ORIGINAL: This research seeks immunohistochemical evidence of beta cell oxidative and nitrosative stress during various stages of T1D and whether such stressors are also present preceding and during the early onset of the disease. Antibodies to nitrotyrosine, a marker of peroxynitrite-mediated cell damage and to 8-hydroxy-2-deoxyguanosine (a marker of DNA oxidation) are being employed in triple-label immunohistochemistry to determine if such processes are activated within the beta cell. Combined immunohistochemistry and histochemistry are being applied to correlate the immunolabelling of such deleterious markers with the extent and spatial distribution of leukocytic infiltrates within the islets and with beta cell co-localization. Studies to date have shown weak to moderate immunolabelling of nitrotyrosine restricted to a majority of the remaining beta cells from 5 T1D cases, being also present in islet beta cells with minimum insulitis. Nitrotyrosine was absent in glucagon cells and the islet leukocytes. It was absent or weakly expressed in islet cells of non-diabetic donors (2 cases). DNA oxidation was observed within islet endocrine cells but was not restricted to beta cells from T1D cases.
The expression of nitrotyrosine and DNA oxidation are being extended to non-diabetic cases with single or two autoantibodies. In line with previous in vitro studies with human islets in culture, the current research is also exploring the possible activation of the pro-inflammatory cytokine-nitric oxide axis within the islets of T1D cases, likely under-expression of anti-oxidant enzymes and whether stressors within the pre-diabetic beta cell lead to beta cell apoptosis and attract the early immune response.
Protocol development, and search for suitable primary antibodies and optimization studies are in progress for the immunohistochemical localization of interleukin-1β, inducible nitric oxide synthase and interferon-α.
ADDENDUM: Despite considerable efforts directed at finding a cure for type 1 diabetes (T1D), the molecular processes that initiate and perpetuate beta cell destruction and how and why beta cells become targets of immune destruction are poorly understood.This knowledge gap and the realization that the underlying processes of T1D and the age of onset of T1D are heterogeneous have compounded progress in finding a cure.In addition, evidence of beta cell dysfunction is present well before clinical onset.
Beta cells have high rates of metabolism, protein synthesis and secrete insulin continuously in a pulsatile fashion and with increased output in response to meals. We propose that the highly metabolic beta cells are more susceptible to environmentally-induced insults, that oxidant-induced beta cell stress, leading to generation of abnormal cellular proteins due to their intrinsic inability to clear excess free radicals. The resulting abnormal proteins compromise beta cell function and act as antigenic targets of immune-mediated destruction.
Environmental factors such as certain viruses or toxins can also lead to excess and uncontrolled generation of reactive oxygen species (ROS) in beta cells, resulting in stress.Thus, sustained beta cell stress may be a key determinant of beta cell damage. In this research, we wish to demonstrate if the remaining beta cells from cases with new-onset T1D and those with longer duration of disease have low levels of three key ROS clearing enzymes, and express detectable levels of interferon-α (IFN-α), a marker of virally-infected beta cells and which my contribute to oxidative stress.The endoplasmic reticulum (ER), a vital subcellular organelle, is responsible for correctly processing and folding of nascent proteins and is exquisitely sensitive to imbalances in oxidative stress. We also wish to examine if oxidative stress disturbs the redox balance and promotes stress within the ER.
Our studies will provide key insights on why beta cells become targets of self-immunity and may pave the way for the development of future therapies aimed at alleviating beta cell stress in the early stages of type 1 diabetes and even prior to its onset and thus, prevent this serious disorder.