Interaction between genetic background and oxidative environmental stimuli in the pathogenesis of human lung disease has been largely unexplored. Biological response to oxidative stress is a key mechanism in numerous inflammatory diseases. In this program, we have begun a number of research initiatives that evolved from our mouse modeling of inflammation and acute lung injury. In the first project, we have extablished strong collaboration with Drs. Francine Kauffmann and Rachel Nadif (INSERM, Paris) to investigate the genetic basis of susceptibility to coal workers pneumoconiosis (CWP) in individuals differentially exposed to environmental oxidants coal dust and cigarette smoke. Drs. Kauffmann and Nadif have undertaken a prospective epidemiologic study in 253 coal miners, which included quantitative phenotypes of response to environmental stimuli that may be involved in CWP, an inflammatory lung disease. Six oxidative stress markers were studied as intermediate phenotypes of response to exposure, including erythrocyte glutathione peroxidase (GSH-Px) and catalase activities. Oxidant exposures studied were smoking habits and cumulative dust exposure assessed by job history and ambient measures of exposure. Disease phenotypes included subclinical computed tomography score at the first survey and X-ray profusion grades twice 5 years apart to assess established CWP. My laboratory obtained blood from each of the individuals recruited to the study and DNA was isolated. We began to investigate the genetic basis for CWP susceptibility by evaluating association of selected phenotypes with polymorphisms in the following classes or categories of genes: innate immunity, inflammation, and antioxidant. Miners were genotyped for common functional single nucleotide polymorphisms (SNPs) in a number of candidate genes and have found significant association of SNPs and disease outcome. Results suggest that interactions of genetic background with environmental exposure and intermediate response phenotypes are important components in the pathogenesis of disease (CWP) in coal miners. ? ? A second project has been established to investigate the genetic basis of susceptibility to adverse outcomes of acute respiratory distress syndrome (ARDS). ARDS is a major acute lung disease in adults and neonates, and is characterized by noncardiogenic edema and inflammation. The mortality rate for ARDS ranges from 50-80%. The incidence of ARDS is not well established, but approximately 2-8 cases per 100,000 are estimated. The mechanisms of susceptibility are unclear, and there are no specific therapies. Our studies in inbred mice have identified the transcription factor NRF2 as a candidate gene for susceptibility to hyperoxic lung injury, a model of ARDS. We have hypothesized, therefore, that loss-of-function polymorphisms in NRF2 predispose to oxidative lung damage associated with ALI/ARDS. To date, the genomic sequence of NRF2 is not well characterized and only a few SNPs have been localized to coding regions within the gene. In the current study, portions of the coding region and 1-kb of the promoter were amplified, sequenced and compared in order to locate NRF2polymorphisms. Subsequently, potentially important identified SNPs were evaluated in vitro for functional relevance NRF2 expression or binding activity. We are using a similar approach to identify and evaluate SNPs in the gene for KEAP1, an intracellular NRF2 inibitory protein. In a parallel investigation we have begun to evaluate SNPs in a target gene of NRF2, the phase II antioxidant NQO1 (NAD(P)H:quinone oxidoreductase1). We have identified novel SNPs in the promoter of NQO1 that have functional relevance to the cellular response to oxidant stimule. Functionally relevant SNPs in NRF2 and NQO1 have been evaluated by association for their importance in disease pathogenesis in a population of ALI patients through a collaboration with Dr. Jason Christie (University of Pennsylvania).? ? Our third project is designed to evaluate genetic mechanisms of host response to respiratory syncytial virus and innate immunity studies in mice and humans. This project is designed to investigate the role of toll-like receptors and antioxidant defense in respiratory syncytial virus (RSV) infection and disease progression. Because TLR4 and NRF2 appear to be critical to protection against RSV immune challenge, a collaboration has been established with Dr. Fernando Polack (Johns Hopkins University) to test the hypothesis that loss of gene function will enhance development of lower respiratory tract infection during primary RSV infection in a cohort of infected infants recruited in Buenos Aires, Argentina.? ? Our fourth project has been designed to investigate the role of antioxidant genes in the pathogenesis of bronchopulmonary dysplasia (BPD) in very low birth weight (VLBW) infants. BPD is the most frequent cause of chronic pulmonary illness in infants. In collaboration with Dr. Fernando Polack, we are prospectively examining the role of candidate antioxidant genes in susceptibility to BPD. We believe that understanding the role of antioxidant genes in susceptiblity to BPD can lead to immmediate identification of susceptible populations requiring a personalized medical approach, evaluation of available gene-targeting antioxidant agents of potential preventive and/or therapeutic value, and extension of these observations to other important oxidant stress-related diseases in the near future (e.g. COPD, lung cancer, asthma).
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