Multiple Sclerosis Treatment and Research -Vitamin D presentations at European Congress Oct 2015

Vitamin D section ECTRIMS 2015
31st European Committee for the Treatment and Research in Multiple Sclerosis (ECTRIMS)
Vitamin D: 89 Abstracts, 8 Webcasts, 43 ePosters, 8 PPT Shared (of 3600 abstracts)
26 of the abstracts are new for 2015 - others are from previous conferences
Full text/video/slides apparently only available to participants
Next Conference Sept 2016: Multiple Sclerosis Conference had scores of Vitamin D presentations - Sept 2016

The following are about 1/3 of the Vitamin D abstracts
Note: you might find some of the actual PDF files by finding the presentation number (from the web) and substituing it in the following URL
I was not successful, but another person was.
http://www.abstractstosubmit.com/ectrims2015/pupload/documents/0_2154.pdf

Environmental factors,

Abstract Category: Invited / Oral LB / Poster LB

Pathogenic mechanisms underlying Multiple Sclerosis development have yet to be clearly identified, but considerable evidence indicates autoimmunity plays an important role in the etiology of the disease. It is generally accepted that autoimmune diseases like MS arise from complex interactions between genetic susceptibility and environmental factors. Although environmental factors unequivocally influencing MS development have yet to be established, accumulating evidence singles out several candidates, including

• sunlight-UV exposure /or Vitamin D deficiency,
• viral infections,
• hygiene,
• high sodium intake, and
• cigarette smoking.

Several investigations indicate 1-25 (OH)2 Vitamin D plays a critical role in shaping T cell response, inducing T cells with immunosuppressive properties. Likewise, helminth infections represent another potential environmental factor exerting immunomodulatory properties. Both epidemiological and experimental data provide evidence to support autoimmune down-regulation secondary to parasite infections in MS patients, through regulatory T and B cell action, with effects extending beyond simple response to an infectious agent. Furthermore, salt has been shown to modulate the differentiation of human and mouse Th17 cells and mice that were fed a high-sodium diet were described to develop more aggressive courses of experimental autoimmune encephalomyelitis. Moreover, different epidemiological studies have demonstrated that Epstein-Barr virus infection confers added risk of developing MS. Proposed mechanisms responsible for this association include: activation and expansion of self-reactive T and B cells, lower threshold for self-tolerance breakdown and improved autoreactive -B cell survival. Finally, epidemiological studies have demonstrated that smoking worsens multiple sclerosis prognosis. Evidence indicate that indoleamine 2,3-dioxygenase activity is reduced in MS patients who smoke, and both degree of expression and renin-angiotensin system activity levels are increased in MS patients who smoked. Both pathways contributed to a significant decrease in the number of CD4+CD25+FoxP3+ regulatory T cells in MS patients who smoked, inducing a pro-inflammatory response. Understanding environmental factors influencing propensity to MS will lead to new and more effective approaches to prevent and treat the disease.

Vitamin D and the development and evolution of permanent black holes among patients with clinically isolated syndrome

Abstract: P799

Objective: To assess the relationship between vitamin D 25(OH)D and irreversible brain tissue damage characterized by the occurrence of persistent T1- hypointensities (permanent black holes-PBHs) in patients with clinically isolated syndrome (CIS) who were followed for 5 years.

Methods: BENEFIT was a randomized trial comparing early versus delayed interferon beta-1b (IFNB-1b) treatment in patients with a first event suggestive of MS (CIS). Serum 25(OH)D concentrations were measured at baseline, 6, 12, and 24 months. 433 of the 468 patients had at least one 25(OH)D measurement and had lesion follow-up for at least 1 year. We calculated a season-adjusted 25(OH)D and estimated the association between the time-dependent cumulative average of 25(OH)D and the number of new PBHs after 6 months. We modeled lesion counts using negative binomial models and logistic regression models to assess the proportion of lesions evolving into PBHs accounting for intra-patient correlation using generalized estimating equations. We also assessed the association between 25(OH)D and number of lesions of a specific type at initial presentation (nodular Gd-enhancement, ring-like Gd-enhancement, T1-isointense T2 lesions or T1-hypointense lesions). Analyses were adjusted for age, sex, treatment, baseline T2 lesions and CIS onset type.

Results: A total of 3789 new lesions developed over the 5 year follow-up period with 383 developing into PBHs. Average 25(OH)D levels were significantly inversely correlated with the number of PBHs from the 6-month to five-year MRI; patients with serum 25(OH)D levels ≥50 nmol/L experienced a 55% lower absolute rate of PBHs (95% CI: 0.29 to 0.71; P=0.006) than those < 50 nmol/L. We also observed a marginal association between serum 25(OH)D and the proportion of T2 lesions evolving into PBHs (0.71; 95% CI: 0.50 to 1.01; P=0.056, comparing patients with ≥50 nmol/L vs those < 50 nmol/L). We found generally similar associations between patients with serum 25(OH)D levels ≥50 nmol/L and the number of each of the 4 lesion types at presentation (for any T1-hypointense lesions: 0.54; 95% CI:0.54-0.91; for T1-isointense lesions: 0.63; 95% CI:0.47-0.85; for nodular Gd+ lesions: 0.67; 95% CI:0.47-0.98; for ring-like-Gd+ lesions: 0.79; 95% CI: 0.47-1.35).

Conclusions: Our results that higher levels of 25(OH)D were associated with lesser accumulation of irreversible brain tissue damage support the importance of adequate vitamin D status in delaying MS progression.
 Download the PDF from Vitamin D Life

Vitamin D and disability in multiple sclerosis patients

Type: e-Poster

Background: Multiple sclerosis (MS) is associated with increasing disability in young adults. Vitamin D plays protective role in bone health and may influence on physical activity.

Goal: The goal of this paper is to investigate the association between serum vitamin D levels, bone health, quality of life (QoL) and the disability status in MS patients.

Methods: 82 patients with relapsing-remitting MS (with or without immunomodulatory treatment) were included in the study. Baseline characteristics, expanded disability status scale (EDSS) and QoL according to EQ-5D scale were examined. Serum vitamin D (VitD) and parathormon (PTH) levels were assessed. T-Score and Z-score parameters were obtained in the dual-energy X-ray absorptiometry (DXA).

Patients were divided into 3 groups:

• VitD deficiency (VitDd for serum level < 12ng/ml),
• VitD insufficiency (VitDi for levels 12-30 ng/ml) and
• normal VitD levels (VitDn for serum levels >30 ng/ml).

PTH, densitometry parameters, EDSS and QoL were compared between the groups.

Results: VitDd, VitDi and VitDn were found in 30, 45 and 7 participants respectively.

There were differences in PTH levels between VitDd, VitDi and VitDn groups: 40,5 pg/ml, 32,9 pg/ml and 26,9 pg/ml (p=0,028), respectively. The differences were also found in complaints about impaired mobility according to EQ-5D scale between VitDd, VitDi and VitDn groups:

• 73,3%,
• 42,2% and
• 28,5% (p=0,013), respectively.

There were no differences between VitD groups in EDSS (p=0,84), T-score (p=0,28) and Z-score (p=0,26).

Conclusions: Although lower Vitamin D concentrations were associated with higher PTH levels and more often complaints about impaired mobility, no differences in densitometry parameters or EDSS scale were observed.

Association of genetic variants to multiple sclerosis by performing whole exome sequencing in a high prevalence family

Type: Poster

Our research aims to shed light on the role of genetic component in Multiple Sclerosis etiology by identifying rare or new gene variants, by whole exome sequencing (WES) in a high prevalence family (HPF).

Infact, the high prevalence of the disease in the family, seems to suggest that the genetic component plays a major role in MS onset. We started selecting from this HPF three patients (a father and two sons) and then we performed the WES. Reads obtained were aligned to reference genome hg19 by BWA aligner, then, GATK tool kit has been used for quality score recalibration near indels regions and to perform variant calling. The study of raw data, executed by Ingenuity Variant Analysis permitted to select those shared between donors and then to filter rare variants with a global Minor Allele Frequency< 5% and those predicted deleterious by SIFT and PolyPhen2. We found 1483 variants on 856 genes. Then we selected all the variants that potentially result as alteration of gene function and probably induce a phenotypic manifestation: homozygous variants, compound heterozygous variants, hemizygous variants and haploinsufficient variants. 832 variants on 288 genes were been found. Starting from this list of genes, we executed further analysis. We firstly focused on variants that may alter pathways or physiologic mechanisms supposed to be related to MS. We found 331 deleterious variants laid on 102 genes involved in the regulation of the immune response and it results that 29 pathways, regulating immune processes (p-value ≤ 0.05, Fisher's exact test), could be potentially altered.

Moreover, we identified variants affecting genes that interact with environmental risk factors. By comparing the starting list of 288 genes with most recent Virus Mentha database, we identified 11 variants affecting 10 genes that interact with EBV. Then, by an analysis performed through String, we found one gene affected by deleterious and homozygous variants interacting with VDR that potentially could alter Vitamin D pathway.

Finally, by comparing a list of damaging and likely damaging variants shared by patients we found variants on 4 genes already related to MS by former GWAS studies: 2 genes, affected respectively by 3 and 2 deleterious variants and 2 genes, affected by non-deleterious variants.

By this analysis, we found altered mechanisms potentially involved in MS, contributing to identification of new targets that could be studied to enlighten MS etiology.

Polyunsaturated fatty acids and the risk of multiple sclerosis

Type: Oral

Background: While polyunsaturated fatty acids (PUFA) have anti-inflammatory properties that could be relevant for multiple sclerosis (MS), previous studies on PUFA and MS risk have yielded inconsistent results.

Objective: To investigate the association between dietary PUFA intake and the risk of MS.

Methods: We prospectively followed 81,757 women from Nurses´ Health Study (1984-2004) and 95,452 women from Nurses´ Health Study II (1991-2009) who were free of MS at baseline. Diet was assessed using a validated food frequency questionnaire every 4 years. Cox proportional hazard regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) in each cohort, adjusting for age, latitude of residence at age 15, ethnicity, body mass index at age 18, supplemental vitamin D intake, cigarette smoking and total energy intake. The effect estimates were then pooled by the inverse of their variance.

Results: During follow-up, we documented a total of 479 incident cases of MS. Higher intake of total PUFA at baseline was associated with a significant lower risk of MS (HR top vs. bottom quintile: 0.66, 95% CI: 0.49-0.90, p trend=0.01). Among the specific types of PUFA, higher intakes of linolenic acid

(HR top vs. bottom quintile: 0.64, 95% CI: 0.47-0.87, p trend=0.002), and linoleic acid (HR top vs. bottom quintile: 0.76, 95% CI: 0.56-1.03, p trend=0.04) were inversely associated with MS risk. Other types of PUFA, including EPA and DHA, were not associated with the risk of the disease. Results from analyses using the cumulative average intake of PUFA during follow up were consistent with the results from the baseline analyses.

Conclusions: Higher intakes of linolenic acid and linoleic acid are inversely associated with MS risk. Confirmation in other studies is needed, but intake of these PUFA may be another modifiable risk factor for MS.

Vitamin D and cognitive performance

8 slides

Background: Recent research data suggest an association between vitamin D level and cognitive performance. A reduced level of vitamin D in the blood has been proposed as well as a risk factor for developing multiple sclerosis (MS).

Objectives: The aim of this study is to examine the relationship between serum levels of vitamin D and cognitive performance in patients with a MS diagnosis.

Method: A total of 91 adults with MS (61 patients with ≥20ng/ml, 30 less than 20ng/ml) were selected from an initial sample (n=214). In order to reduce the number of confounding variables exclusion criteria were: being older than 65 years old, progressive MS phenotypes, clinically isolated syndrome, scores above 10 on any of the subscales of the Hospital Anxiety Depression Scale (HADS), relevant medical condition other than MS, including drug/alcohol abuse.

Rao´s Brief Repeatable Battery, Trail Making Test parts A and B (TMT-A/B), Tower of London (TOL) and some additional verbal fluency tests were administered. Demographic data (sex, age, and years of education), disease duration, MS subtype, Expanded Disability Status Scale (EDSS) were also registered.

Results: Group with vitamin D ≥20ng/ml: 43 women (70.49%) and 18 men, mean age 43,95 years old (Sd: 10.06), mean disease duration:10.43 years (Sd: 6.96), mean EDSS: 1.14 (Sd: 1.03).

Lower vitamin D concentration (< 20ng/ml) group: 21 women (70%) and 9 men, mean age: 43.70 years old (Sd: 11.70), disease duration: 11.60 years (Sd: 8.40), EDSS: 2.30 (Sd: 1.98).

Age, sex and years of education and disease duration did not differ significantly between groups. Lower vitamin D concentrations were associated with a higher EDSS score (p=.014).

Not significant differences have been found regarding cognitive performance (5% significance level).

Conclusion: The association between vitamin D levels and cognitive performance remains unclear. Some studies suggest 10ng/ml as a better threshold than 20ng/ml associated with cognition.

Does air pollution influence risk of relapse in multiple sclerosis?

18 slides, Type: e-Poster

Background: Only few studies have considered possible role of air pollutants in pathogenesis of multiple sclerosis (MS) or of MS relapses. Yet air pollutants have been suggested as an important factor determining solar UVB radiation reaching earth and they could also contribute to low circulating vitamin D levels of inhabitants of polluted areas. Therefore, air pollutants may be involved in MS relapses occurrence through an intermediary effect on vitamin D. A retrospective study in south-western Finland, including 1205 relapses which occurred among 406 patients in the period 1985-1999, has shown a strong relationship between MS relapses and peak amounts in PM10 and SO2+NO2+NO levels with odds ratio greater than 3. Moreover, an ecologic study in Georgia, USA, has showed that MS prevalence at county level was linked to PM10 levels. These two studies led to significant associations, but they suffered from several methodological limitations that may question the results… That's the reason why we decided to assess the potential association between air pollutants and MS relapses in Strasbourg city in North Eastern France.

Objectives: Assess the association between air pollution and relapse occurrence in MS patients.

Methods: All the relapses have been assessed by a neurologist. Regarding air pollution data, NO2, O3, PM10, CO and benzene were available at a small geographical area and on hour basis. A case crossover design has been selected, i.e. the analysis was performed by comparing among each patient the air pollutants levels observed in period where relapses occurred with period without relapses. Lags, i.e. time between pollutants levels measurements and occurrence of a potential relapse, from 1 to 30 days, have been tested using conditional logistic regression models.

Results: A total of 251 patients accounting for 1136 relapses (range 1-30 per patient) in the period 2000-2009 have been included. Mean age at MS clinical onset was 29.8±9.9 years and sex ratio F:M was 2.9. The annualized relapse rate during the study period was 0.78 relapse per patient-year. As expected, strong correlations have been observed between air pollutants. Preliminary analysis showed that relapse occurrence may be influenced by PM10, with a significant but small effect (odds ratio 1.01).

Seasonal variation of acute exacerbations in neuromyelitis optica spectrum disorder

Type: Poster

Background: Environmental factors related to season, including ultraviolet radiation, vitamin D, and infective agents, interplay with genetic factors in the development of autoimmune diseases. In multiple sclerosis, several studies have described the seasonality of relapses, which occur more frequently in spring and summer. However, in neuromyelitis optica spectrum disorders (NMOSD), only a few small cohort studies reported no seasonal variation of clinical exacerbations.

Objectives: We aimed to determine whether seasonal variation of acute NMOSD exacerbation is observed in a large Korean cohort.

Methods: We included 236 consecutive NMOSD patients seropositive for anti-aquaporin-4 antibody from four referral hospitals in Korea from May 2005 to April 2015. Medical records of demographic and clinical findings, including age, gender, dates of each attack and last follow-up were retrospectively reviewed and only clinically documented exacerbations were considered a definite attack in order to avoid ascertainment and recall bias. Attacks were divided into myelitis, optic neuritis, and brain manifestations with attacks presenting more than one kind of symptom being counted repetitively. Seasons were defined as spring (March-May), summer (June-August), autumn (September-November) and winter (December-February). We performed chi-squre tests with a significance level of P < 0.05 to compare the observed numbers of attacks.

Results: A total of 1306 attacks from 236 patients (88% female) were identified for a mean follow-up duration of 125 months. The onset of the disease showed no seasonal pattern of variation. Acute exacerbations were most common in winter (355), followed by summer (336), spring (321), and autumn (294) although the differences were not statistically significant. The frequency of exacerbations was highest in January (147) and lowest in October (88). Among the attack symptoms, myelitis was most common (632), followed by optic neuritis (418) and brain symptoms (221). However, their occurrence did not vary markedly with month or season.

Conclusions: In Korean cohort of NMOSD, acute attacks appear to occur more commonly in winter, particularly in January, although significant seasonality of attacks was not seen. Larger, nationwide data collection is undergoing.

Sunshine, sea, and season of birth: MS incidence in Wales

11 slides, Type: Poster

Background: Exposure to sunlight, proximity to coastal areas, and season of birth have been reported as factors associated with multiple sclerosis (MS). Sunlight exposure in gestation and development is necessary for vitamin D synthesis, and living near the sea is a proxy for access to vitamin D in seafood. This study examines whether geographical factors are related to MS incidence in Wales.

Method: Our main data source was a central MS diagnosis registry of hospital admissions between 2002 and 2013. Geographical information was available, including the lower super output area (LSOA) level of residence. For the 1,909 LSOAs, coastal status, population, longitude/latitude, and average hours of sunlight were obtained. MS incidence was calculated separately for males and females. Poisson regression was used to model MS counts with the geographical characteristics as predictors. Month of birth was compared with the distribution of births in Wales.

Results: There were 3,769 new MS cases between 2002 and 2013, with an incidence of 8.4 (95% CI: 7.95-8.85) among males and 13.32 (12.79-13.85) among females. The Poisson model showed that MS incidence varied directly with latitude, with more northern areas having more cases, and inversely with sunlight, fewer cases with more sunlight hours. There was a significant interaction between coastal status and longitude indicating that proximity to the coast was important only for more eastern areas. MS patients were more likely to be born in the months of April, May and September as compared to the Welsh population.

Discussion: The study supports previous findings regarding the beneficial effect of sunlight in relation to MS. Higher cases of MS in more northern latitudes may suggest other factors in addition to sunlight such as ethnicity. The Coast × Longitude interaction suggests that coastal status is critical in the east, where occupations are more likely to be indoors.

Effect of vitamin D replacement on cognition in multiple sclerosis (MS) patients

Type: Oral

MS is a disease of the central nervous system with genetic and environmental risk factors including low serum 25(OH)D. Recent studies correlated low 25(OH)D levels with cognitive dysfunction in adults. In this study we evaluated the cognitive function after vitamin D supplementation in MS patients with low 25(OH)D (< 25µg/ml) compared to those with normal levels (>35 µg/ml). Demographic and health behavior information were collected on 113 adult MS patients with relapsing-remitting disease stable on interferon-beta. Depression and anxiety were assessed using the Arabic-Hopkins Symptoms Checklist. Cognitive performance was measured, at baseline and 3 months after vitamin D supplementation (10,000 IU daily for 3 months or 50,000 IU weekly for 3 months), using the Arabic-Montreal Cognitive Assessment (MoCA), Stroop Test, Symbol Digit Modalities Test (SDMT) and Brief Visual Memory Test delayed recall (BVMT- DR). Of the recruited subjects, 89 were found eligible with low (< 25, n=41) or normal (>35, n=48) 25(OH)D.
After 3 months, anxiety scores decreased in those with low baseline 25(OH)D and there was an improvement on BVMT immediate (10 and 30 sec), delayed recall (20 min) and MoCA. Interestingly, Stroop and BVMT (10 and 20 sec) scores improved in both groups. At baseline, the low 25(OH)D group scored less on all cognitive tests except Stroop; this difference was significant for SDMT and BVMT-DR. Serum 25(OH)D level correlated positively and significantly with BVMT-DR. Exercise was positively associated with cognitive performance in all tests except the Stroop [BVMT,10 sec r=0.61, 20 sec r=0.64, 30 sec, r=0.70, DR, r=0.54, SDMT, r=0.56, MoCA=0.46; p < 0.05], and correlated strongly with cognitive performance in those with low 25(OH)D [BVMT, 10 sec r=0.94, 20 sec r=0.8, 30 sec, r=0.89, DR, r=0.81, SDMT, r=0.74, MoCA r=0.61; p < 0.01]. The normal baseline 25(OH)D group had lower anxiety scores at baseline. Alcohol consumption correlated positively with SDMT, BVMT and MoCA in the group with low baseline 25(OH)D and with BVMT and SDMT in the normal group. Cognitive performance and anxiety in MS seem to be affected by low 25(OH)D level and improve after vitamin D replacement. There is a positive correlation between exercise and cognitive performance in all subjects, but it is stronger in the group with low 25(OH)D suggesting compensatory exercise role in this group. Of interest is the positive alcohol effect on cognitive performance that needs further exploration.

Vitamin D deficiency and vitamin D receptor polymorphisms as the enviromental risk factors for paediatric multiple sclerosis in Poland

8 slides. Type: e-Poster

Introduction: Recently, growing interest in paediatric multiple sclerosis (MS) is observed. Approximately 3 to 10% of MS patients develop their symptoms before the age of 16. In Poland, the incidence of MS is high and the number of MS individuals and children with MS is estimated as 40,000-60,000 and 1,200-5,000, respectively.

Vitamin D has recently received increased attention for possible role in MS pathogenesis. The polymorphisms of Vitamin D Receptor (VDR), especially FokI and BsmI, are considered as modulators of vitamin D metabolism and its immunomodulating functions.

Aim: To establish the impact of vitamin D supply as well as FokI and BsmI polymorphisms of VDR on the development of CNS demyelinating disorders in Polish children.

Methods: 57 children with definite MS/clinically isolated syndrome (CIS) and 85 healthy controls were included in the study. Serum 25(OH) D concentration and molecular genetic analysis of the FokI and BsmI polymorphisms were examined in all children. The results were correlated with the incidence of MS/CIS.

Findings: In all children, both healthy and suffering from MS/CIS, significant deficiency in vitamin D was found. In children with MS/CIS the deficiency tended to be more severe than in control group. There was no statistical correlation of the level of vitamin D with the incidence of the disease in our patients. The distribution of Fokl and Bsml polymorphisms did not differ significantly between the groups. There were no significant differences between the vitamin D levels in the patients with individual polymorphisms. The polymorphisms did not correlate with demyelination of CNS in our study.

Conclusions: Neither vitamin D level, nor the polymorphisms of VDR exert important influence on the development of demyelinating disorder in vitamin D-deficient population. It cannot be excluded, that vitamin D deficiency is an important population risk factor for MS in Poland. There is an urgent need to improve the vitamin D supply in Polish children.

Vitamin D, race/ethnicity and the risk of multiple sclerosis

7 slides, Type: Poster

Importance: Hypovitaminosis D has been associated with an increased risk of multiple sclerosis (MS) in whites. Whether this is true in darker skinned individuals (who have lower levels of vitamin D) is unknown.

Objective: To determine whether hypovitaminosis D is a risk factor for MS in blacks and Hispanics.

Design: Incident case-control

Setting: Membership of Kaiser Permanente Southern California (KPSC). Data were collected from a structured in-person interview, blood draw and the complete electronic health record.

Participants: Cases with newly diagnosed MS or its precursor, clinically isolated syndrome (CIS), were identified through the multiethnic KPSC Demyelinating Disease Cohort between 2011 and 2014. Controls were matched on age, sex, race/ethnicity and zip code.

Exposure: Deseasonalized serum 25-hydroxyvitamin D levels were modeled as a continuous variable, by quintiles and pre-specified thresholds.

Main outcome measure: MS/CIS analyzed using conditional logistic regression within each racial/ethnic group.

Results: We recruited 457 incident cases of MS/CIS and 472 controls of which 108 cases and 116 controls were black; 146 and 151 Hispanic and 203 and 205 white, non-Hispanic, respectively. 25-hydroxyvitamin D levels were highest in whites followed by Hispanics and blacks (median 70, 55 and 47nmol/L respectively). Higher serum 25-hydroxyvitamin D levels were associated with a lower risk of MS/CIS in whites particularly in those with levels greater than 75nmol/L (OR 0.30, 95% CI 0.17-0.56; p< 0.001 adjusted for obesity, smoking, mononucleosis and family history of MS; reference category < 50nmol/L). No association was found in blacks or Hispanics regardless of how 25-hydroxyvitamin D was modeled.

Conclusions and relevance: 25-hydroxyvitamin D levels appear to be associated with the risk of MS only in whites. A non-causal association cannot be excluded as vitamin D is a better surrogate measure of sunlight exposure in light than dark-skinned individuals. Another possible explanation is complex interactions between vitamin D and genotype as several genes that increase vitamin D bioavailability are more common in blacks. Our findings indicate that vitamin D supplementation to prevent MS is premature. They also underscore the importance of including multiple racial/ethnic groups in studies of vitamin D.

Vitamin D status does not influence disability progression of multiple sclerosis patients over three years follow-up

10 slides, Type: Poster

Introduction: 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 progressive MS patients.

Methods: This retrospective 3-year follow-up study included 554 MS patients with a baseline 25(OH)D serum 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 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 10nmol/L 25(OH)D, p=0.041). Baseline 25(OH)D was not significantly associated with either disability or disability progression, irrespective of MS phenotype.

Discussion and conclusion: Within the physiological range, 25(OH)D status appears to affect the occurrence of relapses in younger MS patients, but does not significantly diminish disability or disability progression. 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 enhances interferon-beta-1b response in multiple sclerosis

Type: Poster

Objective: Determine molecular effect of Vitamin D3 on interferon-beta response in mononuclear cells.

Background: Oral vitamin D has additive effects on IFN-b therapy in reducing MRI lesions and clinical activity in MS. The mechanism is unknown, and may be confounded by IFN elevation of serum vitamin D levels, lifestyle, and the subnormal endogenous IFN signaling present in therapy-naïve MS patients.

Design/methods: MNC from therapy-naïve patients with stable RRMS (N=36), active RRMS (10), progressive MS (21), plus MS patients receiving IFN-b (15) or glatiramer treatment (11), plus other neurologic diseases (OND) (23) and healthy controls (19) were incubated in vitro with 160 U/ml IFN-beta-1b for 30min to 48h ± 100-200 nM Vit D3 (calcitriol; 12-60h pre-incubation). IFN-responsive transcription factors, p-Y-STAT1, p-S-STAT1, and also MxA protein, were measured with flow cytometry and western blots.

Results: Vitamin D enhanced IFN-b induction of p-Y-STAT1 and MxA in MNC from therapy-naïve stable RRMS (p=0.024; 2-fold change), in IFN-b-treated stable RRMS (p=0.026; 1.6-fold change), and in healthy controls (p=0.029; 2-fold change) and in OND (p=0.015; 1.5-fold change). There was less effect of Vit D on IFN responses during exacerbations and progression. Vit D had no effect on IFN response in glatiramer-treated patients. IFN response increased more in T cells than in monocytes.

Conclusions: Vitamin D enhances IFN-beta activation of STAT1 by 100% in untreated patients and by 60% in IFN-treated patients. This increase in interferon signaling may provide a mechanism for benefits of vitamin D in preventing and ameliorating MS, before and during IFN-b therapy. Vitamin D is an inexpensive and safe way to amplify IFN-b's therapeutic benefit.

Sun exposure over the life-course and associations with multiple sclerosis

Type: Oral

Background: Sunlight exposure may influence multiple sclerosis (MS) risk and later health; however its measurement, timing and potential for sun avoidance behaviour have not been well-delineated. We examined sun exposure and MS over the life course.

Methods: In 2012/13, 151 MS incident cases and 235 matched controls from the Nurses' Health Study (NHS) and NHSII completed an environmental-related questionnaire (89% response). Cumulative ambient UV-B exposure pre-MS onset was based on place of residence (latitude, altitude, cloud cover), and expressed as tertiles over the life course (birth, ages 5-15, 16-20, 21-30, 31-40 years). Sun avoidance measures were assessed both pre- and agnostic of MS onset, and included time spent outdoors in winter or summer over the life course (ages 5-15, 16-20, every ten years thereafter), expressed as 'low, intermediate, high,' and sunscreen use. Relative risks (RR) and 95% confidence intervals (CI) were estimated via conditional logistic regression, which in addition to the matching factors, included adjustment for body mass index at age 18, ethnicity, smoking, and supplemental vitamin D intake.

Results: Most participants were Caucasian (377/386; 98%), with a mean MS onset of 39.7 years (SD:9.05, range 16-72). Those women who lived in areas of high (vs. low) ambient UV-B between ages 5-15 years had a 48% lower risk of MS (RR:0.52;95%CI:0.28-0.96). Similar, but non-significant trends were observed across all age groups (pre-MS onset; p>0.05). Neither time spent outdoors in summer nor winter was associated with MS risk (p>0.05). However, when living in areas of low ambient UV-B, with less time spent outdoors in summer at ages 5-15 years, a two-fold increased risk of MS was observed (RR:2.19;95%CI:1.12-4.30). Sun avoidance behavior was apparent in later life (and thus largely after MS onset); by age 50, cases were around 60% less likely to report high (vs low) outdoor exposure (winter RR:0.44;95%CI:0.22-0.90; summer RR:0.40;95%CI:0.21-0.76) relative to controls. Sunscreen use increased with age, but was similar for cases and controls across the life course.

Conclusions: Our results support the hypothesis that low ambient UV-B exposure is a risk factor for MS. Further, sun avoidance behaviour was evident among MS cases in later adulthood which could impact vitamin D status and health in those with MS.

Relationship of headache and vitamin D supplementation in MS patients

9 slides, Type: Poster

There is a growing body of evidence implicating vitamin D deficiency in multiple sclerosis (MS) and migraine headache (MH). Both of these conditions increase in prevalence at higher latitudes and some observational studies have directly linked low serum vitamin D (Vit D) levels to risk of developing MS. Headache is an important comorbidity in multiple sclerosis with higher prevalence of MH and tension type headache (TTH) found in MS populations compared to control groups. We compared prevalence of MH, TTH and use of Vit D supplementation using a survey tool in 203 sequential patients in a multiple sclerosis clinic from December 2009 to May 2010. We hypothesized that supplementation of vitamin D was associated with lower headache prevalence in MS patients.

Our data showed that the prevalence of headache in the Vit D group was lower than the No Vit D group (87.4% vs 93.2%), but this difference was not statistically significant. However, there was a much higher prevalence of headache overall in our patient population than in general population and compared to what has been previously reported in MS patients in the literature. We concluded that supplementation with vitamin D did not correlate with reduction in headache. Future studies would benefit from a larger collection of patient surveys and using serum vitamin D level as a more objective measure of vitamin D deficiency to further investigate the relationship between headache and MS.

Vitamin D levels in multiple sclerosis in correlation to age, sex, EDSS and dosage of substitution. First results of a prospective study

VIEW EPOSTER, 8 slides

Background: Vitamin D in multiple sclerosis (MS) is still controversial discussed regarding the impact of serum levels and possible therapeutic options.

Objectives: Therefore we initiated a non-interventional study in MS patients in our center to address these questions. We now present the design of the study and preliminary data of the included patients since October 2013.

Methods: Since October 2013 the 25-OH vitamin D3 levels (normal range between 20 and 70 µg/l), epidemiological data (sex, age) type of MS (rrMS, spMS, ppMS) EDSS as well as medication have been recorded. In every group of MS type at least 100 patients will be evaluated and observed over a period of two years. Patients with 25-OH vitamin D3 levels below 45 µg/l are allocated randomly to a substitution with either up to 10.000 IU or 20.000 IU weekly. Patients will be followed over at least one year with including 25-OH-vitamin D3 level control.

Results: We present the data of 161 consecutive MS patients, 65 men and 96 women, mean age 53 (20-77) years: The course of disease was relapsing-remitting in 43, secondary progressive in 98 and primary progressive in 17 patients, a CIS diagnosed in three. 113 patients (70 per cent) received disease modifying therapy. Mean EDSS was 5,7 (1,5 - 8). Mean 25-OH vitamin D3 level was low with 19,7 µg/l. Women tended to have higher mean vitamin D levels than men (20,11 vs. 18,7 µg/l). Lower 25-OH vitamin D3 levels were seen in younger patients (median 11,5 µg/l in the age group 30-39 years) than in patients 60-69 years (median 22,5 µg/l). Relatively small differences of mean vitamin D levels were seen in relation to EDSS groups: 17,3 µg/l in patients up to EDSS 3,5, 22,3 µg/l in EDSS 4.0 -5,5, 19,1 µg/l in EDSS 6.0-6,5 and 19,7 µg/l beyond EDSS 7.0. 40 patients substituted with vitamin D were seen again up to March 2014.
High dose substitution with 20.000 IE led to a higher increase of 25-OH vitamin D levels (mean 51,4 µg/l) than weekly doses below 10.000 IE (mean 27,5 µg/l).

Conclusions: 25-OH vitamin D3 levels were below normal (< 20 µg/l) in 103 of our 161 MS patients since yet. High dose substitution led to a higher increase of 25-OH-vitamin D levels than weekly doses below 10.000 IU. In the short run of up to 6 months therapy there were no effects detected on disability progression.

Vitamin D status as a predictor of multiple sclerosis outcome in children with acute demyelinating syndromes: a prospective cohort study

Background: Vitamin D may be a protective etiologic factor for multiple sclerosis (MS). Acute demyelinating syndromes (ADS) of the CNS represent the first clinical attack of MS for some and a monophasic illness for others.

Objectives: We sought to
(i) determine whether vitamin D status, as defined by serum 25-hydroxyvitamin D (25(OH)D) levels, at presentation with pediatric ADS and serially for up to 12 months post-ADS onset were associated with risk of MS and time to MS diagnosis and
(ii) assess factors influencing 25(OH)D at presentation.

Methods: Consecutively recruited participants (< 16 years old) were monitored prospectively from ADS onset at 23 sites participating in the Canadian National Paediatric Demyelinating Disease Network. We used Cox proportional hazards (PH) to determine risk of MS and time to MS diagnosis as a function of serum 25(OH)D levels at ADS onset and in the following year, accounting for potential confounders such as age, body mass index (BMI), sex, season, and HLA-DRB1*15 status. We used multivariable regression models to evaluate factors influencing 25(OH)D levels at ADS onset.

Results: Of 225 eligible participants, 47 children (20.9%) were diagnosed with MS a median of 105 days (IQR 94-222 days) from ADS onset. Higher 25(OH)D levels at ADS onset were associated with a lower risk of MS (adjusted HR/10 nmol/L increase: 0.86; 95% CI: 0.77-0.97), but 25(OH)D levels in serial samples post-ADS were not (HR/10 nmol/L increase: 1.00; 95% CI: 0.89-1.12). Among children with MS, higher 25(OH)D levels at enrollment were associated with a lower hazard of a second clinical attack (adjusted HR per 10 nmol/L increase: 0.82; 95% CI: 0.67-0.99); that is, a longer time to a second attack. In multivariable analyses, higher 25(OH)D levels at ADS onset were associated with summer season, younger age at ADS onset, and intake of supplements containing vitamin D.

Conclusions: Higher serum 25(OH)D at pediatric ADS onset but not during the year thereafter is associated with a lower likelihood of MS outcome and longer time to second attack, suggesting that vitamin D contributes to the inciting biology of MS. Vitamin D supplement intake is a modifiable factor associated with higher 25(OH)D levels, indicating that vitamin D supplementation represents a strategy to improve vitamin D status and possibly reduce risk of MS.

Melatonin contributes to the seasonality of multiple sclerosis relapses (mice)

Type: YSI Session ?

Background and goals: Vitamin D levels are known to affect MS development and course. A seasonal fluctuation on vitamin D levels is observed with a peak in spring-summer and a nadir in autumn-winter. Based on the reported anti-inflammatory effects of vitamin D, MS relapse occurrence is predicted to peak during autumn and winter. However several studies found that MS disease activity is lower during autumn and winter, suggesting that additional factors play a role in MS relapse seasonality. Melatonin production is stimulated by darkness and follows a seasonal pattern with higher levels during autumn and winter. We thus aimed to investigate the relationship between melatonin levels and MS disease activity and to characterize the molecular mechanisms involved.

Design/methods: 119 RRMS patients were recruited from MS clinic. Melatonin levels were determined in urine by ELISA. A Poisson regression model was used to assess melatonin levels and the number of clinical relapses. EAE was induced in C57/B6 mice and CNS-infiltrating cells were assessed by FACS. RAG-1 deficient mice were reconstituted with wild type, MTNR1A, REV-ERBα or NFIL3-deficient CD4+ T cells and immunized for EAE. For in vitro experiments, murine and human naïve T cells were differentiated in Th17 or Tr1, with or without the addition of melatonin.

Results: Seasonal changes in melatonin levels were negatively correlated with MS disease activity. Melatonin administration ameliorated EAE and decreased the number of CNS-infiltrating Th17 cells. Melatonin also interfered with the differentiation of Th17 cells in vitro. Th17 cell differentiation in the presence of melatonin led to an MTNR1A-dependent C/EBPα phosphorylation and the recruitment of C/EBPα to the nr1d1 promoter, with decreased nr1d1 expression and a consequent Nfil3-dependent inhibition of rorc expression. Moreover, melatonin boosted Tr1 cell development and suppressive activity in vitro via Erk1/2 and the transactivation of the il10 promoter by ROR-α. Finally, melatonin reduced RORC and IL17A expression by human CD4+ T cells activated under Th17 polarizing conditions. Concomitantly, melatonin increased IL10 expression and production by human CD4+ T cells activated under Tr1 polarizing conditions.

Conclusions: Melatonin effects on T cells contribute to seasonal variations in MS activity and melatonin-dependent signaling is a potential target for therapeutic intervention in MS.

Neuroprotective effects of calcitriol in autoimmune optic neuritis

Background: Optic neuritis can result in persistent visual impairment due to apoptosis of retinal ganglion cells (RGCs) and degeneration of optic nerve (ON) axons. Therefore, efficient protection of retinal ganglion cells is a major challenge.

Objectives: We assessed the effects of calcitrol, the hormonally active form of vitamin D3, in a rat model of optic neuritis, using myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmune encephalomyelitis (EAE).
Methods: RGCs were retrogradly labelled by stereotactical fluorogold injection in the superior colliculus. After MOG immunization, brown Norway rats were randomly assigned to be treated with either vehicle or calcitriol 1 mg/kg i.p.. On day 8 of clinical manifest EAE, animals were sacrificed and ON histopathology was assessed by Luxol-fast blue (LFB) staining, immunohistochemistry to detect ?-amyloid precursor protein (ß-APP), Bielschowsky's silver impregnation and hematoxylin-eosin (HE) staining. Furthermore RGC density was compared using fluorescence microscopy on flat mounted retinas.

Results: We did not detect significant differences in clinical disease severity, demyelination (LFB: vehicle 72.2% ± 9.582%; calcitriol 79.72% ± 7.334%; p< 0.5421), acute axonal damage (?-APP: vehicle 5.40 ± 1.516; calcitriol 3.939 ± 2.927 positive axons per mm2; p< 0.712), chronic axonal damage (Bielschowsky: vehicle 9.517% ± 1.472%; calcitriol 9.204% ± 1.625%; p< 0.8949), and infiltrating cells (HE: vehicle 2.391 ± 0.2154; calcitriol 2.667 ± 0.2462 cells per mm2; p< 0.4342) in the ON. However, there was a significant lower densitiy of RGCs in vehicle- versus calcitiriol-treated animals. (vehicle 651.6 ± 56.21; calcitriol 926 ± 87.72 cells per mm2; p< 0.0101).

Conclusions: Despite calcitriol did not show any anti-inflammatory effects, https://www.vitad.org/img/icons/book_open.png we found a significant influence on RGC survival. This suggests a direct neuroprotective property of this substance which could be of value in combination with anti-inflammatory drugs.

Does smoking influence MRI disease activity in multiple sclerosis?

7 slides

Background: Smoking is a risk factor for MS and previous studies have reported that smoking affect disease progression. So far, no study has explored the association between smoking and MRI disease activity.

Objectives: To study if smoking is associated with MRI-activity and relapse-rate in MS patients and examine if there is a dose-response correlation between smoking and MRI activity.

Methods: Cohort study of 87 patients based on a multicenter, double-blind, randomized, placebo-controlled trial of patients with RRMS according to the McDonald criteria, followed for 2 years (The OFAMS study). Serum levels of cotinine, a nicotine metabolite that is widely used as a biomarker for recent tobacco use, were analyzed using an immunoassay. Patients were divided into three groups according to mean cotinine level, -levels < 10 for non-smokers, 10-300 for active smokers and levels >300 for heavy smokers. We analyzed serum samples from patients at baseline and month 6, 12, 18 and 24. MRI assessments were made at baseline, months 6, 12 and 24 and included the count of T1 gadolinium enhancing (T1Gd+) lesions, T2 lesions and combined unique activity (CUA; the sum of T1Gd+ lesions and new or enlarging T2 lesions). Associations between cotinine level and MRI activity were assessed by a logistic regression model.

Results: 29 patients had cotinine levels < 10, 9 patients had cotinine levels between 10- 300 and 42 patients had cotinine levels >300. There was no significant association between cotinine levels and MRI activity. For patients with cotinine levels >300 the odds ratio for new T1Gd + lesions were 0.81
(95% CI 0.43-1.55; p=0.526), for new T2 lesions 0.86 (95% CI 0.42-1.75; p=0.672) and for CUA 0.92 (95% CI 0.46-1.84; p=0.816) compared to the non-smokers. Cotinine levels were not associated with clinical disease activity assessed by relapse-rate or EDSS score. The results were not influenced by interferon-beta treatment. Adjustments for gender, age, HLA-DRB1*15 status, body mass index, vitamin D-levels, vitamin A-levels or ENBA-1 IgG titers did not affect the results.

Conclusions: We did not find any association between smoking and MRI- or clinical disease activity. To our knowledge no other study has addressed this issue. Our results indicate that smoking does not directly influence MRI activity or relapse-rate and thus probably increases the risk for disease development and progression through other mechanisms.

Neonatal vitamin D status and risk of multiple sclerosis

9 slides

Background: Vitamin D status at birth may be associated with risk of adult onset multiple sclerosis but this link has not been studied directly.

Objectives: To assess the relation between 25-hydroxyvitamin D concentration at birth and risk of multiple sclerosis.

Methods: This was a population-based case-control study in Sweden including 459 incident cases of multiple sclerosis and 663 controls, randomly drawn from a national population registry and frequency matched on sex, age and residential area. Neonatal 25-hydroxyvitamin D concentrations were measured in dried blood spots from a nationwide biobank, using a highly sensitive liquid chromatography-tandem mass spectroscopy method. Using logistic regression, odds ratios for developing multiple sclerosis were compared between quintiles of neonatal 25-hydroxyvitamin D.

Results: There was no association between neonatal 25-hydroxyvitamin D quintile and risk of multiple sclerosis. The crude odds ratios for the quintiles, in increasing order, were 1.0 (reference), 1.0 (95% CI 0.68 - 1.43), 0.9 (0.65 - 1.37), 1.0 (0.67-1.42), and 1.0 (0.68-1.44). Adjusting for a number of potential confounding factors in early life (month of birth, latitude of birth, breastfeeding) and in adult life (serum 25-hydroxyvitamin D status, sun exposure, vitamin D intake from dairy products, fat fish consumption, smoking, body mass index at 20 years of age) as well as ancestry, multiple sclerosis heredity, and socioeconomic group, did not considerably affect the result.

Conclusions: By observing that vitamin D status at birth is not associated with risk of multiple sclerosis at a broad population level, this study provides no support for the widely discussed and hitherto only indirectly assessed hypothesis regarding the role of vitamin D in the etiology of MS.

Vitamin D in multiple sclerosis; clinical and immunological implications

Background: Vitamin D insufficiency is common among multiple sclerosis (MS) patients, and hypovitaminosis D has been associated with MS risk and disease activity. This finding has been supported by in vitro studies, which have demonstrated anti-inflammatory effects of vitamin D. Moreover synergistic effects of vitamin D and interferon-beta (IFN-?) have been suggested. However, the overall evidence for a beneficial effect of vitamin D in MS is inconclusive.

Objectives: To investigate how screening for vitamin D insufficiency and recommendation on vitamin D3 supplies affect 25(OH)D levels in MS patients and to examine the clinical and immunological implications of this action.

Methods: A cohort of 210 natalizumab-treated relapsing-remitting MS (RRMS) patients was enrolled in the study. In winter 2009/2010 (period 1) patients had a blood sample collected and the procedure was repeated the following winter (period 2). Patients with serum 25(OH)D < 50nmol/l in period 1 were recommended treatment with vitamin D3 (50 to 100µg per day). Information on disease duration, annualized relapse-rate (ARR) and status for neutralizing antibodies (NAbs) to IFN-? was obtained retrospectively from the Danish MS Treatment Register. To asses how changes in 25(OH)D affect the immune system, quantitative real-time PCR analysis of MX1, USP18, PD-1, PD-L1, IFIT1, IFIT2, IL10, IL10RA, FOXP3 and IDO1 was performed on whole blood samples from period 1 and 2 in patients with the highest change in vitamin D level between period 1 and 2, n=30.

Results: Mean 25(OH)D increased significantly from 63 nmol/l (95% CI 58.8-67.7) to 76 nmol/l (95% CI 71.8-80.7) from period 1 to period 2 (p=5.1x10-8). At the same time mean ARR decreased from 0.61 to 0.54. We found a trend between increasing levels of 25(OH)D and decreasing ARR (p=0.07). None of the ten genes examined were associated with changes in serum 25(OH)D levels.

Conclusions: Recommendation of vitamin D supplies to MS patients with 25(OH)D < 50nmol/l resulted in a significant increase in mean 25(OH)D but was not associated with a statistically significant decrease in ARR. Whether this relates to an insufficient increase in 25(OH)D or lack of efficacy of 25(OH)D supplementation remains to be established. Changes in 25(OH)D levels did not affect gene expression of genes previously associated with either endogenous IFN-response, vitamin D or both, which underlines the complexity of vitamin D research in MS.

Vitamin D is associated with degree of disability in patients with fully ambulatory relapsing-remitting multiple sclerosis

8 slides

Background: Vitamin D deficiency is a recognized risk factor for multiple sclerosis (MS) and is associated with increased disease activity. It has also been proposed that the lower the vitamin D levels the higher was the handicap.

Objectives: To refine the links between vitamin D insufficiency and disability in MS patients and investigate the potential prognostic role of vitamin D in MS.
Methods: We performed a retrospective cohort analysis of 181 patients: data included age, gender, age at MS onset, MS type, MS activity, Expanded Disability Status Scale (EDSS) and plasma vitamin-D levels.

Results: Vitamin D levels were significantly higher in relapsing-remitting MS than in progressive forms of MS in multivariate analyses adjusted for age, ethnicity, gender, disease duration and season (p = 0.0487). Overall, there was a negative correlation between vitamin D level and EDSS score (p = 0.0001, r = -0.33). In relapsing-remitting MS, vitamin D levels were only correlated with disability scores for EDSS< 4 (p = 0.0012). Patients with >20ng/mL of vitamin D were 2.78 times more likely to have an EDSS < 4 (p = 0.0011, 95% CI:1.49-5.00).

Conclusions: Data support previous works suggesting that vitamin D deficiency is associated with higher risk of disability in MS. Vitamin D levels also correlated with the degree of disability in fully ambulatory patients with relapsing-remitting MS. These additional results support the pertinence of future studies analysing the interest of an early vitamin D supplementation in MS patients to influence evolution of disability.

Genetic modification of 25(OH)D levels in MS

16 slides

Background: Genome wide association studies provide robust evidence that in the general population, single nucleotide polymorphisms located within genomic regions of the genes DHCR7, GC and CYP2R1 are associated with 25-hydroxyvitamin D (25[OH]D) serum levels. It is not known, whether these results are also applicable to MS patients.

Objectives: Using interferon beta-1b-treated RRMS and CIS subjects from the BEYOND and BENEFIT trials, we analyzed candidate SNPs to assess their impact on 25(OH)D levels. Also, a 25(OH)D genetic risk score was implemented for patient stratification.

Methods: 25(OH)D levels were measured at two to three time points during the trials using an enzyme-linked immunosorbent assay or a chemiluminescence-based immunoassay and seasonally adjusted to derive an averaged 25(OH)D levels for each patient. Eligible data were combined and resulted in 819 unique patients for whom data was available. 25(OH)D values were loge-transformed and modeled as a function of candidate SNPs using Gaussian linear models. The first two ancestry components were included as covariates to correct for population stratification (resulting ?GC~1.03), as were gender, age, treatment and maximum NAb titer under treatment within the period of time 25(OH)D was measured in.

Results: All assessed candidate SNPs were significantly associated with 25(OH)D levels with p ? 0.05. The most significant SNPs were located within the genomic region incorporating gene GC (coding for vitamin D binding protein) with p = 1.05*10-6 for SNP rs17467825. 25(OH)D levels were reduced by 10% comparing one to zero risk allele copies. In the genomic region of gene DHCR7, rs4945008 was the most significantly associated SNP with p=6.1*10-4 and a reduction of 25(OH)D by 7%. With respect to the genomic risk region incorporating gene CYP2R1, the most significant SNP was rs1993116 with p=4.7*10-3 and a reduction of 25(OH)D levels by 6%. The cumulative count of risk alleles per patient was used to derive a 25(OH)D risk score and indicate the odds ratio of being 25(OH)D deficient. For example, the odds ratio for a risk score of 4 relative to risk score 0 is OR=5.29 (p=1.05*10-5).

Conclusions: All candidate SNPs were significantly associated with 25(OH)D levels at the nominal level. The genetic background and derived 25(OH)D risk score of patients and their first degree relatives could help to assess individual risk of 25(OH)D deficiency.

Dose-response and safety of high dose vitamin D supplementation: subgroup analysis of an exploratory randomized double blind placebo controlled trial

11 slides

Background: Increasing evidence links vitamin D deficiency to both susceptibility to, and severity of, multiple sclerosis. We report the dose- response results in a sub-group analysis of a double blind randomized placebo-controlled clinical trial examining the immunological effects of two doses of vitamin D (5000 IU or 10,000 IU daily) compared to placebo over 24 weeks in both healthy control participants and patients presenting with CIS.

Objectives: To assess the dose response, safety and tolerability of high dose vitamin D in healthy control participants over 24 weeks.
Methods: Healthy control participants, aged 25 -40 years, with a 2:1 female to male ratio and no contraindications to high dose vitamin D supplementation were randomized to receive either, placebo, 5000 IU or 10000 IU of vitamin D for a 24 week period in a 1:1:1 fashion. All participants had renal function and serum calcium measured at 4 weekly intervals and vitamin D and PTH at baseline and weeks 4, 8,16, 24. Adverse events were recorded at each visit.

Results: 38 healthy control participants (mean age 30 years (SD:4.5), 26 (68%) women) were recruited from November 2012 to June 2013. Mean baseline serum 25(OH)D was 52 (SD: 24.6) nmol/L and PTH 4.7 (1.7) pmol/L. Using a cut-off of serum 25(OH)D < 50 nmol/L as deficient and 50-72.5 nmol/L as insufficient, 22 (58%) participants were classed as deficient, 7 (18%) insufficient and 9 (24%) had an acceptable serum 25(OH)D level of >72.5nmol/L. After seasonal adjustment of vitamin D levels, 76% participants remained deficient or insufficient at baseline. The greatest mean changes in serum 25(OH)D levels were seen between baseline and 16 weeks of dosing: placebo group: -4.2 (SD: 22.5) nmol/L, 5000 IU group: +83.2 (27.2) nmol/L, 10,000 IU group +154.8 (66.2). Serum levels plateaued after 16 weeks. No increases in serum calcium, urea and creatinine levels were observed despite a maximum vitamin D level of 402 nmol/L achieved. No serious adverse events were reported in the course of this study.

Conclusions: This study adds to the growing evidence that: a) vitamin D deficiency remains highly prevalent in higher latitudes and b) high dose vitamin D supplementation is safe.
EU Clinical Trials Register: EudraCT: 2012-000635-68. ClinicalTrials.gov identifier: NCT01728922

In utero 25-hydroxyvitamin D and risk of multiple sclerosis among offspring in the Finnish Maternity Cohort

8 slides

Background: Adequate vitamin D nutrition is associated with a reduced risk of multiple sclerosis (MS), but whether vitamin D exposure in utero is associated with MS risk later in life is not clear.

Objectives: To measure 25-hydroxyvitamin D (25(OH)D) during pregnancy and determine whether these levels are associated with MS risk in the offspring later in life.
Methods: The Finnish Maternity Cohort (FMC) is comprised of over 800,000 women who have provided a blood sample from at least one pregnancy since 1983 for various pre-natal tests. Children of women in the FMC who were diagnosed with MS before December 31, 2009 were identified by searching the Finnish Hospital Discharge Register for diagnostic codes for MS, and the Social Insurance Register for reimbursements of MS disease modifying drugs. Cases were confirmed by review of the medical records when available and matched to up to 7 controls (n=400) on county of birth, date of sample collection (+/- 60 days), and date of mother's birth (+/- 6 months). 25(OH)D was measured using an enzyme linked immunosorbent assay. Conditional logistic regression was used to estimate the relative risk (RR) and 95% confidence intervals.

Results: We identified a total of 197 individuals diagnosed with MS. Serum 25(OH)D levels could be determined in 193 cases, which were included in the analyses. Among these, 138 were confirmed by review of the medical record, whereas 55 were confirmed based on prescription of MS drugs. Over 70% of serum samples were collected at or before 12 weeks of gestation and 99% of samples were collected prior to 28 weeks gestation. Mean 25(OH)D levels did not differ based on trimester of collection (36.6 nmol/L in first vs. 36.2nmol/L in second, p=0.80). In utero 25(OH)D level was associated with a 41% decreased risk of MS among the offspring (top median=56.8 nmol/L vs. bottom median=21.1 nmol/L quintile of 25(OH)D, RR=0.59 (95%CI: 0.30-1.19), p for trend=0.01). Further adjusting for sex of the offspring attenuated the association though the overall trend remained statistically significant (RR=0.69 (95%CI: 0.33-1.43), p for trend=0.04).

Conclusions: These results support that in utero (early life) exposure to adequate vitamin D levels may be protective against the development of MS later in life.

Risk of multiple sclerosis related to month of birth changes over time

7 slides

Background: Several studies have shown an increased risk of MS in spring births in the northern hemisphere, while the risk is increased in autumn births in the southern hemisphere. Decreased sun exposure in winter pregnancies, with a concomitant reduction in vitamin D levels, has been suggested as the environmental factor explaining this pattern.

Objectives: To determine if the risk of MS associated with season of birth is influenced by time period, gender and disease course.

Methods: Patients born between 1901 and 1990 registered in the Oslo City MS registry (n=1658) were compared to the total Norwegian population born in the same period (n=5105367, Statistics Norway). The patients and controls were subdivided in 30-year cohorts, and gender and disease course (relapsing-remitting, RRMS and primary progressive, PPMS) were analysed separately.

Results: An increased risk of MS for patients born in spring was only found for patients born in the period 1961-90 (p=0.00096) and only for patients with RRMS. There was no difference between females and males.

Conclusions: This study confirms an increased risk of MS in patients born in spring, but shows also that this risk is restricted to patients born after 1960 and to RRMS coinciding with the period of increasing prevalence of MS. This indicates that the risk of MS may be related to a change in environmental factors occurring after 1960 and thus further studies of changes in lifestyle habits triggering MS is warranted.

Significant effect of genetic and environmental factors on 25-hydroxyvitamin D levels in multiple sclerosis

11 slides

Background: Evidence is accumulating supporting a beneficial effect of vitamin D in the pathogenesis of multiple sclerosis (MS). MS patients in general have lower serum levels of 25- hydroxyvitamin D (25(OH)D) than do healthy controls. Two recent genome-wide association studies (GWAS) have shown a significant association between serum levels of 25(OH)D and six single nucleotide polymorphisms (SNPs) in NADSYN1, CYP2R1 and GC; all key genes in vitamin D metabolism. To date, no studies have compared the effect of these SNPs, environmental factors and disease severity on 25(OH)D levels in MS.

Aim: To examine the association between the six GWAS SNPs, specific environmental factors, the Multiple Sclerosis Severity Score (MSSS) and serum 25(OH)D levels in a cohort of Danish MS patients.

Methods: 1530 patients were eligible for the study. Blood samples for 25(OH)D-measurements and comprehensive questionnaires were collected between 2009 and 2012. Genotyping of rs7041, rs2282679, rs3829251, rs12785878, rs2060793 and rs10741657 SNPs was performed by TaqMan allelic discrimination (Life Technologies). Associations between 25(OH)D, SNPs, MSSS, sex, age, month of blood sampling, body mass index (BMI), smoking, fish intake and vitamin D supplements were analysed using univariate ANOVA. Variables with p-values< 0.05 were then analysed by multivariate ANOVA; only patients with complete data were included; n=670. Bonferroni correction was applied to the p-values in the final multivariate model, although explanatory variables with nominal significance (p<0.05) were retained in the model.

Results: As expected month of blood sampling was the strongest factor influencing 25(OH)D (p= 1.83e-24). SNPs in GC (rs7041, p=1.90e-04) and CYP2R1 (rs2060793, p=6.76e-03) were significantly associated with 25(OH)D. Vitamin D supplements (p= 4.54e-12) and sex (p=0.006) also had significant impacts on 25(OH)D levels. No association with MSSS was observed.

Conclusion: We found a significant association between 25(OH)D and SNPs in GC and CYP2R1, vitamin D supplements and sex in a cohort of Danish MS patients. As expected month of blood sampling was the most dominant factor influencing 25(OH)D. In Denmark food is not enriched with vitamin D, and sun exposure is minimal much of the year. As a result of this, vitamin D insufficiency is widespread. This study highlights the need to resolve the controversy surrounding vitamin D supplies to certain risk groups, such as MS patients, in the Danish population.

Vitamin D and pregnancy in multiple sclerosis patients

Background: Vitamin D is an environmental factor thought to be involved in the development and disease course of multiple sclerosis (MS). In relapsing-remitting MS patients, lower serum vitamin D concentrations are associated with a higher relapse risk. Also, in a number of conditions, low vitamin D has been associated with fatigue. Pregnant women are known to be at particular risk for vitamin D insufficiency. Therefore, our objectives were to investigate whether vitamin D status is associated with postpartum relapse, and with quality of life during pregnancy. Furthermore, we investigated if vitamin D levels differ between pregnant MS patients and healthy controls.

Methods: In the Rotterdam Study on Pregnancy in MS (Neuteboom et al.,2010), we prospectively followed 43 pregnant relapsing-remitting MS patients and 21 pregnant healthy controls. All patients were seen before pregnancy, in the late first trimester, in the early third trimester, 4-8 weeks postpartum and 9 months postpartum. The controls had the same visits except for the pre-pregnancy visit. At every visit, blood samples for serum 25-OH-vitamin D measurements were taken. Differences between patients and controls and associations of vitamin D with postpartum relapse, breastfeeding and several quality-of-life items (SF36, MSIS and Guy’s neurological disability scale) were calculated with alpha set at 0.01.

Results: We found significantly higher vitamin D levels in the third trimester and lower levels after delivery (p<0.001 corrected for seasonal influences). There were no significant differences in vitamin D levels between MS patients and healthy controls. We found no association of vitamin D concentrations with the occurrence of postpartum relapse, nor with breastfeeding (p=0.037). In controls, but not in MS patients, a higher vitamin D concentration was associated with better scores on several SF36 quality-of-life measures: general health (p<0.001), social functioning (p=0.004) and mental health (p=0.002), but not with fatigue (p>0.6).

Conclusions: Although low vitamin D has been associated with MS relapse risk, and vitamin D levels are lower after delivery, we did not find an association with postpartum relapse. In pregnant MS patients, vitamin D levels are similar to levels in healthy women and are not associated with quality of life. Therefore, there is no need for MS patients to take more vitamin D supplements during pregnancy than healthy pregnant women.

High-dose 1.25 dihydroxyvitamin D3 stimulates remyelination in the cuprizone model

9 slides

Background: Studies have suggested that vitamin D is not only involved in the pathogenesis of multiple sclerosis (MS), but may also influence disease activity. Several authors have recommended that supplementation of vitamin D should be included in the treatment of the disease. Some studies, however, suggests that high dose vitamin D supplements could have negative effects on the remyelination. By using the cuprizone model for de- and remyelination, we aimed to determine the effects of high dose vitamin D3 on the repair process.

Methods: 48 five week old female C57B1/6 mice were used for this experiment. To induce demyelination 0.2 % cuprizone was added to milled mouse chow. Cuprizone exposure was discontinued after seven weeks. The mice received intra-abdominal injections of 1.25-dihydroxy-vitamin D3 or placebo injections twice a week, from week six and throughout week nine. The animals were sacrificed at four time points, week 6, 7, 8 and 10. The brains were removed and post-fixed in 4% paraformaldehyde. Sections were then stained for myelin (Luxol Fast Blue), oligodendrocytes (NOGO-A), astrocytes (GFAP), microglia (MAC3), T-lymphocytes (CD3) and axonal damage (APP).

Results: After 7 weeks of demyelination, the mice given vitamin D3 had more myelin loss than mice given placebo injections. The remaining myelin in the mice given placebo had, however, an unstructured pattern, typical for damaged myelin. After 3 weeks of remyelination the vitamin D group had more myelin than the placebo group (p=0.001). The vitamin D group had a higher number of mature oligodendrocytes throughout the experiment. Mice that received vitamin D had initially increased astrocytosis (p= 0.043) and microglia activation, compared to the placebo group. However, levels of astrocytosis and microglia activation dropped below the placebo group during the remyelination phase. There were no significant differences in the degree of T-lymphocyte infiltration or axonal damage at any time point.

Discussion and conclusion: High dose vitamin D3 appears to have positive effects on remyelination. It seems that vitamin D stimulates oligodendrocyte maturation and removal of damaged myelin debris, through a stimulating effect on astrocytes and microglia recruitment. In conclusion our results point to a possible new mechanism of action for vitamin D in demyelinating diseases and indicate that high dose vitamin D3 has an essential role in myelin repair.

Sodium intake is associated with increased disease activity in multiple sclerosis

Background: Salt (NaCl) has been recently reported to promote the differentiation of pathogenic T cells and worsen disease in an experimental model of Multiple Sclerosis. However, the relevance of these observations for Multiple Sclerosis and other human immune-mediated disorders is unknown. Here, we investigated the relationship between salt consumption and Multiple Sclerosis disease clinical and radiological activity.

Methods: Sodium intake was calculated in urine samples from a cohort of 70 relapsing-remitting multiple sclerosis patients followed for two years. During the follow-up, clinical and radiological assessment was performed every 3-6 months or in the occurrence of a relapse. The effect of sodium intake in MS disease activity was estimated by regression analysis.

Results: We found a positive correlation between exacerbation rates and sodium intake in a multivariate model adjusted for age, gender, disease duration, smoking status, vitamin D levels, Body Mass Index and treatment. We found an exacerbation rate of 2.75 (95% Confidence Interval CI 1.30-5.81) and 3.95 (95% CI 1.39-11.21) times higher (P=0.001 for Trend) in patients with medium and high sodium intake when compared to low intake group. In addition, individuals with high sodium intake had 3.4 times greater odds of developing a new lesion in the MRI and on average had 8 more T2 lesions on MRI.

Conclusion: Our results suggest that higher sodium is associated with increased clinical and radiological disease activity in Multiple Sclerosis patients.

The effect of vitamin D on disease activity in multiple sclerosis patients on fingolimod

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Background: Multiple studies have demonstrated that higher vitamin D levels have a protective effect against new relapses and MRI lesions in MS. A few recent investigations have replicated this finding in patients on beta-interferon therapy, but it is unknown whether the same effect may be observed with other MS drug types.
Objective: To determine the effect of vitamin D level on outcomes in MS patients treated with fingolimod.

Methods: Seventy-nine subjects with RRMS, age>18, EDSS<5.0, and initiating fingolimod were identified from the CLIMB Study at the Partners MS Center. All subjects had a blood sample drawn on the first day of fingolimod therapy and a second blood sample taken within 3-18 months of starting therapy. Each sample was tested for 25-hydroxy-vitamin D (25(OH)D) concentration (ng/mL) in a clinical laboratory and the mean of the two samples was used for analysis. New relapses and MRI findings were entered prospectively in the Center’s Oracle database by the treating physician, and the data were validated against the longitudinal electronic medical record and neuroradiology reports. The mean (SD) follow-up time for subjects was 1.6 (0.6) years. The primary analysis assessed the association between the time to first relapse and mean 25(OH)D tertile based on the average of the two samples using survival analysis. Secondary analyses investigated the time to relapse or new T1 gadolinium-enhancing lesion as the outcome.

Results: The mean (SD) 25(OH)D was 31.0 (13.3) ng/mL unadjusted, and 31.1 (13.2) when adjusted for season and batch effects. The tertile cut points were 22.3/34.6. There was a trend towards longer time to first relapse in those with higher 25(OH)D (p=0.050; hazard ratio (HR) for an increase of one-tertile; 95% CI: 0.51; 0.26, 1.00). 25(OH)D level significantly predicted event-free (i.e. absence of relapse or new gadolinium-enhancing lesion) survival (p=0.033; HR 0.52; 95% CI 0.29, 0.95).

Conclusions: Higher vitamin D levels are associated with improved clinical and MRI outcomes in MS patients treated with fingolimod. These data suggest that targeting a 25(OH)D level of >35 ng/mL may be optimal for preventing disease activity in MS patients on fingolimod.