Multiscale networks in multiple sclerosis.

Complex diseases such as Multiple Sclerosis (MS) cover a wide range of biological scales, from genes and proteins to cells and tissues, up to the full organism. In fact, any phenotype for an organism is dictated by the interplay among these scales. We conducted a multilayer network analysis and deep phenotyping with multi-omics data (genomics, phosphoproteomics and cytomics), brain and retinal imaging, and clinical data, obtained from a multicenter prospective cohort of 328 patients and 90 healthy controls. Multilayer networks were constructed using mutual information for topological analysis, and Boolean simulations were constructed using Pearson correlation to identified paths within and among all layers. The path more commonly found from the Boolean simulations connects protein MK03, with total T cells, the thickness of the retinal nerve fiber layer (RNFL), and the walking speed. This path contains nodes involved in protein phosphorylation, glial cell differentiation, and regulation of stress-activated MAPK cascade, among others. Specific paths identified were subsequently analyzed by flow cytometry at the single-cell level. Combinations of several proteins (GSK3AB, HSBP1 or RS6) and immune cells (Th17, Th1 non-classic, CD8, CD8 Treg, CD56 neg, and B memory) were part of the paths explaining the clinical phenotype. The advantage of the path identified from the Boolean simulations is that it connects information about these known biological pathways with the layers at higher scales (retina damage and disability). Overall, the identified paths provide a means to connect the molecular aspects of MS with the overall phenotype.

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 disability worsening.

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 branching in vivo. Vitamin D3 supplementation did at large not affect the frequencies of peripheral immune cells.

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-ß-1b (IFN-ß-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.

Impact of treatment on cellular immunophenotype in MS: A cross-sectional study.

OBJECTIVE: To establish cytometry profiles associated with disease stages and immunotherapy in MS. METHODS: Demographic/clinical data and peripheral blood samples were collected from 227 patients with MS and 82 sex- and age-matched healthy controls (HCs) enrolled in a cross-sectional study at 4 European MS centers (Spain, Italy, Germany, and Norway). Flow cytometry of isolated peripheral blood mononuclear cells was performed in each center using specifically prepared antibody-cocktail Lyotubes; data analysis was centralized at the Genoa center. Differences in immune cell subsets were assessed between groups of untreated patients with relapsing-remitting or progressive MS (RRMS or PMS) and HCs and between groups of patients with RRMS taking 6 commonly used disease-modifying drugs. RESULTS: In untreated patients with MS, significantly higher frequencies of Th17 cells in the RRMS population compared with HC and lower frequencies of B-memory/B-regulatory cells as well as higher percentages of B-mature cells in patients with PMS compared with HCs emerged. Overall, the greatest deviation in immunophenotype in MS was observed by treatment rather than disease course, with the strongest impact found in fingolimod-treated patients. Fingolimod induced a decrease in total CD4+ T cells and in B-mature and B-memory cells and increases in CD4+ and CD8+ T-regulatory and B-regulatory cells. CONCLUSIONS: Our highly standardized, multisite cytomics data provide further understanding of treatment impact on MS immunophenotype and could pave the way toward monitoring immune cells to help clinical management of MS individuals.

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-ß1b. 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.

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.