concanavalin-a and Diabetes-Mellitus--Type-1

Recently, it was shown that children at the onset of type 1 diabetes (T1D) have a higher proportion of oligomannose glycans in their total plasma protein N-glycome compared to their healthy siblings. The most abundant complement component, glycoprotein C3, contains two N-glycosylation sites occupied exclusively by this type of glycans. Furthermore, complement system, as well as C3, was previously associated with T1D. It is also known that changes in glycosylation can modulate inflammatory responses, so our aim was to characterize the glycosylation profile of C3 in T1D. For this purpose, we developed a novel high-throughput workflow for human C3 concanavalin A lectin affinity enrichment and subsequent LC-MS glycopeptide analysis which enables protein-specific N-glycosylation profiling. From the Danish Childhood Diabetes Register, plasma samples of 61 children/adolescents newly diagnosed with T1D and 84 of their unaffected siblings were C3 N-glycoprofiled. Significant changes of C3 N-glycan profiles were found. T1D was associated with an increase in the proportion of unprocessed glycan structures with more mannose units. A regression model including C3 N-glycans showed notable discriminative power between children with early onset T1D and their healthy siblings with area under curve of 0.879. This study confirmed our previous findings of plasma high-mannose glycan changes in a cohort of recent onset T1D cases, suggesting the involvement of C3 N-glycome in T1D development. Our C3 glycan-based discriminative model could be valuable in assessment of T1D risk in children.

Topics: Adolescent; Biomarkers; Child; Complement C3; Concanavalin A; Diabetes Mellitus, Type 1; Glycopeptides; Glycoproteins; Humans; Lectins; Mannose; Polysaccharides

Implantation failure is a major hurdle to a successful pregnancy. The high rate of postimplantation fetal loss in nonobese diabetic (NOD) mice is believed to be related to an abnormal decidual production of interferon (IFN)gamma. To address whether diabetes alters the natural events associated with successful implantation, certain morphological and molecular features of uterine receptivity in diabetic NOD (dNOD) mice were examined in normally mated pregnancy and in concanavalin A (ConA)-induced pseudopregnancy. As opposed to normoglycemic NOD (cNOD) mice, dNOD mice expressed retarded maturation of their uterine pinopodes and overexpressed MUC1 mucin at implantation sites (P < 0.001). Uterine production of leukemia inhibitory factor (LIF) and phosphorylation of uterine NFkappaBp65 and STAT3-Ty705 were found to be low (P < 0.01) during Day 4.5 postcoitum, whereas IFNgamma was aberrantly overexpressed. Loss of temporal regulation of progesterone receptor A (PR A) and PR B, together with aberrantly increased expression of the protein inhibitor of activated STAT-y (PIASy) (P < 0.01) and reduced recruitment (P < 0.01) of the latter to nuclear progesterone receptor sites were prominent features of decidualization failure occurring at peri-implantation in dNOD mice. In conclusion, the aberrant expression of endometrial IFNgamma in dNOD mice is associated with a nonreceptive endometrial milieu contributing to peri-implantation embryo loss in type 1 diabetes.

Topics: Abortion, Spontaneous; Animals; Concanavalin A; Diabetes Mellitus, Type 1; Embryo Implantation; Endometrium; Female; Interferon-gamma; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Mucin-1; Pregnancy; Protein Inhibitors of Activated STAT; Receptors, Progesterone; STAT3 Transcription Factor; Transcription Factor RelA

Antigen-specific immunotherapy is expected to be an ideal strategy for treating type 1 diabetes (T1D). We investigated the therapeutic efficacy of a peptide in the leader sequence of preproinsulin, which was selected because of its binding affinity to the MHC I-A(g7) molecule. Preproinsulin-1 L7-24 peptide (L7-24) emulsified in Freund's incomplete adjuvant was administered subcutaneously to NOD mice. Administration of L7-24 increased the proportion of regulatory T cells in the spleen. Splenocytes of NOD mice immunized with this peptide secreted IL-4 and IL-10 in response to L7-24. This peptide also significantly prevented the development of diabetes and cured some newly diabetic NOD mice without recurrence. L7-24 peptide, which has a high affinity for pockets of I-A(g7), induced regulatory T cells and showed therapeutic effects. This peptide may provide a new approach for developing antigen-specific immunotherapy for autoimmune diabetes.

Topics: Amino Acid Sequence; Animals; Autoimmunity; Blood Glucose; Cell Count; Concanavalin A; Diabetes Mellitus, Type 1; Female; Forkhead Transcription Factors; Histocompatibility Antigens Class II; Immunotherapy, Active; Insulin; Interferon-gamma; Interleukin-10; Interleukin-4; Islets of Langerhans; Lymphocyte Activation; Mice; Mice, Inbred NOD; Mice, Knockout; Peptide Fragments; Protein Binding; Protein Precursors; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory; Th2 Cells; Transforming Growth Factor beta; Vaccination

The autoimmunity of type 1 diabetes is associated with T-cell hyperactivity. Current study was designed to examine the effect of circulating ribonucleic acids (RNAs), isolated from type 1 diabetic patients on proliferative, apoptotic and inflammatory potential of rat thymocytes. Rat thymocytes were assayed for proliferating nuclear cell antigen (PCNA), Bcl-2, Bax and NF-κB level, using the flow cytometric and fluorometric assays. Cells were allocated into groups, treated with RNAs purified from plasma of juvenile diabetics, adult type 1 diabetic patients, control healthy children, healthy adult persons, nucleic acids and polynucleotide standards (RNA, polyC, PolyA, PolyIC, and CpG). The upregulation of PCNA and Bcl-2 protein and downregulation of Bax protein and NF-κB was shown when the thymocytes where incubated with RNA purified from plasma of juvenile type 1 diabetic patients. The dysregulation of inflammatory cascade and central tolerance may be a defect in autoimmune diseases related to innate immunity leading to corresponding alteration in adaptive immune response.

Topics: Adolescent; Adult; Animals; bcl-2-Associated X Protein; Cell Proliferation; Cells, Cultured; Child; Child, Preschool; Concanavalin A; Deoxycytosine Nucleotides; Deoxyguanosine; Diabetes Mellitus, Type 1; Humans; Male; Middle Aged; NF-kappa B; Oligonucleotides; Plasma; Poly I-C; Polyribonucleotides; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; RNA; RNA, Ribosomal; Thymus Gland; Young Adult

Diabetes mellitus (DM), one of the commonest metabolic disorders, can impair the function of cells involved in cellular and/or humoral immunity. This study sought to define potential effects upon cell-mediated immune cells due to an acute hyperglycemic state (in vitro) for comparison against those that might be attributable to a diabetic phenotype itself. Peripheral blood mononuclear cells (PBMC) were isolated from ten diabetic patients (5 with Type I disease and 5 with Type II) and 10 healthy controls. The cells were then challenged with 1 of 3 different mitogens (concanavalin A, phytohemagglutinin, pokeweed mitogen) in the presence of differing glucose concentrations (0, 100, 200, 400, or 800 mg/dl), and proliferative responses assessed. Neutrophils (PMNC) from the blood samples, exposed to the same experimental conditions, were analyzed for respiratory burst activity using nitroblue tetrazolium. The results indicated that there was significant inhibition of the proliferative responses to mitogens among the stimulated PBMC and in respiratory burst activity among the PMNC obtained from the diabetic patients. However, these effects were not affected by either the added presence of increasing amounts of exogenous glucose, the type of diabetes the patients had, the length of time the patient had had the disease, or whether or not the patients had been receiving insulin treatments. In contrast, the PBMC from healthy individuals appeared to display dose-trend decreases in responsiveness to mitogens; interestingly, similar effects on their PMNC were not evident. It was thus concluded that in situ ongoing repeated hyperglycemic states caused changes in cells of the immune system that could have been caused by repeated "continuous" exposures to excess sugar. Further studies are needed to more clearly identify hyperglycemia (sugar)-sensitive targets on/in these cells that could contribute to the appearance of the diabetic immunodeficiency in these types of patients.

Topics: Adult; Cell Proliferation; Concanavalin A; Diabetes Mellitus; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Glucose; Humans; Hyperglycemia; Leukocytes, Mononuclear; Male; Middle Aged; Neutrophils; Nitroblue Tetrazolium; Phytohemagglutinins; Pokeweed Mitogens; Respiratory Burst

Heme oxygenase-1 (HO-1) is an enzyme with potent immunoregulatory capacity. To evaluate the effect of HO-1 on autoimmune diabetes, female NOD mice at 9 weeks of age received a single intravenous injection of a recombinant adeno-associated virus bearing HO-1 gene (AAV-HO-1; 0.5 x 10(10)-2.5 x 10(10) viruses/mouse). In a dose-dependent manner, HO-1 transduction reduced destructive insulitis and the incidence of overt diabetes examined over a 15-week period. HO-1-mediated protection was associated with a lower type 1 T-helper cell (Th1)-mediated response. Adaptive transfer experiments in NOD.scid mice demonstrated that splenocytes isolated from AAV-HO-1-treated mice were less diabetogenic. Flow cytometry analysis revealed no significant difference in the percentages of CD4(+)CD25(+) regulatory T-cells between saline-treated and AAV-HO-1-treated groups. However, the CD11c(+) major histocompatibility complex II(+) dendritic cell population was much lower in the AAV-HO-1-treated group. A similar protective effect against diabetes was observed in NOD mice subjected to carbon monoxide (CO) gas (250 ppm CO for 2 h, twice per week). These data suggest that HO-1 slows the progression to overt diabetes in pre-diabetic NOD mice by downregulating the phenotypic maturity of dendritic cells and Th1 effector function. CO appears to mediate at least partly the beneficial effect of HO-1 in this disease setting.

Topics: Adoptive Transfer; Animals; Concanavalin A; Cytokines; Dendritic Cells; Dependovirus; Diabetes Mellitus, Type 1; DNA Primers; DNA, Complementary; Female; Genetic Vectors; Heme Oxygenase-1; Immunohistochemistry; Mice; Mice, Inbred NOD; Mice, SCID; Pancreas; Reverse Transcriptase Polymerase Chain Reaction; Spleen; Th1 Cells

The present study investigated whether Astragalus polysaccharide (APS) possessed immunotherapeutic effects on type 1 diabetes mellitus. Diabetic mice induced by multiple low dose streptozotocin (MLD-STZ) were administered either APS (100, 200, 400 mg/kg body weight) or saline intraperitoneally daily, and sacrificed after 15 or 30 d of treatment. Meanwhile normal mice not treated with STZ nor with APS were offered into non-diabetic group. Blood glucose and serum insulin levels were measured, histologic and morphometric analyses of the pancreas were performed to determine the effect of APS on pancreatic islets. Further investigations on immune changes in spleens were tested by ELISA, semi-quantitative RT-PCR and Western blot. Downregulated blood glucose level, upregulated serum insulin concentration, increased beta cell mass, decreased apoptotic beta cell percentage, downregulation of Th1/Th2 cytokine ratio and upregulation of peroxisome proliferator-activated receptor gamma (PPARgamma) gene expression in spleens were significantly time- and dose-dependent on APS treatment, when compared to saline controls. These results show that APS seems to be helpful to attenuate insulitis and preserve beta cells from apoptosis, but it can't entirely rescue type 1 diabetes mellitus. APS ameliorates both the clinical and histological parameters of the MLD-STZ induced diabetic mice in a long-lasting fashion, most likely through immunoregulatory actions on Th1/Th2 cytokine ratio, strongly associated with PPARgamma gene expression in spleens.

Topics: Adjuvants, Immunologic; Animals; Astragalus propinquus; Blood Glucose; Blotting, Western; Cells, Cultured; Concanavalin A; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Interferon-gamma; Interleukin-4; Islets of Langerhans; Male; Mice; Mice, Inbred C57BL; Polysaccharides; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spleen; Th1 Cells; Th2 Cells; Up-Regulation

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

Topics: Adoptive Transfer; Amino Acid Sequence; Animals; Blood Glucose; Concanavalin A; Corticosterone; Diabetes Mellitus, Type 1; Disease Progression; Female; Islets of Langerhans; Male; Mice; Mice, Inbred NOD; Phenotype; Point Mutation; Radiation Chimera; Receptors, Cell Surface; Receptors, Leptin; Spleen; T-Lymphocyte Subsets

Genetically-engineered cells offer a solution to the cell availability problem in tissue engineering a pancreatic substitute for the treatment of insulin-dependent diabetes. These cells can be non-beta cells, such as hepatocytes or myoblasts, retrieved as a biopsy from the same patient and genetically engineered to secrete recombinant insulin constitutively or under transcriptional regulation. However, the continuous or slowly responsive insulin secretion dynamics from these cells cannot provide physiologic glucose regulation in patients. Our objective consists of using such cells as an insulin source and of regulating insulin release by incorporating a glucose-responsive material, which acts as a control barrier for insulin in a cell-material hybrid device. Experiments were performed with insulinoma betaTC3 cells, HepG2 hepatomas, and C2C12 myoblasts, the latter two genetically-modified to constitutively secrete insulin. The control barrier consisted of concanavalin A (con A)-based glucose-responsive material, which forms a gel at low and a sol at high glucose concentrations. Results demonstrated that the device released insulin at a higher rate in response to glucose challenges. In contrast, a device containing an inert hydrogel instead of glucose-responsive material released insulin at an essentially constant rate, irrespective of the surrounding glucose concentration. Necessary material improvements include increased sensitivity to glucose, so that the material responds to physiologically relevant glucose concentrations, and increased stability. The prospects of developing a properly functional, implantable substitute based on engineered non-beta cells and glucose-responsive material, and the material and device improvements that need to be made prior to in vivo experiments, are discussed.

Topics: Alginates; Animals; Cell Line; Cell Line, Tumor; Cell Survival; Concanavalin A; Diabetes Mellitus, Type 1; Glucose; Glucuronic Acid; Glycogen; Hexuronic Acids; Humans; Hydrogels; Insulin; Insulin Secretion; Membranes, Artificial; Pancreas, Artificial; Phase Transition; Polycarboxylate Cement; Tissue Engineering; Transfection

Viruses are believed to contribute to the pathogenesis of autoimmune type 1A diabetes in humans. This pathogenic process can be modeled in the BBDR rat, which develops pancreatic insulitis and type 1A-like diabetes after infection with Kilham's rat virus (RV). The mechanism is unknown, but does not involve infection of the pancreatic islets. We first documented that RV infection of BBDR rats induces diabetes, whereas infection with its close homologue H-1 does not. Both viruses induced similar humoral and cellular immune responses in the host, but only RV also caused a decrease in splenic CD4(+)CD25(+) T cells in both BBDR rats and normal WF rats. Surprisingly, RV infection increased CD4(+)CD25(+) T cells in pancreatic lymph nodes of BBDR but not WF rats. This increase appeared to be due to the accumulation of nonproliferating CD4(+)CD25(+) T cells. The results imply that the reduction in splenic CD4(+)CD25(+) cells observed in RV-infected animals is virus specific, whereas the increase in pancreatic lymph node CD4(+)CD25(+) cells is both virus and rat strain specific. The data suggest that RV but not H-1 infection alters T cell regulation in BBDR rats and permits the expression of autoimmune diabetes. More generally, the results suggest a mechanism that could link an underlying genetic predisposition to environmental perturbation and transform a "regulated predisposition" into autoimmune diabetes, namely, failure to maintain regulatory CD4(+)CD25(+) T cell function.

Topics: Animals; Antibodies, Viral; Bromodeoxyuridine; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Division; Cells, Cultured; Coculture Techniques; Concanavalin A; Diabetes Mellitus, Type 1; Epitopes, T-Lymphocyte; Female; Genetic Predisposition to Disease; Immunity, Cellular; Interferon-gamma; Lymph Nodes; Lymphocyte Count; Lymphocytosis; Male; Pancreas; Parvoviridae Infections; Parvovirus; Poly I-C; Rats; Rats, Inbred BB; Rats, Inbred WF; Receptors, Interleukin-2; Spleen; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Linomide prevents the development of autoimmune insulitis and insulin-deficient diabetes mellitus in female NOD mice. Linomide prevents development of autoimmune manifestations in other experimentally induced and spontaneous autoimmune diseases as well, but the mechanism of action is unknown. The present report summarizes our investigations on the effect of Linomide on different functional T cell subsets in NOD mice analyzed according to their cytokine profile. Supernatants from cultured splenocytes and peritoneal cells taken from Linomide-treated mice contained lower levels of TNFalpha, IL-1 beta, IFN gamma and IL-12 versus higher levels of IL-4, IL-6 and IL-10 in comparison with supernatants from cultures of untreated mice. Our results suggest that regulation of autoimmunity following oral Linomide administration in NOD mice induces a shift from Th(1) to Th(2) phenotype response, thereby preventing the development of diabetes by active cytokine-induced immunoregulation of T cell subsets, including downregulation of Th(1) and upregulation of Th(2).

Topics: Adjuvants, Immunologic; Animals; Autoimmunity; Concanavalin A; Cytokines; Diabetes Mellitus, Type 1; Female; Hydroxyquinolines; Inflammation; Mice; Mice, Inbred NOD; Spleen; Th1 Cells; Th2 Cells

Indoleamine 2,3-dioxygenase (IDO) catalyzes the breakdown of the amino acid tryptophan into kyneurenine. It has been shown that IDO production by placental trophoblasts prevents the attack of maternal T-cells activated in response to the paternal HLA alleles expressed by the tissues of the fetus. In this article, we show that adenoviral gene transfer of IDO to pancreatic islets can sufficiently deplete culture media of tryptophan and consequently inhibit the proliferation of T-cells in vitro. Experiments in vivo have also demonstrated that transplantation of IDO-expressing islets from prediabetic NOD mouse donors into NODscid recipient mice is associated with a prolongation in islet graft survival after adoptive transfer of NOD diabetogenic T-cells. This protection is attributed to the depletion of tryptophan at the transplantation site beneath the kidney capsule. These results suggest that local modulation of tryptophan catabolism may be a means of facilitating islet transplantation as a therapy for type 1 diabetes.

Topics: Adenoviridae; Animals; Cell Division; Concanavalin A; Culture Media; Diabetes Mellitus, Type 1; Female; Gene Transfer Techniques; Graft Survival; In Vitro Techniques; Indoleamine-Pyrrole 2,3,-Dioxygenase; Insulin; Insulin Secretion; Islets of Langerhans; Islets of Langerhans Transplantation; Mice; Mice, Inbred NOD; Spleen; T-Lymphocytes; Time Factors; Tryptophan; Tryptophan Oxygenase

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.

Topics: Animals; Antigens, CD1; Autoantigens; Concanavalin A; Diabetes Mellitus, Type 1; Female; Galactosylceramides; Glutamate Decarboxylase; Immunoglobulin E; Interferon-gamma; Interleukin-4; Killer Cells, Natural; Ligands; Mice; Mice, Inbred NOD; Mice, Inbred Strains; Mice, Mutant Strains; Spleen; Th2 Cells

Interferon regulatory factor-1 (IRF-1), a transcriptional factor, regulates type I interferon and interferon-induced genes. It was reported that IRF-1 regulates important molecules required for inflammation and immune reactions. To investigate the role of IRF-1 in the development of autoimmune diabetes, we established IRF-1 deficient (IRF-1(-/-)) non-obese diabetic (NOD) mice. IRF-1-deficient C57BL/6J mice were out-crossed to NOD mice, and F1 were backcrossed to NOD mice. At the N8 generation, the heterozygote for IRF-1 mutation was intercrossed and N8F1 was obtained. Out of three NOD genotypes, IRF-1(+/+) and IRF-1(+/-) developed spontaneous diabetes with an incidence of 47% (9/19) and 50% (10/20) by 30 weeks of age, respectively; whereas IRF-1(-/-) did not develop diabetes (0/18, P< 0.01 vs. (+/+) and (+/-)). Histologically, IRF-1(+/+) and IRF-1(+/-) had various degrees of insulitis, but IRF-1(-/-) had no insulitis. In comparison with IRF-1(+/+), the percentage of CD4(+) and Mac-1(+) splenic cells significantly increased, whereas CD3(+), CD8(+) and B220(+) cells decreased in IRF-1(-/-). Furthermore, spleen cell proliferation in response to Con A or murine GAD65 peptide, a major autoantigen of the pancreatic beta-cell, significantly increased, and the IFN-gamma/IL-10 ratio in the culture supernatant significantly decreased in IRF-1(-/-), suggesting Th2 deviation in cytokine balance. These results indicate that IRF-1 plays a key role in developing insulitis and diabetes in NOD mice.

Topics: Animals; Cell Division; Concanavalin A; Crosses, Genetic; Diabetes Mellitus, Type 1; DNA-Binding Proteins; Female; Flow Cytometry; Glutamate Decarboxylase; Immune Tolerance; Interferon Regulatory Factor-1; Islets of Langerhans; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Mutant Strains; Microsatellite Repeats; Peptides; Phosphoproteins; Spleen

Diabetic mellitus is attended by the development of endothelial dysfunction which is suggested to be accompanied with a chronic low-degree of inflammation. During a chronic hepatic inflammatory response, specific changes in glycosylation of the acute phase protein alpha1-acid glycoprotein (AGP) can be detected. In this report we studied the changes in glycosylation of AGP in more detail and evaluated the relation between a change in glycosylation of AGP and urinary albumin secretion in Type I diabetic patients. The glycosylation of AGP, studied by crossed affinity immunoelectrophoresis (CAIE) and high pH anion exchange chromatography with pulse amperometric detection (HPAEC-PAD), showed an increase in alpha3-fucosylation. Staining with an antibody against sialyl Lewis(x) (sLe(x)) implied that part of the alpha3-fucosylation was present in a sLe(x)-conformation. In the group of Type I diabetic patients with increased urinary albumin excretion, a significant increase in alpha3-fucosylation of AGP (p<0.0005) could be detected. Therefore, the increased alpha3-fucosylation of AGP can be used as an additional marker for the development of vascular complications in Type I diabetic patients.

Topics: Adolescent; Adult; Aged; Albuminuria; Carbohydrate Conformation; Chromatography, Ion Exchange; Concanavalin A; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Female; Fucose; Fucosyltransferases; Glycosylation; Humans; Inflammation; Lectins; Male; Middle Aged; Oligosaccharides; Orosomucoid; Sialyl Lewis X Antigen; Statistics as Topic

Various fungal products, such as gliotoxin (GT), have immunomodulating activity, a fact exploited previously by our group for prevention of autoimmune diabetes mellitus in BB/Wor rats. To understand better the immunologic effects in GT-treated rats, splenocytes from 65-day-old prediabetic diabetes-prone rats were phenotypically characterized after chronic treatment with GT. A parallel study examined the direct effects of GT on splenocyte preparations incubated with the mycotoxin. In vitro treatment of splenocytes with GT revealed relative decreases in CD4+ and increases in CD8+ T-cell subsets, whereas in vivo treatment with GT did not result in detectable alterations in relative CD4+ and CD8+ cell subsets. We were unable to show significant effects on NK cells or MHC class II cells. However, in vitro and in vivo GT treatments significantly enhanced the detectable RT6 surface marker, a key regulatory element in autoimmune diabetes pathogenesis. This study showed that GT selectively affects certain lymphocyte subsets, possibly through the mechanism of apoptosis, which was increased in vivo as well as in vitro.

Topics: ADP Ribose Transferases; Animals; Antigens, Differentiation, T-Lymphocyte; Apoptosis; CD4-Positive T-Lymphocytes; CD5 Antigens; CD8-Positive T-Lymphocytes; Concanavalin A; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Gliotoxin; Histocompatibility Antigens Class II; Immunosuppressive Agents; In Situ Nick-End Labeling; In Vitro Techniques; Killer Cells, Natural; Male; Membrane Glycoproteins; Rats; Rats, Inbred BB; Spleen

T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.

Topics: Animals; CD28 Antigens; Concanavalin A; Diabetes Mellitus, Type 1; Immunophenotyping; Mitogens; Rats; Rats, Inbred F344; Receptors, Antigen, T-Cell; Receptors, Interleukin-2; Signal Transduction; T-Lymphocytes; Thy-1 Antigens

UK-114 is a 14-kDa ubiquitous protein recently sequenced by several groups throughout the world. Its activity ranges from being a tumor antigen, a protein synthesis inhibitor or a specific mu-calpain activator. UK-114 shows structural homologies also with proteins of the MHC-1 binding proteins, and heat shock proteins (HSPs). We investigated the possible effects of UK-114 on T helper cells cytokine profile and the development and progression of experimental autoimmune diseases. Homogeneous recombinant UK-114 was used in all experiments. Treatment of Balb/c male mice for two weeks resulted in the increase of IL-4, and the decrease of TNF-alpha, IFN-gamma, and IL-2 release from stimulated splenocytes, suggesting that UK-114 modulates the Th1/Th2 cytokine profile toward Th2. Similar to that observed with HSP60/65, a single pretreatment of Lewis rats with UK-114 significantly blunted the development of adjuvant-induced arthritis, whereas chronic treatment of 4-week-old female NOD mice dose dependently inhibited the development of diabetes.

Topics: Animals; Autoimmune Diseases; Concanavalin A; Cytokines; Diabetes Mellitus, Type 1; Female; Interferon-gamma; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Neoplasm Proteins; Rats; Rats, Inbred Lew; Th1 Cells; Th2 Cells; Time Factors

It has been supposed that beta-cell destruction in man and animals is due to autoreactive T-cells. We used the [51Cr]-release assay to identify the presence of beta-cell reactive cells in the spleen of diabetes-prone BB/OK rats before and after diabetes manifestation as well as in long-term normoglycaemic rats with a reduced diabetes risk of 3%. Splenic mononuclear cells (MNCs) obtained from diabetes-resistant LEW.1W and the majority of long-term normoglycaemic BB/OK rats (86.4%) showed no reactivity to pancreatic islets in vitro. In contrast, beta-cell reactive cells were identified in dependence on age in 30.4-65.0% of 75-120 days old normoglycaemic rats and in relation to diabetes duration (1 and 20 days) in 75.0% and 16.0% of diabetic BB/OK rats. Islet antigen-specific stimulation of splenic MNCs, that showed no spontaneous islet-directed reactivity, resulted in a concentration-dependent activation of cytolytically reactive cells in BB/OK but not in LEW.1W rats. Splenic MNCs derived from all diabetic, from 82.4% of young normoglycaemic and from 46.2% of long-term normoglycaemic BB/OK rats developed an islet-directed reactivity in vitro. Phenotyping of MNCs showed a significant increase of activated IL2R+ T-lymphocytes in diabetic BB/OK rats, but without any correlation to their cytolytic potential in the [51Cr]-release assay. Despite this fact, IL2R+ cells enriched from the pool of MNCs mediated an enhanced [51Cr]-release from islets, indicating their relevance in the beta-cell destruction. These data suggest, that functional reactivity rather than phenotypic characterization of MNCs is useful to identify the existence of beta-cell reactive cells. Furthermore, for screening diabetes risk in young normoglycaemic BB/OK rats besides the detection of beta-cell reactive cells the occurrence of regulatory cells seems to be decisive.

Topics: Animals; Cell Division; Cells, Cultured; Concanavalin A; Diabetes Mellitus, Type 1; Female; Islets of Langerhans; Male; Rats; Rats, Inbred BB; Rats, Inbred Lew; Spleen

Previous studies have shown that anti-gamma-interferon (IFN-gamma) antibody reduces the frequency of autoimmune IDDM in the DP-BB rat. We tested the effects of systemically administered recombinant rat IFN-gamma in both DP-BB and DR-BB rats. Unexpectedly, IFN-gamma markedly reduced the incidence of IDDM as compared with control rats when administered six times per week at a dosage of 280,000 U between ages 30-35 to 105 days or ages 60-64 to 105 days. A lower dosage (28,000 U on alternate days) was also protective when administered to DP-BB rats between birth and age 60 days. However, long-lasting protection against IDDM development over the 1-year study period was achieved only by the highest dosage of IFN-gamma administered from age 30 to 105 days. Ex vivo production of tumor necrosis factor-alpha from splenic lymphoid cells (SLCs) and peritoneal macrophages of the rats treated with IFN-gamma was comparable with that of controls; however, SLCs from the IFN-gamma-treated animals secreted lower amounts of IFN-gamma after stimulation with concanavalin A. IFN-gamma treatment also markedly reduced the frequency of phenotypically activated SLC-expressing class II antigens and interleukin-2 receptor. Finally, in agreement with the observed antidiabetogenic effects, exogenously administered IFN-gamma induced neither insulitis nor IDDM development in DR-BB rats, a subline of DP-BB rats in which autoimmune diabetes rarely occurs spontaneously but can be induced by administration of polyinosinic-polycytidilic acid.

Topics: Aging; Animals; Concanavalin A; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Dose-Response Relationship, Drug; Female; Histocompatibility Antigens Class II; Hypoglycemic Agents; Immunosuppressive Agents; Incidence; Injections, Intraperitoneal; Interferon-gamma; Macrophages, Peritoneal; Male; Phenotype; Random Allocation; Rats; Rats, Inbred BB; Receptors, Interleukin-2; Recombinant Proteins; Spleen; Tacrolimus; Tumor Necrosis Factor-alpha

Alpha-interferon (IFN-alpha) is thought to be important in the pathogenesis of insulin dependent diabetes mellitus (IDDM). However, since potent inducers of IFN-alpha, viruses, have been shown to modulate immune function and autoimmunity, we investigated whether administration of recombinant IFN-alpha (rIFN-alpha) would inhibit the diabetic process in BB rats. The development of diabetes was significantly inhibited by injections of either 10(5) units or 4x10(5) units rIFN-alpha. rIFN-alpha was more effective in preventing disease when injections were initiated at an earlier age (28-30 days vs 35-40 days). Histologic examination revealed a markedly lower degree of insulitis in rIFN-alpha treated rats. The mean total peripheral WBC and differential count, T-cell subsets, peripheral blood NK cell number, splenic NK cell activity, and serum cytotoxic beta cell surface antibody levels were unaltered by rIFN-alpha administration. In vitro incubation with rIFN-alpha inhibited the Con A proliferative response of mononuclear splenocytes of BB rats but not of Sprague Dawley rats. These results document that rIFN-alpha treatment potently prevents diabetes by inhibiting the development of insulitis. This paradoxical diabetes sparing effect may have significant implications for the treatment and prevention of IDDM and towards the understanding the autoimmune process.

Topics: Animals; Concanavalin A; Diabetes Mellitus, Type 1; Disease Models, Animal; Interferon Type I; Leukocyte Count; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Rats; Rats, Inbred BB; Rats, Sprague-Dawley; Recombinant Proteins; Spleen

Poly I:C, an inducer of IFN-alpha and other cytokines, has been used to study the development of diabetes in both the BioBreeding (BB) diabetes prone rat and non-obese diabetic (NOD) mouse animal models of insulin-dependent diabetes mellitus (IDDM). Surprisingly, poly I:C accelerates the disease in the BB rat while inhibiting it in the NOD mouse. Since cytokines can have dose related opposing effects on immune responses, we hypothesized that the paradoxical effect of polyinosinic polycytidylic acid (poly I:C) on diabetes in the two animal models is dose related. Accordingly, we compared the incidence of diabetes and degree of insulitis in diabetes prone BB rats administered saline and poly I:C at doses (0.05 microg/g body weight and 0.1 microg/g body weight) up to 100-fold lower than doses (poly-5 microg/g) previously found to accelerate diabetes. In addition, the non-specific suppressor activity of mononuclear splenocytes from BB rats administered low dose (poly-0.05 microg/g body weight), high dose (poly-5 microg/g body weight), and saline were compared. The development of diabetes was inhibited in rats treated with each dose of poly I:C. The degree of insulitis in poly-I:C treated animals was also less severe. The total white blood cell count and proportion of RT6+ T-cells and each T-cell subset were unaltered by poly I:C. When compared to splenocytes of control animals, splenocytes from poly I:C (0.05 microg/g body weight) treated rats suppressed responder cell proliferation to concanavalin A and alloantigen. However, spleen cells from high dose poly-I:C did not suppress responder cell proliferation to alloantigen. In adoptive transfer studies, the administration of spleen cells from poly-0.05 treated rats decreased the development of diabetes in recipient BB rats. In vitro studies also demonstrated that poly-I:C inhibits the proliferative response of BB rat spleen cells to concanavalin A. The administration of poly-0.05, but not poly-5.0, decreased TNF-alpha mRNA and IL-10 mRNA content in spleen cells. We conclude that poly I:C, at a dose 100 times lower than that required to accelerate diabetes prevents the development of diabetes in BB rates by interfering with the development of insulitis. The induction of suppressor cell activity induced by low dose poly-I:C in vivo and the inhibition of T-cell responses by poly-I:C in vitro suggests that the diabetes sparing activity of poly I:C is mediated by augmented immunoregulatory cell activity. Further st

Topics: Animals; Body Weight; Concanavalin A; Cytokines; Diabetes Mellitus, Type 1; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression; Histocompatibility Antigens Class I; Interferon Inducers; Islets of Langerhans; Leukocyte Count; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Phenotype; Poly I-C; Rats; Rats, Inbred BB; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory

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 NOD mouse is a model of human IDDM, which is characterized by a cell-mediated autoimmune process resulting in spontaneous diabetes. Alpha-interferon (IFN-alpha) is thought to play a pathogenic role in this autoimmune process. We report that recombinant alpha-interferon (rIFN-alpha) administration decreases the development of spontaneous diabetes and the passive transfer of diabetes in NOD mice. Spontaneous diabetes was inhibited by IFN-alpha in a dose-dependent fashion. A dose of as little as 20 x 10(3) U inhibited diabetes development, while a dose of 100 x 10(3) U potently prevented diabetes (14% incidence vs. 70% incidence in control mice). Even at the termination of the experiment, nondiabetic mice administered rIFN-alpha maintained normal glucose tolerance. Islet inflammation was 65% lower in the pancreases of rIFN-alpha mice. rIFN-alpha administration decreased anti-islet effector cell bioactivity of spleen cells without inducing generalized immunosuppression. Passive transfer experiments demonstrated that the decreased anti-islet effector cell activity was not a direct action of rIFN-alpha on these cells. In conclusion, rIFN-alpha potently and paradoxically prevents diabetes by indirectly decreasing anti-islet effector cell activity and in turn the development of insulitis without inducing generalized immunosuppression. This work, which goes against our current understanding of the role of rIFN-alpha in autoimmunity, may have significant implications to further our understanding of the pathogenesis of IDDM and to further the development of novel modes to prevent the disease.

Topics: Animals; Antineoplastic Agents; Cell Division; Concanavalin A; Diabetes Mellitus, Type 1; Female; Interferon Type I; Islets of Langerhans; Male; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Recombinant Proteins; Spleen

In order to study cytokine production profile (IFN-gamma, IL-4 and TNF-alpha) and TCRBV-gene usage of peripheral autoreactive T cells from IDDM patients, we have generated antigen-specific T cell lines with either tetanus toxoid, insulinoma membranes or a single beta-cell protein, recombinant ICA69, which has been shown to be a target of both autoantibodies and T cells in IDDM. By semi-quantitative polymerase chain reaction (PCR) analysis, we have determined the composition of the T cell receptor repertoire of these T cell lines and compared this with the general peripheral repertoire. T cell responses against beta-cell antigens and tetanus toxoid (TT) were shown to be associated with IFN-gamma and TNF-alpha production, suggestive of a Th1-like phenotype of the T-cell lines. The production of IFN-gamma was significantly higher in T-cell lines generated with ISG compared to those generated with TT. The cytokine production profiles of the T-cell lines generated with ICA69 did not provide an obvious explanation for the inverse relation between cellular and humoral responses to this protein observed earlier. Upon stimulation with beta-cell antigens, outgrowth of T cells using a restricted set of TCRBV elements was observed in newly diagnosed IDDM patients. However, this skewing in TCRBV-gene expression was patient-specific rather than antigen-associated, since the T-cell repertoire that is used for the recognition of these antigens was, overall, heterogeneous.

Topics: Adolescent; Antigens; Autoantigens; Child; Child, Preschool; Concanavalin A; Cytokines; Diabetes Mellitus, Type 1; Female; Gene Expression; Genes, T-Cell Receptor beta; Humans; Immunoglobulin Variable Region; Individuality; Insulin; Insulin Secretion; Islets of Langerhans; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Receptors, Antigen, T-Cell, alpha-beta; Stimulation, Chemical; T-Lymphocytes; Tetanus Toxin; Th1 Cells

We studied the effect of peripheral blood mononuclear cells (PBMNC) from insulin-dependent diabetic (IDDM) children on the insulin secretion pattern of the pancreas from recipient athymic mice. PBMNC from healthy controls or IDDM patients in different stages of disease were injected into athymic mice. PBMNC from newly diagnosed IDDM children elicited basal nonfasting hyperglycemia and in vitro inhibition of the first and second phases of glucose-stimulated insulin secretion in recipient mice. Animals injected with cells from chronically IDDM children showed normoglycemia, abnormal tolerance to glucose, and inhibition of first-phase insulin secretion. Mitomycin C treatment of MNC from IDDM patients abolished insulin secretion inhibition in recipient mice. PBMNC from newly diagnosed and chronically IDDM patients showed positive anti-beta-cell cellular immune aggression. Mice injected with cells from patients during the remission period showed normoglycemia and no alteration of insulin secretion patterns. When relapsed to their former clinical stage, injection of the cells significantly inhibited first-phase glucose-induced insulin secretion in recipients. PBMNC from newly diagnosed IDDM patients were found to migrate to the pancreas of recipient mice preferably as compared with cells from controls. Cells from chronically IDDM patients cultured with concanavalin A (Con A) increased insulin secretion inhibition; despite this, cells from children during the remission period cultured with Con A failed to modify insulin secretion in recipients. These results show that injection of PBMNC from diabetic patients leads to insulin secretion impairment in recipient mice pancreas, and provide a basis for the study of mechanisms involved in the onset and modulation of anti-beta-cell cellular immune aggression induced by human PBMNC.

Topics: Adolescent; Animals; Antibiotics, Antineoplastic; Cell Transplantation; Cells, Cultured; Child; Child, Preschool; Concanavalin A; Diabetes Mellitus, Type 1; Female; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitomycin; Monocytes; Pancreas; Radioimmunoassay; Rats; Rats, Wistar

Inbred diabetes-prone (DP) BioBreeding/Worcester (BB/Wor) (RT1u) rats develop spontaneous autoimmune diabetes, which, like human insulin-dependent diabetes mellitus, is mediated by autoreactive T lymphocytes. Breeding studies have shown an absolute requirement for at least one copy of the major histocompatibility complex (MHC) RT1u haplotype for spontaneous diabetes expression. Concanavalin A-activated spleen cells from acutely diabetic DP rats adoptively transfer diabetes only to recipients that express at least one RT1u haplotype. To investigate the basis for the MHC requirement in BB/Wor autoimmunity, diabetes-inducing T cell lines were derived from the spleens of acutely diabetic DP rats. Upon activation in vitro with islet cells, the T cell lines adoptively transfer insulitis and diabetes into young DP recipients and non-diabetes-prone RT1 congenic rat strains that are class IIu. Recipients that are RT1u at only the class I A or C locus, but not at the class II B/D loci, do not develop diabetes after T cell transfer. The adoptive transfer of diabetes by Concanavalin A-activated diabetic DP spleen cells also requires that donor and recipient share class II B/Du gene products. Furthermore, the adoptive transfer of diabetes into MHC class IIu congenic rats is independent of the class I haplotype; i.e., it occurs in the presence of class I Aa Cu or Au Ca gene products. BB/Wor T cells can be activated in vitro for the transfer of diabetes with islet cell antigens and class II-positive but not class IIu-negative antigen-presenting cells. The inductive phase of BB diabetes is therefore MHC class II restricted, and this appears to operate at the level of interaction between inducing T cells and class IIu antigen-presenting cells. These results may explain the well-documented, but not yet understood, MHC class II genetic contribution to insulin-dependent diabetes mellitus pathogenesis, and they may facilitate the development of protocols designed to prevent diabetes onset in susceptible individuals.

Topics: Animals; Antigen-Presenting Cells; Breeding; Cell Line; Concanavalin A; Diabetes Mellitus, Type 1; Disease Models, Animal; Genes, MHC Class II; Haplotypes; Immunotherapy, Adoptive; Islets of Langerhans; Lymphocyte Activation; Rats; Spleen; T-Lymphocytes

Topics: Amino Acid Oxidoreductases; Animals; Concanavalin A; Diabetes Mellitus, Type 1; Enzyme Induction; Guanidines; Immunosuppressive Agents; Islets of Langerhans; Islets of Langerhans Transplantation; Lymphocyte Activation; Macrophages; Nitric Oxide; Nitric Oxide Synthase; Polymerase Chain Reaction; Rats; Rats, Inbred BB; Rats, Inbred F344; RNA, Messenger; Spleen; T-Lymphocytes

Splenic cells from the diabetes-prone BB rat show reduced proliferative responses to concanavalin A (ConA) and other mitogens. This study was undertaken to test whether this reduced lymphoproliferation in the BB rat is mediated by an increased production of nitric oxide (NO) by macrophages. Splenic leukocytes from diabetes-prone BB rats and five strains of control rats (BB-R, Wistar-Furth, Sprague-Dawley, Wistar, and Lewis) were cultured in RPMI-1640 media containing ConA. The leukocytes from BB rats showed reduced [3H]thymidine uptake and increased release of NO compared with the control rats. Partial depletion of macrophages from the culture or incubation with -NG-monomethylarginine (NGMMA), a specific NO synthase inhibitor, markedly augmented ConA-induced proliferation of the splenic leukocytes from BB but not the control rats. Enrichment of BB rat macrophages suppressed the proliferation of BB-R rat spleen cells. Excess L-arginine added to the culture reversed the NGMMA effect. These results suggest that increased production of NO by macrophages is partly responsible for the reduced proliferative responses of splenic leukocytes in the BB rat.

Topics: Analysis of Variance; Animals; Arginine; Cells, Cultured; Concanavalin A; Diabetes Mellitus, Type 1; Lymphocyte Activation; Lymphocytes; Macrophages; Nitric Oxide; omega-N-Methylarginine; Rats; Rats, Inbred BB; Rats, Inbred Lew; Rats, Inbred WF; Rats, Sprague-Dawley; Rats, Wistar; Species Specificity; Spleen

The IL-2 system which involves IL-2 production, IL-2 receptor expression, and response to IL-2, is associated with autoimmune phenomena. Immunological abnormalities including autoimmune phenomena are believed to contribute to the pathogenesis of IDDM. In this study, the production of IL-2, the responses to IL-2 and IL-2 receptor expression by peripheral blood T lymphocytes were compared in IDDM and normal non-diabetic children. The percentage of IL-2 receptor-positive circulating T cells was significantly increased in diabetic children, although IL-2 receptor expression induced by con A stimulation did not differ in the diabetic and control children. IL-2 production was significantly decreased in diabetic children compared with the control children. The response of stimulated T cells to IL-2 did not differ in IDDM and control children. In IDDM, IL-2 production by CD4-positive T lymphocytes within the IL-2 system is thought to be selectively defective. On the other hand, IL-4, which is also produced by CD4-positive T lymphocytes, was increased. Since IL-4 did not suppress IL-2 production, it would seem that the IL-2 producing subset in CD4+HLA-DR+ T cells is decreased in IDDM. These results suggest that in recent onset IDDM, IL-2 receptor-positive circulating T cells require an IL-2 supply.

Topics: Adolescent; Antigens, CD; Cells, Cultured; Child; Child, Preschool; Concanavalin A; Diabetes Mellitus, Type 1; Female; Humans; Interferon-gamma; Interleukin-2; Kinetics; Lymphocyte Activation; Male; Receptors, Interleukin-2; Reference Values; T-Lymphocyte Subsets; T-Lymphocytes

Diabetes-prone (DP) BB rats spontaneously develop a hyperglycaemic condition which closely resembles human insulin-dependent diabetes mellitus (IDDM), both in terms of clinical and histological features. The incidence of IDDM was significantly reduced when these animals were treated with 2 or 4 mg fusidic acid (FA)/day i.m. from day 30 to day 120 of age. In addition, the mean insulitis score was significantly diminished in the animals treated with FA compared to both vehicle-treated and untreated controls. Finally, 2 mg/day of FA i.m. prevented cell proliferation and interferon-gamma secretion from peripheral blood mononuclear cells upon ex vivo stimulation with concanavalin A. The capacity of FA to substantially reduce the incidence of autoimmune diabetes in a well-known animal model of human IDDM supports previous observations regarding the immunosuppressive properties of FA and its potential use in the treatment of human autoimmune diabetes.

Topics: Age Factors; Animals; Autoimmune Diseases; Cell Division; Cells, Cultured; Concanavalin A; Diabetes Mellitus, Type 1; Female; Fusidic Acid; Interferon-gamma; Islets of Langerhans; Male; Rats; Rats, Inbred BB; T-Lymphocytes

Insulin-dependent diabetes mellitus is an autoimmune disease that is characterized by the destruction of insulin-producing beta cells in the islet of Langerhans. We have recently reported that the induction of the disease in nonobese diabetic (NOD) mice can be prevented by a single injection of CFA. In this study, we have explored the cellular basis and the time course of the disease protection. Since CFA contains a mycobacterial cell wall that has adjuvant property, we investigated the protective role of mycobacteria in young NOD mice. Mice injected with Mycobacterium tuberculosis or Mycobacterium bovis (BCG vaccine) at 4 wk of age were also found to be protected from diabetes. We have found that complete protection from diabetes is only achieved by administration of CFA between 4 and 10 wk of age. Draining lymph node cells or spleen cells from CFA-treated NOD mice transfer the protection. Adoptive transfer of spleen cells from CFA-treated mice with spleen cells from acutely diabetic mice delayed the induction of disease into irradiated recipient mice. CFA-treated old NOD mice were also resistant to passive transfer of disease by spleen cells from acutely diabetic mice. Depletion of the Thy 1.2+ cells or CD4(+)-bearing T cells abrogated the protection. However, disease can be induced in the protected mice by cyclophosphamide treatment. We also found that thymocytes from NOD mice responded only weakly to mitogen Con A. CFA treatment, however, restored the ability of these cells to respond to Con A. Finally, our results suggest that T cells induced after CFA treatment of NOD mice prevent both the induction and effector phases of the disease.

Topics: Animals; Concanavalin A; Cyclophosphamide; Diabetes Mellitus, Type 1; Female; Freund's Adjuvant; Immunotherapy, Adoptive; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Phenotype; Spleen; T-Lymphocytes

In a comparative study congenic rat strains bearing either the genetic background of diabetic BB/OK rats and the MHC (RTla) of diabetes-resistant LEW.1A rats (BB.1A/OK) or vice versa carrying the genetic background of LEW.1A rats in combination with the MHC (RTlu) of diabetic BB/OK rats (LEW.1BB/OK) and their parental rat strains BB/OK and LEW.1A were checked for insulin secretion of pancreatic islets, for the number of splenic and peripheral blood mononuclear cells (MNC) as well as for the mitogenic response of splenic MNC. Glucose stimulated insulin secretion of isolated islets of Langerhans was not different in 50, 70 and 100 day-old congenic rats and the progenitor rat strains excluding an impact of the genotype on this beta-cell-specific function. The number of splenic and peripheral blood MNC was reduced in BB/OK and BB.1A/OK rats compared to LEW.1A and LEW.1BB/OK rats. Splenic MNC from BB/OK and BB.1A/OK rats displayed a strongly decreased total [3H]thymidine incorporation under basal conditions as well as upon mitogenic stimulation by ConA in comparison with MNC from LEW.1A and LEW.1BB/OK rats. Thus, the occurrence of lymphopenia and the impairment of mononuclear cell proliferation in BB/OK rats is not related to the RTlu haplotype of the MHC but is linked to non-MHC genes as indicated by the phenotypic expression of these traits in congenic BB.1A/OK rats.

Topics: Animals; Concanavalin A; Crosses, Genetic; Diabetes Mellitus, Type 1; Female; Glucose; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Lymphocyte Activation; Lymphocytes; Male; Rats; Rats, Inbred BB; Rats, Inbred Lew; Species Specificity; Spleen; Thymidine

We studied the effects of anti-CD2 monoclonal antibodies (MAb) on spontaneous and induced autoimmune diabetes mellitus in diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats. In DP rats, all anti-CD2 MAb prevented spontaneous diabetes and the adoptive transfer of diabetes with Con-A--stimulated acute diabetic spleen cells; OX34 prevented Poly I:C induced accelerated onset of diabetes and the adoptive transfer of diabetes with Con-A--stimulated RT6.1+ T cell depleted DR splenocytes. In DP rats, all anti-CD2 MAb except OX53 depleted CD4+ T cells, without depleting natural killer cells or CD8+ T cells. OX34 injected DR rats were profoundly depleted of CD4+ T cells without evidence of decreased CD8+ T cells, but were not protected against the induction of diabetes by RT6.1+ T-cell depletion and Poly I:C injections. We conclude that anti-CD2 MAbs protect against BB/Wor autoimmune diabetes by depleting CD4+ T cells, preventing the activation of effector cells, or by blocking CD2/ligand interaction between effector and target cells.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Autoimmune Diseases; CD2 Antigens; CD4 Antigens; CD8 Antigens; Concanavalin A; Diabetes Mellitus, Type 1; Flow Cytometry; Hyperinsulinism; Immunotherapy, Adoptive; Injections; Lymph Nodes; Poly I-C; Rats; Rats, Inbred BB; Receptors, Immunologic; Spleen; T-Lymphocytes

In most studies the activity of suppressor T cells and the percentage of suppressor/cytotoxic T-lymphocyte subsets in type 1 diabetes have been found to be altered. To determine whether thymosin and insulin in vitro have a role in improving or normalizing these abnormalities, PBMC from 28 patients with type 1 diabetes of various durations were treated with thymosin or insulin and the activity and the percentage of suppressor T cells were detected by using the method of ConA-induced suppressor T cells and WuT8 (suppressor/cytotoxic) monoclonal antibody respectively. Both thymosin and insulin were found to have ability to improve and normalize the ConA-induced suppressor cell activity and the percentage of WuT8 cells in diabetic patients. Data have shown that the lower the activity and the percentage of suppressor T cells, the more intense the effects of both compounds. The strongest effects were found at the concentrations of 10 micrograms/ml thymosin and 10 ng/ml insulin. Thymosin was more effective than insulin. This experiment also suggested that the activated lymphocytes stimulated by mitogens (PHA or ConA) were required when insulin exerted a significant effect on suppressor T cells. We conclude that thymosin and insulin in vitro can exert immuno-regulatory or immunostimulating effects on suppressor T cells.

Topics: Adult; Cells, Cultured; Concanavalin A; Diabetes Mellitus, Type 1; Female; Humans; Insulin; Lymphocyte Activation; Male; Reference Values; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Thymosin; Time Factors

We have recently reported that systemic and chronic administration of recombinant tumour necrosis factor alpha (TNF-alpha), as well as streptococcal preparation (OK-432), inhibits development of insulin-dependent diabetes mellitus (IDDM) in NOD mice and BB rats, models of IDDM. In this study we examined whether serum containing endogenous TNF induced by OK-432 injection could inhibit IDDM in NOD mice. Treatment twice a week from 4 weeks of age with OK-432-injected mouse serum, which contained endogenous TNF (75U), but not IL-1, IL-2 and interferon-gamma (IFN-gamma) activity, reduced the intensity of insulitis and significantly inhibited the cumulative incidence of diabetes by 28 weeks of age in NOD mice, as compared with the incidence in non-treated mice (P less than 0.01) and in mice treated with control serum (P less than 0.02). This inhibitory effect of the serum was diminished, although not significantly, by neutralization of serum TNF activity with anti-mouse TNF antibody. In the mice treated with the serum from OK-432-injected mice, Thy-1.2+ or CD8+ spleen cells decreased (P less than 0.01) and surface-Ig+ (S-Ig+) cells increased (P less than 0.05), whereas the proliferative response of spleen cells to concanavalin A (P less than 0.01) and lipopolysaccharide (P less than 0.05) increased. The results indicate that the inhibition by OK-432 treatment of IDDM in NOD mice was partially mediated by serum factors including endogenous TNF.

Topics: Animals; Concanavalin A; Diabetes Mellitus, Type 1; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immune Sera; Immunization, Passive; Injections, Intravenous; Interferon-gamma; Interleukin-1; Interleukin-2; Lipopolysaccharides; Lymphocyte Subsets; Mice; Mice, Inbred BALB C; Mice, Inbred NOD; Picibanil; Spleen; Tumor Necrosis Factor-alpha

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

We studied the tracking of 51chromium-labeled (resting) and Con A-induced (activated) tritiated thymidine-labeled syngeneic lymphocytes to adrenal, blood, brain, heart, liver, lymph nodes, pancreas, spleen, testis/ovary, thymus and thyroid in prediabetic, nonobese diabetic (NOD) mice and in age- and sex-matched C57BL/6 mice. 51Chromium-labeled cells showed no significant difference between strains in tracking to any tissue except lymph nodes (decreased in NOD vs. C57, P less than .05). Con A incubation resulted in no difference between strains in lymphocyte tracking to lymph nodes, but NOD mice had increased pancreatic tracking with Con A-incubated cells compared to C57 mice (P less than .05). Female NOD mice had reduced thymic tracking (P = .001) compared to C57 controls. Positive selection experiments showed the Con A-responsive cell to be a T cell. Both Lyt2+ (CD8+) and L3T4+ (CD4+) enriched T cell populations showed a labeling response to Con A. After 48 h of Con A incubation, the L3T4+/Lyt2+ ratio increased in splenocytes from NOD mice (P less than .05), whereas it decreased in C57 controls (P less than .01). Over the course of 6 days in culture, NOD splenocytes exposed to Con A characteristically exhibited a delayed expansion of the Lyt2+ population. We conclude that Con A incubation of NOD splenocytes results in T cells that, when reinfused, avoid the thymus and track preferentially to the pancreas. Con A immunomodulation as potential treatment for autoimmune disease warrants further study in this murine model.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Antigens, Ly; Cell Movement; Concanavalin A; Diabetes Mellitus, Type 1; Female; Lymphocytes; Male; Mice; Mice, Inbred C57BL; Organ Size; Species Specificity

Concanavalin A (Con A) administered in vivo inhibited the development of insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic mice. By experimental day 310 only one of 10 Con A-treated animals had developed IDDM compared to six of 11 cohort controls receiving buffered saline and 65% of colony controls. Treatment consisted of an initial 70-day course of Con A at a dose of 10 micrograms/g b. wt., i.p. in sterile saline every other day. After two deaths occurred in the Con A group, treatment was stopped at day 70. The remaining animals were given two 1-week booster doses beginning on experimental days 118 and 189. The single animal in the Con A group that eventually developed IDDM at day 293 had not received an injection for 107 days. Histological examination revealed peri-islet lymphocytosis but no islet infiltration in the Con A group. Surviving control animals showed both peri-islet and islet infiltrates. One-week Con A treatment significantly suppressed in vitro responses of spleen cells to Con A and allogeneic lymphocytes. The treatment increased the frequency of "blast-sized" cells in vivo and decreased the CD4+/CD8+ ratio among resting splenocytes. It is concluded that polyclonal T cell activation by Con A provides protection against autoimmune diabetes in nonobese diabetic mice concomitant with phenotypic and morphologic lymphocyte changes.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Blood Glucose; CD4 Antigens; CD8 Antigens; Concanavalin A; Diabetes Mellitus, Type 1; Female; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL; Pancreas; Phenotype

The immune abnormalities of NOD mice, a model of human type I (insulin-dependent) diabetes, have been postulated to be T-lymphocyte dependent. We measured responsiveness to exogenous interleukin 2 (IL-2) and IL-2 production in spleen mononuclear cells from female NOD/Shi/Kbe mice after stimulating the cells with concanavalin A (ConA blasts) or phytohemagglutinin (PHA blasts). Exogenous IL-2 produced significantly lower proliferative responses in each blast from 3- and 10-wk-old NOD/Shi/Kbe mice than from control strains. IL-2 production in NOD/Shi/Kbe mice was inclined to decrease but not significantly compared with controls. Even sufficient amounts of recombinant IL-2 (rIL-2) or IL-1 (rIL-1), added with mitogens to the preculture medium, failed to provoke normal proliferative responses from NOD/Shi/Kbe mouse cells. To clarify the reason for this defect, we investigated the expression of IL-2 receptors (IL-2Rs) on mitogen-activated cells with anti-IL-2R monoclonal antibody (PC61) and radiolabeled IL-2. Cytofluorometry showed no significant difference between strains in the number of PC61+ ConA and PHA blasts. However, Scatchard analysis with 125I-labeled IL-2 showed that the number of high-affinity IL-2Rs (H-IL-2Rs), the mediators of the biological activity of IL-2, was decreased in NOD/Shi/Kbe mice compared with controls, whereas the number of low-affinity IL-2Rs (L-IL-2Rs) was not different. Separating the L3T4+ and Lyt-2+ populations of T lymphocytes by cell sorting showed both to be deficient in H-IL-2Rs.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Concanavalin A; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Female; Flow Cytometry; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred Strains; Mitogens; Monocytes; Phytohemagglutinins; Receptors, Interleukin-2; Spleen; T-Lymphocytes

It is generally accepted that T lymphocyte-mediated autoimmunity contributes to the pathogenesis of Type 1 diabetes in humans and animals. Using spleen cells from nonobese diabetic (NOD) mice, a model of human Type 1 diabetes, we have analyzed the subset of T lymphocytes by flow cytometry and investigated concanavalin A (Con A)-induced interleukin 2 (IL-2) production and cell proliferation. NOD mice showed a higher percentage of Thy1.2+, L3T4+, and Lyt2+ T lymphocytes than did control ICR mice through the whole age examined. Spleen cells from a large majority of NOD mice were found to generate very low IL-2 production and cell proliferation in response to Con A. However, a few mice preserved their responsiveness to Con A. The following reasons may indicate that macrophage-mediated suppression participates in the deficient function of NOD spleen cells. (a) Macrophage depletion from NOD spleen cells retrieved Con A-induced IL-2 production. (b) Thioglycollate-induced peritoneal exudate cells containing many activated macrophages could completely suppress cell proliferation. (c) Prostaglandin synthetase inhibitor indomethacin reversed the suppression of IL-2 production by macrophages. (d) Conversely, exogenous prostaglandins could show the partial suppression of IL-2 production. These results suggest that activated macrophages suppress the response of NOD spleen cells to Con A mostly through prostaglandins. This impairment may contribute to the pathogenesis of Type 1 diabetes in NOD mice.

Topics: Animals; Cell Division; Concanavalin A; Diabetes Mellitus, Type 1; Female; Immunity, Cellular; Immunosuppression Therapy; Indomethacin; Interleukin-2; Leukocyte Count; Macrophage Activation; Mice; Mice, Inbred Strains; Prostaglandins; Spleen; T-Lymphocytes

The biological basis for autoimmunity and immunoincompetence in the BB rat has yet to be localized. In spite of normal thymic histology, thymocyte subsets and blastogenesis, thymus gland products (thymosins) have yet to be studied. In the present report, thymus gland function was studied by measuring thymosin alpha 1 levels at one time point in the BB rat compared with control rates, and BB rat responses to exogenous thymosin (Thymosin fraction 5) were observed. At five months of age, BB rats had thymosin alpha 1 levels comparable to Lewis and Wistar furth rats. Thymosin fraction 5 increased the ratio of peripheral blood W3/25 positive to OX8 positive cells, but otherwise had no effect on the BB rats' T-cell immunodeficiency, or frequencies of tissue autoantibodies or insulin-dependent diabetes. Although B-lymphocyte counts were normal in BB rats, splenocyte responses to B-lymphocyte mitogens were depressed. However, thymosin fraction 5 improved the BB rat B-lymphocyte blastogenesis to near normal for Mycoplasma neurolyticum. Coupled with our previous work, our results suggest that the immune derangement in the BB rat resides outside the thymus.

Topics: Animals; Autoantibodies; Bacterial Toxins; Concanavalin A; Diabetes Mellitus, Type 1; Disease Models, Animal; Leukocytes; Lymphocyte Activation; Mycoplasma; Rats; Rats, Inbred BB; Rats, Inbred Lew; Rats, Inbred WF; Thymalfasin; Thymosin; Thymus Gland

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Interleukin-2; Islets of Langerhans; Lipopolysaccharides; Lymphocyte Activation; Lymphocytes; Mice; Mice, Mutant Strains; Receptors, Immunologic; Receptors, Interleukin-2

BB rats are prone to develop an autoimmune form of insulin-dependent diabetes mellitus (IDDM) and thyroiditis. Development of autoimmunity is thymus dependent. Previous studies have shown that BB rats lack a population of T cells bearing the RT6 antigen and have very low numbers of suppressor/cytotoxic T cells. In this study, we confirm that BB rats have decreased numbers of phenotypic T suppressor/cytotoxic (Ts/c) cells (OX19+, OX8+ cells) in their lymphoid organs. Moreover, we find that the phenotypic Ts/c cells of BB rats lack apparent cytotoxic activity. These T cells fail to kill allogeneic target cells in a cell-mediated lympholysis assay and fail to generate lectin-dependent cytotoxicity. The addition of interleukin 2, gamma-interferon, and other lymphokines to cultures of BB T cells does not induce functional cytotoxic T lymphocytes. We find that the activated T cells of newly diabetic rats are incapable of killing major-histocompatibility-complex-matched islet cells, despite the ability of these cells to cause IDDM in passive transfer experiments. We conclude that autoimmune disease occurs in BB rats in the absence of functional cytotoxic T cells.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Autoimmune Diseases; Concanavalin A; Diabetes Mellitus, Type 1; Hypersensitivity, Delayed; Interferon-gamma; Interleukin-2; Islets of Langerhans; Lymphocyte Activation; Lymphoid Tissue; Rats; Rats, Mutant Strains; T-Lymphocytes, Cytotoxic

Spontaneous diabetes was fully prevented in 65 BB/hooded (BB/h) highly diabetes-prone hybrid rats that were given five intraperitoneal injections (25 to 30 x 10(6) cells/injection) of fresh splenocytes or concanavalin A (ConA)-activated cultured splenocytes (blasts) from the diabetes-free Wistar-Furth or Long-Evans strains during the first 2 postnatal wk. Rats remained under observation for up to the age of 180-200 days. Of 70 littermate controls that received no cell injections, 63 developed overt diabetes before the age of 180 days. One intraperitoneal injection (25 x 10(6) cells) of splenocytes or blasts given during the first 36 h after birth was not as effective as multiple injections in preventing overt diabetes. Mild insulitis was present in 4 of 59 "protected" rats; small, discrete mononuclear infiltrates in periductular connective tissue and/or between pancreatic acini were observed in 27. Nondiabetic BB/h rats that were protected with splenocytes or blasts from diabetes-free strains had the same degree of lymphopenia in peripheral blood and spleen as age-matched, insulin-treated diabetic BB/h rats, but the level of islet cell surface antibodies in their serum was significantly lower. The same neonatal injections that protected rats from the development of spontaneous diabetes were completely ineffective in preventing the adoptive transfer of diabetes later in life by the injection of blasts from acutely diabetic BB/h rats.

Topics: Acute Disease; Animals; Animals, Newborn; Autoantibodies; Concanavalin A; Diabetes Mellitus, Type 1; Lymphopenia; Rats; Rats, Inbred BB; Rats, Inbred Strains; Rats, Inbred WF; Spleen

Topics: Adolescent; Child; Child, Preschool; Concanavalin A; Diabetes Mellitus, Type 1; Female; Humans; Interleukin-2; Male; Receptors, Immunologic; Receptors, Interleukin-2

Several immunological responses of the spontaneously diabetic BB rat colony in Edinburgh designated (BB/E) have been studied. The proliferative responses to Con A and LPS, ability to make IL-2 and to show NK activity have been studied using diabetic and non-diabetic BB/E rats and normal Wistar rats. Our data suggest that the diabetic animals in the BB/E colony do not have marked deficiencies in any of these parameters. Lymphopenia and depressed T-cell responses do not appear to be a prerequisite for the development of diabetes in the BB/E colony.

Topics: Animals; Concanavalin A; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Disease Models, Animal; Interleukin-2; Killer Cells, Natural; Lipopolysaccharides; Lymphocyte Activation; Rats; Rats, Inbred BB; Spleen

Five Persian Jews were detected with the polyglandular deficiency syndrome (PDS). Primary hypoparathyroidism and hypogonadism were present in each, adrenal insufficiency in two, and insulin-dependent diabetes mellitus and latent hypothyroidism in single subjects. The percentage of T and B cells, and the mononuclear cell response to phytohemagglutinin and Concanavalin A were normal in all five. IgG and IgA levels and the OKT4+/OKT8+ cell ratio were low in one subject. Antinuclear and antithyroid antibodies were present in one subject. HLA-DR5 was present in 4/4, HLA-24 and B5 (B51) in 3/4 subjects. A single case of isolated hypoparathyroidism (IHP) was detected among 12 first degree relatives. HLA antigens B8, DR3, were absent in all of these subjects. Seven non-Iranian Jews with IHP were also examined. HLA A26 or A25 were present in all seven. Persian Jews appear to have a unique variant of PDS.

Topics: Adolescent; Adrenal Insufficiency; Adult; Antibodies, Antinuclear; B-Lymphocytes; Concanavalin A; Diabetes Mellitus, Type 1; Female; HLA Antigens; Humans; Hypogonadism; Hypoparathyroidism; Hypothyroidism; Immunoglobulin A; Immunoglobulin G; Iran; Jews; Lymphocyte Activation; Male; Monocytes; Phytohemagglutinins; Syndrome; T-Lymphocytes

Peripheral blood lymphocytes from 13 patients with established insulin-dependent diabetes mellitus (IDDM) and 2 prediabetic patients were examined for natural killer (NK) and antibody-dependent cellular cytotoxic activities (ADCC), lectin-dependent cellular cytotoxicity (LDCC), interferon- and interleukin-2-induced cytotoxicity, and concanavalin A-induced suppressor-cell activities in comparison with age-matched normal controls. IDDM patients demonstrated normal levels of NK and ADCC activities against K562 and antibody-coated SB target cells, respectively, compared to controls. IDDM patients showed normal levels of LDCC activity. Notable deviations from control values were, however, observed with diabetic lymphocytes in the following systems. Interferon- and interleukin-2-induced NK activities were significantly higher with IDDM lymphocytes than with control cells. IDDM lymphocytes precultured with concanavalin A demonstrated lower NK and ADCC activities than control cells and manifested decreased suppressor effects on the NK activity of normal allogeneic lymphocytes. Lymphocytes from one of two prediabetic patients showed increased NK, ADCC, and LDCC activities in comparison to controls. The increased interferon- and interleukin-2-induced enhancement of NK activity and reduced suppressor activity of lymphocytes from IDDM patients may be involved in the pathogenesis of the disease.

Topics: Antibody-Dependent Cell Cytotoxicity; Concanavalin A; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Female; Glucose; Humans; Insulin; Interferons; Interleukin-2; Killer Cells, Natural; Lectins; Lymphocyte Activation; Male; T-Lymphocytes, Regulatory

Peripheral blood mononuclear cells from patients with Type 1 diabetes mellitus were examined for the proportion of monoclonal antibody-defined T-cell subsets, natural killer (NK) cells, and macrophages and the proliferative response to phytohemagglutinin (PHA), Concanavalin A (Con A), pokeweed mitogen (PWM) and in the autologous mixed lymphocyte reaction (AMLR) and allogeneic mixed lymphocyte reaction (MLR). The in vitro response of purified IL-2 on PHA- and PWM-induced proliferative response was also examined. Total T cells (Leu 1+), helper/inducer phenotype (Leu 3+) T cells, suppressor/cytotoxic phenotype (Leu 2+) T cells, surface Ig+ B lymphocytes and monoclonal antibody-defined monocytes (Mac +) in patient group were comparable to the control group. The Leu 7+ NK cells were, however significantly (p less than 0.05) decreased in the diabetic group. The NK function was also deficient in the diabetic group when compared to healthy non-diabetic controls. The proliferative responses to all 3 concentrations of PHA, PWM, and Con A, and in the MLR were similar in 2 groups. However, the proliferative response in the AMLR was significantly reduced (p less than 0.05) in the diabetic group. Exogenous purified IL-2 failed to induce any enhancement in the PHA- and PWM-induced proliferative response; this was in contrast to control group in which IL-2 enhanced proliferative response to both mitogens. This study demonstrates deficiency of the AMLR, Leu 7+ and of natural killer cell function and unresponsiveness of mitogen-activated T cells to purified IL-2. The significance of these findings is discussed.

Topics: Adolescent; Adult; B-Lymphocytes; Cell Division; Child; Concanavalin A; Diabetes Mellitus, Type 1; Female; Humans; Interleukin-2; Killer Cells, Natural; Lymphocyte Culture Test, Mixed; Macrophages; Male; Phytohemagglutinins; Pokeweed Mitogens; T-Lymphocytes

Impairment of suppressor-cell activity may be important in the pathogenesis and maintenance of insulin-dependent diabetes mellitus (IDDM). In 23 recent-onset IDDM patients, lymphocyte sensitivity in vitro to theophylline was tested both in basal conditions and after improvement of metabolic control. This pharmacologic agent is mainly effective on a lymphocytic subpopulation with phenotypic and functional suppressive features. Peripheral blood lymphocytes from IDDM patients showed a loss of theophylline sensitivity, identified as inhibition of both E-rosette formation and blastogenic response to polyclonal mitogens concanavalin A (ConA) and phytohemagglutinin (PHA). An inverse relationship was demonstrated between the theophylline-induced suppression of ConA blastogenic response and blood glucose and glycosylated hemoglobin levels (P less than .01). Metabolic control seemed to be important even in relation to lymphocyte subpopulation distribution. In IDDM patients we found a significant (P less than .05) reduction of OKT4+ lymphocytes that is correlated with blood glucose and glycosylated hemoglobin levels (P less than .01). The improvement of metabolic control led to recovery of theophylline sensitivity. We suggest a deficiency in a suppressive system that could be involved in IDDM onset and the possible role of metabolic control in the impairment of some immunologic functions reported with this pathologic condition.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Antigens, Surface; Blood Glucose; Concanavalin A; Diabetes Mellitus, Type 1; Female; Glycated Hemoglobin; Humans; Lymphocytes; Male; Phytohemagglutinins; Rosette Formation; T-Lymphocytes, Regulatory; Theophylline

The concentration and degree of glycosylation of serum ferritin was evaluated in type I male diabetic patients at different levels of glycaemic control. Serum ferritin did not appear to be affected by hyperglycaemia, but some patients undergoing photocoagulation had abnormally high levels of serum ferritin. The glycosylated, (concanavalin A binding), proportion of serum ferritin was essentially the same in the control and diabetic groups. The finding that hyperglycaemia does not affect the degree of enzymatic glycosylation of this serum protein is discussed.

Topics: Adolescent; Adult; Carbohydrates; Concanavalin A; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Ferritins; Glycated Hemoglobin; Humans; Male; Middle Aged; Phototherapy

Injections of media conditioned by concanavalin A-activated spleen cells from acutely diabetic rats accelerated the appearance of diabetes in young Bio-Breeding/Worcester (BB/W) rats. Activity was also found in media conditioned by spleen cells from nondiabetic, W-line Wistar Furth and Buffalo rats. Unconditioned media containing mitogen had no activity. Conditioned media also induced diabetes in resistant W-line BB/W rats but not in Wistar Furth rats. A soluble factor may activate a BB lymphocyte population that promotes diabetes.

Topics: Age Factors; Animals; Autoimmune Diseases; Concanavalin A; Culture Media; Diabetes Mellitus, Type 1; Disease Susceptibility; Female; Immunization, Passive; Lymphocyte Transfusion; Male; Rats; Rats, Inbred BB; Rats, Inbred BUF; Rats, Inbred Strains; Rats, Inbred WF; Spleen

Total lymphoid irradiation (TLI) at doses of 2200 rads or greater prevented diabetes in susceptible BB/W rats. Two of 29 (7%) treated rats became diabetic compared with 23 of 39 (59%) controls (P less than 0.001). TLI did not, however, prevent insulitis or thyroiditis in nondiabetic rats, nor did it restore the depressed concanavalin-A responsiveness of BB rat lymphocytes. T-lymphocyte subset proportions were the same in both groups. TLI was associated with significant radiation-related mortality, and nondiabetic TLI-treated rats weighed significantly less than controls. We conclude that TLI is effective in the prevention of BB rat diabetes. However, TLI fails to correct the subclinical immunologic abnormalities of the model and is associated with significant morbidity.

Topics: Animals; Autoimmune Diseases; Concanavalin A; Diabetes Mellitus, Type 1; Dose-Response Relationship, Radiation; Female; Islets of Langerhans; Lymphoid Tissue; Male; Mitosis; Pancreatitis; Rats; T-Lymphocytes; Thyroiditis

The Bio-Breeding/Worcester (BB/W) rat develops spontaneous autoimmune diabetes similar to human insulin-dependent diabetes mellitus. Transfusions of whole blood from the nondiabetic W-line of BB/W rats prevent the syndrome in diabetes-prone recipients. We report three experiments designed to determine which blood component is protective. In all experiments, diabetes-prone BB/W rats 23 to 35 d of age were given four or six weekly intravenous injections. In the first experiment, animals received either saline or transfusions of erythrocytes, white blood cells, or plasma from W-line donors. Diabetes occurred in 7/22 (32%) erythrocyte, 2/27 (7%) white cell, 14/24 (58%) plasma, and 15/27 (56%) saline recipients (P less than 0.001). At 120 d of age, peripheral blood was obtained from nondiabetic rats. Fluorescence-activated cell sorter analysis of OX 19 tagged leucocytes revealed 35% T lymphocytes in white cell recipients (n = 13), compared with 9% in saline recipients (n = 7; P less than 0.001). Responsiveness to concanavalin A was also increased in the white cell group, whereas the frequency of both insulitis and thyroiditis was decreased. In the second experiment, 1/19 (5%) rats transfused with W-line spleen cells developed diabetes, as contrasted with 12/18 (67%) recipients of diabetes-prone spleen cells and 19/31 (61%) noninjected controls (P less than 0.001). In the third experiment, diabetes-prone rats received either W-line blood treated with a cytotoxic anti-T lymphocyte antibody plus complement, untreated blood, or saline. Diabetes occurred in 8/20 (40%), 1/20 (5%), and 13/19 (68%) rats in each group, respectively (P less than 0.001). We conclude that transfusions of W-line T lymphocytes prevent diabetes in the BB/W rat.

Topics: Animals; Autoantibodies; Blood Transfusion; Concanavalin A; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Immunity, Innate; Lymphocyte Activation; Rats; Rats, Inbred Strains; T-Lymphocytes

Autoantibodies, cell-mediated autoimmunity, and impaired suppressor T cell function, suggesting abnormal immunoregulation, have been implicated in the pathogenesis of juvenile-onset insulin dependent diabetes mellitus (IDDM). To examine one of the parameters of immunoregulation, and to explore its relationship to the disease, we tested suppressor cell function in IDDM patients, their clinically healthy relatives, and in normal unrelated controls. 9/15 IDDM had impaired suppressor cell function compared to 1/8 age-matched healthy sibs (p less than 0.04) and to 0/9 unrelated controls (p less than 0.005). There was no correlation between abnormal suppressor cell function and the patient's age, sex, preprandial blood glucose levels, age at the time of diagnosis, or duration of the disease. However, there was a trend for a higher proportion of HLA Dr3 positive diabetics to have abnormal suppressor cell function compared to DR3 negative patients. Impaired suppressor cell function was also found in 5/23 clinically healthy first degree relatives; 4/5 were related to a diabetic who demonstrated abnormal suppressor cell function. These findings raise the possibility that underlying familial, probably genetically determined abnormalities in immunoregulation, acting in concert with other environmental or genetic factors, may contribute to disease susceptibility in IDDM.

Topics: Child; Concanavalin A; Diabetes Mellitus, Type 1; Female; HLA Antigens; Humans; Immune Tolerance; Male; Pedigree; T-Lymphocytes, Regulatory

The proliferative responses of IDDM or NIDDM patients and normal controls to monocomponent pork and beef insulins was assayed over a period of 5 days. No difference was detected in the proliferative responses between the groups. About 25-30% responded to insulin antigens by giving stimulation indices of 1.5 or higher. The exception was the HLA-B8/15 group of patients where no responses (less than or equal to 1.5) were observed. The evidence that the responses detected were insulin specific is discussed, as is the HLA and possible immune response gene linkage.

Topics: Adult; Aged; Animals; Antigens; C-Peptide; Cattle; Concanavalin A; Diabetes Mellitus; Diabetes Mellitus, Type 1; HLA Antigens; HLA-B Antigens; Humans; Insulin; Lymphocyte Activation; Middle Aged; Swine; Tuberculin

Peripheral blood from 25 patients with insulin-dependent diabetes mellitus was examined for any alteration in the proportions and or functions of immunoregulatory T-cell subsets, defined with monoclonal antibodies. Ten of 25 (40%) patients demonstrated deficiency of OKT8+ (suppressor/cytotoxic) T-cells. Eight of these 10 patients had abnormally high ratios of OKT4+/OKT8+ T-cells. Nine of 10 patients with abnormally low proportion of OKT8+ T-cells had deficient concanavalin A-induced suppressor cell activity against the proliferative response of autologous or allogeneic lymphocytes to phytohemagglutinin. No correlation was observed between the deficiency of suppressor T-cells and the control of diabetes. Therefore, it is likely that the deficiency of suppressor cells is related to insulin-dependent diabetes mellitus itself and not to the metabolic changes that are associated with diabetes mellitus. This study demonstrates both quantitative and qualitative deficiency of suppressor T-cells in at least some patients with insulin-dependent diabetes, that might play an important role in the pathogenesis and autoimmune manifestations of a proportion of patients with insulin-dependent diabetes mellitus.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Child; Concanavalin A; Cytotoxicity, Immunologic; Diabetes Mellitus, Type 1; Female; Humans; Leukocyte Count; Male; T-Lymphocytes; T-Lymphocytes, Regulatory

We evaluated antigen-nonspecific (Con A) and antigen-specific (islet cell) activation of suppressor cell function in 11 IDD patients. Compared with healthy controls, IDD was associated with both antigen-specific (n = 11, p less than 0.01) and nonspecific (n = 6, p less than 0.03) suppressor cell hypofunction. The specific defect was not present in NIDD patients and correlated negatively with the duration of the disease (r = -0.6, p less than 0.05). No relationship was found between the degree of specific suppressor cell dysfunction and diabetic control as assessed by glycosylated hemoglobin, plasma glucose values, insulin-binding capacity, or C-peptide determinations. Plasma from IDD lacked anti-suppressor cell activity. Low levels of circulating immune complexes were detected in IDD patients whose disease duration was 1 month or less. Specific suppressor cell hypofunction and/or enhanced helper cell activity in early stages of IDD could be contributing to the formation of islet cell autoantibodies, immune complexes, islet cell injury, and the diabetic state.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Antigen-Antibody Complex; Blood Glucose; Child; Concanavalin A; Diabetes Mellitus, Type 1; Female; Hemoglobins; Histocompatibility; Humans; Insulin; Lymphocyte Activation; Middle Aged; Pancreas; T-Lymphocytes, Regulatory

Percentage binding of 125 I-labelled concanavalin A to erythrocytes in diabetic patients was significantly higher than that in normal subjects (12.2 +/- 2.8 versus 8.1 +/- 1.8%, mean +/- SD, p less than 0.001). Insulin-dependent diabetic patients showed significantly higher concanavalin A binding than non-insulin-dependent diabetic subjects (15.0 +/- 1.4 versus 11.4 +/- 2.5%, p less than 0.01). There was a highly significant correlation between percentage binding of 125I-labelled concanavalin A and glycosylated haemoglobin.

Topics: Adult; Aged; Concanavalin A; Diabetes Mellitus; Diabetes Mellitus, Type 1; Erythrocytes; Female; Glycated Hemoglobin; Humans; Kinetics; Male; Middle Aged; Receptors, Concanavalin A

Lymphocytes of juvenile onset, insulin-dependent diabetics failed to generate suppressor cell activity after preincubation with concanavalin A (Con A). The mean suppression of the autologous proliferative response to phytohemagglutinin (PHA) was 6.1 +/- 7.8% (mean +/- SEM) in the diabetics and 34.0 +/- 4.9% in 9 age-matched healthy controls (p less than 0.01). Cell mixing experiments identified the defect to be in the generation of suppressor cells rather than in the responses to their action. Plasma of insulin-dependent diabetics had no effect on the generation of suppressor activity. In contrast to our findings in insulin-dependent diabetics, Con A preincubation of lymphocytes from 4 maturity onset diabetics induced normal suppression of PHA responses. Defective immunoregulation is present in insulin-dependent diabetes and may underlie autoimmunity.

Topics: Adolescent; Adult; Concanavalin A; Diabetes Mellitus; Diabetes Mellitus, Type 1; Humans; Insulin; Phytohemagglutinins; T-Lymphocytes, Regulatory

Cell-mediated immunity was evaluated in 11 children with diabetes mellitus; six children were evaluated during ketoacidosis and five were evaluated with ketonuria in the absence of acidosis. Five of the six ketoacidotic children had at least one positive delayed-hypersensitivity skin test. Lymphocytes from two ketoacidotic patients were unresponsive to phytohemagglutinin and pokeweed mitogen, and lymphocytes from these two patients plus a third patient were unresponsive to concanavalin A. Lymphocytes from all six patients responded to these three mitogens after one week of therapy. In the five diabetic children without ketoacidosis, lymphocyte responses were normal to all three mitogens. Similarly, the addition of glucose to normal plasma did not alter the lymphocyte transformations of three healthy nondiabetic controls. These data suggest that cell-mediated immunity may be transiently defective in children with acute diabetic ketoacidosis.

Topics: Acute Disease; Adolescent; Child; Concanavalin A; Diabetes Mellitus, Type 1; Diabetic Ketoacidosis; Female; Humans; Immunity, Cellular; Ketone Bodies; Lymphocyte Activation; Lymphocytes; Male; Mitogens; Phytohemagglutinins; Pokeweed Mitogens; Skin Tests