esterase (NS) expression was observed compared to unaffected control tissue. CXCL8 expres-sion and histology scores were included to assess the severity of inflammation. In epithelialcell line CACO-2, IL-1β (10 ng/ml) induced dissociation of tight junction proteins (occludin& ZO1) was observed by immunoblotting, which was prevented upon pre-treatment withmAChR agonists. Treatment with IL-1β enhanced transcellular permeability (4kD dextran)and reduced impedance (ECIS) across the monolayer, which was counteracted by preincuba-tion with acetylcholine (10 μM; P < 0.05), or bethanechol (100 nM; P = 0.009). Theprotective effects of ACh were antagonised by atropine (50 nM). Since the transcription ofmyosin light chain kinase (MLCK) and phosphorylation of myosin light chain were reducedwithout any significant effect on IkB-αβ phosphorylation, thus the effects of cholinergicagonists in the epithelium are postulated to be NF-kB independent. In conclusion, reducedepithelial acetylcholine metabolic enzyme expression in an inflamed colon may contributeto impaired barrier and colon pathogenesis. Acetylcholine restores epithelial barrier functionunder inflammatory conditions.

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Colonic Claudin-1 Overexpression Augments Notch-Signaling andExperimentally-Induced Colitis While Impairs RecoveryJillian Pope, Ajaz A. Bhat, Ashok Sharma, Robert D. Beauchamp, Kay Washington, AmarB. Singh, Punita Dhawan

We along with other laboratories have reported that expression of tight junction protein,claudin-1 is highly upregulated in colon cancer. Claudin-1 expression is also upregulatedin ulcerative colitis and associated carcinogenesis. Further, claudin-1 expression inverselycorrelates with the epithelial differentiation. However, role of claudin-1 in the regulation ofnormal intestinal homeostasis and a causal association with mucosal inflammation remainunexplored. To investigate, we generated Villin-Claudin-1 transgenic (TG) mice whereclaudin-1 was overexpressed in the intestinal epithelium. Analysis at the level of mRNA andprotein expression confirmed claudin-1 over-expression, and also demonstrated decreasedexpressions of claudin-2 (~2 fold) and claudin-7 (~10 fold). Further characterization usingPeriodic Acid Schiff (PAS) staining showed that goblet cell population is significantlydecreased in claudin-1TGmice (33% compared toWT littermates; p<0.001). Neuroendocrinecell population (chromogranin A staining) was not altered while colonocytes (carbonicanyhydrase staining) were markedly increased suggesting disruption of normal colonicepithelial cell differentiation. The Notch-signaling pathway, acts as the molecular switchbetween the secretory and absorptive fates. Indeed, immunoblot analysis using tissue lysatesfromWT and Claudin-1TG mice showed increased expression of cleaved Notch, an indicatorof Notch-activation, and an upstream regulator of Muc-2. Further studies demonstrated asignificant decrease in Muc-2 expression in Claudin-1TG mice. Muc-2 provides defenseagainst the luminal antigens and decrease/loss of its expression associates with enhancedinflammation. Also, in our studies, 3.5% Dextran Sodium Sulfate (DSS) for 7 days inducedcolitis and resulted in a higher degree of epithelial injury and inflammation in Claudin-1TGmice as compared to WT as assessed by CD3 staining, cytokine profile and H&E. Mostimportantly, recovery following the DSS-colitis (mice were exposed to regular drinking waterafter 7 days of DSS-treatment) was markedly impaired in Claudin-1TG mice and we observedhyperplasia in claudin-1TG mice compared to normal regenerative colonic crypts in the WTmice. Further analysis showed that Hes1 expression was significantly increased while muc-2 expression was decreased in claudin-1TG mice either subjected to DSS colitis or therecovering TG mice compared to the WT littermates exposed to similar experimental condi-tions. Taken together, our data suggest a key role of claudin-1 in colonic homeostasis throughthe regulation of epithelial cell lineage commitment. Further, claudin-1 activates Notch-signaling possibly through the activation of MMPs which in turn decreases goblet cellpopulation, Muc-2 expression and increases susceptibility to colonic inflammation.

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Acute Psychological Stress Impairs Small Intestinal Barrier Function inHealthy VolunteersTim Vanuytsel, Sander Van Wanrooy, Shadea Salim Rasoel, Els Houben, Ricard Farré,Lukas Van Oudenhove, Hanne Vanheel, Guy E. Boeckxstaens, Kristin Verbeke, Jan F. Tack

Background: In inflammatory bowel disease (IBD), psychological stress is associated withan increased risk of relapse of disease. An impaired intestinal barrier function has beenimplicated in the pathogenesis of IBD and other chronic gastrointestinal disorders. It isunknown whether psychological stress can induce defects in intestinal permeability inhumans. Aim: To test the effect of acute psychological stress on small intestinal permeability.Methods: Twenty-one volunteers (12 men, 21±0.3 yr), without personal or familial historyof gastrointestinal or atopic disorders and not taking medication known to influence intestinalpermeability, were enrolled in the study. In Vivo small intestinal permeability was determinedbymeasuring the urinary lactulose-mannitol ratio (LMR) in a fractionated 24h urine collectionafter drinking a 150mL solution containing 5g of lactulose and 2g of mannitol. Sampleswere analyzed by HPLC with ELSD-detector. Tests were performed after an 8h fasting periodat 2pm to account for diurnal variation. Four different conditions were tested in each subjectwith an interval of at least 1 week: 1) control; 2) after ingestion of 75 mg of indomethacinthe previous evening and 50 mg 8h before the test; 3) during and after a 30 min anticipatorystress protocol, involving 20 auditory countdown sequences at random intervals with halfof these followed by a painful electrical stimulus in a randomized fashion; 4) during andafter a public speech in an exam situation. Stress-related symptoms were scored by a validatedquestionnaire (STAI-state) and salivary cortisol was determined by ELISA. Data were analyzedby a mixed model analysis with ‘condition' as a within-subject factor. Results: The electrosti-mulation protocol and public speech both increased the total STAI-state score (46.6±2.2and 50.7±2.2 vs. 30.7±1.2; both p<0.0001). Salivary cortisol was only augmented duringpublic speech (17±1.6 vs. 8.4±0.7 ng/ml; p<0.0001). During the first two hours, the urinaryLMR was significantly higher after NSAID (0.073±0.009 vs. 0.031±0.009; p<0.001) andpublic speech (0.058±0.011; p<0.05), but not after the electrostimulation protocol(0.034±0.004; NS) (Fig.1). In the subsequent collections (2-4, 4-6 and 6-24h) no influenceof the test condition could be demonstrated. Subgroup analysis showed no effect of electrosti-mulation or public speech on the LMR in volunteers that had a cortisol less than the 90th

S-811 AGA Abstracts

percentile during this test. However, a significant elevation of the LMR was seen in thevolunteers with a significant cortisol increase during public speech. The STAI-state scoresdid not predict the permeability response to the different conditions. Conclusion: Acutepsychological stress alters small intestinal permeability. This finding provides further insightin the pathogenesis of stress-related chronic gastrointestinal disorders.

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GM-CSF and Il23 Protect the Intestinal Epithelial Barrier From Disruption byTreatment With IFNγAnnette Mach, Sarah C. Glover, John F. Valentine

Introduction: Tight junctions between the epithelial cells lining the intestine create a barrierthat restricts the access of luminal contents to the submucosal environment. Homeostasisof intestinal permeability is a dynamic process and increased intestinal permeability is afeature of intestinal inflammation. Proinflammatory cytokines IFNγ, TNFα, and IL-1β disruptthe tight junctions between intestinal epithelial cells (IEC) resulting in increase permeability.IEC express receptors for GM-CSF and IL23 but little is known about the function. Wehypothesized that GM-CSF and IL23 would have protective effects on the IEC barrier.Aim: To characterize the In Vitro effect of GM-CSF and IL23 treatment of IEC monolayerpermeability. Methods: Caco2 IEC were grown in DMEM and 10% FBS on permeabletranswell inserts with 0.4μ pore size. Confluent monolayers (transepithelial electrical resist-ance (TEER) >250 ohm x cm2) were treated IFNγ 10 to 100 ng/ml, GM-CSF 20 ng/ml,and IL23 100 ng/ml individually and in combination. Alterations in monolayer permeabilitywas determined by monitoring the TEER and confirmed by measurement of the passage ofLucifer yellow (LY) 100μg/ml at 2 hr. Results: Treatment with IFNγ 100 ng/ml decreasedthe TEER (normalized to control) to 0.73±0.07 at 24 hr and 0.68±0.07 at 48 hrs (P<0.0001vs control). Pre-treatment with GM-CSF for 24 hr prior to treatment with IFNγ resulted ina TEER of 0.82±0.07 at 48 hr (P=0.0002 vs IFNγ alone). Treatment with GM-CSF and IFNγsimultaneously also resulted in a greater TEER at 48 hr (0.85±0.065) vs treatment withIFNγ alone (P = 0.0027). Similar results were seen when LY was used as the measure ofmonolayer permeability. Caco2 cells express IL23R following treatment with IFNγ 10 ng/ml but not under basal conditions. Treatment with low dose IFNγ 10 ng/ml did have aslight effect on the TEER, 0.85 at 24 hr. Following 24 hr treatment with low dose IFNγ,treatment with IL23 for 24 hr prior to IFNγ 100 ng/ml resulted in a TEER of 0.92±0.03vs. IFNγ alone 0.76±0.04 (P=0.0096). Similar results were observed if IL23 and IFNγ wereadded simultaneously. Treatment with GM-CSF or IL23 if added to the basal or the apicalchamber were equally protective. Conclusions: Treatment of IEC with GM-CSF before orsimultaneous with IFNγ, protect IECs from the deleterious effects of IFNγ on IEC monolayerpermeability. Although Caco2 cells under basal conditions do not express IL23R, IL23R isinduced by IFNγ at low doses. Treatment with IL23 also protects the IEC barrier functionfrom further insult even if treated with high dose IFNγ. The mechanisms of the protectiveeffects are being investigated.

Tu1363

Evidence for Post-Diverticulitis Irritable Bowel Syndrome (Pdv-IBS):Longitudinal Analysis Reveals Higher Incidence of IBS in DV Cases vs.ControlsErica R. Cohen, Garth Fuller, Roger Bolus, Bradley J. Snyder, Michelle Vu, KamyarShahedi, Rena Shah, Rusha Modi, Mary A. Atia, Nicole Kurzbard, Victoria Sheen, NikhilAgarwal, Marc Kaneshiro, Linnette Yen, Paul Hodgkins, Moshe H. Erder, PoyrungPoysophon, Brennan M. Spiegel

Background: Diverticulitis (DV) is often considered to be an acute illness surrounded byperiods of relative clinical silence. However, evolving data reveal that many DV patientsdevelop long-term bowel symptoms and impaired quality of life long after their DV attack;this suggests that DV may become a chronic bowel disorder in some patients. Previous datareveal that diverticulosis and IBS often co-exist, but there are no longitudinal data linkingacute DV with subsequent development of IBS and related functional bowel disorder (FBD)symptoms. Similar to post-infectious IBS, where an abrupt illness triggers longstandingsymptoms, we hypothesized that acute DVmight also stimulate IBS symptoms. We compared

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