Prognostic value of natural killer cell/T cell ratios for disease activity in multiple sclerosis.

BACKGROUND AND PURPOSE: Natural killer (NK) cells may play a role in multiple sclerosis (MS). Ratios of NK cells to CD4+ T cells have been proposed as a biomarker for the therapeutic effect of stem cell transplantation in MS. The objectives here were to explore the relevance of this ratio in MS patients by analysing NK and T cell subsets, as well as their prognostic value for disease activity. METHODS: Baseline peripheral blood mononuclear cells of 50 relapsing-remitting MS patients, participating in our vitamin D supplementation study (SOLARIUM), were analysed with flow cytometry. Disease activity was measured as new magnetic resonance imaging lesions, relapses and mean plasma neurofilament light chain levels after 48 weeks of follow-up. RESULTS: The proportion of NK cells correlated negatively with CD4+ T cells (R = -0.335, p = 0.001) and interleukin 17A (IL-17A+ ) CD4+ T cells (R = -0.203, p = 0.043). Participants with magnetic resonance imaging activity or relapses displayed lower NK/IL-17A+  CD4+ T cell ratios (p =0.025 and p = 0.006, respectively). The NK/IL-17A+  CD4+ T cell ratio correlated negatively with neurofilament light chain levels (R = -0.320, p = 0.050). Vitamin D supplementation did not affect these ratios. CONCLUSIONS: Our data suggest a protective role of an expanded NK cell compartment compared to the CD4+ T cell subset fractions in relapsing-remitting MS patients. NK/CD4+ T cell ratios may be a prognostic biomarker for disease activity in MS.

Exploring the effect of vitamin D3 supplementation on the anti-EBV antibody response in relapsing-remitting multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) infection and vitamin D insufficiency are potentially interacting risk factors for multiple sclerosis (MS). OBJECTIVES: To investigate the effect of high-dose vitamin D3 supplements on antibody levels against the EBV nuclear antigen-1 (EBNA-1) in patients with relapsing-remitting multiple sclerosis (RRMS) and to explore any underlying mechanism affecting anti-EBNA-1 antibody levels. METHODS: This study utilized blood samples from a randomized controlled trial in RRMS patients receiving either vitamin D3 (14,000 IU/day; n = 30) or placebo ( n = 23) over 48 weeks. Circulating levels of 25-hydroxyvitamin-D, and anti-EBNA-1, anti-EBV viral capsid antigen (VCA), and anti-cytomegalovirus (CMV) antibodies were measured. EBV load in leukocytes, EBV-specific cytotoxic T-cell responses, and anti-EBNA-1 antibody production in vitro were also explored. RESULTS: The median antibody levels against EBNA-1, but not VCA and CMV, significantly reduced in the vitamin D3 group (526 (368-1683) to 455 (380-1148) U/mL) compared to the placebo group (432 (351-1280) to 429 (297-1290) U/mL; p = 0.023). EBV load and cytotoxic T-cell responses were unaffected. Anti-EBNA-1 antibody levels remained below detection limits in B-cell cultures. CONCLUSION: High-dose vitamin D3 supplementation selectively reduces anti-EBNA-1 antibody levels in RRMS patients. Our exploratory studies do not implicate a promoted immune response against EBV as the underlying mechanism.

Illuminating vitamin D effects on B cells--the multiple sclerosis perspective.

Vitamin D is associated with many immune-mediated disorders. In multiple sclerosis (MS) a poor vitamin D status is a major environmental factor associated with disease incidence and severity. The inflammation in MS is primarily T-cell-mediated, but increasing evidence points to an important role for B cells. This has paved the way for investigating vitamin D effects on B cells. In this review we elaborate on vitamin D interactions with antibody production, T-cell-stimulating capacity and regulatory B cells. Although in vitro plasma cell generation and expression of co-stimulatory molecules are inhibited and the function of regulatory B cells is promoted, this is not supported by in vivo data. We speculate that differences might be explained by the B-cell-Epstein-Barr virus interaction in MS, the exquisite role of germinal centres in B-cell biology, and/or in vivo interactions with other hormones and vitamins that interfere with the vitamin D pathways. Further research is warranted to illuminate this tube-versus-body paradox.

Fingolimod in active multiple sclerosis: an impressive decrease in Gd-enhancing lesions.

BACKGROUND: Fingolimod is a disease modifying therapy (DMT) in highly active relapsing remitting multiple sclerosis (RRMS), as is natalizumab. Fingolimod decreases annual relapse rates and gadolinium enhancing lesions on MRI as compared to either interferon beta (IFNß) or placebo. The effect of fingolimod on MRI outcomes compared to natalizumab treatment has not been investigated in (head to head) clinical trials. Clinical experience with natalizumab is much more extended and in general practice often preferred. CASE PRESENTATION: This case describes a 31-year old woman with RRMS, who experienced severe side effects on natalizumab. After a voluntary four months treatment free period, a severe relapse appeared which was treated with prednisone and plasmapheresis; thereafter fingolimod was initiated. In the following months MRI signs improved spectacularly. CONCLUSION: This case suggests that fingolimod might be a good alternative for natalizumab, especially for use in RRMS patients, with highly active, advanced disease, when natalizumab treatment is stopped due to side effects or even after a severe relapse.

NK/T cell ratios associate with interleukin-2 receptor alpha chain expression and shedding in multiple sclerosis.

NK/T-cell ratios predict disease activity in relapsing remitting multiple sclerosis (RRMS). We investigated in 50 RRMS patients whether interleukin-2 receptor alpha-chain (IL-2Rα) expression and shedding associates with NK/T-cell balance, as suggested by daclizumab-trials in RRMS. A subsample (N = 31) was genotyped for IL2RA-associated MS risk SNPs. CD56bright NK-cell/IL-17A+CD4+ T-cell ratios correlated negatively with plasma and PBMC-culture supernatant sIL-2Rα-levels [R = -0.209; p = 0.038 and R = -0.254; p = 0.012, resp.], and with CD4+ T-cell CD25 MFI [R = -0.341; p = 0.001]. Carriers of the rs3118470 risk-allele showed higher sIL-2Rα-levels (P = 0.031) and a lower CD56bright NK-cell/IL-17A+CD4+ T-cell ratio (P = 0.038). Therefore, IL-2Rα may be involved in the interplay between NK-cells and T-cells.

Proportions of circulating transitional B cells associate with MRI activity in interferon beta-treated multiple sclerosis patients.

B-cells contribute to MS pathogenesis. The association of circulating B-cell phenotypes with combined unique active lesions (CUA) on MRI at 48 weeks follow-up was investigated in 50 interferon beta-treated MS patients. Transitional B-cell proportions were lower in participants with CUA at week 0 and 48 [p = 0.004, p = 0.002]. A decrease in circulating anti-EBNA-1 IgG levels between week 0 and 48 associated with absence of CUA [p = 0.047], but not with B-cell profiles. In a multi-factor model for CUA-risk, transitional B-cell proportions contributed independent from NK/T-cell ratio, change in anti-EBNA-1 IgG, and vitamin D supplementation. Transitional B-cells may predict treatment response in MS.

Vitamin D related genetic polymorphisms affect serological response to high-dose vitamin D supplementation in multiple sclerosis.

INTRODUCTION: A poor 25-hydroxyvitamin D (25(OH)D) status is a much replicated risk factor for developing multiple sclerosis (MS), and several vitamin D-associated single nucleotide polymorphisms (SNPs) have been associated with a higher risk of MS. However, studies on the benefit of vitamin D supplementation in MS show inconclusive results. Here, we explore whether vitamin D-associated SNPs and MS risk alleles confound serological response to vitamin D supplementation. METHODS: 34 participants from the SOLARIUM study consented to genotyping, of which 26 had vitamin D data available. The SOLARIUM study randomised relapsing-remitting MS patients to placebo or 14,000 IU vitamin D3 for 48 weeks. Participants were categorised as either 'carriers' or 'non-carriers' of the risk allele for 4 SNPs: two related to D binding protein (DBP) and associated with lower 25(OH)D levels (rs4588 and rs7041), and two related to vitamin D metabolism enzymes CYP27B1 and CYP24A1 and associated with a higher risk of MS (rs12368653; rs2248359, respectively). 25(OH)D levels were determined at baseline and after 48 weeks. RESULTS: The DBP-related SNPs showed no difference in 25(OH)D status at baseline, but carriers of the rs7041 risk allele showed lower 25(OH)D-levels compared to non-carriers after 48 weeks of supplementation (median 224.2 vs. 332.0 nmol/L, p = 0.013). For CYP related SNPs, neither showed a difference at baseline, but carriers of the rs12368653 risk allele showed higher 25(OH)D-levels compared to non-carriers after 48 weeks of supplementation (median 304.1 vs. 152.0 nmol/L, p = 0.014). DISCUSSION: Vitamin D-related SNPs affect the serological response to high-dose vitamin D supplementation. The effects on more common doses of vitamin D, as well as the clinical consequence of this altered response, need to be investigated further.

Correlation of different cellular assays to analyze T cell-related cytokine profiles in vitamin D3-supplemented patients with multiple sclerosis.

Different laboratory approaches have been exploited to analyze an effect of vitamin D3 supplements on T cell cytokine profiles in multiple sclerosis, with poorly reproducible results. We assessed the correlation between intra-cellular flowcytometry analysis of CD4 T cell-enriched CD3+CD8- lymphocytes after PMA/ionomycin stimulation directly ex-vivo or after 72 h pre-stimulation with anti-CD3, and cytokine levels excreted in culture supernatants. Pre-stimulation with anti-CD3 resulted in higher proportions of cells positive for IFN-γ, IL-17 A, IL-4, IL-10 and GM-CSF (all P < 0.001), but not TNF-α. Positive correlation between approaches was highly variable, but most eminent for IFN- γ and IL-4 (R = 0.608-0.612 and R = 0.677-0.777, resp., all P < 0.001). No effect of 16-weeks vitamin D3 supplements on any outcome was found except for a decreased TNF-α concentration in culture supernatants. Choice of immune-assay is, apparently, a relevant confounder for the reproducibility of individual studies.

Vitamin D3 supplementation and the IL-2/IL-2R pathway in multiple sclerosis: Attenuation of progressive disturbances?

Vitamin D3 upregulates IL-2 receptor alpha (IL2RA, CD25)-expression on CD4+ T cells in vitro. We investigated effects of 48-weeks vitamin D3 supplements on CD25-expression by CD4+ T cells of patients with multiple sclerosis (MS). There was no significant difference between the vitamin D3 (n=30) and placebo group (n=23) in IL2RA mRNA-expression by PBMC. Likewise, CD25 cell surface-expression by conventional or regulatory T cells (Treg) did not differ between groups, although Treg CD25-expression and circulating soluble-CD25 levels decreased significantly in the placebo but not vitamin D3-group. We speculate that vitamin D3 may promote the maintenance of CD25-related immune homeostasis in MS.

Stress-Axis Regulation by Vitamin D3 in Multiple Sclerosis.

INTRODUCTION: Multiple sclerosis (MS) has been associated with both a poor vitamin D status and hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis. Since nuclear receptor ligands may regulate each other, we explored the association of vitamin D3 supplements with circadian cortisol levels in a double-blind and placebo-controlled supplementation study. METHODS: Female patients with relapsing-remitting MS received vitamin D3 supplements (4,000 IU/day; n = 22) or placebo (n = 19) during 16 weeks. Salivary cortisol levels, repeatedly measured during the day, and serum 25(OH)D levels were assessed before (T0) and after (T1) this treatment period. RESULTS: Median 25(OH)D levels at T1 were 139.9 (interquartile range 123.5-161.2) and 74.5 nmol/L (58.6-88.1) in the vitamin D3 and placebo group, respectively (p < 0.001). Comparisons within and between groups showed no differences in area under the curve (AUC) and slope of the cortisol day curve. Although the AUC of the cortisol awakening response (CAR, sampling each 15 min the first hour after awakening) showed a reduction over time in the vitamin D3 group [39.16 nmol/L (27.41-42.07) at T0 to 33.37 nmol/L (26.75-38.08) at T1] compared to the placebo group [33.90 nmol/L (25.92-44.61) at T0 to 35.00 nmol/L (25.46-49.23) at T1; p = 0.044], there was no significant difference in AUC of CAR at T1 corrected for baseline AUC of CAR (p = 0.066). CONCLUSION: Suppression of HPA-axis activity by vitamin D3 supplements in non-depressed MS patients may be best reflected by CAR as primary outcome measure. Further studies should address this interaction and its potential implications for the disease course of MS. REGISTRATION: This study was registered on ClinicalTrials.gov (NCT02096133) and EudraCT (2014-000728-97).

Vitamin D3 supplementation in multiple sclerosis: Symptoms and biomarkers of depression.

Depressive symptoms are common in multiple sclerosis (MS), and both depression and MS have been associated with a poor vitamin D status. As cytokine-mediated inflammatory processes play a role in the pathogenesis of both disorders, we hypothesized that vitamin D3 supplementation reduces depressive symptoms in MS via its immunomodulatory properties. In this randomized pilot study relapsing remitting (RR) MS patients received either vitamin D3 supplementation (n=20; 14.000IU/day) or placebo (n=20) during 48weeks. Pre- and post-supplementation depression scores, measured using the Hospital Anxiety Depression Scale (HADS) depression subscale (HADS-D), showed a significant decrease within the vitamin D3 group (median HADS-D 4.0 to 3.0, p=0.02), a trend towards a decrease within the placebo group (median HADS-D 3.0 to 2.0, p=0.06), but no significantly different reductions between groups (p=0.78). Furthermore, no reductions in pro- and anti-inflammatory cytokine balances, secreted by stimulated leukocytes and CD8+ T cells, were found in the vitamin D3 compared to the placebo arm. Therefore, we found no evidence for a reduction of depressive symptoms or related biomarkers upon vitamin D3 supplementation in RRMS patients in this exploratory study. Whether vitamin D3 supplementation is of benefit in manifest depression in MS needs to be assessed by additional studies.

Network of nuclear receptor ligands in multiple sclerosis: Common pathways and interactions of sex-steroids, corticosteroids and vitamin D3-derived molecules.

Sex-steroids, corticosteroids and vitamin D3-derived molecules have all been subject to experimental studies and clinical trials in a plethora of autoimmune diseases. These molecules are all derived from cholesterol metabolites and are ligands for nuclear receptors. Ligation of these receptors results in direct regulation of multiple gene transcription involved in general homeostatic and adaptation networks, including the immune system. Indeed, the distinct ligands affect the function of both myeloid and lymphoid cells, eventually resulting in a less pro-inflammatory immune response which is considered beneficial in autoimmune diseases. Next to the immune system, also the central nervous system is prone to regulation by these nuclear receptor ligands. Understanding of the intricate interactions between sex-steroids, corticosteroids and vitamin D3 metabolites, on the one hand, and the immune and central nervous system, on the other hand, may reveal novel approaches to utilize these nuclear receptor ligands to full extent as putative treatments in multiple sclerosis, the prototypic immune-driven disease of the central nervous system.

A low vitamin D status at diagnosis is associated with an early conversion to secondary progressive multiple sclerosis.

Low circulating 25-hydroxyvitamin D (25(OH)D) levels have been associated with an increased risk of relapses in relapsing remitting multiple sclerosis (RRMS), but an association with disability progression is uncertain. Lower 25(OH)D levels are found in secondary progressive MS (SPMS) when compared to RRMS. We hypothesized that a poor vitamin D status in RRMS is associated with an increased risk of conversion to SPMS. In a retrospective longitudinal study we measured 25(OH)D levels at the start of a 3-year follow-up, and analyzed whether these levels predict the risk of RRMS to SPMS conversion. In 338 RRMS patients, vitamin D status did not predict the 3-year risk of conversion to SPMS (n=51; OR 0.970; p=0.65). However, in diagnostic blood samples of SPMS patients with a relatively short RRMS duration (n=19) 25(OH)D levels were significantly lower (38nmol/L; Q1-Q3: 24-50) than in diagnostic samples of matched RRMS patients with no progression to SPMS ((n=38; 55nmol/L; Q1-Q3: 40-70) (p<0.01). These data indicate an association between a low vitamin D status at the start of RRMS and the early conversion to SPMS. Therefore, time to SPMS conversion is of interest as clinical measure in (follow-up of) clinical vitamin D supplementation studies.

Immune regulatory effects of high dose vitamin D3 supplementation in a randomized controlled trial in relapsing remitting multiple sclerosis patients receiving IFNß; the SOLARIUM study.

Multiple sclerosis (MS) is characterized by a disturbed immune homeostasis and low serum vitamin D levels are associated with an increased disease activity. While vitamin D has been hypothesized to promote the maintenance of immune homeostasis, vitamin D supplementation could be of benefit to patients with MS. The SOLAR study investigated the effects of high dose vitamin D3 supplementation on clinical outcomes in a randomized controlled trial. Here we present the immune regulatory effects, investigated in the SOLARIUM sub-study. Thirty Dutch relapsing remitting (RR) MS patients treated with IFNß-1a received high dose vitamin D3 supplementation and 23 patients received placebo during a period of 48weeks. Lymphocytes were phenotypically characterized by flow cytometry and in vitro cytokine secretion was assessed in the presence or absence of 1,25(OH)2D3 using Luminex technology. Changes in immune regulatory parameters were determined within subjects as well as between treatment groups. The proportion of cells in the immune regulatory cell compartment (nTreg, iTreg and Breg) was not altered upon high dose vitamin D3 supplementation. Proportions of T helper subsets were not affected by vitamin D3, except for the proportion of IL4+ Th cells, which decreased in the placebo but not in the vitamin D3 group. T cell cytokine secretion increased, most pronounced for IL5 and latency activated protein of TGFß, in the placebo group but not in the vitamin D3 group. Lymphocytes remained equally reactive to in vitro 1,25(OH)2D3. In conclusion, high dose vitamin D3 supplementation did not result in a relative increase in lymphocytes with a regulatory phenotype. However, this study supports the hypothesis that vitamin D contributes to the maintenance of immune homeostasis by preventing further disturbance of the T cell compartment early in the disease course of MS.

Vitamin D Status Does Not Affect Disability Progression of Patients with Multiple Sclerosis over Three Year Follow-Up.

BACKGROUND AND OBJECTIVE: The risk of developing multiple sclerosis (MS) as well as MS disease activity is associated with vitamin D (25(OH)D) status. The relationship between the main functional disability hallmark of MS, disability progression, and 25(OH)D status is less well established though, especially not in MS patients with progressive disease. METHODS: This retrospective follow-up study included 554 MS patients with a serum baseline 25(OH)D level and Expanded Disability Status Scale (EDSS) with a minimum follow-up of three years. Logistic regressions were performed to assess the effect of baseline 25(OH)D status on relapse rate. Repeated measures linear regression analyses were performed to assess the effect on disability and disability progression. RESULTS: Baseline deseasonalized 25(OH)D status was associated with subsequent relapse risk (yes/no), but only in the younger MS patients (≤ 37.5 years; OR = 0.872, per 10 nmol/L 25(OH)D, p = 0.041). Baseline 25(OH)D status was not significantly associated with either disability or disability progression, irrespective of MS phenotype. CONCLUSION: Within the physiological range, 25(OH)D status is just significantly associated with the occurrence of relapses in younger MS patients, but is not associated with disability or disability progression over three years follow-up. Whether high dose supplementation to supra physiological 25(OH)D levels prevents disability progression in MS should become clear from long term follow-up of supplementation studies.

Vitamin D effects on B cell function in autoimmunity.

Vitamin D seems to be implicated in the pathophysiology of autoimmune disorders as a natural immune modulator. Beneficial effects of vitamin D have been associated with different cells of the immune system; however, thus far, B cells seem to be somewhat neglected. In this paper, we describe the possible direct effects of vitamin D on B cells, with a focus on antibody production and the more recently identified regulatory B (Breg ) cells. B cells upregulate the vitamin D receptor (VDR) upon activation. Furthermore, due to regulated expression of the metabolizing enzymes CYP27B1 and CYP24A1, B cells have the potential to control the local availability of active vitamin D. B cells, therefore, may participate in vitamin D-mediated immune homeostasis, including plasma cell generation. Whether or not other B cell subsets, such as Breg cells, are equally responsive to vitamin D remains to be established.

[White matter abnormalities following attempted suicide]. / Wittestofafwijkingen na een zelfmoordpoging.

BACKGROUND: Delayed post-hypoxic leukoencephalopathy (DPHL) is a demyelinating disorder characterized by neuropsychiatric symptoms occurring a few days to some weeks following cerebral hypoxia. CASE DESCRIPTION: A 50-year old female patient showed rapidly progressive cognitive deterioration with apathy, mutism and regressive behaviour a few weeks after a suicide attempt with carbon monoxide (CO). This eventually leads to a state of akinetic mutism. Magnetic resonance imaging (MRI) of the brain showed diffuse white matter abnormalities. These MRI findings combined with CO intoxication and the clinical picture were highly suggestive for DPHL. CONCLUSION: This case emphasizes that a neurological cause should be considered if rapidly progressive neuropsychiatric symptoms occur, and that after suspected auto intoxication it is important to take possible hypoxia and its after-effects into consideration. Recognition of DPHL is important so that unnecessary invasive diagnostics and treatment can be avoided. Considering the favorable natural course of DPHL appropriate measures should be taken in order to provide supportive care and rehabilitation.