Effects of trypan blue on thyroid secretion. Inhibition of purified cathepsin D from bovine thyroid.

Previous studies have demonstrated that trypan blue directly inhibits thyroid secretion when the dye is administered in vitro or in vivo. To further study the mechanisms of inhibition, cathepsin D (EC 3.4.23.5) (thyroidal acid proteinase) has been purified from bovine thyroid. Trypan blue inhibited the proteolysis of both 125I-labeled thyroglobulin and 125I-labeled hemoglobin in both crude lysosomal enzyme preparation and purified endopeptidase and the inhibition was competitive. Inhibition was also observed when the dye was allowed to prebind to either purified enzyme or purified substrate. Inhibition of cathepsin D is shown to account for part of the inhibition of thyroid secretion.

Recurrent apathetic hyperthyroidism.

A patient experienced three separate episodes of apathetic hyperthyroidism. Each time, this clinical picture appeared after the patient had been euthyroid for one to three months and within one to three weeks of discontinuing antithyroid drug therapy. This rapid recurrence of apathetic hyperthyroidism represents evidence against the concept that this disorder represents "burnt-out," or exhaustion of, body reserves in long-standing, untreated hyperthyroidism. Initial recognition and diagnosis of thyrotoxicosis was delayed by contamination of her serum from therapeutic radioactive iodine and possible interference with thyroxine radioimmunoassay. The solution to this problem with double isotope counting is discussed.

Differential reactivities of recombinant glycosylated ectodomains of mouse and human thyrotropin receptors with patient autoantibodies.

We expressed the extracellular domain of the mouse TSH receptor (mET-gp) using the baculovirus expression system. The recombinant protein was identified as mET-gp by immunoblotting and N-terminal amino acid sequencing. Carbohydrate analysis of the recombinant protein showed that the protein is glycosylated. Experimental antibodies raised against the extracellular domain of the human TSHr (ETSHr) were assayed for reactivity against mET-gp and glycosylated human ETSHr (ETSHr-gp) in an ELISA and found to be comparable. Similarly, both mET-gp and ETSHr-gp proteins neutralized the TSH binding inhibitory immunoglobulin (TBII) activity of rabbit anti-ETSHr antibodies in a RRA. However, when these proteins were compared for their ability to neutralize TBII and blocking activities (TSBAb) of IgG from patients with thyroid autoimmune disorders, only ETSHr-gp was able to neutralize these activities. In contrast, mET-gp partially neutralized, whereas ETSHr-gp completely neutralized the stimulatory (TSAb) activities of IgG from patients. Analyses of reactivities of these two proteins against a panel of antipeptide and monoclonal antibodies and their protein sequences showed differences in some specific epitopes. These data showed that in spite of significant homology between the two proteins, they exhibit specific epitope differences that are sufficient to cause divergence in their ability to react with patient autoantibodies to TSHr. This suggests that the two proteins might differ in their three-dimensional structure.

Use of osmium-ferrocyanide treatment for improved lysosomal acid trimetaphosphatase reaction and subcellular detail in thyroid follicular cells.

The histochemical reaction for acid trimetaphosphatase in addition to secondary tissue treatment with an osmium-ferrocyanide mixture was used to study lysosomes and phagolysosomes in the mouse thyroid gland. The osmium-ferrocyanide postfixation enhanced reaction product localization, reduced diffuse reaction, and improved membrane contrast. In addition, the ultrathin tissue sections did not require heavy metal staining, thus eliminating potential stain artifacts due to precipitation. In view of the improved tissue preservation and enzyme localization, it is suggested that osmium-ferrocyanide postfixation be used after the acid trimetaphosphatase method.

Effects of trypan blue on thyroid secretion: localization of trypan blue within the colloid space and phagolysosomes of thyroid follicles.

Trypan blue was previously shown to directly inhibit thyroid secretion following TSH stimulation. Inhibition of both colloid droplet formation and thyroglobulin proteolysis was demonstrated. By observing the characteristic bright red fluorescence of the dye-protein complex, we have demonstrated that trypan blue rapidly enters the colloid space and combines with thyroglobulin. In addition, the dye in association with thyroglobulin has been demonstrated within phagosomes and phagolysosomes by centrifugation of the lysosomal (P15) fraction on both sucrose and Percoll density gradients. Lability or latency of the dye with the phagolysosomal contents was demonstrated and the dye was found in association with thyroglobulin by column chromatography. It is proposed that the complexing of trypan blue to thyroglobulin alters its attachment to specific follicular cell receptors, inhibits pinocytosis, and, thus, thyroid hormone secretion.

Effect of therapy on the neutropenia of hyperthyroidism.

Observations in a patient with recurrent hyperthyroidism, each time associated with neutropenia which resolved after therapy, prompted a chart review of other patients referred for radioactive iodine therapy. Of 99 untreated patients, 18 had neutrophil counts of less than 2,000/cu mm. After therapy with either thionamides or 131I, 41 of 53 (77%) evaluable patients had an increase in neutrophil count. Eleven of these evaluable patients had neutropenia before therapy; after therapy, all 11 had an increase in their neutrophil counts into the normal range, with a mean increase of 170%. In one patient, studies on the mechanism of neutropenia indicate that bone marrow production and reserve remain normal and that circulating neutrophils are normally marginated. A decreased neutrophil circulation time may be the cause of neutropenia associated with hyperthyroidism.

Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells.

Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing rhe intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.

Direct effects of trypan blue on thyroid secretion.

Trypan blue directly inhibited in vitro thyroid secretion (butanol soluble 125I release to the media) induced by both thyroid stimulating hormone (TSH) and dibutyryl cAMP. Intracellular colloid droplet counts were also decreased. Inhibition was directly proportional to dye concentration and could be overcome by supramaximal TSH and dibutyryl cAMP. Inhibition could be observed as early as 20 min of incubation, was not increased by preincubation, and could even be demonstrated after TSH in vivo. Trypan blue, in vivo, produced similar inhibition of thyroid secretion. Incubation of 125I-thyroglobulin with lysosomal enzymes revealed inhibition with much lower concentrations of dye. Inhibition of lysosomal enzyme(s) would not appear to explain the marked decreases in colloid droplets, and this may represent two separate effects of trypan blue on thyroid secretion.

Thyroid antibodies in children and adolescents with thyroid disorders.

Thyroid antibodies were determined by three different techniques in the sera of 125 children and adolescents with thyroid disorders and in the sera of 53 short, normal children without thyroid dysfunction. The incidence of antithyroglobulin antibodies in patients with thyroiditis was highest when measured by radioimmunoassay (85%), less than when measured by hemagglutination (24%), and least by antimicrosomal antibodies (7%). No patient who had initially negative serum for RATA subsequently had positive tests during follow-up of five to 24 months, whereas eight of 31 patients with initially negative serum for ATA later developed positive tests. Treatment appeared to have a suppressive effect on RATA, but not on ATA titers, in hypothroid patients with clinical thyroiditis. The incidence of hypothyroidism in the patients with clinical thyroiditis on initial presentation was significant (37%) and suggests that identification of children and adolescents with thyroiditis is important to ensure adequate medical follow-up.

Painless thyroiditis and transient hyperthyroidism without goiter.

Eight women had painless thyroiditis, transient thyrotoxicosis, and low radioactive iodine uptakes but were without goiter; they constituted 15% of all thyrotoxicosis cases that we saw during the past year. Standard antithyroid antibody tests by tanned erythrocyte hemagglutination, complement fixation, and colloid and microsomal fluorescence, if present initally, were only weakly psotivie and became negative by 9 months. However, human antithyroglobulin antibody levels by a sensitive radioimmunoassay were elevated initially in all patients. In general, these radioimmunoassayable antibody levels fell, but they had not returned to normal by 9 months. Serial thyroid function tests and standard antithyroid antibody tests were most compatible with subactue thyroidtis. However, the persistence of radioimmunoassayable antithyroblobulin antibodies and recent reports of histologic evidence of lymphocyte thyroiditis in similar patients with goiter leaves open the possibility that this is a previously unrecognized presentation of chronic lymphocytic thyroiditis. Neither the cause nor the ultimate course of this syndrome is known.