The lavage fluids collected in the OR and "baseline" lavages obtained one week 
earlier will be analyzed for evidence of transgene expression and for evidence that 
transgene expression has altered various clinical parameters (Fig. 9). IRAP 
concentrations will be measured by conventional ELISA, and the presence of IRAP 
protein of the correct size will be further confirmed by Western blotting. To assess 
whether IRAP has altered pathophysiological processes within the joint, we will measure 
leukocyte concentrations, and additionally measure levels of IL-1)3, IL-6, IL-8 and PGE^- 
All of these are induced by IL-1 and are thus down-regulated by IRAP. 
Solid tissue (synovium, sub-synovium and adjacent capsule) will be divided into 
three samples. One will be cut into frozen sections which will be used for in situ 
hybridization to detect the presence of tk message, immunohistochemistry to detect the 
presence of tk protein and standard hematoxylin and eosin staining for histological 
examination. A second sample will be used for extraction of DNA and mRNA for 
detecting the presence of DFG-IRAP-tk genes by PCR, and DFG-IRAP-tk mRNA by 
RT-PCR. A third sample will be placed into organ culture to detect the ganciclovir- 
sensitive production of recombinant IRAP. If insufficient material is available, the first 
two sets of assays will be given priority. 
E^ch patient will undergo complete history and physical examination (H&Ps) at 
2nd-5th joint arthroplasty, and at 2 weeks, 6 weeks, 3 months, 6 months, 1 year, 2 years, 
3 years, 4 years, 5 years, 7 years, 9 years and every two years thereafter. At the 2nd-5th 
MCP joint arthroplasty and 2 weeks, 6 weeks, 3 months, and 1 year visits for follow-up, 
25ml of blood will be drawn and the leukocytes analyzed for the presence of viral DNA 
by PCR. Serial H&Ps and blood draws comprise the extent of patient monitoring for 
this study. 
The remainder of the clinical procedures which might be utilized for this study 
relate to the possibility, though unlikely occurrence, of untoward events. The most likely 
complication of participation in this study would be septic MCP joint induced at the time 
of genetically modified synoviocyte reimplantation. This complication would be clinically 
dealt with in an appropriate fashion on a case-by-case basis depending upon extent, 
location and identity of inciting pathogen. Appropriate antibiotic therapy would be 
initiated immediately. Open drainage of the affected joint(s) and temporary delay of the 
2nd-5th MCP joint arthroplasty would comprise a worst case scenario. 
In rabbit studies, we have failed to detect migration of the transplanted cells from 
the joint into which they were injected. However, as an additional safety measure, the tk 
gene has been included in the vector. In the highly unlikely event that eradication of the 
transduced cells were necessary, ganciclovir would most likely be administered over one 
hour intravenously at a dosage of 5mg/kg every 12 hours for 14 days in a patient with 
normal kidney function. The dosage and response to therapy would be evaluated by the 
infectious disease consultant. 
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Recombinant DNA Research, Volume 19 
