Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with K d values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro 346 was shown to control heme cofactor conformation, whereas Arg 386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.

Objective. To determine cancer risk in a cohort of 459 rheumatoid arthritis (RA) patients treated with methotrexate in community practice. Methods. All RA patients who started methotrexate prior to June 1986 and were attending 1 of 6 rheumatologists were studied. Demographic data were matched to the State Cancer Registry to identify all malignancies (except nonmelanoma skin cancer) for 1983-1998, and to the National Death Index to identify all deaths to the end of 1999. Followup started on the date when methotrexate was started and ended either on the last confirmed date on which the patient was seen by the rheumatologist or at death. Standardized incidence ratios (SIRs) were calculated using state population cancer rates stratified by sex, age (in 5-year groups), and calendar year. Results. There were 4,145 person-years of followup (average 9.3 years). Eighty-seven malignancies were identified (14 before, 64 during, and 9 after the followup period). There was an estimated 50% excess risk of malignancy among methotrexate-exposed RA patients relative to the general population (SIR 1.5, 95% confidence interval [95% CI] 1.2-1.9), with a 3-fold increase in melanoma (SIR 3.0, 95% CI 1.2-6.2), a 5-fold increase in non-Hodgkin's lymphoma (SIR 5.1, 95% CI 2.2-10.0), and an almost 3-fold increase in lung cancer (SIR 2.9, 95% CI 1.6 -4.8). Conclusion. Compared with the general population, methotrexate-treated RA patients have an increased incidence of melanoma, non-Hodgkin's lymphoma, and lung cancer. There may be a role for regular skin cancer screening for all RA patients, particularly those receiving immunosuppressive therapy.

BackgroundInflammation is hypothesized to be a key event in the growth of sporadic vestibular schwannoma (VS). In this study we sought to investigate the relationship between inflammation and tumor growth in vivo using the PET tracer 11C-(R)-PK11195 and dynamic contrast enhanced (DCE) MRI derived vascular biomarkers.MethodsNineteen patients with sporadic VS (8 static, 7 growing, and 4 shrinking tumors) underwent prospective imaging with dynamic 11C-(R)-PK11195 PET and a comprehensive MR protocol, including high temporal resolution DCE-MRI in 15 patients. An intertumor comparison of 11C-(R)-PK11195 binding potential (BPND) and DCE-MRI derived vascular biomarkers (Ktrans, vp, ve) across the 3 different tumor growth cohorts was undertaken. Tissue of 8 tumors was examined with immunohistochemistry markers for inflammation (Iba1), neoplastic cells (S-100 protein), vessels (CD31), the PK11195 target translocator protein (TSPO), fibrinogen for vascular permeability, and proliferation (Ki-67). Results were correlated with PET and DCE-MRI data.ResultsCompared with static tumors, growing VS displayed significantly higher mean 11C-(R)-PK11195 BPND (−0.07 vs 0.47, P = 0.020), and higher mean tumor Ktrans (0.06 vs 0.14, P = 0.004). Immunohistochemistry confirmed the imaging findings and demonstrated that TSPO is predominantly expressed in macrophages. Within growing VS, macrophages rather than tumor cells accounted for the majority of proliferating cells.ConclusionWe present the first in vivo imaging evidence of increased inflammation within growing sporadic VS. Our results demonstrate that 11C-(R)-PK11195 specific binding and DCE-MRI derived parameters can be used as imaging biomarkers of inflammation and vascular permeability in this tumor group.