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Mo1799 Targeting Endovascular Adhesion Molecules With Ultrasound Contrast Agent In Vivo to Characterize Murine DSS-Colitis in Mice
Mo1799
started; the tubing is connected to a perfusion system and the gut mesenterially perfused in a custom designed perfusion chamber, which allows a luminal perfusion and serosal superfusion with a carbogen gassed physiological salt solution. Mesenteric superior artery and the gastrointestinal lumen are perfused with different perfusion media. Acute inflammation in the small intestine is induced by the application of LPS via the intestine's lumen and a cytomix through the mesenteric superior artery. Motility of the small intestine in response to perfusion and inflammatory stimuli can be measured and interpreted by using optical recording and appropriate analysis software. Inflammation has been proven on the transcriptional level at different time points. Furthermore histological stainings were performed. The model is especially valuable for the testing of new compounds intended for the treatment of inflammatory bowel disease. Our perfusion model is used to evaluate the effects of novel anti-inflammatory drugs from fungi. The substances are applied through the luminal perfusion medium to mimic oral ingestion. Anti-inflammatory effects are evaluated in comparison to dexamethasone. Intestinal perfusion is a suitable alternative to various mouse In Vivo inflammatory bowel disease models to assess the effects of new pharmacological treatment strategies. The assessment of drug effects in our In Vitro model can help to accelerate drug development for inflammatory conditions of the intestine.
AGA Abstracts
Targeting Endovascular Adhesion Molecules With Ultrasound Contrast Agent In Vivo to Characterize Murine DSS-Colitis in Mice Markus Brückner, Jörg Stypmann, Dominik Bettenworth, Jan Heidemann INTRODUCTION The course of Dextran Sodium Sulphate (DSS) colitis is routinely monitored by clinical and post mortem analyses, including assessment of colon length and histomorphological changes. Until today, the majority of DSS colitis experiments have a static endpoint requiring euthanization. In humans, the use of image-enhancing ultrasound contrast agents is increasing as they represent a diagnostic tool for a variety of morphological changes insoluble for regular ultrasonography. Concerning echocardiography or ultrasonography of liver lesions, contrast agents are used routinely today. AIM To assess the value of image-enhancing antibody-labeled ultrasound contrast agents against gut-specific endovascular Mucosal Addressin Cellular Adhesion Molecule 1 (MAdCAM-1) and Vascular Cell Adhesion Molecule 1 (VCAM-1) in identyfying inflammatory characteristics of experimental murine DSS-colitis by In Vivo 40 MHz ultrasonography. Findings were correlated with weight loss as an established colitis parameter and to recent findings of In Vivo 40 MHz hydrosonography of the colon. MATERIAL and METHODS Four groups (n = 20) of female C57BL/6 mice received 3% (w/v) DSS in tap water or tap water as control (group A). After administration of DSS for 3, 6 and 9 days (groups B - D), mice were anesthesized and antibodylabeled ultrasound contrast agents against MAdCAM-1 and VCAM-1 were intravenously administered before perfoming 40 MHz abdominal sonography of the colon. Acoustic echoes were measured in the colonic wall using specialized software to quantify severity of DSSColitis in groups A-D. RESULTS 1) The expression of MAdCAM-1 and VCAM-1 as endovascular adhesion molecules is significantly increased in DSS-Colitis as depicted by immunoblot and immunohistochemic analyses. 2) Image-enhancing ultrasound with contrast agents labeled against endovascular MAdCAM-1 and VCAM-1 is able to noninvasively display the course of experimental DSS colitis in mice in a highly specific manner. Results correlate with exposure to DSS over time, with group D showing the highest echogenicity of the bowel wall (p < 0.05). 3) The results are significantly correlated to clinical weight loss in mice and to bowel wall thickness as valuable ultrasonographic characteristics of DSS-Colitis in mice. CONCLUSION Our findings indicate that image-enhancing ultrasound with contrast agents against endovascular MAdCAM-1 and VCAM-1 is a feasible and accurate tool to noninvasively and specifically monitor the course of experimental DSS colitis in mice. Results correlate significantly with weight loss as established parameter of colitis and recent findings in 40 MHz hydrosonography of the inflamed colon.
Mo1802 Role of CD24 in the Development of DSS-Induced Murine Experimental Colitis Inna Naumov, Sarah Kraus, Shiran Shapira, Ilan Aroch, Diana Kazanov, Ehud Ron, Gilad Gitstein, Eran Elinav, Alexei V. Salnikov, Peter Altevogt, Nadir Arber Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory disease with an undefined pathogenesis. Its treatment is effective in only ~50% of the cases. Patients with UC have an increased risk of developing colorectal cancer. We have previously shown that CD24, a mucin-like glycoprotein, is overexpressed in ~90% of colorectal tumors at an early stage. Aim: To investigate the role of CD24 in dextran sulphate sodium (DSS)-induced colitis in 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 were given 2.5-4% DSS in the drinking water for 5 days, followed by 2 days of water. Control mice received water. Animals were sacrificed on day 7 and scored for their disease activity index (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, CD24 KO mice displayed a significant reduction in the pathological scores, reflecting a reduction in the colitis severity. Cytokine analysis by an antibody array demonstrated striking differences in the serum and colon of CD24 KO vs. WT mice, supporting the involvement of CD24 in the inflammatory response. Conclusions: CD24 may have an important role in the induction of colitis in mice. Targeting of CD24 may be potentially useful for the treatment of experimental colitis in mice and may be an important novel target in IBD patients.
Mo1800 Methyl Donor Deficiency Affects Small Intestinal Differentiation and Barrier Function in Rats Aude Bressenot, Shabnam Pooya, Carine Bossenmeyer-Pourie, Guillaume Gauchotte, Adeline Germain, Jean Baptiste Chevaux, Florence Coste, Jean-Michel Vignaud, JeanLouis Gueant, Laurent Peyrin Biroulet
Mo1803
BACKGROUND: Dietary methyl donors (vitamin B12 and folate) and their genetic determinants are associated with Crohn's disease risk. Methyl donor deficiency (MDD) aggravates experimental colitis in rat pups from mothers under methyl deficient diet. However, the effects of MDD on differentiation and barrier function are not known, in the distal and proximal intestine. MATERIALS AND METHODS: We investigated whether MDD may affect development and barrier functions of the proximal and distal small intestine in rat pups from 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, proliferation and 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 crypt apoptosis, loss of enterocyte differentiation in villous and reduction of intestinal phosphatase alkaline production. In both proximal and distal small bowel cleaved caspase-3 immunostaining and Apostain labelling index showed increased crypt apoptosis. Decreased proliferation was observed in crypts of the proximal small bowel with a reduced number of MCM6 and Proliferating Cell Nuclear Antigen (PCNA)-positive cells. This lack of enterocyte differentiation in distal small bowel was associated to impaired expression of β-catenin and decreased β-catenin/E-cadherin interaction. Low Protein Phosphatase 2A expression levels may trigger increased β-catenin degradation. MDD also affected intestinal barrier in proximal small bowel by decreasing Paneth cells number after immunostaining for lysosyme and by reducing goblet cells number and mucus production after immunostaining for mucin-2. CONCLUSIONS: Overall, MDD has dual effects on small intestine by producing dramatic effects on enterocyte differentiation and barrier function in rats. MDD might be a predisposing factor to small bowel Crohn's disease.
Overexpression of N-Acetylglucosaminyltransferase V (GnT-V) Exacerbates Murine Colitis With Dysfunction of Macrophages Mayuko 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 modifications and is catalyzed by the action of several glycosyltransferases to make various complex sugar chains. N-acetylglucosaminyltransferase V (GnT-V) is involved in the biosynthesis of N-linked oligosaccharides in Golgi organelle where it catalyzes N-acetylglucosamine (GlcNAc) by beta-1,6-branching. Previous reports show that the levels of GnT-V expression and beta1,6-branched glycoproteins are increased in cancer cells, and GnT-V promotes tumor growth by inhibition of CD4+ T cells and macrophages, indicating that GnT-V has a pivotal role on host immune responses. The role of GnT-V for intestinal inflammation, however, is not clearly elucidated. The aim of the present study is to clarify the role of GnT-V in murine colitis using GnT-V transgenic (Tg) mice. Methods: Male 8-11-week-old GnT-V Tg and wild-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. 2,4,6Trinitrobenzene 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 μl of 1% TNBS in 50% ethanol on day 8. The mice were sacrificed on day 12. The expression of beta-1,6-branching glycoprotein was investigated by L4-PHA lectin blotting. Fluorescent latex beads were opsonized to peritoneal macrophages (PM) or bone marrow derived macrophages (BMDM) for 1 hour, and phagocytic activity was investigated by calculating the number of fluorescent cells by flow cytometry. To deplete macrophages In Vivo, clodronateliposomes 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 were observed in colon and splenic mononuclear cells of GnT-V Tg mice. In both DSS and TNBS colitis models, GnT-V Tg mice showed severe weight loss and histological damages compared with WT mice. Phagocytic activity of macrophages was significantly impaired in GnT-V Tg mice than in WT mice. When macrophages were depleted during DSS administration, the extent of colitis was unchanged in GnT-V and WT mice, indicating that the exacerbation of colitis in GnT-V was eliminated in absence of macrophages. Conclusions: Overexpression of GnT-V exacerbated murine experimental colitis. Dysfunction of macrophages has a pivotal role in the pathogenesis of colitis in GnT-V mice.
Mo1801 A Model of In Vitro Perfused Mouse Intestine Suitable for Pharmacological Testing of Anti-Inflammatory Drugs Dominik 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 being adequate In Vitro models that can simulate the In Vivo situation are not available yet. Different cell culture models for intestinal cells are very simple to use, but do not come close to the physiology of the small intestine as a whole. Therefore we established a mouse small intestine long time perfusion model primarily designed for pharmacological testing. The most important advantage of our model is its ability to imitate the tissue interaction in inflamed 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. The mesenteric artery is rapidly cannulated and the intestine is mesenterially perfused in situ. During the intestinal resection procedure tissue is cooled and humidified with physiological salt solution. After resection of a 5-6 cm segment of the proximal small intestine with the adhering cannulated mesenterial root, In Vitro longterm mesenterial perfusion is immediately
AGA Abstracts
S-688
started; the tubing is connected to a perfusion system and the gut mesenterially perfused in a custom designed perfusion chamber, which allows a luminal perfusion and serosal superfusion with a carbogen gassed physiological salt solution. Mesenteric superior artery and the gastrointestinal lumen are perfused with different perfusion media. Acute inflammation in the small intestine is induced by the application of LPS via the intestine's lumen and a cytomix through the mesenteric superior artery. Motility of the small intestine in response to perfusion and inflammatory stimuli can be measured and interpreted by using optical recording and appropriate analysis software. Inflammation has been proven on the transcriptional level at different time points. Furthermore histological stainings were performed. The model is especially valuable for the testing of new compounds intended for the treatment of inflammatory bowel disease. Our perfusion model is used to evaluate the effects of novel anti-inflammatory drugs from fungi. The substances are applied through the luminal perfusion medium to mimic oral ingestion. Anti-inflammatory effects are evaluated in comparison to dexamethasone. Intestinal perfusion is a suitable alternative to various mouse In Vivo inflammatory bowel disease models to assess the effects of new pharmacological treatment strategies. The assessment of drug effects in our In Vitro model can help to accelerate drug development for inflammatory conditions of the intestine.
AGA Abstracts
Targeting Endovascular Adhesion Molecules With Ultrasound Contrast Agent In Vivo to Characterize Murine DSS-Colitis in Mice Markus Brückner, Jörg Stypmann, Dominik Bettenworth, Jan Heidemann INTRODUCTION The course of Dextran Sodium Sulphate (DSS) colitis is routinely monitored by clinical and post mortem analyses, including assessment of colon length and histomorphological changes. Until today, the majority of DSS colitis experiments have a static endpoint requiring euthanization. In humans, the use of image-enhancing ultrasound contrast agents is increasing as they represent a diagnostic tool for a variety of morphological changes insoluble for regular ultrasonography. Concerning echocardiography or ultrasonography of liver lesions, contrast agents are used routinely today. AIM To assess the value of image-enhancing antibody-labeled ultrasound contrast agents against gut-specific endovascular Mucosal Addressin Cellular Adhesion Molecule 1 (MAdCAM-1) and Vascular Cell Adhesion Molecule 1 (VCAM-1) in identyfying inflammatory characteristics of experimental murine DSS-colitis by In Vivo 40 MHz ultrasonography. Findings were correlated with weight loss as an established colitis parameter and to recent findings of In Vivo 40 MHz hydrosonography of the colon. MATERIAL and METHODS Four groups (n = 20) of female C57BL/6 mice received 3% (w/v) DSS in tap water or tap water as control (group A). After administration of DSS for 3, 6 and 9 days (groups B - D), mice were anesthesized and antibodylabeled ultrasound contrast agents against MAdCAM-1 and VCAM-1 were intravenously administered before perfoming 40 MHz abdominal sonography of the colon. Acoustic echoes were measured in the colonic wall using specialized software to quantify severity of DSSColitis in groups A-D. RESULTS 1) The expression of MAdCAM-1 and VCAM-1 as endovascular adhesion molecules is significantly increased in DSS-Colitis as depicted by immunoblot and immunohistochemic analyses. 2) Image-enhancing ultrasound with contrast agents labeled against endovascular MAdCAM-1 and VCAM-1 is able to noninvasively display the course of experimental DSS colitis in mice in a highly specific manner. Results correlate with exposure to DSS over time, with group D showing the highest echogenicity of the bowel wall (p < 0.05). 3) The results are significantly correlated to clinical weight loss in mice and to bowel wall thickness as valuable ultrasonographic characteristics of DSS-Colitis in mice. CONCLUSION Our findings indicate that image-enhancing ultrasound with contrast agents against endovascular MAdCAM-1 and VCAM-1 is a feasible and accurate tool to noninvasively and specifically monitor the course of experimental DSS colitis in mice. Results correlate significantly with weight loss as established parameter of colitis and recent findings in 40 MHz hydrosonography of the inflamed colon.
Mo1802 Role of CD24 in the Development of DSS-Induced Murine Experimental Colitis Inna Naumov, Sarah Kraus, Shiran Shapira, Ilan Aroch, Diana Kazanov, Ehud Ron, Gilad Gitstein, Eran Elinav, Alexei V. Salnikov, Peter Altevogt, Nadir Arber Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory disease with an undefined pathogenesis. Its treatment is effective in only ~50% of the cases. Patients with UC have an increased risk of developing colorectal cancer. We have previously shown that CD24, a mucin-like glycoprotein, is overexpressed in ~90% of colorectal tumors at an early stage. Aim: To investigate the role of CD24 in dextran sulphate sodium (DSS)-induced colitis in 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 were given 2.5-4% DSS in the drinking water for 5 days, followed by 2 days of water. Control mice received water. Animals were sacrificed on day 7 and scored for their disease activity index (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, CD24 KO mice displayed a significant reduction in the pathological scores, reflecting a reduction in the colitis severity. Cytokine analysis by an antibody array demonstrated striking differences in the serum and colon of CD24 KO vs. WT mice, supporting the involvement of CD24 in the inflammatory response. Conclusions: CD24 may have an important role in the induction of colitis in mice. Targeting of CD24 may be potentially useful for the treatment of experimental colitis in mice and may be an important novel target in IBD patients.
Mo1800 Methyl Donor Deficiency Affects Small Intestinal Differentiation and Barrier Function in Rats Aude Bressenot, Shabnam Pooya, Carine Bossenmeyer-Pourie, Guillaume Gauchotte, Adeline Germain, Jean Baptiste Chevaux, Florence Coste, Jean-Michel Vignaud, JeanLouis Gueant, Laurent Peyrin Biroulet
Mo1803
BACKGROUND: Dietary methyl donors (vitamin B12 and folate) and their genetic determinants are associated with Crohn's disease risk. Methyl donor deficiency (MDD) aggravates experimental colitis in rat pups from mothers under methyl deficient diet. However, the effects of MDD on differentiation and barrier function are not known, in the distal and proximal intestine. MATERIALS AND METHODS: We investigated whether MDD may affect development and barrier functions of the proximal and distal small intestine in rat pups from 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, proliferation and 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 crypt apoptosis, loss of enterocyte differentiation in villous and reduction of intestinal phosphatase alkaline production. In both proximal and distal small bowel cleaved caspase-3 immunostaining and Apostain labelling index showed increased crypt apoptosis. Decreased proliferation was observed in crypts of the proximal small bowel with a reduced number of MCM6 and Proliferating Cell Nuclear Antigen (PCNA)-positive cells. This lack of enterocyte differentiation in distal small bowel was associated to impaired expression of β-catenin and decreased β-catenin/E-cadherin interaction. Low Protein Phosphatase 2A expression levels may trigger increased β-catenin degradation. MDD also affected intestinal barrier in proximal small bowel by decreasing Paneth cells number after immunostaining for lysosyme and by reducing goblet cells number and mucus production after immunostaining for mucin-2. CONCLUSIONS: Overall, MDD has dual effects on small intestine by producing dramatic effects on enterocyte differentiation and barrier function in rats. MDD might be a predisposing factor to small bowel Crohn's disease.
Overexpression of N-Acetylglucosaminyltransferase V (GnT-V) Exacerbates Murine Colitis With Dysfunction of Macrophages Mayuko 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 modifications and is catalyzed by the action of several glycosyltransferases to make various complex sugar chains. N-acetylglucosaminyltransferase V (GnT-V) is involved in the biosynthesis of N-linked oligosaccharides in Golgi organelle where it catalyzes N-acetylglucosamine (GlcNAc) by beta-1,6-branching. Previous reports show that the levels of GnT-V expression and beta1,6-branched glycoproteins are increased in cancer cells, and GnT-V promotes tumor growth by inhibition of CD4+ T cells and macrophages, indicating that GnT-V has a pivotal role on host immune responses. The role of GnT-V for intestinal inflammation, however, is not clearly elucidated. The aim of the present study is to clarify the role of GnT-V in murine colitis using GnT-V transgenic (Tg) mice. Methods: Male 8-11-week-old GnT-V Tg and wild-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. 2,4,6Trinitrobenzene 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 μl of 1% TNBS in 50% ethanol on day 8. The mice were sacrificed on day 12. The expression of beta-1,6-branching glycoprotein was investigated by L4-PHA lectin blotting. Fluorescent latex beads were opsonized to peritoneal macrophages (PM) or bone marrow derived macrophages (BMDM) for 1 hour, and phagocytic activity was investigated by calculating the number of fluorescent cells by flow cytometry. To deplete macrophages In Vivo, clodronateliposomes 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 were observed in colon and splenic mononuclear cells of GnT-V Tg mice. In both DSS and TNBS colitis models, GnT-V Tg mice showed severe weight loss and histological damages compared with WT mice. Phagocytic activity of macrophages was significantly impaired in GnT-V Tg mice than in WT mice. When macrophages were depleted during DSS administration, the extent of colitis was unchanged in GnT-V and WT mice, indicating that the exacerbation of colitis in GnT-V was eliminated in absence of macrophages. Conclusions: Overexpression of GnT-V exacerbated murine experimental colitis. Dysfunction of macrophages has a pivotal role in the pathogenesis of colitis in GnT-V mice.
Mo1801 A Model of In Vitro Perfused Mouse Intestine Suitable for Pharmacological Testing of Anti-Inflammatory Drugs Dominik 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 being adequate In Vitro models that can simulate the In Vivo situation are not available yet. Different cell culture models for intestinal cells are very simple to use, but do not come close to the physiology of the small intestine as a whole. Therefore we established a mouse small intestine long time perfusion model primarily designed for pharmacological testing. The most important advantage of our model is its ability to imitate the tissue interaction in inflamed 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. The mesenteric artery is rapidly cannulated and the intestine is mesenterially perfused in situ. During the intestinal resection procedure tissue is cooled and humidified with physiological salt solution. After resection of a 5-6 cm segment of the proximal small intestine with the adhering cannulated mesenterial root, In Vitro longterm mesenterial perfusion is immediately
AGA Abstracts
S-688