Reports have suggested that adult mouse bone marrow cells (BMCs) are capable of transdifferentiating into cells with neural characteristics in the central nervous system (CNS) (1, 2). Because side-population (SP) cells within whole bone marrow are hematopoietic stem cells that can reconstitute the BMC population and are capable of differentiating into other types of cells such as cardiac myocytes and endothelial cells (3-5), wecells. Their close association with blood vessels and the lack of morphological features of neural cells suggested that they were hematopoietic. The brains from Rosa26 control mice had robust P-Gal staining in neural as well as blood cells.Because injury increases SP transdifferentiation in other tissue systems (5), we tested whether neural injury would cause donor cells to transdifferentiate into neural-like cells. Four Table 1. Summary of experimental manipulations and results. ND, not done. Type and number of mice receiving injury to the Brains with Type of cells brain donor-derived transplanted neural cells/brains Contusion injury Stab injury No injury analyzed SP cells 2 4 2 0/8* BMCs ND 7 5 0/12* *50 to 100 coronal sections containing more than 106 neural cells were analyzed per brain.surmised that they too would transdifferentiate into neural cells.To test this, 8 C57B1/6 (B6) mice were treated with a lethal dose of irradiation and transplanted with 2 X 103 SP cells derived from the Rosa26 mouse. The Rosa26 mouse carries the LacZ gene that is constitutively expressed in most cells including neural and SP cells and is an unambiguous marker for donor-derived cells (6). Ten to 12 weeks after transplantation, 80 to 95% of the recipient blood cells were LacZ positive. Four months after transplantation, the CNS of two of the recipient mice were inspected for cells derived from the Rosa26 donor with standard X-gal cytohistochemistry. In coronal sections (50 to 100 sections per brain representing more than 106 cells per brain) taken throughout the full extent of the brain, including the olfactory bulbs and cervical spinal cord, the only P-galactosidase (P-Gal)-positive cells detected were a few cells (<5) that were associated with blood vessels. These P-Gal-positive cells had a globular morphology and no processes that would suggest that they were neural mice with SP transplants underwent cortical stab injury, and two underwent cortical contusion injury (7) 4 months after the transplant. Reconstitution of the hematopoietic system is considered stable and complete around 4 months after BMC or SP transplantation. Therefore, cells capable of transdifferentiation should be in place at that time. One month after cortical stab injury and 4 months after contusion injury, no 3-Gal-positive cells were observed in brains (fig. S1) or cervical spinal cords except a rare few associated with blood vessels. We concluded that adult bone marrow SP cells or cells derived from them were incapable of transdifferentiating into neural cells in this experimental system.Because SP cells will reconstitute the BMC p...

C57BI/6 mice infected with mouse hepatitis virus, strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis. Infectious virus can be isolated only from symptomatic mice. In C57BI/6 mice, two CD8+ T cell epitopes within the MHV-JHM surface glycoprotein were previously identified. Here, we show that mutations in the RNA encoding the immunodominant of the epitopes are present in nearly all virus samples isolated from these mice. Mutations are not present in sequences flanking this epitope or in other CD8+ or CD4+ T cell epitopes. Furthermore, analysis of five peptides corresponding to variant epitopes in direct ex vivo cytotoxicity assays showed that each mutation caused a loss of epitope recognition. These results suggest that escape from CD8+ T cell recognition is necessary for enhanced virus replication and development of clinical disease in these MHV-JHM-infected mice.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV-JHM) develop a demyelinating encephalomyelitis several weeks after infection. Astrogliosis and infiltration of inflammatory cells are prominent findings in the brains and spinal cords of infected mice. In this report, astrocytes in infected spinal cords were analyzed for expression of three pleiotropic cytokines, TNF-alpha, IL-1 beta, and IL-6; Type 2 nitric oxide synthase (iNOS); and MHC class I and II antigen. The data show that all three cytokines and iNOS are expressed by astrocytes in chronically infected spinal cords. These activated astrocytes are localized to areas of virus infection and demyelination, although most of the astrocytes expressing these proteins are not MHV-infected. MHC class I and II antigen can be detected in these spinal cords as well, but not in cells with the typical morphology of astrocytes. TNF-alpha, IL-6, and iNOS are also evident in the brains of mice with MHV-induced acute encephalitis, but in marked contrast to the results obtained with the chronically infected mice, most of the cells expressing these cytokines or iNOS had the morphology of macrophages or other mononuclear cells and very few appeared to be astrocytes. Additionally, astrocytes and, most likely, oligodendrocytes are infected in the spinal cords of mice with chronic demyelination. These results are consistent with a role for both viral infection of glial cells and high localized levels of proinflammatory cytokines and nitric oxide in the demyelinating process in mice infected with MHV-JHM. They also show that analogously to the human demyelinating disease, multiple sclerosis, astrocytes are a major cellular source for these cytokines in mice with chronic, but not acute disease.

CD8 ؉ T cells with cytotoxic activity against the surface glycoprotein (S) of mouse hepatitis virus, strain JHM, have been identified in the central nervous system (CNS) of both acutely and chronically infected C57BL/6 mice. In this report, two specific epitopes recognized by these CNS-derived cells were identified, using a panel of peptides chosen because they conformed to the allele-specific binding motif for MHC class I H-2K b and H-2D b. The active peptides encompassed residues 510 to 518 (CSLWNGPHL, H-2D b) and 598 to 605 (RCQIFANI, H-2K b). Both epitopes are located within the region of the S protein previously shown to be prone to deletion after passage in animals. These deleted strains are generally less neurovirulent than the wild-type virus but still are able to cause demyelination. Since C57BL/6 mice become persistently infected more commonly than many other strains of mice, these data are consistent with a role for CD8 ؉ T-cell escape mutants in the pathogenesis of the demyelinating disease. This is the first report of CD8 ؉ T-cell epitope localization within the S protein, the protein most strongly implicated thus far in pathogenesis in the host.

C57BI/6, but not BALB/c, mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a late onset, symptomatic demyelinating encephalomyelitis. In this report, we characterized anti-viral cytotoxic T cells in the central nervous system and spleen during the acute and chronic stages of the MHV infection. The data show that C57BI/6 mice display a cytotoxic T cell (CTL) response to the surface (S) glycoprotein and this response can be demonstrated in lymphocytes isolated from the brains and spinal cords of mice both acutely and persistently infected with MHV-JHM. Thus, the anti-S CTL activity present in the central nervous system of chronically infected animals is not sufficient to prevent the demyelinating process. BALB/c mice have been shown previously to mount a CTL response against the nucleocapsid (N) protein (Stohlman et al., 1992). Since C57BI/6 mice do not mount a response to the N protein, the role of the N-specific response in preventing the late onset disease was assessed using B10.A(18R) mice, recombinant in the H-2 locus. These mice contain the d alleles of the D and L loci and exhibit a CTL response against the N protein. However, unlike the BALB/c mice, these animals develop the late onset symptomatic disease. These results suggest that the N-specific response is partially protective against the development of the demyelinating disease, but that additional factors are also likely to be involved.