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Biomedical subjects

W Fiers

Publications and source records attributed to W Fiers.

At least 235 records · Page 13Linked to original sources
PubMed1989

Tumor necrosis factor-alpha induces the phosphorylation of 28kDa stress proteins in endothelial cells: possible role in protection against cytotoxicity?

Tumor necrosis factor-alpha has been shown to rapidly increase the phosphorylation of three 28 kDa proteins in bovine aortic endothelial cells but not in L929 cells. Tumor necrosis factor-alpha induces the necrosis of the latter cells but not of the former. Arsenite enhanced the phosphorylation of the same 28kDa proteins as tumor necrosis factor-alpha in the endothelial cells. As stress proteins often play a protective role, we suggest that the phosphorylation of these proteins in endothelial cells may be responsible for the resistance of these cells to tumor necrosis factor-alpha.

Animals↗
PubMed1989

Four different interleukin-1 species sensitize to the lethal action of tumour necrosis factor.

We studied the induction of lethal shock by Tumour Necrosis Factor (TNF) in mice and observed a remarkable difference between the effect of human and murine TNF, which could be eliminated by co-administration of sensitizing agents. We identified interleukin-1 (IL1) as a natural sensitizer, rendering mice as susceptible to human TNF as to murine TNF. This IL1 activity was found to be exerted to the same extent both by human and murine IL1-alpha or IL1-beta, and was also different from the sensitization obtained with galactosamine, since these agents had an additive effect. Pretreatment of the animals with indomethacin, a cyclooxygenase inhibitor, provided partial protection against TNF lethality in IL1-sensitized but not in galactosamine-sensitized mice.

Animals↗
PubMed1989

An ongoing in vivo immune response affects the abundancy and differentiation of lymphokine-activated killer cell precursors, but does not influence their broad spectrum target reactivity.

Using a model of local lymph node (LN) immunization, we investigated the effect of in vivo Ir on the generation of lymphokine-activated killer (LAK) cells or their precursors. Ag used for immunization were SRBC, horse RBC, OVA, keyhole limpet hemocyanin, or CFA. Ag-draining LN, in the acute phase of the Ir, did not contain detectable LAK effector activity, nor an enhanced NK activity. After culture for 3 to 5 days in the absence of exogenously added IL-2, immunized LN cells developed a spontaneous LAK-like cytotoxicity. This activity represented a substantial fraction of the IL-2-generated LAK cytolysis and was mediated by a Thy-1+ cell population phenotypically indistinct from IL-2-induced LAK. Inclusion (on day 0 of culture) of antibodies to IL-2, IL-2R, IL-4, IL-6, IFN-gamma, or TNF suggests a marginal involvement of IL-2 and IL-4 in the generation of this response. LAK, induced in vitro by exogenously added IL-2, developed earlier in LN cells immunized with particle Ag (SRBC, horse RBC, and CFA), but not with protein Ag (OVA and keyhole limpet hemocyanin). This effect was not mediated by endogenous IL-4. During further culture time in the presence of a saturating IL-2 concentration, similar levels of LAK activity were generated in naive and immunized LN cells. This agrees with the similar or slightly higher LAK precursor frequencies in immunized versus naive LN as assessed by limiting dilution experiments. Considering the 2.7-fold to 18-fold increase in cell content of the immunized LN, due to a recruitment and expansion of Ag-reactive B and T lymphocytes, a de novo generation of LAK precursors at the site of the Ir, and resulting from the Ir, must be assumed. In conclusion, our results suggest an interrelation between immune reactivity and LAK responses.

Animals↗
PubMed1989

Purification of human interleukin-4 produced in Escherichia coli.

An interleukin-4 (IL4)-encoding cDNA isolated from human splenocytes was used to construct an expression plasmid that directs a high-level synthesis of mature IL4 protein in Escherichia coli. The expression was under the control of the major leftward promoter, pL, of phage lambda and the phage Mu ribosome-binding site. The IL4 protein was present as insoluble inclusion bodies in the bacterial extract. The IL4 could be solubilized in 5 M MgCl2 and was purified to homogeneity by several chromatographic steps. The yield of protein from bacteria ranged between 3 and 5 mg of IL4 protein per gram of wet cells. The specific activity of the recombinant human IL4 was about the same as that of the natural product.

Animals↗
PubMed1989

Acquisition by the murine host of responsiveness toward various neoplastic cell lines, but not toward self, through adoptive transfer of a helper T-lymphocyte clone with antiself specificity.

Self-antigens, when expressed on neoplastic cells, have been shown to exhibit a certain antigenicity. We attempted to apply this antigenicity to enhance antitumor immune responses. Cells from the syngeneic, CD4+, CD8-, helper T-lymphocyte clone TE2 were adoptively transferred to C57BL/6 mice. TE2 lymphocytes recognize a self-antigen on splenocytes that is expressed aberrantly on the neoplastic cell lines EL4/8, EL4/13, B16-BL6, and PG19, all of C57BL/6 origin. Their adoptive transfer led to the rejection by the host of the former neoplastic cells and of 3LL carcinoma cells, administered 2 months later; inocula 40 to 80 times the minimal lethal size were rejected and conveyed to the mice a 10-fold enhanced cytotoxic T-lymphocyte response. Despite the autoimmune responsiveness of the TE2 T-lymphocytes, no graft-versus-host reaction was apparent. This conclusion is based on the absence of a polyclonal B-lymphocyte stimulation in the host, the stable number of residual donor TE2 cells, and the general health of the recipient mice. Consequently, the autoimmune and tumor-responsive TE2 cells, transplanted into the immune environment of the host, exhibit a specificity that is restricted toward neoplastic cells.

Animals↗
PubMed1989

Inflammatory properties of recombinant tumor necrosis factor in rabbit skin in vivo.

We have investigated the ability of recombinant TNF (mouse and human) to produce acute inflammatory lesions in an established experimental model of inflammation. Upon intradermal injection in rabbit skin, TNF, in amounts as low as 3 x 10(-14) mol/site, was found to be very potent at inducing local neutrophil accumulation and neutrophil-dependent oedema formation, thereby fulfilling two important criteria to be considered as an inflammatory mediator. Our findings further indicate that the pro-inflammatory properties of TNF are probably more related to its immediate stimulatory effects on neutrophils rather than to its slow (protein biosynthesis-dependent effects on endothelial cells. Our data thus show that very low amounts of mouse and human recombinant TNF can initiate an acute inflammatory reaction in vivo in rabbit skin and that TNF is able to evoke two of the four cardinal signs of inflammation.

Animals↗
PubMed1989

Circulating interleukin 6 during a continuous infusion of tumor necrosis factor and interferon gamma.

Plasma samples obtained from patients receiving a 24-h continuous infusion of human recombinant (hr)TNF or a combination of two overlapping, 24-h continuous infusions of hrIFN-gamma and hrTNF were analyzed for IL-6 in a sensitive bioassay. A transient appearance of circulating IL-6 was observed with peak levels between 3 and 6 h after the start of the hrTNF infusion. These peak levels correlated quite well with the dose of hrTNF administered (r = 0.86; p less than 0.001). The maximal value observed was 27.5 ng/ml IL-6 in a sample of a patient receiving 545 micrograms/m2 hrTNF. The combination of hrIFN-gamma (200 micrograms/m2) and hrTNF in the infusions resulted in higher IL-6 levels than a comparable dose of hrTNF alone. A maximal value of 23.5 ng/ml IL-6 was observed in a patient receiving 205 micrograms/m2 hrTNF. No IL-6 was found in the plasma of patients during the 12-h infusion with hrIFN-gamma alone, except for two borderline samples.

Drug Administration Schedule↗
PubMed1989

Transformation of Aspergillus niger with the homologous nitrate reductase gene.

A homologous transformation for Aspergillus niger was developed based on the nitrate reductase structural gene niaD. This system offered certain advantages over existing A. niger systems, such as the ease of recipient mutant isolation, absence of abortive transformants, convenient enzyme assay, ease of transformant stability testing, and complete absence of background growth. Transformation frequencies of up to 100 transformants per microgram DNA were obtained with the vector pSTA10 which carries the niaD gene of A. niger. Southern blotting analysis indicated that vector DNA had integrated into the genome of A. niger. Mitotic stability studies demonstrated that while some transformants were as stable as the wild-type (wt), others were markedly less so. No correlation was seen between plasmid integration, mitotic stability and nitrate reductase activity, which was markedly different from wt in only three of the transformants examined.

Aspergillus niger↗
PubMed1989

Different pro-inflammatory profiles of interleukin 1 (IL 1) and tumor necrosis factor (TNF) in an in vivo model of inflammation.

The pro-inflammatory properties of IL 1 and TNF were investigated in an in vivo model of inflammation. IL 1 induced PMN leukocyte accumulation that was slow in onset, reaching a peak rate at 3-4 h and that was inhibitable by Actinomycin D and Cycloheximide. PMN leukocyte emigration was not associated with any significant plasma leakage. In contrast, TNF induced PMN leukocyte accumulation and oedema formation, that were rapid in onset and very short of duration (t1/2 6-10 min). TNF-induced plasma leakage was PMN leukocyte-, but not protein biosynthesis-dependent. The differences in time course and biological profile suggest that IL 1 and TNF exert their pro-inflammatory effects in vivo via different mechanisms.

Disease Models, Animal↗
PubMed1989

Species specificity and involvement of other cytokines in endotoxic shock action of recombinant tumour necrosis factor in mice.

We compared the effects of human rTNF and murine rTNF in murine models of toxicity, esp. the induction of endotoxic shock. As was the case for the antitumour activity, we found a marked difference in activity between these two TNFs. Only murine rTNF was able to cause lethality, while human rTNF needed the synergistic action of sensitizing agents to become lethal. Further experiments, such as the study of IL-6 induction by TNF in mice, allowed us to distinguish two types of TNF effects: those that can equally well be exerted by human rTNF and by murine rTNF (type I effects) and those that can only be exerted by murine rTNF (type II effects). Both types of effects, the "toxic" (a type I effect) and the sensitizing (a type II effect) are needed to produce a lethal outcome. Other cytokines such as IL-1 and IFN-gamma, however, can also exert such a sensitizing effect and consequently lead to a fatal outcome when co-administered with human rTNF.

Animals↗
PubMed1989

Different preparations of natural and recombinant human interleukin-6 (IFN-beta 2, BSF-2) similarly stimulate acute phase protein synthesis and uptake of alpha-aminoisobutyric acid by cultured rat hepatocytes.

1. Rat hepatocytes were cultured for 2 days in Williams E medium containing 1 microM insulin and dexamethasone. 2. Production of five plasma proteins was determined by electroimmunoassay in the media, and amino acid uptake was measured by [alpha-14C]aminoisobutyric acid accumulation in hepatocytes. 3. Supernatants from rat peritoneal macrophages and IL-6/IFN-beta 2/BSF-2 obtained from four different laboratories similarly stimulated synthesis of fibrinogen, alpha 1-cysteine proteinase inhibitor and alpha 2-macroglobulin, as well as [14C]-accumulation in cultured hepatocytes. 4. It is concluded that IL-6 is the principal hepatocyte stimulating factor responsible for typical features of the acute phase response of liver cells.

Acute-Phase Proteins↗
PubMed1989

Lithium chloride potentiates tumor necrosis factor-mediated cytotoxicity in vitro and in vivo.

Tumor necrosis factor (TNF) is cytotoxic for several transformed cell lines in vitro. In the presence of LiCl, the murine fibrosarcoma cell lines L929 and WEHI 164 clone 13 became greater than 10 times more sensitive to TNF-mediated cytotoxicity. The human tumor cell lines BT20 and HeLa D98/AH2 were also responsive to the cytotoxicity-enhancing effect of LiCl. Other monovalent or divalent cations did not affect TNF-mediated cytotoxicity. The potentiating effect of LiCl on TNF cytotoxicity was largely independent of transcription, and LiCl could be added to the cells as early as 2 hr before or as late as 4 hr after TNF without loss of effectiveness. The mechanism by which LiCl increases the cytotoxic response seems to differ from the sensitizing effect of actinomycin D or interferon gamma, since the latter treatments overcame TNF resistance of several cell lines, whereas LiCl did not. Evidence is presented that LiCl acts, either directly or indirectly, via the TNF-activated phospholipase A2 pathway. In nude mice, a combination of TNF and LiCl led to hemorrhagic necrosis and growth inhibition of L929 tumors, whereas little effect was observed when TNF was administered alone. HeLa D98/AH2 tumors also were sensitive to the potentiating effect of LiCl in vivo. We conclude that LiCl enhances the effectiveness of TNF in vitro and in vivo, results that may have therapeutic implications.

Animals↗
PubMed1989

Expression of human and murine interleukin-5 in eukaryotic systems.

A cDNA coding for murine interleukin-5 (IL-5) was isolated from the EL4.ExC5 cell line. With the exception of a single amino acid substitution at position 79 (Arg----His), it is identical to a published sequence. The coding sequence for human IL-5 was synthesized chemically, allowing the introduction of strategically located restriction enzyme cleavage sites. Both cDNAs were expressed in various eukaryotic systems. Deletion of the 3' untranslated region of the murine IL-5 gene led to a 5- to 10-fold increase in expression in Xenopus laevis oocytes and in NIH-3T3 cells. The highest production, however, was obtained in Sf9 cells using a baculovirus vector. Human IL-5 was obtained from transformed Saccharomyces cerevisiae as a secreted, mature form using an in-frame fusion to the leader sequence of alpha-mating type factor, and was purified to homogeneity. In all cases mentioned, IL-5 was found to be glycosylated, and its biological activity was dependent on a 40- to 50-kD homodimer configuration, linked together by disulfide bridges. Deglycosylation did not affect the biological activity. Recombinant human IL-5 is biologically active on some human B-CLL cells (proliferation in the presence of IL-2) and on murine BCl1 cells (proliferation) at a low specific activity (about 1-2 x 10(3) U/mg) and on human eosinophils (eosinophil peroxidase assay) at a high specific activity (at least 5 x 10(6) U/mg). Recombinant murine IL-5 from Sf9 cells has a specific activity of 1-2 x 10(7) U/mg in the BCl1 proliferation assay. An additive effect is seen in the presence of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and a synergistic effect in the presence of murine IL-4.

Animals↗
PubMed1989

A role for interleukin 1 in the in vivo actions of tumor necrosis factor.

We have observed a marked species specificity in the in vivo antitumor, shock-inducing and Interleukin-6 (IL-6)-inducing activities of human recombinant TNF (hrTNF) versus murine rTNF (mrTNF) in mouse models. We also have observed that this species specificity can be abolished when synergistic factors are co-administered. We now present data showing that the profiles of induced IL-6 and TNF after lethal and non-lethal doses of endotoxin are different. These results are discussed in the context of a model of dual signalling for the induction of the major post-endotoxin phenomena that takes into consideration the involvement of Interleukin-1 (IL-1) in the in vivo actions of TNF.

Animals↗
PubMed1988

Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity.

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.

Animals↗
PubMed1988

Endocytic pathway of recombinant murine tumor necrosis factor in L-929 cells.

The fate of TNF after binding to the surface of L-929 cells was followed by using murine rTNF coupled to colloidal gold as a probe. A time-course study using electron microscopy was performed. Our results confirm previous indications obtained from biochemical studies suggesting that TNF is internalized by this cell type. They further directly show that internalization proceeds through the classical receptor-mediated endocytosis pathway, i.e., via clathrin-coated structures and endosomes before accumulation in secondary lysosomes.

Animals↗
PubMed1988

Genes for IFN-beta-2 (IL-6), tumor necrosis factor, and IL-1 are expressed at high levels in the organs of normal individuals.

The gene of a cytokine designated IFN-beta-2, or IL-6, and recently identified as identical to the B cell-stimulatory factor 2, is transcribed at high levels in the spleen, liver, kidney, and peripheral blood leukocytes of normal individuals. The number of IFN-beta-2/IL-6 transcripts present endogenously in normal human tissues (0.6 to 16 copies/cell) is comparable to that present in normal cells induced in vitro with human rTNF. This is in marked contrast to the absence of detectable IFN-beta-1 transcripts (less than 0.0003 copy/cell) in the same samples of human tissue. The expression of the IFN-beta-2/IL-6 gene is closely associated with that of two other cytokines TNF, and IL-1. Thus, significant levels of IFN-beta-2/IL-6, TNF, IL-1 alpha, and IL-1 beta, mRNA were detected in all the samples of normal tissue tested and those samples which contained high levels of IFN-beta/IL-6 mRNA also contained high levels of TNF, and IL-1 beta mRNA. These results suggest that these cytokines may function in consort as regulators of cellular growth and function in normal tissues.

Gene Expression Regulation↗