Ingrid Catz

Lipid microarrays identify key mediators of autoimmune brain inflammation

Recent studies suggest that increased T-cell and autoantibody reactivity to lipids may be present in the autoimmune demyelinating disease multiple sclerosis. To perform large-scale multiplex analysis of antibody responses to lipids in multiple sclerosis, we developed microarrays composed of lipids present in the myelin sheath, including ganglioside, sulfatide, cerebroside, sphingomyelin and total brain lipid fractions. Lipid-array analysis showed lipid-specific antibodies against sulfatide, sphingomyelin and oxidized lipids in cerebrospinal fluid (CSF) derived from individuals with multiple sclerosis. Sulfatide-specific antibodies were also detected in SJL/J mice with acute experimental autoimmune encephalomyelitis (EAE). Immunization of mice with sulfatide plus myelin peptide resulted in a more severe disease course of EAE, and administration of sulfatide-specific antibody exacerbated EAE. Thus, autoimmune responses to sulfatide and other lipids are present in individuals with multiple sclerosis and in EAE, and may contribute to the pathogenesis of autoimmune demyelination.

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.

Antibodies from Inflamed Central Nervous System Tissue Recognize Myelin Oligodendrocyte Glycoprotein

Autoantibodies to myelin oligodendrocyte glycoprotein (MOG) can induce demyelination and oligodendrocyte loss in models of multiple sclerosis (MS). Whether anti-MOG Abs play a similar role in patients with MS or inflammatory CNS diseases by epitope spreading is unclear. We have therefore examined whether autoantibodies that bind properly folded MOG protein are present in the CNS parenchyma of MS patients. IgG was purified from CNS tissue of 14 postmortem cases of MS and 8 control cases, including cases of encephalitis. Binding was assessed using two independent assays, a fluorescence-based solid-phase assay and a solution-phase RIA. MOG autoantibodies were identified in IgG purified from CNS tissue by solid-phase immunoassay in 7 of 14 cases with MS and 1 case of subacute sclerosing panencephalitis, but not in IgG from noninflamed control tissue. This finding was confirmed with a solution-phase RIA, which measures higher affinity autoantibodies. These data demonstrate that autoantibodies recognizing MOG are present in substantially higher concentrations in the CNS parenchyma compared with cerebrospinal fluid and serum in subjects with MS, indicating that local production/accumulation is an important aspect of autoantibody-mediated pathology in demyelinating CNS diseases. Moreover, chronic inflammatory CNS disease may induce autoantibodies by virtue of epitope spreading.

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.

Tolerance induction to myelin basic protein by intravenous synthetic peptides containing epitope P85VVHFFKNIVTP96 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.

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.

Multiple Sclerosis: Validation of MR Imaging for Quantification and Detection of Iron

PURPOSE To investigate the relationship between iron staining and magnetic resonance (MR) imaging measurements in postmortem subjects with multiple sclerosis (MS). MATERIALS AND METHODS Institutional ethical approval was obtained, and informed consent was obtained from the subjects and/or their families. Four MR imaging methods based on transverse relaxation (T2 weighting, R2 mapping, and R2* mapping) and phase imaging were performed by using a 4.7-T system in three in situ postmortem patients with MS less than 28 hours after death and in one in vivo patient 1 year before death. Iron staining with the Perls iron reaction was performed after brain extraction. Region-of-interest measurements from six subcortical gray matter structures were obtained from MR imaging and then correlated with corresponding locations on photographs of iron-stained pathologic slices by using a separate linear least-squares regression in each subject. Iron status of white matter lesions, as determined by staining, was compared with appearance on MR images. RESULTS R2* mapping had the highest intrasubject correlations with iron in subcortical gray matter (R(2) = 0.857, 0.628, and 0.685; all P < .001), while R2 mapping (R(2) = 0.807, 0.615, 0.628, and 0.489; P < .001 and P = .001, .034, and .001, respectively), phase imaging (R(2) = 0.672, 0.441, 0.596, 0.548; all P ≤ .001), and T2-weighted imaging (R(2) = 0.463, 0.582, 0.650, and 0.551; all P < .001) had lower but still strong correlations. Within lesions, hypointense areas on phase images did not always represent iron. A hyperintense rim surrounding lesions on R2* maps was only present with iron staining, yet not all iron-staining lesions had R2* rim hyperintensity. CONCLUSION All four MR imaging methods had significant linear correlations with iron and could potentially be used to determine iron status of subcortical gray matter structures in MS, with R2* mapping being preferred. A reliable method of determining iron status within MS lesions was not established.

Detecting lesions in multiple sclerosis at 4.7 tesla using phase susceptibility‐weighting and T2‐weighting

To demonstrate 4.7 Tesla (T) imaging methods for visualizing lesions in multiple sclerosis in the human brain using phase susceptibility‐weighting and T2 weighting.

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.

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.