A simple competitive enzyme-linked immunosorbent assay for the specific detection of the multiphosphorylated 1-25 ß-casein fragment.

A specific and simple competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine bovine ß-casein phosphopeptides (ß-CPP) in casein phosphopeptides (CPP) or CPP complexes such as casein phosphopeptide amorphous calcium phosphate complexes added into dairy products. The method combines sample pretreatment designed for CPP enrichment and anti-ß-CPP(f(1-25)) monoclonal antibody 1A5 (mAb 1A5). The mAb 1A5 bound specifically to the tryptic phosphopeptides from ß-casein but not from αs1- or αs2-casein. Reactivity was also influenced by the extent of the phosphorylated form of serine residues. Based on the sequence-specific recognition and contribution of phosphorylated serine residues, the epitope of mAb 1A5 was found to reside within the cluster motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu and the surrounding residues in ß-CPP. The competitive ELISA developed here can be used as an alternative to specialised and expensive techniques such as mass spectrometry. In particular, it is suitable for the measurement of CPP or CPP complexes in dairy products, which contain closely related endogenous molecular species.

Influence of long-term consumption of a Lactococcus lactis strain on the intestinal immunity and intestinal flora of the senescence-accelerated mouse.

The senescence-accelerated mouse develops normally until 5-6 months of age and then displays rapid and irreversible advancement of senescence manifesting as clinical signs and gross lesions. To clarify the effect of lactic acid bacteria on the physiological changes with increasing age, heat-killed Lactococcus lactis G50 was administered to 1-month-old senescence-accelerated-prone mouse (SAMP)6 mice for 11 months, a senescence-accelerated mouse strain that develops senile osteoporosis. Mice fed G50 gained more weight than the control mice (not fed G50) during the feeding experiment. Faecal IgA levels in the mice fed G50 at 3 months were higher than those of the control mice but decreased to control levels with increasing age. The numbers of viable cells of Bacteroides sp., Lactobacillus sp., Staphylococcus sp., Enterococcus/Streptococcus sp. and Enterobacteriaceae sp. in faeces were similar for mice fed the G50 and control diets at any age, but strain G50 suppressed the intestinal growth of H2S-producing bacteria. Bone density of the thigh bone did not differ between aged G50 and control mice. Strain G50 would be a beneficial bacterium for the enhancement of intestinal immunity during youth and to suppress the growth of harmful intestinal bacteria. The applicability of strain G50 for the food and animal industries has been proposed in the present study.

Absorption, migration and kinetics in peripheral blood of orally administered ovalbumin in a mouse model.

Intestinal absorption of food proteins is well known, whereas its physiological significance remains to be investigated. Various amounts (1, 10 and 50 mg) of ovalbumin were orally administered to mice and the blood kinetics were subsequently analyzed by two-site ELISA. The blood ovalbumin concentration consistently reached its maximum (7-90 ng/ml) about 20 min after the oral administration and then gradually decreased in a dose-dependent manner. Only intact (45 kDa) and truncated (40 kDa) ovalbumins were always detected in the blood independently of the administration site, intra-stomach or intra-intestine, while various fragments of the protein were observed in the gastrointestinal lumen after the oral administration. Recognition by a specific monoclonal antibody and an acidic shift of its pI value suggested that the 40-kDa truncated ovalbumin was produced by intracellular limited proteolysis at its C-terminus. Such stable absorption and blood kinetics of undigested ovalbumin in normal mice suggest some sort of physiological significance for the intestinal uptake of intact food proteins.

Anti-ageing effect of a lactococcal strain: analysis using senescence-accelerated mice.

The effects of oral administration of a lactococcal strain on physiological changes associated with ageing were investigated using senescence-accelerated mice (SAM). SAM develop normally, but then show an early onset and irreversible advancement of senescence. SAMP6 is a SAM strain that develops osteoporosis with ageing. Oral administration of heat-killed Lactococcus lactis subsp. cremoris H61 (strain H61) to aged SAMP6 mice was associated with reduced bone density loss, a suppression of incidence of skin ulcers and reduced hair loss, compared with controls. Spleen cells from mice fed strain H61 produced more interferon-gamma and IL-12 than those from control mice, suggesting that administration of strain H61 altered immune responses. The numbers of viable cells of Bifidobacterium sp., Bacteroides sp. and Enterococcus sp. in faeces were similar for mice fed the strain H61 and control diets, but counts for Staphylococcus sp. were significantly lower (P < 0.05) in mice fed strain H61. Mice fed strain H61 had similar serum concentrations of thiobarbituric acid-reactive substances as in controls, indicating a lack of effect on lipid peroxidation status. Administration of living cells of strain H61 or fermented milk containing strain H61 was also associated with a suppression of incidence of skin ulcers and reduced hair loss. These results indicate that oral administration of strain H61 has the potential to suppress some of the manifestations associated with ageing.

Transition of ovalbumin to thermostable structure entails conformational changes involving the reactive center loop.

Ovalbumin is a serpin without inhibitory activity against proteases. During embryonic development, ovalbumin in the native (N) form undergoes changes and takes a heat-stable form, which was previously named HS-ovalbumin. It has been known that N-ovalbumin is artificially converted to another thermostable form called S-ovalbumin by heating at an alkaline pH. Here, we characterized further the three ovalbumin forms, N, HS, and S. The epitope of the monoclonal antibody 2B3/2H11, which recognizes N- and HS-ovalbumin but not S-ovalbumin, was found to reside in the region Glu-Val-Val-Gly-Ala-Ser-Glu-Ala-Gly-Val-Asp-Ala-Ala-Ser-Val-Ser-Glu-Glu-Phe-Arg, which corresponds to 340-359 of amino acid residues and is contained in the reactive center loop (RCL). Removal of RCL by elastase or subtilisin mitigated binding of the antibody. Dephosphorylation experiments indicated that the phosphorylated Ser-344 residue located on RCL is crucial for the epitope recognition. We suggest that the shift to the heat-stable form of ovalbumin accompanies a movement of RCL.

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.

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 nonviable 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.

Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues.

We have reported that ovalbumin accumulates without digestion in various tissues during embryonic development of the chicken. There are different types of ovalbumin with respect to thermal stability and one of them, which was named "HS-ovalbumin" in the present study, was found to have a T(m) value of 83 degrees C and to be present dominantly in albumen, egg yolk, amniotic fluid, and serum of fertilized eggs. HS-ovalbumin, arising physiologically from its native form (N-ovalbumin), is reminiscent of the previously described intermediate form appearing during the production processes of the so-called S-ovalbumin, which disappeared shortly in fertilized eggs. We showed that HS-ovalbumin is distinguishable from S-ovalbumin by a monoclonal antibody and also from N-ovalbumin by the stability to heating. At the late stages of development, ovalbumin of amniotic fluid seems to be swallowed through pharynx, carried in the intestine through stomach, and absorbed in the blood. Analyses by monoclonal antibody and heat treatment indicated that the HS-form occupies the largest fraction of ovalbumin that accumulates in the embryonic tissues. The current findings suggest that HS-ovalbumin is crucial for embryogenesis.

New Lactococcus strain with immunomodulatory activity: enhancement of Th1-type immune response.

Few studies exist dealing with the probiotic activity of lactococci, which are commonly used as starter bacteria in the manufacture of many kinds of fermented dairy products. Fifteen strains of the genus Lactococcus were examined for their probiotic activities, such as immunomodulatory effects. Six strains induced the production of cytokines (IL-12, IL-6, and TNF-alpha) in macrophage-like cell line J774.1, and the highest induction was observed with Lactococcus lactis subsp. lactis G50. The cytokine induction in the J774.1 cell line was almost entirely sustained after heat-killing of the strain. Spleen cells from BALB/c mice fed G50 culture produced more IL-12 and IFN-gamma and slightly less IL-4 and IL-6 than the control (i.e., without strain G50), indicating that strain G50 can enhance Th1-type immune response in vivo. The effect of the oral administration of strain G50 on antibody response in mice was also investigated. Mice were immunized with ovomucoid (OVM), a potent egg allergen, and the antibody level in the serum was then determined. The total IgE antibody level in the group treated with strain G50 was significantly lower than that of the control. The response of OVM-specific IgG1 and IgE antibodies tended to be low in the group that was administered strain G50, compared with the response of the control group. These results suggest that strain G50 has an ability to suppress the Th2 response. Thus, Lactococcus lactis subsp. lactis G50 is a potential probiotic strain for the suppression of hypersensitive reactions caused by the Th2 response.

Localization of T cell epitope regions of chicken ovomucoid recognized by mice.

We localized the T cell epitope regions of chicken ovomucoid (OVM), a potent egg allergen, with the overlapping pin-peptides covering the entire sequence of OVM and three strains of mice with different haplotypes. In C3H/He (H-2k) mice, the T cells recognized relatively broad regions on OVM; the dominant regions were 49-93 and 97-114 residues, and the subdominant regions were 7-21, 37-48, 94-96, 115-123 and 145-177 residues. In contrast, a more limited number of T cell epitope regions were localized in BALB/c (H-2d) and C57BL/6 (H-2b) mice. The T cells from BALB/c mice recognized 100-114 and 157-171 residues, and the T cells from C57BL/6 mice recognized only 157-180 residues. These results were confirmed by using peptides separately synthesized and purified on the putative epitope regions. The roles of the carbohydrate moieties and cysteine residues involved in the disulfide bridges of OVM were also examined, and we found that they were not important in recognition by the T cell/antigen presenting cell.

Antigen-specific, CD4+CD25+ regulatory T cell clones induced in Peyer's patches.

Since intestine is exposed to numerous exogenous antigens such as food and commensal bacteria, the organ bears efficient mechanisms for establishment of tolerance and induction of regulatory T cells (T(reg)). Intestinal and inducible T(reg) include T(r)1-like and T(h)3 cells whose major effector molecules are IL-10 and transforming growth factor (TGF)-beta. These antigen-specific T(reg) are expected to become clinical targets to modify the inflammatory immune response associated with allergy, autoimmune diseases and transplantation. In the present study, we characterized the antigen-specific T(reg) induced in the intestine by orally administering high-dose beta-lactoglobulin (BLG) to BALB/c mice. Seven days after feeding, only Peyer's patch (PP) cells among different organs exerted significant suppressive effect on antibody production upon in vitro BLG stimulation. This suppressive effect was also prominent in six BLG-specific CD4(+) T cell clones (OPP1-6) established from PP from mice orally administered with high doses of BLG and was partially reversed by antibodies to TGF-beta. Intravenous transfer of OPP2 efficiently suppressed BLG-specific IgG1 production in serum following immunization, indicating the role of such T(reg) in the systemic tolerance after oral administration of antigen (oral tolerance). OPP clones secrete TGF-beta, IFN-gamma and low levels of IL-10, a cytokine pattern similar to that secreted by anergic T cells. OPP clones bear a CD4(+)CD25(+) phenotype and show significantly lower proliferative response compared to T(h)0 clones. This lower response is recovered by the addition of IL-2. Thus, antigen-specific CD4(+)CD25(+) T(reg), which have characteristics of anergic cells and actively suppress antibody production are induced in PP upon oral administration of protein antigen.

Cytokine production by the murine macrophage cell line J774.1 after exposure to lactobacilli.

Eleven strains of lactobacilli were tested for their ability to induce the murine macrophage-like cell line J774.1 to secrete cytokines. Some of the bacteria tested induce the production of interleukin(IL) 6, IL-12, and tumor necrosis factor a (TNF-alpha) by J774.1 cells. Seven strains also induced the production of IL-10. However, no IL-1beta was produced. Lactobacillus acidophilus TMC 0356 significantly induced the production of more IL-6, IL-10, IL-12, and TNF-alpha than the other bacteria tested (p < 0.0001; ANOVA). These results suggest that lactobacilli can activate macrophages to secrete both inflammatory and anti-inflammatory cytokines. Selected strains might be used to bring about pro or antiinflammatory immune reactions.

Induction of oral tolerance in mice by continuous feeding with beta-lactoglobulin and milk.

We examined the immune response of mice to beta-lactoglobulin (BLG), a potent milk allergen, after continuous feeding of a BLG solution or milk instead of drinking-water. Strong suppression of the anti-BLG antibody response and antigen-specific T cell response were observed in mice fed BLG and milk. Although the profile of the antibody specificity to BLG peptides in mice fed BLG or milk was different from the control, the dominant determinants were still recognized and a limited number of new recognition sites appeared by BLG or milk feeding. These findings suggest that continuous feeding with BLG or milk not only induced tolerance in the periphery, but also priming. In terms of the antigen-specific IgG subclass response, the production of both IgG1 and IgG2a was significantly reduced. The cytokine secretion of interleukin (IL)-2, IFN-gamma and IL-10 was also reduced in the culture supernatants of lymph node cells from the mice fed BLG. The results indicate that the continuous feeding of BLG or milk induces suppression of both Th1- and Th2-dependent responses. This may reflect a state of oral tolerance induced by food ingestion.

Adhesion of lactic acid bacteria to caco-2 cells and their effect on cytokine secretion.

Cytokines secreted by human enterocytes play a critical role in mucosal and systemic immunity. Intestinal microorganisms can influence this secretion. In the present study, 30 strains of lactic acid bacteria were characterized for their adhesion to Caco-2 cells and their potential to stimulate proinflammatory cytokine secretion by this cell line. The bacteria adhered in a strain-dependent manner to Caco-2 cells. Contact with lactobacilli did not result in the production of IL-6 or IL-8. A slight IL-6 and IL-8 production by a Caco-2 cell was detected after exposure to 8 of the tested Bifidobacterium strains. No correlation was found between adhesion and cytokine induction among the bacteria tested. This indicates that lactic acid bacteria, even those with strong adhesive properties, are not very likely to trigger an inflammatory response in human enterocytes.

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-alpha, 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.