Cytokine Profiling in Cerebrospinal Fluid of Patients with Newly Diagnosed Relapsing-Remitting Multiple Sclerosis (RRMS): Associations between Inflammatory Biomarkers and Disease Activity.

Cytokines regulate immune responses and are crucial to MS pathogenesis. This study evaluated pro-inflammatory and anti-inflammatory cytokine concentrations in the CSF of de novo diagnosed RRMS patients compared to healthy controls. We assessed cytokine levels in the CSF of 118 de novo diagnosed RRMS patients and 112 controls, analyzing relationships with time from symptom onset to diagnosis, MRI lesions, and serum vitamin D levels. Elevated levels of IL-2, IL-4, IL-6, IL-13, FGF-basic, and GM-CSF, and lower levels of IL-1ß, IL-1RA, IL-5, IL-7, IL-9, IL-10, IL-12p70, IL-15, G-CSF, PDGF-bb, and VEGF were observed in RRMS patients compared to controls. IL-2, IL-4, IL-12p70, PDGF, G-CSF, GM-CSF, and FGF-basic levels increased over time, while IL-10 decreased. IL-1ß, IL-1RA, IL-6, TNF-α, and PDGF-bb levels negatively correlated with serum vitamin D. TNF-α levels positively correlated with post-contrast-enhancing brain lesions. IL-15 levels negatively correlated with T2 and Gd(+) lesions in C-spine MRI, while TNF-α, PDGF-bb, and FGF-basic correlated positively with T2 lesions in C-spine MRI. IL-6 levels positively correlated with post-contrast-enhancing lesions in Th-spine MRI. Distinct cytokine profiles in the CSF of de novo diagnosed MS patients provide insights into MS pathogenesis and guide immunomodulatory therapy strategies.

Cerebrospinal Fluid-In Gradient of Cortical and Deep Gray Matter Damage in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: A CSF-in gradient in cortical and thalamic gray matter (GM) damage has been found in multiple sclerosis (MS). We concomitantly explored the patterns of cortical, thalamic, and caudate microstructural abnormalities at progressive distances from CSF using a multiparametric MRI approach. METHODS: For this cross-sectional study, from 3T 3D T1-weighted scans, we sampled cortical layers at 25%-50%-75% depths from pial surface and thalamic and caudate bands at 2-3-4 voxels from the ventricular-GM interface. Using linear mixed models, we tested between-group comparisons of magnetization transfer ratio (MTR) and R2* layer-specific z-scores, CSF-in across-layer z-score changes, and their correlations with clinical (disease duration and disability) and structural (focal lesions, brain, and choroid plexus volume) MRI measures. RESULTS: We enrolled 52 patients with MS (33 relapsing-remitting [RRMS], 19 progressive [PMS], mean age: 46.4 years, median disease duration: 15.1 years, median: EDSS 2.0) and 70 controls (mean age 41.5 ± 12.8). Compared with controls, RRMS showed lower MTR values in the outer and middle cortical layers (false-discovery rate [FDR]-p ≤ 0.025) and lower R2* values in all 3 cortical layers (FDR-p ≤ 0.016). PMS had lower MTR values in the outer and middle cortical (FDR-p ≤ 0.016) and thalamic (FDR-p ≤ 0.048) layers, and in the outer caudate layer (FDR-p = 0.024). They showed lower R2* values in the outer cortical layer (FDR-p = 0.003) and in the outer thalamic layer (FDR-p = 0.046) and higher R2* values in all 3 caudate layers (FDR-p ≤ 0.031). Both RRMS and PMS had a gradient of damage, with lower values closer to the CSF, for cortical (FDR-p ≤ 0.002) and thalamic (FDR-p ≤ 0.042) MTR. PMS showed a gradient of damage for cortical R2* (FDR-p = 0.005), thalamic R2* (FDR-p = 0.004), and caudate MTR (FDR-p ≤ 0.013). Lower MTR and R2* of outer cortical, thalamic, and caudate layers and steeper gradient of damage toward the CSF were significantly associated with older age, higher T2-hyperintense white matter lesion volume, higher thalamic lesion volume, and lower brain volume (ß ≥ 0.08, all FDR-p ≤ 0.040). Lower MTR of outer caudate layer was associated with more severe disability (ß = -0.26, FDR-p = 0.040). No correlations with choroid plexus volume were found. DISCUSSION: CSF-in damage gradients are heterogeneous among different GM regions and through MS course, possibly reflecting different dynamics of demyelination and iron loss/accumulation.

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.

Glial cell injury and atrophied lesion volume as measures of chronic multiple sclerosis inflammation.

BACKGROUND: Atrophied lesion volume (aLV), a proposed biomarker of disability progression in multiple sclerosis (MS) and transition into progressive MS (PMS), depicts chronic periventricular white matter (WM) pathology. Meningeal infiltrates, imaged as leptomeningeal contrast enhancement (LMCE), are linked with greater cortical pathology. OBJECTIVES: To determine the relationship between serum-derived proteomic data with the development of aLV and LMCE in a heterogeneous group of people with MS (pwMS). METHODS: Proteomic and MRI data for 202 pwMS (148 clinically isolated syndrome /relapsing-remitting MS and 54 progressive MS (PMS)) were acquired at baseline and at 5.4-year follow-up. The concentrations of 21 proteins related to multiple MS pathophysiology pathways were derived using a custom-developed Proximity Extension Assay on the Olink™ platform. The accrual of aLV was determined as the volume of baseline T2-weighted lesions that were replaced by cerebrospinal fluid over the follow-up. Regression models and age-adjusted analysis of covariance (ANCOVA) were used. RESULTS: Older age (standardized beta = 0.176, p = 0.022), higher glial fibrillary acidic protein (standardized beta = 0.312, p = 0.001), and lower myelin oligodendrocyte glycoprotein levels (standardized beta = -0.271, p = 0.002) were associated with accrual of aLV over follow-up. This relationship was driven by the pwPMS population. The presence of LMCE at the follow-up visit was not predicted by any baseline proteomic biomarker nor cross-sectionally associated with any protein concentration. CONCLUSION: Proteomic markers of glial activation are associated with chronic lesional WM pathology (measured as aLV) and may be specific to the progressive MS phenotype. LMCE presence in MS does not appear to relate to proteomic measures.

Effects of anti-CD20 therapy on circulating and intrathecal follicular helper T cell subsets in multiple sclerosis.

Follicular helper T (Tfh) cells and their interplay with B cells likely contribute to the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). Tfh cells are enriched in cerebrospinal fluid (CSF) in RRMS, but effects of anti-CD20 therapy are unknown. We investigated Tfh cells in controls, untreated and anti-CD20-treated patients with RRMS using flow cytometry. CSF Tfh cells were increased in untreated patients. Compared to paired blood samples, CD25- Tfh cells were enriched in CSF in RRMS, but not in controls. Contrast-enhancing brain MRI lesions and IgG index correlated with CSF CD25- Tfh cell frequency in untreated patients with RRMS. Anti-CD20 therapy reduced the numbers of circulating PD1+ Tfh cells and CD25- Tfh cells, and the frequency of CSF CD25- Tfh cells. The study suggests that CD25- Tfh cells are recruited to the CSF in RRMS, associated with focal inflammation, and are reduced by anti-CD20 therapy.

Epstein-Barr virus-encoded BART9 and BART15 miRNAs are elevated in exosomes of cerebrospinal fluid from relapsing-remitting multiple sclerosis patients.

Epstein-Barr virus (EBV) infection is approved as the main environmental trigger of multiple sclerosis (MS). In this path, we quantified ebv-miR-BART9-3p and ebv-miR-BART15 in exosomes of cerebrospinal fluid (CSF) of untreated relapsing-remitting MS (RRMS) patients in comparison with the control group. Interestingly, patients displayed significant upregulation of ebv-miR-BART9-3p (18.4-fold) and ebv-miR-BART15 (3.1-fold) expression in CSF exosomes. Moreover, the expression levels of hsa-miR-21-5p and hsa-miR-146a-5p were found to be significantly elevated in the CSF samples obtained from the patient group compared to those obtained from the HC group. The levels of Interferon-gamma (IFN-γ), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), transforming growth factor beta (TGF-ß), and tumor necrosis factor-alpha (TNF-α) were observed to be significantly elevated in the serum and CSF exosomes of the patients. The highest increase was observed in TGF-ß (8.5-fold), followed by IL-23 (3.9-fold) in CSF exosomes. These findings are in agreement with the association between EBV infection and inflammatory cytokines induction. Furthermore, the ratios of TGF-ß: TNF-α and TGF-ß: IFN-γ attained values of 4 to 16.4 and 1.3 to 3.6, respectively, in the CSF exosomes of the patients, in comparison to those of the control group. These findings show EBV activity in RRMS patients is different from that of healthy ones. Elevation of ebv-miR-BART9-3p, ebv-miR-BART15, and inflammatory cytokines expression in CSF exosomes in RRMS patients provides a substantial link between EBV activity and the onset of the disease, as well as the transition from EBV infection to MS.

CD138 as a Specific CSF Biomarker of Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The aim of this study was to identify novel biomarkers for multiple sclerosis (MS) diagnosis and prognosis, addressing the critical need for specific and prognostically valuable markers in the field. METHODS: We conducted an extensive proteomic investigation, combining analysis of (1) CSF proteome from symptomatic controls, fast and slow converters after clinically isolated syndromes, and patients with relapsing-remitting MS (n = 10 per group) using label-free quantitative proteomics and (2) oligodendrocyte secretome changes under proinflammatory or proapoptotic conditions using stable isotope labeling by amino acids in cell culture. Proteins exhibiting differential abundance in both proteomic analyses were combined with other putative MS biomarkers, yielding a comprehensive list of 87 proteins that underwent quantification through parallel reaction monitoring (PRM) in a novel cohort, comprising symptomatic controls, inflammatory neurologic disease controls, and patients with MS at various disease stages (n = 10 per group). The 11 proteins that passed this qualification step were subjected to a new PRM assay within an expanded cohort comprising 158 patients with either MS at different disease stages or other inflammatory or noninflammatory neurologic disease controls. RESULTS: This study unveiled a promising biomarker signature for MS, including previously established candidates, such as chitinase 3-like protein 1, chitinase 3-like protein 2, chitotriosidase, immunoglobulin kappa chain region C, neutrophil gelatinase-associated lipocalin, and CD27. In addition, we identified novel markers, namely cat eye syndrome critical region protein 1 (adenosine deaminase 2, a therapeutic target in multiple sclerosis) and syndecan-1, a proteoglycan, also known as plasma cell surface marker CD138 and acting as chitinase 3-like protein 1 receptor implicated in inflammation and cancer signaling. CD138 exhibited good diagnostic accuracy in distinguishing MS from inflammatory neurologic disorders (area under the curve [AUC] = 0.85, CI 0.75-0.95). CD138 immunostaining was also observed in the brains of patients with MS and cultured oligodendrocyte precursor cells but was absent in astrocytes. DISCUSSION: These findings identify CD138 as a specific CSF biomarker for MS and suggest the selective activation of the chitinase 3-like protein 1/CD138 pathway within the oligodendrocyte lineage in MS. They offer promising prospects for improving MS diagnosis and prognosis by providing much-needed specificity and clinical utility. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that CD138 distinguishes multiple sclerosis from other inflammatory neurologic disorders with an AUC of 0.85 (95% CI 0.75-0.95).

Increased IL-22 in cerebrospinal fluid of neuro-behçet's disease patients.

Remitting-Relapsing Multiple Sclerosis (RRMS) and Neuro-Behçet Disease (NBD) are two chronic neuro-inflammatory disorders leading to brain damage and disability in young adults. Herein, we investigated in these patients the cytokine response by beads-based multiplex assays during the early stages of these disorders. Cytokine investigations were carried out on treatment-naive patients suffering from RRMS and NBD recruited at the first episode of clinical relapse. Our findings demonstrate that Cerebrospinal Fluid (CSF) cells from NBD patients, but not RRMS, secrete significant high levels of IL-22 which is associated with elevated IL-22 mRNA expression. We also observed an increase in IL-22 levels in the definite NBD subgroup as compared to the probable NBD one, indicating a clear relationship between elevated IL-22 levels and diagnostic certainty. Interestingly, we found no correlation of IL-22 secretion between CSF and serum arguing about intrathecal release of IL-22 in the CNS of NBD patients. Moreover, we showed by correlogram analysis that this cytokine doesn't correlate with IL-17A, IL-17F and IL-21 suggesting that this cytokine is secreted by Th22 cells and not by Th17 cells in the CSF of NBD patients. Finally, we found elevated levels of IL-6 and a positive correlation between IL and 6 and IL-22 in the CSF of NBD. In conclusion, these results suggest that IL-6 contributes to the production of IL-22 by T cells leading to the exacerbation of inflammation and damage within the CNS of NBD patients.

Matching proposed clinical and MRI criteria of aggressive multiple sclerosis to serum and cerebrospinal fluid markers of neuroaxonal and glial injury.

BACKGROUND: Definitions of aggressive MS employ clinical and MR imaging criteria to identify highly active, rapidly progressing disease courses. However, the degree of overlap between clinical and radiological parameters and biochemical markers of CNS injury is not fully understood. Aim of this cross-sectional study was to match clinical and MR imaging hallmarks of aggressive MS to serum/CSF markers of neuroaxonal and astroglial injury (neurofilament light chain (sNfL, cNfL), and glial fibrillary acidic protein (sGFAP, cGFAP)). METHODS: We recruited 77 patients with relapsing-remitting MS (RRMS) and 22 patients with clinically isolated syndrome. NfL and GFAP levels in serum and CSF were assessed using a single-molecule-array HD-1-analyzer. A general linear model with each biomarker as a dependent variable was computed. Clinical and imaging criteria of aggressive MS, as recently proposed by the ECTRIMS Consensus Group, were modeled as independent variables. Other demographic, clinical or laboratory parameters, were modeled as covariates. Analyses were repeated in a homogenous subgroup, consisting only of newly diagnosed, treatment-naïve RRMS patients presenting with an acute relapse. RESULTS: After adjusting for covariates and multiplicity of testing, sNfL and cNfL concentrations were strongly associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.00008; pcNfL = 0.004) as well as the presence of infratentorial lesions on MRI (psNfL = 0.0003; pcNfL < 0.004). No other clinical and imaging criteria of aggressive MS correlated significantly with NfL or GFAP in serum and CSF. In the more homogeneous subgroup, sNfL still was associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.001), presence of more than 20 T2-lesions (psNfL = 0.049) as well as the presence of infratentorial lesions on MRI (psNfL = 0.034), while cNfL was associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.011) and presence of more than 20 T2-lesions (psNfL = 0.029). CONCLUSIONS: Among proposed risk factors for an aggressive disease course, MRI findings but not clinical characteristics correlated with sNfL and cNfL as a marker of neuroaxonal injury and should be given appropriate weight considering MS prognosis and therapy. No significant correlation was detected for GFAP alone.

Prognostic relevance of the C-X-C motif chemokine ligand 13 and interleukin-8 in predicting the transition from clinically isolated syndrome to multiple sclerosis.

The initial phase of multiple sclerosis (MS), often known as clinically isolated syndrome (CIS), is a critical period for identifying individuals at high risk of progressing to full-blown MS and initiating timely treatment. In this study, we aimed to evaluate the prognostic value of C-X-C motif chemokine ligand 13 (CXCL13) and interleukin-8 (IL-8) as potential markers for CIS patients' conversion to MS. Our study encompassed patients with CIS, those with relapsing-remitting MS (RRMS), and control subjects, with sample analysis conducted on both cerebrospinal fluid (CSF) and serum. Patients were categorized into four groups: CIS-CIS (no MS development within 2 years), CIS-RRMS (conversion to RRMS within 2 years), RRMS (already diagnosed), and a control group (CG) with noninflammatory central nervous system disorders. Results showed significantly elevated levels of CXCL13 in CSF across all patient groups compared with the CG (p < 0.0001, Kruskal-Wallis test). Although CXCL13 concentrations were slightly higher in the CIS-RRMS group, statistical significance was not reached. Similarly, significantly higher levels of IL-8 were detected in CSF samples from all patient groups compared with the CG (p < 0.0001, Kruskal-Wallis test). Receiver operating characteristic analysis in the CIS-RRMS group identified both CXCL13 (area under receiver operating characteristic curve = .959) and IL-8 (area under receiver operating characteristic curve = .939) in CSF as significant predictors of CIS to RRMS conversion. In conclusion, our study suggests a trend towards elevated CSF IL-8 and CSF CXCL13 levels in CIS patients progressing to RRMS. These findings emphasize the importance of identifying prognostic markers to guide appropriate treatment strategies for individuals in the early stages of MS.

Natalizumab promotes anti-inflammatory and repair effects in multiple sclerosis.

BACKGROUND: Multiple sclerosis is an inflammatory and degenerative disease of the central nervous system leading to demyelination and axonal loss. Relapsing-remitting multiple sclerosis (RRMS) is commonly treated by anti-inflammatory drugs, where one of the most effective drugs to date is the monoclonal antibody natalizumab. METHODS: The cerebrospinal fluid (CSF) proteome was analyzed in 56 patients with RRMS before and after natalizumab treatment, using label-free mass spectrometry and a subset of the changed proteins were verified by parallel reaction monitoring in a new cohort of 20 patients, confirming the majority of observed changes. RESULTS: A total of 287 differentially abundant proteins were detected including (i) the decrease of proteins with roles in immunity, such as immunoglobulin heavy constant mu, chitinase-3-like protein 1 and chitotriosidase, (ii) an increase of proteins involved in metabolism, such as lactate dehydrogenase A and B and malate-dehydrogenase cytoplasmic, and (iii) an increase of proteins associated with the central nervous system, including lactadherin and amyloid precursor protein. Comparison with the CSF-PR database provided evidence that natalizumab counters protein changes commonly observed in RRMS. Furthermore, vitamin-D binding protein and apolipoprotein 1 and 2 were unchanged during treatment with natalizumab, implying that these may be involved in disease activity unaffected by natalizumab. CONCLUSIONS: Our study revealed that some of the previously suggested biomarkers for MS were affected by the natalizumab treatment while others were not. Proteins not previously suggested as biomarkers were also found affected by the treatment. In sum, the results provide new information on how the natalizumab treatment impacts the CSF proteome of MS patients, and points towards processes affected by the treatment. These findings ought to be explored further to disclose potential novel disease mechanisms and predict treatment responses.

The role of oligoclonal band count and IgG index in treatment response and disease activity in multiple sclerosis.

BACKGROUND/AIM: Multiple sclerosis (MS) is an inflammatory demyelinating central nervous system (CNS) disease. Among the paraclinical tests, brain and spinal Magnetic Resonance Imaging (MRI) is primarily involved in the diagnosis process, and cerebrospinal fluid (CSF) analysis is fundamental in diagnosing MS and the differential diagnosis. A positive relationship was demonstrated between oligoclonal band (OCB) positivity, CSF band number and immunoglobulin G(IgG) index. The study aimed to evaluate whether the number of OCB can predict disease activity and determine a correlation with the IgG index. METHODS: Our study included 401 MS patients who had relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS), clinic isolated syndrome (CIS), radiologic isolated syndrome (RIS), Neuromyelitis optica spectrum disorder (NMOSD) and Acute disseminated encephalomyelitis (ADEM) with OCB number groups of 2-4, 4-8, 8-12, and 12 and above. RESULTS: No significant correlation was observed between IgG index, pre-and post-treatment EDSS (Expanded Disability Status Scale Scores) and disease-modifying therapies (DMT). Drug response was better in the patient group with band number between 2 and 8 and post-treatment EDSS scores were lower (1.62±0.44). CONCLUSION: The study results suggested that band number may be as valuable as the IgG index and a predictive biomarker for disease activity.

Development of an LC-MS/MS Method to Measure Sphingolipids in CSF from Patients with Multiple Sclerosis.

Multiple sclerosis is an inflammatory and degenerative disease characterized by different clinical courses including relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). A hallmark of patients with multiple sclerosis (pwMS) includes a putative autoimmune response, which results in demyelination and neuroaxonal damage in the central nervous system. Sphingolipids in cerebrospinal fluid (CSF) have been proposed as potential biomarkers reflective of disease activity in pwMS. Hence, sensitive methods to accurately quantify sphingolipids in CSF are needed. In this study, we report the development of a sensitive high-throughput multiplexed liquid chromatography coupled to a tandem mass spectrometry method to perform quantitation on 14 species of sphingolipids in human CSF. We applied this method to measure CSF sphingolipids in healthy controls (n = 10), PPMS (n = 27), and RMS (n = 17) patients before and after ocrelizumab treatment. The median CSF levels of the 14 sphingolipids measured herein was higher in PPMS (17.2 ng/mL) and RMS (17.6 ng/mL) when compared with the healthy controls (13.8 ng/mL). Levels of sphingolipids were decreased by 8.6% at week 52 after treatment with ocrelizumab in RMS patients but not in PPMS patients. Specifically, C16 glucosylceramide (-26%; P = 0.004) and C18 ceramides (-13%; P = 0.042) decreased from baseline in RMS patients. Additionally, in PPMS patients C16 glucosylceramide levels correlated with CSF neurofilament heavy levels at baseline (Rho =0.532; P = 0.004) and after treatment (Rho =0.424; P = 0.028). Collectively, these results indicate that CSF sphingolipid levels are altered in pwMS and treatment with ocrelizumab results in significant shifts in the sphingolipid profile that may reflect a reduction in disease activity supporting further investigation into sphingolipids as tools to monitor disease state. SIGNIFICANCE STATEMENT: This study describes the development of a new method to measure 14 sphingolipid species in CSF. These results demonstrate that sphingolipids levels are elevated in CSF from pwMS compared to healthy controls. Distinct sphingolipid signatures were observed between patients with different clinical disease courses, and these lipid signatures changed after treatment with ocrelizumab, especially in RMS patients. This method enables further investigation into the role of sphingolipids as candidate biomarkers in pwMS and other central nervous system disorders.

High temperature requirement A1 and macrophage migration inhibitory factor in the cerebrospinal fluid; a potential marker of conversion from relapsing-remitting to secondary progressive multiple sclerosis.

BACKGROUND: Predictive and prognostic biomarkers for multiple sclerosis (MS) remain a significant gap in MS diagnosis and treatment monitoring. Currently, there are no timely markers to diagnose the transition to secondary progressive MS (SPMS). OBJECTIVE: This study aims to evaluate the discriminatory potential of the High temperature requirement serine protease (HTRA1)/Macrophage migration inhibitory factor (MIF) cerebrospinal fluid (CSF) ratio in distinguishing relapsing-remitting (RRMS) patients from SPMS patients. METHODS: The MIF and HTRA1 CSF levels were determined using ELISA in healthy controls (n = 23), RRMS patients before (n = 22) and after 1 year of dimethyl fumarate treatment (n = 11), as well as in SPMS patients before (n = 11) and after 2 years of mitoxantrone treatment (n = 7). The ability of the HTRA1/MIF ratio to discriminate the different groups was determined using receiver operating curve (ROC) analyses. RESULTS: The ratio was significantly increased in treatment naïve RRMS patients while decreased again in SPMS patients at baseline. Systemic administrated disease modifying treatment (DMT) only significantly affected the ratio in RRMS patients. ROC analysis demonstrated that the ratio could discriminate treatment naïve RRMS patients from SPMS patients with 91% sensitivity and 100% specificity. CONCLUSION: The HTRA1/MIF ratio is a strong candidate as a MS biomarker for SPMS conversion.

Distinct intrathecal inflammatory signatures following relapse and anti-COVID-19 mRNA vaccination in multiple sclerosis.

BACKGROUND: The role of vaccine-mediated inflammation in exacerbating multiple sclerosis (MS) is a matter of debate. OBJECTIVE: In this cross-sectional study, we compared the cerebrospinal fluid (CSF) inflammation associated with MS relapses or anti-COVID-19 mRNA vaccinations in relapsing-remitting multiple sclerosis (RRMS). METHODS: We dosed CSF cytokines in 97 unvaccinated RRMS patients with clinical relapse within the last 100 days. In addition, we enrolled 29 stable RRMS and 24 control patients receiving COVID-19 vaccine within the last 100 days. RESULTS: In RRMS patients, a negative association was found between relapse distance and the CSF concentrations of the pro-inflammatory cytokines interleukin (IL)-2 (beta = -0.265, p = 0.016), IL-6 (beta = -0.284, p = 0.01), and IL-17 (beta = -0.224, p = 0.044). Conversely, vaccine distance positively correlated with a different set of cytokines including IL-12 (beta = 0.576, p = 0.002), IL-13 (beta = 0.432, p = 0.027), and IL-1ra (beta = 0.387, p = 0.05). These associations were significant also considering other clinical characteristics. No significant associations emerged between vaccine distance and CSF molecules in the control group. CONCLUSION: Vaccine for COVID-19 induces a central inflammatory response in RRMS patients that is qualitatively different from that associated with disease relapse.

Ferroptosis contributes to multiple sclerosis and its pharmacological targeting suppresses experimental disease progression.

Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by central nervous (CNS) demyelination resulting in axonal injury and neurological deficits. Essentially, MS is driven by an auto-amplifying mechanism of inflammation and cell death. Current therapies mainly focus on disease modification by immunosuppression, while no treatment specifically focuses on controlling cell death injury. Here, we report that ferroptosis, an iron-catalyzed mode of regulated cell death (RCD), contributes to MS disease progression. Active and chronic MS lesions and cerebrospinal fluid (CSF) of MS patients revealed several signs of ferroptosis, reflected by the presence of elevated levels of (labile) iron, peroxidized phospholipids and lipid degradation products. Treatment with our candidate lead ferroptosis inhibitor, UAMC-3203, strongly delays relapse and ameliorates disease progression in a preclinical model of relapsing-remitting MS. In conclusion, the results identify ferroptosis as a detrimental and targetable factor in MS. These findings create novel treatment options for MS patients, along with current immunosuppressive strategies.

Cerebrospinal fluid soluble CD27 is associated with CD8+ T cells, B cells and biomarkers of B cell activity in relapsing-remitting multiple sclerosis.

Cerebrospinal fluid (CSF) soluble CD27 (sCD27) is a sensitive biomarker of intrathecal inflammation. Although generally considered a biomarker of T cell activation, CSF sCD27 has been shown to correlate with biomarkers of B cell activity in multiple sclerosis. We analyzed CSF from 40 patients with relapsing-remitting multiple sclerosis (RRMS) and nine symptomatic controls using flow cytometry and multiplex electrochemiluminescence immunoassays. CSF sCD27 levels were increased in RRMS and correlated with IgG index, soluble B cell maturation antigen, cell count, B cell frequency and CD8+ T cell frequency. We provide new data indicating that CSF sCD27 is associated with CD8+ T cells and B cells in RRMS.

Granulocyte activation markers in cerebrospinal fluid differentiate acute neuromyelitis spectrum disorder from multiple sclerosis.

BACKGROUND: Granulocyte invasion into the brain is a pathoanatomical feature differentiating neuromyelitis optica spectrum disorder (NMOSD) from multiple sclerosis (MS). We aimed to determine whether granulocyte activation markers (GAM) in cerebrospinal fluid (CSF) can be used as a biomarker to distinguish NMOSD from MS, and whether levels associate with neurological impairment. METHODS: We quantified CSF levels of five GAM (neutrophil elastase, myeloperoxidase, neutrophil gelatinase-associated lipocalin, matrixmetalloproteinase-8, tissue inhibitor of metalloproteinase-1), as well as a set of inflammatory and tissue-destruction markers, known to be upregulated in NMOSD and MS (neurofilament light chain, glial fibrillary acidic protein, S100B, matrix metalloproteinase-9, intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), in two cohorts of patients with mixed NMOSD and relapsing-remitting multiple sclerosis (RRMS). RESULTS: In acute NMOSD, GAM and adhesion molecules, but not the other markers, were higher than in RRMS and correlated with actual clinical disability scores. Peak GAM levels occurred at the onset of NMOSD attacks, while they were stably low in MS, allowing to differentiate the two diseases for ≤21 days from onset of clinical exacerbation. Composites of GAM provided area under the curve values of 0.90-0.98 (specificity of 0.76-1.0, sensitivity of 0.87-1.0) to differentiate NMOSD from MS, including all anti-aquaporin-4 protein (aAQP4)-antibody-negative patients who were untreated. CONCLUSIONS: GAM composites represent a novel biomarker to reliably differentiate NMOSD from MS, including in aAQP4- NMOSD. The association of GAM with the degree of concurrent neurological impairment provides evidence for their pathogenic role, in turn suggesting them as potential drug targets in acute NMOSD.

Cerebrospinal fluid sulfatide isoforms lack diagnostic utility in separating progressive from relapsing-remitting multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system. The glycosphingolipid sulfatide, a lipid particularly enriched in the myelin sheath, has been shown to be involved the maintenance of this specific membrane structure. Sulfatide in cerebrospinal fluid (CSF) may reflect demyelination, a dominating feature of MS. We investigated the diagnostic utility of CSF sulfatide isoform levels to separate different courses or phenotypes of MS disease. MATERIAL AND METHODS: This was a mono-center, cross-sectional study of relapsing-remitting MS (RRMS) (n = 45) and progressive MS (PMS) (n = 42) patients (consisting of primary PMS (n = 17) and secondary PMS (n = 25)) and healthy controls (n = 19). In total, 20 sulfatide isoforms were measured in CSF by liquid chromatography-mass spectrometry. RESULTS: CSF total sulfatide concentrations, as well as CSF sulfatide isoform distribution, did not differ across the study groups, and their levels were independent of disease course/phenotype, disease duration, time to conversion to secondary PMS, age, and disability in MS patients. CONCLUSION: CSF sulfatide isoforms lack diagnostic and prognostic utility as a biomarker for progressive MS.