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Characteristics of human lymphoblastoid (Namalva) interferon.

Interferon derived from the human lymphoblastoid cell line, Namalva, was fractionated by antibody affinity chromatography into two antigenically distinct interferon subspecies. At least 13% of the total Namalva interferon activity possessed the F antigenic determinant found on human interferon derived from fibroblast cultures, while the bulk of the Namalva interferon activity had the Le antigenic determinant characteristic for human leukocyte interferon. The separated Le and F subspecies of Namalva interferon differed in the degree of their heterospecific activities on bovine cells. The Le moiety resembled crude leukocyte interferon in that it was highly active in bovine cells. The F component of Namalva interferon showed a lower degree of activity in bovine cells, thus resembling crude fibroblast interferon. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by isoelectric focusing, crude Namalva interferon qualitatively resembled crude leukocyte interferon.

Le interferon production by human fibroblasts

Abstract Human leukocyte ( Le ) and fibroblast ( F ) interferons differ in antigenic properties, physicochemical characteristics, and activity on cells of heterologous species. Previous work from this laboratory showed that two human foreskin fibroblast strains can produce either F interferon alone or both F and Le interferons, depending on the nature of the inducer. The present study demonstrates that both the cell type and the inducer determine the proportion of Le interferon produced by human fibroblast strains. Of eight strains tested, the human GM-258 strain, which is trisomic for chromosome 21, produced the highest absolute and relative amounts of Le interferon after Newcastle disease virus (NDV) inoculation. However, there was no correlation between Le interferon production and number of copies of chromosome 21 in other cell strains examined. Although both NDV and vesicular stomatitis virus (VSV) induced a substantial proportion of Le interferon in GM-258 cells, little or no Le interferon was induced in the same cells by polyinosinic-polycytidylic acid (poly(I)·poly(C) under five different conditions. In GM-258 cells induced with NDV, it was possible to partially manipulate the relative proportion of F and Le interferons and the time at which they were made by altering the conditions of induction. The percentage of Le interferon was increased by decreasing the multiplicity of infection or irradiating the virus with ultraviolet light. Kinetics of F and Le interferon production after different modes of NDV inoculation paralleled each other closely. F and Le interferon production was inhibited to similar degrees in the presence of the glycosylation inhibitors 2-deoxy- d -glucose and d -glucosamine. The bovine EBTr cell strain was used for the selective assay of Le interferon. These cells were as sensitive to Le interferon as human diploid fibroblasts. In contrast, less than 0.1% of human F interferon activity crossed the species line in EBTr cells. Le and F interferons produced by NDV-induced GM-258 cells were physically separated by affinity chromatography on immobilized anti- Le and anti- F globulins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the Le interferon component produced in GM-258 cells formed two separate peaks (with molecular weights of approximately 18,000 and 25,000), closely resembling a control Le interferon preparation. In contrast, the F interferon moiety made in GM-258 cells formed a single peak on SDS-PAGE, as did control F interferon (molecular weight approximately 26,000).

Correlation of physicohemical and antigenic properties of human leukocyte interferon subspecies

SummaryHuman leukocyte interferon produced in primary cultures of buffy coat cells and human fibroblast interferon from cultures of the FS-4 foreskin cell strain were subjected to isoelectric focusing in polyacrylamide gels. Leukocyte interferon could be resolved into three major components (pI 5.5, 6.2 and 6.6, respectively) and one minor component (pI 7.0). Fibroblast interferon activity focused in a broad pH range of 6.8–7.8.The isoelectrically distinct subspecies of human leukocyte interferon were isolated and compared as to their antigenic nature, heterospecific antiviral activity in cultures of bovine cells, and apparent molecular weights upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The three major subspecies (pI 5.5, 6.2 and 6.6) were similar in their neutralization by antiserum against whole leukocyte interferon and in their relative heterospecific activities on bovine cells. When analyzed on SDS-PAGE, the component focusing at pH 5.5 migrated to a position corresponding to a molecular weight of 17,500 (Lef), the component with the pI of 6.6 had its major peak corresponding to a molecular weight of 23,000 (Les), while the pI 6.2 component contained a mixture of the two molecular weight species. The minor isoelectric component focusing at pI 7.0 contained interferon with the antigenic specificity of fibroblast (F) interferon. It is concluded that the two major antigenic species of human interferon (Le andF) and two known subspecies of human leukocyte interferon (Les andLef) can be resolved by isoelectric focusing.

Immune interferon induction by a monoclonal antibody specific for human T cells

Abstract OKT3, a monoclonal antibody reactive with a surface antigen shared by all human T lymphocytes, was found to act as a potent interferon (IFN) inducer in cultures of human mononuclear white blood cells. Two other monoclonal antibodies reactive with T-cell subpopulations failed to induce IFN. IFN-inducing activity of OKT3 was similar to that of phytohemagglutinin (PHA). The phorbol ester, 12- O -tetradecanoylphorbol-13-acetate (TPA) had a strong potentiating effect on IFN production stimulated with OKT3 or PHA. IFN produced after stimulation with OKT3, like PHA-induced IFN, had properties characteristic for “immune” IFN (IFN-γ).

Stimulation of lymphokine production by teleocidin, aplysiatoxin, and debromoaplysiatoxin

Previous studies showed that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-gamma) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-gamma and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-gamma during a multistep purification procedure.

[6] Induction of human interferon gamma with phorbol esters and phytohemagglutinin

Publisher Summary This chapter discusses the induction of human interferon gamma with phorbol esters and phytohemagglutinin (PHA). It is well known that immune interferon (IFN-γ) is produced by lymphocytes in response to specific antigens or nonspecific mitogens. The T cell mitogen, PHA has been widely employed as an inducer of IFN-γ in cultures of human mononuclear cells from peripheral blood. 12- O -tetradecanoylphorbol-13-acetate (TPA) (also termed phorbol myristate acetate (PMA)) is a potent tumor-promoting agent exerting hormone-like pleiotropic effects on growth, differentiation, and many other cell functions. Recently, it is shown that the cellular receptor for TPA and related phorbol esters is likely to be protein kinase C. Buffy coats can be employed as a source of cells instead of plateletpheresis residues. However, the average IFN-γ yields obtained from buffy coat-derived cultures are only about half of the yields obtained with cells from plateletpheresis residues. The reason for this difference is not known.

STRUCTURE-FUNCTION STUDIES WITH HUMAN INTERFERON-GAMMA

ABSTRACT Interferon-gamma, produced in cultures of human white blood cells by combined stimulation with a phorbol ester and phytohemagglutinin (PHA), was purified about 1,000-fold in a simple 3-step procedure. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that interferon activity not destroyed by SDS treatment was recovered mainly from two peaks with apparent molecular weights of 20,000 and 25,000. The two molecular weight subspecies, isolated in pure form, were antigenically indistinguishable. Highly purified interferon-gamma was iodinated with the 125-I-Bolton-Hunter reagent. This material bound with a high affinity (Kd 7.5 × 10 −11 M) to human fibroblasts in culture. Unlabeled human interferon-gamma or -beta, but not interferon-alpha, competitively inhibited the specific binding of 125I-interferon-gamma. Crude interferon-gamma preparations had a pronounced cytotoxic effect on HeLa and FS-4 cells, while highly purified interferon-gamma was effective only at high concentrations. Furthermore, only in HeLa cells did it show a greater antiproliferative effect than interferon-alpha or -beta.

[30] Production of human fibroblast interferon in the presence of the glycosylation inhibitor tunicamycin

Publisher Summary This chapter describes procedure that deals specifically with the use of tunicamycin in the production of unglycosylated interferon induced by polyinosinate-polycytidylate [poly(I) . poly(C)] in cultures of human foreskin fibroblasts. A similar procedure with 2-deoxy-o-glucose or o-glucosamine as inhibitor is reported. Production of interferon in FS-4 cells by the superinduction procedure has been described. Only the steps involving tunicamycin treatment of the cell culture are described. Tunicamycin is a complex molecule consisting of one residue of uridine and two residues of N-acetyl-o-glucosamine, with the amino hydrogen in one of these residues substituted by a long-chain fatty acid. Four major components is identified in tunicamycin preparations that differ in fatty acid chain length; the molecular weight of each component is determined to be 970, 984, 998, and 1012, respectively. A more recent study demonstrated that tunicamycin can be resolved into two major and eight minor components by high-performance liquid chromatography (HPLC).