Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT04822038 |
| Other study ID # |
R-2014-3501-105 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
|
| Last updated |
|
| Start date |
June 1, 2017 |
| Est. completion date |
December 31, 2019 |
Study information
| Verified date |
April 2021 |
| Source |
Coordinación de Investigación en Salud, Mexico |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Interventional
|
Clinical Trial Summary
Introduction. The thickening fibrotic of the skin in systemic sclerosis (SSc) could reduce
endogenous availability of Vitamin D by sun exposition. Vitamin D hypovitaminosis have been
described in high prevalence in autoimmune disease as SSc. The cholecalciferol contributes to
improve the balance TH1/Th2/Treg in favor anti-inflammation and anti-fibrotic profile.
Aim. to analyze the effect(s) of short-term cholecalciferol supplementation on cytokine
profile in Th1, Th2, and Treg cells subpopulations in SSc patients.
Method. Randomized clinical trial conduct in patients with SSc (ACR-EULAR 2015) who signed
informed consent. General characteristics, severity of organ involvement scored by Medsger
disease severity scale (MsDSS) and cytokine Th1, Th2 and Treg will be determinate.
All data will be analyzed using SPSS software. It will be used parametric statistics for
normally distributed variables and nonparametric statistics for free distribution.
Description:
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 .
