Increased IgG and oligoclonal bands (OGBs) are found in the cerebrospinal fluid (CSF) of humans with chronic infectious CNS diseases such as neurosyphilis, cryptococcal and tuberculous meningitis, Lyme disease, some viral meningitides, varicella zoster virus vasculopathy, and subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus (MV). Studies in which the specificity of CSF OGBs was analyzed showed that the antibodies were directed against the agent that causes disease (1-8). For example, the OGBs in SSPE CSF and brain are antibodies directed against MV (1). These studies have led to the hypothesis that the oligoclonal IgG in the brain and CSF of patients with a chronic inflammatory CNS disease of unknown etiology, such as multiple sclerosis (MS), sarcoidosis, and Behcet's disease, is antibody directed against the agent that causes disease. Although OGBs are found in 88-100% of CSF from MS patients (9), the corresponding antigens remain unknown (10, 11). Such unanswered questions point to the need for improved techniques to identify disease-relevant antibodies and their cognate antigens and suggest the promise of such techniques in revealing the causes of inflammatory diseases with unknown etiologies.Here, we used laser-capture microdissection (LCM) to isolate individual CD38ϩ plasma cells from the brain of a patient with SSPE. Single-cell RT-PCR was used to amplify individual IgG heavy (H) and light (L) chain sequences expressed by each cell. Based on overrepresented Ig sequences, we constructed functional recombinant antibodies and identified their target antigens.Materials and Methods SSPE Brain. Brain removed from a 14-year-old male SSPE patient 5 h after death was flash-frozen and stored at Ϫ70°C. Sections (7 m) of frozen SSPE brain were prepared on nonplus glass slides (Fisher Scientific) at Ϫ30°C, fixed in acetone for 5 min at Ϫ20°C, and immunostained at 0°C for CD38ϩ cells. Briefly, sections were treated with 0.1% H 2 O 2 for 30 sec, incubated for 2 min with PBS containing 10% goat serum and then with a 1:50 dilution of mouse anti-human CD38 antibody (DakoCytomation, DAKO) for 10 min, rinsed in PBS, and incubated for 5 min with a 1:100 dilution of horseradish peroxidase-conjugated horse anti-mouse antibody (Vector Laboratories) and 5% goat serum in PBS. After rinsing in PBS, sections were incubated for 5 min with DAB substrate (Vector Laboratories), counterstained with hematoxylin, dehydrated in nuclease-free graded alcohols to 100% ethanol, and cleared with two rinses of H 2 0-free xylene. To preserve RNA, all aqueous solutions contained 200 units͞ml RNase inhibitor (Fisher Scientific), and the total time of tissue exposure to aqueous solutions was Ͻ25 min at 0°C. Staining was visualized by light microscopy.LCM and RT-PCR. LCM was performed on a PixCell IIe microscope (Arcturus Engineering, Mountain View, CA) with CapSure HS caps by using a pulse power of 70 mW, a 7.5-m laser spot diameter, pulse duration of 5 ms, and target voltage of 170 mV. Individual CD38ϩ cells ...

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Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.Panning of phage-displayed random peptide libraries allows an unbiased selection of antibody epitopes/mimotopes without preconceptions about the nature of the target antigens (reviewed in reference 23). In central nervous system (CNS) inflammatory diseases, where access to active diseased tissue is limited or where the levels of tissue antigen may be extremely low, phage peptide panning provides an alternative and sensitive avenue for antigen identification. Panning of phage-displayed random peptide libraries has successfully identified rheumatoid factor-specific mimotopes (22) and allergen mimotopes (19) and has mapped both linear and discontinuous viral epitopes recognized by antibodies specific for various infectious agents (5,10,11,16,20).Infectious and inflammatory diseases of the CNS are often characterized by increased intrathecal immunoglobulin G (IgG) synthesis that is seen as discrete bands of oligoclonal IgG when cerebrospinal fluid (CSF) or brain IgG is separated by isoelectric focusing. In CNS infectious diseases, such as subacute sclerosing panencephalitis (SSPE), neurosyphilis, mumps meningitis, progressive rubella panencephalitis, cryptococcal meningitis, and varicella zoster virus vasculitis, the oligoclonal IgG is directed largely against the infectious agent that causes the disease (reviewed in reference 12). Increased CNS IgG synthesis is accompanied by an elevated number of CD19 ϩ B cells and the app...

Increased immunoglobulin G (IgG) and intrathecally produced oligoclonal bands (OGBs) are characteristic of a limited number of inflammatory central nervous system (CNS) diseases and are often directed against the cause of disease. In subacute sclerosing panencephalitis (SSPE), the cause of disease and the target of the oligoclonal response is measles virus (MV). The authors previously showed that clonally expanded populations of CD38+ plasma cells in SSPE brain, the likely source of OGBs, are directed against MV. In characterizing the breadth of the plasma cell reactivities, the authors found that a large proportion of the less abundant plasma cells are also directed against MV. The intrathecal response may be useful in determining the causes of other inflammatory CNS diseases, such as multiple sclerosis, Behcet's disease, and neurosarcoidosis.

Chronic infectious diseases of the central nervous system (CNS) are characterized by intrathecal synthesis of increased amounts of immunoglobulin G (IgG) directed against the agent that causes disease. In other inflammatory CNS diseases such as multiple sclerosis and CNS sarcoid, the targets of the humoral immune response are uncertain. To identify the IgGs expressed by individual CD38+ plasma cells seen in human brain sections, we merged the techniques of laser capture microdissection (LCM) and single-cell RT-PCR. Frozen brain sections from a patient who died of subacute sclerosing panencephalitis (SSPE), were rapidly immunostained and examined by LCM to dissect individual CD38+ cells. After cell lysis, we developed two techniques for reversetranscription (RT) of unpurified total RNA in the cell lysates. The first method performed repeated and rapid freeze-thawing, followed by centrifugation of the cell lysate into tubes for subsequent RT. The second, more successful method performed RT in situ on detergent-solubilized cells directly on the cap surface; subsequent nested PCR identified heavy and light chain sequences expressed by two-thirds of individually isolated plasma cells. These techniques will streamline the identification of gene expression products in single cells from complex tissues and have the potential to identify IgGs expressed in the CNS of inflammatory diseases of unknown etiology.