Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero)

Vero cells, a line of African green monkey kidney cells, failed to produce interferon when infected with Newcastle disease, Sendai, Sindbis, and rubella viruses, although the cells were sensitive to interferon. Further, infection of Vero cells with rubella virus did not result in interference with the replication of echovirus 11, Newcastle disease virus, or vesicular stomatitis virus, even in cultures where virtually every cell was infected with rubella virus. Under the same conditions, BSC-1 cells and other cells of primate origin produced interferon and showed rubella virus interference. The results indicate that the presence of rubella virus in the cell does not in itself exclude multiplication of other viruses and that rubella virus interference appears to be linked to the capability of the cell to produce interferon.     

Regulatory effects of macrophage-secreted factors on T-lymphocyte colony growth.

Human fibroblast and leukocyte interferons were found to suppress lymphocyte mitogenesis induced by optimal doses of phytohemagglutinin and concanavalin A. In certain situations (low doses of mitogen and/or low doses of interferon), however, interferon significantly enhanced mitogenesis. In experiments using varying concentrations of interferon, dose-response curves with different slopes were obtained for fibroblast and leukocyte interferons. The effect of interferon was apparently exerted during early stages of the lymphocyte cell cycle. There was no inhibitory effect of interferon if the lymphocytes were washed with medium before being exposed to mitogen. Interferon increased the binding of radiolabeled mitogens to cells. The results suggest that the immunological effects of interferon are consequences of actions on lymphoid cells. Fibroblast and leukocyte interferons seem to have different modes of action, or to bind differently to target cells. Possible mechanisms for the suppressive and enhancing effects of interferons on lymphoid cells are discussed.     

Induction of fumarase in resting Euglena

Exposure of dark-grown resting (carbon deficient) Euglena to light, ethanol or malate produced a transient increase in the specific activity of fumarase. Fumarase levels decreased 8–12 h after the start of induction and this decrease could not be prevented by additional inducer. During the period of fumarase accumulation, cycloheximide prevented further fumarase synthesis and enzyme levels decreased at a rate comparable to the rate of decline normally observed 8–12 h after the start of induction. Although the addition of ethanol to ethanol-induced cultures or malate to malate-induced cultures 12 or 24 h after the initial induction failed to maintain or induce additional fumarase synthesis, the addition of organic carbon to photoinduced cells 8 or 24 h after light exposure induced additional enzyme synthesis. Additional enzyme synthesis was not induced when ethanol- or malate-induced cells were exposed to light 12 or 24 h after organic carbon addition. Light exposure or ethanol addition failed to induce fumarase synthesis during balanced growth indicating that fumarse inducibility is a property of resting cells.     

Inhibition of Arbovirus Protein Synthesis by Interferon

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.     

Immune interferon produced in vitro as a quantitative indicator of cell-mediated immunity.

The present study was constructed to provide some information on the possibility of utilizing immune interferon as a quantitative indicator of cell-mediated immunity and to clarify some of the nature of immune interferon-producing cells (IIPC). When spleen cells derived from L cell-sensitized mice were co-cultivated with L cells, interferon appeared in the culture fluid. It was shown by additional experiments that the cells responsible for immune interferon production in this system were T-lymphocytes assisted by macrophages. The pattern of kinetics of immune interferon production in vitro was similar to that of migration inhibitory factor or T cell-mediated cytotoxicity.     

Requirement of newly synthesized protein for the priming activity of interferon.

Pretreatment of mouse L cells with interferon (IF) enhanced IF production in response to polyinosinic-polycytidylic acid (poly I-poly C). Post-treatment of cells with IF caused no significant enhancement of IF production. The enhancing effect of IF pretreatment (priming) reached a maximum after incubation with IF (10 or 100 units/ml) for 4-6 hr at 37 C, but this effect was absent when the incubation was done at 4 C. Cells which were incubated for additional several hours at 37 C after IF pretreatment at 4 C did not develop the primed state nor the antiviral state. The presence of protein synthesis inhibitors during the IF pretreatment depressed, though not completely, the development of the primed state. The residual priming effect was lost when the cells were incubated with the inhibitors at 37 C for 2 hr before they were exposed to poly I-poly C. There was no significant difference in the binding rate of poly I-poly C to cells between IF-treated and untreated cells. The degradation rate of cell-bound poly I-poly C and its sensitivity to exogenous pancreatic ribonuclease in the pretreated cells were also similar to those in the untreated cells.     

Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults

Human rotavirus-specific CD4(+) and CD8(+) T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8(+) and CD4(+) T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4(+) and CD8(+) T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4(+) and CD8(+) T cells and the frequencies of IL-13-secreting CD4(+) T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4(+) cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.     

Priming: a Nonantiviral Function of Interferon

No interferon is made by L cells when they are infected with MM virus. However, several thousand units of interferon are produced when interferon-treated L cells are infected with MM virus. We call the conversion of cells, from nonproducers to producers, priming. The time required for cells to become fully primed is dependent on the interferon concentration with which they are incubated. Primed cells produced interferon earlier than normal cells stimulated by other inducers. Cells which were exposed to interferon in the presence of inhibitors of protein synthesis became fully primed yet developed no virus resistance. Also, primed cells produced interferon in response to low concentrations of polyriboinosinic acid . polyribocytidylic acid that did not induce interferon in normal cells. Therefore, priming appears to be a function of interferon separable from its antiviral activity. Several other picornaviruses that failed to induce interferon in L cells, human embryonic lung cells, or monkey kidney cells did induce interferon when these cells had been primed by homologous interferons.     

Requirement of Newly Synthesized Protein for the Priming Activity of Interferon

Pretreatment of mouse L cells with interferon (IF) enhanced IF production in response to polyinosinic-polycytidylic acid (poly I·poly C). Post-treatment of cells with IF caused no significant enhancement of IF production. The enhancing effect of IF pretreatment (priming) reached a maximum after incubation with IF (10 or 100 units/ml) for 4–6 hr at 37 C, but this effect was absent when the incubation was done at 4 C. Cells which were incubated for additional several hours at 37 C after IF pretreatment at 4 C did not develop the primed state nor the antiviral state. The presence of protein synthesis inhibitors during the IF pretreatment depressed, though not completely, the development of the primed state. The residual priming effect was lost when the cells were incubated with the inhibitors at 37 C for 2 hr before they were exposed to poly I·poly C. There was no significant difference in the binding rate of poly I·poly C to cells between IF-treated and untreated cells. The degradation rate of cell-bound poly I·poly C and its sensitivity to exogenous pancreatic ribonuclease in the pretreated cells were also similar to those in the untreated cells.     

Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.     

Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures

Hallum, J. V. (University of Pittsburgh, Pittsburgh, Pa.), and J. S. Youngner. Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures. J. Bacteriol. 92:1047-1050. 1966.-The effect of interferon on single cycles of replication of vesicular stomatitis virus and Mahoney poliovirus ribonucleic acid was studied in chick embryo cell cultures. The results showed that the titer of a given interferon preparation was independent of the input multiplicity of the challenge virus. In addition, the increase in virus yield with increasing virus challenge was a function of the number of infected cells, each of which yielded progeny at a level determined by the concentration of interferon to which the cells were exposed. These findings are not compatible with the concept that increases in the size of the virus challenge reverse or overcome protection of cells by interferon.     

Factors Affecting the Sensitivity of Different Viruses to Interferon

When the sensitivities to interferon of Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) were compared by the plaque reduction method in chick embryo cell cultures, NDV was found to be 45-fold more resistant than VSV. This difference was exaggerated when a multiple-cycle yield inhibition method was employed. In marked contrast, when the same viruses were tested by a single-cycle yield inhibition method, the difference in sensitivity to interferon of the two viruses was virtually eliminated. Further investigation showed that, in chick embryo cells exposed to interferon, the resistance to NDV decayed more rapidly than resistance to VSV. This finding explained the divergent results obtained with the two viruses when single- or multiple-cycle replication techniques were employed. Experiments carried out with L cells showed that cellular antiviral resistance decayed much more slowly in these cells than in chick embryo cells. Consequently, when measured by the plaque reduction method in L cells, no difference was observed in the sensitivity to interferon of VSV and NDV(pi), a mutant of NDV which replicates efficiently in L cells. A procedure is suggested for determining the relative sensitivities to interferon of different viruses under conditions which minimize the role of decay of antiviral resistance in the host cells.     

An antigenic analysis for membranes of Mycoplasma hominis by cross-absorption

Antigenic components at the outer surface membranes of seven serotypes of Mycoplasma hominis were analysed by the mycoplasmacidal reaction and the agglutination during growth reaction. Antibody absorbing capacities of the mycoplasma cells were compared with absorbing capacities of membranes. It was shown that serologically active membrane antigens were mainly heat-labile proteins. No major antigens common to all seven serotypes were detected and each strain had its own specific antigens at the cell surface. Results of analysis indicate that there is a complex antigenic structure exposed in M. hominis and that 7 to 14 cross-reacting antigens may be present at the outer surface in the different serotypes examined. Additional cross-reacting antigens, presumably inner membrane in origin and not exposed at the cell surface, were also demonstrable.     

Interferon gamma promotes survival of lymphoblasts overexpressing 9-O-acetylated sialoglycoconjugates in childhood acute lymphoblastic leukaemia (ALL)

An enhanced linkage-specific 9-O-acetylated sialic acid (9-O-AcSA) on peripheral blood mononuclear cells (PBMC) of children with acute lymphoblastic leukaemia, ALL (PBMC(ALL), 9-O-AcSA+ cells) was demonstrated by using a lectin, Achatinin-H, whose lectinogenic epitope was 9-O-AcSAalpha2-6GalNAc. Our aim was to evaluate the in vitro contributory role of this glycotope (9-O-AcSAalpha2-6GalNAc) towards the survival of these 9-O-AcSA+ cells in ALL patients. For direct comparison, 9-O-AcSA- cells were generated by removing O-acetyl group of 9-O-AcSA present on PBMC(ALL) using O-acetyl esterase. An elevated level of serum interferon gamma (IFN-gamma) in affected children led us to think that PBMC(ALL) are continuously exposed specifically to this cytokine. Accordingly, 9-O-AcSA+ and 9-O-AcSA- cells were exposed in vitro to IFN-gamma. A twofold increased NO release along with inducible NO synthase (iNOS) mRNA expression by the 9-O-AcSA+ cells was observed as compared to the 9-O-AcSA- cells. The decreased viability of IFN-gamma exposed 9-O-AcSA- cells as compared to 9-O-AcSA+ cells were reflected from a 5.0-fold increased caspase-3-like activity and a 10.0-fold increased apoptosis in the 9-O-AcSA- cells when production of NO was lowered by adding competitive inhibitor of iNOS in reaction mixture. Therefore, it may be envisaged that a link exists between induction of this glycotope and their role in regulating viability of PBMC(ALL). Taken together, it is reasonable to hypothesise that O-acetylation of sialic acids on PBMC(ALL) may be an additional mechanism that promotes the survival of lymphoblasts by avoiding apoptosis via IFN-gamma-induced NO production.     

Potentiation of thermal injury in mouse cells by interferon

Mouse cells, when exposed to high temperature (43 degrees), shut off overall protein synthesis and continue to synthesize "heat shock proteins". Such heat shocked cells, upon reincubation at 37 degrees C, recover and proliferate. However, when mouse cells are pretreated with mouse interferon (IFN) and then exposed to 43 degrees, more than 99% of the cell population fail to recover. Synthesis of the major heat shock protein is unaffected in cells treated with IFN. Experiments designed to assess the role of intracellular glutathione (GSH) during cells' recovery from hyperthermia indicated that there is an irreversible depletion of glutathione when IFN treated cells are heat shocked. Neither depletion of GSH, nor potentiation of thermal injury was observed in a IFN-resistant line of mouse cells.     

Intracellular production of virus particles and viral components in NIH/3T3 cells chronically infected with Moloney murine leukemia virus: effect of interferon.

The effect of interferon on the biochemical properties and the maturation process of intracellular viral particles isolated from the cytoplasmic fraction of NIH/3T3 cells chronically infected with Moloney murine leukemia virus was investigated. By labeling these virions with either [35S]methionine or [3H]glucosamine, we demonstrated that they contain the same viral proteins and glycoproteins found in extracellular virions. Interferon treatment was found to reduce the rate of intracellular virus assembly. This effect was not a consequence of an interferon inhibition of viral RNA synthesis or its translation or a consequence of an interference with the posttranslational cleavage processing of viral precursor proteins, since all of these steps were not affected by interferon. However, the reduced rate of virus assembly could be attributed to the inhibition of viral protein glycosylation observed in interferon-treated cells. Nevertheless, despite this reduced rate, virus particles accumulated in interferon-treated cells. This accumulation was probably due to the strong inhibition of their final release from such cells.     

Tilapia are able to withstand long-term exposure to low environmental pH, judged by their energy status, ionic balance and plasma cortisol

Tilapia Oreochromis mossambicus were exposed to water at pH 4·0 for 37 days. The water was acidified slowly over 6 h enabling the animals to acclimate and preventing damage of the gill epithelium. Additional stressors, e.g. aluminium ions and handling stress, were avoided. No mortality or decreased food consumption was observed during the exposure period. No significant changes were observed between the control and acid exposed groups for the energy rich compounds and related parameters, i.e. the adenylate energy charge, the pool of total adenine nucleotides, and the IMP load of white muscle and liver, indicating maintenance of homeostasis. Moreover, there were no significant differences between control groups and acidified groups at 3, 17 and 37 days for plasma sodium, chloride, cortisol and glucose, implying that ionic balance was maintained and that there was no activation of the pituitary-interrenal axis. It is concluded that tilapia can acclimate to water at pH 4·0 when the acidification rate is slow and additional stressors are avoided.     

Interferon-induced proteins in human fibroblasts and development of the antiviral state.

Treatment of human fibroblasts with interferon induces the synthesis of several proteins, as detected by incorporation of [35S]methionine followed by analysis of cell extracts by polyacrylamide gel electrophoresis. The induction of these proteins had features in common with the development of the antiviral effect of interferon, such as (i) sensitivity to actinomycin D and cycloheximide when these compounds were added together with interferon, (ii) insensitivity to actinomycin D if the actinomycin D was added 2 h after the addition of interferon, (iii) similar dependence on interferon concentration, and (iv) species specificity for interferon. When interferon treatment was given in the presence of cycloheximide and actinomycin D was added before the removal of cycloheximide, all four proteins were induced, thus suggesting that their inductions are coordinated. Labeling for 2-h periods at varying time intervals after the addition of interferon revealed that the synthesis of these proteins was induced within a few hours, peaked at different time intervals, and was soon followed by a marked decline, suggesting that the mRNA's for these proteins have short half-lives. Moreover, this decline occurred despite the fact that the cells were continuously exposed to interferon, and there was no measurable loss of interferon activity in the medium. This suggests that the induction of these proteins is transient and is apparently subject to further control.     

Relative Antiviral Resistance Induced in Homologous and Heterologous Cells by Cross-Reacting Interferons

Human cells incubated with human interferon become more resistant to vesicular stomatitis virus (VSV) than to Semliki Forest virus (SFV); monkey cells treated with monkey interferon become more resistant to SFV than to VSV. However, monkey cells incubated with human interferon developed relative antiviral activity identical to that induced by homologous interferon, and human cells developed characteristic human interferon-induced relative antiviral activity when exposed to monkey interferon. Therefore, cross-reacting interferons induce the relative antiviral activity characteristic of the interferon-treated cell rather than the cell of the interferon's origin. This relationship supports the hypothesis that interferon is not itself antiviral but rather induces cells to develop their own antiviral activity.