Administration of 1 mU bovine TSH iv to mice resulted, within 1 hour, in the increase of the serum T4 level from 32 +/- 1.4 ng/ml to 53 +/- 2.6 ng/ml (Mean +/- SE, n = 24). Treatment with 1 mug triiodothyronine (T3) per day, for 10 days, abolished the responsiveness of the thyroid to TSH, as measured by thyroxine (T4) release. Thyroidal response to TSH was measured also in vitro. The basal hormonal release was 4.66 +/- 0.55 ng T4 and 0.98 +/- 0.15 ng T3 per thyroid per 3 h (n = 30). In the presence of bovine TSH (0.2 mU/ml) the hormonal secretion increased 3-fold for T4 and 2.5-fold for T3. Thyroids from mice pretreated with T3 for 10 days showed almost no response to TSH. Partial refractoriness to TSH was already significant 5 days after T3 pretreatment. Responsiveness to TSH was restored 3 days after T3 withdrawal or after 3 daily injections of 10 mU bovine TSH, concomitant with the last 3 days of T3 pretreatment. These results indicated that the prolonged absence of an adequate level of trophic hormone may be the cause of thyroidal unresponsiveness to acute TSH treatment. With 20 mU of TSH, cAMP levels rose from 4 +/- 0.5 picomoles to 80 +/- 9.3 picomoles per thyroid (n = 6). In mice subjected to 10 days of T3 pretreatment the response was markedly reduced: 20 +/- 3 picomoles/ thyroid. Thyroids of the T3-treated mice responded normally to 1 mM DBcAMP in vitro. From these results it was concluded that the impaired responsiveness of the thyroids to TSH occurs at a step prior to cAMP accumulation.
These experiments tested the effects of reserpine on estrogen-induced LH release and the effects of estrogen on gonadotropin-releasing hormone-induced LH release in ovariectomized ewes. Injection of 50, 200, or 500 mug of estradiol benzoate (EB) into progesterone-treated ewes, or of 50 mug of EB into non-treated ewes induced a large surge in serum LH levels approximately 15 h later. Injection of 5 mg/ewe of reserpine 6 h prior to the EB, blocked the LH surge. Reserpine reduced, but did not block the release of LH induced by injection of 20 mug or 40 mug of synthetic gonadotropin-releasing hormone (GnRH). Injection of graded doses (5, 10, or 20 mug) of GnRH into progesterone-treated ewes that had been treated with reserpine, induced increases in serum LH levels that were proportional to the logarithm of the dose. In the final series of experiments both progesterone-treated and non-treated ewes were injected with reserpine and then with either EB or oil. Fifteen hours after receiving EB or oil they were injected with either GnRH or the diluent. GnRH elevated serum LH levels significantly higher in ewes given EB than in those given oil. LH levels in ewes given oil and diluent were not elevated. These data suggest that reserpine blocked EB-induced LH release by an action on the central nervous system and that EB acted directly on the anterior pituitary to increase the response to GnRH.
Balb/C mice were immunized with bTSH (xenogeneic TSH) or extracts of Balb/C pituitaries (containing syngeneic TSH), either intracutaneously with Freund's adjuvant or intrasplenically. After bTSH, all blood samples contained anti-TSH antibodies. Thyroi d\x=req-\ stimulating activity was assayed on FRTL-5 cells. Of 80 sera from immunized mice, 33 induced an increase in 99mTcO4 uptake, and 31/79 induced an increase in thyroidal 3[H]thymidine uptake by thyrocytes. With syngeneic TSH, anti-TSH antibodies were found in 10/19 mice. Immunoglobulins increasing thyrocyte technetium uptake were found in 14/19 mice, and immunoglobulins that stimulated thymidine uptake in thyrocyte DNA were detected in 12/19 sera. After immunization with both types of TSH, sera of some mice showed only one of the two bioactivities. Hybridomas were prepared with splenic lymphocytes of the mice immunized with either of the TSH preparations. These secreted TSH-binding immunoglobulins or immunoglobulins which stimulated thymidine or technetium uptake by the thyrocytes. One of the hybridomas from mice immunized with pituitary extract secreted a monoclonal antibody which stimulated thyroidal thymidine uptake, but inhibited bTSH-induced 99mTcO4 uptake. These findings might suggest that the anti-idiotypic network acting on TSH as an antigen could be involved in the pathogenesis of stimulatory antibodies in thyroid disease.
The time course of the effect of bovine TSH (bTSH) on serum concentrations of thyroxine (T4) and triiodothyronine (T3) was measured in the normal mouse. The basal, unstimulated levels were 3.2+/-1.1 mug/100 ml T4 and 104+/-25 ng/100 ml T3 (mean+/-SD). With doses of bTSH from 0.5 to 100 mU the peak levels of the thyroid hormones were only 2.6 and 1.8 times the basal level for T4 and T3, respectively. With increasing doses of bTSH there was a proportional prolongation of the increased serum levels of thyroid hormones, i.e., about 2 h for 0.5mU to 12 h for 100 mU TSH. The integrated response with time was linearly related to the log dose. This would suggest a control mechanism which prevents excessive concentration of thyroid hormones in the serum. This pattern of response to TSH differs somewhat from that obtained by following radioiodine release in the McKenzie type bio-assay. To avoid the problems of changing blood concentrations of thyroid hormones and TSH, the release of T4 and T3 from the mouse thyroid was measured in vitro. The secretion increased with bTSH concentrations in the range of 0.02-0.8 mU/ml for T4 and 0.02-0.4 mU/ml for T3. The maximal response was 8.8+/-0.5 ng T4/3h/thyroid and 3.6+/-0.3 ng T3/3h/thyroid as against the basal secretion of 2.4+/-0.2ng T4 and 0.8+/-0.1 ng T3 (mean+/-SEM). Further in crease in bTSH concentration was associated with a decreased rate of thyroid hormone release. Thyroidal cAMP accumulation was enhanced with increasing bTSH concentration, even when there was a decrease in secretion. The dichotomy in the dose-response pattern between the two parameters indicated that the effect of high TSH concentrations on the release was induced at a step beyond cAMP accumulation. This was corroborated by the similar pattern of release induced by increasing concentration of DBcAMP. These findings indicate the existence of an intrathyroidal autoregulatory mechanism which prevents excess increase of thyroid hormone levels in the blood.
The purpose of these studies was to examine whether thyroid stimulating antibodies in Graves' patients could arise as auto-antiidiotypic antibodies to endogenous anti-TSH antibodies. The model system chosen was the thyroidectomized mouse, exhibiting an elevated level of endogenous, circulating TSH. Mice were thyroidectomized by 131I administration. Sera samples were drawn 1 to 14 months later. The following activities were measured in the immunoglobulin (Ig) fractions prepared: (a) TSH binding by elisa techniques, (b) iodide pump activity (as measured by 99mTcO4 uptake) and (c) increased [3H]thymidine incorporation into the DNA of FRTL-5 cells. TSH binding Igs were detected in 29/98 mice thyroidectomized for 7\p=n-\14 months. Stimulation of technetium uptake was observed in 59/110 mice and stimulated labeled thymidine uptake in 37/102 mice, beginning eight and nine months after thyroidectomy, respectively. Of the positive animals, 51 showed a single stimulating activity. The incidence and the serum titers of Igs that stimulate technetium uptake increased significantly with time.Indeed, in the group tested 14 months post-thyroidectomy, 75% of the sera were positive for this antibody with a mean titer eightfold higher than the controls. Hybridomas were prepared from the spleen lymphocytes of thyroidectomized mice. Of these, 18 produced 99mTcO4 uptake stimulating Igs,12 [3H]thymidine-uptake stimulating Igs and 18 TSH binding Igs. Most of the hybridomas secreted Igs with a single bioactivity. One monoclonal antibody was isolated which neutralized the bioactivity of bTSH on FRTL-5 cells. 99mTcO4 uptake was decreased by 50% and [3H]thymidine uptake was virtually abolished. These results suggest that the hypothyroid mouse can develop anti-TSH antibodies and thyroid-stimulating antiidiotypic antibodies by an autoimmune process. Several pathological syndromes of the thyroid gland in the human are associated with the appearance of TSH receptor-recognizing immunoglobulins. These could be elicited as primary idiotypes against determinants on the TSH receptor ( 1 ) or as antiidiotypes to previously formed anti-TSH antibodies (abs), in accordance with the immune regulation theory of Jeme (2). The presence of both anti-bovine and anti-human bTSH abs was shown in the sera of some of the thyrotoxic patients, as reviewed by Noh et al. ( 3 ). However, the sequence of appearance of the antibodies could not be shown, neither could their idiotype-antiidiotype relationship be confirmed (4). Several experimental models were set up to examine the possibility that circulating TSH might be recognized by the immune system as nonself, and trigger thyrotoxicosis.Farid and Lo (5) immunized rats with bovine or human TSH and isolated and purified the anti-TSH antibodies which they then injected into rabbits. Some of the antiidiotypic antibodies obtained activated the TSH receptor. Costagliola et al. (6) isolated antiidiotypic abs to a mono¬ clonal anti-hTSH ab, which reacted in various ways with human TSH receptor preparations. Beall et al. (7) ...
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