Thyroid blocking antibodies in thyroiditis

Serum from a woman with a history of Hashimoto's thyroiditis, who had given birth to two children with congenital hypothyroidism, contained potent TSH blocking activity. Immunoglobulin preparation from this serum abolished completely TSH-stimulated cAMP production in human thyroid membranes. The blocking activity was associated with the IgG fraction absorbed to and eluted from a Protein A column. The stimulation of adenylate cyclase by a preparation of thyroid-stimulating antibodies from a patient with Graves' disease was also inhibited by the antibodies. In contrast, no effect was observed upon fluoride-stimulated cAMP production. The data indicate that the antibody activity was directed against the TSH receptor. Immunoglobulin preparations from 22 other patients with Hashimoto's thyroiditis and 16 patients with subacute thyroiditis were examined for the existence of TSH receptor blocking antibodies. A blocking activity was found in two of the 22 Hashimoto patients. No such activity was found in the patients with subacute thyroiditis. It appears that thyroid blocking antibodies sometimes contribute to hypothyroidism associated with Hashimoto's thyroiditis.     

Changes in thyrotropin receptor antibody after subtotal thyroidectomy in Graves' disease: comparison with the degree of lymphocytic infiltration in the thyroid

To evaluate changes in TSH receptor antibody after surgery in Graves' disease and its relationship with the degree of lymphocytic infiltration, serial serum levels of TSH receptor antibody were measured before and after the subtotal thyroidectomy in 50 patients with Graves' disease. In 22 (44%) out of 50 patients, thyrotropin binding inhibitor immunoglobulin (TBII) levels gradually decreased and disappeared completely within 12 months after surgery (TBII disappearing group). Twenty-eight (56%) patients showed persistent TBII activity and their levels were not changed until 12 months after surgery (TBII persistent group). The changes of thyroid stimulating antibody (TSab) levels were very similar to those of TBII in both groups. The thyroidal lymphocytic infiltration was more prominent in the TBII disappearing group. The degree of the decrease of TBII levels after surgery correlated with the grade of thyroidal lymphocytic infiltration. There was no significant difference of TSH receptor antibody (both TBII and TSab) levels between the thyroid and peripheral venous blood. These data suggest that the persistence or disappearance of TSH receptor antibody after surgery may reflect the difference between patients in whom the thyroid is the major site of TSH receptor antibody and those in whom additional sites of TSH receptor antibody synthesis exist.     

A novel bioassay for anti‐thyrotrophin receptor autoantibodies detects both thyroid‐blocking and stimulating activity

Autoantibodies to the thyrotrophin (TSH) receptor (anti‐TSHR) are unique, in that they are involved directly in the pathophysiology of certain autoimmune thyroid diseases (AITD). Thyroid‐stimulating antibodies (TSAb) act as agonists that activate the thyroid gland and cause Graves' disease. Other anti‐TSHR antibodies block TSH and can cause hypothyroidism. Thyroid‐blocking antibodies (TBAb) have not been studied as extensively as TSAb. We developed a TBAb bioassay based on a cell line that expresses a chimeric TSHR. The 50% inhibitory concentration of the chimeric Chinese hamster ovary (CHO)‐Luc cells was more than five‐fold lower compared with the wild‐type CHO‐Luc cells. We tested the performance of this bioassay using a thyroid‐blocking monoclonal antibody K1‐70, established an assay cut‐off and detected TBAb in 15 of 50 (30%) patients with AITD. Interestingly, the assay detects both TSAb and TBAb and measures the net activity of a mixture of both types of antibodies. There was a high correlation (R² 0·9, P < 0·0001) between the results of the TSAb assay and the negative percentage inhibition of the TBAb assay. The TBAb bioassay was approximately 20‐fold more sensitive than a commercially available TSHR binding assay (TRAb). In contrast to TRAb, sera with high levels of TBAb activity were able to be diluted several hundred‐fold and still exhibit blocking activity above the cut‐off level. Thus, this TBAb bioassay provides a useful tool for measuring the activity of anti‐TSHR antibodies and may help clinicians to characterize the diverse clinical presentations of patients with AITD.     

Determination and clinical significance of anti-idiotypic antibody--in model experiments of thyroglobulin and TSH]

Anti-idiotypic (anti-ID) antibody in test serum was determined by the direct binding assay using 125I-anti-human thyroglobulin (hTg). Several positive cases were found in Graves' disease and thyroiditis chronica. Positive anti-ID antibodies could be classified into two types. Type 1 showed the positive anti-hTg antibody and high Tg levels by RIA using double antibody method. Type 2 showed the positive anti-hTg antibody but low Tg levels by RIA. The binding of 125I-hTg to anti-hTg antibody was displaced by anti-ID antibody in type 1, but was not anti-ID antibody in type 2. A case of coexistence of autoantibody to hTSH and auto-anti-ID antibody to anti-hTSH antibody was found. She showed normal thyroid function (T4, T3), but TSH level showed discrepancy by different assay methods. Both autoantibodies for hTSH and for anti-hTSH antibody were demonstrated by the reaction of patient's antibody with both 125I-hTSH and 125I-anti-hTSH (MoAb). These two autoantibodies belong to the polyclonal IgG. The autoantibody for hTSH recognized only the beta-subunit of hTSH. Neither stimulating type of TSH receptor antibody (TRAb) nor blocking type of TRAb interfered with the binding of patient's anti-ID to 125I-anti-hTSH. This binding reaction could be inhibited by the unlabeled hTSH. This anti-ID might represent the internal image of the non-biological active site of TSH molecule, because of absence of thyroid stimulating activity. These anti-ID antibodies may provide evidence supporting a network theory of the immune system.     

Glycosylated ectodomain of the human thyrotropin receptor induces antibodies capable of reacting with multiple blocking antibody epitopes

Recently, we showed that the glycosylated ectodomain of the human thyrotropin receptor (hET-gp) reacts with autoantibodies from autoimmune thyroid disease (AITD) patients' sera. To better understand the effects of glycosylation of thyrotropin receptor (TSHR) in antibody induction, we immunized rabbits with hET-gp protein. The rabbits developed relatively high titers of antibodies with highly potent TSH binding inhibitory immunoglobulin (TBII) and thyroid stimulatory blocking antibody (TSBAb) activities. Both the hET-gp and a nonglycosylated ectodomain of the human TSHR (hETSHR) protein significantly reversed the TBII as well as TSBAb activity. Based on the ability of synthetic peptides to significantly reverse the functional activity of these rabbit antisera, we identified three discrete regions of the TSH R, represented by amino acids 202-221, 292-311 and 367-386, as TBII epitopes and four regions represented by amino acids 352-371, 367-386, 382-401 and 392-415 as TSBAb epitopes. These data demonstrate that rabbit antibodies that bind to amino acids 367-386 mediate their TSBAb activity by inhibiting the binding of TSH to TSHR; whereas, antibodies to regions 352-415, excluding aa 367-386, exert their TSBAb activity by affecting a step subsequent to TSH binding. Coincident with the elevation of TBII and TSBAb activity, serum total T4 levels declined and thus suggested that the antibodies exerted functional effects on thyroid in vivo. Together, these data demonstrate that glycosylated hET-gp protein is a more potent immunogen and it can induce a broader antibody response directed against multiple TBII and TSBAb epitopes.     

Heterogeneity of thyrotropin binding inhibitor immunoglobulins in serum from untreated patients with hyperthyroid Graves' disease.

We have previously established an assay for the simultaneous assessment of thyrotropin (TSH) binding inhibitor immunoglobulin (TBII) and thyroid stimulating autoantibody activities in cultured rat thyroid cells (FRTL-5 cell), and found a discrepancy in some patients with untreated Graves' disease between the activities of TBII measured in FRTL-5 cells (TBII-rc) and in solubilized thyroid membranes (TBII-pm). In three selected patients with untreated Graves' disease, the different dose-response relationship between TBII-rc and TBII-pm clearly indicated the heterogeneous populations of TBII-pm in patients' sera, with different binding affinities for TSH receptor in intact cells.     

大鼠脂肪细胞TSH受体机能研究

目的：研究TSH及Graves'TgC对大鼠脂肪细胞的作用。方法：获取大鼠游离的脂肪细胞。溶脱TSH受体。采用A蛋白尾析柱纯化Graves患者的IgG以脂肪细胞内cAMP浓度及释放到孵化液中甘油浓度作为脂肪分解指标,用cAMP及甘油试剂盒测定cAMP浓度及甘油浓度。结果：1mU／ml的TSll能增加大鼠雅高脂肪细胞的cAMP浓度及甘油的释放。脓苷引起cAMP及甘油的TSH剂量依赖曲线向右移。Gare’TgG抑制[125I]-TSH结合于大鼠脂肪细胞膜上ISH受体,并能刺激雅高脂肪细胞内cAMP的形成。结论：ISH受体功能性表达于脂肪细胞,其生物学效应是通过活化腺耷环化酶而实现的。     

Immunology of the thyrotropin receptor.

Antibodies to the thyrotropin receptor appear to be responsible for hyperthyroidism in Graves' disease. The antibodies, described as thyroid-stimulating antibodies (TSAb) mimic the effects of thyrotropin (TSH) by binding to the TSH receptor and activating adenylate cyclase. TSAb consist of an electrophoretically heterogeneous population of IgG and the thyroid-stimulating site is formed by combination of heavy and light chains in the Fab part of the molecule. Binding studies indicate that the TSAb molecule interacts monovalently with membrane bound TSH receptors and that TSAb consists of an antibody population which shows a restricted heterogeneity with regard to TSH receptor affinity. Studies in patients with Graves' disease and hyperthyroidism indicate that the levels of TSAb correlate well with thyroidal iodine uptake and the absence of pituitary control of thyroid function. However in some patients with ophthalmic Graves' disease or autoimmune thyroiditis there is evidence of serum antibodies which interact with the TSH receptor but are unable to stimulate thyroid function.     

Monoclonal antibodies to the thyrotropin receptor

The thyrotropin receptor (TSHR) is a seven transmembrane G-protein linked glycoprotein expressed on the thyroid cell surface and which, under the regulation of TSH, controls the production and secretion of thyroid hormone from the thyroid gland. This membrane protein is also a major target antigen in the autoimmune thyroid diseases. In Graves' disease, autoantibodies to the TSHR (TSHR-Abs) stimulate the TSHR to produce thyroid hormone excessively. In autoimmune thyroid failure, some patients exhibit TSHR-Abs which block TSH action on the receptor. There have been many attempts to generate human stimulating TSHR-mAbs, but to date, only one pathologically relevant human stimulating TSHR-mAb has been isolated. Most mAbs to the TSHR have been derived from rodents immunized with TSHR antigen from bacteria or insect cells. These antigens lacked the native conformation of the TSHR and the resulting mAbs were exclusively blocking or neutral TSHR-mAbs. However, mAbs raised against intact native TSHR antigen have included stimulating mAbs. One such stimulating mAb has demonstrated a number of differences in its regulation of TSHR post-translational processing. These differences are likely to be reflective of TSHR-Abs seen in Graves' disease.     

Associated expression of CD1 antigen and Fc receptor for IgE on epidermal Langerhans cells from patients with atopic dermatitis.

The presence of Fc receptors for IgE on epidermal Langerhans cells (LC) from patients with atopic dermatitis (AD) was demonstrated by three different types of experiments. Firstly, cell-bound IgE on LC was removed by acid elution and restored by highly purified human myeloma IgE (IgE kappa). Secondly, after pepsin digestion of cell-bound IgE the number of LC staining with anti-human light chain (kappa, lambda) antibodies significantly decreased in contrast to the number of LC staining with anti-human epsilon heavy chain antibody. Thirdly, LC formed rosettes with sheep red blood cells (SRBC) coated with IgE kappa. Epidermal LC from normal non-atopic controls, did not form rosettes with SRBC-IgE. The SRBC-IgE rosette formation could be inhibited by preincubation with IgE kappa and BB10 (MoAb directed against the Fc receptor for IgE on human eosinophils, platelets and macrophages), but also with human IgG, whereas the SRBC-IgG rosette formation could be inhibited neither by IgE kappa nor by BB10. Both the SRBC-IgE and the SRBC-IgG rosette formation could be inhibited by OKT6 (anti-CD1) antibody. The results of inhibition studies with OKT6 antibody on the reconstitution of IgE on epidermal LC after acid elution suggest an associated expression of the CD1 antigen and the Fc receptor for IgE.     

Evidence for two discontinuous regions on the thyrotropin receptor involved in the binding of the monoclonal antibody 34A

The human thyrotropin receptor (hTSHR) is a major autoantigen in thyroid autoimmunity. The aim of this study was to localize the discontinuous epitope recognized by the anti-hTSHR monoclonal antibody (mAb) 34A. We used the phage-displayed peptide technology and selected thirteen 34A-specific mimotopes which could be grouped into four families according to their sequence homologies. Mimotope sequence alignments on the primary sequence of hTSHR allowed us to identify regions 88-100 (family I homologous motif Tx(8)FYNL) and 276-281 (family IV homologous motif DxSYPS) as being putative parts of the discontinuous epitope recognized by the mAb 34A. Interestingly, by using the Spot method, TSH was also found to interact with peptides bearing amino acids from these two regions, suggesting their involvement in TSH/TSHR interaction. Moreover these data are in agreement with the ability of the mAb 34 to displace TSH binding to its receptor. In addition, purified IgG from nine patients with Graves' disease were able to specifically recognize family I-specific mimotopes, whereas those from healthy donors did not. Taken together, our data suggest the involvement of regions 88-100 and 276-281 in the epitope recognized by mAb 34A as well as purified IgG from patients' sera and provide a basis for rational guided mutagenesis.     

Anti-human interleukin 2 receptor monoclonal antibody isotypic switching: Chimeric rat-human antibodies

Univalent and bivalent (2Fab'-Fc) chimeric rat-human antibodies were constructed by chemical coupling (thioether bonds) of Fab' fragments from the 33B31 rat anti-human interleukin 2 receptor ( chain) monoclonal antibody to human IgG or Fc fragments. The purity of the chimeric antibodies obtained after purification was assessed by the sodium dodecyl sulfide-polyacrylamide gel electrophoresis method. The affinity of chimeric antibodies for the human interleukin 2 receptor was determined. The affinity of the 2Fab'-Fc was better (Kd = 0.87 nM) than that of the Fab'-IgG (Kd = 2.04 nM) or the Fab'-Fc chimera (Kd = 3.93 nM). Binding studies on a T-cell clone showed that the percentage of positive cells recognized by chimeric antibodies was comparable to that obtained with unmodified 33B3.1 IgG2a or its corresponding Fab' fragment. In addition, the inhibition of interleukin 2-induced cell proliferation and allogeneic proliferative response by chimeric antibodies was of the same magnitude as that obtained with the rat IgG2a anti-interleukin 2 receptor monoclonal antibody and Fab' fragments. This study shows the possibility of changing the isotype of monoclonal antibodies without important modification to their binding activity. These reagents may offer an alternative to unmodified monoclonal antibodies for therapeutic application.     

Thyrotropin receptor autoantibodies (TSHRAbs): epitopes, origins and clinical significance

Epitopes for > 95% stimulating thyrotropin receptor autoantibodies (TSHRAbs) causally implicated in Graves' disease (Basedow's disease or primary hyperthyroidism) have been identified on on the N-terminal portion of the TSHR extracellular domain, residues 8-165. If the stimulating TSHRAb activity is solely dependent on this region, it is termed homogeneous; if its activity is only largely related to this region, it is termed heterogeneous. The presence of a heterogeneous stimulating TSHRAb in a patient is associated with rapid responses to propylthiouracil or methimazole and may be predictive of long term remission with these oral immunosuppressives. Epitopes for two different Graves' autoantibodies that inhibit TSH binding, TSH binding inhibition immunoglobulins or TBIIs, have also been identified on this region of the TSHR. They do not increase cAMP levels, although one may activate the inositol phosphate, Ca++, arachidonate release signal system. The epitope of blocking TSHRAbs with the ability to inhibit TSH binding (TBII activity), TSH activity, and stimulating TSHRAb activity, and that are causally implicated in the primary hypothyroidism of patients with idiopathic myxedema or some patients with Hashimoto's disease have, in contrast, been largely identified largely on the C-terminal portion of the TSHR extracellular domain, residues 270-395. They have been implicated as important in pregnancy where they attenuate the signs and symptoms of Graves' hyperthyroidism. The appearance of these blocking TSHRAbs during pregnancy in Graves' patients might cause overt or occult hypothyroidism, with resultant effects on fetal development and postnatal intelligence levels. The different TSHRAbs can exist in the same patient at any moment in time, potentially making disease expression a sum of their activities. Assays taking advantage of the epitope mapping findings enable us to detect individual TSHRAbs within a single patient and to better understand their clinical significance.     

A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screening of human hybridoma antibody in culture supernatants

Endogenous immunoglobulin in tissue sections pose a problem in immuno-histochemical techniques employing homologous antibody as primary reagents and enzyme-labelled anti-immunoglobulin for the development. A method for the blocking of endogenous immunoglobulin in human tissue sections by incubation with monomeric pepsin fragments (Fab') of rabbit anti-human immunoglobulin before applying monoclonal antibody was evaluated for the screening of human monoclonal antibody. It was initially demonstrated that Fab' rabbit anti-human IgM and anti-IgG could block endogenous immunoglobulin in human IgM and IgG producing tumors thereby abolishing the binding of subsequently applied peroxidase-labelled anti-IgM or anti-IgG. Frozen sections of human colo-rectal adenocarcinomas show a variable background staining caused by the endogenous immunoglobulin. The background completely disappeared in the IgM system by preincubation with Fab' anti-IgM while the background was clearly reduced but not abolished in the IgG system. A human hybridoma supernatant containing IgM reactive with colo-rectal adenocarcinoma could easily be screened on frozen sections using this method. This approach should be generally useful for the screening of human antibody on human tissue sections.     

Intrathyroidal lymphocytes from non toxic multinodular goiter: no evidence for production of thyroid stimulating antibodies

Although an autoimmune pathogenesis for non toxic goiter has been suggested, reports concerning circulating antibodies to TSH receptor structures have been conflicting. Intra thyroid lymphocytes, capable of secreting IgG, have been shown to be involved in the pathogenesis of Graves' and Hashimoto's diseases; therefore, the ability of conditioned media obtained from intra thyroid lymphocyte culture, and of IgG purified from these media, to stimulate cAMP accumulation and [3H]-Thymidine (TdR) uptake in FRTL-5 cells was investigated. The activity of IgG produced "in vitro" was compared with that of circulating IgG. Thyroid tissue samples were obtained at surgery from 21 patients with non toxic multinodular goiter (MNG), 5 patients with active Graves' disease (GD), and from 10 normal subjects, undergoing neck surgery for non-thyroidal pathology. IgG purified from media of GD lymphocyte cultures stimulated both cAMP accumulation and [3H]-TdR in 5 out of 5 cases: all of the IgG purified from control or MNG lymphocyte culture media was not active in either assay. Circulating IgG did not affect cAMP accumulation or [3H]-TdR in any of the non toxic MNG cases: controls showed no changed at all. However, both activities represented were increased by GD IgG. Conditioned media from intra thyroid lymphocyte cultures significantly inhibited basal cAMP accumulation in 7 out of the 21 non toxic MNG samples and totally abolished the response in all GD patients. [3H]-TdR was not affected by IgG of any of the controls, but it had an inhibitory effect on 8 out of 21 non toxic MNG patients, and significantly stimulated [3H]-TdR in all GD patients. In conclusion, present data demonstrate that intra thyroid lymphocytes from non toxic MNG do not produce antibodies capable of mimicking TSH actions through the adenylate cyclase cascade. Conversely, soluble factors interacting in TSH-mediated functions of FRTL-5 cells are present in conditioned media of intra thyroid lymphocytes of GD and MNG thyroid lymphocytes of GD and MNG thyroid cultures.     

Modulation of Fas-mediated apoptosis of human thyroid epithelial cells by IgG from patients with Graves' disease (GD) and idiopathic myxoedema

The expression of two autoimmune thyroid diseases, GD and idiopathic myxoedema, is associated with antibodies to the thyroid-stimulating hormone (TSH) receptor. Thyroid stimulating antibodies (TSAb) in GD are TSH agonists and cause hyperthyroidism as well as goitre, whereas thyroid stimulation blocking antibodies (TSBAb) in idiopathic myxoedema are TSH antagonists and cause hypothyroidism and thyroid atrophy. We investigated the effect of antibodies to TSH receptor on Fas-mediated apoptosis of thyroid epithelial cells (thyrocytes). Human IgG was isolated from healthy donors, patients with GD and idiopathic myxoedema. Human thyrocytes were obtained from surgical specimens. Thyrocytes were cultured in the presence or absence of human IgG with or without interferon-gamma (IFN-γ) or IL-1β for a specified time. After incubation, we examined the level of cAMP in cultured supernatants and both Fas and Bcl-2 expression on thyrocytes. In addition, we examined anti-Fas-mediated apoptosis of thyrocytes. Fas expression on thyrocytes was significantly down-regulated by Graves' IgG and TSH, although idiopathic myxoedema IgG did not affect Fas expression on thyrocytes. Idiopathic myxoedema IgG abrogated the effect of TSH on both cAMP production and inhibition of Fas expression on thyrocytes. Treatment of thyrocytes with IL-1β or IFN-γ caused a marked augmentation of Fas expression on thyrocytes. The increase of Fas expression of thyrocytes induced by IL-1β or IFN-γ was significantly suppressed in the presence of TSH or Graves' IgG. Anti-Fas-induced apoptosis of thyrocytes was observed in thyrocytes treated with IL-1β or IFN-γ, but was markedly inhibited in the presence of TSH or Graves' IgG. Furthermore, idiopathic myxoedema IgG abrogated most of the inhibitory effect of TSH on Fas-mediated apoptosis of thyrocytes treated with IL-1β or IFN-γ. Bcl-2 expression of thyrocytes did not change after stimulation with TSH, Graves' IgG, idiopathic myxoedema IgG, IL-1β or IFN-γ. These results suggest that TSAb found in Graves' patients may be potentially involved in the development of goitre by inhibition of Fas-mediated apoptosis of thyrocytes. In addition, TSBAb inhibit the action of TSH and increase the sensitivity toward Fas-mediated apoptosis of thyrocytes, inducing thyroid atrophy seen in patients with idiopathic myxoedema.     

Analysis of circulating thyroid-stimulating activities in pregnant women with Graves' disease: differential measurement of activities induced by TSH receptor antibody and hCG]

Thyrotoxicosis in Graves' disease is often aggravated in early pregnancy and this aggravation is associated with postpartum relapse of thyrotoxicosis. To examine whether thyroid-stimulating TSH receptor antibody (TSAb) or human chorionic gonadotropin (hCG), which also has thyroid-stimulating activity (TSA), is responsible for this early aggravation, the respective TSA due to TSAb or hCG were evaluated with a sensitive cAMP accumulation assay using FRTL-5 cells. TSA, which was detectable in all of 11 women in normal early pregnancy, correlated positively with serum hCG level, but was abolished completely by the pretreatment of serum samples with the solid-phase hCG antibody coupled with Sepharose-4B. Total TSA in the model samples of mixture of Graves' and pregnant sera (Gr + Preg), was reduced by the pretreatment with the solid-phase antibody, just corresponding with the reduction in hCG-induced TSA. Total TSA in early pregnant sera in 15 patients with Graves' disease, decreased significantly but was still positive even by the pretreatment with the hCG antibody. Pregnancy-associated changes in TSA was examined serially in a patient with Graves' disease, and hCG-induced TSA increased predominantly along with the serum thyroid hormone in early aggravation period. These data indicate that (1) the respective TSA due to TSAb or hCG can be differentially measured by using the solid-phase hCG antibody and (2) hCG plays an important role for aggravation of Graves' thyrotoxicosis in early pregnancy.     

A novel hypothesis for the etiology of Graves' disease: TSAb may be thyroid stimulating animal IgG-like hormone and TBAb may be the precursor of TSAb

There are doubtful points about the theory that autoimmunity with auto-antibody (Ab) to TSH receptor (R) causes hyperthyroidism in Graves' disease (GD). A main doubtful point is no curative effect of corticosteroid on Graves' hyperthyroidism in spite of curative effect of corticosteroid for all autoimmune diseases. Recently we demonstrated the immunological similarity of TSAb and TBAb-IgG to animal IgGs, except for human (h)IgG, by neutralization and purification of TSAb and TBAb-IgG using (1) heterophilic Ab to animal IgG in GD sera and (2) experimentally generated anti-animal IgG Abs [such as dog (d), bovine (b), porcine (p), and rabbit (rb)]. Furthermore, greater immunological similarity of Fab- and F(ab')(2)-portion of TSAb- and TBAb-IgG to bovine Fab, compared to hFab, was demonstrated using goat anti-bovine F(ab')(2) Ab. Existence of b and p TSH-like portions in the LATS-IgG molecule (probably Fab portion) was suggested by a previous report of neutralization of LATS activity by anti-b- or anti-p-TSH Ab. We suggested the existence of a mammalian animal-TSH-like structure, excepting hTSH, in the TSAb-IgG molecule (probably Fab portion), by discovery of anti-mammalian TSH Ab (such as d, b, p, guinea-pig, rat, whale, except h) in sera of GD. Lately, similar TSHR binding of H- and L-chain of human stimulating monoclonal TSHR Ab (M22)-Fab with TSH-α and-β subunit was reported. This evidence suggests that Fab portion of TSAb has a structure like mammalian TSH, but not hTSH. IgG-λ type of d, horse, b, p, goat, ovine is 95% and IgG-κ type is 5%, while human κ and λ chain is 60:40. Previous report that LATS (TSAb)-IgG composed of predominant λ type is supporting evidence that TRAb-IgG has immunological similarity with these animal IgGs compared to hIgG. We speculate that TSAb-IgG may be referred as a mermaid consisted in face (Fab) and trunk-leg (Fc). Face may be a kind of hormone with animal TSH-like structure and trunk-leg has animal IgG-like structure (in spite of no antibody function). There are many reports for co-existence of TSAb and TBAb-IgG in sera of GD. We reported conversion from TBAb (non-thyroid stimulating type IgG) to TSAb by co-incubation of anti-hIgG Ab (containing anti-animal IgG Ab as a cross-reaction) with TBAb-bound porcine thyroid cells. Thus, we suggest that TBAb may be the precursor form of TSAb.     

Renaissance of the blocking antibody concept in type I allergy

Formation of IgE antibodies against per se harmless antigens (i.e. allergens) is the hallmark and key pathomechanism of type I allergy, a hypersensitivity disease affecting more than 25% of the population. Classical experiments performed more than 65 years ago demonstrated that allergen-specific IgG antibodies, termed blocking antibodies, can antagonize the cascade of allergic inflammation resulting from allergen recognition by IgE antibodies. However, controversial results have questioned the protective role of IgG antibodies in allergic diseases. Here, we review recent data demonstrating that blocking antibodies inhibit allergen-induced release of inflammatory mediators from basophils and mast cells as well as IgE-facilitated allergen presentation to T cells, thus leading to suppression of T cell activation. Furthermore, it has been reported that the development of blocking antibodies is associated with reduced boosts of allergen-specific IgE production in patients receiving allergen-specific immunotherapy. These findings suggest that blocking antibodies have protective activity by inhibiting immediate as well as late inflammatory responses and long-term ameliorating activity on the allergic immune response by antagonizing the underlying IgE production. Induction of blocking antibodies is thus an important mechanism underlying allergen-specific immunotherapy. In addition, passive administration of blocking antibodies may be considered as a potential therapeutic strategy for allergic diseases.     

How one TSH receptor antibody induces thyrocyte proliferation while another induces apoptosis

Thyroid stimulating hormone (TSH) activates two major G-protein arms, Gsα and Gq leading to initiation of down-stream signaling cascades for survival, proliferation and production of thyroid hormones. Antibodies to the TSH receptor (TSHR-Abs), found in patients with Graves' disease, may have stimulating, blocking, or neutral actions on the thyroid cell. We have shown previously that such TSHR-Abs are distinct signaling imprints after binding to the TSHR and that such events can have variable functional consequences for the cell. In particular, there is a great contrast between stimulating (S) TSHR-Abs, which induce thyroid hormone synthesis and secretion as well as thyroid cell proliferation, compared to so called “neutral” (N) TSHR-Abs which may induce thyroid cell apoptosis via reactive oxygen species (ROS) generation.In the present study, using a rat thyrocyte (FRTL-5) ex vivo model system, our hypothesis was that while N-TSHR-Abs can induce apoptosis via activation of mitochondrial ROS (mROS), the S-TSHR-Abs are able to stimulate cell survival and avoid apoptosis by actively suppressing mROS. Using fluorescent microscopy, fluorometry, live cell imaging, immunohistochemistry and immunoblot assays, we have observed that S-TSHR-Abs do indeed suppress mROS and cellular stress and this suppression is exerted via activation of the PKA/CREB and AKT/mTOR/S6K signaling cascades. Activation of these signaling cascades, with the suppression of mROS, initiated cell proliferation. In sharp contrast, a failure to activate these signaling cascades with increased activation of mROS induced by N-TSHR-Abs resulted in thyroid cell apoptosis.Our current findings indicated that signaling diversity induced by different TSHR-Abs regulated thyroid cell fate. While S-TSHR-Abs may rescue cells from apoptosis and induce thyrocyte proliferation, N-TSHR-Abs aggravate the local inflammatory infiltrate within the thyroid gland, or in the retro-orbit, by inducing cellular apoptosis; a phenomenon known to activate innate and by-stander immune-reactivity via DNA release from the apoptotic cells.