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Principal Investigator: JOHN I GALLIN
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2020
Award: $256,927
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <1-Alkyl-2-acetyl-sn-glycerophosphocholine><1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine><2019 novel coronavirus><2019-nCoV><AGEPC><Acetyl Glyceryl Ether Phosphorylcholine><Actin-Binding Protein><Actins><Activator Appliances><Activator Orthodontic Appliances><Age><Alleles><Allelomorphs><Assay><Atheroscleroses><Atherosclerosis><Atherosclerotic Cardiovascular Disease><Bacteria><Binding><Bioassay><Biologic Assays><Biological Assay><Biological Markers><Biomedical Research><Blood Circulation><Blood Neutrophil><Blood Plasma><Blood Polymorphonuclear Neutrophil><Blood leukocyte><Blood monocyte><Bloodstream><Bone Marrow Grafting><Bone Marrow Transplant><Bone Marrow Transplantation><Brain Trauma><Buffers><COVID-19><COVID-19 epidemic><COVID-19 pandemic><COVID19><COVID19 epidemic><COVID19 pandemic><CXC-R4><CXCR-4><CXCR4><CXCR4 gene><Case Fatality Rates><Cell Body><Cell Function><Cell Line><Cell Locomotion><Cell Migration><Cell Movement><Cell Process><Cell physiology><CellLine><Cells><Cellular Function><Cellular Migration><Cellular Morphology><Cellular Motility><Cellular Physiology><Cellular Process><Cellular Regulation><Chemicals><Chronic Granulomatous Disease><Circulation><Clinical><Clinical Research><Clinical Study><Collaborations><Complex><Cytoplasmic Granules><D2S201E><DNA Alteration><DNA Sequence Alteration><DNA Therapy><DNA mutation><Data><Defect><Development><Diagnosis><Disease><Disorder><Doctor of Philosophy><Dysfunction><FB22><Family member><Follow-Up Studies><Followup Studies><Function Activator><Functional disorder><Gelsolin><Gene Transfer Clinical><Generations><Genes><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Genetic mutation><Genetic study><Goals><HM89><HSY3RR><High Throughput Assay><History><Host Defense><Hyper-IgE Syndrome><Hyperimmunoglobulin E Syndrome><Hyperimmunoglobulin E-Recurrent Infection Syndrome><IRAK4><IRAK4 gene><Immune Diseases><Immune Disorders><Immune Dysfunction><Immune 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