Islet cell autoantigens in insulin-dependent diabetes.

A burgeoning number of antigenic targets of the islet cell autoimmunity in IDD have been identified, and more can be anticipated through improved methods for their identification. The challenge for those investigating the pathogenesis of IDD will be to assign the relative importance of these antigens to the development of the disease, and to resolve whether there is a dominant primary immunologic event that is followed by a series of secondary immunizations to a variety of normally sequestered islet cell antigens in the sequence of pathogenic events that culminate in IDD. One interesting observation that may have potential pathogenic implications is the observation that of all islet cell autoantigens described, only two (i.e., 64 kD/GAD, 38 kD) are reactive in their native configurations, implying that recognition of conformational epitopes is most important. This property argues for primary immunizing agents rather than secondary ones after release of denatured antigens and antigenic recognition through their epitopes. Given the complex and multiple physiological functions of islet cells and the continuous variation in their activity, it is reasonable to speculate that the speed of the progression to IDD could vary between individuals with respect to their insulin needs and the relative activities of their islets. Activated islets may express autoantigens that have only limited expression in quiescent islets. The often times striking variation in the severity of insulitis seen in different islets of a single pancreas may be explained by the level of activity of individual islets. Furthermore, disparity in HLA-DR/DQ associations with disease may involve differences in the immunological recognition of autoantigens. Whereas there is still much to learn, it is clear that disease predictability and disease intervention studies have been enhanced through the identification of the islet cell autoantigens in IDD.     

Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is thought to result from the autoimmune destruction of the insulin-producing beta cells of the pancreas. Years before IDDM symptoms appear, we can detect autoantibodies to one or both forms of glutamate decarboxylase (GAD65 and GAD67), synthesized from their respective cDNAs in a bacterial expression system. Individual IDDM sera show distinctive profiles of epitope recognition, suggesting different humoral immune responses. Although the level of GAD autoantibodies generally decline after IDDM onset, patients with IDDM-associated neuropathies have high levels of antibodies to GAD, years after the appearance of clinical IDDM. We note a striking sequence similarity between the two GADs and Coxsackievirus, a virus that has been associated with IDDM both in humans and in experimental animals. This similarity suggests that molecular mimicry may play a role in the pathogenesis of IDDM.     

Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes.

Insulin-dependent diabetes (IDD) results from the autoimmune destruction of the insulin-producing pancreatic beta cells. Autoreactive T-lymphocytes are thought to play a pivotal role in the pathogenesis of IDD; however, the target antigens of these cells, as well as the inductive events in the disease, are unclear. PBMC in persons with or at increased risk for IDD show elevated reactivity to the beta cell enzyme glutamate decarboxylase (GAD). To identify the T-lymphocyte-reactive determinants of GAD, an overlapping set of synthetic peptides was used to stimulate the PBMC from these individuals, PBMC responsiveness to GAD peptides was not restricted to those with IDD, and a number of peptides elicited responses in PBMC. However, the major determinant of GAD recognized by persons at increased risk for IDD was amino acids 247-279, a region which has significant sequence similarity to the P2-C protein of Coxsackie B virus (47% of 15 increased risk [islet cell autoantibody-positive relatives]; 25% of 16 newly diagnosed IDD patients; and 0% of 13 healthy control subjects). Responses to tetanus and insulin antigens were not different between the study groups. In addition, PBMC from individuals responding to GAD peptides within 247-279 also responded to a Coxsackie viral peptide (i.e., P2-C amino acids 32-47), an observation supporting potential molecular mimicry in this immune response. Although the role of environmental agents in the pathogenesis of the disease remains unclear, these cellular immunological findings support the epidemiological evidence suggesting an inductive role for enteroviruses like Coxsackie B in the autoimmunity underlying IDD.     

Prevalence of autoantibodies to the 65- and 67-kD isoforms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

We investigated the presence of autoantibodies to baculovirus-expressed human recombinant 65- and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using [35S]methionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies.     

Detection of pancreatic islet 64,000 M(r) autoantigens in insulin-dependent diabetes distinct from glutamate decarboxylase.

Patients with insulin-dependent diabetes (IDDM) possess antibodies to islet proteins of M(r)-64,000. Potential autoantigens of this M(r) include glutamate decarboxylase (GAD) and 65 kD heat shock protein. We have detected two distinct antibody specificities in IDDM that bind 50,000 M(r) or 37,000/40,000 M(r) proteolytic fragments of 64,000 M(r) proteins. In this study, we investigated relationships of these proteolytic fragments to GAD and heat shock proteins. Polyclonal antibodies to GAD bound 50,000 M(r) fragments of islet antigen. Recombinant GAD65, but not GAD67, blocked binding to this antigen, suggesting that 50,000 M(r) fragments are derived from islet GAD65. In contrast, GAD antibodies did not recognize 37,000/40,000 M(r) fragments, and neither GAD isoforms blocked autoantibody binding to precursors of these fragments. The 37,000/40,000 M(r) fragments, but not the 50,000 M(r) fragments, were detected after trypsin treatment of immunoprecipitates from insulinoma cells that lacked expression of major GAD isoforms. Antibodies in IDDM did not bind native or trypsinized islet heat shock proteins. Thus, IDDM patients possess antibodies to GAD, but also distinct antibodies to a 64,000 M(r) protein that is not related to known GAD isoforms or heat shock proteins.     

A clinical profile of insulin-dependent diabetes with islet cell cytoplasmic and cell surface antibodies

Both islet cytoplasmic and cell surface antibodies (ICA and ICSA) were studied from the onset of diabetes to the honeymoon in an insulin-dependent diabetic patient. The patient, a 24-year-old male, was admitted to the hospital because of ketoacidotic hyperglycemic precoma. Continuous subcutaneous infusion of a small dose of insulin was carried out for a couple of days followed by NPH-insulin injection. The dose was gradually decreased and on the 45th day after the onset, an oral hypoglycemic agent was substituted for insulin. The patient was followed up after discharge from the hospital and his disease was controlled well by diet and a hypoglycemic agent until he caught a common cold. He was then admitted again to the hospital because of hyperglycemia, and insulin injection was performed. Both ICA and ICSA were found independently of each other during the course of the disease. The ICSA, which was quantitatively determined by immunoassay using 125I-protein A, closely paralleled the clinical profile. However, the levels of quantitative ICSA were higher than normal, even though the patient's diabetes reached the honeymoon stage. These results suggest that quantitative ICSA has a strong association with the clinical profile in IDDM, and it may be a "marker" of islet cell damage during the diabetic period or a parameter for diabetic prognosis.     

Islet cell antibodies predict insulin-dependent diabetes in United States school age children as powerfully as in unaffected relatives.

Islet cell antibodies (ICA) in the sera of nondiabetic relatives of patients with insulin-dependent diabetes (IDD) are predictive of the disease, a finding that permits the design of intervention strategies to prevent it. However, 85% or more of patients with new onset IDD have no affected relative. We therefore screened 9,696 schoolchildren between the ages of 5 and 18 yr (mean age 10.7 yr) in Pasco County, Florida for ICA in three surveys during 1984/5, 1987/8, and 1990/1 and have followed them prospectively. Approximately 4,000 of these children have been followed for nearly 8 yr. ICA titers > or = 10 Juvenile Diabetes Foundation units on replicate tests were detected in 57 of the children (0.59%). 10 children have developed diabetes so far, and all had ICA detected beforehand. The likelihood of developing IDD among the ICA-positive children was compared with 2,959 age-matched nondiabetic first degree relatives of IDD probands who were screened for ICA by our laboratory during the same time period and also followed prospectively. Of 103 (3.5%) ICA-positive relatives, 31 have developed IDD. Life table analysis reveals no statistically significant differences in the probability of developing IDD between the ICA-positive schoolchildren and ICA-positive first degree relatives (P = 0.3). The estimated risk of developing IDD by 7 yr in the ICA-positive schoolchildren was 45% (95% confidence interval 15-74%) compared with 43% (confidence interval 22-63%) in the relatives. We conclude that ICA appear to be as predictive of IDD in low-risk schoolchildren as they are in high-risk relatives. These data suggest that it is feasible to predict IDD by screening a general population of schoolchildren for ICA and that those found to be positive could be considered, in addition to relatives, for intervention protocols to prevent the disease.     

Ocular and systemic reactivity to isoprenaline in patients with insulin-dependent diabetes mellitus

There is experimental evidence of decreased β-adrenergic myocardial sensitivity in patients with insulin-dependent diabetes mellitus (IDDM). In the present study we hypothesized that the ocular response to isoprenaline, as a consequence of increased arterial vessel rigidity, might also be blunted in patients with IDDM. We therefore compared the correlation between systemic pulse pressure amplitude (PPA) and fundus pulsation amplitude (FPA) during intravenous isoprenaline administration in 11 otherwise healthy IDDM patients and 11 healthy control subjects. Ocular fundus pulsations were measured by a recently developed laser interferometric method. Isoprenaline increased PPA in both study groups in a dose-dependent way, but the response was significantly less in IDDM patients (at 0.8 μg min⁻¹: + 38% in control subjects, + 27% in IDDM patients, P  < 0.05 between groups). Moreover, a dose-dependent increase in FPA was observed, which again was more pronounced in healthy subjects (at 0.8 μg min⁻¹: + 45% in controls, + 17% in IDDM patients, P  < 0.005 between groups). The regression line between PPA and FPA was very close to the 45° line in healthy subjects, whereas it was significantly flattened in IDDM patients. In conclusion, linear regression between PPA and FPA during isoprenaline suggests arterial stiffening in patients with IDDM. Hence, comparison of systemic PPA and FPA during isoprenaline provocation may be a useful method of estimating changes in arterial capacitance in patients with diabetes mellitus.     

Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes

We previously demonstrated that a spontaneous Th1 response against glutamate decarboxylase (GAD65) arises in NOD mice at four weeks in age and subsequently T cell autoimmunity spreads both intramolecularly and intermolecularly. Induction of passive tolerance to GAD65, through inactivation of reactive T cells before the onset of autoimmunity, prevented determinant spreading and the development of insulin- dependent diabetes mellitus (IDDM). Here, we examined whether an alternative strategy, designed to induce active tolerance via the engagement of Th2 immune responses to GAD65, before the spontaneous onset of autoimmunity, could inhibit the cascade of Th1 responses that lead to IDDM. We observed that a single intranasal administration of GAD65 peptides to 2-3-wk-old NOD mice induced high levels of IgG1 antibodies to GAD65. GAD65 peptide treated mice displayed greatly reduced IFN gamma responses and increased IL-5 responses to GAD65, confirming the diversion of the spontaneous GAD65 Th1 response toward a Th2 phenotype. Consistent with the induction of an active tolerance mechanism, splenic CD4+ (but not CD8+) T cells from GAD65 peptide- treated mice, inhibited the adoptive transfer of IDDM to NOD-scid/scid mice. This active mechanism not only inhibited the development of proliferative T cell responses to GAD65, it also limited the expansion of autoreactive T cell responses to other beta cell antigens (i.e., determinant spreading). Finally, GAD65 peptide treatment reduced insulitis and long-term IDDM incidence. Collectively, these data suggest that the nasal administration of GAD65 peptides induces a Th2 cell response that inhibits the spontaneous development of autoreactive Th1 responses and the progression of beta cell autoimmunity in NOD mice.     

Islet beta cell failure in type 2 diabetes

The major focus of this Review is on the mechanisms of islet beta cell failure in the pathogenesis of obesity-associated type 2 diabetes (T2D). As this demise occurs within the context of beta cell compensation for insulin resistance, consideration is also given to the mechanisms involved in the compensation process, including mechanisms for expansion of beta cell mass and for enhanced beta cell performance. The importance of genetic, intrauterine, and environmental factors in the determination of "susceptible" islets and overall risk for T2D is reviewed. The likely mechanisms of beta cell failure are discussed within the two broad categories: those with initiation and those with progression roles.     

Pre-conception diabetes care in insulin-dependent diabetes mellitus.

Prospective studies of pre-conception diabetes care have confirmed its positive impact on the incidence of malformations by improving glycaemic control. Less information is available on the impact of pre-conception care on maternal and neonatal morbidity. This audit addresses its impact on timing and mode of delivery, incidence of macrosomia and rate of admission to neonatal unit care in addition to sociodemographic factors which may influence attendance at such a service. Attenders were more likely to be in a stable relationship and be non-smokers. They were more likely to book for antenatal care earlier and with a lower glycated haemoglobin. There were no early deliveries (i.e. < 30 weeks) or small for gestational age (SGA) babies in those who attended for pre-conception care and no neonatal deaths. Admission to NNU care was reduced by 50% in those who attended for pre-conception care. Although the rate of macrosomia was reduced, there was no impact on the Caesarian section rate. A pre-conception diabetes clinic may have a positive impact on neonatal morbidity.     

Elimination of islet cell antibodies and glutamic acid decarboxylase antibodies II in a patient with newly diagnosed insulin-dependent diabetes mellitus

Islet cell antibodies and glutamic acid decarboxylase II (GAD II) antibodies have been discussed in the autoimmune pathogenesis of insulin-dependent diabetes mellitus (IDDM). Hence, immunosuppressants, intravenous immunoglobulins, and plasmapheresis have been used in an effort to modulate autoimmune activity and thereby prevent the destruction of pancreatic β-cells. We describe the autoantibody (islet cell antibody and GAD II) kinetics and clinical course in a patient with newly diagnosed IDDM treated with a specific immunoglobulin apheresis technique. Five days after the initial diagnosis a 37-year-old patient with IDDM underwent a series of seven immunoglobulin aphereses. Immunoglobulin (IgG, IgA, IgM), islet cell antibody, GAD II, and C-peptide concentrations were monitored for a time course of 74 days. Daily insulin requirements were recorded. One single immunoglobulin apheresis decreased IgG by 66.2 ± 9.1%, IgA by 66.8 ± 8.7%, and IgM by 57.7 ± 12.9%. GAD II antibodies were reduced by 61.9 ± 12.4%. The islet cell antibody titer declined from 1:32 to 1:4 after the treatment series. There were no relevant changes in the safety parameters determined nor were there any clinical side effects. The efficient decrease in islet cell antibodies and glutamic acid decarboxylase II antibodies in a patient with IDDM encourages further investigations into the impact of this treatment on the clinical course of this autoimmune disorder. J. Clin. Apheresis 12:196–199, 1997. © 1997 Wiley-Liss, Inc.     

HLA antigens in insulin-dependent diabetes mellitus.

Diabetes mellitus is largely determined by genetic factors but environmental factors are necessary to convert genetic susceptibility into overt disease. Studies of twins show that the genetic impact in non-insulin-dependent diabetes mellitus is stronger than in insulin-dependent diabetes mellitus. The genetic factors involved in non-insulin-dependent diabetes mellitus are not known and the outcome of molecular genetic research has so far been disappointing. The major genetic susceptibility to insulin-dependent diabetes mellitus is conferred by genes in the HLA region on chromosome 6. Despite many advances in molecular genetics in insulin-dependent diabetes mellitus the serologically detectable HLA antigens and haplotypes are still the best available markers. This review describes the important developments in immunogenetics in insulin-dependent diabetes mellitus and summarises the main findings from earlier studies. Genetically the potential for primary prevention of insulin-dependent diabetes mellitus already exists and will become a reality as soon as the environmental determinants are identified. A wide application of immunogenetic methods will be needed in the prevention of insulin-dependent diabetes mellitus.     

Beta-adrenoceptor regulation in insulin-dependent diabetes mellitus.

The study was performed to determine whether the regulation of mononuclear leukocyte beta-adrenergic receptors and responses was changed in insulin-dependent diabetes mellitus (IDDM). The concentrations of noradrenaline, the beta-adrenoceptor densities, basal cAMP levels and maximal isoprenaline-induced cAMP responses were the same in the diabetic and healthy subjects. After isoprenaline-promoted receptor internalization and uncoupling, the receptor densities and the responsiveness did not differ. In the control group, a highly significant correlation existed between the number of beta-adrenoceptors and maximal isoprenaline responses, before (r = 0.99, p less than 0.01) and after (r = 0.96, p less than 0.01) receptor internalization and uncoupling. This correlation between receptor densities and responses was not present in the IDDM group, which also showed elevated levels of plasma adrenaline. This study demonstrates that IDDM subjects have an unaltered mechanism of agonist-promoted beta-adrenoceptor internalization, but indicates a partial dysfunction of the beta-adrenoceptor-coupling to adenylate cyclase.     

1型糖尿病患者中胰岛B细胞特异性锌转运蛋白自身抗体检测标准的建立

目的建立1型糖尿病（Type 1 diabetes,T1DM）患者中一种新的自身抗体即锌转运蛋白8自身抗体（ZnT8-Ab）检测标准,并应用于临床上T1DM的预测与诊断。方法采用放射免疫沉淀法检测T1DM患者中ZnT8-Ab的阳性率,结果用ZnT8-Ab指数来表示。以102名健康对照组ZnT8-Ab指数的99．5％百分位点作为正常上限,检测106例T1DM和126例2型糖尿病（T2DM）患者血清中ZnT8．Ab水平滴度。结果该改进的放射免疫沉淀法批内变异系数（CV）为4．8％-10．7％,批间CV为3．9％-14．1％,阳性临界值为0．015,结果判断重复性为100％,用该方法检测T1DM患者ZnT8．Ab阳性率为39．6％（42／106）,T2DM患者为1．6％（2／126）,正常人为1．0％（1／102）,P〈0．001,结果具有显著性差异。结论此种放射免疫沉淀法检测ZnT8．Ab,其灵敏度高,特异性强,可用来预测以及诊断T1DM,结合糖尿病其它自身抗体可提高T1DM和IADA患者的检出率,可在临床上广泛应用。     

Islet cell transplantation for the treatment of diabetes mellitus

Diabetes mellitus is estimated to affect at least 16 million individuals in the United States and 135 million persons worldwide. It is a significant cause of morbidity and early mortality. The related expenses are astronomical with at least 15% of healthcare expenditures in the United States being used for the treatment of diabetes and its complications, a figure that approaches US$100 billion annually. The Diabetes Control and Complications Trial (DCCT) convincingly showed that intensive glucose management delays the onset and slows the progression of diabetic complications. Numerous studies have shown that pancreas transplantation not only delays the onset and progression of diabetic complications, but in some cases reverses some of the effects of diabetes. Human islet cell transplantation provides an alternative, less invasive alternative to whole organ transplantation. Human islet allotransplantation would only exacerbate the organ shortage, as recipients usually require islets from more than one pancreas. Xenotransplantation of porcine islets is a more attractive option; however, the recipient’s immune response to xenografted tissue would be a formidable obstacle. Microencapsulation of the islets is a method of immunoisolation that would prevent the need for immunosuppressive drugs and the risks associated with their long-term use and have the potential to make xenoislet transplantation a clinical reality.