Enhanced T cell responsiveness to citrulline-containing myelin basic protein in multiple sclerosis patients

Myelin basic protein (MBP), a candidate autoantigen in multiple sclerosis (MS), exists in different isoforms and charge isomers generated by differential splicing of exons and by a combination of posttranslational modifications, respectively. These various isoforms and charge isomers of MBP vary in abundance and most likely serve different functions during myelinogenesis and remyelination. The least cationic among the charge isomers of MBP is citrullinated and is referred to as MBP-C8. MBP-C8 is relatively increased in the population of MBP isomers in more developmentally immature myelin and in MS brain tissue. In a previous study, we found that MBP-C8-reactive T cells could be detected in CD4+ T cell lines (TCL) generated with MBP from both MS patients and normal controls. Here, we examined the frequency and peptide specificity of MBP-C8-specific TCL generated with MBP-C8 in MS patients and controls. Ten subjects grouped in five sets, each an MS patient and a control, were studied. In all cases, the MS patient had either a higher overall number of MBP-C8-responding lines, responded with greater sensitivity to the MBP-C8 antigen or both. Few lines responded to the MBP-C8 peptides but, if they did, they appeared to be specific to the carboxyl-half of the MBP-C8 molecule. Given the large amounts of citrullinated MBP in MS brain tissue, a preferential T cell response to MBP-C8 may be involved in the induction and perpetuation of this disease. Multiple Sclerosis (2000) 6 220 - 225     

T cell responses to myelin basic protein in patients with spinal cord injury and multiple sclerosis.

Autoimmune inflammation secondary to myelin destruction may play an inhibitory role in restoration of nerve functions in spinal cord injury (SCI). In this study, we demonstrated that T cells recognizing myelin basic protein (MBP) occurred at a high precursor frequency in patients with SCI, which was compatible to that in patients with multiple sclerosis (MS), a disease of presumed autoimmune pathology. The findings suggest of hyperactivity of MBP-reactive T cells in patients with SCI. MBP-reactive T cell lines derived from patients with SCI exhibited a preferential recognition pattern toward the 81-99 and the 151-169 regions of MBP. There were functional differences in the epitope recognition and cytokine profile between two panels of MBP-reactive T cell lines derived from patients with SCI and patients with MS. The study provides new evidence important for further investigation of the role of the inflammatory component in SCI.     

Target epitopes of myelin basic protein specific T cell lines in multiple sclerosis

A highly efficient new method for the generation of antigen specific T cell lines (TCL) is now available. By this method we established 134 myelin basic protein (MBP) TCL from the peripheral blood of 9 patients with definite multiple sclerosis (n=69) and 8 healthy donors (n=65). The yield of MBP reactive TCL in the two groups was comparable. So far 22 MBP specific TCL from 7 patients and 24 from 7 healthy individuals have been tested for their proliferative response to a panel of four synthetic peptides representing MBP residues 7–26, 80–99, 139–153 and 148–162. Although the peptide sequences did not encompass the whole MBP, the pattern of reactivity to these peptides in patients and controls seems to be similar. Further, when multiple TCL from the same donor were analysed, no dominant recognition emerged. Paper presented at the National Congress at Sorrento in 1991 and selected by the Editorial Board of the Journal     

CSF myelin basic protein in multiple sclerosis

Cerebrospinal fluid (CSF) from 221 patients with multiple sclerosis (MS) and 85 patients with other neurological disorders (OND) was examined using a competitive radioimmunoassay for myelin basic protein (MBP) immunoreactivity. MBP was found in 46 of 55 MS patients (84%) examined within six weeks of relapse but in only 11 of 85 patients (13%) with OND. There was a significant correlation between the concentration of MBP in the CSF and relapse severity in patients seen within four weeks of the onset of symptoms (p less than 0.01). Of 44 patients in remission, MBP was detected in 12, and these patients had a significantly higher tendency to subsequent relapse (p less than 0.05). In 72 patients with progressive disease the presence of MBP in the CSF reflected the confidence of clinical diagnosis. The results of this study suggest that measurement of MBP in the CSF gives an objective method of monitoring disease activity in patient with MS.     

Human T-cell response to myelin basic protein in multiple sclerosis patients and healthy subjects

In order to explore the T-cell repertoire to myelin basic protein (BP) of both multiple sclerosis (MS) patients and healthy subjects (HS), we raised BP reactive T-cell lines from blood mononuclear cells of eight MS patients and five HS. These lines were triggered in vitro by human BP. When analyzing their patterns of recognition of human BP versus heterologous BP, we could observe differences between healthy subjects and MS patients. Whereas T-cell lines from healthy subjects developed a response to heterologous BP, which was in most cases equal or higher than that elicited by human BP, T-cell lines from most MS patients displayed a low response, or no response at all, to one or several of the heterologous BP tested. A low response to bovine BP was only observed in active cases, whereas decreased responses to rat and/or monkey BP were observed both during remission and during active disease. This may indicate that T-cell repertoire to BP in MS patients differs from that of healthy subjects. BP-reactive T-cell clones were obtained by limiting dilution from two healthy subject lines. Their pattern of response to heterologous BP as compared to human BP suggest that T-cells from the same individual can recognize different BP epitopes.     

Urinary myelin basic protein-like material in patients with multiple sclerosis during interferon beta-1b treatment.

OBJECTIVES: To determine levels of urinary myelin basic protein-like material (MBPLM) in patients with multiple sclerosis (MS) openly treated with interferon beta-1b and to correlate these with clinical changes. BACKGROUND: Levels of urinary MBPLM correlate with the presence of the progressive phase of MS and with the disease burden detected on T2-weighted, cranial magnetic resonance imaging. Measurement of urinary MBPLM level may be a feasible test for monitoring or predicting response to therapeutic measures. DESIGN AND METHODS: In a prospective study at one site, 166 patients with MS (131 with relapsing-remitting [RR] and 35 with secondary progressive [SP] disease) were treated for a minimum of 1 year and up to 3 years with interferon beta-1b and underwent assessment for neurologic disability (Expanded Disability Status Scale and Scripps Neurological Rating Scale) and change in disease subtype. Urine samples were obtained at 1219 of 1378 clinic visits, and urinary MBPLM level was determined and related to creatinine level to adjust for renal function. RESULTS: Statistical analysis using the general linear models procedure confirmed previous findings that the level of urinary MBPLM related to urinary creatinine level (MBPLM/creatinine) was higher (P<.001) in patients with SP than RR MS. Of the 131 patients with RR MS, SP disease developed in 13 during the observation period. Compared with those in the RR group, the RR to SP group had a higher level (P<.001) of urinary MBPLM and did not differ from the SP group. CONCLUSIONS: The level of urinary MBPLM is higher in SP MS than RR MS but not in RR MS that converts to SP MS. Level of urinary MBPLM may permit the examination of treatment tested to prevent RR disease from becoming progressive.     

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45-89 peptide fragment, using a [3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15-31, 75-96, 83-96 and 131-141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131-141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the responses in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.     

Cellular hypersensitivity to gangliosides and myelin basic protein in multiple sclerosis

Peripheral blood lymphocytes from patients with multiple sclerosis (MS), other neurological diseases and healthy controls were investigated for the presence of cell-mediated hypersensitivity to brain gangliosides and myelin basic protein using an active E-rosette assay. Sensitivity to myelin basic protein and gangliosides was found in MS patients in acute relapse and with progressive disease, whereas no sensitivity was found in MS patients in remission. Patients with other neurological diseases showed no response to gangliosides, but sensitization to myelin basic protein was found in a patient with leucoencephalopathy and in 4 of 6 stroke patients. Healthy controls did not respond to either antigen. In MS patients a positive correlation was seen between lymphocyte responses to myelin basic protein and to gangliosides. The data suggest that in comparison to gangliosides, myelin basic protein is a weaker stimulator of active rosette-forming cells. Moreover, cellular hypersensitivity to myelin basic protein is not MS-specific and may be present as a consequence of brain damage. However, cellular hypersensitivity to gangliosides appears to be more specific to MS and may be important in the pathogenesis of the disease.     

Intrathecal synthesis of autoantibodies to myelin basic protein in multiple sclerosis

A solid phase radioimmunoassay was used to detect anti-myelin basic protein (MBP) antibodies in the CSF and serum of multiple sclerosis (MS) patients and controls. CSF and serum samples were assayed prior to acid hydrolysis in order to detect free anti-MBP as well as after acid hydrolysis to measure the total (free and bound) amount of antibody. An anti-MBP index controlling for serum levels as well as the degree of breakdown of the blood brain barrier was used to estimate intrathecal synthesis of anti-MBP. MS patients with acute exacerbations or chronically progressive disease have significantly elevated levels of both free and total CSF anti-MBP. The anti-MBP index is also significantly increased in MS patients with both forms of active disease. Anti-MBP antibodies are intrathecally produced in MS patients with active disease.     

IgG reactivity against citrullinated myelin basic protein in multiple sclerosis.

An increased level of citrullinated myelin basic protein (MBP-C8) has been reported in the brains of multiple sclerosis (MS) patients. However, the involvement of the immune response to post-translational modified MBP in the pathophysiology of MS remains speculative. The aim of this study was to compare the levels of immunoglobulin G antibodies to several MBP epitopes, before and after citrullination, in the cerebrospinal fluid (CSF) and sera of MS patients using enzyme-linked immunosorbent assay (ELISA). We analyzed antibody reactivity against various MBP-peptides in the CSF and sera of 60 MS patients, and 30 patients with other neurological diseases (OND) as controls. The peptides tested were: MBP(75-98) (peptide 1), native (peptide 2) and citrullinated (peptide 3) MBP(108-126) (ARG(122)-->Cit(122)), and native (peptide 4) and citrullinated (peptide 5) MBP(151-170) (ARG(159, 170)-->Cit(159, 170)). All selected peptides could support an immune reactivity in CSF and sera of MS and OND patients. A higher reactivity against peptide 4 was found in the CSF of MS patients compared with OND patients (P<0.0001), but not against citrullinated peptides (peptides 3 and 5). However, we observed that the citrullination state of peptide 2 modified the patterns of immune reactivity more markedly in MS patients (P<0.0001) than in OND patients (P<0.02). Although some MBP epitopes could be a potential target in MS, our data did not demonstrate any difference of antibody response to MBP peptides in their citrullinated forms.     

Cross-reactivity with myelin basic protein and human herpesvirus-6 in multiple sclerosis

Viral infections are though to play an important role in the pathogenesis of multiple sclerosis (MS) potentially through molecular mimicry. An identical sequence was found in both myelin basic protein (MBP, residues 96-102), a candidate autoantigen for MS, and human herpesvirus-6 (HHV-6 U24, residues 4-10) that is a suspected viral agent associated with MS. In this study, we showed that greater than 50% of T cells recognizing MBP(93-105) cross-reacted with and could be activated by a synthetic peptide corresponding to residues 1 to 13 of HHV-6 U24 in MS patients. The estimated precursor frequency of these cross-reactive T cells recognizing both peptides, MBP(93-105) and HHV-6 (U24)(1-13), was significantly elevated in MS patients compared with that in healthy controls. These cross-reactive CD4+ T cells represented the same Th1 phenotype as that of monospecific T cells recognizing MBP(93-105). There were increased antibody titers for both peptide HHV-6 (U24)(1-13) and peptide MBP(93-105) in the same patients with MS compared with those in healthy controls, suggesting B-cell sensitization to the antigens in MS patients. The study provides important evidence in the understanding of the potential role of HHV-6 infection/reactivation in the activation of autoimmune reactivity to MBP and its implication in the pathogenesis of MS.     

Adherence of cells to myelin basic protein I. Adherence of red and white blood cells from patients with multiple sclerosis to myelin basic protein

Red blood cells (RBC) and white blood cells (WBC) of patients with multiple sclerosis (MS) show decreased adherence to myelin basic protein (MBP) immobilized on plastic surfaces compared to the binding of cells from patients with other neurological diseases (OND), or such other autoimmune diseases as psoriasis (PS), and to that of healthy controls (HC). No similar phenomenon occurred to basic and non-basic type proteins other than MBP, for example, to histone (HIS), lysozyme (LYS) and ovalbumin (OVA). Thus, decreased adherence of RBC and WBC in MS patients to MBP appears to be a unique feature of the disease if compared with OND or PS.     

Defining interferon beta response status in multiple sclerosis patients

IFN-beta is effective in reducing relapses and magnetic resonance imaging (MRI) lesions in multiple sclerosis (MS). It is assumed that individual therapeutic responses vary, but methods to identify IFN-beta responsiveness have not been validated. Our objective was to evaluate methods to classify IFN-beta responder status using relapses and MRI lesions. Data was analyzed from 172 patients who were followed up in a placebo-controlled clinical trial of IFN-beta1a for 2 years. Patients were classified as responders or nonresponders using (1) the number of relapses during the 2-year trial; (2) the number of new T2 lesions after 2 years; and (3) the number of gadolinium-enhancing lesions at year 1 and year 2 on study. Outcomes included 2-year change in the Expanded Disability Status Scale, Multiple Sclerosis Functional Composite, and brain parenchymal fraction. We found that subgroups with high on-study relapse numbers had more disease progression, differences between responder subgroups were similar in the IFN-beta1a and placebo arms. In contrast, subgroups with high numbers of new MRI lesions had significantly more disease progression only in the IFN-beta1a arm. Baseline characteristics failed to account for differential outcome. New MRI lesion activity during IFN-beta1a treatment correlates with poor response to IFN-beta1a. MRI classification may facilitate rational therapeutic decisions, better clinical trial designs, and studies correlating biomarkers with therapeutic response.     

Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis

T cell sensitization to two myelin components, myelin basic protein (MBP) and myelin proteolipid protein (PLP), may be important to the pathogenesis of multiple sclerosis (MS). Using the limiting dilution assay, we demonstrated that the blood of MS patients had an increased frequency of MBP-reactive T cells compared with normal subjects and patients with other neurological diseases (OND) and rheumatoid arthritis. There was no difference in T cell frequency to a synthetic peptide, PLP139-151, or Herpes simplex virus. Within cerebrospinal fluid (CSF), 37% of IL-2/IL-4-reactive T cell isolates from MS patients responded either to MBP or PLP139-151 while only 5% of similar isolates from OND patients responded to these myelin antigens. The mean relative frequency of MBP-reactive T cells within CSF from MS patients was significantly higher than that of OND patients (22 x 10(-5) cells versus 1 x 10(-5) cells) and was similar to that of MBP reactive T cells within the central nervous system of rats with experimental autoimmune encephalomyelitis. These results lend new support to the hypothesis that myelin-reactive T cells mediate disease in MS.     

Sensitization to myelin basic protein in attacks of multiple sclerosis. A preliminary report.

Recent attempts to detect the presence of antibody to encephalitogenic basic protein in multiple sclerosis have generally been unsuccessful. The utilization of a new system of double immunodiffusion in detecting such antibody in EAE serum prompted us to apply this method in the search of such antibody MS sera. In our preliminary investigation of 67 sera, antibody was detectable in MS sera, but it was most often found during convalescence rather than an acute exacerbation of illness. Antibody was also found in several myasthenic patients, and occasionally in subjects with other neurological disease.     

髓鞘碱性蛋白基因与多发性硬化相关性的研究

目的探讨中国人群中多发性硬化（ＭＳ）的遗传易患性与髓鞘碱性蛋白基因５′端四核苷酸重复序列多态性的相关性。方法以２８例确诊ＭＳ患者及４７名正常对照为研究对象,从外周血白细胞中提取ＤＮＡ,利用聚合酶链反应技术特异性扩增ＭＢＰ基因５′端四核苷酸重复序列,并通过琼脂糖凝胶电泳分析扩增产物的多态性。结果发现四种等位基因,片段大小为１．０６ｋｂ,０．９８ｋｂ,０．６１ｋｂ和０．５４ｋｂ,以前两种多见。１．０６ｋｂ基因片段在女性中出现频率很高,达７３％,ＭＳ组与对照组无差别。男性中１．０６ｋｂ基因片段出现频率ＭＳ组（６９％）明显高于对照组（３９％）。ＭＳ组中等位基因的分布无性别差异。结论中国人群中ＭＢＰ基因第一个外显子５′端四核苷酸重复序列多态位点等位基因的分布有性别差异,１．０６ｋｂ的等位基因片段可能与ＭＳ易患性相关。     

Polyneuropathy associated with interferon beta treatment in patients with multiple sclerosis

Peripheral neuropathy has been reported as a side effect of interferon alpha, but not with interferon beta (IFNbeta) treatment. The authors assessed six patients with multiple sclerosis who developed polyneuropathy, or had exacerbation of previously subclinical neuropathy, during treatment with IFNbeta. In five patients the neuropathy improved after discontinuation of treatment and in two patients it relapsed upon rechallenge.     

Serum autoantibody responses to myelin oligodendrocyte glycoprotein and myelin basic protein in X-linked adrenoleukodystrophy and multiple sclerosis

We analyzed the sera of 51 patients with various phenotypes of X-linked adrenoleukodystrophy (X-ALD), 20 patients with multiple sclerosis (MS) and 22 healthy volunteers for the presence of autoantibodies specific for the recombinant extracellular immunoglobulin-like domain of human myelin oligodendrocyte glycoprotein (rhMOG(Igd)) and myelin basic protein (MBP). Anti-rhMOG(Igd) autoantibodies were significantly more frequent in X-ALD and MS patients as opposed to healthy individuals (p<0.05). Anti-MBP autoantibodies were present in about one-fourth of X-ALD and MS patients but in less than 10% of healthy individuals. Anti-rhMOG(Igd) autoantibody responses were not accompanied by increased T cell reactivity against rhMOG(Igd). These findings may have important implications for the understanding of humoral anti-myelin immunoreactivity in demyelinating diseases of the central nervous system such as X-ALD and MS.