Plasma Of Multiple Sclerosis Patients Research Articles

Acetate correlates with disability and immune response in multiple sclerosis.

BackgroundGut microbiota has been related to multiple sclerosis (MS) etiopathogenesis. Short-chain fatty acids (SCFA) are compounds derived from microbial metabolism that have a role in gut-brain axis.ObjectivesTo analyse SCFA levels in plasma of MS patients and healthy donors (HD), and the possible link between these levels and both clinical data and immune cell populations.MethodsNinety-five MS patients and 54 HD were recruited. Patients were selected according to their score in the Expanded Disability Status Scale (EDSS) (49 EDSS ≤ 1.5, 46 EDSS ≥ 5.0). SCFA were studied in plasma samples by liquid chromatography-mass spectrometry. Peripheral blood mononuclear cells were studied by flow cytometry. Gender, age, treatments, EDSS and Multiple Sclerosis Severity Score (MSSS) were evaluated at the recruitment.ResultsPlasma acetate levels were higher in patients than in HD (p = 0.003). Patients with EDSS ≥ 5.0 had higher acetate levels than those with EDSS≤ 1.5 (p = 0.029), and HD (p = 2.97e–4). Acetate levels correlated with EDSS (r = 0.387; p = 1.08e–4) and MSSS (r = 0.265; p = 0.011). In untreated MS patients, acetate levels correlated inversely with CD4+ naïve T cells (r = − 0.550, p = 0.001) and directly with CD8+ IL-17+ cells (r = 0.557; p = 0.001).ConclusionsPlasma acetate levels are higher in MS patients than in HD. In MS there exists a correlation between plasma acetate levels, EDSS and increased IL-17+ T cells. Future studies will elucidate the role of SCFA in the disease.

Evaluation of pentraxin-3 levels in sera and plasma of multiple sclerosis patients

Evaluation of pentraxin-3 levels in sera and plasma of multiple sclerosis patients

The enteric nervous system is a potential autoimmune target in multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.

Sulfatides in extracellular vesicles isolated from plasma of multiple sclerosis patients.

Extracellular vesicles (EVs) are membrane nanovesicles of diverse sizes secreted by different cell types and are involved in intercellular communication. EVs shuttle proteins, nucleic acids, and lipids that reflect their cellular origin and could mediate their biological function in recipient cells. EVs circulate in biological fluids and are considered as potential biomarkers that could be used to analyze and characterize disease development, course and response to treatment. EVs exhibit specific distribution of glycolipids and membrane organization, but little is known about the biological significance of this distribution or how it could contribute to pathological conditions such as multiple sclerosis (MS). We provide the first description of sulfatide composition in plasma-derived EVs by ultra-high-performance liquid chromatography tandem mass spectrometry. We found that EVs of different sizes showed C16:0 sulfatide but no detectable levels of C18:0, C24:0, or C24:1 sulfatide species. Small EVs isolated at 100,000 × g-enriched in exosomes-from plasma of patients with MS showed a significant increase of C16:0 sulfatide compared with healthy controls. Nanoparticle tracking analysis showed that the particle size distribution in MS plasma was significantly different compared with healthy controls. Characterization of small EVs isolated from MS plasma showed similar protein content and similar levels of exosomal markers (Alix, Rab-5B) and vesicular marker MHC class I (major histocompatibility complex class I) compared with healthy controls. Our findings indicate that C16:0 sulfatide associated with small EVs is a candidate biomarker for MS that could potentially reflect pathological changes associated with this disease and/or the effects of its treatment. © 2016 Wiley Periodicals, Inc.

Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells

Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein–kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders.

Leukocyte Gene Expression and Plasma Concentration in Multiple Sclerosis: Alteration of Transforming Growth Factor-βs, Claudin-11, and Matrix Metalloproteinase-2.

Multiple sclerosis is a neurodegenerative disease characterized by the present of leukocytes in the brain tissue and subsequently the formation of sclerotic plaques. Leukocytes penetration into the blood-brain barrier is related to several factors, such as, the conversion of leukocyte gene expression or plasma characteristics. In this frame, we explore alteration of matrix metalloproteinase-2 (MMP-2), transforming growth factor beta (TGF-β) family, and Claudin-11 (as a main myelin structural protein) in leukocytes and blood plasma of multiple sclerosis patients compared to the normal group. Blood samples were collected from thirteen men affected by MS and fifteen healthy men. Leukocyte gene expression was measured using real-time PCR and plasma parameters were examined by ELISA. The results of this study showed that the gene expression of Claudin-11 was significantly higher in MS group compared with normal. Interestingly, the MMP-2 pattern was similar to Claudin-11 and correlated positively with it. It was observed that, although the expressions of TGF-β1 and TGF-β2 are down-regulated in the leukocytes of subjects with MS, they showed higher levels of these cytokines in blood plasma. The plasma level of TGF-β3 in MS patients was higher than normal and correlated with Claudin-11 concentration. In conclusion, the aberrant pattern of Claudin-11, TGF-βs family, and MMP-2 expression in leukocytes of the MS patients was observed in this study. Moreover, the plasma levels of TGF-βs family increased in the MS group. The findings of this study provide clues for further investigations to assay MS pathogenesis.

Selected Extracellular microRNA as Potential Biomarkers of Multiple Sclerosis Activity—Preliminary Study

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). Four distinct disease courses are known, although approximately 90 % of patients are diagnosed with the relapsing-remitting form (RRMS). The name “multiple sclerosis” pertains to the underlying pathology: the presence of demyelinating plaques in the CNS, in particular in the periventricular region, corpus callosum, cervical spine, and the cerebellum. There are ongoing efforts to discover biomarkers that would allow for an unequivocal diagnosis, assess the activity of inflammatory and neurodegenerative processes, or warn of disease progression. At present, small noncoding RNA particles-microRNA (miRNA, miR) seem to be particularly noteworthy, as they take part in posttranscriptional regulation of expression of various genes. Changes in composition as well as function of miRNA found in body fluids of MS patients are subjects of research, in the hope they prove accurate markers of MS activity. This preliminary study aims to evaluate the expression of selected extracellular microRNA particles (miRNA-let-7a, miRNA-92a, miRNA-684a) in patients experiencing MS relapse and remission, with healthy volunteers serving as a control group and to evaluate the correlation between miRNA expression and selected clinical parameters of those patients. Thirty-seven patients suffering from MS formed two examined groups: 20 patients undergoing relapse and 17 in remission. Thirty healthy volunteers formed the control group. All patients who were subjects to peripheral blood sampling had been hospitalized in the Department of Neurology and Stroke1. Four milliliters of venous whole blood had been collected into EDTA tubes. The basis for the selection of the three particular miRNA investigated in this study (miRNA-let-7a, miRNA-92a, miRNA-684a) was a preliminary bioinformatic analysis of data compiled from several medical databases, including Ovid MEDLINE®, Embase, Cochrane Database of Systematic Reviews (CDSR), miRWalk, and miRBase. The isolation of extracellular microRNA from plasma was carried out using miRNeasy Mini Kit (Qiagen) reagents. The reverse transcription was carried out with TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems), as per manufacturers’ instructions. Standard microRNA TaqMan® tests (Applied Biosystems) were used for miRNA quantification. The qPCR were performed on a 7900 HT Fast Real-Time PCR System (Applied Biosystems) and analyzed using Sequence Detection System 2.3 software. In addition, all patients at the Department of Neurology and Stroke undergo a routine complete blood count with differential. The main objective of this study was to evaluate the expression of selected microRNA (has-miR-let-7a, miR-92a, and miR-648a) in the plasma of patients with MS during a relapse as well as in remission and attempt to correlate the acquired data with clinically relevant parameters of the disease. Finding such correlations may potentially lead to the use of miRNA as a biomarker of MS, which could help diagnose the disease and assess its severity and the efficacy of treatment. The difference in the expression of has-miR-let-7a in the remission group and the control group was statistically significant (p = 0.002). Similarly, the expression of miRNA-648a in patients in remission was significantly different from the expression in the control group (p = 0.02). Analysis of the correlation between the expression of miRNA-92a and the severity of the disease as measured by the EDSS scale in patients undergoing relapse showed significant negative linear correlation (r = −0.54, p = 0.01). Higher miR-648a expression correlated with more frequent flare-ups in the joint group of patients in remission and relapse (p = 0.03). This study is one of the few that demonstrate significantly changed expression of selected extracellular miRNA in plasma of MS patients and correlate those findings with clinical parameters. These observations may suggest that some miRNA subsets may be potential biomarkers for MS activity.

Human Endogenous Retrovirus HERV-Fc1 Association with Multiple Sclerosis Susceptibility: A Meta-Analysis

BackgroundHuman endogenous retroviruses (HERVs) are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS) in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts.MethodsA single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls), Central (883 patients and 692 controls) and Southern (1021 patients and 1359 controls) Spain. Our results were pooled in a meta-analysis with previously published data.ResultsSignificant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: pM-H = 0.0005; ORM-H (95% CI) = 1.27 (1.11–1.45)]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [pM-H = 0.0003, ORM-H (95% CI) = 1.32 (1.14–1.53)].ConclusionAssociation of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts.

Low sensitivity to glucocorticoid inhibition of in vitro Th17-related cytokine production in multiple sclerosis patients is related to elevated plasma lipopolysaccharide levels

Exogenous glucocorticoid plays an important role in controlling clinical relapses of multiple sclerosis (MS), but the response to this treatment differs among patients. In this study, T-cell proliferation and IL-17 production were less sensitive to hydrocortisone (HC) inhibition in MS patients than healthy individuals, mainly in CD8(+) compartment. Furthermore, in vitro IL-17 production was positively related with neurological disability and its release was proportional to IL-23 and IL-6 productions by LPS-activated monocytes. Interestingly, elevated LPS levels were quantified in the plasma of MS patients, and their levels were directly related to in vivo IL-6 production. Finally, HC-resistance in reducing IL-17 production by polyclonally-activated CD8(+) T cells was particularly observed among MS patients with higher in vivo LPS levels. In summary, the results indicate that T-cells derived from MS patients show an enhanced Th17-like phenotype that is directly associated with neurological disability, resistance to glucocorticoid inhibition and elevated bacterial translocation.

Soluble thrombomodulin levels in plasma of multiple sclerosis patients and their implication

Thrombomodulin (TM) on the cell-surface of cerebrovascular endothelial cells (CECs) is released into blood upon CEC damage. TM promotes activation of protein C (APC), an anticoagulant, anti-inflammatory, neuroprotective molecule that protects CECs and impedes inflammatory cell migration across the blood–brain barrier (BBB). Multiple sclerosis (MS) is associated with CEC damage and BBB dysfunction. We evaluated soluble TM (sTM) levels as a biomarker of BBB integrity and whether glatiramer acetate (GA) influenced sTM levels in MS patients. sTM levels quantified by 2-site ELISA from sera of healthy controls and systemic lupus erythematosus (SLE) patients (CEC-damage positive control) were compared with levels from patients with relapsing–remitting (RRMS) or secondary-progressive MS (SPMS), stratified as: RRMS/GA/no relapse, RRMS/GA/in relapse, RRMS no GA/no relapse, RRMS/no GA/in relapse; and SPMS/no GA. Additionally, soluble endothelial protein C receptor (sEPCR) levels were assessed in the non-stratified MS group, SLE patients, and controls. sTM levels were highest in RRMS patients taking GA with or without relapse, followed in decreasing order by SLE, RRMS/no GA/in relapse, SPMS, RRMS/no GA/no relapse, healthy controls. sEPCR levels were highest in MS patients, then SLE, then controls. sTM may be a useful biomarker of BBB integrity in RRMS patients. Further evaluation of sEPCR is needed. The finding that the highest sTM levels were in RRMS patients taking GA is interesting and warrants further investigation.

Oxidative modification of patient's plasma proteins and its role in pathogenesis of multiple sclerosis

BackgroundOxidative stress plays an important role in multiple sclerosis (MS). Objective and methodsThe present study was designed to evaluate the modifications of plasma proteins by estimation markers of oxidative/nitrosative stress: carbonyl groups and 3-nitrotyrosines (3-NT) levels in relapsing-remitting (RR) (n=10) and secondary progressive (SP) (n=10) clinical course of multiple sclerosis. Moreover, we estimated the level of uric acid (UA) in plasma of MS patients. ResultsCompared to controls (n=10), the levels of carbonyl groups in plasma proteins were elevated (P<0.0001) as well in RRMS as in SPMS. The highest concentration of 3-NT was observed in plasma proteins obtained from SPMS patients (P<0.0005). The level of uric acid in plasma was significantly lower in RRMS (P<0.0001) than SPMS. ConclusionThis is the first report which presented differences between SPMS and RRMS patients in 3-NT and protein carbonyl groups in plasma proteins.

Paraoxonase 1 Polymorphisms Are Not Related with the Risk for Multiple Sclerosis

It has been suggested a possible role of oxidative stress and lipid peroxidation in the inflammatory processes and in the pathogenesis of multiple sclerosis. Human serum paraoxonase 1 is a polymorphic enzyme encoded by the gene PON1, located in chromosome 7q21.3, that plays a major role in the metabolism of organophosporus compounds, and in the protection against oxidative stress. Paraoxonase-1 activity has been found decreased in the plasma of multiple sclerosis patients. An association between PON1 polymorphism and the risk of multiple sclerosis has been described in Italians. To investigate the possible association between the PON1 genotype and allelic variants of the polymorphisms L55M and Q192R and the risk for multiple sclerosis in the Spanish Caucasian population; we studied the frequency of the PON1 genotypes and allelic variants in 228 patients with multiple sclerosis and 220 healthy controls using a PCR-RLFP method. The frequencies of the PON1 genotypes and allelic variants did not differ significantly between patients and controls, and were unrelated with gender, age of onset, and course of the disease. The OR (95% confidence intervals) for the variant alleles PON1-55L and PON1-192R were 0.96 (0.73-1.26) and 1.01 (0.76-1.35), respectively. The results of the present study suggest that PON1 polymorphism is not related with the risk for multiple sclerosis in our population.

Proteolytic shedding of the macrophage scavenger receptor CD163 in multiple sclerosis

The scavenger receptor CD163 is selectively expressed on tissue macrophages and human monocytes. CD163 has been implicated to play a role in the clearance of hemoglobin and in the regulation of cytokine production by macrophages. Membrane CD163 can be cleaved by matrix metalloproteinases (MMP) resulting in soluble CD163 (sCD163). In the present report the shedding of CD163 was investigated in multiple sclerosis (MS). An upregulation of plasma sCD163 and a down regulation of membrane CD163 in MS patients compared to healthy controls was observed. The levels of plasma sCD163 correlated with plasma MMP-9 levels in controls, but not in MS patients. Moreover, evidence was obtained for CD163-cleaving MMP activity in plasma of MS patients. Finally, the increased proteolytic shedding of CD163 correlated to reduced plasma levels of circulating inflammatory cytokines. Collectively, our results provide evidence for proteolytic shedding of CD163 in MS and suggest a possible link to cytokine production.

Kynurenine metabolism in multiple sclerosis

Objective--Excitatory amino acid receptors are involved in the normal physiology of the brain, and may play a role in the pathogenesis of neurological disorders such as Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, etc. It has been demonstrated that the blockade of one of these receptors ameliorates the symptoms of experimental allergic encephalomyelitis, an animal model of multiple sclerosis (MS). In a recent study, a decreased level of kynurenic acid was found in the cerebrospinal fluid of patients with MS. The only known endogenous excitotoxin receptor antagonist is the tryptophan metabolite kynurenic acid. Another metabolite is quinolinic acid, which exerts different action: it is an excitotoxin receptor agonist. The ratio of these two metabolites is determined by the activities of kynurenine aminotransferase I and II (KAT I and KAT II). In this study, we measured the activities of these enzymes and the concentration of kynurenic acid in the red blood cells (RBC) and in the plasma of patients with MS. KAT activities were detected both in the RBC and in the plasma. As compared with the control subjects, the KAT I and KAT II activities were significantly higher in the RBC of the patients. The concentration of kynurenic acid is elevated in the plasma of MS patients, and there is a tendency to an elevation in the RBC. These changes may indicate a compensatory protective mechanism against excitatory neurotoxic effects. Our data demonstrate the involvement of the kynurenine system in the pathogenesis of MS, which may predict a novel therapeutic intervention.

Alpha-Tocopherol and NADPH in the Erythrocytes and Plasma of Multiple Sclerosis Patients

Objectives: To investigate the influence of interferon-β-1b (INF-β-1b) therapy on blood antioxidants (α-tocopherol and NADPH) in multiple sclerosis (MS). Patients and Methods: Patients with relapsing-remitting MS (n = 14) have been studied during 6 months of INF-β-1b therapy. α-Tocopherol was determined by HPLC and UV or electrochemical detection; NADPH was quantified spectrophotometrically. Results: The erythrocyte α-tocopherol level was reduced (p < 0.001) before treatment, but had regained the control level by 6 months of therapy. The plasma α-tocopherol/lipid ratios were constant during therapy. Plasma triglyceride levels were transiently increased (p = 0.0270) after 1 month of treatment. INF-β-1b had also induced a transient decrease in NADPH after 1 month, but thereafter the level returned to approximately the initial value (p = 0.0248). Conclusion: INF-β-1b seems to exert a sparing effect toward the erythrocyte α-tocopherol content. The fall in NADPH in parallel to the rise in plasma triglycerides suggests stimulation of fatty acid synthesis by INF-β-1b.

5-31-05 Relationship between magnesium and protein concentrations in plasma of multiple sclerosis patients

5-31-05 Relationship between magnesium and protein concentrations in plasma of multiple sclerosis patients

Quantitation of free amino acids in biological samples by high-performance liquid chromatography : Application of the method in evaluating amino acid levels in cerebrospinal fluid and plasma of patients with multiple sclerosis

An automatic on-line high-performance liquid chromatographic method based on a precolumn derivatization with o-phthalaldehyde has been developed to quantitate levels of free amino acids in cerebrospinal fluid (CSF) and plasma samples from 12 patients with multiple sclerosis (MS) and 12 controls. The analytical method gave reproducible results with relative standard deviations of 0.5–3% for all amino acids. The separation of 24 amino acids was performed on a reversed-phase C 18 column, using two solvents and a multiple-step gradient. Each chromatographic experiment was completed within 40 min. The results showed higher levels of Glu, Gln, Gly and Ala and lower levels of Met, Val, Phe and Lys in plasma of MS patients. In CSF, increased levels of Gln, Arg, Ser and Tyr and decreased levels of Asp, Glu, Met, γ-aminobutyric acid and Phe were found in MS patients, whereas the levels of other amino acids remained more or the less same in both groups.

Relevance of platelet factor four (PF4) plasma levels in multiple sclerosis.

In order to study the role of platelets in Multiple Sclerosis (MS) we assessed, in a group of patients during a quiescent phase of the disease, the plasma levels of beta-thromboglobulin (beta-TG) and platelet factor four (PF4) both in absence of treatment and during administration of aspirin (ASA) at the dose of 50 mg/daily. In the MS patients studied, the basal plasma levels of beta-TG and PF4 were significantly higher than in control subjects. The increase in the beta-TG plasma levels occurred independently of the age, sex and severity of the disease, whereas the modification in the PF4 plasma levels was significantly correlated with the severity of the disease. Administration to the patients of ASA, at the dose that does not affect prostacyclin production, determined a decrease of beta-TG in 77% of the patients. Mean PF4 plasma levels remained unchanged. These results suggest that PF4 in the plasma of MS patients may originate not only from the platelets but also from the mast cells following platelet aggregating factor (PAF) stimulation and immunocomplex formation.