thioguanine-anhydrous and Arthritis--Rheumatoid

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Arthritis, Rheumatoid; Azathioprine; Chlorambucil; Colitis, Ulcerative; Crohn Disease; Cyclophosphamide; Granulomatosis with Polyangiitis; Hepatitis; Humans; Immune Complex Diseases; Immunosuppressive Agents; Infections; Liver Cirrhosis, Biliary; Lupus Erythematosus, Systemic; Mercaptopurine; Methotrexate; Nephrotic Syndrome; Ophthalmia, Sympathetic; Psoriasis; Thioguanine; Uveitis

Topics: Arthritis, Rheumatoid; Azathioprine; Erythrocytes; Female; Humans; Injections, Intravenous; Male; Middle Aged; Thioguanine

The study of T cell clones at the genomic level is expanding our understanding of their role in diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). We have been carrying out genotypic analysis by PCR of hypoxanthine phosphoribosyltransferase (hprt) mutations in these cells. Mutant T cells in the population can be cloned on the basis of their resistance to the cytotoxic drug, 6-thioguanine-(6-TG). A difficulty is that the majority of primary human T cells are capable of only limited growth ex vivo, even in the presence of 'feeder' cells. PCR analysis of DNA from such clones is made difficult by the limited number of viable mutant (drug-resistant) T cells and the large number of dead (drug-sensitive) mononuclear cells and feeder cells. DNA from the 'dead' cells remains sufficiently intact for many weeks in culture and can represent a significant source of background in PCR analysis. Here we describe a method employing hypotonic shock and micrococcal nuclease that reliably eliminates non-viable 6-TG-sensitive cells, allowing the study of the hprt gene in < 200 T cells by PCR.

Topics: Arthritis, Rheumatoid; Cell Survival; DNA Mutational Analysis; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Multiple Sclerosis; Polymerase Chain Reaction; T-Lymphocytes; Thioguanine

The frequency of mutant T cells (FMC) in blood lymphocytes from patients with systemic lupus erythematosus (SLE) was measured by growing cells in the presence and in the absence of 6-thioguanine. Patients with SLE had a spectrum of FMC ranging from normal to about 100 times normal. This high FMC among cells from SLE patients appears to reflect excessive in vivo activation and proliferation during the course of the disease. This represents the first demonstration of such a T cell abnormality in SLE; it supports the hypothesis that SLE T cells demonstrate increased in vivo division and/or survival.

Topics: Adult; Arthritis, Rheumatoid; Cells, Cultured; Female; Humans; Hypoxanthine Phosphoribosyltransferase; Lupus Erythematosus, Systemic; Male; Mutation; Reference Values; T-Lymphocytes; Thioguanine

Topics: Arthritis, Rheumatoid; Azathioprine; Collagen Diseases; Humans; Immunosuppressive Agents; Lupus Erythematosus, Systemic; Mercaptopurine; Methotrexate; Thioguanine