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Slco1b1*1b Haplotype is Associated With Risk of Aspirin Induced Peptic Ulcer and Bleeding
mg of aspirin for cardiovascular diseases who participating in endoscopic surveillance and/ or those who had recent ulcer bleeding were studied. Genotypes of SLCO1B, ABCC2, ABCG2, and MDR1,which encode organic anion transporting polypeptide (OATP)1B, multidrug resistance-associated protein 2 (MRP2), breast cancer resistance protein (BCRP), and Pglycoprotein (P-GP) respectively, were determined by PCR or PCR-RFLP. Results: 500 patients enrolled including 78 with peptic ulcer and 34 with upper GI bleeding. The frequencies of the SLCO1B1 521TT genotype were significantly higher in both the ulcer (p=0.006) and bleeding groups (p=0.005) compared to controls. However, there were no significant associations in other genes' genotypes. After adjustment for significant factors, peptic ulcer history (adjusted OR 2.92, 95% CI 1.54-5.58), chronic renal failure (7.59, 1.89-30.4), co-treatment of NSAIDs (4.23, 1.36-13.3), PPIs (0.15, 0.07-0.36) and ARBs or ACE inhibitors (0.43, 0.24-0.75) and the SLCO1B1 *1b (3.64, 1.81-7.29) were significantly associated with peptic ulcer. Co-treatment of thienopyridine (3.29, 1.50-7.23), PPIs (0.26, 0.10-0.73) and statins (0.30, 0.13-0.71) and the SLCO1B1 *1b (6.20, 1.82-21.2) were significantly associated with bleeding. Conclusions: The SLCO1B1 SNP as well as co-treatment of stains and ARBs was associated with aspirin induced peptic ulcer and upper GI bleeding, and the SLCO1B1*1b haplotype may be useful to identify patients at increased risk for aspirin induced peptic ulcer and upper GI bleeding.
1048 Notch-Hes1 Pathway and TNF-α Synergistically up-Regulates OLFM4 Expression in the Inflamed Mucosa of the Human Intestine Ryuichi Okamoto, Junko Akiyama, Tatsuro Murano, Hiromichi Shimizu, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-α mediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific gene might be required to promote the regenerative response of the inflamed intestinal environment.
1051 COX2 Hypermethylation in H. pylori-Positive NSAID-Induced Gastric Ulcer Patients Hiroshi Yasuda, Yoshiyuki Watanabe, Fumio Itoh [Aim] Cyclooxygenase (COX) plays a critical role in the development of nonsteroidal antiinflammatory drug (NSAID)-induced gastric ulcer. Two isoforms of COX have been identified. In stomach, while constitutively expressed COX1 is a house-keeping gene, inducible COX2 is involed in repair process of mucosal injury. When NSAID inhibits COX1 in gastric mucosa, induced COX2 might produce prostaglandins compensately to avoid mucosal injury. Only COX2 has a CpG island in the promoter area, and is highly silenced by DNA methylation in several gastric neoplasms. We hypothesized that COX2 silencing by hypermethylation could cause NSAID-induced gastric damage in some patients. Our goal is to identify the promoter DNA methylation in COX2 of various gastric mucosa samples. [Methods] DNA was extracted from endoscopic biopy materials collected from gastric antrum. Methylation levels of COX2 were analyzed by quantitative bisulfite-pyrosequencing method. COX2 mRNA expression was measured by real-time PCR in cell lines with/without 5-Aza-dC. [Results] First, We examined COX2 methylation in healthy cases (n=32). In H. pylori (HP) positive cases, COX2 methylation levels were significantly increased when compared with those in HP negative cases (21.9 vs 7.9 %, p<0.001). Next, we examine COX2 methylation in NSAIDinduced gastric ulcer patients (n=12). COX2 methylation levels in HP positive patients were significantly increased when compared with those in HP negative patients (26.2 vs 7.4 %, p<0.029). COX2 methylation levels in HP-positive NSAID-induced gastric ulcer patients were significantly higher than those of HP-positive healthy case (26.2 vs 21.9 %, p<0.016). Finally, we examined whether mRNA expression was silenced by COX2 hypermethylation In Vitro using human gastric carcinoma cell line Kato III. COX2 was densely methylated in these cells (nearly 80%). COX2 mRNA expression was not observed in Kato III cells even after addition of a PKC stimulator α-phorbol 12,13-dibutyrate (PDBu), but restored the expression levels by demethylating agent 5-Aza-dC, and it was enhanced by PDBu. [Conclusion] HP infection caused significant increase in methylation levels of COX2 in gastric mucosa. In addition to transcriptional regulation, COX2 expression was regulated through epigenetic mechanism. COX2 hypermethylation seems to increase vulnerability to NSAIDinduced gastric damage in HP-infected patients.
1049 A Novel Role for IL-27 as a Mediator of Intestinal Epithelial Barrier Protection Mediated via Differential Stat Signaling and Induction of Antibacterial and Anti-Inflammatory Proteins Julia Diegelmann, Torsten Olszak, Burkhard Göke, Stephan Brand Background & Aims: IL-27 is a cytokine that inhibits the development of pro-inflammatory Th17 cells that are involved in the pathogenesis of inflammatory bowel disease (IBD). However, the role of IL-27 in IBD is contradictory and its functions in intestinal epithelial cells (IEC) including its effect on the intestinal barrier have not been investigated so far which was therefore the aim of this study. Methods: Expression studies were performed by qPCR, Western blot and immunohistochemistry. IL-27 target genes were analyzed by microarray in IEC and by qPCR in DSS colitis. Cell restitution was analyzed in wounding assays and cell proliferation was determined by measuring DNA content. Signal transduction was analyzed by Western blot experiments and siRNA transfection. Enzymatic activity was determined photometrically. Results: IEC express both IL-27 receptor subunits IL-27R and gp130. Their expression is up-regulated under proinflammatory conditions In Vitro and in active Crohn's disease (CD) In Vivo. IL-27 activates ERK and p38 MAP kinases as well as Akt, STAT1, STAT3 and STAT6 in IEC. IL-27 significantly enhances cell proliferation and IEC restitution particularly via STAT3 and STAT6. In IEC, IL-27 modulates expression of 428 target genes, including the anti-inflammatory gene indoleamine 2,3-dioxygenase 1 (INDO1) and the antibacterial gene deleted in malignant brain tumor 1 (DMBT1). While the IL-27-induced INDO1 mRNA and protein expression as well as its enzymatic activity is completely dependent on STAT1 signaling, the expression of DMBT1 mRNA and protein is mediated via the p38 and STAT3 pathway. All main IL-27 target genes are up-regulated in IEC isolated from mice with DSS-induced colitis. Mucosal IL-27 and DMBT1 expression are increased in active IBD. Conclusion: For the first time, we characterize IL-27 as a mediator of intestinal epithelial barrier protection mediated via differential STAT signaling and the transcriptional activation of anti-inflammatory and antibacterial target genes.
1052 Effect of H. pylori Eradication on the Long-Term Incidence of Recurrent Ulcer Bleeding in High-Risk Aspirin Users: A 10-Year Prospective Cohort Study Francis K. L. Chan, Jessica Ching, Bing-yee Suen, Yee Kit Tse, Justin C. Wu, Joseph J. Sung BACKGROUND & AIM Among low-dose aspirin (ASA) users with H. pylori (Hp) infection who have a history of ulcer bleeding, the risk of recurrent ulcer bleeding with ASA use after Hp eradication is unknown. This prospective cohort study aimed to investigate the effect of Hp eradication on the long-term incidence of recurrent ulcer bleeding in high-risk ASA users. METHOD Three cohorts of ASA users were prospectively recruited from a single center. The first cohort consisted of ASA users with Hp infection who developed ulcer bleeding. After ulcer healing and confirmed Hp eradication, patients received ASA (80 mg daily) without anti-ulcer prophylaxis (Hp-eradicated cohort). The second cohort consisted of ASA users without Hp infection who developed ulcer bleeding. They received ASA without anti-ulcer prophylaxis after ulcer healing (Hp-negative cohort). The third cohort consisted of new onset ASA users who had no history of ulcer. Drug violation, defined as concomitant use of anti-ulcer drugs, NSAIDs, other anti-platelet drugs, anti-coagulants, and steroids, was monitored during the follow-up period. The primary endpoint was endoscopically confirmed ulcer bleeding. All patients were followed up prospectively until the first occurrence of the primary endpoint, death, or up to 10 years. RESULTS A total of 904 patients were enrolled: 249 in the Hp-eradicated cohort, 118 in the Hp-negative cohort, and 537 in the averagerisk cohort. The Hp negative cohort was terminated prematurely after 4 years due to the high incidence of recurrent ulcer bleeding. The mean age (SD) of the Hp-eradicated cohort and average-risk cohort were 68 (10), and 69 (9), respectively. The adjusted incidence rates (per 100 patient-years) of ulcer bleeding were not significantly different between the Hperadicated cohort (1.09, 95% CI 0.61-1.98) and the average-risk cohort (0.67, 95% CI 0.421.06) among patients receiving ASA alone. In contrast, concomitant use of NSAIDs, steroids, anti-coagulants, and/or other anti-platelet drugs significantly increased the risk of ulcer bleeding in the Hp-eradicated cohort irrespective of anti-ulcer drug use (Table). Overall, the risk of ulcer bleeding was significantly increased in patients older than 70 (incidence rate ratio IRR = 1.98, 95% CI 1.14-3.43). There was no significant interaction between age and cohort in adjusted incidence rates. The cumulative incidence rates of all-cause mortality were 34.5% and 25.9%, respectively (p=0.013). CONCLUSION Among ASA users with Hp
1050 Slco1b1*1b Haplotype is Associated With Risk of Aspirin Induced Peptic Ulcer and Bleeding Akiko Shiotani, Takahisa Murao, Hideaki Tsutsui, Hiroshi Imamura, Ken-ichi Tarumi, Noriaki Manabe, Tomoari Kamada, Hiroaki Kusunoki, Takashi Sakakibara, Ken Haruma Background: In the recent case-control study, we indicated the possible inverse association between peptic ulcer and angiotensin type 1 receptor (AT1R) blockers (ARBs) or HMG-Co A reductase inhibitors (statins). (J Gastroenterol. 2009;44:126-31, 717-25, Dig Dis Sci. 2010 [Epub ahead of print] ). Aims: To evaluate whether the genotypes of uptake and efflux transporters of ARBs and statins relate to the presence of peptic ulcer and/or upper gastrointestinal (GI) bleeding associated with aspirin use. Methods: Patients taking enteric-coated 100
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AGA Abstracts
AGA Abstracts
carriers and is therefore likely to contribute to the increased immune activation as seen in CD patients carrying autophagy-related SNP.
1048 Notch-Hes1 Pathway and TNF-α Synergistically up-Regulates OLFM4 Expression in the Inflamed Mucosa of the Human Intestine Ryuichi Okamoto, Junko Akiyama, Tatsuro Murano, Hiromichi Shimizu, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe Background & Aims: Recent studies have shown that OLFM4 is a robust marker for intestinal epithelial stem cells. However, the precise distribution of OLFM4-expressing cells within the inflamed human intestinal mucosa has never been described. Also, the molecular mechanism regulating its expression within intestinal epithelial cells (IECs), with regard to the inflammatory environment, remains largely unknown. Our previous studies have shown that NotchHes1 pathway is constitutively activated in IECs of the inflamed intestinal mucosa, and function as a key pathway for lineage-specific gene expression. Thus, we planned the present study to identify the distribution of OLFM4-expressing IECs, and also to reveal the underlying molecular mechanism regulating expression of OLFM4, under such an inflammatory environment. Methods: Immunostaining using human intestinal tissues were performed to determine the distribution of OLFM4-expressing cells. The expression level of OLFM4 by IECs in response to various inflammatory cytokines was analyzed by RT-PCR or western blot. Also, tetracycline inducible over-expression of Notch1 intracellular domain (NICD1), or Hes1, was employed to examine the involvement of Notch-Hes1 pathway upon OLFM4 expression. Finally, a series of promoter assay was performed to analyze the transcriptional regulation of the human OLFM4 gene. Results: Immunostaining of normal small intestinal and colonic tissues clearly showed the distribution of OLFM4-positive IECs at the lowest part of the crypt, including the crypt base columnar cells. However, in inflamed intestinal tissues of inflammatory bowel disease patients, increased number of OLFM4-expressing IECs was observed, expanding its distribution to the upper part of the crypt. In LS174T cells, stimulation by TNF-α, but not by IL1-β and IFN-γ, significantly up-regulated the mRNA expression of OLFM4. Also, forced expression of both NICD1 and Hes1 significantly up-regulated mRNA and protein expression of OLFM4. Surprisingly, combination of NICD1 or Hes1 over-expression with TNF-α stimulation had synergistic effect upon up-regulation of OLFM4 mRNA expression, reaching up to 2500 fold increase in LS174T cells. Promoter assays using 5' flanking region of the human OLFM4 gene revealed that such a synergistic effect of TNF-α and Notch-Hes1 pathway is mediated through transcriptional regulation, depending on the proximal NF-κB binding site. Consistently, TNF-α mediated up-regulation of NF-κB-dependent transcriptional activity was significantly enhanced by both NICD1 and Hes1 over-expression in LS174T cells. Conclusion: In the inflamed human intestinal mucosa, TNF-α and Notch-Hes1 pathway synergistically up-regulate expression of OLFM4 in IECs. Such an up-regulation of a stem-cell specific gene might be required to promote the regenerative response of the inflamed intestinal environment.
1051 COX2 Hypermethylation in H. pylori-Positive NSAID-Induced Gastric Ulcer Patients Hiroshi Yasuda, Yoshiyuki Watanabe, Fumio Itoh [Aim] Cyclooxygenase (COX) plays a critical role in the development of nonsteroidal antiinflammatory drug (NSAID)-induced gastric ulcer. Two isoforms of COX have been identified. In stomach, while constitutively expressed COX1 is a house-keeping gene, inducible COX2 is involed in repair process of mucosal injury. When NSAID inhibits COX1 in gastric mucosa, induced COX2 might produce prostaglandins compensately to avoid mucosal injury. Only COX2 has a CpG island in the promoter area, and is highly silenced by DNA methylation in several gastric neoplasms. We hypothesized that COX2 silencing by hypermethylation could cause NSAID-induced gastric damage in some patients. Our goal is to identify the promoter DNA methylation in COX2 of various gastric mucosa samples. [Methods] DNA was extracted from endoscopic biopy materials collected from gastric antrum. Methylation levels of COX2 were analyzed by quantitative bisulfite-pyrosequencing method. COX2 mRNA expression was measured by real-time PCR in cell lines with/without 5-Aza-dC. [Results] First, We examined COX2 methylation in healthy cases (n=32). In H. pylori (HP) positive cases, COX2 methylation levels were significantly increased when compared with those in HP negative cases (21.9 vs 7.9 %, p<0.001). Next, we examine COX2 methylation in NSAIDinduced gastric ulcer patients (n=12). COX2 methylation levels in HP positive patients were significantly increased when compared with those in HP negative patients (26.2 vs 7.4 %, p<0.029). COX2 methylation levels in HP-positive NSAID-induced gastric ulcer patients were significantly higher than those of HP-positive healthy case (26.2 vs 21.9 %, p<0.016). Finally, we examined whether mRNA expression was silenced by COX2 hypermethylation In Vitro using human gastric carcinoma cell line Kato III. COX2 was densely methylated in these cells (nearly 80%). COX2 mRNA expression was not observed in Kato III cells even after addition of a PKC stimulator α-phorbol 12,13-dibutyrate (PDBu), but restored the expression levels by demethylating agent 5-Aza-dC, and it was enhanced by PDBu. [Conclusion] HP infection caused significant increase in methylation levels of COX2 in gastric mucosa. In addition to transcriptional regulation, COX2 expression was regulated through epigenetic mechanism. COX2 hypermethylation seems to increase vulnerability to NSAIDinduced gastric damage in HP-infected patients.
1049 A Novel Role for IL-27 as a Mediator of Intestinal Epithelial Barrier Protection Mediated via Differential Stat Signaling and Induction of Antibacterial and Anti-Inflammatory Proteins Julia Diegelmann, Torsten Olszak, Burkhard Göke, Stephan Brand Background & Aims: IL-27 is a cytokine that inhibits the development of pro-inflammatory Th17 cells that are involved in the pathogenesis of inflammatory bowel disease (IBD). However, the role of IL-27 in IBD is contradictory and its functions in intestinal epithelial cells (IEC) including its effect on the intestinal barrier have not been investigated so far which was therefore the aim of this study. Methods: Expression studies were performed by qPCR, Western blot and immunohistochemistry. IL-27 target genes were analyzed by microarray in IEC and by qPCR in DSS colitis. Cell restitution was analyzed in wounding assays and cell proliferation was determined by measuring DNA content. Signal transduction was analyzed by Western blot experiments and siRNA transfection. Enzymatic activity was determined photometrically. Results: IEC express both IL-27 receptor subunits IL-27R and gp130. Their expression is up-regulated under proinflammatory conditions In Vitro and in active Crohn's disease (CD) In Vivo. IL-27 activates ERK and p38 MAP kinases as well as Akt, STAT1, STAT3 and STAT6 in IEC. IL-27 significantly enhances cell proliferation and IEC restitution particularly via STAT3 and STAT6. In IEC, IL-27 modulates expression of 428 target genes, including the anti-inflammatory gene indoleamine 2,3-dioxygenase 1 (INDO1) and the antibacterial gene deleted in malignant brain tumor 1 (DMBT1). While the IL-27-induced INDO1 mRNA and protein expression as well as its enzymatic activity is completely dependent on STAT1 signaling, the expression of DMBT1 mRNA and protein is mediated via the p38 and STAT3 pathway. All main IL-27 target genes are up-regulated in IEC isolated from mice with DSS-induced colitis. Mucosal IL-27 and DMBT1 expression are increased in active IBD. Conclusion: For the first time, we characterize IL-27 as a mediator of intestinal epithelial barrier protection mediated via differential STAT signaling and the transcriptional activation of anti-inflammatory and antibacterial target genes.
1052 Effect of H. pylori Eradication on the Long-Term Incidence of Recurrent Ulcer Bleeding in High-Risk Aspirin Users: A 10-Year Prospective Cohort Study Francis K. L. Chan, Jessica Ching, Bing-yee Suen, Yee Kit Tse, Justin C. Wu, Joseph J. Sung BACKGROUND & AIM Among low-dose aspirin (ASA) users with H. pylori (Hp) infection who have a history of ulcer bleeding, the risk of recurrent ulcer bleeding with ASA use after Hp eradication is unknown. This prospective cohort study aimed to investigate the effect of Hp eradication on the long-term incidence of recurrent ulcer bleeding in high-risk ASA users. METHOD Three cohorts of ASA users were prospectively recruited from a single center. The first cohort consisted of ASA users with Hp infection who developed ulcer bleeding. After ulcer healing and confirmed Hp eradication, patients received ASA (80 mg daily) without anti-ulcer prophylaxis (Hp-eradicated cohort). The second cohort consisted of ASA users without Hp infection who developed ulcer bleeding. They received ASA without anti-ulcer prophylaxis after ulcer healing (Hp-negative cohort). The third cohort consisted of new onset ASA users who had no history of ulcer. Drug violation, defined as concomitant use of anti-ulcer drugs, NSAIDs, other anti-platelet drugs, anti-coagulants, and steroids, was monitored during the follow-up period. The primary endpoint was endoscopically confirmed ulcer bleeding. All patients were followed up prospectively until the first occurrence of the primary endpoint, death, or up to 10 years. RESULTS A total of 904 patients were enrolled: 249 in the Hp-eradicated cohort, 118 in the Hp-negative cohort, and 537 in the averagerisk cohort. The Hp negative cohort was terminated prematurely after 4 years due to the high incidence of recurrent ulcer bleeding. The mean age (SD) of the Hp-eradicated cohort and average-risk cohort were 68 (10), and 69 (9), respectively. The adjusted incidence rates (per 100 patient-years) of ulcer bleeding were not significantly different between the Hperadicated cohort (1.09, 95% CI 0.61-1.98) and the average-risk cohort (0.67, 95% CI 0.421.06) among patients receiving ASA alone. In contrast, concomitant use of NSAIDs, steroids, anti-coagulants, and/or other anti-platelet drugs significantly increased the risk of ulcer bleeding in the Hp-eradicated cohort irrespective of anti-ulcer drug use (Table). Overall, the risk of ulcer bleeding was significantly increased in patients older than 70 (incidence rate ratio IRR = 1.98, 95% CI 1.14-3.43). There was no significant interaction between age and cohort in adjusted incidence rates. The cumulative incidence rates of all-cause mortality were 34.5% and 25.9%, respectively (p=0.013). CONCLUSION Among ASA users with Hp
1050 Slco1b1*1b Haplotype is Associated With Risk of Aspirin Induced Peptic Ulcer and Bleeding Akiko Shiotani, Takahisa Murao, Hideaki Tsutsui, Hiroshi Imamura, Ken-ichi Tarumi, Noriaki Manabe, Tomoari Kamada, Hiroaki Kusunoki, Takashi Sakakibara, Ken Haruma Background: In the recent case-control study, we indicated the possible inverse association between peptic ulcer and angiotensin type 1 receptor (AT1R) blockers (ARBs) or HMG-Co A reductase inhibitors (statins). (J Gastroenterol. 2009;44:126-31, 717-25, Dig Dis Sci. 2010 [Epub ahead of print] ). Aims: To evaluate whether the genotypes of uptake and efflux transporters of ARBs and statins relate to the presence of peptic ulcer and/or upper gastrointestinal (GI) bleeding associated with aspirin use. Methods: Patients taking enteric-coated 100
S-173
AGA Abstracts
AGA Abstracts
carriers and is therefore likely to contribute to the increased immune activation as seen in CD patients carrying autophagy-related SNP.