Fluid Of Multiple Sclerosis Patients Research Articles

Expanded T lymphocytes in the cerebrospinal fluid of multiple sclerosis patients are specific for Epstein-Barr-virus-infected B cells.

Epstein-Barr virus (EBV) infection has long been associated with multiple sclerosis (MS), but the role of EBV in the pathogenesis of MS is not clear. Our hypothesis is that a major fraction of the expanded clones of T lymphocytes in the cerebrospinal fluid (CSF) are specific for autologous EBV-infected B cells. We obtained blood and CSF samples from eight relapsing-remitting patients in the process of diagnosis. We stimulated cells from the blood with autologous EBV-infected lymphoblastoid cell lines (LCL), EBV, varicella zoster virus, influenza, and candida and sorted the responding cells with flow cytometry after 6 d. We sequenced the RNA for T cell receptors (TCR) from CSF, unselected blood cells, and the antigen-specific cells. We used the TCR Vβ CDR3 sequences from the antigen-specific cells to assign antigen specificity to the sequences from the CSF and blood. LCL-specific cells comprised 13.0 ± 4.3% (mean ± SD) of the total reads present in CSF and 13.3 ± 7.5% of the reads present in blood. The next most abundant antigen specificity was flu, which was 4.7 ± 1.7% of the reads in the CSF and 9.3 ± 6.6% in the blood. The prominence of LCL-specific reads was even more marked in the top 1% most abundant CSF clones with statistically significant 47% mean overlap with LCL. We conclude that LCL-specific sequences form a major portion of the TCR repertoire in both CSF and blood and that expanded clones specific for LCL are present in MS CSF. This has important implications for the pathogenesis of MS.

Interferome perturbation of human brain organoids induces progenitor and neuronal dysfunction seen in multiple sclerosis and autism (P2-3.015)

<h3>Objective:</h3> To characterize the effect of activation of the interferome in human organoids toward understanding human cortical neuronal dysfunction. <h3>Background:</h3> The mechanisms by which maternal infection and neuroinflammation impact human brain development, function, and repair remain unclear. IFN-g is a proinflammatory cytokine found elevated in serum of pregnant mothers with children diagnosed with autism and in Th1 cells in the cerebrospinal fluid of multiple sclerosis patients. <h3>Design/Methods:</h3> Nearly three hundred human cerebral organoids derived from iPSCs were used across all analyses. Organoids were continually exposed to IFN-g and compared to controls for up to 80 days in culture. 10X single-cell transcriptomics was performed at three timepoints, along with immunolabeling and pulse EdU experiments. Bioinformatic and Markov Chain analyses were used to determine the molecular, cellular, and systems level effects of IFN-g. <h3>Results:</h3> IFN-g exposure at low concentration restricts organoid growth across timepoints, diminishes the pool of cycling radial glia progenitors, and disrupts ventricular adherens cytoarchitecture. We find no evidence of cell cycle arrest or apoptosis, but rather an induction of premature neuronal differentiation and acceleration of neuronal maturation, that stems from cell-type-specific transcriptional rewiring by IFN-g, inducing expression of inflammatory and neurogenic genes that persists in all cell lineages. Markov chain modeling demonstrates that IFN-g targets differentiation lineages in a parallel pan-neuronal fashion. Finally, we find that cell-type-specific gene dysregulation induced by IFN-g overlaps with patient derived organoids from genetically linked autism spectrum disorder, and neuronal intrinsic dysregulation observed in cortical neurons in progressive multiple sclerosis. <h3>Conclusions:</h3> Our results show the feasibility of using a human brain organoid to model the effects of neuroinflammation on human brain development and function. This approach reveals a novel inflammatory mechanistic link with autism and identifies a critical network of neuronal identity and synaptic genes perturbed in multiple sclerosis recapitulated in a preclinical model. <b>Disclosure:</b> Ethan Hollingsworth has nothing to disclose. Mr. Julian has nothing to disclose. Fumihiro Watanabe has nothing to disclose. Mr. Reutershan has nothing to disclose. Miss Julian has nothing to disclose. Ms. Martorell Serra has nothing to disclose. Sofia Lizarraga has nothing to disclose. Dr. Hester has nothing to disclose. Dr. Imitola has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis . The institution of Dr. Imitola has received research support from biogen. Dr. Imitola has a non-compensated relationship as a Board Member with National MS Society that is relevant to AAN interests or activities. Dr. Imitola has a non-compensated relationship as a Committee Member with International Society for Stem Cell Research that is relevant to AAN interests or activities.

Lipid profile of cerebrospinal fluid in multiple sclerosis patients: a potential tool for diagnosis

Multiple sclerosis (MS) is a complex multifactorial neuropathology. Although its etiology remains unclear, it has been demonstrated that the immune system attacks myelin, leading to demyelination and axonal damage. The involvement of lipids as one of the main components of myelin sheaths in MS and other demyelinating diseases has been postulated. However, it is still a matter of debate whether specific alteration patterns exist over the disease course. Here, using a lipidomic approach, we demonstrated that, at the time of diagnosis, the cerebrospinal fluid of MS patients presented differences in 155 lipid species, 47 of which were identified. An initial hierarchical clusterization was used to classify MS patients based on the presence of 25 lipids. When a supervised method was applied in order to refine this classification, a lipidomic signature was obtained. This signature was composed of 15 molecules belonging to five different lipid families including fatty acids (FAs). An FA-targeted approach revealed differences in two members of this family: 18:3n3 and 20:0 (arachidic acid). These results reveal a CSF lipidomic signature in MS patients at the time of diagnosis that might be considered as a potential diagnostic tool.

B-cell composition in the blood and cerebrospinal fluid of multiple sclerosis patients treated with dimethyl fumarate

BackgroundB cells may contribute to the immunopathogenesis of multiple sclerosis (MS). Dimethyl fumarate (DMF) has recently been shown to reduce the frequency of memory B cells in blood, but it is not known whether the drug influences the cellular composition in the cerebrospinal fluid (CSF). MethodsA cross-sectional study examining the cellular composition in blood and cerebrospinal fluid (CSF) from 10 patients treated with DMF and 18 patients receiving other disease modifying drugs or no treatment. ResultsPatients treated with DMF had reduced proportions of memory B cells in blood compared to other MS patients (p = 0.0007), and the reduction correlated with treatment duration (rs = −0.75, p = 0.021). In the CSF, the absolute number of mononuclear cells were significantly lower in DMF-treated patients compared to the other patients (p = 0.023), and there was a disproportionate decrease of plasmablasts (p = 0.031). ConclusionThe results of this exploratory study support a B-cell mediated mechanism of action for DMF in both blood and CSF.

RANKL/RANK/OPG Axis Is Deregulated in the Cerebrospinal Fluid of Multiple Sclerosis Patients at Clinical Onset

Objectives: Our study focused on the RANKL (receptor activator of nuclear factor-κB ligand)/RANK/OPG (osteoprotegerin) axis and selected proinflammatory/immunoregulatory upstream mediators in the peripheral blood (PBL) and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. Methods: PBL and CSF were collected from healthy controls (n = 35) and MS patients at the clinical onset of the disease (n = 33). In addition, PBL samples were obtained from relapse-remitting (RR)-MS patients (n = 30). Patients were assessed by means of the expanded disability status scale (EDSS) and routine laboratory parameters. Soluble (s)RANKL and OPG were measured in the CSF and plasma; gene expression was detected for RANKL, RANK, OPG, and selected cytokines/chemokines (interleukin [IL]-4, IL-10, IL-17, CCL2, and CXCL12) in PBL mononuclear cells. Results: The OPG level in the CSF was lower in MS patients at clinical onset than in controls. Moreover, the sRANKL/OPG ratio was higher in the CSF of MS patients at clinical onset and in the plasma of RR-MS patients than in controls. Gene expression of RANKL/RANK/OPG in PBL mononuclear cells was higher only in RR-MS patients. IL-4, CCL2, and CXCL12 were positively correlated and IL-10 was negatively correlated with RANKL/RANK expression. OPG was negatively correlated with EDSS and alkaline phosphatase level. Conclusion: Our study revealed that changes of RANKL/RANK/OPG axis are associated with MS, particularly the decreased OPG level in the CSF at disease onset. Therefore, these factors may serve as disease biomarkers and molecular targets of novel therapeutic approaches.

Immune-related microRNAs are differentially regulated in the cerebrospinal fluid of Multiple Sclerosis patients according to disease activity and their profile mirrors neuroinfectious diseases (P1.404)

Immune-related microRNAs are differentially regulated in the cerebrospinal fluid of Multiple Sclerosis patients according to disease activity and their profile mirrors neuroinfectious diseases (P1.404)

Natalizumab reduces serum pro-angiogenic activity in MS patients.

Angiogenesis has been implicated in the pathobiology of multiple sclerosis (MS). Osteopontin exerts a pro-angiogenetic effect and is increased in body fluid of MS patients. To evaluate the effect of 1year natalizumab treatment on serum pro-angiogenic activity and on plasma osteopontin levels in relapsing (RR) MS patients. Ten RRMS patients scheduled for natalizumab treatment were enrolled and evaluated at baseline and after 1-year natalizumab treatment. Pro-angiogenic activity was assessed by a chick embryo chorioallantoic membrane assay (CAM), osteopontin levels were evaluated by an enzyme-linked immunosorbent assay. Plasma and serum samples of 10 treatment-naïve RRMS and 10 healthy controls (HCs) were used as controls of baseline evaluations. Both treatment-naïve and natalizumab scheduled RRMS patients had higher baseline vessel density (22.0 ± 3.9 and 22.5 ± 2.6, p < 0.0001) and higher osteopontin levels (65.7 ± 24.3ng/ml and 65.9 ± 16.6ng/ml, p = 0.019 and p = 0.029, respectively) than HCs (9.0 ± 2.2; 48.5 ± 7.8ng/ml, respectively). Baseline osteopontin levels and vessel density were significantly correlated (rs = 0.373, p = 0.043). After 1year of treatment, the number of vessels and the osteopontin levels, were significantly reduced (11.9 ± 2.1, p < 0.005; 49.3 ± 20.0ng/ml, p = 0.028). Our results suggest that natalizumab could exert its anti-inflammatory properties also by inhibiting the angiogenetic mechanisms in RRMS patients.

The effect of upper-respiratory viral infection on leukocyte surveillance of the brain

Peripheral infections exacerbate symptoms of many neurological diseases, including the most common autoimmune demyelinating disease of the central nervous system (CNS), multiple sclerosis (MS). We hypothesized that upper-respiratory viral infection induces glial activation and results in immune cell trafficking to the CNS, which may be detrimental to MS patients. To test this hypothesis we inoculated C57BL/6 mice with saline or influenza A virus (Puerto Rico/8/34; PR8). Glial activation and chemokine expression was evaluated by RNA-seq at days 0, 4, and 8 p.i.. Immune cell surveillance over the course of infection was assessed by flow cytometry. Chemokine levels in the cerebrospinal fluid of MS patients in remission and relapse were measured by luminex. Influenza infection caused transcriptomic changes to the cerebellum and spinal cord which was consistent with and glial activation and induced a predominantly A1 reactive astrocyte gene expression profile. Infection increased the expression of Ccl6 and Cxcl5 in the cerebellum and modulated expression of Ccl17, Ccl25, Ccl28, Ccl27a and Cx3cl1 in both the cerebellum and spinal cord. Flow cytometry analysis indicated that infection increased immune surveillance of the brain by monocytes and neutrophils as well as altered the profile of B-cells in the CNS. Finally, we found that CXCL5 was increased in the CSF of patients during relapse compared with those in remission.

Elevated Levels of Proinflammatory Cytokines in Cerebrospinal Fluid of Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease characterized by chronic brain inflammation. Leukocyte infiltration of brain tissue causes inflammation, demyelination, and the subsequent formation of sclerotic plaques, which are a hallmark of MS. Activation of proinflammatory cytokines is essential for regulation of lymphocyte migration across the blood–brain barrier. We demonstrate increased levels of many cytokines, including IL-2RA, CCL5, CCL11, MIF, CXCL1, CXCL10, IFNγ, SCF, and TRAIL, were upregulated in cerebrospinal fluid (CSF), whereas IL-17, CCL2, CCL3, CCL4, and IL-12(p40) were activated in MS serum. Interaction analysis of cytokines in CSF demonstrated a connection between IFNγ and CCL5 as well as MIF. Many cells can contribute to production of these cytokines including CD8 and Th1 lymphocytes and astrocytes. Therefore, we suggest that IFNγ released by Th1 lymphocytes can activate astrocytes, which then produce chemoattractants, including CCL5 and MIF. These chemokines promote an inflammatory milieu and interact with multiple chemokines including CCL27 and CXCL1. Of special note, upregulation of CCL27 was found in CSF of MS cases. This observation is the first to demonstrate CCL27 as a potential contributor of brain pathology in MS. Our data suggest that CCL27 may be involved in activation and migration of autoreactive encephalitogenic immune effectors in the brain. Further, our data support the role of Th1 lymphocytes in the pathogenesis of brain inflammation in MS, with several cytokines playing a central role.

Evaluation of total, ceruloplasmin-associated and type II ferroxidase activities in serum and cerebrospinal fluid of multiple sclerosis patients

Multiple sclerosis (MS) patients have increased brain iron deposition with higher oxidative stress (OxS). These two features can be caused by an inefficient removal of free iron from extracellular compartment. Ferroxidase activity (Feox) exerted by ceruloplasmin (FeoxCp) and by other molecules (FeoxII) appears to have a central role in this process. The aim of this study was to investigate serum and cerebrospinal fluid (CSF) total Feox, FeoxII and FeoxCp activities in MS patients and neurological controls. Serum and CSF Feox activity, FeoxII and FeoxCp activity was measured in 91 relapsing-remitting (RR) MS patients, 79 subjects with other inflammatory neurological disorders (OIND) and 65 with non-inflammatory neurological disorders (NIND), as controls. This study was approved by the Local Committee for Medical Ethics in Research. Serum total Feox activity was lower in MS group than in both NIND and OIND, with only the former control group differing significantly (p<0.001); FeoxII and FeoxCp activities were comparable among the groups. Serum Feox activities were not associated with disease activity as assessed by clinical examination or by Magnetic Resonance Imaging (MRI). Only total Feox activity was detectable in the CSF and was not different in MS compared to either OIND or NIND.In conclusion, a condition of low systemic Feox may increase the susceptibility of MS patients to iron(II) mediated-oxidative damage. This alteration is not reflected in CSF, suggesting that agents endowed with Feox activity might have different impact in iron homeostasis in the central nervous system compared to periphery.

Myelin-Reactive Monoclonal Antibodies from Multiple Sclerosis Patients Cross-React with Nucleoproteins in HEp-2 Lysate

Autoimmune disorders are characterized by appearance of self-reactive species of immune system such as T cells, B cells and antibodies. For the majority of autoimmune pathologies the list of specific autoantigens is known. Myelin basic protein (MBP) is one of the most important autoantigens in multiple sclerosis (MS), which destruction is a hallmark of disease progression. Antibodies toward MBP are found in serum and cerebrospinal fluid of MS patients. Here we investigated whether monoclonal human MBP-specific antibodies selected from MS patients repertoire cross-react with other autoimmune markers. For this purpose we performed Western blot analysis of recombinant anti-MBP antibodies with HEp-2 cell lysate. Our data suggest existence of enhanced level of cross-reactivity of anti-MBP antibodies with ribonucleoprotein A (RNP A), a marker of Sharp’s syndrome and systemic lupus erythematosus, ribosomal P protein (Rib. P-Prot), a marker for systemic lupus erythematosus, and centromere protein A/B (CENP A/B), markers for progressive systemic sclerosis.

Abnormal galactosylation of immunoglobulin G in cerebrospinal fluid of multiple sclerosis patients

Background: Glycosylation alterations have been associated with the development of several human diseases and their animal models, including multiple sclerosis. Objectives: We aimed to determine whether immunoglobulin G galactosylation might be changed in multiple sclerosis. Methods: Immunoglobulin G was isolated from serum and cerebrospinal fluid of patients with multiple sclerosis or viral meningitis and control patients without history of inflammatory or autoimmune disease. A lectin-based assay was used to investigate potential galactosylation modifications of immunoglobulin G. Results and conclusion: Galactosylation of immunoglobulin G isolated from cerebrospinal fluid of control patients was found to be age- and gender-dependent. In addition, immunoglobulin G galactosylation was significantly altered in cerebrospinal fluid but not in serum of multiple sclerosis patients. Furthermore, this modification was correlated with an active progression of multiple sclerosis. Finally, the loss of galactosyl moieties was not simply associated with inflammation as no such change was detected in viral meningitis patients characterized by brain inflammation.

Complement Receptor 2 is increased in cerebrospinal fluid of multiple sclerosis patients and regulates C3 function

Besides its vital role in immunity, the complement system also contributes to the shaping of the synaptic circuitry of the brain. We recently described that soluble Complement Receptor 2 (sCR2) is part of the nerve injury response in rodents. We here study CR2 in context of multiple sclerosis (MS) and explore the molecular effects of CR2 on C3 activation.Significant increases in sCR2 levels were evident in cerebrospinal fluid (CSF) from both patients with relapsing-remitting MS (n=33; 6.2ng/mL) and secondary-progressive MS (n=9; 7.0ng/mL) as compared to controls (n=18; 4.1ng/mL). Furthermore, CSF sCR2 levels correlated significantly both with CSF C3 and C1q as well as to a disease severity measure. In vitro, sCR2 inhibited the cleavage and down regulation of C3b to iC3b, suggesting that it exerts a modulatory role in complement activation downstream of C3.These results propose a novel function for CR2/sCR2 in human neuroinflammatory conditions.

Detection of protein aggregates in brain and cerebrospinal fluid derived from multiple sclerosis patients.

Studies of the properties of soluble oligomer species of amyloidogenic proteins, derived from different proteins with little sequence homology, have indicated that they share a common structure and may share similar pathogenic mechanisms. Amyloid β, tau protein, as well as amyloid precursor protein normally associated with Alzheimer’s disease and Parkinson’s disease were found in lesions and plaques of multiple sclerosis patients. The objective of the study is to investigate whether brain and cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients demonstrate the presence of soluble oligomers normally associated with protein-misfolding diseases such as Alzheimer’s disease. We have used anti-oligomer monoclonal antibodies to immunodetect soluble oligomers in CSF and brain tissues derived from multiple sclerosis patients. In this report, we describe the presence of soluble oligomers in the brain tissue and cerebral spinal fluid of multiple sclerosis patients detected with our monoclonal anti-oligomer antibodies with Western blot and Sandwich enzyme-linked immunosorbent assay (sELISA). These results might suggest that protein aggregation plays a role in multiple sclerosis pathogenesis although further and more refined studies are needed to confirm the role of soluble aggregates in multiple sclerosis.

Effects of natalizumab on oligoclonal bands in the cerebrospinal fluid of multiple sclerosis patients: A longitudinal study

Retrospective studies show that natalizumab modifies oligoclonal immunoglobulin (IgG) bands (OCBs) in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. In this study, we prospectively analyzed both serum and CSF samples from 24 MS patients, before and after 2 years of natalizumab-based therapy. Our results showed complete (55%) or partial (27%) disappearance of the OCBs in CSF samples that were taken after 2 years of therapy. Intrathecal IgG production, represented by the IgG index and IgGLoc, was also quantitatively reduced. Our data showed that natalizumab substantially modulates both intrathecal polyclonal and oligoclonal IgG production: This effect was much more potent than was previously reported.

Decreased Level of sRAGE in the Cerebrospinal Fluid of Multiple Sclerosis Patients at Clinical Onset

Objectives: Receptor for advanced glycation end products (RAGE) ligands/RAGE interactions have been proposed to have a pathogenic role in neuroinflammatory disorders. Our study aimed to assess changes in high-mobility group box (HMGB)1 and its receptor RAGE in peripheral blood (PBL) and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) at the disease onset compared with control subjects. Methods: PBL and CSF were collected from control subjects (n = 30) and MS patients (n = 27) at clinical onset. Soluble RAGE (sRAGE), HMGB1, S100 calcium-binding protein A12 (S100A12), interleukin (IL)-1β and tumor necrosis factor (TNF)-α were measured in the CSF and plasma by enzyme-linked immunosorbent assay. Gene expression in PBL mononuclear cells (PBMCs) was detected by quantitative PCR for RAGE, HMGB1, S100A12 and several proinflammatory/immunoregulatory cytokines. Results: We found a significantly lower expression of IL-10 (p = 0.031) in the PBMCs of MS patients. The level of sRAGE in the CSF of MS patients was lower (p = 0.021), with the ability to discriminate between MS patients and control subjects. Moreover, PBMC gene expression for HMGB1 and S100A12 positively correlated with IL-6. Conclusions: Our study confirmed that the cytokine network is disturbed in PBL and CSF at MS clinical onset. The deregulated HMGB1/RAGE axis found in our study may present an early pathogenic event in MS, proposing sRAGE as a possible novel therapeutic strategy for MS treatment.

Correlation between Oxidatives Stress and Chemokines in the Cerebrospinal Fluid of Multiple Sclerosis Patients (P03.232)

Correlation between Oxidatives Stress and Chemokines in the Cerebrospinal Fluid of Multiple Sclerosis Patients (P03.232)

Non-Classical and Intermediate Monocytes in the Peripheral Blood and Cerebrospinal Fluid of Multiple Sclerosis Patients (P03.228)

Non-Classical and Intermediate Monocytes in the Peripheral Blood and Cerebrospinal Fluid of Multiple Sclerosis Patients (P03.228)

Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients Detected with Peptide Microarrays (P03.229)

Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients Detected with Peptide Microarrays (P03.229)

Increased levels of IL-23 and osteopontin in serum and cerebrospinal fluid of multiple sclerosis patients

Osteopontin (OPN) and interleukin-23 (IL-23) are pro-inflammatory cytokines proposed to play central roles to the development of multiple sclerosis (MS). The aim of this study was to evaluate levels of OPN, IL-23 and other inflammatory cytokines and investigate their relationships in serum and cerebrospinal fluid (CSF) in patients with MS. Fifty one MS patients and 48 patients with non-inflammatory neurological diseases (NIND) were recruited from clinic. The levels of OPN, IL-23, IL-17, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in serum and CSF were determined in each participant. Compared with NIND group, MS patients had significantly elevated levels of OPN, IL-23, IL-17 and TNF-alpha in CSF, and elevated levels of IL-23, IL-17 and TNF-alpha in serum (All P<0.001). In MS patients, OPN and IL-23 were positively correlated with IL-17 (r=0.302, P=0.019; r=0.417, P=0.001, respectively); and IL-23 was positively correlated with EDSS (r=0.329, P=0.019). Both OPN and IL-23 may play pivotal role in development of MS and might be specific markers and therapeutic targets for MS.