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, 11 p47phox-deficient, 1 p67phox-deficient, and 27 gp91phox-deficient subjects. Nucleic acid sequencing determined the specific DNA mutations in 55 patients and family members. This FY, the NML pioneered 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). During FY17, the NML has also provided molecular diagnoses for patients with other immunodeficiencies, for example those found to carry mutations in CXCR4 (11 patients), ITGB2 (8 patients), and C6PD (7 patients). (2) Our group continues its clinical and laboratory studies of the emerging Gram-negative CGD pathogen, Granulibacter bethesdensis. We continue to monitor seropositivity in culture-confirmed patients to evaluate our hypothesis that this organism can establish persistent, clinically unapparent infections. During this period, we have optimized detection of Granulibacter in paraffin-embedded infected mouse tissues and will be expanding these methods to detection of this bacterium in similar human clinical samples. (3) Our protocol, (#10-I-0029 Non-invasive Assessment of Atherosclerosis in Patients with CGD and other Disorders of the Immune System) yielded a publication describing 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. Our current efforts with this protocol involve measurements of 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 NIH CGD patient community includes a large number of female X-CGD carriers of all ages and degrees of lionization. This permits the study of ages of patients more likely to have atherosclerosis to potentially answer the question as to whether different amounts of cells producing ROS correlate with the extent of atherosclerosis. To date, mostly during FY17, we have enrolled 29 carriers. (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 LHD, 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 FY17, we completed our development of several laboratory-scale counterscreens to eliminate false positives and specific inhibitors of upstream signaling pathways. This entire candidate identification and testing pipeline was tested using the well characterized NCATS Pharmaceutical Collection (NPC) and Mechanistic Interrogation Plate (MIP) libraries (4,426 compounds in total). No specific NOX2 inhibitors were identified, however the pipeline was validated and is being scaled up to screen for larger libraries of compounds in the coming FY. In parallel, we are developing collaborations to test bone fide NOX2 inhibitors in animal models of atherosclerosis as well as other conditions where NOX2 plays a role in pathogenesis. 5) During FY17, we have studied the role of plasma gelsolin in 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 inflammatory cells.