Immunomodulating Effects of Supplementation With 25-OH Vitamin D

The study will be carried out with prior authorization from the Local Research and Ethics Committee. Patients entitled to our healthcare services and complying with screening criteria will be included.

Patients with scleroderma included in our database will be invited to participate in the study through a telephone call. Patients complying with the screening criteria and accepting to participate in the study will be given a date.

On the day of the interview, they will be given the informed consent form. Once it has been signed, they will be asked to complete clinical recorder and serum samples were collected for biochemical determinations including Vitamin D status.

Once patients have completed the initial evaluations, another visit will be scheduled two weeks later. Patients with hypovitaminosis D result will be randomly assigned to one of two groups: Group 1. Vitamin D3 supplementation and Group 2. Dietary recommendations. Two groups given instructions to follow dietary recommendations or to receive vitamin D3 supplementation and complete the medical measurements. Additionally, the investigators included SSc patients with vitamin D sufficiency and another one with healthy donors.

On the second visit (four weeks later), clinical data and the following parameters will be recorded: calcium, phosforus, parathyroid hormone by quimioluniscencia 25-hydroxyvitamin D serum status by ELISA, intracellular cytokine (IL-2, INF-γ, IL-4 and IL-10) production from Th1, Th2 and Treg lymphocytes by flow cytometry.

Group 1. Daily oral dosages of 5,000 UI vitamin D3 during 4 weeks.

Group 2. Dietary recommendations according to the recommendations for normal daily intake of vitamin D in food during 4 week.

And additionally, the investigators are including SSc group with vitamin D sufficiency and healthy donors both as comparators.

Each group will be given written instructions regarding the drug use. At the end of the supplementations another visit will be scheduled to check for treatment compliance (remaining capsules will be counted) and patients will complete the second clinical evaluations and calcium, phosforus, parathyroid hormone by quimioluniscencia 25-hydroxyvitamin D serum status by ELISA, intracellular cytokine (IL-2, INF-γ, IL-4 and IL-10) production from Th1, Th2 and Treg lymphocytes by flow cytometry. At the end the statistical analysis will be carried out.

Participants will also be asked to complete a form recording symptoms and potential adverse events.

Lymphocyte Separation Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Ficoll-Hypaque; Sigma-Aldrich) from freshly drawn venous blood. Intracellular cytokine production in PBMCs (1x106 cells/tube) were stimulated at 37ºC for 4h with Ionomicyn calcium salt from Streptomyces conglobates (1μg/ 1 x106 cells, Sigma-Aldrich), Brefeldin A (10μg/ 1 x106 cells, Cayman chemical company) and phorbol-12-myristate-13-acetate (PMA-25ng/1 x106 cells, Sigma-Aldrich).

Cell Surface and Intracellular Staining of T Cells PBMCs were resuspended with (PBS) phosphate-buffered saline (pH 7.2) (Thermo Fisher Scientific) and stained with monoclonal conjugated antibodies with either fluorescein isothiocynate (FITC), peridinin chlorophyll protein complex (PerCP), phyco-erythrin (PE) or allophycocyanin (APC) and directed to CD69 (L/78), CD25, CD3, CD4 (BD PharmigenTM and BD Bioscience) for 20 min. For intracellular staining of IL-4 (BD PharmigenTM), IL-2, INF-γ (BD Fast Immune), FoxP3 (Affymetrix) and IL10 (eBioscinence), cells previously were fixed using fixation buffer (BioLegend) for 20 min at room temperature followed by a washing step with permeabilization buffer (Perm/Wash Buffer BD Pharmigen TM) according to the manufacture's protocols, then staining cells were incubated with monoclonal antibody for 20 min at room temperature and resuspended in PBS buffer. Evaluation of Th1, Th2 and Treg subpopulations by flow cytometry were determinate as IL-2+ and INF-γ+ percentages expression for Th1 lymphocytes, IL-4+ for Th2, and CD25+FoxP3+IL-10+ for Treg within the CD3+CD4+ gate. Data were acquired and analyzed using a four-parameter flow cytometer FACS Calibur using ProCellQuest software (Beckman Coulter; BD Biosciences) and Flowing Software (Version 2.5.1) Cell Imaging Core .