Ocrelizumab Alters Cytotoxic Lymphocyte Function While Reducing EBV-Specific CD8+ T-Cell Proliferation in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The role of B cells in the pathogenic events leading to relapsing multiple sclerosis (R-MS) has only been recently elucidated. A pivotal step in defining this role has been provided by therapeutic efficacy of anti-CD20 monoclonal antibodies. Indeed, treatment with anti-CD20 can also alter number and function of other immune cells not directly expressing CD20 on their cell surface, whose activities can contribute to unknown aspects influencing therapeutic efficacy. We examined the phenotype and function of cytotoxic lymphocytes and Epstein-Barr virus (EBV)-specific immune responses in people with R-MS before and after ocrelizumab treatment. METHODS: In this prospective study, we collected blood samples from people with R-MS (n = 41) before and 6 and 12 months after initiating ocrelizumab to assess the immune phenotype and the indirect impact on cytotoxic functions of CD8+ T and NK cells. In addition, we evaluated the specific anti-EBV proliferative responses of both CD8+ T and NK lymphocytes as surrogate markers of anti-EBV activity. RESULTS: We observed that while ocrelizumab depleted circulating B cells, it also reduced the expression of activation and migratory markers on both CD8+ T and NK cells as well as their in vitro cytotoxic activity. A comparable pattern in the modulation of immune molecules by ocrelizumab was observed in cytotoxic cells even when patients with R-MS were divided into groups based on their prior disease-modifying treatment. These effects were accompanied by a significant and selective reduction of CD8+ T-cell proliferation in response to EBV antigenic peptides. DISCUSSION: Taken together, our findings suggest that ocrelizumab-while depleting B cells-affects the cytotoxic function of CD8+ and NK cells, whose reduced cross-activity against myelin antigens might also contribute to its therapeutic efficacy during MS.

Temporal Relationship Between Serum Neurofilament Light Chain and Radiologic Disease Activity in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Serum neurofilament light chain (sNfL) levels correlate with multiple sclerosis (MS) disease activity, but the dynamics of this correlation are unknown. We evaluated the relationship between sNfL levels and radiologic MS disease activity through monthly assessments during the 24-week natalizumab treatment interruption period in RESTORE (NCT01071083). METHODS: In the RESTORE trial, participants with relapsing forms of MS who had received natalizumab for ≥12 months were randomized to either continue or stop natalizumab and followed with MRI and blood draws every 4 weeks to week 28 and again at week 52 The sNfL was measured, and its dynamics were correlated with the development of gadolinium-enhancing (Gd+) lesions. Log-linear trend in sNfL levels were modeled longitudinally using generalized estimating equations with robust variance estimator from baseline to week 28. RESULTS: Of 175 patients enrolled in RESTORE, 166 had serum samples for analysis. Participants with Gd+ lesions were younger (37.7 vs 43.1, p = 0.001) and had lower Expanded Disability Status Scale scores at baseline (2.7 vs 3.4, p = 0.017) than participants without Gd+ lesions. sNfL levels increased in participants with Gd+ lesions (n = 65) compared with those without (n = 101, mean change from baseline to maximum sNfL value, 12.1 vs 3.2 pg/mL, respectively; p = 0.003). As the number of Gd+ lesions increased, peak median sNfL change also increased by 1.4, 3.0, 4.3, and 19.6 pg/mL in the Gd+ lesion groups of 1 (n = 12), 2-3 (n = 18), 4-9 (n = 21), and ≥10 (n = 14) lesions, respectively. However, 46 of 65 (71%) participants with Gd+ lesions did not increase above the 95th percentile threshold of the group without Gd+ lesions. The initial increase of sNfL typically trailed the first observation of Gd+ lesions, and the peak increase in sNfL was a median [interquartile range] of 8 [0, 12] weeks after the first appearance of the Gd+ lesion. DISCUSSION: Although sNfL correlated with the presence of Gd+ lesions, most participants with Gd+ lesions did not have elevations in sNfL levels. These observations have implications for the use and interpretation of sNfL as a biomarker for monitoring MS disease activity in controlled trials and clinical practice.

Feasibility study to assess lesion repair in relapsing-remitting multiple sclerosis: A randomized controlled pilot clinical trial of domperidone add-on treatment.

BACKGROUND: Identification of therapies to promote repair in multiple sclerosis is challenged by the lack of an accepted trial model and associated outcome measures. The goal of this study was to determine the feasibility of a new trial model that enrolls disease modifying therapy (DMT)-treated relapsing-remitting multiple sclerosis (RRMS) participants who have enhancing lesions on clinically indicated brain MRI, and to explore estimates of lesion repair using MRI. METHODS: This was a single site randomized controlled clinical trial. Recruitment took place between November 2015 and January 2019, with final follow-up in February 2019. DMT-treated RRMS participants aged 18-60 years with at least one gadolinium-enhancing lesion on clinically indicated brain MRI were included. Participants were randomized 2:1 to oral domperidone add-on 10-mg three times daily for 16 weeks or no add-on treatment (control). The primary outcomes were feasibility of the model pre-defined as recruitment of 24 participants within 36 months with a 79 % completion rate, and MRI outcomes of lesion repair measured at 16 and 32 weeks using texture analysis, magnetization transfer imaging (MTI), and diffusion tensor imaging (DTI). The impact of domperidone on serum prolactin at 6 and 16 weeks was also evaluated. RESULTS: Of 237 RRMS participants screened, 17 (14 women) were randomized: 12 to domperidone add-on and 5 to control. All completed the study. Median (range) age was 38.9 (26.7-55.9) years; EDSS was 1.5 (1.0-3.5); and disease duration was 12.9 (2.9-23.3) years. Both groups showed improvement in MRI texture and diffusion fractional anisotropy (FA) at 32 weeks, and the domperidone group demonstrated additional recovery at 16 weeks in both texture and FA. There was no significant group difference in any MRI outcome. Of the 12 domperidone participants, 7 had ≥4x higher serum prolactin than normal. There were no serious adverse events. CONCLUSION: The recruitment target was not met and therefore the trial model was not feasible despite a full completion rate. The imaging techniques performed well, especially MRI texture analysis, suggesting the sample size being sufficient for estimating lesion repair. The main challenge of this trial model may be recruiting gadolinium-enhancing lesions in DMT-treated RRMS participants. Prolactin is safe and may hold promise as a remyelination therapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02493049.

Serum neurofilament light for detecting disease activity in individual patients in multiple sclerosis: A 48-week prospective single-center study.

BACKGROUND: Serum neurofilament light (sNfL) reflects neuroaxonal damage and is now used as an outcome in treatment trials of relapsing-remitting multiple sclerosis (RRMS). However, the diagnostic properties of sNfL for monitoring disease activity in individual patients warrant further investigations. METHOD: Patients with suspected relapse and/or contrast-enhancing lesions (CELs) were consecutively included and performed magnetic resonance imaging (MRI) of the brain at baseline and weeks 28 and 48. Serum was obtained at baseline and 2, 4, 8, 16, 24, and 48 weeks. Neurofilament light concentration was measured using Single molecule array technology. RESULTS: We included 44 patients, 40 with RRMS and 4 with clinically isolated syndrome. The median sNfL level peaked at 2 weeks post-baseline (14.6 ng/L, interquartile range (IQR); 9.3-31.6) and reached nadir at 48 weeks (9.1 ng/L, IQR; 5.5-15.0), equivalent to the median sNfL of controls (9.1 ng/L, IQR; 7.4-12). A baseline Z-score of more than 1.1 (area under the curve; 0.78, p < 0.0001) had a sensitivity of 81% and specificity of 70% to detect disease activity. CONCLUSION: One out of five patients with relapse and/or CELs did not change significantly in post-baseline sNfL levels. The utility of repeated sNfL measurements to monitor disease activity is complementary rather than a substitute for clinical and MRI measures.

Predicting clinical progression and cognitive decline in patients with relapsing-remitting multiple sclerosis: a 6-year follow-up study.

INTRODUCTION: Cognitive impairment occurs from the earliest stages of multiple sclerosis (MS) and progresses over time. The introduction of disease modifying therapies (DMTs) has changed the prognosis for MS patients, offering a potential opportunity for improvement in the cognitive arena as well. MATERIAL AND METHODS: 41 patients with relapsing-remitting multiple sclerosis (MS) were recruited to the study. Thirty patients were available for final follow-up and were included in the analysis. Baseline (BL) brain MRI including volumetry and neuropsychological tests were performed. Blood samples were collected at BL and follow-up (FU) and were tested for: vascular endothelial growth factor (VEGF), soluble vascular cell adhesion molecule-1 (sVCAM1), soluble platelet-endothelial CAM-1 (sPECAM1), and soluble intercellular CAM-1 (sICAM-1). Patients were invited for a final neuropsychological follow-up after a median of 6 years. Disease activity (relapses, EDSS increase, new/active brain lesions on MRI) was analysed between BL and FU. RESULTS: The study group deteriorated in the Rey-Osterrieth Complex Figure (ROCF) test (p = 0.001), but improved significantly in three other tests, i.e. semantic fluency test (p = 0.013), California Verbal Learning Test (CVLT, p = 0.016), and Word Comprehension Test (WCT, p < 0.001). EDSS increase correlated negatively with semantic fluency and WCT scores (r = -0.579, p = 0.001 and r = -0.391, p = 0.033, respectively). Improvements in semantic fluency test and WCT correlated positively with baseline deep grey matter, grey matter, and cortical volumes (p < 0.05, r > 0). Higher EDSS on FU correlated significantly negatively with baseline left and right pallidum, right caudate, right putamen, right accumbens, and cortical volume (p < 0.05, r < 0). No significant relationship was found between the number of relapses and EDSS on FU or neuropsychological deteriorations. Improvements in WCT and CVLT correlated positively with baseline sPECAM1 and sVCAM1 results, respectively (r > 0, p < 0.05). Deterioration in ROCF test correlated significantly with higher levels of baseline VEGF and sVCAM1 (p < 0.05). CONCLUSIONS: Brain volume is an important predictor of future EDSS and cognitive functions outcome. MS patients have a potential for improving in neuropsychological tests over time. It remains to be established whether this is related to successful disease modification with immunotherapy. Baseline volumetric measures are stronger predictors of cognitive performance than relapse activity, which yet again highlights the importance of atrophy in MS prognosis.

The alterations of cerebrospinal fluid TNF-alpha and TGF-beta2 levels in early relapsing-remitting multiple sclerosis.

AIM OF THE STUDY: This study aimed to analyze serum and cerebrospinal fluid (CSF) concentrations of proinflammatory and anti-inflammatory cytokines produced by T regulatory (Treg) cells in early RRMS according to the 2017 McDonald criteria. CLINICAL RATIONALE FOR THE STUDY: Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) with the cytokine network playing an important role. However, there is a continual lack of data regarding the immunopathogenesis of early RRMS, especially according to the 2017 McDonald criteria. MATERIALS AND METHODS: The study groups included early RRMS patients during relapse (n = 18), remission (n = 14), and the control group. The MS diagnosis was established according to the 2017 McDonald criteria. Patients were studied up to 1 year after diagnosis was made. A quantitative test kit based on ELISA was used for cytokine measurement in the serum and CSF. Comparative and correlation analyses between the levels of TNF-α, TGF-ß2, IgG index, and relapse duration were performed. RESULTS: Significantly higher CSF concentrations of TNF-α in both RRMS-relapse and RRMS-remission groups were found compared to the controls (p < 0.01). The CSF levels of TGF-ß2 in the RRMS-relapse group were significantly lower in comparison to the control group (p = 0.01). CONCLUSIONS AND CLINICAL IMPLICATIONS: An inappropriate inflammatory response seems to occur in early RRMS and includes the production of TNF-α and a decrease in TGF-ß2 release suggesting a significant Treg cells role. Further studies on the topic may contribute to developing new disease-modifying drugs and biochemical markers of the disorder.

Blood-based untargeted metabolomics in relapsing-remitting multiple sclerosis revealed the testable therapeutic target.

Metabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 patients with relapsing-remitting multiple sclerosis (RRMS) and 14 healthy age-matched controls. Of 632 known metabolites detected, 60 were significantly altered in RRMS. Bioinformatics analysis identified an altered metabotype in patients with RRMS, represented by four changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these four pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient-derived peripheral blood mononuclear cells showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the antiinflammatory phenotype of monocytes/macrophage in the central nervous system. Our study provided a proof of principle for how a blood-based metabolomic approach using patient samples could lead to the identification of a therapeutic target for developing potential therapy.

Concomitant Expression of IL-6 and TGF-ß Cytokines and their Receptors in Peripheral Blood of Patients with Multiple Sclerosis: The Effects of INFß Drugs.

BACKGROUND: Concomitant signals from IL-6 and TGF-ß have a central role in the Th17 cells development and differentiation, and these cells are the main promoters of demyelinating inflammation in the central nervous system (CNS) resulting in multiple sclerosis (MS). OBJECTIVES: To evaluate the simultaneous IL-6 and TGF-ß gene and their receptor protein expression in patients with Relapsing-Remitting (RR)-MS. MATERIALS AND METHODS: IL-6 and TGF-ß mRNA and their receptor expression on the surface of CD4+T cells were evaluated using real-time PCR (RT-PCR) and flow cytometry, respectively. RESULTS: The IL-6 mRNA expression in patients with RRMS was significantly higher than in the controls (p= 0.019). When patients who did not receive any other treatment were compared with the controls, the significant difference was substantial (p=0.006). The TGF-ß mRNA expression in patients was lower than in the controls (p = 0.03). However, in patients receiving IFNß, it increased compared with the other patients (p= 0.036). There was no difference in cytokine receptor expression between patients and the control group. CONCLUSION: Our data conclude an increase and decrease in mRNA expression levels of IL-6 and TGF-ß in patients with RRMS, respectively. Moreover, there were no significant differences in receptor expression of either cytokines. Based on our data the balance of TGF and IL-6 appears to have a positive impact on the disease control.

Evaluation of S100A12 and Apo-A1 plasma level potency in untreated new relapsing-remitting multiple sclerosis patients and their family members.

Multiple sclerosis is an inflammatory disease of the spinal cord and brain. Receptor for advanced glycation end products and Apolipoprotein A1 (Apo-AI) have been recommended to have a pathogenic role in the neuroinflammatory disorder as multiple sclerosis. The purpose of this research was to measure the plasma levels of S100A12 and Apo-A1 in the first-degree family of relapsing-remitting multiple sclerosis (RRMS) patients. Plasma levels of S100A12 & Apo-A1 were evaluated via enzyme-linked immunosorbent assay in the thirty-five new cases of untreated patients with deterministic RRMS according to the McDonald criteria, twenty-four healthy controls, and twenty-six first-degree members of untreated RRMS patients (called them as high-risk group). The main findings of this study were as follows: the plasma level of S100A12 was significantly lower in the new cases of untreated RRMS (P ≤ 0.05; 0.045) and high-risk (P ≤ 0.05; 0.001) groups. Although the plasma protein level of Apo-A1 was reduced significantly in the high-risk group (P < 0.05, P = 0.003) as compared to the healthy control group, there was no significant difference in the untreated RRMS patients (P = 0.379). The plasma level of vitamin D3 in both RRMS patients and high-risk groups displayed significance reduction, although, there was no significant association between vitamin D and S100A12 & Apo-A1 levels. Given the role of S100A12 and Apo-A1 in the inflammatory process performed in the first-degree family members of the RRMS patients, which revealed a significant decrease in this group, we concluded that they can be considered as one of the contributing factors in the pathogenesis of MS, though more research is needed before assuming them as predictive biomarkers.

CNS endothelial derived extracellular vesicles are biomarkers of active disease in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a complex, heterogenous disease characterized by inflammation, demyelination, and blood-brain barrier (BBB) permeability. Currently, active disease is determined by physician confirmed relapse or detection of contrast enhancing lesions via MRI indicative of BBB permeability. However, clinical confirmation of active disease can be cumbersome. As such, disease monitoring in MS could benefit from identification of an easily accessible biomarker of active disease. We believe extracellular vesicles (EV) isolated from plasma are excellent candidates to fulfill this need. Because of the critical role BBB permeability plays in MS pathogenesis and identification of active disease, we sought to identify EV originating from central nervous system (CNS) endothelial as biomarkers of active MS. Because endothelial cells secrete more EV when stimulated or injured, we hypothesized that circulating concentrations of CNS endothelial derived EV will be increased in MS patients with active disease. METHODS: To test this, we developed a novel method to identify EV originating from CNS endothelial cells isolated from patient plasma using flow cytometry. Endothelial derived EV were identified by the absence of lymphocyte or platelet markers CD3 and CD41, respectively, and positive expression of pan-endothelial markers CD31, CD105, or CD144. To determine if endothelial derived EV originated from CNS endothelial cells, EV expressing CD31, CD105, or CD144 were evaluated for expression of the myelin and lymphocyte protein MAL, a protein specifically expressed by CNS endothelial cells compared to endothelial cells of peripheral organs. RESULTS: Quality control experiments indicate that EV detected using our flow cytometry method are 0.2 to 1 micron in size. Flow cytometry analysis of EV isolated from 20 healthy controls, 16 relapsing-remitting MS (RRMS) patients with active disease not receiving disease modifying therapy, 14 RRMS patients with stable disease not receiving disease modifying therapy, 17 relapsing-RRMS patients with stable disease receiving natalizumab, and 14 RRMS patients with stable disease receiving ocrelizumab revealed a significant increase in the plasma concentration of CNS endothelial derived EV in patients with active disease compared to all other groups (p = 0.001). CONCLUSIONS: For the first time, we have identified a method to identify CNS endothelial derived EV in circulation from human blood samples. Results from our pilot study indicate that increased levels of CNS endothelial derived EV may be a biomarker of BBB permeability and active disease in MS.

Differential Expression of Serum Extracellular Vesicle miRNAs in Multiple Sclerosis: Disease-Stage Specificity and Relevance to Pathophysiology.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its first clinical presentation (clinically isolated syndrome, CIS) is often followed by the development of relapsing-remitting MS (RRMS). The periphery-to-CNS transmission of inflammatory molecules is a major pathophysiological pathway in MS. This could include signalling via extracellular vesicle (EV) microRNAs (miRNAs). In this study, we investigated the serum EV miRNome in CIS and RRMS patients and matched controls, with the aims to identify MS stage-specific differentially expressed miRNAs and investigate their biomarker potential and pathophysiological relevance. miRNA sequencing was conducted on serum EVs from CIS-remission, RRMS-relapse, and viral inflammatory CNS disorder patients, as well as from healthy and hospitalized controls. Differential expression analysis was conducted, followed by predictive power and target-pathway analysis. A moderate number of dysregulated serum EV miRNAs were identified in CIS-remission and RRMS-relapse patients, especially relative to healthy controls. Some of these miRNAs were also differentially expressed between the two MS stages and had biomarker potential for patient-control and CIS-RRMS separations. For the mRNA targets of the RRMS-relapse-specific EV miRNAs, biological processes inherent to MS pathophysiology were identified using in silico analysis. Study findings demonstrate that specific serum EV miRNAs have MS stage-specific biomarker potential and contribute to the identification of potential targets for novel, efficacious therapies.

Serum levels of lipocalin-2 are elevated at early times in African American relapsing remitting multiple sclerosis patients.

Previous studies showed that depleting Liver Kinase-B1 (LKB1) from astrocytes increased inflammatory factors lipocalin-2 (LCN2) and osteopontin (OPN) in EAE. A single nucleotide polymorphism (SNP) in STK11 (encoding LKB1) is a risk factor for MS, suggesting increased LCN2 or OPN contributes to risk. Serum LCN2 and OPN levels in African American female MS patients were higher than healthy controls, and while levels increased with disease duration in cases without the SNP, levels decreased with duration in cases with the SNP. Increased MS risk associated with the STK11 SNP may be due to higher LCN2 or OPN levels at early times.

Impact of Dietary Intervention on Serum Neurofilament Light Chain in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Adapted ketogenic diet (AKD) and caloric restriction (CR) have been suggested as alternative therapeutic strategies for multiple sclerosis (MS), but information on their impact on neuroaxonal damage is lacking. Thus, we explored the impact of diets on serum neurofilament light chain (sNfL) levels in patients with relapsing-remitting MS. METHODS: We retrospectively evaluated a prospective randomized controlled trial of 60 patients with MS who were on a common diet or ketogenic diet or fasting. We examined sNfL levels of 40 participants at baseline and at the end of the study after 6 months using single molecule array assay. RESULTS: sNfL levels were investigated in 9 controls, 14 participants on CR, and 17 participants on AKD. Correlation analysis showed an association of sNfL with age and disease duration; an association was also found between sNfL and the Multiple Sclerosis Functional Composite. AKD significantly reduced sNfL levels at 6 months compared with the common diet group (p = 0.001). DISCUSSION: For clinical or study use, consider that AKD may incline sNfL levels independent of relapse activity up to 3 months after initiation. At 6 months, AKD, which complements current therapies, reduced sNfL levels, therefore suggesting potential neuroprotective effects in MS. A single cycle of seven-day fasting did not affect sNfL. AKD may be an addition to the armamentarium to help clinicians support patients with MS in a personalized manner with tailored diet strategies. TRIAL REGISTRATION INFORMATION: Clinical trial registration number NCT01538355.

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

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

miR-132-3p, miR-106b-5p, and miR-19b-3p Are Associated with Brain-Derived Neurotrophic Factor Production and Clinical Activity in Multiple Sclerosis: A Pilot Study.

Introduction: Brain-derived neurotrophic factor (BDNF) levels are reduced in advanced stages of multiple sclerosis (MS) and may be associated with reduced regenerative capability in progressive MS. This has brought increased attention to factors regulating BDNF production in MS. Our aim was to investigate the link between neurotrophin-regulating microRNAs (miRNA) and disease progression in MS. Materials and Methods: Serum levels of BDNF and peripheral blood mononuclear cell (PBMC) expression levels of miR-132-3p, miR-106b-5p and miR-19b-3p were respectively measured by ELISA and real time PCR in twelve relapsing remitting MS (RRMS) patients, seven secondary progressive MS (SPMS) patients and fourteen healthy controls. Results: Serum BDNF levels were significantly reduced in SPMS patients, while selected miRNAs were significantly upregulated in PBMC of RRMS and SPMS patients. miR-106b-5p and miR-19b-3p respectively showed the highest sensitivity and specificity for MS diagnosis by receiver operating characteristic curve analysis. There was a negative correlation between levels of BDNF and the miRNAs in RRMS. Likewise, levels of BDNF and the investigated miRNAs showed positive and negative correlations respectively with the expanded disability status scale in RRMS and SPMS patients. miR-132-3p and miR-106b-5p levels showed positive correlations with the progression index in SPMS patients. Conclusion: Our results suggest that increased disability is associated with downregulation of miR-132-3p, miR-106b-5p and miR-19b-3p in RRMS patients and putatively promotes increased production of neuroprotective BDNF as a compensatory mechanism. This link between the investigated miRNAs and BDNF in RRMS does not appears to hold for SPMS. This might be one of the factors contributing to reduced regenerative ability in the progressive stage of MS.

Evaluation of Age-Dependent Immune Signatures in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: In MS, an age-related decline in disease activity and a decreased efficacy of disease-modifying treatment have been linked to immunosenescence, a state of cellular dysfunction associated with chronic inflammation. METHODS: To evaluate age-related immunologic alterations in MS, we compared immune signatures in peripheral blood (PB) and CSF by flow cytometry in patients with relapsing-remitting (RR) (PB n = 38; CSF n = 51) and primary progressive (PP) MS (PB n = 40; CSF n = 36) and respective controls (PB n = 40; CSF n = 85). RESULTS: Analysis revealed significant age-related changes in blood immune cell composition, especially in the CD8 T-cell compartment of healthy donors (HDs) and patients with MS. However, HDs displayed a strong age-dependent decline in the expression of the immunoregulatory molecules KLRG1, LAG3, and CTLA-4 on memory CD8 T cells, whereas this age-dependent reduction was completely abrogated in patients with MS. An age-dependent increase in the expression of the costimulatory molecule CD226 on memory CD8 T cells was absent in patients with MS. CD226 expression correlated with disability in younger (≤50 years) patients with MS. CSF analysis revealed a significant age-dependent decline in various immune cell populations in PPMS but not RRMS, suggesting a differential effect of aging on the intrathecal compartment in PPMS. DISCUSSION: Our data illustrate that aging in MS is associated with a dysbalance between costimulatory and immunoregulatory signals provided by CD8 T cells favoring a proinflammatory phenotype and, more importantly, a pattern of premature immune aging in the CD8 T-cell compartment of young patients with MS with potential implications for disease severity.

Serum GFAP and NfL levels in benign relapsing-remitting multiple sclerosis.

OBJECTIVE: We aimed to investigate serum glial fibrillary acidic protein (GFAP) and serum neurofilament light chain (NfL) levels as potential discriminative biomarkers between benign relapsing-remitting multiple sclerosis (BRRMS) and aggressive relapsing-remitting MS (ARRMS). METHODS: Serum GFAP and NfL levels were analyzed in patients with BRRMS (n = 34), ARRMS (n = 29), and healthy controls (n = 14) by using Single Molecule Array (Simoa). Patients with ARRMS had been treated with highly effective disease-modifying treatments (DMT) (fingolimod or natalizumab). RESULTS: Serum GFAP levels in both BRRMS (median 210.19 pg/ml, IQR 163.69-287.19) and in ARRMS (median 188.60 pg/ml, IQR39.23-244.93) were significantly higher (p = 0.035 and p = 0.034, respectively) compared to healthy controls (median 117.93 pg/ml, IQR 60.28-183.83). Serum GFAP levels did not differ between BRRMS and ARRMS. There were no statistical differences in NfL levels between BRRMS, ARRMS and healthy controls. GFAP level was significantly higher (p = 0.04) in BRRMS without DMT (median 216.04 pg/ml, IQR 188.60-274.79) than in those BRRMS patients who had used DMT (median 196.26 pg/ml, IQR 133.33-325.54). CONCLUSIONS: We found elevated levels of serum GFAP in both BRRMS and ARRMS compared to healthy controls, reflecting astrocytic activation. Serum NfL did not differ between BRRMS and ARRMS, probably due to the stable inflammatory phase of the disease and effective DMT use in ARRMS. Single serum NfL and GFAP measurements cannot separate a patient with BRRMS from effectively treated ARRMS after a long history of the disease, thus consecutive samples are needed in the follow-up.