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.
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.
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-g g 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.
SummaryPolysorbate 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 lg/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 Cryconsensus peptide alone. However, there was no significant difference between the effect of Cry-consensus peptide administration on interferon (IFN)-c 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-c 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.
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 antiCry 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.
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 28ΐ, 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.
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