Microstructural MRI Correlates of Cognitive Impairment in Multiple Sclerosis: The Role of Deep Gray Matter

Although cognitive impairment (CI) is frequently observed in people with multiple sclerosis (pwMS), its pathogenesis is still controversial. Conflicting results emerged concerning the role of microstructural gray matter (GM) damage especially when involving the deep GM structures. In this study, we aimed at evaluating whether differences in cortical and deep GM structures between apparently cognitively normal (ACN) and CI pwMS (36 subjects in total) are present, using an extensive set of diffusion MRI (dMRI) indices and conventional morphometry measures. The results revealed increased anisotropy and restriction over several deep GM structures in CI compared with ACN pwMS, while no changes in volume were present in the same areas. Conversely, reduced anisotropy/restriction values were detected in cortical regions, mostly the pericalcarine cortex and precuneus, combined with reduced thickness of the superior frontal gyrus and insula. Most of the dMRI metrics but none of the morphometric indices correlated with the Symbol Digit Modality Test. These results suggest that deep GM microstructural damage can be a strong anatomical substrate of CI in pwMS and might allow identifying pwMS at higher risk of developing CI

Cerebrospinal fluid IgM levels in association with inflammatory pathways in multiple sclerosis patients

Background Intrathecal immunoglobulin M (IgM) synthesis has been demonstrated in the early disease stages of multiple sclerosis (MS) as a predictor factor of a worsening disease course. Similarly, increased cerebrospinal fluid (CSF) molecules related to B-cell intrathecal activity have been associated with a more severe MS progression. However, whether CSF levels of IgM are linked to specific inflammatory and clinical profile in MS patients at the time of diagnosis remains to be elucidated. Methods Using customized Bio-Plex assay, the protein levels of IgG, IgA, IgM, and of 34 other inflammatory molecules, related to B-cell, T-cell, and monocyte/macrophage activity, were analyzed in the CSF of 103 newly diagnosed relapsing-remitting MS patients and 36 patients with other neurological disorders. CSF IgM levels were also correlated with clinical and neuroradiological measures [advanced 3-T magnetic resonance imaging (MRI) parameters], at diagnosis and after 2 years of follow-up. Results A 45.6% increase in CSF IgM levels was found in MS patients compared to controls (p = 0.013). CSF IgM levels correlated with higher CSF levels of CXCL13 (p = 0.039), CCL21 (p = 0.023), interleukin 10 (IL-10) (p = 0.025), IL-12p70 (p = 0.020), CX3CL1 (p = 0.036), and CHI3L1 (p = 0.048) and were associated with earlier age of patients at diagnosis (p = 0.008), white matter lesion (WML) number (p = 0.039) and disease activity (p = 0.033) after 2 years of follow-up. Conclusion IgMs are the immunoglobulins mostly expressed in the CSF of naive MS patients compared to other neurological conditions at the time of diagnosis. The association between increased CSF IgM levels and molecules related to both B-cell immunity (IL-10) and recruitment (CXCL13 and CCL21) and to macrophage/microglia activity (IL-12p70, CX3CL1, and CHI3L1) suggests possible correlation between humoral and innate intrathecal immunity in early disease stage. Furthermore, the association of IgM levels with WMLs and MS clinical and MRI activity after 2 years supports the idea of key role of IgM in the disease course

Changes in cerebrospinal fluid balance of TNF and TNF receptors in naïve multiple sclerosis patients: early involvement in compartmentalised intrathecal inflammation

An imbalance of TNF signalling in the inflammatory milieu generated by meningeal immune cell infiltrates in the subarachnoid space in multiple sclerosis (MS), and its animal model may lead to increased cortical pathology. In order to explore whether this feature may be present from the early stages of MS and may be associated with the clinical outcome, the protein levels of TNF, sTNF-R1 and sTNF-R2 were assayed in CSF collected from 122 treatment-naive MS patients and 36 subjects with other neurological conditions at diagnosis. Potential correlations with other CSF cytokines/chemokines and with clinical and imaging parameters at diagnosis (T0) and after 2 years of follow-up (T24) were evaluated. Significantly increased levels of TNF (fold change: 7.739; p < 0.001), sTNF-R1 (fold change: 1.693; p < 0.001) and sTNF-R2 (fold change: 2.189; p < 0.001) were detected in CSF of MS patients compared to the control group at T0. Increased TNF levels in CSF were significantly (p < 0.01) associated with increased EDSS change (r = 0.43), relapses (r = 0.48) and the appearance of white matter lesions (r = 0.49). CSF levels of TNFR1 were associated with cortical lesion volume (r = 0.41) at T0, as well as with new cortical lesions (r = 0.56), whilst no correlation could be found between TNFR2 levels in CSF and clinical or MRI features. Combined correlation and pathway analysis (ingenuity) of the CSF protein pattern associated with TNF expression (encompassing elevated levels of BAFF, IFN-gamma, IL-1 beta, IL-10, IL-8, IL-16, CCL21, haptoglobin and fibrinogen) showed a particular relationship to the interaction between innate and adaptive immune response. The CSF sTNF-R1-associated pattern (encompassing high levels of CXCL13, TWEAK, LIGHT, IL-35, osteopontin, pentraxin-3, sCD163 and chitinase-3-L1) was mainly related to altered T cell and B cell signalling. Finally, the CSF TNFR2-associated pattern (encompassing high CSF levels of IFN-beta, IFN-lambda 2, sIL-6R alpha) was linked to Th cell differentiation and regulatory cytokine signalling. In conclusion, dysregulation of TNF and TNF-R1/2 pathways associates with specific clinical/MRI profiles and can be identified at a very early stage in MS patients, at the time of diagnosis, contributing to the prediction of the disease outcome

Intrathecal versus peripheral inflammatory protein profile in MS patients at diagnosis: a comprehensive investigation on serum and CSF

Intrathecal inflammation plays a key role in the pathogenesis of multiple sclerosis (MS). To better elucidate its relationship with peripheral inflammation, we investigated the correlation between cerebrospinal fluid (CSF) and serum levels of 61 inflammatory proteins. Paired CSF and serum samples were collected from 143 treatment-naïve MS patients at diagnosis. A customized panel of 61 inflammatory molecules was analyzed by a multiplex immunoassay. Correlations between serum and CSF expression levels for each molecule were performed by Spearman's method. The expression of sixteen CSF proteins correlated with their serum expression (p-value < 0.001): only five molecules (CXCL9, sTNFR2, IFNα2, Pentraxin-3, and TSLP) showed a Rho value >0.40, suggesting moderate CSF/serum correlation. No correlation between inflammatory serum patterns and Qalb was observed. Correlation analysis of serum expression levels of these sixteen proteins with clinical and MRI parameters pinpointed a subset of five molecules (CXCL9, sTNFR2, IFNα2, IFNβ, and TSLP) negatively correlating with spinal cord lesion volume. However, following FDR correction, only the correlation of CXCL9 remained significant. Our data support the hypothesis that the intrathecal inflammation in MS only partially associates with the peripheral one, except for the expression of some immunomodulators that might have a key role in the initial MS immune response

CSF levels of CXCL12 and osteopontin as early markers of primary progressive multiple sclerosis

Background and objectives: To evaluate the extent of intrathecal inflammation in patients with primary progressive MS (PPMS) at the time of diagnosis and to define markers and a specific inflammatory profile capable of distinguishing progressive from relapsing-remitting multiple sclerosis (RRMS). Methods: Levels of 34 pro- and anti-inflammatory cytokines and chemokines in the CSF were evaluated at the diagnosis in 16 patients with PPMS and 80 with RRMS. All patients underwent clinical evaluation, including Expanded Disability Status Scale assessment and a 3T brain MRI to detect white matter and cortical lesion number and volume and global and regional cortical thickness. Results: Higher levels of CXCL12 (odds ratio [OR] = 3.97, 95% CI [1.34-11.7]) and the monocyte-related osteopontin (OR = 2.24, 95% CI [1.01-4.99]) were detected in patients with PPMS, whereas levels of interleukin-10 (IL10) (OR = 0.28, 95% CI [0.09-0.96]) were significantly increased in those with RRMS. High CXCL12 levels were detected in patients with increased gray matter lesion number and volume (p = 0.001, r = 0.832 and r = 0.821, respectively). Pathway analysis confirmed the chronic inflammatory processes occurring in PPMS. Conclusions: At the time of diagnosis, a specific CSF protein profile can recognize the presence of early intrathecal inflammatory processes, possibly stratifying PPMS with respect to RRMS. Elevated CSF levels of CXCL12 and osteopontin suggested a key role of brain innate immunity and glia activity in MS. These molecules could represent useful candidate markers of MS progression, with implications for the pathogenesis and treatment of progressive MS. Classification of evidence: This study provides Class III evidence that CXCL12 and monocyte-related osteopontin may be correlated with PPMS, and IL-10 may be related to RRMS. It is may be correlated due to Bonferroni correction negating the statistical correlations found in the study

The prognostic value of white-matter selective double inversion recovery mri sequence in multiple sclerosis: an exploratory study

Using a white-matter selective double inversion recovery sequence (WM-DIR) that suppresses both grey matter (GM) and cerebrospinal fluid (CSF) signals, some white matter (WM) lesions appear surrounded by a dark rim. These dark rim lesions (DRLs) seem to be specific for multiple sclerosis (MS). They could be of great usefulness in clinical practice, proving to increase the MRI diagnostic criteria specificity. The aims of this study are the identification of DRLs on 1.5 T MRI, the exploration of the relationship between DRLs and disease course, the characterization of DRLs with respect to perilesional normal-appearing WM using magnetization transfer imaging, and the investigation of possible differences in the underlying tissue properties by assessing WM-DIR images obtained at 3.0 T MRI. DRLs are frequent in primary progressive MS (PPMS) patients. Amongst relapsing-remitting MS (RRMS) patients, DRLs are associated with a high risk of the disease worsening and secondary progressive MS (SPMS) conversion after 15 years. The mean magnetization transfer ratio (MTR) of DRLs is significantly different from the lesion without the dark rim, suggesting that DRLs correspond to more destructive lesions

Increased NK Cell Count in Multiple Sclerosis Patients Treated With Dimethyl Fumarate: A 2-Year Longitudinal Study

Background: Dimethyl fumarate (DMF) is a disease-modifying drug for relapsing-remitting multiple sclerosis. Among others, DMF impedes immune activation by shifting the balance between inflammatory and regulatory cell types and by inducing apoptosis-triggered lymphopenia. Although the decrease in lymphocyte count is an early effect of the drug in several patients, the long-term impact on lymphocyte subsets is largely unknown.Methods: We performed a 2-years observational study on total lymphocyte count and subsets thereof by flow cytometry of peripheral blood of 38 multiple sclerosis patients in treatment with DMF. Data were collected at the beginning and after 3, 6, 12, and 24 months of therapy.Results: Total lymphocyte count decreased in relation to time of exposure to DMF. Mean absolute B cell count decreased by 34.1%(p < 0.001) within the first 3months of therapy and then remained stable over time. Mean absolute CD3(+) T cells count decrement reached 47.5% after 12 months of treatment ( p < 0.001). NK cells count showed a heterogeneous trend, increasing by 85.9%( p < 0.001) after 2 years of treatment. CD4(+) T cells and CD8(+) T cells substantially decreased, with a significant increase of CD4(+)/CD8(+) ratio during the first year of therapy.Conclusions: NK cells showed a heterogeneous behavior during DMF treatment with a significant increase over time. Since NK cells may also have a regulatory effect on immune system modulation, their increase during DMF treatment might play a role in the efficacy and safety of the drug

Immune Response after COVID-19 mRNA Vaccination in Multiple Sclerosis Patients Treated with DMTs

The impact of disease-modifying therapies (DMTs) on the immune response to coronavirus disease-2019 (COVID-19) vaccines in persons with multiple sclerosis (pwMS) needs further elucidation. We investigated BNT162b2 mRNA COVID-19 vaccine effects concerning antibody seroconversion, inflammatory mediators' level and immunophenotype assessment in pwMS treated with cladribine (c-pwMS, n = 29), fingolimod (f-pwMS, n = 15) and ocrelizumab (o-pwMS, n = 54). Anti-spike immunoglobulin (Ig)-G detection was performed by an enzyme immunoassay; molecular mediators (GrB, IFN-gamma and TNF-alpha) were quantified using the ELLA platform, and immunophenotype was assessed by flow cytometry. ANCOVA, Student's t-test and Pearson correlation analyses were applied. Only one o-pwMS showed a mild COVID-19 infection despite most o-pwMS lacking seroconversion and showing lower anti-spike IgG titers than c-pwMS and f-pwMS. No significant difference in cytokine production and lymphocyte count was observed in c-pwMS and f-pwMS. In contrast, in o-pwMS, a significant increase in GrB levels was detected after vaccination. Considering non-seroconverted o-pwMS, a significant increase in GrB serum levels and CD4+ T lymphocyte count was found after vaccination, and a negative correlation was observed between anti-spike IgG production and CD4+ T cells count. Differences in inflammatory mediators' production after BNT162b2 vaccination in o-pwMS, specifically in those lacking anti-spike IgG, suggest a protective cellular immune response

CSF TNF and osteopontin levels correlate with the response to dimethyl fumarate in early multiple sclerosis

Background: Disease activity in the first years after a diagnosis of relapsing-remitting multiple sclerosis (RRMS) is a negative prognostic factor for long-term disability. Markers of both clinical and radiological responses to disease-modifying therapies (DMTs) are advocated. Objective: The objective of this study is to estimate the value of cerebrospinal fluid (CSF) inflammatory markers at the time of diagnosis in predicting the disease activity in treatment-naïve multiple sclerosis (MS) patients exposed to dimethyl fumarate (DMF). Methods: In total, 48 RRMS patients (31 females/17 males) treated with DMF after the diagnosis were included in this 2-year longitudinal study. All patients underwent a CSF examination, regular clinical and 3T magnetic resonance imaging (MRI) scans that included the assessment of white matter (WM) lesions, cortical lesions (CLs) and global cortical thickness. CSF levels of 10 pro-inflammatory markers - CXCL13 [chemokine (C-X-C motif) ligand 13 or B lymphocyte chemoattractant], CXCL12 (stromal cell-derived factor or C-X-C motif chemokine 12), tumour necrosis factor (TNF), APRIL (a proliferation-inducing ligand, or tumour necrosis factor ligand superfamily member 13), LIGHT (tumour necrosis factor ligand superfamily member 14 or tumour necrosis factor superfamily member 14), interferon (IFN) gamma, interleukin 12 (IL-12), osteopontin, sCD163 [soluble-CD163 (cluster of differentiation 163)] and Chitinase3-like1 - were assessed using immune-assay multiplex techniques. The combined three-domain status of 'no evidence of disease activity' (NEDA-3) was defined by no relapses, no disability worsening and no MRI activity, including CLs. Results: Twenty patients (42%) reached the NEDA-3 status; patients with disease activity showed higher CSF TNF (p = 0.009), osteopontin (p = 0.005), CXCL12 (p = 0.037), CXCL13 (p = 0.040) and IFN gamma levels (p = 0.019) compared with NEDA-3 patients. After applying a random forest approach, TNF and osteopontin revealed the most important variables associated with the NEDA-3 status. Six molecules that emerged at the random forest approach were added in a multivariate regression model with demographic, clinical and MRI measures of WM and grey matter damage as independent variables. TNF levels confirmed to be associated with the absence of disease activity: odds ratio (OR) = 0.25, CI% = 0.04-0.77. Conclusion: CSF inflammatory markers may provide prognostic information in predicting disease activity in the first years after DMF initiation. CSF TNF levels are a possible candidate in predicting treatment response, in addition to clinical, demographic and MRI variables

Iron homeostasis, complement, and coagulation cascade as CSF signature of cortical lesions in early multiple sclerosis

OBJECTIVE: Intrathecal inflammation, compartmentalized in cerebrospinal fluid (CSF) and in meningeal infiltrates, has fundamental role in inflammation, demyelination, and neuronal injury in cerebral cortex in multiple sclerosis (MS). Since the exact link between intrathecal inflammation and mechanisms of cortical pathology remains unknown, we aimed to investigate a detailed proteomic CSF profiling which is able to reflect cortical damage in early MS. METHODS: We combined new proteomic method, TRIDENT, CSF analysis, and advanced 3T magnetic resonance imaging (MRI), in 64 MS patients at the time of diagnosis and 26 controls with other neurological disorders. MS patients were stratified according to cortical lesion (CL) load. RESULTS: We identified 227 proteins differently expressed between the patients with high and low CL load. These were mainly related to complement and coagulation cascade as well as to iron homeostasis pathway (30 and 6% of all identified proteins, respectively). Accordingly, in the CSF of MS patients with high CL load at diagnosis, significantly higher levels of sCD163 (P < 0.0001), free hemoglobin (Hb) (P < 0.05), haptoglobin (P < 0.0001), and fibrinogen (P < 0.01) were detected. By contrast, CSF levels of sCD14 were significantly (P < 0.05) higher in MS patients with low CL load. Furthermore, CSF levels of sCD163 positively correlated (P < 0.01) with CSF levels of neurofilament, fibrinogen, and B cell-related molecules, such as CXCL13, CXCL12, IL10, and BAFF. INTERPRETATION: Intrathecal dysregulation of iron homeostasis and coagulation pathway as well as B-cell and monocyte activity are strictly correlated with cortical damage at early disease stages