Autoimmune diseases of the thyroid gland are the most common autoimmune diseases in humans, and encompass a wide spectrum of clinical presentations, ranging from Graves' disease (GD), Graves' ophthalmopathy (GO), Hashimoto's thyroiditis, and idiopathic myxedema. (1) GD is characterized by the production of autoantibodies directed against the receptor for the thyroid-stimulating hormone (TSH), frequently leading to increased thyroid function and clinical hyperthyroidism. (2)

GD is the most common cause of hyperthyroidism in iodine-sufficient areas. It typically presents with enlargement of the thyroid gland (goiter), signs and symptoms of excessive thyroid function, ophthalmopathy (which is severe in 3% to 5% of cases), and less frequently pretibial myxedema and acropachy. GD affects approximately two of every 1,000 Americans every year, most of whom are women (male: female ratio is 1:7) in the third or fourth decade of life.

Other immunological features of GD, common to other autoimmune thyroid diseases, are limphocytic infiltration of the thyroid, association with certain haplotypes of the major histocompatibility complex, familial occurrence, and presence of autoantibodies directed against other thyroid antigens such as thyroglobulin and thyro-peroxidase. (3)

TSH-R structure and function

The TSH receptor (TSH-R) is a membrane glycoprotein expressed mainly on thyroid follicular cells. It is a member of the G-protein-coupled, seven-transmembrane receptor superfamily, which also includes the luteinizing hormone and the follicle-stimulating hormone receptors. (4,5) The 10-exon gene encoding the TSH-R, located on chromosome 14, was cloned in 1989. (6) The mature TSH-R protein comprises 744 amino acids and has a molecular weight of 82 kDa. (7) Post-translational modifications are required for expression of functional TSH-R, including glycosylation of six asparagine residues in the extracellular domain. (8) The carbohydrate content of TSH-R can represent up to 30% of its molecular weight.

The mature TSH-R appears first on the plasma membrane as an intact holoreceptor. It is then cleaved onto the cell surface into two subunits that are held together by disulfide bonds. The N-terminal A subunit, encoded by the first nine exons, is extracellular and 395 amino acids long (Figure 1); the C-terminal B subunit, encoded by the tenth and largest exon, forms the seven-transmembrane domain and the intracytoplasmic tail. (4,7,9) The A subunit is responsible for recognition and binding of its ligand, the thyroid-stimulating hormone. TSH is the primary factor that regulates the function of thyroid follicular cells and, ultimately, thyroid hormone secretion.

[FIGURE 1 OMITTED]

In the past few years, several mechanisms of activation of the TSH-R have been proposed. (4) Actually, the most accepted model describes a possible activation of TSH-R through the TSH binding that breaks interactions between the ectodomain and transmembrane domain, inducing a cleaved, "opened," and active conformation with subsequent launching of the intracellular signal. The "closed" conformation represents the inactive state of the TSH-R. (9,10) The transmembrane B subunit leads the intracellular signal via cyclic AMP (cAMP) production through activation of adenylyl cyclase and consequent activation of phosholipase C (PLC) and the protein kinase A signal transduction systems. PLC activation regulates [H.sub.2][O.sub.2] production and thyroglobulin iodination, while phosphorilation of protein kinase A increases iodide uptake, thyroid peroxidase, and thyroglobulin synthesis. The physiological significance of this dual signaling system is unknown. (9)

TRAb production and binding to TSH-R

The TSH-R is today considered the major autoantigen in GD. Recent studies suggest that TSH-R cleavage can lead to the shedding of some of the extracellular A subunits. The shed A subunit may be at the origin of circulating antigenically active TSH-R ectodomain detected in human blood. The shedding of A subunits of TSH-R is probably crucial in breaking peripheral tolerance with induction of GD. (7) It is well demonstrated that TSH-R antibodies (TRAb) show functional heterogeneity. (3) TRAb with functional stimulating activity on TSH-R is designated thyroid-stimulating antibody (TSAb). (11) TSAb can mimic thyrotropin action and stimulate thyroid cells. On the contrary, TSH-blocking antibodies (TSHBAb) can bind to the TSH-R and induce a block of the TSH-mediated activation of thyroid cells. Patients with GD may have both stimulating and blocking autoantibodies. (2,12) The amounts or affinity of various antibodies in a single patient can determine the functional balance on thyroid function. TSAb is predominant in patients with autoimmune hyperthyroidism. Many studies have determined the epitopes on the TSH-R to which TSH and autoantibodies bind. (5) The majority of the epitopes for TSAb are located on the N-terminal region of the extracellular domain, whereas those for TSHBAb are on the C-terminal region of the ectodomain (Figure 1). These findings, however, are difficult to interpret due to the fact that TSAb and TSHBAb can coexist in the blood of the same patient with GD. (3)

TRAb assays

TRAb can be detected by two approaches: in vitro assays detecting the inhibition of radiolabeled TSH to its own receptor (TBII), and biological assays measuring the functional effect (stimulatory or inhibitory) of the TRAb on the TSH-R signaling pathway.

The TSH-binding inhibitory immunoglobulin (TBII) assays traditionally used membrane extracts prepared from porcine thyroid glands, or Chinese hamster ovary cells stably transfected with recombinant human TSH-R. (3,13) More recently, Costagliola, et al, introduced a new solid-phase radioimmunoassay where full-length human TSH-R was produced by DNA recombinant technology and immobilized on test tubes, yielding a superior reproducibility and sensitivity as compared to the above described traditional methods. (14) Overall, TBII assays do not distinguish stimulatory from blocking TRAb. TBII measured by the first two assays is positive in 76% to 95% of patients with Graves' hyperthyroidism, and positively correlates with TSAb activity. In contrast, the new assay raises the sensitivity to 99%, while maintaining an excellent specificity (99% for all TBII assays). (15,16)

The second approach uses cultured rat thyroid cells (FTRL-5), or cells transfected to express the human TSH-R (CHO-R), to measure the production of cAMP upon incubation with the patient serum. TSAb can be detected in the serum of more than 90% of patients with Graves' thyrotoxicosis. The initial TSAb activity averages 200% to 300% as expressed in percent increase of basal cAMP production. These bioassays have some disadvantages due to the complex culture conditions of FTRL-5 cells, and they require purified IgG preparations. The assay using transfected CHO-R cells is more sensitive than those using FTRL-5 cells. TSHBAb detection by bioassay is based on measurements of inhibition of the production of cAMP in cultures of FTRL-5 cells or CHO-R cells. (17)

There are discrepancies among the various methods, likely reflecting the heterogeneous nature of TRAb in terms of function and epitope recognition. (3,18) Recently, Kim, et al, demonstrated that 18.5% of patients with hyperthyroid GD had both TSAb and TSHBAb activities. (19) Thus, sera from most patients with GD contain both TSAb and TSHBAb/TBII activities, and the clinical effect may depend on the relative concentration and affinity of the predominating antibody. (3)

Clinical use of TRAb

The diagnosis of GD is currently based on the presence of symptoms and signs of autoimmune hyperthyroidism (such as tachycardia and goiter), ophthalmopathy, increased thyroid hormones, and reduced TSH levels. (20) Nevertheless, the measurement of TRAb can be extremely useful in the conditions listed in Table 1.

Prediction of relapse after antithyroid drug therapy

The treatment of Graves' thyrotoxicosis includes the use of antithyroid drugs (ATD), such as methimazole, carbimazole, and propyltiouracile; destructive therapy with radioactive iodine; and thyroid surgery. ATD is the preferred treatment modality in most centers outside the United States, and TRAb can be useful for patient management. TRAb titers usually decline in most patients receiving ATD, but the extent of the decline varies substantially. The quick reduction of serum TRAb values until their disappearance after the beginning of relatively low doses of ATD gives a good probability of relapse of the disease. On the other hand, the probability of recurrent thyrotoxicosis is higher in patients who have detectable serum TRAb after prolonged treatment with ATD; and the higher the value, the more likely the patient is to experience recurrence. These two opposite situations present several exceptions, however, and although most patients with undetectable TRAb at the end of treatment are likely to remain euthyroid, some can have a recurrence, and few patients with high values of TRAb at this time can remain euthyroid. (2,20,21,22)

In a retrospective clinical study, Vitti, et al, have identified subgroups of patients with a high or low risk of relapse, taking into account the titer of TRAb and other parameters such as age, gender, goiter, the severity of hyperthyroidism, and the presence of ophthalmopathy. In particular, the combination of patients with a small goiter (<40 mL), low TBII level (<30 U/L), and age >40 years conferred a 45% chance of remission during the five years after completion of a 12- to 24-month course of ATD therapy. In the same cohort of 306 patients with an overall average rate of relapse of 71.6%, patients with a large goiter (>70 mL) and a higher TRAb level (>30 U/L) had less than a 10% chance of remaining in remission within the five years after treatment. (23) Moreover, the presence of TRAb at the end of an ATD course had a high positive predictive value of recurrence of thyrotoxicosis. (2)

Serum titers of TRAb from patients before the initiation of ATD could be helpful in the decision for the treatment. Patients with Graves' thyrotoxicosis with both ATD and radioiodine, who had higher values of TRAb at the time of diagnosis, were likely to have persistently detectable activity regardless of the type of therapy. These data suggest that patients with higher levels of baseline TRAb should probably immediately undergo destructive therapy with radioiodine. (2)

TRAb and radioiodine therapy

TRAb values usually increase in the first trimester after radioiodine therapy in patients with GD as a result of radiation-induced destructive release of thyroid antigens. Starting with the second trimester after radioiodine treatment, TRAb values start to decrease and, in the absence of thyrotoxicosis recurrence, they usually disappear within one year but may persist for several years. The appearance of TRAb in serum with subsequent development of Graves' thyrotoxicosis has rarely been reported in patients with non-toxic or toxic nodular goiter after radioiodine therapy. This event could be explained as a possible thyroid antigens release of the TSH-R due to the radiation damage of follicular cells that can induce the production of TSAb and, as final effect, Graves' thyrotoxicosis. (2)

Even if uncommon, in some patients who quickly became hypothyroid after radioiodine therapy was detected, serum TSHBAb at various titers is associated with the disappearance of TSAb. TRAb detection has no practical routine usefulness after near-total thyroidectomy. (2)

TRAb and thyroid surgery

After surgery, TRAb levels decline and become undetectable in most patients within nine months. The outcome after thyroidectomy is mainly dependent on the residual volume of the gland. Many retrospective studies have shown a correlation between postoperative recurrence of hyperthyroidism and the persistence of TRAb after operation. The current trend of thyroid surgery is to remove an extensive amount of thyroid tissue to prevent recurrences; therefore, TRAb determination is of no help in the management of patients, except in the perspective of subsequent pregnancy. (1)

Graves' disease and pregnancy

As indicated, GD is common in fertile women. Graves' thyrotoxicosis is estimated to occur at a rate of 0.5 to 2 per 1,000 pregnancies. Although uncommon during pregnancy, this association has gained much attention as a complex situation with potential maternal and fetal complications. During pregnancy, serum TRAb usually decreases and a spontaneous remission of GD can occur. These changes reflect the immunosuppressive effect of pregnancy. After delivery, TRAb activity usually increases and can lead to a postpartum Graves' thyrotoxicosis. The possibility of hyperthyroidism may be overlooked because mild clinical signs and symptoms may resemble the manifestations associated with pregnancies. (1)

TRAb--but not thyroid hormones--can cross the placental barrier and induce fetal thyrotoxicosis after the 28th to 30th week of gestation when the thyroid of the fetus is completely developed. Due to the fact that ATD can also cross the placental barrier, they could be helpful in the treatment of fetal hyperthyroidism. Since TRAb production may persist for several years after radical radioiodine or surgical treatment of Graves' thyrotoxicosis, euthyroid women previously treated radically for GD may still have the risk of exposing the fetus to TRAb. (1)

The European Thyroid Association published the following guidelines for measurement of TRAb during pregnancy as the result of an evidence-based symposium:

* In pregnant women with previous GD in remission after ATD treatment, the risk for fetal-neonatal hyperthyroidism is small, and systematic measurement of TRAb is not necessary. Thyroid function should be evaluated during pregnancy to detect an unlikely but possible recurrence. In that case, TRAb assay is mandatory.

* In pregnant women with antecedent GD previously treated with radioiodine or thyroidectomy and regardless of the current thyroid status (euthyroidism with or without thyroxine substitution), TRAb (and eventually also TSAb by bioassay) should be measured early in pregnancy to evaluate the risk for fetal hyperthyroidism. If the level is high, careful monitoring of the fetus is mandatory for the early detection of signs of thyroid hyperfunction (pulse rate >170 bpm, impaired growth rate, oligoamnios, goiter). ATD administration to the mother may be considered to treat the fetal hyperthyroidism.

* In pregnant women who take ATD for GD, to keep thyroid function normal (therapy has been started before or during pregnancy), TRAb should be measured in the last trimester. If the TRAb assay is negative or the level is low, fetal-neonatal hyperthyroidism is unlikely. If antibody levels are high (TBII>40 U/L or TSAb>300%), evaluation of the fetus for hyperthyroidism is mandatory (clinical evaluation and thyroid function tests on cord blood and after four to seven days to detect early and delayed hyperthyroidism). In such situations, the use of the radioimmunoassay method for routine detection of TRAb is recommended. The minority of patients with positive sera should be tested subsequently in stimulation and blocking bioassays. It should be underlined that also TSHBAb can cross the placental barrier of hypothyroid mothers with autoimmune thyroiditis, causing transient fetal-neonatal hypothyroidism. (24) Moreover, TRAb should be detected early in the course of pregnancy in women who have previously given birth to a newborn with hyperthyroidism. (1)

TRAb in patients with less common manifestation of GD

Graves' ophthalmopathy is considered not to be caused by TRAb; however, there is an association between TRAb and GO in several epidemiologic and longitudinal studies. In clinical practice, the detection of TRAb could be helpful in the diagnosis of suspected euthyroid GO. TRAb are positive in 32% to 40% of patients who have euthyroid Graves' disease. (1,2) In some patients, the presence of TRAb may be the only detectable abnormality. Pretibial myxedema is observed in 2% to 5% of patients with GD. Pretibial myxedema often occurs after radioiodine treatment and is associated with ophthalmopathy and with high serum levels of TRAb, although the reason of this correlation is not yet understood. (1)

CE test on CLINICAL UTILITY OF AUTOANTIBODIES DIRECTED AGAINST TSH-R

MLO and Northern Illinois University (NIU), DeKalb, IL, are co-sponsors in offering continuing education units (CEUs) for this issue's article on CLINICAL UTILITY OF AUTOANTIBODIES DIRECTED AGAINST TSH-R. CEUs or contact hours are granted by the College of Health and Human Sciences at NIU, which has been approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E.[R] program (Provider No. 0001) and by the American Medical Technologists Institute for Education (Provider No. 121019; Registry No. 0061). Approval as a provider of continuing education programs has been granted by the state of Florida (Provider No. JP0000496), and for licensed clinical laboratory scientists and personnel in the state of California (Provider No. 351). Continuing education credits awarded for successful completion of this test are acceptable for the ASCP Board of Registry Continuing Competence Recognition Program. After reading the article on page 10 answer the following test questions and send your completed test form to NIU along with the nominal fee of $20. Readers who pass the test successfully (scoring 70% or higher) will receive a certificate for 1 contact hour of P.A.C.E.[R] credit. Participants should allow four to six weeks for receipt of certificates.

The fee for each continuing education test will be $20

All feature articles published in MLO are peer-reviewed.

Learning objectives and CE questions were prepared by Sharon M. Miller, Professor Emeritus, Northern Illinois University, DeKalb, IL.

1. The most common autoimmune disorders involve what gland?

a. Parathyroid.

b. Adrenal.

c. Thyroid.

d. Pineal.

2. Graves' disease (GD) is characterized by all of the following EXCEPT

a. hypothyroidism.

b. hyperthyroidism.

c. goiter.

d. ocular signs and symptoms.

3. TSH receptor protein is located on membranes of what type of thyroid cell?

a. Parafollicular.

b. Follicular.

c. Thyroductal.

d. C cell.

4. The prime autoantigen in Graves' disease is

a. TRAK.

b. TBI.

c. TPO.

d. TSH-R.

5. The A subunit of the TSH-R is responsible for

a. recognizing and binding TSH.

b. producing the "second messenger" cAMP.

c. activating phospholipase C.

d. initiating thyroglobulin synthesis.

6. Shedding of B subunits of TSH-R into the circulation triggers development of Graves' disease.

a. True

b. False

7. Thyroid stimulating antibodies (TSAb)

a. prevent TSH-R B subunit activation.

b. mimic thyrotropin activity.

c. are seldom detected in patient's with GD.

d. are the exclusive thyroid reactive autoantibodies in GD.

8. Thyrotropin receptor antibodies (TRAb)

a. solely stimulate thyroid activity.

b. solely inhibit thyroid activity.

c. exhibit either blocking or stimulatory action.

d. None of the above.

9. Second-generation TBII assays

a. have poor sensitivity but excellent specificity.

b. cannot distinguish stimulatory from blocking antibodies.

c. use membrane extracts from cloned rat thyroid cells.

d. require complex culture and incubation conditions.

10. Biological assays for TSH-R antibodies involve measurement of

a. cAMP production.

b. thyroglobulin iodination.

c. 5'-deiodinase activity.

d. [H.sub.2][O.sub.2] synthesis.

11. Treatments for hyperthyroidism for GD include all of the following EXCEPT

a. antithyroid drugs.

b. radioiodine treatments.

c. L-thyroxine administration.

d. thyroid surgery.

12. A patient whose TRAb titer is elevated after treatment with antithyroid drugs is

a. likely to become euthyroid over time.

b. at high risk of becoming hypothyroid.

c. likely to have disease recurrence.

d. expected to achieve permanent immunological remission.

13. A patient whose serum level of TRAb remains elevated one month after radioiodine therapy is

a. unlikely to achieve permanent immunological remission.

b. at increased risk of thyroid storm.

c. at high risk of becoming hypothyroid.

d. still capable of eventually becoming euthyroid.

14. Following a thyroidectomy, successful postoperative management of the patient depends upon monitoring TRAb levels.

a. True

b. False

15. During pregnancy, TSH receptor antibody levels usually

a. fluctuate unpredictably.

b. remain unchanged.

c. increase.

d. decrease.

16. Fetal thyroid development is completed

a. one month after conception.

b. at the end of the first trimester.

c. by the second trimester.

d. within the third trimester.

17. TSH receptor antibodies may cross the placenta and at highenough concentrations cause fetal-neonatal

a. hyperthyroidism.

b. hypothyroidism.

18. Signs of fetal thyroid hyperfunctioning include all of the following EXCEPT

a. pulse rate less than 170 beats per minute.

b. impaired growth rate.

c. goiter diagnosed by ultrasound.

d. an abnormally small amount of amniotic fluid.

19. Successful treatment of fetal hyperthyroidism is possible if the mother

a. is placed on an iodine-restricted diet.

b. receives radioiodine therapy.

c. receives antithyroid drug treatment.

d. None of the above.

20. A woman diagnosed with Graves' disease has become pregnant. Her physician has prescribed propylthiouracil daily, but the patient has been noncompliant with medication in the past. Measurement of thyroid autoantibodies in the third trimester yields the following results (reference values in parentheses): TRAb level = 52% (<15%); TSAb level = 294% (<180%); TSHBAb = 9% (<25%). Testing of the newborn is most likely to indicate

a. euthyroidism.

b. hypothyroidism.

c. hyperthyroidism.

[GRAPHIC OMITTED]

References

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2. Marcocci C, Chiovato L. Thyroid-directed antibodies. In: Braverman LE, Utiger RD, eds. Werner and Ingbar's The thyroid: A fundamental and clinical text. 8th ed. Philadelphia, PA: JB Lippincott Co.; 2000:415.

3. Cho BY. Clinical applications of TSH receptor antibodies in thyroid diseases. J Korean Med Sci. 2002;17:293-301.

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7. Chistiakov DA. Thyroid-stimulating hormone receptor and its role in Graves' disease. Mol Genet Metab. 2003;80(4):377-88.

8. Graves PN, Pritsker A, Davies TF. Post-translational processing of the natural human thyrotropin receptor: demonstration of more than two cleavage sites. J Clin Endocrinol Metab. 1999;84:2177-2181.

9. Davies TF, Marians R, Latif R. The TSH receptor reveals itself. J Clin Invest. 2002;110:161-164.

10. Wonerow P, Neumann S, Gudermann T, Paschke R. Thyrotropin receptor mutations as a tool to understand thyrotropin receptor action. J Mol Med. 2001;79:707-721.

11. Tonacchera M, Costagliola S, Cetani F, et al. Patient with monoclonal gammopathy, thyrotoxicosis, pretibial myxedema and thyroid-associated ophthalmopathy; demonstration of direct binding of autoantibodies to the thyrotropin receptor. Eur J Endocrinol. 1996; 134:97-103.

12. Saravanan P, Dayan CM. Thyroid autoantibodies. Endocrinol Metab Clin North Am. 2001; 30:315-337.

13. Shewring GA, Rees Smith B. An improved radioreceptor assay for TSH receptor antibodies. Clin Endocrinol (Oxf). 1982;17:409-417.

14. Costagliola S, Morgenthaler NG, Hoermann R, et al. Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves' disease. J Clin Endocrinol Metab. 1999;84:90-97.

15. Morgenthaler NG, Nagata A, Katayama S, Bergmann A, Likata M. Detection of low titre TBII in patients with Graves' disease using recombinant human TSH receptor. Clin Endocrinol (Oxf). 2002;57(2):193-198.

16. Paunkovic N, Paunkovic J. Diagnostic sensitivity of two radio receptor assays (TRAK Assay and TRAK Dyno Human) for detection of TSH receptor antibodies. Nucl Med Rev Cent East Eur. 2003;6(2):119-122.

17. Vitti P, Elisei R, Tonacchera M, et al. Detection of thyroid-stimulating antibody using Chinese hamster ovary cells transfected with cloned human thyrotropin receptor. J Clin Endocrinol Metab. 1993;76:499-503.

18. Kohn LD, Harii N. Thyrotropin receptor autoantibodies (TSHRAbs): epitopes, origins and clinical significance. Autoimmunity. 2003;36(6-7):331-337.

19. Kim WB, Chung HK, Park YJ, et al. The prevalence and clinical significance of blocking thyrotropin receptor antibodies in untreated hyperthyroid Graves' disease. Thyroid. 2000;10:579-586.

20. Davies TF, Roti E, Braverman LE, Degroot LJ. Thyroid controversy--stimulating antibodies. J Clin Endocrinol Metab. 1998;83:3777-3785.

21. Chung H-K, Kim WB, Park DJ, Kohn LD, Tahara K, Cho BY. Two Graves' disease patients who spontaneously developed hypothyroidism after antithyroid drug treatment: characteristics of epitopes for thyrotropin receptor antibodies. Thyroid. 1999;9:393-399.

22. Feldt-Rasmussen U, Schleusener H, Carayon P. Meta-analysis evaluation of the impact of thyrotropin receptor antibodies on long term remission after medical therapy of Graves' disease. J Clin Endocrinol Metab. 1994;78:98-102.

23. Vitti P, Rago T, Chiovato L, et al. Clinical features of patients with Graves' disease undergoing remission after antithyroid drug treatment. Thyroid. 1997;7:369-375.

24. Laurberg P, Nygaard B, Glinoer D, Grussendorf M, Orgiazzi J. Guidelines for TSH-receptor antibody measurements in pregnancy: results of an evidence-based symposium organized by the European Thyroid Association. Eur J Endocrinol. 1998;139:584-586.

By Roberto Rocchi, MD

Roberto Rocchi, MD, specializes in endocrinology and metabolism. He is a clinical and research associate in the Department of Endocrinology at the University of Pisa in Italy from which he graduated medical school, and is currently at Johns Hopkins University's Department of Pathology (Autoimmune Disease Center) and Medicine in Baltimore, MD, as a post-doctoral fellow. He is an active member of EUGOGO (European Group on Graves' Orbitopathy).

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