0041-1337/01/7112-1709/0 TRANSPLANTATION Vol. 71, 1709–1718, No. 12, June 27, 2001 Copyright © 2001 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. Transplantation  ARTICLES NOVEL APPROACHES TOWARD EARLY DIAGNOSIS OF ISLET ALLOGRAFT REJECTION 1 A.M. JAMES SHAPIRO, 2,5 ER GENG HAO, 2 JONATHAN R.T. LAKEY, 2 WALTER J. YAKIMETS, 2 THOMAS A. CHURCHILL, 2 PARASKEVI G. MITLIANGA, 4 GEORGE K. PAPADOPOULOS, 4 JOHN F. ELLIOTT, 3 RAY V. RAJOTTE, 2 AND NORMAN M. KNETEMAN 2 Surgical-Medical Research Institute, Department of Surgery, and Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada; and Laboratory of Biochemistry and Biophysics, Technological Educational Institute of Epirus, Greece Background. The inability to diagnose early rejec- tion of an islet allograft has previously proved to be a major impediment to progress in clinical islet trans- plantation. The need to detect early rejection will be- come even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation. Methods. (a) Canine islet allograft transplant recip- ients were immunosuppressed for 1 month, then ther- apy was withdrawn. Serum glutamic acid decarboxyl- ase antigen (GAD 65 ), an endogenous islet protein, was monitored daily with a CO 2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral- specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glu- cose tolerance tests were followed until graft failure occurred. Results. (a) Although serum monitoring of GAD 65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engi- neered rodent islets demonstrated normal insulin ki- netics in vitro (insulin stimulation index 2.570.2 vs. 2.950.3 for control islets, Pns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet “sentinel signal” could be detected serolog- ically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demon- strated evidence of allograft dysfunction (decline in K G ) with a 2-day lead time to hyperglycemia (2.580.3 vs. 1.630.2%/min, respectively, P<0.001), with an ac- curacy of 89%, sensitivity of 78%, and specificity of 95%. Conclusions. Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunc- tion due to rejection. INTRODUCTION The inability to diagnose early rejection of an islet allograft has been a key factor that until recently had severely ham- pered progress in clinical islet transplantation. This was a major drawback, because without recourse to appropriately directed intensification of immunosuppressive treatment in the background of rejection rates of up to 40 – 60% under glucocorticoid and cyclosporine immunosuppression, the ma- jority of islet grafts were destroyed by their first and final rejection episode. In other forms of transplantation, a tissue biopsy would have provided confirmatory indication for in- tervention and reversal of rejection, after elevations in serum tests (e.g., liver enzymes for liver transplant, creatinine for kidney transplant, amylase, lipase, and/or anodal trypsino- gen for pancreas transplant). If solid organ transplants were thus handicapped, the current 1-year graft survival in excess of 90% enjoyed by selected centers would fall dramatically, and would be predictably of the order of 40 – 60% (1). Recent dramatic progress in clinical outcome of islet trans- plantation has enhanced previous graft survival from 8% at 1 year (2, 3) to 100% with sustained insulin independence in a small, consecutive series of 7 patients undergoing islet-alone transplantation at our center (4, 5). These grafts continue to function, providing sustained normoglycemia without insulin with the longest follow-up currently 21 months, without ev- 1 This study was supported by a Juvenile Diabetes Foundation- Diabetes Interdisciplinary Research Group Grant. A.M.J.S. was sup- ported by a Clinical Fellowship Award, J.R.T.L. by a Postdoctoral Fellowship Award, and N.M.K. by a Scholarship Award from the Alberta Heritage Foundation for Medical Research (AHFMR). G.K.P. received support for GAD 65 assay development from a European Union Biotech Program Grant. 2 Surgical-Medical Research Institute, Department of Surgery, University of Alberta. 3 Department of Medical Microbiology and Immunology, Univer- sity of Alberta. 4 Laboratory of Biochemistry and Biophysics, Technological Edu- cational Institute of Epirus. 5 Address correspondence to: Dr. James Shapiro M.D., FRCS(Eng), FRCSC, 2D4.37 Department of Surgery, University of Alberta Hospi- tals, Mackenzie Health Sciences Center, 8440 - 112 Street, Edmonton, AB, Canada T6G 2B7. E-mail: amjs@powersurfr.com. 1709