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Background: We studied PVC-211 murine leukemia virus (MuLV) (1), a neuropathogenic variant of Friend MuLV, to determine its cellular tropism and distribution in the nervous system of infected rats and the factors that affected disease expression., Experimental Design: Rats from five different strains and mice from 3 strains were inoculated intracerebrally or intraperitoneally from birth to 10 days of age and observed for signs of neurologic disease and tumors for 24 weeks. Nervous system pathology, MuLV gp70 expression, and virus production were evaluated weekly for 4 weeks after perinatal infection of Fisher (F344) rats. Blood-brain-barrier integrity and ultrastructure were evaluated in 21-day-old symptomatic infected rats. Microvessel and mixed glial cell cultures were prepared from brains of infected and uninfected 21-day-old F344 rats and evaluated for virus production, MuLV gp70 expression, and the presence of PVC-211 MuLV DNA., Results: Tremor, ataxia, spasticity, and hindlimb weakness occurred in rats and mice as early as 3 weeks after neonatal infection. Severity, latency, and progression varied among mouse and rat strains but exposure to PVC-211 MuLV before 6 days of age was required for disease expression. Rapid PVC-211 MuLV replication in brain capillary endothelial cells (BCEC) early in the perinatal period was followed by widespread astrogliosis, neuropil vacuolation, and finally, neuronal degeneration in the spinal cord, brainstem, cerebellum, and subcortex. MuLV gp70 expression in vivo increased during infection, was restricted to BCEC, but was not associated with perivascular inflammatory infiltrates. BCEC cultured from microvessel preparations but not astrocytes or microglia in mixed glial cell cultures isolated from infected rats contained PVC-211 MuLV DNA, expressed MuLV gp70, and produced infectious virus., Conclusions: The rapid replication of PVC-211 MuLV that occurs in the nervous system of infected rodents is restricted to BCEC. These infected BCEC appear to play a critical role in initiating the astroglial response in this neurodegenerative process through mechanisms that remain to be defined.

There are a variety of murine hematopoietic progenitor cell lines which are differentiation arrested, but still require growth factors such as interleukin-3 for their continued growth and survival. While oncogenes such as v-myc and v-abl have been demonstrated to abrogate the requirement for exogenous growth factors, none have been shown to have an effect on the differentiation of these cell lines. In this report, we demonstrate that the introduction and expression of Abelson murine leukemia virus into a myeloblast progenitor cell line can promote further differentiation along the monocytic lineage. There is a marked alteration in cell morphology, the acquisition of Mac-1 antigen expression, the induction of nonspecific esterase expression and the induced ability to phagocytize opsonized zymosan. Thus, the expression of Abelson murine leukemia virus protein in interleukin-3-dependent hematopoietic progenitors can provide differentiation-inducing signals in cells which are arrested in differentiation. The potential role of Abelson murine leukemia virus gene products in normal hematopoietic cell differentiation and in transformation is discussed.

Wild mouse ecotropic retrovirus (Cas-Br-M) induced paralysis and non-thymic lymphomas in susceptible NIH Swiss and NFS/N mice. The incidence of paralysis was highest and latency shortest in mice receiving high doses of virus. Lower dose inoculation and inoculation of older mice produced less paralysis with longer latency, but resulted in more lymphomas. However, 10-day-old mice did not develop paralysis and had fewer lymphomas. Anti-Cas-Br-M antibody was detectable in sera from 10-day-old infected mice but not from paralyzed mice. These data suggest that while paralysis and lymphoma may result from different virus-host interactions, the development of immunocompetence may play a role in the age-dependent resistance to Cas-Br-M-associated paralysis and lymphoma in these mice.

Spleen cells taken from mice infected as adults with two different variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, as well as spleen cells taken from mice infected as newborns with Friend murine leukemia virus (F-MuLV) were assayed in a proliferation assay in the presence or absence of the erythroid hormone erythropoietin (Epo). Infection of NIH Swiss mice with SFFV resulted in excessive proliferation of erythroid cells that could still differentiate, and spleen cells taken from these mice were able to incorporate high levels of tritiated thymidine ([3H]dThd) in the absence of Epo, even in the presence of antibodies to Epo. In contrast, the level of proliferation of spleen cells from SFFVA-infected mice, but not those from SFFVP-infected mice, could be greatly enhanced by the addition of Epo to the cultures. Infection of newborn mice with F-MuLV resulted in the generation of Friend mink cell focus-inducing virus, which caused excessive proliferation of erythroid cells that appeared to be blocked in differentiation, resulting in severe anemia. Spleen cells from these mice were unable to proliferate in the absence of Epo. However, when increasing doses of Epo were added to the cultures, the cells proliferated at levels equivalent to the levels seen with SFFV. These results indicate that a proliferation assay based on the incorporation of [3H]dThd into spleen cells in response to Epo can be used as a quantitative means of assessing and comparing the effects of erythroleukemia-inducing retroviruses on the proliferation of their target cells.

The polycythemia-inducing strain of the Friend spleen focus-forming virus (SFFVP) induces an acute erythroleukemia in mice. Erythroid cells from these mice differ from normal erythroid cells in that they can proliferate and differentiate in the apparent absence of the erythroid hormone erythropoietin (Epo). Although it was recently shown that the unique envelope protein encoded by SFFV is responsible for altering the hormonal requirements of erythroid cells for growth and differentiation, the mechanisms by which this occurs is not known. Since the SFFV envelope protein appears to interact with a target present only in erythroid cells and since Epo is specific for these cells, it is possible that the virus is exerting its effect through this hormone. In an effort to ascertain if this is the case, we examined cells from SFFVP-infected mice to determine (a) if they produce Epo or other erythroid growth factors that stimulate erythroid cells to grow in an autocrine-like manner and (b) if they express elevated numbers of Epo receptors that may result in a reduced requirement for the level of Epo needed for growth and differentiation. Our results indicate that SFFVP-infected cells do not secrete Epo or any other erythroid growth factors that could account for the reduced hormonal requirements of these cells. Also, our studies using iodinated Epo in cell binding assays and cross-linking studies indicate that SFFVP-infected cells are not significantly different from normal erythroid cells in the number, affinity, or size of their Epo receptors.

The antibody response of genetically inbred rats to poly(Glu52Lys33Tyr15) is controlled by a complex polygenic system which includes at least two autosomal genes and a sex influence, which may also be genetically determined. The genetic control of the quantity, binding constants, and specificity of the antibody formed linked to the major histocompatibility locus. Factors other than the major genetic ones and the sex influence also affect the quantity of antibody formed, since animals of the same genotype can make significantly different amounts of antibody, depending upon the crosses by which they acquire the major histocompatibility alleles. After immunization with poly(Glu52Lys33Tyr15) the low responders make fewer antibody-producing cells, are not capable of mounting a delayed hypersensitivity reaction to the polypeptide and appear to be deficient in their ability to produce the specific IgM antibody. Immunization of the low responders with antigen aggregated with methylated bovine serum albumin enhances the quantity of antibody formed, increases the binding constants and crossreactivity of the antibody and enhances the delayed hypersensitivity response. In contrast to the findings with the L-amino acid polypeptide, there does not appear to be any genetic control over the antibody response to the D-amino acid enantiomorph poly(DGlu52DLys33DTyr15), which is minimal in all strains.

Strains of genetically inbred rats representative of the known Ag-B groups produced migration inhibitory factor in response to immunization with live bacillus Calmette-Guerin bacilli and intracardiac challenge with old tuberculin; the assays were performed by both in vitro and in vivo methods. None of the strains immunized in this fashion developed delayed hypersensitivity skin reactions to purified protein derivative.

The McDonough strain of feline sarcoma virus (SM-FeSV) contains a viral oncogene, v-fms, transduced from cat cellular genetic sequences designated c-fms. Monoclonal antibodies reactive to antigenic determinants encoded by v-fms were prepared by immunizing rats with live, syngeneic SM-FeSV-transformed cells, and fusing splenic lymphocytes from a tumor-bearing animal with cultured rat myeloma cells. Culture supernatants from hybrids producing antibodies to epitopes encoded by v-fms were identified by immunoprecipitation of radiolabeled polypeptides from SM-FeSV-transformed mink cells. Four positive hybrids were cloned twice in soft agar, established as stable lines, and grown in defined serum-free medium to facilitate purification of homogeneous antibodies. The monoclonal antibodies were used to assay SM-FeSV-specific products by "immunoblotting" of electrophoretically separated proteins, and by fixed-cell immunofluorescence.

An isotopic antiglobulin assay was developed to measure antibodies in "normal" cats to FOCMA, a cell surface antigen associated with immunity to FeLV-induced tumors. The assay was shown to be specific for detecting FOCMA antibody by several criteria including a correlation with cell membrane immunofluorescence and absorption tests. Antibody to FOCMA developed following FeLV infection and was expressed in heterologous species. In combination with an assay for antibody to FeLV p30, the major virion structural protein, 200 cats sera from three different geographic areas were examined for evidences of FeLV-type-C-virus infection. Approximately 17% of all sera examined contained antibody to FOCMA. Antibodies directed to the FeLV p30 were detected in 8 of the sera and only in cats with FOCMA antibody. The correlation in cat sera between antibody to FOCMA and FeLV p30 antigen suggests that both are associated with the same process, infection with FeLV type-C-virus. The relationship of the observed pattern of antibody response is discussed in relation to other horizontally transmitted RNA-containing viruses.

Tumor and normal tissues from domestic cats were examined for FeLV and RD-114 specific nucleic acids and proteins. Virus-positive tissues showed a 100- to 200-fold higher level of FeLV and a 200- to 400-fold higher level of FeLV p30 than virus-negative tumors and normal cat tissues. In contrast, the levels of RD-114 viral RNA and p30 antigen were comparable in the majority of tumorous and normal tissues examined. When the DNA of these tissues was examined for FeLV-related sequences, the virus-positive tumors were found to contain extra copies of FeLV DNA. Virus-negative tumors contained DNA sequences homologous with FeLV but the copy number of these sequences could not be distinguished from normal tissues. The results indicate that in certain FeLV-induced type-C lymphoproliferative diseases, extra type-C sequences not present in normal tissues can be identified.

The mechanism of alkylating agent-induced leukemia is unknown. For the determination of whether chronic alkylating agent treatment of hematopoietic stem cells in vitro was detectably leukemogenic, murine long-term bone marrow cultures (LTBMC) and clonal interleukin 3 (IL-3)-dependent multipotential hematopoietic progenitor cell lines [B6SUtA clone (cl) 27 and Ro cl 3-1] derived from LTBMC were chronically pulse treated in vitro with the alkylating agent melphalan [L-phenylalanine mustard (L-PAM)]. Weekly treatment of C3H/HeJ or CD-1 Swiss mouse LTBMC with 3 X 10(-6)M L-PAM significantly decreased cumulative production of nonadherent granulocytes and granulocyte-macrophage progenitor cells responsive to L-cell or WEH1-3 cell colony-stimulating factor compared to the production seen in untreated control cultures; it also significantly reduced the hematopoietic longevity (13 wk compared to greater than 20 wk for untreated control cultures). Weekly, twice weekly, or daily (3 X 10(-6)M) L-PAM treatment of IL-3-dependent cell lines induced gradual L-PAM adaptation in the absence of a detectable change in the maximum binding capacity of 125I-labeled IL-3. No leukemogenic variants of line B6SUtA cl 27 were detectably induced. However, 3 stably expressed marker chromosomes were induced after 12 months of L-PAM treatment of line B6SUtA cl 27. Thus IL-3-dependent hematopoietic progenitor cells slowly adapt to L-PAM when in suspension culture in vitro. Physiologic expression of drug toxicity in LTBMC may prevent this hematopoietic cell gradual adaptation.