Efficacy of continuous zidovudine infusion at early stages of retroviral infection in mice.

We studied the pharmacokinetics of zidovudine (ZDV) in mice after twice-daily s.c. bolus injections and during continuous infusion with s.c. ALZET mini-osmotic pumps. We also compared the antiretroviral efficacy of these two modes of administration against Friend leukemia virus (FLV) infection. Mice were infected by retro-orbital inoculation of about 50 focus-forming units (ffu) of FLV, and treatment was started 1 or 4 h later with ZDV at 40 mg/kg/day for 5 days. Efficacy was evaluated in terms of spleen weight and/or virus titer (spleen focus assay) on day 21 in comparison with untreated infected mice. In a separate experiment, survival time after infection was also monitored over a 140-day period. Plasma concentrations of ZDV were determined by means of high-performance liquid chromatography. Following bolus administration, the peak plasma ZDV concentration (30.5 mg/ml) was reached within 10 min, and elimination was rapid (mean half-life, 0.7 h). During the continuous infusion, the mean concentration was constant at about 1.2 mg/ml. After 5 days of treatment, continuous ZDV infusion consistently inhibited virus-induced splenomegaly by more than 97%; bolus injections were less effective with inhibition ranging from 13 to 98%. These results suggest that moderate constant levels of ZDV have greater antiretroviral efficacy than intermittent high concentrations.

Prospective study of CD8+ lymphocyte activation in relation to viral load in HIV-infected patients with > or = 400 CD4+ lymphocytes per microliter.

To investigate the temporal relationship between CD8+ lymphocyte phenotypic alterations, the CD4+ T cell decline, and plasma HIV RNA levels during the natural history of HIV infection, 33 treatment-naive HIV-infected patients with > or =400 CD4+ cells/microl were studied prospectively for 3 years. During the study period, 20 patients remained untreated, and only 6 received more than 6 months of therapy. A significant relationship was found between changes in plasma HIV RNA and changes in the proportion of CD38+CD8+ cells. Conversely, the number of CD4+ T cells lost per year was strongly related to the increase in the proportion of CD28-CD8+ T cells. A strong relationship between mean yearly changes in CD4+ T cell numbers and changes in HIV RNA was also observed. CD4+ T cell changes were associated with changes in both viral load and CD8+ T cell activation. These results provide support for the use of both virologic and immunologic parameters for prognosis and management during HIV infection.

Factors influencing zidovudine efficacy when administered at early stages of Friend virus infection in mice.

Strategies for zidovudine (AZT) administration in retrovirus infection may greatly influence treatment efficacy, especially in the case of early intervention. Antiretroviral activity of AZT in mice infected with Friend leukemia virus (FLV) has been investigated using various experimental protocols. Mice were inoculated with FLV and treated with AZT either 1 or 4 h after inoculation. A dose/effect relationship of AZT therapy was established for two different loads of virus inoculum. The effects of treatment duration (5 or 14 days) and route of administration (b.i.d. subcutaneous injection or administration in drinking water) were also evaluated. In all cases AZT therapy suppressed or reduced virus-induced splenomegaly and increased survival time. AZT therapy was more effective when started 1 h rather than 4 h after virus inoculation. A mutual influence between the dosage of the antiviral drug and the virus inoculum size was observed. A 5-day therapy was inadequate to suppress infection. AZT therapy led to similar results whether administered subcutaneously or in drinking water. The present results suggest that AZT efficacy declines when the inoculum size is increased, when the initiation of treatment is delayed and when treatment duration is shortened.

Competitive reverse transcription-polymerase chain reaction for quantifying murine AIDS virus.

The causative agent of murine AIDS (MAIDS) is the defective murine leukemia virus BM5d, that requires the replication-competent ecotropic MuLV (BM5e) helper virus. We developed a competitive quantitative PCR method including specific internal standards to quantify the expression of BM5d in the spleen of infected mice and to characterize BM5d expression kinetics following experimental infection. Specimen RNA was reverse-transcribed and co-amplified with a competitive template containing a gag sequence specific for BM5d that can be discriminated from that corresponding to wild-type cDNA by the presence of a unique restriction site, Bg/II. PCR products were quantified by means of densitometric analysis after ethidium bromide staining of gels. To standardise the RNA extraction and reverse transcription steps, the amount of defective-virus mRNA was compared to a constant copy number of murine beta actin mRNA. LP-BM5 production was measured in the spleen of infected mice. Defective gag mRNA production was compared to that of the ecotropic virus. The mRNA level of the defective virus and the titre of replicative virus increased with the duration of infection, and the amount of defective virus mRNA correlated with the titre of replicating virus.

Absence of involvement of nitric oxide in LP-BM5-induced immunodeficiency syndrome.

To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of L-NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFN gamma + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages, from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by L-NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.

Kinetics of ex-vivo cytokine production by splenocytes during murine acquired immunodeficiency syndrome (MAIDS).

The role of T helper 1 (Th1) and T helper 2 (Th2) responses in the murine acquired immunodeficiency syndrome (MAIDS) is unclear. It has been suggested that differential activation of T cell subsets, particularly a shift to Th2 cytokine production, may be associated with disease progression. To clarify the regulation of the cytokine network in the course of MAIDS, we examined the kinetics of cytokine production by isolated splenocytes. C57/BL6 mice were infected with the LP-BM5 mixture. The spleen cell proliferative response, together with IL-2, IFN-gamma, IL-10 and IL-4 production by unstimulated and ConA or anti-CD3 MoAb-stimulated spleen cells, were determined at various times after inoculation (weeks 1, 3, 6 and 9). Spleen cells isolated from murine leukemia virus complex (LP-BM5) infected mice spontaneously produced significant amounts of IL-2 and IFN-gamma one and three weeks post-infection, compared to uninfected controls. The capacity of isolated T cells to produce the Th1 cytokines IL-2 and IFN-gamma in response to stimulation with ConA and anti-CD3 MoAb decreased after 3 weeks of infection. The fall in IL-2 production ran parallel to the fall in the T cell proliferative response to ConA. IL-10 production in response to ConA and anti-CD3 MoAb increased after three weeks post-inoculation, and followed the reverse kinetic pattern to IFN-gamma and IL-2. In contrast, no significant spontaneous IL-4 production and no increase in IL-4 production in response to ConA or anti-CD3 MoAb occurred during the course of MAIDS, relative to uninfected controls. These results suggest that LP-BM5 infection leads to a fall in Th1 cytokine production rather than a clear switch to Th2 cytokine production.

The effect of drugs on the mortality of mice inoculated with Friend leukaemia virus or toxoplasma gondii.

Infection and tumors provoke substantial changes accompanied with the disbalance of many neuroendocrine factors which in their summarizing effects influence the life span of animals. Our previous results showed enhanced mortality after one injection of morphine in association with Friend leukaemia virus infection. The aim of this study was to examine the effects of some other opioids (pethidine and pentazocine) and an acetylcholine esterase inhibitor neostigmine on the survival of animals under two conditions: (1) Friend leukaemia virus infection which mostly depressed immune functions, and (2) Toxoplasma gondii infection which in general enhanced the immune status. In contrast to our previous observation with morphine, the mortality induced by single doses of pethidine (150 mg/kg) or pentazocine (50-75 mg/kg) was unchanged during the Friend leukaemia virus infection. A single injection of neostigmine (0.42 or 0.56 mg/kg) was significantly more lethal in DBA-2 mice infected with Friend leukaemia virus. Neostigmine in doses of 0.33 and 0.4 mg/kg caused death in 46 % and 57 %, respectively, of animals infected with Toxoplasma gondii which was significantly higher in comparison with only 8 % and 12.5 % in control groups. Pethidine (150 mg/kg) killed 70 % of Toxoplasma gondii infected animals and even 90 % of non-infected mice. Thus, the Friend leukaemia virus and Toxoplasma gondii infections increased toxicity only of some drugs which may, at least partly, be associated with altered immune status during infection and involvement of the cholinergic system.

[Effects of chloroquine on the replication of a murine retrovirus]. / Effets de la chloroquine sur la réplication d'un rétrovirus murin.

The wide use of chloroquine (Cq) for prophylaxis and chemotherapy of malaria in Africa, and the increased spread of AIDS in areas of this continent where malaria is endemic, raised the question of a possible interaction between chloroquine intake and HIV infection. Indeed, hydroxychloroquine and chloroquine itself have been shown to inhibit HIV-1 replication in vitro, hydroxychloroquine being proposed as a potential useful adjunctive therapy in the treatment of HIV-1 infection. On the other hand, chloroquine has been reported to enhance the replication of Semliki forest and encephalomyocarditis viruses in a mouse model. In an attempt to elucidate Cq effect on retroviral replication, we have studied the effect of various concentrations of chloroquine in vitro (0.1 nmol/l to 25 mumol/l) on Friend retrovirus (FV)-infected fibroblasts of mice and in vivo (2 to 30 mg/kg) on FV-infected mice. No reduction in the number of virus foci was found in chloroquine-treated fibroblasts cultures. In chloroquine treated-infected mice, no differences were observed in the spleen weights, except an increase at 10 mg/kg. A decrease in splenocyte virus titer was only observed at 10 and 30 mg/kg. No differences in the median survival time was observed up to 30 mg/kg. The authors concluded that chloroquine seemed to have variable effects on viral replication in vivo depending on the dosage, but has no influence on the course of FV-induced disease.

Effects of morphine on the pathogenesis of murine Friend retrovirus infection.

The immunomodulatory effects of opiates can modify host defenses against infection. We investigated the mechanisms involved in these effects by studying the influence of morphine on the pathogenesis of murine Friend retrovirus infection. The response to this opiate varied greatly according to the treatment schedule. Daily intraperitoneal administration of morphine (50 mg/kg) for 16 to 27 days attenuated pathological manifestations in infected animals without modifying the mortality rate. The protective effect increased proportionately with the duration of treatment and depended on the time of treatment initiation relative to inoculation. Naloxone (100 mg/kg/day i.p.) inhibited the morphine-induced decrease in both splenomegaly and viral titer. Mifepristone--a glucocorticoid receptor inhibitor--had no significant effect on the morphine-induced attenuation of splenomegaly. The influence of the infection on acute morphine toxicity was also analyzed using a nonlethal dose in noninfected mice (200 mg/kg). Susceptibility to morphine increased in parallel to the development of the infection, with mortality rates ranging from 20% on day 14 to 90% on day 21. Simultaneous administration of naloxone (20-100 mg/kg) reduced the mortality rate and postponed death. Administration of mifepristone, terfenadin, phentolamine or propranolol did not modify mortality at the doses used. These findings show that the influence of morphine on the development of Friend virus infection in mice depends on the conditions of administration. The transient protective effect seen in certain conditions of administration appears to be due essentially to the direct effects of morphine on its specific receptors.

Prognostic value of phenotypic alterations in blood lymphocyte subsets in a murine retrovirus-induced immunodeficiency syndrome (MAIDS).

Mice infected with the Duplan strain of murine leukaemia virus (Dup MuLV), a retrovirus, develop a syndrome sharing several features with AIDS, including lymphadenopathy and profound immunodeficiency. We measured the changes in peripheral blood lymphocyte populations and evaluated their predictive value for the outcome of disease in C57Bl/6 mice. Animals were inoculated with Dup MuLV (SC1/Dup MuLV confluent fibroblast supernatant or spleen extract from an infected mouse). Peripheral blood lymphocyte subsets were sequentially monitored for 73 days using flow cytometric analysis and MoAbs directly conjugated to fluorochromes. A striking fall in the Thy1.2+ cell count occurred in diseased animals, mostly affecting the CD8+ cell compartment. At the same time, the percentage of Ly5+ cells was increased. Mice were killed at day 73 and spleen and lymph node lymphocytes were analysed. Phenotypic lymphocyte modifications in peripheral blood were closely related to those in the spleen or lymph nodes. Analysis of Ly6c antigen expression on CD4+ and CD8+ cells showed a selective expansion of the CD8+Ly6c+ subset, which may reflect a state of immune activation. Our results suggest that phenotypic alterations of peripheral blood lymphocytes are a good marker of disease progression in this model and could be a useful criterion to evaluate antiretroviral therapy.

Inhibitory effect of nitric oxide on the replication of a murine retrovirus in vitro and in vivo.

Nitric oxide (NO) exerts microbicidal effects on a broad spectrum of pathogens, including viruses, but its antiretrovirus properties have not yet been described. The purpose of this study was to determine whether NO inhibits murine Friend leukemia virus (FV) replication in vitro and to what extent NO may play a role in defenses against FV infection in mice. Three NO-generating compounds were studied: 3-morpholino-sydononimine (SIN-1), sodium nitroprusside (SNP), and S-nitroso-N-acetylpenicillamine (SNAP). The effects of these three compounds were compared with those of their controls (SIN-1C, potassium ferricyanide, and N-acetylpenicillamine, respectively), which do not generate NO and with that of sodium nitrite (NaNO2). SIN-1, SNP, and SNAP inhibited FV replication in dunni cells in a concentration-dependent manner. In contrast, no significant inhibitory effect was observed with the three controls or NaNO2. Furthermore, the addition of superoxide dismutase did not alter the inhibitory effect of SIN-1, which is also known to generate superoxide anions. No dunni cell toxicity was observed in the range of concentrations tested. We also assessed the effect of NO produced by activated macrophages on FV replication. Macrophages activated by gamma interferon and lipopolysaccharide inhibited FV replication in a concentration-dependent manner. This inhibition was due in part to NO production, since it was reversed by NG-monomethyl L-arginine, a competitive inhibitor of NO synthase. In vivo administration of NG-nitro-L-arginine methyl ester, a competitive inhibitor of NO synthase, significantly increased the viral load in spleen cells of FV-infected mice. These results suggested that NO may play a role in defenses against the murine Friend leukemia retrovirus.

Immune status and survival of opiate- and cocaine-treated mice infected with Friend virus.

In as much as the immunomodulatory effects of opiates and cocaine are known to modify spontaneous host defenses against infection, we investigated the effects of morphine, pentazocine and cocaine on the time course of Friend virus infection in mice. Repeated i.p. injections with increasing doses of morphine hydrochloride (10-100 mg/kg for 10 days before infection, a dose regimen which induced tolerance to the acute antinociceptive effects of the drug, followed by 30 mg/kg for 14 days postinfection) did not increase the mortality due to Friend virus infection. This regimen did not significantly affect the immune response of infected mice assessed in terms of delayed hypersensitivity (ear thickness) and the hemagglutination assay. In contrast, a single challenge with a large dose of morphine (up to 300 mg/kg), which is not lethal in noninfected mice, increased mortality markedly (up to 100%) in infected mice when administered at day 14 or 21 postinfection. Repeated i.p. injections with pentazocine (50 mg/kg b.i.d. for 5 days before infection, followed by 30 mg/kg for 14 days postinfection) had no influence on mortality or immune responses in infected mice; similar results were obtained with a single high-dose injection (up to 100 mg/kg). Lastly, repeated i.p. injections of cocaine, using the same experimental procedure as that for pentazocine, decreased immune responsiveness and slightly increased mortality, whereas a single injection was devoid of lethal effect. These findings suggest that chronic opioid treatment does not lower host resistance to viral infection but that the latter could increase the toxicity of a single high dose of morphine.(ABSTRACT TRUNCATED AT 250 WORDS)