M.J. Welsh and A.E. Smith, RAC Application 
CF Polyp 
CAMP 
t 
DPC 
t 
Ad2/CFTR-1 
Normal Polyp 
CAMP 
Figure 11. Effect of Ad2/CFTR-1 infection on Cl* secretion by CF airway epithelium. 
Tracings are short-circuit current in the presence of mucosal amiloride (10 (iM). 
cAMP agonists (20|iM forskolin and 100 |lM EBMX) and 3 mM diphenylamine-2- 
carboxylate (DPC) were added at times indicated. Response of a normal airway 
epithelium is shown for comparison. 
In our first study, we used two different preparations of Ad2/BGal-l virus: one that was 
purified on a CsCl gradient and then dialyzed against tris-buffered saline to remove the 
CsCl, and a crude unpurified one. Titers of the Ad2/6Gal-l viruses were ~2 x lQ^O PFU in 
the purified preparation and ~ 1 x 10 11 PFU in the crude preparation. Both preparations 
produced B-galactosidase activity in 293 cells. 
Monkeys were anesthetized by intramuscular injection of ketamine (15 mg/kg). We used 
the entire epithelium of one nasal cavity in each monkey. We inserted a foley catheter (size 
10) through each nasal cavity into the pharynx, inflated it with 2-3 ml of air, and then 
pulled the catheter anteriorly to obtain tight posterior occlusion at the posterior choana. 
Both nasal cavities were then irrigated with a solution (~5 ml) of 5 mM dithiothreitol plus 
0.2 U/ml neuraminidase in phosphate-buffered saline (PBS) for five minutes. We used this 
solution to dissolve any residual mucus overlaying the epithelia. (In our first in vitro 
studies with monolayers of human airway epithelia we had used a similar treatment to 
remove mucus. Subsequently, we have found that such treatment is not required.) The 
washing procedure also allowed us to determine whether the balloons were effectively 
isolating the nasal cavity. We then instilled the virus (Ad2/6Gal-l) slowly into the right 
[892] 
Recombinant DNA Research, Volume 16 
