The results of this study demonstrate that RRMS patients had lower plasma 25(OH)D concentrations compared with HC during the winter months. In agreement with our results, previous studies  demonstrated summer/winter differences in vitamin D status between MS patients and HC. In our opinion, differences in plasma 25(OH)D concentrations during summer could not be detected because sun exposure in these months led to an increase in plasma 25(OH)D concentrations in RRMS patients to levels similar to HC.
In this study, the RRMS patients selected were not on disease-modifying drugs (DMD) since we aimed to analyse plasma 25(OH)D concentrations, T-cell reactivity against myelin peptides and the association between vitamin D status and clinical, radiological and immunological variables without the interference of immunomodulatory treatment. The main reasons for patients not receiving DMD were: they did not fulfil the criteria for receiving DMD in our area (two relapses in the previous three years) or did not wish to be treated with DMD.
In this study, PPMS patients had similar plasma 25(OH)D concentrations to HC, as previously reported . Consistent with these observations, a growing body of evidence indicates that distinct pathogenic processes mediate brain damage in different groups of MS patients .
Several efforts have been made to correlate clinical, radiological and immunological MS data with plasma 25(OH)D concentrations Reviewed in . However, a relationship between vitamin D and clinical activity of the disease has not been established. In accordance with our results, a significant correlation of plasma 25(OH)D with gadolinium-enhanced or T2-weighted MRI has not been reported. In contrast, previous studies demonstrated a correlation between low 25(OH)D levels and high EDSS score or an increased risk of relapse [8–10], which concurred with our observation that patients with 25(OH)D insufficiency had higher EDSS scores than those with sufficient 25(OH)D. We believe there are two possible explanations. Low 25(OH)D levels could induce a greater neurological disability due to an aetiopathogenic factor. Disabled patients with high EDSS have loss of autonomy, gait impairment and a more sedentary lifestyle than patients with low EDSS; therefore, they are less likely to be exposed to sun, which could lead to less endogenous synthesis of 25(OH)D.
Furthermore, lower plasma 25(OH)D concentrations were observed in patients with positive T-cell proliferation against myelin peptides who had a higher number of previous relapses and greater disability than unresponsive patients. A limitation of this study is that our cohort was too small to permit a definitive assessment of the clinical outcome measures. However, we believe increasing 25(OH)D levels exert a possible beneficial effect on improving not only immunological variables such as T-cell proliferation against myelin peptides but also the clinical parameters. In this context, previous studies  reported that MS patients treated with vitamin D supplements had a reduced number of relapse events and a persistent reduction in T-cell proliferation compared to controls.
To define the role of vitamin D as an immunomodulatory agent, the relationship between plasma 25(OH)D concentrations and immunological parameters such as proliferation of T-cell reactivity against myelin peptides was analysed in this study. Previously, we demonstrated that T-cell proliferation against a selected mix of 7 myelin peptides was more common in RRMS patients than in HC, which supports their pathogenic significance in MS . Interestingly, patients with positive T-cell proliferation against myelin peptides had low levels of plasma 25(OH)D. Correale et al.  demonstrated by in vitro coculture with 1,25(OH)2D3 that the proliferation of myelin basic protein (MBP)-specific T cells was significantly inhibited. These results suggest that vitamin D may play an immunomodulatory role in T-cell proliferation to myelin peptides. Moreover, previous studies reported an inhibitory effect of vitamin D on Th1 cell function and a beneficial effect on Th2 and Treg cells in vitro . Furthermore, 1,25-(OH)2D proved to exert a beneficial effect on clinical and histological disease features in an experimental allergic encephalomyelitis animal model of MS .
Vitamin D acts through the vitamin D receptor (VDR). Certain polymorphisms of the VDR may modify vitamin D function. Some studies showed that polymorphisms are significantly more common in MS . Nevertheless, other studies found no differences in VDR gene polymorphisms between MS and control groups . A limitation of this study was that we did not determine the polymorphisms of the VDR, which should therefore be assessed in further studies on vitamin D and MS.