Effect of oxygen on the antiviral activity of human interferon in cell culture

Antiviral activity of human lymphocytic interferon under conditions of increased oxygen levels in the cell culture was studied. It was found that oxygen had a capacity for increasing the antiviral effect of human interferon in homologous cells. When 20-80% air was replaced by oxygen the interferon titers on an average amounted to 1:113.4-1:124.8 against 1:29.1 in the control. This means that the average titer of interferon in the experiments with oxygen was 4 times higher than that in the control. On the basis of these data it is recommended using interferon in the form of aerosols in conjunction with oxygen for the treatment of viral respiratory infections.     

Differential inactivation and separation of homologous and heterologous antiviral activity of human leukocyte interferon by a proteolytic enzyme.

Human leukocyte interferon (HuLeIF) can express its antiviral activity on both human and bovine cells. The rates of inactivation of HuLeIF by α-chymotrypsin, as expressed on human and bovine cells, are not the same: the ability to induce activity on human cells is lost significantly more rapidly than the activity detected on bovine cells; usually a margin of greater than one hundred-fold exists after α-chymotrypsin treatment. HuLeIF, when subjected to analysis on 10% SDS-PAGE, can be separated into two molecular weight species, one having apparent molecular weight of approximately 21,000 daltons, the other 18,000 daltons. A more rapidly migrating form (molecular weight 16,500 daltons) can also be isolated, which is considerably more active on bovine cells than on human cells. α-chymotrypsin-treated samples analyzed by SDS-PAGE show a clear separation of the activities expressed on human and bovine cells. The residual activity detected on human cells is isolated only in the 21,000 component while the activity found on bovine cells is recovered only as the 16,500 dalton species.     

Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-alpha A, B, C, D, F) and 2 molecular hybrids between them (IFN-alpha AD(Bg1II) and IFN-alpha DA(Bg1II)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-alpha B, C and F showed comparable antiproliferative activity which was greater than that of IFN-alpha A, D, AD(Bg1II) and DA(Bg1II). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-alpha A, D and AD(Bg1II) were observed to be more protective than IFN-alpha B, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.     

Antiviral and antiproliferative activity of human fibroblast interferon fractions

The affinity chromatography of Human crude beta-interferon preparations on Blue Dextran Sepharose columns resulted in isolation of several fractions with different ratio of antiviral to antiproliferative activities. The results of investigation of two of these fractions are described in this report. The first of them was eluted by 1N NaCl in 0.01 M tris buffer at pH 7.8, the second was eluted by 1 M NaCl, 50% methylethylenglycol in 0.01 M tris-HCl buffer at pH 7.8. The first of the fractions possessed presumably antiproliferative and the second presumably antiviral activity. Both fractions induced the increase of 2'5'-oligoadenylatesynthetase activity in cells although the inducing activity of the first fraction was about 6-fold higher than that of the second one as compared with their antiviral activities. The obtained results indicate that purification of interferon preparation for interferons main antiviral activity may lead to the loss of the great part of antiproliferative material.     

Effect of dibasol and ascorbic acid on the antiviral activity of human interferon in cell culture]

The antiviral effect of human lymphocytic interferon was studied in the primary culture of human embryonic fibroblasts in the presence of dibazol and ascorbic acid. It was found that dibazol and ascorbic acid in concentrations of 5 and 10 gamma/ml respectively were capable of increasing the antiviral effect of human interferon in homological cells. The assays of 13 lots of interferon showed that its average titer in the experiments with ascorbic acid was 2.5 times higher than that in the control. The assays of 21 lots of interferon showed that its average titer in the experiments with dibazol was 3 times higher than that in the control. It is suggested that an increase in the protective properties of interferon in the presence of dibazol and ascorbic acid is connected with their capacity for stimulating the intracellular production of DNA and protein. The data obtained indicate that dibazol and ascorbic acid may be recommended in the complex of therapy and prophylaxis of antiviral infections.     

Apparent dispensability of the carbohydrate moiety of human interferon for antiviral activity.

Human leukocyte and tritium-labeled fibroblast interferons, prepared by induction with Sendai virus and with double-stranded polyinosinic acid.polycytidylic acid respectively, have been studied in relation to the carbohydrate moieties attached to them. These interferons were partially purified by immunoabsorbance and by gel filtration. On treatment with glycosidases, about 80% of the 3H-labeled sugar moieties in this glycoprotein-containing fraction was removed without detectable alteration of the antiviral activity or antibody-binding properties characteristic of interferon. The molecular weight of leukocyte interferon was reduced by about 4000. As others have reported, the heterogeneous character of interferon revealed by isoelectric focusing was greatly reduced by the enzyme treatment.     

Ribonuclease activity in preparations of human leukocyte interferon]

Preparations of human leukocyte interferon obtained by multi-stage purification procedure exhibited ribonuclease activity with the optimum at pH 7.0--7.5. The enzyme possessed the endonuclease action mechanism. Most substances studied for their effect on the RNA-ase activity in human interferon preparations showed many of them to act on the enzyme in the same way as on other ribonucleases. However, dithioerythritie, a reducing agent for disulfide bounds, activated the ribonuclease in the interferon preparation, as distinct from the pancreatic ribonuclease, which was inhibited by this preparation. Patterns of protein and RNA-ase distribution were obtained by electrophoresis in polyacrylamide gel.     

Effect of protein kinase C inhibitors on the antiviral activity of human alpha interferon in herpes simplex virus-infected human neuroblastoma cells.

Pretreatment of human neuroblastoma cells with an inhibitor of protein kinase C (PKC), staurosporine or H-7, prior to the addition of human alpha interferon (HuIFN-alpha), recombinant HuIFN-alpha, or recombinant HuIFN-beta blocked the inhibitory effect of these IFNs on the release of infectious herpes simplex virus type 1 from treated cells. In addition, staurosporine blocked the inhibitory effect of HuIFNs on the expressions of herpes simplex type 1 glycoproteins B, C, and D in treated neuroblastoma cells. Furthermore, addition of HuIFNs resulted in an increased expression of PKC in treated neuroblastoma cells. These results suggest that inhibitors of PKC block the expression of HuIFN-induced genes in treated human neuroblastoma cells. Thus, the activation of PKC is an important step in the HuIFN-treated cells of neuronal origin.     

Antiprotease activity in tears and nasal secretions

Summary A trypsin-inhibitory capacity in tears and nasal secretions is demonstrated. No correlation exists between the serum a₁-AT genotype and the level of trypsin-inhibitory capacity in these secretions. Application of the radial-immunodiffusion technique indicates that the antiprotease activity in tears is different from that associated with a₁ globulin.

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Zusammenfassung Im Nasen- und Tränensekret gelang der Nachweis von Antiproteasen-aktivität (Trypsin-Hemmkapazität). Zwischen dem Serum a₁-AT-Genotyp und dem Ausmaß der Trypsin-Hemmkapazität in diesen Sekreten besteht keine Korrelation. Mittels der Radial-Immunodiffusionstechnik konnte gezeigt werden, daß die Antiproteasenkapazität im Tränensekret immunologisch nicht mit dem Serum-a₁-Antitrypsin identisch ist.

     

Effect of levamisole on interferon production by PHA-stimulated human leukocyte cultures

Mononuclear leukocytes of 10 normal blood donors were cultured in vitro and treated with phytohemagglutinin (PHA-P) and/or levamisole. Interferon-like activity was investigated in the supernatant fluids of the cultures, using VSV as challenge virus. In most of the cases the PHA-stimulated interferon-like activity was slightly but significantly enhanced by levamisole. The antiviral activity produced in the supernatant fluids was characterized as interferon since it was trypsin sensitive, species specific and inhibited by specific antiserum. This interferon has the characteristic sensitivity to PH2 of immune interferon.     

Purification and characterization of human leukocyte interferon components.

Human leukocyte interferon, prepurified either by acid ethanol extraction or by affinity chromatography with antibodies, was further purified by gel filtration in the presence of sodium dodecyl sulfate. Interferon was eluted from gel filtration columns as an apparently homogeneous entity with a molecular weight of 26,600, resulting in an up to 50-fold additional purification during a single step. The antiviral activity could be further resolved into two components by hydroxylapatite adsorption chromatography. The isolated components (A and B) were distinguishable by isoelectric focusing and polyacrylamide gel electrophoresis. The apparent molecular weights were 20,000 to 16,000 and 16,000, respectively. No differences were detected in their susceptibility toward reduction of disulfide bonds by beta-mercaptoethanol. Both could be obtained on a preparative scale with minimal losses in biological activity.     

Recombinant human leukocyte interferon produced in bacteria has antiproliferative activity

Recombinant human leukocyte interferon synthesized by Escherichia coli possesses antiproliferative activity in addition to antiviral activity. When the ability to inhibit multiplication of lymphoblastoid Daudi cells was examined, the growth-inhibitory capacity of recombinant leukocyte interferon was equivalent to that exhibited by crude human leukocyte interferon or by the homogeneous gamma 2 species of leukocyte interferon synthesized by human cells.     

Affinity of human leukocyte interferon for polyribonucleotides.

Human leukocyte interferon (HL-IF)binds to AGPOLY(A)TM, AGPOLY(U)TM and AGPOLY(I)TM. The bound interferon could be displaced from all three polyribonucleotides by including sodium chloride in the eluant. The nature of interaction of HL-IF with polyribonucleotides is electrostatic and not hydrophobic since its binding was not prevented in the presence of 50% ethylene glycol. The binding of HL-IF on AGPOLY(I)TM is stronger at lower pH since an increase in ionic strength is required to displace it.     

Nature of the molecular heterogeneity of human leukocyte interferon.

The existence of two components of human leukocyte interferon has been recently reported. In the present study, the nature of this molecular heterogeneity was explored by affinity chromatography on immobilized micro- and macroligands, ion-exchange chromatography, and molecular sieving. Chromatography on a series of alkyl-agarose adsorbents shows, for the first time, the intrinsic hydrophobicity of human leukocyte interferon. Additionally, the separation of two interferon components is achieved by use of the alkyl-agarose as well as by the omega-aminoalkyl-agarose adsorbents. Clear-cut separation of the two components was also achieved by chromatography on BSA-CH-Sepharose and on DEAE-Bio Gel A. An important feature of these separations is that they do not require the use of denaturing conditions. The molecular weights of the leukocyte interferon components, as determined on Sephadex G-75, are quite similar or identical, approximately 26,000. Thus, the molecular heterogeneity of human leukocyte interferon can be attributed, at least in part, to differences in the hydrophobicity and ionic properties of its two components.