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Biomedical subjects

W Fiers

Publications and source records attributed to W Fiers.

At least 289 records · Page 16Linked to original sources
PubMed1987

Pathology of tumours produced in syngeneic Fischer rats by fibroblast-like cells before and after transfection with oncogenes.

Fischer rat cells before and after transfection with immortalizing and transforming genes produced tumours after s.c., i.p., or i.v. injection of cell suspensions and after s.c. implantation of cellular aggregates in the tail of syngeneic rats. Tumours were described histologically as fibrosarcoma-like. Virtually all tumours were considered macroscopically to be invasive because they adhered to the neighbouring tissues; in many tumours invasion was confirmed microscopically. All types of cells produced lung colonies (artificial metastases) after i.v. injection. Spontaneous metastases (from a primary tumour) were found with some tumours produced by cells before as well as after transfection. Differences in metastasis between various cell types could not be ascribed to variations in the periods of observation, in the minimum tumour-bearing periods, in the latency periods, or in the volume of primary tumours. We concluded that local invasion and spontaneous metastasis are usefull for the characterization of malignancy in experimental fibrosarcoma-like tumours. Since Fischer rat cells produced invasive and sometimes metastatic tumours before transfection, the present data do not show a rôle of immortalizing and transforming genes in the acquisition of invasiveness and metastatic capability.

Abdominal Neoplasms↗
PubMed1987

The toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors.

Tumor necrosis factor (TNF) is a macrophage product under active study as an anticancer drug. However, this agent can be very toxic and has been implicated in the pathogenesis of endotoxic shock. After intravenous injection of human recombinant TNF (4 micrograms/g), growing rats showed an unusual constellation of physiological responses, and all died within 2-4 hr. In 1 hr, TNF caused a sharp fall (2.5 degrees C) in body temperature and a large increase in plasma prostaglandin E2 levels. Blood glucose initially increased, but then a profound hypoglycemia developed by 2 hr. The TNF-treated animals also showed diarrhea, cyanosis, and a severe metabolic acidosis. A single injection of the cyclooxygenase inhibitors indomethacin or ibuprofen before the TNF treatment completely prevented the rapid killing and reduced eventual lethality by 70%. These agents blocked prostaglandin E2 production and prevented the hypothermia, changes in blood glucose, acidosis, and other symptoms. Since similar physiological changes have been reported after endotoxin injection, our data support the suggestion that TNF production is a critical factor in the development of septic shock. These findings also indicate that increased production of prostaglandins or thromboxanes is important in endotoxic shock and argue that cyclooxygenase inhibitors should be useful in its therapy. Indomethacin did not block the cytotoxic effects of TNF in vitro on several transformed cell lines (HeLa, Me 180, or L929). Therefore, combined use of TNF with a cyclooxygenase inhibitor may allow safer administration of high doses of this polypeptide to cancer patients.

Animals↗
PubMed1987

Induction and regulation of mRNA encoding 26-kDa protein in human cell lines treated with recombinant human tumor necrosis factor.

A 26-kDa protein, originally described in human fibroblasts superinduced for interferon beta (IFN-beta) production, and termed IFN-beta 2 by other investigators, is induced by cycloheximide and by a 22-kDa, interleukin 1 (IL-1)-related factor. Although the structure and sequence of the corresponding gene show nonhomology with the IFN-beta gene, the gene is identical to that of B-cell stimulatory factor 2, a human interleukin, and displays a very potent growth and differentiation factor activity for B lymphocytes. In this work we show that IL-1 beta and tumor necrosis factor (TNF) strongly induce the 26-kDa protein in FS-4 fibroblasts and in some transformed cell lines. Addition of cycloheximide to recombinant (r)IL-1 beta and rTNF further enhances the level of 26-kDa-protein mRNA. We determined the kinetics of induction and the amounts of rTNF and rIL-1 beta required for optimal induction of this mRNA in FS-4 cells and in HeLa H21 cells and found that rIL-1 beta is a more efficient inducer of 26-kDa protein mRNA than is TNF. By analyzing the inducibility of the 26-kDa protein gene by rTNF and rIL-1 beta in a series of transformed cell lines that differ in their sensitivity to the cytotoxic action of TNF, we report a direct correlation between the 26-kDa protein mRNA expression and the resistance of these cells to the cytotoxic effect of TNF.

B-Lymphocytes↗
PubMed1987

The 3' untranslated region of the human interferon-beta mRNA has an inhibitory effect on translation.

In vitro-transcribed human interferon-beta (IFN-beta) mRNA, which contains all the sequence of the natural molecule, is poorly translated in a reticulocyte lysate or when injected in Xenopus oocytes. This low level of translation is due to an inhibition by the 5' and 3' untranslated regions (UTRs). Indeed, the replacement of these sequences by those of Xenopus beta-globin mRNA dramatically increases the translational efficiency of the mRNA, especially in oocytes. This phenomenon is not due to a difference in mRNA stability since both native and chimeric mRNAs remain undegraded, at least during the translation period considered. Construction of different chimeric molecules having various combinations of 5' and 3' UTRs from IFN-beta or Xenopus beta-globin mRNA or a small sequence of SP6 polylinker as 5' UTR has revealed that the 3' UTR of IFN-beta in itself has a pronounced inhibitory effect on translation in the two translation systems from animal cells. Indeed, the addition of this 3' UTR at the 3' end of the coding region of a chicken lysozyme mRNA also causes a large decrease of its translational capacity in both systems. However, the nature of the 5' noncoding sequence influences the degree of translation inhibition exerted by the 3' UTR. Remarkably, we observed no difference in translation level when the different mRNAs were tested in a wheat germ extract.

Animals↗
PubMed1987

A broad-host-range expression vector based on the pL promoter of coliphage lambda: regulated synthesis of human interleukin 2 in Erwinia and Serratia species.

We report the construction of a broad-host-range expression vector based on an RSF1010-derived replicon. The vector carries the strong leftward promoter (pL) of coliphage lambda as well as the cI857 allele, which codes for a thermolabile repressor protein. The coding region of mature human interleukin 2, which is preceded by the ner ribosome binding site of phage Mu, was cloned downstream from the pL promoter. The plasmid was introduced into Erwinia and Serratia species by means of mobilization. Heat-inducible synthesis of interleukin 2 protein was obtained, showing that the pL promoter is functional in these genera. As in Escherichia coli, the bulk of the overproduced protein was present in an insoluble form.

Bacteriophage lambda↗
PubMed1987

Recombinant human tumour necrosis factor (rTNF)2 enhances leukotriene biosynthesis in neutrophils and eosinophils stimulated with the Ca2+ ionophore A23187.

Recombinant human tumour necrosis factor alpha (rTNF) did not cause the release of leukotrienes (LT) by human neutrophils and eosinophils. It did, however, prepare neutrophils and eosinophils for enhanced production of LT after activation with the calcium ionophore A23187. The effect was observed with 10(2) to 10(4) units/ml of rTNF which enhanced LTB4 production by ionophore-stimulated neutrophils, by 40 to 400%, while 10(3) to 10(4) units/ml of rTNF increased LTC4 production by ionophore-stimulated eosinophils by 30 to 120%. The enhancement was observed when granulocytes were incubated for 5 to 10 min with rTNF before stimulation with the ionophore. Increased leukotriene production was attributed to the induction of fatty acid hydrolase and/or 5-lipoxygenase activities on the basis of the increment in the total counts released after 3H-arachidonic acid labelling of neutrophils pretreated with rTNF as compared to control medium.

Calcimycin↗
PubMed1987

Toxic effects of recombinant tumor necrosis factor in suckling mice. Comparisons with interferon alpha/beta.

Newborn Swiss and A2G mice were given daily subcutaneous injections for 1 week of highly purified recombinant mouse tumor necrosis factor (TNF) or mouse interferon alpha/beta. Both treatments resulted in inhibition of growth of suckling mice and severe fatty changes and necrosis in the liver. The simultaneous injection of polyclonal antibody to interferon alpha/beta abrogated the effects of interferon but did not block the effects induced by TNF. The kidneys of TNF-treated suckling mice could be distinguished from interferon-treated mice by the absence of glomerular basement membrane abnormalities and the presence of numerous rounded eosinophilic hyaline granules within the cytoplasm of the proximal tubules. Treatment of suckling mice with TNF and interferon alpha/beta induced similar changes in the spleen and thymus. Interferon treatment of suckling A2G mice resulted in the appearance of pulmonary cysts, which were not observed in TNF-treated mice. It is concluded that the pattern of lesions induced in suckling mice by mouse TNF is both similar and different from that induced by mouse interferon alpha/beta.

Animals↗
PubMed1987

Recombinant tumour necrosis factor inhibits malaria parasites in vivo but not in vitro.

As tumour necrosis serum kills malarial parasites in vitro and inhibits the multiplication of some species of Plasmodium in mice, we examined the effect of recombinant mouse tumour necrosis factor (rTNF) on P. yoelii both in vitro and in vivo. Parasites incubated overnight with rTNF showed no loss of viability, but repeated injection of rTNF into infected mice reduced parasitaemia and significantly prolonged survival of mice infected with a lethal variant of the parasite. We conclude that TNF acts on blood-stage malaria in vivo via a host cell and that another molecule in tumour necrosis serum is involved in killing the parasite in vitro.

Animals↗
PubMed1986

Two monokines, interleukin 1 and tumor necrosis factor, render cultured vascular endothelial cells susceptible to lysis by antibodies circulating during Kawasaki syndrome.

Kawasaki syndrome (KS) is an acute febrile illness of early childhood characterized by diffuse vasculitis and marked immune activation. The present study was undertaken to determine whether the acute phase of KS is associated with circulating cytotoxic antibodies directed to target antigens induced on vascular endothelium by the monokines, IL-1, or tumor necrosis factor (TNF). Sera from 20 patients with acute KS, 11 patients in the convalescent phase of KS, and 17 age-matched controls were assessed for complement-dependent cytotoxic activity against 111In-labeled human endothelial cells (HEC), dermal fibroblasts, and vascular smooth muscle cells. Sera from patients with acute KS but not the other subject groups caused significant (p less than 0.01) complement-mediated killing of IL-1- or TNF-stimulated HEC. None of the sera tested had cytotoxicity against control HEC cultures or the other target cell types, with or without IL-1 or TNF pretreatment. Expression of the IL-1- or TNF-inducible target antigens on endothelial cells was rapid and transient, peaking at 4 h and disappearing after 24 h despite continued incubation with monokine. In contrast, we have previously shown that IFN-gamma requires 72 h to render HEC susceptible to lysis with acute KS sera. Serum adsorption studies demonstrated that IL-1- and TNF-inducible endothelial target antigens are distinct from IFN-gamma-inducible antigens. These observations suggest that mediator secretion by activated monocyte/macrophages could be a predisposing factor to the development of vascular injury in acute KS. Although our present observations have been restricted to KS, the development of cytotoxic antibodies directed to monokine-inducible endothelial cell antigens may also be found in other vasculitides accompanied by immune activation.

Antibodies↗
PubMed1986

In vivo anti-tumour activity of recombinant human and murine TNF, alone and in combination with murine IFN-gamma, on a syngeneic murine melanoma.

TNF, a protein released by induced macrophages, is believed to mediate, at least in part, the tumoritoxic effects of activated macrophages. In vitro, it has cytotoxic effects on transformed cells but not on normal cells, and in vivo it causes necrosis of tumours. Recently, both human and murine TNF became available as pure recombinant proteins. Subsequent work confirmed its in vitro cytotoxic activity, selective for transformed cells, and revealed other, non-cytotoxic effects on some normal cells. In vitro, the B16BL6 melanoma cells, syngeneic with C57BL6 mice, are resistant to the cytotoxic effects of rTNF but become sensitive when they are also treated with rIFN-gamma. We report that established, s.c. B16BL6 tumours in vivo can be induced to necrotize and regress by a combined systemic treatment with rTNF and murine rIFN-gamma. Although TNF is not species-specific in vitro, the effects of treatment with human and murine rTNF in vivo are different: with murine rTNF, the synergism with rIFN-gamma is relatively less clear, the addition of IFN-gamma is not necessary to induce regression, toxicity is more pronounced and additional mechanisms of tumoritoxicity could be involved. Relapses are frequent but complete cures have been observed. These results give further evidence in favour of a potential clinical use of TNF in combination therapy, e.g. with IFN-gamma. However, there is still a need to develop better regimens, especially for consolidation, and to continue research in order to understand and limit the toxicity, which could be mediated by the activating effects of TNF on some normal cell types.

Animals↗
PubMed1986

Anti-tumor effects of interferon in mice injected with interferon-sensitive and interferon-resistant Friend leukemia cells. V. Comparisons with the action of tumor necrosis factor.

A number of similarities and dissimilarities in the anti-tumor effects of TNF and interferon alpha/beta have been observed in DBA/2 mice injected with Friend erythroleukemia cells (FLC). Mouse TNF exerted marked anti-tumor effects in mice injected either s.c. or i.p. with FLC lines 3C18 or 3 gamma R8 resistant in vitro to the cytotoxic effects of TNF. Likewise, mouse interferon alpha/beta had anti-tumor activity in mice injected with these FLC, resistant to the action of interferon alpha/beta or gamma in vitro. The results of histopathologic examination and 31P nuclear magnetic resonance analyses of 3C18 FLC s.c. tumors injected with TNF resembled the results previously obtained for 3C18 FLC tumors injected with interferon alpha/beta, although the effects of TNF occurred more rapidly. Injection of mice with antibody to mouse interferon alpha/beta or gamma did not abrogate the anti-tumor effects of TNF in mice injected i.p. with FLC. Our results suggest that in this experimental system the anti-tumor effects of TNF, like interferon alpha/beta, do not result from a direct effect on the tumor cells themselves but are host-mediated.

Animals↗
PubMed1986

Species specificity of human and murine tumor necrosis factor. A comparative study of tumor necrosis factor receptors.

Recombinant murine and human tumor necrosis factor (mTNF and hTNF, respectively) were radioiodinated to high specific activity using a solid-phase lactoperoxidase method. A single class of high affinity receptors for 125I-TNF was identified on TNF-sensitive murine L cells and human HeLa S2 cells. Competitive radioligand binding assays were used to study the species specificity of TNF preparations. Unlabeled hTNF competed 30-fold less effectively than mTNF for binding to L cell receptors, whereas mTNF competed to approximately the same extent as hTNF for binding to HeLa cell receptors. A similar species specificity was observed in cytotoxicity assays; hTNF was more cytotoxic for HeLa cells than mTNF. Conversely, mTNF was more growth inhibitory and cytotoxic for L cells than hTNF. mTNF. and hTNF.receptor complexes were compared by gel filtration chromatography and polyacrylamide gel electrophoresis before and after cross-linking with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES). These complexes eluted in gel filtration at a position corresponding to a globular protein of 350,000 Mr. Gel autoradiographs of the fractions containing cross-linked complexes showed bands of 95,000 and 75,000 Mr as well as small amounts of higher Mr bands. mTNF and hTNF treated with BSOCOES formed cross-linked dimers and trimers. Therefore, we were unable to determine whether the 95,000 and 75,000 Mr bands represented two distinct subunits of receptors or one subunit to which either a dimer or a monomer of TNF was cross-linked. These results demonstrate species specificity in the TNF-receptor interaction. In addition, the affinity labeling studies in two species give an identical pattern for the TNF X receptor complexes, suggesting that the receptors have similar subunit composition.

Animals↗
PubMed1986

Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis.

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.

Animals↗
PubMed1986

Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts.

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.

Amino Acid Sequence↗
PubMed1986

Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon.

We have used the quantitative binding of murine monoclonal antibodies to the surface of cultured human umbilical vein endothelial (HUVE) cells to study the responses of HUVE cells to three different immune mediators: interleukin 1 (IL 1), tumor necrosis factor (TNF), and immune interferon (IFN-gamma). Antibody H4/18, reactive with an endothelial cell-specific activation antigen, does not bind to unstimulated HUVE cells but shows rapidly and transiently inducible binding (peak 4 to 6 hr) to cells stimulated by IL 1 or TNF that declines to basal levels by 24 hr, even in the continued presence of mediator. Binding of H4/18 is unaffected by IFN-gamma. Antibody RR1/1, reactive with intercellular adhesion molecule 1, binds to unstimulated HUVE cells, but binding is rapidly increased (plateau 24 hr) after stimulation by IL 1 or TNF and slowly increased (over several days) by IFN-gamma. In contrast to H4/18 binding, the increase in RR1/1 binding is sustained in the continued presence of mediator. Antibody W6/32, reactive with HLA-A,B antigens, binds to unstimulated HUVE cells and shows gradually progressive increases (over several days) in binding upon treatment with IFN-gamma or TNF. These observations demonstrate that HUVE cells show distinct but overlapping patterns of antigenic modulation in response to three different lymphokines, and suggest that the "activation" of endothelial cells observed in situ may represent a complex integration of several lymphokine-mediated signals.

Antibodies, Monoclonal↗
PubMed1986

Purification, crystallization and preliminary X-ray data of the bacteriophage MS2.

Single crystals of the bacteriophage MS2 have been produced by the vapour diffusion technique in the presence of 1.5% polyethylene glycol 6000 and 0.2 M-sodium phosphate buffer (pH 7.4). These are the first bacteriovirus crystals diffracting to high resolution. The crystal space group is C2 with the unit cell parameters a = 467.9 A, b = 289.5 A, c = 275.6 A and beta = 121.8 degrees. The asymmetric unit contains one half of the virion. The maximum resolution limit of the X-ray diffraction data obtained from these crystals was 2.9 A. The purification of the virus material was done by mild procedures exclusively and involved precipitation with polyethylene glycol 6000 and size exclusion chromatography on Sepharose CL-4B.

Chromatography, Gel↗
PubMed1986

Structural studies of the carbohydrate chains of human gamma-interferon.

Human gamma-interferon (IFN-gamma) was prepared biotechnologically using Chinese hamster ovary cells. These cells were shown to be able to produce glycosylated IFN-gamma. Sugar analysis revealed the presence of Man, Gal, GlcNAc, NeuAc and Fuc residues in a molar ratio of 3.8:2.0:3.5:0.6:0.4 suggesting the occurrence of N-glycosidically linked N-acetyllactosamine type of carbohydrate chains. For structure determination of these chains, the glycoprotein was subjected to the hydrazinolysis procedure, yielding oligosaccharide-alditols. The latter compounds were analysed by 500-MHz 1H-NMR spectroscopy. The carbohydrate material was found to consist of biantennary structures, exhibiting microheterogeneity as to the terminal sialic acids and the core Fuc residue: (Formula: see text). As similar carbohydrates are present on several human secreted proteins, this glycosyl group is not expected to be immunogenic in man. It remains to be established to what extent the carbohydrate chains of this biotechnologically produced IFN-gamma are identical to those of naturally occurring human IFN-gamma.

Animals↗