Delta 9-tetrahydrocannabinol decreases alpha/beta interferon response to herpes simplex virus type 2 in the B6C3F1 mouse

This study was undertaken to determine the effect of delta 9-tetrahydrocannabinol (delta 9-THC) on polyinosinic:polycytidylic acid [poly(I):poly(C)]-induced, and on herpes simplex virus type 2 (HSV-2)-induced, alpha/beta interferon in the B6C3F1 mouse. Animals were administered delta 9-THC, or the diluent, intraperitoneally for 4 consecutive days or at various time intervals prior to administration of the interferon inducer. Poly(I):poly(C) or HSV-2 was injected intravenously on Day 4. Animals receiving poly(I):poly(C) and treated with delta 9-THC at doses ranging from 5 to 100 mg/kg exhibited significantly lower titers of interferon than mice given poly(I):poly(C) and the diluent. Diminished interferon titers occurred in HSV-2-infected animals treated with delta 9-THC in doses exceeding 15 mg/kg when compared to virus-infected animals given the diluent. This suppression of early interferon persisted through 24 hr.     

Effect of a perfluorochemical emulsion on the radiation response of BA1112 rhabdomyosarcomas

The effect of treatment with a perfluorochemical emulsion (Fluosol DA, 20%), carbogen, or the combination of these two agents on the radiation response of BA1112 tumors in WAG/rij rats was examined. Fluosol and carbogen as single agents had only small effects on the tumor cell survival curve. The combination of Fluosol plus carbogen had a larger effect on tumor cell survival, reducing the hypoxic fraction of the tumor from 23 to 1.6%. The amount of sensitization was a function of the Fluosol dose, with maximal augmentation of the radiation response obtained at doses of 7.5-15 ml/kg. Carbogen pretreatments ranging from 5 to 60 min in duration all had similar effects on tumor radiosensitivity. The effect of the perfluorochemical emulsion plus carbogen on the survival of irradiated tumor cells appears to reflect changes in tumor oxygenation, rather than cytotoxic or immunological effects, since the perfluorochemical emulsion (with or without carbogen) had no effect on the viability of cells in unirradiated tumors. These experiments extend previous studies by ourselves and others using mouse tumors to show that the combination of a perfluorochemical emulsion and carbogen breathing can also increase the radiation response of a nonimmunogenic rat tumor.     

Comparison of in vitro and in vivo modulation of myelopoiesis by biological response modifiers

Summary In vitro growth and differentiation of granulocyte-macrophage progenitor cells (GM-CFU-C) requires colony-stimulating factors (CSF), and an in vivo role for CSF has also been proposed. Prostaglandins of the E series (PGE) have been reported to serve as negative feedback regulators of myelopoiesis. Here, we report evidence of augmented CSF secretion by mouse peritoneal Mo (macrophages) and bone marrow cells in vitro upon stimulation with various biological response modifiers (BRMs). Optimal induction of CSF secretion occurred after in vitro treatment of peritoneal Mo and mononuclear bone marrow cells with 50 g/ml poly ICLC (polyriboinosinic-polycytidylic acid poly-L-lysine), 5 g/ml lipopolysaccharide (LPS), or 500 U/ml interferon (IFN_,) for 2 days. The in vitro stimulation of CSF secretion was paralleled by an increase in PGE secretion by Mo and bone marrow cells. The PGE secretion could, however, be selectively blocked by preincubating the cells for 3 h with indomethacin (10^–7 Mol) leaving CFS production intact. In vivo treatment of mice with either maleic anhydride divinyl ether copolymer (MVE-2; 25 mg/kg) or poly ICLC (2 mg/kg) significantly increased levels of CSF in serum, as well as in culture supernatants of in vivo-treated peritoneal Mo and bone marrow cells. The increase in serum CSF levels and in secretion of CSF by peritoneal Mo and bone marrow cells was followed by a dose-dependent increase in GM-CFU-C, in nucleated bone marrow cells, and in peripheral blood leukocytes. The same BRMs also stimulated the secretion of PGE by in vivo-activated peritoneal Mo, but not by bone marrow cells. Pretreatment of the mice with indomethacin (4 mg/kg) almost completely suppressed PGE secretion by peritoneal Mo, but did not change the CSF secretion by peritoneal Mo or bone marrow cells and had no significant effect on bone marrow cellularity. Therefore, MVE-2 and poly ICLC, in addition to their immunomodulatory activity, can also have stimulatory effects on myelopoiesis, presumably mediated through secretion of CSFs. Protection and/or restoration of bone marrow function could thus either provide the opportunity for more extensive chemotherapy or could increase the number of Mo effector cells available for activation against tumor targets.     

Mechanism of the antimutagenic action of interferon: the capacity to protect the human cellular repair system

It has been shown that premutagenic treatment with leukocytic interferon (10, 100 IU/ml) of human peripheral blood lymphocytes cultivated in vitro at the G1-stage of the mitotic cycle results in different cell response to gamma-radiation in doses of 0.5, 1, 2, 4 Gy according to chromosome aberration. The antimutagenic effect failed to be attained with the doses 0.5 and 1 Gy, being maximal at the dose 2 Gy. According to sister chromatid exchanges (SCE) cell pretreatment with interferon leads to a reduction in the effect of gamma-radiation at the dose 2 Gy to the level obtained in the cells after exposure to interferon. In experiments with 4-nitroquinoline-I-oxide, there was a significant decrease in the number of SCE in interferon-treated cells.     

Exploiting tumor-specific defects in the interferon pathway with a previously unknown oncolytic virus

Interferons are circulating factors that bind to cell surface receptors, activating a signaling cascade, ultimately leading to both an antiviral response and an induction of growth inhibitory and/or apoptotic signals in normal and tumor cells. Attempts to exploit the ability of interferons to limit the growth of tumors in patients has met with limited results because of cancer-specific mutations of gene products in the interferon pathway. Although interferon-non-responsive cancer cells may have acquired a growth/survival advantage over their normal counterparts, they may have simultaneously compromised their antiviral response. To test this, we used vesicular stomatitis virus (VSV), an enveloped, negative-sense RNA virus exquisitely sensitive to treatment with interferon. VSV rapidly replicated in and selectively killed a variety of human tumor cell lines even in the presence of doses of interferon that completely protected normal human primary cell cultures. A single intratumoral injection of VSV was effective in reducing the tumor burden of nude mice bearing subcutaneous human melanoma xenografts. Our results support the use of VSV as a replication-competent oncolytic virus and demonstrate a new strategy for the treatment of interferon non-responsive tumors.     

Combined effect of interferon inducers, radiosensitizing and antineoplastic agents on solid tumors

In experiments with mice bearing solid sarcoma 37 a study was conducted on the combined effect of radiation and inductors of endogenous interferon synthesis (IEIS), together with hyperthermia or together with an alkylating and carbomoilating agent, dimethinur. The effect was estimated by the tumor growth coefficient and by the number of animals with the regressed tumors. Po I. polyC was not shown to influence the efficiency of hyperthermia combined with radiation; dextransulphate and tiloron increased the radiosensitizing effect of hyperthermia. Dimethinur aggravated the effect of radiation, but with IEIS used together with dimethynur and radiation, the response of the tumor increased insignificantly as compared to the effect of IEIS together with radiation.     

Double-stranded RNA regulation of DNA synthesis in fibroblasts.

The double-stranded RNA molecule polyinosinic-polycytidylic acid (poly IC) has been found in some studies to have a mitogenic effect on fibroblast proliferation while other studies found poly IC to have an inhibitory effect on proliferation. In this study, we investigated whether a stabilized form of poly IC complexed with poly-L-lysine and carboxymethylcellulose (poly ICLC) had a bidirectional effect on DNA synthesis in fibroblasts from four different cell lines and determined factors that potentially influence this bidirectional effect. In medium containing fetal bovine serum, poly ICLC slightly increased the levels of [3H]thymidine incorporation in growing fibroblasts in three of the four fibroblast cell lines tested, while poly ICLC increased [3H]thymidine incorporation in confluent, quiescent fibroblasts in two of four cell lines. Poly ICLC did not induce DNA synthesis in subconfluent, quiescent or in confluent, quiescent fibroblasts under serum-free conditions. Poly ICLC significantly suppressed serum-induced [3H]thymidine incorporation by quiescent fibroblasts in all cell lines. We conclude that the stimulatory and inhibitory effects of poly ICLC on DNA synthesis are influenced by both the cell line and the presence of serum components in the culture medium but not by population density.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Induction of interferon production with dextran sulfate

Interferon-inducing activity of polyanions (dextran Sulfate, native DNA, polyphosphate, polypentose, polyanetole sulfate, heparin) was studied. Dextran sulfate applied parenterally and enterally was found to be capable of inducing endogenous interferon production in white mice. The maximal titers of interferon (160 units/ml) were seen in the blood serum of mice upon administering the preparation in a dose of 20 mg/kg body mass. No substantial differences were discovered in the time course of interferon production whatever the route of administration.     

Enhanced induction of plasma interferon after subcutaneous administration in rabbits of poly ICLC with albumin

Intravenous administration of poly ICLC in rabbits yields very high plasma interferon (IFN) levels; dosages from 250 to 1000 micrograms/kg body weight are very toxic. Subcutaneous administration of poly ICLC together with a high concentration of human serum albumin potentiates the inducer activity in comparison to poly ICLC in saline. Finally, a dosage of 50 micrograms of poly ICLC in 12% serum albumin is more effective as an IFN inducer than other dosages.     

Effect of remantadine and ribavirin on the production and antiviral action of human interferon types I and II

The results of using human interferon of types I (alpha- and beta-interferons) and II (gamma-interferon) in combination with chemotherapeutic drugs, such as remantadin and ribavirin for the study in human cell cultures are presented. Moderate doses of the drugs (25 microgram/ml) did not eliminate the interferon production. In higher doses (50 microgram/ml) they lowered the interferon production levels 2--3 times. In the presence of ribavirin the level of the interferon production lowering was higher. On the whole the effect of the drugs on production of interferons of types I and II was of a similar character despite the different means of interferon induction. The combined use of interferons of types I and II with the chemotherapeutic drugs in human embryo cultures infected with Semiliki forest virus (SFV) revealed an additive character of the antiviral effect in all combinations tested. The level of the antiviral activity of alpha-, beta- and gamma-interferons against the SFV was practically the same.     

Para-aminobenzoic acid--an interferon inducer]

Para-aminobenzoic acid (PABA) was shown to be an early type interferon inductor. PABA (10 micrograms/ml) induced interferon production in vitro in the cells of human peripheral blood and in vivo in albino mice (10 mg/kg). The results of the study suggested that PABA was able to induce production of interferon-alpha/beta in various immunocyte populations. By its interferonogenic activity PABA was comparable with the known interferon inductors. One of the mechanisms of the previously described in vivo antiherpes action of PABA can be attributed to its interferon inducing activity.     

Radiosensitivity of mouse lip mucosa: influence of anesthesia, carbogen, and a new high O2 carrying perfluorochemical emulsion

The effect of a new perfluorochemical emulsion based on F-66E (54%, w/v) which carries, in combination with carbogen, twice as much oxygen as Fluosol-DA 20% was tested on the radiation response of the lip mucosa of unanesthetized mice. Mice were pretreated with 0.015 ml/g of the F-66E emulsion in the presence of carbogen for 1 h prior to and during irradiation. There was a significant increase in the mortality rate following the highest radiation dose in mice given F-66E emulsion plus carbogen. The reactions of lip mucosa of mice given F-66E emulsion and/or carbogen were not significantly different from that of the control group using three end points (average score, mean peak, incidence of mucosal desquamation), but the peak mucosal reaction was delayed. The radiosensitivity of the mouse lip mucosa to Ethrane, an anesthetic gas inhaled with carbogen, was also tested. The reaction of lip mucosa in the anesthetized mice was significantly greater than that of the control group. There was also a significant increase in the mortality rate following the two highest radiation doses.     

Reversibility of the antiproliferative effect of interferon

The reversibility of the antiproliferative effect of interferon (IFN) and its correlations to the induction of (2',5') oligoadenylate synthetase (2-5A synthetase) activity was studied on NIH/3T3 cells transformed by Moloney murine sarcoma virus. The cells were treated with various doses of mouse beta-IFN. At 72 h after treatment, the cultures were subdivided. While half received fresh doses of IFN, the second half received no IFN. Reversibility of the IFN effect was then followed. Three different parameters as indicators for cell proliferation were used: cell growth, protein synthesis and cloning efficiency. In parallel, the IFN-induced activity of 2-5A synthetase was determined. The data obtained led to the following conclusions. (1) The antiproliferative effect of IFN increases with increased IFN concentration (90-1,800 IU/ml) and with time of treatment, up to 72 h after treatment. (2) The induced activity of 2-5A synthetase increases with a much faster rate, reaching maximum activity at 24 h after treatment with 450 IU/ml. This means that the induction of the enzyme precedes the antiproliferative effects of IFN. (3) There is almost no recovery of the IFN antiproliferative effect following treatment for 72 h with high doses of IFN (1,200-1,800 IU/ml). However, at lower doses, recovery is evident. (4) Removal of IFN after treatment for 3 days with 450 IU/ml resulted in a gradual decrease of 2-5A synthetase activity, reaching the basal level at 72 h after removal. However, there is no reduction of enzyme activity following treatment for 72 h with 1,800 IU/ml of IFN.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Effect of a combination of mild-temperature hyperthermia and nicotinamide on the radiation response of experimental tumors

Ogawa, A., Griffin, R. J. and Song, C. W. Effect of a Combination of Mild-Temperature Hyperthermia and Nicotinamide on the Radiation Response of Experimental Tumors. The effect of mild-temperature hyperthermia and nicotinamide individually or combined on tumor radiosensitivity was investigated with SCK tumors grown s.c. in the right hind limbs of A/J mice. An i.p. injection of nicotinamide at 50-250 mg/kg slightly enhanced the cell killing caused by 10-20 Gy of ionizing radiation as determined by the in vivo/in vitro tumor excision assay. Treatment of tumors with mild-temperature hyperthermia at 41.5 degrees C for 60 min prior to tumor irradiation was significantly more effective than nicotinamide and the combination of nicotinamide and hyperthermia was far more effective than nicotinamide or hyperthermia alone in enhancing radiation-induced cell killing. Radiation-induced tumor growth delay was enhanced by a factor of 1.2 by 50 mg/kg nicotinamide, 2.1 by hyperthermia, and 3.6 by the combination of nicotinamide and hyperthermia. Taking these results and those of our previous studies together, we conclude that mild-temperature hyperthermia increases tumor blood flow and oxygenation and that combining mild-temperature hyperthermia and nicotinamide is more effective than either of these alone in increasing tumor radiosensitivity.     

Suppression of NK-mediated natural resistance by interferon treatment of murine lymphomas

The observation that interferon (IFN) can suppress the NK lytic sensitivity of murine lymphomas in vitro led us to examine the consequences of this treatment on tumor behavior in vivo. Preincubation in IFN suppressed natural resistance to two lymphomas in syngeneic DBA/2 and semisyngeneic BDF1 mice in a dose-dependent manner, measured by the retention of (131I)dUrd-labeled tumor. Poly I:C enhancement of NK-mediated natural resistance in the lung, liver, and peritoneal cavity was also abolished by IFN pretreatment. IFN was, however, ineffective in altering the elimination of the IFN-resistant L1210R lymphoma when compared to its IFN-sensitive variant, L1210S. In DBA/2 mice that were made NK-deficient by treatment with cyclophosphamide or rabbit anti-asialo GM1 antiserum, or in congenitally NK-deficient bg/bg strain mice, IFN-treated tumor and control tumor were rejected equally well. This indicated that the effects of IFN were dependent on the presence of NK cells in these mice, and suggests that the IFN suppressed the sensitivity of the lymphomas to NK cell-mediated host resistance.     

Lipopolysaccharide induction of IgM antibodies to polyadenylic acid in normal mice.

Lipopolysaccharide (LPS) is able to induce autoantibodies reversibly in normal mice. Single or multiple doses of LPS induced a rapid and dose-dependent rise in antibodies to Poly A from 113 ng to 590 ng/ml serum as determined by Millipore filter radioimmunoassay. The response peaked at day 3 and was over by day 21. No response was seen if the LPS was chemically inactivated or injected into genetically nonresponsive mice. Antibodies were specific for Poly A and were not induced by T cell mitogens. Sucrose gradient ultracentrifugation demonstrated only IgM antibody. Neonatal thymectomy altered neither the immunoglobulin class nor quantity of antibody. Neonatal splenectomy did not affect antibody class but reduced the amount produced. No free Poly A could be detected in circulation after LPS stimulation. These findings suggest that normal mice have B lymphocytes capable of limited and reversible autoantibody production.     

Time and dosage dependence of immunoenhancement by murine type II interferon preparations.

Preparations of Type II immune induced Interferon enhanced the plaque forming cell response of mice to sheep red blood cells both in vivo and in vitro. The enhancement of the antibody response was dependent on the dosage of interferon used and the time of administration of interferon. The expression of the antiviral and immuno-enhancing activities of Type II interferon preparations shared several physical-chemical properties, including pH 2 lability and heat stability. The plaque forming cell response to lipopolysaccharide, a T-independent antigen, could not be enhanced by treatment with Type II interferon. In addition, treatment of spleen cell cultures of nude thymic deficient mice with Type II interferon could not cause an enhancement of the plaque forming cell response to lipopolysaccharide. These data suggest that the immunoenhancing effect of Type II interferon on antibody responses is produced by an effect on T lymphocytes in contrast with the immunosuppressive effect which appears to be mediated through an effect on B lymphocytes.     

Elevated natural killer cell-mediated cytotoxicity, plasma interferon, and tumor cell rejection in mice persistently infected with lymphocytic choriomeningitis virus

To assess the effects of chronic virus infection on NK cells, the related phenomena of interferon (IFN) production, NK cell activation, and resistance to tumor implants were studied in mice persistently infected with lymphocytic choriomeningitis virus (LCMV). NK cells from these LCMV-carrier mice displayed augmented killing of the NK-sensitive YAC-1 target cell. They did not lyse the more resistant targets L-929 and P815, whereas NK cells from acutely infected mice efficiently lysed all three cell types. The plasma from LCMV-carrier mice contained an antiviral substance identified as IFN type I, based on species specificity, virus nonspecificity, resistance to pH 2, and sensitivity to antibody to type I IFN. IFN titers in plasma from LCMV-carrier mice were 32 to 64 U/ml, about 20-fold less than those in acutely infected mice. Both the IFN and NK cell levels continuously remained elevated in the LCMV carrier mice up to at least 6 months of age. IFN is known to activate NK cells and to induce their blastogenesis in vivo. As determined by centrifugal elutriation, large NK blast-size cells were isolated from the spleens of acutely infected mice, but not from either normal or LCMV-carrier mice, suggesting augmented NK cell-mediated lysis in the absence of enhanced proliferation. Poly inosinic-cytidylic acid induced high levels of NK cell-mediated cytotoxicity and blastogenesis in both control and LCMV-carrier mice, but IFN was induced to lower levels in carriers as compared with controls. Coincidental with augmented NK cell activity, the LCMV-carrier mice rejected intravenously injected 125IUdR-labeled tumor cells more efficiently than did normal mice. Thus, LCMV carrier mice have low levels of type I IFN, moderately augmented NK cell activity lasting for at least 6 months, and increased resistance to tumor cell implants. This indicates that augmented NK cell-mediated cytotoxicity can be maintained in vivo over prolonged periods of time in the presence of chronic low-level IFN stimulation.