Newswise — Knowing who has, or will develop Type-1 diabetes is of immense clinical significance, even as the number of cases of the much more common Type-2 diabetes skyrocket. Because of technology developed at Antigen Express, the wholly-owned subsidiary of NASDAQ-listed oral insulin delivery company Generex Biotechnology, Antigen Express collaborators are now one step closer to a reliable, highly sensitive diagnostic test that will identify Type-1 diabetes patients in time for early intervention.
This work will be presented at the American Diabetes Association's 68th Scientific Sessions meeting, June 6-10, at the Moscone Convention Center in San Francisco.
Type-1 diabetes results from an immune system gone awry, specifically the immune-mediated destruction of pancreatic beta cells. A key hallmark in the etiology of Type-1 diabetes is T cell recognition of islet cell antigens.
Several proteins are associated with development of Type-1 diabetes, in particular insulin and glutamic acid decarboxylase 65 (GAD65). Unfortunately, a highly sensitive, specific assay for T cells reactive to insulin or GAD65 is still under development, and not available as a diagnostic or prognostic tool to clinicians. Currently, Type I diabetes can only be diagnosed by the presence of antibodies when the disease is already well underway.
Researchers at Antigen Express, in collaboration with investigators at the Institute of Cell and Molecular Science in London, U.K., and at the University Campus Biomedico in Rome, Italy, approached the lack of an assay through a novel approach. Investigators were aware of Antigen Express technology whereby antigenic peptides linked to the "Ii-key" fragment of the MHC class II associated invariant chain enhances the presentation of those antigenic peptides. Ii-Key/peptide 'hybrids' are able to bind directly to MHC class II molecules on the test cell's surface, thus obviating the need for antigen internalization within cells. Earlier studies by Antigen Express suggested that chemically linking the antigenic peptide to Ii-key through a flexible chemical linking agent could improve recognition of antigens, and perhaps lead to a rapid, reliable assay for individuals with, or at risk for, Type-1 diabetes.
The antigen used in the study was GAD65, a protein that frequently triggers the autoimmune response in some diabetics. Researchers linked an antigenic peptide derived from GAD65 to the Ii-Key fragment, which is known to enhance presentation and binding of antigenic peptides directly to MHC Class II receptors on the cell surface.
Investigators isolated peripheral blood mononuclear cells from thirteen patients with Type-1 diabetes, and 17 healthy subjects matched for age. Cells were cultured with either intact GAD65 or GAD65 peptides linked to Ii-Key, then analyzed for release of two cytokines associated with T cell activation: interferon gamma and interleukin-10. Cells from patients with Type I diabetes demonstrated a distinct profile of cytokine production compared to cells from healthy volunteers.
Researchers found that the interferon response to whole GAD65 was significantly greater in diabetic subjects compared to controls (p<0.01), but there was no difference in interleukin-10 responses between the groups. Type-1 diabetics also showed a similarly significant increase in interferon response to GAD65 Ii-Key peptides.
"The results show that Ii-Key/GAD65 hybrids produce a similar response from T cells as does the GAD65 protein," commented Prof. David Leslie of the Department of Diabetes and Metabolism, St. Bartholomew's Hospital, London, U.K. and Antigen Express collaborator. "The cytokine expression profile we observe is helpful from a diagnostic perspective and gives hope that Ii-Key/GAD65 hybrids may have utility as agents to suppress autoimmunity."
T cell reactivity to GAD65 is of particular interest in individuals who develop diabetes later in life, as this helps to differentiate them from the more common Type-2 diabetes. Since treatment and maintenance differ considerably for the two diseases, a validated diagnostic based on GAD65 could put those patients on the right treatment regimen earlier. A test based on â‰ˆ might also be useful in monitoring treatments of clinical-stage diabetes medications.
It could also help identify patients at high risk for developing Type-1 diabetes later in life, or who are at the disease's earliest stages. Several studies are underway that attempt to delay or reverse the onset of Type-1 diabetes by slowing down or even turning off the autoimmune response against insulin and islet cell antigens.