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sMo1799

Targeting Endovascular Adhesion Molecules With Ultrasound Contrast AgentIn Vivo to Characterize Murine DSS-Colitis in MiceMarkus Brückner, Jörg Stypmann, Dominik Bettenworth, Jan Heidemann

INTRODUCTION The course of Dextran Sodium Sulphate (DSS) colitis is routinely mon-itored by clinical and post mortem analyses, including assessment of colon length andhistomorphological changes. Until today, the majority of DSS colitis experiments have astatic endpoint requiring euthanization. In humans, the use of image-enhancing ultrasoundcontrast agents is increasing as they represent a diagnostic tool for a variety of morphologicalchanges insoluble for regular ultrasonography. Concerning echocardiography or ultrasonog-raphy of liver lesions, contrast agents are used routinely today. AIM To assess the value ofimage-enhancing antibody-labeled ultrasound contrast agents against gut-specific endovascu-lar Mucosal Addressin Cellular Adhesion Molecule 1 (MAdCAM-1) and Vascular Cell Adhe-sion Molecule 1 (VCAM-1) in identyfying inflammatory characteristics of experimentalmurine DSS-colitis by In Vivo 40 MHz ultrasonography. Findings were correlated withweight loss as an established colitis parameter and to recent findings of In Vivo 40 MHzhydrosonography of the colon. MATERIAL and METHODS Four groups (n = 20) of femaleC57BL/6 mice received 3% (w/v) DSS in tap water or tap water as control (group A). Afteradministration of DSS for 3, 6 and 9 days (groups B - D),micewere anesthesized and antibody-labeled ultrasound contrast agents against MAdCAM-1 and VCAM-1 were intravenouslyadministered before perfoming 40 MHz abdominal sonography of the colon. Acoustic echoeswere measured in the colonic wall using specialized software to quantify severity of DSS-Colitis in groups A-D. RESULTS 1) The expression ofMAdCAM-1 andVCAM-1 as endovascu-lar adhesion molecules is significantly increased in DSS-Colitis as depicted by immunoblotand immunohistochemic analyses. 2) Image-enhancing ultrasound with contrast agentslabeled against endovascular MAdCAM-1 and VCAM-1 is able to noninvasively display thecourse of experimental DSS colitis in mice in a highly specific manner. Results correlatewith exposure to DSS over time, with group D showing the highest echogenicity of thebowel wall (p < 0.05). 3) The results are significantly correlated to clinical weight loss inmice and to bowel wall thickness as valuable ultrasonographic characteristics of DSS-Colitisin mice. CONCLUSIONOur findings indicate that image-enhancing ultrasound with contrastagents against endovascular MAdCAM-1 and VCAM-1 is a feasible and accurate tool tononinvasively and specifically monitor the course of experimental DSS colitis in mice. Resultscorrelate significantly with weight loss as established parameter of colitis and recent findingsin 40 MHz hydrosonography of the inflamed colon.

Mo1800

Methyl Donor Deficiency Affects Small Intestinal Differentiation and BarrierFunction in RatsAude Bressenot, Shabnam Pooya, Carine Bossenmeyer-Pourie, Guillaume Gauchotte,Adeline Germain, Jean Baptiste Chevaux, Florence Coste, Jean-Michel Vignaud, Jean-Louis Gueant, Laurent Peyrin Biroulet

BACKGROUND: Dietary methyl donors (vitamin B12 and folate) and their genetic determin-ants are associated with Crohn's disease risk. Methyl donor deficiency (MDD) aggravatesexperimental colitis in rat pups from mothers under methyl deficient diet. However, theeffects of MDD on differentiation and barrier function are not known, in the distal andproximal intestine. MATERIALS AND METHODS: We investigated whether MDD may affectdevelopment and barrier functions of the proximal and distal small intestine in rat pupsfrom dams subjected to the MDD during gestation and lactation. Two groups were considered(n=10 for MDD and n=8 for control animals). Cell organization, apoptosis, proliferationand differentiation were studied within proximal and distal small intestine mucosa of rats.RESULTS: A global wall hypotrophy was observed in distal small bowel with increased cryptapoptosis, loss of enterocyte differentiation in villous and reduction of intestinal phosphatasealkaline production. In both proximal and distal small bowel cleaved caspase-3 immuno-staining and Apostain labelling index showed increased crypt apoptosis. Decreased prolifera-tion was observed in crypts of the proximal small bowel with a reduced number of MCM-6 and Proliferating Cell Nuclear Antigen (PCNA)-positive cells. This lack of enterocytedifferentiation in distal small bowel was associated to impaired expression of β-catenin anddecreased β-catenin/E-cadherin interaction. Low Protein Phosphatase 2A expression levelsmay trigger increased β-catenin degradation. MDD also affected intestinal barrier in proximalsmall bowel by decreasing Paneth cells number after immunostaining for lysosyme and byreducing goblet cells number and mucus production after immunostaining for mucin-2.CONCLUSIONS: Overall, MDD has dual effects on small intestine by producing dramaticeffects on enterocyte differentiation and barrier function in rats. MDDmight be a predisposingfactor to small bowel Crohn's disease.

Mo1801

A Model of In Vitro Perfused Mouse Intestine Suitable for PharmacologicalTesting of Anti-Inflammatory DrugsDominik H. Schreiber, Gerhard Erkel, Karl-Herbert Schäfer

Inflammatory bowel diseases constitute a very important entity in gastrointestinal disorders.There are many different animal models available to mimic the disease. For the time beingadequate In Vitromodels that can simulate the In Vivo situation are not available yet. Differentcell culture models for intestinal cells are very simple to use, but do not come close to thephysiology of the small intestine as a whole. Therefore we established a mouse smallintestine long time perfusion model primarily designed for pharmacological testing. Themost important advantage of our model is its ability to imitate the tissue interaction ininflamed intestine more similar to the In Vivo situation than cell culture approaches can do.Before starting the surgical procedure animals are sacrificed by cervical dislocation. Themesenteric artery is rapidly cannulated and the intestine is mesenterially perfused in situ.During the intestinal resection procedure tissue is cooled and humidified with physiologicalsalt solution. After resection of a 5-6 cm segment of the proximal small intestine with theadhering cannulated mesenterial root, In Vitro longterm mesenterial perfusion is immediately

S-688AGA Abstracts

started; the tubing is connected to a perfusion system and the gut mesenterially perfusedin a custom designed perfusion chamber, which allows a luminal perfusion and serosalsuperfusion with a carbogen gassed physiological salt solution. Mesenteric superior artery andthe gastrointestinal lumen are perfused with different perfusion media. Acute inflammation inthe small intestine is induced by the application of LPS via the intestine's lumen and acytomix through the mesenteric superior artery. Motility of the small intestine in responseto perfusion and inflammatory stimuli can be measured and interpreted by using opticalrecording and appropriate analysis software. Inflammation has been proven on the transcrip-tional level at different time points. Furthermore histological stainings were performed. Themodel is especially valuable for the testing of new compounds intended for the treatmentof inflammatory bowel disease. Our perfusion model is used to evaluate the effects ofnovel anti-inflammatory drugs from fungi. The substances are applied through the luminalperfusionmedium tomimic oral ingestion. Anti-inflammatory effects are evaluated in compar-ison to dexamethasone. Intestinal perfusion is a suitable alternative to various mouse In Vivoinflammatory bowel disease models to assess the effects of new pharmacological treatmentstrategies. The assessment of drug effects in our In Vitro model can help to accelerate drugdevelopment for inflammatory conditions of the intestine.

Mo1802

Role of CD24 in the Development of DSS-Induced Murine ExperimentalColitisInna Naumov, Sarah Kraus, Shiran Shapira, Ilan Aroch, Diana Kazanov, Ehud Ron, GiladGitstein, Eran Elinav, Alexei V. Salnikov, Peter Altevogt, Nadir Arber

Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory disease with anundefined pathogenesis. Its treatment is effective in only ~50% of the cases. Patients withUC have an increased risk of developing colorectal cancer. We have previously shown thatCD24, a mucin-like glycoprotein, is overexpressed in ~90% of colorectal tumors at an earlystage. Aim: To investigate the role of CD24 in dextran sulphate sodium (DSS)-induced colitisin CD24 knockout (KO) [HSA -/-] compared to wild-type (WT) [HSA+/+] C57BL/6 mice.Methods: CD24 KO and WT mice were subjected to DSS-induced colitis. The mice weregiven 2.5-4% DSS in the drinking water for 5 days, followed by 2 days of water. Controlmice received water. Animals were sacrificed on day 7 and scored for their disease activityindex (DAI). Results: CD24 KO mice are significantly resistant to DSS-induced colitis,compared to the WT mice, even at high DSS concentrations (3% and 4%). Moreover, CD24KO mice displayed a significant reduction in the pathological scores, reflecting a reductionin the colitis severity. Cytokine analysis by an antibody array demonstrated striking differencesin the serum and colon of CD24 KO vs. WT mice, supporting the involvement of CD24in the inflammatory response. Conclusions: CD24 may have an important role in theinduction of colitis in mice. Targeting of CD24 may be potentially useful for the treatmentof experimental colitis in mice and may be an important novel target in IBD patients.

Mo1803

Overexpression of N-Acetylglucosaminyltransferase V (GnT-V) ExacerbatesMurine Colitis With Dysfunction of MacrophagesMayuko Ishii, Shinichiro Shinzaki, Hironobu Fujii, Norika Tatsunaka, Yoshihiro Kamada,Hideki Iijima, Tetsuo Takehara, Eiji Miyoshi

Background and aims: Glycosylation is one of the most common post-translational modifica-tions and is catalyzed by the action of several glycosyltransferases to make various complexsugar chains. N-acetylglucosaminyltransferase V (GnT-V) is involved in the biosynthesis ofN-linked oligosaccharides in Golgi organelle where it catalyzesN-acetylglucosamine (GlcNAc)by beta-1,6-branching. Previous reports show that the levels of GnT-V expression and beta-1,6-branched glycoproteins are increased in cancer cells, and GnT-V promotes tumor growthby inhibition of CD4+ T cells and macrophages, indicating that GnT-V has a pivotal roleon host immune responses. The role of GnT-V for intestinal inflammation, however, is notclearly elucidated. The aim of the present study is to clarify the role of GnT-V in murinecolitis using GnT-V transgenic (Tg) mice. Methods: Male 8-11-week-old GnT-V Tg andwild-type (WT) C57BL/6 mice were used for experiments. Dextran sulfate sodium (DSS)colitis was generated by adding DSS to the drinking water at the concentration of 3 %.Mice were provided with this water ad libitum for 7 days and sacrificed on day 9. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) colitis was generated by sensitizing with the 2.5%TNBS in 50% ethanol by skin painting on day 1, and intrarectally administrating of 150 μlof 1% TNBS in 50% ethanol on day 8. The mice were sacrificed on day 12. The expressionof beta-1,6-branching glycoprotein was investigated by L4-PHA lectin blotting. Fluorescentlatex beads were opsonized to peritoneal macrophages (PM) or bone marrow derived macro-phages (BMDM) for 1 hour, and phagocytic activity was investigated by calculating thenumber of fluorescent cells by flow cytometry. To deplete macrophages In Vivo, clodronate-liposomes were injected intravenously at 2 days before and 4 days after DSS administration.Results: Increases in beta-1,6-branching on glycoproteins by overexpression of GnT-V wereobserved in colon and splenic mononuclear cells of GnT-V Tg mice. In both DSS and TNBScolitis models, GnT-V Tg mice showed severe weight loss and histological damages comparedwith WT mice. Phagocytic activity of macrophages was significantly impaired in GnT-V Tgmice than in WT mice. When macrophages were depleted during DSS administration, theextent of colitis was unchanged in GnT-V and WT mice, indicating that the exacerbationof colitis in GnT-V was eliminated in absence of macrophages. Conclusions: Overexpressionof GnT-V exacerbated murine experimental colitis. Dysfunction of macrophages has a pivotalrole in the pathogenesis of colitis in GnT-V mice.