PURSE 2005

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Browsing PURSE 2005 by Author "Budhia, S."

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    Analysis of T Cell Rece'ptor β Variable (TCR Vβ)' Region Gene Expression in Sheep with Maedi -Visna Virus ( MVV) Infection
    (University of Peradeniya, 2005-11-10) Kalupahana, A. W.; Budhia, S.; Kalupahana, R. S.; Blacklaws, B. A.
    Maedi-Visna virus (MVV) is a member of the subfamily lentivirinae, which causes a persistent, chronic active inflammatory disease in sheep potentially affecting many body systems. MVV is a macrophage-tropic lentivirus which infects accessory cells of the immune system, leading to lymphocyte proliferation. In order to study the role of CDS+ T lymphocytes in immunity to MVV it would be helpful to be able to identify the presence of virus specific precursor cytotoxic T lymphocytes (pCTL) in cell preparations or tissues. Sheep chronically, experimentally infected with MVV were used as a source of lymphocytes which were analysed for Vf3 RNA expression before and after in vitro stimulation with autologous MVV infected skin cell monolayers to produce active anti-MVV CTL. In this study the method for analysis of the Vf3 gene usage in response to MVV was evaluated. RNA was extracted from CDS+ and CfL cell populations and cDNA was amplified by peR using specific primers for TCR Vf3 gene families. The comparative analysis of each VI' product between the different samples was achieved by densitometric analysis of the signals on the Southern blot membrane after hybridization with specific digoxigenin (DIG) oligonucleotide probes. This study does show that the PCR method used in this study to analyse TCR VI' gene has the potential to use for screening of large numbers of sheep. The development of this method for analysis of the TCR Vf3 repertoire in populations of lymphocytes in sheep is much faster than the three-week CTL culture and assay. In addition the method practised in this study includes a semi-quantitative analysis, which indicates the proportional increase in the level ofTCR Vj3 mRNA in the lymphocytes. We are interested in finding out whether we would be able to find an immunodominant TCR Vf3response to a persistent infection ofmonocytes, macrophages and dendritic cells in the circulation. Sufficient numbers of sheep were analysed to determine any definite trends in TCR Vf3 expression. Using the method practised in this study we were able to suggest four candidates out of 16 genes identified so far namely Vf3 1.2,2.1, 7.1 and 24.1 for future analysis of TCR VI' region usage in sheep against MVV infection. At the same time we were able to exclude the involvement of several genes namely Vj3 4.1,6.1, 10.1, 13.2, 17.1 and 28.1.2 in the CD8+ T cell response to MVV. In conclusion it is necessary to look for other more sensitive methods in future to definitely find which Vf3gene is used by CD8+ T lymphocytes.