Usefulness of the microcolorimetric method of XTT reduction for evaluation of intracellular killing of pathogens

The aim of this study was to evaluate a XTT assay in testing the killing activity of mouse phagocytes in vitro. Live microorganisms converted XTT to water soluble orange formazan in the presence of CQ. Absorption of formazan measured at 492 nm was directly related to the number of viable cells. The percentage of staphylococci killed by granulocytes and macrophages was 10-40% (1 h- and 2 h-respectively), in the cultures containing 10 bacteria/phagocyte. Killing of Candida albicans (1-3 blastospors/phagocyte) was seen after 2 h of incubation. The percentage of Listeria and Mycobacterium killed by phagocytes depended on pathogenicity of the tested strains. The bactericidal activity of phagocytes estimated in the XTT assay and by the CFU method was quite similar.     

In vivo verification of in vitro model of antibiotic treatment of device-related infection

Device-related infections are difficult to treat with antibiotics alone. Standard susceptibility tests do not correlate with treatment success. Therefore, the utility of a pharmacokinetic in vitro model has been evaluated in comparison with the tissue-cage infection model in guinea pigs. The bactericidal activity of 28 treatment regimens has been studied by using three different test strains. In vitro efficacy was defined as reduction in the number of suspended or adherent bacteria, and in vivo efficacy was defined as reduction in the number of bacteria in tissue-cage fluid. Test results between the two models (in vivo and in vitro) correlated well, with correlation coefficients of 0.85 for in vivo efficacy versus in vitro efficacy against suspended bacteria and 0.72 for in vivo efficacy versus in vitro efficacy against adherent bacteria (P < 0.05) for Staphylococcus aureus, 0.96 and 0.82 (P < 0.05) for Staphylococcus epidermidis, and 0.89 and 0.97 for Escherichia coli, respectively. In contrast, standard susceptibility tests, ratios of MICs to trough or peak levels, ratios of the area under the curve to the MIC, or time above the MIC were not predictive for therapeutic outcome in either the in vitro or in vivo model. In both models, the bactericidal activity levels with combination regimens were significantly higher than those with single-drug regimens (P < 0.001). Furthermore, rifampin combinations with either vancomycin, teicoplanin, fleroxacin, or ciprofloxacin were significantly more bactericidal against adherent bacteria than netilmicin combinations with vancomycin or daptomycin (P < 0.01). Thus, in vivo verification of the pharmacokinetic in vitro model correlated well with the animal model. The in vitro model offers an alternative to ther animal model in experiments that screen and assess antibiotic regimens against device-related infections.     

Decreased susceptibility to antibiotic killing of a stable small colony variant of Staphylococcus aureus in fluid phase and on fibronectin-coated surfaces

The frequency of small colony variants of staphylococci associated with persistent, antibiotic resistant and relapsing infections is probably underestimated. These variants demonstrate decreased metabolism, leading to slow growth, increased resistance to cell-wall-active antibiotics, and decreased uptake of aminoglycoside antibiotics. This altered phenotype arises from defects in menadione and haemin biosynthesis resulting in impaired electron transport and decreased ATP concentrations. The recent acquisition of a stable small colony variant (SCV strain JB1), generated from strain 6850 of Staphylococcus aureus, allowed us to study the susceptibilities to antibiotic killing of parent and variant strains. Because differences in susceptibilities have been found between unattached and surface-adherent organisms, we tested both strains in solid and fluid-phase assays. Suspensions of SCV strain JB1 exposed to 8 x MIC of either oxacillin, vancomycin or fleroxacin, exhibited lower reductions in viable counts than the parent strain 6850, especially when high bacterial concentrations (1-2 x 10(7) cfu/mL) of either strain were tested. Susceptibility to antibiotic killing of bacteria attached to fibronectin-coated coverslips was markedly influenced by their growing or nongrowing state on the surface. In the latter condition, surface-bound SCV organisms were highly resistant to the bactericidal action of oxacillin or vancomycin in contrast to the parental strain which was normally eliminated by each antimicrobial agent. In conclusion, the decreased susceptibility of the stable SCV strain of S. aureus to bactericidal concentrations of antibiotics may help to explain the persistence of such organisms in chronic infections.     

Biomaterial-associated staphylococcal peritoneal infections in a neutropaenic mouse model

Adhesion of staphylococcal cells to polyethylene with end point-attached heparin was quantified by bioluminescence. Staphylococcus epidermidis 3380 and the slime-producing S. epidermidis RP12 adhered to the highest extent, and S. lugdunensis 2342 to the least extent. Preincubation of the polymer with dialysis fluid reduced adhesion of S. epidermidis 3380 and RP12 but enhanced that of S. aureus, and preadsorption of the surface with fibronectin decreased subsequent adhesion of S. epidermidis and S. haemolyticus strains. When staphylococci were grown in the presence of a biomaterial their ability to activate peritoneal cells was decreased. The bactericidal activity was impaired, whereas ingestion of opsonized coagulase-negative staphylococci (CNS) strains was unaffected. With S. epidermidis RP12 the presence of biomaterial did not influence either phagocytosis or bactericidal effect of peritoneal cells. After intra-peritoneal challenge with staphylococcal strains, the organ uptake of S. aureus Cowan 1 was increased in normal mice whereas immunosuppressed mice died. CNS strains increased mainly in the peritoneal cavity of immunosuppressed mice. The uptake of bacteria in liver and kidneys was increased with S. epidermidis 3380, S. lugdunensis 2343 and S. schleiferi 667-88. Generally, CNS strains persisted in the peritoneal cavity of both normal and immunosuppressed mice. These data indicate that host defense mechanisms, mainly polymorphonuclear neutrophils, fail to eliminate CNS infections in the peritoneum, and that initial adhesion to an implanted biomaterial may be of lesser importance in the peritoneal cavity than in e.g. catheter-associated infections. There are strain-specific virulence factors of bacteria, and slime producing strains evade the host defense mechanisms more efficiently than non-slime producing strains.     

Penicillin G-induced microbicidal activity of endothelial cells cultured on gelfoam blocks

A body of evidence has surfaced documenting the ability of endothelial cells cultured on monolayers to phagocytose but not kill bacteria. Several years ago, a new three-dimensional endothelial cell culturing model was developed, which simulated the morphology of the endothelium in small vessels and capillaries. Given that endothelial cells may be derived from the same pluripotent stem cells as macrophages, the question of whether endothelial cells might phagocytose and kill bacteria was explored. Endothelial cells grown on Gelfoam blocks exhibited bactericidal activity towards Staphylococcus aureus, reaching maximal killing of > 90% after 2 h. Evidence documents the involvement of bacterial adherence to the plasma membrane of the endothelial cell. This is followed by phagocytosis of S. aureus, leading to intracellular killing. Penicillin G, included in the endothelial cell growth medium, was found to be a critical factor in the bactericidal activity demonstrated by Gelfoam blocks laden with endothelial cells.     

Intraoperative modulation of alveolar macrophage function during isoflurane and propofol anesthesia

BACKGROUND: Alveolar macrophages are a critical part of the defense against pulmonary infection. Thus the authors determined time-dependent changes in alveolar macrophage functions in patients having surgery who were anesthetized with isoflurane or propofol. METHODS: Patients anesthetized with propofol (n = 30) or isoflurane (n = 30) during orthopedic surgery were studied. Alveolar macrophages were harvested by bronchoalveolar lavage immediately, and 2, 4, and 6 h after induction anesthesia and at the end of surgery. The fraction of aggregated and nonviable macrophages was determined. Then phagocytosis was measured by ingestion of opsonized and unopsonized particles. Finally, microbicidal activity was determined as the ability of the macrophages to kill Listeria monocytogenes directly. RESULTS: Demographic and morphometric characteristics of the patients given propofol and isoflurane were similar, as were their levels of pulmonary function and hemodynamic responses. The fraction of alveolar macrophages ingesting opsonized and unopsonized particles, and the number of particles ingested, decreased significantly over time, with the decrease slightly but significantly greater during isoflurane anesthesia. Microbicidal function decreased progressively during anesthesia and surgery, with the decrease almost twice as great during isoflurane compared with propofol anesthesia. The fraction of aggregated macrophages and recovered neutrophils increased over time in the patients given each anesthetic. CONCLUSIONS: Pulmonary immunologic function changed progressively during anesthesia and surgery. The data from this study suggest that pulmonary defenses are modulated by the type of anesthesia and by the duration of anesthesia and surgery.     

Amphotericin B-induced resistance to Pseudomonas aeruginosa infection in mice

We evaluated the effects of amphotericin B (AmB) against Pseudomonas aeruginosa (P. aeruginosa) infection in mice. Pretreatment with 2 mg/kg of AmB 24 hours before infection significantly increased the survival rates of mice intraperitoneally infected with either P. aeruginosa or Escherichia coli. To evaluate the mechanism of this AmB-induced resistance to infection, we conducted a number of experiments. Peritoneal macrophages exposed in vitro to AmB showed superior bactericidal activity compared to that of control macrophages. Interleukin-1 production by peritoneal macrophages from mice pretreated with 2 mg/kg of AmB was significantly higher than that in control mice. Serum tumor necrosis factor level after intravenous injection of P. aeruginosa was also higher in mice pretreated with 2 mg/kg of AmB than in control mice. These data indicate that AmB induces resistance to P. aeruginosa in mice. Furthermore AmB-induced activation of peritoneal macrophages and their production of interleukin-1 and tumor necrosis factor appeared to play important roles in this phenomenon.     

Comparison of neuronal response patterns in the external and central nuclei of inferior colliculus during ethanol administration and ethanol withdrawal

It is known that murine macrophages produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma), and NO mediates the tumoricidal activity of activated macrophages. The present study was designed to investigate whether the intercellular adhesion is necessary for activated rat alveolar macrophages to exert the full cytotoxic effects. Rat alveolar macrophages produced NO dose dependently in response to either LPS or IFN-gamma, and caused DNA fragmentation in rat type II pneumocytes transformed with SV40 (SV40T2). Chemically produced NO also caused the DNA fragmentation and viability loss in SV40T2, and both of them were inhibited by a NO radical scavenger. The cytotoxicity of activated macrophages was reduced by NG-monomethyl-L-arginine, a competitive nitric synthase inhibitor, and neither superoxide dismutase nor catalase modulated the cytotoxicity. Although alveolar macrophages stimulated with either LPS or IFN-gamma caused DNA fragmentation of SV40T2, only LPS increased the intercellular adherence between macrophages and SV40T2. The intercellular adhesion was reduced by both anti-CD18 and anti-CD11a. However, those antibodies did not affect the cytotoxicity of LPS-stimulated macrophages. These results clearly indicate that NO-mediated cytotoxicity is caused predominantly by diffusion of NO, and the beta 2 integrin-mediated intercellular adhesion does not play an important role, if any, in activated macrophage-mediated cytotoxic effects on SV40T2.     

Synergistic effects of mineral fibres and cigarette smoke on the production of tumour necrosis factor by alveolar macrophages of rats

The objective of this study was to evaluate the combined effects of mineral fibres and cigarette smoke on the production of tumour necrosis factor (TNF) by alveolar macrophages. Rats were exposed to cigarette smoke in vivo, and production of TNF by alveolar macrophages was measured in the presence of mineral fibres in vitro. For smoke exposure, rats were divided into two groups. Five were exposed to a daily concentration of 10 mg/m3 of cigarette smoke for an eight hour period, and five rats (controls) were not exposed to smoke. Bronchoalveolar lavage was performed after exposure to smoke and the recovered alveolar macrophages were incubated with either chrysotile or ceramic fibres on a microplate for 24 hours. Activity of TNF in the supernatant was determined by the L-929 fibroblast cell bioassay. When alveolar macrophages were not stimulated by mineral fibres, production of TNF by rats exposed to smoke and unexposed rats was essentially the same. When alveolar macrophages were stimulated in vitro by chrysotile or ceramic fibres, production of TNF by alveolar macrophages from rats exposed to smoke was higher than that by alveolar macrophages from unexposed rats. The findings suggest that cigarette smoke and mineral fibres have a synergistic effect on TNF production by alveolar macrophages.     

Oxidation of low density lipoprotein by iron or copper at acidic pH

Oxidized low density lipoprotein (LDL) may play a significant role in atherosclerosis. We have investigated the effect of pH on the oxidation of LDL by iron or copper. When LDL was oxidized by iron in the presence of cysteine in either Hanks' balanced salt solution (HBSS) or Ham's F-10 medium, an acidic pH greatly decreased the lag period and increased the rate of formation of hydroperoxides and thiobarbituric acid-reactive substances (TBARS), and increased its uptake by macrophages. There was a dose-dependent increase of LDL oxidation at acidic pH in the presence of increasing concentrations of cysteine. When LDL was oxidized by copper in HBSS, an acidic pH increased the lag phase before the rapid formation of conjugated dienes, hydroperoxides, and TBARS, but increased its uptake by macrophages. Similar results were obtained using Ham's F-10 medium. Cysteine (100 microM) inhibited the modification of LDL by copper in HBSS at both pH 7.4 and 5.5 As atherosclerotic lesions may be acidic, these observations may help to explain why LDL oxidation occurs locally at these sites.     

Follicular carcinoma

In vitro assays to quantify killing of bacteria by macrophages provide useful insights into host-pathogen relations. In the present study, we used strains of Yersinia enterocolitica and Escherichia coli which varied in their ability to invade mammalian cells to evaluate these assays. The results showed that 30 min and 24 h after incubation with murine bone marrow-derived macrophages, strains of Y. enterocolitica and E. coli which expressed invasin (an outer membrane protein which allows bacteria to penetrate mammalian cells) achieved significantly greater numbers in macrophages than otherwise isogenic bacteria which lacked this protein (P < 0.01). When the 24-h data were corrected for the number of bacteria ingested by macrophages initially, the differences between invasin-positive and -negative bacteria were no longer evident (P> 0.2). This study has shown (1) that invasin-mediated penetration of macrophages by bacteria is not associated with enhanced intracellular survival, and (2) that invasion of macrophages by bacteria may influence the interpretation of assays for bactericidal capacity unless allowance is made for the number of bacteria ingested during the early phase of the assay.     

The role of alveolar macrophages in Pneumocystis carinii degradation and clearance from the lung

Although studies indicate that alveolar macrophages participate in host defense against Pneumocystis carinii, their role in organism degradation and clearance from the lung has not yet been established. We, therefore, quantified the uptake and degradation of 35S-labeled P. carinii by cultured macrophages, demonstrating significant degradation of P. carinii over 6 h. We further evaluated the role of macrophages in elimination of P. carinii from the living host. Rats received either intratracheal PBS, liposomal PBS (L-PBS), or liposomal dichloromethylene diphosphonate (L-Cl2MDP), a preparation which leads to selective depletion of macrophages. Over 72 h, L-Cl2MDP-treated animals had loss of > 85% of their alveolar macrophages. In contrast, L-PBS-treated rats had cellular differentials identical to rats receiving PBS. Macrophage-depleted rats and controls were next inoculated with P. carinii and organism clearance was determined after 24 h. P. carinii elimination was evaluated with both cyst counts and an ELISA directed against glycoprotein A (gpA), the major antigen of P. carinii. Both assays indicated that macrophage-depleted rats had substantial inpairment of P. carinii clearance compared to L-PBS- or PBS-treated rats. These data provide the first direct evidence that macrophages mediate elimination of P. carinii from the living host.     

Can ECG changes predict the long-term outcome in patients admitted to hospital for suspected acute myocardial infarction?

SETTING: The activity of KRM 1648 (KRM), a new benzoxazinorifamycin, and rifabutin (RBT), alone or in combination with clarithromycin (CLA), was evaluated against Mycobacterium avium complex (MAC) that multiplied in human alveolar macrophages (AM). DESIGN: AM were recovered by bronchoalveolar lavage, incubated in RPMI 1640 medium with 10% human AB serum, infected with four strains of MAC (of non-acquired immune deficiency syndrome [AIDS] origin), and then treated with each drug alone or in combination. After incubation for 7 days, colony forming units in each well were counted on 7H10 agar. RESULTS: Although concentrations between 0.2 microgram/ml and 20 micrograms/ml of both rifamycins showed clear dose-dependent activities against all MAC strains tested, only 20 micrograms/ml of each drug had modest bactericidal effect. In combination with 2.0 micrograms/ml of CLA, however, 0.2 microgram/ml of both drugs caused a bactericidal response against two of the four MAC strains examined. CONCLUSION: According to this human alveolar macrophage model of MAC infection, KRM and RBT in combination with CLA was found to be a promising candidate against human pulmonary MAC infection, and deserves clinical evaluation.     

Studies on transformation in Naegleria gruberi: effects of ions and bacterial suspensions

The presence of electolytes inhibited the transformation of Naegleria gruberi from amoeba to flagellate, the molarity required varying with the salt used, namely 80 mM NaCl, 90 mM KCl, 50 mM CaCl2 or 60 mM MgCl2. Non-electrolytes also prevented this transformation at 250 mM for either sucrose or glucose, and this is known to be an osmotic effect. That the effect of ionic solutions was different was demonstrated by varying the time at which the environemnt was changed from distilled water to salt solution. Experiments with suspensions of either living or heat-killed bacteria in distilled water, together with the supernatants obtained when bacteria were removed by centrifugation, showed that the inhibition of transformation which occurred in bacterial suspensions was not due to any factors produced by the bacteria and present in solution. It appeared that this inhibition was brought about by the physical presence of the bacteria, either living or heat-killed, and some possible interpretations of this 'contact' phenomenon are discussed.     

The immune response to staphylococcal antigens in mice depleted of macrophages by Cl2MDP-liposomes

To investigate the role of macrophages in the induction of the production of antibody to staphylococcal antigens, we used Cl2MDP (clodronate) liposomes as a tool for local macrophage depletion. Macrophage depletion caused in mice by intraperitoneal (i.p.) injection of Cl2MDP liposomes was associated with a reduction in the clearance of Staphylococcus aureus Cowan 1 bacteria from the tissues of infected animals and with a marked decrease in the bactericidal activity of macrophages escaping from the lethal effect of clodronate. Despite the functional defect of macrophages, the mice treated with Cl2MDP liposomes two days before the injection of alpha-toxin (toxoid) or whole heat-killed S. aureus Cowan 1 bacteria, demonstrated an enhancement in the production of anti-staphylococcal alpha-toxin IgM and anti-collagen-binding protein IgG. A similar enhancement of antistaphylococcal antibody synthesis was observed in mice after receiving phosphate buffered saline (PBS) encapsulated in liposomes.     

Nitric oxide: description of a pipeline delivery system

The effect of azelastine hydrochloride (azelastine) on synthesis and release of platelet activating factor (PAF) in alveolar macrophages obtained from asthmatic and non-asthmatic subjects was examined. Alveolar macrophages (AMs) were preincubated with or without azelastine and stimulated with f-Met-Leu-Phe (fMLP, 10 microM) for 15 min. PAF activity was detected by aggregation of washed guinea pig platelets. PAF activity released from alveolar macrophages (AMs) from asthmatics without preincubation of azelastine was 15.97 [2.17] (mean [SD], ng/10(7) cells) in supernatants and 42.52 [10.16] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF activity reduced to 10.71 [2.73] (mean [SD], ng/10(7) cells), 7.86 [0.94], and 3.52 [0.31] in the supernatants, and 35.58 [7.37], 21.57 [4.36], and 14.77 [0.99] (n = 15) in the cell pellets, respectively. PAF activity in non-asthmatic subjects without preincubation of azelastine was 8.55 [1.16] (mean [SD], ng/10(7) cells) in supernatants and 32.64 [3.37] in cell pellets. After preincubation with 10(-8), 10(-6), and 10(-4) M of azelastine, PAF activity reduced to 6.68 [0.78] (mean [SD], ng/10(7) cells), 4.47 [0.51], and 2.97 [0.36] in the supernatants, and 29.53 [3.75], 14.78 [1.95], and 6.16 [0.55] (n = 20) in the cell pellets, respectively. Our results showed that preincubation with azelastine caused a dose-dependent inhibition of intra- and extracellular PAF activity from asthmatic and non-asthmatic macrophages in the same manner.     

In vitro expression and inhibition of procoagulant activity produced by bovine alveolar macrophages and peripheral blood cells

Local and systemic activation of coagulation is frequently associated with bacterial sepsis. The coagulopathy is due, at least in part, to expression of tissue factor (TF) by monocytes and macrophages. The purpose of this study was to evaluate the expression of procoagulant activity by bovine alveolar macrophages, leukocytes and platelets, and to determine the relative potency of three chemical inhibitors of TF expression (pentoxifylline, retinoic acid, and cyclosporin A). Bovine alveolar macrophages were stimulated with lipopolysaccharide (LPS) derived from Pasteurella haemolytica or recombinant bovine tumour nervous factor (TNF) and dose- and time-dependent effects on TF expression were studied. LPS and TNF induced TF expression in alveolar macrophages and LPS treatment of whole blood induced TF expression in mononuclear cells. Neutrophils and platelets also expressed procoagulant activity, but this activity was not inhibited by anti-bovine TF monoclonal antibody. Pentoxifylline (40 mumol/L), retinoic acid (0.01 mmol/L) and cyclosporin A (0.08 mumol/L) inhibited TF expression when added concurrently with LPS or TNF, but not when added 4 h after stimulation. TF mRNA was not detected in unstimulated alveolar macrophages by Northern blot analysis. In contrast, exposure to LPS or TNF for 6 h induced marked expression of TF mRNA, which was inhibited by treatment with pentoxifylline, retinoic acid and cyclosporin A. Expression of TNF by alveolar macrophages stimulated with LPS was also inhibited by these compounds. Our results indicate that procoagulant activity expressed by alveolar macrophages and monocytes is associated with expression of TF, whereas procoagulant activity expressed by neutrophils and platelets is not. The concentrations of pentoxifylline and retinoic acid necessary for inhibition of TF expression in vitro may not be achievable in vivo owing to their toxic effects. However, the in vitro concentration of cyclosporin A that inhibited TF expression did not exceed the plasma concentration observed in humans, and therefore may be useful for inhibition of TF expression in vivo.     

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment

BACKGROUND: Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness and airway inflammation. OBJECTIVE: This study was designed to investigate the effects of the tachykinin NK1 receptor antagonist SR 140333 (Nolpitantium) and the NK2 receptor antagonist SR 48968 (Saredutant) on the activation of alveolar macrophages in the guinea-pig. METHODS: Guinea-pigs sensitized and challenged by ovalbumin administered by aerosol or naive guinea-pigs were exposed by aerosol to the neutral endopeptidase, phosphoramidon and, 15 min later, to substance P. Twenty-four hours later, bronchoalveolar lavages were performed and the cell composition of bronchoalveolar lavage fluids and the arachidonate release from alveolar macrophages stimulated in vitro with fMLP were evaluated. RESULTS: Antigen challenge in sensitized guinea-pigs induced an increase in the total number of cells and granulocytes in the bronchoalveolar lavage fluids that was not reduced by pre-treatment of guinea-pigs with a single dose of SR 140333 or SR 48968 (1 mg/kg). Substance P exposure in phosphoramidon-pretreated guinea-pigs did not induce an increase in the total number of cells. In contrast, antigen or substance P exposure induced a significant increase in the in vitro fMLP-induced arachidonate release from alveolar macrophages. Pre-treatment of the guinea pigs with SR 140333 or SR 48968 did not reduce the increase in arachidonate release from fMLP-stimulated alveolar macrophages from sensitized and challenged guinea-pigs. Pre-treatment of the animals by SR 140333 and SR 48968 reduced the enhanced arachidonate release induced by fMLP from substance P-exposed guinea-pigs. CONCLUSION: The present data demonstrate the importance of NK1- and NK2-receptor stimulation in the development of substance P-induced increased reactivity of alveolar macrophages.     

Oxysterols and TBARS are among the LDL oxidation products which enhance thromboxane A2 synthesis by platelets

In this study, we compared the effects of normal LDL (nLDL) and oxidized LDL (oxLDL) on thromboxane (TXA2) release by platelets triggered by low concentration of thrombin, and we determined which component of oxLDL is responsible for that activation. After oxidation of LDL with copper sulfate, the small molecular weight fraction (< 10 kDa) which was high in TBARS was removed; using Amicon Centriprep-10 concentrator membrane. More than 67% of TBARS in the oxLDL preparation was found in solution while the remaining was covalently attached to the oxLDL particles. OxLDL contained significantly higher levels of oxysterols and TBARS than the nLDL. Platelets preincubated with low concentrations of oxLDL (33-132 micrograms protein/mL) produced significantly higher TXA2 than platelets preincubated with equivalent concentrations of nLDL when triggered with thrombin. Platelets treated with oxLDL also contained significantly higher levels of oxysterols than platelets treated with nLDL. Platelets preincubated with pure cholestanetriol (10 micrograms/mL) contained a high level of cholestanetriol in the membrane, and TXA2 release was significantly increased in these platelets compared to the control platelets. The TBARS in solution also was very potent in enhancing TXA2 release by thrombin-treated platelets. These results indicate that oxysterols and the free TBARS either in solution or covalently attached to the oxLDL particles are partly responsible for the stimulatory effect of oxLDL on TXA2 release by platelets. The present study also showed that this enhancement of TXA2 release was due to activation of phospholipase A2 and to the increase of arachidonic acid liberation from the platelet phospholipids.