Evaluation of T cell stimulation by thyrotropin-receptor epitopes in Graves' disease.

In Graves' disease (GD) immunized T cells reactive to TSH-receptor epitopes contribute to pathogenesis through B cell help, and cytotoxicity. We evaluated T cell responses to synthetic TSH-receptor epitopes in hyperthyroid patients with GD prior to therapy, at 6-8 weeks after radioactive iodine (RAI) administration, or 6-8 months later when euthyroid, and in control subjects. All T cell responses were relatively low as generally found in human autoimmune diseases. Responses in hyperthyroid GD patients were significantly greater than among controls, were augmented 6-8 weeks after RAI treatment, were still present after patients became euthyroid, and did not differ between DR3+ and non-DR3+ patients. Patient's T cells reacted to multiple different epitopes, and reactivity differed depending on the course of the disease and treatment.While certain epitopes most commonly cause T cell reactivity, we did not find evidence for a single or few "dominant" epitopes.

Immunotherapy for medullary thyroid carcinoma by a replication-defective adenovirus transducing murine interleukin-2.

We have evaluated the feasibility of gene transduction using replication-defective adenovirus vector as a novel therapy for medullary thyroid carcinoma (MTC), a thyroid C cell neoplasm. Replication-defective adenoviruses were constructed to express murine interleukin-2 (mIL-2) gene and Escherichia coli beta-galactosidase (beta-gal; lacZ) gene under the control of the human cytomegalovirus (CMV) promoter (AdCMVmIL2, AdCMVbeta-gal) by homologous recombination. The efficiency of transduction was evaluated using AdCMVbeta-gal at different conditions. The gene transduction efficiency was dependent on multiplicity of infection, duration of exposure to the virus, and viral concentration. The expression of functional mIL-2 in transduced tumor cells was verified both in vitro and in vivo. Two cell lines (rat MTC and mMTC) secreted large amounts of functional mIL-2 after transduction, as tested in cytotoxic T lymphocyte (CTL) L-2 cells. When AdCMVmIL2-infected mMTC cells were injected s.c. into their host animals, tumors developed in 2 of 10 animals, in contrast to 9 of 10 animals injected with AdCMVbeta-gal-infected mMTC cells and all 10 animals injected with parental mMTC cells. Moreover protected animals developed a long lasting immunity against mMTC tumor cells and their splenocytes, showing cytotoxicity to parental tumor cells, and active natural killer (NK) cell activity. BALB/c-SCID (severe combined immune deficiency) mice were also used to evaluate the function of NK cells in antitumor activities. No tumor developed in SCID mice injected with AdCMVmIL2-infected cells, whereas all animals injected with either AdCMVbeta-gal-infected or parental mMTC cells developed tumors. Our data indicate that IL-2 production by MTC cells leads to rejection in syngeneic animals and suggest that both cytotoxic T cells and NK cells may play an important role. In addition, transduction of adenoviral vectors into tumor cells produces some nonspecific antitumor effects.

Production of rat Spot14 protein in Escherichia coli.

Hormonal, nutritional and developmental factors modulate, in rat lipogenic tissues, the transcription of the mRNA coding for a protein of unknown function, called Spot14 (S14). The corresponding protein has never been purified from tissues. In this paper, we describe the production of S14 in Escherichia coli. In the absence of available antibodies (Ab) directed against S14 protein, our strategy was to produce this protein by constructing two different recombinant expression vectors. The first recombinant plasmid produced a S14::protein A fusion which was easily purified and then rabbit Ab could be raised against it. The second expression vector directly produced S14. This expression was demonstrated by specific binding of polyclonal Ab directed against the fusion protein. These Ab also recognized a rat-liver protein sharing characteristics of S14.