Rudolf C. Kuppers

IgG subclass distribution of thyroglobulin antibodies in patients with thyroid disease

The IgG subclass distribution of thyroglobulin antibodies (TgAb) has been studied in Hashimoto and Graves’ patients by several investigators with conflicting results, in part explainable by methodological problems. We have recently developed a quantitative ELISA to measure in absolute terms the serum concentration of TgAb subclasses. The aim of the present study was to apply this method in a large series of patients with autoimmune as well as, for the first time, non‐autoimmune thyroid diseases. We examined 28 patients with Hashimotos thyroiditis, 30 with Graves’ disease, 21 with thyroid carcinoma and 18 with non‐toxic goitre, all selected for the presence of TgAbs. The results indicated that TgAbs in thyroid diseases were not restricted to any particular isotype, but comprised all four IgG subclasses. IgG1 was represented similarly in the four groups. The same was true for IgG3, even though its contribution to the total antibody content was very small. IgG4 was the dominant subclass in patients with Graves’ disease, thyroid carcinoma and non‐toxic goitre, probably reflecting a prolonged antigenic challenge. In Hashimotos thyroiditis IgG2 was dominant, possibly because T helper lymphocytes infiltrating the thyroid are typically Th1 type.

Mouse Thyroglobulin: Conservation of Sequence Homology in C-Terminal Immunogenic Regions of Thyroglobulin

cDNA encoding 287 amino acids of the C-terminus of mouse thyroglobulin was cloned and sequenced. The amino acid homology between mouse and rat thyroglobulin was 96%, and was 78% between mouse and human. It was found that mouse thyroglobulin completely shared homology with two thyroiditogenic peptides described by other investigators. These findings are consistent with our hypothesis that in murine experimental thyroiditis, the primary thyroiditogenic epitopes are encoded by mouse-specific regions of thyroglobulin.

Tryptic peptides of human thyroglobulin: I. Immunoreactivity with murine monoclonal antibodies.

Human thyroglobulin (Tg) was treated with trypsin at different concentrations of trypsin/Tg for various incubation times at 37°C using non‐reducing conditions. A ratio of trypsin to Tg of 1:100 (w/w) was optimal to release small peptides that were reactive to murine MoAbs to human Tg. Most peptides were released after only 1 h incubation with trypsin, but these peptides were further degraded at longer incubation times. However, a few small peptides. the largest of which with an apparent molecular weight (MWap) of 40kD, resisted tryptic digestion up to at least 12 h of incubation. These resistant peptides were further degraded by trypsin at 18–24 h of incubation. Tryptic peptides of Tg, released at I h and 4 h of incubation, were analysed for their immunoreactivity to 16 well characterized anti‐Tg MoAbs by Western immunoblot. Patterns of peptide recognition of these MoAbs were generally unique. Eight MoAbs reacted with peptides of MWapof 10–25 kD and above. Four other MoAbs reacted with peptides of MWap of 25 43 kD and above, and the remaining four reacted with peptides of MWap >43kD. Nine of these MoAbs failed to recognize peptides after reduction, suggesting that the MoAbs bind conformation‐dependent epitopes. The above information will promote the development of models relating the structure of Tg to the autoimmune process, and may provide an understanding of those regions of Tg responsible for the induction of autoimmune thyroiditis.

Influence of Diet on the Induction of Experimental Autoimmune Thyroid Disease

Abstract Immunization of CBA/J mice with thryoglobulin (Tg) emulsified in complete Freunds adjuvant induces experimental thyroiditis (EAT), a well-characterized model of Hashimotos disease. Recent studies have suggested that dietary factors play a role in the modulation of the immune response and that diet can have a profound effect on the induction of autoimmune diseases. In this study, we examined the influence of diet on autoimmune thyroiditis in mice. EAT was induced in mice fed ad libitum one of the three diets, a standard maintenance chow (Agway H1000), Purina 5020 Breeding Chow, and Purina 5010 Autoclavable (unautoclaved) Diet. Tg-immunized mice fed the Agway 1000 diet were found to be resistant to the development of autoimmune thyroid disease, with only 4 out of 25 mice developing mild thyroiditis. In contrast, 16 out of 25 mice fed the Purina 5010 diet developed moderate to severe thyroiditis. Mice fed the 5020 diet were partly susceptible: 7 out of 25 developed a mild to moderate thyroiditis. Histologic examination of thyroid glands of diseased mice fed the 5010 and 5020 diets showed marked lymphocytic infiltration with destruction of follicles, compared with mice fed the Agway diet, the latter showing only mild infiltration with preservation of thyroid follicles. Titers of antibody to Tg did not differ among the groups, and there was no significant difference in the IgG isotype subclass usage. The results demonstrate that diet can markedly affect the severity of autoimmune disease in the EAT model. In contrast, diet has little effect on the humoral autoimmune response in this system. These results implicate diet as a factor in the severity of cell-mediated autoimmune destruction and suggest that dietary modification could decrease pathology in some forms of autoimmune disease.

Silicone Binding Immunoglobulins in Human Sera

We have evaluated two recently published test procedures for demonstrating antibodies to silicone. Positive control serum samples provided to us by the original authors were positive in our hands, using the methods described. However, serum samples from patients with certain connective tissue diseases but no history of silicone implants were also positive. The question of silicone-specific antibodies remains unresolved.

The IgG2a antibody response to thyroglobulin is linked to the Igh locus in mouse

The IgG-subclass usage by several strains of mice in the response to immunization with mouse thyroglobulin (mTg) was examined in the experimental autoimmune thyroiditis model. While the subclass usage by most mouse strains was similar, the Ighb allotype-bearing mice consistently produced lower IgG2a levels to mTg. Using CBA-Ighb congenic and recombinant inbred strains of mice, the lower level of IgG2a in the Ighb mouse was mapped to the Igh locus. The regulation of IgG2a appeared to be cis controlled, as the CBA x C57BL/6F1 mouse also produced reduced IgG2a of the Ighb (B6) allotype but not Ighj (CBA) allotype.

Igg Subclass Distribution of Anti-Tg Antibodies Among Thyroid Disease Patients and Their Relatives and in High and Low Responder Mouse Strains

A large body of clinical and experimental evidence points to a genetic predisposition toward the development of autoimmune thyroid disease. During the past several years, work in our laboratory1 as well as by 2 others has resulted in significant progress in sorting out the several genetic factors that influence thyroid autoimmunity in human populations. In these investigations, we have derived considerable benefit from our earlier analyses of the genetic determinants of autoimmune thyroiditis in 3 the Obese strain (OS) chicken.3 This strain of birds develops a spontaneous form of thyroiditis that closely resembles human Hashimoto’s disease. It is marked by extensive mononuclear infiltration of the thyroid gland and production of thyroid-specific autoantibodies, especially to thyroglobulin (Tg). In our studies of the OS chicken, the key finding was that the occurrence of disease is not the result of any single gene, but rather a conglomeration of several unrelated genetic lesions. 4,5 We found no evidence for “disease susceptibility” genes as such. Rather, our findings suggested that genes widely distributed among chickens derived from the original Cornell colony have a number of consequences that, in the aggregate, favor the spontaneous development of autoimmune thyroid disease. We recognize that this view differs substantially from that of many other investigators, who suggest that etiology of autoimmune disease is based on an abnormal gene in the major histocompatibility complex (MHC).6

The frequency of LPS-responsive B cells to autologous and heterologous thyroglobulin

The frequency of precursors within the mouse splenic B cell pool, reactive with mouse thyroglobulin (mTg) was estimated using a limiting dilution assay system. The mean frequency was found to be 1/3900 B cells. The results provide a minimal estimate of the frequency of mTg-reactive B cells. The frequency of mTg-reactive B cells was not influenced by the MHC locus, as both high- and low-responder strains showed similar frequencies. While the frequency of B cells reactive to human Tg was found to be similar to that reactive to mTg, only 20% of the mTg-reactive clones also cross-react with human Tg. Similarly, only 30% of huTg reactive clones were found to react with mTg. Therefore, a large proportion of Tg-reactive antibodies are restricted to self-determinants and not determinants to conserved regions of the Tg molecule.