Phagocytosis and Epithelial Cell Invasion by Crohn's Disease-Associated Adherent-Invasive Escherichia coli Are Inhibited by the Anti-inflammatory Drug 6-Mercaptopurine.

Adherent-invasive Escherichia coli (AIEC) strains are overrepresented in the dysbiotic microbiota of Crohn's disease (CD) patients, and contribute to the onset of the chronic inflammation typical of the disease. However, the effects of anti-inflammatory drugs used for CD treatment on AIEC virulence have not yet been investigated. In this report, we show that exposure of AIEC LF82 strain to amino-6-mercaptopurine (6-MP) riboside, one of the most widely used anti-inflammatory drugs in CD, impairs its ability to adhere to, and consequently to invade, human epithelial cells. Notably, phagocytosis of LF82 treated with 6-MP by human macrophages is also reduced, suggesting that 6-MP affects AIEC cell surface determinants involved both in interaction with epithelial cells and in uptake by macrophages. Since a main target of 6-MP in bacterial cells is the inhibition of the important signal molecule c-di-GMP, we also tested whether perturbations in cAMP, another major signaling pathway in E. coli, might have similar effects on interactions with human cells. To this aim, we grew LF82 in the presence of glucose, which leads to inhibition of cAMP synthesis. Growth in glucose-supplemented medium resulted in a reduction in AIEC adhesion to epithelial cells and uptake by macrophages. Consistent with these results, both 6-MP and glucose can affect expression of cell adhesion-related genes, such as the csg genes, encoding thin aggregative fimbriae (curli). In addition, glucose strongly inhibits expression of the fim operon, encoding type 1 pili, a known AIEC determinant for adhesion to human cells. To further investigate whether 6-MP can indeed inhibit c-di-GMP signaling in AIEC, we performed biofilm and motility assays and determination of extracellular polysaccharides. 6-MP clearly affected biofilm formation and cellulose production, but also, unexpectedly, reduced cell motility, itself an important virulence factor for AIEC. Our results provide strong evidence that 6-MP can affect AIEC-host cell interaction by acting on the bacterial cell, thus strengthening the hypothesis that mercaptopurines might promote CD remission also by affecting gut microbiota composition and/or physiology, and suggesting that novel drugs targeting bacterial virulence and signaling might be effective in preventing chronic inflammation in CD.

Crystal structures of five 6-mercaptopurine derivatives.

The crystal structures of five 6-mercaptopurine derivatives, viz. 2-[(9-acetyl-9H-purin-6-yl)sulfan-yl]-1-(3-meth-oxy-phen-yl)ethan-1-one (1), C16H14N4O3S, 2-[(9-acetyl-9H-purin-6-yl)sulfan-yl]-1-(4-meth-oxy-phen-yl)ethan-1-one (2), C16H14N4O3S, 2-[(9-acetyl-9H-purin-6-yl)sulfan-yl]-1-(4-chloro-phen-yl)ethan-1-one (3), C15H11ClN4O2S, 2-[(9-acetyl-9H-purin-6-yl)sulfan-yl]-1-(4-bromo-phen-yl)ethan-1-one (4), C15H11BrN4O2S, and 1-(3-meth-oxy-phen-yl)-2-[(9H-purin-6-yl)sulfan-yl]ethan-1-one (5), C14H12N4O2S. Compounds (2), (3) and (4) are isomorphous and accordingly their mol-ecular and supra-molecular structures are similar. An analysis of the dihedral angles between the purine and exocyclic phenyl rings show that the mol-ecules of (1) and (5) are essentially planar but that in the case of the three isomorphous compounds (2), (3) and (4), these rings are twisted by a dihedral angle of approximately 38°. With the exception of (1) all mol-ecules are linked by weak C-H⋯O hydrogen bonds in their crystals. There is π-π stacking in all compounds. A Cambridge Structural Database search revealed the existence of 11 deposited compounds containing the 1-phenyl-2-sulfanyl-ethanone scaffold; of these, only eight have a cyclic ring as substituent, the majority of these being heterocycles.

ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics.

Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.

TPMT testing in azathioprine: a 'cost-effective use of healthcare resources'?

This study aimed to critically appraise the current level of economic evidence available for thiopurine S-methyltransferase (TPMT) testing of thiopurine drugs, such as azathioprine. Six economic evaluations of testing were identified, which all recommended that TPMT testing is a cost-effective use of healthcare resources. Critical appraisal, using published guidelines, showed potential limitations in model structures, approaches to data analysis and input parameters, which were mainly based on expert opinion. Where data did exist these were from retrospective studies. To conduct economic evaluations with more robust findings, decision analysts need good quality data for the following key parameters: current prevalence of profound neutropenia among patients prescribed thiopurine drugs; mean length of related hospitalization and clinical outcome; impact of introducing the test on clinical pathways in terms of resource use; and clinical effectiveness data in terms of number of cases of neutropenia averted and subsequent impact on mortality and health-related quality of life. An iterative approach may be used to stimulate the production of a sufficient evidence base for innovative technologies, such as pharmacogenetic testing. Such an iterative approach involves starting with simple models using available existing clinical and resource use data, as in the case of TPMT testing. The use of formal value of information methods may guide the decision whether prospective studies are required to address uncertainties in the key parameters driving the model results. The results from well-designed prospective studies can then be used to populate more complex economic models.