Clinical Studies Of Abnormal Host Defense

结果： (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、11个p67Phox缺陷和16个gp91Phox缺陷的受试者或携带者提供了分子诊断。核酸测序确定了44例患者及其家庭成员的特异性DNA突变。在2020财年，NML继续对5名接受慢病毒介导的基因治疗的CGD患者进行功能研究，以监测该方法纠正白细胞功能缺陷的效果。 同样在20财年，NML为突发事件综合征(CXCR4突变，9例患者)患者提供了分子诊断。与Covid19研究相关的NML活动包括对来自SarsCoV2感染患者的约706个个体样本进行分析，其中包括来自美国国立卫生研究院临床中心和国外中心的患者。 (2)本课题组继续对新出现的革兰氏阴性CGD病原体Granulibacter bethe densis进行临床研究。我们继续监测培养确认的患者和疑似感染革兰氏杆菌的患者的血清阳性率，以评估我们的假设，即这种细菌可以建立持续的、临床上无症状的感染。尽管罕见，但报告的CGD患者中的格兰利氏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样本中细菌的流行情况，并正在对这种微生物的各种临床分离株进行基因研究，以更好地了解发病机制。 (3)我们的方案(#10-I-0029非侵入性评估患有CGD和其他免疫系统疾病的患者的动脉粥样硬化)已经证明了依赖NOX2的ROS对颈动脉血管壁厚度增加的作用，这是动脉粥样硬化的临床前迹象，可以通过颈动脉磁共振成像很容易地检测到。在18财年，我们通过评估34名受试者对X连锁CGD携带者的临床前动脉粥样硬化进行了跟踪研究，从而推进了我们在这一项目上的临床工作。X-CGD携带者通常是健康的，尽管裂解或X染色体失活会导致X-CGD携带者循环中有不同数量的正常细胞和CGD样细胞。在某些情况下，含有野生型等位基因的X染色体在90%-95%的祖细胞中失活，患者可能出现与CGD无法区分的临床表型。对携带者和健康年龄匹配的对照组的研究将检验这一假设，即产生ROS的细胞比例增加与动脉粥样硬化的程度呈正相关。 (4)基于我们的临床研究(10-I-0029)的初步结果，我们一直在与国家先进翻译科学中心(NCATS)的研究人员合作，以确定NOX2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NOX2活性筛选方法，然后由NCATS进行优化，以实现高通量、机器人筛选NOX2抑制剂。到目前为止，我们已经在一次和二次筛选中评估了70,000多种化合物，并正在研究候选铅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NOX2的分析方法，这些方法不依赖于对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)，这种亚基相互作用已知调节活性酶复合体的组装，或者是具有人工激活剂的高纯度酶复合体，其功能是分子定义的分析，而不是整个细胞。到目前为止，这些替代检测方法中有几种处于后期开发阶段。 我们还与NINDS博士合作，对各种NOX酶基因缺陷的小鼠品系进行了研究，以评估它们在创伤性脑损伤模型中的致病作用。该模型还被用于评估铅NOX2抑制剂，目前正在进行进一步的实验，以明确证明NOX2参与了这一过程。 5)在20财年，我们完成了血浆明胶蛋白在炎症过程中控制细胞激活的作用的研究。血浆明胶蛋白是由编码胞浆肌动蛋白结合蛋白的同一基因产生的，明胶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，它另外具有一种功能未知的短肽。血浆明胶蛋白被认为是肌动蛋白和血小板激活因子等促炎刺激的缓冲区，在各种急性护理环境中，血浆明胶蛋白的降低被证明是预后不良的指标。在2020财年，我们发表了我们的发现(Audley等人，炎症)，发现慢性肉芽肿性疾病患者的血浆明胶蛋白降低，可能是这些患者出现过度炎症的原因之一，而且骨髓移植后这种水平可能会上升。 像其他实验室一样，Covid19大流行的影响大大减少了我们的研究活动。然而，我们已经与NIH临床中心和LCIM的其他研究人员合作，从有记录的SARS-CoV2感染患者身上获取样本，以评估这些患者的白细胞形态及其循环中的明胶蛋白水平。这些研究预计将在20财年结束前提交发表。

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 4 p47phox-deficient, 11 p67phox-deficient and 16 gp91phox-deficient subjects or carriers. Nucleic acid sequencing determined the specific DNA mutations in 44 patients and family members. During FY20, the NML continued functional studies on five CGD patients undergoing lentivirus-mediated gene therapy to monitor efficacy of the approach to correct functional defects in leukocytes. Also during FY20, the NML has provided molecular diagnoses for patients with WHIM syndrome (mutations in CXCR4, 9 patients. NML activities related to Covid19 research include measurement of 64 analytes on approximately 706 individual samples from patients with SarsCoV2 infection including patients from the NIH Clinical Center as well as centers abroad. (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 are performing genetic studies on various clinical isolates of this microbe to better understand pathogenesis. (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. (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. Several of these alternative assays are in late-stage development as of now. 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 FY20, 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. Plasma gelsolin is thought to act as a buffer of proinflammatory stimuli such as actin and platelet activating factor and decreases in plasma gelsolin have been shown to be an indicator of a poor prognosis in a variety of acute care settings. During FY20 we published our findings (Audley et al., Inflammation) that plasma gelsolin was decreased in patients with Chronic Granulomatous Disease, possibly contributing to excessive inflammation seen in these patients, and that levels may increase following bone marrow transplant. 6) Like other labs, the impact of the Covid19 pandemic has significantly curtailed our research activities. Nevertheless, we have collaborated with other investigators in the NIH Clinical Center and LCIM to obtain samples from patients with documented infections by SARS-CoV2 to evaluate morphology of leukocytes from these patients and the levels of gelsolin in their circulation. These studies are expected to be submitted for publication before the end of FY20.