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Protein Isolate Attenuated DSSInduced Colitis in a Defined Floral Model
T cell development. Aim: We sought to determine whether deficiencies in regulators of actin polymerization Rac1/2 and of actin bundling L-plastin alter Treg development. In addition, given Rac1 and Rac2 are isoforms with partial redundancy in some cell types, we also sought to tease apart the requirement of either or both in regulatory T cell generation. Methods: Given embryonic lethality of universal Rac1 deletion, Rac1 conditional knockout mice were generated by mating Rac1fl/fl mice with CD11c-Cre mice or Mx-Cre mice (with subsequent induction of Cre by serial polyI:C treatment). Thus, Rac2 knockout (KO), Rac1/ 2 double KO, and L-plastin KO mice were analyzed for Treg proportions in the thymus, spleen, mesenteric and peripheral lymph nodes, and colonic lamina propria (LP). To study effects on colitis propensity, animals were subjected to 5 days of dextran sulfate sodium followed by 5 days of water or 2.5% 2,4,6-trinitrobenzenesulfonic acid (TNBS) preceded by skin pre-sensitization. Results: We found that L-plastin KO mice harbored reductions of 25-50% in Treg proportions in the spleen, mesenteric, and peripheral lymph nodes (p < 0.05) but not thymus or colonic LP. Likewise, Rac2 deficiency leads to a similar pattern of Treg reductions in the periphery to a lesser extent. Mice with concomitant Rac2 deficiency and a conditional deletion in Rac1 in dendritic cells had a more dramatic reduction in Tregs in the periphery. Despite this altered Treg environment, there was no spontaneous colitis; Rac2 KO mice appear protected and L-plastin KO mice have intact propensity for DSS- and TNBS-induced colitis. Conclusions: Regulators of the actin polymerization and bundling play important roles in peripheral Treg development but their absence in animals on a C57/ Bl6 background do not lead to increased susceptibility to chemically-induced colitis, likely due to other immune defects. The particular etiology for altered Treg proportions in Rac1/ 2- and L-plastin-deficient mice, the effect of T-cell specific deletion of Rac1 along with Rac2 deficiency, and mechanism underlying protection against colitis in Rac2 KO mice remain to be elucidated.
The Effect of Pentadecapeptide BPC 157 on Colovesical Fistula Healing Tihomir Grgic, Robert Klicek, Dora Grgic, Jelena Suran, Leonardo Patrlj, Masa Hrelec Patrlj, Sven Seiwerth, Predrag Sikiric Introduction. A colovesical fistulas present an important clinical problems. They usually occur as a complication of diverticulosis, IBD or malignant process. The pentadecapeptide BPC 157 , in clinical trials for IBD therapy, has already shown the effectiveness in healing colocutaneous fistulas ( J. Pharmacol Sci 2008 ) , gastrocutaneous fistulas ( Dig Dis Sci 2009 ). Therefore we suggest it as a possible therapy of colovesical fistulas. Matherials and methods. Wistar Albino rats were randomly assigned, at 5 cm from anocutanous borderline, with the diameter of 3 mm. The surgical procedure was performed according to rules brought by the Local Ethical Committee. Medication of BPC 157 (10ug/kg, 10ng/kg) was per-orally, in drinking water and intraperitoneally, once daily, the first dose immediately after the operation, the last dose at 24 h before sacrifice, at 7, 15, 30 post-operative day, with the biomechanical, functional, macroscopic and microscopic assessment. Results. Biomechanical analysis :we measured pressure of the fistulas rupture for all groups of animals with the system for central venous pressure assesment .In the control group pressures for fistulas rupture were similar for every experimental period : ≤1mL H2O, 1mL±0,3 mL, 2 mL±0,5 ml H2O. In the second group( BPC per-orally in drinking water 10ug/kg and 10ng/kg ) water pressures were 1mL±0,3; 2mL±0,3 and 5mL±0,5 but without fistulas rupture at the and of 30 days experimental period.In the third group( BPC intraperitoneally 10ug/kg and 10ng/kg ) water pressure was increased from 2mL±0,5mL until 5 mL±0,5 at the and of 15 days experimental period. In most animals fistulas were healed at the end of 30 days experimental period. Macroscopic analysis: We measured diameters of fistulas on the colonic and the bladder side.In the control group diameters of fistulas were similar for both sides for all experimental periods : 3mm ±0.5mm on the colon and bladder side,3mm±1mm and 2,5±0,5mm. In the second group fistulas diameter was 3±0,2 mm for both sides ( 7 days ),2mm±0,5 mm for colon and 2,5±0,5 mm for bladder ( 15 days ), 1,5mm±0,5 for colon, 2mm±0,5 mm for bladder side ( 30 days ). In the third group fistulas were reduced : 2,5mm±0,2mm colon, 3mm±0,5 bladder ( 7 days ), 1,5mm±0,5mm colon; 2mm±0,5mm bladder ( 15 days ) and at the end oft he 30 days experimental period in most animals fistulas were healed er reduced diameter at 1mm±0,3mm for both sides.. Conclusion. According to these results the pentadecapeptide BPC 157 could present a new possible pathway in therapy of colovesical fistulas.
Mo1710 GEF-H1 Controls IRF3 Activation by RIG-I Like Receptor Signaling Yun Zhao, Hao-Sen Chiang, Joo Hye Song, Song Liu, Ninghai Wang, Cox Terhorst, HansChristian Reinecker Background and AIM: The activation of type I interferon (IFN) is central to innate host immune defenses against viral pathogens. RIG-I-like receptors (RLR), are important inducers of innate immunity that recognize viral RNA. RLR requires mitochondrial antiviral signaling protein (MAVS), for the activation of TBK1 and IKKε that mediate the phosphorylation of IRF3 for the induction of type I IFNs. GEF-H1 is a member of the Dbl family that is sequestered on microtubules and directs spatiotemporal activation of Rho GTPases. GEFH1 can be activated and released from microtubules by intracellular pathogens. Methods: GEF-H1-deficient mice were generated by targeting Arhgef2 in C57BL/6 ES cells and bone marrow-derived macrophages were isolated to define the role of GEF-H1 in innate immune activation by foreign nucleic acids. Cytokine secretion and mRNA expression were determined in response to HMWpoly(I:C) as a ligand for Mda5 or LMWpoly(I:C) and 5′ppp-dsRNA were used as synthetic ligands for RIG-I. Phosphorylation of GEF-H1, IRF3 and NF-kB-p65 was detected with specific antibodies. IFNβ-, IFIT1 (p561)-, or NF-κB-promoter luciferase reporter constructs were used to define functional protein domains of GEF-H1 for the induction of innate immune responses in HEK293T cells or bone marrow-derived macrophages. Results: IFN-β, IL-6 and TNF-α expression was significantly reduced in GEFH1-deficient macrophages in response to RLR but not TLR signaling. GEF-H1-deficient macrophages failed to activate IRF3 in response to RIG-I and Mda5 activation. In contrast, 5′ppp-dsRNA and HMW poly(I:C) induced similar p65 phosphorylation and IκBα degradation in GEF-H1- or MAVS-deficient and wildtype macrophages. Upon RLR signaling, GEFH1 formed complexes with TBK1 and colocalized with MAVS containing intracellular compartments. Conclusion: Our findings identify GEF-H1 as a new signaling intermediate that is required for foreign nucleic acid recognition. Our data further indicate that distinct mechanisms are responsible for IRF3 and NF-κB activation by RLRs. GEF-H1 functions in the RLR pathway in conjunction with MAVS and TBK1 to enhance the phosphorylation of IRF3 and induction of IFN-β and proinflammatory cytokine expression.
Mo1708 Serum-Derived Bovine Immunoglobulin/ Protein Isolate Attenuated DSSInduced Colitis in a Defined Floral Model Kenneth J. Maas, Meghan Wymore Brand, Abigail Henderson, Christopher J. Detzel, Alan Horgan, Jesse M. Hostetter, Gregory Phillips, Albert Jergens, Michael J. Wannemuehler, Ross Darling, Eric Weaver Background: Inflammatory bowel disease (IBD) has a complex, multifaceted etiology. Contributing factors to the disease include genetic predisposition, diet, microbial antigens, aberrant immunity, and environmental triggers. Alterations in gut microbiota contribute to an exaggerated immune response further complicating the disease. Altered Schaedler flora (ASF) mice, harboring a defined gut microbial population, were used to elucidate host inflammatory responses to the microbiome following dextran sodium sulphate (DSS)-induction of colitis. Serum-derived bovine immunoglobulin/protein isolate (SBI), comprised primarily of immunoglobulins (Igs) is indicated for the clinical dietary management of enteropathy under physician supervision. SBI Igs have been shown to bind and neutralize microbial components and toxins in vitro. We hypothesize that SBI Igs bind whole bacteria or bacterial components and reduce inflammatory responses directed at the gut microbiota through immune exclusion. Methods: ASF mice were gavaged with 40 mg/day for 6 weeks of SBI or the hydrolyzed collagen (HC) control protein. One week after SBI commenced, mice were orally gavaged with 5 x 108 CFU of LF82 (E. coli originally isolated from a Crohn's patient). Low dose DSS (1.5%) was administered in drinking water 3 weeks after E. coli inoculation. Mice were allowed to drink freely for 7 days prior to necropsy. At the time of euthanasia, mice were grossly scored based on luminal blood in cecum and colon, absence of formed fecal pellets, cecal atrophy, and colon length. Colon and cecum sections were taken for fluorescence in-situ hybridization (FISH). Fecal contents were collected for microbiome analysis. Cecal/colon biopsy punches and plasma were collected for cytokine analysis and antibody responses to flora. Histological scoring of cecal/colon sections was performed based on mucosal height, ulceration, inflammation score, edema, stromal collapse and gland hyperplasia. Results: DSS administration resulted in elevated inflammatory scores as measured in macroscopic and microscopic analyses. A significant attenuation of colonic inflammation (p=0.03), cecal stromal collapse (p=0.05), and colonic glandular hyperplasia (p=0.03) in DSS treated mice fed SBI compared to DSS treated mice fed HC was observed. A strong trend for SBI to attenuate DSS induced cecal ulceration p=0.07 and colon stromal collapse p= 0.07 was also observed. Conclusions: Results of this study demonstrate that SBI attenuates histological parameters of inflammation of ASF mice administered DSS, presumably by binding to luminal antigens and excluding them from immune cell stimulation. Future microbiome, FISH, and mucosal cytokine analyses will contribute to understanding the mechanisms by which SBI dampens this immune response.
Mo1711 MiR320 Family Regulates NOD2/CARD15: A New Mechanism for Controlling Inflammation? Maria Pierdomenico, Vincenzo Cesi, Laura Stronati, Manuela Costanzo, Marina Aloi, Salvatore Oliva, Paolo Rossi, Franca Viola, Salvatore Cucchiara Background: Regulation of inflammatory responses is ensured by coordinated and controlled gene expression in immune system cells. One group of gene expression regulators is a class of short single-stranded RNA molecules termed microRNAs (miRNAs). Recent studies have suggested that miR-320 is altered in tissue samples of patients with Inflammatory Bowel Disease (IBD) (1). We performed a bioinformatic analysis showing that miR-320 family may bind the 3'-UTR of NOD2/CARD15 (nucleotide-binding oligomerization domain), the first susceptibility gene associated to Crohn's disease (CD), one of the two main forms of IBD. Interestingly, NOD2 has been previously identified as a functional target of other miRNAs (2). Aims: the present study was aimed at 1) exploring the hypothesis that NOD2/CARD15 is a target of members of the miR-320 family, including miR-320a, -320b, -320c, -320d and 320e, 2) assessing the contribution of the miR-320/NOD2 axis to intestinal inflammation. Methods: the colonic adenocarcinoma cell line, HT29, was transfected with exogenous miR320a, -320b, -320c, -320d, -320e and a miRNA negative control. In addition, miR320 family inhibitor and miRNA inhibitor negative control were transfected to inhibit endogenous miRNAs. In a second experimental setting, cells, transfected as above, were also exposed to IFNγ+TNFα cytomix to induce inflammation. After 48hrs, proteins were extracted and NOD2 expression was assessed by western blot. Results: A strong decrease of NOD2 protein expression was observed after transfections with miR-320a and -320b compared to negative control. Differently, miR-320 family inhibitor induced an increase of NOD2 expression. The exposure to cytomix induced a significant up-regulation of NOD2, which was strongly reduced by transfected miR-320a and -320b. Conclusions: In the present study, we show for the first time that NOD2 expression is under the control of two members of the miR320 family: miR-320a and -320b. Moreover, we show that miR-320a and -320b are able
Mo1709 The Actin Network Is Crucial in Regulatory T Cell Development Yan Song, Romela Marin, Sharon Celeste Morley, David A. Williams, Deanna D. Nguyen Background: Regulators of actin dynamics, such as Rac1 and Rac2, have been recently found to be associated with IBD risk. In animal studies, Rac1 and Rac2 knockout mice have been shown to have altered propensity for colitis in different models. The expression of actin bundling protein L-plastin has been found to be altered in IBD patient mucosal tissues. Given the importance of regulatory T cells (Tregs) in mucosal immune homeostasis and given the actin network is a critical component in TCR signaling, an essential element in Treg generation, we hypothesize that actin regulators play an important role in regulatory
S-641
AGA Abstracts
AGA Abstracts
Mo1707
The Effect of Pentadecapeptide BPC 157 on Colovesical Fistula Healing Tihomir Grgic, Robert Klicek, Dora Grgic, Jelena Suran, Leonardo Patrlj, Masa Hrelec Patrlj, Sven Seiwerth, Predrag Sikiric Introduction. A colovesical fistulas present an important clinical problems. They usually occur as a complication of diverticulosis, IBD or malignant process. The pentadecapeptide BPC 157 , in clinical trials for IBD therapy, has already shown the effectiveness in healing colocutaneous fistulas ( J. Pharmacol Sci 2008 ) , gastrocutaneous fistulas ( Dig Dis Sci 2009 ). Therefore we suggest it as a possible therapy of colovesical fistulas. Matherials and methods. Wistar Albino rats were randomly assigned, at 5 cm from anocutanous borderline, with the diameter of 3 mm. The surgical procedure was performed according to rules brought by the Local Ethical Committee. Medication of BPC 157 (10ug/kg, 10ng/kg) was per-orally, in drinking water and intraperitoneally, once daily, the first dose immediately after the operation, the last dose at 24 h before sacrifice, at 7, 15, 30 post-operative day, with the biomechanical, functional, macroscopic and microscopic assessment. Results. Biomechanical analysis :we measured pressure of the fistulas rupture for all groups of animals with the system for central venous pressure assesment .In the control group pressures for fistulas rupture were similar for every experimental period : ≤1mL H2O, 1mL±0,3 mL, 2 mL±0,5 ml H2O. In the second group( BPC per-orally in drinking water 10ug/kg and 10ng/kg ) water pressures were 1mL±0,3; 2mL±0,3 and 5mL±0,5 but without fistulas rupture at the and of 30 days experimental period.In the third group( BPC intraperitoneally 10ug/kg and 10ng/kg ) water pressure was increased from 2mL±0,5mL until 5 mL±0,5 at the and of 15 days experimental period. In most animals fistulas were healed at the end of 30 days experimental period. Macroscopic analysis: We measured diameters of fistulas on the colonic and the bladder side.In the control group diameters of fistulas were similar for both sides for all experimental periods : 3mm ±0.5mm on the colon and bladder side,3mm±1mm and 2,5±0,5mm. In the second group fistulas diameter was 3±0,2 mm for both sides ( 7 days ),2mm±0,5 mm for colon and 2,5±0,5 mm for bladder ( 15 days ), 1,5mm±0,5 for colon, 2mm±0,5 mm for bladder side ( 30 days ). In the third group fistulas were reduced : 2,5mm±0,2mm colon, 3mm±0,5 bladder ( 7 days ), 1,5mm±0,5mm colon; 2mm±0,5mm bladder ( 15 days ) and at the end oft he 30 days experimental period in most animals fistulas were healed er reduced diameter at 1mm±0,3mm for both sides.. Conclusion. According to these results the pentadecapeptide BPC 157 could present a new possible pathway in therapy of colovesical fistulas.
Mo1710 GEF-H1 Controls IRF3 Activation by RIG-I Like Receptor Signaling Yun Zhao, Hao-Sen Chiang, Joo Hye Song, Song Liu, Ninghai Wang, Cox Terhorst, HansChristian Reinecker Background and AIM: The activation of type I interferon (IFN) is central to innate host immune defenses against viral pathogens. RIG-I-like receptors (RLR), are important inducers of innate immunity that recognize viral RNA. RLR requires mitochondrial antiviral signaling protein (MAVS), for the activation of TBK1 and IKKε that mediate the phosphorylation of IRF3 for the induction of type I IFNs. GEF-H1 is a member of the Dbl family that is sequestered on microtubules and directs spatiotemporal activation of Rho GTPases. GEFH1 can be activated and released from microtubules by intracellular pathogens. Methods: GEF-H1-deficient mice were generated by targeting Arhgef2 in C57BL/6 ES cells and bone marrow-derived macrophages were isolated to define the role of GEF-H1 in innate immune activation by foreign nucleic acids. Cytokine secretion and mRNA expression were determined in response to HMWpoly(I:C) as a ligand for Mda5 or LMWpoly(I:C) and 5′ppp-dsRNA were used as synthetic ligands for RIG-I. Phosphorylation of GEF-H1, IRF3 and NF-kB-p65 was detected with specific antibodies. IFNβ-, IFIT1 (p561)-, or NF-κB-promoter luciferase reporter constructs were used to define functional protein domains of GEF-H1 for the induction of innate immune responses in HEK293T cells or bone marrow-derived macrophages. Results: IFN-β, IL-6 and TNF-α expression was significantly reduced in GEFH1-deficient macrophages in response to RLR but not TLR signaling. GEF-H1-deficient macrophages failed to activate IRF3 in response to RIG-I and Mda5 activation. In contrast, 5′ppp-dsRNA and HMW poly(I:C) induced similar p65 phosphorylation and IκBα degradation in GEF-H1- or MAVS-deficient and wildtype macrophages. Upon RLR signaling, GEFH1 formed complexes with TBK1 and colocalized with MAVS containing intracellular compartments. Conclusion: Our findings identify GEF-H1 as a new signaling intermediate that is required for foreign nucleic acid recognition. Our data further indicate that distinct mechanisms are responsible for IRF3 and NF-κB activation by RLRs. GEF-H1 functions in the RLR pathway in conjunction with MAVS and TBK1 to enhance the phosphorylation of IRF3 and induction of IFN-β and proinflammatory cytokine expression.
Mo1708 Serum-Derived Bovine Immunoglobulin/ Protein Isolate Attenuated DSSInduced Colitis in a Defined Floral Model Kenneth J. Maas, Meghan Wymore Brand, Abigail Henderson, Christopher J. Detzel, Alan Horgan, Jesse M. Hostetter, Gregory Phillips, Albert Jergens, Michael J. Wannemuehler, Ross Darling, Eric Weaver Background: Inflammatory bowel disease (IBD) has a complex, multifaceted etiology. Contributing factors to the disease include genetic predisposition, diet, microbial antigens, aberrant immunity, and environmental triggers. Alterations in gut microbiota contribute to an exaggerated immune response further complicating the disease. Altered Schaedler flora (ASF) mice, harboring a defined gut microbial population, were used to elucidate host inflammatory responses to the microbiome following dextran sodium sulphate (DSS)-induction of colitis. Serum-derived bovine immunoglobulin/protein isolate (SBI), comprised primarily of immunoglobulins (Igs) is indicated for the clinical dietary management of enteropathy under physician supervision. SBI Igs have been shown to bind and neutralize microbial components and toxins in vitro. We hypothesize that SBI Igs bind whole bacteria or bacterial components and reduce inflammatory responses directed at the gut microbiota through immune exclusion. Methods: ASF mice were gavaged with 40 mg/day for 6 weeks of SBI or the hydrolyzed collagen (HC) control protein. One week after SBI commenced, mice were orally gavaged with 5 x 108 CFU of LF82 (E. coli originally isolated from a Crohn's patient). Low dose DSS (1.5%) was administered in drinking water 3 weeks after E. coli inoculation. Mice were allowed to drink freely for 7 days prior to necropsy. At the time of euthanasia, mice were grossly scored based on luminal blood in cecum and colon, absence of formed fecal pellets, cecal atrophy, and colon length. Colon and cecum sections were taken for fluorescence in-situ hybridization (FISH). Fecal contents were collected for microbiome analysis. Cecal/colon biopsy punches and plasma were collected for cytokine analysis and antibody responses to flora. Histological scoring of cecal/colon sections was performed based on mucosal height, ulceration, inflammation score, edema, stromal collapse and gland hyperplasia. Results: DSS administration resulted in elevated inflammatory scores as measured in macroscopic and microscopic analyses. A significant attenuation of colonic inflammation (p=0.03), cecal stromal collapse (p=0.05), and colonic glandular hyperplasia (p=0.03) in DSS treated mice fed SBI compared to DSS treated mice fed HC was observed. A strong trend for SBI to attenuate DSS induced cecal ulceration p=0.07 and colon stromal collapse p= 0.07 was also observed. Conclusions: Results of this study demonstrate that SBI attenuates histological parameters of inflammation of ASF mice administered DSS, presumably by binding to luminal antigens and excluding them from immune cell stimulation. Future microbiome, FISH, and mucosal cytokine analyses will contribute to understanding the mechanisms by which SBI dampens this immune response.
Mo1711 MiR320 Family Regulates NOD2/CARD15: A New Mechanism for Controlling Inflammation? Maria Pierdomenico, Vincenzo Cesi, Laura Stronati, Manuela Costanzo, Marina Aloi, Salvatore Oliva, Paolo Rossi, Franca Viola, Salvatore Cucchiara Background: Regulation of inflammatory responses is ensured by coordinated and controlled gene expression in immune system cells. One group of gene expression regulators is a class of short single-stranded RNA molecules termed microRNAs (miRNAs). Recent studies have suggested that miR-320 is altered in tissue samples of patients with Inflammatory Bowel Disease (IBD) (1). We performed a bioinformatic analysis showing that miR-320 family may bind the 3'-UTR of NOD2/CARD15 (nucleotide-binding oligomerization domain), the first susceptibility gene associated to Crohn's disease (CD), one of the two main forms of IBD. Interestingly, NOD2 has been previously identified as a functional target of other miRNAs (2). Aims: the present study was aimed at 1) exploring the hypothesis that NOD2/CARD15 is a target of members of the miR-320 family, including miR-320a, -320b, -320c, -320d and 320e, 2) assessing the contribution of the miR-320/NOD2 axis to intestinal inflammation. Methods: the colonic adenocarcinoma cell line, HT29, was transfected with exogenous miR320a, -320b, -320c, -320d, -320e and a miRNA negative control. In addition, miR320 family inhibitor and miRNA inhibitor negative control were transfected to inhibit endogenous miRNAs. In a second experimental setting, cells, transfected as above, were also exposed to IFNγ+TNFα cytomix to induce inflammation. After 48hrs, proteins were extracted and NOD2 expression was assessed by western blot. Results: A strong decrease of NOD2 protein expression was observed after transfections with miR-320a and -320b compared to negative control. Differently, miR-320 family inhibitor induced an increase of NOD2 expression. The exposure to cytomix induced a significant up-regulation of NOD2, which was strongly reduced by transfected miR-320a and -320b. Conclusions: In the present study, we show for the first time that NOD2 expression is under the control of two members of the miR320 family: miR-320a and -320b. Moreover, we show that miR-320a and -320b are able
Mo1709 The Actin Network Is Crucial in Regulatory T Cell Development Yan Song, Romela Marin, Sharon Celeste Morley, David A. Williams, Deanna D. Nguyen Background: Regulators of actin dynamics, such as Rac1 and Rac2, have been recently found to be associated with IBD risk. In animal studies, Rac1 and Rac2 knockout mice have been shown to have altered propensity for colitis in different models. The expression of actin bundling protein L-plastin has been found to be altered in IBD patient mucosal tissues. Given the importance of regulatory T cells (Tregs) in mucosal immune homeostasis and given the actin network is a critical component in TCR signaling, an essential element in Treg generation, we hypothesize that actin regulators play an important role in regulatory
S-641
AGA Abstracts
AGA Abstracts
Mo1707