Development of a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry for human urine (RaDPi-U).

Drug screening tests are mandatory in the search for drugs in forensic biological samples, and immunological methods and mass spectrometry (e.g., gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry) are commonly used for that purpose. However, these methods have some drawbacks, and developing new screening methods is required. In this study, we develop a rapid-fire drug screening method by probe electrospray ionization tandem mass spectrometry (PESI-MS/MS), which is an ambient ionization mass spectrometry method, for human urine, named RaDPi-U. RaDPi-U is carried out in three steps: (1) mixing urine with internal standard (IS) solution and ethanol, followed by vortexing; (2) pipetting the mixture onto a sample plate for PESI; and (3) rapid-fire analysis by PESI-MS/MS. RaDPi-U targets 40 forensically important drugs, which include illegal drugs, hypnotics, and psychoactive substances. The analytical results were obtained within 3 min because of the above-mentioned simple workflow of RaDPi-U. The calibration curves of each analyte were constructed using the IS method, and they were quantitatively valid, resulting in good linearity (0.972-0.999) with a satisfactory lower limit of detection and lower limit of quantitation (0.01-7.1 ng/mL and 0.02-21 ng/mL, respectively). Further, both trueness and precisions were 28% or less, demonstrating the high reliability and repeatability of the method. Finally, we applied RaDPi-U to three postmortem urine specimens and successfully detected different drugs in each urine sample. The practicality of the method is proven, and RaDPi-U will be a strong tool as a rapid-fire drug screening method not only in forensic toxicology but also in clinical toxicology.

Invariant natural killer T cells play dual roles in the development of experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) represents an experimental model for human endogenous uveitis, which is caused by Th1/Th17 cell-mediated inflammation. Natural killer T (NKT) cells recognize lipid antigens and produce large amounts of cytokines upon activation. To examine the role of NKT cells in the development of uveitis, EAU was elicited by immunization with a peptide from the human interphotoreceptor retinoid-binding protein (hIRBP1-20) in complete Freund's adjuvant and histopathology scores were evaluated in C57BL/6 (WT) and NKT cell-deficient mice. NKT cell-deficient mice developed more severe EAU pathology than WT mice. When WT mice were treated with ligands of the invariant subset of NKT cells (α-GalCer or RCAI-56), EAU was ameliorated in mice treated with RCAI-56 but not α-GalCer. IRBP-specific Th1/Th17 cytokines were reduced in RCAI-56-treated compared with vehicle-treated mice. Although the numbers of IRBP-specific T cells detected by hIRBP3-13/I-Ab tetramers in the spleen and the draining lymph node were the same for vehicle and RCAI-56 treatment groups, RORγt expression by tetramer-positive cells in RCAI-56-treated mice was lower than in control mice. Moreover, the eyes of RCAI-56-treated mice contained fewer IRBP-specific T cells compared with control mice. These results suggest that invariant NKT (iNKT) cells suppress the induction of Th17 cells and infiltration of IRBP-specific T cells into the eyes, thereby reducing ocular inflammation. However, in sharp contrast to the ameliorating effects of iNKT cell activation during the initiation phase of EAU, iNKT cell activation during the effector phase exacerbated disease pathology. Thus, we conclude that iNKT cells exhibit dual roles in the development of EAU.

Recombinant Fusion Allergens, Cry j 1 and Cry j 2 from Japanese Cedar Pollen, Conjugated with Polyethylene Glycol Potentiate the Attenuation of Cry j 1-Specific IgE Production in Cry j 1-Sensitized Mice and Japanese Cedar Pollen Allergen-Sensitized Monkeys.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent seasonal rhinitis in Japan. A standardized Japanese cedar pollen extract (CPE) containing 1.5-4.2 µg of Cry j 1 is currently the highest-concentration extract available for allergen-specific immunotherapy (SIT) against this pollinosis. Therefore, we developed a PEGylated fusion protein as a more effective SIT vaccine against Japanese cedar pollinosis. METHODS: The fusion protein of major allergens for Japanese cedar pollen, Cry j 1 and Cry j 2, was expressed in Escherichia coli and conjugated with polyethylene glycol (PEG). The purified PEGylated Cry j 1/2 fusion protein (PEG-fusion) was subcutaneously injected four times into Cry j 1- sensitized mice and CPE-sensitized monkeys. The mice were then subcutaneously challenged with Cry j 1 and serum levels of Cry j 1-specific immunoglobulin, and the proliferation and cytokine production of splenocytes were analyzed. The monkeys were intranasally challenged with CPE and analyzed for Cry j 1-specific immunoglobulin levels in plasma. RESULTS: Cry j 1-specific IgE was significantly attenuated in the PEG-fusion-treated group after Cry j 1-challenge and Cry j 1-specific IgG was significantly increased following PEG-fusion treatment in mice and monkeys. Proliferation and Th2-type cytokine production in splenocytes stimulated with Cry j 1 were also reduced in PEG-fusion-treated mice. IL10 and IL2 production were reduced, but not significantly, while IFN-x03B3; was significantly increased in the PEG-fusion-treated group. CONCLUSIONS: A high-dose injection of PEG-fusion appears to be a valid candidate for a safer and more effective vaccine than the conventional SIT extract for Japanese cedar pollinosis.

Increase of regulatory T cells and the ratio of specific IgE to total IgE are candidates for response monitoring or prognostic biomarkers in 2-year sublingual immunotherapy (SLIT) for Japanese cedar pollinosis.

The aims of this study were to examine the therapeutic effects of sublingual immunotherapy (SLIT) and to identify potential biomarkers that would predict the therapeutic response in a randomized, double-blind, placebo-controlled clinical trial. The trial was carried out over two pollinosis seasons in 2007 and 2008. Carry-over therapeutic effects were analyzed in 2009. SLIT significantly ameliorated the symptoms of pollinosis during the 2008 and 2009 pollen seasons. Cry j 1-specific cytokine production in a subgroup of patients with mild disease in the SLIT group was significantly attenuated. The ratio of specific IgE to total IgE before treatment correlated with the symptom-medication score in the SLIT group in 2008. Patients with increased Cry j 1-iTreg in the SLIT group had significantly improved QOL and QOL-symptom scores. In summary, the specific IgE to total IgE ratio and upregulation of Cry j 1-iTreg are candidates for biomarker of the clinical response to SLIT.

Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha gene.

Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT-cell potential and obtain NKT cells with desired function. Here, using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system that preferentially developed functional NKT cells and also identified early NKT progenitors, which first appeared on day 11 as a c-kit(+) population in the cocultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kit(lo/-) on day 14. Interestingly, in the presence of Notch signals, NKT-ES cells differentiated only to thymic CD44(lo) CD24(hi) NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44(hi) CD24(lo) liver NKT cells producing mainly interferon gamma (IFN-gamma) and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT-cell development and for the establishment of NKT-cell therapy.

RCAI-37, 56, 59, 60, 92, 101, and 102, cyclitol and carbasugar analogs of KRN7000: their synthesis and bioactivity for mouse lymphocytes to produce Th1-biased cytokines.

Cyclitol [RCAI-37 (1), 59 (5), 92 (7), and 102 (2)] and carbasugar analogs [RCAI-56 (3), 60 (4), and 101 (6)] of KRN7000 were synthesized through coupling reactions of the corresponding cyclitol or carbasugar derivatives with a cyclic sulfamidate (9) as the key step. Bioassay showed RCAI-56 (3, carbagalactose analog of KRN7000), 59 (5, 1-deoxy-neo-inositol analog), and 92 (7, 1-O-methylated 5) to be remarkably potent stimulants of mouse lymphocytes to produce Th1-biased cytokines, such as interferon-gamma, in vivo. RCAI-60 (4, carbafucose analog) and RCAI-101 (6, 6-O-methylated 3) showed strong bioactivity, on the other hands, RCAI-37 (1, myo-inositol analog) and 102 (2, neo-inositol analog) induced little cytokine production.

Suppression of mast cell degranulation by a novel ceramide kinase inhibitor, the F-12509A olefin isomer K1.

Antigen-induced degranulation of mast cells plays a pivotal role in allergic and inflammatory responses. Recently, ceramide kinase (CERK) and its phosphorylated product ceramide 1-phosphate (C1P) have emerged as important players in mast cell degranulation. Here, we describe the synthesis of a novel F-12509A olefin isomer, K1, as an effective CERK inhibitor. In vitro kinase assays demonstrated that K1 effectively inhibits CERK without inhibiting sphingosine kinase and diacylglycerol kinase. Treating RBL-2H3 cells with K1 reduced cellular C1P levels to 40% yet had no effect on cell growth. Furthermore, treatment with K1 significantly suppressed both calcium ionophore- and IgE/antigen-induced degranulation, indicating that K1 interferes with signals that happen downstream of Ca(2+) mobilization. Finally, we show that K1 affects neither IgE/antigen-induced global tyrosine phosphorylation nor subsequent Ca(2+) elevation, suggesting a specificity for CERK-mediated signals. Our novel CERK inhibitor provides a useful tool for studying the biological functions of CERK and C1P. Moreover, to our knowledge, this is the first report demonstrating that inhibition of CERK suppresses IgE/antigen-induced mast cell degranulation. This finding suggests that CERK inhibitors might be a potential therapeutic tool in the treatment of allergic diseases.

Impaired contact hypersensitivity in macrophage migration inhibitory factor-deficient mice.

To determine whether macrophage migration inhibitory factor (MIF) is required for contact hypersensitivity (CHS) response, MIF-deficient (MIF KO) and wild-type (WT) mice were sensitized with trinitrochlorobenzene (TNCB) or oxazolone on their abdominal skin and challenged on the dorsum skin of one ear 5 days later. Significant ear swelling was observed in the WT mice, but this response was inhibited in the MIF KO mice (p<0.01 for MIF KO vs. WT mice in 24 h). In addition, lymph node cells from hapten-sensitized MIF KO mice showed a decreased capacity for transferring the CHS response. A topical application of TNCB (200 microg) caused a significant decline in epidermal Langerhans cell (LC) density (20.3%; p<0.01 compared with vehicle) 4 h after application in WT mice, but it failed to provoke a significant epidermal LC migration in MIF KO mice (7.4%). By mixed lymphocyte reaction, the T cell proliferative response to alloantigen was significantly decreased in the MIF KO mice compared with WT mice (p<0.005). Taken together, these results indicate that MIF is pivotal in the regulation of cutaneous immune responses and plays a central role in LC migration and T cell proliferation for the CHS response.