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Adherence to vascular endothelium is considered an essential step in the pathogenesis of hematogenously disseminated candidiasis. Platelets have been shown to promote Candida adherence to vascular endothelium in vitro. In contrast, recent studies indicate that platelets may also play a role in the primary host defense against endovascular infection by secretion of alpha granule-derived platelet microbicidal protein (PMP), which possesses both bactericidal and fungicidal activities as well as antiadherence properties. We examined the influences of PMP and the antifungal agent fluconazole on the adherence of Candida albicans to rabbit platelets, as measured by quantitative flow cytometry. In the absence of PMP and fluconazole, adherence of C. albicans to platelets was rapid (complete within 1 min), saturable, and reversible. Following 2 h of exposure to fluconazole at 10x the MIC, platelet binding of C. albicans was substantially reduced (mean reduction, 32.1%; P = 0.08). Similarly, exposure of C. albicans to PMP (range, 0.5 to 5 micrograms/ml) for 2 h (but not 30 min) significantly reduced candidal adherence to platelets 43.1 to 62.1%; (reduction range, P < 0.05). Moreover, exposure of C. albicans to PMP (5 micrograms/ml for 30 min) and then fluconazole (10x the MIC for 2 h) further decreased candidal adherence to platelets in comparison with the adherence after exposure to either agent alone (mean reduction, 57.2%; P = 0.02 and 0.05, respectively). These data demonstrate that PMP and fluconazole individually reduce the ability of C. albicans to bind to platelets in vitro and that the antiadherence activities of fluconazole are augmented by PMP.

Since stress both activates the sympathoadrenal axis and profoundly affects inflammation and inflammatory diseases, many of which are sexually dimorphic, we tested whether the effect of stress on neutrophil recruitment, a primary component of the acute inflammatory response, is sexually dimorphic. The effect of intermittent sound (over 4 days), a nonhabituating stress, on lipopolysaccharide (LPS)-induced recruitment of neutrophils was evaluated in vivo in the rat air pouch model. At 24 h following the last stress exposure, LPS-induced neutrophil recruitment was enhanced in male rats, but not in females. When gonadectomized prepubertally and tested as adults, stress significantly inhibited the magnitude of LPS-induced neutrophil recruitment in males, while it still had no effect in gonadectomized females. In males, following adrenal denervation, the increase in LPS-induced neutrophil recruitment produced by stress was prevented. Since these data suggest that the effect of stress is dependent on the sympathoadrenal axis, we tested the hypothesis that catecholamines mediate the stress effects. In male rats, the effect of stress on LPS-induced neutrophil recruitment was significantly attenuated by continuous administration of the beta-adrenergic receptor antagonist, propranolol (4 mg kg(-1) day(-1)), during sound stress exposure, and administration of isoproterenol (10 nmoles, i.v.) significantly increased neutrophil recruitment in males, an effect that was qualitatively and quantitatively similar to the effect of stress. Propranolol significantly increased neutrophil recruitment in nonstressed female rats, but did not significantly affect neutrophil recruitment in stressed females. These findings indicate a marked male sex hormone-dependent sexual dimorphism in the sympathoadrenal-dependent effect of stress on neutrophil migration, a primary component of the inflammatory response, and suggest that the sympathoadrenal axis contributes to this effect via release of epinephrine.

Bacterial adherence to platelets on the cardiac valve surface is believed to be critical in the induction of infective endocarditis. Recent studies have confirmed that thrombin-activated platelets secrete platelet microbicidal protein (PMP), which can both kill and exert nonlethal antiadherence effects against endovascular pathogens. In the present study, we quantified the influence of antibiotic and/or PMP exposures on in vitro platelet adherence of two Staphylococcus aureus strains, identical by DNA restriction and cell wall protein profiles, that differed in their susceptibility to PMP-induced killing (PMPs or PMPr, respectively). Adherence assays were performed by flow cytometry in the presence of sublethal PMP concentrations (1 to 2.5 micrograms/ml) alone or in combination with ampicillin (AMP) alone, sulbactam (SUL) alone, or AMP plus SUL (AMP-SUL), at levels achievable in serum. Exposure of the PMPs and PMPr S. aureus strains to antibiotics (for 2 h at 37 degrees C) prior to flow cytometry resulted in no substantive changes in the percent adherence to platelets compared with that for S. aureus cells not exposed to antibiotics, except for modestly increased adherence of both PMPs and PMPr cells exposed to AMP-SUL (18.5 and 15.8% increases, respectively). Addition of PMP to antibiotic-S. aureus mixtures (final 30 min) caused a significant decrease in S. aureus adherence to platelets, for both the PMPs and PMPr S. aureus strains, compared with antibiotic exposure alone (e.g., reduction in platelet adherence from 57.9 +/- 8.2% to 12.2 +/- 3.6% for PMPs cells exposed to AMP-SUL and PMP [P = 0.01]). Moreover, addition of PMP following exposure of the PMPs and PMPr strains to AMP-SUL reversed the enhanced bacterium-platelet adherence observed with such antibiotic exposures alone (P < or = 0.005). These data demonstrate that PMP exerts a potent antiplatelet adherence effect which is independent of its microbicidal capacity, rendering S. aureus cells less adherent to platelets in the presence or absence of antibiotics. Reduction of microbial adherence to platelets by PMP alone or with antibiotics provides further insight into the mechanism(s) that may be involved in host defense and antibiotic prophylaxis of infective endocarditis and other endovascular infections.

Streptococcal exopolysaccharides are major virulence factors in the pathogenesis of endocarditis. They promote bacterial adherence to valves and subsequent vegetation formation. Since platelet binding and aggregation by streptococci are postulated mechanisms for endocardial colonization and vegetation production, the effect of exopolysaccharide on binding and aggregation was evaluated by flow cytometry and aggregometry. Streptococcus salivarius D1, a minimal exopolysaccharide producer, bound human platelets extensively (86.8% of bacteria bound by 1 min). S. Salivarius M13 and M15 and Streptococcus mitis M4 produced larger amounts of exopolysaccharide and bound platelets significantly less (52.6%, 51.2%, 52.8%, respectively). Exopolysaccharide also inhibited platelet aggregation: Strains with minimal exopolysaccharide aggregated platelets maximally, while strains with extensive exopolysaccharide failed to induce aggregation. Removal of exopolysaccharide by shearing restored aggregation by these latter strains. Thus, exopolysaccharides can inhibit the binding and aggregation of platelets by streptococci. The virulence associated with exopolysaccharide may result from the inhibition of platelet-mediated interactions that limit disease progression.

Quantitative analyses of Staphylococcus aureus binding to platelets were done using flow cytometry after bacterial exposure to the following treatments: proteases (trypsin, protease K), antibiotics (oxacillin, gentamicin), surface carbohydrate modifiers (sodium periodate, anticapsular antibody), or platelet microbicidal protein. In separate studies, platelets were exposed to a monoclonal antibody to their Fc receptor (Fc gamma RII) before binding was quantified. The percentage of bacteria bound to platelets varied significantly among strains (22.1% +/- 3.8% to 76.4 +/- 3.2%). For all isolates, binding to platelets was rapid, saturable, and reversible, suggesting a receptor-ligand interaction. The following modifiers significantly reduced binding: platelet microbicidal protein (by 32.1% +/- 5.2%; P less than .001), homologous (but not heterologous) anticapsular antibody (by 17.7% +/- 1.9%; P less than .05), sodium periodate (by 36.3% +/- 4.3%; P less than .005), and anti-platelet Fc monoclonal antibody (by 41.5% +/- 4.4%; P less than .002). Collectively, these data suggest that the mechanism(s) involved in S. aureus-platelet binding are complex and multimodal, involving carbohydrate-rich and platelet microbicidal protein-susceptible S. aureus surface ligands as well as the platelet Fc receptor.