into the posterior segment of the left upper lobe via a bronchoscope. After ten 
minutes the virus was removed by gentle aspiration. The animal had pre-existing 
disease in his right lung that was evident on chest X-ray and which revealed collapse 
of his right upper lobe. 
The clinical and laboratory features of B208 are summarized in Appendix I. 
The animal tolerated the procedure well without clinical evidence for acute toxicity. 
The blood hematology and chemistry evaluations were unchanged three days later 
at the time of necropsy. At necropsy, the lungs were removed en bloc, inflation 
fixed in glutaraldehyde, and stained with X-gal in situ. This provided a sensitive, 
specific, and comprehensive analysis of the lung for genetic reconstitution. The 
distribution of lacZ expression was predominantly localized to the segment that was 
instilled with virus. A small area of adjacent lobe contained a localized area of lacZ 
activity. Within the transduced lobe there was extensive expression of lacZ. The 
majority of cells in the alveolus, including type II and type I cells, were positive. It 
was difficult to assess the full extent of gene transfer into macrophages because the 
lungs were irrigated with PBS before fixation. LacZ activity was also found in 
patches in proximal airways of the segment. Low levels of activity were detected in 
the more distal airways. Frozen sections of other organs, including heart, liver, 
spleen, kidney, brain, and testes, were stained in X-gal to evaluate for possible 
dissemination of virus. All extrapulmonary tissues were negative. It was difficult to 
evaluate the lung for pathology related to the adenovirus because the animal had 
pre-existing disease. Diffuse atelectasis and abnormalities of the airway epithelia 
were noted in the lobe contralateral to the adenovirus-treated lobe which had 
demonstrated collapse on chest X-ray. Evaluation of tissue from the adenovirus 
treated segment revealed essentially normal airspace and abnormalities in the 
airway epithelium that were noted in nontreated tissue. A peribronchial 
inflammatory response was noted in some areas of the treated segment. 
Similar studies of the direct instillation of Ad5-CMV-lacZ and Ad5-CB- 
CFTR into the nasal cavity of non-human primates are underway. 
IV.B.2. Possibility of ectopic expression of Ad5-CMV-lacZ 
Because of the possibility that Ad5-CB-CFTR virus could be swallowed by 
patients consequent to nasal administration, the ability of the adenoviral vector to 
transduce gastrointestinal cells was tested. In a series of studies, Ad5-CMV-lacZ 
was lavaged into the stomachs of mice (300 n\, 10^ pfu/ml) and expression of Ad5- 
CMV-lacZ determined by X-gal staining in stomach, small intestine, and large 
intestine. In a series of 8 animals, no expression in stomach or intestines was 
observed. 
To test for the possibility of germline transmission, a volume of Ad5-CMV- 
lacZ (lCr 1 pfu/ml) equal to the volume to be instilled in the nasal cavity of mice 
was injected intravenously into male and female mice (n = 3 each). Two days after 
injection, the testes and ovaries were removed and stained with X-gal. No X-gal 
positive cells were seen in testes or ovaries. In addition, no untoward events were 
seen in the animals consequent to injection of Ad5-CMV-lacZ. 
In a second series of experiments, we infused lacZ adenovirus into the 
venous circulation of 6 male rabbits (10-10^ pfus) and 2 male rhesus monkeys 
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Recombinant DNA Research, Volume 17 
