Assessment of specific beta-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for... Show moreAssessment of specific beta-cell death can be used to determine the quality and viability of pancreatic islets prior to transplantation and hence predict the suitability of the pancreas for isolation. Recently, several groups have demonstrated that unmethylated insulin (INS)-DNA is correlated to beta-cell death in type 1 diabetes patients and during clinical islet isolation and subsequent transplantation. Here, we present a step-by-step protocol of our novel developed method for quantification of the relative amount of unmethylated INS-DNA using methylation sensitive restriction enzyme digital polymerase chain reaction This method provides a novel and sensitive way to quantify the relative amount of beta-cell derived unmethylated INS-DNA in cellular lysate. We therefore suggest that this technique can be of value to reliably determine the purity of an islet preparation and may also serve as a measure of the quality of islets prior to transplantation measuring unmethylated INS-DNA as a reflection of the relative amount of lysed beta-cells. Show less
Vig, S.; Lambooij, J.M.; Zaldumbide, A.; Guigas, B. 2021
Beta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In response to inflammatory signals, beta-cells engage adaptive... Show moreBeta-cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In response to inflammatory signals, beta-cells engage adaptive mechanisms where the endoplasmic reticulum (ER) and mitochondria act in concert to restore cellular homeostasis. In the recent years it has become clear that this adaptive phase may trigger the development of autoimmunity by the generation of autoantigens recognized by autoreactive CD8 T cells. The participation of the ER stress and the unfolded protein response to the increased visibility of beta-cells to the immune system has been largely described. However, the role of the other cellular organelles, and in particular the mitochondria that are central mediator for beta-cell survival and function, remains poorly investigated. In this review we will dissect the crosstalk between the ER and mitochondria in the context of T1D, highlighting the key role played by this interaction in beta-cell dysfunctions and immune activation, especially through regulation of calcium homeostasis, oxidative stress and generation of mitochondrial-derived factors. Show less
