Of the 2,500 children born every year with cystic fibrosis (CF) in the United States, 80% harbor P. aeruginosa by the time they reach 18 years of age. Recurrent Pseudomonas pulmonary infections are the major cause of morbidity and mortality in these patients. Airway structural damage is induced by excessive neutrophil populations in the airway, releasing large amounts of proteases which impair phagocytic killing of organisms. To date, the repertory of anti-inflammatory therapies to limit excessive epithelial cell signaling to inflammatory mediators remains limited and imperfect, which may be partially due to a limited understanding of critical pathways to curb inflammation and their role in the pathogenesis of CF. Evidence suggests that the zinc finger protein, lung kruppel like factor (LKLF), described here for the first time as highly regulated by infection, plays a significant role in the regulation of the airway epithelial cell response to Pseudomonas infection. Based on preliminary data, Dr. Saavedra hypothesizes that a) the transcriptional activator LKLF is cytoprotective in normal airway cells, b) that it suppresses nuclear factor kappa B activation and c) that in cystic fibrosis, overexpression of LKLF is not protective due to alternate apoptotic/death pathway activation.
These aims will be studied with an in vitro epithelial cell air liquid interface culture model system and the well characterized Pseudomonas strain PAO1. The approach will initially utilize functional genomics to define pathways activated by overexpression of this gene utilizing an LKLF plasmid construct. The true focus will be on elucidating gene function with apoptosis assays, luciferase reporter constructs, EMSA experiments and use of various constructs overexpressing genes involved in NF-kappaB activation to ascertain target site of LKLF activity along that pathway. These experiments are designed to further understanding of how epithelial cells drive neutrophil recruitment, knowledge of which may contribute to development of new therapeutic interventions in inflammatory airways diseases such as CF. This project will allow Dr. Saavedra to become an independent investigator and expert in the realm of Pseudomonas and airway epithelial cell biology, via a multi-faceted approach of 75% dedicated laboratory time and didactic training with a special focus on mechanisms of microbial pathogenesis. Outside of the laboratory, 25% clinical time will be spent as an instructor in adult Pulmonary and Critical Care Medicine, taking care of both inpatients and outpatients with CF, with the ultimate goal of a closely knit research and clinical niche as a principal investigator and expert adult CF doctor.
| Saavedra, Milene T; Hughes, Grant J; Sanders, Linda A et al. (2008) Circulating RNA transcripts identify therapeutic response in cystic fibrosis lung disease. Am J Respir Crit Care Med 178:929-38 |
| Saavedra, Milene T; Patterson, Abby D; West, James et al. (2008) Abrogation of anti-inflammatory transcription factor LKLF in neutrophil-dominated airways. Am J Respir Cell Mol Biol 38:679-88 |
| Case, D; Irwin, D; Ivester, C et al. (2007) Mice deficient in galectin-1 exhibit attenuated physiological responses to chronic hypoxia-induced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 292:L154-64 |
