Major histocompatibility genes and type 1 (insulin-dependent) diabetes mellitus.
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Diabetes Mellitus, Type 1↗
Biomedical subjects
L C Harrison
Publications and source records attributed to L C Harrison.
Murine models of autoimmune diabetes: nonspecific cytotoxic lymphocytes derived from pancreatic islets in the presence of IL-2.
Our aim was to derive T lymphocyte lines that specifically recognize islet antigens in murine models of autoimmune diabetes. Islets of Langerhans infiltrated with lymphocytes were isolated either from mice previously injected with multiple low doses of streptozotocin or from NOD-WEHI mice and were cultured in the presence of the T cell growth factor, interleukin 2 (IL-2). With islets from both models of autoimmune diabetes, rapidly proliferating, large granular lymphocytes emerged after 7-10 days and destroyed the islets and other cells such as fibroblasts in the cultures. Cytotoxicity assays showed that these cells were capable of destroying both P815 and YAC-1 tumor cells. In contrast to lymphocytes present initially in the islet infiltrates which express predominantly the L3T4 marker, the large granular lymphocytes were shown to be Ly-2 positive. They also expressed the alpha beta T cell receptor and contained mRNA for the alpha beta T cell receptor demonstrable by in situ hybridization. While morphologically similar to NK cells these large granular lymphocytes bear T cell markers and destroy a broader range of targets. They may represent a minor population of T lymphocytes particularly responsive to IL-2 although other studies show that T cells generally can develop a similar phenotype after prolonged culture with IL-2. The lack of target cell specificity indicates that these IL-2-stimulated large granular lymphocytes are unlikely to mediate the immunopathogenesis of diabetes in these animal models.
Animals↗
Sources of improvement in between laboratory variation in estrogen and progestin receptor measurement using tissue samples during the Australian Quality Assurance Programme.
Tissue and cytosol samples were compared as quality control material for assessment of between and within laboratory error in measurement of estrogen and progestin receptors (ER and PR) in a series of four trials for a total of 17 participating laboratories during the Australasian Quality Assurance Programme. For tissue samples, a substantial reduction in between laboratory CVs for both ER and PR from about 90 to 50% was achieved during the programme. In contrast, for cytosol samples a substantially lower between laboratory CV of about 30% was obtained. Tissue sample heterogeneity could be excluded as a major source of variation between laboratories. The likely source of the observed improvement in CV for tissue samples during the trials was due to a reduction of the initial under-estimation of receptor concentration in tissue samples by some of the participants. Although cytosol preparation from tissue samples was shown to be one major source of error, other sources of error such as the receptor assay itself and the associated protein measurements were identified. It is concluded that fragmented tissue samples are essential for a realistic assessment of between laboratory error in receptor measurements in biopsy material such as obtained from clinical breast cancer samples.
Animals↗
Intercellular adhesion molecule 1 is induced on isolated endocrine islet cells by cytokines but not by reovirus infection.
The selective destruction of the pancreatic islet beta cells in type 1 diabetes mellitus is thought to be mediated by a cellular autoimmune process, possibly triggered by virus infection in genetically susceptible individuals. Because of the potentially important role of cell-cell adhesion in the immune response, we investigated whether cytokine products of mononuclear cells, or virus infection, induced the expression of intercellular adhesion molecule 1 (ICAM-1) on human endocrine islet cells. By flow cytofluorimetry, control islet cells did not express detectable ICAM-1. However, after a 72-hr exposure of islets to interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) (each at 250 units/ml), ICAM-1 was induced on greater than 85% of islet cells. IFN-gamma was 50% more potent than TNF-alpha; together, their effects were additive. Class I major histocompatibility complex (MHC) protein expression, detected on control islet cells, was also stimulated by IFN-gamma and/or TNF-alpha. In contrast, infection with reovirus type 3 did not induce ICAM-1 on islet cells, although it stimulated the expression of class I MHC proteins. By double-label indirect immunofluorescence microscopy, ICAM-1 expression was identified on both beta (insulin-secreting) and delta (somatostatin-secreting) islet cells. Monoclonal antibody to ICAM-1 precipitated protein of Mr 97,000 from [35S]methionine-labeled islets exposed to IFN-gamma and TNF-alpha, but not from control islets. RNA blot analysis revealed a major species of 3.3 kilobases and a minor species of 2.2 kilobases induced in islets exposed to the cytokines. These findings have implications for the molecular mechanisms of beta-cell destruction in type 1 diabetes, in that expression of ICAM-1 by beta cells may facilitate adhesion of antigen-targeted immune cells.
Antibodies, Monoclonal↗
Pancreatic beta cell proliferation in rabbits demonstrated by bromodeoxyuridine labeling.
The purpose of the present study was to quantify beta cell proliferation in rabbits that had undergone subtotal beta cell ablation by labeling cell nuclei undergoing DNA synthesis with bromodeoxyuridine (BUdR). Ten New Zealand white rabbits were compared with five sham-operated controls. The beta cells in the head of the pancreas were destroyed by intravenous alloxan (200 mg/kg) while the tail of the pancreas was temporarily isolated from the systemic circulation by vascular occlusion clamps. On day 7, BUdR (70 mg/kg) was infused prior to killing to label newly synthesised DNA. Immunoperoxidase staining with anti-insulin sera and an anti-islet cell monoclonal antibody confirmed the absence of insulin-producing cells in the head of the pancreas. Beta cell proliferation within the islets of the tail of the pancreas was suggested by the appearance of cells undergoing mitosis and confirmed by BUdR labeling of cell nuclei detected with anti-BUdR monoclonal antibody. The mitotic index was significantly increased compared to control rabbits (p = 0.0235, Mann-Whitney U nonparametric test). This study demonstrates that the BUdR labeling can be used to document beta cell proliferation in vivo in the rabbit model after subtotal beta cell ablation.
Animals↗
Pancreatic islet A2B5- and 3G5-reactive gangliosides are markers of differentiation in rat insulinoma cells.
Rat insulinoma (RIN) cells, in comparison with adult islet cells, are relatively undifferentiated. They secrete low amounts of islet hormones, are unresponsive to glucose, and display pluripotency. A minority of RIN cells react with monoclonal antibodies A2B5 and 3G5 which recognize complex gangliosides on normal islet cells. In order to determine whether the expression of A2B5- or 3G5-reactive gangliosides is modulated during differentiation RIN cells were cultured with various concentrations of sodium butyrate (NaB), a known inducer of cellular differentiation. Expression of A2B5- and 3G5-reactive gangliosides was determined by indirect immunofluorescence and flow cytofluorimetry. NaB exposure resulted in a dose-dependent decrease in cell proliferation over 5 days of 1.5-, 2.9-, and 17.5-fold at 0.5, 1.0 and 3.0 mM, respectively, and a distinct change in cellular morphology. Cells exposed to NaB displayed prominent neurite-like projections. At 3 mM NaB, insulin secretion increased 7.9-fold and the percentage of cells expressing A2B5- and 3G5-reactive gangliosides increased by up to 4.4- and 5.5-fold, respectively. The expression of A2B5- or 3G5-reactive gangliosides per cell also increased, by 2.4- and 1.3-fold, respectively, at 3 mM NaB. These findings demonstrate that the expression of cell surface A2B5- and 3G5-reactive gangliosides is not static but increases with cell differentiation. NaB-treated RIN cells may serve as a model to study the role of gangliosides in the function and lineage relationships of islet cells.
Adenoma, Islet Cell↗
Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.
We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.
Autoradiography↗
Chronic stimulation of glucose transporter gene expression in L6 myocytes mediated via the insulin-like growth factor-1 receptor.
We have used differentiated L6 myocytes to investigate the regulation of glucose transporter gene expression by insulin and insulin-like growth factor-1 (IGF-1). Chronic exposure to insulin (1 microM) or IGF-1 (10 nm) resulted in a 2- to 5-fold stimulation of 3H-2-deoxy-D-glucose uptake and a corresponding increase in the expression of rat brain/HepG2-type glucose transporter mRNA (GTmRNA) and immunoreactive transporter protein. The dose responses to both insulin and IGF-1 for stimulation of glucose uptake were paralleled by the expression of GTmRNA. Glucose uptake and GTmRNA levels were half maximally stimulated by 350 and 100 nM insulin, respectively, or by 2 nM IGF-1. Comparison of receptor occupancy with stimulation of glucose uptake and GTmRNA expression suggests that insulin exerts its effects through the IGF-1 receptor. Fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and phorbol ester had little or no effect on GTmRNA expression. These results demonstrate that the IGF-1 receptor mediates chronic regulation of transporter mRNA expression and protein synthesis and activity in cultured rat muscle cells.
Animals↗
Insulin receptor expression in the Burkitt lymphoma cells Daudi and Raji.
The specific binding of insulin to either intact or Triton-solubilized Daudi cells (a Burkitt lymphoma cell line) was reduced by over 95% compared to that to control IM-9 lymphocytes due to a decrease in receptor number without a change in affinity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that 125I-labeled Daudi cells had reduced amounts (approximately 1/20th) of immunoprecipitable binding (alpha) subunit [mol wt (Mr), 130,000] of the receptor and a relatively abundant 210,000 Mr form not seen in IM-9 cells. The transmembranous (beta) subunit (Mr, 90,000) of the receptor, although not detected by 125I surface labeling, could be phosphorylated and, together with the 210,000 Mr form, exhibited the same 2-fold stimulation of phosphorylation by insulin as that in IM-9 cells. Northern blot hybridization revealed a decrease in Daudi cells of all four major species of insulin receptor mRNA. The Raji cell, another Burkitt lymphoma cell line, also exhibited reduced protein and genetic expression of the insulin receptor, indicating that reduced insulin receptor expression may be representative of other Burkitt lymphoma cell lines.
Blotting, Northern↗
Double-blind controlled trial of azathioprine in children with newly diagnosed type I diabetes.
A double-blind controlled trial of azathioprine (2 mg.kg-1.day-1) was conducted with 49 patients aged 2-20 yr (mean 10.8 yr) who had newly diagnosed type I (insulin-dependent) diabetes. Patients were randomly assigned to receive either azathioprine (n = 24) or placebo (n = 25) for 12 mo, beginning within the 20-day period after diagnosis. Baseline clinical and metabolic characteristics did not differ between the two groups. No patient experienced complete remission, defined as restoration of normal carbohydrate tolerance without other treatment. Partial remission, defined as good metabolic control (hemoglobin A1c less than or equal to 7.9%, preprandial blood glucose less than or equal to 8 mM with an insulin dose of less than 0.5 U.kg-1.day-1), occurred in 10 placebo (40%) and 7 azathioprine (29%) patients at 6 mo and in 4 placebo (16%) and 4 azathioprine (17%) patients at 12 mo (differences not significant). Fasting plasma C-peptide was significantly greater in the azathioprine-treated group at 3 and 6 mo, but this difference was not sustained. C-peptide responses to a standard meal and the frequency of islet cell and insulin antibodies did not differ between the two groups over the 12-mo period. Azathioprine caused no significant side effects. We conclude that in the dosage used, and despite early effects on endogenous insulin secretion, azathioprine alone does not influence the remission phase in children with newly diagnosed type I diabetes.
Azathioprine↗
MHC molecules and beta-cell destruction. Immune and nonimmune mechanisms.
Hyperexpression of major histocompatibility complex (MHC) molecules by islet cells is a prominent, early feature of islet pathology in insulin-dependent diabetes mellitus and concomitant with beta-cell failure after exposure of islets to specific cytokines or viruses. The transgenic expression of a class I MHC gene (H-2Kb) in the beta-cells of either syngeneic or allogeneic mice leads to beta-cell failure by a nonimmune mechanism. Several class II MHC transgenes, with one exception, have the same effect, but the expression of other transgenes that have products that are membrane proteins is not necessarily detrimental. Class I MHC molecules have been shown to interact directly with other membrane proteins. The inappropriate expression of MHC molecules could therefore interfere with key cellular functions. We postulate that the hyperexpression of MHC molecules in the beta-cell, e.g. in response to viruses, is a primary, nonimmune mechanism of beta-cell failure that precedes a secondary autoimmune response.
Animals↗
IFN-gamma and tumor necrosis factor-alpha. Cytotoxicity to murine islets of Langerhans.
We have previously reported that the cytokines IFN-gamma and TNF-alpha each upregulate the expression of class I MHC proteins and, in combination, induce the expression of class II MHC proteins on pancreatic islet cells. IFN-gamma and TNF-alpha are therefore implicated in the immunologic destruction of beta-cells in insulin-dependent diabetes mellitus. The objective of the present study was to define the effects of IFN-gamma and TNF-alpha on the function and viability of murine pancreatic islet beta-cells in vitro. Exposure of islets for 3 days to 200 U/ml of either IFN-gamma or TNF-alpha did not affect glucose-stimulated insulin release, but at higher concentrations (2000 U/ml) of either cytokine there was significant inhibition of glucose-stimulated insulin release. In combination, IFN-gamma and TNF-alpha each at 200 U/ml caused significant inhibition of glucose-stimulated insulin release; at 2000 U/ml glucose-stimulated insulin release was abolished. In time-course experiments, glucose-stimulated insulin release from islets exposed to IFN-gamma and TNF-alpha each at 1000 U/ml was significantly increased at 4-h (twofold increase compared with control islets), decreased back to control levels at 18 h, significantly inhibited by 24 h (threefold decrease compared with control islets), and completely abolished by 48 h. The progressive impairment of beta-cell function mediated by IFN-gamma plus TNF-alpha was associated with morphologic derangement of the islets that were almost totally disintegrated by day 6 of exposure to the cytokines. At day 6, insulin content of the islets was significantly reduced by exposure to TNF-alpha but not IFN-gamma. The combination of IFN-gamma and TNF-alpha resulted in a further dose-dependent depletion in insulin content compared with TNF-alpha alone. The synergistic functional and cytotoxic effects of IFN-gamma and TNF-alpha are consistent with a direct role for these cytokines in the destruction of beta-cells in insulin-dependent diabetes.
Animals↗
Chromatography of serum on Sep-pak C18 corrects falsely elevated vitamin D metabolite levels measured by protein binding assay.
Using a commercial kit method we obtained a vitamin D metabolite level within the normal range in a patient with biopsy-proven osteomalacia. This suggested that the ethanol extraction method employed had not removed serum factors known to falsely elevate the measurement of vitamin D metabolites. We therefore compared the levels measured after ethanol extraction and after purification by chromatography on Sep-pak C18 or Sephadex LH-20. Vitamin D metabolite levels after ethanol extraction of sera correlated with, but were higher than, those after chromatography on Sep-pak C18 cartridges (r = 0.84; 134 +/- 76 vs 76 +/- 46 nmol/l: mean +/- SD; p less than 0.01). Results were similar after chromatography on Sep-pak C18 and Sephadex LH-20 (r = 0.95; 79 +/- 46 vs 68 +/- 41 nmol/l). Sera from 5 patients (4 with osteomalacia, 1 with chronic pancreatitis/malabsorption) had vitamin D metabolite levels in the normal range after ethanol extraction but had low levels after Sep-pak C18 chromatography; four of these sera also had low levels after chromatography on Sephadex LH-20. These findings indicate that chromatography of serum on Sep-pak C18 cartridges corrects falsely elevated vitamin D metabolite levels measured by protein binding assay.
Adult↗
Diabetes in transgenic mice resulting from over-expression of class I histocompatibility molecules in pancreatic beta cells.
A class I histocompatibility gene, H-2Kb, linked to the rat insulin promoter, is overexpressed in the pancreatic beta cells of transgenic mice. The mice, whether syngeneic or allogeneic to the transgene, develop insulin dependent diabetes without detectable T cell infiltration, suggesting a direct, non-immune role for the transgenic class I molecules in the disease process.
Animals↗
DNA probes for presymptomatic diagnosis: keys to biological determinism?
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DNA↗
IFN-gamma induces islet cell MHC antigens and enhances autoimmune, streptozotocin-induced diabetes in the mouse.
To explore the role of the lymphokine, IFN-gamma, in the development of autoimmune-mediated insulin-dependent diabetes, we examined the effects of systemically administered IFN-gamma on the clinical features and pancreatic immunohistology of CBA mice made diabetic with multiple low doses of the pancreatic islet beta-cell toxin, streptozotocin. Mice given streptozotocin and IFN-gamma were significantly more hyperglycemic than those given streptozotocin alone and had significantly decreased body weight. Mice given IFN-gamma alone did not differ in glycemia or weight from vehicle-injected mice. On day 11, Ia proteins were detected on islet cells from mice given streptozotocin and their expression was potentiated by IFN-gamma; they could not be detected on islet cells from mice given IFN-gamma alone or vehicle. H-2K protein expression was increased on islet cells from mice given streptozotocin and was potentiated by IFN-gamma. IFN-gamma alone also increased H-2K protein expression on islet cells compared with vehicle-treated mice. These findings show that IFN-gamma enhances the severity of diabetes in mice given multiple-low doses of streptozotocin, in association with enhanced expression of Ia and H-2K proteins on islet cells. They indicate an important role for IFN-gamma in amplifying the autoimmune process leading to beta-cell destruction in diabetes. The ability of IFN-gamma to worsen autoimmune disease has implications for its use in man.
Animals↗
Diabetic amyotrophy: reassessment of the clinical spectrum.
Diabetic amyotrophy is a syndrome whose recognition may be difficult or delayed. We reviewed thirteen patients with this disorder, all of whom had significant proximal lower limb wasting and weakness as the predominant feature and eleven of whom had pain in the affected limbs. Significant weight loss was common. In nine patients the deficits were largely or totally reversible. Important variations from the classical features were observed. Only five patients displayed asymmetric proximal lower limb wasting, weakness and pain, motor deficits in the remainder being either unilateral or bilateral and symmetrical. The shoulder girdle and arms were also involved in two patients. Proximal limb pain was not invariable, a distal sensory peripheral neuropathy was common, and diabetic control at diagnosis was likely to be good. No prognostic factors were identified. Thus, not all patients with diabetic amyotrophy exhibit the classically-described features. Other than careful clinical examination, a thorough bilateral electromyographic and nerve conduction study remains the most helpful diagnostic test. Appreciation of the clinical spectrum and context of diabetic amyotrophy should facilitate its differentiation from other disorders, including other forms of diabetic neuropathy.
Diabetic Neuropathies↗
Cytokines: an expanding network of immuno-inflammatory hormones.
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Animals↗