Clinical Studies Of Abnormal Host Defense

1)慢性肉芽肿性疾病(CGD)是一种由多组分NADPH氧化酶(吞噬细胞氧化酶，NOX2)复合体突变引起的原发免疫缺陷。在过去的财年中，通过与Douglas Kuhns，PhD(Leidos，Inc.)管理的中性粒细胞监测实验室(NML)的合作，我们使用免疫检测NADPH氧化酶的成分为4个p47Phox缺陷和12个gp91Phox缺陷的受试者提供了分子诊断。核酸测序确定了33名患者和家庭成员的突变，包括最罕见的形式，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。库恩斯博士开发了一种新的方法，依靠数字液滴聚合酶链式反应(手稿正在准备中)来确定p47突变。在本财年，我们与LHD的遗传免疫治疗科合作，展示了纠正CGD患者诱导的多能干细胞的新基因治疗方法(Merling等人，分子疗法2015)。我们目前正在与LHD的人类免疫疾病部门合作，评估几名患者吞噬细胞的先天免疫功能。 2)本课题组继续对新出现的革兰氏阴性CGD病原菌Granulibacter bethe densis进行临床和实验室研究。在2015财年期间，我们筛选了11份CGD样本和15份正常样本，以检测免疫优势抗原G的致密甲醇脱氢酶血清阳性。我们继续监测培养确认的患者的血清阳性反应，以评估我们的假设，即这种微生物可以建立持续的、临床上不明显的感染。 3)在2015财年期间，我们发表了一篇论文，描述了NIH议定书#10-I-0029对患有CGD和其他免疫系统疾病患者的动脉粥样硬化进行非侵入性评估的数据(目前共有85名受试者)。动脉粥样硬化的部分原因是血管系统的炎症，而活性氧自由基(ROS)的过度产生与这种疾病的发病机制有关。我们假设，CGD患者，其吞噬细胞和其他细胞产生的活性氧物种不足，可能受到保护，不会发生动脉粥样硬化。这项研究的主要终点是通过CT、MRI和其他成像方法评估这些和其他先天免疫功能障碍患者颈动脉和冠状动脉中动脉粥样硬化斑块的形成/钙沉积。我们发现，与年龄和性别匹配的对照组相比，CGD患者的颈动脉壁体积显著减小，这是动脉粥样硬化的临床前测量指标。这一发现发表在《循环》(Sibley等人)上。我们继续对这一方案的患者进行研究，并将重点放在X连锁CGD的携带者身上，理由是这些原本健康的人应该具有低于正常水平的ROS，这可能对这组年龄较大的受试者具有保护作用。尽管X连锁CGD携带者的情况与CGD患者不同，一些细胞正常，一些细胞异常，但目前的研究将证明这一假设，即较低水平的ROS产生是保护性的。同时，我们正在与国家先进翻译科学中心(NCATS)合作，开发高通量的NADPH氧化酶活性检测方法，以便大规模筛选NOX2的化学抑制剂。 (4)在正常的PMN中，NOX2的激活导致NADPH的迅速耗尽和缺氧。鉴于氧和NAPDH在代谢中的中心作用，我们在一项初步研究中比较了NOX2激活前后PMN的代谢物，发现PMN代谢发生了意想不到的变化。我们正在对CGD患者的PMN重复这些研究，以确定ROS在这些过程中的确切作用。

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.