Clinical Studies Of Abnormal Host Defense

该项目的目的是研究宿主防御异常的患者;确定其异常的原因;并为其潜在的疾病和与疾病过程相关的危及生命的感染设计有效的治疗方法。LHD在研究吞噬细胞功能异常的患者方面有着悠久的传统。这些研究包括早期描绘中性粒细胞特异性颗粒缺乏症、儿童慢性肉芽肿病（CGD）、白细胞粘附缺陷和高免疫球蛋白E综合征、复发性感染和IRAK 4缺乏症患者的临床、功能和分子缺陷。多年来，我们一直在收集患者队列，并在NIH继续跟踪。目前，我们跟踪了150多例CGD患者，约40例高免疫球蛋白E复发性感染综合征患者，以及30例其他吞噬细胞功能障碍综合征患者，包括白细胞粘附缺陷，周期性中性粒细胞减少症，中性粒细胞特异性颗粒缺乏症和ChediakHigashi综合征和IRAK 4缺乏症。所有这些患者都是研究人员希望从患者身上获得样本的国家资源，并可用于涉及校内或校外科学家的临床研究方案。我们现在有EB病毒转化的B细胞从我们的大多数病人，我们一直很高兴地分享这些B细胞系与其他内部或外部的同事。我们将继续监测和扩大这些患者队列，这些患者将作为异常宿主防御的免疫操纵的长期研究的模型。 今年，我们完成了一项关于乳铁蛋白在预防曲霉菌感染方面的重要性的研究，曲霉菌感染是当今CGD患者最常见的死亡原因。 我们已经表明，中性粒细胞分泌的产品，乳铁蛋白，抑制分生孢子萌发螯合铁，一个关键的生长因子。 CGD患者发生侵袭性曲霉病的风险增加。 然而，尽管每天暴露于烟曲霉分生孢子，许多CGD患者存活数年而无感染，这表明存在针对曲霉菌的非氧化宿主防御。以往的研究表明，中性粒细胞通过氧化机制杀死菌丝和肿胀但不休眠的分生孢子。 我们发现，虽然CGD中性粒细胞不能杀死菌丝，他们的能力，逮捕肿胀的分生孢子的生长是相同的正常中性粒细胞显示非氧化机制在宿主防御这种生物体的作用。(Kol Zarember） 与NIAID临床传染病实验室的Janyce Sugui和June KwongChung合作进行的其他研究调查了正常和CGD中性粒细胞对PMN介导的烟曲霉的杀伤作用。 该项目已经显示了许多参与金属吸收的基因的差异调节。 更好地了解分生孢子如何识别宿主中的低铁水平，以及曲霉菌从宿主和其他来源获得铁的生化方法，可能会为抗真菌药物的开发提供很好的靶点。 使用cDNA微阵列技术来探测真菌对中性粒细胞攻击的反应，我们已经确定了在分生孢子或菌丝中响应于具有完整（正常）或缺陷的氧化杀微生物系统（CGD）的PMN而上调的基因子集。 KwonChung小组已经从曲霉菌模型中敲除了几个假定的毒力基因，我们的实验室分析了这些真菌突变对人类PMN产生呼吸爆发能力的影响。（Kuhns，Gallin，Malech，Holland）. CGD患者的基因测序已经揭示了许多关于NADPH氧化酶系统的分子组成的损伤。 今年，我们对来自约200名CGD患者的基因进行了测序。所有p47 phox缺陷的患者都表现出外显子1的GT缺失，这种突变在该CGD亚组的所有患者中是常见的。 对gp 91 phox缺陷的分析显示，77个不同的缺陷随机分布在整个编码区，其中32个新的突变以前没有报道。 目前正在分析其他CGD亚组中的突变。 我们还注意到遗传信息是CGD患者干扰素γ反应性的重要预测因子。 来自具有错义突变的患者的单核细胞对干扰素γ（响应佛波醇酯PMA的呼吸爆发刺激）有响应，而来自具有无义突变的患者的单核细胞没有响应。 这些研究可能对指导未来的治疗具有重要的治疗意义。 （Gallin，Malech，Holland，Kuhns）. 在其他研究中，贝氏颗粒杆菌是一种感染儿童慢性肉芽肿性疾病（CGD）患者的微生物，但不会在这些患者中诱导显著的感染典型病理学，如肉芽肿性炎症。 贝塞登颗粒杆菌对过氧化氢有较强的抵抗力。此外，与感染CGD患者的其他细菌不同，贝氏颗粒杆菌是中性粒细胞凋亡的不良诱导剂，并且不能诱导正常中性粒细胞中的活性氧。 这些数据表明，贝塞登颗粒杆菌是宿主防御的不良刺激物，这可能使其建立持续感染。（Zarember，Gallin，Holland和Greenberg）

The purpose of this project is to study patients with abnormal host defense; to determine the cause of their abnormality; and to devise effective therapies for their underlying disorder and the lifethreatening infections associated with their disease processes. The LHD has a long tradition of investigating patients with abnormalities of phagocytic cell function. These studies include early delineation of the clinical, functional, and in some cases, the molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease of childhood (CGD), leukocyte adhesion deficiency and the syndrome of hyperimmunoglobulinE and recurrent infections and IRAK4 deficiency. Cohorts of patients have been collected over the years which we continue to follow at NIH. Currently we follow over 150 patients with CGD, about 40 patients with the hyperimmunoglobulinE recurrent infection syndrome, and 30 patients with other phagocyte dysfunction syndromes, including leukocyte adhesion deficiency, cyclic neutropenia, neutrophil specific granule deficiency and ChediakHigashi syndrome and IRAK4 deficiency. All these patients serve as a national resource for investigators desiring samples from patients and are available for clinical research protocols involving intramural or extramural scientists. We now have EB virus transformed B cells from most of our patients and we have been pleased to share these B cell lines with other intramural or extramural colleagues. We continue to monitor and expand these cohorts of patients who serve as models for long term studies of the immunological manipulation of the abnormal host defenses. This year we completed a study of the importance of lactoferrin in protecting against aspergillus infection, the most common infectious cause of mortality in CGD patients today. We have shown that the neutrophil secretory product,lactoferrin,inhibits conidial germination by sequestering iron, a critical growth factor. Patients with CGD have an increased risk for invasive aspergillosis. However, despite daily exposure to Aspergillus fumigatus conidia, many CGD patients survive years without infection, suggesting the existence of nonoxidative host defenses against Aspergillus. Previous studies indicated that PMN kill hyphae and swollen but not resting conidia through oxidative mechanisms. We found that while CGD PMN are unable to kill hyphae, their ability to arrest the growth of swollen conidia was identical to that of normal PMN showing a role for nonoxidative mechanisms in host defenses against this organism. (Kol Zarember) Other studies,done in collaboration with Janyce Sugui and June KwongChung in NIAIDs Laboratory of Clinical Infectious Diseases, investigated PMNmediated killing of Aspergillus fumigatus by normal and CGD neutrophils. This project has shown differential regulation of many genes putatively involved in metal uptake. A better molecular understanding of how conidia recognize low iron levels in a host and the biochemical methods employed by Aspergillus to acquire iron from the host and other sources may well reveal excellent targets for the development of antifungal drugs. Using cDNA microarray technology to probe the responses the fungus makes to neutrophil attack we have identified subsets of genes that are upregulated in either conidia or hyphae in response to PMN with intact(normal)or deficient oxidative microbicidal systems (CGD). Several putative virulence genes have been knocked out of aspergillus models by the KwonChung group and our lab has analyzed the impact of these fungal mutations on the ability of human PMN to generate a respiratory burst. (Kuhns, Gallin, Malech, Holland). Gene seqencing of CGD patients has taught numerous lessions about molecular components of the NADPH oxidase system. This year we have sequenced the genes from our cohort of about 200 CGD patients. All patients with p47phox deficient exhibited a GT deletion in exon 1, a mutation common to nealry all patients within this subgroup of CGD. Analysis of gp91phox deficiency revealed 77 differnt defects randomly distributed through out the coding regions with 32 novel mutations not previously reported. Mutations in the other CGD subgroups are currently being analyzed. We also noted that genetic information is an important preditor of interferon gamma responsiveness of CGD patients. Monocytes from patients with missense mutations are responsive to interferon gamma (stimulation of a respiratory burst in response to the phorbol ester PMA) whereas monocytes from patients with nonsense mutations are not responsive. These studies may have important therapeutic implications for guiding future therapy. (Gallin, Malech, Holland, Kuhns). In other studies Granulibacter bethesdensis, an organism infecting patients with chronic granulomatous disease of childhood (CGD)without inducing significant pathology typical of infection in these patients such as granulomatous inflammation. Granulibacter bethesdensis was shown to be complementresistent and relatively resistent to hydrogen peroxide. In addition, unlike other bacteria that infection CGD patients, Granulibacter bethesdensis was a poor inducer of neutrophil apoptosis and failed to induce reactive oxygen species in normal neutrophils. The data suggest that Granulibacter bethesdensis is a poor stimulator of host defenses and this may allow it to establish persistent infections. (Zarember, Gallin, Holland and Greenberg)