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Oxidized low-density lipoproteins play important roles in the development of atherosclerosis and contain several lipid-derived, bioactive molecules which are believed to contribute to atherogenesis. Of these, some cholesterol oxidation products, referred to as oxysterols, are suspected to favor the formation of atherosclerotic plaques involving cytotoxic, pro-oxidant and pro-inflammatory processes. Ten commonly occurring oxysterols (7alpha-, 7beta-hydroxycholesterol, 7-ketocholesterol, 19-hydroxycholesterol, cholesterol-5alpha,6alpha-epoxide, cholesterol-5beta,6beta-epoxide, 22R-, 22S-, 25-, and 27-hydroxycholesterol) were studied for both their cytotoxicity and their ability to induce superoxide anion production (O2*-) and IL-8 secretion in U937 human promonocytic leukemia cells. Cytotoxic effects (phosphatidylserine externalization, loss of mitochondrial potential, increased permeability to propidium iodide, and occurrence of cells with swollen, fragmented and/or condensed nuclei) were only identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, which also induce lysosomal destabilization associated or not associated with the formation of monodansylcadaverine-positive cytoplasmic structures. No relationship between oxysterol-induced cytotoxicity and HMG-CoA reductase activity was found. In addition, the highest O2*- overproduction quantified with hydroethidine was identified with 7beta-hydroxycholesterol, 7-ketocholesterol and cholesterol-5beta,6beta-epoxide, with cholesterol-5alpha, 6alpha-epoxide and 25-hydroxycholesterol. The highest capacity to simultaneously stimulate IL-8 secretion (quantified by ELISA and by using a multiplexed, particle-based flow cytometric assay) and enhance IL-8 mRNA levels (determined by RT-PCR) was observed with 7beta-hydroxycholesterol and 25-hydroxycholesterol. None of the effects observed for the oxysterols were detected for cholesterol. Therefore, oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever.

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Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, "adaptive" resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P. aeruginosa. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.Aminoglycosides remain invaluable antibiotics in the treatment of severe infections caused by Pseudomonas aeruginosa. However, their in vivo efficacy may be compromised by the development of transiently resistant subpopulations (7,8,15,34). Exposure of susceptible P. aeruginosa to an aminoglycoside classically results in an early and rapid drug concentrationdependent killing followed by a phase of bacterial refractoriness characterized by a slow drug concentration-independent killing (27). This so-called adaptive resistance, which is distinct from the postantibiotic effect and which disappears when the organism is no longer in contact with the aminoglycoside, has been observed in vitro (5,11,19), in animal models of infection (12,40), and in patients with cystic fibrosis (6). Because of its ephemeral and reproducible nature, adaptive resistance is not believed to result from mutational events. Ribosomal alterations and drug inactivation are not considered plausible mechanisms, either, as they would result in specific patterns of susceptibility to aminoglycosides and not cross-resistance to these antibiotics (11). Reduced intracellular accumulation of aminoglycosides, which is concomitant to adaptive resistance, was first interpreted as the consequence of lower drug uptake across the bacterial envelopes (11,19). Supporting this assumption, pleiotropic changes in the protein profiles of the cytoplasmic membrane were detected in drug-exposed bacteria by some investigators (19). However, clear evidence for a substantial decrease in aminoglycoside transport across the inner membrane could not be obtained. The membrane potential ⌬ (the driving force for drug entry) appears to be marginally diminished in adaptively resistant bacteria (19), a finding which agrees well with the observation that surviving bacteria grow normally during the postexposure refractory phase (11,19). These characteristics are opposite of those of another drugrecalcitrant subpopulation also selectable by aminoglycosides, composed of energy-deficient variants (also called small-colony variants) (7, 34).Recently, severa...