thioguanine-anhydrous and Multiple-Sclerosis

Queuine is a modified pyrrolopyrimidine nucleobase derived exclusively from bacteria. It post-transcriptionally replaces guanine 34 in transfer RNA isoacceptors for Asp, Asn, His and Tyr, in almost all eukaryotic organisms, through the activity of the ancient tRNA guanine transglycosylase (TGT) enzyme. tRNA hypomodification with queuine is a characteristic of rapidly-proliferating, non-differentiated cells. Autoimmune diseases, including multiple sclerosis, are characterised by the rapid expansion of T cells directed to self-antigens. Here, we demonstrate the potential medicinal relevance of targeting the modification of tRNA in the treatment of a chronic multiple sclerosis model—murine experimental autoimmune encephalomyelitis. Administration of a de novo designed eukaryotic TGT substrate (NPPDAG) led to an unprecedented complete reversal of clinical symptoms and a dramatic reduction of markers associated with immune hyperactivation and neuronal damage after five daily doses. TGT is essential for the therapeutic effect, since animals deficient in TGT activity were refractory to therapy. The data suggest that exploitation of the eukaryotic TGT enzyme is a promising approach for the treatment of multiple sclerosis.

Topics: Animals; Brain; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Genetic Therapy; Mice, Inbred C57BL; Multiple Sclerosis; Pentosyltransferases; Pyrimidinones; Pyrroles; RNA, Transfer; 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

Analysis of somatic mutation in circulating peripheral blood T cells can be used as an index of in vivo T-cell amplification. The ability to assess the frequency of T cells that survive in vitro in the presence of 6-thioguanine is an index of mutation at the HPRT gene locus. In the absence of exposure to mutational agents, elevations of the HPRT mutant frequency (mF) of T cells reflects errors in DNA replication and repair that have become fixed during the process of cell division. We estimated the mF of T cells in MS patients and controls over a period of 36 months and found that the mF was consistently elevated in MS patients of all clinical subgroups. In the chronic progressive group of MS, the mF increased over a 3-year period and appeared to correlate with the clinical worsening of the disease.

Topics: Adult; Female; Humans; Hypoxanthine Phosphoribosyltransferase; Longitudinal Studies; Male; Multiple Sclerosis; Mutation; Nervous System Diseases; Reference Values; T-Lymphocytes; Thioguanine

Gene mutation in vivo in human T lymphocytes appears to occur preferentially in dividing cells. Individuals with multiple sclerosis (MS) are assumed to have one or more populations of diving T cells that are being stimulated by autoantigens. Mutant T cell clones from MS patients were isolated and tested for reactivity to myelin basic protein, an antigen that is thought to participate in the induction of the disease. The hypoxanthine guanine phosphoribosyltransferase (hprt) clonal assay was used to determine mutant frequency values in MS patients with chronic progressive disease. Eleven of 258 thioguanine-resistant (hprt-) T cell clones from five of the six MS patients who were tested proliferated in response to human myelin basic protein without prior in vitro exposure to this antigen. No wild-type clones from these patients, nor any hprt- or wild-type clones from three healthy individuals responded to myelin basic protein. Thus, T cell clones that react with myelin basic protein can be isolated from the peripheral blood of MS patients.

Topics: Adult; Autoantigens; Cell Division; Clone Cells; Female; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Middle Aged; Multiple Sclerosis; Mutation; Myelin Basic Protein; T-Lymphocytes; Thioguanine; X Chromosome

An autoradiographic assay for 6-thioguanine-resistant (TGr) lymphocytes was used to determine the frequency of in vivo derived variant T lymphocytes in peripheral blood from multiple sclerosis (MS) patients treated with monthly intravenous infusions of 750 mg/m2 of cyclophosphamide (CP). To analyze the time-course of response to CP, the MS patients were studied prospectively. Samples were obtained from the patients before the beginning of CP therapy, 4-5 times during the course of treatment, and, finally, 2 or 3 months after the completion of therapy. 2 weeks after the first CP infusion, the variant frequencies (Vfs) of the MS patients were significantly increased (p less than 0.05) above their pre-treatment values, but by 4 weeks following the first CP infusion the Vfs had fallen to normal or near-normal levels. After subsequent treatments, the frequencies of variant TGr cells were again higher than pre-treatment Vfs. However, within 7-13 weeks after the cessation of CP therapy, the Vfs of all subjects had returned to normal levels. The transient nature of the response indicates rapid in vivo selection against CP-induced TGr mutant cells. The mean pre-treatment Vf of the 4 MS patients who were cigarette smokers was 6.56 X 10(-6) which was significantly greater (p less than 0.05) than the mean Vf (1.52 X 10(-6) of the 4 MS patients who were non-smokers. The mean Vf from 8 assays of healthy non-smokers was 1.92 X 10(-6).

Topics: Autoradiography; Cyclophosphamide; Drug Resistance; Humans; Lymphocytes; Multiple Sclerosis; Mutagens; Prospective Studies; Smoking; Thioguanine; Time Factors