Metabolomic profiling in multiple sclerosis: insights into biomarkers and pathogenesis.

Metabolomics enables the provision of sensitive bio-markers of disease. We performed 800 MHz (1)H-nuclear magnetic resonance (NMR) spectroscopic analyses of cerebrospinal fluid (CSF) specimens to identify biomarkers of multiple sclerosis (MS), yielding reproducible detection of 15 metabolites from MS (n=15) and non-MS (n=17) patients. Mean levels of choline, myo-inositol and threonate were increased, whereas 3-hydroxybutyrate, citrate, phenylalanine, 2-hydroxyisovalerate and mannose were decreased in MS-derived CSF (p<0.05), suggesting alterations to energy and phospholipid metabolism. Multivariate hierarchal cluster analysis indicated a high correlation within the metabolite profiles, significantly clustering samples into the two clinical groups, which was corroborated using principal components analysis. CSF metabolomics have the capacity to yield quantitative biomarkers and insights into the pathogenesis of MS.

Recognition of the immunodominant myelin basic protein peptide by autoantibodies and HLA-DR2-restricted T cell clones from multiple sclerosis patients. Identity of key contact residues in the B-cell and T-cell epitopes.

Myelin basic protein (MBP) may be an important autoantigen in multiple sclerosis (MS), with the MBP(82-100) region being immunodominant for T cells and autoantibodies. The structural requirements for autoantibody recognition were compared to those previously defined for MBP-specific T cell clones. MBP autoantibodies were affinity-purified from central nervous system lesions of 11/12 postmortem cases studied. The MBP(83-97) peptide was immunodominant in all 11 cases since it inhibited autoantibody binding to MBP > 95%. Residues contributing to autoantibody binding were located in a 10-amino acid segment (V86-T95) that also contained the MHC/T cell receptor contact residues of the T cell epitope. In the epitope center, the same residues were important for antibody binding and T cell recognition. Based on the antibody-binding motif, microbial peptides were identified that were bound by purified autoantibodies. Autoantibody binding of microbial peptides required sequence identity at four or five contiguous residues in the epitope center. Microbial peptides previously found to activate T cell clones did not have such obvious homology to MBP since sequence identity was not required at MHC contacts. The similar fine specificity of B cells and T cells may be useful for tolerance induction to MBP in MS.

Intravenous synthetic peptide MBP8298 delayed disease progression in an HLA Class II-defined cohort of patients with progressive multiple sclerosis: results of a 24-month double-blind placebo-controlled clinical trial and 5 years of follow-up treatment.

MBP8298 is a synthetic peptide with a sequence corresponding to amino acid residues 82-98 of human myelin basic protein (DENPVVHFFKNIVTPRT). It represents the immunodominant target for both B cells and T cells in multiple sclerosis (MS) patients with HLA haplotype DR2. Its administration in accordance with the principle of high dose tolerance results in long-term suppression of anti-myelin basic protein (MBP) autoantibody levels in the cerebrospinal fluid (CSF) of a large fraction of progressive MS patients. MBP8298 was evaluated in a 24-month placebo-controlled double-blinded Phase II clinical trial in 32 patients with progressive MS. The objective was to assess the clinical efficacy of 500 mg of MBP8298 administered intravenously every 6 months, as measured by changes in Expanded Disability Status Scale (EDSS) scores. Contingency analysis for all patients at 24 months showed no significant difference between MBP8298 and placebo-treatments (n = 32, P = 0.29). Contingency analysis in an HLA Class II defined subgroup showed a statistically significant benefit of MBP8298 treatment compared with placebo in patients with HLA haplotypes DR2 and/or DR4 (n = 20, P = 0.01). Long-term follow-up treatment and assessment of patients in this responder group showed a median time to progression of 78 months for MBP8298 treated patients compared with 18 months for placebo-treatment (Kaplan-Meier analysis, P = 0.004; relative rate of progression = 0.23). Anti-MBP autoantibody levels in the CSF of most MBP8298 treated patients were suppressed, but antibody suppression was not predictive of clinical benefit. Anti-MBP autoantibodies that reappeared in the CSF of one patient at 36 months, whilst under treatment with MBP8298, were not reactive with the MBP8298 peptide in vitro. The identification of a responder subgroup (62.5% of the patients in this study) enables a more efficient design of a large confirmatory clinical trial of MBP8298. The probability that patients with other less common HLA-DR haplotypes will respond to this treatment should not be ignored.

Synthetic peptide specificity of anti-myelin basic protein from multiple sclerosis cerebrospinal fluid.

Human myelin basic protein (h-MBP) purified from central nervous system (CNS) myelin has a molecular mass of 18.5 kDa and 170 residues. Eighteen synthetic peptides ranging from 8 to 25 residues and covering the length of h-MBP were prepared by the Fmoc method. Antibodies to h-MBP (anti-MBP) were isolated and purified from cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) by two-step affinity chromatography. Purified anti-MBP was reacted with increasing amounts of h-MBP as well as each of the 18 synthetic peptides in an initial liquid phase assay, and then titers of free anti-MBP in the resulting mixtures were determined by a solid phase radioimmunoassay. Purified anti-MBP was neutralized by h-MBP and 6 of the 18 synthetic peptides used in this study. The antibody was completely neutralized by peptides No. 12 (residues: 80-97), No. 15 (residues: 91-106) and No. 56 (residues: 75-95) and was partially neutralized by peptides No. 27 (residues: 61-75), No. 16 (residues: 64-78) and No. 21 (residues: 69-83). The 12 remaining synthetic peptides covering both the amino (residues 1-63) and carboxyl (residues 117-162) terminals of h-MBP did not neutralize purified anti-MBP. These results suggest that anti-MBP purified from CSF of patients with MS have affinity for discontinuous epitopes located between residues 61 and 106 on the h-MBP molecule. Alternatively anti-MBP may be polyspecific recognizing different amino acid sequences.

Increased synthetic peptide specificity of tissue-CSF bound anti-MBP in multiple sclerosis.

Free and bound hydrosoluble protein extracts were prepared from four anatomical areas of a multiple sclerosis (MS) cerebrum and from corresponding anatomical areas of a normal (non-MS) control. Increased levels of IgG and anti-myelin basic protein antibodies (anti-MBP) were detected in all MS samples and they were undetectable in the controls. IgG and anti-MBP from free (unbound) hydrosoluble protein extracts are defined as free IgG and free anti-MBP while IgG and anti-MBP from tissue bound protein extracts are defined as bound IgG and bound anti-MBP. IgG was purified from free protein extracts by protein G Sepharose affinity chromatography and anti-MBP was further isolated from purified IgG by antigen specific (MBP) Sepharose affinity chromatography. Free and bound anti-MBP were reacted with 20 synthetic peptides of human MBP prepared by the Fmoc method. Free anti-MBP, whether in the context of whole protein extracts, or as purified IgG or as purified antibody was completely neutralized by peptides #12, #15, #56 and #56* containing overall residues 75-106, partially neutralized by peptides #27, #16 and #21 containing overall residues 61-83 and did not react with the remaining 13 peptides. Tissue bound anti-MBP was completely neutralized only by peptides #12, #15, #56 and #56* (overall residues 75-106) and showed no reactivity towards the remaining 16 peptides including peptides #27, #16 and #21. Synthetic peptide specificity of free anti-MBP purified from MS cerebrum was identical to previously reported specificity of free anti-MBP from MS cerebrospinal fluid (CSF), while tissue bound anti-MBP, as well as bound anti-MBP from CSF had a more restricted synthetic peptide specificity than free anti-MBP. This suggests that the most likely epitope of anti-MBP is located between residues 84 and 95 of human MBP just proximal to the tri-proline sequence (99-101).

A double antibody radioimmunoassay for myelin basic protein in cerebrospinal fluid.

Levels of myelin basic protein (MBP) in cerebrospinal fluid (CSF) are useful in assessing the extent of demyelination which has occurred, or is occurring, in a variety of human problems associated with demyelination. Thus, an accurate, specific, simple, and consistent test for detecting MBP in CSF is needed. We describe such a test herein. The radioimmunoassay (RIA) described is a double antibody RIA using human MBP, and rabbit anti-MBP as reagents. The test can be done using only 200 microL of unconcentrated CSF. The test was sensitive to 0.1 microgram MBP/L. Recovery of known amounts of MBP was 97-105%, the within-assay and between-assay reproducibility were excellent. The "normal range" for our MBP-RIA was less than 2 micrograms/L, with a "grey area" between 2.0 and 3.0 micrograms/L.

A myelin basic protein antibody cascade in purified IgG from cerebrospinal fluid of multiple sclerosis patients.

Immunoglobulin G (IgG) was purified by affinity chromatography from the CSF of multiple sclerosis (MS) patients and controls. In MS patients, the IgG fraction contains anti-myelin basic protein (anti-MBP), anti-MBP neutralizing antibody and an antibody which inhibits neutralization of anti-MBP. Anti-MBP was detected in patients with acute relapses, anti-MBP neutralizing antibody was present in patients in clinical remission and the inhibiting antibody was detected in patients with chronically progressing MS. A myelin basic protein antibody cascade could be involved in the mechanism of MS.

Neutralization of anti-myelin basic protein by cerebrospinal fluid of multiple sclerosis patients in clinical remission.

Autoantibodies to myelin basic protein (anti-MBP) can be detected in the cerebrospinal fluid (CSF) of MS patients using a solid phase radioimmunoassay. Acute relapses of MS are characterized by an elevated, above unity, free (F) to bound (B) anti-MBP ratio; while in contrast, patients with chronic progressing disease have a low, below unity, free to bound ratio of anti-MBP. As patients with acute relapses enter into remission, the F/B anti-MBP ratio gradually decreases and eventually the level of these autoantibodies becomes undetectable. Neutralization of free anti-MBP was observed in intra- and interpatient experiments in which CSF from MS patients in remission was reacted with CSF of patients with acute relapses. Inhibition of anti-MBP neutralization was produced by CSF from MS patients with chronic progressing disease.

Purification of autoantibodies to myelin basic protein by antigen specific affinity chromatography from cerebrospinal fluid IgG of multiple sclerosis patients. Immunoreactivity studies with human myelin basic protein.

Immunoglobulin G (IgG) was purified by single-step protein A-Sepharose (Pharmacia) affinity chromatography from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and controls. Autoantibodies to myelin basic protein (anti-MBP) were isolated from the purified IgG fraction by two-step antigen specific affinity chromatography. Anti-MBP in the context of whole CSF or in purified form reacts equally to MBP prepared from non-MS or MS brain tissue. Kinetic studies of anti-MBP titers demonstrate that when anti-MBP is reacted with increasing amounts of non-MS or MS MBP, the autoantibody is immunoabsorbed by either antigen in vitro. Immunoabsorption of anti-MBP by MBP or its synthetic peptides may also be possible in vivo as a potential therapeutic tool.

Administration of myelin basic protein synthetic peptides to multiple sclerosis patients.

A double blind Phase 1 clinical research project was conducted in vivo in multiple sclerosis (MS) patients to determine the effect of myelin basic protein (MBP) synthetic peptides on free (F) and bound (B) titers of anti-MBP in cerebrospinal fluid (CSF). Intrathecal administration of peptide MBP75-95, either as a single dose, or as repeated injections for periods up to 10 weeks, produced complete binding-neutralization of F anti-MBP with no change in B levels. A control peptide MBP35-58 had no effect on F and B anti-MBP levels. Intravenous administration of MBP75-95 resulted in significant decline of F and B CSF anti-MBP levels over a period of one month. Administration of MBP synthetic peptides to MS patients either intrathecally or intravenously did not have any adverse neurological effects and systemic complications did not occur. The MBP epitope for MS anti-MBP was further localized to an area between Pro85 and Pro96.

The effect of intrathecal MBP synthetic peptides containing epitope P85 VVHFFKNIVTP96 on free anti-MBP levels in acute relapsing multiple sclerosis.

Acute relapses of multiple sclerosis (MS) are characterized by elevated Free (F)/Bound (B) anti-MBP ratios during the initial phase, followed by a steady decline of F antibody as the recovery/remission phase develops. The (human) MBP epitope for MS anti-MBP is: Pro85-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro96. In phase one clinical research, synthetic peptides (p) containing this epitope, namely pMBP86-95 and/or pMBP82-98, were intrathecally administered to MS patients with monosymptomatic or polysymptomatic relapses to determine the dosage, frequency and duration of administration which will immediately neutralize F circulating CSF anti-MBP. Patients with monosymptomatic relapses required 50 mg of peptide administered daily for 4-5 days. In patients with polysymptomatic relapses, F anti-MBP can be neutralized with dosages between 50 mg peptide daily for 4 days up to 100 mg twice a day for 2 days; however due to the prolonged nature of polysymptomatic relapses, antibody neutralization could not be maintained by these short courses of intrathecal peptide administration. Intravenous administration of these same peptides did not prevent occurrence of future relapses.

Relative frequency of autoantibodies to myelin basic protein and proteolipid protein in optic neuritis and multiple sclerosis cerebrospinal fluid.

Myelin basic protein (MBP) and proteolipid protein (PLP) were purified from non-MS human brain and used in solid phase radioimmunoassays to detect their specific antibodies in cerebrospinal fluid (CSF) of optic neuritis and clinically definite multiple sclerosis (MS) patients. In 53 optic neuritis patients free anti-MBP was elevated in 47 and in 6 of these 47 patients bound anti-MBP was also increased. The remaining 6 patients with undetectable anti-MBP had increased levels of anti-PLP in their CSF. None of these optic neuritis patients had autoantibodies to both antigens. Of 173 MS patients with acute relapses 169 had increased free anti-MBP. Three of the 4 remaining patients with undetectable anti-MBP had increased anti-PLP in their CSF. Of 110 MS patients with chronic progressive disease, 107 had increased CSF anti-MBP and 2 had elevated anti-PLP. Of 87 MS patients in remission, 15 had modestly elevated anti-MBP and none had detectable anti-PLP. Considering the total of 370 clinically definite MS patients with active and inactive disease, 77% had increased CSF anti-MBP and 1% had increased CSF anti-PLP. These findings are suggesting 2 immunochemically distinct forms of MS: a common form with autoantibodies directed against MBP and a more rare form associated with anti-PLP.

Cerebrospinal fluid autoantibodies to myelin basic protein in multiple sclerosis patients. Detection during first exacerbations and kinetics of acute relapses and subsequent convalescent phases.

In order to determine if free (F) and bound (B) levels of autoantibodies to myelin basic protein (anti-MBP) are present from the onset of multiple sclerosis (MS), 201 patients referred to our clinic were clinically divided into a group diagnosed as having an initial MS relapse and a group of non-MS controls. Ninety-four of 106 patients thought to have an initial MS relapse had increased CSF anti-MBP, while only 14 of 95 controls had elevated antibody levels; 9 of these 14 positive controls were subsequently shown to have MS by magnetic resonance imaging and/or clinical follow-up. CSF anti-MBP was more frequently abnormal than 3 estimates of intrathecal IgG synthesis in the group with suspected MS. Kinetics of F and B CSF anti-MBP were determined in a group of 29 patients with clinically definite MS during an acute relapse and 97.4 +/- 54 days later in the subsequent convalescent phase when in clinical remission. F and B anti-MBP levels were highly dependent on the timing of the CSF sampling; generally, as patients entered into clinical remission F anti-MBP declined, B antibody levels rose and F/B anti-MBP ratios initially above unity gradually declined towards zero. These data suggest that anti-MBP may be involved in the mechanism of MS.

Autoantibodies to myelin basic protein within multiple sclerosis central nervous system tissue.

Previous research has demonstrated that free (F) and bound (B) anti-myelin basic protein (anti-MBP) can be detected in the cerebrospinal fluid (CSF) of patients with active multiple sclerosis (MS). The purpose of this report was to determine whether the immunoglobulin G (IgG) isolated from central nervous system (CNS) tissue of MS patients contains anti-MBP. IgG was detected in free and bound hydrosoluble protein extracts obtained from the brain, spinal cord and optic nerves of a patient with clinically definite and neuropathologically confirmed MS. IgG was purified from free protein extracts from brain and spinal cord by Protein G-Sepharose affinity chromatography. Anti-MBP was detected by a solid phase radioimmunoassay (RIA) in all free and bound protein extracts. Anti-MBP was isolated from purified IgG from brain and spinal cord by MBP-Sepharose affinity chromatography. Free anti-MBP in the context of whole protein extracts, within purified IgG or as purified antibody as well as tissue-bound anti-MBP in the context of whole protein extracts was completely neutralized by human MBP (h-MBP) and synthetic peptide No. 56 (residues 75-95 of h-MBP) and did not react with synthetic peptide No. 41 (residues 35-58 of h-MBP). Anti-MBP which has previously been detected in the CSF of MS patients with active disease is also present as free antibody in the extracellular space of MS-central nervous system tissue and in a smaller proportion as tissue-bound antibody.

Optic neuritis anti-myelin basic protein synthetic peptide specificity.

Elevated titers of anti-myelin basic protein (anti-MBP) are highly associated with acute idiopathic unilateral optic neuritis as well as acute relapses of multiple sclerosis (MS). During acute phases of optic neuritis, free/bound antibody ratios are generally above unity, with high titers of free anti-MBP and relatively low or undetectable values of bound antibody. Three to 5 months after the acute phase when the majority of patients have recovered, free/bound anti-MBP ratios are below unity with low titers of free antibody and relatively higher levels of bound anti-MBP. Anti-MBP purified from cerebrospinal fluid of patients with optic neuritis are neutralized by synthetic peptides of human MBP containing overall amino acid residues 61-106 and do not react with synthetic peptides corresponding to residues 1-60 and 107-170. Anti-MBP may either have multiple epitopes in the region corresponding to residues 61-106 or it may react with a discontinuous epitope in this range. The mechanism of the optic nerve demyelination may be associated with anti-MBP binding in situ to MBP in the 61-106 amino acid region.

Tolerance induction to myelin basic protein by intravenous synthetic peptides containing epitope P85 VVHFFKNIVTP96 in chronic progressive multiple sclerosis.

Peptide-based tolerance induction may be useful for antigen-specific immunotherapy of human autoimmune diseases. Induction of tolerance to myelin basic protein (MBP) was examined in a Phase I clinical trial in multiple sclerosis (MS) patients with chronic progressive disease using a peptide that is immunodominant for MBP specific T cells and B cells. Tolerance induction was monitored by quantification of MBP specific autoantibodies in cerebrospinal fluid (CSF). The route of peptide administration was important since only intravenous but not intrathecal or subcutaneous injection induced tolerance to MBP. Following a single intravenous injection of a peptide containing epitope P85VVHFFKNIVTP96, MBP autoantibodies were undetectable for three to four months. Tolerance was more prolonged following a second injection since autoantibodies were low or undetectable after one year in the majority of patients. Duration of tolerance to MBP depended on MHC class II haplotypes of patients; tolerance was long-lived in all patients with disease associated HLA-DR2. No neurological or systemic side effects were observed, regardless of the route of peptide administration. These data demonstrate that intravenous administration of a soluble peptide can result in long-lasting tolerance to an autoantigen in humans.

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.

CSF myelin basic protein levels in acute optic neuritis and multiple sclerosis.

Normal CSF-MBP levels as determined by a RIA were less than 6.2 ng/ml CSF (mean 3.9). Eighty percent of patients with acute optic neuritis have CSF-MBP levels greater than 6.2 ng/ml (mean 7.6 ng/ml CSF). Five of 7 patients with acute internuclear ophthalmoplegia due to an initial exacerbation of demyelination have CSF-MBP levels above 6.2 ng/ml (mean 6.8 ng/ml). Fifty percent of MS patients with chronic progressive disease have CSF-MBP levels above 6.2 ng/ml (mean 6.7 ng/ml). MS patients experiencing monosymptomatic exacerbations show elevated CSF-MBP levels in 75% in 75% of cases (mean 8.2 ng/ml). MS patients experiencing polysymptomatic exacerbations show significantly higher levels of CSF-MBP (mean 22.3 ng/ml) than the patients with monosymptomatic exacerbations. Ninety-five percent of MS patients experiencing polysymptomatic exacerbations have elevated levels of CSF-MBP.

Effect of methylprednisolone on CSF IgG parameters, myelin basic protein and anti-myelin basic protein in multiple sclerosis exacerbations.

Clinical exacerbations of multiple sclerosis (MS) are characterized by elevated levels of cerebrospinal fluid (CSF) myelin basic protein (MBP). The purposes of this study were to determine whether anti-MBP antibodies are present in increased titer in CSF of MS patients with exacerbations, and whether they can be suppressed by the administration of immunosuppressive dosages of methylprednisolone (MP). A solid phase radio-immunoassay (RIA) was used to detect free and total anti-MBP antibodies before and after acid hydrolysis of CSF. In MS exacerbations, the majority of elevated anti-MBP is in the free form. With the exception of subacute sclerosing panencephalitis (SSPE) and some cases of post infectious encephalomyelitis, anti-MBP antibodies are not present in either MS patients in remission or in non-MS controls. Anti-MBP levels remained elevated over a 10 day period when patients are managed by bed rest only or when treated with intravenous (IV) ACTH. IV administration of MP in "high" (160 mg/day) or "mega" (2 g/day) dosages produces a highly significant reduction of both MBP (p less than 0.01) and anti-MBP (p less than 0.001) levels. Total intrathecal IgG synthesis is also significantly suppressed by IV-MP but not by ACTH.

Myelin basic protein: a component of circulating immune complexes in multiple sclerosis.

Myelin basic protein (MBP) is an antigenic component of circulating immune complexes (CIC) in patients with multiple sclerosis (MS). Immune complexes were isolated from the sera by adsorption to Raji cells and then acid eluted. Final identification of MBP from Raji eluates was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by MBP radioimmunoassay (RIA) of gel eluates and by an immunoblot technique.