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Biomedical subjects

W Fiers

Publications and source records attributed to W Fiers.

At least 325 records · Page 18Linked to original sources
PubMed1985

Interferon-gamma is a strong modulator of NK susceptibility and expression of beta 2-microglobulin but not of transferrin receptors of K562 cells.

The human cell line K562 was treated with human natural leukocyte interferon (IFN-alpha) and recombinant immune interferon (IFN-gamma). Cell cultures exposed to both types of IFNs displayed a reduced susceptibility to the cytotoxic activity of human PBL (NK activity). While this effect occurred preferentially at high doses of IFN-alpha, as little as 10 U/ml of IFN-gamma caused a marked decrease in susceptibility to NK-cell-mediated lysis. Using a monoclonal antibody against human beta2-microglobulin (beta2M) a low level of specific binding to K562 cells was detected. The binding increased after treatment with IFN-alpha (1.4-fold) and IFN-gamma (1.7-fold). The expression of transferrin receptors (TR) was not changed significantly. A hybrid cell line between K562 and a Burkitt's lymphoma-derived cell line displayed a similar pattern of response to IFN-alpha and IFN-gamma as did K562, when effects on NK susceptibility, beta2M expression, and TR expression were studied. The Burkitt's lymphoma line PUT showed no consistent changes in expression of beta2M and TR. These results demonstrate that IFN-gamma is highly efficient in modulating the NK susceptibility, and the expression of beta2M on K562. The presented data do not support a role for expression of TR as the only property that determines the degree of NK susceptibility, since there was no correlation between NK susceptibility and TR expression among the cell lines tested or when IFN-treated and untreated cells were compared.

Cell Line↗
PubMed1985

Recombinant interleukin 2 induces immunoglobulin secretion in Staphylococcus aureus Cowan strain I activated human B-cells.

Human B-cells, exhaustively depleted for T-cells, were activated with Staphylococcus aureus Cowan strain I (SAC) and responded to recombinant human interleukin 2 (rIL2) by secretion of immunoglobulin (Ig), as measured by a protein A hemolytic plaque assay. The rIL2, however, had to be present early, since addition later than 24 h after SAC-activation of the B-cells reduced the response to background levels. No clear dose response was observed and Ig-secreting cells (ISC) could be induced even with rIL2 at 0.5 U/ml. The monoclonal antibody anti-TAC prevented the rIL2-promoted induction of ISC. Ig production could be induced in SAC-activated cultures with supernatants of Xenopus laevis oocytes injected with sucrose-gradient-fractionated poly(A+) RNA derived from a stimulated human spleen cell culture. This activity coincided with the IL2 mRNA activity and was well separated from the interferon-gamma mRNA activity. Our results suggest that IL2 is not only a B-cell growth factor but also promotes the differentiation of activated human B-cells towards Ig secretion.

Antibody-Producing Cells↗
PubMed1985

Screening of sera and tumor extracts of cancer patients using a monoclonal antibody directed against human placental alkaline phosphatase.

A sensitive endogenous enzyme immunoassay involving an anti-human placental alkaline phosphatase (PLAP) monoclonal antibody was used in the screening of sera and tumor extracts of patients with various types of cancer. In sera of breast cancer patients an incidence of 5.2% was recorded. This value rose to 43% when tumor extracts were analyzed. For lung and bronchial cancers we found 11.2% seropositive patients. On several occasions a good correlation was observed between the PLAP determinations and the histopathological staging of tumor tissue.

Adult↗
PubMed1985

Alterations upstream from the Shine-Dalgarno region and their effect on bacterial gene expression.

A vector containing the leftward promoter (pL) as transcription initiation signal and a synthetic, easily adaptable translation initiation region have been constructed. We have used the expression system to assess the relevance of sequences upstream from the Shine-Dalgarno (SD) region in the translational-initiation process. To this end, a series of structural variants of the prototype ribosome-binding site were used to direct the synthesis of both mature human fibroblast interferon and beta-galactosidase (beta-gal). It was found that alterations 5' to the SD element can considerably affect the rate of mRNA translation. The observation that the relative efficiency of the various 5'-untranslated regions depends on the downstream coding information implies that secondary (and/or tertiary) structure formation is of major importance in the initiation process. But an mRNA folding, in which the SD and ATG determinant are set free in single-stranded regions, does not unconditionally guarantee an efficient initiation of translation.

Base Sequence↗
PubMed1985

Human dermal fibroblasts present tetanus toxoid antigen to antigen-specific T cell clones.

Cultured human dermal fibroblasts treated with immune interferon express HLA-DR antigens. We report here that DR-positive fibroblasts present tetanus toxoid (TT) to autologous TT-specific monoclonal helper T cells vigorously depleted of monocytes by passage over Sephadex G10 columns followed by treatment with the monoclonal antibodies (mAb) OKM1 and Leu M1 plus complement. The extent of T cell proliferation in response to TT presented by DR-positive fibroblasts was similar to that elicited using monocytes as antigen-presenting cells. The proliferative response was TT dependent, antigen specific, depended upon DR expression by fibroblasts, appeared MHC restricted, and was completely blocked by mouse mAb to HLA-DR but not by mAb to HLA-A,B, or DQ. DR-positive fibroblasts pulsed with TT were similarly effective in antigen presentation. In summary, immune interferon-stimulated human dermal fibroblasts can substitute for classical antigen-presenting cells in antigen-specific proliferative responses. Since fibroblasts are a ubiquitous cell type in the body, they may play a significant role in the immunobiology of the host.

Antibodies, Monoclonal↗
PubMed1985

Defective IFN-gamma production in the human neonate. II. Role of increased sensitivity to the suppressive effects of prostaglandin E.

Analysis of endogenous production and effects of exogenous addition of interleukin 2 (IL 2), leukotrienes (LT), and prostaglandin E (PGE) has been used to investigate the dysregulation responsible for impaired PHA-induced IFN-gamma secretion by cord blood leukocytes (CBL). The addition of LT or IL 2 could not reverse the IFN defect of CBL. The production of these two mediators was found to be normal in CBL cultures. CBL and control leukocytes from adult donors produced comparable amounts of PGE2. In contrast, sensitivity to the suppressive effects of PGE2 on IFN-gamma secretion was much higher with CBL than with control leukocytes. Treatment with indomethacin reversed the IFN-gamma defect with most CBL tested, and the addition of physiologic amounts of PGE2 to indomethacin-treated cultures resulted in a profound impairment of IFN-gamma production similar to that of untreated CBL cultures. Preincubation of CBL for 24 hr before PHA stimulation resulted in restoration of a normal sensitivity to exogenous PGE2, in parallel with correction of the IFN-gamma defect. Our observations suggest that the impairment of IFN-gamma secretion in neonates is not due to deficient amplification circuits, but is the consequence of an exaggerated cellular sensitivity to the suppressive effects of PGE produced endogenously in normal amounts.

Adult↗
PubMed1985

The occurrence of human placental alkaline phosphatase (PLAP) in extracts of normal, benign and malignant tissues of the female genital tract.

In addition to its presence in extracts of carcinomatous tissues from the vulva, endometrium and ovary, PLAP can also be found in tissue extracts of benign conditions of the endometrium and myometrium. We detected slightly elevated levels of PLAP in patients with myoma, raised levels in 2/2 patients with endometrial polyps and high values in 2/2 patients with glandulocystic hyperplasia. Moreover, normal endometrium and fragments of normal Fallopian tube also contained fairly high amounts of endogenous PLAP. These results have important implications with regard to the possible use of PLAP as a tumour marker or of anti-PLAP monoclonal antibodies in radioimmunolocalization or radioimmunotherapy.

Adolescent↗
PubMed1985

Interferons as macrophage-activating factors. III. Preferential effects of interferon-gamma on the interleukin 1 secretory potential of fresh or aged human monocytes.

Human peripheral blood adherent leukocytes incubated with interferon (IFN) of three different species (alpha, beta, or gamma) show an enhanced potential of IL 1 synthesis and secretion that can be revealed by a second signal provided by endotoxins or Poly IC. We have shown that recombinant IFN-gamma, compared with recombinant IFN-alpha or purified IFN-beta, has preferential effects on IL 1 secretion in fresh monocyte cultures. We have observed a progressive and profound loss of the ability of adherent cell cultures to secrete IL 1 upon aging for 4 to 12 days in vitro. IFN-gamma was found to be more efficient than IFN-alpha or -beta at maintaining (when added at the onset of the cultures) or reversing the loss (when added on the fourth day of culture) of the IL 1 secretory function. These observations suggest that the secretion of IFN-gamma during the course of immune responses may have a critical role in feeding back the cascade of interleukins in a loop of amplification, and may thereby regulate macrophage-T lymphocyte interactions.

Adjuvants, Immunologic↗
PubMed1984

Characterization of soluble factors that induce the cytolytic activity and the expression of T cell growth factor receptors of a T cell hybrid.

A rat X mouse T cell hybrid (PC60) proliferates in the absence of T cell growth factor (TCGF) and its cytolytic activity can be induced by culture in mixed leukocyte culture supernatants or concanavalin A-activated rat spleen cell supernatant (CS) to lyse 51Cr-labeled tumor target cells. To characterize the factor(s) responsible for this reversible induction, serum-free CS was fractionated by reverse phase high performance liquid chromatography and by phenyl-Sepharose chromatography. A cytotoxicity-inducing activity (CIA) was separated from TCGF and macrophage-activating factor/interferon-gamma. CIA was found to be a macromolecule with an apparent molecular weight of 12,000-18,000 and a pI of 5.0 and 6.2. Its activity on PC60 cells depended on the addition of TCGF. Thus TCGF may have other effects on T cells than the induction of entry into cell cycle. The number of TCGF surface receptors on PC60 cells was measured using purified 3H-TCGF. TCGF receptors were undetectable on noninduced cells but appeared during induction. The expression of TCGF receptors was not induced either by TCGF or by CIA-containing supernatants or fractions alone, only by a combination of both. These results show that TCGF plays a role in the regulation of the expression of its own receptors.

Animals↗
PubMed1984

Induction of cytolytic cells by pure recombinant human interleukin 2.

Purified recombinant human interleukin 2, produced in Escherichia coli, was sufficient to generate cytolytic cells in concanavalin A-stimulated, T helper cell-depleted (Lyt-1.1-) or accessory cell-depleted (Ia-) murine spleen cell cultures. Moreover, recombinant interleukin 2 (rIL2) was able (even at 0.5 ng/ml) to induce cytolytic cells in undepleted murine spleen cell cultures and undepleted human peripheral blood lymphocyte cultures in the absence of a mitogen. Purified recombinant human interferon-gamma, produced in Chinese hamster ovary cells, did not induce either cytolytic activity or IL2 responsiveness in human peripheral blood lymphocyte cultures. Possibly, the rIL2-induced cytolytic cells are formed as a result of nonspecific secondary cytotoxic T lymphocyte activation.

Animals↗
PubMed1984

Oligonucleotide directed mutagenesis: selection of mutants by hemimethylation of GATC-sequences.

We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an AT----CG transversion in the mut L region of phage lambda. Under optimal conditions, about 50-60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.

Bacteriophage lambda↗
PubMed1984

Potentiation of interferon action by mixtures of recombinant DNA-derived human interferons.

Natural and essentially pure recombinant DNA-derived HuIFN-alpha and HuIFN-gamma were examined for their relative abilities to potentiate interferon action. Potentiation of human interferon's antiviral and antiproliferative activities were studied. The essentially pure recombinant DNA-derived human interferons were found to be as effective in their potentiating interactions as their natural counterparts. The results demonstrate that it is the human interferons themselves which interact to potentiate human interferon's varied activities.

Antiviral Agents↗
PubMed1984

High-level expression of human interferon gamma in Escherichia coli under control of the pL promoter of bacteriophage lambda.

Several recombinant plasmids have been constructed which direct high-level synthesis of mature human interferon gamma (IFN-gamma) in Escherichia coli using the inducible leftward promoter pL of phage lambda followed by a translational initiator region derived either from the phage MS2 replicase gene or the E. coli tryptophan attenuator region. Under these conditions, IFN levels of up to 25% of the total cellular protein can be achieved. The highest levels were obtained when a terminator of transcription was cloned downstream from the IFN-gamma sequence. IFN-gamma was almost entirely found in the initial pellet fraction and not in soluble extracts. Co-induction of the lysis genes derived from phage MS2 or from phage lambda, inserted downstream from the IFN-gamma sequence, did not enhance the biological activity present in the supernatant fraction.

Bacteriophage lambda↗
PubMed1984

Interactions of T lymphocytes with human vascular endothelial cells: role of endothelial cells surface antigens.

We have studied the interactions of peripheral blood T lymphocytes with cultured human vascular endothelial cells, focusing upon endothelial cell surface antigens important for T cell recognition. Under standard culture conditions endothelial cells express class I but not class II major histocompatibility complex (MHC) antigens. However, class II antigens may be induced by activated T cells or T cell products, including the lymphokine immune interferon. Immune interferon concomitantly increases class I antigen expression and causes a change in cell shape. In addition to vascular endothelial cells, we have found that vascular smooth muscle cells and human dermal fibroblasts may also be induced by immune interferon to express class II antigens. All known human class II antigens are induced (i.e. HLA-DR, DC and SB) as is the associated invariant chain. Induced antigen expression in these cells is stable over several days, although mRNA levels decline rapidly upon withdrawal of interferon. Vascular and stromal cell class II antigens are functional, in that they can be recognized by cytolytic and helper T cell clones. Several non-MHC antigens are also involved in the recognition of endothelial and stromal cells by T cells. We propose a model for the role of inducible class II molecules on endothelium and stromal cells in vivo: The induction of class II MHC antigens on endothelial cells, locally mediated by activated T cells, enables endothelium to present an immunogenic cell surface structure, comprised of antigen plus self class II polymorphic determinants, which in turn, serves to recruit additional antigen-specific T cells from the circulation into the site of a developing cell mediated immune response. Class II molecules on stromal cells, also induced locally at the site of a developing response, confers immune accessory function on these cells and may serve to augment and sustain a T cell response.

Antigen-Presenting Cells↗
PubMed1984

Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.

Immune interferon (IFN-gamma) increases the surface expression of HLA-A,B antigens and induces the surface expression of HLA-DR antigens on vascular endothelial cells and dermal fibroblasts. Here we report that IFN-gamma induces parallel expression of two other class II major histocompatibility complex (MHC) antigens, SB and DC. Maximal surface expression of all three antigens is reached in 4-6 days, and HLA-DR and -SB are induced to a higher level of expression than HLA-DC. For all three class II antigens, induction is marked by the de novo appearance of detectable transcripts of class II heavy and light chains and of the non-MHC-encoded invariant chain, suggestive of the transcription of multiple previously silent genes. Class I message levels and antigen expression are also increased by IFN-gamma at similar rates but from initial levels that are 50% of maximal. After removal of IFN-gamma, class II antigen expression persists for at least 4 days, while mRNA levels decrease rapidly. The parallel induction and persistence of the several class II MHC antigens may be important in conferring immune accessory function on vascular and stromal cells.

Endothelium↗
PubMed1984

Purification of recombinant glycosylated human gamma interferon expressed in transformed Chinese hamster ovary cells.

Human IFN-gamma was produced in cultures of a Chinese hamster ovary (CHO) cell line transformed with a combination of plasmids encoding HuIFN-gamma cDNA and mouse DHFR cDNA and subsequently selected for growth in the presence of methotrexate. Confluent monolayers of these cells constitutively secrete HuIFN-gamma into the medium reaching a concentration of 2-5 micrograms/ml; the supernatant of the monolayer could be harvested daily for a period of more than 10 days. IFN-gamma was purified by passing the filtered CHO cell culture medium directly through a phosphocellulose column followed by elution and adsorption on a Con A-Sepharose column. Further concentration on an AMICON PM 10 filter and removal of high mw contaminating proteins with DEAE-Sephacel resulted in a IFN-gamma preparation of more than 99% purity (specific activity of about 10(8) International units per mg of protein). Each liter of CHO conditioned culture medium yielded 1-2 mg pure HuIFN-gamma. Its molecular weight, as determined by gel filtration, is about 50 kD and corresponds to a dimer structure. SDS-polyacrylamide gel electrophoresis indicated the presence of a 21 kD and a 25 kD polypeptide as compared with 17 kD for unglycosylated, bacterially made HuIFN-gamma and consistent with the two glycosylated forms of HuIFN-gamma produced in mitogen-stimulated human lymphocyte cultures.

Animals↗
PubMed1984

Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.

Amino Acids↗