Clinical Studies Of Abnormal Host Defense

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

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 2 p22phox-deficient, 9 p47phox-deficient, 4 p67phox-deficient, and 15 gp91phox-deficient subjects. Nucleic acid sequencing determined the mutations in 46 patients and family members including p40phox deficiency. The NML has also provided molecular diagnosis of other patients with immunodeficiencies due to mutations in RAC2 (1 patient), CXCR4 (3 patients), ITGB2 (1 patient), WDR1 (4 patients), and IRAK4 (4 patients). Dr. Kuhns has continued to refine the application of digital droplet PCR to determining p47 mutations. Due to the presence of 2 pseudogenes that are highly homologous to NCF1, the gene encoding p47phox, direct sequencing is not able to clearly identify mutations in this gene. The digital droplet PCR technique has now been applied to over 150 normal subjects, as well as 163 patients, carriers and kindreds with suspected NCF1 mutations and 193 patients with known CGD mutations in other genes. This year we completed studies of three families of patients describing a new disease impacting the dynamic remodeling of the cellular cytoskeleton of phagocytes due to mutation of WDR1, the gene for actin-interacting protein 1 (Aip1). This disease was shown to have autosomal recessive inheritance and has clinical findings of recurrent bacterial infections and abnormal wound healing with clinically important stomatitis. Allogeneic stem cell transplantation corrected the immunologic defect in one patient. The paper describing this disease has been accepted in Blood, 2016. (2) Our group continues its clinical and laboratory studies of the emerging Gram-negative CGD pathogen, Granulibacter bethesdensis. During FY16, we screened 62 CGD samples and 37 normal samples for seropositivity toward G. bethesdensis methanol dehydrogenase, an immunodominant antigen. We have also been asked by outside researchers to measure seropositivity in suspected Granulibacter patients. We continue to monitor seropositivity in culture-confirmed patients to evaluate our hypothesis that this organism can establish persistent, clinically unapparent infections. During the summer of 2016, our section hosted a high school student who worked to establish an immunofluorescence-based assay to identify Granulibacter in paraffin-embedded clinical specimens and samples from experimentally infected mice. Currently, we are exploiting this method to screen additional NIH patient materials for the presence of Granulibacter as well as examine the tissue and cell tropism of this organism during experimental infections in mice. (3) Last year, we published a paper describing data from NIH Protocol #10-I-0029 Non-invasive Assessment of Atherosclerosis in Patients with CGD and other Disorders of the Immune System. In collaboration with the Department of Radiology, we used carotid magnetic resonance imaging and found evidence that patients with CGD had significantly lower carotid vessel wall volumes than healthy age and sex-matched controls. Carotid vessel wall volume increases early during atherogenesis and is a validated marker of preclinical atherosclerosis. This finding, published in Circulation (Sibley et al.) suggested that the absence of NOX2 activity may be protective against atherosclerosis, at least in the carotid arteries. 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 provides a large numbers of X-CGD carriers and many are older than CGD subjects. 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. In an effort to extend our finding that patients with CGD may be protected from atherosclerosis to the general population, we are 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 LHD, we developed a lab scale-screening assay for NOX2 activity that was further optimized by NCATS for high throughput screening for inhibitors of NOX2. Primary screens of several libraries have been completed and counterscreens using complementary assays are underway prior to detailed evaluation of lead compounds in lab-scale assays to identify mechanisms of action. Novel lead compounds will be tested for protective activity in animal models of atherosclerosis such as the carotid ligation model in collaboration with NHLBI. 4) The blister protocol (90-I0120) is being used to evaluate inflammation in vivo in several populations of patients with rare immunodeficiencies including STAT3 deficiency, CGD, and others. Our efforts to accrue subjects with CGD (currently n=3) in order to examine the development and progression of inflammation in vivo in this patient population have been slow. This year, we collaborated with the Human Immunologic Diseases Unit (LHD) to examine inflammation in a patient with a chemotactic defect. 5) During FY16, 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.