Dysbiosis of the Fecal and Biliary Microbiota in Biliary Tract Cancer.

Characteristic bile duct and gut microbiota have been identified in patients with chronic biliary tract disease. This study aimed to characterize the fecal and bile microbiota in biliary tract cancer (BTC) patients and their relationship. Patients with BTC (n = 30) and benign biliary disease (BBD) without cholangitis (n = 11) were included. Ten healthy, age-matched subjects were also recruited for fecal microbiota comparison. The fecal and bile duct microbiotas were analyzed by sequencing the 16S rRNA gene V3-V4 region. Live bacteria were obtained in the bile from three BTC patients by culture, and metagenomics-based identification was performed. Linear discriminant analysis effect size showed a higher Enterobacteriaceae abundance and a lower Clostridia abundance, including that of Faecalibacterium and Coprococcus, in the BTC patients than in the other subjects. Ten of 17 operational taxonomic units (OTUs) assigned to Enterobacteriaceae in the bile were matched with the OTUs found in the BTC subject fecal samples. Furthermore, a bile-isolated strain possessed the carcinogenic bacterial colipolyketide synthase-encoding gene. Enterobacteriaceae was enriched in the BTC feces, and more than half of Enterobacteriaceae in the bile matched that in the feces at the OTU level. Our data suggests that fecal microbiota dysbiosis may contribute to BTC onset.

Enriched metabolites that potentially promote age-associated diseases in subjects with an elderly-type gut microbiota.

We previously investigated the gut microbiota of 453 healthy Japanese subjects aged 0 to 104 years and found that the composition of the gut microbiota could be classified into some age-related clusters. In this study, we compared fecal metabolites between age-matched and age-mismatched elderly subjects to examine the roles of the gut microbiota in the health of the elderly. Fecal metabolites in 16 elderly subjects who fell into an age-matched cluster (elderly-type gut microbiota group, E-GM) and another 16 elderly subjects who fell into an age-mismatched cluster (adult-type gut microbiota group, A-GM) were measured by CE-TOF-MS. A total of eight metabolites were significantly different between the groups: cholic acid and taurocholic acid were enriched in the A-GM group, whereas choline, trimethylamine (TMA), N8-acetylspermidine, propionic acid, 2-hydroxy-4-methylvaleric acid, and 5-methylcytosine were enriched in the E-GM group. Some metabolites (choline, TMA, N8-acetylspermidine) elevated in the E-GM group were metabolites or precursors reported as risk factors for age-associated diseases such as arteriosclerosis and colorectal cancer. The abundance of some species belongs to Proteobacteria, which were known as TMA-producing bacteria, was increased in the E-GM group and correlated with fecal TMA levels. In vitro assays showed that these elderly-type fecal metabolites suppressed the expression of genes related to tight junctions in normal colonic epithelial cells and induced the expression of inflammatory cytokines in colon cancer cells. These findings suggest that metabolites produced by the aged gut microbiota could contribute to intestinal and systemic homeostasis and could be targeted for preventing aging-associated diseases.

Evaluation of Porcine Intestinal Epitheliocytes as an In vitro Immunoassay System for the Selection of Probiotic Bifidobacteria to Alleviate Inflammatory Bowel Disease.

The use of in vitro systems that allow efficient selection of probiotic candidates with immunomodulatory properties could significantly minimize the use of experimental animals. In this work, we generated an in vitro immunoassay system based on porcine intestinal epithelial (PIE) cells and dextran sodium sulfate (DSS) administration that could be useful for the selection and characterization of potential probiotic strains to be used in inflammatory bowel disease (IBD) patients. Our strategy was based on two fundamental pillars: on the one hand, the capacity of PIE cells to create a monolayer by attaching to neighboring cells and efficiently mount inflammatory responses and, on the other hand, the use of two probiotic bifidobacteria strains that have been characterized in terms of their immunomodulatory capacities, particularly in mouse IBD models and patients. Our results demonstrated that DSS administration can alter the epithelial barrier created in vitro by PIE cells and induce a potent inflammatory response, characterized by increases in the expression levels of several inflammatory factors including TNF-α, IL-1α, CCL4, CCL8, CCL11, CXCL5, CXCL9, CXCL10, SELL, SELE, EPCAM, VCAM, NCF2, and SAA2. In addition, we demonstrated that Bifidobacterium breve M-16V and B. longum BB536 are able to regulate the C-jun N-terminal kinase (JNK) intracellular signalling pathway, reducing the DSS-induced alterations of the in vitro epithelial barrier and differentially regulating the inflammatory response in a strain-dependent fashion. The good correlation between our in vitro findings in PIE cells and previous studies in animal models and IBD patients shows the potential value of our system to select new probiotic candidates in an efficient way.

Metagenomic analyses of the gut microbiota associated with colorectal adenoma.

Recent studies have suggested an association between certain members of the Fusobacterium genus, especially F. nucleatum, and the progression of advanced colorectal carcinoma (CRC). We assessed such an association of the gut microbiota in Japanese patients with colorectal adenoma (CRA) or intramucosal CRC using colonoscopy aspirates. We analyzed samples from 81 Japanese patients, including 47 CRA and 24 intramucosal CRC patients, and 10 healthy subjects. Metagenomic analysis of the V3-V4 region of the 16S ribosomal RNA gene was performed. The linear discriminant analysis (LDA) effect size (LEfSe) method was used to examine microbial dysbiosis, revealing significant differences in bacterial abundances between the healthy controls and CRA or intramucosal CRC patients. In particular, F. varium was statistically more abundant in patients with CRA and intramucosal CRC than in healthy subjects. Here, we present the metagenomic profile of CRA and intramucosal CRC and demonstrate that F. varium is at least partially involved in the pathogenesis of CRA and intramucosal CRC.

Orally administered heat-killed Lactobacillus paracasei MCC1849 enhances antigen-specific IgA secretion and induces follicular helper T cells in mice.

Antigen-specific immunoglobulin (Ig) A plays a major role in host defense against infections in gut mucosal tissue. Follicular helper T (Tfh) cells are located in germinal centers and promote IgA production via interactions with germinal center B cells. Several studies have demonstrated that some lactic acid bacteria (LAB) strains activate the host's acquired immune system, inducing IgA secretion in the intestine. However, the precise molecular mechanisms underlying the effects of LAB on IgA production and Tfh cells are not fully resolved. Lactobacillus paracasei MCC1849 is a probiotic strain isolated from the intestine of a healthy adult. In this study, we investigated the effects of orally administered heat-killed MCC1849 on IgA production in the intestine and on Tfh cell induction in vivo. We found that orally administered MCC1849 induced antigen-specific IgA production in the small intestine, serum and lungs. We also observed that MCC1849 increased the proportion of IgA+ B cells and Tfh cells in Peyer's patches (PPs). In addition, MCC1849 increased the gene expression of IL-12p40, IL-10, IL-21, STAT4 and Bcl-6 associated with Tfh cell differentiation. These results suggest that orally administered MCC1849 enhances antigen-specific IgA production and likely affects Tfh cell differentiation in PPs.

Administration of bovine casein-derived peptide prevents cognitive decline in Alzheimer disease model mice.

There is a growing interest in identifying natural food ingredients that may serve to prevent dementia such as that due to Alzheimer disease (AD). Peptides derived from food proteins have been demonstrated to have various physiological activities such as a hypotensive action. Recent findings have indicated possible associations of hypertension with AD progression, and suggest that angiotensin converting enzyme (ACE) inhibitors with potential to pass through the blood brain barrier (BBB) may reduce the risk of AD. In this study, we investigated the effect of milk peptide (CH-3) on cognitive function in AD model mice. CH-3 contains a tripeptide (methionine-lysine-proline, MKP) that has been found to have a strong ACE inhibitory effect and the potential to pass through the BBB. Adult male ddY mice were used in this study, and an animal model of AD was induced by intracerebroventricular (ICV) injection of Aß1-42. CH-3 (250 mg/kg/day) or MKP (0.5 mg/kg/day) was orally administered every day starting 2 days before ICV injection. At 3 weeks after ICV injection, cognitive function was evaluated by the Morris water maze test. Brain samples were obtained after behavioral testing, and expression of inflammatory cytokines and NADPH oxidase subunits was measured by real-time quantitative RT-PCR. ICV injection of Aß1-42 significantly impaired cognitive function compared with that in PBS-injected mice. Daily administration of CH-3 markedly attenuated this Aß1-42-induced cognitive decline. Aß1-42 injection significantly enhanced the expression of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and p22phox in the mouse hippocampus compared with PBS injection, and showed a tendency to increase the expression of monocyte chemoattractant protein-1 (MCP-1), p47phox and gp91phox, whereas CH-3 treatment markedly reduced Aß1-42-induced TNF-α, MCP-1, iNOS, p47phox and gp91phox expression. Finally, administration of MKP also attenuated Aß1-42-induced cognitive impairment with an increase in cerebral blood flow. The present study demonstrated that repeated oral administration of CH-3 to AD model mice not only improved cognitive function but also suppressed the expression of inflammatory cytokines and production of oxidative stress, and suggests its therapeutic potential for preventing cognitive impairment in AD.

Perioperative supplementation with bifidobacteria improves postoperative nutritional recovery, inflammatory response, and fecal microbiota in patients undergoing colorectal surgery: a prospective, randomized clinical trial.

The use of probiotics has been widely documented to benefit human health, but their clinical value in surgical patients remains unclear. The present study investigated the effect of perioperative oral administration of probiotic bifidobacteria to patients undergoing colorectal surgery. Sixty patients undergoing colorectal resection were randomized to two groups prior to resection. One group (n=31) received a probiotic supplement, Bifidobacterium longum BB536, preoperatively for 7-14 days and postoperatively for 14 days, while the other group (n=29) received no intervention as a control. The occurrences of postoperative infectious complications were recorded. Blood and fecal samples were collected before and after surgery. No significant difference was found in the incidence of postoperative infectious complications and duration of hospital stay between the two groups. In comparison to the control group, the probiotic group tended to have higher postoperative levels of erythrocytes, hemoglobin, lymphocytes, total protein, and albumin and lower levels of high sensitive C-reactive proteins. Postoperatively, the proportions of fecal bacteria changed significantly; Actinobacteria increased in the probiotic group, Bacteroidetes and Proteobacteria increased in the control group, and Firmicutes decreased in both groups. Significant correlations were found between the proportions of fecal bacteria and blood parameters; Actinobacteria correlated negatively with blood inflammatory parameters, while Bacteroidetes and Proteobacteria correlated positively with blood inflammatory parameters. In the subgroup of patients who received preoperative chemoradiotherapy treatment, the duration of hospital stay was significantly shortened upon probiotic intervention. These results suggest that perioperative oral administration of bifidobacteria may contribute to a balanced intestinal microbiota and attenuated postoperative inflammatory responses, which may subsequently promote a healthy recovery after colorectal resection.

Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling.

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-ß and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-ß production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities.

Innovative use of platinum compounds to selectively detect live microorganisms by polymerase chain reaction.

PCR cannot distinguish live microorganisms from dead ones. To circumvent this disadvantage, ethidium/propidium-monoazide (EMA/PMA) and psoralen to discriminate live from dead bacteria have been used for 2 decades. These methods require the use of numerous laborious procedures. We introduce an innovative method that uses platinum compounds, which are primarily used as catalysts in organic chemistry and partly used as anti-cancer drugs. Microorganisms are briefly exposed to platinum compounds in vivo, and these compounds penetrate dead (compromised) microorganisms but not live ones and are chelated by chromosomal DNA. The use of platinum compounds permits clear discrimination between live and dead microorganisms in water and milk (including Cronobacter sakazakii and Escherichia coli) via PCR compared with typically used PMA. This platinum-PCR method could enable the specific detection of viable coliforms in milk at a concentration of 5-10 CFU mL(-1) specified by EU/USA regulations after a 4-h process. For sample components, environmental water contains lower levels of PCR inhibitors than milk does, and milk is similar to infant formula, skim milk and blood; thus, the use of the platinum-PCR method could also prevent food poisoning due to the presence of C. sakazakii in dairy products. This method could provide outstanding rapidity for use in environmental/food/clinical tests. Platinum-PCR could also be a substitute for the typical culture-based methods currently used.

Immunoregulatory effect of bifidobacteria strains in porcine intestinal epithelial cells through modulation of ubiquitin-editing enzyme A20 expression.

BACKGROUND: We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains. OBJECTIVE: The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells. RESULTS: Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells. CONCLUSIONS: We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.

Immunomodulating and anti-infective effects of a novel strain of Lactobacillus paracasei that strongly induces interleukin-12.

Several studies have demonstrated that some strains of lactic acid bacteria (LAB) can elicit natural killer (NK) cell activities via interleukin-12 (IL-12) induction and protect against influenza virus (IFV) infection. LAB strains that strongly induce IL-12 are expected to be effective in protecting against IFV infection. In this study, we screened 85 strains for their ability to induce the in vitro production of IL-12, and Lactobacillus paracasei MoLac-1 most strongly induced IL-12. To examine the immunomodulating effects of MoLac-1, we have performed in vitro studies using murine splenocytes. Heat-killed MoLac-1 cells induced IL-12 and interferon-γ (IFN-γ) production by murine splenocytes. Experiments using splenocytes depleted of various cell populations indicated that macrophages might be a major source of MoLac-1-induced IL-12 secretion. Intracellular staining of IFN-γ suggested that MoLac-1 activated NK cells and induced IFN-γ production by NK cells in vitro. Oral administration of heat-killed MoLac-1 increased the proportion of NK cells in spleen, and ameliorated the symptoms of IFV infection in mice. These results suggest that heat-killed MoLac-1 has the potential to modulate innate immunity and is useful for alleviation of the symptoms of IFV infection.

Effect of the oral intake of yogurt containing Bifidobacterium longum BB536 on the cell numbers of enterotoxigenic Bacteroides fragilis in microbiota.

Enterotoxigenic Bacteroides fragilis (ETBF) strains have been suggested to be associated with acute and persistent diarrheal disease, inflammatory bowel disease and colorectal cancer, although further epidemiological studies are needed for clarification. Here, a pilot study was performed to examine the effect of the oral administration of yogurt supplemented with a probiotic strain on the cell numbers of fecal ETBF in a healthy population. Among 420 healthy adults, 38 subjects were found to be ETBF carriers, giving a prevalence of approximately 9%. Among them, 32 subjects were enrolled in an open, randomized, parallel-group study to ingest yogurt supplemented with a probiotic strain, Bifidobacterium longum BB536 (BB536Y group), for 8 weeks, with milk provided to the control group (milk group). The cell numbers of ETBF and the dominant species of the B. fragilis group were measured by a quantitative PCR method. Compared with the baseline values, there was a significant decrease in the cell number of ETBF at week 8 in the BB536Y group but not in the milk group. Linear mixed models analysis for longitudinal data revealed a significant difference in the changes of ETBF cell number between the two groups during the intervention phase. These results imply the potential of probiotic yogurt for eliminating ETBF in the microbiota, but its clinical significance needs to be evaluated in the future. This is the first report of a possible effect of probiotic intake on ETBF in the microbiota.

Toll-like receptor-2-activating bifidobacteria strains differentially regulate inflammatory cytokines in the porcine intestinal epithelial cell culture system: finding new anti-inflammatory immunobiotics.

A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter assay using a toll-like receptor 2-expressing transfectant (HEK(pTLR2) system) and the mitogenic assay using porcine Peyer's patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyer's patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEK(pTLR2) and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEK(pTLR2) system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.