Preparation of Monoclonal Antibodies Specifically Reacting with the Trichothecene Mycotoxins Nivalenol and 15-Acetylnivalenol via the Introduction of a Linker Molecule into Its C-15 Position.

Nivalenol (NIV) is a trichothecene mycotoxin that is more toxic than deoxynivalenol. It accumulates in grains due to infection with Fusarium species, which are the causative agents of scab or Fusarium head blight. An immunoassay, which is a rapid and easy analytical method, is necessary for monitoring NIV in grains. However, a specific antibody against NIV has not been prepared previously. To establish an immunoassay, we prepared NIV, introduced a linker, and generated antibodies against it. NIV was prepared from a culture of Fusarium kyushuense obtained from pressed barley through chromatographic procedures with synthetic adsorbents and silica gel. NIV was reacted with glutaric anhydride, and the reaction was stopped before mono-hemiglutaryl-NIV was changed to di-hemiglutaryl-NIV. 15-O-Hemiglutaryl-NIV was isolated via preparative HPLC and bound to keyhole limpet hemocyanin (KLH) using the active ester method. Two different monoclonal antibodies were prepared by immunizing mice with the NIV-KLH conjugate. The 50% inhibitory concentration values were 36 and 37 ng/mL. These antibodies also showed high reactivity in a direct competitive enzyme-linked immunosorbent assay and specifically reacted with NIV and 15-acetyl-NIV but not with deoxynivalenol and 4-acetyl-NIV.

Intestinal microbe-dependent ω3 lipid metabolite αKetoA prevents inflammatory diseases in mice and cynomolgus macaques.

Dietary ω3 fatty acids have important health benefits and exert their potent bioactivity through conversion to lipid mediators. Here, we demonstrate that microbiota play an essential role in the body's use of dietary lipids for the control of inflammatory diseases. We found that amounts of 10-hydroxy-cis-12-cis-15-octadecadienoic acid (αHYA) and 10-oxo-cis-12-cis-15-octadecadienoic acid (αKetoA) increased in the feces and serum of specific-pathogen-free, but not germ-free, mice when they were maintained on a linseed oil diet, which is high in α-linolenic acid. Intake of αKetoA, but not αHYA, exerted anti-inflammatory properties through a peroxisome proliferator-activated receptor (PPAR)γ-dependent pathway and ameliorated hapten-induced contact hypersensitivity by inhibiting the development of inducible skin-associated lymphoid tissue through suppression of chemokine secretion from macrophages and inhibition of NF-κB activation in mice and cynomolgus macaques. Administering αKetoA also improved diabetic glucose intolerance by inhibiting adipose tissue inflammation and fibrosis through decreased macrophage infiltration in adipose tissues and altering macrophage M1/M2 polarization in mice fed a high-fat diet. These results collectively indicate that αKetoA is a novel postbiotic derived from α-linolenic acid, which controls macrophage-associated inflammatory diseases and may have potential for developing therapeutic drugs as well as probiotic food products.

Mechanisms underlying enhanced IgA production in Peyer's patch cells by membrane vesicles derived from Lactobacillus sakei.

We analyzed the mechanisms underlying enhanced IgA production in the cells of Peyer's patch cells via membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC 15893. Depletion of CD11c+ cells from Peyer's patch cells suppressed the enhanced IgA production mediated by membrane vesicles. Meanwhile, the stimulation of bone-marrow-derived dendritic cells with membrane vesicles increased gene expression of inducible nitric oxide synthase, retinaldehyde dehydrogenase 2, and several inflammatory cytokines. The production of nitric oxide and interleukin (IL)-6 by membrane vesicle stimulation was induced via Toll-like receptor 2 on bone marrow-derived dendritic cells. Inhibition of inducible nitric oxide synthase and retinaldehyde dehydrogenase 2, as well as the neutralization of IL-6 in Peyer's patch cells, suppressed the enhanced IgA production by membrane vesicle stimulation. Hence, nitric oxide, retinoic acid, and IL-6 induced by membrane vesicles play crucial roles in the enhanced IgA production elicited by membrane vesicles in Peyer's patch cells.

ω3 fatty acid metabolite, 12-hydroxyeicosapentaenoic acid, alleviates contact hypersensitivity by downregulation of CXCL1 and CXCL2 gene expression in keratinocytes via retinoid X receptor α.

ω3 fatty acids show potent bioactivities via conversion into lipid mediators; therefore, metabolism of dietary lipids is a critical determinant in the properties of ω3 fatty acids in the control of allergic inflammatory diseases. However, metabolic progression of ω3 fatty acids in the skin and their roles in the regulation of skin inflammation remains to be clarified. In this study, we found that 12-hydroxyeicosapentaenoic acid (12-HEPE), which is a 12-lipoxygenase metabolite of eicosapentaenoic acid, was the prominent metabolite accumulated in the skin of mice fed ω3 fatty acid-rich linseed oil. Consistently, the gene expression levels of Alox12 and Alox12b, which encode proteins involved in the generation of 12-HEPE, were much higher in the skin than in the other tissues (eg, gut). We also found that the topical application of 12-HEPE inhibited the inflammation associated with contact hypersensitivity by inhibiting neutrophil infiltration into the skin. In human keratinocytes in vitro, 12-HEPE inhibited the expression of two genes encoding neutrophil chemoattractants, CXCL1 and CXCL2, via retinoid X receptor α. Together, the present results demonstrate that the metabolic progression of dietary ω3 fatty acids differs in different organs, and identify 12-HEPE as the dominant ω3 fatty acid metabolite in the skin.

Vitamin B1 Supports the Differentiation of T Cells through TGF-ß Superfamily Production in Thymic Stromal Cells.

Homeostatic generation of T cells, which occurs in the thymus, is controlled at least in part by endogenous cytokines and ligands. In addition, nutritional factors are other key regulators for the homeostasis of host immunity, but whether and how nutrition affects the homeostatic generation of thymocytes remains to be established. Here, we showed that vitamin B1 deficiency resulted in a bias toward the maturation of γδ thymocytes accompanied by decreased differentiation into double-positive thymocytes during thymic involution. These events were mediated through the increased production of TGF-ß superfamily members due to the accumulation of branched-chain α-keto acids in thymic stromal cells. These findings revealed essential roles of vitamin B1 in the appropriate differentiation of T cells through the metabolism of thymic stromal cells.

Maternal ω3 docosapentaenoic acid inhibits infant allergic dermatitis through TRAIL-expressing plasmacytoid dendritic cells in mice.

BACKGROUND: Maternal dietary exposures are considered to influence the development of infant allergies through changes in the composition of breast milk. Cohort studies have shown that ω3 polyunsaturated fatty acids (PUFAs) in breast milk may have a beneficial effect on the preventing of allergies in infants; however, the underlying mechanisms remain to be investigated. We investigated how the maternal intake of dietary ω3 PUFAs affects fatty acid profiles in the breast milk and their pups and reduced the incidence of allergic diseases in the pups. METHODS: Contact hypersensitivity (CHS) induced by 2,4-dinitrofluorobenzene (DNFB) and fluorescein isothiocyanate was applied to the skin in pups reared by mother maintained with diets mainly containing ω3 or ω6 PUFAs. Skin inflammation, immune cell populations, and expression levels of immunomodulatory molecules in pups and/or human cell line were investigated by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. ω3 PUFA metabolites in breast milk and infant's serum were evaluated by lipidomics analysis using LC-MS/MS. RESULTS: We show that maternal intake of linseed oil, containing abundant ω3 α-linolenic acid, resulted in the increased levels of ω3 docosapentaenoic acid (DPA) and its 14-lipoxygenation products in the breast milk of mouse dams; these metabolites increased the expression of TNF-related apoptosis-inducing ligand (TRAIL) on plasmacytoid dendritic cells (pDCs) in their pups and thus inhibited infant CHS. Indeed, the administration of DPA-derived 14-lipoxygenation products to mouse pups ameliorated their DNFB CHS. CONCLUSION: These findings suggest that an inhibitory mechanism in infant skin allergy is induced through maternal metabolism of dietary ω3 PUFAs in mice.

Dietary Omega-3 Fatty Acid Dampens Allergic Rhinitis via Eosinophilic Production of the Anti-Allergic Lipid Mediator 15-Hydroxyeicosapentaenoic Acid in Mice.

The metabolism and generation of bioactive lipid mediators are key events in the exertion of the beneficial effects of dietary omega-3 fatty acids in the regulation of allergic inflammation. Here, we found that dietary linseed oil, which contains high amounts of alpha-linolenic acid (ALA) dampened allergic rhinitis through eosinophilic production of 15-hydroxyeicosapentaenoic acid (15-HEPE), a metabolite of eicosapentaenoic acid (EPA). Lipidomic analysis revealed that 15-HEPE was particularly accumulated in the nasal passage of linseed oil-fed mice after the development of allergic rhinitis with the increasing number of eosinophils. Indeed, the conversion of EPA to 15-HEPE was mediated by the 15-lipoxygenase activity of eosinophils. Intranasal injection of 15-HEPE dampened allergic symptoms by inhibiting mast cell degranulation, which was mediated by the action of peroxisome proliferator-activated receptor gamma. These findings identify 15-HEPE as a novel EPA-derived, and eosinophil-dependent anti-allergic metabolite, and provide a preventive and therapeutic strategy against allergic rhinitis.

Development of a bivalent food poisoning vaccine: augmented antigenicity of the C-terminus of Clostridium perfringens enterotoxin by fusion with the B subunit of Escherichia coli Shiga toxin 2.

Food poisonings caused by Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) occur frequently worldwide; however, no vaccine is currently available. Therefore, we aimed to develop a bivalent vaccine against C. perfringens and STEC infections. Although it has been considered that the C-terminal region of C. perfringens enterotoxin (C-CPE) could be a good vaccine antigen to block the binding to its receptor, it was insufficient for induction of a protective immune response because of the low antigenicity. However, the fusion of C-CPE with Stx2 B subunit (Stx2B) augmented the antigenicity of C-CPE without affecting the antigenicity of Stx2B. Indeed, high levels of C-CPE-specific neutralizing IgG were found in the serum of mice immunized with the fusion protein Stx2B-C-CPE. Additionally, comparable and substantial levels of Stx2B-specific neutralizing IgG were induced in mice receiving Stx2B-C-CPE or Stx2B alone. These antibody responses against C-CPE and Stx2B lasted for at least 48 weeks, which were sufficient for protective immunity in vitro and in vivo, indicating that Stx2B-C-CPE could induce long-term protective immunity. As an underlying mechanism, ex vivo stimulation with Stx2B, but not with C-CPE, induced cytokine production from splenic T cells collected from mice immunized with Stx2B-C-CPE, suggesting that Stx2B-specific, but not C-CPE-specific, T cells were induced by the immunization with Stx2B-C-CPE and plausibly promoted immunoglobulin class switching of both Stx2B- and C-CPE-specific B cells from IgM to IgG. These findings collectively indicate that Stx2B-C-CPE is a T-cell-antigen-supplement-type bivalent vaccine, which could be an efficient against C. perfringens and STEC infections.

Direct Comparison of Manganese Detoxification/Efflux Proteins and Molecular Characterization of ZnT10 Protein as a Manganese Transporter.

Manganese homeostasis involves coordinated regulation of specific proteins involved in manganese influx and efflux. However, the proteins that are involved in detoxification/efflux have not been completely resolved nor has the basis by which they select their metal substrate. Here, we compared six proteins, which were reported to be involved in manganese detoxification/efflux, by evaluating their ability to reduce manganese toxicity in chicken DT40 cells, finding that human ZnT10 (hZnT10) was the most significant contributor. A domain swapping and substitution analysis between hZnT10 and the zinc-specific transporter hZnT1 showed that residue Asn(43), which corresponds to the His residue constituting the potential intramembranous zinc coordination site in other ZnT transporters, is necessary to impart hZnT10's unique manganese mobilization activity; residues Cys(52) and Leu(242) in transmembrane domains II and V play a subtler role in controlling the metal specificity of hZnT10. Interestingly, the His → Asn reversion mutant in hZnT1 conferred manganese transport activity and loss of zinc transport activity. These results provide important information about manganese detoxification/efflux mechanisms in vertebrate cells as well as the molecular characterization of hZnT10 as a manganese transporter.

Properties of Zip4 accumulation during zinc deficiency and its usefulness to evaluate zinc status: a study of the effects of zinc deficiency during lactation.

Systemic and cellular zinc homeostasis is elaborately controlled by ZIP and ZnT zinc transporters. Therefore, detailed characterization of their expression properties is of importance. Of these transporter proteins, Zip4 functions as the primarily important transporter to control systemic zinc homeostasis because of its indispensable function of zinc absorption in the small intestine. In this study, we closely investigated Zip4 protein accumulation in the rat small intestine in response to zinc status using an anti-Zip4 monoclonal antibody that we generated and contrasted this with the zinc-responsive activity of the membrane-bound alkaline phosphatase (ALP). We found that Zip4 accumulation is more rapid in response to zinc deficiency than previously thought. Accumulation increased in the jejunum as early as 1 day following a zinc-deficient diet. In the small intestine, Zip4 protein expression was higher in the jejunum than in the duodenum and was accompanied by reduction of ALP activity, suggesting that the jejunum can become zinc deficient more easily. Furthermore, by monitoring Zip4 accumulation levels and ALP activity in the duodenum and jejunum, we reasserted that zinc deficiency during lactation may transiently alter plasma glucose levels in the offspring in a sex-specific manner, without affecting homeostatic control of zinc metabolism. This confirms that zinc nutrition during lactation is extremely important for the health of the offspring. These results reveal that rapid Zip4 accumulation provides a significant conceptual advance in understanding the molecular basis of systemic zinc homeostatic control, and that properties of Zip4 protein accumulation are useful to evaluate zinc status closely.

Effects of oral contraceptive use on body mass index and blood pressure among female villagers in north-east Thailand.

The use of contraceptives has become prevalent among females in Thailand in the past 20 years, and oral contraceptive use has been suggested to trigger changes in fat intake, energy expenditure, fat metabolism and blood pressure. Based on field investigations of 391 married women aged 20 years or over in Yasothon Province, North-east Thailand, this study aims to elucidate the effects of oral contraceptive use on body mass index (BMI: kg/m2) and blood pressure, taking into account reproductive histories and socioeconomic conditions. The proportion of obese (BMI > or = 25) subjects was high in the age groups 30-39, 40-49 and 50-59, accounting for, respectively, 39.4%, 51.1% and 48.5% of these populations. The proportion of women with hypertension (90/140 mmHg) was 23.7%, 18.5% and 26.2% in the 40-49, 50-59 and 60-69 age groups. Current contraceptive practices in the studied population included sterilization by operation, oral contraception and injection. These methods accounted for 43.0%, 12.8% and 8.2% of the population, respectively. Sociodemographic factors such as reproductive history, years of education and household income were not significantly related to BMI or to blood pressure (ANOVA with age adjustment). In contrast, oral contraceptive users had significantly higher BMIs and diastolic blood pressures (p<0.01, ANOVA with age adjustment). Multiple regression analysis also revealed that oral contraceptive use was a weak but significant contributing factor to both high BMI and blood pressure when sociodemographic factors were taken into account and controlled for statistically. It can thus be concluded that the use of contraceptive pills, which contain oestrogen and progestin and are provided free of charge to Thai women, tend to increase BMI and to elevate blood pressure.