Type 3 autoimmune polyglandular syndrome (APS-3) or type 3 multiple autoimmune syndrome (MAS-3): an expanding galaxy.

BACKGROUND: The number of recognised distinct autoimmune diseases (AIDs) has progressively increased over the years with more than 100 being reported today. The natural history of AIDs is characterized by progression from latent and subclinical to clinical stages and is associated with the presence of the specific circulating autoantibodies. Once presented, AIDs are generally chronic conditions. AIDs have the tendency to cluster and co-occur in a single patient. Autoimmune thyroid diseases (AITD) are the most prevalent of AIDs in the world population, and about one-third of the AITD patients also present with a non-thyroid AID during their life-span. Furthermore, patient with non-thyroid AIDs often presents with a form of AITD as a concurrent condition. Many of the clusters of AIDs are well characterized as distinctive syndromes, while some are infrequent and only described in case reports. PURPOSE: In this review, we describe the wide spectrum of the combinations and the intricate relationships between AITD and the other AIDs, excluding Addison's disease. These combinations are collectively termed type 3 Autoimmune Polyglandular Syndrome (APS-3), also called type 3 Multiple Autoimmune Syndrome (MAS-3), and represent the most frequent APS in the world populations. CONCLUSIONS: Numerous associations of AITD with various AIDs could be viewed as if the other AIDs were gravitating like satellites around AITD located in the center of a progressively expanding galaxy of autoimmunity.

Autoimmune polyendocrine syndrome type 1: an Italian survey on 158 patients.

BACKGROUND: Autoimmune Polyglandular Syndrome type 1 (APS-1) is a rare recessive inherited disease, caused by AutoImmune Regulator (AIRE) gene mutations and characterized by three major manifestations: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CH) and Addison's disease (AD). METHODS: Autoimmune conditions and associated autoantibodies (Abs) were analyzed in 158 Italian patients (103 females and 55 males; F/M 1.9/1) at the onset and during a follow-up of 23.7 ± 15.1 years. AIRE mutations were determined. RESULTS: The prevalence of APS-1 was 2.6 cases/million (range 0.5-17 in different regions). At the onset 93% of patients presented with one or more components of the classical triad and 7% with other components. At the end of follow-up, 86.1% had CH, 77.2% AD, 74.7% CMC, 49.5% premature menopause, 29.7% autoimmune intestinal dysfunction, 27.8% autoimmune thyroid diseases, 25.9% autoimmune gastritis/pernicious anemia, 25.3% ectodermal dystrophy, 24% alopecia, 21.5% autoimmune hepatitis, 17% vitiligo, 13.3% cholelithiasis, 5.7% connective diseases, 4.4% asplenia, 2.5% celiac disease and 13.9% cancer. Overall, 991 diseases (6.3 diseases/patient) were found. Interferon-ω Abs (IFNωAbs) were positive in 91.1% of patients. Overall mortality was 14.6%. The AIRE mutation R139X was found in 21.3% of tested alleles, R257X in 11.8%, W78R in 11.4%, C322fsX372 in 8.8%, T16M in 6.2%, R203X in 4%, and A21V in 2.9%. Less frequent mutations were present in 12.9%, very rare in 9.6% while no mutations in 11% of the cases. CONCLUSIONS: In Italy, APS-1 is a rare disorder presenting with the three major manifestations and associated with different AIRE gene mutations. IFNωAbs are markers of APS-1 and other organ-specific autoantibodies are markers of clinical, subclinical or potential autoimmune conditions.

Practical applications of studies on the TSH receptor and TSH receptor autoantibodies.

Studies on the TSH receptor (TSHR) have numerous practical applications in vitro and in vivo. For example human monoclonal autoantibodies (MAbs) to the TSHR are useful reagents for in vitro diagnostics. Measurement of TSHR autoantibodies (TRAbs) is helpful in diagnosis and management of autoimmune thyroid disease. Currently available highly sensitive and specific assays to measure TRAbs use the human TSHR MAb M22 instead of the TSH. Furthermore, preparations of the human TSHR MAb M22 are useful as the World Health Organisation International Standard for thyroid stimulating antibody and for calibration of the assays for measuring TRAbs. Preparations of thermostabilised TSHR extracellular domain have recently become available and this is likely to have an impact on improvements in specificity testing for TRAb assays. In addition the stable TSHR preparations have practical application for specific immunoadsorption of patient serum TRAbs. Human TSHR MAbs also have promising prospects as new therapeutics. Autoantibodies with TSHR antagonistic activities are "natural" inhibitors of TSHR stimulation and are expected to be helpful in controlling TSHR activity in patients with Graves' disease, Graves' ophthalmopathy and thyroid cancer.

Epidemiology, pathogenesis, and diagnosis of Addison's disease in adults.

BACKGROUND: Addison's disease (AD) is a rare disorder and among adult population in developed countries is most commonly caused by autoimmunity. In contrast, in children genetic causes are responsible for AD in the majority of patients. PURPOSE: This review describes epidemiology, pathogenesis, genetics, natural history, clinical manifestations, immunological markers and diagnostic strategies in patients with AD. Standard care treatments including the management of patients during pregnancy and adrenal crises consistent with the recent consensus statement of the European Consortium and the Endocrine Society Clinical Practice Guideline are described. In addition, emerging therapies designed to improve the quality of life and new strategies to modify the natural history of autoimmune AD are discussed. CONCLUSIONS: Progress in optimizing replacement therapy for patients with AD has allowed the patients to lead a normal life. However, continuous education of patients and health care professionals of ever-present danger of adrenal crisis is essential to save lives of patients with AD.

Mechanisms of Action of TSHR Autoantibodies.

The availability of human monoclonal antibodies (MAbs) to the TSHR has enabled major advances in our understanding of how TSHR autoantibodies interact with the receptor. These advances include determination of the crystal structures of the TSHR LRD in complex with a stimulating autoantibody (M22) and with a blocking type autoantibody (K1-70). The high affinity of MAbs for the TSHR makes them particularly suitable for use as ligands in assays for patient serum TSHR autoantibodies. Also, M22 and K1-70 are effective at low concentrations in vivo as TSHR agonists and antagonists respectively. K1-70 has important potential in the treatment of the hyperthyroidism of Graves' disease and Graves' ophthalmopathy. Small molecule TSHR antagonists described to date do not appear to have the potency and/or specificity shown by K1-70. New models of the TSHR ECD in complex with various ligands have been built. These models suggest that initial binding of TSH to the TSHR causes a conformational change in the hormone. This opens a positively charged pocket in receptor-bound TSH which attracts the negatively charged sulphated tyrosine 385 on the hinge region of the receptor. The ensuing movement of the receptor's hinge region may then cause activation. Similar activation mechanisms seem to take place in the case of FSH and the FSHR and LH and the LHR. However, stimulating TSHR autoantibodies do not appear to activate the TSHR in the same way as TSH.

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 Addison's 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.

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

AIMS: 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. METHODS: Serum samples from 227 children with newly diagnosed Type 1 diabetes and from 101 control children without diabetes were analysed in a retrospective cross-sectional study. One hundred and seventy-one samples from 116 of the patients with diabetes were analysed in a follow-up study at (median) intervals of 1, 3, 5 and 10 years after onset of Type 1 diabetes. ZnT8 autoantibodies were measured using a bridging enzyme-linked immunosorbent assay, while antibodies to glutamic acid decarboxylase, insulinoma antigen 2 and insulin were measured by radioimmunoassays. RESULTS: ZnT8 autoantibodies were detected in 163/227 (72%) of children at Type 1 diabetes onset and in 1/101 (1%) of the control subjects. Sixteen out of 227 (7%) patients with Type 1 diabetes were antibody negative based on three antibodies (glutamic acid decarboxylase, insulinoma antigen 2 and insulin). This false-negative rate was reduced to 10/227 (4.4%) (P < 0.05) after inclusion of ZnT8 autoantibody measurements. Of the children, 142/227 (63%) were positive for at least three antibodies and the most common combination was insulinoma antigen 2, glutamic acid decarboxylase and ZnT8. ZnT8 autoantibody levels decreased over time after Type 1 diabetes onset and the presence and level of ZnT8 autoantibodies correlated with IA-2 autoantibodies. CONCLUSIONS: A ZnT8 autoantibody enzyme-linked immunosorbent assay showed 72% disease sensitivity and 99% specificity at Type 1 diabetes onset. Measurements of ZnT8 autoantibodies are important for Type 1 diabetes diagnosis and should be included in the panel of autoantibodies tested at the onset of Type 1 diabetes.

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.

Thyrotrophin receptor antibody characteristics in a woman with long-standing Hashimoto's who developed Graves' disease and pretibial myxoedema.

CONTEXT: Sequential conversion of Hashimoto's thyroiditis (HT) to Graves' disease (GD) is uncommon. Distinct immune paradigms, paucity of functioning tissue in long-standing HT, and infrequent conversion of blocking (TBAb) to stimulating (TSAb) thyrotrophin receptor antibody (TRAb) may account for this. Molecular and crystal structure analysis helps delineate TSH receptor (TSHR)/TRAb interactions in detail. Such 'fingerprinting' helps determine the behaviour and characteristics of TRAb in longitudinal studies. PATIENT: An 80-year-old woman taking thyroxine for long-standing HT became hyperthyroid. This persisted despite thyroxine withdrawal - free T3 was 7·3 pmol/l (2·6-5·7) and TSH < 0·01 mU/l (0·2-4·5) and TRAb highly positive. She had a goitre (ultrasound - HT), pretibial myxoedema, with mild inactive Graves' orbitopathy. She had RAI treatment and is on thyroxine replacement. MEASUREMENTS AND RESULTS: Blood samples at presentation (A) and 1 year (B) showed high TSAb and TPOAb activity but no TBAb. Experiments involving TSHR mutations confirmed that (i) TRAb had stable characteristics over 1 year; (ii) TSHR mutation R255D caused complete inhibition and (iii) R109A caused marked reduction of cAMP production by M22 (TSHR-stimulating human monoclonal antibody) and A and B; (iv) mutations R80A, E107A and K129A while affecting M22 had little effect on A and B. CONCLUSIONS: The reasons for an immunological paradigm shift in this elderly woman remain speculative. We believe that de-novo TSAb synthesis occurred converting her long-standing HT to GD although the mechanisms responsible remain unexplained. TRAb analysis confirmed stable autoantibody characteristics over 1 year and variable effects of TSHR mutations on TRAb and M22 function.

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 Addison's 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.

TSH receptor - autoantibody interactions.

TSH receptor (TSHR) autoantibodies (TRAbs)activate the TSHR cyclic AMP cascade (stimulating TRAbs) or act as TSHR antagonist (blocking TRAbs), and both types inhibit TSH binding to the TSHR. Isolation of human monoclonal TSHR autoantibodies (stimulating M22 and blocking 5C9) has been a key milestone in studies of the TSHR and TSHR autoimmunity. Comparison of M22 and TSH interactions with the TSHR at the atomic level reveal that M22 heavy and light chains mimic TSH alpha and beta chains, respectively, in the way they bind to the receptor, but the evolutionary forces which have caused this close molecular mimicry are as yet completely unknown. More recently two more human monoclonal antibodies to the TSHR (K1-18 with stimulating and K1-70 with blocking activities) have been isolated from a single blood sample collected from a patient with hypothyroidism who previously presented with hyperthyroidism. K1-18 and K1-70 were derived from different lymphocytes as shown by V region genes analysis. This provides, for the first time, clear proof that a patient can produce both blocking and stimulating TRAbs at the same time. Although it has been postulated that stimulating and blocking TRAbs bind to different regions on the TSHR, our studies showed that antibodies of both types bind well to the TSHR containing only N-terminal amino acids 22-260. Whether TRAbs make contact with other parts of the TSHR in order to produce their biological effects (stimulation or blocking) remains to be elucidated.

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.

Thyroid stimulating autoantibody M22 mimics TSH binding to the TSH receptor leucine rich domain: a comparative structural study of protein-protein interactions.

The TSH receptor (TSHR) ligands M22 (a thyroid stimulating human monoclonal antibody) and TSH, bind to the concave surface of the leucine rich repeats domain (LRD) of the TSHR and here, we show that M22 mimics closely the binding of TSH. We compared interactions produced by M22 with the TSHR in the M22-TSHR crystal structure (2.55 A resolution) and produced by TSH with the TSHR in a TSH-TSHR comparative model. The crystal structure of the TSHR and a comparative model of TSH based on the crystal structure of FSH were used as components to build the TSH-TSHR model. This model was built based on the FSH-FSH receptor structure (2.9 A) and then the structure of the TSHR in the model was replaced by the TSHR crystal structure. The analysis shows that M22 light chain mimics the TSHbeta chain in its interaction with TSHR LRD, while M22 heavy chain mimics the interactions of the TSHalpha chain. The M22-TSHR complex contains a greater number of hydrogen bonds and salt bridges and fewer hydrophobic interactions than the TSH-TSHR complex, consistent with a higher M22 binding affinity. Furthermore, the surface area formed by TSHR residues N208, Q235, R255, and N256 has been identified as a candidate target region for small molecules which might selectively block binding of autoantibodies to the TSHR.

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.

Preparation and testing of diabetes autoantibody controls.

Stocks of the WHO islet cell antibody, GAD(65) antibody, and IA-2 antibody standard (NIBSC 97/550) are now very limited. We have therefore made and tested a series of control preparations in which human monoclonal autoantibodies to IA-2 and to GAD(65) were diluted in antibody-negative human serum to different concentrations. Three different diabetes autoantibody controls (DAC 1-3) were made as was a negative control preparation. Aliquots containing 1 mL of autoantibodies in serum were freeze-dried. After reconstitution (with 1 mL of water) the controls were tested by (125)I-IA-2 immunoprecipitation assay (IPA), (125)I-GAD(65) IPA, GAD(65) Ab ELISA and IA-2 Ab ELISA (kits from RSR Ltd.) and for ICA by immunofluorescence test (IFT). DAC1 is particularly suitable as a control for the (125)I IA-2 IPA; DAC2 is suitable for the (125)I-GAD(65) IPA, GAD(65) Ab ELISA, and IA-2 Ab ELISA; and DAC3 is suitable for the ICA IFT. Freeze-dried preparations showed good stability at 37 degrees C. Reconstituted liquid preparations were stable when stored at 4 degrees C and at 37 degrees C. Availability of an essentially unlimited supply of these reagents should be useful in establishing reproducible and comparable measurements of diabetes autoantibodies in different laboratories using different assays.

Effects of a thyroid-stimulating human monoclonal autoantibody (M22) on functional activity of LH and FSH receptors.

OBJECTIVE: The glycoprotein hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) show low-level cross-reactivity between their respective receptors (R). Patient serum autoantibodies to the thyrotropin receptor (TSHR) do not appear to cross-react with the luteinizing hormone receptor (LHR) or follicle-stimulating hormone receptor (FSHR), although the concentrations of autoantibody with which it is feasible to carry out experiments of this type are limited. Consequently, we have studied the effects of high doses of the thyroid-stimulating human monoclonal autoantibody (M22) on the LHR and FSHR. DESIGN: Chinese Hamster ovary (CHO) cells stably expressing the TSHR, LHR, and FSHR and purified M22 IgG preparations were used in the study. METHODS: CHO-TSHR, CHO-LHR, and CHO-FSHR cells were incubated with bovine TSH (0.1-25mU/mL), human recombinant chorionic gonadotropin (hCG; 0.5-10mU/mL) or human recombinant FSH (100-5000mU/mL) or with M22 IgG (0.001-5.0 microg/mL), and the extracellular cyclic AMP was measured by radioimmunoassay. RESULTS: Cyclic AMP levels increased in a dose-dependent manner after incubation of CHO-TSHR cells with TSH or M22 IgG, and on a molar basis the effects of TSH and M22 were similar. Cyclic AMP stimulation was not detectable in CHO-LHR and CHO-FSHR cells after incubation with M22 IgG, whereas incubation with hCG or FSH, respectively, caused dose-dependent cyclic AMP stimulation. On a molar basis, concentrations of M22 IgG approximately 100x those of FSH causing clear stimulation were ineffective with CHO-FSHR cells. Similarly, molar concentration of M22 IgG 20,000x those of hCG causing clear stimulation had no effect on CHO-LHR cells. CONCLUSIONS: This study shows that at relatively high concentrations, M22 IgG is unable to stimulate cyclic AMP levels in CHO-LHR or CHO-FSHR cells, suggesting that TSHR autoantibodies have greater specificity for the TSHR than TSH itself.

Islet cell, thyroid, adrenal and celiac disease related autoantibodies in patients with Type 1 diabetes from Sri Lanka.

AIMS: The prevalence of islet cell, thyroid, adrenal and celiac disease related autoantibodies in patients with Type 1 diabetes mellitus (Type 1 DM) from Sri Lanka is described. DESIGN AND METHODS: Autoantibodies to glutamic acid decarboxylase 65 (GAD65Ab), protein tyrosine phosphatase IA-2 (IA-2Ab), insulin (IAAb), thyroglobulin (TgAb), thyroid peroxidase (TPOAb), TSH receptor (TRAb), 21-hydroxylase (21-OHAb) and tissue transglutaminase (tTGAb) were measured in 122 Type 1 DM patients who had low C-peptide activity or were >20 yr old at the time of diagnosis and in 100 non-diabetic blood donors. RESULTS: GAD65Ab and/or IA-2Ab were present in 74/122 (60.7%) Type 1 DM subjects with a significantly higher prevalence compared to non-diabetic controls (no. 100) (GAD65Ab-59 vs 4%; IA-2Ab-14 vs 0%; respectively) (p<0.001). The median (inter-quartile range) Type 1 DM duration in antibody positive subjects was 3.3 (0.99-6.9) vs 4.9 (1.7-7.5) yr in antibody negative subjects (p=0.23). IA-2Ab prevalence decreased with disease duration > or =5 yr (19 vs 4%) (p<0.001). There was no difference in the prevalence of TgAb (25 vs 33%)(p=0.21) and TPOAb (22 vs 18%) (p=0.48) in Type 1 DM and non-diabetic subjects. Also, there was no difference in TgAb and TPOAb prevalence in antibody positive Type 1 DM (34.7%) compared to antibody negative Type 1 DM (24.4%) subjects (p=0.24). tTGAb (3/119) and TRAb (1/119) were found in low prevalence and 21-OHAb were not detected. CONCLUSIONS: Diabetes associated autoantibodies were detected in the majority of Type 1 DM subjects, suggesting a major role for autoimmunity in the pathogenesis of Type 1 DM in Sri Lankans. The prevalence of TgAb and TPOAb in Type 1 DM subjects and non-diabetic controls was relatively high and similar in both groups.

Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist.

Analysis of nine mouse monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) with TSH antagonist activity showed that only one of the mAbs (RSR B2) was an effective antagonist of the human thyroid stimulating autoantibody M22. Crystals of B2 Fab were analyzed by x-ray diffraction and a crystal structure at 3.3 A resolution was obtained. The surface charge and topography of the B2 antigen binding site were markedly different from those of the thyroid-stimulating mAb M22 and these differences might contribute to the different properties of the two mAbs. B2 (but not other mouse TSHR-specific mAbs) was also an effective antagonist of thyroid stimulating autoantibody activity in 14 of 14 different sera from patients with Graves' disease. 125I-labeled B2 bound to the TSHR with high affinity (2 x 10(10) L/mol) and patient serum TSHR autoantibodies inhibited labeled B2 binding to the receptor in a similar way to inhibition of labeled TSH binding (r = 0.75; n = 20). Furthermore, labeled B2 binding was inhibited by patient serum TSHR autoantibodies with TSH antagonist activity and also by mouse and human thyroid stimulating mAbs. Overall, mAb B2 is a powerful antagonist of thyroid stimulating autoantibodies (and TSH) thus resembling closely patient serum TSH antagonist TSHR autoantibodies. Furthermore, B2 might have potentially important in vivo applications when tissues containing the TSHR (including those in the orbit) need to be made unresponsive to stimulating autoantibodies.

Isolation and characterisation of a human monoclonal autoantibody to the islet cell autoantigen IA-2.

A hybridoma secreting a human monoclonal autoantibody to the islet cell autoantigen IA-2 was prepared from peripheral lymphocytes of a patient with type 1 diabetes and Graves' disease using EBV infection followed by fusion with a mouse/human hybrid cell line. The monoclonal antibody (M13) is an IgG1/kappa and in an immunofluorescence test M13 at 1 microg/mL showed islet cell antibody reactivity equivalent to 40 JDF units. M13 IgG bound (35)S-labelled IA-2 (26% at 100 microg/mL) and (125)I-labelled IA-2 (34% at 100 microg/mL) in an immunoprecipitation assay and reacted well with IA-2 in western blotting analysis. Amino acids 777-808 in the PTP domain of IA-2 were found to be important for M13 binding in an analysis using modified (35)S-labelled IA-2 proteins. M13 V region genes were from VH1-3, D3-22, JH4b, VKI DPK8/Vd+ and JK3 genes and showed a high replacement/silent mutation ratio for both the heavy (11.0) and the light (6.0) chain genes. Mouse monoclonal antibodies (mMAbs) reactive with at least three different epitopes within IA-2 aa 604-686 corresponding to the juxtamembrane domain were also obtained. F(ab')(2) or Fab from the mMAbs inhibited serum IA-2 autoantibody binding to IA-2 in 20/22 diabetic sera whereas M13 F(ab')(2) caused inhibition in only 6/22 sera. M13 is representative of some patient serum IA-2 autoantibodies and as such provides a useful tool to study autoimmune responses to IA-2.