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S1728 Intestinal Bile Salt Nuclear Receptor FXR Protects From Inflammatory Bowel Disease: Potential Therapeutic Implications
AGA Abstracts
S1728
Although recent clinical studies have demonstrated efficacy in patients with Crohn's disease (CD) using monoclonal antibodies (mAbs) directed against CD49d (e.g. a4 integrin), very little is known regarding the role of this integrin in T-cell trafficking to the GALT, MLNs and gut as well as its role in T-cell activation during the induction of chronic colitis in animal models of CD. A major limitation in defining the role of α4 in T-cell trafficking and activation In Vivo is the fact that α4 deficiency is embryonically lethal. To begin to define the role of this T-cell-associated integrin in the pathogenesis of chronic gut inflammation we subjected a conditional mutant mouse (Mx.cre+α4flox/flox) to multiple (4) injections (ip) of polyI:C lead which ablated ~40-70% of α4 expression on CD4+T cells in these mice. We then sorted CD4+CD45RBhigh cells into α4+ and α4- populations and adoptively transferred these naïve T-cells into RAG-1 deficient (RAG-/-) recipients to assess their ability to induce chronic colitis. We found, using blinded histopathological analysis that α4+→RAG-/developed moderate-to-severe colitis (histopath score=15.67±0.56) at 8 weeks post-transfer whereas α4-→RAG-/- mice exhibited little or no colitis (histopath score=3.89±0.76). Failure to induce colitis was associated with a dramatic decrease (~30-fold) in the numbers of Tcells and myeloid cells within the colonic LP but not in the MLNs or spleens in the α4-→RAG-/- mice when compared to α4+→RAG-/- group. Intracellular cytokine staining demonstrated that the few T cells that infiltrated the colonic LP of the α4-→RAG-/- mice produced similar levels of IL-17 and IFN-γ when compared to the T-cells isolated from the LP of colitic α4+ →RAG-/- animals. In addition, we found similar levels of T-proliferation In Vitro for both the α4+ and α4- T-cell populations. Taken together, our data suggests that α4 is required for trafficking of T-cells to gut (but not the MLNs) where they induce chronic colitis. T-cell-associated α4 is not required for T-cell activation and polarization to disease-producing Th1/Th17 cells (Supported by NIH-PO1 43785).
Intestinal Bile Salt Nuclear Receptor FXR Protects From Inflammatory Bowel Disease: Potential Therapeutic Implications Raffaella Maria Gadaleta, Karel J. Van Erpecum, Bas Oldenburg, Ellen C. Willemsen, Stefania Murzilli, Leo W. Klomp, Peter D. Siersema, Antonio Moschetta, Saskia W. van Mil The bile salt nuclear receptor Farnesoid X Receptor (FXR) was recently implicated in intestinal antibacterial defense and barrier function. We aimed to study its role in pathogenesis of inflammatory bowel diseasE. colitis was induced in Wild Type (WT) and FXR knock-out (ko) mice (n=8 to 10 for each group) by Dextran Sodium Sulphate (DSS: 2.5% in drinking water, 10 days) with or without synthetic FXR ligand 6-Ethyl Chenodeoxycholic Acid (6ECDCA: 5 mg/kg/day, 13 days, starting 3 days before DSS). Colitis symptoms were checked daily and intestinal permeability (FITC-dextran assay), bile salt composition (HPLC), histology and colonic inflammatory gene expression (Q-PCR) determined. mRNA expression of FXR and target genes was determined in IBD patient biopsies. Underlying mechanisms were explored in complementary In Vitro experiments. 6ECDCA-treated WT but not FXR ko mice were protected from DSS-induced colitis, as shown by highly significant reduction of body weight loss, rectal bleeding, colonic shortening, normalization of intestinal permeability, 49% reduction in blinded histological score and 55% reduction in goblet cell loss. Only in 6ECDCA-treated WT mice, mRNA levels of pro-inflammatory genes (IL-1β, IL-6, IL10 and MCP-1) were strongly down-regulated while antibacterial defense gene iNOS was upregulated. 6ECDCA was enriched in bile of both WT and FXR ko mice (9.8% and 5.3% of total bile salts, respectively). However intestinal expression of FXR target genes FGF15 and SHP was increased 4.5- and 19-fold with 6ECDCA treatment only in WT. In patients with quiescent Crohn colitis (n=17), mRNA expression of FXR and SHP was significantly altered compared to patients with ulcerative colitis (n=16) or healthy controls (n=17). In differentiated CaCo2 cells grown on trans-well plates, FXR activation by GW4064 prevented DSS-dependent loss of integrity of the monolayer. In differentiated HT29 cells, TNFαinduced 20-fold increase of IL-1β expression was abolished by GW4064-dependent FXR activation. In reporter assays, the FXR agonist GW4064 prevented TNFα-induced NF-κB activity in HEK293 cells transfected with WT FXR, but no effect was achieved with FXR mutant W469A (defective in Ligand Binding Domain), indicating FXR-mediated inhibition of NF-κB signalling. In conclusion, FXR activation protects against experimental murine colitis, supposedly by preserving the intestinal barrier and inhibiting NF-κB activity. Currently available potent synthetic FXR agonists may offer new therapeutic strategies for inflammatory bowel disease.
S1731 High Fat Diet Drives Immunological Changes in the Gut That Predispose to Development of Experimental Colitis Jesse D. Aitken, Frederic A. Carvalho, Shanthi V. Sitaraman, Andrew T. Gewirtz, Matam Vijay-Kumar Background: Recent epidemiological studies indicate that the incidence of inflammatory bowel diseases (IBD) is disproportionately high in populations that consume Western-style diets rich in fat. As colitis is often thought to result from an aberrant immune response to otherwise harmless ligands, we sought to determine whether high-fat diet might alter the gut immunophysiology of mice in such a way as to predispose them to developing colitis. Methods: Four week old male mice (C57BL6, Jackson Labs) were given either regular diet (RD) (5% energy from fat) or high fat diet (HFD) (60% energy from fat, Research Diets Inc.) for 8 weeks. Subsequently, mice were given water supplemented with (or lacking) 3.5% dextran sodium sulfate (DSS) for a period of 7 days or infected with 1x108 CFU Salmonella Typhimurium (SL3201) via gavage and assayed for standard colitis parameters and mortality, respectively. Results: Mice given HFD exhibited increased epithelial barrier disruption as assayed by FITC-dextran translocation, elevated sera immunoreactivity to bacterial flagellin and, despite substantial weight gain, mild gut inflammation as evidenced by histopathology and myeloperoxidase (MPO) activity. We observed a striking reduction of cleaved caspase 3 expression in the colonic epithelia of HFD-mice which suggests decreased levels of apoptosis. Concomitantly, we observed decreased expression of the proliferative markers Ki-67 and proliferating cell nuclear antigen (PCNA), suggesting reduced epithelial cell division. Relative to mice given RD, HFD-mice exhibited markedly more severe colitis in response to DSS as measured by every parameter analyzed including body weight, incidence of rectal bleeding, colonic MPO and histopathology as well as serum KC, lipocalin-2 and RegIIIγ. Additionally, the HFD-mice displayed increased mortality when infected orally with Salmonella Typhimurium. This outcome correlated with reduced expression of the neutrophil chemoattractant KC in HFD-mice in response to systemically administered flagellin. Conclusion: Elevated levels of dietary fat can alter gut immunophysiology in such a way as to prime the mucosal immune responses to potential triggers of acute colitis. This is likely at least partially due to altered levels of epithelial cell turnover effecting an increase in barrier permeability which, in turn, allows for inordinate translocation of luminal contents to subepithelial tissues. Application of this paradigm to humans predicts that prolonged consumption of HFD may contribute to the development of IBD.
S1729 Colitis-Associated Cancer in Mice Lacking the RNA-Binding Protein APOBEC1 Thomas A. Kerr, Yan Xie, Valerie Blanc, Hitoshi Matsumoto, Rodney D. Newberry, Matthew A. Ciorba, Susan Kennedy, Jianyang Luo, Nicholas O. Davidson Introduction: The AU-rich RNA binding protein Apobec-1 is an RNA-specific cytidine deaminase. Apobec-1 knockout in the Apcmin/+ background reduces intestinal adenoma formation at least in part by attenuating cox-2 mRNA stability. Because the molecular pathways involved in colitis-associated cancer (CAC) may differ from small intestinal adenoma formation, our goal was to examine the role of Apobec-1 in a murine model of CAC. Methods: CAC was examined in control (WT) and Apobec-1-/- mice treated with azoxymethane (AOM) and 3 cycles of dextran sodium sulfate (DSS) over 20 weeks. Short term studies were also undertaken in WT and Apobec-1-/- mice with 7-day 2.5% DSS. Weight change, histology and gene expression (in anorectum and spleen) were quantified. Serum immunoglobulin levels were measured by ELISA. Results: Apobec-1-/- mice displayed increased CAC mortality (56%, n=18) compared to congenic controls (0%, n=11). Surviving Apobec-1-/- mice developed more extensive anorectal polyps (5.2 fold greater surface area, p<0.01). Mice exposed to short-term (7 days) 2.5% DSS lost more weight than WT (7% vs. 3%, p=0.02), with increased colon damage (33%, p=0.04). We also examined activation induced deaminase (AID), the Apobec-1 homolog and ancestral gene that plays a key role in somatic hypermutation and immunoglobulin class switching. Ectopic AID expression has been linked to genotoxicity in CAC and other malignancies. AID was elevated 4 fold vs WT in the anorectum of chow-fed Apobec-1-/- mice (p=0.006) and 39 fold (p=0.02) in DSS treated mice. Splenic AID mRNA was also elevated in untreated Apobec-1-/- mice (5.7 fold, p=0.02) suggesting dysregulated AID expression extends beyond the GI tract, independent of DSS exposure. Elevated AID correlated with increased splenic IgA mRNA (4.1 fold, p=0.002), and serum IgA in both control (1247 vs. 165 μg/ml, p=0.0008) and DSS treated (1241 vs. 55 μg/ml, p=0.0001) mice. Higher anorectal IL-17 mRNA was also found in Apobec-1-/- vs WT mice both at baseline (4.7 fold, p=0.06), and after DSS (7.2 fold, p=0.01). Conclusions: Apobec1-/- mice have baseline activation of gut immune pathways including IgA and IL-17 production. This phenotype is amplified under conditions of acute colitis possibly contributing to excess mortality and CAC in Apobec-1-/- mice. These studies reveal an unanticipated role for Apobec-1 in immune homeostasis and IgA production, and implicate the mucosal immune system in colitis associated cancer development.
S1732 Functional Knock-out of Adenosine a3 Receptors (a3-/-Ar) Alters Intestinal Motility and Protects Mice From Developing DSS - Colitis Tianhua Ren, Iveta Grants, Andrew J. Rozmiarek, Matthew P. McKiernan, Mazin Alhaj, Hamdy H. Hassanain, Omega Griffin, Marlene Jacobson, Wendy L. Frankel, Fievos L. Christofi Background: Pharmacological data suggest activation of adenosine low affinity A3 receptors (A3AR) inhibits the enteric short inter-plexus neural motor reflex triggered by histamine (AJP, 2009 In Press). Oral administration of the A3AR agonist N6-(3-iodobenzyl)-adenosine5'-N-methyluronamide (IB-MECA) protects rodents against developing colitis at doses of 13 mg/Kg/day (IBD;12(8):766-89, 2006) that may not be restricted to actions at A3AR. Beneficial effects of IB-MECA against inflammation have been linked to post-receptor signaling to down-regulate A3AR and attenuate NFκB transcription. Indeed, in ischemia or inflammation models A3AR knockout (A3-/-AR) is protective. Aim: We used a functional A3-/-AR model to test whether the A3AR is involved in regulating DSS colitis and In Vivo motility. Methods: The A3AR was probed by PCR genotyping, western blot and immunochemistry. In Vivo motility was assessed by the bead-latency test (evacuation time) or stoolfrequency (pellets/hr) in WT and A3-/-AR micE. colitis was induced in A3-/-AR or wild-type (WT) age-matched control C57BL/6 mice by 7 days DSS in drinking water (n=6-12 /group). Animals were evaluated in blinded-fashion for histopathology, myeloperoxidase (MPO), colon length/weight ratio, CD4+ cell counts, weight-loss/recovery, diarrhea, and Guaiac-test. Results: Western blot with goat anti-hu-A3 antiserum (1:100 dil) revealed a 66 kDa band in the colon. A3AR-ir is expressed in enteric nerve varicosities and glia and up-regulated in s-100-positive glia in DSS-colitis. A3AR-ir is abolished by immunoabsorption. Bead latency was reduced by 50% in A3-/-AR mice compared to WT (p<0.001) and stool evacuation
S1730 Cd49d (α4 Integrin) is Required for T-Cell Homing to the Colon and Induction of Chronic Gut Inflammation Dmitry V. Ostanin, Fridrik Karlsson, Songlin Zhang, Iurii Koboziev, Laura Gray, Matthew B. Grisham It is well-recognized that T-cell-associated CD49 (α4 integrin) plays an important role for naïve and effector T-cell trafficking to the gut-associated lymphoid tissue(GALT; Peyer's Patches, lymphoid follicles), mesenteric lymph nodes (MLNs) and intestinal tissue. This is thought to be mediated by the specific interactions of the very late antigen-4 (VLA-4; α4β1) and/or lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1; α4β7) with the high endothelial venules in the GALT and MLNs as well as the post-capillary venules of the gut.
AGA Abstracts
S-262
S1728
Although recent clinical studies have demonstrated efficacy in patients with Crohn's disease (CD) using monoclonal antibodies (mAbs) directed against CD49d (e.g. a4 integrin), very little is known regarding the role of this integrin in T-cell trafficking to the GALT, MLNs and gut as well as its role in T-cell activation during the induction of chronic colitis in animal models of CD. A major limitation in defining the role of α4 in T-cell trafficking and activation In Vivo is the fact that α4 deficiency is embryonically lethal. To begin to define the role of this T-cell-associated integrin in the pathogenesis of chronic gut inflammation we subjected a conditional mutant mouse (Mx.cre+α4flox/flox) to multiple (4) injections (ip) of polyI:C lead which ablated ~40-70% of α4 expression on CD4+T cells in these mice. We then sorted CD4+CD45RBhigh cells into α4+ and α4- populations and adoptively transferred these naïve T-cells into RAG-1 deficient (RAG-/-) recipients to assess their ability to induce chronic colitis. We found, using blinded histopathological analysis that α4+→RAG-/developed moderate-to-severe colitis (histopath score=15.67±0.56) at 8 weeks post-transfer whereas α4-→RAG-/- mice exhibited little or no colitis (histopath score=3.89±0.76). Failure to induce colitis was associated with a dramatic decrease (~30-fold) in the numbers of Tcells and myeloid cells within the colonic LP but not in the MLNs or spleens in the α4-→RAG-/- mice when compared to α4+→RAG-/- group. Intracellular cytokine staining demonstrated that the few T cells that infiltrated the colonic LP of the α4-→RAG-/- mice produced similar levels of IL-17 and IFN-γ when compared to the T-cells isolated from the LP of colitic α4+ →RAG-/- animals. In addition, we found similar levels of T-proliferation In Vitro for both the α4+ and α4- T-cell populations. Taken together, our data suggests that α4 is required for trafficking of T-cells to gut (but not the MLNs) where they induce chronic colitis. T-cell-associated α4 is not required for T-cell activation and polarization to disease-producing Th1/Th17 cells (Supported by NIH-PO1 43785).
Intestinal Bile Salt Nuclear Receptor FXR Protects From Inflammatory Bowel Disease: Potential Therapeutic Implications Raffaella Maria Gadaleta, Karel J. Van Erpecum, Bas Oldenburg, Ellen C. Willemsen, Stefania Murzilli, Leo W. Klomp, Peter D. Siersema, Antonio Moschetta, Saskia W. van Mil The bile salt nuclear receptor Farnesoid X Receptor (FXR) was recently implicated in intestinal antibacterial defense and barrier function. We aimed to study its role in pathogenesis of inflammatory bowel diseasE. colitis was induced in Wild Type (WT) and FXR knock-out (ko) mice (n=8 to 10 for each group) by Dextran Sodium Sulphate (DSS: 2.5% in drinking water, 10 days) with or without synthetic FXR ligand 6-Ethyl Chenodeoxycholic Acid (6ECDCA: 5 mg/kg/day, 13 days, starting 3 days before DSS). Colitis symptoms were checked daily and intestinal permeability (FITC-dextran assay), bile salt composition (HPLC), histology and colonic inflammatory gene expression (Q-PCR) determined. mRNA expression of FXR and target genes was determined in IBD patient biopsies. Underlying mechanisms were explored in complementary In Vitro experiments. 6ECDCA-treated WT but not FXR ko mice were protected from DSS-induced colitis, as shown by highly significant reduction of body weight loss, rectal bleeding, colonic shortening, normalization of intestinal permeability, 49% reduction in blinded histological score and 55% reduction in goblet cell loss. Only in 6ECDCA-treated WT mice, mRNA levels of pro-inflammatory genes (IL-1β, IL-6, IL10 and MCP-1) were strongly down-regulated while antibacterial defense gene iNOS was upregulated. 6ECDCA was enriched in bile of both WT and FXR ko mice (9.8% and 5.3% of total bile salts, respectively). However intestinal expression of FXR target genes FGF15 and SHP was increased 4.5- and 19-fold with 6ECDCA treatment only in WT. In patients with quiescent Crohn colitis (n=17), mRNA expression of FXR and SHP was significantly altered compared to patients with ulcerative colitis (n=16) or healthy controls (n=17). In differentiated CaCo2 cells grown on trans-well plates, FXR activation by GW4064 prevented DSS-dependent loss of integrity of the monolayer. In differentiated HT29 cells, TNFαinduced 20-fold increase of IL-1β expression was abolished by GW4064-dependent FXR activation. In reporter assays, the FXR agonist GW4064 prevented TNFα-induced NF-κB activity in HEK293 cells transfected with WT FXR, but no effect was achieved with FXR mutant W469A (defective in Ligand Binding Domain), indicating FXR-mediated inhibition of NF-κB signalling. In conclusion, FXR activation protects against experimental murine colitis, supposedly by preserving the intestinal barrier and inhibiting NF-κB activity. Currently available potent synthetic FXR agonists may offer new therapeutic strategies for inflammatory bowel disease.
S1731 High Fat Diet Drives Immunological Changes in the Gut That Predispose to Development of Experimental Colitis Jesse D. Aitken, Frederic A. Carvalho, Shanthi V. Sitaraman, Andrew T. Gewirtz, Matam Vijay-Kumar Background: Recent epidemiological studies indicate that the incidence of inflammatory bowel diseases (IBD) is disproportionately high in populations that consume Western-style diets rich in fat. As colitis is often thought to result from an aberrant immune response to otherwise harmless ligands, we sought to determine whether high-fat diet might alter the gut immunophysiology of mice in such a way as to predispose them to developing colitis. Methods: Four week old male mice (C57BL6, Jackson Labs) were given either regular diet (RD) (5% energy from fat) or high fat diet (HFD) (60% energy from fat, Research Diets Inc.) for 8 weeks. Subsequently, mice were given water supplemented with (or lacking) 3.5% dextran sodium sulfate (DSS) for a period of 7 days or infected with 1x108 CFU Salmonella Typhimurium (SL3201) via gavage and assayed for standard colitis parameters and mortality, respectively. Results: Mice given HFD exhibited increased epithelial barrier disruption as assayed by FITC-dextran translocation, elevated sera immunoreactivity to bacterial flagellin and, despite substantial weight gain, mild gut inflammation as evidenced by histopathology and myeloperoxidase (MPO) activity. We observed a striking reduction of cleaved caspase 3 expression in the colonic epithelia of HFD-mice which suggests decreased levels of apoptosis. Concomitantly, we observed decreased expression of the proliferative markers Ki-67 and proliferating cell nuclear antigen (PCNA), suggesting reduced epithelial cell division. Relative to mice given RD, HFD-mice exhibited markedly more severe colitis in response to DSS as measured by every parameter analyzed including body weight, incidence of rectal bleeding, colonic MPO and histopathology as well as serum KC, lipocalin-2 and RegIIIγ. Additionally, the HFD-mice displayed increased mortality when infected orally with Salmonella Typhimurium. This outcome correlated with reduced expression of the neutrophil chemoattractant KC in HFD-mice in response to systemically administered flagellin. Conclusion: Elevated levels of dietary fat can alter gut immunophysiology in such a way as to prime the mucosal immune responses to potential triggers of acute colitis. This is likely at least partially due to altered levels of epithelial cell turnover effecting an increase in barrier permeability which, in turn, allows for inordinate translocation of luminal contents to subepithelial tissues. Application of this paradigm to humans predicts that prolonged consumption of HFD may contribute to the development of IBD.
S1729 Colitis-Associated Cancer in Mice Lacking the RNA-Binding Protein APOBEC1 Thomas A. Kerr, Yan Xie, Valerie Blanc, Hitoshi Matsumoto, Rodney D. Newberry, Matthew A. Ciorba, Susan Kennedy, Jianyang Luo, Nicholas O. Davidson Introduction: The AU-rich RNA binding protein Apobec-1 is an RNA-specific cytidine deaminase. Apobec-1 knockout in the Apcmin/+ background reduces intestinal adenoma formation at least in part by attenuating cox-2 mRNA stability. Because the molecular pathways involved in colitis-associated cancer (CAC) may differ from small intestinal adenoma formation, our goal was to examine the role of Apobec-1 in a murine model of CAC. Methods: CAC was examined in control (WT) and Apobec-1-/- mice treated with azoxymethane (AOM) and 3 cycles of dextran sodium sulfate (DSS) over 20 weeks. Short term studies were also undertaken in WT and Apobec-1-/- mice with 7-day 2.5% DSS. Weight change, histology and gene expression (in anorectum and spleen) were quantified. Serum immunoglobulin levels were measured by ELISA. Results: Apobec-1-/- mice displayed increased CAC mortality (56%, n=18) compared to congenic controls (0%, n=11). Surviving Apobec-1-/- mice developed more extensive anorectal polyps (5.2 fold greater surface area, p<0.01). Mice exposed to short-term (7 days) 2.5% DSS lost more weight than WT (7% vs. 3%, p=0.02), with increased colon damage (33%, p=0.04). We also examined activation induced deaminase (AID), the Apobec-1 homolog and ancestral gene that plays a key role in somatic hypermutation and immunoglobulin class switching. Ectopic AID expression has been linked to genotoxicity in CAC and other malignancies. AID was elevated 4 fold vs WT in the anorectum of chow-fed Apobec-1-/- mice (p=0.006) and 39 fold (p=0.02) in DSS treated mice. Splenic AID mRNA was also elevated in untreated Apobec-1-/- mice (5.7 fold, p=0.02) suggesting dysregulated AID expression extends beyond the GI tract, independent of DSS exposure. Elevated AID correlated with increased splenic IgA mRNA (4.1 fold, p=0.002), and serum IgA in both control (1247 vs. 165 μg/ml, p=0.0008) and DSS treated (1241 vs. 55 μg/ml, p=0.0001) mice. Higher anorectal IL-17 mRNA was also found in Apobec-1-/- vs WT mice both at baseline (4.7 fold, p=0.06), and after DSS (7.2 fold, p=0.01). Conclusions: Apobec1-/- mice have baseline activation of gut immune pathways including IgA and IL-17 production. This phenotype is amplified under conditions of acute colitis possibly contributing to excess mortality and CAC in Apobec-1-/- mice. These studies reveal an unanticipated role for Apobec-1 in immune homeostasis and IgA production, and implicate the mucosal immune system in colitis associated cancer development.
S1732 Functional Knock-out of Adenosine a3 Receptors (a3-/-Ar) Alters Intestinal Motility and Protects Mice From Developing DSS - Colitis Tianhua Ren, Iveta Grants, Andrew J. Rozmiarek, Matthew P. McKiernan, Mazin Alhaj, Hamdy H. Hassanain, Omega Griffin, Marlene Jacobson, Wendy L. Frankel, Fievos L. Christofi Background: Pharmacological data suggest activation of adenosine low affinity A3 receptors (A3AR) inhibits the enteric short inter-plexus neural motor reflex triggered by histamine (AJP, 2009 In Press). Oral administration of the A3AR agonist N6-(3-iodobenzyl)-adenosine5'-N-methyluronamide (IB-MECA) protects rodents against developing colitis at doses of 13 mg/Kg/day (IBD;12(8):766-89, 2006) that may not be restricted to actions at A3AR. Beneficial effects of IB-MECA against inflammation have been linked to post-receptor signaling to down-regulate A3AR and attenuate NFκB transcription. Indeed, in ischemia or inflammation models A3AR knockout (A3-/-AR) is protective. Aim: We used a functional A3-/-AR model to test whether the A3AR is involved in regulating DSS colitis and In Vivo motility. Methods: The A3AR was probed by PCR genotyping, western blot and immunochemistry. In Vivo motility was assessed by the bead-latency test (evacuation time) or stoolfrequency (pellets/hr) in WT and A3-/-AR micE. colitis was induced in A3-/-AR or wild-type (WT) age-matched control C57BL/6 mice by 7 days DSS in drinking water (n=6-12 /group). Animals were evaluated in blinded-fashion for histopathology, myeloperoxidase (MPO), colon length/weight ratio, CD4+ cell counts, weight-loss/recovery, diarrhea, and Guaiac-test. Results: Western blot with goat anti-hu-A3 antiserum (1:100 dil) revealed a 66 kDa band in the colon. A3AR-ir is expressed in enteric nerve varicosities and glia and up-regulated in s-100-positive glia in DSS-colitis. A3AR-ir is abolished by immunoabsorption. Bead latency was reduced by 50% in A3-/-AR mice compared to WT (p<0.001) and stool evacuation
S1730 Cd49d (α4 Integrin) is Required for T-Cell Homing to the Colon and Induction of Chronic Gut Inflammation Dmitry V. Ostanin, Fridrik Karlsson, Songlin Zhang, Iurii Koboziev, Laura Gray, Matthew B. Grisham It is well-recognized that T-cell-associated CD49 (α4 integrin) plays an important role for naïve and effector T-cell trafficking to the gut-associated lymphoid tissue(GALT; Peyer's Patches, lymphoid follicles), mesenteric lymph nodes (MLNs) and intestinal tissue. This is thought to be mediated by the specific interactions of the very late antigen-4 (VLA-4; α4β1) and/or lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1; α4β7) with the high endothelial venules in the GALT and MLNs as well as the post-capillary venules of the gut.
AGA Abstracts
S-262