Phenotypically “immature” dendritic cells (DCs), defined by low cell surface CD40, CD80, and CD86 can elicit host immune suppression in allotransplantation and autoimmunity. Herein, we report the most direct means of achieving phenotypic immaturity in NOD bone marrow-derived DCs aiming at preventing diabetes in syngeneic recipients. CD40, CD80, and CD86 cell surface molecules were specifically down-regulated by treating NOD DCs ex vivo with a mixture of antisense oligonucleotides targeting the CD40, CD80, and CD86 primary transcripts. The incidence of diabetes was significantly delayed by a single injection of the engineered NOD DCs into syngeneic recipients. Insulitis was absent in diabetes-free recipients and their splenic T cells proliferated in response to alloantigen. Engineered DC promoted an increased prevalence of CD4+CD25+ T cells in NOD recipients at all ages examined and diabetes-free recipients exhibited significantly greater numbers of CD4+CD25+ T cells compared with untreated NOD mice. In NOD-scid recipients, antisense-treated NOD DC promoted an increased prevalence of these putative regulatory T cells. Collectively, these data demonstrate that direct interference of cell surface expression of the major costimulatory molecules at the transcriptional level confers diabetes protection by promoting, in part, the proliferation and/or survival of regulatory T cells. This approach is a useful tool by which DC-mediated activation of regulatory T cells can be studied as well as a potential therapeutic option for type 1 diabetes.

OBJECTIVE—This study was aimed at ascertaining the efficacy of antisense oligonucleotide-formulated microspheres to prevent type 1 diabetes and to reverse new-onset disease. RESEARCH DESIGN AND METHODS—Microspheres carrying antisense oligonucleotides to CD40, CD80, and CD86 were delivered into NOD mice. Glycemia was monitored to determine disease prevention and reversal. In recipients that remained and/or became diabetes free, spleen and lymph node T-cells were enriched to determine the prevalence of Foxp3+ putative regulatory T-cells (Treg cells). Splenocytes from diabetes-free microsphere-treated recipients were adoptively cotransferred with splenocytes from diabetic NOD mice into NOD-scid recipients. Live-animal in vivo imaging measured the microsphere accumulation pattern. To rule out nonspecific systemic immunosuppression, splenocytes from successfully treated recipients were pulsed with β-cell antigen or ovalbumin or cocultured with allogeneic splenocytes. RESULTS—The microspheres prevented type 1 diabetes and, most importantly, exhibited a capacity to reverse clinical hyperglycemia, suggesting reversal of new-onset disease. The microspheres augmented Foxp3+ Treg cells and induced hyporesponsiveness to NOD-derived pancreatic β-cell antigen, without compromising global immune responses to alloantigens and nominal antigens. T-cells from successfully treated mice suppressed adoptive transfer of disease by diabetogenic splenocytes into secondary immunodeficient recipients. Finally, microspheres accumulated within the pancreas and the spleen after either intraperitoneal or subcutaneous injection. Dendritic cells from spleen of the microsphere-treated mice exhibit decreased cell surface CD40, CD80, and CD86. CONCLUSIONS—This novel microsphere formulation represents the first diabetes-suppressive and reversing nucleic acid vaccine that confers an immunoregulatory phenotype to endogenous dendritic cells.

Glucosamine is a naturally occurring derivative of glucose and is an essential component of glycoproteins and proteoglycans, important constituents of many eukaryotic proteins. In cells, glucosamine is produced enzymatically by the amidation of glucose 6-phosphate and can then be further modified by acetylation to result in N-acetylglucosamine. Commercially, glucosamine is sold over-the-counter to relieve arthritis. Although there is evidence in favor of the beneficial effects of glucosamine, the mechanism is unknown. Our data demonstrate that glucosamine suppresses the activation of T-lymphoblasts and dendritic cells in vitro as well as allogeneic mixed leukocyte reactivity in a dosedependent manner. There was no inherent cellular toxicity involved in the inhibition, and the activity was not reproducible with other amine sugars. More importantly, glucosamine administration prolonged allogeneic cardiac allograft survival in vivo. We conclude that, despite its documented effects on insulin sensitivity, glucosamine possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent.Glucosamine is a naturally occurring sugar that is synthesized by virtually all cells. Upon uptake, glucose is immediately phosphorylated and enzymatically converted into a series of substrates that will be either converted into glycogen, lipids, and proteins or used to generate ATP and CO 2 . 2-3% of glucose 6-phosphate, the immediate intracellular glucose derivative following uptake, is diverted into a pathway known as the hexosamine biosynthesis pathway (1, 2). The rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase is responsible for the commitment of glucose derivatives to the pathway, ultimately resulting in the formation of glycoprotein precursors (3). Glucosamine is not secreted outside of cells, but exogenously added glucosamine is taken up by glucose transporters (GLUT-2 and GLUT-4) and then phosphorylated (4, 5).In 1953, Quastel and Cantero (6) demonstrated that glucosamine possesses tumor-inhibiting activity. Since then, a number of reports have confirmed the tumoricidal activity of glucosamine (7-16). Glucosamine has been shown to inhibit nucleic acid and protein biosynthesis and irreversible damage to organelles in tumor cells, but not in normal cells (8 -15, 17-20).In addition, glucosamine also inhibits platelet aggregation and ATP release induced by Staphylococcus aureus, ADP, epinephrine, and collagen (21). The mechanisms by which glucosamine acts are not completely clear; however, it has been shown to alter the ultrastructure of plasma and intracellular membranes (9, 22), to inhibit membrane transport of nucleosides (14, 15), and reportedly to shift its distribution from glycoproteins to glycolipids (22).O'Neill and Parish (23) demonstrated that monosaccharides, especially amino sugars, are able to inhibit cytotoxic T-lymphocyte function in culture, preventing target lysis in a cytotoxic T-lymphocyte clone-specific manner. Yagita et al. further demonstrated that free hexosamines ...

Aims/hypothesis Reactive oxygen species (ROS) generated during hyperglycaemia are implicated in the development of diabetic vascular complications. High glucose increases oxidative stress in endothelial cells and induces apoptosis. A major source of ROS in endothelial cells exposed to glucose is the NAD(P)H oxidase enzyme. Several studies demonstrated that C-peptide, the product of proinsulin cleavage within the pancreatic beta cells, displays anti-inflammatory effects in certain models of vascular dysfunction. However, the molecular mechanism underlying this effect is unclear. We hypothesised that C-peptide reduces glucose-induced ROS generation by decreasing NAD(P)H oxidase activation and prevents apoptosis Methods Human aortic endothelial cells (HAEC) were exposed to 25 mmol/l glucose in the presence or absence of C-peptide and tested for protein quantity and activity of caspase-3 and other apoptosis markers by ELISA, TUNEL and immunoblotting. Intracellular ROS were measured by flow cytometry using the ROS sensitive dye chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H 2 -DCDFA). NAD(P)H oxidase activation was assayed by lucigenin. Membrane and cytoplasmic levels of the NAD (P)H subunit ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1) (RAC-1) and its GTPase activity were studied by immunoblotting and ELISA. RAC-1 (also known as RAC1) gene expression was investigated by quantitative real-time PCR. Results C-peptide significantly decreased caspase-3 levels and activity and upregulated production of the antiapoptotic factor B cell CLL/lymphoma 2 (BCL-2). Glucose-induced ROS production was quenched by C-peptide and this was associated with a decreased NAD (P)H oxidase activity and reduced RAC-1 membrane production and GTPase activity. Conclusions/interpretation In glucose-exposed endothelial cells, C-peptide acts as an endogenous antioxidant molecule by reducing RAC-1 translocation to membrane and NAD (P)H oxidase activation. By preventing oxidative stress, C-peptide protects endothelial cells from glucose-induced apoptosis.

CD25؉ T-cells obtained from NOD-scid mice reconstituted with ex vivo engineered iDCs and NOD splenocytes expressed significantly higher levels of IL-7R␣ compared with levels in the CD4 ؉ CD25 ؊ subset, especially in diabetessuppressive dendritic cell-administered NOD-scid recipients. Taken together, our data suggest a novel mechanism by which iDCs delay autoimmunity through the CD4 ؉ CD25؉ Treg pathway and suggest IL-7 as a survival factor for these putative Tregs, which express the ␣-chain of its receptor at considerably higher levels than CD4 ؉ CD25 ؊ T-cells. Diabetes 55:158 -170, 2006