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smice (129SvEv) with Enterococcus faecalis (E. faecalis) strain OG1RF revealed increased mRNAexpression levels of the bacterial gelatinase E (GelE) under conditions of experimental colitis.To further characterize the role of bacterial proteases in the pathogenesis of chronic intestinalinflammation, we monoassociated WT and IL-10-/- mice for 15 weeks with E. faecalis strainOG1RF and isogenic mutant strains which lack GelE expression including TX5264 (gelEdeletion) and TX5266 (fsrB deletion). Histopathological analysis (score 0-4) revealed asignificant reduction of distal and proximal colonic tissue pathology (OG1RF: distal 3.2 ±0.2, proximal 2.1 ± 0.3) in the absence of bacterial GelE (TX5264: distal 2.1 ± 0.7, proximal1.0 ± 0.3; TX5266: distal 1.8 ± 0.6, proximal 0.9 ± 0.2, all p<0.001 vs OG1RF). To furtherinvestigate the impact of GelE on transepithelial electrical resistance (TER) and barrierfunction, we used human (T84) and mouse (Ptk6) colonocytes in transwell culture systems.Apical stimulation of the transwell cultures with concentrated conditioned media (CM) ofE. faecalis OG1RF reduced TER values by approximately 75%. In contrast, GelE deficientmutant strains (TX5264 and TX5266) did not affect barrier integrity of the epithelial cellcultures. Consistent with these results, OG1RF but not its isogenic mutant strains translocatedacross the epithelial cell monolayer, supporting the hypothesis that GelE impairs barrierintegrity of the intestinal epithelium. Interestingly, purified proteolytically active GelE didnot reduce TER in the transwell culture system. Additional experiments with purified GelEand CM of the GelE deficient strain (TX5264) again impaired epithelial integrity, suggestingthat additional secreted factors interact with GelE to modulate barrier function In Vitro.Conclusion. E. faecalis-derived gelatinase E contributes to the development of experimentalcolitis in IL-10-/- mice. In combination with other secreted components of this bacterialspecies, GelE lead to the impairment of epithelial barrier integrity, supporting the hypothesisthat bacterial proteases in the gut lumen specifically contribute to the pathogenesis of chronicintestinal inflammation.

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Epithelial PI3K Signaling Is Critical to Epithelial and Innate ImmuneResponses to Citrobacter rodentium InfectionJeffrey Brown, Paul Cheresh, Terrence A. Barrett

Background/Aims: Epithelial phosphoinositol-3 kinase (PI3K) phosphorylation (activation)of Akt (P-Akt) has been implicated in cellular proliferation, growth, and apoptosis. Usingsite-specific antibodies for P-Akt and Akt phosphorylated beta-catenin (P-b-cat), we foundP-Akt and P-b-cat at sites of intestinal stem cell activation in small bowel polyposis (He etal. Nat Genetics 2007). Its role in host defense is less clear. The aim of this study was toidentify a mechanistic link between PI3K, Akt and P-b-cat signaling in host defense duringCitrobacter rodentium infection. Methods/Results: C57BL/6 mice were infected via oral gavagewith C. rodentium and treated with vehicle control (DMSO) or the PI3K inhibitor LY294002(LY) (day 5, 8, 13). Tissues were examined at multiple time points post-infection (day 4,7, 14, 18, 21, 28). Epithelial PI3K-induced P-Akt and P-b-cat activation were assessed byimmmunohistochemistry (IHC) and Western blot of leukocyte-depleted epithelial cells. RealTime PCR demonstrated a significant increase in IL22 expression at day 4 (96-fold) andday 7 (342-fold). By day 14, mice developed distal colitis with marked crypt hyperplasia(6-fold increased BrdU incorporation) and mucin depletion that coincided with reducedbacterial infection and subsequent bacterial clearance. Hyperplastic epithelia had increased“activated” P-Akt and P-b-cat levels (20 and 25-fold, respectively) that localized to lowerand mid-crypt regions by IHC. LY treatment (PI3K inhibition) resulted in a 95% reductionin subsequent IL22 transcription and reduced P-Akt and P-b-cat protein 90%; BrdU stainingwas reduced 60% resulting in 43% less crypt hyperplasia and increased epithelial ulceration.LY treatment delayed clearance of colonic bacteria, increased invasive/systemic infection andweight loss. Real time PCR demonstrated PI3K inhibition had no effect on adaptive immuneresponses (CXCL1, 5, and 9; IL6 and IL1beta) at day 4, 7, and 14. Upon cessation of PI3Kblockade, mice were able to rapidly clear bacteria and recover weight loss with recovery ofepithelial proliferative responses and compensatory increases in cytokine and chemokinemRNA levels at day 18 and 21. Conclusion: C. rodentium infection induces epithelial PI3Ksignaling (increased crypt cell nuclear (Akt-activated) P-b-cat) and epithelial proliferationnecessary for appropriate host defense response and limitation of invasive disease. PI3Kblockade impaired critical early innate immune responses (IL22) with no negative effect onsubsequent adaptive responses. This data suggests epithelial PI3K-induced P-b-cat signalingis required to maintain intestinal host defenses at the mucosal barrier.

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Cholesterol-α-Glucosyltransferase Is Important for Establishing ColonizationBy Helicobacter Hepaticus in Male A/JCr MiceZhongming Ge, Yan Feng, Sureshkumar Muthupalani, Mark T. Whary, James G. Fox

Helicobacter hepaticus (Hh), an enterohepatic helicobacter used in infectious models fordissecting pathogenic mechanisms of human diseases including IBD, hepatocellular carcin-oma and intestinal cancers. Approximately 50% of genes predicted from the genome of Hhare orthologs of Helicobacter pylori (Hp), a gastric pathogen in humans, including a geneencoding cholesterol-α-glucosyltransferase (cgt). It has been reported that exogenously sup-plemented cholesterol amplified In Vitro phagocytosis of Hp by antigen-presenting cells suchas macrophages and dendritic cells, and also enhances Hp-specific T cell responses. Thisenzyme catalyzes conversion of cholesterol into cholesteryl-α-glucoside, thereby protectingHp from phagocytosis, and is essential for In Vivo survival of Hp. To investigate a role ofcgt in the pathogenesis of Hh, we generated two cgt-deficient isogenic mutants (HhcgtM1,HhcgtM2) of Hh 3B1, which were subsequently used to experimentally inoculate 4 to 6-week-old male A/JCr mice. Cecal and hepatic colonization levels of the inoculated micewere measured by real-time quantitative PCR at 4 months post-inoculation. Both mutantswere not detectible in the cecal tissues of all mice previously dosed with the respectivemutants (10 per mutant) but were positive in two livers (one for each mutant); by contrast,9 and 7 out of 10 mice inoculated with Hh were positive for Hh 3B1 in the ceca and livers,respectively. In addition, the mice inoculated with the mutants developed significantly lesssevere hepatic inflammation (P < 0.05) and also produced significantly lower hepatic mRNAlevels of proinflammatory cytokines IFN-γ (P < 0.01) and TNF-α (P ≤ 0.02) compared tothe Hh 3B1-infected mice. Furthermore, the Hh 3B1-infected mice developed significantly

A-22AGA Abstracts

higher Th1-associated IgG2a (P < 0.0001) and Th2-associated IgG1 responses (P < 0.0001)to infection than those dosed with the mutants. Thus, our data indicate that Hh cholesterol-α-glucosyltransferase plays an important role in bacterial colonization of intestine and liver.The finding that two mice infected with the mutants did not develop hepatitis also suggeststhat this enzyme is necessary for inducing hepatic inflammation by Hh in male A/JCr mice.

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Acid and Bile Acids Modulate the Esophageal Squamous Barrier Function ByModulating the Localization of ClaudinsTadayuki Oshima, Junichi Koseki, Karin Gedda, Stefan E. Pierrou, Hiroto Miwa

Background and Aims: There has been no model by which the function of esophagealepithelium can be investigated In Vitro. Here we have established a model for the esophageallike squamous epithelial cell layers by air-liquid interface (ALI) system and investigated theinfluence of acid and bile acids on squamous epithelial barrier function. Methods: Normalhuman bronchial epithelial (NHBE) cells were used in this study. Cells were seed on thecollagen and fibronectin coated inserts. Culture at ALI conditions was initiated by removingthe apical medium. Barrier function was measured by trans-epithelial electrical resistance(TEER) and diffusion of paracellular tracers. Squamous epithelial cell character was confirmedwith the expression of cytokeratin 4 and 13 by western blotting and immunofluorescencestaining. Morphological changes were confirmed by optical and transmission electron micro-scope. After the establishment of this In Vitro system, we examined the influence of acid(pH 2-4) and bile acid (glycocholic acid and taurocholic acid) (0.4-10 mM) against thebarrier created by these cells. Tight junction protein (claudins and tricellulin) expressionand localization was examined by immunofluorescence staining, and by western blotting intotal and in 1% NP-40 soluble and insoluble fractions. Results: The In Vitro ALI culturesystem showed a tight barrier (1500-2500Ω.cm2) previously only achievable with esophagealbiopsies mounted in Ussing chambers (Tobey et al., 2004). Molecular markers for tightjunctions (claudin-1, -4 and -7), and mature esophageal epithelium (cytokeratin-4, -13) areupregulated in the differentiating culture in parallel with functional properties includingdecreased permeability and increased TEER. ALI cultured cells exposed to acid at pH 2 for4 hours on the apical side resulted in a decrease in TEER to 62% at pH 2. On the otherhand, the TEER was increased to 170% with acid at pH 3 (4 hours) in parallel with theincreased detergent insoluble tricellulin. Bile acids did not change the TEER by themselves.However, when the cells are exposed to glycocholic acid (10 mM) or taurocholic acid (10mM) at pH 3 acid, the TEER significantly decreased and the detergent insoluble claudin-7decreased, which may explain the dislocalization of tight junction proteins for the TJ.Conclusions: 1. We have for the first time established esophageal like squamous epithelialcell layers by ALI system In Vitro. 2. Acid plus bile acid stimulation disrupted the barrierfunction by modulating the localization of claudin-7.

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Trefoil Factor-2 and Sodium-Hydrogen Exchanger Share Common Pathways inStimulating Gastric Epithelial Repair of Microlesions In VivoLin Xue, Eitaro Aihara, Marshall H. Montrose

Introduction: Our previous data showed the TFF2 null mice have impaired epithelial repairof (laser-induced) gastric microlesions, including an abnormal regulation of extracellular pHat the gastric surface. Both trefoil factor peptides (TFFs) and sodium-hydrogen exchangetransporter protein (NHEs) have been independently shown to stimulate epithelial woundrepair, potentially by stimulating migration of healthy cells into a wounded area to replacedamaged cells. Therefore we asked whether TFF2 and NHE have overlapping mechanismsof action during gastric epithelial repair In Vivo. Aim: We asked if NHE activity affected theepithelial repair kinetics of TFF2 null and control animals. Methods: Quantitative In Vivoconfocal microscopy of anesthetized mice was used to monitor the time course of repairafter (710nm laser) photodamage induced microlesions (PD-microlesion) to the gastricsurface epithelium in corpus. Wild-type, TFF2+/- mice and TFF2 KO mice were studiedwith/without application of pan-NHE inhibitor EIPA (15 μM) or NHE-1 and NHE-2 inhibitorHOE694 (25 μM) to the luminal perfusate. The expression of NHE subtypes was evaluatedwith Western blot. Results: Measuring the size of PD-microlesions over time, the restitutionrate constant (κ, (s x1000)-1) was slower in TFF2 KO (0.8±0.2 κ, n=6) versus control, WTand TFF2+/- (3.6±0.5 κ, n=9). Interestingly, when the perfusate included NHE inhibitorEIPA or HOE694, the gastric epithelial restitution rate in control mice was also significantlydecreased, to 0.6±0.1 κ (n=3, P<0.05) and 1.1±0.2 κ (n=4, P<0.05), respectively. After PD-microlesion, HOE694 was unable to produce further reduction in restitution rate in TFF2KO mice (1.4±0.5 κ, n=4 P>0.05 v.s. TFF2 KO). The NHE2 protein expression in TFF2KO mice stomach was downregulated, but the NHE1 and NHE3 were not changed. Conclu-sion: Both TFF2 and NHE play important roles In Vivo in epithelial restitution after laserphotodamage induced gastric microlesion. TFF2 may promote cell migration at least partiallythrough a pathway that requires activation of NHE activity. Supported by NIH DK 54940.

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Down-Regulation of 15-Hydroxyprostaglandin Dehydrogenase During GastricUlcer HealingTetsuya Tanigawa, Toshio Watanabe, Hiroshi Tatsuwaki, Hirohisa Machida, HirotoshiOkazaki, Hirokazu Yamagami, Kenji Watanabe, Kazunari Tominaga, Yasuhiro Fujiwara,Nobuhide Oshitani, Tetsuo Arakawa

Background: Prostaglandins (PGs), especially PGE2, play important roles in promotinghealing of gastrointestinal ulcers. The amount of biologically active PGE2 in tissue is deter-mined by the balance of its synthetic enzymes, such as cyclooxygenase (COX), and itscatabolic enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) responsible for bio-logical inactivation of PG. We investigated the expression of 15-PGDH in the stomach andgastric ulcer and the mechanism by which 15-PGDH is regulated during gastric ulcer healing.Methods: In C57BL/6J mice, gastric ulcers were induced by focal serosal application of