We evaluated the effect of the combination of micafungin and polymorphonuclear leukocytes (PMN) against hyphae of Candida albicans and Candida dubliniensis. Micafungin enhanced the PMN oxidative burst dose dependently. The combination was synergistic (C. albicans) or additive (C. dubliniensis); when PMN were pretreated with granulocyte-macrophage colony-stimulating factor, the combination was more effective.When infecting parenchymal tissues, both Candida albicans and Candida dubliniensis switch between blastoconidia and hyphae (10). This constitutes an important virulence factor, probably because hyphae are too large for phagocytosis. Within the tissue, then, the hyphae will be subject both to extracellular attack by phagocytes, mainly neutrophils or polymorphonuclear leukocytes (PMN), and to the drug that is being administered. Therefore, the effects of the combination of new antifungal drugs with the fungicidal functions of these immune cells are clinically relevant. Micafungin (MCFG) is a new candin already licensed in Japan for systemic use. In this study, we have evaluated the activity of MCFG on the PMN oxidative burst and the fungicidal activity of a combination of MCFG with PMN, pretreated or not with granulocyte-macrophage colony-stimulating factor (GM-CSF), against hyphae of C. albicans and C. dubliniensis.The strains used were C. albicans ATCC 90028 and C. dubliniensis CBS 7987. Fungi were seeded from frozen stocks on Sabouraud agar (Merck, Darmstadt, Germany) plates and grown for 3 days. Five colonies were transferred to 10 ml of liquid Sabouraud medium and incubated overnight at 35°C with shaking. After being washed, blastoconidia were counted on a hemocytometer and their concentration was adjusted to 10 ϫ 10 6 blastoconidia/ml of Hanks' balanced salt solution (HBSS) containing 50% fetal bovine serum. Following incubation at 37°C with shaking for 30 to 60 min, 95% of the Candida cells showed pseudohyphae.Drug. MCFG was kindly donated by Fujisawa GmbH, Mü-nich, Germany. MCFG was dissolved at 0.5 mg/ml in saline solution and stored at Ϫ24°C. For the fungicidal-activity experiments, the concentration of 0.05 g/ml was selected in order to achieve a low, but constant, fungicidal effect.Superoxide anion release assay. The oxidative burst evidenced by the production of O 2 Ϫ was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c.Freshly purified PMN were obtained from the German Cancer Research Center, Heidelberg, Germany. Buffy coats from healthy donors were centrifuged over a Ficoll layer, the pellet was resuspended in phosphate-buffered saline, and the erythrocytes were sedimented by use of dextran. Remaining erythrocytes were lysed by incubating the cells in lysis buffer (155 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA; pH 7.4) at 4°C for 15 min. After being washed, the cells were resuspended in HBSS without Ca 2ϩ or Mg 2ϩ and counted on a hemocytometer. PMN were then added at a final concentration of 10 6 cells/ml to 300 l of a suspension containing 50 M ferricytochrome c (fro...
