A single dose of vitamin D enhances immunity to mycobacteria.

RATIONALE: Vitamin D was used to treat tuberculosis (TB) in the preantibiotic era. Prospective studies to evaluate the effect of vitamin D supplementation on antimycobacterial immunity have not previously been performed. OBJECTIVES: To determine the effect of vitamin D supplementation on antimycobacterial immunity and vitamin D status. METHODS: A double-blind randomized controlled trial was conducted in 192 healthy adult TB contacts in London, United Kingdom. Participants were randomized to receive a single oral dose of 2.5 mg vitamin D or placebo and followed up at 6 weeks. MEASUREMENTS AND MAIN RESULTS: The primary outcome measure was assessed with a functional whole blood assay (BCG-lux assay), which measures the ability of whole blood to restrict luminescence, and thus growth, of recombinant reporter mycobacteria in vitro; the readout is expressed as a luminescence ratio (luminescence postinfection/baseline luminescence). IFN-gamma responses to the Mycobacterium tuberculosis antigens early secretory antigenic target-6 and culture filtrate protein 10 were determined with a second whole blood assay. Vitamin D supplementation significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro compared with placebo (mean luminescence ratio at follow-up, 0.57, vs. 0.71, respectively; 95% confidence interval for difference, 0.01-0.25; p=0.03) but did not affect antigen-stimulated IFN-gamma secretion. CONCLUSIONS: A single oral dose of 2.5 mg vitamin D significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro without affecting antigen-stimulated IFN-gamma responses. Clinical trials should be performed to determine whether vitamin D supplementation prevents reactivation of latent TB infection. Clinical trial registered with www.clinicaltrials.gov (NCT 00157066).

Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3.

BACKGROUND: Measurement of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D. METHODS: We used isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for routine determination of 25-OH D2 and D3 with a stable-isotope-labeled internal standard (IS). Serum samples (100 microL) were denatured with methanol-propanol containing IS, vortex-mixed, extracted into hexane, and dried under nitrogen. The reconstituted extract was chromatographed on a BDS C8 HPLC column, and the metabolites and IS were detected by electrospray ionization MS/MS in multiple-reaction monitoring mode. RESULTS: 25-OH D2 and D3 and the IS nearly coeluted, whereas 1alpha-hydroxyvitamin D3 was separated; total run time was 8 min. The interassay CVs for 25-OH D2 were 9.5% and 8.4% at 52 and 76 nmol/L, respectively, and for 25-OH D3 were 5.1% and 5.6% at 55 and 87 nmol/L, respectively. The detection limit of the present method was <4 nmol/L for both metabolites. Method comparison with a commercial RIA measuring total 25-hydroxyvitamin D showed good correlation: y=0.97x - 2.7 nmol/L (r=0.91). The analytical system can assay 100 samples in 12.5 h. CONCLUSIONS: This simple robust interference-free LC-MS/MS assay is suitable for routine measurement of the 25-hydroxy metabolites of vitamins D2 and D3 in human serum. The assay has been in use for 9 months and has been used to assay more than 6000 routine samples.

Advanced glycation end products modulate the maturation and function of peripheral blood dendritic cells.

Advanced glycation end products (AGEs), a complex and heterogeneous group of posttranslational modifications of proteins in vivo, have been widely studied for their involvement in diabetic complications; these complications are largely vascular and accompanied by inflammation. Because dendritic cells (DCs) initiate and modulate inflammatory responses, we hypothesized that AGEs might exert immunomodulatory effects via antigen-presenting DCs. To test this hypothesis, we investigated effects of the AGE peptide, compared with the naked peptide, on maturation, costimulatory molecule expression, and function of DCs in peripheral blood. From flow cytometry, we found a dose-dependent inhibition in CD83 expression on DCs exposed for 2.5 h to each of two synthetic AGE peptides. A similar culture for 24 h additionally produced an inhibition of CD80 expression, whereas exposure to AGEs for 3 days induced a large increase in DC numbers and a concomitant loss of monocyte/macrophages. Exposure of DCs to AGEs resulted in a dose-dependent loss in their capacity to stimulate primary proliferation of allogeneic T-cells. We conclude that AGEs promote development of DCs but that these DCs fail to express maturation markers and lose the capacity to stimulate primary T-cell responses. Effects of AGEs on DCs could be instrumental in the immunological changes associated with diabetes.