Conclusions In summary, activated T cells express CYP27B1 and can convert 25 D3 to 1,25 2D3 in sufficiently high concentrations to affect vitamin D responsive genes when cultured in serum free medium. However, DBP sequesters 25 D3 and inhibits the production of 1,25 2D3 in T cells. To fully exploit the immune regulatory potential of vitamin D, further studies of most the mechanisms that en able the immune system to Inhibitors,Modulators,Libraries exploit 25 D3 and convert it to 1,25 2D3 are required. Methods Chemicals were prepared in anhydrous ethanol and stored at ?80 C. To determine 1,25 2D3 in the medium we used the 1,25 Dihydroxy Vita min D EIA kit from IDS, Tyne and Wear, UK according to the manufacturers instructions. DBP and albumin purified from human serum were from Meridian Life Sciences and Sigma Aldrich, respectively.

Actin, arachidonic acid and ketoconazole were from Sigma Aldrich. Serum and central lymph from mini pigs were provided by the Department for Experimental Medicine, University of Copenhagen, Denmark. Cell culture Mononuclear cells from blood were isolated by Lympho prep Inhibitors,Modulators,Libraries density gradient centri fugation from healthy donors after obtaining informed, written consent in accordance with the Declarations of Helsinki principles for research involving human objects. The study was approved by the local Ethics Committee. Na ve CD4 T cells were isolated, cultured and activated as previously described. The cells were activated for 3 days in serum free X VIVO 15 medium if not otherwise stated. Flow cytometry For flow cytometry analyses of CD38 and TCR expres sion the cells were stained with anti CD38 APC both Inhibitors,Modulators,Libraries from BD Biosciences and analyzed on a FACS Calibur.

Fold change in CD38 surface expression was calculated as mean CD38 fluores cence intensity of cells stimulated in the presence of divided with mean CD38 fluores cence intensity Inhibitors,Modulators,Libraries of cells stimulated in the absence Percent TCR surface expression was calculated as Western blot and Inhibitors,Modulators,Libraries ELISA Western blot analyses were performed as previously described. Following incubation with primary antibody, the membranes were washed, and the pro teins visualized following 60 min incubation at room temperature with HRP conjugated sellckchem rabbit anti mouse Ig, swine anti rabbit Ig or rabbit anti goat Ig using ECL technology. The primary antibodies used were anti ezrin and anti DBP. For band density quantification ECL exposed sheets were analysed in a ChemiDoc MP Imaging System from Bio Rad. Meas urement of the cytokines IL 13 and IFN were deter mined by ELISA according to the manufacturers protocol.