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sreducing the risk of HE-related hospitalizations. Methods: This randomized, double-blind,placebo-controlled trial evaluated rifaximin 550 mg twice daily for 6 months in patientswith a history of HE. Patients with cirrhosis who had ≥2 episodes of HE (Conn score ≥2)within 6 months prior to screening and were currently in remission defined as a Connscore = 0 or 1 were enrolled. Continued therapy with lactulose was permitted in both arms.During the 6 month treatment period, patients were assessed in the clinic and via telephone.A key secondary endpoint was time to first HE related hospitalization (defined as the numberof days from the first dose of study drug to the first hospitalization for an event related toHE). Results: A total of 299 patients were randomized to either rifaximin (n=140) orplacebo (n=159). Demographics and baseline characteristics were similar between the groups.Rifaximin significantly reduced the risk of an HE-related hospitalization by 48% comparedto placebo (hazard ratio=0.521; 95% confidence interval, 0.313-0.868; p=0.01). At 6 monthsthe percentage of patients on rifaximin who had been hospitalized for reasons related toHE was significantly less than placebo (16% vs. 26%, respectively; p=0.04), with a numberneeded to treat of 9 to prevent 1 hospitalization related to HE. Conclusion: In this large,randomized controlled trial, rifaximin at a dose of 1100 mg/d provided significant protectionagainst HE related hospitalizations compared to placebo (48% risk reduction) during the6-month treatment period. This outcome indicated that treatment of only 9 patients isrequired to prevent 1 case of HE-related hospitalization.

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Activation of Host Proteolytic Pathways By Nematode Generated SerineProteases Is a Stereotypic Mechanism of Molecular MimicryJennifer A. Stiltz, Sarah Netzel-Arnett, Luigi Notari, Toni Antalis, Joseph F. Urban, TerezShea-Donohue, Aiping Zhao

Introduction: Enteric nematodes induce a stereotypic increase in mucosal permeability. Weshowed previously that Nippostrongylus brasiliensis (Nb) generate a serine-like protease (WSP)that decreased transepithelial resistance (TEER). Protease-activated receptors (PAR) are pre-sent on the apical and basolateral surface of epithelial cells and activation of apical PAR-2is implicated in the increase in gut permeability associated with a number of pathologies.Aim: To determine if 1) WSP alter permeability via PAR-2 and 2) if WSP is a stereotypicmechanism of molecular mimicry. Methods: Adult Nb or Trichuris muris (Tm) were washedfrom the host's lumen and incubated in medium containing antibiotics for three days.WSP were isolated by passing worm secretions through a benzamidine column. To furthercharacterize these WSP we used casein zymography to identify in-gel activity componentsfrom the column purification followed by an inhibition assay using benzamidine (a specificinhibitor of serine proteases). To determine if WSP generated In Vitro was comparable tothat produced in the lumen during infection, we collected the contents from the smallintestine of Nb-infected WT mice. TEER, an index of permeability, was used to measuremuscle-free segments of jejunum taken from wild type (WT) or PAR-2 KOmice and mountedin microsnapwells. We exposed either buffer or WSP on the luminal side of the jejunumand compared it to the PAR-2 agonist, SLIGRL (500μM). TEER values are expressed as apercent (%) of the initial value. Results: Nematodes generated a 26-kDa protein thatcorresponds to a serine protease, trypsin, whose activity was inhibited by benzamidine ona zymogram gel. To determine if WSP affect mucosal permeability, we performed TEERassays. We found no difference in jejeunal permeability between WT and PAR-2 KO mice(42 ± 2 vs 44 ± 4 Ω×cm2). When compared to vehicle, addition of WSP to the mucosalside of small intestine of WT mice significantly decreased TEER after 180 minutes (90 ±4vs 69±9 %; p<0.05). This was comparable to the reduction in TEER observed after additionof SLIGRL (67±14%). In contrast, there was no decrease in TEER in response to WSP inPAR2 KO mice (91±7 vs. 85±8 %). WSP from Tm also significantly decreased TEER in thesmall intestine (69±9 %). In addition, the concentration and activities of the WSP used forTEER was comparable to that isolated from the intralumenal fluid of infected mice. Conclu-sions:Nematodes generation ofWSP, which activates PAR-2, appears to be commonmechan-ism in infection-induced alterations in mucosal permeability.

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Bacterial Type Three Secretion Protein AvrA Increases p53 Acetylation inIntestinal InflammationYun Zhao, Zhongde Ye, Shaoping Wu, Yinglin Xia, Andrew Steiner, Elaine O. Petrof,Erika C. Claud, Jun Sun

Salmonella is a well-armed opportunistic pathogen that produces a diverse array of pathogenicfactors and causes infection. Salmonella injects virulence proteins, called effectors, into thehost cells. Bacterial effectors can paralyze or reprogram the eukaryotic cell to the benefit ofthe pathogen. AvrA is a newly described Salmonella effector. The function of AvrA and themechanism by which AvrA modulates the host cell signaling is not entirely clear. P53 issituated at the crossroads of a network of signaling pathways that are essential for cell growthregulation and apoptosis induced by genotoxic and non-genotoxic stresses such as microbialinfection. However, it is unknown whether Salmonella induced inflammation involves thep53 pathway. We hypothesized that bacterial AvrA activates the p53 pathway when thehost is under the stress of Salmonella infection. Our data demonstrates that Salmonellaspecifically increases p53 acetylation. We colonized human colonic epithelial cells withwild-type Salmonella or TNF, a proinflammatory cytokine. Western blot data showed thatSalmonella colonization increased the acetylation of p53 at the 373/382 lysines, whereasTNF treatment did not change p53 acetylation. We further demonstrated that cells infectedwith AvrA-sufficient Salmonella have increased p53 acetylation, whereas cells infected withAvrA-deficient Salmonella have less p53 acetylation. Specifically, P53 acetylation sites 373and 382 were regulated by AvrA expression whereas the p53 acetylation site 320 was notaffected. In addition, cotransfection and immunoprecipitation data showed that AvrA hada physical interaction with p53. Biochemical assays in a cell-free system revealed that AvrApossessed acetyltransferase activity and used p53 as a substrate. AvrA mutations at the keyamino acid domains 123, 142, and 179 resulted in loss of this acetyltransferase activity.Functionally, AvrA expression induced cell cycle arrest with increased cell numbers at theG0/G1 phase and decreased cell number at the G2/M phase in the host cells. Correspondingly,intestinal epithelial p53 acetylation was also increased by bacterial AvrA expression in a

A-12AGA Abstracts

mouse model. To our knowledge, this is the first study that provides direct evidence anda mechanism for how a bacterial protein interferes with host responses, such as cell cycle,via p53 acetylation. Bacteria play a key role in intestinal homeostasis. We speculate thatinvestigating the effect of other bacteria on P53 acetylation will provide further insight intobeneficial and pathogenic roles of bacteria important for understanding health as well asdisease states such as IBD and cancers.

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Acute Intestinal Inflammation Decreases Microbial Diversity and Triggers theProliferation of Adherent and Invasive E. coliMelanie Craven, Charlotte E. Egan, Belgin Dogan, Eric Denkers, Sean McDonough, EllenJ. Scherl, Kenneth W. Simpson

Inflammatory bowel disease (IBD) is thought to result from the interaction between immunor-egulatory and environmental factors in a genetically susceptible individual. Recent studieshave detected imbalances in the enteric flora and Adherent and Invasive E. coli (AIEC) inpatients with IBD, and have linked acute enteritis and non-steroidal anti-inflammatory drugs(NSAID) to the development of IBD, although the role of these findings in the pathogenesisof IBD is poorly understood. We examined the effects of acute intestinal inflammationinduced by infectious agents and NSAID on the composition and spatial distribution of theileal mucosal flora. Wildtype C57BL/6 mice were gavaged with Toxoplasma gondii (n=10mice), Giardia muris (n=10), or indomethacin at doses of 0.1 mg/mouse for 5 days (LDI,n=5), or 1mg/mouse for 3 days (HDI, n=5). Ileal tissue was harvested from controls (n=8)and affected mice: 4 and 8 days after T.gondii, 7 and 14 days after Giardia, and 3 and 7days after HDI and LDI respectively. Inflammation was assessed histopathologically by aninvestigator masked to treatment. Composition and spatial distribution of the mucosal florawas evaluated by 16S rDNA sequencing, quantitative PCR, fluorescence in situ hybridization(FISH) and culture. Mucosal E. coli isolates were characterized by phylogroup, genotype(RAPD-PCR, virulence determinants) and pathotype (invasion of Caco-2, persistence in J774macrophages). Infection with T.gondii (8 days post-infection,Toxo8) and HDI resulted insevere ileitis, that was fatal in 2 of 5 HDI mice. The proportion of clones in 16S rDNAlibraries corresponding to Enterobacteriales was significantly greater in Toxo8 (84%, P<0.05)and HDI (90%, P<0.01) than in controls (0%) and other groups (≤2%). The number of E.coli (median copies uidA:18S.106) was higher in Toxo8 (3x106, P<0.01) and HDI (1.7x106,P<0.05) compared to controls (2.6x104). FISH positive mucosally invasive E. coli wereobserved solely in the Toxo8 and HDI groups. E. coli isolates from Toxo8 and HDI werephylogroup B1 and had identical RAPD-PCR profiles. A representative strain (CUMT8) wasserogroup O8:H21, contained few known virulence genes (hcp,gsp,lpfA), and displayed anAIEC pathotype. We conclude that acute ileitis perturbs the enteric microbiota and triggersthe proliferation of adherent and invasive E. coli similar to those described in IBD. Themicrobial alterations induced by acute inflammation may promote and perpetuate intestinalinflammation in an individual susceptible to IBD.

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CD-Associated Adherent-Invasive Escherichia coli LF82 Colonize and InduceSevere Intestinal Inflammation in Transgenic Mice Expressing the HumanCEACAM6 ReceptorNicolas Barnich, Frédéric A. Carvalho, Carlos H. Chan, Clifford P. Stanners, ArletteDarfeuille-Michaud

Background: Abnormal expression of CEACAM6 is observed at the apical surface of theileal epithelium in Crohn's disease (CD) patients and CD ileal lesions are colonized bypathogenic Adherent-Invasive Escherichia coli (AIEC). Ex vivo studies using primary CD ilealenterocytes suggested that CEACAM6 could act as the host cell receptor for AIEC colonizationvia type 1 pili. Aim: We investigated in the CEABAC10 transgenic mice expressing humanCEACAM6, compared to wild-type, the ability of AIEC reference strain LF82 to colonizethe intestinal mucosa and to induce intestinal inflammation. Materials and Methods: MaleCEABAC10 transgenic mice or wild-typemice were orally challenged oncewith 10e9 bacteria.The severity of colitis was assessed by determining disease activity index, survival rate andhistological score. The colonization was assessed by numbering AIEC LF82 bacteria in thestools and associated to intestinal mucosa. CEACAM6 and cytokine expression followinginfection was measured in intestinal mucosa by real-time RT-PCR. Results: AIEC LF82virulent bacteria, but not nonpathogenic E. coli K-12 or nonpiliated LF82 mutants, are ableto persist in the gut of CEABAC10 transgenic mice at 6 days post-infection and AIEC LF82bacteria colocalized with CEACAM6 in the intestinal mucosa. AIEC LF82 bacteria induceda severe colitis in CEABAC10 transgenic mice, but not in wild-type mice, reducing survivalrate, inducing marked weight loss and increased rectal bleeding, greatly lowering stoolconsistency, and significantly increasing erosive lesions, mucosal inflammation and pro-inflammatory cytokines. Interestingly, this was not observed with nonpathogenic E. coli K-12 or nonpiliated LF82 mutants. Finally, AIEC LF82 infection induced increased CEACAM6expression in the intestinal mucosa of transgenic mice. Conclusion: Together, these resultsindicated that CD-associated virulent AIEC LF82 bacteria are able to induce overexpressionof CEACAM6 in order to colonize the intestinal mucosa and to induce intestinal inflammation,reinforcing the hypothesis of the existence of an amplification loop of colonization andinflammation in CD patients.

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Clonal Invasive Escherichia coli Isolates from Inflammatory Bowel Disease(IBD) ResectionsErin K. Okeefe, Edgar C. Boedeker

Background: Escherichia coli (EC) strains with an epithelial cell adherent and invasive pheno-type, and with prolonged survival in macrophages, have been isolated from biopsies ofpatients with early recurrence of Crohn's Disease (CD) and have been designated attachinginvasive E. coli (AIEC). Design: To examine the prevalence of and characteristics of such