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W Fiers

Publications and source records attributed to W Fiers.

At least 91 records · Page 5Linked to original sources
PubMed1996

alpha1-Antitrypsin inhibits the lethal response to TNF in mice.

alpha1-Antitrypsin, an acute-phase reactant in many species, protects significantly against the lethality induced by TNF or endotoxin in mice. The protection is optimal with a single dose of at least 300 microg i.p. or 100 microg i.v. given 2 h before a lethal challenge, either with a low dose of TNF in the presence of galactosamine or a higher dose of murine TNF alone. Under optimal conditions, the drop in body temperature, the release of liver transaminases, and the increase in clotting time are also inhibited. alpha1-Antitrypsin does not protect against a lethal dose of platelet-activating factor. It is suggested that the protection is due to reduced release of platelet-activating factor.

Alanine Transaminase↗
PubMed1996

Functional characterization of the prodomain of interleukin-1beta-converting enzyme.

Interleukin-1beta-converting enzyme (ICE) has been identified as the main protease responsible for maturation of the prodomain of interleukin-1beta. Recently, it was shown to belong to a larger gene family, members of which play an important role in programmed cell death. A common feature of the ICE family proteases is the presence of a prodomain that has been hypothesized to keep the enzyme in an inactive form. Expression analysis in yeast revealed autocatalytic degradation of p45ICE, but not of p30ICE lacking a prodomain. We further demonstrate that p45ICE, in which the critical cysteine has been mutated, is still able to dimerize in vivo. Dimerization requires the prodomain and occurs prior to autoprocessing. These results provide evidence for a regulatory role of the prodomain of ICE.

Animals↗
PubMed1996

ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines.

We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1398-nucleotide 3'-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor alpha (TNFalpha) or interleukin (IL)-1beta but not with IL-4 or interferon gamma (IFNgamma) alone. In addition, when IFNgamma was combined with TNFalpha, IFNgamma inhibited the TNFalpha-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.

Amino Acid Sequence↗
PubMed1996

Synergistic induction of endothelial tissue factor by tumor necrosis factor and vascular endothelial growth factor: functional analysis of the tumor necrosis factor receptors.

Tissue factor expression on the surface of endothelial cells can be induced by tumor necrosis factor (TNF) and vascular endothelial growth factor (VEGF) in a synergistic manner. We have investigated the role of the two different TNF receptors for this synergy. Firstly, stimulation of the 60 kDa TNF receptor (TNFR60) by a mutant of TNF specific for TNFR60 induced responses comparable to wild-type TNF. In contrast, stimulation of TNFR80 by a TNFR80-specific TNF mutein did not result in enhancement of tissue factor expression even in the presence of a suboptimal TNFR60 triggering. Secondly, we tested neutralizing TNF receptor antibodies for inhibition of tissue factor synthesis induced by VEGF and TNF. A TNFR60-specific antibody inhibited tissue factor production over a broad range of TNF concentrations, indicating an essential role of TNFR60 in the TNF/VEGF synergy. In contrast, blocking of TNF binding to TNFR80 strongly inhibited TNF-induced tissue factor expression at low, but less pronounced at high, TNF concentrations. In conclusion, these data are in agreement with a model in which TNFR80 participates in the synergy between VEGF and low concentrations of soluble TNF by passing the ligand to the signalling TNFR60.

Antibodies↗
PubMed1996

Murine IL-12 is involved in Calmette-Guérin bacillus-induced sensitization and is by itself sufficient to sensitize mice to the lethal effects of human TNF1.

Human TNF, a specific agonist of murine (m)TNF-R55, is fairly harmless in healthy mice, but readily induces lethality in animals sensitized by a malignant tumor or a bacterial infection. We here report that IL-12 is both a necessary and sufficient mediator in Calmette-Guérin bacillus-induced sensitization to the lethal effects of hTNF. Evidence for this involvement is provided by the protection observed after anti-mIL-12 Ab treatment and by the fact that Calmette-Guérin bacillus-induced sensitization can be mimicked by repetitive mIL-12 administration. IL-12 exerts this effect by inducing IFN-gamma in NK cells. Tumor-induced sensitization, however, does not seem to involve mIL-12.

Animals↗
PubMed1996

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.

Amino Acid Sequence↗
PubMed1996

A20, an inhibitor of cell death, self-associates by its zinc finger domain.

A20 is a primary response gene which is induced after monocyte adherence or cytokine stimulation of a variety of cells. The A20 protein belongs to a novel class of Cys2/Cys2 zinc finger proteins, and has been characterized as an inhibitor of both apoptotic and necrotic cell death. In order to clarify its molecular mechanism of action, we used the yeast-based two-hybrid system to screen for A20-associated proteins. Here we report that A20 is able to self-associate, and demonstrate that the latter interaction is mediated by its zinc finger domain.

Base Sequence↗
PubMed1996

The oxidative metabolism of glutamine. A modulator of reactive oxygen intermediate-mediated cytotoxicity of tumor necrosis factor in L929 fibrosarcoma cells.

Treatment of the mouse fibrosarcoma cell line L929 with tumor necrosis factor (TNF) induces necrotic cell death. A crucial step in the cytotoxic action mechanism of TNF involves perturbation of mitochondrial functions leading to the formation of reactive oxygen intermediates (ROI). L929 cells have energy requirements adapted to a high proliferation rate. Glutamine (Gln) is utilized as a major energy source and drives mitochondrial ATP formation, while glucose is mainly converted to lactate through glycolysis. We investigated the role of the bioenergetic pathways involved in substrate utilization on the cytotoxic action of TNF and established a link between Gln oxidation and TNF-induced mitochondrial distress. Omission of Gln from the medium desensitizes the cells to TNF cytotoxicity, while the lack of glucose in the medium does not alter the TNF response. Sudden depletion of Gln from the culture medium results in a sharp decline in mitochondrial respiration in the cells, which might explain the decreased TNF responsiveness. However, when L929 cells are adapted to long term growth under conditions without Gln, these so-called L929/Gln- cells have restored respiration, but they still display a decreased sensitivity to TNF cytotoxicity. Thus the TNF responsiveness of L929 cells depends on bioenergetic reactions that are specifically involved in the oxidation of Gln. This is further confirmed by the desensitizing effect of specific inhibitors of these Gln-linked enzyme reactions on TNF cytotoxicity in the parental cells, but not in the L929/Gln- cells. Analysis of the induction of mitochondrial ROI formation by TNF in parental and L929/Gln- cells suggests that the effect of Gln on the sensitivity to TNF cytotoxicity involves a mechanism that renders the mitochondria more susceptible to TNF-induced mediators, resulting in enhanced ROI production and accelerated cytotoxicity.

Animals↗
PubMed1996

Tumour growth results in changes in placental amino acid transport in the rat: a tumour necrosis factor alpha-mediated effect.

The implantation of a fast growing tumour (Yoshida AH-130 ascites hepatoma) to late pregnant rats resulted in no changes in fetal growth, this possibly being associated with an important increase in the fetal uptake of maternal-derived amino acids [Carbó, López-Soriano and Argilés (1995) Endocrinology 136, 3579-3584]. The present investigation was undertaken to see whether the presence of the tumour induced changes in placental transport systems. For alanine transport, although no changes in affinity (Km) were observed, tumour growth resulted in a 192% increase in Vmax in the Na(+)-independent component. Kinetic analysis of the Na(+)-dependent component resulted in two clearly different components: while the low-affinity and high-capacity component was unaffected by tumour growth, the high-affinity, low-capacity component of the tumour-bearing rats showed an important increase in Vmax. (78%). With regard to leucine transport, tumour burden induced important increases in the Na(+)-independent component, not only in Km (262%) but also in Vmax. (189%). Since elevated tumour necrosis factor-alpha (TNF) concentrations have been reported in this kind of tumour model, we performed the same type of transport experiments in rats chronically treated with TNF, the results obtained showing great similarities with those observed with tumour growth. The Vmax. of Na(+)-independent alanine transport was also increased by the cytokine (104%) while no changes were observed in affinity. TNF treatment also induced an increase in the Vmax. (67%) of the Na(+)-dependent (high-affinity, low-capacity) component while no changes in affinity were observed. Concerning leucine kinetics, TNF treatment, as in the case of tumour growth, also increased Km (155%) and Vmax. (72%) associated with Na(+)-independent transport. Interestingly, treatment with the cytokine increased both the Km (43%) and Vmax. (64%) of the Na(+)-dependent component. The inhibition patterns suggest the existence of more that one Na(+)-dependent transport for alanine although the majority of the amino acid is transported through the A system. The results presented suggest that, during gestation, the mother is able to adapt her placental amino acid transport systems to compensate for the nitrogen drainage associated with tumour growth and thus provide the fetus with enough amino acids to allow its normal growth, and that TNF could be responsible for the triggering of this compensatory mechanism.

Alanine↗
PubMed1996

Molecular and immunological characterization of soluble aggregated A/Victoria/3/75 (H3N2) influenza haemagglutinin expressed in insect cells.

A/Victoria/3/75 (H3N2)-derived cDNA coding for a secreted haemagglutinin (HA0s) was cloned into the polyhedrin promoter-based pVL1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 micrograms/ml of secreted HA were obtained following infection of Spodoptera frugiperda-9 cells. Gel filtration revealed the presence in the cell supernatant of immunoreactive HA molecules with varying M(r). The high M(r) fraction (aHA0s) could be purified by Matrex Cellufine Sulphate and Lentil-lectin affinity chromatography, followed by Sephacryl S300 HR gel filtration. aHA0s consisted of aggregated, non-covalently linked subunits which were not cleaved into HA1 and HA2 polypeptides; aHA0s was highly susceptible to trypsin treatment and reacted with two low pH-specific monoclonal antibodies, suggesting that a HA0s consists of monomeric subunits. When the expression medium was adjusted to pH 8.5, no aHA0s was observed, suggesting that aggregation occurred in the cells due to a low intracellular pH. Balb/c mice immunized with purified aHA0s developed high, aHA0s-specific antibody titres. Despite these high titres, almost no binding to trimeric viral HA was observed, and immunized mice were not protected against a challenge with homologous mouse-adapted X47 virus. However, when virus was subjected to low pH, resulting in a profound conformational rearrangement, strong binding was observed. Moreover, binding to the low pH-treated HA of different drift variants, isolated between 1968 and 1989, occurred.

Animals↗
PubMed1996

Recombinant neuraminidase vaccine protects against lethal influenza.

The N2 neuraminidase gene of A/Victoria/3/75 influenza virus was engineered to encode a secretable protein (NAs) by replacing the natural N-terminal membrane anchor sequence with the cleavable signal sequence of the corresponding influenza hemagglutinin gene. Soluble NAs was expressed by a baculovirus/insect cell system and accumulated in the medium at levels between 6 and 8 microgram ml-1. A combination of biochemical and standard chromatographic techniques allowed the purification of NAs to homogeneity. Cross-linking analysis indicated that NAs was partly recovered as an authentic tetrameric protein, while the remaining fraction was composed of dimeric molecules and small amounts of monomeric NAs. Purified NAs was supplemented with low-reactogenic adjuvants and used to immunize mice. After a challenge infection with a lethal dose of homologous mouse-adapted X47 influenza virus, vaccinated animals showed resistance against severe disease symptoms and were protected from lethality. Based on the results of a passive immunization experiment, it may be concluded that performed antibody plays a central role in the mechanism by which vaccination with NAs confers viral protection.

Animals↗
PubMed1996

Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia.

IL-10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice from lipopolysaccharide (LPS)-induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL-10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2-10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL-10, while a high methylprednisolone dose (50 mg/kg) increased LPS-induced IL-10 levels. In parallel, we observed that LPS-induced IL-10 production is TNF-independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0.01 to 100 micrograms/ml) also increased the biosynthesis of IL-10 by LPS-activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL-10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.

Adjuvants, Immunologic↗
PubMed1996

Abnormal glutathione and sulfate levels after interleukin 6 treatment and in tumor-induced cachexia.

Excessive urea excretion associated with a negative nitrogen balance and massive loss of skeletal muscle mass (cachexia) is a frequent life threatening complication in malignancies and HIV infection. As these patients have often elevated interleukin-6 (IL-6) and abnormally low cystine levels, we have now determined the intracellular levels of glutathione and other cysteine derivatives in the liver and muscle tissue of IL-6-treated or tumor-bearing C57BL/6 mice. IL-6 treatment or inoculation of the MCA-105 fibrosarcoma caused a significant increase in hepatic gamma-glutamyl-cysteine synthetase activity and a decrease in the sulfate level, glutamine/urea ratio, and glutamine/glutamate ratio, suggesting that a decrease of the proton generating cysteine catabolism in the liver may increase carbamoyl-phosphate synthesis and urea formation at the expense of net glutamine synthesis. Treatment with cysteine, conversely, caused an increase in sulfate, glutamine/urea ratios, and glutamine/glutamate ratios and may thus be a useful therapeutic tool in clinical medicine. In contrast to the liver, muscle tissue of tumor-bearing mice showed decreased glutathione and increased sulfate levels, suggesting that the cysteine pool may be drained by an increased cysteine catabolism in this tissue. The findings indicate that tumor cachexia is triggered initially by IL-6 and is later sustained by processes driven by an abnormal cysteine metabolism in different organs.-Hack, V., Gross, A., Kinscherf, R., Bockstette, M., Fiers, W., Berke, G., and Dröge, W. Abnormal glutathione and sulfate levels after interleukin 6 treatment and in tumor-induced cachexia.

Animals↗
PubMed1996

The expression of the Photinus pyralis luciferase gene in Staphylococcus aureus Cowan I allows the development of a live amplifiable tool for immunodetection.

We expressed the luc gene, encoding luciferase from Photinus pyralis, in Staphylococcus aureus Cowan I downstream of the plasmid-borne promoter for protein A. Constitutive luciferase synthesis did not impair the growth rate of the host nor did it affect the stability of the plasmid. Light production started immediately after addition of luciferin. The kinetic profile is of the glowing rather than the peak type. Because S. aureus Cowan I produces large quantities of protein A, of which a substantial part becomes covalently attached to rigid cell walls, the bacterial cells could be specifically immobilized on a substrate to which immunoglobulin G molecules were adsorbed either directly or as secondary antibodies. Light production from these cells can be used as a reporter tool for the detection of antigen-antibody complexes. Fourfold amplifications of the emitted signals were obtained by in situ incubation of the bound cells in bacterial growth medium.

Animals↗
PubMed1996

Increased stability of phage T7g10 mRNA is mediated by either a 5'- or a 3'-terminal stem-loop structure.

The mRNA encoding the major capsid protein of phage T7 (T7g10) is highly expressed in Escherichia coli. In common with other highly expressed T7 genes, the 5' end of this mRNA contains a stem-loop structure, while transcription termination at the phage T7 T phi terminator generates a stable 3'-end stem-loop structure. We assessed the influence of these structures on the expression level of T7g10 and on the functional stability of the mRNA. Each one of the 5'- or 3'-hairpin structures was sufficient to increase the functional stability of the T7g10 mRNA more than twofold. A duplication of the 3' T phi-terminator slightly increased the mRNA stability further. Also, differences in the observed functional half-life could be correlated with the expression level of the T7g10 derivatives when these were partially induced. Our data suggest that mRNA stabilization by a 5' stem-loop structure can occur even in the absence of a stem-loop structure that protects RNA against 3' exonucleases.

Bacteriophage T7↗
PubMed1995

Anti-tumor activity of tumor necrosis factor in combination with interferon-gamma is not affected by prior tolerization.

Repetitive administration of low doses of tumor necrosis factor (TNF) results in a state of selective tolerance to some of its effects. We have demonstrated that tolerance does not impair the therapeutic efficacy of TNF against a syngeneic murine B16BL6 melanoma and allows a complete cure. Another study, performed with a distinct tumor model, came to apparently contradictory results. To clarify this, we investigated whether the outcome depended on the tumor type and on the inclusion of interferon-gamma (IFN gamma) in the treatment. Three syngeneic tumors of different histological origin, i.e., B16BL6 melanoma, Lewis lung carcinoma (LLC) and EL4 lymphoma, were compared in C57BL/6 mice. The anti-tumor efficacy of TNF against B16BL6 and EL4 was not impaired in tolerant mice, but the effect of TNF against LLC was slightly, though significantly, reduced. Inclusion of IFN gamma in the treatment regimen, however, abolished this difference and resulted in complete cure for all 3 tumor systems. As therapeutically optimal doses were lethal in normal mice, only tolerance allowed a long-term cure. We conclude that the influence of tolerance on the anti-tumor activity of TNF as a single agent depends on the tumor type; in combination therapy with IFN gamma, however, tolerance allowed us to dissociate lethal toxicity from anti-tumor activity, irrespective of the tumor type tested.

Animals↗
PubMed1995

Versatile, multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: overproduction of human and murine cytokines.

We describe the construction, expression characteristics and some applications of a versatile dual-promoter expression plasmid for heterologous gene expression in Escherichia coli which contains both lambda pL and PT7 promoters. Furthermore, the plasmid is optimized to allow the expression of mature coding sequences without compromising the strength of the highly efficient PT7 or of the T7g10 ribosome-binding site. The effect of the the naturally occurring RNA loops at both the 5' and 3' ends of the T7g10 mRNA on expression was also examined. A double T7 RNA polymerase transcription terminator was inserted to ensure more reliable transcription termination and a higher expression level of the preceding gene. Further improvements involve a clockwise orientation of the promoters to minimize read-through transcription from plasmid promoters, a largely extended multiple cloning site, an antisense phage T3 promoter and a phage f1-derived, single-stranded replication origin. Variants of this plasmid allow for the production of fusion proteins with part of T7g10, a hexahistidine peptide and an enterokinase recognition site. The potential of these expression vectors is demonstrated by comparing the expression levels of a number of mammalian cytokines (human tumor necrosis factor, human immune interferon, human and murine interleukins 2, murine interleukin 4 and murine fibroblast interferon), using these expression plasmids.

Amino Acid Sequence↗