Brahma S. Sharma

Roles of interferon and natural killer cells in the antiviral activity of 7-thia-8-oxoguanosine against Semliki Forest virus infections in mice.

7-Thia-8-oxoguanosine is a novel biological response modifier with broad-spectrum antiviral activity against many DNA and RNA viruses in vivo. Since two of its properties are to induce interferon and to activate natural killer (NK) cells, we investigated the roles of the lymphokine and NK cells in the antiviral activity of the compound against Semliki Forest virus. Antibody to interferon alpha/beta could completely abolish the protective activity of the nucleoside against virus infection in mice, whereas antibodies to interferons beta and gamma could not, indicating that interferon alpha was of major importance to confer protection to the animals. Reduced activation of NK cells was also observed in mice treated with 7-thia-8-oxoguanosine and antibody to interferon alpha/beta. The role of NK cells in the protective activity of the compound was directly assessed in beige mice or in Swiss Webster mice treated with asialo GM1 antibody. In both experiments, the animals were protected from lethal virus infection by treatment with nucleoside. Spleen cells primed by 7-thia-8-oxoguanosine and adoptively transferred to untreated mice could not save them from virus-induced mortality. These three results provide evidence that natural killer cells activated by 7-thia-8-oxoguanosine play a minimal role in protection from acute Semliki Forest virus infections in mice.

Potentiation of the efficacy of murine L1210 leukemia vaccine by a novel immunostimulator 7-thia-8-oxoguanosine: Increased survival after immunization with vaccine plus 7-thia-8-oxoguanosine

SummaryWe have investigated the ability of a novel immunopotentiator, 7-thia-8-oxoguanosine (7T8OG) to increase the efficacy of a weakly immunogenic murine L1210 leukemia vaccine. The vaccine was prepared by irradiating L1210 leukemia cells in a cesium source with a total of 6000-R dose. DBA/2 mice were treated with 150 mg/kg 7T8OG and/or with vaccine consisting of 107 irradiated cells. In combination therapy, mice first received the vaccine and then were injected with 75 mg/kg 7T8OG 2 h and 4 h after vaccination. One week after the last treatment all mice were inoculated with 104 live leukemia cells intraperitoneally. Control, untreated mice (n = 66) injected with 104 live leukemia cells had a mean survival time ± standard error of 10.5±0.2 days. Treating mice (n = 66) with one, two or three doses of 7T8OG administered i.p. 1 week apart did not increase survival (mean survival time = 10.7 days). Mice immunized with one, two or three doses of vaccine had 14.5±1.1, 45.4±6.2 and 68.3±10.6 days mean survival, respectively. 7T8OG-stimulated vaccination increased the survival dramatically. The best survival was noted when the mice were treated with 2× (vaccine + 7T8OG). Immunization of mice (n = 30) with this treatment regimen increased the mean survival to 156±10.0 days. Over 90% of mice that were treated this way had a cumulative survival time greater than 160 days. In contrast, only 12% of the mice immunized twice with the leukemia vaccine alone survived over 160 days. These results suggest a rationale for the use of this immuno-potentiator with various vaccines for a more effective immunization.

Inhibition of pyrimidine metabolism in myeloid leukemia cells by triazole and pyrazole nucleosides.

Two triazole nucleosides, 1 (3-beta-D-ribofuranosyl-1,2,4-triazole-5-carboxamide) and 2 (2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide), and a pyrazole nucleoside, 3 (1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide), were found to inhibit pyrimidine nucleotide biosynthesis in the human myeloid leukemia cell line, K562. Cells treated with these inhibitors released orotate in quantities of 8-35 nmol/10(5) cells/day. Treatment with these compounds caused the K562 cells to accumulate in the S phase of the cell cycle and induced the cells to synthesize hemoglobin.

Successful immunotherapy of murine melanoma metastases with 7-thia-8-oxoguanosine

We have recently reported that a synthetic nucleoside, 7-thia-8-oxoguanosine (7T8OG) is a potent activator of a number of effectors which are involved in anti-tumor immune responses. 7T8OG was found to induce interferon (IFN) production, to activate asialo-GM1 positive (AGM1+) killer cells, and to enhance specific antibody responses. In the present study, we investigated the effect of 7T8OG on growth of the murine pulmonary B16 melanoma and on formation of metastases. C57BL/6 mice were injected i.p. with 50–150 mg/kg 7T8OG before or after i.v. inoculation of B16 melanoma tumor cells, and 17–19 days after tumor inoculation, the number of metastases in the lungs were counted. 7T8OG given systemically in a single or a divided dose 24 h prior to the challenge of tumor cells reduced the number of lung tumor metastases by 89–99% which is highly significant as compared to untreated control (P<0.001). Occasional extra pulmonary tumor growth in the thoracic cavity and neck lymph node was also completely inhibited. The reduction in the number of tumor nodules was dose dependent. A single dose of 150 mg/kg of 7T8OG was also effective in inhibiting the growth of 3–5 day old metastatic tumors. The cytotoxic activity of killer cells inducedin vivo by 7T8OG was completely abolished byin vitro treatment of cells with antiAGM1 antibody plus complement. Administration of anti-AGM1 antibody following the 7T8OG treatment completely abrogated the anti-tumor effect of 7T8OG, resulting in a massive increase in the number of tumor foci in the lungs. Administration of carageenan or silica followed by injection of 7T8OG caused a significant increase (P<0.01) in the number of pulmonary tumor nodules compared to treatment with 7T8OG only. These findings indicate that activated macrophages or perhaps their cytokine (tumor necrosis factor) also contribute to the host tumor defense by 7T8OG.

Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine: I. Activation of natural killer cells in mice

We reported recently that a novel immunomodulator, 7-thia-8-oxoguanosine (7T8OG)2 inhibited formation of pulmonary melanoma metastases (1), prevented against viral infection in mice (2) and potentiated the efficacy of a weakly immunogenic leukemia vaccine (3). Since certain tumor metastases and virus infected cells are targets to natural killer cells (NK cells), we now investigated whether 7T8OG is capable of activating NK cells in mice using NK cell sensitive YAC-1 and B16 and NK cell insensitive P815 targets. CBA/CaJ spleen cells incubated in vitro with 7T8OG at concentrations ranging from 0.005 to 0.5 mM responded with increased NK cell activity (32-62%) compared to controls (4-8%) to YAC-1 targets. Similar levels of augmentation in NK cell activity were observed when 40-168 mg/kg of 7T8OG was administered in vivo. In addition to the spleen, 7T8OG activated NK cells in the bone marrow (BM), the lungs, the liver, and in peritoneal exudate cells (PE). Although 7T8OG elicited activation of NK cells was observed as early as three hours after treatment, the maximal activity was observed after 24 h in the spleen; 12 h in the BM; 48 h in the lungs, and 72 h in PE. Administration of the drug by s.c., i.v., and i.p. routes all induced activation of NK cells in spleen, BM and PE. 7T8OG was found to activate NK cells in seven inbred and an outbred mouse strain, suggesting that the induced cytotoxicity against allogeneic and syngeneic tumor cells is not strain specific as well as independent of MHC restriction. C3H/He, CBA/CaJ and BDF/1 displayed higher levels of increased NK cell activity, whereas AKR mice were low responders. Low concentrations of IL-2 (0.25-5 U/ml) that induce little or no NK cell activity, when used in combination with 7T8OG, elicited significant enhancement of NK cell cytotoxicity. In contrast, IFN and 7T8OG showed no such synergism.

Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine. II. Characterization of induced effector cells and the mechanism of induction.

In a preceding paper we characterized the in vivo and in vitro induction of cytotoxic effector cells elicited by a novel synthetic immuno-stimulator 7-thia-8-oxoguanosine (7T8OG)2. In the present study we further characterized the cells responsible for the induced cytotoxicity and the mechanisms together with the lymphokines mediating the immunological response to 7T8OG. Removal of macrophages from 7T8OG activated spleen cell suspensions by various methods resulted in a significant increase in cytotoxicity to YAC-1 targets. 7T8OG induced effectors did not exert cytotoxic effect on macrophage sensitive P815 target cells. In vivo activated effectors when incubated with anti-asialo-GM1 antibody plus complement lost completely their ability to lyse YAC-1 targets. Together, these findings indicate that the 7T8OG induced effector cells are not macrophage like. Spleen cells from nude mice were readily activated by 7T8OG. The induced effectors were resistant to complement mediated lysis using anti-L3T4, anti-Lyt1 or anti-Lyt2 antibodies. Pretreatment of spleen cells with macrophage depleting agents both, in vitro and in vivo and subsequent activation of cells by 7T8OG resulted in effectors with reduced cytotoxicity. When injected in vivo, 7T8OG induced strong IFN production which paralelled the kinetics of NK cell activation. Furthermore, antibodies to alpha & beta-IFN but not to gamma-IFN diminished the induction of the cytotoxic activity. Although these findings suggest that activation of NK cells by 7T8OG is most likely to be mediated by alpha & beta-IFN involvement of other cytokines can not be ruled out.

Antiviral and Immunoenhancing Properties of 7-Thia-8-Oxoguanosine and Related Guanosine Analogues

7-thia-8-oxoguanosine (TOGuo) is the first reported structure of a family of modified guanosine analogues exhibiting antiviral activity in rodent models. Its spectrum of action includes interferon-sensitive viruses such as alphaviruses, bunyaviruses, corona viruses, flaviviruses, picornaviruses and, to a lesser extent, herpesviruses. Early treatment before or shortly after virus challenge is necessary to protect animals from mortality. The protective effect of TOGuo against Semliki Forest and Punta Toro viruses can be eliminated by co-treatment with antibody to alpha/ beta-interferon. indicating that interferon induction is of prime importance for antiviral activity against these two viruses. Immunological studies indicate that the nucleoside induces alpha-interferon and activates B cells, natural killer cells, antibody-dependent cytotoxic cells and macrophages. TOGuo also has adjuvant activity, since its use in combination with a killed Ll210 leukemia cell vaccine greatly reduced the mortality of mice inoculated with live Ll210 leukemia cells compared with the vaccine used alone. Several related guanosine analogues show similar antiviral and immunoenhancing properties, including 7-methyl-8-oxoguanosine, 7-methyl-8-thioxoguanosine and 7-deazaguanosine. These studies indicate that certain modifications at the 7 and 8 positions of guanosine may confer antiviral and immunostimulatory properties to nucleoside analogues.

Low molecular weight human T-cell response immunopotentiator: α-2'-deoxy-3-deazaguanosine

Abstract We have examined the immunological activity of a unique α-nucleoside analog of 2′-deoxyguanosine in which the pyrimidine ring nitrogen in the 3 positionis replaced by CH [6-amino-1, 5- dihydro -1-(2- deoxy -α- D -erythro- pentofuranosy ) imidazo [4,5-c] pyridinc pyridin -4- one , α- d 3 DGuo , 1 ] and its structural analogs. The α-d3DGuo is not mitogenic to human PBL. It displayed consistently, however, a potent immunoenhancing activity on PHA-induced human lympocyte proliferation at concentrations ranging from 0.0125 mM to 0.4 mM in a dose dependent manner. These findings thus suggest that mitogenecity is not a pre-requisite for the immunoenhancing effect. The maximal potentialting effect of α-d3DGuo is ussually exerted at the bottom range of the dose response to PHA. The magnitude of increase is about the same as that mediated by rIL-2. Similarly, Con A mediated lymphocyte proliferation is markedly enhanced by α-d3DGuo. When added during allogenic MLR, α-d3DGuo also augmented the proliferation of alloreactive T-cells and the magnitude of response was similar to that induced by rIL-2. The α-d3DGuo induced increase in allogeneic response was dependent on concentrationss of both α-d3DGuo alloantigens as noted with T-mitogen induced proliferative responses. The cytototic activity of lymphocytes induced in allogeneic mixed cultures was also augmented by α-d3DGuo. It showed, however, no potentiating ffect on B-lymphocytes proliferation stimulated either with SAC or PWM. The α-d3DGuo is also able to restore, at least partially, the depressed proliferative responses of T-cells to both PHA and Con A. The β-anomer of α-d3DGuo exhibited both immunopotentiating and immunosuppressive effects on T-cell proliferation. Certain structural changes in α-d3DGuo result in the loss of activity.

Generation of killer lymphocytes in vitro against human autologous leukemia cells with soluble bacterial extraxts

SummaryTen patients with acute lymphoblastic leukemia (ALL) were studied to determine the ability of their remission lymphocytes to kill autologous leukemic blasts (ALB) following in vitro exposure to soluble extracts (SE) of BCG, Staphylococcus aureus (SA) or Listeria monocytogenes (LM). Remission lymphocytes from some patients became markedly cytotoxic to ALB after stimulation with BCG-SE, LM-SE, or SA-SE. These bacterially stimulated lymphocytes, although specifically lytic for ALB, were usually not cytotoxic to autologous remission lymphocytes. Bacterial extracts were able to generate killer lymphocytes at low concentrations. Generally, large amounts either had no stimulatant effect or were less stimulating. Bacteria-stimulated lymphocytes of ALL patients were cytotoxic not only to their leukemia cells, but also to leukemia cells from ALL and AML patients who were allogeneic to stimulated lymphocytes.