To determine whether epithelial ion transport is physiologically important for lung water clearance after birth, the sodium transport inhibitor amiloride or its vehicle saline was given intratracheally to newborn full-term guinea pigs before the first breath. Guinea pigs given saline intratracheally breathed normally and had arterial O2 saturations (SaO2) greater than 94%. In contrast, guinea pigs that had an estimated 10(-4) M intra-alveolar concentration of amiloride had chest wall retractions and 88 +/- 3.6% (SD) SaO2 (P less than 0.01). Extravascular lung water (EVLW) per gram of dry lung weight 4 h after birth was significantly greater in newborns that received amiloride (8.3 +/- 1.1, n = 5) than in those that received saline (5.6 +/- 0.9, n = 7, P less than 0.01). The degree of perivascular fluid cuffing at 25 cmH2O inflation was quantitatively similar in amiloride- and saline-treated animals. The effect of amiloride was dose dependent. Intratracheal amiloride did not affect EVLW in 9-day-old guinea pigs. This study demonstrates that intratracheal amiloride before the first breath results in respiratory distress, hypoxemia, and an abnormally high EVLW. Epithelial sodium transport contributes normal lung liquid clearance after birth.

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-Expression using either mode of administration was maxivector formulations are two strategies to obtain gene transmal 24 h after injection and declined to around 10% of day fer to the lung. It is still uncertain, however, which of these 1 levels 2 weeks after injection. For i.v. delivery of DODAC: two modes of delivery will be more effective in the treat-DOPE-DNA complexes multilamellar vesicles were more ment of cystic fibrosis and other lung diseases. In this effective than large unilamellar vesicles in all organs invesstudy, we attempted to optimize formulations of the cationic tigated. Recombinant DNA could be detected in the distal liposome DODAC:DOPE (dioleoyldimethylammoniumlung region following either route of administration. Howchloride:dioleoylphosphatidylethanolamine) complexed to ever, i.t. administration predominantly led to DNA depoplasmids encoding chloramphenicol acetyltransferase for sition in epithelial cells lining the bronchioles, eg in clara i.t. and i.v. injection into CD-1 mice and compared the two cells, whereas i.v. administration resulted in DNA depomethods. Our results showed that both methods conferred sition in the alveolar region of the lung including type II reporter gene expression in the lung that was significantly alveolar epithelial cells. higher relative to injection of plasmid DNA alone.

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.