Richard J. Clatch

Monocytes/macrophages isolated from the mouse central nervous system contain infectious Theiler's murine encephalomyelitis virus (TMEV)

Knowledge of the cells in which Theilers murine encephalomyelitis virus (TMEV) persists is crucial to understanding the pathogenesis of TMEV-induced demyelinating disease; however, it is still uncertain whether oligodendrocytes or macrophages are the primary target for persistence. In this study, mononuclear cells (MNC) isolated directly from central nervous system (CNS) inflammatory infiltrates of TMEV-infected mice on discontinuous Percoll gradients were found to contain infectious TMEV. Macrophages appeared to be the principal MNC infected as determined by two-color immunofluorescence. Infectious center assay and double immunostaining together indicated the presence and possible synthesis of TMEV in approximately 1 in 225 to 1 in 1000 CNS macrophages, with 1 to 7 PFU produced per macrophage. On the basis of these findings, limited replication in macrophages is consistent with the total CNS virus content detected at any time during the persistent phase of the infection as well as the slow pace of the infection.

The Theiler's murine encephalomyelitis virus (TMEV) model for multiple sclerosis shows a strong influence of the murine equivalents of HLA-A, B, and C ☆

Following intracerebral inoculation of Theilers murine encephalomyelitis virus (TMEV), susceptible mouse strains develop a chronic demyelinating disease characterized histologically by mononuclear cell-rich infiltrates in the central nervous system (CNS). An immune-mediated basis for this disease is strongly supported by previous studies demonstrating a correlation between clinical disease susceptibility, the presence of particular H-2 region genotypes, and the development of chronically elevated levels of TMEV-specific, MHC class II-restricted delayed-type hypersensitivity (DTH). The present study compared disease susceptibility in (B10.S X SJL)F1 and (B10.S(26R) X SJL)F1 mice which differ only at the D region of the H-2 complex. The data conclusively demonstrates a major influence for homozygosity of H-2s alleles at the H-2D region (the murine equivalent of the human class I HLA-A, B, and C genes) in determining disease susceptibility, as measured by either clinical or histopathological endpoints. In addition, disease susceptibility strongly correlated with the development of high levels of TMEV-specific DTH in the susceptible (B10.S X SJL)F1 strain. However, disease susceptibility did not appear to correlate with TMEV titers in the CNS, TMEV-specific humoral (ELISA and neutralizing) immune responses, or virus-specific splenic T cell proliferative responses. These findings lend additional support to our hypothesis that CNS myelin damage is mediated by a TMEV-specific DTH response. The possible role of class I-restricted responses in the demyelinating process is discussed and murine TMEV-induced demyelinating disease is compared with experimental allergic encephalomyelitis as relevant animal models for human multiple sclerosis.

Monoclonal antibody-induced inhibition of relapsing EAE in SJL/J mice correlates with inhibition of neuroantigen-specific cell-mediated immune responses☆

Relapsing experimental allergic encephalomyelitis (R-EAE) in SJL/J mice was examined in relation to the development of neuroantigen-specific T cell proliferative (Tprlf) and delayed-type hypersensitivity (DTH) responses. R-EAE was induced by injecting syngeneic mouse spinal cord homogenate in CFA on days 0 and 7 over the shaved flanks of female SJL/J mice. Mice primed in this manner exhibited significant Tprlf and DTH responses specific for both major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP). A time course comparison between the induction of R-EAE and the development of neuroantigen-specific cell-mediated immune (CMI) responses (Tprlf and DTH) revealed that the MBP- and PLP-specific Tprlf and DTH responses peaked prior to the onset of initial clinical symptoms and the DTH responses remained at significant levels throughout the relapsing course of the disease. Monoclonal antibodies were used to determine whether in vivo inhibition of class II-restricted Tprlf and DTH responses correlated with inhibition of R-EAE. In vivo administration of a total of 100 micrograms anti-L3T4 antibody, but not anti-Lyt-2 antibody, resulted in delayed onset and reduced severity of clinical signs of R-EAE concomitant with significantly reduced levels of MBP- and PLP-specific Tprlf and DTH responses. Treatment with a total of 300 micrograms of purified anti-L3T4 resulted in total abrogation of R-EAE induction and neuroantigen-specific CMI. Thus, clinical signs of R-EAE were found to correlate with the activity of neuroantigen-specific, class II-restricted T cells.

Characterization and Specificity of Humoral Immune Responses to Theiler's Murine Encephalomyelitis Virus Capsid Proteins

Humoral antibody responses to Theilers murine encephalomyelitis virus (TMEV) capsid proteins were examined. Rabbit antisera produced against the native BeAn strain of TMEV and against the isolated capsid proteins (VP1, VP2 and VP3) were tested for their ability to bind or neutralize virus and to inhibit the virus-induced haemagglutination of human O+ erythrocytes. Western immunoblotting analysis showed that isolated VP1, VP2 and VP3 each primed for a specific antibody response, but that native virions primed for antibodies specific for VP1 and VP2, but not VP3. Virus neutralization studies revealed that a dominant TMEV neutralizing determinant(s) lay on VP1, as did the haemagglutinating determinant. The possible location of the neutralizing epitopes are discussed on the basis of molecular modelling of the predicted amino acid sequence of TMEV from that of the closely related Mengo virus for which the three-dimensional structure is known.

Fine specificity of T-cell-mediated immune responses of susceptible and resistant strains in Theiler's murine encephalomyelitis virus-induced demyelinating disease

Previous studies have shown a close correlation between susceptibility to Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease and the temporal development of chronically high levels of TMEV-specific, MHC class 11-restricted delayed type hypersensitivity (DTH).’.’ In this study, we examined the specificity of T-cell mediated immune (CMI) responses (DTH and splenic T-cell proliferative [ Tprlf]) in TMEV-infected susceptible SJL mice to other picornaviruses and to the neuroantigens’ myelin basic protein, proteolipid protein, and spinal cord homogenate. Neuroantigen-specific CMI responses were not detected in TMEV-infected mice before, during, or after the onset of clinical signs of demyelination. However, cross-reactive responses to closely related picornaviruses were seen. In contrast, SJL mice with chronic relapsing EAE exhibited significant CMI responses to all neuroantigens tested, but failed to show picornavirus-specific responses. The functionally defined CMI cross-reactivity pattern correlated with amino acid sequence homology between TMEV capsid proteins (VP1, VP2, and VP3) and other picornaviruses, but not with CNS myelin protein^.^.^ These results are consistent with a TMEV-specific, DTH-mediated CNS pathology, but do not support a major role for CNS-specific autoimmune responses in the demyelinating process. HPLC-purified virion proteins were used to elicit CMI responses in both susceptible SJL/J and resistant C57BL/6 mice in order to determine the fine antigenic specificity

Fine specificity of T cell-mediated immune(CMI) responses of susceptible and resistant strains in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease

Using lectin staining of electrophoresed polypeptides from various components of human CNS, we identified a 120kd polypeptide (120kdpp) that is in white matter, but not in gray matter. It binds Peanut Agglutinin (PNA) avidly and it is the only PNA binding protein in CNS myelin. It is not present in PNS myelin and it is not present in the CNS until myellnation has begun. We showed that the 120kdpp is membrane bound, but upon exposure to Phospholipase-C it is released from the membrane indicating that it is anchored to the membrane through a phosphatidylinosltol linkage. We have also shown that the 120kdpp is in ovine myelin and in isolated ovlne oligodendrocytes. It is present in the oligodendrocytes after a few days in culture, but not after 50 days. We have developed a method for isolating the 120kdpp which consists of: a) exposing well washed myelin to Phospholipase-C, b) amonium sulfate precipitation of the released proteins, and c) two sequential runs over a PNA column. This yields the 120kdpp in a pure form as assessed on a silver-stained SDS-PAGE. Hence, we have identified, characterized somewhat and isolated a PNA binding polypeptide that is attached to myelin and oligodendrocyte membranes through a phosphatidylinositol linkage.