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Tu1382 The Role of NHE8 in Intestinal Mucosal Homeostasis
Tu1380 Fistulising Perianal Crohn's Disease - Has Anti-TNF Therapy Improved Outcomes? Nuha A. Yassin, Alan Askari, Linda Ferrari, Omar Faiz, Janindra Warusavitarne, Arun Gupta, Robin K. Phillips, Ailsa Hart
Tu1383
Introduction Anti-tumor necrosis factor (TNF) therapies are currently being used to treat fistulising perianal Crohn's disease (CD). We evaluated the clinical and radiological outcomes of patients with perianal Crohn's fistulas who have been treated with immunomodulators alone as compared with those treated with anti-TNF therapies. Methods A local database of 270 consecutive patients with CD anal fistulas treated at our institution between 2000 and 2014 was established. Demographic and clinical data were recorded in addtion to radiological reports. Results Ninety patients were in the non-anti-TNF group and 180 were treated with anti-TNF therapy (Infliximab or Adalimumab). Clinical response rates were significantly higher in the anti-TNF group (74% vs 62%, p=0.04). Similarly, radiological response rates were higher in the anti-TNF group (56% vs 28%, p<0.01). Cox Regression analysis demonstrated fistula duration (p=0.01) and biologic therapy (p=<0.01) to be significant at the univariate level. At the multivariate level, patients on anti-TNF therapy had a faster radiological response over a 6-year follow-up period (OR=2.25, CI= 1.14-4.46, p= 0.02). A short duration of CD (less than 5 years) contributes to a faster time to clinical response (OR=1.77, CI=1.03-3.05, p=0.04). Treatment with anti-TNF therapy is an independent predictor of radiological response (OR 3.55, CI 1.59-7.91, p<0.01). Patients with L1 luminal disease are 3 times more likely not to go into clinical remission on both univaraite and multivariate analyses (OR=3.08, CI=1.47-6.46, p=0.01). The duration of CD is also a poor predictor of clinical response to therapy (p<0.01). Conclusion Patients on anti-TNF therapy have improved clinical and radiological response rates compared with patients without. Anti-TNF therapy is a positive predictor of radiological response to therapy.
Serologic and Genetic Markers May Predict the Development of Chronic Pouchitis After Pouch Surgery in Ulcerative Colitis Patients Shay Ben-Shachar, Yael Finezilber, Hofit Elad, Henit Yanai, Hagit Tulchinsky, Iris Dotan BACKGROUND: Serologic markers characterize Crohn's disease (CD) and a minority of ulcerative colitis (UC) patients. NOD2 gene single nucleotide variations (SNVs) and NOD2 InsC mutation were found to be associated with CD but not with UC. UC patients undergoing pouch surgery due to disease complications may develop inflammation in the previously normal small bowel constructing the pouch (pouchitis).The NOD2 InsC mutation is more frequent in UC patients developing pouchitis compared to those with an uninflamed pouch. Several molecular similarities between pouchitis and CD were previously detected. We hypothesized that serologic and genetic similarities characterizing CD will be found in patients with pouchitis. METHODS: Samples from IBD patients and controls were obtained. Pouch patients were defined as having a normal pouch (NP) or chronic pouchitis (CP) according to clinical definitions. Anti-glycan antibodies: anti-Saccharomyces cerevisiae (ASCA) anti-laminaribioside, anti-chitobioside, and anti-mannobioside carbohydrate antibodies (ALCA, ACCA and AMCA, respectively), were tested using ELISA. Two missense SNVs of NOD2 gene (R702W/rs2066844, G908R/rs2066845) and the frameshift mutation (1007FS/ rs2066847) were analyzed using polymerase chain reaction. RESULTS: A total of 144 CD, 69 UC, 111 UC patients after pouch surgery (57 NP, 54 CP), and 90 healthy controls were recruited. Demographic data in pouch and UC patients were comparable except for younger age of UC diagnosis in CP patients (21.7±10.3 vs 30 ±12.6 years, respectively, p<0.05). All four serologic markers were significantly increased in CD patients compared with controls (P<0.05). In UC, ALCA and AMCA levels were elevated compared to controls (P=0.023 and P=0.43, respectively). No significant alteration of serologic markers was detected in NP, however in CP both AMCA and ACCA levels were elevated compared to controls (76 and 62 IU vs. 44 and 34 IU, respectively, p=0.001 and P=0.11 respectively). Significant differences in allele frequencies of all tested NOD2 variants were detected in CD (P<0.05). The NOD2 InsC allele frequencies tended to increase in CP as well: 11.2%, 7.6%, 2.9% and 1.7% in CD, CP, UC and NP, respectively). Interestingly, 81% of CD patients with NOD2 1007FS mutation were ASCA positive, compared to only 36% of "wild-type" patients (p<0.001). Only four CP patients with NOD2 1007FS mutation were detected. However, ALCA level was elevated in 50% of those, and normal in all 49 "wild-type" CP patients (P=0.004). CONCLUSIONS: UC patients developing pouchitis after pouch surgery have specific serologic and genetic characteristics, that may predict disease course after surgery. CP and CD patients have serologic and genetic similarities suggesting that the processes causing pouchitis and CD may be similar.
Tu1381 Genome-Wide Association Identifies Multiple Collagenous Colitis Susceptibility Loci Giulia Roda, Joana Torres, Jianzhong Hu, ke hao, Zhongyang Zhang, anli chen, Robert E. Petras, Darrell S. Pardi, Alina C. Iuga, Gabriel Levi, Wenqing Cao, Florian Rieder, Ilyssa O. Gordon, Noam Harpaz, Judy H. Cho, Inga Peter, Jean-Frederic Colombel Background: Microscopic colitis (MC) accounts nowadays for 10-13% of the cases investigated for chronic, non-bloody diarrhea. The etiology of MC remains unknown. As in Inflammatory Bowel Disease (IBD) different components contribute to the pathogenesis of MC such as environmental factors, a genetic predisposition and an aberrant immune response. The HLA associations and the higher occurrence of immune-mediated diseases in patients with collagenous colitis (CC) strongly suggest a role for auto-immunity. The nature of the adaptive local immune responses in the mucosa of CC is still unknown. A decreased amount of CD4+ lymphocytes in the lamina propria has been described. No genome-wide association or fine mapping studies of CC have been conducted. Accordingly we carried out an unbiased genome-wide association study of CC with the aim of identifying genetic loci that are closely associated with CC. Methods: We extracted DNA from ~450 CC formalin-Fixed, paraffinembedded tissue (FFPE) of patients with histologically confirmed CC. DNA samples that passed the quality control (N=295) were genotyped using the Illumina Immunochip array. Existing Immunochip genotyping data on IBD-free individuals (N=1,228) provided by the NIDDK IBD Genetics Consortium were obtained to explore unique pathways associated with CC. Results: Ten loci passed the study-wise statistical significance for association with CC after accounting for the number of independent tests. Among them there were loci previously reported to be associated with that map to chromosomes 5 (IL7R), 6 (PTPRK), 9 (TNFSF15), 17q12 (IKZF, ORMDL3), and 17q12 (ATP6V0A1, STAT3). The most significant association signal we observed genome-wide in CC was near HLA-DQA1, a majot histocompatibilty complex class II implicating CD4+ T cells in CC pathogenesis. Numerous additional SNPs within the MHC region have been significantly associated with CC. A significant association was also identified for CTL4, a marker of the T-regulator cells (Treg), CD44 and ICOS. Conclusion: These preliminary findings support the hypothesis that CC has a genetic component that involves pathways which potentially overlap with those of IBD as well as other pathways that are unique and further support the important role of CD4+ T-cells in CC pathogenesis.
Tu1384 Meconium Ileus: Mucin Secretion in the Cystic Fibrosis Ileum Koryse S. Woodrooffe, Chang Suk Moon, Chandrima Sinha, kavisha arora, Sunitha Yarlagadda, Mehmet Kesimer, Lubna H. Abdullah, Anjaparavanda Naren Introduction: Meconium Ileus, a form of neonatal bowel obstruction that affects neonates with Cystic Fibrosis, is characterized by thick and viscous intestinal mucus. The composition of the mucin glycoprotein polymers that form this thick intestinal mucus is still incompletely understood. Objective: We investigated the intestinal mucin composition and mucus secretion rates from the terminal ileum of wild type mice compared to age matched mice expressing the delta F508 CFTR mutation. Methods: We performed real time PCR for several mucin genes and mucin glycoproteins expression in terminal ileal tissue sections were localized using immunofluorescence. Goblet cells, the mucin secreting cells in the intestinal epithelium, were quantified using Periodic Acid Schiff and Alcian Blue staining of terminal ileum tissue sections. In addition, mucus secretion rates from these tissues were analyzed using Periodic Acid Schiff staining on a nitrocellulose membrane. Results: The terminal ileum from mice with the delta F508 mutation had increased expression of several mucins including MUC 2 as compared to wild type (WT)(p<0.05). Periodic Acid Schiff staining secretion analysis showed increased mucus secretion rates in mice without functional CFTR protein as compared to WT-mice. The number of goblet cells in the terminal ileum of delta F508 mice were higher compared to WT-mice as demonstrated by Periodic Acid Schiff and Alcian Blue staining of terminal ileum tissue sections. The immunofluorescence for MUC2 was increased as well in delta F508 mice. We are currently performing experiments using ileum from Cystic Fibrosis patients. Conclusion: Our studies indicate that meconium ileus observed in individuals with Cystic Fibrosis is likely due to increased mucus secretion. Thus, we believe that differences in the mucin glycoprotein composition along with the enhanced mucus secretion rates may play an important role in the development of the abnormal intestinal mucus in Cystic Fibrosis.
Tu1382 The Role of NHE8 in Intestinal Mucosal Homeostasis Aiping Wang, Jing Li, Yang Zhao, Fayez K. Ghishan, Hua Xu Introduction: NHE8, a newest member of the Na+/H+ exchanger family, is extensively expressed in intestinal epithelial cells. Our previous finding that Mucin2 expression was remarkably impaired, with concomitantly increase in bacterial adhesion to colonic mucosa in NHE8-/- mouse, suggests that NHE8 participates in intestinal mucosal protection. This
S-875
AGA Abstracts
AGA Abstracts
study extended our understanding about the role of NHE8 in mucosal protection. Method: Wild type mice and NHE8-/- mice were treated with DSS. Ileum, proximal and distal colon specimens were collected for pathological scoring, RNA/DNA isolation. Real-time PCR was used to quantify inflammatory cytokine expression. DNA was used to measure bacteria adhered on the epithelial layer. Mesenteric lymph nodes and whole blood were also collected at laparotomies for bacterial translocation study. RNA was purified from wild-type mice and NHE8-/- mice and used to determine the expression of major defensins. Result: DSS treatment in NHE8-/- mice displayed severe symptoms with earlier onset of disease and higher disease activity index compared with that of in wild-type mice. Bacterial translocation occurrence in NHE8-/- mice was remarkably higher than in wild-type mice. Severe histological inflammation was observed in both proximal and distal colon of NHE8-/- mice. Cytokine expression (IL-1β, IL-4, IL-6 and TNFα) was also significantly elevated in the colon in NHE8-/- mice. Meanwhile, the expression of intestinal global defensins and primary cryptdin group of cryptdin-1, cryptdin-related peptide-4c and defensin-22, were decreased in NHE8-/- mice compared to wild-type mice. Conclusion: The dysbiosis of intestinal microbiota and decreased innate defensin expression resulting from the loss of NHE8 function may be responsible for occurrence of bacteria translocation and development of intestinal inflammation. These observations supported that NHE8 plays an important role in maintaining mucosal homeostasis.
Tu1383
Introduction Anti-tumor necrosis factor (TNF) therapies are currently being used to treat fistulising perianal Crohn's disease (CD). We evaluated the clinical and radiological outcomes of patients with perianal Crohn's fistulas who have been treated with immunomodulators alone as compared with those treated with anti-TNF therapies. Methods A local database of 270 consecutive patients with CD anal fistulas treated at our institution between 2000 and 2014 was established. Demographic and clinical data were recorded in addtion to radiological reports. Results Ninety patients were in the non-anti-TNF group and 180 were treated with anti-TNF therapy (Infliximab or Adalimumab). Clinical response rates were significantly higher in the anti-TNF group (74% vs 62%, p=0.04). Similarly, radiological response rates were higher in the anti-TNF group (56% vs 28%, p<0.01). Cox Regression analysis demonstrated fistula duration (p=0.01) and biologic therapy (p=<0.01) to be significant at the univariate level. At the multivariate level, patients on anti-TNF therapy had a faster radiological response over a 6-year follow-up period (OR=2.25, CI= 1.14-4.46, p= 0.02). A short duration of CD (less than 5 years) contributes to a faster time to clinical response (OR=1.77, CI=1.03-3.05, p=0.04). Treatment with anti-TNF therapy is an independent predictor of radiological response (OR 3.55, CI 1.59-7.91, p<0.01). Patients with L1 luminal disease are 3 times more likely not to go into clinical remission on both univaraite and multivariate analyses (OR=3.08, CI=1.47-6.46, p=0.01). The duration of CD is also a poor predictor of clinical response to therapy (p<0.01). Conclusion Patients on anti-TNF therapy have improved clinical and radiological response rates compared with patients without. Anti-TNF therapy is a positive predictor of radiological response to therapy.
Serologic and Genetic Markers May Predict the Development of Chronic Pouchitis After Pouch Surgery in Ulcerative Colitis Patients Shay Ben-Shachar, Yael Finezilber, Hofit Elad, Henit Yanai, Hagit Tulchinsky, Iris Dotan BACKGROUND: Serologic markers characterize Crohn's disease (CD) and a minority of ulcerative colitis (UC) patients. NOD2 gene single nucleotide variations (SNVs) and NOD2 InsC mutation were found to be associated with CD but not with UC. UC patients undergoing pouch surgery due to disease complications may develop inflammation in the previously normal small bowel constructing the pouch (pouchitis).The NOD2 InsC mutation is more frequent in UC patients developing pouchitis compared to those with an uninflamed pouch. Several molecular similarities between pouchitis and CD were previously detected. We hypothesized that serologic and genetic similarities characterizing CD will be found in patients with pouchitis. METHODS: Samples from IBD patients and controls were obtained. Pouch patients were defined as having a normal pouch (NP) or chronic pouchitis (CP) according to clinical definitions. Anti-glycan antibodies: anti-Saccharomyces cerevisiae (ASCA) anti-laminaribioside, anti-chitobioside, and anti-mannobioside carbohydrate antibodies (ALCA, ACCA and AMCA, respectively), were tested using ELISA. Two missense SNVs of NOD2 gene (R702W/rs2066844, G908R/rs2066845) and the frameshift mutation (1007FS/ rs2066847) were analyzed using polymerase chain reaction. RESULTS: A total of 144 CD, 69 UC, 111 UC patients after pouch surgery (57 NP, 54 CP), and 90 healthy controls were recruited. Demographic data in pouch and UC patients were comparable except for younger age of UC diagnosis in CP patients (21.7±10.3 vs 30 ±12.6 years, respectively, p<0.05). All four serologic markers were significantly increased in CD patients compared with controls (P<0.05). In UC, ALCA and AMCA levels were elevated compared to controls (P=0.023 and P=0.43, respectively). No significant alteration of serologic markers was detected in NP, however in CP both AMCA and ACCA levels were elevated compared to controls (76 and 62 IU vs. 44 and 34 IU, respectively, p=0.001 and P=0.11 respectively). Significant differences in allele frequencies of all tested NOD2 variants were detected in CD (P<0.05). The NOD2 InsC allele frequencies tended to increase in CP as well: 11.2%, 7.6%, 2.9% and 1.7% in CD, CP, UC and NP, respectively). Interestingly, 81% of CD patients with NOD2 1007FS mutation were ASCA positive, compared to only 36% of "wild-type" patients (p<0.001). Only four CP patients with NOD2 1007FS mutation were detected. However, ALCA level was elevated in 50% of those, and normal in all 49 "wild-type" CP patients (P=0.004). CONCLUSIONS: UC patients developing pouchitis after pouch surgery have specific serologic and genetic characteristics, that may predict disease course after surgery. CP and CD patients have serologic and genetic similarities suggesting that the processes causing pouchitis and CD may be similar.
Tu1381 Genome-Wide Association Identifies Multiple Collagenous Colitis Susceptibility Loci Giulia Roda, Joana Torres, Jianzhong Hu, ke hao, Zhongyang Zhang, anli chen, Robert E. Petras, Darrell S. Pardi, Alina C. Iuga, Gabriel Levi, Wenqing Cao, Florian Rieder, Ilyssa O. Gordon, Noam Harpaz, Judy H. Cho, Inga Peter, Jean-Frederic Colombel Background: Microscopic colitis (MC) accounts nowadays for 10-13% of the cases investigated for chronic, non-bloody diarrhea. The etiology of MC remains unknown. As in Inflammatory Bowel Disease (IBD) different components contribute to the pathogenesis of MC such as environmental factors, a genetic predisposition and an aberrant immune response. The HLA associations and the higher occurrence of immune-mediated diseases in patients with collagenous colitis (CC) strongly suggest a role for auto-immunity. The nature of the adaptive local immune responses in the mucosa of CC is still unknown. A decreased amount of CD4+ lymphocytes in the lamina propria has been described. No genome-wide association or fine mapping studies of CC have been conducted. Accordingly we carried out an unbiased genome-wide association study of CC with the aim of identifying genetic loci that are closely associated with CC. Methods: We extracted DNA from ~450 CC formalin-Fixed, paraffinembedded tissue (FFPE) of patients with histologically confirmed CC. DNA samples that passed the quality control (N=295) were genotyped using the Illumina Immunochip array. Existing Immunochip genotyping data on IBD-free individuals (N=1,228) provided by the NIDDK IBD Genetics Consortium were obtained to explore unique pathways associated with CC. Results: Ten loci passed the study-wise statistical significance for association with CC after accounting for the number of independent tests. Among them there were loci previously reported to be associated with that map to chromosomes 5 (IL7R), 6 (PTPRK), 9 (TNFSF15), 17q12 (IKZF, ORMDL3), and 17q12 (ATP6V0A1, STAT3). The most significant association signal we observed genome-wide in CC was near HLA-DQA1, a majot histocompatibilty complex class II implicating CD4+ T cells in CC pathogenesis. Numerous additional SNPs within the MHC region have been significantly associated with CC. A significant association was also identified for CTL4, a marker of the T-regulator cells (Treg), CD44 and ICOS. Conclusion: These preliminary findings support the hypothesis that CC has a genetic component that involves pathways which potentially overlap with those of IBD as well as other pathways that are unique and further support the important role of CD4+ T-cells in CC pathogenesis.
Tu1384 Meconium Ileus: Mucin Secretion in the Cystic Fibrosis Ileum Koryse S. Woodrooffe, Chang Suk Moon, Chandrima Sinha, kavisha arora, Sunitha Yarlagadda, Mehmet Kesimer, Lubna H. Abdullah, Anjaparavanda Naren Introduction: Meconium Ileus, a form of neonatal bowel obstruction that affects neonates with Cystic Fibrosis, is characterized by thick and viscous intestinal mucus. The composition of the mucin glycoprotein polymers that form this thick intestinal mucus is still incompletely understood. Objective: We investigated the intestinal mucin composition and mucus secretion rates from the terminal ileum of wild type mice compared to age matched mice expressing the delta F508 CFTR mutation. Methods: We performed real time PCR for several mucin genes and mucin glycoproteins expression in terminal ileal tissue sections were localized using immunofluorescence. Goblet cells, the mucin secreting cells in the intestinal epithelium, were quantified using Periodic Acid Schiff and Alcian Blue staining of terminal ileum tissue sections. In addition, mucus secretion rates from these tissues were analyzed using Periodic Acid Schiff staining on a nitrocellulose membrane. Results: The terminal ileum from mice with the delta F508 mutation had increased expression of several mucins including MUC 2 as compared to wild type (WT)(p<0.05). Periodic Acid Schiff staining secretion analysis showed increased mucus secretion rates in mice without functional CFTR protein as compared to WT-mice. The number of goblet cells in the terminal ileum of delta F508 mice were higher compared to WT-mice as demonstrated by Periodic Acid Schiff and Alcian Blue staining of terminal ileum tissue sections. The immunofluorescence for MUC2 was increased as well in delta F508 mice. We are currently performing experiments using ileum from Cystic Fibrosis patients. Conclusion: Our studies indicate that meconium ileus observed in individuals with Cystic Fibrosis is likely due to increased mucus secretion. Thus, we believe that differences in the mucin glycoprotein composition along with the enhanced mucus secretion rates may play an important role in the development of the abnormal intestinal mucus in Cystic Fibrosis.
Tu1382 The Role of NHE8 in Intestinal Mucosal Homeostasis Aiping Wang, Jing Li, Yang Zhao, Fayez K. Ghishan, Hua Xu Introduction: NHE8, a newest member of the Na+/H+ exchanger family, is extensively expressed in intestinal epithelial cells. Our previous finding that Mucin2 expression was remarkably impaired, with concomitantly increase in bacterial adhesion to colonic mucosa in NHE8-/- mouse, suggests that NHE8 participates in intestinal mucosal protection. This
S-875
AGA Abstracts
AGA Abstracts
study extended our understanding about the role of NHE8 in mucosal protection. Method: Wild type mice and NHE8-/- mice were treated with DSS. Ileum, proximal and distal colon specimens were collected for pathological scoring, RNA/DNA isolation. Real-time PCR was used to quantify inflammatory cytokine expression. DNA was used to measure bacteria adhered on the epithelial layer. Mesenteric lymph nodes and whole blood were also collected at laparotomies for bacterial translocation study. RNA was purified from wild-type mice and NHE8-/- mice and used to determine the expression of major defensins. Result: DSS treatment in NHE8-/- mice displayed severe symptoms with earlier onset of disease and higher disease activity index compared with that of in wild-type mice. Bacterial translocation occurrence in NHE8-/- mice was remarkably higher than in wild-type mice. Severe histological inflammation was observed in both proximal and distal colon of NHE8-/- mice. Cytokine expression (IL-1β, IL-4, IL-6 and TNFα) was also significantly elevated in the colon in NHE8-/- mice. Meanwhile, the expression of intestinal global defensins and primary cryptdin group of cryptdin-1, cryptdin-related peptide-4c and defensin-22, were decreased in NHE8-/- mice compared to wild-type mice. Conclusion: The dysbiosis of intestinal microbiota and decreased innate defensin expression resulting from the loss of NHE8 function may be responsible for occurrence of bacteria translocation and development of intestinal inflammation. These observations supported that NHE8 plays an important role in maintaining mucosal homeostasis.