B. Rees Smith

An enzyme-linked immunoassay for thyroid microsomal antibodies

An enzyme-linked immunoassay (ELISA) for microsomal antibody is described. The method was found to be rapid, sensitive and precise and analysis of 115 serum samples showed good correlation between the ELISA and the conventional tanned red cell haemagglutination test. The presence of thyroglobulin antibody, rheumatoid factor, antinuclear factor or gastric parietal cell antibodies did not interfere in the microsomal antibody ELISA but some sera with mitochondrial antibody activity appeared to cause a non-specific effect. The ELISA was particularly useful for analysing microsomal antibody production by Hashimoto lymphocyte cultures and in some cases antibody synthesis could be studied in the absence of mitogen. The high capacity of the ELISA combined with its sensitivity suggest that it will be a valuable technique for studying microsomal autoantibody activity both in serum and in lymphocyte cultures.

Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

The enhancement of immunoglobulin synthesis by human lymphocytes with lithium

Using a protein A plaque assay to detect immunoglobulin synthesis we have shown that culture of pokeweed mitogen (PWM)-stimulated lymphocytes in the presence of lithium significantly increased IgG production at concentrations of 10−3 to 10−2 mM. Enhanced IgG and IgM synthesis was found in cultures with lithium alone at concentrations of 1–10 mM, levels within the therapeutic range. Significantly higher numbers of spontaneous IgM plaque-forming cells were found in the peripheral blood of patients receiving lithium carbonate therapy than in normal controls suggesting that these findings may have clinical relevance.

Premature Ovarian Failure in Patients with Autoimmune Addison's Disease: Clinical, Genetic, and Immunological Evaluation

DESIGN The design of the study was to investigate the prevalence of the following: 1) premature ovarian failure (POF) in patients with autoimmune Addisons disease (AD); 2) steroid-producing cell antibodies (StCA) and steroidogenic enzymes (17α-hydroxylase autoantibodies and P450 side-chain cleavage enzyme autoantibodies) in patients with or without POF; and 3) the value of these autoantibodies to predict POF. PATIENTS The study included 258 women: 163 with autoimmune polyendocrine syndrome type 2 (APS-2), 49 with APS-1, 18 with APS-4, and 28 with isolated AD. METHODS StCA were measured by an immunofluorescence technique and 17α-hydroxylase autoantibodies and P450 side-chain cleavage enzyme autoantibodies by immunoprecipitation assays. RESULTS Fifty-two of 258 women with AD (20.2%) had POF. POF was diagnosed in 20 of 49 (40.8%) with APS-1, six of 18 (33.3%) with APS-4, 26 of 163 (16%) with APS-2, and none of 28 with isolated AD. In patients with APS-1 and APS-4, POF developed after AD, whereas it preceded AD in patients with APS-2. StCA were detected in 31 of 43 with POF (72%) and 51 of 198 without POF (25.7%). StCA were present in 22 of 38 with APS-1 (57.9%) (11 of 13 with POF); in five of 13 with APS-4 (38.5%) (three of four with POF); in 53 of 162 with APS-2 (32.7%) (17 of 26 with POF), and in one of 28 isolated AD patients (3.6%). Twelve of 13 patients with POF with a duration less than 5 yr (92.3%) and 18 of 25 with duration longer than 5 yr (72%) were StCA positive. Twenty-eight of 31 with POF (90.3%) were positive for at least one steroidogenic antibody. Forty-one women with AD less than 40 yr were followed up for a mean period of 9 yr. Eight of 21 women (38%) positive or seroconverted for steroidogenic autoantibodies developed POF at a mean age of 23 yr (six with APS-1, one with APS-2, and one with APS-4), and none of the 20 patients negative for steroidogenic autoantibodies developed POF. CONCLUSIONS This study indicates that AD is frequently associated with POF and that steroidogenic antibodies are markers of patients with POF. Steroidogenic autoantibodies are predictive markers of POF in patients with AD.

FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity

The crystal structures of the leucine-rich repeat domain (LRD) of the FSH receptor (FSHR) in complex with FSH and the TSH receptor (TSHR) LRD in complex with the thyroid-stimulating autoantibody (M22) provide opportunities to assess the molecular basis of the specificity of glycoprotein hormone-receptor binding. A comparative model of the TSH-TSHR complex was built using the two solved crystal structures and verified using studies on receptor affinity and activation. Analysis of the FSH-FSHR and TSH-TSHR complexes allowed identification of receptor residues that may be important in hormone-binding specificity. These residues are in leucine-rich repeats at the two ends of the FSHR and the TSHR LRD structures but not in their central repeats. Interactions in the interfaces are consistent with a higher FSH-binding affinity for the FSHR compared with the binding affinity of TSH for the TSHR. The higher binding affinity of porcine (p)TSH and bovine (b)TSH for human (h)TSHR compared with hTSH appears not to be dependent on interactions with the TSHR LRD as none of the residues that differ among hTSH, pTSH or bTSH interact with the LRD. This suggests that TSHs are likely to interact with other parts of the receptors in addition to the LRD with these non-LRD interactions being responsible for affinity differences. Analysis of interactions in the FSH-FSHR and TSH-TSHR complexes suggests that the alpha-chains of both hormones tend to be involved in the receptor activation process while the beta-chains are more involved in defining binding specificity.

Autoantibodies and associated T-cell responses to determinants within the 831-860 region of the autoantigen IA-2 in Type 1 diabetes.

B-cells influence T-cell reactivity by facilitating antigen presentation, but the role of autoantibody-secreting B-cells in regulating T-cell responses in Type 1 diabetes is poorly defined. The aims of this study were to characterise epitopes on the IA-2 autoantigen for three monoclonal antibodies from diabetic patients by amino acid substitutions of selected residues of IA-2, establish contributions of these epitopes to binding of serum antibodies in Type 1 diabetes and relate B- and T-cell responses to overlapping determinants on IA-2. The monoclonal antibodies recognised overlapping epitopes, with residues within the 831-860 region of IA-2 contributing to binding; substitution of Glu836 inhibited binding of all three antibodies. Monoclonal antibody Fab fragments and substitution of residues within the 831-836 region blocked serum antibody binding to an IA-2 643-937 construct. IL-10-secreting T-cells responding to peptides within the 831-860 region were detected by cytokine-specific ELISPOT in diabetic patients and responses to 841-860 peptide were associated with antibodies to the region of IA-2 recognised by the monoclonal antibodies. The study identifies a region of IA-2 frequently recognised by antibodies in Type 1 diabetes and demonstrates that these responses are associated with T-cells secreting IL-10 in response to a neighbouring determinant.

Tryptophan hydroxylase autoantibodies as markers of a distinct autoimmune gastrointestinal component of autoimmune polyendocrine syndrome type 1.

BACKGROUND Autoantibodies to tryptophan hydroxylase (TPHAbs) directed against serotonin-producing enterochromaffin cells (EC) have been reported in autoimmune-polyendocrine-syndrome type 1 (APS-1) patients with gastrointestinal dysfunction (GID). Serotonin plays a critical role in enteric function and its peripheral blood levels reflect serotonin release from the gastrointestinal tract. AIMS We test the hypothesis that TPHAbs mark a distinct autoimmune component of APS-1 characterized by an autoimmune attack toward EC, which results in clinical GID. METHODS TPHAbs were measured in 64 APS-1 patients. Endoscopy with gastric (antrum/body) and duodenal biopsy was carried in 16 TPHAbs+ patients (8 with and 8 without GID) and in 2 TPHAbs- patients (without GID). Immunohistochemistry of biopsy specimens was carried out using antibodies to serotonin, chromogranin-A, CD3, CD4, CD8, and CD20. Serotonin serum levels were measured in TPHAbs+ and TPHAbs- patients who had endoscopy. RESULTS Thirty-seven of 64 patients were TPHAbs+ (11/12 with GID and 26/52 without GID; P < .001). Gastric and duodenal biopsies in all 8 TPHAb+ patients with GID showed lymphocytic infiltration with increased CD3+CD8+ intraepithelial lymphocytes and absence of EC. Furthermore, mean serotonin serum levels were below the normal range in TPHAb+ patients with GID (P < .01). In 8 TPHAb+ patients without GID gastric and duodenal biopsies showed different grades of inflammatory infiltration and reduced number of EC. Mean serotonin serum levels were near the lower limit of the normal range. In all TPHAbs+ patients the biopsies showed a reduced number of chromogranin-A positive cells consistent with enteroendocrine cells depletion. TPHAbs- patients without GID showed normal gastrointestinal mucosa and serotonin serum levels. CONCLUSIONS TPHAbs appear to be markers of a distinct autoimmune component of APS-1. Progressive involvement of the gastrointestinal EC leads to the transition from preclinical to clinical disease, characterized by GID and reduced serotonin serum levels.

Sex hormones enhance immunoglobulin synthesis by human peripheral blood lymphocytes

Using a protein A haemolytic plaque assay to detect immunoglobulin synthesis by human peripheral blood lymphocytes cultured with pokeweed mitogen, we have shown a significant increase in IgG plaque-forming cells in the presence of physiological concentrations of testosterone and oestradiol. Higher concentrations of these steroids inhibit plaque-forming cells. There was no difference in the response to testosterone and oestradiol of lymphocytes derived from male subjects or menstruating or post-menopausal females.

The dynamic changes of zinc transporter 8 autoantibodies in Czech children from the onset of Type 1 diabetes mellitus

The prevalence of autoantibodies to zinc transporter 8 (ZnT8) in Czech children at the onset of Type 1 diabetes mellitus and dynamic changes in ZnT8 autoantibody levels during disease progression were studied. The value of ZnT8 autoantibody measurements in diagnosis of Type 1 diabetes was assessed.

Gonadal function in males with autoimmune Addison's disease and autoantibodies to steroidogenic enzymes

Steroidogenic enzyme autoantibodies (SEAbs) are frequently present and are markers of autoimmune premature ovarian failure (POF) in females with autoimmune Addisons disease (AAD). The prevalence and significance of SEAbs in males with AAD have not yet been defined. We studied the prevalence of SEAbs in a large cohort of males with AAD and assessed the relationship between SEAbs positivity and testicular function. A total of 154 males with AAD (mean age 34 years) were studied. SEAbs included autoantibodies to steroid‐producing cells (StCA), detected by immunofluorescence, and steroid 17α‐hydroxylase (17α‐OHAbs) and side chain cleavage enzyme (SCCAbs) measured by immunoprecipitation assays. Gonadal function was evaluated by measuring follicle‐stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (TT), sex hormone‐binding globulin (SHGB), anti‐müllerian hormone (AMH) and inhibin‐B (I‐B). Twenty‐six males, 10 SEAbs(+) and 16 SEAbs(–), were followed‐up for a mean period of 7·6 years to assess the behaviour of SEAbs and testicular function. SEAbs were found in 24·7% of males with AAD, with the highest frequency in patients with autoimmune polyendocrine syndrome type 1 (APS‐1). The levels of reproductive hormones in 30 SEAbs(+) males were in the normal range according to age and were not significantly different compared to 55 SEAbs(–) males (P > 0·05). During follow‐up, both SEAbs(+) and SEAbs(–) patients maintained normal testicular function. SEAbs were found with high frequency in males with AAD; however, they were not associated with testicular failure. This study suggests that the diagnostic value of SEAbs in males with AAD differs compared to females, and this may be related to the immunoprivileged status of the testis.