RESUMEN
Murine cytomegalovirus (MCMV) has been used extensively as an animal model for human cytomegalovirus (HCMV). Understanding drug resistance and its treatment in MCMV may lead to more effective treatments of HCMV disease. Most ganciclovir-resistant HCMV clinical isolates exhibit a decreased capacity to induce ganciclovir phosphorylation (to its biologically active form) in infected cells. Using an MCMV strain resistant to both ganciclovir and cidofovir, the intracellular metabolism of these drugs was studied to determine if MCMV resistance correlates with decreases in drug phosphorylation. The wild-type (WT) MCMV used for comparison was inhibited in plaque reduction assays, by ganciclovir and cidofovir by 50% at 5.1 and 0.24 microM, respectively; the resistant strain was inhibited at 72 and 2.7 microM, respectively. In uninfected, WT, or resistant virus-infected cells, the extent of metabolism of 10 microM ganciclovir or 1 microM cidofovir to intracellular triphosphorylated species was similar. Phosphorylation and catabolism (following drug removal) rates over time were also similar. Intracellular levels of ganciclovir triphosphate and cidofovir diphosphate increased less than two-fold with increasing multiplicity of virus infection. Because few differences in drug phosphorylation between WT and resistant virus-infected cells were found, virus resistance to ganciclovir and cidofovir apparently is not linked to altered drug phosphorylation. Since the viral DNA polymerase is the antiviral target for these compounds, the resistant MCMV is most likely a DNA polymerase mutant.
Asunto(s)
Antivirales/metabolismo , Citosina/análogos & derivados , Ganciclovir/metabolismo , Muromegalovirus/efectos de los fármacos , Organofosfonatos , Compuestos Organofosforados/metabolismo , Animales , Antivirales/farmacología , Cidofovir , Citosina/metabolismo , Citosina/farmacología , Farmacorresistencia Microbiana , Ganciclovir/farmacología , Ratones , Compuestos Organofosforados/farmacología , Fosforilación , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Oligonucleotides consisting of only deoxyguanosine and deoxythymidine were stable in culture and were able to significantly inhibit Friend Murine Leukemia Virus (FMLV) production in acute cell culture assay systems. The oligonucleotides did not share homology with, or possess any complementary (antisense) sequence motifs to the FMLV genome. The guanosine/thymidine-containing oligonucleotides (GTOs) which demonstrated anti-FMLV activity in acute infection assays were synthesized with natural phosphodiester (PD) linkages (backbones). The observed antiviral activities of these oligonucleotides increased significantly when the PD backbone was replaced with a phosphorothioate (PT) backbone. Experiments designed to investigate a potential antiviral mechanism of action demonstrated that oligonucleotides tested were capable of blocking virus adsorption. In addition, GTOs with PD backbones were competitive inhibitors of FMLV reverse transcriptase (RT). When the same experiments were performed using oligonucleotides with PT backbones, all compounds tested demonstrated significant competitive inhibition of FMLV RT. The measured inhibitory activity of all compounds tested in culture assays was enhanced by at least a factor of 10 when the PD linkages were replaced with PT. The enhanced antiviral activity exhibited by the sulfur group on the oligonucleotide backbone, and the lack of any designed, sequence-specific interactions, suggest that a large percentage of the reported antiviral activity of oligonucleotides containing a phosphorothioate backbone is due to factors other than rationally designed, sequence-specific interactions. The ability of GTOs to inhibit FMLV in culture, potentially via a number of different mechanisms, makes this a class of compounds which warrants investigation as therapeutic agents to be used against retroviral infections.
Asunto(s)
Antivirales/farmacología , Desoxiguanosina/farmacología , Virus de la Leucemia Murina de Friend/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Timidina/farmacología , Células 3T3 , Animales , Antivirales/síntesis química , Secuencia de Bases , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Virus de la Leucemia Murina de Friend/fisiología , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Plásmidos , Inhibidores de la Transcriptasa Inversa , Proteínas Virales/análisisRESUMEN
The disease induced by the Friend virus complex (FV) in F1 hybrid mice containing the Rfv-3r/s genotype in the presence of H-2a/a was used to evaluate a variety of immunomodulating substances. In these genetically defined mice, the FV disease results in splenomegaly, early production of high titers of cell-associated and plasma virus, high levels of splenic viral RNA, increased hematocrit, and eventual death. As the disease progresses, reduced levels of infectious virus correlate with development of specific antibody; reduction in T cell populations, increase in B cells, and decrease in T-cell function also occur. The following immunomodulators were evaluated, listed in the order of their ability to inhibit the FV disease: imexon > MVE-2 > human recombinant IFN-alpha A/D > AS101 > ampligen > AM-3 = oxamisole > ImuVert > bropirimine. In fact, bropirimine, used with certain treatment regimens, appeared to enhance the FV disease. These data suggest that certain immunomodulators may have potential value in the treatment of HIV disease, but also indicate that caution should be exercised in their clinical use.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antivirales/uso terapéutico , Virus de la Leucemia Murina de Friend/efectos de los fármacos , Inmunosupresores/uso terapéutico , Leucemia Experimental/tratamiento farmacológico , Adyuvantes Inmunológicos/farmacología , Animales , Antivirales/farmacología , Citosina/análogos & derivados , Citosina/uso terapéutico , Hexanonas/uso terapéutico , Inmunosupresores/farmacología , Leucemia Experimental/inmunología , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos , Poli I-C/uso terapéutico , Poli U/uso terapéuticoRESUMEN
The effects of two immunomodulators were investigated in severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID mice). Both immunomodulators, maleic anhydride divinyl ether (MVE-2) and 4-imino-1,3-diazobicyclo-(3.1.0)-hexan-2- one (imexon), have been previously studied by us in retrovirus-infected mice. To determine the effects of these compounds as they may function in humans, 24 SCID mice were each reconstituted with 20 x 10(6) ficoll-purified lymphocytes from a single donor. Five weeks after reconstitution, the mice received 16 mg/kg/day of MVE-2 intraperitoneally (i.p.) on days 0, 7, and 14 or 110 mg/kg/day of imexon i.p. daily for 14 days. Spleens were removed and splenocytes labeled with monoclonal antibodies for T- and B-cell enumeration as determined by flow cytometry 24 h after final treatment. Imexon-treated mice demonstrated a slight increase in total T cells and T cell subsets compared to control mice. T helper/T suppressor cell ratios in imexon-treated mice were brought to a normal 3:2 ratio compared to placebo-treated mice. Human immunoglobulin levels were markedly increased in imexon-treated mice. MVE-2-treated hu-PBL-SCID mice had significantly reduced numbers of total T cells compared to controls. The T-cell population results using human cells in SCID mice were similar to the effects of these immunomodulators on murine cells in immunologically competent mice.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Hexanonas/farmacología , Inmunoglobulinas/análisis , Copolímero del Pirano/farmacología , Inmunodeficiencia Combinada Grave/inmunología , Subgrupos de Linfocitos T/inmunología , Adyuvantes Inmunológicos/uso terapéutico , Animales , Anticuerpos Monoclonales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Recuento de Células/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Hexanonas/administración & dosificación , Hexanonas/uso terapéutico , Inyecciones Intraperitoneales , Ratones , Ratones SCID , Copolímero del Pirano/administración & dosificación , Copolímero del Pirano/uso terapéutico , Bazo/citología , Bazo/efectos de los fármacos , Subgrupos de Linfocitos T/efectos de los fármacosRESUMEN
The thymidine analog, 2',3'-didehydro-2',3'-dideoxythymidine (D4T), and 3'-azido-3'-deoxythymidine (AZT) were evaluated for activity against Friend virus complex (FV) in Mus dunni cells using a focal immunoenzyme assay. The 50% effective doses were, respectively, 1.2 and 0.1 microM for the two compounds; the 50% cytotoxic doses using trypan blue dye exclusion were 25.4 and > 100 microM. Four FV inhibition experiments with D4T were run in F1 hybrid mice containing the Rfv-3r/s genotype. This mouse strain allows the study of treatment effects on development of specific neutralizing antibodies and on splenomegaly, splenic and plasma virus titers, and splenic viral RNA. In the first experiment, D4T was given by oral gavage (p.o.) three times daily (t.i.d.) for 14 days beginning 4 h post-virus inoculation. All dosages used (187.5, 375, 750 mg/kg/day) significantly inhibited all viral parameters. Other experiments used D4T p.o. twice daily, with dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day or four times daily with a dose of 375 mg/kg/day. No significant disease inhibition was seen using the twice daily treatment schedule, but efficacy was apparent using the four times daily treatment. The final experiment repeated the initial study, extending the t.i.d. treatments to 25 days and using dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day. All but the lowest dose reduced each virus parameter. None of the D4T treatment regimens caused death in toxicity controls, although moderate host weight loss or less weight gain was seen, and variable hematocrit decreases occurred, particularly in mice receiving the highest drug dosage. Inhibition of natural killer (NK) cell activity also was seen in these same animals, but in infected mice, FV-induced decrease in NK cell activity was prevented by D4T treatment. Virus-specific neutralizing antibodies developed in all infected, treated animals. These data indicate D4T has potential as a possible candidate for anti-human immunodeficiency virus evaluations in the clinic.
Asunto(s)
Antivirales/farmacología , Didesoxinucleósidos/farmacología , Virus de la Leucemia Murina de Friend/efectos de los fármacos , Leucemia Experimental/tratamiento farmacológico , Animales , Anticuerpos Antivirales/sangre , Antivirales/administración & dosificación , Antivirales/uso terapéutico , Línea Celular , Didesoxinucleósidos/administración & dosificación , Didesoxinucleósidos/uso terapéutico , Esquema de Medicación , Femenino , Virus de la Leucemia Murina de Friend/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Leucemia Experimental/inmunología , Leucemia Experimental/microbiología , Leucemia Experimental/patología , Masculino , Ratones , Estavudina , Zidovudina/farmacologíaRESUMEN
(B10.A x A/WySn)F1 mice, infected with the Friend virus (FV) complex, were used as a predictive therapeutic model for AIDS. These infected mice exhibit many of the viral and immunologic manifestations of AIDS. Bropirimine (2-amino-5-bromo-6-phenyl-4[3H]pyrimidinone, ABPP) is an immunomodulating compound which has been shown to inhibit other viral infections. Oral (per os treatment) dosages of ABPP ranging from 50 to 400 mg/kg/day for 3 days resulted in increased numbers of infectious centers in the infected mice and increased splenomegaly and percentage of Ig+ (B cells) in spleens of infected and uninfected mice. Decreased percentages of total Thy-1.2+ (total T) cells and L3T4+ (T-helper) cells were seen in both uninfected and infected mice and a slightly decreased percentage of Ly-2+ (T-suppressor/cytotoxic) cells was observed in spleens of the infected mice. No effect on Ly2+ cells in spleens of uninfected mice was found. Intraperitoneal injection, single or multiple, of 20-200 mg/kg ABPP prior to FV injection resulted in increased spleen weights but had no effect on numbers of infectious centers in the spleens or on FV antibody titers in the plasma. Intraperitoneal treatment of uninfected mice with ABPP resulted in slight or no changes in percentages of Thy-1.2+, L3T4+ and Ly-2+ cells. Mice receiving multiple exposures of ABPP had an increase in percentage of splenic B cells and a depressed response to the T cell mitogen PHA. Treatment with ABPP induced the production of interferon (IFN); however, a state of hyporesponsive IFN production was seen following multiple administrations of ABPP. These data suggest that the immunomodulator ABPP may have an enhancing effect on this retroviral disease.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Citosina/análogos & derivados , Virus de la Leucemia Murina de Friend/efectos de los fármacos , Leucemia Experimental/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida , Adyuvantes Inmunológicos/administración & dosificación , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Citosina/farmacología , Modelos Animales de Enfermedad , Inyecciones Intraperitoneales , Inductores de Interferón/administración & dosificación , Inductores de Interferón/farmacología , Ratones , Ratones EndogámicosRESUMEN
Mice infected with various tumor retroviruses have been used as models for evaluating therapeutic substances for the treatment of some cancers, and more recently, for human immunodeficiency virus (HIV) infection, the causative agent of acquired immune deficiency syndrome (AIDS). Consequently, there is a need to determine the ability of biological response modifiers (BRMs) to specifically reduce virus-infected cells, as compared to their non-specific anti-proliferative effects. To address this need, a BRM, imexon, was evaluated in this study using three strains of mice having different Friend virus (FV)-specific immunological capabilities. The first strain, (B10.A x A/WySn)F1, was genetically capable of producing FV-specific neutralization and cytotoxic antibodies, the second, Balb/c, was not, and the third, SCID mice, lacked functional T and B cell immunity. Imexon treatment reduced virally-induced splenomegaly in all 3 strains; however, the concentration of splenic viral infectious centers (IC) were not affected. Since imexon was efficacious in reducing splenomegaly in SCID mice, the mode of action was concluded to not require functional T or B cell immunity. The observation that imexon did not affect splenic IC titers also suggested that imexon did not specifically eliminate virally infected cells, but may have functioned by other mechanisms. This study also demonstrated the use of various mouse strains as a strategy for delineating the modes of action of BRMs against murine retroviral infections.
Asunto(s)
Antivirales/farmacología , Virus de la Leucemia Murina de Friend/efectos de los fármacos , Hexanonas/farmacología , Infecciones por Retroviridae/tratamiento farmacológico , Animales , Linfocitos B/inmunología , Susceptibilidad a Enfermedades , Femenino , Virus de la Leucemia Murina de Friend/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Infecciones por Retroviridae/inmunología , Bazo/citología , Linfocitos T/inmunologíaRESUMEN
Imexon (4-imino-1,4-diazobicyclo-3.1.0-hexan-2-one) was moderately effective in the treatment of a retroviral infection in a genetically defined murine model. The animal model consisted of a Friend virus complex (FV) infection in a hybrid mouse strain, (B10.A x A/WySn)F1, which has similarities with acquired immune deficiency syndrome (AIDS). Intraperitoneal imexon initiated 1 or 3 days after FV inoculation and continued through 13 days after inoculation significantly reduced splenomegaly, splenic cell-free virus titers and viral RNA. Viral infectious centers/10(6) splenocytes and FV titers in the plasma were reduced, though not to a statistically significant level. The effect of imexon on survival was not statistically significant which suggested that the antiviral effects were only transiently effective. Phytohemagglutinin-induced blastogenesis and percent of total T cells, T helper cells and T suppressor/cytotoxic cells in the spleens were increased, and the percentage of B cells decreased by imexon treatment of both FV-infected and uninfected mice. The splenic natural killer cell activity and interleukin-1 production were not markedly affected. Virus specific neutralizing antibody developed in both imexon- and placebo-treated FV-infected mice, although titers were lower in the imexon-treated animals.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Antivirales/farmacología , Virus de la Leucemia Murina de Friend/efectos de los fármacos , VIH-1/efectos de los fármacos , Hexanonas/farmacología , Leucemia Experimental/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Recuento de Células , Femenino , Virus de la Leucemia Murina de Friend/genética , VIH-1/genética , Leucemia Experimental/microbiología , Linfocitos/efectos de los fármacos , Linfocitos/microbiología , Masculino , Ratones , Ratones Endogámicos , Modelos Genéticos , Fitohemaglutininas/farmacología , ARN Viral/análisis , Esplenomegalia/tratamiento farmacológico , Esplenomegalia/microbiologíaRESUMEN
An F1 hybrid mouse strain containing the Rfv-3r/s genotype was inoculated with Friend virus complex (FV) and treated with zidovudine (ZDV) intraperitoneally three times daily for 20 days beginning as early as 10 min after initial viral exposure. This strain of mice develops FV-specific neutralizing antibodies that aid in reducing viremia and splenic virus titers but do not prevent splenomegaly and eventual FV-associated death. The virally exposed mice treated with ZDV did not develop splenomegaly or have detectable viremia after the last drug treatment. On day 21, a single animal had demonstrable virus in the spleen as determined by a focal immunoenzyme assay; 57% had detectable virus at 5 weeks, but non displayed splenic virus after 35 weeks. None of the animals died after the 35-week holding period, compared to 38% dying in placebo-treated mice. To detect low levels of the virus, or potentially latent virus, splenocytes were cocultivated with a cell line known to readily propagate FV, and the cells were subsequently passaged four times to amplify replication of the virus. After amplification, a significant increase was seen in the number of mice testing positive for virus. Thus, ZDV treatment initiated early after virus exposure was effective in preventing FV-induced splenomegaly and death, but did not prevent low levels of persistent retrovirus in the mice.
Asunto(s)
Virus de la Leucemia Murina de Friend/efectos de los fármacos , Leucemia Experimental/tratamiento farmacológico , Zidovudina/uso terapéutico , Animales , Anticuerpos Antivirales/inmunología , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos , Pruebas de Neutralización , Recurrencia , Esplenomegalia/prevención & control , Linfocitos T/efectos de los fármacos , Linfocitos T/microbiología , Viremia/inmunología , Viremia/prevención & control , Cultivo de Virus , Replicación ViralRESUMEN
Infection with the Friend virus complex (FV) in (B10.A x A/WySn)F1 mice containing the Rfv-3r/s genotype results in several disease manifestations analogous to those seen in patients with acquired immune deficiency syndrome, predominantly high levels of specific antibody and low levels of infectious virus with eventual retroviral disease-induced death of the host. Other immunologic manifestations of FV infection in this murine host included inhibition of percent total T, T-helper, and T-suppressor/cytotoxic cells of total splenic lymphocytes and phytohemagglutinin-induced response of spleen cells. Interleukin-1 production was not affected but the numbers of splenic B cells were increased by the infection. 3'-Azido-3'-deoxythymidine (zidovudine, AZT) administered (a) intraperitoneally three times daily for 24 days beginning 4 h after virus inoculation in doses of 60 to 480 mg/kg/day, (b) in drinking water for 22 days beginning 4 h after virus inoculation in doses of 22 to 216 mg/kg/day, or (c) in drinking water for 29 days beginning 6 days after virus inoculation in doses of 22 to 216 mg/kg/day markedly inhibited FV-induced disease. In the mice receiving early-initiated AZT therapy, FV-induced splenomegaly and hematocrit values were inhibited and infectious centers in the spleen and FV titers in the plasma were reduced to below detectable levels at the higher AZT dosage levels. The percent of total T cells in splenic lymphocytes was increased in the infected, AZT-treated mice. In the intraperitoneal experiment, FV disease-induced death was prevented by treatment with all doses of AZT. Neutralizing antibody to FV was significantly reduced in all AZT-treated groups. Toxicologic manifestations of these AZT treatments included splenic enlargement and reduced hematocrit, although all treated, uninfected mice survived the treatments, gained weight, and displayed no significant effects on enumeration of T and B cells.