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Despite the major impact of ROS on human health, their quantification remains difficult and requires an analytical approach, such as the EPR spin trap technique. In this study, a comparative EPR analysis of different macrophage types stimulated for superoxide and nitric oxide production was performed. U937 monocytes, J774A.1, RAW 264.7 and primary mouse (PMM) macrophages were included. In contrast to the U937 cells, all macrophages produced significant EPR signals after stimulation. The use of PMA as stimulator and CM-H as spin probe led to the highest response in EPR signals for detection of O(2)(.-) as nitroxide radical. A combination of LPS and IFN-gamma and the spin trap [Fe(DETC)(2)] turned out to be the best combination for the production and detection of intracellular NO spin adducts. In conclusion, this study established practical experimental conditions for the EPR analysis of O(2)(.-) and NO produced by different types of activated macrophages.

SUMMARYLeishmaniaparasites are able to survive in the macrophage, one of the most hostile environments of the vertebrate host. The present study investigated howLeishmaniainfection influences these host cell defence mechanisms. Macrophages were infected with antimony-susceptible and -resistantLeishmaniastrains. Free radical production inLeishmania-infected macrophages was measured by electron paramagnetic resonance. Apoptosis was detected with fluorescence microscopy using Annexin-V FITC labelling and with Western blotting to detect caspase-3 cleavage. Independent of their drug susceptibility profile or species background, all studiedLeishmaniastrains induced a similar increase in free radical production in macrophages. O2●−production was significantly elevated during phagocytosis of the stationary phase promastigotes. Conversely, NO levels increased later in the infection and none of the strains induced capsase-3 cleavage.Leishmania donovaniinfection led to phosphatidylserine externalization only in RAW 264.7 cells. After an initial burst of O2●−during phagocytosis of promastigotes, amastigotes protect themselves by decreasing the O2●−production to the basal level. An increased NO production was observed 6 h after infection. Finally, induction of cell death is probably not essential in the survival of the parasite within the macrophage.

BackgroundDendritic cells (DCs), professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS). A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (mo)DCs and monocytes in response to oxidative stress.MethodsOxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min). Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H2DCFDA). Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels.Results Tert-BHP increased CM-H2DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress–related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2), an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation.ConclusionsOur results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.

This study demonstrates that PX-6518 possesses potent and broad-spectrum prophylactic and curative efficacy against cutaneous leishmaniasis in the BALB/c mouse model. L. major was the least susceptible species tested and parasitological cure could not be obtained.

The proposed technique allows detection of very low quantities of H. pylori DNA in biological samples. In laboratory animal models, detailed monitoring of infection and complete clearance of infection can be demonstrated thanks to the low detection limit.

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