Thyrocyte migration and histiotypic follicle regeneration are promoted by epidermal growth factor in primary culture of thyroid follicles in collagen gel.

The effect of epidermal growth factor (EGF) on the morphology of porcine thyroid follicles cultured in collagen gel was studied by light and electron microscopy. Treatment with EGF (10 ng/ml) for 1-4 days induced a progressive migration of thyrocytes radiating from the mother follicles into the collagen lattice. Migrating cells were often connected with junctional complexes, thus forming small follicles containing microlumina, in which the limiting portion of the plasma membrane expressed microvilli. Autoradiography of [3H]thymidine incorporation in EGF-treated cultures showed that more than 80% of nuclei in mother follicles and migrating cells were labeled after 4 days. TSH (2 mU/ml) given simultaneously with EGF did not influence the effect of EGF on multiplication, migration, and formation of microfollicles. TSH given for 12 h after 4 days of exposure to EGF was able to induce luminal dilation of mother follicles, but caused no change in the appearance of microlumina. It is concluded that thyrocytes in collagen gel retain epithelial characteristics during prolonged stimulation with EGF, in spite of a migrating response. The whole sequence of events, starting with the intact follicle, i.e. multiplication, migration of thyrocytes, and formation of new follicles, could, thus, be visualized to occur in response to a single mitogen, EGF. EGF might be involved in the generation of new follicles in the intact gland.

Epidermal growth factor modulates thyroid growth and function in culture.

The effect of epidermal growth factor (EGF) on the growth and function of porcine thyroid follicle suspension cultures was examined. When incubated with EGF (0.01-10 ng/ml), the incorporation of [3H]thymidine was markedly stimulated, with the half-maximal effect occurring at about 0.5 ng/ml. Through 6-day experiments, [3H]thymidine incorporation increased almost linearly after an initial lag phase of 1 day. Autoradiographical analysis showed that more than 50% of the nuclei were labeled after 4 days of incubation with EGF (10 ng/ml) compared to control values of 3-7% labeled nuclei. EGF was essentially as effective in serum-free medium as in medium containing 1% fetal calf serum. EGF added at the same concentrations that stimulated incorporation of [3H]thymidine was found to reduce iodide metabolism of the follicles within 30 min of addition; both TSH-stimulated efflux and organification of [125I]iodide were suppressed by the addition of EGF. Since EGF is a physiologically occurring substance, the data suggest that EGF might be a modulator of thyroid growth and function in vivo.

Location of epidermal growth factor receptors on porcine thyroid follicle cells and receptor regulation by thyrotropin.

By using a direct electron microscopic autoradiographical technique, we were able to demonstrate that the epidermal growth factor (EGF) receptor is asymmetrically located on polarized porcine thyroid follicle cells cultured on a collagen gel matrix. More than 80% of autoradiographical grains from [125I]EGF were associated with the basolateral cell surface compared to only about 2.5% at the apical cell margin. EGF stimulated growth only when the collagen gels were floating, indicating a basolateral location of functional EGF receptors. Preincubation with TSH or another stimulator of cAMP synthesis, forskolin, increased binding of [125I]EGF to the cells. Moreover, the subsequent mitogenic response to EGF was potentiated by such pretreatment. The results are in accordance with the hypothesis that TSH potentiates the response to EGF in the stimulation of thyroid growth.

A somatic point mutation in a putative ligand binding domain of the TSH receptor in a patient with autoimmune hyperthyroidism.

Nucleotide sequence analysis of PCR fragments of TSH receptor cDNA synthesized from thyroid RNA of a patient with autoimmune hyperthyroidism, revealed two different sequences in the first position of codon 36. In one of the sequences, there was a C for G substitution leading to the D36----H substitution in the predicted peptide. Both variants were also found in genomic DNA of thyroid tissue. However, only the germ line sequence was found in other tissues representing all three different germ layers. The novel sequence is therefore likely to represent a somatic mutation in the thyroid tissue, of possible relevance for the pathogenesis of the patient's thyroid disorder.

Determination of the frequencies of ten allelic variants of the Wilson disease gene (ATP7B), in pooled DNA samples.

Wilson disease is an autosomal recessive disorder characterised by toxic accumulation of copper in liver, brain and other organs. The disorder is caused by mutations in the ATP7B gene, encoding a copper transporting P-type ATPase. Based on the number of known patients with this diagnosis in Sweden, the prevalence can be estimated to 1 in 250,000 to 300,000, whereas the prevalence of Wilson disease has been estimated to be 1 in 30,000 in other populations. We estimated the prevalence of Wilson disease by determining the Swedish population frequencies of two mutant alleles, making up approximately half the mutations in Swedish Wilson patients, in a large number of DNA samples. In addition we determined the allele frequencies of eight common single-nucleotide polymorphisms (SNPs) in the ATP7B gene. For the analyses we devised two strategies for analysing pooled DNA samples using the quantitative minisequencing method. The two procedures allowed sensitive identification of rare mutant alleles present as a mixture with an excess of the normal allele, as well as accurate estimation of the frequencies of the common SNPs in a large pooled DNA sample.

Thyrotropin modulates EGF receptor function in porcine thyroid follicle cells.

Porcine thyroid follicle cells in monolayer cultures were shown to contain one single class of high-affinity EGF receptors with Kd = 4.5 X 10(-10) M and approximately 20 000-25 000 receptors per cell. Suspension cultures of aggregated follicle cells, exposed to TSH for 3 days, showed a 3-fold increase in [125I]EGF binding. Scatchard analysis demonstrated that this was due to an increase in receptor number. Other cAMP-elevating agents (cholera toxin, dibutyryl cAMP, forskolin) induced a similar effect. In suspension cultures, preincubation with TSH or cholera toxin for 2 days reduced the subsequent [3H]thymidine incorporation. This inhibition was overcome by a low concentration of EGF (0.1 ng/ml). At higher concentrations of EGF (1-10 ng/ml) the incorporation of [3H]thymidine was potentiated 2-3-fold in cultures preexposed to TSH or cholera toxin. The results demonstrate the presence of a high-affinity EGF receptor in porcine thyroid follicle cells. Receptor expression, as well as responsiveness to the mitogenic action of EGF, is modulated in vitro by TSH, through a cAMP-dependent process.

Functional properties of porcine-thyroid follicles in suspension.

Porcine thyroid follicle cells were isolated (about 10(7) cells per gram of tissue) and cultured in small aggregates in agarose-coated culture dishes. The aggregates became arranged into follicle-like structures capable of iodide uptake and organification. In the presence of TSH (0.2 mU/ml), the aggregation of follicles was enhanced, and iodide uptake as well as TSH-stimulated organification of iodide was increased compared with that in the control. In culture, the active iodide metabolism was gradually lost over a 7-day period. This was not due to a disappearance of the TSH-adenylate cyclase system, since cAMP production was retained and stimulated by TSH (half-maximal effect at about 1 mU/ml). Acutely TSH stimulated iodide efflux and iodide organification (half-maximal effect at about 20 microU/ml). The stimulatory effect on organification was transient: within an hour further organification proceeded as in the absence of hormone. The effects on efflux and organification were already maximal at low TSH concentrations, whereas cAMP production was stimulated with up to 50-fold higher TSH levels, i.e. the findings were typical of spare receptors. In the continued presence of epidermal growth factor, a potent mitogen for thyroid cells, the follicles increased in size and contained one single large lumen. Their capability to take up and organify iodide was reduced.

Functional analysis of a variant of the thyrotropin receptor gene in a family with Graves' disease.

Nucleotide sequence analysis of PCR fragments corresponding to the TSH-receptor (TSHR) amplified from genomic DNA collected from the four members of a family, two of which had Graves' thyrotoxicosis, revealed a nucleotide substitution in the first position of codon 36 of the TSH-receptor gene in the two patients. The nucleotide substitution was from G to C, leading to a 36D-->36H change (D36H) in the predicted amino acid sequence of the receptor. The altered sequence was also found in DNA obtained from their mother, but not in DNA from their father. We stably expressed the two receptor variants in NIH 3T3 cells, by transfection of cDNA encoding the wildtype (WT) and D36H variants of the TSHR. Neither the binding of 125I-TSH nor the responsiveness to TSH measured as cAMP formation, appeared to be different in the TSHR-D36H compared to the TSHR-WT. Furthermore, the D36H-receptor also became desensitized when exposed to TSH as did the WT-receptor.

Integrins in thyroid tissue: upregulation of alpha2beta1 in anaplastic thyroid carcinoma.

OBJECTIVE: To evaluate the integrin pattern in the normal thyroid gland and in different pathological disorders including malignant tumors, because the aggressiveness of several malignant tumors correlates with alterations in the expression of one or more integrins. DESIGN: We examined the expression of integrins and E-cadherin immunohistochemically in a large and well-defined sample of normal and pathological human thyroid tissue. METHODS: Cryosections of 58 thyroid tissue specimens from normal tissue, thyrotoxicosis, nodular goiter, oxyphilic adenoma, follicular adenoma, follicular carcinoma, papillary carcinoma and anaplastic carcinoma, and three lymph node metastases were investigated immunohistochemically using monoclonal antibodies specifically recognizing the integrin beta1-, beta4-, alpha1-, alpha2-, alpha3-, alpha5- and alpha6-subunits, or E-cadherin. RESULTS: All thyroid epithelial cells expressed integrin beta1- and alpha3-subunits. Immunostaining of the beta4-subunit and the alpha6-subunits was found only in tumors. The staining pattern in the three lymph node metastases from papillary carcinomas did not differ from that in their primaries. Anaplastic carcinomas demonstrated neoexpression of the integrin alpha2-subunit. E-cadherin was detected in all tissues except anaplastic carcinomas. CONCLUSIONS: Neoexpression of alpha6beta4 was seen in most malignant tumors, whereas alpha2 was exclusively found in anaplastic carcinomas. In the latter, a loss of E-cadherin expression was also seen. These changes in cell adhesion molecule expression strongly suggest an association with the acquisition of proliferative and invasive properties.

Retrospective evaluation of subtotal and total thyroidectomy in Graves' disease with and without endocrine ophthalmopathy.

A retrospective analysis was performed in 173 consecutive patients with Graves' disease (GD) with the principal aim of evaluating the influences of subtotal (N = 157) and total (N = 19) thyroidectomy on postoperative recurrence rates, endocrine ophthalmopathy (EO) and thyrotropin receptor antibody (TSH-R-ab) titres. Postoperatively recurrent disease, identified by increased thyroid hormone levels, occurred in 32 patients (20%) who underwent subtotal resection. These recurrences were associated with over-representation of preoperative EO (p < 0.001) as well as high TSH-R-ab levels postoperatively (p < 0.05-0.01). Subtotal and total resections were followed by an aggravation of preoperative EO in nine (16%) and one (6%), and by a development of EO in two and none of the patients, respectively. Persistently elevated TSH-R-ab titers during thyrostatic therapy became close to normalized in seven (32%) and 15 (88%) of the patients undergoing subtotal or total thyroidectomies, respectively, which illustrates a thyroid tissue dependency of the autoantibody production. Among the total material of 173 patients, altogether 75 cases exhibited persistent or progressive EO and/or TSH-R-ab elevation after more than 1 year of preoperative thyrostatic treatment. In this group, recurrent GD or aggravated EO occurred in 23 (39%) of those operated with subtotal resection and in one (6%) of those undergoing total thyroidectomy (p < 0.05). The results thus indicate that EO, particularly at the time of surgery, and prevailing TSH-R-ab titers are associated with an increased risk of recurrent GD and suggest that patients exhibiting these characteristics should benefit from total rather than subtotal thyroidectomy.

Total thyroidectomy in therapy-resistant Graves' disease.

BACKGROUND: Influences of total thyroidectomy have not been evaluated in patients with severe Graves' disease who might respond less satisfactorily to subtotal thyroid resection. METHODS: Thirty-three patients with Graves' disease underwent total thyroidectomy because of persistent endocrine ophthalmopathy (n = 28) or elevated thyrotrophin receptor antibody titers (n = 25) despite a mean of 2 years of thyrostatic therapy. Moreover, six and four patients had undergone radioiodine treatment and subtotal thyroid resection, respectively. Perioperative findings and complications have been investigated, as have influences on endocrine ophthalmopathy and thyrotrophin receptor antibody titers during a mean of 2.5 postoperative years. RESULTS: Total thyroidectomy substantiated mean thyroid weights of 17 gm, 2.3 hours of operating time, and total blood loss of 264 cc. Vocal cord paralysis and vitamin D-treated hypocalcemia occurred in two and three patients, respectively, and invariably persisted less than 6 months. Normalization of elevated thyrotrophin receptor antibody titers occurred in 86% of patients without radioiodine exposure, and stable or improved signs of endocrine ophthalmopathy were found in 96% of patients examined 6 or more months after the operation. CONCLUSIONS: Total thyroidectomy seems to be a surgically safe procedure in complicated Graves' disease and to provide normalization of therapy-resistant thyrotrophin receptor antibody titers. Because favorable influences might also encompass severe endocrine ophthalmopathy, prospective analysis on its efficiency is warranted.

Abdominal malignancies in patients with Wilson's disease.

BACKGROUND: Wilson's disease is associated with heavy copper overload, primarily in the liver. Copper is a toxic metal, and might be expected to be associated with cancer induction, as iron is in haemochromatosis. However, liver cancer is currently believed to be extremely rare in this disease, and other intra-abdominal malignancies have not been reported. AIM: To assess the frequency of abdominal malignant disease in patients with Wilson's disease on long-term follow-up. DESIGN: Retrospective study in two specialist Wilson's disease clinics: Cambridge/London and Uppsala. METHODS: We reviewed the case records of 363 patients seen at three centres: Addenbrooke's Hospital, Cambridge, 1955-1987; the Middlesex Hospital, London, 1987-2000; and the University Hospital, Uppsala, Sweden, 1966-2002. Patients were grouped by length of follow-up: 10-19 years; 20-29 years; 30-39 years; and 40 years or more. RESULTS: No cancers were seen in patients followed for <10 years. For patients in the 10-19 years group, the frequency was 4.2%; at 20-29 years, it was 5.3%; and at 30-39 years, 15%. No cancers were seen in the 40+ years follow-up group. The cancers consisted of hepatomas, cholangiocarcinomas, and poorly differentiated adenocarcinomas of undetermined primary site. DISCUSSION: Patients with Wilson's disease appear to be vulnerable to the formation of aggressive malignant intra-abdominal tumours during long-term follow-up, irrespective of treatment. Ultrasound scanning of the abdomen seems to be a useful screening procedure.

Long-term treatment of Wilson's disease with triethylene tetramine dihydrochloride (trientine).

Long-term treatment with triethylene tetramine dihydrochloride, (trientine, TETA) was evaluated in 19 patients with Wilson's disease (WD). Two were given the drug as first choice and 17 after treatment with penicillamine. The change was made because of side-effects, lack of improvement or worsening of neurological symptoms. All penicillamine-induced side-effects reverted. Thirteen patients still receive trientine, and the mean total observation time on this treatment is 8.5 years/patient. Seven of the 13 are free from symptoms related to WD, five have mild to moderate neurological symptoms, mainly dysarthria. One patient with neurological symptoms who received trientine from the start of treatment deteriorated rapidly and is now severely dystonic. The symptoms initially worsened and later improved in one patient. All other patients improved during trientine treatment. Three patients died: two from a multifocal cancer including the liver and one non-complier from a ruptured spleen. Two patients underwent liver transplantation for progressive liver failure: one non-complier and one with liver cirrhosis whose liver function deteriorated despite treatment; both are now free from symptoms. Unexpectedly, two patients developed a serious colitis, one with duodenitis as well, that improved after withdrawal of the drug. No other unfavourable effects of trientine were recorded.

Analysis of mutations in exon 1 of the human thyrotropin receptor gene: high frequency of the D36H and P52T polymorphic variants.

The aim of the present study was to investigate the N-terminal part (the translated part of exon 1) of the human thyrotropin receptor (TSHR) for the presence of mutations. Patients with Graves' disease (n = 160) and healthy controls (blood donors; n = 140) were screened using single-stranded conformational polymorphism (SSCP) in combination with restriction enzyme digestion for the two previously known mutations in this part of the receptor, viz. D36H and P52T TSHR-variants. We did not find any novel mutation in this region. However, D36H and P52T variants were found both in the TSHR of Graves' patients and in the healthy controls. The overall frequency of the D36H-receptor variant was 5.0% (15/300) and of the P52T-receptor, 7.3% (22/300). There was no major difference in the frequency for either of the TSHR alleles between the 2 groups. Thus, these 2 polymorphic variants of the TSHR seem to occur in a relatively high frequency in the population.

Personality traits in treated Wilson's disease determined by means of the Karolinska Scales of Personality (KSP).

OBJECTIVE: The aim was to elucidate the personality traits of patients with treated Wilsons disease (WD) in comparison to healthy volunteers. METHOD: Twenty-five WD patients, ten females and 15 males, with a mean age of 35.2 +/- 8.3 years completed the Karolinska Scales of Personality (KSP), a self-report inventory comprising 15 separate scales. The results were compared to a control series comprising 200 men and 200 women drawn from the general population. RESULTS: The patients with treated WD scored significantly lower than the healthy controls on aggressivity-hostility-related scales and the scale measuring Psychic Anxiety. Patients with predominantly hepatic symptoms had the lowest aggressivity-related scores and patients with predominantly neurological symptoms had the lowest Irritability, Guilt and Detachment scores and the highest Impulsiveness and Muscular Tension scores. Both groups scored low on the Somatic Anxiety scale. CONCLUSION: The present results illustrate that patients with treated WD have significant deviations in personality traits, especially in aggressivity-hostility-related scales and Psychic Anxiety, compared to healthy controls when investigated by means of a self-report inventory, the KSP. The deviations were not related to age, age at onset or duration of the disease.

Baroreflex-governed sympathetic outflow to muscle vasculature is increased in hypothyroidism.

Microelectrode recording of muscle nerve sympathetic activity (MSA), which is baroreflex-governed and involved in cardiovascular homeostasis, was made in five patients with hypothyroidism and in seven patients with hyperthyroidism prior to treatment and after recovery to euthyroidism. Patients with hypothyroidism had a higher level of MSA when hypothyroid than after recovery (mean +/- SEM 39.3 +/- 7.1 and 26.8 +/- 7.9 bursts/min, respectively, P less than 0.05), whereas hyperthyroidism was not accompanied by a change in the number of sympathetic bursts/min. The response of MSA to manouvres known to influence the neural outflow via baroreceptors and other receptor inputs was not changed with altered thyroid function. The findings provide direct evidence of an increased sympathetic activity in hypothyroidism, but suggest that other physiological properties of MSA are intact in thyroid disease.

Epidermal growth factor stimulates thyroid follicle neogenesis in collagen gel culture.

The mechanism involved in the neoformation of thyroid follicles is poorly understood. In the present study, whole porcine thyroid follicles were cultured as "miniorgans", embedded within collagen gels. Incubation was performed up to 4 days with or without EGF (10 ng/ml) and TSH (2 mU/ml). A single dose of 3H-thymidine was added at the start of the experiments in some cases. Light microscopy and autoradiography was performed. EGF induced a dramatic migration of follicles cells; these were seen to back out from the mother follicle and formed microfollicles with normal polarity including microvilli at the apical border. As evident from the analysis of 3H-thymidine incorporation, most microfollicles were comprised of newly divided cells. The results infer a new mechanism for the neoformation of follicles in the thyroid.