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Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonistic anti-CD40 antibody 2C10R4, rapamycin, soluble tumor necrosis factor receptor and anti-interleukin 6 receptor antibody induce long-term (≥1 year) tolerance to islet allografts in 5 of 5 nonsensitized, MHC class I-disparate, and one MHC class II DRB allele-matched rhesus macaques. Tolerance in our preclinical model is associated with a regulatory network, involving antigen-specific Tr1 cells exhibiting a distinct transcriptome and indirect specificity for matched MHC class II and mismatched class I peptides. Apoptotic donor leukocyte infusions warrant continued investigation as a cellular, nonchimeric and translatable method for inducing antigen-specific tolerance in transplantation.

Insufficient pancreatic β-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from β-cells in diabetic patients, no pharmacological agents have been described that increase β-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust β-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces β-cell proliferation, increases β-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore β-cell mass, and highlights a tractable pathway for future drug discovery efforts.

Combination therapy with a dipeptidyl peptidase-4 inhibitor (DPP-4i) and a proton pump inhibitor (PPI) raises endogenous levels of GLP-1 and gastrin, respectively, and restores pancreatic β-cell mass and normoglycemia in nonobese diabetic (NOD) mice with autoimmune diabetes. The aim of this study was to determine whether a DPP-4i and PPI combination could increase β-cell mass in the adult human pancreas. Pancreatic cells from adult human pancreas donors were implanted in NOD-severe combined immunodeficient (NOD-scid) mice and the mice were treated with a DPP-4i and a PPI for 16 weeks. Human grafts were examined for insulin content and insulin-stained cells. Graft β-cell function was assessed by intravenous glucose tolerance tests (IVGTT) and by glucose control in human cell-engrafted mice treated with streptozotocin (STZ) to delete mouse pancreatic β-cells. Plasma GLP-1 and gastrin levels were raised to two- to threefold in DPP-4i- and PPI-treated mice. Insulin content and insulin-stained cells in human pancreatic cell grafts were increased 9- to 13-fold in DPP-4i and PPI-treated mice and insulin-stained cells were colocalized with pancreatic exocrine duct cells. Plasma human C-peptide responses to IVGTT were significantly higher and STZ-induced hyperglycemia was more completely prevented in DPP-4i- and PPI-treated mice with grafts than in vehicle-treated mice with grafts. In conclusion, DPP-4i and PPI combination therapy raises endogenous levels of GLP-1 and gastrin and greatly expands the functional β-cell mass in adult human pancreatic cells implanted in immunodeficient mice, largely from pancreatic duct cells. This suggests that a DPP-4i and PPI combination treatment may provide a pharmacologic therapy to correct the β-cell deficit in type 1 diabetes.

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.

In a previous study, we observed that combination therapy of nonobese diabetic (NOD) mice with epidermal growth factor (EGF) and gastrin partially restored pancreatic islet beta-cell mass and reversed hyperglycemia without the use of immunotherapy. Herein we have studied the effects of EGF plus gastrin on recurrent autoimmune responses in diabetic NOD mice transplanted with syngeneic islets. EGF (10 microg/kg) plus gastrin (30 microg/kg) given intraperitoneally (i.p.) once daily to diabetic NOD mice (blood glucose, 23 +/- 2 mmol/L) significantly prolonged the median survival time of NOD islet grafts to 60 days (n = 10 mice) measured as the days until hyperglycemia recurrence (blood glucose > or =12 mmol/L; versus EGF alone to 36 days (n = 10), or gastrin alone, 19 days (n = 10), or vehicle, 11 days (n = 9). At 7-14 days after transplantation insulin-stained beta-cells were much more numerous in islet grafts of EGF plus gastrin-treated mice (13.0 +/- 0.9 x 10(5) cells) versus grafts in vehicle-treated mice (1.0 +/- 0.3 x 10(5) cells). CD45+ leukocytes were significantly reduced in number and surrounded but did not destroy the beta cells in the islets of EGF plus gastrin-treated mice (29 +/- 2 x 10(5) cells) versus those in vehicle-treated mice (57 +/- 3 x 10(5) cells). We concluded that the EGF plus gastrin combination therapy inhibited the recurrent autoimmune response and delayed rejection of syngeneic islet grafts, suggesting a therapeutic role for these peptides in islet transplantation.

Pancreatic islet transplantation as a treatment for type 1 diabetes is limited by human donor tissue availability. We investigated whether the beta-cell mass in human isolated islets could be expanded by treatments with glucagon-like peptide-1 (GLP-1) and gastrin, peptides reported to stimulate beta-cell growth in mice and rats with deficits in beta-cell mass. Human islets with low endocrine cell purity (7% beta-cells, 4% alpha-cells) and abundant exocrine cells (29% duct cells and 25% acinar cells) were implanted under the renal capsule of nonobese diabetic-severe combined immune deficiency (NOD-scid) mice made diabetic with streptozotocin. The mice were treated with GLP-1 and gastrin, separately and together, daily for 5 weeks. Blood glucose was significantly reduced only in mice implanted with human pancreatic cells and treated with GLP-1 plus gastrin. Correction of hyperglycemia was accompanied by increased insulin content in the human pancreatic cell grafts as well as by increased plasma levels of human C-peptide in the mice. Immunocytochemical examination revealed a fourfold increase in insulin-positive cells in the human pancreatic cell grafts in GLP-1 plus gastrin-treated mice, and most of this increase was accounted for by the appearance of cytokeratin 19-positive pancreatic duct cells expressing insulin. We conclude that combination therapy with GLP-1 and gastrin expands the beta-cell mass in human islets implanted in immunodeficient diabetic mice, largely from pancreatic duct cells associated with the islets, and this is sufficient to ameliorate hyperglycemia in the mice.

Islet Neogenesis-Associated Protein (INGAP) is a member of the Reg family of proteins implicated in various settings of endogenous pancreatic regeneration. The expression of INGAP and other RegIII proteins has also been linked temporally and spatially with the induction of islet neogenesis in animal models of disease and regeneration. Furthermore, administration of a peptide fragment of INGAP (INGAP peptide) has been demonstrated to reverse chemically induced diabetes as well as improve glycemic control and survival in an animal model of type 1 diabetes. Cultured human pancreatic tissue has also been shown to be responsive to INGAP peptide, producing islet-like structures with function, architecture and gene expression matching that of freshly isolated islets. Likewise, studies in normoglycemic animals show evidence of islet neogenesis. Finally, recent clinical studies suggest an effect of INGAP peptide to improve insulin production in type 1 diabetes and glycemic control in type 2 diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans by autoreactive T helper 1 (Th1) cells characterized by their cytokine secretory products, interleukin-2 (IL-2) and interferon gamma (IFNgamma). Th1-type cytokines (IL-2 and IFNgamma) correlate with T1D, whereas Th2 (IL-4 and IL-10), Th3 (transforming growth factor beta [TGFbeta]), and T regulatory cell-type cytokines (IL-10 and TGFbeta) correlate with protection from T1D. Paradoxically, however, administrations of Th1-type cytokines (IL-2 and IFNgamma) and immunotherapies that induce Th1-type cytokine responses actually prevent T1D, at least in animal models. Therefore, immunotherapies that inhibit IL-2 production/action will block Th1 cell/cytokine-driven effector mechanisms of pancreatic islet beta-cell destruction; however, anti-IL-2 therapy will not allow immune tolerance to be established. In contrast, immunotherapies that increase IL-2 production/action may correct an immunodeficiency in IL-2 production that appears to underlie the autoimmunity of T1D, thereby restoring immune tolerance to islet beta-cells and prevention of T1D.

Combination therapy with epidermal growth factor (EGF) and gastrin induces beta-cell regeneration in rodents with chemically induced diabetes. We investigated whether EGF plus gastrin could correct hyperglycemia in NOD mice with autoimmune diabetes. Combined treatment with EGF (1 mug/kg) and gastrin (3 mug/kg) for 2 weeks restored normoglycemia after diabetes onset in NOD mice, whereas EGF or gastrin alone did not. Fasting blood glucose remained normal (3.5-6.5 mmol/l) or mildly elevated (<11 mmol/l) in five of six mice (83%) for 10 weeks after EGF plus gastrin treatment was stopped, whereas all mice treated with vehicle or EGF or gastrin alone became severely hyperglycemic (12-35 mmol/l). Pancreatic beta-cell mass was increased threefold and insulin content was increased eightfold in mice treated with EGF plus gastrin compared with pretreatment values. The correction of hyperglycemia correlated significantly with increases in pancreatic beta-cell mass and insulin content. In addition, splenic cells from mice treated with EGF plus gastrin delayed diabetes induction by adoptive transfer of diabetogenic cells into immunodeficient NOD-scid mice, suggesting the induction of immunoregulatory cells in NOD mice treated with EGF plus gastrin. We conclude that a short course of combined EGF and gastrin therapy increases pancreatic beta-cell mass and reverses hyperglycemia in acutely diabetic NOD mice; the impact of this combined therapy may result from the effects of EGF and gastrin on beta-cells, immune cells, or both.

Nuclear and mitochondrial genomes combine in ALR/Lt mice to produce systemically elevated defenses against free radical damage, rendering these mice resistant to immune-mediated pancreatic islet destruction. We analyzed the mechanism whereby isolated islets from ALR mice resisted proinflammatory stress mediated by combined cytokines (IL-1beta, TNF-alpha, and IFN-gamma) in vitro. Such damage entails both superoxide and NO radical generation, as well as peroxynitrite, resulting from their combination. In contrast to islets from other mouse strains, ALR islets expressed constitutively higher glutathione reductase, glutathione peroxidase, and higher ratios of reduced to oxidized glutathione. Following incubation with combined cytokines, islets from control strains produced significantly higher levels of hydrogen peroxide and NO than islets from ALR mice. Nitrotyrosine was generated in NOD and C3H/HeJ islets but not by ALR islets. Western blot analysis showed that combined cytokines up-regulated the NF-kappaB inducible NO synthase in NOD-Rag and C3H/HeJ islets but not in ALR islets. This inability of cytokine-treated ALR islets to up-regulate inducible NO synthase and produce NO correlated both with reduced kinetics of IkappaB degradation and with markedly suppressed NF-kappaB p65 nuclear translocation. Hence, ALR/Lt islets resist cytokine-induced diabetogenic stress through enhanced dissipation and/or suppressed formation of reactive oxygen and nitrogen species, impaired IkappaB degradation, and blunted NF-kappaB activation. Nitrotyrosylation of beta cell proteins may generate neoantigens; therefore, resistance of ALR islets to nitrotyrosine formation may, in part, explain why ALR mice are resistant to type 1 diabetes when reconstituted with a NOD immune system.

Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet beta-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the beta-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 mug/ml), gastrin (1.0 mug/ml), or the combination of EGF and gastrin. beta-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (-3%) or control medium (-62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, beta-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased beta-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases beta-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of beta-cell neogenesis from pancreatic exocrine duct cells.

Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that consumes NAD in response to DNA strand breaks. The PARP inhibitor nicotinamide prevents NAD consumption and protects islet beta-cells from chemically induced necrosis but not cytokine-induced apoptosis. Therefore, it is unclear how nicotinamide protects NOD mice from autoimmune diabetes in which apoptosis is the mode of beta-cell death. To investigate the mechanism of diabetes prevention by PARP inhibition, we studied the effects of a novel, potent PARP inhibitor, PJ34, a phenanthridinone derivative, on diabetes development in NOD mice and on diabetes recurrence in diabetic NOD mice transplanted with syngeneic islets. PJ34 administration from age 5 or 15 weeks significantly decreased insulitis, beta-cell destruction and diabetes incidence, and protection from diabetes continued for 12 weeks after PJ34 therapy was stopped. Similarly, syngeneic islet graft survival was prolonged and outlasted therapy in PJ34-treated mice. Immunohistochemical studies revealed significantly fewer leukocytes in islet grafts of PJ34-treated mice, together with increased apoptosis of these cells and decreased expression of the T helper 1-type cytokine interferon (IFN)-gamma. These results suggest that PARP inhibition protects against autoimmune beta-cell destruction in NOD mice by inducing apoptosis of islet-infiltrating leukocytes and decreasing IFN-gamma expression in the islets.

Background: We have previously reported that matrix metalloproteinase-2 (MMP-2) contributes to myocardial ischemia-reperfusion injury by degradation of troponin I, a regulatory element of the contractile proteins. MMP activities are also tightly regulated by tissue inhibitors of metalloproteinase (TIMPs). The change in TIMPs during acute myocardial ischemia-reperfusion injury is not clear., Methods and Results: Isolated rat hearts were perfused either aerobically for 75 minutes or subjected to 15, 20, or 25 minutes of global, no-flow ischemia followed by 30 minutes of aerobic reperfusion. During reperfusion after ischemia, there was a rapid, enhanced release of TIMP-4, the most abundant TIMP in the heart, into the coronary effluent, as shown both by reverse zymography and Western blot. There was a negative correlation between the recovery of cardiac mechanical function and the release of TIMP-4 during reperfusion in hearts subjected to different durations of ischemia. Immunogold electron microscopy revealed a close association of TIMP-4 with the sarcomeres in aerobically perfused hearts. Moreover, TIMP-4 was present only in thin myofilaments prepared from aerobically perfused hearts but not in ischemic-reperfused hearts. An enhanced MMP activity was shown in ischemic-reperfused hearts by in situ zymography., Conclusions: Loss of TIMP-4 from the cardiac myocyte leads to an increase in net myocardial MMP activity that contributes to acute myocardial stunning injury.

Objective: Pro-inflammatory cytokines depress myocardial contractile function by enhancing peroxynitrite production, yet the mechanism by which peroxynitrite does this is unknown. As matrix metalloproteinases (MMPs) can be activated by peroxynitrite and can proteolytically cleave troponin I in hearts, we determined whether this occurs in cytokine-induced myocardial dysfunction., Methods: Isolated working rat hearts were perfused with buffer containing interleukin-1 beta, interferon-gamma, and tumor necrosis factor-alpha., Results: Cytokines induced a marked decline in mechanical function during 60-120 min of perfusion. This decline was accompanied by increased myocardial inducible NO synthase activity and perfusate dityrosine (a marker of peroxynitrite), compared to control hearts. Before the decline in mechanical function there was enhanced MMP-2 activity in the perfusate. This was accompanied by decreased tissue levels of MMP-2, tissue inhibitor of matrix metalloproteinases-4 and troponin I in cytokine-treated hearts. The collagen content of the heart was not affected by cytokine treatment. A neutralizing anti-MMP-2 antibody or the MMP inhibitors Ro31-9790 or PD166793 attenuated the decline in myocardial function. Moreover, the MMP-2 antibody prevented the decline in myocardial MMP-2 and troponin I levels., Conclusions: Myocardial contractile dysfunction caused by pro-inflammatory cytokines results in MMP-2 activation and a decline in tissue inhibitor of matrix metalloproteinases-4 in the heart. Troponin I is also a target for the proteolytic action of MMP-2 during acute heart failure triggered by pro-inflammatory cytokines. Inhibition of MMPs may be a novel pharmacological strategy for the treatment of acute inflammatory heart disease.

Background: Matrix metalloproteinases are best recognized for their ability to degrade the extracellular matrix in both physiological and pathological conditions. However, recent findings indicate that some of them are also involved in mediating acute processes such as platelet aggregation and vascular tone. The acute contractile defect of the heart after ischemia-reperfusion may involve the proteolytic degradation of the thin filament protein troponin I; however, the protease responsible for this remains obscure., Methods and Results: Here we report that matrix metalloproteinase-2 is colocalized with troponin I within the thin myofilaments of cardiomyocytes in ischemic-reperfused hearts and that troponin I is a novel intracellular target for proteolytic cleavage by matrix metalloproteinase-2. Inhibition of matrix metalloproteinase-2 activity prevented ischemia-reperfusion-induced troponin I degradation and improved the recovery of mechanical function of the heart., Conclusions: These data reveal for the first time a novel molecular mechanism by which matrix metalloproteinase-2 causes acute myocardial dysfunction after ischemia-reperfusion-injury and that matrix metalloproteinase-2 has a biological action within the cell.

Sirolimus is an immunosuppressant that inhibits interleukin (IL)-2 signaling of T-cell proliferation but not IL-2-induced T-cell apoptosis. Therefore, we hypothesized that administration of IL-2, together with sirolimus, might shift T-cell proliferation to apoptosis and prevent autoimmune destruction of islet beta-cells. We found that sirolimus and IL-2 therapy of female NOD mice, beginning at age 10 weeks, was synergistic in preventing diabetes development, and disease prevention continued for 13 weeks after stopping sirolimus and IL-2 therapy. Similarly, sirolimus and IL-2 were synergistic in protecting syngeneic islet grafts from recurrent autoimmune destruction after transplantation in diabetic NOD mice, and diabetes did not recur after stopping sirolimus and IL-2 combination therapy. Immunocytochemical examination of islet grafts revealed significantly decreased numbers of leukocytes together with increased apoptosis of these cells in mice treated with sirolimus and IL-2, whereas beta-cells were more numerous, and significantly fewer were apoptotic. In addition, Th1-type cells (gamma-interferon-positive and IL-2(+)) were decreased the most, and Th2-type cells (IL-4(+) and IL-10(+)) and Th3-type cells (transforming growth factor-beta1(+)) were increased the most in islet grafts of sirolimus and IL-2-treated mice. We conclude that 1) combination therapy with sirolimus and IL-2 is synergistic in protecting islet beta-cells from autoimmune destruction; 2) diabetes prevention continues after withdrawal of therapy; and 3) the mechanism of protection involves a shift from Th1- to Th2- and Th3-type cytokine-producing cells, possibly due to deletion of autoreactive Th1 cells.

Aims/hypothesis: Sirolimus and tacrolimus are immunosuppressive drugs that prevent rejection of pancreatic islet allografts transplanted into patients with Type I (insulin-dependent) diabetes mellitus. This study aimed to determine whether sirolimus and tacrolimus can prevent autoimmune beta-cell destruction, and if so, what the mechanisms of action are., Methods: Sirolimus and tacrolimus were given separately and together to female non-obese diabetic (NOD) mice from age 12 to 35 weeks. Diabetes incidence was determined and pancreatic insulitis and insulin content were measured. Sirolimus and tacrolimus were also given separately and together to diabetic NOD mice from the time of syngeneic islet transplantation until the reappearance of hyperglycaemia. Islet grafts were examined by RT-PCR assay for expression of interferon (IFN)- gamma, interleukin (IL)-2, IL-4, IL-10 and transforming growth factor (TGF)- beta1., Results: Low doses of sirolimus (0.1 mg/kg) and tacrolimus (0.1 mg/kg) were synergistic in reducing insulitis, preserving pancreatic insulin content and preventing diabetes in female NOD mice (8 % diabetes incidence at 35 weeks vs 66 % in vehicle-treated mice). Also, the combination of sirolimus and tacrolimus prolonged syngeneic islet graft survival (median 34 days vs 13 days for vehicle-treated mice). Islet grafts from sirolimus plus tacrolimus-treated mice expressed significantly decreased mRNA contents of Th1-type cytokines (IFN- gamma and IL-2) and the highest ratio of TGF- beta1/IFN- gamma mRNA., Conclusion/interpretation: These findings suggest that combination therapy with sirolimus and tacrolimus prevent autoimmune beta-cell destruction by upregulating expression of the immunoregulatory cytokine, TGF- beta1 and reducing Th1 cytokines (IFN- gamma and IL-2) expressed in the islets. Low-dose sirolimus and tacrolimus combination therapy could warrant consideration for prevention or early treatment of human Type I diabetes.

Nonobese diabetic (NOD) mice develop diabetes and destroy syngeneic islet grafts through an autoimmune response. Because transforming growth factor (TGF)-beta1 downregulates immune responses, we tested whether overexpression of TGF-beta1 by gene transfection of NOD mouse islets could protect beta-cells in islet grafts from autoimmune destruction. NOD mouse islet cells were transfected with an adenoviral DNA expression vector encoding porcine latent TGF-beta1 (Ad TGF-beta1) or the adenoviral vector alone (control Ad vector). The frequency of total islet cells expressing TGF-beta1 protein was increased from 12 +/- 1% in control Ad vector-transfected cells to 89 +/- 4% in Ad TGF-beta1-transfected islet cells, and the frequency of beta-cells that expressed TGF-beta1 was increased from 12 +/- 1% to 60 +/- 7%. Also, secretion of TGF-beta1 was significantly increased in islets that overexpressed TGF-beta1. Ad TGF-beta1-transfected NOD mouse islets that overexpressed TGF-beta1 prevented diabetes recurrence after transplantation into diabetic NOD mice for a median of 22 days compared with only 7 days for control Ad vector-transfected islets (p = 0.001). Immunohistochemical examination of the islet grafts revealed significantly more TGF-beta1+ cells and insulin+ cells and significantly fewer CD45+ leukocytes in Ad TGF-beta1-transfected islet grafts. Also, islet beta-cell apoptosis was significantly decreased whereas apoptosis of graft-infiltrating leukocytes was significantly increased in Ad TGF-beta1-transfected islet grafts. These observations demonstrate that overexpression of TGF-beta1, by gene transfection of NOD mouse islets, protects islet beta-cells from apoptosis and autoimmune destruction and delays diabetes recurrence after islet transplantation.

The proinflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma), are cytotoxic to pancreatic islet beta cells, possibly by inducing nitric oxide and/or oxygen radical production in the beta cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet beta-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1beta, TNFalpha, and IFNgamma. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet beta cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet beta-cell destruction. L-N(G)-monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet beta-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet beta-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.

Cytokines produced by immune system cells that infiltrate pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune (type 1) diabetes mellitus. Because the calcium binding protein, calbindin-D(28k), can prevent apoptotic cell death in different cell types, we investigated the possibility that calbindin-D(28k) may prevent cytokine-mediated islet beta-cell destruction. Using the expression vector BSRalpha, rat calbindin-D(28k) was stably expressed in the pancreatic islet beta-cell line, betaTC-3. Calbindin-D(28k) expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1beta (30 U/ml), TNFalpha (10(3) U/ml), and interferon gamma (10(3) U/ml). The greatest protection was observed in the betaTC-3 cell clone expressing the highest concentration of calbindin-D(28k). Apoptotic cell death was detected by annexin V staining and by the TdT-mediated dUTP-X nick end labeling assay in vector-transfected betaTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D(28k)-overexpressing betaTC-3 cells incubated with cytokines (5-6% apoptotic cells). To address the mechanism of the antiapoptotic effects of calbindin, studies were done to examine whether calbindin inhibits free radical formation. The stimulatory effects of the cytokines on lipid hydroperoxide, nitric oxide, and peroxynitrite production were significantly decreased in the calbindin-D(28k)-expressing betaTC-3 cells. Our findings indicate that calbindin-D(28k), by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of beta-cells. These findings suggest that calbindin-D(28k) may be an important regulator of cell death that can protect pancreatic islet beta-cells from autoimmune destruction in type 1 diabetes.

Activation of poly(ADP-ribose) synthetase (PARS, also termed polyADP-ribose polymerase or PARP) has been proposed as a major mechanism contributing to beta-cell destruction in type I diabetes. In the present study, we have investigated the role of PARS in mediating the induction of diabetes and beta-cell death in the multiple-low-dose-streptozotocin (MLDS) model of type I diabetes. Mice genetically deficient in PARS were found to be less sensitive to MLDS than wild type mice, with a lower incidence of diabetes and reduced hyperglycemia. A potent inhibitor of PARS, 5-iodo-6-amino-1,2-benzopyrone (INH(2)BP), was also found to protect mice from MLDS and prevent beta-cell loss, in a dose-dependent manner. Paradoxically, in the PARS deficient mice, the compound increased the onset of diabetes. In vitro the cytokine combination; interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma inhibited glucose-stimulated insulin secretion from isolated rat islets of Langerhans and decreased RIN-5F cell viability. The PARS inhibitor, INH(2)BP, protected both the rat islets and the beta-cell line, RIN-5F, from these cytokine-mediated effects. These protective effects were not mediated by inhibition of cytokine-induced nitric oxide formation. Inhibition of PARS by INH(2)BP was unable to protect rat islet cells from cytokine-mediated apoptosis. Cytokines, peroxynitrite and streptozotocin were all shown to induce PARS activation in RIN-5F cells, an effect suppressed by INH(2)BP. The present study provides evidence for in vivo PARS activation contributing to beta-cell damage and death in the MLDS model of diabetes, and indicates a role for PARS activation in cytokine-mediated depression of insulin secretion and cell viability in vitro.

Peroxynitrite (ONOO(-)) is a highly reactive oxidant produced by the interaction of the free radicals superoxide (O*-2) and nitric oxide (NO(*)). In a previous study, we found that peroxynitrite is formed in islet beta-cells of nonobese diabetic (NOD) mice. Here, we report that guanidinoethyldisulphide (GED), a selective inhibitor of inducible nitric oxide synthase (iNOS) and scavenger of peroxynitrite prevents diabetes in NOD mice. GED treatment of female NOD mice, starting at age 5 weeks, delayed diabetes onset (from age 12 to 22 weeks) and significantly decreased diabetes incidence at 30 weeks (from 80% to 17%). GED did not prevent pancreatic islet infiltration by leukocytes; however, beta-cells that stained positive for nitrotyrosine (a marker of peroxynitrite) were significantly decreased in islets of GED-treated mice (1+/-1%) compared with vehicle-treated mice (30+/-9%). In addition, GED significantly inhibited nitric oxide and nitrotyrosine formation and decreased destruction of beta-cells in NOD mouse islets incubated in vitro with the combination of proinflammatory cytokines interleukin 1-beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These findings indicate that both superoxide and nitric oxide radicals contribute to islet beta-cell destruction in autoimmune diabetes via peroxynitrite formation in the beta-cells., (Copyright 2001 Academic Press.)

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic beta-cell autoreactive T cells from a Th1 (IFN-gamma) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-gamma, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-gamma to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-gamma. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among beta-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Type 1 (insulin-dependent) diabetes mellitus, like other organ specific autoimmune diseases, results from a disorder of immunoregulation. T cells specific for pancreatic islet beta cell constituents (autoantigens) exist normally but are restrained by regulatory mechanisms (self-tolerant state). When regulation fails, beta cell-specific autoreactive T cells become activated and expand clonally. Current evidence indicates that islet beta cell-specific autoreactive T cells belong to a T helper 1 (Th1) subset, and these Th1 cells and their characteristic cytokine products, IFNgamma and IL-2, are believed to cause islet inflammation (insulitis) and beta cell destruction. Immune-mediated destruction of beta cells precedes hyperglycemia and clinical symptoms by many years because these become apparent only when most of the insulin-secreting beta cells have been destroyed. Therefore, several approaches are being tested or are under consideration for clinical trials to prevent or arrest complete autoimmune destruction of islet beta cells and insulin-dependent diabetes. Approaches that attempt to correct underlying immunoregulatory defects in autoimmune diabetes include interventions aimed at i) deleting beta cell autoreactive Th1 cells and cytokines (IFNgamma and IL-2) and/or ii) increasing regulatory Th2 cells and/or Th3 cells and their cytokine products (IL-4, IL-10 and TGFbeta1).

Aims/hypothesis: A mechanism implicated in pancreatic islet beta-cell destruction in autoimmune diabetes is the binding of the Fas ligand (FasL) on T cells to Fas receptors on beta cells, causing their destruction. Evidence for this mechanism is, however, controversial. The aim of this study was to find whether the Fas ligand contributes to beta-cell death in autoimmune diabetes., Methods: We transplanted syngeneic islets under the renal capsule in non-obese diabetic (NOD) mice and treated the mice with a neutralizing monoclonal antibody to the Fas ligand. Survival of beta cells in islet grafts and phenotypes of graft-infiltrating cells were investigated., Results: We found 58% (7 of 12) of mice treated with anti-Fas ligand antibody were normoglycaemic at 30 days after islet transplantation compared with none (0 of 9) of the mice treated with control antibody. Immunohistochemical analysis of islet grafts showed that infiltration of leucocytes (CD4+ T cells, CD8+ T cells, macrophages and neutrophils) and apoptosis of beta cells in the grafts was significantly decreased in mice treated with anti-Fas ligand antibody. Expression of proinflammatory cytokines (interleukin 1 alpha, tumour necrosis factor alpha and interferon gamma) was not different in islet grafts of mice treated with anti-Fas ligand and control antibodies., Conclusion/interpretation: These findings indicate that Fas ligand-mediated mechanisms play a major part in promoting leucocytic infiltration of islets and beta-cell destruction in autoimmune diabetes.

Aims/hypothesis: Testicular Sertoli cells protect allogeneic islet grafts from rejection after transplantation into animals with chemically induced diabetes. The aims of this study were to determine whether Sertoli cells can protect syngeneic islets from autoimmune destruction after transplantation into non-obese diabetic (NOD) mice and, if so, whether protection is due to Sertoli cell expression of Fas ligand (FasL), believed to be the mechanism that protects against allograft rejection., Methods: We compared the survival of syngeneic islets transplanted under the renal capsule of nonobese diabetic mice, alone and together with purified Sertoli cells prepared from testes of newborn nonobese diabetic mice. Additionally, we examined the composition of the islet and Sertoli cell co-transplants by immunohistochemistry to determine whether islet graft survival correlated with Sertoli cell expression of Fas ligand., Results: Sertoli cell doses of 1, 2 and 4 x 10(6) cells produced a dose-dependent prolongation of median islet graft survival from 11 days (islets alone) to 32 days (islets + 4 x 10(6) Sertoli cells); addition of 8 x 10(6) Sertoli cells to the islet grafts decreased, however, median survival to 8 days. Immunohistochemical analysis of the islet and Sertoli cell co-transplants showed a correlation between Fas ligand expression by Sertoli cells and graft infiltration by neutrophilic leucocytes, leading to islet beta-cell destruction and diabetes recurrence., Conclusion/interpretation: Sertoli cells exert opposing effects on survival of syngeneic islet grafts in nonobese diabetic mice: Fas ligand-dependent neutrophil infiltration and graft destruction, and Fas ligand-independent protection of the graft from autoimmune destruction.

To study the relative roles of CD4(+)and CD8(+)T cells and their cytokine products in autoimmune diabetes development, we selectively depleted CD4(+)and CD8(+)T cells in autoimmune diabetes-prone (DP) biobreeding (BB) rats, by administrations of anti-CD2 and anti-CD8 monoclonal antibody (mAb) respectively. We then analysed cytokine mRNA expression, by PCR assay, in mononuclear leukocytes isolated from islets and spleens of control and mAb-treated DP-BB rats. Depletion of CD4(+)T cells (by anti-CD2 mAb) in blood, spleen and islets prevented diabetes development in DP-BB rats, and depletion of CD8(+)T cells (by anti-CD8 mAb) delayed and significantly decreased diabetes incidence. Depletion of either CD4(+)or CD8(+)T cells completely prevented IFN-gamma mRNA upregulation in islets of DP-BB rats above the low level expressed in islets of diabetes-resistant (DR) BB rats. Also, IL-10 mRNA levels in islets of DP-BB rats were significantly decreased by depletion of either CD4(+)or CD8(+)T cells, whereas the effects of the anti-T cell mAb on mRNA levels of other cytokines in islets (IL-2, IL-4, IL-12p40, and TNF-alpha) were discordant. In contrast, both mAb treatments significantly upregulated IL-4 and TNF-alpha mRNA levels in spleens of DP-BB rats. These results demonstrate that islet infiltration by both CD4(+)and CD8(+)T cells is required for IFN-gamma and IL-10 production in islets and beta-cell destruction. Depletion of either CD4(+)or CD8(+)T cells may prevent beta-cell destruction by decreasing IFN-gamma and IL-10 production in islets and increasing IL-4 and TNF-alpha production systemically., (Copyright 1999 Academic Press.)

Background: Pancreatic islet transplantation is limited because of immune rejection of the transplanted tissue. Long-term survival of allogeneic pancreatic islet grafts in the absence of systemic immunosuppressive agents should be possible by transfecting the islets directly with DNA encoding immunoregulatory molecules. Localized production of these molecules should affect only the immune cells that come into the vicinity of the foreign tissue. We investigated whether local expression of human CTLA4-Ig or soluble human Fas ligand from biolistically transfected mouse islets would have a protective effect on allograft survival., Methods: Isolated CBA (H2k) islets were biolistically transfected using the gene gun. The experimental groups were naked gold particles (n=6), empty vector DNA (n=5), DNA encoding human CTLA4-Ig (n=8), or soluble human Fas ligand (n=5). Secretion of the transfected gene product was confirmed by screening islet culture supernatants for protein production using a sandwich ELISA. The blasted islets were transplanted under the kidney capsule of alloxan-diabetic BALB/c (H2d) recipients., Results: Control grafts survived for 23 days, on average. CTLA4-Ig-transfected islets showed a bimodal distribution: 50% of cases survived > or = 46 days and 50% were similar to the controls. In the soluble human Fas ligand group, 80% of grafts survived > or = 50 days. There was no correlation between graft survival times and pretransplant levels of protein production., Conclusion: Our results indicate that local production of human CTLA4-Ig or soluble human Fas ligand by biolistically transfected islets can promote allograft survival. This approach should be valuable as a potential immunoprotective therapeutic strategy in tissue transplantation.

Insulin-dependent diabetes mellitus (IDDM) is a disease that results from autoimmune destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The autoimmune response against islet beta-cells is believed to result from a disorder of immunoregulation. According to this concept, a T helper 1 (Th1) subset of T cells and their cytokine products, i.e. Type 1 cytokines--interleukin 2 (IL-2), interferon gamma (IFNgamma), and tumor necrosis factor beta (TNFbeta), dominate over an immunoregulatory (suppressor) Th2 subset of T cells and their cytokine products, i.e. Type 2 cytokines--IL-4 and IL-10. This allows Type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islet (insulitis), culminating in beta-cell destruction. Type 1 cytokines activate (1) cytotoxic T cells that interact specifically with beta-cells and destroy them, and (2) macrophages to produce proinflammatory cytokines (IL-1 and TNFalpha), and oxygen and nitrogen free radicals that are highly toxic to islet beta-cells. Furthermore, the cytokines IL-1, TNFalpha, and IFNgamma are cytotoxic to beta-cells, in large part by inducing the formation of oxygen free radicals, nitric oxide, and peroxynitrite in the beta-cells themselves. Therefore, it would appear that prevention of islet beta-cell destruction and IDDM should be aimed at stimulating the production and/or action of Type 2 cytokines, inhibiting the production and/or action of Type 1 cytokines, and inhibiting the production and/or action of oxygen and nitrogen free radicals in the pancreatic islets.

Background: The recurrent autoimmune response to syngeneic pancreatic islet grafts transplanted into nonobese diabetic (NOD) mice is cell-mediated and relatively resistant to cyclosporine (CsA) therapy. Therefore, we asked whether interleukin (IL)-4 and IL-10, cytokines that inhibit cell-mediated immunity, might improve the therapeutic effect of CsA., Methods: We compared the survival of syngeneic islet grafts transplanted into diabetic NOD mice treated with IL-4, IL-10, and CsA, administered as single agents and in combinations. Additionally, we measured mRNA levels of type 1 cytokines (interferon-gamma [IFN-gamma], IL-2, and IL-12), type 2 cytokines (IL-4 and IL-10), and transforming growth factor-beta (TGF-beta) to determine whether graft rejection or survival might correlate with expression of these cytokines in the grafts., Results: CsA (20 mg/kg/day) significantly prolonged islet graft survival (median: 20 days vs. 10 days for vehicle-treated mice). Neither IL-4 (2.5 microg, twice daily), nor IL-10 (10 microg, twice daily) significantly prolonged islet graft survival. By contrast, combination therapy with CsA and IL-10 significantly prolonged islet graft survival (median: 34 days) compared with vehicle-treated mice (median: 10 days), and combination therapy with CsA and IL-4 significantly prolonged islet graft survival (median: 59 days) compared with both vehicle-treated mice (median: 10 days) and mice treated with CsA alone (median: 20 days). Islet grafts from normoglycemic mice treated with CsA plus IL-10, and with CsA plus IL-4, were surrounded but not infiltrated by mononuclear leukocytes and beta cells were intact, whereas islet grafts from mice treated with vehicle, IL-4, IL-10, and CsA (as single agents) were infiltrated by mononuclear leukocytes and fewer beta cells were detected. Polymerase chain reaction analysis of cytokine mRNA expression in islet grafts at 8-12 days after transplantation revealed that CsA decreased mRNA levels of type 1 cytokines (IFN-gamma and IL-12p40), whereas CsA plus IL-10 did not, and CsA plus IL-4 increased mRNA levels of IFN-gamma, IL-12p40, and TGF-beta., Conclusions: These results demonstrate that IL-4, and to a lesser extent IL-10, improves the ability of CsA to prevent autoimmune destruction of beta cells in syngeneic islets transplanted into diabetic NOD mice; however, there is no simple correlation between the protective effects of the different treatment regimens (CsA, CsA plus IL-4, and CsA plus IL-10) and mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and IL-12), type 2 cytokines (IL-4 and IL-10), or TGF-beta in the islet grafts.

Administration of TNF-alpha to autoimmune diabetes-prone nonobese diabetic mice and biobreeding rats inhibits diabetes development; however, the mechanism(s) of diabetes prevention by TNF-alpha has not been established. We used the model of syngeneic islet transplantation into diabetic nonobese diabetic mice to study the effects of TNF-alpha administration on the types of mononuclear cells and cytokines expressed in the islet grafts and on autoimmune diabetes recurrence. Twice daily i.p. injections of TNF-alpha (20 microg/day) from day 1 to day 30 after islet transplantation significantly prolonged islet graft survival; thus, 70% (16 of 23) of mice treated with TNF-alpha were normoglycemic at 30 days after islet transplantation compared with none (0 of 14) of vehicle-treated control mice. Islet grafts and spleens from TNF-alpha-treated mice at 10 days after islet transplantation contained significantly fewer CD4+ and CD8+ T cells, and significantly decreased mRNA levels of type 1 cytokines (IFN-gamma, IL-2, and TNF-beta) than islet grafts and spleens from control mice. Regarding type 2 cytokines, IL-4 mRNA levels were not changed significantly in islet grafts or spleens of TNF-alpha-treated mice, whereas IL-10 mRNA levels were decreased significantly in islet grafts of TNF-alpha-treated mice and not significantly changed in spleens. TGF-beta mRNA levels in islet grafts and spleens were similar in TNF-alpha-treated and control mice. These results suggest that TNF-alpha partially protects beta cells in syngeneic islet grafts from recurrent autoimmune destruction by reducing CD4+ and CD8+ T cells and down-regulating type 1 cytokines, both systemically and locally in the islet graft.

Background: We performed ex vivo adenoviral gene transfer in a mouse pancreatic islet transplant model to test the efficacy of this expression system. We then determined whether adenoviral-mediated expression of mouse interleukin (IL) 4 or IL-10 from transduced syngeneic islet grafts could prevent disease recurrence in diabetic nonobese diabetic (NOD) mice., Methods: An adenoviral vector expressing beta-galactosidase (AdCMV betaGal) was used to transduce BALB/c islets (2.5 x 10(3) plaque-forming units/islet), which were analyzed for glucose responsiveness, islet cell recovery, and efficiency of gene transfer. In vivo function and reporter gene expression were examined with AdCMV betaGal-transduced islet grafts in alloxan-induced diabetic syngeneic recipients. Adenoviruses expressing either IL-4 or IL-10 were used in a similar fashion to infect NOD islets, which were characterized in vitro, as well as transplanted into diabetic syngeneic recipients., Results: In vitro functional studies showed no significant difference between control or transduced islets, with 50+/-4% of AdCMV betaGal-infected islet cells staining positive for beta-galactosidase. Transplant recipients became nomoglycemic within 48 hr after transplant, and, although beta-galactosidase expression decreased over time, it was detectable in the graft for up to 8 weeks. Despite the nanogram quantities of IL-4 or IL-10 produced/day from each graft equivalent in vitro, transduced and transplanted NOD islets failed to prevent disease recurrence., Conclusions: These results suggest that adenoviruses are efficient for at least medium term gene expression from islets in vivo, but neither IL-4 nor IL-10 alone can prevent autoimmune disease recurrence in NOD mice.

Peroxynitrite (ONOO-) is a highly reactive oxidant species produced by the reaction of the free radicals superoxide (O(2).-) and nitric oxide (NO.). Here we report a marked increase in nitrotyrosine (NT), a marker of peroxynitrite, in islet cells from NOD mice developing spontaneous autoimmune diabetes. By using specific antibodies and immunohistochemical methods, we found that NT-positive cells were significantly more frequent in islets from acutely diabetic NOD mice (22 +/- 6%) than in islets from normoglycemic NOD mice (7 +/- 1%) and control BALB/c mice (2 +/- 1%). The NT+ cells in islets were identified to be macrophages and also beta-cells. Most of the beta-cells in islets from acutely diabetic NOD mice were NT+ (73 +/- 8%), whereas significantly fewer beta-cells were NT+ in islets from normoglycemic NOD mice (18 +/- 4%) and BALB/c mice (5 +/- 1%). Also, the percentage of beta-cells in islets from NOD mice (normoglycemic and diabetic) correlated inversely with the frequency of NT+ beta-cells. This study demonstrates for the first time that peroxynitrite, a reaction product of superoxide and nitric oxide, is formed in pancreatic islet beta-cells of NOD mice developing autoimmune diabetes. This suggests that both oxygen and nitrogen free radicals contribute to beta-cell destruction in IDDM via peroxynitrite formation in the islet beta-cells.

Cytokines produced by mononuclear leukocytes infiltrating pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune insulin-dependent diabetes mellitus. Cytokines may damage islet beta-cells by inducing oxygen free radical production in the beta-cells. Lipid peroxidation and aldehyde production are measures of oxygen free radical-mediated cell injury. In the current study, we used a HPLC technique to measure levels of different aldehydes produced in rat islets incubated with cytokines. The cytokine combination of interleukin-1beta (10 U/ml), tumor necrosis factor-alpha (10(3) U/ml), and interferon-gamma (10(3) U/ml), and the oxidant, t-butylhydroperoxide, induced significant increases in islet levels of the same aldehydes: butanal, pentanal, 4-hydroxynonenal (4-HNE), and hexanal. Cytokine-induced aldehyde production was associated with islet beta-cell destruction. Thus, cytokine-induced increases in malondialdehyde (MDA; at 4 h) and 4-HNE (at 8 h) preceded islet cell destruction (at 16 h), and the addition of 4-HNE, hexanal, MDA, and pentanal (1-200 microM) to th islets, but not other aldehydes at similar concentrations, produced dose-dependent destruction of islet beta-cells. Furthermore, an antioxidant (lazaroid U78518E) prevented cytokine-induced increases in 4-HNE, hexanal, and MDA and significantly inhibited cytokine-induced decreases in insulin and DNA in the islets. In contrast, N(G)-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitrite production, but did not prevent cytokine-induced increases in 4-HNE, hexanal, and MDA or decreases in insulin and DNA in the islets. These results suggest that cytokines may damage islet beta-cells by inducing oxygen free radicals, lipid peroxidation, and, consequently, the formation of cytotoxic aldehydes in the islet cells.

Interferon-gamma (IFN-gamma) is implicated as a mediator of islet beta-cell destruction in autoimmune, insulin-dependent diabetes mellitus (IDDM). Because interleukin 12 (IL-12) is a potent inducer of IFN-gamma production, we sought evidence implicating IL-12 in IDDM development. In the present study, we used a reverse transcriptase polymerase chain reaction (RT-PCR) assay to measure IL-12 mRNA expression levels in islets from nonobese diabetic (NOD) mice. Expression of mRNA encoding the p40 chain of IL-12 (IL-12 p40) in mono-nuclear leukocytes isolated from islets of female NOD mice increased progressively from age 5 weeks to diabetes onset (> 13 weeks). By contrast, IL-12 p40 mRNA levels were significantly decreased in islet mononuclear leukocytes, but not spleens, from female NOD mice protected from diabetes by administration of complete Freund's adjuvant (CFA) in early life. In addition, mRNA levels of IL-12 p40, IFN-gamma and IL-2 were significantly decreased in syngeneic islet grafts, but not spleens, from female NOD mice protected from diabetes recurrence by CFA administration at the time of islet transplantation. These findings show that IL-12 gene expression in the insulitis lesion correlates with both primary and recurrent diabetes development in NOD mice, possibly via induction of T helper (Th) 1-type cytokines, IL-2 and IFN-gamma.

Cytokines produced by immune system cells infiltrating pancreatic islets are candidate mediators of islet beta-cell destruction in autoimmune insulin-dependent diabetes mellitus. Cytokine-induced islet beta-cell destruction may be mediated by reactive oxygen intermediates. To determine the possible roles of oxygen free radicals and nitric oxide (NO) as mediators of islet beta-cell destruction, we studied the relationships among cytokine-induced beta-cell destruction, production of malondialdehyde (MDA; an end product of lipid peroxidation), and production of nitrite (the stable end product of NO). The cytokine combination of interleukin-1 beta (50 U/mL), tumor necrosis factor-alpha (10(3) U/mL), and interferon-gamma (10(3) U/mL) induced significant increases in MDA and nitrite and significant decreases in insulin and DNA in islets after 60-h incubation. A novel antioxidant (lazaroid U78518E) significantly inhibited both a strong oxidant. t-butylhydroperoxide, and the combination of cytokines from inducing MDA production, but not from increasing nitrite production in the islets. Also, the lazaroid antioxidant significantly reversed the cytokine-induced decreases in insulin and DNA contents of the islet cultures. In contrast, L-NG-monomethyl arginine, an inhibitor of NO synthase, prevented cytokine-induced nitrite production, but did not prevent cytokine-induced increases in MDA and decreases in insulin and DNA in the islet cultures. In addition, the addition of MDA to the islets produced a dose-dependent decrease in their insulin and DNA contents, and this was only partially prevented by the lazaroid antioxidant. These results suggest that cytokines may be toxic to human islet beta-cells by inducing oxygen free radicals, lipid peroxidation, and aldehyde production in the islets, and that MDA is one of the cytotoxic mediators of cytokine-induced beta-cell destruction.

Inflammatory cytokines and nitric oxide (NO) are candidate mediators of pancreatic islet beta-cell destruction in insulin-dependent diabetes mellitus. In this study, we used a semiquantitative PCR assay to measure levels of messenger RNA (mRNA) expression of the inflammatory cytokines, interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha, and interferon-gamma (IFN gamma), and of the inducible form of NO synthase (iNOS) in mononuclear leukocytes isolated from pancreatic islets of autoimmune diabetes-prone nonobese diabetic (NOD) female mice. We found that mRNA levels of iNOS, IL-1 alpha, and IFN gamma in islet mononuclear leukocytes increased from 5 weeks of age to onset of diabetes ( > 13 weeks of age). To determine whether increased iNOS, IL-1 alpha, and IFN gamma mRNA expressions were related to diabetes development, we compared mRNA levels of these molecules in mononuclear leukocytes from islets of 12 week-old diabetes-prone NOD female mice and three groups of 12-week-old mice with low diabetes risk: NOD female mice injected with complete Freund's adjuvant at 4 weeks of age, NOD male mice, and BALB/c female mice that do not develop diabetes. We found that iNOS, IL-1 alpha, and IFN gamma mRNA levels were higher in mononuclear leukocytes from islets of diabetes-prone NOD female mice than in those from mice correlated with IL-1 alpha and IFN gamma mRNA levels. By using specific antibodies and immunohistochemical methods, we localized iNOS in macrophages as well as in beta-cells of islets from diabetes-prone NOD female mice. These findings suggest that IL-1 alpha and IFN gamma may promote islet beta-cell destruction at least in part by up-regulating iNOS expression an No production by both macrophages and beta-cells in the islets of autoimmune diabetes-prone NOD mice.

Syngeneic pancreatic islet grafts in nonobese diabetic (NOD) mice elicit a cell-mediated autoimmune response that destroys the insulin-producing beta cells in the islet graft. IL-4 and IL-10 are cytokines that inhibit cell-mediated immunity. In this study, we evaluated the effects of IL-4 and IL-10 on the survival of syngeneic pancreatic islets transplanted into diabetic NOD mice. Islet grafts survived beyond 18 days and normoglycemia was maintained in 67% (10 of 15) of mice treated with IL-4 plus IL-10, but in none (0 of 20) of vehicle-injected (control) mice. Also, 40% (6 of 15) of the mice treated with IL-4 plus IL-10 were normoglycemic at 30 days after transplantation, compared with 14% (1 of 7) of the mice treated with IL-4 alone, 8% (1 of 13) of the mice treated with IL-10 alone, and none (0 of 20) of the control mice. Histological examination of grafts at 10 days after transplantation revealed peri-islet accumulations of mononuclear leukocytes and intact islet beta cells in grafts from IL-4 plus IL-10-treated mice, whereas islets were infiltrated by leukocytes and the beta cell mass was greatly reduced in grafts from control mice. Polymerase chain reaction (PCR) analysis of cytokine mRNA expression in the grafts revealed higher levels of IL-2, IFN gamma, and IL-10 mRNA in grafts of diabetic compared with normoglycemic control mice, whereas IFN gamma and TNF alpha mRNA levels were significantly decreased in grafts of IL-4 plus IL-10-treated mice compared with either normoglycemic or diabetic control mice. These results suggest that T helper (Th)1 cells and their cytokine products (IL-2, IFN gamma, and TNF alpha) may promote islet beta cell destructive insulitis and autoimmune diabetes recurrence in syngeneic islet-transplanted NOD mice, and that administration of IL-4 plus IL-10 may inhibit diabetes recurrence by suppressing Th1 cytokine production in the islet grafts.

In previous studies we reported that a single injection of complete Freund's adjuvant (CFA) in early life inhibits the development of autoimmune diabetes in BB diabetes-prone (DP) rats, and that CFA upregulates tumor necrosis factor-alpha (TNF alpha) production by macrophages of DP rats. In this study, we asked if CFA-induced prevention of diabetes in DP rats might be mediated by TNF alpha. Chronic intraperitoneal injection of TNF alpha, 20 micrograms daily from age 50 to 110 days, significantly decreased diabetes incidence in DP rats from 83% (15 of 18) to 37% (seven of 19) at age 110 days. Similarly, a single intraperitoneal injection of CFA, 100 microliters at age 26 days, significantly decreased diabetes incidence from 80% (16 of 20) to 50% (10 of 20) at age 110 days. Prevention by either TNF alpha or CFA was associated with decreased insulitis and preservation of islet beta-cells. By contrast, treatment of DP rats with CFA, plus antiserum to TNF alpha three times a week from age 26 to 110 days, obviated CFA-induced protection against beta-cell destructive insulitis and diabetes. Thus, diabetes incidence was 75% (18 of 24) in DP rats treated with CFA plus anti-TNF alpha antiserum, similar to that in untreated DP rats (80%), but only 44% (eight of 18) in DP rats treated with CFA plus control serum, similar to that in DP rats with CFA alone (50%). These results suggest that TNF alpha may be a mediator of the protective effects of CFA against autoimmune diabetes development in BB rats.

Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet beta-cells following islet infiltration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in beta-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1 beta, IL-2, IL-4, IL-10, and IFN-gamma in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (> 13 wk). However, only IFN-gamma mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-gamma and IL-4 were not different in the four groups of mice. These results suggest that islet beta-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-gamma production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-gamma production in the islets.

The inflammatory cytokines, interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma are cytotoxic to human islet beta-cells in vitro. To determine the possible role of nitric oxide (NO) as a mediator of cytokine-induced islet beta-cell destruction, we studied the relationships between NO production and destruction of human pancreatic islet cells incubated with cytokines in vitro. The cytokine combination of interleukin-1 beta (50 U/mL), tumor necrosis factor-alpha (10(3) U/mL), and interferon-gamma (10(3) U/mL) induced a significant increase in NO production and significant decreases in DNA and insulin contents of the islet cell cultures after a 48-h incubation. L-NG-Monomethyl arginine, an inhibitor of NO synthase, completely prevented cytokine-induced NO production during incubations of 18, 36, 60, and 84 h. Cytokine-induced decreases in DNA and insulin contents of the islet cell cultures, however, were unaffected by the NO synthase inhibitor. Conversely, nicotinamide prevented cytokine-induced islet beta-cell destruction without inhibiting NO production. We conclude that cytokine-induced NO production in human islet cells may be neither necessary nor sufficient to destroy the islet beta-cells and that cytotoxic mechanisms, independent of NO, exist and can be inhibited by nicotinamide.

The cytokines, interleukin 1, tumour necrosis factor, and interferon gamma are cytotoxic to islet beta cells, however, their mechanisms of beta-cell killing are not fully characterized. Since DNA damage is a mechanism of cytokine-induced cell death in some cell types, we sought evidence for cytotoxic effects of cytokines at a nuclear level in islet beta cells by measuring DNA fragmentation in rat islets and islet beta-cell lines. The individual cytokines, interleukin 1 (10 U/ml), tumour recrosis factor (10(3) U/ml) and interferon gamma (10(3) U/ml) inhibited insulin release from rat islets, but did not cause DNA fragmentation or destroy islet cells; by contrast, combination of the three cytokines induced DNA fragmentation and islet-cell death. Cytokine-induced DNA fragmentation preceded cell lysis in islet beta-cell lines (RINm5F, rat insulinoma cells; and NIT-1, NOD/Lt mouse transgenic beta cells), whereas in non-islet cell lines (GH-3, rat pituitary; and PC-12, rat adrenal) the cytokines induced cell lysis and no or late DNA fragmentation. Nicotinamide prevented both DNA fragmentation and destruction of RINm5F islet cells by the cytokines. These findings identify DNA as an early target of cytokine action in islet beta cells, and implicate DNA fragmentation as a mechanism of cytokine-induced beta-cell destruction.

The injection of complete Freund's adjuvant into diabetic nonobese diabetic (NOD) mice at the time of syngeneic islet transplantation prevents monocytic/lymphocytic cell infiltration into the islet graft, Beta-cell destruction, and autoimmune diabetes recurrence. We have used semiquantitative reverse transcriptase-polymerase chain reaction analysis to examine and compare cytokine mRNA expression profiles in islet grafts from complete Freund's adjuvant-injected and control NOD mice. Interleukin 10 mRNA expression was significantly increased whereas interleukin 2 and interferon gamma mRNA levels were significantly decreased in islet grafts from complete Freund's adjuvant-injected mice compared to control mice. Levels of mRNA for interleukin 1 beta, interleukin 4, and tumour necrosis factor alpha were not significantly different in islet grafts from complete Freund's adjuvant-injected and control mice. These findings suggest that a Th1 subset of lymphocytes and their cytokine products, interleukin 2 and interferon gamma, may be involved in the rejection of syngeneic islet grafts and diabetes recurrence in NOD mice, and that the protective effect of complete Freund's adjuvant may result from the induction of interleukin 10 production and consequent down-regulation of Th1 cells and cytokines in the islet graft.

Cytokines produced by immune system cells infiltrating pancreatic islets are candidate mediators of islet beta-cell destruction in insulin-dependent diabetes mellitus. In this study, we examined the role of nitric oxide (NO) as a mediator of cytokine-induced islet beta-cell destruction in a rat insulinoma cell line (RINm5F). The cytokine combination of interleukin-1 beta (IL-1 beta; 10 U/ml), tumor necrosis factor-alpha (10(3) U/ml), and interferon-gamma (10(3) U/ml) induced DNA fragmentation (first detected at 6 h), mitochondrial damage (by 12 h), and death (by 24 h) of RIN cells, whereas the individual cytokines did not have these destructive effects. Also, the cytokine combination of IL-1 beta, tumor necrosis factor-alpha, and interferon-gamma induced a 10-fold increase in NO production by RIN cells, and L-NG-monomethyl arginine, an inhibitor of NO synthase, produced a dose-dependent inhibition of cytokine-induced NO production, DNA fragmentation, and cell destruction. However, IL-1 beta, acting alone, induced a 7-fold increase in NO production without causing DNA fragmentation, mitochondrial damage, or cell destruction. In addition, nicotinamide, a known inhibitor of ADP ribosylation and scavenger of oxygen free radicals, inhibited cytokine-induced DNA fragmentation and cell destruction without affecting NO production. We conclude that stimulation of NO production may be a necessary, but not sufficient, condition for cytokine-induced destruction of islet beta-cells.