1 Third Paediatric Department, Aristotle University, Hippokration Hospital, Thessaloniki GR-54642; 2 First Propedeutic Department of Medicine, Athens University, Athens GR-11527, Greece; 3 Immunocompromised Host Section, Paediatric Oncology Branch, National Cancer Institute, Building 10, Room 13N240, Bethesda, MD 20892, USA
Received 29 July 2002; accepted 9 August 2002
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Abstract |
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Keywords: amphotericin B formulations, Scedosporium, polymorphonuclear leucocytes
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Introduction |
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Deoxycholate AMB (AMBDC), a polyene that interacts with the fungal cell membrane, causing cell death, is often considered the gold standard of antifungal agents. However, its administration is frequently complicated by dose-limiting nephrotoxicity. AMB lipid complex (ABLC) and liposomal AMB (LAMB) are lipid formulations developed to reduce these nephrotoxic complications. Both formulations have fewer adverse reactions than conventional AMBDC.
The main host defences against filamentous fungi consist of tissue and circulating phagocytes. Polymorphonuclear leucocytes (PMNs) are the main mediators of host defence against hyphae of filamentous fungi.4 Hyphae of S. prolificans are damaged by PMNs in a dose-responsive manner.5 AMBDC has variable effects on phagocytes; these have been studied by several investigators,68 including the stimulatory effects of AMBDC at clinically relevant concentrations on macrophages and immunostimulatory effects in mice.8
Improvement of antifungal therapy against Scedosporium spp. is urgently needed. Whether the combination of AMB lipid formulations enhances the antifungal effect of PMNs against these emerging pathogens is not known. We therefore investigated the antifungal effects of human PMNs and AMB lipid formulations alone and in combination against S. prolificans and S. apiospermum.
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Materials and methods |
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The isolate of S. prolificans CM906 used for these studies is the type strain CBS 465.74, stored in the Instituto de Salud Carlos III, Madrid, Spain (kindly donated by Dr Juan Luis Rodriguez-Tudela), which was originally isolated from a case of osteomyelitis. S. apiospermum SA1216 was isolated from a biopsy of an infection of the leg. Both isolates caused fatal infections in experimental animals. The inocula were prepared as described previously.5
AMBDC (Bristol-Myers Squibb, La Grande Nord, Paris, France), ABLC (Liposome Company, Inc., Princeton, NJ, USA) and LAMB (Gilead Sciences, San Dimas, CA, USA) were used. For S. prolificans, the final concentrations of AMBDC, ABLC and LAMB were 0.625, 0.125 and 0.625 µg/mL, and for S. apiospermum they were 0.312, 0.062 and 0.625 µg/mL, respectively. These concentrations were selected as the most appropriate for use from separate doseresponse XTT experiments. In these experiments, different drug concentrations were mixed with hyphae of the two Scedosporium spp. and incubated according to the method described below. The drug concentrations chosen to be used in combination with PMNs were those that achieved <50% activity against the hyphae alone.
The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide sodium salt (XTT dye; Sigma, St Louis, MO, USA) plus coenzyme Q (2,3-dimethoxy-5-methyl-1,4-benzoquinone; Sigma) assay was used as described previously.5 A suspension containing 7.5 x 104 conidia/mL in 200 µL yeast nitrogen base supplemented with 2% glucose (YNB) was plated in each well of a 96 flat-bottomed well cell culture cluster (Corning Inc., New York, NY, USA) and incubated for 18 h at 32°C. YNB was then replaced by RPMI 1640 supplemented with 10% pooled human serum; PMNs were added to appropriate wells at an effector/target ratio of 5:1, and antifungal agents were added at specified concentrations. After incubation at 37°C with 5% CO2 for 3 h, PMNs were lysed by washing three times with H2O and shaking for 5 min at room temperature before adding 150 µL of PBS containing 0.25 mg/mL XTT plus 40 µg/mL coenzyme Q. After incubation at 37°C with 5% CO2 for 1 h, the wells were aspirated dry and 100 µL aliquots were transferred and read in a spectrophotometer at 450 nm. Antifungal activity was calculated as percentage hyphal damage = (1 X/C) x 100, where X is the optical density of test wells and C is the optical density of control wells with hyphae only.
Each experiment was performed with cells of one donor and by use of duplicate or quadruplicate wells for each condition. The average value of these replicate wells was taken as the value for this particular donor/experiment. The averages of each experiment were then used to calculate the mean ± standard error of mean (S.E.M.). Differences between mean values were statistically evaluated by Wilcoxon or MannWhitney non-parametric test, as appropriate. A P value of <0.05 indicated significance.
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Results |
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The effect of ABLC in combination with PMNs on both organisms was also studied (Figure 1b). The antifungal activity of the combination of ABLC and PMNs against S. prolificans and S. apiospermum was significantly greater as compared with PMNs or drug alone. For example, while PMNs damaged 46.4 ± 4.5% of S. prolificans hyphae, the addition of ABLC with PMNs damaged 56.8 ± 4.9%, achieving an enhancement of 22% (P = 0.031). Similarly, while PMNs alone damaged 37.4 ± 8% of S. apiospermum hyphae, the combination of ABLC and PMNs damaged 68 ± 3.6%, achieving an enhancement of 81% (P = 0.013).
The hyphal damage produced by the combination of LAMB and PMNs was also greater than that produced by the drug alone (Figure 1c). Hyphal damage of S. prolificans was 46.7 ± 6.1% for the combination compared with 13.8 ± 5.7% for LAMB alone (P < 0.001). S. apiospermum hyphal damage was 39.3 ± 8.9% with the combination compared with 23.1 ± 9.4% for LAMB alone; however, this difference did not reach significance. The effect of the combination of LAMB and PMNs was similar to that of PMNs alone on both S. prolificans and S. apiospermum hyphae.
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Discussion |
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AMB forms barrel-like pores, which allow ions to traverse the lipid bilayer. This results in depletion of ion gradients leading to electrolyte leakage and cell death. Other proposed mechanisms include lipid peroxidation, inhibition of membrane enzymes, blockade of endocytosis and immune stimulation.8 In the case of ABLC, AMB is concealed within ribbon-like structures. The release of lipases by the fungus breaks down the ribbon-like structures, thus releasing AMB and increasing its local concentration. This could enhance local antifungal activity of AMB and increase the permeability of the fungal membrane to PMN fungicidal products. If Scedosporium spp. release lipases, an enhanced delivery of drug and PMN fungicidal products to the hyphal membrane could ensue, which might account for the additive effect observed.
The complex ribbons may also exert a direct effect on PMNs (e.g. activating them further or increasing their antifungal functions) or on hyphae, rendering them more susceptible to PMNs. AMB has been found to increase the killing of phagocytosed A. fumigatus conidia and Candida albicans blastoconidia by phagocytes. In addition, it enhances their adherence6,7,10 and reduces cell viability. The effects of AMB on superoxide anion production are variable; some authors have found priming or enhancement,7 whereas others have found an inhibitory effect6 or no effect.10
Little is known about the effects of ABLC or LAMB on superoxide anion production by PMNs. As a first attempt to address the mechanism of this combination result, the effect of the three AMB formulations on the oxidative burst of PMNs was studied in our laboratory. None of these formulations affected the superoxide anion produced by PMNs, either unstimulated or stimulated by formylmethionyl-leucylphenylalanine (data not shown). Regardless of the mechanism(s) of these additive effects, the findings of additive antifungal action of ABLC and PMNs described here suggest that interactions between phagocytes and pharmaceutical compounds merit exploration against other filamentous fungi that cause life-threatening infections.
Although granulocyte transfusions are controversial, encouraging reports of efficacy of transfusions of granulocyte-colony stimulating factor-mobilized granulocytes in patients with mycoses refractory to antifungal chemotherapy11 lend support to this strategy in Scedosporium spp. infection. Improved PMN function through withdrawal of immunosuppressants or through enhancement by recombinant cytokines may optimize the management of non-neutropenic patients with infection due to these emerging pathogens.
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Acknowledgements |
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Footnotes |
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References |
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