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AGA Abstracts models of fibrosis suggest an anti-fibrotic benefit. We investigated the efficacy of SPIR and losartan In Vitro, and in 2 rodent models of intestinal fibrosis. Methods: In Vitro, TGFβ induction of CCD-18co human colonic myofibroblasts was used as a model of fibrogenesis. The Grassl Salmonella model of cecal fibrosis and the chronic TNBS model of distal colon fibrosis were used as In Vivo rodent models. Human outcomes were determined through a search of medical records at a US tertiary care hospital. Results: SPIR and its metabolite (canrenone) prevent TGFβ-induced fibrogenesis in human colonic myofibroblasts and prim- ary myofibroblasts, repressing gene expression of αSMA, collagen, and CTGF. Other RAAS pathway inhibitors (aliskiren, enalaprilat, losartan) partially suppressed TGFβ-induced fib- rosis In Vitro, supporting the importance of the RAAS pathway in intestinal fibrosis. However, in two animal models of intestinal inflammation and fibrosis, treatment with SPIR caused significant mortality. In the mouse S. typhimurium infection model, treatment with 0.7mg/ kg/day of SPIR produced 100% mortality (95%CI 0.69-1.00). In the rat TNBS model, SPIR treatment at 20 mg/kg/day (10-fold lower than well tolerated doses in other rat models) produced 44% mortality (95%CI 0.14-0.79). A dose-response experiment in the rat TNBS model demonstrated increased survival with decreasing SPIR dose. Mortality was 33% at 10 mg/kg/day, and 0% at 2.5 or 0.5 mg/kg/day. However, lower doses of SPIR did not alter the development of fibrosis, as determined by gross pathology, histopathology, fibrotic gene expression, and protein expression (αSMA and collagen I). Similar experiments with losartan resulted in no mortality and no improvement in fibrosis. We hypothesized that SPIR increased mortality during intestinal inflammation. To study this in humans, we determined the association between the mortality rate of Clostridium difficile colitis and SPIR therapy. In patients hospitalized with C. difficile, we found an increased risk (OR = 1.88, 95% CI (1.37, 2.60) of death in patients on SPIR. Conclusions: SPIR, while a promising anti-fibrotic In Vitro, induces significant mortality with no fibrosis benefit in 2 rodent models of inflammation and fibrosis. In patients on SPIR with C. difficile, a similar but smaller effect is seen. The human effect could be confounded by comorbidities (CHF, cirrhosis), but further research is needed to determine the safety of continuation of SPIR during C. difficile colitis. Mo1787 The Role of Matriptase, Urokinase-Type Plasminogen Activator, and SERPINE1 in Colitis Pathogenesis Sarah Netzel-Arnett, Marguerite S. Buzza, Terez Shea-Donohue, Aiping Zhao, Alessio Fasano, Toni M. Antalis Inflammatory bowel diseases (IBD) are chronic intestinal disorders whose etiology is not completely understood. Several studies have shown that increased intestinal epithelial per- meability participates in the pathogenesis of IBD. Both matriptase and urokinase-type plasminogen activator (uPA) are serine proteases expressed all along the gastrointestinal (GI) tract and can be inhibited by the IBD associated gene SERPINE1 (PAI-1). Previous studies have shown matriptase plays a critical role in barrier function, however, the molecular mechanism for its role in barrier function remains unknown. Elevated levels of the serine protease inhibitor PAI-1 have previously been reported in IBD patients suggesting impaired protease activity may be important in the pathogenesis of IBD. To determine if matriptase expression levels are also altered in human diseases of the GI tract, we analyzed colonic tissue specimens of IBD patients. We found that matriptase mRNA levels were reduced 50 or 70% in Crohn's disease and ulcerative colitis patient groups, respectively. Matriptase has been shown to activate several known regulators of GI pathophysiology including protease- activated receptor-2 (PAR2), uPA, and pro-hepatocyte growth factor (proHGF) suggesting it may play a functional role in intestinal physiology and diseases of the GI tract. To investigate the role of matriptase, uPA, and PAI-1 in GI pathophysiology we utilized knockout (KO) and hypomorphic mouse strains in a model of intestinal injury and inflammation induced by oral administration of dextran sodium sulfate (DSS). Since the matriptase knockout mouse is perinatal lethal, a matriptase hypomorphic mouse strain expressing less than 1% matriptase mRNA levels and their littermate controls were used in these studies. We found that decreased matriptase expression results in a dramatic increase in susceptibility to an acute DSS-induced colitis injury model as assessed by survival, clinical symptoms, and histopathology. When mice were treated with DSS for 7 days followed by water without DSS, 70% of matriptase hypomorphic mice and 20% of uPA KOs were moribund by day 13, while 100% of control mice and PAI-1 KOs survived DSS-induced injury. Similar survival rates were seen in a chronic model of DSS-induced colitis. The clinical disease scores were significantly higher after day 8 in the hypomorphic and uPA KO groups compared to the control group. The clinical disease scores of PAI-1 KO mice were ameliorated compared to the control group. Histological analyses of DSS-treated mice suggest wound healing is delayed in the matriptase hypomorphs. Molecular analysis of colonic tissues is ongoing but should provide more insight into the functional role of matriptase and the plasminogen activation system in the GI tract. Mo1788 Bilateral Adrenalectomy Potentiates Ulcerogenic and Motility Responses Caused by Indomethacin in Rat Small Intestine Naoko Abe, Kikuko Amagase, Koji Takeuchi Background/Aim: Glucocorticoids, important hormones released from the adrenal cortex, act as the final step in the activation of the hypothalamus-pituitary-adrenal (HPA) system. We recently found that urocortin I (Ucn 1), a corticotropin-releasing factor (CRF) agonist, prevented indomethacin-induced small intestinal lesions mediated by CRF2 receptors. How- ever, it remains unknown whether endogenous glucocorticoids are involved in modulating the mucosal integrity against these lesions. In the present study, we examined the influence of adrenalectomy and mifepristone (glucocorticoid receptor antagonist) against NSAID- induced small intestinal damage in rats, in attempts to investigate the possible involvement of adrenal glucocorticoid in the action of Ucn 1. Methods: Male SD rats without fasting were given indomethacin (3, 10 mg/kg) SC and killed 24 h later to examine hemorrhagic lesions developed in the small intestine. Ucn I (20 μg/kg) was given IV 10 min before the administration of indomethacin. Astressin 2B (CRF2R antagonist: 60 μg/kg) was given IV 10 min before Ucn I. Mifepristone (10 mg/kg) was given PO twice 0.5 h before and 6 h after indomethacin. Bilateral adrenalectomy was performed a week before the experiment. S-650 AGA Abstracts Intestinal motility was measured by a balloon method, while mucus secretion was examined by PAS staining. Enterobacterial count was determined by a culture method. Expression of iNOS mRNA was examined by RT-PCR. Results: Indomethacin, at 10 but not 3 mg/kg, caused multiple hemorrhagic lesions in the small intestine, accompanied by a decrease in mucus secretion and increases in intestinal motility, enterobacterial invasion and iNOS expression. The severity of these lesions was markedly aggravated by adrenalectomy or pretreatment with mifepristone, accompanied by further enhancement of intestinal motility, bacterial invasion as well as iNOS expression. Especially, adrenalectomy markedly potentiated the ulcerogenic and motility responses caused by indomethacin, and severe lesions occurred even at 3 mg/kg of indomethacin, together with marked hepermotility. By contrast, Ucn I prevented these lesions as well as such functional alterations, in an astressin-2B inhibitable manner, but the degree of protection was not significantly mitigated by adrenalectomy or mifepristone. Conclusion: These results suggest that (a) adrenalectomy worsens the intestinal ulcerogenic response to indomethacin, and the increased intestinal motility may be a key element in the mechanism for this aggravation, (b) endogenous glucocorticoids play a role in mucosal defense against indomethacin-induced small intestinal lesions but do not much account for the protective action of Ucn 1. Mo1789 Rebamipide Inhibits Indomethacin-Induced Small Intestinal Injury: Possible Involvement of the Modulation of Bacterial Flora Through Defensin Upregulation Koji Otani, Tetsuya Tanigawa, Toshio Watanabe, Yuji Nadatani, Hirohisa Machida, Hirotoshi Okazaki, Hirokazu Yamagami, Kenji Watanabe, Kazunari Tominaga, Yasuhiro Fujiwara, Tetsuo Arakawa Background: Enterobacteria play important roles in the pathophysiology of non-steroidal anti-inflammatory drug (NSAID)-induced enteropathy. Bacterial flora is regulated by anti- microbial peptides such as defensins. Rebamipide is a widely used mucoprotective drug that protects the gastric mucosa against acute injury caused by various noxious agents. Moreover, recent studies suggest that rebamipide inhibits NSAID-induced small intestinal injury. However, the mechanisms underlying the mucoprotective effect of rebamipide in the small intestine have not been elucidated. Aim: To investigate the effects of rebamipide on indomethacin-induced small intestinal injury and the influence of rebamipide on bacterial flora and the expression of defensin-5. Methods: (1) Indomethacin (10 mg/kg) was orally administered to male C57BL/6J mice after oral administration of rebamipide (100 or 300 mg/kg) or vehicle for 1 week. The small intestines were removed 24 h later. Small intestinal injury was assessed by measuring the area stained with 1% Evans blue, which corresponded to the injured area in the intestinal mucosa. (2) After oral administration of rebamipide (100 or 300 mg/kg) or vehicle for 1 week, the contents of the small intestine were subjected to terminal restriction fragment length polymorphism (T-RFLP) analysis in order to assess the diversity of enterobacteria. (3) After oral administration of rebamipide (100 or 300 mg/ kg) or vehicle for 1 week, defensin-5 mRNA expression in the ileal tissue was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results: (1) Rebamipide inhibited indomethacin-induced small intestinal injury. (2) T-RFLP analysis showed that rebamipide (300 mg/kg) increased the percentage of gram-positive Lactobacillales (80.5% in the vehicle-treated mice and 93.8% in the rebamipide-treated mice) and decreased the percentage of gram-negative Bacteroides (9.0% in the vehicle-treated mice and 2.5% in the rebamipide-treated mice), compared with those in the vehicle-treated controls. (3) The rebamipide-treated mice showed a 1.4-fold increase in defensin-5 mRNA expression in the ileal tissue compared with that in the vehicle-treated control mice. Indomethacin reduced the expression of defensin-5 mRNA in the ileal tissue by 52% of that in the vehicle control, whereas rebamipide reversed the expression of defensin-5 mRNA. Conclusion: These findings suggest that rebamipide inhibits the indomethacin-induced small intestinal injury possibly by modulating bacterial flora in the small intestine through the upregulation of defensins. Mo1790 Intestinal Transport Defects in a Mouse Model of Salmonella-Induced Diarrhea Ronald Marchelletta, Declan F. McCole, Sharon Okamoto, Elise Roel, Rachel Klinkenberg, Donald Guiney, Joshua Fierer, Kim E. Barrett Background: Despite the high incidence of enteritis in non-typhoidal Salmonella infections, little is known of the pathogenesis of diarrhea in this disease. To better understand Salmonella- induced diarrhea, we used a new model of Salmonella enterocolitis in mice carrying a wild- type Nramp1 (Slc11a1) allele, reflecting the level of resistance to Salmonella found in normal humans. Mice were treated with the antibiotic kanamycin prior to infection. The mice showed increased fecal water following infection with kanamycin-reisistant wild-type (wt) S. enterica serovar Typhimurium 14028s, thus recapitulating human diarrhea. The aim of this study was to identify ion transport processes contributing to diarrheal symptoms. Methods: BALB/c.D2 Slc11a1 mice (6-8 weeks) were treated orally with one dose of kanamy- cin 2 days prior to infection with S. Typhimurium (wt) or an invA mutant defective in epithelial cell invasion. Control mice received kanamycin (kana) alone or were untreated. Proximal colon was removed five days after the start of the experiment and partially stripped of muscle layers before mounting in Ussing chambers. Histology was assessed by H & E staining and RNA expression by real-time PCR. Results: H & E staining of infected but not control colons showed mild/moderate inflammation and no evidence of epithelial damage in any treatment group. Mice infected with wild-type (wt) S. Typhimurium showed an increase in baseline short-circuit current (Isc) compared to other treatment groups but this did not reach significance (n=6). cAMP-dependent ion transport responses to forskolin (20 μM) were significantly suppressed in wt-infected (12 ± 4 μA/cm 2 ) vs. untreated mice (43 ±4 μA/cm 2 ; p<0.05, n=5-6). Mice infected with the invA mutant showed a partial reduction that did not reach significance (26 ± 2 μA/cm 2 ; n=9). Ca 2+ -dependent Isc responses to the muscarinic agonist, carbachol (CCh, 300 μM) were also slightly lower in wt-infected (11 ± 5 μA/cm 2 ; n=3) vs. untreated mice (19 ± 5 μA/cm 2 ; n=3). Analysis of ion transport protein mRNA expression revealed a significant reduction in the Cl - :HCO 3 - exchanger, Downregul- ated in Adenoma (DRA) in wt-infected (n=7) vs. untreated (p<0.001; n=8), wt-infected vs. kana (p<0.001; n=6), and wt-infected vs. invA mutant (p<0.01; n=8) (ANOVA followed by
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smodels of fibrosis suggest an anti-fibrotic benefit. We investigated the efficacy of SPIR andlosartan In Vitro, and in 2 rodent models of intestinal fibrosis. Methods: In Vitro, TGFβinduction of CCD-18co human colonic myofibroblasts was used as a model of fibrogenesis.The Grassl Salmonella model of cecal fibrosis and the chronic TNBS model of distal colonfibrosis were used as In Vivo rodent models. Human outcomes were determined through asearch of medical records at a US tertiary care hospital. Results: SPIR and its metabolite(canrenone) prevent TGFβ-induced fibrogenesis in human colonic myofibroblasts and prim-ary myofibroblasts, repressing gene expression of αSMA, collagen, and CTGF. Other RAASpathway inhibitors (aliskiren, enalaprilat, losartan) partially suppressed TGFβ-induced fib-rosis In Vitro, supporting the importance of the RAAS pathway in intestinal fibrosis. However,in two animal models of intestinal inflammation and fibrosis, treatment with SPIR causedsignificant mortality. In the mouse S. typhimurium infection model, treatment with 0.7mg/kg/day of SPIR produced 100% mortality (95%CI 0.69-1.00). In the rat TNBS model, SPIRtreatment at 20 mg/kg/day (10-fold lower than well tolerated doses in other rat models)produced 44% mortality (95%CI 0.14-0.79). A dose-response experiment in the rat TNBSmodel demonstrated increased survival with decreasing SPIR dose. Mortality was 33% at10 mg/kg/day, and 0% at 2.5 or 0.5 mg/kg/day. However, lower doses of SPIR did not alterthe development of fibrosis, as determined by gross pathology, histopathology, fibrotic geneexpression, and protein expression (αSMA and collagen I). Similar experiments with losartanresulted in nomortality and no improvement in fibrosis.We hypothesized that SPIR increasedmortality during intestinal inflammation. To study this in humans, we determined theassociation between the mortality rate of Clostridium difficile colitis and SPIR therapy. Inpatients hospitalized with C. difficile, we found an increased risk (OR = 1.88, 95% CI (1.37,2.60) of death in patients on SPIR. Conclusions: SPIR, while a promising anti-fibrotic InVitro, induces significant mortality with no fibrosis benefit in 2 rodent models of inflammationand fibrosis. In patients on SPIR with C. difficile, a similar but smaller effect is seen. Thehuman effect could be confounded by comorbidities (CHF, cirrhosis), but further researchis needed to determine the safety of continuation of SPIR during C. difficile colitis.
Mo1787
The Role of Matriptase, Urokinase-Type Plasminogen Activator, and SERPINE1in Colitis PathogenesisSarah Netzel-Arnett, Marguerite S. Buzza, Terez Shea-Donohue, Aiping Zhao, AlessioFasano, Toni M. Antalis
Inflammatory bowel diseases (IBD) are chronic intestinal disorders whose etiology is notcompletely understood. Several studies have shown that increased intestinal epithelial per-meability participates in the pathogenesis of IBD. Both matriptase and urokinase-typeplasminogen activator (uPA) are serine proteases expressed all along the gastrointestinal (GI)tract and can be inhibited by the IBD associated gene SERPINE1 (PAI-1). Previous studieshave shown matriptase plays a critical role in barrier function, however, the molecularmechanism for its role in barrier function remains unknown. Elevated levels of the serineprotease inhibitor PAI-1 have previously been reported in IBD patients suggesting impairedprotease activity may be important in the pathogenesis of IBD. To determine if matriptaseexpression levels are also altered in human diseases of the GI tract, we analyzed colonictissue specimens of IBD patients. We found that matriptase mRNA levels were reduced 50or 70% in Crohn's disease and ulcerative colitis patient groups, respectively. Matriptase hasbeen shown to activate several known regulators of GI pathophysiology including protease-activated receptor-2 (PAR2), uPA, and pro-hepatocyte growth factor (proHGF) suggestingit may play a functional role in intestinal physiology and diseases of the GI tract. To investigatethe role of matriptase, uPA, and PAI-1 in GI pathophysiology we utilized knockout (KO)and hypomorphic mouse strains in a model of intestinal injury and inflammation inducedby oral administration of dextran sodium sulfate (DSS). Since the matriptase knockout mouseis perinatal lethal, a matriptase hypomorphic mouse strain expressing less than 1%matriptasemRNA levels and their littermate controls were used in these studies.We found that decreasedmatriptase expression results in a dramatic increase in susceptibility to an acute DSS-inducedcolitis injury model as assessed by survival, clinical symptoms, and histopathology. Whenmice were treated with DSS for 7 days followed by water without DSS, 70% of matriptasehypomorphic mice and 20% of uPA KOs were moribund by day 13, while 100% of controlmice and PAI-1 KOs survived DSS-induced injury. Similar survival rates were seen in achronic model of DSS-induced colitis. The clinical disease scores were significantly higherafter day 8 in the hypomorphic and uPA KO groups compared to the control group. Theclinical disease scores of PAI-1 KO mice were ameliorated compared to the control group.Histological analyses of DSS-treated mice suggest wound healing is delayed in the matriptasehypomorphs. Molecular analysis of colonic tissues is ongoing but should provide moreinsight into the functional role of matriptase and the plasminogen activation system in theGI tract.
Mo1788
Bilateral Adrenalectomy Potentiates Ulcerogenic and Motility ResponsesCaused by Indomethacin in Rat Small IntestineNaoko Abe, Kikuko Amagase, Koji Takeuchi
Background/Aim: Glucocorticoids, important hormones released from the adrenal cortex,act as the final step in the activation of the hypothalamus-pituitary-adrenal (HPA) system.We recently found that urocortin I (Ucn 1), a corticotropin-releasing factor (CRF) agonist,prevented indomethacin-induced small intestinal lesions mediated by CRF2 receptors. How-ever, it remains unknown whether endogenous glucocorticoids are involved in modulatingthe mucosal integrity against these lesions. In the present study, we examined the influenceof adrenalectomy and mifepristone (glucocorticoid receptor antagonist) against NSAID-induced small intestinal damage in rats, in attempts to investigate the possible involvementof adrenal glucocorticoid in the action of Ucn 1. Methods: Male SD rats without fastingwere given indomethacin (3, 10 mg/kg) SC and killed 24 h later to examine hemorrhagiclesions developed in the small intestine. Ucn I (20 μg/kg) was given IV 10 min before theadministration of indomethacin. Astressin 2B (CRF2R antagonist: 60 μg/kg) was given IV10 min before Ucn I. Mifepristone (10 mg/kg) was given PO twice 0.5 h before and 6 hafter indomethacin. Bilateral adrenalectomy was performed a week before the experiment.
S-650AGA Abstracts
Intestinal motility was measured by a balloon method, while mucus secretion was examinedby PAS staining. Enterobacterial count was determined by a culture method. Expression ofiNOS mRNA was examined by RT-PCR. Results: Indomethacin, at 10 but not 3 mg/kg,caused multiple hemorrhagic lesions in the small intestine, accompanied by a decrease inmucus secretion and increases in intestinal motility, enterobacterial invasion and iNOSexpression. The severity of these lesions was markedly aggravated by adrenalectomy orpretreatment with mifepristone, accompanied by further enhancement of intestinal motility,bacterial invasion as well as iNOS expression. Especially, adrenalectomymarkedly potentiatedthe ulcerogenic and motility responses caused by indomethacin, and severe lesions occurredeven at 3 mg/kg of indomethacin, together with marked hepermotility. By contrast, Ucn Iprevented these lesions as well as such functional alterations, in an astressin-2B inhibitablemanner, but the degree of protection was not significantly mitigated by adrenalectomy ormifepristone. Conclusion: These results suggest that (a) adrenalectomy worsens the intestinalulcerogenic response to indomethacin, and the increased intestinal motility may be a keyelement in the mechanism for this aggravation, (b) endogenous glucocorticoids play a rolein mucosal defense against indomethacin-induced small intestinal lesions but do not muchaccount for the protective action of Ucn 1.
Mo1789
Rebamipide Inhibits Indomethacin-Induced Small Intestinal Injury: PossibleInvolvement of the Modulation of Bacterial Flora Through DefensinUpregulationKoji Otani, Tetsuya Tanigawa, Toshio Watanabe, Yuji Nadatani, Hirohisa Machida,Hirotoshi Okazaki, Hirokazu Yamagami, Kenji Watanabe, Kazunari Tominaga, YasuhiroFujiwara, Tetsuo Arakawa
Background: Enterobacteria play important roles in the pathophysiology of non-steroidalanti-inflammatory drug (NSAID)-induced enteropathy. Bacterial flora is regulated by anti-microbial peptides such as defensins. Rebamipide is a widely used mucoprotective drugthat protects the gastric mucosa against acute injury caused by various noxious agents.Moreover, recent studies suggest that rebamipide inhibits NSAID-induced small intestinalinjury. However, the mechanisms underlying the mucoprotective effect of rebamipide inthe small intestine have not been elucidated. Aim: To investigate the effects of rebamipideon indomethacin-induced small intestinal injury and the influence of rebamipide on bacterialflora and the expression of defensin-5. Methods: (1) Indomethacin (10 mg/kg) was orallyadministered to male C57BL/6J mice after oral administration of rebamipide (100 or 300mg/kg) or vehicle for 1 week. The small intestines were removed 24 h later. Small intestinalinjury was assessed by measuring the area stained with 1% Evans blue, which correspondedto the injured area in the intestinal mucosa. (2) After oral administration of rebamipide(100 or 300 mg/kg) or vehicle for 1 week, the contents of the small intestine were subjectedto terminal restriction fragment length polymorphism (T-RFLP) analysis in order to assessthe diversity of enterobacteria. (3) After oral administration of rebamipide (100 or 300 mg/kg) or vehicle for 1 week, defensin-5 mRNA expression in the ileal tissue was determined byreal-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results: (1) Rebamipideinhibited indomethacin-induced small intestinal injury. (2) T-RFLP analysis showed thatrebamipide (300 mg/kg) increased the percentage of gram-positive Lactobacillales (80.5% inthe vehicle-treated mice and 93.8% in the rebamipide-treated mice) and decreased thepercentage of gram-negative Bacteroides (9.0% in the vehicle-treated mice and 2.5% in therebamipide-treated mice), compared with those in the vehicle-treated controls. (3) Therebamipide-treated mice showed a 1.4-fold increase in defensin-5 mRNA expression in theileal tissue compared with that in the vehicle-treated control mice. Indomethacin reducedthe expression of defensin-5 mRNA in the ileal tissue by 52% of that in the vehicle control,whereas rebamipide reversed the expression of defensin-5 mRNA. Conclusion: These findingssuggest that rebamipide inhibits the indomethacin-induced small intestinal injury possiblyby modulating bacterial flora in the small intestine through the upregulation of defensins.
Mo1790
Intestinal Transport Defects in a Mouse Model of Salmonella-Induced DiarrheaRonald Marchelletta, Declan F. McCole, Sharon Okamoto, Elise Roel, Rachel Klinkenberg,Donald Guiney, Joshua Fierer, Kim E. Barrett
Background: Despite the high incidence of enteritis in non-typhoidal Salmonella infections,little is known of the pathogenesis of diarrhea in this disease. To better understand Salmonella-induced diarrhea, we used a new model of Salmonella enterocolitis in mice carrying a wild-type Nramp1 (Slc11a1) allele, reflecting the level of resistance to Salmonella found in normalhumans. Mice were treated with the antibiotic kanamycin prior to infection. The miceshowed increased fecal water following infection with kanamycin-reisistant wild-type (wt)S. enterica serovar Typhimurium 14028s, thus recapitulating human diarrhea. The aim ofthis study was to identify ion transport processes contributing to diarrheal symptoms.Methods: BALB/c.D2 Slc11a1 mice (6-8 weeks) were treated orally with one dose of kanamy-cin 2 days prior to infection with S. Typhimurium (wt) or an invA mutant defective inepithelial cell invasion. Control mice received kanamycin (kana) alone or were untreated.Proximal colon was removed five days after the start of the experiment and partially strippedof muscle layers before mounting in Ussing chambers. Histology was assessed by H & Estaining and RNA expression by real-time PCR. Results: H & E staining of infected but notcontrol colons showed mild/moderate inflammation and no evidence of epithelial damagein any treatment group. Mice infected with wild-type (wt) S. Typhimurium showed anincrease in baseline short-circuit current (Isc) compared to other treatment groups but thisdid not reach significance (n=6). cAMP-dependent ion transport responses to forskolin (20μM) were significantly suppressed in wt-infected (12 ± 4 μA/cm2) vs. untreated mice (43± 4 μA/cm2; p<0.05, n=5-6). Mice infected with the invA mutant showed a partial reductionthat did not reach significance (26 ± 2 μA/cm2; n=9). Ca2+-dependent Isc responses to themuscarinic agonist, carbachol (CCh, 300 μM) were also slightly lower in wt-infected (11 ±5 μA/cm2; n=3) vs. untreated mice (19 ± 5 μA/cm2; n=3). Analysis of ion transport proteinmRNA expression revealed a significant reduction in the Cl-:HCO3
- exchanger, Downregul-ated in Adenoma (DRA) in wt-infected (n=7) vs. untreated (p<0.001; n=8), wt-infected vs.kana (p<0.001; n=6), and wt-infected vs. invA mutant (p<0.01; n=8) (ANOVA followed by
