Prevalence of autoantibodies to thyroperoxidase in patients with various thyroid and autoimmune diseases.

An original radioimmunoassay for quantitation of circulating autoantibodies (aAb) to thyroperoxidase (TPO) proved to be well suited for large scale routine testing. The present study was aimed to assess the prevalence of aAb to TPO in patients with various thyroid and autoimmune disease and, for comparison, in women referred for reproductive disorders and indication of in vitro fertilization. Anti-TPO aAb were measured in sera from 32 healthy subjects and 262 patients thoroughly investigated for thyroid dysfunction. As determined in healthy subjects, the normal level of aAb to TPO in serum ranged from 0.30 to 3.07 mg/l (of affinity-purified) anti-TPO aAb. Anti-TPO and anti-MIC aAb levels were both normal in 115 patients and correlated well (r = 0.835, P less than 0.001) in the remaining 147 patients. Coexistence of normal level of anti-TPO aAb and abnormal level of anti-MIC aAb was found in 4 patients and ascribed to a lack of specificity or sensitivity of the test for anti-MIC aAb. Coexistence of abnormal level of anti-TPO aAb and normal level of anti-MIC aAb was found in 67 patients of whom 62 presented only slightly elevated (3.1 to 10.0 mg/l) anti-TPO aAb concentration; the 5 remaining patients, all with overt thyroid autoimmune disease, showed anti-TPO levels between 10.7 to 100.7 mg/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunopurification and characterization of thyroid autoantibodies with dual specificity for thyroglobulin and thyroperoxidase.

The presence of autoantibodies (aAbs) to thyroglobulin (TG) and thyroperoxidase (TPO) in most of the patients with autoimmune thyroid disease is now well documented. Studies of these aAbs suggested that some, termed TGPO aAbs, could interact with both TG and TPO. This hypothesis was investigated using IgG fraction from a pool of 25 patients' sera with high TG and TPO aAb titres. Immunopurification of TG, TPO and TGPO aAbs was carried out by sequential affinity chromatography using a large quantity of highly purified human TG and TPO. TGPO aAbs, obtained absorption-elution of affinity purified TG aAbs onto a TPO column, were found to represent about 20% of the TG reactive aAbs and 0.23% of the total amount of IgG. Purified TGPO aAbs were characterized and compared to specific TG and TPO aAbs. In contrast to TG and TPO aAbs which recognized only their target antigen, TGPO aAbs showed high affinity interactions with both TG and TPO. As compared to TG aAbs, TGPO aAbs displayed similar affinity for native TG and higher affinity for denatured TG. Compared to TPO aAbs, TGPO aAbs showed lower affinity for both native and denatured TPO. TGPO aAbs also differed from specific TG and TPO aAbs with regard to IgG subclass distribution and antigen fine specificities as determined by monoclonal antibody assisted mapping of TG and TPO surface epitopes. Taken together, these data indicate that TGPO aAbs are effectively present in the serum of patients with autoimmune thyroid disease. TGPO aAbs may be considered as a subpopulation of TG aAbs with the unique property to cross-react with TPO. The existence of aAbs cross-reacting with these functionally and antigenically related thyroid molecules could lead to a re-examination of the emergence of thyroid autoimmunity.     

Significance of thyroglobulin antibodies cross-reactive with thyroperoxidase (TGPO antibodies) in individual patients and immunized mice.

Thyroglobulin (TG) and thyroperoxidase (TPO), both involved in thyroid hormone synthesis, represent major autoantigens in thyroid autoimmune disease. Despite numerous studies, the emergence, pathophysiological significance and role of autoantibodies to TG and TPO remain elusive. The recent identification of a new category of thyroid-specific autoantibody interacting with both TG and TPO (TGPO autoantibodies) offers a new opportunity in the study of thyroid autoimmunity. To gain a better insight into the significance of these TGPO autoantibodies, measurement in individual samples appeared necessary. The unique property of TGPO autoantibodies, simultaneous binding to TG and TPO, was used to set up a sandwich method which combined coated TG and radio-iodinated TPO. This method was found to be strictly specific for TGPO autoantibodies and sensitive enough to assay TGPO autoantibodies in serum. In humans, TGPO autoantibodies were found in most of the sera with high TG and TPO autoantibody titres, but not in sera negative for TG autoantibodies, whatever the TPO autoantibody titre. Furthermore, high TGPO autoantibody titres were found in sera strongly cytotoxic for cultured porcine thyroid cells. However, significant correlation of TGPO autoantibody titre was observed neither with TG and TPO autoantibody titres (n = 48) nor with complement-dependent cytotoxicity (n = 50). TGPO antibody assay was also performed in individual plasma of CBA/J mice immunized with either human TG (n = 6) or human TPO (n = 6). Immunization with TG induced high levels of not only TG but also TGPO antibodies, which exhibited a strong reactivity for TPO and whose binding to TG and TPO was fully inhibited by TG. In contrast, immunization with TPO induced high levels of only specific TPO antibodies accompanied by low levels of specific TG antibodies. In this case TGPO antibodies were not detected. Of note, TG- and TPO-immunized mice mounted an immune response against their own TG, but did not exhibit histological signs of thyroiditis. Large panels of TG and TPO MoAbs were also investigated with this method: 18/25 TG MoAbs and only 1/13 TPO MoAbs were found cross-reactive. Taken together, these data provide evidence that TGPO antibodies are effectively present in individual patients and TG-immunized mice, are different from specific TG and TPO antibodies, and may derive from natural B cell repertoire by autoimmune processes involving TG and not TPO.     

The relative antigen-neutralizing capacity (avidity) of thyroglobulin antibodies in various thyroid diseases as measured by haemagglutination-inhibition with antibody-coated cells

Human sera which in a haemagglutination test with thyroglobulin-coated cells revealed thyroglobulin-antibody titres of 5⁵ or more were tested by a haemagglutination-inhibition technique with cells coated with highly purified rabbit antibody to human thyroglobulin. Theoretical considerations indicated that the titres obtained in this inhibition system would be influenced not only by the concentration of thyroglobulin-antibody, but also by the avidity of the antibody. Therefore, it should be possible to disclose differences in antibody avidity by comparing the inhibiting and agglutinating activity of the sera.

Based on these assumptions, sera from twenty-four patients with lymphadenoid goitre were found generally to contain highly avid thyroglobulin antibodies, although a decrease in avidity could apparently occur during the disease. Similar findings were made with sera from twenty-three patients with primary myxoedema, whereas sera from twelve thyrotoxic patients yielded relatively weaker inhibition reactions and were of a significantly lower avidity. Antibodies of very low avidity were found in five of seven cases with a clinical diagnosis of non-toxic goitre (or in all five cases of histologically verified non-toxic goitre), in two of three patients with cancer of the thyroid, and in two patients with subacute thyroiditis. These differences in reactivity of the thyroglobulin-antibodies may refer to different pathogenic mechanisms eliciting the autosensitization.

     

The measurement of the serum sex-hormone binding globulin in various thyroid diseases

Synthesis of "sex-hormone binding globulin" (SHBG) is influenced by thyroid hormones and its concentration in the serum of female subjects may be a marker of thyroid hormone effect at the peripheral tissue (liver) level. Compared to the levels found in euthyroid females (n = 46), the mean (+/- S.D.) serum SHBG concentration was found elevated in overt hyperthyroidism (Graves' disease: n = 56; 141.6 +/- 37.6 vs. 48.3 +/- 16.2; toxic nodular goiter: n = 16; 119.9 +/- 50.7 vs. 48.3 +/- 16.2 nmol/l; P less than 0.001). In contrast, it was decreased in manifest hypothyroidism (n = 25; 24.9 +/- 14.8 vs. 48.3 +/- 16.2; P less than 0.001). In the group of preclinical hyperthyroidism (n = 43), despite suppressed TSH secretion, the serum value of SHBG was normal (47.4 +/- 16.8), while its serum level approached the lower border of the normal range in subclinical hypothyroidism (n = 10; 33.6 +/- 6.1 vs 48.3 +/- 16.2 nmol/l; P less than 0.01). Data indicate that the pituitary responds more sensitively than the liver to a slight change of the serum thyroid hormone level. During thyroid hormone replacement for hypothyroidism, measurement of serum SHBG may provide help to assess the response of the target organ to the given therapy. In patients with generalized resistance to thyroid hormone, the serum SHBG level is within the normal range (51.3 +/- 9.8 nmol/l), thus, its determination supports the diagnosis of this disease.     

Circulating immune complexes in various thyroid diseases.

In a study of 171 patients with various thyroid diseases, circulating immune complexes (CIC), measured by a C1q solid phase radioassay, were detected in 26% of the patients as compared to 8% of the control subjects. CIC were found in 33--55% of the patients with a well defined thyroid autoimmune disorder (Hashimoto's goitre, asymptomatic thyroiditis, spontaneous myxoedema and Graves' disease) and also in the same proportion of patients with diffuse goitre. CIC were correlated to the presence of serum antibodies to microsomal thyroid antigen but not to their titre. No relationship was observed between CIC and the age or sex of the patients and the presence of exophthalmos, or between CIC and the different thyroid function tests or serum anti-thyroglobulin antibodies. CIC were found in untreated patients as well as in those treated with prednisone, methimazole or thyroxine.     

Thyroid autoantibody synthesis by cultures of thyroid and peripheral blood lymphocytes. II. Effect of thyroglobulin on thyroglobulin antibody synthesis.

The influence of antigen (thyroglobulin, Tg) on Tg antibody synthesis has been investigated using cultures of Hashimoto thyroid and peripheral blood lymphocytes. In cultures of thyroid lymphocytes, Tg antibody synthesis was stimulated by a 24h pulse of Tg (10-100 micrograms/ml) and similar results were obtained using spleen lymphocytes from BALB/c mice immunized with human Tg. In contrast, Tg antibody synthesis by Hashimoto peripheral blood lymphocytes was not affected by similar concentrations of Tg (1-240 micrograms/ml) in the presence or absence of pokeweed mitogen (PWM). However, peripheral blood lymphocytes from two out of nine patients produced increased levels of Tg antibody in the presence of very low concentrations of Tg (50 ng/ml). This increase in Tg antibody production was accompanied by a rise in total IgG synthesis indicating that the response to Tg was polyclonal. On the basis of other unusual features of the lymphocyte cultures from these two patients including a relatively small response to PWM and evidence of circulating plasma cells, it is suggested that sufficient numbers of lymphocytes responsive to Tg are only released into the circulation during active phases of the disease process.     

Immunohistochemical localization of thyroglobulin (Tg), thyroxine (T4), and triiodothyronine (T3) in thyroid diseases

Thyroglobulin (Tg), thyroxine (T4), and triiodothyronine (T3) were detected by the immunoperoxidase method in 51 cases of the following thyroid diseases: euthyroid goiter, Basedow's disease, Hashimoto's thyroiditis, folliculo-papillary carcinoma, follicular carcinoma, anaplastic carcinoma, and medullary carcinoma. Tg, T3, and T4 are present in most of the examined disorders, excepting anaplastic and medullary carcinoma. The pattern of Tg, T3, and T4 distribution between the cases of a benign lesion is rather similar; Tg and T4 are localized both in the epithelium and the colloid; T3 is disposed especially in the follicular cells. The pattern of Tg, T3, and T4 distribution in differentiated carcinomas is more heterogeneous. In half of the cases, T3 distribution resembled that of Tg. In 2 cases, a predominance of T4 was noted. In the other specimens, T3 and T4 showed a weak or a negative immunostaining.     

Thyrotropin binding inhibitory immunoglobulin (TBII) and thyroid diseases using ROC analysis

At attempt to detect the auto-immune origin of hyperthyroidism may be made by measuring thyrotropin binding inhibitory immunoglobulin (TBII) which uses the antibodies ability to inhibit labelled TSH binding to the TSH receptor. This study was carried out on 196 patients of which 128 had autoimmune hyperthyroidism and 68 another thyroid disease. Construction of receiver operating characteristic (ROC) curves allowed us to show the quality of the assay. This method, according to the prevalence of the disease, helped to determine the ideal cut-off of the assay. This cut-off was between 13.5 and 7.5% for prevalences ranging between 10 and 80%. For a 9% cut-off, which corresponded to the group studied, we observed 87.5% sensitivity and 87% specificity. Existence of false positives and false negatives was linked to the assay method which only informed us about the occupation of the TSH binding site and not its physiological activity. However, we concluded this easy to perform assay is a good test for the diagnosis of autoimmune hyperthyroidism.     

Amylase in thyroid tissues from normal and various thyroid diseases. Biochemical and immunohistochemical study

Amylase activity was measured in thyroid tissues of various thyroid diseases and was analysed electrophoretically. Normal thyroid tissues contained significant amounts of amylase (mean +/- SD; 2.71 +/- 1.15 IU/g of tissue), and their amylase isozyme was composed of a majority of salivary type isoamylase and other peculiar isoamylase. The statistical decrease of amylase activities in tissues of Graves' disease under hyperthyroidism, thyroid carcinoma, and most of thyroid adenomas were found (Graves' disease; 1.04 +/- 0.41, carcinoma; 1.49 +/- 1.10, adenoma (except five cases with high activity); 0.88 +/- 0.49 IU/g tissue). Five of 18 cases of adenoma showed strikingly higher amylase activity in their tissues. Electrophoretical patterns of amylase isoenzymes in these five adenoma tissue were different from those of normal thyroid tissues. The cellular localization of amylase in the normal thyroid tissues and the adenoma tissues was also demonstrated immunohistochemically.     

Analysis of carbohydrate residues on human thyroid peroxidase (TPO) and thyroglobulin (Tg) and effects of deglycosylation, reduction and unfolding on autoantibody binding.

The contribution of carbohydrate residues and peptide chain conformation to autoantibody binding sites on human thyroid peroxidase (TPO) and thyroglobulin (Tg) has been investigated. In addition the nature of carbohydrate residues associated with human TPO has been studied. 125I-labelled human TPO and Tg were treated with the following glycosidases: EndoD, EndoH, neuraminidase, O-glycanase, neuraminidase followed by O-glycanase and PNGaseF. Thereafter binding to different sera containing TPO autoantibodies and Tg autoantibodies was assessed using solid phase protein A to separate antibody-bound and free labelled antigens. In addition, labelled Tg and TPO were treated with reducing agent (dithiothreitol) or sodium acetate buffer pH 7.5, 5.5 and 3.2 (followed by neutralisation with 2 M Tris pH 8.3) prior to antibody binding studies. Furthermore, the effect of deglycosylation and treatment with acid buffers on TPO enzyme activity was studied. The nature of carbohydrate residues associated with hTPO was analysed by assessment of the effects of different glycosidases on 125I-TPO mobility on SDS-PAGE followed by autoradiography and by the use of lectins. Deglycosylation of labelled Tg and TPO had no clear effect on Tg and TPO autoantibody binding. Reduction of labelled Tg and TPO resulted in almost complete loss of autoantibody binding with all sera studied. Furthermore, adjusting the pH of labelled TPO or Tg transiently to pH 5.5 lowered autoantibody binding in the case of all the sera and the effect was more marked at pH 3.2. TPO enzyme activity (guaiacol assay) of unlabelled TPO was decreased after treatment with EndoH but not with other glycosidases. The low pH buffers affected unlabelled TPO enzyme activity measured by iodide assay. Treatment of 125I-labelled TPO with EndoH, neuraminidase and PNGaseF caused marked changes in the double band pattern characteristic of TPO on analysis by SDS gel electrophoresis (TPO doublet). Analysis of changes in the mobility of the 2 bands of the doublet after treatment with different glycosidases and binding studies with lectins indicated that both high mannose and complex type sugar residues were associated with hTPO. The high mannose type residues were associated mostly with the lower band of the hTPO doublet whereas complex type residues were associated mostly with the upper band. Overall, our studies indicate that (1) the major autoantibody binding sites on hTPO and hTg are conformational, (2) sugar residues do not appear to be important in forming the autoantibody binding sites on hTPO and hTg, and (3) both high mannose type and complex type sugar residues are associated with hTPO.     

Purification and analysis of bispecific tetrameric antibody complexes.

In order to study the type and yield of immune complexes obtained by the mixing of purified F(ab')2 fragments of rat monoclonal antibodies specific for mouse IgG1 with equimolar amounts of purified mouse IgG1 size exclusion HPLC of the reaction mixture was performed. Immune complexes eluted as a single peak at a position compatible with a tetrameric antibody complex configuration. The yield of tetramers could be increased by incubation of the antibody mixture for several hours at 37 degrees C, indicating a preference of the tetrameric composition over other immune complex compositions. Size exclusion HPLC also showed that greater than 80% of purified tetramers retained their original dimensions after storage for 1 year at 4 degrees C, thus indicating the long-term stability of tetrameric antibody complexes. When complexes were prepared with a mixture of two different mouse IgG1 antibodies, bispecific tetramers were obtained that could be separated from monospecific tetramers using DEAE-HPLC. Purified bispecific antibody complexes of mouse IgG1 anti-CD34 (My10) cross-linked to mouse IgG1 anti-desferal with F(ab')2 rat anti-mouse IgG1 were useful for the purification of cells expressing CD34 from human bone marrow. For this purpose cells were labelled with the antibody complexes, selectively adsorbed onto columns containing desferal coated glass beads and then selectively eluted by treatment with dithiothreitol resulting in reductive cleavage of the disulfide bonds of the F(ab')2 fragments. This relatively simple cell fractionation technique illustrates the unique cross-linking properties of bispecific tetrameric antibody complexes. The procedure appears useful for further studies of hemopoietic cells and bone marrow transplantation.     

Tryptic peptides of human thyroglobulin: II. Immunoreactivity with sera from patients with thyroid diseases.

Tryptic peptides of human thyroglobulin (Tg) were analysed by Western immunoblot for their reactivity to circulating autoantibodies from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and thyroid carcinoma, and from normal human controls. Low molecular weight peptides were released after 4 h incubation of Tg with trypsin. The sera of thyroid disease patients reacted with several peptides, but predominantly bound three peptides with apparent molecular weights (MWap) of 25 kD, 20 kD, and 15 kD; the sera of normal individuals did not bind these fragments of Tg. The pattern of tryptic peptides recognized by the majority of sera from GD patients differed from that recognized by sera from most patients with HT. Autoantibodies from both groups of patients recognized a 15-kD peptide with a high frequency, but the sera from 26/43 (60%) GD patients also recognized a peptide with MWap of 25 kD, whereas the sera from 22/35 (63%) of HT patients recognized a 20-kD peptide. A few sera from patients with thyroid carcinoma reacted with peptides with MWap of 15 and 20-kD, and none bound the 25-kD peptide. The immunoreactivity of autoantibodies in HT sera to the 20-kD peptide paralleled the competitive inhibition of the MoAb 137C1 by these sera. In addition, MoAb 137C1 and Hashimoto's sera showed the same Western immunoblot-binding pattern to Tg tryptic peptides, suggesting that a Hashimoto-associated epitope and the 137C1-binding site are found on the same peptide. These findings suggest that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.