forskolin and IBMX stimulated a rapid rise in 100% of the cells infected the wild type Ad.CB- 
CFTR virus which was quenched when iodide was substituted for NO3. None of the cells that were 
mock infected or infected demonstrated cAMP mediated changes in fluorescence. Cells infected 
with Ad.CB-CFTR at MOI’s ranging from 50 to 500 consistently produced good SPQ responses in 
100% of the cells. 
Experiments were performed to test the feasibility of detecting recombinant derived CFTR in 
primary isolates of CF airway epithelial cells. Cells removed from the main stem bronchus of a 
CF patient were piated in culture and exposed to either Ad.CMV-lacZ or Ad.BA-CFTR at an MOI of 
500. High levels of recombinant derived CFTR were clearly detected in 100% of the Ad.BA- 
CFTR cells (Figure 4). Techniques are being developed for measuring cAMP mediated Cl 
conductance in the primary cultures using the SPQ assay. 
II.B.4. Xenograft model of the Human CF Airway 
A critical evaluation of the safety and efficacy of adenoviral-mediated transfer of the CFTR gene 
to human airway would be greatly simplified if there was an authentic animal model. Critical 
questions relate to the biology of El deleted Ad5 in the context of a human airway and 
parameters necessary to achieve functional correction in this structure. Most currently 
available animal models fall far short. A major problem is that the airway of humans differs 
substantially in function and anatomy from that of other species. For example, many species, 
including rodents, are essentially void of the sites of mucous production found in humans (i.e., 
goblet cells of the surface epithelium and submucosal glands). This may explain why the 
recently described murine models of CF, generated by mutating CFTR in the germ line, have no 
pathology in the lung [Snouwaert et al., 1992]. Another important consideration is the marked 
variation in infection and replication of adenoviruses that is observed in recipient cells from 
different species. 
Our strategy was to develop a model based on the development of a CF human airway xenograft 
established in a nu/nu mouse. A schematic representation of the steps involved in the procedure 
are shown in Figure 5. Primary airway epithelial cells are released from lung tissue obtained 
in the setting of lung transplantation. CF cells are obtained from the bronchi of diseased lung 
that has been removed, and non-CF cells are obtained from proximal airway of the lung that is 
being transplanted into the CF patient. The cells are cultured for several days in the presence of 
antibiotics, released with trypsin, and seeded into the lumen of rat trachea that have been 
stripped of their epithelium by freeze thawing. The seeded grafts are then implanted 
subcutaneously into the flanks of nu/nu mice with the proximal and distal ports of the graft 
open to the environment via tubing ligated to the ends. The grafts are irrigated with normal 
saline biweekly to remove accumulations of mucus. 
The grafts are allowed to mature in vivo for 3 weeks after which a fully differentiated 
pseudostratified epithelium has developed. Light micrographs of GMA embedded tissue from a 
native bronchus and a xenograft are shown in Figure 6. We have now generated 108 grafts from 
18 non-CF bronchial samples and 104 grafts from 13 CF bronchial samples. 
The xenografts have been subjected to extensive morphometric analysis in order to evaluate the 
suitability of the model. Blocks of tissue from a non-CF and CF xenograft were carefully 
examined by transmission electron microscopy. Overall structure of the epithelium, 
distribution of cell types (i.e., ciliated, goblet, basal, and undifferentiated), and ultrastructural 
features of the various types of cells was determined. These findings were compared to similar 
analyses of native main stem bronchus (non-CF and CF) located adjacent to the area from which 
the cells destined for xenografts were removed. Electron micrographs of non-CF and CF 
[814] 
Recombinant DNA Research, Volume 16 
