Research - 2004
Investigate a gene therapy strategy to improve the success of islet (or B cell) transplantation
Illani Atwater, Ph.D.
Sansum Medical Research Institute
Santa Barbara, CA
Current Research to prevent and correct Type 1 diabetes has focused on development of strategies to protect insulin secreting cells from recognition and/or destruction by the immune system. Research on an enzyme known as IDO, (indoleamine 2,3 dioxygenase), showed that this enzyme was responsible for successful pregnancy by keeping levels of the amino acid tryptophan low in the placenta. And when IDO was inhibited in mice, the resulting inflammatory response directed against the embryos caused early abortion. Moreover, in vitro, the activation and proliferation of lymphocytes is profoundly depressed when the culture medium levels of tryptophan are low. These findings have led Dr. Atwater to propose a gene therapy strategy to improve the success of islet (or B cell) transplantation. This is to incorporate the gene for IDO into islets or beta cells, which, upon transplantation, would then deplete their surroundings of tryptophan, which would inhibit approaching lymphocytes, and thus escape immune attack. The development of IDO expressing B cells will then solve the 2 major problems facing transplantation as a cure for diabetes, namely, the long term requirement for immune suppression therapy and the lack of sufficient viable islets.
Moreover, it is important to study how tryptophan affects lymphocytes. This project will attempt to define the role of tryptophan during lymphocyte activation. This will help to engineer B cells able to protect themselves against immune attack. Furthermore, this study will measure differences in the early immune response and in the tryptophan dependency of the response during the normal rejection process in contrast to the specific B cell rejection response, which occurs in type 1 diabetes. It is important to understand the cellular mechanisms of these two processes in order to develop a strategy to cure by protecting B cells from immune destruction after transplantation as well as to preventing diabetes by stopping the autoimmune destruction in Type 1 diabetes.
Antioxidants, Oxidative Stress, and the Prevention of Diabetic Complications
Ira D. Goldfine, M.D.
Professor of Medicine
University of California
San Francisco, CA
This proposal is directed towards understanding the causes of diabetic complications, along with evaluating the ability of antioxidants to prevent or reverse them. Elevated blood sugar levels in people with diabetes cause the late complications of diabetes mellitus including blood vessel damage, nerve damage, and kidney failure. However, effective treatments for complications are not yet available. Evidence now indicates that excess glucose entering the mitochondria causes the formation of injurious oxygen molecules, called ROS (reactive oxygen species), which alter the major building blocks (DNA, proteins, lipids) of the cells. This increase in ROS formation inside cells leads to the activation of biochemical signaling pathways and inflammatory responses that result in permanent damage in the cells of eyes, nerves, kidney, and blood vessels. In this project Dr. Goldfine plans to study cells in culture (blood vessel, kidney, and nerve) to identify the primary stress-activated pathway(s) resulting in cell damage. Dr. Goldfine will be studying the mechanism of action and efficacy of several currently available antioxidants to block cell damage. As a result of these studies, he hopes to uncover new treatment strategies for diabetic complications, and identify molecular targets for new drugs to treat and/or prevent diabetic complications.
Mechanisms of IL-4 Dependent Xenograft Survival in Mice
Ethel J. Gordon, Ph.D.
University of Massachusetts Medical School and Bluefield State College
Worcester, MA and Bluefield, WV
Clinical transplantation for the replacement of damaged human organs is severely limited by the paucity of available organs, and the requirement of lifelong treatment with drugs to prevent the patient’s immune system from rejecting the donated organ. The potential to use organs for tissues from non-human donors to cure human diseases is great, but the scientific hurdles are enormous. The goal of this proposal is to dissect the mechanisms responsible for survival of rat skin and rat islet xenografts in mice. This lab has designed an animal model of transplantation that uses laboratory rats as organ and tissue donors, and that uses laboratory mice as graft recipients. This transfer of tissues from individuals of one species to another is called xenotransplantation. This model promotes survival of the donated organ (xenograft) by means of a transfusion of donor spleen cells (DST), and the injection of an antibody to temporarily blockade T cell activation. In contrast to current clinical practice, this protocol requires no lifelong treatment with drugs to suppress the immune system. In this proposal, Dr. Gordon will test the general hypothesis that Interleukin-4 (IL-4) is strictly required for transplantation tolerance to both rat skin and islet xenografts. This general hypothesis is based on observation that tolerance, as measured by prolonged skin xenograft survival, is induced in IL-4 expressing mice, whereas rapid rejection of skin xenografts occurs in IL-4 knockout (KO) mice.
Pancreatic Development and its Role in Modeling Pancreatic Disease
Dr. Paul Grippo
The pancreas is an organ that is critical for both glucose metabolism and proper digestion. Several debilitating diseases, with no real cure, arise when pancreatic cells become altered and unable to function properly. Diabetes, pancreatitis, and pancreatic cancer have serious health consequences with a greatly reduced quality of life for the patient. There is a link between diabetes and these other pancreatic diseases. For example, diabetics are at greater risk for developing pancreatic cancer, and those with pancreatic cancer often develop diabetic type conditions. The relationship between these different diseases supports interactions among different pancreatic cell types. Furthermore, all pancreatic cells may have arisen from the same stem cell. Thus, understanding pancreatic development is important for investigating pancreatic disease progression. Establishing collaborations among scientists with interests in different pancreatic disease types or in pancreatic development are crucial and currently not being pursued vigorously. Dr. Grippo is organizing a conference that will bring together these groups of scientists in the hope of fostering collaborations aimed at understanding both pancreatic development and disease progression while developing new models of pancreatic diseases. The ultimate goal is to design new treatments for these diseases based on information gathered from combined investigations and/or evaluating these treatments using improved models of these diseases.
Role of Vascular Endothelial Growth Factor and its Receptors in Mediating Increases in Markers of Fibrosis that are Induced by Hyperglycemia and Excess Amino Acids in Rat Mesangial Cells
Emily Carolyn Johnson, Ph.D.
Adjunct Research Scientist and Associate Professor
The Heart Institute of Spokane and Washington State University
Diabetic nephropathy (DN) is a significant problem in diabetes. Although high glucose is considered to be a primary cause of pathological changes in the kidney due to diabetes, dietary protein has also been shown to be an important factor in mediating progression of the disease. Dr. Johnson’s laboratory has recently shown that treatment of rat mesangial cells with an excess of amino acids causes increased production and activation of transforming growth factor beta-1, which has been implicated in the disease processes involved in DN, as well as significant increases in markers of fibrosis (fibronectin and collagen) in rat mesangial cells similar to high glucose. These results suggest that excess amino acids may be involved in controlling the fibrosis seen in the diabetic kidney. Recently, another important growth factor, vascular endothelial growth factor (VEGF), has been shown to increase collagen production in mesangial cells and to be involved in the renal enlargement associated with fibrosis in diabetes. Dr. Johnson proposes to study the effects of excess amino acids on regulation of VEGF and VEGF receptor levels and localization in cultured rat mesangial cells (RMC), and will compare the results to RMC grown in high glucose. In addition, Dr. Johnson will investigate the role of VEGF in mediating the increases in collagen and fibronectin that have been previously demonstrated in her laboratory. This study has significant implications for understanding mechanisms that underlie disease processes involved in DN and may provide information that ultimately leads to therapeutic interventions to prevent the development or retard the progression of diabetic nephropathy.
Identification and Characterization of Genes Triggering Pancreatic B-Cell Formation
Raghavendra G. Mirmira, M.D., Ph.D.
Assistant Professor of Medicine
University of Virginia
Diabetes Mellitus, a disorder that affects 6% of the U.S. population, results from either the complete (type 1) or relative (type 2) deficiency of insulin secretion by the B-cells of the pancreas. The long term objective of this project is to understand the manner in which cells of the pancreas develop into B-cells, and to apply this knowledge to reprogram other cell types to mimic the insulin-producing ability of B-cells. The focus of this proposal is to study which genes are controlled by the transcription factor, Nkx6.1. Transcription factors regulate the production of proteins by controlling their corresponding genes. These proteins, in turn, are ultimately responsible for the development of various cell types in the embryo. Prior studies have shown that when experimental mice lack Nkx6.1, they do not develop B-cells within the pancreas and therefore produce no insulin. Thus, Nkx6.1 must somehow direct the formation of insulin-producing B-cells in the developing embryo. Since Nkx6/1 is also present in humans, we believe that understanding the mechanisms by which this protein works in mice will allow us to know how precursor cells are converted into insulin producing B-cells. This knowledge can then allow us to convert other cell types into insulin producing cells. In this project, Dr. Mirmira proposes to determine which proteins are specifically controlled by Nkx6.1 by studying the genes that encode these proteins. By knowing the identity of the proteins that are controlled by Nkx6.1, one can understand the biochemical processes that occur for a given cell to become a B-cell. This information can eventually be applied to engineering new B-cells for patients with diabetes.
Adiponectin and Cytokine Levels in Children with Diabetes
Alba E. Morales, MD
University of Florida
Although the frequency of type 1 diabetes in children has remained somewhat constant in recent years, the frequency of type 2 diabetes has increased at an alarming rate, reflecting the increasing numbers of overweight and obese children. Historically, the way physicians can tell type 1 from type 2 diabetes has been based strongly on age of patient and presence or absence of obesity. Because childhood obesity is a serious problem, the number of obese patients with type 1 diabetes will rise as well, making it even harder to differentiate between types 1 and 2 diabetes in children. Finding blood markers that might distinguish these two forms of the disease would be very helpful to clinicians. One of the markers that might serve such a purpose is adiponectin, a newly described hormone make only in fat in adults with type 1 diabetes. Because of this difference in the adiponectin levels, Dr. Morales predicts that serum adiponectin levels can be used to distinguish between type 1 and type 2 diabetes also in children. Dr. Morales also plans to test for cytokine levels as theses proteins, (which are small proteins released by cells that affect the behavior of other cells), appear important in the pathogenesis of both type 1 and type 2 diabetes but have not yet been effectively studied in children. By studying the relationship between cytokine levels in healthy children and children with both type 1 and type 2 diabetes, Dr. Morales hopes to further understand the differences and similarities of these diseases. If the role that these important hormones play in all childhood diabetes can be determined, it will then be possible to determine and use the best preventive and therapeutic strategies against this disease.
Beta Cell Inhibitors Act by Disrupting the SNARE Complex
Thomas Schermerhorn, Ph.D.
Kansas State University
This study seeks to understand the cellular mechanisms that regulate the inhibition of insulin release by pancreatic beta cells. Numerous compounds, such as hormones and neurotransmitters, control the rate of insulin secretion. Several potent physiological inhibitors of insulin release, such as norepinephrine, somatostatin, and galanin, have been identified. Although these compounds are chemically diverse, they produce similar inhibitory effects on cellular function. These compounds inhibit molecular signals in the beta cell by decreasing the concentrations of important “second messenger” molecules, such as calcium and cyclic AMP, that couple signals from the external environment to the exocytotic process by which insulin-containing granules are released from the cell. In addition, a potent but poorly understood inhibitory effect occurs at a site in the exocytotic pathway that is beyond the inhibitory effects on calcium and cAMP. Little is known regarding the mechanism of inhibition at the so-called “distal site” except that it is independent of the concentrations of calcium and cAMP and involves heterotrimeric GTP-binding proteins. Dr. Schermerhorn’s lab has identified proteins (synaptobrevin, syntaxin, and SNAP 25), which may represent the distal action of the inhibitory compounds. Dr. Schermerhorn’s studies will further characterize this newly identified action of the inhibitor compounds. Understanding this mechanism can lead to better ways to stimulate insulin secretion in type 2 diabetes.
Treatment of Diabetes Mellitus with Gene Therapy
Douglas Sobel, MD
Professor of Pediatrics
Georgetown University Children’s Medical Center
The goal of the research study is to develop a means to inject a gene construct which will allow non-human primates with diabetes mellitus to again have glucose regulated insulin secretion, that is a cure of their diabetes. A gene construct with a rodent regulator has been developed by Dr. Sobel’s collaborator, Dr. J-W Yoon, that cures diabetes when injected into diabetic rats and mice by allowing liver cells to make insulin in response to alterations of blood glucose. Because this treatment has never been demonstrated to cure diabetes in non-human primates or in any other animals larger than rodents, Dr. Sobel hopes to develop this mode of gene therapy for use in humans. A major aim of the study is to determine if the transfer of gene construct produces functional insulin activity to improve glucose utilization or reverse the diabetic state of insulin deficiency. Specifically, does gene construct transfer:
1. decrease exogenous daily insulin requirement?
2. cause a non-insulin requiring state, (i.e. cure)
3. improve the body’s handling of a glucose load
4. improve glycemic control (which is also dependent on the exogenous insulin administration)
Information obtained in this study will make it possible to understand:
1. the required dose of gene construct which may be considered to perform human studies to cure diabetes
2. the duration of insulin gene expression or cure of the diabetic state, the potential toxicities from this therapy
3. the immunologic responses to the gene construct (rAAV) which may be important to the ability of administering repeated doses of the rAAV
4. the potential toxicity of using this therapy
Thus, this work will provide a basis for future studies for exploring the possibility of a human trial to cure diabetes with gene therapy.
Preparing Trainers for the Stanford's Chronic Disease Self-Management Program for Louisiana State University Medical Systems
Helen L. Sloan, RN, Ph.D.
The University of Louisiana at Lafayette
As a major health problem in America, diabetes is the cause of death and chronic disabilities for over 10 million people, primarily from complications relating to the heart and blood vessels. Although projects to encourage persons with diabetes to perform self-care have been studied among many populations, there have been few studies that address the influence of self-efficacy upon foot care. If older adults with diabetes mellitus do not receive adequate interventions to promote self care of their feet, nurses and other health care providers are failing to reach and reduce the incidence of one of the most devastating complications of diabetes mellitus: lower limb amputations. In Louisiana, over 5% of the adult population has been diagnosed with diabetes, and a statewide analysis of diabetes in 1998 noted that African Americans had 1x greater risk of diabetes. In addition, African Americans suffer particularly high burdens from diabetes with a rate of lower extremity amputation 2.8 xs higher than Caucasians. The purpose of this project is to train “Master Trainers” recruited by the LSU Health Science System directors of nursing from across the state of LA. These trainers will then be instructed by educators from the Stanford Patient Education Research Center at Stanford University School of Medicine, which provides a 7 week, small group interventional program which emphasizes problem solving, decision making, and confidence building. The goal of this initial conference is to prepare “Trainers” as key to a proposal to NIH for later funding, which will evaluate the effectiveness of the self management course with persons diagnosed with diabetes mellitus in a predominately indigent population.