Islet transplantation is capable of restoring normal blood sugar control in patients with insulin dependent diabetes mellitus (DM). In spite of the great success shared by several islet transplant centers, one of limiting factors that has impaired the rapid success of this treatment is the shortage of organs. Donation after Cardiac Death (DCD) is a viable alternative that will aid in reducing this shortage. At present, islets for transplant into patients with type 1 DM are not typically isolated from DCD pancreata. Our goal is to provide a comprehensive assessment of human islets from DCD donors and compare them to islets from Donation after Brain Death (DBD) donors. The specific hypothesis is that islets from DCD donors are functionally equivalent to or even superior to islets from DBD. This hypothesis is based on observations that: a) long terms results of solid organ pancreas transplants with organs from DCD have provided excellent long term glycemia control and normalization of glycosylated hemoglobin; b) it is possible to isolate large number of islets, and to cure type 1 diabetes mellitus with a single donor islet transplant from a DCD pancreas; c) organs from DCD donors are not exposed to the hormonal and pro-inflammatory cytokine release which is typically present in DBD donors due to the physiological effect of brain death. These effects have been shown to contribute to apoptosis and even necrosis of the beta cells.
The specific aims of this study are to compare isolated human islets from DCD donors to those from DBD donors by: 1) characterization of the physiological response to glucose as a rapid potency indicator of the quality of the islet preparation prior to transplantation, 2) determination of the effects of hypoxia on islet viability and function after isolation, 3) determination of the sensitivity of islets to cytokine induced apoptosis. Kinetic analysis of the pyridine nucleotide redox states, intracellular calcium, and mitochondrial membrane potential in response to glucose challenge will be measured. The effects of hypoxia after human islet isolation will be assessed through the quantification of hypoxia inducible transcription factor-1 (HIF-1) and the formation of reactive oxygen species (ROS), nitric oxide, and lipid peroxidation. Measurements of the mechanism of repair such as superoxide dismuatase and catalases upregulation will also be performed. The results obtained in this aim will aid in the development of therapies to protect against hypoxia induced islet death. The percentage of apoptotic islets from DBD and DCD will be assessed immediately post isolation and afterculture with IL-1beta, TNFalpha, and IFNgamma; mimicking the effects of ischemia/reperfusion injury after transplant. Determination of viability and apoptosis will be performed using a novel approach in which islets can be analyzed intact, preserving their complete architecture using Complex Object Parametric Analyzer and Sorter (COPAS) flow cytometry and results will be correlated in vivo. Increasing the donor pool available for islet isolation would significantly increase the availability of this therapy for type 1 diabetic patients. In addition, the development of rapid, accurate, and predictive tests of islet viability and function post isolation will contribute to significant improvements in post transplant success.
| Hanson, Matthew S; Park, Elisa E; Sears, Mallory L et al. (2010) A simplified approach to human islet quality assessment. Transplantation 89:1178-88 |
| Hanson, Matthew S; Steffen, Anja; Danobeitia, Juan S et al. (2008) Flow cytometric quantification of glucose-stimulated beta-cell metabolic flux can reveal impaired islet functional potency. Cell Transplant 17:1337-47 |
| Armann, B; Hanson, M S; Hatch, E et al. (2007) Quantification of basal and stimulated ROS levels as predictors of islet potency and function. Am J Transplant 7:38-47 |
