Effects of aliphatic aldehydes on the growth and patulin production of Penicillium expansum in apple juice.

The effects of 16 aliphatic aldehydes with 3-10 carbons on the growth and patulin production of Penicillium expansum were examined. When P. expansum spores were inoculated into apple juice broth, some alkenals, including 2-propenal, (E)-2-butenal, (E)-2-pentenal, and (E)-2-hexenal, inhibited fungal growth and patulin production. Their minimal inhibitory concentrations were 5, 50, 80, and 80 µg/mL respectively. Vital staining indicated that these alkenals killed mycelia within 4 h. Treatment of the spores with these aldehydes also resulted in rapid loss of germination ability, within 0.5-2 d. On the other hand, aliphatic aldehydes with 8-10 carbons significantly enhanced patulin production without affecting fungal growth: 300 µg/mL of octanal and 100 µg/mL of (E)-2-octenal increased the patulin concentrations in the culture broth by as much as 8.6- and 7.8-fold as compared to that of the control culture respectively. The expression of the genes involved in patulin biosynthesis in P. expansum was investigated in mycelia cultured in apple juice broth containing 300 µg/mL of octanal for 3.5, 5, and 7 d. Transcription of the msas gene, encoding 6-methylsalicylic acid synthase, which catalyzed the first step in the patulin biosynthetic pathway was remarkably high in the 3.5-d and 5-d-old cultures as compared with the control. However, octanal did not any increase the transcription of the msas in the 7-d-old culture or that of the other two genes, IDH and the peab1, in culture. Thus the enhanced patulin accumulation with supplementation with these aldehydes is attributable to the increased amount of the msas transcript.

A new murine model of allergic rhinitis by repeated intranasal Cry j 1 challenge.

To evaluate the long-lasting effects of new therapeutic approaches to allergies, we established a new model of allergic rhinitis by repeated challenges with intranasal Cry j 1, a common Japanese cedar (Cryptomeria japonica) pollen allergen, in B10.S mice. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at 1-week intervals. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally for 5 consecutive days starting 1 day after intranasal histamine pretreatment (challenge-1). We challenged the mice by instilling histamine and Cry j 1 intranasally again 12 weeks later (challenge-2). There were significantly more sneezes after challenge-2 than challenge-1. Cry j 1-specific IgE levels in serum were significantly increased in both challenge-1 and 2 after continuous nasal antigen challenge. Serum levels of anti-Cry j 1 IgE in challenge-2 was 2.3 times higher than after challenge-1. Thus, we have established a new model of seasonal allergic rhinitis in B10.S mice by repeated intranasal antigen challenge, and this model may help elucidate mechanisms of allergic rhinitis and the development of new drugs.

[Multiresidue method for pesticides and veterinary drugs in bovine milk using GC/MS and LC/MS/MS].

A simple, sensitive and selective method with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed to detect 342 pesticides and veterinary drugs contaminating bovine milk at the maximum residue limits (MRLs) defined in the "positive list system". Sample preparation was performed by extracting the analytes with acetonitrile, followed by salting-out with sodium chloride. For some pesticides, the extract was further cleaned up by n-hexane partitioning and PSA cartridge column chromatography. GC/MS-EI or -NCI was used to determine pesticide residues, while LC/MS/MS-ESI was applicable to the determination of pesticide and veterinary drug residues. The variation of the recoveries of these drugs at MRL was relatively wide; however the relative standard deviations of the recovery of each drug were within 28%, suggesting that the present method is good enough for use as a screening test for contaminants at the MRLs. These results show that this method is useful for multiresidue analysis of numerous pesticides and veterinary drugs in bovine milk.

Effect of Cry-consensus peptide, a novel recombinant peptide for immunotherapy of Japanese cedar pollinosis, on an experimental allergic rhinitis model in B10.S mice.

BACKGROUND: We are developing an immunotherapeutic peptide, Cry-consensus peptide, for Japanese cedar pollinosis. Cry-consensus peptide is a recombinant polypeptide containing six major human T-cell epitopes derived from both Cry j 1 and Cry j 2, two major allergens of Japanese cedar pollen. We examined the effect of Cry-consensus peptide on an allergic rhinitis model in B10.S mice, which have one common T-cell epitope in the Cry-consensus peptide. METHODS: B10.S mice were sensitized with Cry j 1/alum, then the Cry-consensus peptide was administered subcutaneously once a week for 5 weeks from the last sensitization. Histamine was dropped in both nostrils (10 microL per nostril) of each mouse on the day before continuous intranasal instillation of Cry j 1. Soon after the final challenge with Cry j 1, the mice were observed for 5 minutes for the resulting number of sneezes. In addition, serum levels of Cry j 1-specific IgE and IgG2a antibody, eosinophil infiltration in nasal tissue, and Cry j 1-specific cytokine production from splenocytes were evaluated. RESULTS: Cry-consensus peptide markedly inhibited Cry j 1-induced sneezes, eosinophil infiltration, and eosinophil peroxidase (EPO) activity in nasal tissue. Cry-consensus peptide inhibited the production of anti-Cry j 1 IgE (Th2-mediated) and significantly enhanced anti-Cry j 1 IgG2a (Th1-mediated). In cytokine production from splenocytes, Cry-consensus peptide significantly decreased in IL-4/IFN-gamma and IL-5/IFN-gamma ratios. CONCLUSIONS: It was concluded that Cry-consensus peptide effectively controlled allergic responses, which results from shifting from a Th2-dominated to a Th1-dominated immune response.

Establishment of an allergic rhinitis model in mice for the evaluation of nasal symptoms.

The purpose of our study was to establish a new model of allergic rhinitis in mice, eliciting symptoms such as sneezing, infiltration of eosinophils into the nasal mucosa, and antigen-specific IgE production. One of the major human T-cell epitopes in Cry j 1, an allergen of Japanese cedar pollen, is also a major murine T-cell epitope in B10.S mice. Thus we tried to establish an allergic rhinitis model in B10.S mice with Cry j 1 as the antigen. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at intervals of 1 week. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally from the day after intranasal histamine pretreatment. Soon after, we counted the number of sneezes. We then evaluated the infiltration of eosinophils into the nasal tissues and also measured the serum levels of antigen-specific IgE antibody. In addition, we confirmed the effects of ketotifen fumarate and dexamethasone hydrochloride on these animals. In Cry j 1-sensitized B10.S mice, sneezes, eosinophil peroxidase (EPO) activity in nasal tissues, and Cry j 1-specific IgE clearly increased after intranasal histamine pretreatment and 5 days of continuous intranasal Cry j 1 challenge. Both ketotifen and dexamethasone inhibited the increase in sneezing, and dexamethasone also inhibited EPO activity and Cry j 1-specific IgE. Thus we succeeded in establishing a new model of allergic rhinitis in Cry j 1-sensitized B10.S mice, which exhibited sneezing, eosinophil infiltration into the nasal mucosa, and Cry j 1-specific IgE production.

Biological assay using T cell response for Cry-consensus peptide designed for the peptide-based immunotherapy of Japanese cedar pollinosis.

INTRODUCTION: Cry-consensus peptide is a linearly linked peptide of T-cell epitopes for the management of Japanese cedar (JC) pollinosis and is expected to become a new drug for immunotherapy. However, the mechanism of T-cell epitopes in allergic diseases is not well understood, and thus, a simple in vitro procedure for evaluation of its biological activity is desired. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 27 JC pollinosis patients and 10 healthy subjects, and cultured in vitro for 4 days in the presence of Cry-consensus peptide and (3)H-thymidine. The relationship between growth stimulation (stimulation index; SI) and antigen-specific IgE levels in serum was also investigated in JC pollinosis patients. Moreover, to confirm the importance of the primary sequence in Cry-consensus peptide, heat-treated Cry-consensus peptide and a mixture of the amino acids of which Cry-consensus peptide is composed, and their (3)H-thymidine uptake was compared with Cry-consensus peptide. Finally, whether Cry-consensus peptide stimulates PBMCs from healthy subjects was investigated. RESULTS: The mean SI of JC patients showed a good correlation with Cry-consensus peptide concentration in the culture medium; however, the SI was independent of the anti-Cry j 1 IgE level. Heat-denatured Cry-consensus peptide retained a PBMC proliferation stimulatory effect comparable to the original Cry-consensus peptide, while the mixture of amino acids constituting Cry-consensus peptide did not stimulate PBMC proliferation. PBMCs from healthy subjects did not respond to Cry-consensus peptide at all. DISCUSSION: These data indicate that the PBMC response of patients suffering from JC pollinosis to Cry-consensus peptide is specific for the sequence of T cell epitopes thereof and may be useful for the evaluation of the efficacy of Cry-consensus peptide in vivo.

Enzyme-linked immunosorbent assay of a linear, recombinant peptide designed for immunotherapy of Japanese cedar pollinosis.

INTRODUCTION: Cry-consensus peptide, a recombinant T-cell epitope peptide for immunotherapy of Japanese cedar pollinosis, is a linear peptide that does not have disulfide bonds because no cysteine residue exists in the molecule. We examined whether a sandwich enzyme-linked immunosorbent assay (ELISA) could be performed for linear peptides such as Cry-consensus peptide. METHODS: The 3-dimensional conformation of Cry-consensus peptide was examined by (1)H NMR analysis. Nineteen monoclonal antibodies (mAbs) that recognized various domains of Cry-consensus peptide were established to use in a sandwich ELISA. The relationship between the recognition sites of mAbs and the sensitivity of the ELISA was investigated to optimize the selection of the combination of the capture and the detection antibodies. ELISA inhibitors in serum and plasma were also studied to improve the stability and the sensitivity of determination. RESULTS: (1)H NMR analysis of Cry-consensus peptide suggested that Cry-consensus peptide molecule had no portions with rigid conformation. The sensitivity of the ELISA showed a good correlation with the distance between the respective binding sites of the capture and the detection antibodies. Human serum albumin and alpha1-acid glycoprotein strongly inhibited the binding of the capture mAb to Cry-consensus peptide in a dose-dependent manner, and heparin also inhibited the binding in the concentration at which it is used as anticoagulant. Taken together, the findings indicated that an optimized method showed good linearity and minimal variation from 0 to 1000 ng/ml of Cry-consensus peptide. DISCUSSION: These data indicate that this method is useful for monitoring Cry-consensus peptide concentrations in plasma or serum.

Cry-consensus peptide, a novel peptide for immunotherapy of Japanese cedar pollinosis, induces Th1-predominant response in Cry j 1-sensitized B10.S mice.

Cry-consensus peptide (CCP) is a newly designed peptide for peptide-based immunotherapy of Japanese cedar pollinosis but its mechanism of efficacy is unknown. We investigated the effect of CCP on Cry j 1-specific Th1/Th2 response in a mice model. Subcutaneous injection of CCP decreased Cry j 1-specific IgE and IgG1 in blood slightly, but the IgG2a level was increased significantly in a dose dependent manner. Splenocytes from these mice were stimulated with Cry j 1 in vitro. This inhibited IL-4, IL-5 and IL-10 secretion significantly, but IFN-gamma secretion was increased. In vitro CCP stimulation of splenocytes from Cry j 1-sensitized mice induced more marked Th1-predominancy of cytokine production than native allergen stimulation. Taken together, these data suggest that one of the mechanisms of CCP is dependent on the modulation of the antigen-specific Th1/Th2 response.

PS80 interferes with the antiallergic effect of Cry-consensus peptide, a novel recombinant peptide for immunotherapy of Japanese cedar pollinosis, at very low concentration through modulation of Th1/Th2 balance.

Polysorbate 80 (PS80 or Tween-80) is often used as an additive to promote the rapid solubilization of pharmaceuticals in aqueous solutions. We investigated whether coinjection of a minimal amount of PS80 had a modulatory effect on the immunotherapeutic effects of Cry (Cryptomeria)-consensus peptide, a novel peptide developed for the therapeutic management of Japanese cedar pollinosis, using a Cry j 1-sensitized mouse model with experimental allergic rhinitis. Subcutaneous challenge with Cry-consensus peptide plus 50 microg/ml of PS80 did not affect the antigen-specific proliferation of splenocytes, but decreased the potency of Cry-consensus peptide to inhibit antigen-specific interleukin (IL)-5 production by the cells significantly in comparison with challenge with Cry-consensus peptide alone. However, there was no significant difference between the effect of Cry-consensus peptide administration on interferon (IFN)-gamma production in the presence and absence of PS80, indicating that PS80 interfered with the T helper 1 (Th1)-dominant T helper balance induced by Cry-consensus peptide challenge. Moreover, the increase in the level of antigen-specific immunoglobulin G2a (IgG2a) induced by Cry-consensus peptide challenge was inhibited slightly but unambiguously by PS80 coinjection. These in vitro experiments indicated that PS80 induces Th2-type differentiation of T helper cells through preferential inhibition of IFN-gamma expression relative to IL-5 expression in splenocytes in a concentration-dependent manner. In naïve mice, sensitization by Cry-consensus peptide with PS80 induced antigen-specific IL-5 production more potently than sensitization by Cry-consensus peptide alone, and when PS80 was added to bone marrow-derived dendritic cells, the endocytosis of fluorescence-labelled Cry-consensus peptide was dramatically inhibited in a concentration-dependent manner. Therefore, we conclude that PS80 has an immunomodulatory effect on the antigen-specific response resulting in a shift towards Th2 predominance with respect to the antigen recognition stage. Taken together, our findings suggest that PS80 might decrease the efficacy of Cry-consensus peptide through modulation of the efficiency of antigen endocytosis and/or of the direction of successive T helper cell differentiation.

An improved method for acetaldehyde determination in blood by high-performance liquid chromatography and solid-phase extraction.

This study reports on an improved method for acetaldehyde (ACH) determination in blood by high-performance liquid chromatography (HPLC). In the case of HPLC analysis, ACH is generally converted to derivatives for ultraviolet detection (for example 2,4-dinitrophenylhydrazine [DNPH] derivative). Nevertheless, elevation of the background during protein precipitation, hydrazone synthesis, or both frequently results in a serious loss of accuracy and precision of the analysis. The method in this study is developed to minimize the increase in nonspecific ACH-DNPH with a view to optimize mainly the synthetic condition of ACH-DNPH. The background is decreased dramatically by gentle deproteination, optimization of the DNPH amount and reaction pH, and reversed-phase solid extraction for the elimination of excess DNPH reagent. The standard curves show good linearity between 0 and 100 microM and minimal background is observed, indicating that the method is useful for monitoring the ACH concentration in blood.