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  • 1
    ISSN: 1432-0428
    Keywords: Insulin antibodies ; insulin structure ; evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary In the present study, we attempted to define possible subpopulations of antibodies which theoretically could be directed against evolutionarily conserved regions of the insulin molecule in sera from insulin-treated diabetic patients using a variety of labelled and unlabelled insulins which differ widely in structure but are very similar in functional properties. Ten high titre human insulin antisera from patients treated with mixed beef-pork insulin were examined. All sera were found to bind 125I-pork insulin better than labelled chicken insulin which bound better than labelled fish insulin. Detailed studies were conducted with four of the antisera using the pork and fish tracers. With two of the antisera, a subpopulation of antibody could be detected with 125I-fish insulin which had similar affinity for both fish and pork insulin, but reacted much less well with guinea pig insulin and the desoctapeptide derivative of porcine insulin. Based on the known properties of these four insulins, the data provide suggestive evidence consistent with the hypothesis that there are subpopulations of antibodies recognizing regions on the insulin molecule that are well conserved, possibly the region involved in the formation of insulin dimers or receptor binding.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Diabetologia 33 (1990), S. 641-648 
    ISSN: 1432-0428
    Keywords: Glucose transporter mRNA ; hexose ; L6 myocyte
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Glucose deprivation of L6 myocytes results in the upregulation of glucose transporter activity, protein and mRNA. We have investigated the downregulation of transporter gene expression by glucose and other hexoses in glucose-deprived L6 myocytes. Glucose transport activity was measured as the uptake of 3H-2-deoxyglucose. Transporter protein and mRNA were detected by immunoblot and Northern blot analysis, respectively, with probes to the rat brain glucose transporter. Glucose deprivation of myocytes, in the absence and presence of insulin, increased 3H-2-deoxyglucose uptake, transporter protein and mRNA levels. Refeeding with glucose reversed the glucose deprivation effects on transport activity and mRNA within 12 h, with half-maximal effects at 1–2 mmol/l glucose. Mannose fully substituted for glucose. Refeeding with the non-metabolisable glucose analogues 2-deoxyglucose and 3-0-methylglucose, or with glucosamine or mannitol, downregulated 3H-2-deoxyglucose uptake but had little or no effect on transporter protein and mRNA expression. In contrast, glucose-6-phosphate markedly increased 3H-2-deoxyglucose uptake but partly downregulated transporter mRNA levels, whereas galactose had a small stimulatory effect on both 3H-2-deoxyglucose uptake and transporter mRNA; neither affected transporter protein levels. The transporter mRNA level was not affected by several metabolites (pyruvate, glyceraldehyde, glycerol) and amino acids (alanine, glutamine). These findings indicate that (i) there are independent pathways for hexose regulation of transport activity, protein and mRNA and (ii) down-regulation of transporter mRNA requires metabolism beyond hexose phosphate whereas glucose uptake may be regulated by direct interaction of hexoses with the transporter.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Diabetologia 40 (1997), S. B50 
    ISSN: 1432-0428
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0428
    Keywords: Type 1 (insulin-dependent) diabetes mellitus ; non-obese diabetic (NOD) mouse ; pancreatic islets ; class I major histocompatibility complex (class I MHC) ; anti-interferon-γ antibodies
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Overexpression of class I major histocompatibility complex (MHC) proteins on pancreatic islet cells is a characteristic of autoimmune Type 1 (insulin-dependent) diabetes mellitus in humans and in animal models. Studies of post-mortem pancreases from humans with Type 1 diabetes suggest that overexpression of class I MHC proteins may precede mononuclear cell infiltration of the islets (insulitis). Pancreatic histology from the earliest stages of human Type 1 diabetes is rarely available. We have used the non-obese diabetic mouse, given cyclophosphamide to accelerate Betacell destruction, to investigate the temporal relationship between the Overexpression of class I MHC protein and mRNA and other pathological changes associated with Beta-cell destruction. Prior to cyclophosphamide, immunoperoxidase staining showed that expression of class I MHC proteins was greater on islet cells and infiltrating inflammatory cells of the non-obese diabetic mouse than on islet cells of other mouse strains, whereas staining on exocrine cells was similar. On day three after cyclophosphamide administration, when insulitis had regressed, islet class I MHC protein expression had diminished. A dramatic increase in class I MHC protein expression occurred between days seven and nine, concomitant with reinfiltration of the islets by mononuclear cells; Overexpression was seen both on islet cells and on surrounding exocrine cells, but only in the presence of mononuclear cell infiltration. By day 21, class I MHC protein Overexpression was again confined to the islets, the exocrine pancreas being free of infiltration. Class I mRNA also increased dramatically by day eight but had virtually returned to normal by day 12. Overexpression of class I MHC protein following cyclophosphamide was prevented by administration of antiinterferon- γ antibody. Expression of class II MHC proteins was not detected on pancreatic cells following cyclophosphamide but was present on infiltrating mononuclear cells. These findings demonstrate a close association between class I MHC protein and mRNA Overexpression and insulitis in non-obese diabetic mice given cyclophosphamide. They are consistent with the view that class I MHC Overexpression is effected by cytokines secreted by activated immunoinflammatory cells. Class I MHC Overexpression should enhance targeting of cytotoxic T cells to Beta cells bearing autoantigen.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-0428
    Keywords: Keywords Pre-clinical Type I diabetes, infants, insulin autoantibodies, GAD antibodies, IA2 antibodies, HLA.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Aims/hypothesis. To determine the sequence of development of islet autoantibodies and their relation to HLA genes in infants at risk for Type I diabetes followed from birth.¶Methods. We followed 357 (189 male, 168 female) infants, with a first degree relative with Type I diabetes for a mean of 3 years from birth. Human leukocyte antigen typing and assays for insulin autoantibodies (IAA), glutamic acid decarboxylase antibodies (GADAb) and tyrosine phosphatase IA2 (IA2Ab) antibodies were done on cord blood, and venous blood was sampled every 6 months for IAA, GADAb and IA2Ab.¶Results. We did not find any antibodies in 263 (73 %) infants; 50 (14 %) were positive for a single antibody once, 19 (5 %) for a single antibody more than once and 25 (7 %) for two or more antibodies. Of the latter, 10 (2.8 % overall) were persistently positive; they had higher frequencies of HLA DR4 (p 〈 0.01) and HLA DR3, 4 (p 〈 0.05). Of the group persistently positive for two or more antibodies four infants developed diabetes. Insulin autoantibodies were the first ones to develop in 64 % of infants with two or more antibodies.¶Conclusion/interpretation. Infants with high risk HLA-DR alleles and multiple antibodies at high risk for diabetes were identified. A much larger group of infants had transient low level increases usually of a single antibody. Whereas transient low level positivity could be attributed to difficulties with assay technique and cut off levels for normality, the results overall support the phenomenon of transient ’self limited' islet autoimmunity in at risk infants. [Diabetologia (2000) 43: 203–209]
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Diabetologia 32 (1989), S. 218-218 
    ISSN: 1432-0428
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-0428
    Keywords: Insulin-dependent diabetes mellitus ; non-obese diabetic (NOD) mouse ; autoreactive T-cell clones ; suppressor T cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The non-obese diabetic (NOD) mouse is a spontaneous model of human insulin-dependent diabetes mellitus. Both CD4+ and CD8+ T cells infiltrate the pancreatic islets of NOD mice prior to beta-cell destruction. T-cell lines isolated from the islets of NOD mice are tools for studying the pathogenesis of insulin-dependent diabetes mellitus. During attempts to generate such lines we isolated an autoreactive CD4+ T-cell line, designated C2, from the ‘insulitis’ lesion of a 20-week-old female non-diabetic NOD/WEHI mouse. Islet T cells were propagated by the addition of interleukin-2 and reexposure every 2 weeks to whole NOD islets and irradiated NOD spleen cells as antigen presenting cells. C2 cells proliferated up to 100-fold upon exposure to NOD antigen presenting cells but did not respond to whole NOD islets or antigen presenting cells from allogeneic mouse strains. Proliferation of C2 cells to NOD antigen presenting cells was blocked by a monoclonal antibody against the unique class II MHC molecule of NOD, I-Ag7. In response to NOD antigen presenting cells, C2 cells secreted Interferon-γ, tumour necrosis factor-α and interleukin-6 but no detectable interleukin-2, interleukin-4 or interleukin-10, a pattern of cytokine secretion more characteristic of Th1 CD4 cells. C2 cells displayed significant cytotoxicity in a redirected lysis assay. To explore a possible role for autoreactive T cells in the pathogenesis of autoimmune diabetes, C2 cells were injected i.v. into female NOD mice that had received cyclophosphamide to accelerate development of diabetes. Although not different at day 10, by day 16 the frequency of diabetes (blood glucose〉15 mmol/l) in mice given cyclophosphamide and C2 cells was significantly less than in mice given cyclophosphamide alone (p〈0.05). These results are consistent with the ‘suppressor’ function of autoreactive T-cells previously reported and with the concept that autoreactive T cells modulate autoimmune beta-cell destruction in the NOD mouse.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Diabetologia 38 (1995), S. 125-126 
    ISSN: 1432-0428
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1432-0428
    Keywords: Keywords NOD mouse ; beta-cell apoptosis ; diabetes ; T cells ; cyclophosphamide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Autoimmune-mediated destruction of pancreatic islet beta cells leads to insulin-dependent diabetes in non-obese diabetic (NOD) mice. Although both direct cytotoxic T cell- and indirect cytokine-, nitric oxide- or free radical-mediated mechanisms induce beta-cell apoptosis in vitro, beta-cell death in vivo in spontaneous autoimmune diabetes is not well-characterized. Furthermore, whether beta cells die gradually, or rapidly in the late pre-clinical stage, is a question of current interest. To investigate beta-cell death in vivo, we measured the frequency and intra-islet localisation of apoptosis, defined as DNA strand breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique, during spontaneous and cyclophosphamide-accelerated diabetes in NOD mice. In spontaneous diabetes, the frequency of apoptosis in islets correlated with the progression of beta-cell destruction with age. Although apoptosis was detected at low frequency within the reduced insulin-positive islet area of pre-diabetic mice at 90 days of age, it was rarely co-localised to beta cells. After acceleration of beta-cell destruction with cyclophosphamide, the frequency of apoptosis reached maximum at 12 days, at which time 3.2 % of apoptotic cells were beta cells. Apoptosis was most frequent in the insulin-negative islet area comprised of mononuclear cell infiltrate and was localized to CD8+ T cells. The rarity of detectable apoptotic beta cells in spontaneous pre-diabetic mice with pronounced insulitis and reduced insulin-positive islet areas most likely reflects the rapid clearance of apoptotic beta cells. Our findings are more consistent with gradual destruction of non-renewable beta-cells in spontaneous diabetes, than with their rapid, accelerated destruction (as after cyclophosphamide) in the late pre-clinical stage. [Diabetologia (1998) 41: 1381–1388]
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1432-0428
    Keywords: Human lymphocytes ; surface labelling ; insulin receptors ; auto-antibodies ; receptor subunits
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Cultured human lymphoblastoid B lymphocytes were surface-labelled with iodine125 and solubilized in 1% Triton X-100 in the presence of protease inhibitors. After purification of labelled glycoproteins by elution from immobilized wheat germ leetin with 0.3 mol/l N-acetyl-D-glucosamine, insulin receptors were quantitatively immunoprecipitated using IgG receptor auto-antibodies. The overall recovery of labelled glycoprotein was 0.02–0.04%; analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography under reducing conditions revealed two major bands with molecular weights of 126,000 and 90,000, and a minor band of 67,000 daltons. The mobilities of both major receptor subunits were increased after treatment with neuraminidase. When lymphocyte receptor binding was ‘down-regulated’ before surface labelling, there was a concomitant decrease in the recovery of both the 126,000 and 90,000 subunits. These data indicate that ‘down-regulation’ of binding probably involves degradation of the receptor molecule.
    Type of Medium: Electronic Resource
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