Radioprotective efficacy of tocopherol succinate is mediated through granulocyte-colony stimulating factor

The purpose of this study was to elucidate the role of granulocyte colony-stimulating factor (G-CSF) induced by α-tocopherol succinate (TS) in protecting mice from total-body irradiation. CD2F1 mice were injected with a radioprotective dose of TS and the levels of cytokine in serum induced by TS were determined by multiplex Luminex. Neutralization of G-CSF was accomplished by administration of a G-CSF antibody and confirmed by cytokine analysis. The role of G-CSF on gastrointestinal tissue protection afforded by TS after irradiation (11 Gy, 0.6 Gy/min of ⁶⁰Co γ-radiation) was determined by analysis of jejunum histopathology for crypt, villi, mitotic figures, apoptosis, and cell proliferation. Our results demonstrate that TS protected mice against high doses of radiation-induced gastrointestinal damage and TS also induced very high levels of G-CSF and keratinocyte-derived chemokine (KC) production in peripheral blood 24 h after subcutaneous administration. When TS-injected mice were administered a neutralizing antibody to G-CSF, there was complete neutralization of G-CSF in circulating blood, and the protective effect of TS was significantly abrogated by G-CSF antibody. Histopathology of jejunum from TS-injected and irradiated mice demonstrated protection of gastrointestinal tissue, yet the protection was abrogated by administration of a G-CSF antibody. In conclusion, our current study suggests that induction of G-CSF resulting from TS administration is responsible for protection from ⁶⁰Co γ-radiation injury.     

Gene with Quantitative Effect on Circulating Interferon Induced by Newcastle Disease Virus

Circulating interferon production, induced by Newcastle disease virus, is about seven times higher in C(57) Black mice than i Balb/c/Gif mice. A Mendelian analysis was carried out and circulating interferon production was measured in reciprocal F(1) hybrids, in the F(2) generation, in progeny of backcrosses of F(1) hybrids to either parent strain, and in second backcross progeny. The results indicate that a single, partly dominant, autosomal factor is responsible for the difference in circulating interferon production between both parent strains.     

Circulating Interferon Production in the Mouse : Origin and nature of cells involved and influence of animal genotype

A radiobiological study of circulating interferon production in the mouse was undertaken in the hope of elucidating the site(s) of circulating interferon production. After total body X-irradiation of the animals, different radiosensitivities of circulating interferon production were observed with different viral inducers. Myxovirus-induced circulating interferon production was especially radiosensitive. Moreover, a study of interferon production in syngeneic and xenogeneic radiochimeras demonstrated that cells producing NDV (Newcastle disease virus)-induced circulating interferon were derived from hematopoietic stem cells. In addition, treatment of mice with antilymphocyte serum significantly reduced NDV- and Sendai virus-induced circulating interferon, as opposed to other inducers. Taken together, these results strongly suggest that the lymphocyte is the major source of myxovirus-induced circulating interferon. A survey of interferon production in 12 inbred mouse strains, using NDV as inducer, revealed the existence of low and high producers. A Mendelian analysis carried out with low producing Balb/c and high producing C57BL indicated that the difference between low and high interferon producers was caused by a single, autosomal, codominant factor.     

Mechanism of Antiviral Activity <Emphasis Type="Italic">in vivo</Emphasis> of Polycarboxylates which induce Interferon Production

SYNTHETIC polycarboxylates have been reported to impart resistance to viral infection to experimental animals^1–8. Injection of these polyanions induces interferon^1–3,5–9, to which it therefore seemed logical to attribute the antiviral effect. The high degree and long duration of protection, however, are not in accord with the low and transitory levels of interferon induced, suggesting that mechanisms other than interferon are involved. Certain polyanions have been found directly to inactivate virus or to inhibit its adsorption to cells¹⁰in vitro. This may delay the development of viral infection in vivo. Stimulation of reticuloendothelial cell activity, as demonstrated by increased phagocytosis induced by pyran copolymer¹¹, may deviate virus from its target cells.     

Effect of human adenovirus on antibody-dependent cellular cytotoxicity (ADCC) in chickens

The influence of human adenovirus type 6 on antibody-dependent cellular cytotoxicity (ADCC) in chickens has been investigated. The cytotoxic effect of peripheral blood mono-nuclear cells of chickens was studied on sheep red blood cells (SRBC) coated with chicken anti-SRBC serum. Cytotoxicity was estimated using a ⁵¹Cr release assay system. A single intravenous injection of the virus enhanced ADCC. ADCC was enhanced 14 to 24 hr after the virus injection, but then decreased and the preinjection level was reached after 36 hr. The capacity for virus-augmented activity was not removed with phagocytic cell depletion. The possible role of interferon induced by the virus in chickens in augmenting ADCC is discussed.     

Anomalous Effects of Heterologous Normal Serum on Interferon Production in Mice

WHILE studying the inhibitory effect of burro anti-mouse lymphocyte serum on the production of interferon in mice¹, we investigated whether rabbit anti-mouse lymphocyte serum (ALS) had similar activity. There was some inhibition of interferon production after intraperitoneal injection of polyinosinic/polycytidylic acid (poly IC) in NIH Swiss strain mice pretreated with three doses of potent rabbit anti-mouse lymphocyte serum. Animals treated with normal rabbit serum, however, showed a similar inhibition of interferon production (Table 1), although normal burro serum had no effect¹.     

Cat Interferon inhibits Feline Leukaemia Virus Infection in Cell Culture

TRANSMISSION of feline leukaemia can be accomplished with tissue extracts from cases which occur naturally¹. Virus particles which are morphologically indistinguishable from the murine and avian C-type viruses are present in cats with the transmitted disease². Feline leukaemia virus (FeLV) replicates in cat cell cultures³ and infected cells are demonstrable by the indirect immunofiuorescent antibody test which detects FeLV group-specific antigen as granular punctate fluorescence in the cytoplasm of acetone fixed cells⁴; this method allows easy quantitation of the antiviral effect of interferon. We report the production and assay of feline interferon using the fluorescent antibody test with FeLV infected cat cell cultures.     

Effect of Interferon on Some Aspects of Transformation by Polyoma Virus

WHEN BHK 21 hamster cells are infected with polyoma virus¹, there is no vegetative growth of virus, but stably transformed cells appear. These transformed cells are more easily transplanted than BHK 21 cells; they initiate their growth cycle in otherwise restrictive cultural conditions such as the absence of serum, high density and suspension; they grow with random orientation and have exposed on their surfaces receptor sites for certain glycoprotein agglutinins^2–5. The proportion of stably transformed cells is low, even after high doses of virus. But a much higher proportion (sometimes all cells) shows abortive transformation—changes characteristic of transformation, but which last only a few days. In suspension cultures, for example, most of the infected cells grow into small colonies of four to thirty-two cells⁶. In surface cultures deprived of serum DNA synthesis is initiated and the cells may then divide at least once⁷: they also temporarily lose the normal parallel orientation and develop the typical random appearance of transformed cells. Moreover, the polyoma nuclear T-antigen and also a surface antigen detected by immunofluorescence, appear temporarily in most polyoma infected BHK 21 cells⁸, while 3T3 cells exposed to SV40 virus show transient exposure of cell surface sites reacting with conconavalin A (ref. 9).     

A virus inhibitor circulating in the blood of chickens,induced byFrancisella tularensis andListeria monocytogenes

Following the intravenous inoculation of chickens with large doses of livingFrancisella tularensis andListeria monocytogenes organisms, a virus inhibitor appeared in their serum. The first traces of the inhibitor were found four hours and the maximum levels eight and 24 hours after inoculation with a bacterial suspension. The administration of heat-inactivated microorganisms did not induced formation of the inhibitor. The dynamics of formation of the inhibitor and its properties resembled those of virus interferon.     

The infectivity and pathogenicity of hepatitis A virus live-attenuated vaccine strain H2 in type I interferon receptor-deficient mice

Hepatitis A virus (HAV) live-attenuated vaccine H2 strain has been approved for clinical use for decades with ideal safety profiles in nonhuman primate models and humans. Recently, type I interferon (IFN) receptor-deficient mice were shown to be susceptible to HAV infection. Herein, we sought to determine the infection and replication dynamics of the H2 in Ifnar^-/- mice that lack type I IFN receptor. Following intravenous injection, the H2 failed to cause obvious clinical symptoms in Ifnar^-/- mice, and no significant upregulation in serum alanine aminotransferase (ALT) levels was observed. Notably, the histopathological examination showed that there were significant focal infiltrations of lymphocytes and neutrophils in the portal area, but no focal necrosis was observed in liver tissues. Viral RNAs sustained in the liver, and the infectious virus could be recovered from the liver tissue until 42 days post-infection. More importantly, H2 infection induced obvious viremia and persistent viral shedding in feces. In addition, robust HAV-specific humoral immune responses were induced in Ifnar^-/- mice. Overall, our study revealed the safety profile of H2 in Ifnar^-/- mice, which not only helps understand the attenuation mechanism of H2, but also expands the application of the Ifnar^-/--/- mouse model for HAV studies.     

Production of Interferon in Mice: Effect of Altered Gaseous Environments

Effects of altered gaseous environments (parabarosis) on interferon production in mice were studied, with Newcastle disease virus (NDV) as the inducer. Increased levels of interferon in lung tissue were observed when mice were exposed to 11% O₂ in N₂ for 3 days before and after, or only after, injection of NDV. However, serum interferon levels remained unchanged. Exposure of mice to 77% O₂ for up to 7 days did not affect the response to interferon induction as assayed in lungs or sera. Interferon levels were significantly depressed in mice exposed to a simulated depth of 213 ft in seawater [with normal partial pressure of O₂ (pO₂) in N₂] for 2 or 4 weeks. Whereas definite depression of interferon was also observed in mice maintained at a simulated altitude of 37,000 ft (with normal pO₂) for 2 weeks, those maintained at the same condition for 4 weeks showed a normal level of interferon. The results obtained with hypoxia are compatible with other reports on the influence of O₂ tension on viral infection. The factors responsible for alterations observed in interferon level in mice kept in normal pO₂, but under altered pressure, have not yet been identified.     

Effect of exogenous interferon on the transplantation reactions of mice]

The authors studied the influence of the serum obtained at various periods after the administration of interferon inductors (New castle disease virus, amino ethylisothiouronium, E. coli endotoxin) on the rate of rejection of the skin or cell transplant of mice C3H and CBA, and also CC57Br. The allogenous skin transplant perished more rapidly; there was also an acceleration of elimination of allogenous lymphoid cells, suppression of colony formation by the cells of allogenous bone marrow in the spleen of the irradiated recipient in administration of the serum obtained at the period of maximal content of interferon induced by the Newcastle disease virus and by amino ethylisothiouronium. The cytotoxic activity of lymphocytes of mice CC57Br against the allogenous target cells rose in the presence of these sera. The serum containing interferon induced with E. coli endotoxin failed to influence the rate of the allotransplant rejection and did not increase the cytotoxic activity of lymphocytes.     

Interferon-Inducing Characteristics of MM Virus

Interferon induction by MM virus in mice and in L cells was studied. In mice the virus readily induced interferon. The time of appearance was dose-dependent. A large virus dose induced interferon by 4 hr, whereas a small dose resulted in interferon production which paralleled virus replication 24 hr after infection. In L cells the interferon-inducing capacity of the virus was rapidly destroyed by ultraviolet light irradiation. Heating (56 C) of the virus, on the other hand, greatly increased its ability to induce interferon. Interferon production could also be increased by prior treatment of the cells with homologous interferon (priming). The increase in interferon production after priming was dependent on the concentration of interferon used for priming, the length of interferon treatment, and the multiplicity of infection. It is suggested that MM virus might be useful for the further study of the mechanisms involved in the production and action of interferon.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

Mouse interferon produced in vivo does not inhibit the development of preimplantation mouse embryos

Mouse embryos were cultured in vitro in medium with serum containing interferon which had been induced in vivo by intravenous administration of polyinosine-polycytidylic acid. Two-cell and blastocyst-stage embryos were incubated for 72 and 24 h respectively before embryo transfer, or fixation to determine cell number. Further, blastocysts were outgrown on coverslips in embryo culture medium with fetal calf serum and interferon. Expression of an intermediate filament protein (Mr 55 000) in blastocyst outgrowths was examined with a monoclonal antibody. Embryos appeared morphologically normal and after treatment the mean cell number did not differ from that of controls. Implantation was unaffected by any of the treatments, but culture before transfer in medium containing mouse serum reduced the number of normal fetuses recovered on Day 14 of gestation compared to those cultured in medium without serum. Exposure to interferon did not modify the expression of filaments in the outgrown blastocyst. It is therefore unlikely that interferon induced by viral infection during pregnancy is responsible for preimplantation embryonic loss.     

Biosynthesis of subviral oncogenic particles (virosomes) in mitochondria of rous sarcoma and Rauscher murine leukemia cells

Summary Experimental evidence for the presence and biosynthesis of subviral, leukemogenic particles in the isolated mitochondria of spleen cells of mice infected with Rauscher murine leukemia (RML) virus is presented. These subviral particles sediment at a density of 1.27–1.29 g/ml and induce splenomegaly and RML three weeks after i.v. or i.p. administration to white mice. Virosomes have been labelled with [³²P]phosphate in the isolated mitochondria from RML spleen cells and high molecular weight (70S) [³²P]RNA has been isolated from these subviral, leukemogenic particles. Rauscher virus group specific antigens were detected by immunodiffusion in the inner membrane and matrix fraction of the mitochondria of RML spleen cells. These results together with our earlier findings strongly suggest that mitochondria of the transformed cells participate in the biosynthesis of RNA tumor viruses. Possible mechanism of the penetration of viral genetic information of RNA tumor viruses into mitochondria of tumor cellsin vivo is discussed.     

Potentiation of the Antiviral Activity of Interferon by Actinomycin D

INTERFERON induces cellular synthesis of the antiviral protein which is probably responsible for conferring antiviral activities. Although this antiviral protein has not been isolated, indirect evidence favours a two-step mechanism in the development of cellular resistance to viruses. Taylor¹ and subsequently Friedman and Sonnabend² and Lockart³ have shown that from 0 to 4 h after the treatment of the cells with interferon, the induction of the antiviral state requires the integrity of the cellular apparatus for protein synthesis. Cassingena et al.⁴ have shown that in somatic mouse-monkey hybrid cells, the genes which code for the production and action of interferon are located at different chromosomal sites.     

Influenza virus-induced interferon production in mouse spleen cell culture: T cells as the main producer.

Interferon production by spleen cells from unimmunized C3H mice challenged in vitro with influenza virus AO/PR8 was investigated. Glass-nonadherent cells (lymphocytes) produced significant levels of interferon, although cocultivation of glass-adherent macrophages was needed for optimal production. Treatment of the cells with antithymocyte serum and complement markedly reduced the interferon production. When glass-nonadherent cells were fractionated on a nylon wool column, the T-cell-enriched fraction consistently produced more interferon than the B-cell-enriched fraction. It is concluded that T cells are an important producer of interferon in spleen cell cultures from normal mice upon challenge with influenza virus, although non-T cells (macrophages and B cells) also may produce interferon under suitable conditions.     

Inverse Relationship of Polyoma Tumour Specific Cell Surface Antigen to H-2 Histocompatibility Antigens

NEOPLASTIC transformation is known to be associated with changes in the strength of normal cellular antigens, but the effect can be either an increase or a decrease. In the former category are Forssman antigens in guinea-pig hepatoma¹ and SV40 transformed cells²; HL-A antigens in leukaemic cells³; and “G” antigen in human tumour cells⁴. On the other hand, the intensity of the expression of mouse H-2 histocompatibility antigens is decreased in TL(+) leukaemia⁵ and methylcholanthrene (MCA)-induced tumours⁶. We set out to tell whether the expression of histocompatibility antigens was also affected by transformation with an oncogenic virus and have found that in tumours induced by polyoma virus, the quantity of H-2 antigens varied inversely with the amount of tumour-specific cell surface antigen.