Possible participation of colloid antigen 2 and abhormone (IgG with hormone activity) for the etiology of Graves' disease.

The theory that antibody (Ab) directed against the TSH receptor (TSHR) (TSHRAb) is the causal factor of Graves' disease seems unlikely. Corticosteroids have not had a curative effect on the hyperthyroidism of Graves' disease despite their effectiveness for other autoimmune diseases. Two kinds of TSHRAb, thyroid-stimulating Ab (TSAb) and thyroid-blocking Ab (TBAb), are known as causal factors of hyperthyroidism and hypothyroidism, respectively. Previously, we reported that TSAb may be thyroid stimulating animal IgG-like hormone and TBAb may be the precursor of TSAb. In this paper we suggested that TBAb (precursor) converts to TSAb (active form) via the action of the protease, colloid antigen 2 (CA2). We speculate that the conversion of TBAb to TSAb is controlled by two factors: the protease and an anti-protease Ab. When anti-protease Ab levels are high, the patient exhibits hypothyroidism due to the increase in TBAb levels caused by neutralization of the protease. When anti-protease Ab levels are negative, the patient's hypothyroidism disappeared by the negative serum TBAb due to increased protease. An immunoglobulin G (IgG) with enzyme activity is known as an abzyme, which may be an undeveloped form. IgG with hormone activity may be likewise called an abhormone, which could also be an undeveloped form. The tumor marker CEA is a known member of the IgG supergene family. Many ancestral versions of proteins may have been produced as an IgG form. Possible participation of colloid antigen 2 and abhormone for the etiology of Graves' disease is suggested.

Antithyroid Drugs Inactivate TSH Binding to the TSH Receptor by their Reducing Action.

Backgroud and Objective: Antithyroid drugs (ATDs) [methylmercaptoimidazole (MMI) and propylthiouracil (PTU) ] are used to treat hyperthyroidism in Graves' disease. The effect of ATDs and reducing agents (mercaptoethanol, dithiothreitol and cysteine) on bovine (b) TSH binding to human (h) and porcine (p) TSH receptor (R) was examined. METHODS AND RESULTS: (1) ATDs was pre-incubated with hTSHR coated tube for 1- 4 h, washed free of ATDs, and then 125I-bTSH binding to hTSHR after 1 h incubation was examined. MMI (10-40 mM) decreased 125I-bTSH binding in a dose-dependent manner and binding decreased proportionally as preincubation time increased from 1 to 4 h. PTU (10mM) slightly decreased binding, When reducing agents were pre-incubated with hTSHR for 2 h, 125I-bTSH binding similarly decreased. (2) Porcine thyroid membrane was pre-incubated with both agents for 2 h. Then, the washed or unwashed membrane was incubated with 125I-bTSH for 1 h. 125I-bTSH binding in both methods decreased. (3) When the effect of ATDs or reducing agents on the biological activity of 125I-bTSH and thyroid stimulating antibody (TSAb) was examined after gel-filtration of 125I-bTSH- and TSAb- treated with both reagents for 1 h, no inactivation was observed. (4) ATDs showed similar reducing action as reducing agents because iodine (I+) was reduced to I- by ATDs. CONCLUSION: ATDs inactivate the TSH-binding site of TSHR by reduction, although ATDs do not inactivate bTSH and TSAb activity. This suggests that TSAb would not stimulate the thyroid due to the inactivation of the TSHR when ATDs are administered to patients with Graves' disease.

A novel method for measuring TBAb activity in TSAb- and TBAb-positive serum.

We reported a conversion assay in which thyroid blocking antibody (TBAb) function as thyroid stimulating antibody (TSAb). TBAb-bound porcine thyroid cells (PTC) were made by incubating TBAb(+) serum and PTC for 1 hour. When these TBAb-bound PTC were incubated 4 h with rabbit anti-human (h) IgG antibody (Ab), cAMP production was high, but when incubated with normal rabbit serum (NRS) cAMP production was low. TBAb-Mnoclonal Ab (MoAb) (KI-70) showed similar conversion. However, when TSAb-MoAb(M22) was assayed, anti-hIgG Ab-produced cAMP was lower than NRS-produced cAMP. When a mixture of M22 and KI-70 was assayed, anti-hIgG Ab-produced cAMP was higher than NRS. Thus, it is possible to determine existence of TBAb in TSAb(+)serum when anti-IgG Ab-produced cAMP is higher than NRS-produced cAMP. In this assay TBAb activity in TSAb(+)serum was scored as positive, gray zone and negative when the difference [anti-hIgG Ab-produced cAMP(%)-NRS-produced cAMP(%)] was >100%, 50-100% and <±50%, respectively. In TSAb(+)sera of Graves' patients with no treatment or anti-thyroid therapy, positive TBAb was 9% (3/33 )and 6.9% (5/72), and gray zone was 18 % (6/33) and 25% (18/72), respectively. A low prevelance of TBAb and low TBAb activity (<200% as cAMP) was found in these Graves' patients. A radioisotope treated Graves' patient showed existence of both TSAb and TBAb at 5 months (NRS, 800% cAMP and anti-IgGAb,1,350% cAMP), and highly positive TBAb (NRS, 180% cAMP and anti-hIgG Ab, 3,200% cAMP) at 30 months. This conversion assay is useful principally for TBAb determination but is also useful for TBAb determination in TSAb(+)serum.

Conversion of TBAb response to TSAb response by anti-human IgG antibody.

Previously, we reported the conversion phenomenon (CP) of thyroid blocking antibody (TBAb) to thyroid stimulating antibody (TSAb) by induced cAMP production during incubation of TBAb-bound porcine thyroid cells (PTC) with rabbit anti-IgG Ab. In the present experiment we examined the CP by TBAb-positive sera with high TSH binding inhibitor immunoglobulin (TBII) activity in primary hypothyroidism. Two patients with extremely high TBII patients; patient No.1 (35 yo male) with TSH 26.5µU/ml, TSAb negative, TBII 4,600 U/L, TBAb100% and patient No.2 (40 yo female) with TSH 4.5µU/ml, TSAb negative, TBII 1,620 U/L, TBAb 99.8% were examined. Cyclic AMP production was examined by 2nd incubation (3h) of anti-IgG Ab with TBAb-bound PTC that was made by 1st incubation (0.5h) of TBAbpositive serum and PTC. When sera (0.001-0.05 ml) of patient No.1 and No.2 were tested, cAMP production showed 980- 3,700% and 570-3,000% in a dose-dependent manner, respectively. Cyclic AMP production was also observed by anti- IgG fragments Ab [(Fab')2, Fab and light chain]. Cyclic AMP production by anti-F(ab')2 was higher than anti-Fab Ab, and cAMP by anti-κ Ab was significantly higher (>3 fold) than anti-λ Ab. Cyclic AMP production by TBAb-positive sera with high TBII activity (35-270 U/L) showed a correlation with serum TBII activity (R=0.76). The fact that all high TBAb-positive sera show the CP of TBAb to TSAb suggests that TSAb activity may be present in TBAb molecule and TBAb may be the precursor of TSAb.

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-ß 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.

Immunological similarity of thyroid stimulating antibody (TSAb) and thyroid blocking antibody (TBAb) with animal IgG.

Previously we reported neutralization and partial purification of TSAb and TBAb activity using heterophilic antibody (Ab) to animal IgG from Graves' disease. Thus, we examined immunological similarity of TSAb and TBAb with animal IgG using experimentally generated anti-animal IgG [dog (d), bovine (b), porcine (p) and rabbit (rb)] Abs. TBII activity of TSAb- and TBAb-positive serum was neutralized by these anti-animal IgG Abs. Applied TSAb- or TBAb-IgG protein (purified by Protein A) on these anti-animal IgG Abs-bound column was found mainly in the unbound fraction (UF) (>65%) and partially in the bound fraction(BF) (<35%). The TBII and TSAb activity of TSAb-IgG in the BF showed significantly higher than the UF. Thus, the ratio of TBII activity (U/L)/mg protein in the BF/UF was high. TBII activity of TBAb-IgG was similarly purified by this column. We examined immunological characteristics of TSAb-and TBAb-Fab or F(ab')2 using rabbit anti-bF(ab')2 Ab. TBII and TSAb activity of TSAb-Fab or- F(ab')2 and TBII activity of TBAb-Fab or -F(ab')2 were neutralized by anti-bF(ab')2 Ab. Partial purification of TSAb- or TBAb-Fab and -F(ab')2 by anti-bF(ab')2 Ab-bound column was also possible. Immunological similarity of TSAb- and TBAb-IgG with animal IgG such as d, b, p, rb by anti-animal IgG Ab, and TSAb- or TBAb-Fab and -F(ab')2 with bFab by anti-bF(ab')2 Ab were demonstrated. These fact suggest that both Fab and Fc portion of TSAb- and TBAb-IgG molecule have immunological similarity with animal IgG.

Neutralization and purification of thyroid stimulating antibody (TSAb) and thyroid blocking antibody (TBAb) by heterophilic antibody to animal IgG in Graves' disease.

There are several reports that sera from Graves' patients contain heterophilic antibody (Ab) to animal IgG such as human anti-mouse antibody (HAMA). We examined the binding of TSAb and TBAb with heterophilic Ab. The binding of animal IgG with patient's IgG was examined by the inhibition of animal IgG on the binding of labeled bovine (b) IgG with patient's IgG. The binding to labeled bIgG was detected in the serum of 5 patients (2.7 %) among 185 patients with Graves' disease. The binding of the labeled bIgG with patient's IgG was inhibited by animal serum or the crude IgG (45% ammonium sulfate fraction of serum)(such as dog, horse, bovine, porcine, goat, ovine, rabbit, guinea-pig, rat, mouse) except human, monkey and chick. This heterophilic Ab which had cross-reaction with mammalian IgG (except human, monkey) was used as human anti-animal IgG Ab. TBII and TSAb activity of TSAb-positive serum, and TBII activity of TBAb-positive serum were neutralized by incubation with this Ab-bound column. Partial purification of TSAb- or TBAb- IgG from Protein A-purified TSAb- or TBAb-IgG was possible using this Ab-bound column. TBII and TSAb activity of TSAb-IgG and TBII activity of TBAb-IgG were neutralized by incubation with rabbit anti-human (h) IgG Ab (having cross-reaction with animal IgG). Further purification of Protein A-purified TSAb-IgG or TBAb-IgG by rabbit anti-hIgG Ab-bound column was impossible. The binding of TSAb and TBAb with heterophlic Ab means that TSAb-and TBAb-specific IgG have immunological similarity with mammalian species IgG compared to human IgG.

Inhibitory effect of thyroid blocking antibody (TBAb) on the thyroid stimulatory effect of human chorionic gonadotropin (HCG) and equine chorionic gonadotropin (ECG).

We examined the inhibitory effect of thyroid blocking antibody (TBAb) on the thyroid stimulating activity of human chorionic gonadotropin (HCG) and equine CG (ECG). Five TBAb positive sera obtained from patients who had been hypothyroid but were currently on T4 treatment. The TSH binding inhibitory immunoglobulin (TBII) activities of the sera were 60-160 IU/L. Inhibition of TSH binding to the TSH receptor (TSHR) [TSH binding inhibition (TBI) activity] of HCG or ECG, and inhibition of TBAb on HCG or ECG-stimulated cAMP production were examined. Both HCG and ECG preparations showed weak TBI activity in the presence of small amounts of protein [bovine serum albumin (BSA)] but were negative in the presence of large amounts of protein [normal human serum (NHS) or BSA]. Four thousand IU/mL of HCG and ECG preparation caused cAMP production similar to 100 microU/mL of bovine (b) TSH. The inhibitory effect of TBAb on cAMP production by this amount of HCG or ECG was then examined. The inhibitory effect of TBAb on cAMP production by HCG and ECG was similar to bTSH, and TBAb positive sera with more than 40 IU/L TBII activity completely blocked cAMP production by HCG, ECG and bTSH. This suggests that common alpha -subunit of both HCG and TSH are involved in the inhibitory effect of TBAb. Previous reports demonstrated that the thyroid stimulating activity of thyroid stimulating antibody (TSAb) was blocked by deglycosylated HCG (competitive antagonist of TSH binding to TSHR). The fact and our present study suggest that TSH, HCG ECG, TSAb and TBAb have a similar binding site (alpha-subunit-mimicking binding site) on the TSH receptor.

The effects of nonionic polymers on thyroid stimulation and TSH receptor binding by the human monoclonal TSH receptor autoantibody M22.

BACKGROUND: Nonionic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), and dextran amplify the ability of thyroid stimulating antibodies (TSAbs) from patients with Graves' disease to stimulate cAMP production in thyroid cells. Therefore we sought to determine if nonionic polymers also augment the effects of the human thyroid stimulating monoclonal antibody (M22) on isolated thyroid cells. METHODS: The ability of nonionic polymers to alter the effects of M22 on certain parameters in porcine thyroid cells was examined. These parameters were augmentation of cAMP production (TSAb activity), inhibition of bovine thyrotropin (bTSH)-induced cAMP production (TBAb activity), and inhibition of bTSH binding to the TSH receptor (TSHR) (TBI activity). RESULTS: Stimulation of cAMP production by M22 in porcine thyroid cells was augmented by PEG, PVA, and dextran in a manner similar to that of Graves' serum. In contrast, TSH-stimulated cAMP production was not increased by nonionic polymers. M22-stimulated cAMP production was completely inhibited by the sera of patients with TBAb activity, and this inhibition was diminished by PEG. M22 and TBI activity in first and second generation assays and this activity was not affected by PEG. Binding of biotin-M22 to TSHR-coated plate wells (third generation assay) was not significantly increased by co-incubation with polymers. PEG augmented the binding of (125)I-M22 to TSHR-coated tubes by twofold, but this was associated with a threefold increase in nonspecific binding. There was no increase in total and nonspecific (125)I-TSH binding. This means that PEG has less than a twofold augmentative effect on (125)I-M22 binding to the TSHR. CONCLUSION: Nonionic polymers have similar effects in augmenting cAMP production in porcine thyroid cells in response to stimulation either by M22 or Graves' disease serum. The mechanism of this effect on the thyroid stimulating activity of M22 is unclear. The hypothesis that nonionic polymers augment M22 thyroid stimulation by increasing the mass of M22-occupied TSH receptors is not supported by the present study.

Increased receptor binding by bovine (b) TSH bound to monoclonal antibody to bTSHbeta-subunit.

TSH receptor (R) binding and cAMP production by bovine (b) TSH-bound to a monoclonal antibody (MoAb) or polyclonal antibody (Ab) to bTSH were examined, using TSH receptor (R) coating tube and porcine thyroid cells. (125) I-bTSH bound-to MoAbs to bTSH(alpha) or discontinuous type MoAb showed TSHR binding (10%) similar to intact (125) I-bTSH. TSHR binding was completely decreased (<2%) when (125) I-bTSH was bound by polyclonal Abs to bTSH(alpha) in Graves' patient or rabbit polyclonal Abs to bTSH. When either of the two MoAb (No. 1 and 2) to bTSH(beta) was bound to (125) I-bTSH, TSHR binding was 4 times higher (40%) compared to intact (125) I-bTSH. Binding of another MoAb (No. 3) caused no increased binding. TSHR binding of intact (125) I-bTSH was decreased from 10% to 2% by excess amounts of bTSH. Binding of (125) I-bTSH bound to MoAb to bTSH(beta) (No. 1 and 2) decreased from 40% to 30% by excess amounts of bTSH. When (125) I-bTSH bound-Fab of MoAb was used, the binding was reduced from 30 to 10% (No. 1) and from 25 to 6% (No. 2), respectively. In contrast, cAMP production by bTSH was decreased by pre-binding of all MoAbs and polyclonal Abs. Binding of (125) I-MoAb to bTSH (beta) to a synthetic peptide array of bTSH (beta) sequence was examined by the radioautography. The epitope of MoAb to bTSH(beta) was suggested to be LPK (beta 42-44) for No. 1, KLF (beta 39-41) for No. 2 and PKYA (beta 43-46) for No. 3, respectively, although the existence of discontinuous epitope could not be ruled out. The increased TSHR binding and the decreased cAMP production by bTSH bound to MoAbs may be due to the conformational change of TSH molecule or TSHR by binding of both bTSH and MoAb.

Sensitive thyroid-stimulating antibody assay with high concentrations of polyethylene glycol for the diagnosis of Graves' disease.

The aim of the present study was to determine the usefulness of a newly developed thyroid-stimulating antibody (TSAb) assay. We developed a highly sensitive TSAb (sTSAb) assay with 22.5% polyethylene glycol-precipitated crude IgG. The thyroid-stimulating hormone (TSH) receptor antibody (TRAb) causes Graves' disease and TRAb has been measured as TSH-binding inhibitor immunoglobulin (TBII) and thyroid-stimulating antibody (TSAb). The TSAb stimulates the thyroid glands and causes hyperthyroidism. In addition to investigating the usefulness of the newly developed sTSAb assay, we also investigated the frequencies of positive TRAb in thyrotoxic patients with subacute thyroiditis, painless thyroiditis or a solitary toxic nodule. We studied 700 untreated Graves' patients with hyperthyroidism and 923 normal controls. We also studied thyrotoxic patients with subacute thyroiditis, painless thyroiditis or a solitary toxic nodule. Conventional TSAb (cTSAb) and sTSAb were measured as TSAb, whereas porcine TBII (pTBII) and human recombinant TBII (hTBII) were measured as TBII. Levels of cTSAb and sTSAb were determined in 923 normal controls and 629 untreated Graves' patients and cTSAb and sTSAb were found to be normally distributed in normal controls, but not in untreated Graves' patients. Receiver operating characteristic (ROC) curve analysis demonstrated that cTSAb and sTSAb had high sensitivity and specificity for Graves' disease. Of the patients investigated, 96.5% of untreated Graves' patients were positive for sTSAb and/or pTBII. Some untreated Graves' patients who were negative for cTSAb were positive for sTSAb. Paired determinations of cTSAb and sTSAb were performed in 146 untreated Graves' patients. A positive correlation was found between cTSAb and sTSAb. Titres of sTSAb were higher than those of cTSAb and sTSAb had high sensitivity. Of the 35 untreated Graves' hyperthyroid patients who were negative for cTSAb, 18 (51%) were positive for sTSAb. Of the 36 untreated Graves' patients who were negative for hTBII, nine (25%) were positive for sTSAb. Some untreated Graves' patients who were negative for cTSAb were positive for sTSAb and some who were negative for hTBII and pTBII were positive for sTSAb. 5. Some thyrotoxic patients with subacute thyroiditis or painless thyroiditis were positive for TRAb. However, the frequency of TRAb-positive patients was low in this group. None of the patients with a solitary toxic nodule was positive for TRAb. In conclusion, sTSAb had higher sensitivity than cTSAb. Graves' patients who were cTSAb negative and hTBII negative could be sTSAb positive. The sTSAb indicates TSAb activity, but pTBII and hTBII do not necessarily do so. We recommended that the sTSAb is used in Graves' patients.

Demonstration of anti-TSH antibody in TSH binding inhibitory immunoglobulin-positive sera of patients with Graves' disease.

OBJECTIVES: The presence of anti-TSH antibodies in Graves' patients with unusually low TSH binding inhibitory immunoglobulin (TBII) has been reported. Recently, we found the first case of an anti-TSH antibody in TBII-positive sera of patients with Graves' disease. The prevalence and immunological specificity of this anti-TSH antibody were examined. DESIGN: The presence of 125I-bovine(b) TSH binding antibody in TBII positive serum was examined by prolonged incubation of more than 1 day because only weak binding occurred after 1 h incubation at 37 degrees C. The clinical course of these patients and binding characteristics of anti-bTSH antibody were examined. RESULTS: The corrected method-TBII activity (%)[1 - (a - b)/(c - d)] x 100 and the standard method-TBII activity (%) [1 - (a - d)/(c - d)] x 100 [a, 125I-bTSH binding with TSH receptor (R) in the presence of test serum; b, 125I-bTSH binding with test serum; c, 125I-bTSH binding with TSH R in the presence of normal serum; d, 125I-bTSH binding with normal serum] were calculated. The corrected method-TBII activity was always higher than the standard method-TBII activity in anti-bTSH antibody-positive serum. Anti-bTSH antibody-positive cases in TBII-positive Graves' disease were found in approximately 1% of Graves' patients. Anti-bTSH antibodies were confirmed as IgG from the increase of precipitated radioactivity by adding rabbit antihuman IgG antibody after the incubation of 125I-bTSH with test serum. These antibodies bind with not only bTSH, bTSH(alpha) and bLH, but also porcine (p)TSH, pTSH(alpha) and pFSH. However, these antibodies did not bind with human TSH. Binding of 125I-bTSH with patient's serum was neither inhibited by other Graves' thyroid stimulating antibody (TSAb), nor thyroid blocking antibody (TBAb) in primary hypothyroidism. CONCLUSIONS: The presence of anti-bTSH antibody in TBII-positive serum of high titre means that TBII-positive sera cannot rule out the absence of anti-bTSH. Thus, determination of 125I-bTSH binding with test serum in TSH receptor assays is necessary to determine the precise TBII activity and to detect anti-bTSH antibody.