Objective-The serum of most neuromyelitis optica (NMO) patients contains autoantibodies (NMO-IgGs) directed against the aquaporin-4 (AQP4) water channel located on astrocyte foot processes in the perivessel and subpial areas of the brain. Our objectives were to determine the source of central nervous system (CNS) NMO-IgGs and their role in disease pathogenesis.Methods-Fluorescence activated cell sorting and single-cell reverse transcriptase PCR were used to identify overrepresented plasma cell immunoglobulin (Ig) sequences in the cerebrospinal fluid (CSF) of an NMO patient after a first clinical attack. Monoclonal recombinant antibodies (rAbs) were generated from the paired heavy and light chain sequences and tested for target specificity and Fc effector function. The effect of CSF rAbs on CNS immunopathology was investigated by delivering single rAbs to rats with experimental autoimmune encephalomyelitis (EAE).Results-Repertoire analysis revealed a dynamic, clonally expanded plasma cell population with features of an antigen-targeted response. Using multiple independent assays, 6 of 11 rAbs generated from CSF plasma cell clones specifically bound to AQP4. AQP4-specific rAbs recognized conformational epitopes and mediated both AQP4-directed antibody-dependent cellular cytotoxicity and complement-mediated lysis. When administered to rats with EAE, an AQP4-specific NMO CSF rAb induced NMO immunopathology: perivascular astrocyte depletion, myelinolysis and complement and Ig deposition.Interpretation-Molecular characterization of the CSF plasma cell repertoire in an early NMO patient demonstrates that AQP4-specfic Ig is synthesized intrathecally at disease onset and directly

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.

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...

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.

Multiple sclerosis (MS) cerebrospinal fluid and brain contain increased IgG and oligoclonal bands. Whether this oligoclonal and polyclonal IgG is directed against a disease-relevant antigen remains unknown. To distinguish between random activation versus a targeted B-cell response, we analyzed the IgG heavy chain variable region (VH) repertoire expressed in different lesions of an acute MS brain. To obtain a representative sample of the VH repertoire, we constructed directional complementary DNA libraries from plaque-periplaque messenger RNA and amplified VH regions from the library by nested polymerase chain reaction. When MS VH sequences were aligned to germline segments, about 60% of different VH sequences in the acute MS brain were VH4 germline segments, significantly greater than the known approximately 20% VH4 germline prevalence. Specific VH sequences were overrepresented and expressed at multiple plaque sites. Within some overexpressed populations, there were distinct sequence differences (clonal variants) indicative of clonal expansion. Alignment of VH sequences to their closest germline counterparts revealed extensive somatic mutation and the preferential accumulation of amino acid replacement mutations in complementarity determining regions. These observations suggest the limited B-cell response found in this acute MS brain was antigen driven.

Programmed cell death is an essential cellular process that occurs in epithelial turnover, neural development, and regulation of cell populations of the immune system. Thymocytes undergo programmed cell death in response to several inductive stimuli, including exposure to glucocorticoids or radiation. This program can be blocked by inhibitors of RNA or protein synthesis; this implies that new proteins are required to execute the death programs. To search for possible death-associated mRNAs, we directionally cloned cDNA representing mRNA from control and dexamethasone-treated thymocytes. These libraries were used to produce ample amounts of DNA and RNA used in subtractive hybridization for the removal of sequences present in both control and induced cells. The remaining unhybridized sequences were selectively amplified by polymerase chain reaction and cloned to produce a library enriched for sequences expressed in death-induced cells. From this library we isolated cDNAs of death-associated mRNAs. One of these mRNAs, RP-8, appears within 1 h after exposure to gamma radiation, and a second mRNA, RP-2, is observed within 2 h. Both of these mRNAs accumulate during a period when a reference mRNA, actin, is declining. RP-2 and RP-8 are no longer detectable after 6 h postinduction, when apoptosis and mRNA degradation are evident in the culture. Sequence analysis of RP-8 cDNA indicates the presence of a zinc finger domain suggestive of a possible DNA regulatory role for the RP-8 protein. cDNA sequence results on RP-2 classify the corresponding protein as an integral membrane protein. We conclude that RP-2 and RP-8 are death-associated mRNAs that should be functionally evaluated in the context of the death process. As previously suggested, it may be that a family of "death genes" is activated by various stimuli depending on the type of cell, in a manner somewhat analogous to the induction of heat shock (stress) protein genes.

IntroductionAntibodies against myelin oligodendrocyte glycoprotein (MOG-IgG) are present in some neuromyelitis optica patients who lack antibodies against aquaporin-4 (AQP4-IgG). The effects of neuromyelitis optica MOG-IgG in the central nervous system have not been investigated in vivo. We microinjected MOG-IgG, obtained from patients with neuromyelitis optica, into mouse brains and compared the results with AQP4-IgG.ResultsMOG-IgG caused myelin changes and altered the expression of axonal proteins that are essential for action potential firing, but did not produce inflammation, axonal loss, neuronal or astrocyte death. These changes were independent of complement and recovered within two weeks. By contrast, AQP4-IgG produced complement-mediated myelin loss, neuronal and astrocyte death with limited recovery at two weeks.ConclusionsThese differences mirror the better outcomes for MOG-IgG compared with AQP4-IgG patients and raise the possibility that MOG-IgG contributes to pathology in some neuromyelitis optica patients.

Neuromyelitis optica (NMO) is an inflammatory disorder mediated by antibodies to aquaporin-4 (AQP4) with prominent blood-brain barrier (BBB) breakdown in the acute phase of the disease. Anti-AQP4 antibodies are produced mainly in the periphery, yet they target the astrocyte perivascular end feet behind the BBB. We reasoned that an endothelial cell–targeted autoantibody might promote BBB transit of AQP4 antibodies and facilitate NMO attacks. Using monoclonal recombinant antibodies (rAbs) from patients with NMO, we identified two that strongly bound to the brain microvascular endothelial cells (BMECs). Exposure of BMECs to these rAbs resulted in nuclear translocation of nuclear factor κB p65, decreased claudin-5 protein expression, and enhanced transit of macromolecules. Unbiased membrane proteomics identified glucose-regulated protein 78 (GRP78) as the rAb target. Using immobilized GRP78 to deplete GRP78 antibodies from pooled total immunoglobulin G (IgG) of 50 NMO patients (NMO-IgG) reduced the biological effect of NMO-IgG on BMECs. GRP78 was expressed on the surface of murine BMECs in vivo, and repeated administration of a GRP78-specific rAb caused extravasation of serum albumin, IgG, and fibrinogen into mouse brains. Our results identify GRP78 antibodies as a potential component of NMO pathogenesis and GRP78 as a candidate target for promoting central nervous system transit of therapeutic antibodies.