Detection of TNF α and Fas ligand mRNA within synovial mononuclear cells by fluorescence in-cell labeling PCR (FICL-PCR)

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Abstract

T cells that infiltrate the synovial lesions of rheumatoid arthritis may play a key role in its pathogenesis. To learn more about their functional nature, we determined the frequency of synovial T cells that harbored the TNF α and Fas ligand transcript by a technique, called Fluorescence In-Cell Labeling Polymerase Chain Reaction (FICL-PCR). The mRNA of interest was detected in fixed cells by the incorporation during PCR of a fluorescein-12-dUTP label following an initial reverse transcription PCR step. Using this technique the CD3 transcript was detected in the T leukemic cell line, MOLT-4, with calculated sensitivity and specificity values of 91% and 100%, respectively. The percentage mean (± S.D.)of TNF α mRNA positive cells and Fas ligand mRNA positive cells in peripheral blood mononuclear cells from 12 rheumatoid arthritis patients were 5.1 2.3% and 4.8 3.1%, respectively. The percentage mean (±S.D.) of TNF α mRNA positive cells and Fas ligand mRNA positive cells among synovial mononuclear cells from six rheumatoid arthritis patients was 16.8 8.3% and 10.8 1.8%, respectively. This result indicates that the cytotoxic T cells expressing TNF α accumulate in rheumatoid arthritic lesions where they may play a pathogenic role.

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