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.

Background: There is limited information about diabetes and thyroid related autoantibodies in children with type 1 diabetes (T1D) or their siblings in Sri Lanka.

Objectives: To 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).

Methods: We selected - (a) consecutive T1D children, and (b) their unaffected siblings of both sexes, from the T1D Registry at Lady Ridgeway Hospital, Colombo.

Cryo-electron microscopy structures of human thyroid peroxidase (TPO) in complex with TPO antibodies.

Determination of the structure of the extracellular domain of human thyroid peroxidase (hTPO) by cryo-electron microscopy (cryo-EM) is described. TPO, purified to homogeneity was complexed with the hTPO monoclonal autoantibody 2G4 Fab and also with a mouse monoclonal TPO antibody 4F5 Fab (which competes with autoantibody binding to TPO). Both complexes were analysed by cryo-EM.

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.

TSH receptor specific monoclonal autoantibody K1-70 targeting of the TSH receptor in subjects with Graves' disease and Graves' orbitopathy-Results from a phase I clinical trial.

Objectives: In Graves' disease (GD), autoantibodies to the thyroid stimulating hormone receptor (TSHR) cause hyperthyroidism. The condition is often associated with eye signs including proptosis, oedema, and diplopia (collectively termed Graves' orbitopathy [GO]). The safety profile of K1-70 (a human monoclonal TSHR specific autoantibody, which blocks ligand binding and stimulation of the receptor) in patients with GD was evaluated in a phase I clinical trial.

Characteristic phenotype of Chinese patients with adult-onset diabetes who are autoantibody positive by 3-Screen ICA™ ELISA.

Aims: To assess the prevalence of diabetes-associated autoantibodies in Chinese patients recently diagnosed with adult-onset diabetes and to evaluate the potential role of the autoantibody markers for characterization of disease phenotype in the patient population.

Methods: The study included 1273 recent-onset adult patients with phenotypic type 2 diabetes mellitus (T2DM). Serum samples were tested using the 3-Screen ICA™ ELISA (3-Screen) designed for combined measurement of GADAb and/or IA-2Ab and/or ZnT8Ab.

Preclinical studies on the toxicology, pharmacokinetics and safety of K1-70 a human monoclonal autoantibody to the TSH receptor with TSH antagonist activity.

Background: The human monoclonal autoantibody K1-70™ binds to the TSH receptor (TSHR) with high affinity and blocks TSHR cyclic AMP stimulation by TSH and thyroid stimulating autoantibodies.

Methods: The preclinical toxicology assessment following weekly intravenous (IV) or intramuscular (IM) administration of K1-70™ in rats and cynomolgus monkeys for 29 days was carried out. An assessment of delayed onset toxicity and/or reversibility of toxicity was made during a further 4 week treatment free period.

Analysis of diabetes-associated autoantibodies in children and adolescents with autoimmune thyroid diseases.

Background Zinc transporter 8 autoantibodies (ZnT8Abs) together with glutamic acid decarboxylase autoantibodies (GADAbs), insulinoma antigen 2 autoantibodies (IA-2Abs) and insulin autoantibodies (IAbs) are markers of type 1 diabetes mellitus (T1DM). We studied the prevalence of ZnT8Ab in children with autoimmune thyroid diseases (AITDs) to assess the association of AITDs and T1DM at the serological level. Methods The study groups consisted of 44 children with Graves' disease (GD), 65 children with Hashimoto's thyroiditis (HT), 199 children with T1DM with or without AITDs and 58 control children.

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain.

The crystal structures of the thyroid-stimulating hormone receptor (TSHR) leucine-rich repeat domain (amino acids 22-260; TSHR260) in complex with a stimulating human monoclonal autoantibody (M22TM) and in complex with a blocking human autoantibody (K1-70™) have been solved. However, attempts to purify and crystallise free TSHR260, that is not bound to an autoantibody, have been unsuccessful due to the poor stability of free TSHR260. We now describe a TSHR260 mutant that has been stabilised by the introduction of six mutations (H63C, R112P, D143P, D151E, V169R and I253R) to form TSHR260-JMG55TM, which is approximately 900 times more thermostable than wild-type TSHR260.

The natural history of autoimmune Addison's disease from the detection of autoantibodies to development of the disease: a long-term follow-up study on 143 patients.

Background: Adrenal cortex autoantibodies (ACAs) 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 to 90% in different studies.

Aim: To assess the predictive value of different parameters in the progression toward AAD in patients with ACA and/or 21OHAb-positive patients with autoimmune polyendocrine syndromes (APS).

Thyrotrophin receptor antibody concentration and activity, several years after treatment for Graves' disease.

Objective: TSH receptor antibodies (TRAb) are responsible for autoimmune hyperthyroid disease (Graves' disease; GD) with TRAb levels tending to decrease following treatment. Measurement of TRAb activity during follow-up could prove valuable to better understand treatment effectiveness.

Study Design: TRAb concentration and stimulating (TSAb) and blocking (TSBAb) activity of patient serum were assessed following different treatment modalities and follow-up length.

A new ELISA for autoantibodies to steroid 21-hydroxylase.

Background: A new ELISA for autoantibodies to steroid 21-hydroxylase (21-OH Ab) is described.

Methods: In the assay test sample autoantibodies form a bridge between 21-OH coated onto the plate well and liquid phase 21-OH-biotin. Bound 21-OH-biotin is detected by the addition of streptavidin peroxidase and colorogenic peroxidase substrate.

Assessment of autoantibodies to interferon-ω in patients with autoimmune polyendocrine syndrome type 1: using a new immunoprecipitation assay.

Background: Measurements of autoantibodies to interferon-ω (IFN-ω) in patients with autoimmune polyglandular syndrome type 1 (APS-1) were performed using a new immunoprecipitation assay (IPA) based on 125I-labeled IFN-ω.

Methods: We have developed and validated a new IPA based on 125I-labeled IFN-ω. Sera from 78 patients (aged 3-78 years) with clinically diagnosed APS-1, 35 first degree relatives, 323 patients with other adrenal or non-adrenal autoimmune diseases and 84 healthy blood donors were used in the study.

Structure and activation of the TSH receptor transmembrane domain.

Purpose: The thyroid-stimulating hormone receptor (TSHR) is the target autoantigen for TSHR-stimulating autoantibodies in Graves' disease. The TSHR is composed of: a leucine-rich repeat domain (LRD), a hinge region or cleavage domain (CD) and a transmembrane domain (TMD). The binding arrangements between the TSHR LRD and the thyroid-stimulating autoantibody M22 or TSH have become available from the crystal structure of the TSHR LRD-M22 complex and a comparative model of the TSHR LRD in complex with TSH, respectively.

Glycosylation pattern analysis of glycoprotein hormones and their receptors.

We have studied glycosylation patterns in glycoprotein hormones (GPHs) and glycoprotein hormone receptor (GPHR) extracellular domains (ECD) from different species to identify areas not glycosylated that could be involved in intermolecular or intramolecular interactions. Comparative models of the structure of the TSHR ECD in complex with TSH and in complex with TSHR autoantibodies (M22, stimulating and K1-70, blocking) were obtained based on the crystal structures of the FSH-FSHR ECD, M22-TSHR leucine-rich repeat domain (LRD) and K1-70-TSHR LRD complexes. The glycosylation sites of the GPHRs and GPHs from all species studied were mapped on the model of the human TSH TSHR ECD complex.

3 Screen islet cell autoantibody ELISA: A sensitive and specific ELISA for the combined measurement of autoantibodies to GAD, to IA-2 and to ZnT8.

3 Screen, a new ELISA for the combined measurement of autoantibodies to GAD, to IA-2 and to ZnT8, has been developed and evaluated. In the assay serum samples were incubated (overnight; 2-8°C) in ELISA plate wells coated with GAD, IA-2 and ZnT8 followed by a wash step and incubation with biotinylated GAD, IA-2 and ZnT8 (1h; 2-8°C,). The assay was completed by addition of streptavidin-peroxidase and tetramethylbenzidine.

3 Screen ELISA for High-Throughput Detection of Beta Cell Autoantibodies in Capillary Blood.

Background: Testing for beta cell autoantibodies is used for wide-scale identification of early stages of type 1 diabetes. This requires suitable screening assays. We aimed to establish screening that utilized a first step assay (3 Screen) able to detect autoantibodies to the target antigens glutamic acid decarboxylase-65 (GAD), insulinoma-associated antigen 2 (IA-2), and zinc transporter 8 (ZnT8) to identify children positive for multiple beta cell autoantibodies.

Organ-specific autoimmunity in relation to clinical characteristics in children with long-lasting type 1 diabetes.

Background: The aim of this study was to assess the prevalence of diabetes and other organ-specific autoantibodies (Ab) associated with various autoimmune conditions, in Polish children with type 1 diabetes mellitus (T1DM).

Methods: In this study 114 patients, aged 13.4 years, with mean diabetes duration 5.

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.

GAD autoantibody affinity in adult patients with latent autoimmune diabetes, the study participants of a GAD65 vaccination trial.

Objective: Patients with latent autoimmune diabetes in adults (LADA) express autoantibodies against the 65-kDa isoform of GAD (GADA). Intervention with recombinant human GAD65 formulated with aluminium hydroxide (GAD-alum) given twice subcutaneously to LADA patients at intervals of 4 weeks was safe and did not compromise β-cell function in a Phase II clinical trial. GADA affinity has been shown to predict progression to type 1 diabetes.

Addison's disease: a survey on 633 patients in Padova.

Objective: Addison's disease (AD) is a rare endocrine condition.

Design: We aimed to evaluate clinical, immunologic, adrenal imaging, and genetic features in 633 Italian patients with AD followed up since 1967.

Methods: Adrenal cortex autoantibodies, presence of other autoimmune and nonautoimmune diseases, nonadrenal autoantibodies, adrenal imaging, and genetic profile for HLA-DRB1 and AIRE were analyzed.

Failure to detect anti-idiotypic antibodies in the autoimmune response to IA-2 in Type 1 diabetes.

The concept that immune responses to self antigens are regulated by anti-idiotypic networks has attracted renewed interest following reports of circulating factors within IgG fractions of serum that impair detection of autoantibodies with autoantigen. Thus, preclearance of sera with bead-immobilised monoclonal autoantibodies to the Type 1 diabetes autoantigen GAD65, or prebinding of serum antibodies to protein A Sepharose prior to addition of antigen, increases immunoreactivity detected in serum samples consistent with the trapping on the beads of anti-idiotypic antibodies that block antibody binding to the autoantigen. The aim of this study was to investigate the presence of anti-idiotypic antibodies to another major target of autoantibodies in Type 1 diabetes, IA-2.

Blocking type TSH receptor antibodies.

TSH receptor (TSHR) autoantibodies (TRAbs) play a key role in the pathogenesis of Graves' disease. In the majority of patients, TRAbs stimulate thyroid hormone synthesis via activation of the TSHR (stimulating TRAbs, TSHR agonists). In some patients, TRAbs bind to the receptor but do not cause activation (blocking TRAbs, TSHR antagonists).