[Thyroid emergencies : Thyroid storm and myxedema coma]. / Schilddrüsennotfälle : Thyreotoxische Krise und Myxödemkoma.

Thyroid emergencies are rare life-threatening endocrine conditions resulting from either decompensated thyrotoxicosis (thyroid storm) or severe thyroid hormone deficiency (myxedema coma). Both conditions develop out of a long-standing undiagnosed or untreated hyper- or hypothyroidism, respectively, precipitated by an acute stress-associated event, such as infection, trauma, or surgery. Cardinal features of thyroid storm are myasthenia, cardiovascular symptoms, in particular tachycardia, as well as hyperthermia and central nervous system dysfunction. The diagnosis is made based on clinical criteria only as thyroid hormone measurements do not differentiate between thyroid storm and uncomplicated hyperthyroidism. In addition to critical care measures therapy focusses on inhibition of thyroid hormone synthesis and secretion (antithyroid drugs, perchlorate, Lugol's solution, cholestyramine, thyroidectomy) as well as inhibition of thyroid hormone effects in the periphery (ß-blocker, glucocorticoids).Cardinal symptoms of myxedema coma are hypothermia, decreased mental status, and hypoventilation with risk of pneumonia and hyponatremia. The diagnosis is also purely based on clinical criteria as measurements of thyroid hormone levels do not differ between uncomplicated severe hypothyroidism and myxedema coma. In addition to substitution of thyroid hormones and glucocorticoids, therapy focusses on critical care measures to treat hypoventilation and hypercapnia, correction of hyponatremia and hypothermia.Survival of both thyroid emergencies can only be optimized by early diagnosis based on clinical criteria and prompt initiation of multimodal therapy including supportive measures and treatment of the precipitating event.

Comparison of Color Flow Doppler Sonography (CFDS) and immunohistologic detection of microvessels for the assessment of the malignancy of thyroid nodules.

The assessment of tumor vascularization by color flow Doppler sonography (CFDS) has been suggested for the distinction between benign and malignant thyroid nodules. Our objective was to investigate if the CFDS results reflect the percentage of histologically determined microvessels in adenomas (As), adenomatous nodules (ANs), and papillary carcinomas (PCs). Tissue sections from 10 adenomas, 8 ANs and 13 PC and surrounding tissue of 10 PCs and 2 benign nodules were immunostained for CD34. A computerized image analysis was used to determine the microvessel density in four hot spots and ten systematically selected fields. Preoperatively CFDS was performed and classified according to Frates et al. We found a consistent percentage increase of CD34 stained microvessels in PCs (83 and 96%) as compared to adenomas and ANs (38 and 49%) determined by the hot spot analysis and systematic field analysis. A ROC analysis on the basis of the histologically determined number of microvessels demonstrated 70% microvessels as an optimal cut point for the diagnosis of PC with the highest sensitivity of 92% and highest specificity of 89%. The analysis of the CFDS-classification IV for the distinction between PCs and adenomas and ANs showed a sensitivity of 62% with a specificity of 100%. The lower sensitivity of the CFDS classification as compared with the immunohistologic determination of the microvessel density indicates that the CFDS classification detects the pathognomonic intranodular microvessels only incompletely. The higher CFDS specificity is most likely due to the detection of other vascular aspects of malignancy in addition to intranodular microvessels.

Selenium supplementation does not decrease thyroid peroxidase antibody concentration in children and adolescents with autoimmune thyroiditis.

In adults, selenium supplementation decreases thyroid peroxidase antibody (TPO Ab) concentrations in patients with autoimmune thyroiditis (AIT). Our aim in this study was to investigate if selenium supplementation decreased TPO Ab and thyroglobulin antibody (Tg Ab) concentrations in children with AIT. Forty-nine patients (33 females) with newly diagnosed AIT and hypothyroidism were randomized to daily oral therapy with levothyroxine alone (group A, n=18), levothyroxine plus 100 microg sodium-selenite (group B, n=13), or levothyroxine plus 200 microg sodium-selenite (group C, n=18). Mean age at diagnosis was 12.2+/-2.2 years. All 49 patients needed a mean levothyroxine dose of 1.6+/-0.5 microg/kg body weight to lower TSH to the treatment goal of 1-2 microU/ml, with no significant difference between groups. At study entry and after 12 months, TPO Ab concentrations were comparable in all three groups. Tg Ab concentrations decreased significantly after 12 months in group A and group C (p=0.03 and p=0.01), but not in group B (p=0.06). It is our conclusion that selenium supplementation with sodium-selenite does not decrease TPO Ab concentrations in children and adolescents, neither given in the reduced dose of 100 microg daily nor given in the "adult" supplementation dose of 200 microg daily.

[Is iodine deficiency still relevant?]. / Das Ziel der WHO wurde verfehlt. Immer noch Jodmangel in Deutschland.

In Germany, iodine deficiency and its consequences is still a problem, although it is of less importance than it was twenty years ago. In accordance with the WHO definition, Germany still belongs among those countries with mild iodine deficiency and too low an intake of iodine. As a result groups at particular risk, such as pregnant and nursing women, must still receive iodine supplementation, since, in the absence of supplemental iodine,the amount of iodine in the mother's milk continues to be below average throughout Germany. Both in private households and in the food industry, the aim is to increase the use of iodized salt to more than go%. This entails no risk of an iodine overdose. The current average daily uptake of iodine of approximately 120 micrograms is responsible neither for the development or progression of an autoimmune disease nor a functional disorder of the thyroid gland.

Release of an endothelial cell growth factor from cultured porcine thyroid follicles.

It has been proposed from in vivo studies that thyroid angiogenesis during thyroid enlargement may be due to paracrine mitogenic factors released by epithelial thyroid cells. To study this paracrine growth regulating communication between thyroid cells and endothelial cells in vitro, culture medium from isolated porcine thyroid follicles was investigated for a growth promoting effect on porcine aortal endothelial cells. Serum-free conditioned medium (CM) from thyroid follicles in suspension culture contains a dose-related mitogenic activity which stimulates endothelial cell growth up to 197%. Stimulation of the thyroid follicles with TSH (1 mU/ml) significantly reduced the mitogenic activity for endothelial cells in CM to 131%. Thyroid hormones had no influence on mitogenic activity in CM. When follicles were treated with iodide (20 microM) during CM production, no proliferation of endothelial cells was observed by this CM. In contrast, CM from epidermal growth factor-treated thyroid follicles significantly enhanced the mitogenic activity for endothelial cells up to 235%. The mitogenic activity was precipitable by saturated ammonium sulfate, showed high affinity to heparin by chromatography on heparin-sepharose, and was abolished after treatment of CM with trypsin. On gel electrophoresis the heparin-binding fraction showed a double band with a mol wt of 15 and 15.5 k. These data show a paracrine mitogenic activity on endothelial cells released by thyroid follicles which is regulated by TSH, epidermal growth factor, and iodide in parallel with the direct effect of these substances on thyroid cell growth. The data suggest that the mitogenic factor is a polypeptide, which belongs to the heparin-binding growth factors.

Skeletal effects of low-dose cyclosporin A in aged male rats: lack of relationship to serum testosterone levels.

The aim of this study was to investigate the skeletal effects of cyclosporin A (CsA) in a dose range relevant to clinical medicine in lumbar vertebral cancellous bone of aged male rats and to correlate these effects with possible changes in serum testosterone levels. Thirty-one 18-month-old male Wistar rats were divided into four weight-matched groups and subcutaneously injected with either 0, 1, 3, or 5 mg of CsA/kg of body weight three times per week After 4 weeks of treatment, all rats were killed after in vivo fluorochrome labeling and the first lumbar vertebrae analyzed by quantitative histomorphometry. Serum was analyzed for total calcium, creatinine, alkaline phosphatase, osteocalcin, parathyroid hormone, total testosterone, and CsA levels. CsA administration resulted in a dose-dependent increase in serum osteocalcin levels and in histomorphometric indices of cancellous bone turnover in the axial skeleton. Furthermore, CsA-treated rats showed a deterioration of vertebral cancellous bone structure with increased discontinuity of the trabecular bone network due to trabecular plate perforations. Serum testosterone levels were not significantly changed by CsA treatment and were uncorrelated to all biochemical or histomorphometric indices of bone turnover. We conclude that the 4-week administration of CsA at doses that are close to those used in transplantation patients induced high turnover osteopenia in the axial skeleton of aged, 18-month-old male rats, and that these effects were likely not mediated by changes in serum testosterone levels.

Evidence that an iodolactone mediates the inhibitory effect of iodide on thyroid cell proliferation but not on adenosine 3',5'-monophosphate formation.

Iodolactone (6-iodo-8,11,14-eicosatrienoic-delta-lactone), an iodinated derivative of arachidonic acid, was found to be synthesized in rat thyroid slices; however, the physiological role of this compound is still unknown. We tried to detect iodolactone in isolated porcine thyroid follicles and investigated the effects of in vitro synthesized iodolactone on epidermal growth factor-induced thyroid cell proliferation and TSH-induced cAMP formation. In vitro synthesis of iodolactone was performed with lactoperoxidase-catalyzed iodination of arachidonic acid in the presence of trace amounts of [125I]- and [3H]arachidonic acid. After purification by silica gel chromatography, HPLC of the reaction products revealed one main peak containing trace amounts of both [125I]- and [3H]arachidonic acid. With gas chromatography-mass spectrometry (GC-MS) a molecular mass of 391 m/z, corresponding to the derivatization product of iodolactone, was found. An ethanol-chloroform extract of isolated thyroid follicles preincubated with KI (10 microM) and arachidonic acid (1 microM) revealed peaks in HPLC and GC comparable with those of in vitro synthesized iodolactone. This indicates the ability of thyroid follicles to form iodolactone. Iodolactone (0.1-1.0 microM) dose-dependently inhibited epidermal growth factor-induced thyroid cell growth. This growth-inhibiting effect of iodolactone was 50-fold more pronounced than the inhibitory effect of KI (4 X 10(-5) microM) on thyroid cell proliferation. In contrast to the effect of iodide, the inhibitory effect of iodolactone on thyroid cell growth could not be abolished by methimazole (1 mM). Basal as well as TSH (0.5 U/liter)-induced cAMP formation were not changed by iodolactone. These experiments suggest a physiological role of iodolactone as a mediator of the known inhibitory effect of iodide on thyroid growth.

Thyroxine-binding globulin: investigation of microheterogeneity.

Preparations of T4-binding globulin (TBG) from human serum was performed using only two affinity chromatography steps. Purity of the protein was demonstrated by a single band in overloaded disc and sodium dodecyl sulfate electrophoresis, equimolar binding to T4, and linearity in sedimentation velocity run. The molecular weight was calculated to be 60,000 +/- 3,000 daltons (n = 3), the sedimentation coefficient was 3.95S, and the Stokes' radius was 37 A. The amino acid composition was found to be in good agreement with the calculations of other authors. By isoelectric focussing (IEF), pure TBG showed four main bands at pH 4.25, 4.35, 4.45, and 4.55 together with several fainter bands. The N-acetylneuraminic acid (NANA) content of the four TBG bands isolated by preparative IEF was found to decrease from 10.2 mol NANA/mol TBG in the band at pH 4.25 to 4.8 mol NANA/mol TBG in the band at pH 4.55. No significant difference in the affinity constants of the TBG bands to T4 was found. The affinity constants for TBG ranged from 3.1 x 10(9) to 7.2 x 10(9) M-1. Sequential kinetic desialylation of pure TBG resulted in a progressive tendency toward one major band at pH 6.0. In native sera, microheterogeneity of TBG was detected after IEF on polyacrylamide gel plates by immunofixation. The typical TBG patterns shown by pure TBG were also found in normal subjects. Characteristic deviations from this pattern were found in the sera of females during estrogen therapy or pregnancy, where there was a gradual increase in density of the band at pH 4.25 and the appearance of an additional band at pH 4.15. In sera from patients with liver disease and elevated TBG levels, there was a fading of the acidic bands, whereas the more alkaline band at pH 4.55 was intensified. It is therefore proposed that microheterogeneity of TBG is caused by differences in NANA content and that variations of TBG patterns in native sera may reflect altered TBG synthesis or degradation. A genetically related microheterogeneity of TBG could not be demonstrated after examination of 800 sera, including 2 families with quantitative TBG deficiency.

Functioning thoracic paraganglioma: association with Von Hippel-Lindau syndrome.

Functioning thoracic paraganglioma (pheochromocytoma) is unusual and therefore suggestive of a pathogenesis distinct from that of sporadic adrenal pheochromocytoma. To determine whether the pheochromocytoma-associated syndromes Von Hippel-Lindau disease (VHL) and multiple endocrine neoplasia type 2 (MEN 2) play a role in the development of thoracic functioning paragangliomas, germline DNA from five unselected patients with this rare tumor was analyzed for mutations in the genes that predispose to VHL and MEN 2. Genetic investigations and further clinical data revealed that three had VHL, with two different germline mutations of the vhl gene, but no individual was affected by MEN 2. Two of the three patients with VHL did not show any additional VHL-associated lesions. This result suggests that VHL should be considered in the differential diagnosis of thoracic pheochromocytoma, as such a diagnosis carries further important implications for the patient and family. Conversely, in patients suspected of a catecholamine-secreting tumor and known VHL, thoracic localization should be considered if an adrenal pheochromocytoma cannot be detected.

Induction of apoptosis in porcine thyroid follicles by transforming growth factor beta1 and epidermal growth factor.

For thyroid cells in culture DNA fragmentation and morphological changes related to apoptosis were first described in dog thyroid cells after deprivation of serum, epidermal growth factor or thyrotropin. With intact porcine thyroid follicles in three-dimensional culture, the effect of deprivation of growth factors and of incubation with transforming growth factor beta1 (TGF-beta1), epidermal growth factor (EGF), thyrotropin (TSH) or insulin-like growth factor I (IGF-I) on the incidence of apoptosis was studied. Thyroid follicles were embedded in growth factor-depleted Matrigel and cultured in serum-free medium with or without growth factors for 7 days followed by incubation for 4, 24 and 72 h with TGF-beta1 (2 or 5 ng/mL). The percentage of apoptotic cells was determined by direct counting in electron-microscopy. Approximately 1% of apoptotic bodies could be detected in unstimulated follicles. This was unchanged in the presence of TSH (1 mU/mL) or IGF (10 ng/mL) but significantly increased up to 3.99 +/- 1.24% with 2 ng/mL of EGF. After incubation with TGF-beta apoptosis increased dose-dependently to 4.05 +/- 0.67% with 2 ng/mL TGF-beta1 and 5.16 +/- 1.75% with 5 ng/mL TGF-beta1. The incidence of necrotic cells remained constant at about 1 to 2%. Preincubation of follicles with 2 ng/mL of EGF followed by incubation with 5 ng/mL TGF-beta1 increased the rate of apoptic bodies up to 13.19 +/- 1.9%. We conclude that growth factor depletion in thyroid follicles in three-dimensional culture does not lead to apoptosis. TGF-beta1, however, induces apoptosis even in quiescent thyroid follicular cells and is significantly more pronounced in growing thyroid cells. EGF, which is a dedifferentiating growth factor for thyroid cells, also induces apoptosis. As EGF enhances TGF-beta1 mRNA and protein in thyroid follicular cells, the induction of apoptosis by EGF might also be due to TGF-beta1.

Prevention of osteoporosis after cardiac transplantation: a prospective, longitudinal, randomized, double-blind trial with calcitriol.

BACKGROUND: Accelerated bone loss is a well-recognized complication after cardiac transplantation (HTx) due to immunosuppressive therapy. The purpose of this prospective, longitudinal, randomized, placebo-controlled, double-blind study was to investigate the effect of calcitriol (1,25-dihydroxyvitamin D3) in the prevention of bone loss and fracture rate after HTx. METHODS: Basic therapy included 1000 mg of calcium daily and sex hormone replacement in hypogonadal patients. A total of 132 patients (111 male, 21 female; mean age: 51+/-10 years; 35+/-25 months after HTx) were randomized to 0.25 microg of calcitriol or placebo. Bone mineral density (BMD, g/cm2; T score, %) of the lumbar spine and x-rays for the assessment of vertebral fractures were performed at baseline and after 12, 24, and 36 months. Biochemical indexes of mineral metabolism were measured every 3 months. RESULTS: Overall BMD was significantly decreased after HTx (T score 87+/-13%). BMD increased continuously within the study period in the calcitriol group (1 year: 2.2+/-4.8%; 2 years: 3.9+/-5.4%; 3 years: 5.7+/-4.4%) as well as in the placebo group (1 year: 1.8+/-4.9%; 2 years: 3.7+/-6.5%; 3 years: 6.1+/-7.8%) without statistical difference between the groups. Fracture incidence was low during the study interval (1 year: 2.0%; 2 years: 3.4%; 3 years: 0%). Hypogonadism (20%) was associated with a lower BMD (78+/-12% vs. 88+/-12%; P<0.01) and a higher increase (35%) after hormone replacement in comparison to normogonadal patients. Increased intact parathyroid hormone and bone resorption markers decreased significantly during therapy. CONCLUSIONS: Calcium supplementation and sex hormone replacement in hypogonadism proved a sufficient long-term prevention therapy to improve decreased BMD and to prevent fractures after HTx. Besides immunosuppression, both concomitant hypogonadism and secondary hyperparathyroidism play a major role in the long-term bone loss and should therefore be monitored and treated adequately. Low-dose calcitriol demonstrated no significant extra benefit regarding BMD and fracture rate in the long-term period after HTx.

Involvement of cyclic AMP, iodide and metabolites of arachidonic acid in the regulation of cell proliferation of isolated porcine thyroid follicles.

Experiments with primary cultures of isolated porcine thyroid follicles were performed in serum-free well-defined medium to investigate different pathways that may be involved in the regulation of thyroid cell growth. The incorporation of [3H]thymidine into DNA within 72 h was about 25-fold with fetal calf serum (FCS, 1%), 20-fold with epidermal growth factor (EGF, 1 ng/ml) and 3.5-fold with insulin (10 micrograms/ml) as compared to controls. Bovine TSH significantly reduced the basal and insulin-induced growth rate at concentrations of 10(-6) to 10(-4) U/ml and 10(-4) U/ml, respectively. Forskolin stimulated cyclic AMP accumulation in thyroid cells and significantly reduced FCS-, EGF- or insulin-induced growth. In contrast, a 2- to 7-fold increase in FCS-, insulin- or EGF-induced growth rate was found, when cyclic AMP formation was inhibited by 2',5'-dideoxyadenosine (DDA). Iodide was stimulatory at low concentrations (1 microM) and inhibitory at higher concentrations (40-80 microM) on FCS-induced growth rate. The inhibitory effect of iodide was blocked by propylthiouracil (PTU), indicating that an iodinated compound is responsible for this effect. Indomethacin, a cyclooxygenase inhibitor, did not inhibit EGF- and insulin-induced growth up to a concentration of 100 microM. However, nordihydroguaiaretic acid (NDGA) and BW-755C, which are lipoxygenase inhibitors, strongly inhibited the growth of thyroid cells at micromolar concentrations. These data clearly show that (1) bovine TSH is not a growth factor for isolated thyroid cells in vitro, (2) thyroid cell proliferation, induced by FCS, EGF and insulin is under negative control of cyclic AMP. (3) Iodide controls dose-dependently thyroid cell growth by iodinated metabolites, probably modulating 2 different pathways: (a) at low iodide concentrations, an iodinated compound enhances the growth rate by inhibition of cyclic AMP formation, and (b) at high concentrations, iodide diminishes the growth rate by inhibiting the response to growth factors. (4) Metabolite(s) of lipoxygenase appear to be involved in intracellular signal transduction evoked by growth factors in thyroid cells.

Preparation of porcine thyroid follicles with preserved polarity: functional and morphological properties in comparison to inside-out follicles.

One of the main problems in establishing isolated thyroid follicles in vitro is their tendency to form inside-out follicles. The reason for this change in polarity is unknown. We describe here a method for the preparation of stable thyroid follicles with preserved polarity for at least 6 days. Isolated follicles were obtained by infusion of collagenase (1.5 mg/ml) dissolved in minimal essential medium into the artery of intact thyroid glands. The morphological and functional properties of these follicles were compared to inside-out follicles. These inside-out follicles were obtained by digestion of minced thyroid tissue in a collagenase (1 mg/ml) solution. The polarity of follicles was proved by morphological criteria. Follicles with preserved polarity did not change polarity for at least 6 days in the presence of 1% or 5% fetal calf serum. As the culture conditions for both preparation methods were identical, we conclude that the preparation method rather than the culture condition is responsible for the preservation of cell polarity of isolated thyroid follicles in our system. Increases in cyclic AMP levels induced either by bovine thyrotropin (10(-3) U/ml) or by isoproterenol (10(-6) M) as well as iodide uptake and organification were rapid and significant in right-side-right follicles but not in inside-out follicles. Therefore the TSH receptor and the beta-adrenergic receptor appear to be exclusively located at the basal membrane of follicular cells. In addition, iodide uptake apparently is unidirectional.

delta-Iodolactones decrease epidermal growth factor-induced proliferation and inositol-1,4,5-trisphosphate generation in porcine thyroid follicles--a possible mechanism of growth inhibition by iodide.

delta-Iodolactone (6-iodo-8,11,14-eicosatrienoic delta-lactone, delta-IL), an iodinated derivative of arachidonic acid, has been shown to be synthesized in thyroid tissue and to inhibit thyroid cell proliferation. It is discussed as a potential mediator of the autoregulatory pathway of iodide in cyclic adenosine-3',5'-monophosphate (cAMP)- and thyrotropin (TSH)-independent growth. We therefore further localized the action of iodide and of delta-IL in isolated porcine thyroid follicles. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) dose dependently stimulated thyroid cell proliferation, which could be inhibited by staurosporin (0.1-10 nmol/l). Iodide (2.5-40 mumol/l) as well as delta-IL (0.5-2 mumol/l) also dose dependently inhibited EGF- and TPA-induced proliferation. As the calcium ionophor A23187 (100 pmol/l) completely abolished the inhibitory effects of iodide and of delta-IL, this may indicate a mechanism of delta-IL at or proximal to the calcium-dependent activation of protein kinase C. The growth inhibitory effect was restricted to delta-iodolactones when delta-IL was compared to 6-iodo-8,11,14,17-eicosatetraenoic delta-lactone and 5-iodo-7,10,13,16,19-docosapentaenoic gamma-lactone. It could not be prevented with propylthiouracil and therefore deiodination and a different iodide action is unlikely. Inositol-1,4,5-trisphosphate (IP3) and cAMP were measured in extracts from isolated porcine thyroid follicles stimulated with EGF (10 ng/ml) or TSH (1.0 U/l) revealing comparable kinetics in IP3 generation, while cAMP formation was only stimulated by TSH. delta-Iodolactone (2 mumol/l) only decreased EGF-induced IP3 formation, whereas TSH-induced IP3 and cAMP formation was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor I messenger ribonucleic acid expression in porcine thyroid follicles is regulated by thyrotropin and iodine.

Insulin-like growth factor I (IGF-I) has been shown to be released from thyrocytes in vitro. We investigated IGF-I mRNA expression during treatment with thyrotropin (TSH), forskolin and potassium iodide (KI) in intact porcine thyroid follicles ex vivo. Porcine thyroid follicles were prepared by collagenase digestion and cultured in the presence of TSH, forskolin or KI. After different incubation times, mRNA was isolated and examined by Northern hybridization with a porcine IGF-I cDNA probe of 405 bp in length. In untreated follicles no IGF-I mRNA was found, whereas in follicles stimulated with TSH an IGF-I mRNA of 7.0 kb was detected after 24 h, which persisted for another 24 h. Forskolin treatment mimicked the TSH effect, indicating that IGF-I mRNA expression may be stimulated by the adenylate cyclase pathway. Preincubation of the porcine follicles with KI decreased dose dependently the TSH-induced IGF-I mRNA expression, with complete inhibition at 10 mumol/l KI. These results suggest that TSH acts via the cAMP pathway to enhance IGF-I mRNA expression, which then may lead to an autocrine IGF-I stimulation. The IGF-I mRNA expression is under negative control of iodide.

Stimulation of thyroid cell proliferation by epidermal growth factor is different from cell growth induced by thyrotropin or insulin-like growth factor I.

Isolated intact porcine thyroid follicles free of contaminating single cells were embedded in "Matrigel", which is a gel-forming basement membrane preparation containing mainly collagen type IV, laminin, heparan sulfate proteoglycans and entactin. Follicles were treated with different growth factors: thyrotropin (TSH), insulin-like growth factor I (IGF-I), epidermal growth factor (EGF) or transforming growth factor beta. Cell proliferation was quantified by counting cell numbers. Morphological studies were done by photodocumentation and analysis of histology by light and electron microscopy. The thyrocytes had the physiological polarity with follicular cell arrangement, microvilli at the apical membrane, desmosomes and tight junctions. The lumen contained colloid. Iodide organification (10.2 +/- 2.1 vs 26.1 +/- 5.8 pmol/10(6) cells; TSH 0.1 mU/ml) and release of thyroid hormones (thyroxine, 1754 +/- 207 vs 2890 +/- 460 pg/10(6) cells; triiodothyronine, 164 +/- 22 vs 412 +/- 106 pg/10(6) cells; TSH, 1mU/ml) were significantly stimulated by TSH. There was no basal growth rate in serum-free medium but proliferation was slightly stimulated with TSH (1 mU/ml; 149 +/- 19%) and in the same order of magnitude with IGF-I (10 ng/ml; 159 +/- 23%) but without follicle neoformation. In contrast, BGF (1.0-5.0 ng/ml) induced thyrocyte proliferation dose dependently three- to sixfold. With BGF up to 2 ng/ml, buds of new follicles formed surrounding pre-existing follicles. With BGF higher than 3 ng/ml, typical papillary structures developed. Transforming growth factor beta inhibited this dedifferentiated growth. A migration of single cells into the gel was never observed. Thus, three-dimensional culture of isolated thyroid follicles in "Matrigel" provides a tool for investigating the regulation of follicular growth and neoformation close to the in vivo situation.

Presence of a soluble inhibitor of thyroid iodination in primary cultures of thyroid cells.

Monolayer cultures of thyroid cells lose their iodide organification capacity a few days before the disappearance of thyroid peroxidase (TPO) activity. The present studies were performed in order to clarify this point. The above mentioned difference was due to the presence of an inhibitor in the monolayer thyroid cells culture, given that total homogenate prepared from confluent cells caused a significant inhibition of activity of TPO from fresh tissue. The inhibitor was localized in the 105000g supernatant of the homogenate of the cell culture, but not in a similar preparation obtained from fresh thyroid. It is thermostable, dialyzable and has a molecular weight of less than 2 kDa. Addition of the inhibitor at the end of the reaction of tyrosine iodination failed to alter the results. This fact suggests that the compound does not destroy the iodinated product. The presence of the cytosolic inhibitor was observed in monolayer thyroid cell cultures of different species (bovine, porcine, rat and human) but not in free follicles cultures.

Regulation of transforming growth factor beta 1 messenger ribonucleic acid expression in porcine thyroid follicles in vitro by growth factors, iodine, or delta-iodolactone.

Transforming growth factor beta 1 (TGF beta 1) is an autocrine growth factor for thyrocytes and is supposed to be the mediator of iodine-induced growth inhibition of thyroid epithelial cells, but this is still controversial. We further investigated this hypothesis using intact porcine thyroid follicles ex vivo in a three-dimensional culture system. In this culture system it has been shown previously that both iodide as well as delta-iodolactone, the putative iodocompound mediating thyroid cell proliferation, inhibit growth of these follicles. We measured the amount of TGF beta 1 mRNA expression in these follicles after treatment either with thyrotropin (TSH), epidermal growth factor (EGF), or transforming growth factor alpha (TGF alpha) for growth stimulation or with inorganic iodine or delta-iodolactone in concentrations known to inhibit growth. TGF beta 1-mRNA was detected by Northern blot analysis. The known major transcript of 2.5 kb was detected in a steady state level up to 48 hours in untreated thyroid follicles. EGF and TGF alpha (5 ng/mL each) enhanced TGF beta 1 mRNA about threefold within 4 and 8 hours. This increase of TGF beta 1 mRNA was slightly decreased by simultaneous incubation with delta-iodolactone (1 microM) or iodide (40 microM KI). In contrast, both TSH (1 mU/mL) and forskolin (16 microM) decreased TGF beta 1 mRNA expression to about 70%, and this effect was abolished when follicles were pretreated with iodide (40 microM KI) in a concentration known to inhibit TSH action on cyclic adenosine monophosphate (cAMP) formation and proliferation. Iodide or delta-iodolactone alone had no significant effect on basal TGF beta 1 mRNA expression. We conclude that the growth inhibitory effect of iodide as well as of delta-iodolactone is not mediated through TGF beta 1 in intact porcine thyroid follicles ex vivo. The stimulatory effect of EGF and TGF alpha on TGF beta 1 expression might be related to extracellular matrix modulation during proliferation.