Clinical Studies Of Abnormal Host Defense

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

(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 1 p22phox-deficient, 9 p47phox-deficient, and 38 gp91phox-deficient subjects. Nucleic acid sequencing determined the specific DNA mutations in 23 patients and family members. This FY, the NML completed development of a novel 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). Overall, this new tool has clarified our understanding of the inheritance of p47phox CGD, and simultaneously show that the NCF1 locus is more fluid than previously appreciated. These findings have been submitted for publication and are currently under review. During FY18, the NML also performed functional studies of CGD patients undergoing lentivirus-mediated gene therapy to monitor efficacy of the approach to correct functional defects in leukocytes. Also during FY18, the NML has provided molecular diagnoses for patients with other immunodeficiencies, for example those found to carry mutations in CXCR4 (8 patients), ITGB2 (1 patient), and Elastase (1 patient). (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. During FY18, we developed a custom antibody to this pathogen and have optimized detection of bacteria in archived paraffin-embedded tissues from culture-confirmed cases of Granulibacter infection. Going forward, we will examine prevalence of bacteria in such specimens from suspected cases and from other diseases with a suspected but as yet unidentified bacterial cause of granulomatous inflammation such as sarcoidosis. (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 anticipate having sufficient numbers of X-carriers and controls to complete statistical analysis of these data in FY19. (4) Based on the findings described in the section above, we initiated a collaboration 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. During FY18, NCATS screened 60,000 compounds and we are currently evaluating leads in several laboratory scale counter-screens to eliminate false positives and specific inhibitors of upstream signaling pathways. Furthermore, in addition to progress in developing mouse assays of atherosclerosis for testing potential leads, we are collaborating with Dr. Dorian McGavern (NINDS) to evaluate compounds in an assay of traumatic brain injury. 5) During FY18, we examined 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 investigating gelsolin levels during inflammation and the contribution of exogenous gelsolin on the activation state of leukocytes in vitro.