in the WT littermates (n#15; p<0.001). Further studies using real time PCR showed, asexpected, several fold increase in the expressions of proinflammatory cytokines includingIL-1α, IL-1β, TNF-α, IFN-γ in the DSS-treated WT mice but not in the DSS-treated Cl-2TGmice suggesting potential immune tolerance/adaptation. Further assessment of prolifera-tion and apoptosis in isolated colonic epithelial cells using BrdU and cleaved caspase-3staining and FACS analysis showed decreased apoptosis (p<0.05) and increased proliferation(p<0.05) in the DSS-treated Cl-2Tg mice compared to the WT littermates. Taken together,our data supports that claudin-2 has a key role in the regulation of colonic homeostasis.Our data further suggests that immune tolerance/adaptation possibly due to the constitutiveincrease in colonic permeability and increased colonic epithelial cell viability underlie theprotection from the DSS-induced colitis in claudin-2 TG mice.

Claudin-2 TG mice are protected from the DSS-colitis. A. % change in body weight in theDSS-treated (4% DSS wt./vol in drinking water) Cl-2TG and WT mice. Body weight ispresented relative to the weight of respective group exposed to water alone; B. Injury scorefor DSS treated WT & Cl-2TG mice (n#15). Injury score for mice exposed to water (eithergroup) was zero; and C. H&E staining showing histological details . mean+SEM,**p<0.01***p<0.001

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In Vivo Constitutive Expression of an IBD Associated Gene TNFSF15 CausesSevere Inflammation and Induces Fibrostenotic Disease in 2 Murine Models ofChronic ColitisRobert Barrett, Xiaolan Zhang, Nicole Yeager, Hon Wai Koon, Mary Ann Nguyen,Piangwarin Phaosawasdi, Chenyang Li, Charalabos Pothoulakis, Stephan R. Targan, DavidQ. Shih

Background: Elevated expression of TNFSF15 (TL1A) is found in inflamed colonic andintestinal mucosa of IBD patients. Furthermore, TL1A haplotype B is associated with itsenhanced production and is also characterized by fibrostenotic disease and need for surgeryin IBD patients. Transgenic (tg) mice with constitutive expression of TL1A in either T cellsor antigen presenting cells (APC) lead to spontaneous ileitis. Aim: To assess whether InVivo constitutive TL1A expression in myeloid and lymphoid cells exacerbate inflammationand cause fibrostenotic disease in 2 murine models of chronic colitis. Methods: Chronicdextran sodium sulfate (DSS) and adoptive T-cell transfer models were used. Disease activityindex (DAI: stool blood, consistency and weight loss), gross inflammation, Histologicalanalyses of H&E, Masson Trichrome, and vimentin stained gut sections were analyzed forinflammation and fibrosis in a blinded fashion. mRNA was measured by RT-PCR and proteinexpression was measured by flow cytometry and ELISA. Results: Compared to wildtype(WT) littermates, overexpression of Tl1a in lymphoid or myeloid cells lead to significantlyenhanced weight loss and worsened DAI. We observed enhanced gross inflammatory scorein the intestine of myeloid Tl1a tg (3.2 for vs. 1.4, p<0.05) and T cell tg (3.5 vs. 1.4, p<0.05)in the chronic DSS model and in the T cell transfer model (3.6 vs. 1, p<0.01). Tl1a tg miceshowed increased histologic inflammation in the intestine, particularly in the ileum of myeloidTl1a tg (8.9 vs. 5.9, p<0.05) and T cell Tl1a tg (9.7 vs. 5.7, p<0.05) for the chronic DSSmodel and adoptive transfer model (11.2 vs. 5.8, p<0.05). Mucosal Tl1a tg T cells or APCwere found to have a more activated phenotype (WT 21%, myeloid tg 37%, myeloid tg46%) and higher IFN-γ expression (WT 1.2%, myeloid tg 2%, T cell tg 3.2%) in bothmodels of chronic colitis. Notably, gross strictures were only present in the Tl1a tg mice:small intestine (30%, up to 3 cm), colon (20%, up to 1 cm) and ureter (10%, 2 mm) withresultant hydronephrosis. Enhanced histologic fibrosis in the Tl1a tg mice were confirmedby increased Masson Trichrome (marker for collagen formation) and vimentin (marker forfibroblast) staining. Consistent with increased fibrosis, increased expression of fibrogenicfactors TGF-β1 and IGF-1 were found in the intestine and colon of tg mice. Conclusion:Tl1a modulates the development of both severe gut mucosal inflammation and markedfibrosis through enhancing Th1 effector functions and expression of pro-fibrogenic factors.

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Pyrosequencing Reveals Irritable Bowel Syndrome Subtype Defined bySpecies-Specific Alterations in the Microbial Gut Environment.Ian B. Jeffery, Paul W. O' Toole, Lena Ohman, Marcus J. Claesson, Jennifer Deane,Eamonn M. Quigley, Magnus Simren

Introduction: Irritable bowel syndrome (IBS) is a common functional gastrointestinal dis-order that may be triggered by enteric pathogens and has also been linked to alterations inthe microbiota and the host immune response. Aim: To perform a detailed analysis of thefecal microbiota in IBS and control subjects and to correlate findings with key clinical andphysiological parameters. Methods: We included 20 patients with IBS (mean age 38 years;14 females; 9 IBS-D, 4 IBS-C, 7 IBS NonC-NonD). GI and psychological symptom severityand quality of life were evaluated with validated questionnaires. Rectal sensitivity was testedwith a rectal barostat, and colonic transit time (radiopaque markers) was measured. Fecalsamples were obtained from the patients and 20 age- and gender-matched healthy subjects.We applied pyrosequencing of over 32,000 16S rRNA gene V4 region amplicons per subjectto characterize the fecal microbiota. Results: The majority of patients had GI symptomseverity ranging from moderate to severe, and 8/20 patients had clinically significant anxietyand/or depression. Rectal hypersensitivity was detected in 6/19 patients, whereas colonictransit was rapid in 6 and slow in 2 patients. Reduced quality of life (mental and/or physical)was seen in 18/19 patients. The microbial data had no association with the clinical or

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physiological phenotype, except for a number of OTUs that were significantly associatedwith a rapid colonic transit time. Clustering analysis of the pyrosequencing data revealedtwo subgroups of IBS patients. These groups were significantly different from each other(p-value: 0.0025) and did not correlate with Rome II or Rome III subtypes or other clinicaldata. Group 1 clustered with the control samples and had no significantly associatedmicrobialfeatures when compared to normal subjects. Group 2 was defined by large microbiome-wide changes characterized by increased prevalence in the proportion of Phylum Firmicutescompared to control subjects, and a depletion of Bacteroidetes. Statistically associated withthis high Firmicutes/ Bacteroidetes (FB) ratio subset, were 114 operational taxonomic units(OTUs) of the phylum Firmicutes. 110 of these were of the Order Clostridiales and 47 and27 of these were of the Genera Blautia and Papillibacter, respectively. These Genera belongto Clostridium cluster XIVa, which, as a cluster, was over-represented in the High FB IBSassociated OTUs (p-value: < 0.001). Conclusions: This detailed analysis of a well-character-ized cohort of IBS patients does not support a pronounced or reproducible IBS-relatedmicrobiome, but rather identified a discrete subset of IBS patients with alterations in theiroverall microbial ecology. No clear association with the clinical and physiological phenotypeof the patients could be detected apart from a modest association with colonic transit.

927

The Cellular Distribution and Expression of ZO-1, Occludin and Claudin-1Were Differentially Altered During Irritable Bowel Syndrome According to theSub-Types and SymptomsNathalie Bertiaux-Vandaële, Stéphanie Beutheu Youmba, Belmonte Liliana, StephaneLecleire, Michel Antonietti, Guillaume Gourcerol, Leroi Anne Marie, Pierre M. Déchelotte,Philippe R. Ducrotté, Moïse Coëffier

Background & aims: Recent studies have suggested that increased intestinal permeabilitymay be involved in the pathophysiology of irritable bowel syndrome (IBS). However thedifferential expression of tight junction proteins according to IBS sub-types and symptomsremained unknown. Thus, we aimed to study the expression of ZO-1, occludin and claudin-1 in the colonic mucosa of patients with IBS. Methods: Fifty IBS patients fulfilling the RomeIII criteria and 31 controls were included. IBS patients were with predominant diarrhea(IBS-D, n=19), with predominant constipation (IBS-C, n=14), with constipation alternatingwith diarrhea (IBS-A, n=15) or unclassified (IBS-U, n=2). IBS symptom intensity was quanti-fied on 10-cm VAS. Tight junction proteins (claudin-1, ZO-1, occludin) were studied byqRT-PCR, western blot and immunofluorescence in immunohistochemistry. The results,expressed as mean ± SD, were compared by nonparametric Mann-Whitney or Kruskal-Wallis with Dunn post-tests, p <0.05 was considered the threshold of significance. Results:ZO-1 and occludin expression was lower in IBS patients compared with controls (0.25±0.27 vs 0.50 ±0.54 and 0.50 ±0.34 vs 1.22 ±1.76; p<0.05), whereas only a trend for adecrease of claudin-1 was observed (0.29 ±0.36 vs 0.32 ±0.23 ; p=0.08). The mRNA levelsremained unaffected. In the subgroup analyses, occludin and claudin-1 expression wasdecreased in IBS-D patients (p<0.05) but not in IBS-C and IBS-A patients whereas ZO-1expression was not modified. The subcellular distribution of these 3 proteins was alteredin IBS-C and IBS-D patients. While the 3 tight junction proteins staining appeared mainlyat the apical of epithelial cells with the typical reticular pattern in the colonic samples ofcontrols, occludin and claudin-1 was mainly cytosolic in the samples of IBS-C patients andcompleted abolished in IBS-D patients. In IBS-C and IBS-D patients, ZO-1 staining was lessapical and more diffuse in the cytoplasm. Occludin (r=0.40, p<0.01) and claudin-1 (r=0.46,p<0.01) expression was correlated with the duration of symptoms. The expression of occludinwas lower in patients with an abdominal pain intensity higher than 6 on the VAS (p<0.05).Conclusions: Occludin and claudin-1 appeared markedly affected in IBS with predominantdiarrhea. In addition, our results suggest that alteration of tight junction proteins may beinvolved in the initiation of IBS and contribute to visceral hypersensitivity.

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The Potency of Constipated IBS Fecal Supernatant to Increase ColonicPermeability in Mice Involves Occludin Enzymatic Degradation Linked toCysteine-Protease ActivityAnita Annahazi, Valérie Bézirard, Mathilde Leveque, Helene Eutamene, Laurent Ferrier,Moïse Coëffier, Philippe R. Ducrotté, Richárd Róka, Krisztina Gecse, Andras I. Rosztoczy,Tamas Molnar, Thierry Piche, Vassilia Theodorou, Tibor Wittmann, Lionel Bueno

Background and aims: We have previously shown that (i) fecal cysteine-protease (CP)activity is elevated only in constipated IBS (IBS-C) patients among IBS subtypes and (ii)repeated mucosal exposure to this luminal factor enhances colorectal sensitivity and increasesintestinal permeability in mice. Since CP are involved in the increase of airway permeabilityby digesting epithelial tight junction proteins, we evaluated the ability of CP in the IBS-Cfecal supernatant to degrade tight junction proteins in mice and T84 cells as well as recombin-ant occludin In Vitro. Methods: Fecal samples and colonic biopsies were collected fromhealthy subjects (n=20 & 5 respectively) and IBS-C patients (n=25 & 5 respectively). CPactivity was assayed using a selective substrate and controlled by a selective CP inhibitor,E64. IBS-C and healthy controls fecal supernatants (FSN) pre-incubated or not with E64,or papain solution (as a control CP) or vehicle (saline) were (i) infused intracolonically (IC)in C57/BL6 mice (0.3 ml for 3h, twice at 12 h interval) or, (ii) added to the apical site of T84cells seeded (5x105 cells/well) in 24-well Fluoblock® plate system. Intestinal permeability tooral FITC-4kD dextran was assessed 4h after the end of the second IC infusion. Permeabilityto FITC-4kD dextran was measured every hour over 24 hours in T84 cells. Expression ofoccludin in mice, human biopsies and T84 cells was evaluated by, western blotting andimmunostaining. Finally, recombinant occludin was incubated in presence of IBS-C, healthycontrols FSN or papain during 15 min, and occludin degradation was assessed by westernblotting. Results: CP activity in IBS-C FSN was significantly increased (p<0.05) vs healthycontrols (2420±718 and 319±91 Δfluo/mg prot). IBS-C FSN and papain IC infusion increasedcolonic permeability in mice compared to healthy controls or saline (403±53 vs 333±43;562±73 vs 341±40 Δfluo/μl respectively) as well as in T84 cells (34% increase vs controlFSN, starting at 15h after application). This effect was markedly reduced by E64 both inmice and T84 cells. Occludin protein levels were 2-fold lower in IBS-C colonic biopsies vs

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