APE2 Promotes AID-Dependent Somatic Hypermutation in Primary B Cell Cultures That Is Suppressed by APE1.

Somatic hypermutation (SHM) is necessary for Ab diversification and involves error-prone DNA repair of activation-induced cytidine deaminase-induced lesions in germinal center (GC) B cells but can also cause genomic instability. GC B cells express low levels of the DNA repair protein apurinic/apyrimidinic (AP) endonuclease (APE)1 and high levels of its homolog APE2. Reduced SHM in APE2-deficient mice suggests that APE2 promotes SHM, but these GC B cells also exhibit reduced proliferation that could impact mutation frequency.

Multiple endocrine neoplasia type 1 deletion in pancreatic alpha-cells leads to development of insulinomas in mice.

The pancreatic alpha- and beta-cells are critical components in regulating blood glucose homeostasis via secretion of glucagon and insulin, respectively. Both cell types are typically localized in the islets of Langerhans. However, little is known about the roles of paracrine interactions that contribute to their physiological functions.

Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection.

Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS.

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors.

Background: Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice.

Results: We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene.

Deciphering von Hippel-Lindau (VHL/Vhl)-associated pancreatic manifestations by inactivating Vhl in specific pancreatic cell populations.

The von Hippel-Lindau (VHL) syndrome is a pleomorphic familial disease characterized by the development of highly vascularized tumors, such as hemangioblastomas of the central nervous system, pheochromocytomas, renal cell carcinomas, cysts and neuroendocrine tumors of the pancreas. Up to 75% of VHL patients are affected by VHL-associated pancreatic lesions; however, very few reports in the published literature have described the cellular origins and biological roles of VHL in the pancreas. Since homozygous loss of Vhl in mice resulted in embryonic lethality, this study aimed to characterize the functional significance of VHL in the pancreas by conditionally inactivating Vhl utilizing the Cre/LoxP system.

Blood glucose levels regulate pancreatic beta-cell proliferation during experimentally-induced and spontaneous autoimmune diabetes in mice.

Background: Type 1 diabetes mellitus is caused by immune-mediated destruction of pancreatic beta-cells leading to insulin deficiency, impaired intermediary metabolism, and elevated blood glucose concentrations. While at autoimmune diabetes onset a limited number of beta-cells persist, the cells' regenerative potential and its regulation have remained largely unexplored. Using two mouse autoimmune diabetes models, this study examined the proliferation of pancreatic islet ss-cells and other endocrine and non-endocrine subsets, and the factors regulating that proliferation.

Dynamic changes in pancreatic endocrine cell abundance, distribution, and function in antigen-induced and spontaneous autoimmune diabetes.

Objective: Insulin deficiency in type 1 diabetes and in rodent autoimmune diabetes models is caused by beta-cell-specific killing by autoreactive T-cells. Less is known about beta-cell numbers and phenotype remaining at diabetes onset and the fate of other pancreatic endocrine cellular constituents.

Research Design And Methods: We applied multicolor flow cytometry, confocal microscopy, and immunohistochemistry, supported by quantitative RT-PCR, to simultaneously track pancreatic endocrine cell frequencies and phenotypes during a T-cell-mediated beta-cell-destructive process using two independent autoimmune diabetes models, an inducible autoantigen-specific model and the spontaneously diabetic NOD mouse.

Immunomodulation of autoimmune diabetes by dendritic cells.

The biology and properties of dendritic cells (DCs) have been intensely studied in the research areas of infectious diseases, tumor immunology, and vaccine development. This unique subset of immune cells has recently also moved to the center of interest for basic and clinical research in autoimmunity, owing not only to the extraordinary importance of DCs in the initiation and sustenance of adaptive immune responses, but also to more recent discoveries about their profound ability to control and downregulate ongoing T-cell responses. We review current progress of using DCs in mice for induction and propagation of autoimmune T-cell responses and their therapeutic potential to dampen or even stop beta-cell-specific autoimmunity.

Cytotoxic T cell-mediated diabetes in RIP-CD80 transgenic mice: autoantigen peptide sensitivity and fine specificity.

Rodent immune-mediated diabetes model studies have advanced understanding of beta cell-specific T cell responses, and the testing of therapeutic approaches. We have used an inducible diabetes model based on rat insulin promotor (RIP)-driven expression of CD80 (B7-1) on pancreatic beta cells. Using these mice, we have established that immunizing with a single autoantigen can promote progressive islet inflammation and eventually T cell-mediated diabetes.

No evidence for mouse pancreatic beta-cell epithelial-mesenchymal transition in vitro.

We used cre/loxP-based genetic lineage tracing analysis to test a previously proposed hypothesis that in vitro cultured adult pancreatic beta-cells undergo epithelial-mesenchymal transition (EMT) to generate a highly proliferative, differentiation-competent population of mesenchymal islet "progenitor" cells. Our results in the mouse that are likely to be directly relevant to the human system show that adult mouse beta-cells do not undergo EMT in vitro and that the mesenchymal cells that arise in cultures of adult pancreas are not derived from beta-cells. We argue that these cells most likely originate from expansion of mesenchymal cells integral to the heterogeneous pancreatic islet preparations.

Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus.

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action.

Beta cell-specific CD80 (B7-1) expression disrupts tissue protection from autoantigen-specific CTL-mediated diabetes.

T cell responses toward pancreatic beta cell autoantigens arise spontaneously or on immunization in many mouse strains, yet sustained islet infiltration and progressive diabetes rarely ensues. Most mouse diabetes models overcome the innocuous coexistence of anti-islet specific T cells and endogenous islets via incompletely understood mechanisms (e.g.

Induction of autoimmune diabetes through insulin (but not GAD65) DNA vaccination in nonobese diabetic and in RIP-B7.1 mice.

Insulin has been used to modify T-cell autoimmunity in experimental models of type 1 diabetes. In a large clinical trial, the effect of insulin to prevent type 1 diabetes is currently investigated. We here show that insulin can adversely trigger autoimmune diabetes in two mouse models of type 1 diabetes, using intramuscular DNA vaccination for antigen administration.