Study population. Twenty MS patients (15 females and 5 males; mean age 35 ± 5 years) and 20 healthy control subjects (15 females and 5 males; mean age 34.1 ± 5 years) were recruited. All MS patients had been diagnosed based on McDonald's criteria [20] and assessed for clinical picture (Expanded Disability Status Scale: EDSS) by clinicians at the MS Research Centre, Sina Hospital, Tehran, Iran. All study participants gave informed consent to provide 10 ml blood samples for the study, which was approved by the local research ethics committee (approval code: IR.TUMS.SPH.REC.1395.756) of Tehran University of Medical Sciences (TUMS).
Sample Preparation. Peripheral blood mononuclear cells (PBMCs) were separated from fresh whole blood by Ficoll- Paque centrifugation (GE Healthcare Life Sciences, Massachusetts, USA), resuspended in RPMI 1640 (Gibco Life Technologies, USA) supplemented with 10 % FCS (Gibco), and used fresh.
EBV and vitamin D preparation. EBV stock was prepared as described previously [21]. Briefly, B95-8 cells were grown 3–4 weeks at 37\(\text{℃}\) with 5% CO2 in RPMI-1640 supplemented with 10 % FCS, penicillin (100 IU) and streptomycin (100 lg/ml) (Biosera, Ringmer, East Sussex, UK) as completed medium 10% (CM-10). After removal of remained cells and debris by centrifugation at 600g for 6 min at 4\(\text{℃}\), the supernatants were passed through a 0.45 µm membrane filter (Millipore, Billerica, USA) and concentrated by Beckman ultracentrifuge (L8-80 M; Beckmann, Palo Alto, CA,USA) at 30,000 g for two hours at 4 \(\text{℃}\). The pellets containing concentrated virus were re-suspended with CM-10 in 1/100 of original volume, passed through a 0.22 µm filter, aliquoted and stored in cryovials at -80°C.
As described previously [22] the infectious capability and the absolute amount of EBV genome was determined. To optimize the virus concentration, PBMCs (with 1µg/ml cyclosporine) and B cells were transformed by different titers of virus and 15\(\times\)106 copies included further in all experiments to minimize inter-assay variations. To determine the optimal vitamin D concentration, the expression of VDR and CYP24A1 was checked after PBMCs treatment with different concentration of vitamin D and 100 nM of vitamin D showed the best expression of VDR and CYP24A1.
EBV stimulation in the absence and the presence of vitamin D. PBMCs were infected with EBV and seeded into 24-well plates (1\(\times\)106 cells/well) (SPL Life Science Co, Phcheon-si, Gyeonggi-do, Korea) and incubated at 37\(\text{℃}\) in a 5% CO2 atmosphere in the presence and the absence of 100 nM recombinant 1,25-(OH)2D (Enzo Life Sciences, Inc., AnnArbor, MI.). After 72 hours, supernatants and cells were collected and stored at -80°C until further use. Our previous study [22] showed 72 hours’ post-infection was considered as optimal time for cells harvesting for the both EBV activity and vitamin D metabolism.
RNA extraction and cDNA synthesis. RNA from PBMCs (both treated and untreated cells) were manually extracted by Trizol according to the manufacturer’s instructions (Roche, Mannheim, Germany). The final RNA pellet were dissolved in 30 µl of RNase and DNase water (Invitrogen). The purity and concentration of total RNA were determined using a Nano Drop ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). 1000 ng of total RNA was used for cDNA synthesis according to the manufacturer’s instructions (Roche, Mannheim, Germany).
Determination of HERV-K18 env and TGF -β and level in PBMCs. Relative quantitative (RQ) RT-PCR was applied to quantify the expression of HERV-K18 env (before and after treated) and TGF–β by using SYBR® GreenER™ qPCR SuperMix Universal (Roche, Mannheim, Germany) on the on the Rotor-Gene® 6000 (Corbett Research, Sydney, Australia). PCR amplification reactions were performed in 25µl reaction mixtures containing cDNA (1:10 fold diluted), 12µl SYBR Green, and 10 pmol of each primer. The reactions were incubated at 95°C for 10 min, followed by of 95°C for 15 s, 57°C for 30 s, and 70°C for 30 s. A melting curve analysis was performed to confirm single gene-specific peaks. The linearity and accuracy of real-time RT-PCR were evaluated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a reference gene, and standard curve derived from amplifying serially diluted pooled cDNA. Biomarker expression patterns were analyzed according to the \({2}^{-\varDelta Ct}\) method [23]. The relative values of each biomarker were expressed as change fold to compare mRNA levels between treatment groups.
Statistical analysis. GraphPad Prism software, version 8 (GraphPad software, Inc, La Jolla, California) was used for plotting graphs and statistical analysis. p values ≤ 0.05 were regarded as significant. Data are presented either with median or mean ± SEM.