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.
The entire varicella-zoster virus (VZV) genome appears to be present in latently infected human ganglia, but the extent of virus DNA transcription is unknown. Conventional methods to study virus gene transcripts by Northern (RNA) blotting are not feasible, since ganglia are small and VZV DNA is not abundant. To circumvent this problem, we prepared radiolabeled cDNA from ganglionic RNA, hybridized it to Southern blots containing VZV DNA, and demonstrated the presence of a transcript within the Sall C fragment of the virus genome (R.
Plaque-periplaque areas from MS brain tissue were explanted and propagated in tissue culture. The same in vitro techniques that successfully rescued measles virus from SSPE brain, papovavirus from PML brain, and HSV from normal human trigeminal ganglia, were applied. MS brain cells were also inoculated into chimpanzees, multiple rodent species, and embryonated hens eggs. No neurologic disease developed in experimentally infected animals, and no cytopathic effect was observed in explanted cells, or after cocultivation or fusion of MS brain cells with indicator cells. Further analysis of explanted and cocultivated cells by indirect immunofluorescence with various antiviral antisera prepared against viruses associated with post-infectious encephalomyelitis, as well as antisera to other ubiquitous viruses, failed to detect viral antigen. Finally, attempts to detect a latent enveloped virus in MS brain cells by 'superinfecting' MS brain cells in culture with vesicular stomatitis virus (VSV) did not reveal a VSV non-neutralizable fraction. Nevertheless, since oligoclonal bands (OGBs) in the CSF of patients with chronic infectious diseases of the CNS are directed against the causative agent, it is likely that OGBs in MS CSF are antibody directed against the agent or antigen that triggered disease. Although the relevant antibody may be scarce relative to irrelevant antibody in MS CSF, and only small amounts of an MS-specific antigen may be present in brain, this report provides a rationale for strategies proposed in our companion report by Owens et al which will allow detection of an MS-specific antigen or its cognate RNA in brain.
Proteomics combines two-dimensional gel electrophoresis and peptide mass fingerprinting and can potentially identify a protein(s) unique to disease. Such proteins can be used either for diagnosis or may be relevant to the pathogenesis of disease. Because patients with multiple sclerosis (MS) have increased amounts of immunoglobulin (Ig) G in their cerebrospinal fluid (CSF) that is directed against an as yet unidentified protein, we are applying proteomics to MS CSF, studies that require optimal separation of proteins in human CSF. We found that recovery of proteins from CSF of MS patients was improved using ultrafiltration, rather than dialysis, for desalting. Resolution of these proteins was enhanced by acetone precipitation of desalted CSF before electrophoresis and by fractionation of CSF using Cibacron Blue sepharose affinity chromatography. Improved protein recovery and resolution will facilitate excision from gels for analysis by peptide mass fingerprinting.
Chronic inflammatory and infectious diseases of the central nervous system (CNS) are characterized by increased IgG and oligoclonal bands (OGBs) in the brain and cerebrospinal fluid (CSF). The OGBs in CNS infectious diseases of known cause have been shown to be directed against the pathogenic agent. In multiple sclerosis (MS), the antigenic specificity of the OGBs is unknown, but could be directed against an infectious agent, an autoantigen, or both. In a molecular approach to identify antigens specific for MS, we constructed directional cDNA expression libraries with mRNA extracted from chronic and acute MS plaques and periplaque white matter. The libraries were: (1) screened to identify clones whose expression products react with MS CSF, but not with CSF from other infectious and inflammatory diseases of the CNS; (2) subtracted by hybridization to mRNA from normal human brain white matter and differentially screened to detect unique MS transcripts; and (3) used as template in polymerase chain reactions to amplify, clone, and sequence IgG heavy and light chain variable regions (VH and VL, respectively) expressed in MS plaques. Analysis of the VH and VL IgG repertoire in MS brain may identify disease-relevant IgG sequences that can be assembled into functional antibodies using recombinant phage technology. Such recombinant antibodies will be useful to probe brain tissue to identify antigens unique to MS.
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