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.

BackgroundDexamethasone (DXM) is commonly used in the management of cerebral edema in patients diagnosed with glioblastoma multiforme (GBM). Bevacizumab (BEV) is FDA-approved for the progression or recurrence of GBM but has not been shown to improve survival when given for newly diagnosed patients concurrently with radiation (RT) and temozolomide (TMZ). Both DXM and BEV reduce cerebral edema, however, DXM has been shown to induce cytokine cascades which could interfere with cytotoxic therapy. We investigated whether DXM would reduce survival of GBM patients in the setting of concurrent TMZ and BEV administration.MethodsWe reviewed the treatment of all 73 patients with GBM who received definitive therapy at our institution from 2005 to 2013 with RT (60 Gy) delivered with concurrent daily TMZ (75 mg/m2). Of these, 34 patients also were treated with concurrent BEV (10 mg/kg every two weeks). Patients received adjuvant therapy (TMZ or TMZ/Bev) until either progression, discontinuation due to toxicity, or 12 months after radiation completion. All patients who had GBM progression with TMZ were offered BEV for salvage therapy, with 19 (56 %) receiving BEV.ResultsWith a median follow-up of 15.6 months, 67 (91.8 %) patients were deceased. The OS for the entire cohort was 15.9 months, while the PFS was 7.7 months. The extent of resection was a prognostic indicator for OS (p = .0044). The median survival following gross tumor resection (GTR) was 22.5 months, subtotal resection (STR) was 14.9 months, and biopsy was 12.1 months. The addition of BEV to TMZ with RT was borderline significantly associated with increased PFS (9.4 vs. 5.1 months, p = 0.0574) although was not significantly associated with OS (18.1 vs. 15.3 months respectively, p = 0.3064). In patients receiving TMZ, DXM use concurrent with RT was a poor prognostic indicator of both OS (12.7 vs. 22.6 months, p = 0.003) and PFS (3.6 vs. 8.4 months, p <0.0001). DXM did not reduce OS in patients who received TMZ and BEV concurrently with RT (22.9 vs 22.8 months, p = 0.4818). On multivariable analysis, DXM use predicted an unfavorable OS hazard ratio (HR) = 1.72, p = 0.045).ConclusionsOur results with TMZ, BEV, and RT are similar to previous studies in terms of PFS and OS. DXM use during RT with concurrent TMZ correlated with reduced OS and PFS unless BEV was administered.

Background: Patient-derived aquaporin 4 (AQP4)-specific recombinant monoclonal antibodies (rAbs) cause neuromyelitis optica (NMO)-specific nervous system injury in animal models.Results: AQP4-specific rAbs bind human AQP4 based on differential sensitivity to loop A and C mutations.Conclusion: AQP4-specific rAbs derived from NMO patients recognize multiple conformational epitopes within the extracellular domains of human AQP4.Significance: High resolution mapping of AQP4 autoantibody epitopes identifies target regions for potential blocking therapies.

Using FACS and single cell reverse transcriptase polymerase chain reaction, we examined the cerebrospinal fluid (CSF) IgG VH repertoires from 10 subjects with a clinically isolated demyelinating syndrome (CIS). B and plasma cell repertoires from individual subjects showed similar VH family germline usage, nearly identical levels of post-germinal center somatic hypermutation, and significant overlap in their clonal populations. Repertoires from 7 of 10 CIS subjects demonstrated a biased usage of VH4 and/or VH2 family gene segments in their plasma or B cell repertoires. V region bias, however, was not observed in the corresponding peripheral blood CD19+ B cell repertoires from 2 CIS subjects or in normal healthy adults. Clinically, subjects with VH4 or VH2 CSF IgG repertoire bias rapidly progressed to definite MS, whereas individuals without repertoire bias did not develop MS after a minimum of 2 years of follow-up (p = 0.01).

BackgroundImproving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock.ResultsIt was found that RG displayed increased accumulation of lignin in leaves and depletion in the stems, antithetic to the trend observed in wild type. Consistent with these measurements, the RG leaf tissue displayed reduced saccharification efficiency whereas the stem saccharification efficiency increased relative to wild type. Reduced lignin was linked to improved saccharification in RG stems, but a chemical shift to greater S:G ratios in RG stem lignin was also observed. Similarities in cellulose content and structure by XRD-analysis support the correlation between increased saccharification properties and reduced lignin instead of changes in the cellulose composition and/or structure.ConclusionAntithetic lignin accumulation was observed in the RG mutant leaf-and stem-tissue, which resulted in greater saccharification efficiency in the RG stem and differential thermochemical product yield in high lignin leaves. Thus, the red leaf coloration of the RG mutant represents a potential marker for improved conversion of stem cellulose to fermentable sugars in the C4 grass Sorghum.

Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus×giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.

Real-time immuno-PCR (RT-IPCR) is a powerful technique that combines ELISA with the specificity and sensitivity of PCR. RT-IPCR of phage-displayed peptides exploits the unique physical associations between phenotype (the displayed peptide) and genotype (the encoding DNA) within the same phage particle. Previously, we identified phage peptides specific for recombinant antibodies (rAbs) prepared from clonally expanded plasma cells in multiple sclerosis (MS) cerebrospinal fluid (CSF) and subacute sclerosing panencephalitis (SSPE) brain. Herein, we applied phage-mediated RT-IPCR to study reactivity of these specific phage peptides for the rAbs. Compared to standard ELISA, which required greater than 10 4 or 10 5 phage particles to detect binding to rAbs, RT-IPCR detected binding with as few as 100 phage particles. RT-IPCR was also superior to ELISA in determining relative affinities of rAbs for phage peptides and was effective in screening MS CSF for IgG reactivity to phage peptides. Phage-mediated RT-IPCR is a rapid, high-throughput technology that avoids the requirement for synthetic peptides and will facilitate the identification of candidate peptides that react with the IgG in MS CSF.