The assessment of human islets for transplantation and the effect of the immunosuppressant rapamycin on MIN-6 cells, rat and human islets

Bell, Ewan (2004) The assessment of human islets for transplantation and the effect of the immunosuppressant rapamycin on MIN-6 cells, rat and human islets. MD thesis, University of Glasgow.

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Human islet transplantation is currently successfully establishing a role in the management of certain patients with type 1 diabetes. For islet transplantation to be successful there must be an adequate supply of high quality islets, which are damaged neither by host immunological defences nor the immunosuppressants used. In an attempt to characterise human islet quality several tests were developed. The static stimulation test assesses beta-cell function and responsiveness to glucose by measuring insulin release. To assess the degree of exocrine contamination of the islet preparation, the amylase content of both the whole pancreas and islet preparation were measured. Measuring the insulin content of both the whole pancreas and islet preparation allowed us to quantify the increase in insulin concentration as a consequence of the islet isolation procedure. The mean exocrine contamination (EC) was 2.5 +/- 0.7%, mean insulin enrichment (IE fold increase) 180 +/- 37 and mean static stimulation index (SSI) was 1.47 +/- 0.08 suggesting that the islet preparations have low levels of exocrine contamination, have significant insulin enrichment and release insulin in response to glucose. Thus the islet isolation procedure is reasonably successful at separating the endocrine from the exocrine pancreas. The curative potential of each islet material was then determined by transplanting representative islet samples to non-obese diabetic mice with severe combined immunodeficiency disease (NOD-SCID) as the biological endpoint in a standardised system. In the first two weeks following transplant, the degree of EC of the islet preparation may have some role in predicting the in-vivo function of islets, with those preparations of highest EC taking longer to restore normoglycaemia than the other groups. However, beyond 14 days post-transplant, EC is of no value in predicting the in-vivo function of islets. Similarly the degree of IE and the SSI have no value in predicting the in-vivo function of islets in this model. Thus, none of the biochemical indices (EC, IE nor SSI) are able to predict the in-vivo effectiveness or function of transplanted islets in the NOD-SCID mouse model. Non-heart beating donors (NHBDs) are generally not deemed suitable for whole organ pancreas donation but could provide a significant additional source of pancreata for islet transplantation if it was demonstrated that NHBD islets functioned no differently from islets isolated from heart-beating donors (HBDs). The recovery of islets from NHBDs was comparable to that of control HBD. In-vitro assessment of NHBD islet function revealed function equivalent to those isolated from HBD, and NHBD islets transplanted to NOD-SCID mice efficiently reversed diabetes. A single donor transplant from a NHBD resulted in a state of stable insulin independence in a type 1 diabetic recipient. Thus, normally functioning islets can be isolated successfully from NHBD pancreata, suggesting that NHBDs may provide an untapped source of pancreatic tissue for preparation of isolated islets for clinical transplantation. Rapamycin (sirolimus) is a macrolide fungicide with immunosuppressant properties that is used in human islet transplantation. Little is known about the effects of rapamycin on islets and MIN-6 cells (mouse clonal insulin-producing cells that are used experimentally as a beta-cell model). Rapamycin had a dose-dependent, time-dependent, glucose-independent deleterious effect on MIN-6 cell viability. At day 1, using the MTT method (this mitochondrial succinate dehydrogenase activity assay is used as an indirect measure of cell viability), 0.01 nM rapamycin reduced cell viability to 83 +/- 6% of control (p<0.05). Using the calcein AM method (a fluorescent marker of live cells), at day 2, 10 nM rapamycin caused a reduction in cell viability to 73 +/- 5% of control (p<0.001). Furthermore, 10 and 100 nM rapamycin caused apoptosis in MIN-6 cells as assessed by the TUNEL assay (apoptotic nicked DNA is fluorescinated and detected at the single cell level by flow cytometry). Compared to control, there was a 3.1 +/- 0.6 fold increase (p<0.01) in apoptosis in MIN-6 cells treated with 10 nM rapamycin. A supra- therapeutic rapamycin concentration of 100 nM significantly impaired glucose- and carbachol-stimulated insulin secretion of rat islets and had a deleterious effect on the viability of rat and human islets. Thus, currently there is no evidence that therapeutic concentrations of rapamycin have any in-vitro deleterious effect on islets.

Item Type: Thesis (MD)
Qualification Level: Doctoral
Keywords: Cellular biology, immunology, pharmacology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Wolf, Prof. Bryan and Shepherd, Prof. James
Date of Award: 2004
Depositing User: Enlighten Team
Unique ID: glathesis:2004-71093
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 05 Aug 2022 13:41
Thesis DOI: 10.5525/gla.thesis.71093
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