Favorable predictive value of thyroid autoimmunity in high aggressive breast cancer.

A high incidence of anti-thyroid antibodies (TAb) has been found in patients with breast cancer (BC). The aim of this study was to evaluate the prognostic value of TAb in a group of 47 women submitted to mastectomy for high malignancy degree BC. All patients were evaluated for thyroid disorders after breast surgery and before any anti-tumoral adjuvant therapy. Five yr after BC diagnosis 31/47 (65.9%) patients were alive (survivors group: SG) and 16/47 (34.1%) were dead (deaths group: DG). The overall prevalence of TAb was 15/47 (31.9%): 14/31 (45.1%) in SG and 1/16 (6.2%) in DG (p=0.008). Five-yr mortality was 15/32 (46.9%) in TAb- and 1/15 (6.7%) in TAb+ patients (p=0.01). Eight out of 47 (17.0%) patients had Hashimoto's thyroiditis and 7 of them (87.5%) were in SG. Estrogen receptor (ER) was measured in 43/47 (91.5%) BC specimens. ER was detected in 19/30 (63.0%) patients in SG and 3/13 (23.1%) in DG (p=0.01). Five-yr mortality was 10/21 (47.6%) in ER- and 3/22 (13.6%) in ER+ patients (p=0.008). Absence of ER expression [odds ratio (OR) 6.54; p=0.006] and absence of TAb (OR 9.37; p=0.03) were related to a higher mortality rate. TAb were detected in 8/21 (38.1%) ER- and in 7/22 (31.8%) ER+ patients; no relation was found between ER expression and TAb positivity (p=ns). Patients with ER+ and TAb+ have a better prognosis and the absence of a significant relationship between these two parameters suggests an independent prognostic role in high malignancy degree BC women.

Thyroid autoimmunity in patients with malignant and benign breast diseases before surgery.

BACKGROUND: Previous studies have demonstrated a high prevalence of thyroperoxidase antibodies (TPOAb) and autoimmune hypothyroidism in breast cancer (BC). These studies have been performed in BC patients generally 20-30 days after mastectomy. It is known that stress may have an influence on the immune system and a relation between stressful events and the onset or worsening of autoimmune thyroid disorders has been reported by several authors. The aim of the study was to evaluate the prevalence of autoimmune thyroid disease in patients with nodular breast disease selected for surgery before any treatment. Our hypothesis was that the high prevalence of thyroid autoimmune disorders in BC is independent of stressful events represented by surgery and/or anaesthetic procedures. METHODS: Our series included 61 consecutive women aged 52.8 +/- 10.2 yrs (mean age +/- s.d.) with nodular breast disease selected for breast surgery: 36 out of 61 of them (59%) had BC and 25 out of 61 had benign breast disease (BBD). Controls included 100 healthy age-matched women. All patients and control subjects were submitted to clinical, ultrasound thyroid evaluation and serum-free thyroxine (FT4), serum-free tri-iodothyronine (FT3), TSH, TPOAb and thyroglobulin antibodies (TgAb) determination. RESULTS: Mean FT3, FT4 and TSH concentration showed no differences between BC patients, BBD patients and controls. The prevalence of TPOAb in BC patients (12/36: 33.33%) was significantly higher than in BBD patients (5/25: 20%) (P < 0.01) and in controls (8/100: 8%) (P < 0.01). Similarly, the prevalence of TgAb in BC patients was 12 out of 36 (33.33%) significantly higher than that detected in BBD patients (4/25: 16%) (P < 0.01) and in controls (12/100: 12%) (P < 0.01). Of the 36 BC patients, 20 showed a diffuse hypoechogenicity of the thyroid gland to ultrasound evaluation, significantly higher than in BBD (7/25: 28%) (P = 0.03). Of the 20 BC patients who showed a hypoechogenic pattern of thyroid gland, 10 (50%) were associated with antithyroid antibodies positivity (TAb). This finding was present in two of seven BBD (28.57%) (P < 0.0001). Only two controls showed focal hypoechogenicity of the thyroid gland. Generally, 24 out of 36 (66.7%) of BC and 9 out of 25 (36%) of BBD (P = 0.02) had signs of thyroid autoimmunity consistent with the hypoechogenic pattern of thyroid gland associated or not with TAb; 2 out of 36 (5.55%) of BC and 1 out of 25 (4%) of BBD patients had autoimmune hypothyroidism and no hypothyroidism was found in controls. CONCLUSIONS: The results of this study confirm the strong relation between thyroid autoimmunity and BC. This finding is independent of stressful events represented by surgery or anaesthetic procedures. The present data call attention to the usefulness of screening for autoimmune thyroid disorders in patients with nodular breast disease selected for surgery.

Serum concentrations of adiponectin and leptin in patients with thyroid dysfunctions.

Thyroid dysfunction is associated with metabolic changes that affect mass and adipocyte function, as well as lipid and carbohydrate metabolism. Adipose tissue performs complex metabolic and endocrine functions. Leptin and adiponectin are two of the most important adipocytokines, both involved in the regulation of intermediate metabolism. The aim of this study was to evaluate the relationships between thyroid status and circulating levels of the two adipose tissue hormones. We studied 15 patients with hyperthyroidism, 15 patients with hypothyroidism and 15 euthyroid subjects, all matched by sex, age and body mass index (BMI). Serum concentrations of free thyroxine, free tri-iodothyronine, thyrotropin, leptin and adiponectin and anthropometric parameters (weight, height, BMI) were assessed. No significant difference was found among the 3 groups, as assessed by Student's t-test, both for adiponectin and leptin. We conclude that metabolic changes associated with thyroid dysfunction are not related to variations in serum levels of adiponectin or leptin.

In vitro assay of thyroid disruptors affecting TSH-stimulated adenylate cyclase activity.

Several natural or synthetic chemicals have been indicated as potential thyroid disruptors. The development of in vitro assays has been recommended to comprehensively assess the potential thyroid disrupting activity of a substance or a complex mixture. In this study, 12 substances suspected for acting as thyroid disruptors were tested for their ability to inhibit TSH-stimulated cAMP production in vitro. Those substances producing an inhibition were further studied to establish the level at which they interfere with this step of thyroid cell function. Using Chinese hamster ovary cells (CHO) transfected with the recombinant human TSH receptor, a dose-dependent inhibition of TSH-stimulated adenylate cyclase activity was produced by 1,1-bis-(4-chlorphenyl)-2,2,2-trichloroethan (DDT), Aroclor 1254 and Melissa Officinalis. All three substances also inhibited the cAMP production stimulated by TSH receptor antibody. Melissa Officinalis produced a significant inhibition of TSH binding to its receptor and of antibody binding to TSH, while no significant changes were produced by Aroclor 1254 or DDT in these assays. These data suggest that principles contained in Melissa Officinalis may block the binding of TSH to its receptor by acting both on the hormone and the receptor itself, while DDT and Aroclor 1254 affect cAMP production mainly at post-receptor step. In conclusion, we have developed a set of in vitro assays that allow investigation into the effect of thyroid disruptors on the TSH-mediated activation of the cAMP cascade. These assays may be useful to identify the mechanism of action of thyroid disruptors, coming beside and supporting animal studies or epidemiological surveys.

Evidence for a role of the type III-iodothyronine deiodinase in the regulation of 3,5,3'-triiodothyronine content in the human central nervous system.

OBJECTIVE: Thyroid hormone is essential for maintaining normal neurological functions both during development and in adult life. Type III-iodothyronine deiodinase (D3) degrades thyroid hormones by converting thyroxine and 3,5,3'-triiodothyroinine (T3) to inactive metabolites. A regional expression of D3 activity has been observed in the human central nervous system (CNS), and a critical role for D3 has been suggested in the regulation of local T3 content in concert with other enzymes. DESIGN: This study was undertaken to further characterize D3 activity in human CNS and to understand its role in the local regulation of T3 content. METHODS: Autoptic specimens from various areas of human CNS were obtained 6--27 h postmortem from 14 donors who died from cardiovascular accident, neoplastic disease or infectious disease. D3 was determined by measuring the conversion of T3 to 3,3'-diiodothyronine. The T3 content was measured by radioimmunoassay in ethanol extracts, using a specific antiserum. RESULTS: High levels of D3 activity were observed in hippocampus and temporal cortex, lower levels being found in the thalamus, hypothalamus, midbrain cerebellum, parietal and frontal cortex, and brain stem. An inverse relationship between D3 activity and T3 content in these areas was demonstrated. CONCLUSIONS: We have concluded that D3 contributes to the local regulation of T3 content in the human CNS.

Serum iodothyronines in the human fetus and the newborn: evidence for an important role of placenta in fetal thyroid hormone homeostasis.

UNLABELLED: The pattern of circulating iodothyronines in the fetus differs from that in the adult, being characterized by low levels of serum T3. In this study, concentrations of various iodothyronines were measured in sera from neonates of various postconceptional age (PA). Results obtained in cord sera at birth (PA, 24-40 weeks), reflecting the fetal pattern, were compared with those found during extrauterine life in newborns of 5 days or more of postnatal life (PA, 27-46 weeks). The main findings are: Starting at 30 weeks of PA, serum levels increase linearly during extrauterine life; and at 40 weeks, they are more than 200% of those measured in cord sera from newborns of equivalent PA. Serum reverse T3 (rT3) levels during fetal life are higher than those measured during extrauterine life; but they significantly decrease, starting at 30 weeks of PA. Serum T3 sulfate (T3S) does not significantly differ between the two groups, showing the highest values at 28-30 weeks of PA, and significantly decreasing at 30-40 weeks. T3S levels are directly correlated with rT3, both in fetal and extrauterine life, whereas a significant negative correlation between T3S and T3 is found only during extrauterine life. IN CONCLUSION: 1) changes in serum concentrations of iodothyronines in umbilical cord and during postnatal life indicate that maturation of extrathyroidal type I-iodothyronine monodeiodinase (MD) accelerates, starting at 30 weeks of PA; 2) high levels of type III-MD activity in fetal tissues prevent the rise of serum T3, whereas they maintain high levels of rT3 during intrauterine life; 3) an important mechanism leading to the transition from the fetal to the postnatal thyroid hormone balance is a sudden decrease in type III-MD activity; iv) because placenta contains a high amount of type III-MD, it is conceivable that placenta contributes to maintain low T3 and high rT3 serum concentrations during fetal life and that its removal at birth is responsible for most changes in iodothyronine metabolism occurring afterwards.

Outcome of thyroid function in Graves' patients treated with radioiodine: role of thyroid-stimulating and thyrotropin-blocking antibodies and of radioiodine-induced thyroid damage.

UNLABELLED: We investigated the interrelationship and the influence of thyroid-stimulating antibodies (TSAb), TSH-blocking antibodies (TSHBAb), and of radioiodine (131I)-induced thyroid damage in the early (within 1 yr) outcome of thyroid function in hyperthyroid patients with Graves' disease (GD) treated with 131I. TSAb, TSHBAb, and ultrasound thyroid volume (as an index of thyroid damage) were simultaneously measured before and at 1, 3, 6, and 12 months after 131I in 31 GD patients. One year after radioiodine, 9.7% of patients were hyperthyroid (Hyper-group), requiring methimazole; 12.9% were euthyroid (Eu-group); and 77.4% were hypothyroid (Hypo-group). Pretreatment thyroid volume in the Eu-group and Hyper-group was significantly greater (P = 0.009) than in the Hypo-group. Pre-131I TSAb levels were higher in the Hyper-group vs. the Hypo-group (P = 0.01) or the Eu-group (P = 0.03). A significant post-131I increase in TSAb levels occurred in 66% of patients developing hypothyroidism but not in those remaining hyperthyroid. After 131I, TSHBAb appeared in 7 patients, in all but one associated with high levels of TSAb. One year after radioiodine: 1) the mean percent reduction in thyroid volume was greater in the Hypo-group (80.7%) or the Eu-group (83.5%) than in the Hyper-group (35.7%) (P = 0.007 and 0.0033 respectively); 2) hypothyroid patients had smaller (P = 0.0058) post-131I thyroids than hyperthyroid patients; and 3) TSAb were still elevated in 75% hypothyroid patients, but all of them had a thyroid volume < or = 8 mL, indicating major postradioiodine gland damage. IN CONCLUSION: 1) the early outcome of thyroid function after 131I for GD is mainly related to pretreatment thyroid volume and to the degree of its reduction after therapy; 2) high TSAb levels before 131I are associated with a relative resistance to therapy; 3) a postradioiodine increase in TSAb levels is related to the development of hypothyroidism; and 4) the concomitant appearance of TSHBAb and disappearance of TSAb are not frequent after 131I and play a role in the development of early postradioiodine hypothyroidism only in a minority of patients.

Autoimmune hypothyroidism and hyperthyroidism in patients with Turner's syndrome.

A high prevalence of autoimmune thyroid disease (AITD) has been described in Turner's syndrome (TS) but the extent of this association is controversial for the prevalence of thyroid autoantibody and the clinical impact of thyroid dysfunction. In this study we searched for thyroid disease and thyroid autoantibodies in patients with TS. Seventy-five unselected TS patients (age range 3-30 years) were studied. Sera were tested for thyroid hormones, thyrotropin (TSH), thyroglobulin (TG-ab) and thyroperoxidase (TPO-ab) antibodies. The TSH-receptor antibodies with thyroid-stimulating (TS-ab) or TSH-blocking activity (TSHB-ab) were measured in the IgG fraction using a bioassay. Ten out of 75 (13.3%) TS patients had AITD: eight had autoimmune thyroiditis (AT) (six with subclinical and two with overt hypothyroidism and one with euthyroidism) and one had Graves' disease. The prevalence of AITD increased significantly (p < 0.05) from the first (15%) to the third (30%) decade of life. The prevalence of TPO-ab and/or TG-ab (20%) was higher (p < 0.05) in TS than in age-matched female controls and increased from the first (15%) to the third (30%) decade of life. Clinical AITD was diagnosed in 46% of TS patients with TPO-ab and/or TG-ab. Thyroid-stimulating antibody was detected in the hyperthyroid patient, and TSHB-ab was found in one of eight patients with hypothyroid AT. It was concluded that: TS patients are at higher than average risk of developing AITD not only in adolescence and adult age but also in childhood; hypothyroidism, mainly subclinical, is the most frequent thyroid dysfunction; elevated TPO-ab and/or TG-ab alone do not imply thyroid dysfunction; TS-ab or TSHB-ab are always associated with thyroid dysfunction although most cases of autoimmune hypothyroidism are not due to the latter antibody.

Transfection with the cDNA of the human thyrotropin receptor of a poorly differentiated rat thyroid cell line (FRT).

A cell line derived from the Fisher rat thyroid (FRT), that does not have functional TSH receptor, was stably transfected with the cDNA of the human TSH receptor (h TSH-R). In wild FRT cells TSH (1-1000 mU/l) was unable to increase cAMP production, while 10-10000 nmol/l forskolin elicited a 10-30 fold cAMP stimulation. Two of the transfected clones were responsive to TSH in terms of cAMP production. In particular, the FRT-R3 transfected clone showed the highest sensitivity to the hormone with a 10 fold cAMP increase over the basal at 100 mU/l TSH. The Northern blot analysis using a 2.4 kbp cDNA probe for the hTSH-R showed a band corresponding to the mRNA of TSH receptor in FRT-R3 cells, but not in wild FRT cells. In both cell types TSH was ineffective in stimulating growth assayed by 3H-thymidine incorporation into DNA. Hybridization with a probe for thyroperoxidase on polymerase chain reaction products after reverse transcription of mRNA showed that FRT-R3, as well as FRT cells, do not have a transcript for thyroperoxidase. In conclusion, the data reported in this paper show that the insertion of the hTSH-R cDNA in the genome of poorly differentiated rat thyroid cells results in the recovery of TSH-dependent adenylate cyclase, but not other differentiated thyroid cell functions.

Use of cells expressing the human thyrotropin (TSH) receptor for the measurement of thyroid stimulating and TSH-blocking antibodies.

A clone of Chinese hamster ovary cells (CHO) transfected with the cDNA of the human thyrotropin (TSH) receptor (CHO-R) was used to optimise assays for TSH receptor antibodies with either thyroid stimulating (TSAb) or TSH blocking (TSH-blocking Ab) activity. The study group included 89 patients with Graves' disease, 38 patients with goitrous Hashimoto's thyroiditis (HT) and 47 subjects with atrophic thyroiditis (AT). In the HT group, 8 patients had subclinical hypothyroidism (HT-SH) and 30 had overt hypothyroidism (HT-H). In the assay for TSAb, CHO-R cells were incubated with 1mg/ml of the immunoglobulin G (IgG) from patients with Grave's disease, while in the assay for TSH-blocking Ab cells were incubated with IgGs from patients with HT or AT alone (1mg/ml), or IgGs plus TSH (10 mU/L). After 2 h of incubation the extracellular cAMP was measured by a RIA. In these conditions a significant stimulation by Graves' IgG was obtained in patients with active hyperthyroidism (33/35, 94% untreated; 21/23, 91% relapsed after a course of medical treatment), in 12/20 (60%) patients euthyroid under methimazole and in 4/11 (36%) euthyroid after a course of antithyroid drugs. TSH-blocking Ab were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and in 16/47 (34.0%) patients with AT. TSAb and TSH-blocking Ab coexisted in 4 IgGs that belonged to patients in whom spontaneous hypothyroidism developed after hyperthyroidism, or viceversa. TSAb and TSH-blocking Ab were also tested on FTRL-5 cells. TSAb were positive in both assays in 43/58 (74%) patients with active Graves' disease, negative in both assays in 3 (5%), negative in FRTL-5 and positive in CHO-R in 11 (19%), negative in CHO-R and positive in FRTL-5 in 1 (1.7%). In FTRL-5 cells TSH-blocking Ab were detected in 1/8 (12.5%) patients with HT-SH, in 5/30 (16.6%) with HT-H and in 15/47 (31.9%) with AT. The results of cAMP stimulation in FRTL-5 and CHO-R showed a fairly good correlation in TSAb (r = 0.60, p < 0.0001) and in TSH-blocking Ab (r = 0.74, p < 0.0001) assays. In conclusion, CHO cells transfected with the cloned human TSH receptor are suitable for the clinical assay of TSAb and TSH-blocking Ab. The sensitivity of this assay is higher than that obtained using FRTL-5 cells, having the additional advantages of expressing the human TSH receptor and requiring less cumbersome procedures for cell culture.

Humoral thyroid autoimmunity is not involved in the pathogenesis of myxedematous endemic cretinism.

A role of thyroid autoimmunity in the pathogenesis of myxedematous endemic cretinism was suggested by reports indicating the presence of thyroid growth-blocking antibodies in the sera of these patients. To check this hypothesis, we searched for TSH receptor antibodies with thyroid growth-blocking or adenylate cyclase (AC)-inhibiting (TSH-blocking) activity in immunoglobulin G (IgG) from 18 euthyroid and 21 hypothyroid endemic cretins living in Italy and Peru. Among hypothyroid cretins, 12 had no palpable goiter. Stages I-III goiters were present in 12 of 18 euthyroid cretins. Controls included 25 euthyroid nongoitrous subjects living in the same endemic regions as cretins, and 10 normal subjects from an iodine-sufficient area. IgG from 4 selected patients with autoimmune atrophic thyroiditis and from 2 neonates with sporadic transient congenital hypothyroidism due to maternal TSH-blocking antibodies were included in the study. The blocking effect of the IgG was assessed in FRTL-5 cells by measuring TSH-stimulated [3H]thymidine incorporation, DNA accumulation, and AC activation. A radioreceptor assay was used to detect TSH-binding inhibiting antibodies (TBIAb). No IgG from hypothyroid endemic cretins without goiter contained TBIAb or inhibited TSH-stimulated cell growth or AC activation. The effect of IgG from hypothyroid nongoitrous cretins did not differ from that produced by IgG from hypothyroid cretins with goiter, euthyroid cretins with or without goiter, or normal controls. In contrast to these results, IgG from patients with autoimmune atrophic thyroiditis and from neonates with sporadic transient congenital hypothyroidism contained TBIAb that inhibited both TSH-stimulated cell growth and AC activation. In conclusion, our results indicate that, similar to other types of endemic cretinism, hypothyroid endemic cretins without goiter do not have TSH receptor antibodies able to inhibit TSH-stimulated thyroid cell growth or function. These observations argue against a role of humoral thyroid autoimmunity in the development of myxedematous endemic cretinism.

The location and the regulation of the type I-iodothyronine 5'-monodeiodinase (type I-MD) in the rat thyroid: studies using a specific anti-type I-MD antibody.

Type I-iodothyronine monodeiodinase (type I-MD) is abundant in thyroid tissue and contributes to the generation of T3 secreted by the gland. The availability of a specific antibody against rat type I-MD (type I-MD Ab) allowed us to directly identify this enzyme in rat thyroid glands, and in a differentiated strain of rat thyroid cells maintained in continuous culture (FRTL-5 cells). FRTL-5 cells maintain many differentiated functions of thyroid cells, including the expression of TSH receptor and thyroid peroxidase. Using an immunohistochemical technique on rat thyroid sections, a clear staining for type I-MD was demonstrated in follicular cells. The degree of immunoreactivity was greater in small follicles containing little amounts of colloid compared to large follicles lined by functionally inactive cells. Using immunofluorescence (IFL), a strong staining for type I-MD was observed in FRTL-5 cells grown in medium containing TSH. Both in vivo and in culture the staining for type I-MD was localised in the cytoplasm of thyroid cells, while nuclei were negative. Interestingly, no surface staining was shown when viable FRTL-5 cells were submitted to the same IFL procedure. TSH deprivation for 7 days was followed by the disappearance of type I-MD. Immunoreactivity for type I-MD was recovered by addition of TSH, forskolin or thyroid stimulating antibody (TSAb) to TSH-deprived FRTL-5 cells. The effect of TSH was prevented by cycloheximide. There was no induction of type I-MD when IGF-I was added to FRTL-5 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of antibodies blocking thyrotropin effect using Chinese hamster ovary cells transfected with the cloned human TSH receptor.

Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) were used to develop an assay to detect thyroid autoantibodies blocking the TSH-dependent cAMP production (TSHBAb). The study group included 38 patients with goitrous Hashimoto's thyroiditis (HT) and 47 subjects with atrophic thyroiditis (AT). In the HT group, 8 patients had subclinical hypothyroidism (HT-SH) and 30 had overt hypothyroidism (HT-H). Thirty normal subjects served as controls. Immunoglobulin G (IgG) was prepared from serum by double chromatography on DEAE-Sephadex. CHO-R cells were seeded in 96-well plates and were cultured for 48 h before the assay in RPMI-1640 medium plus 1 mmol/L glutamine, 10% fetal calf serum, and 0.4 g/L geneticin. In the assay for TSHBAb, CHO-R cells were incubated with IgG alone (0.5-2 mg/ml), TSH alone (0.2-625 mU/L), or IgG plus TSH; all samples were diluted in hypotonic medium containing 0.5 mmol/L isobutylmethylxanthine (IBMX). After 2 h of incubation at 37 degrees C in 5% CO2-95% air atmosphere, TSH-stimulation was quantified by measuring extracellular cAMP by a RIA. IgGs from normal subjects did not significantly modify the stimulation of adenylate cyclase produced by TSH, the results obtained ranging between -30% and +18% (mean +/- SD = -3 +/- 14%). All IgGs producing an inhibition greater than 2SD from the mean of controls (> 25%) were considered positive for blocking antibodies. TSHABAb were detected in 1/8 (12.5%) patients with HT-SH, in 7/30 (23.3%) with HT-H and 16/47 (34.0%) patients with AT.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure to detect thyroid growth-promoting activity in immunoglobulin G of patients with endemic goiter.

The [3H]thymidine incorporation assay in FRTL-5 cells was used to measure thyroid growth-stimulating antibody in the purified immunoglobulin G (IgG) fraction of patients with endemic nontoxic goiter (grade I-III) living in Italy (n = 34) or Peru (n = 37). IgG of euthyroid nongoitrous subjects living in the same endemic area (n = 25) and from an area of sufficient iodine intake were used as controls. Bovine TSH (10 mU/L) and thyroid-stimulating antibody of Graves' disease produced a significant increase in [3H]thymidine incorporation and DNA content in FRTL-5 cells. IgG from Italian or Peruvian patients with endemic goiter produced a small increase in [3H]thymidine incorporation in FRTL-5 cells (131 +/- 54% and 165 +/- 57%, respectively), which was indistinguishable from that obtained with IgG from normal nongoitrous subjects residing in endemic or nonendemic areas (167 +/- 80% and 161 +/- 36%, respectively). For comparison 18 of 25 (72%) IgG of hyperthyroid patients with Graves' disease produced clear-cut increases in [3H]thymidine incorporation (1142 +/- 1065%) and DNA content (219%) in FRTL-5 cells. IgG from patients with endemic goiter, at variance with Graves' IgG, did not cause an increase in DNA in FRTL-5 cells. All Graves' IgG that stimulated [3H]thymidine incorporation in FRTL-5 cells also stimulated cAMP production in this culture system, whereas no adenylate cyclase stimulation was produced by IgG from patients with endemic goiter. The prevalence of thyroglobulin antibody and thyroperoxidase antibody in endemic goiter patients did not differ from that in control subjects residing in the same iodine-deficient area. Our data show that sera of endemic goiter patients are devoid of thyroid growth-stimulating antibody and thyroid-stimulating antibody activities. These observations argue against a direct role of thyroid autoimmunity in the development of goiter in iodine-deficient areas.

Studies on the in vitro cytotoxic effect of amiodarone.

Amiodarone, a potent antiarrhythmic drug, contains 37.2% iodine by weight and may induce either hypo- or hyperthyroidism. The high iodine content of amiodarone may be responsible for both complications, but a cytotoxic effect of the drug on the thyroid resulting in thyroiditis has been reported. In the present study the cytotoxic effect of amiodarone was evaluated in three culture systems with different biological properties: 1) a strain of rat thyroid cells (FRTL-5 cells) that maintains most differentiated functions of normal thyroid cells, including an active iodide pump, but an inability to organify iodide; 2) a line of Chinese hamster ovary (CHO) fibroblasts; and 3) freshly prepared primary cultures of human thyroid follicles (hTF) that trap and organify iodide. Cells were radiolabeled with 51Cr and incubated for 24 h with medium alone, medium plus amiodarone (3.75-200 microM), medium plus an iodinated radiographic contrast agent (sodium diatrizoate; 7.5-200 microM), or medium plus potassium iodide (7.5-300 microM). At concentrations ranging from 75-200 microM, amiodarone induced a significant and dose-dependent release of 51Cr in FRTL-5 cells. In contrast, diatrizoate or KI had no cytotoxic effect on FRTL-5 cells. In the same molar concentrations, amiodarone was also cytotoxic in CHO cells. In hTF, the release of 51Cr produced by amiodarone occurred at a lower concentration (37.5 vs. 75 microM) and was significantly greater than that in FRTL-5 cells. The cytotoxic effect of amiodarone in hTF was partially, but significantly, reduced by methimazole, an inhibitor of iodide organification. In the FRTL-5 cell culture system, amiodarone also produced a dramatic inhibition of TSH-stimulated cell growth. This growth-inhibiting effect of amiodarone was evident at low concentrations (3.75-7.5 mumol/liter) of the drug, which did not produce significant cytotoxicity. In conclusion, 1) amiodarone had a cytotoxic effect in CHO fibroblasts, a nonthyroid cell line; 2) this cytotoxic effect occurred in thyroid cells independent of their ability to organify iodide; 3) however, the toxic effect of amiodarone was greater and occurred at a lower molar concentration in freshly prepared human thyroid follicles that trap and organify iodide; and 4) in the latter culture system, methimazole, an inhibitor of iodide organification, partially, but significantly, reduced the cytotoxic effect of amiodarone. These data suggest that thyroid cytotoxicity produced by amiodarone is mainly due to a direct effect of the drug on thyroid cells, but excess iodide released from the drug may contribute to its toxic action.

Antibodies producing complement-mediated thyroid cytotoxicity in patients with atrophic or goitrous autoimmune thyroiditis.

Thyroid-cytotoxic antibodies (thyroid-cytotoxic Abs) have been described in patients with autoimmune thyroiditis, but their role in the development of hypothyroidism remains to be clarified. In this study, we evaluated the pathogenetic role of thyroid-cytotoxic Abs in 20 patients with atrophic thyroiditis (idiopathic myxedema; AT) and 94 patients with goitrous Hashimoto's thyroiditis (HT). Among patients with HT, 27 were euthyroid (HT-E), 27 had subclinical hypothyroidism (HT-SH), and 40 had overt hypothyroidism (HT-H). Seventeen normal subjects and 8 patients with nonthyroidal illnesses were used as controls (C). To detect thyroid-cytotoxic Abs, human thyroid cells expressing thyroid peroxidase (TPO) were labeled with 51Cr and challenged with the immunoglobulin G (IgG) fraction of serum plus rabbit complement. The cytotoxic effect of IgGs was calculated as the percent specific lysis (% SL), taking into account the lytic effect of complement alone and the maximal lysis produced by a detergent. Most C-IgGs decreased the cytotoxic effect of complement (median % SL, -3.3). IgGs from hypothyroid patients with thyroiditis had a greater cytotoxic effect than C-IgGs, either as a whole group (P < 0.001), or when subdivided according to clinical diagnosis: HT-SH (median % SL, 4.8; P < 0.005), HT-H (%SL, 2.2; P < 0.0001), or AT (%SL, 0.9; P < 0.01). Among patients with HT, the lytic activity of IgGs from patients with subclinical and overt hypothyroidism was higher than that of IgGs from euthyroid patients (P < 0.05). The results of IgGs from euthyroid patients with HT (median % SL, -0.9) did not significantly differ from those of C-IgGs. By taking a cut-off over the upper range of % SL produced by C-IgGs (> 2), the prevalence of thyroid-cytotoxic Abs was 30% in AT, 59% in HT-SH, and 55% in HT-H. However, 37% of euthyroid patients with HT also had thyroid-cytotoxic Abs. No IgG containing TPO antibodies (TPOAb) at low titer (< 40(2)) was cytotoxic. However, the levels of thyroid-cytotoxic Abs did not correlate with TPOAb titers, and preabsorption with TPO only partially abolished the lytic effect of some HT-IgG. These findings suggest that TPO is a target of thyroid-cytotoxic Abs, but other thyroid antigens are also involved in the cytotoxic reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of cAMP accumulation in Chinese hamster ovary cells transfected with the recombinant human TSH receptor (CHO-R): a new bioassay for human thyrotropin.

Circulating TSH bioactivity may vary in several clinical and experimental conditions. Since the reliability of the current methods for the measurement of TSH bioactivity is limited, a new bioassay based on cAMP accumulation in Chinese Hamster Ovary cells transfected with recombinant human TSH receptor (CHO-R) was set up. The sensitivity was 0.3 +/- 0.1, 0.4 +/- 0.1 and 0.01 +/- 0.01 micrograms/L for TSH IRP 80/558, recombinant human TSH and bovine TSH, respectively. Standard curves were parallel, and the intra- and inter-assay coefficients of variation were 13 +/- 1.1% and 22 +/- 1.9%, respectively. LH, FSH, CG and TSH subunits did not stimulate cAMP accumulation up to high concentrations. Circulating TSH was partially purified by immunoaffinity separation and concentrated before being bioassayed. However, plain sera with high TSH levels, such as those from primary hypothyroid patients (PH), could be directly tested in CHO-R bioassay, provided that sera were added at concentrations lower than 10%. TSH from 6 normal subjects had biological to immunological ratio (B/I) ranging from 0.6 to 2.1 (mean +/- SD = 1.4 +/- 0.5). TSH from 6 patients with PH showed bioactivity significantly lower than in normals (B/I = 0.6 +/- 0.3; p < 0.001; range = 0.3-1.1). TSH from 5 patients with central hypothyroidism of hypothalamic origin (CH) had undetectable basal bioactivity (B/I < 0.2), which normalized in only one patient after acute TRH and in all patients after chronic TRH administration. In conclusion, CHO-R cells provide an excellent tool for evaluating TSH bioactivity, owing to high sensitivity, specificity, reproducibility and feasibility of the assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of thyroid-stimulating antibody using Chinese hamster ovary cells transfected with cloned human thyrotropin receptor.

A clone of Chinese hamster ovary (CHO) cells transfected with the cloned human TSH receptor (CHO-R) was used to optimize an assay for thyroid-stimulating antibody (TSAb), measuring adenylate cyclase stimulation by purified immunoglobulin G from patients with Graves' disease. Optimal sensitivity to bovine TSH (1 mU/L) and TSAb was obtained using hypotonic buffer and measuring extracellular cAMP. In time-response experiments, TSAb stimulation was maximal after 2 h of incubation in hypotonic buffer. Under these conditions, a significant stimulation by Graves' immunoglobulin G was obtained with 33 of 35 (94%) samples from patients with untreated Graves' disease and with 21 of 23 (91%) from patients who relapsed after a course of antithyroid drugs. On the other hand, TSAb was detected in only 12 of 20 (60%) patients who were euthyroid during methimazole treatment and in 4 of 11 (36%) who were euthyroid after a course of antithyroid drugs. All samples from Graves' patients were also tested for TSAb activity on FRTL-5 cells. The results of cAMP stimulation in FRTL-5 and CHO-R showed a fairly good correlation (r = 0.60; P < 0.0001). In particular, of the 58 patients with active Graves' disease (35 with untreated hyperthyroidism and 23 relapsed after methimazole), 43 (74%) were positive in both assays, 3 (5%) were negative in both, 11 (19%) were negative in FRTL-5 and positive in CHO-R, and 1 (1.7%) was negative in CHO-R and positive in FRTL-5. In conclusion, CHO cells transfected with the cloned human TSH receptor are suitable for the clinical assay of TSAb. The sensitivity of this assay is higher than that obtained using FRTL-5 cells, having the additional advantages of expressing the human TSH receptor and requiring less cumbersome procedures for cell culture.

Use of FRTL-5 for the study of thyroid antibodies involved in goitrogenesis.

Some authors have suggested a role of autoimmunity in the pathogenesis of iodine deficiency disorders (IDD). For this purpose we have searched for thyroid adenylate cyclase stimulating antibody (TSAb) and thyroid growth stimulating antibody (TGSAb) in patients with endemic goiter (EG) and endemic cretinism (EC). Immunoglobulins G preparations (IgGs) were tested in FRTL-5 cells. TSAb were calculated as percent of cAMP increase over basal production and TGSAb were expressed as percent of increase of 3H-thymidine incorporation and DNA content in FRTL-5 cells. Our results show that IgGs from goitrous patients were devoid of TSAb and TGSAb activities, while in the same conditions IgGs from patients with Graves' disease had the ability to stimulate cAMP production and 3H-thymidine incorporation in FRTL-5 cells. These data argue against a direct role of TSAb and TGSAb in the pathogenesis of IDD.