Fluid Of Multiple Sclerosis Patients Research Articles

Correlations between IL-4, IL-12 levels and CCL2, CCL5 levels in serum and cerebrospinal fluid of multiple sclerosis patients.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Both cytokines and chemokines have been implicated in the pathogenesis of MS. The aim of the study was to assess whether cytokine levels are correlated with chemokine levels during a different stage of relapsing-remitting MS (RR-MS). The study included 53 patients with RR-MS (20 subjects in stable stage and 18 patients with relapse). By ELISA method, the levels of the interleukin-4 (IL-4), interleukin-12 (IL-12), CCL2 and CCL-5 chemokines were measured both in serum and cerebrospinal fluid (CSF) of all patients. The serum IL-4 and IL-12 levels and CSF CCL5 level of patients with stable RR-MS were significantly different from the control level and the IL-12 levels were correlated with CCL5 levels in serum. During the relapse, a significant change in chemokine levels both in serum and CSF and IL-12 in CSF were noted, however no correlations were found between cytokines and chemokines.

Maintenance of B lymphocyte-related clones in the cerebrospinal fluid of multiple sclerosis patients.

A longitudinal study of Ig V gene segments utilized by B cells from the cerebrospinal fluid (CSF) of two patients with multiple sclerosis (MS) was carried out using RT-PCR methodologies. One patient with a relapsing-remitting (RR)-MS was investigated at onset and at relapse, 1 year later. A patient with secondary-progressive (SP)-MS was tested 9 and 13 years after disease onset. Sequence analyses of V(H)DJ(H) segments bearing V(H)3 and V(H)4 that were obtained from Cgamma cDNA genes demonstrated a substantial proportion of shared clones in the samples taken at different times; these clones were identical or closely related, i.e. had the same third complementary determining region (CDR) of the H chain variable region gene (HCDR3) with different mutations in the V(H) segment. Collectively, these data demonstrate that in MS patients there is a strong selective pressure, which could be exerted by antigen (or autoantigen) stimulation, for the maintenance and partial diversification of certain V(H)DJ(H) Cgamma sequences.

Lipoprotein Oxidation, Plasma Total Antioxidant Capacity and Homocysteine Level in Patients with Multiple Sclerosis

Free radical-mediated peroxidation of biological molecules, especially of lipids, is implicated in the pathogenesis of a number of diseases like multiple sclerosis. Low concentration of antioxidant vitamins: beta carotene, retinol, alpha tocopherol and ascorbic acid have been observed in serum or cerebrospinal fluid of multiple sclerosis patients. On the basis of these observations, we studied the potential lipoprotein oxidation and total antioxidant capacity in the pathogenesis of multiple sclerosis. Lipoprotein oxidizability for plasma in vitro, serum levels of autoantibodies against oxidized low-density lipoproteins, plasma total homocysteine levels with vitamin B<PRE>12</PRE> and folate, and plasma total antioxidant capacity were measured in twenty four patients with multiple sclerosis and twenty four healthy sex- and age-matched person as control. In multiple sclerosis patients during an attack, a significant increase in both in vitro lipid oxidizability for plasma and in the levels of autoantibodies against oxidized low-density lipoproteins, and a strong decrease in plasma total antioxidant capacity were detected. Plasma total homocysteine levels were significantly higher in multiple sclerosis patients whose plasma vitamin B<PRE>12</PRE> and folate levels were lower but not statistically significant, than controls. The present study indicates that lipoprotein oxidation may be important factor in the course of multiple sclerosis and in vitro measurements of plasma oxidation kinetics as an indication for lipoprotein oxidation might be useful as an additional tool for the clinical diagnosis of multiple sclerosis.

Antibodies against myelin oligodendrocyte glycoprotein in the cerebrospinal fluid of multiple sclerosis patients

Antibodies against myelin oligodendrocyte glycoprotein (MOG) mediate demyelination in experimental autoimmune encephalomyelitis (EAE) in different animal species and are implicated in the immunopathogenesis of multiple sclerosis (MS). In order to evaluate the anti-MOG response, we have analyzed the cerebrospinal fluids (CSFs) from 44 MS patients and 51 controls, 11 with other inflammatory neurological disorders (OIND) and 40 with non-inflammatory neurological disorders (NIND). The frequency of anti-MOG antibodies positive patients in the MS group (30%) was significantly higher compared to the NIND (8%, p=0.02), but not compared to the OIND group (55%, p=0.228). Interestingly, all six patients with neurosarcoidosis had MOG-specific antibodies in their CSF. Frequency of anti-MOG antibodies was similar in patients with clinically active and stable MS (32% and 26%, respectively; p=0.921). However, in clinically active MS patients, antibody titers were higher in comparison with patients with stable disease, although the difference did not reach the level of statistical significance ( p=0.06). These results further support the potential role of anti-MOG antibodies in the immunopathology of MS in the subset of patients with this disease. Furthermore, our findings suggest for the first time that anti-MOG antibodies could be an accessory diagnostic tool in neurosarcoidosis.

Myelin basic protein-reactive autoantibodies in the serum and cerebrospinal fluid of multiple sclerosis patients are characterized by low-affinity interactions

The presence of autoantibodies to the immunodominant antigen, myelin basic protein (MBP), in the serum and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) has been poorly characterized. Many studies report detectable levels of autoantibodies to myelin basic protein though other studies, using similar techniques, report their absence. We compared a solution-phase assay that has detected clinically relevant autoantibodies in diabetes and other autoimmune diseases to solid phase assays similar to those used in previous reports. The solution-phase assay consistently measured autoantibodies to MBP in serum from human subjects with Semple rabies vaccine (SRV)-induced demyelinating disease and from MBP-immunized animals. A solid phase assay detected MBP autoantibodies in the serum of a fraction of patients with MS. Autoantibodies capable of binding to MBP in the solution-phase were not detected in the CSF or serum of patients with MS. Additional solution-phase measurements revealed that anti-MBP antibodies from individuals with SRV-induced demyelinating disease demonstrated a binding affinity profile consistent with that of polyclonal antibodies with a range of affinities from low to high. In contrast, antibodies to MBP in the serum of MS patients detected by ELISA did not bind soluble MBP in the same assay. These results indicate that the humoral response in patients with MS does not include moderate- or high-affinity autoantibodies to MBP.

Cortisol is increased in postmortem cerebrospinal fluid of multiple sclerosis patients: relationship with cytokines and sepsis.

Hypothalmo-pituitary-adrenal (HPA) axis activity is altered in patients with multiple sclerosis (MS), resulting in elevated basal levels and enhanced response of cortisol in stimulation tests. HPA axis hyperactivation in MS is thought to be the result of complex interactions of genetic, immunologic, and neuroendocrinological mechanisms. In order to investigate whether cytokine levels in the central nervous system are associated with the activation of the HPA axis in MS, we measured cortisol, interleukin (IL)-6, IL-10 and TNF-alpha levels in postmortem cerebrospinal fluid (CSF) of 18 patients with severe MS and 50 controls. We also investigated the cortisol and cytokine levels in the CSF of a group of MS patients and controls who died with sepsis, in order to see whether acute infectious situations affect the association between cortisol and cytokines. The cortisol levels in MS patients were increased by 80% in comparison to controls (p=0.008). There was no difference in IL-6 levels between the groups, while IL-10 and TNF-alpha levels of the majority of subjects were below detection limits. There was a positive correlation between cortisol and IL-6 only in control patients with sepsis (r=0.89, p=0.019), but not within the MS patents with sepsis or MS and control groups without sepsis. Cortisol levels in postmortem serum and CSF were highly correlated (r>0.78, p<0.001). We concluded that the basal level of cortisol is significantly increased in the CSF of MS patients and that IL-6 is not responsible for this rise. The relationship between cortisol and IL-6 in sepsis is discussed.

Elevated serum and cerebrospinal fluid levels of soluble human herpesvirus type 6 cellular receptor, membrane cofactor protein, in patients with multiple sclerosis.

Membrane cofactor protein (CD46) is a member of a family of glycoproteins that are regulators of complement and prevent activation of complement on autologous cells. Recently, CD46 has been identified as the cellular receptor for human herpesvirus Type 6 (HHV-6). Elevated levels of soluble CD46 have been described in several autoimmune disorders, and may be implicated in the pathogenesis of these diseases. As several reports have supported an association of HHV-6 and multiple sclerosis, it was of interest to compare levels of soluble CD46 in the sera of multiple sclerosis patients to that of healthy controls, other neurological disease controls, and other inflammatory disease controls. Using an immunoaffinity column comprised of immobilized monoclonal antibodies to CD46, serum levels of soluble CD46 were found to be significantly elevated in multiple sclerosis patients compared with healthy and other neurological disease controls. Moreover, multiple sclerosis patients who tested positive for HHV-6 DNA in serum had significantly elevated levels of soluble CD46 in their serum compared with those who were negative for HHV-6 DNA. A significant increase in soluble CD46 was also found in the serum of other inflammatory disease controls tested compared to healthy controls. Additionally, a significant correlation was demonstrated between levels of soluble CD46 in the serum and cerebrospinal fluid of multiple sclerosis patients. Collectively, these data suggest that elevated levels of soluble CD46 may contribute to the pathogenesis of inflammatory diseases, including multiple sclerosis.

Accumulation of clonally related B lymphocytes in the cerebrospinal fluid of multiple sclerosis patients.

The accumulation of B lymphocyte clones in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) and patients with other neurological disorders was investigated using PCR technologies. Oligoclonal B cell accumulations were detected in 10 of 10 MS patients, but only in 3 of 10 of the patients with other neurological disorders. Analyses of the Ig V(D)J sequences on the CSF from MS patients disclosed that VH3 and VH4 genes were extensively mutated compared with germline sequences. Moreover, a substantial proportion of the molecular clones analyzed shared the same third CDR of the H chain variable region gene (HCDR3) and the same VH genes, albeit with different numbers and locations of point mutations, thus indicating an ongoing process of intraclonal diversification. A larger number of clonally related VH sequences could be obtained by using a VH3 gene-specific PCR so that genealogical trees depicting the process of diversification could be drawn. Analyses of the Ig V(D)J from the CSF of a patient with viral meningitis and oligoclonal B cell accumulations revealed that VH3 genes were extensively mutated. However, no intraclonal diversification could be observed even using VH3 gene-specific PCR methodologies. Clone-specific PCR and sequencing was used to detect the V(D)J found in the CSF of one MS patient in the PBL of the same patient. Only 1/3 of the V(D)J sequences investigated could be demonstrated in the PBL, indicating that the V(D)J genes utilized by B cells in the CSF are much less represented in the PBL. Collectively, the data suggest that in MS there is a compartmentalized clonal expansion.

Interferon gamma production in blood lymphocytes correlates with disability score in multiple sclerosis patients.

The proinflammatory cytokine interferon gamma (IFG) is elevated in body fluids of multiple sclerosis patients but its variation range is broad. The reason for this wide scatter of IFG production is not yet known. We looked for the relation between clinical parameters such as disability, exacerbation frequency, disease duration, course of the disease and IFG producing blood lymphocytes. Forty-one consecutive, clinically stable multiple sclerosis patients with primary relapsing course of the disease and without immunomodulatory or immunosuppressive treatment in the last 3 months were investigated for IFG in blood lymphocytes by flow cytometry. A significant positive correlation between IFG production and disability (r = 0.45, P<0.01, Spearman's rho coefficient) was found. Pathophysiological implications and therapeutical relevance of this unexpected finding are discussed.

The small sodium-channel blocking factor in the cerebrospinal fluid of multiple sclerosis patients is probably an oligopeptide.

An endogenous factor that is able to reduce the fast transient sodium current of excitable cells has been reported to exist in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. This was confirmed with nine clinically definite MS patients in the acute relapse. In order to purify and chemically identify the factor, microconcentration and gel filtration high-performance liquid chromatography (HPLC) were applied. After each purification step the activity-containing fraction was determined using a biological assay. With all CSFs the activity was contained in the fraction corresponding to 600-800 Da molecular weight, indicating that the factor is chemically homogeneous. The biological activity of the CSF specimens was not correlated to the laboratory CSF data; however, it was correlated to the area under the 210 nm UV light absorption peak in the corresponding chromatogram, i.e. the 600-800 Da MW fraction. As the factor was degradable by acid hydrolysis and a carboxypeptidase, it is suggested that it might be a small peptide.

Human Transaldolase and Cross-Reactive Viral Epitopes Identified by Autoantibodies of Multiple Sclerosis Patients

Abstract Multiple sclerosis is mediated by an autoimmune process causing selective destruction of oligodendrocytes. Transaldolase, which is expressed in the brain selectively in oligodendrocytes, is a target of high affinity autoantibodies in serum and cerebrospinal fluid of multiple sclerosis patients. A three-dimensional model of human transaldolase was developed based on the crystal structure of the enzyme from Escherichia coli. To identify immunodominant epitopes, 33 peptides overlapping human transaldolase by 5 amino acids were synthesized. Ab 12484, raised against enzymatically active human transaldolase, recognized antigenic determinants corresponding to linear epitopes (residues 27–31 and 265–290) and α helices (residues 75–98 and 302–329). Four immunodominant peptides harboring charged amino acid residues with topographically exposed side chains were identified by sera from 13 multiple sclerosis patients with predetermined autoreactivity to transaldolase. Autoantibodies binding to the most prominent human transaldolase epitope, between residues 271 and 285, showed cross-reactivity with Epstein-Barr and herpes simplex virus type 1 capsid-derived peptides. Molecular mimicry between immunodominant autoepitopes and viral Ags may be a decisive factor in directing autoimmunity to transaldolase in multiple sclerosis patients.

Ferritin, transferrin and iron concentrations in the cerebrospinal fluid of multiple sclerosis patients

The concentrations of ferritin, transferrin and iron were measured in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) and control patients. Ferritin levels were significantly elevated in the CSF of chronic progressive active MS patients (4.71±0.54 ng/ml) compared to levels in normal individuals (3.07±0.17 ng/ml). MS patients with active or stable relapsing–remitting disease had ferritin levels that were comparable to those found in normal individuals. There were no significant differences in transferrin or iron levels in the CSF between MS and normal individuals. Both ferritin and transferrin levels were elevated in patients that had high CSF IgG values but not in patients with a high IgG index. Since ferritin binds iron, the increase of CSF ferritin levels in chronic progressive MS patients could be a defense mechanism to protect against iron induced oxidative injury. Ferritin levels could be a laboratory measure that helps to distinguish between chronic progressive and relapsing–remitting MS.

Soluble Fas (Apo-1) levels in cerebrospinal fluid of multiple sclerosis patients

CSF and serum levels of soluble Fas were studied in MS patients, in patients with various neurological diseases and in healthy controls. We did not detect differences in serum sFas levels between MS patients and controls. In CSF, despite sFas levels being similar in all patients studied, a statistically significant correlation between sFas CSF/sFas serum ratio and BBB damage (expressed as albumin CSF/albumin serum ratio) was detected in non-MS neurological disease, but not in MS patients. The normalized ratio (sFas CSF/sFas serum)/(Alb CSF/Alb serum) was significantly increased in MS patients compared to patients with non-inflammatory neurological disease suggesting an intrathecal synthesis of soluble Fas in MS. The percentage of apoptotic mononuclear cells was higher in CSF as compared to peripheral blood; moreover a lower proportion of apoptotic cells was found in CSF of MS patients. The findings lend support to the involvement of sFas in MS pathogenesis and suggest that a lower apoptosis in CSF may be a feature of the disease.

Soluble HLA Class I and Class II Molecule Levels in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients

Increased concentrations of soluble HLA class I and class II molecules (sHLA-I and sHLA-II) have been observed in infectious, inflammatory, and autoimmune diseases. Because autoimmune mechanisms are considered to play a role in the pathogenesis of multiple sclerosis (MS), we decided to dose sHLA-I and sHLA-II in serum and cerebrospinal fluid (CSF) of MS patients comparing their concentrations with those observed in serum and CSF of patients with other neurologic diseases (OND) without evidence of neuroradiologic involvement of central nervous system (CNS) and in serum of healthy donors. The serum concentrations of sHLA-I were higher in both MS and OND patients than in healthy donors ( P < 0.05) whereas sHLA-II serum concentrations were lower in MS patients than in both OND patients and healthy donors ( P < 0.01). Detectable amounts of sHLA-II were observed in the CSF of 45% of MS patients and in CSF of only 6% of OND patients ( P < 0.001). In MS patients a significant correlation between sHLA-I serum and CSF concentrations was observed ( P < 0.01), whereas sHLA-II serum and CSF levels did not correlate. In conclusion, alterations of sHLA-I and sHLA-II serum and CSF concentrations are present in MS patients and could be involved in the induction of enhanced susceptibility to develop MS or in MS pathogenesis.

Interleukin-12 and tumor necrosis factor-α levels in cerebrospinal fluid of multiple sclerosis patients

Concentrations of interleukin (IL)-12 and tumor necrosis factor-α (TNF-α) in cerebrospinal fluid (CSF) were measured in patients with multiple sclerosis (MS) and control patients with non-inflammatory neurological diseases (NIND) by an enzyme-linked immunosorbent assay. TNF-α was detectable in the CSF of 60% of the patients with active MS, none of those with inactive MS and 29% of patients with NIND. CSF concentrations of TNF-α correlated with the degree of disability in MS patients ( P < 0.05). Detectable levels of IL-12 were found in 10% of the MS CSF samples and 18% of NIND CSF samples. There was a significant relationship between CSF concentrations of IL-12 and those of TNF-α in MS patients ( P < 0.05); no relationship was observed between the presence of IL-12 and disease activity or severity. These findings further stress the involvement of T helper 1 type-response within the central nervous system in MS.

High resolution proton MR spectroscopy of cerebrospinal fluid in MS patients. Comparison with biochemical changes in demyelinating plaques

Proton magnetic resonance spectroscopy (1H-MRS) investigation was performed on CSF samples of patients with neurological inflammatory diseases including 53 cases of multiple sclerosis (MS), 12 acute idiopathic polyneuropathies, 20 acute meningitides (10 viral and 10 bacterial). Spectra were compared with those acquired in 18 neurological controls. High CSF lactate levels were found in MS patients during clinical exacerbation of relapsing-remitting course (p = 0.036 vs. neurological controls). In MS patients with MRI evidence of Gd-enhanced plaques CSF lactate was higher than in patients with MRI inactive plaques (p = 0.017). CSF lactate positively correlated with number of CSF mononuclear cells in MS patients with clinical activity (p = 0.05) as well as in MS patients with MRI enhancement (p = 0.003). A comparative 1H-MRS investigation in vivo on localized demyelinating areas confirmed an elevated lactate signal in Gd-enhanced (61%) more frequently than in unenhanced (22%) plaques (p = 0.03). MS patients with high lactate signal in active plaques showed high lactate levels in CSF. Increased CSF lactate was found also in patients with acute meningitis and idiopathic polyneuropathy. These data suggest that changes in lactate levels may depend on anaerobic glycolytic metabolism in activated leukocytes during inflammatory diseases. A decrease of CSF formate levels was found in MS patients during active and inactive clinical phase (p = 0.037, p = 0.05 vs. neurological controls respectively). Formate changes might be related to a disorder of choline-glycine cycle in MS. 1H-MRS in vivo showed significant increase of choline in acute plaques, whereas a decrease of N-acetyl aspartate was found in chronic plaques; these metabolites are undetectable in CSF. CSF glucose levels were lower in bacterial than in viral meningitis (p = 0.014) and in neurological controls (p = 0.05). These observations suggest that 1H-MRS may be able to detect CSF metabolic impairment in neurological inflammatory diseases. In MS some CSF findings reflect metabolic changes occurring in brain demyelinating areas, and they could be useful for evaluation of disease activity in different stages of disease evolution.

Oligoclonal expansion of gamma delta T cells in cerebrospinal fluid of multiple sclerosis patients.

We have used a PCR based method to analyse TCR gamma chain repertoire and clonality of gamma delta T cells in the CSF and blood of II MS patients. Samples collected from nine patients with other neurological diseases were used as a control. Five controls had central nervous system inflammation and four had non-inflammatory processes. We have observed a decreased percentage of gamma delta T cells expressing TCR gamma with V gamma 9 and J gamma P fragments in the CSF samples in comparison with the blood. We did not final clonal expansion of the gamma delta T cells in any control case. Clonal expansion of gamma delta T cells occurred in five of II MS cases in the CSF but not in the blood. Two of these clones expressed TCR gamma rearranged with V gamma 9 and J gamma 1 fragments, two others used V gamma 10 and J gamma P1, and one used V gamma 9 and J gamma P fragments. We found no correlation between clonality and clinical state of patients, duration of the disease or number of cells in CSF. Our study provides additional evidence for the possible role of the gamma delta T cells in the MS pathogenesis.

T cell receptor γδ repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen‐reactive γδ clones

To study the relevance of gamma delta T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) gamma delta repertoire and the antigen reactivity of gamma delta clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype. Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.

Measurement of immune markers in the serum and cerebrospinal fluid of multiple sclerosis patients during clinical remission.

Magnetic resonance imaging of multiple sclerosis (MS) patients often shows active inflammatory lesions despite clinical remission. No immunological marker of disease activity has been identified in these patients. Concentrations of neopterin, interleukin-2 (IL-2), soluble interleukin-2 receptor (sIL-2R) and tumour necrosis factor-alpha (TNF-alpha) were measured in the serum and cerebrospinal fluid of 19 clinically-inactive MS patients and compared with those of 19 non-inflammatory controls. Cerebrospinal fluid (CSF) neopterin concentrations were significantly higher in the MS group than in controls (mean 9.1 mM vs 3.4 nM, P < 0.01) and 10 of 19 MS patients had levels above the control range. This finding provides evidence of ongoing T-cell-directed and interferon-gamma-mediated macrophage activation in the central nervous system. Analysis of IL-2, sIL-2R and TNF-alpha concentrations revealed no significant differences between MS patients and controls. We conclude that CSF neopterin concentration may correlate with disease activity in asymptomatic patients.

Elevated mast cell tryptase in cerebrospinal fluid of multiple sclerosis patients

Multiple sclerosis (MS) lesions are associated with infiltration of T lymphocytes and macrophages that appear to mediate myelin destruction and gliosis (scarring). Mast cells are located perivascularly in the brain, are juxtaposed to neurons, and have been shown to secrete vasoactive and inflammatory mediators in response to neuropeptides and direct nerve stimulation. Mast cells have been previously identified in MS lesions, are activated by myelin basic protein, and can participate in the regulation of blood-brain barrier permeability, as well as in myelin destruction. Here, cerebrospinal fluid from MS patients and controls with other neurologic diseases was assayed for histamine, its major metabolite methylhistamine, and the specific mast cell marker tryptase. Histamine and methylhistamine were not elevated in MS. However, the mast cell specific proteolytic enzyme tryptase was significantly elevated in MS, suggesting that mast cell activation may be involved in the pathophysiology of this disease.