and immunoprecipitation) 
In addition to the evaluation of biologic correction in airway epithelial 
cells removed by brushing, the CFTR- related function of the intact respiratory 
epithelial sheet will be evaluated by quantifying the potential difference 
between the surface of the airway epithelium and the subcutaneous tissues. In 
normal individuals the potential difference is -25 - 1 mV, whereas in CF it is 
> -40 mV (Knowles et al . , 1981). 
To measure the nasal electrical potential an intravenous catheter is placed 
subcutaneously in the forearm and connected to an infusion of Ringers so- 
lution. Less than 1 ml of Ringers solution is infused to flush the catheter 
and establish continuity of the subcutaneous space and the infused solution. A 
second catheter, the "exploring catheter", is filled and constantly perfused 
with Ringers solution. A Y-connection is present in both infusion lines with a 
silver chloride electrode in one of the limbs of each. Both electrodes are 
connected to a battery operated, high impedance volt meter, which is connected 
to a chart recorder. The exploring catheter is placed on the surface of 
epithelia in various locations within the nasal cavity and the electrical 
potential difference between the mucosal surface and the interstitial space is 
measured. Visualization is achieved with a headlight and nasal speculum. To 
measure tracheal and bronchial potential, the equipment is set up in the same 
manner as for measuring nasal potential, the only difference being that the 
infusion for the exploring catheter is connected to a length of polythene 
tubing that can be placed through the suction channel of the bronchoscope and 
then rested on the tracheal or bronchial surface. 
Increased serum levels of tumor necrosis factor, have been correlated with 
exacerbations of cystic fibrosis (Suter et al . , 1989a); this parameter will 
also be followed. 
The biologic efficacy parameters relating to the respiratory epithelium are 
central to the goals of this protocol. In this context, and in recognition 
that not all parameters can be measured at each time point because of 
limitations in biologic materials, inability for a bronchoscopy to be carried 
out appropriately for clinical and/or technical reasons and variability in the 
biologic parameters, the following categorizes the respiratory epithelial- 
related biologic parameters into primary and secondary efficacy parameters. 
All efforts will be focused on obtaining, at a minimum, the primary efficacy 
parameters. The primary parameters will include: expression of normal CFTR 
genotype at the mRNA level (sequence and PCR with specific probes) in nasal 
and bronchial epithelium, expression of CFTR protein ( immunohistochemistry) in 
nasal and bronchial epithelium, and measurement of potential difference across 
the nasal epithelium. The secondary parameters will include: level of total 
CFTR mRNA (quantitative PCR, Northern, in situ hybridization) in nasal and 
bronchial epithelium, secretion of Cl - in response to intracellular elevation 
of cAMP ( 36 C1“ efflux, SPQ dye) in nasal and bronchial epithelium, and 
expression of CFTR protein (immunoprecipitation and phosphorylation with 
kinase, 35 S -methionine metabolic labeling and immunoprecipitation) in nasal 
and bronchial epithelium. 
5. 5. 3. 2 Clinical Efficacy Parameters 
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Recombinant DNA Research, Volume 16 
