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autoantigens and type I diabetesInsulin-dependent diabetes mellitus (IDDM) and glutamic acid decarboxylase (GAD)
Insulin-dependent diabetes mellitus (IDDM), also called type 1 diabetes, is an autoimmune disease in which a person's immune system mounts a response against the β-cells in the pancreas. Destruction of these cells, which normally produce insulin to regulate blood sugar after meals, results in the life-long dependence on insulin injections. Early prevention of autoimmunity has the potential to greatly improve these individuals' quality of life. Furthermore, there has been some link between viral infection and autoimmune disease development and even from the antiviral therapy used to treat HIV[2,3]. Therefore, immunotherapy treatment for IDDM is an important area of research today and for the future.
Immunotherapy treatment initially focused on the use of immunesuppressive agents, such as prednisone, but the effectiveness of these treatments was minimal. Within the last decade or so, research has turned to the oral application of self-antigens or autoantigens to induce immune tolerance. By repeatedly exposing the immune system to these antigens, it is hoped that the body will eliminate B cells that carry antibodies against these antigens, which prevents the destruction of β cells in the pancreas. The most promising methods thus far are the use of insulin, glutamic acid decarboxylase (GAD) and the heat shock protein 60 (Hsp60)-derived peptide 277 as antigens to prime the immune system. The GAD system is discussed below since this is the only antigen that has been produced in transgenic plants and has the greatest scale-up potential, which are important considerations for a smaller country with limited means.
GAD65 is a cell surface protein recognized by T cells
Autoimmunity in IDDM is a complicated process and many different systems have been implicated. Current research suggests that activated T cells, particularly CD4+ and CD8+ cells, are involved in the direct killing of pancreatic cells while cytokines released by Th1 and Th2 cells seem to modulate the severity of the autoimmune response. Th1 cells secrete cytokines such as interferon (IFN)-γ and interleukin-2 (IL-2) that increase the severity of the autoimmune reaction while Th2 cells secrete anti-inflammatory cytokines such as IL-4 and IL-10 that decrease the severity of the response. GAD is implicated as one of the antigens that is recognized as foreign by the immune system and used to target pancreatic cells.
There are two forms of GAD that are produced in the body: GAD67 and GAD65. GAD67 is an intracellular enzyme while GAD65 is a cell surface enzyme; both convert L-glutamic acid into the neurotransmitter GABA. Because of its location on the cell surface, GAD65 can interact with other cells of the body, including immune cells, and is therefore implicated as one of the autoantigens targeted by the immune system in IDDM. This mechanism is supported by the presence of anti-GAD65 antibodies in the bloodstream of IDDM patients and by research showing recognition of GAD65 by CD8+ T cells in the initial stages of IDDM.
Oral tolerance to GAD in immunotherapy
Treatment of IDDM in NOD mice represents an interesting overlap between antibodies and cytokines. One problem with the use of GAD65 to induce tolerance in non-obese diabetic (NOD) mice, a standard laboratory animal used to model type 1 diabetes, is the high concentrations of GAD protein required to do so. In their experimental set-up, Ma et al. compared the ability of GAD65 alone, IL-4 alone and GAD65 with IL-4 to decrease inflammation in pancreatic β cells. They found that GAD65 used in conjunction with IL-4, one of the anti-inflammatory cytokines, and cholera toxin B as the adjuvant in NOD mice showed the lowest levels of inflammation. Furthermore, prolonged treatment of prediabetic NOD mice from 4-30 weeks of age with GAD65 and IL-4 prevented the development of IDDM. The amount of GAD required for oral tolerance, however, is still unclear; there has been some suggestion that the use of ~1mg of GAD is required to induce do so. Overall, experimental results have been very encouraging for the use of GAD in preventing IDDM.
Improvements in GAD production in transgenic plants
GAD has been produced in transgenic potato, tobacco and carrot plants[5,6]. In all of these systems, GAD antigen production is relatively low: for example, GAD65 production in tobacco makes up between 0.01 and 0.04% of total soluble protein. Recently, Avesani et al. developed a new production technique in which a new viral vector was used to bring the human GAD65 protein into transgenic tobacco plants. Potato virus X (PVX) vectors carrying the hGAD65 gene were innoculated into tobacco leaves and yielded a fifty-fold increase in GAD production: GAD produced in transgenic tobacco plants infected with the PVX vector made up 2.16% of total soluble protein. Therefore, it can be seen that transgenic plant systems can easily be altered to increase protein production. These promising results, however, should be confirmed with additional studies before actual development.
IDDM is an autoimmune disease that has profound impacts on an individual's quality of life. Current research has shown some promising alternatives to its treatments, including the use of GAD65 protein in the induction of immune tolerance to prevent the development of IDDM. NOD mouse experiments have been highly successful. Encouragingly, clinical trials on the safety and efficacy of GAD immunomodulation in human patients with latent autoimmune diabetes in adults (LADA) have begun as well. Furthermore, transgenic plant systems have shown their suitability for the production of the autoantigens used in these immunotherapy treatments. The next step would be to confirm the results observed in NOD mouse models in human patients and to begin scaling up antigen production in transgenic plants, or to experiment with promising alternative plant systems for this purpose.
references1) Silverthron DU. Human Physiology: An Integrated Approach, 3rd ed. San Francisco: Benjamin Cummings, 2004.
2) Jun HS, Khil LY, Yoon JW. Role of glutamic acid decarboxylase in the pathogenesis of type 1 diabetes. Cell. Mol. Life Sci. 2002, 59: 1892-1901.
3) French MA, Price P, Stone SF. Immune restoration disease after antiretroviral therapy. AIDS 2004, 18: 1615-27.
4) Raz I, Eldor R, Naparstek Y. Immune modulation for prevention of type 1 diabetes mellitus. Trends in Biotechnology 2005, 23: 128-34.
5) Ma S, Huang Y, Yin Z, Menassa R, Brandle JE, Jevnikar AM. Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4. PNAS 2004, 101: 5680-5685.
6) Avesani L, Falorni A, Tornielli GB, Marusic C, Porceddu A, Polverari A, Faleri C, Calcinaro F, Pezzotti M. Improved in planta expression of human islet glutamic acid decarbodylase (GAD65). Transgenic Research 2003, 12: 203-212.