The sodium iodide symporter is unlikely to be a thyroid/breast shared antigen.

PURPOSE: Anti-thyroid peroxidase (TPO) autoantibodies (TPOAb) seem to be protective for patients with breast cancer (BC). Thyroid and breast tissues both express the sodium iodide symporter (NIS), similarly both have a peroxidase activity, TPO and lactoperoxidase (LPO) respectively. We hypothesize a common immune response to a thyroid/breast shared antigen suggesting three putative mechanisms: (1) TPOAb react to both TPO and LPO, (2) TPO could be expressed in BC and (3) patients with TPOAb could have autoantibodies to NIS (NISAb). Previous studies excluded NISAb that block NIS activity in sera of patients with thyroid autoimmunity (TA) and/or BC. This study investigates neutral NISAb (binding without affecting function). METHODS: Clones of CHO cells stably expressing human NIS (hNIS; CHO-NIS) were isolated following transfection of hNIS in pcDNA3 vector. Expression of hNIS mRNA and surface protein was confirmed by PCR and flow cytometry respectively using a hNIS-mouse-monoclonal-antibody. CHO-NIS and controls transfected with the empty pcDNA3 vector (CHO-Empty) were incubated with 42 heat-inactivated human sera followed by an anti-human-IgG-AlexaFluor488-conjugate: 12 with BC, 11 with TA, 10 with both BC and TA and 9 with non-autoimmune thyroid diseases. The Kolmogorov-Smirnov Test was used to compare the fluorescence intensity obtained with CHO-NIS and CHO-Empty, using sera from six young males as a negative control population. RESULTS: None of the 42 sera were positive for NISAb. CONCLUSIONS: NISAb are rare and NIS is unlikely to be a common thyroid/BC shared antigen. We have recently demonstrated TPO expression in BC tissue and are currently investigating TPOAb cross-reactivity with TPO/LPO.

Does thyroid peroxidase provide an antigenic link between thyroid autoimmunity and breast cancer?

Women with breast cancer (BC) and antithyroid peroxidase (TPO) autoantibodies (TPOAb) have a better prognosis than women lacking TPOAb. Sera from women with TPOAb displayed immunoreactivity to BC tissue by immunofluorescence that was not apparent in women without TPOAb. We hypothesize a BC/thyroid shared antigen that provides a target for humoral or cell-mediated immune activity; candidates include the sodium/iodide symporter (expressed in thyroid and BC), cross-reacting epitopes in TPO and lactoperoxidase (LPO) or TPO itself. As the association is with TPOAb, we investigated TPO expression in BC, breast peritumoral tissue (PT), other tissues (tumoral and not) and thyroid as positive control. Transcripts for known and novel TPO isoforms were detected in BC (n = 8) and PT (n = 8) but at approximately 10(4) -fold lower than in thyroid while in non-BC tumors (n = 5) they were at the limit of detection. TPO was expressed also in adipose tissue (n = 17), 10(3) -fold lower than in thyroid. Full length TPO (Mr 105-110 kDa) was detected in Western blots in the majority of examined tissues; preabsorption of the TPO antibody with recombinant TPO (but not LPO) reduced the signal, indicating specificity. The same occurred with some lower molecular weight bands, which could correspond to smaller TPO transcript isoforms, present in all samples. In conclusion, TPO is weakly expressed in BC and other tissues; this could partly explain the high frequency and protective role of TPOAb in BC patients. Further studies will investigate tissue specificity, function and immunogenicity of the novel TPO variants (some BC-specific) identified.

Failure to demonstrate cell-mediated immune responses to thyroid antigens in Graves' disease using in vitro assays of lymphokine-mediated migration inhibition.

The use of in vitro assays of cell-mediated immunity has provided evidence of a defect in thyroid antigen-specific T suppressor cells in the peripheral blood of patients with autoimmune diseases. We employed a direct assay of T lymphocyte migration inhibition in an attempt to demonstrate cell-mediated immune responses to thyroid antigens in untreated patients with hyperthyroid Graves' disease and examined the influence of (1) a variety of antigen preparations, (2) different assay conditions, and (3) the HLA-DR3 status of normal subjects on this system. There was no significant difference in the migration of lymphocytes from 13 untreated patients with hyperthyroid Graves' disease to a crude 800 X g supernatant antigen preparation of normal human thyroid when incubated at either 20 or 37 C compared with the response of 13 normal subjects. The mean migration index was 57 +/- 22 (+/- SD) in the Graves' patients compared with 65.2 +/- 19.9 in the normal subjects at 20 C and 83 +/- 16.9 in the Graves' patients compared with 86.6 +/- 15.1 in the normal subjects at 37 C. Similarly, there was no significant difference in the migration indices obtained with T cells from Graves' patients and normal subjects using an 800 X g supernatant prepared from the thyroid glands of 2 patients with Graves' disease, incubated at 20 or 37 C (44.0 +/- 22.5 in the Graves' patients compared with 45.7 +/- 12.8 in the normal subjects at 20 C and 67.9 +/- 20.8 in the patients compared with 72 +/- 12.6 in the controls at 37 C). In contrast, the migration indices calculated for 20 C incubation were significantly lower than the corresponding value at 37 C using 800 X g normal thyroid antigen (P less than 0.05) and 800 X g Graves' thyroid antigen (P less than 0.01) in both patient and control groups. An identically prepared uterine antigen produced a similar reduction in the migration indices at 20 C compared with those at 37 C in 6 normal donors (P less than 0.05), but no temperature effect was found when 8 normal subjects were tested with purified protein derivative of tuberculin. The responses of 12 normal individuals to 800 X g supernatants of the normal and Graves' thyroid antigens were not influenced by the HLA-DR3 antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells.

A criticism of current techniques for monitoring changes in T-cell subset numbers over extended periods in individuals with disease states in which such changes might provide insight in the fact that serial samples taken are usually analysed fresh and therefore not in the same assay. To try to overcome this problem we have stored peripheral blood mononuclear cells frozen in liquid nitrogen and thence examined their ability to form sheep red blood cell (E) rosettes and to label with OK monoclonal antibodies. Results obtained show that cell viabilities following freezing and T-cell subset analysis of E-rosette positive cells are no different when fresh or frozen and subsequently thawed peripheral blood mononuclear cells are used.

The thyrotropin receptor as a model to illustrate receptor and receptor antibody diseases.

The thyrotropin receptor (TSHR) has been used as an example to illustrate how disease may be the consequence of: 1. Modifications or inappropriate production of the natural ligand. 2. Production of abnormal agonists or antagonists such as autoantibodies. 3. Modifications in receptor structure resulting in constitutive activation or the absence of activation following ligand binding. 4. Changes in the cellular machinery which transduces the signal from the receptor to the cytoplasmic or nuclear endpoint target. This chapter concentrates on mechanisms (2) and (3). Since the cloning of the TSHR it has been shown that approximately 50% of cases of toxic adenoma can be explained by somatic point mutations in the nucleotide sequence of the receptor gene which causes single amino acid substitutions. The resulting modified TSHR structure constitutively activates adenylate cyclase (via Gs), intracellular cAMP levels are increased and, since cAMP controls both growth and function of the human thyrocyte clonal expansion of the mutated cell ensues. Similarly, activating mutations of the TSH receptor gene in the germline are responsible for hereditary hyperthyroidism with goitre, which is transmitted in the autosomal dominant mode. Changes in receptor primary structure, i.e. a modified autoantigen, do not seem to be responsible for the escape from tolerance which must precede production of thyroid stimulating antibodies (TSAB) which cause hyperthyroid Graves' disease and thyroid blocking antibodies (TBAB) which are responsible for some cases of hypothyroid idiopathic myxoedema. The eukaryotic expression of wild-type, experimentally mutated and chimeric TSHR has enabled some progress in delineating the residues involved in binding TSH, TSAB and TBAB. All three ligands bind numerous discontinuous residues in the extracellular domain of the receptor. The difference between the bioactivity of TSAB and TBAB cannot be explained completely by different binding sites on the receptor. Subtle differences in, for example, glycosylation and sialation of the immunoglobulins may be implicated, since bioactivity of TSH itself seems to depend on these. Attempts to define T cell epitopes have not identified a major immunogenic region. Indeed heterogeneity seems to be a hallmark of TSHR autoantibodies (TRAB). The possibility that thyroid-associated ophthalmopathy and pretibial myxoedema may be receptor antibody diseases is discussed. Further progress awaits large-scale production of TSHR able to bind TSH to facilitate X-ray crystallographic studies, the development of specific T cell clones and the cloning of TSAB autoantibodies.

Stimulation of autologous blood lymphocytes by malignant lymphoma cells and homogenates.

The blastogenic response to autologous blood lymphocytes to whole-cell suspensions and to homogenates obtained from malignant lymphoma tissue has been investigated. Spleens were obtained from patients in whom laparotomy was performed for staging of malignant lymphoma. Cell suspensions prepared from tumour nodules were treated with mitomycin C and allowed to react with separated autologous blood lymphocytes for 6 days. Lymphocyte stimulation was measured by liquid scintillation counting after exposure to 3H-TdR. Cultures were also prepared in which autologous lymphocytes were treated with spleen tumour homogenate. Control experiments used spleens from staging procedures in which no tumour deposits were present, and normal spleens removed incidentally during other operations. In the controls, the uptake of TdR was never more than twice that of unstimulated lymphocytes. Greater degrees of lymphocyte stimulation were seen in 6 out of 14 patients, using whole tumour cells, and in 7 out of 16 patients, using tumour homogenates. The results indicate an antigenic difference between tumour and host cells, and suggest that lymphocytes can react to a tumour-associated antigen.

Prevention and treatment of postpartum Graves' disease.

Postpartum Graves' disease requires differentiation from postpartum thyroiditis and subacute thyroiditis in addition to other causes of hyperthyroidism. This may be done by assessing thyrotropin receptor antibody and radioiodine uptake together with clinical examination and thyroid scanning. The effect of pregnancy on thyroid function causes changes in iodine metabolism, thyroid hormone transport proteins and thyroid gland size. Amelioration of autoimmune disease such as Graves' disease, systemic lupus erythematosus and rheumatoid arthritis is often observed during pregnancy followed by postpartum exacerbation. The immunological effects of pregnancy involve placental factors as well as a transient diversion from T helper (Th) 1 to Th2 T-cell cytokine profile in addition to a change in B-cell lymphopoiesis. Prevention of postpartum Graves' disease by immune strategies which have been experimentally performed to reduce expression of diabetes in the non-obese diabetic mouse are attractive but not currently feasible in humans. Treatment of Graves' disease prior to pregnancy or postpartum with 131I is effective. Therapy with anti-thyroid drugs with or without thyroxine is variably effective.

Analysis of T cell subsets in Graves' disease: alterations associated with carbimazole.

Conflicting data on subpopulations of peripheral blood lymphocytes in patients with autoimmune disease largely reflect variations in methods of study. An investigation was therefore conducted aimed at avoiding this difficulty. Serial samples of peripheral blood mononuclear cells from 42 patients with hyperthyroid Graves' disease were collected at monthly intervals before, during, and for 12 months after a six month course of carbimazole. Samples were stored in liquid nitrogen until completion of the study, when they were thawed and all samples from each patient analysed within the same assay using mouse monoclonal antibodies to human cell subsets and a fluorescence activated cell sorter. Proportions of cytotoxic/suppressor (OKT8) positive cells before treatment (mean 17.4 (SEM 0.8)%) were significantly lower (p less than 0.001) than those in normal controls (29.8 (1.9)%; n = 10) and returned to normal by the end of treatment. In contrast, the proportions of activated T cells (OKIa-OKM1) were significantly raised before treatment as compared with normal (14.4 (0.6)% versus 4.6 (0.8)%; p less than 0.001) and fell to normal by the end of treatment. Proportions of OKT3 and OKT4 positive T cells remained unchanged throughout treatment and in the succeeding 12 months. In patients who relapsed after treatment there was a rise in the proportion of activated T cells and a fall in OKT8 positive T cells, which returned towards normal with retreatment. The explanation for the alterations in numbers of circulating T cells remains to be determined but they may provide a means for predicting more accurately the outcome of Graves' disease after treatment with carbimazole.

Identification of antigenic domains on the human sodium-iodide symporter which are recognized by autoantibodies from patients with autoimmune thyroid disease.

The sodium-iodide symporter (NIS) is a novel autoantigen in autoimmune thyroid disease. In the present study we have characterized the antigenic domains on the human symporter which are recognized by autoantibodies from patients with either Graves' disease (GD) or autoimmune hypothyroidism (AH). Deletion derivatives of complementary DNA (cDNA) encoding the Na(+)/I(-) symporter were constructed using polymerase chain reaction (PCR) amplification. These deletion constructs were translated in vitro with the concomitant incorporation of [(35)S]methionine into the protein products. The reactivity of seven GD and six AH sera, which were known to contain symporter-binding antibodies, to each of the radiolabelled modified symporters was then determined in immunoprecipitation experiments. Analyses of the results obtained in the radiobinding assays suggest the existence of multiple antibody binding sites on human NIS (hNIS), including regions between amino acids (aa) 1--134, 191--286, 290--411, 411--520 and 520--588. Computer prediction of the potential B cell epitopes on the symporter revealed that, apart from aa 134--191, all the epitope domains identified overlapped, at least in part, with areas predicted to be highly antigenic. Interestingly, the antigenic domains represented by aa 191--286, 290--411 and 411--520 include regions of the polypeptide which form putative extracellular domains in the secondary structure model of the rat symporter. No correlation between the recognition of specific epitopes on the human symporter and the type of autoimmune thyroid disease was demonstrated.