Biomedical subjects

W Fiers

Publications and source records attributed to W Fiers.

At least 145 records · Page 8Linked to original sources

PubMed1993

Enhancement of tumor necrosis factor cytotoxicity by lithium chloride is associated with increased inositol phosphate accumulation.

We have previously reported that LiCl increases considerably the cytotoxic activity of TNF towards some transformed cell lines such as L929. Here we show that treatment of these cell lines with the combination of TNF and LiCl leads to the prolonged accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, whereas treatment with TNF or LiCl alone did not. In contrast, both a LiCl-unresponsive TNF-sensitive cell line and TNF-resistant cell lines did not respond with increased accumulation of inositol phosphates (IPn) upon treatment with the combination of TNF and LiCl. Furthermore, the combination of TNF and LiCl induced a transient increase in cytidine diphosphate-diacylglycerol in L929 cells. Increased IPn and cytidine diphosphate-diacylglycerol accumulation preceded the onset of cell killing by approximately 1 h. TNF-mediated cytotoxicity and TNF-induced IPn accumulation were equally sensitive to inhibition by the phospholipase inhibitor neomycin and to stimulation by the protein kinase inhibitor staurosporine. Characterization of the inositol bisphosphate isomers by HPLC analysis revealed that the TNF + LiCl-induced increase in IPn levels was due to activation of a phospholipase C and not of a phospholipase D. In contrast to TNF, several other cytotoxic agents did not increase IPn production upon application in the presence of LiCl. The TNF + LiCl-induced increase in inositol triphosphate suggests a role for intracellular Ca2+ mobilization in TNF action. Moreover, several agents that lower the intracellular Ca2+ concentration inhibited TNF cytotoxicity. In conclusion, our data provide evidence that TNF cytotoxicity and its enhancement by LiCl are mediated by increased IPn accumulation resulting in Ca2+ mobilization.

Animals↗

PubMed1993

Sensitization of tumor cells to tumor necrosis factor action by the protein kinase inhibitor staurosporine.

Tumor necrosis factor (TNF), first described as a cytokine with tumor-necrotizing activity, is now known to be a pleiotropic molecule. The molecular mechanisms responsible for the cytotoxic activity of TNF on malignant cells are still largely unknown. In this study, we report that the protein kinase inhibitor staurosporine (56 to 1500 nM) increases about 500 times the in vitro cytotoxic activity of TNF for several murine and human tumor cell lines. Even some tumor cell lines which are resistant to TNF cytotoxicity could be sensitized to TNF killing by staurosporine. In the L929 fibrosarcoma cell line, staurosporine also enhanced the transcriptional activation of interleukin 6 synthesis by TNF (500-fold stimulation at 56 nM). At the biochemical level, staurosporine increased the TNF-mediated activation of phospholipases C and D and the transcription factor NF-kappa B in L929 cells. The TNF-sensitizing effect of staurosporine does not seem to be mediated by one of the currently known staurosporine-sensitive kinases, as various other inhibitors which also inhibit one or more of these kinases were not synergistic with TNF. Interestingly, staurosporine (1 microgram) also enhanced the in vivo antitumor activity of TNF against a murine tumor model (L929 fibrosarcoma) in athymic nude mice (Swiss-nu/nu; s.c. treatment). These results suggest that TNF responsiveness of tumor cells is regulated by a novel staurosporine-sensitive target and that the combination of TNF and staurosporine may open new strategies of tumor treatment.

Alkaloids↗

PubMed1993

Folding of the MS2 coat protein in Escherichia coli is modulated by translational pauses resulting from mRNA secondary structure and codon usage: a hypothesis.

Possible translational pauses within the coat protein of the RNA bacteriophage MS2 were located on the basis of a distribution plot of rare codons and RNA secondary structure. It appeared that the position of certain codon pauses corresponds with the size of some nascent polypeptide intermediates, which have been isolated from MS2-infected cells. Other accumulated polypeptide intermediates seemed to be related to RNA regions, where double-stranded secondary structures occur, which probably impede the movement of ribosomes during chain elongation. We assume that a discontinuous translation rate is designed to allow optimal folding of this (and other) polypeptide(s).

Capsid↗

PubMed1993

Increased IL-6 production and IL-6-mediated Ig secretion in murine host-vs-graft disease.

BALB/c mice neonatally injected with semiallogenic (A/J x BALB/c)F1 splenocytes develop a host-vs-graft (HVG) reaction between host T cells and donor B cells, resulting in hypergammaglobulinemia, splenomegaly, and increased serum levels of various autoantibodies. This syndrome is associated with a polyclonal activation of the donor-derived B cells. High serum levels of IL-6 were found in 4-wk-old mice undergoing HVG disease (mean +/- SEM, 132 +/- 93 as compared with 12 +/- 2 in control mice, p < 0.05). Also supernatants of spleen cell cultures from HVG mice contained increased levels of IL-6. In situ hybridization and cell depletion experiments demonstrated that host macrophages were responsible for this pathologic IL-6 secretion. The spontaneous in vitro production of autoreactive antibodies by donor B cells from HVG mice was further enhanced by adding human rIL-6, whereas addition of human rIL-1 beta, human rIL-2, murine rIL-4, murine rIL-5, or combinations of these cytokines had no effect. Finally, addition of blocking anti-IL-6 and anti-IL-6 receptor mAb markedly reduced hyper IgG1 production in cultures of spleen cells from HVG mice. These data suggest that an increased production of IL-6 by persistently stimulated host macrophages is involved in the activation of donor B cells leading to HVG disease.

Animals↗

PubMed1993

Recombinant soluble human interleukin-5 (hIL-5) receptor molecules. Cross-linking and stoichiometry of binding to IL-5.

Recombinant soluble human interleukin-5 receptor alpha (shIL-5R alpha) has been expressed in COS-1 cells and in baculovirus-infected cells. The protein was purified from the supernatant by chromatography on concanavalin A-Sepharose, MonoQ, and a final gel filtration step. A chimeric fusion receptor protein (hIL-5R alpha-h gamma 3) was constructed by fusion of the cDNA corresponding to the shIL-5R alpha to the cDNA corresponding to the Fc part of the human IgG C gamma 3 chain, and was expressed in baculovirus-infected insect cells. The chimeric receptor was secreted as a disulfide-linked homodimer, and was purified by protein G affinity chromatography. In a solid-phase binding assay the shIL-5R alpha and the bivalent hIL-5R alpha-h gamma 3 were found to bind hIL-5 with a similar affinity, corresponding to the membrane-bound, low affinity hIL-5R alpha. SDS-polyacrylamide gel electrophoresis of shIL-5R alpha cross-linked to radiolabeled hIL-5, suggested that one shIL-5R alpha molecule binds to one hIL-5 homodimer molecule. Gel filtration studies of the complex formed between the shIL-5R alpha and hIL-5 pointed toward the same stoichiometry of binding. The formation of such a complex could be observed by electrophoresis in native gels. Immunoaffinity chromatography using a non-neutralizing monoclonal antibody directed against hIL-5, followed by size column chromatography, allowed the purification of the complex. The data obtained from the amino acid analysis of the constituents of the complex blotted from an SDS-polyacrylamide gel, and from the amino acid composition of the complex blotted from a native polyacrylamide gel, provided direct evidence that the shIL-5R alpha binds the hIL-5 dimer in a 1:1 ratio.

Animals↗

PubMed1993

Tumor necrosis factor cytotoxicity is associated with phospholipase D activation.

The activation of phospholipase D in different cell lines treated with recombinant human tumor necrosis factor (TNF) has been investigated. When the murine fibrosarcoma cell lines L929 and WEHI164c113, as well as the human promonocytic cell line U937, were prelabeled with [14C]palmitic acid or [3H]arachidonic acid, and treated with TNF in the presence of ethanol, TNF induced the synthesis of [14C]phosphatidylethanol or [3H]phosphatidylethanol, respectively, as the result of a phospholipase-D-catalyzed transphosphatidylation. TNF-induced phospholipase D activity was observed 1 h before the onset of cell killing and gradually increased thereafter. Subclones selected for resistance to TNF cytotoxicity did not show phospholipase D activation in response to TNF. In contrast, when these subclones were treated with TNF in the presence of actinomycin D, TNF cytotoxicity as well as TNF-induced phospholipase D activity could be restored. TNF cytotoxicity and TNF-induced phospholipase D activity were equally modulated by various drugs known to interfere with different steps in the TNF-signaling pathway. Phospholipase D activation was found not to be the result of cell killing per se, as a number of other cytotoxic reagents were unable to activate phospholipase D. Prelabeling of cells with [14C]lysophosphatidylcholine indicated phosphatidylcholine as one of the substrates for TNF-activated phospholipase D. In conclusion, this study demonstrates that TNF-induced cytotoxicity is associated with activation of phospholipase D.

Animals↗

PubMed1993

Interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia.

Because of its ability to efficiently inhibit in vitro cytokine production by activated macrophages, we hypothesized that interleukin (IL) 10 might be of particular interest in preventing endotoxin-induced toxicity. We therefore examined the effects of IL-10 administration before lipopolysaccharide (LPS) challenge in mice. A marked reduction in the amounts of LPS-induced tumor necrosis factor (TNF) release in the circulation was observed after IL-10 pretreatment at doses at low as 10 U. IL-10 also efficiently prevented the hypothermia generated by the injection of 100 micrograms LPS. Finally, pretreatment with a single injection of 1,000 U IL-10 completely prevented the mortality consecutive to the challenge with 500 micrograms LPS, a dose that was lethal in 50% of the control mice. We conclude that IL-10 inhibits in vivo TNF secretion and protects against the lethality of endotoxin in a murine model of septic shock.

Animals↗

PubMed1993

Human TNF mutants with selective activity on the p55 receptor.

The remarkable ability of tumour necrosis factor (TNF), especially in combination with interferon, selectively to kill or inhibit malignant cell lines is so far unmatched by any other combination of cytokines. But clinical trials in cancer patients have on the whole been disappointing, and it has been estimated that a TNF dose would be effective only at 5-25 times the maximum tolerated dose. High TNF concentrations give a much more pronounced antitumour activity in mice, in which murine TNF is about 50-fold more systemically toxic than human TNF. But there is little or no species specificity in cytotoxicity of murine TNF and human TNF on human as well as on murine cell lines. This dual action of TNF may be explained by the existence of two types of receptor for TNF: the smaller, TNF-R55, is present on most cells and particularly on those susceptible to the cytotoxic action of TNF; the larger, TNF-R75, is also present on many cell types, especially those of myeloid origin, and is strongly expressed on stimulated T and B lymphocytes. In mice, human TNF binds only to murine TNF-R55 (ref. 15), which can then mediate cytotoxic activity on malignant cells. As human TNF does not bind to murine TNF-R75, the latter must be responsible for the much enhanced systemic toxicity of murine TNF. Human TNF can, however, become toxic in mice when a second pathway is activated. There is no reciprocal situation in the human system: human and murine TNF bind almost equally well to the two human TNF receptors. Here we describe human TNF mutants that sill interact with the human TNF-R55 receptor but which have largely lost their ability to bind to human TNF-R75. Activation of TNF-R55 is sufficient to trigger cytotoxic activity towards transformed cells. One representative human TNF mutant retains its antitumour activity in nude mice carrying tumours derived from human cancers. Under the appropriate conditions, such human TNF mutants are expected to induce less systemic toxicity in man, while still exerting their direct antitumour effect.

Adenocarcinoma↗

PubMed1993

Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF.

Tumor necrosis factor (TNF) has cytotoxic and gene-inductive activities on several cell types. Previous studies on L929 fibrosarcoma cells have revealed that the mitochondrial electron transport system plays a key role in inducing TNF cytotoxicity, presumably by the formation of reactive oxygen intermediates (ROI). Here we report that mitochondria-derived intermediates are not only cytotoxic but, in addition, function as signal transducers of TNF-induced gene expression. The activation of NF kappa B, which fulfills an important role in TNF-induced gene transcription, could be blocked by interference with the mitochondrial electron transport system. Furthermore, antimycin A, a mitochondrial inhibitor that increases the generation of ROI, potentiated TNF-triggered NF kappa B activation. The dual role of mitochondria-derived intermediates in cytotoxicity and immediate-early gene induction of TNF was further substantiated by isolating L929 subclones which lacked a functional respiratory chain. This depletion of the mitochondrial oxidative metabolism resulted in resistance towards TNF cytotoxicity, as well as in inhibition of NF kappa B activation and interleukin-6 gene induction by TNF. These findings suggest that mitochondria are the source of second messenger molecules and serve as common mediators of the TNF-cytotoxic and gene-regulatory signaling pathways.

Animals↗

PubMed1993

High-level expression, purification, and renaturation of recombinant murine interleukin-2 from Escherichia coli.

A murine interleukin-2 (mIL-2)-encoding cDNA, isolated from a stimulated EL4 mRNA library, was used to construct several expression plasmids directing synthesis of the mature protein in Escherichia coli. The expression was under control of either the PTrp or the PL promoter. Using these systems, a high-level expression of between 10 and 35% of the total cellular protein was obtained. The mIL-2 protein, present as insoluble inclusion bodies, could be solubilized in a chaotropic mixture and was partially purified by preparative gel filtration under denaturing conditions. After renaturation, the protein was further purified to homogeneity by anion-exchange chromatography. Depending on the fermentation, induction, and renaturation conditions, the yield ranged between 0.35 and 1 mg of purified mIL-2/g wet cells. The specific biological activity was about 10(7) units/mg and the endotoxin content < 4 ng/mg pure recombinant protein.

Amino Acid Sequence↗

PubMed1993

LamB as a carrier molecule for the functional exposition of IgG-binding domains of the Staphylococcus aureus protein A at the surface of Escherichia coli K12.

One, two or four IgG-binding domains of the Staphylococcus aureus Protein A (SPA) were inserted into the LamB protein which was expressed under control of the tac promoter. The chimeric proteins were shown to be exposed at the cell surface by analysis of isolated outer membranes and also by testing their functional interaction with IgG molecules. We hereby show that the LamB protein can accept as many as 232 amino acids (four SPA domains) and still be incorporated into the Escherichia coli outer membrane, while maintaining the functional conformation of the inserted SPA polypeptides.

Bacterial Outer Membrane Proteins↗

PubMed1993

Phenotypic effects in Saccharomyces cerevisiae after regulated expression of beta-(1,3)-glucanase from Nicotiana plumbaginifolia.

A recombinant yeast strain was constructed of which the cell wall porosity could be reversibly and conditionally modulated. This strain expresses the Nicotiana plumbaginifolia beta-(1,3)-glucanase under control of the Saccharomyces cerevisiae GAL1 promoter and the mating factor alpha 1 prepro-sequence. The following phenotypic effects were observed after expression of this enzyme: (a) expressed beta-(1,3)-glucanase is toxic to the producing yeast cells, which is reflected by a strong growth inhibition; as beta-(1,3)-glucanase could be detected only inside the cells, it seems to interfere with cell wall growth from within the cell; (b) after induction of glucanase, the recombinant strain lost up to 20% of some periplasmic enzymes, as evidenced by the release of normally periplasmic-associated invertase; (c) preliminary growth in a synthetic medium containing galactose significantly increased the transformation efficiency of the recombinant yeast strain.

Base Sequence↗

PubMed1993

Recombinant secreted haemagglutinin protects mice against a lethal challenge of influenza virus.

Balb/c mice were immunized with 2 x 2 micrograms of purified recombinant secreted haemagglutinin, derived from the A/Victoria/3/75 (H3N2) virus. In the first immunization, Ribi adjuvant was used, while for the booster injection a monophosphoryl lipid A/muramyl dipeptide combination was chosen. Mice immunized in this way were 90-100% protected against a challenge with 20 LD50 of mouse-adapted, homologous virus (strain X47). Bromelain-solubilized haemagglutinin gave only 70% protection under comparable conditions.

Animals↗

PubMed1993

Activation of the nuclear factor kappa B is not sufficient for regulation of tumor necrosis factor-induced interleukin-6 gene expression.

After treatment of L929 cells, a murine fibrosarcoma line, with tumor necrosis factor (TNF), a nuclear kappa B-like transcription factor is rapidly induced as identified by gel shift mobility assays using the kappa B-responsive sequence of the immunoglobulin or interleukin-6 (IL-6) genes as a DNA probe. When induction was carried out under conditions of increased or decreased cytotoxicity, which correlates with altered IL-6 gene expression, nuclear factor kappa B (NF-kappa B) activation was also demonstrated, but the abundance of the protein/DNA complex observed remained unchanged. Also activation of NF-kappa B as a function of time following TNF treatment did not reveal a correlation between the abundance of the protein/DNA complex and the TNF-induced IL-6 mRNA levels. Moreover, in L929 cells resistant to TNF cytotoxicity, the kappa B-like factor still became fully activated by TNF, although the IL-6 gene was only marginally expressed. In conclusion, discrepancies between the abundance of the activated NF-kappa B-like factor and the IL-6 mRNA production upon treatment with TNF indicate that (an) additional transcription factor(s) and/or (a) regulating mechanism(s) is (are) necessary for fine regulation of the level of IL-6 gene expression in response to cytokine stimulation.

Animals↗

PubMed1993

Tumor necrosis factor, its receptors and the connection with interleukin 1 and interleukin 6.

Cytokines are important mediators of the effects observed after the administration of endotoxin. One of them, tumor necrosis factor, is particularly important since it plays a cardinal role in two major endotoxin activities: its antitumor effect and its capacity to induce a systemic inflammatory response syndrome. TNF exerts its activity on a wide variety of target cells by the triggering of two distinct receptor types. TNF-R55 and TNF-R75. They induce distinct intracellular signals but can have cooperative effects. So, their differential triggering or modulation may have clinically relevant consequences. Based upon observations in the mouse, where hTNF does not interact with the TNF-R75 while mTNF triggers both receptor types, we propose that both receptors need to be triggered to obtain lethality after the administration of TNF. Since human TNF has retained antitumor activity, esp. in combination with IFN-gamma, TNF-mutants that are selective agonists for the TNF-R55 might have a broader therapeutic margin. One such human TNF mutant was already shown to be as effective as the wild-type hTNF in a xenograft model. However, several sensitizing agents may mimic TNF-R75 triggering and so make TNF-R55 triggering a lethal challenge. The fact that two such agents, RU38486 and IL-1 have similar effects regarding their kinetics and their capacity to sensitize for the lethality- and IL-6-inducing effect of hTNF may give a hint regarding the mechanism of the sensitizing effect.

Animals↗

PubMed1993

Cell membrane permeabilization and cellular collapse, followed by loss of dehydrogenase activity: early events in tumour necrosis factor-induced cytotoxicity.

Early events in the cytotoxic response to tumour necrosis factor (TNF) of the murine fibrosarcoma cell lines L929 and WEHI164cl13 were assessed on a cell by cell basis using the fluorescent exclusion dye propidium iodide (PI) and analysis by flow cytometry. The rationale of this approach is based on the exclusion of PI by cells with intact membranes. PI-positive cells appeared within a few hours of TNF treatment and further accumulated with time at a TNF dose-dependent rate. Thus, TNF rapidly caused a breakdown of the barrier function of the membrane in these TNF-sensitive fibrosarcoma cell lines. On a time basis, membrane permeabilization was immediately followed by a sudden shrinkage of the cell and was accompanied by cell death, but preceded the inactivation of the mitochondrial succinate dehydrogenase by several hours. The latter enzymatic activity was measured by the MTT chromogenic assay. Cell death was determined on the basis of the capability of individual cells to produce a progeny in a clonogenicity assay. Both membrane permeabilization and cellular collapse were fast events that were completed within a very short time and may represent the direct cause for cell death. Opposed to this, loss of mitochondrial succinate dehydrogenase activity evolved more slowly, was initiated at a later time and apparently represents a post-lethal event, not directly linked to the TNF signal transduction pathway. Finally, the enhancing effect of the protein synthesis inhibitor cycloheximide on the various features of TNF-induced cytotoxicity was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Cyclooxygenase inhibitors prevent the induction of tolerance to the toxic effects of tumor necrosis factor.

Nonsteroidal anti-inflammatory drugs, such as indomethacin (INDO), prevent acute toxicity caused by a single bolus injection of tumor necrosis factor (TNF). However, when given as a pretreatment to repetitive daily injections of TNF, INDO enhances toxicity. We investigated the effect of INDO on the induction of tolerance that develops after repeated injections of TNF, when administered in strictly dose- and schedule-dependent manners. Both INDO and another cyclooxygenase inhibitor, piroxicam, abrogated induction of a protective effect to a subsequent, potentially lethal challenge with TNF. At the same time, INDO blocked glucocorticoid induction by TNF. We conclude that cyclooxygenase inhibitors interfere with the pathway leading to protective tolerance. Hence, caution should be exercised when INDO is included in protocols involving multiple TNF administrations.

Animals↗

PubMed1993

Pap pili as a vector system for surface exposition of an immunoglobulin G-binding domain of protein A of Staphylococcus aureus in Escherichia coli.

Fusion genes between papA, the gene coding for the major Pap pilus subunit, and fragments coding for an immunoglobulin G-binding domain of the Staphylococcus aureus protein A were constructed in such a way that the spa fragments were inserted following either codon 7 or 68 of the coding sequence for the mature portion of PapA. Peptides in the area of amino acids 7 and 68 of PapA are localized at the external side of the pilus. A set of pL expression plasmids containing papA and derivatives suitable for insertion were constructed. A papA gene carrying a spa insert following codon 68 was cloned back into the pap operon. The presence of this altered operon in a bacterial strain allowed the detection of immunoglobulin G-binding activity at the surfaces of the bacterial cells.

Amino Acid Sequence↗