Vitamin D and Disease Severity in Multiple Sclerosis-Baseline Data From the Randomized Controlled Trial (EVIDIMS)

Objective: To investigate the associations between hypovitaminosis D and disease activity in a cohort of relapsing remitting multiple sclerosis (RRMS) and clinically isolated syndrome (CIS) patients. Methods: In 51 RRMS and 2 CIS patients on stable interferon-β-1b (IFN-β-1b) treatment recruited to the EVIDIMS study (Efficacy of Vitamin D Supplementation in Multiple Sclerosis (NCT01440062) baseline serum vitamin D levels were evaluated. Patients were dichotomized based on the definition of vitamin D deficiency which is reflected by a < 30 vs. ≥ 30 ng/ml level of 25-hydroxyvitamin D (25(OH)D). Possible associations between vitamin D deficiency and both clinical and MRI features of the disease were analyzed. Results: Median (25, 75% quartiles, Q) 25(OH)D level was 18 ng/ml (12, 24). Forty eight out of 53 (91%) patients had 25(OH)D levels < 30 ng/ml (p < 0.001). Patients with 25(OH)D ≥ 30 ng/ml had lower median (25, 75% Q) T2-weighted lesion counts [25 (24, 33)] compared to patients with 25(OH)D < 30 ng/ml [60 (36, 84), p = 0.03; adjusted for age, gender and disease duration: p < 0.001]. Expanded disability status scale (EDSS) score was negatively associated with serum 25(OH)D levels in a multiple linear regression, including age, sex, and disease duration (adjusted: p < 0.001). Interpretation: Most patients recruited in the EVIDIMS study were vitamin D deficient. Higher 25(OH)D levels were associated with reduced T2 weighted lesion count and lower EDSS scores

Exploratory investigation of intestinal function and bacterial translocation after focal cerebral ischemia in the mouse

The gut communicates with the brain bidirectionally via neural, humoral and immune pathways. All these pathways are affected by acute brain lesions, such as stroke. Brain-gut communication may therefore impact on the overall outcome after CNS-injury. Until now, contradictory reports on intestinal function and translocation of gut bacteria after experimental stroke have been published. Accordingly, we aimed to specifically investigate the effects of transient focal cerebral ischemia on intestinal permeability, gut associated lymphoid tissue and bacterial translocation in an exploratory study using a well-characterized murine stroke model. Methods: After 60 min of middle cerebral artery occlusion (MCAO) we assessed intestinal morphology (time points after surgery day 0, 3, 5, 14, 21) and tight junction protein expression (occludin and claudin-1 at day 1 and 3) in 12-week-old male C57BI/6J mice. Lactulose/mannitol/sucralose test was performed to assess intestinal permeability 24-72 h after surgery. To investigate the influence of cerebral ischemia on the local immune system of the gut, main immune cell populations in Peyer's patches (PP) were quantified by flow cytometry. Finally, we evaluated bacterial translocation to extraintestinal organs 24 and 72 h after MCAO by microbiological culture and fluorescence in situ hybridization targeting bacterial 16S rRNA. Results: Transient MCAO decreased claudin-1 expression in the ileum but not in the colon. Intestinal morphology (assessed by light microscopy) and permeability did not change measurably after MCAO. After MCAO, animals had significantly fewer B cells in PP compared to naive mice. Conclusions: In a murine model of stroke, which leads to large brain infarctions in the middle cerebral artery territory, we did not find evidence for overt alterations neither in gut morphology, barrier proteins and permeability nor presence of intestinal bacterial translocation

Exploratory Investigation of Intestinal Function and Bacterial Translocation After Focal Cerebral Ischemia in the Mouse

Background and Purpose: The gut communicates with the brain bidirectionally via neural, humoral and immune pathways. All these pathways are affected by acute brain lesions, such as stroke. Brain-gut communication may therefore impact on the overall outcome after CNS-injury. Until now, contradictory reports on intestinal function and translocation of gut bacteria after experimental stroke have been published. Accordingly, we aimed to specifically investigate the effects of transient focal cerebral ischemia on intestinal permeability, gut associated lymphoid tissue and bacterial translocation in an exploratory study using a well-characterized murine stroke model.Methods: After 60 min of middle cerebral artery occlusion (MCAO) we assessed intestinal morphology (time points after surgery day 0, 3, 5, 14, 21) and tight junction protein expression (occludin and claudin-1 at day 1 and 3) in 12-week-old male C57Bl/6J mice. Lactulose/mannitol/sucralose test was performed to assess intestinal permeability 24–72 h after surgery. To investigate the influence of cerebral ischemia on the local immune system of the gut, main immune cell populations in Peyer's patches (PP) were quantified by flow cytometry. Finally, we evaluated bacterial translocation to extraintestinal organs 24 and 72 h after MCAO by microbiological culture and fluorescence in situ hybridization targeting bacterial 16S rRNA.Results: Transient MCAO decreased claudin-1 expression in the ileum but not in the colon. Intestinal morphology (assessed by light microscopy) and permeability did not change measurably after MCAO. After MCAO, animals had significantly fewer B cells in PP compared to naïve mice.Conclusions: In a murine model of stroke, which leads to large brain infarctions in the middle cerebral artery territory, we did not find evidence for overt alterations neither in gut morphology, barrier proteins and permeability nor presence of intestinal bacterial translocation

High-dose Vitamin D Supplementation in Multiple Sclerosis - Results From the Randomized EVIDIMS (Efficacy of Vitamin D Supplementation in Multiple Sclerosis) Trial

Background: Epidemiological, preclinical, and non-interventional studies link vitamin D (VD) serum levels and disease activity in multiple sclerosis (MS). It is unclear whether high-dose VD supplementation can be used as an intervention to reduce disease activity. Objectives: The study aimed to compare the effects of every other day high- (20,400 IU) versus low-dose (400 IU) cholecalciferol supplementation on clinical and imaging markers of disease activity in patients with relapsing-remitting MS or clinically isolated syndrome. Methods: The EVIDIMS (efficacy of vitamin D supplementation in multiple sclerosis) trial was a multicentre randomized/stratified actively controlled explorative phase 2a pilot trial with a double-blind intervention period of 18 months, add on to interferon-β1b. Results: Fifty-three patients were randomized, and 41 patients completed the study. Cholecalciferol supplementation was well tolerated and safe in both arms. After 18 months, clinical (relapse rates, disability progression) and radiographical (T2-weighted lesion development, contrast-enhancing lesion development, brain atrophy) did not differ between both treatment arms. Post-study power calculations suggested that the sample size was too low to prove the hypothesis. Conclusions: The results neither support nor disprove a therapeutic benefit of high-dose VD supplementation but provide a basis for sound sample size estimations in future confirmatory studies. www.clinicaltrials.gov/NCT01440062

Predicting disease severity in multiple sclerosis using multimodal data and machine learning

Background Multiple sclerosis patients would benefit from machine learning algorithms that integrates clinical, imaging and multimodal biomarkers to define the risk of disease activity. Methods We have analysed a prospective multi-centric cohort of 322 MS patients and 98 healthy controls from four MS centres, collecting disability scales at baseline and 2 years later. Imaging data included brain MRI and optical coherence tomography, and omics included genotyping, cytomics and phosphoproteomic data from peripheral blood mononuclear cells. Predictors of clinical outcomes were searched using Random Forest algorithms. Assessment of the algorithm performance was conducted in an independent prospective cohort of 271 MS patients from a single centre. Results We found algorithms for predicting confirmed disability accumulation for the different scales, no evidence of disease activity (NEDA), onset of immunotherapy and the escalation from low- to high-efficacy therapy with intermediate to high- accuracy. This accuracy was achieved for most of the predictors using clinical data alone or in combination with imaging data. Still, in some cases, the addition of omics data slightly increased algorithm performance. Accuracies were comparable in both cohorts. Conclusion Combining clinical, imaging and omics data with machine learning helps identify MS patients at risk of dis- ability worsening.publishedVersio

Der Einfluss der Hypovitaminose D und Vitamin D-Supplementierung auf Krankheitsaktivität und Immunprofil in Patienten mit Multipler Sklerose

Deficient 25-hydroxyvitamin D [25(OH)D] (hypovitaminosis D) levels are associated with increased risk, disease activity and severity, and multiple sclerosis (MS) progression. It remains unclear whether high-dose vitamin D intake ameliorates disease activity and whether it exerts immunologic effects in MS. This PhD project aimed to investigate the: 1) association of hypovitaminosis D with markers of disease severity in MS, 2) effects of high-dose versus low-dose cholecalciferol (vitamin D3) supplementation, on disease activity and brain magnetic resonance imaging (MRI) markers of MS and clinically isolated syndrome (CIS), 3) effects of vitamin D3 on immune cell landscape and N-glycan branching on immune cells. These objectives were addressed in the context of the EVIDIMS study (Efficacy of Vitamin D Supplementation in Multiple Sclerosis), ClinicalTrials.gov identifier, NCT01440062. EVIDIMS was a multicenter, randomized, actively-controlled explorative phase 2a pilot trial with a double-blind vitamin D3 intervention (20,400 international units (IU) vs. 400 IU). Vitamin D3 was administered for 18 months, as an add-on to interferon β1b treatment in 53 MS/CIS patients. We studied the association of clinical and brain MRI features with hypovitaminosis D (Project 1) or with vitamin D3 supplementation (Project 2). In Project 3, the immunologic effects of vitamin D3 supplementation on T, B and natural killer (NK) cell subpopulations were investigated using flow cytometry. Hypovitaminosis D was associated with increased MRI T2-weighted brain lesion counts and clinical disability scores. Vitamin D supplementation did not affect clinical and MRI measures of disease activity longitudinally. Moreover, high-dose supplementation did not affect the frequencies of broad, regulatory/effector immune cell subpopulations, but did reduce N-glycan branching in broad T cells and NK cell subtypes. These projects suggest – in line with the literature - that hypovitaminosis D in MS patients is associated with increased disease activity, while vitamin D intake did not affect clinical outcomes of MS or the immune profile in the blood. It shows, however, that vitamin D regulates N-glycan branching on immune cells.Ein 25-Hydroxyvitamin D Mangel (Hypovitaminose D) ist mit einem erhöhten Erkrankungsrisiko und erhöhter Krankheitsaktivität einer Multiplen Sklerose (MS) verbunden. Es ist unklar, ob eine Vitamin-D Nahrungsergänzung die MS-Krankheitsaktivität reduziert. Das Promotionsprojekt hatte folgende Ziele: Untersuchen der 1) Assoziation von Hypovitaminose D mit MS Erkrankungsschwere, 2) Auswirkungen einer hochdosierten im Vergleich zu einer niedrigdosierten Vitamin D3 Nahrungsergänzung auf die Krankheitsaktivität bei MS und klinisch isoliertem Syndrom (CIS), 3) Auswirkungen von Vitamin D3 auf die Immunzelllandschaft und N-Glycan-Verzweigung auf Immunzellen. Die Untersuchungen wurden im Rahmen der EVIDIMS-Studie (Wirksamkeit der Vitamin-DSupplementierung bei Multipler Sklerose) durchgeführt. EVIDIMS war eine multizentrische, randomisierte, aktiv kontrollierte explorative Phase-2a-Pilotstudie mit einer doppelblinden Vitamin-D3 Intervention (20.400 internationale Einheiten (IE) gegenüber 400 IE). Vitamin D3 wurde 53 MS/CIS-Patienten 18 Monate lang als Ergänzung zur Interferon-β1b-Behandlung verabreicht. Wir untersuchten den Zusammenhang von klinischen und magnetresonanztomographischen (MRT)-Merkmalen der MS mit Hypovitaminose D (Projekt 1) und mit Vitamin D3- Nahrungsergänzung (Projekt 2). Im Projekt 3 wurde die Wirkung der Vitamin D3- Nahrungsergänzung auf T-, B- und Natural Killer (NK) -Zell-Subpopulationen mittels Durchflusszytometrie untersucht. Eine Hypovitaminose D war mit einer erhöhten T2-gewichteten Läsionslast im MRT und der klinischen Behinderung assoziiert. Eine Vitamin-D- Nahrungsergänzung hatte keinen Einfluss auf die klinischen und MRT-Messungen der Krankheitsaktivität. Eine hochdosierte Nahrungsergänzung hatte keinen Einfluss auf die Häufigkeit von Immunzellpopulationen, reduzierte jedoch die N-Glycan-Verzweigung in T-Zellen und NK-Zell-Subtypen. Die Arbeit zeigt, dass Hypovitaminose D bei MS-Patienten mit einer erhöhten Krankheitsaktivität verbunden ist, während die hochdosierte Einnahme von Vitamin D die klinischen Ergebnisse von MS oder das Immunprofil im Blut nicht beeinflusst. Die Regulierung der Verzweigung von N-Glycan auf Immunzellen stellt einen möglichen Mechanismus dar, wie Vitamin D die Aktivität von Immunzellen beeinflusst

Effect of vitamin D supplementation on N-glycan branching and cellular immunophenotypes in MS.

ObjectiveTo investigate the effect of cholecalciferol (vitamin D3) supplementation on peripheral immune cell frequency and N-glycan branching in patients with relapsing-remitting multiple sclerosis (RRMS).MethodsExploratory analysis of high-dose (20&nbsp;400&nbsp;IU) and low-dose (400&nbsp;IU) vitamin D3 supplementation taken every other day of an 18-month randomized controlled clinical trial including 38 RRMS patients on stable immunomodulatory therapy (NCT01440062). We investigated cholecalciferol treatment effects on N-glycan branching using L-PHA stain (phaseolus vulgaris leukoagglutinin) at 6&nbsp;months and frequencies of T-, B-, and NK-cell subpopulations at 12&nbsp;months with flow cytometry.ResultsHigh-dose supplementation did not change CD3+&nbsp;T cell subsets, CD19+ B cells subsets, and NK cells frequencies, except for CD8+&nbsp;T regulatory cells, which were reduced in the low-dose arm compared to the high-dose arm at 12&nbsp;months. High-dose supplementation decreased N-glycan branching on T and NK cells, measured as L-PHA mean fluorescence intensity (MFI). A reduction of N-glycan branching in B cells was not significant. In contrast, low-dose supplementation did not affect N-glycan branching. Changes in N-glycan branching did not correlate with cell frequencies.InterpretationImmunomodulatory effect of vitamin D may involve regulation of N-glycan branching in vivo. Vitamin D3 supplementation did at large not affect the frequencies of peripheral immune cells

Effect of vitamin D supplementation on N‐glycan branching and cellular immunophenotypes in MS

Objective: To investigate the effect of cholecalciferol (vitamin D3) supplementation on peripheral immune cell frequency and N-glycan branching in patients with relapsing-remitting multiple sclerosis (RRMS). Methods: Exploratory analysis of high-dose (20 400 IU) and low-dose (400 IU) vitamin D3 supplementation taken every other day of an 18-month randomized controlled clinical trial including 38 RRMS patients on stable immunomodulatory therapy (NCT01440062). We investigated cholecalciferol treatment effects on N-glycan branching using L-PHA stain (phaseolus vulgaris leukoagglutinin) at 6 months and frequencies of T-, B-, and NK-cell subpopulations at 12 months with flow cytometry. Results: High-dose supplementation did not change CD3+ T cell subsets, CD19+ B cells subsets, and NK cells frequencies, except for CD8+ T regulatory cells, which were reduced in the low-dose arm compared to the high-dose arm at 12 months. High-dose supplementation decreased N-glycan branching on T and NK cells, measured as L-PHA mean fluorescence intensity (MFI). A reduction of N-glycan branching in B cells was not significant. In contrast, low-dose supplementation did not affect N-glycan branching. Changes in N-glycan branching did not correlate with cell frequencies. Interpretation: Immunomodulatory effect of vitamin D may involve regulation of N-glycan branchingin vivo. Vitamin D3 supplementation did at large not affect the frequencies of peripheral immune cells