Cell cultures of spotted souslik--preliminary characterization of their growth and sensitivity to viruses

Cell cultures from the cutaneo-muscular tissue of fetuses (SE), and from lungs (SL), kidneys (SK), testes (STe) of adult spotted sousliks were obtained. Cells from lungs (SL) were viable at 4 degrees C three times longer than fibroblastic cells of human embryos (HEF). At 37 degrees C the growth rate of SL, HEF and L929 cells was similar. However, at 26 degrees C SL cells grew faster than HEF or L929 cells and their population doubling time was shorter. The cultures of SE, SL and SK cells were sensitive to vesicular stomatitis virus (VSV), Newcastle disease virus Radom (NDV-R) and Hertfordshire (NDV-H) strains, to vaccinia virus and herpes simplex virus type 1 and type 2. All these viruses were able to reproduce in souslik cell cultures and caused characteristic CPE.     

Age-related production of spotted souslik (Spermophilus suslicus) interferon

Interferon production by virus stimulated spotted souslik tissue explants in vitro depends on the age of the tissue donor and on the kind of tissue. The interferon titers in foetal liver were 1/120-1/370 and foetal lungs 1/21-1/500 of these observed in autologous organs of adult sousliks after infection in vitro with Newcastle disease virus, Radom strain (NDV-R). After birth interferon response in vivo develops with the age. Nine-day-old suckling sousliks were not able to respond with interferon production to lipopolysaccharide (LPS), but serum interferon levels after intraperitoneal (i.p.) injection of polyinosinic polycytidylic acid complex (poly I. poly C) complexed with diethyl-amino-ethyl-dextran (DEAE-dextran) or after injection of NDV-R were nearly similar to those in adult animals. In tissue however, levels of interferon were always lower than in adults. Young 4-5-week-old sousliks responded to induction with NDV-R and poly I. poly C with DEAE-dextran and tilorone hydrochloride as adults and produced interferon in serum and different organs, however, after induction with LPS serum interferon level did not reach the values observed in serum of adult sousliks.     

Characterization of antiviral activity of spotted souslik (Spermophilus suslicus) interferons

The antiviral activity of three types of spotted souslik interferons So IFN alpha, So IFN beta and So IFN gamma was studied. Fibroblasts of lungs (SL), kidneys (SK) of adult sousliks and skin fibroblasts of foetuses (SE) were equally sensitive to three types of souslik interferons. In contrast to So IFN beta and So IFN gamma, So IFN alpha, exhibited a high (100% of activity in homologous cells) cross species antiviral activity in mouse embryo fibroblasts (MEF) and a low (3% to 10% of the activity) in bovine embryo fibroblasts (BEF) and human embryo fibroblasts (HEF). The development kinetics of antiviral activity not only depended on the interferon type but also on the temperature of incubation. In comparison with So IFN gamma, So IFN alpha and So IFN beta activated earlier the maximal antiviral state. Low incubation temperature (26 degrees C) did not decrease but only delayed the antiviral activity of spotted souslik interferons. Mixed preparations of So IFN gamma with So IFN alpha or So IFN beta exhibited synergistic antiviral activity at physiological (37 degrees C) and low (26 degrees C) temperatures. The development of antiviral activity of So IFN beta as well that of Mu IFN alpha/beta was inhibited by plant lectins which reacted with the cell membrane compounds. All three types of souslik interferons were completely destroyed by trypsin and boiling at 100 degrees C for 1 min. and partially by SDS. Their sensitivity to shaking, beta-mercaptoethanol and mouse antisera against So IFN beta was different in relation to the interferon type.     

Production and initial characterization of spotted souslik (Spermophilus suslicus) interferon

Spotted souslik (Spermophilus suslicus) interferon was induced in different cell cultures with two strains: Random (NDV-R) and Hertfordshire (NDV-H) of Newcastle disease virus. Both strains were good interferon inducers in cultures of lung cells (SL), kidney cells (SK) and embryo cells (SE). There were no significant differences in the amount of interferon produced in cells at different passage levels but the yield of interferon depended on the donor of cells. The highest interferon titers were produced 24h after infection of SK cells with NDV-R at multiplicity of infection 100 or .10 TCID50 per cell. UV-irradiation decreased the interferonogenic properties of NDV-R. Priming with 100 U/ml of 10 U/ml of homologous interferon significantly enhanced interferon production in souslik cells. However, the interferon concentrations higher than 100 U/ml caused a distinct inhibition in the subsequent interferon production ("blocking"). The pretreatment of cells with L-ascorbic acid slightly increases the yield of interferon produced in souslik cells after induction with NDV-H. Calcium chloride at nontoxic concentrations added together with the virus-inducer practically exhibited no effect on the interferon production in SL cells.     

Interferonogenicity of the bacterial lysates in human and murine cell cultures

The antibacterial JRS vaccine was found to be capable of inducing interferon production in human peripheral blood leukocytes and mouse bone marrow cells. The vaccine induced no interferon in L-929 cells or human diploid M-19 cells. Interferon appeared in the culture fluid within 4-6 hours after induction and reached the maximum levels in 18-24 hours. One-hour contact of the vaccine with leukocytes was sufficient for interferon induction. The interferon generated in human blood leukocytes was partially stable at pH 2.2; heating at 56 degrees C for 30 min reduced its activity 4-fold; antiserum to alpha-interferon inhibited it.     

Macrophage Dependence of Polyriboinosinic Acid-Polyribocytidylic Acid-Induced Resistance to Herpes Simplex Virus Infection in Mice

The relative contributions of macrophages and lymphocytes to the induction of resistance to primary herpes simplex virus type 1 (HSV-1) infection by polyriboinosinic-polyribocytidylic acid complex [poly (I:C)] were investigated in C58 mice. The induction of resistance was found to be strongly dependent on macrophages compared to lymphocytes. Macrophage-deficient (silica-treated) mice produced less interferon and were not as responsive to prophylactic treatment of HSV-1 infections with poly (I:C) as were either normal, lymphocyte-deficient (cyclophosphamide-treated), or T-lymphocyte-deficient (anti-thymocyte serum-treated, adult-thymectomized) mice. Silica and cyclophosphamide treatments reduced the therapeutic activity of poly (I:C), whereas T-cell depletion did not have a significant effect. Similarly, the protection of mice with exogenous interferon was markedly reduced in silica-treated mice and moderately reduced in cyclophosphamide-treated mice, but unaffected in T-cell-deficient mice. Furthermore, suppression of HSV-1 plaque formation was obtained by cocultivation of infected mouse fibroblast monolayers with peritoneal (macrophage-rich) cells, but not with splenic (lymphocyte-rich) cells, from poly (I:C)-treated mice. Peritoneal cells did not protect heterologous (human) fibroblasts, suggesting that the protection of mouse embryo fibroblasts is mediated by interferon. Collectively, the data indicate that macrophages are required for the production of poly (I:C)-induced interferon and that macrophages and perhaps B-lymphocytes are important for mediating the protection against HSV-1 infection after interferon has been produced.     

Effect of thymosin on interferon production and antiviral resistance in mice

The 5th and 6th fractions of thymosin, a hormone of the thymus gland, stimulated interferon production both in vivo (experiments in white and CBA mice) and in vitro in CBA mouse splenocytes when different interferon inducers were used (phage dsRNA, poly(G): poly(C), NDV, and mitogens). The highest stimulating effect in vivo was observed with interferon induction 6-8 hours after thymosin administration. An increase in production of both alpha/beta and gamma interferons under the influence of thymosin was observed. Thymosin alone induced no interferon synthesis.     

Distinguishing characteristics of interferon induction with poly(I)-poly(C) and Newcastle disease virus in human cells.

Exposure of a human diploid foreskin cell strain (FS-4) to polyinosinate-polycytidylate [poly(I)·poly(C)] resulted in an early rise of interferon production that peaked at about 4 hr after induction and decreased rapidly thereafter. Irradiation of cells with low to moderate doses of ultraviolet (uv) light immediately before induction with poly(I)·poly(C) increased the amount of interferon produced up to about tenfold. This enhancement was apparently due to interference with the shut-off process which in unirradiated cells leads to early termination of interferon production; in irradiated cells interferon production continued for much longer.Inoculation of FS-4 cells with Newcastle disease virus (NDV) resulted in interferon production that showed a slower rise and peaked only at about 10–15 hr after inoculation. Irradiation of cells at the time of induction with NDV resulted in a dose-dependent decrease of interferon production. However, a small fraction of the total amount of interferon produced in response to NDV, which appeared by about 5 hr after virus inoculation, was resistant to uv. This uv-resistant early peak of NDV-induced interferon was greatly enhanced in cells which 6 hr before virus inoculation had either been induced with poly(I)·poly(C) or incubated with interferon, while the appearance of the major, uv-sensitive peak of NDV-induced interferon was inhibited or delayed after the same treatments. In its characteristics the early peak of NDV-induced interferon resembled the poly(I)·poly(C)-induced interferon response. Poly(I)·poly(C)-induced, as well as the early and late NDV-induced interferons were all neutralized by an antiserum raised against poly(I)·poly(C)-induced interferon, suggesting that they represent products of the same structural gene(s). It is concluded that there may be more than one mechanism of interferon induction by a single virus.     

Interferon inducing activity of polyinosinic acid.

Although poly(I) is generally considered to be inactive as an interferon inducer, we have found several authentic poly(I) preparations to be effective inducers. Their interferon inducing ability varied considerably from one cell system to another. In human diploid fibroblasts, primed with interferon and superinduced by cycloheximide and actinomycin D, all active poly(I) samples proved nearly as effective in inducing interferon as poly(I).poly(C). In primary rabbit kidney cell cultures, the active poly(I) samples were either as active, or 3 to 30 times less active than poly(I).poly(C). In intact rabbits they were 100 times less active than poly(I).poly(C). Except for one particular sample, all active poly(I) preparations were inferior to poly(I).poly(C) when assayed for interferon induction in interferon-treated mouse L cells; in DEAE-dextran-treated L cells, they induced little, if any, interferon. The poly(I) inducers of interferon were considerably more susceptible to degradation by TI ribonuclease, pancreatic ribonuclease and human serum nuclease(s) than was poly(I).poly(C) when assayed under the same conditions. Due to their limited half-life time in biological fluids, poly(I) analogues such as those described here may offer a greater safety margin in clinical use than poly(I).poly(C).     

Regulation of human interferon production. I. Superinduction by 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

The halobenzimidazole riboside, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), a reversible inhibitor of nuclear heterogeneous RNA synthesis, has been shown to enhance (superinduce) polyinosinic:polycytidylic acid (poly(I:C))-induced interferon production in a strain of diploid human fibroblasts (FS-4). Cells exposed for 1 hr to both poly(I:C) (100 μg/ml) and DRB (40 μM), and then treated with DRB for an additional period of 4 or 5 hr, produce two- to fivefold more interferon than cells treated with DRB for 4 or 5 hr only after removal of poly(I:C). Prolonged pretreatment of cells with DRB does not alter interferon yields. These findings substantiate the suggestion that the process whose inhibition is responsible for superinduction is already occurring during the 1-hr period of poly(I:C) induction. DRB-containing cultures (30, 40, or 60 μM) continue to produce interferon for at least 4 days. However, interferon production is gradually shut off at lower DRB concentrations (10 or 20 μM). The prolonged production of interferon in the continuous presence of DRB is due to new synthesis of interferon on mRNA that was transcribed within 3 hr of poly(I:C) induction. The shutoff of interferon production that occurs on removal of DRB correlates closely with the recovery of cellular RNA synthesis. These observations strongly support the hypothesis that interferon superinduction is closely linked to inhibition of RNA synthesis.A drug regimen consisting of treatment of cells with cycloheximide (50 μg/ml) for 6 hr beginning with poly(I:C) induction and with DRB (40 μM) beginning 3 hr after the start of poly(I:C) induction and continuing until the end of the experiment at 24 hr gives as high an interferon yield as a protocol in which cycloheximide treatment is combined with a 2-hr treatment with actinomycin D (1 or 5 μg/ml) beginning 4 hr after the start of poly(I:C) induction.     

Dipyridamole is an interferon inducer

2,6-Bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]-pyrimidine (dipyridamole) induced interferon production in vitro in explanted mouse peritoneal leukocytes and to a lower degree in non-lymphoidal cell cultures of mouse (L cells primary embryo fibroblasts) and human (diploid embryo lung fibroblasts) origin. Dipyridamole induced interferon also in mice after intravenous administration. Peak interferon levels in the blood (128 IU/ml) were attained at 49 hr after injection of 0.1 mg dipyridamole per kg body weight and at 24 and 12 hr after injection of 0.6-1.8 and 16.7 mg/kg respectively. By its pH stability, thermostability and antigenic properties the interferon induced in mice, mouse peritoneal leukocytes and L cells corresponded to IFN-alpha and IFN-beta. This interferon-inducing capacity of dipyridamole may account for its broad-spectrum antiviral effect.     

Regulation of human interferon production stimulated with poly(I) · Poly(C): Correlation between shutoff and hyporesponsiveness to reinduction

The exposure of FS-4 cells to polyinosinate-polycytidylate [poly(I) · poly(C), 2 μg/ml] for 1 hr resulted in rapid interferon production which peaked in 3–4 hr and was rapidly shut off thereafter, declining to low or undetectable levels by 6 hr. Reexposure of cells to 50 μg/ml of poly(I) · poly(C) at 2, 3, 4, or 5 hr after the first induction did not prevent the s shut off of interferon production due to the first induction. However, the restimulated cultures underwent a second round of interferon production peaking at 8–10 hr. The appearance of the second peak could be inhibited with actinomycin D, if the drug was added 3 hr after reexposure to poly(I) · poly(C), i.e., before the actual onset of the second round of interferon production. The second interferon peak resembled the peak seen a after the first induction in that it also showed a rather steep rise and rapid shutoff. The shutoff of the second peak could be prevented by treatment with actinomycin D at 9 hr (5 hr after the second stimulation). A second interferon peak at 9 hr was also seen following a single 1-hr exposure to a high concentration (50 μg/ml) of poly(I) · poly(C). It is suggested that a rapidly turning over repressor system is induced coordinately with interferon. This repressor system causes irreversible inactivation of interferon mRNA and is likely to be responsible for the shutoff of interferon production as well as the phenomenon of hyporesponsiveness to repeated interferon induction.     

Regulation of human interferon production : I. Superinduction by 5,6-dichloro-1-β- -ribofuranosylbenzimidazole

The halobenzimidazole riboside, 5,6-dichloro-1-β- -ribofuranosylbenzimidazole (DRB), a reversible inhibitor of nuclear heterogeneous RNA synthesis, has been shown to enhance (superinduce) polyinosinic:polycytidylic acid (poly(I:C))-induced interferon production in a strain of diploid human fibroblasts (FS-4). Cells exposed for 1 hr to both poly(I:C) (100 μg/ml) and DRB (40 μM), and then treated with DRB for an additional period of 4 or 5 hr, produce two- to fivefold more interferon than cells treated with DRB for 4 or 5 hr only after removal of poly(I:C). Prolonged pretreatment of cells with DRB does not alter interferon yields. These findings substantiate the suggestion that the process whose inhibition is responsible for superinduction is already occurring during the 1-hr period of poly(I:C) induction. DRB-containing cultures (30, 40, or 60 μM) continue to produce interferon for at least 4 days. However, interferon production is gradually shut off at lower DRB concentrations (10 or 20 μM). The prolonged production of interferon in the continuous presence of DRB is due to new synthesis of interferon on mRNA that was transcribed within 3 hr of poly(I:C) induction. The shutoff of interferon production that occurs on removal of DRB correlates closely with the recovery of cellular RNA synthesis. These observations strongly support the hypothesis that interferon superinduction is closely linked to inhibition of RNA synthesis.A drug regimen consisting of treatment of cells with cycloheximide (50 μg/ml) for 6 hr beginning with poly(I:C) induction and with DRB (40 μM) beginning 3 hr after the start of poly(I:C) induction and continuing until the end of the experiment at 24 hr gives as high an interferon yield as a protocol in which cycloheximide treatment is combined with a 2-hr treatment with actinomycin D (1 or 5 μg/ml) beginning 4 hr after the start of poly(I:C) induction.     

Comparison of the properties of antirickettsial activity and interferon in mouse lymphokines

Certain properties of the antirickettsial activity and interferon in lymphokine preparations obtained from concanavalin A-stimulated mouse spleen cells were compared. Both the antirickettsial activity and interferon were relatively stable to heating at 56 degrees C, whereas both activities were destroyed by trypsin, by heating at 80 degrees C, or by exposure to pH 2 for 24 h. Both activities were likewise inhibited after incubation with rabbit antisera to partially purified murine interferon-gamma. In contrast to the mouse lymphokine preparations, which contained both interferon-gamma and antirickettsial activity, a preparation of virus-induced interferons (type I) had no detectable antirickettsial activity. Human foreskin fibroblasts, which were not sensitive to the antirickettsial activity in mouse lymphokines, acquired the ability to inhibit rickettsial growth when they were cocultured with sensitive mouse L929 cells treated with mouse lymphokines. These results are consistent with the idea that the antirickettsial activity in mouse lymphokines is due to interferon-gamma.     

Effect of amphotericin B on the activity of synthetic polyribonucleotide interferon inducers

The authors investigated the intensity of interferon production and the degree of the associated antiviral resistance induced by double-stranded synthetic polyribonucleotide interferon inducers poly(G) . poly(C), poly(G, I) . poly(C) and poly(G, A) . poly(C) in chick embryo cell cultures, continuous diploid human fibroblasts and in mice in the presence of amphotericin B, a macrolide polyene antibiotic enhancing the permeability of plasma membranes for macromolecules. Amphotericin B was found to increase considerably the interferon-inducing and antiviral activity of the above polyribonucleotide complexes in those cell systems where they induced interferon production and antiviral resistance alone, without the antibiotic, but to a lower degree. Amphotericin B did not contribute to the activity of the complexes in those cell systems where they were inert alone. The importance of permeability of the plasma membrane for interferon induction is discussed, and a conclusion is reached that the inertness of the complexes under study in one cell system and their activity in the others are not associated with differences in the permeability of the plasma membranes of different cells for polyribonucleotide interferon inducers.     

Production,purification and characterization of rat interferon

Summary Priming with heterologous mouse interferon, increased production of an antiviral substance induced in rat diploid fibroblasts by Newcastle disease virus. This substance was characterized as an acid stable interferon. This rat interferon exhibited marked cross-species antiviral activity when tested in mouse L929B cells, guinea pig embryo fibroblasts, human fibroblasts and bovine cells but was not active on chick embryo cells.     

Production of interferon by human tumor cell lines

Summary Fourteen continuous human cell lines, including nine derived from tumors and five from non-neoplastic tissues, produced interferon in response to induction with bluetongue virus (BTV), Newcastle disease virus (NDV), and poly(I) · poly(C) complexed with DEAE-dextran. The seven best interferon-producing cell lines (one from a melanoma, five derived from carcinomas, and one SV40-virus-transformed kidney cell line) responded to at least one of the viral inducers with yields of interferon over 1000 units/ml. Because the HT-1376 bladder carcinoma cell line produced high yields of interferon in this survey, and is easily propagated, the optimal conditions for interferon production were investigated, using BTV as the inducer. Interferon yields in 59 inductions over a period of about two years consistently fell within a 6-fold range, and had a geometric mean titer of about 2700 reference units (RU)/ml, representing the production of about 3 RU/10³ cells. This yield is comparable to mean titers of 1 to 10 RU/10³ cells obtained by others with human leukocytes, foreskin cell strains, or the Namalva lymphoblastoid cell line. UV-inactivated BTV at a multiplicity corresponding to 10 PFU/cell was as effective an inducer in the HT-1376 cell line as the fully infectious virus at a multiplicity of 1 PFU/cell. The interferon produced by the HT-1376 epithelial cell line has characteristics similar to the interferon induced by poly(I) · poly(C) in human diploid fibroblasts.These studies clearly demonstrate that many different types of tumor-derived cells have the capacity to produce interferon, and that some equal or surpass the efficiency of diploid cells.With 1 Figure     

Interferon externalization by producing cell before induction of antiviral state.

The mechanism by which poly(I · C) induces the antiviral state in human fibroblast cells was studied using antisera which were selectively reactive with the inducer [poly(I · C)], the product (human fibroblast interferon), and the fibroblast cell surface. Anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Furthermore, when added to the medium of the cells in which interferon synthesis had already been induced by poly(I · C), interferon antiserum prevented the intracellular antiviral state from developing. Antibodies specific to double stranded RNA inhibited the antiviral activity of poly(I · C) whether it was in solution or bound to the cells, but the same antibodies were without effect when added after the induction of the antiviral state was initiated. Treatment with serum directed against the cell surface of human fibroblasts failed to inhibit the antiviral activity of poly (I · C) or of human interferon in these cells. These results indicate that the antiviral effect of poly(I · C) is interferon mediated, and that for the development of the antiviral state, interferon has to interact with the external part of the cellular membrane of the producing cell.     

Interferon induction via peroral administration of high-molecular polynucleotides

High molecular polynucleotides, poly(I).poly(C) and poly(G).poly(C), incorporated into liposomes may be used for serum interferon induction when administered orally. Titres of interferon induced by this method are sufficiently high (320-640 units/ml) and close to those induced by the same preparations administered parenterally (640-1280 units/ml). The use of liposomal polynucleotides results in prolonged (up to 24 hours) circulation of interferon in the blood of mice at a sufficiently high level. Liposomal polynucleotides are low-toxic upon oral intake. The liposome-polynucleotide complex is sufficiently stable on storage, and retains the interferon-inducing activity, if given orally, after 6 months of storage at 4 degrees C in the liquid form.