- Email: [email protected]
T1772 Control of Colonic Neuromuscular Functions By A3 Purinergic Receptors in Normal Colon and in Experimental Colitis
AGA Abstracts
by providing doxycycline in the drinking water. Methods: We generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2 (Tet-On) under control of the 12.4 kb murine intestine-specific Villin promoter. To assess inducibility and tissuespecificity of the newly generated Villin-rtTA2 model, mice were bred with the tetO-H2B/ GFP strain and administered with doxycycline. If the Villin-rtTA2 model functions correctly, the nuclear localized green fluorescent H2B/GFP protein will only be expressed in cells where the rtTA2 protein is expressed and activated by doxycycline. Results: Expression of the H2B/GFP fusion protein was observed exclusively in double transgenic mice upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium (see figure). The Villin-rtTA2 was also found to drive transgene expression in the mouse intestine during embryonic development. Furthermore, by administering different concentrations of doxycycline, we show that the Villin-rtTA2 system drives transgene expression in a dosage-dependent fashion. Conclusions: We have successfully generated a novel intestinalspecific doxycycline-inducible mouse model, providing a valuable tool to study the effect of intestinal-specific expression of basically any gene of interest on intestinal physiology and pathology.
regardless of LBT result. Specifically, the entire group had significantly greater lactulose recovery when compared to controls (0.13±0.13 vs.0.07±0.03; p<0.05). Mannitol recovery was not different between groups. Conclusion: Crude RKB diet increased intestinal permeability independent of LBT result suggesting that this deleterious effect on intestinal permeability may be due to a direct toxic effect of lectin. T1771 A Novel Model for Chronic Mucosal Inflammation in IBD and Periodontitis Helieh S. Oz, Jeffrey L. Ebersole Chronic inflammation at mucosal surfaces generally represents an aberrant mucosal immune response to luminal bacteria. These responses generate an array of oxygen radicals, leading to inflammation, tissue destruction, and loss of function as noted in IBD and Periodontitis. The objectives of this study was to examine the time course of mucosal injury following “oral delivery” of DSS and TNBS for an extended period of 18 weeks reflected by oral and GI chronic inflammatory responses. Methods: Mice were administered DSS in diet biweekly; or received TNBS orally 2 times/ week; and controls received sham treatment. Another experimental group received TNBS or sham injection into subgingival maxillas. Results: Animals tolerated oral applications of DSS and TNBS for the duration of study with no mortality or severe clinical symptoms. DSS-treated animals developed diarrhea during DSS administration and returned to normal during the weeks of abstinence. Animals treated with TNBS orally developed no clinical symptoms. Splenic length and weight increased in DSStreated animals in a time depended fashion (18 wk <0.01) and remained normal in TNBStreated mice (vs Co p>0.05). Colons from DSS-treated mice were significantly shortened (<0.001) and colonic weight increased compared to normal controls or TNBS-treated animals. DSS-treated mice developed extensive dilation of stomach wall, ileum, megacolon and abdominal fat deposits. In addition, DSS-treated animals showed a dysregulated hepatic concentrations of endogenous antioxidants (cystine, cysteine, GSH, adenosine, SAMe). Intriguing, the oral inflammation, followed by DSS and TNBS treatment, resulted in a significant increase in alveolar bone resorption in a time dependent manner. Finally, TNBS sub-mucosal delivery caused a severe local inflammation, granuloma formation and extensive alveolar bone resorption. Conclusions: This murine model of chronic mucosal inflammation result in IBD and periodontitis, may mimics some aspects of dysregulated inflammation and disease progression in IBD patients. Supported by NIH-grants NCRR P20RR020145, NCCAMAT1490 (HO).
T1769 Mucosal Cytokine Levels Normalize Despite Continued Clinical and Histopathologic Evidence of Chronic Colitis in a Murine DSS Model of Colitis Steven Polyak, Lisa R. Dixon, Clive Wasserfall, Annette Mach, Cathryn Mah, Chris G. Kevil, John F. Valentine Background: There is significant interest in differentiating between the physiology of acute inflammation and persistent chronic inflammation; and between early versus late disease in inflammatory bowel disease. The purpose of our study was to determine the differences in clinical disease activity, histopathology and mucosal cytokine levels in the acute versus chronic inflammatory stages in a cycled DSS mouse colitis model. Methods: Acute and chronic colitis was induced in Balb/cJ mice by providing 4% DSS in drinking water for 5 days followed by 7 days of regular water for a total of three successive cycles, followed by two additional weeks on regular water, for a total of 50 days. Mice were sacrificed in groups of 3 (plus their controls) on days 5, 12, 24, 36, 43, and 50. Colitis severity was determined by clinical disease activity scores, validated histopathology scores, and serum amyloid A (SAA) levels. Cytokine levels were determined by the Luminex assay from whole length colon homogenates and serum. Results: Cumulative clinical disease activity index score (04) peaked at 2.5 during the second cycle and gradually improved to a score of 1 by day 50 (controls remained at 0 throughout). Cumulative histopathology scores (0-17) were most severe in the distal colon and peaked at 11 after the first cycle, stayed high through the third cycle, and gradually decreased to only 6 by day 50 (controls ranged between 0-1 throughout). SAA levels peaked during the first cycle; and returned to baseline by day 43. Cytokines IL-6, IL-1a, IL-1b, TNFa, IL-15, IP-10, GCSF, KC, MCP and Rantes all peaked after the first cycle of DSS and quickly normalized despite continued cycling of DSS and elevated clinical and histopathology scores. IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-17 and GMCSF did not significantly change or were not detectable at all time points. These cytokines were also analyzed in serum at each time point, but did not reveal significant differences. Conclusions: Clinical and histopathology scores improved but never returned to baseline in our mouse model despite normalization of systemic inflammatory markers and mucosal cytokines. Serum cytokine levels do not distinguish between acute and chronic colitis in Balb/cJ mice. Interestingly, specific mucosal cytokines that were elevated early in the disease course could not be induced again with repeated dosing of DSS. It is likely that the physiology between acute colitis and persistent chronic colitis is different and this model can be used to discover other pathways involved in the development and maintenance of late disease.
T1772 Control of Colonic Neuromuscular Functions By A3 Purinergic Receptors in Normal Colon and in Experimental Colitis Luca Antonioli, Matteo Fornai, Rocchina Colucci, Narcisa Ghisu, Marco Tuccori, Maria Cecilia Giron, Anna Bin, Chiara Zoppellaro, Ignazio Castagliuolo, Rosa Maria Gaion, Mario Del Tacca, Corrado Blandizzi Introduction: Adenosine regulates complex immune/neuromotor interplays in bowel inflammation. The pharmacological activation of adenosine A3 receptors (A3R) results in beneficial effects on experimental colitis. However, the possible involvement of A3R in the regulation of enteric neuromuscular functions is undetermined. This study investigates the expression of A3R in rat colon and their role in the control of colonic motility, under normal conditions and in the presence of experimental colitis. Methods: Colitis was induced by 2,4-dinitrobenzenesulfonic acid (DNBS) in Sprague-Dawley rats. A3R expression and localization were examined by RT-PCR and immunofluorescence. Colonic longitudinal muscle strips (LMS), obtained from normal or inflamed rats, were suspended in organ baths with Krebs solution, containing guanethidine and antagonists of NK 1, 2 and 3 receptors. LMS were connected to isotonic transducers, and the effects of MRS 1523 (MRS, 0.01-1 μM; A3R antagonist), chloro-IB-MECA (CIB, 0.001-10 μM; A3R agonist), dipyridamole (DIP, adenosine reuptake inhibitor) and adenosine deaminase (ADA) were assayed on contractile responses evoked by electrical stimulation (ES; 0.5 ms, 28 V, 10 Hz) or carbachol (1 μM in the presence of tetrodotoxin). Results: RT-PCR revealed the presence of A3R in normal colon and an increased expression of these receptors in inflamed colonic tissues. The immunofluorescence analysis showed a predominant localization of A3R in myenteric plexus and highlighted an increase in their density following the induction of colitis. MRS enhanced ES-induced contractions in normal LMS (+30.5% at 0.1 μM), while it was less effective in inflamed tissues (+11.7% at 0.1 μM). Upon blockade of neuronal nitric oxide synthase by Nω-propyl-L-arginine to promote cholinergic contractions, the enhancing effects of MRS were not affected (+27.3% and +10.2%). Under incubation with DIP plus ADA to abate extracellular adenosine levels, CIB decreased the cholinergic motor responses of normal LMS to ES (EC50 = 27 nM, Emax= -38%). In LMS from inflamed colon, the potency of CIB did not vary (EC50 = 30 nM), while its inhibitory efficacy was increased (Emax= -57.3%). The inhibitory effects of CIB were antagonized by MRS. CIB and MRS did not affect carbachol-induced contractions. Conclusions: Under normal conditions, adenosine modulates the cholinergic motility of colon via activation of A3R at neural level. The inhibitory control of adenosine is impaired in the presence of bowel inflammation, despite an increase in A3R density, which might occur as a compensatory process to a reduced local availability of the endogenous agonist.
T1770 Effect of Red Kidney Beans On Intestinal Permeability May Be Due to Direct Toxicity Wen-Huan S. Ho, Roberto Aguero, Michel Boivin, Yatin Patel, Henry C. Lin Background and aim: Crude red kidney bean (RKB) rich in the lectin phytohemagglutinin has been reported to induce small intestinal bacterial overgrowth (SIBO) (Gastroenterology 1983;84(3):506) and cause gastrointestinal distress. Phytohemagglutinin added to an intestinal cell culture model decreased transepithelial resistance suggesting increased intestinal permeability. Since SIBO is also associated with increased intestinal permeability, it is not known if the effect of RKB on IP is due to SIBO or a direct toxic effect of lectin. In this study, we tested the hypothesis, in a whole animal model, that RKB-induced increased intestinal permeability may depend on an abnormal lactulose breath test (LBT) result suggesting SIBO. Methods: Twenty-three rats were fed a chow containing 26% RKB for two weeks. Seven control rats were fed regular chow. Animals were gavaged with 15 mg lactulose and 10 mg mannitol in 1ml volume after an 8-hour fast. Breath samples were collected for 3h for LBT and urine was collected for 5 hours for measurement of intestinal permeability as assessed by the urinary recovery of lactulose and mannitol. SIBO was detected by an abnormal LBT using criteria applied in an IBS study (Am J Gastroenterol 2003; 98(2):412) Results: Seven out of 23 rats (30%) fed RKB diet had an abnormal LBT suggesting SIBO. The recovery of lactulose was significantly greater in rats with an abnormal LBT when compared to controls that had a normal LBT result (0.16±0.07vs.0.07±0.03; p<0.005). However, there was no difference in the urinary lactulose recovery in RKB chow-fed rats
AGA Abstracts
T1773 Phylochip Analysis of Enteric Microbiota in An Experimental Model of Irritable Bowel Syndrome Reveals Profound Alterations in Community Composition Tyrrell A. Nelson, Alfred M. Spormann, Mohan Shenoy, David L. Hirschberg, Justin Sonnenburg, Pankaj J. Pasricha Background: Although IBS has been associated with altered intestinal microbial communities, research in this area has been limited both by the sensitivity of the microbiological techniques as well as the lack of a suitable experimental model. The Phylochip is a high-density oligonucleotide microarray containing 500,000 probes capable of identifying the 16SrRNA
A-576
by providing doxycycline in the drinking water. Methods: We generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2 (Tet-On) under control of the 12.4 kb murine intestine-specific Villin promoter. To assess inducibility and tissuespecificity of the newly generated Villin-rtTA2 model, mice were bred with the tetO-H2B/ GFP strain and administered with doxycycline. If the Villin-rtTA2 model functions correctly, the nuclear localized green fluorescent H2B/GFP protein will only be expressed in cells where the rtTA2 protein is expressed and activated by doxycycline. Results: Expression of the H2B/GFP fusion protein was observed exclusively in double transgenic mice upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium (see figure). The Villin-rtTA2 was also found to drive transgene expression in the mouse intestine during embryonic development. Furthermore, by administering different concentrations of doxycycline, we show that the Villin-rtTA2 system drives transgene expression in a dosage-dependent fashion. Conclusions: We have successfully generated a novel intestinalspecific doxycycline-inducible mouse model, providing a valuable tool to study the effect of intestinal-specific expression of basically any gene of interest on intestinal physiology and pathology.
regardless of LBT result. Specifically, the entire group had significantly greater lactulose recovery when compared to controls (0.13±0.13 vs.0.07±0.03; p<0.05). Mannitol recovery was not different between groups. Conclusion: Crude RKB diet increased intestinal permeability independent of LBT result suggesting that this deleterious effect on intestinal permeability may be due to a direct toxic effect of lectin. T1771 A Novel Model for Chronic Mucosal Inflammation in IBD and Periodontitis Helieh S. Oz, Jeffrey L. Ebersole Chronic inflammation at mucosal surfaces generally represents an aberrant mucosal immune response to luminal bacteria. These responses generate an array of oxygen radicals, leading to inflammation, tissue destruction, and loss of function as noted in IBD and Periodontitis. The objectives of this study was to examine the time course of mucosal injury following “oral delivery” of DSS and TNBS for an extended period of 18 weeks reflected by oral and GI chronic inflammatory responses. Methods: Mice were administered DSS in diet biweekly; or received TNBS orally 2 times/ week; and controls received sham treatment. Another experimental group received TNBS or sham injection into subgingival maxillas. Results: Animals tolerated oral applications of DSS and TNBS for the duration of study with no mortality or severe clinical symptoms. DSS-treated animals developed diarrhea during DSS administration and returned to normal during the weeks of abstinence. Animals treated with TNBS orally developed no clinical symptoms. Splenic length and weight increased in DSStreated animals in a time depended fashion (18 wk <0.01) and remained normal in TNBStreated mice (vs Co p>0.05). Colons from DSS-treated mice were significantly shortened (<0.001) and colonic weight increased compared to normal controls or TNBS-treated animals. DSS-treated mice developed extensive dilation of stomach wall, ileum, megacolon and abdominal fat deposits. In addition, DSS-treated animals showed a dysregulated hepatic concentrations of endogenous antioxidants (cystine, cysteine, GSH, adenosine, SAMe). Intriguing, the oral inflammation, followed by DSS and TNBS treatment, resulted in a significant increase in alveolar bone resorption in a time dependent manner. Finally, TNBS sub-mucosal delivery caused a severe local inflammation, granuloma formation and extensive alveolar bone resorption. Conclusions: This murine model of chronic mucosal inflammation result in IBD and periodontitis, may mimics some aspects of dysregulated inflammation and disease progression in IBD patients. Supported by NIH-grants NCRR P20RR020145, NCCAMAT1490 (HO).
T1769 Mucosal Cytokine Levels Normalize Despite Continued Clinical and Histopathologic Evidence of Chronic Colitis in a Murine DSS Model of Colitis Steven Polyak, Lisa R. Dixon, Clive Wasserfall, Annette Mach, Cathryn Mah, Chris G. Kevil, John F. Valentine Background: There is significant interest in differentiating between the physiology of acute inflammation and persistent chronic inflammation; and between early versus late disease in inflammatory bowel disease. The purpose of our study was to determine the differences in clinical disease activity, histopathology and mucosal cytokine levels in the acute versus chronic inflammatory stages in a cycled DSS mouse colitis model. Methods: Acute and chronic colitis was induced in Balb/cJ mice by providing 4% DSS in drinking water for 5 days followed by 7 days of regular water for a total of three successive cycles, followed by two additional weeks on regular water, for a total of 50 days. Mice were sacrificed in groups of 3 (plus their controls) on days 5, 12, 24, 36, 43, and 50. Colitis severity was determined by clinical disease activity scores, validated histopathology scores, and serum amyloid A (SAA) levels. Cytokine levels were determined by the Luminex assay from whole length colon homogenates and serum. Results: Cumulative clinical disease activity index score (04) peaked at 2.5 during the second cycle and gradually improved to a score of 1 by day 50 (controls remained at 0 throughout). Cumulative histopathology scores (0-17) were most severe in the distal colon and peaked at 11 after the first cycle, stayed high through the third cycle, and gradually decreased to only 6 by day 50 (controls ranged between 0-1 throughout). SAA levels peaked during the first cycle; and returned to baseline by day 43. Cytokines IL-6, IL-1a, IL-1b, TNFa, IL-15, IP-10, GCSF, KC, MCP and Rantes all peaked after the first cycle of DSS and quickly normalized despite continued cycling of DSS and elevated clinical and histopathology scores. IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-17 and GMCSF did not significantly change or were not detectable at all time points. These cytokines were also analyzed in serum at each time point, but did not reveal significant differences. Conclusions: Clinical and histopathology scores improved but never returned to baseline in our mouse model despite normalization of systemic inflammatory markers and mucosal cytokines. Serum cytokine levels do not distinguish between acute and chronic colitis in Balb/cJ mice. Interestingly, specific mucosal cytokines that were elevated early in the disease course could not be induced again with repeated dosing of DSS. It is likely that the physiology between acute colitis and persistent chronic colitis is different and this model can be used to discover other pathways involved in the development and maintenance of late disease.
T1772 Control of Colonic Neuromuscular Functions By A3 Purinergic Receptors in Normal Colon and in Experimental Colitis Luca Antonioli, Matteo Fornai, Rocchina Colucci, Narcisa Ghisu, Marco Tuccori, Maria Cecilia Giron, Anna Bin, Chiara Zoppellaro, Ignazio Castagliuolo, Rosa Maria Gaion, Mario Del Tacca, Corrado Blandizzi Introduction: Adenosine regulates complex immune/neuromotor interplays in bowel inflammation. The pharmacological activation of adenosine A3 receptors (A3R) results in beneficial effects on experimental colitis. However, the possible involvement of A3R in the regulation of enteric neuromuscular functions is undetermined. This study investigates the expression of A3R in rat colon and their role in the control of colonic motility, under normal conditions and in the presence of experimental colitis. Methods: Colitis was induced by 2,4-dinitrobenzenesulfonic acid (DNBS) in Sprague-Dawley rats. A3R expression and localization were examined by RT-PCR and immunofluorescence. Colonic longitudinal muscle strips (LMS), obtained from normal or inflamed rats, were suspended in organ baths with Krebs solution, containing guanethidine and antagonists of NK 1, 2 and 3 receptors. LMS were connected to isotonic transducers, and the effects of MRS 1523 (MRS, 0.01-1 μM; A3R antagonist), chloro-IB-MECA (CIB, 0.001-10 μM; A3R agonist), dipyridamole (DIP, adenosine reuptake inhibitor) and adenosine deaminase (ADA) were assayed on contractile responses evoked by electrical stimulation (ES; 0.5 ms, 28 V, 10 Hz) or carbachol (1 μM in the presence of tetrodotoxin). Results: RT-PCR revealed the presence of A3R in normal colon and an increased expression of these receptors in inflamed colonic tissues. The immunofluorescence analysis showed a predominant localization of A3R in myenteric plexus and highlighted an increase in their density following the induction of colitis. MRS enhanced ES-induced contractions in normal LMS (+30.5% at 0.1 μM), while it was less effective in inflamed tissues (+11.7% at 0.1 μM). Upon blockade of neuronal nitric oxide synthase by Nω-propyl-L-arginine to promote cholinergic contractions, the enhancing effects of MRS were not affected (+27.3% and +10.2%). Under incubation with DIP plus ADA to abate extracellular adenosine levels, CIB decreased the cholinergic motor responses of normal LMS to ES (EC50 = 27 nM, Emax= -38%). In LMS from inflamed colon, the potency of CIB did not vary (EC50 = 30 nM), while its inhibitory efficacy was increased (Emax= -57.3%). The inhibitory effects of CIB were antagonized by MRS. CIB and MRS did not affect carbachol-induced contractions. Conclusions: Under normal conditions, adenosine modulates the cholinergic motility of colon via activation of A3R at neural level. The inhibitory control of adenosine is impaired in the presence of bowel inflammation, despite an increase in A3R density, which might occur as a compensatory process to a reduced local availability of the endogenous agonist.
T1770 Effect of Red Kidney Beans On Intestinal Permeability May Be Due to Direct Toxicity Wen-Huan S. Ho, Roberto Aguero, Michel Boivin, Yatin Patel, Henry C. Lin Background and aim: Crude red kidney bean (RKB) rich in the lectin phytohemagglutinin has been reported to induce small intestinal bacterial overgrowth (SIBO) (Gastroenterology 1983;84(3):506) and cause gastrointestinal distress. Phytohemagglutinin added to an intestinal cell culture model decreased transepithelial resistance suggesting increased intestinal permeability. Since SIBO is also associated with increased intestinal permeability, it is not known if the effect of RKB on IP is due to SIBO or a direct toxic effect of lectin. In this study, we tested the hypothesis, in a whole animal model, that RKB-induced increased intestinal permeability may depend on an abnormal lactulose breath test (LBT) result suggesting SIBO. Methods: Twenty-three rats were fed a chow containing 26% RKB for two weeks. Seven control rats were fed regular chow. Animals were gavaged with 15 mg lactulose and 10 mg mannitol in 1ml volume after an 8-hour fast. Breath samples were collected for 3h for LBT and urine was collected for 5 hours for measurement of intestinal permeability as assessed by the urinary recovery of lactulose and mannitol. SIBO was detected by an abnormal LBT using criteria applied in an IBS study (Am J Gastroenterol 2003; 98(2):412) Results: Seven out of 23 rats (30%) fed RKB diet had an abnormal LBT suggesting SIBO. The recovery of lactulose was significantly greater in rats with an abnormal LBT when compared to controls that had a normal LBT result (0.16±0.07vs.0.07±0.03; p<0.005). However, there was no difference in the urinary lactulose recovery in RKB chow-fed rats
AGA Abstracts
T1773 Phylochip Analysis of Enteric Microbiota in An Experimental Model of Irritable Bowel Syndrome Reveals Profound Alterations in Community Composition Tyrrell A. Nelson, Alfred M. Spormann, Mohan Shenoy, David L. Hirschberg, Justin Sonnenburg, Pankaj J. Pasricha Background: Although IBS has been associated with altered intestinal microbial communities, research in this area has been limited both by the sensitivity of the microbiological techniques as well as the lack of a suitable experimental model. The Phylochip is a high-density oligonucleotide microarray containing 500,000 probes capable of identifying the 16SrRNA
A-576