The activities of voriconazole, posaconazole, caspofungin, and anidulafungin against Candida albicans and Candida parapsilosis biofilms were evaluated. In contrast to planktonic cells, the MICs for voriconazole and posaconazole for the biofilms of the two species were high (>256 and >64 mg/liter, respectively) but relatively low for the echinocandins caspofungin and anidulafungin (<1 and <2 mg/liter, respectively).Candida spp. have frequently been associated with the formation of biofilms on biological and inert surfaces, such as intravascular catheters (15). Candida albicans and Candida parapsilosis are the most prevalent species related to the biofilm mode of growth (12). Resistance of Candida biofilms to conventional antifungal agents has been previously documented (2, 10, 16). Given that biofilm-associated infections are very difficult to cure without device removal, the demand for newer, more effective therapies has been developed.The goal of the present study was to examine the activities of newer antifungal agents against C. albicans and C. parapsilosis biofilms and compare them to their corresponding planktonic cells. Voriconazole (VRC; Pfizer, Groton, CT), posaconazole (PSC; Schering-Plough, Kenilworth, NJ), caspofungin (CAS; Merck, Whitehouse Station, NJ), and anidulafungin (AND; Pfizer), were examined.Documented biofilm-producing strains were used, including C. albicans M61, C. albicans GDH2346, and C. parapsilosis PA/71 (4, 5). Aliquots were maintained in 25% glycerol and 75% peptone at Ϫ35°C.Planktonic MICs were determined according to the Clinical and Laboratory Standards Institute M27-A2 method. MICs were determined as the lowest drug concentrations at which a prominent decrease in turbidity was observed, corresponding to ca. 50% inhibition in growth (8-10, 13). The MICs were recorded after incubation for 24 h.Biofilm MIC determination was based on modifications of methods previously described (5, 10, 16). Biofilms were grown on the surface of silicone elastomer disks (Bioplexus Corp., Saticoy, CA), pretreated with fetal bovine serum (Gibco, Paisley, Scotland), in 12-or 96-well plates for 48 h for C. albicans strains and 72 h for C. parapsilosis. In an effort to make our adopted model resemble in vivo conditions, organisms were grown on the surface of a silicone substrate coated with a fetal bovine serum-conditioning film under constant linear shaking (4). Mature biofilms were then incubated in RPMI 1640 containing VRC, PSC, CAS, or AND at doubling dilutions ( Fig. 1 and 2) for 24 h. Drugfree biofilms containing only RPMI 1640 served as controls. Four replicate biofilms were used for each condition. Biofilm MICs were determined as the minimum antifungal drug concentrations that caused Ն50% reduction in the metabolic activity of the biofilms compared to controls (10). Biofilm formation and antifungal activities were assessed by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide (XTT; 0.25 mg/ml) and coenzyme Q (40 g/ ml) assay spectrophotometrically at 450 nm with a reference wa...

The immunomodulatory effects of liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD) on mRNA and protein profiles of five cytokines and chemokines expressed by human monocyte-enriched mononuclear leukocytes (MNCs) were comprehensively evaluated by semiquantitative reverse transcription-PCR and enzyme-linked immunosorbent assays; they were compared to those of deoxycholate amphotericin B (DAMB). mRNAs of interleukin-1␤ (IL-1␤), IL-1 receptor antagonist (IL-1ra), tumor necrosis factor alpha (TNF-␣), monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 1␤ (MIP-1␤) were assessed after treatment of MNCs with each drug for 0.5, 2, 6, and 22 h. The cytokine protein profiles were obtained after incubation of MNCs with the drugs for 2 h (TNF-␣) or 6 h (all the others). In the mRNA studies, DAMB resulted in an early increase of inflammatory cytokines or chemokines IL-1␤, TNF-␣, MCP-1, and MIP-1␤ (2 to 6 h) and in a late increase of antiinflammatory IL-1ra (22 h). ABCD showed a general similar trend of inflammatory gene up-regulation. LAMB and ABLC decreased or did not affect IL-1␤ and TNF-␣, whereas ABLC additionally decreased MIP-1␤. In protein measurement studies, DAMB and ABCD up-regulated production of IL-1␤ (P < 0.05), decreased the IL-1ra/IL-1␤ ratio, and up-regulated the production of MCP-1 and MIP-1␤. In comparison, LAMB and ABLC down-regulated or did not affect the production of these cytokines/chemokines compared to untreated MNCs; furthermore, ABLC tended to increase the IL-1ra/IL-1␤ ratio. These studies demonstrate that amphotericin B formulations differentially affect gene expression and release of an array of proinflammatory and antiinflammatory cytokines that potentially may explain the differences in infusion-related reactions and dosedependent nephrotoxicity as well as modulation of the host immune response to invasive fungal infections.Historically, deoxycholate amphotericin B (DAMB) has been considered the "gold standard" of antifungal therapy, and it remains the drug with the broadest antifungal spectrum (21, 25). However, DAMB causes adverse infusion-related reactions and dose-dependent nephrotoxicity, which are clearly associated with increased morbidity in immunocompromised patients (1,13,19). The lipid-based amphotericin B formulations liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD) have been developed with the goal to decrease toxicity and improve drug tolerance and thus efficacy (17, 38). Patients with neutropenia and invasive fungal infections developed infusion-related adverse reactions less frequently with LAMB than with ABLC and ABCD, whereas nephrotoxic tolerability was improved with all three lipid formulations compared to with DAMB (8,17,22,41).In vivo, amphotericin B-related toxicity has been previously correlated with increased levels in plasma of interleukin-1␤ (IL-1␤), tumor necrosis factor alpha (TNF-␣), and IL-1 receptor antagonis...

The aim of this study was to evaluate the pharmacokinetics and efficacy of posaconazole (PSC) in combination with granulocyte colony-stimulating factor (G-CSF) in a neutropaenic murine model of disseminated zygomycosis (mucormycosis) due to Rhizopus microsporus. Male BALB/c mice were rendered neutropaenic with cyclophosphamide (200 mg kg(-1), intraperitoneally) administered on days -1 and +5 postinfection. Mice were infected with R. microsporus (5 × 10(4) spores ml(-1)) intravenously. Mice were treated with PSC (40 mg kg(-1) day(-1) by gavage) or G-CSF (300 μg kg(-1) day(-1) subcutaneously) or with the combination of PSC and G-CSF. The fungal burden was assessed by culturing the brain, liver, kidneys and lungs. Blood levels of PSC were measured by high performance liquid chromatography. The survival rates were 33%, 27% and 31% for PSC-treated-, G-CSF-treated- and PSC + G-CSF-treated mice, respectively, as compared to 18% for the controls (P = NS). PSC monotherapy and combination therapy significantly reduced the fungal burden in the kidneys, but not in the rest of the organs. Combination therapy was not superior to PSC monotherapy in terms of either survival or reduction in fungal burden. Serum concentrations of PSC were well-above the MIC of PSC for the particular isolate. PSC monotherapy has a modest efficacy against R. microsporus in reducing fungal burden in neutropaenic mice. Combining G-CSF with PSC does not substantially affect the antifungal activity of PSC.

These findings suggest that AMBF have enhancing effects of variable degree on phagocyte-induced hyphal damage of A. fumigatus and F. solani. Other fungicidal mechanisms, perhaps non-oxidative, are more likely to mediate these immunomodulatory effects of AMBF on host defence against the two medically important filamentous fungi.

Invasive aspergillosis is the most frequent opportunistic filamentous fungal infection causing excessive morbidity and mortality in immunocompromised patients (9, 12). Mononuclear phagocytes constitute a prominent component of the host defense against Aspergillus spp. (24). In particular, NADP (NADPH)-dependent production of antifungal compounds, such as superoxide anion (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and H 2 O 2 -dependent intracellular intermediates (DIIs), contributes to phagocyte-induced damage of microorganisms including fungi (2,8,16). While O 2 Ϫ production is an early event of oxidative burst, H 2 O 2 and DIIs consisting of compounds such as hydroxyl radical and HOCl are produced at late steps and are even more powerful oxidizing species.For decades, deoxycholate amphotericin B (DAMB) has been considered to be the cornerstone of antifungal therapy for fungal infections including invasive aspergillosis (1, 21). However, due to frequent infusion-related reactions and doselimiting nephrotoxicity, less-toxic lipid-associated formulations, such as liposomal amphotericin B (LAMB), amphotericin B lipid complex (ABLC), and amphotericin B colloidal dispersion (ABCD), have been developed (5,7,15). These compounds appear to offer a better therapeutic index than DAMB, circumscribing excessive toxicity (5, 7). Although newer azoles and echinocandins have been added to the antifungal armamentarium, amphotericin B formulations remain important agents against invasive aspergillosis.While lipid formulations of amphotericin B differ in their degree of induction of infusion-related reactions, little is known about their immunomodulatory effects when each of them is combined with phagocytes against Aspergillus fumigatus. Specifically, DAMB and ABLC were found to additively augment the fungicidal activity of pulmonary alveolar macrophages against conidia of A. fumigatus (17). However, the studies of the effects of DAMB on oxidative burst of phagocytes as evidenced by O 2 Ϫ production have shown contradictory results (18,22,25,26). To date, there are no reports published on the comparative effects of the four formulations on the antifungal activity of monocytes (MNCs) against A. fumigatus. We therefore investigated whether DAMB, LAMB, ABLC, and ABCD enhance the antifungal activities of monocytes against hyphae of A. fumigatus, as evidenced by monocyte-mediated hyphal damage, production of O 2 Ϫ , and production of H 2 O 2 as well as secondary DIIs, both in response to A. fumigatus.