In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines.
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We show that a combination of the immunosuppressive drugs, vitamin D3 and Dexamethasone, induced human and mouse naive CD4(+) T cells to differentiate in vitro into regulatory T cells. In contrast to the previously described in vitro derived CD4(+) T cells, these cells produced only interleukin (IL)-10, but no IL-5 and interferon (IFN)-gamma, and furthermore retained strong proliferative capacity. The development of these IL-10-producing cells was enhanced by neutralization of the T helper type 1 (Th1)- and Th2-inducing cytokines IL-4, IL-12, and IFN-gamma. These immunosuppressive drugs also induced the development of IL-10-producing T cells in the absence of antigen-presenting cells, with IL-10 acting as a positive autocrine factor for these T cells. Furthermore, nuclear factor (NF)-kappaB and activator protein (AP)-1 activities were inhibited in the IL-10-producing cells described here as well as key transcription factors involved in Th1 and Th2 subset differentiation. The regulatory function of these in vitro generated IL-10-producing T cells was demonstrated by their ability to prevent central nervous system inflammation, when targeted to the site of inflammation, and this function was shown to be IL-10 dependent. Generating homogeneous populations of IL-10-producing T cells in vitro will thus facilitate the use of regulatory T cells in immunotherapy. (+info)
Kidney microsomal 25- and 1alpha-hydroxylase in vitamin D metabolism: catalytic properties, molecular cloning, cellular localization and expression during development.
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Both a 25-hydroxylation and a 1alpha-hydroxylation are necessary for the conversion of vitamin D(3) into the calcium-regulating hormone 1alpha,25-dihydroxyvitamin D(3). According to current knowledge, the hepatic mitochondrial cytochrome P450 (CYP) 27A and microsomal CYP2D25 are able to catalyze the former bioactivation step. Substantial 25-hydroxylase activity has also been demonstrated in kidney. This paper describes the molecular cloning and characterization of a microsomal vitamin D(3) 25- and 1alpha-hydroxylase in kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D(3) and 1alpha-hydroxyvitamin D(3) and, in addition, 1alpha-hydroxylation of 25-hydroxyvitamin D(3). The cDNA encodes a protein of 500 amino acids. Both the DNA sequence and the deduced peptide sequence of the renal enzyme are homologous with those of the hepatic vitamin D(3) 25-hydroxylase CYP2D25. Genomic Southern blot analysis suggested the presence of a single gene for CYP2D25 in the pig. Immunohistochemistry experiments indicated that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. These findings indicate a tissue-specific developmental regulation of CYP2D25. The results from the current and previous studies on renal vitamin D hydroxylations imply that CYP2D25 has a biological role in kidney. (+info)
Interleukin-6 enhances transforming growth factor-alpha mRNA expression in macrophage-like human monocytoid (U-937-1) cells.
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We have previously reported that the human monocytoid cell line U-937-1 constitutively expresses transforming growth factor-alpha (TGF-alpha) and that the steady-state levels of TGF-alpha mRNA as well as TGF-alpha protein release increase when U-937-1 cells are differentiated towards monocytes/macrophages. Interleukin-6 (IL-6), which has been shown to have growth-stimulatory effects on a number of cell types, has recently been shown to enhance TGF-alpha expression in keratinocytes. In the present study we investigated whether TGF-alpha expression in macrophage-like cells could be regulated by IL-6 using U-937-1 cells as a model system of monocyte/macrophage differentiation. U-937-1 cells were differentiated with retinoic acid (RA), vitamin D3 (Vit-D3) or phorbol-12-myristate-13-acetate (PMA) for 4 days and were then treated with human recombinant IL-6 (1000 IU/ml) for up to 24 hr. Northern blot analysis revealed that cells differentiated with PMA, inducing the phenotype of a secretory macrophage, markedly increased their TGF-alpha mRNA levels (2.7-fold) when treated with IL-6; the response was maximal at 6 hr and remained high at 12 hr. The expression of the TGF-alpha gene was accompanied by release of TGF-alpha protein into the cell culture medium, irrespective of differentiating agent, as demonstrated by enzyme-linked immunosorbent assay (ELISA), as well as by surface expression of pro-TGF-alpha as determined by indirect immunofluorescent cytometry. However, the superinduction of the TGF-alpha gene by IL-6 in cells differentiated with PMA was not accompanied by any increase in TGF-alpha protein release or pro-TGF-alpha surface expression. We conclude that since IL-6 causes increased steady-state levels of TGF-alpha mRNA in macrophage-like cells, it may prime these cells for production of this growth factor. Furthermore, we have shown that the IL-6 receptor complex is functional in U-937-1 cells induced to differentiate towards a secretory macrophage by treatment with PMA. (+info)
An estimate of premature cancer mortality in the U.S. due to inadequate doses of solar ultraviolet-B radiation.
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BACKGROUND: There are large geographic gradients in mortality rates for a number of cancers in the U.S. (e.g., rates are approximately twice as high in the northeast compared with the southwest). Risk factors such as diet fail to explain this variation. Previous studies have demonstrated that the geographic distributions for five types of cancer are related inversely to solar radiation. The purpose of the current study was to determine how many types of cancer are affected by solar radiation and how many premature deaths from cancer occur due to insufficient ultraviolet (UV)-B radiation. METHODS: UV-B data for July 1992 and cancer mortality rates in the U.S. for between 1970-1994 were analyzed in an ecologic study. RESULTS: The findings of the current study confirm previous results that solar UV-B radiation is associated with reduced risk of cancer of the breast, colon, ovary, and prostate as well as non-Hodgkin lymphoma. Eight additional malignancies were found to exhibit an inverse correlation between mortality rates and UV-B radiation: bladder, esophageal, kidney, lung, pancreatic, rectal, stomach, and corpus uteri. The annual number of premature deaths from cancer due to lower UV-B exposures was 21,700 (95% confidence interval [95% CI], 20,400-23,400) for white Americans, 1400 (95% CI, 1100-1600) for black Americans, and 500 (95% CI, 400-600) for Asian Americans and other minorities. CONCLUSIONS: The results of the current study demonstrate that much of the geographic variation in cancer mortality rates in the U.S. can be attributed to variations in solar UV-B radiation exposure. Thus, many lives could be extended through increased careful exposure to solar UV-B radiation and more safely, vitamin D3 supplementation, especially in nonsummer months. (+info)
Fluorinated vitamin D analogs to probe the conformation of vitamin D in its receptor complex: 19F-NMR studies and biological activity.
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To investigate vitamin D conformation, specifically the A-ring and seco-B ring parts, in its complex with the vitamin D receptor (VDR), we have previously suggested the use of 19F-NMR spectroscopy and have synthesized three fluorovitamin D derivatives to be used for the study, 4,4-difluoro-1alpha,25-dihydroxyvitamin D3 [4,4-F2-1,25-(OH)2D3, 2a], and (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3 [19-F-1,25-(OH)2D3, 3a, 4a]. In this paper, we examined the 19F-NMR spectra of these and related fluorovitamin D compounds in detail. In the low temperature 19F-NMR spectra, we observed two well-separated rigid conformations of 2a (51:49) and 4a (84:16), while only one conformation was detected with 3a. The two observed conformers of 2a and 4a were respectively assigned to the known alpha- and beta-conformers formed by the flipping of the A-ring where the C(19) exocyclic methylene points to the alpha- and beta-faces. The single conformation observed for 3a was assigned to the alpha-conformer. We detected no other conformation in the 19F-NMR of all vitamin D compounds examined. The effect of solvents on the 19F chemical shifts of fluorovitamin D compounds was found to be small (less than 6.3 ppm). This was much smaller than the difference between the two A-ring conformers (13-30 ppm). Using the dynamic 1H-NMR studies of fluorovitamin D compounds, we determined the free energy of activation for the interconversion between the two conformations of 2a (9.9 kcal/mol) and 4a (10.8, 11.5 kcal/mol). Introduction of the 1alpha-hydroxyl group raised the activation energy about 1 kcal/mol. The affinity for VDR was evaluated, and the relative potency of 2a, 3a and 4a was found to be 1%, 8% and 9%, respectively, of that of 1,25-(OH)2D3 (1). Though the affinity for VDR was considerably reduced in these compounds, the ability to activate gene transcription was similar and remained in the range 30-50% of the effect of 1. This biological information in combination with the NMR properties indicates that 2a and 4a are promising probes for studying the VDR-bound A-ring conformation of vitamin D. (+info)
A 1alpha,25-dihydroxyvitamin D(3) analog enhances regulatory T-cells and arrests autoimmune diabetes in NOD mice.
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Type 1 diabetes is a chronic progressive autoimmune disease characterized by mononuclear cell infiltration, dominated by interleukin-12 (IL-12)-dependent Th1 cells, of the pancreatic islets, with subsequent destruction of insulin-producing beta-cells. Here, we demonstrate that treatment of adult nonobese diabetic (NOD) mice with an analog of 1alpha,25-dihydroxyvitamin D(3), an immunomodulatory agent preventing dendritic cell maturation, decreases lipopolysaccharide-induced IL-12 and gamma-interferon production, arrests Th1 cell infiltration and progression of insulitis, and inhibits diabetes development at nonhypercalcemic doses. Arrest of disease progression is accompanied by an enhanced frequency in the pancreatic lymph nodes of CD4(+)CD25(+) regulatory T-cells that are able to inhibit the T-cell response to the pancreatic autoantigen insulinoma-associated protein 2 and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells. Thus, a short treatment of adult NOD mice with an analog of 1,25-dihydroxyvitamin D(3) inhibits IL-12 production, blocks pancreatic infiltration of Th1 cells, enhances CD4(+)CD25(+) regulatory cells, and arrests the progression of type 1 diabetes, suggesting its possible application in the treatment of human autoimmune diabetes. (+info)
The p38 and JNK pathways cooperate to trans-activate vitamin D receptor via c-Jun/AP-1 and sensitize human breast cancer cells to vitamin D(3)-induced growth inhibition.
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The signaling connection between mitogen-activated protein kinases(MAPKs) and nuclear steroid receptors is complex and remains mostly unexplored. Here we report that stress-activated protein kinases p38 and JNK trans-activate nuclear steroid vitamin D receptor (VDR) gene and increase vitamin D(3)-dependent growth inhibition in human breast cancer cells. Activation of p38 and JNK by an active MAPK kinase 6 stimulates VDR promoter activity independently of the ligand vitamin D(3) and estrogen receptor expression. Moreover, stimulation of the endogenous stress pathways by adenovirus-mediated delivery of recombinant MAPK kinase 6 also activates VDR and sensitizes MCF-7 cells to vitamin D(3)-dependent growth inhibition. Both the p38 and JNK MAPK pathways and the downstream transcription factor c-Jun/AP-1 are required for the VDR stimulation, as revealed by application of their dominant negatives, the specific p38 inhibitor SB203580, and site-directed mutagenesis of the AP-1 element in the VDR promoter. The essential role of the p38 and JNK stress pathways in up-regulation of VDR expression is further confirmed by using the chemical stimulator arsenite. These results establish a signaling connection between the stress MAPK pathways and steroid hormone receptor VDR expression and thereby offer new insights into regulation of cell growth by the MAPK pathways through regulation of vitamin D(3)/VDR activity. (+info)
Treatment of autoimmune diabetes recurrence in non-obese diabetic mice by mouse interferon-beta in combination with an analogue of 1alpha,25-dihydroxyvitamin-D3.
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Autoimmune diabetes recurrence is in part responsible for islet graft destruction in type 1 diabetic individuals. The aim of the present study was to design treatment modalities able to prevent autoimmune diabetes recurrence after islet transplantation in spontaneously diabetic NOD mice. In order to avoid confusion between autoimmune diabetes recurrence and allograft rejection, we performed syngeneic islet transplantations in spontaneously diabetic NOD mice. Mice were treated with mouse interferon-beta (IFN-beta, 1 x 105 IU/day), a new 14-epi-1,25-(OH)2D3-analogue (TX 527, 5 microg/kg/day) and cyclosporin A (CsA, 7.5 mg/kg/day) as single substances and in combinations. Treatment was stopped either 20 days (IFN-beta and CsA) or 30 days (TX 527) after transplantation. Autoimmune diabetes recurred in 100% of control mice (MST 11 days). None of the mono-therapies significantly prolonged islet graft survival. Combining CsA with TX 527 maintained graft function in 67% of recipients as long as treatment was given (MST 31 days, P < 0.01 versus controls). Interestingly, 100% of the IFN-beta plus TX 527-treated mice had normal blood glucose levels during treatment, and even had a more pronounced prolongation of graft survival (MST 62 days, P < 0.005 versus controls). Cytokine mRNA analysis of the grafts 6 days after transplantation revealed a significant decrease in IL-2, IFN-gamma and IL-12 messages in both IFN-beta plus TX 527- and CsA plus TX 527-treated mice, while only in the IFN-beta with TX 527 group were higher levels of IL-10 transcripts observed. Therefore, we conclude that a combination of IFN-beta and TX 527 delays autoimmune diabetes recurrence in islet grafts in spontaneously diabetic NOD mice. (+info)