Quantitative fluorescence measurements of chloride secretion in native airway epithelium from CF and non-CF subjects.
Functional assessment of the efficacy of CFTR gene transfer protocols in humans has previously involved measurement of in vivo potential difference. We have studied whether freshly obtained airway epithelial cells may provide suitable tissue for studies of in vivo gene transfer using fluorescent digital imaging microscopy. Nasal epithelial cells from non-cystic fibrosis subjects (n = 6) and from cystic fibrosis (CF) patients (delta F508: delta F508, n = 5) were obtained by brushing and loaded with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Addition of the cAMP-agonists forskolin (20 microM) and 3-isobutyl-1-methylxanthine (IBMX, 100 microM) produced an increased efflux of iodide from the cells which was significantly (P < 0.05) greater in non-CF than in CF cells. Efflux following addition of the calcium ionophore, ionomycin (100 microM) was similar in both non-CF and CF cells. Liposome-mediated transfection of CF nasal epithelial cells in vitro with CFTR-cDNA restored the cAMP-stimulated efflux to non-CF values. Bronchial epithelial cells from non-CF subjects showed responses to forskolin and ionomycin that were not different to those in non-CF nasal epithelia. These data demonstrate that the assay provides a useful method for assessing correction of abnormal ion transport in non-cultured CF epithelium and is likely to provide a further assay for assessment of in vivo gene transfer efficiency in protocols of gene therapy for CF.
Stern
M
M
Ion Transport Unit, National Heart and Lung Institute, London, UK.
Munkonge
F M
FM
Caplen
N J
NJ
Sorgi
F
F
Huang
L
L
Geddes
D M
DM
Alton
E W
EW
D000328:Adult; D000368:Aged; D001980:Bronchi / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D002712:Chlorides / Q000032:analysis / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000235:genetics*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005456:Fluorescent Dyes; D015316:Genetic Therapy; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008875:Middle Aged; D009297:Nasal Mucosa / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D011806:Quinolinium Compounds; D012016:Reference Values; D014162:Transfection; D014420:Turbinates
C505032:CFTR protein, human; D002712:Chlorides; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D015759:Ionomycin; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium