Protection of mice from Semliki Forest virus infection by lymphocytes treated with low levels of interferon

These studies provide the first evidence that adoptive transfer of syngeneic mouse (BALB/c) lymphocytes treated with low levels of mouse interferon (IFN)-alpha/beta can result in sufficient protection to protect mice from Semliki Forest virus (SFV) infection. Specifically, intraperitoneal inoculation of noncytotoxic lymphocytes treated exogenously with IFN (3 to 50 U/ml), washed exhaustively, and mixed with antibody to IFN-alpha/beta to neutralize any residual or early produced IFN, resulted (after repeated studies) in a 35% to 40% reduction in mortality of mice challenged with SFV (P less than or equal to .01), while inoculation of control lymphocytes had no effect. Direct administration of relatively high levels of IFN-alpha/beta (2,000 U/d) only moderately reduced the mortality (by 20%) in mice. Passive transfer of IFN-treated BALB/c mouse embryo cells also did not protect. The protection could not be attributed to carryover of IFN by the lymphocytes, endogenous IFN induction, enhanced cytotoxicity of endogenous splenocytes or peritoneal leukocytes, or early appearance of antiviral neutralizing antibody. Thus, the most likely cause of the observed protection is consistent with a unique mechanism that can be activated by the IFN-treated lymphocytes.     

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.     

2'-Phosphodiesterase activity in human cell lines treated or untreated with human interferon

2'-Phosphodiesterase activity was investigated, by measuring either the disappearance of (2',5')oligo(adenylate) or the release of 5'AMP, in several human cell lines (RSa, IFr, HEC-1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2'-5')oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2'-phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1-25000 nM). The activity of 2'-phosphodiesterase was dependent on Mg(OAc)2. Our results indicate that in various human cell lines the modulation of (2'-5')oligo(adenylate) metabolism by interferon does not involve an increase of 2'-phosphodiesterase activity.     

Failure to induce an antiviral state in preimplantation bovine embryos treated with interferon

Bovine blastocysts hatched from their zonae pellucidae were cultured for 24 h in the presence or absence of interferon and then challenged with either vesicular stomatitis virus or bluetongue virus to assess the induction of an antiviral state. In contrast to its application to fetal bovine cells, where significant antiviral effects were induced, interferon treatment of embryos failed to reduce virus yield and had no effect on virus-induced cytopathology. This lack of biologic activity of interferon in bovine embryos is similar to that previously observed with undifferentiated murine embryonal carcinoma cells and is probably a manifestation of a more general mechanism regulating gene expression in the early mammalian embryo.     

Effect of mycoplasma on interferon production and interferon assay in cell cultures

Armstrong, D. (The Children's Hospital of Philadelphia, Philadelphia, Pa.), and K. Paucker. Effect of mycoplasma on interferon production and interferon assay in cell cultures. J. Bacteriol. 92:97-101. 1966.-The influence of mycoplasma on the production and action of interferon was studied in cultures of both L and human embryonic kidney (HEK) cells. Mycoplasma hominis 1, the Negroni agent, and the F12 mycoplasma were used for infection of L cells, and M. hominis 1 and M. pneumoniae for inoculation of HEK cells. All strains were capable of multiplication in the culture systems employed. None produced detectable levels of interferon, and responsiveness of the cells to induction of interferon by virus remained unaltered. Infection with mycoplasma did not impair the sensitivity of the cells to the action of interferon, nor was the replication of vesicular stomatitis virus noticeably diminished.     

Generation of lipopolysaccharide-induced interferon in spleen cell cultures

Incubation of murine spleen-cell cultures with lipopolysaccharide (LPS) induces interferon (IF) production. Maximal IF levels are obtained after incubation with 100 g/ml for 10 h. Two inbred mouse strains differing in their ability to generate LPS-induced IF in spleen-cell cultures were used: C3H/eB, which generates high levels of IF (about 60 units/ml), and C3H/HeJ, which fails to generate detectable quantities of IF. In a genetic analysis these strains were hybridized and IF production was determined in spleen-cell cultures from F₁ and F₂ generations, and from backcrosses of F₁ hybrids to parent strains. The results indicate that, in parent strains, a single dominant autosomal gene is responsible for differences in IF production in spleen cultures. LPS-induced IF in spleen-cell cultures resists pH 2 for as long as 48 h, but is labile to heating at 56° C for 30 min. Both macrophages and lymphocytes must be present in cultures for generation of LPS-induced IF. By using mixed cultures of macrophages and lymphocytes from C3H/eB and C3H/HeJ mice, it was shown that macrophages have to interact directly with LPS to enable IF production in the cultures.     

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.     

Formation of immune interferon and blast transformation in stimulated cultures of human lymphocytes

Lymphocyte blast transformation (LBT) and interferon (IF) production were studied in 55 phytohemagglutinin- and dry purified tuberculin-stimulated lymphocyte cultures obtained from normal subjects (7 adults and 24 children). A relationship has been disclosed between LBT and interferon production. However, in some of the culture with marked LBT there occurred a defective IF production. On the contrary, with suppressed PHA-induced transformation, IF production was within normal. The role of the interferon production test for estimation of the functional activity of human lymphocytes is discussed.     

Antiviral activity of preparations of RNA isolated from cells treated with interferon (messenger RNA for an antiviral protein?)]

Chick embryo cultures treated with interferon yielded a biologically active RNA which, upon inoculation into chick embryo cells, created an antiviral condition in them. The level of vesicular stomatitis virus reproduction in such cells was 2-30% of that observed in the cells treated with control RNA. The maximum activity of the experimental RNA was seen 3 hours after the treatment with interferon.     

Dissociation of protein kinase activity and the induction of the antiviral state in a cell line responsive to the antiviral effects of interferon

Interferons or oxidized glutathione were found to induce double-stranded RNA-dependent protein kinase activity in mouse L cells that phosphorylates the α subunit of eukaryotic peptide initiation factor 2. A mixture of leukocyte/fibroblast interferons as well as immune interferon induced the protein kinase and also suppressed virus replication in the L cells. Oxidized glutathione was equally effective in inducing protein kinase activity, but it did not induce an antiviral state in these cells. The data suggest that a simple cause and effect relationship does not exist between protein kinase induction and the establishment of the antiviral state in a cell that is responsive to the antiviral effects of interferon.     

Investigations of genotoxic activity of antimicrobial/antiviral agent FS-1 in human lymphocytes and tumor cell

A very promising antiviral and antimicrobial agent FS-1 was studied for its ability to induce DNA damage and micronuclei in human tumor cell lines HeLa and Caco-2 at concentrations of 200, 500 and 1000 microg/ml without exogenous metabolic activation. The compound was additionally tested for DNA damaging ability in human lymphocytes at concentrations of 200, 400 and 800 microg/ml. Neither DNA damage nor micronucleus formation was observed after treatment of all types of cells with FS-1. Based on these results, FS-1 can be further studied for its safety to humans for potential application in clinical medicine as an antimicrobial/antiviral drug.     

The role of cell membrane in the antiviral effect of interferon

The mechanism of interferon action in human fibroblasts has been studied by use of both antisera to human fibroblast interferon and the antisera to the surface of human fibroblast cell. The anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Interferon antiserum prevented the intracellular antiviral state from developing when added to the medium of the cells in which interferon synthesis had already been induced by poly (I · C). This suggests that development of the antiviral state involves interferon interaction with the external part of the producing cell. Treatment with the serum directed against the surface of human fibroblast cells failed to inhibit the antiviral activity of human interferon in these cells. In addition, the effect of gangliosides on the antiviral activity of human interferon was studied and it was found that human interferon binds to gangliosides and that this interaction leads to inactivation of the antiviral effect of interferon. Pretreatment of human fibroblasts with gangliosides had no effect on the sensitivity of these cells to exogenous interferon.