Measuring adrenal autoantibody response: Interlaboratory concordance in the first international serum exchange for the determination of 21-hydroxylase autoantibodies

n/

The prevalence of diabetes and thyroid related autoantibodies in Sri Lankan children with type 1 diabetes and their unaffected siblings – The utility of a new screening assay

BackgroundThere is limited information about diabetes and thyroid related autoantibodies in children with type 1 diabetes (T1D) or their siblings in Sri Lanka.ObjectivesTo assess in T1D children and their unaffected siblings the prevalence of autoantibodies to (1) glutamic acid decarboxylase (GADA), insulinoma associated antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) using 3 Screen ICA™ (3-Screen) and individual ELISA assays; (2) insulin (IAA); and (3) thyroid peroxidase (TPOA), thyroglobulin (TgA) and the TSH receptor (TSHRA).MethodsWe selected - (a) consecutive T1D children, and (b) their unaffected siblings of both sexes, from the T1D Registry at Lady Ridgeway Hospital, Colombo.ResultsThe median age (IQR) of 235 T1D children and 252 unaffected siblings was 11 (8.4, 13.2) and 9 (5.4, 14.9) years respectively, and the duration of T1D was 23 (7, 54) months. (1) T1D children (a) 79.1% were 3-Screen positive; (b) all 3-Screen positives were individual antibody positive (GADA in 74%; IA-2A 31.1%; ZnT8A 38.7%); (c) and were younger (p=0.01 vs 3-Screen negatives); (d) multiple autoantibodies were present in 45.1%; (e) IA-2A (p=0.002) and ZnT8A (p=0.006) prevalence decreased with T1D duration. (f) TPOA and TgA prevalence was higher in T1D children compared to unaffected siblings (28%, p=0.001 and 31%, p=0.004, respectively). (2) Unaffected siblings (a) 6.3% were 3-Screen positive (p=0.001 vs T1D), and 2.4% were positive for IAA; (b) four subjects had two diabetes related autoantibodies, one of whom developed dysglycaemia during follow-up.ConclusionsThe 3-Screen assay, used for the first time in Sri Lankan T1D children and their siblings as a screening tool, shows a high prevalence of T1D related Abs with a high correlation with individual assays, and is also a helpful tool in screening unaffected siblings for future T1D risk. The higher prevalence of thyroid autoantibodies in T1D children is consistent with polyglandular autoimmunity

THE NATURAL HISTORY OF AUTOIMMUNE ADDISON'S DISEASE FROM THE DETECTION OF AUTOANTIBODIES TO DEVELOPMENT OF THE DISEASE: A LONG FOLLOW-UP STUDY ON 143 PATIENTS

Adrenal cortex autoantibodies (ACA) and/or 21-hydroxylase (21OHAb) are markers of autoimmune Addison's disease (AAD) and progression to overt AAD. The reported cumulative risk of developing AAD varies from 0-90% in different studies. Aim To assess the predictive value of different parameters for progression towards AAD in ACA and/or 21OHAb-positive patients with autoimmune polyendocrine syndromes (APS). Materials and Methods 29 patients with APS-1 and 114 patients with APS-2 or APS-4, were followed-up for a median of 10 years (range 6 months-33 years) and assessed by ACTH test. The risk of AAD was estimated according to age, gender, stage of adrenal dysfunction, associated diseases and antibody titer. Univariate and multivariate Cox proportional hazard models were used for statistical analysis. Results The cumulative risk (CR) of developing AAD was higher in APS-1 patients (94.2%) compared to patients with APS-2/APS-4 (38.7%). The CR was high in both males and females with APS-1 patients, while in patients with APS-2/APS-4 it was high only in males. Stage 1 (increased plasma renin) for patients with APS-1 and Stage 2 (no response of cortisol to ACTH-test) for patients with APS-2/APS-4 were established as the points of no return in the progression to AAD. Adjusted hazard ratio analyses by multivariate Cox model for AAD showed that gender, diseases, adrenal function were independent risk factors for developing clinical AAD. The risk of developing clinical AAD appears to subside after 19 years of follow up. Conclusions A model for estimating the probability to survive free of AAD has been developed and should be a useful tool in designing appropriate follow-up intervals and future therapeutic strategies

From appearance of adrenal autoantibodies to clinical symptoms of addison's disease: Natural history

Abstract Recent progress in the immunopathology field has greatly improved our understanding of the natural history of autoimmune diseases, particularly of Addison's disease. Addison's disease is known to be a chronic illness characterized by adrenocortical gland insufficiency that develops following a long and mainly asymptomatic period, characterized by the presence of circulating autoantibodies directed to adrenal cortex antigens. In this chapter we describe the groups of subjects at risk of developing Addison's disease, together with the diagnostic tests considered the most appropriate for evaluating adrenal function: determination of basal plasma adrenocorticotropic hormone (ACTH) levels, plasma renin activity, plasma aldosterone and cortisol levels, and cortisol levels after intravenous stimulation with ACTH (ACTH test). The employment of specific clinical, immunological and functional criteria in the subjects with autoantibodies to the adrenal cortex allows identifying those at risk of developing overt disease. The independent risk factors for the progression to adrenal failure have also been identified and they contribute to different risks of developing clinical Addison's disease. Based on the risk level, the subjects should be monitored over time to observe early signs of adrenal dysfunction, and start substitutive treatment as soon as possible. For patients presenting with high risk, prevention strategies and trials might be available

Assessment of the shape and molecular size of TSH-TSH receptor complexes

Photoaffinity labelling and analysis under denaturing conditions (SDS-PAGE) have shown that the porcine TSH receptor contains an A subunit (Mr = 47 000) which forms the binding site for TSH and a B subunit (Mr = 25 000) linked to the A subunit by a disulphide bridge. In order to assess the size and shape of the receptor under non-denaturing conditions we have solubilized photoaffinity-labelled porcine TSH receptors using the small micelle-sized detergent sodium deoxycholate and analysed the preparations by sucrose density gradient centrifugation and gel filtration. Under these conditions, the cross-linked TSH-TSH receptor complex showed an s20,w of 6.4 S and a frictional ratio f/f0 of 1.8. These values were consistent with those which might be expected from an elongated protein complex with a molecular weight of about 100 000 (the value obtained by SDS-PAGE). Analysis of another thyroid membrane protein, human thyroid microsomal antigen (Mr = 110 000 by SDS-PAGE) under the same conditions gave an s20,w of 6.0 S and f/f0 = 1.3, suggesting that this protein has a compact structure. The TSH receptor A subunit cross-linked to TSH (Mr = 70000 by SDS-PAGE) gave an s20,w of 4.6 S and f/f0 = 1.8 and these values could be compared with those obtained for the A subunit alone (s20.w =3.6 S; ///q = 1.4; M,, by SDS-PAGE = 47000) and TSH alone (s20,w = 2.6 S; f/f0 = 1.6; Mr = 28000). The data indicate that when TSH (which has an elongated structure) combines with the TSH receptor A subunit (which has a compact structure) the two proteins do not fold into each other extensively but form a structure with an even greater degree of elongation

High prevalence of chronic thyroiditis in patients with polycystic ovary syndrome

Objective: A higher prevalence (26.9% versus 8.3% of controls) of autoimmune thyroiditis (AIT) in polycystic ovary syndrome (PCOS) has been reported in one study to date. We aimed to evaluate the prevalence of clinical, subclinical, potential thyroid autoimmune diseases and other organ-specific autoimmunity in a group of Italian patients with PCOS. Study design: 113 consecutive patients referred to our endocrinology unit as outpatients over 18 months, and diagnosed with PCOS according to the Rotterdam criteria, were included in the study, and 100 age-matched healthy women were enrolled as controls. Each patient was evaluated for family and personal history of autoimmune and non-autoimmune diseases and tested for autoantibodies against thyroperoxidase, thyroglobulin, parietal cells, intrinsic factor, adrenal-cortex, 21-hydroxylase, steroid-producing cells, 17-alpha-hydroxylase, side-chain cleavage enzyme, islet-cells, glutamic-acid decarboxylase, nuclei and mitochondria. All patients had serum TSH, FT4 and FT3 tested and patients with thyroid autoantibodies and/or abnormal TSH levels had an ultrasound thyroid scan. An oral glucose tolerance test and measurements of serum anti-Mullerian hormone (AMH) and inhibin B levels were carried out. Results: AIT was present in 30/113 (27%) patients compared with 8% of controls (p < 0.001). Subclinical hypothyroidism was detected in 13/30 (43%) patients with All; the remaining patients had normal thyroid function. The prevalence of non-thyroid autoantibodies in PCOS patients was not different from controls. AMH concentration was higher in PCOS patients compared to controls, but there was no difference between AIT and non-AIT groups. Conclusions: The prevalence of AIT in patients with PCOS was significantly higher than in controls. No other autoimmune diseases were associated with PCOS. This observation suggests that PCOS patients should be screened for AIT

Bridging-type enzyme-linked immunoassay for zinc transporter 8 autoantibody measurements in adult patients with diabetes mellitus

Aims A bridging-type ELISA for measuring autoantibodies to zinc transporter 8 (ZnT8A) was assessed using samples from different forms of diabetes mellitus. Methods ZnT8A were measured using an ELISA in patients with type 1 diabetes mellitus (T1DM; n = 94), latent autoimmune diabetes of adulthood (LADA; n = 51), type 2 diabetes mellitus (T2DM; n = 59) and healthy blood donors (HBD; n = 200). ZnT8A in ELISA and immunoprecipitation assays (IPA) using ZnT8 dimer (W325/R325) and monomers (W325, R325 and Q325) were compared. Results Inter- and intra-assay coefficients of variation (CV) were 7.1% and 1.7%, respectively (medium ZnT8A) and 8.5% and 2.7%, respectively (high ZnT8A). In the ELISA 51/94 (54.3%) T1DM, 16/51 (31.4%) LADA and 1/59 (1.7%) T2DM sera were ZnT8A positive. ROC analysis of T1DM and HBD for the ELISA showed 54% sensitivity and 99% specificity (cutoff 15 u/mL) and AUC 0.80 (95% CI, 0.74–0.86). ELISA and IPA measurements were in very good agreement (r = 0.856, k = 0.889, n = 204). Measurement of ZnT8A in addition to autoantibodies for GAD, IA-2 and insulin increased antibody positivity in T1DM by 4.3%, from 80.9% to 85.1%. Conclusions The bridging-type ELISA is a convenient and reproducible method for determination of ZnT8A in serum. Measurement of ZnT8A increased autoantibody positivity in adult T1DM

Evaluation of the RSR 3 screen ICA™ and 2 screen ICA™ as screening assays for type 1 diabetes in Sweden

Aim: The study aim was to evaluate the RSR 3 Screen ICA™ and 2 Screen ICA™ for detection of islet cell autoimmunity in healthy Swedish subjects and patients with newly diagnosed type 1 diabetes (T1D). Methods: 3 Screen is designed for combined detection of autoantibodies to glutamic acid decarboxylase (GADA), to the islet antigen IA-2 (IA-2A) and to zinc transporter 8 (ZnT8A), while 2 Screen detects GADA and IA-2A. Serum samples from 100 T1D patients at onset and 200 healthy controls were studied. Results: 3 Screen achieved 93% assay sensitivity and 97.5% specificity, while 2 Screen achieved 91% assay sensitivity and 98.5% specificity. Samples were also tested in assays for individual autoantibodies. There was only one 3 Screen positive healthy control sample (0.5%) that was positive for multiple autoantibodies (IA-2A and ZnT8A). In contrast, most of the 93 3 Screen positive patients were positive for multiple autoantibodies with 72% (67/93) positive for both GADA and IA-2A and 57% (53/93) positive for three autoantibodies (GADA, IA-2A and ZnT8A). Insulin autoantibodies (IAA, measured by radioimmunoassay) were positive in 13 patients and two healthy controls. Conclusion: 3 Screen achieved high sensitivity and specificity, suitable for islet cell autoimmunity screening in a healthy population. In the case of 3 Screen positivity, further assays for GADA, IA-2A and ZnT8A are required to check for multiple autoantibody positivity, a hallmark for progression to T1D. In addition, testing for IAA in children below two years of age is warranted