Emerging biomarkers for improving pregnancy planning in multiple sclerosis.

Background: Patient disability, relapse rate, and age are used for family planning in multiple sclerosis (MS). However, the need for more accurate biomarkers is widely recognized. We aimed to explore the influence of age on neurofilament light chain (sNfL), which reflects acute inflammation; glial fibrillary acidic protein (GFAP), associated with disability progression independent of relapses; and anti-Müllerian hormone (AMH), reflecting ovarian reserve, to provide a tailored family planning strategy. Methods: This case-control study included 95 MS patients and 61 healthy control women (HCW). sNfL and GFAP levels were measured using a sensitive single-molecule array assay. AMH levels were measured by the automated Elecsys® Anti-Müllerian Hormone Assay. Results: We observed no significant differences in AMH values between MS patients and the control group within any of the age-matched categories. Age exhibited a negative correlation with AMH values in both groups, as expected. Nevertheless, our findings suggest a slight tendency toward reduced ovarian reserve in MS patients (rho MS patients = -0.67, p < 0.0001; rho HCW = -0.43, p = 0.0006). Interestingly, among the 76 MS participants under 40 years old, we identified ten individuals (13.1%) with AMH levels below 0.7 ng/ml, indicative of a low ovarian reserve, and an additional six individuals (7.8%) with AMH levels between 0.7 ng/ml and 0.9 ng/ml, suggesting a potential risk of premature ovarian failure. Conversely, sNfL and GFAP levels in the MS group exhibited high variability but showed no significant association with age intervals. Conclusion: We found no significant differences in AMH, sNfL or GFAP values between MS patients and the control group within any of the age-matched categories. The assessment of AMH, sNFL and GFAP levels at MS onset facilitates personalized therapeutic and family planning strategies for childbearing-age women.

Factors Associated with SARS-CoV-2 Infection in Fully Vaccinated Nursing Home Residents and Workers.

Persons living or working in nursing homes faced a higher risk of SARS-CoV-2 infections during the pandemic, resulting in heightened morbidity and mortality among older adults despite robust vaccination efforts. This prospective study evaluated the humoral and cellular immunity in fully vaccinated residents and workers from two nursing homes in Madrid, Spain, from 2020 to 2021. Measurements of IgG levels were conducted in August 2020 (pre-vaccination) and June and September 2021 (post-vaccination), alongside assessments of neutralizing antibodies and cellular responses in September 2021 among the most vulnerable individuals. Follow-up extended until February 2022 to identify risk factors for SARS-CoV-2 infection or mortality, involving 267 residents (mean age 87.6 years, 81.3% women) and 302 workers (mean age 50.7 years, 82.1% women). Residents exhibited a significantly higher likelihood of experiencing COVID-19 before June 2021 compared with nursing staff (OR [95% CI], 7.2 [3.0 to 17.2], p < 0.01). Participants with a history of previous COVID-19 infection showed more significant increases in IgG levels in August 2020, June 2021 and September 2021, alongside an increased proportion of neutralizing antibodies in the most vulnerable individuals. However, IgG decay remained the same between June and September 2021 based on the previous COVID-19 status. During the Omicron variant wave, residents and staff showed a similar rate of SARS-CoV-2 infection. Notably, preceding clinical or immunological factors before receiving three vaccination doses did not demonstrate associations with COVID-19 infection or overall mortality in our participant cohort.

Establishing the best combination of the kappa free light chain index and oligoclonal bands for an accurate diagnosis of multiple sclerosis.

Introduction: The immunoglobulin kappa free light chain (KFLC) index has been proposed as a potentially suitable alternative to oligoclonal IgG bands (OCGB) for diagnosing multiple sclerosis (MS), offering automation and reduced processing time. However, there is no consensus on the preferred approach or how to combine both techniques. Methods: This prospective cohort study aimed to determine the best utilization of OCGB and KFLC index in patients with a clinically isolated syndrome (CIS) followed for at least two years. OCGB and KFLC were assessed using isoelectric focusing and immunoblotting and turbidimetry, respectively. Sensitivity, specificity, and accuracy for diagnosing MS were calculated for each method. Results: The study included 371 patients, with 260 (70.1 %) being women, and a median age of 34.9 (27.8 - 43.9) years. Using a cut-off value of 6.1, the KFLC index demonstrated a sensitivity and specificity of 86.3% and 93.9%, respectively. The sensitivity of OCGB (95.3%) was higher (p < 0.001 vs. KFLC index) and the specificity (100%) was comparable to that of the KFLC index (p = 0.5). The concordance between the methods was not uniform across all patients, with 97.8% agreement in patients with KFLC index ≥ 6.1 and 56.0 % in patients with KFLC index < 6.1. In patients with a KFLC index < 6.1, OCGB still identified 75.0 % of MS patients due to its higher sensitivity. An algorithm using the KFLC index as a screening tool and OCGB as an alternative for patients with a negative KFLC index result achieved an accuracy of 96.3 %. Discussion: Combining the KFLC index and OCGB can provide an easily reproducible and accurate method for diagnosing MS, with OCGB primarily reserved for patients with a KFLC index < 6.1.

Teriflunomide and Epstein-Barr virus in a Spanish multiple sclerosis cohort: in vivo antiviral activity and clinical response.

Background: Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6) have been associated with multiple sclerosis (MS). Teriflunomide is an oral disease-modifying therapy approved for treatment of relapsing forms of MS. In the preclinical Theiler's murine encephalitis virus model of MS, the drug demonstrated an increased rate of viral clearance versus the vehicle placebo. Furthermore, teriflunomide inhibits lytic EBV infection in vitro. Objective: 1. To evaluate the humoral response against EBV and HHV-6 prior to teriflunomide treatment and 6 months later. 2. To correlate the variation in the humoral response against EBV and HHV-6 with the clinical and radiological response after 24 months of treatment with teriflunomide. 3. To analyze the utility of different demographic, clinical, radiological, and environmental data to identify early biomarkers of response to teriflunomide. Methods: A total of 101 MS patients (62 women; mean age: 43.4 years) with one serum prior to teriflunomide onset and another serum sample 6 months later were recruited. A total of 80 had been treated for at least 24 months, 13 had stopped teriflunomide before 24 months, and 8 were currently under teriflunomide therapy but with less than 24 months of follow-up. We analyzed the levels of the viral antibodies titers abovementioned in serum samples with ELISA commercial kits, and the levels of serum neurofilament light chain (Nf-L). Results: Antiviral antibody titers decreased for EBNA-1 IgG (74.3%), VCA IgG (69%), HHV-6 IgG (60.4%), and HHV-6 IgM (73.3%) after 6 months of teriflunomide. VCA IgG titers at baseline correlated with Nf-L levels measured at the same time (r = 0.221; p = 0.028) and 6 months later (r = 0.240; p = 0.017). We found that higher EBNA-1 titers (p = 0.001) and a higher age (p = 0.04) at baseline were associated with NEDA-3 conditions. Thus, 77.8% of patients with EBNA-1 >23.0 AU and >42.8 years (P50 values) were NEDA-3. Conclusion: Treatment with teriflunomide was associated with a reduction of the levels of IgG antibody titers against EBV and HHV-6. Furthermore, higher EBNA-1 IgG titers prior to teriflunomide initiation were associated with a better clinical response.

Blood CD8+ Naïve T-Cells Identify MS Patients with High Probability of Optimal Cellular Response to SARS-CoV-2 Vaccine.

This single-center study included 68 multiple sclerosis (MS) patients who received the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination from one of several approved vaccine preparations in Spain. Blood samples were collected one to three months after the second dose of the vaccine had been administered. Cellular immune responses to the vaccine were assessed using QuantiFERON analysis, and peripheral blood mononuclear cell subsets were assayed using flow cytometry. Response associated with higher percentages of total lymphocytes, naïve CD4+ T-cells (p = 0.028), CD8+ T-cells (p = 0.013), and, mostly, naïve CD8+ T-cells (p = 0.0003). These results were confirmed by analyzing absolute numbers (p = 0.019; p = 0.002, and p = 0.0003, respectively). Naïve CD8 T-cell numbers higher than 17 cells/µL were closely associated with an optimal cellular response to SARS-CoV-2 vaccination (odds ratio: 24.0, confidence interval: 4.8-460.3; p = 0.0001). This finding clearly shows that independent of the treatment received, higher numbers of naïve CD8+ T-cells yield a strong cellular response to SARS-CoV-2 vaccines in MS patients. If this finding is validated with other viruses/vaccines, it could provide a good tool for identifying MS patients undergoing treatment who will develop strong cellular responses to anti-virus vaccines.

Short- and Long-Term Humoral and Cellular Immune Responses to SARS-CoV-2 Vaccination in Patients with Multiple Sclerosis Treated with Disease-Modifying Therapies.

BACKGROUND: This study aimed to evaluate short- and long-term humoral and T-cell-specific immune responses to SARS-CoV-2 vaccines in patients with multiple sclerosis (MS) treated with different disease-modifying therapies (DMTs). METHODS: Single-center observational longitudinal study including 102 patients with MS who consecutively received vaccination against SARS-CoV-2. Serum samples were collected at baseline and after receiving the second dose of the vaccine. Specific Th1 responses following in vitro stimulation with spike and nucleocapsid peptides were analyzed by quantifying levels of IFN-γ. Serum IgG-type antibodies against the spike region of SARS-CoV-2 were studied by chemiluminescent microparticle immunoassay. RESULTS: Patients undergoing fingolimod and anti-CD20 therapies had a markedly lower humoral response than those treated with other DMTs and untreated patients. Robust antigen-specific T-cell responses were detected in all patients except those treated with fingolimod, who had lower IFN-γ levels than those treated with other DMTs (25.8 pg/mL vs. 868.7 pg/mL, p = 0.011). At mid-term follow-up, a decrease in vaccine-induced anti-SARS-CoV-2 IgG antibodies was observed in all subgroups of patients receiving DMTs, although most patients receiving induction DMTs or natalizumab and non-treated patients remained protected. Cellular immunity was maintained above protective levels in all DMT subgroups except the fingolimod subgroup. CONCLUSIONS: SARS-CoV-2 vaccines induce robust and long-lasting humoral and cell-mediated specific immune responses in most patients with MS.

Association of Serum Neurofilament Light Chain Levels at Disease Onset With Disability Worsening in Patients With a First Demyelinating Multiple Sclerosis Event Not Treated With High-Efficacy Drugs.

Importance: The value of serum neurofilament light chain (sNfL) levels for predicting long-term disability in patients with multiple sclerosis (MS) remains controversial. Objective: To assess whether high sNfL values are associated with disability worsening in patients who underwent their first demyelinating MS event. Design, Setting, and Participants: This multicenter cohort study included patients who underwent their first demyelinating event suggestive of MS at Hospital Universitario Ramón y Cajal (development cohort; June 1, 1994, to September 31, 2021, with follow-up until August 31, 2022) and 8 Spanish hospitals (validation cohort; October 1, 1995, to August 4, 2020, with follow-up until August 16, 2022). Exposures: Clinical evaluations at least every 6 months. Main Outcomes and Measures: The main outcomes were 6-month confirmed disability worsening (CDW) and an Expanded Disability Status Scale (EDSS) score of 3. Levels of sNfL were measured in blood samples obtained within 12 months after disease onset using a single molecule array kit. The cutoffs used were sNfL level of 10 pg/mL and a standardized score (z score) of 1.5. Multivariable Cox proportional hazards regression models were used to evaluate outcomes. Results: Of the 578 patients included in the study, 327 were in the development cohort (median age at sNfL analysis, 34.1 years [IQR, 27.2-42.7 years]; 226 female [69.1%]) and 251 patients were in the validation cohort (median age at sNfL analysis, 33.3 years [IQR, 27.4-41.5 years]; 184 female [73.3%]). The median follow-up was 7.10 years (IQR, 4.18-10.0 years). Levels of sNfL greater than 10 pg/mL were independently associated with higher risk of 6-month CDW and an EDSS of 3 in the development cohort (6-month CDW: hazard ratio [HR], 2.39; 95% CI, 1.39-4.12; P = .002; EDSS of 3: HR, 4.12; 95% CI, 2.18-7.77; P < .001) and the validation cohort (6-month CDW: HR, 1.61; 95% CI, 1.07-2.42; P = .02; EDSS of 3: HR, 2.03; 95% CI, 1.23-3.33; P = .005). Highly effective disease-modifying treatments were associated with lower risks of 6-month CDW and an EDSS of 3 in patients with high baseline sNfL values. Conclusions and Relevance: This cohort study found that high sNfL values obtained within the first year of disease were associated with long-term disability worsening in MS, suggesting that sNfL level measurement may help identify optimal candidates for highly effective disease-modifying treatments.

Mitochondrial Impairments in Peripheral Blood Mononuclear Cells of Multiple Sclerosis Patients.

Although impaired mitochondrial function has been proposed as a hallmark of multiple sclerosis (MS) disease, few studies focus on the mitochondria of immune cells. We aimed to compare the mitochondrial function of the peripheral blood mononuclear cells (PBMCs) from MS patients with (M+) and without (M-) lipid-specific oligoclonal immunoglobulin M bands (LS-OCMB), and healthydonors (HD). We conducted an exploratory cross-sectional study with 19 untreated MS patients (M+ = 9 and M- = 10) and 17 HDs. Mitochondrial superoxide anion production and mitochondrial mass in PBMCs were assessed without and with phytohemagglutinin by flow cytometry. The PBMCs' mitochondrial function was analyzed using Seahorse technology. Superoxide anion production corrected by the mitochondrial mass was higher in MS patients compared with HDs (p = 0.011). Mitochondrial function from M+ patients showed some impairments compared with M- patients. Without stimulus, we observed higher proton leak (p = 0.041) but lower coupling efficiency (p = 0.041) in M+ patients; and under stimulation, lower metabolic potential ECAR (p = 0.011), and lower stressed OCR/ECAR in the same patients. Exclusively among M+ patients, we described a higher mitochondrial dysfunction in the oldest ones. The mitochondrial impairments found in the PBMCs from MS patients, specifically in M+ patients, could help to better understand the disease's physiopathology.

Baseline Inflammatory Status Reveals Dichotomic Immune Mechanisms Involved In Primary-Progressive Multiple Sclerosis Pathology.

Objective: To ascertain the role of inflammation in the response to ocrelizumab in primary-progressive multiple sclerosis (PPMS). Methods: Multicenter prospective study including 69 patients with PPMS who initiated ocrelizumab treatment, classified according to baseline presence [Gd+, n=16] or absence [Gd-, n=53] of gadolinium-enhancing lesions in brain MRI. Ten Gd+ (62.5%) and 41 Gd- patients (77.4%) showed non-evidence of disease activity (NEDA) defined as no disability progression or new MRI lesions after 1 year of treatment. Blood immune cell subsets were characterized by flow cytometry, serum immunoglobulins by nephelometry, and serum neurofilament light-chains (sNfL) by SIMOA. Statistical analyses were corrected with the Bonferroni formula. Results: More than 60% of patients reached NEDA after a year of treatment, regardless of their baseline characteristics. In Gd+ patients, it associated with a low repopulation rate of inflammatory B cells accompanied by a reduction of sNfL values 6 months after their first ocrelizumab dose. Patients in Gd- group also had low B cell numbers and sNfL values 6 months after initiating treatment, independent of their treatment response. In these patients, NEDA status was associated with a tolerogenic remodeling of the T and innate immune cell compartments, and with a clear increase of serum IgA levels. Conclusion: Baseline inflammation influences which immunological pathways predominate in patients with PPMS. Inflammatory B cells played a pivotal role in the Gd+ group and inflammatory T and innate immune cells in Gd- patients. B cell depletion can modulate both mechanisms.

Anti-Human Herpesvirus 6 A/B Antibodies Titers Correlate With Multiple Sclerosis-Associated Retrovirus Envelope Expression.

Human endogenous retrovirus W family envelope proteins (pHERV-W ENV/syncytin-1) have been repeatedly associated with multiple sclerosis (MS). Here, we have focused on the study of pHERV-W ENV/syncytin-1 expression levels in MS patients (relapsing and progressive forms) and in healthy donors (HD) and on exploring their possible relationship with Epstein-Barr virus (EBV) and human herpesvirus-6A/B (HHV-6A/B). We included blood samples from 101 MS patients and 37 HD to analyze antiviral antibody titers by ELISA and pHERV-W ENV/syncytin-1 expression levels by flow cytometry as well as by qPCR. Patients with relapsing MS forms showed significantly higher pHERV-W ENV/syncytin-1 protein and gene expression levels than HD. Progressive MS patients also showed significantly higher protein and gene expression levels than both HD and relapsing MS patients. Regarding antiviral antibodies titers, anti-HHV-6A/B IgM levels were positively correlated with pHERV-W ENV/syncytin-1 protein expression levels in patients with relapsing MS, while in the progressive forms patients this correlation was found with anti-HHVA/B IgG levels. Therefore, pHERV-W ENV could be involved in MS pathogenesis, playing a role in relapsing and progressive forms. Besides, anti-HHV-6A/B antibodies positively correlated with pHERV-W ENV expression. Further studies are needed to better understand this possible relationship.

Role of B Cell Profile for Predicting Secondary Autoimmunity in Patients Treated With Alemtuzumab.

Objective: To explore if baseline blood lymphocyte profile could identify relapsing remitting multiple sclerosis (RRMS) patients at higher risk of developing secondary autoimmune adverse events (AIAEs) after alemtuzumab treatment. Methods: Multicenter prospective study including 57 RRMS patients treated with alemtuzumab followed for 3.25 [3.5-4.21] years, (median [interquartile range]). Blood samples were collected at baseline, and leukocyte subsets determined by flow cytometry. We had additional samples one year after the first cycle of alemtuzumab treatment in 39 cases. Results: Twenty-two patients (38.6%) developed AIAEs during follow-up. They had higher B-cell percentages at baseline (p=0.0014), being differences mainly due to plasmablasts/plasma cells (PB/PC, p=0.0011). Those with no AIAEs had higher percentages of CD4+ T cells (p=0.013), mainly due to terminally differentiated (TD) (p=0.034) and effector memory (EM) (p=0.031) phenotypes. AIAEs- patients also showed higher values of TNF-alpha-producing CD8+ T cells (p=0.029). The percentage of PB/PC was the best variable to differentiate both groups of patients. Baseline values >0.10% closely associated with higher AIAE risk (Odds ratio [OR]: 5.91, 95% CI: 1.83-19.10, p=0.004). When excluding the 12 patients with natalizumab, which decreases blood PB/PC percentages, being the last treatment before alemtuzumab, baseline PB/PC >0.1% even predicted more accurately the risk of AIAEs (OR: 11.67, 95% CI: 2.62-51.89, p=0.0007). The AIAEs+ group continued having high percentages of PB/PC after a year of alemtuzumab treatment (p=0.0058). Conclusions: A PB/PC percentage <0.1% at baseline identifies MS patients at low risk of secondary autoimmunity during alemtuzumab treatment.​.

Identification of the Immunological Changes Appearing in the CSF During the Early Immunosenescence Process Occurring in Multiple Sclerosis.

Patients with multiple sclerosis (MS) suffer with age an early immunosenescence process, which influence the treatment response and increase the risk of infections. We explored whether lipid-specific oligoclonal IgM bands (LS-OCMB) associated with highly inflammatory MS modify the immunological profile induced by age in MS. This cross-sectional study included 263 MS patients who were classified according to the presence (M+, n=72) and absence (M-, n=191) of LS-OCMB. CSF cellular subsets and molecules implicated in immunosenescence were explored. In M- patients, aging induced remarkable decreases in absolute CSF counts of CD4+ and CD8+ T lymphocytes, including Th1 and Th17 cells, and of B cells, including those secreting TNF-alpha. It also increased serum anti-CMV IgG antibody titers (indicative of immunosenescence) and CSF CHI3L1 levels (related to astrocyte activation). In contrast, M+ patients showed an age-associated increase of TIM-3 (a biomarker of T cell exhaustion) and increased values of CHI3L1, independently of age. Finally, in both groups, age induced an increase in CSF levels of PD-L1 (an inductor of T cell tolerance) and activin A (part of the senescence-associated secretome and related to inflammaging). These changes were independent of the disease duration. Finally, this resulted in augmented disability. In summary, all MS patients experience with age a modest induction of T-cell tolerance and an activation of the innate immunity, resulting in increased disability. Additionally, M- patients show clear decreases in CSF lymphocyte numbers, which could increase the risk of infections. Thus, age and immunological status are important for tailoring effective therapies in MS.

Predicting Aggressive Multiple Sclerosis With Intrathecal IgM Synthesis Among Patients With a Clinically Isolated Syndrome.

OBJECTIVE: To determine the best method to measure intrathecal immunoglobulin (Ig) M synthesis (ITMS), a biomarker of worse prognosis in multiple sclerosis (MS). We compared the ability for predicting a poor evolution of 4 methods assessing ITMS (IgM oligoclonal bands [OCMBs], lipid-specific OCMBs [LS-OCMBs], Reibergram, and IgM index) in patients with a clinically isolated syndrome (CIS). METHODS: Prospective study with consecutive patients performed at a referral MS center. We used unadjusted and multivariate Cox regressions for predicting a second relapse, Expanded Disability Status Scale (EDSS) scores of 4 and 6, and development of secondary progressive MS (SPMS). RESULTS: A total of 193 patients were included, with a median (interquartile range) age of 31 (25-38) years and a median follow-up of 12.9 years. Among all methods, only OCMB, LS-OCMB, and Reibergram significantly identified patients at risk of some of the pre-established outcomes, being LS-OCMB the technique with the strongest associations. Adjusted hazard ratio (aHR) of LS-OCMB for predicting a second relapse was 2.50 (95% CI 1.72-3.64, p < 0.001). The risk of reaching EDSS scores of 4 and 6 and SPMS was significantly higher among patients with LS-OCMB (aHR 2.96, 95% CI 1.54-5.71, p = 0.001; aHR 4.96, 95% CI 2.22-11.07, p < 0.001; and aHR 2.31, 95% CI 1.08-4.93, p = 0.03, respectively). CONCLUSIONS: ITMS predicts an aggressive MS at disease onset, especially when detected as LS-OCMB. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that lipid-specific IgM oligoclonal bands can predict progression from CIS to MS and a worse disease course over a follow-up of at least 2 years.

Low serum neurofilament light chain values identify optimal responders to dimethyl fumarate in multiple sclerosis treatment.

Serum neurofilament light chains (sNfL) are biomarkers of disease activity in multiple sclerosis (MS), but their value to predict response to treatment, and their association with patient immunological profile, need to be further explored. We studied 80 relapsing-remitting MS patients initiating dimethyl fumarate (DMF) treatment. sNfL levels were explored at baseline and at 3, 6 and 12 months by single molecule array. Blood lymphocyte subsets were measured at baseline and at 6 months by flow cytometry. Patients were followed a year and classified as NEDA (no evidence of disease activity) or ODA (ongoing disease activity). NEDA patients had lower sNfL levels at baseline (p = 0.0001), and after three (p = 0.004) and six (p = 0.03) months of DMF treatment. Consequently, low baseline sNfL values (≤ 12 pg/ml) increased the probability of NEDA (OR 5.8; CI 1.82-15.6; p = 0.002, after correcting by disease activity in the previous year), and associated with significant reductions of central memory CD4+ T lymphocytes, interferon-gamma+ CD8+ T lymphocytes, Natural Killer T cells, and memory B cells upon DMF treatment, being the highest differences in memory B cells (p < 0.0001). This shows that low baseline sNfL values identify MS patients with higher probability of optimal response to DMF and of a reduction in effector immune cells.

Effect of Ocrelizumab in Blood Leukocytes of Patients With Primary Progressive MS.

OBJECTIVE: To analyze the changes induced by ocrelizumab in blood immune cells of patients with primary progressive MS (PPMS). METHODS: In this multicenter prospective study including 53 patients with PPMS who initiated ocrelizumab treatment, we determined effector, memory, and regulatory cells by flow cytometry at baseline and after 6 months of therapy. Wilcoxon matched paired tests were used to assess differences between baseline and 6 months' results. p Values were corrected using the Bonferroni test. RESULTS: Ocrelizumab reduced the numbers of naive and memory B cells (p < 0.0001) and those of B cells producing interleukin (IL)-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNFα) (p < 0.0001 in all cases). By contrast, the proportions of plasmablasts and B cells producing GM-CSF and TNFα increased significantly, suggesting the need for treatment continuation. We also observed a decrease in CD20+ T-cell numbers (p < 0.0001) and percentages (p < 0.0001), and a clear remodeling of the T-cell compartment characterized by relative increases of the naive/effector ratios in CD4+ (p = 0.002) and CD8+ (p = 0.002) T cells and relative decreases of CD4+ (p = 0.03) and CD8+ (p = 0.004) T cells producing interferon-gamma. Total monocyte numbers increased (p = 0.002), but no changes were observed in those producing inflammatory cytokines. The immunologic variations were associated with a reduction of serum neurofilament light chain (sNfL) levels (p = 0.008). The reduction was observed in patients with Gd-enhanced lesions at baseline and in Gd- patients with baseline sNfL >10 pg/mL. CONCLUSIONS: In PPMS, effector B-cell depletion changed T-cell response toward a low inflammatory profile, resulting in decreased sNfL levels.

Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls.

Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future.

Genomic Multiple Sclerosis Risk Variants Modulate the Expression of the ANKRD55-IL6ST Gene Region in Immature Dendritic Cells.

Intronic single-nucleotide polymorphisms (SNPs) in the ANKRD55 gene are associated with the risk for multiple sclerosis (MS) and rheumatoid arthritis by genome-wide association studies (GWAS). The risk alleles have been linked to higher expression levels of ANKRD55 and the neighboring IL6ST (gp130) gene in CD4+ T lymphocytes of healthy controls. The biological function of ANKRD55, its role in the immune system, and cellular sources of expression other than lymphocytes remain uncharacterized. Here, we show that monocytes gain capacity to express ANKRD55 during differentiation in immature monocyte-derived dendritic cells (moDCs) in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). ANKRD55 expression levels are further enhanced by retinoic acid agonist AM580 but downregulated following maturation with interferon (IFN)-γ and lipopolysaccharide (LPS). ANKRD55 was detected in the nucleus of moDC in nuclear speckles. We also analyzed the adjacent IL6ST, IL31RA, and SLC38A9 genes. Of note, in healthy controls, MS risk SNP genotype influenced ANKRD55 and IL6ST expression in immature moDC in opposite directions to that in CD4+ T cells. This effect was stronger for a partially correlated SNP, rs13186299, that is located, similar to the main MS risk SNPs, in an ANKRD55 intron. Upon analysis in MS patients, the main GWAS MS risk SNP rs7731626 was associated with ANKRD55 expression levels in CD4+ T cells. MoDC-specific ANKRD55 and IL6ST mRNA levels showed significant differences according to the clinical form of the disease, but, in contrast to healthy controls, were not influenced by genotype. We also measured serum sgp130 levels, which were found to be higher in homozygotes of the protective allele of rs7731626. Our study characterizes ANKRD55 expression in moDC and indicates monocyte-to-dendritic cell (Mo-DC) differentiation as a process potentially influenced by MS risk SNPs.

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.

Predictive factors and early biomarkers of response in multiple sclerosis patients treated with natalizumab.

There are an increasing number of treatments available for multiple sclerosis (MS). The early identification of optimal responders to individual treatments is important to achieve individualized therapy. With this aim, we performed a multicenter retrospective longitudinal study including 186 MS patients treated with natalizumab who were followed for 2 years. We analyzed the following variables at recruitment: sex, current age, age at disease onset, disease duration, EDSS, number of T2 and Gd + lesions, IgG and IgM oligoclonal bands, HLA class II (DR, DRB, DQA, DQB, and DRB1*15:01), IgG and IgM antibody titers against human herpesvirus 6 (HHV-6) and the antibody response to Epstein-Barr virus (EBV) through the measurement of the anti-EBNA-1 and anti-VCA IgG titers, in relation to clinical response (no relapses or disability progression), and to NEDA-3 (no evidence of disease activity in terms of clinical response and no changes in MRI scans either) after 2-years follow-up. Baseline EDSS score, baseline EBNA-1 IgG titers and percentage change of HHV6 IgG titers between baseline and 6 month visits were significantly different in clinical responders and in NEDA-3 status (all of them remained significant in the multivariate analysis). We identified three variables for the early identification of natalizumab optimal responders in a rapid and cost-effective approach.

Adaptive Features of Natural Killer Cells in Multiple Sclerosis.

Human cytomegalovirus (HCMV) has been recently related with a lower susceptibility to multiple sclerosis (MS). HCMV promotes an adaptive development of NK cells bearing the CD94/NKG2C receptor with a characteristic phenotypic and functional profile. NK cells are proposed to play an immunoregulatory role in MS, and expansion of the NKG2C(+) subset was recently associated with reduced disability progression. To further explore this issue, additional adaptive NK cell markers, i.e., downregulation of FcεRIγ chain (FcRγ) and PLZF transcription factor, as well as antibody-dependent NK cell activation were assessed in controls and MS patients considering HCMV serology and clinical features. In line with previous reports, increased proportions of NKG2C(+), FcRγ(-), and PLZF(-) CD56dim NK cells were found in HCMV(+) cases. However, PLZF(-) NK cells were detected uncoupled from other adaptive markers within the CD56bright subset from HCMV(+) cases and among CD56dim NK cells from HCMV(-) MS patients, suggesting an additional effect of HCMV-independent factors in PLZF downregulation. Interferon-ß therapy was associated with lower proportions of FcRγ(-) CD56dim NK cells in HCMV(+) and increased PLZF(-) CD56bright NK cells in HCMV(-) patients, pointing out to an influence of the cytokine on the expression of adaptive NK cell-associated markers. In addition, proportions of NKG2C(+) and FcRγ(-) NK cells differed in progressive MS patients as compared to controls and other clinical forms. Remarkably, an adaptive NK cell phenotype did not directly correlate with enhanced antibody-triggered degranulation and TNFα production in MS in contrast to controls. Altogether, our results provide novel insights into the putative influence of HCMV and adaptive NK cells in MS.