PubMed Health⌕ Search

Biomedical subjects

J Stankova

Publications and source records attributed to J Stankova.

At least 19 recordsLinked to original sources
PubMed2004

Leukotrienes and isocyanate-induced asthma: a pilot study.

BACKGROUND: The role of leukotrienes (LTs) in the pathophysiology of isocyanate-induced asthma is not well known. OBJECTIVE: We sought to characterize the type of airway inflammation induced by exposure to isocyanates and to investigate whether exposure to isocyanates induced an increase in LT receptor cysteinyl leukotriene ((CysLT)(1), CysLT(2) and leukotriene B(4) receptor (BLT(1))) expression, as well as a release of LT (LTC(4) and leukotriene B(4) (LTB(4))) and IL-8 in both asthmatics with isocyanate-induced asthma and healthy subjects. METHODS: We investigated eight subjects with isocyanate-induced asthma and eight healthy subjects. Both groups underwent specific inhalation challenges to isocyanates in the laboratory. Induced sputum was collected before and after exposure to isocyanates. CysLT(1), CysLT(2) and BLT(1) expression was assessed by flow cytometry, whereas LTC(4), LTB(4) and IL-8 were measured in the sputum supernatants by enzyme immunoassay. RESULTS: Exposure to isocyanates induced an increase in sputum neutrophils only in subjects with occupational asthma. There was a significant increase in CysLT(1) and BLT(1) receptor expression, as well as a release of LTB(4) and IL-8 after exposure to isocyanates compared with the baseline, only in subjects with isocyanate-induced asthma, whereas there was no increase in LTC(4). Exposure to isocyanates did not induce any change in LT receptor expression nor in the levels of LTC(4), LTB(4) and IL-8, in healthy subjects. CONCLUSION: The neutrophilia observed after exposure to isocyanates is likely to be related to the release of LTB(4), probably enhanced by the increased expression of BLT(1) on neutrophils as well as by the release of IL-8. The significance of the increase of CysLT1 receptor expression on neutrophils is unknown and needs further investigation.

Adult↗
PubMed2001

High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation.

Endothelial cells derived from the human umbilical vein (HUVEC) are used to study the mechanisms involved in EC response to various stimuli as well as to investigate the basis of pathological conditions of the vascular system such as altered endothelium permeability, tumor-induced angiogenesis, atherosclerosis and leukocyte extravasation in chronic inflammatory responses. However, investigations of gene involvement related to these conditions have progressed slowly because of the difficulty of transfecting HUVEC with high efficiency. Whereas several technical approaches have been described, they usually result in low levels of transfected cells or they require several steps or sophisticated instrumentation. We describe here a straightforward protocol of transfection of freshly isolated HUVEC that is based on the simple technique of electroporation. Efficiencies of gene transfection greater than 40% were routinely obtained by using a combination of optimized conditions of HUVEC isolation, composition of the electroporation medium and homogeneity of the plasmids. The protocol has been applied to the functional transient transfection of functional genes in HUVEC as illustrated in the case of the cDNA encoding GFP, protein kinase C (alpha and epsilon isotypes) and beta-galactosidase.

Cells, Cultured↗
PubMed2001

Oxidized low-density lipoprotein and peroxisome-proliferator-activated receptor alpha down-regulate platelet-activating-factor receptor expression in human macrophages.

Regulation of the expression of platelet-activating factor (PAF) receptor by atherogenic lipoproteins might contribute to atherogenesis. We show that progressive oxidation of low-density lipoprotein (LDL) gradually inhibits PAF receptor expression on the macrophage cell surface. We tested the effect of oxidized LDL (oxLDL) on PAF receptor expression in human monocytes that do not contain peroxisome-proliferator-activated receptor gamma (PPARgamma), a nuclear receptor activated by oxLDL. OxLDL decreased by 50% (P < or = 0.001) and by 29% (P < or = 0.05) the binding of PAF and the expression of PAF receptor mRNA respectively. Next we demonstrated that progressive oxidation of LDLs significantly activated PPARalpha-dependent transcription in transfected mouse aortic endothelial cells. Finally we demonstrated, in mature macrophages, that fenofibrate (20 microM), a specific PPARalpha agonist, but not the specific PPARgamma agonist BRL49653 (20 nM), significantly decreased both PAF binding and PAF receptor mRNA expression, by 65% and 40% (P < or = 0.001) respectively. Additionally, another PPARalpha agonist, Wy14,643, decreased PAF receptor promoter activity by 70% (P < or = 0.05) in transfected THP-1 cells, suggesting the involvement of the proximal promoter region (-980 to -500) containing a series of four nuclear factor (NF)-kappaB motifs. Thus PPARalpha might be involved in the down-regulation of PAF receptor gene expression by oxLDLs in human monocytes/macrophages. The oxidation of one or more lipid components of LDLs might result in the formation of natural activators of PPARalpha. It is hypothesized that such activators might modulate inflammation and apoptosis upon atherogenesis by decreasing the expression of PAF receptor.

Cells, Cultured↗
PubMed2000

Expression of platelet-activating factor receptor in human carotid atherosclerotic plaques: relevance to progression of atherosclerosis.

BACKGROUND: Human monocyte-derived macrophages synthesize numerous proinflammatory and prothrombotic substances, including lipid mediators, such as platelet-activating factor (PAF), which may play a major role in the onset and perpetuation of atherosclerotic lesions. In addition, both monocytes and macrophages express PAF receptors (PAF-R). The expression of PAF-R is transcriptionally downregulated by oxidized LDL in in vitro primary cultures of monocyte/macrophages. In this study, we evaluated the expression of PAF-R in human carotid plaque tissue, in foam cells isolated from human carotid plaques, and in primary cultures of umbilical smooth muscle cells (SMCs). METHODS AND RESULTS: We show that PAF-R was expressed at low levels in foam cells compared with monocyte/macrophages in plaques, as assessed by immunohistochemical staining and in situ hybridization. In addition, low levels of mRNA were also detected by RT-PCR in isolated human carotid foam cells. A prominent finding of our study was the demonstration that contractile SMCs were positive for PAF-R, and its mRNA was extracted from primary cultures of umbilical SMCs. CONCLUSIONS: As macrophages loose their inflammatory phenotype on transformation into foam cells, they may equally loose their capacity of defense against aggression. We postulate that the diminished expression of PAF-R may be deleterious in the context of plaque formation and progression. The observation that arterial SMCs of contractile phenotype express PAF-R opens new avenues concerning the migration of these cells from media to intima and atherosclerotic plaque formation.

Antigens, Differentiation↗
PubMed1999

Modulation of formyl peptide receptor expression by IL-10 in human monocytes and neutrophils.

IL-10, originally described as a cytokine synthesis inhibitory factor, is secreted by a number of cells of the immune system, including monocytes and T cells. Although IL-10 is being assigned as an immunosuppressive cytokine, our study showed that FMLP-R mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of this cytokine, with maximal (three- to fourfold) stimulation with 10 ng/ml. The effect was rapid, being observable as early as 1 h of treatment with IL-10, maximal between 2 and 4 h, and still evident after 24 h and was associated with an increase of receptor expression on the cell surface as assessed by flow cytometry analysis. Pretreatment of monocytes with actinomycin D completely abrogated the effect of IL-10, suggesting a transcriptional regulation. Moreover, IL-10-treated monocytes showed a significantly enhanced functional responsiveness to FMLP with enhanced (three- to fourfold) chemotaxis and augmented (twofold) intracellular calcium mobilization. In polymorphonuclear neutrophils (PMN), IL-10 also mediated a twofold augmentation of FMLP-R expression. In parallel experiments, we observed that IL-10 could differentially modulate other chemotactic receptors. Hence, we observed that IL-10 augmented two-to threefold platelet-activating factor receptor (PAF-R) expression, whereas it had no significant effect on the fifth component of complement (C5a) receptor (C5a-R) expression. Collectively, our results demonstrate that IL-10 may play an important role in inflammatory process through modulation of chemotactic receptor expression.

Chemotaxis, Leukocyte↗
PubMed1999

Heat-killed Streptococcus suis capsular type 2 strains stimulate tumor necrosis factor alpha and interleukin-6 production by murine macrophages.

Streptococcus suis capsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since mononuclear phagocytes have been suggested to play a central role in the pathogenesis of meningitis, the objective of the present study was to evaluate the capacity of whole killed S. suis type 2 organisms to induce the release of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) by murine macrophages. Induction of cytokines was evaluated in the presence or absence of phorbol ester (phorbol 12-myristate 13-acetate [PMA]) costimulation. Results showed that S. suis type 2 stimulated the production of both cytokines in a concentration- and time-dependent fashion. Although large doses of bacteria were required for maximal cytokine release, titers were similar to those obtained with the lipopolysaccharide (LPS) positive control. An increase in cytokine release was observed with both S. suis and LPS with PMA costimulation. Experiments with cytochalasin-treated macrophages showed that the stimulation of cytokine production was phagocytosis independent. When macrophages were stimulated with an unencapsulated mutant, an increase in TNF production was observed, but the absence of the capsule had no effect on IL-6 production. In fact, whereas purified capsular polysaccharide of S. suis failed to induce cytokine release, purified S. suis cell wall induced both TNF and, to a lesser extent, IL-6. IL-6 secretion probably requires some distinct stimuli which differ from those of TNF. Finally, the S. suis putative virulence factors suilysin and extracellular protein EF showed no cytokine-stimulating activity. The ability of S. suis to trigger macrophages to produce proinflammatory cytokines may have an important role in the initiation and development of meningitis caused by this microorganism.

Animals↗
PubMed1997

Augmented expression of platelet-activating factor receptor gene by TNF-alpha through transcriptional activation in human monocytes.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine produced by activated monocytes and often associated with platelet-activating factor (PAF) during the pathogenesis of many inflammatory and infectious diseases. PAFR is a G-protein-coupled receptor constitutively expressed on monocytes. TNF-alpha (100-400 U/mL) significantly increased PAFR mRNA expression in human monocytes. This increase was seen after 1 h of stimulation and persisted up to 24 h. Actinomycin D pretreatment studies revealed a transcriptional increase in PAFR gene expression without effect on mRNA half-life. [3H]WEB 2086 binding studies showed a significant (43%) increase in specific binding sites in 24-h-treated cells without change in receptor affinity. Increased interleukin-6 production in response to PAF was also found in 24-h TNF-alpha-pretreated monocytes. These observations provide new evidence for TNF-alpha and PAF interactions in human monocytes during inflammatory processes through up-regulation of PAFR expression by TNF-alpha.

Azepines↗
PubMed1997

Changes in apoptosis of human polymorphonuclear granulocytes with aging.

Many alterations with aging occur at the cellular and organic levels in the immune system ultimately leading to a decrease in the immune response. Our aim in the present work was to study apoptosis of polymorphonuclear granulocytes (PMN) with aging under various stimulations since apoptosis might play an important role in several pathologies encountered with aging. The PMN of healthy young (20-25 years) and elderly (65-85 years) subjects were examined after 24 h of sterile culture with and without stimulation. The stimulating agents included: phorbol myristate acetate (PMA), hydrogen peroxide (H2O2), N-formyl-methionyl-leucyl phenylalanine (FMLP), granulocyte-macrophage colony stimulating factor (GM-CSF), reduced glutathione (GSH), lipopolysaccharide (LPS) and interleukin 2 (IL-2). Apoptosis was assessed by traditional staining of the plates, by flow cytometric staining and DNA gel electrophoresis. It was found that without stimulation the susceptibility of PMN to apoptosis was slightly increased with aging. Under various stimulations, such as PMA. H2O2, apoptosis was almost 100%, while the treatment by FMLP, oxLDL and GSH did not change its extent in PMN obtained either from young or elderly subjects. Marked age-related changes were observed in the extent of apoptosis under stimulation with GM-CSF, IL-2 and LPS. These agents were able to significantly prevent apoptosis in PMN of young subjects, while only the GM-CSF was able to slightly modulate it in neutrophils of elderly subjects. From these results, we suggest that changes in apoptosis of PMN with aging could play a role in the increased incidence of certain immune system related pathologies of aging, such as cancer, infections and autoimmune disorders.

Adult↗
PubMed1997

Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor.

The interaction between human endothelial cells (EC) and leukocytes during inflammation is in part mediated through the release of soluble factors. Since platelet-activating factor (PAF) is a potent mediator of inflammatory responses, we investigated the potential of PAF to modulate IL-6 and GM-CSF production by EC. Exposure of these cells to PAF resulted in a concentration-dependent increase in IL-6 production, with a maximum at 10(-10) M PAF. Sequential incubation of EC with PAF and TNF alpha resulted in a synergistic increase of IL-6 production. This effect was specific for PAF since it was prevented by preincubation with the PAF receptor antagonist, WEB 2086. Northern blot analysis revealed enhanced IL-6 mRNA expression in PAF-treated EC. However, the synergy observed in protein synthesis between PAF and TNF alpha was not reflected in IL-6 mRNA accumulation, suggesting a post-translational modulation. Pretreatment of EC with the protein synthesis inhibitor cycloheximide before their exposure to PAF resulted, after washout of the cycloheximide, in a markedly augmented production of IL-6, suggesting a synergy between augmented IL-6 mRNA accumulation by PAF and IL-6 mRNA superinduction by cycloheximide. GM-CSF production by EC was also stimulated by the combined effects of PAF and TNF alpha, but PAF alone did not affect GM-CSF production. Taken together, our data suggest that PAF can stimulate EC to synthesize cytokines, including IL-6 and GM-CSF, which may contribute to local and, possibly, systemic responses during inflammation.

Azepines↗
PubMed1997

Differential signaling pathways in platelet-activating factor-induced proliferation and interleukin-6 production by rat vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) can be induced to proliferate in response to several cytokines and growth factors, including interleukin (IL)-6. Platelet-activating factor (PAF) also has been shown to induce SMC proliferation. Because PAF can stimulate IL-6 production in monocytes, macrophages, and endothelial cells, our study was undertaken to determine whether PAF could induce IL-6 production by SMCs and to define the underlying signaling pathways. Exposure of rat aortic SMCs to picomolar concentrations of PAF resulted in enhanced production of IL-6. The effect was concentration dependent, selective for the active form of PAF, and mediated by specific PAF receptors. Pretreatment of the cells with Bordatella pertussis toxin (PTX) prevented the effect of PAF, suggesting the involvement of alpha i-type subunits of G proteins in the signal-transduction pathway. PAF-dependent IL-6 production was also prevented by inhibition of tyrosine kinases with genistein or erbstatin. Inhibition of eicosanoid production by blocking either phospholipase A2 or cyclooxygenase also abrogated the effect of PAF on IL-6 production. Moreover, inhibition of Ca2+-calmodulin activity with W7 or blocking of calcium channels with verapamil or nifedipine prevented PAF-mediated enhancement of IL-6 production. Whereas PAF-induced signal-transduction pathways leading to IL-6 production and SMC proliferation were partially common, they appeared to diverge downstream of PLA2 activation: inhibition of cyclooxygenase had no effect on proliferation, whereas augmentation of cyclic adenosine monophosphate (cAMP) levels or activation of protein kinase A inhibited proliferation, in contrast to IL-6 production. Our findings suggest a role for PAF in modulating vascular function by stimulating local production of IL-6 by SMCs and promoting their proliferation. The two effects are, however, associated with partially divergent signaling pathways and may not be causally related.

Animals↗
PubMed1996

Modulation of human platelet-activating factor receptor gene expression by protein kinase C activation.

The effect of PMA on early and late regulation of platelet-activating factor receptor (PAF-R) expression was examined in human monocytes. Treatment with 16 nM PMA for 5 min initiated a rapid reduction (50-75%) in [3H]WEB 2086 binding, which was maximal between 5 and 15 min. Scatchard analysis revealed that PMA treatment reduced the number of binding sites to 50% of control cells without an appreciable change in their affinity. In parallel cultures, flow cytometry analysis using anti-PAF-R Abs failed to reveal any significant decrease in the level of PAF-R expression until after 4 h of treatment with PMA. By 24 h, PAF-R expression had declined by 80 to 90%. This PMA-induced down-regulation of surface PAF-R expression was preceded by a rapid down-regulation of PAF-R mRNA expression. PMA-mediated down-regulation could be blocked by the protein kinase C (PKC) inhibitors H-7 and calphostin C. Activation of PKC by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol also resulted in down-regulation of PAF-R mRNA accumulation, whereas the inactive phorbol diester 4alpha-PMA was ineffective. The rapid disappearance of the PAF-R transcripts was associated with decreased stability of receptor mRNA and not with a change in the nuclear transcription rate of the PAF-R gene. These findings indicate that PAF-R gene expression in human leukocytes can be regulated through a PKC-dependent pathway and involves post-transcriptional destabilization of receptor mRNA.

Enzyme Activation↗
PubMed1996

Natural killer and lectin-dependent cytotoxic activities of Kurloff cells: target cell selectivity, conjugate formation, and Ca++ dependency.

Kurloff cells may represent a major component of NK cell activity in the guinea pig. We have pursued to characterize the mechanism of their action. Using murine target cells, we found Kurloff cell cytotoxicity to be selective for the NK-sensitive YAC-1 target cell, with minimal activity against the NK-resistant P815 target cell. In the presence of PHA, but not ConA, cytotoxicity was markedly augmented against both YAC-1 and P815. While effector-target conjugate formation was observed with YAC-1 cells but not P815 cells in control cultures, it was augmented with both target cell types in cultures with PHA. Pretreatment alone with PHA was ineffective, however. NK cell activity of Kurloff cells was dependent on extracellular Ca++ and entry of Ca++ into the effector cells, as demonstrated by abrogation of cytotoxicity when extracellular Ca++ was chelated with EDTA or EGTA, or following treatment with the Ca++ channel blockers verapamil and diltiazem. Furthermore, inhibition of PKC by H7 resulted in significant reduction of Kurloff cell-mediated NK activity, while pretreatment of effector cells with the PKC activator TPA enhanced NK activity. Kurloff cells could also be stimulated to produce serine esterases by contact with target cells or treatment with phorbol ester and ionophore. Finally, a majority of Kurloff cells, identified by the monoclonal antibody 14D1, reacted with the human NK cell marker CD56. Taken together, these data suggest that Kurloff cells have NK-like characteristics and activity, with target cell selectivity, and that their lytic mechanisms involve influx of extracellular Ca++, PKC activation and serine esterase production.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

ET-1 induces IL-6 gene expression in human umbilical vein endothelial cells: synergistic effect of IL-1.

Endothelins are a family of potent vasoactive peptides that have also been implicated in inflammation. To examine the consequence of endothelin stimulation on cytokine production, we explored the effect of endothelin-1 (ET-1) on interleukin-6 (IL-6) gene expression. ET-1 augmented the production of IL-6 in human umbilical vein endothelial cells (HUVEC) by 2- to 5-fold and synergized with IL-1 to induce a 150-fold increase in IL-6 secretion, compared with spontaneous production. The peak of IL-6 production was in the presence of 100 pM ET-1 after 12 h of stimulation. ET-1 augmented IL-6 mRNA expression at the optimal concentration of 100 pM, in the presence of 1 ng/ml of IL-1. ET-1-induced IL-6 mRNA expression was significant within 4 h of stimulation and persisted for 18 h. Similarly, the ETB receptor-specific agonist, IRL-1620, also augmented IL-6 mRNA expression and IL-6 protein production in HUVEC. Our results suggest that endothelin may play a role at the vascular inflammatory site by modulating cytokine production through ETB receptor activation in endothelial cells of human origin.

Cells, Cultured↗
PubMed1996

Modulation of cardiac cell Ca2+ currents by PAF.

Using the whole-cell voltage clamp technique, PAF was found to stimulate T- and L-type Ca2+ currents in a dose dependent manner in both human and chick ventricular single cells. However, PAF had no effect on either the fast sodium current or the delayed outward K+ current in these heart cells. The effect of PAF on both Ca2+ currents was blocked by the PAF receptor antagonist WEB2170. Treatment of single ventricular cells with Pertussis toxin (PTX) prevented stimulation of T- and L-type calcium currents by PAF. These results suggest that there are functional PAF receptors in both human and chick ventricular cells. The stimulation of both types of Ca2+ currents seems to be mediated via a PTX sensitive G-protein.

Animals↗
PubMed1994

Transforming growth factor-beta regulates c-kit message stability and cell-surface protein expression in hematopoietic progenitors.

The cell-surface receptor c-kit and its cognate ligand stem-cell factor (SCF) or steel factor (SLF) are important for the maintenance of hematopoiesis both in vitro and in vivo. Transforming growth factor-beta (TGF-beta) has been shown to be a potent inhibitor of SLF-mediated synergistic growth of murine Lin-Sca-1+ progenitor cells, as well as more committed progenitors. In the present study, we examined the regulation of c-kit mRNA and cell-surface expression by TGF-beta. Among the murine hematopoietic progenitor cells tested, the myeloid cell line FDC-P1 and the mast-cell line MC-6, as well as progenitor-enriched bone marrow cells, constitutively expressed functional cell-surface c-kit. Treatment of these progenitor cell lines and primary progenitor cells with TGF-beta resulted in downregulation of cell-surface c-kit expression. This effect was not a secondary event of cell-cycle status. TGF-beta inhibition was dose- and time-dependent, with 50% inhibition seen between 0.3 to 3 ng/mL TGF-beta and maximal inhibition at 30 ng/mL. Using the FDC-P1 cell line, we observed that the inhibition of cell-surface c-kit expression by TGF-beta is preceded by a marked reduction in c-kit mRNA levels starting 2 hours after TGF-beta treatment, and reaches a maximum by 6 hours. The inhibition in steady-state c-kit mRNA levels is explained, in part, by a decrease in the half-life of c-kit transcripts (2 to 4 hours for control cells v 0.5 to 1.5 hours for TGF-beta-treated cells). These findings suggest that TGF-beta regulates the responsiveness of murine hematopoietic progenitors to SLF through a decrease in c-kit message stability leading to decreased cell-surface expression.

Animals↗