Comparative study of the effect of thiabendazole and fungizone on Histoplasma capsulatum in macrophages.

The antifungal properties of Fungizone (amphotericin B intravenous solution) and thiabendazole on Histoplasma capsulatum within guinea pig macrophages were compared using the staining method and a newly developed plating method to determine the viability of intracellular H. capsulatum. The two methods were compared to determine the effectiveness of Fungizone and thiabendazole on H. capsulatum within macrophages. Fungizone was fungicidal for intracellular H. capsulatum, killing 99.9% of the yeasts at a concentration of 0.5 microgram/ml. There was some indication that non-viable intracellular yeasts were stained which could result in misinterpretation of the effectiveness of Fungizone using the staining method unless the yeasts are very closely examined for staining abnormalities. There was a good correlation between the two methods to demonstrate suppression of the multiplication of intracellular H. capsulatum in macrophages treated with 50 microgram/ml of thiabendazole. Thiabendazole was lethal for some intracellular H. capsulatum.     

The effects of amphotericin B on the interaction of Candida albicans with fibroblast cultures

Sublethal amounts of amphotericin B inhibited the interaction of Candida albicans with cultured fibroblasts. Different C. albicans clinical isolates exhibited varying degrees of sensitivity to the drug, but those isolates that were the most infective in control cultures appeared to be the most resistant to amphotericin B mediated infection inhibition. Although amphotericin B inhibited germ tube formation at the sublethal concentration of 0.3 microgram/mL, lower concentrations inhibited infection without preventing germination. The extent of this latter activity varied with the isolate and amphotericin B concentration and appeared to be related to sublethal effects on germinated yeasts. While amphotericin B effectively prevented new fibroblast infection, it did not dissociate those yeasts which had established an infection before its addition.     

Factors Affecting the Survival of Auxotrophs and Prototrophs of Saccharomyces cerevisiae in Mixed Populations

Moat, Albert G. (Hahnemann Medical College, Philadelphia, Pa.), Isabel J. Barnes, and Eleanor H. McCurley. Factors affecting the survival of auxotrophs and prototrophs of Saccharomyces cerevisiae in mixed populations. J. Bacteriol. 92:297-301. 1966.-The conditions under which the number of yeast prototrophs, as well as respiration-deficient mutants, could be materially decreased, while allowing the survival of auxotrophic mutants in recoverable numbers, have been investigated in detail. Neither the use of carbohydrates other than glucose to prevent development of respiration-deficient mutants, nor treatment with acriflavine to render all surviving wild types respiration-deficient, provided a selective advantage for the auxotrophs. Increased concentrations of the antifungal agents amphotericin B or endomycin, while reducing the number of respiration-deficient mutants, did not significantly increase the final mutant-wild type ratio. A more soluble form of amphotericin B (Fungizone), when used under carefully defined physiological conditions, produced a significant reduction in the number of surviving prototrophs relative to the surviving auxotrophs, without development of respiration-deficient mutants.     

Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells.

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.     

Effect of Amphotericin B on Dopachrome Tautomerase Activity and Other Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 μg/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173–175) for tyrosine hydroxylase.     

Evaluation of Systemic Antifungal Agents in X-Irradiated Mice

The effect of X irradiation on the survival time of animals experimentally infected with pathogenic fungi was studied, and the activity of antifungal agents in pre-irradiated hosts was evaluated. A 24-hr preinfection dose of X irradiation decreased the survival time of mice infected with Cryptococcus neoformans and Histoplasma capsulatum to a greater extent than Candida albicans or Blastomyces dermatitidis infections. Exposure to 400 r caused a significant reduction in the variation (S(2)) survival time of C. albicans or H. capsulatum mouse infections. A single 100-mg/kg dose of 5-fluorocytosine or amphotericin B administered within 24 hr postinfection significantly extended the survival time of mice infected with C. albicans. Delayed treatment with amphotericin B was effective against C. neoformans infections. Four 50-mg/kg doses of 5-fluorocytosine were more effective than a single 200-mg/kg dose against C. neoformans infections. A single dose of amphotericin B provided significant protection when administered 48 hr postinfection against B. dermatitidis in preirradiated mice. A single dose of saramycetin 48 hr postinfection was highly effective against H. capsulatum mouse infections. A 100-mg/kg dose of amphotericin B was only effective against this fungal pathogen when administered within 8 hr postinfection. In vivo activity of the antifungal agents studied was detected within 8 to 14 days. The relative in vivo activity of several antifungal agents indicated the importance of considering their individual pharmacological properties for optimum effectiveness. The experimental model used in this study should be useful for the detection and for the preclinical evaluation of new antifungal agents.     

Varying Virulence in Rabbits Infected with Different Filamentous Types of Histoplasma capsulatum

Histoplasma capsulatum filamentous primary isolates and their subcultures are separable into two distinct colonial types (A and B) having different microscopic characteristics. Yeast forms of the A and B types and the parent (P) strains from which they are derived are microscopically indistinguishable. Critically standardized inocula of living P, A, and B yeasts from one strain of H. capsulatum (G-184) were injected intravenously into 12 rabbits. Each type produced progressively debilitating disease, but in varying degrees. Of the 12 animals, 6 died within 2 to 14 weeks. A persisting copious nasal exudate, beginning at or before 1 week, was cultured weekly at 26 C on Mycosel (BBL) agar. Pure cultures of A and B filamentous type colonies were recovered from exudates of animals receiving A and B yeasts, respectively, whereas both filamentous types were isolated from rabbits injected with P yeasts, with B predominating. Only A and B yeasts thus maintained their filamentous integrity during animal passage. It was noted that dissemination of H. capsulatum through the nares of infected rabbits represents a possible hazard to laboratory personnel heretofore unrecognized. It is also a possible means of cross-infecting or sensitizing or cross-infecting and sensitizing animals housed in the same room, if A and B yeasts prove not to be antigenically identical.     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Amphotericin B susceptibility testing of Candida species by flow cytometry.

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.     

Chronic Cavitary Histoplasmosis of the Lung

Four main clinical types of disease result from infection by the fungus Histoplasma capsulatum: (1) the primary complex; (2) acute pulmonary histoplasmosis; (3) chronic cavitary pulmonary histoplasmosis; (4) disseminated systemic histoplasmosis.Chronic cavitary histoplasmosis of the lung is indistinguishable clinically and radiographically from pulmonary tuberculosis. In this disease, however, the histoplasmin skin test and the histoplasmin complement fixation test are positive in more than 80% of cases and sputum cultures are usually positive for Histoplasma capsulatum. The mortality rate of the chronic cavitary type is greater than 30%. The antibiotic, amphotericin B, is the most effective drug in treatment. If surgical resection of the cavitary lesion is feasible, under amphotericin coverage, this is the treatment of choice.During the past year, two patients with chronic cavitary histoplasmosis illustrating the aforementioned features were diagnosed at the Toronto Hospital for Tuberculosis.     

Comparative glucose utilization rates in separated and mated schistosomes

The rate of phosphorylation of 2-deoxyglucose (2DG) was determined by sequential pulsing of schistosomes (Schistosoma mansoni, S. japonicum, and S. haematobium) with 3H- and 14C-labeled 2-deoxy-D-glucose. Subsequent column chromatographic separation of the neutral [3H]2DG and [14C]2DG from the 3H- and 14C-labeled 2-deoxy-D-glucose 6-phosphate permitted estimation of the quantity of [3H]2DG phosphorylated in 2 min, and the proportion of [14C]2DG phosphorylated in 1 min; thus a phosphorylation rate was determined from a single tissue sample. The relative phosphorylation rate of 2-[3H]2DG to D-1-[14C]glucose (i.e., the phosphorylation coefficient) was also measured in male and female schistosomes. It was demonstrated that even though 2DG is taken up more rapidly than glucose, it is phosphorylated at a much slower rate in both S. mansoni and S. japonicum. In both of these species, mated males phosphorylate 2DG and glucose at a greater rate than do unmated males. Similarly, mated females phosphorylate and consume more glucose than do separated females. In contrast, the phosphorylation coefficient is greater in separated than in mated schistosomes. Intraspecific comparisons suggest that, at reduced substrate concentrations, glucose utilization rates are higher in S. japonicum, intermediate in S. mansoni, and lower in S. haematobium.     

Incidence of yeasts and influence of nystatin on their control in a group of burned children

The incidence of yeasts in the oral cavity, rectum and urine of a population of 60 children hospitalized for treatment of acute second and third degree burns was approximately the same at the time of their admission as would be expected in healthy subjects. After hospitalization, the incidence of yeasts was reduced in the intestinal tract of acute patients who received nystatin orally but increased in the oral cavity. The majority of 418 yeasts were inhibited in vitro by less than 50 units/ml nystatin and only 6 yeasts were resistant to more than 3.1 g/ml amphotericin B. The oral cavity appeared to act as a significant reservoir from which yeasts spread to cause or contribute to the deaths of 2 of 5 patients who died during the study.     

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.     

Effect of the aggregation state of amphotericin B on its interaction with ergosterol

The interaction of amphotericin B with ergosterol was studied in aqueous solutions of propanol. The mode of the interaction was found to be related to the aggregation state of amphotericin B. Ergosterol does not react (or reacts extremely slowly) with monomeric amphotericin B. Traces of a small aggregate, probably a dimer, enable a cooperative reaction. At high concentrations of the dimer, the reaction is immediate and the concentration of amphotericin B complexed with ergosterol is twice as high as the amount of added sterol. The interaction with ergosterol is hindered when the antibiotic is in micellar form. The pharmaceutical form, Fungizone, behaves similarly to the pure amphotericin B. Fungizone's greater solubility in water does not modify either the extent or the mode of interaction with ergosterol.     

Drug—induced alterations in the ultrastructural organization of Histoplasma capsulatum and Blastomyces dermatitidis

Aspects of the fine structure as seen in thin section of yeastlike cells ofHistoplasma capsulatum andBlastomyces dermatitidis exposed to polyenic antibiotics are described and illustrated by electron micrographs. The exposure of log phase yeastlike cells to minimal fungicidal concentrations of both amphotericin B (Fungizone) and hamycin resulted in detectable alterations of the plasma membrane, and, to a lesser extent, the mitochondria. WithH. capsulatum, ultrastructural changes were observed to occur within 1 h exposure to amphotericin B. Marked degenerative changes and plasmolysis were observed to occur within 6 hrs exposure of the yeastlike cells to both polyenes. The observed changes in ultrastructural appearance are compatible with the concept of binding of the polyene with membrane sterol and subsequent damage due to alterations of permeability.     

Trial of glucose versus fat emulsion in preparation of amphotericin for use in HIV infected patients with candidiasis.

OBJECTIVES--To compare the tolerance, efficacy, and pharmacokinetics of amphotericin deoxycholate (Fungizone) prepared in a parenteral fat emulsion (Intralipid 20%) or glucose in HIV patients with candidiasis. DESIGN--Non-blind randomised controlled trial. SETTING--University hospital; tertiary clinical care. PATIENTS--22 HIV positive patients with oral candidiasis. INTERVENTIONS--Amphotericin 1 mg/kg/day given on four consecutive days as a one hour infusion dissolved in either 5% glucose (amphotericin-glucose) or parenteral fat emulsion at a final concentration of 2 g/l fat emulsion (amphotericin-fat emulsion). MAIN OUTCOME MEASURES--Clinical tolerance (fever, chills, sweats, nausea, arterial pressure, and pulse rate); biological tolerance (serum creatinine, electrolyte, and magnesium values); clinical score of candidiasis; and serum concentrations of amphotericin. RESULTS--11 patients were enrolled in each group. All the amphotericin-fat emulsion infusions were given without serious problem whereas four amphotericin-glucose infusions were stopped because of renal impairment (n = 3) or severe chills (n = 2), or both. For patients completing the amphotericin-glucose treatment creatine concentration increased by 42 mumol/l; four of seven patients had at least one creatinine value > or = 133 mumol/l versus one of 11 receiving amphotericin-fat emulsion. Magnesium concentration fell significantly with amphotericin-glucose but not with amphotericin-fat emulsion. Clinical side effects were noted in 36/38 infusions with amphotericin-glucose but 10/44 with amphotericin-fat emulsion. Oral candidiasis score was reduced similarly in both groups. Serum amphotericin concentrations were significantly lower and the volume of distribution of the drug higher after infusion of amphotericin-fat emulsion than after amphotericin-glucose. CONCLUSIONS--Clinical and renal toxicity of amphotericin are reduced when the drug is prepared in fat emulsion. Preparation is simple and cost effective. Its efficacy is similar to that of conventional amphotericin.     

Accumulation of 2-deoxy-D-glucose-6-phosphate as a measure of glucose uptake in the isolated perfused heart: a 31P NMR study

The accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38-60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a K(m) of 17.5mM and V(max) of 1.4 micromol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution.     

Effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae cells. Studies by 13C-, 1H-NMR and biochemical methods

A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells. The effects of various concentrations of amphotericin B on the aerobic [1-13C]glucose metabolism in glucose-grown repressed Saccharomyces cerevisiae cells were studied as a function of time using 13C-, 1H-NMR and biochemical methods. The 13C enrichment of different compounds such as ethanol, glycerol and trehalose were determined by 1H-NMR spectroscopy. In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane. Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1 microM, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases. The 13C relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug. Thus it can be concluded that amphotericin B induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation. Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug. The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase. Without amphotericin B, this ratio (approximately 1.0) is practically constant upon addition of glucose which suggests that the activities of pyruvate carboxylase and citrate synthase are equivalent. By contrast, upon coaddition of 25 mM glucose and 1 microM amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time. It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.     

The effect of amphotericin b derivatives on Leishmania and immune functions

The effects of a water-soluble amphotericin B (AmB)-arabinogalactan (AG) conjugate on several immune functions were investigated. The experiments measured the effects of AmB-AG on (1) release of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), and interferon-gamma (IFN-gamma) from phagocytic cells and (2) cell-mediated immune responses. AmB-AG increased TNF-alpha release from mouse peritoneal macrophages and human monocytes but had no effect on IFN-gamma and NO release. A commercial preparation of nonconjugated AmB (Fungizone) also increased TNF-alpha production, but to a lesser extent than AmB-AG. AG alone had no effect on TNF-alpha production, proving that AmB caused the increased TNF-alpha production. AmB-AG and Fungizone were also tested for their effect on B- and T-cell proliferation. Neither compound altered T-lymphocyte responses to concanavalin A, but both inhibited the stimulation of B lymphocytes by lipopolysaccharides. However, Fungizone showed a stronger inhibitory effect on B cells. Allocytotoxicity was also inhibited by AmB-AG and more strongly by Fungizone. The increased production of TNF-alpha by cells treated with AmB-AG and the lower inhibitory effect of AmB-AG on lymphocyte stimulation and allocytotoxicity, as compared with Fungizone, explain the better therapeutic efficacy of the AmB-polysaccharide conjugate. AmB is active because of its preferential binding to ergosterol rather than cholesterol, the former sterol preferentially present in parasite surface membranes. This is also valid for the axenic amastigotes, which were sensitive to the AmB-AG. Overall, our results suggest that the antileishmanial activity of AmB-AG is mediated both directly and via modulation of immune functions.