Serum thyrotropin receptor antibodies concentrations in patients with Graves' disease before, at the end of methimazole treatment, and after drug withdrawal: evidence that the activity of thyrotropin receptor antibody and/or thyroid response modify during the observation period.

AIM AND METHODS: We performed a quantitative retrospective analysis of serum thyrotropin receptor antibody (TRAb) concentrations measured by a second-generation radioreceptor assay in 58 patients with Graves' disease (GD) at the onset of the disease, at the end of 18 month methimazole (MMI) treatment, and after MMI withdrawal in order to evaluate the correlation between the presence of these antibodies and the relapse of hyperthyroidism. Sixty healthy subjects were enrolled as a control group. RESULTS: Before MMI treatment the best cutoff TRAb value for identifying patients with GD was 1.45 UI/L (specificity, 100%; sensitivity, 98.3%). At the end of MMI treatment, serum TRAb concentrations were significantly lower (p < 0.001) than those measured at baseline, but they were still significantly higher (p < 0.001) than those found in the control subjects. At the end of MMI treatment, 44 patients (75.9%) had positive TRAb values (>1.45 UI/L). After MMI withdrawal (median, 15 months), 34 patients (58.6%) became hyperthyroid, 4 patients (6.9%) became hypothyroid, and 20 patients (34.5%) remained euthyroid. There was a significant correlation between serum TRAb concentrations at the end of MMI treatment and the percentage of patients who became hyperthyroid (r: 0.56; p < 0.001) and the time of appearance of hyperthyroidism (r: -0.38; p = 0.03). All 4 patients with TRAb values below 0.9 UI/L at the end of MMI treatment remained euthyroid throughout the follow-up period. Among the 27 patients who had serum TRAb values higher than 4.4 UI/L, 23 developed hyperthyroidism and 4 hypothyroidism. The TRAb values between 0.9 and 4.4 UI/L did not discriminate between the 27 patients (46.6%) who remained euthyroid from those who had relapse of hyperthyroidism. Thus a different TRAb end of treatment cutoff was calculated to identify patients who became again hyperthyroid. This TRAb cutoff value was 3.85 UI/L (sensitivity, 85.3%; specificity, 96.5%). All but 1 patient who had serum TRAb values above 3.85 UI/L became hyperthyroid after MMI was withdrawn (positive predictive value, 96.7%). In these patients, relapse of hyperthyroidism was independent of the changes in serum TRAb concentrations (r: 0.27; p = 0.15) and occurred after a median period of 8 weeks (range, 4-48). Hyperthyroidism also developed in 5 of 24 patients who had serum TRAb concentrations lower than 3.85 UI/L at the end of MMI treatment. In these 5 patients the relapse of hyperthyroidism occurred after a median period of 56 weeks (range, 24-120) and was always accompanied by an increase in serum TRAb concentrations. CONCLUSIONS: TRAb persist in the blood of most patients with GD after 18 months of MMI treatment. Both the frequency and the time of appearance of hyperthyroidism are closely correlated with serum TRAb concentrations at the end of MMI treatment. Our data would suggest that TRAb maintain stimulating activity after a full course of MMI treatment in the large majority of patients with GD. However, it is likely that the potency of these antibodies and/or the thyroid response to them change during treatment, as suggested by the different values measured in euthyroid control subjects and in euthyroid patients after MMI treatment.     

促甲状腺激素受体,甲状腺过氧化物酶及甲状腺球蛋白基因表达…

为探讨促甲状腺激素受体。甲状腺过氧化物酶及甲状腺球蛋白ｍＲＮＡ在自身免疫甲状腺疾病（ＡＩＴＤ）病人甲状腺组织的表达情况及其相互关系，应用Ｎｏｒｔｈｅｒｎ印记杂交技术观察了ＴＳＨ受体、ＴＰＯ以及ＴＧ ｍＲＮＡ在７例Ｇｒａｖｅｓ病（ＧＤ），２例桥本甲状腺素炎（ＨＴ）病人甲状腺组织中的表达。研究显示：ＴＳＨ受体ｍＲＮＡ与ＴＰＯ和ＴＧ ｍＲＮＡ在ＧＤ组和ＨＴ组表达不一致，ＧＤ组ＴＳＨ受体ｍＲＮＡ与ＴＰＯ和     

Modulation of differentiated function in cultured thyroid cells: thyrotropin control of thyroid peroxidase activity

The activity of thyroid peroxidase (TPO) in primary dog thyroid cell cultures was measured by both guaiacol oxidation and iodide oxidation assays. Whether cultures were initiated in the absence or presence of 50 mU/ml TSH, TPO activity fell in the first 24 h of culture to approximately 10% of the activity in freshly isolated follicles. After 5 days in culture, TPO activity almost completely disappeared in the absence of TSH, whereas in the presence of TSH, TPO activity rebounded to approximately 30% of that in freshly isolated follicles. TSH similarly induced TPO activity in cells that had lost this activity during a 1- to 6-day preincubation period in the absence of hormone. The half-time for the induction of TPO activity was approximately 3 days. Whether TSH was present from the start of culture or added after 5 days of culture without TSH, the half-maximal dose for reinduction of TPO activity was 0.3-0.4 mU TSH/ml. (Bu)2cAMP, 8-bromo-cAMP, forskolin, and cholera toxin all mimicked, either completely or in part, the ability of TSH to induce TPO activity in cells preincubated without hormone. We conclude that, in cultured dog thyroid cells, TPO activity is modulated by chronic TSH stimulation, and that this effect is mediated by cAMP. However, even though TSH stimulates TPO activity in cultured thyroid cells, the fact that there is no comparable restoration of organic iodine formation (as found in previous studies) makes it likely that other aspects of the iodide organification mechanism are altered.     

Heterogeneity of autoantibodies against thyroid peroxidase in autoimmune thyroid disease: evidence against antibodies directly inhibiting peroxidase activity as regulatory factors in thyroid hormone metabolism.

The close relationship between thyroid microsomal antigen and thyroid peroxidase (TPO) is now well established. The present study evaluates the significance of autoantibodies against TPO (anti-TPO-Abs) in the various forms and stages of autoimmune thyroid disease with respect to a possible heterogeneous nature and particularly to their influence on TPO activity. When measured by a RIA using purified human TPO, anti-TPO-Abs were highly correlated with microsomal antibodies determined by enyzme-linked immunosorbant assay (r = 0.96; P less than 0.0001) and with the results of a TPO immunoprecipitation assay using crude microsomal preparations (r = 0.76; P less than 0.001). Relating the results of these assays to the reactivities of patients' sera with thyroid microsomes in immunoblot under nonreducing and reducing conditions, discordant results could be observed in a few cases. Further analysis of these data indicate a heterogeneous nature of anti-TPO-Abs, which react with at least two antigenic domains of the TPO molecule. The comparative analysis of patients with hyperthyroid Graves' disease, patients with Graves' disease in clinical remission, and patients with hypothyroid Hashimoto's thyroiditis revealed no significant differences in the antibody spectrum. When evaluating the direct influence of anti-TPO-Abs on the activity of TPO under a rigorous methodological approach, we found no significant inhibition of the enzymatic activity by any of the sera investigated from patients with autoimmune thyroid disease compared to that in sera from normal controls. In conclusion, the data indicate a heterogeneous nature of anti-TPO-Abs. The spectrum of antigenic epitopes recognized by anti-TPO-Abs seems not to be significantly different in the various forms and stages of autoimmune thyroid disease. The lack of an inhibitory effect of autoantibodies on TPO activity argues against direct binding of autoantibodies to the enzymatic sites of TPO and indicates that they are not important factors in producing thyroid dysfunction in autoimmune thyroid disease.     

The aging thyroid. Relationship between elevated serum thyrotropin level and thyroid antibodies in elderly patients

The relationship of thyroid antibodies and the serum level of thyrotropin in older adults (over age 60) was studied to determine whether thyroid antibodies were a good clue to thyroid failure in elderly persons. Of those with thyroid failure, evidenced by clearly elevated serum thyrotropin values (more than 10 microU/ml), 67 percent had positive antimicrosomal antibody levels, a prevalence much greater (p less than 0.001) than that among those of comparable age with normal thyroid function (18 percent). Nevertheless, one third (33 percent) had thyroid failure without positive antimicrosomal antibody levels; this was true whether or not a low serum thyroxine value was present. Furthermore, of those with positive antimicrosomal antibody levels, most (68 percent) did not have thyroid failure. Thus, although positive antimicrosomal antibody levels occurred more often in elderly patients with thyroid failure than in those with normal thyroid function, a sizable fraction of those with thyroid failure did not have positive antimicrosomal antibody levels. Hence, measurement of thyroid antimicrosomal antibodies is not a good test of early thyroid failure in older patients; direct demonstration of a clearly elevated serum thyrotropin value is a better approach.     

Incidence of postradioiodine immunogenic hyperthyroidism/Graves' disease in relation to a temporary increase in thyrotropin receptor antibodies after radioiodine therapy for autonomous thyroid disease.

BACKGROUND: The aim of the study was to analyze retrospectively the incidence of postradioiodine immunogenic hyperthyroidism/Graves' disease in relation to a temporary increase in TSH-receptor antibodies without overt hyperthyroidism after radioiodine therapy for autonomous thyroid disease. PATIENTS AND METHODS: Between May 2000 and May 2003 all patients (n = 1,357) who had undergone radioiodine therapy for autonomous thyroid disease were retrospectively analyzed for development of postradioiodine immunogenic hyperthyroidism. On pretreatment evaluation 565 of 1,357 patients (41.6%) had unifocal autonomous thyroid disease (UFA), 693 of 1,357 patients (51.1%) had multifocal autonomous thyroid disease (MFA), and 99 of 1,357 patients (7.3%) had diffuse thyroid disease (DISS). Free triiodothyronine (FT(3)), free thyroxine (FT(4)), thyrotropin (TSH), and thyroid antibodies were measured. Ultrasound examinations and thyroid scintigraphy were performed before and after radioiodine therapy. A sensitive assay with the human TSH receptor as antigen was chosen for measurement of the TSH receptor antibody and the study was limited to analysis of data obtained since introduction of this assay. RESULTS: Fifteen of 1,357 patients (1.1%) (UFA, 8/565 = 1.4%; MFA, 6/693 = 0.9%; DISS 1/99 = 1.0%) developed postradioiodine hyperthyroidism between 1 and 13 months after radioiodine therapy with clinically overt hyperthyroidism and an elevation of TSH receptor antibodies. Patients with elevated thyroid peroxidase (TPO) antibodies before radioiodine therapy had an almost 10-fold (6/57 patients =10.5%) higher risk of developing postradioiodine immunogenic hyperthyroidism. Thirteen of 999 patients (1.3%) with antibody measurements after radioiodine therapy (UFA, 2/421 = 0.5%; MFA, 9/494 = 1.8%, DISS, 2/84 = 2.4%) had increased levels of TSH receptor antibodies and, to some extent, TPO antibodies without development of clinically overt hyperthyroidism. CONCLUSIONS: There is an estimated 1.1% risk of developing postradioiodine immunogenic hyperthyroidism/Graves' disease in patients undergoing radioiodine therapy for autonomous thyroid disease and this increases approximately 10-fold when TPO antibody levels are elevated before radioiodine therapy. Furthermore, there is an estimated 1.3% risk of a temporary increase of TSH receptor antibodies after radioiodine therapy for autonomous thyroid disease without development of clinically overt hyperthyroidism.     

Prognostic value of anti-TSH receptor antibodies in Basedow's disease treated with carbimazole

In order to precise their prognostic value, the anti-TSH receptor antibodies (TRAK) have been measured in 70 cases of Graves' disease treated with carbimazole. Results (expressed in percentage of inhibition of labeled TSH binding) were grouped into 7 mean values: before treatment, from 15 days to 3 months, from 3 to 12 months, 2nd, 3rd, 4th and 5th years. For all cases together, a progressive decrease was observed: 40, 37, 29, 19, 14, 10, 7 p. 100. In 16 cases with good outcome, the mean values are significantly lower than those of 29 cases with bad outcome, respectively: 33 vs 46, 29 vs 43, 17 vs 30, 12 vs 27, 11 vs 22, 3 vs 15, 1 vs 11 p. 100. The TRAK titers at whatever t time are correlated to the clinical state at t + 2 years: about 30 p. 100 relapses if the TRAK titer is less than 10 p. 100; about 80 p. 100 relapses if it is 80 p. 100. So, the anti-TSH receptor antibodies have their place among the prognostic parameters of Graves' disease treated with carbimazole.     

Monoclonal antibodies to the thyrotropin receptor: implications for receptor structure and the action of autoantibodies in Graves disease.

Hybridoma cells have been obtained by fusing P3-NS1/1-Ag4-1 mouse myeloma cells with spleen cells from mice immunized with solubilized preparations of the thyrotropin receptor. Five clones were produced that secrete a monoclonal antibody whose binding to thyroid membranes is specifically inhibited by unlabeled thyrotropin. The antibody interacts with functioning thyroid cells in culture but not with nonfunctioning cells; this interaction is prevented by thyrotropin. The antibodies are capable of competitively blocking thyrotropin binding to bovine thyroid membrane preparations; they prevent 125I-labeled thyrotropin binding to a solubilized preparation of the glycoprotein component of the bovine thyrotropin receptor but are unable to inhibit 125I-labeled thyrotropin binding to liposomes containing gangliosides at comparable concentrations. They prevent 125I-labeled thyrotropin binding to rat, bovine, or human (Graves disease) thyroid membrane preparations. They do not stimulate adenylate cyclase activity in thyroid membrane preparations but can inhibit thyrotropin-stimulated iodide uptake by functioning thyroid cells in culture.     

甲状腺刺激抗体对甲状腺细胞过氧化物酶活性影响

目的　探讨甲状腺刺激抗体 (TSAb)对培养的甲状腺细胞甲状腺过氧化物酶 (TPO)活性及其信号传导途径的影响。方法　应用改良愈创木酚法观察TSAb对原代培养人甲状腺细胞TPO活性的影响。结果　 4mg/mlTSAb作用 48h可使甲状腺细胞TPO活性达对照的 3 60 % ;蛋白激酶A(PKA)抑制剂使TSAb作用的TPO活性增加减少近 79.2 % (P <0 .0 1) ,而胞内Ca2 鳌合剂及木黄酮对TPO活性无明显影响。结论　TSAb通过激活cAMP/PKA途径导致TPO活性增加可能是TSAb致Graves病的主要机制之一。     

Characterization of monoclonal antibodies to the human thyrotropin receptor.

We have produced four monoclonal antibodies (mAbs), 34A, 49G, 11E7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thyroid cell line (GEJ), freshly dissociated human and murine thyroid cells, or Chinese hamster ovary cells stably transfected with the hTSH-R gene. These mAbs were obtained after immunization of DBA/1 mice with affinity-purified TSH-binding sites from GEJ cells. Biochemical studies, including sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, Western blot, and immunoprecipitation of solubilized GEJ cell membranes or human thyroid cells showed that most of the mAbs recognized two bands: one located at 46-48 kilodaltons and the other at 86-88 kilodaltons. Inhibition of [125I]hTSH binding to solubilized porcine membranes (TSH-receptor auto-antik?rper assay) or Chinese hamster ovary cell membranes previously transfected with hTSH-R gene showed that mAb 34A recognizes the hTSH-binding site of both receptors. In contrast, mAbs 49G, 11E7, and 12E3 recognize a structure located near the hTSH-binding site. Lastly, the ability of these mAbs to stimulate murine thyroid function was investigated by measuring cAMP production and iodide accumulation. The 34A mAb, which fully competes with [125I]TSH for binding to hTSH-R, was able to induce both functions. Conversely, the 12E3 mAb, which was the least potent inhibitor of [125I]TSH binding to hTSH-R-transfected cells had no effect. A relationship was, therefore, established between the capacity of mAb to hTSH-R to inhibit [125I]hTSH binding and their ability to induce thyroid functions.     

Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease.

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.     

Serum antibodies to gliadin in coeliac disease after gluten withdrawal

Serum gliadin antibodies of the IgA and IgG classes were determined by the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) in 10 adult patients with villous atrophy of the small-intestinal mucosa. After introduction of a gluten-free diet a gradual decrease in serum gliadin antibody levels occurred, reaching statistical significance at 3 months of treatment for the IgA class (p less than 0.01) and at 6 months for the IgG class (p less than 0.05). A decrease of serum gliadin antibody levels after gluten withdrawal was related to an improvement of the intestinal mucosa and should thus be indicative of whether the patient is following the dietary recommendations. However, determination of gliadin antibody levels cannot replace small-intestinal biopsy, as there are a few patients in whom the antibody levels are not related to the morphology of the gut mucosa.     

Anti-thyroid peroxidase antibody in patients with autoimmune thyroid disease: possible identity with anti-microsomal antibody

Thyroid microsomal antigen and peroxidase (TPO) have a close intracellular anatomical relationship, especially in exocytotic vesicles. We considered that antibodies to microsomal antigen might react with TPO and therefore looked for the presence of antibodies against TPO in the serum of patients with autoimmune thyroid disease (AITD). TPO was prepared from Graves' thyroid glands, solubilized by n-octyl glucoside, and its activity was assayed by the guaiacol method. Control sera and sera with a positive microsomal hemagglutination test (MCHA(+) ) were assayed for their ability to precipitate TPO activity by incubation of sera with TPO and protein A. We identified MCHA(+) sera which caused precipitation of TPO activity, and the extent of precipitation was related to the amount of serum added. A significant correlation was present between this anti-peroxidase activity and microsomal antibodies titers, measured by a micro-ELISA method. Affinity columns prepared from immunoglobulins of MCHA(+) sera, coupled to Reacti-Gel (6X), bound TPO activity, whereas using control IgG the recovery in the unbound fraction was high. These data provide evidence of antibodies against thyroid peroxidase in the serum of patients with AITD and suggest a close link between microsomal antigen and thyroid peroxidase.     

Graves' disease and coexisting struma ovarii: struma expression of thyrotropin receptors and the presence of thyrotropin receptor stimulating antibodies.

Struma ovarii is a rare cause of hyperthyroidism and particularly rare in patients with coexisting Graves' disease. We describe a 28-year-old female who presented with symptoms and signs of hyperthyroidism (free thyroxine [FT(4)] 39 pmol/L, thyrotropin [TSH] < 0.05 mU/L) and associated ophthalmopathy, consistent with Graves' disease. The patient relapsed twice: once after initial successful management with carbimazole and subsequently after subtotal thyroidectomy. Radioisotope scanning showed focal uptake bilaterally in the neck and believing this was the source of thyroid hormone excess, carbimazole was restarted. A left ovarian mass was found on ultrasound during the investigation of unrelated nephrotic syndrome resulting from focal segmental glomerulosclerosis. A 555-g struma ovarii was removed surgically. Hypothyroidism developed postoperatively (FT(4) 9.7 pmol/L, TSH 36 mU/L). Circulating TSH receptor stimulating antibodies were positive and immunohistochemical studies confirm the presence of TSH receptors on the struma ovarii. The demonstration of TSH receptors on the struma ovarii increases previous speculation that struma ovarii growth and function may be augmented by the circulating TSH receptor stimulating antibodies of Graves' disease.