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ObjectivesPrevotella copriis considered to be a contributing factor in rheumatoid arthritis (RA). However, in some non-Westernised countries, healthy individuals also harbour an abundance ofP. copriin the intestine. This study investigated the pathogenicity of RA patient-derivedP. copri(P. copriRA) compared with healthy control-derivedP. copri(P. copriHC).MethodsWe obtained 13P.copristrains from the faeces of patients with RA and healthy controls. Following whole genome sequencing, the sequences ofP. copriRAandP. copriHCwere compared. To analyse the arthritis-inducing ability ofP. copri, we examined two arthritis models (1) a collagen-induced arthritis model harbouringP. copriunder specific-pathogen-free conditions and (2) an SKG mouse arthritis model underP. copri-monocolonised conditions. Finally, to evaluate the ability ofP. coprito activate innate immune cells, we performed in vitro stimulation of bone marrow-derived dendritic cells (BMDCs) byP. copriRAandP. copriHC.ResultsComparative genomic analysis revealed no apparent differences in the core gene contents betweenP. copriRAandP. copriHC, but pangenome analysis revealed the high genome plasticity ofP. copri. We identified aP. copriRA-specific genomic region as a conjugative transposon. In both arthritis models,P. copriRA-induced more severe arthritis thanP. copriHC. In vitro BMDC stimulation experiments revealed the upregulation of IL-17 and Th17-related cytokines (IL-6, IL-23) byP. copriRA.ConclusionOur findings reveal the genetic diversity ofP. copri, and the genomic signatures associated with strong arthritis-inducing ability ofP. copriRA. Our study contributes towards elucidation of the complex pathogenesis of RA.

Bile acid resistance is crucial to allow probiotic strains to survive in the gastrointestinal tract and exert health-promoting effects on their hosts. Our aim here was to determine the mechanism of this resistance via a genetic approach by identifying the genes essential for bile acid resistance in Lacticaseibacillus paracasei strain Shirota (LcS). We generated 4649 transposon-inserted lines of L. paracasei YIT 0291, which has the same genome sequence as LcS but lacks the pLY101 plasmid, and we screened them for bile-acid-sensitive mutants. The growth of 14 mutated strains was strongly inhibited by bile acid, and we identified 10 genes that could be involved in bile acid resistance. Expression of these genes was not markedly induced by bile acid, suggesting that their homeostatic expression is important for exerting bile acid resistance. Two mutants, in which the transposon was independently inserted into cardiolipin synthase genes, showed strong growth inhibition. Disruption of the cardiolipin synthase genes in LcS caused decreased cardiolipin production and accumulation of the precursor phosphatidylglycerol in bacterial cells. These data suggest that LcS possesses several mechanisms for exerting bile acid resistance, and that homeostatic cardiolipin production is among the factors most essential for this resistance.

Human DNA present in faecal samples can result in a small number of human reads in gut shotgun metagenomic sequencing data. However, it is presently unclear how much personal information can be reconstructed from such reads, and this has not been quantitatively evaluated. Such a quantitative evaluation is necessary to clarify the ethical concerns related to data sharing and to enable efficient use of human genetic information in stool samples, such as for research and forensics. Here we used genomic approaches to reconstruct personal information from the faecal metagenomes of 343 Japanese individuals with associated human genotype data. Genetic sex could be accurately predicted based on the sequencing depth of sex chromosomes for 97.3% of the samples. Individuals could be re-identified from the matched genotype data based on human reads recovered from the faecal metagenomic data with 93.3% sensitivity using a likelihood score-based method. This method also enabled us to predict the ancestries of 98.3% of the samples. Finally, we performed ultra-deep shotgun metagenomic sequencing of five faecal samples as well as whole-genome sequencing of blood samples. Using genotype-calling approaches, we demonstrated that the genotypes of both common and rare variants could be reconstructed from faecal samples. This included clinically relevant variants. Our approach can be used to quantify personal information contained within gut metagenome data.

Several studies revealed that substantial artificial changes in the gut microbiota resulted in modification of hepatic cytochrome P450 3a (Cyp3a) in mice. Consequently, we hypothesized that “normal” variation of the gut microbiota might also alter hepatic Cyp activity and lead to individual differences in drug metabolism. Therefore, this study investigated the effects of normal gut microbiota variation on hepatic Cyp activity under the same genetic and environmental conditions using ex‐germ‐free mice. Using the feces of three breeder BALB/c mice (Jcl, Slc, and Crj), germ‐free BALB/cYit mice were conventionalized (Yit‐Jcl, Yit‐Slc, and Yit‐Crj). The gut microbiota composition and hepatic Cyp activity of these donors and recipients were evaluated. 16S rRNA sequencing revealed clear differences of the gut microbiota among donors and among recipients. Cyp3a activity was significantly higher in Slc mice than in Jcl and Crj mice. Notably, among recipients, Cyp3a activity was significantly higher in Yit‐Slc and Yit‐Crj mice than in Yit‐Jcl mice. Cyp2b activity was significantly higher in Slc mice than in Jcl and Crj mice. Cyp2b activity was significantly higher in Yit‐Slc mice than in Yit‐Jcl mice. Additionally, in correlation analysis, some genera displayed significant positive or negative correlations with Cyp activity, particular the strong positive correlation between Clostridium sensu stricto 1 with Cyp3a activity. In conclusion, this study demonstrated that normal variation of the gut microbiota affected hepatic Cyp3a and Cyp2b activity, which might result in individual differences of drug metabolism., Using ex‐GF mice (Yit‐Jcl, Yit‐Slc, and Yit‐Crj) conventionalized with feces from mice obtained from three breeders (Jcl, Slc, and Crj), we revealed that normal variation of the gut microbiota could affect hepatic Cyp activity, and several bacterial groups might modulate Cyp activity.

Dietary carbohydrates impact the composition of the human gut microbiota. However, the relationship between carbohydrate availability for individual bacteria and their growth in the intestinal environment remains unclear. Here, we show that the availability of long-chain xylans (LCX), one of the most abundant dietary fibres in the human diet, promotes the growth of Bifidobacterium pseudocatenulatum in the adult human gut. Genomic and phenotypic analyses revealed that the availability of LCX-derived oligosaccharides is a fundamental feature of B. pseudocatenulatum, and that some but not all strains possessing the endo-1,4-β-xylanase (BpXyn10A) gene grow on LCX by cleaving the xylose backbone. The BpXyn10A gene, likely acquired by horizontal transfer, was incorporated into the gene cluster for LCX-derived oligosaccharide utilisation. Co-culturing with xylanolytic Bacteroides spp. demonstrated that LCX-utilising strains are more competitive than LCX non-utilising strains even when LCX-derived oligosaccharides were supplied. In LCX-rich dietary interventions in adult humans, levels of endogenous B. pseudocatenulatum increased only when BpXyn10A was detected, indicating that LCX availability is a fitness determinant in the human gut. Our findings highlight the enhanced intestinal adaptability of bifidobacteria via polysaccharide utilisation, and provide a cornerstone for systematic manipulation of the intestinal microbiota through dietary intervention using key enzymes that degrade polysaccharide as biomarkers.

The bacterial species living in the gut mediate many aspects of biological processes such as nutrition and activation of adaptive immunity. In addition, commensal fungi residing in the intestine also influence host health. Although the interaction of bacterium and fungus has been shown, its precise mechanism during colonization of the human intestine remains largely unknown. Here, we show interaction between bacterial and fungal species for utilization of dietary components driving their efficient growth in the intestine. Next generation sequencing of fecal samples from Japanese and Indian adults revealed differential patterns of bacterial and fungal composition. In particular, Indians, who consume more plant polysaccharides than Japanese, harbored increased numbers of Prevotella and Candida. Candida spp. showed strong growth responses to the plant polysaccharide arabinoxylan in vitro. Furthermore, the culture supernatants of Candida spp. grown with arabinoxylan promoted rapid proliferation of Prevotella copri. Arabinose was identified as a potential growth-inducing factor in the Candida culture supernatants. Candida spp. exhibited a growth response to xylose, but not to arabinose, whereas P. copri proliferated in response to both xylose and arabinose. Candida spp., but not P. copri, colonized the intestine of germ-free mice. However, P. copri successfully colonized mouse intestine already harboring Candida. These findings demonstrate a proof of concept that fungal members of gut microbiota can facilitate a colonization of the intestine by their bacterial counterparts, potentially mediated by a dietary metabolite.

Mucosal barriers segregate commensal microbes from the intestinal epithelia to maintain gut homeostasis. Ly6/Plaur domain-containing 8 (Lypd8), a highly glycosylated glycosylphosphatidylinositol-anchored protein selectively expressed on colonic enterocytes, promotes this segregation by inhibiting bacterial invasion of the inner mucus layer and colonic epithelia. However, it remains unclear whether Lypd8 prevents infection with enteric bacterial pathogens. Here, we demonstrate that Lypd8 strongly contributes to early-phase defense against Citrobacter rodentium, which causes colitis by inducing attachment and effacement (A/E) lesions on colonic epithelia. Lypd8 inhibits C. rodentium attachment to intestinal epithelial cells by binding to intimin, thereby suppressing the interaction between intimin and translocated intimin receptor. Lypd8 deficiency leads to rapid C. rodentium colonization in the colon, resulting in severe colitis with Th17-cell and neutrophil expansion in the lamina propria. This study identifies a novel function for Lypd8 against A/E bacteria and highlights the role of enterocytes as crucial players in innate immunity for protection against enteric bacterial pathogens.

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Objective The intestinal microbiota is involved in the pathogenesis of arthritis. Altered microbiota composition has been demonstrated in patients with rheumatoid arthritis (RA). However, it remains unclear how dysbiosis contributes to the development of arthritis. The aim of this study was to investigate whether altered composition of human intestinal microbiota in RA patients contributes to the development of arthritis. Methods We analyzed the fecal microbiota of patients with early RA and healthy controls, using 16S ribosomal RNA−based deep sequencing. We inoculated fecal samples from RA patients and healthy controls into germ-free arthritis-prone SKG mice and evaluated the immune responses. We also analyzed whether the lymphocytes of SKG mice harboring microbiota from RA patients react with the arthritis-related autoantigen 60S ribosomal protein L23a (RPL23A). Results A subpopulation of patients with early RA harbored intestinal microbiota dominated by Prevotella copri; SKG mice harboring microbiota from RA patients had an increased number of intestinal Th17 cells and developed severe arthritis when treated with zymosan. Lymphocytes in regional lymph nodes and the colon, but not the spleen, of these mice showed enhanced interleukin-17 (IL-17) responses to RPL23A. Naive SKG mouse T cells cocultured with P copri−stimulated dendritic cells produced IL-17 in response to RPL23A and rapidly induced arthritis. Conclusion We demonstrated that dysbiosis increases sensitivity to arthritis via activation of autoreactive T cells in the intestine. Autoreactive SKG mouse T cells are activated by dysbiotic microbiota in the intestine, causing joint inflammation. Dysbiosis is an environmental factor that triggers arthritis development in genetically susceptible mice.

Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8(-/-) mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8(-/-) mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

Ten specific primer sets, for Lactobacillus gasseri, Lactobacillus crispatus, Atopobium vaginae, Gardnerella vaginalis, Mobiluncus curtisii, Chlamydia trachomatis/muridarum, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium angulatum, were developed for quantitative analysis of vaginal microbiota. rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) analysis of the vaginal samples from 12 healthy Japanese volunteers using the new primer sets together with 25 existing primer sets revealed the diversity of their vaginal microbiota: Lactobacilli such as L. crispatus, L. gasseri, Lactobacillus jensenii, Lactobacillus iners, and Lactobacillus vaginalis, as the major populations at 10(7) cells/ml vaginal fluid, were followed by facultative anaerobes such as Streptococcus and strict anaerobes at lower population levels of 10(4) cells/ml or less. Certain bacterial vaginosis (BV)-related bacteria, such as G. vaginalis, A. vaginae, M. curtisii, and Prevotella, were also detected in some subjects. Especially in one subject, both G. vaginalis and A. vaginae were detected at high population levels of 10(8.8) and 10(8.9) cells/ml vaginal fluid, suggesting that she is an asymptomatic BV patient. These results suggest that the RT-qPCR system is effective for accurate analysis of major vaginal commensals and diagnosis of several vaginal infections.

Bacterial translocation is a major cause of multiple organ dysfunction syndrome in critical illness, and its management is an important therapeutic strategy. In this study, we focused on the key factors responsible for bacterial translocation including the intestinal microbiome and investigated the impact of molecular hydrogen therapy as a countermeasure against bacterial translocation in a murine model of sepsis. The experimental protocols were divided into the sham, saline treatment (control), and hydrogen treatment (H2) groups. In the H2 group, 15 mL/kg of hydrogen-rich saline (7 ppm) was gavaged daily for 7 days following cecal ligation and puncture (CLP). In the control group, normal saline was gavaged in the same way. In the results, the 7-day survival rate was significantly improved in the H2 group versus the control group (69% vs. 31%, P 0.05). The incidence of bacterial translocation at 24 h after CLP as assessed by cultivation of mesenteric lymph nodes and blood was significantly decreased in the H2 group versus the control group. Administration of hydrogen-rich saline also prevented the expansion of facultative anaerobic Enterobacteriaceae and ameliorated intestinal hyperpermeability at 24 h after CLP. Intestinal tissue levels of inflammatory mediators such as inducible nitric oxide synthases, tumor necrosis factor α, interleukin (IL)-1β, IL-6, and oxidative stress marker malondialdehyde at 6 h after CLP were down-regulated in the H2 group. These results suggest luminal administration of hydrogen-rich saline, which prevents intestinal dysbiosis, hyperpermeability, and bacterial translocation, could potentially be a new therapeutic strategy in critical illness.

Bifidobacteria are important members of human gut microbiota; however, quantitative data on their early-life dynamics is limited. Here, using a sensitive reverse transcription-qPCR approach, we demonstrate the carriage of eight signature infant-associated Bifidobacterium species (B. longum, B. breve, B. bifidum, B. catenulatum group, B. infantis, B. adolescentis, B. angulatum and B. dentium) in 76 healthy full-term vaginally-born infants from first day to three years of life. About 21% babies carry bifidobacteria at first day of life (6.2 ± 1.9 log10 cells/g feces); and this carriage increases to 64% (8.0 ± 2.2), 79% (8.5 ± 2.1), 97% (9.3 ± 1.8), 99% (9.6 ± 1.6), and 100% (9.7 ± 0.9) at age 7 days, 1, 3 and 6 months, and 3 years, respectively. B. longum, B. breve, B. catenulatum group and B. bifidum are among the earliest and abundant bifidobacterial clades. Interestingly, infants starting formula-feed as early as first week of life have higher bifidobacterial carriage compared to exclusively breast-fed counterparts. Bifidobacteria demonstrate an antagonistic correlation with enterobacteria and enterococci. Further analyses also reveal a relatively lower/ delayed bifidobacterial carriage in cesarean-born babies. The study presents a quantitative perspective of the early-life gut Bifidobacterium colonization and shows how factors such as birth and feeding modes could influence this acquisition even in healthy infants.

The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those for bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is the de facto standard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio’s circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed of Candida albicans or Saccharomyces cerevisiae and a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.

Summary: Foxp3+ regulatory T (Treg) cells, which suppress immune responses, are highly proliferative in vivo. However, it remains unclear how the active replication of Treg cells is maintained in vivo. Here, we show that branched-chain amino acids (BCAAs), including isoleucine, are required for maintenance of the proliferative state of Treg cells via the amino acid transporter Slc3a2-dependent metabolic reprogramming. Mice fed BCAA-reduced diets showed decreased numbers of Foxp3+ Treg cells with defective in vivo proliferative capacity. Mice lacking Slc3a2 specifically in Foxp3+ Treg cells showed impaired in vivo replication and decreased numbers of Treg cells. Slc3a2-deficient Treg cells showed impaired isoleucine-induced activation of the mTORC1 pathway and an altered metabolic state. Slc3a2 mutant mice did not show an isoleucine-induced increase of Treg cells in vivo and exhibited multi-organ inflammation. Taken together, these findings demonstrate that BCAA controls Treg cell maintenance via Slc3a2-dependent metabolic regulation. : Treg cells regulate excess immune responses and are highly proliferative in vivo. Ikeda et al. find that branched-chain amino acids (BCAAs) are essentially required to maintain expansion and the suppressive capacity of Treg cells via Slc3a2 and mTORC1. Keywords: Treg cells, amino acids, immunometabolism, immune regulation, transporter

To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

A sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was developed for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli by using specific primers. Counts of the enteric pathogens spiked in human stools were quantified at the lower detection limit of 10(3) cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 10(5) to 10(6) cells/g stool. The bacterial counts determined by RT-qPCR were almost equivalent to those determined by the culture method and fluorescence in situ hybridization (FISH) during the course of in vitro culture. Bacterial rRNA in the stools was stable for at least 4 weeks when the stools were kept as the suspensions in RNA-stabilizing agent, RNAlater®, even at 37(o) C. These data suggested that the rapid and high sensitive rRNA-targeted RT-qPCR was applicable for the accurate quantification of viable enteric pathogens, such as V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli.

VBNC Vibrio cholerae O139 VC-280 obtained by incubation in 1% solution of artificial sea water IO at 4°C for 74 days converted to the culturable state when co-cultured with CHO cells. Other eukaryotic cell lines, including HT-29, Caco-2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC-280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC-280 was incubated in 1% IO at 4°C for up to 91 days, the number of cells converted by co-culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed.

The growth of plants depends on continuous function of the meristems. Shoot meristems are responsible for all the post-embryonic aerial organs, such as leaves, stems and flowers. It has been assumed that the phytohormone cytokinin has a positive role in shoot meristem function. A severe reduction in the size of meristems in a mutant that is defective in all of its cytokinin receptors has provided compelling evidence that cytokinin is required for meristem activity. Here, we report a novel regulation of meristem activity, which is executed by the meristem-specific activation of cytokinins. The LONELY GUY (LOG) gene of rice is required to maintain meristem activity and its loss of function causes premature termination of the shoot meristem. LOG encodes a novel cytokinin-activating enzyme that works in the final step of bioactive cytokinin synthesis. Revising the long-held idea of multistep reactions, LOG directly converts inactive cytokinin nucleotides to the free-base forms, which are biologically active, by its cytokinin-specific phosphoribohydrolase activity. LOG messenger RNA is specifically localized in shoot meristem tips, indicating the activation of cytokinins in a specific developmental domain. We propose the fine-tuning of concentrations and the spatial distribution of bioactive cytokinins by a cytokinin-activating enzyme as a mechanism that regulates meristem activity.

During a double-blind, randomized, placebo-controlled probiotic trial among 3758 children residing in an urban slum in Kolkata, India, Vibrio cholerae/mimicus was detected in fecal microbiota of healthy children. The importance of this finding in the local, regional and global transmission of cholera is discussed.

Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet.

Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyer's patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyer's patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

Extracellular ATP is released from live cells in controlled conditions, as well as dying cells in inflammatory conditions, and, thereby, regulates T cell responses, including Th17 cell induction. The level of extracellular ATP is closely regulated by ATP hydrolyzing enzymes, such as ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases). ENTPDase1/CD39, which is expressed in immune cells, was shown to regulate immune responses by downregulating the ATP level. In this study, we analyzed the immunomodulatory function of ENTPDase7, which is preferentially expressed in epithelial cells in the small intestine. The targeted deletion of Entpd7 encoding ENTPDase7 in mice resulted in increased ATP levels in the small intestinal lumen. The number of Th17 cells was selectively increased in the small intestinal lamina propria in Entpd7−/− mice. Th17 cells were decreased by oral administration of antibiotics or the ATP antagonist in Entpd7−/− mice, indicating that commensal microbiota-dependent ATP release mediates the enhanced Th17 cell development in the small intestinal lamina propria of Entpd7−/− mice. In accordance with the increased number of small intestinal Th17 cells, Entpd7−/− mice were resistant to oral infection with Citrobacter rodentium. Entpd7−/− mice suffered from severe experimental autoimmune encephalomyelitis, which was associated with increased numbers of CD4+ T cells producing both IL-17 and IFN-γ. Taken together, these findings demonstrate that ENTPDase7 controls the luminal ATP level and, thereby, regulates Th17 cell development in the small intestine.

Pyrosequencing-based 16S rRNA profiling has become a common powerful tool to obtain the community structure of gastrointestinal tract microbiota, but it is still hard to process the massive amount of sequence data into microbial composition data, especially at the species level. Here we propose a new approach in combining the quantitative insights into microbial ecology (QIIME), Mothur and ribosomal database project (RDP) programs to efficiently process 454 pyrosequence data to bacterial composition data up to the species level. It was demonstrated to precisely convert batch sequence data of 16S rRNA V6-V8 amplicons obtained from adult Singaporean fecal samples to taxonomically annotated biota data.

During a double-blind, randomized, placebo-controlled probiotic trial among 3758 children residing in an urban slum in Kolkata, India, Vibrio cholerae/mimicus was detected in fecal microbiota of healthy children. The importance of this finding in the local, regional and global transmission of cholera is discussed.

A sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was developed for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli by using specific primers. Counts of the enteric pathogens spiked in human stools were quantified at the lower detection limit of 10(3) cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 10(5) to 10(6) cells/g stool. The bacterial counts determined by RT-qPCR were almost equivalent to those determined by the culture method and fluorescence in situ hybridization (FISH) during the course of in vitro culture. Bacterial rRNA in the stools was stable for at least 4 weeks when the stools were kept as the suspensions in RNA-stabilizing agent, RNAlater®, even at 37(o) C. These data suggested that the rapid and high sensitive rRNA-targeted RT-qPCR was applicable for the accurate quantification of viable enteric pathogens, such as V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli.

An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) for enumeration of catalase-negative, Gram-positive cocci was established. Subgroup- or species-specific primer sets targeting 16S or 23S rRNA from Enterococcus , Streptococcus , and Lactococcus were newly developed. The RT-qPCR method using these primers together with the previously reported primer sets specific for the Enterococcus genus, the Streptococcus genus, and several Streptococcus species was found to be able to quantify the target populations with detection limits of 10 3 to 10 4 cells per gram feces, which was more than 100 times as sensitive as the qPCR method (10 6 to 10 8 cells per gram feces). The RT-qPCR analysis of fecal samples from 24 healthy adult volunteers using the genus-specific primer sets revealed that Enterococcus and Streptococcus were present as intestinal commensals at population levels of log 10 6.2 ± 1.4 and 7.5 ± 0.9 per gram feces (mean ± standard deviation [SD]), respectively. Detailed investigation using species- or subgroup-specific primer sets revealed that the volunteers harbored unique Enterococcus species, including the E. avium subgroup, the E. faecium subgroup, E. faecalis , the E. casseliflavus subgroup, and E. caccae , while the dominant human intestinal Streptococcus species was found to be S. salivarius . Various Lactococcus species, such as L. lactis subsp. lactis or L. lactis subsp. cremoris , L. garvieae , L. piscium , and L. plantarum , were also detected but at a lower population level (log 10 4.6 ± 1.2 per gram feces) and prevalence (33%). These results suggest that the RT-qPCR method enables the accurate and sensitive enumeration of human intestinal subdominant but still important populations, such as Gram-positive cocci.