Clinical Studies Of Abnormal Host Defense

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The LHD has a long history of investigating patients with abnormalities of phagocytic cell function. These studies include the delineation of clinical, functional, and molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease (CGD), leukocyte adhesion deficiency (LAD), the syndrome of hyperimmunoglobulinE and recurrent infections (Jobs Syndrome) and IRAK4 deficiency. Large cohorts of these patients have been recruited over the years and represent a unique national resource for biomedical research at the NIH. Currently we follow over 150 patients with CGD, about 40 patients with Jobs syndrome, and 30 patients with other phagocyte dysfunction syndromes, including LAD, cyclic neutropenia, neutrophil specific granule deficiency, Chediak-Higashi syndrome, IRAK4 deficiency and NEMO deficiency. We now have EB virus transformed B cells from most of our patients and we have been pleased to share these B cell lines with other intramural or extramural colleagues. We continue to monitor and expand these cohorts of patients who serve as models for long term studies of the clinical consequences of the immune dysfunction in humans. In 2008 we continued our studies of the importance of lactoferrin in protecting against Aspergillus fumigatus infection, the most common infectious cause of mortality in CGD patients today. We found that while CGD PMN are unable to kill Aspergillus hyphae, their ability to arrest the growth of conidia was identical to that of normal PMN showing a role for nonoxidative mechanisms in host defenses against this organism. We then showed that the neutrophil secretory product, lactoferrin, inhibits conidial germination by sequestering iron, a critical growth factor. A postbac IRTA, Anna R. Cruz (who recently left the lab to attend medical school), has continued studying the growth inhibitory properties of iron chelating drugs against Aspergillus fumigatus, alone and in combination with first line antibiotics such as amphotericin B, itraconazole, and fluconazole and demonstrated antifungal synergy in some combinations. Further studies have demonstrated significant antifungal activity of some iron chelators against other fungi (Cryptococcus neoformans, Candida albicans) suggesting an important new avenue for prevention and treatment of fungal infections common to immunocompromised patients. (Anna R. Cruz, 50% effort, Kol Zarember, 30% effort). In 2008 we published other studies (in collaboration with Janyce Sugui and June Kwong Chung, NIH/NIAID/LCID) investigating responses of Aspegillus to attack by normal and CGD neutrophils. We found differential regulation of many genes putatively involved in metal uptake and a variety of other fungal metabolic pathways that may be promising targets for new therapeutic approaches. Putative virulence genes identified by this approach have been deleted and we are pursuing whether they play a role in infection or resistance to human PMN attack. DNA sequencing of CGD patients continues to reveal new information about molecular components of the NADPH oxidase system. In 2008 we completed the sequencing of p47phox and gp91phox genes from about 200 living and deceased CGD patients. Nearly all patients with p47phox deficiency exhibited the same GT deletion in exon 1. Interestingly, analysis of gp91phox deficiency revealed 77 different mutations or deletions randomly distributed through out the coding regions with 32 novel mutations detected. Mutations in p67phox and p40phox CGD patients are currently being analyzed. The sequencing of gp91phox in these patients has revealed an important correlation between genotype and potential efficacy of one of the main prophylactic treatments available in the management of CGD. Previously (N. Engl. J. Med. 324:509-516, 1991), we had shown that treatment of CGD patients with IFN-γ reduced the overall frequency of infections and certain CGD subgroups (p47phoxCGD and a subset of the patients with gp91phox CGD ) responded to IFN γ both in vitro and in vivo whereas others did not (PNAS, 85:4874-4878, 1988). With the sequence data now available, a retrospective analysis revealed that the gp91phox subgroup responsive to IFN-γ represented patients with missense mutations in gp91phox while the gp91phox subgroup unresponsive to IFN-γ represented patients with nonsense mutations. We have recently repeated those studies in vitro, a study that included over 650 analyses of H2O2 production, and showed that CGD patients with missense mutation in gp91phox respond to IFN-γ in vitro (p = 0.045) while patients with nonsense mutations in gp91phox fail to respond to IFN-γ (p = 0.419). Moreover, CGD patients with a GT deletion in exon 1 of p47phox also respond to IFN-γ (p = 0.0009). These findings provide a genetics-based rationale for predicting the efficacy of IFN-γ therapy in the treatment of patients with CGD and may have important therapeutic implications for guiding future therapy. (Gallin, Malech, Holland, Kuhns). Over the past year, the laboratory has expanded its study of Granulibacter bethesdensis, a recently described bacterial pathogen of CGD patients. Granulibacter is remarkably hypostimulatory of the innate immune system, both in terms of weak activation of the NADPH oxidase and poor stimulation of cytokine secretion. With collaborators at the University of Georgia Complex Carbohydrate Research Center, we are purifying and characterizing the biological activity and structure of the LPS. We have isolated a fraction that appears to represent the Lipid A of this organism and that has strongly stimulatory activity in the limulus LPS test but fails to activate human PMN in-keeping with our findings that this microbe may avoid host defenses by using molecular stealth. We have also found that G. bethesdensis is remarkably resistant to complement and antimicrobial peptides. Further studies are underway to understand the pathogenesis of this organism in CGD patients and host defenses against it (Zarember, 40% effort, Anna R. Cruz 50% effort and contributions by David E. Greenberg, LCID). In 2008 we studied two Qatari sisters (5 & 12 yrs.) with an undescribed neutrophil dysfunction. The sisters had frequent severe skin and mucosal ulcerations, recurrent upper and lower respiratory tract infections, and neutropenia. The younger sister had repeated oral infections that ultimately resulted in development of oral strictures. A twin sister of the younger child who had a similar presentation died at age 3 years from a septic event. Although degranulation and staphylocidal activity were not dramatically altered, a profound chemotactic defect in vitro was detected. Microscopic examination of neutrophils from both sisters showed abnormal morphology with herniation of nuclear lobes into membrane-enclosed surface blebs. Neutrophils from both sisters failed to undergo a shape change after treatment with fMLF. Moreover, herniation of nuclear lobes coincided with retraction of the granules into the central region of the cells, resulting in an agranular region in the periphery of the cell. Confocal microscopy, using Alexa Fluor 546-phalloidin to identify filamentous actin and DAPI to localize nuclei, showed that cells with herniated nuclear lobes exhibited increased levels of filamentous actin compared to either patient cells with normal nuclear lobes or to normal neutrophils. Using FACS, a 4-5-fold increase in F-actin staining was detected in patient cells compared to normal controls. The abnormal nuclear morphology, profound defects in chemotaxis and in receptor internalization, and cytoskeletal anomalies detected in these two sisters with recurrent infections represents a novel defect in neutrophils that is likely an autosomal recessive mutation. (Kuhns, Uzel, Gallin and Holland).