Probiotics modulate gut microbiota and health status in Japanese cedar pollinosis patients during the pollen season.

BACKGROUND: Japanese cedar pollinosis (JCP) is a challenging public health problem in Japan. Altered gut microbiota is associated with several diseases, including allergic diseases. However, only a few studies have focused on JCP and the underlying mechanisms for probiotic effects remain unclear. In addition, this study is the first observation of the correlation between the gut microbiota and blood lipid in JCP. METHODS: Faecal samples from JCP subjects were collected before and after treatment with (n = 14) and without (n = 11) LGG-TMC0356-fermented milk for 10 weeks. Gut microbiota composition was characterized from faecal DNA using sequencing of 16S rRNA genes. RESULTS: 16S rRNA-based operational taxonomic unit clustering of the microbiota revealed that LGG-TMC0356-fermented milk significantly altered gut microbiota after 10 weeks of milk consumption, and eight dominant genera of microbes were detected. During the JCP season, the Bacteroidetes/Firmicutes ratio, when compared to baseline, was significantly decreased in subjects at end of the study. Bacteroidetes showed positive correlation with LDL- and HDL-cholesterol levels, whereas Firmicutes showed negative correlation with total cholesterol, LDL- and HDL- cholesterol. CONCLUSIONS: The altered gut microbiota through supplementation of fermented milk containing the study probiotics may be a prospective target for protection against JCP, with beneficial effects on blood lipid levels.

A combination of probiotics and whey proteins enhances anti-obesity effects of calcium and dairy products during nutritional energy restriction in aP2-agouti transgenic mice.

Lactobacillus rhamnosus GG, Lactobacillus paracasei TMC0409, Streptococcus thermophilus TMC1543 and whey proteins were used to prepare fermented milk. For the experiment aP2- agouti transgenic mice were pre-treated with a high-sucrose/high-fat diet for 6 weeks to induce obesity. The obese mice were fed a diet containing 1·2% Ca and either non-fat dried milk (NFDM) or probiotic-fermented milk (PFM) with nutritional energy restriction for 6 weeks. The animals were examined after the treatment for changes in body weight, fat pad weight, fatty acid synthase (FAS) activity, lypolysis, the expression levels of genes related to lipid metabolism, insulin sensitivity in adipocytes and skeletal muscle and the presence of biomarkers for oxidative and inflammatory stress in plasma. It was found that the PFM diet significantly reduced body weight, fat accumulation, and adipocyte FAS activity, and increased adipocyte lipolysis as compared with the effects of the NFDM diet (P<0·05). The adipose tissue gene expression of 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) was significantly suppressed in mice that were fed PFM as compared with those that were fed NFDM (P<0·05). PFM caused a greater up-regulation of skeletal muscle PPARα, PPARδ, uncoupling protein 3 (UCP3) and GLUT4 expression and a significant decrease in the plasma concentration of insulin, malondialdehyde, TNF-α, monocyte chemotactic protein-1 and C-reactive protein as compared with the effects of NFDM (P<0·05). Fermentation of milk with selected probiotics and supplementation of milk with whey proteins may thus enhance anti-obesity effects of Ca and dairy products by the suppression of adipose tissue lipogenesis, activation of fat oxidation in skeletal muscle and reduction of oxidative and inflammatory stress.

Influence of orally administered Lactobacillus GG on respiratory immune response in a murine model of diet-induced obesity.

Mice with diet-induced obesity were fed with Lactobacillus rhamnosus GG (LGG) suspended in saline or saline alone (control mice). Pulmonary mRNA expression of IFN-γ; IFN-α receptor 1; CD247 antigen; killer cell lectin-like receptor subfamily K, member 1; TNF-α; IL-12 receptor ß1 and IL-2 receptor ß, and the proportion of Lactobacillales in feces were significantly greater in the LGG group than in the control mice (P < 0.05 and P < 0.01, respectively). These results suggest that LGG alters the respiratory immunity of obese subjects through having a potent impact on intestinal immunity.

Oral administration of Lactobacillus gasseri TMC0356 stimulates peritoneal macrophages and attenuates general symptoms caused by enteropathogenic Escherichia coli infection.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in human. This study was conducted to investigate the ability of orally administrated probiotic lactobacilli to protect hosts from EPEC infection via enhancement of immune responses. METHODS: Lyophilized Lactobacillus gasseri TMC0356 (TMC0356) was orally administered to Institute of Cancer Research (ICR) mice and Sprague Dawley (SD) rats for 11 and 7 days, respectively. These tested mice and rats were intraperitoneally injected with EPEC. Body weight, general symptoms (piloerection, soft stool, diarrhea, and anal hyperemia), and mortality of the tested mice were observed. Peritoneal macrophages were extracted from peritoneal cavity of tested rats, and their phagocytosis and cytokine production were analyzed. RESULTS: Oral administration of TMC0356 accelerated the disappearance of general symptoms and reduced mortality of EPEC-infected mice in the early phase. Peritoneal macrophages from rats orally administered with TMC0356 showed significant increases in phagocytic activity (p < 0.05) and interleukin (IL)-6 production (p < 0.01) compared to those from control rats. Tumor necrosis factor-α and production of IL-1ß, IL-10, and IL-12 slightly increased, although the changes were not statistically significant. CONCLUSION: These results suggest that some of selected probiotic lactobacilli may, at least partly, protect hosts from EPEC infection by the enhancement of innate immunity of host and attenuate symptoms caused by the infection.

Heat-killed Lactobacillus gasseri TMC0356 protects mice against influenza virus infection by stimulating gut and respiratory immune responses.

This study investigated whether heat-killed Lactobacillus protects host animal against influenza virus infection and stimulates their immunity. Heat-killed Lactobacillus gasseri TMC0356 was orally administered to BALB/c mice for 19 days; the mice were intranasally infected with Flu A/PR/8/34 (H1N1) on day 14, and clinical symptoms were monitored. After 6 days, the mice were sacrificed, and pulmonary virus titres were determined. Splenic activation of natural killer (NK) cells and the mRNA expression of cytokines and other immune molecules in the lung and Peyer's patch (PP) were analysed. Clinical symptom scores of mice orally fed TMC0356 ameliorated significantly (P < 0.01); their pulmonary virus titres decreased significantly compared with those of control mice (P < 0.05); their mRNA expression of interleukin (IL)-12, IL-15 and IL-21 in PP and the pulmonary mRNA expression of IFN-γ, TNF, IL-12a, IL-12rbl, IL-2rb and perforin 1 increased significantly (P < 0.05). Oral administration of heat-killed lactobacilli may protect against influenza virus infection by stimulating local and systemic immune responses. Cellular components of lactobacilli may be pivotal in protecting against viral infection by enhancing gut and respiratory immune responses.

Orally administered heat-killed Lactobacillus gasseri TMC0356 can upregulate cell-mediated immunity in senescence-accelerated mice.

The present study was conducted to test the ability of probiotic lactobacilli to alter age-related immunosenescence in host animals. Senescence-accelerated mouse prone 1 mice were orally fed heat-killed Lactobacillus gasseri TMC0356 (TMC0356) for 4 and 8 weeks at dosages of 10 mg day(-1) after a 16-week period of prefeeding with a standard diet. After 4 and 8 weeks of TMC0356 intervention, splenic activation of natural killer (NK) cells and mRNA expression of cytokines and other immune molecules in the lungs were analysed. After 4 and 8 weeks, splenic NK cell activities were significantly higher in the TMC0356-fed mice compared with control mice (P < 0.05). After 4 weeks, mRNA expression of interleukin-2 and interferon-(α and ß) receptor 1 in lung cells isolated from the TMC0356-fed mice also increased significantly compared with that in lung cells from the control mice (P < 0.05). These results suggest that lactobacilli, especially certain selected strains, might enhance cell-mediated immunity in host animals and thereby alter age-related immunosenescence.

Strain-specific detection by pulsed-field gel electrophoresis of Lactobacillus gasseri TMC0356 in human feces after oral administration of these organisms.

The present study aimed to develop an innovative, strain-specific means of identifying the probiotic Lactobacillus gasseri TMC0356 and to determine whether orally administered TMC0356 could be recovered from the human intestine. High molecular weight genomic DNA was isolated from TMC0356 and 14 reference strains of L. gasseri, including the type strain. The DNA samples were digested with the selected rare-cutting restriction endonucleases SmaI, SacII and ApaI and the resulting fragments separated by pulsed-field gel electrophoresis (PFGE) in a size range between 20 to 290 kb. TMC0356 could be distinguished from the other L. gasseri strains on the basis of the SmaI and SacII macrorestriction patterns. Furthermore, L. gasseri strains isolated from the feces of subjects who had ingested TMC0356 were identical to TMC0356 in the SmaI, SacII and ApaI macrorestriction fragments of digested DNA. These results suggest that PFGE of genomic DNA digested with SmaI, SacII, could be a practical means of identification of TMC0356. Furthermore, these results indicate that ingested TMC0356 can survive in, and colonize, the human intestine.

Diversity of intestinal bifidobacteria in patients with Japanese cedar pollinosis and possible influence of probiotic intervention.

This study was conducted to evaluate the potential association between intestinal bifidobacteria and Japanese cedar pollinosis (JCPsis) and possible influences of probiotic intervention. In this study, fecal samples were the collected from 29 JCPsis patients. The qualitative and quantitative analyses of fecal bifidobacteria were conducted by quantitative real-time PCR with 16S rRNA-gene-targeted species-specific primers before cedar pollen spread and after a 10-week intervention with fermented milk prepared with Lactobacillus GG and L. gasseri TMC0356 during pollen spread. Each JCPsis patient had a unique diversity of bifidobacteria, which varied qualitatively and quantitatively in an individual-dependent manner during pollen spread. The serum IgE concentration of JCPsis patients with more than 3 detectable Bifidobacterium species was significantly lower than that of patients with less than 2 detected species. The prevalence of B. adolescentis, B. longum, and B. catenulatum increased after probiotic intervention, although the changes were not statistically significant. These results suggest that lower diversity of intestinal Bifidobacterium species might be a pathological aspect of JCPsis. The diversity of intestinal bifidobacteria could be a prospective target for using probiotics in the management of IgE-mediated allergic disorders including JCPsis.

Bifidobacterium suppresses IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells.

Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro. The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6-56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41-55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis (P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.

Lactobacillus strains stabilize intestinal microbiota in Japanese cedar pollinosis patients.

A randomized double-blind, placebo-controlled trial was conducted to ascertain the intestinal microbiota-altering properties of LGG and L. gasseri TMC0356 (TMC0356) in Japanese cedar Cryptomeria japonica pollinosis patients. Fecal bacteria communities were examined before and after fermented milk administration using culture, FISH and T-RFLPmethods. Test group subjects showed the presence of LGG and TMC0356 along with a significant increase in fecal lactobacilli (P < 0.001) after giving LGG and TMC0356 fermented milk. Culture and FISH analysis revealed no significant changes in other intestinal bacterial groups. Each subject exhibited a characteristic T-RFLP profile pattern that varied quantitatively and qualitatively with JCP shedding. Profile changes were observed in 53% of placebo group subjects and in 21% of test group subject's post-administration, indicating that LGG and TMC0356 suppressed intestinal microbiota changes in JCPsis patients. The results suggest that intestinal microbiota might be more sensitive to exposure to environmental allergens than expected from the results of general culture method studies. Stabilization of intestinal microbiota by selected probiotic strains such as LGG and TMC0356 could be beneficial to homeostasis of the intestinal microbiota and useful in the management of JCPsis.

Differentiated implication of Lactobacillus GG and L. gasseri TMC0356 to immune responses of murine Peyer's patch.

Lactobacillus GG and L. gasseri TMC0356 were examined for their potential to alter the immune responses of murine PP cells in vitro and in vivo. Lactobacillus GG and L. gasseri TMC0356 characteristically stimulated the production of IL-12, IL-6, IFN-gamma and IgA from isolated PP cells in vitro. Anatomical analysis indicated uptake of these bacteria by the PP tissue after giving orally in mice. Isolated PP cells exposed to Lactobacillus GG in vivo secreted more IFN-gamma, IL-6 and total IgA, whereas those exposed to L. gasseri TMC0356 in vivo did not exhibit altered immune responses in terms of cytokine and IgA production. Therefore, these two bacteria might exhibit different immunodulatory effects in host animals by strain-dependent interaction with gut-associated lymphoid tissues in vivo.

Effect of fermented milk prepared with two probiotic strains on Japanese cedar pollinosis in a double-blind placebo-controlled clinical study.

There has been much interest in the potential of using probiotic bacteria for treating allergic diseases. A double-blind, placebo-controlled study was conducted to examine the effectiveness of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC0356) in alleviating Japanese cedar pollinosis (JCP), a seasonal allergic rhinitis caused by Japanese cedar pollen. Fermented milk prepared with the tested bacteria or placebo yoghurt was administered to 40 subjects with a clinical history of JCP for 10 weeks. Subjective symptoms, self-care measures and blood samples were compared between the two groups. Peripheral blood mononuclear cells (PBMCs) were collected from seven patients with JCP and in vitro cytokine production by the isolated PBMCs was analysed in the presence of heat-killed lactic acid bacteria. Consumption of the fermented milk significantly decreased the mean symptom score for nasal blockage after 9 weeks (P<0.05) and mean symptom-medication scores after 9 and 10 weeks when compared with the placebo group (P<0.01 and P<0.05 respectively). The tested strains of lactic acid bacteria affected cytokine production by isolated PBMCs in vitro in a strain-dependent manner. LGG significantly inhibited IL-4 and IL-5 production by PBMCs stimulated by both Cry j 1 and PHA. TMC0356 only suppressed IL-5 production stimulated by PHA. The fermented milk prepared with LGG and TMC0356 might be beneficial in JCP because of its effect on nasal blockage. The effects of LGG and TMC0356 might arise at least partly from their specific down-regulation of the human Th2 immune response.

Orally administrated Lactobacillus gasseri TMC0356 and Lactobacillus GG alleviated nasal blockage of guinea pig with allergic rhinitis.

Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC0356) were investigated for their ability to alleviate nasal blockage associated with allergic rhinitis using a guinea pig model. The increases in sRaw at 10 min and 5 hr after the exposure of the nasal mucosa to OVA were significantly alleviated in the guinea pigs orally administrated with LGG and TMC0356 compared with those of the control (P<0.05 and P<0.01). The total numbers of leukocytes, particularly eosinophils and neutrophils from the nasal cavity lavage fluid, and the OVA-specific IgE concentration in the serum were also decreased in the guinea pigs orally administrated with LGG and TMC0356, although the decreases were not statistically significant. These results suggest that LGG and TMC0356 can alleviate antigen-induced nasal blockage in earlyphase and late-phase inflammatory responses associated with allergic rhinitis.

Inhibitory effect of Lactobacillus gasseri TMC0356 and Lactobacillus GG on enhanced vascular permeability of nasal mucosa in experimental allergic rhinitis of rats.

The nasal vascular permeability of ovablumin (OVA)-sensitized Brown Norway rats was evaluated by analyzing a brilliant blue concentration in perfusate from the nose after exposure of the nasal mucus to OVA. Oral administration of Lactobacillus GG and L. gasseri TMC0356 significantly inhibited the increase in nasal vascular permeability (P<0.01). The serum IgE of the tested rats also decreased, although the change was not statistically significant. These results indicate that Lactobacillus GG and L. gasseri TMC0356 might alleviate nasal allergic symptoms by suppressing the increase in nasal vascular permeability caused by local inflammation associated with allergic rhnititis.

Preliminary human study for possible alteration of serum immunoglobulin E production in perennial allergic rhinitis with fermented milk prepared with Lactobacillus gasseri TMC0356.

The fermented milk prepared with Lactobacillus gasseri TMC0356 was administered at 200 ml per day for 4 weeks to 15 subjects with high serum IgE levels and perennial allergic rhinitis. The serum total IgE concentration was significantly reduced after 28 days' exposure to the fermented milk (P <0.05) compared to that before the intervention. The serum IgE specific to Acari and those to Japanese cedar pollen also significantly declined (P <0.05). T helper 1 (Th1) cells in the composition of their peripheral blood mononuclear cells (PBMCs) significantly increased after 14 days (P <0.01) and after 28 days (P <0.05). These results suggest that the fermented milk prepared with L. gasseri TMC0356 may alter serum IgE concentration, at least partly by enhancement of Th1 immune responses of the subjects with high concentration of serum IgE. However, further studies are still necessary to know the underlying mechanisms by which the tested fermented milk could influence the host immunity.