Use of gnotobiotic mice to identify and characterize key microbes responsible for the development of the intestinal immune system

Symbiosis between intestinal microbiota and the host animal plays an important role in the homeostasis of host physiology. Since the first production of germ-free rodents in 1945, it has become increasingly clear that the intestinal immune system and the biochemical characteristics of epithelial cells differ greatly between conventional and germ-free rodents. However, questions remain about the types of microbes involved and the precise mechanism by which these microbes affect the host physiology. Here, we review experiments designed to answer these questions with the use of gnotobiotic mice. We have determined suitable biochemical and immunological markers for monitoring microbial effects in these mice. Using these markers, we have found clear differences in epithelial cell glycolipid biosynthesis and intraepithelial lymphocyte dynamics between germ-free and conventional mice. Furthermore, we have identified a key microbe that activates the mucosal immune system in the small intestine. This indigenous bacteria, called segmented filamentous bacteria, is a key symbiont in the host-microbiota interplay, including Th17 cell-inducing activity.

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title: Metagenomic Analysis of the Human Distal Gut Microbiome
abstract: The human intestinal microbiota is composed of 1013 to 1014 microorganisms whose collective genome (“microbiome”) contains at least 100 times as many genes as our own genome. We analyzed ∼78 million base pairs of unique DNA sequence and 2062 polymerase chain reaction–amplified 16S ribosomal DNA sequences obtained from the fecal DNAs of two healthy adults. Using metabolic function analyses of identified genes, we compared our human genome with the average content of previously sequenced microbial genomes. Our microbiome has significantly enriched metabolism of glycans, amino acids, and xenobiotics; methanogenesis; and 2-methyl-d-erythritol 4-phosphate pathway–mediated biosynthesis of vitamins and isoprenoids. Thus, humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes.

title: Colonic bacterial flora: changing understandings in the molecular age.
abstract: The human intestinal microbiota is a complex bacterial consortium that is critical to normal health. The microflora is present at concentrations of 10(11)-10(12) cells/g of intestinal contents; the number of species present may exceed 500, although exact numbers remain to be defined, due in part to the fact that <30% of microorganisms are culturable with current microbiologic methods. Molecular tools based on 16S rDNA sequence similarities such as fluorescent in-situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE), quantitative dot blot hybridization, restriction fragment length polymorphism (RFLP) and large scale 16S rDNA sequencing have helped to overcome limitations of conventional microbiological plating methods in studying the fecal microflora composition. However, these tools are just now beginning to be applied to understand the dynamics of this complex community, and its relationship to diet and human health. There is a need to understand both the limitations of the current data and the importance of moving forward with the best possible molecular and epidemiologic techniques as we deal with these critical questions.

title: Establishment of an Analytical System for the Human Fecal Microbiota, Based on Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNA Molecules
abstract: An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 102 to 104 cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 108 cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RT-qPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log10(6.3 ± 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.

title: Enterotypes of the human gut microbiome
abstract: This corrects the article DOI: 10.1038/nature09944

title: Intestinal microbiota, diet and health
abstract: The human intestine is colonised by 1013 to 1014 micro-organisms, the vast majority of which belong to the phyla Firmicutes and Bacteroidetes. Although highly stable over time, the composition and activities of the microbiota may be influenced by a number of factors including age, diet and antibiotic treatment. Although perturbations in the composition or functions of the microbiota are linked to inflammatory and metabolic disorders (e.g. inflammatory bowel diseases, irritable bowel syndrome and obesity), it is unclear at this point whether these changes are a symptom of the disease or a contributing factor. A better knowledge of the mechanisms through which changes in microbiota composition (dysbiosis) promote disease states is needed to improve our understanding of the causal relationship between the gut microbiota and disease. While evidence of the preventive and therapeutic effects of probiotic strains on diarrhoeal illness and other intestinal conditions is promising, the exact mechanisms of the beneficial effects are not fully understood. Recent studies have raised the question of whether non-viable probiotic strains can confer health benefits on the host by influencing the immune system. As the potential health effect of these non-viable bacteria depends on whether the mechanism of this effect is dependent on viability, future research needs to consider each probiotic strain on a case-by-case basis. The present review provides a comprehensive, updated overview of the human gut microbiota, the factors influencing its composition and the role of probiotics as a therapeutic modality in the treatment and prevention of diseases and/or restoration of human health.

title: TREATMENT OF ULCERATIVE COLITIS BY IMPLANTATION OF NORMAL COLONIC FLORA
abstract: His glomerular filtration rate was 16 ml/min. Significant numbers of Candida albicans (more than 108 organisms/1) were recovered from several midstream urine specimens and from a renal papilla passed per urethrum; blood cultures were sterile. No other organisms were isolated. The candida was fully sensitive to antifungal agents and the patient was given a single dose of fluconazole 3 mg/kg. 24 h and 96 h serum trough levels were 3-8 mg/1 and 2.0 mg/1, respectively (the minimum inhibitory concentration for the strain was 0-8 mg/1). This dose of fluconazole was, therefore, continued every 96 h with regular monitoring. Rapid clinical and microbiological response ensued, urine cultures becoming negative at 4 days and remaining so while the patient was

title: Safety, Tolerability, and Clinical Response After Fecal Transplantation in Children and Young Adults With Ulcerative Colitis
abstract: Colonic dysbiosis contributes to the development of colonic inflammation in ulcerative colitis (UC). Fecal microbial transplantation (FMT) is being proposed as a novel treatment for UC because it can eliminate dysbiosis; however, no prospective data exist. We initiated a pilot study to evaluate feasibility and safety of FMT in children with UC.  Ten children, 7 to 21 years of age, with mild-to-moderate UC (pediatric UC activity index [PUCAI] between 15 and 65) received freshly prepared fecal enemas daily for 5 days. Data on tolerability, adverse events, and disease activity were collected during FMT and weekly for 4 weeks after FMT. Clinical response was defined as decrease in PUCAI by >15, and decrease in PUCAI to <10 was considered clinical remission.  No serious adverse events were noted. Mild (cramping, fullness, flatulence, bloating, diarrhea, and blood in stool) to moderate (fever) adverse events were self-limiting. One subject could not retain fecal enemas. Average tolerated enema volume by remaining 9 subjects was 165 mL/day. After FMT, 7 of the 9 (78%) subjects showed clinical response within 1 week, 6 of the 9 (67%) subjects maintained clinical response at 1 month, and 3 of the 9 (33%) subjects achieved clinical remission at 1 week after FMT. Median PUCAI significantly improved after FMT (P = 0.03) compared with the baseline.  Fecal enemas were feasible and tolerated by children with UC. Adverse events were acceptable, self-limiting, and manageable by subjects. FMT indicated efficacy in the treatment of UC.

title: Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome.
abstract: Alterations in intestinal microbiota are associated with obesity and insulin resistance. We studied the effects of infusing intestinal microbiota from lean donors to male recipients with metabolic syndrome on the recipients' microbiota composition and glucose metabolism. Subjects were assigned randomly to groups that were given small intestinal infusions of allogenic or autologous microbiota. Six weeks after infusion of microbiota from lean donors, insulin sensitivity of recipients increased (median rate of glucose disappearance changed from 26.2 to 45.3 μmol/kg/min; P < .05) along with levels of butyrate-producing intestinal microbiota. Intestinal microbiota might be developed as therapeutic agents to increase insulin sensitivity in humans; www.trialregister.nl; registered at the Dutch Trial Register (NTR1776).

title: Life span, morphology, and pathology of diet-restricted germ-free and conventional Lobund-Wistar rats.
abstract: The effect of germ-free life and dietary restriction (DR) on life span and pathology was investigated in isolator housed germ-free (GF) and conventional (CV) Lobund-Wistar rats fed either ad libitum or restricted to 12 grams per day (70% of adult ad libitum intake) of a natural ingredient diet from weaning. The median length of life of ad libitum CV and GF rats was 31.0 and 33.6 months respectively, while DR increased the median length of life of CV and GF rats to 38.6 and 37.8 months respectively. DR reduced the frequency or postponed the occurrence of diseases which eventually lead to death in the Lobund-Wistar rat. This was especially true of prostate adenocarcinoma, prostatitis, and mammary fibroma. The reduced early food intake and smaller body weight of adult GF rats may be the reason ad libitum fed GF rats live slightly longer than their CV counterparts, but GF life was without additional effect on life span when food intake was restricted.

title: Microbial modulation of innate defense: goblet cells and the intestinal mucus layer.
abstract: The gastrointestinal epithelium is covered by a protective mucus gel composed predominantly of mucin glycoproteins that are synthesized and secreted by goblet cells. Changes in goblet cell functions and in the chemical composition of intestinal mucus are detected in response to a broad range of luminal insults, including alterations of the normal microbiota. However, the regulatory networks that mediate goblet cell responses to intestinal insults are poorly defined. The present review summarizes the results of developmental, gnotobiotic, and in vitro studies that showed alterations in mucin gene expression, mucus composition, or mucus secretion in response to intestinal microbes or host-derived inflammatory mediators. The dynamic nature of the mucus layer is shown. Available data indicate that intestinal microbes may affect goblet cell dynamics and the mucus layer directly via the local release of bioactive factors or indirectly via activation of host immune cells. A precise definition of the regulatory networks that interface with goblet cells may have broad biomedical applications because mucus alterations appear to characterize most diseases of mucosal tissues.

title: Bacterial colonization leads to the colonic secretion of RELMbeta/FIZZ2, a novel goblet cell-specific protein.
abstract: Goblet cells are highly polarized exocrine cells found throughout the small and large intestine that have a characteristic morphology due to the accumulation of apical secretory granules. These granules contain proteins that play important physiologic roles in cellular protection, barrier function, and proliferation. A limited number of intestinal goblet cell-specific proteins have been identified. In this study, we investigate the expression and regulation of RELMbeta, a novel colon-specific gene.



The regulation of RELMbeta messenger RNA expression was determined in LS174T, Caco-2, and HT-29 cell lines in response to stimulation with interleukin 13 and lipopolysaccharide. Quantitative reverse-transcription polymerase chain reaction, immunoblots, and immunohistochemistry were used to examine the expression of RELMbeta in BALB/c and C.B17.SCID mice housed in conventional, germ-free, and gnotobiotic environments.



Messenger RNA for RELMbeta is restricted to the undifferentiated, proliferating colonic epithelium. Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon and cecum with lower levels detectable in the proximal colon. High levels of RELMbeta can be detected in the stool of mice and humans, where it exists as a homodimer under nonreducing conditions. Interestingly, the secretion of RELMbeta is dramatically reduced in germ-free mice. Furthermore, introduction of germ-free mice into a conventional environment results in enhanced expression and robust secretion of RELMbeta within 48 hours.



These studies define a new goblet cell-specific protein and provide the first evidence that colon-specific gene expression can be regulated by colonization with normal enteric bacteria.

title: Microflora modulates endocrine cells in the gastrointestinal mucosa of the rat.
abstract: Gastrointestinal peptides and biogenic monoamines participate in the regulation of gastrointestinal functions. The aim of this study was to examine the influence of the microflora on the distribution of endocrine cells and on the release of gastrointestinal peptides.



A quantitative morphological study using stereological methods was performed in gastrointestinal sections of conventional and germ-free rats. Tissue and plasma concentrations of peptides were measured.



The total volumes of gastrin- and serotonin-immunoreactive cells were significantly increased in the gastric mucosa of germfree rats (P < 0.05), as well as the total volumes of serotonin- and motilin-immunoreactive cells in the ileum (P < 0.05) and serotonin-immunoreactive cells in the colonic mucosa (P < 0.05). The tissue concentration of somatostatin was significantly higher in the jejunum (P < 0.05) and lower in the ileum of germfree rats than in controls (P < 0.05). Plasma glucagon was significantly increased in germfree rats (P < 0.05). The total volume of the fundic mucosa was enlarged in germfree rats (P < 0.05), whereas the total volume, the mucosal thickness, and the number of crypt cells of the colonic mucosa were significantly reduced in these rats compared with controls (P < 0.05).



Our findings suggest that the intraluminal microflora influences the release of biologically active peptides and that it participates in the regulation of gastrointestinal endocrine cells and the epithelial structure.

title: SMALL INTESTINAL MUCOSAL CELL PROLIFERATION AND BACTERIAL FLORA IN THE CONVENTIONALIZATION OF THE GERMFREE MOUSE
abstract: The relationship between intestinal colonization and the small bowel mucosal cellular proliferation rate during conventionalization of the germfree mouse was examined. 16 mice were maintained under standard germfree conditions, and 54 others were conventionalized. Migration of the small bowel epithelial cells was followed by radioautography with administration of tritiated thymidine. Colonization was followed by qualitative and quantitative bacteriological fecal analyses. The percentages of the villi labeled (as determined by cell count) 24, 48, and 72 hr following thymidine administration showed immediate progression in the conventionalized animals from the germfree villus migration time (4 days) toward the conventional villus migration time (2 days). The epithelial migration rate of animals conventionalized for 8 days was comparable to that of conventional animals. After conventionalization, aerobic and anaerobic organisms undergo a period of extensive multiplication; however, 72 hr later the number of these microorganisms cultured in the stool decrease and are similar to those recovered from normal animals. Coliforms and streptococci are recovered in large numbers only in the first days after conventionalization, while the Bacteroides are first recovered in significant numbers on the fifth day of conventionalization. Except for smaller numbers of Bacteroides, the bacterial populations in the stools of the conventionalized animals are qualitatively and quantitatively similar by the eighth day of conventionalization to those of true conventional mice. Adaptive balance between cell proliferation and sloughing, and thus migration rate, begins immediately after conventionalization of germfree animals as bacterial populations establish themselves throughout the gastrointestinal tract, and results in a doubling of the mucosal cell turnover after 8 days. At this time both the small intestinal epithelial cell migration rate and the intestinal microflora are similar to those of conventional animals.

title: The effects of small intestinal colonization by fecal and colonic bacteria on intestinal function in rats.
abstract: The effects of colonic and fecal bacterial proliferation on jejunal function were studied in normal rats and in low-germ rats after intraperitoneal injections of mecamylamine HCl. Jejunal bacteriology, bile salts, ultrastructure, and transport capacity were assessed. Normal rats given mecamylamine for 3 days had increased anaerobic bacteria in the intestinal fluid, and had high concentrations of deconjugated bile salts in the intraluminal contents. Jejunal bacteria were lodged between microvilli without penetrating the cell cytoplasm. However, there was focal cellular damage, including fused microvilli, dilated endoplasmic reticulum, and secondary lysosomes. In the mecamylamine treated normal rats intestinal glucose transport was reduced with an alteration compatible with noncompetitive inhibition. The absorption rates of galactose, fructose, 3-0-methyl-D-glucose, tyrosine, Na, and K were also decreased. In contrast, low-germ mecamylamine-treated rats showed no evidence of either increased anaerobic bacterial proliferation or deconjugation of bile salts, and had none of the fine structural alterations seen in regularly raised rats. Also, the transport of carbohydrates was unaltered. The findings suggest that non-invasive enteric proliferation of colonic and fecal bacterial anaerobes in rats may be associated with deconjugation of bile salts, ultrastructural alterations of the intestinal epithelial cells, and a diminished jejunal transport capacity of carbohydrates and other solutes.

title: Role of bile acids in colorectal carcinogenesis.
abstract: Dietary factors are considered important environmental risk determinants for colorectal cancer development. Epidemiological studies have shown that a high fat (or meat) intake is associated positively and a high starch, fibre (non-starch polysaccharide), vegetable and fruit intake negatively with colorectal cancer incidence. One mechanism by which these effects are possibly exerted is through the metabolism of secondary bile acids. Secondary bile acids are formed after enzymatic deconjugation and dehydroxylation of primary bile acids in the large bowel by anaerobic bacteria. It has been shown that these compounds can have tumour-promoting capacities in animal experiments. In epidemiological studies, colonic cancer risk is related to the faecal bile acid concentration. In serum and bile of patients with colonic adenomas, more deoxycholic acid was detected than in healthy controls. Secondary bile acids are toxic to several cell systems at physiological concentrations. The exact mechanism by which these amphiphilic molecules exert their action is not well understood. It might act through membrane damage, intracellular mitochondrial action or genotoxic effects. So far the evidence that bile acids are involved in colonic carcinogenesis is largely circumstantial. It is, however, well accepted that environmental factors, such as dietary habits influence genetic susceptibility. Bile acids could play a promoting role in this process.

title: Normal gut microbiota modulates brain development and behavior
abstract: Microbial colonization of mammals is an evolution-driven process that modulate host physiology, many of which are associated with immunity and nutrient intake. Here, we report that colonization by gut microbiota impacts mammalian brain development and subsequent adult behavior. Using measures of motor activity and anxiety-like behavior, we demonstrate that germ free (GF) mice display increased motor activity and reduced anxiety, compared with specific pathogen free (SPF) mice with a normal gut microbiota. This behavioral phenotype is associated with altered expression of genes known to be involved in second messenger pathways and synaptic long-term potentiation in brain regions implicated in motor control and anxiety-like behavior. GF mice exposed to gut microbiota early in life display similar characteristics as SPF mice, including reduced expression of PSD-95 and synaptophysin in the striatum. Hence, our results suggest that the microbial colonization process initiates signaling mechanisms that affect neuronal circuits involved in motor control and anxiety behavior.

title: Biosynthesis of microvillus membrane-associated glycoproteins of small intestinal epithelial cells in germ-free and conventionalized mice.
abstract: We studied the effect of intestinal microorganisms on the synthesis of membrane-associated glycoproteins in the upper small intestine by intraperitoneally administering L-[3H]fucose, D-[14C]glucosamine, or L-[3H]leucine to germ-free mice and mice exposed to microorganisms for 4 weeks (conventionalized). The incorporation of the labeled compounds into sucrase-isomaltase complex and maltase was determined by immunoprecipitating Triton X-100-solubilized microvillus membranes with their antibodies. Purified microvillus membranes from germ-free and conventionalized mice differed in the activities of some marker enzymes but not in the number and mobility of the components on SDS-polyacrylamide gel electrophoresis. Maximal incorporation of [3H]fucose and [14C]glucosamine into the microvillus membrane and two enzymes was reached 2-3 h post-injection in both groups, however, the amounts incorporated were larger in conventionalized mice. There was little difference in [3H]leucine incorporation into the total glycoproteins of microvillus membranes between the two groups. Our results suggest that the introduction of microorganisms stimulates the synthesis of sugar chains of microvillus membrane-associated glycoproteins. The enhanced in vitro fucosyltransferase activity in conventionalized mice partly supports this suggestion.

title: The skin microbiome
abstract: (2011) It has been brought to our attention that in Fig. 1 of the original article the morphology and localization of the Demodex mites were not accurate. We have corrected the figure to show a cartoon that is more representative of the straight body and short limbs of these mites, and of their localization in the hair follicle.

title: Appearance of fucolipid after conventionalization of germ-free mice.
abstract: Incorporation of [6-3H]fucose and [1-14C]glucosamine into the lipid fraction of microvillus membrane (MVM) was studied in germ-free and conventionalized mice after intraperitoneal injection of the radioactive precursors. Incorporation of [6-3H]fucose was clearly detected in conventionalized mice but not detectable in germ-free mice. There was no difference in the incorporation of [1-14C]glucosamine between the two groups. The lipid fraction of MVM labeled with [6-3H]fucose showed a spot of slower mobility than asialo GM1 on TLC with autoradiography and it was confirmed to be a fucolipid on abolishing the radiolabeled original spot by alpha-L-fucosidase treatment. These results suggest that the synthesis of the fucolipid is induced by conventionalization of germ-free mice.

title: Presence of asialo GM1 and glucosylceramide in the intestinal mucosa of mice and induction of fucosyl asialo GM1 by conventionalization of germ-free mice.
abstract: Two major neutral glycolipids of the intestinal mucosa were purified by a series of column chromatographies and the structures were determined to be glucosylceramide and asialo GM1 by gas liquid chromatography. The carbohydrate structure of asialo GM1 was also confirmed from the reactivity of the glycolipid to rabbit anti-asialo GM1 antibody by an enzyme linked-immunosorbent antibody. The ceramide portion of both glycolipids had an extremely hydrophilic nature and more than 90% of the ceramide was composed of phytosphingosine and alpha-hydroxy fatty acids. In the previous paper we reported that the induction of a fucolipid in the microvillus membrane of mouse intestinal mucosa by conventionalization was observed on monitoring the incorporation of radiolabeled fucose in vivo. A fucoglycolipid having the same mobility on an autoradiogram as the fucolipid labeled in vivo was produced by in vitro incubation of intestinal asialo GM1 and GDP-[14C]fucose with the mucosal homogenates. Moreover, asialo GM1 prepared from brain gangliosides exhibited a similar ability to accept fucose and it was converted to fucosyl asialo GM1 which moved faster than the product from intestinal asialo GM1. The difference is considered to be due to the ceramide composition. These results suggest that conventionalization can induce the fucosyl asialo GM1 in the microvillus membrane probably through the induction of fucosyltransferase. Understanding the molecular mechanism of interaction between the physiological flora and host is the matter of further study.

title: Presence of phytosphingosine combined with 2-hydroxy fatty acids in sphingomyelins of bovine kidney and intestinal mucosa
abstract: A minor part of sphingomyelins of bovine kidney and small intestine has been shown by mass spectrometry to contain phytosphingosine in the earlier unknown combination with 2-hydroxy fatty acids.

title: Abrupt induction of GDP-fucose: asialo GM1 fucosyltransferase in the small intestine after conventionalization of germ-free mice.
abstract: We studied the time-course of the induction of GDP-fucose: asialo GM1 fucosyltransferase and its product, i.e. fucosyl asialo GM1, of the small intestine after introduction of microorganisms to germ-free mice (conventionalization). We found that the fucosyltransferase activity was abruptly induced and asialo GM1 was converted into fucosyl asialo GM1 within a few days after conventionalization. However, two weeks after conventionalization this enzyme activity dropped to approximately 10−2 level of the maximum value and asialo GM1 appeared again as one of the major glycolipids. These results showed that the microbial colonization in the gut evoked a drastic change of the glycolipid pattern at the intestinal epithelial cell-surface via the induction of a fucosyltransferase.

title: Factors regulating the expression of the neutral glycolipids in the mouse small intestinal mucosa.
abstract: GDP-fucose:asialo GM1 alpha(1-2)fucosyltransferase (FT) is induced in the small intestinal mucosa after microbial contamination of germ-free mice (Umesaki, Y., Sakata, T. and Yajima, T. (1982) . As a result, asialo GM1 glycolipid, a major component of the epithelial cell membrane, drastically converted into fucosyl asialo GM1. There were many other examples in which FT was induced. One was the weaning period for conventional mice. Others included injuries of the small intestine by punctures or administration of cytosine arabinoside, and the injection of protein synthesis inhibitors, such as cycloheximide or emetine, or the soluble fraction of the small intestinal homogenate (SISF). The induction of FT was more rapid after injection of cycloheximide or SISF than after injury, mechanical puncturing or after administration of cytosine arabinoside. The changes in the neutral glycolipids of the small intestine by injection of cycloheximide or SISF were analyzed in detail. FT activity started to increase after approx. 5 h and reached the maximum 10-12 h after injection of cycloheximide or SISF, and rapidly declined thereafter. The conversion of asialo GM1 into fucosyl asialo GM1 started after about 10 h and reached the maximal value 24 h after the treatment. Fucosyl asialo GM1 persisted for a few days, although the FT activity fell to near the basal level. On the other hand, the amount of glucosyl ceramide was constant after these treatments. There was little difference in the time-courses of both the FT activity and the glycolipid conversion between these treatments. In the case of co-injection of cycloheximide and SISF, the effect of both materials on FT activity induction was synergistic. The distribution of FT activity and immunohistochemical staining using anti-fucosyl asialo GM1 antibody along the crypt-villus axis showed a stronger expression of fucosyl asialo GM1 in villus portion, the post-mitotic cell zone, than in the crypt portion. Asialo GM1 was converted into fucosyl asialo GM1 after the induction of FT by the various treatments mentioned above. Especially the effects of cycloheximide and/or SISF on FT induction suggest at least the presence of a regulatory protein(s) which controls the glycolipid expression in the small intestine.

title: Intestinal, segmented, filamentous bacteria in a wide range of vertebrate species
abstract: Segmented, filamentous bacteria (SFBs) form a group of bacteria with similar morphology and are identified on the basis of their morphology only. The relationships of these organisms are unclear as the application of formal taxonomic criteria is impossible currently due to the lack of an in vitro technique to culture SFBs. The intestine of laboratory animals such as mice, rats, chickens, dogs, cats and pigs is known to harbour SFBs. To see whether this extends to other animal species, intestines from 18 vertebrate species, including man, were examined. SFBs were detected with light microscopy in the cat, dog, rhesus monkey, crab-eating macaque, domestic fowl, South African claw-footed toad, carp, man, laboratory mouse and rat, wood mouse, jackdaw and magpie. These results suggest that non-pathogenic SFBs are ubiquitous in the animal kingdom. Among apparently identical animals, there was considerable variation in the degree of SFB colonization. It is suggested that SFB colonization could serve as a criterion of standardization of laboratory animals.

title: 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
abstract: In ex‐germ‐free mice conventionalized by association with fecal microorganisms, the induction of 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 αβ 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, segmented 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 αβTCR‐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.

title: A Model of Host-Microbial Interactions in an Open Mammalian Ecosystem
abstract: The maintenance and significance of the complex populations of microbes present in the mammalian intestine are poorly understood. Comparison of conventionally housed and germ-free NMRI mice revealed that production of fucosylated glycoconjugates and an α1,2-fucosyltransferase messenger RNA in the small-intestinal epithelium requires the normal microflora. Colonization of germ-free mice with Bacteroides thetaiotaomicron, a component of this flora, restored the fucosylation program, whereas an isogenic strain carrying a transposon insertion that disrupts its ability to use L-fucose as a carbon source did not. Simplified models such as this should aid the study of open microbial ecosystems.

title: Immunohistochemical and biochemical demonstration of the change in glycolipid composition of the intestinal epithelial cell surface in mice in relation to epithelial cell differentiation and bacterial association.
abstract: We have previously demonstrated the appearance of fucosyl asialo-GM1 (FGA1) in the small-intestinal epithelial cells of germ-free mice via the induction of GDP-fucose: asialo-GM1 (GA1) alpha(1 leads to 2) fucosyltransferase (FT) after the conventionalization of these animals (Umesaki Y, Sakata T, Yajima  1982). The present study, based on this earlier work, demonstrates the changes in the glycolipid antigens of the small-intestinal epithelial-cell membrane as shown immunohistochemically with specific antibodies raised against asialo GM1 (GA1) and FGA1. In germ-free mice, GA1 was localized both in the villus cells and in the crypt cells. In the process of conventionalization, FGA1 appeared in the villus cells while the GA1 content of these cells was decreased. Four to 5 days after the conventionalization procedure, the fluorescence produced by anti-FGA1 was strongest in the villus cells, while that produced by anti-GA1 was detected only in the crypt cells. At this same time the FT activity of the small-intestinal mucosa was highest, with most of the GA1 apparently being converted into FGA1, as shown in the paper cited above. Thereafter, the GA1 content of both the villus and crypt cells again increased greatly. On the other hand, the fluorescence produced with anti-FGA1 decreased, and could no longer be detected 14 days after conventionalization. The activity of FT, measured biochemically in epithelial cells differentially isolated from the villus tip to the crypt, was greater in the villus than in the crypt region. This confirmed the intense staining with anti-FGA1 that was seen in villus cells. The fluorescence produced by the two anti-glycolipid antibodies used in the study distributed not only in the microvillus membrane but also to some extent in the basolateral membrane. The localization of the respective glycolipids contrasted with that of the glycoprotein sucrase--isomaltase enzyme complex, the fluorescence of which was exclusively confined to the microvillus-membrane side of the villus cells.

title: Superinduction of interferon with metabolic inhibitors: possible mechanisms and practical applications.
abstract: Ten years have passed since cycloheximide was first shown to enhance endotoxin-induced production of interferon in mice, in the first demonstration of what was later called the superinduction of interferon. Various inhibitors of protein and RNA symthesis, as well as combinations of these inhibitors, have been shown to act as superinducing agents of interferon production stimulated by polyriboinosinic-polyribocytidylic acid. The evidence that superinduction is due to the suppression of a mechanism of post-transcriptional regulation of interferon synthesis is rather convincing. The notion (first proposed about six years ago) that this modification is caused by a protein "repressor" remains the most plausible, albeit still unproved, hypothesis. The availability of systems that translate with fidelity interferon messenger RNA isolated from induced cells should prove most useful in the elucidation of post-transcriptional control mechanisms of interferon synthesis and of interferon superinduction. Meanwhile, superinduction has become a useful tool for the production of large quantities of interferon. In particular, this technique has been successfully applied to the production of interferon from human diploid fibroblasts. The clinical potential of this material remains to be critically examined.

title: Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.
abstract: Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues.

title: Colonic mucosa-associated microbiota is influenced by an interaction of Crohn disease and FUT2 (Secretor) genotype
abstract: The FUT2 (Secretor) gene is responsible for the presence of ABO histo-blood group antigens on the gastrointestinal mucosa and in bodily secretions. Individuals lacking a functional copy of FUT2 are known as “nonsecretors” and display an array of differences in susceptibility to infection and disease, including Crohn disease. To determine whether variation in resident microbial communities with respect to FUT2 genotype is a potential factor contributing to susceptibility, we performed 454-based community profiling of the intestinal microbiota in a panel of healthy subjects and Crohn disease patients and determined their genotype for the primary nonsecretor allele in Caucasian populations, W143X (G428A). Consistent with previous studies, we observe significant deviations in the microbial communities of individuals with Crohn disease. Furthermore, the FUT2 genotype explains substantial differences in community composition, diversity, and structure, and we identified several bacterial species displaying disease-by-genotype associations. These findings indicate that alterations in resident microbial communities may in part explain the variety of host susceptibilities surrounding nonsecretor status and that FUT2 is an important genetic factor influencing host–microbial diversity.

title: Fucosyltransferase 2 non-secretor and low secretor status predicts severe outcomes in premature infants.
abstract: To investigate secretor gene fucosyltransferase 2 (FUT2) polymorphism and secretor phenotype in relation to outcomes of prematurity.



Study infants were ≤32 weeks gestational age. Secretor genotype was determined from salivary DNA. Secretor phenotype was measured with H antigen, the carbohydrate produced by secretor gene enzymes, in saliva samples collected on day 9 ± 5. The optimal predictive cutoff point in salivary H values was identified with Classification and Regression Tree analysis. Study outcomes were death, necrotizing enterocolitis (NEC, Bell's stage II/III), and confirmed sepsis.



There were 410 study infants, 26 deaths, 30 cases of NEC, and 96 cases of sepsis. Analyzed by genotype, 13% of 95 infants who were non-secretors, 5% of 203 infants who were heterozygotes, and 2% of 96 infants who were secretor dominant died (P = .01). Analyzed by phenotype, 15% of 135 infants with low secretor phenotype died, compared with 2% of 248 infants with high secretor phenotype (predictive value = 76%, P < .001). Low secretor phenotype was associated (P < .05) with NEC, and non-secretor genotype was associated (P = .05) with gram negative sepsis. Secretor status remained significant after controlling for multiple clinical factors.



Secretor genotype and phenotype may provide strong predictive biomarkers of adverse outcomes in premature infants.

title: Secretor Genotype (FUT2 gene) Is Strongly Associated with the Composition of Bifidobacteria in the Human Intestine
abstract: Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa.

title: The light and dark sides of intestinal intraepithelial lymphocytes
abstract: The intraepithelial lymphocytes (IELs) that reside within the epithelium of the intestine form one of the main branches of the immune system. As IELs are located at this critical interface between the core of the body and the outside environment, they must balance protective immunity with an ability to safeguard the integrity of the epithelial barrier: failure to do so would compromise homeostasis of the organism. In this Review, we address how the unique development and functions of intestinal IELs allow them to achieve this balance.

title: Identical T Cell Clones Are Located within the Mouse Gut Epithelium and Lamina Propria and Circulate in the Thoracic Duct Lymph
abstract: Murine gut intraepithelial (IEL) T cell receptor (TCR)-α/β1 lymphocytes bearing CD8α/β or CD8α/α coreceptors have been shown previously to express different oligoclonal TCR β chain repertoires in the same mouse, in agreement with other evidence indicating that these two populations belong to different ontogenic lineages, with only CD8α/β1 IELs being fully thymus dependent. CD8α/β1, but not CD8α/α1, T lymphocytes are also present in the lamina propria. Here, we show that CD8α/β+ lymphocytes from the lamina propria and the epithelium are both oligoclonal, and that they share the same TCR-β clonotypes in the same mouse, as is also the case for CD4+ T cells. Furthermore, identical T cell clones were detected among CD8α/β1 IELs and CD8α/β1 blasts circulating into the thoracic duct (TD) lymph of the same mouse, whereas TD small lymphocytes are polyclonal. These findings must be considered in light of previous observations showing that T blasts, but not small T lymphocytes, circulating in the TD lymph have the capacity of homing into the gut epithelium and lamina propria. These combined observations have interesting implications for our understanding of the recirculation of gut thymus-dependent lymphocytes and their precursors, and of the events leading up to the selection of their restricted TCR repertoire.

title: Expansion of alpha beta T-cell receptor-bearing intestinal intraepithelial lymphocytes after microbial colonization in germ-free mice and its independence from thymus.
abstract: A large proportion of intestinal intraepithelial lymphocytes (IEL) comprises alpha beta and gamma delta T-cell receptor (TcR)-bearing T cells. The numbers of alpha beta and gamma delta IEL are reported to be very different between germ-free and conventional microbial conditions. In this study, we investigated the kinetics of both types of TcR-bearing cells after microbial colonization in germ-free mice and the influence of thymus deprivation on IEL populations during the microbial association process. Immediately after association with microbes in germ-free animals, the number of alpha beta TcR-bearing IEL gradually increased. Fourteen days after microbial association the number of alpha beta IEL equalled that of gamma delta TcR-bearing IEL. Approximately 1 month after microbial association, the number of alpha beta IEL was several times greater than that of gamma delta IEL, having almost reached the level in conventional mice reared in a conventional animal room after birth. On the other hand the number of gamma delta IEL hardly changed throughout this microbial association process. Two-colour analysis involving anti-alpha beta TcR and anti-Lyt-2 or Lyt-3 antibodies showed that the major fraction of IEL that increased after microbial association comprised alpha beta TcR-bearing T cells expressing CD8 antigen composed of a homodimer of alpha-chains, which was not detected in other gut associated-lymphoid tissues (GALT) such as Peyer's patch, mesenteric lymph node and lamina propria tissue. The number of alpha beta T cells in these GALT increased within 1 week more quickly than that of IEL. The increase in alpha beta IEL after microbial association was not prevented by thymectomy. These results strongly suggest that the progenitors of alpha beta TcR-bearing IEL expand outside the thymus in response to microbial colonization in germ-free mice.

title: Proliferative recruitment of intestinal intraepithelial lymphocytes after microbial colonization of germ‐free mice
abstract: Intraepithelial lymhocytes (IEL), particularly αβ TCR‐bearing IEL (αβ‐IEL), dramatically increase in number after microbial colonization of formerly germ‐free (ex‐GF) mice (Umesaki et al., Immunology 1993. ). 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, γδ‐ and αβ‐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 αβ‐IEL was a little higher than that for γδ‐IEL. The maximal labeling for αβ‐IEL and γδ‐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 αβ‐IEL outnumbered γδ‐IEL during this period. In this case, 75% of αβ‐IEL and 67% of γδ‐IEL were labeled with BrdUrd in 10 days. On the other hand, the apparent half lives of αβ‐IEL and γδ‐IEL were 10 and 20 days, respectively, in CV mice. These results strongly suggest that the number of IEL, particularly αβ‐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 αβ‐IEL to that of γδ‐IEL between germ‐free and conventionalized mice indicates a preferential increase in the αβ‐IEL subset during the course of conventionalization.

title: A step‐wise expansion of intestinal intraepithelial T lymphocytes in association with microbial colonization is defined by sensitivity to cyclosporin A
abstract: Murine intestinal intraepithelial lymphocytes (IELs) consist of T cells bearing αβ‐antigen receptor (αβ‐IELs) and those bearing γδ‐antigen receptor (γδ‐IELs). Although γδ‐IELs outnumber αβ‐IELs in germ‐free (GF) mice, oral inoculation of fecal suspension from conventional (CV) mice into GF mice induced the increase in number of αβ‐IELs, leaving the number of γδ‐IELs unchanged, and the number of αβ‐IELs reached the level of CV mice by 3 weeks after conventionalization. Expansion of αβ‐IELs and increase in their CD44+ subset in conventionalized mice were not affected until 2 weeks after beginning of daily injection of cyclosporin A (CsA). However, further expansion of αβ‐IELs during 2–3 weeks after conventionalization was blocked by injection of CsA. Although the relative constitution of CD4−8−, CD4+ 8−, CD4−8αα+, CD4−8αβ+ and CD4+ 8+ subsets among αβ‐IELs was comparable between control and CsA‐treated groups, CsA injection resulted in the decrease in ratio of high‐density fraction cells to low density fraction cells in IELs. CsA completely abrogated the expansion of T cells in peripheral lymph nodes stimulated by alloantigens in vivo, and proliferation of IELs from GF mice induced by immobilized anti‐αβ‐T‐cell receptor (TCR) monoclonal antibodies (mAb) in vitro was also eliminated by CsA. These results indicate that microbial colonization‐induced expansion of αβ‐IELs is subdivided into two steps: the early phase of expansion takes place via TCR‐non‐mediated pathway and the late phase of expansion requires TCR‐mediated signal transduction.

title: Differential induction of major histocompatibility complex molecules on mouse intestine by bacterial colonization.
abstract: The aim of this study was to determine what factors induce major histocompatibility complex (MHC) molecules on the mouse small intestinal epithelium by using immunohistochemical methods. In germ-free mice, although MHC class I molecules such as H-2K and thymus leukemia antigen (TLa) were expressed on the small intestinal epithelium, class II molecules were absent. The introduction of microorganisms into germ-free mice induced characteristic MHC molecules on the small intestinal epithelial cells. The I-A molecule was induced on the villus tip and crypt epithelial cells 7 days after conventionalization, and the I-E molecule was induced on the mid villus and crypt epithelial cells 14 days after conventionalization. The staining intensity of the H-2K molecules was increased 4 days after conventionalization. In contrast, TLa did not change during conventionalization of germ-free mice. These results suggest that the expression of MHC molecules, except for the TLa, is greatly dependent on the presence of intestinal microorganisms.

title: Physiological roles of gammadelta T-cell receptor intraepithelial lymphocytes in cytoproliferation and differentiation of mouse intestinal epithelial cells.
abstract: In this study we aimed to elucidate the physiological role of gammadelta intraepithelial lymphocytes (IEL) in the mouse intestine. For this purpose, we used T-cell receptor (TCR) Vgamma4/Vdelta5 transgenic mice (KN 6 Tg: BALB/c background, H-2d), and compared the immunological and physiological characteristics of the intestinal tracts of KN 6 Tg and non-transgenic (non-Tg) littermates. In KN 6 Tg littermates, 95% of small intestinal (SI) and large intestinal (LI) IEL expressed gammadelta TCR, and their TCR was replaced by Tg gammadelta TCR. In these mice, class II major histocompatibility complex (MHC) expression was up-regulated in the SI epithelium, compared with the non-Tg littermates, under specific pathogen-free (SPF) conditions. Competitive reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNAs of the I-Ealpha chain on the SI epithelial cells was higher in KN 6 Tg than in non-Tg littermates. However, in the LI, class II MHC molecules were not expressed in either KN 6 Tg or non-Tg littermates. The epithelial cell mitotic index in the SI, but not in the LI, was higher in KN 6 Tg than in non-Tg littermates under SPF conditions. However, differentiation markers for SI epithelial cells, such as alkaline phosphatase and disaccharidase (lactase, maltase and sucrase) activities, were similar in KN 6 Tg and non-Tg littermates. MHC class II molecule expression on the SI epithelium was absent in germ-free (GF) Tg mice, but was induced under SPF conditions, coinciding with the increase of interferon-gamma (IFN-gamma) mRNA in gammadelta TCR SI-IEL. These findings suggest that gammadelta TCR IEL regulate epithelial cell regeneration and class II MHC expression, but not cell differentiation in the SI. However, these functions were not observed in the gammadelta TCR IEL in the LI. In addition, the activation step in the gammadelta TCR SI-IEL is dependent on the presence of gut microflora.

title: 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
abstract: 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.

title: Structure of the intestinal flora responsible for development of the gut immune system in a rodent model.
abstract: The intestinal flora comprising indigenous, autochthonous bacteria is constantly present in the alimentary tract of host animals, including humans. The indigenous bacteria greatly affect the structure and functions of the intestinal mucosa. Studies involving gnotobiotic mice or rats have shown that the presence of limited kinds of intestinal bacteria is responsible for the development of the gut immune system, such as secretory IgA, major histocompatibility complex molecules and intraepithelial lymphocytes. Understanding of the structure of the intestinal flora or the organization of the microbial population in the intestine, based on evaluation of the immunological responses, may clarify its functions in the host animal.

title: Complete genome sequences of rat and mouse segmented filamentous bacteria, a potent inducer of th17 cell differentiation.
abstract: Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E. coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.

title: Differential Roles of Segmented Filamentous Bacteria and Clostridia in Development of the Intestinal Immune System
abstract: 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 αβ T-cell receptor-bearing intraepithelial lymphocytes (αβIEL) increased in SFB-associated mice (SFB-mice) but not in clostridium-associated mice (Clost-mice). There was no great difference in Vβ usage among GF mice, Cvd mice, and these gnotobiotic mice, although the association with SFB decreased the proportion of Vβ6+ cells in CD8β− 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 αβIEL 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 CD8αβ+ cells to that of CD8αα+ cells in αβIEL, 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.

title: Apathogenic, intestinal, segmented, filamentous bacteria stimulate the mucosal immune system of mice
abstract: Segmented filamentous bacteria (SFBs) are apathogenic autochthonous bacteria in the murine small intestine that preferentially attach to Peyer's patch epithelium. SFBs have never been cultured in vitro. We have studied the effects of SFBs on the immune system of the host. Mice monoassociated with SFBs were compared with germ-free mice and with mice without SFBs but with a specific-pathogen-free (SPF) gut flora. SFBs versus no microbial flora raised the number of lymphoid cells in the lamina propria of the ileal and cecal mucosa, raised the number of immunoglobulin A (IgA)-secreting cells in the intestinal mucosa, produced elevated IgA titers in serum and intestinal secretions, and enhanced the concanavalin A-induced proliferative responses of mesenteric lymph node cells. The SPF flora had effects similar to but less pronounced than those mediated by SFBs. The results indicate that SFBs stimulate the mucosal immune system to a greater extent than do other autochthonous gut bacteria.

title: SMALL INTESTINAL MUCOSAL CELL PROLIFERATION AND BACTERIAL FLORA IN THE CONVENTIONALIZATION OF THE GERMFREE MOUSE
abstract: The relationship between intestinal colonization and the small bowel mucosal cellular proliferation rate during conventionalization of the germfree mouse was examined. 16 mice were maintained under standard germfree conditions, and 54 others were conventionalized. Migration of the small bowel epithelial cells was followed by radioautography with administration of tritiated thymidine. Colonization was followed by qualitative and quantitative bacteriological fecal analyses. The percentages of the villi labeled (as determined by cell count) 24, 48, and 72 hr following thymidine administration showed immediate progression in the conventionalized animals from the germfree villus migration time (4 days) toward the conventional villus migration time (2 days). The epithelial migration rate of animals conventionalized for 8 days was comparable to that of conventional animals. After conventionalization, aerobic and anaerobic organisms undergo a period of extensive multiplication; however, 72 hr later the number of these microorganisms cultured in the stool decrease and are similar to those recovered from normal animals. Coliforms and streptococci are recovered in large numbers only in the first days after conventionalization, while the Bacteroides are first recovered in significant numbers on the fifth day of conventionalization. Except for smaller numbers of Bacteroides, the bacterial populations in the stools of the conventionalized animals are qualitatively and quantitatively similar by the eighth day of conventionalization to those of true conventional mice. Adaptive balance between cell proliferation and sloughing, and thus migration rate, begins immediately after conventionalization of germfree animals as bacterial populations establish themselves throughout the gastrointestinal tract, and results in a doubling of the mucosal cell turnover after 8 days. At this time both the small intestinal epithelial cell migration rate and the intestinal microflora are similar to those of conventional animals.

title: Design of species‐specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides–Parabacteroides microbiota
abstract: To develop species‐specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization.

title: Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain
abstract: 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. To elucidate the pathogenesis of enteric inflammation in this new subline. The SPF and germ free (GF) SAMP1/Yit strains were used. Histological, immunological, and microbiological characterisation of the mice with enteric inflammation was performed. 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-ε 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. 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.

title: Randomized controlled trial of the effect of bifidobacteria-fermented milk on ulcerative colitis.
abstract: Alterations of intestinal flora, such as reduction in the concentration of bifidobacteria and increase in that of Bacteroides species, are apparently associated with the severity of ulcerative colitis.



We conducted a randomised clinical trial of the use of a bifidobacteria-fermented milk (BFM) supplement as a dietary adjunct in the treatment of ulcerative colitis.



The subjects were randomly divided into two groups: a group with BFM supplementation (BFM group, 11 subjects) and a control group (control group, 10 subjects). The BFM group was given 100 mL/day of BFM for one year. Colonoscopies, general blood markers and examinations of intestinal flora including the analysis of fecal organic acids were performed at the commencement of the study and after one year.



Exacerbation of symptoms was seen in 3 out of 11 subjects in the BFM group and in 9 out of 10 in the control group. Log rank statistic analysis of the cumulative exacerbation rates showed a significant reduction in exacerbations for the BFM group (p = 0.0184). The analysis of microflora and the organic acids in the feces showed a significant reduction in the relative proportion of B. vulgatus in Bacteroidaceae and butyrate concentration, respectively, after supplementation with BFM, in comparison with before.



Supplementation with the BFM product was successful in maintaining remission and had possible preventive effects on the relapse of ulcerative colitis.

title: Segmented filamentous bacteria in a defined bacterial cocktail induce intestinal inflammation in SCID mice reconstituted with CD45RBhigh CD4+ T cells
abstract: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RBhigh subset of CD4+T cells isolated from the spleen of normal BALB/c mice.  A CD4+CD45RBhigh subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ‐free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen‐free (SPF) bacteria. Mice were evaluated 8–12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD).  After the transfer of the CD4+CD45RBhigh T‐cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO‐1 (zona occludens).  Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB.

title: Interactions between epithelial cells and bacteria, normal and pathogenic.
abstract: Lynn Bry et al . show that the monoassociation of germ-free (GF) mice with wild-type Bacteroides thetaiotaomicron induced expression of an α1,2 fucosyltransferase messenger RNA and production of fucosylated glycoconjugates that were reactive with Ulex europaeus agglutinin I in the epithelial cells

title: Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria
abstract: The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease.

title: The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses.
abstract: Microbiota-induced cytokine responses participate in gut homeostasis, but the cytokine balance at steady-state and the role of individual bacterial species in setting the balance remain elusive. Herein, systematic analysis of gnotobiotic mice indicated that colonization by a whole mouse microbiota orchestrated a broad spectrum of proinflammatory T helper 1 (Th1), Th17, and regulatory T cell responses whereas most tested complex microbiota and individual bacteria failed to efficiently stimulate intestinal T cell responses. This function appeared the prerogative of a restricted number of bacteria, the prototype of which is the segmented filamentous bacterium, a nonculturable Clostridia-related species, which could largely recapitulate the coordinated maturation of T cell responses induced by the whole mouse microbiota. This bacterium, already known as a potent inducer of mucosal IgA, likely plays a unique role in the postnatal maturation of gut immune functions. Changes in the infant flora may thus influence the development of host immune responses.

title: Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine.
abstract: The requirements for in vivo steady state differentiation of IL-17-producing T-helper (Th17) cells, which are potent inflammation effectors, remain obscure. We report that Th17 cell differentiation in the lamina propria (LP) of the small intestine requires specific commensal microbiota and is inhibited by treating mice with selective antibiotics. Mice from different sources had marked differences in their Th17 cell numbers and animals lacking Th17 cells acquired them after introduction of bacteria from Th17 cell-sufficient mice. Differentiation of Th17 cells correlated with the presence of cytophaga-flavobacter-bacteroidetes (CFB) bacteria in the intestine and was independent of toll-like receptor, IL-21 or IL-23 signaling, but required appropriate TGF-beta activation. Absence of Th17 cell-inducing bacteria was accompanied by increase in Foxp3+ regulatory T cells (Treg) in the LP. Our results suggest that composition of intestinal microbiota regulates the Th17:Treg balance in the LP and may thus influence intestinal immunity, tolerance, and susceptibility to inflammatory bowel diseases.

title: Focused Specificity of Intestinal Th17 Cells towards Commensal Bacterial Antigens
abstract: T-helper-17 (TH17) cells have critical roles in mucosal defence and in autoimmune disease pathogenesis. They are most abundant in the small intestine lamina propria, where their presence requires colonization of mice with microbiota. Segmented filamentous bacteria (SFB) are sufficient to induce TH17 cells and to promote TH17-dependent autoimmune disease in animal models. However, the specificity of TH17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T-cell antigen receptor (TCR) repertoire of intestinal TH17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4+ T cells and that most TH17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing TH17 cells, even if SFB-colonized mice also harboured a strong TH1 cell inducer, Listeria monocytogenes, in their intestine. The match of T-cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.

title: Segmented filamentous bacteria antigens presented by intestinal dendritic cells drive mucosal Th17 cell differentiation.
abstract: How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.

title: Segmented filamentous bacteria prevent colonization of enteropathogenic Escherichia coli O103 in rabbits.
abstract: Despite their distribution in the intestines of many mammals, including man, segmented filamentous bacteria (SFB) have not been found in rabbits, nor has any function been identified for these uncultivable microbes. New Zealand White rabbits were infected with rabbit enteropathogenic Escherichia coli O103 (REPEC O103) derivatives, followed up clinically, and randomly killed 1-4 days after inoculation. Intestinal tissue samples were examined by electron and light microscopy to search for SFB and to evaluate REPEC O103 colonization. Twelve of 21 rabbits showed SFB colonization on ileal absorptive villi. The presence of SFB was correlated with lack of REPEC 0103 ileal colonization (P<.01) and disease. Rabbits without SFB were always colonized by this pathogen. SFB appear to inhibit intestinal colonization by REPEC O103 and thus protect against REPEC 0103 disease. SFB colonization in rabbits is also described for the first time.

title: Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut
abstract: The mechanism to maintain homeostasis of the gut microbiota remains largely unknown despite its critical role in the body defense. In the intestines of mice with deficiency of activation-induced cytidine deaminase (AID), the absence of hypermutated IgA is partially compensated for by the presence of large amounts of unmutated IgM and normal expression levels of defensins and angiogenins. We show here a predominant and persistent expansion of segmented filamentous bacteria throughout the small intestine of AID–/– mice. Reconstitution of lamina propria IgA production in AID–/– mice recovered the normal composition of gut flora and abolished the local and systemic activation of the immune system. The results indicate that secretions of IgAs rather than innate defense peptides are critical to regulation of commensal bacterial flora and that the segmented filamentous bacteria antigens are strong stimuli of the mucosal immune system.

title: In situ class switching and differentiation to IgA-producing cells in the gut lamina propria
abstract: One of the front lines of the immune defence is the gut mucosa, where immunoglobulin-α (IgA) is continuously produced to react with commensal bacteria and dietary antigens. It is generally accepted that, after antigenic stimulation in the Peyer's patches, IgA+ lymphoblasts (B220+IgA+) migrate through the lymph and blood circulation, and eventually home to the lamina propria of the intestine. Mice that lack activation-induced cytidine deaminase (AID) are defective in class switch recombination (CSR) and somatic hypermutation. CSR changes the immunoglobulin heavy chain constant region (CH) gene being expressed from Cµ to other CH genes, resulting in a switch of the immunoglobulin isotype from IgM to IgG, IgE or IgA. AID-/- mice also secrete large amounts of immunoglobulin-µ (IgM) into faeces, and accumulate B220-IgM+ plasma cells as well as B220+IgM+ cells in the gut. Here we show that lamina propria B220+IgA+ cells have just completed CSR, as they still express both AID and transcripts from circular DNA that has been ‘looped-out’ during CSR. Lamina propria IgM+ B cells seem to be pre-committed to switching to IgA+ in vitro as well as in vivo. Culturing lamina propria IgM+ B cells together with lamina propria stromal cells enhances preferential switching and differentiation of B cells to IgA+ plasma cells. We conclude that IgA+ cells in the gut lamina propria are generated in situ from B220+IgM+ lymphocytes.

title: Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells.
abstract: Commensal microbes can have a substantial impact on autoimmune disorders, but the underlying molecular and cellular mechanisms remain largely unexplored. We report that autoimmune arthritis was strongly attenuated in the K/BxN mouse model under germ-free (GF) conditions, accompanied by reductions in serum autoantibody titers, splenic autoantibody-secreting cells, germinal centers, and the splenic T helper 17 (Th17) cell population. Neutralization of interleukin-17 prevented arthritis development in specific-pathogen-free K/BxN mice resulting from a direct effect of this cytokine on B cells to inhibit germinal center formation. The systemic deficiencies of the GF animals reflected a loss of Th17 cells from the small intestinal lamina propria. Introduction of a single gut-residing species, segmented filamentous bacteria, into GF animals reinstated the lamina propria Th17 cell compartment and production of autoantibodies, and arthritis rapidly ensued. Thus, a single commensal microbe, via its ability to promote a specific Th cell subset, can drive an autoimmune disease.

title: Gender bias in autoimmunity is influenced by microbiota.
abstract: Gender bias and the role of sex hormones in autoimmune diseases are well established. In specific pathogen-free nonobese diabetic (NOD) mice, females have 1.3-4.4 times higher incidence of type 1 diabetes (T1D). Germ-free (GF) mice lost the gender bias (female-to-male ratio 1.1-1.2). Gut microbiota differed in males and females, a trend reversed by male castration, confirming that androgens influence gut microbiota. Colonization of GF NOD mice with defined microbiota revealed that some, but not all, lineages overrepresented in male mice supported a gender bias in T1D. Although protection of males did not correlate with blood androgen concentration, hormone-supported expansion of selected microbial lineages may work as a positive-feedback mechanism contributing to the sexual dimorphism of autoimmune diseases. Gene-expression analysis suggested pathways involved in protection of males from T1D by microbiota. Our results favor a two-signal model of gender bias, in which hormones and microbes together trigger protective pathways.

title: Naturally transmitted segmented filamentous bacteria segregate with diabetes protection in nonobese diabetic mice
abstract: Vertebrates typically harbor a rich gastrointestinal microbiota, which has coevolved with the host over millennia and is essential for several host physiological functions, in particular maturation of the immune system. Recent studies have highlighted the importance of a single bacterial species, segmented filamentous bacteria (SFB), in inducing a robust T-helper cell type 17 (Th17) population in the small-intestinal lamina propria (SI-LP) of the mouse gut. Consequently, SFB can promote IL-17–dependent immune and autoimmune responses, gut-associated as well as systemic, including inflammatory arthritis and experimental autoimmune encephalomyelitis. Here, we exploit the incomplete penetrance of SFB colonization of NOD mice in our animal facility to explore its impact on the incidence and course of type 1 diabetes in this prototypical, spontaneous model. There was a strong cosegregation of SFB positivity and diabetes protection in females, but not in males, which remained relatively disease-free regardless of the SFB status. In contrast, insulitis did not depend on SFB colonization. SFB-positive, but not SFB-negative, females had a substantial population of Th17 cells in the SI-LP, which was the only significant, repeatable difference in the examined T-cell compartments of the gut, pancreas, or systemic lymphoid tissues. Th17-signature transcripts dominated the very limited SFB-induced molecular changes detected in SI-LP CD4+ T cells. Thus, a single bacterium, and the gut immune system alterations associated with it, can either promote or protect from autoimmunity in predisposed mouse models, probably reflecting their variable dependence on different Th subsets.

title: Host specificity of filamentous, segmented microorganisms adherent to the small bowel epithelium in mice and rats
abstract: Germfree rats and mice were given by gavage samples of ileal homogenates prepared from conventional rats and mice. Filamentous, segmented procaryotes adhered to the small bowel epithelium in the ex-germfree mice only when the homogenate was made from mouse bowel and in the ex-germfree rats only when the inoculum came from rats. Thus, the filamentous microorganisms are host animal specific.

title: Comparison of 16S rRNA sequences of segmented filamentous bacteria isolated from mice, rats, and chickens and proposal of "Candidatus Arthromitus".
abstract: Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. "Candidatus Arthromitus" is proposed as the provisional generic name for this group of organisms.

title: 16S Ribosomal DNA Sequence Divergence of Segmented Filamentous Bacteria with Special Reference to In
abstract: Segmented filamentous bacteria (SFB) have been found in the alimentary tracts of a wide range of animals, where they activate the mucosal immune system. We determined the 16S rDNA sequences of SFBs derived from crab-eating monkey and Fischer 344 (F344) rat, in addition to of that from BALB/c mice ( Umesaki et al, 1995), and compared these sequences with those of SE (Swiss) mice, WU rats and chickens reported by Snel et al. (1994, 1995), with special reference to inter-species and within-species differences of the host animals. SFBs isolated from six different hosts mentioned above exhibited levels of 16S rDNA sequence homology of more than 97%. There was only slight divergence in the 16S rDNA sequence between BALB/c and SE mice, and F344 and WU rats, 0.1 and 0.4%, respectively. However, there was some inter-species divergence, except between monkey and rats, suggesting that SFB isolated from different species of animals belong to different species, as proposed by Snel et al. (1995). The phylogenetic tree of SFB 16S rDNA derived from these six kinds of animals, including crab-eating monkey, does not necessarily suggest the coevolution of SFB and their host animals.

title: Gut Immune Maturation Depends on Colonization with a Host-Specific Microbiota
abstract: Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.

title: Production of Immunoglobulin A Protease by Streptococcus pneumoniae from Animals
abstract: Human isolates of Streptococcus pneumoniae tested by traditional immunochemical methods produce a protease that cleaves human immunoglobulin A1 (IgA1) into Fab and Fc fragments. The protease may be an important virulence factor, but studies of its pathogenetic significance have been hampered by lack of a suitable animal model. Since S. pneumoniae is a respiratory pathogen for several species of animals, we sought to determine whether isolates of this organism from animals with pneumococcal infection, including fatal diplococcal pneumonia, produced an IgA protease. Isolates from six animal species including the mouse, rat, dog, guinea pig, rhesus monkey, and chimpanzee were tested for protease activity against IgA preparations from the mouse, rat, dog, guinea pig, rabbit, rhesus and cynomolgus monkeys, gorilla, and human. Cleavage of IgA was demonstrated by the appearance of Fc fragments in Western blots (immunoblots) treated with specific antisera. All these isolates except that from the guinea pig produced a protease that cleaved IgA of human, rhesus monkey, and gorilla origin. Cleavage was inhibited by 5 mM EDTA. IgA cleavage from the other species could not be demonstrated. Although S. pneumoniae can colonize the respiratory tracts of several animal species, it is a significant pathogen principally of humans and some other primates. Our data suggest that some species of nonhuman primates including the rhesus monkey could be suitable for experimental studies on the significance of IgA protease in the pathogenesis of pneumococcal disease.

title: Actin accumulation at sites of attachment of indigenous apathogenic segmented filamentous bacteria to mouse ileal epithelial cells
abstract: Selective attachment of indigenous, apathogenic, segmented filamentous bacteria to follicle-associated epithelia of terminal ileal Peyer's patches involves both the specialized M cells and normal enterocytes. A striking, highly localized cytoplasmic accumulation of polymerized actin, which resembles that associated with adherence of enteropathogenic Escherichia coli to cultured epithelial cells, occurs at attachment sites of segmented filamentous bacteria. The epithelial response to the attachment of microorganisms may thus involve mechanisms which are the same for diverse microorganisms.

title: A genomic analysis of the archaeal system Ignicoccus hospitalis-Nanoarchaeum equitans
abstract: BackgroundThe relationship between the hyperthermophiles Ignicoccus hospitalis and Nanoarchaeum equitans is the only known example of a specific association between two species of Archaea. Little is known about the mechanisms that enable this relationship.ResultsWe sequenced the complete genome of I. hospitalis and found it to be the smallest among independent, free-living organisms. A comparative genomic reconstruction suggests that the I. hospitalis lineage has lost most of the genes associated with a heterotrophic metabolism that is characteristic of most of the Crenarchaeota. A streamlined genome is also suggested by a low frequency of paralogs and fragmentation of many operons. However, this process appears to be partially balanced by lateral gene transfer from archaeal and bacterial sources.ConclusionsA combination of genomic and cellular features suggests highly efficient adaptation to the low energy yield of sulfur-hydrogen respiration and efficient inorganic carbon and nitrogen assimilation. Evidence of lateral gene exchange between N. equitans and I. hospitalis indicates that the relationship has impacted both genomes. This association is the simplest symbiotic system known to date and a unique model for studying mechanisms of interspecific relationships at the genomic and metabolic levels.

title: Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB).
abstract: Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.

title: The Lifestyle of the Segmented Filamentous Bacterium: A Non-Culturable Gut-Associated Immunostimulating Microbe Inferred by Whole-Genome Sequencing
abstract: Numerous microbes inhabit the mammalian intestinal track and strongly impact host physiology; however, our understanding of this ecosystem remains limited owing to the high complexity of the microbial community and the presence of numerous non-culturable microbes. Segmented filamentous bacteria (SFBs), which are clostridia-related Gram-positive bacteria, are among such non-culturable populations and are well known for their unique morphology and tight attachment to intestinal epithelial cells. Recent studies have revealed that SFBs play crucial roles in the post-natal maturation of gut immune function, especially the induction of Th17 lymphocytes. Here, we report the complete genome sequence of mouse SFBs. The genome, which comprises a single circular chromosome of 1 620 005 bp, lacks genes for the biosynthesis of almost all amino acids, vitamins/cofactors and nucleotides, but contains a full set of genes for sporulation/germination and, unexpectedly, for chemotaxis/flagella-based motility. These findings suggest a triphasic lifestyle of the SFB, which comprises two types of vegetative (swimming and epicellular parasitic) phases and a dormant (spore) phase. Furthermore, SFBs encode four types of flagellin, three of which are recognized by Toll-like receptor 5 and could elicit the innate immune response. Our results reveal the non-culturability, lifestyle and immunostimulation mechanisms of SFBs and provide a genetic basis for the future development of the SFB cultivation and gene-manipulation techniques.

title: The genome of th17 cell-inducing segmented filamentous bacteria reveals extensive auxotrophy and adaptations to the intestinal environment.
abstract: Perturbations of the composition of the symbiotic intestinal microbiota can have profound consequences for host metabolism and immunity. In mice, segmented filamentous bacteria (SFB) direct the accumulation of potentially proinflammatory Th17 cells in the intestinal lamina propria. We present the genome sequence of SFB isolated from monocolonized mice, which classifies SFB phylogenetically as a unique member of Clostridiales with a highly reduced genome. Annotation analysis demonstrates that SFB depend on their environment for amino acids and essential nutrients and may utilize host and dietary glycans for carbon, nitrogen, and energy. Comparative analyses reveal that SFB are functionally related to members of the genus Clostridium and several pathogenic or commensal "minimal" genera, including Finegoldia, Mycoplasma, Borrelia, and Phytoplasma. However, SFB are functionally distinct from all 1200 examined genomes, indicating a gene complement representing biology relatively unique to their role as a gut commensal closely tied to host metabolism and immunity.

title: Timing, Localization, and Persistence of Colonization by Segmented Filamentous Bacteria in the Neonatal Mouse Gut Depend on Immune Status of Mothers and Pups
abstract: As a member of the indigenous gut mucosal microbiota, segmented filamentous bacteria (SFB) colonize the guts of a variety of vertebrates and invertebrates. They are potent microbial stimuli of the gut mucosal immune system. In the small intestines of mice and rats, it has been observed that SFB are absent during the suckling period and appear in high numbers shortly after weaning, then quickly retreat to the cecum and large intestine. In this study, we explored whether this microecological phenomenon resulted from the interaction between SFB and the passively acquired maternal mucosal immunity and/or the actively acquired mucosal immunity. We set up a mouse model by reciprocal crossings and backcrossings of SFB-monoassociated, formerly germ-free, immunocompetent (+/+) BALB/c mice and immunodeficient (scid/scid) mice to produce pups which are either immunocompetent (scid/+) or immunodeficient (scid/scid) and are born either to immunocompetent (scid/+) mothers or to immunodeficient (scid/scid) mothers. We monitored the number of SFB on the mucosa of the small intestine in the four different groups of mice after birth, as well as the level of passively acquired antibodies, the active gut mucosal immune responses, and immunoglobulin A (IgA) coating of SFB in the gut. The results showed that, irrespective of whether the pups were scid/scid or scid/+, SFB could be found earlier on the mucosa of the small intestine in pups born to scid/scid mothers, appearing from day 13 and rapidly reaching a climax around weaning time on day 28, compared to the significantly delayed colonization in the pups of scid/+ mothers, starting from day 16 and peaking around days 28 to 32. After the climax, SFB quickly declined to very low levels in the small intestines of scid/+ pups of either scid/scid mothers or scid/+ mothers, whereas they remained at high levels in scid/scid pups at least until day 70, the last observation time in this study. The dynamic changes in SFB colonization of the small intestines of the different groups of pups may be related to the dynamic changes in the levels of SFB coated with secretory IgA (sIgA), which resulted from the significantly different levels of sIgA obtained from the mothers' milk during the suckling period and, later, of self-produced sIgA in the small intestine. Nevertheless, it is evident that the timing, localization, and persistence of colonization of the neonatal gut by SFB depends on the immune status of both mothers and pups.

title: Bacterial Association in the Gastrointestinal Tract of Beagle Dogs
abstract: Nine male beagle dogs, housed in either a conventional or locked environment for 2.5 years, were killed, and the bacterial flora present in various regions of each gastrointestinal tract was assessed by culture techniques, light microscopy, and scanning electron microscopy. All dogs possessed a complex microflora in their colons; in almost every dog anaerobes predominated. The highest number of bacteria cultured was 1010/g (dry weight) of tissue and contents; highest counts obtained with a Petroff-Hauser counting chamber were 1010/ml (wet weight). Although there was a consistency in the detectable genera, there were also noticeable differences in the flora of dogs housed under different environmental conditions. These differences included qualitative and quantitative changes in the flora as well as alterations in the distribution and localization of microorganisms along the gastrointestinal tract and in the crypts of Lieberkuhn. No bacterial layers were detected on the surfaces of stomach or proximal bowel in any of the dogs. Dogs housed in a conventional, open, environment had bacteria that occurred in layers on their ceca and colons and in their crypts of Lieberkuhn; however, dogs housed under “locked” environmental conditions did not possess them or had them less frequently. Dogs removed from the locked environment and kept (30 days) in conventional housing conditions were the only ones with detectable segmented filamentous microbes in their ilea. This study shows that the microbial flora does not simplify when dogs are housed in a locked environment. Indeed, it may increase in complexity and cause alterations in the bacterial flora that is associated closely with gastrointestinal epithelial cells and crypts of Lieberkuhn.

title: Multifocal granulomatous jejunitis associated with an argyrophilic gram-positive segmented filamentous bacterium in a Holstein cow.
abstract: Multifocal, raised, ulcerated firm nodules accompanied by an intussuscepted area were detected in the jejunum of an 8-year-old Holstein cow. The cut surfaces of the nodules were yellow-white. Microscopically, the lamina propria was expanded by an intense infiltration of epithelioid cells, multinucleate giant cells, macrophages, lymphocytes and neutrophils. Numerous bacteria were found within the granulomatous lesions. These were argyrophilic, gram-positive, periodic acid-Schiff-positive, segmented, rarely branched, elongate filamentous bacteria (2-28 μm in length, 0.2-0.35 μm in diameter). Ultrastructurally, a cell wall with an electron-transparent zone was detected. The present pathogen was clearly different from the argyrophilic, gram-negative, non-segmented, filamentous bacterium previously reported in a Holstein cow with jejunal granuloma. Comparative 16S rDNA gene sequencing analysis revealed that the organism was an unpublished species (GenBank accession number AB539875). This is the first report of bovine jejunal granuloma associated with an argyrophilic gram-positive segmented filamentous bacterium.

title: Segmented filamentous bacteria in the bovine small intestine.
abstract: Segmented filamentous bacteria (SFB) were observed in a 28-day-old calf, attached to the absorptive villi. Morphologically, they were similar to SFB described in other animal species. Because these organisms cannot be cultured, further characterization was not possible. The organisms were confined to the upper third of the absorptive villi and were not seen attached to the follicle-associated epithelium of the Peyer's patch, or observed in the caecum or colon. Although they were often associated with minor lesions, their pathological significance was doubtful. With this report, segmented filamentous bacteria have now been described in virtually all the commercially important livestock and poultry species, in other domestic animals, and in man.

title: Segmented filamentous bacteria associated with lymphoid tissues in the ileum of horses.
abstract: Segmented filamentous bacteria preferentially attached to the follicle-associated epithelium overlying the lymphoid tissue in samples of the terminal ileum from seven horses examined by scanning electron microscopy. The bacteria adhered to the apical membrane of the enterocytes by a holdfast segment. Each filament tended to be of uniform diameter, but the filaments ranged from 0.7 to 1.4 microns in diameter. The bacteria were usually absent from the adjacent villous epithelium.