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Biomedical subjects

M De Ley

Publications and source records attributed to M De Ley.

At least 73 records · Page 4Linked to original sources
PubMed1983

Production of human immune interferon (Hu IFN-gamma) studied at the single cell level. Origin, evidence for spontaneous secretion and effect of cyclosporin A.

A reverse hemolytic plaque assay has been developed which specifically detects secretion of human immune interferon (Hu IFN-gamma) at the single cell level. Unstimulated peripheral blood lymphocytes from healthy adult volunteers spontaneously secreted IFN-gamma. Stimulation of these cells with concanavalin A, phytohemagglutinin, or the UCHT1 monoclonal anti-human T cell antibody significantly increased the number of IFN-gamma-secreting cells. The cell producing IFN-gamma, both spontaneously and after UCHT1 antibody stimulation, is an OKT3+,4+,8-,HLA-DR-T lymphocyte as determined at the single cell level. Finally, cyclosporin A, a potent and selective immunosuppressive drug for T cells, strongly inhibited the secretion of IFN-gamma as assayed at the cell level. This IFN-gamma reverse hemolytic plaque assay has great potential for the further study of IFN-gamma both in physiological and pathological conditions.

Antibodies, Monoclonal↗
PubMed1983

An interferon-beta-like or interferon-inducing protein released by mitogen-stimulated human leukocytes.

Human peripheral blood leukocytes were treated with concanavalin A (Con A) to produce interferon gamma (HuIFN-gamma). On gel filtration this interferon eluted as a protein with a molecular weight of 45000. In addition to this, the culture supernatant contained an interferon-like protein of apparent molecular weight 22000 (22K factor). The antiviral activity of this protein was neutralizable by a highly specific antibody to HuIFN-beta. Yet, the 22K factor differed from classical HuIFN-beta in several characteristics: lack of activity on certain homologous and heterologous cells which are sensitive to HuIFN-beta; lack of affinity for zinc-chelate and Con A-Sepharose columns; failure to bind to an anti-HuIFN-beta antibody column. Moreover, a specific antiserum raised against the 22K factor did not neutralize HuIFN-beta. Two alternative explanations of these findings are proposed: (i) the 22K factor is an interferon whose molecular structure resembles that of the known HuIFN-beta but it is not identical to it, or (ii) the 22K factor is not an interferon but a protein that can induce the production of HuIFN-beta in certain lines of fibroblastoid cells.

Concanavalin A↗
PubMed1983

Factors influencing the production of human immune interferon components.

Human interferon (IFN), induced in leukocyte suspensions by mitogens, contains two antivirally active components (45K and 22K), separable from each other by gel filtration. The 45K component was identified as IFN-gamma; the 22K component is serologically related to IFN-beta, but has different physicochemical and biological properties. Relatively large amounts of 22K were produced in leukocyte suspensions isolated from buffy coats after induction with concanavalin A (ConA); induction of plateletpheresis residues with staphylococcal enterotoxin A (SEA) resulted in the production of almost only IFN-gamma (45K). The addition of foetal calf serum (FCS) to the production medium enhanced the yields of IFN-gamma, while the production of 22K remained unchanged. Incubation of the cell suspension in the absence of mitogen gave only small amounts of IFN, containing mostly 22K. Under various conditions tested, the two components had similar kinetics of production. A significant increase of IFN-gamma production was observed when leukocytes were primed with low concentrations of 22K.

Culture Media↗
PubMed1982

Responsiveness of human cells trisomic for chromosome 21 to the antiviral action of human immune interferon.

Seven human diploid cell strains (three disomic and four trisomic for chromosome 21) were tested for sensitivity to preparations of the three types of human interferon (IFN). Relative to the disomic control strains, the trisomic-21 cells strains were found to be more sensitive to leukocyte (HuIFN-alpha) and to fibroblast IFN (HuIFN-beta). This chromosome 21-controlled increase in sensitivity to HuIFN-alpha and -beta, was not always accompanied by a parallel increase in sensitivity to HuIFN-gamma. In fact, in some D21/T21 comparisons the sensitivity to HuIFN-gamma and that to HuIFN-alpha and -beta diverged, suggesting that the cellular receptor site(s) for the different IFN types may be different. The presence of a high concentration (greater than 2%) of bovine serum in the culture medium inhibited the responsiveness of all cells to HuIFN-gamma but not to HuIFN-alpha or HuIFN-beta.

Animals↗
PubMed1982

Activation of natural cytotoxicity of human peripheral blood mononuclear cells by interferon: a kinetic study and comparison of different interferon types.

Overnight incubation of human peripheral blood mononuclear (PBMN) cells with leucocyte interferon (leucocyte IFN) resulted in a 2-5-fold increase in natural cytotoxicity (NC) against the erythroleukaemic cell line K562. Fibroblast IFN, purified to homogeneity by zinc-chelate chromatography, stimulated NC to the same extent, while partially purified immune IFN was about twice as active. Upon gel filtration of immune IFN, NC stimulating and antiviral activity co-eluted. Treatment of PBMN cells with ammonium chloride buffer (AmCl) abrogated NC nearly completely. Incubation of AmCl-treated cells with leucocyte IFN resulted in a partial regeneration of NC. Kinetic studies revealed that this regeneration required only short exposure to IFN followed by a longer incubation period. The data are interpreted as indicating that in the process of NC activation IFN mainly acts as a trigger for precursor cells to mature into cells with NC.

Ammonium Chloride↗
PubMed1981

Interferon induced in human leukocytes by concanavalin A: isolation and characterization of gamma- and beta-type components.

Interferon (IFN) was induced in suspensions of fresh human leukocytes by incubation with concanavalin A (Con A). The crude supernatant was concentrated and partially purified by adsorption to silicic acid and elution with an ethylene glycol (EG)-containing buffer. Th EG eluate was shown to contain two antivirally active components (tentatively called 22K), separable from each other by gel filtration. The 45K component was strictly species specific, relatively sensitive to acid (pH 2) and poorly neutralized by anti-human IFN-beta (HuIFN-beta) antibody. In all probability, it predominantly contained (a) molecular variant(s) of human gamma-type IFN (HuIFN-gamma ). The 22K component was acid resistant and serologically indistinguishable from HuIFN-beta prepared from fibroblasts. However, in contrast to the latter, it had little activity on bovine kidney cells and no activity on human HEp-2 and monkey Vero cells. A preparation with the same properties was obtained by substituting glycine buffer (pH 2) for EG in the elution of Con A-induced IFN from silicic acid. This IFN failed to adsorb to a zinc chelate column, while HuIFN-beta from fibroblasts can easily be purified by affinity chromatography on this type of column. It was concluded that the Con A-induced IFN contained an acid-resistant component of apparent molecular weight 22000 serologically related to classical HuIFN-beta but differing by certain physicochemical and biological criteria.

Animals↗
PubMed1981

The preparation of antibodies directed against human immune interferon.

Antisera against human immune interferon (HuIFN-gamma) were prepared by immunization of rabbits and a goat with antigens of different degrees of purity: a) crude supernatant from concanavalin A-stimulated human leukocytes; b) a preparation partially purified by adsorption to controlled pore glass; and c) pooled fractions (molecular weight - 45,000) obtained by gel filtration and corresponding to the peak of HuIFN-gamma. Antisera with a relatively high titer were obtained in animals immunized with the latter two antigens. The sera were specific for HuIFN-gamma in that they failed to neutralize preparations of HuIFN-alpha and and HuIFN-beta. From the time course of the antibody titers in each animal it seemed that frequent boosters within a short time interval led to a decrease rather than an increase in neutralizing activity of the sera.

Animals↗
PubMed1979

Human fibroblast interferon for clinical trials: production, partial purification, and characterization.

The production and partial purification of human fibroblast interferon for performing clinical trials is described. The interferon was produced by superinduction (exposure to riboinosinic-ribocytidylic acid, cycloheximide, and actinomycin D) of large numbers of human diploid fibroblast cultures. The yield averaged 750 units per cm(2) of culture area. The interferon was concentrated and purified by a two-step procedure involving acid desorption from controlled-pore glass beads and dialysis against polyethylene glycol. Human plasma protein was added as a stabilizer. The lyophilized end product had a specific activity of 0.5 x 10(6) to 1 x 10(6) units/mg of protein; it could be reconstituted for injection at a concentration of 2 x 10(6) units/ml. The composition of this interferon was characterized by crossed immunoelectrophoresis with polyspecific antibodies prepared against the principal sources of potential contaminants: human serum, calf serum, and normal human fibroblasts. Several components of each source were detected. Although the major component of calf serum, bovine serum albumin, was absent, other minor components were retained by the production and purification sequence. One of the main contaminants of fibroblast origin was found to be fibronectin.

Cells, Cultured↗
PubMed1978

Interferon induction and action in human lymphoblastoid cells infected with measles virus.

Virus replication, cytopathic effect and interferon production were measured in Namalva lymphoblastoid cells infected with measles viruses. Eight virus strains of different origin or passage history were compared. An inverse relationship seemed to exist between the abilities of strains to induce interferon and to replicate to high titres. Two representative strains were found to be highly sensitive to lymphoblastoid cell interferon, when tested in a line of monkey kidney cells (Vero). In contrast, Namalva cells were found to be highly insensitive to autologous interferon when challenged with measles or Semliki Forest virus (SFV).

Cell Line↗
PubMed1975

Intramolecular electron transport in human ferroxidase (caeruloplasmin).

The oxidation of reduced human ferroxidase by molecular O2 was studied in a stopped-flow spectrophotometer. It was shown that the two type 1 copper atoms behave differently in the absence of iron. The effect of iron on the kinetic parameters was investiagted. A working model for intramolecular electron transport in the enzyme is proposed.

Ceruloplasmin↗