Safety studies of LP20 powder produced from heat-killed Lactobacillus plantarum L-137.

The safety of LP20 and its prototype, a powder, with potential use in food, produced from a mixture of dextrin and heat-killed Lactobacillus plantarum L-137, was assessed in an acute study in mice, and in an in vitro bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo mouse micronucleus assay. LP20 prototype was not acutely toxic when administered to male and female Slc:ICR mice by single gavage at 2000mg/kg bw. Dosing was not associated with mortality, clinical signs, changes in bodyweight, or macroscopic abnormalities. The LD(50) in mice was greater than 2000mg/kg bw. There was no evidence of genotoxicity of LP20 in the Ames assay (0-5000microg/plate) or in the in vitro chromosome aberration assay with Chinese hamster lung fibroblasts (0-5000microg/mL). Administration of two consecutive daily doses of 500, 1000, or 2000mg/kg bw by gavage to male Crlj:CD-1 mice was not associated with an increased incidence of micronuclei and did not alter the ratio of polychromatic to normochromatic erythrocytes. These studies show that LP20 powder is not acutely toxic and is without genotoxic activity both in vitro and in vivo.

Antitumor effect of heat-killed Lactobacillus plantarum L-137 through restoration of impaired interleukin-12 production in tumor-bearing mice.

We have previously reported that heat-killed Lactobacillus plantarum L-137 is a potent inducer of interleukin-12 (IL-12) in vivo as well as in vitro in mice. In order to develop effective usage of L. plantarum L-137 for tumor immunotherapy, we examined its antitumor effect against DBA/2 mice inoculated with syngenic P388D1 tumor cells in different treatment schedules. Daily injection of L. plantarum L-137 from the day of tumor inoculation induced a steep increase in plasma IL-12 only after the first injection but not after subsequent injections, and had no effect on tumor growth and survival time. In contrast, daily injection of L. plantarum L-137 from the 7th day after tumor inoculation exerted a marked antitumor effect but such an effect was not evident in mice treated with L. plantarum L-137 twice a week from the 7th day. IL-12 production was considerably impaired at the first injection but steeply increased after the third injection in the mice injected daily with L. plantarum L-137 from the 7th day. Our results suggest that daily administration of L. plantarum L-137 is required to exert an antitumor effect at the late stages of tumor development when IL-12 production is considerably impaired.

Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum.

By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.

Immunopotentiating activity of nigerooligosaccharides for the T helper 1-like immune response in mice.

The immunopotentiating activity of nigerooligosaccharides (NOS), a mixture of nigerose, nigerosyl glucose and nigerosyl maltose, was studied in vitro and in vivo in mice. Mitogen-induced proliferation of splenocytes from normal mice was augmented in a dose-dependent manner by nigerose of NOS. NOS enhanced interleukin 12 (IL-12) and interferon-gamma (IFN-gamma) production by normal splenocytes in the presence of the potent IL-12 inducer, heat-killed Lactobacillus plantarum L-137, in vitro. Consistent with the in vitro finding, L. plantarum L-137-induced IL-12 production and IL-2-induced IFN-gamma production were augmented in mice fed with a 14.6% NOS diet for 2 weeks compared with mice fed with a control diet. Notably, mice fed with the NOS diet showed significantly longer survival time than the control mice after the induction of an endogenous infection by administering 5-fluorouracil in a lethal dose. Taken together, these results suggest that NOS may exert immunopotentiating activity through the activation of an IL-12-dependent T helper 1-like immune response.

Heat-killed Lactobacillus plantarum L-137 suppresses naturally fed antigen-specific IgE production by stimulation of IL-12 production in mice.

BACKGROUND: Food allergy is caused by production of IgE against dietary antigen induced by T(H2) response. IL-12 inhibits T(H2) responses and strongly suppresses IgE production. We have recently established a murine model for IgE production with a predominant T(H2) response induced by feeding antigen. OBJECTIVE: We here show a suppressive effect of heat-killed Lactobacillus plantarum L-137, a potent inducer of IL-12, on IgE production against naturally fed antigen in a murine model. METHODS: The ability of L. plantarum L-137 to induce IL-12 production was examined in vitro and in vivo. DBA/2 mice were fed a casein diet and injected intraperitoneally with L. plantarum L-137 from the beginning of feeding or 2 weeks later. Recombinant mouse IL-12 was also injected 2 weeks after the start of feeding. Casein-specific IgE and IgG1 in plasma were determined by ELISA. RESULTS: L. plantarum L-137 directly induced IL-12 production by the peritoneal macrophages and also stimulated spleen cells to produce both IL-12 and interferon-gamma in vitro. In vivo treatment of L. plantarum L-137 also increased the plasma level of IL-12 in mice. Plasma anti-casein IgG1 and IgE levels were gradually elevated in DBA/2 mice fed a casein diet. Administration of L. plantarum L-137 from the beginning of feeding suppressed the elevation of anti-casein IgE levels, whereas the levels of anti-casein IgG1 were rather augmented by L. plantarum L-137. IL-12 production of the peritoneal macrophages was enhanced, but IL-4 production of concanavalin A (Con A)-stimulated spleen cells was suppressed in the L. plantarum L-137-treated mice compared with control mice fed a casein diet. When L. plantarum L-137 was given from 2 weeks after the start of feeding, anti-casein IgE levels were also significantly suppressed, which was similar to the result found in mice treated with IL-12. CONCLUSION: Our results suggest that L. plantarum L-137, a potent IL-12 inducer, is useful for prevention and treatment of food allergy.

Apoptosis of intestinal intraepithelial lymphocytes induced by exogenous and endogenous glucocorticoids.

To investigate the effect of glucocorticoids on apoptosis in intestinal intraepithelial lymphocytes (i-IEL), we examined the changes of i-IEL followed by in vivo treatment with dexamethasone. The fragmented DNA of i-IEL were significantly increased at 15 hr after dexamethasone treatment and, subsequently, the number of total i-IEL were decreased by day 4 after treatment. Although all subsets of i-IEL including CD8 alpha/alpha(+), CD8 alpha/beta(+), CD4+ and CD4+CD8+ i-IEL were decreased after dexamethasone treatment, CD8 alpha/alpha(+) i-IEL appeared to be relatively resistant to dexamethasone-induced apoptosis. Consistent with the in vivo findings, CD8 alpha/alpha(+) i-IEL exhibited less susceptibility to dexamethasone-induced cell death in vitro than other subsets. To investigate whether this process occurs under physiological conditions, we examined the kinetics of i-IEL after treatment with 15-hr water immersion stress. In mice subjected to water immersion stress, plasma glucocorticoids were remarkably elevated soon after the 15-hr stress. The increase in the fragmented DNA of i-IEL and subsequent decrease in the number of i-IEL were observed in the stressed mice in the same kinetics as seen in the dexamethasone-treated mice. Similar to dexamethasone-induced ell death, CD8 alpha/alpha(+) i-IEL appeared to be relatively resistant to stress-induced apoptosis compared with other i-IEL subsets. The expression level of Bcl-2 was significantly higher in CD8 alpha/alpha(+) i-IEL than in CD8 alpha/beta(+) i-IEL. Our results indicate that i-IEL are subjected to cell death via apoptosis by exogenous and endogenous glucocorticoids and that different sensitivity to steroid-induced apoptosis may exist among i-IEL subsets in relation to their Bcl-2 expression.

Murine model of IgE production with a predominant Th2-response by feeding protein antigen without adjuvants.

Stimulation of systemic antigen-specific IgE production plays an important role in the mediation of food allergy; however, the mechanism of IgE production against food antigens is not fully understood. The development of relevant animal models may help to elucidate the pathogenesis of food allergy. We here show that DBA/2 mice receiving a casein diet without any adjuvant produced high levels of IgE specific for casein, accompanied by predominant Th2-like responses in liver lymphocytes, mesenteric lymph node cells and spleen cells. This model of IgE production produced by feeding protein antigen as a constituent of the diet can be applied to investigate the mechanism of IgE production and to develop reagents for controlling food allergy.

Structural study on an exocellular polysaccharide produced by Lactobacillus helveticus TY1-2.

Lactobacillus helveticus TY1-2 produced an exocellular polysaccharide when it was cultured in reconstituted skim milk. This polysaccharide is a high molecular weight heteropolymer of D-glucopyranosyl, D-galactopyranosyl, and 2-acetamido-2-deoxy-D-glucopyranosyl residues in the molar ratio 3.0:2.8:0.9. The primary structure of the polysaccharide was shown by glycose analysis, methylation analysis, Smith degradation, and NMR spectroscopy to be composed of branched heptasaccharide repeating units having the following structure: [formula: see text]

Consumption of milk from cows immunized with intestinal bacteria influences age-related changes in immune competence in mice.

Milk was obtained from nonimmunized cows and cows immunized with a mixture of various human gut bacteria. Each milk was administered orally to 2-mo-old C57BL/6 mice at a dose of 150 g.kg-1.d-1 for either 6 or 16 mo. The study group had fewer enteric bacteria and a lower concentration of the serum antibodies against enteric bacteria compared with the control group at 8 and 18 mo of age. Furthermore, the study group at 18 mo old had a higher redirected cytotoxicity of intestinal intraepithelial lymphocytes, a higher proliferative response of mesenteric lymph nodes cells against mitogenic or alloantigenic stimulation and a greater ability of the spleen cells to produce anti-sheep erythrocytes IgG antibody after systemic immunization with sheep erythrocytes. A lower level of autoantibodies was observed at 8 mo and 18 mo of age in the study group compared with the control group. These results suggest that the senescence of the murine immune system may be delayed by consumption of milk from immunized cows.

Administration of milk from cows immunized with intestinal bacteria protects mice from radiation-induced lethality.

Oral administration of "immune milk", that had been obtained from cows immunized with a variety of human gut bacteria containing E. coli, S. typhimurium, S. dysenteriae and 23 others, protected AKR/J mice from the lethal effect of radiation, when immune milk was orally given to mice at 150 g kg-1 day-1 for 7 days prior to gamma-irradiation of 8 Gy. Mean survival times were 24.8 days for the group given immune milk but only 16.8 days for the group given control milk from unimmunized cows. Enterobacteriaceae were detected in various organs such as liver, lung and kidney on day 13 after irradiation, whereas the numbers were significantly fewer in the study group as compared with the control group. And fewer number of intestinal Enterobacteriaceae were detected in the study group compared with the control group prior to irradiation. Immune milk also enhanced the mitogenic response to mesenteric lymph node cells, the redirected cytolytic activity of intestinal intraepithelial lymphocytes to P815 tumor cells with anti-CD3 mAb, and in vitro killing activities of the phagocytes in mesenteric lymph nodes to E. coli as compared with control milk. These results suggest that immune milk may reduce the number of bacteria translocating from the intestinal-tract and augment the activities of the gut-associated lymphoid tissues against the invasion of intestinal bacteria, causing protection against the lethal effect of radiation.

Sequential appearance of T-cell receptor gamma delta- and alpha beta-bearing intestinal intra-epithelial lymphocytes in mice after irradiation.

We have previously reported that T-cell receptor (TcR) gamma delta-bearing T cells precede TcR alpha beta-bearing T cells in appearance in the thymus after whole-body irradiation. In the present study, the kinetics of appearance of intestinal intra-epithelial lymphocytes (IEL) was examined in mice after whole-body irradiation with a lethal dose of 9.5 Gy or with a sublethal dose of 6 Gy. The number of CD3+ IEL decreased to the lowest value 4 days after irradiation with 9.5 Gy, and thereafter increased to half as many as the normal level by day 7. Thy-1+TcR alpha beta- IEL and Thy-TcR alpha beta- IEL recovered considerably by day 7 after the irradiation, whereas Thy-1+TcR alpha beta+ IEL and Thy-1+TcR alpha beta+ IEL hardly recovered at this stage. All mice died within 12 days after irradiation with a lethal dose of 9.5 Gy. On the other hand, when irradiation dose was decreased to 6 Gy, all mice survived beyond 40 days after irradiation. The number of CD3+ IEL recovered to the normal level by 10 days after irradiation with 6 Gy. Consistently with the results in mice irradiated with a lethal dose, the first cells to increase in IEL of mice irradiated with a sublethal dose were TcR gamma delta+ IEL expressing Thy-1 antigen. The number of Thy-1+TcR gamma delta+ IEL increased to approximately two-fold as many as that in normal mice by day 10, while TcR alpha beta+ IEL began to increase in number from day 20 after irradiation and recovered to the normal level by day 40 after irradiation. Thus, sequential appearance of TcR gamma delta+ and TcR alpha beta+ IEL was evident after irradiation, similar to that seen in the thymus after irradiation. The IEL on day 10 after a sublethal irradiation, which is composed mainly of Thy-1+TcR gamma delta+ IEL, exhibited a strong cytolytic activity against P815 in the presence of anti-CD3 mAb, suggesting that the early appearing Thy-1+TcR gamma delta+ IEL may play important roles in epithelial immunity at an early stage after irradiation.

Influence of intake of skim milk from cows immunized with intestinal bacterial antigens on onset of renal disease in (NZB x NZW)F1 mice fed ad libitum or restricted in energy intake.

Autoimmune disease-prone (NZB x NZW)F1 (B/W) female mice are a model of human lupus erythematosus. Immune milk, obtained from cows immunized with various intestinal bacterial antigens, was given to B/W mice as a component of diets beginning at 8 wk of age. Diets were fed ad libitum or restricted to 60% of ad libitum energy intake. Controls were fed commercial skim milk. In B/W mice fed ad libitum, the titers of anti-single-stranded DNA antibodies were significantly lower in immune milk-fed mice at 4 and 6 mo of age. Onset of proteinuria was delayed and life span was significantly prolonged by immune milk feeding. Surface phenotypes of the T cells and levels of the responsiveness of lymphocytes to mitogens were not changed by immune milk feeding. The B/W mice restricted to 60% of ad libitum energy intake, which preserved immune responsiveness, had not developed proteinuria by 14 mo of age, irrespective of immune milk feeding or control milk feeding. However, at 10 mo of age, the level of plasma antibodies against intestinal bacteria was significantly higher in energy-restricted mice fed control milk than in those fed immune milk or in mice fed ad libitum.

Failure of T cell receptor V beta negative selection in murine intestinal intra-epithelial lymphocytes.

The intestinal intra-epithelial lymphocytes (IEL) are divided into several subsets on the basis of expression of T cell receptor (TCR) alpha beta and gamma delta, intensity of Thy-1 expression and expression of Lyt-3 chain. To investigate the differentiation pathway of the IEL, we examined the repertoire of V beta segments of T cells in the IEL in BALB/c (H-2d, MIs-1b2a) or AKR/J (H-2k, MIs-1a2b) mice. Among freshly isolated IEL, an appreciable number of T cells bearing V beta 3 or V beta 11, which recognize MIs-2a- or MHC IE-encoded molecules respectively, were detected in BALB/c mice. Similarly, in AKR/J mice, IEL contained appreciable levels of V beta 6-bearing T cells. V beta 3- or V beta 11-bearing T cells in the IEL in BALB/c mice increased to a significant level when incubated with staphylococcal enterotoxin A which specifically stimulates V beta 3- and V beta 11-bearing T cells. Most of IEL without clonal deletion expressed Lyt-2 but not Lyt-3 antigens. Such T cells were hardly detected in other organs, including liver. Our results indicate that TCR alpha beta-bearing intestinal IEL that have not undergone negative selection may have differentiated outside the thymus, presumably at a local site of the intestine and can respond normally to the signal via their TCR.