In diabetes mellitus, β cell destruction is largely silent and can be detected only after significant loss of insulin secretion capacity. We have developed a method for detecting β cell death in vivo by amplifying and measuring the proportion of insulin 1 DNA from β cells in the serum. By using primers that are specific for DNA methylation patterns in β cells, we have detected circulating copies of β cell-derived demethylated DNA in serum of mice by quantitative PCR. Accordingly, we have identified a relative increase of β cell-derived DNA after induction of diabetes with streptozotocin and during development of diabetes in nonobese diabetic mice. We have extended the use of this assay to measure β cell-derived insulin DNA in human tissues and serum. We found increased levels of demethylated insulin DNA in subjects with new-onset type 1 diabetes compared with age-matched control subjects. Our method provides a noninvasive approach for detecting β cell death in vivo that may be used to track the progression of diabetes and guide its treatment.epigenetics | autoimmunity | biomarker

Secondary lymphoid organs (SLOs) include lymph nodes, spleen, Peyer’s patches, and mucosal tissues such as the nasal-associated lymphoid tissue, adenoids, and tonsils. Less discretely anatomically defined cellular accumulations include the bronchus-associated lymphoid tissue, cryptopatches, and isolated lymphoid follicles. All SLOs serve to generate immune responses and tolerance. SLO development depends on the precisely regulated expression of cooperating lymphoid chemokines and cytokines such as LTα, LTβ, RANKL, TNF, IL-7, and perhaps IL-17. The relative importance of these factors varies between the individual lymphoid organs. Participating in the process are lymphoid tissue initiator, lymphoid tissue inducer, and lymphoid tissue organizer cells. These cells and others that produce crucial cytokines maintain SLOs in the adult. Similar signals regulate the transition from inflammation to ectopic or tertiary lymphoid tissues.

The pattern recognition receptor, RAGE, has been shown to be involved in adaptive immune responses but its role on the components of these responses is not well understood. We have studied the effects of a small molecule inhibitor of RAGE and the deletion of the receptor (RAGE−/− mice) on T cell responses involved in autoimmunity and allograft rejection. Syngeneic islet graft and islet allograft rejection was reduced in NOD and B6 mice treated with TTP488, a small molecule RAGE inhibitor (p < 0.001). RAGE−/− mice with streptozotocin-induced diabetes showed delayed rejection of islet allografts compared with wild type (WT) mice (p < 0.02). This response in vivo correlated with reduced proliferative responses of RAGE−/− T cells in MLRs and in WT T cells cultured with TTP488. Overall T cell proliferation following activation with anti-CD3 and anti-CD28 mAbs were similar in RAGE−/− and WT cells, but RAGE−/− T cells did not respond to costimulation with anti-CD28 mAb. Furthermore, culture supernatants from cultures with anti-CD3 and anti-CD28 mAbs showed higher levels of IL-10, IL-5, and TNF-α with RAGE−/− compared with WT T cells, and WT T cells showed reduced production of IFN-γ in the presence of TTP488, suggesting that RAGE may be important in the differentiation of T cell subjects. Indeed, by real-time PCR, we found higher levels of RAGE mRNA expression on clonal T cells activated under Th1 differentiating conditions. We conclude that activation of RAGE on T cells is involved in early events that lead to differentiation of Th1+ T cells.

OBJECTIVE— β-Cell regeneration is a fundamental but elusive goal for type 1 diabetes research. Our objective is to review newer human and animal studies of β-cell destruction and regeneration and consider the implications for treatment of type 1 diabetes. RESEARCH DESIGN AND METHODS— Recent human and animal studies of β-cell destruction and regeneration in type 1 diabetes are reviewed. RESULTS— The loss of β-cells that characterizes type 1 diabetes reflects the net effects of destruction and regeneration. These processes have been examined in the nonobese diabetic (NOD) mouse; uncertainty remains about β-cell dynamics in humans. Islet inflammation stimulates β-cell replication that produces new insulin-positive cells. The regenerative process may tide the loss of overall β-cell function, but it also may enhance the autoimmune attack on β-cells by providing new epitopes. The highest rates of β-cell replication are at the time of diagnosis of diabetes in NOD mice, and if autoimmunity and islet inflammation are arrested, new β-cells are formed. However, the majority of β-cells after treatment with immune modulators such as anti-CD3 monoclonal antibody, and most likely during the “honeymoon” in human disease, are recovered β-cells that had been degranulated but present at the time of diagnosis of diabetes. CONCLUSIONS— Residual β-cells play a significant role for the design of therapeutic trials: they not only may respond to combination therapies that include stimulants of metabolic function but are also the potential source of new β-cells.

The Receptor for Advanced Glycation Endproducts (RAGE) is a scavenger ligand that binds glycated endproducts as well as molecules released during cell death such as S100b and HMGB1. RAGE is expressed on antigen presenting cells where it may participate in activation of innate immune responses but its role in adaptive human immune responses has not been described. We have found that RAGE is expressed intracellularly in human T cells following TCR activation but constitutively on T cells from patients with diabetes. The levels of RAGE on T cells from patients with diabetes are not related to the level of glucose control. It co-localizes to the endosomes. Its expression increases in activated T cells from healthy control subjects but bystander cells also express RAGE after stimulation of the antigen specific T cells. RAGE ligands enhance RAGE expression. In patients with T1D, the level of RAGE expression decreases with T cell activation. RAGE+ T cells express higher levels of IL-17A, CD107a, and IL-5 than RAGE− cells from the same individual with T1D. Our studies have identified the expression of RAGE on adaptive immune cells and a role for this receptor and its ligands in modulating human immune responses.

Type 1 diabetes (T1D) results from immune-mediated destruction of insulin-producing β-cells. The killing of β-cells is not currently measurable; β-cell functional studies routinely used are affected by environmental factors such as glucose and cannot distinguish death from dysfunction. Moreover, it is not known whether immune therapies affect killing. We developed an assay to identify β-cell death by measuring relative levels of unmethylated INS DNA in serum and used it to measure β-cell death in a clinical trial of teplizumab. We studied 43 patients with recent-onset T1D, 13 nondiabetic subjects, and 37 patients with T1D treated with FcR nonbinding anti-CD3 monoclonal antibody (teplizumab) or placebo. Patients with recent-onset T1D had higher rates of β-cell death versus nondiabetic control subjects, but patients with long-standing T1D had lower levels. When patients with recent-onset T1D were treated with teplizumab, β-cell function was preserved (P < 0.05) and the rates of β-cell were reduced significantly (P < 0.05). We conclude that there are higher rates of β-cell death in patients with recent-onset T1D compared with nondiabetic subjects. Improvement in C-peptide responses with immune intervention is associated with decreased β-cell death.

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Diabetes is associated with increased basal hypothalamo-pituitary-adrenal (HPA) activity and impaired stress responsiveness. Previously, we demonstrated that the HPA response to hypoglycemia is significantly impaired in diabetic rats. In this study our goals were to 1) differentiate between the effects of hyperinsulinemia and those of hypoglycemia per se, and 2) establish whether diabetes lowers peak stress responses. Normal and streptozotocin-diabetic rats were subjected to hyperinsulinemic-euglycemic glucose clamps to evaluate central and peripheral responses. These were compared with peak ACTH and corticosterone responses to restraint and hypoglycemia. Hyperinsulinemia increased CRH and vasopressin mRNA, and plasma ACTH and corticosterone in normal and diabetic rats. In normal animals, insulin-induced activation of ACTH and corticosterone was lower than the responses during either restraint or hypoglycemia. In contrast, ACTH and corticosterone activation in diabetic rats was similar with all three stressors. Pituitary-adrenal axis activation in diabetic animals was also much lower compared with that in normal controls. The response to hyperinsulinemia (euglycemia) was associated with increases in glucocorticoid receptor mRNA in the anterior pituitary and paraventricular nucleus. Hippocampal mineralocorticoid receptor mRNA expression was increased in normal, but not in diabetic, animals. We speculate that the ability to appropriately match the HPA response to the potency of a stressor is related to the ability to alter hippocampal mineralocorticoid receptor expression. In diabetes, this ability is impaired; hence, maximal HPA activation is greatly diminished. This is a novel observation that may have important implications in the treatment of impaired counterregulatory mechanisms in human diabetes.