F. Clark

Thyroid-stimulating immunoglobulins in ophthalmic Graves' disease.

Thyroid‐stimulating immunoglobulins (TSI) have been detected by receptor assay in the sera of 43% of patients with ophthalmic Graves’disease. Comparison of the receptor assay studies with thyroid function tests indicated that in several patients the antibodies detected by receptor assay were biologically inactive. In other patients, thyroid function appeared to be under TSI control with hyperthyroidism prevented by autoimmune destruction of the thyroid.

TSH RECEPTOR ANTIBODY SYNTHESIS BY THYROID LYMPHOCYTES

Several indirect observations have indicated that lymphocyte in the thyroid may be an important site of TSH receptor antibody synthesis in Graves’disease and we now describe an investigation of this possibility using improved lymphocyte isolation and TSH receptor antibody assay procedures. Our studies demonstrate that thyroid Iymphocytes spontaneously produce TSH receptor antibody in culture. Furthermore, experiments with mitogen tend to suggest that these cells, in contrast to lymphocytes from lymph nodes draining the thyroid, are part of an active immune response to the TSH receptor.

Studies of thyroglobulin autoantibody synthesis using a micro-ELISA assay

Thyroglobulin autoantibody synthesis by Hashimoto lymphocyte cultures has been studied using an ELISA, a plaque assay and tanned red cell haemagglutination. The ELISA system was found to be the most suitable and using this method IgG-class thyroglobulin antibody synthesis was detectable in cultures of mitogen-stimulated lymphocytes from all 10 Hashimoto patients studied, but not in cultures of lymphocytes from 4 normal donors. The ELISA was also sufficiently sensitive to detect thyroglobulin antibody synthesis in mitogen-free lymphocyte cultures from 4 out of the 10 Hashimoto patients and consequently it should be possible to use this system to study the effect of various pathophysiological factors on autoantibody synthesis which would otherwise be masked by mitogenic stimulation.

The IgG Subclass Distribution of Thyroglobulin Antibody Synthesized in Culture

Thyroglobulin autoantibodies synthesized by Hashimoto lymphocytes in culture and present in serum have been analysed in terms of their IgG subclass distribution. The autoantibodies produced in vitro were frequently IgG4 or IgG1. whether pokeweed mitogen or Epstein‐Barr virus was used to stimulate the cultures, and the subclass distribution of these thyroglobulin antibodies was similar to that observed in the patientsserum. It appears therefore that the antibodies synthesized in vitro in response to polyclonal B‐cell activators resemble those produced in vivo, and it seems likely that both pokeweed mitogen and Epstein‐Barr virus influence the same B‐cell precursors of autoantibody‐synthesizing celts, albeit by different mechanisms.

Thyroid peroxidase and the induction of autoimmune thyroid disease

Animal models of autoimmune thyroid disease are associated with thyroglobulin (Tg) as autoantigen whereas in man the autoimmune response to microsomal antigen/thyroid peroxidase (TPO) appears to play a major role in thyroiditis. Consequently, we have compared the ability of TPO and Tg to induce thyroid autoantibodies and thyroid damage in mice known to be susceptible (CBA/J) or resistant (BALB/c) to thyroiditis induced using murine Tg. Groups of three to five mice were immunized twice using Freunds complete adjuvant with 80–100μg highly purified porcine (p) TPO, pTg, rat (r) Tg, human Tg, bovine serum albumin (BSA) or BSA + 0.2 μg pTg (the level of Tg contamination of TPO). Four weeks after immunization with TPO, plasma from CBA/J (but not BALB/c) mice contained IgG class antibodies which bound to TPO‐coated tubes in the presence or absence of excess Tg (and could therefore be clearly distinguished from Tg antibodies) but there was no evidence of thyroiditis in either strain of mice. In contrast, in CBA/J mice immunized with rTg and, to a lesser extent in mice that had received pTg, thyroid tissue was infiltrated with lymphoid cells and/or neutrophils and antibodies to pTg (but not pTPO) were present. Our observations demonstrate that induction of TPO antibody alone is insufficient to lead to thyroiditis in CBA/J mice. Further, these studies emphasize the complex interactions between MHC and different thyroid antigens in the processes leading to thyroid destruction.

FENCLOFENAC‐SECONDARY EFFECTS UPON THE PITUITARY THYROID AXIS

To elucidate the mechanism of suppression of TSH responsiveness to TRH induced by the initiation of fenclofenac therapy, the early period of drug administration was examined in detail and the effect of the drug during a thyrotrophin releasing hormone infusion was assessed. In addition, the effect of fenclofenac upon the response of ACTH, cortisol, growth hormone and prolactin to insulin‐induced hypoglycaemia was examined. The effect of fenclofenac upon an equilibrium dialysis method for estimating free thyroid hormones was evaluated and was found to be insignificant within the therapeutic concentration range of the drug. A sharp, short‐lived rise in free thyroxine (21·7 ± 2·0 to 26·8 ± 1·9 pmol/1; P <0·03) was observed 60 min after the first dose of fenclofenac. Repeated peaks of free thyroxine during chronic fenclofenac treatment, superimposed upon the previously described steady decline of free and total serum thyroxine, are postulated to cause the observed suppression of TSH release which is present only until free and total serum thyroxine levels reach their nadir. The time course of the changes seen during thyrotrophin releasing hormone infusion suggested that the pituitary suppression was secondary to a rise in free thyroxine. The responses to hypoglycaemia of those pituitary hormones examined were not affected by fenclofenac.

Thyroglobulin Autoantibody IgG Subclasses; Regulation By T Cells

Microsomal and thyroglobulin (Tg) antibodies in patients with autoimmune thyroid disease are usually restricted to subclasses IgGl and/or IgG4 (1,2) and this distribution is likely to reflect the capacity of these antibodies to induce thyroid damage (3).

A technique for the isolation and mitogenic activation of thyroglobulin-specific human B lymphocytes

In previous studies we demonstrated that Hashimoto peripheral blood lymphocytes enriched for thyroglobulin (Tg) binding activity could be activated to secrete increased amounts of Tg antibody by Epstein - Barr virus (EBV) but not by pokeweed mitogen (PWM). We now report an investigation into the requirements for the isolation of Tg receptor positive (TgR+) B cells capable of being stimulated by PWM. The interaction between Hashimoto lymphocytes and Tg coated erythrocytes followed by red cell lysis interfered with the ability of the population to synthesize Tg antibody. However, this could be overcome if the rosettes formed between Tg coated erythrocytes and the Tg receptors on B cells were dissociated by digestion followed by red cell lysis and overnight incubation before the addition of PWM. Using this approach, Hashimoto TgR+ B cells could be stimulated by the mitogen to secrete immunoglobulin with a higher Tg antibody specific activity than unfractionated lymphocytes. Consequently, enriched populations of antigen specific human B cells capable of responding to mitogenic signals can be prepared by a positive selection technique.