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swhich is a glucosinolate precursor of SFN, was orally administered to the mice, at dose of8.5 mg/kg, before and after the injection of IND. SGS is converted to biologically activeSFN in the intestinal lumen by myrosinase activity in the intestinal microflora. Expressionof hemoxygenase-1 (HO-1) was evaluated by real time RT-PCR. Vascular permeability wasassessed by Evans Blue exudation into the mucosa after i.v. injection. Neutrophil activationwas evaluated by myeloperoxidase (MPO) activity. Results: 1. IND treatment caused mucosalinjury in small intestine, accompanied by the increases in the vascular permeability, themucosal MPO activity, and the mucosal invasion of anaerobic enterobacteria. 2.SGS attenu-ated the IND-induced small intestinal injury, and prevented the increases in the vascularpermeability and the MPO activity induced by IND. 3. SGS enhanced HO-1 expression in thesmall intestine, and prevented the increase in mucosal invasion of the anaerobic enterobacteriacaused by IND. Conclusions: These results suggest that (1) orally administered SGS protectssmall intestine from IND-induced injury in mice In Vivo, and that (2) SGS affords protectionof the small intestinal mucosa against IND-induced injury, partly by inducing nrf2-dependentantioxidant enzymes in the mucosa, and partly by inhibiting invasion of the anaerobicenterobacteria into the mucosa.

Mo2047

Hydrogen Sulfide Synthesis During Colonic Inflammation Occurs Primarily viaa Pyridoxal-5'-Phosphate (P5p)-Independent PathwayKyle L. Flannigan, John L. Wallace

Hydrogen sulfide (H2S) is an important endogenous anti-inflammatory mediator, which hasbeen shown to promote resolution of colitis and healing of ulcers. H2S synthesis was believedto occur primarily via two P5P-dependent enzymes (cystathionine-β-synthase (CBS) andcystathionine-γ-lyase (CSE)). In the present study, we examined the possible contributionof a third pathway for H2S synthesis, in the healthy and inflamed colon, which involves theenzymes cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST).Methods: The capacity of tissue to produce H2S was measured from homogenized tissue inthe presence of exogenous substrate and/or inhibitors using a zinc-trapping assay. Productionof H2S via the CAT-3MST pathway was explored using the substrate α-ketoglutarate andthe CAT inhibitors L-aspartate and O-carboxymethyl-hydroxylamine hemihydrochloride(CHH). Colitis was induced in rats by intracolonic administration of dinitrobenzene sulfonicacid (DNBS). Rats were euthanized 3 to 28 days post-DNBS for measurement of H2S synthesis,and for immunofluorescent staining to localize CAT and 3MST expression. Results: ColonicH2S synthesis via the CAT-3MST pathway was 5-fold greater than that via the P5P-dependent(CBS and CSE) pathways (225 ± 59 vs. 42 ± 21 nmol/g/h respectively; p<0.05). In theinflamed colon, H2S synthesis via CAT-3MST was markedly increased (>4-fold) comparedto controls (964 ± 115 vs. 225 ± 59 nmol/g/h, respectively; p<0.05). Colonic H2S synthesiswas greatest when inflammation was most robust (3 days post-DNBS; 1132 ± 88 nmol/g/h) and was suppressed by L-aspartate and CHH (424 ± 153 and 69 ± 34 nmol/g/h, respect-ively; both p<0.005). Immunofluorescence staining revealed that CAT and 3MST were co-localized in the colonic epithelium. Moreover, CAT expression was greatest in epithelialcells adjacent to ulcers (MFI: 1202 ± 63 U vs. 873 ± 105 U in healthy epithelium; p<0.05).By day 28, when colitis had largely resolved, H2S synthesis had decreased to control levels.Conclusions: These data suggest that in both the healthy and inflamed colon CAT-3MSTrepresents the primary pathway for H2S synthesis. Increased CAT expression may accountfor the increased H2S synthesis during inflammation. Given the ability of H2S to promoteresolution of inflammation and healing of mucosal injury, understanding the regulation ofintestinal H2S synthesis may have important implications for the development of noveltherapies for inflammatory bowel disease.

Mo2048

Dual Roles Played by Matrix Metalloproteinase (MMP9) in Citrobacterrodentium-induced Hemorrhagic ColitisSabrina Jeppsson, Maiko Sasaki, Jan-Michael A. Klapproth, Christopher W. Harper, DidierMerlin, Shanthi V. Sitaraman, Pallavi Garg

Background and Aims: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E.coli (EHEC) are well known causative agents of diarrheal disease in humans. EPEC isresponsible for infantile diarrhea whereas EHEC causes hemorrhagic colitis. The molecularpathogenesis of EPEC and EHEC related infections have been well studied. However, theexact mechanisms by which these pathogens cause disease remain unknown. Citrobacterrodentium (CR) is a well described murine bacterial pathogen and CR infection is a robust,relevant In Vivo model that can be used to understand the pathogenicity of EPEC and EHECinduced colitis. It is well known that MMP9 is absent in normal tissues but upregulatedduring inflammation. Aim of the present study was to identify the role played by MMP9 inCR-induced colitis. Methods: Age and gender matched MMP9-/- and littermate wild type(WT) mice of C57/B6 strain were used for the study. One group, including both MMP9-/-and WT animals, were infected with CR and another group, without infection served as acontrol. Half of the animals in each group were sacrificed at days 8 and 20 post-infectionrespectively. Colon tissue was subjected to bacterial colony counts, histological staining,Western blotting and Q-PCR. Results: CR colony count was significantly lower in MMP9-/-mice compared to WT mice (5.81±0.93x103 CFU vs. 23.78±7.2x103 CFU/g, respectively)on day 8 post-infection. Infected MMP9-/- mice also showed a significantly lower colonweight (0.05±0.005 g) compared to WT mice (0.07±0.005 g). MMP9 protein expressionwas increased by 5±0.2-fold at day 8 post-infection with CR in WT mice compared touninfected WT mice. Muc2 expression was significantly lower (48.9±12.9%) in MMP9-/-mice infected with CR compared to uninfected MMP9-/- mice on day 8 post-infection.Hematoxylin & Eosin staining revealed significantly decreased neutrophils infiltration, lesscrypt damage and fewer foci of ulceration in the colonic epithelium of MMP9-/- micecompared to WT mice both infected with CR on day 8 post-infection. On day 20 post-infection, crypt architecture was restored as well as neutrophil infiltration was less indicatingthe recovery from ulceration in both MMP9-/- andWTmice. However, the extent of epithelialdysplasia was greater in MMP9-/- mice compared to WT animals. Ki67 staining indicatedhigher colonic epithelial cell proliferation among MMP9-/- mice compared to WT mice onday 20 post-infection. Thus, at early stages of CR-mediated colitis, MMP9-/- mice were

S-728AGA Abstracts

resistant to inflammation, but at later stage exhibited increased dysplasia and proliferation,suggesting its dual role in the course of the disease. Conclusion: The data suggest that theMMP-9 initially mediates inflammation but later exerts a protective role, implying thatfunction of MMP-9 changes as inflammation advances in CR-mediated colitis.

Mo2049

Repeated Cycles of DSS Inducing a Chronically Relapsing Inflammation: ANovel Model to Study Fibrosis Using In Vivo MRI T2 RelaxometryChristine M. Breynaert, Tom Dresselaers, Jonathan Cremer, Kristel Van Steen, ClémentinePerrier, Severine Vermeire, Paul J. Rutgeerts, Jan L. Ceuppens, Uwe Himmelreich, GertVan Assche

BACKGROUND Most experimental animal models of IBD fail to accurately reflect thechronically relapsing inflammation underlying the complications of human Crohn's disease.This study investigated whether repeated cycles of DSS adequately reflect the effects ofchronic transmural healing. Transmural μMR imaging with In Vivo T2 relaxometry was usedto differentiate transmural changes. METHODS DSS colitis was induced in 6 week-oldC57BL6/J mice: acute colitis mice (n=10) received 7 days of DSS prior to sacrifice. Onecycle mice (n=10) received 1 cycle of 7 days of DSS followed by 2 weeks of normal drinkingwater prior to sacrifice, 2-cycle mice (n=10) 2 cycles and 3-cycle mice (n=9) 3 cycles priorto sacrifice. Control mice (n=7) received normal drinking water only. Six mice per groupwere scanned In Vivo on a 9.4T MRI Bruker system. T2 weighted images and T2 maps ofthe distal colon were recorded. Histograms of the colon wall were created from T2 mapsusing a plugin for ImageJ. After scanning and euthanasia, the distal colon of all mice washarvested for histology and FACS analysis. Collagen deposition was quantified with Martius-Scarlett-Blue staining. RESULTS Colon weight, colon weight/length ratio and macroscopicscore were significantly higher in the 1-, 2- and 3-cycle group compared to the controlgroup (p<0.001), however, no significant difference was observed within the cycling groups.Although all mice in the 2-, and 3-cycle group had a normal DAI score, the macroscopicscore was significantly higher compared to the acute group (p<0.001). CD4+Foxp3+ cellsin blood increased with more cycles of DSS (p<0.001). The number of CD4+Foxp3+ cellsin MLN was higher in the 2- and 3-cycle model compared to the acute model and controls(p<0.001). A higher number of IFNg+ cells in MLN was observed in the 1-, 2- and 3-cyclemodel compared to the acute model p<0.001). The 3-cycle model showed a higher numberof IL17+ cells in MLN compared to the acute model and controls (p=0.0012). No significantdifference was observed within the different models in IL13. Collagen deposition was signific-antly higher in the 2- and 3-cycle model compared to the 1-cycle model (p<0.001). T2mapping of the colon was able to discern between all groups: the increasing number of cyclescorrelated with a gradual regression of T2 values to those of normal colon. CONCLUSIONTheimmune profile of a cycling DSS model with induction of relapse and remission is clearlydifferent from the acute DSS model inducing an adaptive immune response. The chronicrepeated cycles of DSS model opens perspectives to study the effects of healing and fibrosisin a murine model of IBD. In Vivo T2 relaxometry is a promising non-invasive assessmentof inflammation and fibrosis and should be explored in CD patients.

Mo2050

Suppression of MLK3 Signaling Selectively Modulates MAPK and PTENSignaling During Intestinal Mucosal Cell MigrationPavlo L. Kovalenko, Lyudmyla Kunovska, Jian Chen, Kathleen A. Gallo, Marc D. Basson

Mixed-lineage kinase-3 (MLK3) variably influences diverse MAPK pathways that may inturn modulate migration and proliferation in various cell types In Vitro. In a previouspreliminary study, we observed that loss of MLK3 expression reduces intestinal epithelialmucosal healing In Vivo after inducing ulcers by serosal acetic acid In Vivo and MLK3inhibition reduced Caco-2 ERK and JNK signaling In Vitro. We now sought to evaluate otherpotential downstream consequences of MLK3 loss, including MAPK and PTEN signaling,proliferation, and tissue morphology, as all these parameters might be relevant to mucosalwound healing. We compared jejunums from MLK3 knockout and wild type mice In Vivoand assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial cells In Vitroafter MLK3 inhibition. The MLK3 knockout mice exhibited significantly greater thickness