Two-dimensional gel electrophoresis and peptide mass fingerprinting were used to identify proteins in cerebrospinal fluid (CSF) pooled from three patients with multiple sclerosis (MS) and in CSF pooled from three patients with non-MS inflammatory central nervous system (CNS) disorders. Resolution of CSF proteins on three pH gradients (3-10, 4-7 and 6-11) enabled identification of a total of 430 spots in the MS CSF proteome that represented 61 distinct proteins. The gels containing MS CSF revealed 103 protein spots that were not seen on control gels. All but four of these 103 spots were proteins known to be present in normal human CSF. The four exceptions were: CRTAC-IB (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein), and autotaxin t (a phosphodiesterase). It remains unknown whether these four proteins are related to the cause and pathogenesis of MS.
Objective-Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory Bcell clonal expansion are well-characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic.Methods-We produced 53 different human IgG1 monoclonal recombinant antibodies (rAbs) by coexpressing paired heavy-and light-chain variable region sequences of 51 plasma cell clones and 2 B-lymphocyte clones from MS cerebrospinal fluid in human tissue culture cells. Chimeric control rAbs were generated from anti-myelin hybridomas in which murine variable region sequences were fused to human constant region sequences. Purified rAbs were exhaustively assayed for reactivity against myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein by immunostaining of transfected cells expressing individual myelin proteins, by protein immunoblotting, and by immunostaining of human brain tissue sections.Results-Whereas humanized control rAbs derived from anti-myelin hybridomas and antimyelin monoclonal antibodies readily detected myelin antigens in multiple immunoassays, none of the rAbs derived from MS cerebrospinal fluid displayed immuno-reactivity to the three myelin antigens tested. Immunocytochemical analysis of tissue sections from MS and control brain demonstrated only weak staining with a few rAbs against nuclei or cytoplasmic granules in neurons, glia, and inflammatory cells.Interpretation-The oligoclonal B-cell response in MS cerebrospinal fluid is not targeted to the well-characterized myelin antigens myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein.Address correspondence to Dr Gilden, Department of Neurology, University of Colorado Denver School of Medicine, 12700 East 19th Avenue, Mail Stop B182, Aurora, CO 80045. don.gilden@ucdenver.edu. Gregory Owens and Jeffrey Bennett contributed equally to this work.Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article. NIH Public Access Subjects and Methods Multiple Sclerosis and Control PatientsCSF (approximately 20ml) was collected from MS patients (see supplemental Table 1) after informed consent was given. MS diagnosis was made using established international criteria. 14, 15 CD138 + plasma cells and, in some patients, CD19 + B lymphocytes were sorted, and H-and L-chain V regions were amplified, sequenced, and analyzed as described elsewhere. 8,10 Construction of Human IgG1 Recombinant AntibodiesFull-length bivalent IgG1 rAbs expressing an H-chain C-terminal Flag epitope ( Supplementary Fig 1) were produced from H-and L-chain V-region sequences of selected CD138 + and CD19 + clones as described previously. 16 Cloned V-region inserts were sequenced to ensure fidelity of the rAbs. Construction of Chimeric Monoclonal AntibodiesAnti-human MBP (5E3 monoclonal antibody [mAb]) and anti-human MOG monoclonal antibodies (6D7 and 2B7 mAbs) were derived from BALB/c mice immuni...
Single-cell RT-PCR was used to sample CD19+ B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominately polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for ∼70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.
Abstract. We have identified and characterized a new
Objective: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that becomes latent in B-lymphocytes and has been implicated in the pathogenesis of multiple sclerosis (MS). We searched for latent and active EBV infection in MS brain and CSF.Methods: Nested and non-nested real-time PCR were used to detect cell-specific and EBVspecific transcripts in 15 fresh-frozen and 5 formalin-fixed paraffin-embedded MS plaques and in single MS CSF B-lymphocytes and plasma cells. Intrathecal anti-EBV antibody synthesis was measured by ELISA. Immunocytochemistry was used to detect binding of MS CSF and recombinant antibodies (rAbs) generated from clonally expanded plasma cells in MS CSF to EBV-infected cells. Results:No EBV RNA was found in MS CSF B-lymphocytes or plasma cells. In active MS plaques, EBV-encoded RNA (EBER)-1 was the only and rarely detected transcript. The frequency of detected intrathecal anti-EBV antibody synthesis in patients with MS did not differ from that in non-MS inflammatory CNS disease control patients. Anti-EBV antibodies were detected in the CSF of patients with MS, but MS rAbs did not react with EBV. Epstein-Barr virus (EBV) is a common herpesvirus that is widespread in all human populations. EBV is spread orally and is the etiologic agent of infectious mononucleosis. Conclusions:1 Most primary infections are asymptomatic. More than 90% of adults are positive for serum immunoglobulin G (IgG) antibodies to the EBV capsid antigen.2 EBV becomes latent in peripheral blood B cells. EBV infection has been associated with multiple sclerosis (MS). 3In a large meta-analysis, EBV-seropositive individuals were found to have an increased risk for MS (odds ratio [OR] ϭ 13.5). 4 In a subsequent prospective study, a fourfold elevation in serum anti-EBV nuclear antigen (EBNA)-2 antibody titer was associated with a fourfold increased risk of developing MS.5 Further evidence of a link between EBV and MS came from reported enhanced immunoreactivity to EBV-specific proteins BRRF2 and EBNA-1 in serum and CSF of patients with MS, and the demonstration that a small fraction of CSF oligoclonal IgG of 13% of patients with MS was removed by incubation with purified BRRF2 and EBNA-1 proteins.6 Recently, about 90% of B-lymphocytes in active and chronic-active MS perivascular white matter lesions and about 80% of brain-infiltrating plasma cells were reported to be infected with EBV. 7 Immunohistologic detection of latent e-Pub ahead of print on March 10, 2010, at www.neurology.org.
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