Germ-free status and altered caecal subdominant microbiota are associated with a high susceptibility to cow's milk allergy in mice

Studies suggesting that the development of atopy is linked to gut microbiota composition are inconclusive on whether dysbiosis precedes or arises from allergic symptoms. Using a mouse model of cow's milk allergy, we aimed at investigating the link between the intestinal microbiota, allergic sensitization, and the severity of symptoms. Germ-free and conventional mice were orally sensitized with whey proteins and cholera toxin, and then orally challenged with β-lactoglobulin (BLG). Allergic responses were monitored with clinical symptoms, plasma markers of sensitization, and the T-helper Th1/Th2/regulatory-T-cell balance. Microbiota compositions were analysed using denaturing gradient gel electrophoresis and culture methods. Germ-free mice were found to be more responsive than conventional mice to sensitization, displaying a greater reduction of rectal temperature upon challenge, higher levels of blood mouse mast cell protease-1 (mMCP-1) and BLG-specific immunoglobulin G1 (IgG1), and a systemic Th2-skewed response. This may be explained by a high susceptibility to release mMCP-1 even in the presence of low levels of IgE. Sensitization did not alter the microbiota composition. However, the absence of or low Staphylococcus colonization in the caecum was associated with high allergic manifestations. This work demonstrates that intestinal colonization protects against oral sensitization and allergic response. This is the first study to show a relationship between alterations within the subdominant microbiota and severity of food allergy.     

Trypanosoma cruzi: influence of predominant bacteria from indigenous digestive microbiota on experimental infection in mice

To verify the influence of some predominant components from indigenous microbiota on systemic immunological responses during experimental Chagas disease, germ-free NIH Swiss mice were mono-associated with Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus or Peptostreptococcus sp. and then infected with the Y strain of Trypanosoma cruzi. All the mono-associations predominantly induced a Th1 type of specific immune response to the infection by T. cruzi. A direct correlation was observed between a higher survival rate and increased IFN-gamma and TNF-alpha production (P<0.05) in E. faecalis-, B. vulgatus-, and Peptostreptococcus-associated mice. Moreover, higher levels of anti-T. cruzi IgG1 and anti-T. cruzi IgG2a were also found in mono-associated animals after infection. On the other hand, with the exception of E. faecalis-associated mice, mono-association induced a lower IL-10 production after infection (P<0.05) when compared with germ-free animals. Interestingly, spleen cell cultures from non-infected germ-free and mono-associated mice spontaneously produced higher levels (P<0.05) of IL-10 than cultures from infected mono-associated mice, except again for E. faecalis-associated animals. In conclusion, the presence of the components of the indigenous microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in gnotobiotic mice. However, the degree of increase in production of cytokines depends on each bacterial component.     

IgA response to symbiotic bacteria as a mediator of gut homeostasis

Colonization of germ-free mice with a normal gut microbiota elicits bacteria-specific IgA antibody responses. The effects of these responses on microbial and host biology remain poorly defined. Therefore, we developed a gnotobiotic mouse model where the microbiota is reduced to one bacterial species, and the antibody repertoire to a single, monoclonal IgA against the bacterium's capsular polysaccharide. Bacteroides thetaiotaomicron was introduced into germ-free wild-type, immunodeficient Rag1(-/-), or Rag1(-/-) mice harboring IgA-producing hybridoma cells. Without IgA, B. thetaiotaomicron elicits a more robust innate immune response and reacts to this response by inducing genes that metabolize host oxidative products. IgA reduces intestinal proinflammatory signaling and bacterial epitope expression, thereby balancing suppression of the oxidative burst with the antibody's negative impact on bacterial fitness. These results underscore the adaptive immune system's critical role in establishing a sustainable host-microbial relationship. Immunoselection of bacterial epitope expression may contribute to the remarkable strain-level diversity in this ecosystem.     

Gram-negative bacteria aggravate murine small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii

Oral infection of susceptible mice with Toxoplasma gondii results in Th1-type immunopathology in the ileum. We investigated gut flora changes during ileitis and determined contributions of gut bacteria to intestinal inflammation. Analysis of the intestinal microflora revealed that ileitis was accompanied by increasing bacterial load, decreasing species diversity, and bacterial translocation. Gram-negative bacteria identified as Escherichia coli and Bacteroides/Prevotella spp. accumulated in inflamed ileum at high concentrations. Prophylactic or therapeutic administration of ciprofloxacin and/or metronidazole ameliorated ileal immunopathology and reduced intestinal NO and IFN-gamma levels. Most strikingly, gnotobiotic mice in which cultivable gut bacteria were removed by quintuple antibiotic treatment did not develop ileitis after Toxoplasma gondii infection. A reduction in total numbers of lymphocytes was observed in the lamina propria of specific pathogen-free (SPF), but not gnotobiotic, mice upon development of ileitis. Relative numbers of CD4(+) T cells did not differ in naive vs infected gnotobiotic or SPF mice, but infected SPF mice showed a significant increase in the frequencies of activated CD4(+) T cells compared with gnotobiotic mice. Furthermore, recolonization with total gut flora, E. coli, or Bacteroides/Prevotella spp., but not Lactobacillus johnsonii, induced immunopathology in gnotobiotic mice. Animals recolonized with E. coli and/or total gut flora, but not L. johnsonii, showed elevated ileal NO and/or IFN-gamma levels. In conclusion, Gram-negative bacteria, i.e., E. coli, aggravate pathogen-induced intestinal Th1-type immunopathology. Thus, pathogen-induced acute ileitis may prove useful to study bacteria-host interactions in small intestinal inflammation and to test novel therapies based on modulation of gut flora.     

Prevention of gut inflammation by Bifidobacterium in dextran sulfate-treated gnotobiotic mice associated with Bacteroides strains isolated from ulcerative colitis patients

Indigenous Bacteroides strains are closely associated with the occurrence and exacerbation of ulcerative colitis (UC). In this study, we aimed to clarify the effect of Bifidobacterium strains, another major member of colonic bacteria, on the development of gut inflammation using gnotobiotic mouse models associated with Bacteroides strains isolated from UC patients. Dextran sulfate (DSS) administration induced inflammation in the large intestine, in particular of the cecum, in the gnotobiotic mice associated with three strains of Bacteroides vulgatus, judging from the myeloperoxidase activity, occult blood score, and IgG leakage into the intestinal contents. However, the severity of the inflammation was greatly reduced in the gnotobiotic mice associated with both B. vulgatus and Bifidobacterium strains. The severity of the cecal inflammation was well correlated with the concentration of succinic acid in the cecum. Bacteriologically, the density of B. vulgatus strain A (BV-A) greatly decreased and the predominant strain changed from BV-A to BV-B on additional association with Bifidobacterium strains. Among gnotobiotic mice associated with a single B. vulgatus strain, the severity of cecal inflammation in BV-A-associated mice was greater than that in BV-B-associated mice. Each Bifidobacterium strain produced compound(s) more effectively inhibiting the growth of BV-A than BV-B in in vitro culture. Taken together, these results suggest that the severity of DSS-induced gut inflammation is closely associated with a particular B. vulgatus strain, and that Bifidobacterium strains may repress exacerbation of intestinal inflammation through growth inhibition of the B. vulgatus strain.     

The suppressive effect of bifidobacteria on Bacteroides vulgatus,a putative pathogenic microbe in inflammatory bowel disease

Bacteroides, a predominant commensal bacteria in the gut, are thought to be responsible for the development of inflammatory bowel disease (IBD). In the present study, we examined whether or not bifidobacteria suppress B. vulgatus, a representative pathogenic Bacteroides species, in both the coculture system and the gnotobiotic murine model. As a result, Bifidobacterium infantis 1222 highly inhibited the growth of B. vulgatus in the coculture and also significantly suppressed the systemic antibody response raised by B. vulgatus colonizing the gut in gnotobiotic mice. Colonization of the mice by B. vulgatus increased the number of Peyer's patch (PP) cells bearing PNA (peanut agglutinin)+/anti-kappa+ phenotype, which represents plasma cell-like B cells. Moreover, treatment of those B. vulgatus-implanted mice with B. infantis 1222 abrogated such increase in the number of PNA+/anti-kappa+ cells. These results thus suggested that B. infantis 1222 protected the gut epithelial layer including the PP from being invaded by Bacteroides, thereby suppressing the systemic antibody response raised by Bacteroides.     

Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways

Newborns are colonized with an intestinal microbiota shortly after birth, but the factors governing the retention and abundance of specific microbial lineages are unknown. Nursing infants consume human milk oligosaccharides (HMOs) that pass undigested to the distal gut, where they may be digested by microbes. We determined that the prominent neonate gut residents, Bacteroides thetaiotaomicron and Bacteroides fragilis, induce the same genes during HMO consumption that are used to harvest host mucus glycans, which are structurally similar to HMOs. Lacto-N-neotetraose, a specific HMO component, selects for HMO-adapted species such as Bifidobacterium infantis, which cannot use mucus, and provides a selective advantage to B. infantis in vivo when biassociated with B. thetaiotaomicron in the gnotobiotic mouse gut. This indicates that the complex oligosaccharide mixture within HMOs attracts both mutualistic mucus-adapted species and HMO-adapted bifidobacteria to the infant intestine that likely facilitate both milk and future solid food digestion.     

Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents

In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats.     

Oral probiotic bacterial administration suppressed allergic responses in an ovalbumin-induced allergy mouse model

This study investigated whether orally administered probiotic bacteria (Bifidobacterium bifidum and Lactobacillus casei) and a gram-negative bacterium (Escherichia coli) function as allergic immune modulators to prevent food allergy, according to the hygiene hypothesis. C3H/HeJ mice were sensitized with ovalbumin (OVA) and cholera toxin for 5 weeks. After sensitization, the OVA-induced mice that were not treated with bacteria had significantly increased levels of OVA-specific IgE, total IgE, and IgG1 in sera, as well as scab-covered tails. In comparison, groups treated with B. bifidum BGN4 (BGN4), L. casei 911 (L. casei), or Escherichia coli MC4100 (E. coli) had decreased levels of OVA-specific IgE, total IgE, and IgG1, and decreased levels of mast cell degranulation and tail scabs. OVA-specific IgA levels were decreased in BGN4- and L. casei-treated groups. In conclusion, administration of E. coli, BGN4, or L. casei decreased the OVA-induced allergy response. However, a normal increase in body weight was inhibited in the E. coli-treated mice and in the montreated mice groups during allergy sensitization. Thus, BGN4 and L. casei appear to be useful probiotic bacteria for the prevention of allergy.     

Role of Intestinal Bacteria in Ileal Ulcer Formation in Rats Treated with a Nonsteroidal Antiinflammatory Drug

The role of intestinal bacteria in induction and repression of ulcer formation in the ileum of rats treated with one of the nonsteroidal antiinflammatory drugs (NSAIDs), 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl) thiophene (BFMeT), was examined in this study. BFMeT was administered by intragastric gavage once at doses of 500-1,500 mg/kg of body weight to Wistar rats treated with and without antibiotics (bacitracin, neomycin, streptomycin), germ-free rats and gnotobiotic rats, and 72 hr later their gastrointestinal tracts were examined for ulcer formation. A single oral administration of BFMeT induced ileal ulcers in specific pathogen-free rats. However, the rats given antibiotics to reduce the intestinal bacteria had no ulcers. BFMeT-treated germ-free rats and gnotobiotic rats mono-associated with Bifidobacterium adolescentis or Lactobacillus acidophilus also had no intestinal ulcers. However, the drug induced ileal ulcers in gnotobiotic rats mono-associated with Eubacterium limosum or Escherichia coli. An overnight culture of B. adolescentis or L. acidophilus or yogurt containing Bifidobacterium breve and Streptococcus thermophilus, when given as drinking water, inhibited ulcer formation in the ileum of rats treated with BFMeT. Gram staining of the ileal contents of normal rats revealed that 97.4% of the stained microorganisms were Gram-positive rods and only 1.2% were Gram-negative rods. In the group of rats with ulcers induced by BFMeT, the Gram-positive rods decreased by 56.4% and the Gram-negative rods including Escherichia coli, Klebsiella, Proteus and Bacteroides increased by 37.3%. However, in the group of rats administered the Bifidobacterium culture, the Lactobacillus culture or yogurt, the percentages of the Gram-negative rods were decreased. Although Lactobacillus was a major bacterium in the ileum of normal rats, the Gram-negative facultatively anaerobic rods E. coli, Klebsiella and Proteus were increased in the ulcerated ileum of rats treated with BFMeT, suggesting that these bacteria are associated with ulcer formation in rats treated with NSAIDs, and that Lactobacillus and Bifidobacterium inhibit it by repressing the growth of ulcer-inducing bacteria.     

Role of Gastrointestinal Microflora in the Mineral Absorption of Young Adult Mice

A comparison between germfree (GF) and gnotobiotic (GB) mice, inoculated with Bacteroides vulgatus, Eubacterium aerofaciens, Bifidobacterium longum, Enterococcus faecalis, Escherichia coli, and Clostridium perfringens, revealed that the GB mice suffered no deleterious effect on the apparent absorption ratios of Ca and P, and showed a higher apparent absorption ratio of Mg.     

Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium species

Fecal microbiota of 31 breast-fed, 26 mix-fed, and 11 bottle-fed infants were analyzed by using terminal restriction fragment length polymorphism (T-RFLP), and culture method. We first determined the total and cultivated bacterial counts in infant fecal microbiota. Only approximately 30% of bacteria present in fecal microbiota were cultivable while the remainder was yet-to-be cultured bacteria. Sixty-eight fecal samples were divided into two clusters (I and II) by T-RFLP analysis, and then subdivided into five subclusters (Ia, Ib, IIa, IIb and IIc). There was no clear relationship between clusters and feeding method. A proportion of bifidobacteria was detected in the fecal material by PCR method using species-specific primers. The predominant Bifidobacterium spp. was Bifidobacterium longum longum type (43 samples (63.2%)), followed by B. longum infantis type (23 samples (33.8%)) and B. breve (16 samples (23.5%)). The distribution of Bifidobacterium spp. was similar in the three feeding groups. In contrast, the high incidence of B. breve in cluster I, especially subcluster Ia and B. longum longum type in cluster II, especially subcluster IIa and IIc were characterized by T-RFLP method. Our results showed that the colonization of Bifidobacterium spp. in infant feces correlated with the T-RFLP clusters.     

Allergic Patients with Long-Term Asthma Display Low Levels of Bifidobacterium adolescentis

Accumulated evidence suggests a relationship between specific allergic processes, such as atopic eczema in children, and an aberrant fecal microbiota. However, little is known about the complete microbiota profile of adult individuals suffering from asthma. We determined the fecal microbiota in 21 adult patients suffering allergic asthma (age 39.43 ± 10.98 years old) and compare it with the fecal microbiota of 22 healthy controls (age 39.29 ± 9.21 years old) using culture independent techniques. An Ion-Torrent 16S rRNA gene-based amplification and sequencing protocol was used to determine the fecal microbiota profile of the individuals. Sequence microbiota analysis showed that the microbial alpha-diversity was not significantly different between healthy and allergic individuals and no clear clustering of the samples was obtained using an unsupervised principal component analysis. However, the analysis of specific bacterial groups allowed us to detect significantly lower levels of bifidobacteria in patients with long-term asthma. Also, in allergic individuals the Bifidobacterium adolescentis species prevailed within the bifidobacterial population. The reduction in the levels on bifidobacteria in patients with long-term asthma suggests a new target in allergy research and opens possibilities for the therapeutic modulation of the gut microbiota in this group of patients.     

Similar bifidogenic effects of prebiotic-supplemented partially hydrolyzed infant formula and breastfeeding on infant gut microbiota

The aim of the study was to assess the quantitative and qualitative differences of the gut microbiota in infants. We evaluated gut microbiota at the age of 6 months in 32 infants who were either exclusively breast-fed, formula-fed, nursed by a formula supplemented with prebiotics (a mixture of fructo- and galacto-oligosaccharides) or breast-fed by mothers who had been given probiotics. The Bifidobacterium, Bacteroides, Clostridium and Lactobacillus/Enterococcus microbiota were assessed by the fluorescence in situ hybridization, and Bifidobacterium species were further characterized by PCR. Total number of bifidobacteria was lower among the formula-fed group than in other groups (P=0.044). Total amounts of the other bacteria were comparable between the groups. The specific Bifidobacterium microbiota composition of the breast-fed infants was achieved in infants receiving prebiotic supplemented formula. This would suggest that early gut Bifidobacterium microbiota can be modified by special diets up to the age of 6 months.     

婴儿双歧杆菌对花生过敏小鼠模型肠道Th2反应的调节

目的研究婴儿型双歧杆菌对花生过敏小鼠肠道Th2型反应的调节作用。方法通过应用花生蛋白诱导肠道的Th2型反应,建立食物过敏小鼠模型。过敏小鼠灌胃给予婴儿型双歧杆菌（ATCC菌或CGMCC0313-2）或不做处理。然后分离小鼠小肠黏膜CD4＋T细胞或DC,另取肠黏膜组织进行石蜡包埋甲苯胺蓝染色肥大细胞计数,HE染色进行嗜酸细胞和单个核细胞计数,流式细胞检测CD4＋T中Th2（CD4＋IL4＋T）细胞和Treg（CD4＋CD25＋Foxp3＋T）比例,另取CD4＋T进行CFSE标记,与DC共培养4d后流式细胞检测CD4＋T增殖反应,收集细胞培养液ELISA检测IL-4、IL-5和IL—13分泌水平。结果过敏组小鼠Th2型细胞数,CD4＋T细胞增殖反应,IL4、IL-5和IL-13水平,肠黏膜中肥大细胞、嗜酸性细胞和单个核细胞数均明显高于对照组（P〈0．01）,而Treg数目低于对照组（P〈0．01）,婴儿双歧杆菌干预后,婴儿双歧杆菌组Th2型细胞数,IL4、IL-5和IL-13水平,肠黏膜中肥大细胞、嗜酸性细胞和单个核细胞数均明显低于过敏组（P〈0．01）,而Treg数目高于过敏组（P〈0．01）。结论口服婴儿型双歧杆菌可以抑制花生过敏导致的肠道Th2型反应。     

Critical role of preconceptional immunization for protective and nonpathological specific immunity in murine neonates

Expression of Th2 immunity against environmental Ags is the hallmark of the allergic phenotype and contrasts with the Th1-like pattern, which is stably expressed in healthy adults throughout life. Epidemiological studies indicate that the prenatal environment plays an important and decisive role in the development of allergy later in life. Since the underlying mechanisms were unclear, an animal model was developed to study the impact of maternal allergy on the development of an allergic immune response in early life. An allergic Th2 response was induced in pregnant mice by sensitization and aerosol allergen exposure. Both, IgG1 and IgG2a, but not IgE, Abs cross the placental barrier. Free allergen also crosses the placental area and was detected in serum and amniotic fluids of neonatal F(1) mice. These F(1) mice demonstrated a suppressed Th1 response, as reflected by lowered frequencies and reduced levels of IFN-gamma production. Development of an IgE response against the same allergen was completely prevented early in life. This effect was mediated by diaplacental transfer of allergen-specific IgG1 Abs. In contrast, allergic sensitization against a different allergen early in life was accelerated in these mice. This effect was mediated by maternal CD4 and OVA-specific Th2 cells induced by allergic sensitization during pregnancy. These data indicate a critical role for maternal T and B cell response in shaping pre- and postnatal maturation of specific immunity to allergens.     

阿莫西林干预对婴儿优势菌在无菌小鼠体内定植的影响

目的考察阿莫西林干预对婴儿优势菌双歧杆菌及乳杆菌在无菌小鼠体内定植的影响。方法 1日龄Balb/c无菌乳鼠接种婴儿粪便悬液。饲养至7~21日龄灌胃阿莫西林(100mg/kg),对照组在同日龄给予等体积的生理盐水。利用qRT-PCR检测小鼠粪便中双歧杆菌、乳杆菌的含量。结果阿莫西林处理可显著降低乳杆菌(P0.05)、双歧杆菌(P0.05)在无菌小鼠体内定植数量,但停药后饲养至成年(53日龄)二者定植数量与对照组小鼠比较差异无统计学意义(P0.05)。结论哺乳期阿莫西林干预会导致乳杆菌、双歧杆菌在小鼠体内定植量下降,但停药后小鼠饲养至成年二者可达到正常定植量,婴儿菌群定植小鼠模型可以模拟与现有动物模型一致的阿莫西林对双歧杆菌、乳杆菌定植的影响规律。     

Protective effect of bifidus milk on the experimental infection with Salmonella enteritidis subsp. typhimurium in conventional and gnotobiotic mice

The ability of Bifidobacterium bifidum from a commercial bifidus milk to antagonize Salmonella enteritidis subsp. typhimurium in vivo, and to reduce the pathological consequences for the host, was determined using conventional and gnotobiotic mice. Conventional animals received daily, by gavage, 0.1 ml bifidus milk containing about 10(9) cfu B. bifidum and germ-free animals received a single 0.1 ml dose. The conventional and gnotobiotic groups were challenged orally with 10(2) cfu of the pathogenic bacteria 5 and/or 10 d after the beginning of treatment. Control groups were treated with milk. Bifidus milk protected both animal models against the challenge with the pathogenic bacteria, as demonstrated by survival and histopathological data. However, to obtain the protective effect in gnotobiotic animals, the treatment had to be initiated 10 d before the challenge. In experimental and control gnotobiotic mice, Salm. enteritidis subsp. typhimurium became similarly established at levels ranging from 10(8) to 10(9) viable cells g-1 of faeces and remained at these high levels until the animals died or were sacrificed. It was concluded that the protection against Salm. enteritidis subsp. typhimurium observed in conventional and gnotobiotic mice treated with bifidus milk was not due to the reduction of the intestinal populations of the pathogenic bacteria.     

Oral Immunotherapy for Pollen Allergy Using T-Cell Epitope-Containing Egg White Derived from Genetically Manipulated Chickens

Peptide immunotherapy using T-cell epitopes is expected to be an effective treatment for allergic diseases such as Japanese cedar (Cryptomeria japonica; Cj) pollinosis. To develop a treatment for pollen allergy by inducing oral tolerance, we generated genetically manipulated (GM) chickens by retroviral gene transduction, to produce a fusion protein of chicken egg white lysozyme and a peptide derived from seven dominant human T-cell epitopes of Japanese cedar pollen allergens (cLys-7crp). The transgene sequence was detected in all chickens transduced with the retroviral vector. Transduction efficiency in blood cells correlated to transgene expression. Western blot analysis revealed that cLys-7crp was expressed in the egg white of GM hens. Mice induced to develop allergic rhinitis by Cj pollinosis were fed with cLys-7crp-containing egg white produced by GM chickens. Total and Cj allergen (Cry j 1)-specific IgE levels were significantly decreased in allergic mice fed with cLys-7crp-containing egg white compared with allergic mice fed with normal egg white. These results suggest that oral administration of T-cell epitope-containing egg white derived from GM chickens is effective for the induction of immune tolerance as an allergy therapy.