Cellular tropism and localization in the rodent nervous system of a neuropathogenic variant of Friend murine leukemia virus.

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.

Effects of viral specific cytotoxic lymphocytes on the expression of murine leukemia virus induced neurologic disease.

A dose and time related effect on neurologic disease expression followed transfer of viral specific cytotoxic T lymphocytes (CTL) to recipient NFS/N mice previously infected at 2 days of age with Cas-Br-M murine leukemia virus. Cas-Br-M MuLV gp70 was expressed in spleen and capillary endothelial cells in the brain and spinal cord of CTL recipients, but the progression of gliosis, vacuolation, and cell death that followed endothelial cell MuLV gp70 expression in unprotected Cas-Br-M MuLV infected mice was interrupted in protected CTL recipients. A direct cytotoxic effect of CTL on infected brain capillary endothelial or neural cells could not be demonstrated. Reduced levels of infectious MuLV and MuLV gp70 expression in brain following syngeneic CTL transfer early in the course of disease suggest that CTL may function by preventing a time-limited interaction of Cas-Br-M MuLV with a susceptible target cell or receptor critical for neurologic disease induction during the perinatal period.

Ras gene product expression in blood and marrow smears of patients with acute leukemia: importance of fixation.

Activation of ras protooncogenes by any of several possible mutations in codons 12, 13 or 61 has been demonstrated in a variety of human malignancies, including acute non-lymphoblastic leukemia (ANLL). In situ staining for the ras gene product, p21, has been demonstrated in carcinomas of several sites. High levels of p21 expression have been associated with histologic anaplasia in prostate cancer and regional lymph node metastasis in breast cancer. We examined 16 marrow aspirates and blood smears from patients with acute leukemia, predominantly ANLL, and eight controls. Marrow aspirates or blood were smeared on glass slides and fixed immediately in 10% buffered formalin. p21 was examined with avidin-biotin linked immunoperoxidase visualization. Particular attention must be paid to antibody selection and fixation protocol to demonstrate p21, owing to its rapid degradation ex vivo. Three of 16 patients exhibited occasional high p21 expression primarily in leukemic blasts, but in no case were more than 10% of blast cells positive. Normal reticuloendothelial and myeloid cells occasionally exhibited mild to moderately heavy staining, but megakaryocytes, erythroid precursors, lymphocytes and plasma cells were consistently negative. Most patients, 5 normal volunteers and 3 patients with non-malignant disease, exhibited no reactivity, or only a faint blush. These data suggest that while point mutation and concomitant activation of c-N-ras occurs regularly in ANLL, high levels of ras p21 expression are rarely found with this technique.

Retrovirus induced motor neuron degeneration.

Neurotropic retroviruses are capable of infecting and altering the function of dividing populations of neuron-like cells such as the PC-12 cell line. However, histological, immunohistochemical, and ultrastructural studies have failed to implicate direct infection of neurons by MuLV as the etiologic mechanism responsible for MuLV induced neurodegenerative disease. Indirect mechanisms such as the physical or biochemical disruption of endothelial cell basement membranes or the production of toxic cytokines by virus infected cells may play a role in the development of retrovirus induced neurodegeneration.

T-cell detection with monoclonal antibody T101 kits.

A solid-phase immunoadsorption procedure (Quantigen T&B cell kit; Bio-Rad Laboratories, Richmond, Calif.) employing monoclonal antibody T101 detected mean percentages of peripheral blood T cells comparable to those obtained by rosetting with sheep erythrocytes, while lower values were obtained with an indirect immunofluorescence procedure (Cytotag T&B cell kit; Hybritech, Inc., San Diego, Calif.) employing the same antibody. Therefore, T101 binding appears to be more easily detected by solid-phase immunoadsorption than by immunofluorescence microscopy.

Light transmission analysis of lymphocyte activation by mitogens: a new technique.

In vitro lymphocyte transformation was evaluated by laser light transmission cytometry. Criteria are given for optimal application of this system to assessment of total cells and proportion of lymphoblasts in cultures. The light transmission studies revealed that cell numbers in mitogen stimulated cultures increase steadily, reaching a plateau. The percentage of lymphoblasts reached a peak after 4 days culture. The kinetics of [3H]-thymidine incorporation are similar. The increase in total cell number concomitant with decrease in [3H]thymidine incorporation at the fifth and sixth days indicates discordance between the cell proliferation and utilization of exogenous thymidine. The kinetics of total cell numbers and percentage of lymphoblasts suggest that in vitro reversion of lymphoblasts to small lymphocytes occurred. In addition to examining directly the cellular events during blastogenesis with ease, the current method appeared to be more reproducible than the [3H]thymidine incorporation assay.