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Objectives: Tissue concentrations of amphotericin B were determined in autopsy material of patients who had been treated with liposomal amphotericin B or amphotericin B colloidal dispersion (colloidal amphotericin B) for suspected or proven invasive fungal infection., Patients and Methods: Amphotericin B tissue levels were measured in liver, spleen, lung, kidney, and myocardial and brain tissue of 20 patients who had been treated with lipid-formulated amphotericin B, before they died from multi-organ failure. Seven patients had been treated with liposomal amphotericin B (AmBisome) and thirteen with colloidal amphotericin B (Amphocil). Tissue samples were obtained during routine autopsy, homogenized and extracted with methanol. Amphotericin B concentrations were measured using HPLC after purification by solid phase extraction., Results: The highest amphotericin B levels were found in liver and spleen, followed by kidney, lung, myocardium and brain. In the lung higher amphotericin B concentrations were found after treatment with amphotericin B colloidal dispersion than after therapy with liposomal amphotericin B., Conclusions: The choice of lipid formulation may influence amphotericin B penetration into the lung.

Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti-endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation-blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C-based treatment of severe sepsis.

Background: Eosinophil infiltration is a characteristic feature of allergic inflammation. Allergic responses are associated with local activation of the coagulation pathway and accumulation of fibrin., Objective: We tested whether protein C and activated protein C (APC), which are endogenous anti-inflammatory coagulation inhibitors, affect eosinophil function., Methods: Eosinophils were from venous blood of healthy donors. Cell migration and apoptosis were studied by using micropore filter assays and fluorometry, respectively. Receptor expression was investigated by means of RT-PCR and SDS-PAGE of immunoprecipitated protein., Results: Protein C and APC had no significant chemotactic effects on eosinophils. Eosinophils pretreated with protein C or APC showed significantly reduced migration toward chemoattractants. No effect of either protein C preparation was seen in eosinophil apoptosis assays. The inhibiting effect on migration was reversed by an antibody against the endothelial protein C receptor (EPCR). Synthesis of EPCR by eosinophils is suggested by demonstration of receptor mRNA expression and detection of metabolically labeled receptor protein., Conclusions: Data suggest that an EPCR is expressed by eosinophils whose activation with protein C or APC arrests directed migration. Protein C-affected eosinophil chemotaxis is a novel thrombin-independent component of the protein C pathway.