The lactoperoxidase (LPO) system is a significant contributor to airway host defense against bacteria, viruses and fungi and also is a major consumer of airway H2O2 that in turn is associated with airway inflammation. A loss of LPO activity may lead to increased airway infection, while any increase in LPO could have injurious consequences. This application will test the hypothesis that all components of the LPO system, including LPO enzyme expression and secretion, SCN~ transport across the epithelium and H2O2 production, are coordinately regulated and that an absence of the LPO substrate, SCN~, in cystic fibrosis (CF) may contribute to pathogenesis. This hypothesis is supported by a computational model of LPO system activity in airway surface liquid that predicts any unbalance in the components will render the system inadequate for host defense. The hypothesis and model are supported by preliminary data showing: a) CF airway epithelia lack normal transport of LPO's substrate, SCN~, to the apical surface that may contribute to airway infections and inflammation; b) airway epithelia produce H2O2 through Duox, an NADPH oxidase that may be regulated by intracellular [Ca2+]; c) reactive oxygen species (ROS) increase expression of LPO; and d) Duox and LPO mRNAs are up-regulated in CF airway epithelia.
Specific Aim 1 will address regulation of Duox H2O2 production in normal and CF airway epithelial cells by changes in [Ca2+]i, cAMP, SCN~ or proinflammatory and infection related stimuli.
Specific Aim 2 will test the hypothesis that defective SCN~ transport in CF airway epithelia results in decreased SCN~ in CF airway secretions in vivo that then may compromise host defense. SCN~ and peroxidase endproducts will be determined in exhaled breath condensate using liquid chromatography in tandem with electrospray ionization-mass spectrometry.
Specific Aim 3 will test the hypothesis that ROS and/or SCN~ increases LPO mRNA expression in normal and CF airway epithelia by using turnover studies and nuclear runoff assays. The 5' end of LPO mRNA will be mapped by RACE and promoter elements in upstream sequences identified.
Specific Aim 4 will test the hypothesis that depressed CF airway SCN~ levels contribute to neutrophil-mediated oxidative damage and to the phenotype of colonizing bacteria observed in CF airways. Lay Summary: These experiments will provide new information about physiological defects in cystic fibrosis patients that may lead to new therapeutic approaches to treating this disease. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL066125-06
Application #
7195753
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Banks-Schlegel, Susan P
Project Start
2001-03-01
Project End
2010-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
6
Fiscal Year
2007
Total Cost
$330,961
Indirect Cost
Name
University of Miami School of Medicine
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
052780918
City
Coral Gables
State
FL
Country
United States
Zip Code
33146
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