sq-23377 and Diabetes-Mellitus--Type-1

Type 1 diabetes is an autoimmune disease where autoreactive T lymphocytes destroy pancreatic beta cells. We previously reported a defect in CD4+ Tregs cell proliferation and reduced CD4+ Tregs PD-1 expression in patients. Another 'memory-like' regulatory subset, CD8+ Tregs, evaluated as CD8+CD25+FOXP3+, has recently raised interest for their effective suppressive activity. Different CD8+ T cell populations, their proliferation capacity and expression of PD-1 molecule were evaluated by flow-cytometer analysis in newly diagnosed, long-term Type 1 diabetes patients compared to healthy normal donors. Under basal conditions, CD8+ Tregs and CD8+ Teffs were seemingly represented among study groups while there was evidence of diminished expression of PD-1 in Teff subsets of long-term patients. After 3 days of PMA/ionomycin stimulation, patients CD8+ Tregs showed decreased percentage in respect to control group. CD8+ Teffs were instead increased in long-term diabetics versus controls. PD-1+CD8+ Tregs were represented at a much lower percentage in long-term diabetic patients, in respect to controls. Importantly, patients CD8+ Tregs and CD8+ Teffs presented a significant proliferation defect in respect to the control group. In conclusion, our study indicates that a defect of CD8+ Tregs is observed in diabetics. This subset could thus represent a novel target of immunotherapy in patients.

Topics: Adolescent; Adult; Biomarkers; Case-Control Studies; CD8-Positive T-Lymphocytes; Cell Proliferation; Child; Child, Preschool; Diabetes Mellitus, Type 1; Female; Glycated Hemoglobin; Humans; Immunotherapy; In Vitro Techniques; Ionomycin; Male; Pilot Projects; Programmed Cell Death 1 Receptor; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate; Young Adult

Regulatory T-cells (Tregs) have catalyzed the field of immune regulation. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires robust methods for the isolation and expansion of these cells-a need forming the basis for these studies.. Tregs from recent-onset type 1 diabetic patients and healthy control subjects were isolated by fluorescence-activated cell sorting and compared for their capacity to expand in vitro in response to anti-CD3-anti-CD28-coated microbeads and IL-2. Expanded cells were examined for suppressive function, lineage markers and FOXP3, and cytokine production.. Both CD4+CD127(lo/-) and CD4+CD127(lo/-)CD25+ T-cells could be expanded and used as Tregs. However, expansion of CD4+CD127(lo/-) cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4+CD127(lo/-)CD25+ T-cells, especially the CD45RA+ subset, resulted in high yield, functional Tregs that maintained higher FOXP3 expression in the absence of rapamycin. Tregs from type 1 diabetic patients and control subjects expanded similarly and were equally capable of suppressing T-cell proliferation. Regulatory cytokines were produced by Tregs after culture; however, a portion of FOXP3+ cells were capable of producing interferon (IFN)-gamma after reactivation. IFN-gamma production was observed from both CD45RO+ and CD45RA+ Treg populations.. The results support the feasibility of isolating Tregs for in vitro expansion. Based on expansion capacity, FOXP3 stability, and functional properties, the CD4+CD127(lo/-)CD25+ T-cells represent a viable cell population for cellular therapy in this autoimmune disease.

Topics: Adult; Age of Onset; Antigens, CD; CD4-Positive T-Lymphocytes; Cell Division; Cell Proliferation; Cytokines; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Forkhead Transcription Factors; Humans; Immunosuppression Therapy; Ionomycin; Male; Phenotype; Reference Values; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate; Young Adult

To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro-activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4(+) fraction, as purified CD4(+) T-cells prevented the accelerated diabetes. When CD25(+) cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4(+)CD25(+) cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4(+)CD25(+) T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4(+)CD25(-) cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4(+)CD25(+) cells exhibit powerful regulatory potential in rat diabetes.

Topics: Adoptive Transfer; Aging; Animals; CD4 Antigens; Diabetes Mellitus, Type 1; DNA-Binding Proteins; Forkhead Transcription Factors; Gene Expression; Ionomycin; Lymphocyte Activation; Prediabetic State; Rats; Receptors, Interleukin-2; T-Lymphocyte Subsets; Tetradecanoylphorbol Acetate; Transcription Factors

We sought to determine the mechanisms for hyperactivity and abnormal platelet Ca(2+) homeostasis in diabetes. The glycosylated Hb (HbA(1c)) level was used as an index of glycemic control. Human platelets were loaded with Ca- green-fura red, and cytosolic Ca(2+) ([Ca(2+)](i)) and aggregation were simultaneously measured. In the first series of experiments, the platelets from diabetic and normal subjects were compared for the ability to release Ca(2+) or to promote Ca(2+) influx. A potent and relatively specific inhibitor of Na(+)/Ca(2+) exchange, 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), increased the second phase of thrombin-induced Ca(2+) response, suggesting that the Na(+)/Ca(2+) exchanger works in the forward mode to mediate Ca(2+) efflux. In contrast, in the platelets from diabetics, CB-DMB decreased the Ca(2+) response, indicating that the Na(+)/Ca(2+) exchanger works in the reverse mode to mediate Ca(2+) influx. In the second series of experiments we evaluated the direct effect of hyperglycemia on platelets in vitro. We found that thrombin- and collagen-induced increases in [Ca(2+)](i) and aggregation were not acutely affected by high glucose concentrations of 45 mM. However, when the platelet-rich plasma was incubated with a high glucose concentration at 37 degrees C for 24 h, the second phase after thrombin activation was inhibited by CB-DMB. In addition, collagen-stimulated [Ca(2+)](i) response and aggregation were also increased. Thus in diabetes the direction and activity of the Na(+)/Ca(2+) exchanger is changed, which may be one of the mechanisms for the increased platelet [Ca(2+)](i) and hyperactivity. Prolonged hyperglycemia in vitro can induce similar changes, suggesting hyperglycemia per se may be the factor responsible for the platelet hyperactivity in diabetes.

Topics: Amiloride; Blood Glucose; Blood Platelets; Calcium; Collagen; Cytosol; Diabetes Mellitus; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Homeostasis; Humans; In Vitro Techniques; Ionomycin; Platelet Aggregation; Reference Values; Sodium-Calcium Exchanger; Thrombin

Type I diabetes mellitus is a chronic disorder that results from autoimmune destruction of the insulin-producing pancreatic beta cell. The non-obese diabetic mouse is a model of the human autoimmune disease Type I diabetes [1-3]. We have previously shown that ingested type 1 interferon inhibits chronic relapsing experimental autoimmune encephalomyelitis and the adoptive transfer of experimental autoimmune encephalomyelites by T cells, and decreases both antigen-specific and mitogen-induced pro-inflammatory cytokine secretion in this disorder. We therefore tried to determine whether ingested murine interferon alpha inhibits insulinitis and suppresses Type I diabetes mellitus in non-obese diabetic mice. Murine interferon alpha, given daily, decreased islet inflammation and suppressed diabetes. It increased the concanavalin A and ionomycin plus myristic acid palmitic ester-induced production of interleukin 4 and 10 and interferon gamma-secretion in spleen cells from treated mice. Adoptive transfer of unstimulated splenocytes secreting interleukin 4 and interleukin 10 from fed interferon alpha donors suppressed spontaneous diabetes mellitus in recipients. The protective effect of adoptively transferred unstimulated splenocytes shows the presence of ingested interferon alpha-activated regulatory splenic cell populations that may work via increased interleukin 4 or interleukin 10 production. Ingested interferon alpha administered during vulnerable periods in at-risk populations may potentially provide a continuous, convenient, non-toxic and effective treatment for Type I diabetes.

Topics: Adoptive Transfer; Animals; Concanavalin A; Diabetes Mellitus, Type 1; Female; Interferon-alpha; Interferon-gamma; Interleukin-10; Interleukin-4; Interleukins; Ionomycin; Mice; Mice, Inbred NOD; Myristic Acid; Palmitic Acid; Spleen; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

The autoimmune syndrome of the BB rat is associated with a marked increase in glutamine (Gln) metabolism in immune system cells of both diabetes-prone (BBdp) and diabetic (BBd) rats. To test whether inhibition of Gln metabolism prevents diabetes, 17 BBdp received acivicin (1 mg/kg) and 17 received saline subcutaneously every 2 days from age 48 days until diabetes onset or age 186 days. Twenty-seven non-diabetes-prone (BBn) rats served as controls. Acivicin caused some growth effects and a macrocytic anemia, but no other clinical or biochemical side effects. Only one acivicin-treated BBdp became diabetic (age 158 days), compared with saline-treated rats, of which 10 became diabetic and 2 became glucose intolerant (p < 0.001). Insulitis was moderate to severe in 88% of the saline-treated BBdp rats, but minimal in most acivicin-treated BBdp rats. Liver glutamine and glutamate tended to be higher in acivicin- than saline-treated BBdp rats. Acivicin caused no change in the proportions of T or B lymphocytes, NK cells, or macrophage phenotypes in spleen or blood; all BBdp rats were typically lymphopenic. Mitogenic responses of splenocytes in vitro were not affected. The results are consistent with the hypothesis that acivicin, by interfering with Gln metabolism, "targets" activated cells of the immune system and thereby attenuates the process and prevents overt diabetes, without major disturbance of Gln levels or generalized immunosuppression. This prevention is not due to a nutritional-growth retardation effect, as diabetes was prevented in females that showed no such effect.

Topics: Animals; Blood Cell Count; Diabetes Mellitus, Type 1; Drug Interactions; Enzyme Inhibitors; Female; gamma-Glutamyltransferase; Glucose Tolerance Test; Glutamic Acid; Glutamine; Ionomycin; Isoxazoles; Lymphocyte Subsets; Male; Rats; Rats, Inbred BB; Spleen; Tetradecanoylphorbol Acetate

Cells infiltrating the Langerhans' islets of prediabetic NOD females were isolated from 6 weeks to 6 months of age. These cells were assayed at a single-cell level for production of eight different cytokines by intracellular immunofluorescent staining. By in vitro stimulation with PMA and ionomycin for 4 hours the method is enhanced also to detect in vivo preactivated cells. During the early phase of insulitis from 6 to 12 weeks of age, mainly the monokines IL-1 alpha, IL-6, and TNF were detected. After stimulation, also IFN-gamma and low numbers of IL-10 and GM-CSF producing cells could be observed, but no IL-2 or IL-4 was seen. This cytokine pattern correlates with an increasing insulitis, and we suggest that these cytokines are important in attracting inflammatory cells to the islets, and may cause initial beta-cell destruction. During a later phase, between 4 and 6 months, there is a characteristic TH1 cytokine profile with production of IL-2 and IFN-gamma occurring after stimulation, as well as lymphocytes producing TNF, supposedly TNF-beta. During this period IL-10 was very rarely observed, and no IL-4 production could be found throughout the study. This indicates the absence of a TH2 cytokine profile in this lesion. In addition IL-6 production occurs in high frequencies at all ages, also in endocrine islet cells. We interpret this as a stress response caused by the inflammatory lesion. Our findings show that the effector phase in NOD insulitis is TH1 rather than TH2 mediated. We also demonstrate that cytokines, that may cause initial tissue destruction, are produced during the recruitment of inflammatory cells.

Topics: Aging; Animals; Cells, Cultured; Cytokines; Diabetes Mellitus, Type 1; Female; Fluorescent Antibody Technique; Granulocyte-Macrophage Colony-Stimulating Factor; Interferon-gamma; Interleukin-1; Interleukin-10; Interleukin-2; Interleukin-4; Interleukin-6; Ionomycin; Islets of Langerhans; Mice; Mice, Inbred NOD; Microscopy, Fluorescence; Pancreatic Diseases; Tetradecanoylphorbol Acetate; Time Factors; Tumor Necrosis Factor-alpha

Glucose metabolism and respiratory burst were studied in vitro in resident peritoneal macrophages from non-diabetes-prone BB, spontaneously diabetic BB, diabetes-prone BB, and STZ-induced diabetic BBn rats, in the presence or absence of phorbol myristate acetate plus ionomycin. Glycolysis and pentose phosphate pathway activity were increased in BBd compared with BBn cells. PMA plus IONO did not influence glycolysis in BBn macrophages and slightly decreased it in BBd macrophages. In contrast, PMA plus IONO increased the pentose phosphate pathway activity in BBn and BBd macrophages with a much greater increase in BBd cells. The release of O2- was greater in BBd than BBn cells; PMA plus IONO also induced a much greater release of O2- in BBd cells. H2O2 release was undetectable in unstimulated BBn cells, and stimulation by PMA plus IONO caused a small incremental release. In contrast, the release of H2O2 was measurable in unstimulated cells and further increased by 50% in BBd cells with PMA-plus-IONO stimulation. The release of O2- and H2O2 was increased in macrophages from 75-day-old BBdp rats but not in 50-day-old BBdp rats, compared with age-matched BBn rats. No differences were observed in either glucose metabolism or release of O2- and H2O2 between BBn and STZ-BBn cells in the absence or presence of PMA plus IONO. These data suggest that enhanced oxidative metabolism in BBd macrophages is unlikely to be attributable to diabetes per se.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Analysis of Variance; Animals; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Glucose; Glycolysis; Hydrogen Peroxide; Ionomycin; Lactates; Macrophages; Pentose Phosphate Pathway; Prediabetic State; Pyruvates; Rats; Rats, Inbred BB; Superoxides; Tetradecanoylphorbol Acetate

IL-2 receptor positive T-cells from leukocyte-infiltrated pancreatic islets of diabetes prone or acutely diabetic NOD mice were propagated in vitro by culture in interleukin-2 containing medium. Of 13 lines obtained after limiting dilution all were positive for the T-cell marker Thy-1 and for CD8. Considerable heterogeneity in T-cell receptor usage was noted. Seven lines expressed T-cell receptors using V beta 8, one line was positive for V beta 5 and two lines expressed a non V beta 5, non V beta 8 receptor. Finally, two further lines lacked T-cell receptors. None of the cell lines were cytotoxic to islet cells although 10 lines showed non MHC restricted lysis of one or more tumour cells including rat insulinoma cells. We conclude that IL-2 receptor positive CD8+ T-lymphocytes from NOD islets are heterogenous with respect to V beta T-cell receptor usage. The majority of these cells are not cytotoxic to islet cells.

Topics: Animals; Antigens, Surface; CD8 Antigens; Cell Line; Cells, Cultured; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Flow Cytometry; Humans; Ionomycin; Islets of Langerhans; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thy-1 Antigens

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.

Topics: Age Factors; Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Calcium; CD3 Complex; Concanavalin A; Diabetes Mellitus, Type 1; Interleukin-2; Ionomycin; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Receptors, Antigen, T-Cell; Recombinant Proteins; T-Lymphocytes; Tetradecanoylphorbol Acetate