Thyroid Research in Canada

thyrobulletin Vol.13, No.2

At his address to the Annual General Meeting, June 1992, in Moncton, N.B., Dr. Volpé pointed out that thyroid research is a relatively small field in Canada. Three or four million dollars are expended annually on thyroid research and in approximately 15 centres across the country. Unfortunately, the government has cut back on medical research funding, which greatly affects a small field like thyroidology. It is regrettable that a lack of funding threatens to curtail the important progress made by researchers – there has been so much progress in thyroid research. In addition, such cutbacks make it more difficult for Canadian researchers in thyroidology to maintain their significant international presence.

Dr. Volpé’s own research program of over 20 years had been funded by the Medical Research Council. Its ultimate aim is to prevent thyroid disease. His investigations continue in the field of autoimmunity and immunogenetics. A technique to replace defective genes by injecting normal genes attached to safe viruses is showing promise.

Finally, Dr. Volpé pointed out that a recent government decision to cut back 20% of the number of doctors going into each medical field, including thyroidology, is a significant development. In his view, we do not have a surfeit of doctors. However, doctors are not well distributed through all the areas of Canada. Under these circumstances, the Thyroid Foundation’s program of funding fellowships is especially important. Each fellowship starts a new career in thyroidology and assists with important thyroid research. Each dollar contributed to this undertaking is an investment in our health and our future.

The facts presented by Dr. Volpé are cause for concern. It does not appear as though governments will reconsider their present policies in the foreseeable future. As a foundation concerned with thyroid disease, our financial support of thyroid research is most vital at this time of constraint and cut backs.

Lottie Garfield
V.P. Education & Research

The following overviews summarize the latest developments from leading thyroid researchers in Canada.

Research in Autoimmune Disease

Dr. Robert Volpé
The Wellesley Hospital, University of Toronto

For the past 25 years, members of Dr. Volpé’s laboratory have been pursuing the cause of autoimmune thyroid disease, namely Graves’ disease and Hashimoto’s thyroiditis. Their current studies involve the function of lymphocytes – cells of the immune system which are essential in the causation of these diseases. They have been able to show that there is a reduction in the efficiency of suppressor T lymphocytes, which ordinarily suppress antibody production, but are defective in patients with autoimmune thyroid disease. There are a number of chemical intermediaries which are termed cytokines, which Dr. Volpé and his associates are studying in terms of their effects on immune regulation and antibody production.

They have been studying recently how the genes (histocompatibility genes) activate the lymphocytes. Their current hypothesis is that it is here where the abnormality lies, namely that the histocompatibility genes in autoimmune thyroid disease do not properly activate the suppressor T lymphocytes when they present antigen to them. There is now early evidence that this is indeed the case. Moreover, they are studying models of autoimmune thyroid disease in some species of mice that have either no immune system, or a very abnormal immune system, and can be reconstituted with the human immune system. It is possible by using these species of mice to transplant human thyroid tissue into them without rejection and study the abnormal tissue very carefully in the transplanted mouse.

All of the above studies are designed to illuminate the abnormality of the regulation of the immune system which seems to be central to these disorders. They have also shown that the thyroid cells themselves are completely normal to begin with and are mere passive victims to the abnormality of the immune system.

Effect of Thyroid Hormones on Brain Development

Dr. Diego Bellabarba
Université de Sherbrooke, Endocrine Division

Thyroid hormones play an important role in the foetus, since they are essential for the maturation and differentiation of many organs, in particular the brain. The various steps of the hormone action on the foetal brain are not well known. To further elucidate such action, we are performing the following studies in which we will use neurons, which are individual nerve cells, from chick embryo.

We believe that by putting in direct contact the thyroid hormones with cultured neurons we will have a better model for understanding their action on the developing brain. We are investigating the effect of thyroid hormones on the synthesis of certain proteins, called Microtubule associated proteins, or MAPs, which are associated with the formation of neurons and on the activity of calcium channels foetal neurons, and on the activation of membrane proteins (G proteins) of synaptosomes (relating to the point of junction of two neurons). These structures are related to neuron differentiation. Furthermore, we will use antisense olygonucleotides to identify the thyroid hormone receptor more involved in the development and differentiation of neurons.

A better knowledge of thyroid hormone action during foetal development would improve the treatment and prevention of brain lesion in congenital hypothyroidism.

Thyroid-associated Ophthalmopathy from the Investigator’s Point of View

Dr. Jack R. Wall
Montreal General Hospital Research Institute

Thyroid-associated Ophthalmopathy (TAO), as we prefer to call Graves’ ophthalmopathy, is by far the least well understood and most difficult-to-treat problem that occurs in patients with thyroid disease. Recently work has, however, given us important insights into the mechanism of this disease and way in which it may be diagnosed early and treated more efficiently. It is even possible that this disorder will, in the near future, be a preventable disease.

TAO has always been a controversial disorder mainly because earlier investigators were not equipped technologically to carry out the necessary studies to explain why an inflammatory eye disorder should be associated with thyroid autoimmunity. Any theory which claimed to explain this association was eagerly accepted by the medical profession, even though, in retrospect, the known facts did not support the notions. With the passage of time and a much better understanding of the immunological mechanisms of both thyroid disease and the eye inflammation, old theories have been discarded to be replaced by new mechanisms based on sound scientific data.

It now seems likely that ophthalmopathy is linked with thyroid autoimmunity, particularly Graves’ disease, because of immunological cross-reactivity, i.e. the reaction of circulating antibodies with proteins (antigens) in both the thyroid cell and the eye muscle cell. There is good evidence for such cross-reactivity and this idea is gradually being accepted by the Medical and Scientific communities as the likely mechanism for both the development of ophthalmopathy and its association with autoimmune thyroid disease. If this hypothesis is true, the eye disease would occur with, or following, the thyroid autoimmune reaction in all cases. Indeed, when patients with so-called “euthyroid” Graves’ disease, that is ophthalmopathy in the apparent absence of thyroid disease, are studied using sophisticated techniques, all patients can be shown to have thyroid immunological abnormalities or thyroid disease, and isolated ophthalmopathy probably never occurs.

If TAO were the result of a reaction against thyroid antigens treatment of the thyroid disease by either thyroidectomy or radioactive iodine ablation would be expected influence the eye disease favourably. Indeed, much anecdotal evidence dating back many decades is confirmed by prospective studies of patients who are treated in this way. One can show that the immune abnormalities directed against eye muscle and the orbital information decrease when patients are treated with radioiodine. On the other hand, use of antithyroid drugs to treat Graves’ hyperthyroidism, particularly where ophthalmopathy is likely to occur, is not recommended as this can have no effect on the thyroid mass which is the target for the initial autoimmune reaction.

Although there is strong evidence that the extraocular muscle is the main target for the inflammatory action in thyroid-associated ophthalmopathy, there is also evidence for stimulation of the orbital connective tissue and fibroblasts which surround individual muscle fibres and bundles and are scattered throughout the periorbital area. It is likely that two reactions proceed together and that the resulting clinical features, namely exophthalmos, eye muscle damage and orbital inflammatory changes, are a result of the autoimmune reactions int he two tissues. Arguments about whether one or the other is more important have distracted from an understanding of the basic mechanisms of the disease. Certainly, the author’s studies have strongly focused on the eye muscle as being the most important target in this disease.

Although other groups have shown evidence for stimulation of the orbital connective tissue and fibroblasts, it now seems likely that the orbital connective tissue inflammation is secondary to the eye muscle reaction. Indeed fibroblasts are very sensitive to a large number of soluble factors produced in the course of the inflammatory reaction in the orbit and respond accordingly. On the other hand, eye muscle cell death, which can be clearly shown in the laboratory and which is associated with cytotoxic antibodies in patient’s blood, seems very likely to be the main mechanisms for eye muscle damage which characterizes the disease.

One of the main target proteins on the eye muscle cell is a protein characterized as a 64 kDa molecular wt membrane antigen which is the target for antibodies in the serum of patients with TAO. This protein has been cloned and the nuclear material encoding it sequenced so that the recombinant 64 kDa protein is now available for use in antibody tests and for the potential treatment of patients with thyroid eye disease.

We can show, in various antibody tests incorporating recombinant 64 kDa eye muscle membrane protein, that antibodies against this protein are produced very early in the course of the eye disease in patients with Graves’ hyperthyroidism even before they develop evidence for eye inflammatory and in patients with lid lag and retraction, which is likely to be a very early sign of progressive ophthalmopathy. This suggests that by testing all patients with Graves’ hyperthyroidism as soon as diagnosis is made, one can predict the development of the eye disease by identifying these antibodies in their blood.

A Canadian-based study in which patients with Graves’ hyperthyroidism in various centres across the country will be studied by thyroidologists and ophthalmologists, is planned. Serum samples, taken from the patients at each visit, will be sent to our laboratory in Montreal for eye muscle antibody measurement and ophthalmologists will assess the patients for eye signs. In this way, we can confirm that those patients with Graves’ hyperthyroidism who have antibodies to the 64 kDa protein eventually develop eye disease, whereas patients who don’t have this antibody do not. If this is confirmed in such an objective, prospective study, the next step would be to treat such patients with Graves’ hyperthyroidism and antibodies to the 64 kDa protein with a specific therapeutic agent which would prevent the development of eye disease.

Two such possibilities for specific immunosuppressive therapy are monoclonal antibodies which can be produced by injecting mice with the recombinant 64 kDa protein; such monoclonal antibodies can be selected for their ability to bind to the eye muscle in the patient and block access of the cytotoxic antibodies circulating in the blood. The recombinant protein itself, or the part of it which binds to the antibody, could be used to absorb out the antibody in the serum of the patients. In the meantime, a non-specific immunosuppressive agent such as cyclophosphamide or steroid could be used to prevent eye disease with Graves’ hyperthyroidism and antibodies to the protein.

Although it is still several years from such a diagnostic and therapeutic approach, I am convinced that the standard management of patients with Graves’ hyperthyroidism in the future will be to test for antibodies to eye muscle antigens, including the 64 kDa protein, and to treat these patients with antibodies with specific immunosuppressive agents such as described above before the onset of signs and symptoms with the expectation that the eye disease would be treated in its early phases or prevented.

Thyroid Research in Canada – Part 2

Thyrobulletin Vol.13, No.3

Mechanism of Action of Thyroid Hormones on the Central Nervous System

Dr. Jean A. Dussault
Laval University, Quebec

The influence of thyroid hormones on the maturation of the central nervous system (CNS) was first noticed following the clinical observations of infants with congenital hypothyroidism. These infants developed neurological abnormalities and mental retardation. The severity of the disease and its onset appear to play a major role in the evolution of the signs and symptoms. Also, the time when therapy is initiated plays and important role in the complete recovery (neurological and mental) of these infants.

In order to understand the mechanism of action of thyroid hormones on the development of the CNS, the rat was chosen as a model by many investigators. Correlations were established between the effects of thyroid hormones on the maturation of the CNS and the development of thyroid functions in the fetal rat. In the rat, thyroid function begins around the 18th day of gestation (4 days before birth), whereas the maturation of the CNS occurs much earlier. On the other hand, since the different parts of the CNS develop at different intervals, the influence of thyroid hormones and their effects will vary depending on the structure and the cell type studied.

This suggests that not all the cells of the CNS are target cells for thyroid hormones. Up to now, very few target cells have been identified in the developing brain. In our present knowledge, thyroid hormones act through a specific nuclear thyroid hormone receptor: the binding of triiodothyronine (T3) to its specific receptor initiates the synthesis and regulation of a limited number of mRNA coding for specific proteins. However, the mechanism of action of thyroid hormone is actually unknown. Recently, Weinberger et al and Sap et al have reported that the nuclear T3 receptor is a product of the v-erbA oncogene. This opens a new field of investigations in the mechanism of action of thyroid hormones.

We propose to study the influence of thyroid hormones on the maturation of nerve cells in vitro and to study thyroid hormone receptors during brain development.

The results expected will help us to understand why in spite of early intervention, some of the hypothyroid infants are still affected in their neuropsychological development and maybe enable us to improve our means of therapy and intervention.

Actions of TSH and Antibodies on Thyroid Cells

Dr. Jody Ginsberg
University of Alberta, Edmonton

Autoimmune Thyroid disease is associated with antibodies that block or stimulate the natural occurring thyroid stimulator, thyroid stimulating hormone, or TSH. Our laboratory has investigated the actions of TSH and these antibodies on thyroid cells in culture. We have been able to show that TSH activates an enzyme in the thyroid known as protein Kinase C. When this enzyme is activated, thyroid hormone is actually decreased. Although this seems the opposite of what one might expect, we believe that TSH primarily stimulates thyroid hormone formation, but that this additional enzyme is like a brake on a car and prevents over stimulation of the thyroid cell. Preliminary evidence would suggest that stimulating antibodies in patients with Graves’ disease only increase thyroid hormone and do not affect this enzyme. However, blocking antibodies that may occur in patients with Hashimoto’s thyroiditis may act by increasing the activity of this enzyme thus producing hypothyroidism. It is conceivable that in the distant future, drugs which would act on this enzyme may be of value in the treatment of thyroid disease.

An Animal Model for Autoimmune Thyroid Disease

Dr. Nicole Bernard
Montreal General Hospital Research Institute

Dr. Bernard is studying an animal model for autoimmune thyroid disease (ATD). The non obese diabetic (NOD) strain of mouse spontaneously develops diabetes and thyroiditis. The characteristics of autoimmune recognition of the thyroid gland, in this strain, are being assessed and compared with what is known of human ATD (i.e. Hashimoto’s thyroiditis and Graves’ disease). An important focus of the research being carried out is the identification of thyroid molecules that are recognized by immune cells which infiltrate the thyroid gland of these animals. The identification of such molecules may suggest therapeutic approaches to ameliorating or preventing autoimmune attack on the thyroid in humans.

Thyroid Hormone and Gene Regulation

Dr. Norman C.W. Wong
University of Calgary, Alberta

Our primary interest is to understand the mechanisms by which thyroid hormone regulates the activity of specific genes. We are currently attempting to determine factors that control the thyroid hormone responsive apolipo-protein A1 (Apo A1) gene. It is important to study the Apo A1 gene because the protein plays a critical role in mediating a physiologic process that transfers cholesterol from peripheral tissues to the liver for disposal, so called reverse cholesterol transport (RCT). The ability to up regulate this process is expected to be beneficial to patients with hypercholesterolemia induced atherosclerotic vascular disease. Our studies are designed to identify the factors that increase the expression of Apo A1. The results may provide us with an avenue for manipulating Apo A1 gene expression to increase RCT.

Post-partum Thyroiditis: Genetic Susceptibility Factors

Dr. Paul G. Walfish
Mount Sinai, Toronto Hospital

Clinical research studies are continuing on autoimmune thyroid disease and particularly the syndrome of post-partum thyroiditis regarding genetic susceptibility factors as determined from white blood cells. We are using the latest technology in collaboration with Dr. Klaus Badenhoop in Frankfurt, Germany.

The role of thymus-derived (T) cells in autoimmune thyroid disease, particularly Graves’ disease, and the effects of antithyroid drug therapy is also being studied using the latest analytical methods. Studies also continue on the role of needle aspiration biopsy and ultrasound in the detection and management of thyroid cancer and the role of adjunctive radioactive iodine therapy in the management of thyroid cancer.

In basic research, work is progressing to study the molecular actions of thyroid hormone receptors in congenital generalized thyroid hormone deficiency (CTHD) and the familial syndrome of generalized thyroid hormone resistance (GTHR). CTHD, as detected by newborn screening, occurs in 1:3,500 North American infants and is associated with developmental abnormalities in many tissues, particularly the brain. This results in mental retardation if not correctly recognized and treated with thyroid hormone within several months of delivery.

GTHR is a clinical disorder with autosomal dominant inheritance and is associated with one of several possible mutations in the á isoform of the thyroid hormone receptor that results in developmental and functional abnormalities. Our present overall objectives are to:

determine the thyroid hormone receptor interference mechanisms involved in the syndrome of (CTHD) using recombinant yeast DNA technology, and
elucidate the role of thyroid hormone receptors in normal and CTHD during embryonic and neonatal development using transgenic mice models.

Understanding the molecular basis for abnormal thyroid hormone receptor expression and function associated with CTHD and receptor mutations in the syndrome of GTHR will be of considerable medical importance and relevant to many other disease processes that are secondary to either hormonal deficiency or receptor mutations which interfere with normal hormonal receptor function.

Tracking the Thyroid*

Dr. Gregory Becks
The Lawson Research Institute, London, Ontario

For endocrinologist Dr. Gregory Becks, persistence pays off. At a time of financial cutbacks in research funding, Dr. Becks’ proposal for a research project involving the thyroid gland had been turned down by the Medical Research Council (MRC) in previous years. But Dr. Becks kept the research going “on a shoestring” and tried again. This year, the MRC has awarded three-year funding for the research project studying the interactions of insulin-like growth factors and their binding proteins during the growth and development of the thyroid gland.

Dr. Becks also sees the MRC support for the project as a step forward for the state of thyroid research in Canada. “This is not a major research area in Canada – only a handful of researchers are doing thyroid studies. And thyroid disorders such as an overactive or underactive thyroid, goitres, nodules, and thyroid tumours have not been very high profile as a public health issue,” says Dr. Becks.

Yet thyroid disorders affect five per cent of Canadians, account for a significant percentage of visits to endocrinologists, and cause considerable morbidity. Dr. Becks’ research is aimed at leaning more about the role of the growth factors in thyroid abnormalities. “We now know that the thyroid produces its own growth factors, and in a rat goitre model, there are changes in the growth factors when the abnormalities occur. We suspect one of the clues is in the binding proteins, which may inhibit the growth factor actions. Some key questions are: do these changes precede the abnormality? Is there a progression of changes? Are there changes in other growth factors as well?

It is too early to tell whether understanding insulin-like growth factors in the thyroid will help lead to changes in treating thyroid patients, but Dr. Becks is optimistic that the research may eventually point to a therapeutic avenue. “But in the shorter term, we hope to discover whether the insulin-like growth factor binding protein changes are reliable markers for thyroid diseases and whether these markers will help us determine the prognosis for some patients, particularly those with certain types of difficult thyroid tumours.”

Dr. Becks works closely with Dr. David Hill and a number of technicians, students and post-doctoral fellows. The Thyroid Foundation of Canada, which was the first thyroid public awareness organization to be organized in the world, gives support for summer students on the project.

The thyroid research project fits very well with the Lawson Research Institute’s expertise in the area of growth factor research. Both the Divisions of Endocrinology and Metabolism and Pregnancy and Perinatology are involved with growth factor research. “The Lawson Research Institute is a pre-eminent centre in Canada for growth factor research. We also enjoy excellent collaboration with international laboratories, particularly in the U.K. One of our post-doctoral fellows on the project, Dr. Ian Phillips, is now working in Australia, so perhaps we can get something collaborative going there as well.”

Findings concerning the growth factors and binding proteins in the thyroid may have wider implications for research and ultimately patient care, Dr. Becks believes. “The same principles and mechanisms we find at work in the thyroid may apply to the growth and development of other glands in the endocrine system.”

(*This article was published by St. Joseph’s Health Centre, London, Ontario in its Centre Report. Reprinted in Thyrobulletin, Vol.14, No.4.)

Reviewed 2000

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