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Only recently, available microarray methods have been extended to the investigation of such complex pathophysiological conditions as inflammatory disease. Genome analysis gives the possibility of looking for susceptibility gene loci and their allelic combinations. By the analysis of mRNAs (transcriptome), novel players, functional groups of participants and regulatory pathways involved in the inflammation can be revealed. The possibility of comparing several samples is ideal to increase our knowledge about the kinetics of inflammation. The effects and post-receptor signalling events of individual pro- or anti-inflammatory mediators can also be tested. The use of human post mortem samples could yield otherwise inaccessible information on molecular mechanisms of chronic inflammatory diseases. However, the analysis and interpretation of a huge amount of data, the selection of appropriate experimental systems and time points may hide some pitfalls, which emphasize the need to confirm results with independent methods such as e.g. semi-quantitative Northern, real time PCR, RNA-ase protection assay as well as functional studies.

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Histamine is a versatile mediator that, according to in vitro studies, affects the synthesis of inflammatory cytokines and acute-phase proteins. The histidine-decarboxylase knockout (HDC-/-) mouse is a model of in vivo investigation of the physiologic and metabolic integration of the acutephase response. These mice do not synthesise histamine and feeding them with histamine-poor diet they are almost completely histamine-deficient. We compared the serum concentrations of representatives of acute-phase plasma proteins, as well as the levels IL-6 and IL-1alpha in wild type and HDC-/- mice during local (turpentine-induced) or systemic (LPS-induced) inflammation. The level of some acute-phase proteins significantly differed in wild-type and HDC-/- mice while others remained unaffected. The IL-6 levels are also differ in the wild-type and histamine-deficient animals, suggesting that the effect of histamine is attained through IL-6, although direct effect is not disclosed yet.

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Interleukin-6, a multifunctional cytokine upon binding to its receptor on hepatocytes regulates production of acute phase proteins involved in local and systemic inflammation. Gene expression and biosynthesis of IL-6 and its receptor (IL-6 R/gp130) is under complex regulation. Histamine, in addition to its principal role in immediate type hypersensitivity has been described to modulate IL-6 production and expression of IL-6 receptor. In this study, the IL-6 and IL-6 receptor expression was examined in histamine deficient histidine decarboxylase (HDC) knock-out mouse model. Our data suggest that in histamine deficient mice the inducibility of IL-6 is significantly reduced, whilst more IL-6 receptor/gp130 mRNA expresses in the liver than in wild type (HDC(+/+)) mice. These in vivo findings confirm earlier in vitro results and emphasize the efficacy of antihistamines in local IL-6 related processes.

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The biosynthesis of interleukin-6 receptor (IL-6R) and gp130 in vitro was blocked using specific antisense oligonucleotides (ASO) in HepG2 liver cells and the efficacy of various ASOs was tested on the generation of IL-6-induced junB mRNA. We used three ASOs specific for the IL-6 receptor, three specific for gp130 and a control (nonsense) oligonucleotide specific for epsilon-chain of IgE (not expressing in HepG2 cells). Our data indicate that a gp130-specific ASO, g2, was the most effective blocker of IL-6-induced junB mRNA, whilst the IL-6 receptor ASOs alone were ineffective. The mechanism of gene inactivation by ASO treatment was partially elucidated by demonstration of the loss of gp130 mRNA from cells treated with ASOs showing functional efficacy. Our data may help to design antisense oligonucleotides that are effective in therapy (e.g. as anti-inflammatory agents) in the future.

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Histidine decarboxylase (HDC) synthesizes endogenous histamine from histidine in mammals. HDC-deficient mice (HDC-/-), if kept on a histamine-free diet, have no histamine in their tissues. HDC-/- mice show multiple phenotypes. In this study we show that both the constitutively expressed and turpentine-induced level of an acute-phase protein, haptoglobin, is significantly lower in the serum of HDC-/- mice compared to that of wild-type animals. This effect was abolished if HDC gene-targeted mice received histamine-rich food. No differences were found when lipopolysaccharide (LPS) was used to induce the acute-phase reaction. Using specific antibodies to phosphorylated tyrosine, we showed that protein tyrosine phosphorylation (Y-P) of approximately 50- and 26- to 27-kDa liver proteins is significantly decreased in HDC-/- mice, but that the difference was largely diminished if the animals were kept on a histamine-rich diet, suggesting that the phenotype with lower haptoglobin production is diet inducible. Upon in vivo treatment with LPS, Y-P band intensity decreased, regardless of the presence or absence of histamine. Identification of elements of the signalling pathway with decreased phosphorylation may elucidate the molecular background of the effect of endogenous histamine in the hepatic acute-phase reaction.

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The growth factors present during liver regeneration partially overlap with the regulators of the hepatic acute phase response. We analysed the acute phase reaction and changes in soluble cytokine receptors after partial hepatectomy, when tissue injury inducing acute phase reaction and major reduction of liver mass occur simultaneously. Three acute phase proteins and mRNAs were determined by ELISA and northern blot hybridisation in rats. Serum levels of IL-6 and three soluble cytokine receptors (sTNF-alpha R I and II, sIL-6R) were detected by ELIBA or dot-blot assay. Time-course profiles of fibrinogen, alpha(2)-macroglobulin and haptoglobin proteins and mRNA are presented. Elevation of IL-6, soluble TNF-alpha receptors and soluble IL-6 receptor levels were also detected. The time-course of changes in haptoglobin concentration and elevation of soluble cytokine receptors is described by this in vivo experimental system. The results show good correlation with (post)transcriptional activation of immediate and delayed early gene products. These data suggest the involvement of both acute phase proteins and soluble cytokine receptors in the regulation of liver regeneration.

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In rats within the first week of partial hepatectomy reconstruction of the normal histological structure of the liver already starts. To approach the possible role of endogenous glucocorticoids in the process of regeneration we measured the changes in the expression of steroid glucocorticoid receptor gene after various regeneration intervals. After partial hepatectomy, between 0.5 168 hours from the surgery, the gene expression (mRNA) of glucocorticoid receptor was determined by reverse transcription followed by PCR and normalized to that of glycerolphoshate dehydrogenase. Two peaks of glucocorticoid receptor mRNA were detected first, between 3 and 6 hours (first peak) and a second between 24 and 36 hours. Immunoreactive glucocorticoid receptor was detected by immunohistochemistry using monoclonal anti-glucocorticoid receptor. Three days after the surgery immunohistochemical studies showed substantially more immunoreactive GcR protein in the regenerated liver than in the controls. These semiquantitative data provide evidence suggesting elevation of glucocorticoid receptor expression during regeneration of liver at mRNA and protein levels.

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Recent findings on the noncanonical positions of some well-known extracellular mediators and their receptors are reviewed. Peptide hormones (insulin) and/or their binding sites (cell membrane insulin receptor, nuclear insulin receptor); steroid hormones (corticosteroids and estrogens) and their putative membrane receptors are in the scope of this paper. The possible roles of these unusually located receptors in the intracellular signal propagation and physiological responses are also discussed.

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The oral apparatus and body ciliature of untreated control cells bind insulin-gold very rarely. From 1 day to 1 week after insulin pretreatment (hormonal imprinting) an enormous quantity of insulin-gold was bound by the oral cilia, completely filling the region, while the insulin-gold on the body ciliature is scattered. The binding was specific for insulin, since polyethylene glycol (PEG)-gold was not bound at all. The results call attention to the binding and the increasing role of hormonal (insulin) imprinting, and particularly to the marked role of the oral region in this binding. The roles of mucocyst extrusion and specific binding by receptors are discussed.

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Gold-labeled insulin is bound first of all to the cilia of the oral field of Tetrahymena. A primary treatment (hormonal imprinting) with insulin increases the binding capacity even after 24h and makes it more sensitive for appearance a week later, within a minute of giving insulin-gold. The food vacuoles contain insulin-gold in pretreated cells or without pretreatment as well, though in imprinted situations the label can be found in pinocytotic vesicles at the bases of cilia in the oral field. Altogether, a functional difference can be observed between the cilia of the oral and non-oral surfaces of Tetrahymena and hormonal imprinting has a specifying effect on the binding of labeled hormone.

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Tritiated diazepam accumulates mainly in the mitochondria of the unicellular Tetrahymena. This is the case in both a single (the first encounter) and a repeated (one day or a week after the first) administration of the drug. When imprinting of Tetrahymena by diazepam (the first encounter) is followed a week later by the administration of the labelled drug, the membranes of the vesicles, too, show the appearance of label. Regarding the studies presented here, the unicellular Tetrahymena also contain diazepam receptors in the mitochondria as suggested for cells of higher rank animals.

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One hour after treatment with 3H-benzpyrene, grains appeared for the most part above the hepatocellular surface, heterochromatin and dense vacuoles, whereas 24 h later they were associated mainly with the dense vacuoles and smooth-surfaced endoplasmic reticulum and only, a few were still present above the heterochromatin. Neonatal pretreatment (imprinting) with benzpyrene accounted for an earlier appearance of benzpyrene in the hepatocytes relative to the non-pretreated control, but while labelled benzpyrene had practically disappeared from the liver of imprinted rats within 24 h, it still persisted in the liver of control rats at a level approximating the 4 h value.

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One hour after treatment with 3H-benzpyrene, grains appeared for the most part above the hepatocellular surface, heterochromatin and dense vacuoles, whereas 24 h later they were associated mainly with the dense vacuoles and smooth-surfaced endoplasmic reticulum and only, a few were still present above the heterochromatin. Neonatal pretreatment (imprinting) with benzpyrene accounted for an earlier appearance of benzpyrene in the hepatocytes relative to the non-pretreated control, but while labelled benzpyrene had practically disappeared from the liver of imprinted rats within 24 h, it still persisted in the liver of control rats at a level approximating the 4 h value.

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The unicellular Tetrahymena first internalized, then partly released the labelled insulin. Insulin-pretreated (imprinted) Tetrahymena cells behaved differently from non-pretreated cells, in that they retained a greater part of internalized insulin in the cytoplasm. Additional exposure to excessive non-labelled (cold) insulin caused a decrease in intracellular labelled insulin retention. Internalized insulin also entered the nucleus of Tetrahymena, where it was found in a heterochromatic localization.

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Quantitative light and electron microscopic autoradiography demonstrated a dissimilar effect of insulin, prednisolone and diiodothyronine on the incorporation and localization of 3H-uridine in Tetrahymena. After treatment with insulin for 1 h, uridine was rapidly incorporated, and after initial accumulation in the cytoplasm its intracellular level tended to drop below the control. Total uridine incorporation was lower than in the control cells. In Tetrahymena treated with prednisolone or diiodothyronine, uridine incorporation was relatively slow, but the intracellular uridine level increased significantly over the control. Re-exposure to the hormone had no significant influence in the case of insulin, but altered the quantitative relations of uridine incorporation significantly in the case of the morphogenetic hormones prednisolone and T2.

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Insulin imprinting given to the unicellular Tetrahymena considerably increases the uptake and intracellular storage of amino acids even many generations after the actual contact with the hormone. On the other hand, both the first and the second contacts with insulin increase the rate of the excretion of the stored amino acids. On the basis of the results obtained it seems to be possible that both protein synthesis and exocytosis of the Tetrahymena change as an effect of imprinting, either in general or specifically due to the formation of new hormone receptors.

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Live Tetrahymena cells bound 3H-diazepam specifically, as demonstrated by autoradiographic evidence of displacement of about 25% of labeled diazepam in the presence of a 1000-fold amount of cold diazepam. The 3H-diazepam bound to membrane preparations isolated from untreated (control) cells was not displaced by cold diazepam, whereas cells involved in primary interaction (imprinting) with diazepam showed amplification and specificity of diazepam binding in both in vivo (cell suspension) and in vitro (pellicle) systems, as well as displacement of bound label in the presence of 1000-fold cold diazepam. It appears that diazepam induced imprinting and, consequently, also the formation of specific receptors in Tetrahymena.

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Steroid hormones are incorporated by Tetrahymena, and appear intracellularly in both cytoplasmic and intranuclear localizations. At primary interaction with the steroid, incorporation was the greatest at 10 min, whereas at the second interaction it tended to increase with time. The Tetrahymena cells preexposed to a steroid incorporated a greater amount of it at the second exposure, owing presumably to the induction of steroid receptors at the first interaction.

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The thymocytes of newborn rats incorporated considerably greater amounts of labeled corticosterone in culture than those of two or four weeks old rats. The incorporation of label tended to increase up to 5 min of exposure in all age groups, but did not further increase in any during longer exposures. The hormone entered the nucleus of the thymocytes and assumed primarily a heterochromatic localisation.

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