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At diagnosis, most people with type 1 diabetes (T1D) produce measurable levels of endogenous insulin, but the rate at which insulin secretion declines is heterogeneous. To explain this heterogeneity, we sought to identify a composite signature predictive of insulin secretion, using a collaborative assay evaluation and analysis pipeline that incorporated multiple cellular and serum measures reflecting β cell health and immune system activity. The ability to predict decline in insulin secretion would be useful for patient stratification for clinical trial enrollment or therapeutic selection. Analytes from 12 qualified assays were measured in shared samples from subjects newly diagnosed with T1D. We developed a computational tool (DIFAcTO, Data Integration Flexible to Account for different Types of data and Outcomes) to identify a composite panel associated with decline in insulin secretion over 2 years following diagnosis. DIFAcTO uses multiple filtering steps to reduce data dimensionality, incorporates error estimation techniques including cross-validation and sensitivity analysis, and is flexible to assay type, clinical outcome, and disease setting. Using this novel analytical tool, we identified a panel of immune markers that, in combination, are highly associated with loss of insulin secretion. The methods used here represent a potentially novel process for identifying combined immune signatures that predict outcomes relevant for complex and heterogeneous diseases like T1D.

Three-dimensional (3D) pseudoislets (PIs) can be used for the study of insulin-producing β-cells in free-floating islet-like structures similar to that of primary islets. Previously, we demonstrated the ability of islet-derived endothelial cells (iECs) to induce PIs using murine insulinomas, where PI formation enhanced insulin production and glucose responsiveness. In this report, we examined the ability of iECs to spontaneously induce the formation of free-floating 3D PIs using the EndoC-βH1 human β-cell line murine MS1 iEC. Within 14 days, the coculturing of both cell types produced fully humanized EndoC-βH1 PIs with little to no contaminating murine iECs. The size and shape of these PIs were similar to primary human islets. iEC-induced PIs demonstrated reduced dysregulated insulin release under low glucose levels and higher insulin secretion in response to high glucose and exendin-4 [a glucagon-like peptide-1 (GLP-1) analog] compared with monolayer cells cultured alone. Interestingly, iEC-PIs were also better at glucose sensing in the presence of extendin-4 compared with PIs generated on a low-adhesion surface plate in the absence of iECs and showed an overall improvement in cell viability. iEC-induced PIs exhibited increased expression of key genes involved in glucose transport, glucose sensing, β-cell differentiation, and insulin processing, with a concomitant decrease in glucagon mRNA expression. The enhanced responsiveness to exendin-4 was associated with increased protein expression of GLP-1 receptor and phosphokinase A. This rapid coculture system provides an unlimited number of human PIs with improved insulin secretion and GLP-1 responsiveness for the study of β-cell biology.

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.

The myelin sheath that coats axons allows rapid propagation of electrical impulses across the nervous system. Oligodendrocytes (ODs) are myelin-producing cells of the central nervous system (CNS) responsible for wrapping the axons of neurons. Multiple sclerosis (MS) is a demyelinating disease of the CNS identifiable by white and gray matter lesions. These lesions consist of axons that have lost their myelin through an autoimmune response to myelin and ODs. Current treatments for MS target the autoimmune aspect of the disease. However, these immunomodulators do not directly enhance the process of remyelination. The ability to remyelinate lesions can be enhanced by neural progenitor cells that can differentiate into ODs and replace lost myelin, although successful remyelination is complex and dependent on multiple factors. The restoration of lost myelin might protect the axon from degeneration and restore optimal conduction of impulses in MS patients, requiring further research on proremyelinating therapies. The combination of immunomodulators and remyelinating enhancers might be the best course of treatment for many MS patients. This Review discusses demyelination in MS, the mechanisms of remyelination, and current therapies designed to promote remyelination in MS patients.

Background Gestational diabetes mellitus (GDM) affects approximately 7–17 % of all pregnancies and has been recognized as a significant risk factor to neonatal and maternal health. Postpartum, GDM significantly increases the likelihood of developing type 2 diabetes (T2D). While it is well established that insulin resistance and impaired β-cell function contribute to GDM development, the role of active β-cell loss remains unknown. Differentially methylated circulating free DNA (cfDNA) is a minimally invasive biomarker of β-cell loss in type 1 diabetes mellitus. Here we use cfDNA to examine the levels of β-cell death in women with GDM. Methods Second to third-trimester pregnant women with GDM were compared with women with normal pregnancy (PRG), women at postpartum (PP), and non-pregnant (NP) women. Fasting glucose levels, insulin, and C-peptide levels were measured. Serum samples were collected and cfDNA purified and bisulfite treated. Methylation-sensitive probes capable of differentiating between β-cell-derived DNA (demethylated) and non-β-cell-derived DNA (methylated) were used to measure the presence of β-cell loss in the blood. Results GDM was associated with elevated fasting glucose levels (GDM = 185.9 ± 5.0 mg/dL) and reduced fasting insulin and c-peptide levels when compared with NP group. Interestingly, β-cell derived insulin DNA levels were significantly lower in women with GDM when compared with PRG, NP, and PP groups (demethylation index: PRG = 7.74 × 10−3 ± 3.09 × 10−3, GDM = 1.01 × 10−3 ± 5.86 × 10−4, p

In type 1 diabetes (T1D), β-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR) of β-cell-derived DNA in the blood can serve as a biomarker of β-cell death in T1D. Amylin is highly expressed by β-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA) may serve as a biomarker of β-cell death in T1D. β cells showed unique methylation patterns within the amylin coding region that were not observed with other tissues. The design and use of methylation-specific primers yielded a strong signal for demethylated amylin in purified DNA from murine islets when compared with other tissues. Similarly, methylation-specific primers detected high levels of demethylated amylin DNA in human islets and enriched human β-cells. In vivo testing of the primers revealed an increase in demethylated amylin cfDNA in sera of non-obese diabetic (NOD) mice during T1D progression and following the development of hyperglycemia. This increase in amylin cfDNA did not mirror the increase in insulin cfDNA, suggesting that amylin cfDNA may detect β-cell loss in serum samples where insulin cfDNA is undetected. Finally, purified cfDNA from recent onset T1D patients yielded a high signal for demethylated amylin cfDNA when compared with matched healthy controls. These findings support the use of demethylated amylin cfDNA for detection of β-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of β-cell loss.

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.

Thymic B cells represent a numerically minor cell population located primarily at the cortico-medullary junction. Their biological role is unclear. B cell-deficient μMT mice exhibited reduced medullary thymic epithelial cell (mTEC) numbers and reduced MOG and insulin mRNA expression. Lymphotoxin produced by B cells was critical for normal tissue restricted antigen (TRA) expression, suggesting that B cells regulate self-antigens through their production of LT. These results reveal an unexpected role of B cells in mTEC maintenance and expression of TRAs through their production of LT.

OBJECTIVE β-Cell and islet endothelial cell destruction occurs during the progression of type 1 diabetes, but, paradoxically, β-cell proliferation is increased during this period. Altered glucose tolerance may affect β-cell mass and its association with endothelial cells. Our objective was to study the effects of glucose and inflammation on islet vascularity and on β function, mass, and insulin in immunologically tolerant anti-CD3 monoclonal antibody (mAb)-treated and prediabetic NOD mice. RESEARCH DESIGN AND METHODS The effects of phloridzin or glucose injections on β-cells and endothelial cells were tested in prediabetic and previously diabetic NOD mice treated with anti-CD3 mAbs. Glucose tolerance, immunofluorescence staining, and examination of islet cultures ex vivo were evaluated. RESULTS Islet endothelial cell density decreased in NOD mice and failed to recover after anti-CD3 mAb treatment despite baseline euglycemia. Glucose treatment of anti-CD3 mAb–treated mice showed increased islet vascular density and increased insulin content, which was associated with improved glucose tolerance. The increase in the vascular area was dependent on islet inflammation. Increased islet endothelial cell density was associated with increased production of vascular endothelial growth factor (VEGF) by islets from NOD mice. This response was recapitulated ex vivo by the transfer of supernatants from NOD islets cultured in high-glucose levels. CONCLUSIONS Our results demonstrate a novel role for glucose and inflammation in the control of islet vasculature and insulin content of β-cells in prediabetic and anti-CD3–treated NOD mice. VEGF production by the islets is affected by glucose levels and is imparted by soluble factors released by inflamed islets.

A key question in the field of autoimmunity concerns the fact that experimental disease is generally induced more easily with closely related, but not completely identical, tissue-restricted antigens. Here, the possibility that naturally occurring regulatory T cells (Tregs) for self-antigens are more potent than those for related antigens was investigated. The self-antigen specificity of naturally occurring Tregs was tested in experimental autoimmune encephalomyelitis (EAE) induced with mouse (self) or closely related (rat) myelin oligodendrocyte glycoproteins (MOGs). Surprisingly, Treg depletion increased EAE severity in mice immunized with mouse, but not rat, MOG. This increase was associated with increased T-cell activation and infiltration of the central nervous system, as well as increased interleukin (IL)-17 production and a higher ratio of interferon-gamma- to IL-10-producing cells. These data suggest that Tregs are specific for self-antigen and do not "cross-protect" against autoimmunity even when disease is induced with closely related foreign antigens.

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.

Untreated diabetic rats show impaired counterregulation against hypoglycemia. The blunted epinephrine responses are associated with reduced adrenomedullary tyrosine hydroxylase (TH) mRNA levels. Recurrent hypoglycemia further impairs epinephrine counterregulation and is also associated with reduced phenylethanolamine N-methyltransferase mRNA. This study investigated the adaptations underlying impaired counterregulation in insulin-treated diabetic rats, a more clinically relevant model. We studied the effects of insulin treatment on counterregulatory hormones and adrenal catecholamine-synthesizing enzymes and adaptations after recurrent hypoglycemia. Groups included: normal; diabetic, insulin-treated for 3 wk (DI); and insulin-treated diabetic exposed to seven episodes (over 4 d) of hyperinsulinemic-hypoglycemia (DI-hypo) or hyperinsulinemic-hyperglycemia (DI-hyper). DI-hyper rats differentiated the effects of hyperinsulinemia from those of hypoglycemia. On d 5, rats from all groups were assessed for adrenal catecholamine-synthesizing enzyme levels or underwent hypoglycemic clamps to examine counterregulatory responses. Despite insulin treatment, fasting corticosterone levels remained increased, and corticosterone responses to hypoglycemia were impaired in DI rats. However, glucagon, epinephrine, norepinephrine, and ACTH counterregulatory defects were prevented. Recurrent hypoglycemia in DI-hypo rats blunted corticosterone but, surprisingly, not epinephrine responses. Norepinephrine and ACTH responses also were not impaired, whereas glucagon counterregulation was reduced due to repeated hyperinsulinemia. Insulin treatment prevented decreases in basal TH protein and increased PNMT and dopamine β-hydroxylase protein. DI-hypo rats showed increases in TH, PNMT, and dopamine β-hydroxylase. We conclude that insulin treatment of diabetic rats protects against most counterregulatory defects but not elevated fasting corticosterone and decreased corticosterone counterregulation. Protection against epinephrine defects, both without and with antecedent hypoglycemia, is associated with enhancement of adrenal catecholamine-synthesizing enzyme levels.

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.

Chronic leptin administration at pharmacologic doses normalizes food intake and body weight in streptozotocin (STZ)-diabetic rats. We examined the metabolic effects of acute partial physiological leptin restoration in STZ-diabetic rats by using subcutaneous osmotic mini pumps. Groups: (1) Rats infused with vehicle (DV); (2) rats infused with recombinant murine methionine leptin (DL) at 4.5 microg . (kg body weight . d)(-1); (3)pair-fed rats (DP) given a food ration matching that consumed by the DL group. A fourth group of nondiabetic, normal (N) rats was also studied to assess normal metabolic efficiency, hypothalamic-pituitary-adrenal (HPA) activity and sympathoadrenal activity. Following leptin infusion, food consumption by DL rats was significantly lower than in DV rats. Paradoxically, despite a similar food intake to that of the DP group, which demonstrated a 40% reduction in body mass, DL rats increased their initial body weight by approximately 20% (P < .05). Plasma corticosterone and ACTH concentrations were elevated by 2-fold to 3-fold in DL versus N, DP, and DV rats. In the pars distalis, glucocorticoid receptor (GR) mRNA levels were significantly higher in DL and DP rats compared with N and DV rats. Our results suggest that partial restoration of physiologic leptin: (1) successfully reduces hyperphagia while allowing body weight gain in STZ-diabetic rats; (2) increases corticosterone levels in STZ-diabetic rats, which may in turn counteract the anorexic effects of diabetes; and (3) is associated with increased pituitary GR mRNA levels, despite elevated corticosterone levels, suggesting that leptin may interfere with the negative feedback regulation of the HPA axis.

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.

OBJECTIVE β-Cell and islet endothelial cell destruction occurs during the progression of type 1 diabetes, but, paradoxically, β-cell proliferation is increased during this period. Altered glucose tolerance may affect β-cell mass and its association with endothelial cells. Our objective was to study the effects of glucose and inflammation on islet vascularity and on β function, mass, and insulin in immunologically tolerant anti-CD3 monoclonal antibody (mAb)-treated and prediabetic NOD mice. RESEARCH DESIGN AND METHODS The effects of phloridzin or glucose injections on β-cells and endothelial cells were tested in prediabetic and previously diabetic NOD mice treated with anti-CD3 mAbs. Glucose tolerance, immunofluorescence staining, and examination of islet cultures ex vivo were evaluated. RESULTS Islet endothelial cell density decreased in NOD mice and failed to recover after anti-CD3 mAb treatment despite baseline euglycemia. Glucose treatment of anti-CD3 mAb–treated mice showed increased islet vascular density and increased insulin content, which was associated with improved glucose tolerance. The increase in the vascular area was dependent on islet inflammation. Increased islet endothelial cell density was associated with increased production of vascular endothelial growth factor (VEGF) by islets from NOD mice. This response was recapitulated ex vivo by the transfer of supernatants from NOD islets cultured in high-glucose levels. CONCLUSIONS Our results demonstrate a novel role for glucose and inflammation in the control of islet vasculature and insulin content of β-cells in prediabetic and anti-CD3–treated NOD mice. VEGF production by the islets is affected by glucose levels and is imparted by soluble factors released by inflamed islets.

The autoimmune regulator (AIRE) gene encodes a transcription factor involved in the presentation of tissue-restricted antigens during T-cell development in the thymus. Mutations of this gene lead to type 1 autoimmune polyglandular syndrome (APS-1), also termed autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is characterized by the clinical presentation of at least two of a triad of underlying disorders: Addison disease, hypoparathyroidism and chronic mucocutaneous candidiasis. This Review describes the process of positive and negative selection of developing T cells in the thymus and the role of AIRE as a regulator of peripheral antigen presentation. Furthermore, it addresses how mutations of this gene lead to the failure to eliminate autoreactive T cells, which can lead to clinical autoimmune syndromes.

Type 1 diabetes is thought to be caused by an immune-mediated destruction of β cells that occurs over years and continues even after presentation with hyperglycemia. Adaptive immune mechanisms are believed to be primary mediators of this process. Immune memory for the initial process that resulted in β-cell failure, the high frequency of alloreactive T cells, and the nonphysiologic environment into which islets have been transplanted all create obstacles for successful reversal of diabetes with islet replacement. In this chapter we review the immune mechanisms that are thought to be responsible for β-cell destruction and discuss the obstacles that stand in the way of the successful achievement of β-cell replacement.

Recently, we established that hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia were impaired in uncontrolled streptozotocin (STZ)-diabetic (65 mg/kg) rats and insulin treatment restored most of these responses. In the current study, we used phloridzin to determine whether the restoration of blood glucose alone was sufficient to normalize HPA function in diabetes. Normal, diabetic, insulin-treated, and phloridzin-treated diabetic rats were either killed after 8 days or subjected to a hypoglycemic (40 mg/dl) glucose clamp. Basal: Elevated basal ACTH and corticosterone in STZ rats were normalized with insulin but not phloridzin. Increases in hypothalamic corticotrophin-releasing hormone (CRH) and inhibitory hippocampal mineralocorticoid receptor (MR) mRNA with STZ diabetes were not restored with either insulin or phloridzin treatments. Hypoglycemia: In response to hypoglycemia, rises in plasma ACTH and corticosterone were significantly lower in diabetic rats compared with controls. Insulin and phloridzin restored both ACTH and corticosterone responses in diabetic animals. Hypothalamic CRH mRNA and pituitary pro-opiomelanocortin mRNA expression increased following 2 h of hypoglycemia in normal, insulin-treated, and phloridzin-treated diabetic rats but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal MR mRNA in control, insulin-, and phloridzin-treated diabetic rats but not uncontrolled diabetic rats, whereas glucocorticoid receptor mRNA was not altered by hypoglycemia. In conclusion, despite elevated basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that defects in the CRH response may be related to a defective MR response. It is intriguing that phloridzin did not restore basal HPA activity but it restored the HPA response to hypoglycemia, suggesting that defects in basal HPA function in diabetes are due to insulin deficiency, but impaired responsiveness to hypoglycemia appears to stem from chronic hyperglycemia.

The receptor for glycation end products (RAGE) has been previously implicated in shaping the adaptive immune response. RAGE is expressed in T cells after activation and constitutively in T cells from patients with diabetes. The effects of RAGE on adaptive immune responses are not clear: Previous reports show that RAGE blockade affects Th1 responses. To clarify the role of RAGE in adaptive immune responses and the mechanisms of its effects, we examined whether RAGE plays a role in T cell activation in a Th2 response involving ovalbumin (OVA)-induced asthma in mice. WT and RAGE deficient wild-type and OT-II mice, expressing a T cell receptor specific for OVA, were immunized intranasally with OVA. Lung cellular infiltration and T cell responses were analyzed by immunostaining, FACS, and multiplex bead analyses for cytokines. RAGE deficient mice showed reduced cellular infiltration in the bronchial alveolar lavage fluid and impaired T cell activation in the mediastinal lymph nodes when compared with WT mice. In addition, RAGE deficiency resulted in reduced OT-II T cell infiltration of the lung and impaired IFNγ and IL-5 production when compared with WT mice and reduced infiltration when transferred into WT hosts. When cultured under conditions favoring the differentiation of T cells subsets, RAGE deficient T cells showed reduced production of IFNγ but increased production of IL-17. Our data show a stimulatory role for RAGE in T activation in OVA-induced asthma. This role is largely mediated by the effects of RAGE on T cell proliferation and differentiation. These findings suggest that RAGE may play a regulatory role in T cell responses following immune activation.

β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.

We have shown that the receptor for advanced glycation endproducts (RAGE) is required for normal T cell activation and differentiation into Th1 cells, since RAGE-/- T cells show reduced proliferation and IFNγ expression after activation with alloantigens. Here, we examined T cell activation in RAGE-/- and OT-II.RAGE-/- mice in a model of T cell induced asthma. Intranasal OVA challenge of RAGE-/- mice resulted in reduced T cell activation as measured by increased CD62L expression, decreased CD69 expression, and reduced IL5 production. Cellular infiltrates in the lungs of OVA immunized RAGE-/- mice were reduced when compared with WT mice. Intranasal OVA challenge of OT-II mice induced a robust T cell infiltration of the lung, which was reduced in OT-II.RAGE-/- mice. Moreover, cells from mediastinal lymph nodes of OT-II.RAGE-/- mice showed reduced IL2, IL5, IL10, IL12, and IFN? production. Interestingly, purified RAGE-/- T cells showed lower basal levels of Ca2+ flux when compared with WT cells. These levels were not corrected in response to αCD3 stimulation. Reduced Ca2+ flux was associated with reduced expression of the transcription factor T-bet but with no change in GATA-3 expression in RAGE-/- T cells. In summary, these results reveal an unexpected defect in Ca2+ flux of RAGE deficient T cells under basal and activated conditions and reduced T cell activation in OVA-induced asthma, suggesting a novel role for RAGE in T cell signaling.

Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). Minimally invasive biomarkers of MS are required for disease diagnosis and treatment. Differentially methylated circulating-free DNA (cfDNA) is a useful biomarker for disease diagnosis and prognosis, and may offer to be a viable approach for understanding MS. Here, methylation-specific primers and quantitative real-time PCR were used to study methylation patterns of the myelin oligodendrocyte glycoprotein (MOG) gene, which is expressed primarily in myelin-producing oligodendrocytes (ODCs). MOG-DNA was demethylated in O4+ ODCs in mice and in DNA from human oligodendrocyte precursor cells (OPCs) when compared with other cell types. In the cuprizone-fed mouse model of demyelination, ODC derived demethylated MOG cfDNA was increased in serum and was associated with tissue-wide demyelination, demonstrating the utility of demethylated MOG cfDNA as a biomarker of ODC death. Collected sera from patients with active (symptomatic) relapsing-remitting MS (RRMS) demonstrated a higher signature of demethylated MOG cfDNA when compared with patients with inactive disease and healthy controls. Taken together, these results offer a minimally invasive approach to measuring ODC death in the blood of MS patients that may be used to monitor disease progression., Highlights • Currently, there are no molecular biomarkers of multiple sclerosis (MS). • A minimally invasive assay for measuring oligodendrocyte (ODC) cell loss in relapsing-remitting MS is described. • DNA methylation of the myelin oligodendrocyte glycoprotein gene is used as a measure of ODC loss in the blood. Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system. Currently, there are no molecular biomarkers of MS, thereby limiting disease diagnosis, prognosis, and assessment of new clinical interventions. Myelin oligodendrocyte glycoprotein (MOG) is expressed solely by oligodendrocytes (ODCs) as an integral part of the myelin sheath. This report describes a minimally invasive biomarker assay for measuring ODC-derived DNA in the blood of MS patients. It describes the presence of unique DNA methylation patterns in the MOG gene in ODCs, which is used to design methylation-specific primers. Analysis of sera from patients with active relapsing-remitting MS shows an increase in levels of ODC-derived circulating free DNA when compared with inactive disease and healthy controls.