Laboratory Studies of Abnormal Host Defense and Immunoregulatory Diseases

铁螯合剂作为抗真菌药物： 基于我们前几年的研究，鉴定了铁螯合蛋白，乳铁蛋白，作为一种重要的宿主防御曲霉病，我们一直在研究合成铁螯合剂抑制真菌生长的能力。 在2011财年，我们完成了环吡酮和乳铁蛋白作为单独预防性抗真菌药物在免疫抑制WT和CGD小鼠实验性曲霉病中的有效性试验（与June Kwon-Chung（LCID）合作）。 宿主对贝氏颗粒杆菌的防御 我们继续研究了最近描述的CGD患者的细菌病原体贝氏颗粒杆菌。我们已经发现，颗粒杆菌属对人先天免疫系统具有显著的低刺激性，无论是在NADPH氧化酶的弱活化方面还是在细胞因子分泌的不良刺激方面。 该生物体的非典型脂多糖（LPS）的部分纯化和结构表征仍在继续，但已经表明2，3-二氨基-2，3-二脱氧-D-葡萄糖（DAG）脂质A骨架（与更常见的葡糖胺骨架相反）。 已报道含有脂质A的DAG具有许多不寻常的信号传导性质。 此外，未检测到存在于大多数LPS中的核心糖KDO。 与Yossi Shiloach（NIDDK）合作，我们为这种生长缓慢和生物量低的生物体开发了大规模生长方法，可以产生足够的细胞糊用于LPS提取和待定的高分辨率结构研究（与格鲁吉亚大学复杂碳水化合物研究中心的Russ Carlson合作）。 我们还发现G. Bethesdensis对补体和抗微生物肽具有显著抗性，并且与大多数微生物不同，颗粒杆菌抑制中性粒细胞凋亡。 我们正在研究参与这种抑制的途径，并试图确定这是否在发病机制中发挥作用。 正常和CGD中性粒细胞中的MicroRNA表达： 许多mRNA的稳态水平在正常和CGD白细胞之间不同，并且认为在这些患者中观察到的一些临床上明显的炎症和免疫问题可能源于这些差异。 最近，一组小的调节RNA种类（miRNA）已被牵连在转录后微调mRNA的稳定性。我们推测，CGD患者的一些炎症问题可能是由于miRNA的不同表达和随后的mRNA水平失调。 为了解决这个问题，我们正在通过深度测序和微阵列方法分析正常和CGD中性粒细胞中的miRNA水平。 这项研究的结果正在等待，并将与这些细胞之间mRNA表达的已知差异进行比较。 这种调节回路的识别可能有助于识别（没有先前的偏见）治疗干预的潜在靶点。 CGD中的脂质介体生物学： 几项研究表明，脂质介质的代谢在CGD中紊乱，特别是在这种疾病中，前列腺素D2减少，白三烯B4增加，脂质过氧化减少。 在体内，载脂组织细胞早已在CGD中观察到。 我们正在对CGD白细胞的脂质代谢进行系统研究，以确定其他介质，例如新描述的抗炎脂质的消退素类可能在CGD中发生改变。

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.