The Section has achieved significant advances in the areas of augmentation of host defense, molecular detection and pharmacology of these life-threatening infections. Immunopharmacological interactions between innate host defenses and antimicrobial agents are a key component of developing new strategies for augmenting host response against emerging or resistant pathogens. We have extensively characterized the immunopharmacological interactions between phagocytic effector cells (pulmonary alveolar macrophages, monocytes, and neutrophils) of the innate host defense and antifungal agents (polyenes, echinocandins, and triazoles) with and without immunomodulators (IFN-g, GCSF, GMCSF) against several of the key pathogens infecting oncology patients: Aspergillus fumigatus, Fusarium solani, Scedosporium spp., and Zygomycetes. These studies demonstrated new immunopharmacological mechanisms of augmenting host response. For example, we recently demonstrated that the combination of voriconazole and hyphae of Aspergillus fumigatus increases mRNA and protein expression of TLR2 and TNF-alpha, as well as NF-kappaB expression. We have further extended our work in Th1/Th2 dysimmunoregulation of invasive candidiasis to the filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp., and Zygomycetes) with particular focus on IL-15, IL-4, and TGF-beta. Reversal of the Th2 immunophenotypic expression augments host response against these organisms. Harnessing this understanding of the host response has allowed us to develop approaches for assessing expression profiles of genes encoding innate host defense molecules. We have completed the first known kinetic studies of the functional genomic response of innate host defenses of human monocytes to Candida albicans, Aspergillus fumigatus, Rhizopus oryzae, and Fusarium solani. These studies will provide fundamental guidance to investigators worldwide in understanding the coordinated transcriptional responses of their selected genes of interest for further exploration. In order to further understand the host factors contributing to infections in pediatric and adult oncology and HSCT patients, we have completed a series of complementary targeted population-based studies in collaboration with the Centers for Disease Control, Roswell Park Cancer Center, and Childrens Hospital of Philadelphia. We are initiating a multicenter clinical trial to further characterize the proteomic, gene expression, and cytokine profile in patients with focal pneumonias in order to develop a predictive prognostic model. In our ongoing efforts to understand the properties of newer antimicrobial agents for pediatric patients, we are characterizing the plasma pharmacokinetics, safety and efficacy of novel combinations of triazoles, polyenes, and echinocandins, and other antimicrobial agents in predictive rabbit models of experimental pulmonary aspergillosis, subacute disseminated candidiasis, and hematogenous candida meningoencephalitis, as well as pulmonary zygomycosis, scedosporiosis, and fusariosis. In exploring new molecules, our sustained collaboration with the USDA has yielded a series of novel water-soluble agriculturally derived plant molecules with potent antimicrobial activity. We have harnessed powerful mathematical modeling systems to more accurately understand the complicated interaction between double and triple combinations of antimicrobial compounds. We demonstrated through a series of in vitro and in vivo studies with Bliss surface modeling and Loewe additivity models that certain combinations of compounds may be highly antagonistic against experimental invasive pulmonary aspergillosis and probably should not be used in immunocompromised patients. We are able to detect significant interactions documenting synergy or antagonism as a guide to clinical trial development. As a continuity of our efforts to advance the understanding of the infectious diseases supportive care in pediatric oncology patients, we have systematically continued our studies of the safety and plasma pharmacokinetics of key systemic antifungal agents. These studies provide a rational basis for selection of correct dosages that provide plasma levels comparable to those of adults. These studies have demonstrated that approximately one-half of all agents studied required new dosage adjustments in order to optimize plasma-concentration time curves in our pediatric oncology patients. These studies lay the foundation for new adaptive pharmacotherapy strategies to improve outcome, particularly in profoundly immunocompromised pediatric patients where host response is abrogated and clearance of infection dependent upon the principally upon compound. We developed an in vitro model of the human alveolus, consisting of a bilayer of human alveolar epithelial and endothelial cells. An A. fumigatus strain expressing green fluorescent protein was used to study the invasion of the cell bilayer was studied using confocal and electron microscopy. The kinetics of culture, PCR and galactomannan were determined by a series of differential equations. Galactomannan was used to measure the antifungal effect of macrophages and amphotericin B. A mathematical model was developed and results bridged to humans. Galactomannan levels were found to be inextricably linked to Aspergillus invasion, and were a robust measure of the antifungal effect of macrophages and amphotericin B. This new model provides a strategy by which relationships between pathogenesis, immunological effectors and antifungal drug therapy for invasive pulmonary aspergillosis may be further understood. As the diagnosis and therapeutic monitoring of hematogenous Candida meningoencephalitis (HCME) in children is very difficult, we developed a new biomarker of (1-->3)-beta-D-glucan (beta-glucan) in CSF and plasma in non-neutropenic animal models of experimental HCME. CSF beta-glucan assay was significantly more were sensitive than CSF or blood cultures in diagnosis and detection of therapeutic response. Clearance of C. albicans from blood cultures was not predictive of eradication of organisms from CNS; conversely, beta-glucan levels in CSF were predictive of therapeutic response. Initial clinical data suggest that CSF beta-glucan may be a useful biomarker for detection and monitoring of therapeutic response in HCME in children. In order to improve the design and implementation of qPCR for the detection of deeply invasive candidiasis, we also developed a more comprehensive understanding of the kinetics of DNA from C. albicans in vitro and in vivo. These studies of the kinetics of DNA-release by C. albicans collectively demonstrated that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable fraction for detection of C. albicans DNA. Using the rRNA regions, we also have developed a qPCR system for the detection of the key organisms causing life-threatening pulmonary zygomycosis, an emerging infection in patients with cancer and HSCT. These studies have led to the first circulating molecular biomarker of mucormycosis in our four models of invasive pulmonary zygomycosis including Rhizopus, Mucor, and Cunn [summary truncated at 7800 characters]
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