Masaya Sasaki

Comparison of the fecal microbiota profiles between ulcerative colitis and Crohn’s disease using terminal restriction fragment length polymorphism analysis

BackgroundTerminal restriction fragment length polymorphism (T-RFLP) analysis is a powerful tool to assess the diversity of a microbial community. In this study, we performed T-RFLP analysis of the fecal microbiota from patients with ulcerative colitis (UC) and those with Crohn’s disease (CD).MethodsThirty-one patients with UC, 31 patients with CD, and 30 healthy individuals were enrolled. The polymerase chain reaction (PCR) products obtained from the 16S rRNA genes of fecal samples were digested with BslI, and T-RF lengths were determined.ResultsThe fecal microbial communities were classified into 5 clusters. Twenty-eight of the 30 healthy individuals and 17 of the 18 patients with inactive UC were classified into clusters I, II, and III, but these clusters included a small number of patients with active UC and inactive/active CD. In contrast, 8 of the 13 patients with active UC and the majority of CD patients (12 of the 16 patients with inactive CD, and 11 of the 15 patients with active CD) were included in clusters IV and V. Based on the BslI-digested T-RFLP database, the bacteria showed a significant decrease in the Clostridium family in patients with active UC and inactive/active CD. In contrast, Bacteroides were significantly increased in CD patients. No significant differences were observed between patients with active UC and those with active CD.ConclusionThe fecal microbial communities of IBD patients were different from those of healthy individuals. The gut microbiota of patients with inactive UC tended to be closer to that of healthy individuals, suggesting different roles for the fecal microbiota in the pathophysiology of UC and CD.

Physiological and anti-inflammatory roles of dietary fiber and butyrate in intestinal functions.

BACKGROUND We investigated the effects of pectin, a soluble dietary fiber, on the morphological parameters of the small intestine. In addition, we tested the effects of butyrate enemas on dextran sulfate sodium (DSS)-induced experimental colitis. METHODS Male Wistar rats were fed an elemental diet containing 2.5% pectin for 14 days, and several parameters were then determined. DSS-induced colitis was evoked by the oral administration of water containing 3% DSS for 10 days. The butyrate enema (3 mL of 100 mmol/L butyrate per day) was begun 7 days before the DSS treatment. Interleukin (IL)-8 secretion in the human intestinal epithelial cell line HT-29 was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS Pectin feeding induced a significant increase in the villus height and crypt depth in the small intestine. These effects correlated with a significant increase in plasma enteroglucagon levels. Pretreatment with a butyrate enema significantly blocked the development of DSS-induced experimental colitis. In the in vitro experiment, sodium butyrate dose-dependently inhibited tumor necrosis factor (TNF)-alpha-induced IL-8 secretion in HT-29 cells. CONCLUSIONS A trophic effect due to dietary fiber was directly observed. The generation of short-chain fatty acids and the induction of enteroglucagon release might play an important role in this process. Butyrate, one of the major metabolites of dietary fiber, exerted a potent anti-inflammatory effect both in vivo and in vitro. Dietary fiber may therefore play important roles in the regulation of normal and pathological conditions in the intestine.

Dietary fat attenuates the benefits of an elemental diet in active Crohn's disease: a randomized, controlled trial.

Objectives Although an elemental diet has been established as the primary treatment for patients with Crohns disease, the influence of dietary fat on the elemental diet remains unclear. We have designed the first randomized, controlled trial for elemental diets containing different fat percentages in patients with active Crohns disease. Methods Each patient was randomized to receive one of three dose levels of fat in an elemental diet (Elental) for 4 weeks: 10 patients received low fat (3.06 g/day), 10 patients received medium fat (16.56 g/day) and eight patients received high fat (30.06 g/day). The additional fat was composed of long-chain fatty acids. All patients were evaluated using the International Organization of Inflammatory Bowel Disease rating, plus C-reactive protein level and erythrocyte sedimentation rate, which were measured at weekly intervals. Results Although the International Organization of Inflammatory Bowel Disease rating, C-reactive protein level and erythrocyte sedimentation rate in the low-fat group decreased, the values in the medium- and high-fat groups fluctuated during the study. The remission rate after 4 weeks in each group was 80%, 40% and 25% for patients in the low-, medium- and high-fat groups, respectively. Conclusions When the fat consisted of long-chain triglycerides, a high amount of this fat in the elemental diet formula decreased its therapeutic effect against active Crohns disease.

Effects of the Soluble Fibre Pectin on Intestinal Cell Proliferation, Fecal Short Chain Fatty Acid Production and Microbial Population

Aim: Although pectin, a dietary fibre, has been suggested to possess some trophic effects on the intestine, the mechanisms involved remain unclear. This study aimed to evaluate the effects of pectin on rat intestinal cell proliferation and the intraluminal environment. Methods: Control and pectin-fed rats were given a fibre-free elemental diet (ED) and an ED containing 2.5% pectin, respectively. On the 15th day, the length, weight and number of Ki-67-positive cells from each intestinal segment, and the short chain fatty acids (SCFAs) and microbial population in the caecum were measured. Plasma glucagon-like peptide-2 (GLP-2) concentration and GLP-2 receptor (GLP-2R) mRNA levels in the epithelium were also determined. Results: Pectin supplementation resulted in significant increases in the length, weight, and number of Ki-67-positive cells in the ileum, caecum and colon. Although pectin supplementation did not affect the caecal microbial flora that produced SCFAs, the caecal SCFA content was significantly increased. Pectin supplementation also induced an increase in the plasma GLP-2 concentration, but did not affect the GLP-2R mRNA levels in the small intestine. Conclusions: The increases in the caecal SCFAs and plasma GLP-2 levels induced by pectin supplementation may cause mucosal proliferation in the lower intestinal tract.

Preventive efficacy of butyrate enemas and oral administration of Clostridium butyricum M588 in dextran sodium sulfate-induced colitis in rats

Abstract: Butyrate enemas have been reported to be effective in ulcerative colitis. However, long-term use is difficult because of the troublesome procedure and the unpleasant smell. We therefore investigated the effects of the oral administration of Clostridium butyricum M588 (CBM588), an enterobacterium producing butyrate, in dextran sodium sulfate (DSS)-induced colitis in rats. First, we confirmed the effects of pre-treatment with a butyrate enema on DSS colitis. We then studied the efficacy of oral administration of CBM588 which was started 1 week prior to the induction of DSS colitis. In the CBM588 group, the ulcer index and myeloperoxidase (MPO) activity in the distal colon were significantly lower than in the control group. Proliferating cell nuclear antigen (PCNA) immuno-positive cells were increased around the ulcer in the CBM588 group. In regard to the contents of the cecum and colon, the proportions of Lactobacillus and Eubacterium were increased in the cecum in the CBM588 group. Further, there were significant increases of n-butyrate, propionate, and acetate concentrations in the cecum in the CBM588 group. These results indicated that the oral administration of CBM588 alleviated DSS-induced colitis, and may be useful instead of butyrate enema.

Suppression of interleukin-1beta- and tumor necrosis factor-alpha-induced inflammatory responses by leukocytapheresis therapy in patients with ulcerative colitis.

BackgroundTo elucidate the molecular mechanisms involved in the therapeutic effects of leukocytapheresis (LCAP), we investigated the alterations in the cytokine responses of peripheral blood mononuclear cells (PBMCs) before and after LCAP therapy in ulcerative colitis (UC) patients.MethodsTwelve patients with UC who did not respond to steroid therapy were enrolled. Nine patients responded to LCAP therapy, but 3 patients did not show clinical improvement. PBMCs were isolated from peripheral venous blood obtained within 5 min before and after the first and second session of LCAP treatment. Cells were stimulated with interleukin (IL)-1β and tumor necrosis factor (TNF)-α for 24 h, and the levels of secreted IL-8 and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA).ResultsLCAP induced a significant decrease in peripheral lymphocyte, monocyte, and platelet counts. IL-1β- and TNF-α-induced IL-8 and IL-6 secretion was significantly decreased after the first and second LCAP treatments. These responses were associated with inhibitory effects on nuclear factor (NF)-κB DNA-binding activity.ConclusionsLCAP downregulates the IL-1β- and TNF-α-induced inflammatory responses in PBMCs isolated from UC patients. The induction of hyporesponsiveness to proinflammatory cytokines may be an important factor mediating the clinical effects of LCAP in UC patients.

Faecal microbiota profile of Crohn's disease determined by terminal restriction fragment length polymorphism analysis.

Background  Terminal restriction fragment length polymorphism (T‐RFLP) analyses are powerful tools to assess the diversity of complex microbiota. T‐RFLPs permit rapid comparisons of microbiota from many samples.

Elevated Circulating Platelet-Derived Microparticles in Patients with Active Inflammatory Bowel Disease

OBJECTIVES:Platelet-derived microparticles (PDMPs) are active molecules involved in the hemostatic and inflammatory responses. To evaluate the changes in the platelet function in patients with inflammatory bowel disease (IBD), we measured circulating PDMP levels.METHODS:Twenty-five healthy controls, 44 patients with ulcerative colitis (UC), and 43 patients with Crohns Disease (CD) were studied. The PDMP and soluble P-selectin (sP-selectin) levels were measured by enzyme-linked immunosorbent assay (ELISA).RESULTS:In the healthy controls, the PDMP levels were 17.2 ± 6.2 U/mL. Significant differences were not observed between the healthy controls and inactive UC patients (20.8 ± 9.5 U/mL, n = 25) or between the healthy controls and inactive CD patients (17.6 ± 7.8 U/mL, n = 24). In contrast, the PDMP levels were significantly higher in both active UC (49.2 ± 33.6 U/mL, n = 19) and active CD (48.6 ± 42.8 U/mL, n = 19) patients than in the healthy controls. A significant correlation was found between the PDMP levels and the clinical activity indexes (CAI) of UC patients (r = 0.65, p < 0.01, n = 44), and between the PDMP levels and Crohns disease activity indexes (CDAI) (r = 0.72, p < 0.01, n = 43). Elevated PDMP levels in active patients were significantly reduced after remission. A significant correlation was observed between the PDMP levels and the sP-selectin levels (r = 0.60, p < 0.01, n = 122).CONCLUSION:Elevated circulating PDMPs in active IBD patients suggest a role for platelets in the pathogenesis of IBD.

Effect of enteral glutamine on intestinal permeability and bacterial translocation after abdominal radiation injury in rats

We investigated the effect of enteral glutamine on intestinal permeability and bacterial translocation after whole abdominal radiation in rats. Rats irradiated with 10 Gy to the abdomen were randomly divided into a glutamine-free diet group and a glutamine-rich diet (2% glutamine) group. After 3 days of feeding of each diet, the 6-h urinary recovery of polyethylene glycol 4000 was significantly decreased in the glutamine-rich diet group compared to that in the glutamine-free diet group. The 6-h urinary recovery of phenolsulfonphthalein was also decreased in the glutamine-rich diet group, but the difference was not significant. Plasma endotoxin concentration was significantly lower in the glutamine-rich diet group. Twenty-four hour after gavage with14C-labeledEscherichia coli, the detection rate of14C-labeled bacteria in the mesenteric lymph nodes of rats was significantly lower in the glutamine-rich diet group. The adherence of14C-labeledE. coli in the jejunal mucosa was significantly lower in the glutamine-rich diet group than in the glutamine-free diet group, but there were no significant differences in the ileal mucosa. These findings suggest that in rats with intestinal injury induced by irradiation, enteral glutamine maintains the intestinal barrier and reduces bacterial translocation.

Increased aggregation response of platelets in patients with inflammatory bowel disease.

BackgroundPlatelets play an important role in hemostatic and inflammatory responses. To evaluate any potential enhancement of platelet activity in patients with inflammatory bowel disease (IBD), we measured the platelet aggregation responses to various stimuli.MethodsTwenty-two healthy controls, 24 patients with ulcerative colitis (UC) and 25 patients with Crohns Disease (CD) were studied. The aggregation responses induced by three agonists (epinephrine, collagen, and ADP) were measured by an 8-channel aggregometer. The platelet-derived microparticles (PDMP) levels were measured by an enzyme-linked immunosorbent assay.ResultsTwenty-one out of the 22 healthy controls did not respond to epinephrine (0.1 µg/ml), collagen (0.2 µg/ml), or ADP (1.0 µM). Eight out of the 12 active UC patients were sensitive to all agonists, and 4 patients showed increased sensitivity to epinephrine/collagen or epinephrine/ADP. Three out of the 12 inactive UC patients were normal, but 9 of these patients showed increased sensitivity, mainly to epinephrine. Ten out of the 12 active CD patients were sensitive to all agonists, and 2 active CD patients were sensitive to epinephrine/collagen or epinephrine/ADP. Eight out of the 13 inactive CD patients were sensitive to two or all agonists. Even after remission, almost all of the UC and CD patients showed some increased sensitivity to the agonists. The platelet number and the plasma PDMP levels were significantly higher in the active IBD patients than in the control group.ConclusionsPlatelet aggregation responses are enhanced in IBD, even in inactive-phase patients. This increased sensitivity of the platelets may play an important role in the pathophysiology of IBD.