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： （1）慢性肉芽肿性疾病（CGD）是由多组分NADPH氧化酶（吞噬细胞氧化酶，NOX2）复合物中突变引起的主要免疫缺陷。在过去的FY期间，通过与道格拉斯·库恩斯（Douglas Kuhns）博士管理的中性粒细胞监测实验室（NML）合作，我们使用NADPH氧化酶的免疫检测提供了分子诊断，以4 p47phox-ficeient，11 p67phox-deciers，11 p67phox-deeiers和crirss and Crarss或16 gpecient和16 gpec9 ficficectient。 核酸测序确定了44例患者和家庭成员的特异性DNA突变。 在第20财年期间，NML继续对五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功效。 同样在第20财年期间，NML提供了WHIM综合征患者的分子诊断（CXCR4，9名患者的突变。与CoVID19研究有关的NML活性包括对来自NIH临床中心以及外在Centers Centers Centerers and Centerers的大约706个单个样本进行了64个分析。 （2）我们的小组继续对新兴的革兰氏阴性CGD病原体肉芽肿贝丝斯氏菌进行临床研究。我们继续监测培养确认的患者和怀疑患有肉芽杆菌感染的患者的血清阳性，以评估我们的假设，即这种生物可以建立持久性，临床上无声的感染。 尽管很少见，但据报道，CGD患者的肉芽杆菌感染的病例死亡率为30％，这表明需要更多的工作来改善该病原体的诊断和治疗。 我们正在检查可疑病例的标本中细菌的患病率，并正在对该微生物的各种临床分离株进行遗传研究，以更好地理解发病机理。 （3）我们的方案（＃10-I-0029对CGD患者的动脉粥样硬化和免疫系统其他疾病的动脉粥样硬化评估）已经证明，NOX2依赖性ROS对增加颈动脉血管壁厚度的发展的贡献，这是一种可容易地检测到可检测的动脉粥样硬化症状的长主角迹象。 在2018财年期间，我们通过评估34名受试者在测量X连锁CGD载体的临床前动脉粥样硬化的后续研究中通过评估34名受试者进行了临床工作。 X-CGD载体通常是健康的，尽管溶血化或X染色体灭活，但导致X-CGD载体在循环中具有不同数量的正常和CGD样细胞。 在某些情况下，在90-95％的祖细胞中，含有野生型等位基因的X染色体灭活，患者可以与CGD无法区分的临床表型。 对载体和健康年龄匹配对照的研究将检验以下假设：增加产生ROS的细胞比率与动脉粥样硬化的程度正相关。 （4）根据我们的临床研究的最初结果（10-I-0029），我们一直与国家前进转化科学中心（NCAT）的研究人员合作，以鉴定NOX2的化学抑制剂。 使用汤姆·莱托（Tom Leto）在LCIM中开发的细胞系，我们开发了一种用于NOX2活性的实验室尺度筛分测定法，然后由NCAT对NOX2抑制剂进行高吞吐量，机器人筛选进行了优化。 迄今为止，我们已经评估了主要和次要筛选中的70,000多种化合物，并正在研究铅候选者的变体以进行进一步研究。 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. 截至目前，这些替代测定中有几种是在晚期开发中。 我们还对各种NOX酶缺乏遗传缺陷的小鼠菌株进行了研究，以评估其与Dorian McGavern博士（NINDS）合作的创伤性脑损伤模型中的发病机理的贡献。该模型还用于评估铅NOX2抑制剂和进一步的实验，以确定证明NOX2参与此过程的参与正在进行中。 5）在第20财年期间，我们完成了血浆凝胶素在控制炎症过程中细胞活化中的作用的检查。 血浆凝胶素是由编码胞质肌动蛋白结合蛋白Gelsolin的相同基因产生的，该基因在调节细胞形态和运动性中起着至关重要的作用。 血浆形式的不同之处在于它具有未知功能的其他短多肽。 血浆凝胶素被认为是促炎刺激（如肌动蛋白和血小板激活因子）的缓冲液，并且血浆凝胶素的降低已被证明是多种急性护理环境中预后不良的指标。 在20财年期间，我们发表了我们的发现（Audley等人，炎症），慢性肉芽肿性疾病患者的血浆凝胶蛋白减少，可能导致这些患者出现的过度炎症，并且骨髓移植后的水平可能会增加。 6）与其他实验室一样，Covid19大流行的影响大大削弱了我们的研究活动。 然而，我们已经与NIH临床中心和LCIM的其他研究人员合作，从SARS-COV2记录感染的患者中获取样品，以评估这些患者的白细胞形态以及其循环中的凝胶蛋白水平。 预计这些研究将在20财年结束之前提交出版。