[Hypothyreodism. From the latent functional disorder up to coma]. / Hypothyreose. Von der latenten Funktionsstörung zum Koma.

An autoimmune thyroiditis represents the main reason of hypothyroidism, defined as a lack of thyroid hormone. This autoimmune process results in destruction of functioning thyroid follicles. While subclinical or latent hypothyroidism is defined on the basis of laboratory values (an elevation of TSH with normal peripheral hormone levels), the typical signs and symptoms are associated with hypothyroidism. In about 80% of cases antibodies against thyroid peroxidase can be measured, but only in about 40-50% of cases antibodies against thyroglobulin are detectable. If hypothyrodism has been diagnosed, substitution with levothyroxine should be initiated, with the therapeutic goal to decrease TSH level to the lower normal range. In cases of subclinical hypothyroidism, levothyroxine medication should be started in patients with a high TSH value, positive antibodies and/or the typical ultrasound of autoimmune thyroiditis. However, substitution with levothyroxine in any case of elevated TSH values should be avoided.

Sodium/iodide symporter (NIS) and pendrin are expressed differently in hot and cold nodules of thyroid toxic multinodular goiter.

OBJECTIVE: The expression of two iodide transporters, the sodium/iodide symporter (NIS) and pendrin, was analyzed in thyroid tissues of patients with toxic multinodular goiter (TMNG) and non-toxic multinodular goiter (MNG). METHODS: The levels of NIS and pendrin proteins were analyzed in total protein extracts from nodular and non-nodular tissues by Western blot. RESULTS: In tissue samples from TMNG, we found an increased expression of NIS (2.5-fold) in the hot nodules, and similar levels between cold nodules and non-nodular tissues. In contrast, the levels of pendrin were slightly increased in both hot and cold nodules from TMNG, and decreased (about twofold) in cold nodules from MNG. We also noticed that there was no relationship between NIS and pendrin expression. CONCLUSIONS: Our data demonstrate that hot nodules from TMNG express a higher number of iodide transporters (mainly NIS), whereas cold nodules from TMNG, but not from MNG, show levels of the two proteins comparable with normal tissue, suggesting a role in vivo of TSH in maintaining the expression of NIS and pendrin protein in normal thyroid tissue. Finally, different mechanisms are involved in the regulation of NIS and pendrin expression.

Nodular goiter and goiter nodules: Where iodine deficiency falls short of explaining the facts.

While the concept of iodine deficiency (ID) still dominates most discussions about the pathogenesis of multinodular goiter (MNG), the present review focuses on those mechanisms that may cause MNG in the absence of ID. Among the many facets of MNG that cannot simply be explained by ID, are the frequent occurrence of the disease in patients not exposed to ID, the autonomous growth of goiters - often accompanied by subclinical or even overt thyrotoxicosis -, the inverse relationship between goiter size and serum TSH, the multifocal, heterogeneous and nodular growth pattern, the heterogeneity of function with the familiar patchy iodine metabolism on scintiscans, the growth of clonal and polyclonal nodules, the prominent genetic predisposition. Even the notoriously low intrathyroidal iodine concentration - common to endemic as well as to sporadic goiter - is a secondary, rather than a primary event. Thus, the fundamental process of goitrogenesis, is independent from ID but operates through mechanisms innate to the hereditary and acquired heterogeneity among the thyrocytes themselves. In this view, goiter nodules and nodular goiters are true benign neoplasias arising by mechanisms common to all benign endocrine and nonendocrine neoplasms. However, superimposed iodine shortage greatly enhances the incidence of MNG and shifts its clinical appearance toward younger ages by adding one more growth factor - presumably enhanced TSH secretion - to an intrinsically activated growth regulating network.

Absence of sodium/iodide symporter gene mutations in differentiated human thyroid carcinomas.

Decrease or loss of the sodium iodide (Na+/I-) symporter (NIS) activity influences the suitability of using radioiodine to detect and treat metastatic thyroid tissues. In previous studies, the presence of the NIS transcript, albeit at lower expression levels, has been shown in most thyroid differentiated carcinomas. In this study we searched for point mutations or other genetic alterations that may be responsible for an altered function of the NIS protein in tumors that still express NIS transcripts. Tumoral cDNAs derived from seven differentiated thyroid carcinomas (DTC), five papillary and two follicular, were analyzed by direct sequencing after polymerase chain reaction (PCR) amplification of the structural gene of the Na+/I- symporter. Neither mutations nor other genetic abnormalities were detected in any tumor sample examined. The data indicate that mutations or other genetic alterations of the NIS structural gene are not a major cause of the reduced iodide uptake in DTC.

Estrogen promotes growth of human thyroid tumor cells by different molecular mechanisms.

Thyroid tumors are about 3 times more frequent in females than in males. Epidemiological studies suggest that the use of estrogens may contribute to the pathogenesis of thyroid tumors. In a very recent study a direct growth stimulatory effect of 17beta-estradiol was demonstrated in FRTL-5 rat thyroid cells. In this work the presence of estrogen receptors alpha and beta in thyroid cells derived from human goiter nodules and in human thyroid carcinoma cell line HTC-TSHr was demonstrated. There was no difference between the expression levels of estrogen receptor alpha in males and females, but there was a significant increase in expression levels in response to 17beta-estradiol. Stimulation of benign and malignant thyroid cells with 17beta-estradiol resulted in an increased proliferation rate and an enhanced expression of cyclin D1 protein, which plays a key role in the regulation of G(1)/S transition in the cell cycle. In malignant tumor cells maximal cyclin D1 expression was observed after 3 h, whereas in benign cells the effect of 17beta-estradiol was delayed. ICI 182780, a pure estrogen antagonist, prevented the effects of 17beta-estradiol. In addition, 17beta-estradiol was found to modulate activation of mitogen-activated protein (MAP) kinase, whose activity is mainly regulated by growth factors in thyroid carcinoma cells. In response to 17beta-estradiol, both MAP kinase isozymes, extracellular signal-regulated protein kinases 1 and 2, were strongly phosphorylated in benign and malignant thyroid cells. Treatment of the cells with 17beta-estradiol and MAP kinase kinase 1 inhibitor, PD 098059, prevented the accumulation of cyclin D1 and estrogen-mediated mitogenesis. Our data indicate that 17beta-estradiol is a potent mitogen for benign and malignant thyroid tumor cells and that it exerts a growth-promoting effect not only by binding to nuclear estrogen receptors, but also by activation of the MAP kinase pathway.

Increased levels of platelet-derived growth factor in vitreous fluid of patients with proliferative diabetic retinopathy.

UNLABELLED: It is generally accepted that growth factors play an important role in the pathogenesis of proliferative diabetic retinopathy. Since platelet-derived growth factor AB (PDGF AB) is known to be involved in many angiogenetic and proliferative processes, it was the aim of our study to elucidate the role of PDGF AB in the angiogenetic process in proliferative diabetic retinopathy. We measured PDGF AB concentrations in the vitreous of 23 patients with proliferative diabetic retinopathy, 4 of them with additional rubeosis iridis as an indicator of very high vasoproliferative activity. Control measurements were done in 19 patients without diabetic or ischemic eye diseases and also in 4 non-diabetic patients with ischemic proliferative retinopathy with rubeosis iridis. To exclude PDGF remnants in the vitreous due to vitreous bleeding we additionally measured platelet factor 4 concentrations as a stable marker of activated thrombocytes in the vitreous. RESULTS: Significantly elevated concentrations of PDGF AB were found in the vitreous of patients with proliferative diabetic retinopathy, with higher levels in individuals with additional rubeosis iridis compared to controls. However, concentrations of PDGF AB were also elevated in ischemic non-diabetic retinopathy, supporting the concept that ischemia might be a strong stimulator of growth factor production in the retina. Platelet factor 4 was not detectable in any of the vitreous samples included in the study. In summary, our results indicate that the growth factor PDGF plays an important role in the pathogenesis of proliferative diabetic retinopathy, probably in synergistic action with other growth factors like IGF I, IGF II, VEGF and TNF alpha.

Detection of mutations in glycoprotein hormone receptors.

The luteinizing, follicle-stimulating, and thyroid-stimulating hormone receptors belong to the huge family of G-protein-coupled receptors. Identification of either activating or inactivating mutations of these receptors has led to a fundamental improvement in our understanding of glycoprotein hormone/receptor interaction. Furthermore, clinical phenotypes such as precocious puberty, follicle-stimulating hormone (FSH) insensitivity syndrome, and congenital hypthyroidism are now being explained by mutated glycoprotein hormone receptors. Since there is an ongoing worldwide search for certain clinical phenotypes that might be caused by mutations of these receptors, there is a demand for strategies and techniques that can be used to screen patients in a effective and reliable way. This article focuses, therefore, on patient selection and techniques for the detection of mutations of glycoprotein hormone receptors, and compiles useful laboratory protocols to conduct such studies.

Multinodular goitre: 'much more to it than simply iodine deficiency'.

For over a century, multinodular goitre (MNG) has been looked upon as the simple consequence of iodine deficiency. This view is now no longer tenable. Indeed, many characteristics of MNG do not fit with the iodine deficiency concept. For example, nodular goitre is a frequent disease even in those countries where the population is never exposed to iodine shortage. Moreover, neither multinodularity, nor the proverbial heterogeneity of growth and function or the autonomous, thyroid stimulating hormone (TSH)-independent growth of many goitres are compatible with the iodine deficiency concept, let alone subclinical or overt thyrotoxicosis which often complicates the course of a MNG. Recent investigations have led to the conclusion that MNGs are true benign neoplasias that are due to the high intrinsic growth potential of a variable, genetically predetermined fraction of all thyrocytes. Gross and heritable metabolic and functional differences between the individual thyrocytes, from which new follicles are generated during goitrogenesis, are the cause of the often spectacular functional and structural heterogeneity of MNG. Superimposed iodine deficiency changes the epidemiology, but not the basic mechanisms of goitrogenesis. These new pathogenetic concepts have a profound impact on the clinical management of MNG.

[Mutations in the thyrotropin receptor gene in the pathogenesis of toxic thyroid adenomas, toxic goiter nodules and autosomal dominant hyperthyroidism]. / TSH-Rezeptor-Mutationen in der Pathogenese des autonomen Adenoms, der multifokalen Autonomie und der angeborenen Hyperthyreose.

In iodine deficiency regions such as Germany, more than 50% of toxic thyroid adenomas and some toxic goiter nodules harbor activating mutations in the TSH receptor or Gs-alpha gene. These mutations cause cellular hyperfunction and hyperthyroidism but are not sufficient to generate thyroid adenomas and nodules. In the pathogenesis of these tumors, other primary and secondary molecular mechanism are operative, including overexpression of growth factors, their receptors and of signaling proteins. Expression of some of these growth factors is regulated by TSH-dependent pathways. However, iodine deficiency and its effect on thyroid growth remains the most important pathogenetic factor for these diseases. Therefore, toxic adenomas as well as the majority of nonfunctioning and eufunctioning thyroid nodules and adenomas are relatively rare in regions with high iodide intake of the population.

Pathogenesis of toxic thyroid adenomas and nodules: relevance of activating mutations in the TSH-receptor and Gs-alpha gene, the possible role of iodine deficiency and secondary and TSH-independent molecular mechanisms.

In iodine deficiency areas, activating mutations in the TSH receptor and Gs-alpha gene are found in the majority of toxic thyroid adenomas and in some toxic goiter nodules. Since TSH receptor and Gs-alpha gene mutations are very rare in areas with high iodine supply, iodine deficiency has been suspected to favor the occurrence of these mutations by yet unknown pathways. However, TSH receptor and Gs-alpha gene mutations alone are not sufficient to cause toxic adenomas and nodules. There is compelling evidence that other secondary and cAMP-independent mechanisms, including enhanced expression of various growth factors, their corresponding receptors and of signaling proteins, may affect the mutated cell and thus promote cell proliferation and in turn generation of the tumor.

Thyroid nodules in recurrent multinodular goiters are predominantly polyclonal.

Not only thyroid adenomas and carcinomas, but also the majority of single and well delimited goiter nodules, even if morphologically heterogeneous, are of clonal origin. However, it is still unknown whether the nodules of rapidly growing, recurrent goiters are clonal or polyclonal. We investigated by PCR-based analysis of exon 1 of the human androgen receptor gene clonality of nodules grown in recurrent multinodular goiters (MNG) of 14 female patients. The total goiter volume varied widely between 15 ml and 170 ml. The mean age of patients undergoing surgery for recurrent goiter at the time of their first operation was significantly lower with 34.6 +/- 10.9 yr in comparison to 50 consecutive patients who were operated for MNG for the first time (53.7 +/- 13.5 yr). The interval between first and recurrent operation was 18 +/- 8.5 yr. The mean volume of well circumscribed nodules selected for the present investigation was 3.8 +/- 1.4 ml. Assessment of clonality in at least 2 samples of each lesion revealed a polyclonal pattern in 10 out of 14 nodules, whereas only 3 nodules were clonal and in one case the result remained unclear. The unexpected finding that most nodules within MNG, that had re-grown after a first subtotal thyroidectomy, were of polyclonal rather than clonal composition, suggests that these lesions are generated by de novo-proliferation of cohorts of differing thyrocytes sharing the common trait of an exceedingly high intrinsic growth rate or alternatively, by unknown growth stimulating molecular events acting focally on clusters of cells derived from different ancestors. In addition, the relatively young age of patients with recurrent MNG at the time of their first surgery and the comparatively short interval between first and second operation point to a genetic element in the occurrence of growth-prone thyrocytes.

Enhanced G protein activation in IDDM patients with diabetic nephropathy.

Genetic susceptibility contributes significantly to the risk of developing nephropathy in insulin-dependent diabetes mellitus (IDDM). The cellular substrate for this has remained enigmatic. We investigated whether afflicted IDDM patients display an enhanced activation of pertussis toxin (PTX)-sensitive G proteins, a phenomenon which has been demonstrated in patients with essential hypertension. We established immortalised B lymphoblast cell lines from 10 IDDM patients without nephropathy (DC) and 15 IDDM patients with nephropathy (DN). Nephropathy was defined as a persistent albumin excretion rate of more than 20 microg/min (DC 3.9 +/- 5.8, DN 562.3 +/- 539.0 microg/min, respectively). Subjects were matched with regard to age (DC 28.9 +/- 6.5, DN 35.9 +/- 9.9 years), diabetes duration (DC 19.3 +/- 6.9, DN 22.7 +/- 5.8 years) and HbA1c values (DC 8.5 +/- 1.4, DN 8.8 +/- 1.6%). Reactivity of PTX-sensitive G proteins was quantified by measuring platelet-activating factor (PAF)-induced Ca2+ mobilisation (fura 2 method) and by mastoparan-stimulated [35S]GTPgammaS binding. Expression of Galphai proteins was quantified by Western blot analysis. PAF-evoked Ca2+ increases above baseline averaged 77.0 +/- 52.5 nmol/l in DC and 150.7 +/- 61.5 nmol/l in DN (p = 0.005). PAF-evoked Ca2+ increases correlated with stimulated [35S]GTPgammaS binding (r2 = 0.42, p = 0.012). From Western blot analysis an overexpression of Galphai proteins could be excluded in DN. A consequence of the altered metabolic milieu in diabetes is the increased release of vasoactive and proliferative agonists which promote glomerular hyperfiltration, hypertrophy, enhanced matrix deposition, and, finally, glomerulosclerosis. Many of these auto- and paracrine agonists bind to G protein-coupled receptors. Therefore, their cellular effects are reinforced by the enhanced G protein reactivity and increase the propensity to nephropathy in IDDM.

The clonality of nodules in recurrent goiters at second surgery.

INTRODUCTION: Some studies showed that in multinodular goiters clonal and polyclonal nodules coexist. The clonality of nodules in recurrent goiters is, however, still unknown and may contribute to help explain the pathogenesis of this thyroid disease. METHODS AND RESULTS: The clonality of 14 nodules derived from recurrent goiters was assessed by means of an X-chromosome-inactivation method. Of 14 nodules, 10 showed a polyclonal pattern, 3 were clonal and, in 1 case, the result remained unclear. The mean age of the patients with recurrent goiter at the time of their first operation was significantly lower than the mean age of 50 patients who underwent thyroid surgery for the first time over the same period of time (34.6+/-10.9 years vs 53.7+/-13.5 years; P<0.05). The mean interval between first and second operation was 18 years. CONCLUSION: The finding that nodules in recurrent goiters are predominantly polyclonal suggests that these lesions have their origin in a de novo proliferation of different cohorts of thyrocytes due to unknown growth stimulating molecular events.

Overexpression of stimulatory G protein alpha-subunit is a hallmark of most human somatotrophic pituitary tumours and is associated with resistance to GH-releasing hormone.

The heterotrimeric Gs protein-adenylyl cyclase (AC) cascade plays a pivotal role in controlling hormone secretion by endocrine glands. Consequently, deficiency of the alpha-subunit of Gs leads to endocrine hypofunction and hypoplasia in the affected cells whereas AC hyperactivity results from activating point mutations within the Gs-alpha gene. The latter, termed gsp oncogenes, are found primarily in a subset of growth hormone (GH)-secreting human pituitary tumours (somatotrophinomas) and are thus associated with excessive GH secretion. We present here evidence that another type of defect in human somatotrophinomas may be overexpression of the Gs-alpha subunit. Immunohistochemistry using an antibody against recombinant human Gs-alpha revealed high levels of expression in 25 of 39 somatotrophinomas but weak staining in normal human pituitary cells. These results were confirmed by Western blot analysis. Additionally, cholera toxin-mediated ADP-ribosylation in the presence of 32P-labelled NAD+ resulted in an autoradiographic signal intensity which correlated directly with magnitude of immunostaining and amount of antigen shown by Western blot analysis, providing evidence for overexpression of functionally active subunit. Finally, reconstitution assays were applied and directly demonstrated the increased activity of overexpressed Gs-alpha. In vivo, the effect of Gs-alpha on AC activity may be partially counterregulated by high levels of inhibitory G protein that also occurred in these tumours. In culture, GH-releasing hormone (GHRH) had markedly reduced effects on GH secretion by somatotrophinomas exhibiting Gs-alpha overexpression, whereas powerful stimulation occurred in weakly staining tumours. In contrast to these observations with Gs-alpha, immunostaining for the phospholipase C-coupled G11-alpha subunit was relatively weak in all somatotrophinomas studied and synthetic GH-releasing peptide, which acts via a specific G11-coupled receptor, led to powerful and consistent stimulation of GH secretion by different tumours. These results indicate that Gs-alpha overexpression is associated with dysfunction in hormone secretion by some somatotrophinomas.

Excessive activation of tyrosine kinases leads to inhibition of proliferation in a thyroid carcinoma cell line.

Autocrine stimulation of growth is a hallmark of many tumor cell lines. In this work we investigated the synthesis and secretion of growth factors and the expression of their corresponding receptors in HTC-TSHr thyroid carcinoma cells. These cells synthesize epidermal growth factor (EGF) receptors and platelet-derived growth factor beta (PDGF beta) receptors and in addition transforming growth factor alpha (TGF alpha), PDGF-A and PDGF-B chains, respectively. Addition of EGF or PDGF-BB to the culture medium resulted in growth inhibition of HTC-TSHr cells. In contrast, treatment of the cells with low concentrations of neutralizing anti-TGF alpha antibodies or tyrosine kinase inhibitors led to stimulation of cell proliferation. Low concentrations of neutralizing anti-PDGF-B antibodies did not affect growth of the cells. As expected, cell proliferation was inhibited when high concentrations of either neutralizing anti-TGF alpha antibodies or anti-PDGF-B antibodies were applied. PDGF-AA did not influence growth of HTC-TSHr cells. We conclude that growth of HTC-TSHr thyroid carcinoma cells is influenced by two autocrine loops between TGF alpha and EGF receptors and between PDGF-B and PDGF beta receptors. However, our data suggest that excessive activation of tyrosine kinase receptors in these cells results in a relative inhibition rather than stimulation of growth.

Suppression of thyrotropin receptor-G protein-phospholipase C coupling by activation of protein kinase C in thyroid carcinoma cells.

In human thyroid follicular cells TSH exerts its action on growth and function at least via two distinct pathways, the adenylate cyclase cascade and the phospholipase Cbeta (PLCbeta)-mediated inositol phosphate generation. We investigated the effect of TSH on activation of phosphoinositide hydrolysis and inositol phosphate generation by PLCbeta in HTh74 thyroid carcinoma cells that express functional TSH receptors and in HTC-TSHr thyroid carcinoma cells that are devoid of endogenous TSH receptors but express recombinant human TSH receptors. In both cell lines, TSH up to concentrations of 300 mU/ml failed to stimulate myo-inositol 1,4,5-trisphosphate and myo-inositol-tetrakisphosphate generation, but led to a decrease in these compounds within 1 min of stimulation. However, ATP and bradykinin increased concentrations of inositol phosphates in both thyroid carcinoma cell lines. In contrast, in differentiated FRTL5 thyroid cell line and CHO-TSHr cell line expressing recombinant human TSH receptors, TSH elicited a significant increase in myo-inositol 1,4,5-trisphosphate and its metabolic derivatives. However, when HTC-TSHr cells were pretreated with calphostin C or staurosporine, inhibitors of protein kinase C, a TSH concentration of 20 mU/ml enhanced generation of inositol phosphates in these cells. From our data we conclude that in HTC-TSHr and HTh74 thyroid carcinoma cells, the coupling within the TSH receptor-Gq protein-PLCbeta signaling pathway is impaired compared to that in nontransformed cells. It is conceivable that this is at least in part dependent on the level of protein kinase C activation in these cells.

Constitutively active germline mutation of the thyrotropin receptor gene as a cause of congenital hyperthyroidism.

Congenital hyperthyroidism is a rare, transient disease usually caused by transmission of thyrotropin receptor autoantibodies from the mother with Graves' disease to her child. We report a German women and her two sons who had congenital, but persistent hyperthyroidism without any signs of autoimmunity. Direct sequencing of the polymerase chain reaction-amplified exon 10 of the thyrotropin receptor genomic DNA revealed in the mother and both sons a transition of GCC to GTC, resulting in an exchange of alanine 623 to valine. This germline mutation in a highly conserved region of the thyrotropin receptor resulted in a constitutive activation of the cyclic adenosine monophosphate-generating cascade with resulting hyperthyroidism. Analysis of the family for a corresponding BstXI restriction-site polymorphism revealed heterozygosity for this mutation in the affected family members, but not in the father or other relatives. We conclude that whenever congenital hyperthyroidism is persistent and parameters of autoimmunity are absent, a constitutively active thyrotropin receptor mutation should be considered. Treatment appears to require aggressive means such as total thyroidectomy or ablation by 131iodine because two subtotal thyroidectomies in the mother were insufficient to control the disease.

TSH receptor and Gs-alpha gene mutations in the pathogenesis of toxic thyroid adenomas--a note of caution.

The detection of a constitutive activation of the AC cascade by TSH-R and Gs alpha mutations in a number of TTAs should not distract from the large gap in our understanding of the pathogenesis of thyroid tumors. TTAs form only a minor fraction of all thyroid nodules, and even within this small subgroup, activating mutations have been found regionally with a highly variable incidence. If activating mutations were the sole and only cause of TTAs, a homogeneous functional and morphological response of all thyrocytes would ensue. This, however, is not the case. Rather, severe disturbances of the Gs protein-AC cascade regularly occur in TTAs. Even within thyroids affected by activating TSH-R germline mutations, some cell clones proliferate at a faster rate than others, causing nodular growth with time. Moreover, not only functional, but also morphological heterogeneity very frequently evolves even in clonal adenomas. The natural heterogeneity among individual thyrocytes may account for a different functional and proliferative response among cells affected by identical mutations or any other gain-of-function event. The recent findings in toxic adenomas must be taken together with the fact that, in the large majority of all thyroid nodules, iodine metabolism is by no means enhanced, but diminished or absent and that, in this type of tumor, no consistent pattern of growth-stimulating mutational events has yet been identified. The nature, the precise temporal sequence and interaction of the genetic, cytogenetic, and environmental events that cause the very common and often autonomous nodular growth of only a few distinct cell populations within the human thyroid gland remain largely unknown.