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Strategies to Combat Inflammation in Pancreas and Islet Transplantation

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Strategies to Combat Inflammation in Pancreas and Islet Transplantation COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Director of Copyright Services sydney.edu.au/copyright Faculty of Medicine Strategies to combat inflammation in pancreas and islet transplantation Tina Ghoraishi A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy 3rd of September 2014 Declaration This thesis is the result of full time and part time study during the period from February 2007 to August 2014. The work, except where duly acknowledged, is my own, and has not been submitted for any other degree at this, or any other University. In vivo experiments in the porcine brain death model involved the participation of a large number of collaborators at the Victor Chang Cardiac Research Institute. The work in the murine brain death model was performed jointly with Zane Wang, packaging and purification of the rAAV vectors was carried out by Moumita Paul. Tina Ghoraishi August 2014 ii Acknowledgements I would like to express sincere appreciation to the many individuals who have offered support and encouragement throughout this project. First and foremost, my deepest gratitude goes to my supervisor A/Prof Alexandra Sharland, for her constant support and inspiration, for patiently guiding me through this research effort with her generosity and wealth of knowledge. Appreciation is extended to collaborators from the Victor Chang Cardiac Research Institute for their great contribution to development of the porcine brain death model. Special thanks go to Zane Wang and Chuanmin Wang for their endeavours in establishment of murine brain death model. I also would like to sincerely thank Moumita Paul for her efforts in virus preparation, and all her assistance. Additionally, appreciation is expressed to The Transplant Society of Australia and New Zealand, and Professor Christopher Jackson for providing resources. Last but not least, I am very grateful to assistance and support from all the group members; Alexander Bishop, Eithne Cunningham, Miriam Habib, Mamdouh Moawadh and Anar Ganbold. Thank you all, without your valuable contribution this degree would have never been completed! iii For my husband, his love and encouragement keeps me pursuing my dreams. For my mother, her unconditional love and kindness brighten difficult days. For my sisters, for having faith in me. And, to the loving memory of my father, for all-time. iv Contents Abstract ................................................................................................................................. viii List of tables .......................................................................................................................... xii List of figures ....................................................................................................................... xiii List of abreviations .......................................................................................................... xviii CHAPTER 1 ................................................................................................................................ 1 Introduction ............................................................................................................................. 1 1.1 Deceased donor organ transplantation ....................................................................... 2 1.2 Indications for pancreas and islet transplantation ................................................. 2 1.3 Organ quality and the outcomes of pancreas and islet transplantation ........ 10 1.4 Clinical outcomes ............................................................................................................... 16 1.5 The innate immune system............................................................................................. 21 1.6 Brain death ........................................................................................................................... 41 1.7 Donor management/preconditioning ........................................................................ 51 1.8 Experimental strategies to reduce graft inflammation ........................................ 53 1.9 Modulation of immune responses using soluble RAGE ........................................ 59 CHAPTER 2 ............................................................................................................................. 62 Materials and methods ...................................................................................................... 62 2.1 Porcine neonatal islet cell cluster (pNICC) preparation and culture .............. 63 2.2 Porcine Aortic Endothelial Cell isolation from piglet aortas .............................. 79 2.3 Preparing tissue segments for immunohistostaining purposes ....................... 81 2.4 Immunohistostaining methods ..................................................................................... 82 2.5 Brain death induction in pigs and different donor management protocols . 85 2.6 Apoptosis detection Assays ............................................................................................ 91 2.7 Splenocyte isolation from C57BL/6 mouse spleen ................................................ 96 2.8 Brain death induction in C57BL/6 mice ..................................................................... 97 CHAPTER 3 ........................................................................................................................... 113 Inflammation in porcine neonatal islet cell clusters (PNICCs) ......................... 113 3.1 Introduction ...................................................................................................................... 114 3.2 Porcine neonatal islet cell clusters in culture after isolation ......................... 115 v 3.3 Gene expression profiling of porcine neonatal islet cell clusters in culture 117 3.4 Activated protein C and the endothelial protein c receptor ............................ 129 3.5 Obtaining full length EPCR from porcine neonatal islet cell clusters and other cell types ............................................................................................................................ 135 3.6 Porcine aortic endothelial Cell (PAEC) treatment with rHuAPC .................... 145 3.7 Antibody against human EPCR is active on porcine tissue but EPCR protein was not detected in islets ........................................................................................................ 156 3.8 Treatment of pNICCs with recombinant human activated protein C ............ 160 3.9 Discussion .......................................................................................................................... 167 CHAPTER 4 ........................................................................................................................... 170 Hormone resuscitation of the brain-dead donor and its effect on pancreatic inflammation and function ............................................................................................ 170 4.1 Introduction ...................................................................................................................... 171 4.2 Physiological changes in BD pig ................................................................................. 172 4.3 Gene expression in pancreas from brain-dead pigs ........................................... 177 4.4 Measurement of insulin production in pancreatic islets .................................. 186 4.5 Detection of apoptosis in pancreas tissue from brain-dead pigs ................... 190 4.6 Discussion .......................................................................................................................... 203 CHAPTER 5 ........................................................................................................................... 209 Systematic overexpression of esrage and its effects on the pancreas and islets in brain-dead mice ............................................................................................................ 209 5.1 Introduction ...................................................................................................................... 210 5.2 Expression of esRAGE in vivo after inoculation with rAAV esRAGE .............. 211 5.3 Physiological changes and systematic inflammatory response in brain dead mice 215 5.4 Investigation of inflammation in the pancreas post
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