Yvonne G. Lukes

TSH receptor gene expression in retroocular fibroblasts

RNA was isolated from fibroblasts from the retroocular area, from endomysial fibroblasts obtained from orbital lateral rectus muscle, and from abdominal skin fibroblasts. The RNA was reverse transcribed into cDNA which was then used as a template for PCR with primers encompassing a portion (nucleotides 989–1235) of the extra-cellular domain of the human TSH receptor (hTSH-R). A definite 247 BP product was detected from fibroblast RNA by ethidium bromide staining, and was confirmed by hybridization with labelled hTSH-R cDNA. The product had homology with the known TSH-R cDNA. These studies indicate that human fibroblasts can express hTSH-R, and they suggest that a cross reactive immunologic response between anti-hTSH-R and these fibroblast TSH receptors may play a role in the genesis of Graves’ ophthalmopathy.

Ras mutations are uncommon in sporadic thyroid cancer in children and young adults

Mutations in the ras genes (H-ras, K-ras, and N-ras) occur in 10–15% of all human cancers, and commonly arise from single base substitutions at codons 12, 13, or 61. Although ras mutations have been found in adult thyroid cancers, they were absent from the two studies which examined childhood thyroid cancers. Both studies included only children with radiation induced thyroid cancer, and it remains unclear if ras mutations occur in children without radiation exposure. To answer this question, we examined archival tissue blocks from 31 children with papillary thyroid cancer (PTC) 4 with follicular thyroid cancer (FTC), 2 with medullary thyroid cancer (MTC), and 1 with lymphoma (LYM). Only 1 patient with PTC had previous radiation exposure. Genomic DNA was extracted and used for PCR amplification of the ras genes. The PCR products were analyzed by oligospecific hybridization for mutations at codons 12, 13, and 61. Two of the PTCs (6.5%) contained ras mutations. Both patients had class II disease and no history of previous radiation exposure. One patient subsequently developed bone and lung metastases. The patient with lymphoma also had a ras mutation (N-61), but ras mutations were absent from all FTC and MTC. These results suggest that ras mutations are uncommon in spontaneous childhood thyroid cancer, but occur with a frequency similar to that found in previous reports of adult differentiated thyroid cancers. The number of subjects was too small to determine if ras mutations are more common in patients with aggressive papillary thyroid cancer.

Thyroglobulin messenger ribonucleic acid levels in the peripheral blood of children with benign and malignant thyroid disease

Reverse transcriptase–PCR has identified thyroglobulin mRNA (Tg mRNA) in peripheral blood of normal adults and adults with thyroid cancer. However, no children were studied. The primary objective of this study was to determine whether whole blood Tg mRNA levels differ between benign and malignant thyroid disease in children. The secondary goals were to determine whether whole blood Tg mRNA levels vary with age or pubertal development among children with thyroid disease. Whole blood Tg mRNA levels were determined in 38 children (29 girls, nine boys; median age, 14.5 y; range, 4.8–20.4 y) with benign and malignant thyroid disease and correlated with diagnosis, age, pubertal status, thyroid size, and serum levels of free thyroxine, TSH, and Tg protein. Tg mRNA levels ranged from 3.3 to 104 pg Eq/μg total thyroid RNA (mean, 28 ± 20.2 pg Eq/μg total thyroid RNA) and were similar in benign and malignant disorders (p = 0.67). However, in children with previously treated papillary thyroid cancer, Tg mRNA levels directly correlated with total body 131I uptake (p = 0.026) and serum Tg protein (p = 0.037). There was no difference between boys and girls, and no change with pubertal maturation. In children with benign thyroid disease, Tg mRNA levels correlated with serum TSH (p = 0.031), but not with diagnosis, age, Tanner stage, or thyroid size. We conclude that Tg mRNA levels are similar in children with benign and malignant thyroid disease and unchanged by age or pubertal status, but correlated with tumor burden in previously treated papillary thyroid cancer.

Immunogenicity of a unique region of the human thyrotropin receptor

Clarifying the role of the TSH receptor protein in the autoimmune process may be the key to understanding the development of Graves’ disease. In the present study we used a 16 amino acid peptide of the human TSH receptor (hTSHR) to immunize rabbits. A comparable, but theoretically less immunogenic, peptide was injected into other rabbits. The antibody response against these and other peptides, as well as against solubilized human thyroid membrane (TM) and guinea pig fat cell membrane (GPF) proteins, was tested using ELISA and Western blots. The GPF and TM binding pattern of rabbits’ sera was compared to that of Graves’ patients’ sera. We have identified an area of antigenic cross-reactivity between GPF and TM; a 63 kD protein was present in both GPF and TM, and this protein uniformly bound IgG-s of the rabbits’ postimmunization sera and one of eight Graves’ patient’s serum. We have shown that i) a theoretically immunogenic 16 amino acid peptide was indeed highly immunogenic in rabbits, ii) antibodies binding to GPF and TM were detected after immunization, and iii) the peak of thyroid stimulating immunoglobulin activity of sera was followed by a transient elevation of serum triiodothyronine levels. Further studies investigating the immunogenic epitopes of the hTSHR as well as characterizing the 63 kD protein are indicated.

Binding of thyrotropin to selected mycoplasma species: detection of serum antibodies against a specific mycoplasma membrane antigen in patients with autoimmune thyroid disease

Radiolabeled human (hTSH) and bovine (bTSH) thyroid stimulating hormone was shown to bind to five species of Mycoplasma, the wall-less prokaryotes. The maximum binding capacity of 125 I-bTSH to these five species was about 7.9 × 10−13 moles −1.4 × 10−12 moles for 50–100 μg protein with dissociation constants of approximately 1.7 to 2.2 × 10−7M. Approximately 50% of the 125I-bTSH binding was displaced by excess, unlabeled bTSH or hTSH, but labeled bTSH was not effectively displaced by growth hormone, LH, FSH, prolactin, or the beta subunit of hTSH, FSH and LH. Antisera prepared against Mycoplasma gallisepticum and Mycoplasma pneumoniae bound to human thyroid membranes and guinea pig fat cells, suggesting that receptors on human thyroid tissues and on Mycoplasma cells may have similarities in antigenicity. These findings were substantiated by the occurrence of TSH binding to Mycoplasma antisera. Further, sera from three of six patients with Graves’ disease containing antibodies to thyroid tissues also reacted to a 108 Kd polypeptide of Mycoplasma gallisepticum.