T1DM results from the immune-mediated destruction of the body's only insulin producing cells, the pancreatic beta cells. Beta cells comprise only about 2% of the pancreatic cell number and are grouped together into cell clusters with alpha cells (making glucagon), delta cells (making somatostatin), F cells (making pancreatic polypeptide), and other rare cells to form """"""""mini-organs"""""""" called islets of Langerhans, or simply """"""""islets."""""""" Isolated destruction of islet alpha, delta, or F cells has not been described. In contrast, isolated immune-mediated beta cell destruction does occur such that pancreata from T1DM animal models or from patients with newly diagnosed T1DM reveal """"""""islet remnants"""""""", i.e. small islets with few or no beta cells, but apparently normal numbers of the other islet cell types. Over time however, clinical evidence indicates that patients with T1DM also lose alpha cell function suggesting that normal alpha cells are dependent upon normal beta cell function. The """"""""mini-organ"""""""" concept is apt for at least 2 other reasons: (1) the different islet cell types are organized in a typical pattern: beta cells centrally located and the other cells types located more peripherally, and (2) islets consume more pancreatic blood flow (about 20%) than their small mass would suggest. Thus islets are important, not well-understood structures with unique physiological and immunological features, and they represent an Achilles' heel for individuals destined to develop T1DM. This project has six parts, to: (1) improve isolation techniques to more predictably yield high quality islets, (2) improve assays for characterizing isolated islet quality, (3) develop the non-human primate islet transplant model to address important pre-clinical questions, (4) initiate human clinical islet transplant protocols, (5) develop assays for characterizing islet function post-transplant, and (6) develop a renewable islet source. Beginning in 7/99, in collaboration with the Clinical Center's Department of Transfusion Medicine/Cell Processing Unit and Dr. Ricordi (University of Miami's Diabetes Research Institute), islets have been isolated from 27 human pancreata and since 9/00, from 4 rhesus monkeys. Before initiating the isolations, we established two separate laboratories, one for human glands and one for animals, and we trained a cadre of technicians. State of the art islet isolation units typically achieve islet yields of only about half the pancreatic total, and even that level is achieved about half the time. The NIH team matches that islet yield standard but is testing ways to improve it. Currently available assays testing islet viability and function in vitro do not correlate with the imperfect but gold standard assay for in vivo function, i.e. islets transplanted into diabetic NOD-scid mice. We have established the capability to perform the standard in vitro islet function assays (islet insulin release in low- and high-glucose media, and viability assays) and the in vivo NOD-scid transplant model. We also initiated plans to study new in vitro assays including efforts to study islets using microarray techniques, and the regulation of insulin biosynthesis at the translational level. We supply islets to collaborators at USUHS for electrophysiological assays, and to collaborators at Vanderbilt to study islet revascularization and function post-transplant into the portal vein of NOD-scid mice. We initiated rhesus monkey experiments. We studied isolated monkey islets in vitro and transplanted islets into diabetic rhesus monkey recipients. The rhesus monkey islet transplants are designed to address several questions including: (1) the viability and function of NIH-isolated islets, (2) novel transplantation sites (portal vein vice the more easily accessed hepatic artery), (3) the optimal islet mass for transplant, and (4) markers of islet function post-transplant Three rhesus monkey islet transplants were performed using traditional immunosuppressive agents to delay rejection and each achieved insulin independence indicating that NIH-isolated islets are functional. The experiments are difficult however: (1) animals do not readily take the anti-rejection agents so achieving appropriate drug levels is difficult, (2) the animals'diabetes was induced via pancreatectomy leaving them without exocrine function and with malabsorption, (3) the traditional anti-rejection agents employed resulted in one animal dying from pneumonia and (4) the portal vein cannulation currently requires laparotomy for vascular access. We are testing ways to improve the model. One, we are establishing collaboration with University of Maryland investigators who maintain a colony of spontaneously diabetic rhesus monkeys. Two, we are attempting to chemically induce diabetes in such a way so as to prevent toxicity from the typically employed diabetogenic drug, and to prevent the pancreatic insufficiency induced by pancreatectomy. Three, we collaborate with a Yale University interventional radiologist to find less invasive ways of transplanting the islets. Four, we collaborate with a group at Novocell, Inc claiming they can grow islets. If Novocell succeeds in expanding single donor rhesus islet number, we will transplant islets back to the original donor, and to an allogeneic host. All these studies are designed to support islet transplant clinical trials at the NIH. Toward that end, we have: (1) written an islet transplant clinical protocol for individuals with T1DM of at least 5 years duration, and with a clinical evidence of """"""""brittle"""""""" disease, (2) established an FDA IND to administer isolated islets, (3) been granted a variance from the United Network for Organ Sharing (UNOS) to utilize two human pancreata for each recipient, (4) established an islet transplant team consisting of the islet isolation unit, scientists versed in the islet functional assays, interventional radiologists for the portal vein cannulation, nurses, and pharmacists, and (5) established a system for addressing questions from the estimated one million Americans with T1DM, many of whom follow the islet transplant field with great anticipation. We have instituted screening questions to rule out patients clearly ineligible for the study. Despite that filter, we have evaluated 78 patients at the NIH for protocol eligibility; 32 patients have been excluded, 19 patients are currently listed for the protocol, and 17 others are undergoing evaluation. An additional 86 patients cleared the initial screen, completed a more detailed diabetes-related questionnaire, and are awaiting more detailed testing. The clinical trial is scheduled to begin shortly. While clinical islet transplantation has historically only rarely achieved insulin independence one-year post-transplant, the Edmonton group recently reported that 7 consecutive patients were rendered insulin independent. Nevertheless, assays do not currently exist to quantitate the islet mass surviving and/or an incipient anti-islet immune response. Thus, we have initiated efforts to quantitate islet mass post-transplant using simple blood tests, and have established collaboration with Dr. Lafferty who is developing an immunological assay to predict an anti-islet destructive immune response. Even if islet transplantation is developed for more widespread clinical use, the field will still be crippled by the inadequate donor supply. We have therefore initiated studies to test whether pancreatic stem cells can be identified and/or stimulated to proliferate, and we have collaborated with others who claim they have proprietary means for growing islets in vitro (Novocell, see above).
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