Masaru Hiramatsu

Stimulation of the secretion of pro-inflammatory cytokines by Bifidobacterium strains.

To characterize the ability of bifidobacteria to affect the production of macrophage‐derived cytokines, a murine macrophage‐like cell line, J774.1, was cultured in the presence of 27 strains of heat‐inactivated bifidobacteria. Bifidobacterium adolescentis and B. longum, known as adult‐type bifidobacteria, induced significantly more pro‐inflammatory cytokine secretion, IL‐12 and TNF‐α, by J774.1 cells, than did the infant‐type bifidobacteria, B. bifidum, B. breve, and B. infantis (P<0.01). In contrast, B. adolescentis did not stimulate the production of anti‐inflammatory IL‐10 from J774.1 cells as the other tested bacteria did. The results suggest that the adult‐type bifidobacteria, especially B. adolescentis, may be more potent to amplify but less able to down‐regulate the inflammatory response.

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.

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 Peyers 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.

Effect of orally administered non-viable Lactobacillus cells on murine humoral immune responses.

BALB/c mice were immunized intraperitoneally with the food antigen ovalbumin (OVA) while they were fed with Lactobacillus GG heated killed cell preparation. The oral administration of Lactobacillus GG did not appear to modify the antigen‐augmented serum IgE in the tested mice but significantly augmented serum OVA specific IgG in the tested mice fed with a diet containing 0.1% Lactobacillus GG as the non‐viable cell preparation (P<0.05). The fecal OVA specific IgA of the tested mice fed with non‐viable Lactobacillus GG cells was also significantly elevated (P<0.05) compared to those from OVA immunized mice. The spleen cells of mice fed with non‐viable Lactobacillus GG cells secreted more IL‐6 (P<0.01). These results suggest that the non‐viable Lactobacillus GG can augment the systemic and mucosal immune responses in a host animal favoring secretory IgA but not IgE in an adjuvant‐like manner.

Lactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor.

BackgroundFermented milk is considered one of the best sources for efficient consumption of probiotic strains by hosts to promote good health. The purpose of this study was to investigate the effects of orally administering LGG-fermented milk (LGG milk) on intestinal inflammation and injury and to study the mechanisms of LGG milk’s action.MethodsLGG milk and non-LGG-fermented milk (non-LGG milk) were administered through gavage to mice before and during dextran sodium sulfate (DSS)-induced intestinal injury and colitis. Inflammatory/injury score and colon length were assessed. Intestinal epithelial cells were treated with the soluble fraction of LGG milk to detect its effects on the epidermal growth factor receptor (EGFR) and its downstream target, Akt activation, cytokine-induced apoptosis, and hydrogen peroxide (H2O2)-induced disruption of tight junctions.ResultsLGG milk treatment significantly reduced DSS-induced colonic inflammation and injury, and colon shortening in mice, compared to that in non-LGG milk-treated and -untreated mice. The soluble fraction of LGG milk, but not non-LGG milk, stimulated the activation of EGFR and Akt in a concentration-dependent manner, suppressed cytokine-induced apoptosis, and attenuated H2O2-induced disruption of tight junction complex in the intestinal epithelial cells. These effects of LGG milk were blocked by the EGFR kinase inhibitor. LGG milk, but not non-LGG milk, contained two soluble proteins, p40 and p75, that have been reported to promote survival and growth of intestinal epithelial cells through the activation of EGFR. Depletion of p40 and p75 from LGG milk abolished the effects of LGG milk on prevention of cytokine-induced apoptosis and H2O2-induced disruption of tight junctions.ConclusionsThese results suggest that LGG milk may regulate intestinal epithelial homeostasis and potentially prevent intestinal inflammatory diseases through activation of EGFR by LGG-derived proteins.

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.

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‐RFLP methods. 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 subjects 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.

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.

Effects of heat-inactivated Lactobacillus gasseri TMC0356 on metabolic characteristics and immunity of rats with the metabolic syndrome

The present study investigated the potential health-promoting effects of heat-inactivated Lactobacillus gasseri TMC0356 (TMC0356) on the metabolic syndrome (MS) and the probable mechanisms underlying these effects using an MS rat model. For the purpose of the study, sixty Sprague-Dawley rats were randomly divided into five groups: a control group fed a conventional diet, an MS model group fed a high-fat and high-salt (HFS) diet and three TMC0356 test groups (low-, medium- and high-dose groups) fed an HFS diet supplemented with TMC0356 at 41.8, 83.5 and 167.0 mg/kg body weight (BW) per d, respectively. Food intake and BW were measured weekly. Fasting blood glucose (FBG), lipid profiles and blood pressure (BP) were measured at 0, 5, 10 and 15 weeks. Organ coefficients, immune cell counts and serum insulin, adiponectin, C-reactive protein (CRP), IL-6, TNF-α, IgG and secretory IgA levels were measured at the 15th week after diet intervention. The HFS diet increased the BW, liver or fat:BW ratio, FBG, homeostatic model assessment of insulin resistance, adiponectin, serum LDL-cholesterol and total cholesterol levels and BP (P< 0.01). Average food and energy intakes in the three TMC0356 groups were significantly lower than those of the MS model group. All the metabolic indices, except BP, were markedly improved (P< 0.05) by oral administration of low and medium doses of TMC0356. The thymus index in the medium-dose group and lymphocyte, CRP, IL-6, TNF-α and IgG levels in all the three TMC0356 groups were significantly increased (P< 0.05 or P< 0.01) compared with those in the MS model group. These results suggest that TMC0356 can improve the metabolic characteristics of MS rats by suppressing appetite. Additionally, the enhancement of inflammatory immune response may be, at least in part, the mechanism underlying the health-promoting effects of TMC0356 on the MS.