Pathogenesis of Aspergillus fumigatus and the kinetics of galactomannan in an in vitro model of early invasive pulmonary aspergillosis: implications for antifungal therapy.

BACKGROUND: Little is known about the pathogenesis of invasive pulmonary aspergillosis and the relationship between the kinetics of diagnostic markers and the outcome of antifungal therapy. METHODS: An in vitro model of the human alveolus, consisting of a bilayer of human alveolar epithelial and endothelial cells, was developed. An Aspergillus fumigatus strain expressing green fluorescent protein was used. Invasion of the cell bilayer was studied using confocal and electron microscopy. The kinetics of culture, polymerase chain reaction, and galactomannan were determined. Galactomannan was used to measure the antifungal effect of macrophages and amphotericin B. A mathematical model was developed, and results were bridged to humans. RESULTS: A. fumigatus penetrated the cellular bilayer 14-16 h after inoculation. Galactomannan levels were inextricably tied to fungal invasion and were a robust measure of the antifungal effect of macrophages and amphotericin B. Neither amphotericin nor macrophages alone was able to suppress the growth of A. fumigatus; rather, the combination was required. Monte Carlo simulations showed that human dosages of amphotericin B of at least 0.6 mg/kg were required to achieve adequate drug exposure. CONCLUSIONS: This model provides a strategy by which relationships among pathogenesis, immunological effectors, and antifungal drug therapy for invasive pulmonary aspergillosis may be further understood.

Compartmentalized intrapulmonary pharmacokinetics of amphotericin B and its lipid formulations.

We investigated the compartmentalized intrapulmonary pharmacokinetics of amphotericin B and its lipid formulations in healthy rabbits. Cohorts of three to seven noninfected, catheterized rabbits received 1 mg of amphotericin B deoxycholate (DAMB) per kg of body weight or 5 mg of either amphotericin B colloidal dispersion (ABCD), amphotericin B lipid complex (ABLC), or liposomal amphotericin B (LAMB) per kg once daily for a total of 8 days. Following sparse serial plasma sampling, rabbits were sacrificed 24 h after the last dose, and epithelial lining fluid (ELF), pulmonary alveolar macrophages (PAM), and lung tissue were obtained. Pharmacokinetic parameters in plasma were derived by model-independent techniques, and concentrations in ELF and PAM were calculated based on the urea dilution method and macrophage cell volume, respectively. Mean amphotericin B concentrations +/- standard deviations (SD) in lung tissue and PAM were highest in ABLC-treated animals, exceeding concurrent plasma levels by 70- and 375-fold, respectively (in lung tissue, 16.24 +/- 1.62 versus 2.71 +/- 1.22, 6.29 +/- 1.17, and 6.32 +/- 0.57 microg/g for DAMB-, ABCD-, and LAMB-treated animals, respectively [P = 0.0029]; in PAM, 89.1 +/- 37.0 versus 8.92 +/- 2.89, 5.43 +/- 1.75, and 7.52 +/- 2.50 mug/ml for DAMB-, ABCD-, and LAMB-treated animals, respectively [P = 0.0246]). By comparison, drug concentrations in ELF were much lower than those achieved in lung tissue and PAM. Among the different cohorts, the highest ELF concentrations were found in LAMB-treated animals (2.28 +/- 1.43 versus 0.44 +/- 0.13, 0.68 +/- 0.27, and 0.90 +/- 0.28 microg/ml in DAMB-, ABCD-, and ABLC-treated animals, respectively [P = 0.0070]). In conclusion, amphotericin B and its lipid formulations displayed strikingly different patterns of disposition in lungs 24 h after dosing. Whereas the disposition of ABCD was overall not fundamentally different from that of DAMB, ABLC showed prominent accumulation in lung tissue and PAM, while LAMB achieved the highest concentrations in ELF.

Functional genomics of innate host defense molecules in normal human monocytes in response to Aspergillus fumigatus.

Aspergillus fumigatus induces the release of innate immune-related molecules from phagocytic cells early in the course of infection. Little is known, however, about the complex expression profiles of the multiple genes involved in this response. We therefore investigated the kinetics of early gene expression in human monocytes (HMCs) infected with conidia of A. fumigatus using DNA microarray analysis. Total RNA from HMCs at 0, 2, 4, and 6 h was extracted, linearly amplified, hybridized onto Affymetrix HG133 Plus 2.0 gene chips, and analyzed with an Affymetrix scanner. Changes in gene expression were calculated as a ratio of those expressed by infected versus control HMCs. Aspergillus fumigatus induced differential regulation of expression in 1,827 genes (P < 0.05). Genes encoding cytokines and chemokines involved in host defense against A. fumigatus, including interleukin-1beta (IL-1beta), IL-8, CXCL2, CCL4, CCL3, and CCL20, as well as the opsonin long pentraxin 3, were up-regulated during the first 2 to 6 h, coinciding with an increase in phagocytosis. Simultaneously, genes encoding CD14, ficolin1, and MARCO were down-regulated, and genes encoding IL-10 and matrix metalloproteinase 1 were up-regulated. Up-regulation of the genes encoding heat shock proteins 40 and 110 and connexins 26 and 30 may point to novel molecules whose role in the pathogenesis of aspergillosis has not been previously reported. Verification of the transcriptional profiling was obtained for selected genes by reverse transcription-PCR and enzyme immunoassay. Thus, A. fumigatus conidia induced a coordinated expression of genes important in host defense and immunomodulation.

Deoxycholate amphotericin B and amphotericin B lipid complex exert additive antifungal activity in combination with pulmonary alveolar macrophages against Fusarium solani.

Fusarium spp. have emerged as important causes of invasive fungal infections in immunocompromised patients. Rabbit pulmonary alveolar macrophages (PAMs) exhibited fungicidal activity against conidia of Fusarium solani and achieved a time-dependent increase in killing. Neither deoxycholate amphotericin B (DAMB) nor amphotericin B lipid complex (ABLC) exerted a suppressive effect on PAMs by decreasing their conidiocidal activity against F. solani. On the contrary, at a concentration of 0.125 microg ml(-1), ABLC and, to a lesser degree, DAMB additively augmented the fungicidal activity of pulmonary alveolar macrophages against conidia of Fusarium solani.

Use of fluorescent probes to determine MICs of amphotericin B and caspofungin against Candida spp. and Aspergillus spp.

We investigated the utility of mechanism-based fluorescent probes for determination of MICs (FMICs) of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. Amphotericin B was selected as a membrane-active antifungal agent, and caspofungin was selected as a cell wall-active agent. FMICs were also compared to the MIC determined by CLSI (formerly NCCLS) methods. Five isolates per species of Candida albicans, Candida glabrata, Candida parapsilosis, Aspergillus fumigatus, and Aspergillus terreus were studied with either amphotericin B or caspofungin. The fluorescent probes, carboxyfluorescein diacetate (CFDA) for cytoplasmic esterase activity and dihexyloxacarbocyanine iodide (DiOC6) for cell membrane potential, were each added to their respective plates. MICs and FMICs were determined in at least three separate experiments (in duplicate). Fluorescence was measured using a 96-well plate fluorometer. For amphotericin B and caspofungin, the FMIC end point was the lowest concentration of drug at which the percent growth inhibition from treated organisms versus control organisms displayed 80% inhibition for amphotericin B and 50% inhibition for caspofungin as measured by a fluorescent signal. The MIC for amphotericin B was defined as the lowest concentration of antifungal displaying no visible growth for both Aspergillus and Candida spp. The MIC for caspofungin was the lowest concentration of drug that displayed a minimum effective concentration for Aspergillus spp. For Candida spp., the MIC for caspofungin was defined as the concentration at which the antifungal agent significantly inhibits the organism. The FMICs of both antifungals, as measured by the DiOC6 membrane probe, showed good agreement (83% to 100%), within one well dilution, with the MICs against amphotericin B and caspofungin for all species. Also, the FMICs measured by the CFDA cytoplasmic esterase probe reflecting damage due to cell wall or cell membrane showed strong agreement (79 to 100%) with the MICs of both amphotericin B and caspofungin for all species. There was no significant difference in comparisons of MIC and FMIC values (P > or = 0.05). The use of fluorescent probes provides a mechanism-based method of determination of MICs of amphotericin B and caspofungin against Candida spp. and Aspergillus spp. that correlates well with standard methods.

Effects of interleukin-15 on antifungal responses of human polymorphonuclear leukocytes against Fusarium spp. and Scedosporium spp.

Fusarium spp. and Scedosporium spp. have emerged as important fungal pathogens that are frequently resistant to antifungal compounds. We investigated the effects of human interleukin-15 (IL-15) on human polymorphonuclear leukocyte (PMNL) activity against Fusarium solani and Fusarium oxysporum as well as Scedosporium prolificans and Scedosporium apiospermum. IL-15 (100 ng/ml) significantly enhanced PMNL-induced hyphal damage of both Fusarium spp. and S. prolificans after incubation for 22 h (P < 0.01) but not S. apiospermum. In addition, IL-15 enhanced PMNL oxidative respiratory burst evaluated as superoxide anion production in response to S. prolificans but not to the other fungi after 2 h incubation. IL-15 increased interleukin-8 (IL-8) release from PMNLs challenged with hyphae of F. solani and S. prolificans (P< or = 0.04). Release of tumor necrosis factor-alpha was not affected. The species-dependent enhancement of hyphal damage and induction of IL-8 release suggest that IL-15 plays an important role in the immunomodulation of host response to these emerging fungal pathogens.

Expression of genes encoding innate host defense molecules in normal human monocytes in response to Candida albicans.

Little is known about the regulation and coordinated expression of genes involved in the innate host response to Candida albicans. We therefore examined the kinetic profile of gene expression of innate host defense molecules in normal human monocytes infected with C. albicans using microarray technology. Freshly isolated peripheral blood monocytes from five healthy donors were incubated with C. albicans for 0 to 18 h in parallel with time-matched uninfected control cells. RNA from monocytes was extracted and amplified for microarray analysis, using a 42,421-gene cDNA chip. Expression of genes encoding proinflammatory cytokines, including tumor necrosis factor alpha, interleukin 1 (IL-1), IL-6, and leukemia inhibitory factor, was markedly enhanced during the first 6 h and coincided with an increase in phagocytosis. Expression of these genes returned to near baseline by 18 h. Genes encoding chemokines, including IL-8; macrophage inflammatory proteins 1, 3, and 4; and monocyte chemoattractant protein 1, also were strongly up-regulated, with peak expression at 4 to 6 h, as were genes encoding chemokine receptors CCR1, CCR5, CCR7, and CXCR5. Expression of genes whose products may protect monocyte viability, such as BCL2-related protein, metallothioneins, CD71, and SOCS3, was up-regulated at 4 to 6 h and remained elevated throughout the 18-h time course. On the other hand, expression of genes encoding T-cell-regulatory molecules (e.g., IL-12, gamma interferon, and transforming growth factor beta) was not significantly affected during the 18-h incubation. Moreover, genes encoding IL-15, the IL-13 receptor (IL-13Ra1), and CD14 were suppressed during the 18-h exposure to C. albicans. Thus, C. albicans is a potent inducer of a dynamic cascade of expression of genes whose products are related to the recruitment, activation, and protection of neutrophils and monocytes.

Efficacy, safety, and plasma pharmacokinetics of escalating dosages of intravenously administered ravuconazole lysine phosphoester for treatment of experimental pulmonary aspergillosis in persistently neutropenic rabbits.

Ravuconazole is a new antifungal triazole with broad-spectrum activity and a long half-life in plasma. We studied the antifungal efficacy, safety, and pharmacokinetics of ravuconazole lysine phosphoester in escalating dosages for the treatment of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Treatment groups consisted of rabbits treated with ravuconazole at 2.5 (RVC2.5), 5 (RVC5), and 10 (RVC10) mg/kg of body weight/day, rabbits treated with amphotericin B (AMB) at 1 mg/kg/day, or untreated controls. There was a dose-dependent reduction of pulmonary residual fungal burden (CFU per gram) in RVC5-, RVC10-, and AMB-treated rabbits in comparison to untreated controls (P < 0.01, P < 0.001, and P < 0.01, respectively). These findings correlated with progressive galactomannan antigenemia in untreated controls and the RVC2.5-treated rabbits, a lower galactomannan index (GMI) in RVC5- and RVC10-treated rabbits, and a similarly low GMI in AMB-treated rabbits (P < 0.01). Rabbits treated with RVC5, RVC10, and AMB also showed a reduction of organism-mediated pulmonary injury, as measured by infarct scores and lung weights, in comparison to untreated controls (P < 0.001). These results were supported by decreased pulmonary infiltrates detected by computed tomography in RVC5- and RVC10-treated rabbits in comparison to untreated controls (P < 0.05). Survival throughout the entire study was achieved in 95% of RVC5-treated rabbits (P < 0.001), 85% of RVC10-treated rabbits (P < 0.001), and 50% of AMB-treated rabbits (P < 0.05) in comparison to none of the untreated controls. Ravuconazole showed linear plasma pharmacokinetics and a large volume of distribution while maintaining concentrations in plasma above the MIC throughout the dosing interval. There was no evidence of hepatotoxicity or nephrotoxicity among ravuconazole-treated animals. Intravenously administered ravuconazole lysine phosphoester showed dose-dependent efficacy and an excellent safety profile for the treatment of invasive pulmonary aspergillosis in persistently neutropenic rabbits.

Human phagocytic cell responses to Scedosporium apiospermum (Pseudallescheria boydii): variable susceptibility to oxidative injury.

Scedosporium apiospermum (Pseudallescheria boydii) is an emerging opportunistic filamentous fungus that causes serious infections in both immunocompetent and immunocompromised patients. To gain insight into the immunopathogenesis of infections due to S. apiospermum, the antifungal activities of human polymorphonuclear leukocytes (PMNs), mononuclear leukocytes (MNCs), and monocyte-derived macrophages (MDMs) against two clinical isolates of S. apiospermum were evaluated. Isolate SA54A was amphotericin B resistant and was the cause of a fatal disseminated infection. Isolate SA1216 (cultured from a successfully treated localized subcutaneous infection) was susceptible to amphotericin B. MDMs exhibited similar phagocytic activities against conidia of both isolates. However, PMNs and MNCs responded differently to the hyphae of these two isolates. Serum opsonization of hyphae resulted in a higher level of superoxide anion (O(2)(-)) release by PMNs in response to SA54A (amphotericin B resistant) than that seen in response to SA1216 (amphotericin B susceptible; P < 0.001). Despite this increased O(2)(-) production, PMNs and MNCs induced less hyphal damage to SA54A than to SA1216 (P < 0.001). To investigate the potential mechanisms responsible for these differences, hyphal damage was evaluated in the presence of antifungal oxidative metabolites as well as in the presence of a series of inhibitors and scavengers of antifungal PMN function. Mannose, catalase, superoxide dismutase, dimethyl sulfoxide, and heparin had no effect on PMN-induced hyphal damage to either of the two isolates. However, azide, which inhibits PMN myeloperoxidase activity, significantly reduced hyphal damage to SA1216 (P < 0.01) but not to SA54A. Hyphae of SA1216 were slightly more susceptible to oxidative pathway products, particularly HOCl, than those of SA54A. Thus, S. apiospermum is susceptible to antifungal phagocytic function to various degrees. The selective inhibitory pattern of azide with respect to hyphal damage and the parallel susceptibility to HOCl suggests an important difference in susceptibilities to myeloperoxidase products that may be related to the various levels of pathogenicity and amphotericin B resistance of S. apiospermum.

Selective effects of interleukin (IL)-15 on antifungal activity and IL-8 release by polymorphonuclear leukocytes in response to hyphae of Aspergillus species.

The effects of interleukin (IL)-15 on human polymorphonuclear leukocyte (PMNL) activity against Aspergillus fumigatus and Aspergillus flavus were investigated. Pretreatment with IL-15 for 2 h increased PMNL oxidative burst, as measured by superoxide anion (O(2)(-)) release in response to A. fumigatus (P<.05) but not to A. flavus. However, after 22-h, but not 2-h, treatment with IL-15, there was significant enhancement in PMNL-mediated hyphal damage to A. fumigatus. Furthermore, 22-h exposure to IL-15 mediated an enhanced release of IL-8 from PMNLs challenged with hyphae of A. fumigatus and A. flavus (P<.05). In contrast, IL-15 treatment did not affect the release of tumor necrosis factor-alpha from PMNLs. The selective time- and species-dependent enhancement of O(2)(-) production and hyphal damage, as well as its induction of IL-8 release, suggest that IL-15 may play an important role in the immunomodulation of host response to invasive aspergillosis.

Experimental pulmonary aspergillosis due to Aspergillus terreus: pathogenesis and treatment of an emerging fungal pathogen resistant to amphotericin B.

Aspergillus terreus is an uncommon but emerging fungal pathogen, which causes lethal infections that are often refractory to amphotericin B (AmB). In comparison to Aspergillus fumigatus, A. terreus was resistant to the in vitro fungicidal effects of safely achievable concentrations of AmB. These in vitro findings correlated directly with resistance of A. terreus to AmB in experimental invasive pulmonary aspergillosis. Residual fungal pulmonary burden and galactomannan antigenemia demonstrated persistent infection, despite therapy with deoxycholate AmB or liposomal AmB. By comparison, posaconazole and itraconazole resolved GM antigenemia, reduced residual fungal burden, and improved survival. There were no differences in phagocytic host response to A. terreus versus A. fumigatus; however, the rate of conidial germination of A. terreus was slower. The strain of A. terreus with the highest minimum inhibitory and minimum lethal concentration of AmB also had the lowest membrane ergosterol content. The hyphae of A. terreus in vivo displayed distinctive aleurioconidia, which may be a practical microscopic feature for rapid preliminary diagnosis.

Combination therapy in treatment of experimental pulmonary aspergillosis: synergistic interaction between an antifungal triazole and an echinocandin.

Invasive pulmonary aspergillosis is an important cause of morbidity and mortality in immunocompromised patients. Simultaneous inhibition of fungal cell-wall and cell-membrane biosynthesis may result in synergistic interaction against Aspergillus fumigatus. We studied the antifungal activity of micafungin, a new echinocandin, in combination with ravuconazole, a second-generation triazole, against experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. This combination led to significant reductions in mortality (P

Immunomodulation of invasive fungal infections.

Genetic and acquired (disease- or therapy- related) host immune factors increase the risk for IFIs. In addition to antifungal drug therapy, modulation of host defenses by the use of HGFs and IFN-gamma has been supported by extensive in vitro and in vivo preclinical data. Clinical studies on the prevention or the adjunctive therapy of IFIs in combination with antifungal agents are limited, however, and do not allow specific recommendations for their cost-effective use in most of the immunodeficient settings. There is an urgent need to push forward with well-structured, randomized clinical trials to determine optimal dose, duration, and timing for different combinations of immunotherapy and antifungal agents in high-risk patients.

Macrophage colony-stimulating factor enhances phagocytosis and oxidative burst of mononuclear phagocytes against Penicillium marneffei conidia.

The responses of rabbit pulmonary alveolar macrophages (PAMs) and elutriated human monocytes (EHMs) to Penicillium marneffei, an emerging dimorphic fungus that may cause fatal disease in human immunodeficiency virus-infected patients, were studied. PAMs and EHMs comparably phagocytosed conidia of two P. marneffei strains in the presence of serum. Electron microscopy showed intraphagosomal destruction of conidia after 12 h. Serum-opsonized conidia elicited significantly more superoxide anion (O(2)(-)) release from EHMs compared to non-opsonized conidia, but equivalent O(2)(-) amounts to that elicited by serum-opsonized Aspergillus fumigatus conidia. Macrophage colony-stimulating factor (M-CSF) significantly enhanced phagocytosis of P. marneffei conidia by PAMs and EHMs, as shown by light microscopy. Moreover, M-CSF enhanced O(2)(-) production by EHMs in response to both serum-opsonized (P<0.001) and non-opsonized (P=0.03) conidia of A. fumigatus as well as conidia of the P. marneffei isolates (P<0.001 and 0.03). We conclude that M-CSF enhances phagocytosis and oxidative metabolism of mononuclear phagocytes suggesting a potential role for this cytokine in host defense against pulmonary and disseminated P. marneffei infection.

Comparative antifungal activities and plasma pharmacokinetics of micafungin (FK463) against disseminated candidiasis and invasive pulmonary aspergillosis in persistently neutropenic rabbits.

Micafungin (FK463) is an echinocandin that demonstrates potent in vitro antifungal activities against Candida and Aspergillus species. However, little is known about its comparative antifungal activities in persistently neutropenic hosts. We therefore investigated the plasma micafungin pharmacokinetics and antifungal activities of micafungin against experimental disseminated candidiasis and invasive pulmonary aspergillosis in persistently neutropenic rabbits. The groups with disseminated candidiasis studied consisted of untreated controls (UCs); rabbits treated with desoxycholate amphotericin B (DAMB) at 1 mg/kg of body weight/day; or rabbits treated with micafungin at 0.25, 0.5, 1, and 2 mg/kg/day intravenously. Compared with the UCs, rabbits treated with micafungin or DAMB showed significant dosage-dependent clearance of Candida albicans from the liver, spleen, kidney, brain, eye, lung, and vena cava. These in vivo findings correlated with the results of in vitro time-kill assays that demonstrated that micafungin has concentration-dependent fungicidal activity. The groups with invasive pulmonary aspergillosis studied consisted of UCs; rabbits treated with DAMB; rabbits treated with liposomal amphotericin B (LAMB) at 5 mg/kg/day; and rabbits treated with micafungin at 0.5, 1, and 2 mg/kg/day. In comparison to the significant micafungin dosage-dependent reduction of the residual burden (in log CFU per gram) of C. albicans in tissue, micafungin-treated rabbits with invasive pulmonary aspergillosis had no reduction in the concentration of Aspergillus fumigatus in tissue. DAMB and LAMB significantly reduced the burdens of C. albicans and A. fumigatus in tissues (P < 0.01). Persistent galactomannan antigenemia in micafungin-treated rabbits correlated with the presence of an elevated burden of A. fumigatus in pulmonary tissue. By comparison, DAMB- and LAMB-treated animals had significantly reduced circulating galactomannan antigen levels. Despite a lack of clearance of A. fumigatus from the lungs, there was a significant improvement in the rate of survival (P < 0.001) and a reduction in the level of pulmonary infarction (P < 0.05) in micafungin-treated rabbits. In summary, micafungin demonstrated concentration-dependent and dosage-dependent clearance of C. albicans from persistently neutropenic rabbits with disseminated candidiasis but not of A. fumigatus from persistently neutropenic rabbits with invasive pulmonary aspergillosis.

Amphotericin B formulations exert additive antifungal activity in combination with pulmonary alveolar macrophages and polymorphonuclear leukocytes against Aspergillus fumigatus.

Deoxycholate amphotericin B (DAMB) and amphotericin B lipid complex (ABLC) additively augmented the fungicidal activity of pulmonary alveolar macrophages against the conidia of Aspergillus fumigatus. DAMB, ABLC, and liposomal amphotericin B similarly displayed additive effects with polymorphonuclear leukocytes in damaging the hyphal elements of A. fumigatus.