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Presents a study which was conducted to identify loci associated with susceptibility to virus-induced demyelinating disease in the mouse. Materials and methods used; Details on the genetic factors involved in the development of the most common human demyelinating disease; Results and discussion.

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long-term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody-mediated cell adhesion, and neurite outgrowth on HIgM12-coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates its in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases. The human antibody HIgM12 stimulates neurite outgrowth in vitro and promotes function in chronically demyelinated mice, a model of multiple sclerosis. The cellular antigen for HIgM12 was undetermined. Here, we identified polysialic acid attached to NCAM (neural cell adhesion molecule) as the cellular target for HIgM12. This includes glial fibrillary acidic protein (GFAP)-positive mouse astrocytes (GFAP, red; HIgM12, green; DAPI, blue) among other cell types of the central nervous system. These findings indicate a new strategy for the treatment of neuro-motor disorders including multiple sclerosis., (© 2015 International Society for Neurochemistry.)

We used transgenic expression of capsid antigens to Theiler's murine encephalomyelitis virus (TMEV) to study how the immune response to VP1 and VP2 influences spinal cord demyelination, remyelination and axonal loss during the acute and chronic phases of infection. Expression from birth of capsid antigen under the ubiquitin promoter resulted in tolerance to the antigen and absence of an immune response to the respective capsid antigen following virus infection. The transgenic mice were crossed to B10.Q mice normally susceptible to demyelination but which, when compared to FVB mice of the same H2 q haplotype, show poor remyelination. The major finding in this study was that VP1+ and VP2+ animals featured more remyelination at all three chronic time points (90, 180 and 270 dpi) than transgene-negative controls. Interestingly, at 270 dpi, remyelination in VP1+ mice tended to be higher and more complete than that in VP2+ mice. Compared with transgene- negative controls, VP1+ and VP2+ animals showed similar demyelination in but less only late in the disease (270 dpi). The number of mid-thoracic axons at the last time point correlated with the levels of remyelination. The increase in number of axons in VP1+ mice with remyelination was driven by counts in medium- and large-caliber axons. This study supports the hypothesis that expression of viral capsid proteins as self and subsequent genetic deletion of capsid-specific T cells influences the extent of spinal cord remyelination following Theiler's virus-induced demyelination. We propose that VP1- and, to a lesser extent, VP2-specific CD8 + T cells limit and/or prevent the naturally occurring process of remyelination. This finding may have relevance to human multiple sclerosis, as targeted removal of CD8 + T cells specific for a yet-to-be-discovered causative peptide may enhance remyelination and prevent axonal loss in patients.

We used transgenic expression of capsid antigens to Theiler's murine encephalomyelitis virus (TMEV) to study the influence of VP1, VP2 or VP2(121-130) to either protection or pathogenesis to chronic spinal cord demyelination, axonal loss and functional deficits during the acute and chronic phases of infection. We used both mice that are normally susceptible (FVB) and mice normally resistant (FVB.D(b) ) to demyelination. Transgenic expression of VP2(121-130) epitope in resistant FVB.D(b) mice caused spinal cord pathology and virus persistence because the VP2(121-130) epitope is the dominant peptide recognized by D(b) , which is critical for virus clearance. In contrast, all three FVB TMEV transgenic mice showed more demyelination, inflammation and axonal loss as compared with wild-type FVB mice, even though virus load was not increased. Motor function measured by rotarod showed weak correlation with total number of midthoracic axons, but a strong correlation with large-caliber axons (>10µm(2) ). This study supports the hypothesis that expression of viral capsid proteins as self influences the extent of axonal pathology following Theiler's virus-induced demyelination. The findings provide insight into the role of axonal injury in the development of functional deficits that may have relevance to human demyelinating disease., (© 2011 The Authors. Brain Pathology © 2011 International Society of Neuropathology.)

The RNA-dependent RNA polymerase 3D(pol) is required for the elongation of positive- and negative-stranded picornavirus RNA. During the course of investigating the effect of the transgenic expression of viral genes on the host immune response, we evaluated the viral load present in the host after infection. To our surprise, we found that 3D transgenic expression in genetically susceptible FVB mice led to substantially lower viral loads after infection with Theiler's murine encephalomyelitis virus (TMEV). As a result, spinal cord damage caused by chronic viral infection in the central nervous system was reduced in FVB mice that expressed 3D. This led to the preservation of large-diameter axons and motor function in these mice. The 3D transgene also lowered early viral loads when expressed in FVB-D(b) mice resistant to persistent TMEV infection. The protective effect of 3D transgenic expression was not altered in FVB-Rag(-/-).3D mice that are deficient in T and B cells, thus ruling out a mechanism by which the overexpression of 3D enhanced the adaptive immune clearance of the virus. Understanding how endogenously overexpressed 3D polymerase inhibits viral replication may lead to new strategies for targeting therapies to all picornaviruses.

Axon injury is a major determinant of the loss of neurological function in patients with multiple sclerosis. It is unclear, however, whether damage to axons is an obligatory consequence of demyelination or whether it is an independent process that occurs in the permissive environment of demyelinated lesions. Previous investigations into the role of CD8 T cells and perforin in the Theiler murine encephalomyelitis virus model of multiple sclerosis have used mouse strains resistant to Theiler murine encephalomyelitis virus infection. To test the role of CD8 T cells in axon injury, we established a perforin-deficient mouse model on the H-2 major histocompatibility complex background thereby removing confounding factors related to viral biology in this Theiler murine encephalomyelitis virus-susceptible strain. This permitted direct comparison of clinical and pathological parameters between perforin-competent and perforin-deficient mice. The extent of demyelination was indistinguishable between perforin-competent and perforin-deficient H-2 mice, but chronically infected perforin-deficient mice exhibited preservation of motor function and spinal axons despite the presence of spinal cord demyelination. Thus, demyelination is necessary but insufficient for axon injury in this model; the absence of perforin protects axons without impacting demyelination. These results suggest that perforin is a key mediator of axon injury and lend additional support to the hypothesis that CD8 T cells are primarily responsible for axon damage in multiple sclerosis.

Differentiating between injurious and reparative factors facilitates appropriate therapeutic intervention. We evaluated the role of tumor necrosis factor alpha (TNFalpha) in parenchymal brain pathology resolution following virus-induced encephalitis from a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). We infected the following animals with TMEV for 7 to 270 days: B6/129 TNF(-/-) mice (without TNFalpha expression), B6/129 TNFR1(-/-) mice (without TNFalpha receptor 1 expression), and B6/129 TNFR2(-/-) mice (without TNFalpha receptor 2 expression). Normal TNFalpha-expressing controls were TMEV-infected B6, 129/J, B6/129F1 and B6/129F2 mice. Whereas all strains developed inflammation and neuronal injury in the hippocampus and striatum 7 to 21 days postinfection (dpi), the control mice resolved the pathology by 45 to 90 dpi. However, parenchymal hippocampal and striatal injury persisted in B6/129 TNF(-/-) mice following infection. Treating virus-infected mice with active recombinant mouse TNFalpha resulted in less hippocampal and striatal pathology, whereas TNFalpha-neutralizing treatment worsened pathology. T1 "black holes" appeared on MRI during early infection in the hippocampus and striatum in all mice but persisted only in TNF(-/-) mice. TNFR2 [corrected] mediated hippocampal pathology resolution whereas TNFR1 [corrected] mediated striatal healing. These findings indicate the role of TNFalpha in resolution of sublethal hippocampal and striatal injury.

We evaluated the participatory role of human HLA-DR molecules in control of virus from the central nervous system and in the development of subsequent spinal cord demyelination. The experiments utilized intracranial infection with Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that, in some strains of mice, results in primary demyelination. We studied DR2 and DR3 transgenic mice that were bred onto a combined class I-deficient mouse (beta-2 microglobulin deficient; beta2m(0)) and class II-deficient mouse (Abeta(0)) of the H-2(b) background. Abeta(0).beta2m(0) mice infected with TMEV died within 18 days of infection. These mice showed severe encephalomyelitis due to rapid replication of virus genome. In contrast, transgenic mice with insertion of a single human class II major histocompatibility complex (MHC) gene (DR2 or DR3) survived the acute infection. DR2 and DR3 mice controlled virus infection by 45 days and did not develop spinal cord demyelination. Levels of virus RNA were reduced in HLA-DR transgenic mice compared to Abeta(0).beta2m(0) mice. Virus-neutralizing antibody responses did not explain why DR mice survived the infection and controlled virus replication. However, DR mice showed an increase in gamma interferon and interleukin-2 transcripts in the brain, which were associated with protection. The findings support the hypothesis that the expression of a single human class II MHC molecule can, by itself, influence the control of an intracerebral pathogen in a host without a competent class I MHC immune response. The mechanism of protection appears to be the result of cytokines released by CD4(+) T cells.

Epidemiological studies suggest that an environmental factor (possibly a virus) acquired early in life may trigger multiple sclerosis (MS). The virus may remain dormant in the central nervous system but then becomes activated in adulthood. All existing models of MS are characterized by inflammation or demyelination that follows days after virus infection or antigen inoculation. While investigating the role of CD4+ T cell responses following Theiler's virus infection in mice deficient in STAT4 or STAT6, we discovered a model in which virus infection was followed by demyelination after a very prolonged incubation period. STAT4-/- mice were resistant to demyelination for 180 days after infection, but developed severe demyelination after this time point. Inflammatory cells and up-regulation of Class I and Class II MHC antigens characterized these lesions. Virus antigen was partially controlled during the early chronic phase of the infection even though viral RNA levels remained high throughout infection. Demyelination correlated with the appearance of virus antigen expression. Bone marrow reconstitution experiments indicated that the mechanism of the late onset demyelination was the result of the STAT4-/- immune system. Thus, virus infection of STAT4-/- mice results in a model that may allow for dissection of the immune events predisposing to late-onset demyelination in MS.

Lymphotoxin (LT) and tumor necrosis factor (TNF) are important in immune system development and function. LT consists of soluble LT-alpha(3), which binds to TNF-R1 and TNF-R2, and membrane LT-alpha(1)beta(2), which binds to LT-beta-R. We investigated the role of LT and TNF in disease induced by Daniel's (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) since the immune response is believed to be important in both resistance to DA infection as well as mediation of virus-induced demyelination. DA persisted and induced inflammatory demyelination in LT-alpha(-/-) (but not TNF(-/-)) weanling mice of a normally resistant haplotype (C57BL/6), suggesting that LT, but not TNF, is critical for resistance to DA infection. This activity of LT depends on membrane LT-alpha(1)beta(2) and not soluble LT-alpha(3), since DA virus persisted and induced inflammatory demyelination in LT-beta-R(-/-), but not TNF-R1(-/-) or TNF-R2(-/-), mice. The LT-alpha(-/-) and LT-beta-R(-/-) mice failed to mount a virus-specific cytotoxic T cell response. Treatment of weanling C57BL/6 mice with LT-beta-R-Ig, which blocks membrane LT activity, failed to increase susceptibility, suggesting that the LT effect is related to its action on immune system development which is fixed by 3 weeks of age. Our data suggest that membrane LT is important in resistance to DA infection (possibly through interference with CD8+ T cell development and function). There was relatively little demyelination associated with inflammation in LT-alpha(-/-) and LT-beta-R(-/-) mice compared to susceptible SJL mice, suggesting the possibility that LT plays a role in mediating demyelination.

Identifying the role of axonal injury in the development of permanent, irreversible neurologic disability is important to the study of central nervous system (CNS) demyelinating diseases. Our understanding of neurologic dysfunction in demyelinating diseases and the ability to assess therapeutic interventions depends on the development of objective functional assays that can non-invasively measure axonal loss. In this study, we demonstrate in a murine model of progressive CNS demyelination that assessment of the hindlimb width of stride provides a powerful indicator of axonal loss and can dissociate between deficits induced by demyelination versus axonal loss.

Daniel's strain of Theiler's virus (DA) induces a chronic demyelinating disease in the central nervous system (CNS) of susceptible SJL mice, which serves as an excellent model of multiple sclerosis. We previously demonstrated that point mutations near a putative virus receptor-binding site [VP1 99 (Gly to Ser) or 100 (Gly to Asp)] totally attenuate the ability of DA to persist and induce demyelination in SJL mice. The current studies demonstrate that class II-restricted CD4(+) T cells play a major role in clearing VP1 mutant DA viruses from the CNS to prevent demyelination. Infection of SJL CD4((-/-)) mice with DA-VP1-99(Ser) or DA-VP1-100(Asp) resulted in virus persistence and prominent demyelination in the spinal cord. In contrast, infection of SJL CD8((-/-)) mice with DA-VP1-99(Ser) or DA-VP1-100 did not result in virus persistence or demyelination. In addition, no virus-specific cytotoxicity was observed in CNS-infiltrating lymphocytes following infection of SJL mice with VP1 mutant viruses. The mutant DA-VP1-99(Ser) and DA-VP1(100) viruses were in fact neurovirulent when compared to the wild-type DA virus, as they induced an overwhelming encephalitis and early lethality (2 to 4 days postinfection) in mice deficient in the IFN-alpha/beta receptor. Therefore, the nondemyelinating phenotype observed with DA-VP1-99(Ser) and DA-VP1-100(Asp) viruses is dependent in part on the CD4-mediated host immune response., (Copyright 2000 Academic Press.)

Theiler's murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L-dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV-induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H-2b mice. CD40L-mediated interactions were also necessary for development of protective H-2b-restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody- and cell-mediated protective immune responses in distinct phases of TMEV-mediated pathology.

The precise factors involved in the development of a progressive motor dysfunction, a hallmark of immune-mediated demyelinating diseases such as multiple sclerosis, are not well defined. The ability to identify neurologic deficits that result in impaired motor performance early in disease may allow for the identification of therapeutic interventions that slow or eliminate the progression toward a permanent dysfunction. Here we describe the use of three objective, quantitative functional assays (spontaneous activity box, rotarod, and footprint analysis) to detect early neurologic deficits following the initiation of a demyelinating disease with Theiler's murine encephalomyelitis virus (TMEV). The results show that the assays are capable of revealing neurologic deficits at the early stages of the demyelinating disease process. These findings are the first to objectively characterize neurologic function in an animal model of progressive CNS demyelination.

Bone marrow from a normal male pig was transplanted into a related female pig with severe homozygous von Willebrand's disease (vWd). After engraftment the circulating leukocytes were of the male karyotype, and the platelets were strongly positive for von Willebrand factor (vWF) by indirect immunofluorescence. The average level of vWF was 1.96 U/dl and of ristocetin cofactor was 2.8 U/dl. The ear immersion bleeding time before transplantation was consistently more than 15 min and afterwards varied between 5 min and more than 15 min. Transfused vWF corrected the bleeding time at a level of 10 U/dl, which is lower than that required for a von Willebrand pig. We concluded that: the plasmatic compartment is only minimally replenished by the vWF from platelets and megakaryocytes; and the platelet vWF alone only partially corrects the abnormal tests of the hemostatic mechanism in severe vWd.