Redefining the Multiple Sclerosis Severity Score (MSSS): The effect of sex and onset phenotype.

BACKGROUND: The Multiple Sclerosis Severity Score (MSSS) is a widely used measure of the disability progression rate. However, the global MSSS may not be the best basis for comparison between all patient groups. OBJECTIVE: We evaluated sex-specific and onset phenotype-specific MSSS matrices to determine if they were more effective than the global MSSS as a basis for comparison within these subsets. METHODS: Using a large international dataset of multiple sclerosis (MS) patient records and the original MSSS algorithm, we constructed global, sex-specific and onset phenotype-specific MSSS matrices. We compared matrices using permutation analysis. RESULTS: Our final dataset included 30,203 MS cases, with 28.9% males and 6.5% progressive-onset cases. Our global MSSS matrix did not differ from previously published data (p > 0.05). The progressive-onset-specific matrix differed significantly from the relapsing-onset-specific matrix (p < 0.001), with lower MSSS attributed to cases with the same Expanded Disability Status Score (EDSS) and disease duration. When evaluated with a simulation, using an onset-specific MSSS improved statistical power in mixed cohorts. There were no significant differences by sex. CONCLUSION: The differences in the disability accrual rate between progressive- and relapsing-onset MS have a significant effect on MSSS. An onset-specific MSSS should be used when comparing the rate of disability progression among progressive-onset cases and for mixed cohorts.

Effectiveness of Fingolimod versus Natalizumab as Second-Line Therapy for Relapsing-Remitting Multiple Sclerosis in Spain: Second-Line GATE Study.

BACKGROUND: There is a lack of head-to-head studies comparing the efficacy of fingolimod (FIN) and natalizumab (NTZ) as second-line therapy for relapsing-remitting multiple sclerosis (RRMS). METHODS: Multicenter, observational study, in which, information of 388 patients randomly selected and treated with FIN or NTZ in routine clinical practice was retrospectively collected with the main objective of comparing the annualized relapse rate (ARR) over the first year, after FIN or NTZ treatment initiation. RESULTS: Mean ARR during the first year of treatment was 0.28 in FIN group and 0.12 in NTZ group (p = 0.0064); nevertheless, the difference between groups lost statistical significance when the propensity score analysis was performed. Time to disability -progression was similar in both treatment groups (12.3 ± 6.7 months in FIN, and 12.8 ± 0.1 months in NTZ; p = 0.4654). Treatment persistence after the first year of treatment was higher in patients treated with FIN (95%) than in those treated with NTZ (84%; p = 0.0014). CONCLUSIONS: After 12 months of treatment, both FIN and NTZ reduced the ARR, but ARR percent reduction was significantly higher with NTZ. Treatment persistence was higher in patients receiving FIN.

Neurofilament light chain and oligoclonal bands are prognostic biomarkers in radiologically isolated syndrome.

The prognostic role of cerebrospinal fluid molecular biomarkers determined in early pathogenic stages of multiple sclerosis has yet to be defined. In the present study, we aimed to investigate the prognostic value of chitinase 3 like 1 (CHI3L1), neurofilament light chain, and oligoclonal bands for conversion to clinically isolated syndrome and to multiple sclerosis in 75 patients with radiologically isolated syndrome. Cerebrospinal fluid levels of CHI3L1 and neurofilament light chain were measured by enzyme-linked immunosorbent assay. Uni- and multivariable Cox regression models including as covariates age at diagnosis of radiologically isolated syndrome, number of brain lesions, sex and treatment were used to investigate associations between cerebrospinal fluid CHI3L1 and neurofilament light chain levels and time to conversion to clinically isolated syndrome and multiple sclerosis. Neurofilament light chain levels and oligoclonal bands were independent risk factors for the development of clinically isolated syndrome (hazard ratio = 1.02, P = 0.019, and hazard ratio = 14.7, P = 0.012, respectively) and multiple sclerosis (hazard ratio = 1.03, P = 0.003, and hazard ratio = 8.9, P = 0.046, respectively). The best cut-off to classify cerebrospinal fluid neurofilament light chain levels into high and low was 619 ng/l, and high neurofilament light chain levels were associated with a trend to shorter time to clinically isolated syndrome (P = 0.079) and significant shorter time to multiple sclerosis (P = 0.017). Similarly, patients with radiologically isolated syndrome presenting positive oligoclonal bands converted faster to clinically isolated syndrome and multiple sclerosis (P = 0.005 and P = 0.008, respectively). The effects of high neurofilament light chain levels shortening time to clinically isolated syndrome and multiple sclerosis were more pronounced in radiologically isolated syndrome patients with ≥37 years compared to younger patients. Cerebrospinal fluid CHI3L1 levels did not influence conversion to clinically isolated syndrome and multiple sclerosis in radiologically isolated syndrome patients. Overall, these findings suggest that cerebrospinal neurofilament light chain levels and oligoclonal bands are independent predictors of clinical conversion in patients with radiologically isolated syndrome. The association with a faster development of multiple sclerosis reinforces the importance of cerebrospinal fluid analysis in patients with radiologically isolated syndrome.

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.

Optimal response to dimethyl fumarate associates in MS with a shift from an inflammatory to a tolerogenic blood cell profile.

BACKGROUND: The precise mechanism of action of dimethyl fumarate (DMF) treatment in MS remains unknown. OBJECTIVE: To identify the changes in the blood lymphocyte profile of MS patients predicting no evidence of disease activity (NEDA) status after DMF treatment. METHODS: We studied blood lymphocyte subsets of 64 MS patients treated with DMF at baseline and after 6 months of treatment by flow cytometry. NEDA (41 patients) or ongoing disease activity (ODA, 23 patients) were monitored after a year of follow-up. RESULTS: During treatment, all patients experienced an increase in the naive T cells and a decrease in effector memory ones. However, only NEDA patients showed a significant reduction in central memory CD4+ and CD8+ T cells, memory B cells, CD4+ T cells producing interferon (IFN)-gamma, CD8+ T cells producing tumor necrosis factor-alpha (TNF-alpha), and IFN-gamma and B cells producing TNF-alpha. Additionally, they had an increase in regulatory CD56bright cells not observed in ODA group. After treatment, there was a negative correlation between CD56bright cells and CD8+ T cells producing IFN-gamma and TNF-alpha. CONCLUSION: A pro-tolerogenic shift in the blood leukocyte profile associates with an optimal response to DMF in MS.

NLRP3 polymorphisms and response to interferon-beta in multiple sclerosis patients.

We aimed to investigate whether NLR family, pyrin domain containing 3 (NLRP3) polymorphisms are associated with the response to interferon-beta (IFNß) in multiple sclerosis (MS) patients. A total of 14 NLRP3 polymorphisms were genotyped in a cohort of 665 relapsing-remitting MS patients recruited across 5 centers and classified into responders and non-responders according to clinical-radiological criteria after 1 year of IFNß treatment. A meta-analysis failed to demonstrate significant associations between the response to IFNß and NLRP3 polymorphisms. These findings do not support a role of polymorphisms located in the NLRP3 gene and the response to IFNß in MS patients.

Decreased soluble IFN-ß receptor (sIFNAR2) in multiple sclerosis patients: A potential serum diagnostic biomarker.

BACKGROUND: The soluble isoform of the interferon-ß (IFN-ß) receptor (sIFNAR2) could modulate the activity of both endogenous and systemically administered IFN-ß. Previously, we described lower serum sIFNAR2 levels in untreated multiple sclerosis (MS) than in healthy controls (HCs). OBJECTIVE: To assess sIFNAR2 levels in a new cohort of MS patients and HCs, as well as in patients with clinically isolated syndrome (CIS) and with other inflammatory neurological disorders (OIND) and to assess its ability as a diagnostic biomarker. METHODS: The cross-sectional study included 148 MS (84 treatment naive and 64 treated), 87 CIS, 42 OIND, and 96 HCs. Longitudinal study included 94 MS pretreatment and after 1 year of therapy with IFN-ß, glatiramer acetate (GA), or natalizumab. sIFNAR2 serum levels were measured by a quantitative ELISA developed and validated in our laboratory. RESULTS: Naive MS and CIS patients showed significantly lower sIFNAR2 levels than HCs and OIND patients. The sensitivity and specificity to discriminate between MS and OIND, for a sIFNAR2 cutoff value of 122.02 ng/mL, were 70.1%, and 79.4%, respectively. sIFNAR2 increased significantly in IFN-ß-treated patients during the first year of therapy in contrast to GA- and natalizumab-treated patients who showed non-significant changes. CONCLUSION: The results suggest that sIFNAR2 could be a potential diagnostic biomarker for MS.

Lipid-specific immunoglobulin M bands in cerebrospinal fluid are associated with a reduced risk of developing progressive multifocal leukoencephalopathy during treatment with natalizumab.

OBJECTIVE: Progressive multifocal leukoencephalopathy (PML) is a serious side effect associated with natalizumab treatment in multiple sclerosis (MS). PML risk increases in individuals seropositive for anti-John Cunningham virus (JC) antibodies, with prolonged duration of natalizumab treatment, and with prior exposure to immunosuppressants. We explored whether the presence of lipid-specific immunoglobulin M oligoclonal bands in cerebrospinal fluid (CSF; IgM bands), a recognized marker of highly inflammatory MS, may identify individuals better able to counteract the potential immunosuppressive effect of natalizumab and hence be associated with a reduced risk of developing PML. METHODS: We studied 24 MS patients who developed PML and another 343 who did not suffer this opportunistic infection during natalizumab treatment. Patients were recruited at 25 university hospitals. IgM bands were studied by isoelectric focusing and immunodetection. CSF lymphocyte counts were explored in 151 MS patients recruited at Ramon y Cajal Hospital in Madrid, Spain. RESULTS: IgM bands were independently associated with decreased PML risk (odds ratio [OR] = 45.9, 95% confidence interval [CI] = 5.9-339.3, p < 0.0001) in patients treated with natalizumab. They were also associated with significantly higher CSF CD4, CD8, and B-cell numbers. Patients positive for IgM bands and anti-JC antibodies had similar levels of reduced PML risk to those who were anti-JC negative (OR = 1.55, 95% CI = 0.09-25.2, p = 1.0). Higher risk was observed in patients positive for anti-JC antibodies and negative for IgM bands (19% of the total cohort, OR = 59.71, 95% CI = 13.6-262.2). INTERPRETATION: The presence of IgM bands reflects a process that may diminish the risk of PML by counteracting the excess of immunosuppression that may occur during natalizumab therapy.

Adaptive natural killer cell response to cytomegalovirus and disability progression in multiple sclerosis.

BACKGROUND: Human cytomegalovirus (HCMV) causes a highly prevalent infection which may have a multifaceted impact on chronic inflammatory disorders. However, its potential influence in multiple sclerosis (MS) remains controversial. The HCMV-host interaction may induce an adaptive reconfiguration of the natural killer (NK) cell compartment, whose hallmark is a persistent expansion of peripheral NKG2C+ NK-cells. OBJECTIVE: The purpose of this study was to evaluate whether the HCMV-driven NKG2C+ NK-cell expansion is related to the MS clinical course. METHODS: Multicentre analysis of NKG2C expression and genotype according to HCMV serostatus and time of assignment of irreversible disability scores in 246 MS patients prospectively followed up in our institutions. RESULTS: NKG2C expression was unrelated to disease-modifying drugs, remained stable under steady-state conditions, and was higher in HCMV(+) NKG2C(+/+) homozygous individuals. NKG2C+ NK-cell expansion in HCMV(+) patients, as compared to HCMV(+) or HCMV(-) patients with lower NKG2C+ NK-cells proportions, conferred a lower risk of progression in Cox regression analysis (Expanded Disability Status Scale (EDSS)>3.0, hazard ratio (HR)=0.33, 95% confidence interval (CI) 0.15-0.71, p=0.005; EDSS>5.5, HR=0.23, 95% CI 0.07-0.74, p=0.014). Neither HCMV serostatus nor NKG2C genotype appeared to be related to disability progression. CONCLUSIONS: HCMV may exert a beneficial influence on MS, decreasing the risk of disability progression in those patients displaying a virus-driven NKG2C+ NK-cell expansion.

Sperm-associated antigen 16 is a novel target of the humoral autoimmune response in multiple sclerosis.

We have previously identified eight novel autoantibody targets in the cerebrospinal fluid of multiple sclerosis (MS) patients, including sperm-associated Ag 16 (SPAG16). In the current study, we further investigated the autoantibody response against SPAG16-a protein with unknown function in the CNS-and its expression in MS pathology. Using isoelectric focusing, we detected SPAG16-specific oligoclonal bands in the cerebrospinal fluid of 5 of 23 MS patients (22%). Analysis of the anti-SPAG16 Ab reactivity in the plasma of a total of 531 donors using ELISA demonstrated significantly elevated anti-SPAG16 Ab levels (p = 0.002) in 32 of 153 MS patients (21%) compared with all other control groups with 95% specificity for the disease. To investigate the pathologic relevance of anti-SPAG16 Abs in vivo, anti-SPAG16 Abs were injected in mice with experimental autoimmune encephalomyelitis, resulting in a significant disease exacerbation. Finally, we demonstrated a consistent upregulation of SPAG16 in MS brain and experimental autoimmune encephalomyelitis spinal cord lesions, more specifically in reactive astrocytes. We conclude that SPAG16 is a novel autoantibody target in a subgroup of MS patients and in combination with other diagnostic criteria, elevated levels of anti-SPAG16 Abs could be used as a biomarker for diagnosis. Furthermore, the pathologic relevance of anti-SPAG16 Abs was shown in vivo.

Chitinase 3-like 1: prognostic biomarker in clinically isolated syndromes.

Chitinase 3-like 1 (CHI3L1) has been proposed as a biomarker associated with the conversion to clinically definite multiple sclerosis in patients with clinically isolated syndromes, based on the finding of increased cerebrospinal fluid CHI3L1 levels in clinically isolated syndrome patients who later converted to multiple sclerosis compared to those who remained as clinically isolated syndrome. Here, we aimed to validate CHI3L1 as a prognostic biomarker in a large cohort of patients with clinically isolated syndrome. This is a longitudinal cohort study of clinically isolated syndrome patients with clinical, magnetic resonance imaging, and cerebrospinal fluid data prospectively acquired. A total of 813 cerebrospinal fluid samples from patients with clinically isolated syndrome were recruited from 15 European multiple sclerosis centres. Cerebrospinal fluid CHI3L1 levels were measured by enzyme-linked immunosorbent assay. Multivariable Cox regression models were used to investigate the association between cerebrospinal fluid CHI3L1 levels and time to conversion to multiple sclerosis and time to reach Expanded Disability Status Scale 3.0. CHI3L1 levels were higher in patients who converted to clinically definite multiple sclerosis compared to patients who continued as clinically isolated syndrome (P = 8.1 × 10(-11)). In the Cox regression analysis, CHI3L1 levels were a risk factor for conversion to multiple sclerosis (hazard ratio = 1.7; P = 1.1 × 10(-5) using Poser criteria; hazard ratio = 1.6; P = 3.7 × 10(-6) for McDonald criteria) independent of other covariates such as brain magnetic resonance imaging abnormalities and presence of cerebrospinal fluid oligoclonal bands, and were the only significant independent risk factor associated with the development of disability (hazard ratio = 3.8; P = 2.5 × 10(-8)). High CHI3L1 levels were associated with shorter time to multiple sclerosis (P = 3.2 × 10(-9) using Poser criteria; P = 5.6 × 10(-11) for McDonald criteria) and more rapid development of disability (P = 1.8 × 10(-10)). These findings validate cerebrospinal fluid CHI3L1 as a biomarker associated with the conversion to multiple sclerosis and development of disability and reinforce the prognostic role of CHI3L1 in patients with clinically isolated syndrome. We propose that determining cerebrospinal fluid chitinase 3-like 1 levels at the time of a clinically isolated syndrome event will help identify those patients with worse disease prognosis.

Immunoglobulin M oligoclonal bands: biomarker of targetable inflammation in primary progressive multiple sclerosis.

OBJECTIVE: To identify a biomarker distinguishing patients who, despite a primary progressive multiple sclerosis (PPMS) clinical course, may nonetheless benefit from immune therapy. METHODS: The presence or absence of both immunoglobulin (Ig) G and IgM oligoclonal bands (OCB) was blindly examined in paired cerebrospinal fluid (CSF) and serum samples from a large PPMS patient cohort, and related to clinical and imaging evidence of focal inflammatory disease activity. RESULTS: Using both cross-sectional samples and serial sampling in a subgroup of patients followed prospectively as part of the placebo-controlled OLYMPUS study of rituximab in PPMS, we found that the presence of CSF-restricted IgM OCB (but not of IgG OCB) is associated with an active inflammatory disease phenotype in PPMS patients. This finding was confirmed in an independent, multicenter validation cohort. INTERPRETATION: The presence of CSF IgM OCB may be a biomarker for a subset of PPMS patients with more active inflammatory disease, who may benefit from immune-directed treatments.

Consensus definitions and application guidelines for control groups in cerebrospinal fluid biomarker studies in multiple sclerosis.

The choice of appropriate control group(s) is critical in cerebrospinal fluid (CSF) biomarker research in multiple sclerosis (MS). There is a lack of definitions and nomenclature of different control groups and a rationalized application of different control groups. We here propose consensus definitions and nomenclature for the following groups: healthy controls (HCs), spinal anesthesia subjects (SASs), inflammatory neurological disease controls (INDCs), peripheral inflammatory neurological disease controls (PINDCs), non-inflammatory neurological controls (NINDCs), symptomatic controls (SCs). Furthermore, we discuss the application of these control groups in specific study designs, such as for diagnostic biomarker studies, prognostic biomarker studies and therapeutic response studies. Application of these uniform definitions will lead to better comparability of biomarker studies and optimal use of available resources. This will lead to improved quality of CSF biomarker research in MS and related disorders.

Novel cerebrospinal fluid and serum autoantibody targets for clinically isolated syndrome.

Limited information is available on the identity of antigens targeted by antibodies present in cerebrospinal fluid (CSF) of patients with clinically isolated syndrome (CIS). The aim of this study was to identify novel antigens for CIS and investigate their prognostic potential to predict conversion to multiple sclerosis (MS). We applied serological antigen selection (SAS) to identify antigens interacting with antibodies present in the pooled CSF from four CIS patients, who developed MS. Antibody reactivity towards CIS antigens identified by SAS was tested in CSF and serum from patients with CIS (n = 123/n = 108), MS (n = 65/n = 44), and other (inflammatory) neurological diseases (n = 75/n = 38) as well as in healthy control sera (n = 44). Using SAS, a panel of six novel CIS candidate antigens was identified. CSF antibody reactivity was detected in both CIS and relapsing-remitting (RR) MS. Serum reactivity was significantly increased in CIS and RR-MS as compared with controls (p = 0.03). For two antigens, the frequency of antibody-positive patients was higher in CIS patients who converted to MS as compared with CIS patients without conversion. We identified novel CIS antigens to which antibody reactivity was primarily detected in CIS and RR-MS as compared to controls. Possible prognostic potential could be demonstrated for two antigens.

Brain atrophy and lesion load are related to CSF lipid-specific IgM oligoclonal bands in clinically isolated syndromes.

INTRODUCTION: The objective of this work is to study the relationship between the presence of lipid-specific oligoclonal IgM bands (LS-OCMB) in CSF, with both T2 lesion volume (T2LV) accumulation and brain atrophy (percentage change of brain volume-PCBV-and brain parenchyma fraction-BPF) in patients with clinically isolated syndromes (CIS) suggestive of demyelination. METHODS: Twenty-four CIS patients were included in this prospective study. IgG oligoclonal bands (OCGB) and LS-OCMB were determined in paired serum and CSF samples within 3 months since clinical onset. Brain MRI studies were scheduled at baseline, 3 months, first and second years after CIS onset. Differences in T2LV, PCBV and BPF between CIS patients according to the type of OCB were studied. RESULTS: Nine patients had no OCB; 15 had only OCGB, and seven had OCGB + LS-OCMB present in the CSF. LS-OCMB were associated with greater T2LV in all scheduled MRI studies. At the end of follow-up (year 2), it was threefold higher in patients with these antibodies than in those without LS-OCMB (3.95 cm(3) vs. 1.36 cm(3), p = 0.001). At that point, brain atrophy was also higher in patients with LS-OCMB (BPF, 0.73 in LS-OCMB+ patients vs. 0.76 in negative ones, p = 0.03). The rate in brain atrophy was higher in the first group of patients as well. Considering only patients with OCGB, the presence of LS-OCMB was also related to greater T2LV, T2LV increase and a trend towards higher atrophy rate. CONCLUSION: The presence of LS-OCMB in the first event suggestive of demyelination is related to an early increase in lesion load and brain atrophy. These data are in line with prospective studies showing the clinical prognostic value of LS-OCMB.

High frequency of co-infection by Epstein-Barr virus types 1 and 2 in patients with multiple sclerosis.

BACKGROUND: The existence of Epstein-Barr virus (EBV) strains specifically associated with multiple sclerosis (MS) is a matter of controversy. Little is also known about the prevalence of EBV types 1 and 2 in MS patients and the presence of co-infections by both strains. OBJECTIVE: To make EBV strain type assignment and compare the frequencies of types 1, 2 and co-infections by both in MS patients and healthy controls. METHODS: Blood samples from 75 consecutive MS patients and 186 controls were collected. EBV was simultaneously detected and typed using a polymerase chain reaction (PCR) which amplified a strain-specific sequence in the EBV nuclear antigen 2. RESULTS: EBV was detected in 70 out of 75 patients (93.3%) and in 123 of 186 controls (66.1%). Among positive cases, type 1 was found in 6 patients (8.6%) and 40 controls (32.5%), type 2 in 1 patient (1.4%) and 37 controls (30.1%), and dual-infections by both EBV types were detected in 63 patients (90%) and 46 controls (37.4%). Logistic regression models showed that MS was significantly associated with the presence of EBV (p < 0.001) and also with dual type infections (p < 0.001). CONCLUSION: This study provides molecular evidence associating co-infection of type 1 and 2 EBV with MS.

Increased peripheral blood CD5+ B cells predict earlier conversion to MS in high-risk clinically isolated syndromes.

Clinically isolated syndrome patients (CIS) with oligoclonal IgG bands (OCGB) are at high risk for clinically definite multiple sclerosis (MS). However, the outcome for individual patients is unpredictable and the search for reliable blood markers predicting early conversion to multiple sclerosis (MS) has clinical relevance. CD5+ B cells (CD5+Bc) are involved in some autoimmune diseases. This study investigated whether high blood CD5+Bc percentage can predict CIS conversion to MS. Fifty-five consecutive CIS showing OCGB were prospectively studied. Every patient underwent a brain MRI study and a flow cytometry analysis of CD5+Bc percentage. Conversion to MS was studied during follow-up. The CD5+Bc percentage was assessed in 40 controls and a cut-off value of 3.5% (mean+2 SD) was calculated. A blood CD5+Bc percentage above this value predicted earlier conversion to MS in the whole group (hazard ratio [HR]: 3.40; 95% confidence interval [CI]: 1.69-6.68; p=0.0005) and in CIS patients fulfilling three or more Barkhof-Tintoré criteria plus OCGB, who showed higher risk for MS (HR: 3.79; 95% CI: 1.86-15.32; p=0.0018). Multivariate analysis also showed a predictive value for high blood CD5+Bc count (HR: 4.3; 95% CI: 1.9-9.5; p<0.0001). It was concluded that high percentages of CD5+Bc independently associate with increased risk of early conversion to MS in CIS patients with OCGB and Barkhof-Tintoré criteria.

Immunological mechanisms that associate with oligoclonal IgM band synthesis in multiple sclerosis.

We described previously that multiple sclerosis (MS) patients with oligoclonal IgM against myelin lipids (M+) develop an aggressive disease. Our aim was to assess possible mechanisms regulating the production of these antibodies. We studied B cell subsets in 180 patients with MS, and 69 with other neurological diseases. M+ MS patients showed a moderate increase of CD5(+) B-cell percentage in peripheral blood and a considerable augment of these cells in cerebrospinal fluid (CSF) that correlated with intrathecal IgM production. The appearance of CD5(+) B cells into the central nervous system (CNS) was related to increased CXCL13 and TNF-alpha levels in CSF. Moreover, the presence of oligoclonal IgM associated with a SNP at position -376 of the TNF-alpha promoter. These results help to elucidate the B lymphocytes responsible for intrathecal IgM secretion in MS and the origin of this abnormal B-cell response in patients with aggressive MS.

Acetate correlates with disability and immune response in multiple sclerosis.

BACKGROUND: Gut microbiota has been related to multiple sclerosis (MS) etiopathogenesis. Short-chain fatty acids (SCFA) are compounds derived from microbial metabolism that have a role in gut-brain axis. OBJECTIVES: To analyse SCFA levels in plasma of MS patients and healthy donors (HD), and the possible link between these levels and both clinical data and immune cell populations. METHODS: Ninety-five MS patients and 54 HD were recruited. Patients were selected according to their score in the Expanded Disability Status Scale (EDSS) (49 EDSS ≤ 1.5, 46 EDSS ≥ 5.0). SCFA were studied in plasma samples by liquid chromatography-mass spectrometry. Peripheral blood mononuclear cells were studied by flow cytometry. Gender, age, treatments, EDSS and Multiple Sclerosis Severity Score (MSSS) were evaluated at the recruitment. RESULTS: Plasma acetate levels were higher in patients than in HD (p = 0.003). Patients with EDSS ≥ 5.0 had higher acetate levels than those with EDSS≤ 1.5 (p = 0.029), and HD (p = 2.97e-4). Acetate levels correlated with EDSS (r = 0.387; p = 1.08e-4) and MSSS (r = 0.265; p = 0.011). In untreated MS patients, acetate levels correlated inversely with CD4+ naïve T cells (r =  - 0.550, p = 0.001) and directly with CD8+ IL-17+ cells (r = 0.557; p = 0.001). CONCLUSIONS: Plasma acetate levels are higher in MS patients than in HD. In MS there exists a correlation between plasma acetate levels, EDSS and increased IL-17+ T cells. Future studies will elucidate the role of SCFA in the disease.