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 dif ference. We have studied whether freshly obtained airway epithelial ce lls may provide suitable tissue for studies of in vivo gene transfer u sing fluorescent digital imaging microscopy. Nasal epithelial cells fr om non-cystic fibrosis subjects (n=6) and from cystic fibrosis (CF) pa tients (Delta F508; Delta F508, n=5) were obtained by brushing and loa ded with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Addition of the cAMP-agonists forskolin (20 mu M) and 3-isobutyl-1-methylxanthine (IB MX, 100 mu M) produced an increased efflux of iodide from the cells wh ich was significantly (P<0.05) greater in non-CF than in CF cells. Eff lux following addition of the calcium ionophore, ionomycin (100 mu M) was similar in both non-CF and CF cells. Liposome-mediated transfectio n of CF nasal epithelial cells in vitro with CFTR-CDNA restored the cA MP-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 epithelial. These data demonst rate 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 eff iciency in protocols of gene therapy for CF.