Genetic control of antinuclear antibodies in mice infected with Rauscher leukemia virus.

The incidence of antinuclear antibodies after Rauscher leukemia virus inoculation was found to be significantly higher in C57BL/6 than in BALB/c mice and still greater in their F1 hybrids. The relationships among antinuclear antibody incidence, erythroblastic disease, Rauscher leukemia virus production, and the H-2 genotypes were studied in the F1 generation and backcrosses using different virus inocula. The results observed suggest that (a) at least two genes are involved in the control of susceptibility to Rauscher leukemia virus-induced erythroblastosis, one of them probably being H-2 linked, and that (b) a non-H-2-linked gene seems to control, at the same time, induction of antinuclear antibodies, focus-forming virus production in the spleen, and susceptibility to the disease. It can be concluded that C-type viruses play an active role in antinuclear antibody induction.

Evaluation of irradiation-plus-urethan-induced murine leukemia virus "release" using a new method for quantitation of oncornaviruses in tissues.

The quantity of C-type RNA tumor viruses in homogenate-sonicates of thymus-bone marrow tissues of C57BL/6J and RFM/Un mice 10 days after irradiation (X-rays or gamma-rays)-plus-urethan treatments is no greater than that in thymus-bone marrow homogenates from nontreated control mice. These results indicate that the leukemogenic activity, shown to be present in such thymus-bone marrow homogenates at this time after irradiation-plus-urethan treatment, is not due to change in quantity of C-type viruses as has been proposed. Virus quantity in tissues was evaluated by a new procedure that includes use of a microchamber with the sides situated on rotor radii so as to produce a uniform virus-containing sediment of tissue homogenate-sonicate that is evaluated by electron microscopic examination of this sections cut perpendicular to the membrane surface. Samples containing as little as 105 to 106 viruses can be relatively easily counted. Semipurified or purified viruses can also be counted after mixing with a tissue homogenate-bovine serum albumin diluent.

Interleukin-12 administration in retroviral infection of mice increases the potential to produce functional dendritic cells from bone marrow stem cells.

Rauscher leukaemia virus (RLV) infection in mice causes production of lymph node and skin dendritic cells (DC) that fail to stimulate a primary mixed leukocyte reaction (MLR). Treatment of mice with IL-12 around the time of infection results in DC with normal stimulatory function (N.J. Williams, J.J. Harvey, I. Duncan, R.F.G. Booth, S.C. Knight, Cell Immunol. 183 (1988) 121-130). Here we derived DC from mouse bone marrow by culture with granulocyte macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha) for 10-12 days; DC were generated from bone marrow cells taken from normal mice, from mice injected 15 days earlier with RLV or from those receiving RLV plus five daily doses of 100 ng of IL-12 starting 2 days before infection. Infection of the DC with RLV was assessed from nested PCR with doubling dilutions of DNA and the capacity of DC to stimulate a MLR was tested. DC derived from bone marrow of IL-12 treated animals showed at least twice the level of infection with RLV as those from non-treated animals although infection never exceeded 20% of the cells. DC derived from bone marrow of mice given RLV caused negligible stimulation of the MLR but those from mice additionally treated with IL-12 functioned normally. Thus, treatment of mice with IL-12 promoted the potential of stem cells taken 12 days after the last IL-12 injection to develop into functional DC despite increased infection with virus. Treatment of mice with IL-12 may have a long term effect on the potential growth of DC from stem cells which may contribute to the potency of this cytokine in promoting cell mediated immune responses.

A combined assay for the rapid preliminary detection of structural retroviruses.

This report describes the use of equilibrium gradients, RNA dependent DNA polymerase assays and electron microscopy (EM) in a combined assay for the rapid preliminary detection of intact retroviruses in crude preparations. Positive combined assays of platelets from preleukemic patients corresponded with karyotypic abnormalities found in these patients. Reconstruction experiments with Rauscher Leukemia Virus added to buffer or disrupted mouse spleen demonstrated the ease of detecting 10(9) or greater particles/g crude tissue, and the effects of buffer or added protein.

In vitro and in vivo enhancement of ddI activity against Rauscher murine leukemia virus by ribavirin.

Ribavirin has been reported to enhance the activity of ddI against HIV. We explored this enhancement of antiviral activity in Rauscher murine leukemia virus (RMuLV) models in vitro and in vivo. The significant finding in these studies was that combinations of the drugs exhibited virus titer reductions that were greater than would be expected if the drug interactions were simply additive. These effects were designated synergistic by the method of Prichard and Shipman (Prichard, M.N. and Shipman, C., Jr. (1990). A three-dimensional model to analyze drug-drug interaction, Antiviral Res. 14, 181-206). In addition to the antiviral synergy, we also observed some synergistic toxicity in the animal model.

The role of CD4 and CD8 T cells in the graft-versus-leukemia response in Rauscher murine leukemia.

Using a mouse model for MHC-matched unrelated donor transplantation, the relative influences of the CD4 and CD8 T cell subtypes on graft-versus-leukemia (GVL) were examined in a murine erythroleukemia induced in SJL/J mice by the injection of Rauscher virus. Following leukemia induction, the mice were given 9.5 Gy of total body irradiation (TBI) and injected with mixed marrow and spleen cells from normal MHC-matched--but minor histocompatibility mismatched--B10.S donors. Prior to their injection these donor cells were selectively depleted ex vivo for either CD4, CD8 or Thy-1 by exposure to the appropriate monoclonal antibody (MoAb) plus complement. Following transplant the recipients were observed for 20 weeks, along with parallel control groups, for survival, leukemia relapse, graft failure and graft-versus-host disease; 98% of the controls receiving no transplantation therapy died of leukemia. Among the controls that received TBI plus undepleted B10.S cells 30.9% died of leukemia relapse, but another 34.2% survived free of any clinical evidence of their leukemia. Donor cell depletion for Thy-1 increased the relapse to 68.8%, while survival fell to 10.4%. CD8 depletion resulted in a relapse of 55.6%, with a survival of 19.4%. By contrast, CD4 depletion had no effect on relapse, but did significantly increase the incidence of graft failure. At the end of the 20 weeks additional tests were run to determine whether those transplant survivors that had remained leukemia-free were also free of any residual Rauscher virus. Those tests showed that they were not.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimization of the reverse transcriptase assay for the detection of viral burden in mice infected with Rauscher murine leukemia virus.

Rauscher murine leukemia virus induces an erythroleukemia in susceptible strains of mice that is associated with splenomegaly and viremia. This animal model has been used for evaluating the in vivo efficacy of potential anti-HIV agents. The in vivo antiviral activity of therapeutic agents has usually been determined by measuring a reduction in the spleen weights of compound-treated mice or by quantitating viremia with the UV-XC plaque assay. The UV-XC assay, however, is time-consuming and labor-intensive. Virions of Rauscher murine leukemia virus, like other retroviruses, contain the enzyme reverse transcriptase. Quantitating the level of this enzyme in infected mouse sera provides a more rapid measure of viremia in the animal. We have examined the effects of several reagents, including detergent, KCl, EGTA, dGMP, spermine, as well as protease and RNase inhibitors, on the reverse transcriptase assay. The optimized assay method was effective in evaluating the antiviral activity of AZT in the Rauscher murine leukemia virus in vivo model. The assay is also amenable to automation if large numbers of assays are required.

Method for reproducible large-volume production and purification of Rauscher murine leukemia virus.

Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.

Separation of B and C type virions by centrifugation in gentle density gradients.

Murine type B particles were separated from type C (Rauscher leukemia virus) by means of gentle (low-increment rate) density gradients. The best separation was obtained when the density ranged from 1.13 to 1.20 g/cm(3) when sucrose was used and from 1.12 to 1.28 g/cm(3) with CsCl. The buoyant densities of the B and C particle bands in sucrose were 1.18 and 1.16 g/cm(3), respectively. The CsCl gradient gave a better separation with the B particles banding at a density of 1.20 g/cm(3) and with the C particle density little different from its value in sucrose.