thioguanine-anhydrous and Inflammation

Thiopurine analogs and anti-tumor necrosis factor (TNF) agents have dramatically changed the therapeutics of inflammatory bowel diseases (IBD), improving short and long-term outcomes. Unfortunately some patients do not respond to therapy and others lose response over time. The pharmacokinetic properties of these drugs are complex, with high inter-patient variability. Thiopurine analogs are metabolized through a series of pathways, which vary according to the patients' pharmacogenetic profile. This profile largely determines the ratios of metabolites, which are in turn associated with likelihoods of clinical efficacy and/or toxicity. Understanding these mechanisms allows for manipulation of drug dose, aiming to reduce the development of toxicity while improving the efficacy of treatment. The efficacy of anti-TNF drugs is influenced by many pharmacodynamic variables. Several factors may alter drug clearance, including the concomitant use of immunomodulators (thiopurine analogs and methotrexate), systemic inflammation, the presence of anti-drug antibodies, and body mass. The treatment of IBD has evolved with the understanding of the pharmacologic profiles of immunomodulating and TNF-inhibiting medications, with good evidence for improvement in patient outcomes observed when measuring metabolic pathway indices. The role of routine measurement of metabolite/drug levels and antibodies warrants further prospective studies as we enter the era of personalized IBD care.

Topics: Adalimumab; Antibodies; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Azathioprine; Drug Monitoring; Genotype; Humans; Immunologic Factors; Inflammation; Inflammatory Bowel Diseases; Infliximab; Methyltransferases; Phenotype; Purines; Thioguanine; Thionucleosides; Treatment Outcome; Tumor Necrosis Factor-alpha

Topics: Apoptosis; Azathioprine; Case-Control Studies; Crohn Disease; Humans; Inflammation; Intestinal Mucosa; Leukocytes, Mononuclear; Macrophages; Monocytes; Peptidoglycan; rac1 GTP-Binding Protein; Signal Transduction; Thioguanine

In inflammatory bowel disease (IBD), large areas of apparently healthy mucosa lie adjacent to ulcerated intestine. Knowledge of the mechanisms that maintain remission in an otherwise inflamed intestine could provide important clues to the pathogenesis of this disease and provide rationale for clinical treatment strategies. We used kinome profiling to generate comprehensive descriptions of signal transduction pathways in inflamed and noninflamed colonic mucosa in a cohort of IBD patients, and compared the results to non-IBD controls. We observed that p21Rac1 guanosine triphosphatase (GTPase) signaling was strongly suppressed in noninflamed colonic mucosa in IBD. This suppression was due to both reduced guanine nucleotide exchange factor activity and increased intrinsic GTPase activity. Pharmacological p21Rac1 inhibition correlated with clinical improvement in IBD, and mechanistically unrelated pharmacological p21Rac1 inhibitors increased innate immune functions such as phagocytosis, bacterial killing, and interleukin-8 production in healthy controls and patients. Thus, suppression of p21Rac activity assists innate immunity in bactericidal activity and may induce remission in IBD.

Topics: Animals; Biopsy; Crohn Disease; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Enzyme Inhibitors; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hydrolysis; Immunity, Innate; Inflammation; Intestinal Mucosa; Protein Kinases; rac1 GTP-Binding Protein; Remission Induction; Signal Transduction; Thioguanine

Infection-driven inflammation has been implicated in the pathogenesis of ~15-20% of human tumors. Expression of microRNA-155 (miR-155) is elevated during innate immune response and autoimmune disorders as well as in various malignancies. However, the molecular mechanisms providing miR-155 with its oncogenic properties remain unclear. We examined the effects of miR-155 overexpression and proinflammatory environment on the frequency of spontaneous hypoxanthine phosphoribosyltransferase (HPRT) mutations that can be detected based on the resistance to 6-thioguanine. Both miR-155 overexpression and inflammatory environment increased the frequency of HPRT mutations and down-regulated WEE1 (WEE1 homolog-S. pombe), a kinase that blocks cell-cycle progression. The increased frequency of HPRT mutation was only modestly attributable to defects in mismatch repair machinery. This result suggests that miR-155 enhances the mutation rate by simultaneously targeting different genes that suppress mutations and decreasing the efficiency of DNA safeguard mechanisms by targeting of cell-cycle regulators such as WEE1. By simultaneously targeting tumor suppressor genes and inducing a mutator phenotype, miR-155 may allow the selection of gene alterations required for tumor development and progression. Hence, we anticipate that the development of drugs reducing endogenous miR-155 levels might be key in the treatment of inflammation-related cancers.

Topics: Antimetabolites, Antineoplastic; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Genes, Tumor Suppressor; HEK293 Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Inflammation; MicroRNAs; Mutation; Neoplasms; Nuclear Proteins; Protein-Tyrosine Kinases; Thioguanine

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

Topics: Cell Line, Tumor; Coagulants; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Inflammation; Jurkat Cells; Lymphoma, Large B-Cell, Diffuse; Mercaptopurine; Stem Cell Transplantation; Thioguanine; Treatment Outcome