A simple, sensitive, and practical assay for thyroid-stimulating autoantibodies (TSAb) was developed in which cryopreserved porcine thyroid cells were incubated with crude immunoglobulin fractions sedimented from serum with polyethylene glycol. In the assay, 1.4- to 2.0-fold and 6- to 12-fold increases in cAMP released into Hank's medium without NaCl were found at 1 and 10 microU/ml bovine TSH, respectively. TSAb were detected in 41 (97.6%) of 42 patients with untreated hyperthyroid Graves' disease, 29 (55.8%) of 52 patients with hyperthyroid Graves' disease who were euthyroid while taking antithyroid drugs, 22 (78.6%) of 28 patients with euthyroid Graves' disease, and none of the patients with simple goiter, adenomatous goiter, thyroid adenoma, or thyroid cancer tested. TSAb activities measured using porcine thyroid cells significantly correlated with those measured using human thyroid adenoma cells (r = 0.908; n = 46; P less than 0.001). Thyroid-stimulating activity was also detected in 11 (28.9%) of 38 patients with Hashimoto's thyroiditis. However, the activity was considered to be due to TSH in the patients' sera, because it was completely abolished by pretreatment with anti-TSH antibodies. Serum TSH concentrations lower than 50 microU/ml did not affect the assay result. In Graves' disease after cessation of antithyroid drugs, 85.7% (12 of 14) of TSAb-positive patients relapsed, while 77.8% (14 of 18) of TSAb-negative patients remained in remission. Thus, the assessment of TSAb was useful as an index to predict prognosis.

A new sensitive in vitro assay for human thyroid stimulator (HTS) was developed using human thyroid adenoma cells in monolayer culture. After being cultured for 2 days, the cells were incubated in 0.3 ml Hank's solution without 0.8% NaCl (medium 1) and with thyroid stimulator (bovine TSH or 3 mg patient serum immunoglobulin G) at 37 C for 2 h. The cAMP generated in the cells and the medium during the incubation was measured by RIA. The assay was sensitive enough to elicit a 1.7- to 7.9-fold increase in cAMP at a TSH concentration of 10 microU/ml. HTS was detected in 33 (82.5%) of the 40 patients with untreated graves' disease using this assay system. In Hank's solution (medium 2), however, HTS was detected in only 5 (23.8%) of the 21 patients with untreated GRaves' disease. cAMP increment upon stimulation by either TSH or HTS in medium 1 was larger than that in medium 2, and the difference in the response to HTS using the two media was much greater than that in the response to TSH. Therefore, all HTS-immunoglobulin G studies showed higher activity using medium 1 than using medium 2 when expressed as bovine TSH equivalent. Analysis by the Lineweaver-Burk plot of dose-response curves of the effect of TSH and HTS stimulation on cAMP increment showed an increase in the Km upon the addition of NaCl to the medium. A similar inhibitory effect of NaCl (150 mM) was also observed in the assay system of human thyroid adenylate cyclase stimulator using crude plasma membrane fractions. In summary: 1) an assay for HTS measuring cAMP production in cultured thyroid adenoma cells was developed and the assay using low NaCL medium was found to be the most sensitive, and 2) the inhibitory effect of NaCl on the response to HTS was much greater than that on the response to TSH. These data suggest different behaviors of these two stimulators at their receptor sites.

Fifty-three untreated patients with histologically confirmed Hashimoto's thyroiditis were divided into two groups according to the echogenicity of the thyroid. In 25 cases, the echogenicity of the thyroid was almost the same as or less than that of the adjacent muscles. Such cases were classified as group A. The other 28 cases were classified as group B. Mean T4 levels in group A were significantly lower than those in group B. In addition, the cases in group A had abnormally low T4 and abnormally high TSH more frequently than those in group B. In all cases in group A, there was severe degeneration and disappearance of thyroid follicles, whereas in most of the cases in group B, follicles of normal size were generally seen. Hypoechogenicity of the thyroid is a sign suggesting hypothyroidism and severe follicular degeneration.

Immunoglobulin G (IgG) fractions prepared from the serum of 18 patients with primary myxedema, 9 patients with goitrous Hashimoto's thyroiditis, and 14 normal controls were tested for their ability to alter TSH stimulation of cAMP production in cultured human thyroid cells and the binding of TSH to its receptor. When compared with the cAMP increase induced by 0.1 mU/ml bovine TSH in the presence of normal IgG, cAMP accumulation was significantly inhibited (P less than 0.005) by the addition of IgG from patients with primary myxedema. TSH-induced cAMP accumulation was not affected by IgG from patients with goitrous thyroiditis. IgG from patients with primary myxedema also inhibited the cAMP increase induced by thyroid-stimulating immunoglobulins, but not against the increase induced by prostaglandin E1. None of the IgG tested affected the basal level of cAMP. Two potent inhibitory IgG were strongly positive for TSH-binding inhibitor immunoglobulins. Excluding these, no significant correlation was found between the thyroid stimulation-blocking activity and the TSH-binding inhibitory activity. These data suggest the presence of at least two different types of antibodies in primary myxedema which block adenylate cyclase stimulation by TSH and might be responsible for thyroid dysfunction and atrophy.