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Biomedical subjects

A Koj

Publications and source records attributed to A Koj.

At least 19 recordsLinked to original sources
PubMed2005

Transcription factors as targets of the anti-inflammatory treatment. A cell culture study with extracts from some Mediterranean diet plants.

During the inflammatory response at least 2 transcription factors, NF-kappaB and AP-1, are involved in the altered profile of gene expression. We used human hepatoma (HepG2) and human umbilical vein endothelial cells (HUVEC) as a model system: NF-kappaB and AP-1 were activated by the proinflammatory cytokine IL-1 in the absence or presence of 21 selected plant extracts and the effect was evaluated by the electrophoretic mobility shift assay (EMSA). In both types of cells activation of NF-kappaB by IL-1 was significantly inhibited by extracts from Scandix australis and Artemisia alba, whereas extracts from Amaranthus sp., Eryngium campestre, Thymus pulegioides and Reichardia picroides elicited cell-type dependent response. The IL-1-induced AP-1 activation was diminished by extracts from Scandix australis, Amaranthus sp. and Artemisia alba more potently in HUVEC, while extracts from Urospermum picroides and Scandix pecten-veneris in HepG2 cells. Inhibitory activities of plant extracts towards cytokine activated NF-kappaB and AP-1 depend to some extent on the order of addition of IL-1 and plant extract to the cell culture, but the mechanism of action of extract components is not clear: although plant polyphenols may participate they are unlikely to be the only mediators, and MAP kinases were found generally not involved in down-regulation of transcription factors activity by plant extracts.

Acetylcysteine↗
PubMed2003

Regulation of expression of stromyelysin-1 by proinflammatory cytokines in mouse brain astrocytes.

Stromelysin-1 belongs to matrix metalloproteinases responsible for proteolytic degradation of extracellular matrix in many tissues during various diseases, especially those involving inflammation. We studied the induced expression of stromelysin-1 in primary cultures of mouse brain astrocytes stimulated with various cytokines and cellular growth factors. Interleukin-1-beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and a mixture of IL-1, TNF and epidermal growth factor (EGF) significantly increased the level of stromelysin-1 mRNA in mouse astrocytes while interferon-gamma (IFN-gamma) inhibited this response or was without effect. This accumulation of specific mRNA was preceded by activation of two examined transcription factors: NFkappaB and AP-1. However, experiments with known inhibitors of activation of these transcription factors: pyrrolidine dithiocarbamate (PDTC), parthenolide and curcumin, indicate that NFkappaB and AP-1 cannot be solely responsible for the cytokine induced expression of stromelysin-1 gene in mouse astrocytes.

Animals↗
PubMed2002

Cytokines regulate plasminogen activation system in astrocytoma cells.

We report here that human astrocytoma cell line U373-MG is able to express genes of the following components of plasminogen activation system: PA1-1, PN-1, u-PA and t-PA. Treatment of these cells with IL-1beta results in accumulation of PA1-1, PN-1 and u-PA mRNAs, whereas t-PA mRNA remains unaffected. IFNy preferentially enhances PN-1 and PA1-1, EGF enhances PA1-1, u-PA and t-PA expression. Simultaneous addition of anti-inflammatory cytokines IL-4, IL-13 and IL-10 has little effect on the tested components, except induction of u-PA mRNA wich was further enhanced by IL-4. We have confirmed interesting time-dependent regulation of plasminogen activation system by EGF/IFNgamma. Cells stimulated with EGF/IFNgamma show at first increased proteolytic activity but after 24 h inhibition of proteolysis with PA1-1 would prevail. To understand the cooperative effect of EGF and IFNgamma in PA1-1 induction the kinetics of activation of STAT1 was studied. It was found that although EGF alone does not activate STAT1, the STAT1 binding activity in the cells treated with the mixture of EGF/IFNgamma was considerably prolonged. Our results indicate the importance of inflammatory cytokines and EGF in gene regulation of plasminogen activation system in astrocytoma cells.

Astrocytoma↗
PubMed2001

Epidermal growth factor and pro-inflammatory cytokines regulate the expression of components of plasminogen activation system in U373-MG astrocytoma cells.

Cytokines and growth factors that influence both secretion of the extracellular matrix (ECM) proteins and migration of the cells decide about the final outcome of tissue remodelling. We have examined expression of the components of the plasminogen activation system in human astrocytoma U373-MG cells and found that interleukin 1beta (IL-1beta), tumour necrosis factor alpha TNF-alpha), interferon gamma (INF-gamma) and epidermal growth factor (EGF) specifically regulate the expression of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor type 1 (PAI-1) and protease nexin-1 (PN-1). We conclude that EGF and IFN-gamma are new important regulators of the plasminogen activation system in astrocytoma cells and, therefore, may influence turnover of extracellular matrix and migration of cells within the brain.

Amyloid beta-Protein Precursor↗
PubMed2001

Differential effects of oncostatin M and leukaemia inhibitory factor expression in astrocytoma cells.

The effects of the production of two closely related cytokines, oncostatin M (OSM) and leukaemia inhibitory factor (LIF), by astrocytoma cells were investigated using the stable cell line human U373-MG, which expressed and secreted both biologically active polypeptides. The expression of LIF by these cells caused resistance to this cytokine due to loss of the LIF receptor (LIFR), from the cell surface, suggesting its retention. In contrast, cells expressing OSM were stimulated by this cytokine, utilizing an autocrine mechanism, and possessed receptors for OSM, but not LIF, on the cell surface. In these cells the continuous up-regulation of OSM-induced gene expression was found even though the Janus kinase-signal transducer and activator of transcription ('JAK/STAT') pathway was almost exhausted due to long-term autocrine stimulation of the cells by OSM. The amount of LIFR was down-regulated in both LIF- and OSM-producing cells and this effect was not found in wild-type U373-MG cells treated with externally added cytokines. To investigate the mechanism of autocrine stimulation by OSM we constructed a stable cell line expressing a form of OSM that is retained in the endoplasmic reticulum (ER). This biologically active cytokine was not secreted, but was localized in the ER. In addition, it did not stimulate the astrocytoma cells in an autocrine manner. We conclude that expression of LIF causes resistance of astrocytoma cells to this cytokine, whereas expression of OSM leads to autocrine stimulation.

Antigens, CD↗
PubMed2000

Differential responses of hematopoietic and non-hematopoietic cells to anti-inflammatory cytokines: IL-4, IL-13 and IL-10.

Recombinant preparations of human anti-inflammatory cytokines: IL-4, IL-13 and IL-10, inhibited LPS-induced synthesis of TNFalpha and IL-6 in the whole human blood tested in vitro. These cytokines also inhibited LPS-induced IL-6 and TNF mRNA accumulation in isolated human blood monocytes/macrophages. On the other hand, similar concentrations of IL-4 and IL-13 (but not IL-10) enhanced synthesis of IL-6 in cultured human umbilical vein endothelial cells (HUVEC). In human hepatoma HepG2 cells IL-4 and IL-13 (but not IL-10) inhibited IL-6-induced synthesis of haptoglobin. These differential responses to the tested anti-inflammatory cytokines were observed at mRNA and protein levels and may reflect cell specificities in signalling pathways and gene expression. When HUVEC and HepG2 cells were cultured together and stimulated with LPS the addition of IL-4 or IL-13 resulted in the reduction of LPS-induced and IL-6-mediated haptoglobin synthesis. Thus in co-culture the inhibitory effects of IL-4 or IL-13 on HepG2 cells prevail over stimulation of IL-6 synthesis in HUVEC.

Endothelium, Vascular↗
PubMed1998

Termination of acute-phase response: role of some cytokines and anti-inflammatory drugs.

1. The acute-phase response is triggered by changes in intracellular mediators that activate stress-sensitive kinases and transcription factors controlling the synthesis of proinflammatory cytokines such as TNF-alpha, IL-1, IL-8 or IFN-gamma. 2. Natural extinguishing of acute-phase response occurs due to short half-lives of inflammatory mediators and production of anti-inflammatory cytokines such as IL-10, IL-4, IL-13, TGF-beta and some others. 3. Excess proinflammatory cytokines are removed by soluble cytokine receptors and receptor antagonists. 4. Synthesis of proinflammatory mediators and cytokines can be blocked by glucocorticoids, some nonsteroidal anti-inflammatory drugs suppressing cyclooxygenase and by specific inhibitors of cytokine induction. 5. The most promising approach in effective termination of acute-phase response appears to be a combined use of anti-inflammatory cytokines and specific drugs.

Acute-Phase Reaction↗
PubMed1998

Vaccinia virus-regulated acute phase cytokine production in human fibroblasts, U937 cells and endothelium.

The production of acute phase cytokines, interleukin 6 (IL-6), tumour necrosis factor (TNFalpha) and interleukin 1 (IL-1beta), was studied in primary cultures of human skin fibroblasts, human monocytic cell line U937 and primary cultures of human umbilical vein endothelial cells (HUVEC) after in vitro infection with vaccinia virus. Significant increase in IL-6 mRNA followed by enhanced protein secretion into the culture media was found in fibroblasts, U937 cells, and HUVEC. TNFalpha increased production in vaccinia virus infected U937 cells resembled closely the pattern of IL-6 production observed in the infected cells. Transient increase in NF-kappaB binding activity was found in the infected U937 (at 90 min) and endothelial (at 30 min) cells. Vaccinia virus induced cytokine production appeared to be transcriptional.

Acute-Phase Reaction↗
PubMed1998

Rooperol tetraacetate decreases cytokine mRNA levels and binding capacity of transcription factors in U937 cells.

We have previously described inhibition of the synthesis of three acute-phase inflammatory cytokines in human and rat macrophages by acetate esters of rooperol, a dicatechol of plant origin. Analysing the mechanism of anticytokine activity of rooperol, we compared levels of TNFalpha, IL-1beta and IL-6 mRNAs in the human promonocytic U937 cell line pretreated with phorbol myristate acetate (PMA) and incubated with rooperol tetraacetate (RTA) alone or in combination with LPS (500 ng/ml). It was found that 10 microM RTA decreased the levels of cytokine mRNAs both in the presence and absence of LPS, suggesting pretranslational inhibition of cytokine synthesis. Electrophoretic mobility shift analysis (EMSA) showed that RTA may influence cytokine mRNA expression by decreasing the binding activity of transcription factors NF-kappaB and AP-1.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1998

Vaccinia virus-induced changes in cytokine-regulated acute phase plasma protein synthesis by hepatoma cells.

Human hepatoma cell line, HepG2, has been infected with vaccinia virus and synthesis of plasma proteins was determined by electroimmunoassay and corresponding mRNA's measured by Northern blotting. The inhibitory effect of the virus was dose- and time-dependent. Electrophoretic mobility shift assay revealed a decrease in C/EBP binding activities in nuclear extracts isolated from the infected hepatoma cells. Supershift analysis of the C/EBP isoforms showed alpha and beta subunit involvement in DNA binding. The treatment of the cells with interleukin-1, interleukin-6, and dexamethasone at the initial stage of infection appears to delay the virally induced inhibition of host cell protein synthesis. Thus, possible "protective" role of the acute phase cytokines in viral infection is proposed.

Acute-Phase Proteins↗
PubMed1997

Inhibitory effect of di-catechol rooperol on VCAM-1 and iNOS expression in cytokine-stimulated endothelium.

Induced expression of vascular cell adhesion molecule-1 (VCAM-1) and of nitric oxide synthase (iNOS) is believed to play a role in the pathogenesis of atherosclerosis, asthma, as well as other inflammatory disorders. In the current study we examined the effect of the di-catechol rooperol [(E)-1,5-bis (3',4'-dihydroxyphenyl) pent-4-en-1-yne] on the process of microvascular endothelial cell (MME) activation by TNF-alpha and IFN-gamma. We show that rooperol decreases VCAM-1 and iNOS mRNA levels in cytokine-activated MME with subsequent inhibition of VCAM-1 membrane expression as measured by adhesion of P815 cells to MME monolayers, and NO production, as reflected in the nitrite concentration in culture medium. The properties of rooperol now described suggest that rooperol may be an anti-inflammatory agent useful in the treatment of several inflammatory disorders.

Animals↗
PubMed1996

Initiation of acute phase response and synthesis of cytokines.

A variety of injuries, such as bacterial infection or ischemic tissue necrosis, induce systemic acute phase reaction expressed as fever, leukocytosis, release of several hormones, activation of clotting, complement and kinin forming pathways, and drastic increase of synthesis of certain plasma proteins. The reaction is triggered by 'alarm molecules', including free radicals, which activate several stress-sensitive protein kinases (ERK, p38, JNK) in macrophages and other responsive cells. These kinases phosphorylate, usually in a multi-step cascade, transcription factors belonging primarily to C/EBP, NF-kappa B and AP-1 families. Active transcription factors after translocation to nucleus interact with responsive elements in the gene promoters of acute-phase cytokines: tumor necrosis factor-alpha, interleukin-1 and interleukin-6. Enhanced transcription of these genes is usually followed by rapid translation and precursor protein processing leading to the release of biologically active cytokines. Fine tuning of the acute phase response appears to be regulated at all stages: primary signals, kinase cascades, transcription factors, mRNA stability and translation, cytokine precursor processing, secretion and bioavailability. This makes possible designing of specific inhibitors of cytokine synthesis as potential therapeutic drugs.

Acute-Phase Reaction↗
PubMed1996

Cytokine production in human and rat macrophages and dicatechol rooperol and esters.

The ability of dicatechol rooperol and esters to inhibit the production of cytokines in endotoxin-stimulated human alveolar macrophages, human blood monocyte/macrophages, histiocytic cell line U937, and rat alveolar macrophages was examined in vitro. Rooperol derivatives inhibited the production of tumour necrosis factor-alpha, interleukin-1 beta and interleukin-6. Of the esters tested on human cells, rooperol diacetate and tetraacetate were more potent inhibitors of cytokine production (IC50 in the range of 10-20 microM) than rooperol disulphate (IC50 in the range of 25-75 microM). The acetate esters also inhibited cytokine production in rat alveolar macrophages, whereas the sulphate had little effect. Rooperol and acetate esters, in the same concentration range, decreased the production of nitric oxide by rat alveolar macrophages stimulated by endotoxin. These concentrations of rooperol had no effect on cell viability, as indicated by incorporation of 14C-labelled leucine into macrophage proteins and their content of lactate dehydrogenase. The results obtained suggest that rooperol esters are potentially useful antiinflammatory agents.

Animals↗
PubMed1995

Identification of the interleukin-6/oncostatin M response element in the rat tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter.

The rat tissue inhibitor of metalloproteinase 1 (TIMP-1) gene is expressed in rat hepatocytes, and this expression is up-regulated by interleukin 6 (IL-6). We report here the cloning of the 5' flanking region of the rat TIMP-1 gene and identification of an IL-6/oncostatin M (OSM) response element at -64 to -36 which functions in hepatic cells. Within this element we have identified two functional binding sites for transcription factors AP-1 (activatory protein-1) and STAT (signal transducer and activator of transcription). IL-6/OSM stimulation induces binding of a protein, identified as STAT3, to the IL-6/OSM response element, while binding of the AP-1 protein was constitutive. Binding sites for both AP-1 and STAT3 are necessary for full responsiveness of the TIMP-1 promoter to IL-6/OSM, as shown by deletion and mutation analysis. Furthermore, the entire IL-6/OSM response element conferred responsiveness onto a heterologous promoter, whereas this has not been observed when AP-1 and STAT elements were separately tested.

Animals↗
PubMed1995

Expression of a biologically active murine tissue inhibitor of metalloproteinases-1 (TIMP-1) in baculovirus-infected insect cells. Purification and tissue distribution in the rat.

Murine tissue inhibitor of metalloproteinases-1 (mTIMP-1) was expressed in baculovirus-infected insect cells (Sf9). The protein secreted into the culture medium was purified to homogeneity by means of heparin-Sepharose CL-6B and FPLC. The purified protein showed metalloproteinase-inhibitory activity in two independent assays: reverse zymography and inhibition of collagenase activity. Digestion of the recombinant TIMP-1 with peptide-N-glycanaseF revealed that both N-glycosylation sites are used. 125I-mTIMP-1 intraveneously injected into a male Sprague Dawley rat disappeared within 2 min from the circulation. 5 min after injection more than 50% of the 125I-mTIMP-1 were found in the liver and 20% in the kidneys. At later times, trichloroacetic-acid-soluble material accumulated in the intestinal tract.

Amino Acid Sequence↗
PubMed1995

Hepatocyte growth factor and retinoic acid exert opposite effects on synthesis of type 1 and type 2 acute phase proteins in rat hepatoma cells.

Rat hepatoma cells H-35 cultured in serum-free medium were exposed to interleukin-6 (IL-6), interleukin-1 (IL-1), hepatocyte growth factor (HGF), retinoic acid (RA), or a mixture of these factors. Production of acute phase proteins, responding to IL-6 alone (type 2) or to the mixture of IL-6 and IL-1, was assessed by electroimmunoassay and the corresponding mRNAs were compared by Northern blot analysis. HGF enhanced IL-6-induced synthesis of alpha-2-macroglobulin but reduced synthesis of C3 complement and alpha-1-acid glycoprotein. Retinoic acid reduced the response to IL-6 of alpha-2-macroglobulin but enhanced that of alpha-1-acid glycoprotein and especially of C3 complement. In general, changes in protein secretion were correlated with the contents of their corresponding cellular mRNAs. These results indicate that hepatocyte growth factor can enhance basal or IL-6-induced gene expression of type 2 and reduce the expression of type 1 acute phase proteins, whereas the action of retinoic acid is opposite. The modulation of acute phase response by HGF and RA likely involves transcriptional factors and regulatory sequences in the genes coding for these two types of acute phase proteins.

Acute-Phase Proteins↗