Adenovirus vectors are a promising vehicle to deliver cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. However, the value of adenovirus vectors will depend on the ...efficiency with which the vector can correct the defective fluid transport that is though to underlie the pathogenesis of the disease. To address the efficiency of gene transfer, we applied adenovirus vectors expressing CFTR (Ad2/CFTR-1) or beta-galactosidase to the mucosal surface of primary cultures of airway epithelial cells grown as polarized epithelial monolayers on permeable filter supports. These conditions provide a model that reproduces the physiology of the airways in vivo. We found that after adding 1 moi Ad2/CFTR-1 to the mucosal surface, cAMP agonists stimulated fluid secretion that was within the range observed in epithelia from normal subjects. When we measured electrolyte transport, we found that as little as 0.1 moi partially restored cAMP-stimulated Cl- secretion, and at 10 moi Cl- secretion was in the normal range. A related vector encoding beta-galactosidase generated activity in approximately 20% of cells at an moi of 1 and 90% of cells at an moi of 10. These data suggest that Ad2/CFTR-1 is very efficient at restoring normal fluid and electrolyte transport to CF airway epithelia. Thus, they suggest that relatively low input doses could be used for gene transfer to CF airway epithelia.
Serum (IS) was obtained 0.5, 2, 4 or 6 h after inoculating s.c. six rabbits (≈2 kg) in each time period with 1 mg/kg of
Tityus
discrepans (Td) venom; the control was serum obtained from four rabbits ...4 h after injecting them 1 ml s.c. of 0.9% NaCl. IS produced a transient (<25 min) rise in pulmonary artery pressure of isolated and perfused rabbit lungs, other lung parameters were not altered. We found that both scorpion venom and IS produced a ≈50% transient increase of transendothelial electric resistance in cultured tissue human umbilical cord vein. Neither venom nor IS changed the transepithelial electrical resistance of tissue cultured human airway epithelia. The experiments suggest that humoral factors contained in the inoculated serum modify vascular endothelium in a much more effective manner than the venom by itself. These experiments also make it unlikely that vascular endothelium is the source of the humoral factors contained in inflammatory serum.
Cell proliferation, vector titer and accessibility of target cells represent hurdles for efficient gene transfer to lung epithelia in vivo using recombinant murine leukemia (MuLV)-based retroviruses. ...We tested the hypothesis that the pulmonary epithelium is susceptible to retroviral-mediated gene transfer when stimulated to proliferate by a mitogen, keratinocyte growth factor (KGF).
Rats received keratinocyte growth factor (KGF, 2.5 micrograms/g x 4 doses, two consecutive days) intratracheally followed by high titer amphotropic retrovirus expressing beta-galactosidase. Gene transfer was assessed five days later.
KGF stimulated transient proliferation in the bronchiolar and alveolar epithelia (30-40% PCNA positive cells at peak) which decreased to background levels seven days after administration. Gene transfer to epithelia (X-Gal positive cells) occurred more frequently in KGF treated rats, but proliferation exceeded the level of gene transfer. X-gal positive cells were noted in the alveolar epithelium and occasionally in the bronchiolar epithelium. In order to understand the discrepancy between the number of proliferating and transduced cells, primary rat tracheal epithelium cultured at the air-liquid interface was infected from either the apical or basolateral side. Gene transfer was achieved only through basolateral application of vector, suggesting that epithelial polarity represents a barrier to MuLV-based lung gene transfer in vivo.
KGF transiently stimulates epithelial proliferation in vivo, facilitating MuLV-based gene transfer. Retroviral vectors may encounter multiple barriers which have evolved to defend the lung from infections.
The human airway epithelium lines the respiratory tract from the nasal mucosa to the bronchioles. Electrolyte transport by these epithelia is crucial in maintaining the appropriate volume and salt ...composition of the airway surface fluid. When this epithelium becomes functionally impaired, the airways are more prone to respiratory infections. We studied the effect of six common topical agents that are commonly used to treat rhinorrhea and nasal inflammation on the transepithelial resistance, sodium, and chloride transport of primary cultures of human airway epithelia grown at the air-liquid interface. The pharmaceuticals fluticasone propionate, cromolyn sodium, ipratropium bromide, azelastine, oxymetazoline, and normal saline were used and the electrical function of the epithelia was studied in Ussing chambers. Azelastin and ipratropium bromide-treated epithelia were found to have a significant decrease in transepithelial resistance. Both normal saline and fluticasone propionate resulted in significant increases in amiloride-sensitive short circuit currents that reflect sodium transport. Finally, normal saline resulted in a significant increase in bumetanide-sensitive short circuit current that reflects chloride transport across the epithelia. The data presented may explain a mechanism by which some topical pharmaceuticals help reduce rhinorrhea, and may point to some unwanted side effects of some pharmaceuticals on the electrolyte transport of the airway epithelia. In summary, several of the common topical nasal agents alter the electrolyte transport of the nasal airway epithelia. The in vivo significance of these findings is to be determined.
We report the measurement of transepithelial voltage across the nasal epithelium in a neonate with pseudohypoaldosteronism (PHA) type 1. A 5-day-old infant was seen with hyponatremia, hyperkalemia, ...and elevated plasma renin and aldosterone levels. Sweat Cl
- concentration was also increased. Measurements of voltage showed a basal value of zero and the absence of an amiloride-sensitive voltage. However, voltage changed as expected for normal cyclic adenosine monophosphate–stimulated Cl
- transport. These data demonstrate the absence of amiloride-sensitive Na
+ transport across airway epithelia in a neonate with PHA. The findings suggest that measurements of voltage could be of value in the diagnosis of PHA. (J Pediatr 1999;135:786-9)
Gene transfer with integrating vectors such as recombinant retrovirus has the potential to correct inherited lung diseases permanently. As a gene therapy target, the pulmonary epithelium presents ...several challenges to vector delivery in vivo. Many of the host defenses that have evolved to prevent infection from inhaled bacteria or viruses represent potential barriers to gene transfer to the lung. We performed in vitro studies to determine whether two components of the innate immune system of the lung, airway surface fluid and alveolar macrophages, inhibit retroviral gene transfer to airway epithelia. Human alveolar macrophages obtained by bronchoalveolar lavage from normal subjects were left untreated or activated with lipopolysaccharide (LPS) for 3 hr in the presence of subconfluent human bronchial epithelial cells (HBE); than 4 x 10(5) cfu DA-luciferase retrovirus was added. Three days after infection, luciferase activity was measured in cell lysates. When the epithelial cells were co-cultured with LPS-activated macrophages, retroviral gene transfer to HBE cells was reduced by approximately 60%. Nonactivated macrophages decreased the transfection to approximately 55% of control values. In control experiments with either activated or inactivated macrophages but without epithelia, no luciferase activity was detected, suggesting that terminally differentiated alveolar macrophages are not infected by the recombinant retrovirus. Pretreatment of alveolar macrophages with dexamethasone restored gene transfer to approximately 60% of control values. In contrast, incubation of retrovirus with airway surface fluid had no inhibitory effect on gene transfer. These experiments document that AM inhibit retrovirus-mediated gene transfer to airway epithelia in vitro, and may represent a barrier to retroviral gene transfer in vivo. These barriers may be overcome, at least partially, with pharmacological agents.
