M.J. Welsh and A.E. Smith, RAC Application 
d. Brushing and swab of nasal and pharyngeal mucosa for evaluation of an 
inflammatory response. 
Purpose : To determine the presence of an inflammatory response. 
Methods: For brushing, the nasal mucosa will be treated with 5 drops of Afrin 
(oxymetazolone hydrochloride 0.05% solution, Schering-Plough) and up to 1 ml of 2% 
Lidocaine five min before brushing. A cytobrush (used for PAP smears) will be used to 
gently brush the nasal mucosa for approximately 3 sec at the site of administration of the 
recombinant virus. The brush will then be removed and the cells dissociated from the 
brush into 2 ml of phosphate buffered saline. The cell suspension will be kept on ice until 
further use. The nasal mucosa, from each nostril and the pharyngeal mucosa will be 
swabbed with a cotton-tipped applicator. 
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. 
e. Biopsy of nasal epithelium. 
Purpose : The nasal epithelium will be biopsied for evaluation of an inflammatory infiltrate 
into and beneath the epithelium and for evaluation of cytopathic effects. 
Methods : The patient's nose will be anesthetized using, initially, a solution of eight parts 
1% Pontocaine and 2 parts 1/1,000 epinephrine which will be topically applied using 
neurosurgical cottonoid pledgets. The pledgets will be placed under direct vision utilizing 
endoscopic control. After ten minutes the area of the biopsy will be submucosally 
infiltrated with 0.5 ml of 1% xylocaine with 1/100,000 epinephrine, to ensure anesthesia 
and aid hemostasis. Approximately 0.5 cm^ of nasal mucosa will be resected from the area 
of virus application under telescopic control. This area will be identified by its relation to 
certain fixed landmarks in the nose, e.g. anterior end of inferior turbinate, cartilaginous 
nasal septum, membranous nasal septum. Immediately after the biopsy has been taken, a 
further cottonoid soaked in oxymetazoline hydrochloride 0.05% solution, will be placed on 
the biopsied area to effect hemostasis. We do not anticipate having to use nasal packing. 
The patient will be treated postoperatively with Tylenol as required for analgesia. 
The biopsy will be evaluated for the integrity of the epithelium and for inflammatory 
changes by conventional light microscopy, including hematoxalin and eosin stain. Slides 
will be reviewed by an independent pathologist. Electron microscopy will also be 
performed on the section. 
f. Nasal brushing and biopsy for evaluation of vector efficacy. 
Purpose: To evaluate the ability of Ad2/CFTR-1 to produce CFTR mRNA and protein. 
Methods: In cells from the nasal brushings, RT-PCR will be used to assess the presence of 
virally-produced mRNA. Evidence for mRNA production and its location will also be 
obtained by in situ hybridization. 
Evidence of CFTR protein production and location at the apical membrane will be assessed 
by immunocytochemistry using monoclonal antibodies directed against CFTR: Ml -4 and 
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Recombinant DNA Research, Volume 16 
