Fumihiko Sakai

Assay systems for screening food components that have anti-proliferative and anti-invasive activity to rat ascites hepatoma cells : In vitro and ex vivo effects of green tea extract

We have developed in vitro and ex vivo assay systems for screening food components and natural substances that suppress the proliferation and/or invasion of a rat ascites hepatoma cell line of AH109A and have used them to study the effect of green tea extract. AH109A cells were found to penetrate underneath the monolayer of primary cultured mesothelial cells isolated from Donryu rat mesentery in the presence of 10% newborn bovine serum. Green tea extract inhibited this AH109A penetration in a dose dependent manner and also inhibited AH109A proliferation in vitro dose-dependently. Green tea tannin, the major polyphenolic substances in green tea extract, also inhibited the proliferation and invasion of AH109A in vitro in a dose dependent manner. When rat serum obtained 0.5 h after oral intubation of green tea extract was added to the culture media instead of newborn bovine serum at a concentration of 10%, the invasion of AH109A was significantly inhibited as compared to control rat serum (before green tea extract intubation); the inhibitory effect lasted for 1 h and disappeared 3 h after oral intubation of green tea extract, but those rat sera showed no inhibition of AH109A proliferation. These results suggest that green tea extract has an inhibitory effect on the invasion of AH109A both in vitro and ex vivo, but on the proliferation of AH109A only in vitro, and that these assay systems are effective for the screening of food components which inhibit tumor cell proliferation and invasion.

Lactobacillus gasseri SBT2055 Induces TGF-β Expression in Dendritic Cells and Activates TLR2 Signal to Produce IgA in the Small Intestine

Probiotic bacteria provide benefits in enhancing host immune responses and protecting against infection. Induction of IgA production by oral administration of probiotic bacteria in the intestine has been considered to be one reason for this beneficial effect, but the mechanisms of the effect are poorly understood. Lactobacillus gasseri SBT2055 (LG2055) is a probiotic bacterium with properties such as bile tolerance, ability to improve the intestinal environment, and it has preventive effects related to abdominal adiposity. In this study, we have found that oral administration of LG2055 induced IgA production and increased the rate of IgA+ cell population in Peyers patch and in the lamina propria of the mouse small intestine. The LG2055 markedly increased the amount of IgA in a co-culture of B cells and bone marrow derived dendritic cells (BMDC), and TLR2 signal is critical for it. In addition, it is demonstrated that LG2055 stimulates BMDC to promote the production of TGF-β, BAFF, IL-6, and IL-10, all critical for IgA production from B cells. Combined stimulation of B cells with BAFF and LG2055 enhanced the induction of IgA production. Further, TGF-β signal was shown to be critical for LG2055-induced IgA production in the B cell and BMDC co-culture system, but TGF-β did not induce IgA production in a culture of only B cells stimulated with LG2055. Furthermore, TGF-β was critical for the production of BAFF, IL-6, IL-10, and TGF-β itself from LG2055-stimulated BMDC. These results demonstrate that TGF-β was produced by BMDC stimulated with LG2055 and it has an autocrine/paracrine function essential for BMDC to induce the production of BAFF, IL-6, and IL-10.

Lactobacillus helveticus SBT2171 Inhibits Lymphocyte Proliferation by Regulation of the JNK Signaling Pathway

Lactobacillus helveticus SBT2171 (LH2171) is a lactic acid bacterium with high protease activity and used in starter cultures in the manufacture of cheese. We recently reported that consumption of cheese manufactured using LH2171 alleviated symptoms of dextran sodium sulfate (DSS)-induced colitis in mice. In this study, we have examined whether LH2171 itself exerts an inhibitory effect on the excessive proliferation of lymphocytes. We found that LH2171 inhibited the proliferation of LPS-stimulated mouse T and B cells, and the human lymphoma cell lines, Jurkat and BJAB. Cell cycle analysis showed an accumulation of LH2171-treated BJAB cells in the G2/M phase. Further, phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun was reduced by LH2171 in BJAB cells. Subsequently, expression of cell division cycle 2 (CDC2), regulated by the JNK signaling pathway and essential for G2/M phase progression, was inhibited by LH2171. It was also demonstrated that intraperitoneal administration of LH2171 strongly alleviated symptoms of collagen-induced arthritis (CIA) in mice. These findings suggest that LH2171 inhibits the proliferation of lymphocytes through a suppression of the JNK signaling pathway and exerts an immunosuppressive effect in vivo.

Prevention of rotavirus-induced diarrhea by preferential secretion of IgA in breast milk via maternal administration of Lactobacillus gasseri SBT2055.

Objectives: Rotavirus (RV)-induced diarrhea poses a major health problem, particularly to infants. An effective measure to prevent RV infection is to consume breast milk with higher levels of protective IgA. We therefore examined whether Lactobacillus gasseri SBT2055 (LG2055) could augment immumoglobulin (Ig) A levels and reduce the incidence of diarrhea in a mouse model of RV infection. Methods: Female BALB/c mouse dams were fed a diet containing 0.1% heat-treated LG2055 or a control, beginning 4 weeks before mating with male mice and continuing until the experiment ended. One week after mating, female dams were immunized orally with simian RV SA-11. Five days after birth, mouse pups were infected orally with RV and the incidence of diarrhea was determined 4 days later. RV-specific and total IgA were quantified by an enzyme-linked immunosorbent assay. Results: LG2055-fed dams immunized with RV (LG2055/RV) secreted breast milk that significantly lowered the incidence of RV-induced diarrhea in their pups as compared with dams immunized with RV alone (C/RV). The LG2055/RV dams also produced a significantly greater amount of RV-specific IgA in breast milk obtained from the pups’ stomach, but not in feces or Peyers patch cell cultures. In addition, LG2055 stimulated total IgA production in splenocyte cultures from Toll-like receptor (TLR)-4-knockout mice, but not those from TLR-2-knockouts. Conclusions: LG2055-fed dams reduced RV infection in their pups and elevated RV-specific IgA levels in breast milk of stomach origin, the possible mechanism of which may be TLR-2 stimulation by LG2055.

Effects and mechanisms of prolongevity induced by Lactobacillus gasseri SBT2055 in Caenorhabditis elegans.

Lactic‐acid bacteria are widely recognized beneficial host associated groups of the microbiota of humans and animals. Some lactic‐acid bacteria have the ability to extend the lifespan of the model animals. The mechanisms behind the probiotic effects of bacteria are not entirely understood. Recently, we reported the benefit effects of Lactobacillus gasseriSBT2055 (LG2055) on animal and human health, such as preventing influenza A virus, and augmentation of IgA production. Therefore, it was preconceived that LG2055 has the beneficial effects on longevity and/or aging. We examined the effects of LG2055 on lifespan and aging of Caenorhabditis elegans and analyzed the mechanism of prolongevity. Our results demonstrated that LG2055 has the beneficial effects on longevity and anti‐aging of C. elegans. Feeding with LG2055 upregulated the expression of the skn‐1 gene and the target genes of SKN‐1, encoding the antioxidant proteins enhancing antioxidant defense responses. We found that feeding with LG2055 directly activated SKN‐1 activity via p38 MAPK pathway signaling. The oxidative stress response is elicited by mitochondrial dysfunction in aging, and we examined the influence of LG2055 feeding on the membrane potential of mitochondria. Here, the amounts of mitochondria were significantly increased by LG2055 feeding in comparison with the control. Our result suggests that feeding with LG2055 is effective to the extend lifespan in C. elegans by a strengthening of the resistance to oxidative stress and by stimulating the innate immune response signaling including p38MAPK signaling pathway and others.

Lactobacillus gasseri SBT2055 inhibits adipose tissue inflammation and intestinal permeability in mice fed a high-fat diet.

The probiotic Lactobacillus gasseri SBT2055 (LG2055) has anti-obesity effects. Obesity is closely correlated with inflammation in adipose tissue, and maintaining adipose tissue in a less-inflamed state requires intestinal integrity or a barrier function to protect the intestine from the disruption that can be caused by a high-fat diet (HFD). Here, we examined the anti-inflammatory and intestinal barrier-protecting effects of LG2055 in C57BL/6 mice fed a normal-fat diet (NFD), HFD, or the HFD containing LG2055 (HFD-LG) for 21 weeks. HFD-LG intake significantly prevented HFD-induced increases in body weight, visceral fat mass, and the ratio of inflammatory-type macrophages to anti-inflammatory ones in adipose tissue. Mice fed the HFD showed higher intestinal permeability to a fluorescent dextran administered by oral administration and an elevated concentration of antibodies specific to lipopolysaccharides (LPS) in the blood compared with those fed the NFD, suggesting an increased penetration of the gut contents into the systemic circulation. These elevations of intestinal permeability and anti-LPS antibody levels were significantly suppressed in mice fed the HFD-LG. Moreover, treatment with LG2055 cells suppressed an increase in the cytokine-induced permeability of Caco-2 cell monolayers. These results suggest that LG2055 improves the intestinal integrity, reducing the entry of inflammatory substances like LPS from the intestine, which may lead to decreased inflammation in adipose tissue.

Lactobacillus gasseri SBT2055 suppresses fatty acid release through enlargement of fat emulsion size in vitro and promotes fecal fat excretion in healthy Japanese subjects

BackgroundLactobacillus gasseri SBT2055 (LG2055) has been shown to prevent abdominal adiposity, and suppression of lipid absorption is considered a possible mechanism, detail of which, however, are poorly understood. In the present study, we evaluated the effects of LG2055 on fat hydrolysis by determining pancreatic lipase activity and fat emulsion properties in vitro. We also examined whether LG2055 influences fecal fat excretion in humans.MethodsPancreatic lipase activity was investigated in vitro using an artificially prepared fat emulsion and 4-methylumbelliferyl oleate (4-MUO) as substrates. The concentrations of free fatty acids and 4-methylumbelliferone were quantified. Fat emulsion droplet size was measured using a particle size analyzer. The clinical study was performed as a double-blind, randomized, placebo-controlled trial. Subjects consumed 100xa0g of fermented milk (FM)/d, either with or without LG2055 supplementation, for seven days. Fecal samples were collected during three-day pre-observational and FM intake periods and fecal fat levels were determined.ResultsLG2055 dose-dependently suppressed lipase activity in the fat emulsion assay but not in the 4-MUO assay. LG2055 dose-dependently increased fat emulsion droplet size. The effects of LG2055 on lipase activity and fat emulsion properties were increased compared with four other tested strains (Lactobacillus gasseri SBT0317, Lactobacillus gasseri JCM1131T, Lactobacillus. delbrueckii subsp. bulgaricus JCM1002T and Streptococcus thermophilus ATCC19258T). In our clinical study, fecal fat level after FM intake was significantly increased compared with that observed before FM intake in the LG2055-containing active FM group but not the control FM group lacking LG2055.ConclusionsLG2055 increased fat emulsion droplet size, resulting in the suppression of lipase-mediated fat hydrolysis. The influence of LG2055 on the physicochemical properties of fat emulsion provides a mechanism for the probiotic-mediated suppression of lipid absorption and promotion of fecal fat excretion in humans.Trial registrationUMIN000015772

Preventive Effect of Lactobacillus helveticus SBT2171 on Collagen-Induced Arthritis in Mice

We recently reported that the intraperitoneal inoculation of Lactobacillus helveticus SBT2171 inhibited the development of collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA). In the present study, we evaluated the effect of the oral administration of L. helveticus SBT2171 on CIA development and on the regulation of antigen-specific antibody production and inflammatory immune cells, which have been implicated in the development of RA. Both oral administration and intraperitoneal inoculation of L. helveticus SBT2171 reduced joint swelling, body weight loss, and the serum level of bovine type II collagen (CII)-specific antibodies in the CIA mouse model. The intraperitoneal inoculation also decreased the arthritis incidence, joint damage, and serum level of interleukin (IL)-6. In addition, the numbers of total immune cells, total B cells, germinal center B cells, and CD4+ T cells in the draining lymph nodes were decreased following intraperitoneal inoculation of L. helveticus SBT2171. These findings demonstrate the ability of L. helveticus SBT2171 to downregulate the abundance of immune cells and the subsequent production of CII-specific antibodies and IL-6, thereby suppressing the CIA symptoms, indicating its potential for use in the prevention of RA.

Lactobacillus Gasseri Potentiates Immune Response Against Influenza Virus Infection

Abstract Probiotics are live microorganisms that can be found in fermented foods. They exhibit various properties that provide health benefits through improving the immune system. A series of studies have suggested that the administration of lactic acid bacteria stimulates innate immunity, e.g., natural killer cell activity and type I interferon production, as well as adaptive immunity, e.g., specific antibody production against a vaccine. However, few reports have identified LAB strains that stimulate both innate and adaptive immunity simultaneously. Recently, we revealed for the first time that the oral administration of yogurt containing Lactobacillus gasseri SBT2055 (LG2055) in healthy adult subjects potentiated both innate and adaptive immunity, namely vaccine-specific antibody production against A/H1N1 and B influenza viruses. In this chapter, the authors present an overview of the potential of probiotics, particularly LG2055, to improve the immune response against influenza virus infection, and discuss the immunogenic role of probiotics in the clinical arena.

Natural IgG antibody with anti-β-galactosyl specificity suppressed hepatoma cell invasion in culture

The effect of natural IgG antibody recognizing β-galactosyl epitope on hepatoma cell invasion was investigated. Anti-β-galactosyl antibody dose-dependently suppressed hepatoma invasion underneath primarily cultured mesothelial cells monolayer without affecting the proliferation, to the same extent as natural IgG antibody with anti-α-galactosyl specificity, which had already been reported to have an anti-metastatic activity. The inhibitory effect of anti-β-galactosyl antibody was completely canceled by adding lactose (galactose-β-1, 4-glucose) to the medium, indicating that this antibody recognized some antigens with β-galactosyl epitope. Hepatoma cells pretreated with this antibody for 48xa0h showed reduced invasive activity, while the pretreatment of mesothelial cells with the antibody did not affect hepatoma cells invasion. Anti-β-galactosyl antibody also suppressed hepatoma cells adhesion to mesothelial cells monolayer. These results suggest that natural antibody with anti-β-galactosyl specificity may recognize the β-galactosyl epitope in some adhesion-related molecules on hepatoma cells, thus suppressing adhesion and invasion to mesothelial cells monolayer. These results suggest possible therapeutic uses of this antibody in the treatment of metastatic tumors.