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Autophagy Regulates Immune Responses Through Destabilization of the Immunological Synapse
AGA Abstracts
converted to jejunostomy at the time of implantation of GES system (total 42). The 39 patients, who already had a J-tube, continued after GES surgery and 18 patients had a new placement of a J-tube during Enterra surgery. Thus there were a total of 99 patients with J-tubes at the initiation of Enterra Therapy. Mean weight in these patients was 149 ±41 lbs at baseline. Fisher's exact Chi-Square test was used for statistical analysis. Results: Only 9 (11%) of Enterra patients continued to required supplemental nutritional support by J-tube (p=0.032) and 2 still required TPN at the final follow up visit.See table: The weight in all 3 groups of patients significantly increased to 162±43 lbs (p<0.005) at the final visit. Conclusions: 1) Enterra therapy resulted in significant nutrition gains with an 89% reduction in J-tube use and significant increase in weight for GP patients of all etiologies; 2) These data indicate that GES therapy is also recommended to be performed when J-tube placement surgery is being considered in patients with malnutrition and GP.
each) in response to TNF/IFNγ co-treatment (100 ng/ml each, 24 h) in T84 IEC. While cytokine treatment significantly induced the expression of autophagy related genes in PTPN2competent cells, PTPN2 knock-down prevented the cytokine-induced rise in protein levels of beclin-1, ATG5-ATG12 conjugates, IRGM and ATG16L1 (n=3; p<0.05 each). This resulted in impaired levels of autophagy as demonstrated by diminished levels of microtubuleassociated protein 1 light-chain 3 B-II (LC3B-II), a marker of autophagy activity (n=3; p<0.05). LC3B+ vesicles, indicative of autophagosomes, were clearly fewer in number, but larger in size in cytokine-treated PTPN2-deficient cells, indicating defective autophagy, when compared to PTPN2-competent cells (n=3). On a functional level, impaired autophagy in PTPN2-deficient cells correlated with increased apoptosis as shown by elevated levels of cleaved caspase-7 and poly ADP-ribose polymerase (PARP) (n=3; p<0.05). Presence of the CD-associated ATG16L1 variant prevented the cytokine-induced rise in PTPN2 protein (n= 3; p<0.05) and resulted in impaired LC3B-II levels (n=3; p<0.05) and autophagosomes in IEC, similar to PTPN2-deficient cells. These observations were fully confirmed in cytokinetreated HT-29 IEC (n=2). Conclusions: Our results demonstrate that PTPN2 regulates autophagy in IEC in the setting of inflammation. We provide a model of how dysfunction of the CD susceptibility genes, PTPN2 and ATG16L1, may contribute to cell dysfunction and cell death. These events could contribute to the onset as well as the perpetuation of chronic intestinal inflammation, as observed in CD. 1046 Reduced Paneth Cell Antimicrobial Protein Levels Correlate With Activation of the Unfolded Protein Response in the Gut of Obese Individuals Caroline M. Hodin, Froukje J. Verdam, Joep Grootjans, Sander S. Rensen, Cornelis H. Dejong, Wim A. Buurman, Jan Willem Greve, Kaatje Lenaerts Background and aims The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been observed in mice with a deficiency in active Paneth cell α-defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects as a possible cause of the obesity-associated microbiota shift. In addition, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Methods Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI>35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human α-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Results Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 and lysozyme transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Moreover, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Conclusions Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects which may underlie the observed Paneth cell compromise.
1044 Conditional XBP1 Deletion Within the Intestinal Epithelial Cells Results in Activation of Autophagy in Paneth Cells Michal Tomczak, Hyun-Jeong Ko, Eduardo Martinez-Naves, Susan J. Hagen, Arthur Kaser, Richard S. Blumberg Introduction: The unfolded protein response (UPR) is one of the pathways critical to the maintenance of epithelial function and intestinal homeostasis. Genetic defects within components of the UPR (e.g. XBP1, AGR2, ORMDL3) have been linked to development of IBD. Recently, we have shown that some IBD patients carry rare hypomorphic variants of XBP1, consistent with this, conditional deletion of XBP1 (XBPflox/floxVcre) within the intestinal epithelial cells (IEC) results in excessive ER stress, Paneth cell apoptosis and development of spontaneous enteritis. The UPR is closely linked to induction of autophagy, another cellular pathway important to epithelial homeostasis and risk for Crohn's disease (e.g. ATG16L1, IRGM and LRKK2). Here, we have tested if unabated ER stress in the IEC activates autophagy. Methods: Activation of autophagy within the IEC was compared in XBPflox/ floxVcre (KO) mice and XBPflox/flox (WT) littermates. To test the effects of ER stress on autophagy induction In Vitro we silenced XBP1 expression in the MODE-K cell line. The activation of autophagy was assessed by LC3 I/II conversion, electron microscopy (EM) and LC3-GFP expression. Results: EM analysis of small intestinal samples from KO mice showed decreased numbers of Paneth cells with reduced numbers of secretory granules and high quantities of autophagic vacuoles compared to WT littermates. A similar pattern of autophagy activation, but to a lesser extent, was observed in goblet cells and enterocytes in KO mice compared to WT mice. Increased activation of autophagy in IEC of KO mice, compared to WT IEC, was confirmed by biochemical conversions of LC3 I/II. EM evaluation of MODEK cells with stably silenced XBP1 showed higher quantities of autophagic vacuoles compared to sham transfected MODE-K cells. Increased biochemical conversion of LC3 I/II, decreased p62 accumulation and increased LC3-GFP fluorescence was consistent with accumulation of autophagic vacuoles in silenced but not in non-silenced cells. To evaluate if the accumulation of autophagosomes was a result of increased autophagic activity or reduced autolysosomal degradation we treated the MODE-K cells with bafilomycin - an inhibitor of autophagosomelysosome fusion. Bafilomycin treatment increased LC3 I/II conversion showing that the absence of XBP1 resulted in increased levels of autophagic activity rather than reduced lysosomal fusion degradation. Conclusions: Our studies demonstrate that unabated ER stress results in activation of authophagy in Paneth cells. Further studies are needed to determine the role played by activation of autophagy by the UPR in highly secretory cells within the epithelium.
1047 Autophagy Regulates Immune Responses Through Destabilization of the Immunological Synapse Manon E. Wildenberg, Anne Christine W. Vos, Simone C. Wolfkamp, Marjolijn Duijvestein, Auke Verhaar, Anje A. Te Velde, Gijs R. van den Brink, Daniel W. Hommes Various polymorphisms in the autophagy related genes ATG16L1 and IRGM have been associated with the development of Crohn's disease (CD). Although the link between decreased autophagy and an inflammatory disorder like IBD suggests a role for this process in the regulation of immune responses, no data has been available on this topic. Therefore, this study focused on the effects of decreased autophagy on the immunogenicity of dendritic cells (DC). Methods Gene knockdown was achieved in monocyte derived (human) or bone morrow (mouse) DC using siRNA technology. DC-T cell interactions were induced in mixed lymphocytes reactions (MLR), and cytoskeletal changes and interaction times were studied by immunofluorescence and time-lapse microscopy. T cell reactivity was determined by 3H incorporation and cytokine production assays. For patient studies, monocytes were obtained from peripheral blood of CD patients genotyped for rs_2241880. Results ATG16L1-low and IRGM-low DC induced significantly more T cell proliferation in both an allogeneic MLR and an antigen specific proliferation assay. This finding was consistent in human and mouse cells, suggesting a conserved role for autophagy in the regulation of immune responses. No alterations in cytokine production or surface marker expression of autophagylow DC were seen, indicating the immunostimulatory phenotype is not due to increased maturation. However, clear aberrancies occured at the site of contact between DC and T cells, the socalled immunological synapse (IS). Autophagy-low DC displayed increased cytoskeletal polarization towards interacting T cells, and interaction times between DC and lymphocytes were prolonged, pointing to IS hyperstability. To confirm the physiological role of this mechanism, we compared IS formation in CD patients carrying the ATG16L1 risk allele and patients carrying the wild type allele. Indeed, IS formation was increased significantly in homozygous risk allele carriers compared to wild type controls. Finally, autophagy-low DC more effectively induced Th17 polarization without affecting Th1 cells, thus increasing the Th17/Th1 balance. This effect was likely due to the IS hyperstability observed, as destabilizing the synapse pharmacologically had the opposite effect and tilted the balance towards Th1. Conclusion Decreased levels of autophagy results in an increased pro-inflammatory capacity in both human and mouse DC. This effect is regulated through hyperstabilization of the IS, leading to increased T cell activation and tilting of immune responses towards a more Th17 phenotype. This phenotype was confirmed in cells obtained from risk allele
1045 Protein Tyrosine Phosphatase Non-Receptor Type 2 is a Regulator of Authophagosome Function Michael Scharl, Kacper A. Wojtal, Helen M. Becker, Anne Fischbeck, Joba M. Arikkat, Theresa Pesch, Silvia Kellermeier, David L. Boone, Achim Weber, Pascal Frei, Stephan R. Vavricka, Michael Fried, Declan F. McCole, Gerhard Rogler Background: Autophagy is a process of critical importance for the maintenance of cell homeostasis, survival and the regulation of inflammation. Dysregulated autophagy contributes to cell death. Recent studies have associated variants within the gene loci encoding the autophagy genes, autophagy-related 16-like 1 (ATG16L1) and immunity-related GTPase, M (IRGM), as well as the gene encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2), with increased risk of developing Crohn's disease (CD). We have recently shown that PTPN2 inactivates the epidermal growth factor receptor (EGFr) in intestinal epithelial cells (IEC). Of note, EGF inhibits autophagy via a pathway involving EGFr and molecular target of rapamycin (mTOR). Here, we show that PTPN2 regulates autophagy in human IEC in response to pro-inflammatory stimuli. Methods: T84 and HT-29 IEC were used for all studies. Protein analysis was performed by Western blotting and visual imaging by immunofluorescence studies. PTPN2 knock-down was induced by siRNA and ATG16L1 mutation was introduced into IEC using a lentiviral vector. Results: Loss of PTPN2 significantly enhanced activity of EGFr, phosphatidylinositol 3-kinase, AKT and mTOR (n=3, p<0.05
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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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carriers and is therefore likely to contribute to the increased immune activation as seen in CD patients carrying autophagy-related SNP.
converted to jejunostomy at the time of implantation of GES system (total 42). The 39 patients, who already had a J-tube, continued after GES surgery and 18 patients had a new placement of a J-tube during Enterra surgery. Thus there were a total of 99 patients with J-tubes at the initiation of Enterra Therapy. Mean weight in these patients was 149 ±41 lbs at baseline. Fisher's exact Chi-Square test was used for statistical analysis. Results: Only 9 (11%) of Enterra patients continued to required supplemental nutritional support by J-tube (p=0.032) and 2 still required TPN at the final follow up visit.See table: The weight in all 3 groups of patients significantly increased to 162±43 lbs (p<0.005) at the final visit. Conclusions: 1) Enterra therapy resulted in significant nutrition gains with an 89% reduction in J-tube use and significant increase in weight for GP patients of all etiologies; 2) These data indicate that GES therapy is also recommended to be performed when J-tube placement surgery is being considered in patients with malnutrition and GP.
each) in response to TNF/IFNγ co-treatment (100 ng/ml each, 24 h) in T84 IEC. While cytokine treatment significantly induced the expression of autophagy related genes in PTPN2competent cells, PTPN2 knock-down prevented the cytokine-induced rise in protein levels of beclin-1, ATG5-ATG12 conjugates, IRGM and ATG16L1 (n=3; p<0.05 each). This resulted in impaired levels of autophagy as demonstrated by diminished levels of microtubuleassociated protein 1 light-chain 3 B-II (LC3B-II), a marker of autophagy activity (n=3; p<0.05). LC3B+ vesicles, indicative of autophagosomes, were clearly fewer in number, but larger in size in cytokine-treated PTPN2-deficient cells, indicating defective autophagy, when compared to PTPN2-competent cells (n=3). On a functional level, impaired autophagy in PTPN2-deficient cells correlated with increased apoptosis as shown by elevated levels of cleaved caspase-7 and poly ADP-ribose polymerase (PARP) (n=3; p<0.05). Presence of the CD-associated ATG16L1 variant prevented the cytokine-induced rise in PTPN2 protein (n= 3; p<0.05) and resulted in impaired LC3B-II levels (n=3; p<0.05) and autophagosomes in IEC, similar to PTPN2-deficient cells. These observations were fully confirmed in cytokinetreated HT-29 IEC (n=2). Conclusions: Our results demonstrate that PTPN2 regulates autophagy in IEC in the setting of inflammation. We provide a model of how dysfunction of the CD susceptibility genes, PTPN2 and ATG16L1, may contribute to cell dysfunction and cell death. These events could contribute to the onset as well as the perpetuation of chronic intestinal inflammation, as observed in CD. 1046 Reduced Paneth Cell Antimicrobial Protein Levels Correlate With Activation of the Unfolded Protein Response in the Gut of Obese Individuals Caroline M. Hodin, Froukje J. Verdam, Joep Grootjans, Sander S. Rensen, Cornelis H. Dejong, Wim A. Buurman, Jan Willem Greve, Kaatje Lenaerts Background and aims The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been observed in mice with a deficiency in active Paneth cell α-defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects as a possible cause of the obesity-associated microbiota shift. In addition, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Methods Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI>35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human α-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Results Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 and lysozyme transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Moreover, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Conclusions Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects which may underlie the observed Paneth cell compromise.
1044 Conditional XBP1 Deletion Within the Intestinal Epithelial Cells Results in Activation of Autophagy in Paneth Cells Michal Tomczak, Hyun-Jeong Ko, Eduardo Martinez-Naves, Susan J. Hagen, Arthur Kaser, Richard S. Blumberg Introduction: The unfolded protein response (UPR) is one of the pathways critical to the maintenance of epithelial function and intestinal homeostasis. Genetic defects within components of the UPR (e.g. XBP1, AGR2, ORMDL3) have been linked to development of IBD. Recently, we have shown that some IBD patients carry rare hypomorphic variants of XBP1, consistent with this, conditional deletion of XBP1 (XBPflox/floxVcre) within the intestinal epithelial cells (IEC) results in excessive ER stress, Paneth cell apoptosis and development of spontaneous enteritis. The UPR is closely linked to induction of autophagy, another cellular pathway important to epithelial homeostasis and risk for Crohn's disease (e.g. ATG16L1, IRGM and LRKK2). Here, we have tested if unabated ER stress in the IEC activates autophagy. Methods: Activation of autophagy within the IEC was compared in XBPflox/ floxVcre (KO) mice and XBPflox/flox (WT) littermates. To test the effects of ER stress on autophagy induction In Vitro we silenced XBP1 expression in the MODE-K cell line. The activation of autophagy was assessed by LC3 I/II conversion, electron microscopy (EM) and LC3-GFP expression. Results: EM analysis of small intestinal samples from KO mice showed decreased numbers of Paneth cells with reduced numbers of secretory granules and high quantities of autophagic vacuoles compared to WT littermates. A similar pattern of autophagy activation, but to a lesser extent, was observed in goblet cells and enterocytes in KO mice compared to WT mice. Increased activation of autophagy in IEC of KO mice, compared to WT IEC, was confirmed by biochemical conversions of LC3 I/II. EM evaluation of MODEK cells with stably silenced XBP1 showed higher quantities of autophagic vacuoles compared to sham transfected MODE-K cells. Increased biochemical conversion of LC3 I/II, decreased p62 accumulation and increased LC3-GFP fluorescence was consistent with accumulation of autophagic vacuoles in silenced but not in non-silenced cells. To evaluate if the accumulation of autophagosomes was a result of increased autophagic activity or reduced autolysosomal degradation we treated the MODE-K cells with bafilomycin - an inhibitor of autophagosomelysosome fusion. Bafilomycin treatment increased LC3 I/II conversion showing that the absence of XBP1 resulted in increased levels of autophagic activity rather than reduced lysosomal fusion degradation. Conclusions: Our studies demonstrate that unabated ER stress results in activation of authophagy in Paneth cells. Further studies are needed to determine the role played by activation of autophagy by the UPR in highly secretory cells within the epithelium.
1047 Autophagy Regulates Immune Responses Through Destabilization of the Immunological Synapse Manon E. Wildenberg, Anne Christine W. Vos, Simone C. Wolfkamp, Marjolijn Duijvestein, Auke Verhaar, Anje A. Te Velde, Gijs R. van den Brink, Daniel W. Hommes Various polymorphisms in the autophagy related genes ATG16L1 and IRGM have been associated with the development of Crohn's disease (CD). Although the link between decreased autophagy and an inflammatory disorder like IBD suggests a role for this process in the regulation of immune responses, no data has been available on this topic. Therefore, this study focused on the effects of decreased autophagy on the immunogenicity of dendritic cells (DC). Methods Gene knockdown was achieved in monocyte derived (human) or bone morrow (mouse) DC using siRNA technology. DC-T cell interactions were induced in mixed lymphocytes reactions (MLR), and cytoskeletal changes and interaction times were studied by immunofluorescence and time-lapse microscopy. T cell reactivity was determined by 3H incorporation and cytokine production assays. For patient studies, monocytes were obtained from peripheral blood of CD patients genotyped for rs_2241880. Results ATG16L1-low and IRGM-low DC induced significantly more T cell proliferation in both an allogeneic MLR and an antigen specific proliferation assay. This finding was consistent in human and mouse cells, suggesting a conserved role for autophagy in the regulation of immune responses. No alterations in cytokine production or surface marker expression of autophagylow DC were seen, indicating the immunostimulatory phenotype is not due to increased maturation. However, clear aberrancies occured at the site of contact between DC and T cells, the socalled immunological synapse (IS). Autophagy-low DC displayed increased cytoskeletal polarization towards interacting T cells, and interaction times between DC and lymphocytes were prolonged, pointing to IS hyperstability. To confirm the physiological role of this mechanism, we compared IS formation in CD patients carrying the ATG16L1 risk allele and patients carrying the wild type allele. Indeed, IS formation was increased significantly in homozygous risk allele carriers compared to wild type controls. Finally, autophagy-low DC more effectively induced Th17 polarization without affecting Th1 cells, thus increasing the Th17/Th1 balance. This effect was likely due to the IS hyperstability observed, as destabilizing the synapse pharmacologically had the opposite effect and tilted the balance towards Th1. Conclusion Decreased levels of autophagy results in an increased pro-inflammatory capacity in both human and mouse DC. This effect is regulated through hyperstabilization of the IS, leading to increased T cell activation and tilting of immune responses towards a more Th17 phenotype. This phenotype was confirmed in cells obtained from risk allele
1045 Protein Tyrosine Phosphatase Non-Receptor Type 2 is a Regulator of Authophagosome Function Michael Scharl, Kacper A. Wojtal, Helen M. Becker, Anne Fischbeck, Joba M. Arikkat, Theresa Pesch, Silvia Kellermeier, David L. Boone, Achim Weber, Pascal Frei, Stephan R. Vavricka, Michael Fried, Declan F. McCole, Gerhard Rogler Background: Autophagy is a process of critical importance for the maintenance of cell homeostasis, survival and the regulation of inflammation. Dysregulated autophagy contributes to cell death. Recent studies have associated variants within the gene loci encoding the autophagy genes, autophagy-related 16-like 1 (ATG16L1) and immunity-related GTPase, M (IRGM), as well as the gene encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2), with increased risk of developing Crohn's disease (CD). We have recently shown that PTPN2 inactivates the epidermal growth factor receptor (EGFr) in intestinal epithelial cells (IEC). Of note, EGF inhibits autophagy via a pathway involving EGFr and molecular target of rapamycin (mTOR). Here, we show that PTPN2 regulates autophagy in human IEC in response to pro-inflammatory stimuli. Methods: T84 and HT-29 IEC were used for all studies. Protein analysis was performed by Western blotting and visual imaging by immunofluorescence studies. PTPN2 knock-down was induced by siRNA and ATG16L1 mutation was introduced into IEC using a lentiviral vector. Results: Loss of PTPN2 significantly enhanced activity of EGFr, phosphatidylinositol 3-kinase, AKT and mTOR (n=3, p<0.05
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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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carriers and is therefore likely to contribute to the increased immune activation as seen in CD patients carrying autophagy-related SNP.