Altered profiles of intestinal microbiota and organic acids may be the origin of symptoms in irritable bowel syndrome.

BACKGROUND: The profile of intestinal organic acids in irritable bowel syndrome (IBS) and its correlation with gastrointestinal (GI) symptoms are not clear. We hypothesized in this study that altered GI microbiota contribute to IBS symptoms through increased levels of organic acids. METHODS: Subjects were 26 IBS patients and 26 age- and sex-matched controls. Fecal samples were collected for microbiota analysis using quantitative real-time polymerase chain reaction and culture methods, and the determination of organic acid levels using high-performance liquid chromatography. Abdominal gas was quantified by image analyses of abdominal X-ray films. Subjects completed a questionnaire for GI symptoms, quality of life (QOL) and negative emotion. KEY RESULTS: Irritable bowel syndrome patients showed significantly higher counts of Veillonella (P = 0.046) and Lactobacillus (P = 0.031) than controls. They also expressed significantly higher levels of acetic acid (P = 0.049), propionic acid (P = 0.025) and total organic acids (P = 0.014) than controls. The quantity of bowel gas was not significantly different between controls and IBS patients. Finally, IBS patients with high acetic acid or propionic acid levels presented with significantly worse GI symptoms, QOL and negative emotions than those with low acetic acid or propionic acid levels or controls. CONCLUSIONS & INFERENCES: These results support the hypothesis that both fecal microbiota and organic acids are altered in IBS patients. A combination of Veillonella and Lactobacillus is known to produce acetic and propionic acid. High levels of acetic and propionic acid may associate with abdominal symptoms, impaired QOL and negative emotions in IBS.

Randomized placebo-controlled trial assessing the effect of bifidobacteria-fermented milk on active ulcerative colitis.

BACKGROUND: Probiotics are efficacious for treating and maintaining remission of ulcerative colitis. AIM: To conduct a randomized placebo-controlled trial of bifidobacteria-fermented milk supplementation as a dietary adjunct in treating active ulcerative colitis. METHODS: Twenty patients with mild to moderate, active, ulcerative colitis randomly received 100 mL/day of bifidobacteria-fermented milk or placebo for 12 weeks with conventional treatment. RESULTS: Clinical and endoscopic activity indices and histological scores were similar in the two groups before treatment. Although improvements were significant in both groups, the clinical activity index was significantly lower in the bifidobacteria-fermented milk than in the placebo group after treatment. The post-treatment endoscopic activity index and histological score were significantly reduced in the bifidobacteria-fermented milk, but not the placebo group. Increases in faecal butyrate, propionate and short-chain fatty acid concentrations were significant in the bifidobacteria-fermented milk, but not the placebo group. No adverse effects were observed in either group. CONCLUSION: Supplementation with this bifidobacteria-fermented milk product is safe and more effective than conventional treatment alone, suggesting possible beneficial effects in managing active ulcerative colitis. This is a pilot study and further larger studies are required to confirm the result these preliminary results.

Improvement of human faecal flora-associated mouse model for evaluation of the functional foods.

AIMS: Animal models are required for evaluation of the functional foods such as pro/prebiotics exerting effects through the metabolism of the intestinal microflora. The object of this study was to establish new human flora-associated mice reflecting the environment of the human intestinal tract. METHODS AND RESULTS: We inoculated a human faecal suspension into segmented filamentous bacteria (SFB) monoassociated mice as a model system. In both human flora (HF) and SFB-associated mouse (HF-SFB mouse), intestinal characteristics such as the composition of intraepithelial lymphocytes, the expression of major histocompatibility complex (MHC) class II molecules and the number of immunoglobulin A-producing cells in the mucosa was closer to those of conventionally reared mice than was case with human flora-associated mice (HF mice) lacking SFB. Several predominant bacterial groups except lactobacilli in human flora were found in faeces of HF-SFB mice. Lactobacilli established small populations in the gut of HF-SFB mice when administered before inoculation with the human flora. Faecal enzymatic activities and organic acid concentration of HF-SFB mice proportionally reflected those of the donor subject. CONCLUSION: We established a new human flora-associated mouse (HF-SFB mouse), in which intestinal characteristics are normally developed and their major microbial composition reflect the human. SIGNIFICANCE AND IMPACT OF THE STUDY: HF-SFB mice are a valuable model for studying pro/prebiotic effects on the human intestine.

Preventive effects of Bifidobacterium- and Lactobacillus-fermented milk on the development of inflammatory bowel disease in senescence-accelerated mouse P1/Yit strain mice.

BACKGROUND/AIMS: We recently discovered inflammatory bowel disease (IBD) in the ileum and cecum of SAMP1/Yit strain mice under specific pathogen-free (SPF) conditions. To determine the effect of lactic acid bacteria (LAB - Bifidobacterium breve, Bifidobacterium bifidum and Lactobacillus acidophilus)-fermented milk on the prevention of IBD in SAMP1/Yit strain mice, we compared the disease severity on the intestinal inflammation among the group of mice fed saline, unfermented milk or LAB-fermented milk. METHODS: Three-week-old SAMP1/Yit strain mice (n = 72) were subdivided into three groups, that were fed saline, unfermented milk and LAB-fermented milk, respectively. The diets were orally administered daily via a gastric tube. When the mice reached 20 weeks of age, they were sacrificed and the IBD scores were compared among the three groups. RESULTS: Administration of the LAB-fermented milk to SAMP1/Yit strain mice reduced histological injury score, compared with those in saline-treated or unfermented milk-treated SAMP1/Yit strain mice. Ileal tissue weight and myeloperoxidase activity also reduced by treatment of LAB-fermented milk. Moreover, the tissue contents of immunoglobulins such as IgG1 and IgG2a were lower in the inflammatory regions in the SAMP1/Yit strain group fed LAB-fermented milk than that fed saline and unfermented milk. Cytokine-specific ELISA assays indicated the production of T-helper 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha in the culture supernatants of MLN cells in the SAMP1/Yit strain group fed LAB-fermented milk was lower than those fed saline and unfermented milk. On the contrary, the production of interleukin-10 in MLN cells increased by prevention with LAB-fermented milk. CONCLUSION: LAB-fermented milk is beneficial for the treatment of murine IBD and this effect may be modulated through stabilization of the mucosal immunity by LAB.

Differential roles of segmented filamentous bacteria and clostridia in development of the intestinal immune system.

The presence of microflora in the digestive tract promotes the development of the intestinal immune system. In this study, to evaluate the roles of two types of indigenous microbe, segmented filamentous bacteria (SFB) and clostridia, whose habitats are the small and large intestines, respectively, in this immunological development, we analyzed three kinds of gnotobiotic mice contaminated with SFB, clostridia, and both SFB and clostridia, respectively, in comparison with germfree (GF) or conventionalized (Cvd) mice associated with specific-pathogen-free flora. In the small intestine, the number of alpha beta T-cell receptor-bearing intraepithelial lymphocytes (alpha betaIEL) increased in SFB-associated mice (SFB-mice) but not in clostridium-associated mice (Clost-mice). There was no great difference in Vbeta usage among GF mice, Cvd mice, and these gnotobiotic mice, although the association with SFB decreased the proportion of Vbeta6(+) cells in CD8beta- subsets to some extent, compared to that in GF mice. The expression of major histocompatibility complex class II molecules on the epithelial cells was observed in SFB-mice but not in Clost-mice. On the other hand, in the large intestine, the ratio of the number of CD4(-) CD8(+) cells to that of CD4(+) CD8(-) cells in alpha betaIEL increased in Clost-mice but not in SFB-mice. On association with both SFB and clostridia, the numbers and phenotypes of IEL in the small and large intestines changed to become similar to those in Cvd mice. In particular, the ratio of the number of CD8alpha beta+ cells to that of CD8alpha alpha+ cells in alpha betaIEL, unusually elevated in the small intestines of SFB-mice, decreased to the level in Cvd mice on contamination with both SFB and clostridia. The number of immunoglobulin A (IgA)-producing cells in the lamina propria was more elevated in SFB-mice than in Clost-mice, not only in the ileum but also in the colon. The number of IgA-producing cells in the colons of Clost-mice was a little increased compared to that in GF mice. Taken together, SFB and clostridia promoted the development of both IEL and IgA-producing cells in the small intestine and that of only IEL in the large intestine, respectively, suggesting the occurrence of compartmentalization of the immunological responses to the indigenous bacteria between the small and large intestines.

Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.

BACKGROUND: A new subline of the senescence accelerated mouse (SAM) P1/Yit strain has been established which shows spontaneous enteric inflammation under specific pathogen free (SPF) conditions. AIMS: To elucidate the pathogenesis of enteric inflammation in this new subline. METHODS: The SPF and germ free (GF) SAMP1/Yit strains were used. Histological, immunological, and microbiological characterisation of the mice with enteric inflammation was performed. RESULTS: Histologically, enteritic inflammation developed as a discontinuous lesion in the terminal ileum and caecum with the infiltration of many inflammatory cells after 10 weeks of age. the activity of myeloperoxidase, and both immunolocalisation and mRNA expression of inducible nitric oxide synthase increased in the lesion. CD3-epsilon positive T cells, neutrophils, and macrophages were more numerous in the inflamed mucosa of the SAMP1/Yit strain. The GF SAMP1/Yit strain did not show any inflammation in the intestinal wall, by the age of 30 weeks, and the enteritis and caecitis developed 10 weeks after the conventionalisation of the GF SAMP1/Yit strain. CONCLUSION: Enteric inflammation in the ileum and caecum developed in the SAMP1/Yit strain. The pathophysiological characteristics of the disease in this mouse have some similarities to those of human inflammatory bowel disease (IBD). This mouse strain should be a useful model system for elucidating the interaction between the pathogenesis of IBD and the gut microflora.

Proliferative recruitment of intestinal intraepithelial lymphocytes after microbial colonization of germ-free mice.

Intraepithelial lymphocytes (IEL), particularly alpha beta TCR-bearing IEL (alpha beta-IEL), dramatically increase in number after microbial colonization of formerly germ-free (ex-GF) mice (Umesaki et al., Immunology 1993. 79: 32). In this study, the kinetics of expansion of IEL after microbial colonization in ex-GF mice were investigated by the bromodeoxyuridine (BrdUrd) continuous labeling method. In GF mice, gamma delta- and alpha beta-IEL were gradually labeled with BrdUrd, reaching approximately 30% and 15% labeling, respectively, after 10 days of continuous BrdUrd labeling. In conventional (CV) mice, the percentage of BrdUrd-labeled alpha beta-IEL was a little higher than that for gamma delta-IEL. The maximal labeling for alpha beta-IEL and gamma delta-IEL reached 50-60% and 40%, respectively, in 10 days. In the case of conventionalized ex-GF mice, continuous labeling was started 11 days after microbial colonization of GF mice because alpha beta-IEL outnumbered gamma delta-IEL during this period. In this case, 75% of alpha beta-IEL and 67% of gamma delta-IEL were labeled with BrdUrd in 10 days. On the other hand, the apparent half lives of alpha beta-IEL and gamma delta-IEL were 10 and 20 days, respectively in CV mice. These results strongly suggest that the number of IEL, particularly alpha beta-IEL, increases after microbial colonization through recruitment into the cell cycle of a large proportion of IEL or their immediate precursors. The difference in the relative BrdUrd-labeling rate of alpha beta-IEL to that of gamma delta-IEL between germ-free and conventionalized mice indicates a preferential increase in the alpha beta-IEL subset during the course of conventionalization.

Gamma delta TCR-bearing intraepithelial lymphocytes regulate class II major histocompatibility complex molecule expression on the mouse small intestinal epithelium.

Introduction of fecal bacteria into germ-free (GF) Balb/c mice induces class II major histocompatibility complex (MHC) molecules on the small intestinal epithelium. In this study, we elucidated the regulatory mechanisms for the class II MHC molecule induction on the mouse small intestinal epithelium during microbial colonisation of the gut in ex-GF mice. Intraperitoneal injection of interferon-gamma (IFN-gamma) into GF Balb/c mice induced class II MHC expression on the small intestinal epithelial cells. Induction of these molecules was inhibited by peritoneal injection of a monoclonal antibody (mAb) against IFN-gamma on the conventionalisation of GF mice. RNA reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the small intestinal epithelium indicated that the class II transactivator (CIITA), a regulatory factor for the class II MHC gene, and the I-E alpha chain, but not IFN-gamma receptor mRNA, increased during conventionalisation. The induction of class II MHC on the epithelial cells during the conventionalisation of GF C.B-17 scid mice was much lower than that in GF Balb/c mice. Immunocytochemical and RT-PCR analysis showed that both the number of IFN-gamma producing IEL and the level of the IFN-gamma mRNA in gamma delta TCR IEL were very low in the GF state, and gradually increased after microbial colonisation. After in vivo treatment with a mAb against gamma delta TCR, the number of gamma delta TCR-expressing IEL greatly decreased and the expression of class II MHC molecules on the small intestinal epithelium was repressed during the conventionalisation of GF mice. Taken together, these results suggested that gamma delta TCR-bearing IEL modulate class II MHC molecule expression on the small intestinal epithelium through the production of IFN-gamma during microbial colonisation in ex-GF mice.

Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC class II molecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse.

In ex-germ-free mice conventionalized by association with fecal microorganisms, the induction fo major histocompatibility complex class II molecules and fucosylation of asialo GM1 glycolipid occur in the small intestinal epithelial cells (IEC). The intestinal intraepithelial lymphocytes (IEL), especially alpha beta T-cell receptor-bearing ones, also remarkably expand and show cytolytic activity. In this study, we investigated the immunological and physiological characteristics of the small intestine induced by a kind of indigenous bacteria of the small intestine, segmental filamentous bacteria (SFB), among chloroform-resistant intestinal bacteria. Monoassociation of SFB with germ-free mice was confirmed by the determination of the base sequences of polymerase chain reaction products of 16S rRNA genes of the fecal bacteria of these mice and in situ hybridization using fluorescein-labeled probes based on them. SFB increased the number of alpha beta TCR-bearing IEL and induced Thy-1 expression and cytolytic activity of IEL. The induction of MHC class II molecules and fucosyl asialo GM1 glycolipids and the increases in the mitotic activity and the ratio of the number of columnar cells to those of goblet cells also occurred in the small intestinal epithelial cells on monoassociation of these bacteria. SFB are important indigenous bacteria for the development of the mucosal architecture and immune system in the small intestine, at least in mice.

Effects of fecal microorganisms and their chloroform-resistant variants derived from mice, rats, and humans on immunological and physiological characteristics of the intestines of ex-germfree mice.

In order to elucidate the nature of intestinal flora affecting the immunological and physiological parameters of the intestine, we produced several kinds of ex-germfree mice associated with fecal organisms and their chloroform-resistant variants derived from mice, rats, and humans. The phenotypes of intraepithelial lymphocytes were changed to those in conventional mice, particularly the increased positive percentage of alpha beta T-cell-receptor and Thy-1-bearing T cells, on association of the microorganisms (MF) and their chloroform resistant variants (MChl) derived from mice, but not rats and humans, with germfree mice. The cytolytic activity of intraepithelial lymphocytes was expressed only in the MF and MChl groups. The induction of the synthesis of fucosyl asialo GM1 glycolipid, the expression of major histocompatibility complex class II molecule, an increase in the mitotic indices of colonic epithelial cells, and a decrease in lactase activity of the small intestinal epithelial cells also occurred only in the two groups. On the other hand, the cecal size (cecal weight/body weight ratio) was reduced in the mice of all groups examined here, there being approximately the same amount and composition of organic acids, such as acetic acid, butyric acid, and propionic acid, in the cecal contents. Taken together, the results suggest that mouse-specific and chloroform-resistant microorganisms, which are difficult to cultivate at present, may contribute to alteration of the immunological and epithelial characteristics of the mouse intestine. Another factor derived from the intestinal flora, for example, bacterial metabolites such as organic acids, may also affect the cecal size.

Optical memory based on heterodyne-detected accumulated photon echoes.

Optical memory based on heterodyne-detected accumulated photon echoes has been demonstrated in dye-doped polymers. Compared with previously developed time-domain optical memory, writing and reading of higher bit data is possible owing to the superior time resolution of this method.