Association of Levels of CSF Osteopontin With Cortical Atrophy and Disability in Early Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: To evaluate CSF inflammatory markers with accumulation of cortical damage as well as disease activity in patients with early relapsing-remitting MS (RRMS). METHODS: CSF levels of osteopontin (OPN) and 66 inflammatory markers were assessed using an immune-assay multiplex technique in 107 patients with RRMS (82 F/25 M, mean age 35.7 ± 11.8 years). All patients underwent regular clinical assessment and yearly 3T MRI scans for 2 years while 39 patients had a 4-year follow-up. White matter lesion number and volume, cortical lesions (CLs) and volume, and global cortical thickness (CTh) were evaluated together with the 'no evidence of disease activity' (NEDA-3) status, defined by no relapses, no disability worsening, and no MRI activity, including CLs. RESULTS: The random forest algorithm selected OPN, CXCL13, TWEAK, TNF, IL19, sCD30, sTNFR1, IL35, IL16, and sCD163 as significantly associated with changes in global CTh. OPN and CXCL13 were most related to accumulation of atrophy after 2 and 4 years. In a multivariate linear regression model on CSF markers, OPN (p < 0.001), CXCL13 (p = 0.001), and sTNFR1 (p = 0.024) were increased in those patients with accumulating atrophy (adjusted R-squared 0.615). The 10 markers were added in a model that included all clinical, demographic, and MRI variables: OPN (p = 0.002) and IL19 (p = 0.022) levels were confirmed to be significantly increased in patients developing more CTh change over the follow-up (adjusted R-squared 0.619). CXCL13 and OPN also revealed the best association with NEDA-3 after 2 years, with OPN significantly linked to disability accumulation (OR 2.468 [1.46-5.034], p = 0.004) at the multivariate logistic regression model. DISCUSSION: These data confirm and expand our knowledge on the prognostic role of the CSF inflammatory profile in predicting changes in cortical pathology and disease activity in early MS. The data emphasize a crucial role of OPN.

Determinants and Biomarkers of Progression Independent of Relapses in Multiple Sclerosis.

Clinical, pathological, and imaging evidence in multiple sclerosis (MS) suggests that a smoldering inflammatory activity is present from the earliest stages of the disease and underlies the progression of disability, which proceeds relentlessly and independently of clinical and radiological relapses (PIRA). The complex system of pathological events driving "chronic" worsening is likely linked with the early accumulation of compartmentalized inflammation within the central nervous system as well as insufficient repair phenomena and mitochondrial failure. These mechanisms are partially lesion-independent and differ from those causing clinical relapses and the formation of new focal demyelinating lesions; they lead to neuroaxonal dysfunction and death, myelin loss, glia alterations, and finally, a neuronal network dysfunction outweighing central nervous system (CNS) compensatory mechanisms. This review aims to provide an overview of the state of the art of neuropathological, immunological, and imaging knowledge about the mechanisms underlying the smoldering disease activity, focusing on possible early biomarkers and their translation into clinical practice. ANN NEUROL 2024;96:1-20.

CSF levels of Chitinase3like1 correlate with early response to cladribine in multiple sclerosis.

Background: Cladribine has been introduced as a high-efficacy drug for treating relapsing-remitting multiple sclerosis (RRMS). Initial cohort studies showed early disease activity in the first year after drug initiation. Biomarkers that can predict early disease activity are needed. Aim: To estimate cerebrospinal fluid (CSF) markers of clinical and radiological responses after initiation of cladribine. Methods: Forty-two RRMS patients (30F/12M) treated with cladribine were included in a longitudinal prospective study. All patients underwent a CSF examination at treatment initiation, clinical follow-up including Expanded Disability Status Scale (EDSS) assessment, and a 3T MRI scan after 6,12 and 24 months, including the evaluation of white matter (WM) and cortical lesions (CLs). CSF levels of 67 inflammatory markers were assessed with immune-assay multiplex techniques. The 'no evidence of disease activity' (NEDA-3) status was assessed after two years and defined by no relapses, no disability worsening measured by EDSS and no MRI activity, including CLs. Results: Three patients were lost at follow-up. At the end of follow-up, 19 (48%) patients remained free from disease activity. IFNgamma, Chitinase3like1, IL32, Osteopontin, IL12(p40), IL34, IL28A, sTNFR2, IL20 and CCL2 showed the best association with disease activity. When added in a multivariate regression model including age, sex, and baseline EDSS, Chitinase 3 like1 (p = 0.049) significantly increased in those patients with disease activity. Finally, ROC analysis with Chitinase3like1 added to a model with EDSS, sex, age previous relapses, WM lesion number, CLs, number of Gad enhancing lesions and spinal cord lesions provided an AUC of 0.76 (95%CI 0.60-0.91). Conclusions: CSF Chitinase 3 like1 might provide prognostic information for predicting disease activity in the first years after initiation of cladribine. The drug's effect on chronic macrophage and microglia activation deserves further evaluation.

Ocrelizumab reduces cortical and deep grey matter loss compared to the S1P-receptor modulator in multiple sclerosis.

INTRODUCTION: Ocrelizumab (OCR) and Fingolimod (FGL) are two high-efficacy treatments in multiple sclerosis which, besides their strong anti-inflammatory activity, may limit neurodegeneration. AIM: To compare the effect of OCR and FGL on clinical and MRI endpoints. METHODS: 95 relapsing-remitting patients (57 OCR, 38 FGL) clinically followed for 36 months underwent a 3-Tesla MRI at baseline and after 24 months. The annualized relapse rate, EDSS, new cortical/white matter lesions and regional cortical and deep grey matter volume loss were evaluated. RESULTS: OCR reduced the relapse rate from 0.48 to 0.04, FGL from 0.32 to 0.05 (both p < 0.001). Compared to FGL, OCR-group experienced fewer new white matter lesions (12% vs 32%, p = 0.005), no differences in new cortical lesions, lower deep grey matter volume loss (- 0.12% vs - 0.66%; p = 0.002, Cohen's d = 0.54), lower global cortical thickness change (- 0.45% vs - 0.70%; p = 0.036; d = 0.42) and reduced cortical thinning/volume loss in several regions of interests, including those of parietal gyrus (d-range = 0.65-0.71), frontal gyrus (d-range = 0.47-0.60), cingulate (d-range = 0.41-0.72), insula (d = 0.36), cerebellum (cortex d = 0.72, white matter d = 0.44), putamen (d = 0.35) and thalamus (d = 0.31). The effect on some regional thickness changes was confirmed in patients without focal lesions. CONCLUSIONS: When compared with FGL, patients receiving OCR showed greater suppression of focal MRI lesions accumulation and lower cortical and deep grey matter volume loss.

High Levels of Perivascular Inflammation and Active Demyelinating Lesions at Time of Death Associated with Rapidly Progressive Multiple Sclerosis Disease Course: A Retrospective Postmortem Cohort Study.

OBJECTIVE: Analysis of postmortem multiple sclerosis (MS) tissues combined with in vivo disease milestones suggests that whereas perivascular white matter infiltrates are associated with demyelinating activity in the initial stages, leptomeningeal immune cell infiltration, enriched in B cells, and associated cortical lesions contribute to disease progression. We systematically examine the association of inflammatory features and white matter demyelination at postmortem with clinical milestones. METHODS: In 269 MS brains, 20 sites were examined using immunohistochemistry for active lesions (ALs) and perivenular inflammation (PVI). In a subset of 22, a detailed count of CD20+ B cells and CD3+ T cells in PVIs was performed. RESULTS: ALs were detected in 22%, whereas high levels of PVI were detected in 52% of cases. ALs were present in 35% of cases with high levels of PVI. Shorter time from onset of progression to death was associated with increased prevalence and higher levels of PVI (both p < 0.0001). Shorter time from onset of progression to wheelchair use was associated with higher prevalence of ALs (odds ratio [OR] = 0.921, 95% confidence interval [CI] = 0.858-0.989, p = 0.0230) and higher level of PVI (OR = 0.932, 95% CI = 0.886-0.981, p = 0.0071). High levels of PVI were associated with meningeal inflammation and increased cortical demyelination and significantly higher levels of B lymphocytes within the PVI. INTERPRETATION: ALs, a feature of early disease stage, persist up to death in a subgroup with high levels of PVI. These features link to a rapid progressive phase and higher levels of meningeal inflammation and B-cell infiltrates, supporting the hypothesis that chronic inflammation drives progression in MS. ANN NEUROL 2024;95:706-719.

Prognostic performance of blood neurofilament light chain protein in hospitalized COVID-19 patients without major central nervous system manifestations: an individual participant data meta-analysis.

BACKGROUND AND AIMS: To investigate the prognostic value of blood neurofilament light chain protein (NfL) levels in the acute phase of coronavirus disease 2019 (COVID-19). METHODS: We conducted an individual participant data (IPD) meta-analysis after screening on MEDLINE and Scopus to May 23rd 2022. We included studies with hospitalized adult COVID-19 patients without major COVID-19-associated central nervous system (CNS) manifestations and with a measurement of blood NfL in the acute phase as well as data regarding at least one clinical outcome including intensive care unit (ICU) admission, need of mechanical ventilation (MV) and death. We derived the age-adjusted measures NfL Z scores and conducted mixed-effects modelling to test associations between NfL Z scores and other variables, encompassing clinical outcomes. Summary receiver operating characteristic curves (SROCs) were used to calculate the area under the curve (AUC) for blood NfL. RESULTS: We identified 382 records, of which 7 studies were included with a total of 669 hospitalized COVID-19 cases (mean age 66.2 ± 15.0 years, 68.1% males). Median NfL Z score at admission was elevated compared to the age-corrected reference population (2.37, IQR: 1.13-3.06, referring to 99th percentile in healthy controls). NfL Z scores were significantly associated with disease duration and severity. Higher NfL Z scores were associated with a higher likelihood of ICU admission, need of MV, and death. SROCs revealed AUCs of 0.74, 0.80 and 0.71 for mortality, need of MV and ICU admission, respectively. CONCLUSIONS: Blood NfL levels were elevated in the acute phase of COVID-19 patients without major CNS manifestations and associated with clinical severity and poor outcome. The marker might ameliorate the performance of prognostic multivariable algorithms in COVID-19.

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, IFNß, 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.

Prominent role of executive functioning on the Phonemic Fluency Test in people with multiple sclerosis.

OBJECTIVE: Executive functioning (EF) can be one of the earliest, despite under-detected, impaired cognitive domains in patients with multiple sclerosis (pwMS). However, it is still not clear the role of EF on verbal fluency tests given the presence of information processing speed (IPS) deficits in pwMS. METHOD: Performance of a group of 43 pwMS without IPS impairment as measured with the Symbol Digit Modalities Test (SDMT) and a group of 32 healthy controls (HC) was compared on the Phonemic and Semantic Fluency Tests. For each group, we scored the number of words generated (i) in the early time interval (i.e., first 15 sec, semi-automatic process) and (ii) in the late time interval (i.e., from 15 to 60 sec, controlled process). RESULTS: Globally, pwMS produced significantly fewer words than HC on the Phonemic but not on the Semantic Fluency Test. Crucially, in the Phonemic Fluency Test pwMS generated significantly fewer words than HC in the late time interval, whereas no significant difference between the two groups emerged in the early time interval. CONCLUSIONS: These findings suggest that executive dysfunction is the core element on the Phonemic Fluency Test also in pwMS and it deserves attention in both research and clinical practice.

Atypical myelin oligodendrocyte glycoprotein antibody-associated optic neuritis and acute demyelinating polyneuropathy after SARS-CoV-2 infection: Case report and literature review.

Post-infectious immune-mediated neurological complications of Sars-Cov-2 have been increasingly recognized since the novel pandemic emerged. We describe the case of a 74 years-old patient who developed a Myelin Oligodendrocyte Glycoprotein (MOG) antibody-associated unilateral retrobulbar optic neuritis a few weeks after paucisymptomatic COVID-19 disease and, subsequently, after the resolution of the optic neuritis, an acute inflammatory demyelinating polyneuropathy. So far, no cases of these two neurological manifestations have been reported in the same patient. We herein report a case characterized by both manifestations and review the accumulating literature regarding MOG antibody-associated disease following SarsCov-2 infection.

Cortical lesions at diagnosis predict long-term cognitive impairment in multiple sclerosis: A 20-year study.

BACKGROUND AND PURPOSE: Although cognitive impairment (CI) is frequent in multiple sclerosis (MS) patients, few studies (and with conflicting results) have evaluated early predictors of CI in the long term. We aimed at determining associations between early clinical/neuroradiological variables with reference to CI after 20 years of MS. METHODS: We investigated in 170 MS patients the relationship between clinical/magnetic resonance imaging (MRI) data at diagnosis and cognitive status almost 20 years after MS onset. Among others, number and volume of both white matter lesions (WMLs) and cortical lesions (CLs) were evaluated at diagnosis and after 2 years. All MS patients were followed over time and underwent a comprehensive neuropsychological assessment at the end of study. Advanced statistical methods (unsupervised cluster analysis and random forest model) were conducted. RESULTS: CI patients showed higher focal cortical pathology at diagnosis compared to cognitively normal subjects (p < 0.001). Volumes of both WMLs and CLs emerged as the MRI metrics most associated with long-term CI. Moreover, number of CLs (especially ≥3) was also strongly associated with long-term CI (≥3 CLs: odds ratio [OR] = 3.7, 95% confidence interval = 1.8-7.5, p < 0.001), more than number of WMLs; the optimal cutoff of three CLs (area under the curve = 0.67, specificity = 75%, sensitivity = 55%) was estimated according to the risk of developing CI. CONCLUSIONS: These results highlight the impact of considering both white and gray matter focal damage from early MS stages. Given the low predictive value of WML number and the poor clinical applicability of lesion volume estimation in the daily clinical context, the evaluation of number of CLs could represent a reliable prognostic marker of CI.

Slowing processing speed is associated with cognitive fatigue in newly diagnosed multiple sclerosis patients.

OBJECTIVE: To further investigate objective measures of cognitive fatigue (CF), defined as the inability to sustain performance over time, in newly diagnosed multiple sclerosis (MS) patients, by conducting a performance analysis on the Paced Auditory Serial Addition Test (PASAT) based on the type of errors (omissions vs. incorrect responses) committed. METHOD: Sixty-two newly diagnosed patients with MS (pwMS) and 41 healthy controls (HC) completed the PASAT. Analysis of the change in performance during the test was performed by comparing the number of correct responses, incorrect responses, and omissions in the 1st versus the 3rd tertile of the PASAT. RESULTS: A significant decline in accuracy over time was observed to be related to an increment in the number of omissions, significantly more pronounced in pwMS than in HC. No change in the number of incorrect responses throughout the PASAT was observed for either group. CONCLUSIONS: CF can be detected even in newly diagnosed pwMS and might objectively manifest as a progressive increase in omissions during a sustained highly demanding task (i.e., PASAT). This pattern may reflect slowed processing speed and increased fatigue in pwMS. Focusing on omissions on the PASAT instead of correct responses only may improve its specificity as an objective measure of CF.

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-γ and TNF-α) 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.

"Ependymal-in" Gradient of Thalamic Damage in Progressive Multiple Sclerosis.

Leptomeningeal and perivenular infiltrates are important contributors to cortical grey matter damage and disease progression in multiple sclerosis (MS). Whereas perivenular inflammation induces vasculocentric lesions, leptomeningeal involvement follows a subpial "surface-in" gradient. To determine whether similar gradient of damage occurs in deep grey matter nuclei, we examined the dorsomedial thalamic nuclei and cerebrospinal fluid (CSF) samples from 41 postmortem secondary progressive MS cases compared with 5 non-neurological controls and 12 controls with other neurological diseases. CSF/ependyma-oriented gradient of reduction in NeuN+ neuron density was present in MS thalamic lesions compared to controls, greatest (26%) in subventricular locations at the ependyma/CSF boundary and least with increasing distance (12% at 10 mm). Concomitant graded reduction in SMI31+ axon density was observed, greatest (38%) at 2 mm from the ependyma/CSF boundary and least at 10 mm (13%). Conversely, gradient of major histocompatibility complex (MHC)-II+ microglia density increased by over 50% at 2 mm at the ependyma/CSF boundary and only by 15% at 10 mm and this gradient inversely correlated with the neuronal (R = -0.91, p < 0.0001) and axonal (R = -0.79, p < 0.0001) thalamic changes. Observed gradients were also detected in normal-appearing thalamus and were associated with rapid/severe disease progression; presence of leptomeningeal tertiary lymphoid-like structures; large subependymal infiltrates, enriched in CD20+ B cells and occasionally containing CXCL13+ CD35+ follicular dendritic cells; and high CSF protein expression of a complex pattern of soluble inflammatory/neurodegeneration factors, including chitinase-3-like-1, TNFR1, parvalbumin, neurofilament-light-chains and TNF. Substantial "ependymal-in" gradient of pathological cell alterations, accompanied by presence of intrathecal inflammation, compartmentalized either in subependymal lymphoid perivascular infiltrates or in CSF, may play a key role in MS progression. SUMMARY FOR SOCIAL MEDIA: Imaging and neuropathological evidences demonstrated the unique feature of "surface-in" gradient of damage in multiple sclerosis (MS) since early pediatric stages, often associated with more severe brain atrophy and disease progression. In particular, increased inflammation in the cerebral meninges has been shown to be strictly associated with an MS-specific gradient of neuronal, astrocyte, and oligodendrocyte loss accompanied by microglial activation in subpial cortical layers, which is not directly related to demyelination. To determine whether a similar gradient of damage occurs in deep grey matter nuclei, we examined the potential neuronal and microglia alterations in the dorsomedial thalamic nuclei from postmortem secondary progressive MS cases in combination with detailed neuropathological characterization of the inflammatory features and protein profiling of paired CSF samples. We observed a substantial "subependymal-in" gradient of neuro-axonal loss and microglia activation in active thalamic lesions of progressive MS cases, in particular in the presence of increased leptomeningeal and cerebrospinal fluid (CSF) inflammation. This altered graded pathology was found associated with more severe and rapid progressive MS and increased inflammatory degree either in large perivascular subependymal infiltrates, enriched in B cells, or within the paired CSF, in particular with elevated levels of a complex pattern of soluble inflammatory and neurodegeneration factors, including chitinase 3-like-1, TNFR1, parvalbumin, neurofilament light-chains and TNF. These data support a key role for chronic, intrathecally compartmentalized inflammation in specific disease endophenotypes. CSF biomarkers, together with advance imaging tools, may therefore help to improve not only the disease diagnosis but also the early identification of specific MS subgroups that would benefit of more personalized treatments. ANN NEUROL 2022;92:670-685.

Anti-SARS-CoV-2 T-stem cell memory persists in ocrelizumab-treated MS patients.

BACKGROUND: Development of long-lasting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) T-cell responses in persons with multiple sclerosis (pwMS) treated with ocrelizumab is questioned. OBJECTIVE: Investigate antiviral T-cell responses after infection with SARS-CoV-2 in ocrelizumab-treated pwMS. Control groups included ocrelizumab-treated pwMS without SARS-CoV-2 infection, and non-MS individuals with and without SARS-CoV-2 infection. METHODS: Peripheral blood mononuclear cells were stimulated with SARS-CoV-2 peptide pools and T-cell reactivity was assessed by ELISPOT for interferon (IFN)-γ detection, and by multiparametric fluorescence-activated cell sorting (FACS) analyses for assessment and characterization of T-cell activation. RESULTS: ELISPOT assay against the spike and the N protein of SARS-CoV-2 displayed specific T-cell reactivity in 28/29 (96%) pwMS treated with ocrelizumab and infected by SARS-CoV-2, similar to infected persons without MS. This reactivity was present 1 year after infection and independent from the time of ocrelizumab infusion. FACS analysis following stimulation with SARS-CoV-2 peptide pools showed the presence of activation-induced markers (AIMs) in both CD4+ and CD8+ T-cell subsets in 96% and 92% of these individuals, respectively. Within naïve AIM+ CD4+ and CD8+ T-cells, we detected T memory stem cells, suggesting the acquisition of long-term memory. CONCLUSIONS: B-cell depletion using ocrelizumab does not impair the development of long-lasting anti-SARS-CoV-2 T-cell responses.

Two years' effect of dimethyl fumarate on focal and diffuse gray matter pathology in multiple sclerosis.

BACKGROUND: Data on the effect of dimethyl fumarate (DMF) on focal and diffuse gray matter (GM) damage, a relevant pathological substrate of multiple sclerosis (MS)-related disability are lacking. OBJECTIVE: To evaluate the DMF effect on cortical lesions (CLs) accumulation and global and regional GM atrophy in subjects with relapsing-remitting MS. METHODS: A total of 148 patients (mean age 38.1 ± 9.7 years) treated with DMF ended a 2-year longitudinal study. All underwent regular Expanded Disability Status Scale (EDSS assessment), and at least two 3T-magnetic resonance imaging (MRI) at 3 and 24 months after DMF initiation. CLs and changes in global and regional atrophy of several brain regions were compared with 47 untreated age and sex-matched patients. RESULTS: DMF-treated patients showed lower CLs accumulation (median 0[0-3] vs 2[0-7], p < 0.001) with respect to controls. Global cortical thickness (p < 0.001) and regional thickness and volume were lower in treated group (cerebellum, hippocampus, caudate, and putamen: p < 0.001; thalamus p = 0.03). Lower relapse rate (14% vs 40%, p < 0.001), EDSS change (0.2 ± 0.4 vs 0.4 ± 0.9, p < 0.001), and new WM lesions (median 0[0-5] vs 2[0-6], p < 0.001) were reported. No severe adverse drug reactions occurred. CONCLUSIONS: Beyond the well-known effect on disease activity, these results provide evidence of the effect of DMF through reduced progression of focal and diffuse GM damage.

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.

Volume changes of thalamus, hippocampus and cerebellum are associated with specific CSF profile in MS.

BACKGROUND: The underlying pathogenesis of surface-in grey matter abnormalities in MS, demonstrated by both neuropathology and advanced MRI analyses, is under investigation and it might be related to CSF-mediated mechanism of inflammation and/or damage. OBJECTIVE: To examine the link of CSF inflammatory profile with the damage of three regions early-involved in MS and bordering with CSF: thalamus, hippocampus and cerebellum. METHODS: In this longitudinal, prospective study, we evaluated, in 109 relapsing-remitting MS patients, at diagnosis and after 2-year follow-up, the association between the baseline CSF level of 19 inflammatory mediators and the volume changes of thalamus, hippocampus, cerebellar cortex and control regions (globus pallidus, putamen). RESULTS: The multivariable analysis showed that the CXCL13 and sCD163 CSF levels at baseline were independent predictors of thalamus (Rmodel2=0.80; p < 0.001) and hippocampus (Rmodel2=0.47; p < 0.001) volume change after 2-year follow-up. These molecules, plus CCL25, IFN-γ and fibrinogen, were independent predictors of the cerebellar cortex volume loss (Rmodel2=0.60; p < 0.001). No independent predictors of volume changes of the control regions were found. CONCLUSION: Our results indicate an association between the CSF inflammatory profile and grey matter volume loss of regions anatomically close to CSF boundaries, thus supporting the hypothesis of a surface-in GM damage in MS.

Cerebrospinal fluid inflammatory profile of cognitive impairment in newly diagnosed multiple sclerosis patients.

BACKGROUND: The cerebrospinal fluid (CSF) molecular milieu is a marker of diffuse intrathecal inflammation in the meninges that, in turn, targets the grey matter (GM) in multiple sclerosis (MS). Cognitive impairment (CI) is associated with brain damage in MS and is often present early in people with MS (pwMS). OBJECTIVE: To investigate whether a specific CSF inflammatory profile is associated with different degrees of CI in newly diagnosed pwMS. METHODS: Sixty-nine pwMS and 43 healthy controls (HCs) underwent neuropsychological testing. The presence and levels of 57 inflammatory mediators in the CSF were assessed. RESULTS: Apparently cognitively normal (ACN) pwMS had impaired executive functioning compared to HCs but performed better than pwMS with mild and severe CI (mCI and sCI) in all tests. CSF mediators involving innate immunity and immune activation and recruitment, differentiate ACN from pwMS with mCI, while CSF mediators related to B- and T-cell immunity and chemotaxis differentiate both ACN and mCI from those with sCI. CXCL13 was the only molecule that differentiated sCI from mCI pwMS. CONCLUSION: Specific CSF molecular patterns, reflecting the involvement of both innate and adaptive immune responses, are associated with the severity of CI in newly diagnosed pwMS.

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.

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-naïve 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-γ, IL-1ß, 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-ß, IFN-λ2, sIL-6Rα) 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.