Framework conditions facilitating paediatric clinical research.

The use of unlicensed and "off-label" medicines in children is widespread. Between 50-80% of the medicines currently administered to children have neither been tested nor authorized for their use in the paediatric population which represents approximately 25% of the whole European population. On 26 January 2007, entered into force the European Regulation of Paediatric Medicines.

Characterization of human pituitary adenomas in cell cultures by light and electron microscopic morphology and immunolabeling.

The morphology and hormone production of pituitary adenoma cell cultures were compared in order to highlight their characteristic in vitro features. Cell suspensions were prepared from 494 surgical specimens. The 319 viable monolayer cultures were analyzed in detail by light microscopy and immunocytochemistry within two weeks of cultivation.

Effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) release and cell morphology in human pituitary adenoma cell cultures.

Six GH adenomas and three prolactinomas were investigated by light- and electron-microscopic morphological and immunocytochemical methods and the effect of vasoactive intestinal polypeptide (VIP) on growth hormone (GH) and prolactin (PRL) secretion was tested in vitro. The tumour cells of the acromegalic patients revealed both GH and PRL immunoreactivity while prolactinomas showed only PRL activity. All the adenomas stained immunocytochemically also for VIP.

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering.

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration.

Transformation of a human mixed GH-PRL adenoma cell population in response to long-term bromocriptine treatment.

A child was operated 3 times because of a recurrent growth hormone- and prolactin-producing pituitary adenoma. Between the operations she was treated for five years with bromocriptine. The characteristics of the tumour cell population collected after the last operation was now examined by electron microscopy, immunocytochemistry, and in tissue culture and compared to those of the primary tumour cells reported earlier.

Heterogeneous distribution of ecto-Ca-ATPases in primary cultures of human adenohypophyseal cells.

The aim of this study was to investigate the localization of ecto-Ca-adenosine-triphosphatase (ecto-Ca-ATPase) in different parenchymal cells of the human pituitary in tissue culture. The distribution of ecto-ATPases on the surface membrane of a particular parenchymal cell varied with the type of cells in contact with this parenchymal cell; the membrane portions immediately exposed to the medium showed low if any ecto-ATPase activity. These results suggest that ecto-Ca-ATPases of the parenchymal cells may be involved in cell adhesion processes and may be of crucial importance in the organization (in vivo) and reorganization (in vitro) of human adenohypophyseal tissue.

Ecto-ATPases and 5'-nucleotidases in the caveolae of smooth muscle. Enzyme-histochemical evidence may indicate a role for caveolae in neurotransmission.

We have demonstrated the localization of ecto-Ca-ATPase and 5'-nucleotidase activity in the caveolae of smooth muscle cells of guinea pig was deferens and the ileum longitudinal muscle strips with a cerium-precipitation enzyme-cytochemical method. The activities seemed to be strongest in the caveolae. Since the simultaneous presence of the 5'-nucleotidase activity supports the hypothesis that this ecto-Ca-ATPase activity does not have a pump function, but, together with 5'-nucleotidase, may play a role in neurotransmission, these specific membrane invaginations, the caveolae, have a functional relationship with transverse tubules of striated muscle.

Presynaptic ecto- and postsynaptic endo-calcium-adenosine-triphosphatases in synaptosomes: doubts about biochemical interpretation of localization.

Ecto- and endo-Ca-adenosine-triphosphatase (ATPase) activity was identified as electron-dense lead or cerium phosphate precipitate in the rat cortical synaptosomes by transmission electron microscopy and enzyme histochemistry. The formation of the deposit was dependent on the presence of ATP (the substrate), Ca (activator) and levamisole, quercetin or ouabain (inhibitors of different phosphatases and ATPases). Reaction products were found at the external surface of the presynaptic membrane, both surfaces of the postsynaptic membrane, in the synaptic cleft and in the free mitochondrial membranes.

Pathological and experimental investigations in a case of gigantism.

A pituitary adenoma was transsphenoidally removed from a 4.5-year-old girl suffering from gigantism. Prior to the operation both the growth hormone (GH) and the prolactin (PRL) levels in the serum were elevated.

Morphological and functional characterization of cell cultures from adult human adenohypophyses.

Parallel primary cultures have been prepared from dispersed cells of adult human pituitary anterior lobes and can be used as a model system for cell-biological studies. The cultured cells were able to secrete all the known hormones of the adenohypophysis. Bromocriptine administration markedly reduced prolactin release into the medium while slightly enhanced growth hormone release.

On the examination of the pulmonary toxicity of mordenite in rats.

Mordenite, a fibrous-granular type of zeolites, was examined in CFY rats in long term in vivo experiments. After single intratracheal treatment, the lungs, cervical and hilar lymph nodes of the animals were processed at the end of the 1st, 3rd and 6th month and also the 1st year by routine histology, enzyme histochemistry and electron microscopy. After observing the effect of mordenite, dust-storing macrophage foci developed in the interstitium, showing minimal fibrotic tendency by the end of the 1st year.

Specificity of the metal-salt method for the localization of Ca2(+)-ATPase activity studied in rat adenohypophysis tissue in a model system of polyacrylamide gel films.

The histochemical method using the lead-salt technique for the detection of Ca2+)-ATPase activity has been studied quantitatively in rat adenohypophyseal cell homogenates incorporated in polyacrylamide gel films using cytochemical conditions which were applied for the ultrastructural localization of this enzyme (El-sherif and Bácsy, 1988, 1989). Polyacrylamide gel films including cell homogenates were fixed in the presence or absence of calcium chloride, incubated for Ca2(+)-ATPase activity, and the mean integrated absorbance was determined. Omission of Ca2+ or levamisole from the incubation medium, as well as substitution of ATP by beta-glycerophosphate resulted in significantly decreased activity.

Ultrastructural study of the Ca2+-dependent ATPase activity in rat adenohypophyseal cells.

Ca-dependent ATPase activity in the rat anterior pituitary was demonstrated in 50-microns tissue slices of aldehyde-fixed tissue with the medium of Takano et al. (Cell Tissue Res. 243:91.

Cationic ferritin uptake by cultured anterior pituitary cells treated with the proteinase inhibitor, BOC-DPhe-Phe-Lys-H.

Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, BOC-DPhe-Phe-Lys-H. The addition of this tripeptide aldehyde decreased the in vitro release of prolactin to 25% of the control value, while the release of growth hormone in the same cultures decreased to 33% of the control value. Prolactin immunostaining was stronger in semithin sections of proteinase-inhibitor-treated cultures than in control sections.

Proteinase inhibitors while influencing hormone release do not affect cell morphology of hypophyseal cultures.

Cultured cells from adult rat anterior pituitaries and intermediate lobes were treated with proteinase inhibitor substrate analogues (Boc-DPhe-Pro-Arginal [BOC-DPPA], DPhe-Pro-Arginal [DPPA], BOC-DPhe-Leu-Lysinal [BOC-DPLL], BOC-DPhe-Phe-Lysinal [BOC-DPPL]) to elucidate their effect on cell morphology. It was established that BOC-DPPA and DPPA (which in previous studies stimulated alpha-MSH release [6]) caused a slight decrease in the number of immunoreactive secretory granules in melanotrophs. BOC-DPLL, which inhibited growth hormone and prolactin release, did not alter the fine structural features of cultured cells.

Morphological evidence of lysosomal uptake of high-density lipoproteins by rat adrenocortical cells in vitro.

The high-density lipoprotein (HDL) pathway in rat adrenocortical cells was studied at the electron microscopic level in vitro via colloidal gold labelling. Steroid hormone assays were performed to confirm that the cells remained intact, viable, responsive to ACTH under the applied conditions, and to reveal the steroidogenic effect of HDL. The gold-labelled HDL particles (HDL-Au) were observed on the surface of the parenchymal cells, often attached to the membranes of the microvilli, but rarely in coated pits and coated vesicles.

[Lysosomal activity in prolactin cells under the effect of a dopamine agonist].

The influence of bromoergocryptine, a dopamine agonist used in clinical praxis, on hormone degradation by the lysosomal system was studied in the pituitary mammotrophs of female rats. Immunocytochemistry of prolactin (protein A-gold technique) combined with the electron-microscopic visualization of the lysosomal system by its non-specific esterase activity (gold thiol-acetic acid method) revealed that bromoergocryptine administration increases the spatial extent of both the lysosomal and the hormone storage compartments with a considerable overlap and interaction between them. This change in compartmentalization may be effective in eliminating hormone excess and preserving cell integrity after release inhibition.

Dissimilar responsiveness of cultured corticotrophs and melanotrophs to tripeptide aldehydes.

Cultured cells from adult rat anterior pituitaries or intermediate lobes were treated with the proteinase inhibitor tripeptide aldehydes BOC-DPhe-Pro-Arg-H (Boc-fPRH) and DPhe-Pro-Arg-H (fPRH), ovine corticotropin-releasing factor (oCRF), and bromocriptine. One millimolar fPRH stimulated basal, and slightly enhanced oCRF-induced ACTH release by melanotrophs in short-term experiments. The basal release of alpha-MSH was also stimulated by the drug.

A glutamate-sensitive neuronal system originating from the area postrema terminates in and transports acetylcholinesterase to the nucleus of the solitary tract.

The possible cellular mechanism of action of systemically administered monosodium-L-glutamate and the projections of glutamate-sensitive area postrema neurons have been studied in rats. Parenteral administration of monosodium-L-glutamate induced a selective degeneration of a particular population of AChE-containing area postrema neurons. Electron microscopic cytochemistry and X-ray microanalysis revealed the presence of calcium-containing electron-dense deposits in the mitochondria of degenerating area postrema neurons indicating the possible pathogenetic role of an enhanced intracellular calcium level in the mechanism of monosodium-L-glutamate-induced nerve cell degeneration.

DNA synthesis in lactotrophs of primary rat anterior pituitary cell cultures. Effects of thyroliberin, bromocriptine and somatostatin.

Prolactin immunostaining in combination with thymidine autoradiography was used to characterize changes in the DNA-synthesizing activity of lactotrophs in primary monolayer cultures of the rat anterior pituitary gland treated for 3 days with thyroliberin (TRH), somatostatin (SRIF) and bromocriptine (CB 154). The number of lactotrophs labelled with 3H-thymidine within the total pool of labeled pituitary cells was used to estimate DNA synthesis in prolactin-producing cells. TRH (10 ng/ml) stimulated DNA synthesis in the whole population of cultured cells but not in lactotrophs.

Neurotoxin induced nerve cell degeneration: possible involvement of calcium.

Neurotoxin induced nerve cell degeneration has been studied in sensory ganglia of newborn and in the area postrema of adult rats following the administration of the selective sensory neurotoxin, capsaicin and the amino acid excitotoxin, glutamic acid, respectively. Light microscopic histochemical, autoradiographic, electroncytochemical and X-ray microanalytical studies revealed that degeneration of certain small-sized, type B primary sensory neurons, induced by capsaicin, was associated with a marked accumulation of calcium predominantly in mitochondria of the damaged ganglion cells. Similarly, monosodium glutamate treatment resulted in the appearance of calcium-containing electron-dense granules in mitochondria of degenerating area postrema neurons.

Inhibition of growth hormone and prolactin secretion by a serine proteinase inhibitor.

The action of the tripeptide aldehyde t-butyloxycarbonyl-DPhe-Pro-Arg-H (boc-fPR-H), belonging to a family of serine proteinase inhibitors, on the release of immunoreactive prolactin (iPRL) and growth hormone (iGH) has been studied. In rat anterior pituitary cell cultures and pituitary quarters 1 mM boc-fPR-H inhibited basal iPRL and iGH release. Thyroliberin-induced iPRL release by cultured cells was also markedly inhibited with a concomitant accumulation of intra-cellular iPRL.

Lysosomal enzyme activities in hypo- and hypersecretory anterior pituitary cells. A combined immunocytochemical and enzyme cytochemical study.

The involvement of lysosomes in ACTH and prolactin secretion was studied. Lysosomes were visualized in the anterior pituitary by their non-specific esterase (gold thioacetic acid technique) or acid phosphatase (Gomori technique) activity. Corticotrophs and mammotrophs were identified by postembedding immunocytochemistry for their respective hormones.