Isolation of a subspecies of murine interferon antigenically related to human leukocyte interferon.

Various preparations of virus-induced murine tissue culture interferon were found to exhibit some antiviral activity in cultures of human and bovine cells. The heterologous antiviral activity of these murine interferons on both human and bovine cultures was shown to be due to an antigenically distinct murine interferon subspecies. Isolation of the murine interferon species responsible for the heterologous antiviral activities was accomplished by means of antibody affinity chromatography using anti-human leukocyte interferon to selectively remove this interferon species from the bulk of murine antiviral activity. The isolated species, while only accounting for 1–2% of homologous antiviral activity of the unfractionated interferon, was responsible for all the heterologous human and bovine antiviral activity. The antiviral activities of this minor murine interferon component on both murine and human cells were neutralized by antiserum against leukocyte interferon as well as by antiserum against mouse L cell interferon.     

Characterization of a subspecies of mouse interferon cross-reactive on human cells and antigenically related to human leukocyte interferon

Mouse interferon preparations from various virus-induced cell cultures exhibited some antiviral activity (about 0.1% of homologous cell titers) on human cells. When analyzed by SDS-polyacrylamide gel electrophoresis the activity of mouse interferon on mouse cells migrated as two bands with activity peaks at 38,000 and 22,000 daltons, while the activity on human cells was detectable only in the faster migrating portion of the lower molecular weight band, at about 21,000 daltons. When the mouse interferon preparation was chromatographed on a column of immobilized anti-human leukocyte interferon antibodies, the specifically retained interferon was found to migrate in SDS-polyacrylamide gels as a 21,000-dalton species which exhibited similar degrees of activity on both mouse and human cells. Thus, mouse interferon preparations contain a low molecular weight interferon species which is both active on mouse cells and is responsible for all the heterologous antiviral activity on human cells and is antigenically related to human leukocyte interferon. These data suggest significant structural similarities between the active cores of certain interferons from phylogenetically diverse animal species.     

Biological properties of human leukocyte interferon components.

Human leukocyte interferon, purified approximately 1000-fold by affinity chromatography on immobilized anti-interferon globulins and SDS-Sephadex filtration, was resolved into one major and one minor component by adsorption chromatography on hydroxylapatite and electrophoresis in polyacrylamide gels. These components were indistinguishable in their capacity to protect bovine, porcine and murine cells, and the antiviral activities of both were equally susceptible to reduction by beta-mercaptoethanol. They were neutralized to the same degree of rabbit anti-leukocyte interferon but were not neutralized by rabbit antifibroblast interferon serum. Mice immunized with either component developed antibodies to both but failed to form antibodies against human fibroblast interferon. Our present evidence indicates that the two components posses at most only minor structural and antigenic dissimilarities.     

Pharmacokinetic properties of human fibroblast and leukocyte interferon in rabbits.

When rabbits were given intramuscular injections of the same quantities of human leukocyte or fibroblast interferons, the former produced moderately higher levels of circulating interferon. Fibroblast interferon was not cleared faster from circulation, nor was direct inactivation by rabbit blood responsible for this difference.     

The sensitizing properties of the ballast admixtures in human leukocyte interferon

Due to the features of its production technology, human leukocyte interferon (HLI) for intranasal administration is likely to contain ovalbumin (OA). Commercial batches of HLI were tested for the presence of OA responsible for the sensitizing effect of HLI given intranasally. The content of OA in commercial batches of HLI was shown to vary widely and to exceed by far the concentrations having the sensitizing effect. As a result of these studies, the upper limit of OA content was established and the technology of HLI production was modified. These measures permitted production of commercial batches of HLI with OA content not exceeding 0.05 micrograms/ml.     

Effect of glycosylation inhibitors on the production and properties of human leukocyte interferon.

Human leukocyte “buffy coat” suspensions were induced to produce interferon by stimulation with Sendai virus and were incubated during the interferon production period in medium containing various concentrations of glycosylation inhibitors, either d-glucosamine or 2-deoxy-D-glucose. At 80 mMd-glucosamine or at 8 mM 2-deoxy-d-glucose, interferon production was inhibited by approximately 95%; however, at 20 and 2 mM, respectively, these inhibitors allowed interferon production at about 50% of levels produced by leukocytes not treated with the inhibitors. The residual interferons produced in the presence of glycosylation inhibitors were antigenically and biologically indistinguishable from native human leukocyte interferons in terms of neutralization by antisera to native interferons and antiviral activities on cells of heterologous species. However, when analyzed by electrophoresis in SDS-polyacrylamide gels, the interferons produced in the inhibitors exhibited markedly less size heterogeneity than found with native human leukocyte interferons, shifting toward the smaller size forms. Treatment of the native interferon with 0.01 M sodium periodate buffer, pH 4.5 at 4° reduced the size heterogeneity to the lower molecular weight form; such treatment had no detectable effect on the interferons produced in the presence of glycosylation inhibitors.     

Augmentation of human polymorphonuclear leukocyte adherence by interferon

Augmentation of human polymorphonuclear leucocyte adherence by alpha and gamma interferons occurred as early as 2 min after incubation. Enhancement of adherence occurred at optimal concentrations of 100-1,000 IU/ml. There was synergism between alpha and gamma interferons, but not between two subtypes of alpha interferon, on augmentation of adherence, indicating that alpha and gamma interferons act on different receptors on the polymorphonuclear leucocyte. Heat treatment at 65 degrees C for 30 min abolished the effect of interferon on adherence.     

Immune reactions and long-term therapy with human leukocyte interferon

Twenty patients with osteosarcoma were treated with exogenous human leukocyte interferon for periods ranging from 6 to 18 months. Eleven of them remained free from detectable tumour growth during this treatment. Blood samples from all patients were tested for antibodies against interferon and against impurities in the interferon preparations. No patient developed detectable levels of neutralizing antibodies against interferon. All patients formed antibodies against contaminants in the concentrated crude interferon and the partially purified interferon preparation which had been used for treatment.     

Glycosylation of human leukocyte interferon: Effects of tunicamycin

Summary Antiviral activity of interferon secreted by human leukocytes into the culture fluid in the presence of tunicamycin (1–2 µg/ml) was significantly decreased, by 50–60 percent, in comparison to that produced in the absence of the antibiotic. No loss in antiviral activity occurred when tunicamycin was added to already harvested interferon preparations. Some physico-chemical and biological properties of human leukocyte interferon synthesized in the presence of tunicamycin (HL-IF^T) were apparently altered by comparison with those of control preparations of human leukocyte interferon (HL-IF): HF-IF^T had only one molecular weight component of 16,000 daltons in contrast to the two components of HL-IF of 16,000 and 21,000 daltons. HL-IF^T also had a higher apparent hydrophobicity and was less efficiently neutralized by an antibody raised against HL-IF. However, some other properties remained unchanged: isoelectric point, pI about 6; affinity for immobilized polyriboinosinic acid and a spectrum of cross-species antiviral activity. These data support the notion that the major component of HL-IF (70 percent, 16,000 daltons) is apparently nonglycosylated whereas the minor component (30 percent, 21,000 daltons) is glycosylated via saccharide-lipid intermediates.With 3 Figures     

Inhibition of human herpesviruses by combination of acyclovir and human leukocyte interferon.

Human leukocytes interferon in low concentrations (1 to 5 U/ml) enhanced the antiviral effect of acyclovir against herpes simplex virus, varicella-zoster virus, and cytomegalovirus grown in human fibroblasts. This occurred without additive inhibition of the division of human fibroblasts or proliferation of peripheral blood mononuclear cells. The combined antiviral effect was additive against clinical isolates of cytomegalovirus and was synergistic against clinical isolates of the other two viruses. The magnitude of the effect with cytomegalovirus was the same when laboratory and wild-type virus were compared. The persistence of varicella-zoster virus in the presence of acyclovir in infected human cells was also reduced by the addition of interferon.     

Monoclonal antibodies neutralizing human leukocyte acid- and thermolabile interferon alpha

Using a high titred human leukocyte IFN alpha preparation which contained both acid- and thermolabile (AL-IFN alpha) and acid- and thermostable IFN alpha species in 9:1 proportion for immunization of BALB/c mice, five hybridomas secreting monoclonal antibodies that reacted with AL-IFN alpha were obtained. In antiviral and antiproliferative tests on HL-60 cells, their products showed high degree of specificity for AL-IFN alpha. The results suggest that both the "normal" leukocyte AL-IFN alpha and the IFN alpha found in sera of autoimmune and other chronic patients might belong to the same subtype of IFN alpha.     

Characterization of the size and charge heterogeneities of human leukocyte interferon populations

Summary Human leukocyte interferons are separable into two size components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and are separable into two charge-components by DEAE-BioGel A chromatography. However, each of the charge-components resolved by ion-exchange chromatography contained both size-components, when analysed by SDS-PAGE. Thus, there are more than two distinct molecular populations of human leukocyte interferons.With 3 Figures     

Physico-chemical properties of interferon produced by a mixed leukocyte suspension.

Physico-chemical properties of partially purified interferon produced by a mixed culture of human peripheral blood leukocytes following induction with double-stranded RNA extracted from f2 phage infected Escherichia coli were studied. Molecular heterogeneity of the interferon preparation was demonstrated by gel chromatography on a Sephadex G-100 column, disc electrophoresis in polyacrylamide gel and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The first two methods revealed 5 peaks, the latter 7 peaks of interferon activity. There was no difference in the molecular nature of interferon produced by cultures exposed to the inducer for the whole period of incubation and that produced by cultures from which the inducer was removed after a short induction time.     

Augmentation of natural and antibody-dependent cell-mediated cytotoxicity by pure human leukocyte interferon

Augmentation of the cytolytic activity of human natural killer cells and of antibody-dependent cell-mediated cytotoxicity has been attributed to human interferons. With the purification to homogeneity of human leukocyte interferon, it became possible to test directly whether pure interferon could increase the activity of these effector cells. Treatment of purified blood mononuclear cells with pure interferon resulted in substantial increases in natural killer cell activity and in antibody-dependent cell-mediated cytotoxicity. Concentrations of 10–100 units/ml of antiviral activity were sufficient to augment appreciably natural killer cell activity.     

Synthesis of interferon in human leukocyte cells stimulated with influenza virus

Strains of influenza A virus were tested for their ability to induce interferon synthesis in suspension of human leukocytes. Both, active as well as UV-inactivated strains were shown to be weak interferon inducers as compared to Newcastle virus. It appeared that interferon synthesis in human leukocytes was conditioned not by infectivity of the virus but by the presence of its surface antigens--hemagglutinin and neuraminidase.     

Concentration of heterogeneous leukocyte interferon

A comparative study of different methods for concentration of porcine leukocyte interferon active in human cells was carried out. Ultrafiltration in an apparatus TCF-10 was found to be the most effective method of concentration by which preparations of porcine interferon showing antiviral activity up to 1 X 10(6) IU/ml were obtained. The total antiviral activity of concentrated interferon was twice as high as that of the native preparation. This increase of the total initial activity is due to the removal in the process of concentration of an inhibitor of antiviral activity which was isolated and concentrated. The concentrated preparation of porcine leukocyte interferon is now undergoing trials in veterinary and medicine.     

A radioimmunoassay of human leukocyte interferon using protein A-containing Staphylococcus aureus

We report the establishment of a radioimmune assay for human alpha-interferon using highly purified alpha-interferon labeled with iodine-125, a purified rabbit immunoglobulin directed against human interferon and the Cowan strain of Staphylococcus aureus. Radiolabeling conditions used do not change the antigenicity of interferon molecules. The regression of counts bound upon log. dose was found to be linear down to 10 units/ml of alpha-interferon (6-7 X10-12 M). This assay was specific for alpha-interferons derived from human peripheral blood leukocytes and from a continuous line of lymphoblastoid cells. No cross-reaction was found with either human beta-interferon or murine interferon. Neither human serum nor plasma interfered with the assay. Correlation between biological assay and radioimmune assay was found to be significant.