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Su1942 Mucosal Immune System and Its Interaction With Microbiota in Patients With Diverticular Disease
(r=-0.51; P=0.02). In patients with SUDD, there was a positive correlation between macrophage count in the diverticular region and fecal Bacteroides/Prevotella (r=0.79; P=0.03). Conclusions: Mucosal immune activation, characterized mainly by macrophages, is a common characteristic of patients with diverticula. In SUDD the fecal microbiota was significantly depleted of Clostridium cluster IX, Fusobacterium and Lactobacillaceae. Akkermansia and Clostridium class IV abundance was inversely correlated with macrophages in the diverticular region. These data suggest that changes in the intestinal microbiota may play a role in lowgrade intestinal inflammation and symptom generation in DD.
Su1943 Alterations in Intestinal Motility and Gut Microbiota Composition by Maternal Separation Stress in Rats Takaaki Murakami, Kazuhiro Kamada, Katsura Mizushima, Kentaro Suzuki, Yasuki Higashimura, Kazuhiro Katada, Kazuhiko Uchiyama, Tomohisa Takagi, Osamu Handa, Yuji Naito, Yoshito Itoh [Aims] Irritable bowel syndrome (IBS) is a disorder of chronic abdominal pain and abnormal bowel movement. It is not fatal but impairs patients' quality of life. The etiology of IBS is thought to be a complicated arrangement of visceral hypersensitivity, disorder of bowel movement and psychological or social stress. Recently, there have been some reports that the change of microbiota affects the pathophysiology of IBS. In this study, we investigated disorder of bowel movement and the influences of the microbiota in rat maternal separation (MS) stress models. [Methods] The MS were produced by separating neonate Wistar male rats from their mother three hours daily from postnatal day (PD) 2 to PD14. On the other hand, control group rats were not separated from their mother. Each rat was assessed anxietylike behaviors by elevated plus maze test and open field test. Animals at 7-8 weeks of age were intravenously administered 10µg/body of corticosterone releasing factor (CRF) as considered to be appropriate acute stress or saline and were sacrificed three hours after the administration. We compared the number of feces, pathological intestinal inflammation, neural peptides in the colon and the dorsal root ganglions (DRG), and the microbiota in the cecal contents. [Results] Time spent and times enter in open arms (elevated plus maze) and bright area (open field test) were decreased in MS group. The number of feces for 3 hours after CRF injection was significantly increased in MS group to 3.25±0.75 from 0.75±0.47, but not in control group. There were no differences between both groups with or without CRF administration in body weight, pathological inflammation of the colon, neural peptides such as substance P and calcitonin gene related peptide. According to the T-RFLP analyses of microbiota in MS group and control group, the ratio of Bacteroides was (9.4% vs. 4.6%), and Clostridium subcluster XIVa was (23.3% vs. 16.8%) respectively. On the other hand, a ratio of Lactobacillales decreased by maternal separation (26.1% vs. 37.6%). Moreover, CRF administration did not substantially affect the composition of the microbiota in cecal contents both in MS group and control group. [Conclusion] The anxiety-related behavior was increased in MS group animals. MS stress enhanced response of CRF in motility of bowel movement and had an effect on the microbiota composition when it becomes age of adult.
Su1941 Involvement of Gut Microbiota in the Association Between Macrophage and Gastrointestinal Motility Mo Yang, Hirokazu Fukui, Hirotsugu Eda, Mio Odani, Takahisa Yamasaki, Takashi Kondo, Hisatomo Ikehara, Yoshio Ohda, Toshihiko Tomita, Tadayuki Oshima, Jiro Watari, Hiroto Miwa Background: Microbiota in the gut is known to play a pivotal role in host physiology by interacting with immune and neuroendocrine systems in the gastrointestinal tissues. Serotonin (5-hydroxytryptamine; 5-HT) acts as not only a neurotransmitter but also a mediator for immune system in the gastrointestinal tissues. In the present study, we aimed to examine how gut microbiota is involved in gastrointestinal motility, focusing the association between 5-HT expression and macrophage behavior in the gastrointestinal tissues. Methods: Germfree (GF) mice were orally administered a fecal bacterial suspension prepared from specific pathogen-free mice, and then after fecal transplantation (FT) gastrointestinal tissues were obtained from the GF mice at various time points. The expression of mannose receptor (MR; marker for M2 macrophage) and 5-HT was examined by immunohistochemistry. The gastrointestinal transit time (GITT) was measured by administration of carmine red solution. Results: MR-positive cells were observed not only in the lamina propria but also around the myenteric neural cells in the muscle layer throughout the gastrointestinal wall. Both in the lamina propria and muscle layer, the number of MR-positive cells was significantly increased in GF mice with FT than in GF mice without gut microbiota reconstitution. 5HT-positive cells were significantly increased throughout the gastrointestinal wall in GF mice with FT than in GF mice without gut microbiota reconstitution. The GITT was significantly shorter in GF mice with FT than in control GF mice without FT. The GITT tended to be correlated with the number of both MR- and 5-HT-positive cells throughout the gastrointestinal wall. Conclusions: Gut microbiota may accelerate gastrointestinal motility while increasing M2 macrophage and 5-HT-positve endocrine cells throughout the gastrointestinal tract.
Su1944 The Role of Toll-Like Receptor 4 and Mast Cell Function in the Ameliorating Effect of Electroacupuncture on Visceral Hypersencitivity in Rats Juan Yang, Haitao Shi, Shanshan Zhu, Lijuan Mao, Xiaomeng Cui, Yumei Luo, Fei Dai Background: Toll-like receptor 4 (TLR4) is a critical pattern recognition molecule of the innate immune system and associated with some gastrointestinal chronic inflammatory disorders. The mechanism of electroacupuncture (EA) attenuating visceral hypersensitivity remains unclear. We hypothesized that TLR4 contributes to visceral hypersensitivity induced by neonatal colonic irritation and EA improves visceral hypersensitivity by down-regulating the expression of TLR4 in the intestinal wall and mast cell. Aims: This study was to investigate the involvement of TLR4 in the ameliorating effect of EA on visceral hypersensitivity. Methods: Forty Sprague-Dawley rats were randomly divided into five groups: control group, colonic sensitized group (injected with acetic acid during the neonatal period), colonic sensitized + EA group (EA group), colonic sensitized + sham-EA group and colonic sensitized +mast cell stabilizers (mast cell stabilizers treatment group). Visceral sensitivity during colorectal distension (CRD) was assessed by the measurement of abdominal electromyogram (EMG) with the pressures of 20, 40, 60 and 80 mmHg. TLR4 mRNA expression was assessed by real time RT-PCR and TLR4 protein expression in distal colon was confirmed by immunofluorescence. The number and morphology of mast cell in colonic tissue were observed by special staining. In addition, detecting tryptase content by immunohistochemistry indirectly reflected mast cell degranulation. Concentrations of inflammatory cytokine productions and endotoxin in serum were investigated by multiplex technology. Results: (1) EA significantly reduced EMG activity to CRD at different distention pressures (p<0.01, respectively). (2) Compared with baseline, EMG activity to CRD at different distention was significantly decreased in mast cell stabilizers treatment group (p<0.01, respectively). (3) TLR4 protein expressions were significantly increased in the mast cell of colonic tissue in colonic sensitized rats (vs. control, p<0.001). (4) Compared with colonic sensitized group, TLR4 mRNA expression in the colonic tissue, the number of mast cells degranulation, and serum concentrations of TNF-a, IL-1b, IL-8 and endotoxin were significantly decreased in EA group and mast cell stabilizers treatment group. Conclusions: EA ameliorated visceral hypersensitivity in colonic sensitized model may be via decreasing TLR4 expressions localized in the mast cells, resulting in decreased release of the mucosal proinflammator cytokines Key-words: Visceral hypersensitivity, Electroacupuncture, Toll-like receptor 4, Mast cell
Su1942 Mucosal Immune System and Its Interaction With Microbiota in Patients With Diverticular Disease Cesare Cremon, M. Raffaella Barbaro, Eleonora Scaioli, Giovanni Marasco, Antonio Colecchia, Davide Festi, Elena Biagi, Patrizia Brigidi, Vincenzo Stanghellini, Giovanni Barbara Background & Aims: Diverticular disease (DD) is a multifactorial disorder involving environmental, sensory-motor and structural factors. Although changes in gut microbiota with subsequent mucosal immune activation may be hypothesized in the pathophysiology of DD, these mechanisms are unproven. The aim of the present study was to characterize mucosal immune system, fecal and mucosal microbiota, and their correlations in asymptomatic controls (AC), subjects with colonic diverticulosis and symptomatic uncomplicated diverticular disease (SUDD). Methods: A total of 38 subjects were enrolled, including 14 AC, 16 with diverticulosis, and 8 with SUDD. In subjects with diverticula, mucosal biopsies were obtained close to diverticula (diverticular region) and at least 20 cm from the last observed diverticula (distant site). In AC, biopsies were performed at sigmoid and at descending colon. Mucosal immune system was characterized with quantitative immunohistochemistry. Total bacterial DNA from fecal and colonic biopsy samples was extracted using DNeasy Blood & Tissue Mini Kit; bacterial 16S rRNA was amplified using universal primers T7promBact-27-F and Uni-1492-R. The microbiota of all subjects was analyzed by HTF-Microbi.Array, a LDR-microarray-based tool able to reliably approximate the composition of the intestinal microbiota in terms of relative abundance of the most common groups of bacteria. Results: Compared to AC, subjects with diverticula showed a significant increase in macrophage count in diverticular region (P<0.001) and distant site (P<0.05). Compared to diverticulosis, patients with SUDD showed a significantly decreased abundance of fecal members of Clostridium cluster IX (16.0±1.8% vs. 9.9±1.5%; P=0.03), Fusobacterium (1.2±0.2% vs. 0.7±0.2%; P=0.05) and Lactobacillaceae (6.3±1.8% vs. 2.8±0.6%; P=0.05). Compared with AC, biopsies from both distant and diverticular sites in subjects with diverticula showed lower abundance of Enterobacteriaceae (P=0.04) and Bacteroides/Prevotella (P=0.06). In subjects with diverticula, we found a negative correlation between macrophage count in the diverticular region and mucosal Clostridium class IV (r=-0.48; P=0.03) and Akkermansia
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samples, colonic inflammation by qPCR for IL-1 b, and cellular tolerance by modulation of cell excitability of DRG neurons by whole-cell voltage clamp. Depletion of gut bacteria was achieved by oral gavage of an antibiotic cocktail (streptomycin, neomycin and metronidazole (all at 100 mg/kg), and vancomycin (50 mg/kg). Ampicillin (1g/l) was provided in drinking water ad libitum for 10 days. On day 5, a 75 mg morphine or placebo pellet was surgically implanted subcutaneously on the dorsum. RESULTS: The gut permeability was significantly enhanced in chronic morphine treated mice compared to placebo or acute morphine treated mice. The fold increase in CFU was 1.6x in mesenteric lymph node, 1.3x in Liver, 1.1x in Spleen and 1.9x in colon of chronic morphine treated mice compared to placebo. Chronic morphine also resulted in a shift in the bacterial load with a reduction in Bacteroidetes and Firmicutes and increase in enterobacteriales. The expression of Il-1b mRNA in distal colon increased by 2 fold in morphine pelleted mice. Antibiotic (ABX) treatment significantly reduced bacterial load (90%) in both placebo and morphine pelleted mice. The caecum of all ABX treated mice was significantly enlarged and spleen weight reduced by 70%, an effect similar to those reported for "germ-free" mice. The latency in the tail-flick assay was increased to maximum (10sec) in response to morphine challenge (10 mg/kg) in placebo pelleted mice with or without ABX treatment. The latency for tail-flick also increased in morphine pelleted mice with ABX while tolerance was observed in saline treated morphine pelleted mice. Morphine reduced the excitability of small diameter L6 DRG neurons of morphine pelleted ABX treated mice but not in saline treated DRG neurons suggesting that tolerance did not develop following ABX treatment. CONCLUSION: These data demonstrate that gastrointestinal microbiota is an important component in the development of analgesic tolerance to opioids and the bacterial translocation associated with chronic morphine may also explain the enhanced infections in Crohn's patients treated with narcotics
Su1943 Alterations in Intestinal Motility and Gut Microbiota Composition by Maternal Separation Stress in Rats Takaaki Murakami, Kazuhiro Kamada, Katsura Mizushima, Kentaro Suzuki, Yasuki Higashimura, Kazuhiro Katada, Kazuhiko Uchiyama, Tomohisa Takagi, Osamu Handa, Yuji Naito, Yoshito Itoh [Aims] Irritable bowel syndrome (IBS) is a disorder of chronic abdominal pain and abnormal bowel movement. It is not fatal but impairs patients' quality of life. The etiology of IBS is thought to be a complicated arrangement of visceral hypersensitivity, disorder of bowel movement and psychological or social stress. Recently, there have been some reports that the change of microbiota affects the pathophysiology of IBS. In this study, we investigated disorder of bowel movement and the influences of the microbiota in rat maternal separation (MS) stress models. [Methods] The MS were produced by separating neonate Wistar male rats from their mother three hours daily from postnatal day (PD) 2 to PD14. On the other hand, control group rats were not separated from their mother. Each rat was assessed anxietylike behaviors by elevated plus maze test and open field test. Animals at 7-8 weeks of age were intravenously administered 10µg/body of corticosterone releasing factor (CRF) as considered to be appropriate acute stress or saline and were sacrificed three hours after the administration. We compared the number of feces, pathological intestinal inflammation, neural peptides in the colon and the dorsal root ganglions (DRG), and the microbiota in the cecal contents. [Results] Time spent and times enter in open arms (elevated plus maze) and bright area (open field test) were decreased in MS group. The number of feces for 3 hours after CRF injection was significantly increased in MS group to 3.25±0.75 from 0.75±0.47, but not in control group. There were no differences between both groups with or without CRF administration in body weight, pathological inflammation of the colon, neural peptides such as substance P and calcitonin gene related peptide. According to the T-RFLP analyses of microbiota in MS group and control group, the ratio of Bacteroides was (9.4% vs. 4.6%), and Clostridium subcluster XIVa was (23.3% vs. 16.8%) respectively. On the other hand, a ratio of Lactobacillales decreased by maternal separation (26.1% vs. 37.6%). Moreover, CRF administration did not substantially affect the composition of the microbiota in cecal contents both in MS group and control group. [Conclusion] The anxiety-related behavior was increased in MS group animals. MS stress enhanced response of CRF in motility of bowel movement and had an effect on the microbiota composition when it becomes age of adult.
Su1941 Involvement of Gut Microbiota in the Association Between Macrophage and Gastrointestinal Motility Mo Yang, Hirokazu Fukui, Hirotsugu Eda, Mio Odani, Takahisa Yamasaki, Takashi Kondo, Hisatomo Ikehara, Yoshio Ohda, Toshihiko Tomita, Tadayuki Oshima, Jiro Watari, Hiroto Miwa Background: Microbiota in the gut is known to play a pivotal role in host physiology by interacting with immune and neuroendocrine systems in the gastrointestinal tissues. Serotonin (5-hydroxytryptamine; 5-HT) acts as not only a neurotransmitter but also a mediator for immune system in the gastrointestinal tissues. In the present study, we aimed to examine how gut microbiota is involved in gastrointestinal motility, focusing the association between 5-HT expression and macrophage behavior in the gastrointestinal tissues. Methods: Germfree (GF) mice were orally administered a fecal bacterial suspension prepared from specific pathogen-free mice, and then after fecal transplantation (FT) gastrointestinal tissues were obtained from the GF mice at various time points. The expression of mannose receptor (MR; marker for M2 macrophage) and 5-HT was examined by immunohistochemistry. The gastrointestinal transit time (GITT) was measured by administration of carmine red solution. Results: MR-positive cells were observed not only in the lamina propria but also around the myenteric neural cells in the muscle layer throughout the gastrointestinal wall. Both in the lamina propria and muscle layer, the number of MR-positive cells was significantly increased in GF mice with FT than in GF mice without gut microbiota reconstitution. 5HT-positive cells were significantly increased throughout the gastrointestinal wall in GF mice with FT than in GF mice without gut microbiota reconstitution. The GITT was significantly shorter in GF mice with FT than in control GF mice without FT. The GITT tended to be correlated with the number of both MR- and 5-HT-positive cells throughout the gastrointestinal wall. Conclusions: Gut microbiota may accelerate gastrointestinal motility while increasing M2 macrophage and 5-HT-positve endocrine cells throughout the gastrointestinal tract.
Su1944 The Role of Toll-Like Receptor 4 and Mast Cell Function in the Ameliorating Effect of Electroacupuncture on Visceral Hypersencitivity in Rats Juan Yang, Haitao Shi, Shanshan Zhu, Lijuan Mao, Xiaomeng Cui, Yumei Luo, Fei Dai Background: Toll-like receptor 4 (TLR4) is a critical pattern recognition molecule of the innate immune system and associated with some gastrointestinal chronic inflammatory disorders. The mechanism of electroacupuncture (EA) attenuating visceral hypersensitivity remains unclear. We hypothesized that TLR4 contributes to visceral hypersensitivity induced by neonatal colonic irritation and EA improves visceral hypersensitivity by down-regulating the expression of TLR4 in the intestinal wall and mast cell. Aims: This study was to investigate the involvement of TLR4 in the ameliorating effect of EA on visceral hypersensitivity. Methods: Forty Sprague-Dawley rats were randomly divided into five groups: control group, colonic sensitized group (injected with acetic acid during the neonatal period), colonic sensitized + EA group (EA group), colonic sensitized + sham-EA group and colonic sensitized +mast cell stabilizers (mast cell stabilizers treatment group). Visceral sensitivity during colorectal distension (CRD) was assessed by the measurement of abdominal electromyogram (EMG) with the pressures of 20, 40, 60 and 80 mmHg. TLR4 mRNA expression was assessed by real time RT-PCR and TLR4 protein expression in distal colon was confirmed by immunofluorescence. The number and morphology of mast cell in colonic tissue were observed by special staining. In addition, detecting tryptase content by immunohistochemistry indirectly reflected mast cell degranulation. Concentrations of inflammatory cytokine productions and endotoxin in serum were investigated by multiplex technology. Results: (1) EA significantly reduced EMG activity to CRD at different distention pressures (p<0.01, respectively). (2) Compared with baseline, EMG activity to CRD at different distention was significantly decreased in mast cell stabilizers treatment group (p<0.01, respectively). (3) TLR4 protein expressions were significantly increased in the mast cell of colonic tissue in colonic sensitized rats (vs. control, p<0.001). (4) Compared with colonic sensitized group, TLR4 mRNA expression in the colonic tissue, the number of mast cells degranulation, and serum concentrations of TNF-a, IL-1b, IL-8 and endotoxin were significantly decreased in EA group and mast cell stabilizers treatment group. Conclusions: EA ameliorated visceral hypersensitivity in colonic sensitized model may be via decreasing TLR4 expressions localized in the mast cells, resulting in decreased release of the mucosal proinflammator cytokines Key-words: Visceral hypersensitivity, Electroacupuncture, Toll-like receptor 4, Mast cell
Su1942 Mucosal Immune System and Its Interaction With Microbiota in Patients With Diverticular Disease Cesare Cremon, M. Raffaella Barbaro, Eleonora Scaioli, Giovanni Marasco, Antonio Colecchia, Davide Festi, Elena Biagi, Patrizia Brigidi, Vincenzo Stanghellini, Giovanni Barbara Background & Aims: Diverticular disease (DD) is a multifactorial disorder involving environmental, sensory-motor and structural factors. Although changes in gut microbiota with subsequent mucosal immune activation may be hypothesized in the pathophysiology of DD, these mechanisms are unproven. The aim of the present study was to characterize mucosal immune system, fecal and mucosal microbiota, and their correlations in asymptomatic controls (AC), subjects with colonic diverticulosis and symptomatic uncomplicated diverticular disease (SUDD). Methods: A total of 38 subjects were enrolled, including 14 AC, 16 with diverticulosis, and 8 with SUDD. In subjects with diverticula, mucosal biopsies were obtained close to diverticula (diverticular region) and at least 20 cm from the last observed diverticula (distant site). In AC, biopsies were performed at sigmoid and at descending colon. Mucosal immune system was characterized with quantitative immunohistochemistry. Total bacterial DNA from fecal and colonic biopsy samples was extracted using DNeasy Blood & Tissue Mini Kit; bacterial 16S rRNA was amplified using universal primers T7promBact-27-F and Uni-1492-R. The microbiota of all subjects was analyzed by HTF-Microbi.Array, a LDR-microarray-based tool able to reliably approximate the composition of the intestinal microbiota in terms of relative abundance of the most common groups of bacteria. Results: Compared to AC, subjects with diverticula showed a significant increase in macrophage count in diverticular region (P<0.001) and distant site (P<0.05). Compared to diverticulosis, patients with SUDD showed a significantly decreased abundance of fecal members of Clostridium cluster IX (16.0±1.8% vs. 9.9±1.5%; P=0.03), Fusobacterium (1.2±0.2% vs. 0.7±0.2%; P=0.05) and Lactobacillaceae (6.3±1.8% vs. 2.8±0.6%; P=0.05). Compared with AC, biopsies from both distant and diverticular sites in subjects with diverticula showed lower abundance of Enterobacteriaceae (P=0.04) and Bacteroides/Prevotella (P=0.06). In subjects with diverticula, we found a negative correlation between macrophage count in the diverticular region and mucosal Clostridium class IV (r=-0.48; P=0.03) and Akkermansia
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samples, colonic inflammation by qPCR for IL-1 b, and cellular tolerance by modulation of cell excitability of DRG neurons by whole-cell voltage clamp. Depletion of gut bacteria was achieved by oral gavage of an antibiotic cocktail (streptomycin, neomycin and metronidazole (all at 100 mg/kg), and vancomycin (50 mg/kg). Ampicillin (1g/l) was provided in drinking water ad libitum for 10 days. On day 5, a 75 mg morphine or placebo pellet was surgically implanted subcutaneously on the dorsum. RESULTS: The gut permeability was significantly enhanced in chronic morphine treated mice compared to placebo or acute morphine treated mice. The fold increase in CFU was 1.6x in mesenteric lymph node, 1.3x in Liver, 1.1x in Spleen and 1.9x in colon of chronic morphine treated mice compared to placebo. Chronic morphine also resulted in a shift in the bacterial load with a reduction in Bacteroidetes and Firmicutes and increase in enterobacteriales. The expression of Il-1b mRNA in distal colon increased by 2 fold in morphine pelleted mice. Antibiotic (ABX) treatment significantly reduced bacterial load (90%) in both placebo and morphine pelleted mice. The caecum of all ABX treated mice was significantly enlarged and spleen weight reduced by 70%, an effect similar to those reported for "germ-free" mice. The latency in the tail-flick assay was increased to maximum (10sec) in response to morphine challenge (10 mg/kg) in placebo pelleted mice with or without ABX treatment. The latency for tail-flick also increased in morphine pelleted mice with ABX while tolerance was observed in saline treated morphine pelleted mice. Morphine reduced the excitability of small diameter L6 DRG neurons of morphine pelleted ABX treated mice but not in saline treated DRG neurons suggesting that tolerance did not develop following ABX treatment. CONCLUSION: These data demonstrate that gastrointestinal microbiota is an important component in the development of analgesic tolerance to opioids and the bacterial translocation associated with chronic morphine may also explain the enhanced infections in Crohn's patients treated with narcotics