic> of LDH-1 for pyruvate increased substantially (4-fold) with the increase of pH from 5.5 to 7.5, while the <italic>K<sub>m</sub></italic> for NADH decreased slightly along with pH. On the other hand, the <italic>K<sub>m</sub></italic> of LDH-2 for pyruvate was scarcely altered with pH, while the <italic>K<sub>m</sub></italic> of LDH-2 for NADH increased approximately 2-fold with the increase of pH.</p><p>In the presence of 1.5&#x000a0;mM NADH and 20&#x000a0;mM pyruvate, LDH-1 and LDH-2 showed hyperbolic kinetic responses to an increasing concentration of FBP at both pH 5.5 and 7.5. Both enzymes were found to be allosteric ones that require FBP for their catalytic activities. The <italic>K</italic><sub>act</sub> value of LDH-1 was not significantly altered with pH. In contrast, the <italic>K</italic><sub>act</sub> value of LDH-2 was increased 500-fold when pH was shifted from 5.5 to 7.5. Like most bacterial <sc>l</sc>-LDHs, the <italic>Ec. mundtii</italic> LDH-1 was activated by the low concentration of FBP (in a range of 10<sup>&#x02212;7</sup>&#x02013;10<sup>&#x02212;5</sup>&#x000a0;M) at pH 5.5 and pH 7.5. On the other hand, although LDH-2 was activated under a low FBP concentration condition at pH 5.5, a high concentration condition is required for activation at pH 7.5. These results suggest that although the <italic>Ec. mundtii</italic> LDH-1 is classified into the general bacterial <sc>l</sc>-LDH, the LDH-2 from <italic>Ec. mundtii</italic> is an acidophilic enzyme, as are the <italic>Lc. lactis</italic> LDHB <xref rid="b0125" ref-type="bibr">[25]</xref> and the <italic>Lb. casei</italic> <sc>l</sc>-LDH <xref rid="b0050 b0055 b0060" ref-type="bibr">[10&#x02013;12]</xref>.</p><p>Since the <italic>Ec. mundtii</italic> LDH-2 has a higher sequence similarity to the <italic>Lc. lactis</italic> LDHB (71%) than to the <italic>Lb. casei</italic> <sc>l</sc>-LDH (46%), the regulation mechanism of the <italic>Ec. mundtii</italic> LDH-2 may be common with that of the <italic>Lc. lactis</italic> LDHB but different from that of the <italic>Lb. casei</italic> <sc>l</sc>-LDH. In fact, in the presence of a high concentration of FBP, the <italic>Ec. mundtii</italic> LDH-2 and the <italic>Lc. lactis</italic> LDHB commonly exhibit lowered affinities for NADH, but not for pyruvate, with the increase of pH. Furthermore, considering the amino acid sequence similarity, the <italic>Ec. faecalis</italic> LDH-2 is likely to exhibit kinetic properties similar to those of the <italic>Ec. mundtii</italic> LDH-2 and the <italic>Lc. lactis</italic> LDHB. However, the kinetic parameters of the <italic>Ec. faecalis</italic> LDH-2 <xref rid="b0135" ref-type="bibr">[27]</xref> are obviously different from those of the <italic>Ec. mundtii</italic> LDH-2 and the <italic>Lc. lactis</italic> LDHB <xref rid="b0125" ref-type="bibr">[25]</xref>.</p></sec><sec id="s0025"><label>2.3</label><title>Overall structures of ligands-unbound and -bound LDH-2</title><p>We crystallographically analyzed the acidophilic LDH-2 from <italic>Ec. mundtii</italic>. Although we tried to crystallize the LDH-2 in various conditions, we could obtain the measurable diffraction data from only two crystals. One crystal was grown in the absence of any ligands, while the other was grown in the presence of FBP and NADH (<xref rid="t0010" ref-type="table">Table 2</xref>). In both cases, an asymmetric unit in the crystal contains one tetramer (<xref rid="f0015" ref-type="fig">Fig. 3</xref>a and b). The atomic coordinates and structure factors of the ligands-unbound and -bound LDH-2 have been deposited in the Protein Data Bank with accession codes <ext-link ext-link-type="uri" xlink:href="pdb:3WSV" id="ir0035">3WSV</ext-link> and <ext-link ext-link-type="uri" xlink:href="pdb:3WSW" id="ir0040">3WSW</ext-link>, respectively. A secondary structure assignment based on the crystal structures is shown in <xref rid="f0020" ref-type="fig">Fig. 4</xref>. In the crystal structure of the ligands-bound LDH-2, two FBP and four NADH molecules were modeled (<xref rid="f0015" ref-type="fig">Fig. 3</xref>a). The electron densities for the FBP molecules are strong, while the densities for the NADH molecules are relatively weak. Specifically, the densities for the nicotinamide moiety of NADH were poorly defined, probably due to the high mobility.</p><p>Four subunits in the ligands-bound tetramer of the <italic>Ec. mundtii</italic> LDH-2 are similar to one another. They can be superimposed with root mean square (rms) deviations in a range between 0.20 and 0.48&#x000a0;&#x000c5; for the 312&#x000a0;C&#x003b1; atoms. When compared with the active state of the <italic>Bf. longum</italic> <sc>l</sc>-LDH, the residues Val100&#x02013;Leu112 and Val213&#x02013;Arg220a of the <italic>Ec. mundtii</italic> LDH-2 are structurally different (<xref rid="f0025" ref-type="fig">Fig. 5</xref>a). The residues Val100&#x02013;Leu112 protrude away from the main body of the subunit, as found in the inactive state of the <italic>Bf. longum</italic> <sc>l</sc>-LDH. Since this region is one side of the pyruvate-binding pocket, the binding of pyruvate to the active site may induce the structural change of this region. On the other hand, the residues Val213&#x02013;Arg220a in the <italic>Ec. mundtii</italic> LDH-2 are structurally different from the corresponding residues in the <italic>Bf. longum</italic> <sc>l</sc>-LDH because former enzyme lacks two residues on the amino acid sequence (<xref rid="f0005 f0010 f0015 f0020" ref-type="fig">Figs. 1 and 4</xref>). However, without these residues, each subunit of the ligands-bound <italic>Ec. mundtii</italic> LDH-2 is structurally similar to that of the <italic>Bf. longum</italic> <sc>l</sc>-LDH in the active state with rms deviations in a range between 1.1 and 1.2&#x000a0;&#x000c5;. It should be noted that an &#x003b1;-helix (&#x003b1;1/2G in <xref rid="f0020" ref-type="fig">Fig. 4</xref>) consisting of the residues Glu222&#x02013;Lys243 is bent around Asn234, as found in the active state of the <italic>Bf. longum</italic> <sc>l</sc>-LDH.</p><p>It has been suggested that the side-chain orientation of the Arg171 residue is important for discriminating between inactive and active states. The side chain of the Arg171 residue of the <italic>Ec. mundtii</italic> LDH-2, which seems to be important for the binding of pyruvate, points toward the pyruvate-binding site (<xref rid="f0030" ref-type="fig">Fig. 6</xref>a), as seen in the active state of other bacterial <sc>l</sc>-LDHs. Furthermore, the quaternary structure of the ligands-bound LDH-2 is similar to that of the other bacterial <sc>l</sc>-LDHs in the active state, in which a compact tetramer is formed by many inter-subunit interactions (<xref rid="f0035" ref-type="fig">Fig. 7</xref>a and c). Therefore, we defined the quaternary structure of the ligands-bound LDH-2 as an active state.</p><p>For the general allosteric <sc>l</sc>-LDHs, FBP is speculated to shift the equilibrium toward an active state <xref rid="b0045" ref-type="bibr">[9]</xref>, although the sensitivities to FBP are different among the enzymes. In the inactive state (<xref rid="f0035" ref-type="fig">Fig. 7</xref>d), the <italic>Y</italic>-axis-related dimer takes an open conformation, resulting in the expanded shape of the tetramer. In the presence of FBP, the ratio of active state tetramer, in which the <italic>Y</italic>-axis-related dimer is stabilized to take a closed conformation in accordance with the rearrangement of the &#x003b1;-helices in each subunit (<xref rid="f0035" ref-type="fig">Fig. 7</xref>c), is increased. The conformational change from an inactive to an active state of <sc>l</sc>-LDH is not significantly associated with the changes of the secondary structure in each subunit, as shown in <xref rid="t0015" ref-type="table">Table 3</xref> and <xref rid="f0020" ref-type="fig">Fig. 4</xref>.</p><p>The molecular shape of the LDH-2 tetramer without the binding of ligands (<xref rid="f0035" ref-type="fig">Fig. 7</xref>b), determined in the present study, is more compact than that of the ligands-bound tetramer (<xref rid="f0035" ref-type="fig">Fig. 7</xref>a) or that of the other bacterial <sc>l</sc>-LDHs in the active state (<xref rid="f0035" ref-type="fig">Fig. 7</xref>c). For examples, the distances between C&#x003b1; atoms of Arg171 in subunits A and B and between the atoms in subunits A and D in the ligands-unbound tetramer are shorter by 1.8 and 1.2&#x000a0;&#x000c5; than those in the ligands-bound tetramer, respectively. In the four subunits of the ligands-unbound LDH-2, subunits A and C, shown in <xref rid="f0015" ref-type="fig">Fig. 3</xref>b, can be superimposed well with an rms deviation of 0.34&#x000a0;&#x000c5; for the 309&#x000a0;C&#x003b1; atoms; subunits B and D can be also be superimposed well with an rms deviation of 0.15&#x000a0;&#x000c5; for the 312&#x000a0;C&#x003b1; atoms. On the other hand, rms deviations between the other pairs of subunits were calculated to be large values in a range between 2.3 and 2.4&#x000a0;&#x000c5;.</p><p>When the structure of subunit A (or C) is compared with that of subunit B (or D) in the ligands-unbound form of the <italic>Ec. mundtii</italic> LDH-2, three regions including the residues Val54&#x02013;Lys75, Val100&#x02013;Leu112, and Lys233&#x02013;Gly249 are different (<xref rid="f0025" ref-type="fig">Fig. 5</xref>b). Atoms in these regions have high <italic>B</italic>-factors commonly. Specifically, the residues Glu62&#x02013;Leu66 in subunit A (or C) in the ligands-unbound form are invisible in the electron density map. The structures of the residues Val100&#x02013;Leu112 in subunits A (or C) and B (or D) are similar to those of the corresponding residues of the <italic>Bf. longum</italic> <sc>l</sc>-LDH in the inactive and active states, respectively (<xref rid="f0025" ref-type="fig">Fig. 5</xref>). The Val100&#x02013;Leu112 residues may be flexible due to the absence of pyruvate.</p><p>In addition, both subunits A (or C) and B (or D) in the ligands-unbound form are structurally different from the subunits in the ligands-bound form due to the differences in some regions in the subunit (<xref rid="f0025" ref-type="fig">Fig. 5</xref>b). In detail, rms deviations between subunit A (or C) in the ligands-unbound form and subunits in the ligands-bound form were calculated to be large values in a range between 2.2 and 2.4&#x000a0;&#x000c5;, while rms deviations between subunit B (or D) in the ligands-unbound form and subunits in the ligands-bound form ranged from 2.9 to 3.2&#x000a0;&#x000c5;. However, when calculated without the residues Val54&#x02013;Lys75, Val100&#x02013;Leu112, and Lys233&#x02013;Gly249, both subunits A (or C) and B (or D) in the ligands-unbound form can be superimposed on the subunits in the ligands-bound form with rms deviations in a range between 0.74 and 0.82&#x000a0;&#x000c5;. It should be noted that the regions Val54&#x02013;Lys75 and Lys233&#x02013;Gly249 in the ligands-unbound form also take different structures from those in the ligands-bound form (<xref rid="f0025" ref-type="fig">Fig. 5</xref>b). That is, these two regions in the LDH-2 can take three different structures.</p><p>Subunit structure of <sc>l</sc>-LDH can be divided into <italic>N</italic>-terminal NADH-binding domain and <italic>C</italic>-terminal catalytic domain. When the superposition was calculated using the C&#x003b1; atoms in the catalytic domain (The165&#x02013;Arg330) of the <italic>Ec. mundtii</italic> LDH-2 without the residues Lys233&#x02013;Gly249, rms deviations were ranged between 0.66 and 0.75&#x000a0;&#x000c5;, which are slightly smaller than a global superposition without the residues Val54&#x02013;Lys75, Val100&#x02013;Leu112, and Lys233&#x02013;Gly249. The superposition shows an apparent rocking motion of the NADH-binding domain with respect to the catalytic domain (<xref rid="f0025" ref-type="fig">Fig. 5</xref>b). However, although the arrangement of subdomains may contribute to the binding of NADH in a small extent, conformational change of the residues Val54&#x02013;Lys75 is primarily required for the binding.</p><p>Based on the orientation of the side chain of Arg171, subunits A and C are assumed to be in an active state, in which Arg171 points toward the pyruvate-binding site without intra-subunit interactions (<xref rid="f0030" ref-type="fig">Fig. 6</xref>b). On the other hand, subunits B and D are assumed to be in an inactive state, in which Arg171 does not point toward the pyruvate-binding site due to the intra-subunit interactions with Glu175 and Tyr248 (<xref rid="f0030" ref-type="fig">Fig. 6</xref>c). Especially, torsion angles between C&#x003b1; and C&#x003b2; atoms of Arg171 in subunits A and C are different by about 90&#x000b0;, when compared with those in subunits B and D. However, the structure of each subunit in the ligands-unbound LDH-2 is quite different from that of the subunit in the other bacterial <sc>l</sc>-LDHs in either the inactive or active state (<xref rid="f0025" ref-type="fig">Fig. 5</xref>). In fact, even when calculated without the residues Val100&#x02013;Leu112 and Val213&#x02013;Arg220a of the <italic>Ec. mundtii</italic> LDH-2, the subunits in the ligands-unbound form are structurally different from the subunit of the <italic>Bf. longum</italic> <sc>l</sc>-LDH in the active state with rms deviations in a range between 2.4 and 2.7&#x000a0;&#x000c5;, and from the subunit of the <italic>Bf. longum</italic> <sc>l</sc>-LDH in the inactive state with rms deviations in a range between 2.4 and 2.8&#x000a0;&#x000c5;. Importantly, secondary structure analysis using the DSSP program indicated that &#x003b1;-helix content in the ligands-unbound LDH-2 is about 34% on average (<xref rid="t0015" ref-type="table">Table 3</xref>), which is smaller than that in the ligands-bound form or those in the inactive and active states of the other <sc>l</sc>-LDHs. The structure of ligands-unbound LDH-2 seems to be an inactive form, since the NADH- and pyruvate-binding pockets are different from those of the other bacterial <sc>l</sc>-LDHs in the active state, as described below. Therefore, we defined the compact quaternary structure of the ligands-unbound LDH-2 as another inactive state.</p></sec><sec id="s0030"><label>2.4</label><title>Substrate-binding sites</title><p>In the Val54&#x02013;Lys75 region of the active state LDH-2, Gln56&#x02013;Trp72 contains helical structures including an &#x003b1;-helix (&#x003b1;C) and following 3<sub>10</sub>-helix (<xref rid="f0020 f0025 f0030" ref-type="fig">Figs. 4 and 6</xref>a). On the other hand, the corresponding region of subunit B in the inactive state has a shorter &#x003b1;-helix consisting of Ala63&#x02013;Trp72, and the helix is differently oriented from that in the active state (<xref rid="f0030" ref-type="fig">Fig. 6</xref>c). The corresponding region of subunit A in the inactive state is more extended (<xref rid="f0030" ref-type="fig">Fig. 6</xref>b), and some of the residues are invisible in the electron density map. Significantly, the <italic>N</italic>-terminal parts of this region in both subunits A and B commonly take the disordered and extended structures in the inactive state and occupy the binding site for the adenosine moiety of NADH (<xref rid="f0025" ref-type="fig">Fig. 5</xref>b). Therefore, the current inactive state of LDH-2 likely is unable to accommodate NADH, which may be related to the kinetic result that LDH-2 exhibits low affinity for NADH even under the high concentration of FBP (<xref rid="t0005" ref-type="table">Table 1</xref>).</p><p>The Lys233&#x02013;Gly249 region is positioned at one side of the pyruvate-binding pocket. In the active state, the <italic>N</italic>-terminal part of this region (Ala235&#x02013;Lys243) is a <italic>C</italic>-terminal part of the bent &#x003b1;-helix (&#x003b1;1/2G), and the following part takes a rigid loop structure (<xref rid="f0020 f0025 f0030" ref-type="fig">Figs. 4 and 6</xref>a). On the other hand, in the inactive state, the corresponding regions of subunits A and B have no helical structures (<xref rid="f0030" ref-type="fig">Fig. 6</xref>b and c), suggesting that the structures of the pyruvate-binding pockets of subunits A and B in the inactive state are clearly different from those in the active state. Therefore, this compact inactive state likely is unable to bind to pyruvate, as with NADH.</p><p>In summary, the <italic>N</italic>-terminal parts of the two regions (Val54&#x02013;Lys75 and Lys233&#x02013;Gly249) form the &#x003b1;-helices in the active state LDH-2, while the parts take more disordered and extended structures in the compact inactive state LDH-2. It should be noted that the residues Lys233&#x02013;Gly249 of one subunit are positioned near the residues Val54&#x02013;Lys75 of the <italic>X</italic>-axis-related subunit (<xref rid="f0040" ref-type="fig">Fig. 8</xref>a and b), indicating that the binding of NADH to one subunit facilitates the formation of the pyruvate-binding pocket in the <italic>X</italic>-axis-related subunit.</p><p>Interestingly, these two parts in the <italic>Ec. mundtii</italic> LDH-2 contain two acidic residues separated by two residues in common (Glu57 and Glu60 in the former part and Glu238 and Asp241 in the latter, <xref rid="f0020 f0025 f0030" ref-type="fig">Figs. 4 and 6</xref>). These two parts seem to have difficulty forming &#x003b1;-helices under neutral to alkaline pH conditions because the positions of the negatively charged residues are three-dimensionally close after the formation of &#x003b1;-helices. The difficulty in forming the &#x003b1;-helices at the two parts may be related to the pH dependence of the enzyme activation.</p></sec><sec id="s0035"><label>2.5</label><title>Site-directed mutation analysis</title><p>To elucidate the effects of acidic residues on catalytic activity, site-directed mutational analysis was performed. Residues selected to generate the mutation were Glu60 and Asp241. The former and latter residues were replaced by Gln and Asn, respectively. The site-directed mutants were named E60Q and D241N, respectively. The pH profiles of the activities of E60Q and D241N in the presence of 20&#x000a0;mM pyruvate, 1.5&#x000a0;mM NADH, and 0.1&#x000a0;mM FBP are depicted in <xref rid="f0010" ref-type="fig">Fig. 2</xref>. The activity of the mutant E60Q was maximal at pH 5.5 and then gradually decreased in accordance with the increase of pH, similar to the wild-type enzyme. On the other hand, the activity of D241N was consistently high between pH 4.5 and 7.5, similar to the <italic>Ec. mundtii</italic> LDH-1 and other bacterial <sc>l</sc>-LDHs. These results indicate that electrostatic repulsion between Glu238 and Asp241 of the <italic>Ec. mundtii</italic> LDH-2 is an important factor for the regulation of catalytic activity, whereas the repulsion between Glu57 and Glu60 is not.</p><p>Detailed kinetic analysis of D241N, which was done at pH 5.5 and pH 7.5, is summarized in <xref rid="t0005" ref-type="table">Table 1</xref>. The <italic>K<sub>m</sub></italic> values for NADH and pyruvate and the <italic>k</italic><sub>cat</sub> value of the D241N mutant were determined in the presence of 3&#x000a0;mM FBP. The <italic>K<sub>m</sub></italic> values for NADH were substantially lower than those of the parent enzyme (2-fold at pH 5.5 and 4-fold at pH 7.5). Other kinetic parameters of D241N were not significantly different from those of the parent enzyme. Next, we investigated the effect of FBP on the catalytic activity of the D241N mutant in the presence of 1.5&#x000a0;mM NADH and 20&#x000a0;mM pyruvate. Although the wild-type LDH-2 absolutely requires FBP for catalytic activity, its D241N mutant displays enzyme activity even in the absence of FBP, which is approximately 30% of the maximum at either pH 5.5 or pH 7.5. Although the <italic>K</italic><sub>act</sub> value was increased approximately 25-fold by the change of pH from 5.5 to 7.5, the mutant was activated by a rather low concentration of FBP under the neutral pH condition when compared with the wild-type enzyme. These results indicate that, if the region including Glu238 and Asp241 can form the &#x003b1;-helix easily, the enzyme has a stronger affinity for NADH and is active in the absence or in the presence of a low concentration of FBP. We also determined the kinetic parameters of the D241N mutant in the absence of FBP (<xref rid="t0005" ref-type="table">Table 1</xref>). At pH 5.5, the <italic>k</italic><sub>cat</sub>/<italic>K<sub>m</sub></italic> values for NADH and pyruvate were 7.5- and 13-fold lower than those obtained in the presence of FBP, respectively. The <italic>k</italic><sub>cat</sub>/<italic>K<sub>m</sub></italic> values were further decreased at pH 7.5 (for NADH and pyruvate, 34- and 41-fold lower than those obtained in the presence of FBP, respectively). It is considered that the allosteric regulation of the D241N mutant is still dependent on the pH conditions, although the sensitivity to FBP was greatly increased by the mutation.</p></sec><sec id="s0040"><label>2.6</label><title>VUVCD spectroscopic analysis</title><p>The secondary structure contents of wild-type LDH-2 and its D241N mutant at pH 7.5 were estimated by VUVCD analysis, and the results are listed in <xref rid="t0015" ref-type="table">Table 3</xref>. As shown in this table, the &#x003b1;-helix content of wild-type LDH-2 was calculated to be 28.5%, which is close to that in the crystal structure of the ligands-unbound form (the average &#x003b1;-helical content for four subunits is 34.4%). The D241N mutation was found to slightly increase the &#x003b1;-helix content (30.1%). If the residues Ala235&#x02013;Lys243 form an &#x003b1;-helix, the helix content is expected to increase by 2.8%. Formation of an &#x003b1;-helix at this region seems to be not completely induced by the mutation. Since the wild-type LDH-2 and its D241N mutant with a concentration above 0.1&#x000a0;mg&#x000a0;mL<sup>&#x02212;1</sup> had the aggregations in the buffer at a pH lower than 6, VUVCD analysis could not be performed under the weak acidic pH conditions.</p><p>In the CD measurements, absorbance of the solution should be kept as low as possible. The effect of NADH could not be investigated due to the high absorption of the compound. Therefore, we only examined the effect of FBP on the secondary structure contents of the wild-type LDH-2 and its D241N mutant (<xref rid="t0015" ref-type="table">Table 3</xref>). As a result, it was found that the addition of FBP to the D241N mutant slightly increased the &#x003b1;-helix content (30.1% and 32.6% in the absence and presence of 20&#x000a0;mM FBP, respectively, <xref rid="t0015" ref-type="table">Table 3</xref>). The effect of the addition of FBP to wild type (1.1% increase in the &#x003b1;-helix content) is smaller as compared with the case of the wild-type enzyme (2.5%). These observations strongly indicate that the sensitivity to FBP was increased by the mutation. However, the &#x003b1;-helix content of the D241N mutant in the presence of 20&#x000a0;mM FBP, which is the maximum value obtained in the present study, is about 10% lower than that estimated from the crystal structure of the ligands-bound form. Structural change from the inactive to the active state seems to be not completely induced by the addition of FBP.</p></sec></sec><sec sec-type="discussion" id="s0045"><label>3</label><title>Discussion</title><p>More than two <sc>l</sc>-LDH-encoding genes exist in the genome of LAB: one exhibits a major role, whereas the others have minor roles <xref rid="b0125 b0130 b0135" ref-type="bibr">[25&#x02013;27]</xref>. In the present study, we also found that the <italic>Ec. mundtii</italic> LDH-1 is a general <sc>l</sc>-LDH. On the other hand, <italic>Ec. mundtii</italic> LDH-2 is an &#x0201c;alternative <sc>l</sc>-LDH&#x0201d;, like the <italic>Lc. lactis</italic> LDHB <xref rid="b0125" ref-type="bibr">[25]</xref> and the <italic>Ec. faecalis</italic> LDH-2 <xref rid="b0130 b0135" ref-type="bibr">[26,27]</xref>. At this point, it is still unclear why <italic>Ec. mundtii</italic> can efficiently produce <sc>l</sc>-lactic acid. Alternative <sc>l</sc>-LDHs seem to have in common a low affinity for NADH <xref rid="b0125 b0135" ref-type="bibr">[25,27]</xref>. In addition, the <italic>Ec. mundtii</italic> LDH-2 and the <italic>Lc. lactis</italic> LDHB <xref rid="b0125" ref-type="bibr">[25]</xref> have an acidophilic feature.</p><p>The quaternary structure of the ligands-bound form of LDH-2 is similar to that of the active state of the other bacterial <sc>l</sc>-LDHs <xref rid="b0020 b0045 b0070 b0080" ref-type="bibr">[4,9,14,16]</xref>. On the other hand, the crystal structure of the ligands-unbound LDH-2 may be an inactive form, although its quaternary structure is very compact compared to that of other <sc>l</sc>-LDHs in the inactive state <xref rid="b0025 b0045 b0070" ref-type="bibr">[5,9,14]</xref>. In addition, we found that electrostatic repulsion between Glu238 and Asp241 of the <italic>Ec. mundtii</italic> LDH-2 is an important factor for the regulation of catalytic activity. A similar mechanism may be present in the <italic>Lc. lactis</italic> LDHB and the <italic>Ec. faecalis</italic> LDH-2, since the Glu238 and Asp241 residues are conserved in these two alternative <sc>l</sc>-LDHs (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). In the general or non-allosteric bacterial <sc>l</sc>-LDHs, at least one of two residues that correspond to Glu238 and Asp241 in the <italic>Ec. mundtii</italic> LDH-2 is not acidic.</p><p>Considering that the D241N mutant partially exhibits catalytic activity in the absence of FBP, a small part of the <italic>Ec. mundtii</italic> LDH-2 seems to adopt an active state without the help of FBP in the solution state. In fact, the active state of the <italic>Ec. mundtii</italic> LDH-2 is stabilized by an abundance of inter-subunit hydrophilic interactions (120 interactions within a tetramer), although the inactive state has only 48 interactions. The number of inter-subunit interactions is larger than that in the other allosteric bacterial <sc>l</sc>-LDHs (64 interactions in the case of active state of <italic>Bf. longum</italic> <sc>l</sc>-LDH) but smaller than that in the non-allosteric <italic>Lb. pentosus</italic> <sc>l</sc>-LDH (128 interactions).</p><p>It is important to note that most of the inter-subunit interactions in the active state of the <italic>Ec. mundtii</italic> LDH-2 are formed by the residues in the regions Val54&#x02013;Lys75 and Lys233&#x02013;Gly249 (<xref rid="f0040" ref-type="fig">Fig. 8</xref>a and b), which have different structures between inactive and active states. In the inter-subunit interactions, although some of them are common with the other bacterial <sc>l</sc>-LDHs, some are considered to be characteristic to alternative <sc>l</sc>-LDHs. Specifically, in the active state of the <italic>Ec. mundtii</italic> LDH-2, the side chain of Asp68 in one subunit interacts with those of Arg171 and Lys174 in the <italic>X</italic>-axis-related subunit (<xref rid="f0040" ref-type="fig">Fig. 8</xref>a and b), indicating that the Asp68 and Lys174 residues are important in making Arg171 protrude toward the pyruvate-binding pocket. Furthermore, the side chains of Trp72 and Thr170 in one subunit interact with the main-chain carbonyls of Thr166 and Asp68 in the <italic>X</italic>-axis-related subunit, respectively (<xref rid="f0040" ref-type="fig">Fig. 8</xref>a and b). These inter-subunit interactions are not formed in the inactive state except for the hydrogen bonds between Arg171 in subunit A and Asp68 in subunit B (<xref rid="f0040" ref-type="fig">Fig. 8</xref>c and d). Furthermore, it is important to note that the Asp68, Trp72, Thr170, and Lys174 residues are conserved in the alternative <sc>l</sc>-LDHs but not in the other bacterial allosteric <sc>l</sc>-LDHs (<xref rid="f0005" ref-type="fig">Fig. 1</xref>).</p><p>In the inactive tetramer of LDH-2, the <italic>Z</italic>-axis-related subunits superimpose better than the <italic>X</italic>- or <italic>Y</italic>-axis-related subunits. In addition, the Val54&#x02013;Lys75 and Lys233&#x02013;Gly249 regions, which take different structures between inactive and active states, are positioned at the <italic>X</italic>-axis interface. These observations indicate that the Val54&#x02013;Lys75 and Lys233&#x02013;Gly249 regions in each subunit can deform the structure to increase the geometric complementarity at the <italic>X</italic>-axis interface. As far as we know, this is the first repot for the asymmetric tetrameric structure of <sc>l</sc>-LDH. Probably, in the solution state, an inactive tetramer where two AB dimers are related by <italic>Z</italic>-axis is coexist with another tetramer where two AB dimers are related by <italic>Y</italic>-axis. Current structure, which corresponds to the former tetramer, is likely derived from selective crystallization.</p><p>The mechanism of the allosteric regulation of the <italic>Ec. mundtii</italic> LDH-2 can be summarized as follows: under the weak acidic condition, since the electrostatic repulsion between Glu238 and Asp241 is lowered, the Ala235&#x02013;Lys243 residues can form an &#x003b1;-helix easily. The formation of the &#x003b1;-helix at the Ala235&#x02013;Lys243 residues may induce the structural change to generate a long &#x003b1;-helix at residues Gln56&#x02013;Leu67 in the <italic>X</italic>-axis-related subunit, which are three-dimensionally close to the Ala235&#x02013;Lys243 residues, leading to the formation of a pocket for the accommodation of NADH. This proposition is in agreement with the experimental results that the <italic>K<sub>m</sub></italic> value of the <italic>Ec. mundtii</italic> LDH-2 for NADH was lowered with the decrease of pH (<xref rid="t0005" ref-type="table">Table 1</xref>) and that the D241N mutation further decreased the <italic>K<sub>m</sub></italic> values. The formation of &#x003b1;-helices in the two regions may induce the change from the compact inactive state to the expanded inactive or the compact active state, although the expanded inactive state of the <italic>Ec. mundtii</italic> LDH-2 is structurally undetermined.</p><p>The <italic>Ec. mundtii</italic> LDH-2 may dominantly take a compact inactive state under the neutral pH condition, whereas the ratio of the expanded inactive or compact active state would be increased with the decrease of pH. Among the three states, only the expanded inactive state may allow the invasion of FBP, whereas the compact inactive or active state might not be bound to FBP due to the molecular shape (<xref rid="f0035" ref-type="fig">Fig. 7</xref>). Therefore, FBP may act only on the expanded inactive tetramer and induce the change of the quaternary structure to the active state. This proposition explains the result that a higher concentration of FBP is required for catalytic activity in accordance with the increase of pH. However, the CD analysis indicated that induction of the structural change by the addition of only FBP is difficult. Perhaps, in addition to FBP, high concentration of NADH may be needed for the structural change. A long &#x003b1;-helix at the Gln56&#x02013;Leu67 residues would be completely generated after the binding of NADH, although the formation of an &#x003b1;-helix at the Ala235&#x02013;Lys243 residues in the <italic>X</italic>-axis-related subunit may induce the helix formation at the Gln56&#x02013;Leu67 residues.</p><p>The D241N mutant loses the electrostatic repulsion between Glu238 and Asp241, thus the Ala235&#x02013;Lys243 residues can much more easily form the &#x003b1;-helix. As a result, the D241N mutant shows catalytic activity even without FBP or by the addition of a low concentration of FBP. However, the CD analysis also indicated that the ratio of the active state was only slightly increased, even with the addition of a high concentration of FBP. This result again suggests that the high concentration of NADH is needed for the complete structural change.</p><p>Like the <italic>Ec. mundtii</italic> LDH-2, the <italic>Lb. casei</italic> <sc>l</sc>-LDH is also known to be an acidophilic enzyme showing a low sensitivity to FBP under a neutral pH condition <xref rid="b0060 b0065" ref-type="bibr">[12,13]</xref>. Another research group has suggested that the acidophilicity of the <italic>Lb. casei</italic> <sc>l</sc>-LDH is caused by the tendency to adopt an expanded inactive state <xref rid="b0070" ref-type="bibr">[14]</xref>. They insisted that the inter-subunit interactions formed between His20 and Asp264 and between His205 and Glu211, which seem necessary in order to form a compact active state, are not generated in the enzyme under a neutral pH condition. The His205 and Glu211 residues are also present in the <italic>Ec. mundtii</italic> LDH-2 (<xref rid="f0005" ref-type="fig">Fig. 1</xref>), which may be a reason for the acidophilicity found in the D241N mutant in the absence of FBP. However, the His20, His205, Glu211, and Asp264 residues are completely conserved in <italic>Ec. mundtii</italic> LDH-1 (<xref rid="f0005" ref-type="fig">Fig. 1</xref>), which was classified as a general <sc>l</sc>-LDH in this study. Probably, the extraordinary low sensitivity of <italic>Lb. casei</italic> <sc>l</sc>-LDH to FBP has another cause that is currently undetermined.</p><p>The exact role of the acidophilic <italic>Ec. mundtii</italic> LDH-2 is currently unknown. It may act only under a low pH condition or in the presence of a high concentration of FBP and NADH. However, in the present study, we could clarify the allosteric regulation mechanism of the acidophilic LDH-2. Another research group has recently suggested that the computational studies are useful to predict the catalytic properties of <sc>l</sc>-LDHs <xref rid="b0135" ref-type="bibr">[27]</xref>. However, the study seems to be meaningless, since their conclusions are based on the homology models built using the active state structures as a reference. To accurately predict the catalytic properties of <sc>l</sc>-LDHs, consideration of the compact inactive state, shown in <xref rid="f0015" ref-type="fig">Fig. 3</xref>b, is necessary. Furthermore, the knowledge obtained from the present study may be applied to protein engineering if one wishes to add the acidophilic feature to a specific protein.</p></sec><sec sec-type="materials|methods" id="s0050"><label>4</label><title>Materials and methods</title><sec id="s0055"><label>4.1</label><title>Gene cloning, expression, and purification of LDH-1 and LDH-2</title><p>The <italic>ldh-</italic>1 gene was amplified by PCR from <italic>Ec. mundtii</italic> 15-1A genomic DNA with the forward primer, 5&#x02032;-GGAATTC<underline>CATATG</underline>ACTGCAAACGCAGAAAAAAAAG-3&#x02032; (the underline indicates an NdeI site), and the reverse primer, 5&#x02032;-TCCG<underline>CTCGAG</underline>TTCAGCATCCAATTTAGC-3&#x02032; (the underline indicates an XhoI site). The <italic>ldh-</italic>2 gene was amplified by PCR with the forward primer, 5&#x02032;-GGAATTC<underline>CATATG</underline>AAAAAAACAAGTCG-3&#x02032; (the underline indicates an NdeI site), and the reverse primer, 5&#x02032;-TCCG<underline>CTCGAG</underline>GCGTACAGTATCAAGTAC-3&#x02032; (the underline indicates an XhoI site). The amplified DNA fragment including <italic>ldh-</italic>1 or <italic>ldh-</italic>2 was digested with NdeI and XhoI and inserted into the pET-21a(+) vector (Novagen) to generate an expression plasmid for LDH-1 or LDH-2, respectively. Each expression vector was introduced into the <italic>E. coli</italic> BL21(DE3) pLysS strain. To overproduce the enzymes with His<sub>6</sub>-tag at the <italic>C</italic>-terminus, the <italic>E. coli</italic> cells were grown at 28&#x000a0;&#x000b0;C in the LB medium, and the production was induced by 1&#x000a0;mM isopropyl-&#x003b2;-<sc>d</sc>-thiogalactopyranoside. The cells were harvested by centrifugation and frozen until being lysed by sonication. Recombinant LDH-1 and LDH-2 were purified using a Ni(II)-chelated His-Bind Resin (Novagen) according to the standard protocol.</p></sec><sec id="s0060"><label>4.2</label><title>Mutation</title><p>A QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used. To create the E60Q mutant of LDH-2, an LDH-2-expression vector was amplified by PCR with the mutagenesis primer, 5&#x02032;-CGATGTCAACCAAGAAAAAGCA<underline>CAA</underline>GGAGAAGCTTTAGAC-3&#x02032; (the underline indicates the mutation site), and its complement chain. To create the D241N mutant of LDH-2, the vector was amplified with the mutagenesis primer, 5&#x02032;-GCAGCTTATGAAATCATT<underline>AAC</underline>CGTAAAAAAGC-3&#x02032; (the underline indicates the mutation site), and its complement chain. The confirmation of the mutation was done by DNA-sequencing analysis. Each of the mutated vectors was introduced into the <italic>E. coli</italic> BL21(DE3) pLysS strain. Overproduction and purification of the mutated product were done as described above.</p></sec><sec id="s0065"><label>4.3</label><title>X-ray crystallography</title><p>Prior to crystallization, the LDH-2 protein was concentrated to 12&#x000a0;mg&#x000a0;mL<sup>&#x02212;1</sup> using Amicon Ultra (Millipore). Crystals of a ligands-unbound form of LDH-2 were obtained using the sitting-drop vapor diffusion method, with a 1:1&#x000a0;(v/v) ratio of protein solution to precipitant solution. Crystals were successfully formed at 25&#x000a0;&#x000b0;C when the 0.1&#x000a0;M MES&#x02013;NaOH buffer (pH 6.2) containing 7%&#x000a0;(w/v) PEG 20,000 was used as a precipitant solution. Crystals of the ligands-bound form were grown in the presence of 1.5&#x000a0;mM NADH and 1&#x000a0;mM FBP. Initial crystals were obtained by using the 0.1&#x000a0;M sodium citrate buffer (pH 5.3) containing 6%&#x000a0;(w/v) PEG 4000 and 0.2&#x000a0;M ammonium acetate as a precipitant solution. The quality of the crystals was improved by repeating the micro-seeding method.</p><p>The diffraction intensities of the crystals were obtained using synchrotron radiation from the BL26B2 station at SPring-8, Japan. Each crystal was captured in a loop and then frozen in liquid nitrogen. After the delivery of the crystals to the facility, they were placed on goniometer head in a stream of nitrogen gas cooled to 100&#x000a0;K automatically. We obtained the diffraction intensity data of ligands-unbound and -bound crystals up to 2.38 and 2.30&#x000a0;&#x000c5; resolutions, respectively. The diffraction intensities of ligands-unbound crystal were integrated and scaled by HKL2000 <xref rid="b0140" ref-type="bibr">[28]</xref>, while those of ligands-bound crystal were processed by the combination of Mosflm and Scala in the CCP4 program suite <xref rid="b0145" ref-type="bibr">[29]</xref>. The three-dimensional structure of the ligands-unbound LDH-2 was solved by the molecular replacement method using the program Molrep in the CCP4 program suite <xref rid="b0145" ref-type="bibr">[29]</xref>. The starting model used in this study was one subunit in the active state of the <italic>B. stearothermophilus</italic> <sc>l</sc>-LDH (PDB code <ext-link ext-link-type="uri" xlink:href="pdb:1LDN" id="ir0015">1LDN</ext-link>) <xref rid="b0020" ref-type="bibr">[4]</xref>. The model was further refined with simulated annealing and conventional restrained refinement methods using the CNS program <xref rid="b0150" ref-type="bibr">[30]</xref>. A subset of 5% of the reflections was used to monitor the free <italic>R</italic> factor (<italic>R</italic><sub>free</sub>) <xref rid="b0155" ref-type="bibr">[31]</xref>. Each refinement cycle includes the refinement of the positional parameters, individual isotropic <italic>B</italic>-factors, and revision of the model visualized by the program Xfit in the XtalView software package <xref rid="b0160" ref-type="bibr">[32]</xref>. The ligands-bound crystal structure was also determined by molecular replacement using Molrep and refined by CNS. The details of the data collection and refinement statistics are shown in <xref rid="t0010" ref-type="table">Table 2</xref>.</p></sec><sec id="s0070"><label>4.4</label><title>Activity assay of LDH</title><p>The LDH activity was determined by measuring the rate of NADH oxidation at 340&#x000a0;nm (<italic>&#x003b5;</italic>&#x000a0;=&#x000a0;6220&#x000a0;M<sup>&#x02212;1</sup>&#x000a0;cm<sup>&#x02212;1</sup>) in a quartz cell with a path-length of 0.1 or 0.2&#x000a0;mm. All measurements were based on at least duplicate determinations of the reaction rates. To measure the pH profiles of activity, reactions were carried out in a broad-range buffer (50&#x000a0;mM 3,3-dimethylglutaric acid, 50&#x000a0;mM Tris, and 50&#x000a0;mM 2-amino-2-methyl-1,3-propandiol), adjusted at a given pH, containing 5&#x000a0;mM magnesium chloride, 20&#x000a0;mM pyruvate, 1.5&#x000a0;mM NADH, and 0.1&#x000a0;mM FBP at 37&#x000a0;&#x000b0;C. Detailed kinetic analysis was performed using 100&#x000a0;mM MES&#x02013;NaOH (pH 5.5) or HEPES&#x02013;NaOH (pH 7.5) buffer. To determine the <italic>K<sub>m</sub></italic> value for NADH, reactions were carried out in the buffer containing 20&#x000a0;mM pyruvate, 3&#x000a0;mM FBP, and a given concentration of NADH (0&#x02013;1.5&#x000a0;mM). To determine the <italic>K<sub>m</sub></italic> value for pyruvate, reactions were carried out in the buffer containing 1.5&#x000a0;mM NADH, 3&#x000a0;mM FBP, and a given concentration of pyruvate (0&#x02013;20&#x000a0;mM). To determine the <italic>K</italic><sub>act</sub> value, reactions were carried out in the buffer containing 1.5&#x000a0;mM NADH, 20&#x000a0;mM pyruvate, and a given concentration of FBP (0&#x02013;3&#x000a0;mM).</p><p>In any case, the enzymes exhibited a hyperbolic kinetic response to increasing concentrations of pyruvate, NADH, or FBP. Therefore, kinetic data were fitted to Eq. <xref rid="e0005" ref-type="disp-formula">(1)</xref>:<disp-formula id="e0005"><label>(1)</label><mml:math id="M1" altimg="si1.gif" overflow="scroll"><mml:mi>v</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>k</mml:mi></mml:mrow><mml:mrow><mml:mtext>cat</mml:mtext></mml:mrow></mml:msub><mml:mo>&#x000b7;</mml:mo><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:msub><mml:mo>&#x000b7;</mml:mo><mml:mo stretchy="false">(</mml:mo><mml:mi>B</mml:mi><mml:mo>+</mml:mo><mml:mi>X</mml:mi><mml:mo stretchy="false">)</mml:mo></mml:mrow><mml:mrow><mml:mi>A</mml:mi><mml:mo>+</mml:mo><mml:mi>X</mml:mi></mml:mrow></mml:mfrac></mml:math></disp-formula></p><p>In the equation, <italic>v</italic> is the initial velocity, <italic>k</italic><sub>cat</sub> is the catalytic constant, <italic>A</italic> and <italic>B</italic> are constant parameters, <italic>E<sub>t</sub></italic> is the concentration of the enzyme, and <italic>X</italic> is the concentration of pyruvate, NADH, or FBP. In general, <italic>B</italic> was fixed to zero, and <italic>k</italic><sub>cat</sub> and <italic>A</italic>, which means <italic>K<sub>m</sub></italic> (for pyruvate or NADH) or <italic>K</italic><sub>act</sub> (for FBP), were determined by the non-linear least square method. Exceptionally, since the mutant enzyme D241N exhibited catalytic activity in the absence of FBP, <italic>B</italic> was also determined in addition to <italic>k</italic><sub>cat</sub> and <italic>A</italic>. In this case, the ratio of activity in the absence of FBP to the maximum activity is correspondent with <italic>B</italic>/<italic>A</italic>, and <italic>K</italic><sub>act</sub> is expressed as <italic>A</italic>&#x000a0;&#x02212;&#x000a0;2<italic>B</italic>.</p><p>In the case of the D241N mutant, we also determined the <italic>k</italic><sub>cat</sub> and <italic>K<sub>m</sub></italic> values in the absence of FBP. At pH 7.5, the <italic>k</italic><sub>cat</sub> and <italic>K<sub>m</sub></italic> values for NADH and pyruvate could not be determined due to the fact that the enzyme activities did not reach the plateau in the concentration range of substrates. Since the enzymatic activity of the mutant increased linearly related to the increasing concentration of substrates, data for the steady-state kinetics were fitted to Eq. <xref rid="e0010" ref-type="disp-formula">(2)</xref>:<disp-formula id="e0010"><label>(2)</label><mml:math id="M2" altimg="si2.gif" overflow="scroll"><mml:mi>v</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>k</mml:mi></mml:mrow><mml:mrow><mml:mtext>cat</mml:mtext></mml:mrow></mml:msub><mml:mo>&#x000b7;</mml:mo><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:msub><mml:mo>&#x000b7;</mml:mo><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi>m</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac></mml:math></disp-formula></p></sec><sec id="s0075"><label>4.5</label><title>VUVCD measurements</title><p>CD spectroscopy is obviously useful for the estimation of the secondary-structure content of proteins, and the accuracy is improved by the extension of CD measurements to the VUV region below 190&#x000a0;nm <xref rid="b0165 b0170 b0175 b0180 b0185 b0190 b0195" ref-type="bibr">[33&#x02013;39]</xref>. The VUVCD spectra of the <italic>Ec. mundtii</italic> LDH-2 in a 20&#x000a0;mM sodium phosphate buffer (pH 7.5) were measured from 260 to 175&#x000a0;nm using the synchrotron-radiation VUVCD spectrophotometer constructed at Hiroshima Synchrotron Radiation Center and an assembled-type optical cell <xref rid="b0200 b0205" ref-type="bibr">[40,41]</xref> at 25&#x000a0;&#x000b0;C. The path length of the cell was adjusted with a Teflon spacer to 10&#x000a0;&#x003bc;m. The details of the optical devices of the spectrophotometer and the optical cell are available elsewhere <xref rid="b0205" ref-type="bibr">[41]</xref>. All of the VUVCD spectra were recorded with a 0.25-mm slit, a 4-s time constant, a 20-nm&#x000a0;min<sup>&#x02212;1</sup> scan speed, and 4 accumulations. The ellipticity was reproducible within an error margin of 5%, which was mainly attributable to noise and to inaccuracy in the optical path length. The secondary structures of LDH-2 were analyzed using the SELCON3 program <xref rid="b0210" ref-type="bibr">[42]</xref>, which was improved by using the VUVCD spectra down to 160&#x000a0;nm of the 31 reference proteins with known X-ray structures <xref rid="b0180 b0185" ref-type="bibr">[36,37]</xref>. The secondary structures in the crystal form were assigned using the DSSP program <xref rid="b0215" ref-type="bibr">[43]</xref> based on the numbers and positions of hydrogen bonds between peptide groups. In this analysis, the 3<sub>10</sub>-helix was classified as an unordered structure.</p></sec></sec><sec id="s0080"><title>Author contribution statement</title><p>Y.M. planned experiments, performed experiments, analyzed data, and wrote the paper; M.M. performed experiments; K.M. performed experiments, analyzed data, and wrote the paper; K.O., M.N., F.H. and T.K. analyzed data; M.S. planned experiments, analyzed data, and wrote the paper.</p></sec>
Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle
<p> Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.</p>
<contrib contrib-type="author"><name><surname>AKAGI</surname><given-names>Satoshi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>MATSUKAWA</surname><given-names>Kazutsugu</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>TAKAHASHI</surname><given-names>Seiya</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><aff id="aff1"><label>1)</label>Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, Ibaraki 305-0901, Japan</aff><aff id="aff2"><label>2)</label>Multidisciplinary Science Cluster, Life and Environmental Medicine Science Unit, Kochi University, Kochi 783-8502, Japan</aff><aff id="aff3"><label>3)</label>Headquarters, National Agriculture and Food Research Organization, Ibaraki 305-8517, Japan</aff>
The Journal of Reproduction and Development
<p>The somatic cell nuclear transfer (SCNT) technology is expected to be useful for farm animal breeding and research, the production of transgenic animals for biomedical purposes, and the conservation of endangered species. Cattle are probably the most widely used species for SCNT experiments [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>]. Successful production of clones of elite bulls [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r4" ref-type="bibr">4</xref>], cows with high milk performance [<xref rid="r5" ref-type="bibr">5</xref>] and an endangered breed of cattle [<xref rid="r6" ref-type="bibr">6</xref>] has been reported. Furthermore, transgenic cattle such as calves lacking the prion protein [<xref rid="r7" ref-type="bibr">7</xref>] and cows overexpressing casein proteins in their milk [<xref rid="r8" ref-type="bibr">8</xref>] have been produced by SCNT. However, the efficiency of bovine cloning remains low, despite the numerous studies that have been conducted. Nuclear transfer (NT) is a complex multistep procedure including oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation, and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following SCNT. In this review, we discuss some of the factors that affect the developmental ability of bovine SCNT embryos based on our studies as well as other previous reports.</p><sec id="s1"><title>Oocyte Source and Quality</title><p>Oocytes are usually collected from slaughterhouse-derived ovaries or live cows by ovum pick-up (OPU) and used for bovine SCNT after <italic>in vitro</italic> maturation. We examined the developmental ability of NT embryos derived from the cumulus cells of a Japanese black cow using slaughterhouse-derived and OPU-derived <italic>in vitro</italic> matured oocytes. As shown in <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Development of nuclear transfer (NT) embryos derived from cumulus cells of a Japanese black cow using ovum pick-up (OPU)-derived and slaughterhouse-derived oocytes</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" valign="top" colspan="2" rowspan="1">Recipient oocytes</td><td align="center" valign="top" rowspan="2" colspan="1">No. of NT<break/>embryos</td><td align="center" valign="top" rowspan="2" colspan="1">No. of cleaved<break/>embryos (%)</td><td align="center" valign="top" rowspan="2" colspan="1">No. of blastocysts<break/>(%)</td><td align="center" valign="top" rowspan="2" colspan="1">No. of embryos<break/>transferred</td><td align="center" valign="top" rowspan="2" colspan="1">No. of calves<break/>(%)</td><td align="center" valign="top" rowspan="2" colspan="1">No. surviving<break/>&#x0003e; 60 days</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Source</td><td align="center" valign="bottom" rowspan="1" colspan="1">Breed</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Slaughterhouse</td><td align="left" rowspan="1" colspan="1">Unknown</td><td align="center" rowspan="1" colspan="1">89</td><td align="center" rowspan="1" colspan="1">70 (78.7)</td><td align="center" rowspan="1" colspan="1">32 (36.0)</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">5 (25)</td><td align="center" rowspan="1" colspan="1">3 (15)</td></tr><tr><td align="left" rowspan="3" colspan="1">OPU</td><td align="left" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">112</td><td align="center" rowspan="1" colspan="1">101 (90.1)</td><td align="center" rowspan="1" colspan="1">33 (29.5)</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">5 (50)</td><td align="center" rowspan="1" colspan="1">1 (20)</td></tr><tr><td align="left" rowspan="1" colspan="1">Japanese black</td><td align="center" rowspan="1" colspan="1">70</td><td align="center" rowspan="1" colspan="1">64 (91.4)</td><td align="center" rowspan="1" colspan="1">19 (27.1)</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">2 (28)</td><td align="center" rowspan="1" colspan="1">0 (0)</td></tr><tr><td align="left" rowspan="1" colspan="1">Holstein</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">37 (88.1)</td><td align="center" rowspan="1" colspan="1">14 (33.3)</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">3 (100)</td><td align="center" rowspan="1" colspan="1">1 (33)</td></tr></tbody></table></table-wrap>, no significant differences in the cleavage and blastocyst formation rates were observed between oocyte sources (OPU and slaughterhouse). Japanese black cows (same breed as donor cells) and Holstein cows were used as the OPU donors, but the breed of oocyte donors did not affect the <italic>in vitro</italic> developmental ability of SCNT embryos. Sugimura <italic>et al</italic>. also reported no difference in the blastocyst formation rates of SCNT embryos between oocytes from a slaughterhouse and OPU, but follicle-stimulating hormone (FSH) pretreatment of OPU donor cows improved oxygen consumption and <italic>OCT4</italic> and <italic>IFN-&#x003c4;</italic> expression of SCNT embryos to levels similar to fertilized embryos [<xref rid="r9" ref-type="bibr">9</xref>], suggesting that FSH pretreatment of OPU donor cows has a positive effect on oocyte quality. Furthermore, <italic>in vivo</italic>-matured oocytes can be collected by OPU from hormone-treated cows [<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r11" ref-type="bibr">11</xref>]. <italic>In vivo</italic>-matured oocytes are more developmentally competent after <italic>in vitro</italic> fertilization (IVF) than <italic>in vitro</italic>-matured oocytes [<xref rid="r10" ref-type="bibr">10</xref>,<xref rid="r11" ref-type="bibr">11</xref>,<xref rid="r12" ref-type="bibr">12</xref>]. We examined the development of <italic>in vivo</italic>- and <italic>in vitro</italic>-matured oocytes after SCNT [<xref rid="r13" ref-type="bibr">13</xref>]. <italic>In vivo</italic>-matured oocytes collected by OPU from heifers treated with FSH, prostaglandin-F2&#x003b1; and gonadotropin hormone-releasing hormone, and <italic>in vitro</italic>-matured oocytes collected from slaughterhouse-derived ovaries were used as recipient cytoplasts. In accordance with the bovine IVF results [<xref rid="r10" ref-type="bibr">10</xref>,<xref rid="r11" ref-type="bibr">11</xref>,<xref rid="r12" ref-type="bibr">12</xref>], the blastocyst formation rate of <italic>in vivo</italic>-matured oocytes after SCNT was significantly higher than that of <italic>in vitro</italic>-matured oocytes. The pregnancy rate did not differ between <italic>in vivo</italic>- and <italic>in vitro</italic>-matured oocytes. However, a high abortion rate (75% of pregnancies) was observed in SCNT fetuses from <italic>in vitro</italic>-matured oocytes, whereas no subsequent abortions were observed from <italic>in vivo</italic>-matured oocytes. These results suggest that inappropriate oocyte maturation of recipient cytoplasts is one of the factors causing embryonic or fetal loss after NT in cattle.</p><p>In SCNT, donor cells are electrically fused with enucleated recipient oocytes containing a large amount of foreign cytoplasm. Cloned calves with mixed mitochondrial DNA from the donor cell and the recipient oocyte (heteroplasmy) have been reported [<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r15" ref-type="bibr">15</xref>,<xref rid="r16" ref-type="bibr">16</xref>,<xref rid="r17" ref-type="bibr">17</xref>], although the influence of heteroplasmy on the development of SCNT embryos is unclear. Cloned calves can be produced using both oocytes and somatic cells derived from the same cow to avoid cytoplasmic contribution from foreign oocytes (autologous SCNT [<xref rid="r18" ref-type="bibr">18</xref>]). Cloned calves produced in this manner do not exhibit heteroplasmy. Yang <italic>et al</italic>. showed that autologous SCNT embryos resulted in higher developmental rates <italic>in vitro</italic> and <italic>in vivo</italic> compared with heterologous SCNT embryos (donor cell not related to recipient cytoplasm) [<xref rid="r18" ref-type="bibr">18</xref>]. In contrast, reports by other laboratories [<xref rid="r19" ref-type="bibr">19</xref>,<xref rid="r20" ref-type="bibr">20</xref>,<xref rid="r21" ref-type="bibr">21</xref>] have indicated no such positive effect of autologous SCNT. This discrepancy may be because of the influence of individual oocyte donors. The oocyte donor influences the production of blastocysts in bovine IVF [<xref rid="r22" ref-type="bibr">22</xref>] and SCNT [<xref rid="r23" ref-type="bibr">23</xref>]. We examined the developmental ability of autologous SCNT embryos using cumulus cells and oocytes collected from six cows by OPU [<xref rid="r24" ref-type="bibr">24</xref>]. The developmental rates of autologous SCNT embryos to the blastocyst stage varied widely among individual cows (range, 19&#x02013;64%) [<xref rid="r24" ref-type="bibr">24</xref>]. We produced four cloned calves by autologous SCNT. However, two of the calves were stillborn, and the remaining two died 13 days and 150 days after birth and had anomalies at the postmortem examination. These results suggest that it is difficult to improve the birth rate of healthy cloned calves only using both oocytes and somatic cells derived from the same cow.</p></sec><sec id="s2"><title>Cell Cycle Combination</title><p>The cell cycle of the donor cells is an important factor affecting the development of SCNT embryos, because cell cycle co-ordination of donor cells and recipient oocytes is essential to maintain ploidy and prevent DNA damage [<xref rid="r25" ref-type="bibr">25</xref>]. Nonactivated metaphase II (MII) oocytes have been primarily used as recipient cytoplasts for bovine SCNT [<xref rid="r26" ref-type="bibr">26</xref>]. Accordingly, G0- or G1-phase cells of the cell cycle have been used in almost all successful reports [<xref rid="r27" ref-type="bibr">27</xref>], although M-phase cells can also be reprogrammed in MII oocytes [<xref rid="r28" ref-type="bibr">28</xref>]. The efficiency of blastocyst and full-term development was compared between SCNT embryos derived from fibroblast cells at the G0 and G1 phases in several studies [<xref rid="r29" ref-type="bibr">29</xref>,<xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r31" ref-type="bibr">31</xref>,<xref rid="r32" ref-type="bibr">32</xref>]. No significant difference was observed in <italic>in vitro</italic> development between G0- and G1-phase cell SCNT embryos. However, the <italic>in vivo</italic> developmental ability of SCNT embryos tended to be higher for G1-phase cells than that for G0-phase cells [<xref rid="r29" ref-type="bibr">29</xref>,<xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r31" ref-type="bibr">31</xref>,<xref rid="r32" ref-type="bibr">32</xref>]. One study suggested that homogeneous expression among all blastomeres of SCNT embryos derived from G1-phase cells at embryonic gene activation contributes to a higher success rate [<xref rid="r33" ref-type="bibr">33</xref>]. The development of SCNT embryos using pre-activated oocytes has been examined in several studies [<xref rid="r28" ref-type="bibr">28</xref>, <xref rid="r34" ref-type="bibr">34</xref>,<xref rid="r35" ref-type="bibr">35</xref>,<xref rid="r36" ref-type="bibr">36</xref>,<xref rid="r37" ref-type="bibr">37</xref>]. Oocytes activated 6 h before NT stopped developing at the 8-cell stage after NT, regardless of the cell cycle of the donor cells [<xref rid="r28" ref-type="bibr">28</xref>]. However, oocytes within a few hours after activation appear to have a capacity to reprogram the somatic cell nucleus, and this capacity may be largely dependent on the cell cycle stage of the donor cells. Successful production of cloned calves was reported with SCNT embryos using S/G2-phase cells and oocytes activated 2.5 h before NT [<xref rid="r35" ref-type="bibr">35</xref>]. In contrast, no cloned calves were obtained with oocytes activated 2 h before NT when we used G0- and G1-phase cells [<xref rid="r38" ref-type="bibr">38</xref>].</p></sec><sec id="s3"><title>Cell Type and <italic>In Vitro</italic> Culture of Donor Cells</title><p>Cloned cattle have been produced from various somatic cell types. However, it is still unclear which cell type is the most appropriate for bovine SCNT [<xref rid="r27" ref-type="bibr">27</xref>, <xref rid="r39" ref-type="bibr">39</xref>]. Moreover, the differentiation status of somatic cells may have no relationship with cloning efficiency [<xref rid="r40" ref-type="bibr">40</xref>]. Bovine SCNT embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by IVF (approximately 30&#x02013;50%) [<xref rid="r41" ref-type="bibr">41</xref>], although the electric conditions for fusion of enucleated oocytes differs among donor cell types [<xref rid="r42" ref-type="bibr">42</xref>]. However, high embryonic and fetal losses occur after embryo transfer regardless of donor cell type. Because the efficiency of bovine cloning is low, it may be difficult to show significant differences among donor cell types [<xref rid="r43" ref-type="bibr">43</xref>].</p><p>In bovine SCNT, donor cells are usually cultured <italic>in vitro</italic> before being used for NT [<xref rid="r44" ref-type="bibr">44</xref>,<xref rid="r45" ref-type="bibr">45</xref>,<xref rid="r46" ref-type="bibr">46</xref>]. Not only the nuclei of short-term cultured cells but also the nuclei of long-term cultured cells (cultured for 3 months) [<xref rid="r47" ref-type="bibr">47</xref>] or those close to the end of their life span [<xref rid="r48" ref-type="bibr">48</xref>] have the ability to generate live healthy calves after NT. We compared the developmental ability of SCNT embryos using bovine cumulus cells under four different culture conditions (non-culture, maturation culture for 20 h, cycling culture and serum-starved culture) to examine the effect of <italic>in vitro</italic> culture of donor cells on cloning efficiency [<xref rid="r49" ref-type="bibr">49</xref>]. The blastocyst formation rate and blastocyst cell number of SCNT embryos derived from cultured cumulus cells (cycling culture and serum-starved culture) were significantly higher than those of SCNT embryos derived from fresh (non-cultured) cells [<xref rid="r49" ref-type="bibr">49</xref>]. Cell cycle analysis using flow cytometer showed that the relative percentage of fresh cells in the G0/G1 phase of the cell cycle (89.7 &#x000b1; 0.4%) was similar to that of serum-starved cells (90.6 &#x000b1; 0.6%) but lower than that of cycling cells (76.0 &#x000b1; 1.8%) [<xref rid="r49" ref-type="bibr">49</xref>], indicating that the difference in <italic>in vitro</italic> development between fresh and cultured cells did not result from the cell cycle of the donor cells. The same results have also been obtained for goat [<xref rid="r50" ref-type="bibr">50</xref>] and rabbit SCNT [<xref rid="r51" ref-type="bibr">51</xref>]. These results suggest that culture of donor cells increases the efficiency of SCNT embryo production <italic>in vitro</italic>. However, the subsequent viability of blastocyst-stage embryos produced using fresh cells may not be different from that using cultured cells. No difference was observed in the <italic>in vivo</italic> developmental ability of SCNT embryos between fresh and cultured cells, and live calves were obtained from cumulus cells under all culture conditions (<xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> Cloned calves produced by nuclear transfer using cumulus cells under 4 different conditions: (a) cells removed from cumulus-oocyte complexes (COCs) after aspiration of ovarian follicles; (b) cells removed from COCs after <italic>in vitro</italic> maturation; (c) cells cultured in Dulbecco&#x02019;s modified Eagle&#x02019;s medium (DMEM) with 10% fetal bovine serum (FBS) for 3 days after some subculture; and (d) cells cultured in DMEM with 0.5% FBS for an additional 5 days.</p></caption><graphic xlink:href="jrd-60-329-g001"/></fig>) [<xref rid="r49" ref-type="bibr">49</xref>].</p></sec><sec id="s4"><title>Timing of Fusion and Activation</title><p>In SCNT, the lack of sperm-induced fertilization necessitates artificial activation to trigger further development. Direct exposure of chromosomes to nonactivated MII cytoplasm is effective for somatic cell nuclear reprogramming [<xref rid="r28" ref-type="bibr">28</xref>, <xref rid="r52" ref-type="bibr">52</xref>], and nonactivated MII oocytes have been used in almost all successful bovine SCNT reports [<xref rid="r26" ref-type="bibr">26</xref>, <xref rid="r27" ref-type="bibr">27</xref>]. The timing of activation of MII oocytes can be classified into two protocols as follows: (1) activation performed immediately after fusion (simultaneous fusion and activation method, FA) and (2) activation performed several hours after fusion (delayed activation method, DA). Successful production of cloned offspring using SCNT has been reported for both the FA [<xref rid="r45" ref-type="bibr">45</xref>, <xref rid="r47" ref-type="bibr">47</xref>] and DA [<xref rid="r44" ref-type="bibr">44</xref>, <xref rid="r46" ref-type="bibr">46</xref>] methods. Donor chromosomes are exposed to factors present in MII cytoplasm for only a short time in the FA method and for a longer time in the DA method. The DA method improves the <italic>in vitro</italic> development of bovine [<xref rid="r6" ref-type="bibr">6</xref>, <xref rid="r53" ref-type="bibr">53</xref>] and mouse [<xref rid="r54" ref-type="bibr">54</xref>, <xref rid="r55" ref-type="bibr">55</xref>] NT embryos derived from somatic cells at the G0/G1 stage compared with that of the FA method. We compared the developmental ability of bovine fibroblast cell NT embryos produced using different fusion and chemical activation timings to develop an efficient fusion and activation protocol for producing SCNT embryos [<xref rid="r56" ref-type="bibr">56</xref>]. As shown in <xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title><italic>In vitro</italic> development of somatic cell nuclear transfer (SCNT) embryos produced using different fusion and activation timings</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Group</td><td align="center" valign="top" colspan="2" rowspan="1">Hours post IVM<hr/></td><td align="center" valign="top" rowspan="2" colspan="1">No. SCNT<break/>embryos</td><td align="center" valign="top" rowspan="2" colspan="1">No. of cleaved<break/>embryos (%)</td><td align="center" valign="top" rowspan="2" colspan="1">No. of<break/>blastocysts (%)</td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Fusion</td><td align="center" valign="top" rowspan="1" colspan="1">Activation</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">F21A21</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">89</td><td align="center" rowspan="1" colspan="1">60 (67)<sup>cd</sup></td><td align="center" rowspan="1" colspan="1">25 (28)<sup>c</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">F21A24</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">125</td><td align="center" rowspan="1" colspan="1">97 (78)<sup>bc</sup></td><td align="center" rowspan="1" colspan="1">79 (63)<sup>a</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">F21A27</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">96</td><td align="center" rowspan="1" colspan="1">52 (54)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">3 (3)<sup>e</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">F24A24</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">150</td><td align="center" rowspan="1" colspan="1">123 (82)<sup>b</sup></td><td align="center" rowspan="1" colspan="1">59 (39)<sup>bc</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">F24A27</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">134</td><td align="center" rowspan="1" colspan="1">122 (91)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">63 (47)<sup>b</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">F24A30</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">30</td><td align="center" rowspan="1" colspan="1">93</td><td align="center" rowspan="1" colspan="1">63 (68)<sup>cd</sup></td><td align="center" rowspan="1" colspan="1">13 (14)<sup>d</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">F27A27</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">121</td><td align="center" rowspan="1" colspan="1">99 (82)<sup>b</sup></td><td align="center" rowspan="1" colspan="1">49 (41)<sup>bc</sup></td></tr></tbody></table><table-wrap-foot><p><sup>a,b,c,d,e</sup> Values without common characters in the same column of each group differ significantly (P &#x0003c; 0.05, chi-square test).</p></table-wrap-foot></table-wrap>, the <italic>in vitro</italic> development of SCNT embryos was affected by the timing of fusion and chemical activation, and the development of SCNT embryos to the blastocyst stage in the F21A24 group (fusion at 21 h and activation 24 h postmaturation) of the DA method was significantly higher than that in the other groups. However, the development of SCNT embryos activated 6 h after fusion (F21A27 and F24A30 groups) in the DA method was significantly lower than that in the FA method. In reports by Aston <italic>et al</italic>. [<xref rid="r57" ref-type="bibr">57</xref>] and Choi <italic>et al</italic>. [<xref rid="r58" ref-type="bibr">58</xref>], excessive exposure to MII cytoplasm resulted in abnormal chromatin morphology, but SCNT embryos activated less than 2.5 h after fusion resulted in improved nuclear morphology and increased development to the compacted morula/blastocyst stage. These reports and our results suggest that the exposure duration of somatic cell nuclei to oocyte cytoplasm before activation affects the <italic>in vitro</italic> development of SCNT embryos and that excessive exposure to MII cytoplasm results in a poor developmental rate to the blastocyst stage. However, no influence of the duration of exposure to oocyte cytoplasm on the <italic>in vivo</italic> developmental ability has been observed. When we examined the <italic>in vivo</italic> developmental ability of cumulus cell NT embryos and postnatal survivability of cloned calves produced by the DA (F21A24) and FA (F24A24) methods, the pregnancy and calving rates did not differ significantly between the two methods [<xref rid="r59" ref-type="bibr">59</xref>]. In addition, high rates of postnatal mortality were observed in both the methods [<xref rid="r59" ref-type="bibr">59</xref>]. Sung <italic>et al</italic>. obtained similar results using two types of donor cells (cumulus and fibroblast cells) [<xref rid="r60" ref-type="bibr">60</xref>]. In a report by Aston <italic>et al</italic>., the time interval between fusion and activation when using the DA method did not affect the <italic>in vivo</italic> development of SCNT embryos [<xref rid="r57" ref-type="bibr">57</xref>].</p></sec><sec id="s5"><title>Histone Deacetylase Inhibitor (HDACi) Treatment</title><p>Abnormal epigenetic modifications such as aberrant DNA methylation and histone modification have been observed in SCNT embryos [<xref rid="r61" ref-type="bibr">61</xref>,<xref rid="r62" ref-type="bibr">62</xref>,<xref rid="r63" ref-type="bibr">63</xref>,<xref rid="r64" ref-type="bibr">64</xref>]. Therefore, preventing epigenetic errors is expected to lead to improved animal cloning success rates [<xref rid="r65" ref-type="bibr">65</xref>]. Several DNA methylation inhibitors and HDACis have been used to improve the developmental ability of bovine SCNT embryos [<xref rid="r66" ref-type="bibr">66</xref>]. Treatment of donor cells with 5-aza-2&#x02019;-deoxycytidine (5-aza-dC), a DNA methylation inhibitor, did not improve the <italic>in vitro</italic> developmental ability of SCNT embryos [<xref rid="r67" ref-type="bibr">67</xref>,<xref rid="r68" ref-type="bibr">68</xref>,<xref rid="r69" ref-type="bibr">69</xref>], whereas treatment with trichostatin A (TSA) or sodium butyrate, an HDACi, increased the blastocyst formation rate [<xref rid="r67" ref-type="bibr">67</xref>,<xref rid="r68" ref-type="bibr">68</xref>,<xref rid="r69" ref-type="bibr">69</xref>,<xref rid="r70" ref-type="bibr">70</xref>]. However, improvement of full-term development following HDACi treatment of donor cells has not been demonstrated. It was reported from two laboratories in 2006 that TSA treatment of mouse SCNT embryos after NT improved the success rate of mouse cloning [<xref rid="r65" ref-type="bibr">65</xref>, <xref rid="r71" ref-type="bibr">71</xref>]. In these reports, TSA treatments for 9&#x02013;20 h led to a significant increase not only in the blastocyst formation rate but also in the full-term developmental rate [<xref rid="r65" ref-type="bibr">65</xref>, <xref rid="r71" ref-type="bibr">71</xref>]. In addition, Kohda <italic>et al</italic>. showed that the gene expression profile of TSA-treated cloned mice came to resemble that of mice produced by intracytoplasmic sperm injection [<xref rid="r72" ref-type="bibr">72</xref>]. These reports suggest that inhibiting histone deacetylation in SCNT embryos during a short period of culture after NT promotes reprogramming of the donor nucleus in mice. We examined the effects of treatment with HDACis, TSA and scriptaid (SCR), on the <italic>in vitro</italic> development of bovine SCNT embryos using three fibroblast cell lines (L1, L2 and L3) [<xref rid="r73" ref-type="bibr">73</xref>]. As shown in <xref ref-type="fig" rid="fig_002">Fig. 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Development to the blastocyst stage of somatic cell nuclear transfer (SCNT) embryos treated with 5, 50 and 500 nM scriptaid (SCR) or 5 nM trichostatin A (TSA). Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors. <sup>a</sup>Significant difference compared with the control (P &#x0003c; 0.05, chi-square test). Reproduced with permission of the Society for Reproduction and Development from Akagi S, <italic>et al</italic>.: Treatment with a histone deacetylase inhibitor after nuclear transfer improves the preimplantation development of cloned bovine embryos. <italic>J Reprod Dev</italic> 2011; 57: 120&#x02013;126.</p></caption><graphic xlink:href="jrd-60-329-g002"/></fig>, TSA treatment improved blastocyst formation rates of SCNT embryos derived from L1 and L3 but had no effect on the rate of embryos derived from L2. Furthermore, SCR treatment increased the blastocyst formation rates of SCNT embryos derived from L1 and L2, but no significant increase was observed in SCNT embryos derived from L3. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, optimal treatment conditions may differ among donor cell lines. Four laboratories have recently reported the <italic>in vivo</italic> developmental ability of bovine SCNT embryos treated with TSA [<xref rid="r74" ref-type="bibr">74</xref>,<xref rid="r75" ref-type="bibr">75</xref>,<xref rid="r76" ref-type="bibr">76</xref>,<xref rid="r77" ref-type="bibr">77</xref>]. In contrast to the results in mice [<xref rid="r65" ref-type="bibr">65</xref>, <xref rid="r71" ref-type="bibr">71</xref>], treatment of bovine SCNT embryos with TSA alone did not significantly improve the full-term developmental rate [<xref rid="r74" ref-type="bibr">74</xref>,<xref rid="r75" ref-type="bibr">75</xref>,<xref rid="r76" ref-type="bibr">76</xref>]. In contrast, a higher calving rate was observed following a combined treatment with a DNA methylation inhibitor [<xref rid="r77" ref-type="bibr">77</xref>]. Wang <italic>et al</italic>. reported that a combined treatment of both donor cells and SCNT embryos with 5-aza-dC and TSA reduced the methylation levels of the NT blastocyst satellite I sequence to levels similar to those in IVF embryos and increased the cloning efficiency from 2.6 to 13.4% [<xref rid="r77" ref-type="bibr">77</xref>]. However, it is difficult to correct epigenetic abnormalities completely only by treatment with epigenetic modifiers, as various abnormalities including large offspring syndrome have been observed in cloned calves after combined treatment of both donor cells and cloned embryos with 5-aza-dC and TSA as well as untreated cloned calves [<xref rid="r77" ref-type="bibr">77</xref>].</p></sec><sec id="s6"><title>Embryo Aggregation</title><p>The cell number in blastocysts has been used as an indicator of embryo quality [<xref rid="r78" ref-type="bibr">78</xref>]. The cell number in SCNT embryos is lower than that in <italic>in vivo</italic>-derived embryos [<xref rid="r79" ref-type="bibr">79</xref>, <xref rid="r80" ref-type="bibr">80</xref>]. This poor blastocyst quality appears to contribute to the decreased survival rate of SCNT embryos after embryo transfer. Embryo aggregation is a method that enables an increase in the cell number in embryos [<xref rid="r80" ref-type="bibr">80</xref>, <xref rid="r81" ref-type="bibr">81</xref>]. In addition, several studies have indicated that embryo aggregation affects SCNT embryo gene expression [<xref rid="r82" ref-type="bibr">82</xref>,<xref rid="r83" ref-type="bibr">83</xref>,<xref rid="r84" ref-type="bibr">84</xref>]. In mice, aggregation of SCNT embryos at the 4-cell stage led not only to an increase in cell number but also improved <italic>Oct 4</italic> expression, and resulted in eight times higher full-term development compared with single embryos [<xref rid="r80" ref-type="bibr">80</xref>]. In cattle, a high pregnancy rate was observed in embryos aggregated at 4 days after SCNT (day 4) [<xref rid="r85" ref-type="bibr">85</xref>, <xref rid="r86" ref-type="bibr">86</xref>], whereas aggregation of 1-cell stage SCNT embryos did not improve <italic>in vivo</italic> development [<xref rid="r82" ref-type="bibr">82</xref>, <xref rid="r87" ref-type="bibr">87</xref>]. Aggregation of 1-cell stage SCNT embryos resulted in reduced <italic>OCT4</italic> expression compared with IVF embryos [<xref rid="r82" ref-type="bibr">82</xref>], whereas no significant difference in <italic>OCT4</italic> expression was observed between IVF embryos and aggregates of day 2 embryos [<xref rid="r88" ref-type="bibr">88</xref>]. Thus, the timing of aggregation may be important for producing high-quality SCNT embryos. We examined the effect of the timing of aggregation on the development of SCNT embryos [<xref rid="r89" ref-type="bibr">89</xref>]. One-cell stage embryos after activation, 8-cell stage embryos on day 2 or 16- to 32-cell stage embryos on day 4 were used for embryo aggregation after removing the zona pellucida. Irrespective of the timing of aggregation, aggregates of the three SCNT embryos developed to the blastocyst stage at a high rate (aggregates of 1-cell stage embryos, 17/19, 89%; aggregates of day 2 embryos, 23/23, 100%; aggregates of day 4 embryos, 22/22, 100%). Furthermore, a significant increase in cell number was observed in aggregates of three day 2 and day 4 embryos (223 &#x000b1; 30 and 163 &#x000b1; 18, respectively) compared with single SCNT embryos (89 &#x000b1; 6). A significantly higher pregnancy rate was observed after embryo transfer at 60 days of gestation in aggregates of three day 2 or day 4 embryos than in single SCNT embryos; however, a high incidence of abortion and stillbirth was subsequently observed in aggregates (<xref rid="tbl_003" ref-type="table">Table 3</xref><table-wrap id="tbl_003" orientation="portrait" position="float"><label>Table 3.</label><caption><title><italic>In vivo</italic> development of bovine fibroblast cell-nuclear transfer (NT) embryos and aggregates after embryo transfer</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Embryo (time of aggregation)</td><td align="center" rowspan="2" colspan="1">No. of embryos/aggregates<break/>transferred</td><td align="center" colspan="3" rowspan="1">No. of pregnancies<hr/></td><td align="center" rowspan="2" colspan="1">No. of calves (%)</td></tr><tr><td align="center" rowspan="1" colspan="1">Day 30</td><td align="center" rowspan="1" colspan="1">Day 60</td><td align="center" rowspan="1" colspan="1">Day 90</td></tr></thead><tbody><tr><td align="left" valign="middle" rowspan="1" colspan="1">Single NT, zona-intact</td><td align="center" valign="middle" rowspan="1" colspan="1">10</td><td align="center" valign="middle" rowspan="1" colspan="1">3 (30)</td><td align="center" valign="middle" rowspan="1" colspan="1">0 (0) <sup>a</sup></td><td align="center" valign="middle" rowspan="1" colspan="1">0 (0)</td><td align="center" valign="middle" rowspan="1" colspan="1">0 (0)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Aggregate (8-cell stage)</td><td align="center" valign="middle" rowspan="1" colspan="1">11</td><td align="center" valign="middle" rowspan="1" colspan="1">7 (64)</td><td align="center" valign="middle" rowspan="1" colspan="1">6 (55) <sup>b</sup></td><td align="center" valign="middle" rowspan="1" colspan="1">3 (27)</td><td align="center" valign="middle" rowspan="1" colspan="1">2 (18)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Aggregate (16- to 32-cell stage)</td><td align="center" valign="middle" rowspan="1" colspan="1">7</td><td align="center" valign="middle" rowspan="1" colspan="1">4 (57)</td><td align="center" valign="middle" rowspan="1" colspan="1">4 (57) <sup>b</sup></td><td align="center" valign="middle" rowspan="1" colspan="1">1 (14)</td><td align="center" valign="middle" rowspan="1" colspan="1">1 (14)</td></tr></tbody></table><table-wrap-foot><p><sup>a,b</sup> Values without common characters in the same column differ significantly (P &#x0003c; 0.05, Fisher&#x02019;s exact test). Reproduced with permission of the Society for Reproduction and Development from Akagi S, <italic>et al</italic>.: Developmental ability of somatic cell nuclear transferred embryos aggregated at the 8-cell stage or 16- to 32-cell stage in cattle. <italic>J Reprod Dev</italic> 2011; 57: 500&#x02013;506.</p></table-wrap-foot></table-wrap>). These results suggest that aggregation of SCNT embryos may improve the pregnancy rate after embryo transfer but that it cannot reduce the high incidence of fetal loss and stillbirth, which is often observed in bovine SCNT.</p></sec><sec sec-type="conclusions" id="s7"><title>Conclusion</title><p>NT is a complex multistep procedure, and there are many biological and technical factors affecting the development of bovine SCNT embryos. Numerous studies have led to significant improvements in SCNT protocols [<xref rid="r90" ref-type="bibr">90</xref>]; however, the cloning efficiency in terms of healthy cloned calves born still remains low. Failure to reprogram the donor genome is believed to be the main reason for the low cloning efficiency [<xref rid="r91" ref-type="bibr">91</xref>]. Further studies to optimize each step of SCNT together with a better understanding of the reprogramming mechanism are necessary in order to improve the efficiency of bovine cloning.</p></sec>
Effects of Trichostatin A on <italic>In Vitro</italic> Development and DNA Methylation Level of the Satellite I Region of Swamp Buffalo (<italic>Bubalus bubalis</italic>) Cloned Embryos
<p> Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82&#x02013;85%), cleavage (79&#x02013;84%) and development to the 8-cell stage (59&#x02013;65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in <italic>in vitro</italic> fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the <italic>in vitro</italic> developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.</p>
<contrib contrib-type="author"><name><surname>SRIRATTANA</surname><given-names>Kanokwan</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>KETUDAT-CAIRNS</surname><given-names>Mariena</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>NAGAI</surname><given-names>Takashi</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>KANEDA</surname><given-names>Masahiro</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>PARNPAI</surname><given-names>Rangsun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1)</label>Embryo Technology and Stem Cell Research Center and School of Biotechnology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand</aff><aff id="aff2"><label>2)</label>NARO National Institute of Livestock and Grassland Science, Ibaraki 305-0901, Japan</aff><aff id="aff3"><label>3)</label>Division of Animal Life Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan</aff>
The Journal of Reproduction and Development
<p>Although somatic cell nuclear transfer (SCNT) in the buffalo has been attempted throughout the last decade, the success rate of producing buffalo cloned offspring is still low when compared with the bovine. From our previous report, the developmental potential to the blastocyst stage of swamp buffalo cloned embryos was lower than that of bovine cloned embryos [<xref rid="r1" ref-type="bibr">1</xref>]. Increasing the blastocyst rate in swamp buffalo cloned embryos enhances the possibility to obtain viable clones. Multiple factors have been shown to influence the development of cloned embryos. One of the key factors is the incomplete reprogramming involved in abnormal epigenetic remodeling such as DNA methylation and histone modifications [<xref rid="r2" ref-type="bibr">2</xref>,<xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r4" ref-type="bibr">4</xref>]. In theory, assisted reprogramming of the donor nucleus for SCNT should improve embryo development. Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency of several species such as the mouse [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r6" ref-type="bibr">6</xref>], bovine [<xref rid="r7" ref-type="bibr">7</xref>,<xref rid="r8" ref-type="bibr">8</xref>,<xref rid="r9" ref-type="bibr">9</xref>], pig [<xref rid="r10" ref-type="bibr">10</xref>,<xref rid="r11" ref-type="bibr">11</xref>,<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>] and rabbit [<xref rid="r15" ref-type="bibr">15</xref>]. However, TSA treatment in swamp buffalo cloned embryos has not yet been reported. This work was carried out to investigate the effects of TSA on the developmental potential of swamp buffalo cloned embryos.</p><p>One of the epigenetic modifications in embryos is involved in DNA methylation at cytosine residues in CpG dinucleotides [<xref rid="r16" ref-type="bibr">16</xref>]. Hypermethylation in the satellite I region has been shown to influence the developmental potential of bovine and sheep cloned embryos by altering gene expression [<xref rid="r17" ref-type="bibr">17</xref>, <xref rid="r18" ref-type="bibr">18</xref>]. Wee <italic>et al</italic>. (2007) reported that the DNA methylation level in the satellite I region of TSA-treated bovine cells was significantly lower than that of untreated cells [<xref rid="r19" ref-type="bibr">19</xref>]. When using TSA-treated bovine cells as donor cells, the DNA methylation level of blastocysts derived from TSA-treated cells was significantly lower than those of untreated cells. An increased developmental rate to the blastocyst stage of cloned embryos derived from TSA-treated cells was observed. TSA can reactivate a silenced transgene in transgenic pig fibroblast cells by inducing DNA hypomethylation and histone hyperacetylation of the promoter region [<xref rid="r20" ref-type="bibr">20</xref>]. Therefore, based on previous reports, TSA can not only induce histone hyperacetylation but can also induce DNA demethylation.</p><p>To the best of our knowledge, there has been no report on the methylation status of the satellite I region in swamp buffalo <italic>in vitro</italic> fertilized or cloned embryos. The relation between TSA treatment and the change in DNA methylation is not known in buffaloes. Therefore, this is the first study to examine the effect of TSA treatment on DNA methylation levels of swamp buffalo cloned embryos. Study of the methylation status of satellite I should provide useful information concerning the molecular events in epigenetic reprogramming during development of swamp buffalo cloned embryos.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Chemicals and media</title><p>All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.</p></sec><sec><title>Embryo production by somatic cell nuclear transfer</title><p>Donor cell and oocyte preparation, the somatic cell nuclear transfer procedure and <italic>in vitro</italic> embryo culture were performed as describe previously [<xref rid="r1" ref-type="bibr">1</xref>]. Briefly, buffalo oocytes were obtained from local abattoir-derived ovaries and cultured in <italic>in vitro</italic> maturation (IVM; TCM199 supplemented 10% FBS, 50 IU/ml hCG (Intervet, Boxmeer, Netherlands), 0.02 AU/ml FSH (Antrin<sup>&#x000ae;</sup>, Kyoritsu Seiyaku, Tokyo, Japan) and 1 &#x000b5;g/ml 17&#x003b2;-estradiol) medium at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub> in air for 23 h. Then, cumulus cells were removed from cumulus oocytes complexes (COC) by repeat pipetting in 0.2% hyaluronidase. Metaphase-II oocytes with a first polar body (1<sup>st</sup> PB) were incubated in 5 &#x003bc;g/ml cytochalasin B for 5 min. The zona pellucida above the 1<sup>st</sup> PB was cut using a glass needle, and the 1<sup>st</sup> PB and a small amount of cytoplasm were squeezed out through the slit of the zona pellucida. Successful enucleation was performed by staining the squeezed out cytoplasts with 10 &#x000b5;g/ml Hoechst 33342 for 10 min and visualization of the metaphase plate under ultraviolet light. A male buffalo fibroblast donor cell was inserted into the perivitelline space of the enucleated oocyte. The donor cell-cytoplast couplet (DCCC) was fused with electric pulses (26 V, 17 &#x000b5;sec, 2 times) in Zimmermann fusion medium [<xref rid="r21" ref-type="bibr">21</xref>] and washed in Syngro<sup>&#x000ae;</sup> holding medium (Bioniche, Belleville, ON, Canada). At 1 h post fusion, successfully fused DCCCs were activated in 7% ethanol (Carlo Erba Reagents, Val de Reuil, France) in Syngro<sup>&#x000ae;</sup> holding medium for 5 min at room temperature and cultured in mSOFaa medium [<xref rid="r22" ref-type="bibr">22</xref>] supplemented with 1.25 &#x000b5;g/ml cytochalasin D and 10 &#x000b5;g/ml cycloheximide at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub> in air for 5 h. Then the embryos were cultured in mSOFaa medium at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub>, 5% O<sub>2</sub> and 90% N<sub>2</sub> for 2 days. Eight-cell stage embryos were selected and co-cultured with bovine oviductal epithelial cells in mSOFaa medium at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub> in air for 5 days.</p></sec><sec><title>TSA treatment</title><p>TSA treatment was performed as previously reported in the mouse [<xref rid="r5" ref-type="bibr">5</xref>] and bovine [<xref rid="r9" ref-type="bibr">9</xref>]; both reports showed that TSA treatment significantly increased the blastocyst formation rate, resulting in viable cloned offspring. Briefly, DCCCs were separated into three groups after fusion: an untreated group and 25 and 50 nM TSA groups (treated groups). For the TSA-treated groups, DCCCs were placed in Syngro<sup>&#x000ae;</sup> holding medium supplemented with 25 or 50 nM TSA for 1 h. After that, the fused DCCCs were activated and cultured in medium supplemented with 25 or 50 nM TSA for another 9 h [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r9" ref-type="bibr">9</xref>]. For the control group, fused DCCCs were activated and cultured under the same conditions except without TSA supplementation.</p></sec><sec><title>Embryo production by in vitro fertilization</title><p>Sperm preparation and the <italic>in vitro</italic> fertilization procedure were performed as describe previously [<xref rid="r23" ref-type="bibr">23</xref>]. Briefly, frozen spermatozoa were thawed at 37 C for 30 sec. The thawed spermatozoa were loaded into the bottom of a 5 ml culture tube containing 2 ml of modified Tyrode&#x02019;s albumin lactate pyruvate (TALP) [<xref rid="r24" ref-type="bibr">24</xref>] supplemented with 2.5 mM caffeine, 50 &#x000b5;g/ml heparin, 20 mM penicillamine, 10 mM hypotaurine and 20 mM epinephrine and then incubated at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub> in air for 30 min. The 1.5 ml of supernatant was taken out from the culture tube and centrifuged at 500 &#x000d7; <italic>g</italic> for 5 min. The pellet was resuspended in TALP at 4 &#x000d7; 10<sup>6</sup> sperms/ml. After 23 h post IVM, 10 COCs were transferred into 100 ml droplets of sperm supernatant and co-incubated at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub> in air for 24 h. After that, embryos were washed and cultured in mSOFaa medium at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub>, 5% O<sub>2</sub> and 90% N<sub>2</sub> for 1 day. Eight-cell stage embryos were selected and co-cultured with bovine oviductal epithelial cells in mSOFaa medium at 38.5 C under a humidified atmosphere of 5% CO<sub>2</sub> in air for 5 days.</p></sec><sec><title>Analysis of DNA methylation level in the satellite I region</title><p>The DNA methylation levels of IVF and cloned embryos and the fibroblasts used as donor cell were analyzed by the bisulfite method. Genomic DNA was extracted from IVF and cloned embryos (1-cell, 8-cell and blastocyst stages) and from donor cells by conventional DNA extraction. Three embryos were pooled and used for DNA extraction. Bisulfite treatment reactions were performed using an EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) according to the manufacturer&#x02019;s instructions. The satellite I region in bisulfite-treated DNA was amplified by polymerase chain reaction (PCR). The 20 &#x000b5;l reaction mixture consisted of 100 ng DNA sample, 1x PCR buffer (Promega, Madison, WI, USA), 2 mM MgCl<sub>2</sub> (Promega), 0.2 mM dNTPs mix (Fermentas, Burlington, ON, Canada), 0.5 &#x000b5;M of 5&#x02019;-GTATTTTTTTTGGAGTTTTTTGAG-3&#x02019; and 5&#x02019;-ACCATAAAAACCTAACTTCCCTAAC-3&#x02019; and 0.625 units Taq DNA polymerase (Promega). The amplification conditions were as follows: 95 C for 2 min followed by 35 cycles of 95 C for 30 sec, 50 C for 30 sec, 72 C for 30 sec. The final elongation step was performed at 72 C for 5 min. PCR products were ligated with pGEM<sup>&#x000ae;</sup> T-easy vector (Promega). The ligation mixture was transformed into the competent <italic>E. coli</italic> strain DH5&#x003b1; by electroporation. Positive clones were selected by blue-white selection on an LB agar plate containing 100 &#x003bc;g/ml ampicillin, 80 &#x003bc;g/ml 5-bromo-4-chloro-3-indolyl-&#x003b2;-D-galactopyranoside (X-gal) and 0.2 mM isopropyl-&#x003b2;-D-thiogalactopyranoside (IPTG). White colonies were cultured in LB broth with the above antibiotics. Plasmid preparation was performed with PureLink<sup>&#x000ae;</sup> Quick Plasmid Miniprep Kits (Invitrogen, Carlsbad, CA, USA) according to the manufacturer&#x02019;s instruction. DNA sequencing was performed by Macrogen Korea (Seoul, Republic of Korea). DNA methylation analysis was performed with the Quantification tool for Methylation Analysis (http://quma.cdb.riken.jp/top/index.html) [<xref rid="r25" ref-type="bibr">25</xref>].</p></sec><sec><title>Statistical analysis</title><p>All values for cloned embryo development and DNA methylation levels in IVF and cloned embryos and donor cells are presented as the mean &#x000b1; standard deviation (SD). The proportional data were subjected to arcsine transformation before statistical analysis. Transformed values were evaluated using the Statistical Analysis System software (SAS Institute, Cary, NC, USA) with a Completely Randomized Design, and comparison of data means was carried out with Duncan&#x02019;s multiple range test. A p-value of 0.05 or less than (P&#x0003c;0.05) were considered significant.</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>Development potential of cloned buffalo embryos after TSA treatment</title><p>After fusion, DCCCs were separated into three groups: two TSA-treated groups, the 25 and 50 nM TSA groups, and the untreated group. There was no significant difference in fusion rate (82&#x02013;85%) after TSA treatment. The cleavage rate (25 nM <italic>vs</italic>. 50 nM: 79% <italic>vs.</italic> 84%) and rate of development to the 8-cell stage (65% <italic>vs.</italic> 59%) were not significantly different among the treatment groups. However, the rate of development to the morula stage of embryos treated with 25 nM TSA (42.7%) was significantly higher than those of the 50 nM TSA-treated (37.8%) and untreated embryos (34.8%). Moreover, the highest blastocyst formation rate was found in the 25 nM TSA-treated embryos (30.1%) when compared with the other groups, and the difference was significant when compared with the untreated embryos (23.4%, P&#x0003c;0.05) (<xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title><italic>In vitro</italic> development of swamp buffalo cloned embryos after TSA treatment for 10 h</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Treatment</td><td align="center" rowspan="2" colspan="1">No. of<break/>replications</td><td align="center" rowspan="2" colspan="1">Fused (%)</td><td align="center" rowspan="2" colspan="1">Cultured</td><td align="center" rowspan="2" colspan="1">Cleaved (%)</td><td align="center" colspan="3" rowspan="1">No. (%)* embryos developed to<hr/></td></tr><tr><td align="center" rowspan="1" colspan="1">8-cells</td><td align="center" rowspan="1" colspan="1">Morula</td><td align="center" rowspan="1" colspan="1">Blastocyst</td></tr></thead><tbody><tr><td align="center" rowspan="2" colspan="1">0 nM</td><td align="center" rowspan="2" colspan="1">11</td><td align="center" rowspan="1" colspan="1">120/140</td><td align="center" rowspan="2" colspan="1">120</td><td align="center" rowspan="1" colspan="1">96</td><td align="center" valign="top" rowspan="1" colspan="1">60</td><td align="center" rowspan="1" colspan="1">33</td><td align="center" rowspan="1" colspan="1">22</td></tr><tr><td align="center" rowspan="1" colspan="1">(85.1 &#x000b1; 6.4)</td><td align="center" rowspan="1" colspan="1">(81.9 &#x000b1;13.2)</td><td align="center" rowspan="1" colspan="1">(62.9 &#x000b1; 15.0)</td><td align="center" rowspan="1" colspan="1">(34.8 &#x000b1; 8.1)<sup>b</sup></td><td align="center" rowspan="1" colspan="1">(23.4 &#x000b1; 6.0)<sup>b</sup></td></tr><tr><td align="center" rowspan="2" colspan="1">25 nM</td><td align="center" rowspan="2" colspan="1">12</td><td align="center" rowspan="1" colspan="1">117/143</td><td align="center" rowspan="2" colspan="1">117</td><td align="center" rowspan="1" colspan="1">91</td><td align="center" valign="top" rowspan="1" colspan="1">58</td><td align="center" rowspan="1" colspan="1">38</td><td align="center" rowspan="1" colspan="1">26</td></tr><tr><td align="center" rowspan="1" colspan="1">(81.6 &#x000b1; 5.3)</td><td align="center" rowspan="1" colspan="1">(78.6 &#x000b1; 19.1)</td><td align="center" rowspan="1" colspan="1">(65.4 &#x000b1; 13.9)</td><td align="center" rowspan="1" colspan="1">(42.7 &#x000b1; 9.6)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">(30.1 &#x000b1; 8.1)<sup>a</sup></td></tr><tr><td align="center" rowspan="2" colspan="1">50 nM</td><td align="center" rowspan="2" colspan="1">10</td><td align="center" rowspan="1" colspan="1">121/146</td><td align="center" rowspan="2" colspan="1">118</td><td align="center" rowspan="1" colspan="1">96</td><td align="center" valign="top" rowspan="1" colspan="1">57</td><td align="center" rowspan="1" colspan="1">33</td><td align="center" rowspan="1" colspan="1">25</td></tr><tr><td align="center" rowspan="1" colspan="1">(82.9 &#x000b1; 2.2)</td><td align="center" rowspan="1" colspan="1">(84.1 &#x000b1; 11.8)</td><td align="center" rowspan="1" colspan="1">(59.2 &#x000b1; 11.4)</td><td align="center" rowspan="1" colspan="1">(37.8 &#x000b1; 4.6)<sup>b</sup></td><td align="center" rowspan="1" colspan="1"> (25.4 &#x000b1; 4.0)<sup>ab</sup></td></tr></tbody></table><table-wrap-foot><p>* Percentages calculated from the number of cleaved embryos and that developed to each stage. Values are means &#x000b1; SD. Different superscripts within a column indicate significant differences (P &#x0003c; 0.05; Duncan&#x02019;s multiple range test).</p></table-wrap-foot></table-wrap>).</p></sec><sec><title>DNA methylation analysis in IVF and cloned embryos</title><p>The DNA methylation levels of the satellite I region in the IVF and cloned embryos and in the swamp buffalo fibroblasts used as donor cells were analyzed. The methylation level of the donor cells was 73.9% (<xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> DNA methylation level of the satellite I region in buffalo fibroblasts used as donor cells for somatic cell nuclear transfer. Values are means &#x000b1; SD.</p></caption><graphic xlink:href="jrd-60-336-g001"/></fig>). In the cloned embryos, the DNA methylation levels of the untreated embryos (0 nM TSA) at the 1-cell (75.0%), 8-cell (76.5%) and blastocyst stages (67.2%) were not significantly different when compared with that of the donor cells. Moreover, no significant differences were found in the methylation levels of the 25 nM and 50 nM TSA-treated cloned embryos at the 1-cell (75.7 and 73.2%, respectively), 8-cell (66.8 and 71.3%, respectively) and blastocyst stages (64.4 and 61.4%, respectively) when compared with those of the untreated cloned embryos and donor cells. On the other hand, the methylation levels of the IVF embryos were significantly lower compared with those of the TSA-treated and untreated cloned embryos at the 1-cell, 8-cell and blastocyst stages (P&#x0003c;0.01, <xref ref-type="fig" rid="fig_002">Fig. 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> DNA methylation level of the satellite I region in IVF and cloned embryos (0, 25 and 50 nM TSA) at the 1-cell, 8-cell and blastocyst stages. Values are means &#x000b1; SD.</p></caption><graphic xlink:href="jrd-60-336-g002"/></fig>).</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>TSA is one of the chromatin remodeling agents that enhances histone acetylation and DNA demethylation [<xref rid="r26" ref-type="bibr">26</xref>]. In this study, the reconstructed embryos were continuously exposed to TSA for 10 h beginning just minutes after fusion. The TSA concentration of 25 nM was shown to be effective for improving the morula and blastocyst formation rates in swamp buffalo cloned embryos. This is the first report of direct TSA treatment in swamp buffalo cloned embryos. According to previous reports, treatment with 5 nM TSA for 20 h [<xref rid="r7" ref-type="bibr">7</xref>] or 14 h [<xref rid="r8" ref-type="bibr">8</xref>] and with 50 nM TSA for 10 h [<xref rid="r9" ref-type="bibr">9</xref>] in bovine cloned embryos could increase the rate of development to the blastocyst stage. However, the effects of TSA treatment on bovine cloning efficiency have been controversial. Some previous reports have shown that TSA treatment did not improve <italic>in vitro</italic> development of bovine cloned embryos [<xref rid="r27" ref-type="bibr">27</xref>, <xref rid="r28" ref-type="bibr">28</xref>]. Recently, Luo <italic>et al</italic>. (2013) found that treatment of donor cells with 150 or 300 &#x003bc;M TSA increased cleavage and blastocyst formation rates of swamp buffalo cloned embryos [<xref rid="r29" ref-type="bibr">29</xref>]. Treatment with 500&#x02013;1000 nM scriptaid, another histone deacetylase inhibitor, has been shown to improve the developmental potential of handmade river buffalo cloned embryos [<xref rid="r30" ref-type="bibr">30</xref>]. Moreover, treatment of pig cloned embryos with 50 nM TSA for 24 h after activation significantly improved the blastocyst formation rate when compared with untreated embryos, while treatment of donor cells under the same conditions resulted in increases in cleavage rates but did not improve the blastocyst rate when compared with an untreated group [<xref rid="r31" ref-type="bibr">31</xref>]. The conflicting results may be due to the differences in treatment method used (embryo or donor cell treatment), concentration of TSA, timing of TSA treatment, cloning protocol, conditions of embryo culture and species.</p><p>In this study, we focused on DNA methylation of the satellite I region because the difference in DNA methylation level in this region between bovine IVF and cloned embryos was significantly higher than for other satellite regions [<xref rid="r32" ref-type="bibr">32</xref>]. We found that the levels of DNA methylation of the satellite I region in swamp buffalo cloned embryos were significantly higher than those of IVF embryos at every stage analyzed (1-cell, 8-cell and blastocyst stages) and similar to those of fibroblasts used as donor cells. This finding was similar to previous reports in bovines. Yamanaka <italic>et al</italic>. (2011) reported that high DNA methylation levels were found for the satellite I region in bovine fibroblasts used as donor cells [<xref rid="r33" ref-type="bibr">33</xref>]. Also, bovine cloned embryos at the blastocyst stage showed significantly higher DNA methylation levels than those of IVF embryos. Similarly, bovine donor cells were highly methylated in the satellite I region, resulting bovine cloned blastocysts with significantly more methylation than IVF blastocysts [<xref rid="r34" ref-type="bibr">34</xref>]. Moreover, Sawai <italic>et al.</italic> (2010) found that the DNA methylation level of the satellite I region in 50 nM TSA-treated bovine cloned embryos at the blastocyst stage was higher than in IVF embryos. However, there was no significant difference in methylation levels between TSA-treated and untreated cloned embryos [<xref rid="r8" ref-type="bibr">8</xref>]. A previous report suggested that DNA hypermethylation of the satellite I region in cloned embryos was caused by cloning procedures, not the <italic>in vitro</italic> embryo culture procedure [<xref rid="r33" ref-type="bibr">33</xref>]. The higher methylation level of the satellite I region in cloned embryos compared with IVF embryos suggests that reprogramming of donor nuclei was uncompleted, and this likely contributed to low cloning efficiency. These findings are important for understanding methylation changes of genomic satellite sequences during swamp buffalo preimplantation development.</p><p>In conclusion, treatment with 25 nM for 10 h after fusion could increase the blastocyst formation rate of swamp buffalo cloned embryos; however, no beneficial effect on the DNA methylation status of the satellite I region was found in our experiment. This is the first report to examine the effects of TSA treatment on <italic>in vitro</italic> development and DNA methylation level of the satellite I region of swamp buffalo cloned embryos.</p></sec>
An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm <italic>In Vitro</italic>
<p> We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E<sub>2</sub> (PGE<sub>2</sub>) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE<sub>2</sub> at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.</p>
<contrib contrib-type="author"><name><surname>LIU</surname><given-names>Jinghui</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>MAREY</surname><given-names>Mohamed A.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>KOWSAR</surname><given-names>Rasoul</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>HAMBRUCH</surname><given-names>Nina</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>SHIMIZU</surname><given-names>Takashi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>HANEDA</surname><given-names>Shingo</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>MATSUI</surname><given-names>Motozumi</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>SASAKI</surname><given-names>Motoki</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>HAYAKAWA</surname><given-names>Hiroyuki</given-names></name><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>PFARRER</surname><given-names>Christiane</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>MIYAMOTO</surname><given-names>Akio</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1)</label>Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan</aff><aff id="aff2"><label>2)</label>Department of Theriogenology, Faculty of Veterinary Medicine, Damanhur University, Beheira, Egypt</aff><aff id="aff3"><label>3)</label>Department of Anatomy, University of Veterinary Medicine Hannover, Hannover D-30173, Germany</aff><aff id="aff4"><label>4)</label>Department of Clinical Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan</aff><aff id="aff5"><label>5)</label>Department of Basic Veterinary Science, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan</aff><aff id="aff6"><label>6)</label>Genetics Hokkaido Association, Hokkaido 089-0103, Japan</aff>
The Journal of Reproduction and Development
<p>The oviduct is a key component of the female reproductive tract, where essential states such as oocyte maturation, sperm capacitation, fertilization, and initial embryonic development take place [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>]. The oviduct is classically described as a sterile milieu, even though pathogens and endotoxins could invade the mucosal surfaces of the oviduct via the uterus, peritoneal cavity, and follicular fluid. Therefore, the oviduct environment should be equipped with an efficient and strictly controlled immune system [<xref rid="r3" ref-type="bibr">3</xref>]. We have recently shown that polymorphonuclear neutrophils (PMNs), the first line of defense against microorganisms, are present in the bovine oviduct fluid during preovulatory stages. Moreover, the findings of our recent study strongly suggest that the bovine oviduct provides a prostaglandin E<sub>2</sub> (PGE<sub>2</sub>)-rich microenvironment to protect sperm from phagocytosis by PMNs that they possibly face <italic>in vivo</italic>, thereby supporting sperm survival in the oviduct [<xref rid="r4" ref-type="bibr">4</xref>].</p><p>Alpha 1-acid glycoprotein (AGP), a major acute-phase protein produced mainly in the liver, is a single polypeptide chain of 20.4 kDa, with a carbohydrate moiety that accounts for 40% of its total mass [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r6" ref-type="bibr">6</xref>]. Hepatic production and serum concentrations of AGP are increased in response to systemic injury and inflammation [<xref rid="r7" ref-type="bibr">7</xref>]. The precise biological functions of AGP are not completely understood, but numerous activities of potential physiological significance have been described, particularly its effects on immunomodulation and its ability to bind basic drugs [<xref rid="r8" ref-type="bibr">8</xref>]. These activities of AGP have been shown to be mostly dependent on carbohydrate composition, and changes in glycosylation can affect the biological properties of AGP [<xref rid="r9" ref-type="bibr">9</xref>]. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary [<xref rid="r10" ref-type="bibr">10</xref>].</p><p>Therefore, we hypothesized that AGP is secreted locally in the bovine oviduct, and is involved in regulation of the phagocytic activity of neutrophils for sperm.</p><p>To test our hypothesis, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effects of AGP on the phagocytic activity of PMNs for sperm and superoxide production; and 3) the impact of AGP desialylation on PMN phagocytosis for sperm.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Oviduct preparation</title><p>Paired oviducts along with their ipsilateral ovaries were collected from a local slaughterhouse, and they were closed from both ends to prevent leakage or contamination of the oviduct contents. The stages of the estrous cycle were determined macroscopically by assessing ovarian morphology through the observation of color, size, and weight of the corpus luteum, as described previously [<xref rid="r11" ref-type="bibr">11</xref>]. Furthermore, oviducts as well as the attached uteri were macroscopically examined to ensure that they were healthy and free from inflammation. Following these examinations, oviducts were immersed in PBS without Ca<sup>2+</sup> and Mg<sup>2+</sup> (PBS<sup>&#x02013;/&#x02013;</sup>) (Sigma-Aldrich, St. Louis, MO, USA) and supplemented with 0.3% amphotericin B (Sigma-Aldrich), and 0.3% gentamicin (Sigma-Aldrich).</p></sec><sec><title>Primary bovine oviduct epithelial cell (BOEC) isolation and cultivation</title><p>The isolation and cultivation of BOECs was based on the method described previously [<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r13" ref-type="bibr">13</xref>]. Briefly, oviducts were cut and separated from the surrounding connective tissue. BOECs were mechanically dislodged, purified, and cultured in D-MEM/F12 culture medium supplemented with 2.2% NaHCO<sub>3</sub>, 0.1% gentamicin, 1% amphotericin, and 10% fetal calf serum (FCS; BioWhittaker, Walkersville, MD, USA), in 6-well culture dishes (Nalge Nunc International, Roskilde, Denmark) at 38.5 C in 5% CO<sub>2</sub> and 95% air. After monolayer formation, cells were trypsinized (0.05% trypsin EDTA; Amresco, Solon, OH, USA), replated in 12-well culture dishes at a density of 3 &#x000d7; 10<sup>4</sup> cells/ml, and cultured until formation of a subconfluent monolayer. The growing BOEC monolayer was then cultured in medium supplemented with 0.1% FCS and incubated for 24 h with AGP (bovine AGP; Life Diagnostics, West Chester, PA, USA) (0, 1, 10 or 100 ng/ml). Finally, the culture medium was collected and stored at &#x02212;80 C until PGE<sub>2</sub> determination. Finally, 500 &#x003bc;l TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added to the wells, and the cells were collected, placed in 1.5 ml microcentrifuge tubes and then, stored at &#x02212;80 C until RNA extraction. The purity of epithelial cell preparations was evaluated with monoclonal antibodies to cytokeratin (anti-cytokeratin-CK1) and immunostaining. Approximately 98% of the cells were positive for anti-cytokeratin (CK1) antibodies.</p></sec><sec><title>AGP gene expression</title><p>AGP gene expression in PMNs, BOECs and the liver was detected by reverse transcription polymerase chain reaction (RT-PCR), as described previously [<xref rid="r14" ref-type="bibr">14</xref>]. The primers used for PCR were as follows: 5&#x02019;- CCAACCTGATGACAGTGGC-3&#x02019;, forward and 5&#x02019;- GCCGACTTATTGTACTCGGG-3&#x02019;, reverse, for AGP (NM<sub>&#x02011;</sub>001040502, 109 bp) and 5-CCAAGGCCAACCGTGAGAAAAT-3&#x02019;, forward, and 5-CCACATTCCGTGAGGATCTTCA-3&#x02019;, reverse, for &#x003b2;-actin (K00622, 256 bp).</p></sec><sec><title>AGP concentration determination</title><p>Fourteen oviducts at different stages of the estrous cycle (preovulatory, n = 4; postovulatory, n = 5; and mid-luteal stage, n = 5) were very gently flushed with 200 &#x000b5;l PBS<sup>&#x02013;/&#x02013;</sup> and the flushing media were collected into 1.5 ml microcentrifuge tubes. Extreme precautions were taken to avoid any outside contaminations. The flushing media were centrifugated at 1000 <italic>g</italic> for 10 min at 4 C to remove cellular debris. The AGP concentrations were measured directly in the flushing media using an ELISA kit (Uscn Life Science, Wuhan, China) according to the manufacturer&#x02019;s protocol. The AGP concentrations were quantified based on a standard curve with optical density (OD) measurements at 450 nm on an ELISA reader (Multiskan MS plate reader, Thermo Labsystems, Vantaa, Finland). The intra- and inter-assays coefficients of variation (CVs) were 10% and 12%, respectively. The range of the standard curves for these assays was 15.5&#x02013;1000 ng/ml.</p></sec><sec><title>PGE<sub>2</sub> concentration determination</title><p>Previously, we have shown that the bovine oviducts provide a PGE<sub>2</sub>-rich microenvironment to protect sperm from phagocytosis by PMNs [<xref rid="r4" ref-type="bibr">4</xref>]. Therefore, we investigated the effect of AGP on PGE<sub>2</sub> production from cultured BOECs. The BOECs were incubated with AGP (0, 1, 10 or 100 ng/ml) for 24 h and then, the culture medium was collected and used for measuring of PGE<sub>2</sub> concentrations by using a second antibody enzyme immunoassay as previously described [<xref rid="r11" ref-type="bibr">11</xref>]. Thirty-six BOEC supernatants were used for PGE<sub>2</sub> concentration determination. The intra and inter-assays CVs were 7.3 and 11.4%, respectively. The ED<sub>50</sub> was 260 pg/ml, and the range of the standard curves for these assays was 20&#x02013;20000 pg/ml.</p></sec><sec><title>Preparations of PMNs</title><p>Heparinized blood from a multiparous Holstein cow during the luteal stage was collected, and PMNs were isolated as previously described [<xref rid="r15" ref-type="bibr">15</xref>]. The PMNs were suspended at a density of 15&#x000d7;10<sup>6</sup> cells/ml in culture medium supplemented with 0, 1, 10 or 100 ng/ml of AGP (bovine AGP; Life Diagnostics) and incubated at 37 C in 5% CO<sub>2</sub> and 95% air for 4 h with gentle shaking. After PMNs incubation, PMNs were washed 2 times with PBS<sup>&#x02013;/&#x02013;</sup> and used for a phagocytosis assay.</p></sec><sec><title>Preparation of sperm</title><p>In parallel with the PMN preparation, sperm preparation was also carried out. Frozen straws were obtained that contained semen from three highly fertile Holstein bulls of the Genetics Hokkaido Association (Hokkaido, Japan). All semen straws were obtained from a single ejaculate from each bull separately. <italic>In vitro</italic> capacitation was induced by 4 h incubation of bull sperm in modified Tyrode&#x02019;s albumin, lactate, and pyruvate medium (Sp-TALP) supplemented with 10 &#x000b5;g/ml heparin, according to the method previously described [<xref rid="r16" ref-type="bibr">16</xref>, <xref rid="r17" ref-type="bibr">17</xref>]. Capacitation was verified by the induction of acrosome reactions using 100 &#x000b5;g/ml lysophosphatidylcholine for 15 min. Acrosome reactions were detected by performing a dual staining procedure with Trypan blue supravital stain and Giemsa stain as described by Kovacs and Foote [<xref rid="r18" ref-type="bibr">18</xref>]. After the treatment for capacitation, sperm were washed and suspended in Tyrode&#x02019;s medium containing lactate, pyruvate, and HEPES (TL-HEPES) [<xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r20" ref-type="bibr">20</xref>], and they were then used in the phagocytosis assays.</p></sec><sec><title>Coating of plates with desialylated AGP (as-AGP)</title><p>Plates were coated as described previously [<xref rid="r21" ref-type="bibr">21</xref>], with minor modifications. Three 96-well ELISA plates (Nunc, Denmark) were coated with AGP (0, or 10 &#x003bc;g/well) dissolved in 150 &#x003bc;l of PBS for 2 h at 37 C. They were then washed three times with PBS and blocked with 0.1% bovine serum albumin (BSA) in PBS for 2 h at 37 C. Plates were coated with as-AGP by incubating the AGP-coated plates with 200 mU/ml neuraminidase (Streptococcus 6646K, EC 3.2.1.18, Seikagaku, Tokyo, Japan) in 0.1 M PBS (pH 5.2) for 2 h at 37 C. Next, the plates were washed three times with PBS and blocked with 0.1% BSA in PBS for 2 h at 37 C. The plates were washed again three times with PBS before use. PMNs were cultured on the coated plates for 4 h and then, used in the detection of the phagocytic activity and superoxide generation of PMNs in response to sperm.</p></sec><sec><title>Phagocytosis assay</title><p>The phagocytic activity of PMNs for capacitated sperm was assayed according to the method previously described [<xref rid="r22" ref-type="bibr">22</xref>], with minor modifications. Briefly, PMNs incubated for 4 h were suspended in TL-HEPES. A 50 &#x000b5;l aliquot of PMN suspension was mixed with an equal volume of the treated sperm suspension in a 96-well untreated polystyrene microtest plate (Thermo Scientific, Roskilde, Denmark) and incubated at 38 C in 5% CO<sub>2</sub> and 95% air for 60 min with gentle swirling on a test-plate shaker. The final concentrations of PMNs and the treated sperm were 15 &#x000d7; 10<sup>6</sup> and 30 &#x000d7; 10<sup>6</sup> cells/ml, respectively. After incubation, an equal volume of heparin (40 mg/ml in TL-HEPES) was added to facilitate the dissociation of agglutinated PMNs. Subsamples of 75 &#x000b5;l were fixed by adding 25 &#x000b5;l of 2% (v/v) glutaraldehyde. The fixed samples were mounted into glass slides and examined at &#x000d7; 400 magnification using a phase-contrast microscope connected to a digital camera and a computer system (Suite, Leica Microsystems, Wetzlar, Germany). At least 400 PMNs were counted in different areas of the specimens. The percentage of PMNs with phagocytized sperm was recorded as the phagocytosis rate. Quantification of the number of PMNs with phagocytized sperm was performed independently by two observers.</p></sec><sec><title>Superoxide generation determination</title><p>PMNs were incubated with AGP (0 or 100 ng/ml) for 4 h, suspended in TL-HEPES either with or without the treated sperm and directly used for measuring superoxide generation. Briefly, 10 &#x000b5;l luminol reagent (Sigma) was pipetted into a 96-well FluoroNunc plate (Nunc, Roskilde, Denmark). Next, 100 &#x000b5;l of PMNs incubated for 4 h (0.5 &#x000d7; 10<sup>6</sup> cells/ml) and treated sperm (1 &#x000d7; 10<sup>6</sup> cells/ml) were added. Superoxide generation was detected at 425 nm using an AB-2350 Phelios (ATTO, Tokyo, Japan).</p></sec><sec><title>Scanning electron microscopy (SEM)</title><p>Neutrophils either directly phagocytize sperm through cell-cell attachment or entrap them within neutrophil extracellular traps (NETs), structures consisting of neutrophil nuclear DNA and associated proteins, that act to ensnare the sperm and hinder their motility [<xref rid="r23" ref-type="bibr">23</xref>, <xref rid="r24" ref-type="bibr">24</xref>]. We therefore used SEM to investigate the effect of AGP on NET formation by PMNs for sperm entanglement. Basically, PMNs were incubated in culture medium without any stimulation, or with AGP (100 ng/ml) for 4 h, and then a phagocytosis assay was performed by incubation of PMNs (15 &#x000d7; 10<sup>6</sup> cells/ml) together with capacitated sperm (30 &#x000d7; 10<sup>6</sup> cells/ml) for 60 min with gentle swirling on a test-plate shaker. For SEM, each sample, after phagocytosis, was placed onto cover glass coated with 0.1% neoprene in toluene, dried at room temperature, and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB, pH 7.4). After fixation, the samples were washed in PB, postfixed in 1% osmium tetroxide in PB and dehydrated in a graded series of ethanol solutions. The specimens were then freeze-dried with <italic>t</italic>-butyl alcohol using a freeze dryer (ES-2030, Hitachi, Tokyo, Japan). Each dried sample was mounted on a specimen stub with cover glass and sputter-coated with platinum (Pt) (Ion sputter coater E-1045 ion sputter coater, Hitachi High-Technologies, Tokyo, Japan). The specimens were observed using a scanning electron microscope (Hitachi High-Technologies) at an accelerating voltage of 5 kV.</p></sec><sec><title>Statistical analysis</title><p>Data are presented as the mean &#x000b1; SEM. Statistical analyses were performed with StatView 5.0 (SAS Institute, Cary, NC, USA). Statistical significance between groups was determined using a one-way ANOVA followed by multiple comparison tests (Fisher&#x02019;s test for three groups, and Bonferroni&#x02019;s test for more than three groups). Results were considered to be statistically significant at P &#x0003c; 0.05.</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>AGP mRNA expression and the AGP concentration in oviduct fluid</title><p>Our results demonstrate local gene expression of AGP by cultured BOECs <italic>in vitro</italic> that was well comparable to that for the liver and PMNs (<xref ref-type="fig" rid="fig_001">Fig. 1a</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> (a) AGP gene expression in PMNs, BOECs, and the liver. (b) The AGP concentrations per oviduct flush during the estrous cycle. (preovulatory, n = 4, postovulatory, n = 5, and mid-luteal stage, n = 5) (c) Percentage of PMN phagocytosis for sperm treated to induce capacitation <italic>in vitro</italic>. PMNs were incubated for 4 h in culture medium supplemented with 0, 1, 10, or 100 ng/ml AGP and then subjected to a 1-h phagocytosis assay. Numerical values are presented as means &#x000b1; SEM of three experiments. The different letters indicate significant differences between treatments at P &#x0003c; 0.05. (d) The effect of AGP on percentage of superoxide production by PMNs undergoing <italic>in vitro</italic> phagocytosis of sperm treated to induce capacitation. Numerical values are presented as means &#x000b1; SEM of four experiments. The different letters indicate significant differences between the marked treatments at P &#x0003c; 0.05.</p></caption><graphic xlink:href="jrd-60-342-g001"/></fig>). Thus, the local concentrations of AGP in the oviduct fluid were determined. The AGP concentrations in bovine oviduct flushing media were not significantly changed during different stages of the estrous cycle and were ranged from 30&#x02013;60 ng/ml (<xref ref-type="fig" rid="fig_001">Fig. 1b</xref>).</p></sec><sec><title>Dose-dependent effect of AGP on the phagocytic activity and superoxide generation of PMNs for sperm</title><p>The AGP system has immunomodulatory functions [<xref rid="r9" ref-type="bibr">9</xref>], and may be involved in regulation of the local immune response in the bovine oviduct. It is therefore of interest to determine the effect of AGP on PMN-mediated phagocytosis of sperm. A 4-h preexposure of PMNs to AGP (1, 10 or 100 ng/ml) resulted in a dose-dependent decrease in the PMN phagocytosis activity for capacitated sperm (<xref ref-type="fig" rid="fig_001">Fig. 1c</xref>). Moreover, the stimulation of PMNs with 100 ng/ml AGP prior to the superoxide assay reduced superoxide production by PMNs incubated with capacitated sperm compared with unstimulated PMNs (<xref ref-type="fig" rid="fig_001">Fig. 1d</xref>).</p></sec><sec><title>Observation of NET formation by SEM</title><p>During the phagocytosis assay, the addition of sperm to PMNs induced NET formation (<xref ref-type="fig" rid="fig_002">Fig. 2a, b, d, e</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Scanning electron microscopy of sperm phagocytosis by PMNs. The upper panels are images obtained at &#x000d7;1,000 (a, b, c) and the lower panels are those obtained at &#x000d7;2,000, respectively. PMNs were incubated without any stimulant (a, d), or with sperm addition to induce neutrophil extracellular traps (NETs) (b, e). NET formation was suppressed in PMNs incubated with AGP (100 ng/ml) prior to a phagocytosis assay (e, f).</p></caption><graphic xlink:href="jrd-60-342-g002"/></fig>). However, the exposure of PMNs to AGP (100 ng/ml) prior to the phagocytosis assay reduced NET formation by PMNs (<xref ref-type="fig" rid="fig_002">Fig. 2c, f</xref>).</p></sec><sec><title>Effect of desialylated-AGP on phagocytic activity and superoxide production by PMNs for sperm</title><p>The immunomodulatory functions and binding activities of AGP have been shown to be mostly dependent on carbohydrate composition, and changes in the glycosylation of AGP can affect its biological properties [<xref rid="r9" ref-type="bibr">9</xref>]. A four-hour incubation of PMNs on AGP-coated plates prior to the phagocytosis assay resulted in a decrease in the phagocytic activity of PMNs for capacitated sperm (<xref ref-type="fig" rid="fig_003">Fig. 3a</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p> Effect of as-AGP on phagocytic activity and superoxide production by PMNs for sperm . PMNs were incubated for 4 h in coated plates supplemented with culture medium only, AGP, or as-AGP and then subjected to a 1-h phagocytosis assay. (a) The percentage PMNs undergoing phagocytosis of sperm treated to induce capacitation, <italic>in vitro</italic>. The different letters indicate significant differences between the marked treatments at P &#x0003c; 0.001. (b) The percentage of superoxide production by PMNs undergoing <italic>in vitro</italic> phagocytosis of sperm treated to induce capacitation. The different letters indicate significant differences between the marked treatments at P &#x0003c; 0.05. Numerical values are presented as means &#x000b1; SEM of four experiments.</p></caption><graphic xlink:href="jrd-60-342-g003"/></fig>). However, incubation of PMNs in the as-AGP-coated plates resulted in complete abolishment of the suppressive effect of AGP on the phagocytosis of sperm by PMNs (<xref ref-type="fig" rid="fig_003">Fig. 3a</xref>). Moreover, the incubation of PMNs in the as-AGP-coated plates resulted in removal of the suppressive effect of AGP on the superoxide production by PMNs in response to sperm (<xref ref-type="fig" rid="fig_003">Fig. 3b</xref>).</p></sec><sec><title>The effect of AGP on PGE<sub>2</sub> production from cultured BOECs</title><p>Incubation of BOECs with AGP (0, 1, 10, or 100 ng/ml) for 24 hours resulted in stimulation of PGE<sub>2</sub> production in BOECs <italic>in vitro</italic> in a dose-dependent manner (<xref ref-type="fig" rid="fig_004">Fig. 4a</xref><fig orientation="portrait" fig-type="figure" id="fig_004" position="float"><label>Fig. 4.</label><caption><p> (a) The percentage of PGE<sub>2</sub> production in BOEC culture medium supplemented with AGP (0, 1, 10 or 100 ng/ml) for 24 h (n = 9 /group, 100% = 48.1 &#x000b1; 4.1 ng/ml, means &#x000b1; SEM). (b) The effect of AGP (50 ng/ml) and PGE<sub>2</sub> (10<sup>&#x02013;8</sup> M, 3.52 ng/ml) in combination, on the phagocytic activity of PMNs for sperm treated to induce capacitation. Numerical values are presented as means &#x000b1; SEM of four experiments. The different letters indicate significant differences between the marked treatments at P &#x0003c; 0.05.</p></caption><graphic xlink:href="jrd-60-342-g004"/></fig>, P &#x0003c; 0.05).</p></sec><sec><title>The effect of AGP and PGE<sub>2</sub> in combination on the phagocytic activity of PMNs for sperm</title><p>A four-hour incubation of PMNs with the local concentration of AGP detected in oviduct flushing media (50 ng/ml), along with that of PGE<sub>2</sub> (10<sup>-8</sup> M, 3.52 ng/ml, [<xref rid="r4" ref-type="bibr">4</xref>]), resulted in an additive effect in suppression of the phagocytic activity of PMNs for treated sperm (<xref ref-type="fig" rid="fig_004">Fig. 4b</xref>).</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>Bovine AGP is the main acute phase protein; its concentration in the peripheral blood circulation increases from approximately 0.3 mg/ml to 0.9 mg/ml during disease [<xref rid="r25" ref-type="bibr">25</xref>, <xref rid="r26" ref-type="bibr">26</xref>]. AGP is mainly produced by the liver, from which it diffuses into the general circulation [<xref rid="r6" ref-type="bibr">6</xref>]. AGP mRNA was detected in extrahepatic organs such as the lung, kidney, spleen, lymph node, uterus, ovary, placenta, and decidua [<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r27" ref-type="bibr">27</xref>]. Additionally, AGP protein has been previously detected in sow oviduct fluid [<xref rid="r28" ref-type="bibr">28</xref>]. In this study, we have provided the first evidence for the local gene expression of AGP by bovine oviduct epithelial cells <italic>in vitro</italic>. The oviduct flushing media contained AGP in the range of 20&#x02013;60 ng/ml, which is much lower than that seen in bovine plasma. Generally, it has been shown that AGP has immunomodulatory effects [<xref rid="r8" ref-type="bibr">8</xref>, <xref rid="r29" ref-type="bibr">29</xref>,<xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r31" ref-type="bibr">31</xref>]. This prompted us to investigate the effect of AGP on the phagocytosis of sperm by PMNs. Our results show that detectable concentrations of AGP were found in the oviduct flushing media, and AGP dose-dependently suppressed PMN phagocytosis of sperm <italic>in vitro</italic>. Additionally, SEM analysis demonstrated that AGP drastically reduced NET formation, preventing sperm from being fixed and trapped by PMNs, and thus indirectly resulted in the suppression of PMN phagocytosis for sperm.</p><p>Previously, it has been shown that AGP induced a dose-dependent inhibition of superoxide generation in PMNs stimulated by phorbol-12-myristate-13-acetate [<xref rid="r32" ref-type="bibr">32</xref>]. Additionally, the ability of PMNs to release superoxide has been used as an indicator for evaluating their phagocytic activity on sperm [<xref rid="r33" ref-type="bibr">33</xref>]. Our results show that only in the presence of sperm did AGP (100 ng/ml) significantly suppress superoxide release by PMNs. These findings suggest that the AGP secreted in bovine oviducts contributes to the protection and maintenance of sperm survival through the suppression of phagocytic activity and superoxide release by PMNs. In humans, it has been shown that leukocytes are the predominant source of superoxide production in sperm preparations and that the contribution of spermatozoa was either undetectable or was a small fraction of that contributed by leukocytes [<xref rid="r34" ref-type="bibr">34</xref>]. In general, PMNs form NETs for pathogen uptake depending on superoxide release through reactive oxygen species (ROS)-generating pathways [<xref rid="r35" ref-type="bibr">35</xref>]. NADPH decomposition results in the release of superoxide, which is converted to hydrogen peroxide (H2O2) either by superoxide dismutase (SOD) or spontaneously [<xref rid="r35" ref-type="bibr">35</xref>]. The generated H2O2 is then used in the formation of halogenated ROS, such as hypochlorite (OCL-) by myeloperoxidase (MPO), which induces NETs formation [<xref rid="r35" ref-type="bibr">35</xref>]. Therefore, we hypothesize that AGP, via the suppression of superoxide generation, could affect the ROS-generating pathways that lead to suppression of NETs formation, altering the phagocytic behavior of PMNs for sperm.</p><p>The mechanism by which AGP suppresses the phagocytosis of sperm by PMNs is still unknown. The binding and immunomodulatory activities of AGP have been shown to be mostly dependent on its carbohydrate composition [<xref rid="r9" ref-type="bibr">9</xref>]. Importantly, bovine AGP is one of the most heavily glycosylated proteins [<xref rid="r5" ref-type="bibr">5</xref>], and AGP glycosylation is modified during disease [<xref rid="r36" ref-type="bibr">36</xref>]. It is conceivable that the terminal sialic acid residues exposed on the surface of AGP block phagocytosis by binding phagocyte sialic acid-binding immunoglobulin-type lectins (Siglec) [<xref rid="r37" ref-type="bibr">37</xref>]. Therefore, we hypothesized that the sialic acid contributes to the suppressive effect of AGP on PMN phagocytosis for sperm. In fact, our results show that desialylating AGP completely abolished the AGP-suppressive effect on both PMN phagocytosis of sperm and superoxide release. These results are in agreement with previous studies showing that desialylating AGP abolished an AGP hepato-protective effect [<xref rid="r38" ref-type="bibr">38</xref>] and anti-apoptotic activity [<xref rid="r39" ref-type="bibr">39</xref>], whereas hyposialylated AGP completely inhibited the phagocytosis of <italic>E. coli</italic> by feline neutrophils [<xref rid="r40" ref-type="bibr">40</xref>].</p><p>We previously demonstrated that luteinizing hormone stimulates BOECs to secrete PGE<sub>2</sub>, which plays a major role in suppressing the phagocytic activity of PMNs for sperm [<xref rid="r4" ref-type="bibr">4</xref>]. Interestingly, our results showed that AGP dose-dependently stimulated PGE<sub>2</sub> secretion from BOECs. Moreover, AGP and PGE<sub>2</sub>, within the physiological concentrations detected in oviduct flushing media, additively suppressed the phagocytosis of sperm by PMNs. Thus, these findings suggest that AGP not only directly suppresses sperm phagocytosis but also works cooperatively with PGE<sub>2</sub> to suppress the phagocytosis of sperm by PMNs in the bovine oviduct.</p><p>Taken together, our findings suggest that AGP has immunomodulatory functions in the bovine oviduct. It is proposed that under physiological conditions, the local AGP system in the bovine oviduct may aid sperm survival through direct suppression of the phagocytic activity of PMNs for sperm, through reduction of superoxide production by phagocytizing PMNs, and by limiting NET formation.</p><p> Conflict of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.</p></sec>
Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes
<p> The osmolarities of media that are most effective for <italic>in vitro</italic> culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity <italic>in vivo</italic> perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P&#x0003c;0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P&#x0003c;0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for <italic>in vitro</italic> culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.</p>
<contrib contrib-type="author"><name><surname>MIYOSHI</surname><given-names>Kazuchika</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>MIZOBE</surname><given-names>Yamato</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><aff id="aff1"><label>1)</label>Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan</aff><aff id="aff2"><label>2)</label>ART Okamoto Woman&#x02019;s Clinic, Nagasaki 850-0861, Japan</aff>
The Journal of Reproduction and Development
<p>Establishment of <italic>in vitro</italic> culture systems for supporting the early development of mammalian embryos is important in basic research for studying the mechanism controlling their development, as well as for applications such as somatic cell nuclear transfer (SCNT) and gene injection.</p><p>The osmolarity of the culture medium is an important factor for mammalian embryonic development. The osmolarity of mouse oviductal fluid was calculated to be above 340 mOsm [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>]. A similarly high osmolarity was predicted for human fallopian tube fluid based on electron probe measurements [<xref rid="r3" ref-type="bibr">3</xref>]. Direct measurements revealed the osmolarity of mouse oviductal fluid to be 290&#x02013;300 mOsm [<xref rid="r4" ref-type="bibr">4</xref>]. Interestingly, even though the physiological osmolarity is very high, the most effective media for culturing mammalian embryos from the 1-cell to blastocyst stages have very low osmolarities, in the range of 240&#x02013;280 mOsm [<xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r6" ref-type="bibr">6</xref>,<xref rid="r7" ref-type="bibr">7</xref>,<xref rid="r8" ref-type="bibr">8</xref>,<xref rid="r9" ref-type="bibr">9</xref>]. Osmolarities greater than 300 mOsm are detrimental to mammalian embryos [<xref rid="r2" ref-type="bibr">2</xref>, <xref rid="r6" ref-type="bibr">6</xref>, <xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r11" ref-type="bibr">11</xref>]. Porcine zygote medium-3 (PZM-3) [<xref rid="r12" ref-type="bibr">12</xref>] at 260&#x02013;280 mOsm [<xref rid="r13" ref-type="bibr">13</xref>, <xref rid="r14" ref-type="bibr">14</xref>] is the most common culture medium used in pigs for 1-cell embryos produced <italic>in vitro</italic> and <italic>in vivo</italic>, artificially activated oocytes and cloned embryos produced by nuclear transfer. The osmolarity of PZM-3 is much lower than the physiological osmolarity of 318 to 321 mOsm for pig oviductal fluid [<xref rid="r13" ref-type="bibr">13</xref>].</p><p>The survival of embryos at a high physiological osmolarity <italic>in vivo</italic> appears to be due to amino acids such as glycine [<xref rid="r15" ref-type="bibr">15</xref>], glutamine [<xref rid="r16" ref-type="bibr">16</xref>] and &#x003b2;-alanine [<xref rid="r17" ref-type="bibr">17</xref>] that serve as organic osmolytes in the female reproductive tract. Recently, Li <italic>et al.</italic>[<xref rid="r13" ref-type="bibr">13</xref>] reported that the concentrations and composition of free amino acids in the female pig reproductive tract are very different from the formulation of PZM-3. At day 3 after the onset of estrus, the total concentration of free amino acids in oviductal fluid was found to be four times higher than that in PZM-3. Remarkably, the concentration of glycine at that stage was 41 times higher in the fluid than in PZM-3.</p><p>Therefore, inclusion of glycine in the culture medium at the concentrations present in pig oviductal fluid could potentially enable pig oocytes and embryos to develop <italic>in vitro</italic> at the physiological osmolarity. To test this hypothesis, the effects of glycine on the parthenogenetic development of pig oocytes were investigated in hypotonic or isotonic media <italic>in vitro</italic> in the present study.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>In vitro maturation of oocytes</title><p>Pig ovaries were collected from prepubertal gilts at a local slaughterhouse and transported at 32&#x02013;35 C in saline to the laboratory. The follicular contents were recovered by aspiration from the follicles (2&#x02013;5 mm in diameter) using an 18-gauge needle (Terumo, Tokyo, Japan) and a 5-ml disposable syringe (Nipro, Osaka, Japan). The cumulus-oocyte complexes (COCs) were collected from the follicular contents and washed twice with a) HEPES (Nacalai Tesque, Kyoto, Japan)-buffered Tyrode solution supplemented with lactate, pyruvate and polyvinyl alcohol (PVA; Sigma-Aldrich, St. Louis, MO, USA) and b) maturation medium, respectively. The maturation medium was composed of 90% (v/v) TCM-199 with Earle&#x02019;s salts (Gibco BRL, Grand Island, NY, USA) supplemented with 0.91 mM sodium pyruvate (Sigma), 3.05 mM D-glucose (Wako Pure Chemical Industries, Osaka, Japan), 0.57 mM cysteine hydrochloride hydrate (Sigma), 10 ng/ml epidermal growth factor (Sigma), 10 IU/ml eCG (Aska Pharmaceutical, Tokyo, Japan), 10 IU/ml hCG (Aska), 100 &#x000b5;g/ml amikacin sulfate (Meiji Seika, Tokyo, Japan), 0.1% (w/v) PVA and 10% (v/v) pig follicular fluid. Only those COCs possessing a compact cumulus mass and evenly granulated ooplasm were selected. COCs were transferred to droplets (200 &#x000b5;l) of maturation medium in groups of 40 to 50 under paraffin oil (Nacalai Tesque) in a 35-mm polystyrene dish (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and cultured at 38.5 C in an atmosphere of 5% CO<sub>2</sub> in air. After 40 to 42 h in culture, cumulus cells were removed by pipetting with 0.1% (w/v) hyaluronidase (Sigma). Oocytes with polar bodies were selected for further experiments.</p></sec><sec><title>Activation and culture of oocytes</title><p><italic>In vitro</italic>-matured and denuded oocytes were washed twice in activation media composed of 250.3 mM sorbitol, 0.5 mM Ca(CH<sub>3</sub>COO)<sub>2</sub>, 0.5 mM Mg(CH<sub>3</sub>COO)<sub>2</sub> and 0.1% (w/v) bovine serum albumin [<xref rid="r18" ref-type="bibr">18</xref>]. Then, the oocytes were transferred in groups of 30 to 60 to a well of a 4-well plate (Nunc, Roskilde, Denmark) containing 800 &#x000b5;l of the activation medium. The ultrasound probe (8 mm in diameter) of a KTAC-3000 Sonopore (Nepa Gene, Chiba, Japan) was inserted directly into the activation media, and the oocytes were exposed to 2872 kHz at 45 V for 30 sec, with a 10 Hz burst rate and a 30% duty cycle. A miniature stirrer was placed within the wells and stirred at 300 rpm during exposure to ultrasound. Modified PZM-3 (mPZM-3), in which 50 &#x000b5;g/ml gentamicin was replaced with 100 &#x000b5;g/ml amikacin sulfate [<xref rid="r19" ref-type="bibr">19</xref>], was used as the standard medium for oocyte culture. After ultrasound exposure, the oocytes were transferred to 50 &#x000b5;l of culture medium supplemented with 2.2 &#x000b5;g/ml cytochalasin B to prevent extrusion of a second polar body. After incubation for 2 h at 38.5 C in an atmosphere of 5% CO<sub>2</sub>, 5% O<sub>2</sub> and 90% N<sub>2</sub>, the oocytes were transferred into 50 &#x000b5;l of the same medium without cytochalasin B and cultured further. The oocytes were examined for cleavage at day 2 in culture. At day 7, oocytes with blastocoels were placed on slides with drops of mounting media that consisted of glycerol and phosphate-buffered saline (9:1) with 100 &#x000b5;g/ml Hoechst 33342 (Sigma). Coverslips were placed on the oocytes, and the edges were sealed with nail polish. The nuclei were counted under ultraviolet light, and oocytes with 32 or more cells were designated as blastocysts.</p></sec><sec><title>Experimental designs</title><p>In Experiment 1, the effects of glycine in hypotonic or isotonic media (osmolarity adjusted with NaCl) on the parthenogenetic development of oocytes were examined. Oocytes were cultured in mPZM-3 or mPZM-3 with 30 mM NaCl (mPZM-3 + NaCl). The osmolarities of mPZM-3 and mPZM-3 + NaCl were 273 mOsm and 326 mOsm, respectively, when measured using a vapor pressure osmometer (Wescor, Logan, UT, USA). These media were supplemented either with or without 4 mM glycine, since the concentration of glycine in pig oviductal fluid is 4.1 mM at day 3 after estrus [<xref rid="r13" ref-type="bibr">13</xref>], and mPZM-3 already contains 0.1 mM glycine.</p><p>In Experiment 2, the effects of glycine in hypotonic or isotonic media (osmolarity adjusted with sucrose) on the parthenogenetic development of oocytes were examined. Oocytes were cultured in either mPZM-3 or mPZM-3 with 50 mM sucrose (mPZM-3 + sucrose; 318 mOsm), both of which were supplemented with or without 4 mM glycine.</p><p>In Experiment 3, the effects of osmolarity on the parthenogenetic development of oocytes cultured in media supplemented with glycine were tested. Oocytes were cultured in mPZM-3 with 4 mM glycine and NaCl at varying concentrations (0, 15, 30 or 45 mM). The osmolarities of the media with 0, 15, 30 and 45 mM NaCl were 273, 299, 326 and 361 mOsm, respectively.</p><p>In Experiment 4, the effects of varying concentrations of glycine on the parthenogenetic development of oocytes in isotonic media were tested. Oocytes were cultured in mPZM-3 + NaCl supplemented with 0, 1, 2, 4, 6 or 8 mM glycine.</p><p>In Experiment 5, the effects of glycine added in different periods for varying times were investigated on the parthenogenetic development of oocytes in isotonic media. Oocytes were cultured in mPZM-3 + NaCl to which 4 mM glycine was added on different days. In a preliminary experiment, the rates of blastocyst formation of oocytes cultured with glycine during days 1&#x02013;2 or 3&#x02013;4 in culture were significantly higher than those cultured without glycine, although the addition of glycine during days 5&#x02013;7 did not affect oocyte development. Therefore, glycine was only added on one day (day 1, 2, 3 or 4) during culture.</p></sec><sec><title>Statistical analysis</title><p>Experiments were replicated five or six times. All percentage data were subjected to arcsine transformation in each replicate. The transformed values and numbers of cells in blastocysts were analyzed by one-way or two-way ANOVA, followed by Fisher&#x02019;s protected least significant difference test. A probability of P&#x0003c;0.05 was considered statistically significant.</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>Experiment 1</title><p>As shown in <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Effects of glycine and NaCl (to adjust osmolarity) on the parthenogenetic development of pig oocytes in hypotonic or isotonic media<sup>a</sup></title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Medium</td><td align="center" rowspan="2" colspan="1">Glycine</td><td align="center" valign="middle" rowspan="2" colspan="1">No. of oocytes<break/>cultured</td><td align="center" colspan="2" rowspan="1">No. (mean % &#x000b1; SEM)<sup>b</sup> of oocytes developed to<hr/></td><td align="center" rowspan="2" colspan="1">Mean % &#x000b1; SEM of blastocysts<break/>per oocytes cleaved</td><td align="center" rowspan="2" colspan="1">Mean no. &#x000b1; SEM of cells<break/>in blastocysts</td></tr><tr><td align="center" rowspan="1" colspan="1">&#x02265; 2-cell (Day 2)<sup>c</sup></td><td align="center" rowspan="1" colspan="1">Blastocyst (Day 7)<sup>c</sup></td></tr></thead><tbody><tr><td align="left" rowspan="2" colspan="1">mPZM-3</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">119</td><td align="center" rowspan="1" colspan="1">58 (49.0 &#x000b1; 2.6)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">19 (15.9 &#x000b1; 1.7)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">32.9 &#x000b1; 3.8<sup>d</sup></td><td align="center" rowspan="1" colspan="1">42.1 &#x000b1; 2.0</td></tr><tr><td align="center" rowspan="1" colspan="1">+</td><td align="center" rowspan="1" colspan="1">116</td><td align="center" rowspan="1" colspan="1">60 (51.8 &#x000b1; 1.7)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">15 (13.4 &#x000b1; 2.6)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">25.9 &#x000b1; 4.9<sup>d</sup></td><td align="center" rowspan="1" colspan="1">49.9 &#x000b1; 4.0</td></tr><tr><td align="left" rowspan="2" colspan="1">mPZM-3 + NaCl</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">120</td><td align="center" rowspan="1" colspan="1">98 (82.2 &#x000b1; 4.8)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">15 (12.3 &#x000b1; 1.3)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">15.3 &#x000b1; 2.0<sup>e</sup></td><td align="center" rowspan="1" colspan="1">41.3 &#x000b1; 1.9</td></tr><tr><td align="center" rowspan="1" colspan="1">+</td><td align="center" rowspan="1" colspan="1">117</td><td align="center" rowspan="1" colspan="1">90 (77.5 &#x000b1; 4.5)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">27 (23.3 &#x000b1; 0.6)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">30.5 &#x000b1; 1.8<sup>d</sup></td><td align="center" rowspan="1" colspan="1">43.9 &#x000b1; 2.5</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup> Oocytes were cultured in mPZM-3 (273 mOsm) or mPZM-3 with 30 mM NaCl (mPZM-3 + NaCl; 326 mOsm) supplemented with or without 4 mM glycine. <sup>b</sup> Percentage per oocytes cultured. <sup>c</sup> Days in culture. <sup>d&#x02013;e</sup> Values with different superscripts within each column are significantly different (at least P&#x0003c;0.05).</p></table-wrap-foot></table-wrap>, the cleavage rates of oocytes cultured in mPZM-3 + NaCl (77.5&#x02013;82.2%) were significantly (P&#x0003c;0.01) higher than those cultured in mPZM-3 (49.0&#x02013;51.8%), regardless of the presence or absence of glycine. The addition of glycine did not affect the rates of blastocyst formation in oocytes cultured in mPZM-3 (13.4&#x02013;15.9%). Interestingly, the blastocyst formation rate of oocytes cultured in mPZM-3 + NaCl (12.3%) did not significantly differ from those cultured in mPZM-3 with or without glycine (13.4&#x02013;15.9%); however, the blastocyst formation rate of oocytes cultured in mPZM-3 + NaCl with glycine was significantly (P&#x0003c;0.01) higher (23.3%). The proportion of blastocysts formed at day 7 relative to the number of oocytes cleaved at day 2 was calculated. The results showed that the rate of blastocyst formation for oocytes cultured in mPZM-3 + NaCl (15.3%) was significantly (at least P&#x0003c;0.05) lower than for those cultured in mPZM-3 with or without glycine or mPZM-3 + NaCl with glycine (25.9&#x02013;32.9%). The mean numbers of cells (41.3&#x02013;49.9) of the blastocysts were not affected by the addition of either salt or glycine.</p></sec><sec><title>Experiment 2</title><p>As shown in <xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title> Effects of glycine and sucrose (to adjust osmolarity) on the parthenogenetic development of pig oocytes in hypotonic or isotonic media<sup>a</sup></title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Medium</td><td align="center" rowspan="2" colspan="1">Glycine</td><td align="center" valign="middle" rowspan="2" colspan="1">No. of oocytes<break/>cultured</td><td align="center" colspan="2" rowspan="1">No. (mean % &#x000b1; SEM)<sup>b</sup> of oocytes developed to<hr/></td><td align="center" rowspan="2" colspan="1">Mean % &#x000b1; SEM of blastocysts<break/>per oocytes cleaved</td><td align="center" rowspan="2" colspan="1">Mean no. &#x000b1; SEM of cells<break/>in blastocysts</td></tr><tr><td align="center" rowspan="1" colspan="1">&#x02265; 2-cell (Day 2)<sup>c</sup></td><td align="center" rowspan="1" colspan="1">Blastocyst (Day 7)<sup>c</sup></td></tr></thead><tbody><tr><td align="left" rowspan="2" colspan="1">mPZM-3</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">121</td><td align="center" rowspan="1" colspan="1">52 (42.6 &#x000b1; 1.1)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">16 (13.1 &#x000b1; 1.9)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">30.6 &#x000b1; 4.2<sup>d</sup></td><td align="center" rowspan="1" colspan="1">47.8 &#x000b1; 3.9</td></tr><tr><td align="center" rowspan="1" colspan="1">+</td><td align="center" rowspan="1" colspan="1">121</td><td align="center" rowspan="1" colspan="1">51 (41.4 &#x000b1; 4.4)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">11 (8.8 &#x000b1; 2.0)<sup>d,e</sup></td><td align="center" rowspan="1" colspan="1">20.8 &#x000b1; 3.8<sup>e</sup></td><td align="center" rowspan="1" colspan="1">43.6 &#x000b1; 4.0</td></tr><tr><td align="left" rowspan="2" colspan="1">mPZM-3 + sucrose</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">122</td><td align="center" rowspan="1" colspan="1">84 (68.9 &#x000b1; 2.2)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">9 (7.4 &#x000b1; 1.4)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">10.8 &#x000b1; 2.1<sup>f</sup></td><td align="center" rowspan="1" colspan="1">43.2 &#x000b1; 4.1</td></tr><tr><td align="center" rowspan="1" colspan="1">+</td><td align="center" rowspan="1" colspan="1">121</td><td align="center" rowspan="1" colspan="1">81 (66.9 &#x000b1; 1.1)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">24 (19.9 &#x000b1; 0.8)<sup>f</sup></td><td align="center" rowspan="1" colspan="1"> 29.8 &#x000b1; 1.4<sup>d,e</sup></td><td align="center" rowspan="1" colspan="1">45.8 &#x000b1; 3.1</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup> Oocytes were cultured in mPZM-3 (273 mOsm) or mPZM-3 with 50 mM sucrose (mPZM-3 + sucrose; 318 mOsm) supplemented with or without 4 mM glycine. <sup>b</sup> Percentage per oocytes cultured. <sup>c</sup> Days in culture. <sup>d&#x02013;f</sup> Values with different superscripts within each column are significantly different (at least P&#x0003c;0.05).</p></table-wrap-foot></table-wrap>, the cleavage rates of oocytes cultured in mPZM-3 + sucrose (66.9&#x02013;68.9%) were significantly (P&#x0003c;0.01) higher than those cultured in mPZM-3 (41.4&#x02013;42.6%), regardless of the presence or absence of glycine. The addition of glycine did not affect the blastocyst formation rates of oocytes cultured in mPZM-3 (8.8&#x02013;13.1%). However, the blastocyst formation rates of oocytes cultured in mPZM-3 + sucrose were significantly increased (P&#x0003c;0.01) by the addition of glycine (19.9%). The proportion of blastocysts formed at day 7 relative to the number of oocytes cleaved at day 2 was calculated. The results showed that the blastocyst formation rate of oocytes cultured in mPZM-3 + sucrose (10.8%) was significantly (P&#x0003c;0.01) lower than that of those cultured in mPZM-3 (30.6%), although there were no significant differences between these two groups when glycine was added to both media (29.8&#x02013;30.6%). The mean numbers of cells (43.2&#x02013;47.8) in the blastocysts were not affected by the addition of either sucrose or glycine.</p></sec><sec><title>Experiment 3</title><p><xref rid="tbl_003" ref-type="table">Table 3</xref><table-wrap id="tbl_003" orientation="portrait" position="float"><label>Table 3.</label><caption><title> Effects of osmolarity on the parthenogenetic development of pig oocytes in media supplemented with glycine<sup>a</sup></title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Osmolarity (mOsm)</td><td align="center" rowspan="2" colspan="1">No. of oocytes<break/>cultured</td><td align="center" colspan="2" rowspan="1">No. (mean % &#x000b1; SEM)<sup>b</sup> of oocytes developed to<hr/></td><td align="center" rowspan="2" colspan="1">Mean no. &#x000b1; SEM<break/>of cells in blastocysts</td></tr><tr><td align="center" rowspan="1" colspan="1">&#x02265; 2-cell (Day 2)<sup>c</sup></td><td align="center" rowspan="1" colspan="1">Blastocyst (Day 7)<sup>c</sup></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">273</td><td align="center" rowspan="1" colspan="1">108</td><td align="center" rowspan="1" colspan="1">52 (48.1 &#x000b1; 1.7)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">13 (12.0 &#x000b1; 1.8)<sup>d,e</sup></td><td align="center" rowspan="1" colspan="1">46.9 &#x000b1; 4.5</td></tr><tr><td align="left" rowspan="1" colspan="1">299</td><td align="center" rowspan="1" colspan="1">109</td><td align="center" rowspan="1" colspan="1">64 (58.3 &#x000b1; 2.5)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">19 (17.3 &#x000b1; 3.8)<sup>e,f</sup></td><td align="center" rowspan="1" colspan="1">45.8 &#x000b1; 2.9</td></tr><tr><td align="left" rowspan="1" colspan="1">326</td><td align="center" rowspan="1" colspan="1">108</td><td align="center" rowspan="1" colspan="1">81 (74.9 &#x000b1; 4.7)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">25 (23.1 &#x000b1; 2.2)<sup>f</sup></td><td align="center" rowspan="1" colspan="1">43.5 &#x000b1; 1.7</td></tr><tr><td align="left" rowspan="1" colspan="1">361</td><td align="center" rowspan="1" colspan="1">109</td><td align="center" rowspan="1" colspan="1">92 (85.0 &#x000b1; 4.0)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">8 (7.4 &#x000b1; 1.9)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">35.8 &#x000b1; 1.3</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup> Oocytes were cultured in mPZM-3 with 4 mM glycine and different concentrations of NaCl (0, 15, 30 or 45 mM). <sup>b</sup> Percentage per oocytes cultured. <sup>c</sup> Days in culture. <sup>d&#x02013;f</sup> Values with different superscripts within each column are significantly different (at least P&#x0003c;0.05).</p></table-wrap-foot></table-wrap> shows that the cleavage rates of oocytes cultured at 326 mOsm and 361 mOsm (74.9&#x02013;85.0%) were significantly (at least P&#x0003c;0.05) higher than those cultured at 273 mOsm and 299 mOsm (48.1&#x02013;58.3%). The blastocyst formation rate of oocytes cultured at 326 mOsm (23.1%) was significantly (P&#x0003c;0.01) higher than that of those cultured at 273 mOsm or 361 mOsm (7.4&#x02013;12.0%). The mean numbers of cells (35.8&#x02013;46.9) in the blastocysts were not affected by the changes in osmolarities.</p></sec><sec><title>Experiment 4</title><p>As shown in <xref rid="tbl_004" ref-type="table">Table 4</xref><table-wrap id="tbl_004" orientation="portrait" position="float"><label>Table 4.</label><caption><title> Parthenogenetic development of pig oocytes in isotonic media supplemented with different concentrations of glycine<sup>a</sup></title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Concentration of glycine (mM)</td><td align="center" rowspan="2" colspan="1">No. of oocytes<break/>cultured</td><td align="center" colspan="2" rowspan="1">No. (mean % &#x000b1; SEM)<sup>b</sup> of oocytes developed to<hr/></td><td align="center" rowspan="2" colspan="1">Mean no. &#x000b1; SEM of cells<break/>in blastocysts</td></tr><tr><td align="center" rowspan="1" colspan="1">&#x02265; 2-cell (Day 2)<sup>c</sup></td><td align="center" valign="middle" rowspan="1" colspan="1">Blastocyst (Day 7)<sup>c</sup></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">101</td><td align="center" rowspan="1" colspan="1">81 (80.4 &#x000b1; 2.4)</td><td align="center" rowspan="1" colspan="1">16 (16.1 &#x000b1; 1.8)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">46.3 &#x000b1; 2.9</td></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">98</td><td align="center" rowspan="1" colspan="1">76 (76.4 &#x000b1; 4.1)</td><td align="center" rowspan="1" colspan="1">20 (20.6 &#x000b1; 4.3)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">42.3 &#x000b1; 2.1</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">77 (77.8 &#x000b1; 2.8)</td><td align="center" rowspan="1" colspan="1">14 (13.2 &#x000b1; 1.8)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">44.1 &#x000b1; 2.6</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">98</td><td align="center" rowspan="1" colspan="1">72 (74.0 &#x000b1; 2.4)</td><td align="center" rowspan="1" colspan="1">32 (33.6 &#x000b1; 3.7)<sup>e</sup></td><td align="center" rowspan="1" colspan="1">47.5 &#x000b1; 2.4</td></tr><tr><td align="left" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">99</td><td align="center" rowspan="1" colspan="1">70 (71.6 &#x000b1; 3.0)</td><td align="center" rowspan="1" colspan="1">14 (13.6 &#x000b1; 2.0)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">47.2 &#x000b1; 3.7</td></tr><tr><td align="left" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">99</td><td align="center" rowspan="1" colspan="1">72 (73.0 &#x000b1; 2.9)</td><td align="center" rowspan="1" colspan="1">15 (15.4 &#x000b1; 1.6)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">43.3 &#x000b1; 3.4</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup> Oocytes were cultured in mPZM-3 with 30 mM NaCl (326 mOsm). <sup>b</sup> Percentage per oocytes cultured. <sup>c</sup> Days in culture. <sup>d&#x02013;e</sup> Values with different superscripts are significantly different (P&#x0003c;0.01).</p></table-wrap-foot></table-wrap>, the cleavage rates of oocytes were not affected by the different concentrations of glycine in media (71.6&#x02013;80.4%). However, the blastocyst formation rate of oocytes cultured with 4 mM glycine in the medium (33.6%) was significantly (P&#x0003c;0.01) higher than those of the other groups (13.2&#x02013;20.6%). The mean numbers of cells (42.3&#x02013;47.5) in the blastocysts were not affected by the different glycine concentrations.</p></sec><sec><title>Experiment 5</title><p>The results in <xref rid="tbl_005" ref-type="table">Table 5</xref><table-wrap id="tbl_005" orientation="portrait" position="float"><label>Table 5.</label><caption><title> Parthenogenetic development of pig oocytes in isotonic media supplemented with glycine on days 1, 2, 3, 4 or 1&#x02013;7 in culture<sup>a</sup></title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="2" colspan="1">Glycine addition (days in culture)</td><td align="center" rowspan="2" colspan="1">No. of oocytes<break/>cultured</td><td align="center" colspan="2" rowspan="1">No. (mean % &#x000b1; SEM)<sup>b</sup> of oocytes developed to<hr/></td><td align="center" rowspan="2" colspan="1">Mean no. &#x000b1; SEM of<break/>cells in blastocysts</td></tr><tr><td align="center" rowspan="1" colspan="1">&#x02265; 2-cell (Day 2)<sup>c</sup></td><td align="center" valign="middle" rowspan="1" colspan="1">Blastocyst (Day 7)<sup>c</sup></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">None</td><td align="center" rowspan="1" colspan="1">112</td><td align="center" rowspan="1" colspan="1">83 (73.6 &#x000b1; 2.2)</td><td align="center" rowspan="1" colspan="1">15 (13.5 &#x000b1; 1.1)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">46.1 &#x000b1; 3.4</td></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">109</td><td align="center" rowspan="1" colspan="1">73 (66.9 &#x000b1; 4.3)</td><td align="center" rowspan="1" colspan="1">20 (19.2 &#x000b1; 2.8)<sup>d,e</sup></td><td align="center" rowspan="1" colspan="1">46.9 &#x000b1; 2.7</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">110</td><td align="center" rowspan="1" colspan="1">75 (68.5 &#x000b1; 1.7)</td><td align="center" rowspan="1" colspan="1">25 (23.1 &#x000b1; 1.5)<sup>e,f</sup></td><td align="center" rowspan="1" colspan="1">47.5 &#x000b1; 2.9</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">111</td><td align="center" rowspan="1" colspan="1">77 (69.9 &#x000b1; 3.5)</td><td align="center" rowspan="1" colspan="1">28 (25.1 &#x000b1; 1.9)<sup>f</sup></td><td align="center" rowspan="1" colspan="1">50.2 &#x000b1; 2.2</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">111</td><td align="center" rowspan="1" colspan="1">76 (68.0 &#x000b1; 2.8)</td><td align="center" rowspan="1" colspan="1">17 (15.1 &#x000b1; 2.7)<sup>d</sup></td><td align="center" rowspan="1" colspan="1">52.9 &#x000b1; 4.5</td></tr><tr><td align="left" rowspan="1" colspan="1">1&#x02013;7</td><td align="center" rowspan="1" colspan="1">110</td><td align="center" rowspan="1" colspan="1">72 (65.8 &#x000b1; 1.8)</td><td align="center" rowspan="1" colspan="1">25 (22.9 &#x000b1; 1.4)<sup>e,f</sup></td><td align="center" rowspan="1" colspan="1">46.6 &#x000b1; 2.9</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup> Oocytes were cultured in mPZM-3 with 30 mM NaCl (326 mOsm) supplemented with 4 mM glycine in different periods during culture. <sup>b</sup> Percentage per oocytes cultured. <sup>c</sup> Days in culture. <sup>d&#x02013;f</sup> Values with different superscripts are significantly different (at least P&#x0003c;0.05).</p></table-wrap-foot></table-wrap> show that the cleavage rates of oocytes cultured in media with glycine added at days 1, 2, 3 or 4 in culture (66.9&#x02013;69.9%) did not significantly differ from those of oocytes continuously cultured with or without glycine in the media (65.8&#x02013;73.6%). However, the blastocyst formation rates of oocytes cultured in media with glycine added during day 2 or 3 of culture (23.1&#x02013;25.1%) were significantly (P&#x0003c;0.01) higher than those cultured without glycine in the media (13.5%) and did not significantly differ from that of oocytes cultured with continuous glycine in the media (22.9%). In contrast, the blastocyst formation rates of oocytes cultured with glycine in the media during day 1 or 4 in culture (15.1&#x02013;19.2%) did not significantly differ from those cultured without glycine in the medium (13.5%). The mean numbers of cells (46.1&#x02013;52.9) in the blastocysts were not affected by the different time periods of glycine addition to the media.</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>The results of the present study indicate that culture of artificially activated pig oocytes in isotonic media improves cleavage rates but inhibits development beyond the cleavage stages as compared with hypotonic media. Addition of glycine to the isotonic media prevents this inhibition of oocyte development, resulting in improved blastocyst formation rates compared with hypotonic media.</p><p>The results of the present study also show that the NaCl-induced changes in osmolarity from 273 mOsm to 326 mOsm improved the cleavage rates of pig oocytes at day 2 in culture. In contrast, development of oocytes beyond the cleavage stages was inhibited at 326 mOsm compared with 273 mOsm. Similar results were obtained when the osmolarity was changed from 273 mOsm to 318 mOsm by the addition of sucrose. Therefore, it is the change in osmolarity and not NaCl or sucrose specifically that is involved in the development of pig oocytes. These results demonstrate that the optimal osmolarities required before and after early cleavage for the parthenogenetic development of pig oocytes are different. Recent reports have indicated that there is a preference for physiological osmolarities during the first 2 to 3 days of culturing pig <italic>in vitro</italic>-fertilized embryos [<xref rid="r13" ref-type="bibr">13</xref>], activated oocytes [<xref rid="r20" ref-type="bibr">20</xref>,<xref rid="r21" ref-type="bibr">21</xref>,<xref rid="r22" ref-type="bibr">22</xref>] and SCNT embryos [<xref rid="r14" ref-type="bibr">14</xref>, <xref rid="r21" ref-type="bibr">21</xref>, <xref rid="r22" ref-type="bibr">22</xref>], although the oocytes/embryos have to be transferred into hypotonic media subsequently in order to maintain <italic>in vitro</italic> development. The results of the present study are consistent with these reports.</p><p>The main discovery from the present study is that glycine can support the parthenogenetic development of pig oocytes in isotonic media. Mammalian cells regulate their volumes osmotically by importing and exporting intracellular osmolytes to adjust the intracellular osmotic pressure and to maintain size. The first line of defense for cells against an unwanted decrease in volume is to import inorganic ions [<xref rid="r23" ref-type="bibr">23</xref>]. However, in cells that are exposed to chronic hypertonicity, accumulation of a large number of ions is needed for sustaining cell size, which increases intracellular ionic strength but is detrimental to cell viability. In such cases, cells also accumulate small, uncharged compounds termed organic osmolytes, which replace a portion of the intracellular ions, thus reducing the ionic strength while still maintaining the cell volume [<xref rid="r24" ref-type="bibr">24</xref>, <xref rid="r25" ref-type="bibr">25</xref>]. In mice, glycine accumulates to high intracellular levels and seems to function as an organic osmolyte during the earliest stages of embryonic development up to the 2-cell stage [<xref rid="r2" ref-type="bibr">2</xref>, <xref rid="r26" ref-type="bibr">26</xref>, <xref rid="r27" ref-type="bibr">27</xref>]. Steeves <italic>et al</italic>. [<xref rid="r28" ref-type="bibr">28</xref>] demonstrated that early mouse embryos use a mechanism that employs GLYT1 (a neurotransmitter transporter family member) for osmotically regulating the accumulation of glycine. In that study, it was shown that GLYT1 was required for embryonic viability and maintenance of normal cell volumes in oviductal fluid. The results of the present study show that the effects of glycine on oocyte development depend on the osmolarity of the media. Development to the blastocyst stage was enhanced in isotonic media but not in hypotonic media, suggesting that glycine functions as an organic osmolyte to protect pig oocytes against the detrimental effects of increased osmolarity. On the other hand, uptake of glycine in mouse oocytes and embryos by GLYT1 is Na<sup>+</sup> and Cl<sup>&#x02013;</sup> dependent. The reliance of this transport system on the inwardly directed Na<sup>+</sup> and Cl<sup>&#x02013;</sup> gradients allows highly concentration-dependent uptake of glycine and the maintenance of steep concentration gradients across the cell membrane, permitting a high glycine content in oocytes and embryos, even when the extracellular concentration is much lower [<xref rid="r29" ref-type="bibr">29</xref>]. Therefore, it is possible that the concentration of NaCl is also an important factor for oocyte development in the present study. Since GLYT1 activity is also found in early human embryos [<xref rid="r29" ref-type="bibr">29</xref>], the mechanism for glycine-dependent cell volume regulation could be common to mammals. Additional investigation is needed to examine the roles of GLYT1 in pig oocytes and embryos. The results from the present study provide insight into a novel <italic>in vitro</italic> culture system that could be used for the development of pig activated oocytes to the blastocyst stages at physiological osmolarity. When cultured in isotonic media during the first 2 days, the development of pig electrically activated oocytes and SCNT embryos improved because of the reduction in their fragmentation rates [<xref rid="r21" ref-type="bibr">21</xref>]. The same culture conditions also reduced apoptosis in pig SCNT embryos, resulting in improved development [<xref rid="r14" ref-type="bibr">14</xref>]. These results suggest that culture in hypotonic media induces excessive apoptosis and fragmentation in pig oocytes and embryos compared with isotonic media because of unphysiological conditions. Such apoptosis and fragmentation could possibly be prevented, resulting in effective production of blastocysts, as pig oocytes could be continuously cultured in media possessing physiological osmolarity by addition of glycine in the present study. We expect that this culture system could potentially be useful for nonsurgical transfer of <italic>in vitro</italic>-produced embryos into recipient females at the morula and blastocyst stages [<xref rid="r30" ref-type="bibr">30</xref>].</p><p>In the present study, the blastocyst formation rate of pig oocytes was at a maximum when 4 mM glycine was added to the culture media and the osmolarity was adjusted to 326 mOsm, i.e., under physiological conditions that occur in pig oviductal fluid. The final concentration of glycine was 4.1 mM because the medium originally contained 0.1 mM glycine. In contrast, when pig <italic>in vitro</italic>-fertilized embryos were cultured in modified PZM-3 (the concentrations and composition of amino acids were modified to mimic those in pig oviductal fluid at 3 days after estrus), addition of 4.1 mM glycine did not make a significant difference in blastocyst formation rates between 250 mOsm and 330 mOsm [<xref rid="r13" ref-type="bibr">13</xref>]. This contradiction in the results may be attributed to the effects of amino acids (other than glycine), which might have a negative effect on the development of pig embryos when their concentrations are modified.</p><p>The results of the present study also indicated that the effects of glycine on oocyte development vary according to the developmental stage of the oocytes. Development to the blastocyst stage in the isotonic medium was enhanced only when glycine was added during days 2 to 3 of culture, i.e., the 2-cell to 8-cell stages. The net rate of glycine appearance is high in pig oocytes that are <italic>in vitro</italic> matured in TCM-199 and <italic>in vitro</italic> fertilized, but it reaches a nadir at the 2-cell stage after culture for one day [<xref rid="r31" ref-type="bibr">31</xref>]. Therefore, we concluded that addition of glycine is not needed during day 1 of culture because pig oocytes contain an adequate amount of glycine at that time for protection against the detrimental effects of increased osmolarity. However, the glycine in pig oocytes is depleted by the 2-cell stage; therefore, it must be added during days 2&#x02013;3 in culture when the oocytes are not transferred into a hypotonic medium. Even if glycine is added by days 4 to 7 of culture, accumulation of glycine might be too late to effectively replace inorganic ions in pig oocytes.</p><p>In conclusion, we established an effective <italic>in vitro</italic> culture system to support the parthenogenetic development of pig oocytes. This system would be useful in basic research for elucidating the mechanism controlling the development of oocytes. Further investigation is needed to address whether this system would be effective for culturing pig <italic>in vitro</italic>- or <italic>in vivo</italic>-fertilized embryos and SCNT embryos, as per our hypothesis.</p></sec>
Effects of Rapid Cooling Prior to Freezing on the Quality of Canine Cryopreserved Spermatozoa
<p> The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P&#x0003c;0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.</p>
<contrib contrib-type="author"><name><surname>RODENAS</surname><given-names>Carmen</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>PARRILLA</surname><given-names>Inmaculada</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>ROCA</surname><given-names>Jordi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>MARTINEZ</surname><given-names>Emilio Arsenio</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>LUCAS</surname><given-names>Xiomara</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1)</label>Department of Animal Medicine and Surgery, University of Murcia, Espinardo 30100, Spain</aff>
The Journal of Reproduction and Development
<p>In recent years, canine breeding has received increased attention in small animal veterinary practice. Reproductive technologies, such as artificial insemination (AI) and semen conservation, are in high demand not only by breeders but also by owners [<xref rid="r1" ref-type="bibr">1</xref>]. Among these technologies, cryopreservation is especially relevant because it allows the storage of spermatozoa for unlimited periods of time. This is of great interest for dog breeding, primarily because it allows the growth of international commerce in canine semen by minimizing the costs of keeping or transporting live animals and by establishing semen banks for males with a high genetic value [<xref rid="r2" ref-type="bibr">2</xref>]. Due to the increasing interest in cryopreservation technology for this species in the last decade, many studies have been undertaken to optimize the different aspects of canine semen cryopreservation, including variations in extenders, cryoprotectant types and cooling and thawing rates [<xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r6" ref-type="bibr">6</xref>].</p><p>It is well known that the cryopreservation procedure affects sperm quality, although sperm susceptibility differs between species and individuals, which has been associated with the composition of the lipid membranes of spermatozoa [<xref rid="r7" ref-type="bibr">7</xref>]. During the cryopreservation process, spermatozoa undergo different steps that involve important temperature changes, resulting in the well-known phenomenon of &#x0201c;cold shock&#x0201d; [<xref rid="r8" ref-type="bibr">8</xref>]. Specifically, during cryopreservation, spermatozoa are very sensitive to the rapid reduction from room temperature to 5 C [<xref rid="r7" ref-type="bibr">7</xref>]. Although dog spermatozoa are considered relatively resistant to cooling at 5 C [<xref rid="r9" ref-type="bibr">9</xref>], slow cooling rates prior to freezing are generally used for this species [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r10" ref-type="bibr">10</xref>,<xref rid="r11" ref-type="bibr">11</xref>,<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>]. For this reason, our hypothesis was that a rapid cooling rate prior freezing could be used efficiently for dog semen cryopreservation.</p><p>One of the most commonly used cryopreservation protocols for canine spermatozoa is the Uppsala method, which consists of two dilutions before freezing separated by a long cool-down step from room temperature (23 C) to 5 C over 1 to 2 h [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r15" ref-type="bibr">15</xref>,<xref rid="r16" ref-type="bibr">16</xref>]. These equilibration periods correspond to estimated mean cooling rates of approximately 0.15&#x02013;0.3 C/min. Although slow cooling rates are considered optimal, to the best to our knowledge, no studies have been performed to evaluate the effects of a rapid cooling rate and of reducing the cool-down time in the Uppsala protocol. This would allow a considerable reduction in the time required for the semen cryopreservation procedure, which is extremely important from a practical point of view. In fact, successful results have been reported using a rapid cooling rate prior to freezing in other species of mammals [<xref rid="r17" ref-type="bibr">17</xref>, <xref rid="r18" ref-type="bibr">18</xref>].</p><p>In the Uppsala method, an extender containing 3% glycerol is usually used during the cool-down period, resulting in a fractionated glycerol addition protocol [<xref rid="r2" ref-type="bibr">2</xref>, <xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r16" ref-type="bibr">16</xref>]. However, unfractionated glycerol addition at 5 C after slow cooling has also been described [<xref rid="r12" ref-type="bibr">12</xref>]. To establish an optimal rapid protocol for dog semen cryopreservation, we were also interested in evaluating the effects of both inclusion and exclusion of glycerol during the cool-down step in combination with a rapid cooling rate.</p><p>The aim of the present work was to evaluate the effect of a rapid cooling rate prior to freezing with the Uppsala method in combination with a fractionated or unfractionated glycerol addition protocol. For this purpose, sperm quality was assessed in frozen-thawed semen at different times during incubation after thawing.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Animals</title><p>The experimental protocols were conducted in accordance with Directive 2000/63/EU EEC for animal experiments and were reviewed and approved by the Ethics Committee for Experimentation with Animals of the University of Murcia, Spain. Six clinically healthy privately owned dogs (<italic>Canis lupus familiaris</italic>) of different breeds (1 Ibizan hound, 2 Scottish terriers, 1 French bulldog, 1 Jack Russell terrier and 1 Golden retriever) aged 2 to 5 years were used in this study. All dogs were of proven fertility and were trained semen donors, and none of them were preselected according to sperm freezability.</p></sec><sec><title>Semen collection</title><p>Ejaculates were collected by digital manipulation [<xref rid="r19" ref-type="bibr">19</xref>] into calibrated 15 ml Falcon conical tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Only the sperm-rich fraction (SRF) of the ejaculates was processed, while the first and third fractions were discarded. Only samples with a sperm concentration &#x0003e;200 &#x000d7; 10<sup>6</sup> spermatozoa/ml, a total motility &#x0003e; 80% and a normal morphology &#x0003e; 80% were included in the study.</p></sec><sec><title>Chemicals and media</title><p>All of the chemicals used in this study were of analytical grade and, unless otherwise stated, were purchased from Sigma-Aldrich (St. Louis, MO, USA). All media were prepared under sterile conditions in a laminar flow hood (MicroH; Telstar, Terrasa, Spain) using water from a Milli-Q Synthesis System (18 M&#x02126; cm; Milli-Q; Millipore, Billerica, MA, USA).</p><p>The extender used during washing and evaluation of the spermatozoa and as a thawing medium was TRIS-citrate-fructose (TCF; composition: 249 mM Trizma base, 80 mM citric acid, 69 mM fructose, pH 6.7; 324 &#x000b1; 3 mOsm/kg) supplemented with 1 mg/ml penicillin and 1 mg/ml s treptomycin sulfate [<xref rid="r12" ref-type="bibr">12</xref>].</p><p>The cooling and freezing extenders used in this study were previously described for dog semen cryopreservation and were composed of TCF containing 20% egg yolk and variable glycerol concentrations with or without Equex STM Paste (Nova Chemical Sales, Scituate, MA, USA) [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r12" ref-type="bibr">12</xref>]. The compositions of the cooling extenders (C-Ext I and II) and the freezing extenders (F-Ext I and II) are shown in <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Composition of extenders used for semen cooling and freezing</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">C-Ext I</td><td align="center" rowspan="1" colspan="1">C-Ext II</td><td align="center" rowspan="1" colspan="1">F-Ext I</td><td align="center" rowspan="1" colspan="1">F-Ext II</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Trizma base</td><td align="center" rowspan="1" colspan="1">3.025 g</td><td align="center" rowspan="1" colspan="1">3.025 g</td><td align="center" rowspan="1" colspan="1">3.025 g</td><td align="center" rowspan="1" colspan="1">3.025 g</td></tr><tr><td align="left" rowspan="1" colspan="1">Citric acid</td><td align="center" rowspan="1" colspan="1">1.7 g</td><td align="center" rowspan="1" colspan="1">1.7 g</td><td align="center" rowspan="1" colspan="1">1.7 g</td><td align="center" rowspan="1" colspan="1">1.7 g</td></tr><tr><td align="left" rowspan="1" colspan="1">Fructose</td><td align="center" rowspan="1" colspan="1">1.25 g</td><td align="center" rowspan="1" colspan="1">1.25 g</td><td align="center" rowspan="1" colspan="1">1.25 g</td><td align="center" rowspan="1" colspan="1">1.25 g</td></tr><tr><td align="left" rowspan="1" colspan="1">Streptomycin</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td></tr><tr><td align="left" rowspan="1" colspan="1">Penicillin</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td><td align="center" rowspan="1" colspan="1">1 mg/ml</td></tr><tr><td align="left" rowspan="1" colspan="1">Glycerol</td><td align="center" rowspan="1" colspan="1">3%</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">7%</td><td align="center" rowspan="1" colspan="1">10%</td></tr><tr><td align="left" rowspan="1" colspan="1">Equex STM Paste</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">1%</td><td align="center" rowspan="1" colspan="1">1%</td></tr><tr><td align="left" rowspan="1" colspan="1">Egg yolk</td><td align="center" rowspan="1" colspan="1">20%</td><td align="center" rowspan="1" colspan="1">20%</td><td align="center" rowspan="1" colspan="1">20%</td><td align="center" rowspan="1" colspan="1">20%</td></tr><tr><td align="left" rowspan="1" colspan="1">Milli-Q water</td><td align="center" rowspan="1" colspan="1">77 ml</td><td align="center" rowspan="1" colspan="1">80 ml</td><td align="center" rowspan="1" colspan="1">72 ml</td><td align="center" rowspan="1" colspan="1">69 ml</td></tr><tr><td align="left" rowspan="1" colspan="1">pH</td><td align="center" rowspan="1" colspan="1">6.76</td><td align="center" rowspan="1" colspan="1">6.72</td><td align="center" rowspan="1" colspan="1">6.70</td><td align="center" rowspan="1" colspan="1">6.76</td></tr><tr><td align="left" rowspan="1" colspan="1">Osmolarity</td><td align="center" rowspan="1" colspan="1">870 mOsm</td><td align="center" rowspan="1" colspan="1">335 mOsm</td><td align="center" rowspan="1" colspan="1">1570 mOsm</td><td align="center" rowspan="1" colspan="1">1978 mOsm</td></tr></tbody></table><table-wrap-foot><p>Cooling extenders (C-Ext I and II)/freezing extenders (F-Ext I and II).</p></table-wrap-foot></table-wrap>.</p><p>Phosphate-buffered saline solution (PBS: 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH<sub>2</sub>PO<sub>4</sub>, 8.1 mM Na<sub>2</sub>HPO<sub>4</sub>&#x000b7;7H<sub>2</sub>O, pH 6.8; 289 &#x000b1; 4 mOsm/kg) was used to dilute the fluorochromes used for flow cytometric evaluation of sperm viability.</p></sec><sec><title>Freezing method</title><p>The semen was frozen using the Uppsala method, which consisted of 2 dilution steps before freezing [<xref rid="r12" ref-type="bibr">12</xref>]. Briefly, after semen collection, the SRFs were washed by centrifugation (700 <italic>g</italic>/5 min/room temperature; Megafuge 1.0 R, Heraeus, Hanau, Germany). The supernatant was removed, and the resulting sperm samples were pooled or processed individually, depending on the experiment. Samples were diluted at 23 C with a cooling extender (C-Ext I or II) to a concentration of 400 &#x000d7; 10<sup>6</sup> spermatozoa/ml and were then cooled to 5 C using 2 different cooling rates (see the experimental design). Once at 5 C and just prior to freezing, the samples were diluted slowly by adding an equal volume of freezing extender (F-Ext I or II) that was previously cooled to 5 C using the same cooling rate as the corresponding diluted semen samples, resulting in a final concentration of 200 &#x000d7; 10<sup>6</sup> spermatozoa/ml. After 10 min of equilibration with the freezing extender, the semen was packed into 0.5 ml plastic straws (Minit&#x000fc;b, Tiefenbach, Germany) placed horizontally on a rack situated above the surface of LN<sub>2</sub> at a distance of 4 cm in a closed Styrofoam box for 10 min (freezing unit reference no. 15043/0636, Minit&#x000fc;b). Finally, the straws were plunged directly into the LN<sub>2</sub>. The semen straws were kept frozen in LN<sub>2</sub> for at least 2 weeks before being thawed for evaluation. The straws were thawed in a water bath at 70 C for 8 sec, and the content of each straw was immediately diluted in TCF (1:2, v/v) and incubated at 38 C for 150 min.</p></sec><sec><title>Sperm quality assessment</title><p>The motility of the spermatozoa was evaluated objectively using a computer-assisted analysis system (ISAS; Proiser R+D, Paterna, Spain). The samples were analyzed at a concentration of 20 &#x000d7; 10<sup>6</sup> spermatozoa/ml. For each evaluation, an aliquot of 5 &#x000b5;l was placed in a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) that was pre-warmed to 38 C. A minimum of 200 spermatozoa per sample were analyzed. Before the track sequence was analyzed, the trajectory of each spermatozoon was identified and recorded in each field, and each was assessed visually to eliminate possible debris and to decrease the risk of including unclear tracks in the analysis. The sperm motility variable recorded was the overall percentage of the total motility (TM) and the progressive motility (spermatozoa moved fast and progressively; PM) in all samples. Spermatozoa with an average path velocity (VAP) &#x02265; 20 &#x000b5;m/s were considered motile, and spermatozoa with 75% linear movement were designated as spermatozoa with PM, in accordance with the parameters provided by the manufacturer. In the frozen-thawed semen samples, the following quality of movement parameters were measured: curvilinear velocity (VCL; &#x000b5;m/s), straight line velocity (VSL, &#x000b5;m/s), linear coefficient (LIN %) and amplitude of the lateral head displacement (ALH, &#x000b5;m).</p><p>Sperm viability was evaluated by simultaneous cytometric assessment of the plasma and acrosomal membrane&#x02019;s integrity using a triple-fluorescence procedure, as described previously by R&#x000f3;denas <italic>et al.</italic>[<xref rid="r20" ref-type="bibr">20</xref>] and modified slightly. Briefly, 2 &#x000d7; 10<sup>6</sup> spermatozoa were transferred to tubes containing 50 &#x000b5;l of TFC-Ext, 4 &#x000b5;l of H-42 (0.05 mg/ml in PBS), 1 &#x000b5;l of propidium iodide (PI, 0.5 mg/ml in PBS, Invitrogen; Molecular Probes, Eugene, OR, USA) and 2 &#x000b5;l of fluorescein-conjugated peanut agglutinin (PNA-FITC, 20 &#x000b5;g/ml in PBS). The samples were incubated at 38 C in the dark for 10 min, and immediately prior to analysis, 400 &#x000b5;l of TCF was added to each sample. The fluorescence spectra of H-42, PI and PNA-FITC were detected using a 450/50 nm band-pass (BP) filter, a 670 nm long-pass (LP) filter and a 530/30 nm BP filter, respectively. The flow cytometry analyses were performed at room temperature under dimmed light using a BD FACSCanto II flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) equipped with three lasers as the excitation sources: blue (488 nm, aircooled, 20 mW solid state), red (633 nm, 17 mW HeNe) and violet (405 nm, 30 mW solidstate). Data were acquired using BD FACSDiva Software (Becton, Dickinson and Company). Non-spermatozoon events were gated out based on their H-42 fluorescence (DNA content), and acquisitions were stopped after 10000 H-42-positive events. Only the results corresponding to viable spermatozoa (intact plasma and acrosomal membranes; PI&#x02212;/PNA<sup>-</sup>FITC&#x02212;) were included in the results.</p></sec><sec><title>Experimental design</title><p>This study was divided into 2 experiments. Each experiment was replicated 5 times.</p><p>In experiment 1, a total of 30 SRFs from 6 dogs (5 per dog) were collected and centrifuged. The resulting samples from each dog were pooled and divided into 4 aliquots, which were frozen by the Uppsala method, as described above, using 2 different cooling rates (control 0.2 C/min or rapid 2.25 C/min) and 2 glycerol addition protocols (fractionated or unfractionated) in a 2 &#x000d7; 2 factorial design. The rapid cooling rate (2.25 C/min) was predetermined via a preliminary study performed in our laboratory, whereas the control (0.2 C/min) cooling rate was estimated based on previously published studies of dog semen cryopreservation [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r12" ref-type="bibr">12</xref>]. The 2.25 C/min cooling rate was achieved by plunging the tube of extended semen into a 250 ml glass beaker containing 200 ml of water at 4 C (<xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig 1.</label><caption><p> Temperature changes recorded for the semen samples during the cooling procedure at a mean rate of 2.25 C/min. Values are the means &#x000b1; SEM of ten measurements. The values of SEMs were not included because they were too small and it would not be possible to present error bars in a visual manner.</p></caption><graphic xlink:href="jrd-60-355-g001"/></fig>), while a programmable temperature bath (Programmable Model 9612; PolyScience, Niles, IL, USA) was used to perform the control rate of cooling. The temperature of the samples and the water during the cooling procedure was recorded. For addition of the fractionated glycerol, the semen samples were diluted in C-Ext I at 23 C for the cooling step and in F-Ext I at 5 C prior to freezing. For addition of the unfractionated glycerol, the semen samples were diluted in C-Ext II at 23 C for the cooling step and in F-Ext II at 5 C prior to freezing.</p><p>Sperm quality in terms of sperm motility and viability were assessed in fresh samples (SRFs after centrifugation at 23 C) and at 30 and 150 min of incubation at 38 C after sperm thawing. A total of 60 frozen-thawed straws (3 straws per each of the 4 treatments and replicate) were analyzed in experiment 1.</p><p>In experiment 2, a total of 20 SRFs from 4 dogs (5 per dog) were collected and frozen individually using the same cooling rates described in experiment 1 (control 0.2 C/min and rapid 2.25 C/min) in combination with an unfractionated glycerol protocol. The dogs included in this experiment were selected from those described in experiment 1 based on the best fresh sperm quality parameters. Sperm quality was assessed as described in experiment 1. A total of 30 frozen-thawed straws were analyzed for each male in experiment 2 (3 straw per each of the 2 treatments and replicate).</p></sec><sec><title>Statistical analysis</title><p>The data were analyzed using IBM SPSS Statistics for Windows, Version 19.0 (IBM, Armonk, NY, USA). The data were evaluated using the Kolmogorov-Smirnov test to check the assumption of normality and assessed using an ANOVA with a MIXED procedure. The ANOVA model included the fixed effects of the cooling rates, the glycerol addition protocols and their interactions in experiment 1; the male, the cooling rate and their interaction in experiment 2; and the random effects of replication in each experiment for the different incubation times after thawing. When the ANOVA revealed a significant effect, the values were compared using the Bonferroni test. The values reported are expressed as the least square means (LSM) &#x000b1; the standard error of the mean (SEM), and statistical significance was considered at P&#x0003c;0.05.</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>Experiment 1</title><p>The mean concentration, volume and total number of spermatozoa in the pool were 670 &#x000b1; 97 &#x000d7; 10<sup>6</sup> spermatozoa/ml, 5.0 &#x000b1; 0.5 ml and 3370 &#x000b1; 297 &#x000d7; 10<sup>6</sup> spermatozoa, respectively. The sperm quality parameters in the fresh pooled sperm samples were 87.6 &#x000b1; 1.8%, 67.6 &#x000b1; 2.3% and 89.3 &#x000b1; 1.3% for TM, PM and viable spermatozoa, respectively.</p><p>The mean percentages of the TM, PM and viable spermatozoa evaluated at 30 and 150 min after thawing are shown in <xref rid="tbl_002" ref-type="table">Tables 2</xref> and<xref rid="tbl_003" ref-type="table"> 3</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title> Mean percentages of total motility, progressive motility and viability in frozen-thawed semen samples at 30 min after thawing (experiment 1)</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">Glycerol addition protocol</td><td align="center" rowspan="1" colspan="1">Total motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Progressive motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Sperm viability<break/>(%)</td></tr></thead><tbody><tr><td align="left" rowspan="2" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">Fractionated</td><td align="center" rowspan="1" colspan="1">61.5<sup>a</sup></td><td align="center" rowspan="1" colspan="1">44.9<sup>a</sup></td><td align="center" rowspan="1" colspan="1">64.5<sup>a</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Unfractionated</td><td align="center" rowspan="1" colspan="1">66.4<sup>b</sup></td><td align="center" rowspan="1" colspan="1">50.5<sup>b</sup></td><td align="center" rowspan="1" colspan="1">67.7<sup>b</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">Fractionated</td><td align="center" rowspan="1" colspan="1">62.7<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">46.0<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">65.4<sup>ab</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Unfractionated</td><td align="center" rowspan="1" colspan="1">64.7<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">46.0<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">66.3<sup>ab</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">SEM</td><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">1.6</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="3" colspan="1">Probability</td><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="center" rowspan="1" colspan="1">Glycerol addition protocol</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.000</td></tr><tr><td align="center" rowspan="1" colspan="1">Interaction</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><p>Data are the LSM &#x000b1; SEM of five replicates per semen pool. <sup>a&#x02013;b</sup> Different letters within the same column indicate significant differences (P&#x0003c;0.05) among the glycerol addition protocols.</p></table-wrap-foot></table-wrap><table-wrap id="tbl_003" orientation="portrait" position="float"><label>Table 3.</label><caption><title> Mean percentages of total motility, progressive motility and viability in frozen-thawed semen samples at 150 min after thawing (experiment 1)</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">Glycerol addition protocol</td><td align="center" rowspan="1" colspan="1">Total motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Progressive motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Sperm viability<break/>(%)</td></tr></thead><tbody><tr><td align="left" rowspan="2" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">Fractionated</td><td align="center" rowspan="1" colspan="1">53.8<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">39.3<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">62.1<sup>a</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Unfractionated</td><td align="center" rowspan="1" colspan="1">57.8<sup>b</sup></td><td align="center" rowspan="1" colspan="1">42.6<sup>b</sup></td><td align="center" rowspan="1" colspan="1">66.5<sup>b</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">Fractionated</td><td align="center" rowspan="1" colspan="1">52.2<sup>a</sup></td><td align="center" rowspan="1" colspan="1">37.4<sup>a</sup></td><td align="center" rowspan="1" colspan="1">61.7<sup>a</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Unfractionated</td><td align="center" rowspan="1" colspan="1">56.5<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">40.4<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">64.1<sup>ab</sup></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">SEM</td><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.7</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="3" colspan="1">Probability</td><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="center" rowspan="1" colspan="1">Glycerol addition protocol</td><td align="center" rowspan="1" colspan="1">0.001</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.000</td></tr><tr><td align="center" rowspan="1" colspan="1">Interaction</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><p>Data are the LSM &#x000b1; SEM of five replicates per semen pool. <sup>a&#x02013;b</sup> Different letters within the same column indicates significant differences (P&#x0003c;0.05) among the glycerol addition protocols.</p></table-wrap-foot></table-wrap>, respectively. Within the same glycerol addition protocol, use of the rapid or control cooling rate prior to freezing did not significantly affect the percentages of TM, PM and viable spermatozoa at either 30 or 150 min after thawing. The glycerol addition protocol significantly affected the values of the TM, PM and viable spermatozoa (P&#x0003c;0.05) at 30 min and the viable spermatozoa (P&#x0003c;0.05) at 150 min of incubation after thawing in samples frozen using the control cooling rate. The interaction of the cooling rate and the glycerol addition protocol was not significant.</p><p>The quality of movement parameters was not significantly influenced by the cooling rates or by the glycerol addition protocol at any post-thawing evaluation time. The average values of the different experimental groups for VCL, VSL, LIN and ALH ranged from 133.4 to 138.9 &#x000b5;m/sec, from 88.9 to 95.0 &#x000b5;m/sec, from 63.9 to 67.5% and from 4.1 to 4.3 &#x000b5;m for 30 min and from 138.5 to 142.0 to &#x000b5;m/sec, from 82.2 to 86.5 &#x000b5;m/sec, from 60.0 to 62.0% and from 4.7 to 4.9 &#x000b5;m for 150 min, respectively.</p></sec><sec><title>Experiment 2</title><p>The mean sperm concentration, volume and total number of spermatozoa of the different males ranged from 350 to 1200 &#x000d7; 10<sup>6</sup> spermatozoa/ml, from 1.0&#x02013;3.5 ml and 450 to 1320 &#x000d7; 10<sup>6</sup> spermatozoa, respectively. The individual mean values for sperm quality in the fresh semen ranged from 87 to 92.8%, from 60 to 72% and from 84.5 to 95.1% for TM, PM and viable spermatozoa, respectively.</p><p>The individual values for TM, PM and sperm viability in the frozen samples evaluated at 30 and 150 min after thawing are shown in <xref rid="tbl_004" ref-type="table">Tables 4</xref> and<xref rid="tbl_005" ref-type="table"> 5</xref><table-wrap id="tbl_004" orientation="portrait" position="float"><label>Table 4.</label><caption><title> Individual mean percentages of total motility, progressive motility and viability at 30 min after thawing in frozen-thawed semen samples frozen using different cooling rates and an unfractionated glycerol addition protocol (experiment 2)</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">Total motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Progressive motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Sperm viability<break/>(%)</td></tr></thead><tbody><tr><td align="left" rowspan="2" colspan="1">1</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">71.4<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">57.7<sup>a</sup></td><td align="center" rowspan="1" colspan="1">73.2<sup>a</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">74.0<sup>a</sup></td><td align="center" rowspan="1" colspan="1">56.3<sup>a</sup></td><td align="center" rowspan="1" colspan="1">73.8<sup>a</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">2</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">64.4<sup>abc</sup></td><td align="center" rowspan="1" colspan="1">47.8<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">71.0<sup>a</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">60.7<sup>bc</sup></td><td align="center" rowspan="1" colspan="1">45.5<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">68.2<sup>ab</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">3</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">60.1<sup>bc</sup></td><td align="center" rowspan="1" colspan="1">49.9<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">60.5<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">63.7<sup>abc</sup></td><td align="center" rowspan="1" colspan="1">53.7<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">61.0<sup>b</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">4</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">58.7<sup>c</sup></td><td align="center" rowspan="1" colspan="1">46.5<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">65.3<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">60.9<sup>bc</sup></td><td align="center" rowspan="1" colspan="1">43.9<sup>b</sup></td><td align="center" rowspan="1" colspan="1">65.5<sup>b</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">SEM</td><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3.7</td><td align="center" rowspan="1" colspan="1">4.4</td><td align="center" rowspan="1" colspan="1">2.3</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="3" colspan="1">Probability</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">0.001</td><td align="center" rowspan="1" colspan="1">0.000</td><td align="center" rowspan="1" colspan="1">0.000</td></tr><tr><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="center" rowspan="1" colspan="1">Interaction</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><p>Data are the LSM &#x000b1; SEM of five replicates per male. <sup>a&#x02013;c</sup> Different letters within the same column indicates significant differences (P&#x0003c;0.05) among the males.</p></table-wrap-foot></table-wrap><table-wrap id="tbl_005" orientation="portrait" position="float"><label>Table 5.</label><caption><title> Individual mean percentages of total motility, progressive motility and viability at 150 min after thawing in frozen-thawed semen samples frozen using different cooling rates and a non-fractionated glycerol addition protocol (experiment 2)</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">Total motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Progressive motility<break/>(%)</td><td align="center" rowspan="1" colspan="1">Sperm viability<break/>(%)</td></tr></thead><tbody><tr><td align="left" rowspan="2" colspan="1">1</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">45.6</td><td align="center" rowspan="1" colspan="1">34.9</td><td align="center" rowspan="1" colspan="1">69.3<sup>a</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">44.8</td><td align="center" rowspan="1" colspan="1">30.9</td><td align="center" rowspan="1" colspan="1">68.9<sup>a</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">2</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">46.2</td><td align="center" rowspan="1" colspan="1">36.9</td><td align="center" rowspan="1" colspan="1">68.9<sup>a</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">40.2</td><td align="center" rowspan="1" colspan="1">28.9</td><td align="center" rowspan="1" colspan="1">66.9<sup>ab</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">3</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">47.8</td><td align="center" rowspan="1" colspan="1">41.3</td><td align="center" rowspan="1" colspan="1">55.1<sup>bc</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">42.5</td><td align="center" rowspan="1" colspan="1">35.5</td><td align="center" rowspan="1" colspan="1">58.1<sup>b</sup></td></tr><tr><td align="left" rowspan="2" colspan="1">4</td><td align="center" rowspan="1" colspan="1">Control</td><td align="center" rowspan="1" colspan="1">47.4</td><td align="center" rowspan="1" colspan="1">40.2</td><td align="center" rowspan="1" colspan="1">58.9<sup>bc</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Rapid</td><td align="center" rowspan="1" colspan="1">42.8</td><td align="center" rowspan="1" colspan="1">34.5</td><td align="center" rowspan="1" colspan="1">58.7<sup>bc</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">SEM</td><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">4.1</td><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">2.3</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="3" colspan="1">Probability</td><td align="center" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">0.000</td></tr><tr><td align="center" rowspan="1" colspan="1">Cooling rate</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="center" rowspan="1" colspan="1">Interaction</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><p>Data are the LSM &#x000b1; SEM of five replicates per male. <sup>a&#x02013;c</sup> Different letters within the same column indicates significant differences (P&#x0003c;0.05) among the males.</p></table-wrap-foot></table-wrap>, respectively. There were significant differences among the males (P&#x0003c;0.01) for TM, PM and sperm viability at 30 min after thawing and in sperm viability at 150 min after thawing. There were no significant differences in the percentages of TM, PM and viable spermatozoa among the samples that were frozen using different cooling rates at either 30 or 150 min after thawing. The interaction of the male and the cooling rate was not significant.</p><p>The quality of movement parameters was not significantly influenced by the cooling rate at either 30 or 150 min. There were significant differences among the males (P&#x0003c;0.05), and the interaction of the male and the cooling rate was not significant. Values in the different males for VCL, VSL, LIN and ALH ranged from 146.3 to 161.4 &#x000b5;m/sec, from 88.9 to 101.5 &#x000b5;m/sec, from 56.1 to 66.2% and from 4.9 to 5.9 &#x000b5;m for 30 min and from 115 to 155.4 &#x000b5;m/sec, from 67.2 to 86.3 &#x000b5;m/sec, from 54 to 61.1% and from 4.8 to 5.8 &#x000b5;m for 150 min, respectively.</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>To optimize the canine semen cryopreservation protocol by reducing the time necessary for this procedure, we evaluated the effect of a rapid cooling rate protocol on the quality of frozen-thawed spermatozoa. To the best to our knowledge, the present work provides the first evidence that dog spermatozoa are capable of surviving rapid cooling rates (2.25 C/min) before freezing with the Uppsala method. While in the traditional Uppsala protocol, semen reaches 5 C in approximately 90 min, this rapid cooling protocol would support a considerable reduction in the time required for the process of freezing dog spermatozoa, as it allows an interval of approximately 8 min between 23 and 5 C.</p><p>It has been reported that the spermatozoa of several species require a long period of cooling before freezing to develop maximal resistance to the effects of freezing [<xref rid="r21" ref-type="bibr">21</xref>] and to reduce the well-known &#x0201c;cold shock&#x0201d; process [<xref rid="r8" ref-type="bibr">8</xref>]. In dogs, there is a shortage of studies regarding the periods used for cooling to 5 C [<xref rid="r22" ref-type="bibr">22</xref>, <xref rid="r23" ref-type="bibr">23</xref>]. To minimize the cold shock process, the majority of the protocols for dog semen cryopreservation include long cooling periods from 23 C to 5 C. The most frequently employed period is from 60 to 120 min, corresponding to approximate mean cooling rates within the range of 0.15&#x02013;0.3 C/min [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r15" ref-type="bibr">15</xref>,<xref rid="r16" ref-type="bibr">16</xref>, <xref rid="r24" ref-type="bibr">24</xref>]. Our results clearly demonstrated that the use of rapid cooling at a rate of 2.25 C/min prior to freezing provides the same post-thaw sperm quality, in terms of motility and viability, as the values obtained with the use of a control rate of 0.2 C/min. The results for the sperm quality after thawing, in terms of the motility and viability in all of the experimental groups were relatively high, adequate for artificial insemination and comparable with those reported recently in other studies performed in frozen-thawed canine semen using the Uppsala method [<xref rid="r25" ref-type="bibr">25</xref>,<xref rid="r26" ref-type="bibr">26</xref>,<xref rid="r27" ref-type="bibr">27</xref>]. In addition, we did not observe differences among the groups in the quality of the movement parameters VCL, LIN and ALH, which have been described as indicators of a hyperactivated motility pattern and a pre-capacitation status [<xref rid="r28" ref-type="bibr">28</xref>]. Therefore, we suggest that use of the rapid cooling rate did not induce a hyperactivation sperm motility pattern after thawing in comparison with the control cooling rate. Regardless of the experimental group, there was a decrease in sperm quality as a result of incubation after thawing at 38 C, which was especially evident in the percentages of total and progressive motility. Our results are in agreement with those described by other authors [<xref rid="r9" ref-type="bibr">9</xref>] and indicate that frozen semen should be used as quickly as possible after thawing.</p><p>The positive results obtained using the rapid cooling rate prior to freezing in this study are in agreement with other reports on other species of mammals, such as the boar and red deer [<xref rid="r17" ref-type="bibr">17</xref>, <xref rid="r18" ref-type="bibr">18</xref>], showing that dog spermatozoa might be particularly resistant to cold shock during the cooling (to 5 C) process. This resistance of sperm cells to thermal stress may be attributed to the constituents of the plasmatic membrane of canine spermatozoa, which has a low ratio of polyunsaturated to saturated phospholipid fatty acids [<xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r22" ref-type="bibr">22</xref>]. Moreover, it should also be considered that the egg yolk included in the extenders for sperm cryopreservation is responsible for membrane stabilization during the cool-down period [<xref rid="r17" ref-type="bibr">17</xref>, <xref rid="r29" ref-type="bibr">29</xref>, <xref rid="r30" ref-type="bibr">30</xref>], reducing the damage that results from the cold shock process. The results of the present study suggested that the presence of 20% egg yolk in the sperm&#x02019;s surrounding media is also adequate when a rapid cooling rate prior to freezing is used.</p><p>Our results regarding the glycerol addition protocol suggest that a protocol for addition of fractionated or unfractionated glycerol is adequate when canine semen is frozen by the Uppsala method using a rapid cooling rate of 2.25 C/min. Based in our attempt to optimize the procedure as much as possible, we believe that a protocol for addition of unfractionated glycerol at 5 C may be more practical because it is only necessary to prepare an extender containing glycerol. Although we observed significant differences in sperm quality after thawing among the glycerol addition protocols in the samples frozen using the control cooling rate, from a practical point of view, these differences were not biologically and clinically important, being approximately 4&#x02013;5%.</p><p>In clinical practice, the ejaculates from different males are cryopreserved individually, and differences in freezability have been reported among individual dogs [<xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r31" ref-type="bibr">31</xref>,<xref rid="r32" ref-type="bibr">32</xref>]. With this in mind and in an effort to adapt the rapid cryopreservation procedure for a practical application in dog breeding, a second experiment was performed. Based on the results of the first experiment, the feasibility of use of a rapid cooling rate in different males was evaluated in combination with an unfractionated glycerol addition protocol. In this regard, our results suggest that the rapid protocol is also adequate for individual applications because the different dogs evaluated in this study did not exhibit different responses to the rapid cooling rate of 2.25 C/min prior to freezing. Regardless of the cooling rate used, all of the dogs included in our study showed adequate percentages of sperm motility and viability in the frozen-thawed semen to be used for AI.</p><p>As expected and regardless of the cooling rate used, significant differences among individuals in sperm cryosurvival were found in sperm motility and viability at 30 min after thawing. However, these differences in sperm motility were not observed after 150 min of incubation at 38 C. In some dogs, a difference in thermoresistance and a higher decline in sperm motility around the time of incubation after thawing were observed, most likely due to the variations in the metabolic exhaustion of the spermatozoa. One explanation for this fact may be related to the finding that dog ejaculates, as in other species, maintain a constant structure of different motile sperm subpopulations during cold storage [<xref rid="r33" ref-type="bibr">33</xref>], after freezing and after thawing [<xref rid="r15" ref-type="bibr">15</xref>, <xref rid="r34" ref-type="bibr">34</xref>]. These subpopulations, which are not distributed uniformly among male dogs [<xref rid="r35" ref-type="bibr">35</xref>], include spermatozoa that differ in their physiological states, functional integrity and quality of sperm kinematics [<xref rid="r34" ref-type="bibr">34</xref>].</p><p>In conclusion, the results of this study suggest that dog spermatozoa could be cryopreserved using a rapid cooling rate of 2.25 C/min prior to freezing in combination with the Uppsala method, obtaining values of sperm quality comparable with those obtained by using a control cooling rate. Thus, use of this rapid cooling rate allows a considerable reduction in the time spent on the procedure. Moreover, the glycerol addition procedure does not affect the feasibility of a rapid protocol.</p></sec>
REV-ERB&#x003b1; Inhibits the <italic>PTGS2</italic> Expression in Bovine Uterus Endometrium Stromal and Epithelial Cells Exposed to Ovarian Steroids
<p> The nuclear receptor REV-ERB&#x003b1; (encoded by <italic>NR1D1</italic>) has a critical role in metabolism and physiology as well as circadian rhythm. Here, we investigated the possible contribution of clock genes including <italic>NR1D1</italic> to the secretion of prostaglandin F<sub>2</sub>&#x003b1; (PGF<sub>2</sub>&#x003b1;) from bovine uterine stromal (USCs) and epithelial cells (UECs) by modulating the expression of <italic>PTGS2</italic>. The circadian oscillation of clock genes in the cells was weak compared with that reported in rodents, but the expression of <italic>BMAL1</italic>, <italic>PER1</italic>, and <italic>NR1D1</italic> was changed temporally by treatment with ovarian steroids. Significant expression of clock genes including <italic>NR1D1</italic> was detected in USCs exposed to progesterone. <italic>NR1D1</italic> was also significantly expressed in UECs exposed to estradiol. The expression of <italic>PTGS2</italic> was suppressed in USCs exposed to progesterone, while the expression was initially suppressed in UECs exposed to estradiol and then increased after long-term exposure to estradiol. <italic>BMAL1</italic> knockdown with specific siRNA caused a significant decrease in the transcript levels of <italic>NR1D1</italic> and <italic>PTGS2</italic> in USCs, but not in UECs. The production of PGF<sub>2</sub>&#x003b1; also decreased in USCs after <italic>BMAL1</italic> knockdown, while its level did not significantly change in UECs. The transcript level of <italic>PTGS2</italic> was increased by treatment with the antagonist of REV-ERB&#x003b1; in both cell types, but the agonist was ineffective. In these two cell types treated with the agonist or antagonist, the PGF<sub>2</sub>&#x003b1; production coincided well with the <italic>PTGS2</italic> expression. Collectively, these results indicate that REV-ERB&#x003b1; plays an inhibitory role in the expression of <italic>PTGS2</italic> in both bovine USCs and UECs treated with ovarian steroids.</p>
<contrib contrib-type="author"><name><surname>ISAYAMA</surname><given-names>Keishiro</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>CHEN</surname><given-names>Huatao</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>YAMAUCHI</surname><given-names>Nobuhiko</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>HATTORI</surname><given-names>Masa-aki</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1)</label>Department of Animal and Marine Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan</aff>
The Journal of Reproduction and Development
<p>In cows and sheep, luteolysis is induced by prostaglandin F<sub>2</sub>&#x003b1; (PGF<sub>2</sub>&#x003b1;), which is secreted in a pulsatile mode from the uterine endometrium during the late luteal phase to the follicular phase. Progesterone (P<sub>4</sub>), estradiol (E<sub>2</sub>), and oxytocin have been regarded as the critical factors regulating the secretion of PGF<sub>2</sub>&#x003b1; from the endometrium. It has been demonstrated that oxytocin promotes the secretion of PGF<sub>2</sub>&#x003b1; as a pulse generator of its secretion in the endometrium [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>]. However, the critical roles of P<sub>4</sub> and E<sub>2</sub> in the secretion of PGF<sub>2</sub>&#x003b1; are still unclear [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r4" ref-type="bibr">4</xref>]. It is generally accepted that ovarian steroids modulate the sensitivity of the endometrium to oxytocin by regulating the expression of the oxytocin receptor [<xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r6" ref-type="bibr">6</xref>,<xref rid="r7" ref-type="bibr">7</xref>]. Conversely, several studies objected to the role of oxytocin in luteolysis [<xref rid="r8" ref-type="bibr">8</xref>,<xref rid="r9" ref-type="bibr">9</xref>,<xref rid="r10" ref-type="bibr">10</xref>], and it was proposed that oxytocin is not essential for PGF<sub>2</sub>&#x003b1; secretion [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r11" ref-type="bibr">11</xref>]. Therefore, existence of another regulator(s) of the PGF<sub>2</sub>&#x003b1; secretion in the endometrium was postulated [<xref rid="r4" ref-type="bibr">4</xref>], but no regulator has not been identified.</p><p>Prostaglandin G/H synthetase (PTGS) is the key rate-limiting enzyme converting arachidonic acid into PGG2 and PGH2, which are the precursors for PGF<sub>2</sub>&#x003b1; and other metabolites. PTGS has two isoforms, PTGS1 and PTGS2. In the bovine endometrium, PGF<sub>2</sub>&#x003b1; is synthesized mostly by PTGS2 [<xref rid="r12" ref-type="bibr">12</xref>]. The promoter region of the bovine <italic>PTGS2</italic> gene contains the E-box element and REV-ERB&#x003b1;/ROR&#x003b1; response element (RORE), which are the circadian clock-controlled <italic>cis</italic>-regulatory elements.</p><p>The cellular clock components CLOCK and BMAL1 bind to the E-box enhancer and induce expression of the nuclear receptor REV-ERB&#x003b1; (encoded by <italic>NR1D1</italic>), resulting in repression of transcription of <italic>BMAL1</italic> through direct binding to RORE located in the <italic>BMAL1</italic> promoter [<xref rid="r13" ref-type="bibr">13</xref>]. In addition to regulating each other to sustain oscillations, the core clock proteins also entrain the rhythmic expression of numerous genes through binding to the E-box, RORE, and D-box at their promoters, which have been called clock-controlled genes (CCGs) and found to comprise a large family. The peripheral oscillators control the expression of downstream CCGs that are expressed in tissue-specific relationships. REV-ERB&#x003b1; has a critical role in the regulation of metabolism and physiology as well as circadian rhythms [<xref rid="r14" ref-type="bibr">14</xref>]. The cellular level of heme, identified as a physiological ligand for REV-ERB&#x003b1; [<xref rid="r15" ref-type="bibr">15</xref>], oscillates in a circadian manner [<xref rid="r16" ref-type="bibr">16</xref>]. SR8278 recently became available as a synthetic antagonist of REV-ERB&#x003b1; [<xref rid="r17" ref-type="bibr">17</xref>]. Based on recent studies, we raised the possibility that the <italic>PTGS2</italic> gene is a downstream CCG in bovine uterus endometrial cells.</p><p>We reported that circadian rhythmicity is weak in the rat uterus luminal epithelium as revealed by immunohistochemistry, although a strong immunostaining signal of PER2 protein is detected in the epithelial cell layer compared with that in the stromal cell layer [<xref rid="r18" ref-type="bibr">18</xref>]. Since regulation of the circadian clockwork may be different between stromal cells and epithelial cells, the two cell types for the circadian clockwork need to be analyzed separately. In the present study, we extended our recent investigations to explore the possible contribution of the circadian clockwork to the secretion of PGF<sub>2</sub>&#x003b1; from the bovine endometrium by modulating the expression of the <italic>PTGS2</italic> gene.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Isolation and culture of bovine USCs and UECs</title><p>Bovine uteri were collected from a slaughterhouse and transferred to laboratory on ice. The two cell types were isolated from the uteri of cows showing the luteal stage at days 11 to 17 as determined by ovarian morphology [<xref rid="r19" ref-type="bibr">19</xref>]. The caruncles were physically dissected from the endometrium of the bovine uterus. Tissue pieces were treated with 0.1% collagenase (Wako, Tokyo, Japan) at 37 C for 30 min and cultured for 1 week in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% charcoal-stripped FBS (Biological Industries, Kibbutz Beit Haemek, Israel) with 1&#x000d7; antibiotic-antimycotic mixed solution (AA; Nacalai Tesque, Kyoto, Japan). USCs and UECs migrated from the tissue pieces and separately proliferated in a monolayer (<xref ref-type="supplementary-material" rid="pdf_002">Supplementary Fig. 1</xref>: on-line only) [<xref rid="r20" ref-type="bibr">20</xref>]. USCs and UECs in primary culture were separated with trypsin-EDTA. USCs and UECs were peeled with 0.05% and 0.25% trypsin-EDTA, respectively. Both cell types separated (4.0&#x000d7;10<sup>5</sup> cells) were seeded on a 35-mm collagen-coated dish (Iwaki, Tokyo, Japan) with 2 ml DMEM/F12 supplemented with 1&#x000d7;AA. Cells were cultured in a humidified atmosphere of 95% air and 5% CO<sub>2</sub> at 37 C for 48 h prior to other treatments.</p><p>Separated USCs and UECs were rinsed with PBS followed by blocking with blocking solution (2% goat serum in PBS) for 30 min at room temperature. These cells were then incubated for 18 h at 4 C with an anti-vimentin polyclonal antibody (1:200; Nichirei Bioscience, Tokyo, Japan) and an anti-cytokeratin monoclonal antibody (1:200; Sigma-Aldrich, St. Louis, MO, USA) diluted in blocking buffer. Goat serum was substituted for the primary antibody as a negative control. After washing several times with PBS, they were incubated with secondary antibodies (Santa Cruz Biotechnology, CA, USA) and DAPI (Sigma-Aldrich) diluted in blocking solution (1:250) for 1 h at room temperature. Immunostaining was detected under a fluorescence microscope (Nikon, Japan). The USCs were positively immunostained for vimentin (a marker protein of stromal cells) but negatively for cytokeratin (a marker of epithelial cells) (<xref ref-type="supplementary-material" rid="pdf_002">Supplementary Fig. 1</xref>). The UECs were strongly immunostained for cytokeratin, as reported previously [<xref rid="r20" ref-type="bibr">20</xref>].</p></sec><sec><title>Treatment with steroid hormones</title><p>USCs and UECs cultured for 2 days were treated with 100 nM P<sub>4</sub> (Sigma-Aldrich) and 100 nM E<sub>2</sub> (Sigma-Aldrich) dissolved in DMSO that was diluted in a serum-free medium with a final DMSO concentration &#x0003c; 0.1%<sub>,</sub> respectively, for 12 h in DMEM/F12 supplemented with 1&#x000d7; Insulin-Transferrin-Selenium (ITS: Life Technologies, Grand Island, NY, USA), 1&#x000d7; AA and 0.1% bovine serum albumin (BSA; Sigma-Aldrich). After treatment with P<sub>4</sub> or E<sub>2</sub>, each cell was washed with culture medium and synchronized with 10 &#x003bc;M forskolin (Sigma-Aldrich) for 2 h in DMEM/F12 supplemented with 1&#x000d7; ITS, 1&#x000d7; AA and 0.1% BSA. Then, USCs and UECs were further cultured with 100 nM P<sub>4</sub> and 100 nM E<sub>2</sub> in DMEM/F12 supplemented with 1&#x000d7; ITS, 1&#x000d7; AA and 0.1% BSA, respectively, and subjected to each experiment.</p></sec><sec><title>Real-time monitoring of mouse Per1 promoter activity</title><p>The &#x02013;1884/&#x02013;102-bp region upstream of the translation start codon of mouse <italic>Per1</italic> was fused to the luciferase gene in the pGL3-Basic vector (Promega, <italic>Per1-Luc</italic> vector) [<xref rid="r21" ref-type="bibr">21</xref>]. The upstream region includes three E-box sites (&#x02013;146 to &#x02013;151, &#x02013;509 to &#x02013;514, and &#x02013;1255 to &#x02013;1260) and a cAMP response element (CRE, &#x02013;1725 to &#x02013;1732). The <italic>Per1</italic>-<italic>Luc</italic> vector (1.0 &#x003bc;g/dish) was transfected into cultured rat and bovine USCs using Hillymax (Wako). Rat USCs were prepared from uteri at the diestrus stage according to a previous report [<xref rid="r18" ref-type="bibr">18</xref>]. These cells were maintained in serum-free DMEM/F12 supplemented with 0.1 mM luciferin (Wako), 0.1% BSA, 1% ITS, 1&#x000d7;AA and 100 nM P<sub>4</sub> after synchronization for 2 h with 10 &#x003bc;M forskolin. Luciferase activity was chronologically monitored at 37 C with a Kronos Dio AB-2550 luminometer (ATTO, Tokyo, Japan) interfaced with a computer for continuous data acquisition [<xref rid="r21" ref-type="bibr">21</xref>].</p></sec><sec><title>BMAL1-specific siRNA transfection</title><p>The sequence targeting the <italic>BMAL1</italic> mRNA and non-silencing RNA for the bovine was purchased from Sigma-Aldrich. The scrambled sequence for the <italic>BMAL1</italic> siRNA was used as a control. The sequences of RNA oligos used are listed in <xref ref-type="supplementary-material" rid="pdf_001">Supplementary Table 1</xref> (on-line only). USCs and UECs were separately seeded on 35-mm collagen-coated dishes with 2 ml DMEM/F12 supplemented with 1&#x000d7; AA, 1&#x000d7; ITS, and 0.1% BSA. After 24 h in culture, the medium was removed, and the <italic>BMAL1</italic>-specific siRNA and non-silencing RNA diluted in Opti-MEM were transfected into cells using Lipofectamine&#x000ae; RNAiMAX reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer&#x02019;s protocol. Both the <italic>BMAL1</italic>-specific siRNA and non-silencing RNA were used at a final concentration of 100 nM. The cells were maintained with transfection medium for an additional 24 h. The medium was replaced with a medium supplemented with 1&#x000d7; AA, 1&#x000d7; ITS, and 0.1% BSA. Then, USCs and UECs were cultured with 100 nM P<sub>4</sub> and 100 nM E<sub>2</sub>, respectively, for 12 h and synchronized with forskolin.</p></sec><sec><title>Treatment with heme and SR8278</title><p>USCs and UECs were treated with 50 &#x003bc;M heme (Sigma-Aldrich) or 10 &#x003bc;M SR8278 (Sigma-Aldrich) dissolved in DMSO in the presence of steroid hormones after synchronization with forskolin. As a control, each cell was treated with 0.1% DMSO instead of heme and SR8278.</p></sec><sec><title>RNA extraction and RT-qPCR</title><p>Cultured cells were harvested at indicated time points, and total RNA was isolated using an RNeasy Mini Kit (Qiagen) according to the manufacturer&#x02019;s protocol. RNA samples were treated with RNase-free DNase (Qiagen). The cDNAs were generated by RT with Oligo (dT)<sub>15</sub> and Random Primers using a GoTaq&#x000ae; 2-Step RT-qPCR System (Promega, Madison, WI, USA). The primer sets used for the RT-qPCR are listed in <xref ref-type="supplementary-material" rid="pdf_001">Supplementary Table 2</xref> (on-line only). All primer pairs were designed to span introns to prevent amplification of products from genomic DNA. RT-qPCR was performed in a 50-&#x003bc;l volume containing a 20-ng cDNA sample in GoTaq&#x000ae; qPCR Master Mix and 250 nM specific primers with an Mx3000P Real-time qPCR System (Agilent Technologies, Santa Clara, CA, USA) using the parameters described in our previous report [<xref rid="r22" ref-type="bibr">22</xref>]. The relative quantification of gene expression was analyzed from the measured threshold cycles (Ct) using the comparative cycle threshold (&#x00394;Ct) method [<xref rid="r18" ref-type="bibr">18</xref>]. The &#x00394;Ct for each sample was normalized to the average level of the constitutively expressed housekeeping gene <italic>GAPDH</italic>. Gene expression was then normalized to the level of the gene of interest in the control samples.</p></sec><sec><title>PGF<sub>2</sub>&#x003b1; assay</title><p>Culture supernatants were collected at 48 h after synchronization. Then the PGF<sub>2</sub>&#x003b1; contents were measured using EIA kits (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer&#x02019;s instructions. The intra-assay and inter-assay variabilities were &#x0003c; 10%.</p></sec><sec><title>Data analysis and statistics</title><p>Data are expressed as the means &#x000b1; SEM of at least three independent experiments, each performed with duplicate samples. The statistical analyses were performed by one-way ANOVA with Tukey&#x02019;s multiple comparison test or the Student&#x02019;s <italic>t</italic> test, as indicated using the SigmaPlot software (Ver. 12.0; Systat Software, San Jose, CA, USA). Differences were considered significant at P&#x0003c;0.05 or less. Rhythmicity in gene expression was determined by the single Cosinor method using Timing Series Single 6.3 (Expert Soft Tech.) [<xref rid="r23" ref-type="bibr">23</xref>].</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>Bioluminescence activity in rat and bovine USCs transfected with the mouse Per1-Luc vector</title><p>To investigate whether the cellular clockwork functions in bovine uterus cells, we first analyzed mouse <italic>Per1</italic> promoter activity as an indicator of the clockwork. There are three E-box sites and one CRE in the mouse <italic>Per1</italic> promoter region (<xref ref-type="fig" rid="fig_001">Fig. 1A</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> Generation of bioluminescence oscillations by rat and bovine USCs transfected with pGL3 vector containing the mouse <italic>Per1</italic> promoter region after synchronization with forskolin. (A) Schematic of the pGL3 vector containing the mouse <italic>Per1</italic> promoter region (upper). Black bars indicate the location of E-box motifs (&#x02013;146 to &#x02013;151, &#x02013;509 to &#x02013;514, and &#x02013;1255 to &#x02013;1260) and a cyclic-AMP response element (CRE, &#x02013;1725 to &#x02013;1732). (B) Bioluminescence activity was induced in bovine and rat USCs transfected with 1 &#x003bc;g of the constructed vector by synchronization with forskolin. Bioluminescence was monitored in real time in serum-free medium DMEM/F12 supplemented with 0.1 mM luciferin, 0.1% BSA, 1% ITS, 1&#x000d7;AA and 100 nM P<sub>4</sub>. Each value represents the means of three independent determinations.</p></caption><graphic xlink:href="jrd-60-362-g001"/></fig>). The rat USCs exposed to P<sub>4</sub> were used as a positive control, in which a robust circadian clockwork has been confirmed [<xref rid="r18" ref-type="bibr">18</xref>, <xref rid="r24" ref-type="bibr">24</xref>]. Real-time monitoring of bioluminescence clearly revealed that there were clear differences in oscillation profiles between rat and bovine USCs. Rat USCs displayed a sharp peak until approximately 30 min after synchronization. Thereafter, the promoter activity in rat USCs showed stable oscillation for 5 days. Conversely, bovine USCs displayed high activity immediately after synchronization, and then the activity in bovine USCs decreased and showed only several small peaks (<xref ref-type="fig" rid="fig_001">Fig. 1B</xref>).</p></sec><sec><title>Expression of core clock genes in bovine USCs</title><p>We next analyzed the temporal changes of the clock gene transcript levels over the course of 48 h using bovine USCs. After synchronization with forskolin, the clock genes <italic>PER1</italic> and <italic>NR1D1</italic> displayed no significant expression in the absence of P<sub>4</sub> (<xref ref-type="fig" rid="fig_002">Fig. 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Expression profiles of core clock gene transcripts over the course of 48 h in bovine USCs. After synchronization with forskolin, total RNA samples were collected at 6 h interval from cells cultured with (bottom) or without (upper) the presence of P<sub>4</sub>. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to <italic>GAPDH</italic> and expressed relative to the first time point (0 h). Each value represents the mean &#x000b1; SEM of three independent experiments. The statistical analyses were performed by one-way ANOVA with Tukey&#x02019;s multiple comparison tests. * P&#x0003c;0.05 <italic>vs.</italic> 0 h.</p></caption><graphic xlink:href="jrd-60-362-g002"/></fig>). <italic>BMAL1</italic> only showed significantly high expression at 6 h (P&#x0003c;0.05). However, these clock gene transcripts showed no diurnal rhythms. P<sub>4</sub> was added to the culture medium, because the stroma is comprised of progesterone-targeting cells and is fitted to the luteal stage. In the presence of 100 nM P<sub>4</sub>, significant expression of three clock genes was detected (P&#x0003c;0.01) (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). Several peaks of clock gene transcripts were observed until 48 h, but their diurnal rhythms were not significant, except in the case of <italic>PER1</italic> (Cosinor, P=0.0006). The relative expression of <italic>NR1D1</italic> was high in the presence of P<sub>4</sub>, especially at 24 to 48 h (P&#x0003c;0.05).</p></sec><sec><title>Expression of core clock genes in bovine UECs</title><p>We also analyzed the temporal changes in the clock gene transcripts in UECs. After synchronization, the clock genes <italic>PER1</italic> and <italic>NR1D1</italic> displayed no significant expression in the absence of E<sub>2</sub> (<xref ref-type="fig" rid="fig_003">Fig. 3</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p> Expression profiles of core clock gene transcripts over the course of 48 h in bovine UECs. After synchronization with forskolin, total RNA samples were collected at 6 h interval from cells cultured with (bottom) or without (upper) the presence of estradiol. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to <italic>GAPDH</italic> and expressed as relative to the first time point (0 h). Each value represents the mean &#x000b1; SEM of three independent experiments. The statistical analyses were performed by one-way ANOVA with Tukey&#x02019;s multiple comparison tests. * P&#x0003c;0.05 <italic>vs.</italic> 0 h.</p></caption><graphic xlink:href="jrd-60-362-g003"/></fig>), but <italic>BMAL1</italic> displayed significant expression (P&#x0003c;0.05) and diurnal rhythms (Cosinor, P=0.0013). E<sub>2</sub> was also added to the culture medium, because the epithelium is comprised of estrogen-targeting cells. In the presence of 100 nM E<sub>2</sub>, <italic>NR1D1</italic> displayed a significant expression and peaked at around 30 h (P&#x0003c;0.05) (<xref ref-type="fig" rid="fig_003">Fig. 3</xref>). <italic>PER1</italic> also showed significant expression (P&#x0003c;0.01). However, the diurnal rhythms of <italic>PER1</italic> and <italic>NR1D1</italic> were not significant, except in the case of <italic>BMAL1</italic> (Cosinor, P=0.0365).</p></sec><sec><title>Expression of PTGS2 in bovine USCs and UECs</title><p>The expression level of <italic>PTGS2</italic> was investigated in bovine USCs and UECs. After synchronization, <italic>PTGS2</italic> displayed significant expression in both cell types. As shown in <xref ref-type="fig" rid="fig_004">Fig. 4</xref><fig orientation="portrait" fig-type="figure" id="fig_004" position="float"><label>Fig. 4.</label><caption><p> Expression of the <italic>PTGS2</italic> gene in bovine USCs and UECs. After synchronization with forskolin, total RNA samples were collected at 6 h interval from USCs (A) and UECs (B) cultured with or without the presence of ovarian steroids. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to <italic>GAPDH</italic> and expressed as relative to the first time point (0 h). Each value represents the mean &#x000b1; SEM of three independent experiments. The statistical analyses were performed by one-way ANOVA with Tukey&#x02019;s multiple comparison tests. * P&#x0003c;0.05 <italic>vs.</italic> 0 h.</p></caption><graphic xlink:href="jrd-60-362-g004"/></fig>, however, different responses of the two cell types to steroid hormones were observed. In the USCs exposed to P<sub>4</sub>, <italic>PTGS2</italic> displayed significantly high expression and peaked at 6 to 12 h (P&#x0003c;0.01) (<xref ref-type="fig" rid="fig_004">Fig. 4A</xref>). In addition, the diurnal rhythm of the <italic>PTGS2</italic> transcript was significant (Cosinor, P=0.0009). The expression level of <italic>PTGS2</italic> was low at 30 to 48 h in the presence of P<sub>4</sub>, whereas it was high in the absence of P<sub>4</sub>. In the UECs exposed to E<sub>2</sub>, <italic>PTGS2</italic> also showed significant expression and peaked at 48 h (P&#x0003c;0.01) (<xref ref-type="fig" rid="fig_004">Fig. 4B</xref>). However, the diurnal rhythm of the <italic>PTGS2</italic> transcript was not significant. The expression of <italic>PTGS2</italic> was suppressed until 30 h in the presence of E<sub>2</sub> compared with in the absence of E<sub>2</sub>. After long exposure to E<sub>2</sub> (48 h), the <italic>PTGS2</italic> transcript level was dramatically increased (P&#x0003c;0.01).</p></sec><sec><title>Effect of BMAL1 knockdown on the expression of PTGS2 in bovine USCs and UECs</title><p>We used <italic>BMAL1</italic>-specific siRNA to investigate whether the <italic>PTGS2</italic> expression is controlled under BMAL1 transcriptional regulation in the two bovine cell types. BMAL1 associated with CLOCK or NPAS2 promotes the transcription of genes such as <italic>NR1D1</italic> through binding to the E-box at the promoter region. The transfection of <italic>BMAL1</italic>-specific siRNA caused a significant decrease in the <italic>BMAL1</italic> transcript level of both the USCs (P&#x0003c;0.01) and the UECs (P&#x0003c;0.05) (<xref ref-type="fig" rid="fig_005">Fig. 5</xref><fig orientation="portrait" fig-type="figure" id="fig_005" position="float"><label>Fig. 5.</label><caption><p> Expression of the <italic>BMAL1</italic>, <italic>NR1D1</italic> and <italic>PTGS2</italic> gene transcripts in bovine USCs and UECs transfected with <italic>BMAL1</italic>-specific siRNA or non-silencing RNA. USCs (A) and UECs (B) were separately treated with <italic>BMAL1</italic>-specific siRNA (siRNA) or non-silencing RNA (CONT) according to the indicated protocols. The cells were then synchronized with forskolin. Total RNA samples were collected at 30 h for the <italic>BMAL1</italic> transcript and 48 h for the <italic>NR1D1</italic> and <italic>PTGS2</italic> transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to <italic>GAPDH</italic> and expressed relative to the non-silencing RNA group. Each value represents the means &#x000b1; SEM of three independent determinations. The statistical analyses were performed by one-way ANOVA with the Student&#x02019;s <italic>t</italic> test. ** P&#x0003c;0.01; * P&#x0003c;0.05.</p></caption><graphic xlink:href="jrd-60-362-g005"/></fig>). Concomitantly, the <italic>NR1D1</italic> transcript level was significantly decreased in the USCs (P&#x0003c;0.01), while it did not change in the UECs. The <italic>PTGS2</italic> transcript level was also significantly decreased in the USCs (P&#x0003c;0.05). Conversely, the UECs displayed no downregulation of the <italic>PTGS2</italic> transcript after <italic>BMAL1</italic> knockdown.</p></sec><sec><title>Effects of heme and SR8278 on the PTGS2 expression in bovine USCs and UECs</title><p>To further investigate the regulation of <italic>PTGS2</italic> expression, we treated bovine USCs and UECs with the agonist (heme) or antagonist (SR8278) of REV-ERB&#x003b1;. As shown in <xref ref-type="fig" rid="fig_006">Fig. 6</xref><fig orientation="portrait" fig-type="figure" id="fig_006" position="float"><label>Fig. 6.</label><caption><p> Expression of the <italic>PTGS2</italic> gene transcript in bovine USCs and UECs treated with the agonist or antagonist of REV-ERB&#x003b1;. USCs (A) and UECs (B) were separately treated with the agonist (heme) or antagonist (SR8278) of REV-ERB&#x003b1; according to the indicated protocols. Cells were then synchronized with forskolin. Total RNA samples were collected at 30 h for the <italic>BMAL1</italic> transcript and 48 h for the <italic>NR1D1</italic> and <italic>PTGS2</italic> transcripts after synchronization. RT-qPCR analyses of transcript levels were performed using their specific primers. The relative transcript level was normalized to <italic>GAPDH</italic> and expressed as relative to the CONT group. Each value represents the means &#x000b1; SEM of three independent determinations. The statistical analyses were performed by one-way ANOVA with the Student&#x02019;s <italic>t</italic> test. ** P&#x0003c;0.01; * P&#x0003c;0.05.</p></caption><graphic xlink:href="jrd-60-362-g006"/></fig>, the <italic>Ptgs2</italic> transcript level was dramatically increased by SR8278 in both cell types. Conversely, treatment with heme did not alter the expression. During treatment with heme or SR8278, different transcript levels of the clock genes <italic>BMAL1</italic> and <italic>NR1D1</italic> were observed. The <italic>NR1D1</italic> transcript level was greatly increased by SR8278 in the USCs but not in the UECs. However, the <italic>BMAL1</italic> transcript level was not changed by SR8278 in the USCs and UECs, probably due to the absence of the REV-ERB&#x003b1; action. Treatment with heme increased the <italic>NR1D1</italic> transcript levels in both cell types, although their increases were very small. Treatment with heme also increased the <italic>BMAL1</italic> transcript level in the USCs.</p></sec><sec><title>Production of PGF<sub>2</sub>&#x003b1; by bovine USCs and UECs</title><p>To further test whether the <italic>PTGS2</italic> expression is regulated by BMAL1 and/or REV-ERB&#x003b1;, we determined the production of PGF<sub>2</sub>&#x003b1; in culture media after treatment with <italic>BMAL1</italic>-specific siRNA and the agonist or antagonist of REV-ERB&#x003b1;. As shown in <xref ref-type="fig" rid="fig_007">Fig. 7A</xref><fig orientation="portrait" fig-type="figure" id="fig_007" position="float"><label>Fig. 7.</label><caption><p> Production of PGF<sub>2</sub>&#x003b1; by bovine USCs and UECs treated with <italic>BMAL1</italic>-specific siRNA and the agonist or the antagonist of REV-ERB&#x003b1;. USCs and UECs were treated with <italic>BMAL1</italic>-specific siRNA (A) and the agonist or antagonist of REV-ERB&#x003b1; (B) as described in <xref ref-type="fig" rid="fig_004">Figs. 4</xref> and<xref ref-type="fig" rid="fig_005"> 5</xref>. The culture media were collected at 48 h after synchronization with forskolin and assayed for PGF<sub>2</sub>&#x003b1;. Each value represents the means &#x000b1; SEM of three independent determinations. The statistical analyses were performed by one-way ANOVA with the Student&#x02019;s <italic>t</italic> test. ** P&#x0003c;0.01; * P&#x0003c;0.05.</p></caption><graphic xlink:href="jrd-60-362-g007"/></fig>, the level of PGF<sub>2</sub>&#x003b1; significantly decreased in USCs after the transfection of <italic>BMAL1</italic>-specific siRNA (P&#x0003c;0.05), and this was coincident with the decreased transcript level of <italic>PTGS2</italic>. Conversely, the level of PGF<sub>2</sub>&#x003b1; did not significantly change in UECs transfected with <italic>BMAL1</italic>-specific siRNA, in which the transcript level of <italic>PTGS2</italic> remained unchanged. In both cell types treated with heme or SR8278, the production of PGF<sub>2</sub>&#x003b1; was well reflected by the expression of <italic>PTGS2</italic>. As shown in <xref ref-type="fig" rid="fig_007">Fig. 7B</xref>, the level of PGF<sub>2</sub>&#x003b1; increased approximately twofold in the presence of SR8278. Conversely, treatment with heme caused no significant changes in the PGF<sub>2</sub>&#x003b1; level.</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>The regulation of PGF<sub>2</sub>&#x003b1; production in the bovine uterus endometrium during the estrus cycle remains poorly understood, although P<sub>4</sub>, E<sub>2</sub> and oxytocin are well known as the regulatory hormones [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r4" ref-type="bibr">4</xref>, <xref rid="r11" ref-type="bibr">11</xref>]. In the present study, we focused on control of the cellular circadian clockwork related to PGF<sub>2</sub>&#x003b1; production in bovine USCs and UECs. We demonstrated that the nuclear receptor REV-ERB&#x003b1; plays an inhibitory role in PGF<sub>2</sub>&#x003b1; secretion, which is mediated through direct inhibition of <italic>PTGS2</italic> expression in both cell types. We also showed that BMAL1 promotes PGF<sub>2</sub>&#x003b1; secretion as a heterodimer with CLOCK, which is mediated through the transactivation of the <italic>PTGS2</italic> expression in USCs. The secretion of PGF<sub>2</sub>&#x003b1; may be balanced by the inhibitory or stimulatory transcriptional regulation of REV-ERB&#x003b1; and BMAL1/CLOCK, respectively.</p><p>Circadian clockwork systems generate cellular rhythms in physiological functions via identified transcriptional and posttranscriptional regulatory processes. The cellular clock components CLOCK and BMAL1 bind to the E-box enhancer and positively drive the expression of the <italic>Period</italic> genes (<italic>Per1-3</italic>) and the <italic>Cryptochrome</italic> genes (<italic>Cry1-2</italic>). In turn, PER and CRY proteins heterodimerize and undergo phosphorylation. The PER-CRY complexes translocate to the nucleus and repress the activity of CLOCK-BMAL1 heterodimers [<xref rid="r25" ref-type="bibr">25</xref>, <xref rid="r26" ref-type="bibr">26</xref>]. Further adding to the complexity, CLOCK-BMAL1 heterodimers induce expression of the nuclear receptor REV-ERB&#x003b1; (encoded by <italic>NR1D1</italic>), resulting in repression of the transcription of <italic>BMAL1</italic> through direct binding to RORE located in the <italic>BMAL1</italic> promoter [<xref rid="r13" ref-type="bibr">13</xref>]. The conservative transcriptional feedback loop resides in the ovine circadian clock [<xref rid="r27" ref-type="bibr">27</xref>], and the circadian expression of the core clock genes <italic>BMAL1</italic>, <italic>PER2</italic>, and <italic>CRY1</italic> is generated in the ovine liver [<xref rid="r28" ref-type="bibr">28</xref>]. It was thus expected that clock genes would also be driven in oscillatory patterns in bovine uterus endometrial cells, which would be similar to murine cells [<xref rid="r18" ref-type="bibr">18</xref>, <xref rid="r29" ref-type="bibr">29</xref>]. Our present results showed that the circadian clockwork machinery functions in bovine USCs exposed to P<sub>4</sub>, while it was weak compared to that in rat (<xref ref-type="fig" rid="fig_001">Fig. 1B</xref>). We also showed that the bovine <italic>PER1</italic> transcript level exhibited a significant diurnal rhythm in bovine USCs exposed to P<sub>4</sub> (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). However, comprehensively, the diurnal rhythms of the clock gene expression were not significant in bovine USCs and UECs in the presence or absence of steroid hormones, except for <italic>PER1</italic> and <italic>BMAL1</italic> under some conditions (<xref ref-type="fig" rid="fig_002">Figs. 2</xref> and<xref ref-type="fig" rid="fig_003"> 3</xref>). Therefore, it is supposed that the circadian rhythm of clock genes, at least, that we observed may be weak in both USCs and UECs.</p><p>In regard to the oscillation, the superiority of clock genes and strength of circadian oscillation in the uterus may depend on cell types and physiological states. An immunohistochemical analysis revealed that the PER2 protein expression is constitutive in epithelial cells but not in the stromal cells [<xref rid="r23" ref-type="bibr">23</xref>]. In addition, the circadian rhythm of clock genes was reported to be significant for <italic>Cry1</italic> and not for <italic>Per1</italic>, <italic>Per2</italic> or <italic>Bmal1</italic> in the mouse uterus during the late stage of pregnancy [<xref rid="r30" ref-type="bibr">30</xref>]. Moreover, there may be differences among animal species. Most of the clock genes displayed no significant rhythms in bovine lymphocytes [<xref rid="r31" ref-type="bibr">31</xref>], unlike murine lymphocytes [<xref rid="r32" ref-type="bibr">32</xref>].</p><p>In the present study, the diurnal rhythms of clock genes were not particularly significant, but steroid hormones differentially affected the temporal changes in clock gene expression in both the USCs and UECs. In the USCs, the transcript levels of <italic>NR1D1</italic> increased, especially at 24 to 48 h after synchronization in the presence of P<sub>4</sub> (<xref ref-type="fig" rid="fig_002">Fig. 2</xref>). In the UECs, the transcript levels of <italic>NR1D1</italic> and <italic>PER1</italic> changed significantly in the presence of E<sub>2</sub> (<xref ref-type="fig" rid="fig_004">Fig. 4</xref>). As revealed by chromatin immunoprecipitation followed by a massive parallel sequencing (ChIP-seq) analysis coupled with microarrays in the mouse uterus, progesterone receptor binding sites are abundant near the coding regions of clock genes (<italic>Clock</italic>, <italic>Npas2</italic>, <italic>Cry1</italic>, <italic>Per1</italic>, and <italic>Nr1d2</italic>) [<xref rid="r33" ref-type="bibr">33</xref>]. The phase of <italic>Per2</italic> oscillation in the uterus from <italic>Per2</italic>::<italic>Luc</italic> knock-in mice is affected by E<sub>2</sub>, and the <italic>Per2</italic> oscillation in the uterus during the mouse estrus cycle is modulated by fluctuating E<sub>2</sub> and P<sub>4</sub>[<xref rid="r29" ref-type="bibr">29</xref>, <xref rid="r34" ref-type="bibr">34</xref>]. These findings, taken together with our present data, indicate that the expression of clock genes affected by steroid hormones may alter the expression of clock-controlled genes and modulate physiological functions in endometrial cells.</p><p>Here we found that the transcript level of <italic>PTGS2</italic> significantly decreased with circadian oscillation in USCs during exposure to P<sub>4</sub> for 48 h, while its transcript level increased in UECs after exposure to E<sub>2</sub> for 48 h (<xref ref-type="fig" rid="fig_004">Fig. 4</xref>). Consequently, we propose that the <italic>PTGS2</italic> expression in USCs is regulated by the circadian clockwork and P<sub>4</sub>. It is possible that other clock genes that we did not analyze in <xref ref-type="fig" rid="fig_001">Fig. 1</xref> may cause circadian oscillation in the <italic>PTGS2</italic> expression in USCs. Conversely, the circadian clockwork in UECs may not affect the <italic>PTGS2</italic> expression. Incidentally, the transcript level of <italic>PTGS2</italic> significantly increased in the absence of P<sub>4</sub> and E<sub>2</sub> (<xref ref-type="fig" rid="fig_004">Fig. 4</xref>), but the expression may result from the action of the forskolin used as a resetting factor for the clockwork. It is known that <italic>PTGS2</italic> expression is induced by cyclic AMP [<xref rid="r35" ref-type="bibr">35</xref>, <xref rid="r36" ref-type="bibr">36</xref>].</p><p>BMAL1 transactivates the target genes by binding of the heterodimer with CLOCK to the E-box element (5&#x02019;-CACGTG-3&#x02019;) on the promoter region of the responsive clock genes such as <italic>PER</italic>, <italic>NR1D1</italic>, <italic>DBP</italic> and <italic>ROR</italic>&#x003b1; [<xref rid="r26" ref-type="bibr">26</xref>, <xref rid="r27" ref-type="bibr">27</xref>]. There are functional E-box elements in the promoter region of the rat <italic>PTGS2</italic> gene [<xref rid="r37" ref-type="bibr">37</xref>], and our recent study demonstrated that <italic>Bmal1</italic> knockdown causes a significant decrease in the <italic>Ptgs2</italic> expression and PGE<sub>2</sub> production in rat ovarian granulosa cells [<xref rid="r22" ref-type="bibr">22</xref>]. In the promoter region of the bovine <italic>PTGS2</italic> gene, also there are also several E-box sites [<xref rid="r38" ref-type="bibr">38</xref>]. In the present study, <italic>BMAL1</italic> knockdown caused significant decreases in <italic>PTGS2</italic> expression and PGF<sub>2</sub>&#x003b1; production in the USCs in the presence of P<sub>4</sub> (<xref ref-type="fig" rid="fig_005">Figs. 5</xref> and<xref ref-type="fig" rid="fig_007"> 7</xref>). Conversely, <italic>BMAL1</italic> knockdown had no significant effect on the <italic>PTGS2</italic> expression and PGF<sub>2</sub>&#x003b1; production in the UECs in the presence of E<sub>2</sub> (<xref ref-type="fig" rid="fig_005">Figs. 5</xref> and<xref ref-type="fig" rid="fig_007"> 7</xref>). In the UECs, <italic>BMAL1</italic> knockdown also had no effect on <italic>NR1D1</italic> expression. This finding may indicate that BMAL1 transactivation in the <italic>PTGS2</italic> and <italic>NR1D1</italic> genes is weak in UECs exposed to E<sub>2</sub>. Actually, it has been reported that E<sub>2</sub> disrupts the circadian expression of <italic>Per1</italic> and <italic>Per2</italic>, which are promoted by BMAL1, in the liver, kidney and uterus of rats [<xref rid="r39" ref-type="bibr">39</xref>]. In the present results, the synchronization of USCs exposed to P<sub>4</sub> caused a significant increase in <italic>BMAL1</italic> transcripts after 6 h, while the UECs exposed to E<sub>2</sub> displayed no significant increase in <italic>BMAL1</italic> transcripts (<xref ref-type="fig" rid="fig_002">Figs. 2</xref> and<xref ref-type="fig" rid="fig_003"> 3</xref>). These results suggest that the effect of <italic>BMAL1</italic> knockdown is weak in the E<sub>2</sub>-treated UECs, probably through E<sub>2</sub> inhibition of the transcriptional activity of <italic>BMAL1</italic>. Taken together with the present data, these findings suggest that the expression of bovine <italic>PTGS2</italic> is controlled under BMAL1 or its responsive clock genes in USCs, but not UECs, in the presence of ovarian steroids.</p><p>Interestingly, when the transcript level of <italic>NR1D1</italic> was high in the USCs and UECs in the presence of P<sub>4</sub> or E<sub>2</sub>, the transcript level of <italic>PTGS2</italic> was low. We therefore focused on the possible regulation of <italic>PTGS2</italic> expression by REV-ERB&#x003b1;. The bovine <italic>PTGS2</italic> gene has two ROREs in the promoter region within &#x02212;3000 bp upstream from the transcriptional start site (AC_000173). We analyzed the expression of <italic>PTGS2</italic> using the antagonist (SR8278) and agonist (heme) of REV-ERB&#x003b1;. The agonist was ineffective, while the antagonist resulted in increased transcript levels of <italic>PTGS2</italic> in both cell types in the presence of P<sub>4</sub> or E<sub>2</sub> (<xref ref-type="fig" rid="fig_006">Fig. 6</xref>). At least, these results indicate that REV-ERB&#x003b1; represses <italic>PTGS2</italic> gene expression.</p><p>Concomitantly, PGF<sub>2</sub>&#x003b1; production completely coincided with the <italic>PTGS2</italic> transcript level (<xref ref-type="fig" rid="fig_007">Fig. 7</xref>). It is also of interest that the antagonist clearly enhanced the <italic>PTGS2</italic> expression in the UECs, in which the circadian regulation of <italic>PTGS2</italic> was weak as revealed by <italic>BMAL1</italic> knockdown. In bovine UECs, therefore, <italic>PTGS2</italic> expression is dominantly controlled under REV-ERB&#x003b1;, but not the circadian clockwork, in the presence of E<sub>2</sub>. Unlike UECs, however, bovine USCs are controlled under both REV-ERB&#x003b1; and the circadian clockwork. Our finding that treatment with the antagonist increased the transcript level of <italic>NR1D1</italic> in the USCs also supports the autoregulation of <italic>NR1D1</italic> expression as reported previously [<xref rid="r40" ref-type="bibr">40</xref>, <xref rid="r41" ref-type="bibr">41</xref>]. However, it is reasonable that the antagonist had no significant effect on the transcript level of <italic>BMAL1</italic> in the USCs, which has been found to be repressed by REV-ERB&#x003b1; [<xref rid="r13" ref-type="bibr">13</xref>]. Unexpectedly, however, treatment with heme increased the <italic>BMAL1</italic> transcript level in the USCs in the present study, and the mechanism remains to be investigated.</p><p>In conclusion, the circadian oscillation of clock genes is partially weak in bovine USCs and UECs, but ovarian steroid hormones may exert differential influences on the expression of the clock genes and the <italic>PTGS2</italic> gene. <italic>BMAL1</italic> knockdown decreased both the <italic>PTGS2</italic> expression and PGF<sub>2</sub>&#x003b1; production in USCs in the presence of P<sub>4</sub>, indicating circadian regulation of the <italic>PTGS2</italic> expression. However, the <italic>PTGS2</italic> expression in UECs is mostly independent of the circadian clockwork. The <italic>PTGS2</italic> expression was commonly suppressed in both cell types by REV-ERB&#x003b1; in the presence of steroid hormones. The present findings contribute to our understanding of the intercellular mechanisms underlying the <italic>PTGS2</italic> expression and PGF<sub>2</sub>&#x003b1; production in bovine uterus endometrium cells.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="pdf_001"><caption><title>Supplement Table</title></caption><media mimetype="application" mime-subtype="pdf" xlink:href="jrd-60-362-s001.pdf" orientation="portrait" xlink:type="simple" id="d35e1509" position="anchor"/></supplementary-material><supplementary-material content-type="local-data" id="pdf_002"><caption><title>Supplement Figure</title></caption><media mimetype="application" mime-subtype="pdf" xlink:href="jrd-60-362-s002.pdf" orientation="portrait" xlink:type="simple" id="d35e1514" position="anchor"/></supplementary-material></sec>
An Earlier Uterine Environment Favors the <italic>In Vivo</italic> Development of Fresh Pig Morulae and Blastocysts Transferred by a Nonsurgical Deep-uterine Method
<p> This study aimed to evaluate the effect of recipient-donor estrous cycle synchrony on recipient reproductive performance after nonsurgical deep-uterine (NsDU) embryo transfer (ET). The transfers (N=132) were conducted in recipients sows that started estrus 24 h before (&#x02013;24 h; N=9) or 0 h (synchronous; N=31), 24 h (+24 h; N=74) or 48 h (+48 h; N=18) after the donors. A total of 30 day 5 morulae or day 6 blastocysts (day 0=onset of estrus) were transferred per recipient. The highest farrowing rates (FRs) were achieved when estrus appeared in recipients 24 h later than that in the donors (81.1%), regardless of the embryonic stage used for the transfers. The FR notably decreased (P&#x0003c;0.05) when recipients were &#x02013;24 h asynchronous (0%), synchronous (61.3%) or +48 h asynchronous (50%) relative to the donors. No differences in litter size (LS) and piglet birth weights were observed among the synchronous and +24 h or +48 h asynchronous groups. While a +24 h asynchronous recipient was suitable for transfers performed with either morulae (FR, 74.3%; LS, 9.2 &#x000b1; 0.6 piglets) or blastocysts (FR, 84.6%; LS, 9.8 &#x000b1; 0.6 piglets), a + 48 h asynchronous recipient was adequate for blastocysts (FR, 87.5%; LS, 10.4 &#x000b1; 0.7 piglets) but not for morulae (FR, 30.0%; LS, 7.3 &#x000b1; 2.3 piglets). In conclusion, this study confirms the effectiveness of the NsDU-ET technology and shows that porcine embryos tolerate better a less advanced uterine environment if they are nonsurgically transferred deep into the uterine horn.</p>
<contrib contrib-type="author"><name><surname>ANGEL</surname><given-names>Miguel Angel</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>GIL</surname><given-names>Maria Antonia</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>CUELLO</surname><given-names>Cristina</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>SANCHEZ-OSORIO</surname><given-names>Jonatan</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>GOMIS</surname><given-names>Jesus</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>PARRILLA</surname><given-names>Inmaculada</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>VILA</surname><given-names>Jordi</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>COLINA</surname><given-names>Ignacio</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>DIAZ</surname><given-names>Marta</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>REIXACH</surname><given-names>Josep</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>VAZQUEZ</surname><given-names>Jose Luis</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>VAZQUEZ</surname><given-names>Juan Maria</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>ROCA</surname><given-names>Jordi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>MARTINEZ</surname><given-names>Emilio A.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1)</label>Department of Animal Medicine and Surgery, University of Murcia, Espinardo 30100, Spain</aff><aff id="aff2"><label>2)</label>Department of Research and Development, Selecci&#x000f3;n Batall&#x000e9; S.A., Girona 17421, Spain</aff>
The Journal of Reproduction and Development
<p>The success of any embryo transfer (ET) program is significantly influenced by the quality of the embryos, the recipients and/or the interaction of both factors [<xref rid="r1" ref-type="bibr">1</xref>]. Therefore, the degree of synchrony of the estrous cycle (or endometrial status) between the recipients and donors is crucial. Despite this fact, previous studies have yielded variable results, including variation among the species. Pregnancy rates were not compromised when synchrony between the recipient and donor was within 48 h, 24 h and 12 h in the sheep [<xref rid="r2" ref-type="bibr">2</xref>,<xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r4" ref-type="bibr">4</xref>], cow [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r6" ref-type="bibr">6</xref>] and buffalo [<xref rid="r7" ref-type="bibr">7</xref>], respectively. In commercial equine ET programs, it is generally accepted that optimum pregnancy rates are achieved in recipients that have ovulated within a range of 24 h before to 72 h after the donors (reviewed by Stout [<xref rid="r8" ref-type="bibr">8</xref>]). Transfers performed outside these ranges invariably decrease the pregnancy rate and increase embryonic loss (ovine [<xref rid="r2" ref-type="bibr">2</xref>, <xref rid="r3" ref-type="bibr">3</xref>], bovine [<xref rid="r9" ref-type="bibr">9</xref>] and equine [<xref rid="r10" ref-type="bibr">10</xref>]).</p><p>In pigs, high pregnancy rates (over 70%) were achieved when surgical transfers were made to recipients in which the onset of estrus was either synchronous relative to that of the donors or 24 h or 48 h later; in contrast, pregnancy rates dramatically decreased in recipients that were ahead of the donors by the same intervals [<xref rid="r11" ref-type="bibr">11</xref>]. Similarly, other studies that also used surgical transfer indicated certain advantages when transfers were performed on recipients that were in estrus 24 h after the donors [<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r13" ref-type="bibr">13</xref>]. In contrast, studies involving nonsurgical ET into the uterine body have shown that transfers into recipients ovulating 18 to 36 h after the donors resulted in reduced pregnancy rates compared with those performed on recipients that had ovulated within the range of 24 h before to 12 h after the donors [<xref rid="r14" ref-type="bibr">14</xref>]. In addition, using nonsurgical ET and blastocysts produced <italic>in vitro</italic>, Yoshioka <italic>et al</italic>. [<xref rid="r15" ref-type="bibr">15</xref>] found that the pregnancy and farrowing rates did not differ in recipients in which the estrous cycle was delayed 24 to 72 h compared with that of donors, although the efficiency of piglet production was greater when the asynchrony was 24 h. Differences in the transfer procedures used (surgical and nonsurgical), the source of embryos (<italic>in vivo</italic> and <italic>in vitro</italic>), the small sample sizes used and the scarce number of studies performed limit comparison of these studies with contemporary application of newer methods, such as nonsurgical deep uterine (NsDU) ET. This technique was developed from the successful deep-uterine insemination procedure in sows [<xref rid="r16" ref-type="bibr">16</xref>,<xref rid="r17" ref-type="bibr">17</xref>,<xref rid="r18" ref-type="bibr">18</xref>], which demonstrated that it was possible to nonsurgically insert a catheter through the cervix deep into a uterine horn, a concept considered impossible at the time. The insemination technique was thereafter adapted for ET, leading to the creation of a novel and unique procedure for nonsurgical insertion of a catheter deep into a uterine horn of gilts and sows during metaestrus [<xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r20" ref-type="bibr">20</xref>]. The NsDU-ET procedure is simple, safe, rapid and well tolerated by the recipients (reviewed by Martinez <italic>et al</italic>. [<xref rid="r21" ref-type="bibr">21</xref>, <xref rid="r22" ref-type="bibr">22</xref>]). The excellent reproductive performance of the recipients after NsDU transfer of fresh embryos [<xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r23" ref-type="bibr">23</xref>] and the promising results obtained using cryopreserved embryos [<xref rid="r20" ref-type="bibr">20</xref>, <xref rid="r21" ref-type="bibr">21</xref>] represent a fundamental advance to widespread commercial use of ET by the pig industry. However, as with the development of any new technology, it is necessary to reevaluate specific factors that can affect the success rate of ET, since they were based on the use of surgical transfer or nonsurgical transfer into the uterine body.</p><p>Therefore, the objective of this study was to determine the effect of several specific degrees of recipient-donor asynchrony on the reproductive performance of recipients following NsDU-ETs of morulae or blastocysts.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><p>All experimental procedures used in this study were performed in accordance with the 2010/63/EU EEC Directive for animal experiments and were reviewed and approved by the Ethical Committee for Experimentation with Animals of the University of Murcia, Spain.</p><sec><title>Animals</title><p>This work was conducted in a pig genetics company (Selecci&#x000f3;n Batall&#x000e9; S.A., Girona, Spain). Purebred Duroc sows (2&#x02013;6 parities) were selected at weaning and used as donors and recipients. Females were allocated individually to crates in a mechanically ventilated confinement facility under field conditions. They were fed a commercial ration twice per day, and water was provided <italic>ad libitum</italic>.</p></sec><sec><title>Superovulation and detection of estrus</title><p>Weaning was used to synchronize estrus between donors and recipients. To standardize the ET&#x02013;schedule, only sows with a weaning-to-estrus interval of 3 to 4 days were selected as donors and recipients. Superovulation of donors was induced by intramuscular administration of 1,000 IU equine chorionic gonadotropin (eCG; Folligon, Intervet, Boxmeer, The Netherlands) at 24 h after weaning. Only sows with clear signs of estrus at 48&#x02013;72 h post eCG administration were further intramuscularly administered 750 IU of human chorionic gonadotropin (Veterin Corion, Divasa, Farmavic S.A., Barcelona, Spain) at the onset of estrus. Beginning at 2 days after the administration of eCG, estrous was checked twice per day (0700 h and 1700 h) by exposing sows to a mature boar (nose-to-nose contact) and applying manual back pressure. Females that exhibited a standing estrous reflex were considered to be in estrus.</p></sec><sec><title>Artificial insemination and embryo recovery and evaluation</title><p>The sperm&#x02013;rich fractions of the ejaculates were manually collected from healthy sexually mature Duroc boars (2&#x02013;3 years of age) that were fertile and undergoing regular semen collection for commercial AI using liquid semen. The donors were inseminated at 0 h, 24 h and 36 h after the onset of estrus with seminal doses (1.5 &#x000d7; 10<sup>9</sup> spermatozoa in 45 ml) prepared from semen diluted in Beltsville thawing solution (BTS) extender [<xref rid="r24" ref-type="bibr">24</xref>]. The seminal doses were stored for a maximum of 72 h at 18 C.</p><p>Embryo collection was performed in a surgical room located at the farm. The donors were subjected to a midventral laparotomy on days 5 and 6 of the estrous cycle (day 0: onset of estrus) to obtain morulae and unhatched blastocysts, respectively. The donors were sedated by administration of azaperone (2 mg/kg body weight, intramuscular). General anesthesia was induced using sodium thiopental (7 mg/kg body weight, intravenous) and maintained with isoflurane (3.5&#x02013;5%). After exposure of the genital tract, the corpora lutea were counted in the ovaries. Embryos were collected by flushing the tip of each uterine horn with 30 ml of protein-free embryo recovery medium consisting of Tyrode&#x02019;s lactate (TL)-HEPES-polyvinyl alcohol (PVA) (TL-HEPES-PVA) [<xref rid="r25" ref-type="bibr">25</xref>] with some modifications. This medium was composed of 124.3 mM NaCl, 3.2 mM KCl, 2 mM NaHCO<sub>3</sub>, 0.34 mM KH<sub>2</sub>PO<sub>4</sub>, 10 mM Na-lactate, 0.5 mM MgCl<sub>2</sub>&#x000b7;6H<sub>2</sub>O, 2 mM CaCl<sub>2</sub>&#x000b7;2H<sub>2</sub>O, 10 mM HEPES, 0.2 mM Na-pyruvate, 12 mM sorbitol, 0.1% (w/v) PVA, 75 &#x003bc;g/ml potassium penicillin G and 50 &#x003bc;g/ml streptomycin sulfate. Recovered embryos were evaluated under a stereomicroscope at a magnification of &#x000d7; 60 to grade their developmental stage and quality. One&#x02013;cell eggs and poorly developed embryos were classified as unfertilized oocytes and degenerate embryos, respectively. The remaining embryos that exhibited appropriate morphology according to the criteria determined by the International Embryo Transfer Society [<xref rid="r26" ref-type="bibr">26</xref>] were considered viable. Only compacted morulae and/or unhatched blastocysts graded as excellent or good for morphological appearance were classified as transferable. The collected embryos were washed six times in embryo recovery medium and placed in Eppendorf tubes containing 1.5 ml of the same medium in a thermostatically controlled incubator (39 C) and maintained for up to 6 h prior to transfer.</p></sec><sec><title>Nonsurgical deep uterine embryo transfer</title><p>The NsDU&#x02013;ETs were conducted on days 3 to 7 of the estrous cycle in nonhormonally treated synchronous or asynchronous recipients using the method previously described by Angel <italic>et al</italic>. [<xref rid="r23" ref-type="bibr">23</xref>]. Six hours prior to transfer, each recipient received a single intramuscular injection of a long&#x02013;acting amoxicillin suspension (Clamoxyl LA<sup>&#x000ae;</sup>; Pfizer, Madrid, Spain) at a dosage of 15 mg/kg body weight. Recipients were housed in gestation crates in a small room (12 crates) exclusively used for that purpose. The perineal area of the recipients was thoroughly cleaned with soap and water using a different sponge for each sow. The tail of each recipient was covered with a latex glove to protect the vulva from possible contamination. The vulva was then washed and decontaminated (inside and outside) using sterile gauze soaked with chlorhexidine. Commercial nonsurgical ET catheters (DeepBlue<sup>&#x000ae;</sup> ET catheter, Minit&#x000fc;b, Tiefenbach, Germany), which were individually packaged and sterilized, were used for the transfers. Each ET catheter was composed of an AI spirette containing a flexible catheter (FC; 1.8 m length) inside and a protective sanitary sheath outside. Prior to insertion, the inner tubing of the FC was rinsed with 0.3 ml of TL-HEPES-PVA medium at 39 C, and the protective sheath was lubricated with silicone (R&#x000fc;sch Silkospray<sup>&#x000ae;</sup>, Willy R&#x000fc;sch, Kernen-Rommelshausen, Germany). Next, the spirette was inserted through the vulva into the first 20 to 25 cm of the vagina. In this region, the spirette tip was pushed through the sheath and inserted into the cervix. The FC was then moved through the cervical canal and propelled forward along one uterine horn until the length of the FC outside of the recipient was approximately 30 to 40 cm. The FC was flushed with 0.3 ml of TL-HEPES-PVA medium at 39 C using a 1 ml disposable syringe when the tip of the FC reached the uterine body. When the FC was completely inserted into one uterine horn, a 1 ml syringe containing the embryos in 0.1 ml of TL-HEPES-PVA medium was connected to the FC, and the contents were introduced into the FC. Finally, an additional volume of 0.3 ml of TL-HEPES-PVA medium was used to force the embryos out of the FC into the uterus. Correct positioning of the FC was assumed if no bends or kinks in the catheter were present after its removal [<xref rid="r19" ref-type="bibr">19</xref>].</p></sec><sec><title>Experimental design</title><p>A total of 193 donors were selected based on their reproductive history (fertility, 96.1 &#x000b1; 0.9%; litter size, 11.0 &#x000b1; 0.1; parity number, 4.9 &#x000b1; 0.1; and lactation length, 21.3 &#x000b1; 0.1 days). Transfers were conducted in recipients that started estrus 24 h before (&#x02013;24 h; N=9) or 0 (synchronous; N=31), 24 (+24 h; N=74) or 48 (+48 h; N=18) h after the donors. The recipients (N=132) were selected based on their reproductive history and body condition. There were no differences in reproductive history of the recipients assigned to each group (fertility range, 93.7 &#x000b1; 3.3% to 95.1 &#x000b1; 4.1%; litter size range, 9.9 &#x000b1; 0.2 to 10.9 &#x000b1; 0.4; parity number range, 2.4 &#x000b1; 0.1 to 2.7 &#x000b1; 0.2; lactation length range, 21.6 &#x000b1; 0.4 to 22.2 &#x000b1; 0.4 days). Thirty transferable embryos (morulae and unhatched blastocysts) were nonsurgically transferred into one uterine horn of each recipient. Each trial was conducted in separate sessions over a 2-year period and included 18 to 20 donors and 11 to 13 NsDU-ETs. The ovulatory response of the donors was determined by counting the number of corpora lutea in both ovaries. To evaluate the effectiveness of the superovulation treatment, the number of viable and transferable embryos and the number of oocytes and/or degenerated embryos were counted in each donor. The recovery rate was defined as the ratio of the number of embryos and oocytes and/or degenerated embryos recovered to the number of corpora lutea present. The fertilization rate was defined as the ratio of the number of viable embryos to the total number of embryos and oocytes and/or degenerated embryos collected. In addition, the presence of follicular cysts (ovarian structures filled with a transparent liquid, without ovulation signs, and with a diameter &#x0003e;2 cm at the time of laparotomy) and polycystic ovaries (ovaries with more than eight follicular cysts) was recorded for each donor. Starting at 12 days after NsDU-ET, the recipients were evaluated daily for signs of estrus. Pregnancy was diagnosed by ultrasonography on days 20 to 22 post transfer. All pregnant sows were allowed to carry litters to term, and the farrowing rates and litter sizes were recorded. The piglet production efficiency was calculated as the ratio of the number of live&#x02013;born piglets to the number of embryos transferred to all recipients.</p></sec><sec><title>Statistical analysis</title><p>The data were analyzed using IBM SPSS Statistics for Windows, Version 19.0 (IBM, Armonk, NY, USA). The percentage data were compared using the Fisher&#x02019;s exact test. Continuous variables were evaluated using the Kolmogorov&#x02013;Smirnov test to assess the assumption of normality, and groups were compared with analysis of variance or the Student&#x02019;s <italic>t</italic>-test. Post hoc analysis was performed using Bonferroni&#x02019;s test. The coefficient of variation (CV, standard deviation/mean) was used as a measure of variability of the ovulatory response. Differences were considered significant when P &#x0003c; 0.05. Differences among values with 0.05&#x0003c;P&#x0003c;0.10 were accepted as representing tendencies toward differences. The results are expressed as percentages and means &#x000b1; standard error of the mean (SEM).</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><p>Of 193 donors, 184 (95.3%) had embryos on days 5 to 6 post AI, 6 (3.1%) had only oocytes after flushing and 3 (1.5%) had polycystic ovaries with no corpora lutea in their ovaries. The proportion of donors with ovarian cysts was 39.1%, and sows with cysts had 2.4 &#x000b1; 0.2 cysts. The mean ovulation rate was 24.9 &#x000b1; 0.4 corpora lutea (range 11 to 51 corpora lutea, CV=25.4%). The recovery and fertilization rates were 94.8% and 92.8%, respectively, and the mean number of viable embryos and oocytes and/or degenerate embryos obtained in the pregnant sows was 21.9 &#x000b1; 0.4 and 1.7 &#x000b1; 0.4, respectively. The proportion of transferable embryos in relation to the number of viable embryos was 95.0%. The total number of transferable embryos collected from the inseminated donors (N=193) was 3,828, resulting in a donor to recipient ratio of 1.5:1.</p><p>Ten out of 132 ETs (7.6%) were removed from the study due to incorrect insertion of the NsDU-ET catheter. The reproductive performance of recipients after transfers is shown in <xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Effects of the different degrees of synchrony between recipients and donors on the farrowing rates and litter sizes after nonsurgical deep intrauterine transfers of 30 fresh embryos at the morula and/or unhatched blastocyst stages</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" valign="top" rowspan="2" colspan="1"/><td align="center" valign="top" colspan="4" rowspan="1">Synchrony recipients&#x02013;donors (h)*<hr/></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">&#x02212;24</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">+ 24</td><td align="center" valign="top" rowspan="1" colspan="1">+ 48</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Recipients, N</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">31</td><td align="center" rowspan="1" colspan="1">74</td><td align="center" rowspan="1" colspan="1">18</td></tr><tr><td align="left" rowspan="1" colspan="1">Pregnancy rate, N (%)</td><td align="center" rowspan="1" colspan="1">1 (11.1)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">19 (61.3)<sup>b</sup></td><td align="center" rowspan="1" colspan="1">63 (85.1)<sup>c</sup></td><td align="center" rowspan="1" colspan="1">9 (50.0)<sup>ab</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Farrowing rate, N (%)</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">19 (61.3)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">60 (81.1)<sup>b</sup></td><td align="center" rowspan="1" colspan="1">9 (50.0)<sup>a</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Total born (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">10.5 &#x000b1; 0.8</td><td align="center" rowspan="1" colspan="1">9.6 &#x000b1; 0.4</td><td align="center" rowspan="1" colspan="1">9.2 &#x000b1; 1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">Born alive (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">10.1 &#x000b1; 0.8</td><td align="center" rowspan="1" colspan="1">8.9 &#x000b1; 0.4</td><td align="center" rowspan="1" colspan="1">8.6 &#x000b1; 0.9</td></tr><tr><td align="left" rowspan="1" colspan="1">Stillborn (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">0.4 &#x000b1; 0.2</td><td align="center" rowspan="1" colspan="1">0.7 &#x000b1; 0.1</td><td align="center" rowspan="1" colspan="1">0.6 &#x000b1; 0.3</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet birth weight (kg; mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">1.2 &#x000b1; 0.1</td><td align="center" rowspan="1" colspan="1">1.7 &#x000b1; 0.1</td><td align="center" rowspan="1" colspan="1">1.6 &#x000b1; 0.1</td></tr><tr><td align="left" rowspan="1" colspan="1">Sex ratio at birth (%; male/female)</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">47.7/52.3</td><td align="center" rowspan="1" colspan="1">49.0/51.0</td><td align="center" rowspan="1" colspan="1">46.4/53.6</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet production efficiency (%)</td><td align="center" rowspan="1" colspan="1">&#x02013;</td><td align="center" rowspan="1" colspan="1">20.6<sup>d</sup></td><td align="center" rowspan="1" colspan="1">24.0<sup>d</sup></td><td align="center" rowspan="1" colspan="1">14.3<sup>e</sup></td></tr></tbody></table><table-wrap-foot><p>*Recipients in estrus before (&#x02013;) or after (+) donors. <sup>a,b,c,d,e </sup>Different superscripts within the same row indicate differences: <sup>a,b,c </sup>P&#x0003c;0.05; <sup>d,e </sup>P&#x0003c;0.001.</p></table-wrap-foot></table-wrap>. The highest pregnancy and farrowing rates were achieved when estrus in recipients was 24 h later that in the donors (85.1% and 81.1%, respectively), regardless of the embryonic stage (day 5 morulae or day 6 blastocysts) used for the transfers. The pregnancy and farrowing rates decreased (P&#x0003c;0.05) when recipients were synchronous or &#x02013;24 h or +48 h asynchronous relative to the donors. Although no differences in litter sizes, piglet birth weights and sex ratio at birth were observed among the groups, the piglet production efficiency was higher (P&#x0003c;0.001) for the synchronous and +24 h groups.</p><p>The reproductive parameters of the recipients in estrus 0 h and 24 h later than the donors after NsDU transfers of day 5 morulae and day 6 blastocysts are shown in <xref rid="tbl_002" ref-type="table">Tables 2</xref> and<xref rid="tbl_003" ref-type="table"> 3</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title> Reproductive parameters of the synchronous recipients after nonsurgical deep-uterine transfer of 30 fresh porcine embryos</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" rowspan="2" colspan="1"/><td align="center" colspan="2" rowspan="1">Embryonic stage*<hr/></td></tr><tr><td align="center" rowspan="1" colspan="1">Morula</td><td align="center" rowspan="1" colspan="1">Blastocyst</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Recipients, N</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">14</td></tr><tr><td align="left" rowspan="1" colspan="1">Pregnancy rate, N (%)</td><td align="center" rowspan="1" colspan="1">10 (58.8)</td><td align="center" rowspan="1" colspan="1">9 (64.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">Farrowing rate, N (%)</td><td align="center" rowspan="1" colspan="1">10 (58.8)</td><td align="center" rowspan="1" colspan="1">9 (64.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">Total born (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">10.3 &#x000b1; 1.1</td><td align="center" rowspan="1" colspan="1">10.8 &#x000b1; 1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">Born alive (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">9.9 &#x000b1; 1.0</td><td align="center" rowspan="1" colspan="1">10.2 &#x000b1; 0.9</td></tr><tr><td align="left" rowspan="1" colspan="1">Stillborn (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">0.4 &#x000b1; 0.3</td><td align="center" rowspan="1" colspan="1">0.6 &#x000b1; 0.3</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet birth weight (kg; mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">1.2 &#x000b1; 0.2</td><td align="center" rowspan="1" colspan="1">1.2 &#x000b1; 0.2</td></tr><tr><td align="left" rowspan="1" colspan="1">Sex ratio at birth (%; male/female)</td><td align="center" rowspan="1" colspan="1">47.0/53.0</td><td align="center" rowspan="1" colspan="1">48.4/51.6</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet production efficiency (%)</td><td align="center" rowspan="1" colspan="1">19.4</td><td align="center" rowspan="1" colspan="1">21.9</td></tr></tbody></table><table-wrap-foot><p>*Embryos at the morula and unhatched blastocyst stages were collected from donors at days 5 and 6 (day 0=onset of estrus), respectively, and were transferred into the recipients on days 5 and 6 of the cycle, respectively.</p></table-wrap-foot></table-wrap><table-wrap id="tbl_003" orientation="portrait" position="float"><label>Table 3.</label><caption><title> Reproductive parameters of the recipients in estrus 24 h later than the donors after nonsurgical deep-uterine transfer of 30 fresh porcine embryos</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" valign="top" rowspan="2" colspan="1"/><td align="center" valign="top" colspan="2" rowspan="1">Embryonic stage*<hr/></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">Morula</td><td align="center" valign="top" rowspan="1" colspan="1">Blastocyst</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Recipients, N</td><td align="center" rowspan="1" colspan="1">35</td><td align="center" rowspan="1" colspan="1">39</td></tr><tr><td align="left" rowspan="1" colspan="1">Pregnancy rate, N (%)</td><td align="center" rowspan="1" colspan="1">29 (82.8)</td><td align="center" rowspan="1" colspan="1">34 (87.2)</td></tr><tr><td align="left" rowspan="1" colspan="1">Farrowing rate, N (%)</td><td align="center" rowspan="1" colspan="1">26 (74.3)</td><td align="center" rowspan="1" colspan="1">33 (84.6)</td></tr><tr><td align="left" rowspan="1" colspan="1">Total born (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">9.2 &#x000b1; 0.6</td><td align="center" rowspan="1" colspan="1">9.8 &#x000b1; 0.6</td></tr><tr><td align="left" rowspan="1" colspan="1">Born alive (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">8.6 &#x000b1; 0.6</td><td align="center" rowspan="1" colspan="1">9.1 &#x000b1; 0.5</td></tr><tr><td align="left" rowspan="1" colspan="1">Stillborn (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">0.6 &#x000b1; 0.2</td><td align="center" rowspan="1" colspan="1">0.7 &#x000b1; 0.2</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet birth weight (kg; mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">1.9 &#x000b1; 0.1</td><td align="center" rowspan="1" colspan="1">1.5 &#x000b1; 0.1</td></tr><tr><td align="left" rowspan="1" colspan="1">Sex ratio at birth (%; male/female)</td><td align="center" rowspan="1" colspan="1">48.1/51.9</td><td align="center" rowspan="1" colspan="1">49.5/50.5</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet production efficiency (%)</td><td align="center" rowspan="1" colspan="1">21.3<sup>a</sup></td><td align="center" rowspan="1" colspan="1">25.6<sup>b</sup></td></tr></tbody></table><table-wrap-foot><p>*Embryos at the morula and unhatched blastocyst stages were collected from donors at days 5 and 6 (day 0=onset of estrus), respectively, and were transferred into the recipients on days 4 and 5 of the cycle, respectively. <sup>a,b </sup>Different superscripts within the same row indicate differences (P&#x0003c;0.02).</p></table-wrap-foot></table-wrap>, respectively. In both cases, no differences were observed between the groups for any of the parameters evaluated with the exception of the piglet production efficiency, which was higher (P&#x0003c;0.02) for blastocysts in the + 24 h group. <xref rid="tbl_004" ref-type="table">Table 4</xref><table-wrap id="tbl_004" orientation="portrait" position="float"><label>Table 4.</label><caption><title> Reproductive parameters of the recipients in estrus 48 h later than the donors after nonsurgical deep-uterine transfer of 30 fresh porcine embryos</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" rowspan="2" colspan="1"/><td align="center" colspan="2" rowspan="1">Embryonic stage*<hr/></td></tr><tr><td align="center" rowspan="1" colspan="1">Morula</td><td align="center" rowspan="1" colspan="1">Blastocyst</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Recipients, N</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td align="left" rowspan="1" colspan="1">Pregnancy rate, N (%)</td><td align="center" rowspan="1" colspan="1">3 (30.0)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">7 (87.5)<sup>b</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Farrowing rate, N (%)</td><td align="center" rowspan="1" colspan="1">3 (30.0)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">7 (87.5)<sup>b</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Total born (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">7.3 &#x000b1; 2.3<sup>e</sup></td><td align="center" rowspan="1" colspan="1">10.4 &#x000b1; 0.7<sup>f</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Born alive (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">7.0 &#x000b1; 2.0<sup>e</sup></td><td align="center" rowspan="1" colspan="1">9.6 &#x000b1; 0.7<sup>f</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Stillborn (mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">0.3 &#x000b1; 0.3</td><td align="center" rowspan="1" colspan="1">0.8 &#x000b1; 0.5</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet birth weight (kg; mean &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">1.7 &#x000b1; 0.2</td><td align="center" rowspan="1" colspan="1">1.5 &#x000b1; 0.2</td></tr><tr><td align="left" rowspan="1" colspan="1">Sex ratio at birth (%; male/female)</td><td align="center" rowspan="1" colspan="1">52.4/47.6</td><td align="center" rowspan="1" colspan="1">43.7/56.3</td></tr><tr><td align="left" rowspan="1" colspan="1">Piglet production efficiency (%)</td><td align="center" rowspan="1" colspan="1">7.0<sup>c</sup></td><td align="center" rowspan="1" colspan="1">27.9<sup>d</sup></td></tr></tbody></table><table-wrap-foot><p>*Embryos at the morula and unhatched blastocyst stages were collected from donors at days 5 and 6 (day 0=onset of estrus), respectively, and were transferred into the recipients on days 3 and 4 of the cycle, respectively. <sup>a,b,c,d </sup>Different superscripts within the same row indicate differences: <sup>a,b </sup>P&#x0003c;0.05; <sup>c,d </sup>P&#x0003c;0.001. <sup>e,f </sup>Different superscripts within the same row indicate tendencies for differences (P=0.09).</p></table-wrap-foot></table-wrap> shows the reproductive performance after NsDU transfers of morulae and blastocysts in recipients with asynchrony of +48 h. The results indicate that such asynchrony was adequate for transfers performed with blastocysts but not for those with morulae. The farrowing rate (87.5%; P&#x0003c;0.05) and piglet production efficiency (27.9%; P&#x0003c;0.001) were increased in the blastocyst group compared with the morula group (30.0% and 7.0%, respectively). There were no differences between groups in the other parameters evaluated, although the litter size and number of piglets born alive tended (P=0.09) to be higher when the blastocyst stage was used.</p></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>This study confirmed previous reports on the effectiveness of the NsDU-ET technology and provides evidence for the first time that the degree of estrous synchrony between recipients and donors can markedly influence the success of NsDU-ET.</p><p>As previously reported in surgical ET studies in the pig [<xref rid="r11" ref-type="bibr">11</xref>], the pregnancy and farrowing rates dramatically decreased when the recipient started estrus 24 h (&#x02013;24 h) ahead of the donor. Studies in other species have demonstrated that transferring embryos to a more advanced uterus can result in accelerated growth by increasing their rate of cell division [<xref rid="r27" ref-type="bibr">27</xref>, <xref rid="r28" ref-type="bibr">28</xref>], which could fatally modify their subsequent development [<xref rid="r4" ref-type="bibr">4</xref>, <xref rid="r29" ref-type="bibr">29</xref>] and adversely affect the pregnancy outcomes.</p><p>On the other hand, our results clearly demonstrate that increased pregnancy and farrowing rates are achieved when NsDU-ETs are performed on recipients that started estrus 24 h (+24 h) after the donors compared with those obtained using synchronous or +48 h asynchronous recipients. These results partially confirm those achieved in surgical experiments, where increased pregnancy rates and embryonic survival were obtained with transfers performed on recipients in which the onset of estrus was 0, 1 or 2 days later than in donors [<xref rid="r11" ref-type="bibr">11</xref>]. The hypothesis that porcine embryos are tolerant of a less advanced uterine environment has also been supported by other studies using surgical transfers [<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r13" ref-type="bibr">13</xref>, <xref rid="r30" ref-type="bibr">30</xref>]. In contrast, Hazeleger <italic>et al</italic>. [<xref rid="r14" ref-type="bibr">14</xref>] reported that transfers to recipients ovulating 24 h before donors might be optimal for nonsurgical transfers into the uterine body, whereas transfers to recipients ovulating 24 h or more after the donors appear to result in significantly reduced pregnancy rates. Several factors might be accountable for such discrepancies. It has previously been speculated that after surgical transfer, embryos might benefit from younger uterine environments due to anesthesia or surgical trauma [<xref rid="r14" ref-type="bibr">14</xref>]. However, our results using NsDU-ETs mirror those achieved in the surgical experiments but not those obtained with the nonsurgical uterine body ETs, clearly indicating that the surgery is not associated with the tolerance of asynchrony of the embryos. The most plausible explanation for these discrepancies might be the location of embryo deposition, which is related to the ET technique used. In surgical ET and NsDU-ET procedures, the embryos are deposited into the tip or anterior quarter of a uterine horn, respectively [<xref rid="r11" ref-type="bibr">11</xref>, <xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r21" ref-type="bibr">21</xref>, <xref rid="r22" ref-type="bibr">22</xref>]; in contrast, in the nonsurgical ET technique used by Hazeleger&#x02019;s group, the embryos are placed within the uterine body [<xref rid="r14" ref-type="bibr">14</xref>]. Under natural conditions, morulae and blastocysts remain near the tip of the uterine horn until day 6 to 7 of the cycle, progressing subsequently toward the uterine body [<xref rid="r31" ref-type="bibr">31</xref>]. Thus, as it has been demonstrated in studies using surgical ETs [<xref rid="r32" ref-type="bibr">32</xref>], transfer of these embryos to anterior portions of the uterine horns of recipients confers some advantages compared with transfer within the uterine body, likely because the uterine environment in this region is unfavorable for embryos during these stages of development. Regional differences in the uterine environment might also explain the synchrony discrepancies among the abovementioned studies. More research is needed to corroborate this hypothesis.</p><p>The reasons underlying the enhanced tolerance that transferred embryos exhibit for less advanced uterine environments remain to be elucidated. Consistent with Blum-Reckow and Holtz [<xref rid="r13" ref-type="bibr">13</xref>], collection and handling of the embryos and/or short&#x02013;term storage <italic>in vitro</italic> prior to transfers might cause a transitory delay of embryonic development, as occurs under <italic>in vitro</italic> culture conditions [<xref rid="r33" ref-type="bibr">33</xref>, <xref rid="r34" ref-type="bibr">34</xref>], which might increase the chances for embryo survival in a less advanced uterus.</p><p>In our study, a recipient asynchrony of +24 h was satisfactory for NsDU-ETs performed with both morulae and blastocysts. However, interestingly, a recipient asynchrony of + 48 h was adequate for blastocysts but not for morulae. The most likely explanation for this observation is that transfers of day 5 morulae into +48 h asynchronous recipients involve the use of recipients on the third day of the cycle, which means that some of the recipients are still in estrus or shortly after estrus. At that point in the cycle, the hormonal events associated with corpora lutea formation and the endometrial secretory products in the uterine lumen might be inadequate for survival of the embryos at the morula stage. In this context, the concentration of serum progesterone in the recipient at the moment of transfer plays an important role in the success of porcine somatic cell nuclear transfer ET programs [<xref rid="r35" ref-type="bibr">35</xref>]. The importance of progesterone in promoting embryo survival after ET has also been demonstrated in other species [<xref rid="r36" ref-type="bibr">36</xref>, <xref rid="r37" ref-type="bibr">37</xref>].</p><p>Consistent with our previous studies [<xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r23" ref-type="bibr">23</xref>], NsDU-ET catheter insertion did not cause uterine infections in the form of vaginal discharge, reflecting the efficacy of the aseptic measures taken. Furthermore, it did not disturb the animal behavior or the reproductive tract of the recipients (data not shown). Together with the high number of transfers performed, these data indicate that the procedure is safe and well tolerated by the recipients.</p><p>A high proportion of donors (39.1%) had cysts in the ovaries with an average of 2.4 cysts per sow. The high incidence of cysts might be attributed to the superovulation treatment used in this experiment. However, in a previous study performed in the same farm using the same breed, we observed a similar frequency of ovarian cysts in superovulated and non-superovulated donors [<xref rid="r23" ref-type="bibr">23</xref>], indicating that the superovulation treatment was not associated with this problem. In the present study, these cysts were likely nonfunctional and did not interfere with the reproductive cycle as previously reported for single cysts [<xref rid="r38" ref-type="bibr">38</xref>]. The excellent reproductive history and quality of the embryos collected support this hypothesis. Although the origin of these cysts is unclear, they might be attributable to innate characteristics of the Duroc breed.</p><p>The high pregnancy and fertilization rates, high numbers of viable and transferable embryos and low numbers of oocytes and/or degenerated embryos obtained in this study indicate the effectiveness of the superovulation treatment used and confirm the results obtained in our previous study [<xref rid="r23" ref-type="bibr">23</xref>]. The ovulatory response variability found in the present study was high (CV=25.4%). This finding was not abnormal because such variability has been widely reported [<xref rid="r39" ref-type="bibr">39</xref>,<xref rid="r40" ref-type="bibr">40</xref>,<xref rid="r41" ref-type="bibr">41</xref>,<xref rid="r42" ref-type="bibr">42</xref>]. However, the variation observed in this study and our previous study [<xref rid="r23" ref-type="bibr">23</xref>] was low compared with those reported for cyclic gilts and sows, wherein the CVs were approximately 40% [<xref rid="r39" ref-type="bibr">39</xref>,<xref rid="r40" ref-type="bibr">40</xref>,<xref rid="r41" ref-type="bibr">41</xref>,<xref rid="r42" ref-type="bibr">42</xref>]. The different superovulation protocols and the different lines and breeds used among these studies, which can exhibit widely variable superovulatory responses [<xref rid="r21" ref-type="bibr">21</xref>], likely contributed to this discrepancy.</p><p>In conclusion, our results indicate that using NsDU transfers of day 5 morulae and day 6 blastocysts, the ideal recipient should start estrus 24 h after the donors. This asynchrony can be increased to +48 h for transfers performed with day 6 blastocysts. In contrast, the use of synchronous recipients or recipients with heat ahead (&#x02013;24 h) of the donors does not result in adequate pregnancy and farrowing rates. The excellent reproductive performance of the recipients following NsDU-ETs reported in this study represents an important advance for the widespread commercial use of ET by the pig industry.</p></sec>
Epigenetic Modification Agents Improve Genomic Methylation Reprogramming in Porcine Cloned Embryos
<p> Incomplete DNA methylation reprogramming in cloned embryos leads to low cloning efficiency. Our previous studies showed that the epigenetic modification agents 5-aza-2&#x02019;-deoxycytidine (5-aza-dC) or trichostatin A (TSA) could enhance the developmental competence of porcine cloned embryos. Here, we investigated genomic methylation dynamics and specific gene expression levels during early embryonic development in pigs. In this study, our results showed that there was a typical wave of DNA demethylation and remethylation of <italic>centromeric satellite repeat</italic> (<italic>CenRep</italic>) in fertilized embryos, whereas in cloned embryos, delayed demethylation and a lack of remethylation were observed. When cloned embryos were treated with 5-aza-dC or TSA, <italic>CenRep</italic> methylation reprogramming was improved, and this was similar to that detected in fertilized counterparts. Furthermore, we found that the epigenetic modification agents, especially TSA, effectively promoted silencing of tissue specific genes and transcription of early embryo development-related genes in porcine cloned embryos. In conclusion, our results showed that the epigenetic modification agent 5-aza-dC or TSA could improve genomic methylation reprogramming in porcine cloned embryos and regulate the appropriate expression levels of genes related to early embryonic development, thereby resulting in high developmental competence.</p>
<contrib contrib-type="author"><name><surname>HUAN</surname><given-names>Yan Jun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>ZHU</surname><given-names>Jiang</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>WANG</surname><given-names>Hong Mei</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>WU</surname><given-names>Zhan Feng</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>ZHANG</surname><given-names>Ji Guang</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>XIE</surname><given-names>Bing Teng</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>LI</surname><given-names>Jing Yu</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>KONG</surname><given-names>Qing Ran</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>LIU</surname><given-names>Zhong Hua</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>HE</surname><given-names>Hong Bin</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1)</label>Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, China</aff><aff id="aff2"><label>2)</label>College of Life Science, Northeast Agricultural University, Haerbin 150030, China</aff><aff id="aff3"><label>3)</label>College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Taian 271018, China</aff><aff id="aff4"><label>4)</label>Shouguang City Hospital of Chinese Medicine, Weifang 262700, China</aff>
The Journal of Reproduction and Development
<p>Though somatic cell nuclear transfer (SCNT) has been achieved in many species, overall cloning efficiency is still low, and this limits the applications of cloning technology in agriculture, medicine and basic research [<xref rid="r1" ref-type="bibr">1</xref>,<xref rid="r2" ref-type="bibr">2</xref>,<xref rid="r3" ref-type="bibr">3</xref>].</p><p>It is generally believed that low cloning efficiency is mainly due to incomplete epigenetic reprogramming [<xref rid="r4" ref-type="bibr">4</xref>, <xref rid="r5" ref-type="bibr">5</xref>]. To improve epigenetic reprogramming in cloned embryos, various strategies have been used, and epigenetic modification agents, such as 5-aza-dC, TSA, scriptaid and valproic acid, are usually applied and have enhanced the developmental competence of cloned embryos [<xref rid="r6" ref-type="bibr">6</xref>,<xref rid="r7" ref-type="bibr">7</xref>,<xref rid="r8" ref-type="bibr">8</xref>,<xref rid="r9" ref-type="bibr">9</xref>]. Our previous results also show that 5-aza-dC or TSA could improve cloning efficiency [<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r11" ref-type="bibr">11</xref>]. However, the mechanism underlying the developmental improvement of cloned embryos induced by epigenetic modification agents is still poorly understood.</p><p>As the most studied epigenetic modification, DNA methylation could reflect the epigenetic reprogramming degree in cloned embryos; therefore, the mechanism of epigenetic reprogramming induced by SCNT mainly focuses on DNA methylation [<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>]. Previous studies have shown that compared with that of <italic>in vivo</italic> or <italic>in vitro</italic> fertilized embryos, the genome of cloned embryos is usually highly methylated, leading to poor cloning efficiency [<xref rid="r13" ref-type="bibr">13</xref>, <xref rid="r15" ref-type="bibr">15</xref>]. Since epigenetic modification agents could improve the development of cloned embryos, it is thought that DNA methylation reprogramming must be improved in treated embryos. At present, some studies have shown that epigenetic modification agents could rescue the disrupted methylation of imprinting genes [<xref rid="r6" ref-type="bibr">6</xref>, <xref rid="r16" ref-type="bibr">16</xref>]. However, the effect of epigenetic modification agents on global methylation reprogramming during early embryonic development has been unclear.</p><p>Previous studies have shown that the <italic>centromeric satellite repeat</italic> (<italic>CenRep</italic>) methylation level could represent the genomic methylation status [<xref rid="r17" ref-type="bibr">17</xref>]; thus, <italic>CenRep</italic> was selected to test genomic methylation reprogramming during early embryonic development. In this study, we first treated porcine cloned embryos with 5-aza-dC or TSA to enhance their development, then investigated genomic methylation dynamics during early embryonic development and finally tested the transcripts of DNA methyltransferase, tissue specificity, pluripotency, <italic>zygotic genome activation</italic> and blastocyst quality-related genes in embryos.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><p>Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), and disposable and sterile plasticware was obtained from Nunclon (Roskilde, Denmark), unless otherwise stated. All experiments were approved by the Animal Care Commission of Shandong Academy of Agricultural Sciences according to animal welfare laws, guidelines and policies.</p><sec><title>Porcine fetal fibroblast cell (PFF) culture</title><p>PFF culture has been described previously [<xref rid="r11" ref-type="bibr">11</xref>]. Briefly, PFFs were isolated from a 35-day-old fetus. After removal of the head, internal organs and limbs, the remaining tissues were finely minced into pieces, digested with 0.25% trypsin-0.04% ethylenediaminetetraacetic acid solution (GIBCO) and then dispersed in high glucose enriched Dulbecco&#x02019;s modified Eagle&#x02019;s medium (DMEM; GIBCO) containing 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin-streptomycin (GIBCO). The dispersed cells were centrifuged, resuspended and cultured in DMEM. Until confluence, PFFs were digested, centrifuged, resuspended in FBS containing 10% dimethyl sulfoxide, and they were then stored in liquid nitrogen until use. Prior to SCNT, PFFs were thawed and cultured, and they were subsequently used in 3 to 5 passages.</p></sec><sec><title>Oocyte collection and in vitro maturation (IVM)</title><p>Oocyte maturation has been described previously [<xref rid="r11" ref-type="bibr">11</xref>]. Briefly, porcine ovaries were collected from a local slaughterhouse and transported to the laboratory. Follicles were aspirated, and follicular contents were washed with HEPES-buffered Tyrode&#x02019;s lactate. Cumulus-oocyte complexes (COCs) with at least three uniform layers of compact cumulus cells and a uniform cytoplasm were recovered, washed and cultured in maturation medium under mineral oil at 38.5 C in a 5% CO<sub>2</sub> atmosphere and saturated humidity. After 42 h, COCs were vortexed in 1 mg/ml hyaluronidase to remove cumulus cells. Only oocytes with a visible polar body, regular morphology and a homogenous cytoplasm were used.</p></sec><sec><title>IVF and SCNT embryo culture, treatment and collection</title><p>The procedures for porcine IVF and SCNT have been described in one of our previous reports [<xref rid="r18" ref-type="bibr">18</xref>]. Briefly, for IVF, the semen was incubated, resuspended and washed in DPBS supplemented with 0.1% (w/v) BSA. The spermatozoa were diluted with modified Tris-buffered medium (mTBM) to the appropriate concentration. Matured oocytes were washed in mTBM, transferred into fertilization medium and co-incubated with spermatozoa. Then the embryos were washed and cultured in porcine zygote medium-3 (PZM-3) for subsequent development. For SCNT, matured oocytes and PFFs were placed in manipulation medium. After enucleation, donor cells were placed into the perivitelline space. Fusion and activation of the cell-cytoplast complexes were induced by electroporation, and the fusion rate was confirmed by microscopic examination. Then reconstructed embryos were cultured in PZM-3 for subsequent development. The cleavage and blastocyst rates of IVF and SCNT embryos were evaluated at 48 h and 156 h, respectively.</p><p>For 5-aza-dC or TSA treatment [<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r11" ref-type="bibr">11</xref>], cloned embryos were cultured in PZM-3 supplemented with 25 nM (optimized) 5-aza-dC (NT-AZA) or 40 nM (optimized) TSA (NT-TSA) for 24 h, washed and then transferred into PZM-3 for further culture.</p><p>For embryo collection, the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stage embryos in the IVF, NT-CON (cloned), NT-AZA and NT-TSA groups were collected at 6 h, 24 h, 48 h, 72 h and 156 h, respectively.</p></sec><sec><title>Bisulfite sequencing</title><p>Bisulfite sequencing has been reported [<xref rid="r18" ref-type="bibr">18</xref>]. Briefly, pooled samples were digested with Proteinase K and then treated with sodium bisulfite to convert all unmethylated cytosine to uracil using an EZ DNA Methylation-Gold<sup>TM</sup> Kit (Zymo Research) according to the manufacturer&#x02019;s protocol. For semen, the sperm was collected by centrifugation, washed in SMB solution (10 mM Tris-HCl, 10 mM EDTA, 50 mM NaCl and 2% SDS, pH 7.2) and then incubated in SMB solution supplemented with 40 mM dithiothreitol and 0.3 mg/ml Proteinase K at 56 C for 1 h. For samples of 10<sup>3</sup> PFFs, 200 MII oocytes and 150, 80, 30, 20 and 10 pooled zona pellucida-removed embryos at the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages, respectively, digestion was performed in M-Digestion Buffer supplemented with Proteinase K at 50 C for 20 min. After digestion, a CT (cytosine to thymine) conversion reagent was added to purified genomic DNA at 98 C for 10 min and 64 C for 2.5 h. Then the samples were desalted, purified and diluted with M-Elution Buffer. Subsequently, PCR was carried out to amplify <italic>CenRep</italic> (Z75640) using the reported primers [<xref rid="r17" ref-type="bibr">17</xref>] and Hot Start Taq<sup>TM</sup> Polymerase (TaKaRa) with a profile of 94 C for 5 min and 45 cycles of 94 C for 30 sec, 55 C for 30 sec and 72 C for 1 min, followed by 72 C for 10 min. Then the amplified products were verified by electrophoresis and purified using an Agarose Gel DNA Purification Kit (TaKaRa). The purified fragments were cloned into pMD18-T Vector (TaKaRa) and subjected to sequence analysis.</p></sec><sec><title>Quantitative real-time PCR</title><p>Measurement of gene expression with quantitative real-time PCR has been applied in our previous studies [<xref rid="r11" ref-type="bibr">11</xref>, <xref rid="r18" ref-type="bibr">18</xref>]. Briefly, total RNA was extracted from 30 pooled embryos at each stage using an RNeasy Mini Kit (Qiagen) according to the manufacturer&#x02019;s instructions. Reverse transcription was performed using a PrimeScript<sup>&#x000ae;</sup> RT Reagent Kit (TaKaRa) with the following parameters: 37 C for 15 min and 85 C for 5 sec, and the cDNA was stored at &#x02013;20 C until use. For quantitative real-time PCR, reactions were performed in 96-well optical reaction plates (Applied Biosystems) using SYBR<sup>&#x000ae;</sup> Premix ExTaq<sup>TM</sup> II (TaKaRa) and a 7500 Real-Time PCR System (Applied Biosystems) with the following conditions: 95 C for 30 sec and 40 two-step cycles of 95 C for 5 sec and 60 C for 34 sec, followed by a dissociation stage consisting of 95 C for 15 sec, 60 C for 1 min and 95 C for 15 sec. For every sample, the cycle threshold (CT) values were obtained from three replicates. The primers used for amplification of target and internal reference genes are presented in <xref ref-type="supplementary-material" rid="pdf_001">Supplementary Table 1</xref> (on-line only). The relative expression levels of target genes were analyzed using the 2<sup>&#x02212;&#x00394;&#x00394;CT</sup> method.</p></sec><sec><title>Statistical analysis</title><p>Differences in data (mean &#x000b1; SEM) were analyzed with the SPSS statistical software. Statistical analysis of data concerning genomic methylation and gene expression levels was performed with one-way analysis of variance (ANOVA). For all analyses, differences were considered to be statistically significant when P&#x0003c;0.05.</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>Delayed and incomplete genomic methylation reprogramming in porcine cloned embryos</title><p>The <italic>CenRep</italic> methylation statuses of sperm and MII oocytes were examined, and a significant difference was observed (P&#x0003c;0.05), with sperm showing 64.58% methylation and MII oocytes showing 28.87% methylation (<xref ref-type="supplementary-material" rid="pdf_002">Suppl Fig. 1</xref>: on-line only). After fertilization, genomic demethylation was not observed in the 1-cell stage embryos in comparison with the mean methylation of sperm and oocytes (<xref ref-type="fig" rid="fig_001">Fig. 1</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> CenRep methylation status. A: CenRep methylation status at the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON, NT-AZA and NT-TSA embryos; B: dynamic CenRep methylation profiles in the IVF, NT-CON, NT-AZA and NT-TSA groups, respectively; C: CenRep methylation status at the 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON, NT-AZA and NT-TSA embryos. Black and white circles indicate methylated and unmethylated CpG sites, respectively, and gray circles represent mutated and/or single nucleotide polymorphism (SNP) variation at certain CpG sites. The data are expressed as means &#x000b1; SEM. <sup>a&#x02013;c</sup> Values for a given group in columns with different superscripts differ significantly (P &#x0003c; 0.05).</p></caption><graphic xlink:href="jrd-60-377-g001"/></fig> and <xref ref-type="supplementary-material" rid="pdf_002">Supplementary Fig. 1</xref>). In the cleavage stage embryos, <italic>CenRep</italic> methylation displayed a continuous decrease from the 1-cell to 8-cell stage, and significant differences were observed between the 1-cell and 2-cell stages or the 4-cell and 8-cell stages (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>, P&#x0003c;0.05). In blastocysts, the <italic>CenRep</italic> methylation level was higher than that in the 8-cell stage (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>), indicating that genomic remethylation occurred in blastocysts. Over all, IVF embryos displayed a typical wave of genomic demethylation and remethylation.</p><p>The <italic>CenRep</italic> methylation level in PFFs was 52.28%, and after SCNT, no significant differences were found between PFFs and the 1-cell stage embryos (<xref ref-type="fig" rid="fig_001">Fig. 1</xref> and <xref ref-type="supplementary-material" rid="pdf_002">Supplementary Fig. 1</xref>). In cloned embryos, gradual demethylation from the 1-cell stage to blastocyst stage was observed, suggesting that remethylation did not take place in the blastocyst stage (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>). When comparing the methylation levels within individual developmental stages between cloned and IVF embryos, the levels in the 4-cell and 8-cell stage cloned embryos were significantly higher than those in their fertilized counterparts (P&#x0003c;0.05). These results strongly suggested that genomic methylation reprogramming in cloned embryos was delayed and incomplete.</p></sec><sec><title>Epigenetic modification agents improved genomic methylation reprogramming in porcine cloned embryos</title><p>The epigenetic modification agents 5-aza-dC or TSA could significantly enhance the developmental competence of porcine cloned embryos (<xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Development of cloned embryos treated with 5-aza-dC or TSA</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" rowspan="1" colspan="1">Groups</td><td align="center" rowspan="1" colspan="1">No. embryos<break/>(Rep.)</td><td align="center" rowspan="1" colspan="1">No. embryos cleaved<break/>(% &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">No. blastocysts<break/>(% &#x000b1; SEM)</td><td align="center" rowspan="1" colspan="1">Blastocyst cell numbers<break/>(mean &#x000b1; SEM) <sup>&#x00026;</sup></td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">NT-CON</td><td align="center" rowspan="1" colspan="1">242 (5)</td><td align="center" rowspan="1" colspan="1">209 (85.79 &#x000b1; 0.95)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">50 (20.50 &#x000b1; 0.70)<sup>a</sup></td><td align="center" rowspan="1" colspan="1">37 &#x000b1; 3 (n=49)</td></tr><tr><td align="left" rowspan="1" colspan="1">NT-AZA</td><td align="center" rowspan="1" colspan="1">247 (5)</td><td align="center" rowspan="1" colspan="1">223 (89.88 &#x000b1; 1.14)<sup>b</sup></td><td align="center" rowspan="1" colspan="1">67 (27.30 &#x000b1; 1.24)<sup>b</sup></td><td align="center" rowspan="1" colspan="1">37 &#x000b1; 2 (n=55)</td></tr><tr><td align="left" rowspan="1" colspan="1">NT-TSA</td><td align="center" rowspan="1" colspan="1">238 (5)</td><td align="center" rowspan="1" colspan="1">210 (88.82 &#x000b1; 1.12)<sup>ab</sup></td><td align="center" rowspan="1" colspan="1">118 (50.71 &#x000b1; 2.21)<sup>c</sup></td><td align="center" rowspan="1" colspan="1">38 &#x000b1; 2 (n=76)</td></tr></tbody></table><table-wrap-foot><p><sup>&#x00026;</sup> Blastocyst cell numbers of less than 16 or blastocysts used for molecular analysis (10 or 40 blastocysts in the NT-AZA or NT-TSA group, respectively) were not included. <sup>a&#x02013;c</sup> Values in the same column with different superscripts differ significantly (P &#x0003c; 0.05).</p></table-wrap-foot></table-wrap> and <xref ref-type="supplementary-material" rid="pdf_002">Supplementary Fig. 2</xref>: on-line only, P&#x0003c;0.05). Here, the alterations of genomic methylation levels in these treated cloned embryos were investigated. In the NT-AZA group, the 1-cell stage embryos did not significantly differ from PFFs, the 1-cell to 8-cell stage embryos underwent DNA demethylation in a gradual fashion, with the level of methylation in the 2-cell stage in particular significantly lower than that in the 1-cell stage (P&#x0003c;0.05), and blastocysts displayed remethylation in comparison with 8-cell stage embryos, which was similar to the pattern in the IVF group (<xref ref-type="fig" rid="fig_001">Fig. 1</xref> and <xref ref-type="supplementary-material" rid="pdf_002">Supplementary Fig. 1</xref>). When comparing the methylation status within individual developmental stages between the NT-AZA and NT-CON or IVF groups, genomic demethylation was shifted earlier in 2-cell stage, 4-cell and 8-cell stage embryos in the NT-AZA group, with the levels of methylation being significantly lower than those in the NT-CON group (P&#x0003c;0.05), and no significant differences were observed between the NT-AZA and IVF group during early embryonic development. These results indicated that 5-aza-dC could improve genomic methylation reprogramming in cloned embryos.</p><p>In the NT-TSA group, similar DNA methylation dynamics to the NT-AZA or IVF group were observed (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>). When comparing the differences between the NT-TSA and NT-AZA groups, the NT-TSA group showed a methylation pattern that was much more similar to that of the IVF group, as the NT-AZA group underwent faster genomic demethylation and slower genomic remethylation, though no significant differences were observed. Overall, our results showed that the epigenetic modification agents 5-aza-dC and TSA rescued the disrupted genomic methylation reprogramming in cloned embryos.</p></sec><sec><title>Epigenetic modification agents improved the expression levels of genes related to early embryo development</title><p>To further explore the mechanism underlying the improved development of cloned embryos treated with 5-aza-dC or TSA, the transcript levels of early embryonic development-related genes in the <italic>zygotic genome activation (ZGA) and</italic> blastocyst stages were investigated (<xref ref-type="fig" rid="fig_002">Fig. 2</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Transcript levels of early embryo development-related genes at the zygotic genome activation and blastocyst stages of IVF, NT-CON, NT-AZA and NT-TSA embryos. The transcript abundance for each gene (Thy1 and Col5a2 in cloned embryos) in IVF embryos was considered the control. The data are expressed as means &#x000b1; SEM. <sup>a&#x02013;c</sup> Values for a given gene at a certain stage in columns with different superscripts differ significantly (P &#x0003c; 0.05).</p></caption><graphic xlink:href="jrd-60-377-g002"/></fig>). Compared with the IVF group, the NT-CON group displayed a significantly higher transcript level of <italic>Dnmt1</italic> and lower expression levels of <italic>Oct4</italic> and <italic>Eif1a</italic> at the ZGA stage (P&#x0003c;0.05), and significantly lower transcripts of <italic>Dnmt3a</italic>, <italic>Oct4</italic>, <italic>Nanog</italic>, <italic>Sox2</italic> and <italic>Cdx2</italic> at the blastocyst stage (P&#x0003c;0.05), suggesting that <italic>zygotic genes were not</italic> effectively activated and that the blastocyst quality was poor in cloned embryos.</p><p>In the NT-AZA group, the mRNA expression levels of early embryonic development-related genes was improved in comparison to those in the NT-CON group, showing significantly higher transcripts of <italic>Eif1a</italic>, <italic>Nanog</italic> and <italic>Sox2</italic> and lower expression of <italic>Dnmt1</italic> and <italic>Col5a2</italic> at the ZGA stage (P&#x0003c;0.05), and significantly higher mRNA expression of <italic>Nanog</italic>, <italic>Sox2</italic>, <italic>ATP1b1</italic> and <italic>Cdx2</italic> at the blastocyst stage (P&#x0003c;0.05). When compared with the IVF group, the NT-AZA group displayed no significant alterations of <italic>Eif1a</italic> and <italic>Cdx2</italic>, suggesting that 5-aza-dC could improve <italic>ZGA</italic> and blastocyst quality, though significantly lower expression levels of <italic>Oct4</italic> at the ZGA stage and <italic>Dnmt3a</italic> and <italic>Oct4</italic> at the blastocyst stage were still observed (P&#x0003c;0.05).</p><p>In the NT-TSA group, significant downregulation of <italic>Dnmt1</italic> and tissue-specific gene transcripts at the ZGA stage and upregulation of <italic>Nanog</italic> and <italic>Sox2</italic> expression levels at the ZGA stage and DNA methyltransferase, pluripotency and blastocyst quality-related gene transcripts at the blastocyst stage were observed in comparison with the NT-CON group (P&#x0003c;0.05). When compared with 5-aza-dC, TSA was more effective for gene expression regulation, showing significant silencing of the tissue-specific genes at the ZGA stage and activation of <italic>Dnmt3a</italic>, pluripotency and blastocyst quality-related genes at the blastocyst stage (P&#x0003c;0.05). Furthermore, in comparison to those in the IVF group, significant upregulation of pluripotency and blastocyst quality-related gene transcripts was observed in the NT-TSA group (P&#x0003c;0.05), though the <italic>Dnmt3a</italic> transcript level at the blastocyst stage was still significantly lower (P&#x0003c;0.05). Thus, these above results showed that treating cloned embryos with 5-aza-dC or TSA improved the transcription levels of genes related to early embryonic development.</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>Our study showed that treating porcine cloned embryos with 5-aza-dC or TSA could enhance genomic methylation reprogramming and regulate the appropriate transcript levels of early embryonic development-related genes in cloned embryos, thereby resulting in improvement of the development of porcine cloned embryos.</p><p>It is generally believed that epigenetic modification agents could improve nuclear reprogramming, and previous studies have shown that 5-aza-dC or TSA could enhance the developmental competence of cloned embryos [<xref rid="r6" ref-type="bibr">6</xref>, <xref rid="r16" ref-type="bibr">16</xref>, <xref rid="r19" ref-type="bibr">19</xref>]. However, the mechanism underlying the improvement of development is poorly studied. In this study, we investigated genomic methylation reprogramming in cloned embryos. In comparison with IVF embryos, cloned embryos took on a process of delayed demethylation without remethylation, suggesting that incomplete methylation reprogramming may be one cause of the developmental block or lethality of cloned embryos [<xref rid="r13" ref-type="bibr">13</xref>]. As for the reason for incomplete methylation reprogramming in cloned embryos, it is possible that there is a mechanism that causes the donor cell nucleus to preserve its methylation pattern, making oocyte-specific factors incompletely reprogram its nucleus [<xref rid="r20" ref-type="bibr">20</xref>]. When 5-aza-dC or TSA were applied, genomic methylation reprogramming was improved and was similar to that in IVF counterparts, showing a typical pattern of demethylation and remethylation. These findings may provide explanations for the observations that 5-aza-dC or TSA enhanced the developmental competence of cloned embryos. As for the improvement of genomic methylation reprogramming, it is possible that 5-aza-dC was incorporated into the genome and that TSA modified the chromatin structure [<xref rid="r4" ref-type="bibr">4</xref>]. Of course, other mechanisms also exist [<xref rid="r4" ref-type="bibr">4</xref>, <xref rid="r11" ref-type="bibr">11</xref>]. In regard to the differences in genomic methylation reprogramming between the NT-AZA and NT-TSA group, one possible explanation is that the manners of genomic methylation regulation induced by 5-aza-dC or TSA are different, and regulation of histone modification possibly fits better with genomic demethylation and remethylation in cloned embryos [<xref rid="r6" ref-type="bibr">6</xref>, <xref rid="r21" ref-type="bibr">21</xref>]. The results concerning embryonic development and the numbers of born and live piglets per surrogate (data not shown) could confirm this explanation. As to how genomic methylation is processed to achieve unimpaired reprogramming in the NT-AZA or NT-TSA group, more information is needed to clarify the mechanism.</p><p>In view of the genomic methylation dynamics during early embryonic development, our results also suggest that the partially progressive demethylation possibly results from replication-related passive demethylation and that active demethylation may not occur, even though traditional bisulfite sequencing could not distinguish between 5-methylcytosine and 5-hydroxymethylcytosine [<xref rid="r17" ref-type="bibr">17</xref>, <xref rid="r22" ref-type="bibr">22</xref>]. Due to the important role of 5-hydroxymethylcytosine in somatic nuclear reprogramming [<xref rid="r23" ref-type="bibr">23</xref>], new technologies such as oxidative bisulfite sequencing will be applied to clarify the genomic demethylation mechanism during embryonic development.</p><p>DNA methylation reprogramming is thought to be possibly associated with gene transcription regulation [<xref rid="r14" ref-type="bibr">14</xref>, <xref rid="r24" ref-type="bibr">24</xref>]. Our study showed that the transcription level of <italic>Dnmt1</italic> at the ZGA stage in the NT-CON group was significantly higher than that in the IVF group, while the <italic>Dnmt3a</italic> transcript level was significantly downregulated at the blastocyst stage, possibly explaining the cause of failure of DNA demethylation and remethylation in the NT-CON group [<xref rid="r13" ref-type="bibr">13</xref>]. Incomplete genomic methylation reprogramming would lead to the disturbed expression levels of genes related to early embryonic development, showing continuous expression of tissue-specific genes, no effective activation of pluripotent genes and downregulation of blastocyst quality-related gene expression in cloned embryos, thereby resulting in low cloning efficiency [<xref rid="r25" ref-type="bibr">25</xref>, <xref rid="r26" ref-type="bibr">26</xref>]. When cloned embryos were treated with 5-aza-dC or TSA, the expression levels of <italic>Dnmt1</italic> and <italic>Dnmt3a</italic> in the treatment groups, especially the NT-TSA group, were improved and were much closer to those in the IVF group (<xref ref-type="supplementary-material" rid="pdf_002">Supplementary Fig. 3</xref>: on-line only), suggesting that DNA methylation reprogramming in the NT-AZA and NT-TSA groups would be facilitated [<xref rid="r27" ref-type="bibr">27</xref>], and our results showed that genomic methylation reprogramming was improved in the NT-AZA and NT-TSA groups. The results of gene transcription showed that the gene expression patterns in the NT-AZA and NT-TSA groups were also appropriate, which was strongly consistent with the improved genomic methylation reprogramming. Previous studies reported that appropriate transcription of these early embryonic development-related genes is essential for cloned embryo development [<xref rid="r28" ref-type="bibr">28</xref>]. Thus, we speculate that the improvement of developmental competence of cloned embryos is probably due to the rescued genomic methylation reprogramming enhancing the restoration of the expression levels of early embryonic development-related genes. Certainly, not all the gene transcription levels were consistent with the overall DNA methylation status during embryonic development, as each gene has its own specific methylation pattern. At present, these gene methylation patterns in cloned embryos have not been well elucidated, and they are very worthy of investigation.</p><p>In conclusion, our results showed that treating porcine cloned embryos with 5-aza-dC or TSA improved genomic methylation reprogramming and regulated the appropriate transcripts of genes related to early embryonic development, thereby resulting in improvement of the development of porcine cloned embryos.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="pdf_001"><caption><title>Supplement Table</title></caption><media mimetype="application" mime-subtype="pdf" xlink:href="jrd-60-377-s001.pdf" orientation="portrait" xlink:type="simple" id="d35e777" position="anchor"/></supplementary-material><supplementary-material content-type="local-data" id="pdf_002"><caption><title>Supplement Figure</title></caption><media mimetype="application" mime-subtype="pdf" xlink:href="jrd-60-377-s002.pdf" orientation="portrait" xlink:type="simple" id="d35e782" position="anchor"/></supplementary-material></sec>
Histone H4 Modification During Mouse Spermatogenesis
<p> The core histone is composed of four proteins (H2A, H2B, H3 and H4). Investigation of the modification patterns of histones is critical to understanding their roles in biological processes. Although histone modification is observed in multiple cells and tissues, little is known about its function in spermatogenesis. We focused on the modification patterns of histone H4 during murine spermatogenesis. We demonstrated that the individual N-terminal sites of H4 show different modification patterns during the differentiation of male germ cells. The methylation pattern varied depending on the residues that were mono-, di-, or tri-methylated. All the H4 modifications were high during the meiotic prophase, suggesting that histone H4 modification plays an important role during this stage of spermatogenesis. Elongating spermatids showed increased acetylation of histone H4, which may be associated with a histone-to-protamine substitution. Our results provide further insight into the specific relationship between histone H4 modification and gene expression during spermatogenesis, which could help to elucidate the epigenetic disorders underlying male infertility.</p>
<contrib contrib-type="author"><name><surname>SHIRAKATA</surname><given-names>Yoshiki</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>HIRADATE</surname><given-names>Yuuki</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>INOUE</surname><given-names>Hiroki</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>SATO</surname><given-names>Eimei</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>TANEMURA</surname><given-names>Kentaro</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><aff id="aff1"><label>1)</label>Laboratory of Animal Reproduction and Development, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan</aff><aff id="aff2"><label>2)</label>National Livestock Breeding Center, Fukushima 961-8511, Japan</aff>
The Journal of Reproduction and Development
<p>Epigenetics is the study of mitotically or meiotically heritable changes in gene expression or cellular phenotype that are caused by mechanisms other than changes in the underlying DNA sequence [<xref rid="r1" ref-type="bibr">1</xref>]. As an epigenetic mechanism, histone modification is as important as DNA methylation and plays essential roles in gene inactivation and activation. The core histone is composed of four proteins (H2A, H2B, H3 and H4), and their N-terminal ends can be chemically modified by methylation, acetylation, and phosphorylation [<xref rid="r2" ref-type="bibr">2</xref>]. These modifications change the chromatin structure, thereby influencing gene expression [<xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r4" ref-type="bibr">4</xref>,<xref rid="r5" ref-type="bibr">5</xref>]. Histone methylation is associated with transcriptional activation and inactivation, depending on the histone N-terminal residues involved [<xref rid="r6" ref-type="bibr">6</xref>]. Histone acetylation is associated with the activation of gene expression [<xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r8" ref-type="bibr">8</xref>], while histone phosphorylation is related to transcriptional activation. Histone modification is a reversible process catalyzed by enzymes such as methyltransferases, demethylases, acetyltransferases and deacetyltransferases [<xref rid="r9" ref-type="bibr">9</xref>, <xref rid="r10" ref-type="bibr">10</xref>].</p><p>During the process of female germ cell differentiation, histone modification patterns undergo dramatic changes [<xref rid="r11" ref-type="bibr">11</xref>,<xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r15" ref-type="bibr">15</xref>,<xref rid="r16" ref-type="bibr">16</xref>] that play an important role in oocyte maturation and oogenesis. In comparison, the effect of histone modification on the differentiation of male germ cells remains understudied.</p><p>Spermatogenesis is a very unique cell differentiation process, in that it consists of gene recombination, meiosis and the exchange from histones to protamines. Histone modification patterns during spermatogenesis have been reported to perform specific roles [<xref rid="r17" ref-type="bibr">17</xref>, <xref rid="r18" ref-type="bibr">18</xref>]. Although the role of histone H3 modifications during spermatogenesis has been examined [<xref rid="r19" ref-type="bibr">19</xref>], the functions of the other histone proteins are unknown. Therefore, our research focused on the histone H4 protein. Histone H4 can be acetylated at lysine 5, 8, 12 and 16 and methylated at arginine 3 and lysine 20 on its N-terminal tail. Histone H4 modifications are involved in the regulation of chromatin structure, protein-protein interactions and transcriptional activity through the nuclear hormone receptor [<xref rid="r20" ref-type="bibr">20</xref>,<xref rid="r21" ref-type="bibr">21</xref>,<xref rid="r22" ref-type="bibr">22</xref>,<xref rid="r23" ref-type="bibr">23</xref>]. In this study, we used immunohistochemical techniques to analyze the modification of histone H4 during spermatogenesis.</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Animals</title><p>We purchased 12-week-old male C57/BL6 mice from SLC (Shizuoka, Japan). The mice were anesthetized with 2,2,2-tribromoethanol. Their testes were surgically removed, fixed with the methacarn fixative (methanol:chloroform:acetic acid = 6:3:1), treated with 100% ethanol and xylene, and then embedded in paraffin. The care and use of all the mice conformed to the Regulations for Animal Experiments and Related Activities at Tohoku University.</p></sec><sec><title>Antibodies</title><p>Mouse monoclonal antibodies against histone H4 mono-methylation (H4me, sc-134221; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and histone H4 tri-methylation (H4me3, sc-134216; Santa Cruz Biotechnology) and rabbit polyclonal antibodies against histone H4 lysine 5 acetylation (H4K5ac, sc-34264; Santa Cruz Biotechnology), histone H4 lysine 8 acetylation (H4K8ac, sc-8661-R; Santa Cruz Biotechnology), histone H4 lysine 12 acetylation (H4K12ac, sc-8661-R; Santa Cruz Biotechnology) and histone H4 lysine 20 di-methylation (H4K20me2, #9759S; Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies. Alexa Fluor 488-labeled anti-mouse secondary antibodies (Invitrogen, Life Technologies, Carlsbad, CA, USA) were used against H4me and H4me3. Alexa Fluor 594-labeled donkey anti-rabbit secondary antibodies (Invitrogen) were used against H4K5ac, H4K8ac, H4K12ac and H4K20me2.</p></sec><sec><title>Immunohistochemistry</title><p>Paraffin-embedded sections (10 &#x000b5;m) were mounted on glass slides. The sections were deparaffinized with xylene, dehydrated with ethanol and subsequently incubated with HistoVT One (Nacalai Tesque, Kyoto, Japan) at 90 C for 30 min to mediate antigen retrieval. The sections were then washed with distilled water, incubated with Blocking One (Nacalai Tesque) at 4 C for 1 h and subsequently incubated with primary antibodies at 4 C overnight [the primary antibodies were added to Blocking One (Nacalai Tesque) and phosphate-buffered saline-mixed liquor (diluted 1:200)]. After incubation, the sections were incubated with Alexa Fluor 488-labeled anti-mouse and Alexa Fluor 594-labeled anti-rabbit secondary antibodies (diluted 1:1000) at 4 C for 3 h. The nuclei were counterstained with Hoechst 33342 (diluted 1:5000; Molecular Probes, Eugene, OR, USA). The stained images were obtained using an LSM-700 confocal laser microscope (Carl Zeiss; Oberkochen, Germany), and the fluorescent brightness was analyzed with the ZEN2010 software in conjunction with the LSM-700 microscope. The stage of each seminiferous tubule was determined following the criteria described previously [<xref rid="r24" ref-type="bibr">24</xref>].</p></sec><sec><title>Comparison of brightness of fluorescence</title><p>We used the ZEN-2010 software in conjunction with the LSM-700 microscope and analyzed the mean of fluorescence intensity in the sperm cells. Fluorescence brightness was classified into 255 levels. We categorized the intensity levels over 200 as &#x0201c;strong,&#x0201d; those between 100 and 200 as &#x0201c;moderate,&#x0201d; those below 100 as &#x0201c;weak&#x0201d; and those that were extra low as &#x0201c;negative.&#x0201d;</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>Histone H4 lysine 5, 8 and 12 acetylation (H4K5ac, H4K8ac and H4K12ac)</title><p>H4K5ac, H4K8ac and H4K12ac showed dynamic changes during the differentiation of male germ cells (<xref ref-type="fig" rid="fig_001">Figs. 1</xref> and<xref ref-type="fig" rid="fig_002"> 2</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> Immunohistochemical analysis of H4K5ac and H4K8ac. The signals represent nuclear (A, A&#x02019;, D, D&#x02019;, G, G&#x02019;, J, J&#x02019;, M, M&#x02019;) histone H4 lysine 5 acetylation (H4K5ac) (B, E, H, K, N) and histone H4 lysine 8 acetylation (H4K8ac) (B&#x02019;, E&#x02019;, H&#x02019;, K&#x02019;, N&#x02019;). Stage VIII seminiferous tubules are shown (A&#x02013; C, A&#x02019;&#x02013;C&#x02019;) (G&#x02013;I, G&#x02019;&#x02013;I&#x02019;). Stage I&#x02013;V (D&#x02013;F, D&#x02019;&#x02013;F&#x02019;), stage X (J&#x02013;L, J&#x02019;&#x02013;L&#x02019;) and stage XII (M&#x02013;O, M&#x02019;&#x02013;O&#x02019;) seminiferous tubules are shown. The scale bars represent 50 &#x000b5;m (A, A&#x02019;, B, B&#x02019;, C, C&#x02019;) and 10 &#x000b5;m (F, F&#x02019;, I, I&#x02019;, L, L&#x02019;, O, O&#x02019;). P-SPC, pachytene spermatocyte; PL-SPC, preleptotene spermatocyte; L-SPC, leptotene spermatocyte; Z-SPC, zygotene spermatocyte; R-SPD, round spermatid; and E-SPD, elongated spermatid . </p></caption><graphic xlink:href="jrd-60-383-g001"/></fig>). In spermatogonia, the levels of H4K5ac, H4K8ac and H4K12ac were moderately intense. In preleptotene and leptotene spermatocytes, H4K5ac, H4K8ac and H4K12ac were highly acetylated (<xref ref-type="fig" rid="fig_001">Fig. 1H, K, H&#x02019;, K&#x02019;</xref>; <xref ref-type="fig" rid="fig_002">Fig. 2N, R</xref><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Immunohistochemical analysis of H4 tri-methylation and H4 lysine 12 acetylation. The signals represent nuclear (A, E, I, M, Q, U) histone H4 lysine 12 acetylation (H4K12ac) (B, F, J, N, R, V) and histone H4 tri-methylation (H4me3) (C, G, K, O, S, W). Stage VIII seminiferous tubules are shown (A, B, C, D). Stage I (E, F, G, H), stage V (I, J, K, L), stage VIII (M, N, O, P), stage X (Q, R, S, T) and stage XII (U, V, W, X) seminiferous tubules are shown. The scale bars represent 50 &#x000b5;m (A, B, C, D) and 10 &#x000b5;m (H, L, P, T, X). P-SPC, pachytene spermatocyte; PL-SPC, preleptotene spermatocyte; L-SPC, leptotene spermatocyte; Z-SPC, zygotene spermatocyte; R-SPD, round spermatid; and E-SPD, elongated spermatid.</p></caption><graphic xlink:href="jrd-60-383-g002"/></fig>). The immunostaining intensity was similar in zygotene spermatocytes (<xref ref-type="fig" rid="fig_001">Fig. 1N, N&#x02019;</xref>; <xref ref-type="fig" rid="fig_002">Fig. 2V</xref>). The expression of H4K5ac, H4K8ac and H4K12ac gradually decreased in pachytene spermatocytes (<xref ref-type="fig" rid="fig_001">Fig. 1E&#x02013;K, E&#x02019;&#x02013;K&#x02019;</xref>; <xref ref-type="fig" rid="fig_002">Fig. 2F&#x02013;R</xref>). However, H4K5ac, H4K8ac and H4K12ac were highly acetylated during meiosis (<xref ref-type="fig" rid="fig_001">Fig. 1N, N&#x02019;</xref>; <xref ref-type="fig" rid="fig_002">Fig. 2V</xref>). In spermatids, H4K5ac, H4K8ac and H4K12ac were detected in only a portion in step 1&#x02013;8 spermatids and were highly acetylated in steps 9&#x02013;12 spermatids (<xref ref-type="fig" rid="fig_001">Fig. 1K, N, K&#x02019;, N&#x02019;</xref>; <xref ref-type="fig" rid="fig_002">Fig. 2R, V</xref>). No acetylation of the histone H4 N-terminal tails was observed in spermatids after step 13.</p></sec><sec><title>Histone H4 tri-methylation (H4me3)</title><p>H4me3 showed specific exchange during spermatogenesis (<xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Modification pattern of histone H4 during mouse spermatogenesis</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" rowspan="3" colspan="1">Spermatogonium</td><td align="center" colspan="11" rowspan="1">Staining intensity<hr/></td></tr><tr><td align="center" colspan="8" rowspan="1">Spermatocyte<hr/></td><td align="center" colspan="3" rowspan="1">Spermatid<hr/></td></tr><tr><td align="center" valign="top" rowspan="1" colspan="1">A+B</td><td align="center" valign="top" rowspan="1" colspan="1">PL</td><td align="center" valign="top" rowspan="1" colspan="1">L</td><td align="center" valign="top" rowspan="1" colspan="1">Z</td><td align="center" valign="top" rowspan="1" colspan="1">PI</td><td align="center" valign="top" rowspan="1" colspan="1">PVIII</td><td align="center" valign="top" rowspan="1" colspan="1">PX</td><td align="center" valign="top" rowspan="1" colspan="1">Meiosis</td><td align="center" valign="top" rowspan="1" colspan="1">Steps 1&#x02013;8</td><td align="center" valign="top" rowspan="1" colspan="1">Steps 9&#x02013;12</td><td align="center" valign="top" rowspan="1" colspan="1">Steps 13&#x02013;16</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">H4K5ac</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">n</td></tr><tr><td align="left" rowspan="1" colspan="1">H4K8ac</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">n</td></tr><tr><td align="left" rowspan="1" colspan="1">H4K12ac</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">n</td></tr><tr><td align="left" rowspan="1" colspan="1">H4me3</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">n</td></tr><tr><td align="left" rowspan="1" colspan="1">H4K20me2</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">m</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">n</td></tr><tr><td align="left" rowspan="1" colspan="1">H4me</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">w</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">w m</td><td align="center" rowspan="1" colspan="1">s</td><td align="center" rowspan="1" colspan="1">n</td></tr></tbody></table><table-wrap-foot><p>PL, preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; PI, pachytene spermatocyte stage I; PVIII, pachytene spermatocyte stage VIII; PX, pachytene spermatocyte stage X; s, strong intensity; m, moderate intensity; w, weak intensity; n, negative.</p></table-wrap-foot></table-wrap>). It showed a weak staining intensity in preleptotene and leptotene spermatocytes (<xref ref-type="fig" rid="fig_002">Fig. 2O, S</xref>), high intensity staining in zygotene spermatocytes and moderate intensity staining in pachytene spermatocytes (<xref ref-type="fig" rid="fig_002">Fig. 2G, K, O, S</xref>). High staining intensity was observed during meiosis (<xref ref-type="fig" rid="fig_002">Fig. 2W</xref>). In the case of round spermatids, a highly modified state was observed in only a portion of the spermatids (<xref ref-type="fig" rid="fig_002">Fig. 2G, K, O</xref>), and the staining intensity of H4me3 gradually increased in step 9 to 12 spermatids (<xref ref-type="fig" rid="fig_002">Fig. 2S, W</xref>). However, no stained spermatids were observed after step 13.</p></sec><sec><title>Histone H4 lysine 20 di-methylation (H4K20me2)</title><p>H4K20me2 varied dynamically during spermatogenesis [<xref rid="r25" ref-type="bibr">25</xref>]. Its staining was moderate in spermatogonia, high at the preleptotene spermatocyte stage and similar in leptotene and zygotene spermatocytes (<xref ref-type="fig" rid="fig_003">Fig. 3N, R, V</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p> Immunohistochemical analysis of histone H4 lysine 20 di-methylation and mono-methylation. The signals represent nuclear (A, E, I, M, Q, U) histone H4 lysine 20 di-methylation (H4K20me2) (B, F, J, N, R, V) and histone H4 mono-methylation (H4me) (C, G, K, O, S, W). Stage VIII seminiferous tubule is shown (A, B, C, D). A stage I (E, F, G, H), stage V (I, J, K, L), stage VIII (M, N, O, P), stage X (Q, R, S, T) and stage XII (U, V, W, X) seminiferous tubules are shown. The scale bars represent 50 &#x000b5;m (A, B, C, D) and 10 &#x000b5;m (H, L, P, T, X). P-SPC, pachytene spermatocyte; PL-SPC, preleptotene spermatocyte; L-SPC, leptotene spermatocyte; Z-SPC, zygotene spermatocyte; R-SPD, round spermatid; and E-SPD, elongated spermatid.</p></caption><graphic xlink:href="jrd-60-383-g003"/></fig>). A highly modified state was observed in early pachytene spermatocytes, and this decreased during pachytene stage (<xref ref-type="fig" rid="fig_003">Fig. 3F, J, N, R</xref>). The expression level of H4K20me2 increased again from diplotene stage to meiotic prophase (<xref ref-type="fig" rid="fig_003">Fig. 3V</xref>). In round spermatids, H4K20me2 was detected in only a portion of the spermatids and showed a weak intensity by step 9 (<xref ref-type="fig" rid="fig_003">Fig. 3F, J, N, R, V</xref>). H4K20me2 was not observed in spermatids by step 13.</p></sec><sec><title>Histone H4 mono-methylation (H4me)</title><p>H4me showed weak expression in spermatogonia. The immunostaining intensity of H4me increased in preleptotene spermatocytes, was similar in leptotene and zygotene spermatocytes (<xref ref-type="fig" rid="fig_003">Fig. 3O, S, W</xref>), and decreased in pachytene spermatocytes (<xref ref-type="fig" rid="fig_003">Fig. 3G, K, O, S</xref>). The expression of H4me increased during meiosis (<xref ref-type="fig" rid="fig_003">Fig. 3W</xref>) and decreased in round spermatids. However, the staining intensity gradually increased in spermatids at around step 8 (<xref ref-type="fig" rid="fig_003">Fig. 3O</xref>). This state was maintained in spermatids until step 12 but was not observed by step 13.</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>In this study, we demonstrated that modification of the histone H4 N-terminal tails undergoes dramatic changes in male germ cells during spermatogenesis. Spermatocyte development consists of the preleptotene, leptotene, zygotene, pachytene and diplotene stages. Each of the spermatocyte stages shows specific patterns of H4 modification. In preleptotene spermatocytes, the expression of H4me, H4K20me2, H4K5ac, H4K8ac and H4K12ac was high, and that of H4me3 was relatively low. The patterns of staining intensity were similar in leptotene and zygotene spermatocytes. DNA and histone proteins are synthesized in the preleptotene stage [<xref rid="r26" ref-type="bibr">26</xref>], and histone acetylation is usually associated with transcriptionally active events [<xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r8" ref-type="bibr">8</xref>]. Therefore, it is possible that H4 acetylation is involved in these events during this phase. In preleptotene and leptotene spermatocytes, the histone acetylation state was found to be relatively low compared with that in elongating spermatids [<xref rid="r27" ref-type="bibr">27</xref>, <xref rid="r28" ref-type="bibr">28</xref>]. However, since we defined strong intensity as an intensity above a certain fluorescence brightness level, our results showed that both spermatocytes and elongating spermatids had strong intensities. The histone H3 acetylation state in spermatocytes is known to be relatively high [<xref rid="r19" ref-type="bibr">19</xref>]. Additional investigation of the relationship between histone acetylation and spermatocyte differentiation is necessary.</p><p>The expression of H4K5ac, H4K8ac, K4K12ac and H4me decreased in spermatocytes during the pachytene stage, but H4K20me2 and H4me3 were in a highly modified state. However, H4K20me2 decreased in staining intensity until the late pachytene stage, whereas H4me3 remained relatively high during the pachytene stage. The transcriptional activity is thought to be low in pachytene spermatocytes [<xref rid="r26" ref-type="bibr">26</xref>, <xref rid="r29" ref-type="bibr">29</xref>, <xref rid="r30" ref-type="bibr">30</xref>], and it may be associated with H4me3.</p><p>During meiosis, all the histone H4 N-terminal tails showed relatively high levels of modification. Since the loss of histone methyltransferase in mouse spermatogenesis is inhibited during meiosis [<xref rid="r31" ref-type="bibr">31</xref>,<xref rid="r32" ref-type="bibr">32</xref>,<xref rid="r33" ref-type="bibr">33</xref>], it is possible that histone modification plays an important role during the meiotic phase. Histone methylation is associated with both transcriptional activation and inactivation, depending on the histone N-terminal residue involved, while acetylation is associated with transcriptional activation [<xref rid="r6" ref-type="bibr">6</xref>, <xref rid="r34" ref-type="bibr">34</xref>]. Both methylation and acetylation states exist in meiosis. The conflicting patterns of histone modification observed in many cells such as embryonic stem cells and spermatogonia may be associated with totipotency [<xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r35" ref-type="bibr">35</xref>]. Since numerous biological events are involved in male germ cell meiosis, the conflicting histone modification patterns need further examination.</p><p>We found moderate modification of only a portion of the spermatids from step 1 to step 8. Only H4me increased in expression from step 1 to step 8. The nuclei were agglutinated gradually in round spermatids, and then gene expression was generally repressed. These results suggest that low levels of H4 acetylation are perhaps associated with the round spermatid state.</p><p>The staining intensity of H4K5ac, H4K8ac, H4K12ac and H4me increased in spermatids from steps 9 to 12. However, those of H4K20me2 and H4me3 were maintained in a lower state. In particular, histone H4 acetylation increased dramatically at around step 10 of the spermatid stage. Histone-to -protamine exchange is known to take place during this period [<xref rid="r36" ref-type="bibr">36</xref>]. Although little is known about the mechanism of this exchange, histone acetylation is probably associated with this event [<xref rid="r28" ref-type="bibr">28</xref>, <xref rid="r37" ref-type="bibr">37</xref>]. Histone acetylation may also contribute to weakening of the binding between DNA and histone and may be involved in histone-to-protamine exchange. Multiple modification patterns depending on mono-, di-, and tri-methylation may also contribute to the histone-to-protamine exchange.</p><p>During spermiogenesis, histone proteins are substituted by transition proteins and then subsequently by protamines. However, a small amount of histone proteins is retained in spermatids, and chemical modification of these histones plays an important role in sperm formation [<xref rid="r38" ref-type="bibr">38</xref>]. Nevertheless, the retention of histone H4 modifications was not observed in our experiments.</p><p>Comparison of the modification patterns of histones H3 and H4 showed that the H3 and H4ac patterns were similar; however, the patterns of H4me3 differed from those of H3K4me3 and H3K27me3. Thus, it is possible that H4me3 and H3 play different roles during murine spermatogenesis [<xref rid="r19" ref-type="bibr">19</xref>, <xref rid="r25" ref-type="bibr">25</xref>].</p><p>In this study, we used immunohistochemical methods to show that specific patterns of histone H4 modification are present during murine spermatogenesis. These results can provide further insight into the genetic control (e.g., chromatin remodeling, telomere repair, meiosis and histone-to-protamine exchange) of spermatogenesis and shed light on the epigenetic disorders that involve histone H4.</p></sec>
Expression and Activation of Mitogen-activated Protein Kinases in Matured Porcine Oocytes under Thermal Stress
<p> In this study, we determined the expression and activation of p38 MAPK in matured porcine oocytes subjected to heat shock (HS). When MII oocytes were heated, only the phosphorylated p38 levels relative to the total p38 levels decreased (P &#x0003c; 0.01) after HS, but no clear relationship with HS treatments was observed in the ERK, JNK and p90<sup>rsk</sup> expressions of matured oocytes. To confirm p38 activation in matured oocytes, immunocytochemical staining was performed to localize its expression and distribution in the ooplasm, and the results were largely consistent with previous Western blot analyses. Moreover, when matured oocytes were co-cultured with a P38 MAPK inhibitor, SB203580, for 4 h at 41.5 C, the activation of its immediate downstream substrate MAPKAPK-2 was not inhibited within any of the treatment groups. It appears that the MAPKAPK2 levels increased only under prolonged culture (HS4h and C4h) compared with the control group. In conclusion, p38 activity in porcine oocytes was decreased after exposure to HS and prolonged culture. These alterations of p38 and activation of MAPKAPK2 may be associated with porcine oocyte viability under HS conditions, and a potential cross-talk between p38 MAPK and other signaling cascades may exist, which warrants additional investigation.</p>
<contrib contrib-type="author"><name><surname>YEN</surname><given-names>Shih-Ying</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>TSENG</surname><given-names>Jung-Kai</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>CHUANG</surname><given-names>Show-Mei</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>CHEN</surname><given-names>Shuen-Ei</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>JU</surname><given-names>Jyh-Cherng</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref><xref ref-type="aff" rid="aff7"><sup>7</sup></xref><xref ref-type="aff" rid="aff8"><sup>8</sup></xref></contrib><aff id="aff1"><label>1)</label>Department of Animal Science, National Chung Hsing University, Taichung 402, Taiwan, ROC</aff><aff id="aff2"><label>2)</label>School of Optometry, Chung Shan Medical University, Taichung 402, Taiwan, ROC</aff><aff id="aff3"><label>3)</label>Department of Ophthalmology, Chung Shan Medical University Hospital, Taichung 402, Taiwan, ROC</aff><aff id="aff4"><label>4)</label>Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan, ROC</aff><aff id="aff5"><label>5)</label>Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan, ROC</aff><aff id="aff6"><label>6)</label>Core Laboratory for Stem Cells, Medical Research Department, China Medical University Hospital, Taichung 40447, Taiwan, ROC</aff><aff id="aff7"><label>7)</label>Agriculture Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan, ROC</aff><aff id="aff8"><label>8)</label>Department of Biomedical Informatics, College of Computer Science, Asia University, Taichung, Taiwan, ROC</aff>
The Journal of Reproduction and Development
<p>Elevated ambient temperatures have long been recognized as a major cause of reduced reproductive performance in domestic livestock during hot seasons in tropical and subtropical areas [<xref rid="r1" ref-type="bibr">1</xref>]. Similar seasonal or environmental effects on birth defects are also identified in humans [<xref rid="r2" ref-type="bibr">2</xref>]. Although the underlying mechanisms causing low embryo survival and conception rates are not fully understood, based on <italic>in vitro</italic> studies, temperature elevation retards embryo development and alters the morphologies of nuclei and cytoskeletons of mammalian oocytes [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r4" ref-type="bibr">4</xref>].</p><p>Fully grown mammalian oocytes are arrested at the prophase of the first meiotic division, which is termed the germinal vesicle (GV) stage. In response to the physiologic LH surge, the oocyte undergoes GV breakdown (GVBD), extrudes the first polar body, and proceeds into metaphase II (MII); at that point, it undergoes second meiotic arrest until fertilized by sperm or activated by other stimuli. Signal transduction within oocytes is mostly via activation or deactivation of specific protein kinases by phosphorylation or dephosphorylation; these are among the most important mechanisms regulating meiosis. These processes are largely controlled by various kinase molecules, such as maturation promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) [<xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r6" ref-type="bibr">6</xref>]. Activation of MPF enables eukaryotic cells to enter into metaphase [<xref rid="r7" ref-type="bibr">7</xref>]. Extracellular signal-regulated kinases (ERK1 and ERK2), which are members of the canonical MAPK family, are activated in response to various extracellular signaling molecules, notably growth factors, via an upstream small G-protein Ras. In addition, Jun kinases (JNK) and p38, collectively known as stress-activated protein kinases (SAPKs) constitute two other kinase families, which are also induced by extracellular cues [<xref rid="r8" ref-type="bibr">8</xref>, <xref rid="r9" ref-type="bibr">9</xref>]. These signaling pathways play critical roles in regulation and determination of cell growth, proliferation, differentiation, and/or apoptosis under physiologic and stress conditions. However, their functions and activation profiles during oocyte development are largely unknown. Nevertheless, p38 has been associated with various cellular stress responses, e.g., hyperosmolarity, ultraviolet radiation, inflammatory cytokines and endotoxins. In that regard, p38 is released in response to various physiologic cues, including growth factors, mitogens and FSH. Furthermore, it is also involved in various processes during differentiation, proliferation, and survival of somatic cell lineages [<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r11" ref-type="bibr">11</xref>]. It appears that p38 in porcine oocytes is activated around GVBD and remains active at and throughout the MI-MII transitional stage (Yen <italic>et al</italic>., unpublished data). The objective of the current study was to determine the expression and activation of p38 and other MAPKs after in vitro maturation (IVM) of porcine oocytes, with or without exposure to external heat shock (HS).</p><sec sec-type="materials|methods" id="s1"><title>Materials and Methods</title><sec><title>Oocyte collection and IVM</title><p>Porcine ovaries were stored in saline (35&#x02013;37 C) and transported to the laboratory in an insulated container within 1 h after slaughter. Cumulus-oocyte complexes (COCs) were collected by aspirating ovarian surface follicles (3 to 6 mm in diameter). The recovered COCs were then matured (20 to 30 COCs/100 &#x003bc;l) in North Carolina State University-23 (NCSU-23) medium supplemented with 10% follicular fluid, cysteine (0.1 mg/mL, C-5182, Sigma-Aldrich, St. Louis, MO, USA), EGF (10 ng/ml, E-4127, Sigma-Aldrich) and gonadotropins (hCG, 10 IU/ml, Sigma-Aldrich, A; PMSG, 10 IU/ml, Sigma-Aldrich) for the first 22 h of culture at 39 C in an incubator containing 5% CO<sub>2</sub> in air [<xref rid="r12" ref-type="bibr">12</xref>]. Thereafter, medium was replaced with gonadotropin-free NCSU-23 medium, and the oocytes were cultured continuously for another 20 h prior to HS treatment. Matured oocytes were selected by visualization of polar body extrusion at 42 h after the onset of IVM [<xref rid="r13" ref-type="bibr">13</xref>].</p></sec><sec><title>Treatments with HS and kinase inhibitor SB203580</title><p>Matured oocytes were randomly allocated to three HS groups and two control groups. In the former, oocytes were subjected to 41.5 C for 1, 2 or 4 h, whereas in the latter, oocytes were cultured at 39 C for 0 (C0h) or 4 h (C4h) without HS, as described previously [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r14" ref-type="bibr">14</xref>]. Inhibition of p38 was done with 10 &#x003bc;M of SB203580 (Calbiochem; San Diego, CA, USA), a specific inhibitor for p38, added to the IVM medium at various stages of culture.</p></sec><sec><title>Western blotting</title><p>Analysis of protein expression was conducted as described previously [<xref rid="r15" ref-type="bibr">15</xref>], and the following antibodies were purchased from Santa Cruz Biotechnology Inc., CA, USA, unless mentioned otherwise. Briefly, oocytes were rinsed in DPBS-PVA immediately after treatments and then collected in sodium dodecyl sulfate (SDS) sample buffer containing 100 mM Tris/HCl (pH 6.8), 5% 2-mercaptoethanol, 3% SDS, 4% glycerol and 0.1% bromophenol blue. All samples (50&#x02013;150 oocytes per sample) were boiled for 5 min and stored at &#x02013;80 C. Oocyte samples were subjected to electrophoresis in 10% (V/V) polyacrylamide/SDS gels [<xref rid="r15" ref-type="bibr">15</xref>,<xref rid="r16" ref-type="bibr">16</xref>]. The resolved proteins were transferred to nitrocellulose membranes, which were blocked with 10% chicken serum in TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 0.1% Tween 20 for 1 h and then incubated with rabbit polyclonal anti-phospho-p38 MAPK (1:100, #9211), anti-phospho-ERK (1:500, #9101), anti-phospho-JNK (1:100, #9251) antibodies or rabbit polyclonal anti-human p38 (1:200, C-20), anti-ERK (1:500, K-23), anti-JNK (1:500, #9252) and mouse monoclonal anti-RSK antibodies (1:100, E-1) at 4 C overnight. Membranes were washed three times (10 min/each) with TBST (20 mM Tris, PH 7.4, 500 mM NaCl, 0.05% Tween-20) and then incubated with secondary antibody (1:10000, anti-rabbit immunoglobulin horseradish peroxidase) for 1 h at room temperature. After three washes for 10 min each, proteins were detected with a Super<sup>&#x000ae;</sup>Signal West Pico Chemiluminrescent Substrate Kit (Pierce Biotechnology, Inc., Rockford, IL, USA). The intensity of each band was measured by using densitometry (Scion Image software for Windows).</p></sec><sec><title>Immunocytochemical staining</title><p>Oocytes were washed twice in DPBS-PVA and then fixed in DPBS-PVA containing 4% paraformaldehyde and 0.2% Triton X-100 for 40 min at room temperature. Thereafter, fixed samples were washed twice in DPBS-PVA for 15 min and stored overnight in 1% BSA in DPBS-PVA (BSA-DPBS-PVA) at 4 C prior to staining. The next day, oocytes were blocked with 10% goat serum (Dako A/S, Glostrup, Denmark) in DPBS-PVA-BSA for 45 min and then incubated in DPBS-PVA-BSA containing rabbit polyclonal anti-phospho-p38 antibody (1:100, #9211, Santa Cruz Biotechnology) at 4 C overnight. After three washes in PBS-PVA-BSA, oocytes were incubated in DPBS-PVA-BSA containing Alexa Fluor 488-labeled goat anti-rabbit IgG (1:300; Molecular Probes Inc., Eugene, OR, USA) for 40 min at room temperature, and then the chromosomes were stained with Hoechst 33342 (10 &#x003bc;g/ml, Sigma-Aldrich). Negative control images were obtained by omitting the first antibody during staining. Following a complete washing, oocytes were mounted on slides with mounting medium (50% DPBS, 50% Glycerol, 25 mg/ml NaN<sub>3</sub>) and observed under an Olympus epifluorescence microscope (AX-70). The intensity of p-p38 expression in oocytes was analyzed with the ImageJ software [<xref rid="r17" ref-type="bibr">17</xref>].</p></sec><sec><title>Experimental designs</title><p>Experiment 1&#x02013; Expression and activation of MAPKs in matured oocytes after HS: For an initial screening of matured oocytes, three members of the MAPK family, i.e., ERK, JNK and p38 MAPK and their related downstream molecules, were examined.</p><p>After 42 h of IVM, cumulus cells were removed from COCs, and matured oocytes were randomly allocated to five treatment groups, i.e., two control groups (C0h and C4h, 39 C) and three HS groups (HS1h, HS2h or HS4h, at 41.5 C) for analyses by Western blotting.</p><p>Experiment 2&#x02013; Subcellular localization of phosphorylated p38 of matured oocytes: Based on the observations in Experiment 1, confirmation of activated p-p38 in matured porcine oocytes was performed by immunocytochemistry to visualize its expression and distribution in heat-shocked (at 41.5 C for 1, 2 and 4 h) oocytes, with non-heat-shocked (39 C) oocytes being used for comparison. Oocytes were stained with Hoechst stain and the secondary antibody but without the primary antibody to serve as the negative control. The fluorescence intensity was further quantified using the ImageJ software for analysis.</p><p>Experiment 3&#x02013; Detection of p38 downstream MAPKAPK2 expression under HS conditions: The p38 inhibitor, SB203580 (10 &#x003bc;M), was added to the IVM medium during the culture period; thereafter, matured oocytes were fixed and stained for determination of nuclear status. To determine whether HS-influenced activation of MAPKAPK2 is mediated by p38 in matured porcine oocytes, denuded MII oocytes were randomly cultured with or without SB203580 in the C4h, C0h, HS1h, HS2h or HS4h groups and then collected for phosphorylated MAPKAP2 analysis by Western blotting.</p></sec><sec><title>Statistical analyses</title><p>All data from Western blotting and immunocytochemical staining were analyzed by ANOVA using the General Linear Model (GLM) procedure in the Statistical Analysis System software [<xref rid="r18" ref-type="bibr">18</xref>] and then subjected to Tukey&#x02019;s test. Percentile data were analyzed by Chi-square. For all statistical analyses, significance was set at P &#x0003c; 0.05.</p></sec></sec><sec sec-type="results" id="s2"><title>Results</title><sec><title>Experiment 1: Expression and activation of MAPKs in matured oocytes after HS</title><p>With various durations of HS treatment, activation of ERK1/2 and its downstream target molecules, p90rsk and JNK, in matured oocytes was not significantly different among treatment groups (<xref ref-type="fig" rid="fig_001">Figs. 1</xref> and<xref ref-type="fig" rid="fig_002"> 2</xref><fig orientation="portrait" fig-type="figure" id="fig_001" position="float"><label>Fig. 1.</label><caption><p> Expressions of ERK and its downstream p90rsk in matured porcine oocytes after <italic>in vitro</italic> heat shock at 41.5 C for 0, 1, 2 or 4 h. (<italic>a</italic>) A representative immunoblot of p-ERK, total ERK (44 and 42 kDa, respectively) and p90rsk (upper panel, 90 kDa). There were no significant differences among treatment groups. (<italic>b</italic>) There was no significant difference among treatment groups in relative activity of ERK (p-ERK/total ERK). Data are expressed as folds of the C0h group. Each lane of the SDS-PAGE gels contained 50 oocytes (four replicates). Bars indicate means &#x000b1; SEM. M, marker.</p></caption><graphic xlink:href="jrd-60-388-g001"/></fig><fig orientation="portrait" fig-type="figure" id="fig_002" position="float"><label>Fig. 2.</label><caption><p> Expression of JNK in matured porcine oocytes after <italic>in vitro</italic> heat shock at 41.5 C for 0, 1, 2 or 4 h. (<italic>a</italic>) A representative immunoblot of JNK (upper panel) and total JNK (lower panel, 54 and 46 kDa, respectively). There were no significant differences among treatment groups in either panel. Data are expressed as folds of the C0h group. Each lane of the SDS-PAGE gel contained 100 oocytes (four replicates). Bars indicate means &#x000b1; SEM. M, marker.</p></caption><graphic xlink:href="jrd-60-388-g002"/></fig>). However, the total p38 levels in all treatments (including the C4h group) were increased (P &#x0003c; 0.05) compared with in the control (C0h) group (<xref ref-type="fig" rid="fig_003">Fig. 3a</xref><fig orientation="portrait" fig-type="figure" id="fig_003" position="float"><label>Fig. 3.</label><caption><p> Level of p-p38 in matured porcine oocytes in control (non-heat-shocked group; maintained at 39 C) and heat-shocked (HS, 41.5 C) groups. (<italic>a</italic>) Representative immunoblot of p-p38 (upper panel) and total p38 (lower panel, 43 kDa). The p-p38 activity was decreased (P &#x0003c; 0.05) in the HS1h group and then slightly rebounded as the duration of HS increased. The total p38 level was increased when the duration of HS was prolonged (P &#x0003c; 0.05). (<italic>b</italic>) The relative activities of p-p38 in all the HS groups were reduced (P &#x0003c; 0.05) compared with the C0h group. Each lane of the SDS-PAGE gel contained 150 oocytes (five replicates). <sup>a, b</sup>Bars without a common superscript differed (P &#x0003c; 0.05). Bars indicate means &#x000b1; SEM. M, marker.</p></caption><graphic xlink:href="jrd-60-388-g003"/></fig>). In contrast, its relative activities (p-p38/total p38) were significantly reduced throughout the time courses of the HS (HS1h, HS2h and HS4h) and prolonged culture (C4h) groups, with no significant differences among them compared with the C0h group (<xref ref-type="fig" rid="fig_003">Fig. 3</xref>). Similarly, the p-p38 signal was clearly reduced in the HS1h group (<xref ref-type="fig" rid="fig_004">Fig. 4B</xref><fig orientation="portrait" fig-type="figure" id="fig_004" position="float"><label>Fig. 4.</label><caption><p> Subcellular localization of p-p38 in matured porcine oocytes in the non-heat-shocked (39 C) and heat-shocked (41.5 C) groups. Green dots are p-p38 labeled by immunocytochemical staining. (A&#x02013;E) Expressions of p-p38 in matured oocytes after heat shock for 0 h (A), 1 h (B), 2 h (C) or 4 h (D) and the p-p38 level in matured oocytes cultured at 39 C for 4 h (E). Panels A&#x02019;&#x02013;E&#x02019; are negative controls (without primary antibody), and insets are chromatin/chromosome (blue) structures stained by Hoechst 33342. Scale bar, 10 &#x003bc;m.</p></caption><graphic xlink:href="jrd-60-388-g004"/></fig>) compared with those in the HS0h (<xref ref-type="fig" rid="fig_004">Fig. 4A</xref>), HS2h (<xref ref-type="fig" rid="fig_004">Fig. 4C</xref>), HS4h (<xref ref-type="fig" rid="fig_004">Fig. 4D</xref>) and C4h (<xref ref-type="fig" rid="fig_004">Fig. 4E</xref>) groups. The p-p38 activity, quantified by the intensity of immunocytochemical staining, of matured porcine oocytes was significantly decreased in the HS1h group but subsequently recovered in the HS2h, HS4h and prolonged culture (C4h) groups (<xref ref-type="fig" rid="fig_005">Fig. 5</xref><fig orientation="portrait" fig-type="figure" id="fig_005" position="float"><label>Fig. 5.</label><caption><p> Quantification of p-p38 activity in matured porcine oocytes after heat shock (HS, 41.5 C). (<italic>a</italic>) a&#x02013;d, p-p38 levels in matured oocytes after HS for 1 h (HS1h, a), 2 h (HS2h, b) or 4 h (HS4h, c) and oocytes cultured at 39 C for 4 h (C4h, d). Panels a&#x02019;&#x02013;d&#x02019; show the p-p38 levels quantified by the ImageJ software. (<italic>b</italic>) The intensity of p-p38 decreased in the HS1h group and then increased in the HS2h or HS4h groups after prolonged HS (P &#x0003c; 0.05). <sup>a-c</sup> Bars without a common superscript differed (P &#x0003c; 0.05). Bars indicate means &#x000b1; SEM (five replicates, five oocytes/replicate).</p></caption><graphic xlink:href="jrd-60-388-g005"/></fig>).</p></sec><sec><title>Experiment 2: Subcellular localization of phosphorylated p38 in matured oocytes</title><p>The expression and distribution of p-p38 in matured porcine oocytes after HS (41.5 C) were visualized, and the results are shown in <xref ref-type="fig" rid="fig_004">Fig. 4</xref>. A normal matured oocyte (without HS, 39 C) has a homogeneous distribution of p-p38 throughout the ooplasm. In the heat-shocked groups, p-p38 first reduced in the HS1h group and then largely recovered in both the prolonged HS (H2h and H4h) and culture (C4h) group as expressed by the immunocytochemical localization and its fluorescence intensity when analyzed by ImageJ (<xref ref-type="fig" rid="fig_005">Fig. 5</xref>).</p></sec><sec><title>Experiment 3: Detection of p38 downstream MAPKAPK2 expression under HS conditions</title><p>As shown in <xref ref-type="fig" rid="fig_006">Fig. 6</xref><fig orientation="portrait" fig-type="figure" id="fig_006" position="float"><label>Fig. 6.</label><caption><p> Expression of p-MAPKAPK2 in matured porcine oocytes cocultured with SB203580 under HS (41.5 C) conditions. (a) A representative immunoblot of p-MAPKAPK2 (49 kDa) derived from matured oocytes cultured in 10 &#x003bc;M SB203580 plus HS conditions for various durations. (b) Expression of p-MAPKAPK2 was increased depending on HS duration in SB203580-free groups but was generally increased under the prolonged culture conditions, especially in the HS4h and C4h groups, which show significantly higher intensities than the C0h group when the intensity of each band was analyzed by the ImageJ software. Five replicates were performed and each lane of the SDS-PAGE gel contained 150 oocytes. <sup>a, b, c, d</sup> P &#x0003c; 0.05. Bars indicate means &#x000b1; SEM. M, marker.</p></caption><graphic xlink:href="jrd-60-388-g006"/></fig>, when denuded matured oocytes were co-cultured with SB203580 and then subjected to HS at 41.5 C for various periods of time, the expression of phosphorylated MAPKAPK-2 (an immediate downstream molecule of p38) was not inhibited in any of the HS and C4h groups and only increased under the prolonged cultured conditions regardless of treatment with the inhibitor. Expression of p-MAPKAPK2 was only significantly increased in the HS4h and C4h groups compared with the control (C0h).</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>It is well known that p38 MAPK is one of the mitogen-activated protein kinases that are associated with cellular responses stimulated by stresses, cytokines, HS, osmotic shock and radiation and are mainly involved in cell differentiation and apoptosis. Although MAPKs have been implicated in oocyte maturation in several species (<italic>Xenopus</italic>, [<xref rid="r19" ref-type="bibr">19</xref>]; mice, [<xref rid="r6" ref-type="bibr">6</xref>]; pigs, Yen <italic>et al</italic>., unpublished data), there is very limited information regarding the role of p38 in HS-induced signaling in oocytes of domestic livestock [<xref rid="r20" ref-type="bibr">20</xref>]. This study is apparently the first report regarding the role of p38 in matured porcine oocytes subjected to HS.</p><p>In mammalian cells, the p38 pathway is activated by various stresses or agonists, e.g., tyrosine kinase or cytokine receptor activators [<xref rid="r21" ref-type="bibr">21</xref>]. It is likely that there are multiple cellular sensing mechanisms and that their signals eventually converge to the p38 signaling pathway, although no specific membrane receptors for heat have been reported. Heat, UV light and osmotic stimuli can trigger hydrolysis of membrane sphingomyelins by sphingomyelinase which in turn increases cellular ceramide, a second messenger, to activate SAPKs [<xref rid="r22" ref-type="bibr">22</xref>, <xref rid="r23" ref-type="bibr">23</xref>]. Also, Jun kinases and apoptosis signal-regulating kinase-1 (ASK1), an upstream molecule of p38, were found to be associated with regulation of apoptosis in somatic cells [<xref rid="r24" ref-type="bibr">24</xref>]. Based on our immunocytochemical assay, activation of p38 decreased only in the HS1h group, although it subsequently rebounded when <italic>in vitro</italic> HS was prolonged. Therefore, we inferred that the apoptotic pathway might have been initiated due to the long duration of HS. This was entirely consistent with our previous studies, in which we detected a drastic reduction in developmental competence of embryos subjected to &#x0003e;2 h of <italic>in vitro</italic> HS [<xref rid="r13" ref-type="bibr">13</xref>, <xref rid="r14" ref-type="bibr">14</xref>, <xref rid="r25" ref-type="bibr">25</xref>]. It has also been reported that apoptosis of bovine oocytes can be induced by <italic>in vitro</italic> HS [<xref rid="r26" ref-type="bibr">26</xref>, <xref rid="r27" ref-type="bibr">27</xref>], one of the severe cellular stresses that has been shown to cause embryonic loss to occur <italic>in vitro</italic>[<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r25" ref-type="bibr">25</xref>, <xref rid="r28" ref-type="bibr">28</xref>] and possibly <italic>in vivo</italic>[<xref rid="r29" ref-type="bibr">29</xref>]. However, the molecular and cellular bases of heat-induced cell injuries, particularly the deleterious effects of HS on oocyte or embryo viability, remain poorly understood.</p><p>Alternatively, short-term mild HS conditions could enhance thermotolerance of various types of cells, organisms and even mammalian embryos [<xref rid="r30" ref-type="bibr">30</xref>, <xref rid="r31" ref-type="bibr">31</xref>]. However, the mode of how it is triggered may be important. For instance, transient activation of JNK or p38 (or both) is usually associated with cell survival or differentiation, whereas sustained activation of these kinases caused apoptosis [<xref rid="r32" ref-type="bibr">32</xref>,<xref rid="r33" ref-type="bibr">33</xref>,<xref rid="r34" ref-type="bibr">34</xref>]. Based on previous reports, HS may desensitize the p38 pathway, and the desensitization kinetics may be associated with development of thermotolerance in some organisms [<xref rid="r35" ref-type="bibr">35</xref>, <xref rid="r36" ref-type="bibr">36</xref>]. In addition, HS-induced p38 desensitization was reported to be closely related to heat-induced thermotolerance in Chinese hamster cells [<xref rid="r35" ref-type="bibr">35</xref>]. Based on our kinase assays, when matured oocytes were heated or prolonged cultured, the relative activity of p38 was drastically decreased compared with in the control groups (<xref ref-type="fig" rid="fig_003">Fig. 3</xref>). However, we could not completely exclude the possibility that along with an extended duration of HS or <italic>in vitro</italic> culture, oocytes may be physiologically adaptive or dysfunctional in terms of the reduction of the relative p38 activity over time. The existence of the confounding effects of heat shock with a prolonged culture period may not be overlooked, although a mild short-term heat shock could enhance the physiologic parameters (such as intracellular calcium concentrations) or metabolic responses of oocytes or embryos [<xref rid="r37" ref-type="bibr">37</xref>, <xref rid="r38" ref-type="bibr">38</xref>]. Conversely, direct HS applied to MII oocytes had no immediate influences in the HS and control groups on activation of p90rsk, ERK and JNK (<xref ref-type="fig" rid="fig_001">Fig. 1</xref>). Consequently, oocytes became apoptotic under longer HS conditions, at least partially due to no significant activation of the ERK signaling pathway, which has a potential role in mediating cell division, migration and survival.</p><p>Activation of the p38 pathway can also promote phosphorylation of small heat shock protein 27 (Hsp27) [<xref rid="r35" ref-type="bibr">35</xref>, <xref rid="r39" ref-type="bibr">39</xref>,<xref rid="r40" ref-type="bibr">40</xref>,<xref rid="r41" ref-type="bibr">41</xref>], which is catalyzed by MAPKAPK2, a serine-protein kinase immediately downstream from p38 [<xref rid="r40" ref-type="bibr">40</xref>, <xref rid="r42" ref-type="bibr">42</xref>]. Activation of Hsp27 stabilizes cellular actin filaments during environmental insults and mediates dynamic changes in actin filaments in response to p38 activation. [<xref rid="r43" ref-type="bibr">43</xref>,<xref rid="r44" ref-type="bibr">44</xref>,<xref rid="r45" ref-type="bibr">45</xref>,<xref rid="r46" ref-type="bibr">46</xref>]. In the present study (Experiment 3), treatment with SB203580 under HS conditions failed to alter MAPKAPK2 activation, and the phosphorylated MAPKAPK2 activity decreased after a short period of HS, although it finally (and unexpectedly) rebounded under prolonged HS and culture conditions (<xref ref-type="fig" rid="fig_006">Fig 6, P</xref> &#x0003c; 0.05). Induction of thermotolerance with a parallel increase of Hsps was found to inhibit heat-induced apoptosis in many cell types [<xref rid="r47" ref-type="bibr">47</xref>,<xref rid="r48" ref-type="bibr">48</xref>,<xref rid="r49" ref-type="bibr">49</xref>,<xref rid="r50" ref-type="bibr">50</xref>]. However, when the concentration of Hsp27 was abnormally elevated, it caused erroneous actin polymerization, leading to cell blebbing or apoptosis [<xref rid="r51" ref-type="bibr">51</xref>]. We therefore reasoned that the increased MAPKAPK2 activity might have caused the elevation of Hsp27/25, which, in turn, could have initiated apoptosis under the extended HS conditions. Given the increased MAPKAPK2 activity during the prolonged HS and even when matured oocytes were incubated with the kinase inhibitor, it seemed that there may be different upstream activators or alternative pathways in addition to p38 signaling. Murai <italic>et al.</italic>[<xref rid="r52" ref-type="bibr">52</xref>] also reported that a short-term stress-hypersensitive PC12m3 mutant cell induced p38 activation, whereas prolonged stress induced activation of JNK. However, the responsiveness of these kinases under HS mainly depended on the cell types.</p><p>In this study, we demonstrated that p38 was the only MAPK examined that responded to the HS signal of matured oocytes. We inferred that spatial and temporal alteration of p38 activation appeared to regulate HS-induced signaling of matured porcine oocytes and that apoptosis might be amplified depending on the p38 MAPK/MAPKAPK2 signaling cascades. However, the changes in the developmental competence of matured porcine oocytes in relation to their apoptotic gene expression after HS require further investigation.</p></sec>
Is the Function of the Porcine Sperm Reservoir Restricted to the Ovulatory Period?
<p> The uterotubal junction (UTJ) and caudal isthmus are recognized as a functional pre-ovulatory sperm reservoir (SR). Spermatozoa are released from the SR in a complex and concerted action. However, whether this functionality is restricted only to the ovulatory period is still open to debate. Our study was aimed to analyze the presence of spermatozoa within the UTJ (SR), isthmus (ISTH) and ampulla (AMP) after laparoscopic intrauterine insemination (LIUI) either in the peri- (PERI) or post-ovulatory (POST) period or at mid cycle (MID). Each uterine horn of estrus synchronized gilts (n=12) was inseminated with 20 ml sperm (29.5&#x000d7;10<sup>6</sup> cells/ml). Oviducts were recovered 7 h after LIUI and separated into the UTJ, ISTH and AMP, and sections were flushed with 10 ml PBS+EDTA solution. After centrifugation, the sperm pellet was evaluated by &#x0010c;e&#x00159;ovsk&#x000fd; staining. The median sperm numbers in the PERI, POST and MID groups were 578, 171 and 789 in the UTJ; 545, 233 and 713 in the ISTH; and 496, 280 and 926 in the AMP, respectively, and there were differences between the POST and MID groups (P&#x0003c;0.05) but not between the oviductal sections of each group (P&#x0003e;0.05). Compared with the MID group, the percent of intact sperm cells was higher (P&#x0003c;0.01) in the PERI and POST groups (32.8 <italic>vs.</italic> 66.4 and 76.8<italic>%</italic>). Also, the percentages of aberrations in the acrosome and tail were higher (P&#x0003c;0.05) in the MID group. Based on this, it can be assumed that the sperm reservoir is active during different phases of the estrus cycle. However, the mid-cycle oviduct environment considerably impairs sperm cell quality.</p>
<contrib contrib-type="author"><name><surname>BR&#x000dc;SSOW</surname><given-names>Klaus-Peter</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>EGERSZEGI</surname><given-names>Istvan</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>R&#x000c1;TKY</surname><given-names>Jozsef</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><aff id="aff1"><label>1)</label>Institute of Reproductive Biology, Leibniz Institute for Farm Animal Biology (FBN), 18196 Dummerstorf, Germany</aff><aff id="aff2"><label>2)</label>Research Institute for Animal Breeding, Nutrition and Meat Science, National Agricultural Research and Innovation Centre, 2053 Herceghalom, Hungary</aff>
The Journal of Reproduction and Development
<p>In the pig, the uterotubal junction (UTJ) and caudal isthmus act as a functional tubal sperm reservoir (SR) that ensures the availability of viable spermatozoa for fertilization [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r2" ref-type="bibr">2</xref>]. In the SR, most spermatozoa maintain a normal ultrastructure and viability [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r4" ref-type="bibr">4</xref>]. A number of concerted factors may explain formation of the SR, including the narrowed lumen [<xref rid="r5" ref-type="bibr">5</xref>], viscous mucus [<xref rid="r6" ref-type="bibr">6</xref>], lower temperature [<xref rid="r7" ref-type="bibr">7</xref>], local enzymatic and ionic milieu [<xref rid="r8" ref-type="bibr">8</xref>], selective binding of spermatozoa to the epithelium [<xref rid="r9" ref-type="bibr">9</xref>] and specific tubal fluid components [<xref rid="r10" ref-type="bibr">10</xref>].</p><p>The sequential release of spermatozoa from the SR towards the site of fertilization (i.e., ampulla-isthmic-junction) is suggested to be a complex and concerted process including opening of the lumen by a decrease in hormonally driven endosalpingeal edema, dissolution of hyaluronan (HA)-rich mucus, hyperactive sperm motility, increased flow of tubal fluid and redirected oviductal contractions [<xref rid="r11" ref-type="bibr">11</xref>]. However, oocyte signals, follicular fluid components and temperature gradients are also involved in the process of (peri-) ovulatory sperm release in pigs [<xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref>,<xref rid="r14" ref-type="bibr">14</xref>,<xref rid="r15" ref-type="bibr">15</xref>].</p><p>It remains unknown, if the function of the SR is restricted only to the ovulatory period. Therefore, the aim of our study was to analyze the presence of spermatozoa within the UTJ, isthmus (ISTH) and ampulla (AMP) either in the peri- (PERI) or postovulatory (POST) period or at mid cycle (MID). Also, the quality of spermatozoa was assayed in terms of their morphology and acrosome integrity. Since insemination of sows out of estrus bears some difficulties, we used laparoscopic intrauterine insemination (LIUI) to ensure appropriate application of semen at these different time points of the estrus cycle. The LIUI approach has been successfully applied in previous studies [<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r16" ref-type="bibr">16</xref>, <xref rid="r17" ref-type="bibr">17</xref>].</p><p>Altogether, 12 gilts were successfully inseminated by means of LIUI, and altogether 24 oviducts were surgically recovered and dissected. Of 72 oviduct sections, 71 could be flushed, and sperm cells were recovered from all oviduct sections except for two (97.2%).</p><p>There was a considerable variation in the number of sperm cells recovered from different oviduct sections (<xref rid="tbl_001" ref-type="table">Table 1</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title> Sperm concentration (mean, median, minimum and maximum values) in different oviduct sections after LIUI in the peri- (PERI) and postovulatory (POST) periods and at mid cycle (MID)</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="center" valign="top" rowspan="1" colspan="1">Oviduct section</td><td align="center" valign="top" rowspan="1" colspan="1">Sperm concentration</td><td align="center" valign="top" rowspan="1" colspan="1">PERI</td><td align="center" valign="top" rowspan="1" colspan="1">POST</td><td align="center" valign="top" rowspan="1" colspan="1">MID</td></tr></thead><tbody><tr><td align="center" rowspan="3" colspan="1">UTJ</td><td align="center" rowspan="1" colspan="1">Mean &#x000b1; SE</td><td align="center" rowspan="1" colspan="1">3932 &#x000b1; 2348</td><td align="center" rowspan="1" colspan="1">676 &#x000b1; 390<sup>a</sup></td><td align="center" rowspan="1" colspan="1">9221 &#x000b1; 8269<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Median</td><td align="center" rowspan="1" colspan="1">578</td><td align="center" rowspan="1" colspan="1">171<sup>a</sup></td><td align="center" rowspan="1" colspan="1">789<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Min &#x02013; max</td><td align="center" rowspan="1" colspan="1">0&#x02013;17261</td><td align="center" rowspan="1" colspan="1">41&#x02013;3141</td><td align="center" rowspan="1" colspan="1">97&#x02013;67063</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="center" rowspan="3" colspan="1">ISTH</td><td align="center" rowspan="1" colspan="1">Mean &#x000b1; SE</td><td align="center" rowspan="1" colspan="1">952 &#x000b1; 382</td><td align="center" rowspan="1" colspan="1">402 &#x000b1; 213<sup>a</sup></td><td align="center" rowspan="1" colspan="1">1770 &#x000b1; 842<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Median</td><td align="center" rowspan="1" colspan="1">545</td><td align="center" rowspan="1" colspan="1">233<sup>a</sup></td><td align="center" rowspan="1" colspan="1">713<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Min &#x02013; max</td><td align="center" rowspan="1" colspan="1">37&#x02013;3205</td><td align="center" rowspan="1" colspan="1">15&#x02013;1830</td><td align="center" rowspan="1" colspan="1">78&#x02013;6850</td></tr><tr><td align="left" colspan="5" rowspan="1"><hr/></td></tr><tr><td align="center" rowspan="3" colspan="1">AMP</td><td align="center" rowspan="1" colspan="1">Mean &#x000b1; SE</td><td align="center" rowspan="1" colspan="1">1022 &#x000b1; 456</td><td align="center" rowspan="1" colspan="1">353 &#x000b1; 106<sup>a</sup></td><td align="center" rowspan="1" colspan="1">2172 &#x000b1; 1460<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Median</td><td align="center" rowspan="1" colspan="1">496</td><td align="center" rowspan="1" colspan="1">280<sup>a</sup></td><td align="center" rowspan="1" colspan="1">926<sup>b</sup></td></tr><tr><td align="center" rowspan="1" colspan="1">Min &#x02013; max</td><td align="center" rowspan="1" colspan="1">0&#x02013;3604</td><td align="center" rowspan="1" colspan="1">73&#x02013;973</td><td align="center" rowspan="1" colspan="1">97&#x02013;12320</td></tr></tbody></table><table-wrap-foot><p><sup>a,b</sup> P&#x0003c;0.05 for comparisons within rows.</p></table-wrap-foot></table-wrap>). There were significantly (P&#x0003c;0.05) increased mean and median cell numbers in all segments of the oviduct after insemination in the peri-ovulatory period and at mid-cycle compared with those for postovulatory LIUI. However, the number of spermatozoa did not differ between the oviduct sections (UTV <italic>vs.</italic> ISTH <italic>vs</italic>. AMP) of each insemination group. However, we found a tendency (P=0.079) for an increasing median number of sperm cells towards the ampulla in the POST group.</p><p>Insemination during the luteal phase of the estrus cycle resulted in a lower percent (P&#x0003c;0.05) of intact spermatozoa compared with LIUI in the peri- and postovulatory periods (<xref rid="tbl_002" ref-type="table">Table 2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title> Assessment of sperm quality at different time points (PERI, POST and MID)</title></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" valign="top" rowspan="1" colspan="1"/><td align="center" valign="top" rowspan="1" colspan="1">Intact spermatozoa<break/>(%)</td><td align="center" valign="top" rowspan="1" colspan="1">Damaged acrosome<break/>(%)</td><td align="center" valign="top" rowspan="1" colspan="1">Tail aberration<break/>(%)</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">PERI</td><td align="center" rowspan="1" colspan="1">66.4 &#x000b1; 6.0<sup>a</sup></td><td align="center" rowspan="1" colspan="1">29.3 &#x000b1; 6.5<sup>a,b</sup></td><td align="center" rowspan="1" colspan="1">17.4 &#x000b1; 4.0<sup>a,b</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">POST</td><td align="center" rowspan="1" colspan="1">76.8 &#x000b1; 3.5<sup>a</sup></td><td align="center" rowspan="1" colspan="1">16.2 &#x000b1; 2.6<sup>a</sup></td><td align="center" rowspan="1" colspan="1">7.0 &#x000b1; 1.7<sup>a</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">MID</td><td align="center" rowspan="1" colspan="1">32.8 &#x000b1; 3.9<sup>b</sup></td><td align="center" rowspan="1" colspan="1">38.6 &#x000b1; 2.7<sup>b</sup></td><td align="center" rowspan="1" colspan="1">28.2 &#x000b1; 4.3<sup>b</sup></td></tr></tbody></table><table-wrap-foot><p><sup>a,b</sup> P&#x0003c;0.05 for comparisons within columns.</p></table-wrap-foot></table-wrap>). As well, aberrations of the acrosome (swollen or lacking acrosome) and tail (folded, bent or broken tail) were more often present in the MID group.</p><p>There is not too much data regarding the sperm cell distribution within the porcine oviduct after mating or insemination. Rigby [<xref rid="r18" ref-type="bibr">18</xref>] found an average of 10<sup>6</sup> spermatozoa in the UTJ and only about 1500 in the isthmus 6 h after insemination. Viring [<xref rid="r19" ref-type="bibr">19</xref>] reported on 500, 2.6 and 0.3 &#x000d7;10<sup>3</sup> sperm cells in the UTJ, isthmus and ampulla, respectively, at the same time point. In other studies [<xref rid="r20" ref-type="bibr">20</xref>,<xref rid="r21" ref-type="bibr">21</xref>,<xref rid="r22" ref-type="bibr">22</xref>], a larger proportion of spermatozoa was found in the UTJ compared with other oviduct sections. In these studies, insemination was performed in the preovulatory period, but oviducts were flushed at different intervals (24 h after insemination [<xref rid="r21" ref-type="bibr">21</xref>, <xref rid="r22" ref-type="bibr">22</xref>] or 6-8 h before ovulation, during ovulation or 6-8 h after ovulation [<xref rid="r20" ref-type="bibr">20</xref>]). Therefore, there is some difficulty in comparing the real number of sperm cells counted between the previous and present studies. Furthermore, there is great variation between gilts in the number of spermatozoa that can be attributed to the boar used [<xref rid="r20" ref-type="bibr">20</xref>] but also to the different methods of sperm number analysis (B&#x000fc;rker or Neubauer hemocytometer compared with sperm cell count after &#x0010c;e&#x00159;ovsk&#x000fd; staining in our study). No significant differences were found in our study regarding the (mean and median) number of sperm cells within the oviduct section, regardless of the time point of insemination. However, the majority of spermatozoa (63.5, 47.3 and 70.0% in the PERI, POST and MID groups, respectively) remained in the UTJ. A larger proportion of spermatozoa was also present in the UTJ and the lower isthmus in other studies [<xref rid="r20" ref-type="bibr">20</xref>, <xref rid="r22" ref-type="bibr">22</xref>, <xref rid="r23" ref-type="bibr">23</xref>]. These results confirm that these sections act as a functional tubal sperm reservoir.</p><p>It was previously shown that spermatozoa are located at specific sites within the SR environment [<xref rid="r24" ref-type="bibr">24</xref>] and that there are two sperm subpopulations, one with epithelial contact and one without such contact [<xref rid="r4" ref-type="bibr">4</xref>]. Since a large number of spermatozoa can be recovered by flushing, these authors suggest that most of the spermatozoa are in the oviduct lumen and loosely attached to the luminal surface. Thus, it can be assumed that the majority of the spermatozoa present in the oviduct were flushed out in our present study.</p><p>The question of whether the function of the SR is restricted to the ovulatory period can be answered only using a special insemination protocol. We used the laparoscopic intrauterine insemination (LIUI) approach [<xref rid="r15" ref-type="bibr">15</xref>, <xref rid="r16" ref-type="bibr">16</xref>]. This allowed us to ensure application of sperm into the genital tract even at mid cycle. Interestingly, we found similar numbers and distributions of sperm cells when insemination was done at mid cycle compared with the peri-ovulatory period. Additionally, the sperm concentrations in all oviduct sections were higher (P&#x0003c;0.05) after MID insemination in comparison to postovulatory sperm application. So we can speculate first of all that the nature of the SR to store and to release spermatozoa is active during the whole estrus cycle. Differences in the sperm concentration within the oviduct sections can be explained probably by differences in hormone milieu. It was previously shown that signals from the preovulatory follicle due to the transfer of steroids, prostaglandins and peptides via the subovarian plexus by a countercurrent mechanism are involved in the function of the SR [<xref rid="r12" ref-type="bibr">12</xref>, <xref rid="r25" ref-type="bibr">25</xref>,<xref rid="r26" ref-type="bibr">26</xref>,<xref rid="r27" ref-type="bibr">27</xref>]. There is experimental evidence [<xref rid="r25" ref-type="bibr">25</xref>] that injection of progesterone into the oviductal serosa at the UTJ increased the release of spermatozoa, leading to polyspermic fertilization. However, it was previously shown [<xref rid="r28" ref-type="bibr">28</xref>] that the progesterone concentration within the oviductal fluid was similar prior to and after ovulation, i.e., during the peri- and postovulatory periods. In the systemic blood, the progesterone level was two times higher after ovulation, and the difference between the oviductal and blood plasma concentrations was about four times higher after ovulation. However, this cannot explain the lower number of spermatozoa in the postovulatory period. On the other hand, since the luteal phase of the porcine estrus cycle is characterized by increased progesterone levels [<xref rid="r29" ref-type="bibr">29</xref>, <xref rid="r30" ref-type="bibr">30</xref>], these concentrations could mimic the signal to release (a higher number of) spermatozoa into the oviduct in the MID group.</p><p>Abnormal spermatozoa, e.g., those with a damaged acrosome, tail aberrations and protoplasmatic droplets, are to some extent common in boar semen [<xref rid="r31" ref-type="bibr">31</xref>]. In the SR, most of the sperm population with epithelial contact maintained intact plasma membranes during the preovulatory period and showed acrosome reacted-like membrane changes during the postovulatory period [<xref rid="r4" ref-type="bibr">4</xref>]. Flushing of oviducts in the pre-, peri- and postovulatory period revealed that 68, 51 and 46% of spermatozoa had an intact plasma membrane [<xref rid="r20" ref-type="bibr">20</xref>]. In our study, the proportions of intact spermatozoa were 66 and 77% after peri- and postovulatory insemination, respectively, whereas after mid-cycle insemination, the proportion fell to 33%, (P&#x0003c;0.05). Acrosome and tail abnormalities were also significantly higher in the MID group. Without doubt, the histo-architecture [<xref rid="r32" ref-type="bibr">32</xref>] and the milieu of the porcine oviduct diverge in different periods of the estrus cycle [<xref rid="r33" ref-type="bibr">33</xref>,<xref rid="r34" ref-type="bibr">34</xref>,<xref rid="r35" ref-type="bibr">35</xref>,<xref rid="r36" ref-type="bibr">36</xref>,<xref rid="r37" ref-type="bibr">37</xref>]. Therefore, the mid-cycle oviduct environment is not physiologically proper for sperm cells, and as a result, the quality of spermatozoa is influenced. However, further studies should highlight in more detail the functionality of the SR and of sperm-oviduct interaction.</p><p>In conclusion, our study revealed that the sperm reservoir functions during different phases of the estrus cycle. However, mid-cycle oviduct environment considerably impairs sperm cell quality.</p><sec sec-type="methods" id="s1"><title>Methods</title><sec><title>Animals and animal treatment</title><p>All procedures involving animal handling and treatment were approved by the Committee for Animal Use and Care of the Agricultural Ministerial Department of Mecklenburg-Vorpommern, Germany.</p><p>Altogether, 12 Landrace gilts (9 months old, with mean body weights of 138 kg) were included in the trial. Estrus was synchronized in all gilts by 15 days of feeding with Regumate&#x000ae; (16 mg altrenogest/day/gilt; MSD Animal Health, Unterschleissheim, Germany). Twenty-four hours after the last Regumate&#x000ae; feeding (0800 h), each animal received a single intramuscular injection of 850 IU equine chorionic gonadotropin (eCG; Pregmagon&#x000ae;, IDT Biologika, Dessau-Tornau, Germany). Ovulation was induced 80 h later by administration of 500 IU human chorionic gonadotropin (hCG; Ovogest&#x000ae;, MSD Animal Health, Unterschleissheim, Germany).</p></sec><sec><title>Laparoscopic insemination and oviduct and sperm recovery</title><p>Laparoscopic intrauterine insemination (LIUI) into each uterine horn was performed with 20 ml of extended, fresh boar semen (29.5&#x000d7;10<sup>6</sup> spermatozoa/ml; motility 80%; extender: AndroStar<sup>&#x000ae;</sup> Plus, Minit&#x000fc;b, Tiefenbach, Germany). Semen was collected from the same proven AI Pietrain boar (AI Station BVN, Malchin, Germany). LIUI was performed as described previously [<xref rid="r15" ref-type="bibr">15</xref>, <xref rid="r17" ref-type="bibr">17</xref>]. Briefly, general anaesthesia in gilts was induced with ketamine (17.25 mg/kg BW, Ursotamin&#x000ae;, Serumwerk Dessau, Germany) and azaperone (1.2 mg/kg BW, Stresnil&#x000ae;, Elanco Animal Health, Bad Homburg, Germany) and animals were fixed in a dorsal position. A pneumoperitoneum with CO<sub>2</sub> was automatically produced (Endo Tech, Munich, Germany). Thereafter, three trocar cannulas (Karl Storz, Tuttlingen, Germany) were inserted into the abdomen for 0&#x000b0; optics (ETB, Berlin, Germany) and grasping forceps (NeoMed, Gutach/Bleibach, Germany). All laparoscopic handling was observed on a video monitoring system (NeoMed, Gutach/Bleibach, Germany). For insemination, the uterine horn was carefully fixed with atraumatic forceps, and the uterine wall was punctured approximately 10 cm caudal to the uterotubal junction with a trocar that was 2.5 mm in diameter. Under visual control, a 2.2 mm catheter (R&#x000dc;SCH feeding tube, W. R&#x000fc;sch AG, Kernen, Germany) connected to a 20 ml syringe was inserted through the trocar cannula about 3 cm into the uterine lumen in the direction towards the tip of the uterine horn, and then semen was deposited. The insemination procedure was repeated at the opposite uterine horn.</p><p>LIUI was performed at three different time points of the estrus cycle, i.e., in the peri-ovulatory (PERI; 31 h post hCG, n=4 gilts) or postovulatory period (POST; 79 h post hCG, n=4) or at mid cycle (MID; day 9 of the estrus cycle, n=4). Seven hours after LIUI, gilts were subjected to ovariohysterectomy, and the oviducts (n=24) were dissected into three segments: the caudal isthmus and uterotubal junction (UTJ), cranial isthmus (ISTH) and ampulla (AMP). Each section was flushed with 10 ml PBS containing 1.78 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA). Flushed fluids were centrifuged twice at 500 <italic>g</italic> (for 10 and for 7 min). Supernatants were removed, and the volume of sperm pellets was measured with a pipette. From each sample, 10 &#x000b5;l of the sperm pellet were taken to prepare smears. Smears were stained according to the method of &#x0010c;e&#x00159;ovsk&#x000fd; [<xref rid="r38" ref-type="bibr">38</xref>] and the total number of spermatozoa was counted from stained smears of each 10 &#x000b5;l sample. The total sperm cell number per flushing was determined based on this number and the volume of the sperm pellet, respectively. Sperm cell morphology (intact and acrosome and tail aberrations) and acrosome integrity were evaluated by analyzing at least 200 spermatozoa.</p></sec><sec><title>Statistical analysis</title><p>Two replications with six gilts each were performed. Calculation of means and standard deviations, as well as of median values and 25th and 75th percentiles was carried out using the software package SigmaPlot 11.0 (Systat Software, San Jose, CA, USA). One-way ANOVA followed by Tukey&#x02019;s test was used to compare the results. Differences of P&#x0003c;0.05 were considered significant.</p></sec></sec>
The Dpp/TGF&#x003b2;-Dependent Corepressor Schnurri Protects Epithelial Cells from JNK-Induced Apoptosis in <italic>Drosophila</italic> Embryos
Could not extract abstract
<contrib contrib-type="author" id="au1"><name><surname>Beira</surname><given-names>Jorge&#x000a0;V.</given-names></name><email>[email protected]</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff2" ref-type="aff">2</xref><xref rid="cor1" ref-type="corresp">&#x02217;</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Springhorn</surname><given-names>Alexander</given-names></name><xref rid="aff3" ref-type="aff">3</xref><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Gunther</surname><given-names>Stefan</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Hufnagel</surname><given-names>Lars</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Pyrowolakis</surname><given-names>Giorgos</given-names></name><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Vincent</surname><given-names>Jean-Paul</given-names></name><email>[email protected]</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="cor2" ref-type="corresp">&#x02217;&#x02217;</xref></contrib><aff id="aff1"><label>1</label>Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK</aff><aff id="aff2"><label>2</label>Department of Cell and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK</aff><aff id="aff3"><label>3</label>BIOSS Centre for Biological Signalling Studies and Institute for Biology I, University of Freiburg, Sch&#x000e4;nzlestrasse 18, 79104 Freiburg, Germany</aff><aff id="aff4"><label>4</label>Spemann Graduate School of Biology and Medicine, Research Training Program GRK 1104, Albert Ludwigs University, 79104 Freiburg, Germany</aff><aff id="aff5"><label>5</label>European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany</aff>
Developmental Cell
<sec id="sec1"><title>Introduction</title><p id="p0030">Signaling by c-Jun N-terminal kinase (JNK) mediates one of the major stress response pathways (<xref rid="bib8" ref-type="bibr">Chen, 2012</xref>, <xref rid="bib39" ref-type="bibr">Stronach and Perrimon, 1999</xref>). Indeed, activation of JNK signaling often boosts or triggers apoptosis (<xref rid="bib10" ref-type="bibr">Dhanasekaran and Reddy, 2008</xref>, <xref rid="bib18" ref-type="bibr">Igaki, 2009</xref>, <xref rid="bib22" ref-type="bibr">Lepp&#x000e4; and Bohmann, 1999</xref>). JNK can exert its proapoptotic effect through phosphorylation of Jun, a component of the AP-1 transcriptional activator, or of other cellular proteins (<xref rid="bib5" ref-type="bibr">Bogoyevitch and Kobe, 2006</xref>). It is important to note, however, that JNK signaling does not always trigger apoptosis (<xref rid="bib43" ref-type="bibr">Weston and Davis, 2007</xref>) and has been shown to control nonapoptotic processes such as cytoskeletal rearrangements (<xref rid="bib16" ref-type="bibr">Homsy et&#x000a0;al., 2006</xref>), cell migration (<xref rid="bib33" ref-type="bibr">R&#x000ed;os-Barrera and Riesgo-Escovar, 2013</xref>), and cell proliferation (<xref rid="bib35" ref-type="bibr">Shaulian and Karin, 2002</xref>) during development and regeneration. It is generally thought that the cellular context or the activity of other signaling pathways determines whether JNK signaling leads to apoptosis or not. In well-documented instances, this involves downregulation or blunting of JNK signaling itself, e.g., through the activity of Gadd45&#x003b2;, an NF-&#x003ba;B-induced factor (<xref rid="bib9" ref-type="bibr">De Smaele et&#x000a0;al., 2001</xref>, <xref rid="bib29" ref-type="bibr">Papa et&#x000a0;al., 2004</xref>), or by Puckered, a feedback inhibitor of JNK signaling (<xref rid="bib25" ref-type="bibr">McEwen and Peifer, 2005</xref>). Mechanisms that dampen JNK&#x02019;s proapoptotic influence without affecting core pathway activity have also been documented. For example, in the developing <italic>Drosophila</italic> eye, mitogen-activated protein kinase phosphorylates and destabilizes Hid, a proapoptotic protein transcriptionally activated by JNK signaling in this tissue (<xref rid="bib4" ref-type="bibr">Bergmann et&#x000a0;al., 1998</xref>). Another documented process involves transcriptional repression of <italic>hid</italic>, which would otherwise be overactivated by JNK in response to irradiation damage (<xref rid="bib23" ref-type="bibr">Luo et&#x000a0;al., 2007</xref>). Non-cell-autonomous protective mechanisms could also be at work. For example, the transcriptional modulator Schnurri limits radiation-induced tissue damage by recruiting macrophages through activation of the PDGF-related growth factor Pvf1 (<xref rid="bib19" ref-type="bibr">Kelsey et&#x000a0;al., 2012</xref>). All the aforementioned mechanisms have been shown to operate in response to cellular stress. However, so far, little is known about the regulatory processes that prevent JNK from causing apoptosis during normal development. One well-characterized nonapoptotic JNK-dependent developmental process is the morphogenetic movement of dorsal closure (<xref rid="bib13" ref-type="bibr">Glise and Noselli, 1997</xref>, <xref rid="bib17" ref-type="bibr">Hou et&#x000a0;al., 1997</xref>, <xref rid="bib31" ref-type="bibr">Riesgo-Escovar and Hafen, 1997</xref>). Here, we set out to investigate the molecular mechanisms that prevent JNK from activating apoptosis during this process.</p><p id="p0035">Dorsal closure is a morphogenetic movement that closes a large gap left on the embryo&#x02019;s dorsal side after germ band retraction. It involves the concerted movement of the dorsal epidermis toward the midline and requires both JNK and Dpp signaling. One current view is that JNK signaling at the leading edge promotes expression of Decapentaplegic (Dpp, a transforming growth factor &#x003b2; [TGF-&#x003b2;] homolog), which, in turn, orchestrates the cell shape changes required for dorsal closure (<xref rid="bib11" ref-type="bibr">Fern&#x000e1;ndez et&#x000a0;al., 2007</xref>, <xref rid="bib33" ref-type="bibr">R&#x000ed;os-Barrera and Riesgo-Escovar, 2013</xref>). It is likely that a protective mechanism is at work at the leading edge since JNK does not trigger apoptosis there. Additional evidence that the dorsal epidermis is protected from apoptosis came from the analysis of <italic>crumbs</italic> (<italic>crb</italic>) (abbreviated as <italic>crb</italic> in genotypic descriptions) mutants, where JNK target genes are upregulated in response to loss of apicobasal polarity (<xref rid="bib20" ref-type="bibr">Kolahgar et&#x000a0;al., 2011</xref>). In such embryos, most epidermal cells undergo apoptosis, except in an approximately ten-cell-wide band of dorsal cells, despite strong activation of JNK signaling there. It appears, therefore, that the protective mechanism acts over a broader domain than just within the leading edge (see <xref rid="fig1" ref-type="fig">Figures 1</xref>A and 1B for a diagram of the relevant region of the embryonic epidermis).<fig id="fig1"><label>Figure&#x000a0;1</label><caption><p>Epithelial Disruption Triggers Canonical JNK Signaling, which, in Turn, Activates <italic>reaper</italic> Transcription in the Ventral&#x000a0;and Lateral Epidermis of <italic>Drosophila</italic> Embryos</p><p>(A and B) Diagram of <italic>Drosophila</italic> embryos at stage&#x000a0;(st.) 11 and stage 12/13, indicating the key epidermal domains: the dorsal (D) edge (deep red), the dorsal epidermis (faded red), the lateral (L) epidermis (green), and the ventral epidermis (purple). as, amnioserosa.</p><p>(C&#x02013;F) Expression of <italic>reaper</italic> in control (<italic>crb</italic><sup><italic>2</italic></sup><italic>/+</italic>) and <italic>crumbs</italic> mutant (<italic>crb</italic><sup><italic>2</italic></sup><italic>/ crb</italic><sup><italic>2</italic></sup>) embryos at stages 11 (C and D) and 12/13 (E and F), as indicated. In&#x000a0;homozygous <italic>crumbs</italic> mutants, segmental upregulation is seen in the ventrolateral (V) epidermis, while in the dorsal (D) epidermis (inside white dotted lines), it remains silent. mRNA, messenger RNA.</p><p>(G&#x02013;J) A fluorescent reporter of JNK activity (<xref rid="bib7" ref-type="bibr">Chatterjee and Bohmann, 2012</xref>) is active at the edge of the dorsal epidermis of control embryos (<italic>crb</italic><sup><italic>2</italic></sup><italic>/+</italic>) and throughout the epidermis of homozygous <italic>crumbs</italic> mutant embryos (<italic>crb</italic><sup><italic>2</italic></sup><italic>/ crb</italic><sup><italic>2</italic></sup>). Engrailed (En) immunoreactivity provides positional landmarks along the anterior-posterior axis. Embryonic stages are indicated.</p><p>(K&#x02013;N) Expression of <italic>reaper</italic> is not activated in <italic>crumbs</italic> mutant embryos that also lack <italic>jra</italic> (jun) or <italic>kayak</italic> (fos). As positive controls, <italic>crumbs</italic> mutant embryos were stained in parallel (data not shown). Genotypes and stages are indicated.</p><p>Scale bar, 200&#x000a0;&#x003bc;m.</p></caption><graphic xlink:href="gr1"/></fig></p><p id="p0040">Most apoptosis in <italic>Drosophila</italic> requires the <italic>H99</italic> locus (<xref rid="bib44" ref-type="bibr">White et&#x000a0;al., 1994</xref>), which comprises the three main proapoptotic genes: <italic>reaper</italic>, <italic>hid</italic>, and <italic>grim</italic>. Among these, <italic>reaper</italic> is the most likely mediator of the response to epithelial disruption since it is upregulated in <italic>crb</italic> mutant embryos in a pattern prefiguring that of caspase immunoreactivity (<xref rid="bib20" ref-type="bibr">Kolahgar et&#x000a0;al., 2011</xref>). Moreover, overexpression of Puckered, a phosphatase that inhibits JNK signaling prevents <italic>reaper</italic> upregulation, as well as apoptosis, in <italic>crb</italic> mutants (<xref rid="bib20" ref-type="bibr">Kolahgar et&#x000a0;al., 2011</xref>). These observations suggested that loss of apicobasal polarity triggers JNK signaling (through an unknown mechanism), which, in turn, causes <italic>reaper</italic> expression and, hence, apoptosis. However, in the dorsal epidermis, JNK signaling does not activate <italic>reaper</italic> expression. Here, we show the molecular mechanism underpinning such protection and thus explain how JNK can control cell migration without triggering apoptosis.</p></sec><sec id="sec2"><title>Results and Discussion</title><sec id="sec2.1"><title>Activation of <italic>reaper</italic> by Canonical JNK Signaling Mediates Apoptosis in Response to Epithelial Disruption</title><p id="p0045">In <italic>crb</italic> mutant embryos, <italic>reaper</italic> is strongly upregulated (<xref rid="fig1" ref-type="fig">Figures 1</xref>C&#x02013;1F) in a pattern similar to that of apoptosis (<xref rid="app2" ref-type="sec">Figures S1</xref>A and S1B available online), while the other two main proapoptotic genes, <italic>hid</italic> and <italic>grim</italic>, remain largely silent (<xref rid="bib20" ref-type="bibr">Kolahgar et&#x000a0;al., 2011</xref>)).&#x000a0;No activated caspase immunoreactivity was detectable in <italic>rpr</italic><sup><italic>87</italic></sup> <italic>crb</italic> double mutant embryos (<xref rid="app2" ref-type="sec">Figure&#x000a0;S1</xref>C), confirming the essential role of <italic>reaper</italic> and highlighting the need to uncover the mechanisms that activate <italic>reaper</italic> expression following loss of apicobasal polarity. As suggested previously (<xref rid="bib20" ref-type="bibr">Kolahgar et&#x000a0;al., 2011</xref>), JNK signaling is likely involved. Indeed, JNK signaling, as measured with a transcriptional reporter (AP-1&#x000a0;&#x0003e; GFP) (<xref rid="bib7" ref-type="bibr">Chatterjee and Bohmann, 2012</xref>) was strongly activated in <italic>crumbs</italic> mutant embryos (<xref rid="fig1" ref-type="fig">Figures 1</xref>G&#x02013;1J; <xref rid="mmc2" ref-type="supplementary-material">Movie S1</xref>). Moreover, little <italic>reaper</italic> transcription was detectable in <italic>crumbs</italic> embryos that also lack <italic>jra</italic> or <italic>kayak</italic>, which encode the two components of AP-1, Jun, and Fos (<xref rid="fig1" ref-type="fig">Figures 1</xref>K&#x02013;1N; see <xref rid="app2" ref-type="sec">Figures S1</xref>F&#x02013;S1I for <italic>reaper</italic> expression in the single mutants), and this was associated with a near-absence of apoptosis, as reported by activated caspase-3 immunoreactivity (<xref rid="app2" ref-type="sec">Figures S1</xref>D and S1E). Notably, JNK signaling does not seem to necessarily cause apoptosis. In the ventral epidermis, the patterns of <italic>reaper</italic> expression and apoptosis (highlighted with anti-activated caspase) appeared to mirror the early segmental activation of JNK signaling (compare <xref rid="fig1" ref-type="fig">Figure&#x000a0;1</xref>D with <xref rid="app2" ref-type="sec">Figure&#x000a0;S1</xref>B), suggesting a relatively straightforward, likely causal, relationship there. However, neither <italic>reaper</italic> expression nor apoptosis were significantly activated in the dorsal epidermis, even at the dorsal edge, where JNK signaling is particularly active both in wild-type and <italic>crumbs</italic> mutants. What is the mechanism that protects the dorsal epidermis from the proapoptotic effect of JNK?</p></sec><sec id="sec2.2"><title>Dpp Signaling Prevents JNK from Activating <italic>reaper</italic> Expression in the Dorsal Epidermis</title><p id="p0050">One feature of the dorsal epidermis is that it is under the influence of Dpp, a member of the BMP family of secreted growth factors (<xref rid="bib15" ref-type="bibr">Hamaratoglu et&#x000a0;al., 2014</xref>). Indeed, phospho-Smad (p-Smad) immunoreactivity (a mark of Dpp signaling) (<xref rid="bib40" ref-type="bibr">Tanimoto et&#x000a0;al., 2000</xref>) was detectable in this region before and during the time when JNK is active (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>A). Therefore, Dpp signaling could prevent JNK signaling from activating <italic>reaper</italic> expression both in the dorsal epidermis of <italic>crumbs</italic> mutant embryos and, physiologically, at the dorsal edge of wild-type embryos. This was tested in embryos expressing a Dpp RNA interference (RNAi)-encoding transgene (<xref rid="app2" ref-type="sec">Supplemental Information</xref>) under the control of the ubiquitous <italic>tubulin-gal4</italic> driver. This led to reduced signaling as indicated by the loss of p-Smad immunoreactivity from stage 11 onward (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>B). In these embryos, a band of <italic>reaper</italic> transcription was observed at the dorsal edge (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>C), where JNK is known to be activated in the wild-type (see <xref rid="fig1" ref-type="fig">Figure&#x000a0;1</xref>I). A similar result was seen in embryos lacking zygotic (but not maternal) activity of <italic>thickveins</italic> (<italic>tkv</italic>), which encodes an essential Dpp receptor (<xref rid="bib27" ref-type="bibr">Nellen et&#x000a0;al., 1994</xref>) (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>D). Expression of <italic>reaper</italic> was also seen in the approximately ten-cell-wide dorsal region in <italic>tkv crumbs</italic> double mutants (as well as in the rest of the epidermis; <xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>F), suggesting that Dpp signaling prevents <italic>reaper</italic> expression throughout the dorsal epidermis. Caspase immunoreactivity became detectable throughout the epidermis of <italic>tkv crumbs</italic> mutants (<xref rid="app2" ref-type="sec">Figure&#x000a0;S1</xref>J), consistent with the notion that repression of <italic>reaper</italic> expression by Dpp signaling translates into anti-apoptotic activity.<fig id="fig2"><label>Figure&#x000a0;2</label><caption><p>Dpp Signaling Counteracts JNK-Dependent Activation of <italic>reaper</italic> via the Transcriptional Repressor Schnurri</p><p>(A and B) p-Smad immunoreactivity, seen in control embryos (A), is not detectable in embryos carrying both UAS-dpp[RNAi] and <italic>tubulin-Gal4</italic> (B), confirming the effectiveness of this RNAi transgene. Anti-Engrailed and DAPI were used as morphological landmarks. En, Engrailed; st., stage.</p><p>(C) In embryos expressing UAS-dpp[RNAi] under the control of <italic>tubulin-Gal4</italic>, <italic>reaper</italic> expression becomes upregulated at the dorsal edge of the epidermis. Such embryos are often misshapen probably because of interference with the patterning activity of Dpp.</p><p>(D) Expression of <italic>reaper</italic> is also upregulated at the dorsal edge of homozygous <italic>thickveins (tkv)</italic> mutants.</p><p>(E) Upregulation of <italic>reaper</italic> in the dorsal and lateral epidermis of <italic>schnurri</italic> mutants. Expression can be seen along the whole dorsal edge but is segmental elsewhere. The basis of the segmental pattern is unknown.</p><p>(F) Upregulation of <italic>reaper</italic> throughout most of the epidermis of <italic>thickveins crumbs</italic> double mutants.</p><p>(G) No <italic>reaper</italic> upregulation is detected in <italic>schnurri kayak (fos)</italic> double mutants, demonstrating the requirement for canonical JNK signaling in <italic>reaper</italic> activation.</p><p>(H) Expression of <italic>reaper</italic> is upregulated throughout the epidermis of <italic>schnurri crumbs</italic> double mutants in a pattern that is roughly the sum of those seen in the single mutants.</p><p>Scale bar, 200&#x000a0;&#x003bc;m.</p></caption><graphic xlink:href="gr2"/></fig></p></sec><sec id="sec2.3"><title>Schnurri Prevents <italic>reaper</italic> Transcription and Apoptosis</title><p id="p0055">Dpp signaling is mediated by the Mad complex, which can activate or repress target genes depending on the sequence context and other cofactors (<xref rid="bib2" ref-type="bibr">Affolter and Basler, 2007</xref>). The best characterized instance of repression by Dpp signaling is that of the <italic>brinker</italic> gene, which occurs via silencer elements where the Mad complex recruits the corepressor Schnurri (<xref rid="bib2" ref-type="bibr">Affolter and Basler, 2007</xref>, <xref rid="bib30" ref-type="bibr">Pyrowolakis et&#x000a0;al., 2004</xref>). We therefore asked if Schnurri could mediate the repression of <italic>reaper</italic> by Dpp. Indeed, <italic>reaper</italic> transcription was upregulated at the dorsal edge of <italic>schnurri</italic> mutant embryos (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>E; <xref rid="app2" ref-type="sec">Figures S1</xref>O&#x02013;S1R). In these embryos, <italic>reaper</italic> expression was also seen to extend segmentally in the lateral region, a feature not seen in Dpp-RNAi-expressing embryos (compare <xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>E with <xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>C), perhaps because Schnurri also has Dpp-independent activity, as suggested by (<xref rid="bib19" ref-type="bibr">Kelsey et&#x000a0;al., 2012</xref>). In any case, the <italic>reaper</italic>-repressive role of Schnurri in the dorsal epidermis was confirmed in <italic>schnurri crumbs</italic> double mutants, where <italic>reaper</italic> transcription was strongly upregulated in both the dorsal and ventral regions (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>H). No <italic>reaper</italic> upregulation was seen in <italic>schnurri kayak (fos)</italic> double mutants (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>G), showing that JNK signaling is an essential positive input to <italic>reaper</italic> transcription at the dorsal edge, as well as in segmentally repeated lateral domains, where the JNK sensor appears not to be sufficiently sensitive to detect activity. It is worth noting that staining of <italic>schnurri</italic> and <italic>schnurri crumbs</italic> embryos with anti-activated caspase confirmed that the repression of <italic>reaper</italic> transcription by Schnurri is needed to suppress apoptosis (<xref rid="app2" ref-type="sec">Figures S1</xref>K and S1L). As AP-1 and Schnurri are both transcriptional regulators, we next sought to address whether the opposing influences of Schnurri and JNK signaling converge directly on the <italic>reaper</italic> promoter.</p></sec><sec id="sec2.4"><title>Binding Sites Upstream of the <italic>reaper</italic> Promoter Integrate the Effects of Dpp and JNK</title><p id="p0060">The promoter region of <italic>brinker</italic> that mediates Schnurri-dependent repression has been extensively characterized, and mutation analysis identified an essential 16-base-pair (bp) repressor sequence (<xref rid="bib30" ref-type="bibr">Pyrowolakis et&#x000a0;al., 2004</xref>). Similar elements are found&#x000a0;at &#x0223c;350 positions in the <italic>Drosophila</italic> genome, defining a consensus sequence: GRCGNCNNNNNGTCTG (<xref rid="bib30" ref-type="bibr">Pyrowolakis et&#x000a0;al., 2004</xref>). Two related sites were identified upstream of <italic>reaper</italic>, in a region that is conserved in the 12 sequenced <italic>Drosophila</italic> species. In all these species, the proximal site (SE<sub>p</sub>) is flanked on either side by a predicted AP-1-binding site, making it a potential regulatory element. Because SE<sub>p</sub> is not an exact match to the consensus Schnurri binding site, we used an electrophoretic mobility shift assay (EMSA) to test whether it is recognized by Schnurri, using the previously characterized site from the <italic>brinker</italic> gene (cSE) (<xref rid="bib30" ref-type="bibr">Pyrowolakis et&#x000a0;al., 2004</xref>) as a positive control. Recombinant Schnurri protein induced a supershift of the Mad-Medea-DNA complex in both cases, although to a lesser extent with SE<sub>p</sub> than with cSE (<xref rid="app2" ref-type="sec">Figure&#x000a0;S2</xref>A). Thus, we conclude that SE<sub>p</sub> is recognized by Schnurri/Mad/Medea and could therefore mediate the repressive influence of Dpp on <italic>reaper</italic> expression. By extension, the module comprising SE<sub>p</sub> and the two putative AP-1 sites could integrate the influence of Dpp and JNK signaling on <italic>reaper</italic> expression. To test this hypothesis in&#x000a0;vivo, we made a reporter construct comprising 5.5 kb of sequence including this module and the basal <italic>reaper</italic> promoter, upstream of a GFP complementary DNA (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>A). This reporter (<italic>rpr-GFP</italic>) and the variants described later were introduced by PhiC31-mediated integration at the same genomic location to allow comparison without confounding influence from position effects. The wild-type reporter was essentially silent in wild-type embryos (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>B), as expected, since <italic>reaper</italic> expression is barely detectable during normal embryogenesis. By contrast, in <italic>crumbs</italic> mutants, <italic>rpr-GFP</italic> became segmentally upregulated in the ventrolateral&#x02014;but not dorsal&#x02014;epidermis (double-headed arrow in <xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>C), thus mirroring the activity of the endogenous <italic>reaper</italic> gene in this background (compare to <xref rid="fig1" ref-type="fig">Figure&#x000a0;1</xref>F). Critically, like the endogenous <italic>reaper</italic> gene, this reporter became active in the dorsal epidermis of <italic>schnurri</italic> mutants (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>D). Such dorsal expression was segmentally modulated (see <xref rid="app2" ref-type="sec">Figure&#x000a0;S2</xref>B for GFP staining alone), resembling the pattern of endogenous <italic>reaper</italic> transcription in <italic>schnurri</italic> mutants (<xref rid="fig2" ref-type="fig">Figure&#x000a0;2</xref>E). Also, like endogenous <italic>reaper</italic>, the reporter was widely and strongly activated in the epidermis of <italic>schnurri crumbs</italic> double mutants (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>E). These observations suggest that Schnurri mediates repression of the reporter. To&#x000a0;test the contribution of the predicted Schnurri binding site, a mutation was introduced in the reporter (ATCGTCTCGCCGTCTG &#x02192; ATCGTCTCGCTTTCTG), thus creating <italic>rpr[&#x00394;Shn]-GFP</italic> (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>A). This mutation was found to abrogate formation of the Mad-Medea-DNA complex in&#x000a0;vitro (see SEm in <xref rid="app2" ref-type="sec">Figure&#x000a0;S2</xref>A), suggesting that <italic>rpr[&#x00394;Shn]-GFP</italic> would no longer be subject to repression by Schnurri. Indeed, this transgene became activated in wild-type embryos in the dorsal epidermis (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>F). One must point out that this dorsal activity of <italic>rpr[&#x00394;Shn]-GFP</italic> did not appear before stage 13, 1&#x02013;2&#x000a0;hr later than the appearance of <italic>rpr-GFP</italic> in <italic>schnurri</italic> mutants (compare <xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>F to <xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>D). Also, unlike <italic>rpr-GFP</italic> in <italic>schnurri</italic> mutants, <italic>rpr[&#x00394;Shn]-GFP</italic> was active in segmentally repeated ventral domains at stage 13. Despite these differences (see further discussion in the legend of <xref rid="app2" ref-type="sec">Figure&#x000a0;S2</xref>), the upregulation of <italic>rpr-GFP</italic> in <italic>schnurri</italic> mutants and the expression of <italic>rpr[&#x00394;Shn]-GFP</italic> in the dorsal epidermis of otherwise wild-type embryos (where <italic>rpr-GFP</italic> is silent) are consistent with the notion that Schnurri represses <italic>reaper</italic> expression in the dorsal epidermis, thus allowing JNK signaling to control epithelial migration without triggering apoptosis.<fig id="fig3"><label>Figure&#x000a0;3</label><caption><p>The <italic>reaper</italic> Promoter Integrates Inputs from JNK and Dpp Signaling</p><p>(A) Diagram of the reporter constructs that were tested in transgenic embryos; 5.5 kb of the <italic>reaper</italic> promoter were used to create <italic>rpr-GFP</italic>. Predicted binding sites for Schnurri or AP-1 were mutated to generate the variants listed. Details of the mutations are indicated in the text.</p><p>(B) The unmutated reporter (<italic>rpr-GFP</italic>) is almost silent in wild-type embryos. En, Engrailed.</p><p>(C) In <italic>crumbs</italic> mutant embryos, the same reporter is active in the ventrolateral, but not the dorsal (double-headed arrow), epidermis.</p><p>(D) By contrast, in <italic>schnurri</italic> mutants, <italic>rpr-GFP</italic> becomes active in the dorsal epidermis, suggesting that Schnurri is an essential repressive factor.</p><p>(E) Embryos lacking both <italic>crumbs</italic> and <italic>schnurri</italic> upregulate <italic>rpr-GFP</italic> throughout much of the epidermis.</p><p>(F) Mutation of the predicted Schnurri binding site leads to upregulation of the reporter in the dorsal epidermis wild-type embryos at stage 13.</p><p>(G) In <italic>crumbs</italic> mutant embryos, <italic>rpr[&#x00394;Shn]-GFP</italic> upregulation is seen in the dorsal epidermis (double-headed arrow) as well as in the ventrolateral epidermis, consistent with the notion that Schnurri contributes to preventing <italic>reaper</italic> expression in the dorsal epidermis of <italic>crumbs</italic> embryos.</p><p>(H&#x02013;J) Upregulation of the <italic>reaper</italic> reporter in <italic>crumbs</italic> mutants requires the two predicted AP-1 binding sites. Deletion of either site leads to reduced expression while the double mutant reporter (<italic>rpr[&#x00394;AP1</italic><sup><italic>D</italic></sup><italic>; &#x00394;AP1</italic><sup><italic>P</italic></sup><italic>]-GFP</italic>) is silent.</p><p>(K) Gamma-irradiated embryos carrying the <italic>rpr[&#x00394;AP1</italic><sup><italic>D</italic></sup><italic>; &#x00394;AP1</italic><sup><italic>P</italic></sup><italic>]-GFP</italic> transgene express GFP throughout, showing that this reporter is functional.</p><p>All embryos are shown at stage 11 except for (F), which shows a stage 12&#x02013;13 embryo. Engrailed (En), shown in white, provides an indication of the embryos&#x02019; overall morphology.</p></caption><graphic xlink:href="gr3"/></fig></p><p id="p0065">We next assessed the function of the two predicted AP-1 binding sites by mutating them (TGACTCATA &#x02192; TGACATTTA) (<xref rid="bib7" ref-type="bibr">Chatterjee and Bohmann, 2012</xref>) individually or as a pair in the GFP reporter (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>A) and assessing their activity in the ventral epidermis of <italic>crumbs</italic> embryos, where the wild-type reporter is strongly activated. Mutating one or the other site reduced activation, while the double mutant reporter (<italic>rpr[&#x00394;AP1</italic><sup><italic>P</italic></sup><italic>;&#x00394;AP1</italic><sup><italic>D</italic></sup><italic>]-GFP</italic>) had no detectable activity in <italic>crumbs</italic> mutants (<xref rid="fig3" ref-type="fig">Figures 3</xref>H&#x02013;3J; wild-type background in <xref rid="app2" ref-type="sec">Figure&#x000a0;S2</xref>E). This reporter is nevertheless functional. It was activated by irradiation (<xref rid="fig3" ref-type="fig">Figure&#x000a0;3</xref>K), consistent with the presence of a p53-response element previously shown to mediate the response to irradiation (<xref rid="bib6" ref-type="bibr">Brodsky et&#x000a0;al., 2000</xref>). It is worth pointing out here that <italic>reaper</italic> expression was still activated in <italic>crb p53</italic><sup><italic>5A-1-4</italic></sup> double mutants (P.F. Langton and J.-P.V., unpublished data), indicating that the response to loss of epithelial integrity does not require p53. Overall, our results show that the two predicted AP-1 binding sites contribute redundantly to <italic>reaper</italic> upregulation in <italic>crumbs</italic> mutants. They also show that JNK signaling acts directly on the <italic>reaper</italic> promoter and not via a relay mechanism. We conclude that a small regulatory module allows JNK to trigger apoptosis, except in dorsal cells that are protected by Dpp signaling.</p></sec><sec id="sec2.5"><title>Schnurri Ensures the Survival of Dorsal Edge Cells during Dorsal Closure</title><p id="p0070">JNK and Dpp signaling have extensively been shown to orchestrate dorsal closure. So far, attention has been focused on the role of these pathways in triggering the cell shape changes required for the dorsal epidermis to spread over the amnioserosa and meet at the dorsal midline (<xref rid="bib11" ref-type="bibr">Fern&#x000e1;ndez et&#x000a0;al., 2007</xref>, <xref rid="bib16" ref-type="bibr">Homsy et&#x000a0;al., 2006</xref>, <xref rid="bib32" ref-type="bibr">Riesgo-Escovar et&#x000a0;al., 1996</xref>). Our results suggest an additional role for Dpp signaling during dorsal closure, namely, to ensure the survival of leading edge cells. We propose that such a protective mechanism is needed because of the proapoptotic influence of JNK signaling. Accordingly, the &#x0201c;dorsal open phenotype&#x0201d; of <italic>schnurri</italic> mutants (and possibly other Dpp pathway mutants) would not only be caused by the failure of dorsal edge cells to migrate but also by their reduced survival. To evaluate the contribution of the latter, we assessed the extent of tissue loss in fixed and live <italic>schnurri</italic> mutant embryos, which, as previously reported (<xref rid="bib3" ref-type="bibr">Arora et&#x000a0;al., 1995</xref>, <xref rid="bib14" ref-type="bibr">Grieder et&#x000a0;al., 1995</xref>, <xref rid="bib37" ref-type="bibr">Staehling-Hampton et&#x000a0;al., 1995</xref>), do not undergo dorsal closure. Cursory observation suggests that, in <italic>schnurri</italic> mutant embryos, the dorsal hole that appears at the end of germband retraction gapes open over time (<xref rid="fig4" ref-type="fig">Figures 4</xref>A and 4B; <xref rid="app2" ref-type="sec">Figures S1</xref>Q and S1R). This was confirmed by confocal live imaging of <italic>schnurri</italic> mutants carrying <italic>spider-GFP</italic> as a marker of cell outlines (<xref rid="app2" ref-type="sec">Figure&#x000a0;S3</xref>D; <xref rid="mmc3" ref-type="supplementary-material">Movie S2</xref>). To test if gaping of the dorsal hole could be due to tissue shrinkage, stage &#x0223c;13 <italic>schnurri</italic> mutant embryos were stained with anti-Cut, which marks dorsal and ventral clusters of peripheral neurons along the D-V axis (<xref rid="fig4" ref-type="fig">Figures 4</xref>A&#x02013;4D; <xref rid="app2" ref-type="sec">Figures S3</xref>A&#x02013;S3C), and the number of Cut-positive cells in each cluster was counted. While cell number in the dorsal cluster remained constant in wild-type embryos, it decreased significantly in <italic>schnurri</italic> mutants (<xref rid="fig4" ref-type="fig">Figure&#x000a0;4</xref>E). The number of ventral cells was relatively unaffected in both genotypes. It appears, therefore, that the dorsal epidermis is preferentially eliminated prior to this stage. Next, we used multiview light-sheet microscopy (MuVi-SPIM; <xref rid="bib21" ref-type="bibr">Krzic et&#x000a0;al., 2012</xref>) to achieve in toto imaging of <italic>schnurri</italic> mutants and control embryos carrying histone-red fluorescent protein (histone-RFP) (<xref rid="fig4" ref-type="fig">Figures 4</xref>H&#x02013;4M). In <italic>schnurri</italic> mutants, cell debris could be seen around the dorsal edge, and many macrophages scurried around (<xref rid="mmc4" ref-type="supplementary-material">Movies S3</xref> and <xref rid="mmc5" ref-type="supplementary-material">S4</xref>). Moreover, the number of epidermal nuclei became reduced, compared to that in control embryos (<xref rid="app2" ref-type="sec">Figure&#x000a0;S4</xref>). These observations confirm that, during stages 12&#x02013;14, the dorsal epidermis of <italic>schnurri</italic> mutants progressively shrinks (<xref rid="app2" ref-type="sec">Figures S4</xref>A&#x02013;S4I), likely by apoptosis (see also <xref rid="app2" ref-type="sec">Figures S1</xref>K, S1Q, and S1R). Consistent with the involvement of apoptosis, the dorsal hole did not appear to gape open in <italic>schnurri</italic> mutants that also lacked <italic>reaper</italic> (<xref rid="fig4" ref-type="fig">Figure&#x000a0;4</xref>G), even though these embryos failed to complete dorsal closure. As shown in <xref rid="fig4" ref-type="fig">Figure&#x000a0;4</xref>H, the JNK reporter remains active throughout tissue shrinkage (<xref rid="fig4" ref-type="fig">Figures 4</xref>F&#x02013;4G), suggesting that, as dorsal edge cells are eliminated, adjacent epidermal cells activate JNK. Taken together, our results suggest that the dorsal open phenotype of <italic>schnurri</italic> mutants is a combined consequence of tissue loss by apoptosis and lack of cell migration over the amnioserosa.<fig id="fig4"><label>Figure&#x000a0;4</label><caption><p>Dorsal Closure and Epithelial Loss in Wild-Type and <italic>schnurri</italic> Mutant Embryos</p><p>(A and B) Homozygous <italic>schnurri</italic> mutant embryos fail to achieve dorsal closure. A heterozygous embryo of similar stage is shown as control.</p><p>(C and D) The reduced number of Cut-positive cells in the dorsal cluster of <italic>schnurri</italic> mutant embryos suggests that the dorsal epidermis shrinks during the time when dorsal closure takes place in control embryos.</p><p>(E) Number of Cut-positive cells in the dorsal (D) and ventral (V) clusters of <italic>schnurri</italic> homozygotes and control heterozygotes at stage &#x0223c;13. A significant loss of cells is seen in the dorsal cluster (Student&#x02019;s t test, <sup>&#x02217;</sup>p&#x000a0;&#x0003c; 0.0001). WT, wild-type; n.s., not significant. Error bars indicate SEM.</p><p>(F and G) <italic>schnurri</italic> and <italic>schnurri reaper</italic> mutant embryos carrying the JNK reporter. Gaping of dorsal hole is more pronounced in <italic>schnurri</italic> mutant (F) than in <italic>schnurri reaper</italic> mutant (G). En, Engrailed.</p><p>(H&#x02013;M) Still images from MuVi-SPIM recordings (before, during, and after closure). Approximate times from the beginning of germband retraction are shown to allow comparison between the mutant and control samples. Control (H&#x02013;J) and <italic>schnurri</italic> mutant (K&#x02013;M) embryos carrying histone-RFP as a nuclear marker are shown. While dorsal cells migrate over the amnioserosa in the wild-type, these cells are progressively lost in <italic>schnurri</italic> mutants.</p><p>See also <xref rid="mmc4" ref-type="supplementary-material">Movies S3</xref> and <xref rid="mmc5" ref-type="supplementary-material">S4</xref>.</p></caption><graphic xlink:href="gr4"/></fig></p><p id="p0075">Most functions of Dpp signaling are achieved through Schnurri-dependent repression of the transcriptional repressor Brinker. However, as we have shown, inhibition of <italic>reaper</italic> by Schnurri is direct and, hence, likely Brinker independent. By&#x000a0;contrast, the migration of dorsal edge cells is mediated by Brinker repression, since <italic>schnurri brinker</italic> double mutant larvae have a sealed dorsal midline (<xref rid="bib24" ref-type="bibr">Marty et&#x000a0;al., 2000</xref>, <xref rid="bib42" ref-type="bibr">Torres-Vazquez et&#x000a0;al., 2001</xref>). It is interesting that the dorsal epidermis of these double mutants is much reduced in surface area compared to that of wild-type embryos or <italic>brinker</italic> single mutant embryos, which have expanded dorsal fates (in <xref rid="bib42" ref-type="bibr">Torres-Vazquez et&#x000a0;al., 2001</xref>, compare Figure&#x000a0;2D to Figures 2A and 2B). We suggest that <italic>schnurri brinker</italic> double mutant embryos complete dorsal closure despite tissue loss, perhaps because a sufficient number of cells are able to migrate before undergoing apoptosis. To assess directly whether Brinker has any impact on <italic>reaper</italic> expression, we performed gain- and loss-of-function experiments (<xref rid="app2" ref-type="sec">Figures S3</xref>E&#x02013;S3I). No ectopic <italic>reaper</italic> expression was seen in embryos lacking or overexpressing <italic>brinker</italic> (<xref rid="app2" ref-type="sec">Figures S3</xref>G and S3H). Moreover, Brinker overexpression did not prevent <italic>reaper</italic> expression in <italic>crumbs</italic> mutant embryos (<xref rid="app2" ref-type="sec">Figures S3</xref>E and S3F), while overexpressing Schnurri did (<xref rid="app2" ref-type="sec">Figures S3</xref>E and S3I). We conclude that Brinker does not affect <italic>reaper</italic> expression and that Schnurri contributes to dorsal closure through two parallel routes: first, by repressing Brinker and, hence, allowing the derepression of genes regulating the cytoskeletal functions required for cell migration (<xref rid="bib11" ref-type="bibr">Fern&#x000e1;ndez et&#x000a0;al., 2007</xref>, <xref rid="bib16" ref-type="bibr">Homsy et&#x000a0;al., 2006</xref>); and second, by repressing <italic>reaper</italic>, thus ensuring survival of the dorsal edge cells in the face of JNK&#x02019;s proapoptotic pressure. Our results highlight the central role of Dpp and Schnurri in cell survival, extending observations on Dpp in imaginal discs (<xref rid="bib1" ref-type="bibr">Adachi-Yamada et&#x000a0;al., 1999</xref>, <xref rid="bib12" ref-type="bibr">Gibson and Perrimon, 2005</xref>, <xref rid="bib26" ref-type="bibr">Moreno et&#x000a0;al., 2002</xref>, <xref rid="bib36" ref-type="bibr">Shen and Dahmann, 2005</xref>) and TGF&#x003b2; in vertebrates (<xref rid="bib34" ref-type="bibr">Sabapathy et&#x000a0;al., 1999</xref>, <xref rid="bib41" ref-type="bibr">Taya et&#x000a0;al., 1999</xref>).</p></sec><sec id="sec2.6"><title>Conclusions</title><p id="p0080">In many contexts, JNK signaling leads to apoptosis, perhaps a remnant of this pathway&#x02019;s ancestral function in stress response (<xref rid="bib33" ref-type="bibr">R&#x000ed;os-Barrera and Riesgo-Escovar, 2013</xref>). JNK signaling may have started to regulate cytoskeletal functions in multicellular organisms to facilitate delamination or extrusion of defective cells. It is conceivable that such a regulatory relation might have subsequently been coopted to control other migratory behaviors such as those required for dorsal closure. Evidently, this would have necessitated coevolution of a protective, anti-apoptotic mechanism. At the dorsal edge of the epidermis, this is mediated by Dpp, which, intriguingly, is itself under the control of JNK signaling. Therefore, under the right circumstances, JNK contributes to the mechanism that counteracts its own AP-1-dependent proapoptotic pressure. The outcome of the regulatory network linking JNK and Dpp to <italic>reaper</italic> must be finely balanced, since a mild increase in JNK signaling (e.g., in <italic>puckered</italic> heterozygous embryos) triggers <italic>reaper</italic> expression (<xref rid="bib20" ref-type="bibr">Kolahgar et&#x000a0;al., 2011</xref>). Unlike the ventral epidermis, the dorsal epidermis seems prone to expressing Dpp in response to JNK, perhaps because of earlier expression in this region or through the action of additional regulators. This is likely to ensure the survival of dorsal edge cells during their migration. As we have shown, the opposing influences of Dpp and JNK are played out within the regulatory region of <italic>reaper.</italic> The anti-apoptotic activity of Dpp is mediated by Schnurri, a protein that could have more general anti-apoptotic activity since mammalian Schnurri has been shown to dampen cell death during T&#x000a0;cell development (<xref rid="bib38" ref-type="bibr">Staton et&#x000a0;al., 2011</xref>). The function of this or similar regulatory modules in other tissues, contexts, and models can thus yield a broader understanding of the balance between apoptosis and survival at the intersection of signaling pathways.</p></sec></sec><sec id="sec3"><title>Experimental Procedures</title><p id="p0085">Details on materials and methods can be found in the <xref rid="app2" ref-type="sec">Supplemental Information</xref>. These include a full list of the <italic>Drosophila</italic> strains and antibodies as well as step-by-step staining protocols for immunofluorescence and in&#x000a0;situ hybridization. For gamma irradiation, embryos were collected for 4&#x000a0;hr, aged a further 4&#x000a0;hr, and introduced in a gamma-cell irradiator for a 4,000&#x000a0;rad exposure (<xref rid="bib28" ref-type="bibr">Nordstrom et&#x000a0;al., 1996</xref>). The embryos were allowed to recover for 2&#x000a0;hr at 25&#x000b0;C before further analysis. The <italic>reaper</italic> reporter constructs were created by standard molecular biology with primers listed in the <xref rid="app2" ref-type="sec">Supplemental Information</xref>. They were introduced into the <italic>Drosophila</italic> genome by PhiC31-mediated integration into PBac[yellow[+]-attP-9A] VK00027 (Bloomington <italic>Drosophila</italic> Stock Center # 9744, on chromosome III). EMSAs were largely performed as described elsewhere (<xref rid="bib30" ref-type="bibr">Pyrowolakis et&#x000a0;al., 2004</xref>). Live embryo imaging was performed either by classical confocal or MuVi-SPIM. For classical confocal microscopy, we used a Leica SP5 microscope equipped with a resonant scanner and a 20&#x000d7; (NA, 0.8) water immersion objective. For MuVi-SPIM, we used a custom-built set-up (two Nikon 10&#x000d7; 0.3-NA illumination objective lenses and two Nikon 25&#x000d7; 1.1-NA detection lenses) and protocols as described elsewhere (<xref rid="bib21" ref-type="bibr">Krzic et&#x000a0;al., 2012</xref>). The custom-modified Hamamatsu Flash 4 cameras were operated in the Lightsheet Readout Mode to reject scattered photons. Details on image processing and data visualization are provided in the <xref rid="app2" ref-type="sec">Supplemental Information</xref>.</p></sec>
Effects of oral anticoagulation with various INR levels in deep vein thrombosis cases
<sec><title>Aim</title><p>In order to avoid the complications associated with thromboembolic disease, patients with this condition typically are placed on long-term anticoagulant therapy. This report compares bleeding complications in this patient population by level of achieved INR.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><p>During the 6-year period between January 1997 and January 2003, 386 patients with venous thromboembolism of the lower extremities were admitted to the Cardiovascular Surgery Outpatient Clinic of Alsancak State Hospital. Of the 386 patients, 198 (51.2%) were women, and the average age was 52.3 years. All diagnoses of venous thromboembolism were confirmed by means of Doppler ultrasonography. Further investigation showed occult neoplasms in 22 (5.6%) of the cases. We excluded the patients with occult disease, and the remaining 364 constituted our study population.</p></sec><sec><title>Results</title><p>Oral anticoagulation was standardized at 6 months' duration in all cases. We divided the patients into two groups. Group I consisted of 192 patients (52.7%) with INR values between 1.9 and 2.5; Group II comprised 172 patients with INR values between 2.6 and 3.5. Complications in each group were assessed and compared. The minor hemorrhage rate was 1.04% in Group I and 4.06% in Group II. The major hemorrhage rate was also 1.04% in Group I and was 6.3% in Group II. We determined that the complication rates for both minor and major hemorrhage were significant in patients with INR values above 2.5.</p></sec><sec><title>Conclusion</title><p>Oral anticoagulation must be followed closely in patients with venous thromboembolism. Higher INR levels are associated with significant increases in hemorrhage and associated complications. INR values of 2.0 to 2.5 are sufficient for long-term anticoagulant therapy, ensuring ideal anticoagulation levels and minimizing the complication rate.</p></sec>
<contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Yetkin</surname><given-names>Ufuk</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Karabay</surname><given-names>&#x000d6;zalp</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>&#x000d6;nol</surname><given-names>Hakan</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib>
Current Controlled Trials in Cardiovascular Medicine
<sec><title>Background</title><p>Deep vein thrombosis (DVT) is the presence in lower-extremity veins of a thrombus that obstructs the branches and blood flow. The natural course of DVT therapy is associated with significant morbidity and mortality rates [<xref ref-type="bibr" rid="B1">1</xref>]. Standard therapy for acute DVT consists of heparin followed by oral warfarin [<xref ref-type="bibr" rid="B2">2</xref>]. To monitor therapy with coumadin anticoagulants, prothrombin time (PT) was the standard until as recently as the past decade [<xref ref-type="bibr" rid="B3">3</xref>]. Because the therapeutic range of PT depends on many factors, comparing patient results and assessing the significance of those results can be difficult. Therefore, the World Health Organization (WHO) developed an international standardization system known as the international normalized ratio (INR) [<xref ref-type="bibr" rid="B4">4</xref>].</p><p>We monitored anticoagulation therapy and compared bleeding complications among outpatients with lower-extremity DVTs who were taking oral anticoagulants (OACs) and who exhibited two different ranges of INR values.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><p>Between January, 1997 and January, 2003, 386 patients with a preliminary diagnosis of venous thromboembolism were admitted to the Cardiovascular Surgery Outpatient Clinic at Alsancak State Hospital. Of these patients, 198 (51.2%) were women and 188 (48.4%) were men. The average age of all patients was 52.3 years (range, 23&#x02013;81 years). In all cases, the diagnosis of venous thromboembolism was confirmed by means of Doppler ultrasonography (USG). When admitted, no patients in the study group were known to have malignant disease as the etiologic factor of DVT. We excluded patients with superficial thrombophlebitis, thrombosis, and recurrent DVT. To investigate for tumors and establish the diagnoses, we used chest radiographs and laboratory tests (erythrocyte sedimentation rate, C-reactive protein, complete blood count, urea, creatinine, serum glutamic oxaloacetic transaminase [SGOT], serum glutamic pyruvic transaminase [SGPT], gamma glutamyl transferase [gamma GT], alkaline phosphatase [ALP], bilirubin, electrophoresis, total protein, alpha fetoprotein [&#x003b1;-FP], fibrinogen, cancer antigen 19-9 [CA-19-9], and in men prostate-specific antigen [PSA]). Abdominal ultrasonography was also used if indicated. We identified previously unrecognized neoplasms in 22 (5.6%) patients (Table <xref ref-type="table" rid="T1">1</xref>). We referred these patients to oncology clinics and limited our study population to the remaining 364 patients. Of this group, 42 (11.5%) were hospitalized for an average of 5.2 days and received intravenous (IV) heparin plus oral anticoagulants. We combined low-molecular-weight heparin (LMWH) with oral anticoagulants for the other patients and followed them in our outpatient clinic. For long-term anticoagulation we used the same drug (coumadin) and monitored results as INR values.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Characteristics of patients admitted with lower extremity venous thromboembolism prediagnosis and found to have various occult malignancies</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left">System with occult malignancy</td><td align="center">Cases</td></tr></thead><tbody><tr><td align="left">Ovarian Ca</td><td align="center">6</td></tr><tr><td align="left">Colon Ca</td><td align="center">5</td></tr><tr><td align="left">Bladder Ca</td><td align="center">5</td></tr><tr><td align="left">Lung Ca</td><td align="center">3</td></tr><tr><td align="left">Prostate Ca</td><td align="center">2</td></tr><tr><td align="left">Extensive genital Ca + peritonitis carcinomatosa</td><td align="center">1</td></tr><tr><td align="left">Total</td><td align="center">22</td></tr></tbody></table></table-wrap></sec><sec><title>Results</title><p>We standardized oral anticoagulant therapy for a 6-month period in all patients and followed them at our outpatient clinic with bleeding profile tests. We classified the patients according to INR values. The INR level was between 1.9 and 2.5 in Group I and between 2.6 and 3.5 in Group II. Group I comprised 192 (52.7%) patients; Group II consisted of 172 (47.3%) patients. During the 6-month control period, no mortality or major morbidity such as pulmonary embolus occurred in either group, and we found no recurrent DVT among the 343 (94.2%) patients during that period. In Group I, two patients (1.04%) experienced minor hermorrhagic complications (subconjunctival hemorrhage and epistaxis), and two patients (1.04%) had major hemorrhagic complications (gastrointestinal [GI] bleeding and hemarthrosis). In Group II, seven patients (4.06%) had minor hemorrhagic complications including skin necrosis in three, epistaxis in three, and subconjunctival hemorrhage in three. Eleven patients (6.3%) in Group II experienced major hemorrhagic complications including GI bleeding in six, vaginal bleeding in three, hemarthrosis in one, and gluteal hematoma in one (Table <xref ref-type="table" rid="T2">2</xref>). We noted no instances of noncompliance and no major hematologic problems such as hemolysis during follow-up. Among six patients with GI bleeding, endoscopy demonstrated peptic ulcer disease in four (66.6%) and acute erosive gastritis in two (33.3%). In three patients with vaginal bleeding, two cases were due to myoma uteri and one was a result of chronic cervicitis. None of the hemorrhagic complications was secondary to malignancy in either Group I or Group II. The patients in Group II, whose INR values were between 2.6 and 3.5, had significantly more minor and major bleeding complications than did patients in Group I.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Complications in both groups, classified due to INR levels.</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="center">Group I (INR: 1.9&#x02013;2.5) (192 Patients)</td><td align="center">%</td><td align="center">Group II (INR: 2.6&#x02013;3.5) (172 Patients)</td><td align="center">%</td></tr></thead><tbody><tr><td align="left">Mortality</td><td align="center">-</td><td></td><td align="center">-</td><td></td></tr><tr><td align="left">Pulmonary thromboembolism</td><td align="center">-</td><td></td><td align="center">-</td><td></td></tr><tr><td align="left">Recurrent DVT</td><td align="center">-</td><td></td><td align="center">-</td><td></td></tr><tr><td align="left">Minor hemorrhage complications</td><td align="center">2</td><td align="center">1.04</td><td align="center">7</td><td align="center">4.06</td></tr><tr><td align="left">&#x02003;Subconjunctival hemorrhage</td><td align="center">1</td><td align="center">0.52</td><td align="center">1</td><td align="center">0.58</td></tr><tr><td align="left">&#x02003;Epistaxis</td><td align="center">1</td><td align="center">0.52</td><td align="center">3</td><td align="center">1.7</td></tr><tr><td align="left">&#x02003;Skin necrosis</td><td align="center">-</td><td align="center">-</td><td align="center">3</td><td align="center">1.7</td></tr><tr><td align="left">Major hemorrhagic complications</td><td align="center">2</td><td align="center">1.04</td><td align="center">11</td><td align="center">6.3</td></tr><tr><td align="left">&#x02003;GI bleeding</td><td align="center">1</td><td align="center">0.52</td><td align="center">6</td><td align="center">3.4</td></tr><tr><td align="left">&#x02003;Hemarthrosis</td><td align="center">1</td><td align="center">0.52</td><td align="center">1</td><td align="center">0.58</td></tr><tr><td align="left">&#x02003;Vaginal bleeding</td><td align="center">-</td><td></td><td align="center">3</td><td align="center">1.7</td></tr><tr><td align="left">&#x02003;Gluteal hematoma</td><td align="center">-</td><td></td><td align="center">1</td><td align="center">0.58</td></tr></tbody></table><table-wrap-foot><p>DVT: Deep vein thrombosis GI: Gastrointestinal</p></table-wrap-foot></table-wrap></sec><sec><title>Discussion</title><p>In 1977, the Committee on Thrombosis and Hemostasis and the National Institute of Biological Standards and Control (NIBSC) in London prepared a thromboplastin material from human brain. This material was coded as "67/40" and was accepted as an international reference preparation by the WHO [<xref ref-type="bibr" rid="B5">5</xref>]. Because PT results are dependent upon a number of factors, rates and activity percentages with different thromboplastins may be inconsistent for an individual patient. The WHO introduced an international standardization system [<xref ref-type="bibr" rid="B6">6</xref>], and in 1983 the Expert Committee on Biological Standardization of the WHO accepted the international normalized ratio (INR) for PT standardization (Table <xref ref-type="table" rid="T3">3</xref>).</p><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>INR formulation</p></caption><table frame="hsides" rules="groups"><tbody><tr><td align="left">&#x02022; INR: prothrombin time rate<sup>ISI</sup></td><td></td></tr><tr><td align="left">&#x02022; Example = found in patient</td><td align="left">PT: 17.9 second</td></tr><tr><td align="right">normal</td><td align="left">PT: 12.2 second</td></tr><tr><td></td><td align="left">ISI: 2.3</td></tr><tr><td align="left">&#x02022; INR: (17.9 / 12.2)<sup>2.3 </sup>= 2.4</td><td></td></tr></tbody></table><table-wrap-foot><p>&#x02022; INR: International normalized ratio; PT: Prothrombin time; ISI: International Sensitivity Index.</p></table-wrap-foot></table-wrap><p>Since 1983, INR values have been calculated based on randomized study results involving laboratory evaluation of OAC therapy. Based on these study findings, the optimal therapeutic range was narrowed. In 1989, a new guide was prepared by the American College of Chest Physicians. We are now using that guide [<xref ref-type="bibr" rid="B7">7</xref>].</p><p>Warfarin is the most frequently used OAC because of its perfect bioavailability and consistent effect in both acute and chronic disease [<xref ref-type="bibr" rid="B8">8</xref>]. Today, INR values are used at many institutions to evaluate OAC therapy (2,4,5,7,9). Anticoagulants are used in two different concentrations: low doses for INR values between 2.0 and 2.5 and high doses for INR values between 2.5 and 3.5. A low-dose regimen is used in the treatment of venous thrombosis or pulmonary embolus as well as prophylactically to prevent venous thrombosis. High-dose regimens are recommended for high-risk patients with mechanical prosthetic cardiac valves. Low doses have been shown to be safer than high doses and just as effective for following patients who undergo DVT therapy [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B10">10</xref>].</p><p>In patients whose INR levels exceed 2.5, the incidence of hemorrhagic complications increases significantly [<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B11">11</xref>]. An occult GI lesion accounted for 70% of the GI bleeds, and in 10%, bleeding during anticoagulant therapy was due to malignancy [<xref ref-type="bibr" rid="B4">4</xref>]. Even with ideal anticoagulant doses, GI and genitourinary bleeding are seen as a result of underlying disease [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B12">12</xref>]. In our study, GI bleeding occurred in both groups, and vaginal bleeding occurred as a complication in Group II (Table <xref ref-type="table" rid="T2">2</xref>).</p><p>Hasenkam and associates proposed an INR between 2.0 and 2.7, even for patients with valve replacements, to minimize the incidence of bleeding as a complication and the risk of thromboembolus [<xref ref-type="bibr" rid="B13">13</xref>].</p><p>Many studies address the effectiveness of LMWH in treating DVT, and this form of heparin therapy should be seriously considered. LMWH eliminates many problems related to the use of nonfractionated IV heparin [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B14">14</xref>]. Its bioavailability is high, and the once-daily subcutaneous injection regimen is sufficient because of its longer half-life. LMWH decreases major hemorrhagic complications and thrombocytopenia as well as the incidence of osteoporosis associated with heparin administration. The single LMWH injection without the necessity of monitoring anticoagulation offers a very attractive treatment method. DVT can be treated without hospitalization, and patients can return to daily activities and to work much more quickly [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. We used LMWH for 88.5% of our patients.</p><p>In conclusion, OAC therapy must be closely monitored during both acute and chronic periods. To compare and standardize results reported by different centers, values should be reported as INR. Bleeding and related complications increase as INR levels are increased. We suggest that, ideally, the anticoagulant dosage should be adjusted to achieve an INR between 2 and 2.5 in patients with DVT. This dose level minimizes complications and is sufficient to control anticoagulant therapy.</p></sec>
The Genetic Architecture of Parallel Armor Plate Reduction in Threespine Sticklebacks
<p>How many genetic changes control the evolution of new traits in natural populations? Are the same genetic changes seen in cases of parallel evolution? Despite long-standing interest in these questions, they have been difficult to address, particularly in vertebrates. We have analyzed the genetic basis of natural variation in three different aspects of the skeletal armor of threespine sticklebacks <italic>(Gasterosteus aculeatus):</italic> the pattern, number, and size of the bony lateral plates. A few chromosomal regions can account for variation in all three aspects of the lateral plates, with one major locus contributing to most of the variation in lateral plate pattern and number. Genetic mapping and allelic complementation experiments show that the same major locus is responsible for the parallel evolution of armor plate reduction in two widely separated populations. These results suggest that a small number of genetic changes can produce major skeletal alterations in natural populations and that the same major locus is used repeatedly when similar traits evolve in different locations.</p>
<contrib contrib-type="author" equal-contrib="yes"><name><surname>Colosimo</surname><given-names>Pamela F</given-names></name><xref ref-type="aff" rid="aff1"> <sup>1</sup> </xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Peichel</surname><given-names>Catherine L</given-names></name><xref ref-type="aff" rid="aff2"> <sup>2</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Nereng</surname><given-names>Kirsten</given-names></name><xref ref-type="aff" rid="aff1"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Blackman</surname><given-names>Benjamin K</given-names></name><xref ref-type="aff" rid="aff1"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Shapiro</surname><given-names>Michael D</given-names></name><xref ref-type="aff" rid="aff1"> <sup>1</sup> </xref></contrib><contrib contrib-type="author"><name><surname>Schluter</surname><given-names>Dolph</given-names></name><xref ref-type="aff" rid="aff3"> <sup>3</sup> </xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Kingsley</surname><given-names>David M</given-names></name><email>[email protected]</email><xref ref-type="aff" rid="aff1"> <sup>1</sup> </xref></contrib>
PLoS Biology
<sec id="s1"><title>Introduction</title><p>The number and type of genetic changes that control morphological and physiological changes during vertebrate evolution are not yet known. The evolutionary history of threespine sticklebacks <italic>(Gasterosteus aculeatus)</italic> provides an unusual opportunity to directly study the genetic architecture of adaptive divergence in natural populations. At the end of the last ice age, marine sticklebacks colonized newly formed freshwater environments throughout the Northern Hemisphere. Over the last 10,000 to 15,000 years, these fish have adapted to a wide range of new ecological conditions, giving rise to diverse populations with striking differences in morphology, physiology, and behavior (<xref rid="pbio-0020109-Bell2" ref-type="bibr">Bell and Foster 1994</xref>). Major changes in the bony armor have evolved repeatedly in different locations, and several hypotheses have been proposed to explain this morphological transformation, including response to changes in calcium availability (<xref rid="pbio-0020109-Giles1" ref-type="bibr">Giles 1983</xref>), stream gradients (<xref rid="pbio-0020109-Baumgartner1" ref-type="bibr">Baumgartner and Bell 1984</xref>), or temperature, salinity, or other factors that may vary in parallel with climate (<xref rid="pbio-0020109-Heuts1" ref-type="bibr">Heuts 1947</xref>; <xref rid="pbio-0020109-Hagen4" ref-type="bibr">Hagen and Moodie 1982</xref>); or exposure to different types of predators (<xref rid="pbio-0020109-Hagen2" ref-type="bibr">Hagen and Gilbertson 1973a</xref>; <xref rid="pbio-0020109-Moodie1" ref-type="bibr">Moodie et al. 1973</xref>; <xref rid="pbio-0020109-Reimchen2" ref-type="bibr">Reimchen 1992</xref>; <xref rid="pbio-0020109-Reimchen3" ref-type="bibr">Reimchen 1995</xref>).</p><p>Three distinctive patterns of body armor, now known as the &#x0201c;lateral plate morphs,&#x0201d; have been recognized as one of the most distinguishing characteristics in sticklebacks since at least the early 1800s (<xref rid="pbio-0020109-Cuvier1" ref-type="bibr">Cuvier and Valenciennes 1829</xref>). Most marine sticklebacks have a continuous row of bony plates that covers the lateral side of the body from head to tail (the &#x0201c;complete morph&#x0201d;; see marine fish in <xref ref-type="fig" rid="pbio-0020109-g001">Figure 1</xref>). In contrast, many freshwater sticklebacks show substantial reductions in total plate number, developing either as &#x0201c;partial morphs,&#x0201d; which lose plates in the middle of the row (not shown), or as &#x0201c;low morphs,&#x0201d; which retain only a few plates at the anterior end (see Paxton benthic and Friant California [lower animal] fish in <xref ref-type="fig" rid="pbio-0020109-g001">Figure 1</xref>). The anterior plates present in low morphs are the first to form during larval development. In contrast, the middle plates absent in partial morphs are the last to form during normal development (<xref rid="pbio-0020109-Igarashi1" ref-type="bibr">Igarashi 1964</xref>; <xref rid="pbio-0020109-Igarashi2" ref-type="bibr">Igarashi 1970</xref>; <xref rid="pbio-0020109-Bell1" ref-type="bibr">Bell 1981</xref>). Thus, the adult plate patterns of low and partial morphs resemble early developmental stages of plate patterns in complete morphs, and paedomorphosis has been proposed as a possible explanation for the repeated evolution of low and partial morphs from completely plated ancestors (<xref rid="pbio-0020109-Bell1" ref-type="bibr">Bell 1981</xref>).</p><fig id="pbio-0020109-g001" position="float"><label>Figure 1</label><caption><title>Mapping the Genetic Basis of Lateral Plate Reduction in Different Natural Populations of Threespine Sticklebacks</title><p>Crossing a completely plated Japanese marine fish with a low-plated fish from Paxton Lake, British Columbia, produced a mixture of complete, partial, and low morph phenotypes in F2 progeny animals (Cross 1). In contrast, crossing a completely plated fish and a low-plated fish from an inland freshwater stream in Friant, California, produced only complete and low-plated progeny (Cross 2). Red dots show the geographic origins of the populations studied. Scale bars equal 1 cm. <italic>AA</italic>, <italic>Aa</italic>, and <italic>aa</italic> refer to genotypes at <italic>Gac4174</italic> (a microsatellite marker) near the major plate locus on LG 4. The genotype at <italic>Gac4174</italic> is missing in ten of the 360 F2s in Cross 1. All fish were stained with alizarin red to reveal bony structures.</p></caption><graphic xlink:href="pbio.0020109.g001"/></fig><p>This dramatic variation in lateral plate patterning has led to repeated efforts to determine the genetic basis of the major plate morphs. Previous studies have shown that plate morphs are reproducibly inherited in the laboratory and that crosses between different morphs generate relatively simple ratios of the three major phenotypes among the progeny. Based on these qualitative results, at least six different genetic models have been proposed for lateral plate patterns in sticklebacks. The simplest models proposed a single major locus with alternative alleles <italic>(A</italic> and <italic>a)</italic> (<xref rid="pbio-0020109-Munzing1" ref-type="bibr">Munzing 1959</xref>; <xref rid="pbio-0020109-Avise1" ref-type="bibr">Avise 1976</xref>). The <italic>A</italic> allele was first proposed to be incompletely dominant to the <italic>a</italic> allele, generating either complete <italic>(AA),</italic> partial <italic>(Aa),</italic> or low-plated <italic>(aa)</italic> fish (<xref rid="pbio-0020109-Munzing1" ref-type="bibr">Munzing 1959</xref>). In other populations, the <italic>A</italic> allele may be completely dominant to the <italic>a</italic> allele, producing either complete <italic>(AA, Aa)</italic> or low-plated <italic>(aa)</italic> fish, but no partials (<xref rid="pbio-0020109-Avise1" ref-type="bibr">Avise 1976</xref>). More complicated models have proposed two major loci controlling plate inheritance (with alternative alleles <italic>A, a</italic> and <italic>B, b</italic>). In one of these models, both major loci contribute equally to plate phenotype, and the total number of <italic>A</italic> and <italic>B</italic> alleles determines whether fish develop as complete (three or more <italic>A</italic> or <italic>B</italic> alleles), partial (two <italic>A</italic> or <italic>B</italic> alleles), or low-plated fish (one or less <italic>A</italic> or <italic>B</italic> allele) (<xref rid="pbio-0020109-Hagen3" ref-type="bibr">Hagen and Gilbertson 1973b</xref>). Additional models have proposed either epistatic interactions between a single major locus and one modifier locus, or the presence of more than two alternative alleles at the major locus to account for variant results in some populations (<xref rid="pbio-0020109-Ziuganov1" ref-type="bibr">Ziuganov 1983</xref>; <xref rid="pbio-0020109-Banbura1" ref-type="bibr">Banbura 1994</xref>). All of these models were proposed before the development of genomewide genetic markers for sticklebacks (<xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. 2001</xref>) and have never been tested by linkage mapping. In this study, we take advantage of these recently developed tools to examine the genetic basis of variation in lateral plate phenotypes in natural populations of sticklebacks.</p></sec><sec id="s2"><title>Results</title><p>To directly analyze the number and location of genetic loci that control plate phenotypes, we crossed a completely plated marine fish with a low-plated benthic fish from Paxton Lake, British Columbia. Three hundred sixty progeny from a single F2 family (Cross 1) were examined in detail for the pattern, number, and size of lateral plates and then genotyped for the inheritance of different alleles at 160 polymorphic loci distributed across all linkage groups. The segregation of plate phenotypes was compared to the segregation of all genetic markers using quantitative trait loci (QTL) analysis (MapQTL; <xref rid="pbio-0020109-van2" ref-type="bibr">van Ooijen et al. 2002</xref>). Significance thresholds for detecting linkage were chosen using conservative criteria for genomewide linkage mapping in noninbred populations (log likelihood ratio [LOD] score &#x02265; 4.5; <xref rid="pbio-0020109-van1" ref-type="bibr">van Ooijen 1999</xref>).</p><p>When plate morph was scored as a qualitative trait, a highly significant QTL on linkage group (LG) 4 was detected (LOD = 117; <xref ref-type="table" rid="pbio-0020109-t001">Table 1</xref> and <xref ref-type="fig" rid="pbio-0020109-g002">Figure 2</xref>). The genotype of the QTL on LG 4 was highly predictive of the major plate morph that developed in a fish. Almost all fish that carried two alleles from the complete morph grandparent in the LG 4 region (hereafter referred to as <italic>&#x0201c;AA&#x0201d;</italic> animals) showed the complete pattern, whereas fish that carried two alleles from the low morph grandparent in this region (hereafter referred to as <italic>&#x0201c;aa&#x0201d;</italic> animals) showed the low pattern. In contrast, most fish with one allele from the complete grandparent and one allele from the low grandparent (hereafter referred to as <italic>&#x0201c;Aa&#x0201d;</italic> animals) developed as either complete or partial fish (see <xref ref-type="fig" rid="pbio-0020109-g001">Figure 1</xref>).</p><fig id="pbio-0020109-g002" position="float"><label>Figure 2</label><caption><title>Comparison of QTL Positions for Different Traits</title><p>LOD scores are shown as a function of genetic distance along different stickleback linkage groups. QTL affecting qualitative plate pattern (red line), total plate number (black lines), or plate size (blue lines) show similar shapes on several linkage groups, suggesting that the same or linked genes control multiple aspects of plate phenotype. Points in LOD plots correspond to the following microsatellite markers from left to right along each linkage group: (A) LG 4: <italic>Pitx2 (Stn220), Stn38, Gac62, Stn42, Gac4174, Stn45, Stn183, Stn46, Stn47, Stn184, Stn39;</italic> (B) LG 7: <italic>Stn70, Stn72, Stn76, Stn71, Stn78, Stn79, Stn75, Stn81, Stn80 Stn82, Pitx1;</italic> (C) LG 10<italic>: Stn119, Stn120, Stn211, Stn121, Stn124, Stn23, Stn125;</italic> (D) LG 25: <italic>Stn212, Stn213, Stn214, Stn215, Stn216, Gac1125, Stn217;</italic> (E) LG 26: <italic>Stn218, Stn219, Bmp6, Stn222, Stn223</italic>. Note that markers <italic>Stn183</italic> and <italic>Stn184</italic> from LG 18 in the Priest Lake cross (<xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. 2001</xref>) map together with LG 4 markers in the larger Cross 1.</p></caption><graphic xlink:href="pbio.0020109.g002"/></fig><table-wrap id="pbio-0020109-t001" position="float"><label>Table 1</label><caption><title>Summary of QTL Affecting Lateral Plate Phenotypes in Cross 1</title></caption><graphic xlink:href="pbio.0020109.t001"/><table-wrap-foot><fn id="nt101"><p>All QTL that exceed the genomewide significance threshold (LOD &#x02265; 4.5) are shown with their respective LG, maximum LOD score, and PVE at the most closely linked microsatellite marker. Each trait was initially mapped in the large panel of F2 animals. Because plate number is dominated by the phenotypic effect of the major locus on LG 4, we have separately listed the phenotypic effects of the plate number modifier QTL within all major genotypic classes near the major locus (<italic>AA</italic>, <italic>Aa</italic>, and <italic>aa</italic> animals). These results are shown even when they do not exceed the LOD &#x02265; 4.5 threshold, in order to facilitate comparison of the effects of significant modifiers in different genetic backgrounds. Mean plate number and size measurements were calculated for progeny that inherited either two marine alleles <italic>(MM)</italic>, one marine and one benthic allele <italic>(MB)</italic>, or two benthic alleles <italic>(BB)</italic> at the microsatellite most closely linked to each QTL. Plate number is the sum of plate counts on both sides of the body. Plate width and plate height were measured in millimeters at the positions indicated in <xref ref-type="fig" rid="pbio-0020109-g002">Figure 2</xref>, summed for both sides of the body, and standardized by overall body length and body depth, respectively. Statistical analysis was done using one-way ANOVA. *significantly different from <italic>MM</italic> mean (<italic>p</italic> &#x0003c; 0.05), **highly significantly different from <italic>MM</italic> mean (<italic>p</italic> &#x02264; 0.0001), <sup>#</sup>significantly different from <italic>MB</italic> mean (<italic>p</italic> &#x0003c; 0.05), <sup>##</sup> highly significantly different from <italic>MB</italic> mean (<italic>p</italic> &#x02264; 0.0001), &#x0201c;n/a&#x0201d; indicates &#x0201c;not applicable.&#x0201d; </p></fn></table-wrap-foot></table-wrap><p>When total plate number was scored, the same major LG 4 chromosome region accounted for more than 75% of the total variance in plate number of F2 fish. Three additional QTL were detected that had significant effects on plate number in <italic>Aa</italic> animals (<xref ref-type="table" rid="pbio-0020109-t001">Table 1</xref>; see <xref ref-type="fig" rid="pbio-0020109-g002">Figure 2</xref>). Increasing the number of benthic alleles at any of the individual modifiers led to a reduction in mean total plate number, even in the heterozygous state (<xref ref-type="table" rid="pbio-0020109-t001">Table 1</xref>). Increasing the number of benthic alleles at the three modifiers considered together caused a more than 2-fold reduction in mean plate number of <italic>Aa</italic> animals, largely accounting for whether <italic>Aa</italic> fish developed as either complete, partial, or low morphs (<xref ref-type="fig" rid="pbio-0020109-g003">Figure 3</xref>A and <xref ref-type="fig" rid="pbio-0020109-g003">3</xref>D). Increasing the number of benthic alleles at the same modifier loci also led to a 2-fold reduction in the mean plate number of <italic>aa</italic> animals but had relatively little effect on the plate number of <italic>AA</italic> animals (<xref ref-type="fig" rid="pbio-0020109-g003">Figure 3</xref>B and <xref ref-type="fig" rid="pbio-0020109-g003">3</xref>C). Taken together, these results suggest that at least four different loci influence lateral plate phenotypes in this cross. Homozygosity at the major locus largely determines whether fish develop as low <italic>(aa)</italic> or complete <italic>(AA)</italic> morphs, while the modifier loci affect the actual number of plates, particularly in <italic>Aa</italic> and <italic>aa</italic> animals.</p><fig id="pbio-0020109-g003" position="float"><label>Figure 3</label><caption><title>Cumulative Effects of Freshwater Alleles on the Number, Pattern, and Size of Lateral Plates in Cross 1</title><p>Increasing the total number of Paxton benthic freshwater alleles at modifier QTL on LGs 7, 10, and 26 significantly reduces plate number in animals with one marine (complete morph) and one Paxton benthic (low morph) allele near the major QTL on LG 4 (<italic>Aa</italic> progeny) (A). The same modifier QTL have little effect on fish with two marine alleles near the major QTL (<italic>AA</italic> animals) (B) and smaller phenotypic effects on animals with two benthic alleles near the major QTL (<italic>aa</italic> animals) (C). Increasing the number of benthic alleles also significantly increases the proportion of <italic>Aa</italic> fish whose overall plate pattern is classified as partial instead of complete (D). (E&#x02013;F) show plate size effects. Increasing the number of benthic alleles at plate size QTL on LGs 4, 7, and 25 significantly reduces mean plate width of F2 progeny (E). (F) shows the schema of plate size measurements. Lateral plates are shown numbered from anterior to posterior. Error bars in (A&#x02013;E) represent standard error.</p></caption><graphic xlink:href="pbio.0020109.g003"/></fig><p>The size of individual lateral plates varies significantly between different stickleback populations (<xref rid="pbio-0020109-Miller1" ref-type="bibr">Miller and Hubbs 1969</xref>; <xref rid="pbio-0020109-Avise1" ref-type="bibr">Avise 1976</xref>). Although this trait has not been systematically analyzed in previous stickleback crosses, studies of meristic characters in other vertebrates suggest that the size and number of repeating skeletal elements can be controlled separately (<xref rid="pbio-0020109-Christians1" ref-type="bibr">Christians et al. 2003</xref>). When height and width of specific plates were analyzed, we detected three QTL that accounted for a significant percentage of plate size variability in the cross (<xref ref-type="table" rid="pbio-0020109-t001">Table 1</xref>; see <xref ref-type="fig" rid="pbio-0020109-g002">Figure 2</xref>). Increasing the number of benthic alleles at these loci led to a progressive reduction in plate size (<xref ref-type="fig" rid="pbio-0020109-g003">Figure 3</xref>E). Two of the three plate size QTL mapped to the same chromosome regions that also affected plate morph or plate number, suggesting that the pattern, number, and size of plates may be controlled by the same or linked genes on LGs 4 and 7 (see <xref ref-type="fig" rid="pbio-0020109-g002">Figure 2</xref>). In contrast, the QTL affecting lateral plate size mapped to different locations than most QTL controlling the size of dorsal spine and pelvic structures (<xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. 2001</xref>; <xref rid="pbio-0020109-Shapiro1" ref-type="bibr">Shapiro et al. 2004</xref>), suggesting that the size of different bones are controlled separately in the stickleback skeleton.</p><p>Some of the differences in previously published models of stickleback plate genetics could be due to different genetic mechanisms operating in different populations. To compare the genetic architecture of armor plate patterning in a separate population located over 1300 km from Paxton Lake, we crossed fish from an unusual stickleback population in Friant, California, which is largely dimorphic for complete and low fish with very few partials. A cross between a Friant complete and a Friant low-plated fish resulted in nearly equal numbers of complete and low progeny (see <xref ref-type="fig" rid="pbio-0020109-g001">Figure 1</xref>, Cross 2), consistent with previous crosses from this population (<xref rid="pbio-0020109-Avise1" ref-type="bibr">Avise 1976</xref>). Genotyping studies with microsatellite markers linked to the major and minor QTL defined above showed very tight concordance between lateral plate phenotype and genotype near the same major locus on LG 4 that was seen in Cross 1 (LOD = 11.1). All fish with an inferred <italic>Aa</italic> genotype at the major locus on LG 4 were completely plated in this cross, suggesting that <italic>Aa</italic> fish develop more plates in Cross 2 than in Cross 1. This could be due to differences in the dominance relationship of the particular alleles at the LG 4 locus in the Friant population (<xref rid="pbio-0020109-Avise1" ref-type="bibr">Avise 1976</xref>), or to modification of dominance by the different genetic backgrounds in the two crosses. Although the number of animals in Cross 2 was small, significant differences in the mean total plate count of low fish could also be detected in animals that inherited different alleles at microsatellites linked to two of the modifier QTL detected in Cross 1 (alternative alleles at <italic>Stn210</italic> on LG 7: mean total plate counts 14.9 &#x000b1; 0.31 vs. 14.0 &#x000b1; 0.23, <italic>p</italic> = 0.0204; alternative alleles at <italic>Stn219</italic> on LG 26: 14.8 &#x000b1; 0.26 vs. 13.9 &#x000b1; 0.31 plates, <italic>p</italic> = 0.0352). Overall, these results suggest that both plate morph and plate number are controlled by similar chromosome regions in different populations.</p><p>To further test whether the same major locus on LG 4 controls armor plate reduction in both populations, we carried out genetic complementation crosses between two low female fish from Friant and one low male fish from Paxton Lake. All 84 progeny developed as low morphs, suggesting that the low-plated phenotype in both populations is likely to be due to the same major locus on LG 4.</p></sec><sec id="s3"><title>Discussion</title><sec id="s3a"><title>QTL Architecture</title><p>This study reports the first genomewide linkage mapping of lateral plate phenotypes in crosses between major stickleback plate morphs. Our results confirm previous suggestions that dramatic changes in lateral plate patterning can be controlled by one locus of major effect (<xref rid="pbio-0020109-Munzing1" ref-type="bibr">Munzing 1959</xref>; <xref rid="pbio-0020109-Avise1" ref-type="bibr">Avise 1976</xref>). This major locus on LG 4 can cause a greater than 5-fold change in total plate number and is sufficient to switch the overall morphology of a fish between the complete, partial, and low-plated states. The dramatic phenotypic effects of this locus likely explain why three types of sticklebacks have long been recognized in natural populations (<xref rid="pbio-0020109-Cuvier1" ref-type="bibr">Cuvier and Valenciennes 1829</xref>). Further molecular studies will be required to determine whether there are one or multiple mutations in the LG 4 region that account for the major QTL.</p><p>Plate number within the complete, partial, and low morphs also varies between fish from different locations. Previous studies suggest that sticklebacks with small changes in plate number show differential survival when exposed to predators, suggesting that selection may fine tune the exact number of plates in different environments (<xref rid="pbio-0020109-Hagen2" ref-type="bibr">Hagen and Gilbertson 1973a</xref>; <xref rid="pbio-0020109-Moodie1" ref-type="bibr">Moodie et al. 1973</xref>; <xref rid="pbio-0020109-Reimchen2" ref-type="bibr">Reimchen 1992</xref>). We have identified three modifier QTL that cause changes in plate number within all morphs but are unlinked to the major locus. The individual phenotypic effects of these QTL can be as small as a single plate per side (<xref ref-type="table" rid="pbio-0020109-t001">Table 1</xref>), while the combined mean effects of the QTL can be as large as 15 plates per side (see <xref ref-type="fig" rid="pbio-0020109-g003">Figure 3</xref>A). The number of modifier QTL is larger than predicted in previous models. We suspect that this is because of the general difficulty of predicting genetic architecture from simple phenotypic ratios of progeny in crosses that are segregating more than one or two genes. The magnitude of the phenotypic effects of the modifiers, their linkage relationships, and interactions with the major locus could not be predicted accurately from previous studies, highlighting the value of genomewide linkage mapping for studying the genetic architecture of major morphological variation in natural populations.</p><p>Postglacial freshwater stickleback populations are thought to be derived from completely plated marine ancestors (<xref rid="pbio-0020109-Bell2" ref-type="bibr">Bell and Foster 1994</xref>). At all of the plate QTL detected in Cross 1, the net effect of the freshwater alleles from the Paxton benthic grandparent is to cause a progressive reduction in the size or number of armor plates (<xref ref-type="table" rid="pbio-0020109-t001">Table 1</xref>). All of the QTL that affect plate morph or plate number also have significant effects in the heterozygous state, showing that plate reduction is likely to evolve through semiadditive genetic changes, rather than through purely recessive or purely dominant mutations. Theoretical studies suggest that semiadditive mutations can be fixed more quickly than purely recessive or dominant mutations when they begin at low frequency, although the overall probability of fixation also depends on whether the mutations arise de novo or are originally present in a founder population (<xref rid="pbio-0020109-Crow1" ref-type="bibr">Crow and Kimura 1970</xref>; <xref rid="pbio-0020109-Orr1" ref-type="bibr">Orr and Betancourt 2001</xref>). Strong selection on a small number of chromosome regions that have large, semiadditive effects may help explain how dramatic changes in lateral plate patterns have evolved relatively quickly in postglacial stickleback populations.</p></sec><sec id="s3b"><title>Parallel Evolution</title><p>Our mapping and complementation results suggest that the same major locus on LG 4 causes major changes in plate pattern in both the Paxton benthic and Friant populations. Phenotypic reduction of lateral plates almost certainly evolved separately in these different locations, given the geographic distance between them (over 1300 km), the presence of completely plated fish in the marine environment separating the sites, and previous studies showing that sticklebacks in nearby lakes have independent mitochondrial haplotypes (<xref rid="pbio-0020109-Taylor1" ref-type="bibr">Taylor and McPhail 1999</xref>). Additional complementation crosses between low-plated fish from Friant and other California populations (<xref rid="pbio-0020109-Avise1" ref-type="bibr">Avise 1976</xref>; unpublished data), Paxton benthic fish and pelvic-reduced fish from Iceland (<xref rid="pbio-0020109-Shapiro1" ref-type="bibr">Shapiro et al. 2004</xref>), and low-plated populations from British Columbia and Japan (<xref rid="pbio-0020109-Schluter1" ref-type="bibr">Schluter et al. 2004</xref>) also produce low-plated progeny. Thus genetic changes at the same major locus may underlie low-plated phenotypes at numerous locations around the world.</p><p>The present study provides the first genetic mapping evidence that some of the chromosome regions controlling smaller quantitative variation in plate number may also be used repeatedly in different populations. The QTL on LG 26 in Cross 1 maps to a similar position as a QTL influencing plate number within low morph fish from Priest Lake, British Columbia (<xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. 2001</xref>). This QTL is also associated with significant variation in plate number of low morphs of the Friant population (Cross 2), suggesting that this chromosomal region on LG 26 contributes to plate number variation in at least three different populations: Paxton, Priest, and Friant sticklebacks.</p><p>Recent studies suggest that the same genes are also used repeatedly when pigmentation and larval cuticle phenotypes have evolved in parallel in different fly populations (<xref rid="pbio-0020109-Gompel1" ref-type="bibr">Gompel and Carroll 2003</xref>; <xref rid="pbio-0020109-Sucena1" ref-type="bibr">Sucena et al. 2003</xref>) or when melanism has evolved independently in birds and mammals (reviewed in <xref rid="pbio-0020109-Majerus1" ref-type="bibr">Majerus and Mundy 2003</xref>). Repeated use of particular genes may thus be a common theme in parallel evolution of major morphological changes in natural populations of both invertebrates and vertebrates.</p><p>Why might some genes be used preferentially when similar phenotypes evolve in parallel in wild populations? Alleles that cause plate reduction may already be present at low frequency in marine populations. In that case, parallel phenotypic evolution could occur by repeated selection for the same preexisting alleles in different freshwater locations. Alternatively, some genes may be particularly susceptible to de novo mutations, either because of the size or structure of coding and regulatory regions, or the presence of hotspots for recombination, insertion, or deletion. Finally, only a limited number of either old or new mutations may actually be capable of producing a specific phenotype without also causing deleterious effects on fitness. Mutations with the largest positive selection coefficients will be fixed most rapidly in evolving populations, and this may lead to parallel selection for mutations in the same genes in different populations.</p><p>A major goal for future work will be to identify the actual genes and mutations that cause parallel evolution of adaptive traits in wild sticklebacks. This study identifies specific markers that are closely linked to chromosome regions that control the pattern, number, and size of lateral plates. With the recent development of BAC libraries and physical maps of the stickleback genome, it should be possible to use forward genetic approaches to identify the genes responsible for the repeated evolution of major morphological transformations in stickleback armor (<xref rid="pbio-0020109-Kingsley1" ref-type="bibr">Kingsley et al. 2004</xref>). Cloning and sequencing of such genes will make it possible to determine the molecular mechanisms that underlie parallel evolution in natural populations and should provide new insight into the nature of genetic, genomic, developmental, and ecological constraints that operate as new characteristics appear during the adaptive evolution of vertebrates.</p></sec></sec><sec id="s4"><title>Materials and Methods</title><sec id="s4a"><title/><sec id="s4a1"><title>Fish crosses and husbandry</title><p>For Cross 1, a wild-caught, completely plated marine female from Onnechikappu stream on the east coast of Hokkaido Island, Japan, was crossed to a wild-caught, low-plated benthic male from Paxton Lake, British Columbia. Both parents showed morphologies typical of the marine and benthic populations at their respective collecting sites. The specific populations were chosen because the large average body size of both parents and the estimated divergence between eastern and western Pacific Ocean fish (<xref rid="pbio-0020109-Orti1" ref-type="bibr">Orti et al. 1994</xref>) were expected to help maximize the size of the progeny, the number of offspring per clutch, and the informativeness of microsatellites and other markers for genetic mapping. F1 progeny were raised to maturity in 30-gallon aquaria and were mated in pairs. Approximately 2600 F2 progeny were raised to a standard length of greater than 28 mm under the same conditions (30-gallon aquaria in a single 18&#x000b0;C room with 16 hours of light and eight hours of dark per day and twice daily feeding of brine shrimp or frozen blood worms). Although limited phenotypic plasticity has been reported for development of some trophic characters in sticklebacks (<xref rid="pbio-0020109-Day1" ref-type="bibr">Day et al. 1994</xref>), previous studies have shown that differences in plate number of wild-caught sticklebacks are stable and reproducible when fish are raised under laboratory conditions (see, for example, <xref rid="pbio-0020109-Hagen1" ref-type="bibr">Hagen 1967</xref>). A total of 360 full siblings from a single F2 family were used for genotypic and phenotypic analysis in this study. For Cross 2, one wild-caught, completely plated female from Friant, California, was crossed to one wild-caught, low-plated male from Friant, California. A total of 58 F1 progeny were raised to a standard length of greater than 28 mm in a ZMOD (Marine Biotech, Beverly, Massachusetts, United States). For the complementation cross, two wild-caught, low-plated females from Friant were crossed to one wild-caught, low-plated benthic male from Paxton Lake, British Columbia. At total of 84 F1 progeny were raised to a standard length of greater than 28 mm in 30-gallon aquaria.</p></sec><sec id="s4a2"><title>Genotyping</title><p>Genotyping of microsatellite markers was performed and analyzed essentially as described in <xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. (2001)</xref>. Some PCR products were analyzed on a 48-capillary array on an ABI3730xl with GeneMapper v3.0 software and GeneScan 500 LIZ (Applied Biosystems, Foster City, California, United States) used as an internal size standard. A total of 160 markers were analyzed in Cross 1, including 144 previously described microsatellite markers (<xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. 2001</xref>), the genes <italic>Pitx1</italic>, <italic>Pitx2 (Stn220),</italic> and <italic>Tbx4 (Stn221),</italic> (<xref rid="pbio-0020109-Shapiro1" ref-type="bibr">Shapiro et al. 2004</xref>) and 13 new markers: <italic>Bmp6</italic> gene and 12 additional microsatellites <italic>(Stn210&#x02013;219, 222&#x02013;223).</italic> A polymorphism within the 3&#x02032; UTR of the <italic>Bmp6</italic> gene was genotyped using single strand conformation polymorphism analysis with MDE Gel Solution (BioWhittaker Molecular Applications, Rockland, Maine, United States). PCR bands were visualized using autoradiography. PCR conditions were the same as for the microsatellite markers except 2.5 mM MgCl<sub>2</sub> and 10% DMSO were used. Primers for <italic>Bmp6</italic> genotyping are: Bmp6F1: 5&#x02032; CCCGGTTT<italic>AA</italic>ATCCTCATCC and Bmp6R1: 5&#x02032; AGGAGGTGATTGACAGCTCG.</p></sec><sec id="s4a3"><title>Morphological analysis and QTL mapping</title><p>Fish were stained with alizarin red to detect skeletal structures as described in <xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. (2001)</xref>. Lateral plates were counted on both sides of each fish. For QTL mapping, the total plate number of both sides was used. Plate width was measured on the first lateral plate located under the first dorsal spine and above the ascending process of the pelvis. Plate height was measured on the lateral plate posterior to the last plate that is under the second dorsal spine and touching the ascending process. These correspond to plate positions 5 and 8 in previous nomenclature (<xref rid="pbio-0020109-Reimchen1" ref-type="bibr">Reimchen 1983</xref>). All measurements were done with Vernier calipers accurate to 0.02 mm and had repeatabilities of 1.1% &#x000b1; 0.9% (SD)(plate width) and 3.9% &#x000b1; 2.9% (SD)(plate height). Plate width and height measurements on both sides of the body were summed and standardized by body length and depth, respectively. Similar QTL were detected when residuals from regressions of plate width and height on standard body length and depth were mapped. When raw plate width and height measurements were used, we also detected one additional significant QTL on LG 19 (plate width: LG 19, LOD = 5.42, 7.3 percent variance explained [PVE]; plate height: LG 19, LOD = 7.3, 11.6 PVE). Standard body length itself maps to LG 19 (LOD = 10, 13 PVE). The LG 19 effect on plate size is not significant when plate measurements are normalized by standard body length, suggesting that the LG 19 QTL is a general body size QTL, while the other size QTLs (<xref ref-type="table" rid="pbio-0020109-t001">Table 1</xref>; see <xref ref-type="fig" rid="pbio-0020109-g002">Figure 2</xref>) act on plate size separately from total body size.</p><p>All morphological traits in Cross 1 were analyzed with MapQTL 4.0 (<xref rid="pbio-0020109-van2" ref-type="bibr">van Ooijen et al. 2002</xref>) using the same parameters as described by <xref rid="pbio-0020109-Peichel1" ref-type="bibr">Peichel et al. (2001)</xref>. Microsatellite markers that were closely linked to QTL detected in Cross 1 were genotyped in all Cross 2 animals (<italic>Gac4174, Stn40,</italic> and <italic>Stn47</italic> on LG 4; <italic>Stn210, Stn71,</italic> and <italic>Stn76</italic> on LG 7; <italic>Stn211</italic> and <italic>Stn121</italic> on LG 10; and <italic>Stn218, Stn219,</italic> and <italic>Stn222</italic> on LG 26). LOD scores between LG 4 markers and the major plate locus in Cross 2 were calculated using Map Manager v2.6.6 (<xref rid="pbio-0020109-Manly1" ref-type="bibr">Manly 1993</xref>). Mean total plate numbers in low-plated fish that inherited different alleles at microsatellite loci on LGs 4, 7, 10, and 26 were compared using one-way ANOVA (Statview v5.0.1, SAS Institute Inc., Cary, North Carolina, United States).</p></sec></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><p>The GenBank accession numbers for the <italic>Bmp6</italic> gene is AY547294 and for the 12 additional new microsatellites <italic>Stn 210&#x02013;219, 222&#x02013;223</italic> are BV102488&#x02013;BV102499.</p></sec>
Single Locus Controls Majority of Armor Evolution in Two Populations of Sticklebacks
Could not extract abstract
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PLoS Biology
<p>The astounding diversity of life&#x02014;different body shapes and sizes, physiologies, and behaviors&#x02014;stems from the accumulation of genetic changes through the process we call evolution. But catching a glimpse into the process of evolution at the gene level is difficult, mostly because significant changes to the plant and animal species of today happened a long time ago. Nevertheless, biologists are keen to understand exactly how evolution progresses. For example, how many genes must be altered before noticeable shifts in appearance can be seen? Is evolution the result of changes in many genes with small additive effects, or of just a few mutations that exert a strong influence?<xref ref-type="fig" rid="pbio-0020143-g001"/> </p><fig id="pbio-0020143-g001" position="float"><caption><title>Complete and low armor stickleback morphs, Friant, California</title></caption><graphic xlink:href="pbio.0020143.g001"/></fig><p>To tackle these questions, Pamela Colosimo and colleagues turned to threespine stickleback fish, a longtime favorite model system of evolutionary biologists because of its relatively youthful evolutionary history. At the end of the last ice age 10,000 years ago, when glaciers all over the Northern Hemisphere began to melt, small populations of these originally marine-dwelling fish became trapped in newly formed lakes. There, isolated stickleback colonies adapted to new ecological conditions&#x02014;different predators, food availability, water chemistry, and temperature&#x02014;and now look distinctly different from their marine ancestors. One of the most obvious changes in appearance is in their body armor&#x02014;they come in three distinct types, or &#x0201c;morphs.&#x0201d; Marine sticklebacks are covered from head to tail with rows of tightly packed boney plates (a complete morph), while those found in freshwater lakes have fewer body plates (a partial morph) or almost none at all (a low morph). Colosimo and colleagues found that a single region of the genome is largely responsible for the dramatic changes in plate morph, and that this is true for two widely separated populations of independently evolving freshwater sticklebacks.</p><p>To uncover the genomic regions that affect armor, Colosimo's team crossed fully armored marine sticklebacks from Japan with deep-water, or benthic, low morph fish from Paxton Lake in British Columbia, Canada. They then &#x0201c;mapped&#x0201d; the full genome of second generation offspring using 160 known genetic markers, or loci, as guideposts for distinct regions of the genome&#x02014;loci that are inherited along with differences in the overall type of plating, and individual plate number and size.</p><p>The team found that one such locus explained 75% of the variation in plate morphs. Offspring that carried two alleles&#x02014;versions of the gene&#x02014;from their marine grandparents, genotype <italic>AA,</italic> were almost always fully plated. Those that inherited two copies of the allele from their benthic progenitors, <italic>aa,</italic> were mostly low morphs with very little plating. And <italic>Aa</italic> heterozygous fish (with one allele from each population) had mostly full or partial plates. Colosimo and colleagues also found three other regions in the genome that significantly affected the number and size of plates. These modifiers had an additive effect&#x02014;the more benthic alleles inherited, the fewer and smaller the plates; more marine alleles caused a trend toward greater armor.</p><p>But is this genetic architecture the same for every independently evolving population of lake-bound sticklebacks in North America? Or did the geographically isolated freshwater groups loose their plates through mutations in different genes? Colosimo and colleagues mapped the genome of a population of sticklebacks from Friant, California, which is 800 miles away from Paxton Lake, and found that the same major locus seemed to be controlling plate morph there as well. Crossing a low morph from Friant with a low morph from Paxton yielded only offspring with very little armor. Further, some of the modifiers uncovered in the Paxton fish were also acting on the Friant sticklebacks. So, though these two populations of fish have been separated for 10,000 years, loss of armor in both groups probably stemmed from changes in the same genetic pathway.</p><p>Without knowing the precise sequence of these genes, it is impossible to tell exactly how and when the alleles that reduce armor arose. Small numbers of individuals with genes causing less plating could have been present in ancestral populations of marine sticklebacks when they were originally locked in newly formed lakes. Alternatively, reduced armor could have arisen independently in different lakes following isolation if, for example, some genes that control armor are predisposed to mutation, or certain armor-related mutations are more advantageous than others. But however it happened, this study clearly shows that dramatic morphological evolution can result from a small number of genetic changes. Further study of this classic system should provide a detailed picture of the genes involved, and of the molecular events that underlie morphological changes in natural populations evolving in new environments.</p>
Gene prediction using the Self-Organizing Map: automatic generation of multiple gene models
<sec><title>Background</title><p>Many current gene prediction methods use only one model to represent protein-coding regions in a genome, and so are less likely to predict the location of genes that have an atypical sequence composition. It is likely that future improvements in gene finding will involve the development of methods that can adequately deal with intra-genomic compositional variation.</p></sec><sec><title>Results</title><p>This work explores a new approach to gene-prediction, based on the Self-Organizing Map, which has the ability to automatically identify multiple gene models within a genome. The current implementation, named RescueNet, uses relative synonymous codon usage as the indicator of protein-coding potential.</p></sec><sec><title>Conclusions</title><p>While its raw accuracy rate can be less than other methods, RescueNet consistently identifies some genes that other methods do not, and should therefore be of interest to gene-prediction software developers and genome annotation teams alike. RescueNet is recommended for use in conjunction with, or as a complement to, other gene prediction methods.</p></sec>
<contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Mahony</surname><given-names>Shaun</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>McInerney</surname><given-names>James O</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Smith</surname><given-names>Terry J</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Golden</surname><given-names>Aaron</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib>
BMC Bioinformatics
<sec><title>Background</title><p>Computational gene prediction methods have yet to achieve perfect accuracy, even in the relatively simple prokaryotic genomes. Problems in gene prediction centre on the fact that many protein families remain uncharacterised. As a result, it seems that only approximately half of an organism's genes can be confidently predicted on the basis of homology to other known genes [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B3">3</xref>], so <italic>ab initio </italic>prediction methods are usually employed to identify many protein-coding regions of DNA.</p><p>Currently, the most popular prokaryotic gene-prediction methods, such as GeneMark.hmm [<xref ref-type="bibr" rid="B4">4</xref>] and Glimmer2 [<xref ref-type="bibr" rid="B5">5</xref>], are based on probabilistic Markov models that aim to predict each base of a DNA sequence using a number of preceding bases in the sequence. These methods are undoubtedly very successful, with published sensitivity rates between 90% and 99% for most prokaryotic genomes. However, as the sensitivity rates of the methods rise, specificity generally tends to fall, and while the application of sophisticated post-processing rules can correct many false-positive predictions, no method has yet achieved 100% accuracy. This is especially the case in the more complex eukaryotic gene-finding problem, where less than 80% of exons in anonymous genomic sequences are correctly predicted by current methods [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B8">8</xref>].</p><p>For the foreseeable future it does not seem that the exact set of genes in any organism can be automatically predicted by any single computational method. In practice, this has meant that the best predictions are to be found by combining evidence from two or more independent methods [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B9">9</xref>]. Genome annotation teams often compare the evidence offered by multiple gene-finders in order to predict the gene complement of a given genome. Because of the degree of 'manual' annotation that now takes place in the major genome sequencing centres, a gene-prediction tool will be of practical use if it can exclusively predict genes that other gene-finders cannot.</p><p>Many <italic>ab initio </italic>gene-prediction methods are based on single models of protein-coding regions and therefore make the implicit assumption that all protein-coding regions within a particular genome will share similar statistical properties. However, evidence has mounted that single gene models of intrinsic coding measures are no longer fully satisfying [<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B11">11</xref>]. The problem with single model methods centres on the degree of oligonucleotide composition variation that exists within most genomes. On the codon level, intra-genomic variation in codon bias has long been correlated with expression level [<xref ref-type="bibr" rid="B12">12</xref>]. Counterbalancing the translational selection theory of codon bias is the effect of mutational bias [<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>]. Many other, often more subtle levels of variation have been recognised over the years, with many disparate evolutionary pressures shown to be acting on codon usage bias [<xref ref-type="bibr" rid="B15">15</xref>]. For example, strand-specific codon usage biases have often been recognised [<xref ref-type="bibr" rid="B16">16</xref>-<xref ref-type="bibr" rid="B20">20</xref>], leading to more general studies of correlation between the location of the gene on the genome and codon bias [<xref ref-type="bibr" rid="B21">21</xref>], and the more specific discovery of a A+T skewed bias near the replication terminus of bacterial genomes [<xref ref-type="bibr" rid="B22">22</xref>]. Other effects shown to shape codon usage are gene length [<xref ref-type="bibr" rid="B23">23</xref>] and selection at the amino acid level [<xref ref-type="bibr" rid="B24">24</xref>]. It has also been suggested that content variation can occur at the exon level in eukaryotic genes, the possibility existing that some exons in a gene may have different codon usage patterns to others [<xref ref-type="bibr" rid="B1">1</xref>]. Given that some of the above pressures on codon usage have only recently been discovered, it is likely that some more subtle patterns have yet to be recognised, and therefore it is difficult to predict the level of compositional variation that will be present in an anonymous genomic sequence.</p><p>The need for gene-finding methods that can overcome the problems presented by intra-genomic variation was recognised and addressed in the case of prokaryotic genomes by GeneMark-Genesis [<xref ref-type="bibr" rid="B25">25</xref>], which derives two models for each genome according to typical and atypical codon usage clusters in that genome. This increase in the number of gene models led to an increase in accuracy of the GeneMark method. While Hayes &#x00026; Borodovsky experimented with a third ('highly-typical') codon usage cluster and an associated model in some cases, they did not see the need to further sub-cluster the atypical codon usage set in order to make even more models. Overly sub-clustering the training data would not be useful in the case of Markov-based methods, as the data contained in each sub-cluster may not allow for a good estimation of model parameters. However, generating more specific models for subtle patterns found in the training set can only be advantageous if it can be done in a way that minimises loss of overall accuracy and produces no extra false-positive predictions.</p><p>This paper aims to show how the Self-Organizing Map neural network algorithm can be used to automatically identify the major trends in oligonucleotide variation in a genome, and in doing so provide multiple gene models for use in gene prediction. It will be explained that this approach is an effective solution to the problem of intra-genomic variation. Specific examples of genes predicted only by this method are offered, thus demonstrating the usefulness of the approach in genome annotation.</p><p>A further advantage of using the Self-Organizing Map for gene prediction is the ability of the algorithm to use complex descriptors as measures of gene coding potential. We demonstrate this ability using relative synonymous codon usage (RSCU) as our measure of gene coding potential. Unlike other gene coding measures, RSCU is not based on the absolute frequency of k-mers, but instead describes the codon choice for each amino acid. Markov chains based on the RSCU measure would have transition probabilities that are conditioned on the underlying amino-acids. Although theoretically possible [<xref ref-type="bibr" rid="B26">26</xref>], the practical computation of such Markov chains would give rise to major difficulties. Therefore, the ability of our approach to make use of a sophisticated gene coding descriptor such as RSCU is a distinct advantage of our approach over Markov model based methods.</p></sec><sec><title>Implementation</title><sec><title>Coding measure</title><p>In this study, relative synonymous codon usage (RSCU) vectors are used as the measure of protein-coding potential for a given window of sequence. The RSCU value for a codon 'i' is defined as:</p><p><inline-graphic xlink:href="1471-2105-5-23-i1.gif"/></p><p>where Obs<sub>i </sub>is the observed number of occurrences of codon 'i', and Exp<sub>i </sub>is the expected number of occurrences of the same codon (based on the number of times the relevant amino acid is present in the gene and the number of synonymous alternatives to 'i', assuming a uniform choice of synonymous codons). In order to make the data more compatible with the mathematical methods used, the log of each RSCU<sub>i </sub>value is found so that the resulting value is positive if the codon is used more than expected in that gene, and negative if the codon is used less than expected. Values were capped at &#x000b1; 10, and set to 0 in the case of the non-occurrence of an amino acid in the sample. Taking the RSCU values for each of the codons with synonymous alternatives (and ignoring the 3 stop codons and the Trp and Met codons), each sample can be represented by a vector of 59 values.</p></sec><sec><title>Self-Organizing Map</title><p>The Self-Organizing Map (SOM) is based around the concept of a lattice of interconnected nodes, each of which contains a model. The models begin as random values, but during the iterative training process they are modified to represent different subsets of the training set. In this work for example, the training set and the lattice node models are 59-dimensional RSCU vectors, and the models change during training to become similar to common or repeated patterns in the training set. The algorithm is fully described elsewhere [<xref ref-type="bibr" rid="B27">27</xref>], but we briefly summarize for our context:</p><p>(1) A vector (<italic>X</italic><sub><italic>i</italic></sub>), corresponding to a gene's RSCU values, is loaded from the training dataset.</p><p>(2) The lattice node is found whose model vector most closely resembles the input pattern. This node is denoted the 'winning node'.</p><p>(3) The winning node's model, <italic>W </italic>(as well as a certain number of 'neighbourhood bubble' node models) is changed to be more similar to the input vector by the equation:</p><p><italic>W</italic><sub><italic>new </italic></sub>= <italic>W</italic><sub><italic>old </italic></sub>+ &#x003b7; (<italic>X</italic><sub><italic>i </italic></sub>- <italic>W</italic><sub><italic>old</italic></sub>) &#x000a0;&#x000a0;&#x000a0; (2)</p><p>(4) If all the vectors in the training dataset are processed, we say that an epoch has been completed. In this study, all SOMs are trained for 3000 epochs.</p><p>The 'neighbourhood bubble' mentioned in step 3 is a group of nodes centered at the winning node. The radius of this bubble is initialised to be large and is linearly decreased during training until only the winning node's model is changed. Changing the models on the winning node's neighbours allows the clustering of similar patterns. The learning rate (&#x003b7;) in step 3 is initialised close to 1 and is also linearly decreased during training until it is held constant at a predefined fraction. The linear decrease in learning rate means that each node's model will not get changed as much or as often as training progresses. Two recognised phases of training result; an ordering phase where the lattice takes its general shape, and a convergence phase where the nodes get more specialised to respond to specific patterns.</p><p>In this work, similarity between two vectors is measured by finding the cosine of the angle between them. A cosine of 1 represents exactly similar vectors while a cosine of 0 represents exactly dissimilar vectors.</p><p>The SOM is used mainly in data visualisation, as it can be effectively used to reduce high-dimensional data to a two dimensional map. One of the main strengths of the method is the ability to automatically cluster similar patterns in its training set. In the context of codon usage data, the SOM has been previously used to cluster genes on the basis of similar codon usage [<xref ref-type="bibr" rid="B28">28</xref>-<xref ref-type="bibr" rid="B30">30</xref>]. However, the previous studies have concentrated on identifying genes with atypical codon usage and hypothesising their origin as horizontally transferred genes. It has since been shown that atypical codon usage is not sufficient evidence to show that a gene has origins in horizontal transfer events [<xref ref-type="bibr" rid="B22">22</xref>,<xref ref-type="bibr" rid="B31">31</xref>]. In contrast, this study uses the fact that once a SOM has been trained using codon usage information, the nodes of the SOM encapsulate models that are representative of the major codon usage patterns within the training set.</p><p>If a new sequence is inputted to a trained SOM, we can easily be told which node's model is most similar to this new sequence, and most importantly, how similar. The similarity (cosine) score is then converted to the probability that the sequence is protein-coding. This is achieved by finding the mean cosine score received by a set of random length, random sequence genes that are generated using the same nucleotide bias as the mutational bias found in the genome. Using the mean score, each similarity score can be converted to a z-score, which is in effect the probability that the sequence is not a random sequence.</p></sec><sec><title>Using the SOM to find genes</title><p>Separate SOMs are trained for each of the 15 genomes under test. The SOMs are each 15&#x000d7;15 nodes in size and trained for 3000 cycles. Finding genes via homology search is usually the first step to be carried out in a genome annotation process, so our training sets consist of all genes in the relevant organism that were previously confirmed by homology searches and are also at least 750 bp long. Note that unlike other gene-finding methods, no statistical knowledge of non-coding DNA is necessary as part of the SOM's training.</p><p>In analysing an entire genome sequence, a sliding window is used to split each of the six reading frames into small samples. The default window size is 110 triplets, which has been chosen as a balance between having a window size long enough to evaluate a meaningful RSCU vector, and short enough to predict short genes. Each window is offset from the next by 10 triplets. An arbitrary probability score of 0.1 is used as the threshold for deciding if a sequence was protein-coding, and all samples that scored higher than 0.1 are recorded as predictions. If a stop codon lies in the sample, the gene prediction is annotated as having ended at that point. Note, however, that no effort is made to find stop codons if they are not within the prediction, and no effort whatsoever is made to find any start codons in the prediction.</p></sec><sec><title>Post-processing the predictions</title><p>Once all the samples are processed, some simple post-processing is carried out. Naturally, all same-frame concurrent predictions are merged. Predictions that are totally overlapped by another prediction are deleted if they are less than 75% the length of the other. Similarly, any prediction in which more than half its length is overlapped is deleted if it is less than half the length of the other prediction. Alternatively, any prediction that is less than 90% as long as the overlapping prediction and receiving a lower score is deleted. Finally, any prediction that is overlapped on both ends to a total overlap of at least 70% is also deleted. A prediction size of 75 codons was found by trial and error to be the smallest gene-coding region that could accurately be found using RescueNet.</p><p>While the above rules aim to delete smaller erroneous predictions, it is recognised that the loose nature of the rules leave room for many other overlapping predictions. However, it was found that in many overlapping cases it was difficult to decide which prediction to delete. Therefore, the best solution is to leave both predictions rather than misleading an annotator by giving only one, possibly erroneous, prediction.</p><p>In assessing the accuracy of our method, we had to take into account that our method will not predict most start sites, and some stop sites, exactly. We assume that our method will be of most use to annotation teams who rigorously inspect the results of our method in conjunction with the results of other gene prediction programs. Such annotation teams base their final genome annotation on widespread evidence, so the fact that our method may produce inexact start and stop sites will not be a major disadvantage. Therefore, a correct prediction is defined here as one that predicts more than 50% of an annotated gene in the correct frame. This criterion means that only predictions that are useful to annotators are considered to be correct.</p></sec></sec><sec><title>Results and discussion</title><sec><title>Evaluating accuracy rates</title><p>Previous studies discuss the possibility that the GenBank annotation of various genomes may be incomplete or incorrect in some cases [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B32">32</xref>]. Since many GenBank annotations are not experimentally corroborated, this possibility remains strong. Large-scale benchmarking of gene-prediction algorithms is therefore difficult, because few 'gold standard' annotations exist for prokaryotic genomes. Also, in most cases hypothetical gene annotations in the public databases have their roots in the predictions of an <italic>ab initio </italic>method, thus biasing any comparison of accuracy in favour of the particular method used in the annotation of that genome. However, for the purpose of defining accuracy in this study we must assume that all GenBank annotations are correct and complete. Sensitivity (Sn) is defined here as the percentage of GenBank gene records that are predicted correctly by our method. Specificity (Sp) is defined as the percentage of total RescueNet gene predictions that are correct.</p><p>Table <xref ref-type="table" rid="T1">1</xref> shows the results of RescueNet's predictions in 15 genomes. All results were generated using the default settings described above. Sensitivity and specificity values for each genome are shown, along with sensitivity values for those genes that are above the prediction length threshold of 75 codons (225 bp) and sensitivity values for those genes that have database matches.</p><p>Sequence data used in this study include the following 15 genomes and associated published genes available from the GenBank database: <italic>A. aeolicus </italic>[<xref ref-type="bibr" rid="B33">33</xref>], <italic>B. subtilis </italic>[<xref ref-type="bibr" rid="B34">34</xref>], <italic>Buchnera sp. </italic>[<xref ref-type="bibr" rid="B35">35</xref>], <italic>B. burgdorferi </italic>[<xref ref-type="bibr" rid="B36">36</xref>], <italic>C. jejuni </italic>[<xref ref-type="bibr" rid="B37">37</xref>], <italic>D. radiodurans </italic>(chromosome 1) [<xref ref-type="bibr" rid="B38">38</xref>], <italic>E. coli </italic>[<xref ref-type="bibr" rid="B39">39</xref>], <italic>H. influenzae </italic>[<xref ref-type="bibr" rid="B40">40</xref>], <italic>H. pylori </italic>[<xref ref-type="bibr" rid="B41">41</xref>], <italic>M. genitalium </italic>[<xref ref-type="bibr" rid="B42">42</xref>], <italic>M. jannaschii </italic>[<xref ref-type="bibr" rid="B43">43</xref>], <italic>R. solanacearum </italic>[<xref ref-type="bibr" rid="B44">44</xref>], <italic>S. coelicolor </italic>[<xref ref-type="bibr" rid="B45">45</xref>], <italic>Synechocystis sp. </italic>[<xref ref-type="bibr" rid="B46">46</xref>], and <italic>Y. pestis </italic>[<xref ref-type="bibr" rid="B47">47</xref>]. These genomes were chosen to be representative of a wide range of GC content.</p></sec><sec><title>High G+C content genomes</title><p>Three of the genomes tested have very high G+C content (<italic>D. radiodurans</italic>, <italic>R. solanacearum </italic>and <italic>S. coelicolor</italic>). High G+C content genomes present a problem to many gene-finding methods because of the relative infrequency of randomly occurring stop codons. The scarcity of stop codons has the effect of a large number of long, overlapping ORFs occurring in the sequence, relatively few of which are actually protein-coding. Many of the current gene-finders fail to discriminate accurately between coding and non-coding ORFs in this type of situation.</p><p>In our method, the relatively high specificity in each high G+C content genome suggests that RescueNet may have advantages in their annotation (see Table <xref ref-type="table" rid="T1">1</xref>). To illustrate a case where RescueNet may be of practical use, we can consider the ORF annotated as DR1142 (see Figure <xref ref-type="fig" rid="F1">1</xref>) from <italic>D. radiodurans</italic>. This ORF is annotated to be protein-coding on the basis of the Glimmer2 prediction only. The RescueNet prediction in this area overlaps DR1142, but on the opposite strand. This type of situation, where a RescueNet prediction directly contradicts a GenBank/Glimmer2 annotation, occurs at least 23 times in the <italic>D. radiodurans </italic>genome. It is entirely possible that the Glimmer2 predictions are wrong in some of these cases, and the RescueNet predictions correct, but this cannot be proven without biochemical characterisation of the relevant gene. However, in the specific case of the DR1142 annotation, the RescueNet prediction has a much stronger database match than the GenBank annotation, and so has a high possibility of being correct.</p><p>Another interesting pointer to the advantages of RescueNet in high G+C content genomes is the substantially higher percentage of genes with database matches that are correctly predicted by RescueNet (see Table <xref ref-type="table" rid="T1">1</xref>). In <italic>D. radiodurans</italic>, for example, 92.54% of genes with database matches are correctly predicted by RescueNet compared with only 84.28% of the total GenBank gene annotations. These figures suggest that hypothetical genes that are predicted only by Markov-based methods are poorly recognised by RescueNet, possibly because many hypothetical genes in high G+C content genomes may in fact be false gene predictions.</p></sec><sec><title>Predicting the location of frameshifts</title><p>The general location of frameshifts within a gene sequence can be found by our method. Two features of our approach facilitate this. Firstly, even though the overall codon usage of a frameshifted gene could seem unusual, the two coding sections of the gene should each retain the organism's native codon usage. Secondly, our approach does not require that a prediction be bounded by a start and a stop codon. The sliding window used in our algorithm can therefore predict the correct coding frames each side of the frameshift.</p><p>In an interesting example in Figure <xref ref-type="fig" rid="F1">1</xref>, two RescueNet predictions overlap the <italic>D. radiodurans </italic>gene DR1143 in such a way that it seems that there may be a frameshift that extends the protein-coding region of the gene past the annotated stop codon. In fact, combining the two RescueNet predictions offers a better database match to the same genes that the original annotation matches. This increases the possibility that the actual gene contains an authentic frameshift or at least that the extra RescueNet prediction is an evolutionary artefact.</p><p>Figure <xref ref-type="fig" rid="F2">2</xref> shows another example of frameshifts which are detected by RescueNet. In this case, the <italic>H. influenzae </italic>genes HI0218 and HI0220 both contain frameshifts, but both are handled by RescueNet's predictions. Note that the GeneMark algorithms are known to show the location of frameshifted regions in much the same manner as we have described, but our approach has required no modification to our basic algorithm in order to facilitate the prediction of frameshifted genes.</p></sec><sec><title>Comparison with a Markov-based method</title><p>There may be a perception that any method using codon usage as the coding measure will only give predictions that are a subset of the predictions given by a Markov-based method that uses a 4<sup>th </sup>or 5<sup>th </sup>order model. To counter this argument, we compared the predictions of our method in two genomes (<italic>H. influenzae </italic>and <italic>H. pylori</italic>) to those of the web-based version of GeneMark.hmm 2.1 for prokaryotes <ext-link ext-link-type="uri" xlink:href="http://opal.biology.gatech.edu/GeneMark/gmhmm2_prok.cgi"/>, which generated results using two models; the 'typical' and 'atypical' models.</p><p>The published sensitivity of GeneMark.hmm in the <italic>H. influenzae </italic>genome (96.2%, see [<xref ref-type="bibr" rid="B4">4</xref>]) is higher than that of our method, and the published specificity (89.8%) is lower, so GeneMark.hmm should give more predictions overall for this genome. However, 11 <italic>H. influenzae </italic>genes are predicted correctly by our method which are not predicted by GeneMark.hmm using 5<sup>th </sup>order models, and 14 genes are predicted correctly by our method which are not predicted by GeneMark.hmm using 4<sup>th </sup>order models. In the <italic>H. pylori </italic>genome, GeneMark.hmm has again a higher sensitivity and a lower specificity (94.0% &#x00026; 91.3% respectively), but even more genes are exclusively predicted by our method; 25 genes as compared to the 5<sup>th </sup>order GeneMark.hmm models and 30 genes as compared to the 4<sup>th </sup>order models. Although these genes represent a small proportion of the total number of genes in the respective organisms, the fact that they are only predicted by RescueNet gives some indication of the advantage of using RescueNet in conjunction with other gene prediction methods.</p></sec><sec><title>Possible future improvements</title><p>RSCU is only one of many possible criterion with which to measure coding potential (see [<xref ref-type="bibr" rid="B48">48</xref>] for a review of others). In-phase hexamers are accepted as the most accurate k-mer frequency based measure of coding potential, and so their use as the coding measure in a Self-Organizing Map may offer improvement in accuracy over RescueNet. However, the larger space dimension of the hexamer coding measure may force a larger sliding window to be used and therefore the use of hexamers could actually decrease the precision of gene prediction.</p><p>The future use of alternative coding measures with our approach may also help to overcome difficulties in recognising genes that are reputed to be horizontally transferred in origin. Horizontally transferred genes would be more likely to have dissimilar codon usage patterns to other genes in the genome. Since our approach currently relies on the codon usage patterns it finds in the training set, it is unlikely to mark areas of unseen codon usage as protein-coding regions. Note, however, we are not suggesting all genes that were not recognised by our approach are of horizontally transferred origin. There are many explanations for a gene displaying atypical codon usage, and codon usage cannot be used as an accurate indicator of horizontal transfer.</p><p>There may be other ways to improve the accuracy of our method. The current implementation has a rather simple post-processing step that does not rely on modifying the prediction in order to include start or stop codons. While the practise of not constraining a prediction to be bound by a start and stop codon stands in stark contrast to other methods, we did not wish to lengthen or shorten any predictions artificially, since doing so can mislead annotation teams (especially in start site annotation). Relatively simple post-processing steps may, in fact, be advantageous. Our predictions represent a raw account of regions of the genome that display typical or native codon usage patterns, and this in itself may be of interest to annotation teams who use codon usage plots as the basis for some genomic feature annotations.</p></sec></sec><sec><title>Conclusion</title><p>Gene-finding in prokaryotic genomes is still not a completely solved problem, partly because current methods use a limited number of models to represent the training data. In this paper, we have introduced an alternative, independent approach to the problem. The Self-Organizing Map approach has the potential to overcome the issue of variation in the statistical properties of the training set data, and can automatically train a representative number of gene-models, depending on the degree of variation within the training data.</p><p>While the current implementation of our approach produces lower raw sensitivity scores in comparison to established Markov-based techniques, we have clearly shown that our method can predict some genes that other methods cannot. We have also demonstrated advantages in annotating the traditionally 'difficult' high G+C content genomes. Annotation teams who are concerned with the complete and accurate annotation of a sequenced genome should find our method useful when used alongside other gene-finding methods. The relatively high specificity of our method, coupled with the independent nature of the algorithm, should make it a useful tool in confirming the predictions of other software programs and in some cases pointing out areas of conflicting or contradictory predictions that are worthy of further examination.</p></sec><sec><title>Availability</title><p>Project name: RescueNet</p><p>Project home page: <ext-link ext-link-type="uri" xlink:href="http://bioinf.nuigalway.ie/RescueNet/"/></p><p>Operating systems: Windows, Linux, IRIX, Digital Unix. Source code also available.</p><p>Programming Language: C++</p><p>Licence: GNU GPL</p><p>Restrictions to use by non-academics: Please contact the authors.</p></sec><sec><title>Authors' contributions</title><p>SM conceived of the study, and designed, implemented and tested the RescueNet software. JMcI, TS and AG supervised, and participated in the design of, the study. All authors read and approved the final manuscript.</p></sec>
A Hidden Markov Model method, capable of predicting and discriminating &#x003b2;-barrel outer membrane proteins
<sec><title>Background</title><p>Integral membrane proteins constitute about 20&#x02013;30% of all proteins in the fully sequenced genomes. They come in two structural classes, the &#x003b1;-helical and the &#x003b2;-barrel membrane proteins, demonstrating different physicochemical characteristics, structure and localization. While transmembrane segment prediction for the &#x003b1;-helical integral membrane proteins appears to be an easy task nowadays, the same is much more difficult for the &#x003b2;-barrel membrane proteins. We developed a method, based on a Hidden Markov Model, capable of predicting the transmembrane &#x003b2;-strands of the outer membrane proteins of gram-negative bacteria, and discriminating those from water-soluble proteins in large datasets. The model is trained in a discriminative manner, aiming at maximizing the probability of correct predictions rather than the likelihood of the sequences.</p></sec><sec><title>Results</title><p>The training has been performed on a non-redundant database of 14 outer membrane proteins with structures known at atomic resolution; it has been tested with a jacknife procedure, yielding a per residue accuracy of 84.2% and a correlation coefficient of 0.72, whereas for the self-consistency test the per residue accuracy was 88.1% and the correlation coefficient 0.824. The total number of correctly predicted topologies is 10 out of 14 in the self-consistency test, and 9 out of 14 in the jacknife. Furthermore, the model is capable of discriminating outer membrane from water-soluble proteins in large-scale applications, with a success rate of 88.8% and 89.2% for the correct classification of outer membrane and water-soluble proteins respectively, the highest rates obtained in the literature. That test has been performed independently on a set of known outer membrane proteins with low sequence identity with each other and also with the proteins of the training set.</p></sec><sec><title>Conclusion</title><p>Based on the above, we developed a strategy, that enabled us to screen the entire proteome of <italic>E. coli </italic>for outer membrane proteins. The results were satisfactory, thus the method presented here appears to be suitable for screening entire proteomes for the discovery of novel outer membrane proteins. A web interface available for non-commercial users is located at: <ext-link ext-link-type="uri" xlink:href="http://bioinformatics.biol.uoa.gr/PRED-TMBB"/>, and it is the only freely available HMM-based predictor for &#x003b2;-barrel outer membrane protein topology.</p></sec>
<contrib id="A1" contrib-type="author"><name><surname>Bagos</surname><given-names>Pantelis G</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Liakopoulos</surname><given-names>Theodore D</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Spyropoulos</surname><given-names>Ioannis C</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" corresp="yes" contrib-type="author"><name><surname>Hamodrakas</surname><given-names>Stavros J</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib>
BMC Bioinformatics
<sec><title>Background</title><p>Integral membrane proteins are divided into two distinct structural classes, the &#x003b1;-helical membrane proteins and the &#x003b2;-barrel membrane proteins. The former class is the more abundant and well studied, since proteins of that type are located mostly in the cell membranes of both prokaryotic and eukaryotic organisms, performing a variety of biologically important functions. Proteins of that class have their membrane spanning regions forming &#x003b1;-helices, which consist mainly of hydrophobic residues [<xref ref-type="bibr" rid="B1">1</xref>]. A variety of algorithms and computational techniques have been proposed for the prediction of the transmembrane segments of &#x003b1;-helical membrane proteins, with high accuracy and precision. The members of the latter class (&#x003b2;-barrel membrane proteins) are located in the outer membrane of gram-negative bacteria, and presumably in the outer membrane of chloroplasts and mitochondria. The members of that class are having their membrane spanning segments formed by antiparallel &#x003b2;-strands, creating a channel in a form of a barrel that spans the outer membrane [<xref ref-type="bibr" rid="B2">2</xref>]. The first known members of that class were found to be the bacterial trimeric porins, forming water-filled channels that mediate the passive transport of ions and small molecules through the outer membrane [<xref ref-type="bibr" rid="B2">2</xref>]. During the last few years, more &#x003b2;-barrel proteins were found in the bacterial outer membrane, and a number of structures have been solved in atomic resolution [<xref ref-type="bibr" rid="B2">2</xref>]. These proteins perform a wide variety of functions such as active ion transport, passive nutrient uptake, membrane anchoring, adhesion, and catalytic activity. A large number of pathogens are actually bacteria belonging to the gram negative bacteria class. Considering additionally the important biological functions in which outer membrane proteins are involved in, it is not a surprise that those proteins attract an increased medical interest. This is confirmed by the continuously increasing number of completely sequenced genomes of gram-negative bacteria deposited in the public databases. On the other hand, the extensive study of the structure of transmembrane &#x003b2;-barrel proteins, could reveal special aspects of the process of protein folding, and give us useful insights on protein structure and function. For the reasons mentioned above, there is clearly a need to develop computational tools for predicting the membrane spanning strands of those proteins, and also discriminating them from water-soluble proteins when searching entire genomes.</p><p>In contrast to the &#x003b1;-helical membrane proteins, whose membrane spanning segments can be identified by statistical methods, neural networks, or Hidden Markov Models with high accuracy, this task is more difficult in the case of the &#x003b2;-barrel membrane proteins of the outer membrane. This is due to the lack of a clear pattern in their membrane spanning strands, such as the stretch of 15&#x02013;30 consecutive hydrophobic residues or the Positive Inside rule, which occur in the &#x003b1;-helical proteins. Furthermore, discrimination of transmembrane strands from other &#x003b2;-strands, forming &#x003b2;-barrel structures in water-soluble proteins, is even more difficult. The reason for that is the fact that water-soluble proteins that form &#x003b2;-barrel structures, share (up to a certain degree) common features with the transmembrane strands of the bacterial outer membrane proteins, such as amphipathicity.</p><p>A few approaches have been made, in the direction of predicting the transmembrane strands of outer membrane proteins and/or identifying those proteins when searching large data sets; they are based on study of the physicochemical properties of the &#x003b2;-strands, such as hydrophobicity and amphipathicity [<xref ref-type="bibr" rid="B3">3</xref>], statistical analyses based on the amino acid composition of the known structures [<xref ref-type="bibr" rid="B4">4</xref>], or machine learning techniques like neural network predictors [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>], and Hidden Markov Models [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>]. Recently, a method based on a sequence profile-based HMM [<xref ref-type="bibr" rid="B8">8</xref>], requiring as input evolutionary information derived from multiple alignments, achieved the highest accuracy.</p><p>In this work we developed a Hidden Markov Model method based solely on the amino acid sequence, without the requirement of evolutionary information. The model is cyclic, and captures the structural characteristics of the transmembrane &#x003b2;-strands of the outer membrane proteins. For training and evaluating the model, we compiled a non-redundant dataset of 14 outer membrane proteins with structure known at atomic resolution (Table <xref ref-type="table" rid="T1">1</xref>, see Materials and methods section), and tested it with a jacknife procedure. The model is also used for discriminating outer membrane proteins, in large-scale genome analyses.</p></sec><sec><title>Results and discussion</title><p>The results obtained from the model are presented in <xref ref-type="supplementary-material" rid="S1">additional file 1</xref> and Table <xref ref-type="table" rid="T2">2</xref> (for a definition of the transmembrane, TM, segments, see the Materials and methods section). Table <xref ref-type="table" rid="T2">2</xref> shows the results obtained comparing the predicted strands and topologies with the PDB annotation and also those comparing with the manual annotation used for training (see Materials and methods). The model correctly predicts the location of 96.3% of the transmembrane strands (206 out of 214). Two (2) additional strands were predicted correctly but slightly misplaced from their observed positions. These misplaced strands, which belong to the proteins with PDB codes 1PRN and 2POR, were the only strands that have been falsely predicted (false positives). The total number of correctly predicted topologies (correct prediction of &#x003b2;-strands and orientation of the loops) is 10 and, when counting the misplaced strands, 11 out of 14 proteins in the training set. When the comparison against the manual annotation used for training was performed, it was noted that there is one additional strand (the second TM strand of 1I78), which is predicted slightly misplaced. The model has also been tested with the well-known jacknife procedure. The jacknife procedure consists of removing a protein from the training set, training the model with the remaining proteins and performing the test on the protein removed. This process is tandemly repeated for all proteins in the training set, and the final prediction accuracy summarizes the outcome of all independent tests. Thus, this procedure is regarded as most appropriate for the assessment of a prediction method based on independent training and test data.</p><p>The result of the jacknife test concerning the correctly predicted TM strands was 204 out of 214 (95.3%), with 12 over-predicted strands. The overall number of correctly predicted topologies was 9 out of 14. When counting the predicted misplaced strands, the number of correctly predicted topologies raises to 10 out of 14. Once again when comparing against the manual annotation, the second TM strand of 1I78, is predicted slightly misplaced.</p><p>The per-residue accuracies and correlation coefficients (see <bold>Materials and methods</bold>) for both the self-consistency and jacknife tests are listed also in Table <xref ref-type="table" rid="T2">2</xref>, with respect to either the PDB annotation or the manual annotation used for training. Apparently, the significantly lower percentages reported in the case of comparison with the PDB annotation, is a clear consequence of the fact that the strands extend in some cases far beyond the lipid bilayer. These strands could not have been predicted as transmembrane along their entire length, and our model predicts only the part of the strand that it is inserted into the membrane. 1I78 is a perfect example of such a case since all of its strands are clearly extending far beyond the membrane by 8 or more residues. In addition to the self-consistency and jacknife test, we performed another independent test. We divided the training set in two datasets of seven proteins each and used the one for training and the other for testing. This procedure was repeated 5 times, choosing randomly 7 different proteins each time, and the results concerning the per-residue accuracy and the correlation coefficient were in the range 0.78 &#x02013; 0.80 and 0.57 &#x02013; 0.71 respectively. We also tested the performance of the model on the Neisserial Surface Protein A (Nspa) [<xref ref-type="bibr" rid="B9">9</xref>], the Outer Membrane Enzyme Pagp from <italic>E. coli </italic>[<xref ref-type="bibr" rid="B10">10</xref>], and the Outer Membrane Cobalamin Transporter (Btub) from <italic>E. coli </italic>[<xref ref-type="bibr" rid="B11">11</xref>] The structures of these proteins have been very recently solved, they have not been included in the training set, and they do not show any significant homology with any protein of the training set. For NspA, and BtuB the model correctly locates all the transmembrane strands and the proteins' full topologies, whereas for Pagp we get two over-predicted strands. For the three proteins the per-residue accuracy is 90.9% and the correlation coefficient is 0.78.</p><p>When we tested the ability of the model to discriminate between outer membrane and globular proteins, the percentage of the correctly classified outer membrane proteins (at a fixed threshold) was 88.8% whereas the percentage of correctly classified globular proteins was 89.2%, (Figure <xref ref-type="fig" rid="F1">1</xref>). The absolute value of the score threshold obtained this way was 2.995, with values lower than that indicating the possibility of the protein being an outer membrane protein.</p><p>Furthermore, we developed a protocol that allowed us to apply the newly developed method to search the complete proteome of <italic>E. coli </italic>[<xref ref-type="bibr" rid="B12">12</xref>] for &#x003b2;-barrel membrane proteins. The protocol consists of three steps: In the first step we perform a search using the PRED-CLASS algorithm [<xref ref-type="bibr" rid="B13">13</xref>], with the aim of identifying and removing &#x003b1;-helical membrane proteins. According to the PRED-CLASS prediction, 1157 proteins out of 5361 were classified as transmembrane and hence they were removed. In the second step, the remaining proteins were filtered with the SignalP program, for finding the secreted proteins, since it is apparent that the majority, if not all, of the outer membrane proteins posses a signal peptide sequence necessary for the translocation to the outer membrane. In order not to discard potential candidates for being identified as outer membrane proteins, we applied both versions of SignalP, the Neural Network [<xref ref-type="bibr" rid="B14">14</xref>] and the Hidden Markov Model [<xref ref-type="bibr" rid="B15">15</xref>], and if at least one of them indicated the presence of a signal peptide, the protein was not discarded. Then, the signal peptide predicted by the Neural Network algorithm of SignalP was removed, since this algorithm is more accurate than the Hidden Markov algorithm in locating the correct splicing site from all the candidate proteins. This procedure resulted to 978 proteins. In the third and final step, the remaining 978 protein sequences were submitted to our HMM predictor having set the discrimination score at the pre-specified threshold mentioned above. In total, 236 proteins scored below the threshold for the outer membrane proteins (after excluding fragments and sequences shorter than 60 residues) accounting for 4.4% of the complete proteome. Among the top scoring proteins, 42 are well known outer membrane proteins according to the existing annotation, including many fimbrial proteins, usher proteins and transporters, and 87 were proteins whose annotation was "putative" but suggested that their localization was to the outer membrane. The number of proteins whose annotation suggested that they were misclassified, including a lot of periplasmic proteins and enzymes, was 34, whereas the "putative" misclassified proteins were 23. Finally, the remaining 50 proteins were hypothetical proteins or proteins with completely unknown function. Apparently, the 57 over-predictions probably are resulting from the fact that outer membrane proteins are only a small fraction compared to the whole proteome. Since it is believed that outer membrane proteins constitute around 2&#x02013;4% of the complete proteomes, it is natural that even a method with 99% of correct predictions, will result in a large number of false positives. Nevertheless, given the constrains mentioned above, this method clearly offers a useful tool for the automatic annotation of entire proteomes, since the false positives could be easily removed considering other sequence characteristics.</p><p>Comparing our method with the best method proposed so far for the prediction of transmembrane &#x003b2;-barrel proteins by Martelli et al. [<xref ref-type="bibr" rid="B8">8</xref>], as well as with the HMM method proposed by Liu et al. [<xref ref-type="bibr" rid="B7">7</xref>], the following points should be mentioned. The HMM-profile based method by Martelli et al. [<xref ref-type="bibr" rid="B8">8</xref>], uses as input the evolutionary information included in multiple alignments. The method proposed here, uses as input only the amino acid sequence of the protein, hence it is computationally simpler. Even though our method does not outperform the profile based HMM method by Martelli et al. [<xref ref-type="bibr" rid="B8">8</xref>] in the per residue accuracy, when it comes to the number of correctly predicted transmembrane strands and overall topologies, the two methods are practically equivalent. The same argument holds for the case of the discriminative power of the two methods, since the percentage of correctly classified &#x003b2;-barrel proteins was 84%, and percentage of correctly classified water soluble proteins was 90% as reported in [<xref ref-type="bibr" rid="B8">8</xref>], showing that better results can be obtained even without the use of evolutionary information. Concerning the method proposed by Liu et al, which uses as input single sequences, the results about strand localization and overall topology assignment are also comparable with our method, but no discrimination could be performed between outer membrane proteins and soluble ones in their method, thus requiring a separate method for the discrimination purposes. Furthermore in Liu et al [<xref ref-type="bibr" rid="B7">7</xref>], no overall measures of accuracy were reported.</p><p>Both methods mentioned above use HMMs with architectures quite similar to the model shown here, with minor differences, and this is not a surprise. For the sake of argument, the two most successful methods for the prediction of transmembrane segments of &#x003b1;-helical membrane proteins use a similar architecture; that architecture reflects the most obvious way to map the biological features of transmembrane proteins to the mathematical formalism of the Hidden Markov Model. Finally, the methodology that we used for the training and the decoding is completely different from those used by Martelli et al. [<xref ref-type="bibr" rid="B8">8</xref>], and Liu et al. [<xref ref-type="bibr" rid="B7">7</xref>]. Our model was trained according to the Conditional Maximum Likelihood criterion, which differs significantly from the Maximum Likelihood training scheme, performed with the Baum-Welch algorithm, by the two methods mentioned. For the decoding, Martelli et al use the so-called posterior decoding method, with the aid of a dynamic programming algorithm, whereas Liu et al, rely on the traditional Viterbi algorithm. Even when in our tests the N-best decoding does not outperform significantly the Viterbi decoding (data not shown), when it comes to newly discovered proteins, the option to perform decoding with the best method available is a clear advantage.</p><p>When our method is compared against the methods developed by Zhai and Saier [<xref ref-type="bibr" rid="B3">3</xref>] and Wimley [<xref ref-type="bibr" rid="B4">4</xref>], we observe that none of the above methods controlled for the number of false positives and false negatives, since they were not validated statistically. They both report the finding of a number of predicted outer membrane proteins, for which the genome annotation suggested localization to the outer membrane. The fact that we report 236 predicted outer membrane proteins in <italic>E. coli </italic>proteome, compared to 118 in [<xref ref-type="bibr" rid="B3">3</xref>] and 200 in [<xref ref-type="bibr" rid="B4">4</xref>], reflects the fact that we chose to retain the threshold obtained from cross-validation. Clearly, in real life applications using our method we could lower the threshold and obtain fewer predictions (&#x0003c;200), with the cost of loosing 5&#x02013;10 outer membrane proteins.</p></sec><sec><title>Conclusions</title><p>We present here a novel method, based on a Hidden Markov Model, for the prediction of the transmembrane &#x003b2;-strands of the outer membrane proteins of Gram-negative bacteria, and for the discrimination of these proteins from globular proteins. To our knowledge, a Hidden Markov Model trained with a discriminative method is applied for the first time in molecular biology for such a task. We show here that we can achieve predictions at least equally successful to other existing methods, without the use of evolutionary information. We also showed that the method is powerful when used for discrimination purposes, as it can discriminate outer membrane proteins from water soluble proteins in large datasets with a high accuracy, suggesting that it is a very reliable solution for screening entire genomes of Gram negative bacteria, for the discovery of novel &#x003b2;-barrel proteins as possible drug targets. Compared to other single sequence methods (for both discrimination and strand prediction) our method is unambiguously the best currently available. Compared to multiple sequence methods (requiring evolutionary information) our method achieves comparable results. Clearly, our method combines equally, higher rates for both strand localization and sequence discrimination, from any existing method. A web server running the application is located in our laboratory and it is the only HMM-based application currently freely available, making our method accessible to scientists around the world. The user may submit a sequence in FASTA format, and has the option to choose between decoding by either the N-best algorithm, the standard Viterbi algorithm or posterior decoding with a dynamic programming algorithm (Figure <xref ref-type="fig" rid="F2">2</xref>). The output consists of the prediction for the transmembrane strands (Figure <xref ref-type="fig" rid="F3">3</xref>). Optionally the user may obtain a graphical plot showing the posterior probabilities in a 3-state mode (extracellular, periplasmic and transmembrane), which may be useful in the case of ambiguously defined topologies. The application also returns the score used for discrimination purposes thus, helping the user to identify possible &#x003b2;-barrel outer membrane proteins.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>The Hidden Markov Model</title><p>Hidden Markov Models have been extensively used for pattern recognition problems, with the most known example found in the speech recognition methodology [<xref ref-type="bibr" rid="B16">16</xref>]. Hidden Markov Models have been used in bioinformatics during the last few years for generating probabilistic profiles for protein families [<xref ref-type="bibr" rid="B17">17</xref>], the prediction of transmembrane helices in proteins [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>], the prediction of signal peptides and their cleavage sites [<xref ref-type="bibr" rid="B15">15</xref>], the prediction of genes [<xref ref-type="bibr" rid="B20">20</xref>] and recently for the prediction of the transmembrane &#x003b2;-strands [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>]. An excellent introduction of those applications in molecular biology is the book of Durbin <italic>et al </italic>[<xref ref-type="bibr" rid="B21">21</xref>] whose notation will follow hereafter.</p><p>The Hidden Markov Model is a probabilistic model consisting of several states, connected by means of the transition probabilities, thus forming a markov process. If we consider an aminoacid sequence of a protein with length <italic>L</italic>, denoted by:</p><p><bold>x </bold>= <italic>x</italic><sub>1</sub>, <italic>x</italic><sub>2</sub>,..., <italic>x</italic><sub><italic>L</italic></sub>,</p><p>with a labeling (corresponding to transmembrane, intracellular and extracellular regions):</p><p><bold>y </bold>= <italic>y</italic><sub>1</sub>, <italic>y</italic><sub>2</sub>,..., <italic>y</italic><sub><italic>L</italic></sub></p><p>then, the transition probability for jumping from a state <italic>k </italic>to a state <italic>l </italic>is defined as:</p><p>&#x003b1;<sub><italic>kl </italic></sub>= <italic>P</italic>(&#x003c0;<sub><italic>i </italic></sub>= <italic>l</italic>|&#x003c0;<sub><italic>i</italic>-1 </sub>= <italic>k</italic>)</p><p>Where &#x003c0; is the "path" in the particular position of the amino acid sequence (i.e. the sequence of states, as opposed to the sequence of symbols). Each state <italic>k </italic>is associated with a distribution of emission probabilities, meaning the probabilities that any particular symbol could be emitted by the current state. Assuming an alphabet &#x003a3;, consisting of the symbols corresponding to the 20 amino acids, the probability that a particular amino-acid <italic>b </italic>is emitted from state <italic>k </italic>is defined as:</p><p><italic>e</italic><sub><italic>k</italic></sub>(<italic>b</italic>) = <italic>P</italic>(<italic>x</italic><sub><italic>i </italic></sub>= <italic>b</italic>|&#x003c0;<sub><italic>i </italic></sub>= <italic>k</italic>)</p><p>The term 'hidden' is justified by the fact that when one observes the emitted symbols he cannot see the underlying states, thus the true state process is hidden from the observer. The total probability of the observation sequence given the model, <italic>P</italic>(<italic>x</italic>|&#x003b8;), is computed using the efficient Forward algorithm [<xref ref-type="bibr" rid="B16">16</xref>], whereas the joint probability of the sequence and the labeling denoted by <italic>P</italic>(<italic>x</italic>,<italic>y</italic>|&#x003b8;), by its trivial modification proposed by Krogh [<xref ref-type="bibr" rid="B22">22</xref>].</p></sec><sec><title>Training and decoding algorithms</title><p>Traditionally, the parameters of a Hidden Markov Model are optimized according to the Maximum Likelihood criterion [<xref ref-type="bibr" rid="B16">16</xref>],</p><p><inline-graphic xlink:href="1471-2105-5-29-i1.gif"/></p><p>A widely used algorithm for this task is the efficient Baum-Welch algorithm (also known as Forward-Backward) [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B23">23</xref>], which is a special case of the Expectation-Maximization (EM) algorithm, proposed for Maximum Likelihood (ML) estimation for incomplete data [<xref ref-type="bibr" rid="B24">24</xref>]. The algorithm, updates iteratively the model parameters (emission and transition probabilities), with the use of their expectations, computed with the use of the Forward and Backward algorithms. Convergence to at least a local maximum of the likelihood is guaranteed. The main disadvantage of ML training is that it is not discriminative. In this work, we used Conditional Maximum Likelihood (CML) training for labeled data, as proposed by Krogh [<xref ref-type="bibr" rid="B25">25</xref>]. The Conditional Maximum Likelihood criterion is:</p><p><inline-graphic xlink:href="1471-2105-5-29-i2.gif"/></p><p>This kind of training, often referred to as discriminating training, seeks to maximize the probability of the correct prediction, i.e. the probability of the labeling <bold>y </bold>for a given sequence <bold>x </bold>and a model &#x003b8;. The parameters of the model (transition and emission probabilities) are updated simultaneously, using the gradients of the likelihood function as described in [<xref ref-type="bibr" rid="B26">26</xref>], and the training process terminates when the likelihood does not increase beyond a pre-specified threshold. To reduce the number of the free parameters of the model, and thus improve the generalization capability, states expecting to have the same emission probabilities, were tied together (Figure <xref ref-type="fig" rid="F4">4</xref>). Furthermore, to avoid overfitting, the iterations started from emission probabilities corresponding to the initial amino-acid frequencies observed in the known protein structures and small pseudocounts were added in each step.</p><p>The decoding was performed using the N-best algorithm [<xref ref-type="bibr" rid="B27">27</xref>] (Figure <xref ref-type="fig" rid="F5">5</xref>), as formulated in [<xref ref-type="bibr" rid="B25">25</xref>]. This algorithm is a heuristic that attempts to find the most probable labeling of a given sequence, as opposed to the well-known Viterbi algorithm [<xref ref-type="bibr" rid="B16">16</xref>], which guarantees to find the most probable path of states. Since there are several states contributing to the same labeling of a given sequence (as in our case), the N-best algorithm will always produce a labeling with a probability at least as high as that computed by the Viterbi algorithm, in other words it always returns equal if not better results. Its main drawback is the memory requirements and computational complexity, resulting in a slowdown of the decoding process. For the purpose of discrimination, the information included in the prediction of the putative TM segments is not sufficient, since a prediction for a transmembrane strand could occur even in globular proteins. Thus, there is need for a global predictor reflecting the overall fit of the query sequence to the model. This predictor is the negative log-likelihood of the sequence given the model, as computed by the Forward algorithm and normalized for the length of the sequence. Thus, the statistical score used for discrimination is:</p><p><inline-graphic xlink:href="1471-2105-5-29-i3.gif"/></p><p>where <italic>L </italic>is the length of the sequence. We studied the proportion of correctly classified proteins as a function of the discrimination score used as the threshold. We defined the optimal threshold as the value that maximizes that function. Proteins with score values below the threshold should be declared as beta-barrel membrane proteins. All algorithms and tools used throughout this work have been implemented by the authors, using the Java programming language by Sun Microsystems.</p></sec><sec><title>The model architecture</title><p>The model that we used is cyclic, consisting of 61 states, (Figure <xref ref-type="fig" rid="F4">4</xref>). The architecture has been chosen so that it could fit as much as possible to the limitations imposed by the known structures. The model consists of three "sub-models" corresponding to the three desired labels to predict, the TM (transmembrane) strand sub-model and the inner and outer loops sub-models respectively. The TM strand model incorporates states to model the special architecture of the transmembrane strands. Thus, there are states that correspond to the core of the strand and the aromatic belt located at the lipid bilayer interface. Furthermore, other states correspond to the amino acid residues facing the bilayer (the external side of the barrel) and the residues facing the barrel interior. All states are connected with the appropriate transition probabilities in order to be consistent with the known structures (i.e. to ensure appropriate length distributions and to model the alternating pattern of hydrophobic-non hydrophobic residues, corresponding to the external-internal residues of the barrel). The minimum allowed length for a transmembrane strand is 7 residues, whereas the maximum is 17.</p><p>The inner and outer loops are modeled with a "ladder" architecture, whereas at the top of the outer loop there is a self transitioning state corresponding to residues too distant from the membrane; these cannot be modeled as loops, hence that state is named "globular". The "inner" loop sub-model has no corresponding "globular" state, reflecting the fact that inner loops are significantly shorter than the outer ones, since none of the known structures possesses an inner loop longer than twelve residues. In order to capture the fact that all known structures are having their N-terminal tail falling into the periplasmic space (the "inside" with respect to the outer membrane) we allowed the begin state of the model to be followed only by states belonging to the inner loop or to TM strands directing to the external side of the outer membrane. Finally, we allowed a self-transitioning absorbing state to follow the inner loop states, in order to correctly model sequences that have a long C-terminus falling in the periplasmic space. States expected to have the same emission probabilities are tied together.</p></sec><sec><title>Training and testing sets</title><p>The training set that we used has been compiled with consideration of the SCOP classification [<xref ref-type="bibr" rid="B28">28</xref>]. In particular, we selected all PDB codes from SCOP that belong to the fold "Transmembrane beta-barrels", and obtained the corresponding structures from the Protein Data Bank (PDB) [<xref ref-type="bibr" rid="B29">29</xref>]. For variants of the same protein, we kept the structure solved at the highest resolution, and we removed multiple chains, keeping only one chain for each structure. The sequences of the remaining structures have been submitted to a redundancy check, removing chains with a sequence identity above some threshold. We considered two sequences as being homologues, if they demonstrated an identity above 30% in a pairwise alignment, in a length longer than 80 residues. For the pairwise local alignment we used BlastP [<xref ref-type="bibr" rid="B30">30</xref>] with default parameters, and the homologous sequences were removed implementing Algorithm 2 from Hobom et al [<xref ref-type="bibr" rid="B31">31</xref>]. The remaining 14 outer membrane proteins constitute our training set (Table <xref ref-type="table" rid="T1">1</xref>). The structures of TolC [<xref ref-type="bibr" rid="B32">32</xref>] and alpha-hemolysin [<xref ref-type="bibr" rid="B33">33</xref>], were not included in the training set for the following reasons: TolC is a mixed beta-barrel and alpha-helical protein which spans both the outer membrane and the periplasmic space of gram negative bacteria. Three TolC protomers assemble to form a continuous, solvent accessible conduit, a "channel-tunnel" over 140 &#x000c5; long. Each monomer of the trimer contributes 4 &#x003b2;-strands to the 12 strand &#x003b2;-barrel. Alpha-hemolysin secreted from <italic>S. aureus </italic>is active as a transmembrane heptamer, where the transmembrane domain is a 14-strand antiparallel &#x003b2;-barrel, in which two strands are contributed by each monomer. Both structures are not included in the fold "transmembrane beta-barrel" of the SCOP database. In summary, the set includes proteins being monomeric, dimeric or trimeric, with a number of TM &#x003b2;-strands ranging from 8 to 22, and is representative of the known functions of outer membrane proteins. As an independent test set of outer membrane proteins, we chose the dataset used in the validation of the PSORT-B algorithm [<xref ref-type="bibr" rid="B34">34</xref>], consisting of 377 proteins. This set was also checked for redundancy with the same criteria mentioned above, and the closest homologues were removed along with the proteins showing similarity to at least one protein from the training set, leaving us with 119 outer membrane proteins. To test the discriminative power of the model we used an additional dataset of globular proteins, with 3-dimensional structures deposited in PDB [<xref ref-type="bibr" rid="B29">29</xref>]. This set was compiled using the PAPIA [<xref ref-type="bibr" rid="B35">35</xref>] server, with the sequence similarity threshold set to 25%, and excluding membrane proteins, proteins with a length lower than 80 residues, and proteins with at least one unidentifiable residue in the sequence; finally we came up with 1100 sequences of such globular proteins.</p><p>It is noteworthy that even in structures known at atomic resolution, the exact boundaries of the TM strands are not obvious, and in some situations the PDB annotations for the strands are clearly extending far beyond the membrane. Since our primary objective was to predict the TM segments of the strands rather than the entire &#x003b2;-strands, the model was trained to identify these particular segments. It is well known that discriminative training algorithms are very sensitive to data mislabeling, thus the training could not have been performed with labels based on the PDB annotation for the TM-strands. In [<xref ref-type="bibr" rid="B18">18</xref>], an automated method for re-labeling the data was proposed, but in our case since the training data set was limited we chose a manual approach. For the training purposes, the labels for the TM segments were set manually, by identifying the aromatic belts of the barrel [<xref ref-type="bibr" rid="B2">2</xref>] after inspection of the 3-dimensional structures of the proteins of the training set using molecular graphics. All residues contained between the two aromatic belts of each &#x003b2;-strand of the &#x003b2;-barrel were set to define a TM segment, including the residues of the belts. In cases where the aromatic belt residues of a &#x003b2;-strand are not clearly defined, neighbouring &#x003b2;-strands of the &#x003b2;-barrel helped in the "belt" identification. The resulting dataset used for training is shown in <xref ref-type="supplementary-material" rid="S1">additional file 1</xref> (second column, TM).</p></sec><sec><title>Measures of accuracy</title><p>To assess the accuracy of the predictions, we used several measures. For the transmembrane strand predictions we calculated the number of correctly predicted strands (True Positives, TP), the number of missed strands (False Negatives, FN) and the number of the over-predicted strands (False Positives, FP). We also calculated the total number of correctly predicted topologies, i.e. when both the strand localization and the loops topology have been predicted correctly. As measures of the accuracy per residue, we report here both the total fraction of the correctly predicted residues (Q<sub>&#x003b2;</sub>) in a two-state model (transmembrane versus non-transmembrane), and the well known Matthews Correlation Coefficient (C<sub>&#x003b2;</sub>) [<xref ref-type="bibr" rid="B36">36</xref>]. The comparisons have been performed against our manual annotation of the TM segments to show the efficiency of the model as well as against the PDB annotation for the transmembrane strands, for demonstration purposes. We feel that this had to be done in order to allow a fair comparison with other published methods [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B8">8</xref>], since in each one of the published methods the comparisons were performed against the PDB annotation.</p></sec></sec><sec><title>Authors' contributions</title><p>PB carried out the design of the algorithms and the model, the data collection, the training and testing procedures, and also participated in the implementation. TL implemented most of the algorithms, and also participated in the model design. IS created the web interface. SH coordinated the whole project, suggesting the general directions and innovating features of the method. All authors have read and accepted the final manuscript.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="S1"><caption><title>Additional File 1</title><p>5.63 kb, a list of the observed &#x003b2;-strands taken from PDB [<xref ref-type="bibr" rid="B29">29</xref>], the manually annotated transmembrane segments (TM), and the predicted transmembrane segments of the 14 proteins of the training set.</p></caption><media xlink:href="1471-2105-5-29-S1.txt" mimetype="text" mime-subtype="plain"><caption><p>Click here for file</p></caption></media></supplementary-material></sec>
Mig12, a novel Opitz syndrome gene product partner, is expressed in the embryonic ventral midline and co-operates with Mid1 to bundle and stabilize microtubules
<sec><title>Background</title><p>Opitz G/BBB syndrome is a genetic disorder characterized by developmental midline abnormalities, such as hypertelorism, cleft palate, and hypospadias. The gene responsible for the X-linked form of this disease, <italic>MID1</italic>, encodes a TRIM/RBCC protein that is anchored to the microtubules. The association of Mid1 with the cytoskeleton is regulated by dynamic phosphorylation, through the interaction with the &#x003b1;4 subunit of phosphatase 2A (PP2A). Mid1 acts as an E3 ubiquitin ligase, regulating PP2A degradation on microtubules.</p></sec><sec><title>Results</title><p>In spite of these findings, the biological role exerted by the Opitz syndrome gene product is still unclear and the presence of other potential interacting moieties in the Mid1 structure prompted us to search for additional cellular partners. Through a yeast two-hybrid screening approach, we identified a novel gene, <italic>MIG12</italic>, whose protein product interacts with Mid1. We confirmed by immunoprecipitation that this interaction occurs <italic>in vivo </italic>and that it is mediated by the Mid1 coiled-coil domain. We found that <italic>Mig12 </italic>is mainly expressed in the neuroepithelial midline, urogenital apparatus, and digits during embryonic development. Transiently expressed Mig12 is found diffusely in both nucleus and cytoplasm, although it is enriched in the microtubule-organizing center region. Consistently with this, endogenous Mig12 protein is partially detected in the polymerized tubulin fraction after microtubule stabilization. When co-transfected with Mid1, Mig12 is massively recruited to thick filamentous structures composed of tubulin. These microtubule bundles are resistant to high doses of depolymerizing agents and are composed of acetylated tubulin, thus representing stabilized microtubule arrays.</p></sec><sec><title>Conclusions</title><p>Our findings suggest that Mig12 co-operates with Mid1 to stabilize microtubules. Mid1-Mig12 complexes might be implicated in cellular processes that require microtubule stabilization, such as cell division and migration. Impairment in Mig12/Mid1-mediated microtubule dynamic regulation, during the development of embryonic midline, may cause the pathological signs observed in Opitz syndrome patients.</p></sec>
<contrib id="A1" contrib-type="author"><name><surname>Berti</surname><given-names>Caterina</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Fontanella</surname><given-names>Bianca</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Ferrentino</surname><given-names>Rosa</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" corresp="yes" contrib-type="author"><name><surname>Meroni</surname><given-names>Germana</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib>
BMC Cell Biology
<sec><title>Background</title><p>Opitz syndrome (OS) is a congenital disorder affecting primarily midline structures (MIM 145410 and 300000). OS patients usually present with facial anomalies, including hypertelorism and cleft lip and palate. OS also includes laryngo-tracheo-esophageal (LTE), cardiac, and genitourinary abnormalities. These symptoms show high variability even within the same family [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B5">5</xref>]. OS is a heterogeneous disease with an X-linked (Xp22.3) and an autosomal locus (22q11.2) [<xref ref-type="bibr" rid="B6">6</xref>]. The gene responsible for the X-linked form, <italic>MID1</italic>, has been identified [<xref ref-type="bibr" rid="B7">7</xref>]. In male OS patients, mutations have been found scattered throughout the entire length of the <italic>MID1 </italic>gene, suggesting a loss of function mechanism at the basis of this developmental phenotype. Females carrying a mutated <italic>MID1 </italic>allele usually show only hypertelorism, likely as the result of differential X-inactivation [<xref ref-type="bibr" rid="B7">7</xref>-<xref ref-type="bibr" rid="B11">11</xref>]. Interestingly, during embryonic development the murine and avian orthologs of the <italic>MID1 </italic>gene show an expression pattern that, although not highly restricted, correlates with the tissues affected in OS. Within these tissues, the mouse and chick <italic>Mid1 </italic>transcripts are preferentially enriched in areas of active proliferation [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>]. Recently, the chick <italic>Mid1 </italic>gene has been shown to be involved in the Sonic Hedgehog pathway during the establishment of the molecular left/right asymmetry in early embryonic avian development [<xref ref-type="bibr" rid="B14">14</xref>].</p><p><italic>MID1 </italic>encodes a protein belonging to the tripartite motif family and is composed of a RING domain, two B-Box domains, a coiled-coil region, together forming the tripartite motif, followed by a fibronectin type III (FNIII) and an RFP-like domain [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. The tripartite motif family, also known as TRIM or RBCC, comprises multi-domain-proteins involved in the definition of cellular compartments [<xref ref-type="bibr" rid="B17">17</xref>]. Mid1 self-interacts and forms high molecular weight complexes that are anchored to the microtubules throughout the cell cycle [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. The most frequent <italic>MID1 </italic>alterations found in OS patients affect the C-terminal portion of the protein. Mutants that reproduce these mutations show an altered microtubule association [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. The association of the wild-type protein with microtubules is dynamic and is regulated by its phosphorylation status: dephosphorylation of Mid1, upon interaction with the &#x003b1;4 regulatory subunit of phosphatase 2A (PP2A) [<xref ref-type="bibr" rid="B20">20</xref>], displaces Mid1 from microtubules [<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B22">22</xref>]. It has also been reported that Mid1 functions as an E3 ubiquitin ligase, regulating the microtubular PP2A catalytic subunit degradation upon interaction with &#x003b1;4. PP2A degradation, in turn, controls the phosphorylation status of yet to be identified microtubule-associated-proteins (MAPs) [<xref ref-type="bibr" rid="B23">23</xref>].</p><p>We have identified a novel Mid1 interacting protein through yeast two-hybrid screening. This novel protein is expressed in the midline during development and co-operates with Mid1 to stabilize the microtubules.</p></sec><sec><title>Results</title><sec><title>Identification of Mig12 as a novel Mid1 partner</title><p>To date, insights on the function of Mid1 in the cell have emerged from its interaction with the &#x003b1;4 subunit of phosphatase 2A (PP2A), however, the role of Mid1 in the pathogenesis of OS is still undetermined [<xref ref-type="bibr" rid="B21">21</xref>-<xref ref-type="bibr" rid="B24">24</xref>]. To get clues on possible biological function of Mid1, we searched for additional partners by screening a fibroblast two-hybrid library. MidM, a construct encompassing the C-terminal half of <italic>MID1</italic>, was used as a bait. This region, which comprises the coiled-coil, the FNIII repeats and the RFP-like domain of <italic>MID1</italic>, appears to be involved in the anchorage to microtubules [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. We obtained 6 positive clones, three of which were of different lengths, belonging to a unique transcript. The largest fragment had an ORF of 514 bp, the shortest of 432 bp. We used BLAST <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/BLAST"/> against the nr and EST databases and we found perfectly matching clones covering an ORF of 546 bp. We derived the complete sequence from the deposited transcripts and amplified the entire cDNA. We performed an interaction-mating assay to confirm the binding. Both the full-length and the largest original clone obtained from the library specifically interact with the entire Mid1 protein (MidA) (Fig. <xref ref-type="fig" rid="F1">1A</xref>). We also found positive interaction with portions of the Mid1 protein: MidD (coiled-coil), MidH (RING-B-boxes-Coiled-coil) and with MidM, the construct used to screen the library. No interaction was observed with <italic>MID1 </italic>constructs that lack the coiled-coil region (MidF and MidC, Fig. <xref ref-type="fig" rid="F1">1A</xref>). The identified clone does not interact with other members of the TRIM family (TRIM19/PML, TRIM25/RFP, TRIM29/ATDC) that share structural homology with Mid1 [<xref ref-type="bibr" rid="B17">17</xref>] (data not shown).</p><fig position="float" id="F1"><label>Figure 1</label><caption><p>Identification of a novel Mid1 partner. (<bold>A</bold>) Interaction-mating assay that confirms Mid1-Mig12 interaction in yeast. B42 fl, Mig12 full-length fused to the B42 activation domain; B42 or, the largest original Mig12 clone fused to the B42 activation domain; LexA Mid, constructs encompassing different <italic>MID1 </italic>domains fused to the LexA DNA binding domain: A, full-length; C, BB; D, CC; F, RFP-like; H, R-BB-CC; M, CC-FNIII-RFP-like. Both the full-length and the original Mig12 clones specifically interact with the entire Mid1 protein and with some of its truncated mutants, MidD, MidH and MidM, as shown by yeast turning blue on X-gal plates and growing on plates lacking leucine (Leu), only when galactose (Gal), and not glucose (Glu), is used as carbon source. Abbreviations: BB, B-box1 and B-box2 domains; CC, coiled-coil domain; FNIII, fibronectin type III repeat; R, RING domain. (<bold>B</bold>) Amino acid sequence of human (h) and mouse (m) MIG12 and comparison with the zebrafish G12 and the human SPOT14 proteins. Amino acids that are identical at least in the human and murine Mig12 are in bold. Conserved amino acids are indicated in gray. The human and mouse MIG12 share 90% of similarity and 88% of identity. The hMIG12 and the zebrafish protein share 56% of similarity and 46% of identity, whereas the homology with the human SPOT-14 protein is 49% and 31%, respectively. There is a gap of 25 aa that are not present in the zebrafish and SPOT14 proteins. (<bold>C</bold>) Co-immunoprecipitation experiments showing Mid1-Mig12 interaction. Western blot (WB) analysis using anti-Mid1 and anti-HA antibodies after immunoprecipitation of HEK293 cells transiently transfected with different combination of MycGFP-tagged Mid1 (MGFP-MID1) and an HA-tagged Mig12 (HA-MIG12); + and - indicate the constructs transfected in each lane. The antibodies used for the immunoprecipitations (IP) are indicated. Mid1 indicates the band corresponding to the endogenous protein. Ig, immunoglobulins. In some experiments, we detected a trace amount of MGFP-Mid1 immunoreactivity in cells transformed with only MGFP-Mid1 and immunoprecipitated with the anti-HA antibody. This signal was always much less than that seen when both tagged constructs were transfected together. (<bold>D</bold>) The same as in (C) using the MGFP-MidM, MGFP-MidH and MGFP-MidD mutant fusions, instead of the full-length protein, in the co-transfections and an anti-Myc antibody for Western blot analysis.</p></caption><graphic xlink:href="1471-2121-5-9-1"/></fig><p>The full-length sequence matches with various anonymous human (hypothetical protein STRAIT11499, NM_021242; FLJ10386, AK001248) and mouse (AL671335, AK090003, and NM_026524 RIKEN) complete cDNA sequences and several ESTs in the databases. The human gene is located in Xp11.4 and is composed of two exons, one of which encompasses the entire coding region. The mouse gene is located in the A1.1 region of the X chromosome. The human (GenBank accession no. BK001260) and mouse (GenBank accession no. AY263385) cDNAs encode a 182- and a 181-residue-protein, respectively, displaying no known domains with the exception of a low score coiled-coil region at the C-terminus of the protein. This Mid1 interactor records the highest homology with the zebrafish 'Gastrulation specific protein G12' (NP_571410), a protein with unknown function [<xref ref-type="bibr" rid="B25">25</xref>], and with the mammalian SPOT-14 (NM_003251), a protein involved in the metabolism of fatty acids [<xref ref-type="bibr" rid="B26">26</xref>,<xref ref-type="bibr" rid="B27">27</xref>]. The novel transcript was dubbed <italic>MIG12 </italic>for <underline>M</underline>id1 <underline>i</underline>nteracting <underline>G12</underline>-like protein, after the similarity with the <italic>Danio rerio </italic>protein. Figure <xref ref-type="fig" rid="F1">1B</xref> shows the alignment of the human and mouse Mig12, the zebrafish G12, and the human SPOT14 proteins.</p><p>To confirm that the two proteins also interact <italic>in vivo</italic>, we transiently transfected a MycGFP-tagged version of <italic>MID1 </italic>(MGFP-Mid1) and an HA-tagged version of <italic>MIG12 </italic>(HA-Mig12) in HEK293 cells and immunoprecipitated using either anti-Mid1 or anti-HA antibodies. Immunoprecipitation of Mid1 in the co-transfected sample pulls down the HA-Mig12 protein (right panel) and, <italic>vice versa</italic>, the immunoprecipitation of Mig12 using the anti-HA antibody pulls down the MGFP-Mid1 protein (left panel) (Fig. <xref ref-type="fig" rid="F1">1C</xref>). An unrelated polyclonal antibody and a different anti-tag monoclonal antibody (anti-FLAG) did not pull down the two proteins (data not shown), confirming the specificity of Mid1-Mig12 interaction. Moreover, Mig12 transfected alone is also pulled down by immunoprecipitation of the endogenous Mid1 protein (Fig. <xref ref-type="fig" rid="F1">1C</xref>). The interaction mating experiments suggest that the coiled-coil region of Mid1 is necessary and sufficient for the binding to Mig12. MGFP tagged versions of MidM, MidH, and MidD were co-transfected with HA-MIG12 in HEK293 cells and immunoprecipitated with either anti-Myc or anti-HA antibodies. The three constructs, all encompassing the coiled-coil region, are able to bind Mig12 further confirming that, also <italic>in vivo</italic>, this region is sufficient for Mid1-Mig12 interaction (Fig. <xref ref-type="fig" rid="F1">1D</xref>).</p></sec><sec><title>Mig12 is mainly expressed in the developing CNS midline</title><p>Since Mid1 is implicated in a developmental disorder, to support a physiologically relevant interaction between Mig12 and Mid1 we analyzed the mRNA expression of <italic>Mig12 </italic>during embryonic development. The Mig12 clone originally obtained from the two-hybrid screening was used as a probe to perform mRNA <italic>in situ </italic>hybridization on mouse embryos at several embryonic stages. A ubiquitous expression pattern was found both on section and in whole mount experiments from embryonic day 9.5 (E9.5) up to E11.5. At E11.5, we detected a diffuse staining in the central nervous system (CNS) and a more restricted signal in the developing limbs by whole-mount <italic>in situ </italic>hybridization (Fig. <xref ref-type="fig" rid="F2">2A</xref>, a). An even more restricted expression pattern is observed at E14.5 when high transcript levels are detected in specific compartments (Fig. <xref ref-type="fig" rid="F2">2A</xref>, b). The strongest expression is observed in the developing central nervous system and is particularly evident in the coronal sections through the hindbrain region (Fig. <xref ref-type="fig" rid="F2">2B</xref>, a&#x02013;c). The signal is observed in the neuroepithelium of the cerebellar primordia (Fig. <xref ref-type="fig" rid="F2">2B</xref>, a,b), of the pons (Fig. <xref ref-type="fig" rid="F2">2B</xref>, a, b, e), and of the medulla oblongata (Fig. <xref ref-type="fig" rid="F2">2B</xref>, c). The ventricular hindbrain signal is mainly confined to the ventral midline (Fig. <xref ref-type="fig" rid="F2">2B</xref>, a, b, c). This medial expression is maintained throughout the central canal of the spinal cord extending through the floor and roof plates (Fig. <xref ref-type="fig" rid="F2">2B</xref>, d). In the telencephalon, <italic>Mig12 </italic>signal is present in the ventricular zone of the telencephalic vesicles (Fig. <xref ref-type="fig" rid="F2">2B</xref>, f). Within the nervous system, <italic>Mig12 </italic>transcript is also detected in the dorsal roots and in the trigeminal ganglia (Fig. <xref ref-type="fig" rid="F2">2A</xref>, b; <xref ref-type="fig" rid="F2">2B</xref>, d). At this stage, expression of <italic>Mig12 </italic>is also observed in several additional organs. The transcript is observed in the interdigital web in both the developing hind- and forelimbs at E11.5 (Fig. <xref ref-type="fig" rid="F2">2A</xref>, a). At E14.5, as the development of the limbs proceeds, <italic>Mig12 </italic>transcript is detected in the perichondrium of the digits (Fig. <xref ref-type="fig" rid="F2">2B</xref>, g). The other organs expressing <italic>Mig12 </italic>include the left and right thyroid lobes and the parathyroid glands (Fig. <xref ref-type="fig" rid="F2">2B</xref>, h); the phallic part of the urogenital sinus (Fig. <xref ref-type="fig" rid="F2">2B</xref>, i); the anal canal (rectum) and the epithelium lining the lumen of the bladder (data not shown). Interestingly, many of the sites that show high <italic>Mig12 </italic>levels also express the <italic>Mid1 </italic>transcript [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>] and are affected in OS patients [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B11">11</xref>].</p><fig position="float" id="F2"><label>Figure 2</label><caption><p>Mig12 expression analysis during embryonic development. (<bold>A</bold>) Whole mount <italic>in situ </italic>hybridization on E11.5 mouse embryo showing expression in the central nervous system and in the developing limbs (blue signal, a). Coronal and sagittal sections of E14.5 entire mouse embryos (white signal) (b). (<bold>B</bold>) Details of coronal (a, b, c, d, h) and sagittal (e, f, g, i) sections of E14.5 mouse embryos. Strong Mig12 expression (red signal) is observed in isthmal (a), pontine (a, b, e) and medulla oblongata (c) neuroepithelia, and it is maintained throughout the entire region of the spinal cord central canal (d). Expression is also observed in dorsal root ganglia (d). Mig12 transcript is detected in the telencephalon at the level of the ventricular zone (f). Signal is also present in other organs: in the perichondrium of the digits (g); in the thyroid (th) and parathyroid (pth) glands (h), and in the phallic part of the urogenital sinus (i). Abbreviations: CB, cerebellum; ccn, central canal neuroepithelium; drg, dorsal root ganglia; IS, isthmus; isn, isthmal neuroepithelium; M, medulla oblongata; mn, medulla oblongata neuroepithelium; P, pons; pc, perichondrium; pnn, pontine neuroepithelium; pth, parathyroid glands; SC, spinal cord; T, telencephalon; th, thyroid gland; us, urogenital sinus; vz, ventricular zone.</p></caption><graphic xlink:href="1471-2121-5-9-2"/></fig></sec><sec><title>Mid1 recruits Mig12 on the microtubules</title><p>Transient expression of either MGFP- or HA-tagged Mig12 reveals a diffuse distribution of the protein in Cos7 as well as in other cell lines (U2OS, HeLa, NIH3T3). To exclude a tag-driven mislocalization, we also transfected a non-tagged version of Mig12: the specific anti-Mig12 antibody reveals a distribution comparable to that of the tagged versions. Mig12 is present in both the nucleus and the cytoplasm and the relative abundance in the two compartments is variable (Fig. <xref ref-type="fig" rid="F3">3A</xref>).</p><fig position="float" id="F3"><label>Figure 3</label><caption><p>Immunofluorescence analyses reveal co-localization of Mid1 and Mig12 within the cell. (<bold>A</bold>) Immunofluorescence analysis after transient expression of MGFP-Mig12 (upper panel), HA-Mig12 (middle panel) and untagged Mig12 (lower panel) in Cos7 cells, revealing a diffuse distribution of the protein, in both the nucleus and the cytoplasm. (<bold>B</bold>) Co-expression of both Mid1 and Mig12 leads to co-localization of the two proteins in cytoplasmic bundles. Standard fluorescence microscopy shows formation of bundles only in Mid1 (left panels) and Mig12 (right panel) co-expressing cells. The arrow indicates a single transfected cell where Mid1 shows the classical distribution along normal interphase microtubules. (<bold>C</bold>) The co-localization is confirmed by confocal microscopy analysis in which HA-Mid protein is visible as a red signal and MGFP-Mig12 protein as a green signal; co-localization is indicated as a yellow signal in merged images. (<bold>D</bold>) Co-localization is also observed using the HA-Mig12 construct (middle panels) together with either a Mid1 OS truncated mutant (GFP-Mid1 1331insA) or a Mid1 mutant (GFP-MidD) retaining the coiled-coil domain, both localized in cytoplasmic bodies. No co-localization is observed when HA-TRIM19/PML protein is co-expressed with GFP-Mig12. The right panels represent the merged images.</p></caption><graphic xlink:href="1471-2121-5-9-3"/></fig><p>Mid1 is associated with microtubules during the entire cell cycle [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. An example of its distribution is shown in figure <xref ref-type="fig" rid="F3">3B</xref> (arrow, upper panel), where Mid1 co-localizes with the normal radial interphase microtubules. Interestingly, when co-expressed in the same cell, Mid1 and Mig12 form bundles within the cytoplasm (Fig. <xref ref-type="fig" rid="F3">3B</xref>). Mig12 usually also maintains a diffused distribution whose extent depends on its expression level. As shown in the lower panels, the observed bundles show variable thickness and shape that depend on the expression levels of the two proteins. Nevertheless, these bundles are only present when the two proteins are co-expressed. In our experimental conditions we do not observe the formation of bundles in cells transfected with only Mid1 (Fig. <xref ref-type="fig" rid="F3">3B</xref>, arrow). The co-localization of Mid1 and Mig12 within the bundles has been confirmed by confocal microscopy analysis (Fig. <xref ref-type="fig" rid="F3">3C</xref>).</p><p>We investigated the distribution of Mig12 in cells co-transfected with mutant Mid1 proteins that are not anchored to the microtubules. Mid1 C-terminal OS mutants localize to cytoplasmic bodies [<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. These mutant forms, that retain the coiled-coil region, are able to recruit Mig12 within these structures (Fig. <xref ref-type="fig" rid="F3">3D</xref>, upper panels). The same is observed using a construct that drives the expression of only the coiled-coil domain of Mid1 (Fig. <xref ref-type="fig" rid="F3">3D</xref>, middle panels). This recruitment is not observed when other TRIM proteins, that share the same domain composition of Mid1, are expressed with Mig12. This is demonstrated by co-transfections of Mig12 with TRIM19/PML (Fig. <xref ref-type="fig" rid="F3">3D</xref>, lower panels), TRIM5 or TRIM27 (data not shown). These results confirm that Mid1, through its coiled-coil domain, is able to specifically recruit Mig12 to different structures within the cell.</p><p>Since Mid1 is a microtubular protein, we asked whether the bundles observed in cells co-expressing Mig12 and Mid1 are structures of microtubular nature. Co-localization of tubulin with the bundles, in immunofluorescence experiments, demonstrates that these structures are microtubule arrays rearranged by overexpression of the two proteins and that are often present as continuous or fragmented perinuclear rings (Fig. <xref ref-type="fig" rid="F4">4A</xref>).</p><fig position="float" id="F4"><label>Figure 4</label><caption><p>Mid1 and Mig12 co-sediment with microtubules. (<bold>A</bold>) Immunofluorescence analysis in Cos7 cells co-transfected with HA-Mid1 (left panels) and MGFPMig12 (middle panel) proteins. Coincidence of the bundles with microtubules is revealed using monoclonal antibodies against &#x003b2;-tubulin (right panel). These images show the different thickness and distribution of the bundles. (<bold>B</bold>) Cos7 cells were transfected with either MGFPMid and HA-Mig12 (left panel) or HA-Mig12 alone (right panel). Lysates (L) from cells were supplemented with 40 &#x003bc;M taxol to stabilize polymerized microtubules. After sedimentation on sucrose cushion, supernatant (S) and pellet (P) fractions were assayed for the presence of Mid1, Mig12, and tubulin using appropriate antibodies. In the co-transfection (left panel) both Mid1 and Mig12 were detected in the pellet together with the polymerized microtubules. As expected Mig12 is also present in the soluble fraction where neither Mid1 nor the tubulin are found. Mig12 is found partially associated with the polymerized tubulin fraction also in the single HA-Mig12 transfected cells (right panel). (<bold>C</bold>) Western blot analysis using the anti-Mig12 antibody reveals a 24 KDa protein in two different cell lines lysates (1, Cos7; 2, HeLa cells). To confirm specificity, incubation with the primary antibody was also performed in the presence of either the fusion protein used to immunize rabbits (GST-Mig12) or an unrelated fusion protein (GST-ur). (<bold>D</bold>) Detection of endogenous Mig12 in the polymerized microtubule fraction (+ taxol) in HeLa cells and as control in the non-treated sample (-taxol); legend as in (A). (<bold>E</bold>) Single Mig12 transfected Cos7 cells show partial localization with microtubules, particularly in the MTOC region (upper panels) and at the mitotic spindle poles (lower panels).</p></caption><graphic xlink:href="1471-2121-5-9-4"/></fig><p>To confirm these data, we performed microtubule sedimentation after taxol treatment in cells co-transfected with both Mid1 and Mig12. After fractionation on a sucrose cushion, the supernatant and the pellet containing the polymerized tubulin were assayed by immunoblot for the presence of both proteins. Mig12 and Mid1 are recovered in the pellet, where tubulin is also found. Mig12, as expected, is also present in the supernatant. This result further indicates that the bundles observed in immunofluorescence experiments are of microtubular nature (Fig. <xref ref-type="fig" rid="F4">4B</xref>, left panel). A control protein that does not associate with the microtubules, spastin &#x00394; N [<xref ref-type="bibr" rid="B28">28</xref>], is not present in the microtubule fraction, confirming that the presence of Mig12 in the pellet is not due to contamination during the sedimentation process (data not shown). Moreover, the presence of Mig12 in the pellet, as well as that of tubulin, is lost when the cells are not treated with the microtubule stabilization agent, taxol (data not shown). Thus, when overexpressed, Mid1 and Mig12 have the ability to rearrange interphase radial microtubules into these structures.</p><p>Interestingly, singly transfected Mig12 also partially sediments with the microtubular pellet, as expected to a lesser extent than the double transfectant (Fig. <xref ref-type="fig" rid="F4">4B</xref>, right panel). Since the affinity purified anti-Mig12 antibody we produced allows the specific detection of the endogenous protein in immunoblot experiments in cell line lysates, as shown in figure <xref ref-type="fig" rid="F4">4C</xref>, we carried out sedimentation of polymerized microtubules in HeLa cells to test the presence of endogenous Mig12 in the microtubule pellet. These results indicate that the protein, likely by interacting with endogenous Mid1 protein, is at least partially associated with microtubules (Fig. <xref ref-type="fig" rid="F4">4D</xref>). A closer look at some single transfected cells reveals indeed a partial co-localization of Mig12 with the microtubules, also in the absence of exogenous Mid1 (Fig. <xref ref-type="fig" rid="F4">4E</xref>). Some filaments are observed over the diffuse staining and in many cells enrichment of Mig12 protein in the MTOC region is evident (Fig. <xref ref-type="fig" rid="F4">4E</xref>, upper panels) as well as partial co-localization with the mitotic spindle (Fig. <xref ref-type="fig" rid="F4">4E</xref>, lower panels).</p></sec><sec><title>Mid1 and Mig12 induce stable microtubule bundles</title><p>To better understand the nature of these microtubule arrays, we asked what happens to the Mid1-Mig12 bundles upon disruption of the microtubular architecture. Cells were co-transfected and exposed to nocodazole, a microtubule-depolymerizing agent, for 1 hour before fixation and then analyzed by immunofluorescence. The filaments observed after overexpression of the two proteins were more resistant to the drug compared to control microtubules (Fig. <xref ref-type="fig" rid="F5">5A</xref>). In contrast, cells overexpressing only Mid1 show complete disruption of the microtubular apparatus, which is consistent with the absence of bundles (Fig. <xref ref-type="fig" rid="F5">5A</xref>, arrow). Partial disruption of the Mid1-Mig12 bundles was observed only after longer exposure to nocodazole (4 h, data not shown).</p><fig position="float" id="F5"><label>Figure 5</label><caption><p>Mid1 and Mig12 together stabilize the microtubules. (<bold>A</bold>) Nocodazole treatment does not disrupt the Mid1/Mig12 generated bundles of tubulin, whereas it disrupts the microtubules in Mid1 single transfected cells (arrow). (<bold>B</bold>) The bundles represent stable microtubules as demonstrated by perfect coincidence with the anti-acetylated tubulin antibody signal (blue).</p></caption><graphic xlink:href="1471-2121-5-9-5"/></fig><p>Modification of tubulin subunits by acetylation marks older microtubules and therefore indicates those that are more stable [<xref ref-type="bibr" rid="B29">29</xref>]. Specific antibodies to acetylated tubulin decorate the Mid1-Mig12 induced nocodazole-resistant bundles, thus indicating stable microtubules (Fig. <xref ref-type="fig" rid="F5">5B</xref>). The ability to stabilize the microtubules is not a characteristic of cells overexpressing Mig12 alone: in fact, treatment with nocodazole does not reveal any residual microtubular structures in these cells (data not shown).</p><p>These data suggest that Mig12 co-operates with Mid1 to stabilize microtubules. The Mid1-Mig12 microtubule-stabilizing effect might be implicated in specific processes during the development of the midline systems that are affected in Opitz syndrome patients.</p></sec></sec><sec><title>Discussion</title><p>The role of the Opitz syndrome gene product, Mid1, in the pathogenesis of this human disorder is still unclear [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B24">24</xref>]. We now present data that support a role of Mid1 in the regulation of microtubule dynamics. We report the identification of a novel gene, <italic>MIG12</italic>, that encodes a Mid1 interacting protein. <italic>MIG12 </italic>shares high sequence homology with a zebrafish gene product, the 'gastrulation protein G12', which is expressed in a narrow window of time during <italic>D. rerio </italic>gastrulation [<xref ref-type="bibr" rid="B25">25</xref>]. A Mig12 paralog in mammals, SPOT14, is a nuclear protein that responds to the thyroid hormone and regulates lipid synthesis [<xref ref-type="bibr" rid="B26">26</xref>,<xref ref-type="bibr" rid="B27">27</xref>]. However, the mechanism of action for both G12 and SPOT14 is still unknown. Further, the absence of recognizable domains in its peptide sequence does not allow any <italic>a priori </italic>hypothesis on <italic>MIG12 </italic>function to be drawn.</p><p>The expression pattern of <italic>Mig12 </italic>during embryonic development is consistent with that of <italic>Mid1 </italic>[<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>]. Furthermore, this pattern overlaps with tissues whose development is defective in OS [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B9">9</xref>,<xref ref-type="bibr" rid="B11">11</xref>]. The strong expression in the midline of the developing central nervous system might be related to the neurological signs found in a high number of patients that manifest agenesis or hypoplasia of the corpus callosum and of the cerebellar vermis, and mental retardation. Moreover, expression of <italic>Mig12 </italic>in the rostral medial CNS could also be involved in the determination of proper craniofacial formation. It is well known that factors expressed in the CNS midline are implicated in resolving a single eye field into two lateral fields, an event that determines the head midline width and the face traits as reviewed in [<xref ref-type="bibr" rid="B30">30</xref>,<xref ref-type="bibr" rid="B31">31</xref>]. One of these, Sonic hedgehog (Shh), plays a crucial role in the ventral midline neural tube patterning and regulates the morphogenesis of a variety of midline and lateral organs. It is interesting to note the recent association of the <italic>Mid1 </italic>gene and the Shh pathway in the early midline and laterality specification in the chicken [<xref ref-type="bibr" rid="B14">14</xref>]. Interference with the correct Mig12-Mid1 pathway might be responsible for the craniofacial defects observed in OS. Expression in the embryonic urogenital and anal apparatus is also reminiscent of defects observed in OS, hypospadias and imperforate or ectopic anus. In addition, we can parallel the inter-digit <italic>Mig12 </italic>expression observed in the mouse embryos with OS manifestations, as we observed syndactyly in a <italic>MID1</italic>-mutated patient [<xref ref-type="bibr" rid="B11">11</xref>]. The low frequency of mutations in <italic>MID1 </italic>and the high variability of the phenotype in OS patients suggest the involvement of other genes in the OS phenotype. It is plausible that other proteins involved in the Mid1 pathway are implicated in the heterogeneity of OS (or in other syndromes showing clinical overlap with OS) and Mig12 might well be a candidate.</p><p>When Mig12 is over-expressed, it barely decorates microtubules with a signal almost imperceptible due to its diffused distribution in the cytoplasm. Accordingly, endogenous Mig12 is partially found associated with the polymerized tubulin fraction in cell lysates. Interestingly, when co-expressed with Mid1 it induces the formation of microtubule bundles. This effect is not observed when Mid1 is expressed alone. Mid1 specifically recruits Mig12 to the microtubules and the consequent induction of bundles could be explained by the propensity of both proteins, Mid1 [<xref ref-type="bibr" rid="B18">18</xref>] and Mig12 (CB, GM, unpublished results), to homo-interact. The formation of multimers might tether a high number of microtubule interacting moieties that, in turn, mediate and favor the association of parallel microtubule arrays. The shape and location of these microtubule bundles is variable within the cell: perinuclear rings, sub-cortical bundles and a roundish mass in the MTOC region. In some cases, we also observed fragmentation of these thick microtubular structures (CB, GM, unpublished results) that might suggest the involvement of a putative microtubule severing activity [<xref ref-type="bibr" rid="B32">32</xref>]. These microtubule bundles are resistant to depolymerizing agents, such as nocodazole, and are composed of acetylated tubulin and therefore represent stable microtubules. This bundling and stabilizing effect has been observed for other microtubule binding proteins, in particular microtubule-associated-proteins (MAPs) and other proteins involved in mitotic spindle organization, cytokinesis and the control of cell motility such as, PRC1, NuMA, CLASPs, and many others [<xref ref-type="bibr" rid="B33">33</xref>-<xref ref-type="bibr" rid="B36">36</xref>]. It is worth noting that recently two proteins sharing homology with the C-terminal half of Mid1, Mir1 and GLFND that have a coiled-coil-FNIII-RFP-like structure, have been shown to bundle and stabilize microtubules [<xref ref-type="bibr" rid="B37">37</xref>,<xref ref-type="bibr" rid="B38">38</xref>]. So far, we have no indications on the behavior of Mid1-Mig12 complexes during mitosis. Mid1 decorates the mitotic spindle [<xref ref-type="bibr" rid="B18">18</xref>] and Mig12, when transfected alone, appears to be both associated with the spindle poles and diffused within the cell. We have never observed mitotic cells overexpressing both proteins. Whether this is due to interference with the division process is still to be clarified.</p><p>The bundling effect observed in our over-expression system probably reflects a weaker and finely tuned-regulated process in physiological conditions. The shuttling of Mig12 between nucleus and cytoplasm might also be dynamically regulated and, in certain conditions, segregation in the nucleus might be necessary to prevent interference with the interphase microtubule network. Mid1 might recruit Mig12 to microtubules only when needed. It is possible that phosphorylation of Mid1 [<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B22">22</xref>] and/or putative post-translational modifications of Mig12 might regulate their physiological association and the subsequent stabilization of the microtubule network. The ultimate aim of the regulation of microtubule stability and dynamics involving the Mid1-Mig12 pathway is still to be elucidated and may be connected to cell cycle progression or cell migration, events known to require microtubule stabilization [<xref ref-type="bibr" rid="B39">39</xref>]. Alteration of either process can be seen as possible causes of pathological signs in OS. <italic>Mig12</italic>, as well as <italic>Mid1</italic>, appears to be preferentially expressed in highly proliferating embryonic fields (e.g., the ventricular zone of the developing brain). Nevertheless, these are also cells that, after mitosis has been completed, are committed to migrate. The zebrafish gastrulation protein G12 is expressed in a restricted lineage characterized by extensive cell migration [<xref ref-type="bibr" rid="B25">25</xref>]; it is tempting to speculate that this process could be the one implicated in the pathogenesis of the Opitz syndrome.</p></sec><sec><title>Conclusions</title><p>We have reported the identification of a novel Opitz syndrome gene product interacting protein, Mig12, that co-operates with Mid1 to stabilize microtubules. These data are consistent with the role of Mid1 in microtubule dynamics. Mid1, in fact, controls MAP phosphorylation through the regulation of PP2A microtubular levels [<xref ref-type="bibr" rid="B23">23</xref>] and Mig12 may participate in this pathway. During embryonic development of midline structures, impairment in Mid1-Mig12-mediated microtubule dynamics regulation might be detrimental and lead to Opitz syndrome.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Plasmid constructs</title><p>The <italic>MID1 </italic>expression vectors MycGFP-MID1 and HA-MID1 have already been reported [<xref ref-type="bibr" rid="B18">18</xref>]. The <italic>MID1 </italic>deletion mutants, MidC, MidD, MidF, MidH, and MidM have been excised from HA-pCDNA3 vectors [<xref ref-type="bibr" rid="B18">18</xref>] and cloned EcoRI/XhoI in the two-hybrid vectors pJG4-5 and pEG202 [<xref ref-type="bibr" rid="B40">40</xref>]. Full-length <italic>MIG12 </italic>cDNA was generated by PCR amplification, using specific primers designed on ESTs sequences, from NIH3T3 total RNA as template. The PCR product was then cloned into EcoRI and XhoI sites in the eukaryotic expression vectors pcDNA3, pcDNA3-MGFP and pcDNA3-HA. Both Myc-GFP and HA tags are positioned at N-terminus region of <italic>MIG12 </italic>coding region. Full-length <italic>MIG12 </italic>was also cloned in the pJG4-5 two-hybrid vector fused to the B42 activation domain [<xref ref-type="bibr" rid="B40">40</xref>].</p></sec><sec><title>Yeast two-hybrid screening</title><p>The two-hybrid screening was performed using MIDM (CC-FNIII-RFP-like) cloned in pEG202 vector that contains the LexA DNA-binding domain. The bait was transformed into the yeast strain EGY48 that was subsequently transformed with an NIH3T3 cDNA library cloned into pJG4-5, containing the B42 activation domain. Transformants (5 &#x000d7; 10<sup>6 </sup>independent clones) were seeded on plates containing either X-gal or lacking Leucine to select positive clones that have activated both LexA driven reporter genes (lacZ and LEU2). Interaction mating assay to confirm the positivity was performed using the same system and two different yeast mating types (EGY48 MAT &#x003b1; and EGY42 MAT a) as described [<xref ref-type="bibr" rid="B40">40</xref>].</p></sec><sec><title>Cell culture and transfection</title><p>Monkey Kidney Cos-7 cells and HEK 293T cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37&#x000b0;C in a 5% CO<sub>2 </sub>atmosphere. All transfections were carried out by calcium phosphate precipitation [<xref ref-type="bibr" rid="B41">41</xref>]. In a typical transfection experiment 20 &#x003bc;g of expression vector were used per 15-cm dish. For immunofluorescence experiments, using chamber-slides (8 wells, Nunc), 0.5 &#x003bc;g DNA/well were transfected.</p></sec><sec><title>Immunoprecipitation, Immunoblot, and Antibodies</title><p>In co-immunoprecipitation experiments 4.5 &#x000d7; 10<sup>6 </sup>HEK 293T cells per 15-cm dish were seeded. 60 h after transfection cells were collected, washed and extracted with RIPA buffer (150 mM NaCl, 1% Igepal, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl pH 8) supplemented with protease inhibitors (Roche). Extracts were sonicated and centrifuged at 10000 <italic>g </italic>for 10 min at 4&#x000b0;C to remove cell debris. The supernatants were immunoprecipitated with either 6 &#x003bc;g of anti-HA antibody, 500 &#x003bc;l anti-Myc (9E10) hybridoma supernatant or 8 &#x003bc;g anti-Mid1 polyclonal antibody (H35) [<xref ref-type="bibr" rid="B18">18</xref>], for 3 h at 4&#x000b0;C and the immuno-complexes collected with protein A-Sepharose beads for 30 min. The beads were washed six times with RIPA buffer and proteins eluted from the beads by boiling in SDS loading buffer. Proteins were separated on either 10% or 12% SDS PAGE and blotted onto PVDF membranes (Amersham). The membranes were rinsed in methanol and blocked in TTBS (20 mM Tris-HCl pH 7, 50 mM NaCl and 0.1% Tween-20), 5% dry milk. Incubation with the primary antibodies was performed using anti-c-Myc monoclonal antibody (1:5 dilution), anti-HA monoclonal antibody (Roche) (1:500 dilution) and anti-Mid1 polyclonal antibody (1:250 dilution) in TTBS, 5% dry milk. Antibody binding was detected with a secondary anti-mouse or anti-rabbit IgG coupled with horseradish peroxidase, followed by visualization with the Enhanced Chemiluminescence Kit (Amersham). A specific anti-Mig12 antiserum has been raised against a full-length Mig12 protein fused to GST and produced in bacteria. Affinity purification of the antibody was performed with the GST-Mig12 covalently attached to a CNBr-activated sepharose column using standard procedures. To perform competition experiments, 20 &#x003bc;g of the same protein were used to compete the binding in immunoblot analysis. As non-specific competitor, the same amount of an unrelated GST fusion protein (Mid1 RING domain) was used.</p></sec><sec><title>Immunofluorescence</title><p>Cos7 cells were grown on chamber-slides (8 wells, Nunc) in DMEM, 10% FBS, and transfected as described. After 36 h, cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature, permeabilized with 0.2% Triton X-100/PBS for 30 min, blocked with normal serum for 1 h and incubated for 3 h with the primary antibodies and 1 h with the appropriate secondary antibodies. The following primary antibodies were used: protein A-purified polyclonal anti-Mid1 (1:200 dilution), monoclonal anti-&#x003b2;-tubulin (1:250 dilution) (Molecular Probes), monoclonal anti-HA (CA25) antibody (1:250 dilution) (Roche), monoclonal anti-acetylated tubulin (1:200 dilution) (Sigma). The following secondary antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibodies alone or both tetramethylrhodamine isothiocyanate (TRITC) conjugated anti-rabbit and FITC conjugated anti-mouse-antibodies (1:100 dilution) (Dako). For confocal microscopy, Cy3-conjugated anti-mouse antibody was used (1:200 dilution) (Amersham). When indicated, nocodazole in DMSO was added at the final concentration of 40 &#x003bc;M for 1 h at 37&#x000b0;C before fixation.</p></sec><sec><title>Microtubule binding assay</title><p>Cells were harvested either 48 hours post-transfection (Cos7 cells) or when at 80% confluence (non-transfected HeLa cells) and lysed in PEM-DNNA buffer (80 mM PIPES pH 6.8, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, 150 mM NaCl, 1% Igepal) supplemented with protease inhibitors, at 4&#x000b0;C for 1 hr. The lysate was centrifuged at 610 <italic>g </italic>for 10 min at 4&#x000b0;C. Cytosol was then purified by successive centrifugations at 10,000 <italic>g </italic>for 10 min, at 21,000 <italic>g </italic>for 20 min and at 100,000 <italic>g </italic>for 1 hr at 4&#x000b0;C. Each supernatant was then supplemented with 2 mM GTP (Roche) and 40 &#x003bc;M taxol (Molecular Probes) and incubated at 37&#x000b0;C for 30 min. Corresponding samples without taxol were also prepared. Each sample was layered over a 15% sucrose cushion and centrifuged at 30,000 <italic>g </italic>for 30 min at 30&#x000b0;C to sediment polymerized microtubules. The resulting supernatants were saved and the pellets were suspended in an equal volume of sample buffer for electrophoresis and immunoblot analysis.</p></sec><sec><title>RNA in situ hybridization</title><p>One of the original clones obtained from the screening (540 bp fragment whose 5' corresponds to nt 113 of the MIG12 coding region) was linearized with the appropriate restriction enzymes to transcribe either sense or antisense <sup>35</sup>S-labeled riboprobe. Mouse embryo tissue sections were prepared and RNA <italic>in situ </italic>hybridization experiments performed as previously described [<xref ref-type="bibr" rid="B42">42</xref>]. Autoradiographs were exposed for 2 days. Slides were then dipped in Kodak NTB2 emulsion and exposed for 14&#x02013;21 days. In the micrographs red represents the hybridization signal and blue shows the nuclei stained with Hoechst 33258 dye. Whole-mount <italic>in situ </italic>hybridization was performed using the same probe and following the protocol described in [<xref ref-type="bibr" rid="B43">43</xref>].</p></sec></sec><sec><title>Authors' contributions</title><p>CB carried out the two-hybrid screening, the RNA <italic>in situ </italic>hybridization analysis, the immunoprecipitation and immunofluorescence studies. BF produced the anti-Mig12 specific antibody and performed the microtubule sedimentation experiments. RF provided assistance in the cloning and preparation of the vectors. GM coordinated the study and wrote the paper. All authors read and approved the final manuscript.</p></sec>
Evolutionary and functional relationships within the DJ1 superfamily
<sec><title>Background</title><p>Inferences about protein function are often made based on sequence homology to other gene products of known activities. This approach is valuable for small families of conserved proteins but can be difficult to apply to large superfamilies of proteins with diverse function. In this study we looked at sequence homology between members of the DJ-1/ThiJ/PfpI superfamily, which includes a human protein of unclear function, DJ-1, associated with inherited Parkinson's disease.</p></sec><sec><title>Results</title><p>DJ-1 orthologs in a variety of eukaryotic species cluster together in a single group. The most closely related group is the bacterial ThiJ genes. These are kinases involved in the biosynthesis of thiamine, a function that has been dispensed with evolutionarily in most eukaryotes where thiamine is an essential nutrient. The similarity with other characterized members of the superfamily, including proteases, is more remote. This is congruent with the recently solved crystal structures that fail to demonstrate the presence of a catalytic triad required for protease activity.</p></sec><sec><title>Conclusion</title><p>DJ-1 may have evolved from the bacterial gene encoding ThiJ kinase. However, as this function has been dispensed with in eukaryotes it appears that the gene has been co-opted for another function.</p></sec>
<contrib id="A1" contrib-type="author"><name><surname>Bandyopadhyay</surname><given-names>Sourav</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" corresp="yes" contrib-type="author"><name><surname>Cookson</surname><given-names>Mark R</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib>
BMC Evolutionary Biology
<sec><title>Background</title><p>Mutations in DJ-1 have been described recently that are associated with recessively inherited Parkinson's disease (PD). Evidence to date suggests that the mutations cause disease by a loss of function mechanism. The reported mutations either delete several exons and result in an effective gene knockout [<xref ref-type="bibr" rid="B1">1</xref>] or are point mutations that destabilize the protein [<xref ref-type="bibr" rid="B2">2</xref>]. Therefore, the normal cellular function of DJ-1 is a critical piece of information in understanding how these mutations cause PD. DJ-1 has a number of reported functions, including cellular transformation [<xref ref-type="bibr" rid="B3">3</xref>], transcriptional effects [<xref ref-type="bibr" rid="B4">4</xref>], control of mRNA stability [<xref ref-type="bibr" rid="B5">5</xref>] and response to oxidative stress [<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>] and it is unclear quite how all of these relate to the pathways involved in PD [<xref ref-type="bibr" rid="B8">8</xref>].</p><p>One way to understand protein function is to find other proteins of known function with sequence or structural homology. This approach has helped in understanding of other PD proteins; parkin was found to be an E3 protein-ubiquitin ligase based on homology to other proteins with similar domain structures [<xref ref-type="bibr" rid="B9">9</xref>]. DJ1 shows sequence homology to a number of proteins that contain a ThiJ domain, including protein chaperones [<xref ref-type="bibr" rid="B10">10</xref>], catalases [<xref ref-type="bibr" rid="B11">11</xref>] proteases [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B13">13</xref>] and the ThiJ kinases [<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. Previous analyses have suggested that the ThiJ domain may be a member of the large glutamine amidotransferase (GAT) superfamily [<xref ref-type="bibr" rid="B11">11</xref>]. Crystal structures of DJ-1 [<xref ref-type="bibr" rid="B16">16</xref>-<xref ref-type="bibr" rid="B20">20</xref>] and other members of this DJ-1/ThiJ/PfpI superfamily including the protease PH1704 [<xref ref-type="bibr" rid="B13">13</xref>] have been reported. The proteins have an overall &#x003b1;/&#x003b2; sandwich structure, arranged similarly to the Rossman fold, which is also present in members of the GAT superfamily [<xref ref-type="bibr" rid="B11">11</xref>]. The structure is similar to another protein of much lower sequence homology, the <italic>E coli </italic>chaperone Hsp31 [<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B20">20</xref>].</p><p>The multitude of functional groupings within the DJ-1/ThiJ/PfpI superfamily limits our ability to make predictions about the cellular role of the human ortholog. A putative catalytic cysteine, cys106, is present, which has led to the suggestion that DJ-1 may be a protease [<xref ref-type="bibr" rid="B17">17</xref>]. However, structural data generally argues against DJ-1 having protease activity as the invariant catalytic triad seen in other cysteine proteases is present but in an unfavorable conformation [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B20">20</xref>]. On the other hand, one recent report suggests human DJ-1 possesses weak protease activity [<xref ref-type="bibr" rid="B21">21</xref>], disputing another claim of chaperone activity [<xref ref-type="bibr" rid="B20">20</xref>]. In an attempt to gain further insight to possible roles of DJ-1 we performed a detailed analysis of several hundred sequences of the DJ-1/ThiJ/PfpI superfamily members. These include orthologs (sequences that are separated by speciation) and paralogs, i.e., those that are separated by other types of rearrangements. Surprisingly, we found that the nearest homologous sequences are the bacterial ThiJ genes, suggesting that DJ-1 may have evolved from thiamine synthesis genes that have been dispensed with in eukaryotes.</p></sec><sec><title>Results</title><p>Using human DJ-1 as a seed sequence for PSI-BLAST, we identified 311 sequences of proteins with significant homology (see <xref ref-type="supplementary-material" rid="S1">additional file 1</xref> for a list of all the sequences we identified). Within this large group there are several distinct subgroups supported by bootstrap analysis. Proteins with similar annotations across different species generally clustered together into distinct clades (Figure <xref ref-type="fig" rid="F1">1</xref>). Those proteins annotated as DJ-1 clustered into a specific node that included several eukaryotic species, which are likely to be orthologs of each other. As expected, primate members (<italic>Homo sapiens </italic>and <italic>Cercopithecus aethiops</italic>, 100% identity) clustered together, with progressively lower similarity to rodent (<italic>Rattus norvegicus</italic>, <italic>Mus musculus </italic>and <italic>Mesocricetus auratus</italic>; 91&#x02013;97% identity) or other vertebrate (<italic>Gallus gallus</italic>, <italic>Salmo salar </italic>and <italic>Xenopus laevis</italic>; 80&#x02013;89% identity) homologues. Within each of two invertebrate species with reported sequences (<italic>Caenorhabditis elegans </italic>and <italic>Drosophila melanogaster</italic>) there appear to be two distinct DJ-1 paralogs, each with about 40% identity to the human protein. The closest grouping to the eukaryote DJ-1 orthologs is the ThiJ family of 4-methyl-5(&#x003b2;-hydroxyethyl)-thiazol monophosphate biosynthesis enzymes, which we have analyzed in more detail (discussed below).</p><fig position="float" id="F1"><label>Figure 1</label><caption><p><bold>Cladogram of the DJ-1/ThiJ/PfpI superfamily. </bold>Consensus maximum likelihood tree with branch distances corresponding to level of bootstrap support. Known structures are highlighted along with the corresponding PDB identifier. From this tree it is clear that the DJ-1 superfamily contains proteins with diverse functions and that the DJ-1 cluster is most similar to the ThiJ subgroup. Group labels are guided by the annotation of the constituent sequences (for more details see text). Unlabeled clusters had a majority of sequences with unknown or disparate function. Sequence identifiers and files for the construction of this tree can be found in the supplemental information. Numbers in parantheses indicate percentage identities; the first number identity within the group, the second is the identity with human DJ-1.</p></caption><graphic xlink:href="1471-2148-4-6-1"/></fig><p>Outside of the DJ-1/ThiJ group, there are a number of distinct clades that have at least one member whose function is known. Of these, three can be separated from the DJ-1/ThiJ proteins by the presence of diagnostic structural elements. Firstly, a series of plant homologues group together and appear to be paralogs as both have a duplicated DJ-1/ThiJ (Pfam PF01965) domains, as described Chinese cabbage [<xref ref-type="bibr" rid="B22">22</xref>]. These proteins, from <italic>Arabidopsis thaliana </italic>and <italic>Oryza sativa </italic>and <italic>Brassica rapa subsp. pekinensis</italic>, are annotated as ThiJ or protease-related, but cluster close to the ThiJ family. Secondly, there are a number of bacterial proteins containing a catalase domain and a DJ-1/ThiJ domain. These are large subunit catalases (EC 1.11.1.6), the structure of one of which has been solved [<xref ref-type="bibr" rid="B11">11</xref>]. Thirdly, another prominent clade includes the AraC type transcriptional regulators from bacteria. These proteins can be defined by presence of one or more helix-turn-helix (HTH) motifs in the C-terminal portion of the protein. The HTH motif is thought to mediate DNA binding, whilst the ThiJ-like domain may be an amidase, although this is unproven.</p><p>Other families have a single DJ-1/ThiJ domain with variable extensions at the C- or N-termini. A major grouping includes two proteases from thermophilic bacteria, PfPI and PH1704 whose ATP-independent protease activity has been demonstrated [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B23">23</xref>]. The crystal structure has been solved for PH1704 [<xref ref-type="bibr" rid="B13">13</xref>]. This protein is hexameric, in contrast to dimeric human DJ-1 and this difference in oligomer formation is mediated by differences at the C-terminal of the two proteins [<xref ref-type="bibr" rid="B18">18</xref>]. The proteases that grouped together in this analysis all lack the most C-terminal &#x003b1;-helix found in DJ-1, and thus are also likely to be hexameric, distinguishing them from the DJ-1/ThiJ clade.</p><p>Several proteins annotated as sigma cross-reacting proteins cluster together. This family has a unique conserved composition (91.4% identity) that distinguishes it from neighboring families. This group also appears to be most similar to a larger family that includes <italic>E coli </italic>Hsp31, a chaperone [<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B20">20</xref>], which we have annotated as ThiJ/PfPI-like proteins including chaperones. A <italic>Saccharomyces cerevisiae </italic>protein, YDR533C, whose transcription is up regulated when yeast cells enter the quiescent state after carbon starvation or in the presence of misfolded proteins [<xref ref-type="bibr" rid="B24">24</xref>,<xref ref-type="bibr" rid="B25">25</xref>], is also present in this larger clade. Further analysis (see discussion) supports this group as having protease activities and we have annotated this family as ThiJ/PfpI-like protease/chaperones. How distinct this grouping is from the PfpI-like proteases is unclear, and they might be regarded as a single group. However, identity between these groups is only moderate (approximately 20%), therefore we have annotated them separately (fig <xref ref-type="fig" rid="F1">1</xref>). The sigma cross-reacting proteins have a distinct ElbB domain (COG3155.1), related to ThiJ but having a moderate overall homology. Hence we have kept these as a separate branch. The structure of a member from <italic>E coli </italic>has been solved (pdb entry 1OY1) and is dimeric.</p><p>To further assess the similarities and differences between the DJ-1 and ThiJ families, we extracted the sequences and realigned them. The resulting cladogram is shown in figure <xref ref-type="fig" rid="F2">2</xref>. The member of the prokaryotic ThiJ enzymes with highest homology to eukaryotic DJ-1 proteins is from <italic>Leptospira interrogans</italic>, which has a 42% identity with human DJ-1. This emphasizes the high degree of conservation between these two groups and indicates that there is likely to be structural conservation. Figure <xref ref-type="fig" rid="F3">3</xref> and <xref ref-type="fig" rid="F4">4</xref> shows a multiple alignment of the DJ-1 and ThiJ families. There are a series of key residues that have been suggested to be important in DJ-1 function which are well conserved between these two groups (see discussion).</p><fig position="float" id="F2"><label>Figure 2</label><caption><p><bold>The DJ1 and ThiJ families </bold>Cladogram of the alignment of the sequences belonging to the ThiJ and DJ-1 subgroups. Bootstrap support for this neighbor-joining tree is labeled at the vertices and the sequences are identified by their species name and accession number. The eukaryotic DJ-1 family members are boxed in blue to highlight their distinctness from the bacterial ThiJ proteins.</p></caption><graphic xlink:href="1471-2148-4-6-2"/></fig><fig position="float" id="F3"><label>Figure 3</label><caption><p><bold>Amino acid conservation of the DJ-1/ThiJ homologues </bold>Multiple alignment of sequences within the DJ-1/ThiJ family shows high homology and presence of a number of absolutely conserved amino acids. Bars below each residue indicate the degree of conservation. As in (a), the eukaryotic DJ-1 family is boxed in blue for clarity.</p></caption><graphic xlink:href="1471-2148-4-6-3"/></fig><fig position="float" id="F4"><label>Figure 4</label><graphic xlink:href="1471-2148-4-6-4"/></fig></sec><sec><title>Discussion</title><p>The aim of this study was to compare sequence homologies between the clearly identifiable DJ-1 homologues and other members of this superfamily whose function or activity is known. The results of a search using PSI-BLAST yielded many genes with significant homology to human DJ-1. We were particularly interested in examining whether sequence analysis would support the previous suggestions that DJ-1 is a chaperone [<xref ref-type="bibr" rid="B20">20</xref>] or a protease [<xref ref-type="bibr" rid="B21">21</xref>]. Our analysis provides some degree of separation of members of the DJ-1/ThiJ superfamily with these functions.</p><p>Although human DJ-1 does not contain a strong catalytic Cys-His-Asp/Glu triad found in proteases such as PH1704, C106 and H126 have been suggested to contribute a catalytic diad [<xref ref-type="bibr" rid="B19">19</xref>]. C106 is absolutely conserved in all of the ThiJ and DJ-1 sequences, as it is within most members of the superfamily (data not shown). However, H126 is conserved only within the DJ-1 family. All higher eukaryotic members have an equivalent histidine with the exception of one of the <italic>Drosophila </italic>genes, which has a phenylalanine. H126 is probably not involved in catalysis, based on the 1.1 &#x000c5; crystal structure [<xref ref-type="bibr" rid="B18">18</xref>], and the significance of conservation of this residue is therefore unclear.</p><p>Further evidence that the DJ-1/ThiJ families would have only minor protease activity comes from examination of the sequence around this conserved cysteine. In the protease family, a consensus sequence AI<underline>C</underline>HGP is found. In the case of PH1704, the equivalent Cys100/His101 pair form part of the catalytic triad [<xref ref-type="bibr" rid="B13">13</xref>]. In contrast, the equivalent sequence in human DJ-1 is AI<underline>C</underline>AGPT, and is conserved in all DJ-1 homologues. These data are consistent with the lack of protease activity in different assays [<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B20">20</xref>]. The AI<underline>C</underline>AGPT sequence may, however, contribute to the weak protease activity reported recently <italic>in vitro </italic>[<xref ref-type="bibr" rid="B21">21</xref>]. <italic>E coli </italic>Hsp31 has both protease and chaperone activities [<xref ref-type="bibr" rid="B20">20</xref>], and contains the sequence SL<underline>C</underline>HGP. This made us analyze all the members of the protease/chaperone family (as annotated in figure <xref ref-type="fig" rid="F1">1</xref>). The consensus sequence is [Aliphatic] [Aliphatic]CH [SAG], with the cys/his pair being invariant. As all known proteases contain adjacent Cys/His residues whereas substitution with Cys/X is found in all non-protease members, we predict that most of the "Hsp31-like proteases/chaperones" will have protease activity. However, our analysis supports the contention that DJ-1 has only minor protease activity. Further experimental evidence to assess the physiological relevance of this weak activity is required.</p><p>It should be noted that using PSI-BLAST with human DJ-1 as a seed sequence has limitations. DJ1 and related proteins represent part of the much larger type I glutamine amidotransferases (GATase; Pfam PF00117) superfamily based on structure and sequence similarities [<xref ref-type="bibr" rid="B11">11</xref>]. No type I GATase enzymes were identified with the methods we have used. This might not be a substantial limitation as it is not possible to integrate all members of such large and divergent superfamilies in a single tree without loss of predictive value [<xref ref-type="bibr" rid="B26">26</xref>]. Equally, there may be important groups of enzymes within the DJ-1/ThiJ/PfPI superfamily, distinct from type I GATases, that have not been highlighted that could be instructive for finding the function of DJ-1. An example is the phosphoribosylformylglycinamidine synthases (FGAM synthases; Pfam PF02700, EC 6.3.5.3). There are at least 50 enzymes with similar annotations within the DJ1/PfPI superfamily annotated in the public database (PF01965 at <ext-link ext-link-type="uri" xlink:href="http://www.sanger.ac.uk/Software/Pfam/index.shtml"/>). The public domain superfamily was constructed using 44 seed sequences, including an FGAM synthase, and identifies a larger grouping (497 sequences) than found in our analysis (311 unique sequences). This is likely due to the generation of a more specific sequence searching profile than the more broadly inclusive methods used to form Pfam families. The sequence identity between FGAM synthases and human DJ-1 is comparable to those between human DJ-1 and some proteins in the <xref ref-type="supplementary-material" rid="S1">additional file 1</xref>. Therefore, the limits of the DJ-1 "superfamily" are unclear and the dataset generated in this study may represent the most tractable set of similar sequences rather than the largest possible grouping.</p><p>One area that this analysis has allowed us to highlight is the degree of conservation of specific residues that are mutated in PD. As noted previously [<xref ref-type="bibr" rid="B1">1</xref>], Leucine 166, which is mutated to proline, is highly conserved throughout the DJ-2 proteins and ThiJ enzymes (with the exception of a phenylalanine in <italic>Fusobacterium nucleatum</italic>). It appears that L166P, in the penultimate &#x003b1;-helix of human DJ-1, destabilizes the protein [<xref ref-type="bibr" rid="B2">2</xref>] perhaps by disrupting this &#x003b1;-helix. Another putative mutation, A104T [<xref ref-type="bibr" rid="B27">27</xref>], is almost completely conserved throughout all DJ-1 and ThiJ members. The site of the M26I mutation [<xref ref-type="bibr" rid="B28">28</xref>] is also absolutely conserved in all vertebrate orthologs, although a Leucine is present in invertebrates and in the ThiJ enzymes.</p></sec><sec><title>Conclusions</title><p>Our analyses demonstrate that the eukaryotic DJ-1 and prokaryotic ThiJ families are closely related. However, they also demonstrate the difficulty of predicting function based on sequence. ThiJ was cloned as an enzyme in the biosynthesis of thiamine in <italic>E coli </italic>[<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B15">15</xref>]. As thiamine is an essential vitamin for many eukaryotes, presumably another use for the gene family has evolved. The mechanistic details of the enzymatic reaction of ThiJ have not been fully elucidated, but it catalyses a phosphorylation reaction of hydroxymethylpyrimidine phosphate, a precursor to thiamine [<xref ref-type="bibr" rid="B14">14</xref>]. An equivalent kinase activity has not been detected in human DJ-1 [<xref ref-type="bibr" rid="B18">18</xref>]. In contrast, human DJ-1 has been suggested to have either chaperone [<xref ref-type="bibr" rid="B20">20</xref>] or a weak protease activity [<xref ref-type="bibr" rid="B21">21</xref>]. A tentative conclusion is that as ThiJ activity was dispensed with, the eukaryotic DJ-1 orthologs have converged on a function that was present in one of the archaic paralogs, namely protein chaperone activity. However, equally feasible is that the major function of DJ-1 is in binding RNA [<xref ref-type="bibr" rid="B5">5</xref>] or an unrecognized function. The role of the conserved cysteine residue, catalytic in other members of the family, is unclear.</p></sec><sec sec-type="methods"><title>Methods</title><p>We performed homology search using iterative PSI-BLAST [<xref ref-type="bibr" rid="B29">29</xref>] using human DJ-1 as the seed sequence (NP_009193.2). PSI-BLAST was performed using default parameters from the NCBI site. The search converged in 7 iterations and the results were trimmed for duplicates and hypothetical results. The resulting 311 sequences were then aligned using CLUSTALW. The results were also aligned with T-COFFEE [<xref ref-type="bibr" rid="B30">30</xref>] and SAM 3.4 [<xref ref-type="bibr" rid="B31">31</xref>], neither of which offered a significant difference in quality (data not shown).</p><p>In order to assess the similarity and quality of subgroups in this alignment, different trees were first made with 1,000 bootstrap replicates using neighbor joining on all three alignment methods from CLUSTALW, T-COFFEE and SAM3.4. Each of these methods gave similar subgroups. Subsequently, the final consensus tree was constructed by maximum likelihood using protpars of the PHYLIP package (version 3.5 c, distributed by J Felsenstein, Department of Genetics, University of Washington, Seattle) with 100 bootstrap replicates. This second method adjusted the position of the subgroups relative to each other compared with the neighbor joining but did not change the overall subgroup membership. For figure <xref ref-type="fig" rid="F2">2</xref>, the TREEVIEW program [<xref ref-type="bibr" rid="B32">32</xref>] was used to render the tree used for figure <xref ref-type="fig" rid="F1">1</xref>. The subgroup containing human DJ-1 was extracted by removing the most specific tree with 100% bootstrap support containing human DJ-1 and its neighbors, the ThiJ group. The resultant subgroup was realigned using T-COFFEE and visually inspected and altered for corrections. One sequence was removed to obtain a higher quality semi-gapless alignment.</p></sec><sec><title>Authors' Contributions</title><p>SB performed all of the multiple alignments and tree constructions. MRC participated in the study design and drafted the manuscript.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="S1"><caption><title>Additional File 1</title><p>Text file containing all sequences and accession numbers used to align the tree in figure <xref ref-type="fig" rid="F1">1</xref>. The numbering of sequences in this file also identifies proteins in the tree in <xref ref-type="supplementary-material" rid="S1">additional file 2</xref>.</p></caption><media xlink:href="1471-2148-4-6-S1.txt" mimetype="text" mime-subtype="plain"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S2"><caption><title>Additional File 2</title><p>A phylogenetic tree of the DJ-1 superfamily as in figure <xref ref-type="fig" rid="F1">1</xref>, but with individual proteins labeled (numbering as for <xref ref-type="supplementary-material" rid="S1">additional file 1</xref>). New Hampshire Extended format can be viewed using TreeView, which can be downloaded from <ext-link ext-link-type="uri" xlink:href="http://taxonomy.zoology.gla.ac.uk/rod/treeview.html"/>.</p></caption><media xlink:href="1471-2148-4-6-S2.nhx" mimetype="text" mime-subtype="plain"><caption><p>Click here for file</p></caption></media></supplementary-material></sec>
The phylogeny of the social wasp subfamily Polistinae: evidence from microsatellite flanking sequences, mitochondrial COI sequence, and morphological characters
<sec><title>Background</title><p>Social wasps in the subfamily Polistinae (Hymenoptera: Vespidae) have been important in studies of the evolution of sociality, kin selection, and within colony conflicts of interest. These studies have generally been conducted within species, because a resolved phylogeny among species is lacking. We used nuclear DNA microsatellite flanking sequences, mitochondrial COI sequence, and morphological characters to generate a phylogeny for the Polistinae (Hymenoptera) using 69 species.</p></sec><sec><title>Results</title><p>Our phylogeny is largely concordant with previous phylogenies at higher levels, and is more resolved at the species level. Our results support the monophyly of the New World subgenera of Polistini, while the Old World subgenera are a paraphyletic group. All genera for which we had more than one exemplar were supported as monophyletic except <italic>Polybia </italic>which is not resolved, and may be paraphyletic.</p></sec><sec><title>Conclusion</title><p>The combination of DNA sequences from flanks of microsatellite repeats with mtCOI sequences and morphological characters proved to be useful characters establishing relationships among the different subgenera and species of the Polistini. This is the first detailed hypothesis for the species of this important group.</p></sec>
<contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Ar&#x000e9;valo</surname><given-names>Elisabeth</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Zhu</surname><given-names>Yong</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Carpenter</surname><given-names>James M</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Strassmann</surname><given-names>Joan E</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib>
BMC Evolutionary Biology
<sec><title>Background</title><p>A robust phylogenetic tree is of paramount importance as a beginning point for many kinds of evolutionary studies [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B4">4</xref>]. Understanding the evolutionary history of a character can allow us to tease apart factors important for adaptation to a specific environmental condition from those due to history [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>]. However, obtaining accurate phylogenetic relationships can sometimes be difficult. Historically, morphological traits were used in combination with the principles of cladistics [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>] to elucidate phylogenetic relationships. Morphological traits require specialists who can detect sometimes minute differences between species, and such expertise requires years of training.</p><p>Nucleic acid sequence information, primarily from mitochondrial DNA (mtDNA) has allowed us to make enormous strides towards resolving phylogenies during the last twenty years [<xref ref-type="bibr" rid="B9">9</xref>-<xref ref-type="bibr" rid="B15">15</xref>]. But these sequence regions have the disadvantage that they are often not evolving at ideal rates to reveal histories of desired traits. The branching pattern of gene trees and species trees are not necessarily overlapping in time and proceed concurrently. Also, evolution of mtDNA genes might not necessarily correspond to the speciation process [<xref ref-type="bibr" rid="B4">4</xref>]. Mitochondrial DNA genes are essentially single linkage groups and might not reveal the actual whole organism phylogeny [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>].</p><p>For nearly as long as sequence data have been available, there has been a heated debate about which kinds of data, molecular or morphological, provide the most accurate result when topologies obtained from different data sets are in conflict. Molecular data are undoubtedly considered by many to be more reliable than morphology (i.e., more likely to produce a "true" tree, e.g. [<xref ref-type="bibr" rid="B18">18</xref>]). It is clear that the perception of morphological data as inferior to molecular data also extends to value judgments on the quality of a particular morphological analysis based on how well the results of that analysis conform to the results of molecular analyses (e.g. [<xref ref-type="bibr" rid="B19">19</xref>]). Such standards are usually not applied to independent molecular analyses, which are also often in conflict with each other (e.g. [<xref ref-type="bibr" rid="B20">20</xref>]). No single particular data set may completely conform with our best estimate based on total evidence. The best estimator for a species phylogeny might therefore not necessarily depend on the sole use of one set of characters (morphological or molecular), but on a combination of different characters that can prove to be a better estimator of species trees [<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B21">21</xref>-<xref ref-type="bibr" rid="B24">24</xref>].</p><p>Nuclear markers are independent from mitochondrial markers, and can be independent from each other if they are scattered through the genome. Nuclear genes represent a largely untouched resource for molecular systematics [<xref ref-type="bibr" rid="B25">25</xref>]. However, few nuclear genes, besides some rDNA sequences, have been used in phylogenetic studies [<xref ref-type="bibr" rid="B26">26</xref>]. The earliest molecular studies in Hymenoptera focused almost entirely on mitochontrial 16S rDNA [<xref ref-type="bibr" rid="B27">27</xref>-<xref ref-type="bibr" rid="B32">32</xref>]. More recently, the nuclear 28S rDNA gene has been used successfully for analyses of relationships within superfamilies, but with less success at higher taxonomic levels [<xref ref-type="bibr" rid="B33">33</xref>-<xref ref-type="bibr" rid="B36">36</xref>]. It has also been applied to social wasps [<xref ref-type="bibr" rid="B37">37</xref>], but its use is controversial [<xref ref-type="bibr" rid="B38">38</xref>]. Microsatellite loci are abundant, single copy, and widely distributed throughout the genome [<xref ref-type="bibr" rid="B39">39</xref>-<xref ref-type="bibr" rid="B41">41</xref>]. Few studies have used microsatellites to reconstruct phylogenies although they have been widely used for population genetic studies (e. g. [<xref ref-type="bibr" rid="B42">42</xref>-<xref ref-type="bibr" rid="B46">46</xref>]). Though their sequences are short and the repeat regions themselves are highly variable, sequence information from several microsatellite flanking regions can be combined for robust, informative and easily aligned characters that can help resolve difficult phylogenetic relationships. The single copy flanking sequences are much less variable than are numbers of repeats themselves, making them easier to align.</p><p>Here we present a phylogeny for a very important group of social wasps. Social insects of the order Hymenoptera constitute a highly diverged group, with cosmopolitan distributions. Because of the existence of different castes, division of labor, altruism, and a haplodiploid genetic system, these social insects represent a unique system to study kin selection [<xref ref-type="bibr" rid="B47">47</xref>-<xref ref-type="bibr" rid="B50">50</xref>]. The subfamily Polistinae (paper wasps) is the second largest of six subfamilies of the family Vespidae, and is entirely composed of social wasps. It is made up of four tribes (Polistini, Epiponini, Mischocyttarini and Ropalidiini), with 943 species ([<xref ref-type="bibr" rid="B51">51</xref>], unpublished). This group probably arose in the mid- to late Jurassic. Carpenter [<xref ref-type="bibr" rid="B51">51</xref>] has estimated that this group diverged from other Vespidae at the breakup of Gondwana, as much as 140 million years ago. A molecular study, which used microsatellite loci to establish the conservation and polymorphisms across different species of this subfamily, discovered interesting results for divergence times among species [<xref ref-type="bibr" rid="B52">52</xref>]. The divergence time for <italic>Polistes bellicosus </italic>and <italic>P. annularis </italic>was estimated to be between 10 to 80 million years. The divergence time for the entire tribe is larger than among the species of the genus <italic>Polistes </italic>and smaller than for the subfamilies of Vespidae (between 80 and 175 million years), and Vespinae and Polistinae probably split in the Mid Jurassic [<xref ref-type="bibr" rid="B52">52</xref>]. The tribe Polistini is the only one with a cosmopolitan distribution (Table <xref ref-type="table" rid="T1">1</xref>). The genus <italic>Polistes </italic>was formerly subdivided into 12 different subgenera, either distributed in the New World (<italic>Fuscopolistes</italic>, <italic>Aphanilopterus</italic>, <italic>Palisotius</italic>, <italic>Epicnemius</italic>, <italic>Onerarius</italic>) or Old World (<italic>Gyrostoma</italic>, <italic>Stenopolistes</italic>, <italic>Nygmopolistes</italic>, <italic>Megapolistes</italic>, <italic>Polistella</italic>, <italic>Sulcopolistes </italic>and <italic>Polistes </italic>sensu stricto). More recently, seven of these subgenera have been synonymized [<xref ref-type="bibr" rid="B53">53</xref>], with one subgenus for the New World species (<italic>Aphanilopterus</italic>) and three for the Old World species (<italic>Gyrostoma</italic>, <italic>Polistella </italic>and <italic>Polistes </italic>sensu stricto). The New World subgenera were synonymized because two were found to be paraphyletic, a result not supported by the present study. The other three tribes, on the other hand, are either distributed in the New World (Epiponini, Mischocyttarini) or the Old World (Ropalidiini).</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>List of Tribe (including subgenera, in the case of the Polistini tribe), species, locality and collector for all the specimens used in the study.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold>Tribe/Subgenera</bold></td><td align="left"><bold>Species</bold></td><td align="left"><bold>Locality of Origin</bold></td><td align="left"><bold>Donor <sup>1</sup></bold></td></tr></thead><tbody><tr><td align="left">Polistini/<italic>Fuscopolistes</italic></td><td align="left"><italic>Polistes bellicosus</italic></td><td align="left">USA</td><td align="left">EA</td></tr><tr><td align="left">Polistini/<italic>Fuscopolistes</italic></td><td align="left"><italic>Polistes apachus</italic></td><td align="left">USA</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Fuscopolistes</italic></td><td align="left"><italic>Polistes aurifer</italic></td><td align="left">USA</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Fuscopolistes</italic></td><td align="left"><italic>Polistes carolina</italic></td><td align="left">USA</td><td align="left">PS/JES/DCQ</td></tr><tr><td align="left">Polistini/<italic>Fuscopolistes</italic></td><td align="left"><italic>Polistes dorsalis</italic></td><td align="left">Texas</td><td align="left">EA</td></tr><tr><td align="left">Polistini/<italic>Fuscopolistes</italic></td><td align="left"><italic>Polistes metricus</italic></td><td align="left">USA</td><td align="left">EA</td></tr><tr><td align="left">Polistini/<italic>Fuscopolistes</italic></td><td align="left"><italic>Polistes fuscatus</italic></td><td align="left">USA</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes annularis</italic></td><td align="left">USA</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes buyssoni</italic></td><td align="left">Chile</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes canadensis</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes lanio</italic></td><td align="left">Argentina</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes cavapyta</italic></td><td align="left">Argentina</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes exclamans</italic></td><td align="left">USA</td><td align="left">EA</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes simillimus</italic></td><td align="left">Bolivia</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes crinitus americanus</italic></td><td align="left">Puerto Rico</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes versicolor</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Polistini/<italic>Aphanilopterus</italic></td><td align="left"><italic>Polistes instabilis</italic></td><td align="left">S. Texas</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Polistini/<italic>Palisotius</italic></td><td align="left"><italic>Polistes major</italic></td><td align="left">USA</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Epicnemius</italic></td><td align="left"><italic>Polistes cinerascens</italic></td><td align="left">Argentina</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Onerarius</italic></td><td align="left"><italic>Polistes carnifex</italic></td><td align="left">Mexico</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Polistini/<italic>Nygmopolistes</italic></td><td align="left"><italic>Polistes tenebricosus</italic></td><td align="left">Malaysia</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Megapolistes</italic></td><td align="left"><italic>Polistes rothneyi</italic></td><td align="left">Japan</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Megapolistes</italic></td><td align="left"><italic>Polistes jokahamae</italic></td><td></td><td align="left">FI</td></tr><tr><td align="left">Polistini/<italic>Polistella</italic></td><td align="left"><italic>Polistes stigmus</italic></td><td align="left">Australia</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Polistella</italic></td><td align="left"><italic>Polistes snelleni</italic></td><td align="left">Japan</td><td align="left">JMC</td></tr><tr><td align="left">Polistini/<italic>Polistella</italic></td><td align="left"><italic>Polistes japonicus</italic></td><td></td><td align="left">FI</td></tr><tr><td align="left">Polistini/<italic>P. sensu stricto</italic></td><td align="left"><italic>Polistes nimphus</italic></td><td align="left">Cavriglia</td><td align="left">EA, FZ</td></tr><tr><td align="left">Polistini/<italic>P. sensu stricto</italic></td><td align="left"><italic>Polistes chinensis</italic></td><td></td><td align="left">FI</td></tr><tr><td align="left">Polistini/<italic>P. sensu stricto</italic></td><td align="left"><italic>Polistes dominulus</italic></td><td align="left">Italy</td><td align="left">EA, FZ</td></tr><tr><td align="left">Polistini/<italic>P. sensu stricto</italic></td><td align="left"><italic>Polistes biglumis bimaculatus</italic></td><td align="left">M. Genevre</td><td align="left">MCL</td></tr><tr><td align="left">Polistini/<italic>P. sensu stricto</italic></td><td align="left"><italic>Polistes gallicus</italic></td><td align="left">Florence</td><td align="left">EA, FZ</td></tr><tr><td align="left">Polistini/<italic>P. sensu stricto</italic></td><td align="left"><italic>Polistes marginalis</italic></td><td align="left">South Africa</td><td align="left">JMC</td></tr><tr><td align="left">Mischocyttarini</td><td align="left"><italic>Mischocyttarus alfkenii</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Mischocyttarini</td><td align="left"><italic>Mischocyttarus immarginatus</italic></td><td align="left">Costa Rica</td><td align="left">JMC</td></tr><tr><td align="left">Mischocyttarini</td><td align="left"><italic>Mischocyttarus melanarius</italic></td><td align="left">La Trinidad</td><td align="left">UM</td></tr><tr><td align="left">Mischocyttarini</td><td align="left"><italic>Mischocyttarus mexicanus</italic></td><td align="left">USA</td><td align="left">JMC</td></tr><tr><td align="left">Mischocyttarini</td><td align="left"><italic>Mischocyttarus pallidipectus</italic></td><td align="left">Costa Rica</td><td align="left">JMC</td></tr><tr><td align="left">Mischocyttarini</td><td align="left"><italic>Mischocyttarus phthisicus</italic></td><td align="left">Puerto Rico</td><td align="left">JMC</td></tr><tr><td align="left">Mischocyttarini</td><td align="left"><italic>Mischocyttarus mastigophorus</italic></td><td align="left">Costa Rica</td><td align="left">JMC</td></tr><tr><td align="left">Ropalidiini</td><td align="left"><italic>Belonogaster juncea colonialis</italic></td><td></td><td align="left">MK</td></tr><tr><td align="left">Ropalidiini</td><td align="left"><italic>Parapolybia varia</italic></td><td></td><td align="left">ST</td></tr><tr><td align="left">Ropalidiini</td><td align="left"><italic>Ropalidia latebalteata</italic></td><td align="left">Malaysia</td><td align="left">JMC</td></tr><tr><td align="left">Ropalidiini</td><td align="left"><italic>Ropalidia socialistica</italic></td><td></td><td align="left">FI</td></tr><tr><td align="left">Ropalidiini</td><td align="left"><italic>Ropalidia fasciata</italic></td><td></td><td align="left">FI</td></tr><tr><td align="left">Ropalidiini</td><td align="left"><italic>Ropalidia sp</italic>.</td><td align="left">Malaysia</td><td align="left">ST</td></tr><tr><td align="left">Ropalidiini</td><td align="left"><italic>Ropalidia romandi</italic></td><td></td><td align="left">FI</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Apoica pallens</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Agelaia multipicta</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Angiopolybia pallens</italic></td><td align="left">Peru</td><td align="left">CRS</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Parachartergus colobopterus</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Parachartergus fraternus</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Protopolybia exigua</italic></td><td align="left">Venezuela</td><td align="left">CRH</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Protonectarina sylveirae</italic></td><td align="left">Brazil</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Brachygastra augusti</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Brachygastra mellifica</italic></td><td align="left">South Texas</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Brachygastra lecheguana</italic></td><td align="left">Argentina</td><td align="left">JMC</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Brachygastra bilineolata</italic></td><td align="left">Trinidad</td><td align="left">JMC</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia occidentalis</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia ignobilis</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia emaciata</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia rejecta</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia raui</italic></td><td align="left">Panama</td><td align="left">JMC</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia ruficeps</italic></td><td align="left">Argentina</td><td align="left">JMC</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia scrobalis</italic></td><td align="left">French Guiana</td><td align="left">JMC</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Polybia affinis</italic></td><td align="left">French Guiana</td><td align="left">JMC</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Synoeca septentrionalis</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Metapolybia cingulata</italic></td><td align="left">Venezuela</td><td align="left">JES/DCQ</td></tr><tr><td align="left">Epiponini</td><td align="left"><italic>Epipona niger</italic></td><td align="left">Venezuela</td><td align="left">CRH</td></tr></tbody></table></table-wrap><p>The subfamily Polistinae is characterized by two major behavioral groups, based on their mode of colony founding and mechanism of reproductive dominance [<xref ref-type="bibr" rid="B54">54</xref>,<xref ref-type="bibr" rid="B55">55</xref>]. Independent founding is characterized by small, simply constructed nests without a protective paper envelope, founded by a single queen or few queens, without the assistance of workers [<xref ref-type="bibr" rid="B56">56</xref>,<xref ref-type="bibr" rid="B57">57</xref>]. By contrast, swarm founding species have large colonies initiated by swarms consisting of multiple queens and a large number of workers [<xref ref-type="bibr" rid="B55">55</xref>].</p><p>The independent-founding group is made up of five genera: <italic>Polistes, Mischocyttarus, Belonogaster, Parapolybia</italic>, and some species of <italic>Ropalidia</italic>. Colonies are initiated by one or several inseminated egg-layers and no workers. Egg layers go through a solitary phase after insemination (few days to months). Soon after nest initiation, one fertilized female becomes the sole egg-layer. These groups of species are characterized by small colony sizes (rarely more than 50 adult wasps) and the lack of a protective envelope on the nest [<xref ref-type="bibr" rid="B56">56</xref>,<xref ref-type="bibr" rid="B58">58</xref>,<xref ref-type="bibr" rid="B59">59</xref>]. Reproductive dominance in this group is at least partly maintained through aggression by the queen.</p><p>The swarm-founding group is made up of 20 genera of the tribe Epiponini, the genus <italic>Polybioides</italic>, and some <italic>Ropalidia </italic>species. Colonies are initiated by a large number of workers and a small number of egg-layers. There is no colony phase without workers. Workers choose the site and built the nest while egg-layers wait. Swarm founding wasps characteristically have large colony sizes (usually over 50 adult wasps), and nests are often provided with a protective envelope. Reproductive dominance in this group is obtained through worker aggression on potential queens, and there is a tendency towards morphological castes in some but not most species [<xref ref-type="bibr" rid="B50">50</xref>,<xref ref-type="bibr" rid="B55">55</xref>].</p><p>Many behavioral, ecological and evolutionary questions in this group could be asked in a phylogenetic framework including characteristics of nest structure [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B60">60</xref>,<xref ref-type="bibr" rid="B61">61</xref>], reproductive strategies [<xref ref-type="bibr" rid="B62">62</xref>-<xref ref-type="bibr" rid="B64">64</xref>], and solutions to potential conflicts of interest among colony members [<xref ref-type="bibr" rid="B57">57</xref>,<xref ref-type="bibr" rid="B65">65</xref>,<xref ref-type="bibr" rid="B66">66</xref>].</p><p>For the present study, we used a combination of nuclear (three microsatellite loci flanking regions and a coding system generated by the different repeat motifs, with priming sites largely conserved throughout the Polistinae subfamily; [<xref ref-type="bibr" rid="B52">52</xref>], adult morphological [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B53">53</xref>,<xref ref-type="bibr" rid="B67">67</xref>], larval [<xref ref-type="bibr" rid="B68">68</xref>-<xref ref-type="bibr" rid="B76">76</xref>], behavioral [<xref ref-type="bibr" rid="B77">77</xref>] and mitochondrial (COI) characters to obtain a better understanding of the relationships in this group. A study by Zhu et al. [<xref ref-type="bibr" rid="B78">78</xref>] used a subset of the characters and species presented in this study. The use of these characters was to reconstruct the evolution of microsatellite repeats within the Polistinae subfamily. For the completion of that study, the best tentative tree available at that time was used in order to establish the evolution of these microsatellite repeats in an explicitly phylogenetic perspective.</p><p>With this study we investigate two phylogenetic hypotheses. First, we wanted to test whether or not the subgenera of <italic>Polistes </italic>of Richards [<xref ref-type="bibr" rid="B79">79</xref>] are monophyletic. For this, we used 33 species of <italic>Polistes </italic>and 36 species in the outgroup, the Epiponini, Ropalidiini and Mischocyttarini. Second, we evaluated the phylogenetic relationships within and among the other three Polistinae tribes, Epiponini, Ropalidiini and Mischocyttarini, using the 33 species of the tribe Polistini as an outgroup.</p></sec><sec><title>Results</title><sec><title>Polistini</title><p>Morphological characters alone showed little resolution in the Polistini at the species level, instead supporting most of the subgenera as monophyletic &#x02013; but not all (Fig. <xref ref-type="fig" rid="F1">1</xref>). New World species as a whole were monophyletic with a bootstrap support of 75% while Old World species were paraphyletic. Of the subgenera, <italic>Polistes sensu stricto</italic>, <italic>Megapolistes, Polistella, Epicnemius </italic>and <italic>Fuscopolistes </italic>were all supported as monophyletic with bootstraps of 64%, 95%, 74%, 63% and 61% respectively. But there was no resolution within these groups, and <italic>Aphanilopterus </italic>was not resolved as a group.</p><fig position="float" id="F1"><label>Figure 1</label><caption><p>Bootstrap analysis of morphological characters with 1000 replications for the 33 species of the Polistini, using 36 species of Mischocyttarini, Ropalidiini, and Epiponini as outgroups.</p></caption><graphic xlink:href="1471-2148-4-8-1"/></fig><p>The COI, and microsatellite combined characters supported the subgenera <italic>Megapolistes, Fuscopolistes</italic>, and <italic>Aphanilopterus </italic>with bootstraps of 93%, 93%, and 70% respectively (Figure <xref ref-type="fig" rid="F2">2</xref>). Both <italic>Polistella </italic>and <italic>Polistes sensu stricto </italic>were mostly supported at 50% and 66%, but each left out one species. There was also considerable resolution within the supported subgenera. Thus, the independent molecular and morphological trees offered resolution in both different and overlapping parts of the tree and, where they were resolved, were not in conflict.</p><fig position="float" id="F2"><label>Figure 2</label><caption><p>Bootstrap analysis of the combined molecular characters (COI and microsatellite sequences) with 1000 replications for 33 species of the Polistini, using 36 species of Mischocyttarini, Ropalidiini, and Epiponini as outgroups.</p></caption><graphic xlink:href="1471-2148-4-8-2"/></fig><p>The overall combined tree topology (Figure <xref ref-type="fig" rid="F3">3</xref>) is much more resolved than what was observed with the independent-character phylogenies alone (Figs. <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>). The New World clade is monophyletic with 62% bootstrap support. Although the support for this same clade was 75% in the morphological characters tree (Fig. <xref ref-type="fig" rid="F1">1</xref>), the combined tree provides a much more resolved phylogeny at the species level. The clade for the Old World <italic>Polistes </italic>is paraphyletic. All the subgenera for which we have 2 or more exemplars are supported. The Old World subgenera, <italic>Megapolistes, Polistella</italic>, and <italic>Polistes sensu stricto</italic> are supported with bootstraps of 99%, 93% and 92% respectively. The New World subgenera, <italic>Fuscopolistes, Aphanilopterus</italic> and <italic>Epicnemius</italic> are supported with bootstraps of 99%, 96% and 70% respectively.</p><fig position="float" id="F3"><label>Figure 3</label><caption><p>Bootstrap analysis of all characters with 1000 replications for the 33 species of the Polistini, using 36 species of Mischocyttarini, Ropalidiini, and Epiponini as outgroups.</p></caption><graphic xlink:href="1471-2148-4-8-3"/></fig></sec><sec><title>Epiponini, Mischocyttarini, Ropalidiini</title><p>Morphological characters alone supported the Old World Ropalidiini and the New World Epiponini and Mischocyttarini with bootstraps of 64%, 85% and 91% respectively (Figure <xref ref-type="fig" rid="F4">4</xref>). All of the genera for which we had more than one species were also supported except for <italic>Polybia</italic>. There was little resolution within genera except for <italic>Brachygastra</italic>. However most genera were only represented by a few species.</p><fig position="float" id="F4"><label>Figure 4</label><caption><p>Bootstrap analysis of morphological characters with 1000 replications for 36 of the species of Mischocyttarini, Ropalidiini, and Epiponini using 33 species of Polistini as outgroups.</p></caption><graphic xlink:href="1471-2148-4-8-4"/></fig><p>The combined COI and microsatellite characters were largely uninformative on their own, though there was some grouping of epiponine species and resolution within the monophyletic <italic>Mischocyttarus </italic>(Figure <xref ref-type="fig" rid="F5">5</xref>).</p><fig position="float" id="F5"><label>Figure 5</label><caption><p>Bootstrap analysis of molecular characters with 1000 replications for the 36 species of Mischocyttarini, Ropalidiini, and Epiponini using 33 species of Polistini as outgroups.</p></caption><graphic xlink:href="1471-2148-4-8-5"/></fig><p>The combined tree is much more resolved than either separate analysis, and bootstrap supports are greater (Figure <xref ref-type="fig" rid="F6">6</xref>). Monophyly of Ropalidiini, Mischocyttarini, and Epiponini is supported at bootstrap levels of 83%, 95%, and 90% respectively. However the relationships between the tribes are not resolved. All genera for which we have more than one species are supported except for <italic>Polybia </italic>which is broken into three groups, among which relationships are not resolved (Figure <xref ref-type="fig" rid="F6">6</xref>). Relationships among genera within Ropalidiini and Epiponini are generally not well resolved. The basal genus of the Epiponini is unresolved.</p><fig position="float" id="F6"><label>Figure 6</label><caption><p>Bootstrap analysis of all characters with 1000 replications for the 36 species of Mischocyttarini, Ropalidiini, and Epiponini using 33 species of Polistini as outgroups.</p></caption><graphic xlink:href="1471-2148-4-8-6"/></fig></sec></sec><sec><title>Discussion</title><sec><title>Relationships in the Polistini</title><p>The combined character phylogeny is partly congruent with the earlier phylogeny proposed by Carpenter in 1996, though there are some differences (Figure <xref ref-type="fig" rid="F7">7</xref>). First, note that the earlier tree is not bootstrapped; Figure <xref ref-type="fig" rid="F1">1</xref> is the bootstrapped tree for morphological characters, based on the present sample of species. Results from both studies on the monophyletic origin of the New and Old World Polistini subgenera only found support for the monophyletic origin of the New World Polistini (Fig. <xref ref-type="fig" rid="F3">3</xref>). In both studies, the Old World Polistini subgenera represent a paraphyletic group. However, the present study showed <italic>Polistes sensu stricto </italic>as the most basal subgenus (with a bootstrap support of 98%), in contrast to Carpenter [<xref ref-type="bibr" rid="B53">53</xref>], where it was the sister group of the New World subgenera (with a bootstrap support of 89%, Fig. <xref ref-type="fig" rid="F7">7</xref>). The present study found the sister group of the New World subgenera to be the clade of <italic>Polistella, Nygmopolistes</italic>, and <italic>Megapolistes</italic>. Several subgenera were absent from the present study (viz. <italic>Gyrostoma</italic>, <italic>Stenopolistes </italic>and <italic>Sulcopolistes</italic>) and it is possible that their absence affected character polarizations obtained within <italic>Polistes</italic>.</p><fig position="float" id="F7"><label>Figure 7</label><caption><p>Comparison of the subgenera of <italic>Polistes </italic>phylogeny obtained by Carpenter's 1996 morphological study (Fig. 2.8) and the phylogeny obtained in the present study, including all morphological and molecular characters. Species included from each subgenus are listed in Table <xref ref-type="table" rid="T1">1</xref>.</p></caption><graphic xlink:href="1471-2148-4-8-7"/></fig><p>In contrast to Carpenter's [<xref ref-type="bibr" rid="B53">53</xref>] study, the present results support monophyly of the subgenus <italic>Aphanilopterus </italic>in the strict sense, and of the subgenus <italic>Epicnemius</italic>, albeit based on a smaller number of species.</p><p><italic>Polistes sensu stricto </italic>is unique in the Polistinae for having social parasites, species that kill their host queen so her workers can rear the brood of the social parasite [<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B80">80</xref>-<xref ref-type="bibr" rid="B82">82</xref>]. The three social parasite species are monophyletic and most closely related to <italic>P. nimphus </italic>and <italic>P. dominulus</italic>, and less close to <italic>P. gallicus and P. biglumis </italic>[<xref ref-type="bibr" rid="B12">12</xref>,<xref ref-type="bibr" rid="B82">82</xref>]. The present study agrees that the latter two species are sister species, less close to <italic>P. nimphus </italic>and <italic>P. dominulus</italic>, but this study does not put these two as sister species. The earlier study did not include morphological characters, and did not include either <italic>P. chinensis </italic>or <italic>P. marginalis </italic>which could account for the differences [<xref ref-type="bibr" rid="B82">82</xref>].</p></sec><sec><title>Relationships in the Epiponini, Mischocyttarini, Ropalidiini</title><p>The combined character phylogeny is largely congruent with the earlier phylogeny proposed by Carpenter in 1991 (Figure <xref ref-type="fig" rid="F8">8</xref>). Depending on the tribe, one or the other is more resolved, but not in major disagreement. Within the tribe Epiponini, of the polytomies observed in the Carpenter's 1991 topology, one was resolved: <italic>Agelaia </italic>and <italic>Angiopolybia </italic>are sister-groups (Fig. <xref ref-type="fig" rid="F8">8</xref>). The genus <italic>Polybia </italic>in the present study does not disagree with Carpenter et al.'s [<xref ref-type="bibr" rid="B67">67</xref>] conclusion that it was paraphyletic: in the present study it is unresolved, with <italic>P. </italic>(<italic>Formicicola</italic>) <italic>rejecta, P. (Trichinothorax) affinis </italic>and <italic>P. (Pedothoeca) emaciata </italic>separated from the main <italic>P. sensu stricto </italic>clade for the genus. Note that the present study is based on a broader sample of genera than in [<xref ref-type="bibr" rid="B67">67</xref>].</p><fig position="float" id="F8"><label>Figure 8</label><caption><p>Comparison of the Polistinae subfamily phylogeny obtained by Carpenter's 1991 morphological study and the phylogeny obtained in the present study, including all morphological and molecular characters.</p></caption><graphic xlink:href="1471-2148-4-8-8"/></fig><p>In a series of papers Strassmann and Queller argued that a form of worker control of sex ratios was general for the Epiponini [<xref ref-type="bibr" rid="B40">40</xref>,<xref ref-type="bibr" rid="B83">83</xref>]. This form of worker control meant that colonies did not produce a new generation of queens until the colony had reduced in queen number to one or perhaps no queens. This is important to sociality because such a queen reduction before requeening maintains high relatedness within colonies. The species that they studied this cyclical oligogyny in were <italic>Parachartergus colobopterus, Protopolybia exigua, Brachygastra mellifica, Polybia emaciata </italic>and <italic>Polybia occidentalis </italic>[<xref ref-type="bibr" rid="B63">63</xref>,<xref ref-type="bibr" rid="B83">83</xref>-<xref ref-type="bibr" rid="B86">86</xref>]. In addition West- Eberhard documented either cyclical oligogyny or some of its features in three additional genera, <italic>Agelaia, Synoeca </italic>and <italic>Metapolybia </italic>[<xref ref-type="bibr" rid="B87">87</xref>-<xref ref-type="bibr" rid="B89">89</xref>]. Taken together, these studies come from all of the four basal-most lineages of the Epiponini, and also cover 4 of the 6 lineages in our best sampled clade, the one containing <italic>Polybia </italic>(Figure <xref ref-type="fig" rid="F7">7</xref>). Thus, it seems they are justified in claiming the generality of this form of worker control for the tribe.</p></sec><sec><title>Importance of molecular and morphological characters</title><p>The inclusion of several genetic markers (mitochondrial and nuclear), improved the resolution of the trees obtained from morphology alone. The COI sequence alone did not resolve <italic>Polistes </italic>as a group; combining it with the microsatellites did. While COI and the microsatellite flanking regions did not resolve <italic>Polistella</italic>, adding the repeat regions did so. The use of microsatellite flanking regions and the presence/absence coding system for the repeated regions of the microsatellites proved to be useful in the resolution of some of the branches.</p><p>For the last fifteen years, a considerable rise in the use of molecular markers has been observed. The majority of the studies have exclusively included molecular data from mtDNA genes. Even when the information gathered for those studies has been informative, problems with the use of this type of molecular data (saturation synonymous sites with mtDNA; constrains imposed on RNA sites by secondary structure, length variation, etc.) have been considered [<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B90">90</xref>]. Unfortunately, few nuclear markers have been used in phylogenetic systematic studies in the past. The search for new, powerful, informative-single copy nuclear markers has increased our wealth of information in systematic studies. At the present time just a few more nuclear markers have been used for insect phylogenetic studies [<xref ref-type="bibr" rid="B26">26</xref>,<xref ref-type="bibr" rid="B91">91</xref>]. More genes are needed for testing phylogenetic hypotheses. Meanwhile, the use of microsatellite flanking regions represents a valuable tool as nuclear single-copy markers. The wealth of microsatellite data is constantly growing across different taxa. The use of these regions will represent a powerful molecular tool in future studies.</p></sec></sec><sec sec-type="methods"><title>Methods</title><sec><title>Species and specimens used</title><p>We used a total of 69 species representing the four tribes of the subfamily Polistinae (Table <xref ref-type="table" rid="T1">1</xref>). Specimens were either donated by colleagues, or were in our own collections. These specimens included 33 species from the tribe Polistini, 7 from the Mischocyttarini, 7 from the Ropalidiini and 22 from the Epiponini. We used the Polistini as the outgroup for the Epiponini+Mischocyttarini+Ropalidiini. We used the latter three tribes as the outgroup for the analysis of the Polistini.</p></sec><sec><title>Morphological and molecular characters</title><p>We used a total of 932 characters, including 95 morphological and behavioral characters (all morphological characters and character states from Carpenter's studies [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B53">53</xref>,<xref ref-type="bibr" rid="B67">67</xref>] examined on pinned specimens for this study; larval and behavioral characters and states from citations given in appendixes 1 and 2); a 488 base pair (bp) fragment of the mtDNA COI gene (appendix 3) [<xref ref-type="bibr" rid="B92">92</xref>]; a 311 bp fragment of single-copy nuclear flanking sequence of three microsatellite loci (Pbe216AAG [137 bp], Pbe269bAAG [75 bp], and, Pbe411AAT [99 bp] (appendix 4) [<xref ref-type="bibr" rid="B52">52</xref>,<xref ref-type="bibr" rid="B93">93</xref>], and 38 characters representing the different trinucleotides in the repeat region (not their number; appendix 5 [<xref ref-type="bibr" rid="B78">78</xref>]). The repeat regions varied considerably across the four different tribes and across the different subgenera in the tribe Polistini. The coding system for the repeat region was based on presence or absence of each repeat motif and resulted in16 characters for Pbe216AAG, 15 characters for Pbe269bAAG, and 7 characters for Pbe411AAT. For example, the first four characters at Pbe216AAG were GGA, AAA, TAA, GCA, all substituted for AAG within the repeat region [<xref ref-type="bibr" rid="B78">78</xref>]. We did not count times a specific motif was repeated because this character is so variable its evolutionary history would be unclear.</p></sec><sec><title>DNA extraction, amplification and sequencing for COI, and microsatellites</title><p>We extracted DNA using protocol Strassmann I [<xref ref-type="bibr" rid="B94">94</xref>]. Prior to DNA extractions, we stored all specimens either at -80&#x000b0;C or in 100% ethanol at room temperature. PCR reactions followed protocols as in Wilcox et al. [<xref ref-type="bibr" rid="B95">95</xref>] for the mtDNA fragment, and Zhu et al. [<xref ref-type="bibr" rid="B78">78</xref>] for the microsatellite loci. Sequencing PCR products were completed directly with thermo sequenase radiolabeled <sup>33</sup>P (Thermo Sequenace radiolabeled terminator cycle sequencing Kit, Amersham Life Science). DNA sequences were run on denaturing polyacrylamide gels, dried and exposed to X-ray film. All new DNA microsatellite sequences were deposited in Genbank under accession numbers AF119623-AF119660, AF120355-AF120455. COI sequences were deposited in Genbank under accession numbers AY382200-AY382265. The aligned DNA matrix is available at TreeBASE <ext-link ext-link-type="uri" xlink:href="http://www.treebase.org/treebase/index.html"/>, submission ID number SN1595.</p></sec><sec><title>Sequence alignment</title><p>The sequences for both mtDNA (COI) and for nuclear regions (microsatellite flanking regions) were aligned based on the criterion of maximum parsimony, using the program Malign, specifying a 3:1 change:gap ratio [<xref ref-type="bibr" rid="B96">96</xref>]. We occasionally adjusted alignments by eye when Malign gave poor results.</p></sec><sec><title>Systematic analysis techniques</title><p>We used Paup* 4.0b10 [<xref ref-type="bibr" rid="B97">97</xref>], Nona [<xref ref-type="bibr" rid="B98">98</xref>], and Winclada [<xref ref-type="bibr" rid="B99">99</xref>] to construct the phylogeny, with equal weight for all base-pair substitutions (including the mtDNA COI sequence, [<xref ref-type="bibr" rid="B100">100</xref>]). For the COI gene, the analysis was completed with the use of the three coding positions weighted equally and with the first and second coding positions only. The phylogenetic trees were not in conflict by excluding the third coding position.</p><p>We completed the phylogenetic analyses following the principles of maximum parsimony, maximum likelihood and distance analysis. As the overall topology was not in disagreement by applying different algorithms, we are only presenting the results obtained based on maximum parsimony. All parsimony analyses were run using approximate searches, due to the large number of species and characters. The runs consisted of 1000 replications of the parsimony ratchet [<xref ref-type="bibr" rid="B101">101</xref>] as implemented in Winclada, with 10% character reweighting and holding one tree; 500 replications at the "default amb-poly+" setting and 500 at "amb = poly-." Resulting trees were read into Nona and swapped on with "max*" (TBR branch swapping). Nona was also used to carry out 1000 random addition sequences as a check. Maximum parsimony trees were first estimated individually for each of the different data sets (morphological and molecular), and then in combination. Gaps were treated as missing characters. Bootstrap analyses with 1000 replications were also run for each data set. Paup* runs gave similar results as those with Winclada and Nona.</p><p>Table <xref ref-type="table" rid="T2">2</xref> shows the number of parsimoniously informative characters and constant characters for the each different set of data used in the study, for the 69 species used.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Numbers of parsimoniously informative and constant characters used in the study. From left to right, for the morphological characters only, and for each of the three types of molecular characters.</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="left">Morphological</td><td align="left">COI</td><td align="left">Microsatellite Flanking Regions</td><td align="left">Repeat Region</td></tr></thead><tbody><tr><td align="left">Parsimoniously Informative</td><td align="left">95</td><td align="left">207</td><td align="left">64</td><td align="left">25</td></tr><tr><td align="left">Constant</td><td align="left">0</td><td align="left">204</td><td align="left">203</td><td align="left">3</td></tr><tr><td align="left">Total # of Characters</td><td align="left">95</td><td align="left">488</td><td align="left">311</td><td align="left">39</td></tr></tbody></table></table-wrap></sec></sec><sec><title>Authors' contributions</title><p>EA carried out a portion of the molecular genetic studies, participated in the sequence alignment, analysis of the data, and drafted the manuscript. YZ carried out a portion of the molecular genetic studies and participated in the sequence alignment. JMC provided the morphological characters, participated in the analyses and writing. JES conceived of the study, and participated in its design, coordination and writing. All authors read and approved the final manuscript.</p></sec>
Upstream plasticity and downstream robustness in evolution of molecular networks
<sec><title>Background</title><p>Gene duplication followed by the functional divergence of the resulting pair of paralogous proteins is a major force shaping molecular networks in living organisms. Recent species-wide data for protein-protein interactions and transcriptional regulations allow us to assess the effect of gene duplication on robustness and plasticity of these molecular networks.</p></sec><sec><title>Results</title><p>We demonstrate that the transcriptional regulation of duplicated genes in baker's yeast <italic>Saccharomyces cerevisiae </italic>diverges fast so that on average they lose 3% of common transcription factors for every 1% divergence of their amino acid sequences. The set of protein-protein interaction partners of their protein products changes at a slower rate exhibiting a broad plateau for amino acid sequence similarity above 70%. The stability of functional roles of duplicated genes at such relatively low sequence similarity is further corroborated by their ability to substitute for each other in single gene knockout experiments in yeast and RNAi experiments in a nematode worm <italic>Caenorhabditis elegans</italic>. We also quantified the divergence rate of physical interaction neighborhoods of paralogous proteins in a bacterium <italic>Helicobacter pylori </italic>and a fly <italic>Drosophila melanogaster</italic>. However, in the absence of system-wide data on transcription factors' binding in these organisms we could not compare this rate to that of transcriptional regulation of duplicated genes.</p></sec><sec><title>Conclusions</title><p>For all molecular networks studied in this work we found that even the most distantly related paralogous proteins with amino acid sequence identities around 20% on average have more similar positions within a network than a randomly selected pair of proteins. For yeast we also found that the upstream regulation of genes evolves more rapidly than downstream functions of their protein products. This is in accordance with a view which puts regulatory changes as one of the main driving forces of the evolution. In this context a very important open question is to what extent our results obtained for homologous genes within a single species (paralogs) carries over to homologous proteins in different species (orthologs).</p></sec>
<contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Maslov</surname><given-names>Sergei</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Sneppen</surname><given-names>Kim</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Eriksen</surname><given-names>Kasper Astrup</given-names></name><xref ref-type="aff" rid="I3">3</xref><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Yan</surname><given-names>Koon-Kiu</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib>
BMC Evolutionary Biology
<sec><title>Background</title><p>Biological processes are rarely performed by single isolated molecules. Instead, they typically involve a coordinated activity of many molecules forming a neighborhood in biomolecular networks. Changes in these networks are thus coupled to the evolution of new functions and functional relationships in the organism. Gene duplication is an important source of raw material for the molecular evolution [<xref ref-type="bibr" rid="B1">1</xref>]. Immediately after a duplication event the pair of freshly duplicated genes is thought to be identical in both sequences and functional roles in the cell. However, with time their properties including their positions within molecular networks diverge. Here we quantify this divergence in the baker's yeast <italic>Saccharomyces cerevisiae </italic>using several recent system-wide data sets. To this end we measure: 1) The similarity of positions of duplicated genes in the transcription regulatory network [<xref ref-type="bibr" rid="B2">2</xref>] given by the number of transcription regulators that regulate both of them; 2) The similarity of the set of binding partners [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>] of their protein products, and their ability to substitute for each other in knock-out experiments [<xref ref-type="bibr" rid="B5">5</xref>]. These measures reflect, correspondingly, the upstream and downstream properties of molecular networks around duplicated genes. We then repeat this analysis using species-wide data on protein interaction networks in a bacterium <italic>Helicobacter pylori </italic>[<xref ref-type="bibr" rid="B6">6</xref>] and a fruit fly <italic>Drosophila melanogaster </italic>[<xref ref-type="bibr" rid="B7">7</xref>], as well as a systematic RNAi gene inactivation assay [<xref ref-type="bibr" rid="B8">8</xref>] in a nematode worm <italic>Caenorhabditis elegans</italic>.</p></sec><sec><title>Results and discussion</title><sec><title>Divergence of the upstream transcriptional regulation of duplicated genes in <italic>S. cerevisiae</italic></title><p>The first measure of the divergence of duplicated genes compares sets of their transcriptional regulators. Such a set contains information about different conditions under which a given gene is expressed, and thus reflects the spectrum of its functional roles in the cell. To quantify the similarity of transcriptional regulation of a pair of genes we use "regulatory overlap" &#x003a9;<sub><italic>req </italic></sub>given by the number of transcription factors that bind to upstream regulatory regions of <italic>both </italic>these genes (see Fig. <xref ref-type="fig" rid="F1">1</xref> for a general illustration). The information about gene duplications used in this study was extracted from the list of all pairs of paralogous (evolutionary related) proteins found in the yeast genome by the blastp program [<xref ref-type="bibr" rid="B9">9</xref>] with a conservative 10<sup>-10 </sup>E-value cutoff (see Methods for more details). The system-wide data for the transcription regulatory network in yeast was taken from the chip-on-chip experiment by Lee <italic>et al</italic>. [<xref ref-type="bibr" rid="B2">2</xref>] which investigated in-vivo binding patterns between 106 yeast transcription factors and upstream regulatory regions of all 6270 yeast genes. Fig. <xref ref-type="fig" rid="F2">2A</xref> shows the distribution of the regulatory overlap for different values of the percent identity (PID) of amino acid sequences of paralogous proteins. From this figure one can see that the regulatory overlap has a tendency to decrease as a function of PID. While multiple overlaps dominate the distribution for PID &#x02265; 80%, they gradually disappear at lower PIDs.</p><fig position="float" id="F1"><label>Figure 1</label><caption><p>The illustration of a concept of overlap in a molecular network. For a pair of paralogs the overlap &#x003a9; is defined as the number of their common neighbors in the network. In the case of a transcription network the (upstream) regulatory overlap &#x003a9;<sub><italic>reg </italic></sub>is given by the number of transcription factors regulating both paralogs, while for the physical interaction network the interaction overlap &#x003a9;<sub><italic>int </italic></sub>counts their common binding partners. The pair of paralogs used in this illustration has the overlap &#x003a9; = 2 out of the total of 5 distinct neighbors of the pair. That corresponds to a normalized overlap of 2/5 = 0.40.</p></caption><graphic xlink:href="1471-2148-4-9-1"/></fig><fig position="float" id="F2"><label>Figure 2</label><caption><p>Divergence of the upstream transcriptional regulation of duplicated genes in yeast. A) The distribution of the regulatory overlap &#x003a9;<sub><italic>reg </italic></sub>of paralo-gous proteins. The y-axis &#x02013; &#x003a9;<sub><italic>reg </italic></sub>&#x02013; is the number of transcription factors that cis-regulate <italic>both </italic>genes encoding a given pair of paralogous proteins. The x-axis is the percent identity (PID) of amino acid sequences of these two proteins. The colorbar shows the likelihood of finding a given &#x003a9;<sub><italic>reg </italic></sub>in a given 10% PID bin (note the logarithmic scale). The data describing the yeast regulatory network were taken from the whole genome chip-on-chip binding assay of 106 transcription factors [<xref ref-type="bibr" rid="B2">2</xref>], while the list of pairs of paralogous proteins was obtained by the whole genome blastp search (see Methods for more details.). B) The PID dependence of the average regulatory overlap &#x003a9;<sub><italic>reg </italic></sub>normalized by the total number of regulators of either one or the other paralog. Relative error bars are estimated by the inverse square root of the total number of shared regulators in a given PID bin. The solid line is the best fit to the exponential form: &#x003a9;<sub><italic>reg </italic></sub>~ exp(&#x003b3; PID) with &#x003b3; = 0.03 (3% change in &#x003a9;<sub><italic>reg </italic></sub>for every 1% change in PID.) The dashed horizontal line at 0.015 is a null-model expectation of the normalized overlap of two randomly selected proteins (not necessarily paralogs).</p></caption><graphic xlink:href="1471-2148-4-9-2"/></fig><p>Fig <xref ref-type="fig" rid="F2">2B</xref> shows the average value of the regulatory overlap as a function of PID. The regulatory overlap in this plot is normalized by a proxy to the ancestral connectivity of a gene, estimated as the total number of distinct transcription factors that are involved in regulation of at least one of the pair of proteins (see Fig <xref ref-type="fig" rid="F1">1</xref>). The correlation between the normalized regulatory overlap &#x003a9;<sub><italic>reg </italic></sub>and the PID is highly statistically significant: the Pearson correlation is 0.34 (P-value around 10<sup>-70 </sup>for 2275 data points). Even for the lowest value of PID = 20% the average &#x003a9;<sub><italic>reg </italic></sub>significantly exceeds its value in non-paralogous proteins. One interesting feature of the graph in Fig. <xref ref-type="fig" rid="F2">2B</xref> is that even pairs of proteins whose amino acid sequences are 100% identical to each other on average have only about 30% overlap in their upstream regulation. Such low regulatory overlap of recently duplicated genes can be partially attributed to false positives and false negatives present in the dataset of Ref. [<xref ref-type="bibr" rid="B2">2</xref>] (see Methods for extended discussion.) It might also be sometimes caused by an incomplete duplication of the upstream regulatory region of a gene, or by a burst of very rapid evolution of the regulatory region immediately following the duplication event. The second feature of the Fig. <xref ref-type="fig" rid="F2">2B</xref> is a gradual decline of the average regulatory overlap over the whole range of sequence similarities. The data in Fig. <xref ref-type="fig" rid="F2">2B</xref> can be fitted with an exponential decay with a rate corresponding to an average 3% loss of common regulators of a paralogous pair for every 1% decrease in their amino acid sequence identity. Thus already at PID = 80% about half of the common regulations present at PID = 100% are lost. The decline in the regulatory overlap at lower PIDs clearly visible in Fig. <xref ref-type="fig" rid="F2">2A,2B</xref> is in accord with a recently published analysis [<xref ref-type="bibr" rid="B11">11</xref>] of similarity between microarray profiles of paralogs. In fact, due to a more direct information about transcriptional regulation contained in the chip-on-chip dataset of Ref. [<xref ref-type="bibr" rid="B2">2</xref>] compared to microarray experiments, our analysis extends the gradual decline to much lower PID than was detected in Ref. [<xref ref-type="bibr" rid="B11">11</xref>]. After we submitted this manuscript another group of authors [<xref ref-type="bibr" rid="B12">12</xref>] has reported a rapid decline in the number of shared regulatory motifs of duplicated genes. This study, carried out as a function of a much faster silent substitution rate <italic>K</italic><sub><italic>s</italic></sub>, nicely complements our own findings. Indeed, in their analysis Papp <italic>et al</italic>. [<xref ref-type="bibr" rid="B12">12</xref>] logarithmically binned the <italic>K</italic><sub><italic>s </italic></sub>into four broad bins: below 0.01, 0.01&#x02013;0.1, 0.1&#x02013;1, and above 1. Since the reliability of the measured silent substitution rate dramatically decreases at high values of <italic>K</italic><sub><italic>s</italic></sub>, the whole long-time behavior (i.e. that for PID &#x0003c; 75% which in yeast roughly corresponds to <italic>K</italic><sub><italic>s </italic></sub>&#x0003e; 1) of the regulatory overlap remained inaccessible to the analysis of Ref. [<xref ref-type="bibr" rid="B12">12</xref>].</p></sec><sec><title>Divergence in downstream functional roles of duplicated genes in <italic>S. cerevisiae</italic></title><p>The rate of divergence between sets of <italic>upstream </italic>transcriptional regulators of paralogous proteins has an obvious <italic>downstream </italic>counterpart: it is the rate at which paralogous transcription factors loose their downstream targets. Unfortunately, an attempt to quantify this rate using the same dataset that we used above for the rate of upstream divergence would be limited to only 4 paralogous pairs formed by 106 transcriptional regulators studied in Ref. [<xref ref-type="bibr" rid="B2">2</xref>]. In general, relatively small number of paralogous transcription factors in any given species makes it difficult to go beyond just describing anecdotal cases in such an analysis. Thus in the remaining part of this study we concentrate on another measure of the downstream divergence, systematically comparing functional roles of duplicated (paralogous) proteins. The functional similarity of a pair of proteins is in part reflected in the "interaction overlap" &#x003a9;<sub><italic>int </italic></sub>given by the number of other proteins that physically interact with both of them (See Fig. <xref ref-type="fig" rid="F1">1</xref>). In our study we use the system-wide information about protein-protein physical interactions obtained by combining two high-throughput two-hybrid experiments [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>]. Fig <xref ref-type="fig" rid="F3">3A</xref> shows the average value of the interaction overlap &#x003a9;<sub><italic>int </italic></sub>between pairs of paralogous proteins as a function of PID &#x02013; their amino-acid similarity. Again &#x003a9;<sub><italic>int </italic></sub>typically decreases with decreasing PID, reflecting the gradual loss/change of binding partners of proteins in the course of evolution. A similar analysis, but as a function of the silent substitution rate (<italic>K</italic><sub><italic>s</italic></sub>) was previously reported by Wagner [<xref ref-type="bibr" rid="B13">13</xref>]. In agreement with that study, we find that paralogous proteins are more likely to share interaction partners than one expects by pure chance alone (see the caption to the Fig. <xref ref-type="fig" rid="F3">3</xref>). Our set of yeast paralogs contains 189 paralogous pairs such that both paralogs physically interact with at least one other protein in the combined dataset of Refs. [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>]. Out of these pairs 60 (30%) share at least one interaction partner. The correlation between the &#x003a9;<sub><italic>int </italic></sub>and the PID in the combined two-hybrid dataset is highly statistically significant: the Pearson correlation is 0.36 (P-value around 5 &#x000d7; 10<sup>-6 </sup>for 189 data points). We also find that in yeast the divergence in the set of binding partners becomes systematic only for PID &#x0003c; 70%, while above 70% it remains roughly constant in both Uetz [<xref ref-type="bibr" rid="B3">3</xref>], Ito [<xref ref-type="bibr" rid="B4">4</xref>], and combined datasets (Fig. <xref ref-type="fig" rid="F3">3A</xref>).</p><fig position="float" id="F3"><label>Figure 3</label><caption><p>Divergence of downstream functions of duplicated genes in the baker's yeast <italic>S. cerevisiae</italic>. A. The average value of the interaction overlap &#x003a9;<sub><italic>int </italic></sub>&#x02013; the number of physical interaction partners shared by a pair of paralo-gous proteins &#x02013; as a function of the similarity of their amino acid sequences. The physical interaction data are taken from the set of Uetz <italic>et al</italic>. [<xref ref-type="bibr" rid="B3">3</xref>] (open circles), the core dataset of Ito <italic>et al</italic>. [<xref ref-type="bibr" rid="B4">4</xref>] (diamonds), and the non-redundant combination of the two (filled circles). Note the apparent plateau for PID's between 70% and 100% in all three datasets. Solid lines are guides for the eye. A randomly selected (usually non-paralogous) pair of proteins in the combined dataset on average has &#x003a9;<sub><italic>int </italic></sub>around 8 &#x000d7; 10<sup>-3 </sup>(off-limits in this figure). All data points at all PIDs are significantly above this null-model value. B. The fraction of essential (lethal null-mutant) proteins among all proteins tested in Ref. [<xref ref-type="bibr" rid="B5">5</xref>] as a function of PID to their most similar paralog in the yeast genome. Proteins with no paralogs (singletons) are binned at 0% PID. Note the apparent plateau between 50% and 100% PID. The inset (note the change of scale on the y-axis) shows the fraction of essential proteins in the subset of all proteins known to be localized in the yeast nucleus [<xref ref-type="bibr" rid="B17">17</xref>]. Here the effect becomes even more pronounced so that all 18 nuclear proteins protected by a paralog with at least 50% similarity were found to be non-essential.</p></caption><graphic xlink:href="1471-2148-4-9-3"/></fig><p>An alternative way to quantify the extent of divergence/redundancy of duplicated genes is to examine phenotypes of of null-mutants lacking one of them. A systematic gene-deletion study in yeast [<xref ref-type="bibr" rid="B10">10</xref>] was recently used [<xref ref-type="bibr" rid="B14">14</xref>] to compare the fraction of essential genes (so that their null-mutants have lethal phenotype) between genes with and without paralogs in the genome. It was found that the fraction of essential genes is approximately 4 times higher among singleton genes than among ones protected by a highly similar paralog. It was also demonstrated that such protection by a paralog persists down to rather low levels of its amino-acid sequence similarity (PID) with the deleted protein. In Fig. <xref ref-type="fig" rid="F3">3B</xref> we confirm these findings using a more recent and larger systematic study [<xref ref-type="bibr" rid="B5">5</xref>] of viability of null-mutants in yeast as well as demonstrate that the magnitude of this protective effect is the strongest in the nucleus, where the largest fraction of essential proteins resides. Notice that the fraction of essential proteins (especially that of nuclear proteins) shows a dramatic increase as the PID to their closest paralog falls below 50%. Thus paralogous proteins with sequence similarity above 50% can typically substitute for each other.</p><p>Having presented different measures of upstream and downstream divergence of duplicated genes in yeast <italic>S. cerevisiae </italic>we are now in a position to discuss them in a wider context. Comparing Fig. <xref ref-type="fig" rid="F2">2B</xref> to Figs <xref ref-type="fig" rid="F3">3A,3B</xref> one concludes that changes in the upstream regulation of duplicated genes happen more readily than changes in their downstream function. The overlap in the set of binding partners (Fig. <xref ref-type="fig" rid="F3">3A</xref>) and the ability of duplicates to substitute for each other (Fig. <xref ref-type="fig" rid="F3">3B</xref>) remain virtually constant down to PID of 70%, at which point their average regulatory overlap has dropped to about 40% of its maximum (Fig. <xref ref-type="fig" rid="F2">2B</xref>). To summarize: our results indicate that duplicated genes would still have the ability to partially substitute for downstream functions of each other even at the time when the repertoire of their regulatory connections has already substantially changed from its ancestral state before the duplication. Such genes would be less constrained in evolving new functions [<xref ref-type="bibr" rid="B15">15</xref>], and thus would contribute to a greater evolutionary plasticity of the network.</p></sec><sec><title>Functional redundancy of paralogous proteins from RNAi experiments on <italic>C. elegans</italic></title><p>One might expect the protective effect of paralogs to be unique to single-celled organisms such as yeast. Indeed, in multicellular organisms duplicated proteins are often expressed only in specific tissues and therefore unable to substitute for each other. However, using a systematic study of RNAi (RNA Interference) phenotypes in a nematode worm <italic>C. elegans </italic>[<xref ref-type="bibr" rid="B8">8</xref>] we found such protection [<xref ref-type="bibr" rid="B16">16</xref>] to be equally strong in this multicellular organism (See Fig. <xref ref-type="fig" rid="F4">4</xref>). As in Fig. <xref ref-type="fig" rid="F3">3B</xref>, the x-axis in Fig. <xref ref-type="fig" rid="F4">4</xref> is PID &#x02013; the similarity of amino acid sequences between a given protein and its closest related paralog (all singleton proteins without paralogs are clumped into the 0% PID bin). The y-axis is the fraction of tested proteins whose elimination by the RNAi technique was found [<xref ref-type="bibr" rid="B8">8</xref>] to give rise to a nonviable phenotype (embryonic or larval lethality or sterility). In worm the protection of having a paralog starts to gradually weaken for PID &#x0003c; 70%. In both worm and yeast there seems to be a four-fold drop in the fraction of essential proteins between PID = 0% and 100%.</p><fig position="float" id="F4"><label>Figure 4</label><caption><p>Protective effect of paralogs in a nematode worm <italic>C. elegans</italic>. The fraction of essential (non-viable RNAi phenotype [<xref ref-type="bibr" rid="B8">8</xref>]) proteins among all tested worm proteins as a function of PID to their most similar paralog in the worm genome. Note the apparent plateau between 70% and 100% PID. The plot in the inset shows the fraction of essential proteins among all RNAis tested in Ref. [<xref ref-type="bibr" rid="B8">8</xref>], while that in the main panel drops RNAis that are predicted [<xref ref-type="bibr" rid="B8">8</xref>] to target mRNA products of more than one gene. Note that while the graph in the main panel is qualitatively similar to that in Fig. <xref ref-type="fig" rid="F3">3B</xref>, in the inset the fraction of essential proteins at PID = 100% rises to its level for singleton proteins. Thus when mRNAs of highly similar paralogs are eliminated along with the targeted mRNA, the protective effect of paralogs totally disappears.</p></caption><graphic xlink:href="1471-2148-4-9-4"/></fig><p>In the inset to Fig. <xref ref-type="fig" rid="F4">4</xref> we kept all successfully cloned genepairs, while in the main panel we dropped those genepairs whose product was predicted [<xref ref-type="bibr" rid="B8">8</xref>] to target mRNA product of more than one gene in the genome (see Methods for more details). It is instructive that the fraction of essential genes as a function of PID shown in the inset to Fig. <xref ref-type="fig" rid="F4">4</xref> has a well pronounced minimum around PID = 70% and then subsequently starts to rise for higher values of PID. The tentative explanation for this behavior is that unlike single-gene deletion technique used in yeast, the RNAi technique is based on RNA complementarity and can eliminate several different mRNAs with similar sequences. Therefore, paralogous genes with nearly identical DNA sequences prove to be useless from the point of view of protection against RNAi since their mRNA products would be eliminated at nearly the same rate as the intended targets. This neatly explains why in the inset to Fig. <xref ref-type="fig" rid="F4">4</xref> the fraction of nonviable phenotypes for genes with a 100% identical paralog in the genome approaches that of unprotected genes without paralogous partners (keep in mind that in this plot we use amino acid sequence identity of proteins and not of their mRNA precursors.) This observation also reinforces the point of view that the decline in the fraction of essential genes vs PID shown in Figs <xref ref-type="fig" rid="F3">3B</xref>,<xref ref-type="fig" rid="F4">4</xref> is indeed caused by protective effects of paralogs and cannot be explained by a possible tendency of nonessential genes to duplicate more frequently.</p></sec><sec><title>Divergence of physical interactions of paralogous genes in <italic>H. pylori </italic>and <italic>D. melanogaster</italic></title><p>The analysis of evolution of molecular networks advocated in this paper requires a <italic>large </italic>(preferably genome-wide) and <italic>unbiased </italic>(i.e. no anthropogenic selection present in databases) dataset describing a molecular network in a given species. Apart from yeast, which is arguably the best studied model organism, system-wide two-hybrid physical interaction assays were published for a simple bacterium <italic>Helicobacter pylori </italic>[<xref ref-type="bibr" rid="B6">6</xref>], and a fly <italic>Drosophila melanogaster </italic>[<xref ref-type="bibr" rid="B7">7</xref>]. In Fig. <xref ref-type="fig" rid="F5">5</xref> we used these two datasets to quantify the decay of the average interaction overlap as a function of amino-acid sequence similarly (see Fig. <xref ref-type="fig" rid="F3">3A</xref> for the same analysis in yeast.) The correlation between &#x003a9;<sub><italic>int </italic></sub>and PID is highly statistically significant in both cases: the Pearson correlation of 0.43 (P-value around 3 &#x000d7; 10<sup>-4 </sup>for 65 data points) for <italic>H. pylori</italic>, and 0.19 (P-value around 10<sup>-26 </sup>for 2843 data points) in <italic>D. melanogaster</italic>. Our basic conclusions agree for all quite diverse organisms used in this study: paralogous proteins are much more likely to share binding partners than expected by pure chance alone. Furthermore, the number of common interaction partners goes down as PID of their amino acid sequences decreases. In the yeast and <italic>H. pylori </italic>we see the evidence of an initial plateau at which the average overlap appears to be independent of PID. On the other hand in the fly there is no evidence of such plateau, which makes the average rate of loss of common binding partners (about 4.5% for every 1% of change in PID) quite high in this organism. However, in the absence of system-wide data on transcription factors' binding in the fly and <italic>H. pylori </italic>we could not quantify rates of upstream changes in these two organisms, and consequently cannot compare them to the corresponding downstream rates.</p><fig position="float" id="F5"><label>Figure 5</label><caption><p>Divergence of physical interaction neighborhoods of duplicated genes in a bacterium <italic>H. pylori </italic>and a fly <italic>D. melanogaster</italic>. The average value of the interaction overlap &#x003a9;<sub><italic>int </italic></sub>of paralogous proteins in H. pylori (A) and D. melanogaster (B) as a function of the amino acid sequence similarity. The physical interaction data are taken from Ref. [<xref ref-type="bibr" rid="B6">6</xref>] for <italic>H. pylori </italic>(A) and from Ref. [<xref ref-type="bibr" rid="B7">7</xref>] for <italic>D. melanogaster</italic>. Note the apparent plateau for PID's between 50% and 100% in panel A and its absence in panel B. Dashed horizontal lines show the average interaction overlap of a random (usually non-paralogous) pair of proteins. The solid line in B is the best fit to the exponential form: &#x003a9;<sub><italic>int </italic></sub>~ exp(&#x003b3;<sub><italic>FLY</italic></sub><italic>PID</italic>) with &#x003b3;<sub><italic>fly </italic></sub>= 0.045.</p></caption><graphic xlink:href="1471-2148-4-9-5"/></fig></sec></sec><sec><title>Conclusions</title><p>The evolution of a biological organism modifies it on multiple levels ranging from sequences of individual molecules, to their coordinated activity in the cell (molecular networks), all the way up to the phenotype of the organism itself. While its manifestations both on the level of protein sequences and phenotypes are reasonably well documented, the data needed to quantify evolutionary changes taking place on the level of molecular networks have appeared only very recently. Systematic experiments such as high-throughput two hybrid assays of protein-protein interactions [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>], chip-on-chip studies of whole-genome binding of a large number of transcription factors [<xref ref-type="bibr" rid="B2">2</xref>], and whole-genome assays of inactivations of single genes [<xref ref-type="bibr" rid="B5">5</xref>] or proteins [<xref ref-type="bibr" rid="B8">8</xref>] allowed us to go beyond describing particular cases of evolution of molecular networks and look at its large scale dynamics.</p><p>For all molecular networks studied in this work we found that even the most distantly related paralogous proteins with amino acid sequence identities around 20% on average have more similar positions within a network than a randomly selected pair of proteins. That means that some pairs of paralogous proteins at least partially retain their functional redundancy for extremely long time after the duplication event.</p><p>Our results also indicate that the genetic regulation of paralogous proteins changes faster than both their amino acid sequences and the set of their protein interactions partners. It is tempting to extend this observation to pairs of homologous proteins in different species (orthologs) that diverged from each other as a result of a speciation (as opposed to a gene duplication) event. This would help to explain how species with very similar gene contents can evolve novel properties on a relatively short timescale. However, such an inter-species comparison of molecular networks has to wait for the appearance of whole-genome data on molecular networks in closely related model organisms.</p></sec><sec sec-type="methods"><title>Methods</title><p>As a source of information about yeast duplicated genes we use the dataset consisting of 3909 pairs of paralogous yeast proteins. This set was obtained by blasting all yeast proteins against each other with a conservative E-value cutoff of 10<sup>-10 </sup>and leaving only pairs in which the aligned region constituted at least 80% of the length of a longer protein. This prevented the appearance of pairs of multidomain proteins paralogous over only one of their domains. We further curated this dataset by removing 72 known [<xref ref-type="bibr" rid="B17">17</xref>] transposable elements and all their paralogs (108 proteins all together). That left us with 2299 paralogous pairs formed by 1596 yeast proteins (about 25% of the genome). These pairs are characterized by a broad and relatively uniform distribution of the percent identity (PID) of amino acid sequences ranging from 20% to 100% (See Fig. <xref ref-type="fig" rid="F6">6</xref>) The histogram in the Fig. <xref ref-type="fig" rid="F6">6</xref> is binned at 5% PID (as the data used to plot the Fig. <xref ref-type="fig" rid="F2">2B</xref>), and one can see that even in the least represented bins there are over 40 paralogous pairs providing sufficient statistics for our analysis. Our set of all possible pairs of paralogous proteins contains some redundant information especially for large protein families. Indeed, a family of, say, 4 proteins would contribute (4&#x000b7;3)/2 = 6 paralogous pairs to our analysis, while it contains at most 3 true duplicated pairs. However, in the situation where the data describing molecular networks are incomplete and noisy such redundancy is rather beneficial by providing better statistics. We have verified that apart from somewhat larger errorbars all our quantitative findings remained virtually unchanged when we repeated our analysis of upstream regulations in yeast using only 938 pairs of putative duplicated proteins. These pairs were obtained from the full set of 2299 paralogous pairs by the detailed phylogenetic analysis of individual families. It is also worthwhile to note that while the average number <italic>K</italic><sub><italic>s </italic></sub>of silent substitutions per substitution site in a pair of duplicated genes is commonly used as a proxy of the time elapsed since the duplication event [<xref ref-type="bibr" rid="B1">1</xref>], the PID (or <italic>K</italic><sub><italic>a </italic></sub>&#x02013; the number of non-silent substitutions per site &#x02013; related to PID via PID = 100 exp(-2<italic>K</italic><sub><italic>a</italic></sub>)) is rather a crude estimate of the extent of their functional similarity. Hence, our analysis emphasizes function-dependent rather than time-dependent divergence between paralogous proteins.</p><fig position="float" id="F6"><label>Figure 6</label><caption><p>The histogram of amino acid sequence identities (PID) of 2299 pairs of paralogous yeast proteins used in our study.</p></caption><graphic xlink:href="1471-2148-4-9-6"/></fig><p>The system-wide data describing the transcription regulatory network of yeast was taken from the Ref. [<xref ref-type="bibr" rid="B2">2</xref>], which reports the so-called "chip-on-chip" study of in-vivo binding of 106 transcription factors to upstream regulatory regions of genes encoding all 6270 of yeast proteins. Since the number of transcriptional regulators in this dataset is quite large, the probability that by pure chance the same transcription factor would be incorrectly detected among upstream regulators of <italic>both </italic>duplicated genes is small (of order of 1%). Thus the contribution of false positives of the dataset of Ref. [<xref ref-type="bibr" rid="B2">2</xref>] to the regulatory overlap &#x003a9;<sub><italic>reg </italic></sub>is quite insignificant. This allowed us to use a P-value cutoff equal to 10<sup>-2 </sup>(12854 regulations) less conservative than the 10<sup>-3 </sup>cutoff (4418 regulations) of Lee <italic>et al</italic>. [<xref ref-type="bibr" rid="B2">2</xref>]. On the other hand, false positives (if present in the data) could significantly affect the average number of regulatory inputs of individual proteins used to normalize the regulatory overlap in Fig. <xref ref-type="fig" rid="F2">2B</xref>. However, we found that both the initial drop and the rate of exponential decay of the <italic>normalized </italic>regulatory remains virtually unchanged when Fig. <xref ref-type="fig" rid="F2">2B</xref> is repeated for different values of the P-value cutoff ranging from 10<sup>-2 </sup>to 10<sup>-4 </sup>(data not shown). In the same range of P-values the average number of regulations per gene changes six-fold (from 2 to 0.33)! This suggests that false positives are not a significant part of the experimental dataset of Ref. [<xref ref-type="bibr" rid="B2">2</xref>] at least up to 10<sup>-2</sup>, and validates the robust nature of parameters extracted from the Fig. <xref ref-type="fig" rid="F2">2B</xref>. In the analysis shown in Fig. <xref ref-type="fig" rid="F2">2</xref> we have dropped 3 paralogous pairs sharing the same intergenic sequence since by design of the chip-on-chip experiment [<xref ref-type="bibr" rid="B2">2</xref>] such pairs would have 100% regulatory overlap. We also checked that Fig. <xref ref-type="fig" rid="F2">2A</xref> does not change significantly if one limits the analysis to genes without diverging promoters ensuring that a given intergenic could possibly regulate only one gene.</p><p>As a source of information about binding partners of yeast proteins we combined the data from two independent high-throughput two-hybrid experiments: the core dataset of Ito <italic>et al</italic>. [<xref ref-type="bibr" rid="B4">4</xref>] (806 interactions among 797 proteins) and the extended Uetz <italic>et al</italic>. dataset [<xref ref-type="bibr" rid="B3">3</xref>], downloaded from the website of this group (1446 interactions among 1340 proteins). The resulting network consists of 1734 proteins joined by 2111 non-redundant interactions. Using this combined dataset we found that even 100% identical proteins share on average only 30% of their binding partners. However, unlike for upstream regulation, the set of interaction partners of a protein is fully determined by its amino acid sequence. Therefore, an imperfect overlap in the set of binding partners of identical proteins has to be attributed to false positives/negatives inevitably present in high-throughput two-hybrid experiments. The relatively high rate of false negatives in genome-wide two-hybrid experiments is further corroborated by the fact that datasets used in our study coming from two independent experiments [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>] have only 141 interactions in common. The abundance of missing interactions makes the normalization of the interaction overlap impractical. That was the reason why unlike in Fig. <xref ref-type="fig" rid="F2">2B</xref> in Fig. <xref ref-type="fig" rid="F3">3A</xref> we used the raw (unnormalized) interaction overlap. To make sure that differences between Figs. <xref ref-type="fig" rid="F2">2B</xref> and <xref ref-type="fig" rid="F3">3A</xref> are not caused by differences in normalization we repeated them using various normalization schemes as well as altogether unnormalized (data not shown). We found that apart from the overall scale of the y-axis, changes in normalization do not affect exponential decay parameters of Figs <xref ref-type="fig" rid="F2">2B</xref>,<xref ref-type="fig" rid="F3">3A</xref>.</p><p>The system-wide data on viability of <italic>S. cerevisiae </italic>null-mutants used in our study was obtained from Ref. [<xref ref-type="bibr" rid="B5">5</xref>] in which 1103 essential (non-viable null-mutants) and 4678 non-essential (viable null-mutants) yeast proteins were reported. The lists of viable and non-viable null-mutants as discovered in Ref. [<xref ref-type="bibr" rid="B5">5</xref>] were downloaded from the Saccharomyces Genome Database [<xref ref-type="bibr" rid="B17">17</xref>].</p><p>Our analysis of protective effects of paralogs in <italic>C. elegans </italic>is based on the set of 15587 viable and 1170 non-viable (embryonic or larval lethality or sterility) RNAi phenotypes reported in [<xref ref-type="bibr" rid="B8">8</xref>]. The information about worm paralogs is obtained from the EuGenes database [<xref ref-type="bibr" rid="B18">18</xref>] and consists of 30036 paralogous pairs involving 10071 worm proteins (blastp with 10<sup>-30 </sup>cutoff and no requirements on the length of aligned region). In Fig. <xref ref-type="fig" rid="F4">4</xref> we used 13884 RNAi phenotypes for which we were able to uniquely map the genepair name to the worm protein name used in EuGenes.</p><p>The two-hybrid assay of protein-protein inetractions in <italic>H. pylori </italic>[<xref ref-type="bibr" rid="B6">6</xref>] used in Fig. <xref ref-type="fig" rid="F5">5A</xref> contains 1465 interactions between 732 proteins, while there are only 260 paralogous pairs involving 140 proteins. As in yeast this set was obtained by blasting all protein sequences found in the fully sequenced genome against each other with a conservative E-value cutoff of 10<sup>-10 </sup>and leaving only pairs in which the aligned region constituted at least 80% of the length of a longer protein.</p><p>Finally, our analysis of the interaction overlap between paralogous proteins in <italic>D. melanogaster </italic>is based on the full dataset of the high-throughput two-hybrid experiment [<xref ref-type="bibr" rid="B7">7</xref>]. It consists of 20671 protein-protein physical interactions involving 7002 of fly proteins obtained in. To generate Fig. <xref ref-type="fig" rid="F5">5B</xref> we also used the set of 16713 paralogous pairs involving 2827 fly proteins.</p></sec><sec><title>Authors Contributions</title><p>SM, KS, and KAE contributed to both the ideas and writing of the manuscript in close collaboration. Koon-Kiu Yan has generated blastp datasets for <italic>H. pylori</italic>, yeast, and fly, as well as performed the analysis of RNAi experiment shown in Fig. <xref ref-type="fig" rid="F4">4</xref>. All authors read and approved the manuscript.</p></sec>
A prospective longitudinal <italic>in vivo </italic><sup>1</sup>H MR spectroscopy study of the SIV/macaque model of neuroAIDS
<sec><title>Background</title><p>The neurological complications of HIV infection remain poorly understood. Clinically, <italic>in vivo </italic><sup>1</sup>H magnetic resonance spectroscopy (MRS) demonstrates brain injury caused by HIV infection even when the MRI is normal. Our goal was to undertsand the dynamics of cerebral injury by performing a longitudinal <italic>in vivo </italic><sup>1</sup>H MRS study of the SIV/macaque model of neuroAIDS.</p></sec><sec><title>Results</title><p>Eight rhesus macaques were infected with SIVmac251 and serially imaged with MRI and <sup>1</sup>H MRS to terminal AIDS or the endpoint of 2 years. During acute infection, there were stereotypical brain MRS changes, dominated by a significant elevation of the Cho/Cr ratio in the frontal cortex. Subsequently, brain metabolic patterns diverged between animals. There was an elevation of basal ganglia Cho/Cr four weeks post-inoculation in 2 animals that developed SIV encephalitis (p = 0.022). Metabolite ratios averaged across all 8 animals were not significantly different from baseline at any time point after 2 weeks post inoculation. However, linear regression analysis on all 8 animals revealed a positive correlation between a change in frontal lobe Cho/Cr and plasma viral load (P &#x0003c; 0.001, R = 0.80), and a negative correlation between NAA/Cr in the basal ganglia and the plasma viral load (P &#x0003c; 0.02, R = -0.73). No MRI abnormalities were detected at any time.</p></sec><sec><title>Conclusions</title><p>After infection with SIV, macaque brain metabolism changes in a complex manner that is dependent on brain region, host factors and viral load. An elevation of basal ganglia Cho/Cr 4 weeks after SIV infection may be marker of a propensity to develop SIV encephalitis. Elevations of Cho/Cr, often observed in CNS inflammation, were associated with increased plasma viral load during acute and chronic infection. Evidence of neuronal injury in the basal ganglia was associated with increased plasma viral load in the chronic stage of infection. These observations support the use of drugs capable of controlling the viral replication and trafficking of virus into the CNS, and may help explain the reduction in incidence of HIV-associated dementia in the era of HAART despite the inability of most of those drugs to effectively enter the CNS.</p></sec>
<contrib id="A1" contrib-type="author"><name><surname>Fuller</surname><given-names>Robert A</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Westmoreland</surname><given-names>Susan V</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Ratai</surname><given-names>Eva</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Greco</surname><given-names>Jane B</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A5" contrib-type="author"><name><surname>Kim</surname><given-names>John P</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A6" contrib-type="author"><name><surname>Lentz</surname><given-names>Margaret R</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A7" contrib-type="author"><name><surname>He</surname><given-names>Julian</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A8" contrib-type="author"><name><surname>Sehgal</surname><given-names>Prabhat K</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A9" contrib-type="author"><name><surname>Masliah</surname><given-names>Eliezer</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A10" contrib-type="author"><name><surname>Halpern</surname><given-names>Elkan</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A11" contrib-type="author"><name><surname>Lackner</surname><given-names>Andrew A</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib><contrib id="A12" corresp="yes" contrib-type="author"><name><surname>Gonz&#x000e1;lez</surname><given-names>R Gilberto</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib>
BMC Neuroscience
<sec><title>Background</title><p>The pathogenesis of brain injury in HIV infection remains incompletely understood. Elucidation of the pathogenesis of HIV brain injury is confounded by major uncertainties in humans, such as time of infection, variable treatments, and lack of access to brain tissue for evaluation. Neuroimaging has helped in understanding HIV related brain injury, and in recent years <italic>in vivo </italic><sup>1</sup>H MR spectroscopy has emerged as amongst the most informative method [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B11">11</xref>]. Brain MRS abnormalities in HIV have been widely reported, but studies have been limited to the chronic stages of HIV infection. To our knowledge, only one longitudinal MRS study of untreated, chronically HIV infected subjects have been reported [<xref ref-type="bibr" rid="B12">12</xref>]. Brain abnormalities that may occur during the acute and subacute stages as well as their relationship to chronic HIV infection are unknown.</p><p>The SIV-infected macaque is an excellent animal model for studying the neuropathogenesis of HIV-related brain injury [<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>], and <sup>1</sup>H MRS may be used repeatedly in the same animal to follow brain abnormalities from early to late stages. <italic>In vivo </italic>macaque brain <sup>1</sup>H MR spectra are similar to humans [<xref ref-type="bibr" rid="B14">14</xref>], and <italic>post mortem </italic>MRS studies from SIV-infected macaques have revealed metabolic abnormalities similar to those observed in chronically HIV-infected human brains by <italic>in vivo </italic>MRS [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. Significantly, cerebral injury is observed by <italic>in vivo </italic><sup>1</sup>H MRS [<xref ref-type="bibr" rid="B17">17</xref>] as well as by <italic>in vitro </italic><sup>1</sup>H MRS and neuropathology shortly after SIV infection [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. It is not known whether full recovery occurs, or whether these cerebral abnormalities persist into the subacute and chronic periods. One puzzle is that while virtually all SIV-infected macaques demonstrate cerebral injury acutely, fewer than a third ultimately develop SIV encephalitis (SIVE) [<xref ref-type="bibr" rid="B18">18</xref>].</p><p>Our previous observations of early brain injury in the SIV-infected macaque led to the investigation reported here [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. Our previous MRS studies were performed only on <italic>post mortem </italic>frontal cortex samples using solution <sup>1</sup>H MRS of brain extracts [<xref ref-type="bibr" rid="B15">15</xref>] or high resolution magic angle spinning <sup>1</sup>H MRS and neurohistochemistry of brain tissue samples [<xref ref-type="bibr" rid="B16">16</xref>]. Those studies revealed evidence of frontal cortex neuronal injury with declines of the MRS neuronal marker N-acetylaspartate (NAA) [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>], and the neurohistochemcial neuronal markers synaptophysin and calbindin. [<xref ref-type="bibr" rid="B16">16</xref>] An important limitation of this previous work was that the brain samples were only from animals that had been euthanized within 2 weeks of infection, or at terminal AIDS. Thus, the events that may occur in the brain and are detectable by MRS between the very early and late periods of infection were unknown, and form, in part, the rationale for the present study design. Another issue arising from our previous in vitro studies was that we failed to detect expected increases in the choline-containing compounds (Cho) and myo-inositol (MI) that are commonly seen HIV infected patients [<xref ref-type="bibr" rid="B5">5</xref>-<xref ref-type="bibr" rid="B11">11</xref>].</p><p>In the study that we report here, we sought to understand whether brain injury in the SIV-infected macaque is detectable by <italic>in vivo </italic><sup>1</sup>H MRS in the subacute and chronic periods after infection, whether there are metabolic brain markers that predict the development of SIV encephalitis, and whether there are relationships between traditional clinical markers and brain MRS abnormalities. In a cohort of 8 macaques, we noninvasively evaluated the effect of SIVmac251 infection on the macaque brain by MRI and <sup>1</sup>H MRS in a prospective, longitudinal manner.</p></sec><sec><title>Results</title><p>The longitudinal <italic>in vivo </italic><sup>1</sup>H MRS study that we report here is an extension of a study in which 15 rhesus macaques were inoculated intravenously with SIVmac251 and imaged during the first month of infection [<xref ref-type="bibr" rid="B17">17</xref>]. Seven of the animals were euthanized within the first month of infection, and the remaining 8 macaques were followed longitudinally until euthanized because of terminal AIDS or the two-year study endpoint. We define 3 time periods with respect to infection: acute (within a month), subacute (between 1 and 3 months), chronic periods (beyond 3 months). Details of animal age, survival and pathological findings are summarized in Table <xref ref-type="table" rid="T1">1</xref>. Two of the 8 animals developed SIV encephalitis (SIVE) as defined by the presence of perivascular infiltrates of macrophages and multinucleated giant cells. Four of the 8 animals where terminated before 2 years after SIV inoculation due to terminal AIDS. Two of the 4 early-terminated animals were the ones that also had SIVE. The other 2 animals that were terminated early had evidence of mild CNS pathology, but not SIVE. Four macaques survived to the designated two-year end point, and none displayed CNS pathology. Obvious neurological deficits such as paralysis or seizures were not observed in any of the animals by the veterinarians caring for the animals. Formal neurological examinations were not performed.</p><p>Serial MR imaging and spectroscopy studies were performed before and after SIV inoculation on a clinical MR instrument (1.5 T). A total of 57 scans were performed on the cohort. The number of scans that were performed in an individual animal varied from 4 to 10 scans, depending on time of survival. A neuroradiologist reviewed all brain MR images. No brain MRI abnormalities such as masses, abscesses, fluid collections, hemorrhage, white matter signal abnormalities or infarcts were detected in any animal before or at any time after SIV infection. <italic>In vivo </italic><sup>1</sup>H MR spectroscopy was performed using a protocol that has been validated for multicenter studies of HIV infected patients [<xref ref-type="bibr" rid="B11">11</xref>]. We have previously reported the details of the normal macaque brain MR spectrum that is very similar to the human brain MR spectrum [<xref ref-type="bibr" rid="B14">14</xref>]. An axial T2 weighted MR image demonstrating frontal lobe voxel placement, and an MR spectrum from that location is shown in figure <xref ref-type="fig" rid="F1">1</xref>. The macaque brain MR spectrum demonstrates high quality in terms of spectral resolution and signal-to-noise ratio, and is similar to human MRS data obtained using the same protocol [<xref ref-type="bibr" rid="B11">11</xref>].</p><p>We examined changes in the brain in MRS resonances commonly designated as N-acetylaspartate (NAA), choline (Cho), myoinositol (MI) and creatine (Cr). MR spectra were obtained from the frontal cortex, centrum semiovale and basal ganglia, before infection and at multiple time points after infection. The time points include 2 weeks, 4 weeks, 3 months, 13 months, 15 months, 17 months, 19 months and 24 months post inoculation (pi). The average values from all animals imaged at each time point for the major metabolite ratios from each anatomic location acquired across the duration of the study are displayed in figures <xref ref-type="fig" rid="F2">2</xref>, <xref ref-type="fig" rid="F3">3</xref> and <xref ref-type="fig" rid="F4">4</xref>. While each animal was scanned for up to 2 years, the study took place over 3 years. Instrumental stability was confirmed through analysis of MRS data from a phantom containing NAA, Cr, Cho and MI acquired the same day as the animal studies. Across the 3 years of the study, the standard deviation of the phantom Cho/Cr was 2.7%, the NAA/Cr standard deviation was 3.0%, and the MI/Cr standard deviation was 5.8%. Regression analysis demonstrated a slope of &#x0003c;0.001% for each metabolite ratio versus time, i.e., there was no detectable change over time in phantom metabolite ratios. These data confirm instrumental stability across the 3 year duration of the study.</p><p>We found in this cohort a significant increase in frontal lobe Cho/Cr 2 weeks post inoculation (p &#x0003c; 0.05), a time coincident with viral neuroinvasion and increased numbers of perivascular macrophages [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B20">20</xref>]. These changes were also observed in the entire 15 animal cohort of the acute SIV infection study [<xref ref-type="bibr" rid="B17">17</xref>]. This was followed by a decrease to below baseline at four weeks post inoculation (p &#x0003c; 0.01) as shown in figure <xref ref-type="fig" rid="F2">2</xref>. Subsequent to the acute period, some variation in Cho/Cr with time is apparent in figure <xref ref-type="fig" rid="F2">2</xref>, but we found no statistically significant difference between pre-infection mean values and any other post infection time point mean values in any of the brain regions. We did not detect statistically significant differences in NAA/Cr (figure <xref ref-type="fig" rid="F3">3</xref>) and MI/Cr (figure <xref ref-type="fig" rid="F4">4</xref>) in any of the brain regions under investigation after SIV infection compared to pre-infection values at any time point. In the 15 animal cohort studied during the first month after SIV infection, we detected statistically significant transient decline in NAA/Cr and an elevation in MI/Cr in the frontal cortex. [<xref ref-type="bibr" rid="B17">17</xref>] Restricting data analysis to the 8 animals followed longitudinally did not provide sufficient statistical power to detect significant changes in NAA/Cr or MI/Cr.</p><p>A major goal of this study was to investigate whether there are metabolic brain markers that predict the development of SIV encephalitis. We found that there was a significant difference in the Cho/Cr ratio in the basal ganglia (p = 0.022) between encephalitic and non-encephalitic animals at four weeks post-inoculation as shown in figure <xref ref-type="fig" rid="F5">5</xref>. In both groups of animals there was an initial rise in Cho/Cr within 2 weeks of infection, but the elevated levels persisted at 4 weeks only in those animals that eventually developed encephalitis. In all other animals, the Cho/Cr ratio dropped substantially after the period of peak viremia (11&#x02013;13 dpi). After 3 months, however, the Cho/Cr values of the encephalitic animals were indistinguishable from those of the non-encephalitic SIV-infected macaques.</p><p>A further goal in this prospective study was to evaluate relationships between changes in blood/plasma measurements and brain metabolite changes. For this evaluation, only blood/plasma samples obtained within 24 hours of scanning were considered. The relationship between a change in frontal Cho/Cr and plasma viral load is shown in figure <xref ref-type="fig" rid="F6">6a</xref>. We observed a significant correlation between these parameters (p &#x0003c; 0.001, r = 0.80). Evaluating MRS data collected after the acute phase (&#x0003e;30 days post-inoculation), a significant negative correlation was found between the change in basal ganglia NAA/Cr and the plasma viral load (p &#x0003c; 0.02, r = -0.73). This relationship is illustrated in figure <xref ref-type="fig" rid="F6">6b</xref>.</p><p>We did not detect other correlations between brain metabolite ratios and other blood/plasma measurements. In particular, we did not find a correlation between lymphocyte subsets (CD4+, CD8+) and brain MRS measurements. However, we did observe substantial variations in the levels of these lymphocyte subsets over time in the individual macaques. The most notable changes were declines in CD4+ lymphocytes to very low levels in the 2 animals that developed SIVE.</p></sec><sec><title>Discussion</title><p>This is the first longitudinal neuroimaging study of human or nonhuman primates that begins before infection by an immunodeficiency virus, and continues through to the development of encephalitis. In a cohort of 8 rhesus macaques, we serially performed <italic>in vivo </italic><sup>1</sup>H MR spectroscopy and MRI with the goals of demonstrating whether brain injury in the SIV-infected macaque is detectable noninvasively, whether there are metabolic brain markers that predict the development of SIV encephalitis, and whether there are relationships between traditional clinical markers and brain <sup>1</sup>H MRS abnormalities. While MRI appeared normal, novel metabolic abnormalities were observed, and they provide new insights into the response of the CNS to SIV infection that are relevant to HIV infection in humans.</p><p>The <sup>1</sup>H MRS changes seen shortly after infection were expected. Previous work from our laboratory documented similar findings by MRS both <italic>in vivo </italic>[<xref ref-type="bibr" rid="B17">17</xref>] and in <italic>post mortem </italic>studies [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. Our observations during the subacute and chronic stages of SIV infection are new. Our most interesting finding was an elevation of Cho/Cr in the basal ganglia four weeks post-inoculation in the 2 animals destined to develop SIVE, whereas the other 6 animals had basal ganglia Cho/Cr values below baseline (figure <xref ref-type="fig" rid="F5">5</xref>).</p><p>SIV-induced neuropathological changes are variable with SIV encephalitis developing in 25% of infected macaques [<xref ref-type="bibr" rid="B18">18</xref>], similar to the incidence of encephalitis in HIV-infected humans, and the rate of SIVE in the present study. It is not clear why there is a spectrum of individual responses to infection with the same virus. The most likely explanations include viral mutation and the differences in host factors. Throughout the course of infection there is a high rate of mutation [<xref ref-type="bibr" rid="B21">21</xref>-<xref ref-type="bibr" rid="B24">24</xref>]. Our data supports an important role for host factors; at 4 weeks pi, when minimal viral mutation is expected, we found a clear difference in the basal ganglia Cho/Cr ratio between those macaques destined to develop SIVE and the others.</p><p>We suggest that the observation of persistent basal ganglia Cho/Cr elevation during the first month after infection is a marker of host susceptibility to SIVE. If confirmed, it may have important implications for design of further research, and potentially in the clinical management of patients. To our knowledge, the only other biomarker with the potential to predict encephalitis is elevated levels of macrophage chemoattractant protein-1 (MCP-1) in the CSF for development of SIVE [<xref ref-type="bibr" rid="B25">25</xref>]. However, the MCP-1 finding was observed in an accelerated SIVE model that employs 2 viruses, results in a high rate of SIVE, and is quite different from the human situation. If persistence of elevated cerebral Cho/Cr is found to also be a predictor of a neurocognitive disorder in HIV-infected patients it could become an important clinical tool. For example, it may be used to identify individuals that may benefit from early, CNS-targeted or more aggressive administration of antiviral, anti-inflammatory, and neuroprotective agents.</p><p>Prior studies have demonstrated viral neuroinvasion in the weeks following infection [<xref ref-type="bibr" rid="B20">20</xref>,<xref ref-type="bibr" rid="B26">26</xref>]. Brain abnormalities have been documented by <italic>in vivo </italic><sup>1</sup>H MRS [<xref ref-type="bibr" rid="B17">17</xref>], <italic>in vitro </italic><sup>1</sup>H MRS [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>], and neuropathology [<xref ref-type="bibr" rid="B16">16</xref>] in post-mortem tissues obtained within 2 weeks of infection. In the present study, we again observed abnormalities <italic>in vivo</italic>, specifically, elevation of frontal lobe Cho/Cr at the time of peak viremia (2 weeks pi), and a diminution of this ratio to below baseline at 4 weeks pi, after the immune system responded to the virus. An elevation of Cho/Cr is often observed in CNS inflammatory processes, and is seen in HIV infected patients before evidence of neuronal injury or neurocognitive symptoms [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B27">27</xref>]. While a marked astrogliosis was observed in post-mortem tissue [<xref ref-type="bibr" rid="B16">16</xref>], elevations of the choline-containing metabolites in the <italic>in vitro </italic>MR spectra were not [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. Thus, <italic>in vivo </italic>Cho/Cr elevation appears to be related to astrogliosis, but levels of choline-containing compounds measured by MRS <italic>in vitro </italic>do not correlate. These findings are curious, and suggest that there is a poorly understood contribution to the Cho resonance measured by <sup>1</sup>H MRS <italic>in vivo </italic>that requires further study.</p><p>Our observation of a positive linear correlation between plasma viral load and changes in frontal Cho/Cr during both the acute and chronic periods (figure <xref ref-type="fig" rid="F6">6a</xref>) is a new finding that may help to illuminate the CNS response to SIV. The brain viral inflammatory process induced by the virus may progress in at least 2 ways [<xref ref-type="bibr" rid="B28">28</xref>]. The virus may enter the CNS shortly after infection, and continue to replicate there, producing a chronic, progressive inflammatory process that ultimately leads to neurodegeneration. Alternatively, virus may be largely cleared from the CNS after the initial viremia is controlled by the immune system, and cerebral inflammation returns only when there is loss of immunological control and recurrence of viremia. Our findings illustrated in figure <xref ref-type="fig" rid="F6">6a</xref> support a third proposition: brain inflammation tracks, and is directly related to blood levels of the virus which may peak from time to time, even before the onset of AIDS.</p><p>We did not detect reduced NAA/Cr as a function of time after infection in this longitudinal study (figure <xref ref-type="fig" rid="F3">3</xref>). We have previously observed declines in NAA/Cr 2 weeks after infection using <italic>in vivo </italic>[<xref ref-type="bibr" rid="B17">17</xref>] and <italic>in vitro </italic><sup>1</sup>H MRS [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. The most likely explanation is that SIVE was infrequent, and CNS disease was mild on average across the 8 animals. Thus NAA changes were too subtle to be detected in this small cohort by <italic>in vivo </italic><sup>1</sup>H MRS at 1.5 T. Indeed in our previous <italic>in vivo </italic><sup>1</sup>H MRS study [<xref ref-type="bibr" rid="B17">17</xref>], we found changes in NAA at 2 weeks, but a total of 15 animals were studied. This conclusion is also supported by human studies. The imaging protocol we employed in this study is identical to one used in a multicenter study of HIV infected subjects [<xref ref-type="bibr" rid="B11">11</xref>]. That study detected a small difference in NAA/Cr, but required 15 HIV- and 38 neurosymptomatic, HIV+ subjects [<xref ref-type="bibr" rid="B11">11</xref>]. Other <italic>in vivo </italic>MRS human studies demonstrating decreased NAA/Cr performed at 1.5 T also employed larger numbers and more advanced disease [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B8">8</xref>,<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B27">27</xref>,<xref ref-type="bibr" rid="B29">29</xref>,<xref ref-type="bibr" rid="B30">30</xref>].</p><p>Variable progression of CNS disease in individual primates may also explain our inability to detect NAA/Cr change as a function of time after infection. If CNS injury in AIDS is characterized by different rates of neuronal injury progression alternating with periods of remission, the averaging of measurements at specific time points within a small cohort would mask evidence of brain injury. We mitigated the effects of temporal averaging by comparing brain metabolite levels with plasma viral load in individual animals, and found evidence of brain injury in the subacute and chronic periods of infection. As illustrated in figures <xref ref-type="fig" rid="F6">6a</xref> and <xref ref-type="fig" rid="F6">6b</xref>, SIV viremia significantly correlated with elevated frontal lobe Cho/Cr and decreased basal ganglia NAA/Cr. These correlations between plasma viral load and brain metabolites during the chronic phase of SIV infection suggests that CNS injury is dependent on the presence of the virus, most likely transmitted into the CNS by the trafficking of SIV-infected monocytes [<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B31">31</xref>-<xref ref-type="bibr" rid="B33">33</xref>].</p><p>The issue of variable response by different brain regions in response to SIV infection merits comment. Such variation in HIV-infected individuals has been reported by many groups [<xref ref-type="bibr" rid="B4">4</xref>-<xref ref-type="bibr" rid="B11">11</xref>]. We also find regional variations in the SIV infected macaque brain. We found that at 2 weeks pi the changes in Cho/Cr ratio appear greater in the frontal cortex than other areas. We do not know the situation in the human condition because such studies have not been reported in the early post infection stage. After the 2 week period, neuronal injury was more evident in basal ganglia than in other brain regions in the macaque, similar to human studies [<xref ref-type="bibr" rid="B11">11</xref>]. It is possible that the tropism of the virus may change with time. That is, it is possible that early metabolic changes are prominent in the cortical regions and only later are they more prominent in the basal ganglia.</p></sec><sec><title>Conclusions</title><p>We monitored the brains of SIV-infected rhesus macaques with <italic>in vivo </italic><sup>1</sup>H MR spectroscopy longitudinally for up to two years. While no MRI abnormalities were detected, metabolic changes were observed by MRS. During the first 2 weeks of infection, we observed stereotypical brain MRS abnormalities. Thereafter we found a divergence in brain metabolic patterns amongst the animals. We found that an elevation of Cho/Cr in the basal ganglia four weeks after inoculation identified animals destined to develop SIVE, an observation that may have important clinical implications. Beyond the 2 week post inoculation, we did not find statistically significant differences in metabolite ratios averaged across all 8 animals between baseline and any specific time point. Yet we found a statistically significant positive correlation between frontal cortex Cho/Cr and viral load, and a statistically significant negative correlation between basal ganglia NAA/Cr and viral load. Thus, both elevations of the Cho/Cr ratio, often observed in CNS inflammatory processes, and evidence of neuronal injury, reduced NAA/Cr, were associated with increased plasma viral load. These observations indicate brain metabolic changes in the SIV infected macaque are driven by plasma viral load, and not by time after infection. Our findings that metabolic indicators of both cerebral inflammation and neuronal injury correlate with viral levels in the periphery also underscore the need to employ therapeutic regimens capable of controlling the propagation and trafficking of virus into the CNS. It may also help explain the reduction in incidence of HIV-associated dementia in the era of HAART despite the inability of most of those drugs to effectively enter the CNS.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>Animals and viral inoculation</title><p>Eight juvenile rhesus macaques (<italic>Macaca mulatta</italic>) were inoculated intravenously with SIVmac251 (50 ng p27/kg) [<xref ref-type="bibr" rid="B34">34</xref>]. Animals were housed at the New England Primate Research Center, Southborough, MA and transported to the MGH NMR Center, Charlestown, MA for imaging. In preparation for imaging, the animals were initially tranquilized with ketamine or telazol and anesthetized with sodium pentobarbital. All animals were housed according to the standards of the American Association for Accreditation of Laboratory Animal Care. Investigators abided by the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal resources, National Research Council. Animals were euthanized by an overdose of sodium pentabarbitol when they reached terminal AIDS or at the 2-year end point of the study. Postmortem pathological examinations were performed on all animals. The determination of encephalitis was based upon the presence of perivascular macrophages and multinucleated giant cells in the brain and detection of viral nucleic acids by in situ hybridization of three different brain regions, as described below.</p></sec><sec><title>Magnetic resonance imaging and spectroscopy</title><p>Each macaque had MRI and MRS performed before infection, and at multiple time points after being inoculated with SIVmac251. All macaques were scanned at 2 weeks post inoculation (11 days post inoculation (dpi) or 13 dpi), 4 weeks post inoculation (25 dpi or 27 dpi), 3 months post inoculation (mpi), 13 mpi, 15 mpi, 17 mpi, 19 mpi and 24 mpi. Since no study of this type had ever been reported, the rationale for this imaging schedule was based on our previous limited investigations of <italic>post mortem </italic>brain samples [<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. We define 3 time periods with respect to infection: acute (within a month), subacute (between 1 and 3 months), chronic periods (beyond 3 months). Brain imaging was performed on a clinical 1.5 Tesla General Electric (Milwaukee, Wisconsin) Signa scanner with operating system Horizon 5.6 or 8.3, and a linear extremity coil.</p><p>The MRI portion of the exam consisted of T1 weighted sagittal images, followed by dual-echo, proton-density and T2 weighted axial images. Axial images were used to localize a 1.5 cm &#x000d7; 1.5 cm &#x000d7; 1.5 cm spectroscopy voxel in 3 locations: frontal cortex with the voxel centered on the interhemispheric fissure; deep white matter with the voxel centered on centrum semiovale; and basal ganglia with the voxel centered on the putamen. On repeat imaging, the animal head was positioned as in the previous study using three-plane scout imaging and iterative repositioning. In this manner, voxel placement was highly reproducible. With the exception of a slightly smaller voxel size, the MR spectroscopy protocol was identical to the one employed in multicenter human HIV studies that is characterized by high intersite and temporal reproducibility [<xref ref-type="bibr" rid="B11">11</xref>].</p><p>After animal imaging, MRS was performed on a phantom containing known concentrations of major brain metabolites (N-acetylaspartate, creatine, choline and myo-inositol). Phantom MR spectral data was employed to assess instrument stability. The protocol uses automated PROBE-P spectroscopy package, which consists of a PRESS sequence (TE = 35 msec, TR = 3000 msec) with CHESS water suppression [<xref ref-type="bibr" rid="B35">35</xref>,<xref ref-type="bibr" rid="B36">36</xref>].</p><p>All spectra were processed off-line with the SAGE-GE spectral analysis program [<xref ref-type="bibr" rid="B37">37</xref>] to determine intensities of N-acetylaspartate (NAA), choline-containing compounds (Cho), and myoinositol (MI) resonances, relative to the creatine/phosphocreatine (Cr) resonance which served as internal intensity reference.</p></sec><sec><title>Plasma viral loads</title><p>Virion associated SIV RNA in plasma was quantified by using a real-time reverse transcription-PCR assay on an Applied Biosystems Prism 7700 sequence detection system as described previously [<xref ref-type="bibr" rid="B38">38</xref>,<xref ref-type="bibr" rid="B39">39</xref>]. Results are averages of duplicate determinations. Analyses of viral RNA levels were performed by Drs. Jeffrey Lifson and Michael Piatak at SAIC-Frederick.</p></sec><sec><title>In situ hybridization</title><p>We examined frontal cortex, basal ganglia, and hippocampus/temporal cortex by SIV riboprobe <italic>in situ </italic>hybridization (ISH) for viral nucleic acid, as previously described [<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B40">40</xref>]. The ISH utilizes a DIG-labeled antisense riboprobe provided by Drs. V. Hirsch and C. Brown (National Institutes of Health, Rockville, MD) [<xref ref-type="bibr" rid="B41">41</xref>]. Tissues were microwave pretreated and hybridized overnight at 45&#x000b0;C with 10 ng of antisense riboprobe. Hybridized probe was detected with alkaline phosphatase-conjugated anti-DIG antibody using standard immunologic methods. Controls consisted of sense probes, which had been labeled with digoxigenin at the same time as the anti-sense probe, and matched tissues from uninfected, age-matched control macaques.</p></sec><sec><title>Flow cytometry</title><p>Lymphocytes were isolated from blood and stained for flow cytometric analysis as previously described [<xref ref-type="bibr" rid="B42">42</xref>]. Samples were immunoreacted with monoclonal antibodies against human CD4 (Ortho Diagnostics), CD8 (Becton Dickinson), and anti-rhesus CD3 (courtesy of J. Wong, Mass General Hospital) directly conjugated to either FITC, PE, PerCP, or APC. Samples were analyzed using a Vantage flow cytometer and Cell Quest Software (Becton Dickinson).</p></sec><sec><title>Statistical analysis</title><p>T tests assuming equal variances were used in comparisons between encephalitic and non-encephalitic animals. Prior to this, the validity of assuming equal variances was assessed. Correlations between cerebral metabolite ratios, flow cytometry, and SIV plasma viral load were calculated by regression analysis after adjusting all values relative to baseline measurements. Additionally ANOVA and Student-Newman-Keuls tests were utilized for the determination of trends.</p></sec></sec><sec><title>Authors' contributions</title><p>RAF participated in imaging experiments, participated in image analysis and drafted the manuscript. SVW participated animal care and conducted pathologic evaluations. ER helped supervise and conduct imaging experiments, participated in image analysis and helped conduct statistical analysis. JBG supervised and participated imaging experiments, participated in image analysis, conducted statistical analysis. JPK participated in imaging experiments, image data analysis and statistical analysis. MRL helped supervise and conduct imaging experiments, participated in image analysis and helped conduct statistical analysis. JH participated in animal care during imaging experiments, and participated in image analysis. PVS supervised animal care and participated in pathologic evaluations. EM participated in the design of the study, and participated in pathologic evaluations. EH supervised and helped conduct statistical analysis. AAL participated in the design of the study, supervised animal care and pathologic evaluations. RGG conceived of the study, participated in its design and directed its execution. All authors read and approved the final manuscript.</p></sec>
Possible mechanism(s) for relaxant effect of aqueous and macerated extracts from <italic>Nigella sativa </italic>on tracheal chains of guinea pig
<sec><title>Background</title><p>In previous studies, the relaxant, anticholinergic (functional antagonism) and antihistaminic effects of <italic>Nigella sativa </italic>have been demonstrated on guinea pig tracheal chains. To elucidate the other mechanisms responsible for the relaxant effect of this plant, its inhibitory effect on the calcium channel was examined in this study.</p></sec><sec><title>Results</title><p>The inhibitory effects of both concentrations of diltiazem in all three groups of experiments were significantly greater than those of saline (p &#x0003c; 0.01 to P &#x0003c; 0.001). The inhibitory of two larger concentrations of aqueous extracts in group 1 and 2 were significantly greater than those of saline (p &#x0003c; 0.01 to P &#x0003c; 0.001). The effect of two larger concentrations of macerated extract in group 1 and all concentrations of this extract in group 2 were also significantly greater than those of saline (p &#x0003c; 0.01 to P &#x0003c; 0.001). However, the extract of <italic>Nigella sativa </italic>did not show any inhibitory effect in group 3. There was a significant correlation between inhibitory effect and increasing concentrations for both extracts and diltiazem in groups 1 and 2 (p &#x0003c; 0.05 to p &#x0003c; 0.005).</p></sec><sec><title>Conclusion</title><p>Although the extracts of <italic>Nigella sativa </italic>showed inhibitory effects on pre-contracted tracheal chains in the presence of both ordinary and calcium free Krebs solution, the absence of inhibitory effects of the extracts on KCl induced contraction of tracheal chains suggest that the calcium channel blocking effect of this plant dose not contribute to the relaxant effect of this plant on the tracheal chains of guinea pigs.</p></sec>
<contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Boskabady</surname><given-names>Mohammad H</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Shirmohammadi</surname><given-names>Batool</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Jandaghi</surname><given-names>Parastoo</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Kiani</surname><given-names>Sahar</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib>
BMC Pharmacology
<sec><title>Background</title><p><italic>Nigella sativa </italic>L. (Ranunculaceae) is a grassy plant with green to blue flowers and small black seeds, which grows in temperate and cold climate areas. The seeds of <italic>Nigella sativa </italic>contain thymoquinone, monotropens such as <italic>p</italic>-cymene and &#x003b1;-pinene [<xref ref-type="bibr" rid="B1">1</xref>], Nigellidine [<xref ref-type="bibr" rid="B2">2</xref>], Nigellimine [<xref ref-type="bibr" rid="B3">3</xref>] and a saponin [<xref ref-type="bibr" rid="B4">4</xref>].</p><p>Several therapeutic effects including those on digestive disorders, gynaecology, and also anti-asthma and dyspnea have been described for the seeds of <italic>Nigella sativa </italic>in ancient Iranian medical books [<xref ref-type="bibr" rid="B5">5</xref>]. <italic>Nigella sativa </italic>has long been known for its medical use as an antispasmodic, especially against gastrointestinal disorders or respiratory ailments, in many countries.</p><p>There is evidence of relaxant effects of volatile oil from this plant on different smooth muscle including rabbit aorta [<xref ref-type="bibr" rid="B6">6</xref>], rabbit jejunum [<xref ref-type="bibr" rid="B7">7</xref>], and isolated tracheal muscles of guinea pigs [<xref ref-type="bibr" rid="B8">8</xref>]. Mahfous and El-Dakhakhny (1960) reported that the volatile oil from <italic>Nigella sativa </italic>protected guinea pigs against histamine-induced bronchospasm, but it did not affect histamine H<sub>1 </sub>receptors in isolated tissues [<xref ref-type="bibr" rid="B9">9</xref>]. However, in an <italic>in vivo </italic>study, increasing respiratory rate and intra tracheal pressure of guinea pigs due to i.v. administration of volatile oil from <italic>Nigella sativa </italic>has been demonstrated [<xref ref-type="bibr" rid="B10">10</xref>]. In our recent studies, a relaxant effect for this plant was shown [<xref ref-type="bibr" rid="B11">11</xref>]. In addition the anticholinergic, and histamine H<sub>1 </sub>receptor blocking effects of this plant on isolated guinea pig tracheal chains were demonstrated by performing concentration response curves to methacoline and histamine in the presence of saline and plant extracts [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>]. In both studies the plant extracts caused rightward shift in concentration response curves to methacoline and histamine.</p><p>To elucidate the other mechanism(s) responsible for the observed bronchodilatory effect of <italic>Nigella sativa</italic>, the inhibitory effect of aqueous and macerated extracts of this plant on the calcium channel of guinea pig tracheal chains was examined in this study.</p></sec><sec><title>Results</title><sec><title>Inhibitory effect</title><p>In group 1 experiments the inhibitory effects of 5 &#x003bc;M concentration of diltiazem and 0.5 g% and 1 g% concentrations of both extracts were significantly greater than that of saline (p &#x0003c; 0.05 to p &#x0003c; 0.001, Table<xref ref-type="table" rid="T1">1</xref>). In group 2 experiments the inhibitory effect of both concentrations of diltiazem, all concentrations of macerated extract and 0.5 g% and 1 g% concentrations of aqueous extracts were significantly greater than the effect of saline (p &#x0003c; 0.5 to p &#x0003c; 0.001, Table <xref ref-type="table" rid="T1">1</xref>). In group 3 experiments only both concentrations of diltiazem showed significant inhibitory effect compared to saline (p &#x0003c; 0.001 for both cases, Table <xref ref-type="table" rid="T1">1</xref>).</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Inhibitory effect of three cumulative concentrations of aqueous and macerated extracts from <italic>Nigella sativa</italic>, two concentrations of diltiazem and saline in three groups of experiments on pre-contracted tracheal chain of guinea pigs.</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="left" colspan="10">Inhibitory effect</td></tr><tr><td></td><td colspan="10"><hr></hr></td></tr><tr><td align="left">Experimental design</td><td></td><td align="left">Group 1</td><td align="left">St.Dif <italic>vs </italic>S.</td><td align="left">St. Dif <italic>vs </italic>D</td><td align="left">Group 2</td><td align="left">St.Dif <italic>vs </italic>S.</td><td align="left">St. Dif <italic>vs </italic>D.</td><td align="left">Group 3</td><td align="left">St.Dif <italic>vs </italic>S.</td><td align="left">St. Dif <italic>vs </italic>D.</td></tr></thead><tbody><tr><td align="left">Sline</td><td align="left">1 ml</td><td align="left">-2.43 &#x000b1; 0.90</td><td></td><td></td><td align="left">-1.93 &#x000b1; 0.74</td><td></td><td></td><td align="left">-0.83 &#x000b1; 1.35</td><td></td><td></td></tr><tr><td align="left">Diltiazem</td><td align="left">1 &#x003bc;M</td><td align="left">12.72 &#x000b1; 2.80</td><td align="left">NS</td><td></td><td align="left">34.30 &#x000b1; 2.02</td><td align="left">P &#x0003c; 0.01</td><td></td><td align="left">58.57 &#x000b1; 8.36</td><td align="left">P &#x0003c; 0.001</td><td></td></tr><tr><td></td><td align="left">5 &#x003bc;M</td><td align="left">19.67 &#x000b1; 6.00</td><td align="left">P &#x0003c; 0.05</td><td></td><td align="left">62.67 &#x000b1; 9.62</td><td align="left">P &#x0003c; 0.001</td><td></td><td align="left">90.71 &#x000b1; 6.21</td><td align="left">P &#x0003c; 0.001</td><td></td></tr><tr><td align="left">Aqueous extract</td><td align="left">0.25 g%</td><td align="left">15.23 &#x000b1; 3.80</td><td align="left">NS</td><td align="left">NS</td><td align="left">19.16 &#x000b1; 4.59</td><td align="left">NS</td><td align="left">NS</td><td align="left">-2.24 &#x000b1; 0.75</td><td align="left">NS</td><td align="left">P &#x0003c; 0.001</td></tr><tr><td></td><td align="left">0.5 g%</td><td align="left">22.87 &#x000b1; 3.66</td><td align="left">P &#x0003c; 0.05</td><td align="left">NS</td><td align="left">35.56 &#x000b1; 7.99</td><td align="left">P &#x0003c; 0.01</td><td align="left">NS</td><td align="left">-1.85 &#x000b1; 0.60</td><td align="left">NS</td><td align="left">P &#x0003c; 0.001</td></tr><tr><td></td><td align="left">1 g%</td><td align="left">35.65 &#x000b1; 10.45</td><td align="left">P &#x0003c; 0.001</td><td></td><td align="left">47.58 &#x000b1; 8.67</td><td align="left">P &#x0003c; 0.001</td><td></td><td align="left">-1.56 &#x000b1; 0.55</td><td align="left">NS</td><td></td></tr><tr><td align="left">Macerate extract</td><td align="left">0.25 g%</td><td align="left">16.20 &#x000b1; 2.67</td><td align="left">NS</td><td align="left">NS</td><td align="left">25.53 &#x000b1; 1.67</td><td align="left">P &#x0003c; 0.05</td><td align="left">NS</td><td align="left">-2.50 &#x000b1; 0.80</td><td align="left">NS</td><td align="left">P &#x0003c; 0.001</td></tr><tr><td></td><td align="left">0.5 g%</td><td align="left">21.50 &#x000b1; 4.26</td><td align="left">P &#x0003c; 0.05</td><td align="left">NS</td><td align="left">41.06 &#x000b1; 5.98</td><td align="left">P &#x0003c; 0.001</td><td align="left">NS</td><td align="left">-1.70 &#x000b1; 0.65</td><td align="left">NS</td><td align="left">P &#x0003c; 0.001</td></tr><tr><td></td><td align="left">1 g%</td><td align="left">28.80 &#x000b1; 5.26</td><td align="left">P &#x0003c; 0.001</td><td></td><td align="left">52.67 &#x000b1; 8.07</td><td align="left">P &#x0003c; 0.001</td><td></td><td align="left">-1.65 &#x000b1; 0.7</td><td align="left">NS</td><td></td></tr></tbody></table><table-wrap-foot><p>Values are presented as percentage change in proportion to maximum contraction obtained due to contractile agents and quoted as mean &#x000b1; SEM. Group 1: experiments on tracheal chains contracted by 10 &#x003bc;M methacholine in the presence of ordinary Krebs solution. Group 2: experiments on tracheal chains contracted by 10 &#x003bc;M methacholine in the presence of calcium free Krebs solution. Group 3: experiments on tracheal chains contracted by 60 mM KCl in the presence of ordinary Krebs solution (for each group, n = 6). St. Dif: Statistical difference, NS: non significant difference, S: saline, D: diltiazem.</p></table-wrap-foot></table-wrap></sec><sec><title>Differences between the effect of diltiazem and extracts</title><p>There was no significant difference between the effects of extracts from <italic>Nigella sativa </italic>with those of diltiazem in group 1 and 2 experiments. However, the effects of all concentrations of aqueous and macerated extracts were significantly lower than those of diltiazem in group 3 (p &#x0003c; 0.001 for all cases).</p></sec><sec><title>Differences of the inhibitory effects between three groups of experiments</title><p>The inhibitory effects of all concentrations of macerated extract in group 2 and those of diltiazem in groups 2 and 3 were significantly greater than the effects obtained in group 1 (p &#x0003c; 0.05 to p &#x0003c; 0.001, Fig <xref ref-type="fig" rid="F1">1</xref>). The inhibitory effects of all concentrations of aqueous extract in group 2 were also non-significantly greater than those of group 1 (Fig <xref ref-type="fig" rid="F1">1</xref>). However, the inhibitory effects of all concentrations of extracts in group 3 were significantly lower than those of group 1 and 2 experiments (p &#x0003c; 0.001 for all cases, Fig <xref ref-type="fig" rid="F1">1</xref>).</p><fig position="float" id="F1"><label>Figure 1</label><caption><p>Inhibitory effect of different concentrations of aqueous and macerated extracts from <italic>Nigella sativa</italic>, diltiazem, and saline on pre-contracted tracheal chains of guinea pigs. group 1 (methacholine induced contraction of guinea pig tracheal chains in the presence of ordinary Krebs solution, blue colour), group 2 (methacholine induced contraction of guinea pig tracheal chains in the presence of calcium free Krebs solution, green colour) and group 3 (KCl induced contraction of guinea pig tracheal chains in the presence of ordinary Krebs solution, red colour). (a) low concentration, (b) medium concentration, and (c) high concentration, for diltiazem only 2 concentrations (low and medium) were examined. Statistical differences between the results of group 1 with those of group 2 and 3; NS: non significant difference *: p &#x0003c; 0.05, **: p &#x0003c; 0.01, ***: p &#x0003c; 0.001. Statistical differences between the results of group 2 with those of group 3, +: p &#x0003c; 0.05, ++: p &#x0003c; 0.01, +++: p &#x0003c; 0.001. Aqueous and macerated extracts did not show any inhibitory effect in group 3 experiments compared to the effect of saline.</p></caption><graphic xlink:href="1471-2210-4-3-1"/></fig></sec><sec><title>Relationship between concentrations and inhibitory effect</title><p>In group 1, only the inhibitory effect of 0.25 g% vs 1 g% aqueous extract was statistically significant (p &#x0003c; 0.05). However, in group 2, the differences in inhibitory effects between all concentrations of two extracts except that of 0.5 g% vs 1 g% macerated extract were significant (p &#x0003c; 0.05 to p &#x0003c; 0.001). The inhibitory effects of two concentrations of diltiazem were also significantly different in group 2 and 3 (p &#x0003c; 0.05 and p &#x0003c; 0.001 respectively). In addition there were significant correlations between increasing concentrations and inhibitory effects for both extracts in group 1 and 2 experiments (p &#x0003c; 0.05 to p &#x0003c; 0.005, Table <xref ref-type="table" rid="T2">2</xref>).</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Correlation between the inhibitory effects of extracts from <italic>Nigella sativa </italic>with concentrations in groups 2 and 3 experiments.</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="left" colspan="2">Aqueous extract</td><td align="left" colspan="2">Macerated extract</td></tr><tr><td></td><td colspan="2"><hr></hr></td><td colspan="2"><hr></hr></td></tr><tr><td align="left">Experimental Groups</td><td align="left">R</td><td align="left">p value</td><td align="left">R</td><td align="left">P value</td></tr></thead><tbody><tr><td align="left">Group 1</td><td align="left">0.49</td><td align="left">p &#x0003c; 0.05</td><td align="left">0.48</td><td align="left">P &#x0003c; 0.05</td></tr><tr><td align="left">Group 2</td><td align="left">0.558</td><td align="left">p &#x0003c; 0.05</td><td align="left">0.62</td><td align="left">P &#x0003c; 0.005</td></tr></tbody></table></table-wrap></sec></sec><sec><title>Discussion</title><p>The bronchodilatory effect seen for <italic>Nigella sativa </italic>in our previous study [<xref ref-type="bibr" rid="B11">11</xref>] might be produced due to several different mechanisms. With regard to bronchodilatory effect of calcium channel blockers [<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>], one possible mechanism responsible for the bronchodilatory effect of this plant could be the inhibitory effect of this plant on calcium channels. This effect of the aqueous and macerated extracts from <italic>Nigella sativa </italic>was therefore examined on isolated guinea pig tracheal preparations in the present study.</p><p>In group 1 experiments (contracted tracheal chains by 10 &#x003bc;M methacholine in the presence of ordinary Krebs solution) both extracts and diltiazem showed concentration-dependent inhibitory effects compared with that of saline.</p><p>To examine whether the inhibitory effects of extracts seen in group 1 were due to inhibitory effect on calcium channels or not, the inhibitory effects of extracts and diltiazem were re-examined on contracted tracheal chains in the presence of calcium-free Krebs solution in group 2. The results of this group also showed concentration-dependent inhibitory effects of both extracts and diltiazem on methacholine-induced contraction of tracheal chains.</p><p>With regard to the inhibitory effect of KCl on calcium channels [<xref ref-type="bibr" rid="B15">15</xref>], the inhibitory effects of different concentrations of extracts and diltiazem were also examined on KCl induced contraction of tracheal chains (group 3 experiments). In this group of experiments extracts from <italic>Nigella sativa </italic>did not show any inhibitory effect on KCl induced contraction. However, diltiazem showed concentration-dependent inhibitory effect on KCl induced contraction of tracheal chains.</p><p>Although, the results of group 1, and mainly group 2, may indicate an inhibitory effect of the extracts from <italic>Nigella sativa </italic>as claimed in a previous study [<xref ref-type="bibr" rid="B16">16</xref>]. However, the results of group 3 clearly showed that the extracts from this plant have no inhibitory effects on calcium channels of guinea pig tracheal chains. The cause for methacholine induced contraction of tracheal chains in group 2 is unclear to us, but the most probable explanation is the role of stored calcium in smooth muscle cytoplasm in this phenomenon. A previous study also showed contraction of tracheal smooth muscle in the presence of calcium-free Krebs solution [<xref ref-type="bibr" rid="B16">16</xref>]. The mechanism of inhibitory effect of diltiazem on methacholine induced contraction of tracheal chains in the presence of calcium free buffer (group 2) is perhaps due to inhibition of increase in sarcoplasmic calcium concentration released from sarcoplasmic reticulum through IP3 induced calcium release [<xref ref-type="bibr" rid="B17">17</xref>].</p><p>In our previous study a rightward shift of concentration-response curves of CaCl<sub>2 </sub>in tracheal chains of guinea pigs has been demonstrated for aqueous extract of <italic>Nigella sativa</italic>, indicating a calcium channel inhibitory effect of this extract [<xref ref-type="bibr" rid="B18">18</xref>]. However, the results of the present study showed that aqueous and macerated extracts have no inhibitory effect on calcium channels. The cause of rightward shift in CaCl<sub>2 </sub>concentration-response curves of our previous study [<xref ref-type="bibr" rid="B18">18</xref>] may be due to the relaxant effect of this plant on tracheal chains.</p><p>While KCl affects calcium channels [<xref ref-type="bibr" rid="B15">15</xref>] and with regard to bronchodilatory effects of calcium channel blockers [<xref ref-type="bibr" rid="B13">13</xref>,<xref ref-type="bibr" rid="B14">14</xref>], these findings showed the absence of a blocking effect of the extracts from <italic>Nigella sativa </italic>on calcium channels. The absence of obvious relaxant effects of aqueous and macerated extracts from this plant in group 3 and the relatively potent relaxant effect of this extract in groups 1 and 2 experiments may also indicate an opening effect of these extracts on potassium channels because the bronchodilatory effects of potassium channel opening has been demonstrated previously [<xref ref-type="bibr" rid="B19">19</xref>]. If the aqueous and macerated extracts from <italic>Nigella sativa </italic>had a potassium channel opening effect, they would not have relaxant effects on tracheal chains contracted by KCl, while they could show relaxant effect when the tracheal chain was contracted by methacholine. In fact, the results of groups 1 and 2 may support this effect of aqueous and macerated extracts. Therefore, the absence of inhibitory effects of extracts in group 3 experiments may suggest the absence of any effects on calcium channels and/or existence of an opening effect of potassium channels for these extracts from <italic>Nigella sativa</italic>. However, multiple substances may present in the extracts leading to its pharmacological effects. The extracts may contain one or more substances affecting calcium channels, but their effects are masked by other substances. Therefore, the calcium channel inhibitory effects of different fractions should be tested in future studies.</p><p>Although both aqueous and macerated extracts are aqueous in nature, the methods of extraction for the two extracts are different. For preparing aqueous extract the seed should be exposed to water steam (100&#x000b0;C) for 18&#x02013;24 h. But for macerated extract it should be macerated with water at room temperature for 48 h while shaking intermittently. Therefore the ingredients of two extracts may be different. In aqueous extract more substances may be extracted, but some ingredients are destroyed due to the high temperature. However, the effect of two extract was not statistically different.</p><p>The cause of a small contraction effect of saline in all 3 groups and extracts in group 3 is presumably the continuous effects of the contractile substances (methacholine or KCl). Therefore, whenever extracts and diltiazem show inhibitory properties, they also inhibit this slight concentration effect.</p><p>The results of our previous studies [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>] and present study suggest that anticholinergic, histamine H<sub>1 </sub>inhibitory, and probable potassium channel opening effects of <italic>Nigella Sativa </italic>may contribute to the bronchodilatory effect of this plant. The other possible mechanisms responsible for the bronchodilatory effect of <italic>Nigella sativa </italic>are as follow: stimulation of &#x003b2;-adrenegic receptors [<xref ref-type="bibr" rid="B20">20</xref>], stimulation of inhibitory non-adrenergic non-cholinergic nervous system (NANC) or inhibition of stimulatory NANC [<xref ref-type="bibr" rid="B21">21</xref>], methylxanthine activity of the plant [<xref ref-type="bibr" rid="B22">22</xref>], and inhibition of phosphodiesterase [<xref ref-type="bibr" rid="B23">23</xref>]. The contribution of these mechanisms and the importance of those seen in our studies in the bronchodilatory effects of <italic>Nigella sativa </italic>should be clarified in further studies. In the present study only direct <italic>in vitro </italic>effective concentrations are shown and examined. Therefore to discover the applied effective concentrations or dosages of the extract more <italic>in vivo </italic>studies are required which is clearly dependent upon the administration route. In addition, the fractionation of the extracts and study of effective fraction are also needed for this purpose.</p><p>In conclusion, the results of the present study show that although the extracts of <italic>Nigella sativa </italic>showed inhibitory effects on pre-contracted tracheal chains in the presence of both ordinary and calcium free Krebs solutions, the absence of inhibitory effect of the extracts on KCl induced contraction of tracheal chains suggest that aqueous and macerated extracts have no inhibitory effect on calcium channels. Therefore, the calcium channel blocking effect of this plant dose not contribute to the relaxant effect of this plant on tracheal chains of guinea pigs. However, the results of this study suggested a potassium channel opening effect for the extracts from <italic>Nigella sativa </italic>that may contribute on the relaxant effect of this plant on tracheal chains of guinea pigs.</p></sec><sec sec-type="materials|methods"><title>Material and Methods</title><sec><title>Plant and extracts</title><p><italic>Nigella sativa </italic>was collected from Torbat Heydarieh (north east Iran) in the spring of 2002, and dried at room temperature in the absence of sunlight. The plant was identified by botanists in the herbarium of Ferdowsi University of Mashhad; and the specimen number of the plant is 293-0303-1 <italic>Nigella sativa</italic></p><p>The aqueous extract was prepared as follows: Fifty grams of the chopped, dried plant was extracted with 300 ml distilled water by suxhelat apparatus. Three hundred millilitres distilled water were added to a glass balloon in the lower part of suxhelat apparatus which was over a heater. The heater was set to boil distilled water continuously. The water steam flow through the middle part of the apparatus contained plant powder. The steam was then cooled and converted to liquid as it passed through a tube in the upper portion of the suxhelat with a helix tube inside through which tap water flowed. By means of aside tube, then added to the balloon in the lower part. This procedure was continued for 18&#x02013;24 h until the liquid water returning to balloon through the side tube became colourless. For macerated extract, the same amount of plant was macerated with 300 ml distilled water (on a shaker) for 48 hr. The solvent of both extracts were then removed under reduced pressure until the extract volumes reached 20 ml. Plant ingredient concentration in the final extracts was 10% W/W in both extracts. The amount of thymoquinone, thymohydroquinone and thymol in the essential oil of <italic>Nigella sativa </italic>is 0.53 %, 0.77 % and 0.91 % respectively [<xref ref-type="bibr" rid="B24">24</xref>]. However, there is no information regarding the amount of main constituents in aqueous and macerated extracts of this plant but their amount in extracts seems to be lower than in the essential oil.</p></sec><sec><title>Tissue preparations</title><p>Male guinea pigs (400&#x02013;700 g) were killed by a blow on the neck and trachea removed. Each trachea was cut into 10 rings (each containing 2&#x02013;3 cartilaginous rings). All the rings were then cut open opposite the trachealis muscle, and sutured together to form a tracheal chain [<xref ref-type="bibr" rid="B25">25</xref>].</p><p>Tissue was then suspended in a 10 ml organ bath (organ bath 61300, Bio Science Palmer-Washington, Sheerness, Kent U.K.) containing Krebs-Henseleit solution of the following composition (mM): NaCl 120, NaHCO<sub>3 </sub>25, MgSO<sub>4 </sub>0.5, KH<sub>2</sub>PO<sub>4 </sub>1.2, KCl 4.72 and dextrose 11.</p><p>The Krebs solution was maintained at 37&#x000b0;C and gassed with 95 % O<sub>2 </sub>and 5 % CO<sub>2</sub>. Tissue was suspended under an isotonic tension of 1 g and allowed to equilibrate for at least 1 h while it was washed with Krebs solution every 15 min.</p></sec><sec><title>Protocols</title><p>The inhibitory effect of three different concentrations of aqueous and macerated extracts from <italic>Nigella sativa </italic>(0.25 g%, 0.5 g%, and 1 g% from each extract) and two concentrations of diltiazem (1 and 5 &#x003bc;M) in comparison with negative control (saline, 1 ml) on calcium channels was examined. To produce three concentrations of each extract 0.25 ml, 0.25 ml, and 0.5 ml of 10 W/V concentrated extract and for diltiazem 0.1 and 0.4 ml of 10 mM concentrated solution were added on a 10 ml organ bath at 5 min intervals.</p><p>In each experiment the effect of three cumulative concentrations from each extract, two cumulative concentrations of diltiazem <italic>or </italic>saline on contracted tracheal smooth muscle were measured after exposing tissue to the solution for 10 min. A decrease in tone was considered as an inhibitory effect and expressed as positive percentage change in proportion to maximum contraction obtained due to contractile agents.</p><p>The inhibitory effects of different solutions were tested with three different experimental designs as follows:</p><p>1. On tracheal chains contracted by 10 &#x003bc;M methacholine hydrochloride in the presence of ordinary Krebs solution (Sigma Chemical Ltd UK), (group 1 experiments).</p><p>2. On tracheal chains contracted by 10 &#x003bc;M methacholine hydrochloride in the presence of calcium free Krebs solution (group 2 experiments).</p><p>3. On tracheal chains contracted by 60 mM KCl in the presence of ordinary Krebs solution (group 3 experiments).</p><p>The inhibitory effects in three groups of experiments were examined in three different series of tracheal chains (for all groups, n = 6). All of the experiments were performed randomly with a 1 h resting period of tracheal chains between each two experiments while washing the tissues every 15 min with Krebs solution. In all experiments responses were recorded on a kymograph (ET8 G-Boulitt, Paris) and were measured after fixation. The study was approved by the ethical committee of Mashhad University of Medical Sciences.</p></sec><sec><title>Statistical analysis</title><p>The data of the inhibitory effect of different experiments were expressed as mean &#x000b1; SEM. The data of inhibitory effects of different concentrations of extracts, diltiazem, and saline were compared using ANOVA test in each group. The effect of each concentration of extracts and diltiazem between three groups was also compared using ANOVA. The inhibitory effect of different extracts were related to the concentrations of the solutions using least square regression. Significance was accepted at p &#x0003c; 0.05.</p></sec></sec>
Comparison of ESTs from juvenile and adult phases of the giant unicellular green alga <italic>Acetabularia acetabulum</italic>
<sec><title>Background</title><p><italic>Acetabularia acetabulum </italic>is a giant unicellular green alga whose size and complex life cycle make it an attractive model for understanding morphogenesis and subcellular compartmentalization. The life cycle of this marine unicell is composed of several developmental phases. Juvenile and adult phases are temporally sequential but physiologically and morphologically distinct. To identify genes specific to juvenile and adult phases, we created two subtracted cDNA libraries, one adult-specific and one juvenile-specific, and analyzed 941 randomly chosen ESTs from them.</p></sec><sec><title>Results</title><p>Clustering analysis suggests virtually no overlap between the two libraries. Preliminary expression data also suggests that we were successful at isolating transcripts differentially expressed between the two developmental phases and that many transcripts are specific to one phase or the other. Comparison of our EST sequences against publicly available sequence databases indicates that ESTs from the adult and the juvenile libraries partition into different functional classes. Three conserved sequence elements were common to several of the ESTs and were also found within the genomic sequence of the carbonic anhydrase1 gene from <italic>A. acetabulum</italic>. To date, these conserved elements are specific to <italic>A. acetabulum</italic>.</p></sec><sec><title>Conclusions</title><p>Our data provide strong evidence that adult and juvenile phases in <italic>A. acetabulum </italic>vary significantly in gene expression. We discuss their possible roles in cell growth and morphogenesis as well as in phase change. We also discuss the potential role of the conserved elements found within the EST sequences in post-transcriptional regulation, particularly mRNA localization and/or stability.</p></sec>
<contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Henry</surname><given-names>Isabelle M</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Wilkinson</surname><given-names>Mark D</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Hernandez</surname><given-names>J Marcela</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Schwarz-Sommer</surname><given-names>Zsuzsanna</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib><contrib id="A5" contrib-type="author"><name><surname>Grotewold</surname><given-names>Erich</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A6" contrib-type="author"><name><surname>Mandoli</surname><given-names>Dina F</given-names></name><xref ref-type="aff" rid="I5">5</xref><email>[email protected]</email></contrib>
BMC Plant Biology
<sec><title>Background</title><p>High-throughput sequencing of partial cDNAs, or expressed sequence tags (ESTs), provides relatively fast and cost-effective access to the gene expression profile of an organism [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>]. EST libraries provide access to the population of genes transcribed, making analyses of ESTs informative in determining which genes are expressed at specific developmental ages, in specific tissues, or under specific environmental conditions.</p><p>EST analyses are especially useful when studying organisms for which little sequence data exists and for which sequencing of the genome is either not planned, or not easily feasible due to genome size. To date, there is little genomic data available for the Chlorophytes (green algae), a group far more diverse and evolutionarily divergent than all land plants combined. From this group, only <italic>Chlamydomonas reinhardtii </italic>has been the object of an extensive EST project [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>]. Genomic information from this project proved critical to elucidating the function, biosynthesis, and regulation of the photosynthetic apparatus [<xref ref-type="bibr" rid="B4">4</xref>].</p><p><italic>Acetabularia acetabulum </italic>(Fig. <xref ref-type="fig" rid="F1">1</xref>), also known as the "Mermaid's Wineglass", is a giant unicellular green alga whose size and complex life cycle make it an attractive model system for understanding morphogenesis and subcellular localization [<xref ref-type="bibr" rid="B5">5</xref>]. Reaching 3 cm in height at maturity, this unicell contains just a single diploid nucleus for most of its life cycle. It undergoes a complex morphogenetic program, most of which takes place at the apex [<xref ref-type="bibr" rid="B6">6</xref>], centimeters away from the nucleus. Classic experiments on <italic>A. acetabulum </italic>[<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>] provided the first compelling evidence for the role of the nucleus in morphogenesis and for the existence of "products of the nucleus", later presumed to be mRNAs [<xref ref-type="bibr" rid="B9">9</xref>].</p><p>The life cycle of <italic>A. acetabulum </italic>is composed of several developmental phases (Fig. <xref ref-type="fig" rid="F1">1</xref>). Like multicellular land plants, juvenile and adult phases of <italic>A. acetabulum </italic>are temporally sequential, but morphologically distinct [<xref ref-type="bibr" rid="B10">10</xref>]. Juvenile phase comprises the first centimeter of growth while adult phase comprises the remaining 2 to 3 cm [<xref ref-type="bibr" rid="B10">10</xref>]. Juvenile whorls of hairs are stacked closer to each other along the stalk, and the branching pattern of the hairs within each whorl is simpler than in adults [<xref ref-type="bibr" rid="B10">10</xref>]. Physiologically, these two phases differ as well. For example, juveniles grow well in crowded conditions and poorly at low population densities, while adults grow well only at low population densities. Similar to land plants, the transition between phases is associated with a change in the reproductive competence of the apex [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>]. In <italic>A. acetabulum</italic>, adult apices are competent to produce a terminal reproductive whorl, the cap, while juvenile apices are not (J Messmer and DF Mandoli, unpublished). At the molecular level however, the difference is gene expression patterns between adult and juvenile phases are virtually unknown.</p><p>To reveal differences in gene expression between adult and juvenile phases, we constructed two subtracted EST libraries from <italic>A. acetabulum</italic>. These libraries were designed to contain transcripts specific to one phase or the other, presumably enriched in transcripts involved in morphogenesis or phase change. We randomly sequenced and analyzed 941 ESTs from these two libraries. Our analyses of these sequences indicate that juvenile and adult phases differ significantly in their gene expression patterns. We also identified 3 consensus sequences, shared mainly by adult ESTs, that have identity with introns and the 3'UTR from carbonic anhydrase genes we previously cloned [<xref ref-type="bibr" rid="B13">13</xref>]. We discuss the potential role of these conserved elements in mRNA post-transcriptional regulation, particularly mRNA localization and/or stability.</p></sec><sec><title>Results</title><sec><title>General characterization of the ESTs</title><p>Suppressive subtractive hybridization, or SSH [<xref ref-type="bibr" rid="B14">14</xref>], results in the isolation and amplification of mRNAs present in one population (the tester population) and absent in the other (the driver population). Using SSH, we created two subtracted libraries, one putatively enriched in juvenile-specific transcripts and one putatively enriched in adult-specific transcripts (<xref ref-type="supplementary-material" rid="S1">Additional file 1</xref>). From now on, we will refer to these libraries as the "juvenile library" and the "adult library", respectively.</p><p>To test the differential expression of the ESTs, 96 clones from each library were randomly chosen and spotted in the same pattern onto two nylon membranes. Each replicate membrane was hybridized with one of two probes, created either from adult or juvenile mRNA samples (Fig. <xref ref-type="fig" rid="F2">2</xref>). Out of the 96 randomly-chosen, putative juvenile clones, 53 were only expressed in juveniles, 13 were expressed at a higher level in juveniles than in adults, 5 were expressed at similar levels in adult and juveniles and 25 did not generate any signal with either probe (Fig <xref ref-type="fig" rid="F2">2</xref>., top panels). Out of the 96 randomly-chosen, putative adult clones, 44 were expressed only in adults, 14 were expressed at a higher level in adults than in juveniles, 10 were expressed at similar levels in adults and juveniles, 5 were expressed at a higher level or only in juveniles and 23 did not generate any signal with either probe (Fig <xref ref-type="fig" rid="F2">2</xref>., bottom panels). In addition, differential expression of three clones was confirmed by virtual northern blots (data not shown). Virtual Northern blots differ from Northern blots in that phase-specific cDNA is blotted on the nylon membranes instead of mRNA [<xref ref-type="bibr" rid="B15">15</xref>]. These data provide solid preliminary evidence that the SSH was successful at isolating many transcripts differentially expressed in these two phases.</p><p>In total, 604 and 601 ESTs were sequenced from the adult and juvenile libraries respectively. Sequences containing no insert or unreliable data (as evidenced by the sequence trace) were excluded, leaving 478 ESTs from the adult library and 463 ESTs from the juvenile library for further analysis. Sequences were cleaned <italic>in silico </italic>of contaminating fragments (vector and primer sequences; see Materials and Methods). For 87% (411) of the adult clones and 83% (392) of the juvenile clones, this single-pass sequencing provided the complete sequence of the insert, i.e. vector sequence bordered both ends of the insert. ESTs ranged from 68 bps to 855 bps in length. On average, juvenile clones were longer than adult clones, averaging 474 bps and 408 bps respectively.</p><p>Due to the way the libraries were created (<xref ref-type="supplementary-material" rid="S2">Additional file 2</xref>), some ESTs in the final library contained either a polyA or polyT tract [<xref ref-type="bibr" rid="B16">16</xref>]. These tracts originated from the polyA tail of the corresponding original mRNAs, indicating that these ESTs probably contained untranslated regions. Because the ESTs were not cloned directionally, sequences containing polyA or polyT tracts were obtained according to which strand was sequenced. The occurrence of these tracts was significantly higher in the juvenile than in the adult library (40 vs 15% respectively).</p></sec><sec><title>Redundancy and overlap between the two libraries</title><p>All ESTs were aligned and partitioned into clusters (see Material and Methods for details). All ESTs that were not part of a cluster remained singletons (279 adult and 233 juvenile ESTs). A consensus sequence was derived from clusters containing two or more ESTs. The juvenile ESTs formed 77 clusters and the adult ESTs formed 84 clusters. Clusters contained up to 14 ESTs (Fig. <xref ref-type="fig" rid="F3">3</xref>). This probably over-estimates the true number of clusters, as non-overlapping ESTs would be placed into two or more separate clusters or remain singletons even if they originated from the same initial mRNA. In addition, it is possible that sequences that only differed because of sequencing errors or regions of poor sequence quality were not clustered together.</p><p>Clusters containing ESTs from both libraries were labeled as "mixed clusters". Only 2 such mixed clusters were found, representing a mere 0.3% of the total number of clusters (Fig. <xref ref-type="fig" rid="F3">3</xref>). Thus, the overlap between the two libraries is minimal, providing additional evidence that SSH probably successfully isolated ESTs specific to each developmental phase.</p></sec><sec><title>Gene functions of the ESTs</title><p>All ESTs or cluster sequences were analyzed for homology using BLASTN, TBLASTX and InterPro (see Materials and Methods). Hits with E values that were &#x0003c;1.00E-06 for BLASTN and TBLASTX searches were considered significant. In total, ESTs representing only 162 clusters or ESTs produced significant BLAST hits, 144 of which were associated with a putative gene function (Table <xref ref-type="table" rid="T1">1</xref>). 45% of these putative functions were independently confirmed by InterPro searches. Interestingly, the only two mixed clusters were both associated with the large subunit ribosomal RNA (rrnL) gene, the only chloroplast encoded gene found in our analyses.</p><p>All singletons and clusters were also analyzed for homologies using BLASTX against the <italic>Arabidopsis thaliana </italic>protein database [<xref ref-type="bibr" rid="B17">17</xref>] and using TBLASTX against the current draft of the <italic>Chlamydomonas reinhardtii </italic>genome [<xref ref-type="bibr" rid="B18">18</xref>]. For both searches, hits with E values &#x0003c; 10E-06 were considered significant. In general, the same sequences produced significant hits against each of the different databases (Table <xref ref-type="table" rid="T1">1</xref>). This independently confirmed the results of the first searches and the low percentage of coding sequences within our ESTs. In total, 178 ESTs (only 26%) produced significant hits in at least one of the BLAST searches (Table <xref ref-type="table" rid="T1">1</xref>).</p><p>The 178 ESTs or clusters that produced significant hits were sorted into functional categories according to the classification scheme developed for plants [<xref ref-type="bibr" rid="B19">19</xref>] (Fig. <xref ref-type="fig" rid="F4">4</xref>). The largest functional class from both libraries contained genes associated with photosynthesis ("energy" in Fig. <xref ref-type="fig" rid="F4">4</xref>). In general, a higher percentage of juvenile ESTs have functions related to transcription, and protein synthesis, transport and storage while a higher percentage of adult ESTs have functions related to cell structure (Fig. <xref ref-type="fig" rid="F4">4</xref> and <xref ref-type="supplementary-material" rid="S3">Additional file 3</xref>).</p></sec><sec><title>Phylogenetic analysis of the ESTs</title><p>To assess if the putative functions of the ESTs also occurred in land plants or in other green algae, we identified (for each of the 178 ESTs that generated significant BLAST hits) the sequence giving the lowest E value ("best match") in the BLAST searches and the organism to which this sequence belongs. As expected, most of these "best-match" sequences belong either to <italic>Chlamydomonas reinhardtii </italic>or the <italic>Streptophyta </italic>(land plants). This is not surprising, as the number of sequences available for most green algal lineages remains extremely limited. Specifically, <italic>A. acetabulum </italic>belongs to the class <italic>Ulvophyceae </italic>for which very little sequence information is available [<xref ref-type="bibr" rid="B20">20</xref>].</p></sec><sec><title>Conserved sequences within the ESTs</title><p>Some ESTs showed similarity to each other over short regions. These ESTs clustered into three groups of 5, 9 and 2 sequences respectively (Fig. <xref ref-type="fig" rid="F5">5</xref>). The length of the common stretch of sequence varies between 30&#x02013;70 bps for group 1, 45&#x02013;90 bps for group 3 and 170 to 250 bps for group 2 (Fig. <xref ref-type="fig" rid="F5">5</xref>). Within each group, these ESTs showed no similarity to each other outside of these regions but within these regions, the level of identity was high. Most of these ESTs belonged to the adult library. None of these 16 ESTs produced any relevant BLAST hit, making it difficult to predict whether or not they contain coding sequences. Among the sequences sharing the second consensus sequence (Fig. <xref ref-type="fig" rid="F5">5c</xref>), all but two of the ESTs ended with a polyA or polyT tract, indicating that they probably contain 3' UTRs. None of the ESTs sharing the first or third consensus sequences (Fig. <xref ref-type="fig" rid="F5">5b</xref> or <xref ref-type="fig" rid="F5">5d</xref>) ended with a polyA or polyT tract. These 3 conserved elements may be specific to <italic>A. acetabulum</italic>, because they were not found in any other sequence in Genbank (nucleotide database) except for the carbonic anhydrase 1 and 2 (CA1 and CA2) genes from <italic>A. acetabulum </italic>[<xref ref-type="bibr" rid="B13">13</xref>]. All three of these conserved sequences fell in the non-coding regions of the two CA genes, either in introns or the 3'UTRs (Fig. <xref ref-type="fig" rid="F5">5a</xref>).</p></sec></sec><sec><title>Discussion</title><sec><title>Adult and juvenile phases in A. acetabulum differ significantly in gene expression</title><p>The expression analysis (Fig. <xref ref-type="fig" rid="F2">2</xref>) and the fact that there is virtually no overlap between the two libraries suggest that the subtraction succeeded in isolating differentially expressed transcripts.</p><p>The 941 ESTs were organized into 675 independent clusters or singletons. Although this number is probably an overestimate &#x02013; non-overlapping ESTs originating from the same transcript may partition to different clusters or remain singletons &#x02013; these ESTs only represent a portion of all the ESTs present in the libraries. These data provide strong evidence that the adult and juvenile phases in <italic>A. acetabulum </italic>differ significantly in gene expression and that a large number of genes are probably phase-specific.</p></sec><sec><title>Physiological differences between the two developmental phases</title><p>The functions of the transcripts expressed at the different phases partition differently into functional classes (Fig. <xref ref-type="fig" rid="F4">4</xref>). Given that the libraries were created such that only ESTs specific to one developmental phase would be isolated, the distribution of gene functions among the ESTs is not expected to reflect that of a typical photosynthetic cell, but merely the functions that are specific to one developmental phase or the other.</p><p>Juveniles seem to devote much of their unique gene expression to transcription, protein synthesis, transport and storage, consistent with the general idea that juveniles are fast growing, more dedicated to growth than morphogenesis. During juvenile phase, the unicells increase about 10-fold in height (from &#x0003c;1 mm to 1 cm) in 2 weeks and are not competent to make a cap. On the other hand, adults increase in height about 3-fold, another 3 centimeters. Adult cells are much more complex than juvenile cells, with numerous whorls of hair that are highly branched. There is thus an increased requirement for cell wall synthesis during the adult phase. Adults also are competent to execute complex cap morphogenesis, and are preparing for nuclear divisions, nuclei transport and gametogenesis [<xref ref-type="bibr" rid="B21">21</xref>]. There is not a large increase in cytoplasmic volume between adult and juveniles because most of the volume within an adult is occupied by a central vacuole (Ngo <italic>et al</italic>., submitted). This more complex adult development is consistent with a lower percentage of transcripts dedicated to protein synthesis and a higher percentage of transcripts involved in cell structure (e.g. cytoskeletal proteins, enzymes involved in cell wall synthesis or maintenance, histones).</p><p>A surprisingly high number of ESTs from both libraries are associated with photosynthesis (<xref ref-type="supplementary-material" rid="S3">Additional file 3</xref>, class 2). For example, five ESTs were putative homologs of the rbcS protein. It is possible that these ESTs truly come from different transcripts. It is also possible that they originate from the same transcript but have not been clustered together because they do not overlap or because of regions of poor sequence quality. To address this question, we aligned these sequences to the <italic>A. acetabulum </italic>rbcS mRNA sequence present in Genbank (Fig. <xref ref-type="fig" rid="F6">6</xref>). We observed in each EST or cluster, a region of high sequence identity to the <italic>Aa</italic>-rbcS sequence (identity varied between 84 and 89% at the nucleotide level). Two of the clusters (cn115 and cn116) were identical except for sequence ambiguities, which were frequent enough for these two sequences not to be clustered together. With the exception of cn116 and cn115, the other sequences were different enough from each other and from the <italic>Aa</italic>-rbcS sequence to conclude that they did not actually originate from the same transcript. In support of this hypothesis, regions outside of these fragments of very high sequence identity could not be aligned (&#x0003c;50% identity). At the amino acid level, the sequence identity between these regions (white boxes in Fig. <xref ref-type="fig" rid="F6">6</xref>) and the <italic>Aa</italic>-rbcS sequence was almost complete, varying between 94 and 96%. A closer look at the sequences confirmed that most of the nucleotide differences present in the ORFs occurred at the third position of a codon, often resulting in the conservation of the amino acid sequence. Finally, one of the clusters (cn115) ends with a polyA tract, indicating the end of the 3'UTR, while the transcript that generated cluster 146 seems to possess a much longer 3'UTR that contains no polyA tract. Taken together, these results suggest that there may be at least 3 different loci coding for the rbcS protein in <italic>Acetabularia </italic>(cn169, J538 and <italic>Aa</italic>-rbcS, Fig <xref ref-type="fig" rid="F6">6</xref>). Confirmation of these surprising results with different methods and detailed expression analysis of the different loci will be of great interest. A similar analysis of the chlorophyll a/b binding proteins might also determine whether different proteins are expressed during different phases and whether they are functionally distinct and/or differentially regulated. The LHC (light-harvesting complex) binding proteins form a very large family that has been best characterized in land plants [<xref ref-type="bibr" rid="B22">22</xref>]. Analysis of the 22 ESTs with identity to LHC binding proteins from land plants will improve our currently poor knowledge of this gene family in algae.</p></sec><sec><title>Putative gene functions of particular interest</title><p>Immuno-cytochemistry has been used to visualize tubulin and actin proteins in <italic>A. acetabulum </italic>during development [<xref ref-type="bibr" rid="B23">23</xref>,<xref ref-type="bibr" rid="B24">24</xref>]. Actin microfilaments were found in thalli of all ages forming continuous, parallel bundles along the entire stalk. Microtubules, conversely, could not be detected in the alga prior to meiosis. Microtubules were detected during reproduction, surrounding haploid nuclei as they are transported up into the cap. Consistent with these results, our data suggest that juveniles express actin transcripts (but not tubulin) while adults express both alpha and beta tubulin (but not actin) transcripts, presumably in preparation for reproduction.</p><p>Two of our ESTs were putative expansin homologues. Expansins promote cellulose walls extension in land plants. Typical of the "mannan weeds", the wall of the diplophase of <italic>A. acetabulum </italic>is predominantly a para-crystalline mannan framework (Dunn <italic>et al</italic>., submitted). Only gametangia are enclosed in a cellulosic wall, itself surrounded by the mannan wall of the cap. Consistent with this, our results indicate that the expansin gene was expressed during adult but not juvenile phase. So far, expansins have only been found in land plants [<xref ref-type="bibr" rid="B25">25</xref>] where expansin acts within the cell wall and is activated by an acidic pH [<xref ref-type="bibr" rid="B26">26</xref>]. If these transcripts code for expansin proteins that play a role in loosening walls in <italic>A. acetabulum</italic>, it would be interesting to see if their mechanism of action is similar to that in land plants, and whether their substrate is also a cellulose wall.</p><p><italic>A. acetabulum</italic>, for most of its life cycle, contains only one nucleus, which is located in the rhizoid. This nucleus undergoes replication at the end of adult phase, during reproduction [<xref ref-type="bibr" rid="B27">27</xref>]. At this juncture, there is a tremendous need for nucleotides and histones to make the millions of haploid nuclei needed for gametogenesis [<xref ref-type="bibr" rid="B21">21</xref>]. Consistent with this, histone mRNAs were found only in the adult library (<xref ref-type="supplementary-material" rid="S3">Additional file 3</xref>, class 9). It would be interesting to look more deeply into when these transcripts are expressed and how this organism is able to produce histone proteins in such high quantities in such a short period of time.</p><p>Finally, one of the ESTs has homology to an argonaute protein (<xref ref-type="supplementary-material" rid="S3">Additional file 3</xref>, class 11, E value to <italic>Oryza sativa </italic>argonaute of 5e-15). Argonaute proteins are highly conserved and play a major role in RNA interference in animals (a.k.a. quelling in fungi or post-transcriptional gene silencing in plants [<xref ref-type="bibr" rid="B28">28</xref>]). These processes are involved in the silencing of specific genes via double stranded RNA [<xref ref-type="bibr" rid="B29">29</xref>] and their importance in post-transcriptional regulation is just starting to be deciphered. Argonaute proteins have been found in land plants, ciliates, animals and fungi but, to the best of our knowledge, this EST is the first identified algal sequence of an argonaute protein.</p></sec><sec><title>Why most of the ESTs do not correspond to any previously described sequences</title><p>We can think of three reasons why only 28.6% of the ESTs, a particularly low number, were assigned a putative homolog based on BLAST and InterPro searches. First, these are subtracted libraries, created with the objective of identifying rare, phase-specific transcripts or transcripts involved in morphogenesis, apical growth, or phase change. Hence, these ESTs should include fewer housekeeping transcripts, abundant transcripts, or transcripts common to both phases or to other organisms.</p><p>Second, the ESTs were generated by a reverse transcriptase using a poly-T primer that often does not generate full-length cDNAs. Our libraries therefore tend to be enriched in 3' ends of the transcripts, which contain non-coding sequences and which would not be recognized in homology searches. The high percentage of ESTs containing a polyA or polyT stretch supports this hypothesis.</p><p>Finally, <italic>A. acetabulum </italic>belongs to the order <italic>Dasycladale</italic>s, in the green algal class <italic>Ulvophyceae</italic>, for which very little sequence data is currently available. Before the addition of our ESTs, only 73 DNA sequences from <italic>A. acetabulum </italic>were available in Genbank, representing just 37 different genes. Although complete genomes of several land plants and green algae are now at least partially available, it is plausible that most of the <italic>A. acetabulum </italic>sequences are too divergent from those of other algae or land plants to be recognized as orthologs when entered in BLAST searches [<xref ref-type="bibr" rid="B30">30</xref>]. To test this hypothesis, we raised the cut-off value for the BLASTN and BLASTX searches against the Genbank databases from 10E-06 to 10E-03. Most additional hits obtained originated from algal or land plant sequence as opposed to a random distribution of the organisms represented in Genbank. This supports the hypothesis that these ESTs are probably homologous to these algal or plant sequences but too divergent for the homologies to be trusted.</p></sec><sec><title>Do adult and juvenile transcripts differ in structure? Insights into post-transcriptional regulation</title><p>Curiously, 40% of the juvenile clones but only 15% of the adult clones end with a polyA or polyT tract. If these tracts correspond to the mRNA polyA tail, then these ESTs contain some or all of the 3' untranslated regions (3' UTR) of the transcript from which they originated. We have diagrammed hypotheses explaining the differential occurrence of these tracts in adult versus juvenile clones (Fig. <xref ref-type="fig" rid="F7">7</xref>). The first explanation presumes an artifact of the techniques used to create the libraries. ESTs result from the amplification of cDNA fragments that have been digested by <italic>Rsa</italic>I, each <italic>Rsa</italic>I fragment having an equal chance of being amplified and cloned. If the adult cDNAs were more completely digested than the juvenile cDNAs, then the adult cDNAs would have generated a higher number of ESTs, a lower proportion of which would contain polyA tracts (Fig. <xref ref-type="fig" rid="F7">7a</xref>). A second hypothesis presumes differential mRNA length: if adult cDNAs were, on average, longer than juvenile cDNAs, each adult cDNA would produce more ESTs, yielding a lower proportion of ESTs containing the polyA tract. Adult cDNAs could be longer if on average they have longer coding sequences (Fig. <xref ref-type="fig" rid="F7">7b</xref>) or longer 3'UTRs (Fig. <xref ref-type="fig" rid="F7">7c</xref>). If they have longer 3'UTRs, the proportion of coding sequences as well as the proportion of ESTs with polyA tracts will be higher in juvenile ESTs than in adult ESTs, consistent with our findings.</p><p>Why would 3' UTRs be longer in adult transcripts than in juvenile transcripts? In adult <italic>A. acetabulum</italic>, growth and morphogenesis occurs almost exclusively at the stalk apex, centimeters away from the unique nucleus located in the rhizoid. Therefore, aspects of post-transcriptional regulation, such as mRNA stability and mRNA localization, are probably very important to the regulation of gene expression in these unicells. Indeed, more than half of the transcripts (9/16) studied to date in <italic>A. acetabulum </italic>are localized to one end or the other of the unicell, most often to its apex [<xref ref-type="bibr" rid="B31">31</xref>-<xref ref-type="bibr" rid="B33">33</xref>]. To achieve this localization, each transcript must contain cis-acting elements within its sequence, also called 'zipcodes' [<xref ref-type="bibr" rid="B34">34</xref>]. In yeast and animal cells, 'zipcodes' are part of the 3' UTR of the localized transcripts [<xref ref-type="bibr" rid="B35">35</xref>]. Also, considering the rate at which mRNA molecules move along cytoskeletal elements along the stalk of <italic>A. acetabulum </italic>[<xref ref-type="bibr" rid="B36">36</xref>,<xref ref-type="bibr" rid="B37">37</xref>], to reach the apex, any mRNA must be at least three days old, classifying them among the "ultra-stable" mRNA species [<xref ref-type="bibr" rid="B38">38</xref>]. In plants, the cis-acting elements responsible for stability of an mRNA molecule are also located in its 3' UTR [<xref ref-type="bibr" rid="B38">38</xref>]. Transcripts 3' UTR might therefore play an important role in the regulation of gene expression in this species, especially in adults.</p><p>To achieve these stability and localization patterns, adult mRNAs probably contain several post-transcriptional regulatory elements within their 3' UTRs, potentially explaining why these would be longer. What are these regulatory elements? The fact that three conserved elements (Fig. <xref ref-type="fig" rid="F5">5</xref>) were found within several unrelated ESTs, most of which originate from the adult library is promising. These 3 elements also appear to be specific to <italic>A. acetabulum </italic>and are located in a non-coding region of the carbonic anhydrase gene, whose transcript is apically localized [<xref ref-type="bibr" rid="B13">13</xref>]. Five of the ESTs containing the second conserved element also contain a polyA tract, suggesting that these ESTs may code for 3' UTRs. The first and second conserved elements fall within introns of <italic>AaCA1 </italic>(Fig. <xref ref-type="fig" rid="F5">5</xref>). It is possible that these sequence elements of <italic>AaCA1 </italic>are part of alternatively spliced introns and sometimes contained in the mature mRNAs produced from this gene. Future research will focus on elucidating the function of these conserved elements and their spatial expression during development.</p></sec></sec><sec><title>Conclusion</title><p>These results presented here provide strong evidence supporting the hypothesis that adult and juvenile phases in <italic>A. acetabulum </italic>differ significantly in gene expression patterns and that a large number of genes are phase-specific. Our next goal is to identify among these genes those that might be involved in morphogenesis or phase change. The ESTs from the two phases also partition into different functional classes, underlining further the physiological differences between the two phases. Finally, we identified conserved elements within the EST sequences. While the functional significance of these conserved elements remains to be elucidated, it is tempting to suggest that these sequences might be involved in the post-transcriptional regulation of these transcripts, possibly in sub-cellular localization and/or stability.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Culture of A. acetabulum</title><p>Unicells were grown in artificial seawater until they reached the desired developmental age. Axenic cultures were obtained by decontaminating mature caps and then using the axenic gametangia they housed for mating [<xref ref-type="bibr" rid="B21">21</xref>]. Zygotes were grown in sterile artificial seawater, Ace27, which is identical to Ace25 [<xref ref-type="bibr" rid="B39">39</xref>] except that the KCl prestock was purified over a chelex-100 column and it contains urea hydrogen peroxide at a final concentration of 10<sup>-15 </sup>M. Cultures were grown under cool white fluorescent lights at a photon flux density of 170 &#x003bc;mol m<sup>-2 </sup>s<sup>-1 </sup>on a 14 h light/10 h dark photoperiod, at 21&#x000b0;C &#x000b1; 2&#x000b0;C and repeatedly diluted to suit their developmental age [<xref ref-type="bibr" rid="B21">21</xref>].</p></sec><sec><title>mRNA extraction</title><p>Juveniles were harvested by filtration and adults were harvested using sterile dental tools. The unicells were dried briefly on a Kimwipe, and weighed on aluminum foil. Packets of algae of the same age were flash-frozen in liquid nitrogen. 7.15 g of juveniles (approximately 18,000 unicells) and 18.2 g of adults (approximately 4,000 unicells) were ground to a fine powder under liquid nitrogen. The powder was transferred to Oakridge tubes containing extraction buffer (0.1 to 0.2 g of ground unicells/ml extraction buffer). RNA was extracted according to Chang <italic>et al</italic>. [<xref ref-type="bibr" rid="B40">40</xref>].</p></sec><sec><title>Suppressive Subtractive hybridization (SSH)</title><p>cDNA synthesis and SSH were performed according to the manufacturer's recommendations using the PCR cDNA Synthesis Kit (Clontech Laboratories, Inc.) and the PCR-Select cDNA Subtraction Kit (Clontech Laboratories, Inc.) respectively. A summary of the steps involved in SSH and a more detailed figure of the formation of the ESTs from mRNA can be found in <xref ref-type="supplementary-material" rid="S1">Additional file 1</xref> and <xref ref-type="supplementary-material" rid="S2">2</xref>.</p></sec><sec><title>Cloning of the ESTs to make the libraries</title><p>DNA was precipitated using a standard ethanol precipitation protocol [<xref ref-type="bibr" rid="B41">41</xref>]. In order to add 3' A-overhangs to the PCR products for subsequent cloning, the DNA was resuspended into 25 &#x003bc;l of PCR reaction cocktail (2.5 &#x003bc;l of 10X buffer, 1.5 &#x003bc;l MgCl<sub>2</sub>, 2 &#x003bc;l 10 mM dNTPs, 18.875 &#x003bc;l water and 0.125 &#x003bc;l Taq polymerase (Promega)) and incubated at 72&#x000b0;C for 8&#x02013;10 minutes. The DNA was precipitated again [<xref ref-type="bibr" rid="B41">41</xref>] and resuspended in TE to the starting volume of the DNA amplification reaction. Following the manufacturer's recommendations, each library was cloned into 2 different cloning vectors using the AdvanTAge&#x02122; PCR Cloning Kit (Clontech Laboratories, Inc., now a discontinued product) and the TOPO&#x02122;-TA Cloning Kit (Invitrogen).</p></sec><sec><title>Dot blot and virtual Northern blot analysis of the libraries</title><p>The quality of subtraction was controlled as recommended by the PCR-Select protocol provided by Clontech. PCR-amplified inserts of 96 randomly picked clones from both libraries were duplica-spotted onto nylon membranes and hybridized with the radioactively labeled subtraction mix from both subtractions. In addition, differential expression of cDNA inserts of three clones was confirmed by virtual northern blots using SMART cDNA synthesis (Clontech) [<xref ref-type="bibr" rid="B15">15</xref>]. The clones used in these dot blots and virtual northern blots were not sequenced and are not part of the following sequence analysis.</p></sec><sec><title>EST sequencing</title><p>Colonies were randomly picked from each library using sterile toothpicks. Plasmid DNA from each colony was isolated and eluted with 2 &#x000d7; 40 &#x003bc;l of elution buffer (Plasmid Miniprep Kit, Qiagen).</p><p>DNA sequencing was carried out at the Plant-Microbe Genomics Facility, Ohio State University. The sequencing reactions were prepared by mixing 400 ng of plasmid DNA and 4 pmol of primer (M13F (5'-GTAAAACGACGGCCAG-3') or M13R (5'-CAGGAAACAGCTATGAC-3') with water for a total volume of 10 &#x003bc;l. Next, 2 &#x003bc;l of BigDyeTerminator mixture, version 2 (Applied Biosystems), 4 &#x003bc;l BetterBuffer (The Gel Company) and 4 &#x003bc;l water were added. The cycling parameters were those recommended by the manufacturer except that the reactions were run for 35 cycles instead of 25. The reactions were cleaned up with Millipore Multiscreen/Sephadex columns, according to the manufacturers recommendations (Millipore Technical Note TN053). The resulting 20 &#x003bc;l of clean sequencing reaction product (in water) was placed in an Applied Biosystems 3700 DNA Analyzer for separation and analysis.</p></sec><sec><title>Sequence analysis</title><sec><title>Sequence preparation</title><p>Each clone was sequenced once using the M13 forward primer. If the sequence was of poor quality, the clone was sequenced again using the M13 reverse primer. Using Sequencher (Gene Codes Inc.), each nucleotide sequence was cleaned <italic>in silico </italic>of contaminating vector or primer sequence individually by aligning the EST sequence to that of the vector and those of the primers used in the creation of the libraries (nested PCR primer 1 (5'-TCGAGCGGCCGCCCGGGCAGGT-3') and nested PCR primer 2 (5'-AGCGTGGTCGCGGCCGAGGT-3'). These steps insured that the remaining sequence was devoid of contaminating DNA fragments that could potentially generate erroneous hits in BLAST searches [<xref ref-type="bibr" rid="B16">16</xref>]. A high proportion of the sequences also contained polyA or polyT tracts. These DNA fragments were also removed <italic>in silico </italic>from the corresponding sequences before performing homology searches.</p></sec><sec><title>Homology searches</title><p>Each EST was queried as follows:</p><p>- BLASTN searches [<xref ref-type="bibr" rid="B42">42</xref>] (database searched: nr (non-redundant nucleotide sequences)) [<xref ref-type="bibr" rid="B43">43</xref>].</p><p>- TBLASTX searches [<xref ref-type="bibr" rid="B42">42</xref>] (database searched: nr (non-redundant nucleotide sequences), genetic code: 6 (used by ciliates and <italic>Dasycladales </italic>[<xref ref-type="bibr" rid="B44">44</xref>]), defaults were used for the rest of the parameters) [<xref ref-type="bibr" rid="B43">43</xref>].</p><p>- InterPro searches [<xref ref-type="bibr" rid="B45">45</xref>] queries protein motifs databases from the European Bioinformatics Institute [<xref ref-type="bibr" rid="B46">46</xref>]. InterPro hits were mapped to the Gene Ontology [<xref ref-type="bibr" rid="B47">47</xref>].</p><p>- BLASTX searches against the <italic>Arabidopsis thaliana </italic>database [<xref ref-type="bibr" rid="B17">17</xref>].</p><p>- TBLASTX searches against the June 2003 draft of the <italic>Chlamydomonas reinhardtii </italic>genome [<xref ref-type="bibr" rid="B18">18</xref>].</p></sec><sec><title>Clustering of the ESTs</title><p>Clustering and alignments of the ESTs were performed using StackPack software (Electric Genetics, Cape Town, South Africa) [<xref ref-type="bibr" rid="B48">48</xref>,<xref ref-type="bibr" rid="B49">49</xref>]. Clustering was performed in two phases. The first phase used the 'd2' algorithm, which is part of the 'd2' cluster [<xref ref-type="bibr" rid="B50">50</xref>,<xref ref-type="bibr" rid="B51">51</xref>]. The second phase used Phrap [<xref ref-type="bibr" rid="B52">52</xref>].</p><p>The analysis was run with the following parameters:</p><p>d2_cluster: word_size = 6, similarity_cutoff = 0.96, minimum_sequence_size = 50, window_size = 150 and reverse_comparison = 1.</p><p>Phrap: old_ace = 1, vector_bound = 0, trim_score = 20, forcelevel = 0, penalty = -2, gap_init = -4, gap_ext = -3, ins_gap_ext = -3, del_gap_ext = -3, maxgap = 30, flags = -retain_duplicates.</p></sec><sec><title>Organization of the data</title><p>Bioinformatics scripts and the database systems used to store and query sequence/annotation data were provided by the Specialized Plant Resources in Informatics and Genomics (SPRIG) project <ext-link ext-link-type="uri" xlink:href="http://bioinformatics.org/sprig"/>, in particular, the SPRIG generic EST database and support script [<xref ref-type="bibr" rid="B53">53</xref>].</p></sec><sec><title>Availability of the sequences</title><p>The EST sequences analyzed in this study have been submitted to dbEST division of Genbank under accession numbers: CF 258288 to CF259228.</p></sec></sec></sec><sec><title>List of abbreviations</title><p>3' UTR: 3' untranslated region</p><p>BLAST: Basic local alignment search tool</p><p>SSH: Suppressive subtractive hybridization</p><p>AaCA1: Carbonic anhydrase 1 from <italic>Acetabularia acetabulum</italic></p><p>EST: Expressed Sequence Tag</p><p>RTase: Reverse transcriptase.</p></sec><sec><title>Authors' contributions</title><p>I.H. prepared some of the plasmid DNAs, participated in the bioinformatics analysis of the sequences and the design of the study, analyzed the results and drafted the manuscript. M.W. performed the bioinformatics analysis of the sequences and created the EST database. Z.S-S. created the subtracted libraries. E.G. and J.M.H. coordinated the sequencing and performed the initial analysis. E.G. participated in the design of the study. D.M. conceived of and initiated the project, prepared some of the plasmid DNAs and participated in the design and coordination of the study. All authors read, reviewed and approved the final manuscript.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="S1"><caption><title>Additional File 1</title><p>Creation of subtracted libraries. Summary of the steps involved in the creation of subtracted libraries.</p></caption><media xlink:href="1471-2229-4-3-S1.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S2"><caption><title>Additional File 2</title><p>cDNA synthesis and <italic>Rsa</italic>I digestion. Detailed description of the steps involved in the formation of double stranded cDNA from mRNA and in the digestion of the cDNA population. This procedure generates a population of ESTs, some of which contain a polyA tract and the size of the ESTs thus depends on the position of the <italic>Rsa</italic>I restriction sites. Non-interrupted lines represent DNA strands and dashed lines represent RNA strands. The gray boxes represent the primers used for these amplification steps. A more detailed description of these steps can be found in Henry and Mandoli [<xref ref-type="bibr" rid="B16">16</xref>].</p></caption><media xlink:href="1471-2229-4-3-S2.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S3"><caption><title>Additional File 3</title><p>Function and name of the ESTs that produced significant BLAST hits. All ESTs that produced significant hits were classified according to their function and following the classification scheme developed for plants [<xref ref-type="bibr" rid="B19">19</xref>].</p></caption><media xlink:href="1471-2229-4-3-S3.pdf" mimetype="application" mime-subtype="pdf"><caption><p>Click here for file</p></caption></media></supplementary-material></sec>
Finding qualitative research: an evaluation of search strategies
<sec><title>Background</title><p>Qualitative research makes an important contribution to our understanding of health and healthcare. However, qualitative evidence can be difficult to search for and identify, and the effectiveness of different types of search strategies is unknown.</p></sec><sec sec-type="methods"><title>Methods</title><p>Three search strategies for qualitative research in the example area of support for breast-feeding were evaluated using six electronic bibliographic databases. The strategies were based on using thesaurus terms, free-text terms and broad-based terms. These strategies were combined with recognised search terms for support for breast-feeding previously used in a Cochrane review. For each strategy, we evaluated the recall (potentially relevant records found) and precision (actually relevant records found).</p></sec><sec><title>Results</title><p>A total yield of 7420 potentially relevant records was retrieved by the three strategies combined. Of these, 262 were judged relevant. Using one strategy alone would miss relevant records. The broad-based strategy had the highest recall and the thesaurus strategy the highest precision. Precision was generally poor: 96% of records initially identified as potentially relevant were deemed irrelevant. Searching for qualitative research involves trade-offs between recall and precision.</p></sec><sec><title>Conclusions</title><p>These findings confirm that strategies that attempt to maximise the number of potentially relevant records found are likely to result in a large number of false positives. The findings also suggest that a range of search terms is required to optimise searching for qualitative evidence. This underlines the problems of current methods for indexing qualitative research in bibliographic databases and indicates where improvements need to be made.</p></sec>
<contrib id="A1" corresp="yes" contrib-type="author"><name><surname>Shaw</surname><given-names>Rachel L</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Booth</surname><given-names>Andrew</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Sutton</surname><given-names>Alex J</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Miller</surname><given-names>Tina</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A5" contrib-type="author"><name><surname>Smith</surname><given-names>Jonathan A</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib><contrib id="A6" contrib-type="author"><name><surname>Young</surname><given-names>Bridget</given-names></name><xref ref-type="aff" rid="I5">5</xref><email>[email protected]</email></contrib><contrib id="A7" contrib-type="author"><name><surname>Jones</surname><given-names>David R</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A8" contrib-type="author"><name><surname>Dixon-Woods</surname><given-names>Mary</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib>
BMC Medical Research Methodology
<sec><title>Background</title><p>The important contribution that qualitative research can make to our understanding of health and healthcare is increasingly well-recognised [<xref ref-type="bibr" rid="B1">1</xref>]. However, it is often difficult to find [<xref ref-type="bibr" rid="B2">2</xref>]. The progress that has been made on the development of indexing systems on bibliographic databases for quantitative study designs, including randomised controlled trials and case control studies, has not been as rapid for qualitative research [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>]. The issue of searching for qualitative research is becoming increasingly important as interest in incorporating qualitative research into systematic reviews grows [<xref ref-type="bibr" rid="B5">5</xref>]. Conducting a thorough search is a distinguishing characteristic of systematic reviews, and there is a need for reviewers to be able to demonstrate the comprehensiveness and reproducibility of their searches [<xref ref-type="bibr" rid="B6">6</xref>].</p><p>The aim of literature searching is to optimise the ability of the search to identify relevant articles, and to exclude irrelevant articles, for a specifically defined research question. Search strategies can be evaluated in terms of their comprehensiveness in identifying relevant literature, known as 'recall', a property which can be likened to the sensitivity of a screening test. Search strategies can also be evaluated in terms of the extent to which the records identified are found to be truly relevant, known as 'precision', a property which can be likened to the positive predictive value of a screening test [<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B8">8</xref>]. A range of strategies is available for searching for qualitative research, but few have been formally evaluated, and little is known about the effectiveness of different search strategies, particularly across different bibliographic databases.</p><p>We selected the example area of support for breast-feeding as an area where qualitative research is likely to be of particular value, where there was likely to be a substantial body of qualitative research, and where there was an existing systematic review which had used recognised search terms for the subject area [<xref ref-type="bibr" rid="B9">9</xref>]. The search strategy for this existing review had been approved by the Cochrane Collaboration, and had used search terms that were designed to identify research on support for breast-feeding but were neutral to methodology; our aim was to identify research on support breast-feeding which had used a qualitative methodology. We evaluated the recall and precision of three electronic strategies for searching for qualitative research on support for breast-feeding across six bibliographic databases.</p></sec><sec sec-type="methods"><title>Methods</title><p>Using recognised search terms for support for breast-feeding derived from a previous Cochrane systematic review in the area, we searched for qualitative research on support for breast-feeding using six electronic bibliographic databases: MEDLINE, EMBASE, CINAHL, British Nursing Index, ASSIA and Social Sciences Citation Index. These databases represented the disciplines of medicine, nursing, and social sciences. We evaluated three electronic search strategies: using thesaurus terms; using free-text terms; and using broad-based terms.</p><sec><title>Strategy 1: Using thesaurus terms</title><p>Indexing systems on electronic literature databases involve systems of controlled keywords (known as thesaurus terms or subject headings) that are used to categorise each record stored. Medline, for instance, uses Medical Subject Headings (MeSH), a highly structured thesaurus. The thesaurus terms that we used varied for each database according to their indexing system. For example, qualitative research is indexed on MEDLINE as "Qualitative Research" or "Nursing Methodology Research", while on CINAHL their subject heading "Qualitative Studies" is complemented by more detailed terms, including "Phenomenological Research" and "Grounded Theory".</p></sec><sec><title>Strategy 2: Using free-text terms</title><p>This strategy was based on using free-text terms that might specifically identify qualitative research. This strategy searches the titles, abstracts and keywords of records held in the databases. We used over 40 commonly used qualitative research methodology terms, including "ethnograph$", "lived experience$", "narrative analysis", "grounded theory" and "glaser adj2 strauss$". These terms were derived through reference to existing methodology filters for qualitative research [<xref ref-type="bibr" rid="B4">4</xref>] and through our own expertise in information retrieval.</p></sec><sec><title>Strategy 3: Using broad-based terms</title><p>This strategy was based on using three broad free-text terms, "qualitative", "findings" and "interview$" and the thesaurus term "Interviews". These terms were selected as a result of unpublished research shared with the project team [<xref ref-type="bibr" rid="B10">10</xref>].</p><p>The terms used in all three strategies were purposively chosen in order to maximise the comprehensiveness or sensitivity of searching for qualitative methodologies. The thesaurus and free-text strategies included terms that are often associated with quantitative rather than qualitative research, including "questionnaire" and "attitude". It was necessary to include these terms because pilot work suggested that they may also be used by bibliographic databases to classify qualitative studies, even though many qualitative researchers might not choose to use such terms to describe their work.</p></sec><sec><title>Relevance</title><p>The "yield" of the searching strategies was assessed as the total number of records identified by each strategy. The total initial yield of all three strategies across the six databases was used as a proxy for the population of qualitative studies in the area of support for breast-feeding. It was not within the scope of our study to determine the "true" population: establishing the "true" population would have required a "gold standard" method of retrieving qualitative evidence, which currently does not exist, and would also have required additional techniques including hand-searching of relevant core journals. Initial analyses indicated that the references were likely to be so widely scattered across journals as to make this a logistically impractical task.</p><p>"Relevance" of records identified by the searches was defined by whether the records were relevant to the topic of breast-feeding support and whether they used a recognised qualitative methodology. The judgements of relevance were made by experts in qualitative research and the topic area (RS, TM, JS, BY, MDW and SB). All judgements of relevance were based on the abstracts for the records, or, where these were unavailable (in 23% of cases), full-text articles were obtained. Any ambiguities or difficult cases were settled by consensus.</p></sec></sec><sec><title>Results</title><sec><title>Recall: potentially relevant studies ("tested positive")</title><p>The total initial yield across the three strategies was 7420 records (see Table <xref ref-type="table" rid="T1">1</xref>). This figure was calculated following elimination of duplicate records that were found in more than one database or by more than one strategy, and also records that referred to non-human research. The total initial yield contained all potentially relevant records. We assessed recall by calculating the proportion of records from each strategy that were potentially relevant, or, in an analogy with screening tests, the proportion of records for each strategy that "tested positive" for being both about support for breast-feeding and using a qualitative methodology.</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Recall of each strategy across databases</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="left">Potentially relevant ("tested positive") records identified by each strategy</td><td align="left">Recall: Potentially relevant records ("tested positive") as a percentage (number) of total initial yield</td></tr></thead><tbody><tr><td align="left">1)Thesaurus</td><td align="left">3537</td><td align="left">47.6% (3537/7420)</td></tr><tr><td align="left">2)Free-text</td><td align="left">3451</td><td align="left">46.5% (3451/7420)</td></tr><tr><td align="left">3)Broad-based</td><td align="left">3912</td><td align="left">52.7% (3912/7420)</td></tr><tr><td align="left">Total initial yield (duplicates and non-human eliminated)</td><td align="left">7420</td><td align="left">-</td></tr></tbody></table></table-wrap><p>The broad-based strategy identified 3912 potentially relevant records and had a recall rate of 52.7%, suggesting that if a record was both potentially qualitative and potentially about breast-feeding, its chances of being identified by this strategy were 52.7% (3912/7420). The thesaurus and free-text strategies had recall rates of 47.6% (3537/7420) and 46.5% (3451/7420), respectively. No single strategy was sufficiently comprehensive to identify all of the total initial yield (7420 records). This suggests that relying on one strategy alone risks missing potentially relevant records.</p></sec><sec><title>Precision: actually relevant studies ("diagnosed positive")</title><p>The total initial yield of 7420 potentially relevant records was subject to abstract screening by subject matter and methodology, with the aim of identifying a total number of records, as well as the number of records for each strategy, judged relevant to both breast-feeding support and qualitative research (Table <xref ref-type="table" rid="T2">2</xref>). The search strategies had identified 2608 records that were relevant to the content area &#x02013; breast-feeding support &#x02013; but were judged not relevant to qualitative methodology. On the other hand, records reporting qualitative research and judged not relevant to breast-feeding support totalled 26. The 262 records that were judged relevant to the criteria both of content area and methodology were published between 1976 and 2002 in over 100 journals.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Precision of each strategy across databases</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="left">Actually relevant records retrieved ("diagnosed positive") by each strategy</td><td align="left">Precision: Actually relevant records ("diagnosed positive") as a percentage (number) of potentially relevant records for that strategy</td></tr></thead><tbody><tr><td align="left">1)Thesaurus</td><td align="left">191</td><td align="left">5.4% (191/3537)</td></tr><tr><td align="left">2)Free-text</td><td align="left">172</td><td align="left">4.9% (172/3451)</td></tr><tr><td align="left">3)Broad-based</td><td align="left">187</td><td align="left">4.7% (187/3912)</td></tr><tr><td align="left">Total actually relevant records (duplicates eliminated)</td><td align="left">262</td><td align="left">-</td></tr></tbody></table></table-wrap><p>We were able to use the judgements of relevance to assess precision, calculated as the proportion of actually relevant records ("diagnosed positive", in screening terms) divided by the number of records that "tested positive" for relevance for that strategy. The precision of all strategies was poor: the thesaurus strategy precision rate was highest at 5.4% (191/3537), suggesting that if a record was initially identified as being about support for breast-feeding and using a qualitative methodology, its chances of being subsequently found to meet these criteria were 5.4%. The precision of the free-text strategy was 4.9% (172/3451) and of the broad-based strategy was 4.7% (187/3912). Overall, 96% ((7420-262)/7420) of the potentially relevant records retrieved were judged irrelevant to breast-feeding support and qualitative methodology. Furthermore, the most successful strategy, using thesaurus terms, only identified 72.9% (191/262) of the total actually relevant records found by all three strategies combined. This suggests that relying on one strategy alone would miss actually relevant records.</p></sec></sec><sec><title>Discussion</title><p>Our study has evaluated three strategies for searching for qualitative research in a specific topic area known to be rich in qualitative research across six databases. A limitation of the study is that it was not possible for practical reasons to identify the "true" population of qualitative research in the area of breast-feeding. For example, to conduct hand-searching of journals, it would have been necessary to search almost 30 years of more than 20 journals to retrieve just half of relevant records identified through our electronic searches. Nonetheless, our findings do have important implications for those seeking to produce documented and transparent searching strategies for qualitative research. Our findings suggest that any of the strategies we have used can identify potentially relevant qualitative studies. However, relying on any one strategy is likely to miss relevant records. This suggests that a combination of searching strategies (using thesaurus terms and free-text terms) is required in order to maximise recall &#x02013; the number of potentially relevant articles retrieved. The strategies used in this study have poor precision, meaning that few articles initially identified will prove relevant on grounds of methodology.</p><p>Clearly a high recall, high precision search strategy would constitute the optimal literature-searching technique, but trade-offs between precision and recall are unavoidable [<xref ref-type="bibr" rid="B11">11</xref>]. Given that we did attempt to maximise recall by using a very wide range of search terms, it was perhaps not surprising that the precision of all three strategies was poor, with 96% of the total initial yield of records identified being judged not relevant. These findings suggest that the task of screening abstracts to identify qualitative research is likely to remain daunting.</p><p>The precision of searching for qualitative research could be improved in a number of ways. It is particularly disappointing that current thesaurus terms used by bibliographic database indexing systems are of limited value when searching for qualitative literature [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B12">12</xref>]. If indexing systems designed specifically to identify qualitative research were available to bibliographic database providers, optimal search strategies would be enabled. Authors of qualitative studies could assist this process by making explicit their study design, and by using structured abstracts (where instructions to authors allow) [<xref ref-type="bibr" rid="B13">13</xref>].</p><p>The problem of poor indexing is compounded by the absence of abstracts for many qualitative studies: 23% of the records screened for our study did not have abstracts stored in the databases. The complex task of determining whether a study is qualitative is made almost impossible when no abstract is provided. The only way in this instance of confirming whether a study uses qualitative methodology is to obtain the full-text article, adding an expensive and time consuming extra layer to the review process.</p></sec><sec><title>Conclusions</title><p>Better indexing by databases and explicit qualitative methodology descriptors from authors are required to make searching for qualitative evidence more efficient and effective. Until improvements are made, those searching for qualitative research must be aware that the price of designing a high recall search strategy is poor precision. Search strategies may need to be over-inclusive so as not to miss any potentially relevant records but this necessitates a time consuming and costly process of screening for very small return. These findings are especially significant for those engaged in the increasingly widespread practice of incorporating qualitative research in systematic reviews.</p></sec><sec><title>Competing interests</title><p>None declared.</p></sec><sec><title>Authors' contributions</title><p>AB and AS designed the study and AB wrote the initial drafts of the search strategies. All authors were involved in the development of the strategies and RS conducted the literature searches using these search strategies. RS, TM, JS, BY, DJ, MDW and SB carried out the abstract screening. The analysis of recall and precision was performed by RS. RS wrote the initial draft of the paper and all authors contributed to and approved the final manuscript.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1471-2288/4/5/prepub"/></p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="S1"><caption><title>Additional File 1</title><p>Breast-feeding support search terms. This contains the Cochrane breast-feeding support search terms. These terms remained constant for all three qualitative methods strategies. Modifications were made for each database to accommodate the different thesaurus terms in each.</p></caption><media xlink:href="1471-2288-4-5-S1.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S2"><caption><title>Additional File 2</title><p>Qualitative methodology Search strategies. This includes the search terms for used in the three qualitative methods search strategies. Each strategy was modified to suit each of the six electronic bibliographic databases making a total of 18 search strategies (on six databases using three qualitative methodology variations).</p></caption><media xlink:href="1471-2288-4-5-S2.doc" mimetype="application" mime-subtype="msword"><caption><p>Click here for file</p></caption></media></supplementary-material></sec>
Family physician views about primary care reform in Ontario: a postal questionnaire
<sec><title>Background</title><p>Primary care reform initiatives in Ontario are proceeding with little information about the views of practicing family physicians.</p></sec><sec sec-type="methods"><title>Methods</title><p>A postal questionnaire was sent to 1200 randomly selected family physicians in Ontario five months after the initial invitation to join the Ontario Family Health Network. It sought information about their practice characteristics, their intention to participate in the Network and their views about the organization and financing of primary care.</p></sec><sec><title>Results</title><p>The response rate was 50.3%. While many family physicians recognize the need for change in the delivery of primary care, the majority (72%) did not expect to join the Ontario Family Health Network by 2004, or by some later date (60%). Nor did they favour capitation or rostering, 2 key elements of the proposed reforms. Physicians who favour capitation were 5.5 times more likely to report that they expected to join the Network by 2004, although these practices comprise 5% of the sample.</p></sec><sec><title>Conclusions</title><p>The results of this survey, conducted five months after the initial offering of primary care reform agreements to all Ontario physicians, suggest that an 80% enrollment target is unrealistic.</p></sec>
<contrib id="A1" equal-contrib="yes" corresp="yes" contrib-type="author"><name><surname>Hunter</surname><given-names>Duncan JW</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I2">2</xref><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A2" equal-contrib="yes" contrib-type="author"><name><surname>Shortt</surname><given-names>Samuel ED</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I2">2</xref><xref ref-type="aff" rid="I3">3</xref><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib><contrib id="A3" equal-contrib="yes" contrib-type="author"><name><surname>Walker</surname><given-names>Peter M</given-names></name><xref ref-type="aff" rid="I5">5</xref><email>[email protected]</email></contrib><contrib id="A4" equal-contrib="yes" contrib-type="author"><name><surname>Godwin</surname><given-names>Marshall</given-names></name><xref ref-type="aff" rid="I2">2</xref><xref ref-type="aff" rid="I3">3</xref><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib>
BMC Family Practice
<sec><title>Background</title><p>Since the inception of Canadian medicare in the late 1960s there have been recurrent cycles, albeit largely fruitless, of demands for innovation to primary care delivery models [<xref ref-type="bibr" rid="B1">1</xref>]. Increasingly, however, the need to review and revise the delivery of primary care has assumed new urgency and is currently seen as a core health policy issue in the developed world and beyond [<xref ref-type="bibr" rid="B2">2</xref>]. Primary care reform has been advocated by provincial and national commissions in Canada as fundamental to health system restructuring [<xref ref-type="bibr" rid="B3">3</xref>-<xref ref-type="bibr" rid="B6">6</xref>]. In Ontario, two main models of reformed primary care (reformed fee-for-service and global capitation) have been initiated as pilot projects and evaluated[<xref ref-type="bibr" rid="B7">7</xref>]. In 2001 the Ontario government launched the Ontario Family Health Care Network, intended to enlist 80% of practitioners by the year 2004 into a redesigned model of delivering primary care. The key elements in this model include: patient rostering, capitation payment with added incentives for prevention and other targeted services, provision of out-of-hours service and tele-triage, and extensive use of electronic medical records and linkages[<xref ref-type="bibr" rid="B8">8</xref>].</p><p>The move towards changes in the delivery of primary care appears to be a top-down process driven by provincial Ministries of Health. What do 'grass-roots' practitioners feel about change? A study in 2001 found that only five percent of Ontario family physicians, in practice eight to ten years, believed primary care reform would have a favourable effect on their practices[<xref ref-type="bibr" rid="B9">9</xref>]. A subsequent study of physicians who were eligible for, but did not participate in, a primary care reform pilot project identified many concerns about the impact of a new system on: practice routines, working conditions, financial arrangements and loss of autonomy[<xref ref-type="bibr" rid="B10">10</xref>].</p><p>Currently in Ontario the long-standing Community Health Centre and Health Services Organization programs, and recent primary reform sites, account for only about five percent of family physician practices[<xref ref-type="bibr" rid="B1">1</xref>]. This small number, along with evidence that practitioners are sceptical about current reforms, suggests that the recruitment target may be unrealistic. The aims of this study are: 1) to determine whether family physicians intended to participate in the Ontario Family Health Care Network; and 2) to identify factors that may influence their decision.</p></sec><sec sec-type="methods"><title>Methods</title><p>A cross-sectional survey of family physicians in Ontario was carried out in July 2002. A two page questionnaire, along with a covering letter and a pre-paid return envelope, was sent to 1200 family physicians, randomly selected from a list of 5200 members of the Ontario College of Family Physicians. The college represents 65% of all practicing family physicians in the province of Ontario. Non-responders were sent a second questionnaire one month later. The questionnaire sought information about physicians (age, sex, years in practice), practice characteristics (type, size, remuneration, percent of time spent in clinical practice, use of computer, out-of-hours service) and their level of agreement with several statements related to primary care reform in Ontario (Table <xref ref-type="table" rid="T1">1</xref>). The level of agreement ranged from: strongly agree, agree, slightly agree, slightly disagree, disagree, to strongly disagree. The wording of statements is presented in Table <xref ref-type="table" rid="T1">1</xref>. To test for sampling bias, responder characteristics (age, sex, years in practice) were compared with the Ontario responses to the 2001 National Family Physician Workforce Survey [<xref ref-type="bibr" rid="B11">11</xref>].</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Statements used to elicit family physician views about primary care reform in Ontario.</p></caption><table frame="hsides" rules="groups"><tbody><tr><td align="left">I understand the Ontario Family Health Network well enough to make informed decisions about my involvement</td></tr><tr><td align="left">I expect to be part of the Ontario Family Health Network by 2004</td></tr><tr><td align="left">I expect to be part of the Ontario Family Health Network at some time after 2004</td></tr><tr><td align="left">The current system for the organization of primary care delivery in Ontario needs to be changed</td></tr><tr><td align="left">The current system for the financing of primary care delivery in Ontario needs to be changed</td></tr><tr><td align="left">A capitation based formula for funding physician services would improve primary care in Ontario</td></tr><tr><td align="left">A roster that links a patient to a single care provider would improve primary care in Ontario</td></tr><tr><td align="left">Appropriate financial incentives would enhance preventive interventions in primary care</td></tr><tr><td align="left">Patients should always have access to extended weeknight and weekend office hours</td></tr><tr><td align="left">A telephone health line staffed by a qualified nurse is a good resource to direct patients to appropriate care</td></tr><tr><td align="left">I would like to see computer systems replace most of the paper systems in my practice</td></tr></tbody></table></table-wrap><p>Data analysis consisted of frequency distributions and the calculation of odds ratios. The variables were re-coded as follows: age-group [less than 44 vs. 45+ years], years in practice [less than 9 years vs. 10 years or greater], type of practice [solo vs. group, community health centre, health services organization, other], remuneration [fee-for-service vs. capitation, salary, other], size of practice [less than 1500 vs. 1500 or more patients], percent time in clinical practice and [less than 75% vs. greater than 75%]. Level of agreement was re-coded as either agreement [strongly agree, agree, slightly agree] or disagreement [slightly disagree, disagree, strongly disagree].</p><p>All analyses were conducted using procedures written in SAS[<xref ref-type="bibr" rid="B12">12</xref>]. Ethics approval was received from the Queen's University Health Sciences and Affiliated Teaching Hospitals Research Ethics Board.</p></sec><sec><title>Results</title><p>Of the 1200 family physicians sent a questionnaire, 50 were not in practice so the final study population was 1150. 565 questionnaires were not returned and a further 8 were returned without a response so that the final response rate was 50.2% (577/1150). The mean age of responders was 44 years, with the majority of responders (39%) aged between 35&#x02013;44 years (Table <xref ref-type="table" rid="T2">2</xref>). There were slightly more men (55%) than women (45%). Responders had practiced for an average of 15 years, with 38% of family physicians reporting that they had practiced for less than 9 years. Most practices differed from the type proposed by the Network (72%). Table <xref ref-type="table" rid="T3">3</xref> shows a comparison of sample characteristics with those of the Ontario portion of the National Family Physician Workforce Survey. Responders to the present survey were more likely to be younger, to be women and to have practiced for less time than the Ontario responders to the national survey.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Characteristics of respondents and their practices, family physicians in Ontario, 2002.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" colspan="2"><bold>Characteristic</bold></td><td align="center"><bold>N</bold></td><td align="center"><bold>(%)</bold></td></tr></thead><tbody><tr><td align="left" colspan="2"><bold>Age group (missing = 66)</bold></td><td></td><td></td></tr><tr><td></td><td align="left">25&#x02013;34 years</td><td align="right">101</td><td align="right">19.8</td></tr><tr><td></td><td align="left">35&#x02013;44 years</td><td align="right">198</td><td align="right">38.8</td></tr><tr><td></td><td align="left">45&#x02013;54 years</td><td align="right">155</td><td align="right">30.3</td></tr><tr><td></td><td align="left">55&#x02013;64 years</td><td align="right">57</td><td align="right">11.2</td></tr><tr><td align="left" colspan="2"><bold>Sex</bold></td><td></td><td></td></tr><tr><td></td><td align="left">Females</td><td align="right">259</td><td align="right">44.9</td></tr><tr><td></td><td align="left">Males</td><td align="right">318</td><td align="right">55.1</td></tr><tr><td align="left" colspan="2"><bold>Years in practice (missing = 90)</bold></td><td></td><td></td></tr><tr><td></td><td align="left">0&#x02013;9 years</td><td align="right">186</td><td align="right">38.2</td></tr><tr><td></td><td align="left">10&#x02013;19 years</td><td align="right">168</td><td align="right">34.5</td></tr><tr><td></td><td align="left">20&#x02013;29 years</td><td align="right">107</td><td align="right">22</td></tr><tr><td></td><td align="left">30&#x02013;39 years</td><td align="right">26</td><td align="right">5.3</td></tr><tr><td align="left" colspan="2"><bold>Practice tyype (missing = 4)</bold></td><td></td><td></td></tr><tr><td></td><td align="left">Solo</td><td align="right">169</td><td align="right">29.5</td></tr><tr><td></td><td align="left">Group</td><td align="right">318</td><td align="right">55.5</td></tr><tr><td></td><td align="left">Community Health Centre</td><td align="right">27</td><td align="right">4.7</td></tr><tr><td></td><td align="left">Health Services Organization</td><td align="right">18</td><td align="right">3.1</td></tr><tr><td></td><td align="left">Other</td><td align="right">41</td><td align="right">7.2</td></tr><tr><td align="left" colspan="2"><bold>Primary Clinical Income (missing = 4)</bold></td><td></td><td></td></tr><tr><td></td><td align="left">Fee for service</td><td align="right">464</td><td align="right">81</td></tr><tr><td></td><td align="left">Capitation</td><td align="right">29</td><td align="right">5.1</td></tr><tr><td></td><td align="left">Salary</td><td align="right">58</td><td align="right">10.1</td></tr><tr><td></td><td align="left">Other</td><td align="right">22</td><td align="right">3.8</td></tr><tr><td align="left" colspan="2"><bold>Size of practice (missing = 25)</bold></td><td></td><td></td></tr><tr><td></td><td align="left">&#x0003c;500 patients</td><td align="right">21</td><td align="right">3.8</td></tr><tr><td></td><td align="left">500&#x02013;1000 patients</td><td align="right">61</td><td align="right">11.1</td></tr><tr><td></td><td align="left">1001&#x02013;1500 patients</td><td align="right">116</td><td align="right">21</td></tr><tr><td></td><td align="left">1501&#x02013;2000 patients</td><td align="right">144</td><td align="right">26.1</td></tr><tr><td></td><td align="left">2001&#x02013;2500 patients</td><td align="right">90</td><td align="right">16.3</td></tr><tr><td></td><td align="left">&#x0003e;2500 patients</td><td align="right">120</td><td align="right">21.7</td></tr><tr><td align="left" colspan="2"><bold>Percent time in clinical practice (missing = 2)</bold></td><td></td><td></td></tr><tr><td></td><td align="left">&#x0003c;25%</td><td align="right">12</td><td align="right">2.1</td></tr><tr><td></td><td align="left">25&#x02013;50%</td><td align="right">29</td><td align="right">5.1</td></tr><tr><td></td><td align="left">51&#x02013;75%</td><td align="right">103</td><td align="right">17.9</td></tr><tr><td></td><td align="left">76&#x02013;100%</td><td align="right">431</td><td align="right">75</td></tr><tr><td align="left"><bold>Use computer (missing = 12)</bold></td><td></td><td></td><td></td></tr><tr><td></td><td align="left">Billing only</td><td align="right">169</td><td align="right">29.9</td></tr><tr><td></td><td align="left">Billing, scheduling &#x00026; registration</td><td align="right">337</td><td align="right">59.5</td></tr><tr><td></td><td align="left">Full electronic record</td><td align="right">59</td><td align="right">10.4</td></tr><tr><td align="left"><bold>Out of hours service*</bold></td><td></td><td></td><td></td></tr><tr><td></td><td align="left">Use on-call physician</td><td align="right">357</td><td align="right">61.9</td></tr><tr><td></td><td align="left">Sign-out to emergency</td><td align="right">180</td><td align="right">31.2</td></tr><tr><td></td><td align="left">Sign-out to walk-in clinic</td><td align="right">154</td><td align="right">26.7</td></tr><tr><td></td><td align="left">Week-day evening hours</td><td align="right">155</td><td align="right">26.9</td></tr><tr><td></td><td align="left">Week-end evening hours</td><td align="right">84</td><td align="right">14.6</td></tr></tbody></table><table-wrap-foot><p>* More than one response allowed, so numbers don't add up to 577.</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Comparison of responders characteristics with the Ontario component of the National Family Physician Workforce Survey, 2001.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left" colspan="2"><bold>Characteristic</bold></td><td align="center"><bold>N</bold></td><td align="center"><bold>(%)</bold></td><td align="center"><bold>N</bold></td><td align="center"><bold>(%)</bold></td></tr></thead><tbody><tr><td align="left" colspan="2"><bold>Age</bold></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td align="left">25&#x02013;34 years</td><td align="right">101</td><td align="right">19.8</td><td align="right">760</td><td align="right">8.7</td></tr><tr><td></td><td align="left">35&#x02013;44 years</td><td align="right">198</td><td align="right">38.8</td><td align="right">2629</td><td align="right">29.7</td></tr><tr><td></td><td align="left">45&#x02013;54 years</td><td align="right">155</td><td align="right">30.3</td><td align="right">2958</td><td align="right">33.5</td></tr><tr><td></td><td align="left">55&#x02013;64 years</td><td align="right">57</td><td align="right">11.2</td><td align="right">1606</td><td align="right">18.4</td></tr><tr><td></td><td align="left">65+</td><td align="right">--</td><td align="right">--</td><td align="right">781</td><td align="right">8.9</td></tr><tr><td></td><td align="left">Missing</td><td align="right">66</td><td></td><td align="right">214</td><td></td></tr><tr><td align="left" colspan="2"><bold>Sex</bold></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td align="left">Females</td><td align="right">259</td><td align="right">44.9</td><td align="right">2778</td><td align="right">31.8</td></tr><tr><td></td><td align="left">Males</td><td align="right">318</td><td align="right">55.1</td><td align="right">5951</td><td align="right">68.2</td></tr><tr><td></td><td align="left">Missing</td><td align="right">0</td><td></td><td align="right">112</td><td></td></tr><tr><td align="left" colspan="2"><bold>Years in practice</bold></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td align="left">0&#x02013;9 years</td><td align="right">186</td><td align="right">38.2</td><td align="right">1699</td><td align="right">19.4</td></tr><tr><td></td><td align="left">10&#x02013;19 years</td><td align="right">168</td><td align="right">34.5</td><td align="right">2827</td><td align="right">32.3</td></tr><tr><td></td><td align="left">20&#x02013;29 years</td><td align="right">107</td><td align="right">22</td><td align="right">2424</td><td align="right">27.7</td></tr><tr><td></td><td align="left">30&#x02013;39 years</td><td align="right">26</td><td align="right">5.3</td><td align="right">1297</td><td align="right">14.8</td></tr><tr><td></td><td align="left">40+ years</td><td align="right">--</td><td></td><td align="right">497</td><td align="right">5.7</td></tr><tr><td></td><td align="left">Missing</td><td align="right">90</td><td></td><td align="right">98</td><td></td></tr></tbody></table></table-wrap><p>The frequency distribution of practice characteristics is presented in Table <xref ref-type="table" rid="T2">2</xref>. The majority of family physicians (56%) worked in group practices, while the remainder worked in solo practices (30%), 'other' practices (7%), community health centres (5%), or health services organizations (3%). A substantial majority (81%) reported that they were remunerated on a fee-for-service basis &#x02013; of the remainder, 10% were salaried, 5% were paid on a capitation basis and 4% were 'other'. Practice sizes were: less than 500 patients (4%); 500&#x02013;1000 patients (11%), 1001&#x02013;1500 patients (21%), 1501&#x02013;2000 patients (26%), 2001&#x02013;2500 patients (16%) and greater than 2500 patients (22%). The majority of family physicians (75%) spent between 76&#x02013;100% of their time in clinical practice. All family physicians reported using a computer in their practice; 60% for billing, scheduling and registration; 30% for billing and only 10% reported that they kept full electronic records.</p><p>The level of agreement with statements about primary care reform is presented in Table <xref ref-type="table" rid="T4">4</xref>. A substantial majority (72%) of physicians did not expect to join the Network by 2004 or by some later date after 2004 (60%). If the intermediate category, 'slightly disagree' was included, these estimates rose to 84% and 76% respectively. The proportion of persons who reported that they never expected to join the network was 82%. Thirty six percent of responders either agreed or strongly agreed with the statement that they did not understand the Network well enough to make an informed decision about it. Forty eight percent of responders agreed or strongly agreed that the current system for the organization of primary care delivery required change and many responders (60%) agreed or strongly agreed with the statement that the financing of primary care requires change. Nevertheless, few responders agreed with the statement that capitation (10%) or rostering (23%) would improve primary care in Ontario. Many responding physicians (68%) believed that appropriate incentives would enhance prevention. Thirty percent of responders agreed or strongly agreed that patients should always have access to extended office hours, while 42% agreed or strongly agreed that patients should have access to a telephone health line. Half the responders (51.9%) agreed or strongly agreed that they would like to see computer systems replace papers systems in their practice.</p><table-wrap position="float" id="T4"><label>Table 4</label><caption><p>Percent level of agreement with statements about primary care reform.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold>Statement</bold></td><td align="left"><bold>Strongly agree</bold></td><td align="left"><bold>Agree</bold></td><td align="left"><bold>Slightly agree</bold></td><td align="left"><bold>Slightly disagree</bold></td><td align="left"><bold>Disagree</bold></td><td align="left"><bold>Strongly disagree</bold></td></tr></thead><tbody><tr><td></td><td align="left"><bold>%</bold></td><td align="left"><bold>%</bold></td><td align="left"><bold>%</bold></td><td align="left"><bold>%</bold></td><td align="left"><bold>%</bold></td><td align="left"><bold>%</bold></td></tr><tr><td colspan="7"><hr></hr></td></tr><tr><td align="left"><bold>I understand the Ontario Family Health Network well enough to make informed decisions about my involvement (n = 574)</bold></td><td align="left">8.9</td><td align="left">27.7</td><td align="left">18.3</td><td align="left">9.1</td><td align="left">24.4</td><td align="left">11.7</td></tr><tr><td align="left"><bold>I expect to be part of the Ontario Family Health Network by 2004 (n = 562)</bold></td><td align="left">3.1</td><td align="left">4.3</td><td align="left">8.7</td><td align="left">12.1</td><td align="left">33.3</td><td align="left">38.6</td></tr><tr><td align="left"><bold>I expect to be part of the Ontario Family Health Network at some time after 2004 (n = 549)</bold></td><td align="left">1.8</td><td align="left">5.8</td><td align="left">16.4</td><td align="left">16.4</td><td align="left">27.7</td><td align="left">31.9</td></tr><tr><td align="left"><bold>The current system for the organization of primary care delivery in Ontario needs to be changed (n = 568)</bold></td><td align="left">14.4</td><td align="left">33.1</td><td align="left">26.8</td><td align="left">7.8</td><td align="left">12.7</td><td align="left">5.3</td></tr><tr><td align="left"><bold>The current system for the financing of primary care delivery in Ontario needs to be changed (n = 567)</bold></td><td align="left">25.2</td><td align="left">34</td><td align="left">19.6</td><td align="left">6.2</td><td align="left">10.2</td><td align="left">4.8</td></tr><tr><td align="left"><bold>A capitation based formula for funding physician services would improve primary care in Ontario (n = 556)</bold></td><td align="left">3.4</td><td align="left">6.5</td><td align="left">17.3</td><td align="left">14.6</td><td align="left">31.7</td><td align="left">26.6</td></tr><tr><td align="left"><bold>A roster that links a patient to a single care provider would improve primary care in Ontario (n = 564)</bold></td><td align="left">6</td><td align="left">16.5</td><td align="left">27.5</td><td align="left">9.6</td><td align="left">22.2</td><td align="left">18.3</td></tr><tr><td align="left"><bold>Appropriate financial incentives would enhance preventive interventions in primary care (n = 567)</bold></td><td align="left">23.5</td><td align="left">44.4</td><td align="left">19.6</td><td align="left">3.4</td><td align="left">6.4</td><td align="left">2.8</td></tr><tr><td align="left"><bold>Patients should always have access to extended weeknight and weekend office hours (n = 568)</bold></td><td align="left">4.4</td><td align="left">25.9</td><td align="left">22.2</td><td align="left">13.2</td><td align="left">24.5</td><td align="left">9.9</td></tr><tr><td align="left"><bold>A telephone health line staffed by a qualified nurse is a good resource to direct patients to appropriate care (n = 569)</bold></td><td align="left">9</td><td align="left">33.2</td><td align="left">28.3</td><td align="left">8.6</td><td align="left">13.9</td><td align="left">7</td></tr><tr><td align="left"><bold>I would like to see computer systems replace most of the paper systems in my practice (n = 563)</bold></td><td align="left">22.6</td><td align="left">29.3</td><td align="left">20.4</td><td align="left">8.5</td><td align="left">13</td><td align="left">6.2</td></tr></tbody></table></table-wrap><p>Odds ratios and their associated 95% confidence intervals are presented in Table <xref ref-type="table" rid="T5">5</xref>. Younger physicians were less likely (OR = 0.62) to agree that rostering would improve primary care and that patients should have access to extended hours (OR = 0.49). Physicians in practice 9 years or less, were 1.6 times more likely to expect to join the Network by 2004, or after 2004 (OR = 1.67). Solo practice physicians were less likely (OR = 0.65) to agree with the idea of extended hours and of computers replacing paper systems (OR = 0.41). Physicians in small practices were less likely (0.64) to understand the reforms and to favour telephone health lines (OR = 0.65). They were 1.49 times more likely to report that they expected to join the Network after 2004. Those physicians remunerated on a fee-for-service basis were less likely to agree with statements about joining by 2004 (OR = 0.25), or after 2004 (OR = 0.47), that the organization required changing (OR = 0.29), favour capitation (OR = 0.27), rostering (OR = 0.34), financial incentives for prevention (OR = 0.41), or extended access for patients (OR = 0.58). Those who derived less than 75% of their income from clinical practice, were less likely to understand the reforms (0.65), much more likely to agree that the financing of primary care required changing (OR = 2.38), agree with both capitation (OR = 1.83) and rostering (OR = 1.77), and favour extended access for patients (OR = 1.58).</p><table-wrap position="float" id="T5"><label>Table 5</label><caption><p>Statistically significant relationship between characteristic of family physicians and their level of agreement with statements about primary care reform, odds ratios with their 95% confidence intervals.</p></caption><table frame="hsides" rules="groups"><thead><tr><td></td><td align="left"><bold>Age group</bold></td><td align="left"><bold>Years in practice</bold></td><td align="left"><bold>Practice type</bold></td><td align="left"><bold>Size of practice</bold></td><td align="left"><bold>Primary clinical Incomee</bold></td><td align="left"><bold>Percenttime in clinical practice</bold></td></tr></thead><tbody><tr><td></td><td align="left"><bold>&#x0003c; 44 vs. 45+ years</bold></td><td align="left"><bold>0&#x02013;9 vs. 10+ years</bold></td><td align="left"><bold>Solo vs. all others *</bold></td><td align="left"><bold>&#x0003c;1500 vs. 1500+ patients</bold></td><td align="left"><bold>Fee-for-service vs. all others**</bold></td><td align="left"><bold>&#x0003c;75% vs. 75%+</bold></td></tr><tr><td colspan="7"><hr></hr></td></tr><tr><td align="left"><bold>Understand the network enough to make informed decision about involvment</bold></td><td></td><td></td><td></td><td align="left">0.64 (0.46&#x02013;0.90)</td><td></td><td align="left">0.65 (0.45&#x02013;0.95)</td></tr><tr><td align="left"><bold>Expect to be part of network by 2004</bold></td><td></td><td align="left">1.60 (1.03&#x02013;2.50)</td><td></td><td></td><td align="left">0.25 (0.16&#x02013;0.40)</td><td></td></tr><tr><td align="left"><bold>Expect to be part of network afer 2004</bold></td><td></td><td align="left">1.67 (1.14&#x02013;2.45)</td><td></td><td align="left">1.49 (1.03&#x02013;2.16)</td><td align="left">0.47 (0.31&#x02013;0.73)</td><td></td></tr><tr><td align="left"><bold>Oorganization of primary care needs changing</bold></td><td></td><td></td><td></td><td></td><td align="left">0.29 (0.15&#x02013;0.55)</td><td></td></tr><tr><td align="left"><bold>Financing primary care needs changing</bold></td><td></td><td></td><td></td><td></td><td></td><td align="left">2.38 (1.37&#x02013;4.14)</td></tr><tr><td align="left"><bold>Capitation would improve primary care</bold></td><td></td><td></td><td></td><td></td><td align="left">0.27 (0.17&#x02013;0.41)</td><td align="left">1.83 (1.24&#x02013;2.72)</td></tr><tr><td align="left"><bold>Rostering would improve primary care</bold></td><td align="left">0.62 (0.45&#x02013;0.87)</td><td></td><td></td><td></td><td align="left">0.34 (0.27&#x02013;0.53)</td><td align="left">1.77 (1.21&#x02013;2.56)</td></tr><tr><td align="left"><bold>Financial incentives would preventive care</bold></td><td></td><td></td><td></td><td></td><td align="left">0.41 (0.18&#x02013;0.93)</td><td></td></tr><tr><td align="left"><bold>Patients should have weeknight and weekend access</bold></td><td align="left">0.49 (0.35&#x02013;0.69)</td><td></td><td align="left">0.65 (0.45&#x02013;0.93)</td><td></td><td align="left">0.58 (0.38&#x02013;0.89)</td><td align="left">1.58 (1.08&#x02013;2.33)</td></tr><tr><td align="left"><bold>Telephone health line is a good resource</bold></td><td></td><td></td><td></td><td align="left">0.65 (0.45&#x02013;0.95)</td><td></td><td></td></tr><tr><td align="left"><bold>Favours computer systems to replace paper</bold></td><td></td><td></td><td align="left">0.41 (0.28&#x02013;0.61)</td><td></td><td></td><td></td></tr></tbody></table><table-wrap-foot><p>* All other type of practices includes: group, community health centre, health services organization and other. ** All other type of primary income includes:capitation, salary and other.</p></table-wrap-foot></table-wrap><p>Three possible explanations for a physicians intention to join the Network were assessed. Neither a lack of understanding of the reform initiative (OR = 1.5, 95% C.I.s 0.96&#x02013;2.3), nor the possibility that physicians were already practising in a manner similar to that proposed by the Network, were statistically significant explanations for the stated intention to join the Network (OR = 1.3, 95% C.I.s 0.6&#x02013;2.9). The main factor associated with a physicians intent to join by 2004 was related to method of payment &#x02013; physicians who favour capitation were 5.5 times more likely to report that they expected to join the Network by 2004 (O.R. = 5.5, 95% C.I.s 3.5&#x02013;8.7).</p></sec><sec><title>Discussion</title><p>This study has described the views of physicians about primary care reform in Ontario. While many physicians recognize the need for change in both the organization and financing of primary care, the majority of physicians do not expect to join the Network, and half of them do not understand the Network enough to make an informed decision about participating. Physicians are divided on issues such as patient rostering and extended hours, although many support the idea of financial incentives for preventive interventions or a telephone health line. Whether or not a physician was remunerated on a fee-for-service basis strongly influenced their views on reform. Physicians on fee-for-service were less likely to: report that they would join the Network; believe that the organization of primary care required changing; support rostering or capitation; favour financial incentives for prevention; or support extended access for patients. Physicians who spent less than 75% of their work in clinical practice were more likely to agree that the financing of primary care required changing, and to favour both capitation, rostering and extended access for patients. Physicians who favour capitation were 5.5 times more likely to report that they intended to join the Network by 2004. Although many responders did not understand the Network, the only statistically significant explanation was that physicians did not support capitation.</p><p>The limitations of this study ought to be considered before any conclusions be drawn. These results may be limited by the representativeness of the sample and by the reliability and validity of the questions used to determine views about reform. The representativeness of this sample is influenced by sampling bias and response bias. Our survey was based on a 20% random sample of the Ontario College of Family Physicians, that represents 65% of practicing family physicians in Ontario. This sampling frame excluded general practitioners who are not certified in Family Medicine, but who are eligible to participate in the reforms. These physicians are likely to be older than certified family physicians and may hold different views from those sampled in this study. The extent that this exclusion may bias these results is unknown, although that it may be minimal is suggested by the finding that non-certified general practitioners were equally likely to join a pilot primary care reform site as those who were members of the Ontario College of Family Physicians [<xref ref-type="bibr" rid="B10">10</xref>]. Responders in our survey were more likely to be younger, to be women and to have practised for less time, than physicians who responded (53%) to the Ontario portion of the National Family Physician Workforce Survey. While, we acknowledge the possibility of sampling bias, we feel that the views of younger physicians may be more relevant because they are more likely to be the ones who are targets of the reform initiative. The low response rate (50%) was expected as it is well known that busy clinicians frequently do not complete questionnaires. Nevertheless, our response rate is comparable to that of the National Family Physician Workforce survey (53%) and a recently published survey of Ontario family physicians (47%) [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B13">13</xref>].</p><p>Second, the way that the questions were phrased could bias the results. The questions used to determine the views of physicians were developed by the authors for this study and were not tested for their reliability and validity. Nevertheless, the questions were developed by the authors, whose expertise ranged from questionnaire design (DH), to primary care research (MG) and family medicine (SS, MG) and reflect the stated objectives of the Ontario initiative. We believe they had face validity. Interpretation of these results was based on the extent that responders agreed or strongly agreed with the statements. Depending on whether the "slightly agreed" category was included, interpretation of the results may differ &#x02013; for example the majority agree with all but three statements (those about whether physicians expected to join the network and about capitation). A third limitation is that the survey was conducted a few months after the initiative was launched. The diffusion of innovations tends to have an initial latent phase before rapidly accelerating adoption. While it is possible that our survey only reflects this latency period, the fact that more than a year after the Network launch, only 5% of eligible physicians have joined, suggests that we have captured long-term intentions.</p><p>What are the implications of these findings for reform initiatives in Ontario? The majority of family physicians reported that they were unlikely to participate in the Network because they were opposed to capitation and patient rosters. In a study of the pilot phase of Ontario primary care reform physicians in Ontario's capitated Health Service Organizations were no more likely to join than fee-for-service physicians[<xref ref-type="bibr" rid="B10">10</xref>]. In contrast, we found that a physicians support for capitation was associated with their intent to join the Network. Capitation payment is a key element in primary care restructuring not only in Canada but also in many foreign jurisdictions[<xref ref-type="bibr" rid="B14">14</xref>]. It was the funding method officially endorsed by the Health Services Restructuring Commission in 1999 and is the dominant element in the Network remuneration scheme[<xref ref-type="bibr" rid="B15">15</xref>]. Nevertheless, in Ontario almost 95% of physicians are currently paid by fee-for-service[<xref ref-type="bibr" rid="B1">1</xref>]. For them, capitation may be associated with the unpopular principle in Ontario's Health Service Organizations where payment for patients attending a source of primary care outside the practice is withheld for that month. It has also been suggested that capitation may lead to loss of autonomy [<xref ref-type="bibr" rid="B16">16</xref>]. Finally, it may be that Ontario family physicians disapprove of capitated practice because they just don't like change, a view supported by their reaction to many elements in contemporary health reform [<xref ref-type="bibr" rid="B9">9</xref>]. Opposition to capitation is not confined to Ontario practitioners. In the USA, physicians who were introduced to partial capitation funding had strong negative views of the method and, while these views tended to moderate with time, capitation continued to be rated far below fee-for-service [<xref ref-type="bibr" rid="B17">17</xref>,<xref ref-type="bibr" rid="B18">18</xref>].</p><p>Currently the vast majority of family physicians operate on a fee-for-service basis, with a significant number in solo practice. The decision not to be part of the network does not mean the physician will not be providing service. This may inhibit the incorporation of innovations, such as nurse practitioners or the electronic medical record, into existing practices with attendant implications for health human resources and quality of care respectively. Moreover, existing models of practice lack the capacity to address distributional issues. If all citizens were rostered to specific practices, there would be competition for patients. Some physicians now located in the urban south might be forced under such a capitated payment scheme to migrate to less serviced areas in order to acquire an adequate list of patients.</p><p>Many physicians reported that they did not fully understand the Network and may be unaware that it allows for a blended payment scheme including both capitation and fee-for-service. Since physicians may equate primary care reform largely with a switch to capitated payment, the successful recruitment to the Network will require the education of physicians about payment. Borrowing from strategy used to introduce Health Services Organizations, government suggested a process to compare the fee-for-service billings of potential recruits with their possible earnings under Network rules [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B16">16</xref>]. Additionally, they need to emphasize that a selection of retained fee-for-service billing codes and the system of target achievement bonuses brings the Network's payment formula closer to a blended scheme favoured by many physicians [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. The key message, however, is that experience from many countries confirms that primary care reform does not succeed without the active support of the physicians involved [<xref ref-type="bibr" rid="B14">14</xref>]. The alternative is to see the Network fail to attract widespread participation, as did its Health Services Organization and Community Health Centre predecessors [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B20">20</xref>]. Since the launch of the Ontario Family Health Network, government has introduced a variant of fee-for-services with a number of incentives, known as family health groups that are proving more popular than the family health networks.</p></sec><sec><title>Conclusions</title><p>The results of this survey, conducted five months after the initial offering of primary care reform agreements to all Ontario physicians, suggest that a 80% enrolment target is unrealistic.</p></sec><sec><title>Competing interests</title><p>None declared.</p></sec><sec><title>Authors' contributions</title><p>DH was the principal investigator of the study and takes responsibility for the integrity of the work as a whole. SEDS, PW, and MG made substantial contributions to the conception and design of the study, to the acquisition of data, and to the analysis and interpretation of data. All authors critically reviewed drafts of the manuscript and have approved the final version.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1471-2296/5/2/prepub"/></p></sec>
Expression of the 60 kDa and 71 kDa heat shock proteins and presence of antibodies against the 71 kDa heat shock protein in pediatric patients with immune thrombocytopenic purpura
<sec><title>Background</title><p>Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by platelet destruction resulting from autoantibodies against platelet proteins, particularly platelet glycoprotein IIb/IIIa. Heat shock proteins (Hsp) have been shown to be major antigenic determinants in some autoimmune diseases. Antibodies to Hsps have also been reported to be associated with a number of pathological states.</p></sec><sec sec-type="methods"><title>Methods</title><p>Using western blot, we measured the levels of the 60 kDa heat shock protein (Hsp60) and of the inducible 71 kDa member of the Hsp70 family (Hsp71) in lymphocytes and the presence of antibodies against these hsps in plasma of 29 pediatric patients with ITP before the treatment and in 6 other patients before and after treatment.</p></sec><sec><title>Results</title><p>Interestingly only one out of 29 patients showed detectable Hsp60 in lymphocytes while this heat shock protein was detected in the 30 control children. Hsp71 levels were slightly lower in lymphocytes of patients with ITP than in controls (1567.8 &#x000b1; 753.2 via 1763.2 &#x000b1; 641.8 integrated optical density (IOD) units). There was a small increase of Hsp71 after recovery from ITP. The titers of plasma antibodies against Hsp60 and Hsp71 were also examined. Antibodies against Hsp71 were more common in ITP patients (15/29) than in control children (5/30). The titer of anti-Hsp71 was also higher in children patients with ITP. The prevalence of ITP children with antibodies against Hsp71 (51.7%) was as high as those with antibodies against platelet membrane glycoproteins (58.3%).</p></sec><sec><title>Conclusions</title><p>In summary, pediatric patients with ITP showed no detectable expression of Hsp60 in lymphocytes and a high prevalence of antibody against Hsp71 in plasma. These changes add to our understanding of the pathogenesis of ITP and may be important for the diagnosis, prognosis and treatment of ITP.</p></sec>
<contrib id="A1" equal-contrib="yes" contrib-type="author"><name><surname>Xiao</surname><given-names>Chengfeng</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" equal-contrib="yes" contrib-type="author"><name><surname>Chen</surname><given-names>Sheng</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Yuan</surname><given-names>Mingchun</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>Ding</surname><given-names>Fuyue</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A5" contrib-type="author"><name><surname>Yang</surname><given-names>Dongliang</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A6" contrib-type="author"><name><surname>Wang</surname><given-names>Ruibo</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A7" contrib-type="author"><name><surname>Li</surname><given-names>Jianxin</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A8" contrib-type="author"><name><surname>Tanguay</surname><given-names>Robert M</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib><contrib id="A9" corresp="yes" contrib-type="author"><name><surname>Wu</surname><given-names>Tangchun</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib>
BMC Blood Disorders
<sec><title>Background</title><p>Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by a low platelet count secondary to accelerated platelet destruction by antiplatelet antibodies that generally recognize platelet membrane glycoproteins (GPs) [<xref ref-type="bibr" rid="B1">1</xref>]. The triggering or immunogenic stimulus involved and the role of antiplatelet antibodies in the disease remain unclear. There is an unbalanced immune response due to inflammation (i.e., viral infection, autoimmunity), or to exposure to environmental agents (i.e., drug, H<sub>2</sub>O<sub>2</sub>) [<xref ref-type="bibr" rid="B2">2</xref>-<xref ref-type="bibr" rid="B6">6</xref>]. Whether the stimulus is endogenous, i.e. truly "self" or exogenous ("non-self") is unknown. Some recent evidence argues for an immune-mediated mechanism in ITP-increased HLA-DR expression, defects in cellular and humoral immunity, and specific autoantibody production. There are several forms of management of ITP ranging from drugs such as corticosteroids, a variety of immunosuppressants and immunoglobulins to splenectomy [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>]. However some patients may be receiving unnecessary treatment especially in the case of pediatric patients [<xref ref-type="bibr" rid="B8">8</xref>] as the cause and etiopathogenesis of ITP remain unknown.</p><p>Heat shock proteins (Hsp) are highly conserved proteins found in prokaryotes and eukaryotes. Induction of Hsps can be triggered by many stresses. These include exposure to supraoptimal temperatures (heat shock) and to various chemicals (xenobiotics, drugs, growth factors, hormones). Pathological states incurring during viral, bacterial or parasitic infections, fever, inflammation, malignancy or autoimmunity, can also induce an increase in synthesis of Hsps [<xref ref-type="bibr" rid="B9">9</xref>-<xref ref-type="bibr" rid="B12">12</xref>]. Many Hsps are also expressed at low levels under normal physiological conditions. Hsps are usually grouped into several families (Hsp110, Hsp90, Hsp/Hsc70 (Hsc: Heat shock cognate), Hsp60, Hsp47 and the small Hsps (Hsp10-30) on the basis of their apparent molecular masses after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Hsps can act as intracellular chaperones of naive, aberrantly folded or mutated proteins, as stimulators of cytokine signal transduction cascades, as well as in cytoprotection against the stress stimuli described above [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B13">13</xref>-<xref ref-type="bibr" rid="B15">15</xref>]. A role of Hsps in the inflammatory response is suggested by different findings. Hsps participate in cytokine signal transduction and in the control of cytokine gene expression [<xref ref-type="bibr" rid="B16">16</xref>]. Hsps enhance antigen presentation to T lymphocytes, can be displayed on the surface of cells and may be important in targeting cytotoxic cells [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B17">17</xref>]. Moreover, recent investigations also indicate that Hsps have the ability to modulate the cellular immune response since as cellular chaperones, they participate in transport through the various cellular compartments. Thus Hsps may act in transfer of peptides between cellular compartments, and in binding endogenous antigenic peptides and transporting them to the major histocompatibility complexes [<xref ref-type="bibr" rid="B18">18</xref>-<xref ref-type="bibr" rid="B20">20</xref>]. Hsps have also been reported to be involved in tumorogenesis and in inflammation [<xref ref-type="bibr" rid="B21">21</xref>-<xref ref-type="bibr" rid="B23">23</xref>].</p><p>Many investigations have shown that Hsps and autoantibodies to these proteins play a role in the pathogenesis and/or prognosis of certain diseases [<xref ref-type="bibr" rid="B24">24</xref>-<xref ref-type="bibr" rid="B31">31</xref>]. Since an important component of ITP is mediated by autoantibodies, we decided to examine if antibodies against Hsps could be detected in pediatric patients with ITP. We also checked if ITP was accompanied by fluctuations in the levels of Hsp60, of the inducible member of the Hsp70 family referred to here as Hsp71 and if the expression of these Hsps changed after recovery from ITP. The data show that Hsp60 is not detectable in lymphocytes of most pediatric ITP patients. Whether the expression of Hsp60 was much lower or totally absent in the ITP children is unknown at the moment. Finally these patients show a high prevalence of antibody against Hsp71 that may be of clinical significance.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Subjects and groups</title><p>The present study comprised two different groups. The first ITP group consisted of 29 ITP pediatric patients aged from 7 to 10 years (male: 18, female: 11) and was conducted from March to November of 2000 in Wuhan. The second group consisted of 6 different ITP pediatric patients aged from 7 to 10 years and was conducted in March and April of 2001. All patients were diagnosed according to criteria and principles of management of ITP defined by the Chinese Association of Hematology. This diagnosis is mainly based on the medical history, symptoms and signs, physical examination, peripheral blood smear, and bone marrow aspiration. Patients who were referred with bleeding complaints (easy bruising, purpura, mucosal bleeding) and had isolated thrombocytopenia with no history of other clinical conditions that can cause thrombocytopenia were included in our investigation. At the time of the ITP diagnosis, patients were excluded from further study if they had a history or clinical findings of antiphospholipid syndrome, systemic lupus erythematosus, other autoimmune disorders, acquired immunodeficiency syndrome, or malignancies. The control group comprised 30 children (15 males, 15 females) aged from 7 to 8 years (mean age: 7.5) who had no diseases after physical examination and had no history of respiratory and virus infection in the preceding two weeks. We also measured changes of Hsp71 levels in lymphocytes of the second group of 6 patients with ITP at admission and after recovery. Treatments consisted of dexamethasone administration followed by prednisone. None of the patients received blood transfusion.</p></sec><sec><title>Evaluation of health status</title><p>Health status was evaluated in all patients and controls using a questionnaire and by clinical examination and laboratory determinations. The questionnaire designed according to diagnostic criteria and principles of ITP, with emphasis on disease history including family history, symptoms, use of drug and food, was answered by the child and/or parent. The clinical examination included a general examination (signs), weight, height, pulse, oral temperature, electrocardiography and chest x-ray. Laboratory tests included the regular analyses of blood and urine (haemoglobin, blood typing, platelet and white blood cell count) and, hepatic functional tests (GPT, GOT, hepatitis B antigens and antibodies).</p></sec><sec><title>Detection of Hsp60 and Hsp71 in lymphocytes</title><p>Blood samples were collected from all pediatric ITP patients before any treatment was given. Additional blood samples were collected from the second group of 6 patients after treatment. About 5 ml of venous blood were collected and divided into two aliquots, one to separate plasma and lymphocytes for Hsp analysis and the other aliquot for the routine laboratory tests mentioned above. Plasma from heparinized whole blood was collected, sedimented cells suspended in 3.0 ml of normal saline and lymphocytes were isolated using Ficoll-Hypaque (Biochemical Reagent Co., Shanghai, China). The collected lymphocytes were washed twice with PBS, counted and the concentrations of lymphocytes adjusted to 10,000 per ul with PBS. 10 ul of lymphocyte suspension was centrifuged and cells lysed in SDS-sample buffer by boiling at 100&#x000b0;C for 5 min, and proteins loaded on SDS-PAGE gels as described previously [<xref ref-type="bibr" rid="B28">28</xref>-<xref ref-type="bibr" rid="B30">30</xref>]. After electrophoresis, proteins were transferred electrophoretically to nitrocellulose membranes [<xref ref-type="bibr" rid="B28">28</xref>-<xref ref-type="bibr" rid="B30">30</xref>,<xref ref-type="bibr" rid="B32">32</xref>]. Transfer was monitored by staining with Ponceau Red. The membrane was saturated with blocking buffer (PBS containing 5% skim milk powder) for 1 h at 37&#x000b0;C with gentle agitation and washed with PBS-0.05% Tween 80 for 5 min. It was then incubated at 37&#x000b0;C for 1 h with gentle agitation with rabbit anti-human Hsp60 or anti-human Hsp71 antibodies [see [<xref ref-type="bibr" rid="B32">32</xref>]] diluted 1:1000 in PBS containing 5% skim milk powder. After washing the membranes six times (6 &#x000d7; 10 min) with 10 ml PBS-0.05% Tween 80, horseradish peroxydase (HRP) conjugated goat anti-rabbit IgG in blocking buffer (1:1000) was added and the membranes further incubated at 37&#x000b0;C for 1 h. Membranes were finally washed four times with 10 ml PBS-0.05% Tween 80. The presence of Hsp60 and Hsp71 was revealed using DAB (3,3-diaminobenzidine tetrahydrochloride) buffer for 3&#x02013;5 min [<xref ref-type="bibr" rid="B29">29</xref>,<xref ref-type="bibr" rid="B30">30</xref>,<xref ref-type="bibr" rid="B32">32</xref>]. Hsp60 and Hsp71 were quantified using an imaging densitometer (Shimadzu CS-930, Shimadzu, Japan) and integrated optical density (IOD) was used to represent the relative levels of these two proteins.</p></sec><sec><title>Determination of antibodies to Hsp60 and Hsp71 in plasma of subjects</title><p>Recombinant human Hsp60 was obtained through the expression of human Hsp60 cDNA in Escherichia coli BL2 (DE3) cells using a pET vector. Recombinant inducible human Hsp71 was obtained through the expression of the human cDNA coding for the inducible Hsp71 in NaCl-induced E. coli GJ1168 cells using pET30 as expression vector [<xref ref-type="bibr" rid="B32">32</xref>,<xref ref-type="bibr" rid="B33">33</xref>]. Approximately 10&#x02013;15 &#x003bc;g of recombinant human Hsp60 or Hsp71 was loaded on each SDS-PAGE gel without combs and transferred electrophoretically to nitrocellulose membranes [<xref ref-type="bibr" rid="B28">28</xref>]. The band containing Hsp60 or Hsp71 was cut into 2 mm &#x000d7; 3 mm pieces and marked with a small red dot on the protein side of the membrane. The pieces were placed in individual wells of an ELISA plate, rinsed with PBS, and saturated with 100 &#x003bc;l of blocking buffer (PBS containing 5% skim milk powder) for 1 h at 37&#x000b0;C with gentle agitation and washed with PBS-0.05% Tween 80 for 5 min. The plasma diluted 1:10, 1:20, 1:40 or 1:80 in 100 &#x003bc;l PBS containing 5% skim milk powder was incubated with the nitrocellulose membrane pieces at 37&#x000b0;C for 2 h with gentle agitation. After washing the membranes six times (10 min each) with 200 &#x003bc;l PBS-0.05% Tween 80, 100 &#x003bc;l of HRP-conjugated goat anti-human IgG (Sigma) in blocking buffer (1:2500) was added and the incubation continued at 37&#x000b0;C for 1 h. Membranes were washed six times (10 min) with 200 &#x003bc;l PBS-0.05% Tween 80. The presence of antibodies to Hsp60 or Hsp71 was revealed using DAB (3,3-diaminobenzidine tetrahydrochloride) buffer for 3&#x02013;5 min. A brown band on the membrane piece is regarded as positive and no colour as negative [<xref ref-type="bibr" rid="B30">30</xref>].</p></sec><sec><title>Statistical analyses</title><p>Results were expressed as the mean &#x000b1; standard deviation (SD) of n samples. Where appropriate, the probability of significant differences between 2 groups for quantity was determined by the unpaired Student t test. Comparison between groups for frequency of an event was done by the Chi-square test/Fisher exact test. <italic>P </italic>&#x0003c; 0.05 was considered significant. All data were analysed using Excel 2000 (Microsoft Corporation, Seattle, USA) and the Statistical Package for Social Sciences (SPSS Inc, Chicago, USA) software.</p></sec></sec><sec><title>Results</title><sec><title>Clinical features of ITP pediatric patients and treatment</title><p>A blood routine was performed on all pediatric patients with ITP, As expected, the number of white blood cells (7.4 &#x000b1; 3.3 &#x000d7; 10<sup>9</sup>/L) and the concentration of haemoglobin (107 &#x000b1; 16 g/L, range 93&#x02013;138) while a bit low in some patients with ITP, did not differ significantly from that of controls (range 112&#x02013;140 g/l) and were within normal values (4 to 10 &#x000d7; 10<sup>9</sup>/L and 110 to 160 g/L respectively). However the mean number of platelets (32 &#x000b1; 26, range 1.0 to 82.0 &#x000d7; 10<sup>9</sup>/L) was significantly lower than its normal value (100 to 300 &#x000d7; 10<sup>9</sup>/L) and is in the abnormal range. There was no difference in the number of platelets between male and female patients with ITP. Table <xref ref-type="table" rid="T1">1</xref> summarizes the clinical information on the 29 individual pediatric patients. Most patients (23/29) presented with acute ITP. Treatment was initiated with dexamethasone followed by prednisone in all cases. Intravenous gamma globulin (IVIG) was only given in very severe cases. Vitamin C was also provided depending on the condition of the patient (data not shown).</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Summary of data obtained for individual pediatric patients with ITP</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="right">NO</td><td align="left">Gender</td><td align="right">Age</td><td align="center">Diagnosis</td><td align="right">BPC</td><td align="right">Hsp71</td><td align="left">Hsp60</td><td align="right">Anti-Hsp71</td><td align="right">Anti-Hsp60</td></tr></thead><tbody><tr><td align="right">1</td><td align="left">F</td><td align="right">7</td><td align="right">acute</td><td align="right">76</td><td align="right">900</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">2</td><td align="left">M</td><td align="right">7</td><td align="right">acute</td><td align="right">38</td><td align="right">2120</td><td align="left">ND</td><td align="right">20</td><td align="right">-</td></tr><tr><td align="right">3</td><td align="left">F</td><td align="right">7</td><td align="right">acute</td><td align="right">5</td><td align="right">1410</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">4</td><td align="left">F</td><td align="right">8</td><td align="right">acute</td><td align="right">43</td><td align="right">860</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">5</td><td align="left">M</td><td align="right">8</td><td align="right">acute</td><td align="right">2</td><td align="right">990</td><td align="left">ND</td><td align="right">20</td><td align="right">-</td></tr><tr><td align="right">6</td><td align="left">M</td><td align="right">8</td><td align="right">acute</td><td align="right">33</td><td align="right">700</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">7</td><td align="left">M</td><td align="right">8</td><td align="right">acute</td><td align="right">2</td><td align="right">1200</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">8</td><td align="left">M</td><td align="right">8</td><td align="right">acute</td><td align="right">1</td><td align="right">1580</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">9</td><td align="left">F</td><td align="right">8</td><td align="right">acute</td><td align="right">9</td><td align="right">2490</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">10</td><td align="left">F</td><td align="right">8</td><td align="right">acute</td><td align="right">46</td><td align="right">1910</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">11</td><td align="left">M</td><td align="right">8</td><td align="right">acute</td><td align="right">6</td><td align="right">1250</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">12</td><td align="left">M</td><td align="right">8</td><td align="right">acute</td><td align="right">56</td><td align="right">1570</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">13</td><td align="left">M</td><td align="right">9</td><td align="right">acute</td><td align="right">50</td><td align="right">1020</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">14</td><td align="left">M</td><td align="right">9</td><td align="right">acute</td><td align="right">82</td><td align="right">900</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">15</td><td align="left">F</td><td align="right">9</td><td align="right">acute</td><td align="right">19</td><td align="right">1000</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">16</td><td align="left">M</td><td align="right">9</td><td align="right">acute</td><td align="right">18</td><td align="right">1600</td><td align="left">ND</td><td align="right">40</td><td align="right">80</td></tr><tr><td align="right">17</td><td align="left">M</td><td align="right">9</td><td align="right">acute</td><td align="right">64</td><td align="right">2590</td><td align="left">DET</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">18</td><td align="left">M</td><td align="right">10</td><td align="right">acute</td><td align="right">23</td><td align="right">1790</td><td align="left">ND</td><td align="right">20</td><td align="right">-</td></tr><tr><td align="right">19</td><td align="left">F</td><td align="right">10</td><td align="right">acute</td><td align="right">67</td><td align="right">880</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">20</td><td align="left">F</td><td align="right">10</td><td align="right">acute</td><td align="right">26</td><td align="right">4000</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">21</td><td align="left">M</td><td align="right">10</td><td align="right">acute</td><td align="right">44</td><td align="right">1850</td><td align="left">ND</td><td align="right">-</td><td align="right">40</td></tr><tr><td align="right">22</td><td align="left">F</td><td align="right">10</td><td align="right">acute</td><td align="right">73</td><td align="right">2210</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">23</td><td align="left">M</td><td align="right">10</td><td align="right">acute</td><td align="right">11</td><td align="right">2600</td><td align="left">ND</td><td align="right">20</td><td align="right">10</td></tr><tr><td align="right">24</td><td align="left">M</td><td align="right">7</td><td align="right">chronic</td><td align="right">4</td><td align="right">1230</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">25</td><td align="left">M</td><td align="right">8</td><td align="right">chronic</td><td align="right">4</td><td align="right">880</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">26</td><td align="left">M</td><td align="right">8</td><td align="right">chronic</td><td align="right">25</td><td align="right">1700</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">27</td><td align="left">F</td><td align="right">9</td><td align="right">chronic</td><td align="right">73</td><td align="right">1030</td><td align="left">ND</td><td align="right">-</td><td align="right">-</td></tr><tr><td align="right">28</td><td align="left">M</td><td align="right">10</td><td align="right">chronic</td><td align="right">5</td><td align="right">1300</td><td align="left">ND</td><td align="right">80</td><td align="right">-</td></tr><tr><td align="right">29</td><td align="left">F</td><td align="right">10</td><td align="right">chronic</td><td align="right">4</td><td align="right">1900</td><td align="left">ND</td><td align="right">20</td><td align="right">-</td></tr></tbody></table><table-wrap-foot><p>M: Male; F: Female; BPC: blood platelets counts; ND: not detected; DET: detected; -: negative. All blood samples were collected before medication was started.</p></table-wrap-foot></table-wrap></sec><sec><title>Levels of Hsp60 and Hsp71 in lymphocytes</title><p>Aberrant expression of Hsps has been associated with many disease states [<xref ref-type="bibr" rid="B24">24</xref>,<xref ref-type="bibr" rid="B25">25</xref>]. We therefore determined the levels of Hsp60 and the inducible Hsp71 in lymphocytes of patients with ITP by Western blot in order to find out if there was any association between Hsp expression and ITP. Figure <xref ref-type="fig" rid="F1">1</xref> shows examples of Western blots for Hsp60 and Hsp71 in lymphocytes of pediatric patients with ITP and in controls. As can be seen, Hsp60 was not detected in lymphocytes of pediatric patients with ITP while there was a variable but detectable expression of this Hsp in all controls (Fig. <xref ref-type="fig" rid="F1">1A</xref>). Throughout the present investigation, we found only one patient out of 29 (Patient no 17, Table <xref ref-type="table" rid="T1">1</xref>) who had a detectable level of Hsp60, while this Hsp was detected in all 30 individuals from the control group. The expression of Hsp60 is significantly different (<italic>P </italic>&#x0003c; 0.0001) between ITP and control groups. Figure <xref ref-type="fig" rid="F1">1B</xref> shows the expression level of Hsp71 in the same patients with ITP and in controls. The relative values of Hsp71 were quantified using an imaging densitometer and are given in IOD units in Table <xref ref-type="table" rid="T1">1</xref>. Patients with ITP tended to have a slightly lower level of Hsp71 (1567.8 &#x000b1; 753.2 IOD units) but this difference with controls (1763.2 &#x000b1; 641.8 IOD units) was not statistically significant. Patients with the acute form had a higher mean value (1627 &#x000b1; 786 IOD units, range 700&#x02013;4000) than those with the chronic form (1340 &#x000b1; 391 IOD units, range 880&#x02013;1900). This difference was not significant (<italic>P </italic>= 0.398).</p><fig position="float" id="F1"><label>Figure 1</label><caption><p><bold>Detection of Hsp60 and Hsp71 in lymphocytes of patients with ITP and in controls by Western blot </bold>A: Examples of detection of Hsp60. Lane 1:Protein marker; Lane 2&#x02013;8: Patients with ITP; Lane 9&#x02013;14: controls. B: Examples of detection of Hsp71. Lane 1:Protein marker; Lane 2&#x02013;8: Patients with ITP; Lane 9&#x02013;14: controls.</p></caption><graphic xlink:href="1471-2326-4-1-1"/></fig></sec><sec><title>Changes of Hsp60 and Hsp71 in lymphocytes before and after recovery</title><p>To find out if changes in Hsp levels were related to the state of the disease, we compared Hsp60 and Hsp71 levels in lymphocytes of a second group of six pediatric patients with ITP at admission to the hospital (before) and upon release after treatment. These six patients were hospitalized for approximately 3 weeks and were given treatments similar to the other ITP patients. None of these 6 patients showed expression of Hsp60 either before or after treatment. In the case of Hsp71, there were changes and the level of Hsp71 was higher after three weeks of treatment for ITP (1951 &#x000b1; 753 IOD units) than before (1160 &#x000b1; 727 IOD units) (<italic>P </italic>= 0.094) (Table <xref ref-type="table" rid="T2">2</xref>). This increase was accompanied by an increase of platelet count (from 35.0 &#x000b1; 30.8 to 94.5 &#x000b1; 69.5 &#x000d7; 10<sup>9</sup>/L, <italic>P </italic>= 0.084) (Table <xref ref-type="table" rid="T2">2</xref>). We also analysed the changes in the Hsp71 levels and platelet counts in these patients. As illustrated in Figure <xref ref-type="fig" rid="F2">2</xref>, five of the six patients showed an increase in the expression levels of Hsp71 and in platelet counts after treatment while a decrease of platelet counts was noted in one patient.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Changes of blood platelets counts and Hsp71 levels in pediatric patients with ITP at admission and after treatment</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="center"><bold>NO</bold></td><td align="center"><bold>Gender</bold></td><td align="center"><bold>Age</bold></td><td align="center"><bold>Diagnosis</bold></td><td align="center" colspan="2"><bold>At admission</bold></td><td align="center" colspan="2"><bold>After treatment</bold></td></tr><tr><td></td><td></td><td></td><td></td><td colspan="4"><hr></hr></td></tr><tr><td></td><td></td><td></td><td></td><td align="center"><bold>BPC</bold></td><td align="center"><bold>Hsp71</bold></td><td align="center"><bold>BPC</bold></td><td align="center"><bold>Hsp71</bold></td></tr></thead><tbody><tr><td align="center">1</td><td align="center">F</td><td align="center">10</td><td align="center">acute</td><td align="center">73</td><td align="center">1030</td><td align="center">99</td><td align="center">1850</td></tr><tr><td align="center">2</td><td align="center">M</td><td align="center">10</td><td align="center">acute</td><td align="center">6</td><td align="center">200</td><td align="center">69</td><td align="center">3060</td></tr><tr><td align="center">3</td><td align="center">F</td><td align="center">8</td><td align="center">acute</td><td align="center">29</td><td align="center">1000</td><td align="center">230</td><td align="center">2600</td></tr><tr><td align="center">4</td><td align="center">M</td><td align="center">10</td><td align="center">acute</td><td align="center">6</td><td align="center">730</td><td align="center">37</td><td align="center">1800</td></tr><tr><td align="center">5</td><td align="center">F</td><td align="center">10</td><td align="center">acute</td><td align="center">75</td><td align="center">1090</td><td align="center">23</td><td align="center">1790</td></tr><tr><td align="center">6</td><td align="center">M</td><td align="center">8</td><td align="center">acute</td><td align="center">57</td><td align="center">1310</td><td align="center">73</td><td align="center">2210</td></tr></tbody></table><table-wrap-foot><p>M: Male; F: Female; BPC: blood platelets counts. Hsp71 values are given in integrated optical density units (see methods)</p></table-wrap-foot></table-wrap><fig position="float" id="F2"><label>Figure 2</label><caption><p>Changes in the association of Hsp71 versus platelets count in patients with ITP before and after treatment.</p></caption><graphic xlink:href="1471-2326-4-1-2"/></fig></sec><sec><title>Presence of plasma antibodies against Hsp60 and Hsp71 in patients and controls</title><p>The presence of antibodies against Hsps has been reported in a number of diseases and it has been suggested that Hsps might be involved in the pathogenesis of the diseases and/or be of use for disease prognosis [<xref ref-type="bibr" rid="B28">28</xref>-<xref ref-type="bibr" rid="B31">31</xref>,<xref ref-type="bibr" rid="B34">34</xref>]. Since there were no available data for pediatric patients with ITP, we next determined if these patients had antibodies to the inducible Hsp71 and to Hsp60. As shown in Table <xref ref-type="table" rid="T3">3</xref> and <xref ref-type="table" rid="T4">4</xref>, antibodies against Hsp60 and Hsp71 were found in both normal children and in ITP patients. Over 50% of ITP patients had a positive reaction to Hsp70 as compared to 16.7% of controls (<italic>P </italic>&#x0003c; 0.05) (Table <xref ref-type="table" rid="T3">3</xref>). Moreover the titer of these antibodies was higher with more than half of the patients (9/15) showing a positive reaction at a dilution of 1:80. Figure <xref ref-type="fig" rid="F3">3</xref> shows a comparison of platelet number in the 29 ITP patients with the dilution of antibodies against Hsp71. Interestingly, the platelet counts of ITP patients showing no antibody against Hsp71 are significantly higher than those positive for antibody against this Hsp (42.8 &#x000b1; 27.4 vs 21.4 &#x000b1; 21.7 &#x000d7; 10<sup>9</sup>/L, <italic>P </italic>&#x0003c; 0.05). Finally as shown in table <xref ref-type="table" rid="T4">4</xref>, there are no differences in the incidence of antibodies against Hsp60 between patients with ITP and matched controls (<italic>P </italic>&#x0003e; 0.05).</p><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Presence of plasma antibodies against Hsp71 in patients with ITP and controls.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold>Groups</bold></td><td align="left"><bold>Number of children</bold></td><td align="left" colspan="6"><bold>Dilution of antibodies against Hsp71 (n and Percentage)</bold></td></tr><tr><td></td><td></td><td colspan="6"><hr></hr></td></tr><tr><td></td><td></td><td align="left"><bold>negative</bold></td><td align="left"><bold>positive</bold></td><td align="left"><bold>1:10</bold></td><td align="left"><bold>1:20</bold></td><td align="left"><bold>1:40</bold></td><td align="left"><bold>1:80</bold></td></tr></thead><tbody><tr><td align="left">Control</td><td align="left">30</td><td align="left">25 (83.3)</td><td align="left">5(16.7)</td><td align="left">0(0)</td><td align="left">3(10.0)</td><td align="left">0(0)</td><td align="left">2 (6.7)</td></tr><tr><td align="left">ITP</td><td align="left">29</td><td align="left">14(48.3) *</td><td align="left">15(51.7) *</td><td align="left">0(0)</td><td align="left">5(17.2)</td><td align="left">1(3.4)</td><td align="left">9 (31.0) *</td></tr></tbody></table><table-wrap-foot><p>*P &#x0003c; 0.05, compared with control; n: represents the number of children at different dilutions of antibodies against Hsp71.</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T4"><label>Table 4</label><caption><p>Presence of plasma antibodies against Hsp60 in patients with ITP and control.</p></caption><table frame="hsides" rules="groups"><thead><tr><td align="left"><bold>Groups</bold></td><td align="left"><bold>Number of children</bold></td><td align="left" colspan="6"><bold>Dilution of antibodies against Hsp60 (n and Percentage)</bold></td></tr><tr><td></td><td></td><td colspan="6"><hr></hr></td></tr><tr><td></td><td></td><td align="left"><bold>negative</bold></td><td align="left"><bold>positive</bold></td><td align="left"><bold>1:10</bold></td><td align="left"><bold>1:20</bold></td><td align="left"><bold>1:40</bold></td><td align="left"><bold>1:80</bold></td></tr></thead><tbody><tr><td align="left">Control</td><td align="left">30</td><td align="left">28(93.3)</td><td align="left">2(6.7)</td><td align="left">0(0)</td><td align="left">0(0)</td><td align="left">1(3.3)</td><td align="left">1(3.3)</td></tr><tr><td align="left">ITP</td><td align="left">29</td><td align="left">26(89.7)</td><td align="left">3(10.3)</td><td align="left">1(3.4)</td><td align="left">0(0)</td><td align="left">1(3.4)</td><td align="left">1 (3.4)</td></tr></tbody></table><table-wrap-foot><p>n: represents the number of children at different dilutions of antibodies against Hsp60.</p></table-wrap-foot></table-wrap><fig position="float" id="F3"><label>Figure 3</label><caption><p>Distribution of platelet counts (&#x000d7;10<sup>9</sup>/L) of the 29 ITP patients versus the titer of antibodies against Hsp71.</p></caption><graphic xlink:href="1471-2326-4-1-3"/></fig><p>The presence of antibodies against Hsp71 did not seem to be related to the form of ITP. Three of the six patients with the chronic form showed antibodies to Hsp71 as compared to 13 out of the 23 patients presenting with the acute form. The majority of patients in both groups had titers of anti-Hsp71 of 1:80 (66% for chronic and 61% for acute) (see Table <xref ref-type="table" rid="T1">1</xref>).</p></sec></sec><sec><title>Discussion</title><p>Many investigations have shown that the aberrant expression of Hsps or the presence of antibodies against Hsps may be involved in the pathogenesis of many diseases and/or may be of use for prognosis and treatment [<xref ref-type="bibr" rid="B24">24</xref>,<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B28">28</xref>-<xref ref-type="bibr" rid="B31">31</xref>,<xref ref-type="bibr" rid="B34">34</xref>]. ITP is an autoimmune diseases caused by platelet destruction resulting from autoantibodies against platelet surface proteins, particularly platelet glycoprotein IIb/IIIa. However, how antibodies against platelets are produced and how platelets are destroyded remains unknown. Hsps may be of importance in both immune and inflammatory responses. Whether patients with ITP exhibit different levels of Hsps and antibodies against Hsps has only now been examined.</p><p>Our data on a group of 29 pediatric patients with ITP show that there is a slightly lower level of Hsp71 (although not statistically significant) in lymphocytes of patients compared with controls (1567.8 &#x000b1; 753.2 versus 1763.2 &#x000b1; 641.8 IOD units). Interestingly, Hsp60 was only detected in 1 of the 29 pediatric patients with ITP in a western blot assay under conditions where this Hsp was detected in all controls. These data are likely to be of clinical interest for the following reasons. Firstly, Hsps can be induced by a wide variety of environmental stresses and pathological stimuli such as viral, bacterial or parasitic infections, fever, inflammation, malignancy and autoimmunity. Many of these conditions may contribute to the pathogenesis of ITP and some can also cause an imbalance of the immune response and autoimmune diseases [<xref ref-type="bibr" rid="B2">2</xref>-<xref ref-type="bibr" rid="B6">6</xref>]. Interestingly, we did not find any induction of Hsps in these patients. Secondly, induced Hsps can protect cells, organs and organisms against many damages caused by these stresses [<xref ref-type="bibr" rid="B35">35</xref>-<xref ref-type="bibr" rid="B44">44</xref>]. A low level or absence of Hsps might render cells more sensitive to such stresses.</p><p>Since Hsps also play an important role in the immune response, they could serve as an early warning to the host's immune response during the onset of disease [<xref ref-type="bibr" rid="B45">45</xref>]. Hsps require no adjuvant to confer immunogenicity to bound peptides as if they possessed an intrinsic "danger" signature [<xref ref-type="bibr" rid="B46">46</xref>]. This "danger signal" alerts the immune system to the death of a cell under stress and the role of Hsps as protein carriers allows the immune effector cells to survey the peptides released by the stressed cell and to activate against new or unrecognized peptides carried by Hsps [<xref ref-type="bibr" rid="B18">18</xref>]. Hsp60 and Hsp71 not only shuttle immunogenic peptides [<xref ref-type="bibr" rid="B47">47</xref>] but can also activate antigen-presenting cells [<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B19">19</xref>]. These studies demonstrating the importance of Hsps in immune response, indicate that a low expression of Hsps (i.e absence of Hsp60 in lymphocytes of patients with ITP) may be related to the formation of autoantibodies against platelets. An emerging body of data supports a role for Hsps in the inflammatory response: Hsps participate in cytokine signal transduction and in the control of cytokine gene expression [<xref ref-type="bibr" rid="B16">16</xref>], enhance antigen presentation to T lymphocytes, and Hsps displayed on the surface of cells are important for targeting cytotoxic cells [<xref ref-type="bibr" rid="B17">17</xref>]. Vabulas et al. [<xref ref-type="bibr" rid="B46">46</xref>] reported that endocytosed human and chlamydial Hsp60 use toll-like receptor 2 and 4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. The present investigation shows a decrease of Hsp60 in patients with ITP. Whether the decrease in Hsp60 occurs before or during its pathogenesis and is an inducing factor or simply an effect is unknown.</p><p>Given the presence of significant differences in Hsp levels and since the immune response to Hsps can be modulated, modifying the immune response to Hsps may be a potential new treatment option for patients with ITP. However at this stage the possible roles of Hsps in the etiopathogenesis or treatment of ITP remain to be further investigated.</p><p>The present study also shows that antibodies against the inducible Hsp71 are particularly prevalent in pediatric patients with ITP. 51.7% of patients had antibodies to Hsp71 and more than half of those were still positive at dilutions of 1:80. Berchtold et al. [<xref ref-type="bibr" rid="B1">1</xref>] reported that rates of autoantibodies against platelet membrane glycoproteins in children with acute and chronic ITP were 26.7%, 58.3% respectively. Thus the high prevalence of antibodies to Hsp71 is significant and merits further analysis considering that the diagnosis for ITP is basically based on exclusion [<xref ref-type="bibr" rid="B8">8</xref>]. Some possible reasons for the production of autoantibodies against Hsps are: 1) genetic factors; 2) viral infection; 3) denaturation and release of Hsps as a result of cell damage and 4) the presence of anti-specific lymphocytes. No obvious genetic diseases were documented in the pediatric patients with ITP. However, there were frequent histories of viral or bacterial infection and treatment with some drugs in pediatric patients before the occurrence of ITP.</p></sec><sec><title>Conclusions</title><p>Many investigations suggest that autoantibodies against Hsps are important in the generation, formation and prognosis of diseases [<xref ref-type="bibr" rid="B28">28</xref>-<xref ref-type="bibr" rid="B31">31</xref>,<xref ref-type="bibr" rid="B34">34</xref>,<xref ref-type="bibr" rid="B48">48</xref>]. Our previous results show that the high prevalence of antibodies against Hsp71 in benzene-poisoned workers was associated with a decrease in white blood cells and with an increase in activities of serum superoxide dismutase and lymphocyte DNA damage [<xref ref-type="bibr" rid="B28">28</xref>]. There is an immunological cross-reaction between microorganisms and autoantigens of human as Hsps show a high degree of amino acid sequence homology between different species from prokaryotes to humans [<xref ref-type="bibr" rid="B48">48</xref>]. Gross et al. [<xref ref-type="bibr" rid="B49">49</xref>] showed that a 66 kDa protein purified from rabbit reticulocyte lysate, which seems to be involved in platelet destruction, could promote the recycling of Hsp70. However, the mode of action of autoantibody is complex, involving modulation of the expression and function of Fc receptors, interference with the activation of complement and the cytokine network, provision of antiidiotypic antibody, and effects on the activation, differentiation, and effector functions of T and B cells [<xref ref-type="bibr" rid="B50">50</xref>]. Therefore it will be important to understand the mechanisms involved in the generation of antibodies against both Hsp71 and platelets, and to understand their role in the pathogenesis of ITP, and whether they can be used for diagnosis, prognosis and/or treatment of ITP.</p></sec><sec><title>Competing interests</title><p>We declare that there is no competing interest in this work.</p></sec><sec><title>Authors' contributions</title><p>CX performed the immuno assays, SC the Western blots together with DY in addition to participating to discussion with MY and FD. MY designed the experiments and was responsible of patient diagnosis with FD. JL carried out the sampling with RW in addition to performing some laboratory analysis. RMT and TW initiated the project, designed the experiments and wrote the manuscript together with DY.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1471-2326/4/1/prepub"/></p></sec>
Exacerbated inflammatory cellular immune response characteristics of HAM/TSP is observed in a large proportion of HTLV-I asymptomatic carriers
<sec><title>Background</title><p>A small fraction of Human T cell Leukemia Virus type-1 (HTLV-I) infected subjects develop a severe form of myelopathy. It has been established that patients with HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) show an exaggerated immune response when compared with the immunological response observed in HTLV-I asymptomatic carriers. In this study the immunological responses in HAM/TSP patients and in HTLV-I asymptomatic carriers were compared using several immunological assays to identify immunological markers associated with progression from infection to disease.</p></sec><sec sec-type="methods"><title>Methods</title><p>Immunoproliferation assays, cytokine levels of unstimulated cultures, and flow cytometry analysis were used to evaluate the studied groups. Nonparametric tests (Mann-Whitney U test and Wilcoxon matched-pairs signed ranks) were used to compare the difference between the groups.</p></sec><sec><title>Results</title><p>Although both groups showed great variability, HAM/TSP patients had higher spontaneous lymphoproliferation as well as higher IFN-&#x003b3; levels in unstimulated supernatants when compared with asymptomatic carriers. Flow cytometry studies demonstrated a high frequency of inflammatory cytokine (IFN-&#x003b3; and TNF-&#x003b1;) producing lymphocytes in HAM/TSP as compared to the asymptomatic group. This difference was accounted for mainly by an increase in CD8 cell production of these cytokines. Moreover, the HAM/TSP patients also expressed an increased frequency of CD28-/CD8+ T cells. Since forty percent of the asymptomatic carriers had spontaneous lymphoproliferation and IFN-&#x003b3; production similar to HAM/TSP patients, IFN-&#x003b3; levels were measured eight months after the first evaluation in some of these patients to observe if this was a transient or a persistent situation. No significant difference was observed between the means of IFN-&#x003b3; levels in the first and second evaluation.</p></sec><sec><title>Conclusions</title><p>The finding that a large proportion of HTLV-I carriers present similar immunological responses to those observed in HAM/TSP, strongly argues for further studies to evaluate these parameters as markers of HAM/TSP progression.</p></sec>
<contrib id="A1" contrib-type="author"><name><surname>Santos</surname><given-names>Silvane Braga</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A2" contrib-type="author"><name><surname>Porto</surname><given-names>Aur&#x000e9;lia Fonseca</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A3" contrib-type="author"><name><surname>Muniz</surname><given-names>Andr&#x000e9; Luiz</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A4" contrib-type="author"><name><surname>de Jesus</surname><given-names>Am&#x000e9;lia Ribeiro</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>[email protected]</email></contrib><contrib id="A5" contrib-type="author"><name><surname>Magalh&#x000e3;es</surname><given-names>Elza</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A6" contrib-type="author"><name><surname>Melo</surname><given-names>Ailton</given-names></name><xref ref-type="aff" rid="I2">2</xref><email>[email protected]</email></contrib><contrib id="A7" contrib-type="author"><name><surname>Dutra</surname><given-names>Walderez O</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>[email protected]</email></contrib><contrib id="A8" contrib-type="author"><name><surname>Gollob</surname><given-names>Kenneth J</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>[email protected]</email></contrib><contrib id="A9" corresp="yes" contrib-type="author"><name><surname>Carvalho</surname><given-names>Edgar M</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I5">5</xref><email>[email protected]</email></contrib>
BMC Infectious Diseases
<sec><title>Background</title><p>Human T cell leukemia virus-type 1 (HTLV-I) infects an estimated 10 to 20 million people worldwide, making it a serious public healthy problem. The HTLV-1 infection has a high prevalence in Brazil, and Salvador, the capital of the state of Bahia, has the highest prevalence of HTLV-1 in the country in blood donors (1,35%) [<xref ref-type="bibr" rid="B1">1</xref>,<xref ref-type="bibr" rid="B2">2</xref>]. It is estimated that 95% of HTLV-I infected individuals are asymptomatic carriers. A small percentage of infected individuals (2 to 5%) develop adult T cell leukemia/lymphoma (ATL) [<xref ref-type="bibr" rid="B3">3</xref>,<xref ref-type="bibr" rid="B4">4</xref>] or a chronic inflammatory disease, involving the central nervous system, termed HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) [<xref ref-type="bibr" rid="B5">5</xref>,<xref ref-type="bibr" rid="B6">6</xref>]. One of the most important immunological observations of HTLV-I infection is the demonstration that lymphocytes spontaneously proliferate <italic>in vitro </italic>in the absence of stimulus [<xref ref-type="bibr" rid="B7">7</xref>]. It has been shown that both infected CD4+ and CD8+ lymphocytes infiltrate spinal cord and peripheral blood and produce cytokines such IFN-&#x003b3;, TNF-&#x003b1; and IL-6, which are considered important inflammatory mediators of the tissue damage in HAM/TSP [<xref ref-type="bibr" rid="B8">8</xref>-<xref ref-type="bibr" rid="B10">10</xref>]. Extensive previous studies have compared the immunological response of asymptomatic HTLV-I carriers to that of HAM/TSP patients [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B12">12</xref>]. Additionally, a recent study [<xref ref-type="bibr" rid="B13">13</xref>] has emphasized that the percentage of HTLV-I carriers that develop other immunological abnormalities, including HAM/TSP, is much higher than that previously cited in the literature. The aim of the present study is to compare in HTLV-I asymptomatic carriers, and in HAM/TSP patients, the spontaneous lymphoproliferative response and cytokine production, the overall <italic>ex vivo </italic>T cell activation states, and the production of immunoregulatory cytokines by CD4+ and CD8+ T cells. The documentation that some HTLV-I carriers have immunological alteration similar to that observed in HAM/TSP suggests that potential markers of disease progression may be determined in HTLV-I infection.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Patients selection and neurological exam</title><p>Patients were selected from the HTLV-I clinic of the Hospital Universit&#x000e1;rio Professor Edgard Santos, Federal University of Bahia, Brazil. The diagnosis was confirmed by Western blot (HTLV blot 2.4, Genelabs, Singapore). Seventeen patients with HAM/TSP were selected based on WHO criteria and thirty-six HTLV-I asymptomatic carriers were referred from two blood banks. Exclusion criteria included the use of antiviral drugs or immunomodulators in the previous 90 days, helminth infection, co-infection with HIV, HCV or hepatitis B and presence of other neurologic diseases. Motor dysfunction was determined by Osame's Motor Disability Score (OMDS) [<xref ref-type="bibr" rid="B14">14</xref>] and Expanded Disability Status Scale (EDSS) [<xref ref-type="bibr" rid="B15">15</xref>]. Patients with HAM/TSP had a marked neurological impairment with EDSS &#x02265; 3 and OMDS &#x02265; 1 and all asymptomatic subjects had OMDS and EDSS of zero. Seronegative normal donors were also referred from the same blood banks and used as negative controls. The Ethical Committee of the Hospital Universit&#x000e1;rio Professor Edgard Santos approved this study and informed consent was obtained from all prospectively enrolled patients.</p></sec><sec><title>Cell preparation and proliferation assay</title><p>Peripheral blood mononuclear cells (PBMC) were isolated and cultivated in RPMI 1640 (Gibco, Grand Island, NY, USA) plus 10 % heat inactivated human AB Rh+ serum (Sigma Chemical Co., St. Louis, MO), antibiotics and glutamine. A total of 2 &#x000d7; 10<sup>5 </sup>cells/mL were incubated at 37&#x000b0;C in 5 % CO<sub>2 </sub>atmosphere in a 96 well flat-bottom microtiter plates. The cells were kept unstimulated (media alone) and after 5 days, the cultures were pulsed with <sup>3</sup>H-Thymidine (1 &#x003bc;Ci/well) for a final 6&#x02013;16 hours period, and then harvested. The <sup>3</sup>H-Thymidine uptake was measured using a LKB beta scintillation counter. The average of counts per minute (cpm) was plotted and PHA (1.0 &#x003bc;g/mL, Sigma) was used as a positive control in the proliferation assay.</p></sec><sec><title>Cytokine determination</title><p>PBMC were adjusted to 3 &#x000d7; 10<sup>6 </sup>cells/mL in RPMI 1640 plus 10 % serum AB Rh+. The cells were cultured unstimulated or stimulated with PHA (5 &#x003bc;g/mL) and all cultures were incubated at 37&#x000b0;C in 5 % CO<sub>2 </sub>atmosphere for 72 hours until supernatants were collected. IFN-&#x003b3;, TNF-&#x003b1;, IL-5 and IL-10 levels were measured by sandwich ELISA technique (R &#x00026; D system, Minneapolis, MN).</p></sec><sec><title>Ex vivo staining of lymphocyte profiles</title><p>2 &#x000d7; 10<sup>5 </sup>PBMC from HTLV-I patients were incubated with FITC, PE, or Cychrome-labeled antibody solutions for 20 minutes at 4&#x000b0;C. After staining, preparations were washed with 0.1% sodium-azide PBS, fixed with 2% formaldehyde in PBS and kept at 4&#x000b0;C until data acquisition using a FACScalibor (Becton Dickinson, San Jose, CA). The antibodies used were all directly conjugated either for FITC, PE, or Cychrome and consisted of: Ig controls, anti-CD4, CD8, CD28, CD69 (Pharmingen, San Diego, CA) and anti-CD62L (Caltag, Burlingame, CA).</p></sec><sec><title>Single cell cytoplasmic cytokine staining</title><p>Briefly, 2 &#x000d7; 10<sup>5 </sup>PBMC were cultured in RPMI 1640 plus 5% AB Rh+ serum in 96 well plates. Based on preliminary results all the cytokine staining was performed after 20 hours of incubation. During the last 4 hours of culture, Brefeldin-A (1 &#x003bc;g/mL) was added to the culture. The cells were then washed and centrifuged using ice-cold PBS plus sodium-azide, stained for surface markers and fixed using 2% formaldehyde. The fixed cells were then permeablized with a solution of Saponin and stained, for 30 minutes at 4&#x000b0;C, using anti-cytokine mAbs directly conjugated with PE (IFN-&#x003b3;, TNF-&#x003b1;, IL-4 and IL-10) (Pharmingen). Preparations were then washed, fixed and analyzed using a FACScalibor. In all cases the cells were double stained for cytokine and for cell surface markers. In all cases, 30,000 gated events were acquired for later analysis due to the low frequency of positive events being analyzed.</p></sec><sec><title>Statistical analysis</title><p>A nonparametric Mann-Whitney U test and Wilcoxon matched-pairs signed rank tests were used to evaluate differences between the groups. An alpha (&#x003b1;) of 5% (<italic>p </italic>&#x0003c; 0.05) was considered for statistical significance. Lymphocytes were analyzed for their intracellular cytokine expression patterns and frequencies and for surface markers using the program Cell Quest. Statistical analysis was performed using the ANOVA "comparison of all pairs" contained in the statistical program from SAS, JMP.</p></sec></sec><sec><title>Results</title><p>The mean age of the seventeen myelopathy patients was 53 &#x000b1; 16 (mean &#x000b1; SD) years and of the thirty-six HTLV-I healthy carriers was 39 &#x000b1; 11 years. To determine if HAM/TSP patients and asymptomatic subjects produce different levels of secreted cytokines, IFN-&#x003b3;, TNF-&#x003b1;, IL-10 and IL-5 were measured in supernatants of unstimulated cultures of HTLV-I infected groups and compared with negative controls. There was a high variability in IFN-&#x003b3; levels in asymptomatic carriers (Figure <xref ref-type="fig" rid="F1">1A</xref>). The mean and SD of IFN-&#x003b3; levels in myelopathy patients (4,246 &#x000b1; 2,924 pg/mL, range: 375 to 10,750), was higher than that observed in asymptomatic carriers (1,362 &#x000b1; 1,408 pg/mL range: 15 to 6,995) or in negative controls (1 &#x000b1; 4 pg/mL), <italic>p </italic>= 0.0001, Mann-Whitney U test. There was also a tendency for higher TNF-&#x003b1; levels in HAM/TSP patients (378 &#x000b1; 316 pg/mL) when compared with levels observed in asymptomatic carriers (259 &#x000b1; 366 pg/mL) or in negative controls (60 &#x000b1; 63 pg/mL), <italic>p </italic>= 0.06. No differences between IL-5 levels (151 &#x000b1; 141 versus 166 &#x000b1; 231 pg/mL), <italic>p </italic>= 0.17 and IL-10 levels (70 &#x000b1; 66 versus 94 &#x000b1; 110 pg/mL), <italic>p </italic>= 0.9, were observed between HTLV-I infected groups or negative controls (2 &#x000b1; 2 versus 2.6 &#x000b1; 10), Figure <xref ref-type="fig" rid="F1">1B</xref>.</p><p>The lymphoproliferative assays performed using PBMC from ten HAM/TSP patients and eleven asymptomatic individuals showed that lymphoproliferation was higher in HAM/TSP than HTLV-I asymptomatic carriers. The five day spontaneous proliferation of the HAM/TSP group gave a mean and SD of 21,404 &#x000b1; 30,859 cpm (range: 919 to 102,242), while the asymptomatic HTLV-I group had a mean and SD of 3,365 &#x000b1; 5,188 cpm (range: 139 to 18,169). The magnitude of the responses was higher in HAM/TSP than in HTLV-I carriers (p = 0.006) although a great variability of the spontaneous lymphoproliferation had been observed in both groups (date not shown).</p><p>Based on IFN-&#x003b3; production, the immunological responses in 40% of the HTLV-I carriers were similar to that observed in patients with HAM/TSP (p &#x0003e; 0.05). These individuals had IFN-&#x003b3; levels higher than 1,322 pg/mL, representing the mean minus one standard deviation of the IFN-&#x003b3; levels obtained in HAM/TSP patients. To determine if the cytokine levels in HTLV-I carriers was reflecting a transient or persistent situation, IFN-&#x003b3; levels were measured between 6 months to one year (with a mean of eight months) after the first evaluation. No significant differences were observed between the means of IFN-&#x003b3; levels from fourteen asymptomatic carriers in the first and second evaluation (1,723 &#x000b1; 450 and 1,670 &#x000b1; 1,396, respectively), p = 0.67. HAM/TSP patients (n = 11) also had similar levels in the first and second evaluation (5,771 &#x000b1; 3,365 versus 4,718 &#x000b1; 2,427, respectively), p = 0.17, Wilcoxon matched-pairs signed ranks test (Figure <xref ref-type="fig" rid="F2">2</xref>).</p><p>To determine the activation states and relative lymphocyte proportions, as well as the cellular sources of immunoregulatory cytokines, four HAM/TSP patients and eight asymptomatic carriers were randomly selected and analyzed using flow cytometry. To measure lymphocyte activation and regulation, the markers CD69 and CD28 respectively, where used in conjunction with CD4 and CD8 in separate staining protocols. The relative proportions of CD4 and CD8 cells expressing the adhesion molecule, CD62L was also determined for both groups. Figure <xref ref-type="fig" rid="F3">3A</xref> demonstrates that the HAM/TSP group expressed a significantly higher frequency of CD8+ T cells (21.4 &#x000b1; 3.3) than the asymptomatic group (9.8 &#x000b1; 1.7). A higher frequency (51.9 &#x000b1; 3.5) of the hyper-activated, CD28-/CD8+ T cells within the CD8+ T cell population in HAM/TSP than in asymptomatic group (35.2 &#x000b1; 7.4) was also observed. There was no difference in CD69 and CD62L lymphocyte populations between both HTLV-I infected groups (data not shown).</p><p>To further investigate the differences in cytokine profile between the HAM/TSP and asymptomatic groups, flow cytometry was performed to determine the frequency of T cells producing IFN-&#x003b3;, TNF-&#x003b1;, IL-4 and IL-10. A significant increase in the frequency of lymphocytes expressing TNF-&#x003b1; and IFN-&#x003b3; was seen in the HAM/TSP group (12 &#x000b1; 6 and 5 &#x000b1; 0.3, respectively) as compared to the asymptomatic group (4 &#x000b1; 2 and 1 &#x000b1; 0.5, respectively) (Figure <xref ref-type="fig" rid="F3">3B</xref>). Moreover, CD8+ T cells where the major cellular source responsible for the difference in IFN-&#x003b3; producing cells between the two groups. The frequency of CD8+ T cells producing TNF-&#x003b1; was higher in HAM/TSP as compared to asymptomatic carriers, with both CD4+ and CD8+ T cells contributing equally to the difference seen in TNF-&#x003b1; production (Figure <xref ref-type="fig" rid="F3">3C</xref>). In contrast, no difference was seen for the frequency of cells producing IL-4 or IL-10 (data not shown). While there was no difference regarding the frequency of CD4+ and CD8+ T cells producing IFN-&#x003b3; within asymptomatic carriers (0.5 &#x000b1; 0.4 vs. 0.5 &#x000b1; 0.3), there was a significant difference (p &#x0003c; 0.05) in the frequency of CD4 cells producing IFN-&#x003b3; in HAM/TSP (1.4 &#x000b1; 0.5) compared to the CD8+ T cells (3.6 &#x000b1; 0.5).</p></sec><sec><title>Discussion</title><p>The present study shows that lymphocytes from patients with HAM/TSP displayed higher spontaneous proliferation and IFN-&#x003b3; synthesis, a higher frequency of TNF-&#x003b1; and IFN-&#x003b3; producing lymphocytes and a significant increase in the deregulated T cell population, CD28-/CD8+, as compared to those from HTLV-I asymptomatic carriers.</p><p>The pathogenesis of HAM/TSP is not completely understood. Although initially considered a rare and late complication of infection, the disease has been identified in children, and in some cases, a rapidly progressive disease has been observed [<xref ref-type="bibr" rid="B16">16</xref>]. Increased proviral load, pro-inflammatory cytokines and the expansion of HTLV-I tax-specific CD8+ cytotoxic T lymphocytes, both in cerebrospinal fluid and in peripheral blood, have been associated with the central nervous system involvement in patients with HAM/TSP [<xref ref-type="bibr" rid="B17">17</xref>-<xref ref-type="bibr" rid="B21">21</xref>]. A recent report [<xref ref-type="bibr" rid="B13">13</xref>] established a cohort of HTLV-I asymptomatic carries to study clinical events and documented an increased frequency of abnormalities, including a case of HAM/TSP.</p><p>Evaluation of immunological responses in patients with HAM/TSP and asymptomatic carries is important for the understanding of the pathogenesis of the HAM/TSP, and to identify early immunological markers associated with progression from infection to disease. This study indicates a higher and significant spontaneous lymphoproliferation and IFN-&#x003b3; production in HAM/TSP patients as compared to HTLV-I asymptomatic carriers. Additionally, lymphocyte responses were quite variable, and in some asymptomatic carriers the lymphocyte proliferation and IFN-&#x003b3; production were similar to those found in patients with myelopathy. These data induce us to observe if the parameters could be considered good markers of disease progression. This is in agreement with our previous observations that asymptomatic carriers can be divided in high and low producers according to IFN-&#x003b3; levels [<xref ref-type="bibr" rid="B22">22</xref>]. A great variability of immune response observed in about 40% of HTLV-I infected subjects was not reflecting a temporary situation, since the IFN-&#x003b3; levels were relatively constant within an individual over time (eight months). During this period of time no neurological manifestation was documented and no other disease that could alter the immune response was observed.</p><p>Many studies have demonstrated that IFN-&#x003b3; and TNF-&#x003b1; produced in large quantities contribute to tissue damage of the central nervous system [<xref ref-type="bibr" rid="B23">23</xref>,<xref ref-type="bibr" rid="B24">24</xref>], and are likely involved in the pathogenesis of several infections and non-infections disease. The flow cytometric determination of cytokine producing lymphocytes extend the observations made in supernatants of lymphocytes cultures showing a significant increase in the frequency of IFN-&#x003b3; and TNF-&#x003b1; producing cells in patients with HAM/TSP. This difference was accounted for both CD4+ and CD8+ T cells for the TNF-&#x003b1; producing cells, and mainly by CD8+ T cells for IFN-&#x003b3; producing lymphocytes. Since CD4+ T cells are the main source of IFN-&#x003b3; in HTLV-I carriers [<xref ref-type="bibr" rid="B22">22</xref>,<xref ref-type="bibr" rid="B25">25</xref>,<xref ref-type="bibr" rid="B26">26</xref>] this data may indicate that during the evolution from asymptomatic to myelopathy there is a switch from CD4+ to CD8+ in relation to the main cell producing IFN-&#x003b3;. These findings support studies suggesting that CD8+ T cells may play an important role in the pathogenesis of HAM/TSP [<xref ref-type="bibr" rid="B27">27</xref>].</p><p>Previous studies have shown that CD28-/CD8+ cells display high cytotoxic activity, playing an important role in the pathology associated with viral diseases [<xref ref-type="bibr" rid="B28">28</xref>,<xref ref-type="bibr" rid="B29">29</xref>]. Additionally, recent studies have shown that HIV-1 can incorporate CD28 and the acquisition of this specific host surface glycoprotein modulates the virus life cycle [<xref ref-type="bibr" rid="B30">30</xref>]. Moreover, the role of CD28-/CD8+ T cells in HAM/TSP needs to be further investigated since these cells may induce cell damage and / or death in infections disease [<xref ref-type="bibr" rid="B31">31</xref>].</p><p>In conclusion, these results show an exacerbated type 1 immune response in HAM/TSP patients, characterized by elevated IFN-&#x003b3; production, an increased frequency of TNF-&#x003b1; and IFN-&#x003b3; producing lymphocytes, and by an increase in the frequency of CD28-/CD8+ T cells. Given that cellular activation and pro-inflammatory cytokine production are likely directly involved in the pathogenesis of HAM/TSP disease, longitudinal studies of HTLV-I infected asymptomatic carriers who present with high lymphoproliferative response, high production of IFN-&#x003b3; and TNF-&#x003b1; and high expression of CD28-/CD8+ T cells should be conducted to determine the frequency of disease progression in this group. The identification of markers of HAM/TSP progression will allow for earlier initiation of current therapeutic interventions, and hopefully delay the fast and progressive development of the motor disability observed in HTLV-I infected individuals.</p></sec><sec><title>Competing interests</title><p>None declared.</p></sec><sec><title>Authors' contributions</title><p>SBS carried out the immunological studies, performed statistical analysis and drafted the manuscript. ALM, EM and AM participated in the coordination of neurological evaluations. AFP was involved in clinical evaluation of the patients. ARJ participated in the design and statistical analysis. WOD and KJG carried out FACS analysis and EMC conceived the study and participated in its design and coordination.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1471-2334/4/7/prepub"/></p></sec>