Our laboratory routinely grows synovial fibroblasts obtained from human, 
rheumatoid synovia, and are have shown these to be susceptible to transduction by 
MFG-IRAP. 
In conjunction with Theresa Whiteside, Ph.D., director of the Adoptive Immuno 
Therapy Laboratory, and a co-investigator on this proposal, we are optimizing the 
conditions for culturing human synoviocytes grown in defined medium (Basal Fibroblast 
Growth Medium, Clonetics) supplemented with human type AB serum, and for 
transducing them with MFG-IRAP-tk. 
(vi) Human Svnovial Fluid Analysis 
Malyak et al 1993 (31) recently reported mean levels of 17.1 +/- 12.6ng/ml IRAP 
in 13 human rheumatoid synovial fluids. Fluids from three other RA patients had no 
detectable IRAP. In a similar study. Miller et al (32) reported synovial fluid IRAP levels 
in Lyme arthritis between 0 and 26ng/ml. 
We have also analyzed a series of human rheumatoid and non-rheumatoid 
synovial fluids for the presence of IRAP. In agreement with the literature (31,32), we 
have also found detectable IRAP concentrations. Initially, we feared that this would 
compromise our ability to measure expression of transgene expression in human 
rheumatoid fluids. However, Western blotting has repeatedly shown that the IRAP 
present in human rheumatoid fluids migrates analogously on SDS-PAGE, appearing as a 
band of approximately 50 kDa in M.W. (fig. 18). The transgene product, however, runs 
authentically at approximately 25 kDa. When rheumatoid synovial fluid is mbced with 
transgene IRAP, a Western blot reveals two bands, one at 50 kDa and the other at 25 
kDa, confirming the authenticity of this distinction (fig. 18). 
Preliminary data using a bioassay based upon the mitogenic response of a mouse 
thymocyte cell line, D.IO to IL-1, suggest that the high M.W. IRAP present in 
rheumatoid fluid has reduced bioactivity. This would explain why the relatively small 
increment in IRAP concentrations produced in the human trial (see section 6-C, p. 21) 
produced a measurable clinical effect. It also permits optimism that the IRAP of the 
correct M.W. synthesized by DFG-IRAP-tk infected cells will produce a measurable 
biological effect. 
Recombinant DNA Research, Volume 19 
[439] 
