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Basic Research: THYROID AUTOIMMUNITY
Thyroid diseases, especially Hashimotos’ thyroiditis and Graves’ disease attract international interest, since they are widely spread, particularly after the Chernobyl accident, throughout the general population (more than 500.000 patients in Greece). Furthermore, thyroid diseases are characterised by high morbidity, as a large number of patients usually have to take thyroxine for their whole life and require frequent medical attention, resulting in increased medical costs. Therefore, new methods for prevention and early diagnosis of the diseases are urgently required. At the basic research level, thyroiditis offers an excellent model of an organ-specific autoimmune disorder and can be used in the study of aberrations of immunoregulation leading to the generation of autoreactive T cells (hypothyroidism) or pathogenic autoantibodies (hyperthyroidism). The use of animal models of thyroiditis in the study of organ specific autoimmune diseases offers important advantages, as the gland is easily accessible and the main autoantigens are known. Our Laboratory is the only one in Greece working on autoimmune thyroid diseases in animal models, in collaboration with Prof. G. Carayanniotis (Endocrinology Section, Faculty of Medicine, Memorial University of Newfoundland, St John’s, Canada).
The main goal of the research activities of the Laboratory is the molecular analysis of thyroglobulin, the main autoantigen of the thyroid gland, (identification of B- and T- cell epitopes) and the development of animal models of human autoimmune thyroid diseases, in order to understand the aetiopathogenesis of autoimmune thyroid diseases and to develop reagents and methods for the early diagnosis and treatment.
 1. Development of experimental autoimmune thyroiditis (ΕΑΤ)
Hashimoto’s thyroiditis is characterised by infiltration and destruction of the thyroid gland by autoreactive Τ lymphocytes. Thyroglobulin (Tg), the main autoantigen of the gland, is a large (homodimer, 660 kDa) glycoprotein and the mapping of its epitopes, which are recognised by Τ lymphocytes, is particularly difficult. Up to date, only thirteen pathogenic epitopes of mouse Tg have been identified, which are recognised by Τ-lymphocytes and are all characterised as non-immunodominant.
Experimental autoimmune thyroiditis (ΕΑΤ), which is induced by immunization of mice with Tg or Tg-peptides, mimics the human disease. EAT is genetically controlled by H-2 complex. Strains of Η-2k και Η-2s haplotype are characterised as high responders, as far as the induction of EAT by Tg immunization is concerned, whereas strains of Η-2b και Η-2d are characterised as low responders. Our goal is to identify new pathogenic peptides, which are immunodominant within the sequence of 2748 aminoacids of the macromolecule. For the last few years we have been studying a 20mer human Tg peptide, namely p2340 (aa2340-59), which was found to be recognised by antibodies derived from sera of patients suffering from Graves’ disease. By using algorithms it was found that this peptide encompasses sequences that possibly bind to Ek MHC molecules and contain T-cell epitopes. The above hypothesis was experimentally verified. Moreover, p2340 was found to be pathogenic, inducing EAT in AKR/J mice ((H-2k haplotype). Given that murine Ek MHC class II molecules are analogue to human DR3 molecules, the above peptide’s pathogenicity was examined in DR3 transgenic mice; after peptide immunization, EAT was developed in these mice. Subsequently, the pathogenicity of p2340 was correlated to other MHC haplotypes. It is the first time that a pathogenic Tg-peptide induces EAT not only in (H-2k mice (AKR/J and CBA/J) but also in H-2d mice (BALB/c). Our future plans include the identification of the minimal T-cell epitope encompassed in the above peptide, by the use of a T-cell clone specific for p2340. To achieve this goal, the specific clone will be developed and characterised (cytokine release, MHC restriction, TCR genes). In parallel, the in vivo and in vitro antigen processing of mouse Tg will be studied, in order to identify dominant T-cell epitopes.
2. Development of an animal model of Graves’ disease
Graves’ disease (GD) is an autoimmune syndrome precipitated by antibodies against the thyrotropin receptor (TSH-R) on the surface of thyroid cells. These antibodies mimic the action of the thyrotropin hormone, frequently leading to hyperstimulation of thyroid cells and increased production of thyroxine in the bloodstream. An animal model that reproduces consistently most GD symptoms has not yet been developed, mainly because TSH-R is expressed in low quantities on thyrocytes and cannot be purified in large amounts required for animal studies. Research in this area is highly competitive on an international level. Our strategy is to develop plasmid constructs containing the hTSH-R gene, alone or accompanied by other genes (encoding for cytokines or for costimulatory molecules) and immunize mice (intradermal injection) with these constructs (naked DNA). This method is more efficient than those based on the use of protein antigens, as it allows the expression of TSH-R molecule on the surface of antigen-presenting cells in mice, in the same way as the normal receptor is expressed on thyroid cells. Furthermore, as proven by numerous studies for the production of vaccines, the immunization method with the use of genes allows the stimulation of helper and cytotoxic T-lymphocytes.
Applied Research: DEVELOPMENT OF IMMUNOASSAYS FOR DIAGNOSTIC AND OTHER PURPOSES – COLLABORATION WITH THE PHARMACEUTICAL INDUSTRY
Through participation in various applied research programs (NATO, EPET II, FAIR-EU) in collaboration with the Immunology Laboratory of “Agios Savas” Hospital and the Greek pharmaceutical industry (Farmalex and Elpen), our Laboratory has acquired the know-how for the development of new immunological products (reagents and immunoassays with a wide range of applications). Amongst the immunoassays developed are:
Diagnostic immunoassays for viral infections (e.g. hepatitis Β, HIV1/2) and autoimmune diseases (e.g. thyroid diseases)
Immunoassays for the measurement of various biomolecules (e.g. cytokines)
Immunoassays for the measurement of biochemical markers related to the quality of meat supplied to the EU market
At present, the Immunology Laboratory concentrates on the development of new diagnostic immunoassays for autoimmune thyroid diseases and of immunoassays for the measurement of biochemical markers related to meat quality.
ΕDUCATION
1.Training in Immunology (laboratory work) of under- and post-graduate students
2.Supervision of PhD theses in collaboration with Greek Universities
3.Organisation and participation in the Educational Programme of the Hellenic Pasteur Institute consisting of a Postgraduate Training Seminar on “new molecular diagnostic techniques applied in infectious and autoimmune diseases” and an annual series of lectures on various topics by Greek scientists.
4.Organisation and participation in the educational activities of other institutions (Universities/post-graduate programmes, Ministry of Public Health, Scientific Societies).
5.Compilation of notes and laboratory methodology used in seminars as well as contributions to or translation of educational books. |