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.

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.

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).

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.

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.

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.

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.

Serum and cerebrospinal fluid BDNF concentrations are associated with neurological and cognitive improvement in multiple sclerosis: A pilot study.

BACKGROUND: Biomarkers of disease activity have been intensively studied in multiple sclerosis (MS) but knowledge on predictors of disability improvement is limited. The aim of this pilot study was to explore whether increased brain-derived neurotrophic factor concentrations in serum and CSF (sBDNF/cBDNF) precede neurological and cognitive improvement in MS. METHODS: In this pilot, monocentric prospective cohort study we collected serum/CSF samples at baseline together with EDSS (n = 36) and cognitive testing (n = 34) in patients with relapsing-remitting/primary progressive MS or clinically isolated syndrome. BDNF was assessed in serum and CSF with a single molecule array (SIMOA) HD-1 analyser (Quanterix). Twelve months later EDSS and cognitive testing were repeated. BDNF concentrations of patients with vs. without disability or cognitive improvement (disability improvement: decrease in EDSS ≥ 0.5; cognitive improvement: average z-score increase in neuropsychological performance ≥ 0.5) were compared using univariate ANOVAs adjusting for covariates. RESULTS: Compared to subjects without, patients with disability improvement had higher sBDNF at baseline (q = 0.04). Subjects with cognitive improvement had higher cBDNF at baseline than those without cognitive improvement (q = 0.004). Secondary analysis demonstrated significant correlations between sBDNF and EDSS change (q = 0.036), cBDNF and average z-score change (q = 0.04) and cBDNF and number of cognitive tests with improvement (q = 0.04), while controlling for covariates. CONCLUSIONS: Our findings suggest a possible role for BDNF in neurological and cognitive improvement in MS. These findings have to be confirmed in a larger sample but they already highlight the potential of BDNF as a biomarker for disability improvement and neuroplasticity in MS.

Cerebrospinal fluid neurofilament light chains and CXCL13 as predictive factors for clinical course of multiple sclerosis.

AIM: The aim of this study was to identify whether NfL and CXCL13 cerebrospinal fluid (CSF) concentrations at diagnostic lumbar puncture can predict the course of multiple sclerosis (MS) in terms of relapses, higher expanded disability status scale (EDSS) and magnetic resonance imaging (MRI) activity. METHODS: We conducted a single-centre prospective observational cohort study at the MS center, University Hospital Ostrava, Czech Republic. CSF NfL (cNfL) and CXCL13 concentrations were examined (ELISA method) in patients with clinically isolated syndrome (CIS) and relapsing-remitting MS (RRMS) at the time of diagnostic lumbar puncture. RESULTS: A total of 44 patients with CIS or early RRMS were enrolled, 31 (70.5%) of whom were women. The median age at the time of CSF sampling was 31.21 years (IQR 25.43-39.32), and the follow-up period was 54.6 months (IQR 44.03-59.48). In the simple and multiple logistic regression models, CXCL13 levels did not predict relapses, MRI activity or EDSS > 2.5. Similarly, cNfL concentrations did not predict relapses or MRI activity in either model. In the multiple regression, higher cNfL levels were associated with reaching EDSS > 2.5 (odds ratio [OR] 1.002, 95% confidence interval [CI] 1.000 to 1.003). CONCLUSIONS: Our data did not confirm cNfL and/or CXCL13 CSF levels were predictive factors for disease activity such as relapses and MRI activity at the time of diagnostic lumbar puncture in patients with RRMS. While cNfL CSF levels predicted higher disability only after adjustment for other known risk factors, elevated CSF CXCL13 did not predict higher disability at all.

The comparative analysis of selected interleukins and proinflammatory factors in CSF among de novo diagnosed patients with RRMS.

OBJECTIVES: Cytokines play a key role in neuroinflammation, which is present in every subset of multiple sclerosis (MS). The aim of the study was to assess levels of selected interleukins and proinflammatory factors in cerebrospinal fluid (CSF) among patients diagnosed with relapsing-remitting multiple sclerosis (RRMS). METHODS: One hundred eighteen patients diagnosed de novo with RRMS were enrolled in the study. We analysed the relationships between selected cytokines' levels depending on the age at diagnosis, time from the first symptoms to diagnosis and presence of MRI lesions. RESULTS: Among the study group the levels of IL-5 and IL-13 increased with the age at the diagnosis of MS. The concentration of IL-10 was lower in group of patients over the age of 35. The levels of IFN-γ, TNF-α, IL-5, IL-10 and IL-15 increased with the longer time from the first symptoms to diagnosis. Positive correlations were found between the levels of IL-2 and IL-12, IL-17, IL-4, IL-1RA as well as IL-1 and IL-4, IL-17. The concentration of IL-5 correlated positively with IL-4, IL-9 and IL-13. The level of IL-10 increased with IL-6 and IL-9 concentrations. A negative correlation was found for IL-10 and IL-4. In turn, between IL-13 and both IL-5 and IL-9, the relationship was positive. The level of IL-2 was significantly higher among patients without gadolinium-enhanced (Gd(+)) MRI lesions. CONCLUSIONS: The results of the study provide new insight into the role of selected molecules in the development of inflammation in MS. It might be crucial in planning the most adequate immunomodulatory therapy.

The alterations of cerebrospinal fluid TNF-alpha and TGF-beta2 levels in early relapsing-remitting multiple sclerosis.

AIM OF THE STUDY: This study aimed to analyze serum and cerebrospinal fluid (CSF) concentrations of proinflammatory and anti-inflammatory cytokines produced by T regulatory (Treg) cells in early RRMS according to the 2017 McDonald criteria. CLINICAL RATIONALE FOR THE STUDY: Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) with the cytokine network playing an important role. However, there is a continual lack of data regarding the immunopathogenesis of early RRMS, especially according to the 2017 McDonald criteria. MATERIALS AND METHODS: The study groups included early RRMS patients during relapse (n = 18), remission (n = 14), and the control group. The MS diagnosis was established according to the 2017 McDonald criteria. Patients were studied up to 1 year after diagnosis was made. A quantitative test kit based on ELISA was used for cytokine measurement in the serum and CSF. Comparative and correlation analyses between the levels of TNF-α, TGF-ß2, IgG index, and relapse duration were performed. RESULTS: Significantly higher CSF concentrations of TNF-α in both RRMS-relapse and RRMS-remission groups were found compared to the controls (p < 0.01). The CSF levels of TGF-ß2 in the RRMS-relapse group were significantly lower in comparison to the control group (p = 0.01). CONCLUSIONS AND CLINICAL IMPLICATIONS: An inappropriate inflammatory response seems to occur in early RRMS and includes the production of TNF-α and a decrease in TGF-ß2 release suggesting a significant Treg cells role. Further studies on the topic may contribute to developing new disease-modifying drugs and biochemical markers of the disorder.

MIF in the cerebrospinal fluid is decreased during relapsing-remitting while increased in secondary progressive multiple sclerosis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is involved in the function of both the innate and adaptive immune systems and in neuroprotection and has recently been implicated in multiple sclerosis (MS). OBJECTIVES: Determination of MIF levels in the cerebrospinal fluid (CSF) of patients with distinct subtypes of MS and the cellular localization of MIF in human brain tissue. METHODS: The levels of MIF were investigated in CSF from patients with clinically isolated syndrome (CIS) (n = 26), relapsing-remitting MS (RRMS) (n = 22), secondary progressive MS (SPMS) (n = 19), and healthy controls (HCs) (n = 24), using ELISA. The effect of disease-modifying therapies in the RRMS and SPMS cohorts were examined. Cellular distribution of MIF in the human brain was studied using immunochemistry and the newly available OligoInternode database. RESULTS: MIF was significantly decreased in treatment-naïve CIS and RRMS patients compared to HCs but was elevated in SPMS. Interestingly, MIF levels were sex-dependent and significantly lower in women with CIS and RRMS. MIF expression in the human brain was localized to neurons, astrocytes, pericytes, and oligo5 oligodendrocytes but not in microglia. CONCLUSION: The finding that MIF was decreased in newly diagnosed CIS and RRMS patients but was high in patients with SPMS may suggest that MIF levels in CSF are regulated by local MIF receptor expression that affects the overall MIF signaling in the brain and may represent a protective mechanism that eventually fails.

Neuroinflammation Is Associated with GFAP and sTREM2 Levels in Multiple Sclerosis.

Background: Astrocytes and microglia play an important role in the inflammatory process of multiple sclerosis (MS). We investigated the associations between the cerebrospinal fluid (CSF) levels of glial fibrillary acid protein (GFAP) and soluble triggering receptors expressed on myeloid cells-2 (sTREM-2), inflammatory molecules, and clinical characteristics in a group of patients with relapsing-remitting MS (RRMS). Methods: Fifty-one RRMS patients participated in the study. Clinical evaluation and CSF collection were performed at the time of diagnosis. The CSF levels of GFAP, sTREM-2, and of a large set of inflammatory and anti-inflammatory molecules were determined. MRI structural measures (cortical thickness, T2 lesion load, cerebellar volume) were examined. Results: The CSF levels of GFAP and sTREM-2 showed significant correlations with inflammatory cytokines IL-8, G-CSF, and IL-5. Both GFAP and sTREM-2 CSF levels positively correlated with age at diagnosis. GFAP was also higher in male MS patients, and was associated with an increased risk of MS progression, as evidenced by higher BREMS at the onset. Finally, a negative association was found between GFAP CSF levels and cerebellar volume in RRMS at diagnosis. Conclusions: GFAP and sTREM-2 represent suitable biomarkers of central inflammation in MS. Our results suggest that enhanced CSF expression of GFAP may characterize patients with a higher risk of progression.

Identification and Clinical Validation of Key Extracellular Proteins as the Potential Biomarkers in Relapsing-Remitting Multiple Sclerosis.

Background: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by autoimmunity. No objective clinical indicators are available for the diagnosis and prognosis of MS. Extracellular proteins are most glycosylated and likely to enter into the body fluid to serve as potential biomarkers. Our work will contribute to the in-depth study of the functions of extracellular proteins and the discovery of disease biomarkers. Methods: MS expression profiling data of the human brain was downloaded from the Gene Expression Omnibus (GEO). Extracellular protein-differentially expressed genes (EP-DEGs) were screened by protein annotation databases. GO and KEGG were used to analyze the function and pathway of EP-DEGs. STRING, Cytoscape, MCODE and Cytohubba were used to construct a protein-protein interaction (PPI) network and screen key EP-DEGs. Key EP-DEGs levels were detected in the CSF of MS patients. ROC curve and survival analysis were used to evaluate the diagnostic and prognostic ability of key EP-DEGs. Results: We screened 133 EP-DEGs from DEGs. EP-DEGs were enriched in the collagen-containing extracellular matrix, signaling receptor activator activity, immune-related pathways, and PI3K-Akt signaling pathway. The PPI network of EP-DEGs had 85 nodes and 185 edges. We identified 4 key extracellular proteins IL17A, IL2, CD44, IGF1, and 16 extracellular proteins that interacted with IL17A. We clinically verified that IL17A levels decreased, but Del-1 and resolvinD1 levels increased. The diagnostic accuracy of Del-1 (AUC: 0.947) was superior to that of IgG (AUC: 0.740) with a sensitivity of 82.4% and a specificity of 100%. High Del-1 levels were significantly associated with better relapse-free and progression-free survival. Conclusion: IL17A, IL2, CD44, and IGF1 may be key extracellular proteins in the pathogenesis of MS. IL17A, Del-1, and resolvinD1 may co-regulate the development of MS and Del-1 is a potential biomarker of MS. We used bioinformatics methods to explore the biomarkers of MS and validated the results in clinical samples. The study provides a theoretical and experimental basis for revealing the pathogenesis of MS and improving the diagnosis and prognosis of MS.