Assessment of linezolid prescriptions in three French hospitals.

The use of linezolid to treat gram-positive cocci infections is increasing in France. Linezolid is approved in pneumonia and complicated skin and soft tissue infections. Overuse and misuse of linezolid can favor the emergence and spreading of linezolid-resistant strains. We aimed to assess the appropriateness of linezolid use in French hospitals. This is a multicenter, retrospective study conducted in three tertiary care hospitals. Appropriateness of linezolid indications and adequacy (composite score concerning dosage, route of administration and blood monitoring) were assessed. Over a three-month period, all prescriptions of linezolid were extracted and analyzed by two independent infectious disease experts. Among the 81 initial prescriptions that were evaluated, indication was appropriate in 48% of cases. Among those, 51% complied with international guidelines. Fifty-seven percent of the prescriptions were adequate regarding dosage, route of administration and blood monitoring. Overall, 23% of prescriptions combined both appropriateness and adequacy. The most frequent reasons for inappropriateness were the possibility of choosing narrower-spectrum antibiotics and the empirical use of linezolid in severe sepsis or septic shock. Initial treatment was the most frequently appropriate in bone and joint infection cases (p = 0.001). Our study shows that even if modalities of use were mostly correct, appropriateness of linezolid indications is low. Educational programs are mandatory to improve practices, as well as clinical studies to better assess the efficacy and safety of linezolid in clinical situations other than pneumonia or complicated skin and soft tissue infections.

Pefabloc, 4-[2-aminoethyl]benzenesulfonyl fluoride, is a new, potent nontoxic and irreversible inhibitor of PAF-degrading acetylhydrolase.

We report here that 4-[2-aminoethyl]benzenesulfonyl fluoride (Pefabloc SC, Pefabloc), a new irreversible serine proteinase inhibitor, efficiently inhibits both human and rat platelet activating factor (PAF)-degrading acetylhydrolase (acetylhydrolase). Indeed, low concentrations of Pefabloc (0.1 mM) rapidly and totally inactivate both human plasma-, VLDL-, IDL-, LDL- and HDL-associated acetylhydrolase, and in addition, acetylhydrolase synthesized and released by human adherent monocytes in culture, as well as rat brain cytosolic acetylhydrolase. By contrast, Pefabloc only minimally inhibited the phospholipase A2 (PLA2) activity from Naja naja and from porcine pancreas. In addition, Pefabloc is relatively nontoxic, stable and convenient to use. Henceforth, Pefabloc may replace both DFP and PMSF and therefore constitutes a useful and valuable tool in future studies of acetylhydrolase.

Phagocytic activation induces formation of platelet-activating factor in human monocyte-derived macrophages and in macrophage-derived foam cells. Relevance to the inflammatory reaction in atherogenesis.

Monocyte-derived macrophages and macrophage-derived foam cells in arterial tissue may undergo phagocytic activation and thereby contribute to an inflammatory reaction. We have investigated the effect of phagocytic activation on the formation of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF-acether, PAF), a proinflammatory phospholipid, in human monocyte-derived macrophages (macrophages) and in cholesterol-loaded macrophage foam cells (foam cells). Adherent human monocyte-derived macrophages were transformed into foam cells upon incubation with acetylated low-density lipoproteins (Ac-LDL). Such foam cells characteristically displayed a markedly increased content of cholesteryl esters compared with macrophages (4.3 +/- 1.3 microgram/microgram DNA and 0.2 +/- 0.3 microgram/microgram DNA, n = 5, respectively). After phagocytic stimulation with serum-opsonized zymosan (OPZ), both macrophages and foam cells synthesized PAF transiently with maximal production (0.5-1.1 pmol PAF/microgram DNA, n = 5, corresponding to 4.0-8.8 pmol PAF/10(6) cells, as assessed by bioassay) occurring approximately 15 min after stimulation. A major fraction of the synthesized PAF remained cell-associated; such PAF was composed mainly of the hexadecyl (16:0 PAF, approximately 75%) and the octadecenyl (18:1 PAF) species and of trace amounts of octadecyl (18:0 PAF), as assessed by reverse-phase liquid chromatography. Addition of exogenous 16:0 lyso-PAF alone triggered PAF formation (0.9-1.7 pmol PAF/microgram DNA, after 15 min of cellular stimulation); simultaneous cellular stimulation with OPZ and 16:0 lyso-PAF increased PAF formation in an additive manner. Acetyltransferase, the enzyme which acetylates the precursor lyso-PAF and transforms it into PAF, displayed elevated activity both in macrophages and in foam cells, attaining 83-240 pmol PAF formed per min per mg DNA (n = 4); such elevated activity was not increased by OPZ-stimulation. The activity of acetylhydrolase, the PAF-degrading enzyme, was similar in macrophages and in foam cells, and varied between 120 pmol and 320 pmol PAF degraded per min per mg DNA (n = 5). Cell-associated acetylhydrolase activity was increased significantly by 40+/-15 % (P < 0.003, n = 5) after 15 - 30 min of activation with OPZ compared with non-stimulated cells and may account for the rapid decrease in cellular PAF content observed approximately 30 min after stimulation. These studies have established that metabolism of PAF in foam cells closely resembles that in macrophages, and thus PAF metabolism is largely independent of cellular cholesterol content. Moreover our data are consistent with the hypothesis that both macrophages and macrophage-derived foam cells upon phagocytic-activation constitute a significant transient source of PAF at inflammatory sites in the arterial intima where this phospholipidic mediator may exert potent proatherogenic and prothrombotic effects.

PAF-degrading acetylhydrolase is preferentially associated with dense LDL and VHDL-1 in human plasma. Catalytic characteristics and relation to the monocyte-derived enzyme.

In human plasma, platelet activating factor (PAF)-degrading acetylhydrolase (acetylhydrolase) is principally transported in association with LDLs and HDLs; this enzyme hydrolyzes PAF and short-chain forms of oxidized phosphatidylcholine, transforming them into lyso-PAF and lysophosphatidylcholine, respectively. We have examined the distribution, catalytic characteristics, and transfer of acetylhydrolase activity among plasma lipoprotein subspecies separated by isopycnic density gradient ultracentrifugation; the possibility that the plasma enzyme may be partially derived from adherent monocytes has also been evaluated. In normolipidemic subjects with Lp(a) levels < 0.1 mg/mL, acetylhydrolase was associated preferentially with small, dense LDL particles (LDL-5; d = 1.050 to 1.063 g/mL) and with the very-high-density lipoprotein-1 subfraction (VHDL-1; d = 1.156 to 1.179 g/mL), representing 23.9 +/- 1.7% and 20.6 +/- 3.2%, respectively, of total plasma activity. The apparent Km values for PAF of the enzyme associated with such lipoproteins were 89.7 +/- 23.4 and 34.8 +/- 4.5 mumol/L for LDL-5 and VHDL-1, respectively: indeed, the Km value for LDL-5 was some 10-fold higher than that of the light LDL-1, LDL-2, and LDL-3 subspecies, whereas the Km of VHDL-1 was some twofold greater than those of the HDL-2 and HDL-3 subspecies. Furthermore, when expressed on the basis of unit plasma volume, the Vmax of the acetylhydrolase associated with LDL-5 was some 150-fold greater than that in LDL-1 (d = 1.019 to 1.023 g/mL). No significant differences in the pH dependence of enzyme activity or in sensitivity to protease inactivation, sulfydryl reagents, the serine protease inhibitor Pefabloc, or the PAF antagonist CV 3988 could be detected between apo B-containing and apo A-I-containing lipoprotein particle subspecies. Incubation of LDL-1 (Km = 8.4 +/- 2.6 mumol/L) and LDL-2 (d = 1.023 to 1.029 g/mL; Km = 8.4 +/- 3.3 mumol/L) subspecies with LDL-5, in which acetylhydrolase had been inactivated by pretreatment with Pefabloc, demonstrated preferential transfer of acetylhydrolase to LDL-5. Acetylhydrolase transferred to LDL-5 from the light LDL subspecies exhibited a Km of 9.4 +/- 2.2 mumol/L, a value characteristic of the particle donors. Finally, acetylhydrolase (Km = 23.4 +/- 7.6 mumol/L) released by adherent human monocytes in culture was found to bind preferentially to small, dense LDL subspecies upon incubation of Pefabloc-inactivated plasma with monocyte supernatant.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue factor pathway inhibitor activity associated with LDL is inactivated by cell- and copper-mediated oxidation.

Human plasma contains a multivalent, Kunitz-type proteinase inhibitor termed tissue factor pathway inhibitor (TFPI), which is a specific inhibitor of the action of the factor VII(a)-tissue factor complex in coagulation. A major fraction of plasma TFPI is transported in association with LDL. Because LDL may undergo oxidation in the arterial wall during atherogenesis, we examined the effect of copper- and cell-mediated oxidative modification on TFPI activity associated with LDL. Oxidation mediated by copper ions resulted in a significant inactivation of LDL-associated TFPI (60% to 72% at 24 hours with 2.5 mumol/l CuCl2). The inactivation of TFPI was strongly negatively correlated with both an increase in the net electrical charge of LDL (r = -.80, P < or = .0001) and with the production of thiobarbituric acid-reactive substances (r = -.78, P < or = .0001) and lipid peroxides (r = -.80, P < or = .0001). Cell-mediated oxidation, involving incubation of LDL for 48 hours with either monocyte-like THP1 cells or human monocytes in Ham's F-10 medium, effected a significant decrease (64% and 75%, respectively) in LDL-associated TFPI activity. By contrast, prolonged exposure of LDL to purified soybean lipoxygenase (5000 U/mL) was less effective in inactivating TFPI (47% reduction after incubation for 72 hours at 37 degrees C). We subsequently investigated the mechanism(s) that may underlie such inactivation. Oxidation of LDL is accompanied by the generation of various aldehydes, including malondialdehyde and 4-hydroxynonenal. Chemical modification with these aldehydes revealed a significant inverse correlation between the progressive loss of TFPI activity and both the increase in net electrical charge (r = -.90, P < or = .0001) and the derivatization of free amino acid residues of LDL (r = -.90, P < or = .0001). Specific chemical modification of lysine amino groups by acetylation similarly led to inactivation of LDL-associated TFPI activity. TFPI activity was almost totally abolished (< 1.4%) when the TNBS reactivities of acetylated LDL, malondialdehyde-modified LDL, and 4-hydroxynonenal-modified LDL were 31%, 21%, and 43% that of native LDL, respectively. Our data demonstrate that expression of LDL-associated anticoagulant activity is markedly decreased as a consequence of the oxidative process, and suggest that the progressive aldehydic derivatization of apo B of LDL, and of the associated TFPI protein, may contribute to this phenomenon. Because tissue factor is overexpressed in the atheromatous plaque, it may exert a marked local procoagulant effect.(ABSTRACT TRUNCATED AT 400 WORDS)

PAF-acether-degrading acetylhydrolase in plasma LDL is inactivated by copper- and cell-mediated oxidation.

In peripheral blood, native low-density lipoprotein (LDL) is a major carrier of acetylhydrolase, the enzyme that hydrolyzes the sn-2 acetate of PAF-acether, converting it to lyso PAF-acether. By controlling the level of PAF-acether, the acetylhydrolase may regulate the biologic effects of this potent inflammatory and thrombotic mediator. The biologic oxidation of LDL appears to underlie its atherogenicity. We report here that oxidative modification of LDL led to progressive loss of associated acetylhydrolase activity. Reductions of approximately 90% and 40% of acetylhydrolase activity occurred respectively in LDL oxidized for 24 hours by copper ions (2.5 mumol/L) in phosphate-buffered saline and in LDL incubated with human monocyte-like THP1 cells in Ham's F-10 medium. Acetylhydrolase activity decreased as a function of the degree of LDL oxidation and was correlated with an increase in net negative charge and in the content of thiobarbituric acid-reactive substances (r = -.94 and r = -.88, respectively; P < or = .001). The acetylhydrolase of mildly oxidized LDL displayed a similar Km for PAF-acether compared with native LDL, whereas its Vmax was lower. Thus, acetylhydrolase conserved its affinity for PAF-acether, whereas a nondefined and noncompetitive inhibitor, apparently produced during oxidation, might account for the observed loss in enzymatic activity. Acetylhydrolase activity was totally recovered in LDL modified by both acetylation and malondialdehyde.(ABSTRACT TRUNCATED AT 250 WORDS)