Abteilung Innere Medizin Ii a Comparison of Gene
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ABTEILUNG INNERE MEDIZIN II Schwerpunkt Endokrinologie und Diabetologie A COMPARISON OF GENE EXPRESSION IN TOLERATED AND REJECTED ISLET GRAFTS BY GENE MICROARRAY ANALYSIS VERGLEICH DER GENEXPRESSION IN TOLERIERTEN UND ABGESTOSSENEN INSELTRANSPLANTATEN MITTELS MICROARRAY-ANALYSE aus der MEDIZINISCHEN UNIVERSITÄTSKLINIK ABTEILUNG INNERE MEDIZIN II Schwerpunkt Endokrinologie und Diabetologie der ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG I. BR. in Kooperation mit dem DIABETES INSTITUTE FOR IMMUNOLOGY AND TRANSPLANTATION DER UNIVERSITY OF MINNESOTA INAUGURAL-DISSERTATION ZUR ERLANGUNG DES MEDIZINISCHEN DOKTORGRADES DER MEDIZINISCHEN FAKULTÄT DER ALBERT-LUDWIGS-UNIVERSITÄT FREIBURG I. BR. 2003 TOBIAS BERG BERNKASTEL-KUES DEKAN: Prof. Dr. med. Josef Zentner 1. GUTACHTER: Prof. Dr. med. Martin Reincke 2. GUTACHTER: Prof. Dr. med. Lars Christian Rump JAHR DER PROMOTION: 2005 FOR BRITTA A comparison of gene expression in tolerated and rejected islet grafts by gene microarray analysis INTRODUCTION ........................................................................................................ 6 MATERIAL AND METHODS ................................................................................... 11 Animals.................................................................................................................................... 11 Transplant protocol................................................................................................................ 11 Treatment ............................................................................................................................. 11 Bone marrow transplantation ............................................................................................... 11 Islet isolation and transplantation......................................................................................... 12 Islet graft function and harvest............................................................................................. 13 Microarray hybridization...................................................................................................... 13 RNA isolation....................................................................................................................... 13 Reverse transcription............................................................................................................ 14 In-vitro transcription ............................................................................................................ 15 Array hybridization, scanning & analysis............................................................................ 15 Real-time RT-PCR to monitor gene expression .................................................................. 18 Gene expression analysis by RNase Protection Assay ........................................................ 20 RESULTS................................................................................................................. 21 Course of transplantation and histology .............................................................................. 21 General overview of microarray analysis result.................................................................. 22 T cell genes .............................................................................................................................. 28 Costimulation and adhesion molecules ................................................................................ 29 Complement system and innate immunity........................................................................... 29 Cytokines and cytokine receptors......................................................................................... 30 Chemokines and chemokine receptors................................................................................. 32 Confirmation of microarray results ..................................................................................... 34 DISCUSSION ........................................................................................................... 37 SUMMARY............................................................................................................... 41 ZUSAMMENFASSUNG ........................................................................................... 42 REFERENCE LIST................................................................................................... 43 ACKNOWLEDGEMENTS ........................................................................................ 52 PUBLICATIONS....................................................................................................... 54 LEBENSLAUF ......................................................................................................... 55 Introduction Seite -6- Introduction Type I diabetes is a serious condition that affects approximately one million individuals in the United States (55) and about 174,000 people (2000) in Germany (43). Other estimates are even higher especially if latent autoimmune diabetes in adults is considered as well. It has long been known that most cases of type I diabetes are caused by a T cell-regulated autoimmune disease with immune-mediated destruction of the insulin-producing β-cells in the islets of Langerhans in the pancreas. Signs for this are the development of islet-antigen specific antibodies in the serum of type I diabetes patients (9) and the incidental transfer of diabetes from one individual to another by bone marrow transplantation (31). The survival of a patient with type I diabetes depends on a lifelong replacement therapy with insulin, so that until insulin became available, type I diabetes was a lethal disease (7). Modern intensified insulin replacement therapy can prolong the life-span of patients with type I diabetes, but their life expectancy is still limited by the earlier or later occurring onset of diabetes complications. Even with intensified insulin therapy glucose control cannot be completely normalized and glucose control is one of the most important factors for the long-term prognosis of a type I diabetic patient (4; 61). An additional approach to the treatment of type I diabetes is prancreas transplantation which has first been performed by Richard Lillehei and William D. Kelly at the University of Minnesota in 1966 (28) and has developed into a successful method for the treatment of diabetic patients under certain circumstances such as hypoglycemia unawareness or end-stage renal disease, where a combined pancreas-kidney transplant has advantages in comparison to kidney transplantation alone (20; 59). A different approach for restoring islet function in a diabetic patient is the transplantation of isolated islets, which is a promising perspective for the cure of type I diabetes without invasive surgical procedures. An islet transplant is basically performed by harvesting a donor pancreas and extracting islets from it. The islet extraction for the purpose of human transplantation is today done using the automated method for pancreatic islet isolation (30; 47). A special blend of collagenase enzyme is pumped down the pancreatic duct in a controlled perfusion system designed to cleave islets from their acinar restraining matrix. The pancreas is then transferred to a recirculating Ricordi digestion chamber. After extensive washing, islets are purified on a continuous density gradient in a cell apheresis centrifuge system. The transfer of the islets into the patient can be carried out without surgery under local anesthetic using radiological guidance. Islets are implanted into the portal vein where they embolize to the liver, nest and Introduction Seite -7- develop a new blood supply. The procedure can be completed as a day case, with patients discharged from hospital in less than 24 hours (54). Donor Recipient Figure 1: Systematic overview of islet transplantation procedure. Steps of the process are the procurement of the pancreas (usually from a brain-dead cadaver donor), the islet isolation procedure with enzymatic and mechanic disruption of the pancreas followed by a density gradient separation and finally the transplantation by portal vein infusion with ultrasonographic or radiological guidance or in a surgical procedure. Figure from the homepage of the Diabetes Institute for Immunology and Transplantation. (www.diabetesinstitute.org) Although it has been successfully used as autotransplantation procedure for the treatment of patients undergoing total pancreatectomy (48) and despite occasional successes (3), islet transplantation for type I diabetes had not been a very successful procedure for quite a long time. In the transplants performed between 1990 and 1997 only 37 % of all transplanted islet grafts survived one year with insulin independence in 11 % of all transplanted patients. In the transplants from the period from 1998 to 2000 there was an improvement in graft survival to 55 % at one year, but insulin independence was only achieved in 14 % of all transplanted patients (10). But recently the field of islet transplantation has made significant progress. A.M. James Shapiro and Jonathan R.T. Lakey in Edmonton achieved insulin independence and established normoglycemia in a very high percentage of patients with type 1 diabetes (53). Introduction Seite -8- 82% of them have maintained independence from insulin at the 1-year mark (49). The success can be explained by the use of a sufficient number of isolated human islets, which in this protocol requires the isolation