An immunohistochemical investigation of the expression of somatostatin receptor subtypes - should therapeutic trials be performed to determine the efficacy of somatostatin analogs in treating advanced thyroid malignances?

UNLABELLED: Somatostatin and its analogs through the specific receptor are known to demonstrate antiproliferative, anti-angiogenic and pro-apoptotic actions. The presence of SSTR-1-5 has not been sufficiently explored in poorly differentiated and undifferentiated thyroid tumors. The aim was to investigate the SSTR subtypes expression in these aggressive thyroid tumors. The study also discusses the usefulness of SSTR analogs as an alternative to conventional forms of therapy. METHODS: The analysis was performed by immunohistochemistry on the 14 archived poorly differentiated and 4 anaplastic thyroid carcinomas. A group of benign thyroid pathologies consisting of 11 patients was also included. RESULTS: SSTR-1, 2A, 2B, 3 and 5 were found to be expressed both in benign and malignant thyroid diseases, while SSTR-4 was not. Expression of SSTR-1 and SSTR-5 was found in samples with poorly differentiated thyroid tumors with a score of at least 2.0 being recorded in 10 tumors (71.4%). For SSTR-2A the same or higher score was noted in 5/14 (35.7%), for SSTR-2B in 4/14 (28.6%) and for SSTR-3 in 3/14 (21.4%) samples. SSTR-1, 2B and 5 were found to have a score of at least 2.0 in all undifferentiated thyroid tumors. Immunostaining of SSTR-2A and 3 was observed in 50% of samples. The immunopositive reactions were observed both in the membranes and cytoplasm of the thyroid cancer' cells. In some cases positive immunostaining was localized also in the endothelium of intrathyroidal blood vessels. CONCLUSIONS: The somatostatin multiligand analogs or selective agonists could be considered alternatives to conventional therapeutic agents in aggressive thyroid tumors.

Evaluation of hyperprolactinaemia with the use of the intervals for prolactin after macroforms separation.

Macroprolactin (MaPRL) - a complex of monomeric prolactin (PRL) with immunoglobulin G, may be a cause of laboratory diagnosed hyperprolactinaemia. To quantify MaPRL, a precipitation with polyethylene glycol may be performed. This method involves calculating of recovery ratio but the cut-off value is not precisely determined. Moreover, it is proposed that the assessment of macroprolactinaemia should include also the evaluation of real PRL concentration which means the level of the hormone after macroforms separation. The study included 245 patients with hyperprolactinaemia, in whom precipitation was performed. A recovery ratio ≤40% indicated macroprolactinaemia. The real PRL concentrations of the studied subjects were compared with reference ranges suggested by the assay manufacturer and with new intervals for PRL after macroforms separation. On the base of the recovery ratio after the precipitation, macroprolactinaemia was detected in 21 persons. In these patients true hyperprolactinaemia (elevation of real PRL concentration above manufacturer's reference ranges) was noted in 9 cases. Among 224 patients with a recovery >40%, real PRL concentration turned out to be within the manufacturer's reference range (pseudohyperprolactinaemia) in 36 persons. The new intervals for PRL after macroforms separation were about 20% lower than the manufacturer's reference ranges. After applying new ranges in patients with macroprolactinaemia, true hyperprolactinaemia was observed in 14 persons, while in the group without MaPRL dominance, pseudohyperprolactinaemia was noted in 5 patients. The use of the recovery ratio only to recognize macroprolactinaemia may lead in some subjects to the misclassification of the results. For that reason the assessment of the PRL concentration after macroforms separation that can help to distinguish true hyperprolactinaemia and pseudohyperprolactinaemia, seems to be reasonable. To evaluate the real PRL concentration, the reference intervals suggested by the manufacturer of immunoassay might be used. However, possibly better means to diagnose patients with hyperprolactinaemia accurately is using an appropriate range for the concentration of PRL after macroforms separation.

Effects of somatostatin-14 and the receptor-specific somatostatin analogs on chromogranin A and alpha-subunit (alpha-SU) release from "clinically nonfunctioning" pituitary adenoma cells incubated in vitro.

The aim of the study was to examine the effect of somatostatin (SST) and its analogs on the release of chromogranin A (CgA) and alpha-subunit (alpha-SU) from clinically non-functioning pituitary adenomas incubated in vitro. Seven pituitary macroadenomas surgically removed were investigated. All of the tumors were diagnosed before surgery as non-functioning, but they expressed either gonadotropins or their subunits as detected by immunohistochemistry. Two tumors additionally expressed prolactin and growth hormone. All adenomas also expressed chromogranin A (CgA) and at least 3 of 5 subtypes of somatostatin receptors. The cells isolated from the examined tumors were exposed in vitro to either native SST-14 or the following receptor-specific SST analogs: BIM-23926 (agonist of sst1 receptor), BIM-23120 (agonist of sst2 receptor), BIM-23206 (agonist of sst5 receptor) and BIM23A387 (somatostatin/dopamine chimera). The concentration of CgA was measured by means of ELISA method and of alpha-SU was measured by an immunoradiometric method. It was found that the exposure on SST-14 resulted in the decrease of CgA and alpha-SU release from tumor cells in majority of samples, and the effect on CgA was positively correlated with the expression of sst3 and also with the sst2A/sst2B expressions ratio. The inhibitory effect of SST-14 on CgA and alpha-SU seems also to correlate negatively with the expression of sst2B. CgA inhibition also correlates positively with sst5 expression. Among the other compounds studied, only the sst2 agonist decreased the release in all the investigated samples. The remaining substances (agonists of sst1 and sst5 and SST/DA chimera) produced the divergent changes (increased or decreased release, depending on the sample). The data suggest that the inhibition of CgA (and possibly of alpha-SU) release by SST is mediated via subtypes sst2A, sst3 and sst5, whereas sst2B subtype may induce the opposite effect.

The effect of selective sst1, sst2, sst5 somatostatin receptors agonists, a somatostatin/dopamine (SST/DA) chimera and bromocriptine on the "clinically non-functioning" pituitary adenomas in vitro.

The aim of the work was to investigate the effects of somatostatin analogs acting selectively on sst1 (BIM-23926), sst2 (BIM-23120) and sst5 (BIM-23206) receptor subtypes on the viability of "clinically non-functioning" pituitary adenomas in vitro. The effects of native SST (SST-14), a SST/DA chimera (BIM-23A387) and a D(2)-dopamine receptor agonist bromocriptine (BC) were also examined. The study was performed on 10 surgically removed pituitary macroadenomas, diagnosed before surgery as "non-functioning". A part of each tumor was mechanically dispersed and digested with collagenase to isolate the tumoral cells. Another part of each tumor was fixed, embedded in paraffin and immunostained to reveal the pituitary hormones and SST receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, sst5). The tumoral cell suspensions were incubated for 24 h with the substances mentioned above. The quantity of viable cells was estimated using the EZ4U system. The results were compared with the immunohistochemical evaluation of the hormonal profile of adenoma and the sst receptor subtype immunoreactivities present. The findings indicate that selective sst1, sst2 and sst5 receptors agonists, SST/DA chimera and D(2)-dopamine receptor agonist bromocriptine affect the viability of some, but not all, "clinically non-functioning" pituitary adenomas in vitro. The most effective was bromocriptine. The investigated somatostatin analogs including SST/DA chimera exerted roughly similar inhibitory effects. Further studies are needed to fully evaluate the potential usefulness of these compounds in the pharmacological treatment of "non-functioning" pituitary tumors.

Medullary thyroid carcinoma (MTC)--clinical and molecular aspects on the basis of own experience.

In our clinic 19615 patients were operated over 25 years on for goiter. Malignant thyroid neoplasms were found in 1049 (5.3%) patients including 875 (83.4%) women and 174 (16.6%) men. Sixty two adult patients (42 women and 20 men were operated on for medullary thyroid carcinoma (MTC). Thyroid cancer was diagnosed in this group pre or intraoperatively in 44 (71%) patients and postoperatively, on histologic examination, in 18 (29%) patients. These patients were reoperated. Radical operations (total thyroidectomy with regional lymph node removal) were conducted in 43 (69.3%) patients and palliative ones in 19 (30.7%) patients. After MTC surgery, MEN 2A (MTC and an adrenal tumor) were diagnosed by means of imaging techniques (USG, CT) in 6 (9.7%) patients. All adrenal tumors were unilateral. Five of these patients were operated, and pheochromocytoma was confirmed by histopathologic examination. Two years after the MTC operation, 1 women was lost to follow-up. After a year, she was admitted to hospital for severe hypertension and died of cerebral hemorrhagia. Pheochromocytoma was revealed by autopsy. All patients were treated complementarily after the MTC operation. Different combinations of teleradiotherapy, chemotherapy and substitutive doses of levothyroxine were used. Ten (23.2%) of 43 patients operated radically were reoperated 1-3 years after the first operation due to loco-regional tumor recurrence. Radical reoperations were performed in 4 patients, and palliative ones in 6. Over a 0.5-23-year follow-up period, 26 (41.9%) patients died, including 20 of cancer, and 6 of other reasons. Four out of 36 living patients have clinical or biochemical symptoms of neoplastic disease. The follow-up period of MEN 2 patients operated on ranged from 1 to 6 years. Up to now, no tumor in the second adrenal gland has been diagnosed in any of these patients. Genetic (molecular) tests performed in 31 out of 36 living patients revealed mutations of RET gene in 4 (12.9%).

Thyroid cryotherapy in an experimental rat model-topography of temperature during therapy and functional results.

The aim of this study was to investigate post cryotherapy thyroid function status of normal rat thyroid tissue and to determine the topography of temperature of cryotreated tissues and of tissues adjacent to them. Nitrous oxide cryotherapy was performed in 40 male Wistar rats. They were divided into four groups of 10. In group I, the right thyroid lobe was subjected to cryotherapy and the left lobe was not frozen. In group II, both thyroid lobes were cryotreated. In group III, the right lobe was frozen and the left lobe was excised. In group IV, the thyroid was subjected to neither cryotherapy nor surgery. During cryotherapy, the temperature in various places of the thyroid and in the surrounding tissues was measured. Serum thyrotropin concentrations were determined before an experiment and 4 weeks after in all rats. The results of temperature measurements proved that it is possible to limit cryotherapy to certain areas of thyroid tissue and to spare the neighboring tissues, because they are not subjected to temperatures that are damaging. The effectiveness of cryotherapy was confirmed by functional effect. Cryotherapy changed function of thyroid tissue. There was a statistically significant difference between mean baseline and follow-up concentrations in rats of groups II and III. In both groups hypothyroidism occurred post cryotherapy.

Pituitary-adrenocortical responses to the chronic administration of granulocyte colony-stimulating factor in rats.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor, but it may play a role in the regulation of the neuroendocrine system activity. Only few data are available about its possible influence on the pituitary gland. We have recently reported an acute stimulatory effect of G-CSF (and of GM-CSF) on adrenocorticotropic hormone (ACTH) secretion in rats in vivo. The purpose of the present study was to evaluate whether chronic administration of G-CSF affects ACTH and corticosterone secretion and growth processes of the rat anterior pituitary gland and adrenal cortex in vivo. We have demonstrated that G-CSF (at a dose of 10.0 microg/kg body weight (BW)) injected s.c. once daily (for 7 days), stimulated both ACTH and corticosterone secretion. Simultaneously, G-CSF treatment did not change the total anterior pituitary cell proliferation as revealed by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). On the other hand, proliferative activity of corticotrophs, detected in the sections of the anterior pituitary using double-labeling. was significantly increased after treatment with G-CSF. Moreover, this growth factor induced an increase in the proliferation ratio in the entire adrenal equatorial section. These findings suggest an involvement of G-CSF in the regulation of pituitary-adrenal axis and support the hypothesis of bidirectional associations between the immune system and the endocrine glands.

Influence of granulocyte-macrophage colony stimulating factor on pituitary-adrenal axis (PAA) in rats in vivo.

We have studied the in vivo influence of granulocyte-macrophage colony stimulating factor (GM-CSF) on blood plasma concentration of adrenocorticotropic hormone (ACTH) and corticosterone in Wistar rats. The administration of 10 micrograms/kg b.w. GM-CSF at 45 (P < 0.01), 90 (P < 0.01) and at 45 (P < 0.001), 90 (P < 0.001) and 180 min (P < 0.001) increased the secretion of ACTH and corticosterone, respectively. Prolonged administration of 10 micrograms/kg b.w. of GM-CSF increased the ACTH (P < 0.001) and corticosterone (P < 0.001) concentration in blood plasma. We have also found that chronic treatment with 10 micrograms/kg b.w. of GM-CSF increased the proliferative activity of corticotrophs (P < 0.05), but it did not significantly change the total cell proliferation in the anterior pituitary gland. Moreover, this cytokine increased cell proliferation of the adrenal cortex (P < 0.001). These experiments suggest that GM-CSF activates the pituitary-adrenal axis and support the hypothesis of bidirectional associations between the immune and neuroendocrine systems.

Inhibitory effects of fumagillin and its analogue TNP-470 on the function, morphology and angiogenesis of an oestrogen-induced prolactinoma in Fischer 344 rats.

The process of angiogenesis occurs in many physiological states, but it is also essential for the growth of solid tumours and metastasis formation. An abnormal arterial vascularization has been shown in prolactin-secreting pituitary adenomas induced by prolonged treatment with oestrogens in Fischer 344 (F344) rats. It is thought that anti-angiogenic agents might be useful in therapy for these tumours. Fumagillin and its analogue TNP-470 are known to inhibit endothelial cell proliferation selectively, but their effect on lactotroph cell secretory function and prolactinoma formation has not yet been described. The aim of the present study was to examine the effects of fumagillin and TNP-470 on prolactin secretion, and morphological and vascular changes within the anterior pituitary in long-term oestrogen-treated male F344 rats in vivo and in vitro. As expected, 7 weeks after s.c. implantation of Silastic tubes containing 10 mg diethyl-stilboestrol (DES), a very high rise in serum prolactin levels was found. Both angiogenesis inhibitors injected s.c. at doses of 10 mg/kg body weight for 24 days attenuated the stimulatory effect of DES on prolactin production and release. They also diminished prolactin cell density and inhibited cell proliferation expressed as the number of anterior pituitary cells labelled with bromodeoxyuridine (BrdU), but the effect of TNP-470 was minor compared with fumagillin. Both angioinhibitors suppressed neo-vascularization within the anterior pituitary with similar potency but, on the other hand, they did not affect DES-induced increases in prolactin secretion from cultured rat pituitary cells and cell proliferation in vitro. In conclusion, our results provide strong evidence for the anti-tumour and anti-prolactin activity of angiogenesis inhibitors in the experimentally oestrogen-induced pituitary adenoma; this might be mediated indirectly through the inhibition of angiogenesis.

[Calcitonin level in cerebrospinal fluid of patients with amyotrophic lateral sclerosis]. / Stezenie kalcytoniny w plynie mózgowo-rdzeniowym chorych na stwardnienie zanikowe boczne.

Several data indicate that the dysfunction of some neuropeptide function may play a role in the pathogenesis of the amyotrophic lateral sclerosis. Therefore, the authors decide to determine a concentration of one of them in CSF, namely calcitonin. Calcitonin is widely distributed in CNS, including both anterior and posterior horns of the spinal cord. It was confirmed with immunohistochemical assays and an examination of the human CSF. Calcitonin concentration in CSF has been assayed in 12 patients with amyotrophic lateral sclerosis and in 12 patients of the control group. Calcitonin concentrations in CSF have been measured with RIA technique, using appropriate kits manufactured by Mallinckrodt Dgn. Mean calcitonin CSF concentration in patients with amyotrophic lateral sclerosis was 448. +/- 74.3 pg/ml, and was lowered in comparison with that in the control group, i.e. 613.9 +/- 147.2 pg/ml. The results confirm the authors' previous reports on the reduced content of some neuropeptides in CSF of patients with amyotrophic lateral sclerosis and suggest a possible calcitonin role in the pathogenesis of this disease.

Effects of omeprazole on the number of immunoreactive gastrin- and somatostatin-cells in the rat gastric mucosa.

The effects of omeprazole--an inhibitor of gastric acid secretion--on gastrin (G)- and somatostatin (D)-cell density in the gastric antral mucosa epithelium in rats were examined, following a 5-day treatment. It was found that omeprazole increased the density of G-cells, whereas it decreased the density of D-cells. That effect was probably independent of hypergastrinaemia, since it could not be blocked by a simultaneous treatment with proglumide--a gastrin receptor blocker. It is concluded that the observed phenomenon is a direct result of a lower gastric acidity, as a consequence of omeprazole treatment.

Effect of mesulergine on prolactin secretion and dopamine D2 receptors-adaptive changes in diethylstilbestrol-induced hyperplasia of the rat anterior pituitary.

Mesulergine (N,N-dimethylsulphamide-N'-1,6-dimethyl-ergoline-8 alpha-yl) is an active semisynthetic ergot derivative with lower antiprolactin potency compared with bromocriptine or pergolide. Since no data are yet available on the effects of mesulergine on pituitary dopamine receptors, the present study has been designated to elucidate the influence of this drug on prolactin secretion in vivo and in vitro and 3H-spiperone binding by the anterior pituitary gland in female Wistar rats with experimentally induced hyperprolactinemia. Three weeks after bilateral ovariectomy and subcutaneous implantation of silastic tubes, containing 10 mg of diethylstilbestrol, a dramatic rise in serum prolactin levels was observed (1.67 +/- 0.23 vs. 80.82 +/- 3.80 ng/ml; P less than 0.001). Mesulergine attenuated the stimulatory effect of diethylstilbestrol on serum prolactin level in a time- and dose-dependent fashion. At concentration range between 10(-5) and 10(-7) M it also inhibited prolactin secretion from cultured rat pituitary cells to the medium during 180 min incubation in a dose-dependent manner. Scatchard analyses performed on the in vitro 3H-spiperone binding kinetics in a dispersed anterior pituitary cell culture, prepared from the pituitaries from rats treated for four weeks with diethylstilbestrol, showed that chronic mesulergine treatment (in dose of 3.0 mg/kg injected s.c. for 10 days) induced a significant decrease in the number of dopamine D2-binding sites (Bmax 28.00 +/- 4.20 vs. 42.80 +/- 4.76 fmol/10(6) cells; P less than 0.01) without any changes in D2-receptor affinity. Our results suggested that antiprolactin activity of mesulergine in vivo and in vitro is probably associated with agonistic effect of this drug on D2-dopamine receptors.

The effect of mesulergine on prolactin secretion and anterior pituitary cells morphology in diethylstilboestrol-treated female Wistar rats.

Mesulergine (Cu32-085) is an active semisynthetic ergot alkaloid with unusual biphasic antagonistic-agonistic effect on dopamine (DA) turnover in the rat striatum. The present study has been made to elucidate the influence of the long-term treatment of this drug on prolactin secretion and prolactin cells morphology in the female Wistar rats with experimentally-induced hyperprolactinemia. Additionally, the effect of this drug was compared with bromocriptine and pergolide activity, applied in the same experimental conditions. It has been shown that prolonged mesulergine treatment attenuated the stimulatory effect of stilboestrol on prolactin secretion in vivo. It also decreased mean prolactin cells density, above all cells and lactotroph mitotic indexes, estimated in immunohistochemically-stained slides. However, antiproliferative activity of Cu 32-085 was weaker, when compared with bromocriptine and pergolide.

Effect of amantadine on prolactin secretion, pituitary DNA synthesis and 3H-spiperone binding in male estrogen-treated rats.

Amantadine, a well-known antiviral agent, causing an increase in dopamine synthesis, release and the inhibition of re-uptake of noradrenaline and dopamine in central and peripheral catecholaminergic neurons, is successfully used in the treatment of Parkinson's disease. In the present paper, we have studied the effect of various doses of amantadine on in vivo prolactin secretion and the incorporation of 3H-thymidine and 3H-spiperone binding by the anterior pituitary gland of long-term diethylstilboestrol-treated male Wistar rats. Four weeks after a subcutaneous implantation of Silastic tubes containing 10 mg of diethylstilboestrol, a dramatic rise in serum prolactin levels was observed, accompanied by an increased uptake of 3H-thymidine by DNA anterior pituitary cells. Amantadine, given in the subcutaneous doses of 50, 5 and 0.5 mg/kg of body weight attenuated the stimulatory effect of stilboestrol on serum prolactin concentration in a dose-dependent fashion. On the other hand, the incorporation of 3H-thymidine into DNA pituitary cells in all the groups of amantadine-treated rats was only slightly suppressed. In an additional experiment, Scatchard analyses were performed on the in vitro 3H-spiperone binding kinetics in a dispersed anterior pituitary cell culture prepared from the pituitaries of 6-week diethylstilboestrol-treated rats. It has been found that amantadine injected in the dose of 5 mg/kg of body weight for 14 days induced a twofold decrease in the density of dopamine D2 binding sites (36.6 +/- 9.4 vs. 70.3 +/- 3.4 fmol/10(6) cells; p less than 0.02), while the apparent affinity of the receptors was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)