The influence of low doses of amphotericin B on the capacity of human monocytes to kill Candida albicans was investigated. Killing rates were quantified by a novel flow cytometric assay and were found to be 37% 3% (standard error of the mean) after 3 h. Preincubation of monocytes for 6 to 20 h with low concentrations of amphotericin B (0.2 ,ug/ml) Despite its toxicity, amphotericin B has remained the prime drug for treating life-threatening fungal infections. This agent binds with high affinity to ergosterol. Its fungicidal action is generally ascribed to an enhancement of membrane permeability as a result of the formation of ion channels (5, 16) and perhaps also because of oxidative damage of membrane constituents (4, 6). Unfortunately, amphotericin B also binds to cholesterol in mammalian cell membranes and elicits severe toxic effects. Many efforts have therefore been devoted to establishing treatment regimens that combine the merits of antifungal activity with a minimum of untoward side effects. The possibilities of local amphotericin B application are also receiving increased attention. Aerosol application, first described nearly 20 years ago (23), and low-dose intravenous application are emerging as prophylactic measures against pulmonary fungal infections in neutropenic patients (13,20,29).General belief holds that the beneficial effect of amphotericin B derives mainly from its direct fungicidal action. In addition, the drug reportedly can act on macrophages to stimulate [3H]thymidine uptake (5) and the respiratory burst (34, 35), promote tumor necrosis factor secretion (9), and enhance the tumoricidal (8, 26) and microbicidal (3,24,26,32) actions of these cells. The mechanisms underlying these processes and the relevance thereof remain unclear.We performed with a high degree of accuracy (18,19). The assays were first introduced for quantifying granulocyte phagocytic function and have now been adapted to the analysis of monocytes. In the studies described here, we showed that monocytes preincubated for hours with low doses of amphotericin B acquire a remarkably augmented capacity to kill ingested Candida albicans. This effect appears to be due to the intracellular accumulation of the drug rather than to a nonspecific stimulation of the cells. Potentiation of the fungicidal capacity of monocytes is likely to be an important novel determinant underlying the therapeutic efficacy of amphotericin B. MATERIALS AND METHODSMicroorganisms. An isolate of C. albicans from our diagnostic laboratory or an amphotericin B-resistant mutant, kindly provided by H. Dermoumi, Essen, Germany, was cultured at 37°C in tryptic soy broth for 16 to 20 h. A total of 1.5 ml of yeast cell cultures was centrifuged and the pelleted cells were resuspended in 1 ml of saline and incubated with 2 ,uM bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester (BCECF-AM; Becton Dickinson, Heidelberg, Germany) for 30 min in an Eppendorf Thermomixer 5436
Two colorimetric methods that use Alamar Blue or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for assaying the in vitro activities of antifungal agents have been described. We report that both tests performed similarly when the antifungal activity of amphotericin B against Candida albicans was determined. However, only the MTT test generated interpretable data when Aspergillus fumigatus was used. Because conventional in vitro susceptibility testing of fungi is fraught with methodological pitfalls, the recent development of colorimetric tests based on measurements of metabolic activity is both welcome and promising. Alamar Blue is an oxidation-reduction indicator for eukaryotic cells, and its utility for assessing the viabilities of yeasts has been demonstrated (1, 8-10). A second approach, based on the detection of mitochondrial activity, was first described by Tellier et al. (12), who used 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5carboxanilide in a macrodilution assay for Candida albicans. An improved microtiter assay based on a menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reaction was subsequently developed in our laboratory (5). The test appeared to be applicable to both C. albicans and Aspergillus fumigatus. Information on the utility of the Alamar Blue test for assessing the activities of antifungal agents against A. fumigatus is lacking, so a comparison of the two tests was undertaken in the present study. C. albicans 0815 and 8166 were from our diagnostic laboratory. Strain R64, exhibiting a relative resistance to amphotericin B, was provided by H. Dermoumi, Institute of Microbiology, Essen, Germany, and strain 3059 was a gift from E. Liehl,
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