Document Detail


Pseudomonas aeruginosa infection destroys the barrier function of lung epithelium and enhances polyplex-mediated transfection.
MedLine Citation:
PMID:  17638571     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Challenged by the lack of success of experimental gene therapy of cystic fibrosis, we set out to investigate one of the potential causes of this failure, the barrier function of the airway epithelium and the way this is affected by bacterial infection. In an in vitro model of the airway epithelium we determined the effect of Pseudomonas aeruginosa or Escherichia coli on the transfection efficiency of polyethylenimine (PEI)-plasmid DNA complexes, carrying a luciferase gene, as well as on the barrier function of the epithelial cell layer, using transepithelial resistance (TER), cytotoxicity, bacterial transmigration, and morphological appearance as parameters. The level of luciferase expression was more than one order of magnitude higher in the cells which, before transfection, were incubated with P. aeruginosa. TER was strongly reduced by P. aeruginosa, whereas E. coli had no effect. Pseudomonas aeruginosa also effectively destroyed the structure of the tight junctions, as visualized by immunostaining of the zonula occludens. By the same token, small but significant numbers of P. aeruginosa cells were found to migrate through the epithelial layer, whereas no E. coli cells were observed at the transcompartment of the wells. Release of lactate dehydrogenase from the epithelial cells, a parameter of cell damage, occurred in a dose-dependent manner on incubation with P. aeruginosa, but not with E. coli. To evaluate the relevance of these results for the in vivo situation, we infected C57BL/6 mice with P. aeruginosa or E. coli 48 hr before transfecting them intratracheally with PEI-DNA polyplexes. Infection with P. aeruginosa caused a 5-fold increase in luciferase expression whereas infection with E. coli had no effect. Fluorescence microscopy of lung sections, after administration of fluorescein isothiocyanate-labeled polyplexes, showed that prior treatment with P. aeruginosa effectuated penetration of the complexes deeper into the epithelium than in untreated animals. In P. aeruginosa-treated animals fluorescence was detected not only in the airway epithelium itself but also in the parenchyma. We conclude that infection with P. aeruginosa causes disruption of the tight junctions between the cells and thus of the barrier function of the epithelium. As a consequence, PEI-DNA complexes injected intratracheally into infected animals gain access to the basolateral side of the cells and to spaces across the epithelial lining, giving rise to substantially increased transfection efficiency.
Authors:
Joanna Rejman; Sante Di Gioia; Alessandra Bragonzi; Massimo Conese
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Human gene therapy     Volume:  18     ISSN:  1043-0342     ISO Abbreviation:  Hum. Gene Ther.     Publication Date:  2007 Jul 
Date Detail:
Created Date:  2007-08-02     Completed Date:  2007-12-07     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9008950     Medline TA:  Hum Gene Ther     Country:  United States    
Other Details:
Languages:  eng     Pagination:  642-52     Citation Subset:  IM    
Affiliation:
Institute for Experimental Treatment of Cystic Fibrosis, San Raffaele Scientific Institute, 20132 Milan, Italy.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cells, Cultured
Escherichia coli Infections / physiopathology
Genetic Vectors*
Humans
Male
Mice
Mice, Inbred C57BL
Plasmids / genetics*
Polyethyleneimine / metabolism*
Pseudomonas Infections* / physiopathology
Pseudomonas aeruginosa*
Respiratory Mucosa / metabolism*,  microbiology
Tight Junctions / metabolism,  pathology*
Transfection
Chemical
Reg. No./Substance:
9002-98-6/Polyethyleneimine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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