Professor Len Harrison is a Senior Principal Research Fellow of the National Health and Medical Research Council (NH&MRC), Australia. He is head of the Autoimmunity and Transplantation Division at the Walter and Eliza Hall Institute of Medical Research in Melbourne and is Professor/Director of the Burnet Clinical Research Unit and Department of Immunology and Allergy at the Royal Melbourne Hospital. He has authored 440 research publications, chapters and reviews on the actions of hormones and immune mechanisms of disease. His research is focused on preventing diabetes.
Professor Harrison has received a number of awards for research, including a C.J. Martin Fellowship from the NH&MRC, the Wellcome (Glaxo) Australia Medal, the Susman Prize from the Australasian College of Physicians, the Kellion Medal from the Australian Diabetes Society and the Rumbough Award for scientific excellence from the Juvenile Diabetes Research Foundation (JDRF) International. His service commitments have included: the Medical Research Committee and the Health Research Ethics Committee of the NH&MRC, Secretary and President of the Australian Diabetes Society, Director of the Australian Society for Medical Research, Chairman of the Scientific Review Committee of the JDRF, member of the JDRF Research Advisory Board, Chair of the Professional Advisory Panel of JDRF Australia and President of the Immunology of Diabetes Society.
He is married to fellow researcher, Dr. Margo Honeyman and his interests outside medicine and research are family, writing, farming and tennis.
Abstract: Type 1 diabetes (T1D) is caused by T cell-mediated destruction of the pancreatic insulin-producing beta cells. While the role of CD4(+) T cells in the pathogenesis of T1D is accepted widely, the epitopes recognized by pathogenic human CD4(+) T cells remain poorly defined. None the less, responses to the N-terminal region of the insulin A-chain have been described. Human CD4(+) T cells from the pancreatic lymph nodes of subjects with T1D respond to the first 15 amino acids of the insulin A-chain. We identified a human leucocyte antigen-DR4-restricted epitope comprising the first 13 amino acids of the insulin A-chain (A1-13), dependent upon generation of a vicinal disulphide bond between adjacent cysteines (A6-A7). Here we describe the analysis of a CD4(+) T cell clone, isolated from a subject with T1D, which recognizes a new HLR-DR4-restricted epitope (KRGIVEQCCTSICS) that overlaps the insulin A1-13 epitope. This is a novel epitope, because the clone responds to proinsulin but not to insulin, T cell recognition requires the last two residues of the C-peptide (Lys, Arg) and recognition does not depend upon a vicinal disulphide bond between the A6 and A7 cysteines. The finding of a further CD4(+) T cell epitope in the N-terminal A-chain region of human insulin underscores the importance of this region as a target of CD4(+) T cell responses in human T1D.
Abstract: OBJECTIVE: In a prospective birth cohort study, we followed infants who had a first-degree relative with type 1 diabetes to investigate the relationship between early growth and infant feeding and the risk of islet autoimmunity. RESEARCH DESIGN AND METHODS: Infants with a first-degree relative with type 1 diabetes were identified during their mother's pregnancy. Dietary intake was recorded prospectively to determine duration of breast-feeding and age at introduction of cow's milk protein, cereals, meat, fruit, and vegetables. At 6-month reviews, length (or height) and weight, antibodies to insulin, GAD65, the tyrosine phosphatase-like insulinoma antigen, and tissue transglutaminase were measured. Islet autoimmunity was defined as persistent elevation of one or more islet antibodies at consecutive 6-month intervals, including the most recent measure, and was the primary outcome measure. RESULTS: Follow-up of 548 subjects for 5.7 +/- 3.2 years identified 46 children with islet autoimmunity. Weight z score and BMI z score were continuous predictors of risk of islet autoimmunity (adjusted hazard ratios 1.43 [95% CI 1.10-1.84], P = 0.007, and 1.29 [1.01-1.67], P = 0.04, respectively). The risk of islet autoimmunity was greater in subjects with weight z score >0 than in those with weight z score < or =0 over time (2.61 [1.26-5.44], P = 0.01). Weight z score and BMI z score at 2 years and change in weight z score between birth and 2 years, but not dietary intake, also predicted risk of islet autoimmunity. CONCLUSIONS: Weight gain in early life predicts risk of islet autoimmunity in children with a first-degree relative with type 1 diabetes.
Abstract: Natural killer T cells (NKT) are a regulatory subset of T lymphocytes whose frequency in peripheral blood is highly variable within the human population. Lower than normal NKT frequencies are associated with increased predisposition to a number of diseases, including type 1 diabetes and some forms of cancer, raising the possibility that an increased frequency may be protective. However, there is little or no understanding of how high NKT frequencies arise or, most importantly, whether the potential exists to boost and maintain NKT levels for therapeutic advantage. Here, we provide a detailed functional and phenotypic characterization of the NKT compartment of a human donor with NKT levels approximately 50 times greater than normal, including an analysis of NKT in her immediate family members. The study focuses upon the characteristics of this donor and her family, but demonstrates more broadly that the size and flexibility of the NKT niche is far greater than envisioned previously. This has important implications for understanding how the human NKT compartment is regulated, and supports the concept that the human NKT compartment might be expanded successfully for therapeutic benefit.
Abstract: Type 1 diabetes (T1D) satisfies many of the criteria for an autoimmune disease. The impact of the environment to promote the development of T1D and the ability to identify individuals at risk for T1D years before clinical presentation afford lessons for other autoimmune diseases, in regard to gene-environment interactions and the potential for rational approaches to pre-clinical diagnosis and prevention. Public health measures aimed at the modern pro-inflammatory environment are required to stem the rising tide not only of T1D but other autoimmune and chronic inflammatory disorders. In the non-obese diabetic (NOD) model of spontaneous autoimmune diabetes, compelling evidence indicates that adaptive autoimmunity to the pancreatic beta cell is initially targeted against proinsulin. Proof-of-principle studies in the NOD mouse, which established that insulin and proinsulin peptides could be applied as tools to induce immune tolerance and protect against diabetes development, await successful translation to at-risk humans. Initial trials of insulin-specific immunotherapy in humans show promise and reveal ways of optimising this approach that are also applicable to other autoimmune diseases.
Abstract: OBJECTIVE: The rising incidence of type 1 diabetes has been attributed to environment, implying a lesser role for genetic susceptibility. However, the rise could be accounted for by either more cases with classic high-risk genes or by cases with other risk genes. Separately, for any degree of genetic susceptibility, age at presentation may decrease in a permissive environment. To examine these possibilities, human leukocyte antigen (HLA) class II DRB1 genes known to confer risk for type 1 diabetes were analyzed in relation to year of birth and age at diagnosis over the last five decades. RESEARCH DESIGN AND METHODS: Caucasoid subjects (n = 462) diagnosed with type 1 diabetes before age 18 between 1950 and 2005 were DRB1 genotyped. RESULTS: Mean +/- SD age at diagnosis, 8.5 +/- 4.5 years, did not differ across decades. Recent diagnosis was associated with a lower proportion but unchanged incidence of the highest-risk DRB1 genotype DR3,4 (2000-2005, 28% vs. 1950-1969, 79%; P < 0.0001) and a higher proportion of lower-risk genotypes DR4,X and DR3,X (2000-2005, 48% vs. 1950-1969, 20%; P = 0.0002). The frequency of the DRX,X genotype was low (<or=3%) across decades. Recent birth was associated with a lower age at diagnosis for lower risk DR3,3 and DR4,4 (P < 0.0001) and DR4,X (P < 0.0001) and DR3,X (P = 0.015) genotypes but not for DR3,4. CONCLUSIONS: The rising incidence and decreasing age at diagnosis of type 1 diabetes is accounted for by the impact of environment on children with lower-risk HLA class II genes, who previously would not have developed type 1 diabetes in childhood.
Abstract: T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.
Abstract: PURPOSE OF REVIEW: To summarize the relevance of the 'accelerator hypothesis' to type 1 diabetes pathogenesis and examine if recent evidence supports the hypothesis. The 'accelerator hypothesis' proposes 'three processes in type 1 diabetes which variably accelerate the loss of beta cells through apoptosis: constitution, insulin resistance and autoimmunity'. RECENT FINDINGS: Insulin resistance is an independent risk factor for progression to clinical type 1 diabetes in people with islet autoimmunity. Higher bodyweight is also associated with type 1 diabetes development although no longitudinal studies have simultaneously assessed bodyweight and insulin resistance in preclinical diabetes. Currently, there is no evidence for the view that accelerated beta-cell apoptosis is due to insulin resistance in the pathogenesis of type 1 diabetes. SUMMARY: Insulin resistance accelerates development of type 1 diabetes in people with islet autoimmunity and insulin deficiency. The increasingly 'obesogenic' environment which promotes insulin resistance could account for the rising incidence of type 1 diabetes.
Abstract: Steroid hormones induce changes in gene expression by binding to intracellular receptors that then translocate to the nucleus. Steroids have also been shown to rapidly modify cell function by binding to surface membrane receptors. We identified a candidate steroid membrane receptor, the progestin and adipoQ receptor (PAQR) 10, a member of the PAQR family, in a screen for genes differentially expressed in mouse pancreatic beta-cells. PAQR10 gene expression was tissue restricted compared with other PAQRs. In the mouse embryonic pancreas, PAQR10 expression mirrored development of the endocrine lineage, with PAQR10 protein expression confined to endocrine islet-duct structures in the late embryo and neonate. In the adult mouse pancreas, PAQR10 was expressed exclusively in islet cells except for its reappearance in ducts of maternal islets during pregnancy. PAQR10 has a predicted molecular mass of 29 kDa, comprises seven transmembrane domains, and, like other PAQRs, is predicted to have an intracellular N-terminus and an extracellular C-terminus. In silico analysis indicated that three members of the PAQR family, PAQRs 9, 10, and 11, have a candidate mitochondrial localization signal (MLS) at the N-terminus. We showed that PAQR10 has a functional N-terminal MLS and that the native protein localizes to mitochondria. PAQR10 is structurally related to some bacterial hemolysins, pore-forming virulence factors that target mitochondria and regulate apoptosis. We propose that PAQR10 may act at the level of the mitochondrion to regulate pancreatic endocrine cell development/survival.
Abstract: Mitochondria of pancreatic beta-cells are potential targets of intrinsic and extrinsic apoptotic pathways in the autoimmune pathogenesis of type 1 diabetes. We aimed to investigate whether cytokine- and FasLigand (FasL)-induced apoptosis is associated with impaired mitochondrial transmembrane potential (Deltapsim) in the pancreatic beta-cell line NIT-1. NIT-1 cells were exposed to the interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma) cytokine combination to induce apoptosis in vitro. Low concentrations of cytokines resulted in Deltapsim impairment, and increasing concentrations had only a minor additional effect. Treatment with the inducible nitric oxide synthase (iNOS) inhibitor Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME) prevented cytokine-mediated Deltapsim impairment, implying that cytokines affect Deltapsim via nitric oxide. The broad-spectrum caspase inhibitor Z-VAD(Ome)-FMK (ZVAD) revealed dichotomic actions. In the presence of ZVAD, cytokine-induced nitrite generation was increased but cell death and Deltapsim impairment were reduced. Deltapsim impairment was also reduced by inhibitors of caspases 1, 6 and 8. Induction of Fas by IL-1beta/IFN-gamma coupled with activation by Super-FasL augmented cytokine-induced cell death. We observed a clear dominance of cytokine- over FasL-induced effects on Deltapsim. Our findings show that IL-1beta/IFN-gamma cytokines have a strong effect to impair Deltaym and prime beta-cells for apoptosis via the intrinsic pathway mediated by iNOS and caspases. Furthermore, at least in NIT-1 cells, the extrinsic FasL/Fas pathway has only a minor additive effect on cytokine-induced Deltapsim impairment.
Abstract: Immune tolerance to self-antigens is physiological. Given a repertoire of self-reactive, potentially pathogenic lymphocytes, therapeutic options to diminish autoimmune disease risk include deletion, reduced activation or increased regulation of self-reactive lymphocytes by means that mimic or promote physiological mechanisms of immunity. Vaccination with self-antigen to promote self-antigen-specific tolerance, 'negative vaccination', may represent the most specific and potentially safest means of averting autoimmune disease. This strategy is therapeutically effective in inbred rodent models but its translation in humans has failed to meet expectations. This failure can be attributed to the use of suboptimal dosage regimens in end-stage disease, as well as other factors. This review focuses on vaccination against self-antigen in type 1 diabetes, an autoimmune disease unique in that individuals at risk can be identified years before clinical presentation. Moreover, the spontaneously diabetic non-obese diabetic mouse, which mimics human type 1 diabetes in many ways, has provided 'proof-of-concept' for negative vaccination. Recent trials of a nasal insulin vaccine in humans at risk of type 1 diabetes provide evidence of tolerance induction as a basis for clinical efficacy.
Abstract: OBJECTIVE: Beta-cell function in type 1 diabetes clinical trials is commonly measured by C-peptide response to a secretagogue in either a mixed-meal tolerance test (MMTT) or a glucagon stimulation test (GST). The Type 1 Diabetes TrialNet Research Group and the European C-peptide Trial (ECPT) Study Group conducted parallel randomized studies to compare the sensitivity, reproducibility, and tolerability of these procedures. RESEARCH DESIGN AND METHODS: In randomized sequences, 148 TrialNet subjects completed 549 tests with up to 2 MMTT and 2 GST tests on separate days, and 118 ECPT subjects completed 348 tests (up to 3 each) with either two MMTTs or two GSTs. RESULTS: Among individuals with up to 4 years' duration of type 1 diabetes, >85% had measurable stimulated C-peptide values. The MMTT stimulus produced significantly higher concentrations of C-peptide than the GST. Whereas both tests were highly reproducible, the MMTT was significantly more so (R(2) = 0.96 for peak C-peptide response). Overall, the majority of subjects preferred the MMTT, and there were few adverse events. Some older subjects preferred the shorter duration of the GST. Nausea was reported in the majority of GST studies, particularly in the young age-group. CONCLUSIONS: The MMTT is preferred for the assessment of beta-cell function in therapeutic trials in type 1 diabetes.
Abstract: Cells with the phenotype of intraepithelial lymphocytes (IEL) are present systemically and have been implicated in immune regulation. To determine whether IEL undergo homeostatic proliferation and migrate from the small intestine, we analysed the fate of congenic IEL transferred into lymphopenic mice. Donor IEL homed to the small intestinal epithelium, where they expanded in an IL-15-dependent manner and expressed CD69, CD44 and CD103; proliferation did not occur in the spleen, the main other site of IEL detection early after transfer. By 12 days after transfer, a small proportion of intestinal IEL had up-regulated the trafficking molecule CD62L. Four weeks after transfer, donor IEL with a CD69-CD44hiCD103- phenotype similar to memory T cells were present in spleen and other extra-intestinal sites. Treatment of mice with blocking antibody to CD62L reduced appearance of cells in mesenteric lymph nodes; treatment with FTY720, a sphingosine 1-phosphate receptor agonist that blocks egress of T cells from lymph nodes, reduced appearance of cells in spleen. The distribution of TCR alphabeta and gammadelta IEL varied between organs, alphabeta IEL being predominant. IEL proliferation and emigration under lymphopenic conditions suggests similar IEL turnover, albeit at a lower level, under physiological conditions.
Abstract: OBJECTIVE: To determine the relationship of intravenous (IVGTT) and oral (OGTT) glucose tolerance tests abnormalities to diabetes development in a high-risk pre-diabetic cohort and to identify an optimal testing strategy for detecting preclinical diabetes. STUDY DESIGN: Diabetes Prevention Trial-Type 1 Diabetes (DPT-1) randomized subjects to oral (n = 372) and parenteral (n = 339) insulin prevention trials. Subjects were followed with IVGTTs and OGTTs. Factors associated with progression to diabetes were evaluated. RESULTS: Survival analysis revealed that higher quartiles of 2-hour glucose and lower quartiles of first phase insulin response (FPIR) at baseline were associated with decreased diabetes-free survival. Cox proportional hazards modeling showed that baseline body mass index (BMI), FPIR, and 2-hour glucose levels were significantly associated with an increased hazard for diabetes. On testing performed within 6 months of diabetes diagnosis, 3% (1/32) had normal FPIR and normal 2-hour glucose on OGTT. The sensitivities for impaired glucose tolerance (IGT) and low FPIR performed within 6 months of diabetes diagnosis were equivalent (76% vs 73%). CONCLUSIONS: Most (97%) subjects had abnormal IVGTTs and/or OGTTs before the development of diabetes. The highest sensitivity is achieved using both tests.
Abstract: Studies on the thymic ontogeny of naturally arising CD4(+)CD25(+) regulatory T cells (TR cells) are complicated by the contamination of recirculating cells from the periphery (both activated CD4(+) T and TR cells). We investigated TR cells in anti-CD4 antibody transgenic (Tg) (GK) mice that continuously deplete peripheral CD4 T cells but not thymocytes so that the generation of thymic TR cells and their developmental requirement can be accurately assessed. We show that in the thymuses of mice that lack peripheral CD4(+) cells, TR cells were present but were fewer in number compared with wild-type (WT) mice. Therefore, we show that peripheral TR cells do re-enter the thymus, comprising 20% of TR cells in the normal thymus. TR cells from both WT and GK mice expressed Foxp3 and GITR, and suppressed the proliferation of CD25(-)CD4(+) T cells. Furthermore, the co-stimulation requirements for TR generation were evaluated in mice with or without peripheral CD4 cells. Splenic TR cells in CD40L(-/-) mice and CTLA4Ig Tg mice were fewer compared with WT mice. Mice deficient in both co-stimulatory pathways had further reduction in splenic TR cells. Unlike the periphery, the reduction in thymic TR cells was only seen for CD40L(-/-) but not for CTLA4Ig Tg mice. Therefore, we found that the co-stimulation requirements for the thymic development of TR cells differed from those for peripheral homeostasis.
Abstract: Peripheral tolerance is required to prevent autoimmune tissue destruction by self-reactive T cells that escape negative selection in the thymus. One mechanism of peripheral tolerance in CD8(+) T cells is their activation by resting dendritic cells (DC). In contrast, DC can be "licensed" by CD4(+) T cells to induce cytotoxic function in CD8(+) T cells. The question that then arises, whether CD4(+) T cell help could impair peripheral tolerance induction in self-reactive CD8(+) T cells, has not been addressed. In this study we show that CD4(+) T cell activation by resting DC results in helper function that transiently promotes the expansion and differentiation of cognate CD8(+) T cells. However, both the CD4(+) and CD8(+) T cell populations ultimately undergo partial deletion and acquire Ag unresponsiveness, disabling their ability to destroy OVA-expressing pancreatic beta cells and cause diabetes. Thus, effective peripheral tolerance can be induced by resting DC in the presence of CD4(+) and CD8(+) T cells with specificity for the same Ag.
Abstract: Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (T(reg)) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4+ T(reg) that suppressed diabetes development, both after adoptive cotransfer with "diabetogenic" spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4+ CD25+ T(reg), CD4+ T(reg) induced by proinsulin DNA were both CD25+ and CD25- and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of T(reg) and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective T(reg), but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity.
Abstract: Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5' of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of "immunological self" and, by analogy with the thymus, may be implicated in peripheral immune tolerance.
Abstract: Type 1 diabetes (T1D) is characterized by immune responses against several autoantigens expressed in pancreatic beta cells. T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) can induce T1D in NOD mice. However, whether immune responses to multiple autoantigens are caused by spreading from one to another or whether they develop independently of each other is unknown. As cytotoxic T cells specific for IGRP were not detected in transgenic NOD mice tolerant to proinsulin, we determined that immune responses against proinsulin are necessary for IGRP-specific T cells to develop. On the other hand, transgenic overexpression of IGRP resulted in loss of intra-islet IGRP-specific T cells but did not protect NOD mice from insulitis or T1D, providing direct evidence that the response against IGRP is downstream of the response to proinsulin. Our results suggest that pathogenic proinsulin-specific immunity in NOD mice subsequently spreads to other antigens such as IGRP.
Abstract: Neonatal thymectomy (NTX) impairs T cell regulation and leads to organ-specific autoimmune disease in susceptible mouse strains. In the NOD mouse model of spontaneous type 1 diabetes, we observed that NTX dramatically accelerated autoimmune pancreatic beta cell destruction and diabetes. NTX had only a minor effect in NOD mice protected from diabetes by transgenic expression of the beta cell autoantigen proinsulin in APCs, inferring that accelerated diabetes after NTX is largely due to failure to regulate proinsulin-specific T cells. NTX markedly impaired the development of intraepithelial lymphocytes (IEL), the number of which was already reduced in euthymic NOD mice compared with control strains. IEL purified from euthymic NOD mice, specifically CD8alphaalpha TCRgammadelta IEL, when transferred into NTX-NOD mice, trafficked to the small intestinal epithelium and prevented diabetes. Transfer of prototypic CD4+CD25+ regulatory T cells also prevented diabetes in NTX-NOD mice; however, the induction of these cells by oral insulin in euthymic mice depended on the integrity of TCRgammadelta IEL. We conclude that TCRgammadelta IEL at the mucosal interface between self and nonself play a key role in maintaining peripheral tolerance both physiologically and during oral tolerance induction.
Abstract: OBJECTIVE: Latent autoimmune diabetes in adults (LADA) is defined as adult-onset diabetes with circulating islet antibodies but not requiring insulin therapy initially. Diagnosing LADA has treatment implications because of the high risk of progression to insulin dependency. Currently, there are no recommendations for islet antibody testing in adult-onset diabetes. In this study, we aimed to develop a clinical screening tool to identify adults at high risk of LADA who require islet antibody testing. RESEARCH DESIGN AND METHODS: Subjects with LADA (n = 102, GAD antibody [GADA]+) and type 2 diabetes (n = 111, GADA-) (aged 30-75 years) were interviewed retrospectively. The clinical features documented were age of onset, acute symptoms of hyperglycemia, BMI, and personal and family history of autoimmune disease. Any clinical feature that was significantly more frequent in LADA was designated as a distinguishing clinical feature. In each subject, a "LADA clinical risk score," based on the total number of distinguishing features, was calculated. A prospective study of adults with newly diagnosed diabetes (n = 130) was used to determine whether the LADA clinical risk score could identify LADA. RESULTS: In the retrospective study, five clinical features were more frequent in LADA compared with type 2 diabetes at diagnosis: 1) age of onset <50 years (P < 0.0001), 2) acute symptoms (P < 0.0001), 3) BMI <25 kg/m2 (P = 0.0004), 4) personal history of autoimmune disease (P = 0.011), and 5) family history of autoimmune disease (P = 0.024). In the prospective study, the presence of at least two of these distinguishing clinical features (LADA clinical risk score > or =2) had a 90% sensitivity and 71% specificity for identifying LADA and a negative predictive value for a LADA clinical risk score < or =1 of 99%. CONCLUSIONS: At least two distinguishing clinical features are found in a majority of patients with LADA at diagnosis and can be used to identify adults with diabetes at higher risk for LADA.
Abstract: The nature of the T-cell response to antigen is governed by the activation state of the antigen-presenting dendritic cell (DC). Immature or resting DCs have been shown to induce T-cell responses that may protect against the development of autoimmune disease. Effectively harnessing this "tolerogenic" effect of resting DCs requires that it be disease-specific and that activation of DCs by manipulation ex vivo is avoided. We reasoned that this could be achieved by transferring in vivo partially differentiated myeloid progenitor cells encoding a disease-specific autoantigen. With the aim of preventing autoimmune diabetes, we transferred myeloid progenitor cells encoding proinsulin into NOD mice. Bone marrow (BM) was cultured in granulocyte macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-beta1, a cytokine combination that expands myeloid cells but inhibits terminal DC differentiation, to yield Gr-1(+)/CD11b(+)/CD11c(-) myeloid progenitor cells and a minor population of CD11c(+)/CD11b(+)/CD86(lo) immature DCs. After transfer, Gr-1(+) myeloid cells acquired the characteristics of resting DCs (CD11c(+)/MHC classII(int)/CD86(lo)/CD40(lo)). Gr-1(+) myeloid cells generated from transgenic NOD mice that expressed proinsulin controlled by a major histocompatibility complex (MHC) class II promoter, but not from wild-type NOD mice, transferred into 4-week-old female NOD mice significantly suppressed diabetes development. The transfer of DC progenitors encoding a disease-specific autoantigen is, therefore, an effective immunotherapeutic strategy that could be applied to humans.
Abstract: We previously described differences in the 50% protective dose and isotype-specific antibody secreting cell (ASC) responses to US and Russian influenza A cold-adapted (ca) donor strains in the lungs of BALB/c mice [Wareing MD, Watson JM, Brooks MJ, Tannock GA. Immunogenic and isotype-specific responses to Russian and US cold-adapted influenza A vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57, and A/Ann Arbor/6/60 (H2N2) in mice. J Med Virol 2001;65(1):171-7]. A/Leningrad/134/17/57(Len/17-ca) was shown to be a superior immunogen to A/Leningrad/134/47/57-ca (Len/47-ca), which, in turn, was superior to A/Ann Arbor/6/60-ca (AA-ca) but no other comparative data exist. In order to extend our findings and determine a means for selecting the most immunogenic ca influenza A vaccine, the intracellular cytokine responses by CD4+ and CD8+ T cells to AA-ca, Len/47-ca and Len/17-ca and their respective wild-type parental viruses were compared in mice. Day 5 after infection with Len/17-ca, when levels of IL-2, -4 and -10 were highest in the mediastinal lymph nodes (MLN) and lungs, was chosen as the optimum time to harvest lymphocytes and 72 h was determined to be the optimum re-stimulation period for lymphocytes by APCs. Under these conditions, the frequency of CD4+ and CD8+ cells expressing cytokines was highest in the lungs compared with the MLN. A dominant IL-6 response was induced, although all virus strains induced a Th1/Th2 cytokine profile. While the CD8+ cytokine response appeared non-specific, the cytokine response elicited in the lungs by CD4+ cells to Len/17-ca-inoculation was greater than that induced by Len/47-ca, or AA/ca. The CD4+ cytokine response in the lungs may be a useful measure of immunogenicity to determine the most effective influenza reassortant for inclusion in vaccines.
Abstract: The homeodomain transcription factor Pdx1 is essential for pancreas development. To investigate the role of Pdx1 in the adult pancreas, we employed a mouse model in which transcription of Pdx1 could be reversibly repressed by administration of doxycycline. Repression of Pdx1 in adult mice impaired expression of insulin and glucagon, leading to diabetes within 14 days. Pdx1 repression was associated with increased cell proliferation predominantly in the exocrine pancreas and upregulation of genes implicated in pancreas regeneration. Following withdrawal of doxycycline and derepression of Pdx1, normoglycemia was restored within 28 days; during this period, Pdx1(+)/Ins(+) and Pdx(+)/Ins(-) cells were observed in association with the duct epithelia. These findings confirm that Pdx1 is required for beta-cell function in the adult pancreas and indicate that in the absence of Pdx1 expression, a regenerative program is initiated with the potential for Pdx1-dependent beta-cell neogenesis.
Abstract: 'Latent autoimmune diabetes in adults' (LADA) is the term coined to describe adults who have a slowly progressive form of autoimmune or type 1 diabetes that can be treated initially without insulin injections. The diagnosis of LADA is currently based on three clinical criteria: (1) adult age at onset of diabetes; (2) the presence of circulating islet autoantibodies, which distinguishes LADA from type 2 diabetes; and (3) insulin independence at diagnosis, which distinguishes LADA from classic type 1 diabetes. The prevalence of LADA in adults presenting with non-insulin-requiring diabetes is approximately 10%. Recognition of LADA expands the concept and prevalence of autoimmune diabetes, but LADA remains poorly understood at both a clinical and research level. In this perspective, we review the nomenclature, diagnostic criteria, genetics, pathology and therapy of LADA, to arrive at recommendations that might advance knowledge and management of this form of diabetes.
Abstract: The autoimmune process that destroys the insulin-producing pancreatic beta cells in type 1 diabetes (T1D) is targeted at insulin and its precursor, proinsulin. T cells that recognize the proximal A-chain of human insulin were identified recently in the pancreatic lymph nodes of subjects who had T1D. To investigate the specificity of proinsulin-specific T cells in T1D, we isolated human CD4(+) T cell clones to proinsulin from the blood of a donor who had T1D. The clones recognized a naturally processed, HLA DR4-restricted epitope within the first 13 amino acids of the A-chain (A1-13) of human insulin. T cell recognition was dependent on the formation of a vicinal disulfide bond between adjacent cysteine residues at A6 and A7, which did not alter binding of the peptide to HLA DR4. CD4(+) T cell clones that recognized this epitope were isolated from an HLA DR4(+) child with autoantibodies to insulin, and therefore, at risk for T1D, but not from two healthy HLA DR4(+) donors. We define for the first time a novel posttranslational modification that is required for T cell recognition of the insulin A-chain in T1D.
Abstract: We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its alpha(6) integrin and alpha-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells.
Abstract: T-cell clones are valuable tools for investigating T-cell specificity in infectious, autoimmune and malignant diseases. T cells specific for clinically-relevant autoantigens are difficult to clone using traditional methods. Here we describe an efficient method for cloning human autoantigen-specific CD4+ T cells pre-labelled with CFSE. Proliferating, antigen-responsive CD4+ cells were identified flow cytometrically by their reduction in CFSE staining and single cells were sorted into separate wells. The conditions (cytokines, mitogens and tissue culture plates) for raising T-cell clones were optimised. Media supplemented with IL-2+IL-4 supported growth of the largest number of antigen-specific clones. Three mitogens, PHA, anti-CD3 and anti-CD3+anti-CD28, each stimulated the growth of similar numbers of antigen-specific clones. Cloning efficiency was similar in flat- and round-bottom plates. Based on these findings, IL-2+IL-4, anti-CD3 and round-bottom plates were used to clone FACS-sorted autoantigen-specific CFSE-labelled CD4+ T cells. Sixty proinsulin- and 47 glutamic acid decarboxylase-specific clones were obtained from six and two donors, respectively. In conclusion, the CFSE-based method is ideal for cloning rare, autoantigen-specific, human CD4+ T cells.
Abstract: A dendritic cell (DC) imbalance with a marked deficiency in CD4- 8+ DC occurs in non-obese diabetic (NOD) mice, a model of human autoimmune diabetes mellitus. Using a NOD congenic mouse strain, we find that this CD4- 8+ DC deficiency is associated with a gene segment on chromosome 4, which also encompasses non-MHC diabetes susceptibility loci. Treatment of NOD mice with fms-like tyrosine kinase 3 ligand (FL) enhances the level of CD4- 8+ DC, temporarily reversing the DC subtype imbalance. At the same time, fms-like tyrosine kinase 3 ligand treatment blocks early stages of the diabetogenic process and with appropriately timed administration can completely prevent diabetes development. This points to a possible clinical use of FL to prevent autoimmune disease.
Abstract: The epidermal growth factor (EGF) family is implicated in the development and function of multiple cells and organs, including the pancreas. We used a serum-free, low-cell density culture system to investigate the effect of EGFs on fetal pancreas cells. By RT-PCR, the EGF receptors ErbB 1-3 were detected in the developing mouse pancreas between embryonic day (E) 13.5 and E17.5, whereas ErbB4 was not detected until E17.5. The presence but not absence of the basement membrane glycoprotein laminin-1, betacellulin, and to a lesser extent EGF, transforming growth factor alpha, heparin binding EGF, and epiregulin induced E15.5 pancreatic cells to proliferate and form cystoid and solid colonies. These results demonstrate that laminin-1 and EGF signaling pathways interact to promote pancreas development.
Abstract: Type 1 diabetes (T1D) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the pancreatic islets. Primary prevention by traditional 'positive' vaccination awaits evidence that infectious agents trigger T1D. The pre-clinical phase of T1D, in which at-risk individuals can be infected by the presence of autoantibodies to islet antigens, is a window for secondary prevention. The Holy Grail of therapy is 'negative' vaccination to induce immune tolerance against disease-specific autoantigens that drive immune-mediated pathology. This can be achieved by administering autoantigen via a 'tolergenic' (e.g., muscosal, intradermal) route, cell (e.g., resting dendritic cell), mode (e.g., with blockade of c0-stimulation molecules) or form (as an 'altered peptide ligand'). Although effective in rodent models of autoimmune disease, these strategies have so far been disappointing in humans. This review discusses the prospects of vaccination to prevent T1D, focusing on autoantigen-specific mucosal tolerance.
Abstract: T lymphocytes (pivotal in many inflammatory pathologies) are targets for glucocorticoid hormone (GC). How TCR-mediated activation and GC signaling via glucocorticoid receptor (GR) impact on T-cell fates is not fully defined. We delineated here the expression of a recently identified glucocorticoid-induced TNF receptor (GITR) induced by GC and by TCR-mediated T-cell activation in GC receptor (GR)-deficient mice (GR-/-). We also compared the action of GC on GITR+ and GITR- T cells by monitoring apoptosis, proliferation and cytokine production stimulated by anti-CD3 antibody. By using GR-/- mice, we observed that the development of GITR+ T cells (both in thymus and periphery) is not dependent upon GR signaling. This contradicts the implication of GITR's name reflecting GC induction. TCR-mediated T-cell activation induced GITR expression in both GR+/+ and GR-/- cells. Somewhat unexpectedly, there was very modest GITR upregulation on GR+/+ T cells by a range of GC doses (10(-8) to 10(-6) M). Constitutive expression of GITR by a subset of CD4+ cells did not significantly render them resistant to GC-induced cell death. However, TCR-induced GITR upregulation on GR+/+ T cells was correlated with resistance to GC-mediated apoptosis suggesting that GITR, in conjunction with other (as yet unidentified) TCR-induced factors, protects T cells from apoptosis. Thus, even though GC is a potent inducer of apoptosis of T cells, activated T cells are resistant to GC-mediated killing. Meanwhile, although GC suppressed anti-CD3-induced cytokine production, cell proliferation was unaffected by GC in GR+/+ mice. GR deficiency has no effect on anti-CD3-induced cytokine production and proliferation. Our findings also have implications for GC treatment in that it would be more difficult to abrogate an ongoing T-cell mediated inflammatory response than to prevent its induction.
Abstract: AIMS/HYPOTHESIS: Glucose homeostasis is determined by an interplay between insulin secretion and insulin action. In type 1 diabetes, autoimmune destruction of pancreatic beta cells leads to impaired insulin secretion. However, the contribution of impaired insulin action (insulin resistance) to the development of type 1 diabetes has received little attention. We investigated whether insulin resistance was a risk factor for progression to type 1 diabetes. METHODS: Islet-antibody-positive first-degree relatives of type 1 diabetes probands were followed for 4.0 years (median). Insulin secretion was measured as first-phase insulin response (FPIR) to intravenous glucose. Insulin resistance was estimated by homeostasis model assessment of insulin resistance (HOMA-R). We compared subjects who progressed (n=43) and subjects who did not progress (n=61) to diabetes, including 21 pairs matched for age, sex, islet antibodies and FPIR. RESULTS: Progressors had higher insulin resistance relative to insulin secretion at baseline (median HOMA-R : FPIR 0.033 vs 0.013, p<0.0001). According to Cox proportional hazards analysis, islet antibody number, FPIR, fasting plasma glucose, fasting serum insulin, HOMA-R and log(HOMA-R : FPIR) were each predictive of progression to diabetes. However, log(HOMA-R : FPIR) (hazard ratio 2.57 per doubling, p<0.001) was the only metabolic variable independently associated with progression. In the matched comparison, progressors had higher fasting glucose, fasting insulin, HOMA-R and HOMA-R : FPIR, both at baseline and during the follow-up pre-clinical phase. CONCLUSIONS/INTERPRETATION: Relatives positive for islet antibodies who progress most rapidly to diabetes have a subtle disturbance of insulin-glucose homeostasis years before the onset of symptoms, distinguished by greater insulin resistance for their level of insulin secretion. Taking steps to reduce this insulin resistance could therefore delay the development of type 1 diabetes.
Abstract: In type 1 diabetes, autoimmune inflammation of pancreatic islets of Langerhans ('insulitis') results in destruction of insulin-producing beta cells. Cytokines released from islet-infiltrating mononuclear cells are known to be cytotoxic both directly and by upregulating Fas for FasL-induced apoptosis. To investigate the role of caspase-3, a major effector of apoptosis in beta-cell death, we asked whether cytokine- and/or FasL-induced apoptosis was associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Measurement of caspase-3 activity using a fluorogenic cleavage assay was validated in NOD mouse thymocytes undergoing dexamethasone (Dex)-induced apoptosis. For cytokine-induced apoptosis, NIT-1 cells or islets were exposed to IL-1 beta and IFN-gamma for 24 h. Caspase-3-like activity was increased 2.1+/-0.7 and 2.4+/-0.9-fold in lysates of cytokine-treated NIT-1 cells and NOD mouse islets, respectively. However, NIT-1 cells exhibited 2.1% (4.7 pg active caspase-3/microg protein) and islets 0.8% (1.9 pg active caspase-3/microg protein) of the active caspase-3 content observed in Dex-treated thymocytes (225.1 pg active caspase-3/microg protein). After 24 h cytokine-exposure, the percentage of Fas-positive NIT-1 cells increased from 1.4+/-1.1 to 29.7+/-11.6%. Addition of FasL for a further 3 h increased caspase-3-like activity an additional 1.8-fold in cytokine-treated NIT-1 cells. In summary, exposure of NOD mouse insulinoma cells or islets to IL-1 beta and IFN-gamma for 24 h induced caspase-3-like activity that, in the case of insulinoma cells at least, can be further enhanced by interaction of cytokine-induced Fas receptor with FasL. Compared to thymocytes, insulinoma cells and islets from NOD mice were characterised by low basal and cytokine-induced caspase-3 activity.
Abstract: The ability to measure proliferation of autoantigen-specific T cells is critical for the evaluation of cellular immune function. Using a novel, sensitive, CFSE-based assay, we were able to directly quantitate autoantigen-specific CD4(+) T cell proliferation. However, peripheral blood cells from healthy, pre-diabetic and diabetic donors exhibited overlap in responses to glutamic acid decarboxylase (GAD65) and proinsulin (PI). This indicates that autoantigen-induced CD4(+) T cell proliferation in a functionally complex cell population may not discriminate disease in the general population. Clear discrimination was found between diabetic and healthy sibs, suggesting the need to standardize the genetic and environmental background. In addition, the ability of the CFSE assay to allow analysis of the phenotype and function of autoantigen-responsive T cells may improve discrimination.
Abstract: Mutations in the triple PDZ domain-containing protein harmonin have been identified as the cause of Usher deafness syndrome type 1C. Independently, we identified harmonin in a screen for genes expressed in pancreatic beta cells. Using a yeast two-hybrid assay, we show that the first PDZ domain of harmonin interacts with a novel protein, designated harp for harmonin-interacting, ankyrin repeat-containing protein. This interaction was confirmed in an over-expression system and in mammalian cells, and shown to be mediated by the three C-terminal amino acids of harp. Harp is expressed in many of the same epithelia as harmonin and co-localization of native harp and harmonin was demonstrated by confocal microscopy in pancreatic duct epithelium and in a pancreatic beta-cell line. Harp, predicted molecular mass 48 kDa, has a domain structure which includes three ankyrin repeats and a sterile alpha motif. Human harp maps to chromosome 16, and its mouse homologue to chromosome 7. Sequences with similarity to harp include the sans gene, mutations of which are responsible for deafness in the Jackson shaker 2 (js) mutant mouse and in human Usher syndrome type 1G. The functional domain structures of harp and harmonin, their interaction under native conditions and their co-localization suggest they constitute a scaffolding complex to facilitate signal transduction in epithelia.
Abstract: OBJECTIVE: Mucosal administration of insulin retards development of autoimmune diabetes in the nonobese diabetic mouse model. We conducted a double-blind crossover study in humans at risk for type 1 diabetes to determine if intranasal insulin was safe, in particular did not accelerate beta-cell destruction, and could induce immune effects consistent with mucosal tolerance. RESEARCH DESIGN AND METHODS: A total of 38 individuals, median age 10.8 years, with antibodies to one or more pancreatic islet antigens (insulin, GAD65, or tyrosine phosphatase-like insulinoma antigen 2) were randomized to treatment with intranasal insulin (1.6 mg) or a carrier solution, daily for 10 days and then 2 days a week for 6 months, before crossover. The primary outcome was beta-cell function measured as first-phase insulin response (FPIR) to intravenous glucose at 0, 6, and 12 months and then yearly; the secondary outcome was immunity to islet antigens, measured monthly for 12 months. RESULTS: No local or systemic adverse effects were observed. Diabetes developed in 12 participants with negligible beta-cell function at entry after a median of 1.1 year. Of the remaining 26, the majority had antibodies to two or three islet antigens and FPIR greater than the first percentile at entry, as well as beta-cell function that generally remained stable over a median follow-up of 3.0 years. Intranasal insulin was associated with an increase in antibody and a decrease in T-cell responses to insulin. CONCLUSIONS: Results from this pilot study suggest that intranasal insulin does not accelerate loss of beta-cell function in individuals at risk for type 1 diabetes and induces immune changes consistent with mucosal tolerance to insulin. These findings justify a formal trial to determine if intranasal insulin is immunotherapeutic and retards progression to clinical diabetes.
Abstract: The beta-cell mass in the adult pancreas possesses the ability to undergo limited regeneration following injury. Identifying the progenitor cells involved in this process and understanding the mechanisms leading to their maturation will open new avenues for the treatment of type 1 diabetes. However, despite steady advances in determining the molecular basis of early pancreatic development, the identification of pancreatic stem cells or beta-cell progenitors and the molecular mechanisms underlying beta-cell regeneration remain unclear. Recent advances in the directed differentiation of embryonic and adult stem cells has heightened interest in the possible application of stem cell therapy in the treatment of type 1 diabetes. Drawing on the expanding knowledge of pancreas development, beta-cell regeneration and stem cell research, this review focuses on progenitor cells in the adult pancreas as a potential source of beta-cells.
Abstract: The results of trials in which autoantigens have been fed to individuals affected by autoimmune diseases - multiple sclerosis, rheumatoid arthritis and type 1 diabetes - have been disappointing in terms of clinical improvement. This is in striking contrast to the results in experimental rodent models of these diseases. The outcome of the recent DPT-1 trial testing oral insulin in individuals at risk of type 1 diabetes was also disappointing, in contrast to the effects of oral insulin in the non-obese diabetic (NOD) mouse model of type 1 diabetes. However, it is premature to conclude that mucosal tolerance works only in in-bred rodents and not in humans with autoimmune disease. Except for oral insulin in DPT-1, the human trials were performed in individuals with end-stage disease when this form of immune regulation might not be expected to be effective. Importantly, in no trial was an immune response to the autoantigen documented, to demonstrate that the dose was at least bioavailable. Furthermore, mucosal autoantigen administration is a 'double-edged sword' and in rodents can lead not only to regulatory and protective immunity but also to pathogenic, tissue-destructive immunity and exacerbation of autoimmune disease. When suppression of autoimmune disease is observed it may be because autoantigen was administered under conditions which minimize induction of pathogenic immunity. Thus, clinical protocols for mucosal autoantigen administration may need to be modified to favor induction of regulatory immunity. In this short review, we discuss recent studies in autoimmune diabetes-prone NOD mice indicating that with novel modifications mucosal autoantigen administration could be harnessed to prevent type 1 diabetes in humans.
Abstract: The non-obese diabetic (NOD) mouse is a unique and invaluable model of autoimmune disease, in particular type 1 diabetes. Bone marrow transplantation as a therapy for type 1 diabetes has been explored in NOD mice. NOD mice require higher doses of conditioning irradiation for successful allogeneic bone marrow transplantation, suggesting that NOD hematopoietic cells are radioresistant compared to those of other mouse strains. However, studies of hematopoietic reconstitution in NOD mice are hampered by the lack of mice bearing a suitable cell-surface marker that would allow transferred cells or their progeny to be distinguished. In order to monitor hematopoietic reconstitution in NOD mice we generated congenic NOD mice that carry the alternative allelic form of the pan-leukocyte alloantigen CD45. Following irradiation and congenic bone marrow transplantation, we found that the myeloid lineage was rapidly reconstituted by cells of donor origin but substantial numbers of recipient T lymphocytes persisted even after supra-lethal irradiation. This indicates that radiation resistance in the NOD hematopoietic compartment is a property primarily of mature T lymphocytes.
Abstract: In the pathogenesis of autoimmune type 1 diabetes, the apoptosis receptor Fas appears de novo on the surface of insulin-producing beta-cells. Fas expression is thought to be induced by proinflammatory cytokines, such as IL-1beta, interferon-gamma (IFNgamma), and TNFalpha, released by islet-infiltrating mononuclear cells. To determine whether beta-cells can modulate their sensitivity to apoptosis at the level of Fas, we investigated the effect of Fas ligand (FasL) on surface expression of Fas in NIT-1 insulinoma cells from nonobese diabetic (NOD) mice prone to autoimmune diabetes and islet cells from NOD and nonautoimmune BALB/c mice. In NIT-1 insulinoma cells, Fas expression induced by the cytokine combination IL-1beta and IFNgamma was reduced in the presence of FasL, whereas in islet cells Fas expression was unaffected by FasL. The effect of FasL on NIT-1 cells was evident during and after the induction of Fas expression by IL-1beta and IFNgamma. Thus, FasL down-regulates cytokine-induced Fas expression in NOD mouse-derived NIT-1 cells, but not in NOD or BALB/c mouse islets. The ability of NIT-1 cells to down-regulate Fas receptor in response to ligation is similar to that of a variety of tumor cells, which may use this mechanism to escape destruction by cytotoxic T cells. Islets apparently cannot protect themselves against FasL-induced apoptosis by down-regulating the Fas receptor. Understanding how NIT-1 insulinoma cells down-regulate Fas receptor in response to ligation by FasL has therapeutic implications for protecting normal beta-cells in autoimmune type 1 diabetes.
Abstract: Bone marrow or hematopoietic stem cell transplantation is a potential treatment for autoimmune disease. The clinical application of this approach is, however, limited by the risks associated with allogeneic transplantation. In contrast, syngeneic transplantation would be safe and have wide clinical application. Because T cell tolerance can be induced by presenting antigen on resting antigen-presenting cells (APCs), we reasoned that hematopoietic stem cells engineered to express autoantigen in resting APCs could be used to prevent autoimmune disease. Proinsulin is a major autoantigen associated with pancreatic beta cell destruction in humans with type 1 diabetes (T1D) and in autoimmune NOD mice. Here, we demonstrate that syngeneic transplantation of hematopoietic stem cells encoding proinsulin transgenically targeted to APCs totally prevents the development of spontaneous autoimmune diabetes in NOD mice. This antigen-specific immunotherapeutic strategy could be applied to prevent T1D and other autoimmune diseases in humans.
Abstract: Insulin is a major target of the autoimmune response associated with destruction of pancreatic beta cells in type 1 diabetes. A peptide that spans the junction of the insulin B chain and the connecting (C) peptide in proinsulin has been reported to stimulate T cells from humans at risk for type 1 diabetes and autoimmune diabetes-prone NOD mice. Here we show that proinsulin B24-C36 peptide binds to I-A(g7), the MHC class II molecule of the NOD mouse, and, after intranasal administration, induces regulatory CD4(+) T cells that, in the absence of CD8(+) T cells, block the adoptive transfer of diabetes. Curiously, however, intranasal B24-C36 did not inhibit development of spontaneous diabetes in treated mice. We then determined that B24-C36, and its core sequence B25-C34, bind to K(d), the NOD mouse MHC class I molecule, and elicit CD8(+) CTLs. When the CD8(+) T lymphocyte epitope was truncated at the C34 valine anchor residue for binding to K(d), the residual CD4(+) T cell epitope, B24-C32/33, significantly inhibited diabetes development after a single intranasal dose. This study identifies a novel CTL epitope in proinsulin and demonstrates that the therapeutic potential of a "tolerogenic" autoantigen peptide can be compromised by the presence of an integral CTL epitope.
Abstract: The ultimate goal of any treatment for autoimmune diseases is antigen- and/or site-specific suppression of pathology. Autoaggressive lymphocytes need to be eliminated or controlled to prevent tissue damage and halt the progression of clinical disease. Strong evidence is emerging that the induction of regulatory T (T(Reg)) cells by autoantigens can suppress disease, even if the primary, initiating autoantigens are unknown and if inflammation is progressive. An advantage of these autoreactive T(Reg) cells is their ability to act as bystander suppressors and dampen inflammation in a site-specific manner in response to cognate antigen expressed locally by affected tissues. In this review, we consider the nature and function of such antigen-specific T(Reg) cells, and strategies for their therapeutic induction are discussed.
Abstract: We aimed to generate T-cell clones specific for human pre-proinsulin. An HLA DQ8, CD4+ T-cell clone that recognised a 10mer (C65-A9) peptide from pre-proinsulin was isolated. Further analysis revealed that the clone responded neither to recombinant proinsulin nor to re-synthesised C65-A9 peptide. Analysis of the original peptide revealed minor contamination (<0.5%) with an N-terminal Fmoc adduct. This peptide was synthesised and shown to stimulate the clone. Thus, Fmoc-modified peptides, which are common contaminants in synthetic peptides, can stimulate human CD4+ T-cells. This finding has important implications for the use of synthetic peptides in screening and epitope mapping studies and their use as vaccines in humans.
Abstract: We describe a strategy for identifying ligands of human leukocyte antigen (HLA) class I molecules based on a peptide library-mediated in vitro assembly of recombinant class I molecules. We established a microscale class I assembly assay and used a capture ELISA to quantify the assembled HLA-peptide complexes. The identity of the bound ligands was then deduced by mass spectrometry. In this method, HLA complexes assembled in vitro in the presence of components of a mixture of peptides were immunoprecipitated and the bound peptide(s) identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This process of epitope extraction is robust and can be used with complex mixtures containing in excess of 300 candidate ligands. A library of overlapping peptides representing all potential octamers, nonamers and decamers from human preproinsulin was synthesized using unique library chemistry. Peptides from the library were used to initiate assembly of recombinant HLA-B8, HLA-B15 and HLA-A2, facilitating the identification of candidate T-cell epitopes from preproinsulin.
Abstract: The Melbourne Prediabetes Family Study, a prospective study of first-degree relatives of people with type 1 diabetes (T1D), provided an opportunity to examine late-onset autoimmune diabetes within the context of a family history of T1D. We compared genetic, immunologic, and clinical features in relatives of people with T1D, who developed early- versus late-onset diabetes.
Abstract: Type 1 diabetes (T1D) is an autoimmune disease characterised by immunity to pancreatic beta-cell autoantigens, associated with beta-cell destruction leading to insulin deficiency and hyperglycaemia. The rigorous definition of an autoimmune disease requires evidence that an autoantigen elicits pathological immune responses. Using criteria for the pathogenicity of an autoantigen, we examine the evidence for proinsulin as an autoantigen in T1D. We conclude that proinsulin satisfies these criteria. As a corollary, proinsulin is a potential immunotherapeutic tool for the prevention of T1D.
Abstract: Regulatory anti-diabetogenic T cells (T(reg)) can be induced by the mucosal administration of insulin or proinsulin peptides, in the non-obese diabetic (NOD) mouse model of autoimmune type 1 diabetes. Naso-respirtory insulin (which avoids insulin degradation) induces CD8+ alpha(alpha) TCR gamma(delta) T(reg) whereas peptides that bind to the NOD MHC class II molecule, I-Ag7, insulin B9-23 and proinsulin B24-C36, induce CD4+ T(regs) Following naso-respiratory delivery of insulin to NOD mice increased numbers of CD8+ gamma(delta) T cells expressing interleukin (IL)10 are detected in the pancreatic lymph nodes. Neonatal (3 day) thymectomy (NTX) dramatically accelerates diabetes development in NOD mice, associated with lymphopaenia and a block in the maturation of mucosal intrepithelial lymphocytes (IEL), especially extrathymic-derived CD8+ alpha(alpha) TCR gamma(delta) IEL. Regulatory anti-diabetogenic T cells cannot be elicited by naso-respiratory insulin in NTX-NOD mice. Reconstitution of NTX-NOD mice with CD8+ alpha(alpha) TCR gamma(delta) T cells prevents diabetes. CD8+ gamma(delta) T(reg) are conceivably physiological and insulin-specific, induced by exposure to insulin in maternal milk. These findings infer an immunoregulatory role for extrathymic-derived IEL, developing under the influence of the thymus and conditioned by early exposure to the exogenous environment.
Abstract: The ability to measure proliferation of rare antigen-specific T cells among many bystanders is critical for the evaluation of cellular immune function in health and disease. T-cell proliferation in response to antigen has been measured almost exclusively by 3H-thymidine incorporation. This method does not directly identify the phenotype of the proliferating cells and is frequently not sufficiently sensitive to detect rare autoantigen-specific T cells. To overcome these problems, we developed a novel assay for antigen-specific human T-cell proliferation. Peripheral blood mononuclear cells (PBMC) were labelled with the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) and cells that proliferated in response to antigen, with resultant reduction in CFSE intensity, were measured directly by flow cytometry. This assay was more sensitive than 3H-thymidine incorporation and detected the proliferation of rare antigen-specific CD4(+) T cells at 10-fold lower antigen concentrations. It also allowed the phenotype of the proliferating cells to be directly determined. Using the CFSE assay we were able to measure directly the proliferation of human CD4(+) T cells from healthy donors in response to the type 1 diabetes autoantigens glutamic acid decarboxylase (GAD) and proinsulin (PI).
Abstract: Chemically-induced diabetic mice and spontaneously diabetic NOD mice have been valuable as recipients for experimental islet transplantation. However, their maintenance often requires parenteral insulin. Diabetogenic chemicals can be cytotoxic to the host's immune system and to other organs some of which are often used as the transplant site. Procurement of diabetic cohorts in the NOD mouse is problematic due to variability in the age of disease onset. We show that RIP-Kb mice, which spontaneously develop non-immune diabetes due to over-expression of the H-2Kb heavy chain in beta cells, offer many advantages as islet transplant recipients. Diabetes is predictable with a relatively narrow range of onset (4 wk) and blood glucose levels (23.0 +/- 4.0 mmol/l for 39 males at 6 weeks of age). The diabetes is mild enough so that most diabetic mice can be maintained to 40 weeks of age without parenteral insulin. This consistency of diabetes avails that outcomes of intervention can be interpreted with confidence.
Abstract: Aberrant dendritic cell (DC) development and function may contribute to autoimmune disease susceptibility. To address this hypothesis at the level of myeloid lineage-derived DC we compared the development of DC from bone marrow progenitors in vitro and DC populations in vivo in autoimmune diabetes-prone nonobese diabetic (NOD) mice, recombinant congenic nonobese diabetes-resistant (NOR) mice, and unrelated BALB/c and C57BL/6 (BL/6) mice. In GM-CSF/IL-4-supplemented bone marrow cultures, DC developed in significantly greater numbers from NOD than from NOR, BALB/c, and BL/6 mice. Likewise, DC developed in greater numbers from sorted (lineage(-)IL-7Ralpha(-)SCA-1(-)c-kit(+)) NOD myeloid progenitors in either GM-CSF/IL-4 or GM-CSF/stem cell factor (SCF)/TNF-alpha. [(3)H]TdR incorporation indicated that the increased generation of NOD DC was due to higher levels of myeloid progenitor proliferation. Generation of DC with the early-acting hematopoietic growth factor, flt3 ligand, revealed that while the increased DC-generative capacity of myeloid-committed progenitors was restricted to NOD cells, early lineage-uncommitted progenitors from both NOD and NOR had increased DC-generative capacity relative to BALB/c and BL/6. Consistent with these findings, NOD and NOR mice had increased numbers of DC in blood and thymus and NOD had an increased proportion of the putative myeloid DC (CD11c(+)CD11b(+)) subset within spleen. These findings demonstrate that diabetes-prone NOD mice exhibit a myeloid lineage-specific increase in DC generative capacity relative to diabetes-resistant recombinant congenic NOR mice. We propose that an imbalance favoring development of DC from myeloid-committed progenitors predisposes to autoimmune disease in NOD mice.
Abstract: The innate immune system existed prior to the emergence of adaptive immunity in sharks and higher vertebrates. Homologues of many mammalian innate immune-system elements such as the toll-like receptors exist in species as distant as Drosophila. Selective pressure has led to the development of highly conserved, soluble, and cell-surface receptors that recognize functionally essential molecules shared by microbial pathogens. It is thought that molecular patterns that exquisitely distinguish pathogenic cells from mammalian cells are recognized. Therefore, it would seem unlikely that innate immune-system elements should recognize mammalian tissues. However, there is increasing evidence to suggest that this is the case and that innate immunity promotes rejection of transplanted mammalian tissues, particularly those from other species (xenografts). Evidence for innate recognition of mammalian grafts, the nature of this recognition, and the bi-directional interactions between innate and adaptive immunity that contribute to graft rejection are discussed in this review, with the emphasis on nonvascular xenografts.
Abstract: Antigen administration via oral and other mucosal routes can suppress systemic immunity to the antigen and has been used to prevent experimental autoimmune disease. This approach may prove ineffective or even harmful if it leads to a concomitant induction of cytotoxic T lymphocytes (CTLs), and indeed, mucosal administration of the model antigen ovalbumin (OVA) has been shown to elicit CTL activation while simultaneously inducing oral tolerance. Here we show that induction by oral OVA of CTLs in wild-type mice, and of diabetes in mice expressing OVA transgenically in pancreatic beta cells, can be prevented by transiently blocking the CD40 ligand (CD40L). However, CD40L blockade did not diminish oral tolerance, as measured by suppression of systemic OVA-primed T cell proliferation, IFN-gamma secretion, and Ab production. Consistent with these findings, mice lacking CD40 expression could be orally tolerized to OVA. Transient CD40L blockade therefore dissociates pathogenic from protective immunity and should enhance the efficacy and safety of oral tolerance for preventing autoimmune disease.
Abstract: Pancreatic islet xenotransplantation has been advocated as a way of overcoming the shortage of human donor tissue for the treatment of type 1 diabetes. However, the potent immune response against xenografts is a major barrier to their use. We show that a short course of the anti-CD45RB antibody, MB23G2, prolongs survival of fetal pig pancreas grafts in mice. To investigate this effect further we used an i.p. xenograft model in which both donor pig cells and host inflammatory cells can be expediently recovered and analyzed. Graft prolongation was associated with reduced T cell and macrophage infiltration, and reduced production of both T(h)1 and T(h)2 cytokines at the graft site. Graft survival was further increased and T cell infiltration further reduced by combining anti-CD45RB antibody with co-stimulation blockade. The primary effect of anti-CD45RB antibody may be on CD4 T cells, in keeping with the marked reduction in T cell cytokine production in both spleen and graft sites. This concurs with previous studies in allogeneic models that indicate that this antibody perturbs T cell responses by modifying signaling via the TCR. In addition, anti-CD45RB treatment led to reduced expression of LFA-1 and CD62 ligand (CD62L) on CD4 T cells, independent of antigenic challenge. LFA-1 may enhance co-stimulation, and both LFA-1 and CD62L are involved in T cell trafficking. Their reduced expression provides an explanation why the T cell pool is reduced in lymph nodes. We conclude that modulation of inflammation against xenografts by anti-CD45RB antibody is due to effects on both T cell priming and trafficking.
Abstract: Type 1 or insulin-dependent diabetes is an autoimmune disease that causes the selective destruction of insulin-secreting beta cells in the pancreatic islets. Although this is a polygenic disease, with at least 20 genes implicated, the dominant susceptibility locus maps to the major histocompatibility complex (MHC), both in humans and in rodent models. However, in spite of progress on several fronts, the molecular pathology of autoimmune diabetes remains incompletely defined. Major areas of research include environmental trigger factors, the identification and role of beta-cell antigens in inducing and maintaining the autoimmune response, and the nature of the pathogenic and protective lymphocytes involved. In this review, we will focus on these areas to highlight recent advances in understanding the pathogenesis of autoimmune diabetes, drawing extensively on insights gained by studying the non-obese diabetic (NOD) mouse.
Abstract: Extracellular signals that guide pancreas cell development are not well characterized. In an in vitro culture system of dissociated pancreas cells from the E15.5 mouse fetus we show that, in the presence of the extracellular matrix protein laminin-1, bone morphogenetic proteins (BMPs-4, -5 and -6) promote the development of cystic epithelial colonies. Transforming growth factor beta1 (TGF-beta1) and activin A antagonise this effect of BMP-6 and inhibit colony formation. Histological analysis revealed that the colonies are composed of E-cadherin-positive epithelial cells, which in localised areas are insulin positive. The colonies also contain occasional glucagon-positive cells, but no somatostatin- or alpha-amylase-positive cells. These findings indicate that members of the TGF-beta superfamily regulate pancreas epithelial cell development and can promote the formation of islet-like structures in vitro.
Abstract: Previous studies on the contribution of T cell-dependent antibody (Ab) to non-vascular xenograft rejection have yielded conflicting results, being confounded by the presence of recipient T cells and the use of different tissues and immunizing regimens to generate Ab. In the present study, the effect of adoptive transfer of Ab on fetal pig pancreas (FPP) and pig PK15 cell xenografts was examined in T cell-deficient severe combined immune deficiency (SCID) mice. T cell-dependent Abs raised by hyperimmunization with different cell types and by FPP transplantation were compared. Ab raised by hyperimmunization with pig thymocytes exhibited strong binding to pig thymocytes and PK15 cells but did not transfer FPP rejection. IgG1 and IgM, but not IgG3, Abs bound strongly to FPP exocrine and connective tissue, whereas binding to endocrine cells in vitro and in vivo was weak or absent. This pattern of Ab binding was similar to that observed after transplanting FPP into BALB/c mice. Furthermore, serum recovered from BALB/c mice 20 days after FPP transplantation bound strongly to non-endocrine but not endocrine cells and did not transfer FPP rejection. In contrast, serum from mice hyperimmunized with PK15 cells bound strongly to PK15 cells and transferred rejection of intraperitoneal PK15 cells. Furthermore, this serum contained IgG1 and IgM Abs that bound strongly, and IgG3 Abs that bound weakly, to endocrine cells in FPP, and also transferred rejection of FPP in SCID mice. These results indicate that endocrine cells express low concentrations of xenoreactive Ab epitopes and that high Ab concentrations and/or high avidity Abs are required for sufficient endocrine cell binding to cause damage and rejection in the immunodeficient mouse model. Such Abs are not elicited by transplanting FPP into immunocompetent mice. Nevertheless, a contribution of Ab to rejection in immunocompetent mice cannot be excluded.
Abstract: Type 1 diabetes has been associated with an increased frequency of activated T cells and T-cell hyperactivity to non-specific and disease-specific stimuli including the islet autoantigen glutamic acid decarboxylase 65 (GAD). To address whether T-cell hyperactivity is genetic or acquired we measured whole blood cytokines in vitro in response to GAD or tetanus in 18 identical twin pairs, nine discordant for type 1 diabetes. In addition, the activity of 2', 5' oligoadenylate synthetase (OAS) in blood mononuclear cells was measured as a marker of viral infection. Interleukin-2 (IL-2) basally and IL-2 and interferon-gamma (IFN-gamma) in response to GAD, were detected more frequently and at higher levels in diabetic compared to non-diabetic twins. IL-10 was not different between groups. OAS activity was increased in diabetic compared to non-diabetic twins and showed a correlation with basal IL-2 and GAD-stimulated IFN-gamma and IL-10. These findings suggest that T-cell hyperactivity in type 1 diabetes is an acquired trait and could reflect persisting virus expression.
Abstract: OBJECTIVE: Serum islet antibodies signify increased risk for type 1 diabetes (T1D). Knowledge of the relationship between age and seroconversion would guide screening for at-risk individuals. We aimed to determine the effectiveness of islet antibody screening in early childhood, in particular the proportion of negative children who subsequently seroconverted. METHODS: We identified 554 children with a first-degree relative with T1D who had tested negative for islet cell antibodies (ICA) and insulin autoantibodies (IAA) when first screened at a mean age of 7.2 yr. Of 423 who were eligible, 350 consented to re-testing for ICA and IAA and antibodies to glutamic acid decarboxylase (GADAb) and tyrosine phosphatase-like insulinoma antigen IA-2 (IA2Ab) at a mean age of 11.1 yr. GADAb and IA2Ab were measured in 239 of the initial stored samples. RESULTS: Of the 350 children who tested negative at first screening, 12 (3.4%) subsequently seroconverted, becoming positive for ICA (n = 4), IAA (n = 7), GADAb (n = 6) or IA2Ab (n = 2). Of 239 initially negative for ICA and IAA, 8/239 (3.3%) now tested positive for GADAb (n = 7) or IA2Ab (n = 1). Four of these children were positive for GADAb in both tests; the one child initially positive for IA2Ab only was positive for all four antibodies 4.6 yr later and developed diabetes. CONCLUSION: Screening for ICA and IAA failed to identify 2-3% of genetically at-risk children who subsequently developed islet antibodies. Testing for GADAb and IA2Ab would not have avoided this. Maximizing the sensitivity of detecting risk for T1D requires repeat screening for islet antibodies throughout childhood.
Abstract: Tissue function is regulated by the extracellular microenvironment including cell basement membranes, in which laminins are a major component. Previously, we found that laminin-1 promotes differentiation and survival of pancreatic islet cells. Here we characterize the expression pattern of laminins and their integrin receptors in adult pancreas. Although they are expressed in the basement membrane of acinar cells and duct epithelium, no laminin chains examined were detected extracellularly in the pancreatic islets. In contrast to laminin beta(1)- and gamma(1)-chains, the alpha(1)-chain, unique to laminin-1, was not detected. Laminin-10 (alpha(5)beta(1)gamma(1)) was expressed in acinar tissue, whereas laminins-2 (alpha(2)beta(1)gamma(1)) and -10 were expressed in the blood vessels. The laminin connector molecule, nidogen-1, had a distribution similar to that of laminin beta(1) and gamma(1), whereas fibulin-1 and -2, which compete with nidogen-1, were mostly confined to blood vessels. Integrin subunits alpha(6) and alpha(3) were detected in acinar cells and duct epithelial cells, but alpha(6) was absent in islet cells. Integrin alpha(6)beta(4) was detected only in duct cells, alpha(6)beta(1) in both acinar and ductal cells, and alpha(3)beta(1) in acinar, duct, and islet cells. These findings are a basis for further investigation of the role of extracellular matrix molecules and their receptors in pancreas function.
Abstract: Cell lineage development is a finely tuned process of proliferation and differentiation, survival and apoptosis, that is regulated by numerous extracellular signals. Here we review some of the extracellular signals--including insoluble cell-cell and extracellular matrix-cell interactions, as well as soluble factors--that appear critical for pancreatic beta-cell development. Knowledge of how these signals control the development of pancreatic endocrine stem/precursor cells into fully functional insulin-secreting beta cells is a platform for the restoration of beta-cell function and the cure therapy of type 1 diabetes.
Abstract: Rotavirus infection in children at risk of developing type 1 diabetes has been temporally associated with development of pancreatic islet autoantibodies. In this study, nonobese diabetic mice were shown to be susceptible to rhesus rotavirus infection and pancreatic islets from nonobese diabetic mice, nonobese diabetes-resistant mice, fetal pigs, and macaque monkeys supported various degrees of rotavirus growth. Human rotaviruses replicated in monkey islets only. This islet susceptibility shows that rotavirus infection of the pancreas in vivo might be possible.
Abstract: A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.
Abstract: Characterizing the interaction between major histocompatibility complex (MHC) molecules and antigenic peptides is critical for understanding immunity and developing immunotherapies for autoimmune diseases and cancer. To identify the peptide binding motif and predict peptides that bind to the human MHC classII molecule HLA-DR4(*0401), we applied a fuzzy neural network (FNN) capable of extracting the relationship between input and output. Analysis of the peptide binding motif revealed that the hydrophilicity of the position 1 residue located on the N-terminal side of the nonamer (9mer) was the most important variable and that the van der Waals volume and hydrophilicity of the position 6 residue and the hydrophilicity of the position 7 residue were also important variables. The estimation accuracy (A(ROC) value) was high and the binding motif extracted from the FNN agreed with that derived experimentally. This study demonstrates that FNN modeling allows candidate antigenic peptides to be selected without the need for further experiments.
Abstract: Circulating antibodies to pancreatic beta-cell antigens are markers of islet autoimmunity. In first-degree relatives of persons with type 1 diabetes, the levels and range of antigen specificities of these islet antibodies reflect the risk for clinical diabetes. However, in the general population, in which the disease prevalence is up to 30-fold lower, the predictive value of islet antibodies is correspondingly less. Islet antibody assays are primarily research tools to identify 'prediabetic' individuals for secondary prevention trials, but can also discriminate type 1 diabetes in several clinical situations. Loss of first-phase insulin response (FPIR) to intravenous glucose signifies imminent diabetes, but FPIR is normal in most islet-antibody-positive individuals. The contribution of a single FPIR measurement to risk assessment is therefore limited, but rate of fall of FPIR may be a useful predictor. Although beta cells are destroyed by autoreactive T cells, the assay of islet antigen-reactive T cells is not routine. Genetically, the major histocompatibility complex encoding human leukocyte antigen (HLA) alleles accounts for about 50% of familial clustering of type 1 diabetes. HLA typing is not diagnostic, but can be used to differentiate high- from low-risk individuals, e.g. at birth. While 'preclinical' diagnosis raises important medical and ethical questions, an optimized screening strategy provides a basis for counselling and follow-up. Recent knowledge of disease mechanisms and 'proof-of-principle' in the non-obese diabetic (NOD) mouse model justify expectations that type 1 diabetes is preventable, and even intervention that only delays onset of clinical diabetes is likely to be cost-effective.
Abstract: The expression of pro(insulin) in the thymus may lead to the negative selection of pro(insulin) autoreactive T cells and peripheral tolerance to this autoantigen in type 1 diabetes (T1D). We investigated whether proinsulin is expressed in the thymus of young nonobese diabetic (NOD) mice, whether T cells from naive NOD female mice at weaning are reactive to mouse proinsulin, and the role of proinsulin as a pathogenic autoantigen in T1D. Proinsulin II mRNA transcripts were detected in the thymus of 2-wk-old NOD mice at similar levels to other control strains. Despite this expression, proinsulin autoreactive T cells were detected in the periphery of 2- to 3-wk-old naive NOD mice. Peripheral T cells reactive to the insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, and islet cell Ag p69 autoantigens were also detected in these mice, indicating that NOD mice are not tolerant to any of these islet autoantigens at this young age. T cell reactivities to proinsulin and islet cell Ag p69 exceeded those to GAD67, and T cell reactivity to proinsulin in the spleen and pancreatic lymph nodes was directed mainly against a p24-33 epitope that spans the B chain/C peptide junction. Intraperitoneal immunization with proinsulin perinatally beginning at 18 days of age delayed the onset and reduced the incidence of T1D. However, s.c. immunization with proinsulin initiated at 5 wk of age accelerated diabetes in female NOD mice. Our findings support the notion that proinsulin p24-33 may be a primary autoantigen epitope in the pathogenesis of T1D in NOD mice.
Abstract: BACKGROUND: The ability to manipulate the development of pancreatic insulin-producing beta cells has implications for the treatment of type 1 diabetes. Previously, we found that laminin-1, a basement membrane trimeric glycoprotein, promotes beta-cell differentiation. We have investigated the mechanism of this effect, using agents that block the receptors for laminin-1, alpha6 integrin, and alpha-dystroglycan (alpha-DG). MATERIALS AND METHODS: Dissociated cells from 13.5-day postcoitum (dpc) fetal mouse pancreas were cultured for 4 days with laminin-1, with and without monoclonal antibodies and other agents known to block integrins or alpha-DG. Fetuses fixed in Bouin's solution or fetal pancreas cells fixed in 4% paraformaldehyde were processed for routine histology and for immunohistology to detect hormone expression and bromodeoxyuridine (BrdU) uptake. RESULTS: Blocking the binding of laminin-1 to alpha6 integrin with a monoclonal antibody, GoH3, abolished cell proliferation (BrdU uptake) and doubled the number of beta cells. Inhibition of molecules involved in alpha6 integrin signaling (phosphotidylinositol 3-kinase, F-actin, or mitogen-activated protein kinase) had a similar effect. Nevertheless, beta cells appeared to develop normally in alpha6 integrin-deficient fetuses. Blocking the binding of laminin-1 to alpha-DG with a monoclonal antibody, IIH6, dramatically decreased the number of beta cells. Heparin, also known to inhibit laminin-1 binding to alpha-DG, had a similar effect. In the presence of heparin, the increase in beta cells in response to blocking alpha integrin with GoH3 was abolished. CONCLUSIONS: These findings reveal an interplay between alpha6 integrin and alpha-DG to regulate laminin-1-induced beta-cell development. Laminin-I had a dominant effect via alpha-DG to promote cell survival and beta-cell differentiation, which was modestly inhibited by alpha6 signaling.
Abstract: Type 1 diabetes (T1D; or insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease with both genetic and environmental components. In addition to the human leukocyte antigen (HLA) complex, the single major genetic contributor of susceptibility, an unknown number of other unidentified genes are required to mediate disease. Although many loci conferring susceptibility to T1D have been mapped, their identification has proven problematic due to the complex nature of this disease. Our strategy for finding T1D susceptibility genes has been to test for human homologues of loci implicated in diabetes-prone NOD (non-obese diabetic) mice, together with application of biologically relevant stratification methods. We report here a new susceptibility locus, IDDM18, located near the interleukin-12 (IL-12)p40 gene, IL12B. Significant bias in transmission of IL12B alleles was observed in affected sibpairs and was confirmed in an independent cohort of simplex families. A single base change in the 3' UTR showed strong linkage disequilibrium with the T1D susceptibility locus. The IL12B 3' UTR alleles showed different levels of expression in cell lines. Variation in IL-12p40 production may influence T-cell responses crucial for either mediating or protecting against this and other autoimmune diseases.
Abstract: Administration of antigens via mucosal routes, such as orally or intranasally, can induce specific immunological tolerance and has been used as a rational basis for the treatment of autoimmune diseases, including type 1 diabetes. Recently, however, orally delivered antigens were shown to induce CD8 cytotoxic T-lymphocytes (CTLs) capable of causing autoimmune diabetes. In this report, we have examined several mucosal routes for their ability to induce CTLs and autoimmune diabetes, with the aim of identifying approaches that would maximize tolerance and minimize CTL generation. In normal C57BL/6 mice, ovalbumin (OVA) delivered by either the oral or nasal routes or by aerosol inhalation was able to prime CTL immunity in both high- and low-dose regimens. To address the relevance of these CTLs to autoimmune disease, OVA was given to mice that transgenically expressed this antigen in their pancreatic beta-cells. Irrespective of antigen dose or the route of delivery, mucosal OVA triggered diabetes, particularly after intranasal administration. These findings suggest that CTL immunity is likely to be a consequence of mucosal antigen delivery, regardless of the regimen, and should be considered in the clinical application of mucosal tolerance to autoimmune disease prevention.
Abstract: Nonvascularized xenograft rejection is T cell mediated, but is dependent on initial macrophage (Mphi) infiltration. We developed an i.p. transplant model to define the roles of Mphi and T cells in xenograft rejection. Nonobese diabetic or BALB/c mice were injected i.p. with xenogeneic, allogeneic, or syngeneic cells, and the responding cells in subsequent lavages were assessed by flow cytometry and adoptive transfer. Neutrophils and monocytes/elicited Mphi were rapidly recruited in response to xenogeneic pig (PK15 or spleen) cells and, to a significantly lesser extent, allogeneic cells. These innate responses preceded T cell infiltration and occurred in their absence in SCID mice. Syngeneic cells induced negligible neutrophil or Mphi responses. Neutrophils and Mphi induced by xenogeneic cells in SCID mice stimulated T cell recruitment after transfer to immunocompetent mice. T cells in turn were required for Mphi activation and xenogeneic cell rejection. Thus, Mphi harvested from immunocompetent but not SCID mice injected with xenogeneic cells expressed activation markers and rejected xenogeneic cells when transferred into SCID mice. These findings demonstrate the interdependent roles of Mphi and T cells in xenograft rejection. The requirement for Mphi reflects their ability to mount a rapid, local innate response that stimulates T cell recruitment and, having received T cell help, to act as direct effectors of rejection.
Abstract: Cytotoxic CD8 T lymphocytes (CTL) are effectors of pancreatic islet beta-cell destruction in type 1 diabetes but, with the exception of a single report, CTL to islet antigen peptides have not been identified. We used autologous blood monocyte-derived dendritic cells to elicit HLA-A2-restricted CTL to a peptide, MVWESGCTV (aa 797-805), that is contiguous with a dominant CD4 T-cell epitope in the islet antigen tyrosine phosphatase IA-2. IA-2 peptide-specific CTL activity measured as 51Cr release from autologous lymphoblasts was detected in 2/6 islet antibody-positive relatives at high risk for type 1 diabetes but also in 2/6 closely HLA-matched controls. All subjects had CTL activity to an HLA-A2-restricted Epstein-Barr virus peptide. CTL to the IA-2 self-peptide were therefore not disease-specific, consistent with other evidence that autoreactive T cells are present in healthy individuals.
Abstract: The MHC class II molecule I-Ag7 is essential for the development of insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse but the requirements for peptide binding to I-Ag7 are still controversial. We have now isolated I-Ag7-binding phage from a large phage display library encoding random nonamer peptides. Ninety peptide-encoding regions of phage eluted from I-Ag7 were sequenced and >75% of the corresponding synthetic peptides bound to I-Ag7. Peptide alignment led to the identification of position-specific anchor residues. Hydrophobic (V and P) and positively charged (K) residues were highly enriched at P6 and positively charged (R and K), aromatic (Y) or hydrophobic (L) residues at P9. In addition, small amino acid residues (G and A) were enriched at P7 and G at P8. The primary anchors at P6 and P9 defining the phage-derived motif were present in most high-affinity I-Ag7-binding peptides from IDDM candidate antigens but only in < or =25% of peptides that were low-affinity binders or failed to bind to I-Ag7. A comparison of these results with the proposed motifs for peptide binding to I-Ag7 validates the one we have previously described.
Abstract: Mucosal delivery of soluble antigen induces systemic tolerance and has been applied to the prevention of autoimmune diseases. We have studied mucosal tolerance in autoimmune diabetes using the non-obese diabetic mouse model. Treatment of prediabetic mice with the pancreatic islet autoantigen insulin, by aerosol or intranasal delivery, reduces the incidence of diabetes and is associated with induction of CD8 (alpha alpha) gamma delta T cells, small numbers of which prevent adoptive transfer of diabetes. We examine the evidence for gamma delta T cells in mucosal tolerance and discuss possible mechanisms underlying the induction and action of insulin-induced CD8 gamma delta regulatory T cells. CD8 gamma delta cells constitute the most abundant subpopulation of intraepithelial lymphocytes (IELs), the major lymphoid cell compartment and first line of cellular immune defence in the mucosa. Induction of regulatory CD8 gamma delta T cells requires conformationally intact but not biologically active insulin. In contrast, intranasal (pro)insulin peptide, or oral insulin which is degraded in the gut, induces CD4 regulatory cells. Regulatory gamma delta T cells secrete interleukin-10 in pancreatic lymph nodes, which could account for the antidiabetic and bystander suppressor effect of naso-respiratory insulin. The physiological role of gamma delta IELs in maintaining peripheral self-tolerance deserves further study.
Abstract: Although innate immunity evolved to combat pathogens, increasing awareness of a pivotal role in driving and shaping adaptive immunity has prompted this review on the role of innate immunity in graft rejection. We present evidence that grafts, especially xenografts, elicit innate responses, required for adaptive immunity. Particular attention is paid to studies by ourselves and others demonstrating the important role of innate immunity in T-cell trafficking. The mechanisms by which grafts elicit innate immunity are a fertile subject for further investigation and an important target for therapeutic intervention.
Abstract: Death of pancreatic beta cells is the final step in the pathogenesis of type 1 diabetes before it becomes clinically apparent. Applying recent basic research about how cells die to the clinical problem of diabetes is a current opportunity and challenge. To date, perforin is the only factor definitely implicated in beta-cell killing in the non-obese diabetic (NOD) mouse model, although some perforin-deficient NOD mice develop diabetes. Our results suggest that other factors that cause beta-cell death remain to be identified.
Abstract: Studies of two post-mortem pancreata of children at the onset of type I diabetes have suggested activation and expansion of islet infiltrating T cells by a superantigen. We present the first reported case of a superantigen mediated disease, toxic shock syndrome (TSS), occurring at the diagnosis of type I diabetes. A 12-year-old girl presented with TSS and newly diagnosed diabetes with ketoacidosis. At presentation she was unconscious, febrile and hypotensive, with a desquamating erythematous rash and Kussmaul breathing. During resuscitation, her renal impairment, diarrhoea, thrombocytopaenia and ketoacidosis resolved. Vaginal discharge and blood cultures grew Staphylococcus aureus. T cell studies at 2 weeks after diagnosis detected a high level of spontaneous and islet antigen-specific proliferation with associated interleukin-10 production compared to human leucocyte antigen DR matched controls.
Abstract: Environmental agents are proposed to play a role in triggering or exacerbating pancreatic islet autoimmunity in people genetically predisposed to type 1 diabetes. However, with few exceptions, these agents remain enigmatic. Clues to environmental agents may be found by investigating population/geographic clusters or 'hotspots' of high disease incidence. We were alerted to a small community where the incidence of type 1 diabetes appeared to be five-fold higher than expected. Because type 1 diabetes is now recognized to have a subclinical phase during which anti-islet antibodies can be detected, we aimed to identify and characterize a reservoir of children with subclinical disease in this community. Venous blood samples were collected from 1906/2347 (81%) local school children during one week. Islet cell antibodies (ICAs) were detected in 122 (6.4%) children, 18 (0.9%) being high titer (> or = 20 Juvenile Diabetes Foundation units (JDFu)). On retest, 15 months later, the majority of low titer ICAs were undetectable, whereas high-titer ICAs persisted. The latter were found in two distinct age-related, ethnically similar groups. The younger group, aged 6-9 yr, had antibodies to insulin (IAAs), glutamic acid decarboxylase (GAD) and tyrosine phosphatase IA2 in addition to ICA, human leukocyte antigen (HLA) genes associated with susceptibility to type 1 diabetes, and lower first-phase insulin responses (FPIRs) to intravenous glucose. The older group, aged 13-16 yr, the age cohort of the index clinical cases, had few antibodies other than ICA, non-susceptibility HLA genes and normal FPIRs. During follow-up, three children, all from the younger group with multiple antibodies and FPIRs less than the first percentile, developed diabetes 4, 6 and 7 yr after screening. The finding of two age groups of subclinical disease suggests that if environmental agents triggered islet autoimmunity they did not act constantly on the community. Furthermore, the absence of multiple autoantibodies and/or HLA susceptibility genes in the older group, the source of index clinical cases, implies they are a residual subgroup with slow or absent progressive beta-cell destruction. This study illustrates that the natural history of type 1 diabetes may be elucidated by analyzing age-related subclinical disease in the general population.
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.
Abstract: Pancreatic islet autoimmunity leading to type 1 diabetes could be triggered by viruses in genetically susceptible individuals. Rotavirus (RV), the most common cause of childhood gastroenteritis, contains peptide sequences highly similar to T-cell epitopes in the islet autoantigens GAD and tyrosine phosphatase IA-2 (IA-2), suggesting T-cells to RV could trigger islet autoimmunity by molecular mimicry. We therefore sought an association between RV infection and islet autoantibody markers in children at risk for diabetes who were followed from birth. There was a specific and highly significant association between RV seroconversion and increases in any of these antibodies: 86% of antibodies to IA-2, 62% to insulin, and 50% to GAD first appeared or increased with increases in RV IgG or IgA. RV infection may therefore trigger or exacerbate islet autoimmunity in genetically susceptible children.
Abstract: The application of self-antigens as therapeutic tools is validated in inbred animal models of autoimmune disease. Mechanisms of antigen-induced tolerance (apoptosis, anergy, regulatory T cells and immune deviation) are being clarified in relation to the properties of antigens and the modes and routes of their delivery. Mucosa-mediated tolerance remains the predominant mode of antigen-specific therapy but awaits demonstration of clinical efficacy in human autoimmune disease.
Abstract: We have cloned a member of the STE20/SPS1 protein kinase family from a transformed rat pancreatic beta cell line. SPAK (STE20/SPS1-related, proline alanine-rich kinase) belongs to the SPS1 subfamily of STE20 kinases and is highly conserved between species. SPAK is expressed ubiquitously, although preferentially in brain and pancreas. Biochemical characterization of SPAK catalytic activity demonstrates that is a serine/threonine kinase that can phosphorylate itself and an exogenous substrate in vitro. SPAK is immunoprecipitated from transfected mammalian cells as a complex with another, as yet uncharacterized, serine/threonine kinase which is capable of phosphorylating catalytically-inactive SPAK and myelin basic protein in an in vitro kinase assay. SPAK specifically activates the p38 pathway in cotransfection assays. Like MST1 and MST2, SPAK contains a putative caspase cleavage site at the junction of the catalytic domain and the C-terminal region. Full-length SPAK is expressed in the cytoplasm in transfected cells, while a mutant corresponding to caspase-cleaved SPAK is expressed predominantly in the nucleus. The similarity of SPAK to other SPS1 family members, its ability to activate the p38 pathway, in addition to its putative caspase cleavage site, provide evidence that SPAK may act as a novel mediator of stress-activated signals. Oncogene (2000) 19, 4290 - 4297
Abstract: The hypothesis that early exposure to cow's milk or lack of breast-feeding predisposes to type 1 diabetes remains controversial. We aimed to determine prospectively the relationship of, first, duration of exclusive breast-feeding and total duration of breast-feeding, and second, introduction of cow's milk protein as infant formula, cow's milk, or dairy products, to the development of islet antibodies in early life. Some 317 children with a first-degree relative with type 1 diabetes were followed prospectively from birth for 29 months (4-73). Mothers kept a home diary and answered infant feeding questionnaires at 6-month intervals. No systematic feeding advice was given. Insulin autoantibodies (normal range <5.5%), anti-GAD antibodies (<5.0 U), and anti-IA2 antibodies (<3.0 U) were measured at 6-month intervals. Cox proportional hazards model of survival analysis detected no significant difference between children who did not develop islet antibodies (225 of 317 [71%]), children with one islet antibody raised once (52 of 317 [16.4%]), children with one antibody raised repeatedly (18 of 317 [5.7%]), or children with two or more antibodies raised (22 of 317 [6.9%]), in terms of duration of exclusive breast-feeding, total duration of breast-feeding, or introduction of cow's milk-based infant formulas, cow's milk, or dairy products (relative risk: 0.91-1.09). Four of the children with two or more islet antibodies developed type 1 diabetes. We conclude that there is no prospective association between duration of breast-feeding or introduction of cow's milk and the development of islet autoimmunity in high-risk children.
Abstract: The hypothesis that early exposure of the infant to cow's milk (or lack of breast-feeding) predisposes the child to type 1 diabetes dates from the 1980s. It has important implications, but remains controversial because the evidence on which it is based has been indirect and is open to criticism. Two meta-analyses of multiple studies in which diabetes prevalence was associated retrospectively with infant feeding revealed only a marginal increase in relative risk. Two recent prospective studies found no apparent association between development of antibodies to islet antigens and feeding patterns in high-risk infants with a first-degree type 1 diabetic relative. Studies reporting increased humoral and cellular immunity to cow's milk proteins in children with type 1 diabetes often lack appropriate controls and standardization and do not, in themselves, establish a causal connection to disease pathogenesis. A review of published data leads to the conclusion that increased immunity to cow's milk proteins is not disease-specific, but reflects genetic predisposition to increased immunity to dietary proteins in general, associated with the HLA haplotype A1-B8-DR3-DQ2 (A1*0501, B1*0201), which also predisposes to celiac disease and selective IgA deficiency. We suggest that the cow's milk hypothesis could be productively reframed around mucosal immune function in type 1 diabetes. Breast milk contains growth factors, cytokines, and other immunomodulatory agents that promote functional maturation of intestinal mucosal tissues. In the NOD mouse model, environmental cleanliness may influence diabetes incidence through mucosal mechanisms, and exposure of the mucosa to insulin (present in breast milk) induces regulatory T-cells and decreases diabetes incidence. The mucosa is a major immunoregulatory barrier, and cow's milk happens to be the first dietary protein it encounters. The basic question is whether impaired mucosal immune function predisposes to type 1 diabetes.
Abstract: Extracellular factors that regulate the growth and differentiation of cell lineages in the pancreatic primordia are poorly understood. Identification of these factors for pancreatic islet beta-cells could open new avenues for the treatment of insulin-dependent diabetes. We developed a low cell density serum-free culture system for dissociated pancreatic cells from the 13.5-day mouse fetus and investigated the effects of extracellular matrix proteins on differentiation of islet cells. After 4 days in culture, total cell number decreased by two-thirds, but insulin-positive beta-cell number increased 10-fold. Both of collagens I and IV inhibited cell survival (by >50%), whereas fibronectin had no effect. In the presence of soluble laminin-1, however, the number of beta-cells increased linearly by 60-fold without an increase in the total cell number; glucagon-positive cell number was unchanged, and somatostatin and pancreatic polypeptide-positive cells were not detected. The effect of laminin-1 was completely blocked by a monoclonal rat anti-laminin-1 antibody. In the presence of laminin-1, the thymidine analogue, BrdU, was incorporated into only 2.5% of cells, which were mainly insulin-negative at days 1-3. Laminin-1 appeared, therefore, to induce differentiation of beta-cells from precursor cells in day-13.5 fetal pancreas. Laminin-1 was shown to be expressed in the epithelial basement membrane of the 13.5- to 17.5-day fetal pancreas. These findings provide the first evidence of a role for laminin-1 to promote differentiation of pancreatic beta-cells.
Abstract: MOTIVATION: Prediction methods for identifying binding peptides could minimize the number of peptides required to be synthesized and assayed, and thereby facilitate the identification of potential T-cell epitopes. We developed a bioinformatic method for the prediction of peptide binding to MHC class II molecules. RESULTS: Experimental binding data and expert knowledge of anchor positions and binding motifs were combined with an evolutionary algorithm (EA) and an artificial neural network (ANN): binding data extraction --> peptide alignment --> ANN training and classification . This method, termed PERUN, was implemented for the prediction of peptides that bind to HLA-DR4(B1*0401). The respective positive predictive values of PERUN predictions of high-, moderate-, low- and zero-affinity binders were assessed as 0.8, 0.7, 0.5 and 0.8 by cross-validation, and 1.0, 0.8, 0.3 and 0.7 by experimental binding. This illustrates the synergy between experimentation and computer modeling, and its application to the identification of potential immunotherapeutic peptides. AVAILABILITY: Software and data are available from the authors upon request. CONTACT: vladimir@wehi.edu. au
Abstract: Cytokine production in human whole blood exhibits diurnal rhythmicity. Peak production of the pro-inflammatory cytokines IFN-gamma, TNF-alpha, IL-1 and IL-12 occurs during the night and early morning at a time when plasma cortisol is lowest. The existence of a causal relationship between plasma cortisol and production is suggested by the finding that elevation of plasma cortisol within the physiological range by the administration of cortisone acetate results in a corresponding fall in pro-inflammatory cytokine production. Cortisol may not be the only neuroendocrine hormone that entrains cytokine rhythms; other candidates include 17-hydroxy progesterone, melatonin and dihydroepiandrostene dione (DHEAS). The finding of diurnal cytokine rhythms may be relevant to understanding why immuno-inflammatory disorders such as rheumatoid arthritis or asthma exhibit night-time or early morning exacerbations and to the optimisation of treatment for these disorders. Diurnal rhythmicity of cytokine production also has implications for the timing of blood samples drawn for diagnostic T-cell assays. Finally, diurnal rhythmicity of immune function suggests that the nature of an immune response, for example in response to vaccination, may be modified by the time of day of antigen administration and raises the possibility that immune responses could be therapeutically manipulated by co-administration of immuno-regulatory hormones such as glucocorticoids.
Abstract: OBJECTIVES: To determine the utility of various autoantibodies in predicting progression to clinical diabetes in first-degree relatives of patients with type 1 diabetes mellitus. PARTICIPANTS: 3315 first-degree relatives of patients with type 1 diabetes (1161 parents, 1206 siblings and 948 offspring) recruited through diabetes clinics, private endocrinologists, Diabetes Australia and the Juvenile Diabetes Foundation. MAIN OUTCOME MEASURES: Prevalence of islet cell antibodies (ICA) levels > or = 20 JDFu, insulin autoantibodies (IAA) levels > 100 nU/mL, and antibodies to glutamic acid decarboxylase (GADAb) and tyrosine phosphatase IA2 (IA2Ab); change in beta cell function over time; and development of clinical diabetes. RESULTS: 2.6% of relatives had elevated ICA levels, 1.3% had elevated IAA levels and 0.3% had both. High ICA levels were significantly more frequent in siblings than in offspring or parents, and were more frequent in relatives younger than 20 years. GADAb were detected in 68% and IA2Ab in 57% of relatives with elevated ICA and/or IAA levels. Diabetes developed in 33 relatives (25 siblings, 2 offspring and 6 parents). Before diagnosis of clinical diabetes, high ICA levels were detected in 18 (58%), high IAA levels in 7 (23%), both in 5 (15%), and either in 19 (61%); GADAb were detected in 26 (84%), IA2Ab in 13 (42%), both in 11 (35%), and either in 28 (90%). First phase insulin release (FPIR) less than 50 mU/L was very strongly associated with progression to diabetes. In relatives with FPIR initially greater than 50 mU/L who eventually developed diabetes, there was a gradual and continuous reduction in FPIR over time before diagnosis. CONCLUSIONS: Type 1 diabetes can be diagnosed in the preclinical stage. The recently described antibodies to glutamic acid decarboxylase and tyrosine phosphatase IA2 appear superior to ICA as screening tools for the preclinical diagnosis of type 1 diabetes.
Abstract: Pancreatic islet beta cell destruction leading to insulin-dependent diabetes mellitus (IDDM) is believed to be mediated by a T-helper 1 (T(H)1) lymphocyte response to islet antigens. In the mouse, T(H)1 (IL-2, IFN-gamma) and T(H)2 (IL-4, -5, -6, -10) responses are associated with the generation of IgG2a and IgG1 subclasses, respectively. The equivalent human subclasses have not been defined. Because the IgG subclass response to an antigen may be a potentially useful marker of T(H)1/T(H)2 immune balance we measured IgG subclass antibodies to glutamic acid decarboxylase (GAD), a major islet autoantigen in IDDM, in 34 newly-diagnosed IDDM patients and in 28 at-risk, first-degree relatives of people with IDDM. In the newly-diagnosed patients, total IgG antibodies to GAD were detected in 74% (25/34); IgG1 and/or IgG3 were significantly more frequent than IgG4 or IgG4/IgG2 (14/34 versus 5/34, p = 0.01). GAD antibody-negative patients were significantly younger (p = 0.01). In 15 at-risk relatives who had not progressed to clinical diabetes after a median of 4.5 years, 10 had IgG2 and/or IgG4 antibodies compared to only 3/13 progressors (p = 0.02). Total IgG and IgG2 antibodies were higher in non-progressors. Non-progressors were older than progressors (p = 0.01), and relatives with IgG2 and/or IgG4 responses were also older (p = 0.01). These results suggest that IgG subclass antibodies to GAD may contribute to diabetes risk assessment in islet antibody relatives.
Abstract: Clinical features of certain immuno-inflammatory disorders such as rheumatoid arthritis and asthma exhibit diurnal fluctuation, which could be related to diurnal rhythmicity of pro-inflammatory cytokine production. To investigate the latter, the authors performed measurements of lipopolysaccharide (LPS)-stimulated whole blood, interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-12 production in 13 healthy volunteers over 24 h. These cytokines exhibited distinct diurnal rhythms that peaked in the early morning and were inversely related to the rhythm of plasma cortisol. Elevation of plasma cortisol within the physiological range by administration of cortisone acetate, 25 mg at 21.00, markedly suppressed IFN-gamma, TNF-alpha, IL-1 and IL-12 production, but not the later early morning rise of endogenous plasma cortisol. Suppression of cytokine production was temporally dissociated from changes in numbers of circulating mononuclear cells. Regulation of pro-inflammatory cytokine production by plasma cortisol has potential therapeutic implications. In contrast to standard schedules, a small, late evening, dose of glucocorticoid to suppress the diurnal increase in pro-inflammatory cytokine production could alleviate early morning inflammatory symptoms and minimize side-effects.
Abstract: We describe a case of multicentric reticulohistiocytosis complicated by central retinal vein thrombosis and trigeminal neuropathy. A variety of treatment modalities were tried in this patient. Both skin disease and arthritis responded to low dose methotrexate over 8 years of followup. Graduated compression gloves produced an excellent cosmetic improvement in the disfiguring skin lesions.
Abstract: MHCPEP (http://wehih.wehi.edu.au/mhcpep/) is a curated database comprising over 13 000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains the peptide sequence, its MHC specificity and where available, experimental method, observed activity, binding affinity, source protein and anchor positions, as well as publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using WWW or FTP.
Abstract: The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. T-cell epitope peptides in autoantigens have potential diagnostic and therapeutic applications, and they may hold clues to environmental agents with similar sequences that could trigger or exacerbate autoimmune disease. We identified 13 epitope peptides in IA-2 by measuring peripheral blood T-cell proliferation to 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 in six at-risk type 1 diabetes relatives selected for HLA susceptibility haplotypes. The dominant epitope, VIVMLTPLVEDGVKQC (aa 805-820), which elicited the highest T-cell responses in all at-risk relatives, has 56% identity and 100% similarity over 9 amino acids (aa) with a sequence in VP7, a major immunogenic protein of human rotavirus. Both peptides bind to HLA-DR4(*0401) and are deduced to present identical aa to the T-cell receptor. The contiguous sequence of VP7 has 75% identity and 92% similarity over 12 aa with a known T-cell epitope in glutamic acid decarboxylase (GAD), another autoantigen in type 1 diabetes. This dominant IA-2 epitope peptide also has 75-45% identity and 88-64% similarity over 8-14 aa to sequences in Dengue, cytomegalovirus, measles, hepatitis C, and canine distemper viruses, and the bacterium Haemophilus influenzae. Three other IA-2 epitope peptides are 71-100% similar over 7-12 aa to herpes, rhino-, hanta- and flaviviruses. Two others are 80-82% similar over 10-11 aa to sequences in milk, wheat, and bean proteins. Further studies should now be carried out to directly test the hypothesis that T-cell activation by rotavirus and possibly other viruses, and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and (for rotavirus) GAD.
Abstract: BACKGROUND: Host macrophages are abundant within fetal pig pancreas xenografts undergoing rejection, but their role is unknown. Therefore, we examined the effect of host macrophage depletion on xenograft rejection. METHODS: Nonobese diabetic (NOD) mice were given clodronate-loaded liposomes intravenously to deplete macrophages. Controls received phosphate-buffered saline (PBS) or PBS-liposomes. General immune status was assessed after 2, 3, and 7 days by (1) fluorescence-activated cell sorter analysis of peripheral blood, spleen, and lymph node cells, (2) immunohistochemistry on spleens, and (3) mixed lymphocyte reaction. Organ-cultured fetal pig pancreas was transplanted under the kidney capsule of NOD mice 3 days after clodronate or PBS injection. Grafts were assessed histologically at 4, 5, 6, and 8 days after transplantation. RESULTS: Splenic macrophages and peripheral blood monocytes were depleted 2 days after clodronate treatment but had recovered within 11 days. T cell, B cell, and dendritic cell numbers were normal in spleen, peripheral blood, and lymph nodes of clodronate-treated mice, and T cells and antigen-presenting cells from these mice functioned normally in mixed lymphocyte reaction. Clodronate treatment markedly reduced graft infiltration by macrophages, T cells, and eosinophils at 4, 5, and 6 days after transplantation, and was associated with maintenance of endocrine cell viability and insulin expression. However, all grafts were rejected 8 days after transplantation, concordant with reappearance of splenic macrophages. CONCLUSIONS: Short-term, specific depletion of macrophages markedly delayed cellular infiltration and rejection of xenografts. The results provide the first evidence that macrophages promote T-cell infiltration and rejection of fetal pig pancreas xenografts in NOD mice.
Abstract: Dendritic cells (DC) present Ag to naive T cells and are therefore pivotal in shaping immune responses. DC may either immunize or tolerize T cells. Humans with pancreatic islet autoimmunity at high risk for insulin-dependent diabetes mellitus (IDDM) present the opportunity to investigate DC in autoimmune disease. We compared DC phenotype and function in 12 euglycemic, asymptomatic IDDM relatives with islet autoimmunity and controls matched for age, sex, and MHC class II alleles. DC were generated from adherent peripheral blood cells by culture with granulocyte/macrophage-CSF and IL-4. The yield of DC was significantly lower in IDDM relatives than in controls. While the DC phenotype, HLA-DR+CD14-, was expressed by > or =90% of the cells generated from relatives and controls, the proportion of cells that expressed CD1a and the costimulator molecules CD80 (B7-1) and CD86 (B7-2) was significantly lower in IDDM relatives. In addition, B7-1 and B7-2 expression per cell was significantly lower in IDDM relatives. These phenotypic changes were accompanied by reduced stimulation of autologous CD4 cells by DC from IDDM relatives. Similar findings were obtained in three recently diagnosed IDDM patients. These findings indicate that impairment of DC phenotype and function is a marker of islet autoimmunity and are consistent with a role for impaired DC function in the pathogenesis of autoimmune disease.
Abstract: Efficiency of presentation of a peptide epitope by a MHC class I molecule depends on two parameters: its binding to the MHC molecule and its generation by intracellular Ag processing. In contrast to the former parameter, the mechanisms underlying peptide selection in Ag processing are poorly understood. Peptide translocation by the TAP transporter is required for presentation of most epitopes and may modulate peptide supply to MHC class I molecules. To study the role of human TAP for peptide presentation by individual HLA class I molecules, we generated artificial neural networks capable of predicting the affinity of TAP for random sequence 9-mer peptides. Using neural network-based predictions of TAP affinity, we found that peptides eluted from three different HLA class I molecules had higher TAP affinities than control peptides with equal binding affinities for the same HLA class I molecules, suggesting that human TAP may contribute to epitope selection. In simulated TAP binding experiments with 408 HLA class I binding peptides, HLA class I molecules differed significantly with respect to TAP affinities of their ligands. As a result, some class I molecules, especially HLA-B27, may be particularly efficient in presentation of cytosolic peptides with low concentrations, while most class I molecules may predominantly present abundant cytosolic peptides.
Abstract: Activation of T cells requires recognition by T-cell receptors of specific peptides bound to major histocompatibility complex (MHC) molecules on the surface of either antigen-presenting or target cells. These peptides, T-cell epitopes, have potential therapeutic applications, such as for use as vaccines. Their identification, however, usually requires that multiple overlapping synthetic peptides encompassing a protein antigen be assayed, which in humans, is limited by volume of donor blood. T-cell epitopes are a subset of peptides that bind to MHC molecules. We use an artificial neural network (ANN) model trained to predict peptides that bind to the MHC class II molecule HLA-DR4(*0401). Binding prediction facilitates identification of T-cell epitopes in tyrosine phosphatase IA-2, an autoantigen in DR4-associated type1 diabetes. Synthetic peptides encompassing IA-2 were tested experimentally for DR4 binding and T-cell proliferation in humans at risk for diabetes. ANN-based binding prediction was sensitive and specific, and reduced the number of peptides required for T-cell assay by more than half, with only a minor loss of epitopes. This strategy could expedite identification of candidate T-cell epitopes in diverse diseases.
Abstract: 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.
Abstract: Insulin-dependent diabetes mellitus (IDDM) is preceded by the presence of antibodies against islet proteins including a protein tyrosine phosphatase (PTP) designated IA-2. Recently, we cloned a novel PTP named IAR which shares 43% sequence identity with IA-2 and is recognised by antibodies from a majority of patients with IDDM. The aim of the present study was to determine whether IAR antibodies (IAR Ab) or IA-2 antibodies (IA-2 Ab) are associated with progression to IDDM in first-degree relatives "at-risk" for IDDM (operationally defined as those with islet cell antibodies [ICA] > or = 20JDFU or insulin autoantibodies [IAA] > or = 100 nU/ml), and to examine combinations of IAR Ab and IA-2 Ab in these subjects. The sensitivity and specificity of these antibodies were also examined in patients with recent-onset IDDM. Using Cox's Proportional Hazards Model, the number of siblings with IDDM was associated with progression to IDDM in "at-risk" relatives, but other covariables (age, sex, number of affected offspring or parents) were not significantly associated. Using number of affected siblings as a covariable, both IAR and IA-2 antibodies were significantly associated with progression to IDDM (p < 0.005). Combinations of both antibodies, however, did not result in a significantly stronger association with progression to IDDM. The threshold of positivity for IAR Ab (0.5 units) and IA-2 Ab (3.0 units) assays was adjusted to give the same specificity (97.9%) for each assay in 144 healthy control subjects, to allow standardised comparisons. Levels of IAR Ab and IA-2 Ab were strongly correlated in 53 recent-onset IDDM patients (r = 0.70, p < 0.0001) but 11.3% had IAR Ab in the absence of IA-2 Ab and 16.9% had IA-2 Ab in the absence of IAR Ab. The sensitivity for IDDM (defined as the proportion of IDDM patients positive) was 56.6% for IAR Ab and 62.3% for IA-2 Ab. We conclude that there is considerable overlap in IA-2 Ab and IAR Ab positivity, although either antibody can occur independently in IDDM patients. Both IAR Ab and IA-2 antibodies are associated with progression to IDDM in first-degree relatives at-risk of IDDM, but the use of IAR and IA-2 antibodies in combination are not significantly more strongly associated with progression than single antibodies. IAR Ab may play an important role in the prediction of IDDM.
Abstract: BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.
Abstract: The recent cloning and recombinant expression of novel islet autoantigens [glutamic acid decarboxylase (GAD) 65 and islet-cell autoantibody 512 (ICA512)] has made possible the determination of whether the quantitative expression of autoantibodies to these molecules is correlated with age of diabetes onset and rate of progression to diabetes, similar to insulin autoantibodies (IAAs). We measured autoantibodies reacting with GAD65 (GAD65AA), ICA512 (ICA512AA), and insulin in patients who recently had received a diagnosis of diabetes and in first-degree relatives prospectively identified and then followed because of the expression of high titers of ICA. Levels of IAAs (but not GAD65AA or ICA512AA) correlated inversely with age at diagnosis of diabetes and directly with time to diabetes onset among the ICA-positive relatives. In multiple linear regression models, the level of IAAs remained a significant predictor of the time to diabetes after allowing for first-phase insulin secretion. The unique and dramatic association of IAAs with progression to diabetes suggests that IAAs contribute directly to disease pathogenesis or that levels of IAAs are influenced uniquely by the process, leading--at different rates in different prediabetic individuals--to type I diabetes. In addition, the linear regression model described (involving two variables, first-phase insulin secretion and levels of IAAs) aids in the prediction of time to diabetes among ICA-positive relatives.
Abstract: T cells recognize peptide epitopes bound to major histocompatibility complex molecules. Human T-cell epitopes have diagnostic and therapeutic applications in autoimmune diseases. However, their accurate definition within an autoantigen by T-cell bioassay, usually proliferation, involves many costly peptides and a large amount of blood. We have therefore developed a strategy to predict T-cell epitopes and applied it to tyrosine phosphatase IA-2, an autoantigen in IDDM, and HLA-DR4(*0401). First, the binding of synthetic overlapping peptides encompassing IA-2 was measured directly to purified DR4. Secondly, a large amount of HLA-DR4 binding data were analysed by alignment using a genetic algorithm and were used to train an artificial neural network to predict the affinity of binding. This bioinformatic prediction method was then validated experimentally and used to predict DR4 binding peptides in IA-2. The binding set encompassed 85% of experimentally determined T-cell epitopes. Both the experimental and bioinformatic methods had high negative predictive values, 92% and 95%, indicating that this strategy of combining experimental results with computer modelling should lead to a significant reduction in the amount of blood and the number of peptides required to define T-cell epitopes in humans.
Abstract: OBJECTIVES: To determine if peptides containing the 'shared epitope' sequence, QKRAA, from either endogenous, HLA-DR beta 1 (0401), or exogenous, Escherichia coli dnaJ, sources activate T cells in recent onset rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cell (PBMC) proliferative and whole blood cytokine responses to shared epitope containing peptides from DR beta 1 (0401) and E coli dnaJ, to control peptides from DR beta 1 (0402) and hsp40 and to the recall antigen, tetanus toxoid, were tested in 20 untreated, recent onset RA subjects, 20 HLA, age, and sex matched healthy controls and 18 other subjects with inflammatory arthritis. PBMC proliferative responses to a second E coli dnaJ peptide (with the shared epitope at the N-terminus) and two peptides from type II collagen with high affinity for DR4(0401) were tested in a further 16 recent onset RA and 17 control subjects. RESULTS: PBMC proliferation and whole blood interferon gamma or interleukin 10 production in response to the shared epitope containing and control peptides were not different between the disease and control groups. On the other hand, compared with controls, RA subjects had significantly higher proliferation to a collagen II (aa 1307-1319) peptide, but significantly lower proliferation and interferon gamma production to tetanus toxoid. CONCLUSION: Recent onset RA subjects had no demonstrable increase in peripheral blood T cell reactivity to shared epitope containing peptides. However, a proportion had increased T cell reactivity to a peptide of similar length from a candidate RA autoantigen, collagen type II. Their impaired responses to tetanus are in keeping with evidence for general T cell hyporesponsiveness in RA.
Abstract: T cells of the vertebrate immune system recognise peptides bound by major histocompatibility complex (MHC) molecules on the surface of host cells. Peptide binding to MHC molecules is necessary for immune recognition, but only a subset of peptides are capable of binding to a particular MHC molecule. Common amino acid patterns (binding motifs) have been observed in sets of peptides that bind to specific MHC molecules. Recently, matrix models for peptide/MHC interaction have been reported. These encode the rules of peptide/ MHC interactions for an individual MHC molecule as a 20 x 9 matrix where the contribution to binding of each amino acid at each position within a 9-mer peptide is quantified. The artificial intelligence techniques of genetic search and machine learning have proved to be very useful in the area of biological sequence analysis. The availability of peptide/MHC binding data can facilitate derivation of binding matrices using machine learning techniques. We performed a simulation study to determine the minimum number of peptide samples required to derive matrices, given the pre-defined accuracy of the matrix model. The matrices were derived using a genetic search. In addition, matrices for peptide binding to the human class I MHC molecules, HLA-B35 and -A24, were derived, validated by independent experimental data and compared to previously-reported matrices. The results indicate that at least 150 peptide samples are required to derive matrices of acceptable accuracy. This result is based on a maximum noise content of 5%, the availability of precise affinity measurements and that acceptable accuracy is determined by an area under the Relative Operating Characteristic curve (Aroc) of > 0.8. More than 600 peptide samples are required to derive matrices of excellent accuracy (Aroc > 0.9). Finally, we derived a human HLA-B27 binding matrix using a genetic search and 404 experimentally-tested peptides, and estimated its accuracy at Aroc > 0.88. The results of this study are expected to be of practical interest to immunologists for efficient identification of peptides as candidates for immunotherapy.
Abstract: MHCPEP is a curated database comprising over 9000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains the peptide sequence, its MHC specificity and, when available, experimental method, observed activity, binding affinity, source protein, anchor positions and publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using WWW, FTP or Gopher.
Abstract: This study describes a novel method of inhibiting T-cell function by the use of peptides rationally designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence involved with TCR receptor assembly. The most effective peptide (core peptide, CP) modulating in vitro and in vivo T-cell function contained nine amino acids two of which, lysine and arginine, were hydrophilic and separated by four hydrophobic amino acids. CP without chemical modification or conjugation was able to enter non-T and T cells. Conjugation of CP at the carboxyl terminus with palmitic acid resulted in a greater inhibition of T-cell interleukin-2 (IL-2) production in vitro than peptide alone. When examined for effects in vivo, CP reduced clinical signs of inflammation in three T cell-mediated disease models including adjuvant-induced arthritis, experimental allergic neuritis, and cyclophosphamide-induced diabetes in NOD/Lt(F) mice. This peptide or its analogues has potential as a therapeutic agent in human inflammatory and autoimmune disorders.
Abstract: IDDM in humans and in nonobese diabetic (NOD) mice is a T-cell-dependent autoimmune disease in which the beta-cells of the pancreatic islets are destroyed. Several putative beta-cell autoantigens have been identified, but insulin and its precursor, proinsulin, are the only ones that are beta-cell specific. (Pro)insulin may be a key autoantigen in IDDM. To address the role of proinsulin in the development of IDDM, we generated NOD mice transgenic for the mouse proinsulin II gene driven off a major histocompatibility complex (MHC) class II promoter to direct expression of the transgene to MHC class II bearing cells, including those in the thymus, with the aim of deleting proinsulin-reactive T-cells. The mononuclear cell infiltration of the islets (insulitis) is almost completely absent, and diabetes is prevented in these transgenic NOD mice. The mononuclear cell infiltration of the salivary glands (sialitis) and immune responses to ovalbumin (OVA) are not altered, indicating that the protective effect of the transgene is specific for islet pathology and not due to general immunosuppression. We conclude that autoimmunity to proinsulin plays a pivotal role in the development of IDDM.
Abstract: One of several possible mechanisms for the HLA-disease association is HLA-related polymorphism of cytokine expression. However, with the exception of the tumor necrosis factors, no evidence has been found for a relationship between HLA alleles and cytokine expression. This may be because cytokine responses to commonly employed mitogens are neither antigen nor HLA dependent, and responses to recall antigens are dominated by the effect of prior antigen exposure. We reasoned that responses to alloantigens would be independent of prior antigen exposure and may therefore reveal subtle HLA-related variations in cytokine production. Here we demonstrate HLA Class II-related polymorphism of IFN-gamma production in the MLR performed between 32 subjects by a novel whole-blood method. HLA DR1, 2, and 6 were associated with high, whereas DR 3, 4, 5, and 7 were associated with low IFN-gamma production. Interestingly, DQ alleles with which these DR alleles are in linkage dysequilibrium, DQ1 and DQ2 and 3, were also associated with high and low IFN-gamma production, respectively. Ranking of HLA alleles according to whole-blood IFN-gamma production in response to mitogen or recall antigens was similar to that in the MLR, although individual allele-related differences did not reach statistical significance. TNF-alpha production was significantly higher in DR3-positive than in DR3-negative subjects, in accord with previous studies. These findings suggest that HLA Class II alleles, particularly at the DQ locus, or alternatively, genes in linkage with them, regulate IFN-gamma expression by T cells. The finding of HLA allele-related polymorphism of IFN-gamma production corroborates other lines of evidence that regulation of IFN-gamma expression contributes to HLA-associated susceptibility to immunoinflammatory diseases, in particular insulin-dependent diabetes and multiple sclerosis.
Abstract: Dendritic cells (DC), with potentially important clinical applications, have been generated from human peripheral blood monocytes in the presence of GM-CSF and IL-4 (G4 DC). In the present report we show that DC with a novel phenotype can be generated from blood adherent mononuclear cells in the presence of GM-CSF and IL-7 (G7 DC). Adherent cells from PBMC, cultured in GM-CSF (600 U/ml) and IL-7 (6 U/ml), were transformed over 7 days into cells with DC morphology, at a yield of 1.2-1.6 x 10(6) per 10(7) PBMC. G7 DC not only expressed class I and class II MHC, CD1a, CD11c, CD23, CD40, CD54, CD58, CD80, CD86 and CD95, like G4 DC, but also CD21, which is the complement receptor type 2, a ligand for CD23 and a receptor for EBV and IFN-alpha. G7 DC were at least one log more effective in the autologous MLR and at least two logs more effective in the allogeneic MLR, than PBMC. They elicited proliferative responses of CD4 T cells to tetanus toxoid and CD8 T cells to an EBV peptide, and stronger T-cell cytotoxicity to EBV peptide than G4 DC. Expression of CD21 by G7 DC suggests that IL-7 delivers a distinct signal to DC precursors and that G7 DC may be functionally distinct.
Abstract: Pancreatic islet beta-cell destruction leading to insulin-dependent diabetes mellitus (IDDM) is an autoimmune T cell-mediated process. Peripheral blood T cells, which proliferate to islet antigens such as glutamic acid decarboxylase (GAD), (pro)insulin or tyrosine phosphatase IA-2, can be detected in at-risk, first degree relatives of people with IDDM. However, cross-sectional studies cannot define the relationship between T cell responses and progression to IDDM. Longitudinal studies were therefore undertaken on 50 at-risk, first degree relatives tested at least yearly for up to 4 years, during which time five developed IDDM. Peripheral blood T cell responses to a GAD67(aa208-404)-glutathione-S-transferase (GST) fusion protein, GST, insulin and tetanus toxoid were measured, together with antibodies to islet cells, GAD, insulin and IA-2. High levels of antibodies to GAD or insulin were generally associated with low T cell responses to these antigens. Relatives who developed IDDM were characterized by high levels of antibodies to insulin and/or islet cells, and high T cell responses to GAD67-GST and tetanus, but not insulin, in the 24 months before clinical diagnosis. Cross-sectionally, T cell responses to GAD67(aa208-404)-GST and to full-length GAD65-GST were highly correlated (r=0.75, P<0.002). In conclusion, increased cellular immunity to the mid region of GAD67 was a marker of late pre-clinical IDDM, but appears to reflect a more general, transient state of cellular immune hyperresponsiveness.
Abstract: OBJECTIVE: Circumstantial evidence links retroviruses (RVs) with human autoimmune diseases. The aim of the present study was to obtain direct evidence of RV gene expression in rheumatoid arthritis (RA). METHODS: Synovial samples were obtained from patients with RA, patients with osteoarthritis (OA), and normal control subjects. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using synovial RNA and primers to conserved sequences in the polymerase (pol) genes of known RVs. RESULTS: PCR products (n = 857) were cloned and sequenced. Multiple pol transcripts, many with open reading frames, were expressed in every sample. Sequences were aligned and classified into 6 families (F1-F6) that contained 33 groups of known and unknown endogenous RVs (ERVs), each distinguished by a specific, deduced peptide motif. The frequency of sequences in each family was similar between RA, OA, and normal synovial tissue, but differed significantly in RA synovial fluid cells. F1 sequences (undefined, but related to murine and primate type C RVs) were lower in frequency, F2 (ERV-9-related), F4 (HERV-K-related), and F6 (HERV-L-related) sequences were higher in frequency, and F3 (RTVL-H-related) sequences were not detected, in the RA synovial fluid cells compared with the RA synovial tissues. CONCLUSION: Multiple ERVs are expressed in normal and diseased synovial compartments, but specific transcripts can be differentially expressed in RA.
Abstract: A knowledge of beta cell-specific gene expression provides a basis for identifying proteins potentially involved in beta cell function and pathology. To identify candidate beta cell-specific genes, we applied the PCR-based subtractive hybridization technique of representational difference analysis (RDA) to the mouse SV40-transformed endocrine cell lines, betaTC3 and alphaTC1. Following three successive subtractions of alphaTC1 complementary DNA from betaTC3 complementary DNA, difference products were cloned into pUC19 and nucleotide sequences determined. Comparison of 91 sequences against the databases identified 11 known and 8 novel genes. Known genes included previously reported beta cell-specific genes, insulin I/II and islet amyloid polypeptide, as well as other non-beta cell-specific genes such as those for insulin-like growth factor II, selenoprotein P, neuronatin, prohormone convertase, and type 1 protein kinase A regulatory subunit. By Northern blot hybridization, expression of the majority of known and novel genes was restricted to betaTC3 cells. Novel genes BA-12, -13, -14, and -18 were expressed not only in betaTC3 cells, but also in normal pancreatic islets and a limited number of other tissues. The deduced amino acid sequence of BA-14 showed significant homology with members of the cadherin superfamily indicating that BA-14 may encode a cadherin-like molecule potentially involved in beta cell adhesion events during islet ontogeny. In betaTC3 cells, none of the novel genes were regulated at the RNA level by high glucose. However, in parallel studies, transcription of BA-12 was significantly increased by both sodium butyrate and nicotinamide, suggesting that this gene may play a role in pancreatic beta cell growth and/or differentiation. In this study, we have demonstrated that cRDA is an effective strategy for systematically mapping differences in gene expression between two related but functionally-distinct endocrine cells. Its application to experimental animal models of islet-cell regeneration may facilitate the discovery of potential factors that mediate beta cell growth and differentiation.
Abstract: The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.
Abstract: Diurnal rhythmicity is a characteristic of neuroendocrine pathways but is less understood in relation to immune function. We asked whether cellular (type 1) or humoral (type 2) immune responses or type 1/type 2 balance exhibit diurnal rhythmicity in healthy humans, and, if so, whether this is related to plasma levels of cortisol or melatonin, two hormones with immunomodulatory actions. LPS- or tetanus-stimulated human whole blood IFN-gamma and IL-10 production, and the IFN-gamma/IL10 ratio exhibited significant diurnal rhythmicity. The IFN-gamma/IL-10 ratio peaked during the early morning and correlated negatively with plasma cortisol and positively with plasma melatonin. IFN-gamma and, to a lesser extent, IL-10 production was sensitive to inhibition by exogenous cortisone; the IFN-gamma/IL-10 ratio decreased by >70% after the administration of oral cortisone acetate (25 mg). Our findings support the concept that plasma cortisol and possibly melatonin regulate diurnal variation in the IFN-gamma/IL-10 ratio. As IFN-gamma and IL-10 have opposing effects on cellular immunity, changes in their balance would be anticipated to impose diurnal rhythmicity on cellular immunity. This implies that the nature of an immune response, e.g., to vaccination, may be modified by the time of day of Ag presentation and could be therapeutically manipulated by the administration of cortisol or melatonin.
Abstract: The rosetting of T cells by sheep erythrocytes is mediated through the interaction of the CD2 molecule on T cells with T11TS, a molecule on sheep erythrocytes homologous to lymphocyte function-associated antigen-3 (LFA-3, CD58). We cloned a T11TS cDNA from sheep leucocyte mRNA which encodes a soluble molecule comprising the distal D1 and the D2 extracellular domains, but not the transmembrane domain. cDNA for this soluble D1 + D2 form of sheep LFA-3 (sLFA-3) was expressed in Escherichia coli and the properties of the purified recombinant protein were assessed by inhibition of T-cell rosette formation. sLFA-3 inhibited rosette formation, but its activity was low, 50% inhibition occurring at 25 micrograms/ml, consistent with the observed low binding avidity of fluorescein isothiocyanate (FITC)-labelled sLFA-3, sLFA-3 was made multimeric to increase its affinity, by crosslinking biotinylated sLFA-3 to streptavidin-biotinylated dextran complexes. The binding of crosslinked sLFA-3 multimers, tested by fluorescence-activated cell sorting (FACS) analysis, was significantly increased compared to sLFA-3 monomers. Competition with monoclonal antibodies demonstrated that multimeric sLFA-3 bound to the T11(1) epitope on CD2. The multimeric form of sLFA-3 was significantly more potent than the monomer in inhibiting proliferation of human T cells in response to purified protein derivative (PPD), tetanus toxoid (TT) or allogeneic cells. Multimeric sLFA-3 might, therefore, have potential as an immunotherapeutic agent to inhibit and/or anergize antigen-specific T-cell responses.
Abstract: Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in beta-cell destruction and immune regulation. A major target of beta-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of beta-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1 beta (1 ng/ml, 24 h), tumour necrosis factor alpha (TNF-alpha; 200 units/ml, 24 h) or interferon gamma (IFN-gamma; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-alpha (1000 units/ml), TNF-beta (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor beta 2 (TGF-beta 2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1 beta, TNF-alpha or IFN-gamma was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1 beta and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1 beta, TNF-alpha or IFN-gamma by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of beta-cell destruction in IDDM.
Abstract: Insulin-dependent diabetes mellitus (IDDM) is a multigenic autoimmune disease. An IDDM susceptibility gene was mapped to chromosome 2q34. This gene may act early in diabetogenesis, because "preclinical" individuals also showed linkage. Human leukocyte antigen (HLA)-disparate, but not HLA-identical, sibs showed linkage, which was even stronger in families with affected females. The genes encoding insulin-like growth factor-binding proteins 2 and 5 were mapped to a 4-megabase pair interval near this locus. These results indicate the existence of a gene that acts at an early stage in IDDM development, screening for which may identify a specific subset of at-risk individuals.
Abstract: In NOD mice, autoimmune recognition and destruction of pancreatic islet beta-cells appear to be independently regulated: all mice develop cellular infiltration of the islets (insulitis), but not all develop diabetes. The destructive potential of the insulitis lesion may depend on the balance between the two CD4+ T-cell subsets. TH1 and TH2, that mediate cellular-cytotoxic and humoral responses, respectively. With a semi-quantitative reverse transcriptase-PCR assay, we examined whether the disease process was reflected in the profiles of TH1 (IL-2, IFN-gamma and IL-12) and TH2 (IL-4, IL-6 and IL-10) cytokine mRNAs expressed in pancreata of NOD mice. Pancreata rather than isolated islets were examined to minimize manipulation ex vivo to preserve the expression of cytokine transcripts in vivo. At age 6 weeks, when 70% of mice had insulitis, all cytokine transcripts were detected in most pancreata, and their expression levels corresponded to the degree of insulitis. Similarly, during induction of diabetes with cyclophosphamide all transcripts were detected and levels corresponded with the degree of insulitis. In one-year-old mice without diabetes, all transcripts were detected but levels did not correspond to the degree of insulitis. Thus, in pancreata of NOD mice with different degrees of insulitis, we were unable to demonstrate, at the RNA level, polarisation of cytokine expression into either a TH1 or TH2 profile. This finding does not, however, exclude expression of distinct cytokine transcripts by immuno-inflammatory cells within the islet lesion, which might be revealed by in situ hybridization.
Abstract: Cellular immune hyporesponsiveness can be induced by the presentation of soluble protein antigens to mucosal surfaces. Most studies of mucosa-mediated tolerance have used the oral route of antigen delivery and few have examined autoantigens in natural models of autoimmune disease. Insulin is an autoantigen in humans and nonobese diabetic (NOD) mice with insulin-dependent diabetes mellitus (IDDM). When we administered insulin aerosol to NOD mice after the onset of subclinical disease, pancreatic islet pathology and diabetes incidence were both significantly reduced. Insulin-treated mice had increased circulating antibodies to insulin, absent splenocyte proliferation to the major epitope, insulin B chain amino acids 9-23, which was associated with increased IL-4 and particularly IL-10 secretion, and reduced proliferation to glutamic acid decarboxylase, another islet autoantigen. The ability of splenocytes from insulin-treated mice to suppress the adoptive transfer of diabetes to nondiabetic mice by T cells of diabetic mice was shown to be caused by small numbers of CD8 gamma delta T cells. These findings reveal a novel mechanism for suppressing cell-mediated autoimmune disease. Induction of regulatory CD8 gamma delta T cells by aerosol insulin is a therapeutic strategy with implications for the prevention of human IDDM.
Abstract: Many studies have examined the role of age, islet cell antibodies (ICAs), insulin autoantibodies (IAAs), and first-phase insulin responses (FPIRs) to an intravenous glucose tolerance test (IVGTT) as markers of risk of progression to IDDM, but a large data set is required for the analysis of the interactions between these markers. The Islet Cell Antibody Register Users Study (ICARUS) register includes 456 first-degree relatives with ICA levels > or = 5 JDF U confirmed in a reference laboratory, 108 of whom have progressed to IDDM in the course of prospective follow-up. Analysis of this data set confirmed the importance of the loss of FPIR, high ICA titer, coexistence of IAA, and young age in enhancing the risk of progression to the disease. The influence of any given marker of risk is modified by the presence or absence of the other markers. Cox regression analysis performed in a subset of 217 subjects for whom IVGTT, ICA, and IAA data were available showed that risk was most strongly associated with loss of FPIR; IAA and ICA titer contributed equally to the model, while age was also an independent risk determinant.
Abstract: MHCPEP is a curated database comprising over 6000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains peptide sequence, MHC specificity and when available, experimental method, observed activity, binding affinity, source protein, anchor positions, as well as publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using Gopher, FTP or WWW.
Abstract: Endogenous retroviruses (ERVs) are estimated to comprise up to 1% of human DNA. While the genome of many ERVs is interrupted by termination codons, deletions or frame shift mutations, some ERVs are transcriptionally active and recent studies reveal protein expression or particle formation by human ERVs. ERVs have been implicated as aetiological agents of autoimmune disease, because of their structural and sequence similarities to exogenous retroviruses associated with immune dysregulation and their tissue-specific or differentiation-dependent expression. In fact, retrovirus-like particles distinct from those of known exogenous retroviruses and immune responses to ERV proteins have been observed in autoimmune disease. Quantitatively or structurally aberrant expression of normally cryptic ERVs, induced by environmental or endogenous factors, could initiate autoimmunity through direct or indirect mechanisms. ERVs may lead to immune dysregulation as insertional mutagens or cis-regulatory elements of cellular genes involved in immune function. ERVs may also encode elements like tax in human T-lymphotrophic virus type I (HTLV-I) or tat in human immunodeficiency virus-I (HIV-I) that are capable of transactivating cellular genes. More directly, human ERV gene products themselves may be immunologically active, by analogy with the superantigen activity in the long terminal repeat (LTR) of mouse mammary tumour viruses (MMTV) and the non-specific immunosuppressive activity in mammalian type C retrovirus env protein. Alternatively, increased expression of an ERV protein, or expression of a novel ERV protein not expressed in the thymus during acquisition of immune tolerance, may lead to its perception as a neoantigen. Paraneoplastic syndromes raise the possibility that novel ERV-encoded epitopes expressed by a tumour elicit immunity to cross-reactive epitopes in normal tissues. Recombination events between different but related ERVs, to whose products the host is immunologically tolerant, may also generate new antigenic determinants. Frequently reported humoral immunity to exogenous retrovirus proteins in autoimmune disease could be elicited by cross-reactive ERV proteins. A review of the evidence implicating ERVs in immune dysfunction leads to the conclusion that direct molecular studies are likely to establish a pathogenic role for ERVs in autoimmune disease.
Abstract: There is increasing interest in the use of glutamic acid decarboxylase antibodies (GADAbs) for identification of subjects at increased risk of developing insulin-dependent diabetes mellitus (IDDM). However, considerable variation exists between laboratories in the reported frequency of GADAb in various clinical situations, and disease sensitivity and specificity have not yet been compared between assays. An international workshop was held in which 101 coded freeze-dried sera, including 39 from subjects with newly diagnosed IDDM, 32 from healthy control subjects, 4 from nondiabetic subjects with Graves' disease, and 4 from islet cell antibody-positive subjects, were analyzed in 52 assays (radiobinding assay [RBA], 26; enzyme-linked immunosorbent assay [ELISA], 19; and enzymatic immunoprecipitation assay [EIP], 7). The mean sensitivity for RBAs (76.2%) was higher than for ELISAs (36.5%) and EIPs (49.9%) (P < 0.01). The mean specificity was similar for each assay format (RBA, 89.4%; ELISA. 89.4%; and EIP, 92.3%). The lower sensitivities of the ELISA and EIP were predominantly due to the inability of these assays to detect low levels of GADAb in IDDM. To convert results to standard units, standard curves were constructed using duplicate dilutions of the anti-glutamic acid decarboxylase monoclonal antibody MICA 3 and serum from a patient with stiff-man syndrome (SMS). Curves could be derived in 28 assays using the MICA 3 serum and in 29 using the SMS serum. The mean coefficients of variation between assays for disease and control samples were 45% when results were converted to MICA units, 77% for SMS units, and 76% for SD scores.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: IDDM results from the immune-mediated destruction of pancreatic islet beta cells. Clinicopathologic heterogeneity in IDDM is reflected in part by the wide age range over which the onset of clinical symptoms can occur, after months to years of subclinical "insulitis." Because MHC genes play a critical role in immune function we studied their possible contribution to IDDM heterogeneity by analyzing HLA profiles of 194 IDDM patients in relation to their age at diagnosis. Restriction of HLA-DR heterogeneity was observed in patients diagnosed before age 21 years. Frequencies of DR3 and DR3/4 were highest in the < or = 6-year-old age group and thereafter declined with increasing age at diagnosis. In contrast, the frequency of DR4 remained increased up to age 30 years at diagnosis. DR7, normally considered to be a neutral allele, was like DR2 and DR5, significantly decreased in patients diagnosed before age 21 years. The A30-B18-DR3 haplotype was significantly increased in the < or = 6-year-old age group, A1-B8-DR3 was increased in the > or = 31-year-old group. B62-DR4 was increased only in the > 12-year-old age group. In DR4 patients the frequency of DQ8 was increased across all age groups. A sex difference was observed in those diagnosed at < or = 12 years of age, with an excess of females in the DR3+/DR4- group and males in the DR3-/DR4+ group. An association of DPB1 with IDDM was revealed by an increased frequency overall of DPB1*0301 and/or DPB1*0401, being more pronounced in patients diagnosed at > 20 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: OBJECTIVES. To investigate whether circulating levels of interleukin 1 beta (IL-1 beta) or soluble interleukin 2 receptor (sIL-2R) reflect clinical disease status and response to therapy in scleroderma. METHODS. Plasma IL-1 beta and serum sIL-2R were measured by ELISA in 19 patients with limited cutaneous scleroderma (9 with extraesophageal internal organ involvement), 5 patients with diffuse cutaneous scleroderma and internal organ involvement, and 11 healthy controls, as well as serially over 12 months in 4 patients with scleroderma treated with cyclosporine. RESULTS. IL-1 beta levels were similar in scleroderma and control subject groups. sIL-2R levels were significantly higher in subjects with scleroderma involving internal organs (elevated in 93%), and correlated with erythrocyte sedimentation rate. sIL-2R levels decreased over 12 months in 2 of 4 patients taking cyclosporine in whom other variables remained unchanged. CONCLUSIONS. Elevated serum sIL-2R is a marker of internal organ involvement in scleroderma and warrants further investigation in assessing disease prognosis and response to therapy.
Abstract: The measurement of cytokines produced by activated T cells refines assessment of cellular immune function and facilitates whole blood T cell assays. The latter approximate conditions in vivo and obviate the need to purify blood mononuclear cells. We have investigated the parameters of the whole blood assay in humans to standardize and optimize the detection of tetanus-specific T cell cytokine responses. Optimal conditions include the use of undiluted whole blood, an incubation time of 36-48 h and a minimum of delay between venesection and incubation of the blood with antigen. Blood should be drawn at a standard time of day to minimize inter-assay variation due to diurnal rhythmicity in cytokine production. Interferon-gamma or interleukin-2 are specific and reliable readouts; other cytokines can be measured to further characterize the TH1 and TH2 elements of the T cell responses, although tetanus-stimulated IL-4 production is detected in only a minority of healthy individuals. The whole blood assay is a potentially valuable tool for assessing cellular immune function and screening for antigen-reactive T cells in humans.
Abstract: BACKGROUND: Individuals at risk for insulin-dependent diabetes mellitus (IDDM), with an affected first-degree relative, can be identified by the presence of islet cell antibodies (ICA). ICA-positive relatives progress at variable rates to IDDM and identification of those at highest risk is a prerequisite for possible preventative treatment. Those who develop IDDM may exhibit less genetic heterogeneity than their ICA-positive or ICA-negative relatives. Specific human leucocyte antigen (HLA) genes predispose to IDDM but could also influence the rate of progression of preclinical disease. Therefore, by comparing HLA antigen frequencies between first-degree relatives, we sought to identify HLA genes that influence progression to IDDM. MATERIALS AND METHODS: HLA antigen frequencies were compared in 68 IDDM, 53 ICA-positive, and 96 ICA-negative first-degree relatives from 40 Caucasoid families. Predictions were tested in a panel of 270 unrelated IDDM subjects. HLA typing was performed serologically (HLA class I and II) and by sequence-specific oligotyping (11th International Histocompatibility Workshop protocol) (HLA class II). ICA tests were measured by an internationally standardized indirect immunofluorescence assay. RESULTS: In general, known susceptibility class II HLA antigens increased in frequency and known protective class II HLA antigens decreased in frequency, from ICA-negative to ICA-positive to IDDM relatives. Thus, DR4 and DQ8 increased whereas DR2 and DQ6 decreased; DR3 and DQ2 increased from ICA-negative to ICA-positive relatives, but not further in IDDM relatives. The high-risk DR3, 4 phenotype increased across the three groups; DR4,X was unchanged, and DR3,X and DRX,X both decreased. The HLA class I antigen, A24, occurred more frequently in ICA-positive relatives who developed IDDM and, in 270 unrelated IDDM subjects, was associated with an earlier age at diagnosis of IDDM in those with the lower risk class II phenotypes DR4,4 and DR3,X. CONCLUSIONS: HLA-DR3 and DQ2 predispose to islet autoimmunity, but not development of clinical IDDM in the absence and DR4 and DQ8. DR4 and DQ8 predispose to the development of clinical IDDM in ICA-positive relatives, in combination with DR3-DQ2 and other haplotypes but not when homozygous. HLA-A24 is significantly associated with rapid progression to IDDM in ICA-positive relatives and with an earlier age at clinical diagnosis. Analysis of IDDM families reveals that HLA genes not only predispose to islet autoimmunity but influence progression to clinical disease. The findings have implications for identifying high-risk relatives as candidates for possible preventative therapy.
Abstract: gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter, synthesised from glutamate by glutamate decarboxylase (GAD), in the central nervous system. Two forms of GAD, designated GAD 65 and GAD 67, are encoded by distinct genes and have been demonstrated in the mammalian brain. GABA has been postulated to be synthesised in neurons of the enteric nervous system (ENS), but evidence for its role as an enteric neurotransmitter is equivocal. We therefore aimed to determine whether GAD 65 and GAD 67 messenger RNAs (mRNAs) and proteins were expressed in the ileum of mice, rats and guinea pigs. Using an RNase protection assay, both GAD 65 and GAD 67 mRNAs were detected in the rodent small intestine. Antisera specific for GAD 65 or GAD 67, used in immunoblot analyses, revealed GAD 65-like and GAD 67-like immunoreactivity in rat and guinea pig ileum. Anti-GAD 65 antisera detected a major band of 65 kDa. Anti-GAD 67 antisera detected a major band of 55 kDa, which probably represented a breakdown product, and a minor band of 67 kDa. Analysis of immunoblot extracts of rat and guinea pig ileum revealed more GAD 67-like than GAD 65-like immunoreactivity. GAD enzymatic activity was high in the rat and guinea-pig brain, and low in the whole and dissected ileum. These results demonstrate that both GAD 65 and GAD 67 genes are transcribed and translated in the ileum of three rodent species and lend indirect support to the postulate that GABA is synthesised by neurons of the ENS and intestinal endocrine cells.
Abstract: OBJECTIVE--To determine whether the reproducibility of the first-phase insulin response (FPIR) measured during an intravenous glucose tolerance test is improved by the use of a lower glucose dose or retrograde sampling from an arterialized hand vein. RESEARCH DESIGN AND METHODS--Previous studies have suggested that the high within-subject variation of FPIR measurement of up to 110% could be reduced by sampling from a retrograde cannulated and arterialized hand vein opposite to the cubital fossa vein through which the glucose was injected or by the use of a lower dose of glucose. Two low-dose (glucose, 5 g/m2 injected over 30 s) and two standard Islet Cell Antibody Registry Users Study (ICARUS) (glucose, 0.5 g/kg injected over 3 min) tests were performed on seven normal subjects at 2-week intervals. Samples were collected simultaneously from the cubital fossa vein, through which the glucose was injected, and from a retrograde cannulated, contralateral hand vein that was arterialized by heating. FPIR was expressed as the sum of the insulin measurements 1 and 3 min after the completion of the glucose injection and as the area under the insulin curve between 0 and 10 min. RESULTS--Responses to the mean sum of serum insulin concentrations at 1 and 3 min after intravenous glucose were significantly lower for the low-dose test (mean 94 mU/l) than for the high-dose test (mean 184 mU/l) for samples taken from the arm (P < 0.05); mean 0- to 10-min insulin areas were 367 and 596 mU/l for low- and high-dose tests, respectively (P < 0.05). Within-subject coefficients of variation for samples from the hand or the arm ranged from 0.33 to 17.5% and 1.3 to 38% for successive ICARUS and low-dose tests, respectively. Reproducibility, measured by the coefficient of variation between successive tests for each protocol, was not significantly different using samples taken from the arm or the contralateral hand. CONCLUSIONS--The intravenous glucose tolerance test is reproducible when performed by the same operator over a short time span. Reproducibility is not significantly improved by sampling from an arterialized, retrograde cannulated, contralateral hand vein. There is no case for changing the present ICARUS protocol to incorporate retrograde cannulation or low-dose (5 g/m2) glucose.
Abstract: The distribution of DRB1*04 alleles and DRB1/DQB1 haplotypes was analysed in 57 DR4+ caucasoid subjects with insulin-dependent diabetes mellitus (IDDM) and 96 DR4+ healthy controls selected on the basis of DR serology, and the findings were analysed in relation to age at diagnosis of IDDM. DNA samples were amplified using specific DR and DQ primers and hybridized with sequence-specific oligonucleotide probes. A significantly increased combined frequency of DRB1*0401 and 0402 was observed in IDDM subjects aged < or = 12 years at diagnosis (allele frequency 88.4% compared with 62.0% in controls, P < 0.025). There was a non-significant increase in DRB1*0401 and 0402 in IDDM subjects < or = 12 years when compared with IDDM subjects > 12 years (P < 0.1). DRB1*0404 was decreased in the total IDDM subject group compared with controls (4.8% vs. 19.0%, P < 0.025) but did not reach statistical significance in the individual age at diagnosis groups. In contrast, the frequency of DQB1*0302 was increased uniformly across both ages at diagnosis groups. In controls DRB1*0401 occurred in haplotype association with DQB1*0301 in a significantly greater frequency than with DQB1*0302. However, 95.0% of DRB1*0401 IDDM subjects were DQB1*0302. DRB1*0404, which was decreased in frequency in IDDM subjects, occurred in association significantly more frequently with DQB1*0302 in controls. These results imply that DRB1 and DQB1 have independent roles as HLA susceptibility genes in IDDM. DQB1 may have a permissive role whereas DRB1 could influence the rate at which underlying disease progresses to clinical IDDM.
Abstract: BACKGROUND: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24-36) that bears marked similarity to a peptide of GAD65 (amino acids 506-518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T cell reactivity to these two peptides in subjects at risk for IDDM. MATERIALS AND METHODS: Subjects at risk for IDDM were islet cell antibody (ICA)-positive, first degree relatives of people with insulin-dependent diabetes. Peripheral blood mononuclear cells from 10 pairs of at-risk and HLA-DR matched control subjects were tested in an in vitro proliferation assay. RESULTS: Reactivity to both proinsulin and GAD peptides was significantly greater among at-risk subjects than controls (proinsulin; p < 0.008; GAD; p < 0.018). In contrast to reactivity to the GAD peptide, reactivity to the proinsulin peptide was almost entirely confined to the at-risk subjects. CONCLUSIONS: This is the first demonstration of T cell reactivity to a proinsulin-specific peptide. In addition, it is the first example of reactivity to a minimal peptide region shared between two human autoimmune disease-associated self antigens. Mimicry between these similar peptides may provide a molecular basis for the conjoint autoantigenicity of proinsulin and GAD in IDDM.
Abstract: Glutamic acid decarboxylase (GAD), a target of both autoantibodies and autoreactive T-cells in insulin-dependent diabetes (IDD), exists as two homologous forms, GAD65 and GAD67. GAD65 is preferentially expressed in human islets and recognized by autoantibodies in IDD, but which form primarily elicits GAD autoimmunity is unknown. GAD67 gene expression in human islets has been demonstrated only by the polymerase chain reaction. We, therefore, quantitatively compared the expression of each GAD gene in human islets and mapped the binding of autoantibodies to recombinant human GAD67 by enzyme-linked immunosorbent assay. In ribonuclease protection assays, both forms of GAD messenger RNA (mRNA) were detected in human islets, although GAD65 mRNA was 200 times more abundant than GAD67 mRNA. Immunoblotting of islets with GAD form-specific antisera revealed GAD65, but not GAD67. By in situ hybridization and immunohistochemistry, GAD65 mRNA and protein were localized to islets, predominantly, but not entirely, to beta-cells; GAD67 mRNA and protein were undetectable. Thus, although GAD67 protein expression was undetectable in human islets, the GAD67 gene is transcribed, albeit weakly. Antibodies that recognized multiple epitopes in recombinant GAD67 were found in 20% of sera from ICA positive "at risk" first degree relatives of IDD subjects and recent-onset IDD subjects. The majority of GAD67 epitopes were mapped within the mid- and C-terminal thirds of the protein, a region that is highly conserved in GAD65. Although GAD67 may share cross-reactive epitopes with GAD65, these findings do not exclude the possibility that autoimmunity to GAD arises as a consequence of the aberrant up-regulation of GAD67 in human islets.
Abstract: OBJECTIVE. To identify synovial antigens that bind to serum antibodies from subjects with recent-onset rheumatoid arthritis (RA) (< 6 months of synovitis). METHODS. Soluble and insoluble fractions of Triton X-100 extracts of RA and normal synovial tissue, and normal spleen and placenta, were immunoblotted with sera from 27 patients with recent-onset RA, 13 autoimmune disease control subjects, and 13 blood bank control donors. Bound immunoglobulin was probed with 125I-labeled protein A. RESULTS. Antibodies in the sera of 20 (74%) patients with recent-onset RA recognized at least 1 of 5 antigens in both a disease- and tissue-specific and nonspecific manner. Anti-La antibodies, usually associated with primary Sjögren's syndrome, were detected in 2 sera. Eight sera had increased reactivity to an IgG heavy and light chain dimer. There was strong binding of 8 sera with a 35-kd doublet and of 3 sera with a 55-kd species in RA and normal synovial lysates (insoluble fractions). Two sera uniquely recognized a 45-kd protein only in the RA synovial lysate (soluble fraction). CONCLUSION. IgG antibodies in the sera of patients with recent-onset RA show positive immunoblots for 3 novel synovial antigens of 35-kd, 55-kd, and 45-kd, as well as for 2 previously characterized antigens (La and IgG). Thus, a variety of synovial antigens appear to be recognized by B cells even early in the clinical course of RA.
Abstract: Rheumatoid arthritis (RA) remains one of the most common, puzzling and poorly treated diseases of humans. However, a surge of interest in the biology of chronic inflammation and in the design of more-potent and specific inhibitors of pro-inflammatory pathways heralds an optimistic era for the treatment of RA. A recent symposium provided a multidisciplinary perspective on the current status of such studies.
Abstract: Glutamic acid decarboxylase antibodies (GADAbs) are being increasingly used in clinical and research programs for the prediction and classification of insulin-dependent diabetes mellitus (IDDM). A number of different assay formats for the measurement of GADAbs have been reported, but the degree of concordance between assays is unknown. In this study, GADAbs were measured on 16 coded sera in 34 assays to examine concordance between GADAb assays and establish the feasibility of an international GADAb standard of measurement unit. The 16 lyophilized coded samples consisted of sera from healthy control subjects (n = 2), IDDM patients (n = 3), a patient with polyendocrine autoimmunity (n = 1), and duplicate dilutions of plasmapheresis serum from a patient with stiff-man syndrome (SMS). A high level of concordance was found in the ranking of GADAb levels (P = 0.99, Friedman's test) in the samples. Thirteen (38%) assays could reproducibly distinguish dilutions of SMS serum and detect GADAbs in all IDDM and polyendocrine autoimmunity sera tested. Although assessed on only four samples, disease specificity was 100% in 29 assays. The majority of assays that immunoprecipitated radiolabeled GAD gave high results for sensitivity and specificity. Enzyme-linked immunosorbent assays and assays using immunofluorescence were generally less sensitive. Several assays, in particular those measuring GAD enzymatic activity immunoprecipitated in fluid phase from rat brain homogenate, showed a prozone-like phenomenon in the SMS dilution curve. Interpolation of results from a standard curve into workshop units resulted in relatively low scatter in samples with lower levels of GADAbs. Hence, the use of an international reference serum to enable comparison of results between laboratories appears feasible.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: MHCPEP is a curated database comprising over 4000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains the source protein (when known), an estimate of binding affinity and critical anchor residues (if identified), and is fully referenced. The present format of the database allows test string matching searches. The database can be accessed via Internet using gopher.
Abstract: Recently, assessment of T cell function has been refined by the ability to measure cytokines produced by activated T cells. We developed a whole blood assay to detect antigen-activated T cells that produce IFN-gamma. With this assay we have found a large circadian variation in tetanus- (acrophase 00(00) p < 0.001) and PPD- (acrophase 00(08) p < 0.001) stimulated IFN-gamma production. IFN-gamma production is inversely correlated with plasma cortisol (r = -0.5), suggesting that variation in IFN-gamma production may be secondary to circadian variation in plasma cortisol levels (acrophase 11(06)). The demonstration of circadian rhythmicity in antigen-stimulated IFN-gamma production is relevant to the diagnostic use of whole blood assays and, in addition, may have implications for the therapy of immuno-inflammatory diseases.
Abstract: The receptor protein for thyrotrophin (thyroid-stimulating hormone; TSH) is associated with a glycosphingolipid moiety. The protein belongs to the family of receptors that couple to guanine nucleotide binding proteins; the glycosphingolipid contains sialic acid and belongs to the family of gangliosides. This report defines the structure of the receptor ganglioside in the Fisher rat thyroid cell line (FRTL-5). Receptor protein was purified by TSH affinity chromatography from FRTL-5 cells, biosynthetically labelled with [3H]galactose and [3H]glucosamine, and resolved by SDS-PAGE. A single radiolabelled band of Mr approximately 80 kDa, corresponding to the predicted size of the cloned receptor, contained ganglioside. Gangliosides were extracted from unlabelled receptor protein after SDS-PAGE and probed on TLC plates with 125I-labelled Limax flavus agglutinin or the B subunit of cholera toxin, before and after digestion with Vibrio cholerae sialidase or beta-galactosidase. The TSH receptor (TSH-R) ganglioside belongs to the gangliotetraose family, having sialic acid attached to both galactose molecules. Its sialic acid is devoid of negative charge because of the formation of internal esterlactones. Its structure is lactonized N-acetylneuraminyl-(alpha 2-->3)galactosyl(beta 1-->3)-N-acetylgalactosaminyl(beta 1-->4)-[N-acetylneuraminyl(alpha 2-->3)]galactosyl(beta 1-->4)glucosyl(beta 1-->1)ceramide (GDla-lactone). Ganglioside lactones have not been previously described as components of thyroid cells. They are highly rigid and are more likely than their parent structures to serve as molecular recognition sites and elicit immunoreactivity. Identification of this unique ganglioside intimately associated with the TSH receptor implies that it has an integral role in receptor structure and function.
Abstract: To determine whether the predictive value of islet cell antibodies (ICA) and insulin autoantibodies (IAA) is increased by measurement of glutamic acid decarboxylase antibodies (GADAb) in first-degree relatives of patients with insulin-dependent diabetes mellitus (IDDM), we measured GADAb in those developing IDDM and in relatives found to be ICA- or IAA-positive in our family screening study. First-degree relatives (n = 2904) were followed for 2.4 (median, range 0.04-5.8) years. Of the subjects developing IDDM, 11/14 (78%) had ICA > or = 20JDF units, 1/14 (7%) had IAA > or = 100 nU/ml and 6/14 (43%) had GADAb (> or = 460 nU/ml, measured by precipitation of enzymatic activity). Of the four subjects with ICA < 20 and IAA < 100 nU/ml who developed IDDM, one had elevated GADAb. Significant inhibition of GAD enzymatic activity by serum immunoglobulins, a potential cause of false-negative results in our immunoprecipitation assay, was not detected in seven subjects who developed IDDM in the absence of GADAb. Sixty-nine of the 2904 subjects with ICA > or = 20 or IAA > or = 100 were followed for 3.1 (median range 0.1-5.4) years. Survival analysis showed that diabetes-free survival in this group was not influenced significantly by GADAb positivity. In conclusion, GADAb in the absence of ICA and IAA are uncommon in first-degree relatives who progress to IDDM and the presence of GADAb does not increase the risk for IDDM in ICA- or IAA-positive relatives.
Abstract: Tumour necrosis factor (TNF) has been implicated in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). To investigate a possible role for TNF in IDDM we compared endogenous TNF production in two lines of non-obese diabetic (NOD) mice, NOD/Lt and NOD/WEHI, that have a high and low incidence of diabetes, respectively. Preliminary experiments had shown that the lower syngeneic mixed lymphocyte reaction (SMLR) in NOD/Lt mice could be corrected by TNF-alpha. Plasma TNF-alpha was measured in 8 week-old female non-diabetic mice primed with 1000 units IV of murine interferon gamma (IFN-gamma) followed after 3 hours by 5 micrograms IV of lipopolysaccharide (LPS). Two hours later plasma was collected and TNF measured by ELISA. Plasma TNF in NOD/Lt mice was 9.2 +/- 2.4 ng/ml (mean +/- SEM, n = 16) compared to 2.5 +/- 0.5 ng/ml in NOD/WEHI mice (n = 15) and 7.6 +/- 1.0 ng/ml in BALB/c mice (n = 14). Time course studies demonstrated higher levels of both immunoreactive and bioactive TNF in NOD/Lt compared to NOD/WEHI mice up to 4 hours post-stimulation. A separate group of female NOD/Lt mice had IFN-gamma/LPS-stimulated plasma TNF-alpha measured at 10 weeks and were followed to age 30 weeks. The mean stimulated plasma TNF-alpha level was consistently higher in those mice that developed diabetes compared to those that remained non-diabetic, the difference being significant when mice were 21 weeks of age. These results suggest that endogenous TNF-alpha production may be a trait marker of IDDM susceptibility.
Abstract: Antibodies to glutamic decarboxylase (GADAb) are present in insulin-dependent diabetes (IDD) but their association with age and sex and their temporal profile in relation to disease onset have not been fully documented. We have examined the association between GADAb and islet cell antibodies (ICA), age and sex, and have cross-sectionally and longitudinally measured the levels of GADAb before and after diagnosis of IDD. GADAb were measured by allowing serum immunoglobulin prebound to protein A Sepharose to precipitate GAD enzymatic activity from a fetal pig brain extract. GADAb levels were above the normal range (mean + 3SD of healthy controls, 460 nU/ml) in 19/44 (43%) at-risk subjects (ICA positive first degree relatives of persons with IDD), 35/108 (32%) recent-onset IDD subjects and 22/46 (47%) established IDD subjects. When analysed according to age and sex, GADAb levels were significantly higher (P < 0.05) in post-pubertal females in at-risk, recent-onset and established IDD groups. There was a significant association between GADAb and ICA > 20 in both first degree relatives (P < 0.001) and recent-onset subjects (P < 0.01) and GADAb were uncommon in the absence of ICA. Levels of GADAb were similar in at-risk, recent-onset and established IDD subjects and GADAb status remained stable in all but 2/41 at-risk subjects followed for 17 (mean, range 3-33) months. In conclusion, GADAb levels are strongly influenced by age, sex and ICA status, and generally remain stable in at-risk subjects and after the onset of clinical IDD.
Abstract: Dexfenfluramine, a serotonin agonist with effects on the central nervous system (CNS), lowers blood glucose in patients with non-insulin-dependent mellitus (NIDDM). Previous studies using the hyperinsulinemic clamp have shown that dexfenfluramine improves insulin action on both stimulation of glucose uptake and inhibition of hepatic glucose production (HGP). Since the central nervous system can influence glucose tolerance in ways that may not be detected using a clamp procedure, we investigated the effects of dexfenfluramine on glucose kinetics during an oral glucose tolerance test (OGTT) in patients with NIDDM. Glucose kinetics were measured basally and during an OGTT using a double isotope technique and the modified one-pool model of the glucose system. After a 4-week run-in period, studies were performed before, after two 15 mg doses, and then after 4 weeks on 15 mg twice daily in 10 subjects with NIDDM. Fasting-plasma glucose was significantly lower after 4 weeks on dexfenfluramine (P < 0.01) as was plasma glucose at both 1 and 2 h during the OGTT (P < 0.05). The lower plasma glucose was associated with a reduction in HGP both basally (P < 0.01) and during the 1st hour of the OGTT (P < 0.05). There was no change in peripheral glucose uptake. Plasma insulin levels were unaltered, but plasma glucagon was lower after 1 month of treatment. We conclude that dexfenfluramine improves fasting-blood glucose and oral glucose tolerance predominantly by reducing hepatic glucose production.
Abstract: Autoantibodies to glutamic acid decarboxylase (GAD) are present in humans before and after the onset of clinical insulin-dependent diabetes (IDD). The non-obese diabetic (NOD) mouse, a model of human IDD, develops mononuclear cell infiltration of the pancreatic islets ('insulitis') associated particularly in females with T cell-mediated destruction of the islet beta cells. In NOD mice of both sexes we detected serum antibodies to GAD (GAD Ab) that precipitate mouse brain GAD enzymatic activity. Antibodies in NOD sera also precipitate a M(r) 65,000 protein from Triton X-100 extracts of 35S-methionine-labelled NOD islets, identical in size to that precipitated by a monoclonal antibody to GAD. GAD Ab were not detected in other mouse strains. There were significant differences in the frequency, level and age at initial detection of GAD Ab between females of the NOD/Lt and NOD/WEHI lines, previously shown to have a higher and lower incidence of diabetes, respectively. Comparing NOD/Lt (n = 26) and NOD/WEHI (n = 20) females, in which diabetes occurred in 38% and 20% by 150 days, the frequency of elevated GAD Ab was 50 vs. 80%, the mean maximum GAD Ab level 21.1 vs. 30.6% and the mean age at which GAD Ab were first detected 94 vs. 45 days. No significant differences in these parameters were observed between male mice of either line. There was a significant negative correlation between the level of GAD Ab and the degree of insulitis in female mice from both lines. GAD Ab were not a prerequisite for the development of diabetes. In 7 of 10 female mice the onset of diabetes was preceded by a decrease of GAD Ab levels into the normal range. These findings indicate that, while GAD is a target of autoimmunity in the NOD mouse, GAD Ab do not necessarily correlate with the development of diabetes. Indeed, the difference between the two NOD lines and the inverse relationship with insulitis suggests that a strong humoral response to GAD may be associated with a less destructive pathology, as proposed in humans 'at-risk' for IDD.
Abstract: 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-gamma, tumour necrosis factor-alpha 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: Glutamic acid decarboxylase (GAD) is a pancreatic islet autoantigen in insulin-dependent diabetes (IDD). Two forms of GAD, GAD65 and GAD67, have been identified in brain but human islets have been reported to express only GAD65. We have isolated full length GAD67 cDNA by polymerase chain reaction (PCR) cloning from human pancreas. Sequence analysis reveals only four nucleotide differences between human pancreas and brain GAD67, two of which result in amino acid changes. These differences are probably individual-specific and reflect allelic variation. Using mRNA from isolated human islets a partial sequence was obtained by PCR cloning that was identical to the midregion of pancreas GAD67 cDNA. GAD67 has previously been shown to be a target of both autoantibodies and autoreactive T cells in IDD. The presence of GAD67 in human pancreas implies that this form of GAD, as well as GAD65, has a pathogenic role in IDD.
Abstract: Glutamic acid decarboxylase (GAD) in pancreatic beta cells is an autoantigen in insulin-dependent diabetes (IDD). We measured immunity to GAD in 31 first-degree relatives of IDD patients judged to be at risk of developing IDD themselves because of the presence of islet-cell antibodies. We found that in most of the subjects GAD autoimmunity was either predominantly humoral or predominantly cellular. High concentrations of circulating autoantibodies that precipitate native GAD activity were associated with low proliferation of peripheral-blood T cells to recombinant GAD; conversely, low concentrations of autoantibody to GAD were associated with high T-cell proliferation to GAD. Although T-cell proliferation was measured in the presence of autologous serum, GAD autoantibodies did not have a blocking effect in vitro. This dichotomy of the immune response to GAD defined heterogeneity within at-risk relatives and could have prognostic importance. We postulate that, if GAD is a pathogenetic autoantigen, sensitisation to beta-cell GAD is more likely to lead to IDD when the immune response deviates towards the expansion of autoreactive T cells rather than towards generation of autoantibodies. This idea is consistent with evidence that beta-cell destruction is mediated by T cells and that high concentrations of GAD antibodies are associated with slower progression to clinical disease.
Abstract: Human actin binding protein (ABP) links specific membrane glycoproteins to cytoskeletal actin microfilaments. In human platelets and leukocytes, ABP directly links, respectively, the membrane glycoproteins GPIb and the high-affinity Fc receptor for IgG (Fc gamma IR) to cytoskeletal actin microfilaments. Similar interaction between the thyrotropin (TSH) receptor and ABP in endocrine cells might explain the rapid and profound disruption of actin microfilaments induced by TSH in cultured thyroid follicular cells. By screening a thyroid lambda gt11 cDNA expression library with serum from a Graves disease patient, we identified a clone encoding a protein, designated truncated ABP (TABP), that shares extensive homology (approximately 70%) with ABP. TABP is a truncated ABP-like protein with an open reading frame of 195 aa that encodes a protein of approximately 21 kDa. TABP lacks an actin binding domain but contains two predicted beta-sheet repeats within which is a putative dimerization domain and between which lies a putative glycoprotein binding site containing a consensus site for phosphorylation by Ca(2+)-calmodulin kinase II. TABP contains a unique C-terminal insertion within which lies a hydrophobic predicted membrane-associated region, absent from ABP. Although TABP mRNA is expressed widely, immunoblot analysis demonstrated the presence of TABP antibodies specifically in the sera of a minority of subjects with autoimmune thyroid disease. A 24-residue sequence of similarity was identified between the TSH receptor and platelet glycoprotein GPIb alpha that may represent a transmembrane ABP binding site. We suggest, therefore, that signal transduction by TSH in the thyroid involves direct linkage of the TSH receptor to actin microfilaments by ABP and that TABP may interact with ABP to mediate TSH-induced actin microfilament disruption.
Abstract: Target antigens defined by autoantibodies in IDDM include insulin, a putative glycolipid that reacts with islet cell antibodies, and a 64,000-M(r) protein recently identified as glutamic acid decarboxylase. In addition, some IDDM sera that contain antibodies to glutamic acid decarboxylase also coprecipitate a 38,000-M(r) protein from islets. This study used a high titer anti-38,000-M(r) serum to screen bacteriophage lambda cDNA expression libraries and identified human islet and placental clones encoding jun-B, the nuclear transcription protein, of predicted 38,000 M(r). Peripheral blood T-cells exhibited significant proliferation in response to a recombinant fragment of jun-B (amino acids 1-180) in 12 of 17 (71%) recent-onset IDDM subjects, 8 of 16 (50%) ICA-positive first-degree relatives of IDDM subjects who were at risk, 3 of 12 (25%) other autoimmune disease subjects, and 0 of 10 healthy control subjects. Proliferation to tetanus toxoid did not differ significantly between the groups. Responses to jun-B were not related to age, sex, or human leukocyte antigen status. Thus, autoreactive T-cells identify a novel antigen, p38 jun-B, in IDDM and appear to indicate subjects at risk for the development of clinical disease.
Abstract: Glutamic acid decarboxylase (GAD) catalyzes synthesis of the inhibitory neurotransmitter gamma-amino butyric acid. Two homologous forms of GAD encoded by separate genes have been cloned from rat brain, with predicted protein sizes of 67 and 65 kilodaltons. GAD is present outside the brain, and pancreatic islet GAD is believed to be a target of autoimmunity in insulin-dependent diabetes mellitus. However, peripheral expression of the two GAD genes is incompletely characterized. We, therefore, investigated GAD expression in peripheral tissues, including pancreas, of mouse and rat. cDNAs encoding GAD 67 and GAD 65 were cloned from mouse brain and shown to be 95% homologous with the rat sequences. RNase protection assay using specific cRNA probes demonstrated expression of both GAD forms in freshly harvested pancreas and testis. Levels of both GAD mRNAs were greater in rat than mouse pancreas. GAD 67 mRNA was more abundant than GAD 65, and both were localized to islet beta-cells by in situ hybridization. In testis, both GAD mRNAs were localized to spermatocytes. Additionally, GAD 67, but not GAD 65, mRNA was detected in mouse and rat spleen and mouse liver. Thus, both GAD genes are expressed in peripheral tissues, with GAD 67 mRNA being more abundant under physiological conditions. The expression of both GAD 67 and GAD 65 genes specifically in islet beta-cells indicates that both GAD forms are candidate autoantigens in rodent models of insulin-dependent diabetes mellitus.
Abstract: Autoantibodies that react with the nuclear enzyme topoisomerase I (Topo I) are used as a diagnostic marker of diffuse scleroderma. To better define immune reactivity to Topo I, antibody epitopes in two patient populations were analyzed using recombinant Topo I proteins. Two overlapping partial cDNA clones encoding the complete amino acid sequence of Topo I were isolated from human placenta. Using the polymerase chain reaction, specific regions of Topo I were amplified and cloned into the pGEX expression vectors. To map Topo I epitopes, recombinant fusion proteins were analyzed by immunoblotting with 66 anti-Topo I sera from Thai and Australian patients with diffuse scleroderma. Six distinct epitope regions were identified along the length of the 765 amino acid enzyme. Almost all sera contained antibodies that recognized the midregion of Topo I (amino acids 453-560), as well as antibodies to one of more of the other epitope regions. Sixty percent of the sera contained antibodies that recognized a COOH-terminal epitope region (amino acids 658-765) encompassing the active site of the enzyme. This subset of Topo I antibodies could be responsible for the inhibition of enzymatic activity previously reported in vitro. Heterogeneous patterns of reactivity with the six Topo I epitope regions were observed, although over half the sera could be assigned to one of six distinct patterns. In general, antibodies in the Thai sera reacted more strongly with the six epitope regions. Furthermore, two of the epitope regions reacted exclusively with Thai sera, suggesting a degree of racial or geographical specificity in the autoantibody response to Topo I. The identification of multiple epitopes in Topo I conforms with the polyclonal autoantibody response to intracellular Ag found in other multisystem autoimmune diseases and is presumed to be driven by the presentation of multiple peptides from Topo I itself.
Abstract: Glutamic acid decarboxylase (GAD) has been shown to be a target of autoantibodies in insulin-dependent diabetes (IDD). Two forms of GAD, with molecular weights of 67,000 and 65,000, have been cloned from separate genes. As pancreatic islet beta cell destruction DD is an autoimmune process mediated by T cells, we sought to determine if recombinant GAD67 was recognized by T cells in IDD subjects and particularly their first-degree relatives with islet cell antibodies known to be at risk for IDD. The central regions of human islet and brain GAD67 (amino acids 208-404) were cloned as fusion proteins with glutathione-S-transferase (GST). Proliferation of peripheral blood T cells in the presence of recombinant GAD67 was significantly higher in both at-risk relatives and recent-onset IDD subjects than in other autoimmune disease subjects and human histocompatibility leukocyte antigen (HLA)-matched healthy controls. Thus, 12 of 29 (41%) at-risk relatives and 11 of 29 (38%) recent-onset IDD subjects responded to GAD67, compared with 1 of 7 (14%) other autoimmune disease subjects and 1 of 23 (4%) HLA-matched controls. T cell responses to GST alone or to tetanus toxoid were not different between the groups. These findings demonstrate that GAD67 is a target autoantigen of T cells in IDD and suggest the possibility that GAD-reactive T cells may delineate asymptomatic subjects at increased risk for IDD.
Abstract: The overexpression of major histocompatibility complex (MHC) class I molecules in endocrine epithelial cells is an early feature of autoimmune thyroid disease and insulin-dependent diabetes mellitus, which may reflect a cellular response, e.g., to viruses or toxins. Evidence from a transgenic model in pancreatic beta cells suggests that MHC class I overexpression could play an independent role in endocrine cell destruction. We demonstrate in this study that the transgenic overexpression of an allogeneic MHC class I protein (H-2Kb) linked to the rat thyroglobulin promoter, in H-2Kk mice homozygous for the transgene, leads to thyrocyte atrophy, hypothyroidism, growth retardation, and death. Thyrocyte atrophy occurred in the absence of lymphocytic infiltration. Tolerance to allogeneic class I was revealed by the reduced ability of primed lymphocytes from transgenic mice to lyse H-2Kb target cells in vitro. This nonimmune form of thyrocyte destruction and hypothyroidism recapitulates the beta-cell destruction and diabetes that results from transgenic overexpression of MHC class I molecules in pancreatic beta cells. Thus, we conclude that overexpression of MHC class I molecules may be a general mechanism that directly impairs endocrine epithelial cell viability.
Abstract: To obtain a better understanding of the immune response to Epstein-Barr virus (EBV), we measured the cytokines tumour necrosis factor (TNF)-alpha/beta, interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the conditioned medium of peripheral blood mononuclear cells from 10 healthy adults before and at 48 h and at 1, 2, 3 and 4 weeks following infection in vitro with EBV. Cultures were examined for regression of outgrowths of nascent virus-transformed B cells, and populations of cells in the cultures were analysed by flow cytometry. TNF-alpha/beta was not detected in infected or non-infected cultures. In infected cultures assayed at the nominated times, the highest levels of IL-2 were detected at 48 hours, IFN-gamma at 1 week, IL-6 at 2 weeks and GM-CSF between 2 and 4 weeks. IL-6 and GM-CSF, but not IL-2 or IFN-gamma, were detected in non-infected cultures but at lower levels than in infected cultures. Nine of the 10 healthy adults showed regression of outgrowths of virus-transformed B cells and, of these, seven had antibodies to the EBV capsid antigen (VCA). Strong regression was associated with sequential increases in IL-2, IFN-gamma, and low levels of IL-6 and GM-CSF. Absent or weak regression was associated with an undetectable level of IL-2, a low level of IFN-gamma, high levels of IL-6 and GM-CSF and an increased frequency of cells bearing the phenotype CD20 and HLA-DR in the final weeks of culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: To detect serum antibodies to GAD in subjects with IDDM, three recombinant mBGAD 67 peptides encompassing the full-length protein were used in an ELISA. In this study 7 of 9 (78%) preclinical IDDM subjects (ICA+ first-degree relatives of a person with IDDM) and 6 of 13 (46%) recent-onset IDDM subjects, but no subjects with Graves' disease (n = 10) or scleroderma (n = 10), nor healthy nondiabetic control subjects (n = 10) had antibodies that reacted with one or more of the recombinant mBGAD peptides. We found no preferential reactivity with any recombinant peptide. Although only 3 preclinical subjects and 1 recent-onset subject had antibodies to all three mBGAD peptides, the results indicate that mBGAD 67 contains at least three B-cell autoepitopes. Compared with an immunoprecipitation assay of native human brain GAD, the ELISA detected 5 of 6 (83%) preclinical and 6 of 6 (100%) recent-onset IDDM subjects. The ELISA should facilitate screening to evaluate the role of autoimmunity to GAD in the development of IDDM.
Abstract: The gene for the insulin receptor has been assigned to chromosome 19 near the breakpoint of the translocation t(1;19) which occurs in 25% of pre-B-cell leukemias. Insulin receptors in a pre-B-cell leukemia cell line (ACV) with t(1;19) were found to have 2-fold higher affinity for insulin, 5-fold higher basal and insulin-stimulated beta sub-unit autophosphorylation, and 2-fold higher basal and 4-fold higher insulin-stimulated beta sub-unit kinase activity on the synthetic peptide poly(Glu,Tyr), compared to receptors in a B-cell line (ADD) with normal karyotype from the same patient. ACV cells had a novel 13-kb receptor mRNA species and expressed a DNA polymorphism localized to the tyrosine kinase domain of the receptor gene. These findings suggest that t(1;19) in the ACV cell may result in rearrangement of the insulin receptor gene and translation of a receptor with enhanced tyrosine kinase activity.
Abstract: We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease.
Abstract: The development of insulin-dependent diabetes mellitus (IDDM) is associated with circulating antibodies to a pancreatic islet protein of MW 64,000 (64 kDa), reported to be glutamic acid decarboxylase (GAD). To investigate the antigenic properties of the 64 kDa antigen/GAD in IDDM we employed fetal pig pro-islets, a convenient source of islet antigens and an alternative to adult human islets for transplantation. Pro-islets contained a 64 kDa protein precipitated by antibodies in the sera of 9/14 (64%) at-risk IDDM, 12/33 (36%) recent-onset IDDM and 4/12 (33%) established IDDM patients and in 1/18 (5%) healthy control subjects. In addition, a 38 kDa protein was co-precipitated with the 64 kDa protein by 6/14 (43%) at-risk IDDM, 5/33 (12%) recent-onset IDDM, 1/12 (8%) established IDDM patient sera and 1/18 (5%) control subject serum. Both 64 kDa and 38 kDa antigens were specific to pro-islets; neither was detected in fetal pig thyrocytes, hepatocytes or splenocytes. The majority of the 64 kDa protein was co-precipitated with GAD enzymatic activity from pro-islets by either IDDM sera or a sheep anti-GAD serum. Previously, we showed that peripheral blood mononuclear cells (PBMC) from over half the subjects defined as being at risk for IDDM proliferate in response to fetal pig pro-islets. Proliferative responses of PBMC from pre-diabetic subjects to a crude extract of pro-islets were therefore measured before and after depletion of GAD by adsorption of the extract against GAD-1 monoclonal antibody. In 5/10 at-risk subjects, T cell stimulation indices were greater than the control mean + 2 SD and decreased in four by > 50% after depletion of GAD. In summary, a 64 kDa protein with the properties of GAD is present in fetal pig pro-islets and is recognized by both antibodies and T cells in a significant proportion of subjects at risk for early IDDM. Some subjects with anti-64 kDa antibodies also have antibodies that co-precipitate a 38 kDa pro-islet protein. Prospective studies of T cell reactivity in at-risk IDDM subjects are required to delineate the role of GAD and other candidate autoantigens in the initiation and progression of beta cell destruction.
Abstract: A major goal of research into IDDM has been the identification of the 'causative antigen'. As described in this article by Len Harrison, this reductionist aim is confounded by the fact that numerous candidate islet cell antigens have been described. He scrutinizes the credentials of these candidates and discusses the problem of using autoantibodies to identify causative antigens in a T-cell-mediated disease.
Abstract: The destruction of pancreatic islet beta cells in insulin-dependent diabetes mellitus (IDDM) is thought to be T cell mediated. To directly identify islet-reactive T cells in asymptomatic, "preclinical" IDDM individuals with islet cell antibodies (ICA), proliferation of peripheral blood mononuclear cells (PBMC) was measured in the presence of sonicated fetal pig proislets. Stimulation indices (mean +/- SD) for [3H]thymidine uptake by PBMC cultured with sonicated proislets were: preclinical IDDM subjects (n = 22) 6.10 +/- 6.50, recent-onset IDDM subjects (n = 29) 3.66 +/- 3.35, Graves' disease subjects (n = 6) 2.17 +/- 0.93, scleroderma subjects (n = 4) 1.65 +/- 0.19 and normal control subjects (n = 14) 1.63 +/- 0.62. 68% (15/22) of preclinical IDDM, 41% (12/29) of recent-onset IDDM and 17% (1/6) of Graves' disease subjects had T cell reactivity greater than the mean + 2 SD of controls. T cell reactivity to proislets was tissue specific, and greater in magnitude and frequency than to human insulin. The majority of preclinical subjects with ICA greater than 20 Juvenile Diabetes Foundation (JDF) units (12/15, 80%) or antibodies to a 64-kD islet autoantigen (11/15, 73%) had significant T cell reactivity to proislets. ICA greater than 40 JDF units, a strong prognostic marker for progression to clinical IDDM, was an absolute index of T cell reactivity. Overall, the frequency of T cell reactivity in preclinical subjects, 68% (15/22), was comparable to that of ICA greater than 20 JDF units or 64-kD antibodies. Greater T cell reactivity to proislets in preclinical subjects accords with the natural history of autoimmune beta cell destruction. The direct assay of islet-reactive T cells in peripheral blood may have prognostic significance for the development of clinical IDDM and should facilitate identification of the primary target autoantigen(s).
Abstract: Insulin-dependent diabetes mellitus (IDDM) is associated with serum antibodies that precipitate a 64-kDa pancreatic islet cell protein reported to be glutamic acid decarboxylase (GAD; glutamate decarboxylase, EC 4.1.1.15). Previously, antibodies to GAD were found in the rare neurological disorder stiff man syndrome. To demonstrate directly antibodies to GAD, enzymatically active GAD was first purified from fresh human cerebellum. Brain GAD activity was precipitated by noninhibitory antibodies in the sera of 16/26 (62%) subjects defined as having preclinical IDDM (islet cell antibody-positive first-degree relatives of a person with IDDM), 3/13 (23%) with recent-onset IDDM, and 3/3 with the stiff man syndrome. In addition, sera of 5/26 (19%) preclinical and 2/13 (15%) recent-onset IDDM subjects contained antibodies that precipitated GAD but inhibited its activity. Thus, overall, 21/26 (81%) preclinical and 5/13 (38%) recent-onset IDDM subjects had antibodies that precipitated GAD activity. Antibodies to GAD were not detected in sera from subjects with other autoimmune diseases (n = 29) or healthy controls (n = 14). GAD affinity-purified to homogeneity (specific activity, 58 units/mg) was specifically immunoprecipitated as a single 60-kDa species by the IDDM sera. In an ELISA incorporating whole mouse brain GAD captured by the GAD-6 monoclonal antibody the frequencies of GAD antibodies for all subject groups were indistinguishable from those found by precipitation of human brain enzymatic activity. We conclude that (i) GAD is an (auto)antigen in a majority of subjects operationally defined as having preclinical IDDM, (ii) pancreatic islet and brain GAD are likely to be cross-reactive, and (iii) the majority of GAD antibodies are directed away from the catalytic site of the brain enzyme. The lower frequency of GAD antibodies in recent-onset IDDM subjects indicates either that immunoreactivity is lost with near-total beta-cell destruction or that GAD antibodies denote a low risk of progression to clinical disease.
Abstract: The binding of [3H]dopamine to platelet membranes has been examined in an attempt to identify the putative dopamine-uptake mechanism of the platelet. [3H]Dopamine has been shown to bind to a 42,000 Da glycoprotein in platelet membrane with high affinity (Kd = 22.6 nM) and binding of [3H]dopamine was competed for by dopamine, molecules with catechol moieties, 5-hydroxytryptamine, GSH and ascorbic acid. Differences in pharmacological profile and molecular mass suggest that [3H]dopamine does not bind to a known receptor, a neuronal-type dopamine transporter or the platelet 5-hydroxytryptamine-uptake site. It is proposed that this novel binding site for dopamine, which has been purified 1000-fold from particulate platelet membrane, is likely to be a component of the dopamine-uptake mechanism of the human platelet.
Abstract: Human islets were isolated by collagenase digestion and tissue culture from pancreata obtained from organ donor subjects and dispersed islet cells were prepared from hand-picked islets. Islet cell surface antibodies (ICSA), detected by indirect immunofluorescence on isolated islet cells, were present in sera from nine of 22 (41%) subjects with recent-onset insulin-dependent diabetes mellitus (IDDM) and three of 11 (27%) control subjects. Sera had been heat inactivated, adsorbed against a human B lymphoblastoid cell line (IM-9) and tested in the presence of 4% bovine serum albumin. However, with a double labelling technique, we were unable to show that ICSA were specific for beta cells. Of the nine ICSA-positive IDDM sera, three stained both beta and non-beta cells, three beta cells only and three non-beta cells only; the three ICSA-positive control sera stained both beta and non-beta cells. There was no apparent relationship between ICSA and standardised measurements of islet cell antibodies (ICA) and insulin autoantibodies (IAA). These results lead us to question whether, despite previous reports, ICSA are specific for beta cells or indeed for IDDM.
Abstract: OBJECTIVE--To compare the magnitude and reproducibility of the FPIR measured during two different IVGTT protocols in nondiabetic subjects. RESEARCH DESIGN AND METHODS--Nine control subjects each had two pairs of IVGTTs with either a 4-min infusion of 0.5 g/kg glucose or a 1-min infusion of 0.3 g/kg glucose. Blood glucose and serum insulin were measured before and 1, 2, 3, 5, and 10 min after completion of the glucose infusion. The FPIR was measured with either 1 + 3-, 2 + 3 + 5-, or 1 + 3 + 5-min serum insulins, areas under the insulin curve (0-5 or 0-10 min), or the ratio of serum insulin to blood glucose area. RESULTS--The FPIR was higher in eight of nine subjects with the short-infusion test, but the within-subject variation of the two methods was identical. Reproducibility was not significantly improved with an integrated insulin area or insulin-to-glucose ratio measurement. CONCLUSIONS--Reproducibility of the FPIR measured during IVGTT is not significantly affected by the duration of the glucose infusion. However, the magnitude of the difference in FPIR observed between the two protocols highlights the need for standardization of the methodology if the IVGTT is to be used in studies of the preclinical stage of IDDM.
Abstract: Immunoblotting was optimized to detect autoantibodies to TSH receptors from human and porcine thyroid tissue and to determine their epitope specificity. Autoantibodies to putative TSH receptor proteins in thyroid particulate membranes were detected in approximately 35% of sera from patients with Graves' disease. However, despite modifications to increase immunoblotting sensitivity and specificity, only a minority (less than 15%) of Graves' disease sera contained autoantibodies that identified epitopes within TSH affinity-purified human or porcine receptor proteins. In these sera there was no correlation between the TSH receptor antibody titre, determined by radioreceptor assay, and receptor epitope reactivity. The sensitivity of immunoblotting was limited by reduced transfer of purified receptor from the gel. However, in addition, the inability to immunoblot the purified receptor with a majority of Graves' sera, under conditions designed to enhance receptor renaturation, appears to reflect a strict conformational requirement for immunoreactivity. Immunoblotting of purified receptors therefore has a limited application in detecting, and defining the epitope reactivity of, TSH receptor autoantibodies.
Abstract: Twenty-five children aged 2-14 years (mean age 8.39 +/- 0.78 years) were studied prospectively during the first year after the diagnosis of type 1 diabetes. Of their clinical and metabolic features at diagnosis, only age showed a significant independent relationship with endogenous C-peptide production during the first year. Age was correlated with higher values for basal and stimulated plasma C-peptide at 7-14 days after diagnosis, at 6 months and at 12 months. At diagnosis, age was also associated with a higher value for HbA1c and a lower prevalence of insulin antibodies. C-peptide production peaked at 3 months and thereafter declined. Mean HbA1c and insulin requirement were both minimal at 6 months. At diagnosis, there were significant inverse relationships between basal C-peptide production and both insulin dose and HbA1c and between stimulated C-peptide production and HbA1c. Basal and stimulated C-peptide production were inversely related to insulin dose at 6 and 12 months. Stimulated C-peptide was higher at 12 months in children retaining islet cell antibodies. These findings confirm the importance of age as a predictor of residual beta-cell function in type 1 diabetes and indicate that older children present clinically following a slower course of beta cell destruction.
Abstract: Cytomegalovirus (CMV) is an important pathogen in persons who are immunocompromised or who have received organ transplants. The rate of symptomatic CMV infection is relatively higher after liver transplantation, and CMV hepatitis may be difficult to differentiate from rejection or cholangitis without recourse to liver biopsy and tissue culture. In this study we have used the polymerase chain reaction (PCR) to amplify and detect CMV-DNA sequences directly from the saliva and urine of liver transplant recipients. The procedure is simple, rapid and specific, and was found to be as sensitive as tissue culture for the diagnosis of CMV.
Abstract: A simple, direct assay for T-lymphocyte reactivity to islet antigen(s) in human insulin-dependent diabetes mellitus (IDDM) should facilitate preclinical diagnosis and the evaluation of intervention therapy to avert autoimmune-mediated beta-cell destruction. In subjects with preclinical or clinical IDDM, we measured the reactivity of peripheral blood mononuclear cells (PBMCs) incubated over 6 days with either adult human islets or fetal pig proislets, or other fetal pig tissues, and with human insulin. With islets, the stimulation index (SI) of [3H]thymidine uptake by PBMCs exceeded the mean + 2SD of control subjects in 6 of 6 preclinical subjects (SI 8.7 +/- 3.7), 7 of 11 clinical subjects (SI 5.2 +/- 3.4), and 1 of 12 control subjects (SI 2.7 +/- 1.7); with insulin, the responses were less in frequency and magnitude, being 4 of 6 (2.7 +/- 1.6), 3 of 11 (2.2 +/- 1.1), and 0 of 12 (1.20 +/- 0.55), respectively. The mean responses to islets of PBMCs from preclinical and clinical subjects differed significantly from control subjects (P less than 0.02 by 2-tailed Kruskal-Wallis test). Secretion of granulocyte macrophage colony-stimulating factor by PBMCs over 6 days was assayed in the preclinical group and generally paralleled the uptake of [3H]thymidine. PBMC reactivity to islets appeared to be at least as sensitive a marker of preclinical IDDM as autoantibodies to a 64,000-Mr protein, presumably the enzyme glutamic acid decarboxylase, in fetal pig proislets. In conclusion, islet-reactive T lymphocytes in subjects with preclinical and clinical IDDM can be identified in bulk culture of PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The tyrosine kinase of the insulin receptor can be activated by trypsin treatment. The concomitant abolition of insulin binding has been postulated to result from proteolytic destruction of the receptor. A discrepancy between the decrease in insulin binding and receptor immunoreactivity after trypsin treatment led us to investigate more closely the structure of the trypsin-treated receptor. After trypsin treatment of the CHOT cell line, which over-expresses transfected human insulin receptors, insulin binding was significantly decreased, but reactivity with five alpha-subunit monoclonal antibodies was either unaffected or only moderately decreased, indicating that the alpha-subunit was substantially intact. Examination of receptor structure after trypsin treatment, receptor autophosphorylation and gel electrophoresis revealed a single band at 110 kDa in non-reduced gels, comprising a small fragment (21 kDa) of the alpha-subunit linked to the beta-subunit by class II disulphides. When the receptor was radio-labelled with 125I, two additional alpha-subunit bands of 142 kDa and 81 kDa (composed of identical reduced bands) were observed on non-reduced gels, which contained disulphide-linked (class I) fragments. All fragments could be precipitated by antibodies to both alpha- and beta-subunits. However, only antibodies directed towards the N-terminus of the receptor could immunoblot trypsin-treated fragments. Thus activation of the receptor tyrosine kinase by trypsin occurs after cleavage, but not loss of the alpha-subunit. This finding has implications for the mechanism of transmembrane activation of the receptor kinase by insulin.
Abstract: We report partial nucleotide sequences of the human enzyme glutamic acid decarboxylase (GAD) from brain and pancreatic islets which encode the middle 180 amino acids of GAD. The brain and islet GAD sequences display a high degree of sequence homology with the equivalent region of other mammalian brain GAD cDNAs. Alignment of the brain and islet GAD sequences showed that there were 45 nucleotide differences which, at the translational level, would result in seven amino acid substitutions. These results which suggest that different isomeric forms of human GAD exist in brain and pancreas may be relevant to the pathogenesis of stiff man syndrome (SMS) and insulin-dependent diabetes mellitus (IDDM), respectively, two distinct but associated clinical disorders in which GAD is the target of autoantibodies.
Abstract: 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 Beta-cell 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 12.2+ effected by cytokines secreted by activated immuno-inflammatory cells. Class I MHC overexpression should enhance targeting of cytotoxic T cells to Beta cells bearing autoantigen.
Abstract: Plasma cortisol and 11-deoxycortisol were measured in 30 depressed patients and 110 normal volunteers before and after a 1.0 mg dexamethasone suppression test (DST). Post-dexamethasone plasma cortisol, 11-deoxycortisol and the cortisol/11-deoxycortisol ratio were significantly higher in the depressives compared to the controls, even when age and sex were taken into account. Pre-dexamethasone plasma cortisol, post-dexamethasone cortisol, 11-deoxycortisol and their ratio were significantly higher in the cortisol nonsuppressors than in the suppressors. The measurement of post-dexamethasone 11-deoxycortisol and the ratio did not differentiate between endogenous and reactive depression. Using the normative data, we explored several methods for determining a criterion value to define abnormal post-dexamethasone plasma 11-deoxycortisol and the cortisol/11-deoxycortisol ratio in depressed patients. All showed poor sensitivity and a low positive predictive value for depression. The measurement of 11-deoxycortisol thus does not enhance the clinical utility of the DST.
Abstract: Gangliosides were extracted from purified human and porcine thyrotropin (TSH) receptors (TSH-R) and were detected by probing with an 125I-labeled sialic acid-specific lectin, Limax flavus agglutinin. Gangliosides copurified with human and porcine TSH-R migrated between monosialoganglioside GM1 and disialoganglioside GD1a. Ceramide glycanase digestion of the purified human TSH-R-associated glycolipid confirmed its ganglioside nature. It was resistant to Vibrio cholerae sialidase, which digests all gangliosides except GM1, but was sensitive to Arthrobacter ureafaciens sialidase, which digests all gangliosides including GM1. These findings indicate that the human TSH-R contains ganglioside that belongs to the galactosyl(beta 1----3)-N-acetylgalactosaminyl (beta 1----4)-[N-acetylneuraminyl(alpha 2----3)]galactosyl(beta 1----4) glucosyl(beta 1----1)ceramide (GM1) family. Its intimate association with receptor protein implies a key role for ganglioside in the structure and function of the TSH-R.
Abstract: Pancreatic beta cell destruction in the non-obese diabetic (NOD) mouse is mediated by T lymphocytes and macrophages and accelerated by cyclophosphamide. We purified pancreatic T lymphocytes from the NOD mouse for comparative phenotypic and functional analysis with T lymphocytes from spleen, peripheral blood and regional lymph nodes. Pancreatic T lymphocytes from NOD-Wehi mice, which have an incidence of spontaneous diabetes of less than 5%, had a CD4:CD8 ratio of 1.25 +/- 0.23 compared with 2.44 +/- 0.31 for peripheral blood lymphocytes. After cyclophosphamide, the CD4:CD8 ratio of pancreatic lymphocytes increased to 2.30 +/- 0.24 at day 7. T lymphocytes bearing IL-2 receptors increased two- to three-fold in number and their secretion of GM-CSF/IL-3 and IFN-gamma increased to a maximum on day 7. Pancreatic insulin content and mRNA levels declined sharply between days 10 and 12, at which time the majority of pancreatic T lymphocytes in hyperglycaemic mice were CD8+ (CD4:CD8 ratio 0.63 +/- 0.04 compared to 4.14 +/- 1.05 in peripheral blood). The pancreatic T lymphocyte CD4:CD8 ratio in prediabetic NOD-Lt mice, which have an incidence of spontaneous diabetes of about 60% at 150 days, was similar to that in untreated NOD-Wehi mice, but 25% of their pancreatic CD8 T lymphocytes were IL-2-receptor positive. Thus, significant changes in the phenotype of NOD pancreatic T lymphocytes following cyclophosphamide were not reflected in peripheral blood or spleen T lymphocytes. The earliest change after cyclophosphamide was an increase in activated, predominantly CD4+ T lymphocytes; with the development of beta cell destruction and hyperglycaemia, pancreatic T lymphocytes were, as in human IDDM, predominantly CD8+.
Abstract: In insulin dependent diabetes mellitis (IDDM) beta cell destruction is associated with infiltration of the pancreatic islets by T lymphocytes and macrophages. Cytokine products from the infiltrating immunocytes not only have powerful immunoregulatory actions but also are capable of impairing islet cell functions and have thus been postulated to assume a central role in mediating anti-beta cell immunity and beta cell destruction. In an effort to explore further the role of cytokines in the pathogenesis of IDDM, we examined clinical, metabolic and pathological features of NOD/Wehi mice injected intraperitoneally with multiple doses of IFN-gamma and/or TNF-alpha. Blood glucose profiles were not significantly altered by injection of cytokines alone or in combination. Except for a hypoglycaemic rebound in mice injected with TNF-alpha, arginine stimulation tests revealed no disturbances in islet secretory function in cytokine injected mice. Compared with vehicle and cytokines alone, injection of IFN-gamma + TNF-alpha was associated with a variety of clinical and pathological changes including abdominal distention, piloerection, ascites, oedema, thymic atrophy, splenic enlargement and pancreatic distention. Histological examination of the pancreas in these mice revealed moderate to severe pancreatitis which included focal haemorrhagic necrosis, oedema and polymorphonuclear and mononuclear cell infiltration. The islets in these mice appeared normal morphologically and when stained for insulin. The injection of IFN-gamma + TNF-alpha, and to a lesser extent TNF-alpha alone, was associated with a significant reduction in the severity of insulitis. Examination of pancreatic MHC-class I and class II molecule expression revealed in mice given IFN-gamma + TNF-alpha, as compared with controls, significant and uniform induction of both these molecules on ductal and acinar cells; low level MHC-class II expression was also detectable on beta cells in these mice. MHC-class I molecules which were expressed at high levels by beta cells in control mice did not appear to change following administration of the cytokines alone or in combination. We conclude that despite their immunostimulatory actions in vitro and in other models in vivo, systemic administration of the cytokines IFN-gamma and/or TNF-alpha to NOD/Wehi mice does not activate or enhance, and may actually suppress, anti-beta cell immunity in this model.
Abstract: Glucose deprivation increases the steady-state levels of mRNA for the rat brain/HepG2-type glucose transporter (GLUT1) in L6 myocytes. Glucose deprivation also inhibits N-linked glycosylation. We therefore investigated a possible relationship between inhibition of glycosylation and GLUT1 expression in cultured L6 myocytes by determining the effects on GLUT1 expression of known inhibitors of glycosylation, namely tunicamycin, 2-deoxyglucose and glucosamine. All conditions prevented incorporation of [3H]mannose into TCA-precipitable myocyte protein and resulted in a 2- to 5-fold increase in the level of GLUT1 mRNA detected on Northern blots. Glucose deprivation and tunicamycin treatment caused an approx. 2-fold increase in GLUT1 mRNA half-life. GLUT1 protein, detected on immunoblots, accumulated 10- to 20-fold in response to all glycosylation inhibitors, with apparent molecular masses of 40 kDa after glucose deprivation, 42 kDa after 2-deoxyglucose and 38 kDa after glucosamine or tunicamycin treatments, compared to 45-50 kDa in glucose-fed cells. However, glucose deprivation was the only condition in which the rate of 2-deoxy-[3H]glucose uptake increased (3- to 5-fold). These results demonstrate a direct correlation between inhibition of glycosylation and the induction of GLUT1 mRNA and protein expression and suggest that the stability of GLUT1 mRNA is controlled by a signal associated with glycosylation.
Abstract: Experimental studies in vitro suggest that cytokines are important mediators in the pathogenesis of autoimmune insulin-dependent diabetes mellitus (IDDM). However, there is little evidence for the role of cytokines in vivo, either in humans or in the spontaneous animal models of IDDM such as the NOD mouse or BB rat. To address this question, we used the model of cyclophosphamide (CYP)-induced autoimmune diabetes in the NOD/Wehi mouse to examine for (a) the production of IFN-gamma and IL-6 from isolated islets, and (b) the effect of anti IFN-gamma or anti IL-6 monoclonal antibodies on the development of diabetes. After cyclophosphamide, the majority of these mice develop of mononuclear cell infiltrate (insulitis) which by 10-14 d is associated with beta cell destruction. IFN-gamma activity at low levels (2.7 +/- 0.3 U/ml) could be detected only in culture supernatants from islets isolated at day 7 post-cyclophosphamide. In contrast, IL-6 activity progressively increased from 457 +/- 44 U/ml at day 0 to 6,020 +/- 777 U/ml at day 10. Culture of islets with anti-CD3 monoclonal antibody resulted in a significant increase in IFN-gamma activity from 41 +/- 7 U/ml at day 0 to 812 +/- 156 U/ml at day 10. Mice given either anti-IFN-gamma or anti-IL-6 antibody had a significantly reduced (P less than 0.001) incidence of diabetes and especially with IFN-gamma, decreased severity of insulitis. We conclude that IFN-gamma and IL-6 have essential roles in the pathogenesis of pancreatic islet beta cell destruction in this model.
Abstract: Forty-one assays were analysed at the 3rd International Workshop on the standardisation of islet cell antibodies. Analysis of precision demonstrated assays consistently detecting blind duplicates within one doubling dilution and capable of discriminating one doubling dilution differences in islet cell antibody concentration. Some assays, however, reported duplicates discrepantly by more than seven doubling dilutions, and consequently could not distinguish even large quantities of islet cell antibodies. Precision was best in assays from laboratories which had participated in all three Standardisation Workshops and was not dependent upon methodology. The use of the Juvenile, Diabetes Foundation reference islet cell antibody standard and standard curves reduced the scatter of results, and was best amongst assays with better precision. Twenty-seven assays reported all ten blood donor sera as negative. However, 14 assays did not, and specificity (negativity in health) was less than 50% in three assays. Low specificity was strongly associated with poor precision. The detection limit of assays ranged from less than 5 to 50 JDF units and was partially dependent upon methodology. Assays incorporating extended incubation had the lowest detection limits without a decrease in the specificity of the ten blood donor sera. Precise quantification is fundamental for the standardisation and comparability of islet cell antibodies. Precise quantitative assays have been identified and reference standards and common units established.
Abstract: Chronic exposure of L6 myocytes to insulin/IGF-1 or glucose deprivation results in an increase in the level of brain-type glucose transporter (GLUT1) mRNA. We have investigated the effects of insulin and glucose deprivation on GLUT1 mRNA stability. The half-life of GLUT1 mRNA in control cells was 2-2.5 h. Insulin increased GLUT1 mRNA levels by 5- to 10-fold, and GLUT1 mRNA half-life and transcription by 2-fold. Glucose deprivation increased GLUT1 mRNA level by 2- to 4-fold and half-life by 2-fold. The effects of insulin and glucose deprivation on GLUT1 mRNA stability were additive. Cycloheximide partially blocked the induction of GLUT1 mRNA by insulin but not by glucose deprivation. GLUT1 mRNA was decreased to basal levels within 12h following insulin withdrawal or glucose refeeding. Cycloheximide did not block this de-induction, suggesting that insulin and glucose deprivation do not increase GLUT1 mRNA expression by inhibiting the synthesis of ribonucleases. These findings indicate that insulin/IGF-1 increases both GLUT1 mRNA stability and transcription by both protein synthesis-dependent and independent mechanisms, whereas glucose deprivation enhances GLUT1 mRNA stability by mechanisms independent of de novo protein synthesis.
Abstract: The initial step in the action of thyrotropin (TSH) is its binding to the TSH receptor. TSH receptor antibodies are detected in up to 90% of patients with Graves' disease. Serial measurements of TSH receptor antibodies in patients with Graves' hyperthyroidism are helpful in predicting relapse. The TSH receptor was purified using affinity chromatography on wheat germ lectin agarose and TSH-agarose. Using an immunoblotting technique to characterize the TSH receptor, it was found to be an oligomeric glycoprotein consisting of three noncovalently bound subunits of Mr approximately 70,000, approximately 50,000 and approximately 35,000 which on reduction yield a single subunit of Mr approximately 25,000.
Abstract: Evidence from epidemiological and histopathologic studies in humans with autoimmune type 1 (insulin-dependent) diabetes suggests that beta-cell destruction within the islets of Langerhans progresses through a number of stages. In this review we draw on recent experimental evidence in an attempt to define the molecular pathology of these stages. Stage 1 is postulated to be initiated by modification of the beta cell by virus, chemical or other factors, leading to the production of interferon-alpha, hyperexpression of major histocompatibility complex (MHC) class I molecules and induction of MHC class II molecules. Experiments in transgenic mice suggest that overexpression of MHC molecules is in itself detrimental to beta-cell function. Shedding of antigen(s) from dying beta cells in combination with hyperexpression of MHC molecules may be a powerful immunogenic stimulus. Stage 2 commences with infiltration of the islets by immuno-inflammatory cells (termed insulitis). It is proposed that production of cytokines from the infiltrating cells induces "phenotypic switching" in beta cells, with further upregulation of MHC molecules and the induction of intracellular adhesion molecule-1 expression and interleukin-6 production. Together, these properties are seen as a prerequisite for the presentation of autoantigen by beta cells to adherent T lymphocytes and autoimmune activation. The final stage encompasses autoimmune-mediated destruction of the beta cells by the targeted delivery of cytotoxic cytokines and other mediators.
Abstract: High titer IgG autoantibodies to the 70-kDa polypeptide component (p70) of the U1 ribonucleoprotein (RNP) complex occur in the sera of patients with mixed connective tissue disease, SLE, and related rheumatic diseases. To gain insight into the pathogenesis and diversity of this antibody response we have used recombinant DNA technology to map the linear B cell epitopes on p70. A full length 1.7-kb cDNA clone encoding p70 was isolated from a human placental library and restriction fragments or polymerase chain reaction-generated fragments of the gene subcloned into the bacterial expression vector pGEX. Purified fusion proteins representing specific regions of p70 were immunoblotted with a panel of 70 anti-(U1)RNP+ sera containing anti-p70 antibodies. Six epitopes, four major (A, B, C, and F) and two minor (D and E) were mapped and were located throughout the molecule. The anti-(U1)RNP sera displayed heterogeneity in their pattern of reactivity to the six epitopes although reactivity to epitope C was more frequently associated with SLE rather than mixed connective tissue disease. The identification of multiple B cell epitopes on p70 is consistent with the concept that this self Ag drives the autoantibody response.
Abstract: Cellular autoimmunity is thought to be primarily responsible for the selective destruction of islet beta cells in Type I diabetes. Why the T lymphocyte reacts to self and recognizes the beta cell as foreign, as against the other endocrine islet cells, is unknown. One key issue is whether the beta cell itself is capable of presenting autoantigen(s) and thereby breaking T lymphocyte tolerance. In this paper we discuss current concepts of antigen presentation and relate these to recent findings from our laboratory, suggesting that the beta cell can be induced to display many of the phenotypic properties of classical antigen-presenting cells, including induction of MHC and ICAM-1 expression and production of IL-6. Finally, a model is presented which provides a new view of the initiation and perpetuation of autoimmune beta-cell destruction in Type I diabetes.
Abstract: To investigate the relationship between surface ganglioside expression and pancreatic islet cell differentiation, we examined the function of five rat insulinoma (RIN-m5F) sublines A4, A6, A7, A10, and A12 selected for increased expression of A2B5-reactive gangliosides, as well as a subline AlGh, low in A2B5- but high in 3G5-reactive ganglioside expression. Class I major histocompatibility (MHC) protein expression was also measured in the sublines because of our previous finding that class I proteins were preferentially expressed on human insulinoma tissue compared with differentiated islet cells. In comparison with parental RIN-m5F cells, subline A12 displayed a 7.6-fold increase in A2B5 expression and a 3.4-fold increase in the number of A2B5 positive cells (81% vs. 24%). A2B5 expression was increased 1.3-, 5.4-, 5.4-, and 6.9-fold on A4, A6, A7, and A10 sublines, respectively. In contrast, AlGh cells, which had comparable A2B5 expression to parental cells, displayed a 2.9-fold increase in 3G5 expression and an 8-fold increase in the number of 3G5 positive cells (72% vs. 9%). Insulin secretion and content increased with increasing A2B5 expression. On day 2, secretion was 1.2-, 8-, 8-, 21-, and 18-fold higher and content 1.4-, 4-, 5-, 26-, and 33-fold higher for A4, A6, A7, A10, and A12 cells, respectively, compared to parental cells. There was a direct association between expression of A2B5 and the level of insulin messenger RNA (mRNA) in the sublines. Neither glucagon nor somatostatin was detected in any subline. The AlGh subline secreted and contained less insulin than parental cells. Fully differentiated adult rat islet cells, the majority of which are beta-cells, contained a lower number of 3G5 (12%) than A2B5 (57%) positive cells. Compared to parental cells, class I MHC proteins were decreased 4-fold on A12 cells, but increased 1.5-fold on AlGh cells. We conclude that, at least in RIN cells, the expression of A2B5-reactive ganglioside expression is associated directly, and class I MHC protein expression indirectly, with beta-cell differentiation.
Abstract: 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.
Abstract: We have generated rat insulinoma (RIN) sublines AlGh, m5F, A12, A13 and AhGh with increasing surface expression of the A2B5 ganglioside, a marker of differentiation. We asked whether the capacity of the sublines to differentiate was related to their stage of differentiation, as is characteristic of cells within the normal beta-cell lineage. To answer this, we measured the effect of the differentiation inducer sodium butyrate (NaB, 1 mM) on proliferation, insulin content, secretion and biosynthesis, and the expression of A2B5 and 3G5 gangliosides by the sublines. Six days after exposure to NaB, cell numbers/dish ranged from (1-3) x 10(6) compared to (4-6) x 10(6) in control cultures. By day 2, AlGh, m5F, A12, A13 and AhGh cells exposed to NaB contained 1.5-, 1.4-, 1.4-, 1.2- and 1.0-fold higher amounts of insulin, respectively, and by day 6, 3.6-, 2.3- and 1.0-fold higher, and 1.2- and 2.4-fold lower, amounts of insulin, respectively, than control cells. After 2 days, insulin secretion from AlGh, m5F, A12, A13 and AhGh cells was 1.7-, 1.0-, 1.5-, 1.0- and 1.0-fold higher, respectively, and the rate of (pro)insulin biosynthesis 1.7-, 2.3-, 1.3-, 1.0- and 1.0-fold higher, respectively, than control cells. After 6 days, A2B5 ganglioside expression was increased 3-, 1.9- and 2-fold on m5F, A12 and A13 cells, respectively, but was not significantly altered on AlGh and AhGh cells. 3G5 ganglioside expression was increased 1.5- and 8.4-fold, respectively, on AlGh and m5F cells, but was unaltered on A12, A13 and AhGh cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: When 20 female anorexic in-patients were investigated with weekly DSTs, 10 had an abnormal result at initial testing. There was no identifiable relationship between severity of weight loss and DST status; % ideal body weight was no different between suppressors and non-suppressors. There was no consistent relationship between normalisation of the DST response and weight gain. Depressive symptoms were common, with half the patients scoring 20 or more on the HRSD. Plasma ACTH concentrations before and after the DST were normal. There was a significant negative correlation between plasma dexamethasone concentrations and pre- and post-dexamethasone plasma cortisol concentrations.
Abstract: There is evidence that tumor necrosis factor (TNF) may be a proliferation and differentiation factor for B lymphocytes. We found that three of four lymphoblastoid cell lines (ADD, IM-9, W1) secreted TNF-beta and expressed TNF receptors, whereas one pre-B cell leukemia and six Burkitt's lymphoma cell lines had no detectable TNF secretion and, except for one Burkitt's cell line (LOU), very low expression of TNF receptors. When IM-9, W1 or LOU cells were cultured over seven days in the presence of either TNF-alpha or antiserum to TNF-beta there was no difference between their growth rate, endogenous TNF-beta secretion or immunoglobulin secretion compared to untreated cells. These findings indicate that TNF does not have a universal role as an autocrine growth factor in transformed B lymphocytes.
Abstract: High-titre IgG antibodies against the immunodominant 70-kDa protein of the (U1)ribonucleoprotein (RNP) complex are present in virtually 100% of patients with mixed connective tissue disease (MCTD), and less commonly in a variety of other autoimmune rheumatic diseases. As T-cell 'help' is assumed to be required for this potentially pathogenic form of immune response, investigations to define T-cell epitopes on the 70-kDa protein were undertaken. In prior studies we expressed the 70-kDa protein and a number of its fragments, spanning most of the molecule, as recombinant fusion proteins using the pGEX expression-vector system. These fusion proteins were used as antigens in the epitope mapping studies reported here. PBMC were isolated from patients with (U1)RNP-positive rheumatic diseases and from both normal controls and rheumatologic patients with other autoantibody reactivities, including those to Ro, La and dsDNA. Reactivity to the purified 70-kDa protein was assayed by thymidine incorporation and was evident only in anti-(U1)RNP positive patients but was not restricted to MCTD patients, being present also in patients with SLE and rheumatoid arthritis. The stimulation indices (SIs) observed were in the two- to five-fold range. Using the 70-kDa protein fragments, a T-cell stimulatory epitope was localized to the C-terminal 63 amino acids of the autoantigen. A T-cell line, derived from PBMC of a (U1)RNP positive patient with MCTD, also reacted predominantly with this C-terminal fragment but with an SI of approximately 15-fold. Thus, we have demonstrated the presence and specificity of autoreactive T lymphocytes to a defined peptide epitope in systemic rheumatic disease.
Abstract: IFN-gamma and TNF-alpha injure the pancreatic beta-cell and may be involved in the pathogenesis of autoimmune type 1 diabetes. Because the induction of IL-6 appears to be an important host cell response to injury, we have examined whether IL-6 is produced by murine pancreatic islets or rat insulinoma (RIN-m5F) cells after their exposure to IFN-gamma and TNF-alpha. Islet culture supernatants contained detectable IL-6 activity which was increased 6-fold when islets were exposed to IFN-gamma and 40- and 115-fold when islets were exposed to TNF-alpha and TNF-alpha + IFN-gamma, respectively. A mAb against murine IL-6 abolished (control and IFN-gamma) or significantly reduced (TNF-alpha and TNF-alpha + IFN-gamma) the IL-6 activity in islet supernatants. The magnitude for the effects of IFN-gamma and TNF-alpha on the production of IL-6 from mouse islets was found to be both time and dose dependent. Northern blot hybridization analysis of islet total cytoplasmic RNA with a cDNA probe to murine IL-6 revealed a band at 1.3 kb, the intensity of which increased in islets exposed to IFN-gamma + TNF-alpha. IL-6 activity was also detected in culture supernatants from RIN-m5F cells exposed to TNF-alpha + IFN-gamma. Islets cultured with rIL-6 secreted higher levels of insulin compared with control islets. Pancreatic islet cells, in all probability beta-cells, produce IL-6, the expression of which is up-regulated by IFN-gamma and/or TNF-alpha. In addition to a possible role in regulating pancreatic beta-cell function we propose that IL-6 produced by the pancreatic beta-cell may act as a costimulator for autoreactive B and T lymphocytes in autoimmune diabetes.
Abstract: Insulin-dependent (type 1) diabetes mellitus (IDDM) is due to the selective autoimmune-mediated destruction of pancreatic beta cells possibly initiated by viruses. To elucidate the possible role of viruses and cytokines in the pathogenesis of IDDM, we have examined the effect of reovirus infection on beta cell major histocompatibility complex (MHC) expression and the effect of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on beta cell function in vitro. Infection of RIN-m5F (rat insulinoma) cells with reovirus-1 or reovirus-3 was associated with a tenfold increase in class 1 MHC protein and mRNA expression. Reovirus infection did not induced the expression of class 11 MHC by RIN-m5F cells. Exposure of reovirus to ultraviolet light almost completely abolished its ability to induce class 1 MHC protein expression on infected cells. Murine islets cultured for 3 days with IFN-gamma and/or TNF-alpha had a significantly reduced insulin response to glucose, which was more marked with a combination of the cytokines. During 6 days of culture in IFN-gamma plus TNF-alpha islets underwent noticeable degeneration associated with an 80% reduction in insulin content. These findings together with previous data suggest viruses and cytokines may have multiple roles in beta cell destruction, indirectly through enhanced MHC protein expression and directly through functional impairment and loss of viability.
Abstract: The very low expression of insulin receptors in the Burkitt lymphoma cell Raji was increased 2-fold, 6-fold and 10-fold after 1, 2 and 3 days, respectively, by incubation with the differentiation inducer sodium butyrate. Insulin receptor number was increased without a change in receptor affinity, in association with an increase in the receptor alpha and beta subunits detected after cell-surface labelling and immunoprecipitation. Expression of cell-surface class I and II human leukocyte antigens, the intercellular adhesion molecule-1 and the CD38 leukocyte antigen was also increased, consistent with B cell differentiation. Butyrate effects were not unspecific, as the binding of tumour necrosis factor and growth hormone and the expression of the B cell markers CD20, B5 and CD21 was not increased. The low expression of insulin receptors on Raji cells is therefore a reflection of the less differentiated state of these cells compared to lymphoblastoid cells.
Abstract: The specific binding of insulin to 7 different Burkitt lymphoma cell lines containing chromosomal translocations t(8;14), t(8;2) and t(8;22) was markedly decreased when compared to binding to lymphoblastoid cells of normal karyotype derived from Burkitt lymphoma patients or the human IM-9 lymphoblastoid line. The number of insulin-binding sites on intact Burkitt cells was decreased by greater than 90% compared to lymphoblastoid cells, with no change in affinity. This decrease in binding was paralleled by reduced amounts of insulin receptor alpha (Mr 130,000) and beta (Mr 95,000) subunits detected by cell-surface-labelling and insulin receptor mRNA transcripts, indicating that transcription of receptor mRNA is decreased in Burkitt cells compared to lymphoblastoid cells and/or that receptor mRNA is less stable. Burkitt cells displayed negligible insulin-stimulated beta subunit auto-phosphorylation, which could reflect either their decreased number of receptors or a defect in signal transduction. Structural analysis also revealed that the Burkitt cells had an increase in a precursor form (Mr 210,000) of the receptor, suggesting that decreased expression of the receptor may be associated with defective processing. Four Burkitt cell lines with t(8;14) also had reductions of 45-100% in expression of class-1 major histocompatibility (MHC) antigens. The expression of insulin receptors in both Burkitt and lymphoblastoid cells correlated with the expression of class-1 MHC antigens. There was also an inverse correlation between the expression of c-myc and both insulin receptors and class-1 MHC antigens. As the insulin receptor is absent on resting B cells and is induced after cell activation, the decrease in receptor expression on Burkitt cells may reflect their less activated phenotype compared to lymphoblastoid cells.
Abstract: Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Isolated human and mouse pancreatic islet cells and the rat insulinoma cell line RIN-m5F were used to examine the ability of recombinant interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to regulate the expression of the class I and class II major histocompatibility (MHC) surface proteins and mRNA in beta-cells. Each cytokine increased significantly the expression of class I MHC proteins as determined by double indirect immunofluorescence microscopy and flow cytofluorimetric analysis. In the RIN-m5F cells, this increase in surface expressed class I MHC proteins was mirrored by an increase in the level of class I MHC mRNA. The order of potency of the cytokines on class I MHC expression was TNF-alpha plus IFN-gamma greater than or equal to IFN-gamma greater than or equal to TNF-alpha. While IFN-gamma or TNF-alpha alone were without effect, in combination they were found to induce class II MHC proteins on 30-40% of human or murine beta-cells. In contrast, IFN-gamma plus TNF-alpha did not induce detectable class II MHC proteins or mRNA in the RIN-m5F cells. These findings indicate that 1) TNF-alpha, in addition to IFN-gamma, upregulates the expression of beta-cell class I MHC proteins and mRNA, and 2) more than one signal is required for the induction of class II MHC proteins on beta-cells. The ability of IFN-gamma plus TNF-alpha to induce class II MHC proteins on only a fraction of the normal beta-cell population and not on RIN-m5F cells suggests that this response is related to the differentiation state of the beta-cell.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: We have shown in previous studies that the TA10-subtype of HLA-DQw3 is significantly increased in HLA-DR4 type I (insulin-dependent) diabetic patients. Data presented in this article indicate that this association only occurs in heterozygous DR3/4 patients and not in DR1/4 patients. Because there is an interactive effect of both DR3/4 and DR1/4 in type I diabetes, the data indicate that the contribution of DR4 haplotypes varies depending on the haplotype borne on the homologous chromosome. In addition, the frequency of the B44-DR4 haplotype was shown to be decreased in DQw3 (TA10-) diabetic subjects who were DR3/4 compared with those who were DR1/4 or DR4/x (a pooled group of patients with different DR alleles). This finding suggests that the decrease in the B44-DR4 haplotype in type I diabetes is not solely dependent on its linkage disequilibrium with the DQw3 (TA10+) allele but suggests there is an additional effect exerted independently.
Abstract: Viruses are implicated in the pathogenesis of beta-cell destruction in type I (insulin-dependent) diabetes. The aim of our study was to investigate whether reovirus 1 or reovirus 3, which are known to infect beta-cells and induce autoimmunity in susceptible mice, could alter the expression of the major histocompatibility complex (MHC) proteins by human beta-cells and rat insulinoma RINm5F cells. Forty-eight hours after infection of either human beta-cells or RINm5F cells with reovirus 1 or reovirus 3, cytopathic effects were noted. By flow-cytofluorometric analysis, infected RINm5F cells exhibited a seven- to eightfold increase in the surface expression of class I MHC proteins. Upregulation of class I MHC proteins on reovirus 3-infected RINm5F cells was inhibited by 80% after preexposure of the virus to reovirus 3 antiserum. When analyzed by double-indirect immunofluorescence microscopy, human beta-cells infected with reoviruses 1 or 3 also exhibited markedly increased levels of class I MHC proteins. Reovirus infection of human beta-cells or RINm5F cells was not accompanied by the induction of class II MHC proteins. These findings suggest that 1) in addition to direct cytopathic effects, reovirus infection may contribute to beta-cell destruction by increasing expression of class I MHC proteins and therefore reactivity with cytotoxic T-lymphocytes; and 2) some viruses may increase MHC protein expression independent of and before the action of cytokines (e.g., interferon-gamma and tumor necrosis factor) released by immunoinflammatory cells.
Abstract: 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.
Abstract: 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.
Abstract: In an attempt to improve the diagnostic value of measuring antibodies to islet cell cytoplasmic antigen, coded sera were distributed to 38 laboratories and results were returned for analysis. Comparison between laboratories revealed that results for some individual sera differed by up to nine doubling dilutions and even within laboratories duplicate samples could differ by six doubling dilutions. By including dilutions of sera it was possible to draw a standard curve for each laboratory and this revealed major variations in shape, slope and intercept. However, using each laboratory's standard curve and converting results to units, a substantial improvement was obtained. The approach described improves standardisation and will permit laboratories to identify poor precision and/or any systematic change in assay performance.
Abstract: A possible immunogenetic basis for diabetes in chronic pancreatitis was explored by studying 19 patients with both disorders, most of whom required treatment with insulin. In contrast to patients with insulin-dependent (Type 1) diabetes, patients with diabetes and chronic pancreatitis had residual beta cell function but blunted C-peptide responses to intravenous glucagon, absence of circulating islet cell antibodies, and HLA-DR types similar to control subjects and patients with chronic pancreatitis without diabetes. Diabetes complicating chronic pancreatitis is therefore not associated with the biochemical or immunogenetic markers characteristic of Type 1 diabetes.
Abstract: We have recently shown that interferon-gamma (IFN-gamma) markedly upregulates the expression of the class I major histocompatibility proteins on pancreatic beta cells and have therefore postulated that interferon-gamma may enhance cytotoxic lymphocyte-mediated beta cell damage in insulin-dependent diabetes mellitus. To further explore the interaction between interferon-gamma and the pancreatic beta cell we have used the RIN-m5F insulinoma line to define the effects of interferon-gamma on major histocompatibility protein expression, (pro)insulin and protein synthesis and cell growth. Interferon-gamma induced a dose-dependent increase in the expression of the class I major histocompatibility proteins on the RIN-m5F cells, the maximal increase (10-fold) being seen at an interferon-gamma concentration of 1 U/ml. The induction of class I proteins by interferon-gamma was nearly completely abolished by cycloheximide. Expression of class II (Ia) proteins was not detected either in the presence or absence of interferon-gamma. (Pro)insulin and protein synthesis were decreased by 60% and 40%, respectively, in RIN-m5F cells cultured with interferon-gamma (10 U/ml). Furthermore, the growth of RIN-m5F cells was significantly inhibited, and corresponding changes in cell morphology were evident, after 3 days of exposure to interferon-gamma (10 U/ml). These findings indicate that, in addition to its potential role in amplifying cytotoxic T cell activity against the pancreatic beta cell, IFN-gamma may also directly inhibit beta cell function and growth. Several mechanisms could therefore account for an ability of IFN-gamma to compromise beta cell function and contribute to the pathogenesis of insulin-dependent diabetes.
Abstract: Conditioned serum-free media from cultured human, bovine and rodent endothelial cells contained binding proteins with high affinity for the insulin-like growth factors (IGFs). After partial purifications on heparin or Multiplication Stimulating Activity (MSA)-affinity columns, 2 species of binding protein were identified, a major protein having Mr approximately 35,000 and a minor 22-28,000 protein. The binding proteins had greater affinity for IGF-I than IGF-II with no affinity for insulin or proinsulin. Substantial amounts of the binding proteins remained cell-associated, loosely bound to the outer cell surface of the endothelial cell. Binding protein(s) from human endothelial cells cross-reacted with antibodies to the 53,000 dalton acid-stable human serum binding protein. Production of endothelial binding proteins was not stimulated by growth hormone or insulin. We conclude that endothelial cells in culture produce large quantities of specific IGF binding proteins. Such binding proteins should be relevant in understanding the complex metabolism and function of the IGFs in the intact host.
Abstract: We have reported that sera from a majority of patients with non-insulin-dependent diabetes mellitus (NIDDM) inhibit insulin-stimulated lipogenesis (3-3H-glucose conversion to 3H-lipid) in rat adipocytes to a greater extent than control sera or sera from patients with insulin-dependent diabetes mellitus (IDDM). This effect was apparently due to a circulating, low molecular weight (Mr) substance soluble in acid/ethanol and resistant to proteases. We now describe the further characterization of this inhibitor isolated uniquely from NIDDM sera. Size exclusion chromatography of acid/ethanol extracts of sera on a Bio-Rad P2 column revealed the presence of a Mr 300-400 inhibitor of insulin-stimulated lipogenesis in 32 (70%) of 46 NIDDM sera but not in 9 IDDM or 12 control sera. NIDDM sera that contained the low Mr inhibitor had significantly elevated haemoglobin A1c levels compared with those without the inhibitor (14.1 +/- 2.6% vs 11.5 +/- 2.7%, p less than 0.001). In rat adipocytes, the low Mr inhibitor at a dilution equivalent to 1:40 unextracted serum, reduced maximal insulin-stimulated lipogenesis by 33%, 14C-6-glucose oxidation by 70% and 14C-1-glucose oxidation by 25%. In contrast, insulin-stimulated 2-deoxy-D-(1-3H) glucose uptake was unaffected. In the presence of the inhibitor, insulin sensitivity (insulin dose required for half-maximal response) was unchanged for lipogenesis but decreased for 14C-6-glucose oxidation and increased for 14C-1-glucose oxidation. In the presence of control sera, the low Mr inhibitor decreased basal lipogenesis (a measure of serum insulin-like activity) and its effect on insulin-stimulated lipogenesis was unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: A sensitive microassay using sera from which endogenous bound insulin was removed by acid-stripping was employed to screen for insulin antibodies in 48 newly-diagnosed Type 1 diabetic children and 20 age-matched controls. Using intact diabetic sera, the binding of 125I-insulin exceeded the upper control range in 16/48 (33%); when the sera were acid-stripped at pH 4.2 for 30 minutes at 4 degrees C, binding to the 16 positive sera increased further and in another five sera became significant, making a total of 21/48 positive (44%). Children 5 years of age or less had a higher prevalence of insulin antibodies. Islet cell antibodies, assayed by indirect immunofluorescence, were detected in equal fractions of insulin antibody-positive and -negative sera (76% versus 77%). Assay of insulin antibodies using acid-stripped sera under the conditions described significantly increases the sensitivity of insulin antibody detection. Insulin antibodies appear to be a specific marker of islet autoimmunity, but compared to islet cell antibodies are relatively insensitive, and may be a marker for a particular subgroup of diabetics.
Abstract: The sera of type I (insulin-dependent) diabetic subjects are reported to contain autoantibodies against a 64,000-Mr protein identified in [35S]methionine biosynthetically labeled pancreatic islet cells. We have attempted to localize this autoantigen to the surface of the beta-cell and to define its properties. Sera from 10 newly diagnosed type I diabetic subjects, including five of the index sera originally used to identify the autoantigen, were shown to specifically precipitate a reduced protein of 67,000 Mr from Triton-solubilized, surface 125I-labeled cultured adult human islet and rat insulinoma (RINm5F) cells but not from fresh rat spleen cells. Further characterization revealed that this protein was bovine serum albumin (BSA) adsorbed to cells from fetal calf serum (FCS)-supplemented culture medium and precipitated by BSA antibodies present in many diabetic sera. No labeled proteins were specifically precipitated when surface 125I-labeled and solubilized human islet or RINm5F cells were precleared with anti-BSA immunoglobulins or when cells were first cultured in human serum. In contrast, a 64,000-Mr protein, clearly not BSA, was precipitated by diabetic globulins from human islets but not from RINm5F cells labeled with [35S]methionine. In addition, a protein of the same size as well as proteins of approximately 35,000, 43,000, 140,000, and 200,000 Mr were specifically precipitated by diabetic globulins from freshly isolated human islets solubilized in Triton X-100 and then labeled with 125I. These findings suggest that the 64,000-Mr antigen is not expressed on the surface of human islet cells, at least in culture, and therefore question its relevance as a target for islet cell surface antibodies in initiating beta-cell damage.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: We examined the effect of interferon-gamma (IFN gamma) on expression of the major histocompatibility proteins on cultured human islet cells isolated from adult and fetal pancreas and from an insulinoma. While the pancreatic beta-cells from different sources varied in their responses to IFN gamma, in all instances the expression of HLA-A,B,C protein was increased. Pancreatic beta-cells did not express HLA-DR protein, before or after culture of the islets in IFN gamma, although HLA-DR protein expression was induced on some non-beta-cells. These findings are at variance with those reported with thyroid follicular cells, in which IFN gamma induced expression of HLA-DR. We, therefore, conclude that the interaction between the immune and the endocrine systems may be endocrine cell specific. The up-regulation of HLA-A,B,C protein on beta-cells by IFN gamma provides a mechanism for enhanced targetting to the beta-cells of autoreactive cytotoxic T-lymphocytes and, hence, for amplifying beta-cell destruction.
Abstract: Whereas the expected decrease in the binding of 125I-insulin to erythrocytes 4.5 h after a standard mixed breakfast was seen in six young nonobese normoglycemic controls (delta % bound/free = 2.0 +/- 0.5 SEM), 10 obese hyperglycemic subjects showed an increase (0.54 +/- 0.43) and eight obese normoglycemic subjects of similar age and weight, no change in binding (0 +/- 0.31), both being significantly different from the controls (p less than 0.05) but not from each other. Of the total 18 obese subjects, the 11 with fasting hyperinsulinemia (23 +/- 1.4 mU/l) showed an increase in binding of 0.82 +/- 0.40, significantly different (p less than 0.05) from the decrease of 0.30 +/- 0.25 shown by the seven normoinsulinemic (11 +/- 1.4 mU/l) subjects, despite their similar age, weight, and blood glucose concentrations. These changes in binding were explained by alterations in receptor affinity. Thus, an anomalous postprandial increase in erythrocyte insulin receptor affinity occurs in some obese subjects with or without diabetes, and is related to hyperinsulinemia rather than hyperglycemia.
Abstract: We previously identified two forms of the insulin-like growth-factor-I (IGF-I) receptor in human placenta: a lower-affinity form reactive with an autoantiserum (B-2) to the insulin receptor and a higher-affinity non-immunoreactive form [Jonas & Harrison (1985) J. Biol. Chem. 260, 2288-2294]. Evidence is now presented that the lower-affinity immunoreactive forms are convertible into higher-affinity non-immunoreactive forms via reduction of receptor disulphide bonds. Treatment of placental membranes with increasing concentrations of dithiothreitol (DTT): (1) converted native Mr-290 000 heterotetrameric IGF-I receptors into Mr-180 000 dimers (determined by chemical cross-linking of 125I-IGF-I with disuccinimidyl suberate); (2) increased 125I-IGF-I binding, owing to an increase in receptor affinity; and (3) abolished the reactivity of Triton-solubilized IGF-I receptors with antiserum B-2 and transformed the curvilinear plot of IGF-I binding to a linear form. In isolated complexes between receptor and B-2 antibody, DTT increased 125I-IGF-I binding and released a single class of higher affinity IGF-I receptors of Mr 180,000. Thus DTT-treated IGF-I receptors have similar properties to the higher-affinity non-immunoreactive forms of the native receptor, except that reduced dimeric forms are not detected by cross-linking of 125I-IGF-I to native membranes. Cleavage of the inter-dimeric disulphide bonds is therefore not a prerequisite for higher-affinity binding or loss of immunoreactivity. These observations suggest that the thiol redox state of the IGF-I receptor in vivo is an important determinant of receptor conformation and therefore of the biological responses to IGF-I.
Abstract: Insulin receptors purified from human placenta by sequential affinity chromatography on wheat germ lectin-agarose and insulin coupled to 1,1'-carbonyldiimidazole-activated agarose (CDI-agarose) retained full binding activity but bound a greater than predicted amount of 125I-labeled insulin-like growth factor I (IGF-I). IGF-I and multiplication-stimulating activity (MSA; the rat homologue of IGF-II) were equipotent in displacing either 125I-labeled IGF-I or 125I-labeled MSA from the purified receptors; insulin was 5-15 times more potent. Competitive binding studies indicated that this IGF binding activity could not be explained by cross-reaction with classical insulin receptors or by coelution of IGF-I or IGF-II receptors. Instead, it was due to a minor population of discrete atypical insulin receptors (6-18% total insulin receptors) with moderately high affinity (Kd = 2-4 X 10(-9) M) for IGF-I and MSA. These receptors were not an artifact of insulin-CDI-agarose chromatography, since they were present in wheat germ lectin-agarose-purified preparations and could also be purified from insulin-succinyldiaminodipropylamino-agarose. Affinity labeling with 125I-labeled MSA revealed that these atypical receptors had the same binding subunit (Mr 140,000) as classical insulin and IGF-I receptors. They displayed intermediate reactivity with polyclonal and monoclonal antibodies to the insulin and IGF-I receptors. It is therefore likely that insulin receptors purified by immunoadsorption would also contain atypical insulin receptors. The finding of more than one type of insulin receptor might relate to the slight variations in the cDNA nucleotide sequences and the multiple mRNA species reported for the insulin receptor [Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758].
Abstract: Antibodies against the gram negative enteric bacterium Yersinia enterocolitica have been found in a high proportion of persons with autoimmune thyroid disorders, especially in those with Graves' disease or hyperthyroidism (Shenkman & Bottone, 1981). There is strong evidence that Graves' disease is caused by receptor autoantibodies which mimic the bioeffects of thyroid stimulating hormone (TSH) on the thyroid (Manley, Knight & Adams, 1982). Recently, saturable binding sites for TSH were demonstrated in Y. enterocolitica under non-physiological conditions (Weiss et al., 1983). We have characterized TSH binding sites on Y. enterocolitica under physiological conditions and studied their interaction with Graves' immunoglobulins (Ig's). Saturable and specific binding of receptor-purified 125I-TSH to lysozyme/EDTA-treated Y. enterocolitica (serotype 03) was demonstrated under both non-physiological and physiological conditions. Scatchard binding plots were linear indicating a single class of binding site (Kd 1 X 10(-7) M, maximum of 30,000 binding sites per cell). In the presence of Graves' Ig's the binding of 125I-TSH to Y. enterocolitica was significantly inhibited. Graves' Ig's also precipitated a protein of relative molecular mass (Mr) 64,000 from Triton-solubilized, 125I-labelled Y. enterocolitica, similar in size to one of the proteins precipitated by Graves' Ig's from human thyroid membranes. These findings are consistent with the hypothesis that thyroid autoimmunity may be triggered by bacterial infection via a mechanism involving crossreactivity at the level of the TSH receptor. They also suggest that elements of mammalian endocrine systems are highly conserved and have a function in prokaryotes.
Abstract: The structure of the insulin receptor in intact human erythrocytes was defined using the techniques of disuccinimidyl suberate (DSS) cross-linking of 125I-insulin and surface [125I]iodination followed by receptor immunoprecipitation. In contrast to a recent report, we found the erythrocyte insulin receptor to be similar in structure to that in classic target tissues for insulin, consisting of at least three species of molecular weight approximately 295,000, 265,000, and 245,000, containing disulfide-linked subunits of molecular weight approximately 130,000 and 95,000. The interconversion of the three oligomeric forms could mediate changes in receptor affinity as postulated in other tissues. The 95,000 subunit was detected by immunoprecipitation only if surface iodination was performed in a Tris/Hepes buffer using lodogen and not if phosphate-buffered saline or lactoperoxidase iodination was used. These findings indicate that the lack of a bioeffect of insulin in erythrocytes is not explained by a gross defect in the structure of their insulin receptors. The apparent identity of the insulin receptor structure in erythrocytes and insulin target tissues provides a firmer basis for the use of erythrocytes in some circumstances to reflect insulin receptor status.
Abstract: The effects of intravenous conscious sedation and general anesthesia on a number of stress variables were compared in healthy patients undergoing oral surgery. In the intravenous sedation group only the levels of plasma growth hormone and prolactin rose significantly. In the general anesthesia group significant rises were seen in heart rate; systolic blood pressure; mean arterial pressure; levels of plasma adrenaline, adrenocorticotropic hormone, cortisol, and prolactin; and levels of blood glucose, all indicative of a stress response. This study demonstrates that oral surgery under intravenous conscious sedation with local analgesia is associated with less patient stress than is oral surgery under general anesthesia.
Abstract: Prior studies on weight change and hypothalamic-pituitary-adrenal (HPA) axis functioning are reviewed. Data on 58 depressed and eight anorexic patients is presented. No significant difference in the frequency of cortisol non-suppression in the dexamethasone suppression test (DST) was found between depressed patients with a history of weight loss and those without, nor between depressed patients who lost weight during their first week in hospital and those who did not. Mean weight loss of suppressors did not significantly differ from that of non-suppressors. Of 12 patients whose DST normalised during their stay in hospital, only four gained weight. Five anorexics who were non-suppressors were less than 70% of their ideal body weight (IBW), while three suppressor anorexics were greater than or equal to 70% IBW. These results indicate that mild to moderate weight change is not a significant influence on DST response in depression.
Abstract: The role of bromocriptine in the treatment of non-functioning pituitary tumours is not yet defined. Patients with these tumours who present with visual field defects usually undergo immediate surgery. Three consecutive patients are reported: each had rapid improvement in their visual field defects following bromocriptine 7.5 mg/d before surgery. Histological and immunohistochemical examination confirmed that their tumours were non-functioning chromophobe adenomas. Their responses indicate that bromocriptine may have a role in the management of some patients with non-functioning pituitary tumours.
Abstract: To examine whether products of the immune system interact with the pancreatic beta-cell, rat insulinoma cells (RIN-m5F line) were cultured in the presence of conditioned medium from concanavalin A-activated mouse spleen cells (CAS medium). Indirect immunofluorescence and flow cytometry revealed that after culture in CAS medium, RIN-m5F cells had an 8- to 10-fold increase in class I major histocompatibility complex (MHC) proteins, whereas class II MHC proteins remained undetectable, and the level of insulin and/or insulin-like growth factor 1 receptors was unchanged. The stimulation of class I MHC expression on RIN-m5F cells by CAS medium could be mimicked by recombinant interferon-gamma. Analysis of 125I-surface-labeled cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that in the presence of CAS medium, there was a major increase in the expression of proteins of 48,000, 32,000, and 12,000 Mr and a minor increase in proteins of 17,000 and 9,000 Mr. Precipitation with monoclonal antibody identified the 48,000- and 12,000-Mr proteins as the class I MHC protein and beta 2-microglobulin, respectively. The ability of lymphokine-conditioned medium to increase the expression of RIN-m5F cell surface proteins, including the class I MHC proteins, provides a potential mechanism for enhancing the immune-mediated destruction of the beta-cell.
Abstract: Autoantibodies to thyroxine (T4) and tri-iodothyronine (T3) were found in a 21-yr-old thyrotoxic woman who had paradoxically low T4 and T3 as measured in the 'Amerlex' solid phase radio-immunoassay. Though rare, thyroid hormone antibodies should be suspected if anomalous thyroid function results are obtained and alternate assay procedures used.
Abstract: HLA-A,B,C and DR typing was performed on 108 Caucasian type I diabetic patients, 68 being Gm typed. The expected association with B8, B18, Bw62, DR3 and DR4 was observed as well as an excess of DR3/4 heterozygotes. DR2 was decreased in frequency. In the total patient group, no Gm association was observed but when the patients were subgrouped according to HLA type, HLA/Gm interactive effects were seen. An increase in Gm(1,3;5) was observed in DR3 positive, DR4 negative patients. This association occurred predominantly in females (compared with DR4 and DR3/4 patients of the same Gm phenotype who were predominantly male). Further genetic heterogeneity was identified within DR3/4 patients. Within this group, Bw62 was increased (strongly suggestive of Bw62-DR4 haplotypes) within B8, Gm heterozygotes compared with B8, Gm homozygotes. This finding can be interpreted as indicating a three-way interaction between genes on two HLA haplotypes and Gm-linked genes. These results reflect the genetic heterogeneity and complexity of insulin-dependent diabetes mellitus and explain in part the previous failure of simple genetic models to adequately explain inheritance patterns observed.
Abstract: Two species of insulin-like growth factor-I (IGF-I) receptors in human placenta have been delineated on the basis of their immunoreactivity with an autoantiserum (B-2) to the insulin receptor. When all the IGF-I binding sites in solubilized human placenta were assayed by polyethylene glycol precipitation, a curvilinear Scatchard plot was obtained which could be resolved into two single classes of binding sites: one immunoprecipitable by B-2 IgG and the other, nonimmunoprecipitable. The B-2 reactive sites bound IGF-I with lower affinity (Kd = 7.1 X 10(-10) M) than the B-2 nonreactive sites (Kd = 2.1 X 10(-10) M) and cross-reacted more readily with insulin, the IGF-I/insulin-binding potencies being congruent to 120 and congruent to 1100, respectively. Both receptor subtypes bound IGF-I with congruent to 30-fold higher affinity than multiplication-stimulating activity, and, after affinity cross-linking with 125I-IGF-I, migrated as specific reduced bands of Mr = 138,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit sizes of the B-2 reactive IGF-I receptor were similar to those of the insulin receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors immunoprecipitated by autoantiserum B-2 or autoantiserum B-10 (which recognizes only insulin receptors) revealed, in both cases, specific reduced bands of Mr = 130,000 and 90,000; the same bands were also seen after sequential precipitation with B-10 and B-2 antisera to enrich the proportion of IGF-I receptors recovered. The presence of two distinct binding and immunoreactive species of IGF-I receptors in human placenta raises the possibility that cell- or tissue-specific isotypes of the IGF-I receptor could mediate the different biological actions of IGF-I.
Abstract: Six obese, non-insulin-dependent diabetic subjects were studied before and 3 mo after treatment with the sulfonylurea gliclazide, 40-80 mg b.i.d. Fasting plasma glucose fell significantly from 13.4 +/- 1.6 (SEM) to 8.6 +/- 1.2 mmol/L, accompanied by a significant reduction from 40.6 +/- 3.7 to 29.8 +/- 2.8 mM X h of the plasma glucose response to 75 g oral glucose. Fasting plasma insulin showed a nonsignificant increase from 24.8 +/- 2.0 to 31.3 +/- 2.3 mU/L. The percent specific binding of tracer 125I-insulin to erythrocytes and monocytes did not change significantly (from 9.8 +/- 1.7 to 8.5 +/- 0.7 for erythrocytes and 1.7 +/- 0.3 to 1.6 +/- 0.4 for monocytes). Glucose utilization was measured at three levels of insulin infusion (40, 100, and 300 mU/kg/h) by the euglycemic clamp technique. Overall there was a significant (P less than 0.05) increase in the disappearance rate (Rd) and metabolic clearance rate (MCRg) for glucose at the two higher insulin infusion rates (MCRg: 3.3 +/- 0.7 to 5.1 +/- 0.7 and 5.9 +/- 0.9 to 7.9 +/- 0.9 ml/kg/min), but not at the lowest infusion rate (MCRg: 3.6 +/- 0.8 to 3.3 +/- 0.6). Thus, the chronic hypoglycemic effect of gliclazide in obese diabetic subjects was associated with an improvement in insulin-mediated glucose utilization at high plasma insulin concentrations. This enhanced effect of insulin after gliclazide treatment was not accompanied by increased monocyte or erythrocyte insulin binding, which suggests that it was due to potentiation of postbinding insulin-sensitive pathways.
Abstract: 'Secondary failure' of oral hypoglycemics, in non-insulin dependent diabetics, has been attributed to dietary non-compliance, inadequate drug dosage, metabolic stress, or true drug failure. Progressive loss of beta cell function is a suggested mechanism for true drug failure but on the basis of little documented evidence. In view of this, we have measured basal and glucagon-stimulated C-peptide levels, human leukocyte antigen (HLA) types, and islet cell antibodies in 20 non-insulin dependent diabetics with 'secondary failure' of oral agents. There were 16 females and four males with a mean ideal body weight of 1.30 units and mean duration of diabetes of 9.5 years. Fasting insulin (mean +/- SD: 15.1 +/- 10.6 mU/l) and fasting C-peptide (2.3 +/- 1.2 micrograms/l) were normal or slightly elevated in all but one patient. Mean C-peptide increased from 2.3 +/- 1.2 micrograms/l to 3.5 +/- 2.2 micrograms/l (152% over basal) 6 minutes after 1 mg i.v. glucagon. In 15 patients the C-peptide response was greater than 130% of basal. Islet cell cytoplasmic antibodies were detected in only two patients. The distribution of HLA types was not significantly different from a control population, with no increase in DR3 or DR4. Thus, absolute insulin deficiency is uncommon in non-insulin dependent diabetics with 'secondary failure' of oral hypoglycemic agents and such patients do not exhibit the immuno-genetic markers of insulin-dependent diabetes.
Abstract: The clinicopathologic features of an adult with insulinoma and pancreatic islet cell hyperplasia, who presented with hyperinsulinemic hypoglycemia are reported, together with in vitro studies on the patient's pancreatic islets. Islet cell hyperplasia with ductal proliferation and budding and beta cell degranulation was demonstrated by immunochemical means. The in vitro studies of cultured hyperplastic islet cells support the clinicopathologic features. Thus, in comparison with control islets maintained in culture for up to 14 days, hyperplastic islets could be cultured for up to 60 days, during which time cell overgrowth required subculture on three occasions. Furthermore, in contrast to control islets the release of both insulin and somatostatin from cultured hyperplastic islets was refractory to glucose, glucagon, and tolbutamide; theophylline was the only secretagogue to stimulate insulin and somatostatin release from hyperplastic islets in vitro. Indirect immunofluorescence revealed the presence of islet cell surface autoantibodies in the plasma of this patient reactive with both normal human islets and a rat insulinoma line (RIN-m5F). These studies demonstrate the proliferative capacity and relatively undifferentiated functional state of hyperplastic islets in vitro. They provide further evidence that islet cell division is capable of being stimulated in adult life. The pathogenic significance of islet cell surface autoantibodies in hyperplastic islet cell disease and insulinoma warrants further investigation.
Abstract: In order to obtain an appropriate tissue model to study human diabetes we isolated islet cells from pancreata obtained from brain-dead, heart-beating kidney donor subjects by collagenase dispersion and tissue culture. The presence of viable islet cells was confirmed by both immunofluorescence staining and hormone release experiments. Insulin and somatostatin release were determined on culture day 3 or 4 when amylase measurements indicated an absence of functional exocrine cells. Glucose, alpha-ketoisocaproic acid, theophylline, glucagon, and tolbutamide each stimulated insulin release 2- to 3-fold and somatostatin release 1.5- to 2-fold. Epinephrine and somatostatin both inhibited glucose-stimulated insulin release. Successful subculture of islet cells was achieved after dispersion of primary cultures with dispase. Subcultured islet cells released insulin into the medium during a subsequent 8-day period and when challenged with glucose responded with a 1.6-fold increase in insulin output. Cells cultured on glass coverslips were used to detect, by indirect immunofluorescence, islet cell surface antibodies (ICSA) in the sera of patients with insulin-dependent diabetes mellitus. Of 15 sera from patients with newly diagnosed insulin-dependent diabetes mellitus 9 were ICSA positive, whereas all of 10 control sera were negative; in contrast, using rat insulinoma cells only 4 diabetic sera were positive, as well as 2 control sera. These findings demonstrate the functional viability of adult human islet cells in primary and secondary culture. Cultured human islet cells are a novel, sensitive, and specific system for detecting ICSA and for studying autoimmune effects, and provide a potential source of islet cells for transplantation.
Abstract: The structure of naturally-formed covalent disulphide-linked complexes between insulin and its receptor was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. To prevent destabilization of disulphide bonds at alkaline pH the standard discontinuous electrophoresis conditions were changed to a continuous buffer system at pH 7.0. 125I-insulin was first bound to either rat adipocytes or human placental membranes for 10 min at 37 degrees C. After washing, non-dissociable radioactivity was extracted from cells or membranes in Triton X-100 and immunoprecipitated with an antiserum (B-2) to the insulin receptor. Electrophoresis of the immune precipitate revealed the two smaller of the three reported species of native insulin receptor (Mr values approx. 350 000, 290 000 and 260 000); in addition, a species of Mr 200 000 was also frequently observed in adipocytes. When non-dissociable 125I-insulin was chemically crosslinked to adipocytes or placental membranes, prior to solubilization and immunoprecipitation, all three species of the native receptor were labelled; after reduction, only a single species of Mr 130000 was observed. These findings indicate that disulphide exchange of insulin occurs with the Mr 130000 (alpha) binding subunit within partially reduced species of the native, oligomeric receptor. The degree of disulphide binding of insulin could therefore depend on the relative abundance of partially reduced receptor species and on the redox state of the cell membrane.
Abstract: A 21-yr-old moderately obese woman with hirsutism, acanthosis nigricans, and oligomenorrhoea was diagnosed as having polycystic ovary syndrome. Despite hyperinsulinemia, binding of insulin to her red cells was within the range for normal, young adult subjects. Her serum did not bind or degrade [125I]insulin or alter its binding to fat cells, and was negative for insulin receptor antibodies. However, her serum caused a dose-dependent inhibition of insulin-stimulated lipogenesis (conversion of [3-3H]glucose to [3H]lipid) in rat fat cells significantly greater than that produced with control serum (relative potency, 3.5:1) and (at a 1:20 dilution) markedly impaired the response of both lipogenesis and 2-deoxy-D-glucose uptake to maximum concentrations of insulin. After the patient was treated with clomiphene for 4 months, her menses resumed, hair growth slowed, fasting blood glucose and plasma insulin concentrations decreased, and serum inhibitory activity decreased to the control range. Serum inhibitory activity was stable to freezing and thawing and to heating at 56 C for 30 min, and could be extracted into acid-ethanol. By dialysis, its mol wt was below 1000, whereas by ultracentrifugation, it was above 3500; both high and low mol wt forms were detected after Sephadex G-50 gel chromatography of serum, suggesting that the inhibitor was of low mol wt but loosely bound to a higher mol wt component in serum. These findings indicate that insulin resistance in this patient with acanthosis nigricans and polycystic ovaries could be attributed to a circulating low mol wt inhibitor of postbinding insulin action.
Abstract: Azathioprine (2 mg/kg) was given, in addition to routine insulin treatment, to alternate patients presenting with recent-onset type I diabetes. Treated (N = 13) and untreated (N = 11) patients did not differ significantly at diagnosis with respect to age, duration of symptoms, body weight, blood glucose, hemoglobin A1c, or presence of ketosis. Eight patients were treated for 12 mo, three elected to stop treatment at 6 mo, and treatment was stopped in two because of side effects. Seven treated patients had a remission compared with one untreated patient. At 12 mo these seven patients were distinguished by significantly higher basal and glucagon-stimulated levels of C-peptide (1.98 +/- 0.52 and 3.88 +/- 0.34 micrograms/L, respectively) compared with the other six treated patients (0.93 +/- 0.52 and 1.32 +/- 0.85 microgram/L, respectively), and by the persistence of islet cell cytoplasmic antibodies. Remissions were not sustained in the 1-2 yr after treatment, although relapsed patients required less insulin for control. These results corroborate those from nonrandomized trials using cyclosporine and suggest that protracted treatment with nonspecific immunosuppressive drugs may be necessary to avert insulin dependence.
Abstract: A novel affinity gel, consisting of insulin coupled to 1,1'-carbonyldiimidazole-activated agarose (CDI-agarose), was used to purify insulin receptors from human placenta to homogeneity. This affinity gel is reproducibly prepared and is reported to have a number of advantages over the standard cyanogen bromide activated supports, such as ease and simplicity of coupling and minimal ligand leakage and non-specific binding. Insulin receptors in Triton X-100-solubilized microsomal membranes were purified 2,000-fold by sequential affinity chromatography on wheat germ lectin-agarose and insulin-CDI-activated agarose. They have one of the highest specific insulin-binding capacities (6 nmol/mg protein) reported and can be calculated to have a binding valence of two on the basis that the molecular weight of the oligomeric receptor is 300-350,000.
Abstract: Cell surface antigens of the major histocompatibility complex (MHC) play a crucial role in the initiation of immune reactions. To investigate whether the expression of MHC antigens on pancreatic islet cells could be altered, we have cultured mouse islets in the presence of interferon-gamma (IFN-gamma) and subsequently examined the levels of MHC antigen by indirect immunofluorescence using monoclonal antibodies. IFN-gamma induced a 10-fold increase in H-2K antigen expression on islet cells, the percentage of cells with detectable H-2K expression increasing from 24% to 98%. The effects of IFN-gamma on H-2D and la antigen expression were less marked, with only a twofold increase in mean fluorescence levels, the percentage of cells with detectable levels of expression increasing from 10% to 48% and 5% to 16%, respectively. Using double-indirect immunofluorescence, it was demonstrated that IFN-gamma enhanced expression of H-2K and H-2D antigens on beta-cells. However la-positive beta-cells were undetectable in the presence or absence of IFN-gamma. The ability of IFN-gamma to induce increased expression of H-2 antigens on beta-cells may represent a mechanism for targeting immune (cytotoxic) reactions to beta-cells, e.g., in autoimmune insulitis or allograft rejection.
Abstract: "Postreceptor" insulin resistance in persons with non-insulin-dependent diabetes (NIDDM) could be due to an intrinsic defect in insulin-sensitive pathways or to the action of a circulating inhibitor. Since evidence for the former is lacking, we have addressed the question of a circulating inhibitor by examining the effect of plasma and plasma extracts from NIDDM subjects on the lipogenic response of rat adipocytes to insulin. A majority (77%) of plasma samples (1:20 dilution) from unselected, treated NIDDM subjects (N = 69) inhibited insulin-stimulated conversion of 3-3H-glucose to 3H-lipid in rat adipocytes to a greater extent than did control samples (N = 24). The mean +/- SD inhibition by NIDDM plasma (81 +/- 21%) was significantly greater (P less than 0.01) than by control plasma (50 +/- 14%). Diabetic and, to a lesser degree, control plasma both caused a significant decrease in the maximal response of lipogenesis to insulin. Inhibitory activity was extracted into acid/ethanol, present in the flow of a Sep-pak C18 column, heat-stable (56 degrees C for 30 min [plasma], 80 degrees C for 30 min [acid/ethanol]), resistant to proteases, and dialyzable through 1000-dalton-mol wt exclusion dialysis tubing. The inhibition by NIDDM plasma or partially purified inhibitor could not be explained by the presence of insulin antibodies, insulin receptor antibodies, other inhibitors of insulin binding, or the concentrations of known counterregulatory factors. There was no correlation between inhibitory activity and plasma glucose (r = 0.26), insulin (r = 0.33), C-peptide (r = 0.26), or HbA1c (r = 0.26).(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The thyrotropin (TSH) receptor is a putative target for autoantibodies in Graves' hyperthyroidism and therefore, should be capable of being identified, isolated, and structurally characterized by immunological means. To this end, four sera from patients with hyperthyroidism, three of which inhibited the binding of 125I-TSH to Triton-solubilized human thyroid membranes, were used to isolate TSH receptors by immunoprecipitation. To account for an effect of TSH binding or receptor occupancy on the ability of Graves' immunoglobulins to precipitate TSH receptors, two approaches were taken: (a) specific 125I-TSH binding activity was measured after solubilized thyroid membranes had been incubated with Graves' sera followed by precipitation with Staphylococcus protein A ("receptor depletion"); (b) TSH binding sites were labeled with 125I-TSH and the complexes were precipitated using Graves' sera and Staphylococcus protein A ("receptor precipitation"). The three sera which inhibited 125I-TSH binding depleted 125I-TSH binding activity between 30-80%. Preformed complexes between Staphylococcus protein A and immunoglobulins in these sera were also able to deplete 125I-TSH binding activity. However, after receptor depletion, the one serum that did not inhibit 125I-TSH binding was associated with a significant increase in 125I-TSH binding. All four sera specifically precipitated 80-100% of receptors identified by prelabeling with 125I-TSH. The dilutions of sera that precipitated 50% of 125I-TSH-receptor complexes ranged from 1:150-1:20. Complexes were partially precipitated by high concentrations of control sera (1:20), but the relative potency of control sera was at least fourfold less than Graves' sera. Immunoprecipitates of 125I-labeled thyroid membranes were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography to reveal Graves'-specific bands of reduced molecular weights of 100-110,000, 80-90,000, and 70-75,000. These bands were similar to those obtained from 125I-labeled thyroid membranes purified by TSH affinity chromatography. Thus, Graves' immunoglobulins: (a) precipitate unoccupied and occupied TSH receptors, (b) in one case, neither inhibit binding nor immunodeplete the unoccupied receptor but immunoprecipitate 125I-TSH-receptor complexes, suggesting that binding of TSH may initiate an interaction between the binding site and a separate immunoreactive molecule, and (c) identify the molecular structure of Graves' autoantigens, putatively, the TSH receptor.
Abstract: We describe a patient with bronchial asthma whose respiratory symptoms worsened during treatment and rechallenge with amiodarone. Laboratory studies on cultured pulmonary cells demonstrated an anti-beta-adrenergic effect of amiodarone not due to inhibition of beta-adrenergic receptor binding. Amiodarone should be used with caution in patients with obstructive pulmonary disease.
Abstract: The subunit structure of receptors for insulin and insulin-like growth factor-1 has been demonstrated on a rat insulinoma cell, RIN-m5F, grown in continuous culture. The specific binding of [125-I]-insulin-like growth factor-1 (2.9 +/- 0.42%; mean +/- SEM) to the RIN-m5F cell was significantly greater than of [125I]-insulin (0.7 +/- 0.2%). Immunoprecipitation of receptors from [125I]-surface labeled cells and analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography revealed reduced subunits of Mr 130,000 and 90,000, identical to those for both insulin and insulin-like growth factor-1 receptors on classic target tissues. An apparent abundance of insulin-like growth factor-1 over insulin receptors could be due to downregulation of the latter by secreted insulin. These findings support a role for insulin and insulin-like growth factor-1 receptors in regulating metabolic or growth functions in the beta cell.
Abstract: We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects.
Abstract: A fraction of the insulin specifically bound to adipocytes undergoes a disulfide interchange with its receptor (Clark, S., and Harrison, L. C. (1982) J. Biol. Chem. 257, 12239-12244). In order to test the hypothesis that this covalent modification is a relevant step in insulin action, we have examined the relationship between disulfide binding of insulin and several insulin bioeffects, using sulfhydryl group blocking reagents as probes. The half-time of disappearance of disulfide linked insulin-receptor complexes (I-(S-S)-R) was rapid (4.5 min), consistent with their hypothesized role. A cell-impermeable reagent 5,5'-dithiobis(nitrobenzoic acid) (DTNB) had no effect on specific insulin binding but caused a dose-dependent decrease in both I-(S-S)-R and insulin-stimulated glucose transport. DTNB also inhibited the effect of insulin to stimulate glucose oxidation and to inhibit epinephrine-stimulated cyclic AMP production. In cultured IM-9 lymphocytes, insulin-induced down regulation of its receptor was decreased by 75% in the presence of 1 mM DTNB. Receptor antibodies stimulated adipocyte glucose transport maximally but their effect, unlike that of insulin, was not inhibited by DTNB. These findings suggest that receptor sulfhydryl groups are required for insulin action and support the notion that their interchange with insulin is a necessary step in activation of postreceptor pathways.
Abstract: Phosphorylation of the insulin receptor of isolated rat adipocytes in response to insulin has been studied. Immunoprecipitation of adipocyte membrane protein demonstrated increased incorporation of 32P after exposure to insulin for 15 min, but this was dependent on the presence of physiological concentrations of Ca2+ and Mg2+. Autoradiography of solubilized immunoprecipitated membrane protein after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that most of the 32P incorporation occurred in a band corresponding to Mr 95 000, which has been identified previously as the beta-subunit of the insulin receptor. 32P incorporation was inhibited by 2,4-dinitrophenol and trifluoperazine. It is suggested that insulin-receptor phosphorylation is an energy-requiring process that is Ca2+-dependent and may be modulated by calmodulin. Phosphorylation may proceed independently of glucose transport.
Abstract: 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 lectin 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.
Abstract: Autoantibodies to the insulin receptor are a rare cause of insulin-resistant diabetes, but when they occur they produce a profound clinical syndrome. These antibodies block insulin binding, immunoprecipitate solubilized insulin receptors, and their acute effect is to mimic the biological effects of insulin. However, prolonged exposure of cells to these antibodies produces a state of insulin resistance. Since the antigen to which the antibody is directed is relatively well-characterized, many of the observations in this syndrome can serve as a model for elucidating molecular mechanisms in other diseases with antibodies against membrane components. The autoantibodies to the insulin receptor have also provided valuable probes in the study of insulin receptor structure and insulin action.
Abstract: Atopic individuals (with asthma, allergic rhinitis or atopic eczema) have impaired sensitivity to beta-adrenergic agents. After the finding of antibodies to the beta-adrenergic receptor in the serum of a subject with allergic rhinitis, coded sera from atopic and control subjects were assayed for immunoglobulins that inhibited the specific binding of 125I-labelled hydroxybenzylpindolol to beta-receptors in mammalian lung membranes. Antibodies were present in nine of 60 subjects: 3/19 normal control subjects, 1/9 pre-allergic, 4/17 asthma, 0/8 allergic rhinitis, and 1/7 cystic fibrosis patients. Antibodies of the IgG class in these sera were also demonstrated by indirect precipitation of solubilized lung beta-receptors. The autonomic sensitivity of the nine antibody-positive subjects (Ab+) was compared with that of antibody-negative subjects (Ab-). The Ab+ subjects required 15.0 +/- 1.9 ng isoprenaline (isoproterenol) kg-1 min-1 i.v. to increase pulse pressure by at least 22 mmHg (Ab-, 7.7 +/- 0.4; n = 20; P less than 0.001), and 12.4 +/- 1.8 ng isoprenaline kg-1 min-1 i.v. to increase plasma cyclic AMP concentrations by 50% (Ab-, 8.08 +/- 0.62; n = 13; P less than 0.02). Ab+ subjects required 2.06 +/- 0.3% phenylephrine to dilate their pupils (Ab-, 2.55 +/- 0.08; n = 57; P less than 0.05) and 0.61 +/- 0.08% carbachol to constrict their pupils (Ab-, 0.78 +/- 0.03%; n = 57; P less than 0.05). A role for autoantibodies as beta-receptor antagonists was further supported by showing that human lung cells (VA-13 line) cultured in the presence of globulins from Ab+ subjects had a markedly impaired cyclic AMP response to isoprenaline. These results suggest that autoantibodies to beta-receptors play a pathogenetic role in asthma and related disorders. They have important implications for the concept of autoimmunity.
Abstract: 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.
Abstract: 125I-insulin was specifically cross-linked to membranes of human cultured lymphocytes (IM-9 line) using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and autoradiography of this preparation revealed a major 125I-labeled band with an apparent mol wt of 130,000 and minor bands of about 300,000 and 95,000. Labeling of these bands was inhibited by incubation of membranes with either unlabeled insulin or autoantibodies to the insulin receptor. The bands were also observed after the 125I-insulin cross-linked preparation was solubilized and immunoprecipitated with a panel of autoantibodies to the insulin receptor. However, immunoprecipitation of the 125I-insulin-receptor complex was inhibited by preincubation with excess unlabeled insulin. Finally, 125I-Fab fragments of mol wt 50,000 prepared from anti-receptor antibodies and cross-linked to membranes were resolved into a major complex of mol wt 180,000 and a minor band of 125,000. Neither band was observed when 125I-Fab fragments were cross-linked to membranes in the presence of an excess of unlabeled insulin. These findings indicate that autoantibodies to the insulin receptor are directed against the insulin binding subunits of an oligomeric receptor.
Abstract: [125] Insulin binding to its receptors was studied on circulating cells from 11 patients (8 females and 3 males) with lipoatropic diabetes. The patients ranged in age from 9-54 yr. All were insulin resistant, as evidenced by fasting hyperinsulinemia and insulin tolerance tests. Nine patients were evaluated by specific [125] insulin binding to monocytes. Three different patterns of receptor abnormalities were observed: 3 patients demonstrated decreased binding due to decreased binding capacity, 2 patients revealed normal tracer binding with decreased receptor affinity,, and 4 patients had normal or increased insulin binding. [125] Insulin binding to erythrocytes in 9 cases demonstrated similar heterogeneities of initial binding. In most cases there was a good correlation between the binding with erythrocytes and monocytes, although decreased affinity was not observed in the red blood cells. There was no obvious correlations between the nature of the receptor defect and the clinical patterns in these patients. Heterogeneity in insulin binding was even observed among affected members of the same family. Antibodies to the insulin receptor were not detected in any of these patients by either binding inhibition or immunoprecipitation assays. These data suggest that the pathogenesis of the insulin resistance in lipoatropic diabetes is heterogeneous and may involve both receptor and postreceptor abnormalities.
Abstract: We have identified the subunits of the insulin receptor in cultured human lymphocytes (IM-9 line) by biosynthetic labeling with [35S]methionine and specific precipitation with autoantibodies against the insulin receptor. IM-9 lymphocytes were cultured with [35S]methionine and extracted with Triton X-100. Insulin receptors were concentrated and purified 20-fold by chromatography of the cell extract on wheat germ agglutinin-agarose, and then specifically precipitated by receptor antibodies after addition of a second antibody. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed specific precipitation of two major bands with molecular weights of 130,000 and 90,000. Both species were precipitated by receptor antibodies from four different patients with the syndrome of extreme insulin resistance and acanthosis nigricans. In accord with previous data that insulin bound to receptor reduces the affinity of receptor for anti-receptor antibody, we found that preincubation of the wheat germ-purified cell extract with insulin (1.7 microM) prior to immunoprecipitation caused a decrease in the appearance of both species. The decrease in insulin binding seen after incubation of the lymphocytes with insulin for 12 hr ("down regulation") was associated with a decrease in the labeling of both the 130,000 and 90,000 bands. The apparent molecular weight of both subunits was decreased after pretreatment with mixed glycosidases. In conclusion, we have biosynthetically labeled the insulin receptor with [35S]methionine and showed that the receptor consists of two major glycoprotein subunits with apparent molecular weights of 130,000 and 90,000.
Abstract: A large number of glucose-monitoring systems suitable for home use are now available. The Glucochek, an early model (Mk I) and a later (Mk II), the Stan Clark RAHC, the Glucometer, and 20-800 BM glycemie strips were evaluated with regard to accuracy, precision, model variability and operator variability before a particular system was recommended for patient use. Whole blood glucose, on samples samples taken in the Diabetic Clinic of The Royal Melbourne Hospital, Melbourne, was measured with the system under test and in the Biochemistry Department. Accuracy was indicated by the mean of the differences between the two results, and precision by the standard deviation of these differences-the closer these results to zero, the better the system. The 20-800 BM Glycemia strips gave the best results in the hands of an experienced operator, but showed the greatest interoperator differences. These differences decreased when a machine-based system was employed. The Glucochek Mk I did not perform satisfactorily. All the systems tested showed a marked decrease in accuracy and precision when blood glucose levels were greater than 15.0 mmol/L. These results show that a machine is not a necessary part of a home glucose-monitoring system; that patients on home glucose-monitoring must be trained and their results checked against a reference method initially and, ideally, at regular intervals; that home glucose-monitoring in patients with marked hyperglycaemia unreliable.
Abstract: Autoantibodies to beta 2-adrenergic receptors have been identified in the serum of one patient with allergic rhinitis ("hay fever") and two patients with asthma. The antibodies precipitate solubilized dog lung beta receptors in an indirect immunoprecipitation assay and inhibit the specific binding of iodine-125-labeled iodohydroxybenzylpindolol to membrane-associated receptors from dog lung, calf lung, and human placenta. Ligand binding to canine heart beta 1 receptors is not affected by the antibodies.
Abstract: The New Zealand Obese (NZO) mouse was studied as a potential model for autoimmune diabetes. NZO mice develop obesity, glucose intolerance, and insulin resistance, and have low-titer IgM antibodies to the insulin receptor. It is shown that they have circulating antibodies to both native DNA and denatured, single-stranded DNA. The antibody levels are higher in females, and, up to 6 mo of age, are comparable to those found in the related NZB X NZW F1 (NZB/W) mouse, a model for systemic lupus erythematosus. After 6 mo of age the antibody levels in NZO mice fall toward normal, in contrast to the persistently elevated levels in NZB/W mice. NZB/W mice are known to succumb to immune complex-mediated proliferative glomerulonephritis before 1 yr of age, whereas NZO mice survive. NZO kidneys exhibit light microscopic features of both diabetic and lupus nephropathies: glomerular proliferation, mesangial deposits, mild basement membrane thickening, glomerulosclerosis, eosinophilic nodules in some glomeruli, occasional hyalinization of the glomerular arterioles, and healing arteriolar inflammation. These changes are associated with glomerular deposition of immunoglobulin, especially IgM, in a granular pattern on fluorescent staining. The NZO mouse, therefore, has evidence of a generalized immune disorder and provides a model for studying the relationship between autoimmunity, obesity, and diabetes.
Abstract: Insulin receptors in human tissue undergo marked changes in both their concentration and their affinity for insulin. In general, alterations in receptor affinity are associated with rapidly changing metabolic environments and can occur within hours, whereas alterations in receptor concentration appear to require longer time periods for their induction. The association of a given type of receptor alteration (i.e., change in affinity or concentration) with a given clinical state indicates the presence of distinct modulators of the insulin-receptor interaction. We have presented evidence for 2 specific modulators, i.e., insulin itself and anti-insulin-receptor antibodies. In several clinical states, especially those associated with changes in receptor affinity, receptor alterations are unrelated to either ambient insulin levels or anti-receptor antibodies, suggesting the presence of several as yet unknown mediators of the insulin receptor. The direct metabolic consequences of these receptor events are not well established. In several states, the receptor alteration correlated quite well with the clinical sensitivity of the whole organism to insulin, indirectly implicating the receptor as the major control point for insulin sensitivity. In contrast, specific examples were cited in which receptor events are not consonant with observed biologic responses to insulin, thereby suggesting a predominance of postreceptor processes. Finally, it should be emphasized that the study of hormone receptors and their relationship to disease states is in the formative stage. With new and improved methodology we hope we will be able to investigate the entire pathway of insulin action at its target tissues, from the initial receptor binding to the final biologic effect.
Abstract: A sensitive and specific radioimmunoassay for the insulin receptor has been developed employing receptor autoantibodies from the serum of a patient with insulin-resistant diabetes. The assay detects insulin binding sites at concentrations as low as 0.1 nanomolar; distinguishes between receptors originating from human placental membranes, human lymphoblastoid cells, and mouse liver membranes; and measures the receptor independently of its binding function. Down-regulation, or loss of binding after exposure to insulin, is associated with loss of immunoreactive receptor.
Abstract: The insulin receptor for human placental membranes has been solubilized in Triton X-100 and its properties have been examined in detail. Binding of [125 I]iodoinsulin to the soluble receptor is markedly inhibited by increas-ng concentrations of Triton X-100, due to a fall in receptor affinity. In 0.02--0.10% Triton X-100, the soluble receptor exhibits all the essential characteristics of the intact or particulate receptor. These include strict specificity for insulin and its analogues, increase in steady state binding with decrease in temperature, a pH optimum at 7.8--8.0, and negatively cooperative site-site interactions. The initial association rate of [125 I]iodoinsulin and the soluble receptor is a direct function of temperature, but the level of steady-state binding is lower at higher temperatures due to a marked increase in dissociation rate. Scatchard binding plots are curvilinear and show a large increase in affinity at 4 C with no change in total binding capacity (R0); increased binding to the particulate placental membrane at 4 C is due chiefly to an increase in R3. Negative cooperatively in the soluble receptor has been confirmed by kinetic experiments; thus, the dissociation of [125I]iodoinsulin from the receptor in the presence of "infinite" dilution is accelerated in the presence of 10(-8) M unlabeled insulin. The apparent molecular weight of the placental receptor, determined by gel filtration on 6% agarose, is approximately 300,000. These studies show that the basic properties of the insulin receptor do not depend on it being an integral conponent of the cell membrane.
Abstract: The syndrome of ataxia telangiectasia is associated with glucose intolerance and insulin resistance. We examined the status of insulin receptors on circulating monocytes and on cultured fibroblasts from two siblings with ataxia telangiectasia and severe insulin resistance. 125I-insulin binding to monocytes of the two patients consistently demonstrated an 80 to 85 per cent decrease in receptor affinity. In contrast, the defect in receptor affinity was not expressed on the patients' cultured fibroblasts or on monocytes or fibroblasts obtained from unaffected family members. Whole plasma and immunoglobulin-enriched fractions of plasma from the patients inhibited the normal binding of insulin to its receptors on cultured human lymphocytes (IM -9 line) and on human placental membranes. We conclude that the insulin resistance in the two siblings with ataxia telangiectasia was associated with defects in the affinity of the receptors for insulin, probably caused by circulating inhibitors of insulin binding.
Abstract: The role of the surrounding membrane structure on the binding characteristics of the insulin receptor was studied by using several digestive enzymes. The effects observed with particulate membrane preparations are compared with those from soluble receptor preparations. beta-Galactosidase and neuraminidase had no effect on insulin binding to either particulate or soluble receptors from human placentae. Exposure to 2 units of phospholipase C/ml increased insulin binding to particulate membranes, but was without effect on the soluble receptor preparation. The increase in binding to particulate membranes was shown to be due to an increase in apparent receptor number. After 5 min exposure to 500 microgram of trypsin/ml there was an increase in insulin binding to the particulate membrane fraction, owing to an increase in receptor affinity. After 15 min exposure to this amount of trypsin, binding decreased, owing to a progressive decrease in receptor availability. In contrast, this concentration of trypsin had no effect on the solubilized receptor preparation. Because of the differential effects of phospholipase C and trypsin on the particulate compared with the solubilized receptor preparations, it is concluded that the effects of these enzymes were due to an effect on the surrounding membrane structure. Changes in receptor configuration due to alterations within the adjoining membrane provide a potential mechanism for mediating short-term alterations in receptor function.
Abstract: A 34 year-old woman with Werner's syndrome has been studied in the light of the current concept that this disorder is a model of premature aging. Endocrine function assays revealed an abnormal glucose tolerance and in vivo insulin insensitivity after prednisolone, and ovarian failure. Immune function assays revealed hypo-responsiveness in skin tests for delayed hypersensitivity, a poorly sustained IgG anti-body response after immunization with flagellin, and a low count of colony-forming T lymphocytes in blood. Cultured fibroblasts had a very limited capacity to replicate in vitro, in comparison with donors of similar age and, moreover, 85% of glucose-6-phosphate dehydrogenase in the patient's cultured fibroblasts was heat-stable at 60 degrees C compared with 100% for a healthy control. Cell receptors (for insulin) were examined by insulin binding to isolated fat-cells, with the finding that fat-cells were abnormally large for the patient's size, and their receptor density was low. The findings from the study point to a genetic defect in Werner's syndrome which, in its effect on particular tissues, may simulate features of aging, but the disease is not a true model of premature aging.
Abstract: Specific binding of insulin to microsomal membranes from the placentae of insulin-dependent diabetics was significantly decreased when compared with normals. This was due to an apparent decrease in the concentration of insulin receptors.
Abstract: A computer-processed postal questionnaire was devised to detect hypothyroidism in patients treated previously for thyrotoxicosis with radioiodine. In a study of 232 patients treated with 131I at the Royal Melbourne Hospital between four and ten years previously, the sum of symptomatic answers in the questionnaire was a sensitive discriminator of hypothyroidism, and allowed 80% of euthyroid patients to be excluded from further assessment. Questions concerned with general well-being and energy, voice and skin changes, showed the highest sensitivity and specificity. The combination of these questions alone was an effective means of identifying hypothyroidism, with a sensitivity and specificity comparable to the more sophisticated technique of discriminant function analysis. Hypothyroidism had an incidence of between 20% and 35% six to eight years after 131I therapy and was related to a smaller initial goitre size for a given dose of 131I. This postal questionnaire, in conjunction with a computer-based automatic recall system, promises to be an efficient and reliable screening tool for the detection of hypothyroidism in the increasing number of patients "at risk" following 131I therapy.
Abstract: 1. The rate of oxidation of [1-14C]glucose to 14CO2 was examined in subcutaneous adipose tissue from fifteen obese non-diabetic subjects and from eleven obese maturity-onset diabetic patients. Production of 14CO2, measured in the basal state and in the presence of insulin, was significantly correlated with mean cell size in both the non-diabetic and the diabetic subjects, independent of age, relative weight and fasting plasma insulin concentration. 2. Comparison of the regressions of glucose oxidation rates on mean cell size indicated: (i) that insulin produced a significant increase in activity over the basal value in both groups, and (ii) that basal and insulin-stimulated activity were both significantly lower in diabetic than in non-diabetic adipose tissue.
Abstract: The effect of varying concentrations of insulin on 1-14C-glucose conversion to 14CO2 was measured in subcutaneous adipose tissue samples obtained from 16 obese human subjects (10 nondiabetic, 6 diabetic). An index of insulin sensitivity in vitro, Kins, was calculated as the concentration of insulin stimulating one-half maximal 14CO2 production. An index of insulin sensitivity in vivo, Kitt, was calculated as the rate constant for decrease in blood glucose after rapid intravenous administration of 0.05 U/kg insulin. There was, over-all, a significant correlation between Kins and Kitt, indicating that insulin sensitivity of 1-14C-glucose oxidation by adipose tissue in vitro reflects the general state of sensitivity of glucose metabolism to insulin in vivo in obese human subjects. The mean values for both Kins and Kitt in the nondiabetic subjects were significantly different from those in the diabetic subjects, indicating greater sensitivity to insulin in the former group. The nondiabetic group was also distinguished by a significantly greater plasma insulin:blood glucose ratio in the oral glucose tolerance test. These results support the view that tissue insulin sensitivity as well as pancreatic beta cell response play an important role in determining glucose tolerance in obesity.
Abstract: 1. The anorexic agent mazindol and its major metabolite 46-034 (Sandoz) in high concentrations ( greater than 0-4 mM) abolished basal and insulin-stimulated conversion of 1-14C-glucose to 14CO2 by rat isolated fat cells. 2. High concentrations (1mM) also inhibited specific binding of 125 I-insulin to fat cells. 3. The observed effects appeared to be due in part to perturbation of the plasma membrane since there was a rise in the lactate dehydrogenase content of the incubation medium, increased 125I-insulin degradation and a reduction in cellular tritiated water space. 4. These effects are unlikely to be relevant to the therapeutic action of mazindol.
Abstract: This study examined the relationship between receptor binding of insulin in a metabolically significant target tissue in vitro and sensitivity to insulin in vivo in obese human subjects. Specific insulin binding was measured at 24 degrees C in isolated enlarged fat cells obtained from 16 patients, by observing the effect of increasing concentrations of unlabeled insulin on the binding of [125I]insulin. Scratchard plots of the binding data were curvilinear with an upward concavity, similarity shaped, and essentially parallel. Kinetic studies on the dissociation of [125I]insulin from fat cells indicated that these curvilinear Scratchard plots could be explained by the presence of site:site interactions of the negative cooperative type. Differences in binding between individual patients were predominantly due to differences in the numbers of receptor sites whether expressed in relation to cell number, cell volume, or cell surface area. These findings were not accounted for by differences in [125I]insulin degradation. Acute exposure of adipose tissue to insulin in vitro had no significant effect on [125I]insulin binding to isolated cells. The number of receptor sites was directly correlated with insulin sensitivity in vivo, measured as the rate constant (Kitt) for the fall in blood glucose after intravenous insulin, and was inversely correlated with the level of fasting plasma insulin. These findings corroborate those from other studies using human mononuclear leukocytes and various tissues from the obese mouse, which indicate that decreased insulin binding is a characteristic feature of insulin resistance in obesity.
Abstract: Oral administration of a single dose of the anorectic agent mazindol to obese subjects led to a significant improvement in oral glucose tolerance and a concomitant reduction in insulin secretion, but had no effect on the blood glucose and plasma insulin responses to glucose given intravenously. Mazindol, when given to obese subjects in conjunction with a hypocaloric diet, was associated with progressive weight loss and reduction in the fasting levels of blood glucose, plasma insulin, serum triglyceride, and serum cholesterol. When oral glucose tolerance was retested after 16-20 wk, blood glucose and plasma insulin responses were significantly decreased compared with initial control values. It is concluded that one effect of mazindol, when given acutely, is to impair absorption of glucose from the gut. Changes in carbohydrate metabolism after chronic administration of mazindol are entirely consistent with weight loss, although a separate effect of the drug cannot be excluded.
