In vitro activity: Suplatast tosilate has an inhibitory effect on antibody production in isolated mouse splenic and human peripheral blood B cells with IC50 ＞100 nM. Suplatast tosilate inhibits mouse and human cytokine production, IFN-γ, IL-2, IL-4, IL-5 and IL-10 with an IC50 ＞100 nM. The induction of Cryj1-dependent IgE synthesis mediated by SN-4 is suppressed in a concentration-dependent manner by Suplatast tosilate (1 and 10 μg/ml) in autologous B cells. IL-4 production, both stimulated by SN-4 with Cryj1 in the presence of autologous APC for 3 days and produced by PHA-stimulated PBMC from normal donors, are effectively inhibited in a concentration-dependent manner by Suplatast tosilate (1 and 10 μg/mL). Suplatast tosilate significantly inhibits the expression of CD1a, CD80, and CD86 on immature dendritic cells (DCs) and of CD1a, CD80, CD83, and CD86 on mature DCs. Suplatast tosilate also significantly inhibits the secretion of CCL17, IL-12p70, and IL-12p40; however, the secretion of IL-10 is not affected. The proliferative responses of allogeneic CD4(+) T cells to Suplatast tosilate-treated DCs are suppressed. Moreover, Suplatast tosilate-treated DCs has an impaired capacity to stimulate CD4(+) T cells to produce IFN-gamma and IL-5.

Kinase Assay: The cultures for cytokine production are set up at 37 °C as follows: the mixtures of SN-4 and autologous APC (each 1 × 105 cells/well) are cultured for 3 days with 50 μg/mL of Cry j1 in a total volume of 0.2 mL in round-bottomed micro plates; PBMC (2 × 10 5 cells/well) from normal donors are cultured for 24 hours with 10 μg/ml of PHA in a total volume of 0.2 mL in flat-bottomed, 96-well micro plates; and purified T cells (1 × 105 cells/well) from normal donors are cultured in a total volume of 1 ml for 24 hours with anti-CD3 mAb that have been immobilized on flat-bottomed, 24-well plates at a concentration of 5 μg/ml. Cytokines in the culture supernatants are quantitatively assayed by the following commercially available kits: IL-4 and IFN-γ. The sensitivity of the assay is 30 pg/mL for IL-4 and 1 U/mL for IFN-γ.

Cell Assay: Allogeneic responder CD4+ T cells obtained from healthy subjects are purified from PBMCs by negative depletion of CD14+, CD16+, CD19+, CD56+, and CD8+ cells using magnetic cell separation system micro-beads and columns. After 7 days of culture with GM-CSF and IL-4, iDCs differentiated from monocytes in the presence or absence of Suplatast tosilate (10 and 100 mg/ mL) are co-cultured with purified 1×10 5 allogeneic CD41 T cells at varying ratios of iDCs in 96-well round bottomed culture plates for 5 days. Cells are pulsed with 1 μCi of 3H-methylthymidine for the last 8 hours of the culture period. Incorporated radioactivity is counted with a liquid scintillation counter, and proliferative responses are expressed as the mean 3 H-methylthymidine incorporation (counts per minutes) of triplicate wells_SEM. Proliferation of DCs alone or CD4+ T cells alone is also assessed.

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