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Biomedical subjects

K M Stuhlmeier

Publications and source records attributed to K M Stuhlmeier.

At least 19 recordsLinked to original sources
PubMed2000

Activation and regulation of Hsp32 and Hsp70.

Endothelial cells (EC) play a key role in the propagation of inflammatory responses. Better understanding of inflammatory processes in EC might provide new ways of controlling inflammation. We report here that the known antioxidant pyrrolidinedithiocarbamate (PDTC) leads to time and dose dependent activation of heat-shock protein 70 (Hsp70) as well as Hsp32 in EC. We further demonstrate that PDTC activates heat-shock factor 1 (HSF1), one of several transcription factor involved in the upregulation of heat-shock proteins. And more importantly, we demonstrate that Hsp32 as well as Hsp70 can be upregulated independently of the transcription factor nuclear factor kappaB (NF-kappaB). The presented data provide further insight into the mechanism of Hsp32 and Hsp70 regulation, as well as further distinguishing these genes from other so called 'protective genes' whose upregulation depends on the activation of NF-kappaB. These findings indicate that Hsp32 and Hsp70 might be ideal candidates among protective genes. Hsp32 and Hsp70 provide many desirable protective effects but, being independent of NF-kappaB, would leave open the option to interfere with the upregulation of proinflammatory genes by modulating the activation of NF-kappaB.

Adenoviridae↗
PubMed1999

Camouflaging endothelial cells: does it prolong graft survival?

Camouflaging antigens on the surface of cells seems an appealing way to prevent activation of the immune system. We explored the possibility of preventing hyperacute rejection by chemically camouflaging endothelial cells (EC). In vitro as well as in vivo experiments were performed. First, the ability of mPEG coating to prevent antibody-antigen interactions was evaluated. Second, we tested the degree to which mPEG coating prevents activation of EC by stimuli such as TNF-alpha and LPS. Third, in vivo experiments were performed to test the ability of mPEG coating to prolong xenograft survival. We demonstrate that binding of several antibodies to EC or serum proteins can be inhibited by mPEG. Furthermore, binding of TNF-alpha as well as LPS to EC is blocked since mPEG treatment of EC inhibits the subsequent up-regulation of E-selectin by these stimuli. However, in vivo experiments revealed that currently this method alone is not sufficient to prevent hyperacute rejection.

Animals↗
PubMed1999

Ibuprofen: new explanation for an old phenomenon.

Nuclear factor-kappaB (NF-kappaB) translocation from the cytoplasm into the nucleus and the subsequent DNA binding is an essential prerequisite in the up-regulation of many pro-inflammatory genes, e.g. tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). The anti-inflammatory drug ibuprofen, thought to exert its beneficial effects mainly by suppressing the production of eicosanoids, inhibited the up-regulation of the pro-inflammatory cytokines IL-1beta and TNF-alpha. This effect was independent of the described potential of ibuprofen as a cyclooxygenase inhibitor. Ibuprofen inhibited the activation and translocation of the key transcription factor NF-kappaB by blocking the degradation of inhibitor-kappaBalpha, a protein that forms a complex with NF-kappaB, thereby preventing the release and subsequent translocation of NF-kappaB into the nucleus and the expression of inflammatory cytokines. The presented data offer a new explanation for the anti-inflammatory effect of ibuprofen.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Accommodation.

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Animals↗
PubMed1998

Extracellular ATP and ADP activate transcription factor NF-kappa B and induce endothelial cell apoptosis.

Inflammation within the vasculature is associated with endothelial cell (EC) perturbation, loss of vascular ATP-diphosphohydrolase activity, and platelet microthrombus formation with release of ATP and ADP into the micro-environment. The nature and effects of purinergic stimulation of EC under these circumstances remain largely undetermined. ATP and ADP activated EC transcribed mRNA from certain transcription factor NF-kappa B target genes and expressed E-selectin protein on cell membranes. Band shift analysis and reporter assays confirmed the activation of NF-kappa B in response to both ATP and ADP. Apoptosis was shown to occur in response to purinergic signaling, potentially through the activation of P2z/P2x7 receptors. Induction of EC activation responses and apoptosis in response to stimulation with ATP and ADP is associated with activation of NF-kappa B.

Adenosine Diphosphate↗
PubMed1998

Importance of glucose-6-phosphate dehydrogenase activity for cell growth.

The intracellular redox potential, which is determined by the level of oxidants and reductants, has been shown to play an important role in the regulation of cell growth. The principal intracellular reductant is NADPH, which is mainly produced by the pentose phosphate pathway through the actions of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and by 6-phosphogluconate dehydrogenase. Previous research has suggested that an increase in G6PD activity is important for cell growth. In this article, we suggest that G6PD activity plays a critical role in cell growth by providing NADPH for redox regulation. The results show the following: 1) inhibition of G6PD activity abrogated growth factor stimulation of [3H]thymidine incorporation in all cell lines tested; 2) overexpression of G6PD stimulated cell growth, as measured by an increase in [3H]thymidine incorporations as compared with cells transfected with vector alone; 3) inhibition of G6PD caused cells to be more susceptible to the growth inhibitory effects of H2O2; 4) inhibition of G6PD led to a 30-40% decrease in the NADPH/NADP ratio; and 5) inhibition of G6PD inhibited cell anchorage and significantly decreased the growth-related stimulation of tyrosine phosphorylation.

3T3 Cells↗
PubMed1997

Arachidonic acid influences proinflammatory gene induction by stabilizing the inhibitor-kappaBalpha/nuclear factor-kappaB (NF-kappaB) complex, thus suppressing the nuclear translocation of NF-kappaB.

Arachidonic acid (AA), through its myriad metabolites, is involved in inflammation in a number of ways. AA is produced and released by several cell types, including endothelial cells (EC), and acts on a variety of cells. EC activation plays a key role in inflammation presumably by modulating the immune response through up- or down-regulation of several genes. We have previously shown that AA and its nonmetabolizable analogue, 5,8,11,14-eicosatetraynoic acid (ETYA), inhibit up-regulation of proinflammatory genes in EC. In the present study we identify a mechanism to explain the inhibitory effects: AA and ETYA both inhibit the translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus by blocking the degradation of the inhibitor of NF-kappaB (IkappaB) and thus stabilizing the IkappaB/NF-kappaB complex. To investigate the mechanism whereby AA inhibits up-regulation of genes encoding proinflammatory mediators, we examined the ability of ETYA to inhibit tumor necrosis factor-alpha (TNF-alpha) mediated phosphorylation and degradation of IkappaBalpha. Western blot analysis revealed that preincubation of EC with ETYA for 40 min prior to stimulation with TNF-alpha inhibits the phosphorylation and degradation of IkappaBalpha. These findings establish a mechanism by which AA inhibits nuclear translocation of NF-kappaB and thereby explaining its modulatory role in the induction of proinflammatory genes.

Animals↗
PubMed1997

The effect of 5,8,11,14-eicosatetraynoic acid on endothelial cell gene expression.

The endothelium plays a key role in inflammation, hemostasis and organ rejection. We report here that a synthetic polyunsaturated fatty acid, 5,8,11,14-eicosatetraynoic acid (ETYA), selectively inhibits the up-regulation of several genes on endothelial cells. ETYA suppresses endothelial cell activation by inhibiting the up-regulation of adhesion molecules like E-selectin. A runoff assay for E-selectin demonstrated that the suppression is at the level of transcription. The fact that ETYA inhibits E-selectin upon stimulation with a diverse group of stimuli like lipopolysaccharide, tumor necrosis factor-alpha or phorbol 12-myristate 13-acetate, suggests that ETYA does not exert its effect by modifying membrane-bound receptors. The messenger RNA for interleukin-8 and glyceraldehyde phosphate dehydrogenase are not affected. Pre-treatment of endothelial cells with ETYA also prevents the adherence of monocytes to tumor necrosis factor-alpha-stimulated cells.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1997

Human monocytes activate porcine endothelial cells, resulting in increased E-selectin, interleukin-8, monocyte chemotactic protein-1, and plasminogen activator inhibitor-type-1 expression.

Monocytes (Mo) are thought to be important effector cells in early xenograft rejection. Effects of Mo-endothelial cell (EC) interactions on EC activation in vitro were studied by coculturing human Mo or human monocytoid cell lines, U937 and THP-1, with porcine EC. Without preactivation, U937 cells and Mo induced mRNA for the EC-specific adhesion receptor, E-selectin, expressed only on activated cells, after 2 hr. Surface protein was maximal when equal numbers of EC and Mo were cocultured. Increased mRNA expression of the chemokines, interleukin-8 and monocyte chemotactic protein-1, and the antifibrinolytic protein plasminogen activator inhibitor type-1, confirmed EC activation. Like E-selectin, plasminogen activator inhibitor type-1 mRNA was rapidly induced and returned to baseline after 24 hr, whereas chemokine gene expression was slower and more prolonged. Interleukin-1 receptor antagonist failed to modulate induction of E-selectin. Soluble tumor necrosis factor (TNF) alpha receptor inhibited E-selectin induced by TNF alpha, but not by U937 cells, and mRNA and protein on EC in Mo-EC mixtures cocultured at 1:1 ratios were not significantly reduced. The TNF alpha inhibitor did reduce E-selectin expression (30-40%), as well as induced chemokine gene expression (80%), at higher Mo-EC ratios. Despite this, minimal TNF alpha was detectable in supernatants. These results, along with the transwell experiments that confirmed a requirement for Mo-EC contact, suggest that membrane-bound TNF alpha may be involved. Thus, Mo-EC interactions in the porcine to human combination activated several EC functions, suggesting that initial Mo contact with the vessel wall of a xenogeneic graft may promote leukocyte recruitment, inflammation, and maintenance of thrombus, resulting in eventual organ destruction.

Animals↗
PubMed1996

Selective suppression of endothelial cell activation by arachidonic acid.

Endothelial cell (EC) activation plays a key role in inflammation, thrombosis and organ rejection. Normally, EC are in a quiescent state in which their function is to prevent coagulation and thrombosis, and to participate in the regulation of leukocyte migration from the bloodstream into the tissue. Upon activation with cytokines or other stimuli, EC up-regulate a number of genes, including E-selectin (ELAM-1), intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, interleukin (IL)-1, IL-8, tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), MCP-1 (monocyte chemoattractant protein-1) and endothelial cell inducible gene (ECI-6). Arachidonic acid (AA) is produced by several cell types, including EC, and acts on various cells. We report here that AA inhibits the up-regulation of some, but not all genes that are induced with EC activation in a dose-dependent manner. AA suppresses TNF-alpha, IL-1 alpha, LPS or PMA-induced E-selectin expression, as well as mRNA accumulation of E-selectin, ICAM-1 and IL-8 stimulated by TNF-alpha. The inhibition appears to be at the level of transcription. At the same time under the same conditions AA does not, repress mRNA accumulation for PAI-1, ECI-6, MCP-1 and VCAM-1. We suggest that the induced expression of AA with EC activation may result in a negative feedback loop regulating further activation.

Animals↗
PubMed1994

Selective inhibition of E-selectin, ICAM-1, and VCAM in endothelial cells.

Endothelial cells, as they normally exist in the vasculature as quiescent cells, perform several functions. In an inflammatory response, endothelial cells are activated to up-regulate a number of genes, including E-selectin (ELAM-1), VCAM-1, ICAM-1, interleukin (IL)-1, IL-8 and plasminogen activator inhibitor-1 (PAI-1). Very little is known about factors that regulate the activation process. We describe here that a heat-stable protein, normally present in the alpha-globulin fraction of serum, inhibits induced expression of E-selectin, ICAM-1, and VCAM-1 in vitro and also impedes the accumulation of mRNA for these molecules. Inhibition of E-selectin, the only gene tested in this respect, is at the level of transcription. At the same time, the alpha-globulins do not, under the same conditions, repress mRNA accumulation for IL-1, IL-8, or PAI-1. The effect of the inhibitor does not relate to constraints on function of nuclear-factor kappa B, the induced activity of which is not interfered with at the early time points at which the suppression of these three genes is seen.

Alpha-Globulins↗
PubMed1992

A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate.

A fluorometric microplate assay was established for the detection of respiratory burst activity in phagocytic cells by assessing oxidation of 2',7'-dichlorofluorescin-diacetate (DCFH-DA). This method is based on flow cytometric studies by Bass et al. (J. Immunol. 130 (1983) p. 1910) describing intracellular detection of DCFH oxidation due to the presence of hydrogen peroxides. In the present study we have adapted the assay for use in microtiter plates to determine the amount of extracellular reactive oxidative products. DCFH-DA, granulocytes and stimuli (phorbol myristate acetate, n-formyl-methionyl-leucylphenylalanine, concanavalin A) were added to microtiter plates and after incubation at 37 degrees C, the development of fluorescence intensity was read in a fluorescence concentration analyzer (FCA, Baxter). Calibration of fluorescence units recorded by the FCA was achieved by comparison with defined amounts of fluorescent DCF. The change in measured fluorescence was linear with cell density over the range of 2 x 10(5)-1 x 10(6) cells/well. Cumulative DCF generation in individual wells could be recorded non-destructively at frequent intervals for time course measurements. Results from FCA measurements correlated perfectly with the FACS analysis of the same samples (r = 0.99). In conclusion, this assay can be useful for screening monoclonal antibodies recognizing cell surface structures possibly involved in signal transduction as well as for testing phagocytes for their capacity to release reactive oxidative intermediates.

Flow Cytometry↗
PubMed1992

Cytokine release and dynamics of leukocyte populations after CD3/TCR monoclonal antibody treatment.

Cytokine release and clinical side effects resulting from the use of OKT3 and BMA 031 monoclonal antibodies in the treatment of kidney graft recipients were evaluated and compared. The rise observed in serum levels of interferon gamma. TNF alpha, and IL-8 was similar after administration of either monoclonal antibody. Furthermore, both OKT3 and BMA 031 resulted in rapid disappearance not only of virtually all T cells, but also of substantial percentages of all major leukocyte populations from the circulation; this effect is probably due to cytokine release activating endothelial cells and thereby causing extravasation even of leukocytes not specifically recognized by the administered antibodies. Evidence has thus been obtained that BMA 031 is as potent as OKT3 in inducing unequivocal signs of T cell activation in vivo. However, while OKT3 therapy was accompanied by adverse side effects in our study as in previous ones, we saw no such reactions in any of the patients receiving BMA 031. This contrast might be due to different mechanisms of leukocyte activation possibly inducing other mediators in the case of OKT3, which then, in combination with the cytokines, could generate treatment-associated morbidity.

Antibodies, Monoclonal↗