RESUMO
PURPOSE: Vitamin D may induce tolerance in the intestinal immune system and has been shown to regulate the phenotype of tolerogenic intestinal dendritic cells (DCs) in vitro. It is unknown whether vitamin D supplementation affects human intestinal DCs in vivo, and we aimed to investigate the tolerability and effect on intestinal CD103+DCs of high-dose vitamin D3 treatment in healthy subjects. METHODS: Ten healthy subjects received a total of 480,000 IU oral vitamin D3 over 15 days and colonic biopsies were obtained before and after intervention by endoscopy. Lamina propria mononuclear cells (LPMCs) were isolated from the biopsies, stained with DC surface markers and analysed with flow cytometry. Snap-frozen biopsies were analysed with qPCR for DC and regulatory T cell-related genes. RESULTS: No hypercalcemia or other adverse events occurred in the test subjects. Vitamin D decreased the number of CD103+ DCs among LPMCs (p = 0.006). Furthermore, vitamin D induced mRNA expression of TGF-ß (p = 0.048), TNF-α (p = 0.006) and PD-L1 (p = 0.02) and tended to induce IL-10 expression (p = 0.06). Multivariate factor analysis discriminated between pre- and post-vitamin D supplementation with a combined increased qPCR expression of PD1, PD-L1, TGF-ß, IL-10, CD80, CD86, FOXP3, NFATc2 and cathelicidin. CONCLUSION: High-dose vitamin D supplementation is well tolerated by healthy subjects and has a direct effect on the CD103+ DCs, local cytokine and surface marker mRNA expression in the colonic mucosa, suggestive of a shift towards a more tolerogenic milieu.
Assuntos
Colecalciferol/administração & dosagem , Colo/citologia , Células Dendríticas/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Células Dendríticas/imunologia , Relação Dose-Resposta a Droga , Voluntários Saudáveis , Humanos , Mucosa Intestinal/citologia , Linfócitos T ReguladoresRESUMO
Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença de Crohn/tratamento farmacológico , Receptores de Calcitriol/biossíntese , Vitamina D/uso terapêutico , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/biossíntese , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Adulto , Idoso , Antígenos CD28/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Placebos , RNA Mensageiro/biossíntese , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptor X Retinoide alfa/biossíntese , Receptor X Retinoide alfa/genética , Adulto JovemRESUMO
Gap-junctional communication and expression of gap junction-forming proteins were investigated in normal human prostate epithelial cells and in several malignant prostate cell lines. In comparison with normal cells, gap-junctional communication in malignant cells, as assayed by the transfer of 443-Da fluorescent tracer Lucifer yellow, was either reduced or not detected. Malignant cells expressed mRNA transcripts for connexin (Cx) 43, whereas normal cells expressed mRNA transcripts for Cx32 and Cx40. In both normal and malignant cells, gap-junctional communication was enhanced twofold to fivefold by treatment with forskolin, an agent known to increase intracellular levels of cAMP. Immunocytochemical staining with a Cx43-specific antibody revealed that in malignant cells this enhancement correlated with the number of gap junctions and occurred without any qualitative or quantitative alteration in Cx43 mRNA or protein. Moreover, western blot analyses showed that both control and forskolin-treated malignant cells expressed only one form of Cx43. Our data suggest that gap-junctional communication in both normal and malignant prostate cells may be regulated by hormones that work via a cAMP-dependent signal transduction pathway. Thus, both normal and malignant cells offer a new experimental model system in which interactions between a hormonal form of cellular communication and intercellular communication mediated via gap junctions can be studied.
Assuntos
AMP Cíclico/fisiologia , Próstata/citologia , Neoplasias da Próstata/patologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Calnexina , Comunicação Celular , Compartimento Celular , Colforsina/farmacologia , Células Epiteliais , Junções Comunicantes , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , RNA Mensageiro/genética , RNA Neoplásico/genética , Solubilidade , Células Tumorais CultivadasRESUMO
Potentiation of chemical toxicity by hypoxia was studied in confluent hepatocyte monolayers. Addition of either hydroperoxyarachidonic acid (50 micrograms), leukotriene C4 (10 micrograms), or calcium ionophore A23187 (1.8 micrograms) to hepatocyte monolayers followed by incubation in 2% oxygen for 24 h killed 95% of the hypoxic cells, but was without effect on the normoxic cells. The greater than 10-fold increase in toxicity of A23187 suggests that hypoxic cells are less able to regulate intracellular calcium. The increased toxicity of hydroperoxyarachidonic acid and leukotriene C4 may be due to a related reduction in activity of protective enzymes.
Assuntos
Ácidos Araquidônicos/farmacologia , Calcimicina/farmacologia , Leucotrienos , Peróxidos Lipídicos/farmacologia , Fígado/citologia , Oxigênio/farmacologia , SRS-A/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Leucotrieno B4/farmacologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
Activation or inhibition by cytochrome b5 of benzphetamine N-demethylation was studied in micelle-reconstituted systems containing cytochrome P-450 LM2, NADPH-cytochrome P-450 reductase, and dilauroyl-phosphatidylcholine. The effects of cytochrome b5 were critically dependent on both protein:protein and lipid:protein ratios. A 200% stimulation of N-demethylation by cytochrome b5 was obtained at cytochrome P-450 reductase:cytochrome P-450 ratios similar to those in microsomes, compared to only a 20% stimulation at a ratio of 1:1. At lipid:protein ratios less than 50:1, the addition of cytochrome b5 caused significant inhibition of benzphetamine N-demethylation. Such an inhibition could be partially reversed by increasing phospholipid content of micelles and was not seen in vesicle-reconstituted systems at cytochrome b5:cytochrome P-450 ratios of 1:1 or lower. At high cytochrome P-450 reductase:cytochrome P-450 ratios, addition of cytochrome b5 did not alter the efficiency (80%) with which NADPH was utilized: however, at ratios similar to those in microsomes, an increase in efficiency from 42% to 80% was observed. The function of cytochrome b5 was interpreted in terms of a model in which inhibition of cytochrome P-450-mediated reactions results from changes in phospholipid-protein interactions and activation occurs via facilitation of electron transfer between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane.