Antibodies to the protein tyrosine phosphatases IAR and IA-2 are associated with progression to insulin-dependent diabetes (IDDM) in first-degree relatives at-risk for IDDM.

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.

The Melbourne Pre-Diabetes Study: prediction of type 1 diabetes mellitus using antibody and metabolic testing.

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.

Cloning and characterization of islet cell antigen-related protein-tyrosine phosphatase (PTP), a novel receptor-like PTP and autoantigen in insulin-dependent diabetes.

Cloning of the cDNA encoding a novel human protein- tyrosine phosphatase (PTP) called islet cell antigen-related PTP (IAR) predicts a receptor-like molecule with an extracellular domain of 614 amino acids containing a hydrophobic signal peptide, one potential N-glycosylation site, and an RGDS peptide which is a possible adhesive recognition sequence. The 376-amino acid intracellular region contains a single catalytic domain. Recombinant IAR polypeptide has phosphatase activity. Northern blot analysis shows tissue-specific expression of two IAR transcripts of 5.5 and 3. 7 kilobases, which are most abundant in brain and pancreas. The IAR PTP is homologous in its intracellular region to IA-2, a putative PTP that is an insulin-dependent diabetes mellitus (IDDM) autoantigen. IAR is also reactive with IDDM patient sera. IAR and IA-2 may distinguish different populations of IDDM autoantibodies since they identify overlapping but nonidentical sets of IDDM patients. Thus IAR is likely to be an islet cell antigen useful in the preclinical screening of individuals for risk of IDDM.

Cytokine regulation of glutamate decarboxylase biosynthesis in isolated rat islets of Langerhans.

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.

Islet cell antibodies and antibodies against glutamic acid decarboxylase in newly diagnosed adult-onset diabetes mellitus.

This study aimed to determine the prevalence of antibodies against glutamic acid decarboxylase (anti-GAD) and islet cell antibodies (ICA) in relation to beta-cell function in adults newly-diagnosed with diabetes mellitus. beta-cell function was assessed in adults aged 25-70 years newly-diagnosed with diabetes mellitus (n = 84) and control subjects (n = 34) using a 1.6 MJ mixed meal test procedure. beta-cell function was evaluated by the true insulin (defined as immunoreactive insulin minus proinsulin) response to the mixed meal test. Subjects were classified on the basis of the area under the true insulin curve (normal 16830-107700 pmol min/I) and the sum of the 30 and 60 min incremental response (normal 285-3295 pmol/I). The prevalence of anti-GAD and ICA was determined using radioimmunoprecipitation and indirect immunofluorescence, respectively. Twelve (14%) of the study cohort were insulin deficient showing little or no true insulin release. Of the insulin deficient individuals, seven (58%) subjects were anti-GAD antibody positive, compared with eleven (15%) of the subjects without insulin deficiency (P < 0.001). Seven (58%) insulin deficient subjects were ICA positive, whereas only two (3%) non-insulin deficient subjects were ICA positive (P < 0.001). Eight (67%) of the insulin deficient individuals had anti-GAD or ICA, compared with twelve (17%) of those who were not insulin deficient (P < 0.001). The positive predictive values for insulin deficiency of anti-GAD and ICA were 39 and 78% respectively. The sensitivity of both antibodies for detecting insulin deficiency was 50%. The specificity for detecting insulin deficiency was 85% for anti-GAD and 97% for ICA. Positivity for both anti-GAD and ICA gave a specificity and positive predictive value for insulin deficiency of 99%, and a sensitivity of 50%. Nearly one in seven adults presenting with diabetes mellitus as a new diagnosis are insulin deficient using our criteria. Loss of beta-cell function in two thirds of individuals who are insulin deficient can be identified by anti-GAD and ICA. Early detection of these immune markers of beta-cell damage creates the potential for immune modulation to limit such damage.

Disease sensitivity and specificity of 52 assays for glutamic acid decarboxylase antibodies. The Second International GADAB Workshop.

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)

Do glutamic acid decarboxylase antibodies improve the prediction of IDDM in first-degree relatives at risk for IDDM?

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.

High level of concordance between assays for glutamic acid decarboxylase antibodies. The First International Glutamic Acid Decarboxylase Antibody Workshop.

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)

Antibodies to glutamic acid decarboxylase in at-risk and clinical insulin-dependent diabetic subjects: relationship to age, sex and islet cell antibody status, and temporal profile.

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.

Inverse relation between humoral and cellular immunity to glutamic acid decarboxylase in subjects at risk of insulin-dependent diabetes.

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.

Plasma proinsulin in recently diagnosed type 2 diabetes mellitus.

We measured levels of immunoreactive insulin (IRI) and proinsulin using a sensitive and specific immunoradiometric assay (IRMA), in non-obese recently diagnosed type 2 diabetic subjects. The proinsulin IRMA showed full cross reaction with intact proinsulin, des 31-32 proinsulin and des 64-65 proinsulin, but no reaction with insulin or C-peptide. In a group of 41 recently diagnosed non-obese Caucasian type 2 diabetic subjects (study group), mean fasting proinsulin levels were greater than that of 40 age and body mass index (BMI) matched controls (study group 17.4 +/- 2.0, controls 10.1 +/- 0.9 pmol-1, P < 0.001). Following a standard test meal, 30 and 60 min levels were not significantly different between groups but 90- and 120-min proinsulin levels were elevated in the study group. The maximum proinsulin to IRI ratio was 20% in the fasting state and did not differ between study and control groups. Proinsulin profiles were similar in subjects receiving oral hypoglycaemic agents and those on dietary treatment alone. Proinsulin did not correlate with indices of glycaemic control, total cholesterol, triglycerides or HDL cholesterol, but a relationship was observed with IRI before and after the meal. These results suggest that increased secretion of insulin precursors of low biological activity occurs in non obese recently diagnosed type 2 diabetic subjects under reasonably good glycaemic control; in such individuals the elevated levels of these precursors are approximately in proportion to the increase in IRI and are less than those reported in some previous studies.

Aminopropylidine diphosphonate (APD) in mild primary hyperparathyroidism: effect on clinical status.

Mild hypercalcaemia associated with primary hyperparathyroidism has been increasingly recognized with the use of automated biochemical screening. Management is often difficult as symptoms are often absent or non-specific. Accordingly, we employed the hypocalcaemic effect of the diphosphonate APD to assess the effect of an acute fall in plasma calcium on indices of general well being, blood pressure, and vasoactive hormones in patients with mild primary hyperparathyroidism. Ten patients were studied in a randomized single blind, placebo-controlled cross-over study, using 30 mg APD intravenously or control saline infusion, over 2 h. Metabolic measurements, formal tests of muscle strength and cognitive function, and a standardized questionnaire were assessed 7 days after infusions. Albumin corrected plasma calcium was significantly lower (mean 2.49 +/- 0.04 SEM mmol/l) after APD when compared to control values (2.70 +/- 0.06 mmol/l, P less than 0.001). Twenty-four-hour urinary calcium, plasma magnesium and absolute monocyte count decreased significantly, whereas plasma parathyroid hormone increased after APD (P less than 0.05). There was no significant change in hypercalcaemic symptoms, muscle strength or cognitive function, and blood pressure, renin, aldosterone and atrial natriuretic peptide did not change. Side-effects, when they occurred, were mild. It is concluded that APD is a safe and effective means of lowering plasma calcium in mild primary hyperparathyroidism, but these acute reductions are associated with little or no improvement in clinical status in these patients.

Plasma corticotrophin-releasing factor and vasopressin responses to hypoglycaemia in normal man.

The plasma cortisol, ACTH, AVP and corticotrophin-releasing factor (CRF) responses to insulin-induced hypoglycaemia were investigated in six normal men using a controlled, randomized, cross-over design. Hormonal concentrations were determined following insulin or saline injection. The maximum cortisol response was seen at 90 min while plasma ACTH, AVP and CRF concentrations peaked at 45 min following insulin injection. The responses of the insulin-treated and control groups were compared by assessing the incremental response from baseline (pre-injection) to peak hormone levels. A significant increase was observed for each hormone following insulin injection. The mean of the incremental responses between 30 and 120 min in each subject was also statistically greater for each hormone in the insulin-treated group when compared with the control group. These results are consistent with the hypothesis that AVP and CRF are both physiological mediators of ACTH secretion induced by a hypoglycaemic stress.

ACTH deficiency: problems in recognition and diagnosis.

Corticotrophin (ACTH) deficiency is an important cause of a potentially lethal form of adrenocortical failure. Difficulties can arise in making the diagnosis, especially when secretion of other pituitary trophic hormones is normal. Presenting features of seven patients with ACTH deficiency, in whom the diagnosis was difficult for a variety of reasons, are reported and discussed. Two patients had a normal cortisol response to synthetic ACTH. The possibility of ACTH deficiency should be considered in any patient presenting with weight loss, vomiting, muscular fatigue and stiffness, hyponatraemia or hypoglycaemia.