Clinical Studies Of Abnormal Host Defense

结果如下： （一） 慢性肉芽肿病（CGD）是由多组分NADPH氧化酶（吞噬细胞氧化酶，NOX 2）复合物突变引起的原发性免疫缺陷。在过去的财政年度，通过与道格拉斯库恩斯博士（Leidos，Inc.）管理的中子监测实验室（NML）合作，我们对2例p47 phox缺陷型和27例gp 91 phox缺陷型受试者或携带者使用NADPH氧化酶组分的免疫检测进行了分子诊断。 核酸测序确定了46例患者和家庭成员的特异性DNA突变。 在2019财年，NML发表了关于NCF 1（编码p47 phox的基因）测序的基于数字液滴PCR的方法的论文，由于存在两个非常相似的假基因（NCF 1B和NCF 1C），这在以前一直是一个严重的诊断挑战。 在2019财年，NML还对5名接受慢病毒介导的基因治疗的CGD患者进行了功能研究，以监测该方法纠正白细胞功能缺陷的有效性，该手稿目前正在审查中。 同样在2019财年，NML为其他免疫缺陷患者提供了分子诊断，例如发现携带CXCR 4（1例患者）、G6 PD（6-磷酸葡萄糖脱氢酶，2例患者）和ELANE（中性粒细胞弹性蛋白酶，2例患者）突变的患者。 NML正在积极研究来自Chediak Higashi综合征，RAC 2突变，PI 3 K缺陷和DOCK 11突变患者的细胞中的中性粒细胞功能。 （二） 我们的小组继续其新兴的革兰氏阴性CGD病原体，贝塞登颗粒杆菌的临床研究。我们继续监测培养证实的患者和怀疑有颗粒杆菌感染的患者的血清阳性，以评估我们的假设，即这种微生物可以建立持续的，临床上沉默的感染。 虽然罕见，但CGD患者中报告的颗粒杆菌感染的病死率为30%，这表明需要更多的工作来改善这种病原体的诊断和治疗。 我们正在检查疑似病例和其他疾病（如结节病）标本中细菌的流行情况，疑似但尚未确定肉芽肿性炎症的细菌原因。 （三） 我们的方案（#10-I-0029患有CGD和其他免疫系统疾病的患者中动脉粥样硬化的非侵入性评估）已经证明了NOX 2依赖性ROS对颈动脉血管壁厚度增加的发展的贡献，颈动脉壁厚度增加是动脉粥样硬化的临床前体征，使用颈动脉磁共振成像可以容易地检测到。 在2018财年期间，我们通过评估X连锁CGD携带者临床前动脉粥样硬化的随访研究中的34名受试者，推进了该项目的临床工作。 X-CGD携带者通常是健康的，尽管Lyonization或X染色体失活导致X-CGD携带者在其循环中具有不同数量的正常和CGD样细胞。 在某些情况下，当含有野生型等位基因的X染色体在90-95%的祖细胞中失活时，患者可以呈现与CGD无法区分的临床表型。 对携带者和健康年龄匹配对照的研究将检验产生ROS的细胞比例增加与动脉粥样硬化程度呈正相关的假设。 我们正在完成对这些数据的统计分析。 （四） 根据我们临床研究的初步结果（10-I-0029，，），我们一直在与国家转化科学推进中心（NCATS）的研究人员合作，以确定NOX 2的化学抑制剂。 使用Tom Leto在LCIM中开发的细胞系，我们开发了一种实验室规模的NOX 2活性筛选试验，然后由NCATS优化，用于高通量，机器人筛选NOX 2抑制剂。 到目前为止，我们已经在初级和二级筛选中评估了70，000多种化合物，并正在研究主要候选化合物的变体以供进一步研究。 鉴于第一代初筛中假阳性化合物的高比率，我们正在积极开发几种替代的NOX 2检测方法，这些方法不依赖于完整细胞中酶活性的间接测量，而是专注于亚基相互作用（结合）已知其调节活性酶复合物或高度纯化的酶复合物与人工活化剂的组装，所述人工活化剂作为分子上定义的检测而不是整个细胞。 我们还与Dorian McGavern博士（NINDS）合作，对各种NOX酶遗传缺陷的小鼠品系进行了研究，以评估其对创伤性脑损伤模型发病机制的贡献。该模型也被用于评估主要的NOX 2抑制剂，并正在进行进一步的实验，以明确证明NOX 2在这一过程中的参与。 第五章） 在2019财年，我们完成了对血浆凝溶胶蛋白在炎症过程中控制细胞活化的作用的研究。 血浆凝溶胶蛋白是由编码细胞溶质肌动蛋白结合蛋白（凝溶胶蛋白）的同一基因产生的，该蛋白在细胞形态和运动的调节中起着至关重要的作用。 血浆形式的不同之处在于它具有一个额外的功能未知的短多肽。 其他研究人员的研究已经确定了凝溶胶蛋白在炎症调节中的作用，并作为先天防御的积极贡献者。 我们正在准备一份手稿，描述几个患者群体炎症期间的凝溶胶蛋白水平，以及外源性凝溶胶蛋白对体外白细胞活化状态的贡献，通过FACS评估活化标志物、细胞毒性、细胞凋亡和粘附。

Results: (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 2 p47phox-deficient, and 27 gp91phox-deficient subjects or carriers. Nucleic acid sequencing determined the specific DNA mutations in 46 patients and family members. During FY19, the NML published its paper on a digital droplet PCR-based approach to the sequencing of NCF1, the gene encoding p47phox, that has previously been a serious diagnostic challenge due to the presence of two closely similar pseudogenes (NCF1B and NCF1C). During FY19, the NML also performed functional studies on five CGD patients undergoing lentivirus-mediated gene therapy to monitor efficacy of the approach to correct functional defects in leukocytes, this manuscript is currently under review. Also during FY19, the NML has provided molecular diagnoses for patients with other immunodeficiencies, for example those found to carry mutations in CXCR4 (1 patient), G6PD (Glucose 6-phosphate dehydrogenase, 2 patients), and ELANE (neutrophil elastase,2 patients). The NML is actively studying neutrophil function in cells from patients with Chediak Higashi syndrome, RAC2 mutations, PI3K deficiency and DOCK11 mutations. (2) Our group continues its clinical studies of the emerging Gram-negative CGD pathogen, Granulibacter bethesdensis. We continue to monitor seropositivity in culture-confirmed patients and patients suspected of having a Granulibacter infection to evaluate our hypothesis that this organism can establish persistent, clinically silent infections. Although rare, reported Granulibacter infections in CGD patients have a case fatality rate of 30% suggesting that more work is required to improve diagnosis and treatment of this pathogen. We are examining the prevalence of bacteria in specimens from suspected cases and from other diseases, such as sarcoidosis, with a suspected but as yet unidentified bacterial cause of granulomatous inflammation. (3) Our protocol, (#10-I-0029 Non-invasive Assessment of Atherosclerosis in Patients with CGD and other Disorders of the Immune System) has already demonstrated the contribution of NOX2-dependent ROS to the development of increased carotid vessel wall thickness, a preclinical sign of atherosclerosis that is readily detectable using carotid magnetic resonance imaging. During FY18, we have advanced our clinical efforts on this project by evaluating 34 subjects in a follow up study of measuring preclinical atherosclerosis in carriers of X-linked CGD. X-CGD carriers are generally healthy although lyonization, or X-chromosome inactivation, results in X-CGD carriers having different numbers of normal and CGD-like cells in their circulation. In some cases, where the X-chromosome containing the wild-type allele is inactivated in 90-95% of progenitor cells, the patients can present with a clinical phenotype indistinguishable from CGD. The study of carriers and healthy-age match controls will test the hypothesis that increasing ratios of cells producing ROS positively correlate with the extent of atherosclerosis. We are completing the statistical analysis of these data. (4) Based on the initial results of our clinical study (10-I-0029, , we have been collaborating with investigators at the National Center for Advancing Translational Sciences (NCATS) to identify chemical inhibitors of NOX2. Using a cell line developed by Tom Leto in the LCIM, we developed a lab scale-screening assay for NOX2 activity that then optimized by NCATS for high throughput, robotic screening for inhibitors of NOX2. To date, we have evaluated over 70,000 compounds in primary and secondary screens and are working on variants of lead candidates for further study. Given the high rate of false-positive compounds in the first generation primary screen, we are actively developing several alternative assays for NOX2 that do not rely on indirect measurements of enzyme activity in intact cells but rather focus either on subunit interactions (binding) that are known to regulate assembly of the active enzyme complex or a highly purified enzyme complex with artificial activators that function as a molecularly defined assay instead of a whole cell. We have also performed studies of mouse strains that are genetically deficient in various NOX enzymes to evaluate their contributions to pathogenesis in a model of traumatic brain injury in collaboration with Dr. Dorian McGavern (NINDS). This model has also been used to evaluate lead NOX2 inhibitors and further ongoing experiments to definitively prove the involvement of NOX2 in this process are underway. 5) During FY19, we completed our examination of the role of plasma gelsolin in controlling cellular activation during inflammation. Plasma gelsolin is produced by the same gene that encodes the cytosolic actin-binding protein, gelsolin, that plays a crucial role in the regulation of cellular morphology and motility. The plasma form differs in that it possesses an additional short polypeptide of unknown function. Studies by other investigators have identified a role for gelsolin in the regulation of inflammation and as a positive contributor to innate defenses. We are preparing a manuscript describing gelsolin levels during inflammation in several patient populations as well as the contribution of exogenous gelsolin on the activation state of leukocytes in vitro assessed via FACS for activation markers, cytotoxicity, apoptosis, and adherence.