M.J. Welsh and A.E. Smith, RAC Application 
Ad2-ORF6/PGK-CFTR 
Item 5 - Clinical Protocol 
one day after administration. If there is viral replication, we expect that after an initial decline, the titer of 
live virus in the nasal swab would increase. We will determine whether wild-type or recombinant virus is 
produced by culture on HeLa cells, by restriction enzyme analysis, and by sequencing. 
Swab of nasal mucosa for evaluation of an inflammatory response . 
Purpose : To determine the presence of an inflammatory response. 
Methods'. The nasal mucosa from each nostril will be swabbed with a cotton-tipped applicator. The cells 
will be dissociated into 2 ml of phosphate buffered saline. The cell suspension will be kept on ice until 
further use. Evidence of an inflammatory response will be assessed by cytological examination of 
cytospin preparations of the cells using Wright stain. The cell differential count will be determined for 
each specimen. Cell morphology and cytopathic effects will be evaluated using the PAP stain. 
Transepithelial electrical potential difference across the nasal epithelium . 
Purpose: To assess the electrophysiological consequences of treatment. Patients with CF have an 
abnormally increased transepithelial electrical potential difference across the nasal epithelium (Vt) (12,24). 
In addition, the response of Vt to several agents that regulate transport is abnormal. The abnormalities in 
patients with CF are well characterized and relatively easy to measure. Correction of the abnormality 
would indicate that administration of the recombinant virus has corrected the CF electrolyte transport 
defect in the nasal epithelium. 
Methods: Measurement of the nasal Vt is safe, easy, and not invasive (12,24). The Vt across the nasal 
epithelium will be measured using techniques similar to those previously described (12,41). A 23 gauge 
subcutaneous needle connected with sterile normal saline solution to a silver/silver chloride pellet (E.W. 
Wright, Guilford, CT) is used as a reference electrode. The exploring electrode is a size 8 rubber catheter 
(modified Argyle® Foley catheter, St Louis, MO) with one side hole at the tip. The catheter is filled with 
Ringer's solution containing (in mM), 135 NaCl, 2.4 KH2PO2, K2HPO4, 1.2 CaCl2, 1.2 MgCl2, and 
10 Hepes (titrated to pH 7.4 with NaOH) and is connected to a silver/silver chloride pellet Voltage will 
be measured with a voltmeter (Keithley Instruments INC., Cleveland, OH) connected to a strip chart 
recorder (Servocorder, Watanabe Instruments, Japan). Prior to the measurements, the silver /silver 
chloride pellets are connected in series with the Ringer's solution; the pellets are changed if the recorded 
Vt is greater than ±4 mV. The rubber catheter is introduced into the nostril under telescopic guidance 
(Hopkins Telescope, Karl Storz, Tuttlingen West Germany) and the side hole of the catheter is placed 
next to the inferior nasal turbinate. Basal Vt will be recorded until no changes in Vt are observed after 
slow intermittent 100 (il/min infusion of the Ringer’s solution. Once a stable baseline is achieved, 200 fil 
of a Ringer's solution containing 100 jjM amiloride (Merck and Co. Inc., West Point, PA) will be 
instilled through the catheter and changes in Vt will be recorded until no further changes are observed 
after intermittent installations. Finally, 200 |il Ringer's solution containing 100 }iM amiloride plus 10 |iM 
terbutaline (Geigy Pharmaceuticals, Ardsley, NY) will be instilled and the changes in Vt recorded. The 
entire procedure will take between 15-30 minutes. 
Mucosal fluid for adenovirus antibody . 
Purpose: To test for the presence on the mucosal surface of IgA or IgG antibody to the recombinant 
adenoviruses (types 2 and 5) and to test for neutralizing antibodies to these serotypes. 
Methods: We will obtain a washing of the nasal surface to test for the presence of specific and 
neutralizing antibody. 
Pulmonary function tests . 
Purpose: To assess patient's pulmonary function. 
Methods: Spirometry, lung volumes, and diffusing capacity will be measured in the Pulmonary Function 
Laboratory at the University of Iowa Hospitals and Clinics using standard techniques. 
Arterial blood gases . 
Purpose: To assess patient's pulmonary function. 
Methods: Blood will be obtained by radial artery puncture and PO2, PCO2, and pH will be measured in 
the Pulmonary Function Laboratory at the University of Iowa Hospitals and Clinics. 
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Recombinant DNA Research, Volume 18 
