Clinical Studies Of Abnormal Host Defense

1)慢性肉芽肿病（CGD）是由多组分NADPH氧化酶（吞噬细胞氧化酶，NOX2）复合物突变引起的原发性免疫缺陷。在过去的一年中，通过与Douglas Kuhns博士（Leidos, Inc.）管理的中性粒细胞监测实验室（NML）的合作，我们对4名p47phox缺陷和12名gp91phox缺陷受试者进行了NADPH氧化酶成分的免疫检测，提供了分子诊断。核酸测序确定了33名患者及其家庭成员的突变，包括最罕见的p40phox缺乏症。NML还为其他因CXCR4、ITGB2、WDR1和PADI4突变而导致免疫缺陷的患者提供了分子诊断。Kuhns博士开发了一种依靠数字液滴PCR（手稿准备）来确定p47突变的新方法。在本年度，我们与LHD的基因免疫治疗部门合作，展示了新的基因治疗方法来纠正来自CGD患者的诱导多能干细胞（Merling等人，Molecular therapy 2015）。我们目前正在与LHD的人类免疫疾病部门合作，评估来自几名患者的吞噬细胞的先天免疫功能。

1) Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the multicomponent NADPH oxidase (phagocyte oxidase, NOX2) complex. During the past FY, through collaboration with the Neutrophil Monitoring Laboratory (NML) managed by Douglas Kuhns, PhD ( Leidos, Inc.), we provided molecular diagnoses using immunodetection of components of the NADPH oxidase for 4 p47phox-deficient- and 12 gp91phox-deficient subjects. Nucleic acid sequencing determined the mutations in 33 patients and family members including the rarest form, p40phox deficiency. The NML has also provided molecular diagnosis of other patients with immunodeficiencies due to mutations in CXCR4, ITGB2, WDR1, and PADI4. Dr. Kuhns has developed a novel approach to determining p47 mutations relying on digital droplet PCR (manuscript in prep). During this FY, we collaborated with the Genetic Immunotherapy Section of the LHD to demonstrate novel gene therapy approaches to correct induced pluripotent stem cells from CGD patients (Merling et al., Molecular Therapy 2015). We are currently collaborating with the Human Immunological Disease Unit of the LHD to evaluate innate immune function in phagocytes from several patients. 2) Our group continues its clinical and laboratory studies of the emerging Gram-negative CGD pathogen, Granulibacter bethesdensis. During FY15, we screened 11 CGD samples and 15 normal samples for seropositivity toward G. bethesdensis methanol dehydrogenase, an immunodominant antigen. We continue to monitor seropositivity in culture-confirmed patients to evaluate our hypothesis that this organism can establish persistent, clinically unapparent infections. 3) During FY15, we published a paper describing data from NIH Protocol #10-I-0029 Non-invasive Assessment of Atherosclerosis in Patients with CGD and other Disorders of the Immune System (current total = 85 subjects). Atherosclerosis is caused, in part, by inflammation in the vasculature and over production of reactive oxygen species (ROS) has been implicated in pathogenesis of this disease. We hypothesized that CGD patients, who have deficient production of reactive oxygen species by their phagocytes and other cells, may be protected from developing atherosclerosis. The primary endpoint of this study was the assessment of atherosclerotic plaque formation/calcium deposition in the carotid and coronary arteries by CT, MRI and other imaging methodologies, in these and other patients with in-born disorders of immune function. We found significantly smaller carotid vessel wall volumes, a pre-clinical measure of atherosclerosis, in CGD patients compared to age- and sex-matched control subjects. This finding was published in Circulation (Sibley et al.). We continue to study patients on this protocol and have focused on carriers of X-linked CGD reasoning that these otherwise healthy individuals should have subnormal amounts of ROS and that this may be protective in this somewhat older cohort of subjects. This current effort will prove the hypothesis that lower levels of ROS production are protective although the situation in the X-linked CGD carriers differs from the CGD patients in that some cells are normal and some are abnormal. Simultaneously, we are collaborating with the National Center for Advancing Translational Sciences (NCATS), involving the development of high throughput assays for NADPH oxidase activity to permit large-scale screening of chemical inhibitors of NOX2. (4) In normal PMN, activation of NOX2 results in a rapid (within seconds to minutes) depletion of NADPH and hypoxia. Given the central roles of oxygen and NAPDH in metabolism, we compared the metabolomes of PMN before and after activation of NOX2 in a pilot study and found unexpected alterations in PMN metabolism. We are repeating these studies in PMN from patients with CGD to determine the exact role of ROS in these processes.