The purpose of this project is to study patients with abnormal host defense; to determine the cause of their abnormality; and to devise effective therapies for their underlying disorder and the lifethreatening infections associated with their disease processes. The LHD has a long tradition of investigating patients with abnormalities of phagocytic cell function. These studies include early delineation of the clinical, functional, and in some cases, the molecular defects of patients with neutrophil specific granule deficiency, chronic granulomatous disease of childhood (CGD), leukocyte adhesion deficiency and the syndrome of hyperimmunoglobulinE and recurrent infections and IRAK4 deficiency. Cohorts of patients have been collected over the years which we continue to follow at NIH. Currently we follow over 150 patients with CGD, about 40 patients with the hyperimmunoglobulinE recurrent infection syndrome, and 30 patients with other phagocyte dysfunction syndromes, including leukocyte adhesion deficiency, cyclic neutropenia, neutrophil specific granule deficiency and ChediakHigashi syndrome and IRAK4 deficiency. All these patients serve as a national resource for investigators desiring samples from patients and are available for clinical research protocols involving intramural or extramural scientists. 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 immunological manipulation of the abnormal host defenses.? ? This year we completed a study of the importance of lactoferrin in protecting against aspergillus infection, the most common infectious cause of mortality in CGD patients today. We have shown that the neutrophil secretory product,lactoferrin,inhibits conidial germination by sequestering iron, a critical growth factor. Patients with CGD have an increased risk for invasive aspergillosis. However, despite daily exposure to Aspergillus fumigatus conidia, many CGD patients survive years without infection, suggesting the existence of nonoxidative host defenses against Aspergillus. Previous studies indicated that PMN kill hyphae and swollen but not resting conidia through oxidative mechanisms. We found that while CGD PMN are unable to kill hyphae, their ability to arrest the growth of swollen conidia was identical to that of normal PMN showing a role for nonoxidative mechanisms in host defenses against this organism. (Kol Zarember)? ? Other studies,done in collaboration with Janyce Sugui and June KwongChung in NIAIDs Laboratory of Clinical Infectious Diseases, investigated PMNmediated killing of Aspergillus fumigatus by normal and CGD neutrophils. This project has shown differential regulation of many genes putatively involved in metal uptake. A better molecular understanding of how conidia recognize low iron levels in a host and the biochemical methods employed by Aspergillus to acquire iron from the host and other sources may well reveal excellent targets for the development of antifungal drugs. Using cDNA microarray technology to probe the responses the fungus makes to neutrophil attack we have identified subsets of genes that are upregulated in either conidia or hyphae in response to PMN with intact(normal)or deficient oxidative microbicidal systems (CGD). Several putative virulence genes have been knocked out of aspergillus models by the KwonChung group and our lab has analyzed the impact of these fungal mutations on the ability of human PMN to generate a respiratory burst. (Kuhns, Gallin, Malech, Holland).? ? Gene seqencing of CGD patients has taught numerous lessions about molecular components of the NADPH oxidase system. This year we have sequenced the genes from our cohort of about 200 CGD patients. All patients with p47phox deficient exhibited a GT deletion in exon 1, a mutation common to nealry all patients within this subgroup of CGD. Analysis of gp91phox deficiency revealed 77 differnt defects randomly distributed through out the coding regions with 32 novel mutations not previously reported. Mutations in the other CGD subgroups are currently being analyzed. ? We also noted that genetic information is an important preditor of interferon gamma responsiveness of CGD patients. Monocytes from patients with missense mutations are responsive to interferon gamma (stimulation of a respiratory burst in response to the phorbol ester PMA) whereas monocytes from patients with nonsense mutations are not responsive. These studies may have important therapeutic implications for guiding future therapy. (Gallin, Malech, Holland, Kuhns).? ? In other studies Granulibacter bethesdensis, an organism infecting patients with chronic granulomatous disease of childhood (CGD)without inducing significant pathology typical of infection in these patients such as granulomatous inflammation. Granulibacter bethesdensis was shown to be complementresistent and relatively resistent to hydrogen peroxide. In addition, unlike other bacteria that infection CGD patients, Granulibacter bethesdensis was a poor inducer of neutrophil apoptosis and failed to induce reactive oxygen species in normal neutrophils. The data suggest that Granulibacter bethesdensis is a poor stimulator of host defenses and this may allow it to establish persistent infections. (Zarember, Gallin, Holland and Greenberg)
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