The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractant and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as associations of loss of function mutations in the corresponding human genes in human disease cohorts. In FY10 we reported discoveries in the following areas: 1. West Nile virus pathogenesis;2. severe congenital neutropenia;and 3.) invasive candidiasis. 1.) We found that among healthy individuals donating blood, homozygous mutation CCR5D32 is a risk factor for symptoms associated with WNV infection but not for infection per se. This extends our previous work in this area that identified this genotype as a risk factor for symptomatic WNV infection in patients presenting with symptomatic illness to a health care professional. The new study was based on comprehensive WNV testing of 35 million blood donations by the American Red Cross from the start of the screening program through July, 2008. The recent work is important because it provides new insight into the mechanism by which CCR5 deficiency increases risk of symptomatic WNV disease. The WNV work is particularly challenging because this is an ongoing emerging epidemic in which it is difficult to obtain useful samples. The overall results are scientifically important because they identify the first genetic risk factor for WNV disease and the first beneficial role in humans for CCR5. The significance extends beyond WNV to HIV/AIDS since CCR5 is exploited by HIV to enter target cells and since CCR5 antagonists are now being used in patients with HIV/AIDS. The new data suggest that blocking CCR5 with a drug may increase the risk of symptomatic WNV disease should treated patients become infected with WNV. 2.) In FY10 we reported preclinical data indicating that the most common mutation found in patients with the rare immunodeficiency known as WHIM syndrome is a gain of function mutation in the chemokine receptor CXCR4, and that the FDA-approved CXCR4 antagonist Mozobil is a full antagonist at this mutant receptor. This is important because it justifies clinical trials of this agent in patients with WHIM syndrome. In FY10 we obtained IRB approval to conduct such a trial and have identified 10 WHIM patients and recruited one to the trial. 3.) In FY10 we described the first two US patients with a novel immunodeficiency due to mutations inactivating glucose-6-phosphatase C3 (G6PC3), the only G6Pase isoform expressed in neutrophils. We found that these patients present with frequent severe bacterial infections due to neutropenia that occurs despite abundant mature neutrophils in the bone marrow. We demonstrated that the neutrophil stimulating factor G-CSF is highly effective in correcting neutropenia and reducing the risk of infection. We also presented evidence that patient neutrophils have extraordinarily high levels of CXCR4, which may explain why these cells are retained in the bone marrow, and we provided proof of principle in a mouse model of G6PC3 deficiency that Mozobil can be effective in correcting neutropenia. 4.) In FY10 we reported the first detailed survey of organ specific immunity in a mouse model of fatal bloodstream infection with the fungus Candida albicans. The work demonstrated that the fungus is rapidly cleared from the blood and distributed to all organs tested, but thereafter events vary greatly depending on the organ with regard to fungal proliferation/clearance, leukocyte accumulation, leukocyte organization within the organ and pathology. The scientific importance of this study is that it emphasizes the concept that pathology is a balance between host defense and dysregulated inflammation. Understanding the setpoint between appropriate and excessive immune responses may be useful in fine-tuning responses with drugs.
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