Current theories of CF pathogenesis predict different predisposing "local environmental" conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Psuedomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O 2 consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.
Current theories of CF pathogenesis predict different predisposing "local environmental" conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Psuedomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O 2 consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.
We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.
Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.Among the most serious manifestations of CF are chronic pulmonary infections with the bacterium P. aeruginosa. The basis for hypersusceptibility of CF patients to this bacterium is not well understood, and the role of mutant CFTR, if any, is not clear. Binding and internalization of respiratory pathogens by epithelial cells followed by desquamation could be an important mechanism for clearing bacteria from the lung. This mechanism has been shown to be important in protecting against bladder infections (1).To investigate whether the most common and severe CFTR mutation (ΔF508) affected uptake of P. aeruginosa, we performed bacterial invasion assays (2) with four cell lines: CFT1, an airway epithelial cell line derived from a CF patient homozygous for ΔF508 CFTR and that is transformed with human papilloma virus 18 E6/E7; CFT1-ΔF508, which expresses a third copy of ΔF508 CFTR introduced by a retrovirus; CFT1-LC3, which expresses a control gene (β-galactosidase) introduced by the same retrovirus; and CFT1-LCFSN, which expresses a functional wild-type human CFTR gene (3). We tested a standard laboratory strain of P. aeruginosa, designated PAOI, and two nonmucoid, LPSsmooth clinical isolates from CF patients (4). Compared with CFT1-LCFSN cells, the three lines expressing ΔF508 CFTR internalized significantly fewer bacterial cells (Fig. 1A). The ΔF508 mutation causes inefficient processing of CFTR, a defect that is partially corrected if the cells are grown at 26°C (5). When epithelial cells were cultured for 72 hours at 26°C there were no longer significant differences in uptake of the P. aeruginosa strains by the cells expressing wild-type or mutant CFTR (Fig. 1B). Because the overall uptake of bacteria at 26°C was low, we performed additional experiments with cells grown for 72 hours at 26°C in which the invasion assay was performed at 37°C for 3 hours, conditions under which surface expression of mutant ΔF508 CFTR is maintained (5). No significant difference in bacterial cell uptake was measured (Fig. 1C), and overall amounts of internalization approached those of the CFT1-LCFSN cells at 37°C. Returning cells expressing ΔF508 CFTR to 37°C for 24 hours after growth for 72 hours at 26°C removes CFTR from the cell surface (5); under these conditions internalization of the bacterial strains ...
The success of adenoviral vectors for gene therapy of lung disease in cystic fibrosis (CF) depends on efficient transfer of the complementary DNA encoding the correct version of the cystic fibrosis transmembrane regulator (CFTR) to the affected columnar epithelial cells lining the airways of the lung. Pre-clinical studies in vitro suggest that low doses of adenovirus vectors carrying this CFTR cDNA can correct defective Cl- transport in cultured human CF airway epithelia. Here we use mice carrying the disrupted CF gene to test the efficacy of this transfer system in vivo. We find that even repeated high doses can only partially (50%) correct the CF defect in Cl- transport in vivo and do not correct the Na+ transport defect at all. We investigated this discrepancy between the in vivo and in vitro transfer efficiency using CF mouse and human samples, and found that it reflects a difference in the susceptibility to adenovirus-5 transduction of the epithelial cell types dosed in vivo (columnar) and in vitro (basal-cell-like). These studies indicate that more efficient adenoviral gene-transfer vectors and/or refinement of dosing strategies are needed for therapy of CF lung disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.