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Animal models of Graves’ disease
Abstract
Graves' disease (GD) is an autoimmune condition in which goitre and hyperthyroidism are induced by thyroid stimulating antibodies (TSAB) which mimic the action of thyrotrophin (TSH). The target of the autoimmune response is the thyrotrophin receptor (TSHR) and, since its cloning, a number of differing approaches have been adopted in an attempt to develop an animal model of GD. Methods in which synthetic peptides or fragments of the receptor produced in bacteria or insect cells have been injected into animals together with immunological adjuvants have had only limited success in inducing some of the signs and symptoms of GD. Genetic immunisation resulted in thyroiditis in the majority, but TSAB formation in only a minority, of treated inbred mice. Transfer of receptor in vitro primed T cells to syngeneic naive recipients, with priming either using a bacterial fusion protein or genetic immunisation, induced destructive thyroiditis in non-obese diabetic (NOD) mice but lymphocytic thyroiditis in BALBc mice. Furthermore, the orbits of 17/22 of the BALBc animals, but not the NOD animals, with thyroiditis had orbital changes similar to those seen in thyroid eye disease. TSAB and elevated thyroxine levels were induced in AKR/N mice injected with fibroblasts expressing the full length human TSHR and murine major histocompatibility complex (MHC) class II homologous to the recipient mice. No thyroiditis was induced but preliminary results from a different group using the same protocol suggest that receptor autoantibodies and thyroid dysfunction could be transferred using T cells primed in vitro with the receptor and MHC-II expressing cells. The majority of the studies described above have studied inbred mouse strains. In a novel departure, the NMR outbred strain has been treated by genetic immunisation with very promising results, including the induction of increased thyroxine levels in 4/30 female mice, accompanied by TSAB in addition to thyroiditis, and with signs of hyperactivity and orbital pathology. This review discusses the various protocols together with the information regarding the pathogenesis of GD which each has contributed, and concludes with an evaluation of how close we are to mimicking this polygenic, multifactorial disease.
... Mouse models of GD allow us to better understand the disease and to investigate novel therapeutic approaches. Over the years, many scholars at home and abroad have tried to prepare GD animal models by using various methods, but the results have not been satisfactory [16]. Currently, the most common method for constructing a GD model is to immunize BALB/c mice with an adenovirus or plasmids expressing TSHR. ...
Background:
The objective was to study targeted therapies using a biologically active monoclonal antibody against intracellular adhesion molecule-1 (ICAM-1 mAb) and an siRNA targeting thyroid-stimulating hormone (TSH) receptor (TSHR) in a BALB/c mouse model of Graves' disease (GD).
Material and methods:
An improved method for establishing a stable model of GD in BALB/c mice was developed by immunization with pcDNA 3.1/TSHR 289 and electroporation (EP). The mice in which GD was successfully established were divided into a nontreated control group, which was treated with continuous immunization, and treated groups, which were treated with the siRNA and ICAM-1 mAb. Normal mice were included as a blank group. These groups were used to compare the effects of treatment with the ICAM-1 mAb and siRNA.
Results:
The two novel treatments markedly improved weight loss, serum thyroxine (T4) levels, thyroid-stimulating hormone antibody (TSAb) levels, thyroid-stimulating blocking antibody (TSBAb) levels and thyroid uptake of 99mTcO4 in GD model mice. Compared with the siRNA treatment, treatment with the ICAM-1 mAb produced more obvious benefits. The differences in the posttreatment indexes between the two treatment groups were statistically significant (p < 0.05).
Conclusions:
These preliminary data suggest that both the biologically active ICAM-1 mAb and the siRNA targeting TSHR were effective. The ICAM-1 mAb exerted a better therapeutic effect than the siRNA targeting TSHR. Both treatments showed potential efficacy as novel treatments for GD and may therefore represent therapeutic options in addition to the existing drugs or interventions.
... The relationships have been established between genetic variations in TSHR gene and thyroid diseases, such as autoantibody-mediated and genetic variant-induced hyperactivation or repression of TSHR, causing hyper-or hypo-thyroidism. For instance, in patients with Graves' disease or autoimmune-related hypothyroidism, such correlations have been extensively investigated and comprehensively reviewed during the past decades [15][16][17][18][19][20][21][22][23][24][25]. These topics are not included in this review. ...
The thyroid stimulating hormone (TSH) and its cognate receptor (TSHR) are of crucial importance for thyrocytes to proliferate and exert their functions. Although TSHR is predominantly expressed in thyrocytes, several studies have revealed that functional TSHR can also be detected in many extra-thyroid tissues, such as primary ovarian and hepatic tissues as well as their corresponding malignancies. Recent advances in cancer biology further raise the possibility of utilizing TSH and/or TSHR as a therapeutic target or as an informative index to predict treatment responses in cancer patients. The TSH/TSHR cascade has been considered a pivotal modulator for carcinogenesis and/or tumor progression in these cancers. TSHR belongs to a sub-group of family A G-protein-coupled receptors (GPCRs), which activate a bundle of well-defined signaling transduction pathways to enhance cell renewal in response to external stimuli. In this review, recent findings regarding the molecular basis of TSH/TSHR functions in either thyroid or extra-thyroid tissues and the potential of directly targeting TSHR as an anticancer strategy are summarized and discussed.
... Animal models can be invaluable in this context and numerous attempts have been made to develop a robust model of induced Graves' orbitopathy in mice. [Au: Edit to the following sentence OK? Minor changes] Many of these models, however, were only able to induce Graves' disease, with no signs of Graves' orbitopathy [133][134][135] . In one instance researchers developed a BALB/c based mouse model that had some aspects of Graves' orbitopathy 136 , but this was not reproducible in different centres, which suggests a role for environmental variables, including micro-organisms, interfering with Graves' orbitopathy pathogenesis 137 . ...
Graves orbitopathy, also known as thyroid eye disease or thyroid-associated orbitopathy, is visually disabling, cosmetically disfiguring and has a substantial negative impact on a patient’s quality of life. There is increasing awareness of the need for early diagnosis and rapid specialist input from endocrinologists and ophthalmologists. Glucocorticoids are the mainstay of treatment; however, recurrence occurs frequently once these are withdrawn. Furthermore, in >60% of cases, normal orbital anatomy is not restored, and skilled rehabilitative surgery is required. Clinical trials have shown that considerable benefit can be derived from the addition of antiproliferative agents (such as mycophenolate or azathioprine) in preventing deterioration after steroid cessation. In addition, targeted biologic therapies have shown promise, including teprotumumab, which reduces proptosis, rituximab (anti-CD20), which reduces inflammation, and tocilizumab, which potentially benefits both of these parameters. Other strategies such as orbital radiotherapy have had their widespread role in combination therapy called into question. The pathophysiology of Graves orbitopathy has also been revised with identification of new potential therapeutic targets. In this Review we provide an up-to-date overview of the field, outline the optimal management of Graves orbitopathy and summarize the research developments in this area to highlight future research questions and direct future clinical trials. Graves orbitopathy has a negative impact on a patient’s quality of life. This Review provides an overview of the field and outlines the optimal management of Graves orbitopathy. The authors also highlight future research questions to direct future clinical trials.
... Hyperthyroidism model can be established also by cyanide (one of the major environmental pollutants termed as thyroid disruptors) (Daniel et al., 2014) and recombinant thyroid stimulating antibodies (TSAB) to induce graves' disease (Ludgate, 2000), but the effects of these materials on the pharmacokinetic and pharmacodynamics of thyroid hormones as well as the other body tissues are unclear. ...
Background and Aims: Hyperthyroidism promotes cardiovascular diseases (CVDS) development and progression. Aldosterone, a key mediator of cardiac inflammation, oxidative stress and fibrosis, may be activated in hyperthyroidism. We aimed to evaluate the impact of hyperthyroidism on aldosterone levels and myocardial oxidants/antioxidants, inflammatory and fibrotic markers in a rat model of hyperthyroidism, and to test if use of spironolactone (an aldosterone antagonist) will attenuate these changes. Method: Adult Wistar rats were randomized into four groups; controls, spironolactone treated rats (Spir, 50mg/kg/day), hyperthyroid rats (Hyper, intraperitoneal levothyroxine 0.3mg/kg/day), and spironolactone treated hyperthyroid rats (Hyper+Spir) for 4 weeks. Blood pressure (Bp), serum and cardiac levels of aldosterone, oxidants/antioxidants, inflammatory and fibrotic markers were
measured. Result: Levothyroxine increased serum thyroid hormones and increased Bp, heart rate and heart to body weight ratio. Relative to control, serum aldosterone levels were increased in Hyper and Hyper+Spir groups. In parallel, cardiac lipid peroxides and serum endothelin-1 were increased whereas cardiac super oxide dismutase, catalase, glutathione, and matrix metalloproteinase -2 were reduced in Hyper group. Spironolactone use reduced serum thyroid hormones and improved cardiac lipid peroxides and metalloproteinase -2 levels. Use of spironolactone decreased serum nitrite levels and increased cardiac SOD and glutathione. Cardiac levels of aldosterone, endothelin-1, transforming growth factor beta and nitrite were
similar among all groups. Conclusions: Hyperthyroidism was associated with an increase in aldosterone and oxidant/inflammatory biomarkers. Use of spironolactone enhanced antioxidant defenses and reduced thyroid hormones. Aldosterone antagonists may serve as potential drugs to attenuate development of cardiac disease in hyperthyroid patients.
Keywords: Spironolactone; Hyperthyroidism; Aldosterone; Cardiovascular disease; Oxidative stress; Fibrosis; Inflammation
... Exophthalmia (also called exophthalmus, proptosis, or exorbitism) is a bulging of the eye out of the orbit (Ludgate, 2000). Exophthalmos can be either bilateral as in hyperthyroidism (Graves' disease) or unilateral as is often seen in an orbital tumors that push the eyeball out of the orbit. ...
All diseases in humans, animals and birds cause physiological, biological, anatomical, morphological, pathological and behavioral changes that entail the appearance of certain symptoms. These symptoms can be seen with the naked eye; heard with ear; felt with hand or they may be associated with certain odors that can be snorted with nose or they can be detected by clinical and/or laboratory means to know their causative agents. The vets need to have tremendous scientific knowledge, practical expertise and sensory skills to understand the symptoms associated with animal diseases to achieve proper disease diagnosis. Since animals do not communicate nor they can explain their pains and sufferings; the vet must has good communication skills to retrieve information from the animals' companion. The vet also needs sensory skills to understand the behavioral changes associated with diseases, interpret and analyze the symptoms so that he can reach an optimum diagnosis.
The symptoms of diseases that can be seen with the eye in equines are countless for examples symptom of tetanus, rabies, Monday sickness, tympany and lameness as well as many other diseases. In ruminants, you can see the symptoms of so many diseases; of which but not limited to; ruminal tympany, abdominal hernias, udder edema, uterine prolapse, dystocia etc. In birds, you can see the symptoms of Newcastle disease, duck viral hepatitis, curled toe paralysis and Marek’s disease etc.
The symptoms of the disease that cause pain make the animal screaming, moaning or grunting. Any other disease which is accompanied by cough or snoring sounds its sounds can be heard. The symptoms of some diseases are associated with odors like bovine ketosis where acetone odor could be smelt. The rot and foul odors also associate with some inflammation. You can also feel the increased pulse rate, tumor, hematoma, abscess and fractures with hands.
Clinical and laboratory examinations help the vet to interpret these symptoms because they explain the physiological, biological, anatomical and pathological changes that take place within the animal's body, body fluids, tissues and organs. If the vet is able to link these changes and symptoms smoothly; undoubtedly he will reach the ideal diagnosis of the disease and then he can treat the diseased animal properly and/or he can give an accurate prognosis.
The objective of this book is to provide veterinary students and veterinarians with simple basics of clinical examination of farm animals; enable them to deal with the animal in case of illness; achieve a tentative diagnosis; and to identify the samples that can be sent to the laboratory for further confirmation of the diagnosis. This book concentrates mainly on veterinary clinical examination of farm animals and focuses on the common diseases that affect the body's various systems. The book also gives some hints about pet animals' examination when necessary.
... Several GD mouse models have been developed using different immunization protocols with no signs of concomitant eye disease as previously reviewed [4,7,8]. Ludgate and colleagues established a TSHR-induced GO model by genetic immunization, i.e., injecting a TSHR expression plasmid [9]. ...
Background: Variation in induced models of autoimmunity has been attributed to the housing environment and its effect on the gut microbiota. In Graves’ disease (GD), autoantibodies to the thyrotropin receptor (TSHR) cause autoimmune hyperthyroidism. Many GD patients develop Graves’ orbitopathy or ophthalmopathy (GO) characterized by orbital tissue remodeling including adipogenesis. Murine models of GD/GO would help delineate pathogenetic mechanisms, and although several have been reported, most lack reproducibility. A model comprising immunization of female BALBc mice with a TSHR expression plasmid using in vivo electroporation was reproduced in two independent laboratories. Similar orbital disease was induced in both centers, but differences were apparent (e.g., hyperthyroidism in Center 1 but not Center 2). We hypothesized a role for the gut microbiota influencing the outcome and reproducibility of induced GO.
Results: We combined metataxonomics (16S rRNA gene sequencing) and traditional microbial culture of the intestinal contents from the GO murine model, to analyze the gut microbiota in the two centers. We observed significant differences in alpha and beta diversity and in the taxonomic profiles, e.g., operational taxonomic units (OTUs) from the genus Lactobacillus were more abundant in Center 2, and Bacteroides and Bifidobacterium counts were more abundant in Center 1 where we also observed a negative correlation between the OTUs of the genus Intestinimonas and TSHR autoantibodies. Traditional microbiology largely confirmed the metataxonomics data and indicated significantly higher yeast counts in Center 1 TSHR-immunized mice. We also compared the gut microbiota between immunization groups within Center 2, comprising the TSHR- or βgal control-immunized mice and naïve untreated mice. We observed a shift of the TSHR-immunized mice bacterial communities described by the beta diversity weighted Unifrac. Furthermore, we observed a significant positive correlation between the presence of Firmicutes and orbital-adipogenesis specifically in TSHR-immunized mice.
Conclusions: The significant differences observed in microbiota composition from BALBc mice undergoing the same immunization protocol in comparable specific-pathogen-free (SPF) units in different centers support a role for the gut microbiota in modulating the induced response. The gut microbiota might also contribute to the heterogeneity of induced response since we report potential disease-associated microbial taxonomies and correlation with ocular disease.
Keywords: Graves’ orbitopathy, Graves’ disease, Induced animal model, Gut microbiota, TSHR, Metataxonomics, Orbital adipogenesis, Firmicutes
... It is widely accepted that the study of animal models of disease provides an improved understanding of the underlying pathogenesis, and may be used for evaluating novel therapeutic strategies. An appropriate animal model of GD would include the following features: i) Elevated T 4 and/or reduced TSH levels, ii) TSHR-associated biologically active antibodies, iii) alterations in thyroid architecture, iv) lymphocytic thyroiditis, v) clinical signs of hyperthyroidism including weight loss, vi) orbital alterations similar to those in thyroid eye disease, and vii) lymphocytic infiltration in the pretibial skin (24). With the exception of pathological alterations of eyes and pretibial skin, the additional five clinical characteristics were observed in the model established in the present study. ...
The present study aimed to develop a stable Graves' disease (GD) model in BALB/c mice by immunization and electroporation (EP). A total of 90 mice were divided into experimental (n=50), control (n=20) and blank (n=20) groups. The recombinant plasmid pcDNA3.1/thyroid‑stimulating hormone (TSH) receptor 268 was constructed and injected into the bilateral gastrocnemius of experimental group mice at weeks 1, 4, 7 and 10. Equal volumes of saline were injected into the control and blank groups at the same time. The experimental and control groups were subjected to EP at the same time and location to enhance immunization. The levels of total serum thyroxine (T4) and serum TSH were examined by radioimmunoassay and immunoradiometric assay, respectively. The levels of serum thyrotropin receptor N‑terminal (TRAb N) and C‑terminal (TRAb C) antibodies were assessed by ELISA. Whole body pertechnetate (99mTcO4‑) imaging was performed. Mouse weight and thyroid morphology and pathology were analyzed. The GD BALB/c mouse model was successfully established, with a positive rate of 79.17% (38/48). T4 levels increased from baseline levels of 12.05±4.23 to 52.51±23.58 ng/ml by week 12 (P<0.0001). TSH levels decreased from baseline levels of 5.53±2.78 to 1.43±0.89 µIU/ml by week 12 (P<0.0001). TRAb N antibody levels increased from baseline levels of 0.006±0.002 to 0.278±0.106 mIU/ml by week 12 (P<0.0001). TRAb C antibody levels increased from baseline levels of 11.111±2.808 to 46.701±26.436 arbitrary units/ml by week 12 (P<0.0001). At week 21, TSH levels remained reduced compared with pre‑immunization levels (P<0.0001). Although T4, and TRAb N and C levels decreased, they remained increased compared with preimmunization levels (P<0.0001, P<0.0001, P=0.001). There were no significant alterations in antibody levels between the control and blank groups. Following four immunizations, the uptake of 99mTcO4‑ by the thyroid was significantly increased in the experimental group. The mean weight of the experimental mice was significantly reduced compared with the control and blank groups (all P<0.0001). Furthermore, the thyroid glands of the immunized mice were enlarged and exhibited lymphocyte infiltration, fewer colloid nodules and an increased height of epithelial cells. In conclusion, by injecting recombinant plasmid pcDNA3.1/TSHR268 and EP, a GD mouse model was successfully established.
... The strong IL-4 and IL-10 immunoreactivity in the thyroids of animals with orbital involvement suggests that a Th2 autoimmune response to the TSH.R is required for the development of GO. Novel experiments using NMR outbred mice (more comparable with the outbred nature of humans) treated by genetic immunization with TSH.R complementary DNA, demonstrated increased T 4 levels, TSAb as well as TBII and TBAb, thyroiditis and orbital pathology in a small minority of the female mice (14). The animal experiments so far indicate that orbital pathology seems to depend on sex, genetic background, and a Th2 response to the TSH.R, but does not require TSAbs. ...
... The animal model used in this study does not aim to mimic Graves' disease but to investigate the specific effects of high-circulating levels of T 3 on B-cells. In animal models of Graves' disease, it is difficult to analyze the effects of high-circulating levels of TH on B-cell physiology due to the interference of autoimmune B lymphocyte activity (Ludgate 2000). In our model of T 3 -injected mice, serum T 3 reached values threefold to fourfold higher than euthyroid levels. ...
The effects of hyperthyroidism on B cell physiology are still poorly known. In this study, we evaluated the influence of high circulating levels of T3 on bone marrow, blood and spleen B cell subsets, more specifically on B cell differentiation to plasma cells, in C57BL/6 mice daily injected with T3 for fourteen days. As analyzed by flow cytometry, T3-treated mice exhibited increased frequencies of pre-B and immature B cells and decreased percentage of mature B cells in the bone marrow, accompanied by an increased frequency of blood B cells, splenic newly-formed B cells and total CD19+ B cells. T3 administration also promoted an increase in the size and cellularity of the spleen as well as in the white pulp areas of the organ, as evidenced by histological analyses. In addition, a decreased frequency of splenic B220+ cells correlating with an increased percentage of CD138+ plasma cells was observed in the spleen and bone marrow of T3-treated mice. Using ELIspot assay, an increased number of splenic immunoglobulin-secreting B cells from T3-treated mice was detected ex vivo. Similar results were observed in mice immunized with hen egg lysozyme and aluminum adjuvant alone or plus treatment with T3. In conclusion, we provide evidence that high circulating levels of T3 stimulate plasmacytogenesis favoring an increase of plasma cells in the bone marrow, a long-lived plasma cell survival niche. These findings suggest that a possible stimulatory effect on plasma cell differentiation could occur in untreated patients with Graves' disease.
... Several animal models of the hyperthyroidism of GD have been developed over the past 2 decades using various vectors to deliver TSHR (8,9). Although no convincing evidence of GO was reported in any of these models, in most, the orbits were not carefully examined. ...
... Since, the fish is able to absorb small molecules dissolved in the surrounding water through their chorion, skin and gills, the administration of drugs in minute quantities is straightforward (Parng, 2005;Rubinstein, 2006). Further, the strong synteny (with overlapping orthologs/analogs) of the genomes and 100% identical nature of the protein functional domains (where drugs bind) between zebrafish and human (Parng, 2005;Parng et al., 2002;Prescott and Yost, 2002) has made this fish a close vertebrate model to study human diseases and is largely employed for in vitro assays in drug/pharmaceutical discovery research (Dooley and Zon, 2000;Ma, 2004;Alestrom et al., 2006). ...
Effects of ibuprofen (a non-selective COX inhibitor) on the embryonic development, hatching success, larval growth, behavioral pattern and survival competence were studied in Danio rerio. Embryos at 2/4 celled stage were exposed to graded doses (0, 1, 5, 10, 50 and 100μg/L distilled water) of ibuprofen in triplicate sets (n=30). The experiment was repeated thrice. The results indicate that developing embryos tolerated lower (1 and 5μg/L) doses of the drug readily but, exposure to higher doses (>10μg/L) caused retarded development, decreased hatching rate and growth, cardiac anomalies, spinal curvature, pectoral fin malformation and behavioral alterations resulting in greater mortality of experimental embryos. This study suggests that, ibuprofen which is marketed as over-the-counter (OTC) drug is embryotoxic at least at higher (>10μg/L) dose level to zebrafish embryos.
... Our study in Swiss outbred mice resulted in a lower induction rate with this protocol (7 . 5% (3 out of 40)), contributed by additional factors such as genetic makeup of mouse strains and environmental differences between animal laboratories (Ludgate 2000). However, TRAB were commonly detected using various assays, demonstrating the dissociation between thyroid status and immunological responses against the antigen. ...
Animal models of Graves' disease have been generated in recent years with various vaccination protocols using wild-type TSH receptor. In this study, we report the findings of genetic immunization of Swiss outbred mice with three different mutated human TSH receptor plasmids, each containing one constitutive activating mutation located at the ectodomain (S281N), exoloop (I486F), and transmembrane segment (D633H) respectively. Although the overall rate of thyrotoxicosis in the mice was < 10%, anti-TSH receptor antibodies could be detected in many animals by flow cytometry, radioreceptor assay, and functional bioassays using recombinant human TSH receptor. Mice injected with plasmids harboring activated mutants (S281N and D633H) showed production of predominantly stimulating antibodies, whilst those treated with wild-type receptor plasmids generated mainly blocking sera. Most of these antibodies displaced radiolabeled bovine TSH, and their epitopes, independent of functional characteristics, were mapped to the first 271 amino acids of the TSH receptor. This supports recent findings that binding of stimulatory or blocking antibodies lie in close proximity within the leucine-rich repeat region.
У статті узагальнено та проаналізовано матеріал багаторічних досліджень деяких аспектів патогенезу, клінічного перебігу, лікування, особливостей довготривалого спостереження хворих на автоімунну офтальмопатію (АО). АО — автоімунне захворювання, що характеризується комплексним ураженням тканин орбіти та супроводжується змінами функціонування органу зору. Патогенез АО тісно пов’язаний із патологією щитоподібної залози (ЩЗ), у 90% випадків АО поєднується з дифузним токсичним зобом (ДТЗ), і лише в 10% із тиреоїдитом Хашимото, але може перебігати і як незалежне захворювання. За різними даними, АО зустрічається з частотою від 5 до 20% усіх випадків ДТЗ. Жінки хворіють у 4 рази частіше ніж чоловіки, переважно у віці від 40 до 60 років. Цитокіни беруть активну участь у розвитку АО при ДТЗ. Так, було виявлено гіперекспресію інтерлейкінів (ІЛ-1β, ІЛ-2, ІЛ-4, ІЛ-5, ІЛ-6, ІЛ-8 та ІЛ-10), фактора некрозу пухлин (ФНП), які зростають у кілька разів при активній стадії АО, однак, залишаються дискусійними питання щодо специфічності цитокінів при АО, ще й досі недостатньо вивченим залишається патогенез АО, ефективність різних методів лікування цієї недуги. Селен та вітамін D3 беруть участь у розвитку автоімунних захворювань, питання ролі змін рівня селену та вітаміну D3 в крові хворих на АО та участь їх у патогенезі змін у тканинах орбіт до кінця не з’ясоване. Актуальною проблемою є дослідження результатів лікування АО за ДТЗ та створення новітніх схем профілактики й лікування. Мета — на підставі аналізу клініко-імунологічних особливостей перебігу захворювання та оцінки віддалених результатів, оптимізувати лікування ДТЗ з АО. Об’єкт дослідження: віддалені результати лікування хворих на ДТЗ з АО, процеси лікування АО. Матеріал та методи: анкетування, клінічні, магніторезонансна томографія (МРТ), ультразвукове дослідження (УЗД) орбіт, гормональні, імуноферментні, біохімічні. Результати. Проведено обстеження хворих на ДТЗ з АО. Визначення функціонального стану ЩЗ показало, що еутиреоз мав місце в 65% пацієнтів, гіпертиреоз у 18%, а субклінічний гіпертиреоз у 17%, показник стимулюючих антитіл до рецептора тиреотропного гормону (рТТТ) вірогідно перевищував нормальні значення. Рівень прозапальних цитокінів — ІЛ-1β вірогідно (р<0,05) зростав у пацієнтів із ДТЗ, порівняно з контрольною групою здорових осіб. У хворих на активну стадію АО концентрація ІЛ-1β вірогідно (p<0,05) перевищувала цей показник у групі пацієнтів без АО, що може вказувати на специфічність цього цитокіну як маркера активності запального авто- імунного процесу в орбітах. Більшість хворих на АО мали III-IV стадію перебігу за класифікацією NOSPECS (No symptoms or signs, Only signs, Soft tissue symptoms and signs, Proptosis, Extraocular muscle involvement, Corneal involvement and Sight loss due to optic neuropathy) і шкалою клінічної активності (Clinical Activity Score, CAS) — 5,2 бала (активну форму АО). УЗД орбіт виявило, що у всіх хворих структура очних м’язів була гіпоехогенною та наявне підвищення рівня ФНП-α). Порівняння розмірів м’язів орбіти за даними УЗД та МРТ виявило значущі відмінності, товщина внутрішнього, зовнішнього, верхнього та нижнього прямих м’язів, як правої, так і лівої орбіти, були вірогідно більшими за даними МРТ порівняно з УЗД (p<0,05). Після хірургічного лікування ДТЗ з АО рівень антитіл до рТТГ (АТрТТГ) та ФНП-α вірогідно зменшується вже через 6 місяців, а мінімального рівня вони досягали через 24 місяці. У 14,3% хворих значно зменшуються клінічні прояви АО, що проявлялося в зникненні спонтанного ретробульбарного болю, періорбітального набряку, гіперемії кон’юнктиви та хемозу. Проведено лікування хворих з активним поступальним перебігом АО на тлі ДТЗ селективним імуносупресантом мофетилмікофенілатом (2-морфоліноетиловий ефір мікофенольної кислоти, МФК). Отримано виразний позитивний ефект відносно зменшення проявів активності АО за шкалою САS, лікування приводило до зменшення рівня АТрТТГ та С-реактивного білка. Висновки. Розроблено, удосконалено та апробовано сучасний алгоритм діагностики та лікування ДТЗ з АО, що дасть змогу персоналізовано підходити до планування програм спостереження та лікування хворих.
Background: Variation in induced models of autoimmunity has been attributed to the housing environment and its effect on the gut microbiota. In Graves' disease (GD), autoantibodies to the thyrotropin receptor (TSHR) cause autoimmune hyperthyroidism. Many GD patients develop Graves' orbitopathy (GO) characterized by orbital tissue remodeling including adipogenesis. Murine models of GD/GO would help delineate pathogenetic mechanisms and although several have been reported most lack reproducibility. A model comprising immunization of female BALBc mice with a TSHR expression plasmid using in vivo electroporation, was reproduced in two independent laboratories. Similar orbital disease was induced in both centers, but differences were apparent (e.g., hyperthyroidism in Center 1 but not Center 2). We hypothesized a role for the gut microbiota influencing the outcome and reproducibility of induced GO.
Results: We combined metataxonomics (16S rRNA gene sequencing) and traditional microbial culture of the intestinal contents from the GO murine model, to analyze the gut microbiota in the two centers. We observed significant differences in alpha, beta-diversity and in the taxonomic profiles, e.g. Operational Taxonomic Units (OTUs) from the genus Lactobacillus were more abundant in Center 2, Bacteroides and Bifidobacterium counts were more abundant in Center 1 where we also observed a negative correlation between the OTUs of the genus Intestinimonas and TSHR autoantibodies. Traditional microbiology largely confirmed the metataxonomics data and indicated significantly higher yeast counts in Center 1 TSHR-immunized mice. We also compared the gut microbiota between immunization groups within the Center 2, comprising the TSHR or βgal control immunized mice and naïve untreated mice. We observed a shift of the TSHR immunized mice bacterial communities described by the beta-diversity weighted Unifrac. Furthermore, we observed a significant positive correlation between the presence of Firmicutes and orbital-adipogenesis specifically in TSHR-immunized mice.
Conclusions: The significant differences observed in microbiota composition from BALBc mice undergoing the same immunization protocol in comparable specific-pathogen free (SPF) units in different centers support a role for the gut microbiota in modulating the induced response. The gut microbiota might also contribute to the heterogeneity of induced response since we report potential disease-associated microbial taxonomies and correlation with ocular disease.
Since the 1970s, the role of infectious diseases in the pathogenesis of Graves' disease (GD) has been an object of intensive research. The last decade has witnessed many studies on Yersinia enterocolitica, Helicobacter pylori and other bacterial organisms and their potential impact on GD. Retrospective, prospective and molecular binding studies have been performed with contrary outcomes. Until now it is not clear whether bacterial infections can trigger autoimmune thyroid disease. Common risk factors for GD (gender, smoking, stress, and pregnancy) reveal profound changes in the bacterial communities of the gut compared to that of healthy controls but a pathogenetic link between GD and dysbiosis has not yet been fully elucidated. Conventional bacterial culture, in vitro models, next generation and high-throughput DNA sequencing are applicable methods to assess the impact of bacteria in disease onset and development. Further studies on the involvement of bacteria in GD are needed and may contribute to the understanding of pathogenetic processes. This review will examine available evidence on the subject.
The animal models used in the investigations on thyroid help to study the pathogenic mechanisms leading to the appearance of disorders of this gland, and give the opportunity to find new treatments and ways of prevention. The objective of this paper was to provide a detailed information on the biomodels of diseases affecting the thyroid, starting from a bibliographic review on this topic, including those obtained in a spontaneous or induced way, and explaining the environmental factors influencing them. To conclude, the advantages of these biomodels for the researchers devoted to study this branch of endocrinology were stressed, although they are not an exact reflex of what happens in man and they do not necessarily have all the distinctive features of thyroid pathology.
Introduction:
Graves' disease (GD) is an autoimmune disorder responsible for 60-90% of thyrotoxicosis, with an incidence of 1 to 2 cases per 1000 population per year in England. Graves' orbitopathy (GO) is the most frequent extrathyroidal manifestation, not provoked directly by abnormal thyroid hormone levels, but by the consequence of the underlying autoimmune process. The aetiology of autoimmune disorders is due to an interplay between susceptibility genes and environmental factors, such as infections and stress. What triggers the autoimmune reaction to a specific site of the body is not yet clearly understood. The lack of knowledge in GD and GO pathogenesis implicates therapies that only limit damage but do not prevent disease onset.
Material and methods:
We performed on PubMed and the Cochrane Library a literature search for the articles published until July 2016 by using the search terms 'graves disease' and 'microbiome', 'orbitopathy' and 'autoimmune pathogenesis'. Reference lists of relevant studies were hand-searched for additional studies.
Conclusion:
In this scenario, a Marie Sklodowska-Curie funded project INDIGO ( http://www.indigo-iapp.eu/ ) is investigating the role of the gut bacteria in GD and GO pathogenesis. The gut is the first and the widest area of bacteria access, with the highest concentration of T cells in the human body and trained to react to microorganisms. Interestingly, all the environmental factors involved in GD and GO pathogenesis can alter the balance within the microorganisms located in the gut, and influence the immune system, in particular the proportions of regulatory Treg and inflammatory TH17 cells. It is hoped that investigating GD and GO pathogenesis from this novel aspect will identify new targets for prevention and treatment.
Graves’ disease (GD) is an autoimmune syndrome characterized by hyperthyroidism and a diffusely enlarged thyroid gland. The most prominent extrathyroidal manifestation of this thyroid disease is thyroid-associated or Graves’ ophthalmopathy (GO), a medically incurable and chronic autoimmune process that affects all orbital tissue compartments and leads to various eye complications such as discomfort, lid edema and retraction, proptosis, extraocular muscle dysfunction, diplopia, and sight loss. Various degrees of GO occur in 80-90% of patients with GD. In most instances, the orbital problems appear within 18 months after diagnosis of thyroid disease. The close clinical association of GD with GO and pretibial dermopathy (PTD), a less frequent extrathyroidal manifestation, suggests a common antigen for these affected tissues (1,2). Enlargement of extraocular muscle bodies together with an increase of orbital connective/fatty tissue within the bony orbits is responsible for most of the orbital problems in patients with severe active GO. This enlargement is caused by marked infiltration of immunocompetent cells, such as macrophages, T lymphocytes and some B cells, and by abundant quantities of collagen and hydrophilic glycosaminoglycans (GAGs). The inflammatory process is likely to be driven by T cells, which access and inflltrate the retro-orbital space after interaction with several adhesion molecules. Once recruited, T cells release numerous cytokines capable of stimulating cell proliferation, GAG synthesis, recruitment of new fat cells from orbital adipose precursor cells, and expression of various immunomodulatory molecules by orbital preadipocyte fibroblasts (3). Although these mechanisms may sufficiently explain various aspects of how GO may evolve and be propagated, the primary antigen for this autoimmune process has remained elusive.
Thyroid stimulating hormone (TSH; thyrotropin) is the primary factor for regulating both differentiated function and growth of thyroid follicular epithelial cells (1). The action of TSH is initiated by its binding to TSH receptor (TSHR) on the basolateral site of the thyroid cell plasma membrane, which transduces signals through Gs-cAMP and, to a lesser extent, Gq-phospholipase C cascades. In pathological terms, TSHR involves thyroid autoimmunity and oncogenesis. Thus, TSHR, as well as thyroid peroxidase (TPO) and thyroglobulin (TG), is a target autoantigen in human autoimmune thyroid diseases such as Graves’ disease and Hashimoto’s thyroiditis. Autoantibodies against TSHR stimulate thyroid cells (stimulatory-type autoantibody) or block TSH action (blocking-type autoantibody) (2). Ectopic expression of TSHR may be involved in extrathyroidal manifestations of Graves’ disease such as ophthalmopathy and pretibial myxedema (3). Furthermore, gain-and loss-of-function mutations of the receptor have been found in hyperfunctioning adenoma/congenital nonautoimmune hyperthyroidism and congenital hypothyroidism, respectively (4,5).
Objective: To study the antigenicity of human throtropin receptor (hTSHR) amino terminus (amino acid 29-280) and its association with Graves' disease. Methods: Total thyroid RNA was prepared from human normal thyroid tissue. RNA was then reversely transcripted and cDNA was subjected to PCR amplification. PCR product was cloned into pcDNA3.1 and the recombinant plasmid was named pcDNA3.1/hTSHR188-940bp. Balb/c mice were immunized with pcDNA3.1/hTSHR188-940bp. The levels of serum thyroxin, anti-TSHR antibody (TRAb) and thyroid stimulating antibody (TSAb) were measured, and the pathological changes of thyroid tissue were also observed. Results: A 753 bp fragment encoding hTSHR ectodomain amino end was obtained after PCR amplification. Confirmed by Hind III restriction enzyme digestion and DNA sequencing, pcDNA3.1/hTSHR188-940bp had been constructed successfully, with the correct sequence and direction of hTSH188-940bp. In the Balb/c mice treated With pcDNA3.1/hTSHR 188-940 bp, elevated TRAb in week 6 (0.148 ± 0.018) were observed compared with those at week 0(0.106 ± 0.006, P < 0.01), and kept a higher level till week 10(0.134 ± 0.011, P< 0.01). T4 and TSAb index values were significantly increased in week 10. Serum T4 concentration increased from (41.02 ± 7.97) μg/L in week 0 to (62.20 ± 12.77) μg/L in week 10 (P < 0.01); TSAb index values rose from 0.864 ± 0.076 at week 0 to 1.392 ± 0.615 (P < 0.01). Thyroid pathological examination showed that proliferated thyroid follicular epithelial cells and follicular capacity increased. Inflammatory cells were occasionally found. Conclusions: There are antigen epitopes in hTSHR ectodomain amino acid 29-280, which can stimulate the production of TSAb. And the latter induces hyperthyroidism and Graves' disease like manifestations. It suggests that hTSHR ectodomain amino acid 29 ∼ 280 is closely associated with Graves' disease, and maybe one of important etiological factors leading to the disease.
Thyroid-associated ophthalmopathy(TAO) is an orbital disease that can affect the patients' external appearance and result in progressive visual regression. The establishment of animal model of TAO will be helpful for research and treatment of TAO. TAO is generally considered to have an autoimmune etiology and is thought to result from a complex interplay of genetic and environmental factors. The most promising approaches to development of more convincing models of TAO is to utilize the cells transferred with the homologous major histocompatibility complex class II molecule and the full-length human or murine TSHR to immunize AKR/N mice, and transfer the TSHR primed T cells to naive syngeneic recipients, and use full-length human TSHR to genetically immunize NMRI outbred mice.
Autoimmune thyroid diseases (AITD) cover the spectrum from hypothyroid Hashimoto’s thyroiditis (HT) to hyperthyroid Graves’ disease (GD). The main autoimmune targets are thyroglobulin (TG), thyroid peroxidase (TPO) and the thyrotropin receptor (TSHR). Autoantibodies and specific T cells directed against all three autoantigens can be detected in the circulation of HT and GD patients and also in a significant proportion of the healthy population. In AITD, as in other autoimmune conditions, the central question remains how is immune tolerance overcome? In vivo models, mostly induced in rodents, have contributed to our understanding of the mechanisms operating and many hundreds of papers, spanning from 1956 to the present day, have been published describing the results obtained. Most of the information has been derived from experimental autoimmune thyroiditis (EAT) models induced with TG, an antigen that, based on current knowledge, seems to be of lesser importance in human AITD. Nevertheless, many of the basic precepts underlying autoimmunity, for example the importance of the major histocompatability complex (MHC) II and the existence of immunoregulatory T cells, have been identified using TG-based models, and these are described. Reports based on induction of disease with TPO, the driving antigen in HT, are not very numerous but include a seminal paper that clearly demonstrates the redundancy of autoantibodies and the central role of T cells in pathogenesis. In contrast, autoantibodies to the TSHR cause GD and much of the chapter is devoted to models attempting to mimic GD, and these have been the subject of considerable effort since its cloning in 1998. This has culminated with the recent publication of monoclonal antibodies with thyroid stimulating activity (TSAB) either measured in vitro or in vivo.
The thyroid-stimulating hormone receptor (TSHR, or thyrotropin receptor) is a family A G protein-coupled receptor. It not only binds thyroid-stimulating hormone (TSH, or thyrotropin) but also interacts with autoantibodies under pathological conditions. The TSHR and TSH are essential for thyroid growth and function and thus for all thyroid hormone-associated physiological superordinated processes, including metabolism and development of the central nervous system. In vitro studies have found that the TSHR permanently stimulates ligand-independent (constitutive) activation of Gs, which ultimately leads to intracellular cAMP accumulation. Furthermore, a vast variety of constitutively activating mutations of TSHR-at more than 50 different amino acid positions-have been reported to enhance basal signaling. These lead in vivo to a "gain-of-function" phenotype of nonautoimmune hyperthyroidism or toxic adenomas. Moreover, many naturally occurring inactivating mutations are known to cause a "loss-of-function" phenotype, resulting in resistance to thyroid hormone or hyperthyrotropinemia. Several of these mutations are also characterized by impaired basal signaling, and these are designated here as "constitutively inactivating mutations" (CIMs). More than 30 amino acid positions with CIMs have been identified so far. Moreover, the permanent TSHR signaling capacity can also be blocked by inverse agonistic antibodies or small drug-like molecules, which both have a potential for clinical usage. In this chapter, information on constitutive activity in the TSHR is described, including up- and downregulation, linked protein conformations, physiological and pathophysiological conditions, and related intracellular signaling.
: The pathophysiology of thyroid eye disease (TED) is complex and incompletely understood. Orbital fibroblasts (OFs) seem to be the key effector cells that are responsible for the characteristic soft tissue enlargement seen in TED. They express potentially pathogenic autoantigens, such as thyrotropin receptor and insulin-like growth factor-1 receptor. An intricate interplay between these autoantigens and the autoantibodies found in Graves disease may lead to the activation of OFs, which then leads to increased hyaluronan production, proinflammatory cytokine synthesis, and enhanced differentiation into either myofibroblasts or adipocytes. Some of the OFs in TED patients seem to be derived from infiltrating fibrocytes. These cells originate from the bone marrow and exhibit both fibroblast and myeloid phenotype. In the TED orbit, they may mediate the orbital expansion and inflammatory infiltration. Last, lymphocytes and cytokines are intimately involved in the initiation, amplification, and maintenance of the autoimmune process in TED.
: A 43-year-old man with a high-grade glioma involving the cerebellar nodulus showed a near-complete suppression of periodic alternating nystagmus (PAN) in the lateral decubitus position to either side. This positional modulation of PAN is consistent with suppression of the velocity storage mechanism by head position changes (tilt dumping) and is supportive of the role of the velocity storage mechanism in generating PAN.
Environmental Factors
in Autoimmune Endocrinopathies
Summary
The autoimmune endocrinopathies include a wide range of diseases affecting one or more endocrine
glands. While a strong genetic predisposition underlies their development, environmental factors are
also involved in their pathogenesis. These environmental agents include infections, therapeutic drugs,
chemicals, and radiation. A firm relationship between these environmental agents and autoimmune diseases
is difficult to establish as exposure to these agents often precedes onset of disease by a considerable
margin. Animal models have helped considerably to establish a cause/effect relationship. The mechanisms
by which autoimmunity may be initiated include changes in autologous antigens, alterations in immune
regulation, or altered gene expression. Environmental factors alter the immune responses depending on the
genetic susceptibility of the host and may be regulated by the quality, quantity, and duration of exposure.
Key Words: Environmental factors, diabetes, thyroid diseases.
From: Contemporary Endocrinology: Autoimmune Diseases in Endocrinology
Edited by: A. P. Weetman © Humana Press, Totowa, NJ
Selection is the major force affecting local levels of genetic variation in species. The availability of dense marker maps offers new opportunities for a detailed understanding of genetic diversity distribution across the animal genome. Over the last 50 years, cattle breeds have been subjected to intense artificial selection. Consequently, regions controlling traits of economic importance are expected to exhibit selection signatures. The fixation index (Fst ) is an estimate of population differentiation, based on genetic polymorphism data, and it is calculated using the relationship between inbreeding and heterozygosity. In the present study, locally weighted scatterplot smoothing (LOWESS) regression and a control chart approach were used to investigate selection signatures in two cattle breeds with different production aptitudes (dairy and beef). Fst was calculated for 42 514 SNP marker loci distributed across the genome in 749 Italian Brown and 364 Piedmontese bulls. The statistical significance of Fst values was assessed using a control chart. The LOWESS technique was efficient in removing noise from the raw data and was able to highlight selection signatures in chromosomes known to harbour genes affecting dairy and beef traits. Examples include the peaks detected for BTA2 in the region where the myostatin gene is located and for BTA6 in the region harbouring the ABCG2 locus. Moreover, several loci not previously reported in cattle studies were detected.
Luciano Adorini – BioXell, Milan, Italy
The autoimmune origin of thyroid diseases, such as Hashimoto's thyroiditis and Graves’ disease was clearly established more than 50 years ago. Since then, considerable progress has been made in understanding immunological mechanisms leading to autoimmune thyroid diseases. The current status of knowledge in the field is clearly summarized in the review by Nicole Parish and Anne Cooke, leading scientists who have made important contributions in experimental autoimmune thyroiditis. This ground-work should facilitate the implementation of appropriate immunointervention protocols targeting key elements in the autoimmune response, although progress in this area has been, so far, relatively slow.
19 women with premature ovarian failure that were seen at the physician's office due to secondary hypergonadotropic amenorrhea were studied to determine the existence of some degree of ovarian endocrine reserve. A detailed medical history was taken and complementary examinations were made to define the cause. The basal levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), Prolactin (Prl), and Thyrotropin (TSH) were determined.The steroideal ovarian reserve was also determined by the dynamic test of hypophyseal inhibition of gonadotropins (Gn) with ethynyl estradiol and, later, it was estimulated with gonadotropic menopausic hormone (GMH).The basal levels of FSH, LH, E2, testosterone (T) and androstenedione (A'd) were calculated during inhibition and after the stimulation with GMH. It was found that the mean basal levels of FSH were higher than those of LH, the levels of E2 were low and Prl was normal on analyzing them as a group. The mean basal level of E2 was low and there was no increase of its mean levels after the stimulation with GMH. The mean basal levels of T were normal, unlike A'd that was low. No rise was observed poststimulation on being analyzed as a group. It was proved that 52.6 % of these patients have an ovarian estrogenic reserve, whereas only 20 % have an androgenic reserve. There were no important differences in the results of the dynamic test on dividing patients into 2 groups, according to the chronological age, time of amenorrhea and cause of POF, excepting that the reserve of T that was only found in those of idiopathic cause, which was not observed in those of immunological cause.
The thyrotropin or thyroid stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors (GPHRs), a sub-family of family A G-protein coupled receptors (GPCRs). The TSHR is of great importance for the growth and function of the thyroid gland. The TSHR and its endogenous ligand TSH are pivotal proteins with respect to a variety of physiological functions and malfunctions. The molecular events of TSHR regulation can be summarized as a process of signal transduction, including signal reception, conversion and amplification. The steps during signal transduction from the extra- to the intracellular sites of the cell are not yet comprehensively understood. However, essential new insights have been achieved in recent years on the interrelated mechanisms at the extracellular region, the transmembrane domain and intracellular components. This review contains a critical summary of available knowledge of the molecular mechanisms of signal transduction at the TSHR, for example, the key amino acids involved in hormone binding or in the structural conformational changes that lead to G-protein activation or signaling regulation. Aspects of TSHR oligomerization, signaling promiscuity, signaling selectivity, phenotypes of genetic variations and potential extra-thyroidal receptor activity are also considered, as these are relevant to an understanding of the overall function of the TSHR, including physiological, pathophysiological and pharmacological perspectives. Directions for future research are discussed.
Objective:
To compare the efficacy of different expression vectors, target genes, and immunization procedures in transfecting mice via liposome to construct murine model of Graves disease.
Methods:
We linked pCDNA3.1(+) and pUBC to full-length human TSHR and TSHR A subunit cDNA to yield four plasmids, which were later injected intramascularly or subcutaneously into female Balb/c mice via liposome. The blood anti-TSHR antibody (TRAb) were determined and the body weight were measured after each immunization. Serum thyroid hormone levels were measured after the animals were sacrificed.
Results:
In mice immunized with pUBC, no significant variance with control in weight nor serum TRAb concentration was observed. Weight gain in pCDNA3.1(+) group was significantlyly slower than controls (p<0.05), and serum TRAb concentration was also significantly elevated. In pCDNA group, animals immunized with TSHR A subunit (TSHRA subgroup) as the target gene revealed even significantly slower weight gain (p<0.001) and even faster TRAb elevation than those immunized with full length TSHR. Significantly higher FT4 (p=0.023) was observed in TSHRA and TSHR subgroups, which was reversely correlated to weight gain, but no significant difference (p>0.05) in FT3 was observed. Weight gain and TRAb concentration mainly varied in the later period of immunization.
Conclusions:
Immunization with pCDNA3.1(+) and TSHR A subunit gene together with higher immunization frequency increases the chance of model induction. Furthermore, FT4 is a better indicator for assessing the thyroid function in this model.
The thyrotropin receptor (TSHR) A-subunit has been reported to be a critical autoantigen in the generation of thyroid-stimulating antibodies, thereby causing Graves' disease (GD). However, immune mechanisms associated with GD animal models induced by TSHR A-subunit are poorly understood until now.
Female BALB/c mice (n = 23) were randomly divided into two groups, and GD presentation was monitored following injection with either 50 μl phosphate-buffered saline containing 10(9) particles of adenovirus expressing the human TSHR A-subunit (Ad-TSHR289) or the Ad-LacZ control. Expressions of CD40, CD40L, CD80, CD86, CD28, CTLA-4, FOXP3 and IL-17A in various tissues were assessed by quantitative RT-PCR and immunohistochemical assays.
Compared with control group, mice of the hyperthyroid group showed significant elevation of expression in the thyroid of CD40 and CD86, expression in the heart of CD28, CD40 and CD40L and expression in the liver of CD28, CD40 and CD86. Conversely, there was significantly diminished expression of CTLA-4 in the thymus of mice in the hyperthyroid group. Expression of all genes examined was not significantly different in the spleens of mice from either of the groups and CD40L and FOXP3 expression was not detected in the thyroids of hyperthyroid mice.
The expression profile of multiple immune-related molecules differed in mice in the GD group following Ad-TSHR289 immunization, suggesting that these molecules played a potential role in GD pathogenesis.
取得学位：博士(医学)，報告番号：甲第2119号，学位授与年月日：平成14年12月31日,学位授与年：2002
We aimed to establish and extend the characterization of murine models of thyroiditis and Graves’ ophthalmopathy, induced by transfer of TSH receptor (TSHR) primed T cells. Experiments were performed in a different animal unit but using female BALB/cbyJico mice from the same supplier as previously. We report our findings together with a reevaluation of the earlier studies. In the first experiment, genetic immunization or TSHR fusion protein induced TSHR antibodies in all nine mice. Some of the antibodies functioned as thyroid-stimulating antibodies and/or TSH binding inhibiting Igs with two of seven mice having elevated T4. Thyroiditis and orbital changes were absent. Splenocyte transfer induced no immune response in naive BALB/cbyJico recipients. Subsequently genetic immunization or fusion protein-treated mice were maintained in either local or Brussels conditions (water, chow, and bedding). TSHR antibodies were induced in nine of nine Brussels (with decreased T4 in one of nine) but five of nine local mice. No thyroiditis or orbital changes were induced, but misleading fixation artefacts in extraocular muscles were noted. Nonspecific in vitro stimulation induced more CD-4+/IL-4+ cells in Brussels maintained. TSHR stimulation produced a significant increase in IL-4 secretion in six of nine local but one of seven Brussels mice. Thyroids from many TSHR-treated and control mice contained ectopic thymus. Our results confirm that thyroiditis is required for disease transfer but indicate the heterogeneity in TSHR-induced immune response in an inbred strain. Ectopic thymus can masquerade as thyroiditis, and care is required to avoid muscle artefacts. Because neither animal unit is pathogen free, microbial environment may contribute to determining TSHR-induced responses.
Previous studies have indicated pre-existing subclinical Graves' disease (GD) in many patients with the scintigraphic diagnosis of disseminated thyroid autonomy (DISA) or toxic multinodular goitre (TMG) type A. After radioiodine (RAI) treatment, an increase or the induction of TSH-receptor antibodies (TRAbs) in patients with GD or TMG has been repeatedly reported.
In the present study, we investigated whether RAI could induce TRAbs in patients with TMG in whom pre-existing GD was excluded with highly sensitive TBII and TSAB assays. Therefore, TRAbs, anti-thyroperoxidase antibodies (anti-TPO-Abs) and anti-thyroglobulin antibodies (anti-TG-Abs) were determined in 43 consecutive patients at the nuclear medicine outpatient clinic with the scintigraphic diagnosis of toxic adenoma (TA; n = 20) or TMG type A (n = 11) or type B (n = 12) before and after RAI treatment. After RAI therapy, we detected TRAbs in 36 % (4 of 11) of patients with TMG type A only, whereas TRAbs were not detectable in patients with TMG type B or in patients with TA. Furthermore, 3 of the 4 patients with detectable TRAbs after RAI showed positive anti-TPO-Abs before RAI therapy. These findings provide further evidence for pre-existing GD in patients with TMG type A or DISA as previously suggested. Therefore, patients with TMG type A and high anti-TPO-Abs seem to be at increased risk of developing TRAbs or side-effects such as relapse of hyperthyroidism or thyroid associated ophthalmopathy. These patients therefore require more frequent evaluation after RAI treatment.
In this work we report a novel method to efficiently induce a murine model of Graves' hyperthyroidism. Inbred mice of different strains were immunized by i.m. injection with adenovirus expressing thyrotropin receptor (TSHR) or beta-galactosidase (1 x 10(11) particles/mouse, three times at 3-wk intervals) and followed up to 8 wk after the third immunization. Fifty-five percent of female and 33% of male BALB/c (H-2(d)) and 25% of female C57BL/6 (H-2(b)) mice developed Graves'-like hyperthyroidism with elevated serum thyroxine (T(4)) levels and positive anti-TSHR autoantibodies with thyroid-stimulating Ig (TSI) and TSH-binding inhibiting Ig (TBII) activities. In contrast, none of female CBA/J (H-2(k)), DBA/1J (H-2(q)), or SJL/J (H-2(s)) mice developed Graves' hyperthyroidism or anti-TSHR autoantibodies except SJL/J, which showed strong TBII activities. There was a significant positive correlation between TSI values and T(4) levels, but the correlations between T(4) and TBII and between TSI and TBII were very weak. TSI activities in sera from hyperthyroid mice measured with some chimeric TSH/lutropin receptors suggested that their epitope(s) on TSHR appeared similar to those in patients with Graves' disease. The thyroid glands from hyperthyroid mice displayed diffuse enlargement with hypertrophy and hypercellularity of follicular epithelia with occasional protrusion into the follicular lumen, characteristics of Graves' hyperthyroidism. Decreased amounts of colloid were also observed. However, there was no inflammatory cell infiltration. Furthermore, extraocular muscles from hyperthyroid mice were normal. Thus, the highly efficient means that we now report to induce Graves' hyperthyroidism in mice will be very useful for studying the pathogenesis of autoimmunity in Graves' disease.
Graves' disease (GD) is a polygenic autoimmune thyroid syndrome. Some of the genes implicated in its pathogenesis may encode thyroid-stimulating hormone receptor (TSHR) and estrogen receptors 1 (ESR1) and 2 (ESR2). We examined dinucleotide repeat polymorphisms in the ESR1 and ESR2 genes and D727E amino acid substitution in the TSHR gene for possible association with GD in a Russian population.
The polymorphic regions of the target genes were amplified by polymerase chain reaction (PCR) on the basis of genomic DNA isolated from blood of 78 unrelated Russian patients with GD and 93 control subjects. To detect the D727E TSHR polymorphism, the PCR product was additionally digested with Eco72I restriction endonuclease. The genotype and allele frequencies in the groups studied were compared by c2 test. The odds ratios and 95% confidence intervals (CI) were calculated to assess the strength of the relationship between the polymorphisms tested and GD.
For polymorphic dinucleotide microsatellites at ESR1 and ESR2, no significant difference was observed in allele frequencies between affected and nonaffected patients. For the D727E TSHR polymorphism, the E allele and the DE genotype were significantly more frequent (p<0.0001) in patients with GD than in control subjects.
The D727E variant of the TSHR gene is associated with Graves' disease in a Russian population. The E727 allele and the heterozygous D727E genotype are related to higher risk of the disease. No association with GD was found for polymorphic microsatellites of the ESR1 and ESR2 gene.
The past decade has witnessed great progress in our understanding of Graves' opthalmopathy (GO), although its precise immunopathogenesis remains an enigma. Several clinical studies have provided a more rational basis for treatment of this distressing disease, which significantly lowers the quality of life. A management plan tailored to the patient's needs can be devised according to the severity and activity of the eye disease. In active GO, immunosuppression might be considered. The combination of intravenous pulses of methylprednisolone and retrobulbar irradiation improves eye changes in 88% of patients, and is well tolerated. Once the disease has become inactive, rehabilitative surgery could be performed (orbital decompression, strabismus surgery and eyelid surgery, in that order). The patient should be reassured that functional and cosmetic improvement of eye changes is feasible, but restoration can require one to two years. To a certain extent, refraining from smoking prevents the development or worsening of GO.
Organ-specific or endocrine autoimmune diseases are complex, polygenic afflictions the penetrance of which is heavily dependent on various environmental influences. Important target tissues are the thyroid, the islets of Langerhans, gastric parietal cells and steroid-producing cells in the adrenal and ovary. The etiology of these diseases remains to be clarified. The pathogenesis is strongly associated with autoimmune phenomena. None of the current treatment approaches provides a cure; rather they represent replacement therapy. An important objective in the treatment of endocrine/organ-specific autoimmune diseases is the detection of individuals at risk for the development of such diseases and the development of interventions to prevent an outbreak of the diseases. This requires an exquisite knowledge of the early etio-pathogenic stages of these diseases. This review concentrates on the usefulness of animal models for a precise understanding of these very early stages. It must be emphasized that studying animal models cannot answer all the problems presented by endocrine/organ-specific autoimmune diseases as seen in the clinic. It must be expected - considering the different etiologies in the different animal models (see below) - that the causes of the diseases in the human and the involvement of various genes and environmental factors may also vary. Yet, particularly in the study of the pre-autoimmune phases of the diseases, there is hardly any alternative to the study of animal models. Only limited series of experiments can be carried out in human subjects at risk to develop such diseases. Moreover, a general semblance (blueprint) of the etio-pathogenesis found in the animal models can lead the way for human studies. Efforts to understand the patho-physiology of the early stages of endocrine/organ-specific autoimmune diseases have mainly involved animal models that "spontaneously" develop such diseases. Of these the bio-breeding diabetes-prone (BB-DP) rat and the non-obese diabetes (NOD) mouse are the most well studied, yet many studies have also been carried out in the obese strain (OS) chicken. Apart from these spontaneous models there are animal models that are induced by environmental perturbations (viruses, toxic substances), by thymectomy procedures or by genetic manipulations, e.g., the RIP-LCMV model and the BDC 2.5 TCR mouse model. A general blueprint has emerged from the studies into the early stages of the pathogenesis of endocrine/organ-specific autoimmune diseases in these animal models: animals at risk to develop endocrine/organ-specific autoimmune diseases show various pre-autoimmune aberrancies in their target glands, T cells, macrophages (Mphi) and dendritic cells (DC). The presumably aberrant target cells, T cells, DC and Mphi need to interact abnormally before autoimmune disease can fully develop. In this abnormal interaction additional aberrancies in other regulatory systems may play a role in a further exacerbation of the self-directed immune response, such as defects in the hypothalamus pituitary adrenal (HPA) axis system. The various aberrancies are partly genetically determined by a variety of separate genes, particularly MHC-related genes, but they may also be environmentally induced (e.g., via viruses, high iodine diet, and other experimental manipulations). Recently evidence has been gathered for pre-autoimmune aberrancies similar to the animal models in the DC/ Mphi compartment and the HPA axis in humans at risk to develop endocrine/organ-specific autoimmune diseases. However, analogous pre-autoimmune abnormalities in human target glands or in T cell function have not yet been found with certainty. We believe that animal models of endocrine/organ-specific autoimmune disease still hold immense promise for the discovery of pathways, genes and environmental factors that determine the development of endocrine/organ-specific autoimmune diseases. Animals affected by such diseases provide a unique opportunity to uncover disease-associated pathways, which are complicated to define in man.
The non-obese diabetic (NOD) mouse is a model of spontaneous type-1 diabetes used in the field of diabetes research. This study looked at the adrenal glands of NOD and control mice both indirectly in vivo for hormone secretion, and directly in vitro for histological examination. Adrenal glands were taken from NOD mice, of both sexes, at different ages and corticosterone and adrenocorticotropic hormone (ACTH) plasma levels evaluated by radioimmunoassay. There was evidence of lymphocytic infiltration of the adrenal glands, which however, was not accompanied by changes in corticosterone levels. There was a reduction in ACTH levels with age (R2 = 0.98). Mice from other strains (TFW, CBA and Balb/c) showed no lymphocytic infiltration in the adrenal glands and had lower levels of corticosterone than NOD mice of similar ages, but the differences were not significant. In conclusion, since the NOD mouse shows histological signs of adrenalitis, thyroiditis, sialitis and parathyroiditis, this animal can be regarded as a model to investigate mechanisms involved in diffuse lymphocytic infiltration of peripheral endocrine glands (polyendocrine autoimmunity). In addition, if diabetes in the NOD mouse is the result of a polyendocrine disorder rather than a process specific for diabetes, then this finding may have implications for attempts to prevent type-1 diabetes in humans.
In an attempt to develop an animal model for thyroid-associated ophthalmopathy (TAO) we have genetically immunized BALB/c and outbred (CD-1) mice with cDNAs encoding the thyroid and eye muscle shared protein G2s and full length human thyrotropin receptor (TSHr). Firstly, BALB/c mice were immunized with cDNAs for G2s and the TSHr, alone or in tandem with cDNAs for interleukin (IL)4 or IL12. Control mice were immunized with empty vehicle only. Sera from the great majority of experimental mice contained antibodies against a G2s fusion protein and the flavoprotein (Fp) subunit of mitochondrial succinate dehydrogenase, the "64 kDa protein", with the greatest levels being found at sacrifice (17 wk). Antibody levels in mice immunized with G2s + TSHr or G2s + IL12 were generally higher than those in mice immunized with G2s only. TSHr antibodies (TRAb), measured as TSH binding inhibition, were detected in only two mice. On histological examination of the orbits, mild edema, eye muscle fiber separation and mast cell infiltration in and around the eye muscles were found in the majority of experimental mice, but not in control mice. Splenocytes were transferred from selected G2s-immunized mice to normal syngeneic litter mates. None of the transfer mice had serum antibodies against G2s, Fp or TSHr but their orbital tissue showed the same degree of mast cell infiltration as primary mice. No major histological changes were observed in the thyroid or other skeletal muscle in either primary or transfer mice. Similar results were observed in CD-1 mice although, overall, the model was better expressed than in BALB/c mice. In these mice, serum anti-G2s antibody levels were not significantly different between the various experimental groups except at 16 wk, when they were slightly greater than in control animals. Anti-Fp antibodies were detected at 12, 14 and 16 wk, in all experimental groups, including those immunized with G2s only, and were greatest in mice immunized with TSHr alone. TRAb levels were greatest in mice immunized with both G2s and the TSHr in the presence of TL4, but not IL12. The finding of negative anti-G2s but positive anti-Fp antibodies in some CD-1 mice suggests that eye muscle damage and Fp release must have been mediated by T lymphocytes, rather than antibodies, targeting G2s or some other as yet unidentified cell membrane antigen. Histological changes in the orbit were similar to those observed in BALB/c mice although mast cell numbers were greater, in both primary and transfer mice. Overall, the greatest histological changes were observed in CD-1 mice immunized with both G2s + TSHr + IL4. None of the animals became overtly hyperthyroid or hypothyroid during the course of the study although several of the CD-1 mice had abnormal TSH or T4 levels. These results indicate that we have established a valid model for human ophthalmopathy using the novel thyroid and eye muscle expressed protein G2s, now recognized as a fragment of the winged-helix transcription factor Foxp1, and TSHr, and that G2s and the TSHr are both primary antigens in TAO. Reactivity against a TSHr-like protein may be the first event leading to ophthalmopathy in humans with TAO and experimental mice and eye muscle damage may result from autoimmunity against G2s and Fp as a result of "antigen spreading".
Various thyrocyte, monocyte, macrophage, DC and T cell abnormalities exist in the animal models of spontaneously developing autoimmune thyroiditis and in patients with autoimmune thyroid disease. An aberrant interaction between such abnormal thyrocytes, abnormal professional antigen-presenting cells (APC) and abnormal T cells forms the basis for the atypical autoimmune reaction targeting thyroid antigens. In the atypical interaction more than one gene and various environmental factors are involved. The genetic and environmental factors must act together to induce full-blown disease. Although there is a general blueprint for the development of destructive autoimmune thyroiditis, thyrocyte and immune cell abnormalities differ between the various animal models and the various forms of autoimmune thyroid disease (either associated with type 1 diabetes, associated with bipolar disorder or not associated). This tells us that there are different etio-pathogenic forms of destructive autoimmune thyroiditis. Whether such heterogeneity is also the case for the etio-pathogenesis of Graves' disease remains unknown. Animal models of spontaneously developing Graves' disease would be helpful in unraveling this question. If indeed there are various etio-pathogenic routes in different patients that lead to destructive autoimmune thyroiditis, then tailor-made therapeutic approaches need to be carried out in attempts to correct the underlying immune abnormalities in individual patients or to prevent the development of destructive autoimmune thyroiditis in individuals at risk. While in some forms of destructive autoimmune thyroiditis (f.i. those associated with bipolar disorder) immune suppression should be the first choice of intervention, other forms (f.i. those associated with type 1 diabetes) may benefit from immune stimulation in certain pre-stages of the disease (to restore f.i. the faulty APC function characteristic of this condition). Obviously a more precise determination of the spectrum of cell-mediated immune abnormalities is required in individual cases of destructive autoimmune thyroiditis, before therapies that aim at correcting the immune abnormalities can be tested successfully.
Intramuscular injection with plasmid DNA encoding the human thyrotropin receptor (TSHR) has been known to elicit symptoms of Graves' disease (GD) in outbred but not inbred mice. In this study, we have examined, firstly, whether intradermal (i.d.) injection of TSHR DNA can induce hyperthyroidism in BALB/c mice and, secondly, whether coinjection of TSHR- and cytokine-producing plasmids can influence the outcome of disease. Animals were i.d. challenged at 0, 3 and 6 weeks with TSHR DNA and the immune response was assessed at the end of the 8th or 10th week. In two experiments, a total of 10 (67%) of 15 mice developed TSHR-specific antibodies as assessed by flow cytometry. Of these, 4 (27%) mice had elevated thyroxine (TT4) levels and goitrous thyroids with activated follicular epithelial cells but no evidence of lymphocytic infiltration. At 10 weeks, thyroid-stimulating antibodies (TSAb) were detected in two out of the four hyperthyroid animals. Interestingly, in mice that received a coinjection of TSHR- and IL-2- or IL-4-producing plasmids, there was no production of TSAbs and no evidence of hyperthyroidism. On the other hand, coinjection of DNA plasmids encoding TSHR and IL-12 did not significantly enhance GD development since two out of seven animals became thyrotoxic, but had no goitre. These results demonstrate that i.d. delivery of human TSHR DNA can break tolerance and elicit GD in inbred mice. The data do not support the notion that TSAb production is Th2-dependent in murine GD but they also suggest that codelivery of TSHR and Th1-promoting IL-12 genes may not be sufficient to enhance disease incidence and/or severity in this model.
Cytokines play a key role in the development of Graves' ophthalmopathy (GO). These molecules are produced in the orbit of GO patients by infiltrating inflammatory cells as well as orbital fibroblasts. Locally produced cytokines stimulate fibroblast proliferation and their production of glycosaminoglycans, which result in accumulation of extracellular matrix and oedema with consequent proptosis. In addition to these direct effects, cytokines can modulate the immune reaction in GO by increasing major histocompatibility complex (MHC) class II, adhesion molecules, CD40, prostaglandin and heat shock protein expression in the orbit, thereby having a role in localising and augmenting the inflammatory response.
Graves' hyperthyroidism can be induced in mice or hamsters by novel approaches, namely injecting cells expressing the TSH receptor (TSHR) or vaccination with TSHR-DNA in plasmid or adenoviral vectors. These models provide unique insight into several aspects of Graves' disease: 1) manipulating immunity toward Th1 or Th2 cytokines enhances or suppresses hyperthyroidism in different models, perhaps reflecting human disease heterogeneity; 2) the role of TSHR cleavage and A subunit shedding in immunity leading to thyroid-stimulating antibodies (TSAbs); and 3) epitope spreading away from TSAbs and toward TSH-blocking antibodies in association with increased TSHR antibody titers (as in rare hypothyroid patients). Major developments from the models include the isolation of high-affinity monoclonal TSAbs and analysis of antigen presentation, T cells, and immune tolerance to the TSHR. Studies of inbred mouse strains emphasize the contribution of non-MHC vs. MHC genes, as in humans, supporting the relevance of the models to human disease. Moreover, other findings suggest that the development of Graves' disease is affected by environmental factors, including infectious pathogens, regardless of modifications in the Th1/Th2 balance. Finally, developing immunospecific forms of therapy for Graves' disease will require painstaking dissection of immune recognition and responses to the TSHR.
In Graves' disease, the overstimulation of the thyroid gland and hyperthyroidism are caused by autoantibodies directed against the TSH receptor (TSHR) that mimics the action of TSH. The establishment of an animal model is an important step to study the pathophysiology of autoimmune hyperthyroidism and for immunological analysis. In this study, we adopted the technique of electroporation (EP) for genetic immunization to achieve considerable enhancement of in vivo human TSHR (hTSHR) expression and efficient induction of hyperthyroidism in mice. In a preliminary study using beta-galactosidase (beta-gal) expression vectors, beta-gal introduced into the muscle by EP showed over 40-fold higher enzymatic activity than that introduced via previous direct gene transfer methods. The sustained hTSHR mRNA expression derived from cDNA transferred by EP was detectable in muscle tissue for at least 2 wk by RT-PCR. Based on these results, we induced hyperthyroidism via two expression vectors inserted with hTSHR or hTSHR289His cDNA. Consequently, 12.0-31.8% BALB/c mice immunized with hTSHR and 79.2-95.7% immunized with hTSHR289His showed high total T(4) levels due to the TSHR-stimulating antibody after three to four times repeated immunization by EP, and thyroid follicles of which were hyperplastic and had highly irregular epithelium. Moreover, TSHR-stimulating antibody surprisingly persisted more than 8 months after the last immunization. These results demonstrate that genetic immunization by in vivo EP is more efficient than previous procedures, and that it is useful for delineating the pathophysiology of Graves' disease.
Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves', including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves' patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90-165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves' TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9-165 (Mc1+2), 90-165 (Mc2), or 261-370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc1+2 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves' TSHRAbs, but it is important also in their formation in a mouse model of Graves' disease.
Normal human serum inhibits 125 I-thyrotropin binding to plasma membranes from bovine and human thyroids, from guinea pig retro-orbital tissue, and from human adipocytes. The inhibitor activity is measurable both by its ability to prevent 125I-thyrotropin binding and by its ability to reverse 125I-thyrotropin previously bound to the membranes. It is thermolabile, nondialyzable, cannot be precipitated by centrifugation at 100,000 x g, and does not fractionate as a γ-globulin. The inhibitor activity appears to result from the interaction of a serum component with the 125I-thyrotropin, since preincubation of normal human sera or partially purified fractions from these sera with 125I-thyrotropin results in the formation of a thyrotropin adduct which is measurable by the forward displacement of the 125 I-thyrotropin on gel filtration columns. The formation of the thyrotropin adduct is associated with a 74% decrease in binding when equimolar concentrations of 125I-thyrotropin or the 125I-thyrotropin adduct are incubated with membranes. The formation of the 125I-thyrotropin adduct is specific in that it is prevented by unlabeled thyrotropin but not by unlabeled glucagon, insulin, prolactin, growth hormone, human chorionic gonadotropin, follicle-stimulating hormone, or luteinizing hormone at equal or 3- to 10-fold higher concentrations than unlabeled thyrotropin. In contrast to these results, preincubation of the membranes with normal human serum or with a partially purified inhibitor preparation from this serum does not result in a measurable inhibitor-membrane complex since centrifugation and resuspension of the membranes in buffer alone restores their binding activity to normal levels. The partially purified inhibitory component in normal human sera does not stimulate the adenylate cyclase activity of thyroid plasma membranes when tested in the absence of thyrotropin. The potential functional significance of such a serum-thyrotropin binding component is discussed.
Graves disease is an autoimmune thyroid disease characterized by the presence of antibodies against the thyrotropin receptor (TSHR), which stimulate the thyroid to cause hyperthyroidism and/or goiter. By immunizing mice with fibroblasts transfected with both the human TSHR and a major histocompatibility complex class II molecule, but not by either alone, we have induced immune hyperthyroidism that has the major humoral and histological features of Graves disease: stimulating TSHR antibodies, thyrotropin binding inhibiting immunoglobulins, which are different from the stimulating TSHR antibodies, increased thyroid hormone levels, thyroid enlargement, thyrocyte hypercellularity, and thyrocyte intrusion into the follicular lumen. The results suggest that the aberrant expression of major histocompatibility complex class II molecules on cells that express a native form of the TSHR can result in the induction of functional anti-TSHR antibodies that stimulate the thyroid. They additionally suggest that the acquisition of antigen-presenting ability on a target cell containing the TSHR can activate T and B cells normally present in an animal and induce a disease with the major features of autoimmune Graves.
Thyroid associated ophthalmopathy (TAO) is generally considered to have an autoimmune pathogenesis but the target antigen has yet to be identified. It is most frequently associated with Graves' disease and there is some logic in assuming that the same antigen, the thyrotropin receptor (TSHR), is the common link. Previous studies, mostly PCR based, aimed at investigating TSHR transcripts in the orbit, have yielded conflicting results, although there is circumstantial evidence for their presence in orbital fat. In this study, we have examined adult human adipose and muscle tissues from various locations, initially by PCR and subsequently by northern blot. We obtained the expected 610bp product in normal intestinal and orbital fat but not skeletal muscle, following two rounds of PCR amplification but only when reverse transcription used a TSHR specific primer. In northern blots, despite loading all of the RNA obtained from total normal orbital fat contents, TSHR transcripts were at the limit of detection and similarly for large samples of intestinal fat. The exception was RNA obtained from TAO orbital fat, in which TSHR transcripts of 4.6 and 1.7kb were clearly visible, as in the thyroid. We conclude that normal adult adipose tissues contain low levels of TSHR transcripts. In TAO, TSHR transcripts are elevated probably due to an increased number of cells, in particular of preadipocytes in orbital adipose tissue.
We recently showed that immunization of AKR/N mice with murine fibroblasts transfected with the thyrotropin receptor (TSHR) and a murine major histocompatibility complex (MHC) class II molecule having the same H-2(k) haplotype, but not either alone, induces immune thyroid disease with the humoral and histological features of human Graves', including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs which cause hyperthyroidism and thyrotropin-binding-inhibiting immunoglobulins (TBIIs). Immunization of 5 different mouse strains that share the H-2(k) haplotype, but differ in their genetic background, results in different TBII titers, indicating non-MHC genetic effect on TSHRAb formation. In addition, immunization of C3H/He mice induced TBII formation even in the absence of aberrant class II, unlike AKR/N mice. However, the mice did not develop hyperthyroxinemia characteristic of the presence of stimulating TSHRAbs. Aberrant class II expression is, therefore, necessary for the development of a full Graves' syndrome with stimulating TSHRAbs; and immune recognition mechanisms by which TBIIs and stimulating TSHRAbs develop are differently regulated.
The generation of Abs recognizing the native structure of the human thyrotropin receptor (hTSHR) has been difficult because there is currently no method allowing the purification of correctly folded Ag in the amounts required by classical immunization protocols. The majority of Abs made against the hTSHR react preferentially with denatured molecules. We report that a humoral response against the native hTSHR, compatible with mAb production, is elicited in mice by immunization with a DNA construct encoding the receptor. BALB/c mice were inoculated in the anterior tibialis muscle with 100 microg of plasmid DNA harboring the hTSHR cDNA. Eleven weeks after the first injection, 10 mice of 14 showed by FACS analysis a strong IgG response against the hTSHR expressed at the surface of Chinese hamster ovary cells. A clear TSH-binding inhibiting Ig and thyrotropin-blocking Ab activity (competition with TSH binding and TSH activity, respectively) was demonstrated in the majority of sera tested. One serum exhibited a clear stimulating activity. Despite the maintenance of normal circulating free T4 levels in all mice, these bioactivities persisted until 18 wk, in which mice were sacrificed, their thyroids were examined histologically, and spleens from two animals were used for mAb production. All mice displayed a severe lymphocytic infiltration of their thyroids, composed mostly of activated B cells. Three mAbs were produced against conformational epitopes of the hTSHR. We conclude that genetic immunization is an efficient method of generating Abs recognizing the native structure of the hTSHR and a new way of inducing thyroiditis in mice murine.
In order to replicate a recently described murine model of Graves' disease, we immunized AKR/N (H-2k) mice i.p., every 2 weeks, with either a clone of fibroblasts expressing both the human TSH receptor (hTSHR) and murine major histocompatibility complex (MHC) class II molecules or with fibroblasts expressing the MHC class II molecules alone. Mice were bled, and their thyroid hormone levels measured, at 6, 12, and up to 18 weeks after the first immunization. Between 11-12 weeks after immunization, a significant number of mice began to die spontaneously and were found to have developed large goiters. Thirty to 40% of mice immunized with hTSHR transfected fibroblasts showed markedly increased serum T3 and T4 hormone levels by 12 weeks compared with controls, with the highest thyroid hormone levels being T3: 420 ng/dl (normal < 70) and T4: 16.5 microg/dl (normal < 5). The murine serum demonstrated the presence of antibodies to the TSHR, as evidenced by inhibition of labeled TSH binding to the hTSHR, and these sera had in vitro thyroid stimulating activity. Many of the hyperthyroid mouse exhibited weight loss and hyperactivity and, on examination, their thyroids had the histological features of thyroid hyperactivity including thyroid enlargement, thyroid cell hypertrophy, and colloid droplet formation--all consistent with Graves' disease. In contrast, a small number of mice (< 5%) developed hypothyroidism with low serum T4 levels and markedly increased TSH concentrations and evidence of thyroid hypoplasia. Both hyperthyroidism and hypothyroidism were successfully transferred to naive mice using ip cells of immunized mice. Surprisingly, hypothyroidism occurred in many recipient mice even after transfer from hyperthyroid donors. These results confirmed that immunization with naturally expressed hTSHR in mammalian cells was able to induce functional TSHR autoantibodies that either stimulated or blocked the mouse thyroid gland and induced hyperthyroidism or thyroid failure. Furthermore, both blocking and stimulating antibodies coexisted in the same mice as evidenced so clearly by the transfer of hypothyroidism from hyperthyroid mice. The addition of a Th2 adjuvant (pertussis toxin) caused approximately 50% of the animals to become hyperthyroid beginning early at 9 weeks, whereas a Th1 adjuvant (CFA) delayed the disease onset such that only 10% were hyperthyroid by 12 weeks. As with human autoimmune thyroid disease, the T cell control of this murine model may be critical and requires more extensive investigation.
In previous studies we have transferred thyroiditis to naive BALB/c and NOD mice with human thyrotropin (TSH) receptor (TSHR)-primed splenocytes. Because the TSHR has been implicated in the pathogenesis of thyroid eye disease (TED) we have examined the orbits of recipients of TSHR-primed T cells, generated using a TSHR fusion protein or by genetic immunization. In the NOD mice, 25 of 26 animals treated with TSHR-primed T cells developed thyroiditis with considerable follicular destruction, numerous activated and CD8+ T cells, and immunoreactivity for IFN-gamma. Thyroxine levels were reduced. Thyroiditis was not induced in controls. None of the NOD animals developed any orbital pathology. Thirty-five BALB/c mice received TSHR-primed spleen cells. Thyroiditis was induced in 60-100% and comprised activated T cells, B cells, and immunoreactivity for IL-4 and IL-10. Autoantibodies to the receptor were induced, including TSH binding inhibiting Igs. A total of 17 of 25 BALB/c orbits displayed changes consisting of accumulation of adipose tissue, edema caused by periodic acid Schiff-positive material, dissociation of the muscle fibers, the presence of TSHR immunoreactivity, and infiltration by lymphocytes and mast cells. No orbital changes or thyroiditis were observed in control BALB/c mice. We have induced orbital pathology having many parallels with human TED, only in BALB/c mice, suggesting that a Th2 autoimmune response to the TSHR may be a prerequisite for the development of TED.
Immunization of AKR/N mice with murine fibroblasts, transfected with the TSH receptor (TSHR) and a murine major histocompatibility complex class II molecule having the same H-2k haplotype (but not either alone), induces immune thyroid disease with the humoral and histological features of human Graves’, including the presence of two different TSHR antibodies (TSHRAbs): stimulating TSHRAbs, which cause hyperthyroidism; and TSH-binding-inhibiting immunoglobulins. The primary functional epitope for both types of antibodies in Graves’ patients is on the N-terminal portion of the extracellular domain of the TSHR, residues 25 to 165; most require residues 90–165 to express TSHRAb activity, as evidenced in studies using chimeras of the TSHR and lutropin-choriogonadotropin receptor (LH-CGR). To evaluate the role of this region of the TSHR in the formation of Graves’ TSHRAbs, we immunized AKR/N mice with fibroblasts transfected with three human TSHR chimeras with residues 9–165 (Mc1+2), 90–165 (Mc2), or 261–370 (Mc4) substituted by equivalent residues of the rat LH-CGR. Mice immunized with the Mc1+2 and Mc2 chimeras, with the N-terminal portion of the extracellular domain of the TSHR substituted by LH-CGR residues, did not develop TSHRAbs. Mice immunized with the Mc4 chimera, having a major portion of the C-terminal portion of the extracellular domain of the TSHR replaced by comparable LH-CGR residues, can develop TSHRAbs. The results suggest that the N-terminal segment of the TSHR extracellular domain is not only a critical functional epitope for Graves’ TSHRAbs, but it is important also in their formation in a mouse model of Graves’ disease.
Lymphomatous goiter (struma lymphomatosa) first described by Hashimoto in 19121, was shown to be an autoimmune disorder 30years ago2. In the sameyear an autoimmune thryoiditis was first induced experimentally by immunization with autologous thyroid extract and complete Freund's adjuvant3. Among the many insights gainedfrom the study of such experimentally induced thyroiditis3–5 is the notion that susceptibility to it is under a genetic control which influences both the reactivity of the immune systems and the properties of the thyroid gland7. This is supported by the appearance of autoimmune thyroiditis in genetically restricted animal populations. Here Georg Wick and his colleagues review recent studies of the Obese strain (OS) of chickens and compare the pathogenesis of theirspontaneous autoimmune thyroiditis with that of Hashimoto's disease.
Graves' disease (GD) develops as a result of a complex interaction between genetic susceptibility genes and likely environmental factors. Most epidemiological data support an important genetic contribution to the development of GD. The concordance rate of GD in monozygotic twins is 30–60% and in dizygotic twins 3–9%, and thyroid autoantibodies have been reported in up to 50% of the siblings of patients with GD. For many years now, HLA studies have consistently shown an increased frequency of HLA-DR3 in Caucasian patients with GD; but with only a risk ratio of 3–5. However, recent advances in human genome mapping techniques have enabled the study of many other candidate genes. Of these additional, non-HLA genes, only CTLA-4 has been consistently found to be associated with GD. Using a linkage based approach which only detects highly significant susceptibility genes we have recently reported preliminary results which demonstrated that a marker located ∼25 cM from the TSH receptor gene on chromosome 14q31 is linked to GD and in the same vicinity as the IDDM-11 locus. Such results, if confirmed, may signal the presence of a gene family related to endocrine autoimmunity on chromosome 14q31.
In this study we analyzed the proliferative response to the extracellular domain of thyrotropin receptor (TSHR-ECD) of T-cell lines raised from healthy subjects. We found high frequencies of cell lines reactive to TSHR-ECD, ranging from 12% to 37%. The response of the cell lines to a set of overlapping peptides of TSHR-ECD showed that the most recognized epitopes by T lymphocytes are on the C-terminal portion. In particular, the regions of residues 360-396 and 258-277 are immunodominant in T-lymphocyte reactivity. A group of cell lines specific for the peptides of TSHR-ECD lost the response to the peptides during time in culture. However, these lines were still responsive to TSHR extracellular domain. The cloning of one of these lines showed three types of T-cell clones: (1) CD4+ clones (n = 4) highly responsive to the TSHR-ECD; (2) CD4+ clones (n = 4) low responsive to TSHR-ECD; (3) CD8+ clones (n = 9) not responsive to TSHR-ECD. The first group of clones was stable during time in culture, while the second group was characterized by the loss of the specific response to TSHR-ECD after some weeks from the first analysis. The observation of a spontaneous anergy in the second group of CD4+ clones suggests that mechanisms of control of the lymphocyte response to TSHR-ECD could be activated in vitro.
An antiserum raised against an alignment of amino acid-(32-56), termed TSHRP-1, in the extracellular domain of human thyrotropin (TSH) receptor was used to identify the TSH receptor-like substance in plasma of Graves' disease. The dilution curve of plasma TSHRP-1-like immunoreactivity was observed in a manner parallel to the standard synthetic peptide curve in radioimmunoassay, and its molecular weight estimated approximately 60 kDa. The amounts of TSHRP-1-like immunoreactivity were significantly higher in Graves' plasma than those in plasma of normal and hypothyroid patients due to Hashimoto's thyroiditis. The present results indicate that human peripheral blood possesses a soluble form of the extracellular domain of TSH receptor which may contribute to the pathophysiology of Graves' disease.
Recently, we have shown that the thyrotropin (TSH) binding regions of human thyrotropin receptor (TSHR) reside in two areas within residues 12-44 and 308-344. Serial antisera were raised against four overlapping synthetic peptides representing these two regions of TSHR (peptides 12-30, 24-44, 308-328, and 324-344) and were investigated for their ability to stimulate or block the cultured porcine thyroid cells. In addition, serum concentrations of triiodothyronine (T3) and thyroxine (T4) in serial sera obtained from each rabbit were examined. It was shown that residues of 12-30 and 324-344 of TSHR, respectively, are the site (at least a part of the site) where stimulating (TSAb) and blocking type (TSBAb) immunoglobulins are directed.
To produce an immune reaction against a foreign protein usually requires purification of that protein, which is then injected into an animal. The isolation of enough pure protein is time-consuming and sometimes difficult. Here we report that such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. This is achieved using a hand-held form of the biolistic system which can propel DNA-coated gold microprojectiles directly into cells in the living animal. Genetic immunization may be time- and labour-saving in producing antibodies and may offer a unique method for vaccination.
We have synthesized four peptides (P2, P4, E3 and P1) corresponding to different segments of human thyrotropin (TSH) receptor. We have obtained antibodies by immunizing them to chickens, and antibodies are evaluated for their thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. None of the antibodies had TSAb activity. Antibodies against human TSH receptor specific region such as P2 and P4 (P2: No. 372-397, P4: No. 341-358) had TSBAb and TBII activities. Anti-E3 antibody (E3: the third putative extracellular loop, No. 649-661) had only TSBAb activity. Anti-P1 antibody (P1: high homology with pig LH/CG receptor, No. 398-417), however, had none. These results suggest that anti-TSH receptor antibodies to different antigenic epitopes show heterogeneity in their biological activities.
This chapter describes epitopes that are considered as “functional epitopes”—that is, portions or fragments of a protein that are able to bind to antibody in an immunoassay—and not as “contact epitopes” or “energetic epitopes” identifiable only in structural studies. The most common way of classifying epitopes is into continuous and discontinuous epitopes. The label “continuous epitope” is given to any short linear peptide fragment of an antigen that is able to bind to antibodies raised against the intact protein. In most cases, these antibodies cross-react only weakly with linear peptide fragment of the antigen. A more extreme viewpoint has been advocated, according to which all continuous epitopes represent “unfoldons”—that is, unfolded regions of the antigen that cross-react only with antibodies specific for the denatured protein. Such antibodies may be present in antisera raised against the protein, because some of the antigen molecules used for immunization is denatured or they could be obtained by immunization with peptide fragments.
NOD mice develop spontaneous insulin-dependent diabetes mellitus (IDDM) associated with infiltration of pancreatic islets with mononuclear cells. Islet infiltration results in autoimmune destruction of insulin-secreting beta-cells. Because in humans and BB rats diabetes is often associated with autoimmune thyroid disease (ATD), the NOD mouse model was examined for evidence of thyroiditis and serum antibodies reactive with mouse thyroid membrane antigens (MTMAs). The incidence of thyroiditis was 77% in mice greater than 180 days old, 67% in mice 61-180 days old, 72% in mice 31-60 days old, 74% in mice 21-30 days old, 78% in mice 11-20 days old, and 90% in mice less than or equal to 10 days old. NOD mice less than or equal to 30 days old had less-severe thyroiditis than animals greater than 180 days old. There was no significant different in severity of thyroiditis between any of the other age-groups tested. The incidence of thyroiditis was not increased in diabetic compared with nondiabetic animals, nor was an association found between thyroiditis and sex. The high incidence of thyroiditis in the less than or equal to 30-day-old age-group indicates that infiltration of lymphocytes into the thyroid can precede initiation of insulitis in this model. Although both thyroiditis and insulitis in NOD mice began early (by the 1st and 2nd mo of life, respectively), no significant association between infiltration of these two organs was noted in individual mice.(ABSTRACT TRUNCATED AT 250 WORDS)
We have produced antibodies against a peptide corresponding to the unique N-terminal segment (amino acid residues 29-57) in the extracellular domain of the human thyrotropin (TSH) receptor by immunizing it to rabbits, and evaluated for their thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. Antibody raised in rabbit B showed significant TSAb activity but not TSBAb activity. In contrast, antibody raised in rabbit A lacked TSAb activity but possessed TSBAb activity. None of these antibodies had TBII activity. These results indicate that TSH receptor antibody can successfully mimic the action of TSH and also suggest that the N-terminal region of TSH receptor is substantially associated with both TSAb and TSBAb activities, but not parallel to TBII activity.
This review considers recent developments in our understanding of the properties of TRAb, particularly measurement of the antibodies and their sites of action and synthesis. Two new assay methods have allowed considerable improvements in the sensitivity, specificity, precision, and ease of measuring TRAb. In particular: 1) receptor assays based on inhibition of receptor-purified labeled TSH binding to detergent-solubilized TSH receptors and 2) bioassays based on stimulation of cAMP release from monolayer cultures of isolated thyroid cells. Detailed studies with the two assays indicate that TSH receptor antibodies nearly always act as TSH agonists in patients with a history of Graves' hyperthyroidism. Studies in areas of dietary iodine sufficiency suggest that measurement of the antibodies at various stages in the course of treating Graves' disease can be of value in predicting the outcome of therapy. However, in areas of iodine deficiency, difficulties in the ability of patients' thyroid tissue to recover from the effects of antithyroid drugs may prevent the receptor antibodies from causing a relapse of thyrotoxicosis. Consequently, the predictive value of receptor antibody measurements would be expected to be lower in these geographical areas. Although patients with a history of Graves' hyperthyroidism nearly always have TRAb which act as TSH agonists, about 20% of patients with frank hypothyroidism due to autoimmune destruction of the thyroid have TRAb which act as TSH antagonists (blocking antibodies). There is some evidence that these blocking antibodies can cause hypothyroidism particularly in the neonate. With regard to the site of synthesis of TRAb, there is now direct evidence that they are synthesized by thyroid lymphocytes, particularly the lymphocytes in close proximity to thyroid follicular cells. This is consistent with the well established effects of antithyroid treatment (drugs, radioiodine, or surgery) on TRAb levels in addition to their effects on thyroid hormone synthesis. Recent studies using affinity labeling with 125I-labeled TSH have enabled elucidation of the structure of the TSH receptor. TSH receptors in human, porcine, and guinea pig thyroid tissue have a two-chain structure in which the TSH binding site is formed on the outside surface of the cell membrane by a water-soluble A subunit (Mr approximately 50 K). The A subunit is linked by a disulfide bridge and weak noncovalent bonds to the amphiphilic B subunit (Mr approximately 30 K). This subunit, which penetrates the lipid bilayer, probably forms the site for interaction of the receptor with the regulatory subunits of adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)
Monoclonal antibodies have been produced that recognize the native human thyrotropin receptor by using a sensitive screening protocol based on flow cytofluorimetry combined with recombinant eukaryotic cells expressing high levels of the full-length functional receptor. The more standard screening method of ELISA preferentially selected antibodies that only reacted with the denatured receptor. Mice were immunized with recombinant receptor produced in either eukaryotic or prokaryotic systems; after screening and cloning, three stable hybridoma lines were established. An IgM antibody (7B5) produced in response to the eukaryotic material recognized only the native receptor (by flow cytofluorimetry) and did not react with denatured material on ELISA or immunoblotting, suggesting that its epitope is conformational. In contrast, two IgG1 antibodies (2C11 and 3B12) produced in response to the prokaryotic material recognized both native and denatured receptor (by flow cytofluorimetry, immunoprecipitation and immunoblotting). The use of different recombinant constructs in the immunoblotting procedure allowed the epitopes for both the IgG1 antibodies to be assigned to the region 125-369. None of the antibodies stimulated production of cAMP by recombinant cells expressing the full-length functional receptor, but one of the IgG1 antibodies (2C11) did inhibit binding of radiolabelled thyrotropin to these same cells. These antibodies, and others that can now be produced with this screening protocol, will help define the relationship between structure and function of this important receptor.
We examined whether mice, immunized with TSH receptor (TSH-R) peptides, which are known to be T-cell epitopes in patients with Graves' disease, would show thyroid-stimulating antibody (TSAb). We immunized DBA/1J mice with TSH-R peptide amino acids 132-150, 145-163, 158-176, and 172-186 and with a pool of these four peptides. The antibodies produced in these mice were evaluated by measurement of TSAb activity using Chinese hamster ovary cells expressing human TSH-R. Seven of 20 mice showed TSAb activity that could be partially blocked with TSH-R peptides. To assess the role of T-cell epitope-specific T-cells in the production of TSAb, we transferred a T-cell line developed from a TSAb-positive mouse to other syngeneic DBA/1J mice. Two of 4 recipient mice showed TSAb activities. These findings suggest that specific T-cell epitopes of TSH-R play a crucial role in the production of TSAb.
Splenocytes from female BALB/c mice immunized with a recombinant extracellular domain of the human TSH receptor (ETSHR) were used to generate a panel of 23 hybridomas that produce TSHR-specific monoclonal antibodies (mAbs). All mAbs were of the immunoglobulin G (IgG) isotype and belonged to different subclasses, including IgG1, IgG2a, and IgG2b. The antibodies bound to the ETSHR with relatively high affinity, and several of them blocked the binding of [125I]TSH to the TSHR, with some showing better blocking than others. Competitive binding studies with a subgroup of 4 biotinylated mAbs showed at least 3 different binding specificities. To determine the TSHR epitopes to which these mAbs were binding, we tested them against 37 overlapping synthetic peptides that span the entire ETSHR. mAb 47, which did not block TSH binding, bound to an epitope represented by amino acid residues 22-30. mAb 28, which had a TSH binding inhibitory index of 20%, bound to an epitope represented by amino acids 32-41. However, mAbs 37 and 49, with TSH binding inhibitory index values of 39% and 43%, respectively, showed no significant reactivity with any of the peptides, suggesting that they react with a conformational epitope. Together, these studies showed that mAbs with discrete binding specificities can interact with either linear or conformational epitopes and block TSH binding. The availability of these mAbs should facilitate identification of fine structures of the TSHR that are relevant for its function as well as pathogenesis of a number of thyroid disorders mediated by antibodies to TSHR.
We constructed a transgenic mouse model that mimics the human autoimmune disease multiple sclerosis in its spontaneous induction and pathology. Transgenic mice were constructed expressing genes encoding a rearranged T cell receptor specific for myelin basic protein (MBP). T cell tolerance was not induced in the periphery, and functional, autoreactive T cells were found in the spleen and lymph nodes of these mice. Transgenic mice developed experimental allergic encephalomyelitis (EAE) following immunization with MBP and adjuvant plus pertussis toxin as well as with administration of pertussis toxin alone. Spontaneous EAE can develop in transgenic mice housed in a non-sterile facility but not in those maintained in a sterile, specific pathogen-free facility. This model system affords a unique opportunity to dissect the genetic and environmental variables that may contribute to the development of spontaneous autoimmune disease.
We recently expressed the extracellular domain of the human TSHR (ETSHR) protein using a baculovirus expression system and purified it to homogeneity. The ETSHR specifically binds both TSH and antibodies to TSHR. In the present study, C57BL/6J, SJL/J, BALB/cJ and B10BR.SgSnJ mice were immunized with the recombinant ETSHR or an equivalent amount of control antigen. All strains of mice produced high titers of antibody against the TSHR protein which were capable of blocking the binding of TSH to native TSHR. However, only BALB/cJ mice showed significantly elevated levels of thyroxine in their sera compared to the control mice. Similarly, BALB/cJ mice primed with ETSHR and then challenged with thyroid membranes showed significantly elevated levels of thyroxine. In addition, histopathological examination of thyroid glands from affected mice showed morphological changes characterized by hydropic and subnuclear vacuolar changes and focal scalloping, with no apparent inflammation or glandular destruction. Moreover, mice with elevated thyroxine levels showed increased in vivo thyroidal uptake of 131Iodine. Together, these data suggest that BALB/cJ mice are susceptible to the induction of hyperthyroxinemia.
The development of autoimmunity was investigated after repeated immunizations with human thyrotropin receptor (hTSH-R) of five congenic strains of female and male mice. After each immunization, free T3 levels and antibodies to hTSH-R and to six peptides of the hTSH-R were assayed. Our results showed that H-2s and H-2q female mice developed features of autoimmunity such as antibody responses to hTSH-R and to hTSH-R peptides, transient variations in the levels of free T3 thyroid hormone, and lymphocytic infiltrations in their thyroid glands. Concerning the antibody responses to hTSH-R peptides, we found that peptide P1 (352-366) contained a major B cell epitope. Furthermore, strain-specific B cell epitope was exemplified by peptide 92 (12-30) and two male- and female-specific B cell epitopes were located in peptides 91 (32-46) and 93 (316-330), respectively. These features appeared rather related to hyperthyroidism.
In a preliminary study, we observed the production of TSH binding-inhibiting (TBII) and thyroid-blocking (TBAb) antibodies accompanied by lymphocytic infiltration of the thyroid in a pool of male BALB/c mice immunized with the extracellular domain (ECD) of the human TSH receptor (TSHR) expressed as a maltose-binding protein (MBP) fusion in bacteria. In the present study we evaluated the humoral response to the same antigenic preparation in a new series of individual male and female BALB/c mice immunized ip on day 0 with 100 micrograms MBP-ECD and days 25, 39, and 53 with 50 micrograms MBP-ECD in an adjuvant composed of aluminum oxide, magnesium hydroxide, and Bordetella pertussis vaccine. Mice immunized with MBP served as control. Individual sera and immunoglobulins were tested for TBII, TBAb, and thyroid-stimulating antibodies (TSAb) on days 0, 32, 46, and 60, and total circulating T4 levels were measured by RIA. Animals were killed on day 120, their thyroids were examined histologically, the infiltrates were characterized using monoclonal antibodies specific for T-cells (total, activated, helper, and suppressor), B-cells, and macrophages. Sera and immunoglobulins G of the MBP-treated control group were all negative for TSAb, TBAb, and TBII activity. The receptor-immunized mice, despite having high titers of antibodies to the immunogen in an enzyme-linked immunosorbent assay, displayed a heterogeneous response in terms of biological activity, with 3 of 7 female and 4 of 8 male mice having TBAb/TBII activities that persisted and whose activity increased throughout the experiment. No significant TSAb antibody activity was observed. Total T4 levels were also heterogeneous even before immunization, but 9 of 15 MBP-ECD-treated mice had levels below the normal range after immunization, and 7 of these also had TBII/TBAb activities. At the end of the experiment, only 4 of the MBP-ECD-treated female mice survived, but all of them had a severe lymphocytic infiltration of their thyroid, composed mostly of activated T-cells, although B-cells and macrophages were also present. A similar infiltrate was seen in 4 of 8 male MBP-ECD-treated mice. No infiltrate was observed in male or female MBP-treated mice. The model described demonstrates the feasibility of using the TSHR as an immunogen to overcome tolerance and mimics some characteristics of human autoimmune disease of the thyroid.
The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21-414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting. To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
A cDNA encoding the extracellular domain of human TSHr (ETSHr) was expressed in large quantities using the baculovirus expression system. Maximum level of protein was produced at 60 hr post-infection and represented approximately 20% of the total cellular protein. The identity of the protein as ETSHr was confirmed by Western blot using antibodies to synthetic peptides derived from the TSHr. The protein has an apparent molecular weight of 50 kDa and is larger than the predicted size of 44 kDa, suggesting that the protein is glycosylated. Polyclonal antibodies raised in rabbits against gel purified ETSHr blocked the binding of 125I-TSH to native TSHr in solubilized porcine thyroid membranes in a radioreceptor assay. The availability of this antigenically active protein will facilitate further characterization of the protein and analysis of immune response against TSHr in experimental animals as well as in patients with autoimmune Graves' disease.
Two groups of 10 Balbc by Jico male mice were immunised on days 0, 15 and 35, with the extra cellular domain (ECD) of the human thyrotropin receptor (TSH-R) expressed as a fusion protein in bacteria (group 1) or with the maltose binding protein (MBP) fusion partner alone (group 2). Blood was obtained on days 0, 22, 32, 42 and 49 and samples from the individual animals pooled for each group. Serum and immunoglobulin (IgG) preparations were tested, using CHO cells expressing the human TSH-R (JP26 and JP09) for thyroid stimulating (TSAb); thyroid blocking (TBAb) and thyrotropin binding inhibiting (TBII) activities. Neither serum nor IgGs were found to contain TSAb at any time point. TBII activity was present in the serum of both groups on day 32 and in group 1 only on day 49; when the test was performed on IgGs, only the MBP-ECD day 49 preparation remained significantly positive for TBII (p < 0.005). Significant TBAb activity was present in both the serum and IgG of group 1 day 49 (p < 0.005) and to a lesser extent on 42 (p < 0.02). Following the second immunisation (day 15) both groups and had decreased circulating T4 levels (p < 0.05) when compared with day 0 in each case. Group 2 were unaffected by the third immunisation on day 35 but the MBP-ECD group again had significantly decreased T4 levels (p < 0.02) compared with MBP day 49 and (p < 0.03) when compared with MBP-ECD day 0. Histological examination of thyroids from group 1 animals revealed extensive vascularisation and an atypical lymphoblastoid infiltration which was not observed in control mice. These preliminary results indicate that care is required in interpreting data since a non-receptor antigen was shown to decrease circulating thyroxine and serum from these animals had apparent TBII like activity. However, the results obtained with the IgGs suggest that receptor autoantibodies can be induced by immunising with the human TSH-R, in addition, the immunised mice show histological evidence for the development of thyroiditis.
The thyrotropin (TSH) receptor in human thyroid glands has been shown to be cleaved into an extracellular alpha subunit and a transmembrane beta subunit held together by disulfide bridges. An excess of the latter component relative to the former suggested the shedding of the ectodomain. Indeed we observed such a shedding in cultures of human thyrocytes and permanently transfected L or Chinese hamster ovary cells. The shedding was increased by inhibitors of endocytosis, recycling, and lysosomal degradation, suggesting that it was dependent on receptor residency at the cell surface. It was slightly increased by TSH and phorbol esters, whereas forskolin and 8-bromo-cyclic AMP were without effect. Decreasing the serum concentration in cell culture medium enhanced the shedding by an unknown mechanism. The shedding of the TSH receptor alpha domain is the consequence of two events: cleavage of the receptor into alpha and beta subunits and reduction of the disulfide bridge(s). The complete inhibition of soluble TSH receptor shedding by the specific inhibitor BB-2116 indicated that the cleavage reaction is catalyzed probably at the cell surface by a matrix metalloprotease. This shedding mechanism may be responsible for the presence of soluble TSH receptor alpha subunit in human circulation.
In a previous study, we have described the induction of thyroid blocking (TBAB) and thyrotropin binding inhibiting antibodies accompanied by thyroiditis in female BALBc mice (H2d) immunised with the extra-cellular domain (ECD) of the human thyrotropin receptor (TSHR) expressed as a maltose binding protein (MBP) fusion. In the present study we have investigated the response induced in mice of varying MHC haplotype. Two groups of female NOD (H2g), CBA (H2k) and C57 (H2b) mice were immunised intra-peritoneally with MBP-ECD or MBP on days 0 (100 micrograms), 15, 30 and 43 (50 micrograms). Blood samples from individual mice were obtained on days 0, 22, 36 and 50 and assessed for thyroid binding inhibiting immunoglobulins (TBII), thyroid stimulating (TSAB) and TBAB. On day 50 the treated mice and five age/sex matched NOD mice were sacrificed, their thyroids removed, examined histologically and any infiltrate characterised. Induction of antibodies to the ECD was tested by ELISA in which plates had been coated with either MBP-ECD or an ECD-protein A fusion. All of the mice developed a strong antibody response to the relevant immunogen but none of them contained TBII, TSAB or TBAB activities. No lymphocytic infiltration of the thyroid glands of the CBA or C57 mice was observed. In contrast, all of the NOD mice displayed severe thyroiditis, whilst one of seven MBP-treated mice had moderate infiltration and none of five untreated controls. Immunohistochemical analysis revealed that the infiltrate was predominantly activated T helper cells with little evidence of B cells or the cytokines IL-10 or IL-4, indicating that a Th1 response had been induced, contrary to our findings in BALBc mice which mount a Th2 response. In conclusion we have shown that the type and extent of response induced by immunising with the TSHR varies in mice of differing genetic background. H2d mice develop thyroiditis and TBAB/TBII, H2g mice develop thyroiditis in the absence of functional TSHR antibodies, whilst H2b and H2k mice are resistant.
In previous studies we have induced TSH binding-inhibiting Igs and thyroiditis in BALB/c mice and thyroiditis alone in NOD mice immunized with the extracellular domain of the human TSH receptor produced as a maltose-binding protein fusion in bacteria (MBP-ECD). In this study, our aim was to determine whether thyroiditis can be transferred to syngeneic naive recipients with in vivo and in vitro primed spleen cells. Groups of 6-week-old female BALB/c and NOD mice were immunized ip with MBP-ECD in an adjuvant of alum plus attenuated Bordetella pertussis toxin, on days 0 (100 micrograms), 14, 28, and 35 (50 micrograms). These mice (in vivo primed) and nontreated age- and sex-matched controls were killed on day 43, and their spleens and thyroids were removed, the latter to verify the induction of thyroiditis in the antigen-treated mice. Splenocytes were disrupted mechanically and cultured at 3 x 10(6)/ml in RPMI supplemented with 20 micrograms/ml MBP-ECD for 48-64 h. After this in vitro priming, some of the splenocytes received no further treatment, but a portion was fractionated into a CD4+-enriched population. Groups of 6-week-old female BALB/c and NOD mice were immunized into the tail vein with 100-200 microliters PBS containing approximately 10(5)-10(7) unfractionated T cells (both in vivo primed and not) and CD4+-enriched (in vivo primed) splenocyte populations. The animals were killed 16 days later, and their thyroids were examined histologically and by immunohistochemistry. In addition, levels of antibody to the MBP-ECD priming antigen were assessed by enzyme-linked immunosorbent assay in the antigen- and spleen-treated mice. In the donor animals, in vivo priming resulted in an extensive lymphocytic infiltration of the thyroids in both BALB/c and NOD mice and follicular destruction in the latter. There was no evidence of thyroiditis in all 9 BALB/c mice and all 4 NOD mice who received unfractionated T cells from mice that had not been primed in vivo. In contrast, transfer of MBP-ECD in vivo primed unfractionated T cells resulted in thyroiditis in 9 of 13 BALB/c mice and 5 of 6 NOD mice; similarly, the equivalent CD4+-enriched population produced extensive thyroiditis in 2 of 3 BALB/c mice and all three NOD mice. The most striking difference between the antigen- and spleen-treated mice was in the quantity of the infiltrate, which was much greater in the latter and extended throughout the thyroid glands of these animals. In common with mice treated directly with the MBP-ECD antigen, the infiltrates of both BALB/c and NOD recipient mice contained large numbers of activated T cells expressing the receptor for interleukin-2, and macrophages and dendritic cells were plentiful, particularly in the BALB/c mice, in which B cells and interleukin-10-positive T cells were also present. The most abundant infiltrates, containing numerous CD8+ T cells and follicular destruction, were observed in NOD mice receiving primed unfractionated T cells or CD4+-enriched T cells. In contrast to the donors, none of the recipient animals had circulating antibodies to the MBP-ECD antigen. In conclusion, we have shown that it is possible to transfer thyroiditis with spleen cells from mice primed in vivo with a human TSH receptor preparation. Furthermore, the thyroiditogenic activity appears to reside in the CD4+ population.
To understand the role of glycosylation on autoantibody reactivity, we expressed cDNA encoding amino acid residues 22 to 416 of the human thyrotropin receptor (TSHR), along with the baculovirus-encoded glycoprotein 67 signal sequence (ETSHR-gp) in insect cells. N-terminal sequence analysis revealed that the signal peptide was cleaved and confirmed the identity of ETSHR-gp protein. The molecular mass of the ETSHR-gp protein was 63 kDa and was higher than the expected molecular mass of 45 kDa, suggesting that the protein was glycosylated. Carbohydrate analysis showed that the protein was glycosylated and that mannose was the major oligosaccharide. A nonglycosylated recombinant ETSHR protein expressed earlier in our laboratory neutralized TSH-binding-inhibitory Ig (TBII) activity in the sera of rabbits immunized with the protein but did not neutralize TBII activity in the sera of patients. In contrast, the glycosylated ETSHR-gp protein neutralized TBII activity in the sera of both experimental animals and patients with autoimmune thyroid disorders. Furthermore, only the ETSHR-gp protein completely neutralized the activities of stimulatory and blocking Abs in the sera of patients with hyperthyroidism and hypothyroidism, respectively. These data clearly show that glycosylated ETSHR-gp, but not the nonglycosylated ETSHR protein, can react with autoantibodies in patients' sera and that it has the epitopes required for the binding of TBII, thyroid stimulatory Abs, and thyroid stimulatory blocking Abs. Moreover, these data suggest that glycosylation might be an important determinant of autoantigenicity of human TSHR.
We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.
The growth and function of the thyroid are controlled by thyrotropin1 through the activation of its receptor, which belongs to the large family of G protein–coupled receptors. Despite the extreme diversity of their ligands, all receptors from this family have a common molecular architecture: seven transmembrane segments, three extracellular loops, three intracellular loops, an extracellular amino terminal, and an intracytoplasmic carboxy terminal (Figure 1). The glycoprotein hormone receptors constitute a subfamily that is characterized mainly by a particularly long amino-terminal extracellular domain that confers binding specificity.1,21 The thyrotropin receptor is encoded by 10 exons spread over 58 kilobases on . . .
We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.
Thyroid eye disease (TED) has an autoimmune etiology, but the nature of the autoantigen that is the target of the initiating event remains unknown. A number of candidates have been proposed based on Western blotting, library screening, and deduction from sequence similarity. A strong favorite is the thyrotropin receptor (TSHR), which is the target of the thyroid stimulating antibodies (TSAB) of Graves' disease (GD). We have recently demonstrated TSHR transcripts in orbital adipose tissue from a patient with TED by Northern blot, transcripts in normal adipose tissue being at the limit of detection. We have shown that the transcripts are translated into protein by immunohistochemical analysis using two monoclonal antibodies to the TSHR generated by genetic immunization. TSHR immunoreactivity is associated with elongated cells with the appearance of a fibroblast, often adjacent to clusters of adipocytes, in orbital biopsies from patients with TED but not in strabismus or pseudotumor biopsies. In animal studies, we have transferred thyroiditis to naive BALBc and NOD mice, using T cells primed to the human TSHR, either using the receptor expressed as a bacterial fusion protein or by genetic immunization. The BALBc develop a Th2-type response to the receptor, but the NOD a Th1-type with thyrocyte destruction. Orbital pathology, edema, infiltration by mast cells and lymphocytes, and adipose accumulation was also induced in 68% of the BALBc but none of the NOD mice. Together these data indicate that the preadipocyte expresses the TSHR and that a Th2 autoimmune response to the receptor may be an initiating event in TED.
Grave's disease (GD) is characterized by pathogenic autoantibodies to the human thyrotropin receptor (hTSH-R), and is frequently associated with a lymphocytic infiltrate of the thyroid gland. In attempts to establish a murine model of GD, we and others have previously shown that immunization of mice with recombinant preparations of the hTSH-R ectodomain induces high titres of specific antibodies, which, however, are not pathogenic, nor is the response accompanied by the development of thyroiditis. Since earlier reports identified the serological immunodominant determinants within the N- and C-terminal regions of hTSH-R ectodomain, we reasoned that immunization of mice with truncated fragments of ectodomain lacking these dominant regions might result in skewing of the response to other determinants of the molecule, with consequent induction of immunopathological features present in GD. We show here that multiple challenge of BALB/c mice with an amino acid fragment of residues 43-282 generates antibodies directed at hTSH-R peptides 37-56, 157-176, 217-236 and 232-251. This reactivity pattern is distinct from that induced previously with the whole ectodomain of hTSH-R in BALB/c animals. Thyroid function remained unaffected in these mice, suggesting that pathogenic antibodies were not being induced. Interestingly, some animals developed lymphocytic infiltration of the thyroid gland, clearly indicating the presence of pathogenic T cell determinants within the 43-282 fragment. Challenge with the related fragment 43-316 produced the same pattern of serological response to the synthetic peptides as fragment 43 282, but was not accompanied by thyroiditis. The results demonstrate: (i) the presence of thyroiditogenic determinants within hTSH-R, and (ii) that these pathogenic determinants are likely to be cryptic, as their effect is exhibited only when the hierarchy of immunodominance within hTSH-R is drastically altered.
The thyrotropin receptor (TSHR) is the major autoantigen of human Graves' disease. In order to define the antigenicity of the TSHR in a defined model, we examined the immune response of BALB/c mice to immunization with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25-50 microg of insect cell expressed, purified and refolded, mTSHR-ecd in alum adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. Control mice received wild-type baculovirus-infected insect cell protein lysate, in a similar way. After 28 days, murine serum contained high titres of antibodies specific to mTSH-ecd and their titres continued to increase over 90 days. Antibody epitope mapping, using 26 peptides spanning the human TSHR-ecd, showed that a variety of regions of the ectodomain were antigenic. The earliest epitope included aa 22-41, but later two regions of reactivity were noted clustered towards the mid portion and carboxyl terminus of the ectodomain. The murine TSHR autoantibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of blocking the native TSHR. We showed that these TSHR antibodies acted, in vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with the hTSHR. Hence, the antibody response to mTSHR-ecd was potentially antagonistic in its influence on the TSHR. Assessment of thyroid function in the immunized mice showed a fall in serum total T3 by 90 days and markedly elevated murine TSH levels (from 64.0 to 239.6 ng/ml), confirming the onset of thyroid failure. However, thyroid histology remained grossly normal. These data demonstrate that mTSHR-ecd is a potent antigen with three major immunogenic regions. The induced mTSHR-Abs blocked TSH action in vivo and reduced murine thyroid function.
We have characterized 10 monoclonal antibodies (Mabs) to recombinant murine thyrotropin receptor extracellular domain (mTSHR-ecd). Affinity purified mTSHR-ecd (amino acids 22-415), expressed in a baculovirus-insect cell system, was refolded in vitro and used to hyperimmunize female Balb/c mice. Spleens were removed 10 days after a final boost of 25 microg mTSHR-ecd intraperitoneally and intravenously, and the cells were fused to SP-2 cells and cloned. Hybridoma supernatants were screened by enzyme-linked immunosorbent assay (ELISA) with folded mTSHR-ecd antigen. Ten of 18 higher affinity hybridomas were selected at random and ascites fluids prepared. Nine of the monoclonals were of IgG 1 isotype, and one was IgM. Five Mabs (M3, M4, M5, M6, and M9) inhibited the binding of 125I-TSH to functional hTSHR expressed on Chinese hamster ovary (CHO) cells, and four (M1, M3, M5, and M9) blocked the TSH-stimulated generation of cyclic adenosine monophosphate (cAMP), using the same cells. The remaining Mabs appeared to be neutral in their interaction with native TSHR. The Mabs were also compared for their reactivity to mTSHR-ecd under folding (ELISA) and unfolding (reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) conditions. Most Mabs demonstrated reactivity to both conformational (folded) and linear (unfolded) forms of mTSHR-ecd, suggesting that they were generated primarily against linear epitopes although one Mab (M4) showed affinity for only folded antigen indicating a preference for a conformational epitope. Mapping the Mab epitopes using 26 overlapping peptides spanning the human (h)TSHR-ecd showed that 6 bound peptide 397-415, 1 bound peptide 352-371, and 1 peptide 22-41. These epitope mapped Mabs to the mTSHR-ecd, both receptor blocking and receptor neutral, will provide further insight into the structure-function of the TSHR ectodomain.
We used the secreted TSH receptor (TSHR) ectodomain variant TSHR-289 (truncated at amino acid residue 289 with a 6-histidine tail) to investigate properties of TSHR autoantibodies in Graves' disease. Sequential concanavalin A and Ni-chelate chromatography extracted milligram quantities of TSHR-289 (approximately 20-40% purity) from the culture medium. Nanogram quantities of this material neutralized the TSH binding inhibitory activity in all 15 Graves' sera studied. We generated a mouse monoclonal antibody (mAb), 3BD10, to partially purified TSHR-289. Screening of a TSHR complementary DNA fragment expression library localized the 3BD10 epitope to 27 amino acids at the N-terminus of the TSHR, a cysteine-rich segment predicted to be highly conformational. 3BD10 preferentially recognized native, as opposed to reduced and denatured, TSHR-289, but did not interact with the TSH holoreceptor on the cell surface. Moreover, mAb 3BD10 could extract from culture medium TSHR-289 nonreactive with autoantibodies, but not the lesser amount (approximately 25%) of TSHR-289 molecules capable of neutralizing autoantibodies. Although the active form of TSHR-289 in culture medium was stable at ambient temperature, stability was reduced at 37 C, explaining the mixture of active and inactive molecules in medium harvested from cell cultures. In conclusion, studies involving a TSHR ectodomain variant indicate the exquisite conformational requirements of TSHR autoantibodies. Even under "native" conditions, only a minority of molecules in highly potent TSHR-289 preparations neutralize patients' autoantibodies. Therefore, Graves' disease is likely to be caused by even lower concentrations of autoantibodies than previously thought. Finally, reciprocally exclusive binding to TSHR-289 by human autoantibodies and a mouse mAb with a defined epitope suggests that the extreme N-terminus of the TSHR is important for autoantibody recognition.
The genetic susceptibility to Graves disease. Bailliere's Clinical Endocrinology and Metabolism
- Y Tomer
- Tf Davies
Tomer Y & Davies TF. The genetic susceptibility to Graves disease.
Bailliere's Clinical Endocrinology and Metabolism 1997 11 431–
450.
Anti-lymphocyte monoclonal antibody treatment of multiple sclerosis suppresses disease activity but induced Graves' disease
- Aj Coles
- Mg Wing
- G Hale
- H Waldmann
- Ap Weetman
- Das Compston
- Vkk Chatterjee
46 Coles AJ, Wing MG, Hale G, Waldmann H, Weetman AP,
Compston DAS & Chatterjee VKK. Anti-lymphocyte monoclonal
antibody treatment of multiple sclerosis suppresses disease
activity but induced Graves' disease. Journal of Endocrinological
Investigation 1998 21 S5.
Generation of a murine model of Graves' disease by genetic immunization of outbred mice with human TSH receptor
- S Costagliola
- J Pohlenz
- S Refetoff
- G Vassart
37 Costagliola S, Pohlenz J, Refetoff S & Vassart G. Generation of a
murine model of Graves' disease by genetic immunization of
outbred mice with human TSH receptor. Journal of Endocrinological
Investigation 1998 21 S4.
Evidence for thyrotopin receptor antibodies which are not TSH agonists/antagonists in thyroid associated ophthalmopathy
- Ms Crisp
- Ap Weetman
- M Ludgate
19 Crisp MS, Weetman AP & Ludgate M. Evidence for thyrotopin
receptor antibodies which are not TSH agonists/antagonists in
thyroid associated ophthalmopathy. Journal of Endocrinological
Investigation 1997 20 (Suppl 5) 58.