Theiler's murine encephalomyelitis virus infection induces early expression of c-fos in astrocytes.

We have determined whether Theiler's murine encephalomyelitis virus (TMEV), a picornavirus that produces demyelination in genetically susceptible strains of mice, induces c-fos in pure quiescent cultures of mouse brain astrocytes. As observed in Northern blots, the expression of this immediate early gene increases in a dose-dependent manner, with its expression peaking at a multiplicity of infection of 100. The expression of c-fos is transient, peaking after 30 min and disappearing 2 h after infection. The virus is quickly internalized at 37 degrees C upon binding to its specific receptor located at the cell surface and is actively replicated in the cytoplasm of the astrocytes, as demonstrated by FACS flow cytometry. Using the same technique, nuclear translation of c-fos mRNA is also shown. The specificity of viral induction is demonstrated by its neutralization with TMEV-specific antibodies and by the fact that only viral particles and not purified protein components VP1, VP2, and VP3 induced proto-oncogene expression. This rapid induction of c-fos in astrocytes could be the first stage in the infection of these central nervous system cell populations by TMEV. The biological relevance of these findings is assessed by the demonstration of c-fos activation after viral infection in vivo.     

Cytotropism of Theiler's murine encephalomyelitis viruses in oligodendrocyte-enriched cultures

Summary The cytotropism of two strains, GDVII and DA, of Theiler's murine encephalomyelitis viruses (TMEV) was studied in the oligodendrocyte-enriched murine neural cell cultures. Both GDVII and DA caused cytopathic effects in the neural cell cultures, and double immunostaining for galactocerebroside (Gal-Cer), a marker molecule for oligodendrocyte, and viral antigens disclosed a dual expression of Gal-Cer and viral antigens in over 80% of cells in both cultures 24h after infection with either GDVII or DA. The kinetics of cell-free and cell-associated infectivity were not significantly different between two cultures. These in vitro observations suggest that neither replication in oligodendrocyte nor cell-associated infectivity is a sole factor in discriminating those two subgroups of TMEV with regard to the demyelinating activity, and that virus cell binding may play an important role in virus persistence and TMEV-induced demyelination.     

Tumour necrosis factor-alpha induces translocation of protein kinase C in tumour necrosis factor-sensitive cell lines.

In this study we investigated whether the anti-proliferative effect of tumour necrosis factor-alpha (TNF-alpha) was associated with the activation of protein kinase C (PKC), using PANC-1 cells (TNF-alpha sensitive) and LoVo cells (TNF-alpha resistant). In combination with 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, TNF-alpha caused marked inhibition of the growth of LoVo cells. Inhibition of PANC-1 cell growth by TNF-alpha was blocked by pretreatment with TPA for 24 hr, along with down-regulation of PKC activity. Intracellular translocation of PKC from cytosol to membrane was induced by TNF-alpha treatment in PANC-1 cells but not in LoVo cells.     

Innate immune response induced by Theiler's murine encephalomyelitis virus infection

Although the causative agents of human multiple sclerosis (MS) are not known, it is suspected that a viral infection may be associated with the initiation of the disease. Several viral disease models in mice have been studied to understand the pathogenesis of demeylination. In particular, Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) has been extensively studied as a relevant model. Various cytokines and chemokines are produced upon viral infection by different cell types, including antigen-presenting cells (APCs) such as macrophages; dendritic cells (DCs); and glial cells, such as astrocytes, microglia, and oligoden-drocytes. The upregulation of the corresponding molecules are also found in MS and are likely to play an important role in the protection and/or pathogenesis of chronic inflammatory demyelinating disease. In this review, the type of cells and molecules, gene-activation mechanisms as well as their potential roles in protection and pathogenesis will be discussed.     

The Candida albicans phospholipomannan induces in vitro production of tumour necrosis factor-alpha from human and murine macrophages.

We have previously identified a Candida albicans 14,000-18,000 MW antigen reacting with anti-beta-1,2-linked oligomannosides antibodies as being a phospholipomannan (PLM). Because of the structural similarities between the C. albicans PLM and lipophosphoglycans from various microbial pathogens known to be potent tumour necrosis factor-alpha (TNF-alpha) inducers, we investigated the PLM ability to induce TNF-alpha. Incubation of human monocytic cells THP-1 with PLM led to dose-dependent production of TNF-alpha that was significantly increased by prestimulation of the cells with interferon-gamma (IFN-gamma). Production of TNF-alpha by macrophages under PLM stimulation was confirmed by using macrophages elicited from the mouse peritoneal cavity. In all investigated conditions, PLM-induced TNF-alpha production differed significantly in both kinetics and dose dependence from lipopolysaccharide (LPS) induction used as control. It appears, therefore, that the C. albicans PLM shares functional homologies with microbial lipophosphoglycans identified as pathogenicity factors, although prestimulation of the target cells was required for the PLM-derived opportunistic pathogen to trigger the cytokine network.     

Neonatal infection with the Daniels strain of Theiler's murine encephalomyelitis virus

The clinical and pathologic manifestation of Theiler's murine encephalomyelitis are age related. Animals infected during the first week of life die of a fulminant encephalitis analogous to human poliomyelitis. By contrast, animals infected within 2 and 4 weeks of age survive but develop chronic relapsing demyelination and persistent infection of the central nervous system. The neonatal infection results in widespread necrosis beginning with neuronal vacuolar degeneration followed by inflammatory infiltrates. Electron microscopy reveals paracrystalline arrays of 27-nm viral particles characteristic of picornaviruses within neurons and macrophages. In addition, oligodendrocytes show reactive changes and intracytoplasmic vacuoles. Immunoperoxidase studies show viral antigen primarily localized within neurons of the cerebral cortex, basal ganglia, hippocampus, and anterior horn cells. Viral antigen is found within the apical dendrites and axonal projections of hippocampal pyramidal cells suggesting that Theiler's murine encephalomyelitis may travel intraaxonally.     

Persistent Theiler's murine encephalomyelitis virus infection in mice depends on plaque size

Theiler's murine encephalomyelitis virus (TMEV) is an enteric pathogen of mice which causes acute and chronic neurological disorders in the natural host. When brain-derived stocks of TMEV isolates are adapted to cell culture they predominantly form either large or small plaques. In this study the type of central nervous system (CNS) infection (acute versus chronic) and the associated disease occurring in mice inoculated intracerebrally with large and small plaque strains of TMEV was investigated. Large and small plaque strains of TMEV were found to vary in virulence, type of neurological disease produced and ability to establish persistent CNS infection in mice. Two large plaque strains, GDVII and FA viruses, were highly virulent, produced acute encephalitis, but were cleared from the nervous systems of surviving animals. Therefore, it appears that these large plaque variants do not cause persistent CNS infection in mice. In contrast, five small plaque strains, DA, WW, TO4, Yale and BeAn8386 viruses, were relatively avirulent, usually produced no illness during the first month after inoculation, but readily established persistent CNS infection in mice. Persistently infected mice later developed demyelinating disease. Having identified strains of TMEV that differ regarding their ability to persist, we now hope to be able to exploit this difference in elucidating the basic mechanism(s) of TMEV persistence.     

Theiler's murine encephalomyelitis: a model of demyelination and persistence of virus

Theiler's murine encephalomyelitis virus (TMEV) causes immune-mediated demyelination in susceptible mice which is similar to human demyelinating disorders such as multiple sclerosis. In addition, the picornavirus persists within the central nervous system throughout the course of the chronic demyelinating disease. This article reviews the neuropathology, virology, immunology, and molecular biology of the model system. We analyze the possible mechanisms by which this virus induces demyelination and persists in the nervous system. Finally, we provide a hypothesis that the specificity of primary white matter destruction in the TMEV model depends on immune-sensitized cells which interact with viral antigen plus major histocompatibility complex (MHC) antigens on the surfaces of oligodendrocytes or myelin sheaths.     

Is Theiler's murine encephalomyelitis virus infection of mice an autoimmune disease?

Viruses can initiate disease by many different means. Direct viral, immune mediated and host factors all play important parts. Molecular mimicry or having cross-reacting determinants that result in immune responses which have the potential to cause damage can be incorporated into this framework. Here, autoimmune responses generated by virus infection have been presented in relation to these other parameters. The cross-reacting immune response originally generated by virus would have to be directed toward or involve a disease inducing site such as an EAE (encephalitogenic), thyroiditis, or diabetogenic site. If the cross-reaction took place at a nondisease inducing site, the ensuring immune response may result in the production of autoantibodies, however no disease would occur. In other systems autoantibodies can potentiate an ongoing inflammatory response. This may be the case that is described here with Theiler's murine encephalomyelitis virus infection. Lastly, viruses having common determinants with MHC determinants may modify immune responses leading to immunosuppression and allowing virus to persist. In addition, similar determinants may lead to disease by an alternative route. For example, we have described a region of human cytomegalovirus that has a common determinant with HLA DR beta chain. This region is associated with diabetes in humans (Todd et al. 1988). Thus, many factors are involved in the outcome of disease induction by viruses of which autoimmunity is one.     

Altered cell growth and morphology in a BHK-21 cell mutant that lacks a receptor for Theiler's murine encephalomyelitis virus

The receptor for Theiler's murine encephalomyelitis virus (TMEV) remains unknown. In vitro, BHK-21 cells are permissive to infection by TMEV. Selecting mutants of BHK-21 cells produced a cell line (BHKR-) resistant to infection by TMEV. Viral persistence was ruled out by immunofluorescent staining for viral antigens. BHKR- cells were nonpermissive to infection even at high multiplicities of infection. In contrast, cells were able to support one round of virus replication when transfected with infectious TMEV RNA. Binding studies indicated that TMEV was unable to attach to these cells. These data are consistent with the BHKR- cells lacking a receptor for TMEV. Interestingly, BHKR- cells were larger in size and had a significant lag in growth after subculture versus BHK-21 cells. This suggests that the TMEV receptor on BHK-21 cells could play an important role in cell growth and morphology under physiologic conditions. BHKR- cells should facilitate the search for TMEV receptors.     

Modulation of epidermal Langerhans' cell frequency by tumour necrosis factor-alpha.

During the induction phase of skin sensitization, dendritic cells (DC), many of which bear high levels of antigen, accumulate in lymph nodes draining the site of exposure. These DC derive from epidermal Langerhans' cells (LC) which are induced to migrate from the skin, via the afferent lymphatics, to lymph nodes. We demonstrated previously that intradermal exposure of mice to homologous, but not human, recombinant tumour necrosis factor-alpha (TNF-alpha) also causes an accumulation of DC in draining nodes, the implication being that the local production of this cytokine by epidermal cells provides one stimulus for LC migration. In the present study we have examined the influence of dermal TNF-alpha on the frequency of LC within the epidermis. Intradermal injection of mice with 25 ng or greater murine recombinant TNF-alpha caused a significant reduction in LC numbers within 30 min of exposure. The same treatment did not influence the frequency of Thy-1+ epidermal DC. The density of LC was unaffected by the same amount of human TNF-alpha of comparable specific activity or by murine granulocyte-macrophage colony-stimulating factor (GM-CSF). These data provide additional evidence that TNF-alpha provides an important signal for the migration of LC from the epidermis.     

Hepatic expression of tumour necrosis factor-alpha in chronic hepatitis B virus infection.

AIM--To determine the hepatic expression of tumour necrosis factor-alpha (TNF alpha) in patients with chronic hepatitis B virus (HBV) infection. METHODS--Frozen liver biopsy sections from 19 patients with chronic HBV infection were studied, 12 of whom were HBeAg positive and 10 serum HBV DNA positive. Hepatic expression of TNF alpha was determined using immunohistochemistry. RESULTS--Only infiltrating mononuclear cells showed immunoreactive staining for TNF alpha (median 2, range 0-3; n = 19) which appeared as diffuse positive staining material in the cytoplasm. Patients with active liver disease, assessed histologically and biochemically, had a higher level of expression, both in the number of TNF alpha positive cells and the proportion of TNF alpha positive infiltrating mononuclear cells. There was no correlation between the expression of TNF alpha and serological parameters of viral infection (HBeAg and HBV DNA status and HBV DNA concentrations). CONCLUSION--Hepatic expression of TNF alpha is increased in chronic HBV infection and is related to the activity of liver disease and not to the level of HBV replication.     

Glucocorticoid exposure alters the pathogenesis of Theiler's murine encephalomyelitis virus during acute infection

Previous research has shown that chronic restraint stress exacerbates Theiler's virus infection, a murine model for CNS inflammation and multiple sclerosis. The current set of experiments was designed to evaluate the potential role of glucocorticoids in the deleterious effects of restraint stress on acute CNS inflammatory disease. Exposure to chronic restraint stress resulted in elevated levels of corticosterone as well as increased clinical scores and weight loss (Experiment 1). In addition, corticosterone administration alone exacerbated behavioral signs of TMEV-induced sickness (i.e. decreased body weight, increased symptoms of encephalitis, and increased mortality) and reduced inflammation in the CNS (Experiment 2). Infected subjects receiving exogenous corticosterone showed exacerbation of acute phase measures of sickness and severe mortality as well as decreased viral clearance from CNS (Experiment 3). These findings indicate that corticosterone exposure alone is sufficient to exacerbate acute CNS inflammatory disease.     

Split tolerance of Thl and Th2 cells in tolerance to Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) produces a chronic, inflammatory demyelinating disease in susceptible mouse strains that is used as a model for multiple sclerosis. Because disease susceptibility correlates temporally with the development of virus-specific delayed-type hypersensitivity (DTH) responses, we studied methods and mechanisms by which virus-specific DTH could be specifically inhibited. The intravenous injection of UV-inactivated TMEV coupled to syngeneic splenocytes via a carbodiimide linkage (TMEV-SP), prior to immunization, induced a significant degree of tolerance in virus-specific helper (T_h) cells as determined by decreased DTH and T cell proliferative responses, and decreased interleukin (IL)-2 and interferon (IFN)-Y protein and mRNA levels. In contrast to the reduced levels of T_hl-specific lymphokine mRNA levels, IL-4-specific mRNA levels in response to virus stimulation were not affected in tolerant mice. Surprisingly, the total anti-TMEV antibody response in DTH tolerant mice was enhanced 20-100-fold over sham-tolerized controls and was composed of reduced levels of anti-virus IgG2a, but dramatically increased levels of anti-virus IgGl. The “split-tolerance” was antigen specific, dependent on the concentrations of TMEV and carbodiimide used in the coupling procedure, and varied with the number of coupled syngeneic splenocy tes administered. The fixative effects of carbodiimide on antigen-presenting function were necessary for the induction of DTH tolerance with TMEV-SP, since intravenous administration of virus coupled to splenocytes via a biotin-avidin linkage led to enhanced virus-specific antibody responses, but was unable to inhibit DTH unless concomitantly fixed with carbodiimide. Collectively, the data indicate that T_hl cells (mediating DTH, IL-2 and IFN-γ production, and helper function for IgG2a production) were specifically anergized, with concomitant stimulation of T_h2 cells (producing IL-4 and mediating helper function for IgGl antibody production).     

Serological evidence that Mus musculus is the natural host of Theiler's murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) infection is maintained in mouse colonies by fecal-oral spread (with no apparent role for persistent central nervous system infection) from an acutely infected animal to another. Therefore, serological methods offer the principal way to assess infection in mice and related rodent populations. Infection of mouse colonies with TMEV appears to be worldwide, yet no systematic serologic studies have been reported. In this study, enzyme-linked immunoassay and neutralization analysis of sera from feral Mus musculus obtained from four locations in the United States and one in Russia revealed antibodies to purified TMEV and two linear viral peptide epitopes in more than 50% of the sera derived from the five different locations. A similar analysis of sera from 26 species of related rodents trapped at multiple locations in North America and Europe indicated the presence of anti-TMEV antibodies only in a small proportion of water and bank voles that belong to a different subfamily. These results indicate that Mus musculus is the natural host of TMEV.     

Association of L* protein of Theiler's murine encephalomyelitis virus with microtubules in infected cells

We used an antibody raised against a synthetic peptide corresponding to amino acid residues 70-88 for characterizing the L* protein of Theiler's murine encephalomyelitis virus (TMEV), which is only synthesized in DA subgroup strains from an alternative AUG and is out of frame with the viral polyprotein; evidence suggests that L* protein is critical to viral persistence, demyelination, and growth in murine macrophage cell lines. It was synthesized with kinetics similar to that of other viral proteins, although less in amount. After synthesis, it remained stable in the cytoplasm and was not incorporated into virions. Immunofluorescent staining and immunoblotting of microtubule preparations demonstrated that it is associated with microtubules. Expression of L* protein also demonstrated that the 5' one third of the coding region may be responsible for the association. The association of L* protein with microtubules may be important in the disease-inducing and in vitro characters of L* protein.     

Anti-adhesion molecule therapy in Theiler's murine encephalomyelitis virus-induced demyelinating disease

We examined the role of leukocyte function-associated antigen (LFA)-1 and its counter-receptor intercellular adhesion molecule (ICAM)-1, one of the most important pairs of adhesion molecules, in the development of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Immunohistochemical study showed hyper-expression of ICAM-1 on vascular endothelial cells and expression of LFA-1 on mononuclear infiltrating cells in the spinal cords of TMEV-infected mice. Treatment with mAb to ICAM-1 and/or LFA-1 molecules resulted in significant suppression of the development of demyelinating disease, both clinically and histologically, with down-regulation in the CNS of the respective adhesion molecules after treatment. In mice treated with these mAb, the specific delayed-type hypersensitivity and T cell proliferative responses for TMEV were decreased. The production of tumor necrosis factor-alpha and IFN-gamma in spleen cells was also decreased, but IL-4 production remained unchanged. These data suggest that ICAM-1/LFA-1 interaction is critically involved in the pathogenesis of TMEV-IDD and that antibodies to these adhesion molecules could be a novel therapeutic approach to the treatment of demyelinating diseases such as human multiple sclerosis.      

Cachexia and tumour necrosis factor-alpha in cytomegalovirus infection.

Although cachexia is a common feature of cytomegalovirus infection, little is known about its cause. To explore any contributory role that tumour necrosis factor-alpha (TNF) might have the serum concentrations of TNF in eight patients who developed CMV disease after liver transplantation were investigated. All patients exhibited pronounced and long lasting increases in TNF serum concentrations. Increased endogenous TNF concentrations were associated with weight loss and anorexia. In contrast, liver transplant recipients without CMV disease showed no weight loss.