Abstracts 113

#137 9.6

EVIDEHCE OF CYTOLYTIC LYI~.IPHOCYTE MEDIATED CIIROIUC REJECTIOH I]FI TLI A[TD CYCLOSPORIHE TREATED BABOO]:IE WITt{ LO]:!GTERM SUEVIVI]:IG IIETEROTOPIC MOUKEY HEART XEITOGRAFTE. A. LTorin, P l. Roslin, ~.I. Coons, R. Tranbaugh, Departments of Hedicine, Anatomy & Cell Biology and Surgery, gUllY Brooklyn, i'lY Longterm survival (>100 days) of concordant .-:enografts has been difficult to achieve. ~,Jeut i l izedTLIcombined withcyclosporine(CyA) 125-150 rag'/day and prednisone (pred) 5 mg/day in a heterotopic monL:ey heart to baboon xenograft model. This combined approach resulted in >37>3",>10e,,180,>430 and 5a0 days of graft survival. In contrast, TLI induced survival of 27 and 2~ days and CyA-prednisone induced graft survival of 17,18 and 63 days. TL! t reated recipients and CyA-pred treated animals had evidence of vasculitis, in ters t i t ia l hemorrhage and cellular infi l trates on necroscopy, and early high t i ters (1/128) of cytotoxic antibody (CDC). Recipients receiving combined TLI and CyA-pred had no detectable CDC during the first 10{} days af ter t ransplantat ion. Recipients with late rejecting grafts at 18(~ and 540 days had no evidence of vasculitis and hemorrhage despite detection of CDC (within 2 mos. of rejection). Cytolytic T lymphocytes, CTL (against 51 Cr labeled monlzey lymptmcytes) were detected (46,47% cytotorici ty, E:T 100:1) in the heart xenografts from the later two recipients at rejection whereas no CTL activity was detected in peripheral blood. F, ubsequently, TLI-CyA animals with early evidence of moderate to severe cellular rejection (cell infi l t rates on biopsy, reduced function on L~UGA scans) were treated with an increased dose of Cy# (250 rag/day) with significant decrease in rejection phenomena. These results suggest (1) that the barrier to t ransplantat ion in concordant primate species may be overcome by eomhined immunosuppression with TLI and CyA and (2) that late xenograft failure in this model is due to CTL which can be inhibited by optimal CyA t rea tment .

#138 9.7

HLA TYPING VERSUS DNAANALYSIS FOR DETERMINING GENETIC RELATIONSHIPS, GL Rosner, TC Li and AA Zachary, Cleveland Clinic Foundation, Cleveland, OH.

A battery of three VNTR (variable number of tandem repeat) probes, TBQ7 (DIOS28), YNH24 (D2S44) and CMMI01 (DI4SI3) (Promega Corp., Madison, Wl) were used to analyze 97 cases of disputed paternity, and results were compared to HLA typings performed in our laboratory. DNAwas extracted, restriction digested with Hae III endonuclease, electrophoresed and transferred to nylon membranes. Each 32P-labelled probe was hybridized sequentially to assure proper allele assignment. Determination of exclusions was by visual inspection of coelectrophoresed mixtures of DNA from the child and alleged father. All 3]. exclusions by HLAtypingwere confirmed using one or more probes. Paternity was suggested when the child's paternal band could not be discriminated from one of the alleged father's bands. Measurement of restriction fragment sizes determined by handmeasurement and 3-point standard curves were compared to those using a talking digitizer (DNASTAR, Madison, WI), which simultaneously calibrates gel images using multiple standard lanes, and calculates band size. Each method had variances of less than 2%. Following a conservative method of allele assignment, allele frequencies for each probe were estimated using racially distinct populations of unrelated individuals. For Caucasians (N=128), each probe defined at least 20 alleles. The heterozygosity at each locus was >93%. Cumulative power of exclusion of 99.85% and probability of paternity of 96%-99+% were obtained. Therefore, DNAanalysis performs aswell as HLA typing and offers advantages, any tissue sample can be used t DNA is stable, and a single, direct technology can be utilized for separate genetic marker systems.