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Gut Hormone Profile is Altered in Patients With Chronic Idiopathic Constipation
(2.2 folds) . Conclusions: VIP deficient mice exhibited significantly lower levels of colonic inflammation than wild type mice. Given that both PACAP and VIP are able to bind with the same affinity to the VIP1 and VIP2 receptors and induce signaling, in the absence of VIP, PACAP doesn't equally activate the VIP receptors. Even though VIP has been reported to stimulate anti-inflammatory cytokine release in T lymphocytes, these data suggests that VIP plays an important pro-inflammatory role in the colonic inflamed mucosa. Su1714 Gut Hormone Profile is Altered in Patients With Chronic Idiopathic Constipation Meagan Gray, Shilpa C. Reddy, Keith C. Falkner, Laura A. Buchanan, Jennifer Eversmann, Matthew C. Cave, Gerald W. Dryden, John M. Wo Gut hormones are known to affect GI motility. Glucagon-like peptide 1 (GLP-1), leptin, and peptide YY (PYY) delay gastric emptying, while ghrelin causes acceleration. Furthermore, the gut microbiota has been described in irritable bowel syndrome with an increase of proinflammatory cytokines. Gut hormone and cytokines data for constipation are lacking. Aim: To determine the association of gut hormones and cytokines with physiologic parameters of chronic idiopathic constipation. Methods: A prospective study was performed in patient with chronic constipation by Rome III criteria. Subjects with GI surgery and secondary constipation were excluded. Asymptomatic volunteers served as controls. Fasting serum gut hormones [amylin, ghrelin, leptin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP-1), pancreatic polypeptide (PP), peptide YY (PYY)] and cytokines [tumor necrosis factor α (TNFa), macrophage chemoattractant protein-1 (MCP-1), interleukin-8 and five others] were analyzed by xMAP Luminex technology using a Milliplex analyzer (Millipore, Billerica, MA). Wireless motility capsule (SmartPill®), lactulose breath test and quantitative DNA PCR of nine predominant fecal bacteria were obtained in constipation subjects. Non-parametric Mann-Whitney U tests were utilized with SPSS. Results: 20 constipation subjects (mean age 46 yrs, 85% F, mean BMI 28.2) and 11 normal controls were enrolled. Results of gut peptide and selected cytokines are shown in table. Ghrelin, leptin and PYY levels were significantly higher in constipation subjects compared to controls. Cytokine profiles were similar. Within the 20 constipation subjects, 40% had delayed colonic transit (≥44 hrs in M or ≥59 hrs in F) and 30% had delayed small bowel transit (≥6 hrs) by wireless motility capsule (1 subject had both). Delayed colon transit was not associated with a change in gut hormone or cytokines. Delayed small bowel transit was associated with elevated the leptin (p=0.036) and PYY levels (p=0.007) seen in the constipation subjects compared to controls. Gut hormones and cytokines were not associated with small intestinal bacterial overgrowth or fecal bacteria quantification/subtype. Conclusions: 1) Patients with chronic constipation have altered gut hormone profile. 2) However, these changes may be dependent on presence of delayed small bowel transit and not by colonic inertia. 3) Further studies are needed to determine the relationship between segmental GI transit and potential gut hormone feedback pathways in constipation.
Su1712 Localization of Transient Receptor Potential Melastatin Type-8 (TRPM8) in Normal Mouse Large Intestine and Its Up-Regulation in Inflammatory Bowel Disease Model Takuji Hosoya, Kenjiro Matsumoto, Kimihito Tashima, Toshihiko Murayama, Syunji Horie BACKGROUND & AIMS: Transient receptor potential melastatin type-8 (TRPM8) is a nonselective cation channels expressed in primary sensory neurons, and is activated by innocuous cold (or cool, < 27 degrees C), icilin, and menthol. In the recent year, it was reported that the mRNA for TRPM8 channels was expressed in the rat gastric fundus. Thus, we speculated that TRPM8 channels may be involved in gut function. However, the distribution of TRPM8 channels in large intestine under physiological and pathological conditions has been hardly studied. In the present study, we investigated the localization of TRPM8 channels in large intestine under physiological state and the alternation of TRPM8-epressing neurons using experimental colitis model mice. METHODS: TRPM8-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification in C57BL/6J mice distal, transverse, and proximal colon. Colitis was induced by 3 % dextrin sulfate sodium as drinking water. Calretinin, c-Kit, calcitonin gene related peptide (CGRP) and substance P (SP) were detected by normal indirect staining with the corresponding specific antibodies. RESULTS: In immunohistochemical study, TRPM8-positive area in the distal colon was the largest among the distal, transverse, and proximal colon. TRPM8immunoreactive neurons were found in the mucosa, submucosal and muscle layers. Nonneuronal TRPM8-immunoreactivities were found in muscle layer. Next, double labeling studies on TRPM8 were carried out using the neuronal marker calretinin, interstetial cells of Cajal (ICC) marker c-Kit and neuronal peptides such as CGRP and SP. Calretininnimmunoreactivities were found in the mucosa, submucosal, and muscle layer. TRPM8immuoreactive nerve fibers colocalized with calretinin in axons located in mucosa. CGRP or SP-immunoreactivities were found in the mucosa, submucosal, and muscle layers. TRPM8immunoreactive nerve fibers partly colocalized with CGRP and SP axons located in mucosa. c-Kit-positive cells were located in the deep muscular plexus, circular muscle, myenteric plexus and longitudinal muscle. Double-labeling with c-Kit revealed that TRPM8 neurons existed in the adjacent of ICC, but did not colocalize in each layer. Finally, by using colitis model mice, we compared TRPM8-immunoreactivities under physiological state with under inflammatory state. The number of TRPM8-positive neurons was increased in mucosa of IBD model mice, but not in muscle layer. CONCLUSION: These results indicate that TRPM8 channels are widely localized and expressed on both peptidergic and non-peptidergic axon in mouse large intestine. Increased expression of TRPM8 channels may be involved in pathology of colitis. Su1713 VIP Deficiency Results in a Protective Effect in a Murine Model of Dextran Sulfate Sodium (DSS) Induced Colitis John P. Vu, Mulugeta Million, Muriel H. Larauche, James Waschek, Yvette Tache, Joseph R. Pisegna, Patrizia M. Germano
*In pg/ml; Data as medians (25th-75th percentile) Su1715
Introduction: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a poorly understood pathogenesis. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and (Vasoactive Intestinal Peptide) VIP potently regulate the immune system through the activation of PAC1, VIP1 and VIP2 receptors (Cell Mol Biol. 2003 Mar;49(2):127-42. Review). Using our Dextran Sulfate Sodium (DSS) induced colitis model, we have previously shown that PAC1-/- deficient mice, totally lacking the high affinity receptor for PACAP, have higher levels of colonic mucosa inflammation and mortality than wild type (WT) control mice with the same genetic background. While only PACAP acts through PAC1, both PACAP and VIP bind with the same affinity to VIP1 and VIP2 receptors activating the same cAMP pathway. Aim: In our knock out model of VIP mice to ascertain the role that VIP plays in modulating colonic inflammation and cytokine release following DSS-colitis induction. Methods: Colitis was induced in 6 adult (WT), and 6 adult VIP -/- deficient, C57BL/6 mice with 2.5% DSS given orally for 5 days. Measurements of water intake, body weight, observations of mice activity and stool appearance were made at baseline and daily thereafter for eleven consecutive days. On the 11th day the animals were sacrificed and the intact colon from each mouse was dissected, examined macroscopically, the weight and length were measured and their ratio was scored in a blinded fashion. MPO (Myeloperoxidase) assay was performed on samples from the proximal and distal colon for each mouse. qPCR was utilized to measure the levels of expression of IL-6, and Actin in samples of the entire colon for each animal. Results: VIP -/- mice showed a significant lower loss of body weight percentage ( 1.7 ± 2), p<0.03, compared to WT mice (11.5±7), following treatment with DSS. The colonic length to weight ratio was very significantly higher in the VIP-/- group (36.4± 1.4), p< 0.0001, indicating a lower level of inflammation, compared to the WT group (28.3±1.8). qPCR data analyses revealed in the WT group IL-6 was increased
Regionally-Dependent Responses to Both Nerve Stimulation and to Motilin in Human Isolated Esophagus, Stomach, Small and Large Intestine John Broad, Samrat Mukerjee, George Boundouki, Frances Hughes, Kesava R. Mannur, Charles H. Knowles, George E. Dukes, Gareth J. Sanger BACKGROUND Translation from cell-based assays requires human native tissue assays. We characterised motor nerve responses to electrical field stimulation (EFS) in human distal esophagus, gastric fundus and antrum, duodenum, terminal ileum and ascending and descending colon, before surveying the actions of motilin, previously described in the stomach (Broad et al 2010, Neurogastroenterol Motil 22, 84). METHODS Tissues were collected at surgery for cancer or obesity, following informed consent. After removing the mucosa, strips were cut parallel to the circular muscle and suspended between 2 platinum ring electrodes in tissue baths (Kreb's; 5% CO2 in O2; 37°C; 1g tension) for isometric recording. EFS (1-20Hz; 0.5ms pulse width, 50V) was applied for 10s every 1 min. RESULTS Responses to EFS varied but were abolished by TTX 1μM and unless stated, contractions were attenuated by atropine 1μM and relaxations abolished by the nitric oxide synthase inhibitor L-NAME 300μM. In esophagus, contractions were evoked during EFS at all frequencies (29/29 strips, n=9 patients) and again on termination of EFS (aftercontractions: AC; 26/29 strips at 5Hz). In fundus and antrum contractions were only observed during EFS (95 strips at 5Hz, n=22 and 150 strips, n=21). For each of these tissues contraction amplitudes were frequency-dependent but in the intestine responses to EFS were complex. In duodenum and terminal ileum relaxations usually occurred at 1-5Hz (eg. 5/5 tissues at 5Hz, n=2
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AGA Abstracts
AGA Abstracts
signaling and TRPV1 channels were performed using rectum. RESULTS: In DSS induced colitis model mice, disease activity index, tissue inflammation and amount of 5-HT was gradually increased during 7 days of DSS treatment. The visceral hyperalgesia to mechanical distension was observed on day 7 but not on day 4. The number of 5-HT3 receptorexpressing nerve fibers is significantly increased in mucosa on day 7. On the other hand, number of the 5-HT4 receptor-expressing nerve fibers is significantly decreased on day 7. No significant alteration in the number of 5-HT3 and 5-HT4 receptors was detected on day 4. Progress of the inflammation led to down regulation of SERT immunoreactivities with concomitant increases in 5-HT- and TPH-1-positive cell numbers. 5-HT- and TPH-1-positive cells were widely distributed in whole mucosa on day 0 and 4. On day 7, these cells were mainly detected in upper part of mucosa. A significant 2-fold increase in number of TRPV1expressing nerve fibers was found in mucosa on day 7. Non-neuronal TRPV1-immunopositive cells were observed on day 7. There is no clear alteration of the immunoreactivities of the 5-HT signaling and the TRPV1 channels in muscle layers of rectum in colitis model mice. CONCLUSION: These results suggest that increased 5-HT, 5-HT3 receptors, TRPV1 channels and decreased 5-HT4 receptors are associated with the pathological conditions of the inflammatory bowel disease.
Su1712 Localization of Transient Receptor Potential Melastatin Type-8 (TRPM8) in Normal Mouse Large Intestine and Its Up-Regulation in Inflammatory Bowel Disease Model Takuji Hosoya, Kenjiro Matsumoto, Kimihito Tashima, Toshihiko Murayama, Syunji Horie BACKGROUND & AIMS: Transient receptor potential melastatin type-8 (TRPM8) is a nonselective cation channels expressed in primary sensory neurons, and is activated by innocuous cold (or cool, < 27 degrees C), icilin, and menthol. In the recent year, it was reported that the mRNA for TRPM8 channels was expressed in the rat gastric fundus. Thus, we speculated that TRPM8 channels may be involved in gut function. However, the distribution of TRPM8 channels in large intestine under physiological and pathological conditions has been hardly studied. In the present study, we investigated the localization of TRPM8 channels in large intestine under physiological state and the alternation of TRPM8-epressing neurons using experimental colitis model mice. METHODS: TRPM8-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification in C57BL/6J mice distal, transverse, and proximal colon. Colitis was induced by 3 % dextrin sulfate sodium as drinking water. Calretinin, c-Kit, calcitonin gene related peptide (CGRP) and substance P (SP) were detected by normal indirect staining with the corresponding specific antibodies. RESULTS: In immunohistochemical study, TRPM8-positive area in the distal colon was the largest among the distal, transverse, and proximal colon. TRPM8immunoreactive neurons were found in the mucosa, submucosal and muscle layers. Nonneuronal TRPM8-immunoreactivities were found in muscle layer. Next, double labeling studies on TRPM8 were carried out using the neuronal marker calretinin, interstetial cells of Cajal (ICC) marker c-Kit and neuronal peptides such as CGRP and SP. Calretininnimmunoreactivities were found in the mucosa, submucosal, and muscle layer. TRPM8immuoreactive nerve fibers colocalized with calretinin in axons located in mucosa. CGRP or SP-immunoreactivities were found in the mucosa, submucosal, and muscle layers. TRPM8immunoreactive nerve fibers partly colocalized with CGRP and SP axons located in mucosa. c-Kit-positive cells were located in the deep muscular plexus, circular muscle, myenteric plexus and longitudinal muscle. Double-labeling with c-Kit revealed that TRPM8 neurons existed in the adjacent of ICC, but did not colocalize in each layer. Finally, by using colitis model mice, we compared TRPM8-immunoreactivities under physiological state with under inflammatory state. The number of TRPM8-positive neurons was increased in mucosa of IBD model mice, but not in muscle layer. CONCLUSION: These results indicate that TRPM8 channels are widely localized and expressed on both peptidergic and non-peptidergic axon in mouse large intestine. Increased expression of TRPM8 channels may be involved in pathology of colitis. Su1713 VIP Deficiency Results in a Protective Effect in a Murine Model of Dextran Sulfate Sodium (DSS) Induced Colitis John P. Vu, Mulugeta Million, Muriel H. Larauche, James Waschek, Yvette Tache, Joseph R. Pisegna, Patrizia M. Germano
*In pg/ml; Data as medians (25th-75th percentile) Su1715
Introduction: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract with a poorly understood pathogenesis. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and (Vasoactive Intestinal Peptide) VIP potently regulate the immune system through the activation of PAC1, VIP1 and VIP2 receptors (Cell Mol Biol. 2003 Mar;49(2):127-42. Review). Using our Dextran Sulfate Sodium (DSS) induced colitis model, we have previously shown that PAC1-/- deficient mice, totally lacking the high affinity receptor for PACAP, have higher levels of colonic mucosa inflammation and mortality than wild type (WT) control mice with the same genetic background. While only PACAP acts through PAC1, both PACAP and VIP bind with the same affinity to VIP1 and VIP2 receptors activating the same cAMP pathway. Aim: In our knock out model of VIP mice to ascertain the role that VIP plays in modulating colonic inflammation and cytokine release following DSS-colitis induction. Methods: Colitis was induced in 6 adult (WT), and 6 adult VIP -/- deficient, C57BL/6 mice with 2.5% DSS given orally for 5 days. Measurements of water intake, body weight, observations of mice activity and stool appearance were made at baseline and daily thereafter for eleven consecutive days. On the 11th day the animals were sacrificed and the intact colon from each mouse was dissected, examined macroscopically, the weight and length were measured and their ratio was scored in a blinded fashion. MPO (Myeloperoxidase) assay was performed on samples from the proximal and distal colon for each mouse. qPCR was utilized to measure the levels of expression of IL-6, and Actin in samples of the entire colon for each animal. Results: VIP -/- mice showed a significant lower loss of body weight percentage ( 1.7 ± 2), p<0.03, compared to WT mice (11.5±7), following treatment with DSS. The colonic length to weight ratio was very significantly higher in the VIP-/- group (36.4± 1.4), p< 0.0001, indicating a lower level of inflammation, compared to the WT group (28.3±1.8). qPCR data analyses revealed in the WT group IL-6 was increased
Regionally-Dependent Responses to Both Nerve Stimulation and to Motilin in Human Isolated Esophagus, Stomach, Small and Large Intestine John Broad, Samrat Mukerjee, George Boundouki, Frances Hughes, Kesava R. Mannur, Charles H. Knowles, George E. Dukes, Gareth J. Sanger BACKGROUND Translation from cell-based assays requires human native tissue assays. We characterised motor nerve responses to electrical field stimulation (EFS) in human distal esophagus, gastric fundus and antrum, duodenum, terminal ileum and ascending and descending colon, before surveying the actions of motilin, previously described in the stomach (Broad et al 2010, Neurogastroenterol Motil 22, 84). METHODS Tissues were collected at surgery for cancer or obesity, following informed consent. After removing the mucosa, strips were cut parallel to the circular muscle and suspended between 2 platinum ring electrodes in tissue baths (Kreb's; 5% CO2 in O2; 37°C; 1g tension) for isometric recording. EFS (1-20Hz; 0.5ms pulse width, 50V) was applied for 10s every 1 min. RESULTS Responses to EFS varied but were abolished by TTX 1μM and unless stated, contractions were attenuated by atropine 1μM and relaxations abolished by the nitric oxide synthase inhibitor L-NAME 300μM. In esophagus, contractions were evoked during EFS at all frequencies (29/29 strips, n=9 patients) and again on termination of EFS (aftercontractions: AC; 26/29 strips at 5Hz). In fundus and antrum contractions were only observed during EFS (95 strips at 5Hz, n=22 and 150 strips, n=21). For each of these tissues contraction amplitudes were frequency-dependent but in the intestine responses to EFS were complex. In duodenum and terminal ileum relaxations usually occurred at 1-5Hz (eg. 5/5 tissues at 5Hz, n=2
S-479
AGA Abstracts
AGA Abstracts
signaling and TRPV1 channels were performed using rectum. RESULTS: In DSS induced colitis model mice, disease activity index, tissue inflammation and amount of 5-HT was gradually increased during 7 days of DSS treatment. The visceral hyperalgesia to mechanical distension was observed on day 7 but not on day 4. The number of 5-HT3 receptorexpressing nerve fibers is significantly increased in mucosa on day 7. On the other hand, number of the 5-HT4 receptor-expressing nerve fibers is significantly decreased on day 7. No significant alteration in the number of 5-HT3 and 5-HT4 receptors was detected on day 4. Progress of the inflammation led to down regulation of SERT immunoreactivities with concomitant increases in 5-HT- and TPH-1-positive cell numbers. 5-HT- and TPH-1-positive cells were widely distributed in whole mucosa on day 0 and 4. On day 7, these cells were mainly detected in upper part of mucosa. A significant 2-fold increase in number of TRPV1expressing nerve fibers was found in mucosa on day 7. Non-neuronal TRPV1-immunopositive cells were observed on day 7. There is no clear alteration of the immunoreactivities of the 5-HT signaling and the TRPV1 channels in muscle layers of rectum in colitis model mice. CONCLUSION: These results suggest that increased 5-HT, 5-HT3 receptors, TRPV1 channels and decreased 5-HT4 receptors are associated with the pathological conditions of the inflammatory bowel disease.