Iron Chelators as antifungal drugs: Based on our previous years investigations identifying the iron-chelating protein, lactoferrin, as an important host defense against aspergillosis, we have been studying the ability of synthetic iron chelators to inhibit fungal growth. In FY11, we completed our tests of the efficacy of ciclopirox and lactoferrin as invidual prophylactic antifungal drugs in experimental aspergillosis in immunosuppressed WT and in CGD mice (collaboration with June Kwon-Chung (LCID). Host defenses against Granulibacter bethesdensis We have continued to study Granulibacter bethesdensis, a recently described bacterial pathogen of CGD patients. We have found that Granulibacter is remarkably hypostimulatory of the human innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. Partial purification and structural characterization of an atypical lipopolysaccharide (LPS) of this organism continue but already indicate a 2,3-diamino-2,3-dideoxy-D-glucose (DAG) lipid A backbone (as opposed to the far more common glucosamine backbone). DAG containing Lipid A have been reported to have many unusual signaling properties. Furthermore, KDO, a core sugar present in most LPS, was not detected. In collaboration with Yossi Shiloach (NIDDK), we developed large-scale growth methods for this slow-growing and low-biomass organism that allowed generation of sufficient cell paste for LPS extraction and pending, high-resolution structural studies (with Russ Carlson at the University of Georgia Complex Carbohydrate Research Center). We have also found that G. bethesdensis is remarkably resistant to complement and antimicrobial peptides and that, unlike most microbes, Granulibacter inhibits neutrophil apoptosis. We are examining the pathways involved in this inhibition and attempting to ascertain whether this plays a role in pathogenesis. MicroRNA expression in normal and CGD neutrophils: Steady-state levels of many mRNAs differ between normal and CGD leukocytes and it is thought that some of the clinically apparent inflammatory and immune problems seen in these patients may stem from these differences. Recently, a group of small regulatory RNA species (miRNA) has been implicated in post-transcriptional fine-tuning of mRNA stability. We hypothesize that some of the inflammatory problems seen in patients with CGD might be due to different expression of miRNA and the subsequent dysregulation of mRNA levels. To address this, we are analyzing miRNA levels in normal and CGD neutrophils by both deep sequencing and microarray approaches. Results of this study are pending and will be compared to known differences in mRNA expression between these cells. Identification of such regulatory circuits may be useful in recognizing (without prior biases) potential targets for therapeutic intervention. Lipid Mediator Biology in CGD: Several studies have demonstrated that metabolism of lipid mediators is deranged in CGD, specifically that in this disease, there is a decrease in prostaglandin D2, and increase in leukotriene B4, and a decrease in lipid peroxidation. In vivo, lipid-laden histiocytes have long been seen in CGD. We are undertaking a systematic study of lipid metabolism in CGD leukocytes to identify what other mediators, for example the newly described resolvin class of anti-inflammatory lipids might be altered in CGD.
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