Clinical Studies Of Abnormal Host Defense

宿主防御异常的临床研究

• 批准号: 10272012
• 负责人: JOHN I GALLIN
• 金额: $ 25.69万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10272012
• 关键词: 2019-nCoV; Actin-Binding Protein; Actins; Activator Appliances; Age; Alleles; Atherosclerosis; Bacteria; Binding; Biological Assay; Biological Markers; Biomedical Research; Blood Circulation; Bone Marrow Transplantation; Buffers; COVID-19; COVID-19 pandemic; CXCR4 gene; Case Fatality Rates; Cell Line; Cell physiology; Cells; Cellular Morphology; Chemicals; Chronic Granulomatous Disease; Clinical; Clinical Research; Collaborations; Complex; Cytoplasmic Granules; DNA Sequence Alteration; Data; Defect; Development; Diagnosis; Disease; Doctor of Philosophy; Family member; Follow-Up Studies; Functional disorder; Gelsolin; Generations; Genes; Genetic study; Goals; Host Defense; IRAK4 gene; Immune System Diseases; Immune system; Individual; Infection; Inflammation; Inflammatory; Investigation; Job' s Syndrome; Laboratories; Lead; Leukocyte-Adhesion Deficiency Syndrome; Leukocytes; Link; Magnetic Resonance Imaging; Measurement; Measures; Mediating; Microbe; Modeling; Molecular; Molecular Diagnosis; Monitor; Morphology; Mouse Strains; Multienzyme Complexes; Mutation; Myeloid Cells; NADPH Oxidase; National Institute of Neurological Disorders and Stroke; Natural History; Nox enzyme; Nucleic acid sequencing; Organism; Oxidases; Pathogenesis; Patients; Phagocytes; Plasma; Platelet Activating Factor; Play; Prevalence; Prevalence Study; Process; Protocols documentation; Publications; Publishing; Recording of previous events; Reporting; Research; Research Activity; Research Personnel; Robotics; Role; Sampling; Specimen; Stimulus; Subfamily lentivirinae; Testing; Therapeutic; Thick; Translational Research; Traumatic Brain Injury; United States National Institutes of Health; Variant; WHIM syndrome; Work; X Chromosome; X Inactivation; acute care; base; biomedical resource; cell growth regulation; cell motility; clinical center; clinical phenotype; cohort; congenital immunodeficiency; enzyme activity; experimental study; gene therapy; high throughput screening; immunoregulation; improved; inhibitor/antagonist; laboratory experiment; lead candidate; macrophage; monocyte; neutrophil; neutrophil cytosol factor 67K; new technology; novel therapeutics; outcome forecast; pathogen; polypeptide; pre-clinical; recruit; recurrent infection; screening; seropositive; small molecule inhibitor; stem cells

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.

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4例p47 phox缺陷型、11例p67 phox缺陷型和16例gp 91 phox缺陷型受试者或携带者采用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44例患者和家庭成员的特异性DNA突变。 在FY 20期间，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有效性。 同样在2010财年，NML为WHIM综合征患者提供了分子诊断（CXCR 4突变，9例患者。 与Covid 19研究相关的NML活动包括测量来自SarsCoV 2感染患者（包括NIH临床中心和国外中心的患者）的约706份个体样本中的64种分析物。 （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遗传研究，以更好地了解发病机制。 （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。 （四） 根据我们临床研究（10-I-0029）的初步结果，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。 到目前为止，这些替代测定中的一些处于后期开发阶段。 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2020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。 血浆凝溶胶蛋白被认为是促炎刺激物如肌动蛋白和血小板活化因子的缓冲剂，并且血浆凝溶胶蛋白的减少已被证明是各种急性护理环境中不良预后的指标。 在FY 20期间，我们发表了我们的研究结果（Audley et al.，炎症），慢性肉芽肿病患者的血浆凝溶胶蛋白降低，可能导致这些患者中观察到的过度炎症，并且在骨髓移植后水平可能增加。 六、 与其他实验室一样，Covid 19大流行的影响大大减少了我们的研究活动。 尽管如此，我们还是与NIH临床中心和LCIM的其他研究人员合作，从有记录的SARS-CoV 2感染患者中获取样本，以评估这些患者的白细胞形态和循环中凝溶胶蛋白的水平。 这些研究预计将于2020财年结束前提交发表。