Serum biomarker levels predict disability progression in patients with primary progressive multiple sclerosis.

BACKGROUND: We aimed to investigate the potential of serum biomarker levels to predict disability progression in a multicentric real-world cohort of patients with primary progressive multiple sclerosis (PPMS). METHODS: A total of 141 patients with PPMS from 18 European MS centres were included. Disability progression was investigated using change in Expanded Disability Status Scale (EDSS) score over three time intervals: baseline to 2 years, 6 years and to the last follow-up. Serum levels of neurofilament light chain (sNfL), glial fibrillar acidic protein (sGFAP) and chitinase 3-like 1 (sCHI3L1) were measured using single-molecule array assays at baseline. Correlations between biomarker levels, and between biomarkers and age were quantified using Spearman's r. Univariable and multivariable linear models were performed to assess associations between biomarker levels and EDSS change over the different time periods. RESULTS: Median (IQR) age of patients was 52.9 (46.4-58.5) years, and 58 (41.1%) were men. Median follow-up time was 9.1 (7.0-12.6) years. Only 8 (5.7%) patients received treatment during follow-up. sNfL and sGFAP levels were moderately correlated (r=0.43) and both weakly correlated with sCHI3L1 levels (r=0.19 and r=0.17, respectively). In multivariable analyses, levels of the three biomarkers were associated with EDSS changes across all time periods. However, when analysis was restricted to non-inflammatory patients according to clinical and radiological parameters (n=64), only sCHI3L1 levels remained associated with future EDSS change. CONCLUSIONS: Levels of sNfL, sGFAP and sCHI3L1 are prognostic biomarkers associated with disability progression in patients with PPMS, being CHI3L1 findings less dependent on the inflammatory component associated with disease progression.

Association of magnetic resonance imaging phenotypes and serum biomarker levels with treatment response and long-term disease outcomes in multiple sclerosis patients.

BACKGROUND AND PURPOSE: The aim was to evaluate whether magnetic resonance imaging (MRI) phenotypes defined by inflammation and neurodegeneration markers correlate with serum levels of neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in relapsing-remitting multiple sclerosis (RRMS) patients; and to explore the role of radiological phenotypes and biomarker levels on treatment response and long-term prognostic outcomes. METHODS: Magnetic resonance imaging scans from 80 RRMS patients were classified at baseline of interferon-beta (IFNß) treatment into radiological phenotypes defined by high and low inflammation and high and low neurodegeneration, based on the number of contrast-enhancing lesions, brain parenchymal fraction and the relative volume of non-enhancing black holes on T1-weighted images. Serum levels of NfL and GFAP were measured at baseline with single molecule array (Simoa) assays. MRI phenotypes and serum biomarker levels were investigated for their association with IFNß response, and times to second-line therapies, secondary-progressive MS (SPMS) conversion and Expanded Disability Status Scale (EDSS) 6.0. RESULTS: Mean (SD) follow-up was 17 (2.9) years. Serum NfL levels and GFAP were higher in the high inflammation (p = 0.04) and high neurodegeneration phenotypes (p = 0.03), respectively. The high inflammation phenotype was associated with poor response to IFNß treatment (p = 0.04) and with shorter time to second-line therapies (p = 0.04). In contrast, the high neurodegeneration phenotype was associated with shorter time to SPMS (p = 0.006) and a trend towards shorter time to EDSS 6.0 (p = 0.09). High serum NfL levels were associated with poor response to IFNß treatment (p = 0.004). CONCLUSIONS: Magnetic resonance imaging phenotypes defined by inflammation and neurodegeneration correlate with serum biomarker levels, and both have prognostic implications in treatment response and long-term disease outcomes.

Development and multi-center validation of a fully automated digital immunoassay for neurofilament light chain: toward a clinical blood test for neuronal injury.

OBJECTIVES: Neurofilament light chain (NfL) has emerged as a promising biomarker for detecting and monitoring axonal injury. Until recently, NfL could only be reliably measured in cerebrospinal fluid, but digital single molecule array (Simoa) technology has enabled its precise measurement in blood samples where it is typically 50-100 times less abundant. We report development and multi-center validation of a novel fully automated digital immunoassay for NfL in serum for informing axonal injury status. METHODS: A 45-min immunoassay for serum NfL was developed for use on an automated digital analyzer based on Simoa technology. The analytical performance (sensitivity, precision, reproducibility, linearity, sample type) was characterized and then cross validated across 17 laboratories in 10 countries. Analytical performance for clinical NfL measurement was examined in individual patients with relapsing remitting multiple sclerosis (RRMS) after 3 months of disease modifying treatment (DMT) with fingolimod. RESULTS: The assay exhibited a lower limit of detection (LLoD) of 0.05 ng/L, a lower limit of quantification (LLoQ) of 0.8 ng/L, and between-laboratory imprecision <10 % across 17 validation sites. All tested samples had measurable NfL concentrations well above the LLoQ. In matched pre-post treatment samples, decreases in NfL were observed in 26/29 RRMS patients three months after DMT start, with significant decreases detected in a majority of patients. CONCLUSIONS: The sensitivity characteristics and reproducible performance across laboratories combined with full automation make this assay suitable for clinical use for NfL assessment, monitoring in individual patients, and cross-comparisons of results across multiple sites.

Increased cytomegalovirus immune responses at disease onset are protective in the long-term prognosis of patients with multiple sclerosis.

OBJECTIVE: It remains unclear whether viral infections interfere with multiple sclerosis (MS) disease progression. We evaluated the prognostic role of antibody responses toward viruses determined at disease onset on long-term disease outcomes. METHODS: Humoral immune responses against Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1, viral capsid antigen (VCA) and early antigen, and toward cytomegalovirus (HCMV), human herpesvirus 6 and measles were investigated in a cohort of 143 patients with MS for their association with long-term disability and inflammation disease outcomes. RESULTS: Median (IQR) follow-up was 20 (17.2-22.8) years. In univariable analysis, increased HCMV levels were associated with a lower risk to Expanded Disability Status Scale 4.0 (HR 0.95; 95% CI 0.91 to 0.99; p=0.03), to develop a secondary progressive MS (HR 0.94; 95% CI 0.90 to 0.99; p=0.02) and to first-line treatment (HR 0.98; 95% CI 0.96 to 0.99; p=0.04). High HCMV IgG levels were associated with a longer time to first-line treatment (p=0.01). Increased immune responses against EBV-VCA were associated with higher risk for first-line (HR 1.45; 95% CI 1.12 to 1.88; p=0.005) and second-line treatments (HR 2.03; 95% CI 1.18 to 3.49; p=0.01), and high VCA IgG levels were associated with shorter time to first-line (p=0.004) and second-line (p=0.02) therapies. EBNA1-specific IgG levels correlated with disease severity (0.17; p=0.04) and with an increased relapse rate during follow-up (relapse rate 1.26; 95% CI 1.03 to 1.56; p=0.02) that remained stable in multivariable analysis. CONCLUSIONS: These results indicate that elevated immune responses against HCMV at disease onset have protective effects on long-term disability and inflammation disease outcomes. Our data also indicate that increased immune responses against EBV in early phases may influence long-term disease prognosis.

The CYP24A1 gene variant rs2762943 is associated with low serum 1,25-dihydroxyvitamin D levels in multiple sclerosis patients.

BACKGROUND AND PURPOSE: Vitamin D is considered to play a role in multiple sclerosis (MS) etiopathogenesis. A polymorphism in the CYP24A1 gene, rs2762943, was recently identified that was associated with an increased MS risk. CYP24A1 encodes a protein involved in the catabolism of the active form of vitamin D. The immunological effects of carrying the rs2762943 risk allele were investigated, as well as its role as genetic modifier. METHODS: Serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D (1,25(OH)2 D) were measured in a cohort of 167 MS patients. In a subgroup of patients, expression levels of major histocompatibility complex class II and co-stimulatory molecules were determined by flow cytometry, and serum levels of pro-inflammatory (interferon gamma, granulocyte macrophage colony-stimulating factor, C-X-C motif chemokine ligand 13) and anti-inflammatory (interleukin 10) cytokines and neurofilament light chain were measured by single-molecule array assays. The effect of the rs2762943 polymorphism on disease activity and disability measures was evaluated in 340 MS patients. RESULTS: Compared to non-carriers, carriers of the rs2762943 risk allele were characterized by reduced levels of 1,25(OH)2 D (p = 0.0001) and elevated levels of interferon gamma (p = 0.03) and granulocyte macrophage colony-stimulating factor (p = 0.008), whereas no significant differences were observed for the other markers. The presence of the rs2762943 risk allele had no significant impact on disease activity and disability outcomes during follow-up. However, risk allele carriers were younger at disease onset (p = 0.04). CONCLUSIONS: These findings suggest that the CYP24A1 rs2762943 polymorphism plays a more important role in MS susceptibility than in disease prognosis and is associated with lower 1,25(OH)2 D levels and a heightened pro-inflammatory environment in MS patients.

Serum neurofilament light chain levels predict long-term disability progression in patients with progressive multiple sclerosis.

ObjectiveThere is a lack of sensitive and specific biomarkers for use in progressive multiple sclerosis (MS). The study aimed to assess the potential of serum neurofilament light chain (sNfL) levels as biomarker of disability progression in patients with progressive MS. METHODS: We performed a prospective observational cohort study in 51 patients with progressive MS who participated in a 2-year phase II single-centre, randomised, double-blind, placebo-controlled trial of interferon-beta. Mean (SD) follow-up duration was 13.9 (6.2) years. Levels of sNfL were measured using a single molecule array immunoassay at baseline, 1, 2 and 6 years. Univariable and multivariable analyses were carried out to evaluate associations between sNfL levels and disability progression at short term (2 years), medium term (6 years) and long term (at the time of the last follow-up). RESULTS: A sNfL cut-off value of 10.2 pg/mL at baseline discriminated between long-term progressors and non-progressors with a 75% sensitivity and 67% specificity (adjusted OR 7.8; 95% CI 1.8 to 46.4; p=0.01). Similar performance to discriminate between long-term progressors and non-progressors was observed using age/body mass index-adjusted sNfL Z-scores derived from a normative database of healthy controls. A cut-off increase of 5.1 pg/mL in sNfL levels between baseline and 6 years also discriminated between long-term progressors and non-progressors with a 71% sensitivity and 86% specificity (adjusted OR 49.4; 95% CI 4.4 to 2×103; p=0.008). CONCLUSIONS: sNfL can be considered a prognostic biomarker of future long-term disability progression in patients with progressive MS. These data expand the little knowledge existing on the role of sNfL as long-term prognostic biomarker in patients with progressive MS.

CSF chitinase 3-like 1 is associated with iron rims in patients with a first demyelinating event.

BACKGROUND: Chronic active lesions with iron rims have prognostic implications in patients with multiple sclerosis. OBJECTIVE: To assess the relationship between iron rims and levels of chitinase 3-like 1 (CHI3L1), neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in patients with a first demyelinating event. METHODS: Iron rims were identified using 3T susceptibility-weighted imaging. Serum NfL and GFAP levels were measured by single-molecule array assays. CSF (cerebrospinal fluid) CHI3L1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Sixty-one patients were included in the study. The presence of iron rims was associated with higher T2 lesion volume and higher number of gadolinium-enhancing lesions. In univariable analysis, having ⩾2 iron rims (vs 0) was associated with increased CSF CHI3L1 levels (ß = 1.41; 95% confidence interval (CI) = 1.10-1.79; p < 0.01) and serum NfL levels (ß = 2.30; 95% CI = 1.47-3.60; p < 0.01). In multivariable analysis, however, only CSF CHI3L1 levels remained significantly associated with the presence of iron rim lesions (ß = 1.45; 95% CI = 1.11-1.90; p < 0.01). The presence of ⩾2 iron rims was not associated with increased serum GFAP levels in univariable or multivariable analyses. CONCLUSION: These findings support an important contribution of activated microglia/macrophages to the pathophysiology of chronic active lesions with iron rims in patients with a first demyelinating event.

NLRP3 inflammasome as prognostic factor and therapeutic target in primary progressive multiple sclerosis patients.

Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.

Cerebrospinal fluid mitochondrial DNA levels in patients with multiple sclerosis.

The role of cerebrospinal fluid (CSF) mitochondrial DNA (mtDNA) levels as biomarker in multiple sclerosis (MS) is unknown. We determined CSF mtDNA levels in a cohort of 237 individuals, including patients with MS and clinically isolated syndrome (CIS), inflammatory and non-inflammatory neurological controls, and cognitively healthy controls (HC). mtDNA concentration was measured by droplet digital polymerase chain reaction. CSF mtDNA levels were increased in all pathological conditions compared with HC, though no differences were observed between relapse-onset and progressive MS clinical forms, CIS patients and neurological controls. These findings do not support the determination of CSF mtDNA levels as a useful biomarker in MS clinical practice.

MRI phenotypes with high neurodegeneration are associated with peripheral blood B-cell changes.

Little is known about the mechanisms leading to neurodegeneration in multiple sclerosis (MS) and the role of peripheral blood cells in this neurodegenerative component. We aimed to correlate brain radiological phenotypes defined by high and low neurodegeneration with gene expression profiling of peripheral blood mononuclear cells (PBMC) from MS patients. Magnetic resonance imaging (MRI) scans from 64 patients with relapsing-remitting MS (RRMS) were classified into radiological phenotypes characterized by low (N = 27) and high (N = 37) neurodegeneration according to the number of contrast-enhancing lesions, the relative volume of non-enhancing black holes on T1-weighted images, and the brain parenchymal fraction. Gene expression profiling was determined in PBMC using microarrays, and validation of selected genes was performed by polymerase chain reaction (PCR). B-cell immunophenotyping was conducted by flow cytometry. Microarray analysis revealed the B-cell specific genes FCRL1, FCRL2, FCRL5 (Fc receptor-like 1, 2 and 5 respectively), and CD22 as the top differentially expressed genes between patients with high and low neurodegeneration. Levels for these genes were significantly down-regulated in PBMC from patients with MRI phenotypes characterized by high neurodegeneration and microarray findings were validated by PCR. In patients with high neurodegeneration, immunophenotyping showed a significant increase in the expression of the B-cell activation markers CD80 in naïve B cells (CD45+/CD19+/CD27-/IgD+), unswitched memory B cells (CD45+/CD19+/CD27+/IgD+), and switched memory B cells (CD45+/CD19+/CD27+/IgD-), and CD86 in naïve and switched memory B cells. These results suggest that RRMS patients with radiological phenotypes showing high neurodegeneration have changes in B cells characterized by down-regulation of B-cell-specific genes and increased activation status.

Circulating EZH2-positive T cells are decreased in multiple sclerosis patients.

BACKGROUND: Recent studies in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), suggest an involvement of the histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) in important processes such as cell adhesion and migration. METHODS: Here, we aimed to expand these initial observations by investigating the role of EZH2 in MS. mRNA expression levels for EZH2 were measured by real-time PCR in peripheral blood mononuclear cells (PBMC) from 121 MS patients (62 untreated and 59 receiving treatment) and 24 healthy controls. RESULTS: EZH2 expression levels were decreased in PBMC from untreated patients compared to that from controls, and treatment significantly upregulated EZH2 expression. Expression of miR-124 was increased in MS patients compared to controls. Blood immunophenotyping revealed EZH2 expression mostly restricted to CD4+ and CD8+ T cells, and circulating EZH2+ CD4+ and CD8+ T cells were decreased in untreated MS patients compared to controls. CD8+ T cells expressing EZH2 exhibited a predominant central memory phenotype, whereas EZH2+ CD4+ T cells were of effector memory nature, and both T cell subsets produced TNF-α. EZH2+ T cells were enriched in the cerebrospinal fluid compartment compared to blood and were found in chronic active lesions from MS patients. EZH2 inhibition and microarray analysis in PBMC was associated with significant downregulation of key T cell adhesion molecules. CONCLUSION: These findings suggest a role of EZH2 in the migration of T cells in MS patients. The observation of TNF-α expression by CD4+ and CD8+ T cells expressing EZH2 warrants additional studies to explore more in depth the pathogenic potential of EZH2+-positive cells in MS.

Exome sequencing study in patients with multiple sclerosis reveals variants associated with disease course.

BACKGROUND: It remains unclear whether disease course in multiple sclerosis (MS) is influenced by genetic polymorphisms. Here, we aimed to identify genetic variants associated with benign and aggressive disease courses in MS patients. METHODS: MS patients were classified into benign and aggressive phenotypes according to clinical criteria. We performed exome sequencing in a discovery cohort, which included 20 MS patients, 10 with benign and 10 with aggressive disease course, and genotyping in 2 independent validation cohorts. The first validation cohort encompassed 194 MS patients, 107 with benign and 87 with aggressive phenotypes. The second validation cohort comprised 257 patients, of whom 224 patients had benign phenotypes and 33 aggressive disease courses. Brain immunohistochemistries were performed using disease course associated genes antibodies. RESULTS: By means of single-nucleotide polymorphism (SNP) detection and comparison of allele frequencies between patients with benign and aggressive phenotypes, a total of 16 SNPs were selected for validation from the exome sequencing data in the discovery cohort. Meta-analysis of genotyping results in two validation cohorts revealed two polymorphisms, rs28469012 and rs10894768, significantly associated with disease course. SNP rs28469012 is located in CPXM2 (carboxypeptidase X, M14 family, member 2) and was associated with aggressive disease course (uncorrected p value < 0.05). SNP rs10894768, which is positioned in IGSF9B (immunoglobulin superfamily member 9B) was associated with benign phenotype (uncorrected p value < 0.05). In addition, a trend for association with benign phenotype was observed for a third SNP, rs10423927, in NLRP9 (NLR family pyrin domain containing 9). Brain immunohistochemistries in chronic active lesions from MS patients revealed expression of IGSF9B in astrocytes and macrophages/microglial cells, and expression of CPXM2 and NLRP9 restricted to brain macrophages/microglia. CONCLUSIONS: Genetic variants located in CPXM2, IGSF9B, and NLRP9 have the potential to modulate disease course in MS patients and may be used as disease activity biomarkers to identify patients with divergent disease courses. Altogether, the reported results from this study support the influence of genetic factors in MS disease course and may help to better understand the complex molecular mechanisms underlying disease pathogenesis.

Matrix metalloproteinase 9 is decreased in natalizumab-treated multiple sclerosis patients at risk for progressive multifocal leukoencephalopathy.

OBJECTIVE: To identify biomarkers associated with the development of progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS) patients treated with natalizumab (NTZ). METHODS: Relapsing-remitting MS patients who developed PML under NTZ therapy (pre-PML) and non-PML NTZ-treated patients (NTZ-ctr) were included in the study. Cryopreserved peripheral blood mononuclear cells and serum samples collected at baseline, at 1- and 2-year treated time points, and during PML were analyzed for gene expression by RNA sequencing and for serum protein levels by Luminex and enzyme-linked immunosorbent assays, respectively. RESULTS: Among top differentially expressed genes in the RNA sequencing between pre-PML and NTZ-ctr patients, pathway analysis revealed a high representation of genes belonging to the following categories: proangiogenic factors (MMP9, VEGFA), chemokines (CXCL1, CXCL5, IL8, CCL2), cytokines (IL1B, IFNG), and plasminogen- and coagulation-related molecules (SERPINB2, PLAU, PLAUR, TFPI, THBD). Serum protein levels for these candidates were measured in a 2-step manner in a screening cohort and a validation cohort of pre-PML and NTZ-ctr patients. Only matrix metalloproteinase 9 (MMP9) was validated; in pre-PML patients, MMP9 protein levels were significantly reduced at baseline compared with NTZ-ctr patients, and levels remained lower at later time points during NTZ treatment. INTERPRETATION: The results from this study suggest that the proangiogenic factor MMP9 may play a role as a biomarker associated with the development of PML in MS patients treated with NTZ. Ann Neurol 2017;82:186-195.

Role of high mobility group box protein 1 (HMGB1) in peripheral blood from patients with multiple sclerosis.

BACKGROUND: High mobility group box protein 1 (HMGB1) is a transcriptional regulator that is receiving increasing attention in autoimmune disorders including multiple sclerosis (MS). Here, we investigated the role of HMGB1 in the peripheral blood compartment from MS patients. METHODS: HMGB1 mRNA expression levels were determined by PCR in peripheral blood mononuclear cells (PBMC) of 29 healthy controls and 57 untreated MS patients (26 with relapsing-remitting MS - RRMS, 13 with secondary progressive MS - SPMS, and 18 with primary progressive MS - PPMS). HMGB1 protein levels were measured by ELISA in serum samples from 18 HC and 37 untreated MS patients (13 with RRMS, 14 with SPMS, and 10 with PPMS). RESULTS: HMGB1 expression levels were increased in PBMC from the whole MS group compared with controls (P = 0.03). Further stratification of the MS group revealed higher expression levels in PBMC from patients with relapse-onset MS, and differences were statistically significant for RRMS patients compared with PPMS patients and controls (P = 4 × 10(-5) and P = 0.005, respectively) and also for SPMS patients compared with PPMS patients (P = 0.001). HMGB1 serum levels were increased in the whole MS group compared with controls (P = 2 × 10(-4)). In MS clinical forms, the highest HMGB1 serum levels were observed in RRMS patients, and differences were statistically significant compared to PPMS patients (P = 5 × 10(-5)), SPMS patients (P = 0.001), and controls (P = 0.001). CONCLUSIONS: These results point to a role of HMGB1 mRNA and protein levels as disease activity biomarkers to discriminate the more inflammatory relapse-onset MS forms, particularly RRMS, from the less inflammatory PPMS form of the disease.

Association of Complement Factors With Disability Progression in Primary Progressive Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The complement system is known to play a role in multiple sclerosis (MS) pathogenesis. However, its contribution to disease progression remains elusive. The study investigated the role of the complement system in disability progression of patients with primary progressive MS (PPMS). METHODS: Sixty-eight patients with PPMS from 12 European MS centers were included in the study. Serum and CSF levels of a panel of complement components (CCs) were measured by multiplex enzyme-linked immunosorbent assay at a baseline time point (i.e., sampling). Mean (SD) follow-up time from baseline was 9.6 (4.8) years. Only one patient (1.5%) was treated during follow-up. Univariable and multivariable logistic regressions adjusted for age, sex, and albumin quotient were performed to assess the association between baseline CC levels and disability progression in short term (2 years), medium term (6 years), and long term (at the time of the last follow-up). RESULTS: In short term, CC played little or no role in disability progression. In medium term, an elevated serum C3a/C3 ratio was associated with a higher risk of disability progression (adjusted OR 2.30; 95% CI 1.17-6.03; p = 0.040). By contrast, increased CSF C1q levels were associated with a trend toward reduced risk of disability progression (adjusted OR 0.43; 95% CI 0.17-0.98; p = 0.054). Similarly, in long term, an elevated serum C3a/C3 ratio was associated with higher risk of disability progression (adjusted OR 1.81; 95% CI 1.09-3.40; p = 0.037), and increased CSF C1q levels predicted lower disability progression (adjusted OR 0.41; 95% CI 0.17-0.86; p = 0.025). DISCUSSION: Proteins involved in the activation of early complement cascades play a role in disability progression as risk (elevated serum C3a/C3 ratio) or protective (elevated CSF C1q) factors after 6 or more years of follow-up in patients with PPMS. The protective effects associated with C1q levels in CSF may be related to its neuroprotective and anti-inflammatory properties.

Serum Levels of CXCL13 Are Associated With Teriflunomide Response in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: To identify biomarkers associated with treatment response in patients with multiple sclerosis (MS) treated with the oral therapies teriflunomide, dimethyl fumarate (DMF), and fingolimod. METHODS: Serum levels of IL-6, IL-17, TNF-α, granulocyte-macrophage colony-stimulating factor, IL-10, interferon-gamma (IFN-γ) IL-1ß, and chemokine ligand 13 (CXCL13) were measured at baseline and 12 months with single molecule array (Simoa) assays in a cohort of patients with MS treated with teriflunomide (N = 19), DMF (N = 22), and fingolimod (N = 25) and classified into "no evidence of disease activity" (NEDA) and EDA patients after 1 year of treatment. RESULTS: Serum CXCL13 and TNF-α levels were significantly decreased after treatment with teriflunomide in NEDA compared with EDA patients after 1 year of treatment (p = 0.008 for both cytokines). These findings were validated in an independent cohort of patients with MS treated with teriflunomide (N = 36) and serum CXCL13, and TNF-α levels were again significantly reduced in NEDA patients (p < 0.0001 for CXCL13 and p = 0.003 for TNF-α). CXCL13, but not TNF-α, showed good performance to classify NEDA and EDA patients according to a cut-off value of 9.64 pg/mL based on the change in CXCL13 levels between baseline and 12 months, with a sensitivity of 75% and specificity of 82% in the original cohort, and sensitivity of 65.4% and specificity of 60% in the validation cohort. DISCUSSION: Altogether, these results point to CXCL13 as a treatment response biomarker to teriflunomide in relapsing-remitting patients with MS, and the change in CXCL13 levels during the first year of treatment can be used in clinical practice to identify optimal responders to teriflunomide.

Molecular signature associated with cladribine treatment in patients with multiple sclerosis.

Introduction: Little is known about the molecular profiling associated with the effect of cladribine in patients with multiple sclerosis (MS). Here, we aimed first to characterize the transcriptomic and proteomic profiles induced by cladribine in blood cells, and second to identify potential treatment response biomarkers to cladribine in patients with MS. Methods: Gene, protein and microRNA (miRNA) expression profiles were determined by microarrays (genes, miRNAs) and mass spectrometry (proteins) in peripheral blood mononuclear cells (PBMCs) from MS patients after in vitro treatment with cladribine in its active and inactive forms. Two bioinformatics approaches to integrate the three obtained datasets were applied: (i) a multiomics discriminant analysis (DIABLO - Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies); and (ii) a multi-stage integration of features selected in differential expression analysis on each dataset and then merged. Selected molecules from the in vitro study were quantified by qPCR ex vivo in PBMCs from MS patients receiving cladribine. Results: PBMCs treated in vitro with cladribine were characterized by a major downregulation of gene, protein, and miRNA expression compared with the untreated cells. An intermediate pattern between the cladribine-treated and untreated conditions was observed in PBMCs treated with cladribine in its inactive form. The differential expression analysis of each dataset led to the identification of four genes and their encoded proteins, and twenty-two miRNAs regulating their expression, that were associated with cladribine treatment. Two of these genes (PPIF and NHLRC2), and three miRNAs (miR-21-5p, miR-30b-5p, and miR-30e-5p) were validated ex vivo in MS patients treated with cladribine. Discussion: By using a combination of omics data and bioinformatics approaches we were able to identify a multiomics molecular profile induced by cladribine in vitro in PBMCs. We also identified a number of biomarkers that were validated ex vivo in PBMCs from patients with MS treated with cladribine that have the potential to become treatment response biomarkers to this drug.

DNA-based vaccines for multiple sclerosis: current status and future directions.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that mainly affects young adults between 20 and 40 years of age and leads to significant disability. Although MS is considered to be an immune-mediated disorder, current immunosuppressive therapies fail to inhibit disease progression, and some of them are associated with serious adverse reactions. DNA vaccination is a strategy of immunization based on the injection of genes encoding for target proteins. Depending on the route as well as the dosage of administration, exposure to certain molecules may either stimulate effector responses or induce immune tolerance. A large body of data from the animal model, experimental autoimmune encephalomyelitis (EAE), has demonstrated efficacy of DNA vaccination at inhibiting the disease through the induction of basic tolerizing mechanisms such as anergy, clonal deletion, immune deviation, or induction of regulatory cells. Interestingly, recent phase I and II clinical trials in MS with DNA vaccines have shown positive results in reducing MRI-measured disease activity in patients with relapse-onset MS, and inducing antigen-specific tolerance to myelin-specific B and T cells. Thus, DNA vaccines represent a promising therapeutic approach for MS which also seem to overcome the safety concerns raised by other currently tested therapeutic strategies. Here, we will review existing data from MS and EAE studies on DNA vaccination and discuss on further optimization of the DNA technology in order to improve treatment efficacy.

Treatment with MOG-DNA vaccines induces CD4+CD25+FoxP3+ regulatory T cells and up-regulates genes with neuroprotective functions in experimental autoimmune encephalomyelitis.

BACKGROUND: DNA vaccines represent promising therapeutic strategies in autoimmune disorders such as multiple sclerosis (MS). However, the precise mechanisms by which DNA vaccines induce immune regulation remain largely unknown. Here, we aimed to expand previous knowledge existing on the mechanisms of action of DNA vaccines in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), by treating EAE mice with a DNA vaccine encoding the myelin oligodendrocyte glycoprotein (MOG), and exploring the therapeutic effects on the disease-induced inflammatory and neurodegenerative changes. METHODS: EAE was induced in C57BL6/J mice by immunization with MOG35₋55 peptide. Mice were intramuscularly treated with a MOG-DNA vaccine or vehicle in prophylactic and therapeutic approaches. Histological studies were performed in central nervous system (CNS) tissue. Cytokine production and regulatory T cell (Treg) quantification were achieved by flow cytometry. Gene expression patterns were determined using microarrays, and the main findings were validated by real-time PCR. RESULTS: MOG-DNA treatment reduced the clinical and histopathological signs of EAE when administered in both prophylactic and therapeutic settings. Suppression of clinical EAE was associated with dampening of antigen (Ag)-specific proinflammatory Th1 and Th17 immune responses and, interestingly, expansion of Treg in the periphery and upregulation in the CNS of genes encoding neurotrophic factors and proteins involved in remyelination. CONCLUSIONS: These results suggest for the first time that the beneficial effects of DNA vaccines in EAE are not limited to anti-inflammatory mechanisms, and DNA vaccines may also exert positive effects through hitherto unknown neuroprotective mechanisms.