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The Amsterdam autoimmune thyroid disease cohort
Strieder, T.G.A.
Publication date 2008 Document Version Final published version
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Citation for published version (APA): Strieder, T. G. A. (2008). The Amsterdam autoimmune thyroid disease cohort.
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Download date:26 Sep 2021 THE AMSTERDAM AUTOIMMUNE THYROID DISEASE COHORT
Thea Strieder
ISBN: 978-90-8559-472-7
Printed by Optima Grafische Communicatie, Rotterdam, the Netherlands
The Amsterdam AutoImmune Thyroid Disease cohort
Theodora Gerarda Ansfrida Strieder Thesis, University of Amsterdam With summary in Dutch
© 2008 by Theodora Gerarda Ansfrida Strieder, Amsterdam, the Netherlands All rights reserved. Copyright of the published articles is with the corresponding journal or otherwise with the author. No part of this thesis may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording or any information storage or retrieval systems, without written permission of the author.
Funding and financial Support: This study was supported by grant NWO-950-10-626 from the Netherlands Organization for Scientific Research, The Hague, the Netherlands. The Margarete Markus Foundation in London, England, supported financially the serological tests for Yersinia enterocolitica . The Geestelijke Gezondheidzorg Midden-Brabant supported one-month leave for the preparation of this manuscript. Financial support for the printing of this thesis has been kindly provided by Psy4You.
Amsterdam, 2008 The Amsterdam AutoImmune Thyroid Disease
Cohort
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
Prof.dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel op dinsdag 16 december 2008, te 12:00 uur door
Theodora Gerarda Anfrida Strieder Geboren te Amersfoort Promotiecommissie
Promotores: Prof. Dr. W.M. Wiersinga Prof. Dr. J.G.P Tijssen
Overige leden: Prof. Dr. R.J.M. ten Berge Prof. Dr. H.A. Drexhage Prof. Dr. J.B.L. Hoekstra Dr. J.B. Reitsma Prof. Dr. J. Smit Prof. Dr. A.H. Schene
Faculteit der Geneeskunde
Paranimfen: Jane van Ams Jaap Willem Cornelese Elk einde is een nieuw begin Contents Chapter 1. General Introduction 9
Chapter 2. The environment and autoimmune thyroid disease 17 Prummel MF, Strieder T, Wiersinga WM Eur J Endocrinol 2004; 150: 605-618
Chapter 3. Risk factors for and prevalence of thyroid disorders in a 49 cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease Strieder TG, Prummel MF, Tijssen JG, Endert E, Wiersinga WM Clin Endocrinol 2003; 59: 396-401
Chapter 4. Increased prevalence of antibodies to enteropathogenic Yersinia 65 enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease Strieder TG, Wenzel BE, Prummel MF, Tijssen JG, Wiersinga WM Clin Exp Immunol 2003; 132: 278-282
Chapter 5. Stress is not associated with thyroid peroxidase autoantibodies in 79 euthyroid women Strieder TG, Prummel MF, Tijssen JG, Brosschot JF, Wiersinga WM Brain Behav Immun 2005; 19:203-206
Chapter 6. A reduced IL2R (CD25) expression level in first and second degree 89 female relatives of autoimmune thyroid disease patients. A sign of a poor capability to preserve tolerance? Strieder TG, Drexhage HA, Lam-Tse WK, Prummel MF, Tijssen JG, Wiersinga WM Autoimmunity 2006; 39: 93-98
Chapter 7. Prediction of Progression to Overt Hypothyroidism or Hyperthyroidism 105 in Female Relatives of Patients with Autoimmune Thyroid Disease using the Thyroid Events Amsterdam (THEA) Score Strieder TGA, Endert E, Wenzel BE, Tijssen JGP, Wiersinga WM Arch Intern Med. 2008;168:1657-1663
Chapter 8. Environmental factors involved in the development of overt autoimmune 121 hypothyroidism and hyperthyroidism: evidence from a prospective nested case-control study Strieder TGA, Tijssen JGP, Wenzel BE, Brosschot JF, Wiersinga WM Submitted for publication Chapter 9. General discussion 139
Summary 163
Samenvatting (summary in Dutch) 171
Dankwoord (acknowledgements) 179
Curriculum Vitae 184
General Introduction 1|
General Introduction
Chapter 1 General Introduction
Background
In 1956 Roitt and Doniach described antibodies against thyroglobulin in serum of patients with Hashimoto disease (1). Also in 1956 Adams and Purves discovered long acting thyroid stimulator (LATS) in serum of patients with Graves’ disease, the first description of thyroid stimulating autoantibodies (2). In the same year Rose and Witebsky generated experimental hypothyroidism in rabbits by immunization with thyroid homogenate (3). A concept of autoimmune thyroid disease (AITD) was born. Autoimmune thyroid disease especially affects women. The prevalence of AITD is 6 to 8x higher in females than in males. The annual incidence of hyperthyroidism and hypothyroidism in the general adult population is estimated as 1 to 2 and 3 to 4 cases respectively per 1000 women (4). AITD has shown a tendency to cluster within families. Both Graves’ disease and Hashimoto’s disease may occur in the same family (5). Relatives have a higher risk of developing AITD than the general population (6). Twin studies indicate that genetic factors account for about 70% of the risk to develop AITD (7,8). Consequently environmental factors may contribute for about 30%. Although AITD has a very low mortality, it is associated with significant morbidity, both physically and mentally, and leads to diminished quality of life (9). Lifelong treatment is the rule rather than the exception. In AITD the immune system produces autoantibodies that attack the thyroid gland thereby either stimulating thyroid cells to produce an excess of thyroid hormone or destroying thyroid hormone producing cells, resulting in Graves’ hyperthyroidism (GH) and Hashimoto’s hypothyroidism (HH) respectively. When hyperthyroidism emerges, the patient complains of loss of energy, severe weight loss, palpitations, excessive perspiration, accelerated bowel activity with diarrhoea, goiter, and agitation or nervositas. Frequently, ophthalmopathy occurs. When hypothyroidism develops, the patient complains of hoarsening of voice, loss of energy, loss of hair, weight gain, dry and thick skin, and intolerance for low temperature. Either way, if not recognized and properly treated autoimmune thyroid dysfunction results in impaired cognitive function and mood disorders.
11 Chapter 1
Aim of the study
In order to evaluate the interaction between familial background, immunological and environmental factors involved in the etiology of AITD we assembled a cohort of 1 st or 2 nd degree female relatives of patients with proven AITD. Firstly, the study was designed to assess environmental and immunological determinants of AITD in this population at risk. Investigated were the pattern of familial occurrence of AITD, environmental factors such as stressful life events and daily hassles in combination with affect state, smoking, pregnancy, estrogen medication, iodine intake, and Yersinia infection. Also associations between thyroid function and immunological factors were investigated such as thyroid stimulating immunoglobulins (TSI), thyroid peroxidase antibodies (TPO-Ab) and thyroglobulin antibodies (Tg-Ab). The second aim was to investigate the possibility to predict the development of overt AITD within 5 years, in this study population at risk. We hypothesized that it would be possible to delineate significant environmental and immunological determinants for AITD in this study. Knowing the family history of AITD combined with these immunological and environmental risk factors we hypothesized that we could construct a prediction model for AITD in female relatives of patients with proven AITD.
Study design
Females willingly to participate in our study, were recruited from all over the Netherlands through patients visiting our department, through advertisements in local newspapers, and through thyroid patient self-support associations. All subjects were seen at our Institution and gave their informed consent. We screened 1003 female subjects with at least one 1st or 2 nd degree relative with documented autoimmune hyper- or hypothyroidism, who were in self-proclaimed good health, without a history of thyroid disease, and between 18 and 65 years of age. We checked the medical history of the affected relative(s) regarding the autoimmune nature of their thyroid disease after obtaining their informed consent. Autoimmune thyroid disease for this purpose was defined as documented hyper- or hypothyroidism in the presence of autoantibodies against thyroid peroxidase (TPO), TSH-receptor autoantibodies, histology compatible with autoimmune thyroiditis, or thyroid eye disease.
12 General Introduction
In 200 of the 1003 screened subjects absolute proof of the autoimmune nature of the thyroid disease in at least one family member was lacking, leaving 803 participants to be included in the presented cohort study. After excluding the 13 females with overt hyper- or hypothyroidism at baseline, 790 first or 2 nd degree female relatives of AITD patients were eligible for follow-up. Endpoints of follow-up were the development of overt hyperthyroidism or hypothyroidism (cases) defined by abnormal TSH values in combination with abnormal fT4 or fT3 values in plasma. Endpoints were assessed every year for 5 years. At each visit blood was sampled for assay of TSH, fT4, TPO-Ab, Tg-Ab, and TSI. Also information was collected on smoking habits, use of oral contraceptives or other estrogens, the number of pregnancies, exposure to iodine excess, Yersinia enterocolitica infection and the amount of stress exposure. Based on the expectation to assemble a cohort of 1000 females a power calculation had been performed. About 50% of the annual incidence of hyper- or hypothyroidism in adult women is of autoimmune nature. This points to an estimated annual incidence of 0.2 % for overt autoimmune thyroid disease (AITD) in women. About ten out of every 1000 women will develop overt AITD within a time span of five years. We assumed an eightfold increase in incidence in female relatives of patients with AITD relative to the general population. When thousand 1 st or 2 nd degree female relatives of patients with proven AITD are included the expected number of females with newly diagnosed overt AITD after five years of follow-up will be 80. When 160 controls are selected, than the study will have a 80% power to identify risk factors with an odds ratio of at least 2.25, provided that the prevalence of the risk factor in the control group is at least 25%. For risk factors with a lower prevalence (e.g. 10%), a power of 80 % will be achieved when the odds ratio is somewhat higher (e.g. 3.0).
Outline of this thesis
In chapter 2 an overview of known environmental factors and their possible pathogenic mechanisms in relation to AITD is described. In chapter 3 baseline characteristics of the assembled cohort are presented and the observed risk factors for and prevalence of AITD in the cohort are described. In chapter 4 the hypothesis is that infection with Yersinia Enterocolitica (YE) is more prevalent in the cohort than in control women of the Dutch population. It was also investigated whether YE infection was more prevalent in the euthyroid participants of the cohort with TPO-Ab then in those without TPO-Ab.
13 Chapter 1
In chapter 5 the hypothesis is that exposure to stressful life events results in TPO-Ab production. Stressful life events have been shown to precipitate GH but the evidence was criticized for recall bias. The role of stress in the pathogenesis of HH has not well been studied. A possible association between exposure to stress and the prevalence of TPO Ab is looked into by relating exposure to stress measured by selfreported questionnaires validated for the Dutch situation to the presence of TPO Ab in still euthyroid subjects at study entrance. In chapter 6 the hypothesis is that T cells in the peripheral blood will have signs of activation in the early stages of thyroid autoimmunity. Cell membrane expression of several relevant molecules are studied in peripheral lymphocytes by measuring the fluorescence of these cells after incubation with marker specific fluorescence; a contrast was made between TPO-Ab positive and negative euthyroid subjects in a random sample of the cohort at study entrance. In chapter 7 the hypothesis is that familial, immunological and environmental factors precede conversion to overt AITD so that a predictive model can be constructed. We evaluate whether we can predict from baseline characteristics who will develop clinically overt AITD in the next five years. Subsequently a prediction model of progression to overt hypo- or hyperthyroidism in euthyroid female relatives of patients with autoimmune thyroid disease by the THEA (THyroid Events Amsterdam) score is presented. In chapter 8 the hypothesis is that particular immunological and environmental factors will change in the years preceding the development of overt AITD analysed in a nested case- control study in the 5 years follow-up of the cohort. In the general discussion (chapter 9), an overview of the above mentioned studies is presented, and their scientific and clinical implications are discussed.
14 General Introduction
References
1. Roitt IM, Doniach D, Campbell PN, Hudson RV. Auto-antibodies in Hashimoto's disease (lymphadenoid goitre). Lancet 1956; 271:820-821. 2. Adams DD, Purves HD. Abnormal responses in the assay of thyrotropins. Proc Univ Otago Sch Med 1956; 34:11–12. 3. Rose NR, Witebsky E. Studies on organ specificity. V. Changes in the thyroid glands of rabbits following active immunization with rabbit thyroid extracts. J Immun 1956; 76:417–427. 4. Vanderpump MP, Tunbridge WM, French JM, Appleton D, Bates D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Tunbridge F, et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol 1995; 43:55-68. 5. Somers EC, Thomas SL, Smeeth l, Hall AJ. Autoimmune diseases co-occurring within individuals and within families: a systematic review. Epidemiology 2006; 17:202-217. 6. Chopra IJ, Solomon DH, Chopra U, Yoshihara E, Terasaki PI, Smith F. Abnormalities in thyroid function in relatives of patients with Graves' disease and Hashimoto's thyroiditis: lack of correlation with inheritance of HLA-B8. J Clin Endocrinol Metab 1977; 45:45-54. 7. Brix TH, Kyvik KO, Christensen K, Hegedus L. Evidence for a major role of heredity in Graves’ disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001; 86: 930-934 25. 8. Brix TH, Kyvik KO, Hegedus L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 2000; 85:536–539. 9. Watt T, Groenvold M, Rasmussen AK, Bonnema SJ, Hegedüs L, Bjorner JB, Feldt- Rasmussen U. Quality of life in patients with benign thyroid disorders. Eur J Endocrinol 2006; 154:501-510. 10. Flynn RWV, MacDonald TM, Morris AD, Jung RT, Leese GP. The Thyroid epidemiology, audit, and research study: thyroid dysfunction in the general population. J Clin Endocrinol Metab. 2004; 89: 3879-3884.
15
The environment and 2| AutoImmune Thyroid Diseases
Invited Interview European Journal of Endocrinology 2004; 150: 605-618
Mark F Prummel Thea Strieder Wilmar M Wiersinga
The environment and autoimmune thyroid diseases
Chapter 2 The environment and autoimmune thyroid diseases
Abstract
Genetic factors play an important role in the pathogenesis of autoimmune thyroid disease (AITD) and it has been calculated that 80% of the susceptibility to develop Graves’ disease is attributable to genes. The concordance rate for AITD among monozygotic twins is, however, well below 1 and environmental factors thus must play an important role. We have attempted to carry out a comprehensive review of all the environmental and hormonal risk factors thought to bring about AITD in genetically predisposed individuals. Low birth weight, iodine excess and deficiency, selenium deficiency, parity, oral contraceptive use, reproductive span, fetal microchimerism, stress, seasonal variation, allergy, smoking, radiation damage to the thyroid gland, viral and bacterial infections all play a role in the development of autoimmune thyroid disorders. The use of certain drugs (lithium, interferon-a, Campath-1H) also increases the risk of the development of autoimmunity against the thyroid gland. Further research is warranted into the importance of fetal microchimerism and of viral infections capable of mounting an endogenous interferon-a response.
19 Chapter 2
Introduction
Graves’ hyperthyroidism, Hashimoto’s hypothyroidism and post-partum thyroid dysfunction are common disorders. They have an autoimmune origin and are therefore also alluded to as autoimmune thyroid disease (AITD). Like other organ-specific autoimmune endocrinopathies, e.g. type I diabetes mellitus (IDDM), they have a multifactorial etiology. Genes are certainly involved and in order to develop AITD a subject will have a certain genetic susceptibility, probably involving multiple genes of which only a few have been identified. Most notably, certain human leukocyte antigen (HLA)-DR genes determine this genetic susceptibility, but there are other genes involved and AITD thus has a polygenetic background. Nevertheless, non-genetic (environmental, hormonal) factors must also play an important etiologic role, because the concordance rate for AITD in monozygotic twins is not 100%. Another argument is that immigrants coming from countries with a low incidence of autoimmune diseases will adopt the incidence rate of the new country. For instance, type I diabetes mellitus is 10 times more frequent in Pakistanis living in the UK than in those living in Pakistan (1). In recent years, a number of excellent reviews have been published on the genetic background of AITD (2–6). Here we will attempt to review the environmental factors that may be involved in the development of AITD (Table 1). In this review we will consider both Graves’
Table 1 . Environmental factors involved in the etiology of AITD
Environmental factor Mechanism Phenotype
Low birth weight Insufficient thymic maturation TPO antibodies Iodine excess No escape from Wolff-Chaikoff effect HT Jod-Basedow GD Selenium deficiency Unknown: viral infections? HT Longer reproductive span Estradiol effect? HT Oral contraceptives Protective TPO antibodies Fetal michrochimerism Male cells in thyroid elicit antithyroid attack HT and GD Stress Upregulation HPA axis GD Allergy Unknown: high IgE levels GD Smoking Hypoxia? High IgE levels GD; esp. GO Yersinia enterocolitica infection Moleculair mimicry GD
See text for explanation and references. GD, Graves’ disease; HT, Hashimoto’s thyroiditis; GO, Graves’ ophthalmopathy. disease and Hashimoto’s thyroiditis. Despite their different phenotype, they do share some homology. First, autoantibodies against thyroid peroxidase (TPO) are common in both diseases. Secondly, Graves’ disease and Hashimoto’s thyroiditis appear to run in the same families and thus share a common genetic background (7, 8).
20 The environment and autoimmune thyroid diseases
Fetal growth
Reduced fetal growth is a risk factor for several common disorders, such as chronic heart disease (9), and famine exposure during fetal life is associated with subsequent glucose intolerance during adult life (10). Prenatal malnutrition is associated with a lower thymic and splenic weight (11), and this may cause earlier maturation of the thymus resulting in a decline in T suppressor cells (12). Indeed, Phillips et al. (12) found that among 305 women aged 60– 71 years born in the UK the presence of TPO antibodies was positively related to lower birth weights (but not to weight at 1 year of age). The prevalence of TPO antibodies was 2.4 times higher in women with a birth weight of less than 5.5 lbs (2.49 kg) compared with those with a higher birth weight. In a twin study, the same group found that among monozygous twins, the smaller twin had higher levels of TPO antibodies (13). Because of the genetic identity of monozygous twins, this study strongly suggests that certain intrauterine factors causing reduced fetal growth are the first environmental risk factors for AITD in later life. However, this was not confirmed in another twin study where birth weight was not found to be a determinant for clinically overt AITD (14).
Iodine intake
Iodine intake seems to influence the prevalence rates of hyper- and hypothyroidism. In areas with sufficient iodine intake, hypothyroidism is more common than in iodine-deficient regions (15), whereas the overall prevalence of thyrotoxicosis is greater in iodine-deficient areas (16). Looking at the different causes of hyperthyroidism, Graves’ hyperthyroidism as the cause of thyrotoxicosis is seen more frequently in iodine-replete areas (17), and TPO antibodies as a marker for impending thyroid failure are more prevalent in iodine-deficient regions (18). Excessive iodine intake can cause dysthyroidism, especially in patients with underlying autoimmune thyroiditis (19). Due to a failure to escape from the Wolff–Chaikoff effect, iodine excess can cause hypothyroidism and/or goiter, but if autonomously functioning nodules or a subclinical form of Graves’ disease are present, it can also induce hyperthyroidism (Jod–Basedow effect) (20, 21). Both phenomena are thought to lead to some thyroid destruction and hence presentation of thyroidal antigens to the immune system leading to an autoimmune reaction (22). It thus appears that iodine intake is indeed a risk factor for the development of AITD. This is in agreement with animal studies showing that a high
21 Chapter 2 iodine intake aggravates autoimmune thyroiditis in several genetically susceptible animal strains (23–25).
Selenium intake
Selenium is a trace mineral and an essential nutrient for selenocysteine synthesis and is also called the 21st amino acid. It is incorporated into 35 selenoproteins, mostly enzymes (26). Selenium also has a marked influence on the immune system and selenium deficiency is associated with a greater susceptibility for viral infections such as the Coxsackie virus (27), possibly because T-lymphocytes have an important functional need for selenium (26). In addition, selenium acts as an antioxidant and reduces free radical formation. It plays an essential role in thyroid hormone synthesis, because two enzymes involved in thyroid hormone production are selenoproteins: the deiodinases and glutathione peroxidase (28). Selenium deficiency leads to a variety of symptoms including a higher miscarriage rate (29) and a higher cancer mortality rate (26). Selenium intake in Europe is lower than in the United States and in many countries it is below the UK reference nutrient intake of 75 mg/day. Sources of selenium are crab, other shellfish and fish, but alternative sources such as wheat are relatively low in selenium content because of the low selenium availability in European soils (26). Low selenium blood levels are associated with increased thyroid volume and with thyroid hypoechogenicity, a marker for lymphocytic infiltration (30). In agreement with this finding, a recent double-blind randomized trial in patients with subclinical hypothryoidism showed that treatment with 200 mg sodium selenite caused a significant decrease in TPO antibody titers (as well as an increase in quality of life), without affecting thyroid hormone status (31). In another randomized trial in patients with subclinical hypothyroidism who were treated with thyroxine supplementation, addition of 200 mg selenium methionine led to a significant decrease in TPO antibody concentrations (32).
Hormonal influences:
Female sex One of the most striking characteristics of organspecific autoimmune diseases is its female preponderance. The female:male ratio for Graves’ disease and Hashimoto thyroiditis is 5– 10:1 (33). The reason for this is unclear and genetic factors must play a role, although it is
22 The environment and autoimmune thyroid diseases noteworthy that Hashimoto’s thyroiditis is very prevalent among girls with Turner’s syndrome (XO karyotype), but not in men with Klinefelter’s syndrome (XXY karyotype) (6). The influence of the X chromosome is thus limited and hormonal influences may also be operative in the induction of AITD. It is interesting to note the female preponderance in nonautoimmune-mediated thyroid disease, such as multinodular goiter, but this is outside the scope of this review.
Oral contraceptives Another link to explain the sex difference would be the use of oral contraceptives or hormone replacement therapy (HRT). The latter, however, was found not to be associated with either subclinical hypothyroidism or the presence of TPO antibodies (39), although in one case report an exacerbation of eye symptoms was seen in a woman with Graves’ ophthalmopathy starting HRT (40). As for oral contraceptives, used by over 100 million women worldwide (41), there are remarkably few studies on their use and the development of AITD and in contrast to what one intuitively may think their use seems to protect against AITD. In an early large study among 46 000 women, cases of hypo- or hyperthyroidism together were seen less frequently among oral contraceptive users than in controls (relative risk (RR), 0.68; 95% confidence interval (CI), 0.52–0.85) (42). Two large population-based studies found that thyroid volume was smaller in oral contraceptive users than in controls (43, 44). We found that estrogen use protected against the development of hyperthyroidism, independently of the number of previous pregnancies (7). This is in agreement with the observation that the use of contraceptives had a protective effect for the development of Graves’ disease (odds ratio (OR), 0.68; 95% CI, 0.49–0.93), but not for Hashimoto’s thyroiditis (45).
Parity Silent thyroiditis frequently occurs in the post-partum period, hence the name post-partum thyroid dysfunction, but Graves’ disease is also often seen in the first months post-partum. During pregnancy, the immune system is suppressed with a fall in the T-helper/suppressor- cell ratio, whereas in the first post- partum months T-cell activation occurs and thyroid autoantibody production rises (34). The immune suppression during pregnancy suggests that high levels of estradiol (E2) may prevent autoimmunity, which is indeed true in several animal models for T-helper (Th)-mediated diseases (35). This immune suppression is associated with a decrease in the severity of Th1-mediated autoimmune diseases such as type I diabetes mellitus, rheumatoid arthritis and multiple sclerosis, whereas systemic lupus
23 Chapter 2 erythematosus (SLE) often worsens or remains unchanged during pregnancy (36). This has been attributed to a shift in the Th1/Th2 balance towards Th2 immunity to protect the fetus. This paradigm has recently been challenged and does not explain why Graves’ disease, as a clearly autoantibodymediated disease, also abates during pregnancy. On the other hand, the hyperprolactinemia of the post-partum period suggests that prolactin may act as an immunostimulant, although prolactin levels are also clearly elevated during pregnancy. In a large survey among 1877 subjects, hyperprolactinemia was found not to be associated with AITD (37). It is possible, therefore, that parity itself is responsible for the gender difference in AITD, but no relation could be found between Hashimoto’s thyroiditis and parity (38). In this study, however, a lower risk for Hashimoto’s thyroiditis was found in subjects with a later age at menarche ( ≥15 years) and a higher risk with a later age at menopause ( ≥51 years), resulting in a higher risk for Hashimoto’s thyroiditis in women with longer reproductive spans.
Fetal microchimerism
Lastly, a new concept has emerged that may explain the female preponderance: fetal microchimerism. This involves the transfer of fetal cells into the maternal circulation. These fetal cells can persist for a long time (46), and the consequences of the presence of semi- allogeneic cells for autoimmunity are currently being explored, also in the field of AITD (47). Imaizumi et al. (48) found fetal cells in the thyroid glands of 12/46 (46%) of Tg-immunized pregnant mice as compared with only a small number in 2/10 (20%) of control pregnant mice. The same group then found that fetal cells were more often present in thyroid glands of patients affected by Graves’ disease than in nodular thyroids (49). Klintschar et al. (50) found intrathyroidal fetal cells in 8/17 (47%) Hashimoto patients compared with only 1/25 controls. This is an exciting new discovery, and it may be that these engrafted semi-allogeneic cells trigger autoimmunity towards the organ in which they live and it has now been implicated in several other autoimmune diseases including systemic sclerosis and Sjögren’s syndrome (47, 51).
Stress
Stress has a profound influence on the immune system through neuroendocrine networks (52, 53). During stress the hypothalamo–pituitary–adrenal (HPA) axis becomes activated, which
24 The environment and autoimmune thyroid diseases would imply that stress as an immunosuppressive effect. However, it is becoming clear that stress and corticosteroids have a differential effect on Th1 and Th2 cells, driving the immune system towards a Th2 response. It thus suppresses cellular immunity and facilitates the persistent presence of certain viruses (such as Coxsackie B), while humoral immunity is enhanced. This may explain why certain autoimmune diseases are often preceded by severe stress (54, 55), and Graves’ disease seems to be one of them. The possible relation between stress and Graves’ hyperthyroidism was noted in the early descriptions by Parry, Graves and von Basedow. Later it was noted that there was always a major increase in the occurrence of Graves’ disease during wartime, a condition called ‘Kriegsbasedow’ (56). For example, the incidence of Graves’ disease in Denmark became 4- fold higher in 1942 as compared with 1940 (57). A good recent example for this is the increase in Graves’ disease during the civil war in Yugoslavia (58). However, there are exceptions because no increased frequency of Graves’ disease was found in Belfast during the civil unrest there (59). Apart from war, the association has also been studied in a number of formal case–control studies. The first study from Sweden established an association between negative life events in the year preceding the diagnosis of Graves’ hyperthyroidism (60). This was later confirmed by various other studies (61–63). However, these case–control studies can and have been criticized because of their retrospective nature, the influence of recall bias and the fact that hyperthyroidism itself is associated with increased anxiety (64, 65). Nevertheless, treatment with a benzodiazepine reduced the relapse rate in a retrospective study from 74% in untreated patients to 29% in treated patients (66). In a recent prospective study, it was shown that four personality traits (hypochondria, depression, paranoia and mental fatigue) were positively related to the relapse rate after antithyroid drugs in Graves’ disease, and that stressful life events correlated with the titer of thyroid-stimulating hormone (TSH)-receptor antibodies (67). Another case– control study found that Graves’ disease patients had had a significantly greater number of stressful life events than patients with toxic nodular goiter or controls (the latter two groups were not different from each other in terms of stressful life events) (68). Whether stress is also related to Hashimoto’s disease is unknown, but we could not find a relationship between stressful life events and daily hassles with the presence of TPO antibodies in euthyroid subjects (203).
Seasonal variation
25 Chapter 2
The incidence of myxedema coma is higher in the winter (provoked by lower ambient temperatures), whereas thyrotoxicosis is more often diagnosed in the warmer periods of the year (69, 70). The seasonality of thyrotoxicosis may not be related to the warmer temperatures (71), but to the fact that milk (in the UK the major source of iodine) contains more iodine in winter than in summer (72). Another factor responsible for seasonal differences may be the seasonal variation in viral infections or in allergen exposure.
Allergy
Allergic diseases (being Th2 disorders) and autoimmune diseases (Th1 mediated) are usually considered as the opposites in immune reactions, but this contention is now less evident because allergy-associated mechanisms can contribute to the pathogenesis of autoimmune diseases such as multiple sclerosis (73). A recent study showed that there is an association between the presence of wheezing as a measure of asthma and the occurrence of type I diabetes (74). Similarly, an association was found between an allergic constitution (asthma, atopic eczema) and AITD with OR values of 2.54 (95% CI, 1.16–5.57) and 2.95 (95% CI, 1.37–6.34) (75). Furthermore, there is a correlation between elevated levels of immunoglobulin E (IgE) and a slower decrease in TSH-receptor autoantibody levels in patients with Graves’ disease (76). Patients with elevated IgE levels also have a lower chance of remission of Graves’ disease after antithyroid drug treatment: remission levels of 20/41 (49%) versus 53/66 (80%; P ¼ 0.0014) were reported in patients with elevated and normal levels of IgE respectively (77). In addition, patients with a relapse of Graves’ hyperthyroidism had a higher rate of allergic rhinitis attacks (34%) than those who went into remission (7%) (78). The same authors reported on a TPO-antibody-positive patient who developed Graves’ disease shortly after a severe allergic rhinitis due to an allergy to Japanese cedar pollen, with a concomitant rise in IgE levels, and suggested that allergic rhinitis is another risk factor for Graves’ disease (79). There is also an association between another allergic disease, chronic urticaria, and Hashimoto’s thyroiditis (80). TPO and/or Tg autoantibodies were found more frequently in patients with chronic urticaria and angioedema (11.7%) than in controls (3.7%) (81), confirming an earlier report that found that 14% of urticaria patients had evidence for thyroid autoimmunity, more than statistically expected (82).
26 The environment and autoimmune thyroid diseases
Smoking
Apart from being a risk factor for cardiovascular diseases and lung carcinoma, cigarette smoking also has an influence on the immune system. Smoking induces a polyclonal activation of both B and T cells enhancing interleukin (IL)-2 production (83); it can also stimulate the HPA axis (84). Smoking (including passive smoking) increases serum IgE levels (85) and increases the risk of allergic symptoms (86). smoking may also increase the presentation of antigens by damaging cells and this mechanism has been proposed in the pathogenesis of Goodpasture’s syndrome (83). It may also explain why anti-heat shock protein (hsp)72 antibodies are more frequently found in smokers than in non-smokers (87). Smoking also appears to induce the production of several cytokines such as soluble IL (sIL)- 2-receptor (88), sIL-1-receptor antagonist (89), soluable Intracellular Adhesion Molecule (sICAM)-1 (90), and IL-4 but not interferon-γ (IFN-γ) (91). Smoking is linked to autoimmune diseases and increases the risk for rheumatoid arthritis, with an RR of 3.8 (92). It is also associated with Graves’ hyperthyroidism with an RR of 2.62 (95% CI, 2.01–3.38) (93), but it is especially related to Graves’ ophthalmopathy as was first reported by Hägg & Asplund (94). In our own study (95), we found an RR for ophthalmopathy of 7.7 (95% CI, 4.3–13.7), and the RR increased significantly from 2.5 for mild eye disease to 27.2 for severe eye disease (95). Similar results were obtained by others, with an RR for ophthalmopathy of 4.66 (95% CI, 3.46–6.27) in Italy (96) and 8.15 (95% CI, 2.81–23.64) in Taiwan (97). In most studies a dose–response relationship between smoking and disease severity was found (98–101). In a recent meta-analysis, the overall OR associated with smoking was 4.40 (95% CI, 2.88–6.73) (93). If smoking increases the risk for Graves’ ophthalmopathy via immunological mechanisms, one would expect it to be also related to autoimmune hypothyroidism. Although one study found an RR of 3.9 (95% CI, 1.6–9.1) (102), a meta-analysis could not confirm this: OR, 1.71 (95% CI, 0.87–3.39) (93). On the other hand, smoking was found to be a risk factor for the development of post-partum thyroid dysfunction: OR, 1.97 (95% CI, 1.23–3.17) (93). We recently found that smoking is negatively associated with the presence of TPO antibodies in euthyroid females and thus seems to protect against autoimmune thyroiditis (7). The association between smoking and Graves’ disease is further underscored by the fact that smoking increases the risk for a relapse of Graves’ hyperthyroidism (103, 104). Smoking also increases the chances of an exacerbation of the eye disease after treatment with 131I, and it
27 Chapter 2 reduces the efficacy of radiotherapy and corticosteroid treatment of the ophthalmopathy (105, 106). The reason for the strong association of smoking with Graves’ Ophthalmopathy is largely unknown (107). Hypoxia may play a role (108), because fibroblasts show a significant increase in proliferation and glycosaminoglycan production when cultured under hypoxic circumstances (109). Nicotine itself may also be involved, since nicotine addition to cultured orbital fibroblasts increased the expression of HLA-DR (110).
Drugs
Several drugs are known to induce AITD in genetically predisposed individuals, but the mechanisms by which they have this effect are different (Table 2).
Table 2. Drugs associated with the induction of AITD.
Drug Mechanism Phenotype
Amiodarone Thyroid damage, iodine excess Uncertain: HT HAART Changes in CD4+ cells GD Campath-1H Decrease in Th1/Th2 ratio GD IFN-α Stimulation of ADCC HT Stimulation of Th1 cells IL-2 Activation of T cells GD For explanations and references see text. HAART, highly active antiretroviral therapy; IFN, interferon; IL, interleukin; ADCC, antibody-dependent cellular cytotoxicity; HT, Hashimoto’s thyroiditis; GD, Graves’ disease.
Amiodarone
Thyroid dysfunction is a frequent side- effect of amiodarone, occurring in approximately 15% of patients (111). Neither amiodarone-induced hypothyroidism nor thyrotoxicosis are autoimmune mediated, although both do occur more frequently in females with thyroid antibodies (112). Whether amiodarone can induce autoimmunity is uncertain (111, 113). An early report that amiodarone induced a transient presence of TPO antibodies (114), could not be confirmed by others (115, 116).
Antiretroviral therapy
28 The environment and autoimmune thyroid diseases
Highly active antiretroviral therapy (HAART) has been found to be associated with Graves’ disease, occurring 16–19 months after initiation of different combinations of indinavir, stavudine, lamivudine and ritonavir (117). It may be related to HAART- induced changes in CD4 T cells (118).
Campath-1H
This humanized anti-CD52 monoclonal antibody induced Graves’ disease in one-third of patients with multiple sclerosis treated with this compound (119). The reason for this is unknown, but since multiple sclerosis is not associated with AITD and the patients in whom Graves’ disease occurred were not predisposed to the development of AITD (they lacked TPO antibodies), the effect should be related to the antibody. Campath-1H suppresses Th1 lymphocytes and thus shifts the Th1/Th2 balance towards antibody production and hence apparently towards a humoral immune response against the TSH-receptor (22).
IFN-ααα
IFN-α is widely used in the treatment of hepatitis C virus infection (120). Unlike IFN-g (121), it is strongly associated with the induction of AITD (122). Risk factors for the development of autoimmune thyroid dysfunction include the female sex (RR, 4.4; 95% CI, 3.2–5.9) and the pretreatment presence of TPO antibodies (RR, 3.9; 95% CI, 1.9–8.1) (122). IFN-a treatment can induce three types of thyroid dysfunction: autoimmune hypothyroidism, destructive thyroiditis and hyperthyroidism. These can occur at any time after the start of treatment with a median of 17 weeks (123). Hypothyroidism is slightly more frequent than thyrotoxicosis, and in the large majority of cases it is of autoimmune origin leading to permanent thyroid failure in approximately 60% of patients (124, 125). Graves’ hyperthyroidism is the cause of thyrotoxicosis in about half of the patients; the rest suffer from silent thyroiditis. IFN-α is a type I interferon (like IFN-β, but not IFN-γ which is a type II IFN) and stimulates Th1 development (126). It has strong antiviral activity by promoting HLA-I class I expression leading to recognition of virus-infected cells by cytotoxic T-lymphocytes (127). It also enhances antibody-dependent cell-mediated immunity by upregulating Fc-receptor density on lymphoid cells (128). Since infections with various viruses stimulate endogenous IFN-α
29 Chapter 2 production (129), we postulated that viral infections may also precipitate AITD via this IFN pathway (see below) (122).
IL-2
IL-2 is used in the treatment of HIV infection and in metastatic renal carcinoma and melanoma. Its pleiotropic immune effects include activation of T cells and among them autoreactive lymphocytes (130). IL-2 is involved in autoimmunity and it was shown recently that labeled IL-2 could be used to visualize sites of autoimmune inflammation in the pancreas of pre-diabetics and in Hashimoto’s thyroiditis patients (131). Shortterm IL-2 administration induces an increase in serum thyroxine (T4), 3,5,30-triiodothyronine (T3) and TSH levels, probably via a direct central stimulation of the pituitary (132); long-term use is associated with hypothyroidism, occurring in as many as 16% of patients (130). However, in most of these patients other therapies (lymphokine-activated killer (LAK) cell infusion) were used concomitantly and the hypothyroidism was not always of autoimmune origin (133, 134).
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
GM-CSF may activate mature lymphocytes and hence aggravate or induce autoimmunity against the thyroid (130). Nevertheless, such a side-effect has been reported only rarely (135). In one study among 25 patients, only two patients with pre-existing TPO antibodies suffered from transient hypothyroidism (136). In another study, however, no thyroid dysfunction was found among 20 patients treated with GM-CSF despite the fact that two had positive antithyroidal antibodies (137).
Irradiation
Irradiation of the thyroid gland may expose thyroidal antigens to the immune system and thus induce autoimmunity by stimulation of dendritic cells (138). Both external irradiation and internal irradiation by 131I are associated with AITD.
External irradiation
30 The environment and autoimmune thyroid diseases
External irradiation is a clear risk factor for the induction of thyroid cancer, but also of hypothyroidism. In a large series of 1677 patients irradiated to the neck because of Hodgkin’s disease, hypothyroidism was found in 47% after a median of 4.0 (0.2–23.7) years after treatment (139). This is probably caused by damage to the gland and is not autoimmune mediated. However, external irradiation is also associated with Graves’ disease, occurring in 3.3% of the patients in the same study. This was confirmed in another study, where Graves’ disease was diagnosed in 5% of 1791 irradiated patients; an 8-fold greater incidence rate than in controls (140). The reason for this association may lie in the exposure of TSH-receptor protein to the immune system and this may also be the reason that external neck irradiation also enhances the risk for Graves’ ophthalmopathy (141–144).
131I therapy
Radioactive iodine is frequently used in the treatment of Graves’ hyperthyroidism and multinodular goiter. In the last decade, it has become clear that it can induce the occurrence of Graves’ hyperthyroidism in patients treated for (non-)toxic multinodular goiter (145, 146). This complication typically occurs after 3–6 months and is seen in 4–5% of cases; it occurs in parallel to an increase in TSH-receptor autoantibodies (147, 148). Interestingly, TSH- receptor antibodies were not induced in ten patients without this complication, indicating that this induction only occurs in otherwise – genetically – predisposed individuals (147).
Environmental radiation (nuclear fall-out)
In addition, environmental radiation exposure such as occurred after the dropping of the nuclear bombs on Nagasaki and Hiroshima, or the Chernobyl nuclear plant accident, may also damage the thyroid and expose antigen to the immune system. Indeed, the survivors of the atomic bomb on Nagasaki not only have an increased risk of thyroid cancer, but also of antibody- positive hypothyroidism (149). The same appears to be true for the people exposed to the Chernobyl fallout. In one case–control study, the OR for the development of TPO antibodies was 6.89 (95% CI, 3.17–14.99) and was higher in girls (9.64) than in boys (4.19) (150). This was confirmed in another case– control study, where 18.9% of children in the exposed area had TPO antibodies versus only 5% of controls from a non-exposed region in southwestern Russia (151). There was no difference in thyroid volume or function. However, there are also a number of studies that failed to find an association with TPO antibodies
31 Chapter 2
(152–155). Nevertheless, when Eheman et al. (156) reviewed the literature they concluded that low-dose environmental radiation exposure may be associated with the development of AITD.
Viral infections
In view of the association between IFN-α and AITD, it has been suggested that viruses causing high endogenous IFN-α levels may also be associated with the induction of AITD. One such virus is the Coxsackie B virus, which has been implicated in the induction of type I or insulin-dependent diabetes mellitus (IDDM). Evidence of a recent Coxsackie B infection was found more frequently in children who developed IDDM than in controls (157–159). In another study, 39/56 (70%) patients with IDDM of recent onset had high IFN-α levels and in half of them the Coxsackie B virus could be detected, while the virus was absent in IDDM patients with low IFN-α levels (160). In line with this, IFN-α induction by injection of polyinosinic polycytidylic acid (Poly IC) could induce IDDM in a rat strain that does not spontaneously develop IDDM (161). IFN-α may thus act as a non-specific stimulus of the induction of autoimmunity. However, whether AITD is associated with viral infections is unknown since no studies like those mentioned above have been done in this field. Only congenital rubella infection, a strong risk factor for IDDM (162), is known to be associated with the presence of TPO antibodies in children, but this syndrome is very rare (163). In addition, there have been reports on the presence of retroviral sequences and proteins in thyroid glands from patients with AITD such as the gag protein from the human foamy virus (HFV) (164). The importance of this virus is doubtful, because HFV sequences can be found in blood lymphocytes from both Graves’ disease patients and healthy controls (165). Viruses are thought to induce De Quervain’s thyroiditis; however, this is not an utoimmune condition but rather an inflammatory disorder with high levels of C-reactive protein (166).
Bacterial infections
Several autoimmune diseases have been linked to bacterial infections, including Graves’ disease (Table 3) (167). There are several hypotheses to explain this association. The first implies molecular mimicry (168). Bacterial pathogens can have an antigen sharing homology with a self-antigen and an immune reaction against the bacterial antigen may then lead to a
32 The environment and autoimmune thyroid diseases
Table 3. Some examples of autoimmune disease linked to bacterial infections via molecular mimicry; adapted from Ebringer & Wilson (167)
Disease Autoantigen Bacterial pathogen Rheumatic fever Cardiac myosin Streptococcus pyogenes Ankylosing spondylitis HLA-B27 Klebsiella pneumoniae Rheumatoid arthritis Type XI collagen Proteus mirabilis Rheumatoid arthritis Hsp 60 Mycobacterium tuberculosis Graves’ disease TSH-receptor Yersinia enterocolitica
breakdown of self-tolerance resulting in autoimmunity. This mimicry is not restricted to similarity in amino acid sequences. An autoreactive T cell line derived from a patient with multiple sclerosis, recognizing myelin basic protein (the utoantigen in multiple sclerosis) presented by a certain HLA-DR 2b protein, also recognized an Epstein–Barr virus peptide (with no homology to myelin basic protein) presented by a different HLA- DR 2a molecule (169). Here, it was not the two antigens but the two antigen–HLA complexes that shared the homology (170). The hsps present on bacteria, but also expressed by human cells in response to inflammation and other stresses (171), provide another link between bacterial infections and autoimmunity (172). T-cell reactivity against hsp60 present on Salmonella typhimurium is thought to cause reactive arthritis because of crossreactivity (173). A link with thyroid autoimmunity may be suggested by the observation that Graves’ disease patients have higher levels of anti-hsp72 antibodies than controls (87, 174). Another explanation linking autoimmunity to bacterial infections is the release of sequestered antigens by local infection and inflammation (175). In this respect it seems worth noting that TSH-receptor protein is expressed by intestinal lymphocytes (176, 177). Whether bacterial infections play a role in AITD has not been studied, with one noticeable exception: Yersinia enterocolitica.
Y. enterocolitica infection
This is an intestinal Gram-negative pathogen from the same family as the notorious Y. pestis (178). It mostly causes a self-limiting enterocolitis, but may persist as a low-grade infection of the mesenteric lymph nodes characterized by the persistence of antibodies against Yersinia outer membrane proteins (YOPs) (179, 180). This may be common, because in one case– control study approximately 25% of both cases (with chronic fatigue syndrome) and controls had IgG anti-YOP antibodies (179).
33 Chapter 2
In the 1970s two studies reported a higher prevalence of Y. enterocolitica (especially serotype O:3) antibodies in Graves’ disease patients (50 and 66% respectively) than in controls (28 and 8% respectively) (181, 182). These findings prompted an investigation into the possibility of shared antigens with the thyroid and it was found that Y. enterocolitica had specific binding sites for TSH in the 1028M range (183). These binding sites were also recognized by TSH-receptor autoantibodies (184). Antibodies against YOPs raised in rabbits displaced TSH from binding to TSH-receptor protein, and these antibodies stained thyroid epithelial cells in immunohistochemistry (185, 186). Cellular immunity is also involved, because Y. enterocolitica can inhibit the migration of lymphocytes from patients with Graves’ disease (181), and in a mouse model Y. enterocolitica acts as a superantigen (187). The cross-reacting protein(s), at first thought to be the TSH-receptor itself, has not been identified yet, but appears to have conformational homology with the TSH-receptor, and one may be hsp70 (188). Others have found two low molecular weight envelope proteins (of 5.5 and 8 kDa) that are cross-reactive with the extracellular part of the TSH-receptor (189). The protein(s) do not seem to be Y. enterocolitica specific, since TSH binding sites were also found on other intestinal pathogens (190). Y. enterocolitica infections are common. In a large Danish study, 8.3% of 48857 patients with bacterial enteritis had a Y. enterocolitica infection (191). In Canada, the annual incidence of Y. enterocolitica infections is 3/100 000 subjects (192); in The Netherlands the yearly incidence is 1.2/100 000 inhabitants (193). In view of the high incidence of AITD, Y. enterocolitica infections may thus play a role in its development. With more specific assays using YOPs, there is indeed an association between antibodies against YOPs and AITD. IgA antibodies are thought to indicate that the primary immune response is mounted in the gut, and not in the thyroid, suggesting the Y. enterocolitica infection is causative (194). In a German study, IgG class antibodies were found in 72% of Graves’ patients and in 66% of patients with Hashimoto’s thyroiditis as compared with 35% in controls; IgA antibodies were found in respectively 33, 37 and 11% (195). In Greece, 25% of Hashimoto patients had IgG antibodies and 2.8% had IgA antibodies, compared with 2 and 0% respectively in controls (196). A higher incidence of Y. enterocolitica antibodies in Graves and Hashimoto patients than in controls was also found in Japan and Turkey (197, 198). However, there are also studies that could not confirm these findings and found a similar rate of seropositivity in AITD patients and controls (199, 200). We recently found that 40% of 803 female relatives of patients with documented AITD had IgG antibodies against Y. enterocolitica YOPs (22% had IgA antibodies), as compared with only 24% of controls (13% had IgA antibodies), but
34 The environment and autoimmune thyroid diseases the presence of these antibodies was unrelated to the presence of thyroid autoimmunity (201). We hypothesized that this high rate of probably persisting, low-grade Y. enterocolitica infections in relatives of AITD patients is related to a particular genetic make-up facilitating Y. enterocolitica infections independently from conferring a risk for AITD.
Concluding remarks
AITD is a polygenetic disease and currently only a few genes have been identified as causing AITD, all with a rather low RR which is seldom higher than 3.0. Nevertheless, it has been calculated that 79% of the susceptibility to develop Graves’ disease can be attributed to genetic factors, leaving 21% for environmental factors (202). Reviewing these non-genetic factors, it appears that multiple environmental factors are involved in the induction of AITD in genetically predisposed individuals (Table 4).
Table 4. Odds ratio (OR) and 95% confidence intervals (95% CI) of several environmental factors associated with the occurrence of AITD.
Environmental factor (phenotype) OR 95% CI Cases/controls Reference Low birth weight 5.5 1.0-30.1 113/190 Philips (12) (<5.5 lb; Tg antibodies) High selenium levels 0.2 0.06-0.7 Logistic regression Derumeaux (300) (hypoechogenicity) High age at menopause 3.0 2.0-6.0 47/47 Philips et al. (38) (>50 years;HT) Use of oral contraceptives (GD) 0.68 0.49-0.93 617/617 Vestergaard et al. (45) Fetal microchimerism (HT) 21.3 2.3-195 17/25 Klintshar et al. (50) (GD) 5.3 0.58-48 27/10 Ando et al. (49)) Stress (GD) 3.37 2.4-4.7 387/524 Winsa et al. (60) Sonino et al. (61) Kung (62) Smoking (GD) 3.3 2.1-5.2 949/5781 Vestergaard (93) (GO) 4.4 2.9-6.7 768/1775 Allergy (relapse rate GD) 4.3 1.8-10.1 44/73 Komiya et al. (77) Y. enterocolitica * (GD) 4.3 3.2-5.9 245/749 Bech et al. (181) Shenkman&Bottone(182) Wenzel et al. (195) Corapcioglu et al. (198) Arscott et al. (199) * Studies reporting positive IgA antibodies, or antibodies against serogroup O:3. GD, Graves’ disease; GO, Graves’ opthalmopathy; HT, Hashimoto’s thyroiditis.
It follows that there must be an interplay at work between different genes and different environmental factors. For instance, the post- partum period is a clear risk factor for Graves’ disease but cannot explain its occurrence in males and only a minority of women will
35 Chapter 2 develop Graves’ disease in the post partum period. In other words, one gene may predispose for AITD in general while a second gene may dictate whether childbirth will precipitate its onset or not, while in another woman with the same first susceptibility gene the trigger may lie in a stress-coping gene. This would explain the rather low OR values of individual – genetic and environmental – risk factors: a specific environmental risk factor may have a very large RR in a person with a certain genetic make- up. This implies that the true importance of both genes and environment can only be discerned when studied in conjunction. Such an approach requires a much larger sample size and probably multi-center cooperation. The good news is, however, that we now have powerful computers to perform the necessary multivariate analyses. It also means that we need a much more rigorous phenotype definition. Environmental risk factors are more likely to be important in older patients with AITD than in younger ones, their influence may also differ between patients who come from a family of AITD patients and isolated cases, or between males and females. This does not mean that a further search for specific risk factors is useless. When reviewing all factors, one of the most promising is fetal microchimerism. To date this has been limited to the search for remnants of male fetuses (the Y chromosome), but female fetuses are likely to have the same impact. This implies further studies into the genetic make-up of partners of AITD patients. A second area holding promise is the importance of viral infections in the induction of AITD. They appear to be of importance in the induction of IDDM, lead to an endogenous surge in IFN-a (a clear risk factor for AITD when administered as a drug) and are more likely to occur in selenium deficiency (which is itself another risk factor for AITD). The ultimate goal of this research is to find a feasible way of preventing the occurrence of AITD; for this we will need progress both in delineating the genetic background and in clarifying the precipitating environmental factors.
36 The environment and autoimmune thyroid diseases
References
1. Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. New England Journal of Medicine 2002 347 911–920. 2. Gough SCL. The immunogenetics of Graves’ disease. Current Opinion in Endocrinology and Diabetes 1999 6 270–276. 3. Tait KF & Gough SC. The genetics of autoimmune endocrine disease. Clinical Endocrinology 2003 59 1–11. 4. Vaidya B, Kendall-Taylor P & Pearce SH. The genetics of autoimmune thyroid disease. Journal of Clinical Endocrinology and Metabolism 2002 87 5385–5397. 5. Gough SC. The genetics of Graves’ disease. Endocrinology and Metabolism Clinics of North America 2000 29 255–266. 6. Barbesino G & Chiovato L. The genetics of Hashimoto’s disease. Endocrinology and Metabolism Clinics of North America 2000 29 357–374. 7. Strieder TG, Prummel MF, Tijssen JG, Endert E & Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clinical Endocrinology 2003 59 396–401. 8. Dayan CM & Daniels GH. Chronic autoimmune thyroiditis. New England Journal of Medicine 1996 335 99–107. 9. Forsen T, Eriksson JG, Tuomilehto J, Osmond C & Barker DJ. Growth in utero and during childhood among women who develop coronary heart disease: longitudinal study. British Medical Journal 1999 319 1403–1407. 10. Roseboom TJ, van der Meulen JH, Ravelli AC, Osmond C, Barker DJ & Bleker OP. Effects of prenatal exposure to the Dutch famine on adult disease in later life: an overview. Molecular and Cellular Endocrinology 2001 185 93–98. 11. Winick M & Noble A. Cellular response in rats during malnutrition at various ages. Journal of Nutrition 1966 89 300–306. 12. Phillips DI, Cooper C, Fall C, Prentice L, Osmond C, Barker DJ et al. Fetal growth and autoimmune thyroid disease. Quarterly Journal of Medicine 1993 86 247–253. 13. Phillips DI, Osmond C, Baird J, Huckle A & Rees-Smith B. Is birthweight associated with thyroid autoimmunity? A study in twins. Thyroid 2002 12 377–380. 14. Brix TH, Kyvik KO & Hegedus L. Low birth weight is not associated with clinically overt thyroid disease: a population based twin case-control study. Clinical Endocrinology 2000 53 171–176. 15. Laurberg P, Pedersen KM, Hreidarsson A, Sigfusson N, Iversen E & Knudsen PR. Iodine intake and the pattern of thyroid disorders: a comparative epidemiological study of thyroid abnormalities in the elderly in Iceland and in Jutland, Denmark. Journal of Clinical Endocrinology and Metabolism 1998 83 765–769. 16. Laurberg P, Nohr SB, Pedersen KM, Hreidarsson AB, Andersen S, Bulow P et al. Thyroid disorders in mild iodine deficiency. Thyroid 2000 10 951–963. 17. Laurberg P, Pedersen KM, Vestergaard H & Sigurdsson G. High incidence of multinodular toxic goitre in the elderly population in a low iodine intake area vs. high incidence of Graves’ disease in the young in a high iodine intake area: comparative surveys of thyrotoxicosis epidemiology in East-Jutland Denmark and Iceland. Journal of Internal Medicine 1991 229 415–420. 18. Pedersen IB, Knudsen N, Jorgensen T, Perrild H, Ovesen L & Laurberg P. Thyroid peroxidase and thyroglobulin autoantibodies in a large survey of populations with mild and moderate iodine deficiency. Clinical Endocrinology 2003 58 36–42.
37 Chapter 2
19. Wiersinga WM & Braverman LE. Iodine-induced thyroid disease. In Contemporary Endocrinology: Diseases of the Thyroid, edn 2,pp 347–362. Ed. LE Braverman. Totowa, NJ: Humana Press Inc., 2003. 20. Roti E & Uberti ED. Iodine excess and hyperthyroidism. Thyroid 2001 11 493–500. 21. Markou K, Georgopoulos N, Kyriazopoulou V & Vagenakis AG. Iodine-induced hypothyroidism. Thyroid 2001 11 501–510. 22. Weetman AP. Autoimmune thyroid disease: propagation and progression. European Journal of Endocrinology 2003 148 1–9. 23. Ruwhof C & Drexhage HA. Iodine and thyroid autoimmune disease in animal models. Thyroid 2001 11 427–436. 24. Sundick RS, Bagchi N & Brown TR. The role of iodine in thyroid autoimmunity: from chickens to humans: a review. Autoimmunity 1992 13 61–68. 25. Lam-Tse WK, Lernmark A & Drexhage HA. Animal models of endocrine/organ-specific autoimmune diseases: do they really help us to understand human autoimmunity? Springer Seminars in Immunopathology 2002 24 297–321. 26. Rayman MP. The importance of selenium to human health. Lancet 2000 356 233–241. 27. Beck MA, Shi Q, Morris VC & Levander OA. Rapid genomic evolution of a non- virulent coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates. Nature Medicine 1995 1 433–436. 28. Zimmermann MB & Kohrle J. The impact of iron and selenium deficiencies on iodine and thyroid metabolism: biochemistry and relevance to public health. Thyroid 2002 12 867–878. 29. Barrington JW, Lindsay P, James D, Smith S & Roberts A. Selenium deficiency and miscarriage: a possible link? British Journal of Obstetrics and Gynaecology 1996 103 130–132. 30. Derumeaux H, Valeix P, Castetbon K, Bensimon M, Boutron-Ruault MC, Arnaud J et al. Association of selenium with thyroid volume and echostructure in 35- to 60-year-old French adults. European Journal of Endocrinology 2003 148 309–315.1 31. Gartner R, Gasnier BC, Dietrich JW, Krebs B & Angstwurm MW. Selenium supplementation in patients with autoimmune thyroiditis decreases thyroid peroxidase antibodies concentrations. Journal of Clinical Endocrinology and Metabolism 2002 87 1687–1691. 32. Duntas LH, Mantzou E & Koutras DA. Effects of a six month treatment with selenomethionine in patients with autoimmune thyroiditis. European Journal of Endocrinology 2003 148 389–393. 33. Vanderpump MP, Tunbridge WM, French JM, Appleton D, Bates D, Clark F et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clinical Endocrinology 1995 43 55–68. 34. Stagnaro-Green A, Roman SH, Cobin RH, el Harazy E, Wallenstein S & Davies TF. A prospective study of lymphocyte-initiated immunosuppression in normal pregnancy: evidence of a T-cell etiology for postpartum thyroid dysfunction. Journal of Clinical Endocrinology and Metabolism 1992 74 645–653. 35. Draca S. Is pregnancy a model how we should control some autoimmune diseases? Autoimmunity 2002 35 307–312. 36. Beagley KW & Gockel CM. Regulation of innate and adaptive immunity by the female sex hormones oestradiol and progesterone. FEMSImmunology and Medical Microbiology 200338 13–22. 37. Vanderpump MP, French JM, Appleton D, Tunbridge WM & Kendall-Taylor P. The prevalence of hyperprolactinaemia and association with markers of autoimmune thyroid
38 The environment and autoimmune thyroid diseases
disease in survivors of the Whickham Survey cohort. Clinical Endocrinology 1998 48 39–44. 38. Phillips DI, Lazarus JH & Butland BK. The influence of pregnancy and reproductive span on the occurrence of autoimmune thyroiditis. Clinical Endocrinology 1990 32 301– 306. 39. Massoudi MS, Meilahn EN, Orchard TJ, Foley TP Jr, Kuller LH, Costantino JP et al. Prevalence of thyroid antibodies among healthy middle-aged women. Findings from the thyroid study in healthy women. Annals of Epidemiology 1995 5 229–233. 40. Ogard CG, Ogard C & Almdal TP. Thyroid-associated orbitopathy developed during hormone replacement therapy. Acta Ophthalmologica Scandinavica 2001 79 426–427. 41. Petitti DB. Clinical practice. Combination estrogen–progestin oral contraceptives. New England Journal of Medicine 2003 349 1443–1450. 42. Frank P & Kay CR. Incidence of thyroid disease associated with oral contraceptives. British Medical Journal 1978 2 1531. 43. Knudsen N, Bulow I, Laurberg P, Perrild H, Ovesen L & Jorgensen T. Low goitre prevalence among users of oral contraceptives in a population sample of 3712 women. Clinical Endocrinology 2002 57 71– 76. 44. Barrere X, Valeix P, Preziosi P, Bensimon M, Pelletier B, Galan P et al. Determinants of thyroid volume in healthy French adults participating in the SU.VI.MAX cohort. Clinical Endocrinology 2000 52 273–278. 45. Vestergaard P, Rejnmark L, Weeke J, Hoeck HC, Nielsen HK, Rungby J et al. Smoking as a risk factor for Graves’ disease, toxic nodular goiter, and autoimmune hypothyroidism. Thyroid 2002 12 69–75. 46. Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S & DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. PNAS 1996 93 705– 708. 47. Khosrotehrani K & Bianchi DW. Fetal cell microchimerism: helpful or harmful to the parous woman? Current Opinion in Obstetrics and Gynecology 2003 15 195–199. 48. Imaizumi M, Pritsker A, Unger P & Davies TF. Intrathyroidal fetal microchimerism in pregnancy and postpartum. Endocrinology 2002 143 247–253. 49. Ando T, Imaizumi M, Graves PN, Unger P & Davies TF. Intrathyroidal fetal microchimerism in Graves’ disease. Journal of Clinical Endocrinology and Metabolism 2002 87 3315–3320. 50. Klintschar M, Schwaiger P, Mannweiler S, Regauer S & Kleiber M. Evidence of fetal microchimerism in Hashimoto’s thyroiditis. Journal of Clinical Endocrinology and Metabolism 2001 86 2494–2498. 51. Badenhoop K. Intrathyroidal microchimerism in Graves’ disease or Hashimoto’s thyroiditis: regulation of tolerance or alloimmunity by fetal–maternal immune interaction. European Journal of Endocrinology 2003 (in press). 52. Besedovsky HO & del Rey A. Immune-neuro-endocrine interactions: facts and hypotheses. Endocrine Reviews 1996 17 64–102. 53. Sternberg EM. Neuroendocrine regulation of autoimmune/inflammatory disease. Journal of Endocrinology 2001 169 429–435. 54. Elenkov IJ & Chrousos GP. Stress hormones, Th1/Th2 patterns, pro/anti-inflammatory cytokines and susceptibility to disease. Trends in Endocrinology and Metabolism 1999 10 359–368. 55. Elenkov IJ & Chrousos GP. Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity. Annals of the New York Academy of Sciences 2002 966 290–303. 56. Rosch PJ. Stressful life events and Graves’ disease. Lancet 1993 342 566–567.
39 Chapter 2
57. Gorman CA. A critical review of the role of stress in hyperthyroidism. In The Thyroid Gland, Environment and Autoimmunity, pp 191–200. Eds HA Drexhage, JJM de Vijlder &WM Wiersinga. Amsterdam: Elsevier Science Publishers, 1990. 58. Paunkovic N, Paunkovic J, Pavlovic O & Paunovic Z. The significant increase in incidence of Graves’ disease in eastern Serbia during the civil war in the former Yugoslavia (1992 to 1995).Thyroid 1998 8 37– 41. 59. Hadden DR & McDevitt DG. Environmental stress and thyrotoxicosis.Absence of association. Lancet 974 2 577–578. 60. Winsa B, Adami HO, Bergstrom R, Gamstedt A, Dahlberg PA, Adamson U et al. Stressful life events and Graves’ disease. Lancet 1991 338 1475–1479. 61. Sonino N, Girelli ME, Boscaro M, Fallo F, Busnardo B & Fava GA. Life events in the pathogenesis of Graves’ disease. A controlled study. Acta Endocrinologica (Copenh) 1993 128 293–296. 62. Kung AW. Life events, daily stresses and coping in patients with Graves’ disease. Clinical Endocrinology 1995 42 303–308. 63. Radosavljevic VR, Jankovic SM & Marinkovic JM. Stressful life events in the pathogenesis of Graves’ disease. European Journal of Endocrinology 1996 134 699–701. 64. Dayan CM. Stressful life events and Graves’ disease revisited. Clinical Endocrinology 2001 55 13–14. 65. Chiovato L & Pinchera A. Stressful life events and Graves’ disease. European Journal of Endocrinology 1996 134 680–682. 66. Benvenga S. Benzodiazepine and remission of Graves’ disease. Thyroid 1996 6 659–660. 67. Fukao A, Takamatsu J, Murakami Y, Sakane S, Miyauchi A, Kuma K et al. The relationship of psychological factors to the prognosis of hyperthyroidism in antithyroid drug-treated patients with Graves’ disease. Clinical Endocrinology 2003 58 550–555. 68. Matos-Santos A, Nobre EL, Costa JG, Nogueira PJ, Macedo A, Galvao-Teles A et al. Relationship between the number and impact of stressful life events and the onset of Graves’ disease and toxic nodular goitre. Clinical Endocrinology 2001 55 15–19. 69. Wiersinga WM. Environmental factors in autoimmune thyroid disease. Experimental and Clinical Endocrinology and Diabetes 1999 107 (Suppl 3) S67–S70. 70. Westphal SA. Seasonal variation in the diagnosis of Graves’ disease. Clinical Endocrinology 1994 41 27–30. 71. Phillips DI, Barker DJ & Morris JA. Seasonality of thyrotoxicosis. Journal of Epidemiology and Community Health 1985 39 72–74. 72. Phillips DI, Nelson M, Barker DJ, Morris JA & Wood TJ. Iodine in milk and the incidence of thyrotoxicosis in England. Clinical Endocrinology 1988 28 61–66. 73. Pedotti R, De Voss JJ, Steinman L & Galli SJ. Involvement of both ‘allergic’ and ‘autoimmune’ mechanisms in EAE, MS and other autoimmune diseases. Trends in Immunology 2003 24 479–484. 74. Stene LC & Nafstad P. Relation between occurrence of type 1 diabetes and asthma. Lancet 2001 357 607–608. 75. Moens HJ, Wiersinga WM & Drexhage HA. Association between autoimmune thyroid disease, atopy, and urticaria? Lancet 1984 2 582–583. 76. Sato A, Takemura Y, Yamada T, Ohtsuka H, Sakai H, Miyahara Y et al. A possible role of immunoglobulin E in patients with hyperthyroid Graves’ disease. Journal of Clinical Endocrinology and Metabolism 1999 84 3602–3605. 77. Komiya I, Yamada T, Sato A, Kouki T, Nishimori T & Takasu N. Remission and recurrence of hyperthyroid Graves’ disease during and after methimazole treatment when assessed by IgE and interleukin 13. Journal of Clinical Endocrinology and Metabolism 2001 86 3540–3544.
40 The environment and autoimmune thyroid diseases
78. Hidaka Y, Amino N, Iwatani Y, Itoh E, Matsunaga M & Tamaki H. Recurrence of thyrotoxicosis after attack of allergic rhinitis in patients with Graves’ disease. Journal of Clinical Endocrinology and Metabolism 1993 77 1667–1670. 79. Hidaka Y, Masai T, Sumizaki H, Takeoka K, Tada H & Amino N. Onset of Graves’ thyrotoxicosis after an attack of allergic rhinitis. Thyroid 1996 6 349–351. 80. Rottem M. Chronic urticaria and autoimmune thyroid disease: is there a link? Autoimmunity Review 2003 2 69–72. 81. Turktas I, Gokcora N, Demirsoy S, Cakir N & Onal E. The association of chronic urticaria and angioedema with autoimmune thyroiditis. International Journal of Dermatology 1997 36 187–190. 82. Leznoff A & Sussman GL. Syndrome of idiopathic chronic urticaria and angioedema with thyroid autoimmunity: a study of 90 patients. Journal of Allergy and Clinical Immunology 1989 84 66–71. 83. George J, Levy Y & Shoenfeld Y. Smoking and immunity: an additional player in the mosaic of autoimmunity. Scandinavian Journal of Immunology 1997 45 1–6. 84. McAllister-Sistilli CG, Caggiula AR, Knopf S, Rose CA, Miller AL & Donny EC. The effects of nicotine on the immune system. Psychoneuroendocrinology 1998 23 175–187. 85. Ronchetti R, Macri F, Ciofetta G, Indinnimeo L, Cutrera R, Bonci E et al. Increased serum IgE and increased prevalence of eosinophilia in 9-year-old children of smoking parents. Journal of Allergy and Clinical Immunology 1990 86 400–407. 86. Weitzman M, Gortmaker S, Walker DK & Sobol A. Maternal smoking and childhood asthma. Pediatrics 1990 85 505–511. 87. Prummel MF, Van Pareren Y, Bakker O & Wiersinga WM. Antiheat shock protein (hsp)72 antibodies are present in patients with Graves’ disease (GD) and in smoking control subjects. Clinical and Experimental Immunology 1997 110 292–295. 88. Prummel MF, Wiersinga WM, Van der Gaag R, Mourits MP & Koornneef L. Soluble IL- 2 receptor levels in patients with Graves’ ophthalmopathy. Clinical and Experimental Immunology 1992 88 405–409. 89. Hofbauer LC, Muhlberg T, Konig A, Heufelder G, Schworm HD & Heufelder AE. Soluble interleukin- 1 receptor antagonist serum levels in smokers and nonsmokers with Graves’ ophthalmopathy undergoing orbital radiotherapy. Journal of Clinical Endocrinology and Metabolism 1997 82 2244–2247. 90. Wakelkamp IM, Gerding MN, van der Meer JW, Prummel MF & Wiersinga WM. Smoking and disease severity are independent determinants of serum adhesion molecule levels in Graves’ ophthalmopathy. Clinical and Experimental Immunology 2002 127 316–320. 91. Byron KA, Varigos GA & Wootton AM. IL-4 production is increased in cigarette smokers. Clinical and Experimental Immunology 1994 95 333–336. 92. Heliovaara M, Aho K, Aromaa A, Knekt P & Reunanen A. Smoking and risk of rheumatoid arthritis. Journal of Rheumatology 1993 20 1830–1835. 93. Vestergaard P. Smoking and thyroid disorders–a meta-analysis. European Journal of Endocrinology 2002 146 153–161. 94. Hagg E & Asplund K. Is endocrine ophthalmopathy related to smoking? British Medical Journal 1987 295 634–635. 95. Prummel MF & Wiersinga WM. Smoking and risk of Graves’ disease. Journal of the American Medical Association 1993 269 479–482. 96. Bartalena L, Martino E, Marcocci C, Bogazzi F, Panicucci M, Velluzzi F et al. More on smoking habits and Graves’ ophthalmopathy. Journal of Endocrinological Investigation 1989 12 733–737.
41 Chapter 2
97. Chen YL, Chang TC & Chen CJ. Influence of smoking on Graves’ disease with or without ophthalmopathy and nontoxic nodular goiter in Taiwan. Journal of the Formosan Medical Association 1994 93 40–44. 98. Shine B, Fells P, Edwards OM & Weetman AP. Association between Graves’ ophthalmopathy and smoking. Lancet 1990 335 1261–1263. 99. Winsa B, Mandahl A & Karlsson FA. Graves’ disease, endocrine ophthalmopathy and smoking. Acta Endocrinologica 1993 128 156–160. 100. Tellez M, Cooper J & Edmonds C. Graves’ ophthalmopathy in relation to cigarette smoking and ethnic origin. Clinical Endocrinology 1992 36 291–294. 101. Pfeilschifter J & Ziegler R. Smoking and endocrine ophthalmopathy:impact of smoking severity and current vs lifetime cigarette consumption. Clinical Endocrinology 1996 45 477–481. 102. Nystrom E, Bengtsson C, Lapidus L, Petersen K & Lindstedt G. Smoking–a risk factor for hypothyroidism. Journal of Endocrinological Investigation 1993 16 129–131. 103. Orgiazzi J & Madec AM. Reduction of the risk of relapse after withdrawal of medical therapy for Graves’ disease. Thyroid 2002 12 849–853. 104. Glinoer D, de Nayer P & Bex M. Effects of l-thyroxine administration,TSH-receptor antibodies and smoking on the risk of recurrence in Graves’ hyperthyroidism treated with antithyroid drugs: a double- blind prospective randomized study. European Journal of Endocrinology 2001 144 475–483. 105. Bartalena L, Marcocci C, Tanda ML, Manetti L, Dell’Unto E, Bartolomei MP et al. Cigarette smoking and treatment outcomes in Graves ophthalmopathy. Annals of Internal Medicine 1998 129 632–635. 106. Eckstein A, Quadbeck B, Mueller G, Rettenmeier AW, Hoermann R, Mann K et al. Impact of smoking on the response to treatment of thyroid associated ophthalmopathy. British Journal of ophthalmology 2003 87 773–776. 107. Bartalena L, Pinchera A & Marcocci C. Management of Graves’ ophthalmopathy: reality and perspectives. Endocrine Reviews 2000 21 168–199. 108. Weetman AP. Determinants of autoimmune thyroid disease. Nature Immunology 2001 2 769–770. 109. Metcalfe RA & Weetman AP. Stimulation of extraocular muscle fibroblasts by cytokines and hypoxia: possible role in thyroid-associated ophthalmopathy. Clinical Endocrinology 1994 40 67–72. 110. Mack WP, Stasior GO, Cao HJ, Stasior OG & Smith TJ. The effect of cigarette smoke constituents on the expression of HLA-DR in orbital fibroblasts derived from patients with Graves ophthalmopathy. Ophthalmic and Plastic Recon structive Surgery 1999 15 260–271. 111. Wiersinga WM. Amiodarone and the thyroid. In Handbook of Experimental Pharmacology, vol 128, pp 225–287. Eds AP Weetman & A Grossman. Berlin: Springer-Verlag, 1997. 112. Trip MD, Wiersinga W & Plomp TA. Incidence, predictability, and pathogenesis of amiodarone- induced thyrotoxicosis and hypothyroidism. American Journal of Medicine 1991 91 507–511. 113. Daniels GH. Amiodarone-induced thyrotoxicosis. Journal of Clinical Endocrinology and Metabolism 2001 86 3–8. 114. Monteiro E, Galvao-Teles A, Santos ML, Mourao L, Correia MJ, Lopo TJ et al. Antithyroid antibodies as an early marker for thyroid disease induced by amiodarone. British Medical Journal 1986 292 227–228.
42 The environment and autoimmune thyroid diseases
115. Trip MD, Duren DR & Wiersinga WM. Two cases of amiodaroneinduced thyrotoxicosis successfully treated with a short course of antithyroid drugs while amiodarone was continued. British Heart Journal 1994 72 266–268. 116. Weetman AP, Bhandal SK, Burrin JM, Robinson K & McKennaW. Amiodarone and thyroid autoimmunity in the United Kingdom. British Medical Journal 1988 297 33. 117. Gilquin J, Viard JP, Jubault V, Sert C & Kazatchkine MD. Delayed occurrence of Graves’ disease after immune restoration with HAART. Highly active antiretroviral therapy. Lancet 1998 352 1907–1908. 118. Weetman AP. Graves’ disease. New England Journal of Medicine 2000 343 1236–1248. 119. Coles AJ, Wing M, Smith S, Coraddu F, Greer S, Taylor C et al. Pulsed monoclonal antibody treatment and autoimmune thyroid disease in multiple sclerosis. Lancet 1999 354 1691–1695. 120. Lauer GM & Walker BD. Hepatitis C virus infection. New England Journal of Medicine 2001 345 41–52. 121. Bhakri H, Sriskandan K, Davis T, Pettingale K & Tee D. Recombinant gamma interferon and autoimmune thyroid disease. Lancet 1985 2 457. 122. Prummel MF & Laurberg P. Interferon-alpha and autoimmune thyroid disease. Thyroid 2003 13 547– 551. 123. Okanoue T, Sakamoto S, Itoh Y, Minami M, Yasui K, Sakamoto M et al. Side effects of high-dose interferon therapy for chronic hepatitis C. Journal of Hepatology 1996 25 283–291. 124. Fattovich G, Giustina G, Favarato S & Ruol A. A survey of adverse events in 11,241 patients with chronic viral hepatitis treated with alfa interferon. Journal of Hepatology 1996 24 38–47. 125. Koh LK, Greenspan FS & Yeo PP. Interferon-alpha induced thyroid dysfunction: three clinical presentations and a review of the literature. Thyroid 1997 7 891–896. 126. Farrar JD & Murphy KM. Type I interferons and T helper development. Immunology Today 2000 21 484–489. 127. Parkin J & Cohen B. An overview of the immune system. Lancet 2001 357 1777–1789. 128. Burman P, Totterman TH, Oberg K & Karlsson FA. Thyroid autoimmunity in patients on long term therapy with leukocyte-derived interferon. Journal of Clinical Endocrinology and Metabolism 1986 63 1086–1090. 129. Biron CA. Interferons alpha and beta as immune regulators–a new look. Immunity 2001 14 661–664. 130. Vial T & Descotes J. Immune-mediated side-effects of cytokines in humans.Toxicology 1995 105 31–57. 131. Signore A, Picarelli A, Annovazzi A, Britton KE, Grossman AB, Bonanno E et al. 123I- interleukin-2: biochemical characterization and in vivo use for imaging autoimmune diseases. Nuclear Medicine Communications 2003 24 305–316. 132. Witzke O, Winterhagen T, Saller B, Roggenbuck U, Lehr I, Philipp T et al. Transient stimulatory effects on pituitary-thyroid axis in patients treated with interleukin-2. Thyroid 2001 11 665–670. 133. Atkins MB, Mier JW, Parkinson DR, Gould JA, Berkman EM & Kaplan MM. Hypothyroidism after treatment with interleukin- 2 and lymphokine-activated killer cells. New England Journal of Medicine 1988 318 1557–1563. 134. Meloni G, Trisolini SM, Capria S, Torelli GF, Baldacci E, Torromeo C et al. How long can we give interleukin-2? Clinical and immunological evaluation of AML patients after 10 or more years of IL2 administration. Leukemia 2002 16 2016–2018.
43 Chapter 2
135. Hansen PB, Johnsen HE & Hippe E. Autoimmune hypothyroidism and granulocyte- macrophage colony-stimulating factor. European Journal of Haematology 1993 50 183– 184. 136. Hoekman K, von Blomberg-van der Flier BM, Wagstaff J, Drexhage HA & Pinedo HM. Reversible thyroid dysfunction during treatment with GM-CSF. Lancet 1991 338 541– 542. 137. Van Hoef ME & Howell A. Risk of thyroid dysfunction during treatment with G-CSF. Lancet 1992 340 1169–1170. 138. Goodnow CC. Pathways for self-tolerance and the treatment of autoimmune diseases. Lancet 2001 357 2115–2121. 139. Hancock SL, Cox RS & McDougall IR. Thyroid diseases after treatment of Hodgkin’s disease. New England Journal of Medicine 1991 325 599–605. 140. Sklar C, Whitton J, Mertens A, Stovall M, Green D, Marina N et al. Abnormalities of the thyroid in survivors of Hodgkin’s disease: data from the Childhood Cancer Survivor Study. Journal of Clinical Endocrinology and Metabolism 2000 85 3227–3232. 141. Jacobson DR & Fleming BJ. Graves’ disease with ophthalmopathy following radiotherapy for Hodgkin’s disease. American Journal of the Medical Sciences 1984 288 217–220. 142. Wasnich RD, Grumet FC, Payne RO & Kriss JP. Graves’ ophthalmopathy following external neck irradiation for nonthyroidal neoplastic disease. Journal of Clinical Endocrinology and Metabolism 1973 37 703–713. 143. Jackson R, Rosenberg C, Kleinmann R, Vagenakis AG & Braverman LE. Ophthalmopathy after neck irradiation therapy for Hodgkin’s disease. Cancer Treament Reviews 1979 63 1393–1395. 144. Loeffler JS, Tarbell NJ, Garber JR & Mauch P. The development of Graves’ disease following radiation therapy in Hodgkin’s disease. International Journal of Radiation Oncology, Biology, Physics 1988 14 175–178. 145. Soule J & Mayfield R. Graves’ disease after 131I therapy for toxic nodule. Thyroid 2001 11 91–92. 146. Niepomniszcze H, Pitoia F, Goodall C, Manavela M & Bruno OD. Development of Graves’ hyperthyroidism after radioiodine treatment for a toxic nodule: is the hyperthyroidism always triggered by 131I therapy? Thyroid 2001 11 991. 147. Nygaard B, Knudsen JH, Hegedus L, Scient AV & Hansen JE. Thyrotropin receptor antibodies and Graves’ disease, a sideeffect of 131I treatment in patients with nontoxic goiter. Journal of Clinical Endocrinology and Metabolism 1997 82 2926–2930. 148. Huysmans AK, Hermus RM, Edelbroek MA, Tjabbes T, Oostdijk, Ross HA et al. Autoimmune hyperthyroidism occurring late after radioiodine treatment for volume reduction of large multinodular goiters. Thyroid 1997 7 535–539. 149. Nagataki S, Shibata Y, Inoue S, Yokoyama N, Izumi M & Shimaoka K. Thyroid diseases among atomic bomb survivors in Nagasaki. Journal of the American Medical Association 1994 272 364–370. 150. Pacini F, Vorontsova T, Molinaro E, Kuchinskaya E, Agate L, Shavrova E et al. Prevalence of thyroid autoantibodies in children and adolescents from Belarus exposed to the Chernobyl radioactive fallout. Lancet 1998 352 763–766. 151. Vermiglio F, Castagna MG, Volnova E, Lo PV, Moleti M, Violi MA et al. Post- Chernobyl increased prevalence of humoral thyroid autoimmunity in children and adolescents from a moderately iodine- deficient area in Russia. Thyroid 1999 9 781–786. 152. Kerber RA, Till JiE, Simon SL, Lyon JL, Thomas DC, Preston- Martin S et al. A cohort study of thyroid disease in relation to fallout from nuclear weapons testing. Journal of the American Medical Association 1993 270 2076–2082.
44 The environment and autoimmune thyroid diseases
153. Yoshimoto Y, Ezaki H, Etoh R, Hiraoka T & Akiba S. Prevalence rate of thyroid diseases among autopsy cases of the atomic bomb survivors in Hiroshima, 1951–1985. Radiation Research 1995 141 278–286. 154. Morimoto I, Yoshimoto Y, Sato K, Hamilton HB, Kawamoto S, Izumi M et al. Serum TSH, thyroglobulin, and thyroidal disorders in atomic bomb survivors exposed in youth: 30-year follow-up study. Journal of Nuclear Medicine 1987 28 1115–1122. 155. Fujiwara S, Carter RL, Akiyama M, Akahoshi M, Kodama K, Shimaoka K et al. Autoantibodies and immunoglobulins among atomic bomb survivors. Radiation Research 1994 137 89–95. 156. Eheman CR, Garbe P & Tuttle RM. Autoimmune thyroid disease associated with environmental thyroidal irradiation. Thyroid 2003 13 453–464. 157. Lonnrot M, Korpela K, Knip M, Ilonen J, Simell O, Korhonen S et al. Enterovirus infection as a risk factor for beta-cell autoimmunity in a prospectively observed birth cohort: the Finnish Diabetes Prediction and Prevention Study. Diabetes 2000 49 1314– 1318. 158. Lonnrot M, Salminen K, Knip M, Savola K, Kulmala P, Leinikki P et al. Enterovirus RNA in serum is a risk factor for beta-cell autoimmunity and clinical type 1 diabetes: a prospective study. Childhood Diabetes in Finland (DiMe) Study Group. Journal of Medical Virology 2000 61 214–220. 159. Sadeharju K, Hamalainen AM, Knip M, Lonnrot M, Koskela P, Virtanen SM et al. Enterovirus infections as a risk factor for type I diabetes: virus analyses in a dietary intervention trial. Clinical and Experimental Immunology 2003 132 271–277. 160. Chehadeh W, Weill J, Vantyghem MC, Alm G, Lefebvre J, Wattre P et al. Increased level of interferon-alpha in blood of patients with insulin-dependent diabetes mellitus: relationship with coxsackievirus B infection. Journal of Infectious Diseases 2000 181 1929–1939. 161. Ellerman KE & Like AA. Susceptibility to diabetes is widely distributed in normal class IIu haplotype rats. Diabetologia 2000 43 890–898. 162. Moriyama H & Eisenbarth GS. Genetics and environmental factors in endocrine/organ- specific autoimmunity: have there been any major advances? Springer Seminars in Immunopathology 2002 24 231–242. 163. Tomer Y & Davies TF. Infection, thyroid disease, and autoimmunity. Endocrine Reviews 1993 14 107–120. 164. Wick G, Trieb K, Aguzzi A, Recheis H, Anderl H & Grubeck-Loebenstein B. Possible role of human foamy virus in Graves’ disease. Intervirology 1993 35 101–107. 165. 165 Lee H, Kim S, Kang M, Kim W & Cho B. Prevalence of human foamy virus-related sequences in the Korean population. Journal of the Biomedical Sciences 1998 5 267– 273. 166. Pearce EN, Farwell AP & Braverman LE. Thyroiditis. New England Journal of Medicine 2003 348 2646–2655. 167. Ebringer A & Wilson C. HLA molecules, bacteria and autoimmunity. Journal of Medical Microbiology 2000 49 305–311. 168. Albert LJ & Inman RD. Molecular mimicry and autoimmunity. New England Journal of Medicine 1999 341 2068–2074. 169. Lang HL, Jacobsen H, Ikemizu S, Andersson C, Harlos K, Madsen L et al. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nature Immunology 2002 3 940–943. 170. Wekerle H & Hohlfeld R. Molecular mimicry in multiple sclerosis. New England Journal of Medicine 2003 349 185–186.
45 Chapter 2
171. Pockley AG. H eat shock proteins as regulators of the immune response. Lancet 2003 362 469–476. 172. Purcell AW, Todd A, Kinoshita G, Lynch TA, Keech CL, Gething MJ et al. Association of stress proteins with autoantigens: a possible mechanism for triggering autoimmunity? Clinical and Experimental Immunology 2003 132 193–200. 173. Lo WF,Woods AS, DeCloux A, Cotter RJ, Metcalf ES & Soloski MJ. Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens. Nature Medicine 2000 6 215– 218. 174. Paggi A, Di Prima MA, Paparo BS, Pellegrino C, Faralli AR, Sinopoli MT et al. Anti 70 kDa heat shock protein antibodies in sera of patients affected by autoimmune and non- autoimmune thyroid diseases. EndocrineResearch 1995 21 555–567. 175. Davidson A & Diamond B. Autoimmune diseases. New England Journal of Medicine 2001 345 340– 350. 176. Wang J, Whetsell M & Klein JR. Local hormone networks and intestinal T cell homeostasis. Science 1997 275 1937–1939. 177. Shanahan F. A gut reaction: lymphoepithelial communication in the intestine. Science 1997 275 1897–1898. 178. Bottone EJ. Yersinia enterocolitica: overview and epidemiologic correlates. Microbes and Infection 1999 1 323–333. 179. Swanink CM, Stolk-Engelaar VM, van der Meer JW, Vercoulen JH, Bleijenberg G, Fennis JF et al. Yersinia enterocolitica and the chronic fatigue syndrome. Journal of Infection 1998 36 269–272. 180. de Koning J, Heesemann J, Hoogkamp-Korstanje JA, Festen JJ, Houtman PM & van Oijen PL. Yersinia in intestinal biopsy specimens from patients with seronegative spondyloarthropathy: correlation with specific serum IgA antibodies. Journal of Infectious Diseases 1989 159 109–112. 181. Bech K, Clemmensen O, Larsen JH, Thyme S & Bendixen G. Cellmediated immunity of Yersinia enterocolitica serotype 3 in patients with thyroid diseases. Allergy 1978 33 82– 88. 182. Shenkman L & Bottone EJ. Antibodies to Yersinia enterocolitica in thyroid disease. Annals of Internal Medicine 1976 85 735–739. 183. Weiss M, Ingbar SH, Winblad S & Kasper DL. Demonstration of a saturable binding site for thyrotropin in Yersinia enterocolitica. Science 1983 219 1331–1333. 184. Heyma P, Harrison LC & Robins-Browne R. Thyrotrophinv (TSH) binding sites on Yersinia enterocolitica recognized by immunoglobulins from humans with Graves’ disease. Clinical and Experimental Immunology 1986 64 249–254. 185. Wenzel BE, Peters A & Zubaschev I. Bacterial virulence antigens and the pathogenesis of autoimmune thyroid diseases (AITD). Experimental Clinical Endocrinology and Diabetes 1996 104 (Suppl 4) 75–78. 186. Wenzel E, Franke TF, Heufelder AE & Heesemann J. Autoimmune thyroid diseases and enteropathogenic Yersinia enterocolitica. Autoimmunity 1990 7 295–303. 187. Stuart PM & Woodward JG. Yersinia enterocolitica produces superantigenic activity. Journal of Immunology 1992 148 225–233. 188. Wenzel BE, Heesemann J, Heufelder A, Franke TF, Grammerstorf S, Stemerowicz R et al. Enteropathogenic Yersinia enterocolitica and organ-specific autoimmune diseases in man. Contributions to Microbiology and Immunology 1991 12 80–88. 189. Zhang H, Kaur I, Niesel DW, Seetharamaiah GS, Peterson JW, Justement LB et al. Yersinia enterocolitica envelope proteins that are crossreactive with the thyrotropin receptor (TSHR) also have B- cell mitogenic activity. Journal of Autoimmunity 1996 9 509–516.
46 The environment and autoimmune thyroid diseases
190. Byfield PG, Davies SC, Copping S, Barclay FE & Borriello SP. Thyrotrophin (TSH)- binding proteins in bacteria and their cross-reaction with autoantibodies against the human TSH receptor. Journal of Endocrinology 1989 121 571–577. 191. Helms M, Vastrup P, Gerner-Smidt P & Molbak K. Short and long term mortality associated with foodborne bacterial gastrointestinal infections: registry based study. British Medical Journal 2003 326 357. 192. Lee MB & Middleton D. Enteric illness in Ontario, Canada, from 1997 to 2001. Journal of Food Protection 2003 66 953–961. 193. van Pelt W, de Wit MA,Wannet WJ, Ligtvoet EJ, Widdowson MA & van Duynhoven YT. Laboratory surveillance of bacterial gastroenteric pathogens in The Netherlands, 1991–2001. Epidemiology and Infection 2003 130 431–441. 194. Toivanen P & Toivanen A. Does Yersinia induce autoimmunity? International Archives of Allergy and Immunology 104 107–111. 195. Wenzel BE, Heesemann J, Wenzel KW & Scriba PC. Antibodies to plasmid-encoded proteins of enteropathogenic Yersinia in patients with autoimmune thyroid disease. Lancet 1988 1 56. 196. Chatzipanagiotou S, Legakis JN, Boufidou F, Petroyianni V & Nicolaou C. Prevalence of Yersinia plasmid-encoded outer protein (Yop) class-specific antibodies in patients with Hashimoto’s thyroiditis. Clinical Microbiology and Infection 2001 7 138–143. 197. Asari S, Amino N, Horikawa M & Miyai K. Incidences of antibodies to Yersinia enterocolitica: high incidence of serotype O5 in autoimmune thyroid diseases in Japan. Endocrinology Journal 1989 36 381– 386. 198. Corapcioglu D, Tonyukuk V, Kiyan M, Yilmaz AE, Emral R, Kamel N et al. Relationship between thyroid autoimmunity and Yersinia enterocolitica antibodies. Thyroid 2002 12 613–617. 199. Arscott P, Rosen ED, Koenig RJ, Kaplan MM, Ellis T, Thompson Net al. Immunoreactivity to Yersinia enterocolitica antigens in patients with autoimmune thyroid disease. Journal of Clinical Endocrinology and Metabolism 1992 75 295–300. 200. Resetkova E, Notenboom R, Arreaza G, Mukuta T, Yoshikawa N & Volpe R. Seroreactivity to bacterial antigens is not a unique phenomenon in patients with autoimmune thyroid diseases in Canada. Thyroid 1994 4 269–274. 201. Strieder TG, Wenzel BE, Prummel MF, Tijssen JG & Wiersinga WM. Increased prevalence of antibodies to enteropathogenic Yersinia enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease. Clinical and Experimental Immunology 2003 132 278–282. 202. Brix TH, Kyvik KO, Christensen K & Hegedus L. Evidence for a major role of heredity in Graves’ disease: a population-based study of two Danish twin cohorts. Journal of Clinical Endocrinology and Metabolism 2001 86 930–934 203. Strieder TG, Prummel MF, Tijssen JG, Brosschot JF, Wiersinga WM. Stress is not associated with thyroid peroxidase autoantibodies in euthyroid women. Brain Behav Immun. 2005 193 203-6.
47
Risk factors for and prevalence of thyroid disorders in a cross-sectional study among 3| healthy female relatives of patients with AutoImmune Thyroid Diseases
Clincal Endocrinology 2003; 59: 396-401
Thea G.A. Strieder Mark F. prummel Jan G.P. Tijssen Eric Endert Wilmar M. Wiersinga
Baseline characteristics of the Amsterdam AITD cohort
Chapter 3 Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid diseases
Summary
Objective: Autoimmune thyroid disease (AITD) is a common disorder especially in women, and both genetic and environmental factors are involved in its pathogenesis. We wanted to gain more insight into the contribution of various environmental factors. Therefore, we started a large prospective cohort study in subjects at risk of developing AITD, for example healthy female relatives of AITD patients. Here we report on their baseline characteristics. Subjects: Only first- or second-degree female relatives of patients with documented AITD were included. Measurements : Smoking habits, oestrogen use, pregnancy history, and iodine exposure were assessed by questionnaires, and correlated to the thyroid function and antibody status. Results: Of 803 subjects, 440 came from families with more than one patient with documented AITD. Of these families, 33% had documented cases of both Graves’ disease and Hashimoto’s thyroiditis. Although the subjects were in self-proclaimed good health, 3.6% were found to have hypothyroidism (overt disease in 1.3%) and 1.9% had hyperthyroidism (overt disease in 0.4%). These patients were older than the euthyroid subjects and were mostly positive for thyroid peroxidase (TPO) antibodies. Oestrogen use was associated with a lower rate of hyperthyroidism [relative risk (RR) 0.169; 95% confidence interval (CI) 0.06- 0.52]; whereas having been pregnant was associated with a higher relative risk for hyperthyroidism (RR 6.88; 95% CI 1.50-30.96). Of the 759 euthyroid subjects, 24% had TPO antibodies. Smoking and oestrogen use were negatively correlated with the presence of TPO antibodies. In the euthyroid subjects, TPO antibody titre correlated positively with TSH levels (r=0.386; P<0.001). Conclusions : The high prevalence of evidence for autoimmune thyroiditis at baseline supports the importance of genetic factors in its pathogenesis. The co-occurrence of Hashimoto’s thyroiditis and Graves’ disease within one family suggests a common genetic basis for these diseases. Oestrogen use is associated with a lower risk, and pregnancy with a
51 Chapter 3 higher risk for developing hyperthyroidism. The positive correlation between TPO antibody titres and TSH levels in euthyroid subjects suggests that TPO antibodies are indeed a marker of future thyroid failure.
Introduction
Thyroid disease is common, especially in women (Canaris et al. , 2000). The prevalence of hypothyroidism, both subclinical and overt, among adult females from all age groups ranges from 3.0 to 7.5% (Wang & Crapo, 1997). It is seen more frequently in elderly women. Parle et al. (1991) found an elevated TSH in 11.6% of 683 women above the age of 60 years, roughly one-half of these women had evidence of thyroid autoimmunity. Hypothyroidism is more prevalent in areas of high iodine intake like Iceland, with a prevalence of 18% in elderly women. (Laurberg et al. , 1998). The prevalence of hyperthyroidism in the general female population ranges from 0.9% (Konno et al. , 1993) to 4.4% (Hollowell et al. , 2002). It is more frequent in elderly women and Parle et al. (1991) found a prevalence of 6.3%, although anti- thyroid antibodies were found in only 5.6% of these hyperthyroid females. The annual incidence of hypothyroidism in females has been estimated as 3.5/1000, while every year 0.8/1000 females will develop hyperthyroidism (Vanderpump et al., 1995). The aetiology of autoimmuny thyroid disease (AITD), encompassing Graves' hyperthyroidism and Hashimoto's thyroiditis, is multifactorial (Weetman & McGregor, 1994). Genetics play an important role in the development of AITD (Vaidya et al. , 2002). The concordance rate for Graves' disease is much higher in monozygotic twins (35%) than in dizygotic twins (3%) (Brix et al. , 2001) and the same applies to Hashimoto's hypothyroidism: 55% vs. 0% (Brix et al. , 2000). In addition, many patients with AITD have family members also affected by this disorder. In a Hungarian study, 5.3% of patients with Graves' disease had a sibling with the same disorder (Stensky et al. 1985). Using this and other studies, Vaidya et al. , (2002) have estimated the λs for Graves’ disease to be approximately 8. The inheritance of AITD is polygenetic and only few of the susceptibility genes have so far been identified (Vaidya et al. , 2002). It has been estimated that 79% of the liability to develop AITD can be attributed to genetics (Brix et al. , 2001) and therefore there are other environmental and hormonal risk factors involved (Weetman & McGregor, 1994). So far, the importance of these environmental and genetic factors has been studied taking them one at a time and not all together. Therefore, we decided to embark on a prospective cohort study in which we will follow for 5 years a large
52 Baseline characteristics of the Amsterdam AITD cohort group of healthy females who are at increased risk of developing AITD because they come from families with at least one relative with documented AITD. Here we report on the base- line characteristics of this cohort.
Subjects and methods
We screened 1003 female subjects with at least one first- or second-degree relative with documented autoimmune hyper- or hypothyroidism, who were in self-proclaimed good health, without a history of thyroid disease, and between 18 and 65 years of age. The subjects were recruited through patients visiting our department, through advertisements in local newspapers and through thyroid patient self-support associations. After obtaining informed consent, we checked the medical history of the affected relative(s) regarding the autoimmune nature of their thyroid disease. Autoimmune thyroid disease was defined as documented hyper- or hypothyroidism in the presence of autoantibodies against thyroid peroxidase (TPO), TSH-receptor autoantibodies, histology compatible with autoimmune thyroiditis, or thyroid eye disease. In 200 of the 1003 included subjects the autoimmune nature of thyroid disease in their family could not be ascertained, leaving 803 participants to be included in the present study. All subjects were seen at our Institution and after giving informed consent they were subjected to a brief medical examination and blood was drawn to assess thyroid status and to collect DNA. All subjects were asked to fill in questionnaires through which information was collected on smoking habits (current and past), use of oral contraceptives or other oestrogens (current and past), exposure to iodine excess, and the number of pregnancies. Current smoking was defined as smoking now, or having stopped smoking within 1 year of the study visit. For this study, subclinical hyperthyroidism was defined as a TSH value of <0.40 mU/L, whereas overt hyperthyroidism was diagnosed if the subject also had an free T4 (fT4) value of >20.1 pmol/L, or a total T3 level of >2.75 nmol/L. Subclinical hypothyroidism was defined as a TSH value of >5.70 mU/L, overt hypothyroidism was diagnosed if there was also an fT4 level of <9.3 pmol/L. These reference values were established in a different cohort of 75 women, recruited from the general population by advertisement and in self-proclaimed good health and without a history of thyroid disease. These 75 women were between 20 and 69 years of age, and there were 15 females in each decade, resulting in a mean age of 42.
53 Chapter 3
In all subjects fT4 (using time-resolved fluoroimmunoassay, Delfia, Turku, Finland) and TSH (Delfia) were measured, as well as antibodies against TPO and thyroglobulin (Tg) using a chemiluminescence immunoassay (LUMI-test, Brahms, Berlin, Germany). TPO and Tg antibody levels of >100 kU/L were considered to be positive. TSH-receptor antibodies were measured by radioreceptor assay as TSH-binding inhibiting immunoglobulins (TBII, TRAK, Brahms, Berlin, Germany) and considered positive if >12 U/L. Total T3 was measured by and in-house radio-immunoassay (RIA) in those subjects with a suppressed TSH level. The study was approved by the local medical ethics committee. Statistical analysis was carried out with the SPSS10 package, using unpaired Student t-tests or Mann-Whitney U-tests to compare means or medians. To compare rates among different groups, we used χ² -test or Fisher’s exact test. Multiple logistic regression analysis was carried out to identify independent risk factors.
Results
Family history The majority of our subjects (713; 89%) had at least one first-degree relative with documented AITD, whereas 90 (11%) had at least one second-degree relative with AITD. Four-hundred and forty subjects came from 233 families with more than one patient with documented AITD. In 47% of these families, only Graves’ hyperthyroidism was found and in 21% all affected relatives had Hashimoto’s hypothyroidism. However, in 33% both Graves’ and Hashimoto’s disease was diagnosed in the same family.
Prevalence of thyroid dysfunction Of the 803 subjects seen at base-line and in self-proclaimed good health, 29 (3.6%) were found to be hypothyroid, 10 of whom had overt disease (1.3%). Of these ten, seven had permanent thyroid failure (all had high levels of TPO antibodies). Fifteen patients (1.9%) had hyperthyroidism, with 3 subjects having overt disease (0.4%). In only one of these latter three, Graves’ hyperthyroidism was diagnosed with high TBII levels; the remaining patients had silent thyroiditis.
Risk factors for thyroid dysfunction The risk factors studied here are shown in Table 1. years of age, and there were 15 females in each decade, resulting in a mean age of 42.
54 Baseline characteristics of the Amsterdam AITD cohort
Table 1. Characteristics of 803 females with at least one relative with documented autoimmune thyroid disease, grouped for thyroid status at study entry
All Hypothyroid Euthyroid Hyperthyroid P-value hypothyroid vs euthyroid hyperthyroid vs euthyroid
n 803 29 (3.6%) 759 (94.5%) 15 (1.9%)
Age (mean ± SD) 36 + 12 43 + 12 36 + 12 44 + 10 0.003 0.02 TSH (median+interquartile range) 1.7 (1.2-2.5) 8.8 (7.6-11.8) 1.7 (1.2-2.4) 0.13 (0.0-0.33) 0.000 0.000 fT4 (median+interquartile range) 12.7 (11.3-14.3) 10.4 (8.5-11.9) 12.8 (11.5-14.4) 13.4 (11.2-16.2) 0.000 0.378 TPO-Ab pos 216 (27%) 25 (86%) 183 (24%) 8 (53%) 0.000 0.009 Tg-Ab pos 66 (8%) 7 (24%) 56 (7%) 3 (20%) 0.001 0.10ª TBII pos 3 (1%) 0 (0%) 2 (0%) 1 (7%) 1.00ª 0.06ª Smoking ever 463 (58%) 20 (69%) 436 (57%) 7 (47%) 0.217 0.403 Smoking current 280 (35%) 12 (42%) 266 (35%) 2 (13%) 0.484 0.101ª Estrogen ever 726 (90%) 28 (97%) 688 (81%) 10 (67%) 0.225 0.002 Estrogen current 360 (45%) 10 (34%) 348 (46%) 2 (13%) 0.225 0.016ª Gravida ever 415 (52%) 18 (62%) 384 (51%) 13 (87%) 0.279 0.006 Iodine intake excess 56 (7%) 1 (3%) 55 (7%) 0 (0%) 0,435ª 0,279ª
P-value for age was obtained by Students’ t-test, p-values for TSH and freeT4 were obtained by Mann-Whitney U-test, P-values for the other variables by χ² test or if appropriate by Fishers exact testª. Hypothyroidism is defined as a TSH>5,7mE/l, hyperthyroidism as a TSH<0,4mE/l.
55 Chapter 3
Patients with hypo- or hyperthyroidism were older than the euthyroid group. The strongest risk factor for both hyper- and hypothyroidism was the presence of TPO autoantibodies. Hyperthyroid subjects had a significantly lower exposure to exogenous oestrogens then the euthyroid subjects, and hyperthyroid subjects had been pregnant more often then the euthyroid subjects. Neither smoking history nor iodine excess in the previous year was found to be associated with dysthyroidism. Using multiple logistic regression analysis, having been pregnant was associated with an increased risk for hyperthyroidism, with an odds ratio (OR) of 6.88 (95% confidence interval (CI) 1.50-30.96), whereas exogenous oestrogen use was found to be associated with a decreased risk for hyperthyroidism (Table 2).
Table 2. Predictive value of some risk factors for the presence of hypothyroidism or hyperthyroidism in 803 females with at least one relative with documented autoimmune thyroid disease
Determinants Odds ratio (95% CI)
Hypothyroid Hyperthyroid
age 1.059 (1.027-1.093) 1.006 (0.955-1.060) smoking, ever 1.299 (0.484-3.487) 0.892 (0.276-2.885) smoking, current 1.619 (0.731-3.583) 0.46 (0.10-2.12)
estrogen use, ever 4.232 (0.552-32,425) 0.169 (0.055-0.523) estrogen use, current 0.889 (0.375-2.104) 0.356 (0.073-1.726) gravida, ever 0.519 (0.197-1.368) 6.88 (1.5-30.958 ) iodine excess 0.332 (0.043-2.545) -
Bold type, significant risk factor
Table 4. Predictive value of some risk factors for the presence of TPO antibodies in 759 euthyroid females with at least one relative with documented autoimmune thyroid disease
Determinant Odds ratio (95% CI)
age 1.037 (1.022-1.051) smoking, ever 1.024 (0.681-1.540)
smoking, current 0.688 (0.480-0.986)
estrogen use, ever 0.578 (0.345-0.967) estrogen use, current 0.821 (0.565-1.193) gravida, ever 0.726 (0.477-1.107)
iodine excess 0.561 (0.287-1.096)
Bold type, significant risk factor
56 Baseline characteristics of the Amsterdam AITD cohort
Risk factors for the presence of TPO antibodies Although the majority of the hyper- and hypothyroid subjects had TPO antibodies, this marker for thyroid autoimmunity was also present in 24% of the euthyroid subjects (Table 3). When we looked at this latter group, the presence of TPO antibodies was associated with age and having been pregnant; the absence of TPO antibodies was associated with smoking and exogenous oestrogen use. Smoking habits and exogenous oestrogen use were both found to be independently associated with a reduced risk for TPO antibody positivity (Table 4).
Table 3. Age, hormonal status, and risk factors in 759 euthyroid females with at least one relative with documented autoimmune thyroid disease, by TPO antibody status
TPO-Ab pos = 100 kU/L TPO-Ab neg < 100 kU/L P-value
n 183 (24.1%) 576 (75.9%) Age (mean ± SD) 40 + 12 34 + 12 0.000
TSH (median+interquartile range) 2.3 (1.5-3.3) 1.6 (1.1-2.2) 0.000
fT4 (median+interquartile range) 12.5 (11.2-13.8) 12.9 (11.6-14.5) 0.161 Smoking ever 96 (52%) 340 (59%) 0.117
Smoking current 46 (25%) 220 (38%) 0.001
Estrogen use ever 155 (85%) 535 (93%) 0.001 Estrogen use current 64 (35%) 284 (49%) 0.001
Gravida ever 106 (58%) 278 (48%) 0.023
Iodine intake excess 12 (7%) 43 (6%) 0.68
P-value for age was obtained by Student t-test, P-values for TSH and fT4 were obtained by Mann-Whitney U- test, P-values for the other variables were obtained by χ² test.
TPO antibodies in relation to Tg antibodies and thyroid function. Twenty-seven percent of the 803 subjects had TPO antibodies, whereas Tg antibodies were found in only 8% of the females. The large majority of the Tg antibody positive persons also had TPO antibodies; Tg antibodies as the only marker of AITD were found in only 3%. Interestingly, in the euthyroid subjects the TPO antibody titres were found to be correlated with increasing TSH levels, indicating the beginning of thyroid failure: Pearson correlation r=0.386, P<0.001 (Fig. 1).
57 Chapter 3
Figure 1. Box and Wisker plots of TSH values grouped according to increasing TPO antibody (TPO-Ab) levels in 759 euthyroid relatives of patients with documented AITD, showing a positive correlation: Pearson correlation coefficient 0.386, P<0.001
7 6 5 4 3 TSH (mU/L) TSH 2 1 0 <50 50-100 100-1,000 1,000-5,000 5,000-10,000 >10,000 n=492 n=84 n=101 n=58 n=12 n=12 TPO-Ab (kU/L)
Discussion
This follow-up cohort study was initiated to determine risk factors involved in newly incident cases of AITD. To increase the likelihood of diagnosing new patients during a 5-year follow- up, we included subjects who had at least one relative with documented AITD and who were in self-proclaimed good health. Indeed, evidence for autoimmune thyroiditis was found in a fairly large proportion of subjects: 227/803 (28.3%) had TPO autoantibodies. In comparison, in the National Health and Nutrition Examination Survey (NHANES III), 14.6% of the females without known thyroid disease had TPO antibodies (Hollowell et al. , 2002). In the first Wickham survey, 10.3% of the females had cytoplasmic antibodies (Tunbridge et al., 1977), and using a more sensitive assay a prevalence of 17.8% was found among female blood donors in the UK (Prentice et al. , 1990). Our observed prevalence of 27% among first- and second-degree relatives of AITD patients thus lies between the prevalence in the general population and prevalence rates of 48% (Phillips et al ., 1990) and 43% (Prentice et al. , 1990) in first-degree relatives. Our findings are thus in agreement with the notion that subjects with a family history of AITD are at increased risk for thyroid disease (Vaidya et al. , 2002; Brix et al ., 1998; Chopra et al., 1977; Carey et al., 1980 ; Tamai et al., 1990). Even though the 803 participants were in self-proclaimed good health, 44 (5.5%) had an abnormal TSH value, of whom 29 (3.6%) had hypothyroidism (19 subclinical, 10 overt disease) and 15 (1.9%) were hyperthyroid (12 subclinical, 3 overt). Of the 759 participants
58 Baseline characteristics of the Amsterdam AITD cohort with a normal thyroid function, 183 (24%) had TPO antibodies and in this group the presence of TPO antibodies correlated significantly with TSH levels (r=0.386, P<0.001). This finding suggests that the presence of TPO antibodies is an early indicator of impending thyroid failure. This corroborates with studies showing that TPO antibody titres are related to the degree of lymphocytic infiltration of the thyroid gland (Yoshida et al., 1978). All our subjects had a family history of at least one relative with documented AITD. As could be expected, 440 subjects (coming from 233 families) had more than one affected relative. Forty-six percent of these 233 families had cases of hyperthyroidism alone, and 21% only of hypothyroidism alone. Interestingly, in 34% of these families, patients with Graves’ disease and Hashimoto’s disease were found. This finding suggests that these two different types of AITD may have a common genetic background, because family members usually do not share environmental factors during their adult life but they do share some genes. We therefore suggest that environmental factors may dictate whether a subject with a certain genetic make- up will develop hypo- or hyperthyroidism, although, strictly speaking, the association might be the reverse. Although delineating the relative importance of several environmental factors can be done more reliably in the future prospective follow-up of this cohort, the cross-sectional findings in our volunteers at study entry already provide some insight into some of these risk factors. First, both hyper- and hypothyroid subjects were older than their euthyroid counterparts, and second 86% of the hypothyroid cases had detectable levels of TPO antibodies. Interestingly, TPO antibodies were also present in 54% of the hyperthyroid patients. Oestrogen use (current or past) was -independently from other factors- associated with a lower risk for hyperthyroidism, and was not associated with the occurrence of hypothyroidism. There are surprisingly few studies on the association between thyroid disease and oestrogen use. In one such study, current contraceptive use was found to protect against thyroid disease in general (thyrotoxicosis, myxedema, or goiter) (Frank and Kay, 1978). These authors could not find a relationship between thyroid disease and parity, whereas we found that the hyperthyroid cases more often reported a pregnancy than the other two groups. It may be argued that subjects who have been pregnant are using oestrogens less often, but multiple logistic regression analysis showed that oestrogen use and pregnancy history were independently associated with hyperthyroidism with a relative risk of 0.17 for oestrogen use and of 6.88 for pregnancy. In agreement with one other study, we could not find any association between parity and hypothyroidism (Phillips et al. , 1990b).
59 Chapter 3
Apart from TPO antibodies and age, we could not detect other factors increasing the risk for hypothyroidism. However, oestrogen use was associated with a lower rate of occurrence of TPO antibodies in the 759 euthyroid subjects (relative risk 0.58). Unexpectedly, we also found that smokers were less likely to have TPO antibodies than non-smokers. Smoking is a risk factor for the development of Graves’ hyperthyroidism, (Prummel & Wiersinga, 1993) and it is difficult to understand why smoking would protect against the development of TPO antibodies. We can only speculate that the absence of TPO antibodies in subjects at risk of developing Graves’ disease may increase the likelihood to become hyperthyroid. Only a prospective study will help to gain insight into this. There are obvious limitations of this study. Although our cohort will be followed prospectively, some of the current base-line findings are retrospective in nature, as exposure to certain risk factors was assessed retrospectively for the previous year. For example, iodine excess was assessed by history taking and this may explain why we could not find a relationship with the occurrence of hypo- or hyperthyroidism. Another important limitation is the fact that this is a selected group of subjects, from which we excluded relatives with known AITD. This means that certain established risk factors will not be detected in this first cross- sectional analysis, because they will already have precipitated thyroid disease in some relatives. Such risk factors might become apparent in a future prospective analysis. Despite these limitations, our study strongly supports the importance of genetic factors in the development of AITD in view of the high prevalence at base-line of autoimmune thyroiditis in this large group of subjects with a positive family history. Regarding this family history, it was striking to note that indeed both Graves’ and Hashimoto’s disease can occur in the same family. Our study also supports the importance of TPO antibodies as a marker for future thyroid failure in view of the positive correlation between TSH values and TPO antibody titres in the euthyroid subjects.
60 Baseline characteristics of the Amsterdam AITD cohort
Acknowledgements This study was financially supported by a grant from the Netherlands Organization for Scientific Research (NWO-950-10-626).
61 Chapter 4
References 1. Brix TH, Kyvik KO & Hegedus L. (1998) What is the evidence of genetic factors in the etiology of Graves' disease? A brief review. Thyroid, 8, 627-634. 2. Brix TH, Kyvik KO, Christensen K & Hegedus L. (2001) Evidence for a major role of heredity in Graves' disease: a population-based study of two Danish twin cohorts. Journal of Clinical Endocrinology and Metabolism, 86, 930-934. 3. Brix TH, Kyvik KO & Hegedus L. (2000) A population-based study of chronic autoimmune hypothyroidism in Danish twins. Journal of Clinical Endocrinology and Metabolism, 85, 536-539. 4. Canaris GJ, Manowits NR, Mayor G &, Ridgway EC. (2000) The Colorado thyroid disease prevalence study. Archives of Internal Medicine, 160, 526-534. 5. Carey C, Skosey C, Pinnamaneni KM, Barsano CP & DeGroot LJ. (1980) Thyroid abnormalities in children of parents who have Graves’ disease: possible pre-Graves’ disease. Metabolism, 29, 369-376. 6. Chopra IJ, Solomon DH, Chopra U, Yoshihara E, Terasaki PI & Smith F. (1977) Abnormalities in thyroid function in relatives of patients with Graves’ disease and Hashimoto’s thyroiditis: lack of correlation with HLA-B8. Journal of Clinical Endocrinology and Metabolism, 45, 545-554. 7. Frank P & Kay CR. (1978) Incidence of thyroid disease associated with oral contraceptives. British Medical Journal, ii, 1531. 8. Hollowell JG, Staehling NW, Flanders WD, Hannon WH, Gunter EW, Spencer CA & Braverman LE. (2002) Serum TSH, T 4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). Journal of Clinical Endocrinology and Metabolism, 87, 489-499. 9. Laurberg P, Pedersen KM, Hreidarsson A, Sigfusson N, Iversen E & Knudsen PR. (1998) Iodine intake and the pattern of thyroid disorders: A comparative epidemiological study of thyroid abnormalities in the elderly in Iceland and in Jutland, Denmark. Journal of Clinical Endocrinology and Metabolism, 83, 765-769. 10. Parle JV, Franklyn JA, Cross KW, Jones SC & Sheppard MC. (1991) Prevalence and follow-up of abnormal thyrotrophin (TSH) concentrations in the elderly in the United Kingdom. Clinical Endocrinology 34, 77-83. 11. Phillips D, McLachlan S, Stephenson A, Roberts D, Moffitt S, McDonald D, Ad'hiah A, Stratton A, Young E, Clark F, Beever K, Bradbury J & Rees-Smith B. (1990) Autosomal dominant transmission of autoantibodies to Thyroglobulin and Thyroid Peroxidase. Journal of Clinical Endocrinology and Metabolism, 70, 742-746. 12. Phillips DIW, Lazarus JH & Butland BK. (1990a) The influence of pregnancy and reproductive span on the occurrence of autoimmune thyroiditis. Clinical Endocrinology 32, 301-306. 13. Prentice LM, Phillips DI, Sarsero D, Beever K, McLachlan SM & Smith BR. (1990) Geographical distribution of subclinical autoimmune thyroid disease in Britain: A study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinologica , 123, 493-498. 14. Prummel MF & Wiersinga WM.. (1993) Smoking and the risk of Graves' disease. JAMA, 269, 479-82. 15. Stenszky V, Kozma L, Balazs C, Rochlitz S, Bear JC & Farid NR. (1985) The genetics of Graves' disease: HLA and disease susceptibility. Journal of Clinical Endocrinology and Metabolism, 61, 735-740. 16. Tamai H, Kasagi K, Morita T, Hidaka A, Kuma K, Konishi J, Kumagai LF & Nagataki S. (1990). Thyroid response, especially to thyrotropin-binding inhibiting immunoglobulins in
62 Baseline characteristics of the Amsterdam AITD cohort
euthyroid relatives of patients with Graves’ disease: a clinical follow-up. Journal of Clinical Endocrinology and Metabolism, 71, 210-215. 17. Tunbridge WMG, Evered DC, Hall R, Appleton D, Brewis M, Clark F, Grimley Evans J, Young E, Bird T & Smith PA. The spectrum of thyroid disease in a community: The Whickham survey. Clinical Endocrinology, 7, 481-493. 18. Vaidya B, Kendall-Taylor P & Pearce SHS. (2002) Genetics of endocrine disease. The genetics of autoimmune thyroid disease. Journal of Clinical Endocrinology and Metabolism, 87, 5385-5397. 19. Vanderpump MPJ, Tunbridge WMG, French JM, Appleton D, Bates D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Tunbridge F & Young ET. (1995) The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clinical Endocrinology, 43, 55-68. 20. Wang C & Crapo LM. (1997) The epidemiology of thyroid disease and implications for screening. Endocrinology and Metabolism Clinics of North America 26, 189-218. 21. Weetman AP & McGregor AM (1994) Autoimmune thyroid disease: Further developments in our understanding. Endocrine Reviews, 15, 788-830. 22. Yoshida H, Amino N, Yagawa K, Uemura K, Satoh M, Miyai K & Kumahara Y. (1978) Association of serum antithyroid antibodies with lymphocytic infiltration of the thyroid gland: studies of seventy autopsied cases. Journal of Clinical Endocrinology and Metabolism, 46,859-862.
63
Increased prevalence of antibodies to enteropathogenic Yersinia enterocolitica 4| virulence proteins in relatives of patients with AutoImmune Thyroid Disease
Clinical Experimental Immunology 2003, 132: 278-282
Thea G.A. Strieder Björn E. Wenzel Mark F. Prummel Jan G.P. Tijssen Wilmar M. Wiersinga
Autoimmune thyroid disease and Yersnia enterocolitica
Chapter 4 Increased prevalence of antibodies to enteropathogenic yersinia enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease
Abstract
Infections have been implicated in the pathogenesis of a number of autoimmune diseases, and Yersinia enterocolitica (YE) might play a role in the development of autoimmune thyroid disease (AITD). Clinical evidence in support of this hypothesis has been inconclusive. We reasoned that looking earlier in the natural course of AITD might enhance chances to find evidence for YE infection. Consequently we determined seroreactivity against YE in subjects at risk to develop AITD, i.e. in 803 female relatives of AITD patients in self proclaimed good health. As comparison group we used 100 healthy women who participated in a program for reference values. IgG and IgA antibodies to virulence-associated outer membrane proteins (YOPs) of YE were measured by a specific assay. Serum thyroid peroxidase antibodies (TPO-Ab) as indicators of AITD were considered to be positive at levels of >100 kU/l. The prevalence of YOP IgG-Ab was higher in AITD relatives than in controls (40.1% vs 24%, p=0.002), and the same was true for YOP IgA-Ab (22% vs 13%, p<0.05). 44 of the 803 AITD relatives had an increased or decreased plasma TSH, and 759 were euthyroid as evident from a normal TSH; the prevalence of YOP-Ab did not differ between these three subgroups. TPO-Ab were present in 10% of controls and in 27% of the AITD relatives (p<0.001). The prevalence of TPO-Ab in the euthyroid AITD relatives was not different between YOP IgG-Ab positive and negative subjects (23.3% vs 24.7%, NS), nor between YOP IgA-Ab positive and negative subjects (21.2% vs 24.9%, NS). In conclusion, healthy female relatives of AITD patients have an increased prevalence of YOP antibodies, which however is not related to the higher prevalence of TPO antibodies in these subjects. The findings suggest a higher rate of persistent YE infection in AITD relatives. Susceptibility genes for AITD may also confer a risk for YE infection.
67 Chapter 4
Introduction
Autoimmune thyroid disease (AITD) is viewed as a multifactorial condition in which the development of the autoimmune response against thyroidal antigens is facilitated by a particular but still incompletely known polygenetic background and presumably provoked by environmental factors. Identified environmental factors are iodine intake, smoking, stress, pregnancy, estrogens and possibly infections. Infections have been implicated in the pathogenesis of a number of autoimmune diseases like Coxsackievirus P2-C in type 1 diabetes mellitus [1], but their role in AITD is disputed. The best studied infection in AITD is that with Yersinia enterocolitica (YE). The membrane of YE has specific TSH binding sites [2], and infection with YE gives rise to antibodies against these TSH binding sites which recognize and stimulate the TSH receptor of human thyroid membranes [3,4]. Conversely, Graves IgG bind to YE membranes [5]. Thus, YE infection by molecular mimicry with self antigens may induce crossreactive TSH receptor antibodies and crossreactive T-cells, leading to AITD. YE furthermore acts as a superantigen [6], and may result via induction of V-gene restricted T-cells in polyclonal stimulation of autoreactive T-cells, again contributing to the development of AITD. Whereas the laboratory data provide a solid base for assuming an etiologic role of YE infection in AITD, clinical evidence in support of this hypothesis has been inconclusive as conflicting results are reported on the frequency of YE infection in AITD patients compared to controls as summarized in Table 1 [7-12]. How are these discrepant results explained? Geographical differences in exposure to YE might be one reason. Differences in the applied methods to measure YE seroreactivity could be another. Serologic evidence of YE infection can be obtained by the agglutination reaction, which however becomes rapidly negative. All pathogenic Yersinia species harbour a 70 kb plasmid encoding for the virulence conferring outer membrane proteins (YOPs) [7,8]. A more suitable method therefore is the demonstration of specific IgA and IgG antibodies against YOPs by ELISA or immunoblots. Methodological differences, however, are unlikely accountable for the sharp contrast between positive and negative studies when similar methods are used. A third possibility is that the studies were done too late in the course of the disease. Indeed, all reported studies in the literature are cross-sectional in nature, investigating serum samples of patients who already had developed full blown Graves’ hyperthyroidism or Hashimoto’s hypothyroidism and who mostly had been treated for months to years. Although
68 Autoimmune thyroid disease and Yersnia enterocolitica
Table 1. Summary of literature data on the frequency of Yersinia enterocolitica (YE) infection in patients with autoimmune thyroid disease compared to controls
Reference Country Applied method for Frequency of YE infection YE seroreactivity Graves’ disease Hashimoto’s disease Controls
[7] Germany Western blot IgG 60-81%* 66%* 35% IgA 29-39%* 37%* 12%
[8] Greece ELISA IgG - 25.4%* 1.9% IgA - 2.8%* 0%
[9] USA Western blot IgG 96% 55.5% 70.8%
[10] Canada Western blot IgG >90% >90% >90% ELISA IgG Not different from controls agglutination O3, O9 All -ve All -ve All -ve
[11] Japan agglutination O3 27.1% 40.0% 29.4% O5 81.4%* 91.1%* 58.9% O6 28.6% 40.0%* 24.7% O9 37.1% 51.1%* 29.9%
[12] Turkey agglutination O3 53.8%* 38.1%* 17.5% O5 29.2%* 15.8%* 7.5% O8 44.6%* 17.5%* 7.5% O9 40.0%* 22.8%* 10.0%
*Significantly different from controls. it has been reported that IgA and IgG antibodies in newly diagnosed patients with Graves' hyperthyroidism are not observed until four weeks after diagnosis [15], patients rarely remember any symptoms of a recent Yersinia infection [10]. We hypothesized that looking earlier in the natural course of the disease (i.e. when thyroid function is still normal but thyroid antibodies are already present) might increase chances to find evidence for YE infection. For, IgA antibodies appear after 10 days postinfection, followed by IgG antibodies. IgA reactivity decreases rapidly after 3-6 months, whereas a decrease of IgG is markedly retarded. In chronic infection persistent IgA and IgG reactivity is seen [7]. To pursue this further, the question is how to find subjects with subclinical AITD. Female relatives of patients with Graves’ hyperthyroidism or Hashimoto’s hypothyroidism are clearly at risk to develop AITD in view of their gender and family history [16,17]. We have assembled a large group of such subjects in the Amsterdam AITD cohort study, designed as a long-term prospective follow-up study to get more insight in genetic and environmental factors involved in the pathogenesis of AITD [18]. Here we report the results at study entrance for YE seroreactivity, which was assayed blindly with respect to the thyroid state. As
69 Chapter 4 comparison we recruited healthy euthyroid volunteers. Serum samples of both groups were collected over the same period of time.
Subjects and Methods
The Amsterdam AITD cohort comprises 803 female subjects between 18 and 65 years of age with at least one 1st or 2nd degree relative with documented autoimmune hyper- or hypothyroidism; they had no personal history of thyroid disease and were in self-proclaimed good health. All subjects were seen at our institution, and blood was drawn after obtaining informed consent; plasma and serum samples were stored at –20° C until assay. As comparison group we used 100 female subjects between 20 and 69 years of age, who were recruited through advertisements in local newspapers to participate in an ongoing program of our institution for delineating reference values of endocrine function tests. They were also in self-proclaimed good health, and had no history of thyroid disease. Blood samples were collected over the same period of time as those of the Amsterdam AITD cohort, and processed in the same manner. The study was approved by the institutional Committee on medical ethics in Amsterdam. In all subjects free thyroxine (FT4, time-resolved fluoroimmunoassay, Delfia, Turku, Finland) and thyrotropin (TSH, Delfia) were measured, as well as antibodies against thyroid peroxidase (TPO-Ab, chemiluminescence immunoassay, LUMI-test, Brahms, Berlin, Germany). Reference values of FT4 are 9.3-20.1 pmol/l and of TSH 0.4-5.7 mU/l. TPO-Ab were considered to be positive at levels of >100 kU/l; inter-assay variation at 70 kU/l is 15%, and at 600 kU/l 5.7%. Specific IgG and IgA antibodies against purified plasmid-encoded virulence associated YOPs of YE serotype O9 (LCR) in sera were demonstrated by immunoblotting with a YOP-Ab assay (AID, Strassberg, Germany). In short, antigens (25,34,36,37,39,40,46,48 kDa) are blotted onto nitrocellulose. Sera are diluted 1:51 in PBS-Tween and incubated with the antigen-coated nitrocellulose strips overnight at 22°C. The IgG and IgA antibody-antigen complexes formed are quantified after immunostaining with the AID-Scan-System. Controls are included in each assay run, using human acute sera (culture-positive YE infection) containing antibodies to the YOPs. Test sera are judged positive if at least three bands (IgG) or two bands (IgA) are seen in immunoblotting at a level greater than 10% (IgG) or 5% (IgA) of reference standards. The inter-assay variation of the YOP-Ab assay is <3% according to
70 Autoimmune thyroid disease and Yersnia enterocolitica the manufacturer. The YOP-Ab assay was performed without prior knowledge of thyroid function tests or the presence of TPO-Ab in the serum samples. The significance of differences between groups was analyzed with the X 2 test, or with Fisher exact test in case of small numbers. P-values are two tailed.
Results
The prevalence of TPO antibodies and of IgG and IgA antibodies against YOPs were higher in AITD relatives than in controls (Table 2). The AITD relatives were on average nine years younger than control women, but the higher prevalence of YOP antibodies in AITD relatives was similar in all age groups (Table 3).
Table 2. Prevalence of antibodies against thyroid peroxidase (TPO-Ab) and against virulence-associated YOPs of Yersinia enterocolitica (YOP IgG-Ab and YOP IgA-Ab) in sera of 803 female relatives of patients with autoimmune thyroid disease (AITD) and 100 healthy female controls
n Age TPO-Ab YOP IgG-Ab YOP IgA-Ab
Controls 100 44±15 10 (10%) 24 (24%) 13 (13%) AITD relatives 803 36±12 216 (27%)‡ 322 (40.1%)* 176 (22%**)
AITD relatives Hypothyroid 29 43±12 25 (86%) 10 (34.5%) 7 (24.1%) Euthyroid 759 36±12 183 (24%)‡ 305 (40.2%)* 165 (21.7%)** Hyperthyroid 15 44±10 8 (53%) 7 (46.7%) 4 (26.7%)
‡P<0.001, * P=0.002, ** P<0.05 vs controls.
Table 3 . Prevalence per age group of antibodies against virulence-associated YOPs of Yersinia enterocolitica (YOP IgG-Ab and YOP IgA-Ab) in sera of 803 female relatives of patients with autoimmune thyroid disease
Age n YOP IgG-Ab n (%) YOP IgA-Ab n (%)
18-29 yr 295 123 (41.7%) 69 (23.4%) 30-39 yr 215 88 (40.9%) 45 (20.9%) 40-49 yr 170 64 (37.6%) 36 (21.2%) 50-59 yr 94 36 (38.3%) 18 (19.1%) 60-69 yr 29 11 (37.9%) 8 (27.6%)
The majority (94.5%) of AITD relatives were euthyroid as evident from a normal TSH, but 3.6% were hypothyroid (plasma TSH >5.7 mU/l) and 1.9% were hyperthyroid (plasma TSH <0.4 mU/l). The prevalence of YOP antibodies did not differ between the euthyroid,
71 Chapter 4 hypothyroid and hyperthyroid AITD relatives (Table 2). The relative risk in AITD female relatives is 1.57 (95% CI 1.09-2.25) for the presence of YOP IgG-Ab, and 1.68 (95% CI 1.00-2.84 for YOP IgA-Ab. The relative risk in the subgroup of euthyroid AITD relatives is similar: 1.67 (95% CI 1.17-2.40) for YOP IgG-Ab, and 1.67 (95% CI 0.99-2.83) for YOP IgA-Ab. We next examined the determinants of the presence of YOP antibodies in the subgroup of euthyroid AITD relatives (Table 4).
Table 4 . Characteristics of 759 euthyroid healthy female relatives of patients with autoimmune thyroid disease according to the presence or absence in serum of antibodies against virulence-associated YOPs of Yersinia enterocolitica
n age yr TSH TPO-Ab > 100 kU/l (x±SD) ( mU/l)* n (%) YOP IgG-Ab Positive 305 35±12 1.6 (1.2-2.3) 71 (23.3%) Negative 454 36±12 1.8 (1.3-2.5) 112 (24.7%)
YOP IgA-Ab Positive 165 35±12 1.8 (1.2-2.3) 35 (21.2%) Negative 594 35±12 1.7 (1.2-2.5) 148 (24.9%)
*Median and interquartile range.
Subjects with YOP IgG-Ab were not different from those without YOP IgG-Ab with respect to age, plasma TSH, and prevalence of TPO antibodies. The same was true for YOP IgA-Ab. The presence of YOP IgG-Ab and IgA-Ab was also not related to smoking behaviour, estrogen medication, parity, or a history of iodine excess (data not shown). Two of the euthyroid AITD relatives had TSH binding inhibitory immunoglobulins (TBII) in their serum, but YOP IgG and IgA antibodies were absent in both.
Discussion
The main finding of the present paper is a higher prevalence of IgG and IgA antibodies to released virulence-associated outer membrane proteins (YOPs) of Yersinia enterocolitica in female relatives of patients with autoimmune thyroid disease than in control women. The validity of this finding depends heavily on the characteristics of the control group: are these comparable to the AITD relatives? All investigated subjects were in self-proclaimed good health and of female sex, but the control women were on average eight years older. However, the difference in age is unlikely to account for the difference of 16.1% (95% CI 16.0-16.2) in the prevalence of YOP IgG-Ab between both groups, nor for the difference of 9% (95% CI
72 Autoimmune thyroid disease and Yersnia enterocolitica
8.9-9.1) in the prevalence of YOP IgA-Ab, because the presence of YOP antibodies was similar in all age groups. Blood samples of both groups were collected over the same time period, excluding bias due to seasonal variation in Yersinia infection. Control women orginated from Amsterdam and surrounding areas, but AITD relatives came from all over The Netherlands. Confounding bias due to some variation in places of residence seems unlikely as no specific geographic distribution of Yersinia infections has been reported in The Netherlands. Measurement bias can be ruled out because all assays of YOP antibodies were done ‘blindly’ using one particular method. The groups differed in thyroid function: control women were all euthyroid, but 5.5% of the female AITD relatives were either hypo- or hyperthyroid. However, thyroid function by itself does not explain the difference in YOP antibodies between both groups: the prevalence of YOP antibodies was equally high in hypo-, hyper- and euthyroid AITD relatives, and the difference in prevalence of YOP antibodies between euthyroid AITD relatives and controls was the same as between the whole group of AITD relatives and controls. Can the higher prevalence of YOP antibodies in AITD relatives be explained from the difference in prevelance of AITD between both groups? The presence of serum TPO antibodies is a good marker for the existence of chronic autoimmune thyroiditis [19]. According to this criterion AITD was present in 10% of control women, a figure in good agreement with the prevalence of AITD in the adult female population [20]. As expected, we found a much higher prevalence of AITD of 27% in the female AITD relatives. Nevertheless, the difference in prevalence of AITD between both groups cannot be held accountable for the difference in prevalence of YOP antibodies as the presence of YOP antibodies in euthyroid relatives was not related at all to the presence of TPO antibodies. One may argue that our cut- off value of >100 kU/l in the TPO-Ab assay as criterion for a positive TPO-Ab test, is too high. For, the detection limit of the assay is 50 kU/l, and values between 50 and 100 kU/l were observed in 103 out of the 759 euthyroid AITD relatives. Although the biologic significance of these intermediate values is less well established, accepting all values greater than 50 kU/l as a positive TPO-Ab test result does not alter the picture: the prevalence of TPO-Ab in the YOP IgG-Ab positive and IgG-Ab negative subjects is then 35.1% and 39.4% respectively (not significantly different), and in the YOP IgA-Ab positive and IgA-Ab negative subjects 29.7% and 39.9% (p=0.017). These figures show an even lower frequency of TPO-Ab in YOP IgA-Ab positive than in IgA-Ab negative subjects. Our findings led us to conclude that the higher prevalence of YOP antibodies in AITD relatives is not related to the higher prevalence of AITD in these subjects.
73 Chapter 4
The lack of association between seroreactivity against YE and AITD argues against Yersinia infection as a causal factor contributing to the development of AITD. We found no support for our initial hypothesis that chances to observe such an association would be enhanced by looking earlier in the natural course of AITD, i.e. when thyroid function is still normal but thyroid antibodies are already present. The limitations of the present study are, however, the same as of those reported so far in the literature: they are all cross-sectional in nature. It cannot be completely excluded that during prospective long-term follow-up, as foreseen in our Amsterdam AITD cohort study, the occurrence of TPO antibodies or an abnormal thyroid function bears a temporal relationship to a previous rise of YOP antibodies. Such an observation would strengthen a cause-and-effect relationship. Having ascertained the validity of the comparison group and the lack of an association between YOP antibodies and AITD, how must the higher prevalence of YOP antibodies in AITD relatives be interpreted? Yersinia virulence plasmids encoding for YOPs have not been found in other enterobacteriacaea. The measured YOP antibodies are apparently specific for YE, and a fair proportion of our investigated subjects must have experienced YE infection. Indeed, Yersiniosis seems a rather common disease in The Netherlands [7]. The lower prevalence of YOP IgA-Ab relative to IgG-Ab indicates that YE infection in our adult subjects must have occurred somewhere in the past and not in recent times. In line with this are demographic data of 261 Dutch patients with enteric forms of Yersiniosis [7]: 40.2% had occurred in the age group of 0-15 years, 18% between 16 and 25 years, and 41.8% in patients olders than 25 years. Our data do not necessarily indicate a higher incidence of YE infection in AITD relatives. This is because, in 65% of patients with yersiniosis, the infection is self-limiting, but in 35% a chronic infection persists with high titres of YOP IgG-Ab and IgA-Ab due to continuous antigenic stimulation from the plaques of Peyer and other lymph nodes still harbouring YE [21]. Why YE infection may persist in some subjects but is self-limiting in others must be determined by host factors. Experimental animal models suggest that the immune state is an important determinant [22]. In human and in mice, a relationship has been noted between the infection pattern and the HLA-B27 antigen [23,24]. Following this line of reasoning, it could be that the higher prevalence of YOP antibodies in AITD relatives is not caused by a higher incidence of YE infection per se , but by a higher rate of persistent YE infection caused by a particular genetic make-up. Some of the susceptibility genes for AITD (like those of the HLA family and CTLA-4) may also confer a risk for persistence of YE infection. For, unlike individuals constantly exposed to infections, individuals living under hygienic conditions (as
74 Autoimmune thyroid disease and Yersnia enterocolitica in the Netherlands) are poor regulators of immune responses in general. According to the ‘hygiene hypothesis’ the decreased infections and antigenic pressure of a westernized lifestyle may result in an increased incidence of allergic and organ-specific autoimmune diseases [25,26]. We consider this possibility as the most attractive explanation of our results.
ACKNOWLEDGEMENTS The study was financially supported by grants from the Dutch Medical Research (NWO 950- 10-626) and the Margarete Markus Charity Foundation, London (MMMG2000).
75 Chapter 4
References
1. Albert LJ, Inman RD. Molecular mimicry and autoimmunity. N Engl J Med 1999; 341: 2068-2074. 2. Weiss M, Ingbar SH, Winblad S, et al. Demonstration of a saturable binding site for thyrotropin in Yersinia enterocolitica. Science 1983; 219: 1331-1333. 3. Wolf MW, Misaki T, Bech K, et al. Immunoglobulins of patients recovering from Yersinia enterocolitica infections exhibit Graves' disease-like activity in human thyroid membranes. Thyroid 1994; 1: 315-320. 4. Luo G, Fan J-L, Seetharamniah GS, et al. Immunization of mice with Yersinia enterocolitica leads to the induction of antithyrotropin receptor antibodies. J Immunology 1993; 151: 922-928. 5. Heyma P, Harrison LC, Robins-Browne R. Thyrotrophin (TSH) binding sites on Yersinia enterocolitica recognized by immunoglobulins from humans with Graves' disease. Clin Exp Immunol 1986; 64: 249-254. 6. Stuart PM, Woodward JG. Yersinia enterocolitica produces superantigenic activity. J Immunol 1992; 148: 225-233. 7. Stolk-Engelaar VMM, Hoogkamp-Korstanje JAA. Clinical presentation and diagnosis of gastrointestinal infections by Yersinia enterocolitica in 261 Dutch patients. Scand J Infect Dis 1996; 28: 571-575. 8. Heesemann J, Eggers C, Schröder J. Serological diagnosis of Yersiniosis by immunoblot technique using virulence - associated antigens of enteropathogenic Yersiniae. Contr Microbiol Immunol 1987; 9: 285. 9. Wenzel BE, Heeseman J, Wenzel KW, et al. Antibodies to plasmid-encoded proteins of enteropathogenic Yersinia in patients with autoimmune thyroid disease. Lancet 1988; ii: 56. 10. Chatzipanagiotou S, Legakis JN, Boufidou F, et al. Prevalence of Yersinia plasmid- encoded outer protein (YOP) class-specific antibodies in patients with Hashimoto's thyroiditis. Clin Microbiol Infect 2001; 7: 138-143. 11. Arscott P, Rosen ED, Koenig RJ, et al. Immunoreactivity to Yersinia enterocolitica antigens in patients with autoimmune thyroid disease. J Clin Endocrinol Metab 1992; 75: 295-300. 12. Resetkova E, Notenboom R, Arreaza G, et al. Seroreactivity to bacterial antigens is not a unique phenomenon in patients with autoimmune thyroid diseases in Canada. Thyroid 1994; 4: 269-274. 13. Asari S, Amino N, Horikawa M, et al. Incidence of antibodies to Yersinia enterocolitica: high incidence of serotype O5 in autoimmune thyroid diseases in Japan. Endocrinol Jap 1989; 36: 381-386. 14. Çorapçio ğlu D, Tonyukuk V, Kiyan M, et al. Relationship between thyroid autoimmunity and Yersinia enterocolitica antibodies. Thyroid 2002; 12: 613-617. 15. Wenzel BE, Heeseman J, Wenzel KW, et al. Patients with autoimmune thyroid diseases have antibodies to plasmid encoded proteins of enteropathogenic Yersinia. J Endocrinol Invest 1988; 11: 139-140. 16. Chopra IJ, Solomon DH, Chopra U, et al. Abnormalities in thyroid function in realtives of patients with Graves' disease and Hashimoto's thyroiditis: lack of correlation with inheritance of HLA-B8. J Clin Endocrinol Metab 1977; 45: 545-554. 17. Tamai H, Kasagi K, Morita T, et al. Thyroid response, especially to thyrotropin binding inhibitory immunoglobulins in euthyroid relatives of patients with Graves' disease: a clinical follow-up. J Clin Endocrinol Metab 1990; 71: 210-215.
76 Autoimmune thyroid disease and Yersnia enterocolitica
18. Wiersinga WM, Prummel MF, Strieder TGA. Environmental factors in healthy women at risk for autoimmune thyroid disease. In: Peter F, Wiersinga W, Hostalek U, eds. The thyroid and environment. Stuttgart: Schatbauer, 2000: 179-184. 19. Yoshida H, Amino N, Yagawa M, et al. Association of serum antithyroid antibodies with lymphocytic infiltration of the thyroid gland: studies of seventy autopsied cases. J Clin Endocrinol Metab 1978; 46: 859-862. 20. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination survey (NHANES III). J Clin Endocrinol Metab 2002; 87: 489- 499. 21. Hoogkamp-Korstanje JAA, Koning J de, Heeseman J, et al. Influence of antibiotics on IgA and IgG response and persistence of Yersinia enterocolitica in patients with Yersinia-associated spondylarthropathy. Infection 1992; 20: 53-57. 22. Curfs JHAJ, Meis JFGM, Lee HAL van der, et al. Persistent Yersinia enterocolitica in three rat strains. Microbiol Pathol 1995; 19: 57-63. 23. Toivanen A, Granfors K, Lahesmaa-Rantala R, et al. Pathogenesis of Yersinia- triggered reactive arthitis: immunological, microbiological and clinical aspects. Immunol Rev 1985; 86: 47-70. 24. Heesemann J, Gaeda K, Autenrieth IB. Experimental Yersinia enterocolitica infection in rodents: a model for human yersioniosis. APMIS 1993; 101: 417-429. 25. Wills-Karp M, Santeliz J, Karp CL. The germless theory of allergic disease: revisiting the hygiene hypothesis. Nature Rev Immunology 2001; 1: 69-75. 26. Yazdanbakhsh M, Kremsner PG, Ree R van. Allergy, parasites and the hygiene hypothesis. Science 2002; 296: 490-494.
77
Stress is not associated with thyroid peroxidase autoantibodies in euthyroid 5| women
Brain, Bahavior and Immunity 2005, 19: 203-206
Thea G.A. Strieder Mark F. Prummel Jan G.P. Tijssen Jos F. Brosschot Wilmar M. Wiersinga
Stress and TPO autoantibodies
Chapter 5 Stress is not associated with thyroid peroxidase autoantibodies in euthyroid women
Abstract
Objective: Multiple genes and environmental factors play a role in the etiology of Autoimmune Thyroid Disease (AITD). In Graves’ hyperthyroidism, stress is such an environmental factor, but whether it plays a role in Hashimoto’s hypothyroidism is unknown. We used validated questionnaires to evaluate an association between TPO antibodies, an early marker for AITD, and self-reported stress. Subjects and methods: Recently Experienced Stressful Life Events, Daily Hassles and mood (tendency to report positive and negative affects) were assessed in 759 euthyroid subjects. Results: TPO antibodies were found in 183/759 (24%) of subjects. The TPO-Ab positive subjects were older (39.7 ±12 vs. 34.2 ±12 years; p<0.001) than the TPO-Ab negative subjects, but the number of daily hassles (24 ±14 vs. 25 ±14; p= 0.24), the number of stressful life events (10 ±6 vs. 11 ±6; p=0.09) and the scores on the affect scales (22.1 ±7.4 vs. 22.2 ±7.3; p=0.89 for negative affect and 38.2 ±5.1 vs. 38.3 ±5.3; p=0.91 for possitive affect) were similar in TPO-Ab positive and TPO-Ab negative subjects. Conclusions: We found no association between recently experienced stressful life events, daily hassles or mood and the presence of TPO antibodies in euthyroid subjects.
81 Chapter 5
Introduction
The etiology of autoimmune thyroid disease (AITD), encompassing Graves' hyperthyroidism and Hashimoto’s hypothyroidism is multifactorial. It has been estimated that 79% of the liability to develop Graves’ disease can be attributed to genes (Brix et al. , 2001); therefore other risk factors must play a role (Weetman & McGregor, 1994). Among environmental factors, stress has been implicated as a risk factor for the development of Graves’ disease (Winsa at al ., 1991; Dayan, 2001). Stress has a profound effect on the immune system (reviewed by Elenkov and Chrousos, 2002) and we have reported previously that self-reported life stress influences the number of circulating T lymphocytes (Brosschot et al ., 1994). We therefore wondered whether stress might also be associated with Hashimoto´s thyroiditis. The earliest event in Hashimoto´s thyroiditis is the occurrence of TPO antibodies and we therefore compared stress/levels between 183 women with TPO antibodies and 576 without.
Subjects and methods
The study cohort consisted of the 759 female subjects (age18-65 years at study entrance) from the Amsterdam AITD cohort who were biochemically euthyroid at study entrance (Strieder et al., 2003). All subjects were in self-proclaimed good health, without a history of thyroid disease. They were asked to complete three questionnaires. The Dutch questionnaire on Recently Experienced Stressful Life Events (Brosschot et al. , 1994; van de Willige et al. , 1985) counts the total number of major life events experienced in the past 12 months (checklist of 60 possible events). The respondent scores separately the amount of pleasantness and unpleasantness associated with each experienced life event, rated on a scale from zero (meaning no (un) pleasantness at all) to four (a huge amount of (un) pleasantness). From this scale, the total amount of pleasantness and unpleasantness is calculated (maximal score 240 for each). When the amount of pleasantness exceeds the amount of unpleasantness the event is categorized as being pleasant and vice versa, yielding the total number of (un) pleasant events (maximum 60). The Dutch Everyday Problem Checklist, a validated version of the Daily Hassles Scale (Kanner et al. , 1981; Vingerhoets et al. , 1989, 1996), consists of 114 items concerning daily hassles experienced in the last 2 months. It also measures the intensity of each hassle on a scale from zero to three, yielding the number of hassles experienced and the total intensity of these hassles (maximum 342).
82 Stress and TPO autoantibodies
The Positive and Negative Affect Schedule (PANAS; Watson et al. , 1988) measures the current mood, in terms of positive and negative affect. It consists of 22 mood states (11 positive, 11 negative) and the respondent is asked to report whether she is affected by each of these states on a scale from 1 (not at all) to 5 (a lot). This yields the tendency to report positive and negative affect states both on a scale from 11 to 55. Information concerning the use of psychotropic medication was obtained via a medical questionnaire. In addition, educational level (on a 4-point scale) and employment (having a paid job) status was also obtained. Blood samples were drawn and coded to ensure confidentiality and serum was stored at -20 °C until assay. The concentration of antibodies against TPO and thyroglobulin (Tg) was measured using a chemiluminescence immunoassay (LUMI-test, Brahms, Berlin, Germany), and a value of ≥ 100 kU/L was defined as positive. Concentration of free thyroxine was assayed by time-resolved fluoroimmunoassay (fT4, Delfia, Turku, Finland) and concentration of thyrotropin by immunoradiometric assay (TSH, Delfia). For this study, euthyroidism was defined as a TSH value between 0.40 and 5.70 mU/L (Strieder et al., 2003). The study was approved by the local Medical Ethics committee and all subjects gave their written informed consent. Statistical analysis was done using the SPSS 10.0 package. Mean values were compared between subjects with and those without TPO antibodies using two-sided, unpaired Student´s t test, median values using Mann-Whitney U test; rates were compared using χ² (or if appropriate Fishers’ exact) tests. Because age is a known risk factor for a positive TPO antibody status (Strieder et al. , 2003) comparison between groups was adjusted for the age difference using linear regression analysis.
Results
Subjects with TPO antibodies as compared to those without were older and had higher TSH levels, whereas free T4 values were similar in both groups as reported previously (Strieder et al ., 2003). There were no differences in educational level, employment status, or the use of psychotropic drugs (Table 1). As for the stress variables (Table 2), we observed no differences in the various stress measurements between subjects with and without TPO antibodies. Women with TPO antibodies reported a lower number of pleasant events and less total pleasantness than females without TPO antibodies, but this was fully accounted for by the age difference. We also did not find any relation between stress and the level or presence
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Table 1. Characteristics of 759 female euthyroid relatives of AITD patients
TPO-Ab pos TPO-Ab neg p Value
N 183 576
Age (mean+SD) in years 39.7 ± 12 34.2 ± 12 <0.001
TSH (median+range) in mU/l 2.3 (0.51-5.70) 1.6 (0.42-5.70) <0.001
FreeT4 (median+range) in pmol/l 12.5 (7.6-42.7) 12.9 (7.2-21.5) 0.16
Highest obtained level of education
- elementary school (low level of education) 6 (3%) 20 (3%) 0.90
- high school (medium level of education) 55 (30%) 139 (24%) 0.11
- high school (high level of education) 65 (36%) 246 (43%) 0.09
- university (very high level of education) 56 (31%) 170 (30%) 0.78
Employed 123 (67%) 404 (70%) 0.45
On psychotropic drugs 4 (2.2%) 12 (2.1%) 0.9
On psychotropic drugs in the previous year 9 (5%) 20 (3.5%) 0.4 p-Value for age was obtained by Students’ t test, p-values for TSH and freeT4 were obtained by Mann-Whitney U test, p-values for the other variables were obtained by χ² test. TPO-Ab positive: TPOAb > 100kU/l. of Tg antibodies (data not shown). There were no other differences among both groups in the three questionnaires.
Discussion
The rationale for our study was the observation that patients with Graves´ disease experienced more negative life events than controls in several case-control studies (Kung, 1995; Matos- Santos et al ., 2001; Winsa et al ., 1991), and that stress has a profound effect on the immune system (Brosschot et al ., 1994; Elenkov and Chrousos, 2002; Sternberg, 2001). We hypothesized that stress may also be associated with the presence of TPO antibodies, an early marker for Hashimoto´s thyroiditis (Prummel and Wiersinga, 2002). We used two different and well-validated questionnaires and found no relation between and antibody status in 759 euthyroid females. One source of bias could have been the Affect State because mood may alter the recall of stressful events. We therefore also assessed the Affect State using the PANAS questionnaire and found no differences in mood between the two groups, also not after correction for age. Age, however, was a definite confounder. The TPO positive women were 5 years older than
84 Stress and TPO autoantibodies
Table 2. Experienced stress in TPO-Ab positive and TPO-Ab negative euthyroid subjects (values as mean with SD between parentheses)
TPO-Ab pos TPO-Ab neg p Value
N 183 576
Recent Life Events Observed Corrected* Observed Observed Corrected**
- total number of life events 10.3 (6.1) 11.2 11.2 (6.2) 0.09 0.97
- number of unpleasant events 4.6 (3.5) 4.9 4.7 (3.4) 0.68 0.68
- number of pleasant events 4.5 (3.5) 5.1 5.2 (3.5) 0.02 0.66
- amount of total unpleasantness 15.1 (11.0) 16.2 16.7 (12.4) 0.13 0.68
- amount of total pleasantness 15.9 (11.4) 16.2 18.9 (12.6) 0.01 0.38
Daily Hassles
- total number of daily hassles 23.8 (13.6) 25.0 25.2 (14.1) 0.24 0.83
- intensity per hassle 1.3 (0.4) 1.3 1.3 (0.4) 0.52 0.64
- total intensity of all hassles 32.3 (22.9) 34.2 35.4 (25.5) 0.15 0.57
Positive and Negative Affect Schedule Scale - tendency to report negative 22.1 (7.4) 22.1 22.2 (7.3) 0.89 0.88 feelings - tendency to report positive 38.2 (5.1) 38.3 38.3 (5.3) 0.91 0.91 feelings
* Corrected for age by linear regression analysis. ** p Value after correction for age. the TPO negative subjects and van de Willige et al. (1985) have shown that differences in age are associated with the experienced amount and type of the studied indicators of self-reported stress (the older one becomes the less pleasant and less unpleasant events one experiences). This is in agreement with our findings that the older TPO positive women reported less life events and daily hassles than the younger TPO negative women (Table 2). Another possible confounding factor is the unknown duration of the presence of TPO antibodies, whereas we measured stress experienced in the preceding 2-12 months. Although even if TPO antibodies were developed a longer time ago, one may have expected a relation between stress and the concentration of TPO antibodies which we also could not establish. Nevertheless, to completely rule out a role of stress a prospective study in TPO antibody negative women developing TPO antibodies is needed.
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Acknowledgment The study was financially supported by a grant from the Dutch Medical Research (NWO 950- 10-626).
86 Stress and TPO autoantibodies
References
1. Bricks TH, Civic KO, Christensen K & Heeds L, 2001. Evidence for a major role of heredity in Graves' disease: a population-based study of two Danish twin cohorts. Journal of Clinical Endocrinology and Metabolism. 86, 930-934. 2. Brosschot JF, Benschop RJ, Godaert GL, Olff M, De Smet M, Heijnen CJ, Ballieux RE, 1994. Influence of life stress on immunological reactivity to mild psychological stress. Psychosom Medicine. May-Jun; 56(3):216-24. 3. Dayan CM, 2001. Stressful life events and Graves' disease revisited. Clinical Endocrinology (Oxf). Jul; 55(1):13-4. 4. Kanner AD, Coyne JC, Schaefer C, Lazarus RS, 1981. Comparison of two modes of stress measurement: daily hassles and uplifts versus major life events. Journal of Behavioral Medicine. Mar; 4(1):1-39. 5. Keung, AWC, 1995. Life events, daily stresses and coping in patients with Graves' disease. Clinical Endocrinology (Oxf.), 42; 303-308. 6. Matos-Santos A, Nobre EL, Costa JG, Nogueira PJ, Macedo A, Galvao-Teles A, de Castro JJ, 2001. Relationship between the number and impact of stressful life events and the onset of Graves' disease and toxic nodular goitre. Clinical Endocrinology (Oxf). Jul; 55(1):15-9. 7. Prummel, M.F., Wiersinga, W.M., 2002. Autoimmune Thyroid Diseases. In: Gill R.G. (Ed.), Immunologically Mediated Endocrine Diseases. Modern Endocrinology, Lippincott Williams & Wilkins, Philadelphia, pp. 373-396. 8. Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM, 2003. Risk Factors for and prevalence of thyroid disorders in healthy female relatives of patients with autoimmune thyroid diseases (AITD): The Amsterdam AITD cohort. Clinical Endocrinology (Oxf). Sep; 59(3):396-401. 9. Vingerhoets AJJM, Jeninga AJ and Menges LJ, 1989. Het meten van chronische en alledaagse stressoren: II. Eerste onderzoekservaringen met de Alledaagse Problemenlijst (APL). [The measurement of daily hassles and chronic stressors: The development of the Everyday Problem Checklist (EPCL,Dutch: APL)]. Gedrag Gezondh 17, pp. 10–17. 10. Vingerhoets AJJM, van Tilburg MAL, 1994. Alledaagse Problemenlijst (APL). Swets & Zeitlinger, Test publishers, Lisse. 11. Watson D, Clark LA, Tellegen A, 1988. Development and validation of brief measures of positive and negative affect: the PANAS scales. Journal of Pers Soc Psychology. Jun; 54(6):1063-70. 12. Weetman AP & McGregor AM, 1994. Autoimmune thyroid disease: Further developments in our understanding. Endocrine Reviews. 15, 788-830. 13. van de Willige G, Schreurs P, Telligen B, Zwart F, 1985. Het meten van ‘life events’: Vragenlijst Recent Meegemaakte Gebeurtenissen. Ned. Tijdschrift voor de Psychologie. 40; 1-19. 14. Winsa B, Adami HO, Bergstrom R, Gamstedt A, Dahlberg PA, Adamson U, Jansson R, Karlsson A, 1991. Stressful life events and Graves' disease. Lancet. Dec 14.
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A reduced Il2R (CD25) expression level in first and second-degree 6| female relatives of AITD patients. A sign of a poor capability to preserve tolerance?
Autoimmunity 2006; 39: 93-98
Thea G.A. Strieder Hemmo A. Drexhage Wai K Lam-Tse Mark F. prummel Jan G.P. Tijssen Wilmar M. Wiersinga
Low IL-2R expression in relatives of thyroid autoimmune patients
Chapter 6 A reduced Il2R (CD25) expression level in first and second-degree female relatives of AITD patients. A sign of a poor capability to preserve tolerance?
Abstract
There is room for immune markers other than TPO-Abs to identify an increased risk to develop autoimmune thyroid disease (AITD). Our aim was to test the hypothesis that activation of CD4 + T cells is such a marker in relatives of AITD patients, who have an increased risk to develop AITD. We established a controlled study on 20 TPO-Ab positive and 20 TPO-Ab negative euthyroid female relatives. All these cases had at least one 1 st or 2 nd degree relative with a documented autoimmune hyper- or hypothyroidism in whom we studied the percentages of circulating subsets of activated (MHC class-II, CD25 (Il-2R), CD71, CD69+) CD4+ T cells and the level of the soluble (s)-IL2R in serum. We found that euthyroid female relatives did not show an activation of their T cell system, but a reduced expression of CD25 on CD4+ T cells. The level of the shed IL-2R in serum was also lower in comparison with levels found in healthy control females. A reduced T cell activity was found in both TPO-Ab positive and negative relatives. In conclusion , female relatives with at least one 1 st or 2 nd degree relative with an AITD show signs of a reduced expansion capability of their T cell pool. It is hypothesized that this reduced expansion capability may affect T cell tolerance mechanisms more than T effector mechanisms.
91 Chapter 6
Introduction
The etiology of autoimmune thyroid disease (AITD), encompassing Graves’ hyperthyroidism and Hashimoto’s thyroiditis, is multifactorial [1]. Genetics play an important role in the development of AITD, illustrated by the concordance rate for Graves’ disease and Hashimoto’s hypothyroidism in monozygotic twins, which is much higher than in dizygotic twins [2,3]. In addition, many patients with AITD have family members affected by this disorder. However, it has been estimated that at best 79% of the liability to develop AITD can be attributed to genetics [3] and, therefore, environmental and hormonal risk factors must also be involved [4]. A follow-up cohort study (the Amsterdam AITD Cohort)was initiated todetermine risk factors involved in new cases of AITD. To increase the likelihood of diagnosing new patients during a 5-year follow-up, subjects were included who had at least one relative with documented AITD and who were in self-proclaimed good health. In a previously reported base-line study, evidence for autoimmune thyroiditiswas found in a fairly large proportion of euthyroid relatives (i.e. relatives with a serumTSH within the normal range): 183/759 (24%) of such relatives had autoantibodies to thyroid peroxidase (TPO). This value lies between the prevalence value in the general population (10–15%) and prevalence rates of 48% [5] and 43%[6] previously reported in first-degree relatives of AITD patients. It was also reported that the TPOantibody titre in the euthyroid subjects of our study cohort correlated positively with TSH levels (r = 0.386; P < 0.001) [7]. In addition 86% of 29 hypothyroid relatives detected in the cohort (i.e. relatives with a TSH above the normal range) had detectable levels of TPO antibodies. Taken together, the findings of the Amsterdam AITD Cohort are in line with the notion that subjects with a family history of AITD are at increased risk for AITD and that TPO-Abs are reasonably good markers to predict such development [8–10]. Interestingly and unexplainably, smoking and estrogen use were negatively correlated with the presence of TPO antibodies in the Amsterdam AITD Cohort [7]. Although TPO-Abs are reasonably good markers for the development of AITD, there is room for immune markers other than the abs to establish the increased chance to develop AITD. Such immune markers are likely to be found in the T cell system. Thyroid antigen specific CD4+ T cells and CD8+ T cells are required for the thyroid autoimmune reaction to develop [11]. Autoreactive CD8+ T cells are capable of directly killing thyrocytes, while autoreactive CD4+ T cells have various helper functions. Autoreactive CD4+ Thelper2 cells (producing IL-4 and IL-5) help autoreactive B cells to switch to IgG- autoantibody producing plasma cells, whilst CD4+ Thelper1 cells (producing IFN-γ) activate
92 Low IL-2R expression in relatives of thyroid autoimmune patients macrophages to destroy thyrocytes. Apart from these immune stimulating and effector functions, T cells are able to down-regulate immune responses and at present various subsets of T cells are recognized as being capable of exerting such immune regulatory function [12,13]. For this report a controlled study was initiated in which the apportioning of the various circulating T cell subsets was studied in 20 TPO-Ab positive and 20 TPO-Ab negative euthyroid female relatives, randomly selected out of the cohort of the 803 characterized subjects rom the Amsterdam AITD Cohort [7] at baseline. Subjects were stratified by age, current smoking habits and current estrogen medication. Apart from measuring the circulating subsets of T helper cells (CD3+ CD4+ ), the T cytotoxic cells (CD3+CD8+ ) and the T helper memory cells (CD4+CD45RO+), the studies concentrated on the determination of T cell activation markers on the CD4+ T cells in the circulation, i.e. of MHC class-II, CD25 (the IL2 receptor), CD71 (the transferring receptor) and CD69, since a previous study found that a raised number of circulating activated T cells, i.e. cells expressing MHC class-II, is indicative for a progression of TPO-Ab positive pregnant women to post-partum thyroiditis [14]. The level of the soluble (s)-IL2R in the serum of these relatives was also tested since this molecule represents the shed CD25 from T cells and can easily be determined.
Subjects and Methods
Subjects
Subjects in the present study are a. Forty euthyroid female subjects selected from the 803 subjects of the Amsterdam AITD Cohort (7). The subjects in the Amsterdam AITD cohort have at least one 1 st or 2nd degree relative with a documented autoimmune hyper- or hypothyroidism and are in self-proclaimed good health, without a history of thyroid disease, and between 18 and 65 years of age. Current pregnancy is an exclusion criterion. Information on smoking habits (current and past), use of oral contraceptives or other estrogens (current and past) is available, because all subjects have been asked to fill in questionnaires at the time blood samples (to obtain serum and circulating mononuclear leukocytes to be stored) were collected. Current smoking is defined as smoking now, or having stopped smoking within one year prior to visiting our clinic. Current estrogen usage is defined as presently on exogenous estrogen medication.
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The actual selection of the 40 subjects from the cohort was carried out as follows: initially 10 euthyroid TPO-Ab positive relatives were randomly selected from the cohort. For each selected subject all possible euthyroid TPO Ab negative counterparts matched for age +/- 5 years, smoking status and current estrogen use were searched. If more than one possible match existed a single match was randomly selected. This procedure was repeated, but starting by randomly selecting 10 euthyroid TPO-Ab negative subjects and matching TPO-Ab positives to these subjects. Of two of the selected subjects, stored blood cells later appeared not to be viable and, therefore, only the results of 38 subjects could be analyzed and will be provided here. The mean age of the selected subjects was 36,8 years (range 20-58 years). Twenty of the 38 studied relatives were smokers, 20 of the 38 used oral contraceptives. b. Female controls in self-proclaimed good health, excluding afflictions such as infections, asthma and endocrine disorders, consisted of Dutch interns, hospital and laboratory staff and healthy female twins selected as controls for a non-related twin study. The family history needed to be negative for thyroid disorders and subjects positive for TPO-Abs were excluded from the study. Serum and mononuclear leukocytes needed to be collected in the same time period (1997-2003) as those of the relatives of the AITD patients using the same density isolation and storage protocol. Stratification for smoking habits and estrogen usagehad not takenplace in the healthy controls. From 65 of such healthy female subjects (ages 22–56, mean age 36.8 years) serum was available, of these only 5 were smokers and 22 used estrogens. Stored mononuclear leukocytes were available from 9 other healthy females (ages 22–62 years,mean age 38.6 years), of these 2 were smokers and 3 used estrogens.
The local Medical Ethics committees of our Institutions had approved the study. All subjects gave written informed consent.
Blood samples Serum samples were stored at -20 0C until determination of the study parameters. Mononuclear cells were obtained from heparinized blood by using Ficoll (Pharmacia, Uppsala, Sweden; density 1.077 g/ml) density gradient centrifugation. The isolated cells were frozen with 10% dimethylsulfoxide (Sigma-Aldrich Chemie, Steinheim, Germany) in RPMI 1640 medium with 25mM HEPES and L-glutamine (BioWhittaker Europe, Verviers, Belgium) containing ultraglutamine (UG) (2mM, BioWhittaker), 100U/ml penicillin
94 Low IL-2R expression in relatives of thyroid autoimmune patients
(BioWhittaker), 100µg/ml streptomycin (BioWhittaker) and 10% heat inactivated fetal calf serum (BioWhittaker) and stored at –70 oC until analysis. TSH, fT4, TPO Ab In all subjects free Thyroxine (fT4; time-resolved fluoroimmunoassay, Delfia, Turku, Finland) and thyrotropin (TSH; Delfia) levels were measured, as well as antibody titers against TPO using a chemiluminescence immunoassay (LUMI-test, Brahms, Berlin, Germany). For this study, euthyroidism was defined as a TSH value of ≥0.40 mU/L and ≤5.70 mU/L, in combination with a fT4 value of ≥9.3 pmol/L and ≤20.1 pmol/L. TPO antibody levels of ≥100 kU/L were considered to be positive.
Soluble IL-2R T cell activation was determined by measuring serum soluble IL-2 receptor (sIL-2R). The soluble IL-2R levels were measured by automatic Immulite chemiluminescent enzyme immunimetric assay (DPC, Los Angeles, CA, USA) according to the manufacturer’s instruction. Enzyme immunoasay (ELISA) was used to determine the serum levels of soluble (s) ICAM-1 (Bio-source ELISA KH5412 high sensitive, Camarillo, CA, USA).
Flowcytometry Marker staining and analysis were done at the same time in the same institution, i.e. the Erasmus MC, on all frozen mononuclear cell samples using the same protocol and FACS analysis settings. The following monoclonal antibodies (mAbs) were used as control mAbs for flowcytometry: anti-IgG1 Fluorescein isothiocyanate (FITC), anti-IgG1 Phycoerythrin (PE), anti-IgG1 Peridin chlorophyl protein (Percp) (all three 1:10, Becton Dickinson (BD), San Jose, CA, USA), anti-CD3 FITC (1:20, BD), anti-CD8 PE (1:20, BD), anti-CD4 Percp (1:10, BD), anti- CD25 FITC (1:10, BD), anti-HLA-DR PE (1:200, BD), anti-CD28 FITC (1:10, Immunotech, Marseille, France), anti-CD45RO PE (1:5, Dako Cytomation, Glostrup, Denmark), anti-CD71 FITC (1:10, BD), and anti-CD69 PE (1:100, Serotec, Oxford, England). Thawed mononuclear cells were incubated for double staining with 25 µl mAb / 10 5 cells (of a FITC-labeled antibody (Ab), of a PE-labeled Ab, and/or 25 _L of a PerCP-labeled Ab) for 15 minutes and washed twice with phosphate buffered saline (PBS) pH 7,4 (Biowittaker) with 0,1% bovine serum albumin (BSA) (Bayer, Kankakee, IL, USA).
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Routinely 10,000 cells in the lymphocyte or monotypic gate were measured immediately following cell staining using a FACScan flowcytometer and analyzed using CellQuestPro (BD). The background staining was determined by the staining of the cells with the isotype antibody controls alone and the threshold for background positivity staining was arbitrarily set between 1 and 2% of cells positive for the isotype control. Using this fixed treshold the percentage of lymphocytes positive for the specific anti-CD antibody was determined and the arbitrarily fixed background staining (obtained with the isotype control of 1-2%) was subtracted from the values found with the specific CD staining. The data obtained was mainly expressed as mean ± SD percentages of positive lymphocytes found in the individual patients of the various patient and control group.
Statistics Because of the abnormal distribution of the variables, statistical analyses were performed using the nonparametric two independent-sample-tests; the Mann Whitney U test. With use of a standard statistical software package (SPSS 11.0) a two-tailed significance value was determined between cases and controls. A p-value of <0.05 was considered as statistically significant.
Results
Soluble IL2-R levels in serum As expected and as a result of the randomization and stratification procedure, healthy first- and seconddegree female relatives of AITD patients with TPOAb were no different from those without TPO-Ab regarding age, current smoking behavior and current estrogen medication. As described previously, the TPO Ab positive subjects had slightly, but significantly higher TSH levels (Table I and [7]). Figure 1 shows, that the healthy relatives had statistically significantly reduced serum levels of sIL- 2R as compared to that of the healthy female controls. This reduced sIL-2R level was present in both TPOAb positive and TPO-Ab negative relatives. Interestingly there was a statistically significant difference between smoking and non- smoking relatives. While the non- smoking relatives had significantly reduced serum sIL-2R levels as compared to our healthy controls, smoking relatives had near normal serum levels of sIL-2R (Figure 1). It is of note that a large proportion of our healthy control females were non-smokers (55 out of 62, i.e. 89%). Probably due to the low number of smokers in the
96 Low IL-2R expression in relatives of thyroid autoimmune patients
Table I. Age, thyroid status and the outcomes of various lymphocyte phenotypic studies in 9 healthy female controls and 38 female euthyroid relatives of AITD patients grouped for TPO antibody status (n ¼ 19 in each group)
a In years, medians ± standard deviations are given. b Means and ranges are given. c There exists a statistical significant difference between this value and the 1.6 value of the TPO-Ab negative relatives (see text). d Percentages of lymphocytes positive for the given markers, means and standard deviations are given. healthy control group it was not possible to detect a statistical significant difference between the smoking and nonsmoking healthy controls: smoking and non-smoking healthy controls
Figure 1. The serum soluble IL2-receptor (R) levels (pg/ml) in healthy female controls (n = 65), and 1st and 2nd degree female relatives of AITD patients (relatives, in total n = 38). Female relatives are subdivided in TPO-Ab positive (n = 19) and TPO-Ab negative (n = 19) female relatives, smoking (n = 18) and nonsmoking (n = 20) female relatives and female relatives using (n = 20) and not using (n = 18) oral estrogens. See for further explanations text. “a” represents a p-value of <0.05 versus healthy controls. “b” represents a p-value of <0.05 versus smoking relatives.
97 Chapter 6 showed sIL-2R levels of 434 (mean) ± 68 (standard deviation) and 461 ± 190 pg/ml respectively (data not shown in Figure 1). Other studies generally show that smoking raises the level of serum sIL-2R in healthy individuals [15–17]. Estrogen usage had no effects on the serum level of sIL-2R levels and both estrogen using female relatives and non-users of estrogen showed reduced s-IL-2R levels and there was no statistically significant difference in sIL-2R serum levels between these two groups of relatives (Figure 1). There was also no such difference between estrogen using female healthy controls and non-estrogen using healthy controls: levels of 461 ± 190 pg/ml (n = 22) and 434 ± 68 pg/ml (n = 43) were found in these healthy control groups respectively (data not shown in Figure 1).
Figure 2. The percentage of circulating lymphocytes expressing CD4CD25 in female healthy controls (n = 9) and in 1st and 2 nd degree female relatives of AITD patients (relatives, in total n = 38). Female relatives are subdivided in TPO-Ab positive (n = 19) and TPO-Ab negative (n = 19) female relatives, smoking (n = 18) and non-smoking (n = 20) female relatives and female relatives using (n = 20) and not using (n = 18) oral estrogens. See for further explanations text. “a” represents a p value of ,0.05 versus healthy controls, “b” represents a p value of ,0.05 versus relatives not using oral estrogens
98 Low IL-2R expression in relatives of thyroid autoimmune patients
Sub-sets of circulating T cells, in particular that of CD4 + CD25 + T cells The percentage of CD3+CD4+ (helper) T cells was reduced in the circulation of the female relatives of AITD patients (Table I, mean 47.4 ± 12.6%) as compared to the percentage found in the healthy females (i.e. mean 56.7 ± 6.7%), but this difference was not considered to be statistically significant ( p = 0.23). Within the subset of CD4+ T helper cells the percentage of T helper cells expressing the IL2-R (CD4+CD25+ cells) was, however, significantly reduced in the female relatives of AITD patients (Figure 2). Percentages CD4+CD25+ cells were equally low in the TPO- Ab positive and TPOAb negative female relatives. In addition smoking and non-smoking relatives both showed reduced percentages of CD4+CD25+ cells (Figure 2) and there was no statistically significant difference in percentages of CD4+CD25+ cells in the smoking and non-smoking female relatives. Therewas such a difference, however, in the smoking and non-smoking healthy control subjects, who showed values of 17.4 ± 1,2% (n = 2) and 11.1 ± 3,5% (n = 7) respectively ( p < 0.01, data not shown in Figure 2) supporting the view that smoking activates the CD25 expression on T cells (see also previous remarks on sIL-2R levels in smoking healthy subjects and [15–17]).The CD4+CD25+ T cell subset was significantly lower in female relatives using estrogens versus that of female relatives not taking estrogens, while both groups werelower as compared to thevalues found in the healthy controls (Figure 2). In regard to comparison with the healthy controls it must be noted that our female control group took oral estrogens in one third of all cases, but in this group we were unable to detect a statistically significantly difference between the estrogen non-using female healthy controls (11.4 ± 3.7%, n = 6) and the estrogen-using healthy controls (14.7 ± 4.8%, n = 3). Subsets of CD3+CD8+ (cytotoxic) T cells, CD4+CD4+5RO (memory) T cells, CD4+ MHCclass- II + , CD4+CD69+ and CD4+CD71+ T cells were not different between female relatives and healthy controls (Table I).
Discussion
This study shows that euthyroid females with an heightened risk of developing thyroid autoimmunity, i.e. females with at least one 1 st or 2 nd degree relative with a documented autoimmune hyper- or hypothyroidism, exhibited a reduced serum level of sIL-2R and reduced percentages of circulating CD4+ T cells expressing the sIL-2R (CD25) as compared to healthy control females. It is important to note that these signs of reduced T cell activity were evident in all female relatives, irrespective of their TPO-Ab status.
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The data must thus be viewed as contradictory to a concept that the T cell system is activated in high-risk female relatives of AITD patients. The data support a notion of a low expansion activity of the T cell system in these relatives, since IL-2 is particularly involved in the proliferation of T cells and the expansion of the T cell pool . Hence, the data reported here can be taken to support the following point of view: the lower the T cell system is triggered to expand via IL-2/IL-2R interactions, the higher the risk of developing thyroid autoimmunity. Can such a view be supported with literature data? The textbook view of the IL-2/IL-2R interaction still remains that it is the most critical cytokine for promoting the clonal expansion of T cells and hence a stimulator of effector T cell functions. This percieved importance of IL-2/IL-2R interactions has made this cytokine and its receptor a prime target for interventions to suppress unwanted immune responses such as those occurring in autoimmunity. Therefore, an attempt has been made to knock-out genes for IL-2 or for its receptorin autoimmune-prone mice in order to cure the mice from disease. (18, 19).An unexpected and counterintuitive result occured:the knock-out mice were not cured from autoimmunity, but in all cases had an aggravation of their autoimmunity and died of massive lympho-proliferative syndrome. These experiments introduced the new and current concept of experts that IL-2/IL-2R interactions are essential earlier for autoimmune responses to end than to be initiated (19). To explain this concept, it was at first assumed that the IL-2/IL-2R interaction was a key element for activation-induced T cell death (AITCD) of autoreactive T cells. In such a view, a failure of an optimal IL-2/IL-2R interaction (as is likely the case in the relatives due to the low IL-2R expression) hampers an appropriate check of (in this case thyroid-) autoreactive T cells escaped from thymic negative selection in the periphery (19). However, the IL2/IL-2R system might also be linked to another mechanism of tolerance induction, i.e. that induced via CD4 + CD25 + T regulator (reg) cells (13). The actual T cells with a regulatory function form a sub- group within the CD4 +CD25 + T cell population, develop in the thymus, are “anergic” to TCR ligation, but capable of suppressing the activation of (bystander) CD4 + and CD8 + cells. Ligation of surface molecules such as CD152 (CLTA-4) and GITR, as well as secretion of anti-inflammatory cytokines, such as TGF-β, IL-4 and IL-10, have all been implicated in the regulation or mediation of the suppressive activity of CD4 +CD25 + Treg cells (13). Is it possible that the low percentages of CD4 +CD25 + T cells found in the female relatives are a reflection of low numbers of this specific subset of thymus-derived T reg cells? Clearly more experiments are needed in the relatives focussing on the actual functional immune suppressive
100 Low IL-2R expression in relatives of thyroid autoimmune patients capability of the cells and on additional marker analyses (CTLA-4, GITR and Fox-P3) to reliably identify the suppressor character of the T cells. A weak point of this study is that subgroups of smoking and estrogen-taking relatives and healthy controls were rather small and, (in the case of the healthy individuals), not evenly distributed. Nevertheless some conclusions may be drawn. Interestingly, relatives who smoked had near normal sIL-2R serum levels and slightly raised percentages of circulating CD4+CD25 + T cells (such raised percentages were also present in the smoking healthy controls). It is well known that smoking activates the IL-2/IL-2R system [15–17]. Hence smoking may have corrected the reduced T cell functions in the relatives of AITD patients, including the defective T cell tolerance mechanisms. The smoking subgroup of relatives is indeed special, because it was previously reported that smoking is associated with a reduced risk in these relatives to be positive for TPO-Abs [7]. With regard to estrogen usage, female relatives taking estrogens showed particularly reduced percentages of circulating CD4 +CD25 + T cells, which was not reflected by an extra reduced serum sIL-2R level in this estrogen taking women. This observation is in accordance with the viewthat estrogens are immune suppressive in autoimmune diseases [20] and that they suppress IL-2 and the IL-2R [21]. However, effects of estrogen usage were not evident in our healthy controls.The observation also does not support the above expressed view of “the lower the T cell system is triggered to expand via IL-2/IL2R interactions, the higher the risk to develop thyroid autoimmunity”, since estrogen taking women have a lower chance of developing thyroid autoimmunity [7]. Clearly estrogen-sensitive mechanisms other than T cell tolerance mechanisms must be operative in preventing thyroid autoimmunity in these female relatives. In conclusion, the study shows that females with an heightened risk of developing thyroid autoimmunity, i.e. females with at least one 1 st or 2 nd degree relative with a documented autoimmune hyper- or hypothyroidism, exhibited various signs of a reduced T cell activation, in particular signs of a reduced expansion of the T cell pool.
Acknowledgements The excellent technical assistance of Harm de Wit is gratefully acknowledged. We are also grateful for the selection of autoimmune thyroid patients by Dr A. Berghout (Zuiderziekenhuis, Rotterdam). This work was supported by Dutch Medical Research NWO grants 950-10-626 and 903-40-193 and a grant of the Dutch Foundation for Diabetic Research 96.606.
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References
1. Tomer Y. Genetic dissection of familial autoimmune thyroid diseases using whole genome screening. Autoimmun Rev. 2002; 1:198-204. 2. Brix TH, Kyvik KO, Christensen K, Hegedus L. Evidence for a major role of heredity in Graves' disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab. 2001; 86:930-4. 3. Brix TH, Kyvik KO, Hegedus L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab. 2000; 85 :536-9. 4. Prummel MF, Strieder T, Wiersinga WM. The environment and autoimmune thyroid diseases. Eur J Endocrinol. 2004; 150 :605-18. 5. Phillips DI, McLachlan SM, Smith BR, Bradbury J, Beever K. Autoantibodies to thyroglobulin and thyroid peroxidase in blood spots measured with a sensitive, direct radioimmunoassay. Clin Chem. 1990; 36 :823-4. 6. Prentice LM, Phillips DI, Sarsero D, Beever K, McLachlan SM, Smith BR. Geographical distribution of subclinical autoimmune thyroid disease in Britain: a study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinol (Copenh). 1990; 123 :493-8. 7. Strieder TG, Prummel MF, Tijssen JG, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol (Oxf). 2003; 59 :396-401. 8. Kuijpens JL, Pop VJ, Vader HL, Drexhage HA, Wiersinga WM. Prediction of post partum thyroid dysfunction: can it be improved? Eur J Endocrinol. 1998; 139 :36-43. 9. Muller AF, Drexhage HA, Berghout A. Postpartum thyroiditis and autoimmune thyroiditis in women of childbearing age: recent insights and consequences for antenatal and postnatal care.Endocr Rev. 2001; 22 :605-30. 10. Premawardhana LD, Parkes AB, John R, Harris B, Lazarus JH. Thyroid peroxidase antibodies in early pregnancy: utility for prediction of postpartum thyroid dysfunction and implications for screening. Thyroid. 2004; 14 :610-5. 11. McLachlan SM, Rapoport B. Autoimmune hypothyroidism: T cells caught in the act. Nat Med. 2004; 10:895-6. 12. Jonuleit H, Schmitt E. The regulatory T cell family: distinct subsets and their interrelations. J Immunol. 2003; 171:6323-7. 13. Thompson C, Powrie F. Regulatory T cells. Curr Opin Pharmacol. 2004; 4:408-14. 14. Kuijpens JL, De Haan-Meulman M, Vader HL, Pop VJ, Wiersinga WM, Drexhage HA. Cell-mediated immunity and postpartum thyroid dysfunction: a possibility for the prediction of disease? J Clin Endocrinol Metab. 1998; 83:1959-66. 15. Tollerud DJ, Kurman CC, Nelson DL, Brown LM, Maloney EM, Blattner WA. Related racial variation in serum-soluble interleukin-2 receptor levels: a population-based study of healthy smokers and nonsmokers. Clin Immunol Immunopathol. 1994; 70:274-9. 16. Apibal S, Srisurapanon S, Kisukapan P, Worapongpaiboon S, Uneanong S, Kupatawintu P, Chitaganone S, Saenghirunwattana S, Rattanasrithong S. The effects of cigarette smoking on peripheral blood leukocytes and lymphocyte subpopulations: an urban population-based study in Thailand. J Med Assoc Thai 2000; 83:S109–S113. 17. Jiao L, GanD, Fang Y. Effects of smoking on interleukin-2 and its receptor system in human peripheral blood.Wei Sheng Yan Jui 1998; 27:385–388. 18. Ehrhardt RO, Ludviksson BR. When immunization leads to autoimmunity: chronic inflammation as a result of thymic and mucosal dysregulation in IL-2 knock-out mice. Int Rev Immunol. 1999;18:591-612.
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19. Malek TR. The main function of IL-2 is to promote the development of T regulatory cells. J Leukoc Biol. 2003; 74:961-5. 20. Jansson L, Holmdahl R. Estrogen-mediated immunosuppression in autoimmune diseases. Inflamm Res 1998;47:290–301. 21. McMurray RW, Ndebele K, Hardy KJ, Jenkins JK. 17-b-estradiol suppresses IL-2 and IL- 2 receptor. Cytokine 2001;14:324–333.
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Prediction of progression to Overt Hypothyroidism or Hyperthyroidism in
female relatives of patients with 7| AutoImmune Thyroid Disease using the Thyroid Events Amsterdam (THEA) Score
Archives of Internal Medicine 2008;168(15):1657-1663
Thea G.A. Strieder Jan G.P. Tijssen Björn E. Wenzel Erik Endert Wilmar M. Wiersinga
Prediction of progression using the THEA Score
Chapter 7 Prediction of Progression to Overt Hypothyroidism or Hyperthyroidism in Female Relatives of Patients With Autoimmune Thyroid Disease Using the Thyroid Events Amsterdam (THEA) Score
Summary
Background: Genetic and environmental factors are involved in the pathogenesis of autoimmune thyroid disease (AITD). Family members of patients with AITD are at increased risk for AITD, but not all will develop overt hypothyroidism or hyperthyroidism. Our goal was to develop a simple predictive score that has broad applicability and is easily calculated at presentation for progression to overt hypothyroidism or hyperthyroidism within 5 years in female relatives of patients with AITD. Methods: We conducted a prospective observational cohort study of 790 healthy first- or second-degree female relatives of patients with documented Graves or Hashimoto disease in the Netherlands. Baseline assessment included measurement of serum thyrotropin (TSH), free thyroxine (FT4), and thyroid peroxidase (TPO) antibody levels as well as evaluation for the presence and levels of Yersinia enterocolitica antibodies. We also gathered data on family background, smoking habits, use of estrogen medication, pregnancy, and exposure to high levels of iodine. In follow-up, thyroid function was investigated annually for 5 years. As main outcome measures, termed events, we looked for overt hypothyroidism (TSH levels >5.7 mIU/L andFT4 levels <0.72 ng/dL) or overt hyperthyroidism (TSH levels <0.4 mIU/L and FT4 levels >1.56 ng/dL). Results: The cumulative event rate was 7.5% over 5 years. The mean annual event rate was 1.5%. There were 38 hypothyroid and 13 hyperthyroid events. Independent risk factors for events were baseline findings for TSH and TPO antibodies in a level-dependent relationship (for TSH the risk already starts to increase at values >2.0 mIU/L) and family background (with the greatest risk attached to subjects having 2 relatives with Hashimoto disease). A numerical score, the Thyroid Events Amsterdam (THEA) score, was designed to predict events by weighting these 3 risk factors proportionately to their relative risks (maximum score, 21): low (0-7), medium (8-10), high (11- 15), and very high (16-21). These THEA scores were associated with observed event rates of 2.7%, 14.6%, 27.1%, and 76.9%, respectively.
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Conclusions: An accurate simple predictive score was developed to estimate the 5-year risk of overt hypothyroidism or hyperthyroidism in female relatives of patients with AITD. However, in view of the small number of observed events, independent validation of the THEA score is called for.
Introduction
Autoimmune thyroid disease (AITD) occurs more often in women than in men and frequently runs in families. Siblings of patients with AITD are clearly at risk, as evident from a sibling risk ratio (λs) ranging from 5.9 to higher than 10 ( λs represents the ratio of the risk for developing AITD in a sibling of a patient with AITD beyond the risk for AITD present in the general population). [1,2] Twin studies suggest that genetic factors account for about 70% of the risk of contracting AITD and that environmental factors contribute about 30% to the risk.[3-6] Subjects with family members who have AITD are obviously at increased risk for AITD. It is thus understandable that a frequently asked question by patients with AITD is “Will mydaughter ormysister also get this disease?” The physician is likely to answer that indeed the risk in family members —especially in women—is increased, but the physician will have difficulties in giving a more precise estimate of the risk. To obtain a quantitative risk estimate, we undertook a prospective observational study in a cohort of healthy first- or seconddegree female relatives of patients with AITD who were observed for 5 years. The baseline characteristics of this cohort have been reported previously.[7] We aimed to design a simple score to predict the risk for developing overt hypothyroidism or hyperthyroidism based on the results of thyroid function tests, family history, and exposure to some environmental insults at study entrance. METHODS Study design and participants
The study was approved by the institutional review board of the Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. The original cohort consisted of 803 healthy female subjects who had at least 1 first- or second-degree relative with documented autoimmune hyperthyroidism or hypothyroidism. All subjects were in self-proclaimed good health with no history of thyroid disease and were between 18 and 65 years old. The autoimmune nature of the thyroid disease in the subjects’ relatives was ascertained by letters from their treating physicians. Results of thyroid function tests revealed overt
108 Prediction of progression using the THEA Score hypothyroidism in 10 subjects and overt hyperthyroidism in 3 subjects, leaving 790 subjects to be included in the present study.[7] All subjects were seen at our institution. After informed consent was obtained, blood was drawn for thyroid function tests, thyroid autoantibody assays, and assays for antibodies against Yersinia enterocolitica outer membrane proteins (YOPs). The rationale for determining the presence and/or levels of YOP antibodies is that Y enterocolitica membranes have specific thyrotropin (TSH) binding sites. Infection with Y enterocolitica induces the production of antibodies against these sites that recognize and stimulate TSH receptors in human thyrocytes. All subjects completed questionnaires on smoking habits (current and past), use of oral contraceptives or other estrogens (current and past), number of pregnancies, and exposure to high levels of iodine in the previous year. Current smoking was defined as smoking at the time of the study visit or having stopped smoking within 1 year of the study visit. Participants were asked to return annually to our institution for evaluation of TSH and free thyroxine (FT4) levels in their serum. Study follow-up time was 5 years, and all events reported in this study occurred within this 5-year period. Event in this study is defined as the development of overt hypothyroidism (TSH levels >5.7 mIU/L and FT4 levels <0.72 ng/dL) or overt hyperthyroidism (TSH levels <0.4 mIU/L and FT4 >1.56 ng/dL). To convert FT4 to picomoles per liter, multiply by 12.871.
Laboratory measurements Serum TSH and FT4 levels were measured using time-resolved fluoroimmunoassay (Delphia, Turku, Finland). Reference levels were 0.4 to 5.7 mIU/L for TSH and 0.72 to 1.56 ng/dL for FT4. 7 Total triiodothyronine (T3) levels were measured by an in-house radioimmunoassay (reference values, 84.4-178.6 ng/ dL) to exclude T3 toxicosis in subjects with TSH levels lower than 0.4 mIU/L but normal FT4 levels. Presence and/or levels of antibodies against thyroid peroxidase (TPO) and thyroglobulin were measured using a chemiluminescence immunoassay (LUMI-Test; Brahms, Berlin, Germany), and results were considered positive at levels higher than 100 kU/L. Thyrotropinreceptor antibodies were measured by radioreceptor assay as TSH- binding inhibitory immunoglobulins (TBII) (first generation TRAK assay; Brahms), and results were considered positive at higher than 12 U/L. The presence and/or levels of specific IgG and IgA antibodies against purified plasmid-encoded associated YOPs of Y enterocolitica serotype 09 in serum samples were demonstrated by immunoblotting with a YOP antibody assay (AID, Strassberg, Germany), as described previously.[8] To convert T3 to nanomoles per liter, multiply by 0.0154.
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Statistical analysis
Statistical analysis was carried out with the SPSS software, version 10 (Chicago, Illinois). To compare event rates among groups, we used Cox regression analysis. Multiple logistic regression analysis was carried out to identify independent risk factors for events. A multivariate model for prognostication of risk for an event was initially developed incorporating baseline characteristics that tested independently in a univariate logistic regression model. Variables associated with P< .05 were retained in the final model. From the logistic regression model, a simplified model was obtained by putting weights to individual risk factors proportional to regression coefficients. The simplified model, called the Thyroid Events Amsterdam (THEA) score, allowed easy calculation of a predictive score.
Results
Baseline characteristics of the cohort are summarized in Table 1 . All participants had serum FT4 concentrations within the reference range, but 19 subjects had subclinical hypothyroidism (2.4%), and 13 subjects had subclinical hyperthyroidism (1.6%). With respect to family background, 608 of the 790 participants came from families with 2 proven patients with AITD (77%); both of these first- or second-degree relatives in each case had either the same AITD (either Graves hyperthyroidism or Hashimoto hypothyroidism for both relatives), or one had Graves disease, and the other had Hashimoto disease. In 182 of the 790 participants, we obtained proof of AITD in only 1 first or second-degree relative (23%). Although each of these participants reported 2 relatives with thyroid disease, information obtained from the treating physician of the second relative indicated either non-AITD (eg, nontoxic goiter or thyroid cancer) or hypothyroidism or hyperthyroidism of unspecified cause, and these cases were labeled as indeterminate . Seven participants used antihypertensive drugs, and 1 used oral antidiabetic agents; none used lithium salts or amiodarone. During the 5-year observation period, 136 of the 790 participants were lost to follow-up (17.2%), the loss occurring rather evenly over the whole study period, with an average annual loss of 27 participants. Characteristics of the dropouts were similar to those of the whole group (data not shown).
Occurrence of events
110 Prediction of progression using the THEA Score
Events occurred in 51 of the 790 participants (6.5%): 38 developed overt hypothyroidism (4.8%), and 13 developed overt hyperthyroidism (1.7%). The cause of hypothyroidism was Hashimoto disease in 34 cases and postpartum thyroiditis in 4 subjects. At the time of diagnosis of hypothyroidism, the median (range) concentration of serum TSH was 14.8 (6.3- 16.9) mIU/L, and or FT4, it was 0.55 (0.15-0.71) ng/dL. At baseline, 7 of the 38 subjects with hypothyroidism had serum TSH levels higher than 5.7 mIU/L; 30 had TPO antibody
Abbreviations: Ab, antibody; AITD, autoimmune thyroid disease; FT4, free thyroxine; TBII, TSH-binding inhibitory immunoglobulins; Tg, thyroglobulin; TPO, thyroid peroxidase; TSH, thyrotropin; YOP, Yersinia enterocolitica outer membrane protein. SI conversion factor: To convert FT4 to picomoles per liter, multiply by 12.871. aFor the definition of normal thyroid function, see the “Methods” section. bUnless otherwise indicated, data are reported as number (percentage) of subjects. cFor the definitions of positive findings, see the “Methods” section. dPresence and type of AITD in 2 of the subject’s first- or second-degree relatives.
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Abbreviation: AITD, autoimmune thyroid disease. aFor the definition of normal thyroid function, see the “Methods” section. b All data are reported as number of applicable subjects/total number of possible subjects (percentage). cPresence and type of AITD in 2 of the subject’s first- or second-degree relatives.
Figure 1. Cumulative event rate over 5 years in a euthyroid cohort of 790 women with first- or second-degree relatives with proven autoimmune thyroid disease.
levels higher than 100 kU/L; and none had TBII levels higher than 12 U/L. The cause of hyperthyroidism was Graves disease in 11 subjects (1 had Graves ophthalmopathy), postpartum thyroiditis in 1 subject, and silent thyroiditis in 1 subject. No cases of T3 toxicosis were encountered. At the time of diagnosis of hyperthyroidism, the median (range)
112 Prediction of progression using the THEA Score
Abbreviations: AITD, autoimmune thyroid disease; CI, confidence interval; HR, hazard ratio; NA, not applicable; TPO, thyroid peroxidase; TSH, thyrotropin; YOP, Yersinia enterocolitica outer membrane protein. aFor the definition of normal thyroid function, see the “Methods” section. bData are reported as percentage (number of applicable subjects/total number of possible subjects); the overall event rate for all subjects was 6.5% (51 of 790). cBy Cox regression analysis. dExcluding the lowest TSH category (_0.4 mIU/L). ePresence and type of AITD in 2 of the subject’s first- or second-degree relatives. serum TSH level was 0.02 (<0.01 to 0.13) mIU/L, and for FT4, it was 3.2 (1.6 to >5.4) mg/dL. At baseline, 2 of the 13 subjects with hyperthyroidism had serum TSH levels lower than 0.4 mIU/L; [9] had TPO antibody concentrations higher than 100 kU/L; and none had TBII levels higher than 12 U/L. Hypothyroid events occurred more often in subjects having 1 or 2 relatives with Hashimoto disease (28 of 386 [7.3%]) than in subjects having 1 or 2 relatives with Graves disease (19 of 599 [3.2%]) ( P=.004). Hyperthyroid events, however,
113 Chapter 7 did not occur more often in subjects having 1 or 2 relatives with Graves disease (10 of 599 [1.7%]) than in subjects having 1 or 2 relatives with Hashimoto disease (6 of 386 [[1.6%]) (P> .10) ( Table 2 ). Figure 1 depicts the cumulative event rate in the population at risk, which was 7.5% over 5 years. The mean annual event rate was 1.5%.
Risk factors for events
Table 3 lists hazard ratios of baseline characteristics for the occurrence of an event, as etermined by Cox regression analysis. Identified risk factors are serum TSH level, TPO antibody concentration, and family background. A TSH outside the reference range of 0.4 to 5.7 mIU/L at study entrance clearly constituted a risk, but the risk started to increase already at TSH levels above 2.0 mIU/L. A level-dependent effect was also observed for TPO antibody concentrations: the higher the concentration, the greater the hazard ratio. Having 2 relatives with Hashimoto disease also enhances the risk. Increasing age might be associated with a higher event rate, but this trend was not significant. Age, according to multiple logistic regression analysis, was not an independent risk factor; nor was thyroglobulin antibody presence or concentration. The 2 subjects with positive TBII findings had normal TSH values at baseline and remained euthyroid. No environmental factors significantly affected hazard ratios.
Predictive score for events
A simplified model was obtained from the logistic regression model by putting weights to individual risk factors proportional to their relative risks. This model allowed the calculation of a predictive score called theTHEA score ( Table4 ). The operational value of the THEA score was evaluated by comparison of the observed and expected event rates ( Table 5 ). The good agreement between both event rates is taken as a measure for the validity of the THEA score. Event rates increased significantly as the THEA score increased ( P<.001 for trend by χ² analysis) ( Figure 2 ).
114 Prediction of progression using the THEA Score
Abbreviations: AITD, autoimmune thyroid disease; Abbreviations: AITD, autoimmune thyroid disease; THEA, Thyroid Events Amsterdam; TPO, thyroid THEA, Thyroid Events Amsterdam; TPO, thyroid peroxidase; TSH, thyrotropin. peroxidase; TSH, thyrotropin. aFor the definition of normal thyroid function, see the aFor the definition of normal thyroid function, see “Methods” section. the “Methods” section. bData are reported as percentage (number of applicable subjects/total number of possible subjects).
We observed in our at-risk female population a cumulative event rate of 7.5%, with an annual incidence rate of 1.5%. Overt hypothyroidism occurred in 38 subjects, and overt hyperthyroidism in 13 subjects during the 5-year follow-up of the 790 participants. The hypothyroidism incidence rate in our population was 9.6/1000/y; the hyperthyroidism incidence rate was 3.3/1000/y. Incidence rates in the general female population have been reported in the prospective DanThyr study by Bülow Pedersen et al [9] (comparing 2 regions of Denmark with moderate and mild iodine deficiency [urinary iodine levels, 45 µg/L in Aalborg and 61 µg/L in Copenhagen]) in the prospective Whickham study by Vanderpump et al,[10] and in the retrospective Tayside study by Flynn et al.[11] Both the Whickham and the Tayside studies were conducted in England, a country considered to be iodine sufficient (mean urinary iodine excretion level, 141 µg/L).[12] The incidence rates of hypothyroidism in these 4 areas were 0.44, 0.61, 3.5, and 4.98 per 1000 women per year, respectively. Incidence rates of hyperthyroidism were 1.49, 1.02, 0.80, and 0.77 per 1000 women per year, respectively. To convert iodine to nanomoles per liter, multiply by 7.880. These epidemiologic data support the notion that higher levels of dietary iodine decrease the incidence of hyperthyroidism at the expense of an increased incidence of hypothyroidism. The Netherlands is considered to be a country of dietary iodine sufficiency, with a median
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Figure 2. The Thyroid Events Amsterdam (THEA) score used to predict hypothyroid and hyperthyroid events over 5 years in a euthyroid cohort of 790 women with first- or second-degree relatives with proven autoimmune thyroid disease. Event rates increased significantly, as measured by the χ² test, as the THEA score increased (P<.001 for trend). urinary iodine excretion level of 154 µg/L.[13] Our incidence figures are therefore best compared with those in the United Kingdom: in our at-risk female population, the incidence rate of overt hypothyroidism is 1.9 to 2.7 times higher than that in the general female population in the United Kingdom, and the incidence rate of hyperthyroidism is 4.1 to 4.3 times higher. These figures once again demonstrate that subjects with a family history of AITD are at increased risk for thyroid disease. The risk in siblings of parents with AITD is much higher than that observed in the present study (with reported λs as high as 11.6 for Graves disease and 28.0 for Hashimoto thyroiditis).[2] This finding is understandable because relatives with AITD in our study could be children, brothers, sisters, parents, grandparents, uncles, or aunts of the participants. Our figures thus fit nicely between the lower risk in the general population and the higher risk in siblings. In the present study, we found that independent risk factors are serum TSH level (increased risk at TSH values <0.4 mIU/L and >2.0 mIU/L, still within the reference range), high TPO antibody concentration, and presence of a family history of AITD (with the greatest risk attached to subjects having 2 relatives with Hashimoto disease). These findings are in perfect agreement with the prospective Whickham study,[10] which reported that the risk to develop hypothyroidism sharply increased at TSH levels above 2 mIU/L, independently of TPO antibody levels. The agreement in these findings underscores the notion that TSH levels between 0.4 and 2.0 mIU/L are associated with optimal thyroid health.[14,15]The progressively increasing risk with increasing TPO antibody concentrations, independent of TSH levels, has also been
116 Prediction of progression using the THEA Score observed in the Whickham study.[10] Advancing age is associated with a higher concentration of TPO antibodies,[10] which likely explains why age in our study was not an independent risk factor. In the Whickham study, the agespecific hazard rates for the development of hypothyroidism (but not for hyperthyroidism) sharply increase when the patients are older than 60 years.[10] In our study, the trend of a higher event rate in older age was nonsignificant, probably owing to the small number of subjects in the age group 60 to 65 years (only 30 subjects). In the Whickham study,[10] a positive family history of thyroid disease was not associated with increased odds of developing hypothyroidism. This absent relationship might be explained by evaluation of the general population in that study and by the fact that not all patients with AITD have a positive family history of thyroid disease (eg, in Graves disease, family history of AITD is reported in only 60% of cases).[1] In our study, limited to families with first- or second-degree relatives with AITD, we observed the greatest risk for hypothyroid events in participants with 2 relatives with Hashimoto disease; this was not the case for hyperthyroid events, nor did participants with 2 relatives with Graves disease represent such a high risk. A baseline concentration of higher than 100 kU/L for TPO antibodies was found with equal frequency in subjects having hypothyroid and hyperthyroid events (79% vs 69%) ( P> .10), just as the median (range) baseline TPO antibody concentration was similar in the 2 groups: 1890 (370 to >10 000) kU/L vs 1060 (340 to >10 000) kU/L ( P>.10). Regarded as early markers of thyroid autoimmunity, the presence of TPO antibodies is common to both Graves and Hashimoto disease. In healthy euthyroid women, genetic factors account for 72% of the risk for positive TPO antibody findings[6]; according to this estimate, 28% of the liability should be caused by environmental factors. However, none of the environmental factors to which the participants of our study were exposed at study entrance contributed to the risk for developing an event. This is not to deny that the investigated environmental factors might have played a role in developing overt hypothyroidism or hyperthyroidism. Exposure might have changed between the time of study entrance and time of the event. However, given the similarity in the baseline prevalence of TPO antibodies in subjects developing hypothyroid and hyperthyroid events, and in view of the dependency of the hypothyroid event rate on family background (3 times higher in cases of 2 relatives with Hashimoto disease)—and keeping in mind that the hyperthyroid event rate was not preferentially influenced by the number of relatives with Graves or Hashimoto disease —it is tempting to speculate that subjects genetically predisposed to AITD first develop TPO antibodies and then develop hypothyroidism unless exposure to smoking and stressful life events provokes development of Graves hyperthyroidism. The 3 independent risk
117 Chapter 7 factors for events allowed us to compose a predictive score for the development of overt hypothyroidism or hyperthyroidism within 5 years in euthyroid women with first- or second- degree relatives with AITD. This THEA score apparently gives a fairly accurate estimate of the risk. Its advantages are the easy calculation and the broad applicability. The family members of patients with AITD can be reassured of a very high likelihood of maintaining euthyroidism in the next 5 years if they have a low score (0-7). If the THEA score is higher, reassessment after 1 year might be warranted. The THEA score might be very useful for young women of AITD families who want to become pregnant in the next few years.[16] Screening seems to be indicated for these women in view of the high abortion rate associated with the presence of TPO antibodies. The abortion rate can be lowered by treatment with levothyroxine.[17] Our study has several limitations. The THEA score is derived from a female population and may be not applicable to men. Although a large sex difference is unlikely, the score should be validated in a male population. Prediction of events by the THEA score is restricted to a time span of 5 years, but undoubtedly many more events will occur after that period. In clinical practice, reassessment of the THEA score after 5 years might be indicated. Another important limitation of our study is the relatively low number of events, especially hyperthyroid events, which probably renders the THEA score less accurate. The small number of observed events may raise concern about possible overfitting of our model. Consequently, there is a need to validate the THEA score in other populations. Finally, the precise genetic background in 182 of the 1580 relatives could not be ascertained (11.5%); an unknown proportion of these indeterminate relatives might still have Graves or Hashimoto disease, thereby affecting the weights assigned to family background in the THEA score. This bias is likely to be small in view of its limited contribution to the maximum THEA score. The uncertainty is acceptable because it reflects the reality of day-to- day practice in which it is not always possible to determine with certainty the autoimmune nature of thyroid disease in family members. Inclusion of indeterminate relatives in our study permits application of the THEA score also in subjects with only 1 known relative with AITD.
118 Prediction of progression using the THEA Score
References FERENCES 1. Tomer Y, Davies TF. Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev . 2003;24(5):694-717. 2. Villanueva R, Greenberg DA, Davies TF, Tomer Y. Sibling recurrence risk in autoimmune thyroid disease. Thyroid . 2003;13(8):761-764. 3. Brix TH, Kyvik KO, Hegedu¨s L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab . 2000;85(2):536-539. 4. Brix TH, Kyvik KO, Christensen K, Hegedu¨s L. Evidence for a major role of heredity in Graves’ disease: a population-based twin study of two Danish twin cohorts. J Clin Endocrinol Metab . 2001;86(2):930-934. 5. Ringold DA, Nicoloff JT, Kesler M, Davis H, Hamilton A, Mack T. Further evidence for a strong genetic influence on the development of autoimmune thyroid disease: the California twin study. Thyroid . 2002;12(8):647-653. 6. Hansen PS, Brix TH, Iachine I, Kyvik KO, Hegedu¨s L. The relative importance of genetic and environmental effects for the early stages of thyroid autoimmunity: a study of healthy Danish twins. Eur J Endocrinol . 2006;154(1):29-38. 7. Strieder TG, Prummel MF, Tijssen JG, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol (Oxf ) . 2003;59(3):396-401. 8. Strieder TG, Wenzel BE, Prummel MF, Tijssen JG, Wiersinga WM. Increased prevalence of antibodies to enteropathogenic Yersinia enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease. Clin Exp Immunol . 2003;132(2):278-282. 9. Bu¨low Pedersen I, Knudsen N, Jorgensen T, Perrild H, Ovesen L, Laurberg P. Large differences in incidences of overt hyper- and hypothyroidism associated with a small difference in iodine intake: a prospective comparative registerbased population survey. J Clin Endocrinol Metab . 2002;87(10):4462-4469. 10. Vanderpump MP, Tunbridge WM, French JM, et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham survey. Clin Endocrinol (Oxf ) . 1995;43(1):55-68. 11. Flynn RW, MacDonald TM, Morris AD, Jung RT, Leese GP. The thyroid epidemiology, audit, and research study: thyroid dysfunction in the general population. J Clin Endocrinol Metab . 2004;89(8):3879-3884. 12. International Council for Control of Iodine Deficiency Disorders. West and Central Europe assesses its iodine nutrition. IDD News . 2002;8(4):51-55. 13. Wiersinga WM, Podoba J, Srbecky M, van Vessem M, van Beeren HC, Platvoet-ter Schiphorst MC. A survey of iodine intake and thyroid volume in Dutch schoolchildren: reference values in an iodine-sufficient area and the effect of puberty. Eur J Endocrinol . 2001;144(6):595-611. 14. Surks MI, Goswami G, Daniels GH. The thyrotropin reference range should remain unchanged. J Clin Endocrinol Metab . 2005;90(9):5489-5496. 15. Wartofsky L, Dickey RA. The evidence for a narrower thyrotropin reference range is compelling. J Clin Endocrinol Metab . 2005;90(9):5483-5488. 16. Pearce SH, Leech NJ. Toward precise forecasting of autoimmune endocrinopathy. J Clin Endocrinol Metab . 2004;89(2):544-547. 17. Negro R, Formoso G, Magnieri T, Pezzarossa A, Dazzi D, Hassan H. Levothyroxine treatment in euthyroid pregnant women with autoimmune thyroid disease:effects on obstetrical complications. J Clin Endocrinol Metab . 2006;91(7):2587-2591.
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Environmental factors involved in the development of overt
autoimmune hypothyroidism and 8| hyperthyroidism: evidence from a prospective nested case -control
Thea G.A. Strieder Jan G.P. Tijssen Björn E. Wenzel Jos F. Brosschot Wilmar M. Wiersinga
Environmental factors involved in the development of overt AITD
Chapter 8 Environmental factors involved in the development of overt autoimmune hypothyroidism and hyperthyroidism: evidence from a prospective nested case-control study
Abstract
Context . Genetic and environmental factors are involved in the pathogenesis of autoimmune thyroid disease (AITD). Evidence for involvement of environmental stimuli has been obtained mostly by association in retrospective cross-sectional case-control or population-based studies, open to potential (recall) bias. Objectives. To evaluate prospectively the role of certain environmental factors (Yersinia infection, smoking, pregnancy, oral estrogens, stress) in the development of overt autoimmune hypo- or hyperthyroidism in women who were euthyroid at study entrance. Desing, setting and subjects. The design is that of a nested case-control study within the prospective observational Amsterdam AITD cohort-study, in which 790 healthy euthyroid women with at least one 1st or 2nd degree relative with documented AITD were followed for five years. Thyroid function and exposure to environmental stimuli were assesed annually. Outcomes. Contrast in environmental exposure between cases (overt hypothyroidism – TSH>5.7mU/l and FT4<9.3pmol/l – or overt hyperthyroidism – TSH<0.4mU/l and FT4>20.1pmol/l -; also referred to as events) and controls (matched for age and duration of follow-up). Results. At baseline, exposure to environmental factors was not different between the 51 cases (38 hypothyroid and 13 hyperthyroid subjects) and their 102 controls. The frequency of Yersinia infection and oral estrogen use remained similar between cases and controls, but at the time of event hypothyroid cases were less often current smokers (p=0.083) and more often in the postpartum period (p=0.006) than their controls, whereas hyperthyroid cases had been pregnant more frequently (p=0.063). Exposure to stress at study entrance, at one year before event and at the timeof the event was not different in hypothyroid and hyperthyroid cases as compared to their respective controls. Conclusion. The data suggest that cessation of smoking and the postpartum period are related to autoimmune hypothyroidism, whereas autoimmune hyperthyroidism is less frequent in women who have never been pregnant. Stress exposure was not related to either hypo- or
123 Chapter 8 hyperthyroidism, but the limitation of this prospective observational study is its limited sample size.
Introduction
The etiology of autoimmune thyroid diseases is multifactorial. Genetic and environmental factors are involved, and it has been estimated that about 79% of the liability to develop Graves’ disease or Hashimoto’s disease can be attributed to genetic factors (1-3). Many environmental factors have been identified such as smoking, stress, pregnancy, exogenous estrogens and possibly certain infections (e.g. with Yersinia enterocolitica) (4). The evidence that these environmental factors are indeed determinants of autoimmune thyroid disease has been obtained mostly in a retrospective manner in cross-sectional case-control or population- based studies. The well established association between e.g. stress and Graves’ hyperthyroidism is based on studies evaluating stress exposure at the time of referral when the patients were still hyperthyroid or later when the patients had become euthyroid. Neither approach is optimal as hyperthyroidism itself may induce stress, and delaying the evaluation until euthyroidism has been reached may increase recall bias. Undoubtedly, prospective studies in subjects at risk to develop autoimmune thyroid disease would be much more reliable in assesing whether or not the observed associations between environmental factors and autoimmune thyroid disease signify a cause and effect relationship. The Amsterdam AITD cohort study is a prospective observational follow-up study designed to determine risk factors for the development of AITD. To increase the likelihood of diagnosing new cases of overt Graves’ hyperthyroidism or Hashimoto’s hypothyroidism during a 5-year follow-up, we included only euthyroid women who had at least one relative with documented AITD and who were in self-proclaimed good health. The baseline characteristics of this cohort has been reported previously (5). Now we report the results of a nested case-control study in the cohort, contrasting exposure to a number of environmental factors (which were assessed annually) between cases (subjects in whom overt autoimmune hypo- or hyperthyroidism had occurred ) and controls (subject who had not developed overt hypo- or hyperthyroidism, matched for age and duration of follow-up).
Subjects and methods
124 Environmental factors involved in the development of overt AITD
Subjects The original Amsterdam AITD cohort consisted of 803 female subjects between 18 and 65 years of age, without a history of thyroid disease, who were in self-proclaimed good health and had at least one first- or second- degree relative with documented autoimmune hyper- or hypothyroidism. At study entrance thyroid function tests revealed overt hyper- or hypothyroidism in 13 participants, leaving 790 subjects for follow-up (5). During a five year follow-up subjects were seen annually at our institution. At each visit blood was withdrawn for laboratory tests, and subjects were asked to fill in questionnaires. Endpoints of the study were either completion of the 5-yr follow-up period, or the development of overt hypothyroidism (defined as TSH>5.7mU/l and FT4<9.3pmol/l) or overt hyperthyroidism (defined as TSH<0.4mU/l and FT4>20.1pmol/l) called cases or events. There occurred 38 cases of overt autoimmune hypothyroidism and 13 cases of overt autoimmune hyperthyroidism, as reported elsewhere (6). For each of these 51 cases two controls were selected from the remaining subjects in the cohort. Controls were matched for age at study entrance, and the analysis of their data was limited up to the same visit at which the cases had reached their endpoint, thereby guaranteeing that both groups in this nested case-control study had been exposed to environmental factors for precisely the same follow-up period. The study was approved by the Institutional Review Board of the Academic Medical Center, and all participants gave their informed consent prior to enrollment in the study.
Laboratory measurements . Serum TSH and FT4 were measured using time-resolved fluoroimmunoassays (Delfia hTSH and Delfia FT4 respectively, Wallac Oy, Turku, Finland). Reference values are for TSH 0.4- 5.7mU/l and for FT4 9.3-20.1pmol/l as described previously (5). Total T3 was measured by an in-house radioimmunoasay (reference values 1.30-2.75nmol/l), only in subjects with a TSH<0.4mU/l but a normal FT4 to exclude the existence of T3 toxicosis. Thyroid peroxidase (TPO) antibodies and thyroglobulin (Tg) antibodies were measured by chemiluminescence immunoassays (LUMI-test anti-TPO and LUMI-test anti-Tg respectively, Brahms, Berlin, Germany). Improved versions of both assays became available during follow- up: detection limits of these new assays were for TPO-Ab 30kU/l and for Tg-Ab 20kU/l. TPO-Ab concentrations obtained with the old assay were multiplied by a factor 0.72 to obtain comparative values in the new assay. TPO-Ab and Tg-Ab concentrations were considered to be positive at values of > 100kU/l. TSH receptor antibodies were determined as TSH binding inhibitory immunoglobulins (TBII) using the TRAK assay (Brahms, Berlin, Germany);
125 Chapter 8 detection limits in the 1st and 2nd generation TRAK assays were 5 and 1U/l respectively, and values above 12 and 1.5U/l respectively were considered as positive. Specific IgG and IgA antibodies against purified plasmid-encoded virulence-associated Yersinia outer membrane proteins (YOP) of Yersinia enterocolitica serotype O9 were demonstrated by immunoblotting with a YOP-Ab assay (AID, Strassberg, Germany) as described previously (7). Sera are considered positive if at least three bands (IgG) or two bands (IgA) are seen in immunoblotting at a level >10% (IgG) or >5% (IgA) of reference standards.
Questionnaires At each visit participants were asked to fill in questionnaires on smoking habits, pregnancy, estrogen treatment, stress and iodine excess in the previous year. Current smoking was defined as smoking now or having stopped smoking within one year before the study visit. Current pregnancy was defined as being pregnant at the time of the study visit, and postpartum as termination of pregnancy in the previous year. Current use of estrogens was defined as using estrogens at the time of the study visit, or having stopped taking estrogens in the previous year. The Dutch questionnaire on Recently Experienced Stressful Life Events (8,9) counts the total number of major life events experienced in the last 12 months (checklist of 60 possible events). The respondent scores separately the amount of pleasantness and unpleasantness associated with each experienced life event, rated on a scale from zero (meaning no (un)pleasantness at all) to four (a huge amount of (un)pleasantness). From this scale, the total amount of pleasantness and unpleasantness is calculated (maximal score 240 for each). When the amount of pleasantness exceeds the amount of unpleasantness the event is categorized as being pleasant and vice versa, yielding the total number of (un)pleasant events (maximum 60). The Dutch Everyday Problem Checklist, a validated version of the Daily Hassles Scale (10- 12), consist of 114 items concerning daily hassles experienced in the last two months. It also measures the intensity of each hassle on a scale from zero to three, yielding the number of hassles experienced and the total intensity of these hassles (maximum 342). The Positive and Negative Affect Schedule (PANAS) (13) measures the current mood, in terms of positive and negative affect. It consists of 22 mood states (11 positive, 11 negative) and the respondent is asked to report whether she is affected by each of these states on a scale from 1 (not at all) to 5 (a lot). This yields the tendency to report positive and negative affect states both on a scale from 11 to 55.
126 Environmental factors involved in the development of overt AITD
Statistical analysis Statistical analysis was carried out with the SPSS10 package. Values are given as mean ± SD for age, but as median and interquartile range for all other parameters. Differences between cases and controls were evaluated by Students’t test for age, by Mann-Whitney U-test for TSH, FT4, TPO-Ab and Tg-Ab, and by X2 test or if appropriate Fisher’s exact test for the other parameters. A p-value of <0.05 was considered to indicate significant differences between groups.
Results
The average time period between study entrance and the occurrence of an event was three years. At baseline, the 38 hypothyroid cases had already higher TSH, lower FT4 and higher TPO-Ab and Tg-Ab serum concentrations than their 76 controls, and the difference between both groups persisted at one year before occurrence of the event (Table 1). In contrast, neither TSH nor FT4 values differed between the 13 hyperthyroid cases and their 26 controls at baseline or at one year before the event, but the prevalence of TPO-Ab and Tg-Ab was higher in cases than in controls. TBII were present in only one participant at study entrance, and its frequency did not differ between cases and controls. The frequency of Yersinia YOP-IgG and YOP-IgA antibodies was also not different between cases and controls, but in the follow-up the proportion of participants having YOP-IgG or YOP-IgA antibodies decreased in both groups. A history of iodine excess in the year preceding a visit was obtained in a few cases and controls, without significant differences in frequency between both groups.
Smoking Smoking status didn’t differ between cases and controls at study entrance or at one year before the event (Table 2). At the time of the event, however, there were less current smokers in hypothyroid cases than in controls. (5 out of 38 (13%) cases vs 21 out of 76 (28%) controls, p=0.083), This was not so in the hyperthyroid subgroup (2 out of 13 (15%) cases vs 7 out of 26 (27%) controls, p=0.7).
Pregnancy and oral estrogens Cases and controls did not differ with respect to pregnancies at study entrance or at one year before event, but there were more cases in the postpartum period than controls at the time of the event (Table 2). This was exclusively due to more hypothyroid cases in the postpartum
127 Chapter 8
Table 1. Comparison of characteristics between patients who developed clinically overt auto-immune thyroid disease (AITD) and their corresponding controls matched for age at time of study entrance, at consecutive time points of follow-up, in a nested case-control study of 153 women with 1 st or 2 nd degree relatives with proven AITD derived from the prospective THyroid Event Amsterdam cohort.
Characteristics Hypothyroidism Hyperthyroidism Cases Controls P-value Cases Controls P-value N 38 76 13 26
At study entrance - Age in years 37 38 ± 12 0.96 42 42 ± 13 0.97 - TSH mU/l 4.2 (2.7-5.7) 1.6 (1.1-2.5) <0.001 1.6 (1.1-2.2) 1.5 (1.2-2.2) 0.85 - fT4 pmol/l 10.7 (9.7-11.6) 12.8 (11.3-14.5) <0.001 13.3 (12.0-14.1) 14.1 (12.0-15.7) 0.34 - TPO Antibody positive 79 % 15 % <0.001 77 % 35 % 0.02 - TPO Antibody kU/l 1123 (304-2239) 25 (<30-49) <0.001 545 (<30-2265) <30 (<30-155) 0.01 - Tg Antibody positive 26 % 10 % 0.01* 15 % 8 % 0.46* - Tg Antibody kU/l 75 (40-238) 8 (<20-29) <0.001 38 (23-108) 17 (<20-41) 0.03 - TBII positive 0 % 0 % 1.00 0 % 4 % 1.00* - YOP-IgG Antibody positive 40 % 42 % 0.79 54 % 62 % 0.66 - YOP-IgA Antibody positive 21 30 % 0.30 31 35 % 0.81
One year before occurrence of event - Age in years 40 ± 12 40 ± 12 0.95 43 ± 14 43 ± 14 0.98 - TSH mU/l 4.0 (2.6-6.1) 1.6 (1.1-2.5) <0.001 1.5 (0.9-2.1) 1.6 (1.3-2.2) 0.57 - fT4 pmol/l 10.6 (9.5-12.0) 13.0 (11.9-14.1) <0.001 13.1 (12.1-14.1) 13.5 (11.5-15.3) 0.95 - TPO Antibody positive 87 % 18 % <0.001 83 % 42 % 0.05 - TPO Antibody kU/l 1076 (666-2326) 25 (<30-42) <0.001 560 (<30-1742) 36 (<30-91) 0.04 - Tg Antibody positive 45 % 21 % <0.001 15 % 12 % 1.09 - Tg Antibody kU/l 123 42-235 10 (<20-32) <0.001 46 (21-133) 15 (<20-57) 0.09 - TBII positive 5 % 0 % 0.11* 0 % 0 % 1.00* - YOP-IgG Antibody positive 26 % 30 % 0.66 46 % 42 % 0.82 - YOP-IgA Antibody positive 16 % 29 % 0.12 8 % 35 % 0.12*
Data are given as means ± SD or medians with interquartile range or proportions, P-value of cases versus controls, * = Fisher’s exact test. Time of event: visit when diagnosis was made or confirmed. TSH: thyroid stimulating hormone, fT4: free T4, TPO: thyroid peroxidase, Tg: thyroglobuline, TBII: TSH-receptor antibodies, YOP: Yersinia enterocolitica outer membrane proteins.
128 Environmental factors involved in the development of overt AITD
Table 2. Comparison of exposure to environmental factors between patients who developed clinically overt auto-immune thyroid disease (AITD) and their corresponding controls matched for age at time of study entrance, at consecutive time points of follow-up, in a nested case-control study of 153 women with 1 st or 2nd degree relatives with proven AITD derived from the prospective THyroid Event Amsterdam cohort. Exposure Hypothyroidism Hyperthyroidism Cases Controls P-value Cases Controls P-value N 38 76 13 26 % % % % Smoking behaviour At study entrance - never 53 43 39 31 - quitters 29 28 46 38 - current smokers 18 29 0.22 15 31 0.45* One year before occurrence of event - never 53 43 39 31 - quitters 29 29 46 38 - current smokers 18 28 0.28 15 31 0.45* At time of occurrence of event - never 53 43 39 31 - quitters 34 29 46 42 - current smokers 13 28 0.08 15 27 0.69*
Pregnancies At study entrance - never 58 45 15 42 0.15* - in the past 39 49 69 54 - current 0 2 8 0 - postpartum 3 4 1.00* 8 4 One year before occurrence of event - never 45 41 8 38 0.06* - in the past 39 53 84 58 - current 11 2 0 0 - postpartum 5 4 1.00* 8 4 At time of occurrence of event - never 34 41 8 38 0.06* - in the past 42 54 77 58 - current 3 1 0 0 - postpartum 21 4 0.006* 15 4
Oral estrogen use At study entrance - never 13 16 8 12 - in the past 34 46 69 58 - recent quitters - - - - - current users 53 38 0.14 23 30 0.72* One year before occurrence of event - never 13 13 8 12 - in the past 34 40 69 54 - recent quitters 21 12 15 15 - current users 32 35 0.68 8 19 0.64* At time of occurrence of event - never 11 12 8 12 - in the past 37 46 84 65 - recent quitters 16 9 0 4 - current users 21 33 0.19 8 19 0.64*
Data are given as proportions with P-value of cases versus controls, * = Fisher’s exact test. Time of event: visit when diagnosis was made or confirmed. Quitters: used to until previous visit. Current: after previous visit and/or at present visit.
129 Chapter 8 period (8 out of 38 (21%) cases vs 3 out of 76 (4%) controls, p=0.006), not to more hyperthyroid cases (2 out of 13 (15%) cases vs 1 out of 26 (4%) controls, p=0.5). The proportion of women who had never been pregnant was lower in hyperthyroid cases than in their controls both one year before the event and at the time of the event (1 out of 13 (8%) cases vs 10 out of 26 (38%) controls, p=0.063); this was not so in the hypothyroid subgroup. The use of oral estrogens didn’t differ between cases and controls at any time. (Table 2).
Stress At study entrance, no differences between cases and controls were observed in recent life events, daily hassles, or affect scales (Table 3); only the amount of total pleasantness was lower in hypothyroid cases than in their controls. During follow-up, the total number of recent experienced life events decreased in controls but not in cases; the difference in (un)pleasant events and amount of total (un) pleasantness between cases and controls did not reach statistical signature at any time point, except a lower number of pleasant events in the hyperthyroid cases one year before the diagnosis of hyperthyroidism. Stress from daily hassles did not differ between cases and controls at any time. The mood states as assessed by the PANAS did not differ between cases and controls during follow-up, although there was a tendency to report less frequently negative feelings in the hypothyroid cases at time when diagnosis was made.
Discussion
The aim of our case-control study nested in the observational Amsterdam cohort study was to evaluate in a prospective manner the involvement of certain environmental factors in the development of overt autoimmune hypo- or hyperthyroidism (called events, or cases in the present study). As reported elsewhere, 51 cases were observed during follow-up, consisting of 38 hypothyroid and 13 hyperthyroid events occurring on average three years after study entrance (6). In our nested case-control study, controls had the same sex (all female) and by matching the same age and duration of follow-up as the cases. Exposure time to environmental factors was thus similar in cases and controls. At study entrance, cases had higher TSH and lower FT4 serum concentrations than controls whereas TPO-Ab concentrations were higher in both hypothyroid and hyperthyroid cases than their respective controls. The data are in agreement with the previously reported finding that baseline serum TSH and TPO-Ab are independent predictors of progression to overt
130 Environmental factors involved in the development of overt AITD hypo- or hyperthyroidism within five years (6). Of interest is the notion that the absolute change in TSH between baseline and one year before the occurence of events was greater in the hypothyroid cases than in their controls ( ∆TSH 1.1 (0.5-2.1) vs 0.4 (0.1-0.9) mU/l, p<0.000) but not in the hyperthyroid cases vs their controls ( ∆TSH 0.2 (0.0-0.5) vs 0.3 (0.0- 0.6) mU/l, p=0.6). Likewise, the absolute change in TPO-Ab values between baseline and one year before the occurence of events was greater in the hypothyroid cases than their controls (∆TPO-Ab 385 (85-853 vs <30 (<30-<30) kU/l, p<0.000), but not in the hyperthyroid cases vs their controls ( ∆TPO-Ab<30 (<30-242) vs <30 (<30-48), p=0.8). The data suggest that progression towards overt hypothyroidism is a gradual process taking several years, but that in contrast overt hyperthyroidism develops faster in terms of months rather than years. The incidence of Yersinia YOP-IgG and YOP-IgA antibodies decreased during follow-up, to a similar extent in both hypothyroid and hypothyroid cases and their respective controls. The data do not support involvement of Yersinia enterocolitica infections in the development of overt autoimmune hypo- or hyperthyroidism. Smoking has clearly been established as a risk factor for the development of Graves’ hyperthyroidism (and even more so for Graves’ ophthalmopathy), but its association with hypothyroidism has been disputed (4,14). In the present study the proportion of current smokers was lower in cases than in controls but only at the time of the event, not at baseline or one year before the event. In view of the marginal statistical significance the data might be interpreted cautiously that discontinuation of smoking promotes the development of hypothyroidism. This interpretation is in line with recent observations that smoking protects against the development of TPO-Ab (5,15) and also against the development of hypothyroidism. With respect to pregnancies, cases were more often in the postpartum period at the time of the event than controls, which was seen only in the hypothyroid cases and due to the occurence of postpartum thyroiditis. The proportion of women who had never been pregnant was lower in the hyperthyroid cases than in their controls, that is hyperthyroid cases had experienced pregnancies more often. This is compatible with the notion that a sizable proportion of patients with Graves’ hyperthyroidism develop Graves’ disease after pregnancy (5,16). The use of oral estrogens (oral contraceptive pills) was not related in our study to the occurrence of hypo- or hyperthyroidism, although a few studies in the past indicate a protective effect of estrogens on the development of hyperthyroidism (5,17,18).
131 Chapter 8
This is the first study in which exposure to stress has been assessed in a prospective manner in order to evaluate the role of stress in the pathogenesis of overt autoimmune hypo- and hyperthyroidism. A number of case-control studies have reported a relationship between stress and the onset of Graves’ hyperthyroidism (19-24), but the results have always been obtained in a retrospective manner (asking patients for stressful life events in the year prior to diagnosis, at a time when they were still hyperthyroid or even later when euthyroidism was restored), making all studies liable to recall bias. Moreover, it could not be ruled out that a higher number of stressful life events in the year preceding the diagnosis of Graves’ hyperthyroidism was the consequence rather than the cause of thyroid hormone excess. Our data do not favour stress as one of the causal factors in Graves’ hyperthyroidism because overall differences in the three stress questionnaires between cases and controls at study entrance and during follow-up were either nonexistent or of marginal significance. However, we cannot exclude the possibiblity that we did not detect an effect of stress in view of the very limited sample size of our hyperthyroid cases (n=13). Daily hassles might be more annoying and more stress generating than recent life events, which can be either pleasant or unpleasant. In this respect it is relevant to mention two prospective studies in patients with Graves’ hyperthyroidism, who were inverstigated when euthyroid on antithyroid drug treatment. Higher scores of daily hassles were observed in patients who experienced recurrent hyperthyroidism after discontinuation of antithyroid drugs than in patients who remained euthyroid (26, 27). We also did not observe that stress exposure was associate dwith the development of autoimmune hypothyroidism. Whereas the total number of recent experienced life events decreased over time in the hypothyroid controls, this was not so in the hypothyroid cases. Differences between cases andd controls however did not reach significance at any time point, nor were there differences in the average exposure to stresssors in the period from study enbtrance until the occurence of the event (data not shown). Only two papers have evaluated the role of stressors in the development of autoimmune hypothyroidism (17, 28). No association was found,but stress was evaluated just once in both studies: in one paper the disease was already diagnosed (17), and in the other at 8 weeks postpartum in order to relate life events over the preceeding year to postpartum dysfunction (28). The strength of our study is its study design, allowing assesment of environmental factors prospectively without knowledge of thyroid function and guaranteeing cases and controls were exposed to environmental factors for precisely the same time period. A weakness of our study is the limited number of subjects who converted from euthyroidism to overt
132 Environmental factors involved in the development of overt AITD hypothyroidism (n=38) or hyperthyroidism (n=13). A larger number of cases is in all likelihood requested to more accurately delineate the role of stress and the use of oral estrogens. Nevertheless, even in this limited sample size significant differences between cases and controls were observed in exposure to tobacco smoke and pregnancy, allowing the conclusion that smoking protects against hypothyroidism, and that never been pregnant protects against hyperthyroidism.
ACKNOWLEDGMENTS The study was supported by a grant from the Netherlands Organization for Scientific Research (NWO 950-10-626). The Margarete Markus Foundation in London, U.K., supported financially the serological tests for Yersinia.
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Table 3. Comparison of scores on three stress questionnaires (Recent Experienced Life events, Daily Hassles, PANAS) between patients who developed clinically overt auto-immune thyroid disease (AITD) and their corresponding controls matched for age at time of study entrance, at consecutive time points of follow-up, in a nested case-control study of 153 women with 1 st or 2 nd degree relatives with proven AITD derived from the prospective THyroid Event Amsterdam cohort.
Exposure Hypothyroidism Hyperthyroidism P- P- Cases Controls Cases Controls value value Recent Experienced Life events At study entrance (n) 38 76 13 26 - total number of life events 7.5 (4.0-15.3) 10.0 (7.0-13.0) 0.14 7.0 (7.0-10.0) 8.5 (6.0-11.3) 0.98 - number of unpleasant events 2.0 (1.0-6.0) 4.0 (2.3-6.0) 0.13 4.0 (2.0-4.5) 4.0 (2.0-6.0) 0.83 - number of pleasant events 3.0 (1.0-6.3) 4.0 (3.0-7.0) 0.13 3.0 (2.5-5.0) 4.0 (3.0-7.0) 0.45 - amount of total unpleasantness 8.0 (3.0-19.4) 13.0 (9.0-22.8) 0.04 11.0 (8.5-18.5) 9.5 (5.8-16.0) 0.28 - amount of total pleasantness 12.5 (4.8-25.3) 17.0 (10.0-25.0) 0.20 14.0 (8.5-18.5) 15.5 (9.5-21.8) 0.64 One year before occurrence of event (n) 31 62 12 24 - total number of life events 8.0 (3.0 – 12.0) 7.0 (5.0 – 12.0) 0.68 6.5 (3.3-9.0) 8.0 (5.0-11.5) 0.38 - number of unpleasant events 3.0 (1.0 – 6.1) 4.0 (2.0 – 6.3) 0.38 4.0 (2.0-7.1) 2.5 (1.0-6.1) 0.44 - number of pleasant events 3.0 (1.0 – 6.0) 2.1 (2.0 – 5.0) 0.82 1.5 (1.0-3.8) 3.0 (2.0-6.8) 0.04 - amount of total unpleasantness 10 0 (3.0 – 22.0) 12.6 (6.0 – 22.0) 0.23 11.1 (5.0-19.1) 8.0 (3.0-15.8) 0.51 - amount of total pleasantness 10.0 (4.0 – 17.0) 9.2 (6.0 – 17.0) 0.74 7.0 (4.3-10.5) 11.5 (6.3-22.8) 0.06 At time of occurrence of event (n) 20 40 9 18 - total number of life events 8.5 (5.3 – 12.0) 6.0 (4.0 – 10.8) 0.20 9.0 (1.5-11.5) 7.0 (5.0-12.8) 0.63 - number of unpleasant events 3.0 (1.0 – 5.0) 3.0 (2.0 – 5.0) 0.94 2.0 (0.0-6.5) 3.0 (2.0-6.8) 0.38 - number of pleasant events 4.1 (2.3 – 6.8) 3.0 (1.0 – 5.0) 0.09 2.0 (0.5-7.5) 3.0 (2.0-5.0) 0.67 - amount of total unpleasantness 9.0 (3.0 – 20.0) 10.1 (5.3 – 18.9) 0.63 6.0 (0.5-27.6) 9.1 (4.8-20.8) 0.63 - amount of total pleasantness 14.3 (10.3 – 23.0) 9.0 (4.0 – 17.8) 0.11 8.0 (1.0-24.5) 10.5 (7.0-16.5) 0.67
134 Environmental factors involved in the development of overt AITD
Daily Hassles At study entrance (n) 38 76 13 26 - total number of daily hassles 18.0 (15.0-28.0) 22.0 (12.0-35.0) 0.50 24.0 (12.5-27.5) 21.5 (8.8-38.3) 0.71 - intensity per hassle 1.2 (1.0-1.4) 1.3 (1.0-1.6) 0.43 1.5 (1.3-1.9) 1.1 (1.0-1.8) 0.12 - total intensity of all hassles 23.5 (15.0-34.1) 28.3 (15.3-46.0) 0.18 29.0 (12.0-53.3) 28.0 (10.3-54.3) 0.78 One year before occurrence of event (n) 31 62 12 24 - total number of daily hassles 20.0 (13.0 – 29.0) 23.0 (14.0 –34.0) 0.31 21.0 (14.5 – 27.8) 19.0 (13.3 – 32.3) 0.92 - intensity per hassle 1.3 (1.0 – 1.6) 1.3 (1.0 – 1.7) 0.84 1.4 (1.3 – 1.7) 1.3 (0.9 – 1.6) 0.28 - total intensity of all hassles 24.0 (16.0 – 47.0) 26.0 (16.0 – 45.4) 0.69 31.5 (19.6 – 42.5) 23.5 (11.3 – 42.5) 0.33 At time of occurrence of event (n) 25 50 9 18 - total number of daily hassles 20.0 (12.0 – 33.0) 22.0 (13.0 –37.0) 0.76 16.0 (10.0 – 29.5) 27.5 (16.8 – 49.0) 0.11 - intensity per hassle 1.4 (0.9 – 1.5) 1.3 (1.0 – 1.6) 0.17 1.1 (1.0 – 1.7) 1.3 (1.3 – 1.8) 0.40 - total intensity of all hassles 24.0 (11.0 – 42.5) 27.0 (16.8 – 48.3) 0.42 19.0 (11.5 – 48.0) 41.0 (19.3 – 61.8) 0.19 PANAS At study entrance (n) 38 76 13 26 - tendency to report negative feelings 21.5 (17.8-27.0) 21.0 (16.3-27.0) 0.67 22.0 (18.0-27.0) 21.5 (15.8-26.5) 0.70 - tendency to report positive feelings 39.1 (35.8-43.0) 38.0 (35.0-42.0) 0.34 39.0 (38.0-41.5) 40.5 (36.0-43.3) 0.68 One year before occurrence of event (n) 35 70 13 26 - tendency to report negative feelings 18.0 (15.0 – 26.0) 21.0 (15.8 – 26.0) 0.63 19.0 (15.0 – 21.5) 18.5 (14.8 – 25.8) 0.60 - tendency to report positive feelings 36.0 (33.0 – 42.6) 37.6 (34.97– 41.5) 0.38 39.3 (34.0 – 42.8) 39.6 (35.5 – 41.5) 0.64 At time of occurrence of event (n) 23 46 9 18 - tendency to report negative feelings 16.0 (14.0 – 21.0) 20.0 (15.0 – 26.3) 0.07 19.0 (15.0 – 25.0) 17.5 (12.5 – 24.5) 0.67 - tendency to report positive feelings 37.1 (35.0 – 42.0) 38.0 (35.0 – 40.1) 0.84 41.0 (31.9 – 41.7) 38.1 (34.8 – 41.8) 0.86
Data are given as medians with interquartile range between parenthesis. P-value of cases versus controls. Time of event: visit when diagnosis was made or confirmed. Cases and matched controls with missing values are omitted from the analysis. PANAS: positive and negative affect scedule.
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REFERENCES
1. Brix TH, Kyvik KO, Christensen K, Hegedus L. Evidence for a major role of heredity in Graves’ disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001; 86:930-934. 2. Brix TH, Kyvik KO, Hegedus L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 2000; 85:536-539. 3. Ringold DA, Nicoloff JT, Kesler M, Davis H, Hamilton A, Mack T. Further evidence for a strong genetic influence in the development of autoimmune thyroid disease: the Californian twin study. Thyroid 2002; 12:647-653. 4. Prummel MF, Strieder T, Wiersinga WM. The environment and autoimmune thyroid diseases. Eur J Endocrinol 2004; 150:605-618. 5. Strieder TGA, Prummel MF, Tijssen JGP, Endert E, Wiersinga WM. Risk factors for and prevalence of thyroid disorders in a cross-sectional study among healthy female relatives of patients with autoimmune thyroid disease. Clin Endocrinol 2003; 59:396- 401. 6. Strieder TGA, Thijssen JGP, Wenzel BE, Endert E, Wiersinga WM. Prediction of progression to overt hypo- or hyperthyroidism in euthyroid female relatives of patients with autoimmune thyroid disease by the THEA (Thyroid Events Amsterdam) score. JAMA 2007, submitted for publication. 7. Strieder TGA, Wenzel BE, Prummel MF, Tijssen JGP, Wiersinga WM. Increased prevalence of antibodies to enteropathogenetic Yersinia enterocolitica virulence proteins in relatives of patients with autoimmune thyroid disease. Clin Exp Immunol 2003; 132:278-282. 8. Van de Willige G, Schreurs P, Telligen B, Zwart F. Het meten van ‘life events’: Vragenlijst Recent Meegemaakte Gebeurtenissen. Ned Tijdschr Psychol 1985; 40:1- 19. 9. Brosschot JF, Benschop RJ, Godaert GL, Olff M, De Smet M, Heynen CJ, Ballieux RE. Influence of life stress on immunological reactivity to mild psychogenic stress. Psychosom Med 1994; 56:216-224. 10. Kanner AD, Coyner JC, Schaefer C, Lazarus RS. Comparison of two modes of stress measurement: daily hassles and uplifts versus major life events. J Behav Med 1981; 4:1-39. 11. Vingerhoets AJJM, Jeninga AJ, Menges LJ. Het meten van chronische en alledaagse stressoren: II. Eerste onderzoekservaringen met de Alledaagse Problemenlijst (APL). Gedrag Gezondh 1989; 17:10-17. 12. Vingerhoets AJJM, Ratliff-Crain J, Jabaaij L, Menges LJ, Baun A. Self-reported stressors, symptoms complaints, and psychological functioning. J Psychosom Res 1996; 40:177-190. 13. Watson D, Clark LA, Tellegen A. Development and validation of brief measures of positive and negative affect: the PANAS scales. J Pers Soc Psychol 1988; 54:1063- 1070. 14. Vestergaard P. Smoking and thyroid disorders – a meta-analysis. Eur J Endocrinol 2002; 146:153-161. 15. Belin RM, Astor BC, Powe NR, Ladenson PW. Smoke exposure is associated with a lower prevalence of serum thyroid antibodies and thyrotropin concentration elevation and a higher prevalence of mild thyrotropin concentration suppression in the third National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2004; 89: 6077-6086.
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16. Asvold BO, Bjoro T, Nilsen TI, Vatten LJ. Tobacco smoking and thyroid function: a population-based study. Arch Int Med 2007; 167: 1428-1432. 17. Martin-du Pan RC. Triggering role of emotional stress and childbirth. Unexpected occurrence of 98 cases of Graves’ disease compared to 95 cases of Hashimoto thyroiditis and 97 cases of thyroid nodules. Ann Endocrinol (Paris) 1998; 59:107-112. 18. Frank P, Kay CR. Incidence of thyroid disease associated with oral contraceptives. Brit Med J 1978; 2:1531. 19. Vestergaard P, Rejnmark L, Weeke J et al. Smoking as a risk factor for Graves’ disease, toxic nodular goiter, and autoimmune hypothyroidism. Thyroid 2002; 12: 69- 75. 20. Winsa B, Adami H-O, Bergström R, Stressful life events and Graves’ disease. Lancet 1991; 338:1475-1479. 21. Sonino N, Girelli ME, Boscaro M. Life events in the pathogenesis of Graves’ disease: a controlled study. Acta Endocrinol 1993; 128:293-296. 22. Kung AWC. Life events, daily stresses and coping in patients with Graves’ disease. Clin Endocrinol 1995; 42: 303-308. 23. Radosavljevic VR, Jankovic SM, Marinkovic JM. Stressful life events in the pathogenesis of Graves’ disease. Eur J Endocrinol 1996; 134: 699-701, 24. Yoshiuchi K, Kumano H, Nomura S et al. Stressful life events and smoking were assiciated with Graves’ disease in women, but not in men. Psychosomatic Med 1998; 60: 182-185. 25. Matos-Santos A, LacerdaNobre E, Costa JGE et al. Relationship between the number and impact of stressful life events and the onset of Graves’ disease and toxic nodular goitre. Clin Endocrinol 2001; 55:15-19. 26. Yoshiuchi K, Kumano H, Nomura S et al. Psychosocial factors influencing the short- term outcome of antithyroid drug therapy in Graves’ disease. Psychosomatic Med 1998; 60:592-596. 27. Fukao A, Takamatsu J, Murakami Y et al. The relationship of psychological factors to the prognosis of hyperthyroidism in antithyroid drug-treated patients with Graves’ disease. Clin Endocrinol 2003; 58:550-555. 28. Oretti RG, Harris B, Lazarus JH, Parkes AB, Crownshaw T. Is there an association between life events, postnatal depression and thyroid dysfunction in thyroid antibody positive women? Int J Psychiatry 2003; 49:70-76.
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General Discussion 9|
General Discussion
Chapter 9 General Discussion
A. FAMILY BACKGROUND B. AGE C. BIOCHEMICAL TESTS D. ENVIRONMENTAL FACTORS E. IMMUNOPATHOGENESIS F. CLINICAL APPLICABILITY G. FUTURE RESEARCH
A. Family background
Our Amsterdam AITD cohort was composed of 803 adult women in self declared good health, with documented proof of one or more 1st or 2nd degree relatives with AITD. At study entrance, the prevalence of autoimmune hypothyroidism was 3.6% (overt hypothyroidism 1.3%, subclinical hypothyroidism 2.3%) and of autoimmune hyperthyroidism 1.9% (overt hyperthyroidism 0.4%, subclinical hyperthyroidism 1.5%) (§3). Our prevalence figures can be compared with those obtained in the general population. For a proper comparison, confounding factors like age, sex and ambient iodine intake should be taken into account. Our cohort was assembled in The Netherlands, an iodine-replete country (1). Because prevalence figures are substantially influenced by ambient iodine intake (2) we focussed on available surveys done in iodine-sufficient countries like the U.K. (3) and the U.S.A. (4,5), excluding studies done in iodine-deficient regions (6,7). The prevalence of unsuspected overt hypothyroidism in the Whickham survey among adult women in the U.K. was 0.3% and of subclinical hypothyroidism 7.5% (3). In the Colorado study (mean age 56 yr, 56% females and 44% males) the prevalence figures in subjects not taking thyroid hormone medication are: overt hypothyroidism 0.4%, subclinical hypothyroidism 8.5%, overt hyperthyroidism 0.1%, subclinical hyperthyroidism 0.9% (4). The NHANES III study in the U.S.A. reports the following prevalences in the disease-free population (that is the total investigated population except those who were pregnant or taking estrogens and those who reported to have thyroid disease, goiter, or thyroid medication): overt hypothyroidism 0.2%, subclinical hypothyroidism in 3.9%, overt hyperthyroidism 0.2%, subclinical hyperthyroidism
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0.2% (not specified for sex) (5). The prevalence figures in our Amsterdam AITD cohort are three times higher than those in the general population, except the prevalence of subclinical hypothyroidism which was about three times lower. The latter can be explained by the difference in age: the mean age of our cohort is 36 yr, 20 years younger than e.g. the median age of 56 yr in the Colorado study; it is well known that the occurrence of subclinical hypothyroidism increases with advancing age (3,4,5). It thus seems reasonable to conclude that the prevalence of autoimmune thyroid dysfunction in the Amsterdam AITD cohort is higher than in the general female population. The finding was to be expected in view of the familial clustering of AITD (8). It must be bear in mind that the prevalence in our study may have been influenced by unintentional selection in the recruitment of participants. In the Amsterdam AITD cohort study we followed 790 women for 5 years (leaving out the 13 subjects with clearly overt thyroid dysfunction at study entrance). The cumulative incidence of overt hypothyroidism and overt hyperthyroidism in the cohort was 7.5% (§7). The calculated hypothyroidism incidence rate was 9.6 /1000 women years in our population, and the hyperthyroidism incidence rate was 3.3 /1000 women years. These incidence figures can be compared to those in two studies from the U.K., the prospective Whickham study (9) and the retrospective Tayside study (10); we did not consider the Danthyr study performed in iodine-deficient Denmark (2). The incidence rates of hypothyroidism in the two English studies were 3.5 and 4.98 /1000 women years, and of hyperthyroidism 0.80 and 0.77 /1000 women years, respectively. The incidence of overt hypothyroidism in our Amsterdam AITD cohort is thus 1.9-2.7 times higher than that in the general female population, and that of overt hyperthyroidism is 4.1-4.3 times higher. The figures once again demonstrate that subjects with a family history of AITD are at increased risk for thyroid disease. The risk in siblings of parents with AITD (with reported λs-values as high as 11.6 for Graves’ disease and 28.0 for Hashimoto’s disease) (8) is much higher than observed in the Amsterdam AITD cohort. This is understandable because relatives with AITD in our study could be children, brothers, sisters, parents, grandparents, uncles or aunts of the participants. Our figures thus fit nicely between the lower risk in the general population and the higher risk in siblings.
The autoimmune nature of thyroid disease in family members of the Amsterdam AITD cohort participants was ascertained by letters from physicians who had treated those family members. This proved to be a slow and tedious process. Definitive evidence of having at least one relative with AITD was obtained in all participants, but definitive evidence of having two or more relatives with AITD was obtained in 55% of participants at study entrance (§3) and in
142 General Discussion
77% of participants in the 5-yr follow-up (§7). The difference is explained by the ongoing efforts of retrieving information about family members, treated often decades ago. The collected data suggest there are AITD families in whom only Graves’ disease or only Hashimoto’s disease occurs. However, we cannot be absolutely sure about this because we had not always the opportunity to investigate all possibly affected family members. Nevertheless, it is certain from our data that Graves’ and Hashimoto’s disease coexist in some AITD families. This suggests a common genetic background. Indeed particular HLA and CTLA-4 polymorphisms have been identified as susceptibility factors for both diseases (11,12). Slow but steady progress is being made in the recognition of other susceptibility genes which are linked specifically to either Graves’ or Hashimoto’s disease (13). Twin studies indicate that genetic factors contribute for about 70% to the risk of developing AITD (14,15). In subjects with a certain genetic make-up environmental factors may determine whether they will develop Graves’ hyperthyroidism or Hashimoto hypothyroidism. The specific nature of the familial background was an independent risk factor for the occurrence of autoimmune hypo- or hyperthyroidism in the 5-yr follow-up, and therefore included in the predictive THEA-score (§7). Hypothyroid events occurred more often in subjects having one or two relatives with Hashimoto disease than in subjects having one or two relatives with Graves’ disease (7.3% vs 3.2%). Hyperthyroid events however, did not occur more often in subjects having one or two relatives with Graves’ disease than in subjects having one or two relatives with Hashimoto disease (1.7% vs 1.6%). Our data are in partial agreement with those of a large U.K. study (16): in Hashimoto hypothyroidism, a family history of hypothyroidism was more common than hyperthyroidism (42.1% vs 22.8% in affected females), but in Graves’ hyperthyroidism a family history of hyperthyroidism in any relative was more frequent than hypothyroidism (30.1% vs 24.4% in affected females). The discrepancy with regard to Graves’ disease between our cohort and the U.K. study could be due to the much larger sample size of the U.K. study; on the other hand, The U.K. study is liable to bias because a family history of thyroid dysfunction was recorded on the basis of subject recall and not checked objectively as in the Amsterdam AITD cohort.
B. Age
The mean age of the Amsterdam AITD cohort was 36 yr at study entrance, but participants who at that time already had an abnormal TSH were older (43 yr in the hypothyroid group and 44 yr in the hyperthyroid group) (§3). Age was an independent risk factor for hypothyroidism
143 Chapter 9 but not for hyperthyroidism in our cohort. This is in accordance with surveys in the general population, all reporting a higher prevalence of hypothyroidism with advancing age (3,4,5); the relation was not observed for hyperthyroidism (9). The mean age at diagnosis of hypothyroidism and hyperthyroidism (e.g. 57 yr and 48 yr respectively in the Whickham survey) in the general population is higher than in our cohort. However, these population- based studies include all causes of thyroid dysfunction. In this respect a more proper comparison is with the U.K. study also dealing exclusively with autoimmune hypo- and hyperthyroidism (16): the median age at diagnosis of both Hashimoto hypothyroidism and Graves’ hyperthyroidism was 41 yr (for both sexes), a figure close to our observed ages of 43 and 44 yr. The peak ages for diagnosis of both Graves’ and Hashimoto disease in the U.K. study were the fourth to sixth decades. Higher age was an independent determinant of the presence of TPO antibodies in the euthyroid participants of the Amsterdam AITD cohort at baseline. This finding is in complete agreement with the higher prevalence of TPO antibodies with advancing age in the disease- free population of the NHANES III study (5,17). In the 5-yr follow-up of the Amsterdam AITD cohort, age was not an independent risk factor for the occurrence of autoimmune hypo- or hyperthyroidism according to multiple logistic regression analysis, and consequently was not included in the predictive THEA-score (§7). The trend to a higher event rate in older age was nonsignificant, probably due to the small number of subjects in the age group 60 to 65 yr and an overriding effect of higher TPO antibodies concentration with advancing age (9). The mean age at which participants in our cohort developed overt hypothyroidism or hyperthyroidism during follow-up, was 41 yr and 44 yr respectively (§8): again, very close to the median age of 41 yr in the U.K. study (16). It is interesting to mention that in the U.K. study the median age in women who had Graves’ hyperthyroidism, was lower in those with a family history of AITD than in those without (38 yr vs 43 yr), and the same was true for women with Hashimoto hypothyroidism (38 yr vs 48 yr). The U.K. study also reports an inverse relationship between the number of relatives with thyroid dysfunction and age at diagnosis, but this has yet not been assessed in our cohort.
C. Biochemical tests
C.1. Thyroid function The prevalence of an abnormal TSH in the Amsterdam AITD cohort at baseline was 5.5% (§3). In the disease-free population of NHANES III the prevalence of an abnormal TSH was
144 General Discussion
7.1% for all ages, falling to 4.9% when excluding subjects with thyroid antibodies (17). Among our participants with a normal TSH at baseline, TSH was higher in subjects with TPO antibodies than without TPO antibodies (2.3 vs 1.6 mU/l), although FT4 was not different between both groups (§3). This interesting finding has later been confirmed in other studies (17). The presence of TPO antibodies indicate the existence of chronic lymphocytic thyroiditis, which may slightly compromise thyroid hormone synthesis and release; it results in a slight change of TSH towards higher values but still within the normal reference range. FT4 values were similar irrespective of the presence or absence of TPO antibodies; this can be explained by the exquisite sensitivity of pituitary thyrotrophs to respond to minute changes in circulating FT4, as illustrated by the log-linear relationship between TSH and FT4. In the 5-yr follow-up of the Amsterdam AITD cohort TSH (but not FT4) levels at baseline were an independent risk factor for the occurrence of overt hypo- or hyperthyroidism, and therefore included in the predictive THEA-score (§7). The risk was dose-dependent, and not restricted to TSH levels outside the reference range (two of the 13 hyperthyroid cases (15.4%) had a baseline TSH <0.4mU/l, and seven of the 38 hypothyroid cases (18.4%) had a baseline TSH >5.7mU/l. The risk clearly increased at TSH above 2.0 mU/l, that is at values within the customary reference range. The cut-off of 2.0 mU/l is exactly the same as observed in the twenty-year follow-up of the Whickham survey, in which the probability of developing hypothyroidism increased sharply at baseline TSH values >2.0 mU/l irrespective of the presence of thyroid antibodies (9). The finding has two important implications. First, optimal thyroid health might be associated with TSH levels between 0.4 and 2.0mU/l. Secondly, the upper normal limit of TSH could or should be lowered to 2.0mU/l. The latter notion has been strengthened by the observation that in reference populations the distribution of TSH values is not normal but skewed to higher values. Proponents and opponents of a narrower TSH reference range hold each other in balance (18,19). We ourselves, are of the opinion that the upper normal limit of TSH should not be lowered to 2.0 mU/l: it will label a sizable proportion of the population as being abnormal, thereby creating a huge burden to health services, without any proof that medical intervention in subjects with TSH values between 2.0 and 4.0 mU/l improves long-term health outcomes (20). The nested case-control study in the 5-yr follow-up of the Amsterdam AITD cohort demonstrated higher TSH and lower FT4 values in the hypothyroid cases than in controls, both at baseline and one year before the event (§8). The absolute change in TSH values in this time period was greater in cases than in controls (∆TSH 1.1 [0.5-2.1] vs 0.4 [0.1-0.9] mU/l, p <0.000). In contrast, TSH and FT4 values were not different between hyperthyroid cases and
145 Chapter 9 their controls, neither at baseline nor at one year before the event. The absolute change in TSH in this time period also did not differ between cases and controls ( ∆TSH 0.2 [0.0-0.5] vs 0.3 [0.0-0.6] mU/l, p=0.6). No differences were found in absolute changes in FT4 between hypothyroid/hyperthyroid cases and their respective controls. The findings suggest that progression towards overt autoimmune hypothyroidism is a gradual process taking several years, but that in contrast overt autoimmune hyperthyroidism develops faster in terms of months rather than years.
C.2. Thyroid antibodies The prevalence of TPO-Ab was 27% and of Tg-Ab 8% in the Amsterdam AITD cohort at study entrance. The prevalence was higher in subjects with an elevated TSH (TPO-Ab 86%, Tg-Ab 24%) than in subjects with a suppressed TSH (TPO-Ab 53%, Tg-Ab 20%; in subjects with a normal TSH the prevalence of TPO-Ab was 24% and of Tg-Ab 7% (§3). Our observed overall prevalence of 27% of TPO-Ab among 1st and 2nd degree relatives of AITD patients lies nicely in between the lower prevalence of TPO-Ab in the general population (10.3% in the Whickham survey, 13.0% in NHANES III) (3,5) and the higher prevalence of TPO-Ab in first-degree relatives of AITD patients (43-48%) (21,22). The findings once again underline the notion that subjects with a family history of AITD are at increased risk for thyroid antibodies. Twin studies (23) and linkage analysis (24) confirm the strong genetic contribution to the development of thyroid antibodies. The lower prevalence of Tg-Ab in comparison with TPO-Ab is in agreement with population studies. Also, the lower prevalence of TPO-Ab and Tg-Ab in hyperthyroid subjects in comparison with hypothyroid subjects is well known. Of interest is the dose-dependent effect of TPO-Ab on TSH values in the participants with a normal TSH, as discussed above in section C.1. TPO-Ab > 100 kU/l (but not Tg-Ab) constitute an independent dose-dependent risk factor for the occurrence of overt thyroid dysfunction in the 5-yr follow-up of the Amsterdam AITD cohort, and are consequently included in the productive THEA-score (§7). This was true for both autoimmune hypothyroid and hyperthyroid events. Baseline TPO-Ab >100 kU/l were observed in 30 of the 38 hypothyroid cases (79%) and in 9 of the 13 hyperthyroid cases (69%). The data are in good agreement with the 20-yr follow-up of the Whickham survey (9): in women, the odds ratios with 95% confidence intervals were 8 (3-20) if serum TSH alone was raised, 8 (5-15) if thyroid antibodies alone were positive, and 38 (22-65) if serum TSH was raised and thyroid antibodies were positive. The fact that Tg-Ab in our cohort did not contribute to the risk, is likely related to the unknown biologic relevance (if any at all) of Tg-
146 General Discussion
Ab; in contrast, TPO-Ab contribute in the immunopathogenesis of AITD by complement- fixation and antibody-dependent cell-mediated cytotoxixity. The nested case-control study in the 5-yr follow-up of the Amsterdam AITD cohort demonstrated higher TPO-Ab and Tg-Ab levels in hypothyroid cases than in controls, both at study entrance and at one year before the event (§8). The same was true for the hyperthyroid cases and their controls, although less pronounced for Tg-Ab. The absolute change in TPO- Ab over this time period was greater in hypothyroid cases than in their controls ( ∆TPO-Ab 385 [85-853] vs <30 [<30-<30], p<0.000), but not different between hyperthyroid cases and their controls ( ∆TPO-Ab <30 [<30-242] vs [<30-48], p=0.8). Like mentioned in section C.1 for TSH, the data suggest a gradual slow development of autoimmune hypothyroidism, whereas overt autoimmune hyperthyroidism develops much faster. The latter notion is further substantiated by the finding that one year before the occurrence of autoimmune hyperthyroidism causative antibodies against the TSH receptor (TBII) were not demonstrable. In line is the very low prevalence of TBII in the general population as well as in our cohort.
D. Environmental factors
D.1. Smoking The prevalence of ever smoking was 58% and of current smoking 35% in the Amsterdam AITD cohort at study entrance. The figures for ever smoking among participants with an elevated, normal or suppressed TSH are 69%, 57% and 47%, and for current smoking 42%, 35%, and 13% respectively; differences between those with an abnormal and a normal TSH were not significant. The prevalence of current smoking (but not of ever smoking) among the euthyroid subjects with a normal TSH was lower in those with TPO-Ab than in those without TPO-Ab (25% vs 38%, p<0.001) (§3). Smoking behavior at study entrance did not contribute to the risk of developing overt hypo- or hyperthyroidism in the 5-yr follow-up of the Amsterdam AITD cohort (§7). However, smoking behavior did change in some subjects during follow-up, and in the nested case-control study there were less current smokers in the hypothyroid cases than in their controls (13% vs 28%, p=0.08) at the time of the event; this was not observed in the hyperthyroid cases and their controls (§8). Of particular interest is the finding that current smoking appears to be protective against the development of TPO-Ab. Subsequent studies have confirmed this original observation. Data from the NHANES III population published in 2004 indicated that fewer smokers (11%, 95% CI 10-13%) had TPO-Ab and/or Tg-Ab compared to nonsmokers (18%, 95 CI 17-19%) (25).
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The relationship persisted when analyzing the presence of one antibody independently of the status of the other antibody. The odds of having thyroid antibodies in serum was lower by 1.1% for every 10 ng/ml increase in serum cotinine after adjustment for confounders. A recent Danish population study published in 2008 also reports a negative association between smoking and the presence of TPO-Ab and Tg-Ab (26). Odds ratio’s were in the order of 0.5- 0.6 (comparable to the OR of 0.688 in our cohort), with no difference in the risk between moderate and heavy smokers neither between never and ex-smokers. One may thus ask the question if smoking decreases the risk on hypothyroidism. Studies in the past have shown conflicting results, and a meta-analysis in 2002 could not prove a negative association between smoking and hypothyroidism although it detected a trend (27). A Danish study from 2002 found a lower prevalence of mild hypothyroidism among smokers compared to nonsmokers (2.6% vs 5.3%) with an adjusted odds ratio of 0.47 (95% CI 0.33- 0.67) (28). In the NHANES III smokers had less frequently an elevated TSH value (>4.5mU/l) compared with nonsmokers (2.6%, 95% CI 2.0-3.2% vs 5.5%, 95% CI 4.7-6.3%) (25). A recent Norwegian population study from 2007 reported a lower prevalence of overt and subclinical hypothyroidism among current smokers compared to never smokers (odds ratio’s are 0.60, 95% CI 0.38-0.95 for overt hypothyroidism and 0.54, 95% CI 0.45-0.66 for subclinical hypothyroidism, both in women) (29). The studies do not distinguish between the various causes of hypothyroidism, but most likely the majority will be due to chronic lymphocytic thyroiditis. In line with these recent studies is our nested case-control study showing a protective effect of smoking on the development of overt autoimmune hypothyroidism, although the p value of 0.08 was just not significant most likely due to a limited sample size. The available data strongly suggest that the preventive effect of smoking on hypothyroidism is – at least partly – explained by the preventive effect of smoking on the development of TPO-Ab. In sharp contrast to the findings in hypothyroidism are the many studies showing that smoking is a risk factor for Graves’hyperthyroidism (with an odds ratio of 1.9, 95% CI 1.1- 3.2 in a particular case-control study) (30). We did not observe this association in our cohort studies, most likely due to the small number of subjects with a suppressed TSH. Thus the peculiar situation arises that refraining from smoking increases the risk on Graves’ disease. Nevertheless the recommendation should be to stop smoking in view of the many other adverse health outcomes associated with smoking.
D.2. Exogenous estrogens
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Exogenous estrogens in our studies comprise both oral contraceptive pills and hormone replacement therapy. The prevalence of ever estrogen use in the Amsterdam AITD cohort at baseline was lower in participants with a suppressed TSH than with a normal TSH (67% vs 81%), and this was also true for current estrogen use (13% vs 46%) (§3). No differences existed between those with an elevated and a normal TSH. Ever estrogen use (but not current estrogen use) was an independent (protective) risk factor for hyperthyroidism according to multiple logistic regression analysis (OR 0.169, 95% CI 0.055-0.523). In the subjects with a normal TSH, the prevalence of ever and current estrogen use was lower in the absence than in the presence of TPO-Ab, but only ever estrogen use was an independent (protective) risk factor for TPO-Ab (OR 0.578, 95% CI 0.345-0967) (§3). In the 5-yr follow-up of the Amsterdam AITD cohort estrogen medication at baseline had no predictive value for the occurrence of overt hypo- or hyperthyroidism (§7). Also in the nested case-control study evidence for a modulating effect of estrogen use on the occurrence of an event was not found (§8). Oral contraceptives are used by over 100 million women worldwide, but there are remarkably few studies on their use and the development of AITD. An early large study among 46000 women found that the incidence of hypo- and hyperthyroidism together was lower in oral contraceptive users than in controls (RR 0.68, 95% CI 0.52-0.85) (31). Although we did not find a protective effect of estrogens in the 5-yr follow-up of the Amsterdam AITD cohort (which has still a limited sample size), the prevalence of Graves’ hyperthyroidism at baseline was lower in estrogen users independent of the number of previous pregnancies. This is in agreement with the observation that the use of contraceptives had a protective effect for the development of Graves’ disease (odds ratio 0.68, 95% CI 0.49-0.93), but not for Hashimoto disease (32). With regard to the effect of estrogens on TPO-Ab, our finding has not been confirmed in a recent population study reporting no association between thyroid antibodies and the use of oral contraceptives, although women aged 60-65 years receiving hormone replacement therapy now or previously had a lower prevalence of Tg-Ab, but not of TPO-Ab (33).
D.3. Pregnancy The prevalence of having been pregnant in the Amsterdam AITD cohort at study entrance was higher in participants with a suppressed TSH than with a normal TSH (87% vs 51%), and pregnancies were an independent risk factor for hyperthyroidism according to multiple logistic regression analyses (odds ratio 6.88, 95% CI 1.50-30.96) (§3). The prevalence of
149 Chapter 9 pregnancies was not associated with an elevated TSH, nor was pregnancy an independent risk factor for the presence of TPO-Ab in subjects with a normal TSH at baseline (§3). A history of pregnancy at baseline did not contribute to the risk of developing overt autoimmune hypo- of hyperthyroidism in the 5-yr follow-up of the Amsterdam AITD cohort (§7). In the nested case-control study parity did not differ between cases and controls at study entrance (§8). However, at the time of the event there were more hypothyroid cases in the postpartum period than controls (21% vs 4%), whereas the proportion of women who had never been pregnant was lower in hyperthyroid cases than in their controls (8% vs 38%), both at one year before the event and at the times of the event. Our data suggest that the postpartum period carries a risk for autoimmune hypothyroidism, and that never been pregnant protects against autoimmune hyperthyroidism. The year after delivery puts the woman at risk for AITD, as illustrated by the high incidence of postpartum thyroiditis (5.2% in a Dutch population study) (34). Late development (after five years or more) of permanent hypothyroidism occurs in 25% of patients with postpartum thyroiditis (35). The prevalence of postpartum Graves’ disease (both persistent and transient) is estimated at 11% of those with postpartum thyroid dysfunction and 0.54% of the general population (36). Recent large population-based studies report conflicting results on the association between parity and AITD. An Australian study found (after adjustment for age) that women who had previously been pregnant did not have a significantly increased risk of positive thyroid antibodies or a raised or reduced TSH compared with women who had never been pregnant (37). Likewise, a Danish study failed to demonstrate an association between previous pregnancy, parity and thyroid antibodies (33). In contrast, a German study did find an association between parity and AITD (38). Women with at least one pregnancy had increased odds for AITD (OR 4.6, 95% CI 1.4-15.1) compared to women who had never been pregnant. Similar results were obtained using positive TPO-Ab (OR 1.8, 95% CI 1.0-3.3) as separate dependent variable or using number of birth as alternate independent variable.
D.4. Yersinia enterocolitica The prevalence of Yersinia enterocolitica (YE) infection in the Amsterdam AITD cohort at study entrance is higher than in controls, both for YOP IgG-Ab (40% vs 20%) and for YOP IgA-Ab (22% vs 13%) (§4). The prevalence of these antibodies did not differ between subjects with a normal or abnormal TSH, nor was it associated with the presence of TPO-Ab. YOP antibodies at study entrance did not predict the occurrence of overt thyroid dysfunction in the 5-yr follow-up of the Amsterdam AITD cohort (§7). In the nested case-control study
150 General Discussion the proportion of subjects positive for YOP-IgG and YOP-IgA did not differ between hypothyroid or hyperthyroid cases and their respective controls, neither at baseline nor at one year before the event (§8). Infection with YE has been shown to produce a lipoprotein that can cross-react with the TSH receptor and thus can act as a potential trigger of thyroid autoimmunity, in particular Graves’ disease (39). Our Amsterdam AITD cohort studies, both at baseline and during follow-up, did not provide any evidence that YE infection is causally related to AITD. The observed higher prevalence of YOP antibodies in our cohort relative to a control group, suggesting a higher rate of persistent YE infection in AITD relatives, could be explained by assuming that susceptibility genes for AITD may also confer a risk for YE infection. Our results are in agreement with a more recent Danish twin study, reporting that YE infection does not confer an increased risk of thyroid antibodies (40). The same group of authors, however, concluded in their latest paper that it is too early to dismiss YE infection in the etiology of Graves’ disease (41). In a classical case-control study the prevalence of YOP- IgG and YOP-IgA in Graves’ disease was higher than in controls (51% vs 35% and 49 vs 34% respectively). Similar results were found in twin pairs discordant for Graves’ disease. Odds ratio’s were significantly increased. Clearly, the issue has not been settled yet, and longitudinal studies examining temporal relationships are required.
D.5. Iodine excess Iodine excess in the Amsterdam AITD cohort was assessed according to the participant’s history of taking iodine-containing nutritional supplements, kelp tablets, seaweed, iodine containing drugs like amiodarone, or iodine-containing contrast agents. The prevalence of selfreported iodine excess over the preceding year excess was 7% at baseline, with no differences between participants with an abnormal or normal TSH, nor in the normal TSH group between those with or without TPO-Ab (§3). Iodine excess at baseline was not a predictive factor for the occurrence of an event in the 5-yr follow-up of the Amsterdam AITD cohort (§7). The occurrence of an event was also not related to exposure to iodine excess in the preceding years as evident from the nested case-control study (§8). This is not to deny that cases of iodine-induced hypothyroidism and Jod-Basedow do occur. Their incidence in the general population is very low, and iodine excess accounts for only a small proportion of all hypothyroid and hyperthyroid patients. Amiodarone carries a rather high risk for induction of thyroid dysfunction, but none of the cohort participants used this drug.
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D.6. Stress Stress exposure in the Amsterdam AITD cohort was assessed by three validated questionnaires: the Recently Experienced Stressful Life Events (counting the number and impact of major life events experienced in the past 12 months), the Dutch Everyday Problem Checklist (counting the number and intensity of daily hassles experienced in the last 2 months), and the positive and negative affect schedule (the PANAS, measuring the current mood in terms of positive and negative effect). Among the euthyroid subjects with a normal TSH of the Amsterdam AITD cohort at study entrance, stress scores were not different between those with or without TPO-Ab (§5). Stress exposure in the 5-yr follow-up of the Amsterdam AITD cohort was not associated with the occurrence of autoimmune hypo- or hyperthyroidism as evident from the nested case-control study (§8). We rejected our hypothesis that stress might be associated with the presence of TPO-Ab, an early marker of AITD. Likewise we did not find evidence for a higher stress exposure preceding the onset of overt autoimmune hypothyroidism. There are only two other studies on a possible association between stress and Hashimoto disease. A cross-sectional French study concluded that major stressors did not have any triggering role in Hashimoto disease (42). An English study in pregnant women did not report an excess of life events in women who developed postpartum thyroid dysfunction, and there was no difference in the number of life events between antibody positive and antibody negative women (43). Stress was evaluated just once in both studies: when the disease was clearly diagnosed (42) and at 8 weeks postpartum (43). In this respect our longitudinal study is more reliable, although equally negative in its conclusions. The absence of an effect of stress exposure on the occurrence of autoimmune hyperthyroidism in our nested case-control study is most likely due to the small number (n=13) of hyperthyroid cases. Indeed, most of the recent case-control studies have supported stress as a factor that affects the onset and clinical course of Graves’ disease (44). However, these studies have been retrospective in nature and liable to recall bias. There is another interesting issue related to the PANAS scores in our cohort, although highly speculative. The PANAS scores of cohort participants at study entrance are higher than those observed in a non-clinical sample broadly representative of the general adult female UK population (n=537, mean age 42.9 ± 15.7 yr) (45): median negative affect scores in our study vs the UK study are 21 and 15 respectively, and median positive affect scores are 39 and 31 respectively. In most clinical contexts, the concern will be whether negative affect scores (reference range 10-42) are unusually high, and whether positive affect scores (range 10-50)
152 General Discussion are unusually low. A high positive affect score points to neuroticism, a low negative affect score is typical for a depressed affect. The high positive and negative affect scores in our AITD cohort can be interpreted as a reactive affect balance style (46).
E. Immunopathogenesis
The Amsterdam AITD cohort study was primarily designed to assess which environmental factors contribute to the development of AITD, not to investigate the mechanism by which identified environmental factors modulate thyroid autoimmunity. Nevertheless, the cohort allowed doing some studies providing further insight in the immunopathogenesis of AITD as described in §6. Key element in the immune response is the presentation of antigen (bound to MHC class II molecules on antigen-presenting cells) to T cell receptors on CD4+ T-cells. Formation of the complex is followed by activation of the CD4+ T-cell, a process that involves expression of interleukin-2 (IL-2) receptor and autocrine stimulation by IL-2 release and leads to T-cell proliferation, secretion of other cytokines, and thus the development of effector function. Activated CD4+ T-cells can, depending on their pattern of cytokine secretion, develop into type 1 helper (Th1) cells (leading to a destructive process as seen in organ-specific endocrinopathies) or type 2 helper (Th2) cells promoting antibody production. Other T-cell subsets exist. In the past suppressor functions have been assigned to the typically cytotoxic CD8+ T-cells, but the most promising regulatory T-cell subset (specifically with regard to autoimmune diseases) is CD4+CD25+ (IL-2 receptor). A defect in CD4+CD25+ T regulatory cells breaks the immunological tolerance (47). A mutation in the FOXP3 gene encoding for a transcription factor expressed on CD4+CD25+ T-cells, is associated with a decreased number of T regulatory cells (48,49). Against this background we investigated whether activated CD4+ T-cells could be an early marker of AITD. To this end we investigated participants of the Amsterdam AITD cohort who were euthyroid at study entrance, contrasting those with and without TPO-Ab (§6). We observed a reduced expression of CD25 on CD4+ T-cells and lower levels of soluble IL-2 receptors in serum in our female relatives of AITD patients as compared to controls; there were no differences between TPO-Ab positive and negative women. Our study suggests that women at risk for AITD exhibit various signs of a reduced T-cell activation, in particular signs of a reduced expansion of the T-cell pool, also already in those without thyroid antibodies.
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In this study we made some further observations on the effect of smoking and estrogen use on the immune response. Smoking activates the IL-2/IL-2R system, and smokers in our study (in contrast to nonsmokers) had near normal sIL-2R serum levels and a slightly higher percentage of CD4+CD25+ T-cells. Hence smoking may have corrected the reduced T-cell functions in our AITD relatives, in line with the protective effect of smoking on the presence of TPO-Ab (see D.1). Estrogens suppress IL-2 and the IL-2R, and are immunosuppressive in many autoimmune diseases (see D.2). In our AITD relatives current estrogen use was associated with lower percentages of CD4+CD25+ T-cells compared to nonusers, but this was not reflected by extra reduced sIL-2R levels. These findings are in contrast with a study reporting that estrogens drive expansion of the CD4+CD25+ regulatory T-cell compartment (50). The weak point of our study, however, is the small size of subgroups.
F. Clinical applicability
The clinical applicability of our findings in the Amsterdam AITD cohort is described in §7. An accurate simple predictive score (called the Thyroid Events Amsterdam or THEA score) was developed to estimate the 5-yr risk of overt autoimmune hypothyroidism or hyperthyroidism in female relatives of AITD patients. The numerical THEA score predicts events by weighting the three independent risk factors: TSH, TPO-Ab, and family background. The higher the THEA score, the higher the risk on developing overt thyroid dysfunction within 5 years. The THEA score can be useful in day-to-day clinical practice. For, many patients with AITD ask their treating physician about the risk that their daughters or sisters will also get this disease. Assessment of the THEA score in family members allows answering this question in an evidence-based quantitative manner. At low THEA scores (0-7) subjects can be reassured of a very high likelihood of maintaining a normal thyroid function in the next 5 years. If THEA scores are higher, reassessment after 1 yr might be warranted. The THEA score might be very useful for young women of AITD families who want to become pregnant in the next few years. Screening of thyroid function and thyroid antibodies seems to be indicated in these women in view of adverse pregnancy outcomes for mother and child when thyroid function is abnormal (51), but also when thyroid function is normal but TPO-Ab are present (52). Thyroxine treatment should be considered under these circumstances. The high abortion rate among euthyroid pregnant women with TPO-Ab is reduced to the abortion rate in TPO-Ab
154 General Discussion negative women by levothyroxine treatment, as reported in a recent randomized clinical trial (53). It is unknown whether the THEA-score is also applicable in male AITD relatives. Very likely this will be the case, although the weights attached to TSH and TPO-Ab concentrations in the THEA score might be different in view of their higher odds for developing hypothyroidism in males than in females in the 20-yr follow-up of the Whickham study (9): in males, the odds ratio’s are 44 (95% CI 19-104) for raised serum TSH alone, 25 (95% CI 10-63) for positive thyroid antibodies alone, and 173 (95% CI 81-370) for both raised serum TSH and positive thyroid antibodies (for comparison with the odds in females, see section C.2). However, the overall risk to develop AITD is much lower in males. Therefore, one might speculate that at a given THEA-score the risk to develop overt hypothyroidism in the next 5 years will be greater in men than in women.
G. Future research
The Amsterdam AITD cohort provides many opportunities for future research studies. First, many more cases of overt hypo- and hyperthyroidism will occur among the participants who were still clinically euthyroid at the end of the study. An extended follow-up would be very valuable. Second, better characterization of T-cell subsets (especially of regulatory T-cells) might provide deeper insight in the immunopathogenesis of AITD at its early stages. Third, assessment of polymorphisms in susceptibility genes for AITD and incorporating these genetic risk factors in the THEA-score, may enhance the predictive value of the THEA-score. Fourth, the cohort allows studying gene-environment interactions. Such studies have scarcely been done in the field of thyroid autoimmunity. A notable exception is a recent study on genetic polymorphisms associated with cigarette smoking and the risk of Graves’ disease (54). Last but not least, the cohort opens up the possibility to do preventive intervention studies, Prevention of disease is better than treatment of disease, but has so far hardly been given any attention in AITD. The ideal population for preventive intervention would be subjects at risk for AITD but in whom thyroid function is still normal. The Amsterdam AITD cohort has identified such subjects: female relatives of AITD patients, in whom TSH is normal but TPO-Ab are already present. The question then is, which kind of preventive measure should be tested in a prospective controlled trial apart from advice promoting healthy lifestyles. Intervention with selenium in these early stages of thyroid autoimmunity might be a very attractive option. Selenium plays a role in the immune system, and acts as an anti-
155 Chapter 9 oxidant (55). Selenium deficiency results in increased H2O2 levels in the thyroid gland, presumably increasing the activity and immunogenecity of TPO. Indeed in subjects on thyroxine replacement TPO-Ab concentrations fall upon selenium supplementation, as shown in several randomized clinical trials (56, 57, 58). Selenium supplementation has also a favourable effect on postpartum thyroid status in pregnant women with thyroid antibodies (59). It remains to be seen, however, if selenium supplementation in euthyroid subjects with TPO-Ab can prevent the development towards subclinical and overt autoimmune hypothyroidism.
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References
1. Wiersinga WM, Podoba J, Srbeoky M, et al. A survey of iodine intake and thyroid volume in Dutch schoolchildren: reference values in an iodine-sufficient area and the effect of puberty. Eur J Endocrinol 2001; 144: 595-611. 2. Bülow Pedersen I, Knudsen N, Jorgensen T, et al. Large differences in incidences of overt hyper- and hypothyroidism associated with a small difference in iodine intake: a prospective comparative register-based population survey. J Clin Endocrinol Metab. 2002; 87: 4462-4469. 3. Tunbridge WM, Evered DC, Hall R, et al. The spectrum of thyroid disease in a community: the Whickham survey. Clin Endocrinol 1977; 481-493. 4. Canaris GJ, Manowitz NR, Mayor G, Ridgway EC. The Colorado thyroid disease prevalence study. Arch Intern Med 2000; 160: 526-534. 5. Hollowell JG, Staehling NW, Hannon WH, et al. Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab. 2002; 87: 489-499. 6. Aghini-Lombardi F, Antonangeli L, Martino E, et al. The spectrum of thyroid disorders in an iodine-deficient community: The Pescopagano Survey. J Clin Endocrinol Metab 1999; 84: 561-566. 7. Knudsen N, Jorgensen T, Rasmussen S, et al. The prevalence of thyroid dysfunction in a population with borderline iodine deficiency. Clin Endocrinol 1999; 51: 361-367. 8. Villanueva R, Greenberg DA, Davies TF, Tomer Y. Sibling recurrence risk in autoimmune thyroid disease. Thyroid 2003; 13: 761-764. 9. Vanderpump MP, Tunbridge WM, French JM et al. The incidence of thyroid disorders in the community: a twenty-year follow-up of the Whickham Survey. Clin Endocrinol. 1995; 43: 55-68. 10. Flynn RWV, MacDonald TM, Morris AD, Jung RT, Leese GP. The Thyroid epidemiology, audit, and research study: thyroid dysfunction in the general population. J Clin Endocrinol Metab. 2004; 89: 3879-3884. 11. Ban Y, Tomer Y. The contribution of immune regulatory and thyroid specific genes to the etiology of Graves’ and Hashimoto’s diseases. Autoimmunity. 2003; 36: 367-379. 12. Ban Y, Greenberg DA, Davies TF et al. Linkage analysis of thyroid antibody production: evidence for shared susceptibility to clinical autoimmune thyroid disease. J Clin Endocrin Metab. First published ahead of print June 17, 2008 as doi: 10.1210/jc.2008-03647. 13. Jacobson EM, Tomer Y. The CD40, CTLA-4, thyroglobulin, TSH receptor, and PTPN22 gene quintet and its contribution to thyroid autoimmunity: back to the future. J Autoimmunity 2007; 28: 85-98. 14. Brix TH, Kyvik KO, Christensen K, Hegedus L. Evidence for a major role of heredity in Graves’ disease: a population-based study of two Danish twin cohorts. J Clin Endocrinol Metab 2001; 86: 930-934 25. 15. Brix TH, Kyvik KO, Hegedus L. A population-based study of chronic autoimmune hypothyroidism in Danish twins. J Clin Endocrinol Metab 2000; 85: 536-539. 16. Manji N, Carr-Smith ID, Boelaert K, et al. Influences of Age, Gender, Smoking, and Family History on Autoimmune Thyroid Disease Phenotype. J Clin Endocrinol Metab 2006; 91: 4873-4880. 17. Surks MI, Hollowell JG. Age-specific distribution of serum thyrotropin and antithyroid antibodies in the US population: implications for the prevalence of subclinical hypothyroidism. J Clin Endocrinol Metab 2007; 92: 4575-4582.
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18. Wartofsky L, Dickey RA. Controversy in clinical endocrinology: the evidence for a narrower thyrotropin reference range is compelling. J Clin Endocrinol Metab 2005; 90: 5483-5488. 19. Surks MI, Goswami G, Daniels GH. Controversy in clinical endocrinology: the thyrotropin reference range should remain unchanged. J Clin Endocrinol Metab 2005; 90: 5489-5496. 20. Brabant G, Beck-Peccoz P, Jarzab B et al. Is there a need to redefine the upper normal limit of TSH? Eur J Endocrinol 2006; 154: 633-637. 21. Prentice LM, Philips DI, Sarsero D et al. Geographical distribution of subclinical autoimmune disease in Britain: a study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinol 1990; 123: 493- 498. 22. Philips D, McLachlan S, Stephenson A et al. Autosomal dominant transmission of autoantibodies to thyroglobulin and thyroid peroxidase. J Clin Endocrinol Metab 1990; 70: 742-746. 23. Brix TH, Hansen PS, Kyvik KO, Hegedus L. Aggregation of thyroid autoimmune thyroid disease is mainly due to genes: a twin study. Clin Endocrinol 2004; 60: 329-334. 24. Ban Y, Greenberg DA, Davies TF et al. Linkage analysis of thyroid antibody production: evidence for shared susceptibility to clinical autoimmune thyroid disease. J Clin Endocrinol Metab. First published ahead of print June 17, 2008 as doi: 10.1210/jc.2008- 0364. 25. Belin RM, Astor BC, Powe NR, Ladenson PW. Smoke exposure is associated with a lower prevalence of serum thyroid autoantibodies and thyrotropin concentration elevation and a higher prevalence of mild thyrotropin concentration suppression in the third National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab. 2004; 89: 6077-6086. 26. Pedersen IB, Laurberg P, Knudsen N et al. Smoking is negatively associated with the presence of thyroglobulin antibody and to a lesser degree with thyroid peroxidase antibody in serum: a population study. Eur J Endocrinol 2008; 158: 367-373. 27. Vestergaard P. Smoking and thyroid disorders – a meta-analysis. Eur J Endocrinol 2002; 146: 153-161. 28. 28 Knudsen N, Bulow I, Laurberg R. et al. The high occurrence of thyroid multinodularity and low occurrence of subclinical hypothyroidism among tobacco smokers in a large population study. J Endocrinol. 2002; 175: 571-576. 29. 29. Asvolk BO, Bjoro T, Nilsen TI, Vatten LJ. Tobacco smoking and thyroid function: a population-based study. Arch Int Med 2007; 167: 1428-1432. 30. Prummel MP, Wiersinga WM. Smoking and risk of Graves’ disease. JAMA 1993; 269: 479-482. 31. 31. Frank P, Kay CR. Incidence of thyroid disease associated with oral contraceptives. Brit Med J 1978; 2: 1531. 32. 32. Vestergaard P, Rejnmark L, Weeke J et al, Smoking as a risk factor for Graves’ disease, toxic nodular goiter, and autoimmune hypothyroidism. Thyroid 2002; 12: 69-75. 33. Bülow Pedersen I, Laurberg P, Knudsen N et al. Lack of association between thyroid autoantibodies and parity in a population study argues against microchimerism as a trigger of thyroid autoimmunity. Eur J Endocrinol 2006; 154: 39-45. 34. 34. Kuypens JL, Pop VJ, Vader HL et al. Prediction of postpartum thyroid dysfunction: can it be improved? Eur J Endocrinol 1998; 139: 36-43. 35. Othman S, Philips DIW, Parkes AB et al. A long-term follow-up of postpartum thyroiditis. Clin Endocrinol 1990; 32: 559-564.
158 General Discussion
36. Hidaka H, Tamaki H, Iwatani Y et al. Prediction of postpartum onset of Graves’ thyrotoxicosis by measurement of thyroid stimulating antibody in early pregnancy. Clin Endocrinol 1994; 41: 15-20. 37. Walsh JP, Bremner AP, Bulsara MK et al. Parity and the risk of autoimmune thyroid disease: a community-based study. J Clin Endocrinol Metab 2005; 90: 5309-5312. 38. Friedrich N, Schwartz S, Thonack J et al. Association between parity and autoimmune thyroiditis in a general female population. Autoimmunity 2008; 174-180. 39. Gangi E, Kapatral V, El Azami El-Idrissi M et al. Characterization of a recombinant Yersinia enterocolitica lipoprotein; implications for its role in autoimmune response against thyrotropin receptor. Autoimmunity 2004; 37: 515-520. 40. Hansen PS, Wenzel BE, Brix TH, Hegedus L. Yersinia enterocolitica infection does not confer an increased risk of thyroid antibodies: evidence from a Danish twin study. Clin Exp Immunol 2006; 146: 32-38. 41. Brix TH, Hansen PS, Hegedus L, Wenzel BE. Too early to dismiss Yersinia enterocolitica infection in the aetiology of Graves’ disease: evidence from a twin case-control study. Clin Endocrinol 2008; 69: 491-496. 42. 42. Martin-du Pan RC. Triggering role of emotional stress and childbirth. Unexpected occurrence of Graves’ disease compared to 96 cases of Hashimoto thyroiditis and 97 cases of thyroid nodules. Ann Endocrinol 1998; 59: 107-112. 43. 43. Oretti KG, Harris B, Lazarus JH et al. Is there an association between life events, postnatal depression and thyroid dysfunction in thyroid antibody positive women? Int J Soc Psychiatry 2003; 49: 70-76. 44. 44. Mizokami T, Li AW, El-Kaissi S, Wall J. Stress and thyroid autoimmunity. Thyroid 2004; 14: 1047-1055. 45. 45. Crawford JR, Henry JD. The positive and negative affect schedule (PANAS): construct validity, measurement properties and normative data in a large non-clinical sample. Brit J Clin Psychol 2004; 43: 245-265. 46. Hassett AL, Simonelli LE, Radvanski DC, Buyske S, Savage SV, Sigal LH. The relationship between affect balance style and clinical outcomes in fibromyalgia. Arthritis Rheum. 2008; 59:833-40. 47. Kriegel MA, Lohmann T, Gabler C, Blank N, Kalden JR, Lorenz H-M. Defective suppressor function of human CD4+CD25+ regulatory T cells in autoimmune polyglandular syndrome type II. J Exp Med 2004; 199:1285-1291. 48. Hori S, Nomura T, Sakaguchi S Control of regulatory Tcell development by the transcription factor Foxp3. Science 2003; 299:1057–1061. 49. Chen Y, Cuda C, Morel L. Genetic determination of T cell help in loss of tolerance to nuclear antigens. J Immunol 2005; 174:7692–7702. 50. Polanczyk MJ, Carson BD, Subramanian S, Afentoulis M, Vandenbark AA, Ziegler SF, Offner H. Cutting edge: estrogen drives expansion of the CD4+CD25+ regulatory T cell compartment. J Immunol. 2004; 173:2227-2230. 51. Abalovich M, Amino N, Barbour LA, Cobin RH, De Groot LJ, Glinoer D, Mandel SJ, Stagnaro/Green A. Management of thyroid dysfunction during pregnancy and postpartum: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2007; 92:S1- S47. 52. Prummel MF, Wiersinga WM. Thyroid autoimmunity and miscarriage. Eur J Endocrinol. 2004; 150:751-755. 53. Negro R, Formoso G, Mangieri T et al. Levothyroxine treatment in euthyroid pregnant women with autoimmune thyroid disease: effects on obstetrical complications. J Clin Endocrinol Metab. 2006; 91:2587-2591.
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54. Bufalo NE, Santos RB, Cury AN et al. Genetic polymorphisms associated with cigarette smoking and the risk of Graves’ disease. Clin Endocrinol 2008; 68: 982-987. 55. Rayman MP. The importance of selenium to human health. Lancet 2000; 356: 233-241. 56. Gartner R, Gasnier BC, Dietrich JW et al. Selenium supplementation in patients with autoimmune thyroiditis decreases thyroid peroxidase antibodies concentrations. J Clin Endocrinol Metab 2002; 87: 1687-1691. 57. Duntas LH, Mantzou E, Koutras DA. Effect of a six month treatment with selenomethionine in patients with autoimmune thyroiditis. Eur J Endocrinol 2003; 148: 389-393. 58. Turker O, Kumanlioglu K, Karapolat I et al. Selenium treatment in autoimmune thyroiditis: 9-month follow-up with variable doses. J Endocrinol 2006; 190: 151-1. 59. Negro R, Greco G, Mangieri T et al. The influence of selenium supplementation on postpartum thyroid status in pregnant women with thyroid peroxidase autoantibodies. J Clin Endocrinol Metab 2007; 92: 1263-1268.
160
Summary
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Summary
Summary
Aim of this thesis
Autoimmune thyroid disease (AITD) encompassing Graves’ hyperthyroidism and Hashimoto hypothyroidism, is a common disorder especially in women, and both genetic and environmental factors are involved in its pathogenesis. The main aim of this thesis was to investigate whether or not it is possible to delineate familial, environmental and immunological risk factors for AITD in order to predict the development of AITD. To this end a prospective study was carried out in 803 healthy female relatives of patients with documented proof of AITD, known as the Amsterdam AITD Cohort. The data of this study provide new insights in the prevalence, incidence and risk factors of AITD. Knowing the family history of AITD combined with the found immunological and environmental risk factors we constructed a prediction model for the development of overt hypo- or hyperthyroidism in female relatives of patients with proven AITD.
Summary of the results
In chapter 2 we present a review of known environmental factors involved in the pathogenesis of AITD.
In chapter 3 we report on baseline characteristics of the Amsterdam AITD Cohort. The females with thyroid dysfunction were older than the euthyroid subjects and age appeared to be an independent risk factor for hypothyroidism. Estrogen use was associated with a lower risk, and pregnancy with a higher risk for having hyperthyroidism. The prevalence of autoimmune thyroid dysfunction in our Amsterdam AITD cohort was higher than in the general female population, thereby demonstrating that first- or second- degree female relatives of patients with proven AITD are indeed at higher risk for AITD. The observed prevalence of TPO antibodies in our cohort (24%) fits in nicely between that in the general female population (ca. 10%) and that found in first-degree relatives of AITD patients (ca. 45%). In the euthyroid subjects a dose-dependent relationship was found between serum TSH and TPO antibody level. Smoking and estrogen use were negatively correlated with the presence of TPO antibodies.
165 Summary
The high prevalence of AITD at baseline supports the importance of particular factors in its pathogenesis. To a certain extent interaction between familial background and environmental factors may determine whether Graves’ or Hashimoto’s disease will develop.
In chapter 4 we investigate the possible relationship between AITD and infection with Yersinia Enterocolitica (YE). YE might play a role in the development of AITD, whilst infection with YE produces a lipoprotein that can cross-react with the TSH receptor. Clinical evidence in support of this hypothesis has been inconclusive. We determined IgG and IgA antibodies to virulence-associated outer membrane proteins (YOPs) of YE. At study entrance the prevalence of YE infection was higher in AITD relatives than in controls. However the prevalence of YOP Ab did not differ between AITD relatives with a normal or abnormal TSH. Also the prevalence of TPO-Ab in the euthyroid AITD relatives was not different between YOP Ab positive and negative subjects. In conclusion, healthy female relatives of AITD patients have an increased prevalence of YOP antibodies, but this phenomenon is not related to the presence of TPO antibodies or abnormal thyroid function. Susceptibility genes for AITD may also confer a risk for YE infection.
In chapter 5 we looked for evidence of involvement of environmental stress exposure on AITD. Recently Experienced Stressful Life Events, Daily Hassles, and mood (tendency to report positive and negative affects) were assessed in 759 euthyroid subjects and related to TPO antibody status as early marker of AITD. The number and impact of stressful life events, the number and impact of daily hassles, and the scores on the affect scales were similar in TPO- Ab positive and TPO-Ab negative subjects. We conclude that the exposure to stress is not related to the presence of TPO antibodies in euthyroid women.
In chapter 6 we searched for markers on peripheral blood lymphocytes in early stages of AITD. Our aim was to test the hypothesis that activation of CD4+ T cells is such a marker in healthy relatives of AITD patients. We performed a controlled study on 20 TPO-Ab positive and 20 TPO-Ab negative euthyroid female relatives in whom we studied the percentages of circulating subsets of activated (MHC class-II, CD25 (IL-2R), CD71 or CD69+) CD4+ T cells and the level of the soluble (s)-IL2R in serum. We found that euthyroid female relatives did not show an activation of their T cell
166 Summary system, but instead a reduced expression of CD25 on CD4+ T cells. The level of the shed IL2R in serum was also lower in comparison with levels found in healthy control females. A reduced T cell activity was found in both TPO-Ab positive and negative relatives. Interestingly, current smoking seemed to correct for this reduction. In conclusion, female relatives with at least one 1st or 2nd degree relative with an AITD show signs of a reduced expansion capability of their T cell pool. It is hypothesized that a defect in T regulatory cells promotes thyroid autoimmunity.
In chapter 7 we describe the prediction of progression to overt hypothyroidism or hyperthyroidism in female relatives of patients with AITD using the Thyroid Events Amsterdam (THEA) Score. Family members of patients with AITD are at increased risk for AITD, but not all will develop overt hypothyroidism or hyperthyroidism. Our goal was to develop a simple predictive score that has broad applicability and is easily calculated at presentation for progression to overt hypothyroidism or hyperthyroidism within 5 years. We conducted a prospective observational cohort study of 790 healthy first- or second-degree female relatives of patients with documented Graves or Hashimoto disease in the Netherlands. Baseline assessment included measurement of serum thyrotropin (TSH), free thyroxin (FT4), and thyroid peroxidase (TPO) antibody levels as well as assay of Yersinia enterocolitica antibodies. We also gathered data on family background, smoking habits, use of oestrogen medication, pregnancy, and exposure to high levels of iodine. In the follow-up, thyroid function was investigated annually for 5 years. As main outcome measures, termed events , we looked for overt hypothyroidism or overt hyperthyroidism. The cumulative event rate was 7.5% over 5 years. The mean annual event rate was 1.5%. There were 38 hypothyroid and 13 hyperthyroid events. Independent risk factors for events were baseline TSH and TPO antibodies in a dose-dependent relationship (for TSH the risk already starts to increase at values >2.0 mIU/L) and family background (with the greatest risk attached to subjects having 2 relatives with Hashimoto disease). A numerical score, the Thyroid Events Amsterdam (THEA) score, was designed to predict events by weighting these 3 risk factors proportionately to their relative risks (maximum score, 21): low (0-7), medium (8-10), high (11-15), and very high (16-21). These THEA scores were associated with observed event rates of 2.7%, 14.6%, 27.1%, and 76.9%, respectively. An accurate simple predictive score was developed to estimate the 5-year risk of overt hypothyroidism or hyperthyroidism in female relatives of patients with AITD. However, in
167 Summary
view of the small number of observed events, independent validation of the THEA score is called for.
In chapter 8 we evaluate prospectively the involvement of environmental factors in the development of overt autoimmune hypothyroidism and hyperthyroidism. Evidence for involvement of environmental stimuli has been obtained mostly by association in retrospective cross-sectional case-control or population-based studies, open to potential (recall) bias. Attempting to avoid potential bias we performed a nested case-control study within the prospective observational Amsterdam AITD cohort-study, in which 790 healthy euthyroid women with at least one 1st or 2nd degree relative with documented AITD were followed for five years. Thyroid function and exposure to environmental stimuli were assessed annually. Contrast in environmental exposure was made between females who developed overt AITD and females who did not (matched for age and duration of follow-up). At baseline, exposure to environmental factors was not different between the 51 cases (38 hypothyroid and 13 hyperthyroid subjects) and their 102 controls. The frequency of Yersinia infection and oral estrogen use remained similar between cases and controls. But at the time of event hypothyroid cases were less often current smokers (p=0.083) and more often in the postpartum period (p=0.006) than their controls, whereas hyperthyroid cases had been pregnant more frequently (p=0.063). Exposure to stress at study entrance, at one year before event and at the time of the event was not different in cases as compared to their respective controls. A rather intriguing and yet still unexplained finding was the elevated affect state of our subjects compared to data from the general female population, reflecting a reactive affect balance style. The data suggest that cessation of smoking and the postpartum period are related to autoimmune hypothyroidism, whereas autoimmune hyperthyroidism is less frequent in women who have never been pregnant. Stress exposure was not related to either hypo- or hyperthyroidism. The limitation of this prospective observational study is its limited sample size.
Finally, in the general discussion ( chapter 9 ) an overview of the above mentioned studies is presented, and their scientific and clinical implications are discussed.
168 Summary
In short, the results of our studies point to an increased risk for developing AITD in the context of a positive familial background of AITD. The risk to develop overt autoimmune hypo- or hyperthyroidism in the next 5 years can be estimated with the Thyroid Events Amsterdam (THEA) Score. A number of environmental factors (like smoking, estrogen use, and pregnancy) modulate the risk.
169
Samenvatting
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Samenvatting
Samenvatting
Doel van dit proefschrift
Autoimmuun schildklierziekte (AITD) bestaande uit Graves’ hyperthyreoidie en Hashimoto hypothyreoidie, is een veelvoorkomende ziekte, vooral bij vrouwen, en zowel genetische als omgevingsfactoren zijn betrokken in de pathogenese. Het voornaamste doel van dit proefschrift was te onderzoeken of het mogelijk is om familiaire, omgevings- en immunologische risicofactoren voor AITD te onderscheiden om zo de ontwikkeling van AITD te kunnen voorspellen. Hiertoe werd een prospectieve studie uitgevoerd in 803 gezonde vrouwelijke familieleden van patiënten met schriftelijk bewijs van AITD, bekend staand als het Amsterdam AITD Cohort. De data van deze studie leveren nieuwe inzichten op aangaande de prevalentie, incidentie en risicofactoren van AITD. Op basis van het familiaire patroon van AITD gecombineerd met de gevonden immunologische en omgevingsfactoren hebben we een voorspellend model ontwikkeld voor het ontstaan van manifeste hypo- en hyperthyreoidie in vrouwelijke familieleden van patiënten met een bewezen AITD.
Samenvatting van de resultaten
In hoofdstuk 2 presenteren we een overzicht van bekende omgevingsfactoren die betrokken zijn bij de pathogenese van AITD.
In hoofdstuk 3 rapporteren we over uitgangskarakteristieken van het Amsterdam AITD Cohort. De vrouwen met een afwijkende schildklierfunctie waren ouder dan de euthyreote deelneemsters en leeftijd bleek een onafhankelijke risicofactor voor hypothyreoidie. Oestrogeengebruik was geassocieerd met een lager risico, en zwangerschap met een hoger risico voor het hebben van hyperthyreoidie. De prevalentie van afwijkende schildklierfunctie in ons Amsterdam AITD Cohort was hoger dan dat in de algemene vrouwelijke bevolking, daarmee aantonend dat 1 e en 2 e-graads
173 Samenvatting vrouwelijke verwanten van patiënten met bewezen AITD inderdaad een verhoogd risico lopen voor AITD. De geobserveerde prevalentie van TPO-antistoffen in ons cohort (24%) past goed tussen het percentage in de algemene vrouwelijke bevolking (ca.10%) en dat gevonden in 1 e-graads verwanten van AITD patiënten (ca. 45%). In de euthyreote deelneemsters werd een dosisafhankelijke relatie gevonden tussen serum TSH en TPO-antistoftiter. Roken en oestrogeengebruik waren negatief gecorreleerd met de aanwezigheid van TPO- antistoffen. De hoge prevalentie van AITD bij aanvang ondersteunt het belang van specifieke factoren in de pathogenese. Tot op zekere hoogte kan interactie tussen familiair voorkomen van AITD en omgevingsfactoren bepalen of de ziekte van Graves’of Hashimoto zich gaat ontwikkelen.
In hoofstuk 4 onderzoeken we de mogelijke relatie tussen AITD en infectie met Yersinia Enterocolitica (YE). YE kan een rol spelen in de ontwikkeling van AITD, gezien het feit dat er bij infectie met YE een lipproteïne wordt geproduceerd dat kan kruisreageren met de TSH- receptor. Klinische evidentie welke deze hypothese ondersteunt, is tegenstrijdig. Wij bepaalden IgG- en IgA-antistoffen tegen virulentiegeassocieerde buitenmembraan eiwitten (YOP’s) van YE. De prevalentie van YE infectie was hoger in AITD verwanten dan in controlepersonen. Toch verschilde de prevalentie van YOP-Ab niet tussen AITD verwanten met een normaal of afwijkend TSH. Ook de prevalentie van TPO-Ab in euthyreote AITD verwanten was niet verschillend tussen YOP-Ab positieve of YOP-Ab negatieve deelneemsters. Concluderend: gezonde vrouwelijke verwanten van AITD patiënten hebben een verhoogde prevalentie van YOP-antistoffen, maar dit fenomeen is niet gerelateerd aan de aanwezigheid van TPO- antistoffen of aan een abnormale schildklierfunctie. Genen die de overdraagbare gevoeligheid voor ontwikkelen van AITD bepalen kunnen ook een risico in zich houden van kwetsbaarheid voor infectie met YE.
In hoofdstuk 5 hebben we gezocht naar bewijs van betrokkenheid van blootstelling aan omgevingsstress bij AITD. Recent meegemaakte gebeurtenissen, dagelijkse kleine irritaties en affect (neiging om positief of negatief gekleurd te rapporteren) werden bepaald in 759 euthyreote deelneemsters en gerelateerd aan TPO-antistof status als een vroege marker van AITD. Het aantal en de impact van recent meegemaakte gebeurtenissen, het aantal en de impact van dagelijkse kleine
174 Samenvatting irritaties en de scores op de affectstijl schalen waren gelijk in TPO-Ab positieve en TPO-Ab negatieve deelneemsters. We concluderen dat blootstelling aan stress niet gerelateerd is aan de aanwezigheid van TPO- antistoffen in euthyreote vrouwen.
In hoofdstuk 6 zoeken we naar markers op perifere bloed lymfocyten voor vroege stadia van AITD. Ons doel was om de hypothese te testen dat activatie van CD4+ T-cellen zo’n marker is in gezonde verwanten van AITD patiënten. We voerden een gecontroleerde studie uit in 20 TPO-Ab positieve en 20 TPO-Ab negatieve euthyreote vrouwelijke verwanten, waarbij wij de percentages van circulerende subsets van geactiveerde (MHC klasse-II, CD25(IL-2R), CD71 of CD69+) CD4+ T-cellen en het niveau van (s)-IL2R in serum bestudeerden. We vonden dat euthyreote vrouwelijke verwanten geen activatie van hun T-cel systeem lieten zien, maar daarentegen een gereduceerde expressie van CD25 op CD4+ T-cellen. Het niveau van de soluble IL2 receptor was ook lager in vergelijking met niveaus gevonden in gezonde controle vrouwen. Een gereduceerde T-cel activatie werd gevonden bij zowel TPO-Ab positieve als TPO-Ab negatieve verwanten. Interessant is dat roken deze reductie lijkt te corrigeren. Concluderend, vrouwelijke verwanten met tenminste één 1 e of 2 e graads familielid met AITD laten tekenen zien van een gereduceerde expansiecapaciteit van hun T-cel systeem. Gehypothetiseerd wordt dat een defect in regulatoire T-cellen schildklier autoimmuniteit bevordert.
In hoofdstuk 7 beschrijven we de predictie van progressie naar manifeste hypothyreoidie of hyperthyreoidie in vrouwelijke verwanten van patienten met AITD daarbij gebruik makend van de Thyroid Events Amsterdam ( THEA) score. Familieleden van patiënten met AITD hebben een verhoogd risico op het ontwikkelen van AITD, maar niet allen gaan een manifeste hypothyreoidie of hyperthyreoidie ontwikkelen. Onze doelstelling was om een simpele voorspellende score te ontwikkelen voor progressie naar manifeste hypothyreoidie of hyperthyreoidie, welke een brede toepasbaarheid heeft en gemakkelijk te berekenen is. We voerden een prospectieve observationele cohort studie uit onder 790 gezonde 1 e en 2 e graads vrouwelijke verwanten van patiënten met een gedocumenteerde ziekte van Graves of Hashimoto in Nederland. Uitgangswaardes van schildklierstimulerend hormoon (TSH), vrij thyroxine (fT4), schildklierperoxidase (TPO) antistoffen als ook Yersinia enterocolitica antistoffen werden gemeten en vastgesteld. Ook verzamelden we informatie over het familiair
175 Samenvatting patroon van voorkomen van AITD, rookgewoontes, gebruik van oestrogenen, zwangerschappen, en blootstelling aan hoge jodiumconcentraties. Vervolgens werd gedurende 5 opeenvolgende jaren de schildklierfunctie jaarlijks onderzocht. Als belangrijkste uitkomstmaten, events genoemd, werd gekeken naar het ontstaan van manifeste hypothyreoidie of manifeste hyperthyreoidie. De cumulatieve event rate was 7,5 % over 5 jaren. De gemiddelde jaarlijkse event rate was 1,5 %. Er waren 38 hypothyreote en 13 hyperthyreote events. Onafhankelijke risicofactoren voor het optreden van een event waren uitgangswaardes van TSH en TPO antistoffen in een dosisafhankelijke relatie (aangaande TSH begint het risico al te stijgen bij waardes boven de 2 mIU/l ) en familieachtergrond (met het grootste risico verbonden aan deelneemsters die 2 verwanten met de ziekte van Hashimoto hebben). Een numerieke score om events te voorspellen, de Thyroid Events Amsterdam (THEA) score werd ontworpen door proportionele weging van deze 3 risicofactoren ten opzichte van hun relatieve risico’s (maximum score, 21): low (0-7), medium (8-10), high (11-15), and very high (16-21). Deze THEA scores waren geassocieerd met geobserveerde event rates van respectievelijk 2,7%, 14,6%, 27,1% en 76,9%. Een accurate en simpele voorspellende score werd ontwikkeld om het 5-jaars risico op manifeste hypothyreoidie en manifeste hyperthyreoidie in te schatten bij vrouwelijke verwanten van patiënten met AITD. Echter, gelet op het kleine aantal van geobserveerde events, is er een onafhankelijke validatie van de THEA score wenselijk.
In hoofdstuk 8 evalueren we de betrokkenheid van omgevingsfactoren bij de ontwikkeling van manifeste autoimmuun hypothyreoidie en hyperthyreoidie. Bewijs voor betrokkenheid van omgevingsstimuli is meestal verzameld in retrospectieve dwarsdoorsnede case-control studies of op populatieniveau uitgevoerde associatiestudies, die vatbaar zijn voor potentiele (recall) bias. In een poging potentiële bias te vermijden voerden we een nested case-controle studie uit binnen het prospectieve Amsterdam AITD Cohort, waarin 790 gezonde euthyreote vrouwen, met op zijn minst één 1 e en 2 e graads familielid met gedocumenteerde AITD, gedurende 5 jaar werden gevolgd. Schildklierfunctie en blootstelling aan omgevingsfactoren werden jaarlijks vastgelegd. Contrast betreffende blootstelling aan omgevingsfactoren werd gemaakt tussen vrouwen die manifeste AITD ontwikkelden en vrouwen die dat niet deden (gematched voor leeftijd en duur van follow-up).
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Ten tijde van de basismeting was blootstelling aan omgevingsfactoren niet verschillend tussen de 51 events (38 hypothyreote en 13 hyperthyreote deelneemsters) en de bijbehorende controles. De frequentie van Yersinia infectie en oraal oestrogeen gebruik bleef gelijk tussen events en controles. Maar ten tijde van het optreden van manifeste ziekte waren hypothyreote events minder vaak actuele rokers (p=0,083) en vaker in de postpartum periode (p=0.063) dan de bijbehorende controles. Blootstelling aan stress ten tijde van studieentree, 1 jaar voor het optreden van de events en ten tijde van het optreden van de events, verschilde niet tussen de cases en de bijbehorende controles. Een nogal intrigerende en nog niet verklaarde bevinding waren de verhoogde affectscores van onze deelneemsters vergeleken met data uit de algemene vrouwelijke bevolking, passend bij een geactiveerde affectbalance stijl. De data suggereren dat stoppen met roken en de postpartum periode gerelateerd zijn aan hypothyreoidie, terwijl autoimmuun hyperthyreoidie minder vaak voorkomt bij vrouwen die nooit zwanger zijn geweest. Blootstelling aan stress was niet gerelateerd aan zowel hypothyreoidie als hyperthyreoidie. De beperking van deze observationele cohort studie is de lage sample size.
In de algemene discussie ( hoofdstuk 9 ) wordt een overzicht gegeven van bovengenoemde studies, en worden de wetenschappelijke en klinische implicaties besproken.
Kort gezegd wijzen de resultaten van onze studies op een verhoogd risico om AITD te ontwikkelen in de context van een positieve familiaire belasting voor AITD. De kans op het ontwikkelen van manifeste autoimmuun hypo- of hyperthyreoidie in de komende 5 jaar kan geschat worden met de Thyroid Event Amsterdam (THEA) score. Een aantal omgevingsfactoren (zoals roken, oestrogeen gebruik en zwangerschap) moduleren dit risico.
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Dankwoord Curriculum Vitae |
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Allereerst wil ik alle vrouwen die aan dit onderzoek deelnamen hartelijk bedanken voor hun medewerking, want zonder hun deelname was dit onderzoek niet mogelijk geweest. Gedurende de hele onderzoeksperiode waren zij belangenloos bereid jaarlijks vanuit heel Nederland en ook Belgie af te reizen naar het AMC te Amsterdam om daar bloed te doneren en vele vragenlijsten in te vullen voor de jaarlijkse follow-up. Ook de Schildklierstichting Nederland en de Nederlandse Vereniging van Graves Patiënten wil ik bedanken voor hun ondersteuning bij het enthousiast maken van al die familieleden van AITD patiënten voor deelname aan dit onderzoek. NWO wil ik bedanken voor de subsidiëring van dit onderzoek. Prof. Dr. Wilmar Wiersinga, beste Wilmar, jij bent niet alleen mijn promotor, maar ook de initiator en motor achter dit project. Jij gaf mij de kans dit onderzoek op jouw afdeling uit te voeren. Jouw enthousiasme, betrokkenheid bij het onderzoek, suggesties voor de artikelen en getoond geduld waren erg prettig. Onze expeditie op Oud en Nieuw naar een varkensfokkerij in het zuiden van het land om drie generaties DNA te verzamelen was ook een bijzondere manier van onderzoek doen. Om nooit te vergeten. Jij hebt me enthousiast gemaakt en gehouden om het medisch specialist zijn te combineren met het doen van wetenschappelijk onderzoek. Hartelijk dank voor je inspirerende en enthousiasmerende begeleiding! Prof. Dr. Jan Tijssen, beste Jan, mijn mede-promotor, zuiverder begeleiden van epidemiologische en statistische interpretatie van de gevonden data kan ik me niet indenken. Je kon als geen ander je vinger op de zere plek leggen. Toen je uiteindelijk je fiat gaf aan de laatste interpretaties wist ik dat het goed zat. Ook jou wil ik hartelijk danken voor je inspirerende begeleiding! Dr. Mark Prummel, beste Mark, helaas heb je het eindresultaat van ons onderzoek niet meer mee mogen maken, maar postuum wil ik je toch bedanken voor al je tijd en inspanningen. Je gaf kritisch en opbouwend commentaar, was erg betrokken en altijd bereid om te overleggen. Zonder jouw inzet was dit onderzoek niet tot stand gekomen. Prof. H. Drexhage, beste Hemmo, het was een verrijking voor ons allen om in jouw immunologische keuken te mogen kijken en heel wat uurtjes tussendoor gedurende het Rotterdamse opleidingsjaar met je samen te werken aan onderbouwing van ons begrip rond de immunogenese van AITD.
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Prof. Björn Wenzel, beste Björn, in Yersiniologie kent men wel geografische beperkingen, maar geen afstand was je te ver om laboratoriumonderzoek uit te voeren naar de staat van Yersinia infectie bij al onze deelneemsters. Ook was je een van de organisatoren van voor mij relevante wetenschappelijke conferenties in Lübeck of omgeving rond schildklieronderzoek, waar verschillende onderzoeksinstituten en gastsprekers in klein comité met elkaar van gedachten konden wisselen over hun onderzoeksresultaten. Stuwende krachten achter dit project waren ook de afdelingssecretaressen Marga Boerigter en Renée Kostelijk voor hun hulp bij en bewaken van de logistiek en de datainvoer, Martine van Vessem, de doktersassistente van de functiekamer Endocrinologie, en Mieke Reedijk, onze gepensioneerde laborante, voor het helpen uitvoeren en begeleiden van alle bloedafnames en lichamelijk onderzoek. Mieke van Beeren en Marjan Platvoet, beiden in het bijzonder, maar ook Eric Endert, Onno Bakker en alle andere betrokken medewerkers van het Endolab, bij deze bedankt voor het beschikbaar stellen van al jullie expertise en laboratoriumfaciliteiten. DNA-isolatie en lymfocyten-isolatie behoren sindsdien ook tot mijn doktersarsenaal. Ook wil ik hier noemen de als zeer prettig ervaren samenwerking met de medewerkers van het immunologisch lab van de Erasmus universiteit en in het bijzonder Harm de Wit, die samen met collega-arts Wai Kwan Tse mij in korte tijd het tot leven brengen van ingevroren bloedcellen bij brachten, een waar wonder om mee te maken. Mijn collega-promovendi en –post-docs, wil ik bedanken voor hun steun, gezelligheid en luisterend oor. Iedereen die bijgedragen heeft aan de tot stand koming van dit proefschrift wil ik hierbij bedanken. Het was een plezier om samen te werken. Prof. Dr. R.J.M. ten Berge, Prof. Dr. H. A. Drexhage, Prof. Dr. J.B.L.Hoekstra, Dr. J.B. Reitsma, Prof. Dr. J. Smit, Prof. Dr. M.W. Schene, hartelijk dank voor uw deelname aan mijn promotiecommissie.
Mijn familie, vrienden en alle anderen die de afgelopen jaren belangstellend voor mijn proefschrift waren, hartelijk dank voor jullie warme betrokkenheid. Jullie belangstelling heeft mij goed gedaan en geholpen om vol te houden. Lieve pap en de laatste jaren ook mam, jullie warmte, steun en vertrouwen in mij hebben er voor gezorgd dat ik alle mogelijkheden om te studeren altijd heb aangepakt en ook dit proefschrift heb weten te voltooien. Helaas hebben jullie de afronding niet meer mee mogen maken. Bedankt voor alles! Annemiek en JaapWillem, mijn zus en broer, Jane, Saskia en Jaqueline, mijn beste vriendinnen, bedankt dat jullie altijd zo belangstellend zijn voor alles wat ik doe en de vele
182 Dankwoord gezellige momenten die we samen hebben. Geweldig dat Jaap en Jane op dit belangrijke moment aan mijn zijde staan als mijn paranimfen. Last but not least, lieve Tommy, Naomi, Nigel, Ruby en Esmee, jullie warmte, liefde en steun betekenen heel veel voor mij. Ik ben ongelofelijk trots op ons dat we met zijn allen de hele gang van het tot stand komen van dit proefschrift tot een goed eind hebben gebracht, want het was me wel een klus, zeg! Nu kunnen we samen een stuk onbezorgder genieten van alle mooie dingen die het leven ons te bieden heeft.
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Curriculum Vitae
Thea Strieder werd geboren op 31 maart 1958 te Amersfoort. In 1977 behaalde zij het Atheneum-ß-diploma aan de RKSG Het Eemlandcollege te Amersfoort. In hetzelfde jaar begon zij met de studie Geneeskunde aan de Vrije Universiteit van Amsterdam. Tijdens haar opleiding tot arts leerde zij haar partner Milton Dorothea kennen en werden hun twee kinderen Naomi (1983) en Nigel (1986) geboren. In januari 1988 behaalde zij haar artsexamen. Vanaf medio 1988 was zij achtereenvolgens werkzaam als verslavingsarts, arts jeugdgezondheidszorg (JGZ) en gemeentelijk lijkschouwer. Vanaf mei 1996 was zij halftime werkzaam als onderzoeker en halftime als arts JGZ GGGD Amsterdam, vanaf 1999 halftime als onderzoeker en halftime districtsarts Ouder&Kindzorg thuiszorg Eemland, het latere Acare. In 2002 was zij een half jaar werkzaam als AGNIO Kindergeneeskunde in het AMC in afwachting van een aanvullende onderzoeksbeurs. Tijdens haar dienstverband bij de GGGD heeft zij de JGZ vertegenwoordigd in de Ondernemingsraad van de GGGD. In haar Acare tijd was zij bestuurslid (secretaris) van de LFC (Landelijke Federatie Consultatiebureau-artsen) en later van de AJN (Artsen Jeugdgezondheidszorg Nederland). Vanaf mei 1996 tot en met december 2003 werd een prospectief cohort onderzoek uitgevoerd naar determinanten van autoimmuun schildklierziekten (projectleiders Prof. Dr. W.M. Wiersinga, Prof. Dr. J.G.P. Tijssen, en wijlen Dr. M.F. Prummel), waarvan de resultaten in dit proefschrift beschreven staan. Tijdens deze onderzoeksperiode werd nauw samengewerkt met Prof. Dr. H. Drexhage, immunoloog van het ErasmusMC te Rotterdam, en Prof. Dr. B.E. Wenzel, biochemicus aan de Universiteit van Lubeck. Vanaf januari 2004 was zij werkzaam als arts in opleiding tot psychiater achtereenvolgens op de afdeling Kinder & Jeugd Psychiatrie (KJP) van het Erasmus MC- Sophia te Rotterdam (opleider: Prof. Dr. F. Verhulst), de PAAZ-afdeling van het Elisabeth ziekenhuis te Tilburg (opleider: Drs. J. van Laarhoven) en bij de GGZ Midden -Brabant te Tilburg (opleider: Prof. Dr. P. Hodiamont, in 2008 opgevolgd door Dr. C. Rijnders). Het laatste half jaar van haar opleiding tot psychiater van maart tot september 2008 was zij werkzaam bij de afdeling MGGZ (opleider: Drs. W. Tuinebreijer) van de GGD Amsterdam en heeft zij dit proefschrift kunnen afronden. Inmiddels is zij sinds oktober 2008 werkzaam op de Kinderkliniek van de afdeling KJP van het Erasmus MC-Sophia te Rotterdam (opleider: Prof. Dr. F. Verhulst) als psychiater in opleiding voor Kinder & Jeugdpsychiater.
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