M.J. Welsh and A.E. Smith, RAC Application 
Conclusion 
The results of these studies indicate that the Ad2/CFTR-1 virus directs expression of CFTR 
mRNA and protein in vivo in primate respiratory epithelial cells. As in the study with the 
Ad2/GGal-l, we found that there was some cross contamination of the control nostril; 
again, this most likely is a result of physical movement of the virus from one side to the 
other. As with Ad2/GGal-l, we were unable to quantitate expression of CFTR in monkey 
nasal cells since we are unsure of the sensitivity of the immunocytochemical assay. It 
should be noted, however, that using the same antibody to detect CFTR in normal human 
airway tissue endogenous protein is exceedingly difficult to detect with certainty (32). 
c. Ad2/BGal-l applied to the bronchial epithelium. 
Procedure 
For this study, we used 2 monkeys (G & H). As of late September, they have been 
followed for 5 weeks. 
The monkeys were anesthetized by intramuscular injection of ketamine (15 mg/kg) and 0.5 
mg of atropine. Flexible fiberoptic bronchoscopy was performed after instilling 1 ml of 1% 
lidocaine over the vocal cords. An airway lavage catheter (Baxter 792017, a modified 
pulmonary artery catheter with two balloons separated by 15 mm and two ports that end 
between the two balloons), was inserted through the bronchoscope channel. The monkeys 
were placed in the left lateral decubitus position to decrease shunt, and the distal balloon 
was inflated with 2 ml of air at the level of the take off of the right middle lobe. The 
proximal balloon was then inflated below the take off of the right upper lobe. This 
procedure isolated a small area of the bronchus intermedius. Ad2/GGal-l (1.6 x 10 8 PFU in 
0.5 ml) was then instilled through one of the inter-balloon ports and flushed with 0.25 ml of 
tris-buffered saline (TBS). After 15 min the balloons were deflated, allowing the 
Ad2/GGal-l virus preparation to run by gravity into the right middle lobe and the right 
lower lobe. The bronchoscope was then removed and the monkeys recovered from 
anesthesia. 
To obtain bronchial epithelial cells, the monkeys were anesthetized as described above, and 
flexible fiberoptic bronchoscopy was performed. A 3 mm cytology brush (Bard) was 
advanced through the bronchoscope and a small area of the bronchus intermedius and the 
left main bronchus was brushed gently for about 3 seconds. The resulting cells were 
dislodged from brushes into 2 ml of sterile PBS. Bronchoalveolar lavage was performed by 
wedging the tip of the bronchoscope in the right middle lobe bronchus. An aliquot of 20 ml 
of sterile PBS at room temperature was infused. Fluid was then withdrawn by hand suction 
into a syringe. The return from the 20 ml infusion ranged between 8 and 14 ml. 
Results 
The cell suspension was processed as described above in 3. a. X-Gal stains failed to reveal 
any blue cells at any point of the study. We believe this may have been due to the low 
sensitivity of the chromogenic staining technique. Therefore, for the samples from day 14, 
we elected to use a fluorescent assay, employing FDG (fluorescein di-G-D- 
galactopyranoside. Molecular Probes Inc) as the reporter for the presence of G- 
galactosidase activity. The use of FDG in a fluorescence-activated cell analysis is reported 
to be several orders of magnitude more sensitive than chromogenic assays (142). When 
analyzed by a fluorescence-activated cell sorter, bronchial epithelium from Monkey H 
revealed a bimodal distribution of fluorescence intensity consistent with G-galactosidase 
activity in at least 10% of the cells. No G-galactosidase activity was detected in bronchial 
Recombinant DNA Research, Volume 16 
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