Molecular manipulation of autoantibody testing in type 1 diabetes: two for one.
Explore the source record for details and available documents.
Autoantibodies↗
Biomedical subjects
A Lernmark
Publications and source records attributed to A Lernmark.
Alternative splicing of GAD67 results in the synthesis of a third form of glutamic-acid decarboxylase in human islets and other non-neural tissues.
Two forms of glutamic-acid decarboxylase (GAD) have been identified in mammalian tissues: a 65-kDa form (GAD65) and a 67-kDa form (GAD67). Alternate splicing produces one or two smaller variants of GAD67 in the brain of embryonic mice and rats. Additionally, a short, heretofore unidentified transcript homologous to GAD67 has been detected in human testis RNA. Because GAD, the enzyme responsible for gamma-aminobutyric acid production and a key autoantigen in type I diabetes, has unclear function in non-neural tissue, it is important to understand its pattern of expression. Unlike GAD65, GAD67 is not produced in human pancreatic islets. Here, we describe a novel splice variant of GAD67 that is produced in human islets, testis, adrenal cortex, and perhaps other endocrine tissues, but not in brain. This transcript directs the synthesis of a protein without GAD enzymatic activity: GAD25. A unique peptide sequence at the carboxyl terminus of GAD25 is highly conserved between mice, rats, and humans. We conclude that humans produce a third form of GAD in non-neural tissues and that human islets, although they do not synthesize full-length GAD67, do express this shortened variant.
Alternative Splicing↗
Rapid-onset type 1 diabetes with pancreatic exocrine dysfunction.
Explore the source record for details and available documents.
Adult↗
The World Health Organization International Collaborative Study for islet cell antibodies.
AIMS/HYPOTHESIS: Islet cell autoantibodies are a specific marker for Type 1 (insulin-dependent) diabetes mellitus. Standardisation of islet cell antibodies and the uniform reporting in International units is critical to research and the development of assays for islet cell autoantibodies as diagnostics. METHODS: The suitability of a candidate serum to serve as the international standard for islet cell antibodies was studied by 19 participants in 8 countries. In addition, the purpose was to investigate whether the serum could also serve as a standard for antibodies to the 65000 Mr isoform of glutamic acid decarboxylase (GAD65) and islet antigen-2 (IA-2). Control sera were included in the study to assess the validity of the various assay systems. The sera were lyophilized to World Health Organization criteria and the candidate serum assigned the ampoule code number 97/550. RESULTS: The use of 97/550 was shown to notably reduce inter laboratory variability in the measurement of islet cell antibodies. In addition, there was a pronounced reduction in inter laboratory variability in the measurement of GAD65 and IA-2 antibodies. CONCLUSIONS/INTERPRETATION: On the basis of the results reported here and with agreement of the participants, the preparation 97/550 has been established by the World Health Organization Expert Committee on Biological Standards for establishment as the first international standard for islet cell antibodies, with an assigned potency of 20 international units. In addition, 97/550 can serve as an international reference reagent for specific GAD65 antibodies, with an assigned potency of 100 units. It can also serve as a National Institute of Biological Standards and Control (NIBSC) reference reagent for IA-2 antibodies for evaluation of assays for this material.
Autoantibodies↗
Evaluation of a novel radioimmunoassay using 125I-labelled human recombinant GAD65 for the determination of glutamic acid decarboxylase (GAD65) autoantibodies.
Autoantibodies to the islet cell 65-kilodalton isoform of glutamic acid decarboxylase (GAD65) are present in most patients with type 1 diabetes mellitus years before the clinical manifestation of the disease. GAD65 autoantibodies are also present in a subset of patients with type 2 diabetes who frequently become insulin dependent. In the present study, we evaluated a new, commercially available radioimmunoprecipitation assay for measuring GAD65 autoantibodies using 125I-labelled human recombinant GAD65. Results obtained with this assay were compared with those obtained by a reference assay based on 35S-labelled recombinant GAD65. Analyses were performed on 67 patients with type I diabetes, 350 with type 2 diabetes, and 150 apparently healthy individuals. An excellent agreement was found between the results obtained by the 125I-GAD65 assay and those obtained by the reference method. The receiver operating characteristic (ROC) curve analysis was used to evaluate the sensitivity and specificity of the two assays. The sensitivity of each assay was determined from the results of the 67 type 1 patients, while the specificity was based on the 150 healthy individuals. Based on the ROC curves, the two assays appeared identical, with a sensitivity of 84% and a clinical specificity of 98%. In conclusion, based on our results, this simple, one-step centrifugation, high-capacity 125I-GAD65 assay has the same sensitivity and specificity as the reference assay and is highly suitable to detect GAD65 autoantibodies in human samples.
Antibody Specificity↗
Ketoacidosis in young adults is not related to the islet antibodies at the diagnosis of Type 1 diabetes mellitus--a nationwide study.
AIMS: To test the hypothesis that there is lower prevalence of islet antibodies in subjects with newly diagnosed Type 1 diabetes mellitus in young adulthood than in children is associated with less severe diabetes at time of diagnosis. METHODS: This investigation was based on a nationwide study (Diabetes Incidence Study in Sweden) of 15-34-year-old newly diagnosed diabetic subjects. During 1992-1993, all diabetic subjects (excluding secondary and gestational diabetes) were reported on standardized forms, with information about clinical characteristics at diagnosis. The study examined islet cell antibodies (ICA) by indirect immunofluorescence, and autoantibodies to glutamic acid decarboxylase (GADA), tyrosine phosphatase-like antigen (IA-2A) and insulin (IAA) as well as C-peptide by radioimmunoassay. RESULTS: Blood samples were available from 78 patients with diabetic ketoacidosis (DKA) and 517 non-acidotic patients. The prevalence of ICA (63% vs. 57%), GADA (63% vs. 66%), IA-2A (35% vs. 44%) and IAA (20% vs. 15%) were very similar in patients with or without DKA. The median levels of the four autoantibodies did not differ between the two groups. High blood glucose (P < 0.001) and low C-peptide levels (P < 0.001) were the only parameters found to be related to DKA. CONCLUSIONS: The similarities in findings of newly diagnosed diabetic patients with or without DKA regarding ICA, GADA, IA-2A and IAA suggest that there is no relationship between the expression of antigenicity and the severity of beta-cell dysfunction. The lower prevalence of the four autoantibodies in 15-34-year-old diabetic subjects compared with previous findings in children is not explained by misclassification of diabetes type.
Adolescent↗
Newly diagnosed latent autoimmune diabetes in adults (LADA) is associated with low level glutamate decarboxylase (GAD65) and IA-2 autoantibodies. Diabetes Incidence Study in Sweden (DISS).
A quantitative assay with microSepharose was used to determine GAD65Ab and IA-2Ab levels in 771 population-based patients diagnosed with diabetes mellitus at 15 to 34 years of age, and in 828 matched controls. Among the patients, 587 (76%) were classified with type I, 108 (14%) with type II, and 76 (10%) with unclassifiable diabetes. The levels above normal demonstrated a prevalence of GAD65Ab in 66% of type I diabetes, 50% of type II diabetes and 54% of unclassifiable patients and for IA-2Ab in 40%, 17% and 21%, respectively. Among the autoantibody-positive sera, the LADA patients had a lower GAD65Ab index (median 0.19, p < 0.0001) and IA-2Ab index (median 0.28, p < 0.0001) than the type I patients (median 0.37 and 0.66). Patients with unclassifiable diabetes had a GAD65Ab (median 0.43) or IA-2Ab (median 0.63) index which was not different from the type I diabetes patients. Our data demonstrate that young adult new-onset LADA patients have low level GAD65Ab and IA-2Ab. The low-level autoantibodies may signify a less aggressive beta-cell autoimmunity, which may explain why these patients are often classified with type II or non-insulin-dependent diabetes.
Adolescent↗
Rel B is an early marker of autoimmune islet inflammation in the biobreeding (BB) rat.
Because the development of insulitis and diabetes is predictable in Lyp/Lyp congenic BB rats, we have characterized early islet inflammation in these rats to determine the cell subsets involved in the onset of autoimmune insulitis. Pancreas sections from prediabetic Lyp/Lyp, Lyp/+ and +/+ rats were analyzed by immunohistochemistry. We found W3/25+ cells in the exo- and endocrine tissue from all three genotypes, but intraislet insulitis was never found in Lyp/+ or +/+ rats. The onset of massive, intraislet B- and T-cell infiltration in Lyp/Lyp rats was preceded by Rel B+ cells in and around the islets, followed by ED1+ monocytes/macrophages. Rel B+ cells were more frequent in the parafollicular cortex of pancreatic lymph nodes from Lyp/Lyp than from Lyp/+ and +/+ rats. In the Lyp/Lyp thymus, we found significantly increased expression of IL-12p40 messenger RNA (mRNA; p<0.001), located in the Rel B-protein-rich corticomedullary junction. The NF-KB/Rel B complex specifically transactivates genes involved in antigen presentation in dendritic cells. Rel B+ cells in the islets may therefore mark the onset of autoimmune insulitis and antigen-specific activation of autoreactive T cells in the lymph nodes of diabetes prone Lyp/Lyp BB rats. In the thymus, Rel B+ cells may support the Lyp-dependent development of self-reactive thymocytes by activation of cytokine expression.
Age Factors↗
Prognostic factors for the course of beta cell function in autoimmune diabetes.
This study presents a 2-yr follow-up of 281 patients, aged 15-34 yr, diagnosed with diabetes between 1992 and 1993. At diagnosis, 224 (80%) patients were positive for at least one of the following autoantibodies: islet cell antibodies (ICAs), glutamic acid decarboxylase antibodies (GADAs), or tyrosine phosphatase antibodies (IA-2As); the remaining 57 (20%) patients were negative for all three autoantibodies. At diagnosis, C-peptide levels were lower (0. 27; 0.16-0.40 nmol/L) in autoantibody-positive patients compared with autoantibody-negative patients (0.51; 0.28-0.78 nmol/L; P: < 0. 001). After 2 yr, C-peptide levels had decreased significantly in patients with autoimmune diabetes (0.20; 0.10-0.37 nmol/L; P: = 0. 0018), but not in autoantibody-negative patients. In patients with autoimmune diabetes, a low initial level of C-peptide (odds ratio, 2. 6; 95% confidence interval, 1.7-4.0) and a high level of GADAs (odds ratio, 2.5; 95% confidence interval, 1.1-5.7) were risk factors for a C-peptide level below the reference level of 0.25 nmol/L 2 yr after diagnosis. Body mass index had a significant effect in the multivariate analysis only when initial C-peptide was not considered. Factors such as age, gender, levels of ICA or IA-2A or insulin autoantibodies (analyzed in a subset of 180 patients) had no effect on the decrease in beta-cell function. It is concluded that the absence of pancreatic islet autoantibodies at diagnosis were highly predictive for a maintained beta-cell function during the 2 yr after diagnosis, whereas high levels of GADA indicated a course of decreased beta-cell function with low levels of C-peptide. In autoimmune diabetes, an initial low level of C-peptide was a strong risk factor for a decrease in beta-cell function and conversely high C-peptide levels were protective. Other factors such as age, gender, body mass index, levels of ICA, IA-2A or IAA had no prognostic importance.
Adolescent↗
Recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different GAD antibody-positive phenotypes.
Autoantibodies against the smaller isoform of glutamic acid decarboxylase (GAD) are markers for Type 1 diabetes. GAD65 autoantibody (GAD65Ab)-positive individuals in the general population are, however, mostly at low risk of developing Type 1 diabetes, suggesting that GAD65Ab phenotypes may be associated with different underlying pathogenic processes. The aim of this study was to test the hypothesis that Type 1 diabetes patients (n = 243; group I), GAD65Ab-positive healthy individuals (n = 28; group II), and healthy first-degree relatives of Type 1 diabetes patients (n = 41; group III) have antibody phenotypes that recognize different GAD65 epitopes. Sera from groups I-III were tested for their binding to GAD65 and GAD67, as well as six different GAD65/67 fusion proteins. Regardless of group, sera reactive to both GAD65 and GAD67 showed broader epitope reactivity than GAD65-specific sera. Furthermore, Type 1 diabetes patients showed a more restricted epitope binding than healthy individuals and first-degree relatives, demonstrating significantly less binding to the N-terminal part of GAD65 and to GAD67. Our analysis demonstrates that the N-terminal part is essential for full antibody binding to GAD65, in particular, to the middle epitope. It is suggested that Type 1 diabetes is associated with restricted GAD65Ab epitope specificity.
Adolescent↗
Antibodies to glutamic acid decarboxylase and peripheral nerve function in type 1 diabetes.
Autoimmune mechanisms have been implicated in the pathophysiology of diabetic neuropathy. We studied the association between glutamic acid decarboxylase (GAD65) and islet cell (IA-2) autoantibodies as well as autoantibodies to the autonomic nervous system and peripheral nerve function in recent onset type 1 diabetes. Thirty-seven patients (27 females and 10 males) enrolled 2-22 months after diagnosis. Humoral factors, glycemic control, and peripheral nerve function were measured annually for 3 yr. Patients with high GAD65Ab had worse glycemic control and higher insulin requirements. Patients with high GAD65Ab had slower motor nerve conduction velocities in the median, ulnar, and peroneal nerves (P < 0.025 for each nerve). The mean motor nerve conduction velocity Z scores at the time of the third evaluation was 0.341 +/- 0.25 for the low GAD65Ab patients and -0.600 +/- 0.25 for the high GAD65Ab patients (P < 0.01). Similar differences between the low and high GAD65Ab groups were observed for F wave latencies, thermal threshold detection, and cardiovascular autonomic function. The composite peripheral nerve function Z scores in the low GAD65Ab patients were 0.62 +/- 11, 0.71 +/- 0.19, and 0.21 +/- 0.14 at the first, second, and third evaluations, significantly different from those in the high GAD65Ab patients in whom they were -0.35 +/- 0.15, -0.46 +/- 0.18, and -0.42 +/- 0.16 (P < 0.001). In summary, GAD65Ab in patients with recent onset type 1 diabetes are associated with worse glycemic control and slightly worse peripheral nerve function. Although the latter remained within normal limits and none of the patients had clinical neuropathy, the GAD65Ab-related differences in composite peripheral nerve function were highly significant (P < 0.001) and could not be attributed to GAD65Ab-related differences in glycemic control.
Adolescent↗
GAD65-specific autoantibodies enhance the presentation of an immunodominant T-cell epitope from GAD65.
GAD65 autoantibodies (GAD65Ab) are highly prevalent in type 1 diabetes, but their functional role in the pathogenesis of the disease and their relationship to T-cell reactivity to GAD65 is still unclear. We tested the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. T-cell hybridoma T33.1, which recognizes the GAD65 274-286 epitope in the context of HLA-DRB 1*0401, was incubated with antigen-presenting cells exposed to recombinant human GAD65 alone or complexed with GAD65Ab' or GAD65Ab- sera. Stimulation of the T33.1 hybridoma was greatly enhanced by multiple GAD65Ab+ sera. The enhancement effect was most prominent with sera from patients with high GAD65 autoantibody levels. Sera from GAD65Ab- subjects had no effect. The correlation between T-cell stimulation and GAD65Ab levels was not absolute, suggesting that other variables such as autoantibody recognition of different regions of GAD65 and variable effects on processing of the 274-286 epitope may contribute. Uptake of antibody-complexed GAD65 was Fc receptor (FcR)-mediated because the enhancement of presentation was inhibited by monoclonal antibodies against FcR. Our results support the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. Increased antigen uptake and heterogeneity in the autoantibody specificity may provide a mechanism for antibody-facilitated T-cell response influencing the progression of type 1 diabetes.
Adolescent↗
Stability of disease-associated antibody titers in pregnant women with type 1 diabetes with or without residual beta-cell function.
Explore the source record for details and available documents.
Adolescent↗
The length of the CTLA-4 microsatellite (AT)N-repeat affects the risk for type 1 diabetes. Diabetes Incidence in Sweden Study Group.
CTLA-4 is important to down-regulating T cell responses and has been implicated in type 1 (insulin dependent) diabetes mellitus in both linkage and association studies. The aim of our study was to relate the polymorphic (AT)n microsatellite in the 3' untranslated sequence of the CTLA-4 gene to diabetes risk. We studied 616 consecutively diagnosed 0-34 year-old Swedish patients and 502 matched controls by PCR-based genotyping fo determine the length of the 3'-end (AT)n repeat region of the CTLA-4 gene and categorizing alleles as predominantly monomorphic short (S) or highly polymorphic (in length) long (L) alleles. The odds of type 1 diabetes of subjects with the L/L genotype was estimated to be 1.84 times that of subjects with the S/S genotype (95% CI 1.44-2.73, p=0.002). Further analysis of the long alleles, partitioned into intermediate (I) length and very long (VL) alleles, suggested that L alleles act recessively in conferring diabetes risk (p=0.0009). This study suggests that the 3'-end (AT)n repeat region of the CTLA-4 gene represents a recessive risk factor for type 1 diabetes.
Abatacept↗
A diabetogenic gene prevents T cells from receiving costimulatory signals.
T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.
Animals↗
[Vaccination against type 1 diabetes at the clinical trial stage].
The occurrence of type I diabetes is associated with autoimmunity against insulin, glutamate decarboxylase (GAD65) and the transmembrane protein IA-2. Experiments in the spontaneously diabetic NOD mouse and the BB rat have shown early autoantigen treatment to confer protection from diabetes. The question remains of whether human type I diabetes can be prevented by autoantigen 'vaccination'.
Animals↗
Islet autoantibodies in cord blood from children who developed type I (insulin-dependent) diabetes mellitus before 15 years of age.
Islet autoantibodies are early markers for Type I (insulin-dependent) diabetes mellitus. The aim of this study was to establish whether islet autoantibodies were present at birth in children who developed Type I diabetes before 15 years of age. Cord blood sera from 81 children who developed Type I diabetes between 10 months and 14.9 years of age were tested for glutamic acid decarboxylase autoantibodies (GAD65Ab), islet cell antigen 512 autoantibodies (ICA512Ab), insulin autoantibodies (IAA) all by quantitative radioligand binding assays and islet cell autoantibodies (ICA) by indirect immunofluorescence. Cord blood sera from 320 randomly selected matched children were controls. The children who developed Type I diabetes had an increased frequency of cord blood islet autoantibodies compared with control subjects: Glutamic acid decarboxylase autoantibodies were detected in 6% (5/81) patients and 2% (5/320) control subjects (p = 0.03); islet cell antigen 512 autoantibodies in 5% (4/73) patients and 1% (4/288) control subjects (p = 0.06); insulin autoantibodies (IAA) in 0% (0/79) patients and 0.3% (1/320) control subjects (p = 0.36); and islet cell autoantibodies in 10% (8/81) patients compared with 0.6% (2/320) control subjects (p = 0.0001). Taken together, 17% (14/81) patients had one or more islet autoantibody compared with 4% (12/320) control subjects (p = 0.0001). Whereas none of the control children had more than one antibody, 4% (3/81) children who later developed Type I diabetes were double positive (p = 0.002). Although glutamic acid decarboxylase autoantibodies' concentrations in cord-blood correlated to those in the mothers' blood at the time of delivery, no corresponding correlation was found for the other two types of autoantibodies. The increased frequency of cord blood islet autoantibodies suggests that the Type I diabetes process could already be initiated in utero.
Adolescent↗
Towards a World Health Organization (WHO) approved standard sample for islet cell antibodies, GAD65 and IA-2 autoantibodies.
Explore the source record for details and available documents.
Autoantibodies↗