ovalbumin and Cardiovirus-Infections

Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler's murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45

Topics: Animals; Antigens; Antigens, CD19; B-Lymphocytes; Brain; Capsid Proteins; Cardiovirus Infections; CD8-Positive T-Lymphocytes; Cell Separation; Cell Survival; Centrifugation, Density Gradient; Collagen Type IV; Collagenases; Disease Models, Animal; Epitopes; Female; Flow Cytometry; Hyaluronan Receptors; Lymphocyte Activation; Male; Mice, Inbred C57BL; Ovalbumin; Povidone; Selectins; Silicon Dioxide; Theilovirus

Cross-presentation of cell-associated antigens plays an important role in regulating CD8+ T cell responses to proteins that are not expressed by antigen-presenting cells (APCs). Dendritic cells are the principal cross-presenting APCs in vivo and much progress has been made in elucidating the pathways that allow dendritic cells to capture and process cellular material. However, little is known about the signals that determine whether such presentation ultimately results in a cytotoxic T cell (CTL) response (cross-priming) or in CD8+ T cell inactivation (cross-tolerance). Here we describe a mechanism that promotes cross-priming during viral infections. We show that murine CD8alpha+ dendritic cells are activated by double-stranded (ds)RNA present in virally infected cells but absent from uninfected cells. Dendritic cell activation requires phagocytosis of infected material, followed by signalling through the dsRNA receptor, toll-like receptor 3 (TLR3). Immunization with virus-infected cells or cells containing synthetic dsRNA leads to a striking increase in CTL cross-priming against cell-associated antigens, which is largely dependent on TLR3 expression by antigen-presenting cells. Thus, TLR3 may have evolved to permit cross-priming of CTLs against viruses that do not directly infect dendritic cells.

Topics: Animals; Antigen Presentation; Cardiovirus Infections; Chlorocebus aethiops; Cross-Priming; Dendritic Cells; Encephalomyocarditis virus; Endosomes; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Ovalbumin; Poly I-C; Receptors, Cell Surface; RNA, Double-Stranded; RNA, Messenger; T-Lymphocytes, Cytotoxic; Toll-Like Receptor 3; Toll-Like Receptors; Vero Cells

Theiler's murine encephalomyelitis virus (TMEV) infection of the central nervous system (CNS) induces a chronic, progressive demyelinating disease in susceptible mouse strains characterized by inflammatory mononuclear infiltrates and spastic hind limb paralysis. Our lab has previously demonstrated a critical role for TMEV- and myelin-specific CD4(+) T cells in initiating and perpetuating this pathology. It has however, also been shown that the MHC class I loci are associated with susceptibility/resistance to TMEV infection and persistence. For this reason, we investigated the contribution of CD8(+) T cells to the TMEV-induced demyelinating pathology in the highly susceptible SJL/J mouse strain. Here we show that beta2M-deficient SJL mice have similar disease incidence rates to wild-type controls, however beta2M-deficient mice demonstrated earlier onset of clinical disease, elevated in vitro responses to TMEV and myelin proteolipid (PLP) epitopes, and significantly higher levels of CNS demyelination and macrophage infiltration at 50 days post-infection. beta2M-deficient mice also displayed a significant elevation in persisting viral titers, as well as an increase in macrophage-derived pro-inflammatory cytokine mRNA expression in the spinal cord at this same time point. Taken together, these results indicate that CD8(+) T cells are not required for clinical or histologic disease initiation or progression in TMEV-infected SJL mice. Rather, these data stress the critical role of CD4(+) T cells in this capacity and further emphasize the potential for CD8(+) T cells to contribute to protection from TMEV-induced demyelination.

Topics: Amino Acid Sequence; Animals; Antigens, Viral; beta 2-Microglobulin; Brain; Capsid; Capsid Proteins; Cardiovirus Infections; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Cytokines; Demyelinating Diseases; Epitopes; Female; Genetic Predisposition to Disease; Hypersensitivity, Delayed; Inflammation Mediators; Macrophages; Mice; Mice, Inbred Strains; Mice, Knockout; Molecular Sequence Data; Myelin Proteolipid Protein; Ovalbumin; Peptide Fragments; RNA, Messenger; Spinal Cord; Theilovirus

Theiler's murine encephalomyelitis virus (TMEV) infection produces a chronic inflammatory disease of the spinal cord white matter, with striking similarities to both experimental allergic encephalomyelitis (EAE) and human multiple sclerosis (MS). The first phase of demyelination in this model appears to be dependent on a delayed-type hypersensitivity (DTH) response to viral antigens, driven by CD4+, Th1 lymphocytes. Macrophages, recruited in the infected CNS, would be responsible for most of the myelin damage. Recently, new populations of CD4+ lymphocytes were demonstrated in infected mice, this time with specificity for myelin antigens, particularly PLP. This suggests that, in the chronic phase of the disease, an autoimmune mechanism of demyelination, similar to EAE, may participate in the process of myelin destruction. The present study represents a first step in exploring the functional activity of these anti-myelin lymphocytes that emerge during the chronic phase of the disease. Lymphocytes were removed from chronically infected animals, they were stimulated with the major PLP encephalitogenic epitope for SJL/J mice, and they were added to organotypic myelinated spinal cord cultures for different lengths of time. Results show that lymphocytes stimulated with the major PLP epitope have a powerful capacity for demyelinating these cultures, while MBP stimulated lymphocytes and lymphocytes from control animals do not. This study, suggests that the anti-myelin response that emerges during the chronic phase of the infection is functionally active. A similar phenomenon of epitope spreading from virus to organ specific antigens may take place in humans and be involved in a number of immune-mediated diseases, including MS.

Topics: Animals; Cardiovirus Infections; Cells, Cultured; Chronic Disease; Demyelinating Diseases; Encephalitis; Epitopes; Immunization; Lymphocytes; Mice; Mice, Inbred Strains; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Organ Culture Techniques; Ovalbumin; Theilovirus