Bedaquiline susceptibility testing of Mycobacterium tuberculosis in an automated liquid culture system.

OBJECTIVES: The objective of this study was to evaluate the performance of the BACTEC MGIT960 system to test the susceptibility to bedaquiline for Mycobacterium tuberculosis complex. METHODS: We determined the quality control (QC) range of bedaquiline using the M. tuberculosis H37Rv reference strain and the epidemiological cut-off (ECOFF) in MGIT960 and on Middlebrook 7H11 agar (M7H11) using 47 strains from bedaquiline treatment-naive patients. The accuracy of MGIT960 was evaluated versus M7H11 using 74 'probably susceptible to bedaquiline' and 18 'probably resistant to bedaquiline' strains. Repeatability and reproducibility of MGIT960 were assessed using five strains showing different resistance levels. RESULTS: The QC range for the H37Rv strain was between 0.125 and 0.50 mg/L. The WT MIC distribution ranged from ≤0.03 to 1.00 mg/L in MGIT960 and from ≤0.008 to 0.25 mg/L on M7H11 with suggested ECOFFs of 1.00 and 0.25 mg/L, respectively. Applying these ECOFFs, the probably susceptible and probably resistant strains were distinguishable by both methods, albeit with only a 2-fold increased MIC for one of the resistant strains compared with the ECOFF. Intermethod agreement to classify the isolates was excellent (100%). All replicates in the repeatability and reproducibility experiments fell within the normal range. CONCLUSIONS: The MGIT960 system proved to be highly stable, reproducible and accurate relative to the M7H11 agar method for determining the bedaquiline MIC. The small margin between the suggested ECOFF and the lowest MIC for the mutant strains risks making both methods prone to discordant results. Further validation in clinical settings linked to treatment outcome data is needed.

Mitochondrial uncoupling protein 2 mediates temperature heterogeneity in atherosclerotic plaques.

AIMS: Rupture-prone atherosclerotic plaques show an elevated temperature, but a molecular explanation for this phenomenon is unknown. Here, we investigated whether mitochondrial uncoupling protein 2 (UCP2) could be involved because this protein is a macrophage homologue of thermogenin in brown fat tissue. METHODS AND RESULTS: Immunohistochemistry, western blotting, and real-time quantitative polymerase chain reaction were used to detect UCP2 expression in human and rabbit atherosclerotic plaques. Temperature was measured in plaques with thermography catheters and in cultured cells with precision thermometers. UCP2 was abundantly expressed in subendothelial macrophages of atherosclerotic plaques but not in deeper layers of the plaque. Ex vivo temperature measurements in atherosclerotic rabbit thoracic aorta demonstrated a correlation between local plaque temperature, total macrophage mass, and UCP2 expression. In vitro, chemical uncoupling of macrophages with sodium cyanide resulted in heat production (DeltaT = 0.13 +/- 0.04 degrees C vs. controls). Also, overexpression of UCP2 in cultured cells led to a similar increase in temperature. CONCLUSION: Our findings provide evidence that temperature heterogeneity in atherosclerotic plaques is at least in part attributed to UCP2 expression in macrophages. The heat generated might be used to detect unstable, macrophage-rich, atherosclerotic plaques via thermography.

Attenuated atherogenesis in apolipoprotein E-deficient mice lacking amyloid precursor protein.

OBJECTIVE: Recent evidence suggests that amyloid precursor protein (APP) is overexpressed in atherosclerosis-prone regions of mouse aorta. We therefore investigated in the present study whether APP has a role in the progression and composition of atherosclerotic plaques. METHODS AND RESULTS: Apolipoprotein E-deficient (apoE(-/-)) mice were crossbred with animals lacking APP (APP(-/-)). After 16 weeks on a Western-type diet, apoE(-/-) and APP(-/-)/apoE(-/-) mice showed similar cholesterol levels. However, atherosclerotic plaque size was significantly reduced in the distal thoracic aorta (90% reduction) and abdominal aorta (75% reduction) of APP(-/-)/apoE(-/-) mice as compared to apoE(-/-). Plaques at the level of the aortic valves were not different in size, but showed a more stable phenotype in APP(-/-)/apoE(-/-) mice, as indicated by a reduced macrophage content, an increased amount of collagen and a thicker fibrous cap. CONCLUSION: Our findings provide evidence that lack of APP attenuates atherogenesis and leads to plaque stability.

Uncoupling protein 2-mediated thermogenesis in vulnerable atherosclerotic plaques.

Timely detection of vulnerable atherosclerotic plaques is of the utmost importance to prevent acute clinical events, such as unstable angina, myocardial infarction and sudden death. Advanced plaques show a dense infiltration of macrophages, of which a subpopulation strongly expresses uncoupling protein 2 (UCP2). This protein uncouples adenosine triphosphate (ATP) production from mitochondrial respiration and thereby converts the loss of potential energy in heat production. In early atherosclerotic lesions, UCP2 seems to fulfil an atheroprotective effect by reducing reactive oxygen species (ROS) production and/or by inhibiting monocyte recruitment. Because the amount of macrophages in these lesions is limited, effects on plaque temperature cannot be detected. As the macrophage content of the plaque increases and the plaque progresses toward an unstable phenotype, ROS production is overwhelming, and possibly cannot be counteracted by the antioxidant properties of UCP2. However, the increasing number of UCP2-positive macrophages inside advanced plaques correlates with increased plaque temperature. This thermogenic effect can be detected by intravascular thermography and may be indicative of rupture-prone regions. Thus, UCP2 expression in macrophages in advanced plaques can be considered a biochemical link between plaque temperature and vulnerability.

mRNA but not plasmid DNA is efficiently transfected in murine J774A.1 macrophages.

Previous studies demonstrated that macrophages are difficult to transfect. In the present study, we investigated whether J774A.1 macrophages can be efficiently transfected using nucleofector technology. Nucleofection of J774A.1 macrophages with mRNA resulted in transfection efficiencies up to 75% without cell death as compared to control pulsed macrophages. In contrast, introduction of DNA into J774A.1 cells caused apoptosis without expression of the gene of interest. Our results show that mRNA nucleofection is a new high-speed transfection method for macrophages.