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Objective: Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell-depleting agents, as well as attempts seeking diabetes prevention, are being considered.Research Design and Methods: Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset.Results: The murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell-dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets.Conclusions: These findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention. [ABSTRACT FROM AUTHOR]

Objective: A progressive decline in insulin responses to glucose was noted in individuals before the onset of type 1 diabetes. We determined whether such abnormalities occurred in prediabetic NOD mice-the prototypic model for human type 1 diabetes.Research Design and Methods: Morning blood glucose was measured every other day in a cohort of NOD females. Glucose tolerance and insulin secretion were measured longitudinally by intraperitoneal glucose tolerance tests in NOD/ShiLtJ and BALB/cJ mice 6 to 14 weeks of age. Arginine-stimulated insulin secretion and insulin sensitivity were assessed during intraperitoneal arginine or intraperitoneal insulin tolerance tests.Results: During prediabetes, NOD females displayed a progressive increase in glucose levels followed by an acute onset of hyperglycemia. First-phase insulin responses (FPIRs) during the intraperitoneal glucose tolerance test (IPGTT) declined before loss of glucose tolerance in NOD. The failure of FPIR could be detected, with a decline in peak insulin secretion during IPGTT. Arginine-stimulated insulin secretion remained unchanged during the study period. The decline in insulin secretion in NOD mice could not be explained by changes in insulin sensitivity.Conclusions: There was an impressive decline in FPIR before changes in glucose tolerance, suggesting that impairment of FPIR is an early in vivo marker of progressive β-cell failure in NOD mice and human type 1 diabetes. We portend that these phenotypes in NOD mice follow a similar pattern to those seen in humans with type 1 diabetes and validate, in a novel way, the importance of this animal model for studies of this disease. [ABSTRACT FROM AUTHOR]

Beta-cell apoptosis appears to represent a key event in the pathogenesis of type 1 diabetes. Previous studies have demonstrated that administration of the serine proteinase inhibitor alpha1-antitrypsin (AAT) prevents type 1 diabetes development in NOD mice and prolongs islet allograft survival in rodents; yet the mechanisms underlying this therapeutic benefit remain largely unclear. Herein we describe novel findings indicating that AAT significantly reduces cytokine- and streptozotocin (STZ)-induced beta-cell apoptosis. Specifically, strong antiapoptotic activities for AAT (Prolastin, human) were observed when murine insulinoma cells (MIN6) were exposed to tumor necrosis factor-alpha. In a second model system involving STZ-induced beta-cell apoptosis, treatment of MIN6 cells with AAT similarly induced a significant increase in cellular viability and a reduction in apoptosis. Importantly, in both model systems, treatment with AAT completely abolished induced caspase-3 activity. In terms of its activities in vivo, treatment of C57BL/6 mice with AAT prevented STZ-induced diabetes and, in agreement with the in vitro analyses, supported the concept of a mechanism involving the disruption of beta-cell apoptosis. These results propose a novel biological function for this molecule and suggest it may represent an effective candidate for attempts seeking to prevent or reverse type 1 diabetes. [ABSTRACT FROM AUTHOR]

Recently, we identified in normally type 1 diabetes-prone NOD/LtJ mice a spontaneous new leptin receptor (LEPR) mutation (designated Lepr(db-5J)) producing juvenile obesity, hyperglycemia, hyperinsulinemia, and hyperleptinemia. This early type 2 diabetes syndrome suppressed intra-islet insulitis and permitted spontaneous diabetes remission. No significant differences in plasma corticosterone, splenic CD4(+) or CD8(+) T-cell percentages, or functions of CD3(+) T-cells in vitro distinguished NOD wild-type from mutant mice. Yet splenocytes from hyperglycemic mutant donors failed to transfer type 1 diabetes into NOD.Rag1(-/-) recipients over a 13-week period, whereas wild-type donor cells did so. This correlated with significantly reduced (P < 0.01) frequencies of insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein-reactive CD8(+) T-effector clonotypes in mutant mice. Intra-islet insulitis was also significantly suppressed in lethally irradiated NOD-Lepr(db-5J)/Lt recipients reconstituted with wild-type bone marrow (P < 0.001). In contrast, type 1 diabetes eventually developed when mutant marrow was transplanted into irradiated wild-type recipients. Mitogen-induced T-cell blastogenesis was significantly suppressed when splenic T-cells from both NOD/Lt and NOD-Lepr(db-5J)/Lt donors were incubated with irradiated mutant peritoneal exudate cells (P < 0.005). In conclusion, metabolic disturbances elicited by a type 2 diabetes syndrome (insulin and/or leptin resistance, but not hypercorticism) appear to suppress type 1 diabetes development in NOD-Lepr(db-5J)/Lt by inhibiting activation of T-effector cells. [ABSTRACT FROM AUTHOR]

Recent increases in life expectancy for patients with cystic fibrosis (CF) have paradoxically been accompanied by an increasing incidence of CF related diabetes (CFRD), which dramatically worsens prognosis. By a number of immunological parameter, CF alone is associated with a T[sub H]2 bias; potentially contributing to decreased macrophage host defense and hence, increasing the risk for prolonged infections. We tested the hypothesis that the development of hyperglycemia would exacerbate this T[sub H]2 bias through analysis of cystic fibrosis transmembrane conductance regulator deficient (CFTR-/-) mice; an animal model of CF. Nine CFTR-/-, 5 C57BL/6J, and 6 FVB/NJ mice received streptozotocin (STZ) (single dose of 100mg/kg) to induce hyperglycemia, while 8 CFTR-/-, 5 C57BL/6J, and 10 FVB/NJ mice received lactated fingers (LR). Four wk later, splenocytes were isolated, stimulated with Con-A, and cytokine production (48 hr) as well as stimulation indices (SI; 72 hr) determined. Four wk following STZ, CFTR-/- mice had greater fasting glucoses (CFTR.-/-288.4 ±97.4 mg/dL, C57BL/6J 154.2 ± 19.4, FVB/NJ 188 ± 42.26, p<0.05). SI of STZ-treated CFTR-/- mice were elevated compared to LR-treated CFTR-/- mice (p<0.05) as well as both STZ-treated and LR-treated control strains. Fasting glucose levels of STZ-treated CFTR-/- mice correlated with splenocyte proliferation (P<0.003). In terms of T[sub H]2 bias, concentrations of stimulated IL-10 were markedly elevated following the development of hyperglycemic induction in CFTR-/- mice compared to all controls (P<0.05). In addition, IL-4 was greater in CFTR-/- mice compared to controls (P<0.05), but no difference was observed between STZ and LR-treated CFTR-/- mice. These studies indicate hyperglycemia may indeed exacerbate the clinical course in CFRD by enhancing T[sub H]2 responses and provide additional, yet novel support for efforts seeking to maximize diabetes management in patients with this disorder. [ABSTRACT FROM AUTHOR]

Assessment of pancreas cell type composition is crucial to the understanding of the genesis of diabetes. Current approaches use immunodetection of protein markers, for example, insulin as a marker of β-cells. A major limitation of these methods is that protein content varies in physiological and pathological conditions, complicating the extrapolation to actual cell number. Here, we demonstrate the use of cell type-specific DNA methylation markers for determining the fraction of specific cell types in human islet and pancreas specimens. We identified genomic loci that are uniquely demethylated in specific pancreatic cell types and applied targeted PCR to assess the methylation status of these loci in tissue samples, enabling inference of cell type composition. In islet preparations, normalization of insulin secretion to β-cell DNA revealed similar β-cell function in pre-type 1 diabetes (T1D), T1D, and type 2 diabetes (T2D), which was significantly lower than in donors without diabetes. In histological pancreas specimens from recent-onset T1D, this assay showed β-cell fraction within the normal range, suggesting a significant contribution of β-cell dysfunction. In T2D pancreata, we observed increased α-cell fraction and normal β-cell fraction. Methylation-based analysis provides an accurate molecular alternative to immune detection of cell types in the human pancreas, with utility in the interpretation of insulin secretion assays and the assessment of pancreas cell composition in health and disease., (© 2024 by the American Diabetes Association.)

Although B cells reactive with islet autoantigens are silenced by tolerance mechanisms in healthy individuals, they can become activated and contribute to the development of type 1 diabetes. We previously demonstrated that high-affinity insulin-binding B cells (IBCs) occur exclusively in the anergic (B ND ) compartment in peripheral blood of healthy subjects. Consistent with their activation early in disease development, high-affinity IBCs are absent from the B ND compartment of some first-degree relatives (FDRs) as well as all patients with autoantibody-positive prediabetes and new-onset type 1 diabetes, a time when they are found in pancreatic islets. Loss of B ND IBCs is associated with a loss of the entire B ND B-cell compartment consistent with provocation by an environmental trigger or predisposing genetic factors. To investigate potential mechanisms operative in subversion of B-cell tolerance, we explored associations between HLA and non-HLA type 1 diabetes-associated risk allele genotypes and loss of B ND s in FDRs. We found that high-risk HLA alleles and a subset of non-HLA risk alleles (i.e., PTPN2 [rs1893217], INS [rs689], and IKZF3 [rs2872507]), relevant to B- and T-cell development and function are associated with loss of anergy. Hence, the results suggest a role for risk-conferring alleles in perturbation of B-cell anergy during development of type 1 diabetes., (© 2018 by the American Diabetes Association.)

Despite decades of research in humans and mouse models of disease, substantial gaps remain in our understanding of pathogenic mechanisms underlying the development of type 1 diabetes. Furthermore, translation of therapies from preclinical efforts capable of delaying or halting β-cell destruction has been limited. Hence, a pressing need exists to identify alternative animal models that reflect human disease. Canine insulin deficiency diabetes is, in some cases, considered to follow autoimmune pathogenesis, similar to NOD mice and humans, characterized by hyperglycemia requiring lifelong exogenous insulin therapy. Also similar to human type 1 diabetes, the canonical canine disorder appears to be increasing in prevalence. Whereas islet architecture in rodents is distinctly different from humans, canine pancreatic endocrine cell distribution is more similar. Differences in breed susceptibility alongside associations with MHC and other canine immune response genes parallel that of different ethnic groups within the human population, a potential benefit over NOD mice. The impact of environment on disease development also favors canine over rodent models. Herein, we consider the potential for canine diabetes to provide valuable insights for human type 1 diabetes in terms of pancreatic histopathology, impairment of β-cell function and mass, islet inflammation (i.e., insulitis), and autoantibodies specific for β-cell antigens., (© 2017 by the American Diabetes Association.)

The introduction of β-cell autoantigens via the gut through Lactococcus lactis (L. lactis) has been demonstrated to be a promising approach for diabetes reversal in NOD mice. Here we show that a combination therapy of low-dose anti-CD3 with a clinical-grade self-containing L. lactis, appropriate for human application, secreting human proinsulin and interleukin-10, cured 66% of mice with new-onset diabetes, which is comparable to therapy results with plasmid-driven L. lactis Initial blood glucose concentrations (<350 mg/dL) and insulin autoantibody positivity were predictors of the stable reversal of hyperglycemia, and decline in insulin autoantibody positivity was an immune biomarker of therapeutic outcome. The assessment of the immune changes induced by the L. lactis-based therapy revealed elevated frequencies of CD4 + Foxp3 + T cells in the pancreas-draining lymph nodes, pancreas, and peripheral blood of all treated mice, independent of metabolic outcome. Neutralization of cytotoxic T-lymphocyte antigen 4 and transforming growth factor-β partially abrogated the suppressive function of therapy-induced regulatory T cells (Tregs). Ablation or functional impairment of Foxp3 + Tregs in vivo at the start or stop of therapy impaired immune tolerance, highlighting the dependence of the therapy-induced tolerance in mice with new-onset diabetes on the presence and functionality of CD4 + Foxp3 + T cells. Biomarkers identified in this study can potentially be used in the future to tailor the L. lactis-based combination therapy for individual patients., (© 2017 by the American Diabetes Association.)

Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and β-cell and α-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb(+), n = 18) and without islet autoantibodies (AAb(-), n = 61). Insulitis was observed in four of four donors (100%) with type 1 diabetes duration of ≤1 year and two AAb(+) donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = -0.58, P = 0.01) but not with age at disease onset. Residual β-cells were observed in all type 1 diabetes donors with insulitis, while β-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin(+) islets compared with 2% of insulin(-) islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45(+), CD3(+), CD4(+), CD8(+), and CD20(+) cell numbers within the insulitis (r = 0.53-0.73, P = 0.004-0.04), but not CD68(+) or CD11c(+) cells. The presence of β-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The rapid rise in the incidence of type 1 diabetes (T1D) suggests the involvement of environmental factors including viral infections. We evaluated the association between viral infections and T1D by profiling antiviral antibodies using a high-throughput immunoproteomics approach in patients with new-onset T1D. We constructed a viral protein array comprising the complete proteomes of seven viruses associated with T1D and open reading frames from other common viruses. Antibody responses to 646 viral antigens were assessed in 42 patients with T1D and 42 age- and sex-matched healthy control subjects (mean age 12.7 years, 50% males). Prevalence of antiviral antibodies agreed with known infection rates for the corresponding virus based on epidemiological studies. Antibody responses to Epstein-Barr virus (EBV) were significantly higher in case than control subjects (odds ratio 6.6; 95% CI 2.0-25.7), whereas the other viruses showed no differences. The EBV and T1D association was significant in both sex and age subgroups (≤12 and >12 years), and there was a trend toward early EBV infections among the case subjects. These results suggest a potential role for EBV in T1D development. We believe our innovative immunoproteomics platform is useful for understanding the role of viral infections in T1D and other disorders where associations between viral infection and disease are unclear., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Most natural history models for type 1 diabetes (T1D) propose that overt hyperglycemia results after a progressive loss of insulin-secreting β-cell mass and/or function. To experimentally address this concept, we prospectively determined morning blood glucose measurements every other day in multiple cohorts (total n = 660) of female NOD/ShiLtJ mice starting at 8 weeks of age until diabetes onset or 26 weeks of age. Consistent with this notion, a majority of mice that developed diabetes (354 of 489 [72%]) displayed a progressive increase in blood glucose with transient excursions >200 mg/dL, followed by acute and persistent hyperglycemia at diabetes onset. However, 135 of the 489 (28%) diabetic animals demonstrated normal glucose values followed by acute (i.e., sudden) hyperglycemia. Interestingly, diabetes onset occurred earlier in mice with acute versus progressive disease onset (15.37 ± 0.3207 vs. 17.44 ± 0.2073 weeks of age, P < 0.0001). Moreover, the pattern of onset (i.e., progressive vs. acute) dramatically influenced the ability to achieve reversal of T1D by immunotherapeutic intervention, with increased effectiveness observed in situations of a progressive deterioration in euglycemia. These studies highlight a novel natural history aspect in this animal model, one that may provide important guidance for the selection of subjects participating in human trials seeking disease reversal., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

An increasing number of therapies have proven effective at reversing hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), yet situations of successful translation to human T1D are limited. This may be partly due to evaluating the effect of treating immediately at diagnosis in mice, which may not be reflective of the advanced disease state in humans at disease onset. In this study, we treated NOD mice with new-onset as well as established disease using various combinations of four drugs: antithymocyte globulin (ATG), granulocyte-colony stimulating factor (G-CSF), a dipeptidyl peptidase IV inhibitor (DPP-4i), and a proton pump inhibitor (PPI). Therapy with all four drugs induced remission in 83% of new-onset mice and, remarkably, in 50% of NOD mice with established disease. Also noteworthy, disease remission occurred irrespective of initial blood glucose values and mechanistically was characterized by enhanced immunoregulation involving alterations in CD4+ T cells, CD8+ T cells, and natural killer cells. This combination therapy also allowed for effective treatment at reduced drug doses (compared with effective monotherapy), thereby minimizing potential adverse effects while retaining efficacy. This combination of approved drugs demonstrates a novel ability to reverse T1D, thereby warranting translational consideration., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

T-cell responses directed against insulin-secreting pancreatic β-cells are the key events highlighting type 1 diabetes (T1D). Therefore, a defective control of T-cell activation is thought to underlie T1D development. Recent studies implicated a B7-like negative costimulatory protein, V-set domain-containing T-cell activation inhibitor-1 (VTCN1), as a molecule capable of inhibiting T-cell activation and, potentially, an important constituent in experimental models of T1D. Here, we unravel a general deficiency within the VTCN1 pathway that is shared between diabetes-prone mice and a subset of T1D patients. Gradual loss of membrane-tethered VTCN1 from antigen-presenting cells combined with an increased release of soluble VTCN1 (sVTCN1) occurs in parallel to natural T1D development, potentiating hyperproliferation of diabetogenic T cells. Mechanistically, we demonstrate that the loss of membrane-tethered VTCN1 is linked to proteolytic cleavage mediated by the metalloproteinase nardilysin. The cleaved sVTCN1 fragment was detected at high levels in the peripheral blood of 53% T1D patients compared with only 9% of the healthy subjects. Elevated blood sVTCN1 levels appeared early in the disease progression and correlated with the aggressive pace of disease, highlighting the potential use of sVTCN1 as a new T1D biomarker, and identifying nardilysin as a potential therapeutic target., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The dysregulation of receptor tyrosine kinases (RTKs) in multiple cell types during chronic inflammation is indicative of their pathogenic role in autoimmune diseases. Among the many RTKs, vascular endothelial growth factor receptor (VEGFR) stands out for its multiple effects on immunity, vascularization, and cell migration. Herein, we examined whether VEGFR participated in the pathogenesis of type 1 diabetes (T1D) in nonobese diabetic (NOD) mice. We found that RTK inhibitors (RTKIs) and VEGF or VEGFR-2 antibodies reversed diabetes when administered at the onset of hyperglycemia. Increased VEGF expression promoted islet vascular remodeling in NOD mice, and inhibition of VEGFR activity with RTKIs abrogated the increase in islet vascularity, impairing T-cell migration into the islet and improving glucose control. Metabolic studies confirmed that RTKIs worked by preserving islet function, as treated mice had improved glucose tolerance without affecting insulin sensitivity. Finally, examination of human pancreata from patients with T1D revealed that VEGFR-2 was confined to the islet vascularity, which was increased in inflamed islets. Collectively, this work reveals a previously unappreciated role for VEGFR-2 signaling in the pathogenesis of T1D by controlling T-cell accessibility to the pancreatic islets and highlights a novel application of VEGFR-2 antagonists for the therapeutic treatment of T1D.

Objective: Previous studies have noted a specific association between type 1 diabetes and insufficient levels of vitamin D, as well as polymorphisms within genes related to vitamin D pathways. Here, we examined whether serum levels or genotypes of the vitamin D-binding protein (VDBP), a molecule key to the biologic actions of vitamin D, specifically associate with the disorder., Research Design and Methods: A retrospective, cross-sectional analysis of VDBP levels used samples from 472 individuals of similar age and sex distribution, including 153 control subjects, 203 patients with type 1 diabetes, and 116 first-degree relatives of type 1 diabetic patients. Single nucleotide polymorphism (SNP) typing for VDBP polymorphisms (SNP rs4588 and rs7041) was performed on this cohort to determine potential genetic correlations. In addition, SNP analysis of a second sample set of banked DNA samples from 1,502 type 1 diabetic patients and 1,880 control subjects also was used to determine genotype frequencies., Results: Serum VDBP levels were highest in healthy control subjects (median 423.5 µg/mL [range 193.5-4,345.0; interquartile range 354.1-]586), intermediate in first-degree relatives (402.9 µg/mL [204.7-4,850.0; 329.6-492.4]), and lowest in type 1 diabetic patients (385.3 µg/mL [99.3-1,305.0; 328.3-473.0]; P = 0.003 vs. control subjects). VDBP levels did not associate with serum vitamin D levels, age, or disease duration. However, VDBP levels were, overall, lower in male subjects (374.7 µg/mL [188.9-1,602.0; 326.9-449.9]) than female subjects (433.4 µg/mL [99.3-4,850.0; 359.4-567.8]; P < 0.0001). It is noteworthy that no differences in genotype frequencies of the VDBP polymorphisms were associated with serum VDBP levels or between type 1 diabetic patients and control subjects., Conclusions: Serum VDBP levels are decreased in those with type 1 diabetes. These studies suggest that multiple components in the metabolic pathway of vitamin D may be altered in type 1 diabetes and, collectively, have the potential to influence disease pathogenesis.

Objective: Glucagon-like peptide-1 (GLP-1) and gastrin promote pancreatic beta-cell function, survival, and growth. Here, we investigated whether GLP-1 and gastrin can restore the beta-cell mass and reverse hyperglycemia in NOD mice with autoimmune diabetes., Research Design and Methods: Acutely diabetic NOD mice were treated with GLP-1 and gastrin, separately or together, twice daily for 3 weeks. Blood glucose was measured weekly and for a further 5 weeks after treatments, after which pancreatic insulin content and beta-cell mass, proliferation, neogenesis, and apoptosis were measured. Insulin autoantibodies were measured, and adoptive transfer of diabetes and syngeneic islet transplant studies were done to evaluate the effects of GLP-1 and gastrin treatment on autoimmunity., Results: Combination therapy with GLP-1 and gastrin, but not with GLP-1 or gastrin alone, restored normoglycemia in diabetic NOD mice. The GLP-1 and gastrin combination increased pancreatic insulin content, beta-cell mass, and insulin-positive cells in pancreatic ducts, and beta-cell apoptosis was decreased. Insulin autoantibodies were reduced in GLP-1- and gastrin-treated NOD mice, and splenocytes from these mice delayed adoptive transfer of diabetes in NOD-scid mice. Syngeneic islet grafts in GLP-1 -and gastrin-treated NOD mice were infiltrated by leukocytes with a shift in cytokine expression from interferon-gamma to transforming growth factor-beta1, and beta-cells were protected from apoptosis., Conclusions: Combination therapy with GLP-1 and gastrin restores normoglycemia in diabetic NOD mice by increasing the pancreatic beta-cell mass and downregulating the autoimmune response.

Objective: Antilymphocyte serum can reverse overt type 1 diabetes in NOD mice; yet, the therapeutic parameters and immunological mechanisms underlying the ability for this agent to modulate autoimmune responses against beta-cells are unclear, forming the rationale for this investigation., Research Design and Methods: A form of antilymphocyte serum, rabbit anti-mouse thymocyte globulin (mATG), was utilized in a variety of in vivo and in vitro settings, each for the purpose of defining the physiological, immunological, and metabolic activities of this agent, with particular focus on actions influencing development of type 1 diabetes., Results: We observed that mATG attenuates type 1 diabetes development in an age-dependent fashion, only proving efficacious at disease onset or in the late pre-diabetic phase (12 weeks of age). When provided at 12 weeks of age, mATG reversed pancreatic insulitis, improved metabolic responses to glucose challenge, and rapidly increased frequency of antigen-presenting cells in spleen and pancreatic lymph nodes. Surprisingly, mATG therapy dramatically increased, in an age-dependent fashion, the frequency and the functional activity of CD4(+)CD25(+) regulatory T-cells. Adoptive transfer/cotransfer studies of type 1 diabetes also support the concept that mATG treatment induces a stable and transferable immunomodulatory repertoire in vivo., Conclusions: These findings indicate that an induction of immunoregulation, rather than simple lymphocyte depletion, contributes to the therapeutic efficacy of antithymocyte globulin and suggest that time-dependent windows for the ability to delay or reverse type 1 diabetes exist based on the capacity to enhance the functional activity of regulatory T-cells.

Regulatory T-cells (Tregs) play a critical role in maintaining dominant peripheral tolerance. Previous characterizations of Tregs in type 1 diabetes have used antibodies against CD4 and alpha-chain of the interleukin-2 receptor complex (CD25). This report extends those investigations by the addition of a more lineage-specific marker for Tregs, transcription factor forkhead box P3 (FOXP3), in subjects with type 1 diabetes, their first-degree relatives, and healthy control subjects. With inclusion of this marker, two predominant populations of CD4(+)CD25(+) T-cells were identified: CD4(+)CD25(+)FOXP3(+) as well as CD4(+)FOXP3(-) T-cells expressing low levels of CD25 (CD4(+)CD25(LOW)FOXP3(-)). In all study groups, the frequency of CD4(+)CD25(+)FOXP3(+) cells was age independent, whereas CD4(+)CD25(LOW)FOXP3(-) cell frequencies strongly associated with age. In terms of additional markers for delineating cells of Treg lineage, FOXP3(+) cells were CD127(-) to CD127(LOW) whereas CD25(+) cells were less restricted in their expression of this marker, with CD127 expressed across a continuum of levels. Importantly, no differences were observed in the frequency of CD4(+)CD25(+)FOXP3(+) T-cells in individuals with or at varying degrees of risk for type 1 diabetes. These investigations suggest that altered peripheral blood frequencies of Tregs, as defined by the expression of FOXP3, are not specifically associated with type 1 diabetes and continue to highlight age as an important variable in analysis of immune regulation.

A spontaneous single-base mutation in the leptin receptor of type 1 diabetes-prone NOD/LtJ mice (designated as Lepr(db-5J)) produced a glycine640valine transversion in the extracellular domain. All mutant mice became obese and hyperinsulinemic at weaning, with 70-80% developing early-onset hyperglycemia. However, these obese diabetic mice continued to gain weight without insulin therapy. Spontaneous diabetes remission was observed in all obese females and a subset of obese males. Insulitis was largely limited to islet perimeters, with intraislet insulitis infrequently observed. In 17 obese males (age 39 weeks), we observed phenotypic heterogeneity, including full remission from hyperglycemia (24%), intermediate hyperglycemia with elevated body weight (41%), and severe hyperglycemia and weight loss (35%). The remitting normoglycemic and intermediate hyperglycemic phenotypes were associated with extensive beta-cell hyperplasia. Unlike the extensive intraislet insulitis present in diabetic lean NOD/Lt mice, the severe obese diabetic phenotype was associated with islet atrophy without extensive intraislet insulitis. These results indicated that the manipulation of the leptin/leptin receptor axis may provide a novel means of downregulating autoimmunity in type 1 diabetes and confirmed a role for leptin as a mediator in the development of this disease in NOD mice.

Islet transplantation represents a potential cure for type 1 diabetes, yet persistent autoimmune and allogeneic immunities currently limit its clinical efficacy. For alleviating the autoimmune destruction of transplanted islets, newly diagnosed NOD mice were provided a single intramuscular injection of recombinant adeno-associated viral vector encoding murine IL-10 (rAAV-IL-10) 4 weeks before renal capsule delivery of 650 syngeneic islets. A dose-dependent protection of islet grafts was observed. Sixty percent (3 of 5) of NOD mice that received a transduction of a high-dose (4 x 10(9) infectious units) rAAV-IL-10 remained normoglycemic for at least 117 days, whereas diabetes recurred within 17 days in mice that received a low-dose rAAV-IL-10 (4 x 10(8) infectious units; 5 of 5) as well as in all of the control mice (5 of 5 untreated and 4 of 4 rAAV-green fluorescent protein-transduced). Serum IL-10 levels positively correlated with prolonged graft survival and were negatively associated with the intensity of autoimmunity. The mechanism of rAAV-IL-10 protection involved a reduction of lymphocytic infiltration as well as induction of antioxidant enzymes manganese superoxide dismutase and heme oxygenase 1 in islet grafts. These studies support the utility of immunoregulatory cytokine gene therapy delivered by rAAV for preventing autoimmune disease recurrence in transplant-based therapies for type 1 diabetes.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAV-delivered transgenes did not interfere with islet cell insulin production and were expressed in both beta- and non-beta-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance longterm graft survival.