Protective effects of Enterococcus faecium strain R30 supplementation on decreased muscle endurance under disuse in rats.

NEW FINDINGS: What is the central question of this study? Does Enterococcus faecium strain R30 (R30), a new lactic acid bacterial strain for supplementation, attenuate shifts in the typology of whole muscle fibres from slow- to fast-twitch by altering the autonomic nervous system in atrophied skeletal muscles? What is the main finding and its importance? R30 supplementation may attenuate the shifts in the typology of whole muscle fibres from slow- to fast-twitch fibres by upregulating peroxisome proliferator-activated receptor-γ coactivator-1α and activating the calcineurin-nuclear factor of activated T-cells signalling pathway, thus ameliorating the decrease in muscle endurance associated with disuse. ABSTRACT: Enterococcus faecium strain R30 (R30), a new lactic acid bacterial strain for supplementation, was hypothesized to attenuate shifts in the typology of whole muscle fibres from slow- to fast-twitch fibres in atrophied skeletal muscles. We further postulated that the prevention of slow-to-fast fibre shifts would suppress the decreased muscle endurance associated with atrophy. To evaluate the protective effects of R30, we analysed slow-to-fast fibre shifts and disuse-associated reduced muscle endurance. R30 was administered to rats with an acclimation period of 7 days before hindlimb unloading (HU) for 2 weeks. The composition ratio of the fibre type and the expression levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), calcineurin and nuclear factor of activated T-cells (NFAT) were measured. Muscle endurance was evaluated at the end of the 2-week HU period in an in situ environment. R30 supplementation suppressed the slow-to-fast fibre switch and decreased the HU-induced expression of PGC-1α proteins and the deactivation of the calcineurin-NFAT pathway. Furthermore, R30 prevented a decrease in HU-associated muscle endurance in calf muscles. These results indicate that R30 supplementation may attenuate the shifts in the typology of whole muscle fibres from slow- to fast-twitch fibres via the upregulation of PGC-1α and the activation of the calcineurin-NFAT signalling pathway, thereby ameliorating the decrease in muscle endurance associated with disuse.

Acute effects of lactic acid-fermented and enzyme-digested soybean on protein synthesis via mTOR signaling in the skeletal muscle.

Protein-containing nutrients result in the efficient hypertrophy of muscles by increasing muscle protein synthesis. Soybean is often ingested by athletes or individuals who exercise; however, it takes very long to be absorbed. Lactic acid-fermented and enzyme-digested (LFED) soybean is absorbed faster than untreated soybean. This study aims at determining muscle protein synthesis after ingesting a single bolus of soybean or LFED soybean produced by lactic acid bacteria and protease digestion. Eight-week-old overnight-fasted ICR mice were administered powdered or LFED soybean. Mice were euthanized at 7, 15, 30, 60, 90, and 120 min after soybean intake. We have demonstrated that LFED soybean administration was quicker in stimulating muscle protein synthesis by activating mammalian target of rapamycin (mTOR) signaling than orally ingesting untreated soybean in the gastrocnemius muscle. These results suggested that LFED soybean is a more efficient source of nutrition for muscle hypertrophy than untreated soybean.

Effects of Pediococcus acidilactici R037 on Serum Triglyceride Levels in Mice and Rats after Oral Administration.

The biological effects of heat-killed Pediococcus acidilactici R037 (R037) were evaluated when orally administered in mice and rats. Oral R037 administration at a daily dose of 10 and 100 mg/kg for 3 wk dose-dependently reduced fasting and non-fasting serum triglyceride concentrations in KK-Ay/TaJcl mice, a model of type II diabetes, obesity, hypercholesterolemia, and hypertriglyceridemia. Serum levels of free fatty acids in the 100 mg/kg group tended to decrease (not statistically significant), and total cholesterol levels remained unchanged. Treatment with R037 resulted in a significant decrease in blood glucose (at 100 mg/kg) and liver weight (at 10 and 100 mg/kg), and a small body weight gain (at 100 mg/kg) as compared to those in control mice. In addition, oral R037 administration at 100, 200, and 400 mg/kg/d for 1 wk dose-dependently suppressed the increase in serum triglyceride levels in Wistar rats after oral fat loading. Moreover, intraduodenal injection of 120 mg of R037 in Wistar rats suppressed gastric vagal nerve activity (GVNA) indicating suppression of intestinal digestion and absorption of food, and suppression of appetite. The R037 injection potentiated epididymal white adipose tissue sympathetic nerve activity (WAT-SNA) and tended to potentiate pancreatic sympathetic nerve activity (PSNA), suggesting that R037 activated lipolysis. Taken together, these findings indicate that R037 lowers serum triglycerides, possibly through suppressing intestinal absorption and potentiating lipolytic pathways. R037 may be useful for primary prevention of coronary artery diseases in subjects with mild or borderline dyslipidemia in combination with lifestyle changes.

Enterococcus faecium strain R30 increases red blood cell velocity and prevents capillary regression in the soleus of hindlimb-unloaded rats via the eNOS/VEGF pathway.

OBJECTIVE: A chronic decrease in neuromuscular activity results in atrophy and capillary regression in skeletal muscles. The purposes of this study were to determine the effects of Enterococcus faecium strain R30 (R30) administration on (i) the hemodynamics of the rat soleus muscle, and (ii) the capillary regression normally associated with HU. METHODS: Experiment 1: The VRBC was measured for up to 1 hour after administration of R30 with or without the ß-blocker propranolol. Experiment 2: R30 was administered daily to control and HU rats for 2 weeks. Mean capillary luminal diameter, volume, and the levels of eNOS and VEGF protein were measured. RESULTS: Experiment 1: VRBC was faster 20, 40, and 60 minutes after than before the administration of R30: This effect was suppressed by propranolol administration. Experiment 2: R30 administration during HU increased capillary luminal diameter and volume and eNOS and VEGF protein levels in the soleus of HU rats. CONCLUSIONS: The results suggest that R30 increases VRBC in the soleus muscle via muscle sympathetic nerve activity (Experiment 1) and that R30 supplementation lessens the capillary regression normally associated with HU via the eNOS/VEGF pathway (Experiment 2).

Oral administration of lactic acid bacteria isolated from traditional South Asian fermented milk 'dahi' inhibits the development of atopic dermatitis in NC/Nga mice.

We investigated the suppressive effect of lactic acid bacteria (LAB) isolated from traditional South Asian fermented milk 'dahi' on the development of atopic dermatitis (AD) using NC/Nga AD model mice. In the initial evaluation, we confirmed the effect of LAB on serum total IgE using ovalbumin (OVA)-induced type 1 allergy model mice. Forty-one bacterial strains isolated from dahi were evaluated for their ability to induce interleukin (IL)-12 production and suppress IL-4 production in splenocytes obtained from OVA-sensitized mice. Of the 41 strains tested, Lactobacillus delbrueckii subsp. lactis R-037 exhibited the greatest IL-12 induction, suggesting that it is a potent Th1 inducer. Oral administration of heat-treated R-037 significantly suppressed the elevation of serum total IgE in OVA-induced type 1 allergy model mice. In NC/Nga AD model mice, oral administration of heat-treated R-037 reduced inflammatory auricular thickness and alleviated the AD clinical score although the effect on serum total IgE level was unclear. Histopathological findings showed a tendency toward improvement of inflammation. Hyperkeratosis in particular showed improvement in dermatitis skin lesions. These results suggest that oral administration of R-037 may alleviate AD.

Effect of novel monoclonal antibodies on LIF-induced signaling in chicken blastodermal cells.

Leukemia-inhibitory factor (LIF) is indispensable for maintaining the undifferentiated state when propagating mouse embryonic stem (ES) cells. We previously cloned chicken LIF (chLIF) cDNA and demonstrated that it maintained chicken ES cell cultures in an undifferentiated state. Here, we developed two monoclonal antibodies, HUL-1 and HUL-2, against chLIF, which specifically recognized recombinant chLIF (rchLIF) produced by Escherichia coli and Chinese hamster ovary K1 cells, in enzyme-linked immunosorbent assays and Western blot analysis. In addition, HUL-2 inhibited the phosphorylation of signal transducer and activator of transcription 3 by rchLIF in chicken blastodermal cells (CBCs), but not that of mitogen-activated protein kinase kinase. Furthermore, the addition of HUL-2 to CBC cultures resulted in embryoid bodies forming earlier than in normal cultures. These results indicated that HUL-2 recognized not only rchLIF but also native chLIF, and suggested that CBCs in culture produce LIF, which functions in autocrine signaling.

Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state.

Mouse embryonic stem (ES) cells can be maintained in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), a member of the interleukin-6 cytokine family. In other mammals, this is not possible with LIF alone. Chicken ES-like cells (blastodermal cells) have only been cultured with mouse LIF because chicken LIF was not available. However the culture system is imperfect and chicken ES-like cells equivalent to mouse ES cells were not observed. In the present study, we cloned the cDNA-encoding chicken LIF using mRNA subtraction and RACE methodology. The chicken LIF cDNA encodes a protein with approximately 40% sequence identity to mouse LIF. It has 211 amino acids including a putative N-terminal signal peptide of 24 residues. Chicken blastodermal cells were cultured in the presence of bacterially expressed chicken LIF or mouse LIF. The expression of alkaline phosphatase and embryonal carcinoma cell monoclonal antibody-1 and stage-specific embryonic antigen-1 and the activation of STAT3 were examined, all of which are indices of the undifferentiated state. Exposure in the blastodermal cells to recombinant chicken LIF but not to mouse LIF maintained the expression of these various markers. After 9 days of incubation, the blastodermal cells formed cystic embryoid bodies in the presence of mouse LIF but not in the presence of recombinant chicken LIF. We conclude that chicken LIF is able to maintain chicken ES cell cultures in the undifferentiated state.

A high-molecular-weight mite antigen (HM1) fraction aggravates airway hyperresponsiveness of allergic mice to house dusts and whole mite cultures.

BACKGROUND: The house dust mite Dermatophagoides farinae is the most common aeroallergen causing human allergic asthma. Previously, we demonstrated that a high-molecular-weight allergenic fraction (HM1), which was abundant in D. farinae extracts, induced a proliferative response of T cells from healthy donors. The induction was mediated through the activation of macrophages without MHC class II restriction. In this study, we investigate whether HM1 influences the development of airway inflammation in murine models of asthma. METHODS: BALB/c mice were injected twice intraperitoneally with D. farinae fecal extract (Dff) at an interval of 5 days. They were exposed daily to aerosolized antigen (group 1: Dff, group 2: HM1, group 3: HM1-depleted Dff and group 4: PBS) for 10 days. The effect of HM1 on their airway inflammation was evaluated by measuring acetylcholine-induced airway hyperresponsiveness and inflammatory cell infiltration in lung tissue. RESULTS: The inhalation of the whole fecal extract or the HM1 fraction induced airway hyperresponsiveness which was detectable after 24 h and was maintained for as long as 120 h. The inhalation of extract depleted of the HM1 fraction induced hyperresponsiveness measured at 24 h but this was not maintained for 120 h. Macrophage infiltration was significantly prolonged in mice inhaling the whole extract and the HM1 fraction compared to the HM1-depleted extract. CONCLUSION: The inhalation of the high-molecular-weight HM1 fraction of D. farinae prolonged airway hyperresponsiveness and macrophage inflammation in a mouse model of hypersensitivity. The results indicate that the HM1 fraction which can induce T cell proliferation through macrophage activation may play a role in the duration of airway responsiveness.

Induction of a proliferative response of T cells by a high-molecular antigen in Dermatophagoides farinae feces.

BACKGROUND: We have previously demonstrated that high-molecular mite antigen (HM1) from Dermatophagoides farinae feces is an allergen which binds to mite-allergic patients IgE. HM1 also induced a proliferative response in lymph node cells from mite-immunized mice as well as nonimmunized mice. In the present study, we demonstrated that HM1 induced T cell proliferation and investigated the HM1-stimulated T cell proliferative pathways using nonallergic human peripheral blood mononuclear cells (PMBC). METHODS: Blood samples were obtained from 10 healthy donors. Using primary culture, T cell response stimulated with HM1 was performed on purified T cells, CD19+ cell-depleted PBMC and CD11b+ cell-depleted PBMC. In addition, interleukin (IL)-5 and interferon (IFN)-gamma produced by mite-allergic and healthy donors stimulated with HM1 were estimated by enzyme immunoassay. RESULTS: T cell proliferation was detected only in CD19+ cell-depleted PBMC. When T cells were cocultured with CD11b+ cells they recovered their proliferative response to HM1. In addition, the pathway of HM1-stimulated T cell proliferation did not involve HLA class II restriction. Both activated CD11b+ cells and their conditioned media were needed to induce HM1-stimulated T cell proliferation. Furthermore, HM1 induced IFN-gamma production in both healthy and allergic donors. CONCLUSION: The high-molecular mite antigen, HM1, induced a proliferative response of T cells in healthy as well as allergic donors, without HLA class II restriction. Our results suggest that further investigation of HM1 could constitute a valuable avenue of research into complex allergic diseases.

Toward elucidating the full spectrum of mite allergens--state of the art.

Our research has focused on the molecular design of immunotherapeutic vaccines and the advancement of mite-allergy diagnosis. Here, we describe the research history of the major group 1 and group 2 allergens, immunoelectrophoretic analyses covering the complete spectrum of mite allergens, our results on allergens with distinctive characteristics (a conjunctival congestion-eliciting antigen [LM2], an immunotherapeutic antigen [HM2] with high efficacy and without definite adverse reactions, and a potent T-cell stimulatory antigen [HM1] with secretion of IFN-gamma), the full spectrum and immunochemical properties of the major and other important mite allergens (including our newly described allergens: a pan-allergen [tropomyosin, group 10], a potent T-cell stimulatory allergen [M-177, apolipophorin, group 14] and its peptide fragments Mag1 and Mag3, a moderate IgE-binding allergen [gelsolin/villin, group 16], an EF-hand Ca2+-binding allergen [group 17], and a less IgE-binding allergen [heat shock protein 70]), and prospects for the development of immunotherapeutic and diagnostic agents.