Cyclophosphamide-accelerated Diabetes Research Articles

An Immunohistochemical Study on the Distribution and Frequency of T Regulatory Cells in Pancreatic Islets of NOD Mice During Various Stages of Spontaneous and Cyclophosphamide-Accelerated Diabetes

To determine if there is an abrupt change in the frequency of intraislet T regulatory (Treg) cells in female nonobese diabetic (NOD) mice preceding and following spontaneous diabetes and during cyclophosphamide-accelerated disease. The frequency of intraislet CD4-positive Treg cells was analyzed between days 21 and 250, at onset, and at 1, 2, and 3 weeks of diabetes by dual-label immunohistochemistry. Tissues were also analyzed between days 0 and 14 after injection of cyclophosphamide or diluent. In the spontaneous group, intraislet Treg cells were first observed on day 30 in CD4 T cells and increased from day 45. There was no statistical difference in the frequency of Treg cells in nondiabetic NOD mice on days 45, 60, and 90. A sustained frequency at and after 1, 2, and 3 weeks of diabetes was also observed. In the cyclophosphamide group, there was a sharp decline in the frequency of Treg cells on day 4, which remained lower on day 7 but increased by days 11 and 14. During spontaneous diabetes and after onset, the frequency of intraislet CD4-positive Treg cells remains unchanged. They may possess diminished immunoregulatory function and thus unable to counteract the increasing tempo of immune-mediated beta-cell destruction.

Dried leaf extract ofOlea europaeaameliorates islet-directed autoimmunity in mice

The health-promoting effects of various constituents of the olive tree (Olea europaea) are mainly associated with hypoglycaemic and insulin-sensitising activities and have been widely demonstrated in the metabolic syndrome and type 2 diabetes. However, their biological activity in autoimmune type 1 diabetes (T1D) is poorly characterised. Therefore, the influence of O. europaea-derived components present in dry olive leaf extract (DOLE) was examined in two established preclinical models of human T1D, which differ in some aspects of diabetogenesis: multiple low-dose streptozotocin-induced diabetes in susceptible C57BL/6 and CBA/H mouse strains; cyclophosphamide-accelerated diabetes in non-obese diabetic mice. In both T1D models, in vivo administration of DOLE significantly reduced clinical signs of diabetes (hyperglycaemia and body weight loss) and led to complete suppression of histopathological changes in pancreatic islets. In line with these, insulin expression and release were restored in DOLE-treated mice. Interestingly, inducible NO synthase expression and NO production were significantly elevated in peripheral tissues but were down-regulated within the local environment of the endocrine pancreas. This interference was reflected in NO-mediated suppression of T lymphocyte proliferation and lower production of the proinflammatory cytokines interferon-gamma, IL-17 and TNF-alpha in the spleen, with subsequent blockade of beta-cell destruction. The results suggest that DOLE interferes with development of autoimmune diabetes by down-regulating production of proinflammatory and cytotoxic mediators. Therefore, the potential use of a DOLE-enriched diet for prophylaxis/treatment of human T1D, and possibly other autoimmune diseases, is worthy of further investigation.

Ectopic PDX-1 expression in liver ameliorates type 1 diabetes

Type 1 diabetes mellitus (T1DM) results from a specific autoimmune mediated destruction of the pancreatic β-cells. PDX-1 induced developmentally redirected liver cells were suggested to restore the ablated pancreatic function in chemically induced diabetes. However, developmentally redirected liver cells, may have acquired along with the desired β-cell characteristics and functions, also undesired sensitivity to autoimmune attack and therefore may be inefficient in ameliorating T1DM. This study analyzes whether subjects with β-cell autoimmunity could benefit from Ad-CMV-PDX-1 gene therapy. Using the model of cyclophosphamide-accelerated diabetes in non-obese diabetic (CAD-NOD) mice, we report that recombinant adenovirus mediated PDX-1 gene therapy, ameliorates hyperglycemia in CAD-NOD mice. Our data demonstrate that 43% of the overtly diabetic CAD-NOD mice treated with Ad-CMV-PDX-1 became normoglycemic and maintained a stable body weight. Ectopic PDX-1 expression induced pancreatic gene expression and insulin production in the mice livers. The amelioration of hyperglycemia, in PDX-1 treated diabetic mice was associated with an immune modulation manifested by Th1 to Th2 shift in the autoimmune T-cell response to antigens associated with NOD diabetes. Thus, liver-to-pancreas transdifferentiation ameliorates T1DM in a process which is associated with a concomitant modulation of the autoimmune attack. Our findings suggest a beneficial therapeutic effect of the PDX-1 gene therapy for treating autoimmune type 1 diabetes mellitus (T1DM).

Histopathological Changes in Insulin, Glucagon and Somatostatin Cells in the Islets of NOD Mice During Cyclophosphamide-accelerated Diabetes: A Combined Immunohistochemical and Histochemical Study

The cyclophosphamide model of accelerated diabetes in the NOD mouse is a useful model of insulin-dependent diabetes mellitus (IDDM). Knowledge on the progressive destruction of beta cells and the fate of other islet endocrine cell-types in this model is sparse. We employed immunohistochemistry and histochemistry, to study temporal changes in islet cell populations, insulitis and glucose transporter-2 expression during cyclophosphamide administration. Cyclophosphamide was administered to day 95 female NOD mice and the pancreas studied at days 0 ( = day 95), 4, 7, 11 and 14 after treatment and in age-matched control mice. At day 0, a majority of the endocrine cells were insulin-positive. Glucagon and somatostatin cells were mostly in the islet periphery and also internally. In the cyclophosphamide group, insulitis was moderate at day 0, declined at day 4 but increased progressively from day 7. The extent of insulitis in treated mice which were diabetes-free at day 14 was comparable to age-matched control mice. From day 11, the marked increase in insulitis correlated with a reciprocal decline in the extent of insulin immunostained islet area. At day 14, the mean insulin area per islet was markedly less in diabetic mice than in age-matched non-diabetic treated and controls. At diabetes, some islets showed co-expression of glucagon and insulin. Our studies suggest that the mean number of glucagon or somatostatin cells per islet does not vary during the study. Glucose transporter-2 immunolabelling was restricted to beta cells but declined in those adjacent to immune cells. We conclude that in the cyclophosphamide model, there is specific and augmented destruction of beta cells immediately prior to diabetes onset. We speculate that the selective loss of glucose transporter-2 shown in this study suggests the existence of a deleterious gradient close to the immune cell and beta cell surface boundary.

Immunohistochemical Demonstration of Nitrotyrosine, a Biomarker of Oxidative Stress, in Islet Cells of the NOD Mouse

Reactive oxygen species and nitric oxide generated within the beta cell or by intraislet immune cells may be major mediators of beta cell death during insulin-dependent diabetes mellitus. Here, the intraislet expression of nitrotyrosine, a biomarker of oxidative damage, and its cellular sources were examined in the islets of NOD mice at various stages of spontaneous and cyclophosphamide-accelerated diabetes. At day 30, nitrotyrosine-positive cells were undetectable; they were rare at day 40, being expressed in only a few beta cells and in macrophages located in the periphery of some islets. At day 90 and at onset of diabetes, an increasing number of macrophages and beta cells were nitrotyrosine positive. In the cyclophosphamide group at day 4, the number of beta cells and macrophages with positive immunolabeling declined slightly from day 0 (day of cyclophosphamide injection = day 90). This pattern of immunolabeling increased gradually by day 7 but increased markedly by days 11 and 14 after cyclophosphamide administration. In the spontaneous and cyclophosphamide groups, macrophages were the predominant source of nitrotyrosine and were present within the intraislet and periislet regions. Nitrotyrosine labeling was observed in a significant number of beta cells but less than in immunolabeled macrophages. We conclude that protein nitration in beta cells and macrophages is an important feature of the diabetogenic process in the NOD mouse. Whether this deleterious process also occurs during the very early stages of the disease and preceding insulitis is unclear.

Functional immunomics: microarray analysis of IgG autoantibody repertoires predicts the future response of mice to induced diabetes.

One's present repertoire of antibodies encodes the history of one's past immunological experience. Can the present autoantibody repertoire be consulted to predict resistance or susceptibility to the future development of an autoimmune disease? Here, we developed an antigen microarray chip and used bioinformatic analysis to study a model of type 1 diabetes developing in nonobese diabetic male mice in which the disease was accelerated and synchronized by exposing the mice to cyclophosphamide at 4 weeks of age. We obtained sera from 19 individual mice, treated the mice to induce cyclophosphamide-accelerated diabetes (CAD), and found, as expected, that 9 mice became severely diabetic, whereas 10 mice permanently resisted diabetes. We again obtained serum from each mouse after CAD induction. We then analyzed, by using rank-order and superparamagnetic clustering, the patterns of antibodies in individual mice to 266 different antigens spotted on the chip. A selected panel of 27 different antigens (10% of the array) revealed a pattern of IgG antibody reactivity in the pre-CAD sera that discriminated between the mice resistant or susceptible to CAD with 100% sensitivity and 82% specificity (P = 0.017). Surprisingly, the set of IgG antibodies that was informative before CAD induction did not separate the resistant and susceptible groups after the onset of CAD; new antigens became critical for post-CAD repertoire discrimination. Thus, at least for a model disease, present antibody repertoires can predict future disease, predictive and diagnostic repertoires can differ, and decisive information about immune system behavior can be mined by bioinformatic technology. Repertoires matter.

Immunolocalization of caspase-3 in pancreatic islets of NOD mice during cyclophosphamide-accelerated diabetes.

Apoptosis may be a major mechanism of beta cell loss during insulin-dependent diabetes mellitus. Caspase-3 is a key enzyme involved in the terminal steps of this death process. Here, the intra-islet expression of caspase-3 in the NOD mouse was examined immunohistochemically following acceleration of the disease with cyclophosphamide. Female NOD mice were treated at day 95 with cyclophosphamide, and caspase-3 expression in pancreatic sections was studied at days 0, 4, 7, 11, and 14 and compared with age-matched control tissue. In the treated group at day 0, caspase-3 labeling was seen in several peri-islet macrophages and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme. From day 7, caspase-3 expression began to increase in intra-islet macrophages and reached a peak at days 11 and 14, when a small number of CD4 and CD8 T cells also showed positive labeling. Beta cell expression of caspase-3 at days 11 and 14 was rare. At this stage, several intra-islet immune cells with positive labeling for the enzyme coexpressed either Fas or interleukin-1beta. Only a small proportion of intra-islet caspase-3 cells showed apoptotic nuclei judged by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We conclude that, during cyclophosphamide-accelerated diabetes, the predominant caspase-3 immunolabeling in intra- and extra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for their elimination. The virtual absence of caspase-3 immunolabeling in most beta cells even during the height of beta cell loss supports the need for developing other markers of early beta cell apoptosis in the NOD mouse.

Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524-543 reduces cyclophosphamide-accelerated diabetes.

NOD mice constitute a model for studying the prevention of human autoimmune type 1 diabetes. Glutamic acid decarboxylase (GAD) could be a key antigen involved in this disease, and GAD65 peptide 524-543 has been implicated in early T cell response in young NOD mice. We performed two i.p. injections of GAD peptide 524-543 (100 micrograms at each injection), together with Freund's incomplete adjuvant (FIA), into female NOD mice at 30 and 45 days old. Diabetes was accelerated 2 weeks later by a single injection of cyclophosphamide (CY), which acts against suppressive mechanisms. Treatment with GAD 524-543 peptide delayed the onset of diabetes and reduced its incidence (28% versus 60%; P < 0.001) compared with control mice injected with FIA alone, or GAD peptide 534-553, or an irrelevant peptide. In the same group, the severity of lymphocytic inflammation of pancreatic islets was reduced (P < 0.03). Up to 3 months after peptide injections, a strong splenocytic proliferative response occurred in immunized NOD mice against the immunizing peptide alone (but not against a panel of seven other GAD65-derived peptides). After peptide challenge of splenocytes in vitro, protection against CY-accelerated diabetes was associated with higher peptide-specific production of T helper type 2 (Th2)-associated interleukins 4 and 10, whereas Th1-associated interferon-gamma and IL-2 were proportionally less represented. During contransfer, T splenocytes from GAD 524-543-immunized mice were able to reduce the capacity of T cells from diabetic donors to transfer the disease adoptively (P < 0.01), demonstrating the generation of cellular mechanisms that actively suppress the disease. It is concluded that immunization of NOD mice with GAD65 peptide 524-543 can counteract CY-accelerated diabetes, possibly through active cellular suppression linked to a shift of Th1/Th2 balance toward the production of Th2 cytokines such as IL-4 and IL-10. This study provides additional support for the notion that GAD, and more precisely its epitope 524-543, could be one of the key targets for the pathogenesis of type 1 diabetes in NOD mice, as well as for the efficacy of disease-specific peptide therapy in type 1 diabetes.

Autoantigen-specific protection of non-obese diabetic mice from cyclophosphamide-accelerated diabetes by vaccination with dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells involved in the initiation of primary immune responses and the preservation of peripheral tolerance. The aim of this study was to develop a DC vaccine for autoantigen-specific prevention of autoimmune diabetes. Splenocytes from diabetes-prone NOD mice were cultured in conditioned media to obtain a homogeneous DC sub-population for vaccination experiments. These cells were used to modulate autoimmune responses in NOD mice after synchronization of diabetes with cyclophosphamide. After immunisation with insulin-pulsed DCs the incidence of diabetes, the insulitis grade and the cytokine production was examined. The long-term culture of splenocytes resulted in the generation of a cell line, termed NOD-DC1, which have a phenotype of myeloid DCs (CD11c, CD11b, DEC-205), express MHC class II and co-stimulatory molecules (CD40, CD80, CD86). The NOD-DC1 cells have preserved functional activity shown by the detection of a high antigen uptake capacity, the induction of a mixed lymphocyte reaction and stimuli-dependent IL-6 and TNF-alpha secretion. Vaccination with insulin-pulsed NOD-DC1 cells results in an antigen-specific prevention of diabetes. This was mediated by a reduction of the severity of insulitis and a decrease of T helper 1 effector cells. We describe the generation of a DC line which confers protection from diabetes in an antigen-specific way. Our data shows that autoantigen-loaded DCs can induce strong immunoregulatory effects supporting the hypothesis that DCs are promising candidates to develop novel vaccines for the prevention of autoimmune diabetes.

Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1beta-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.

DNA Vaccination with Heat Shock Protein 60 Inhibits Cyclophosphamide-Accelerated Diabetes

Nonobese diabetic (NOD) mice spontaneously develop diabetes as a consequence of an autoimmune process that can be inhibited by immunotherapy with the 60-kDa heat shock protein (hsp60), with its mycobacterial counterpart 65-kDa (hsp65), or with other Ags such as insulin and glutamic acid decarboxylase (GAD). Microbial infection and innate signaling via LPS or CpG motifs can also inhibit the spontaneous diabetogenic process. In addition to the spontaneous disease, however, NOD mice can develop a more robust cyclophosphamide-accelerated diabetes (CAD). In this work, we studied the effect on CAD of DNA vaccination with constructs encoding the Ags human hsp60 (phsp60) or mycobacterial hsp65 (phsp65). Vaccination with phsp60 protected NOD mice from CAD. In contrast, vaccination with phsp65, with an empty vector, or with a CpG-positive oligonucleotide was not effective, suggesting that the efficacy of the phsp60 construct might be based on regulatory hsp60 epitopes not shared with its mycobacterial counterpart, hsp65. Vaccination with phsp60 modulated the T cell responses to hsp60 and also to the GAD and insulin autoantigens; T cell proliferative responses were significantly reduced, and the pattern of cytokine secretion to hsp60, GAD, and insulin showed an increase in IL-10 and IL-5 secretion and a decrease in IFN-gamma secretion, compatible with a shift from a Th1-like toward a Th2-like autoimmune response. Our results extend the role of specific hsp60 immunomodulation in the control of beta cell autoimmunity and demonstrate that immunoregulatory networks activated by specific phsp60 vaccination can spread to other Ags targeted during the progression of diabetes, like insulin and GAD.

Polyethylene glycolated recombinant TNF receptor I improves insulitis and reduces incidence of spontaneous and cyclophosphamide-accelerated diabetes in nonobese diabetic mice.

We have conducted three studies to examine the role of TNFalpha in islet destruction in female nonobese diabetic mouse (NOD) mice, a model of human autoimmune diabetes, using polyethylene glycolated (PEGylated) soluble TNF receptor type I (PEG sTNF-RI) as TNFalpha antagonist. PEG sTNF-RI (3 mg/kg, sc) was given every other day to NOD mice from age wk 8 for 12 wk (study 1), from age wk 12 for 8 wk (study 2), or from age wk 8 for 3 wk, with cyclophosphamide (6 mg/mouse) injected at wk 9 to accelerate the onset of diabetes (study 3). Diabetic incidence was reduced (control vs. PEG sTNF-RI) from 68.7% (11 of 16) to 18.3% (3 of 16) in study 1, from 84.6% (11 of 13) to 28.5% (4 of 14) in study 2, and from 66.6% (8 of 12) to 23.1% (3 of 13) in study 3, respectively. The incidence of insulitis was also reduced from 91.6% (11 of 12) to 12.5% (2 of 16) in study 1 and from 100% (7 of 7) to 16.6% (2 of 12) in study 2 by PEG sTNF-RI. PEG sTNF-RI also largely preserved islet insulin content, reduced mRNA of inducible nitric oxide synthase and IL-6 in pancreases, and lowered plasma corticosterone, glycerol, and free fatty acid levels. These results confirm a pathogenic role of TNFalpha in mediating insulitis in NOD mice and suggest the prophylactic and therapeutic potential of PEG sTNF-RI for human autoimmune diabetes.

Unexpected sensitivity of nonobese diabetic mice with a disrupted poly(ADP-Ribose) polymerase-1 gene to streptozotocin-induced and spontaneous diabetes.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that consumes NAD in response to DNA strand breaks. Its excessive activation seems particularly deleterious to pancreatic beta-cells, as exemplified by the complete resistance of PARP-1-deficient mice to the toxic diabetes induced by streptozotocin. Because of the possible implication of this enzyme in type 1 diabetes, many human trials using nicotinamide, an inhibitor of PARP-1, have been conducted either in patients recently diagnosed or in subjects highly predisposed to this disease. To analyze the role of this enzyme in murine type 1 diabetes, we introgressed a disrupted PARP-1 allele onto the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain. We showed that these mice were protected neither from spontaneous nor from cyclophosphamide-accelerated diabetes. Surprisingly they were also highly sensitive to the diabetes induced by a single high dose of streptozotocin, standing in sharp contrast with C57BL/6 mice that bear the same inactivated PARP-1 allele. Our results suggest that NOD mice are characterized not only by their immune dysfunction but also by a peculiarity of their islets leading to a PARP-1-independent mechanism of streptozotocin-induced beta-cell death.

Fas and Fas ligand immunolocalization in pancreatic islets of NOD mice during spontaneous and cyclophosphamide-accelerated diabetes.

During insulin-dependent diabetes mellitus, immune cells which infiltrate pancreatic islets mediate beta cell destruction over a prolonged asymptomatic prediabetic period. The molecular mechanisms of beta cell death in vivo remain unresolved. At least two major molecular processes of destruction have been proposed. One involves the Fas-FasL (Fas-Fas ligand) system and the other, the perforin pathway. Here, dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of Fas and FasL in the NOD mouse, during spontaneous diabetes (days 21, 40 and 90) and following acceleration of diabetes with cyclophosphamide (days 0, 4, 7, 11 and 14 after cyclophosphamide administration). The expression of the proteins was correlated with advancing disease. FasL was expressed constitutively in most beta cells but not in glucagon or somatostatin cells or islet inflammatory cells and paralleled the loss of insulin immunolabelling with advancing disease. It was also expressed in beta cells of non-diabetes prone CD-1 and C57BL/6 mice from a young age (day 21). Strong immunolabelling for Fas was first observed in extra-islet macrophages and those close to the islet in NOD and non-diabetes-prone mice. During spontaneous and cyclophosphamide diabetes, it was observed in a higher proportion of islet infiltrating macrophages than CD4 and CD8 T cells, concomitant with advancing insulitis. In cyclophosphamide-treated mice, the proportion of Fas-positive intra-islet CD4 and CD8 T cells at day 14 (with and without diabetes) was considerably higher than at days 0, 4, 7 and 11. At days 11 and 14, a proportion of Fas-positive intra-islet macrophages co-expressed interleukin-1beta and inducible nitric oxide synthase. Fas was not detectable in beta cells and other islet endocrine cells during spontaneous and cyclophosphamide induced diabetes. Our results show constitutive expression of FasL in beta cells in the NOD mouse and predominant expression of Fas in intra-islet macrophages and to a lesser extent in T cells prior to diabetes onset. Interleukin-1beta in intra-islet macrophages may induce Fas and inducible nitric oxide synthase expression in an autocrine and paracrine manner and mediate beta cell destruction or even death of some macrophages and T cells. However, other mechanisms of beta cell destruction during spontaneous and cyclophosphamide-accelerated diabetes and independent of Fas-FasL, require examination.

Changing patterns of cell surface mono (ADP-ribosyl) transferase antigen ART2.2 on resting versus cytopathically-activated T cells in NOD/Lt mice.

ART2.2 is a mouse T-cell surface ectoenzyme [mono (ADP-ribosyl) transferase] shed upon strong activation. We analysed temporal changes in ART2.2 expression in unmanipulated and cyclophosphamide-treated NOD/Lt mice compared with diabetes-resistant control strains. We used NAD, the ART2.2 substrate, to test whether ART-mediated ADP-ribosylation could retard diabetogenic activation of islet-reactive T cells in vitro. ART2.2 and CD38, another NAD-utilizing enzyme, were measured by flow cytometry. ADP-ribosylation from ethano-NAD was followed by flow cytometry using a reagent specific for etheno-ADP ribose. Although mature NOD CD4 + and C D8 + T cells expressed ART2.2, this expression was delayed in young NOD mice when compared with control strains. This ontological delay at 3 weeks of age correlated with an early burst of CD25 expression unique to NOD splenic T cells. This pattern was reproduced in cyclophosphamide-accelerated diabetes in young NOD/Lt males, wherein a retarded repopulation of ART2.2 T cells in spleen and islets correlated with development of heavy insulitis and diabetes. NAD inhibited anti-CD3 induced activation of splenic T cells in vitro and also retarded killing of beta-cell targets by NOD islet-reactive CD8 effectors in vitro at concentrations equal to or greater than 1 micromol/l. Evidence suggested that CD38 on B lymphocytes competes with ART2.2 for substrate needed by B lymphocytes for ADP ribosylation. ART2.2 on T cells may not simply mark the resting state, but could also contribute to it via ADP-ribosylation.

Effects of plasmid DNA injection on cyclophosphamide-accelerated diabetes in NOD mice.

Type 1 diabetes results in most cases from the destruction of insulin-secreting beta cells by the immune system. Several immunization methods based on administration of autoantigenic polypeptides such as insulin and glutamic acid decarboxylase (GAD) have been used to prevent autoimmune diabetes in the non-obese diabetic (NOD) mouse. In the work presented here, a gene-based approach was taken for a similar purpose. A plasmid carrying different cDNAs was used to investigate the effects of injecting naked DNA on cyclophosphamide-accelerated diabetes in female NOD mice. Four-week-old animals received intramuscular injections of plasmid DNA encoding either intracellular GAD, a secreted form of GAD, or a secreted form of a soft coral luciferase. Monitoring of glycosuria and hyperglycemia indicated that injection of plasmid DNA encoding secreted GAD and secreted luciferase could prevent and delay diabetes, respectively. In contrast, injection of DNA encoding intracellular GAD did not suppress the disease significantly. Analysis of anti-GAD IgG(1) antibody titers in animal sera indicated that diabetes prevention after injection of GAD-encoding DNA was possibly associated with increased Th2-type activity. These results suggest that cellular localization of GAD is a factor to consider in the design of GAD-based genetic vaccines for the prevention of autoimmune diabetes.

Immunoexpression of interleukin-1beta in pancreatic islets of NOD mice during cyclophosphamide-accelerated diabetes: co-localization in macrophages and endocrine cells and its attenuation with oral nicotinamide.

During insulin-dependent diabetes mellitus, islet invading immune cells destroy beta cells over a prolonged asymptomatic pre-diabetic period. Cytokines synthesised and secreted by specific immune cells within the islet infiltrate may be crucial effectors of beta cell destruction or protection during the disease. Interleukin-1beta may be a key cytokine which may act in concert with other cytokines in initiating and/or promoting beta cell destruction. We have examined this hypothesis in NOD mice by assessing the intra-islet expression and co-localization of interleukin-1beta at different time-points following cyclophosphamide administration. We have also tested the effects of long-term oral nicotinamide given to NOD mice in suppressing intra-islet expression of the cytokine in this accelerated model. Cyclophosphamide was administered to day 95 female NOD mice. Pancreatic tissues were examined by dual-label confocal immunofluorescence microscopy for the expression and co-localization of interleukin-1beta at days 0, 4, 7, 11 and at onset of diabetes (day 14). Diabetes developed in 7/11 mice 14 days after administration of cyclophosphamide while nicotinamide completely prevented the disease. At day 0, interleukin-1beta immunolabelling was observed in selective intra-islet macrophages, several somatostatin cells and in a few beta cells. However, at day 4, it was seen mostly in somatostatin and some beta cells. At day 7, an increasing number of interleukin-1beta cells were observed within the islets and co-localized to several somatostatin cells, beta cells and macrophages. The mean number of intra-islet interleukin-1beta cells reached a peak at day 11 and was significantly higher than at day 7 (p = 0.05) and at day 14 (onset of diabetes; p = 0.03). At day 11, interleukin-1beta immunolabelling was also present in selective macrophages which co-expressed inducible nitric oxide synthase. At onset of diabetes, some macrophages, residual beta cells and somatostatin cells showed immunolabelling for the cytokine. Exposure of NOD mice to oral nicotinamide was associated with a considerably reduced expression of interleukin-1beta cells within the islet at day 11 (p = 0.002). We conclude that cylophosphamide treatment enhances the expression of interleukin-1beta in selective macrophages, somatostatin and beta cells during the course of the disease. Its expression reaches a maximum immediately prior to onset of diabetes. Interleukin-1beta present in intra-islet macrophages, somatostatin and beta cells may influence its expression by autocrine and paracrine means. Interleukin-1beta expression within islet macrophages may also up-regulate inducible nitric oxide synthase within the same macrophage or adjacent macrophage populations. These intra-islet molecular events may corroborate with other local cytotoxic processes leading to beta cell destruction. Oral nicotinamide may attenuate intra-islet expression of interleukin-1beta and thus inducible nitric oxide synthase during prevention of Type 1 diabetes in this animal model. The expression of interleukin-1beta in specific islet endocrine cell-types shown in this study requires further investigation.

Apoptosis and beta-cell destruction in pancreatic islets of NOD mice with spontaneous and cyclophosphamide-accelerated diabetes.

Autoimmune-mediated destruction of pancreatic islet beta cells leads to insulin-dependent diabetes in non-obese diabetic (NOD) mice. Although both direct cytotoxic T cell- and indirect cytokine-, nitric oxide- or free radical-mediated mechanisms induce beta-cell apoptosis in vitro, beta-cell death in vivo in spontaneous autoimmune diabetes is not well-characterized. Furthermore, whether beta cells die gradually, or rapidly in the late pre-clinical stage, is a question of current interest. To investigate beta-cell death in vivo, we measured the frequency and intra-islet localisation of apoptosis, defined as DNA strand breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique, during spontaneous and cyclophosphamide-accelerated diabetes in NOD mice. In spontaneous diabetes, the frequency of apoptosis in islets correlated with the progression of beta-cell destruction with age. Although apoptosis was detected at low frequency within the reduced insulin-positive islet area of pre-diabetic mice at 90 days of age, it was rarely co-localised to beta cells. After acceleration of beta-cell destruction with cyclophosphamide, the frequency of apoptosis reached maximum at 12 days, at which time 3.2 % of apoptotic cells were beta cells. Apoptosis was most frequent in the insulin-negative islet area comprised of mononuclear cell infiltrate and was localized to CD8+ T cells. The rarity of detectable apoptotic beta cells in spontaneous pre-diabetic mice with pronounced insulitis and reduced insulin-positive islet areas most likely reflects the rapid clearance of apoptotic beta cells. Our findings are more consistent with gradual destruction of non-renewable beta-cells in spontaneous diabetes, than with their rapid, accelerated destruction (as after cyclophosphamide) in the late pre-clinical stage.

Pancreatic expression of B7 co-stimulatory molecules in the non-obese diabetic mouse.

Expression of the co-stimulatory molecule B7-1 (CD80) on pancreatic beta cells can overcome peripheral T cell tolerance in transgenic models of autoimmune disease. This study aimed to determine if aberrant B7-1 or B7-2 (CD86) expression on pancreatic beta cells is involved in the pathogenesis of autoimmune diabetes in non-obese diabetic (NOD) mice. Immunohistochemical analysis of NOD pancreas sections revealed no evidence of B7-1 or B7-2 expression on pancreatic beta cells at any stage prior to the onset of either spontaneously arising or cyclophosphamide-accelerated diabetes. Likewise, the NOD-derived NIT-1 beta cell line did not express surface B7 or B7-1 mRNA either constitutively or following exposure to IFN-gamma and TNF-alpha, two cytokines known to be present in the insulitis lesion of NOD mice, or cAMP which can induce B7-1 expression on B cells. Both B7-1 and B7-2 were, however, highly expressed on the majority of islet-infiltrating inflammatory cells in NOD mice between days 7 and 12 after the administration of cyclophosphamide which results in accelerated beta cell destruction. Likewise B7-1 and B7-2 were extensively expressed on islet-infiltrating cells present at the time of diabetes onset in NOD SCID mice with adoptively transferred diabetes. By immunohistochemistry and flow cytometry, it was determined that the phenotype of B7+ cells in the pancreas of NOD mice 9 days after cyclophosphamide included a mixture of macrophages and both CD4+ and CD8+ T cells. B7-2 was also expressed on islet-infiltrating cells in the spontaneously occurring diabetes of female NOD mice, but the levels of B7-1 expression were low in comparison with the accelerated models of diabetes. RIP-IL-2 transgenic mice, which have extensive islet infiltration but no autoimmune beta cell destruction, also had virtually no B7-1 expression and a minority of B7-2-expressing inflammatory cells. Thus, the activation of beta cell-specific T cells in NOD mice does not appear to be a result of aberrant expression of B7 on the beta cells. Expression of B7-1 and B7-2 on islet-infiltrating cells is, however, associated with autoimmune beta cell destruction, suggesting a role for the B7-CD28 interaction in this process.