With the objective to determine whether coronary arteries from cardiac allografts from patients with chronic rejection contain clonally ex- panded T-cell receptor+ (TCR+) T cells, we carried out sequence analysis of -chain TCR transcripts from these coronary arteries, after PCR amplification, cloning and sequencing. RT-PCR was carried out using either VI subgroup (V2, V3, V4, V5) or VII subgroup (V9) specific primers as 5'-end amplification primers and a C-specific primer as the 3'-end amplification primer. Sequence analysis revealed that substantial proportions of identical VI and VII subgroup TCR transcripts were present in three of five patients examined. The V9J2.1C1 transcript (CDR3: ALWETAQELGKKI) accounted for twenty of twenty (100%) in-frame VII subgroup transcripts from one patient (GA09). In contrast, peripheral blood mononuclear cells from normal donors were used as a methodological control and were unique in general when compared to each other, in a manner typical of polyclonal populations of T cells. These findings suggest that TCR+ T cells can undergo clonal expansion in vivo in response to as-yet- undefined antigen(s) expressed in the coronary arteries of cardiac allografts from patients with chronic rejection. These TCR+ T cells may play a role in the immunopathogenesis and the progression of chronic rejection. 100 INCREASED EXPRESSION OF SINGLE-STRAND DNA-BINDING PROTEINS REPRESSES ALPHA-MYOSIN HEAVY CHAIN (MHC) GENE EXPRESSION IN HEART FAILURE J. Raman, 1 M.P. Gupta, 1 M. Gupta, 2 V. Jeevanandam, 11 Dept of Cardiac & Thoracic Surgery, The University of Chicago Medical Center, Chicago, IL; 2 Dept of Physiology, University of Illinois- Chicago, Chicago, IL Background: The power out-put generated by the heart is determined by expression of myosin heavy chain (MHC) isoforms. Loss of alpha- MHC in the myocardium is known to be a major cause of reduced contractile function in the failing heart. The mechanism of reduced expression of A-MHC is not understood. Methods: We had previously identified a conserved purine-rich repres- sor element (PNR) in the promoter region of the A-MHC gene, which controls cardiac muscle specific expression. After screening a mouse heart cDNA library, three proteins that bound to the A-MHC gene PNR element, were cloned and characterized, namely Pets (20kda double strand DNA-binding proteins of the Ets family), Pur-A and Pur-B (40kda single strand DNA-binding protein). Western blot analysis was performed in failing animal hearts in rabbits (n=5), and rats (n=5) in addition to explanted failing human hearts (n=7). Adenovirus vectors expressing full-length Pur-A or Pur-B proteins were infected into primary cultures of rat cardiac myocytes to analyze the effect of these proteins on expression of endogenous A-MHC genes. Results: High levels of Pur-A and Pur-B were found in failing hearts but not Pets, by Western blot analysis. Over-expression of Pur-B had a marked negative regulatory effect (80%) on the expression of A-MHC transcripts. Pur-A had a mild repressive effect (30%). When both Pur-A and Pur-B were expressed in combination, a synergistic negative regulatory effect on A-MHC gene expression was noticed. Conclusions: Single strand DNA-binding proteins, Pur-A and Pur-A contribute to reduced expression of A-MHC transcripts during heart failure. This may prove a potential target for heart failure intervention. 101 CHARACTERIZATION OF REGULATORY CD8CD28- T CELLS IN HEART TRANSPLANT RECIPIENTS A.I. Colovai, 1 M. Mirza, 1 G. Vlad, 1 S. Wang, 1 R. Cortesini, 2 N. Suciu-Foca, 11 Department of Pathology, Columbia University, New York, NY; 2 Servizio Trapianti d’Organo, Universita degli Studi di Roma “La Sapienza”, Rome, Italy Regulatory CD8+CD28- T cells (Ts) generated in vitro were demonstrated to suppress the activation and proliferation of T helper cells (Th) induced by allogeneic cells. This effect requires cell-to-cell contact, is antigen-specific and results in Th anergy. To study the population of CD8+CD28- T cells present in vivo, flow cytometry was performed on whole blood specimens obtained from 25 heart transplant recipients. A significant expansion of CD8+CD28- T cells was found in transplant recipients as compared to normal individuals (p=0.005). Expression of CD38, HLA-DR, and perforin positive cells within the CD8+CD28- subset was significantly higher in transplant patients than in normal controls, yet there was no correlation between the expression of these markers and acute rejection. Expression of the CD27 marker, however, was significantly higher within CD8+CD28- T cells from patients without rejection as compared to patients in rejection (p=0.005), indicating that the memory-like CD8+CD28-CD27+ subset comprises regula- tory cells. CD8+CD28- T cells isolated from patients with or without rejection did not display cytotoxic activity against donor cells and showed high expression of the killing inhibitory receptor CD94. This study identifies the phenotypic changes which occur within the CD8+ T cell subset in heart transplant recipients, compared to normal individuals, and opens new avenues for the induction of specific immunosuppresion in transplantation. 102 OPTIMIZATION OF A NOVEL LOW-STENGTH ELECTROPORATION SYSTEM FOR EX VIVIO GENE TRANSFER IN HUMAN HEART L. Sen, G. Guanggen, H. Russell, H. Furukawa, J. Xu, S. Zhang, H. Laks, Surgery, UCLA Medical Center/UCLA School of Medicine, Los Angeles, CA Electroporation (EP) is commonly used for in vitro gene transfection of cell lines and primary cultures, by applying short duration, high intensity (200-2000 volts/cm) electric field pulses to cells. Recently, we invented a novel low-strength EP system for ex vivo gene transfer in human heart. We developed a device consisted of two electrode arrays directly in contact with the endocardium and epicardium in order to achieve uniform gene transfer to the entire adult human heart. To optimize the EP parameters for achieving maximum gene transfer efficiency and lowest cardiac adverse effect, the human explanted hearts from patients with end-stage heart failure under- went orthotopic heart transplantation were used. The heart was mounted on a customized Langendorff perfusion apparatus, then human recombinant IL-10 gene was infused through intracoronary. The EP was applied during gene infusion for 20 minutes. With 10 volt/cm EP field strength, the maximum gene transfer efficiency was achieved, but associated with a mild cardiac contractility reduction in 15% of hearts. However, with 1-5 volt/cm electric field strength, no cardiac adverse effects was observed. The optimized pulse length was 1-10 ms, and the optimized number of pulse 1-20. The burst- interval 2 min prevented myocytes damage. With this optimized EP, the homogeneous electropermeabilization in whole heart was observed within 3 minuets after the beginning of EP using propidium iodide, a membrane-impermeant fluorescent probe. A significant increase in IL-10 mRNA level in myocardium of both ventricles was S106 Abstracts The Journal of Heart and Lung Transplantation January 2003