TSH receptor specific monoclonal autoantibody K1-70TM 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 K1-70TM (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. PATIENTS AND STUDY DESIGN: Eighteen GD patients stable on antithyroid drug medication received a single intramuscular (IM) or intravenous (IV) dose of K1-70TM during an open label phase I ascending dose, safety, tolerability, pharmacokinetic and pharmacodynamic (PD) study. Immunogenic effects of K1-70TM were also determined. RESULTS: K1-70TM was well-tolerated in all subjects at all doses and no significant immunogenic response was observed. There were no deaths or serious adverse events. Increased systemic exposure to K1-70TM was observed following a change to IV dosing, indicating this was the correct dosage route. Expected PD effects occurred after a single IM dose of 25 mg or single IV dose of 50 mg or 150 mg with fT3, fT4, and TSH levels progressing into hypothyroid ranges. There were also clinically significant improvements in symptoms of both GD (reduced tremor, improved sleep, improved mental focus, reduced toilet urgency) and GO (reduced exophthalmos measurements, reduced photosensitivity). CONCLUSIONS: K1-70TM was safe, well tolerated and produced the expected PD effects with no immunogenic responses. It shows considerable promise as a new drug to block the actions of thyroid stimulators on the TSHR.

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. METHODS: Sixty-six subjects were recruited following treatment with carbimazole (n = 26), radioiodine (n = 27) or surgery (n = 13). TRAb, TPOAb, TgAb and GADAb were measured at a follow-up visit as well as bioassays of TSAb and TSBAb activity. RESULTS: Forty-five per cent of all patients remained TRAb-positive for more than one year and 23% for more than 5 years after diagnosis, irrespective of treatment method. Overall, TRAb concentration fell from a median (IQR) of 6.25 (3.9-12.7) to 0.65 (0.38-3.2) U/L. Surgery conferred the largest fall in TRAb concentration from 11.4 (6.7-29) to 0.58 (0.4-1.4) U/L. Seventy per cent of TRAb-positive patients were positive for TSAb, and one patient (3%) was positive for TSBAb. TRAb and TSAb correlated well (r = 0.83). In addition, 38/66 patients were TgAb-positive, 47/66 were TPOAb-positive and 6/66 were GADAb-positive at follow-up. CONCLUSIONS: TRAb levels generally decreased after treatment but persisted for over 5 years in some patients. TRAb activity was predominantly stimulatory, with only one patient demonstrating TSBAb. A large proportion of patients were TgAb/TPOAb-positive at follow-up. All treatment modalities reduced TRAb concentrations; however, surgery was most effective.

Structure of full-length TSH receptor in complex with antibody K1-70™.

Determination of the full-length thyroid-stimulating hormone receptor (TSHR) structure by cryo-electron microscopy (cryo-EM) is described. The TSHR complexed with human monoclonal TSHR autoantibody K1-70™ (a powerful inhibitor of TSH action) was detergent solubilised, purified to homogeneity and analysed by cryo-EM. The structure (global resolution 3.3 Å) is a monomer with all three domains visible: leucine-rich domain (LRD), hinge region (HR) and transmembrane domain (TMD). The TSHR extracellular domain (ECD, composed of the LRD and HR) is positioned on top of the TMD extracellular surface. Extensive interactions between the TMD and ECD are observed in the structure, and their analysis provides an explanation of the effects of various TSHR mutations on TSHR constitutive activity and on ligand-induced activation. K1-70™ is seen to be well clear of the lipid bilayer. However, superimposition of M22™ (a human monoclonal TSHR autoantibody which is a powerful stimulator of the TSHR) on the cryo-EM structure shows that it would clash with the bilayer unless the TSHR HR rotates upwards as part of the M22™ binding process. This rotation could have an important role in TSHR stimulation by M22™ and as such provides an explanation as to why K1-70™ blocks the binding of TSH and M22™ without activating the receptor itself.

The epidemiology of benzodiazepine-related toxicity in Ontario, Canada: a population-based descriptive study.

OBJECTIVES: Despite the widespread use of prescription benzodiazepines, there are few studies examining trends and patterns of benzodiazepine-related toxicity. We describe the epidemiology of benzodiazepine-related toxicity in Ontario, Canada. METHODS: We conducted a population-based, cross-sectional study of Ontario residents who had an emergency department visit or hospitalization for benzodiazepine-related toxicity between January 1, 2013 and December 31, 2020. We reported annual crude and age-standardized rates of benzodiazepine-related toxicity overall, by age, and by sex. In each year, we characterized the history of benzodiazepine and opioid prescribing among people who experienced benzodiazepine-related toxicity, and reported the percentage of encounters with opioid, alcohol, or stimulant co-involvement. RESULTS: Between 2013 and 2020, there were 32,674 benzodiazepine-related toxicity encounters among 25,979 Ontarians. During this period, the crude rate of benzodiazepine-related toxicity declined overall, from 28.0 to 26.1 per 100,000 population (age-standardized rate: 27.8 to 26.4 per 100,000), but increased among young adults aged 19 to 24 (39.9 to 66.6 per 100,000 population). Moreover, by 2020, the percentage of encounters associated with active benzodiazepine prescriptions had declined to 48.9%, while the percentage of encounters that had opioid, stimulant, or alcohol co-involvement rose to 28.8%. CONCLUSION: Benzodiazepine-related toxicity has declined in Ontario overall, but has increased among youth and young adults. Furthermore, there is growing co-involvement of opioids, stimulants, and alcohol, which may reflect the recent emergence of benzodiazepines in the unregulated drug supply. Multifaceted public health initiatives comprising harm reduction, mental health supports, and promotion of appropriate prescribing are needed to reduce benzodiazepine-related harm.

RéSUMé: OBJECTIFS: Malgré l'utilisation généralisée des benzodiazépines sur ordonnance, peu d'études portent sur les tendances et les schémas de toxicité de ces médicaments. Nous décrivons l'épidémiologie de la toxicité liée aux benzodiazépines en Ontario, au Canada. MéTHODE: Nous avons mené une étude populationnelle transversale des résidentes et résidents de l'Ontario ayant visité le service des urgences ou été hospitalisés pour toxicité liée aux benzodiazépines entre le 1er janvier 2013 et le 31 décembre 2020. Nous avons rapporté globalement, par âge et par sexe les taux annuels de toxicité liée aux benzodiazépines, bruts et standardisés pour l'âge. Pour chaque année, nous avons caractérisé les antécédents de prescription de benzodiazépines et d'opioïdes chez les personnes ayant présenté une toxicité liée aux benzodiazépines, et rapporté le pourcentage de rencontres présentant une co-implication avec les opioïdes, l'alcool ou les stimulants. RéSULTATS: Entre 2013 et 2020, il y a eu 32 674 rencontres pour toxicité liée aux benzodiazépines avec 25 979 Ontariens et Ontariennes. Durant cette période, le taux brut de toxicité liée aux benzodiazépines a baissé dans l'ensemble, passant de 28 à 26,1 pour 100 000 habitants (taux standardisé pour l'âge : 27,8 à 26,4 p. 100 000), mais il a augmenté chez les jeunes adultes de 19 à 24 ans (de 39,9 à 66,6 p. 100 000). De plus, en 2020, le pourcentage de rencontres associées à des ordonnances actives de benzodiazépines avait baissé à 48,9 %, tandis que le pourcentage de rencontres présentant une co-implication avec les opioïdes, les stimulants ou l'alcool avait augmenté à 28,8 %. CONCLUSION: La toxicité liée aux benzodiazépines a diminué en Ontario dans l'ensemble, mais elle a augmenté chez les jeunes et les jeunes adultes. De plus, cette toxicité présente une co-implication croissante avec les opioïdes, les stimulants ou l'alcool, ce qui peut refléter l'émergence récente des benzodiazépines dans l'approvisionnement non réglementé en drogues. Des initiatives de santé publique multidimensionnelles incluant la réduction des méfaits, le soutien en santé mentale et la promotion de la prescription appropriée sont nécessaires pour réduire les méfaits liés aux benzodiazépines.

Social Work Practice During COVID-19: Client Needs and Boundary Challenges.

While information and communication technologies (ICTs) permeated social work practice long before the onset of COVID-19, the abrupt need to close non-essential workplaces resulted in an unparalleled incorporation of digital technology into practice across the globe. The onset of COVID-19 occurred during phase two of research in which we were investigating social workers' informal use of ICT with clients. Prior to COVID-19, we were conducting interviews with practitioners and clients from four agencies serving diverse client populations in a large city in Canada. With the onset of COVID-19, we adapted to the COVID-19 context and amended the questions to investigate ICT use during the pandemic. In addition, with ethics approval, we conducted second interviews with practitioners interviewed prior to COVID-19 with a revised guide to address the pandemic context; and we continued to recruit and interview practitioners and clients using an amended interview guide incorporating pandemic-related questions. The sample comprised 27 practitioners and 22 clients. Eleven practitioners participated in interviews prior to and during COVID-19. Analysis of transcribed interviews revealed that the COVID-19 context had led to a paradigm shift in practitioners' ICT use, with two key themes identified: (1) boundary challenges and (2) clients' diverging ICT needs. We discuss these themes and present implications for policy and practice in a post-COVID-19 world.

Blocking the Thyrotropin Receptor with K1-70 in a Patient with Follicular Thyroid Cancer, Graves' Disease, and Graves' Ophthalmopathy.

Background: We report the therapeutic use of K1-70™, a thyrotropin receptor (TSHR) antagonist monoclonal antibody, in a patient with follicular thyroid cancer (FTC), Graves' disease (GD), and Graves' ophthalmopathy (GO). Methods: A 51-year-old female patient, who smoked, presented in October 2014 with FTC complicated by GD, high levels of TSHR autoantibodies with high thyroid stimulating antibody (TSAb) activity, and severe GO. K1-70 was administered at 3 weekly intervals with the dose adjusted to block TSAb activity. Her cancer was managed with lenvatinib and radioiodine therapy. Results: Following initiation of K1-70 therapy, TSAb activity measured in serum decreased and GO (proptosis and inflammation) improved. On K1-70 monotherapy during the pause in lenvatinib, several metastatic lesions stabilized while others showed progression attenuation compared with that before lenvatinib therapy. Conclusions: These observations suggest that blocking TSHR stimulation with K1-70 can be an effective treatment for GO and may also benefit select patients with FTC and GD.

Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample.

OBJECTIVE: Patients who appear to have both stimulating and blocking TSHR autoantibodies in their sera have been described, but the two activities have not been separated and analysed. We now describe the isolation and detailed characterization of a blocking type TSHR monoclonal autoantibody and a stimulating type TSHR monoclonal autoantibody from a single sample of peripheral blood lymphocytes. DESIGN, PATIENTS AND MEASUREMENTS: Two heterohybridoma cell lines secreting TSHR autoantibodies were isolated using standard techniques from the lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies (160 units/l by inhibition of TSH binding). The ability of the two new monoclonal antibodies (MAbs; K1-18 and K1-70) to bind to the TSHR and compete with TSH or TSHR antibody binding was analysed. Furthermore, the effects of K1-18 and K1-70 on cyclic AMP production in Chinese hamster ovary cells (CHO) cells expressing the TSHR were investigated. RESULTS: One MAb (K1-18) was a strong stimulator of cyclic AMP production in TSHR-transfected CHO cells and the other (K1-70) blocked stimulation of the TSHR by TSH, K1-18, other thyroid-stimulating MAbs and patient serum stimulating type TSHR autoantibodies. Both K1-18 (IgG1 kappa) and K1-70 (IgG1 lambda) bound to the TSHR with high affinity (0.7 x 10(10) l/mol and 4 x 10(10) l/mol, respectively), and this binding was inhibited by unlabelled K1-18 and K1-70, other thyroid-stimulating MAbs and patient serum TSHR autoantibodies with stimulating or blocking activities. V region gene analysis indicated that K1-18 and K1-70 heavy chains used the same V region germline gene but different D and J germline genes as well as having different light chains. Consequently, the two antibodies have evolved separately from different B cell clones. CONCLUSIONS: This study provides proof that a patient can produce a mixture of blocking and stimulating TSHR autoantibodies at the same time.

Preclinical studies on the toxicology, pharmacokinetics and safety of K1-70TM 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. The pharmacokinetic parameters of K1-70™ and the effects of different doses of K1-70™ on serum thyroid hormone levels in the study animals were determined in rats and primates after IV and IM administration. RESULTS: Low serum levels of T3 and T4 associated with markedly elevated levels of TSH were observed in the study animals following IV and IM administration of K1-70™. The toxicological findings were attributed to the pharmacology of K1-70™ and were consistent with the hypothyroid state. The no observable adverse effect level (NOAEL) could not be established in the rat study while in the primate study it was 100 mg/kg/dose for both males and females. CONCLUSIONS: The toxicology, pharmacodynamic and pharmacokinetic data in this preclinical study were helpful in designing the first in human study with K1-70™ administered to subjects with Graves' disease.

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. These six mutations did not affect the binding of human TSHR monoclonal autoantibodies or patient serum TSHR autoantibodies to the TSHR260. Furthermore, the response of full-length TSHR to stimulation by TSH or human TSHR monoclonal autoantibodies was not affected by the six mutations. Thermostable TSHR260-JMG55TM has been purified and crystallised without ligand and the structure solved at 2.83 Å resolution. This is the first reported structure of a glycoprotein hormone receptor crystallised without ligand. The unbound TSHR260-JMG55TM structure and the M22 and K1-70 bound TSHR260 structures are remarkably similar except for small changes in side chain conformations. This suggests that neither the mutations nor the binding of M22TM or K1-70TM change the rigid leucine-rich repeat domain structure of TSHR260. The solved TSHR260-JMG55TM structure provides a rationale as to why the six mutations have a thermostabilising effect and provides helpful guidelines for thermostabilisation strategies of other soluble protein domains.

A human monoclonal autoantibody to the thyrotropin receptor with thyroid-stimulating blocking activity.

BACKGROUND: Human monoclonal autoantibodies (MAbs) are valuable tools to study autoimmune responses. To date only one human MAb to the thyrotropin (TSH) receptor (TSHR) with stimulating activity has been available. We now describe the detailed characterization of a blocking type human MAb to the TSHR. METHODS: A single heterohybridoma cell line was isolated from the peripheral blood lymphocytes of a patient with severe hypothyroidism (TSH 278 mU/L) using standard techniques. The line stably expresses a TSHR autoantibody (5C9; IgG1/kappa). Ability of 5C9 to bind and compete with 125I-TSH or TSHR antibodies binding to the TSHR was tested using tubes coated with solubilized TSHR. Furthermore, the blocking effects of 5C9 on stimulation of cyclic AMP production was assessed using Chinese hamster ovary (CHO) cells expressing the wild-type human TSHR or TSHRs with amino acid mutations. MAIN OUTCOME: 5C9 IgG bound to the TSHR with high affinity (4 x 10(10) L/mol) and inhibited binding of TSH and a thyroid-stimulating human monoclonal autoantibody (M22) to the receptor. 5C9 IgG preparations inhibited the cyclic AMP-stimulating activities of TSH, M22, serum TSHR autoantibodies and thyroid-stimulating mouse monoclonal antibodies. Furthermore 5C9 reduced the constitutive activity of wild-type TSHR and TSHR with some activating mutations. The effect of different amino acid mutations in the TSHR on 5C9 biological activity was studied and TSHR Lys129Ala or Asp203Ala completely abolished the ability of 5C9 to block TSH-mediated stimulation of cyclic AMP production. CONCLUSIONS: The availability of 5C9 provides new opportunities to investigate the binding and biological activity of TSHR blocking type autoantibodies including studies at the molecular level. Furthermore, monoclonal antibodies such as 5C9 may well provide the basis of new drugs to control TSHR activity including applications in thyroid cancer and Graves' ophthalmopathy.

Primary healthcare renewal in Canada: a glass half empty?

Primary healthcare renewal was an important government initiative arising in the early 21st century. This sector of the healthcare system in Canada had been under-resourced and ignored for decades. Recent changes include the development of salaried models for physician care, the use of other professionals in primary care, the integration of inter-professional teams, funding for information management systems and some incentives to provide directed primary care services. However, these changes are limited by a lack of overall policy direction to drive innovation, the absence of a shift in the locus of control of healthcare, a lack of education for healthcare providers to support inter-professional team-based practices and a failure to be more accountable to the Canadian public's needs. Without these innovations, the primary healthcare system will again be overwhelmed by future healthcare needs. Based on these limitations, we question whether this renewal represents lasting change in primary healthcare or a band-aid solution to the continued issue of primary healthcare delivery.

TSH receptor antibodies.

The discovery of thyroid-stimulating autoantibodies by Adams and Purves 50 years ago was one of the most important observations in the history of thyroidology. Since that time, the thyroid-stimulating hormone receptor (TSHR) has been shown to be the antigen recognized by these autoantibodies (1974) and the receptor cloned (1989). More recently, different mouse monoclonal antibodies (MAbs) to the TSHR have been produced, culminating in 2002 in the preparation of mouse and hamster MAbs with strong thyroid-stimulating activity. Further, in 2003 a human MAb to the TSHR (M22) with the characteristics of patient thyroid-stimulating autoantibodies was described. M22 has been particularly useful in advancing our knowledge of the TSHR and TSHR autoimmunity, including the development of new assays for TSHR autoantibodies (2004) and determination of a high-resolution (2.55 A) crystal structure of the TSHR leucine-rich domain in combination with M22 (2007). The structure shows that M22 positions itself on the TSHR in an almost identical way to the native hormone TSH but the evolutionary forces that have resulted in production of a common autoantibody that mimics the actions of TSH so well are far from clear at this time. Very recently, a human MAb (5C9) with the characteristics of blocking-type patient serum TSHR autoantibodies has been isolated (2007). Studies on how 5C9 interacts with the TSHR at the molecular level are planned and should provide key insights as to the differences between TSHR autoantibodies with blocking and with stimulating activities. Also, 5C9 and similar MAbs have considerable potential as drugs to inhibit TSHR stimulation by autoantibodies. Further, now the M22-TSHR structure is known at the atomic level, rational design of specific low-molecular-weight inhibitors of the TSHR-TSHR autoantibody interaction is feasible.

Molecular interactions between the TSH receptor and a Thyroid-stimulating monoclonal autoantibody.

OBJECTIVE: To study the molecular interactions between the thyroid-stimulating hormone (TSH) receptor (TSHR) and a human thyroid-stimulating monoclonal autoantibody (M22). DESIGN: Amino acid mutations were introduced in the variable region gene sequences of M22 and the wild-type (WT) or mutated M22 Fab expressed in Escherichia coli. The ability of WT or mutated M22 Fab to inhibit binding of (125)I-TSH or (125)I-M22 to the TSHR and to stimulate cyclic adenosine monophosphate (AMP) production in Chinese hamster ovary cells expressing WT TSHRs was studied. Mutated TSHRs were also used in these studies in combination with WT or mutated M22 Fab to further identify interacting residues in the TSHR-M22 complex. MAIN OUTCOME: Out of 11 amino acid changes in the heavy chain (HC) of M22, 7 had an effect on M22 Fab biological activity, while in the case of 1 mutation the Fab was not expressed. In particular, stimulating activity of M22 Fab mutated at HC residues, D52, D54, and Y56 was markedly reduced. Mutation of M22 light chain (LC) D52 also reduced M22 Fab stimulating activity, while mutations at two further residues (LC D51 and LC D93) showed no effect. Reverse charge mutations at M22 HC D52 and TSHR R80 provided experimental evidence that these two residues interacted strongly with each other. CONCLUSION: Mutation of both the TSHR and M22 Fab has allowed identification of some residues critical for the receptor-autoantibody interaction. This approach should lead to detailed mapping of the amino acids important for M22 biological activity.

Crystal structure of the TSH receptor in complex with a thyroid-stimulating autoantibody.

OBJECTIVE: To analyze interactions between the thyroid-stimulating hormone receptor (TSHR) and a thyroid-stimulating human monoclonal autoantibody (M22) at the molecular level. DESIGN: A complex of part of the TSHR extracellular domain (amino acids 1-260; TSHR260) bound to M22 Fab was prepared and purified. Crystals suitable for X-ray diffraction analysis were obtained and the structure solved at 2.55 A resolution. MAIN OUTCOME: TSHR260 comprises of a curved helical tube and M22 Fab clasps its concave surface at 90 degrees to the tube length axis. The interface buried in the complex is large (2,500 A(2)) and an extensive network of ionic, polar, and hydrophobic bonding is involved in the interaction. There is virtually no movement in the atoms of M22 residues on the binding interface compared to unbound M22 consistent with "lock and key" binding. Mutation of residues showing strong interactions in the structure influenced M22 activity, indicating that the binding detail observed in the complex reflects interactions of M22 with intact, functionally active TSHR. The receptor-binding arrangements of the autoantibody are very similar to those reported for follicle-stimulating hormone (FSH) binding to the FSH receptor (amino acids 1-268) and consequently to those of TSH itself. CONCLUSIONS: It is remarkable that the thyroid-stimulating autoantibody shows almost identical receptor-binding features to TSH although the structures and origins of these two ligands are very different. Furthermore, our structure of the TSHR and its complex with M22 provide foundations for developing new strategies to understand and control both glycoprotein hormone receptor activation and the autoimmune response to the TSHR.

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. However, the mechanism by which the TMD of the TSHR and the other glycoprotein hormone receptors (GPHRs) becomes activated is unknown. METHODS: We have generated comparative models of the structures of the inactive (TMD_In) and active (TMD_Ac) conformations of the TSHR, follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) TMDs. The structures of TMD_Ac and TMD_In were obtained using class A GPCR crystal structures for which fully active and inactive conformations were available. RESULTS: Most conserved motifs observed in GPCR TMDs are also observed in the amino acid sequences of GPHR TMDs. Furthermore, most GPCR TMD conserved helix distortions are observed in our models of the structures of GPHR TMDs. Analysis of these structures has allowed us to propose a mechanism for activation of GPHR TMDs. CONCLUSIONS: Insight into the mechanism of activation of the TSHR by both TSH and TSHR autoantibodies is likely to be useful in the development of new treatments for Graves' disease.

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. The areas on the surfaces of GPHs that are known to interact with their receptors are not glycosylated and two areas free from glycosylation, not involved in currently known interactions, have been identified. The concave faces of GPHRs leucine-rich repeats 3-7 are free from glycosylation, consistent with known interactions with the hormones. In addition, four other non-glycosylated areas have been identified, two located on the receptors' convex surfaces, one in the long loop of the hinge regions and one at the C-terminus of the extracellular domains. Experimental evidence suggests that the non-glycosylated areas identified on the hormones and receptors are likely to be involved in forming intramolecular or intermolecular interactions.

A sensitive non-isotopic assay for acetylcholine receptor autoantibodies.

BACKGROUND: Detection and measurement of serum acetylcholine receptor autoantibodies (AChRAb) are useful in the diagnosis and management of myasthenia gravis (MG). An immunoprecipitation assay (IPA) based on AChR labelled with 125I-alpha bungarotoxin is widely used for measurement of AChRAb, but a non-isotopic assay of sensitivity and specificity comparable to IPA is not available as yet. METHODS: A new AChRAb ELISA, which is based on the ability of AChRAb to compete with 3 different AChR monoclonal antibodies (MAbs 1-3) for binding sites on affinity purified fetal and adult-type AChR preparations, is described. The sensitivity and specificity of the ELISA were assessed by comparing assay results with a conventional IPA. RESULTS: There was good agreement between the IPA and the ELISA for measurement of AChRAb (r = 0.85; n = 83; p <0.001). 76/83 MG sera were positive in the ELISA, whilst 72/83 were positive by IPA. Eight sera were positive in the ELISA (inhibition range 18%-46%) but negative by IPA (0.33-0.47 nmol/L toxin bound) and 4 sera were negative in the ELISA (inhibition range -1% to15%) whilst positive by IPA (0.56-2.9 nmol/L toxin bound). Overall 80/83 (96%) of the MG sera were AChRAb positive in one or both assays. In addition, all 191 control serum samples which were negative for AChRAb by IPA were below or equal to 16% of inhibition in our ELISA. The AChRAb ELISA also showed good inter-assay and intra-assay precision. CONCLUSION: The AChRAb ELISA we have described has sensitivity and specificity at least as high as our current radioactive IPA. It has good precision and good handling characteristics making it suitable for routine use.

Estimation of serum TSH receptor autoantibody concentration and affinity.

We have used the human monoclonal TSH receptor (TSHR) autoantibody (M22) as a labeled ligand in competition with individual patient TSHR autoantibodies (TRAb) to estimate their serum concentrations and affinities. TSHR coated tubes, (125)I-labeled M22 IgG and Fab, and patient sera IgG and Fab were used in these studies. In 15 patients with Graves' disease, TRAb concentrations ranged from 50 to 500 ng/mL of serum (5- 60 parts per million of total serum IgG) and TRAb IgG affinities from 3.0 +/- 1.0-6.7 +/- 1.54-10(10) L/mol (mean +/- SD; n=3). Fab fragment affinities were similar to those of intact IgG. Serum TRAb with blocking (TSH antagonist; 4 patients) activity had similar affinities (3.0 +/- 0.25-7.2 +/- 2.2-10(10) L/mol) to TRAb IgG from patients with Graves' disease, but blocking TRAb concentrations were higher (1.7 - 27 mg/mL of serum). The concentrations of TRAb that we observed in the sera of the 15 Graves' patient (0.33 - 3.3 nmol/L) can be compared with that of circulating TSH. In particular, a serum TSH concentration of 100mU/L (0.7 nmol/L) is in the same range as the concentrations of TRAb we observed. Such a TSH concentration (similar to that observed after injection of 0.9 mg of recombinant human TSH) would be expected to cause a similar degree of thyrotoxicosis as seen in Graves' disease. Consequently, the thyroid-stimulating potencies (i.e., activity per mol) of patient serum TRAb and human TSH appear to be of a similar magnitude in vivo as well as in vitro. Overall, our results indicate that serum TRAb affinities are high and show only limited variations between different sera whereas concentrations of the autoantibodies vary widely.

Effects of TSH receptor mutations on binding and biological activity of monoclonal antibodies and TSH.

The effects of an extensive series of mutations in the TSH receptor (TSHR) leucine-rich domain (LRD) on the ability of thyroid-stimulating monoclonal antibodies (TSMAbs) and TSH to bind to the receptor and stimulate cyclic AMP production in TSHR-transfected CHO cells has been investigated. In addition, the ability of a mouse monoclonal antibody with blocking (i.e., antagonist) activity (RSR-B2) to interact with mutated receptors has been studied. Several amino acids distributed along an extensive part of the concave surface of the LRD were found to be important for binding and stimulation by the thyroid-stimulating human MAb M22 but did not appear to be important for TSH binding and stimulation. Most of these amino acids important for M22 interactions were also found to be important for the stimulating activity of six different mouse TSMAbs and a hamster TSMAb. Furthermore, most of these same amino acids were important for stimulation by TSHR autoantibodies in a panel of sera from patients with Graves' disease. Amino acid R255 was the only residue found to be unimportant for TSH stimulation but critical for stimulation by all thyroid-stimulating antibodies tested (23 patient serum TSHR autoantibodies, M22, and all seven animal TSMAbs). About half the amino acids (all located in the N-terminal part of the LRD) found to be important for M22 activity were also important for the blocking activity of RSR-B2 and although the epitopes for the two MAbs overlap they are different. As the two MAbs have similar affinities, their epitope differences are probably responsible for their different activities. Overall our results indicate that different TSMAbs and different patient sera thyroid-stimulating autoantibodies interact with the same region of the TSHR, but there are subtle differences in the actual amino acids that make contact with the different stimulators.