Epitopes of immunoreactive myelin basic protein in human cerebrospinal fluid

To define in more detail the features of the immunoreactive myelin basic protein (MBP) present in cerebrospinal fluid (CSF) of humans following acute injury to central nervous system myelin, the epitopes of MBP recognized by three different antisera, each capable of detecting immunoreactive MBP in CSF, were examined. All three antisera reacted well with human MBP and human MBP peptide 45-89. Only in radioimmunoassays in which the MBP peptide 45-89 served as the radioligand could clearly elevated values of immunoreactive MBP be measured in CSF specimens from 5 patients with multiple sclerosis during or immediately after an exacerbation. The two antisera that reacted well with MBP peptide 80-89 resulted in higher levels of immunoreactive MBP measured in CSF. An epitope present in human MBP peptide 80-89 but sharing a conformation with both MBP and MBP peptide 45-89 is present in CSF following acute central nervous system myelin damage in multiple sclerosis.     

Human myelin basic protein peptide 69-89: immunochemical features and use in immunoassays of cerebrospinal fluid

The characteristics and heterogeneity of the myelin basic protein (MBP)-like material appearing in cerebrospinal fluid (CSF) after acute central nervous system (CNS) myelin injury are unresolved. Antigenic material containing an epitope in the carboxyl-terminal portion of human MBP peptide 45-89 (from the intact molecule of 170 residues) is a prominent species of the MBP-like material present. In an effort to define further the MBP-like material in CSF and to enhance its detection, a modified double-antibody radioimmunoassay has been developed using a radioligand of human MBP synthetic peptide 69-89. This assay is more sensitive with results paralleling those of previously used MBP assays for CNS myelin damage. Results with this assay provide additional confirmation of the presence of an epitope of MBP in the decapeptide of MBP 80-89 but in a conformation simulating that of intact MBP in CSF after CNS myelin injury. Unexpected buffer effects were noted to influence the immunochemical behavior of some of the small peptides of MBP.     

The presence of immunoreactive myelin basic protein peptide in urine of persons with multiple sclerosis

A polyclonal antiserum has been produced that can detect nanogram amounts of myelin basic protein (MBP)-like material in unconcentrated human urine. The urinary immunoreactive material is cross-reactive with human MBP peptides 45-89 and 69-89, dialyzable, heat resistant, and is not artifact of either degradation of radioligand or salt effect. An octapeptide, MBP peptide 82-89, was demonstrated to be the smallest peptide containing the main epitope against which this antiserum was directed. This epitope differed from the major epitope recognized by antisera detecting MBP-like material in cerebrospinal fluid, implying that the MBP-like material is altered, presumably degraded, in the kidney. Results of gel filtration and high-performance liquid chromatography suggested a size of 1,000 daltons or less and a charge similar to that of human MBP peptide 80-89. In a group of 39 persons with multiple sclerosis, 48 with other neurological diseases, and 26 normal control subjects, the concentration of urinary MBP-like material, related to the concentration of urinary creatinine, was significantly higher in the multiple sclerosis group (22.0 ng MBP-like material/mg creatinine) than in the other neurological diseases or control groups, in which the values were 7.0 and 3.9 ng MBP-like material/mg creatinine, respectively. Variations in the level of MBP-like material appearing in the urine may provide a clinically feasible test for myelin damage. The precise identification of the chemical nature of the urinary MBP-like material may also furnish a means for further analyzing the in vivo catabolism of the potentially autoantigenic MBP.     

Detection of myelin basic protein-like material in cerebrospinal fluid of multiple sclerosis patients by immunoblot assay.

Myelin basic protein (MBP)-like material in 15 cerebrospinal fluid (CSF) samples from patients with multiple sclerosis (MS) was analyzed by isoelectric focusing (IEF) followed by immunoblot assay using rabbit antiserum against human MBP- peptide 69-89, which contains the dominant epitope for MBP-like material. Samples from seven of 10 MS patients with disease in the exacerbation stage showed one band and in three other samples, a number of faint bands also appeared in the alkaline pH region in addition to the one band. CSF from five MS patients whose disease was in remission showed no detectable bands. Our results are consistent with those obtained by quantitative assay, reported in the literature.     

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.     

CSF myelin basic protein, IgG and IgM levels in 101 MS patients before and after treatment with high-dose intravenous methylprednisolone

A total of 101 patients (62 women; 39 men) with definite MS were treated with 1000 mg methylprednisolone (MP) intravenously for 10 consecutive days. Immediately before and after MP treatment clinical scoring (Kurtzke's Expanded Disability Status Scale) and CSF analysis were performed. Before MP treatment CSF MBP, IgG and IgM immunoglobulin levels (CSF Ig, index and intrathecal synthesis) were significantly elevated. The mean CSF MBP levels were significantly higher in the relapsing-remitting (RR) and chronic progressive MS patients with relapses (CP + RR) than in the CP group without a RR disease course, respectively 2.1, 2.3 and 1.5 micrograms/l. A weak positive correlation was found between CSF MBP level and EDSS in the RR MS group (r = 0.34). CSF MBP was significantly correlated with IgM index (r = 0.36), IgM synthesis (r = 0.26), but not with the IgG levels. Therefore demyelination seems to be related to intrathecal IgM production. After MP treatment mean (median) EDSS decreased from 4.4 (4.0) to 3.3 (3.0). Except for Q albumin and IgM index, all CSF immunoglobulin levels decreased significantly after MP. The mean CSF MBP returned to reference values. In the RR group the decrease in CSF MBP was significantly correlated with the change in EDSS (r = 0.39). CSF MBP seems to be a good parameter for disease activity in relapsing MS. Following treatment CSF MBP was found to be related with the change in IgM index (r = 0.30). MP treatment reduces CSF MBP and intrathecal IgM in a similar way indicating a relation between these 2 parameters.     

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8.

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.     

Diagnosis of multiple sclerosis: comparison of the Poser criteria and the new McDonald criteria

OBJECTIVES: A confident and accurate diagnosis of multiple sclerosis (MS) is important, but a specific diagnostic test for the disease does not exist. The traditional diagnostic criteria of Poser et al. were published in 1983, and recently, McDonald et al. recommended new criteria for the diagnosis of MS. PATIENTS AND METHODS: In this study these two diagnostic schemes were compared by prospectively applying both of them to 76 patients with clinical features suggesting a new diagnosis of MS. RESULTS: Using the Poser criteria, 29 patients (38%) were classified as clinically definite and 35 patients (46%) as laboratory definite MS. According to the new McDonald criteria, MS was diagnosed in 39 (52%) patients, 37 patients (48%) had 'possible MS'. All patients with a clinically definite MS with the Poser criteria were also given the diagnosis of MS as recommended by McDonald et al. Of those 35 patients with laboratory definite MS according to Poser et al., four patients could be classified as having MS with the McDonald criteria, 89% of them had 'possible MS'. Conversely, 75% of the 39 patients, who fulfilled the new McDonald criteria for MS were assigned to the category of clinically definite MS according to the Poser criteria, and 83% of the patients with a 'possible MS' using the McDonald criteria, had a laboratory definite MS with the Poser criteria. CONCLUSION: MS according to the McDonald criteria was diagnosed more often than 'clinically definite MS' according to Poser et al., but combining the categories of clinically and laboratory definite MS, the diagnosis of MS could clearly be established more frequently using the Poser criteria.     

NBI-5788, an altered MBP83-99 peptide, induces a T-helper 2-like immune response in multiple sclerosis patients

We assessed the immune response induced in multiple sclerosis (MS) patients who had received NBI-5788, an altered peptide ligand (APL) designed from an immunodominant region (83-99) of the neuroantigen myelin basic protein (MBP) (5, 10, or 20 mg subcutaneously weekly for 4 weeks). The mean frequency of NBI-5788-responsive T cells (stimulation index > 3) in MS patients treated with the APL was 35.8 +/- 12.8% (n = 7) compared with a mean frequency of 6.2 +/- 1.3% (n = 7) for the untreated patients. The mean frequency of whole MBP-responsive T cells in MS patients treated with the APL was not significantly different from that of untreated patients (16.4 +/- 5.7% vs 18.0 +/- 6.3%, respectively). NBI-5788-reactive T-cell lines generated from NBI-5788-treated patients exhibited an increased frequency of cross-reactivity with MBP peptide 83-99 compared with NBI-5788-reactive lines from control MS patients. Cytokine secretion by APL-reactive T-cell lines from NBI-5788-treated MS patients was more frequently T-helper 2-like compared with T-cell lines from untreated MS patients. These results demonstrate that subcutaneous administration of a soluble APL based on the MBP peptide 83-99 in MS patients can induce an APL-reactive immune response in which T lymphocytes cross-reactive with the immunodominant neuroantigen MBP secrete anti-inflammatory cytokines. The significant heterogeneity in immune response between individuals indicates the need for clinical laboratory correlation during the course of therapeutic trials.     

Application of the new McDonald criteria to patients with clinically isolated syndromes suggestive of multiple sclerosis

Traditionally, multiple sclerosis (MS) has been diagnosed on the basis of clinical evidence of dissemination in time and space. Previously, it could not be diagnosed in patients with single clinical episodes of demyelination known as clinically isolated syndromes. New diagnostic criteria from the International Panel of McDonald and colleagues incorporate MRI evidence of dissemination in time and space to allow a diagnosis of MS in patients with clinically isolated syndromes. From clinical and MRI examinations performed prospectively at baseline, 3 months, 1 year, and 3 years of follow-up, the frequency of developing MS was ascertained by the application of both the new McDonald criteria and the Poser criteria for clinically definite MS. The specificity, sensitivity, positive and negative predictive value, and accuracy of the new criteria for the development of clinically definite MS were assessed. At 3 months, 20 of 95 (21%) patients had MS with the McDonald criteria, whereas only 7 of 95 (7%) had developed clinically definite MS. After 1 year, the corresponding figures were 38 of 79 (48%) and 16 of 79 (20%), and after 3 years, they were 29 of 50 (58%) and 19 of 50 (38%). The development of MS with the new MRI criteria after 1 year had a high sensitivity (83%), specificity (83%), positive predicative value (75%), negative predictive value (89%), and accuracy (83%) for clinically definite MS at 3 years. Use of the new McDonald criteria more than doubled the rate of diagnosis of MS within a year of presentation with a clinically isolated syndrome. The high specificity, positive predictive value, and accuracy of the new criteria for clinically definite MS support their clinical relevance.     

Specificity of antibody-dependent lymphocyte cytotoxicity against cerebral tissue constituents in multiple sclerosis

In patients with multiple sclerosis (MS) there is not only an antibody-dependent lymphocyte cytotoxicity (ADLC) against basic protein of myelin (MBP), as was demonstrated earlier, but also against encephalitogenic peptide, cerebrosides and gangliosides. The reaction against cerebrosides and gangliosides is not specific for MS; it is also frequently positive in patients with other neurological diseases (OND), syphilis and rheumatoid arthritis. The ADLC against encephalitogenic peptide shows a very high specificity for MS. Out of 35 cases with OND, 23 had a positive result with MBP, but only one with encephalitogenic peptide. Patients with syphilis and rheumatoid arthritis reacted negatively without exception. According to this, the encephalitogenic peptide is a highly specific antigen for MS; MBP shows less specificity. The ADLC against the encephalitogenic peptide shows a markedly increased sensitivity in MS compared with MBP. Specificity and frequency of positive findings with encephalitogenic peptide support the pathogenic significance of the reaction in MS.     

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.     

Cerebrospinal fluid levels of myelin basic protein-like material and soluble interleukin-2 receptor in multiple sclerosis

The presence, level and disease activity relationships of soluble interleukin-2 receptor (sIL-2R) in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients are unresolved. We measured CSF immunoreactive myelin basic protein (MBP), a marker of acute myelin damage, and sIL-2R levels in the CSF from 11 patients with active relapsing remitting (RR) MS, five with stable RR MS, eight with chronic progressive (CP) MS, five with other neurologic diseases, and three normal controls. No measurable (less than 100 units/ml) sIL-2R was present in any of the samples. Conversely, MBP levels were elevated in the active RR group compared to the other four groups. These results indicate that, at the sensitivity of assays currently available, levels of CSF sIL-2R do not correlate with the diagnosis or disease activity of MS.     

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.     

Fatigue in multiple sclerosis: relationship to depression, disability, and disease pattern

In order to investigate the associations between fatigue and depression, disability, and disease subtype, 207 individuals with clinically definite Multiple Sclerosis (MS) were administered the Fatigue Severity Scale and the Zung Self-rating Depression Scale during a regular clinic appointment. Their current level of disability was established using the Expanded Disability Status Scale. Fatigue and depression were highly correlated (r=0.58), even when the depression measure was corrected for items overlapping with fatigue and other symptoms or consequences of MS (r=0.44). Fatigue and disability were also correlated (r=0.33). Multiple regression revealed that both depressed mood and disability were significant predictors of fatigue, together accounting for approximately 23% of the variance in patients' self-reported fatigue. The combined groups of primary progressive (n=45) and secondary progressive patients (n=25) appeared to have higher fatigue scores than relapsing-remitting patients (n=137). However, an analysis of covariance revealed that this apparent difference was in fact attributable almost exclusively to differences in disability among the three subtypes of MS. Other reports of differences in fatigue between subtypes of MS should be re-examined in light of this finding.     

Electrophysiological disorders in multiple sclerosis and optic neuritis

Visual evoked response (VER), auditory brainstem evoked response (ABER), somatosensory evoked response (SSER), blink reflex and electronystagmographic (ENG) investigative methods were applied to a group of 89 patients with Multiple Sclerosis (MS) and Optic Neuritis (ON). The MS patients were classified as definite (n = 31), probable (n = 31) and possible (n = 27). The aim of this study was to determine the diagnostic value of the five electrophysiological tests in MS. VER and ABER recordings were found to reveal the highest number of asymptomatic abnormalities (33 and 31 percent respectively). The combination of VER, ABER and ENG revealed all possible electrophysiological disorders. As these tests are completely non-invasive it is proposed, that a combination of two of these three tests is useful for the detection of a second silent lesion in patients with suspected MS showing purely spinal signs (VER, ENG, ABER) and/or a history of uncomplicated ON (ABER, ENG).     

Myelin basic protein-responsive blood T lymphocytes in patients with multiple sclerosis

Previous studies from our laboratory showed the development of circulating T lymphocytes sensitized to myelin basic protein (MBP) in guinea pigs challenged with MBP. Also, lymphocytes sensitized to MBP were found in patients with multiple sclerosis (MS). In this report, we describe the kinetics of MBP-sensitized lymphocytes in a longitudinal study (140-316 days) of seven MS patients using the MBP-stimulated active rosette-forming T cell assay (MBP-ARFC). Expressed as the ratio of MBP-ARFC over ARFC (early and 37 degrees C stable rosette-forming T lymphocytes without added antigen), the results show a considerable degree of variation in the levels of MBP-ARFC. Although the levels of ARFC during the study period were relatively unchanged for each patient, increases in the MBP-ARFC/ARFC ratios were associated with the development of neurological symptoms of disease. The results of this study demonstrate the development of T-cell-mediated immunity to MBP in patients with MS. Detection of MBP-sensitive cells was possible during the course of the disease. The level of sensitivity was influenced by the clinical status, degree of neurological deficit, and particular treatment course.     

CSF B – lymphocyte chemoattractant (CXCL13) in the early diagnosis of acute Lyme neuroborreliosis

Recent studies have suggested a diagnostic role of the B-lymphocyte attracting chemokine (CXCL13) in the cerebrospinal fluid (CSF) in Lyme neuroborreliosis (LNB). Our aim was to evaluate diagnostic accuracy of CSF CXCL13 in a cohort of 59 consecutive patients referred to hospital for suspected LNB. Thirty-seven patients were classified as definite LNB and used as the reference standard. Seven were classified as probable, and seven as possible LNB. Eight patients did not fulfil case definitions and were used as controls.At presentation, CSF CXCL13 was elevated in all patients with definite LNB, as compared to a positive CSF B. burgdorferi (Bb) antibody index (AI) in 33 of 37. Pre-treatment sensitivity of elevated CSF [corrected] Bb Al [corrected] was 100 % (95 % CI = 91-100) and 89 % [corrected] (95 % CI = 75-96) respectively (p = 0.053). Among the eight control patients, CSF CXCL13 was normal in five and only slightly elevated in three, and Bb AI was negative in five. Specificity of CSF CXCL13 and Bb AI was similar 63 % (95 % CI = 31-86) (p = 1.0).CSF CXCL13 was elevated in 6/7 patients with probable LNB and 3/7 patients with possible LNB. Bb AI was negative in all these 14 patients.An additional control group consisted of 31 patients with multiple sclerosis (MS), 11 with non-inflammatory neurological diseases, and ten with verified non-Lyme meningitis and high CSF cell count. CSF CXCL13 was slightly elevated in 15 MS patients, and in nine meningitis patients. Mean CSF CXCL13 was higher in definite LNB (3524 ng/g CSF protein) than in MS (27 ng/g) and non-Lyme meningitis (23 ng/g) (p < 0.001).Four months post-treatment CSF CXCL13 was normalized in 82 % of patients with definite LNB, as compared to a negative Bb AI in 10 % (p < 0.001).CSF CXCL13 may be a useful supplement in early diagnosis of acute LNB.     

The diagnosis of multiple sclerosis. Contribution of non-clinical tests

A group of 89 patients in whom multiple sclerosis (MS) has been clinically diagnosed with varying degrees of certainty, and 25 patients with optic neuritis (ON), were subjected to the following electrophysiological tests: visual evoked response (VER), auditory brainstem-evoked response (ABER), somatosensory-evoked response (SSER), blink reflex and electronystagmography (ENG). All these patients also underwent computerized tomography (CT scan) and analysis of cerebrospinal fluid (CSF). A new diagnostic procedure is proposed, combining optimum detection of definite MS with optimally economical use of the above-mentioned non-clinical tests. The results for the MS patients show that definite MS can be diagnosed much more frequently (72%) if abnormal results in the above-mentioned tests are accepted as evidence of a (subclinical) CNS lesion. Application of the clinical diagnostic criteria of McAlpine yielded "definite MS" only in 27% of our patient material. Our diagnostic criteria showed evidence for MS in 36% of the patients clinically diagnosed as having ON. The test results were inconclusive as regards the possibility of the remaining ON patients developing MS in the future.