Strains of Epstein-Barr virus infecting multiple sclerosis patients.

Both epidemiological and experimental studies have indicated that the ubiquitous herpesvirus Epstein-Barr virus (EBV) plays a role in the pathogenesis of multiple sclerosis (MS). Some features of MS epidemiology, such as the decline in risk among migrants from high to low MS prevalence areas, suggest the presence of variant EBV strains that increase MS risk. The objective of this study was to investigate whether genetic variability in EBV is associated with MS. Genes encoding for two EBV antigens (EBNA1 and BRRF2) were sequenced in EBV isolates from 40 MS patients and a similar number of control subjects. These viral antigens were chosen for analysis because they are known to stimulate atypical immune responses in MS. Extensive sequence polymorphism was observed within the EBNA1 and BRRF2 genes in isolates from both MS patients and controls. Interestingly, several single nucleotide polymorphisms within the EBNA1 gene, and one within the BRRF2 gene, were found to occur at marginally different frequencies in EBV strains infecting MS patients versus controls. Although this study does not find a simple causal relationship between EBV strains and the occurrence of MS, the existence of haplotypes that occur at different frequencies in MS patients versus controls may provide an area for future study of the role of EBV strain variation in multiple sclerosis.

Decreased T cell reactivity to Epstein-Barr virus infected lymphoblastoid cell lines in multiple sclerosis.

OBJECTIVE: To investigate T cell and antibody immunity to Epstein-Barr virus (EBV) in multiple sclerosis (MS). METHODS: Immunoglobulin G (IgG) immunity to EBV nuclear antigen 1 (EBNA1) and viral capsid antigen was measured by enzyme linked immunosorbent assays, and T cell immunity was assessed using enzyme linked immunospot assays to measure the frequency of peripheral blood mononuclear cells (PBMC) producing interferon gamma in response to autologous EBV infected B cell lymphoblastoid cell lines (LCL) in 34 EBV seropositive healthy subjects and 34 EBV seropositive patients with MS who had not received immunomodulatory therapy in the previous 3 months. RESULTS: Patients with MS had increased levels of anti-EBNA1 IgG but a decreased frequency of LCL specific T cells compared with healthy subjects. Using purified populations of CD4(+) T cells and CD8(+) T cells, we showed that the LCL specific response resides predominantly in the CD8(+) population, with a frequency 5-7-fold higher than in the CD4(+) population. The decreased CD8(+) T cell response to LCL in MS was not caused by decreased HLA class I expression by LCL, and LCL from MS patients could be killed normally by HLA matched EBV specific cytotoxic CD8(+) T cell clones from healthy subjects. Furthermore, the decreased CD8(+) T cell immunity to EBV was not due to a primary defect in the function of CD8(+) T cells because EBV specific cytotoxic CD8(+) T cell lines could be generated normally from the PBMC of patients with MS. CONCLUSION: This quantitative deficiency in CD8(+) T cell immunity to EBV might be responsible for the accumulation of EBV infected B cells in the brains of patients with MS.

Antigen-induced IL-17 response in the peripheral blood mononuclear cells (PBMC) of healthy controls.

IL-17 is a T cell cytokine with a complex and important role in the immune system. It has been detected in rheumatoid arthritis (RA) synovial membrane and found to stimulate the production of the proinflammatory cytokines IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. To date, there are few data available on the agents that stimulate IL-17 production. We therefore investigated the in vitro IL-17 response to a variety of mitogens and antigens, and compared the IL-17 response to interferon-gamma (IFN-gamma), IL-4, IL-10 and TNF-alpha. In this study we used a type-0 antigen, tetanus toxoid (TT), a type-1 antigen, PPD from Mycobacterium tuberculosis, a potential type-2 rye grass (RG) antigen (Lol I) and an autoantigen SS.B (La), to stimulate PBMC from healthy controls. Cytokine mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction and cytokine protein measured using specific ELISA techniques, while the frequency of IL-17-producing T cells was determined by flow cytometry. The mitogens concanavalin A, phytohaemagglutinin and phorbol myristate acetate/ionomycin induced a significant increase in IL-17, with the highest levels being produced by anti-CD3/anti-CD28 stimulation. The antigens TT and PPD significantly increased IL-17 mRNA expression over time, but failed to have such an effect at the protein level. IL-17 protein was also detectable in both antigen-specific (TT, SS. B) and non-specific T cell clones, but at levels lower than IFN-gamma. IL-17 production did not correlate with either the type-1 cytokine IFN-gamma or TNF-alpha or the type-2 cytokine IL-4 or IL-10 at either the mRNA or protein level.

Intraarticular variability of synovial membrane histology, immunohistology, and cytokine mRNA expression in patients with rheumatoid arthritis.

OBJECTIVE: To assess the variability of synovial histology, immunohistology, and cytokine mRNA expression at different sites within the knee joints of subjects with rheumatoid arthritis receiving slow acting antirheumatic drugs. The effects of intraarticular bupivacaine and adrenaline, and a comparison of synovial fluid cell and synovial membrane cytokine expression, were also investigated. METHODS: Arthroscopically directed synovial biopsies were taken at 3 or 4 predetermined sites from the knee joints of 11 patients. Histology for synovial lining layer, sublining cellularity and vascularity, and immunohistology for T cells, T cell subsets, and macrophages were assessed. Messenger RNA expression of interleukins 1beta, 2, 4, 6, 8, 10, granulocyte-monocyte colony stimulating factor, tumor necrosis factor-alpha, and interferon-gamma was detected using the reverse transcription/polymerase chain reaction technique. RESULTS: Synovial histology, immunohistology, and cytokine mRNA expression did not vary significantly. CD8 cell immunohistology was variable. Intraarticular bupivacaine and adrenaline did not change synovial characteristics. Synovial fluid cell and membrane cytokine expression did not match in 35% of comparisons. CONCLUSION: Biopsies from the suprapatellar pouch, medial gutter, and cartilage-pannus junction will provide a representative sample of synovial membrane pathology in patients with rheumatoid arthritis.