Characterization of Plaque-Sized Variants of Daniel's (DA) Strain in Theiler's Virus-Induced Epilepsy.

Epilepsy is a complex neurological disease characterized by recurrent seizures. Patients with viral encephalitis have a 16-fold increased risk of developing epilepsy, and this risk can persist for about 15 years after the occurrence of initial viral infection. Theiler's murine encephalomyelitis virus (TMEV) infection induces a well-characterized experimental model of epilepsy in C57BL/6 mice. In response to intracerebral (I.C.) injection of Daniel's (DA) strain of TMEV, there is vigorous immune response, which is detrimental to neurons and contributes to acute seizures, rendering mice susceptible to epilepsy. A comparative in vivo challenge study with either one of the two variants of the DA strain, small (DA-DS) or large (DA-CL) plaque forming variants, revealed differences in the diseases they induced in C57BL/6 mice. Compared to DA-CL-, DA-DS-infected mice exhibited significantly more seizures, higher clinical scores, neuroinflammation, and neuronal damage (mainly in the CA1-CA2 regions of hippocampus). Moreover, the brains of DA-DS infected mice contained approximately five-fold higher virus than those of DA-CL infected mice. A sequence comparison of the DA-CL and DA-DS genome sequences showed mutations in the leader (L) and L* proteins of DA-CL variant, which may be the cause of attenuating phenotype of DA-CL variant in the C57BL/6 mouse model of epilepsy.

Social disruption alters pain and cognition in an animal model of multiple sclerosis.

Although pain and cognitive deficits are widespread and debilitating symptoms of multiple sclerosis (MS), they remain poorly understood. Theiler's murine encephalomyelitis virus (TMEV) infection is an animal model of MS where disease course is exacerbated by prior stressors. Here chronic infection coupled with prior social stress increased pain behavior and impaired hippocampal-dependent memory consolidation during the demyelinating phase of disease in SJL mice. These results suggest that the TMEV model may be useful in investigating pain and cognitive impairments in MS. However, in contrast to prior Balb/cJ studies, stress failed to consistently alter behavioral and physiological indicators of disease course.

Neonatal experience interacts with adult social stress to alter acute and chronic Theiler's virus infection.

Previous research has shown that neonatal handling has prolonged protective effects associated with stress resilience and aging, yet little is known about its effect on stress-induced modulation of infectious disease. We have previously demonstrated that social disruption stress exacerbates the acute and chronic phases of the disease when applied prior to Theiler's virus infection (PRE-SDR) whereas it attenuates disease severity when applied concurrently with infection (CON-SDR). Here, we asked whether neonatal handling would protect adult mice from the detrimental effects of PRE-SDR and attenuate the protective effects of CON-SDR on Theiler's virus infection. As expected, handling alone decreased IL-6 and corticosterone levels, protected the non-stressed adult mice from motor impairment throughout infection and reduced antibodies to myelin components (PLP, MBP) during the autoimmune phase of disease. In contrast, neonatal handling X PRE/CON-SDR elevated IL-6 and reduced corticosterone as well as increased motor impairment during the acute phase of the infection. Neonatal handling X PRE/CON-SDR continued to exacerbate motor impairment during the chronic phase, whereas only neonatal handling X PRE-SDR increased in antibodies to PLP, MOG, MBP and TMEV. Together, these results imply that while handling reduced the severity of later Theiler's virus infection in non-stressed mice, brief handling may not be protective when paired with later social stress.

Social disruption induced priming of CNS inflammatory response to Theiler's virus is dependent upon stress induced IL-6 release.

Chronic social disruption stress (SDR) exacerbates acute and chronic phase Theiler's murine encephalomyelitis virus (TMEV) infection, a mouse model of multiple sclerosis. However, the precise mechanism by which this occurs remains unknown. The present study suggests that SDR exacerbates TMEV disease course by priming virus-induced neuroinflammation. It was demonstrated that IL-1ß mRNA expression increases following acute SDR; however, IL-6 mRNA expression, but not IL-1ß, is upregulated in response to chronic SDR. Furthermore, this study demonstrated SDR prior to infection increases infection related central IL-6 and IL-1ß mRNA expression, and administration of IL-6 neutralizing antibody during SDR reverses this increase in neuroinflammation.

Neonatal maternal separation alters immune, endocrine, and behavioral responses to acute Theiler's virus infection in adult mice.

Previous studies have established a link between adverse early life events and subsequent disease vulnerability. The present study assessed the long-term effects of neonatal maternal separation on the response to Theiler's murine encephalomyelitis virus infection, a model of multiple sclerosis. Balb/cJ mouse pups were separated from their dam for 180-min/day (180-min MS), 15-min/day (15-min MS), or left undisturbed from postnatal days 2-14. During adolescence, mice were infected with Theiler's virus and sacrificed at days 14, 21, or 35 post-infection. Prolonged 180-min MS increased viral load and delayed viral clearance in the spinal cords of males and females, whereas brief 15-min MS increased the rate of viral clearance in females. The 15-min and 180-min MS mice exhibited blunted corticosterone responses during infection, suggesting that reduced HPA sensitivity may have altered the immune response to infection. These findings demonstrate that early life events alter vulnerability to CNS infection later in life. Therefore, this model could be used to study gene-environment interactions that contribute to individual differences in susceptibility to infectious and autoimmune diseases of the CNS.

A comparison of the neurotropism of Theiler's virus and poliovirus in CBA mice.

Theiler's murine encephalomyelitis virus (TMEV) and poliovirus infect the central nervous system (CNS) and cause neurological damage. The exact route by which TMEV and polioviruses enter the CNS remains, for the most part, unknown, although the neural and/or the hematogenous pathway have both been postulated. To explore these hypotheses, this research focuses on both the site of entry and the pathway used to invade the CNS. Following different inoculation sites of the GDVII strain of Theiler's virus or Lansing Type 2 poliovirus in CBA mice, the incidence of paralysis and/or encephalitis was evaluated on the basis of clinical signs and histopathology. The forms of paralysis displayed corresponded to the site of viral inoculation. Following intramuscular (i.m.), intraperitoneal (i.p.), and footpad routes of injection, bilateral and or contralateral paralyses were observed for both TMEV and poliovirus. In mice injected intratongue and in the hypoglossal nerve, tongue paralysis or paralysis of the forelimb, which progressed to bilateral forelimb paralysis, was observed, additionally the penis of most infected males was protruded. Intracranial (i.c.) injections with type II poliovirus strain resulted in forelimb paralysis. Intravenous (i.v.), injections with TMEV also resulted in forelimb paralysis. Thus Lansing Type II poliovirus and TMEV infections of CBA mice, result in similar incidence of paralysis and histopathological findings.

Restraint stress decreases virus-induced pro-inflammatory cytokine mRNA expression during acute Theiler's virus infection.

Stressful life events have been associated with the onset and/or exacerbation of multiple sclerosis (MS). Our previous studies have indicated that restraint stress (RS) reduces inflammation and virus-induced chemokine expression in the Theiler's virus-induced demyelination (TVID) model of MS. Here we report that RS significantly reduced the virus-induced interferon-gamma mRNA levels in the brain. Additionally, mRNA levels of lymphotoxin-beta, tumor necrosis factor-alpha, and interferon-gamma in the brain were negatively correlated with viral titers in the brain. These results indicated an immunosuppressive effect of stress during early TVID causing impaired viral clearance, which may be a potential exacerbating factor for later demyelination.

Alterations in chemokine expression following Theiler's virus infection and restraint stress.

Restraint stress (RS) applied to mice during acute infection with Theiler's virus causes corticosterone-induced immunosuppression. This effect was further investigated by measuring chemokine changes in the spleen and central nervous system (CNS) using an RNase Protection Assay. mRNAs for lymphotactin (Ltn), interferon-induced protein-10 (IP-10), MIP-1 beta, monocyte chemoattractant protein-1 (MCP-1) and TCA-3 were detected in the spleen at day 2 pi, but not in the brain of CBA mice infected with Theiler's virus. Ltn, IP-10 and RANTES were elevated in both the spleen and the brain at day 7 pi, and were significantly decreased by RS in the brain. RS also resulted in decreased inflammation within the CNS.

Social stress alters the severity of acute Theiler's virus infection.

Our laboratory has previously shown that restraint stress resulted in decreased Theiler's virus-induced CNS inflammation, while exacerbating illness behaviors during the acute phase of disease. In contrast, social disruption stress (SDR) applied prior to infection led to the development of glucocorticoid (GC) resistance, and these animals developed more severe disease course, with increased inflammation. However, when SDR was applied concurrent with infection, GC resistance fails to develop, disease course is less severe and inflammation was moderate. These results suggest that the effects of SDR on Theiler's virus infection are dependent upon the timing of SDR application in relation to infection.

The effects of restraint stress on the neuropathogenesis of Theiler's virus infection II: NK cell function and cytokine levels in acute disease.

Psychological stress is thought to play an important role in multiple sclerosis. We have been investigating the role of restraint stress in Theiler's virus infection in mice as a model for multiple sclerosis. We have previously determined that restraint stressed CBA mice had higher levels of mortality following infection with Theiler's virus. We proposed that this was due to high levels of stress-induced corticosterone, which resulted in decreased numbers of circulating lymphocytes, decreased inflammatory cell infiltrates into the brain and consequently decreased viral clearance from the central nervous system (CNS). The effect of restraint stress on the innate immune response to Theiler's virus is further investigated in the current study. Restraint stressed mice developed clinical signs of encephalitis, thymic atrophy, and adrenal hypertrophy. Decreased numbers of circulating lymphocytes and increased numbers of neutrophils were observed in the stressed mice. Stressed mice also had lower numbers of spleen cells which correlated with the decreased numbers of lymphocytes in circulation. Restraint stress caused elevations in serum tumor necrosis alpha (TNF-alpha). Virus-induced natural killer cell (NK) cytotoxic activity was significantly reduced in restrained mice at one day post infection which may account for the reduced viral clearance from the CNS. These data suggest that stress-induced immunosuppression of cytolytic NK cell activity may account in part for the reduced ability to clear virus from the CNS and increased mortality observed in this model.

Ovine IFN-tau modulates the expression of MHC antigens on murine cerebrovascular endothelial cells and inhibits replication of Theiler's virus.

Interferon-beta (IFN-beta) has been used successfully to treat patients with relapsing-remitting multiple sclerosis (MS). IFN-tau is a new class of type I IFN that is secreted by the trophoblast and is the signal for maternal recognition of pregnancy in sheep. IFN-tau has potent immunosuppressive and antiviral activities similar to other type I IFN but is less cytotoxic than IFN-alpha/beta. The current investigation concerns the effect of recombinant ovine IFN-tau (rOvIFN-tau) on the modulation of MHC class I and II expression on cloned mouse cerebrovascular endothelial (CVE) cells. IFN-tau induced tyrosine phosphorylation of Stat1 and upregulated the expression of MHC class I on CVE. One proposed action by which type I IFN reduce the relapse rate in MS is via interference with IFN-gamma-induced MHC class II expression. IFN-tau was shown to downregulate IFN-gamma-induced MHC class II expression on CVE and, hence, may be of potential therapeutic value in downregulating inflammation in the central nervous system (CNS). IFN-tau did not upregulate the expression of MHC class II on CVE. IFN-tau also inhibited the replication of Theiler's virus in CVE. These in vitro results suggest that IFN-tau may be of therapeutic value in the treatment of virus-induced demyelinating disease.

The effects of restraint stress on the neuropathogenesis of Theiler's virus infection: I. Acute disease.

Restraint stress was found to have a profound effect on the acute phase of Theiler's virus infection. Increased mortality rates were observed in restrained CBA mice infected with the BeAn strain of Theiler's virus. In addition, restrained mice developed higher CNS viral titers than infected/nonrestrained mice. Thymic atrophy was observed in both infected and uninfected restrained mice. Decreased microgliosis, perivascular cuffing, and astrocytosis were observed in restrained mice compared to nonrestrained infected mice at 7 days postinfection. Restraint-stressed mice also developed decreased numbers of lymphocytes and increased numbers of neutrophils in the blood. The mechanism proposed for these alterations involves stress-induced corticosterone, which causes immunosuppression, decreased trafficking of inflammatory cells in the CNS, and, consequently, increased viral replication.

The use of medication for relapse prevention in substance dependence disorders.

Substance abuse is a significant problem in itself and can greatly complicate the symptomatology and treatment of comorbid psychiatric disorders. In the article, the authors review literature concerning the use of medication to prevent relapse to substance abuse or decrease substance use. Five different general strategies are employed for this purpose: 1) use of a drug with pharmacological properties similar to the substance of concern (i.e., agonist or substitution therapy); 2) use of a receptor antagonist to block or lessen the effects of the substance of concern; 3) use of a medication that produces a conditioned aversive reaction to the substance of concern; 4) use of a medication to reduce the reinforcing properties of the substance of concern; and 5) use of a substance to increase the metabolism or clearance of the substance of concern from the body. The authors review pharmacological treatments that have been studied for the treatment of dependence on the following types of substances: alcohol, sedative-hypnotics, opioids, stimulants, nicotine, hallucinogens, cannabis, inhalants, anabolic steroids, phencyclidine, and designer drugs. The article ends with a brief discussion of the importance of including psychosocial and behavioral interventions in any substance abuse treatment program.

Suicidal ideation and attempts: a longitudinal investigation of children of depressed and well mothers.

OBJECTIVE: This study uses a prospective longitudinal design to examine suicidality (ideation, plans, attempts, and completions) in children and adolescents, to compare suicidality in the offspring of depressed and well mothers, and to identify correlates and predictors of suicidality. METHOD: Two children (n = 192) from each of the families in an ongoing longitudinal study of the offspring of mothers with major depressive disorder (n = 42), with bipolar disorder (n = 26), or without past or current psychiatric diagnosis (n = 30) were studied. Assessment of suicidality, based on diagnostic interviews, was made when the younger of the sibling pairs were approximately 6, 9, and 14 years of age and older siblings were approximately 6, 9, 13, and 18 years of age. RESULTS: Children of depressed mothers were more likely to report suicidal thoughts or behaviors than were children of well mothers (particularly the older sibling cohort). Developmental trajectories of suicidality differed for offspring of mothers with major depressive disorder and bipolar disorder. Links were found between lifetime reports of suicidality and the adolescent's mood problems (e.g., hypomanic behavior), coping strategies, and parental rejection. Also, child's and mother's suicidality were related. CONCLUSIONS: These findings have implications for planning interventions targeted at preventing suicide in youth.

Nitroaromatic munition compounds: environmental effects and screening values.

Available data on the occurrence, transport, transformation, and toxicity of eight nitroaromatic munition compounds and their degradation products, TNT, TNB, DNB, DNA, 2-ADNT, RDX, HMX, and tetryl were used to identify potential fate in the environment and to calculate screening benchmarks or safe environmental levels for aquatic and terrestrial organisms. Results of monitoring studies revealed that some of these compounds persist at sites where they were produced or processed. Most of the compounds are present in soil, sediment, and surface water or groundwater at military sites. Soil adsorption coefficients indicate that these chemicals are only moderately adsorbed to soil and may leach to groundwater. Most of these compounds are transformed by abiotic or biotic mechanisms in environmental media. Primary transformation mechanisms involve photolysis (TNT, RDX, HMX, tetryl), hydrolysis (tetryl), and microbial degradation (TNT, TNB, DNB, DNA, 2-ADNT, and HMX). Microbial degradation for both nitro and nitramine aromatic compounds involves rapid reduction of nitro groups to amino groups, but further metabolism is slow. With the exception of DNB, complete mineralization did not usually occur under the conditions of the studies. RDX was resistant to microbial degradation. Available ecotoxicological data on acute and chronic studies with freshwater fish and invertebrates were summarized, and water quality criteria or ecotoxicological screening benchmarks were developed. Depending on the available data, criteria/benchmarks were calculated according to USEPA Tier I or Tier II guidelines. The munitions chemicals are moderately to highly toxic to freshwater organisms, with chronic screening values < 1 mg/L. For some chemicals, these low values are caused by inherent toxicity; in other cases, they result from the conservative methods used in the absence of data. For nonionic organic munitions chemicals, sediment quality benchmarks were calculated (based on Kow values and the final chronic value) according to USEPA guidelines. Available data indicate that none of the compounds is expected to bioconcentrate. In the same manner in which reference doses for humans are based on studies with laboratory animals, reference doses or screening benchmarks for wildlife may also be calculated by extrapolation among mammalian species. Chronic NOAELs for the compounds of interest were determined from available laboratory studies. Endpoints selected for wildlife species were those that diminish population growth or survival. Equivalent NOAELs for wildlife were calculated by scaling the test data on the basis of differences in body weight. Data on food and water intake for seven selected wildlife species--short-tailed shrew, white-footed mouse, meadow vole, cottontail rabbit, mink, red fox, and whitetail deer--were used to calculate NOAELs for oral intake. In the case of TNB, a comparison of toxicity data from studies conducted with both the white-footed mouse and the laboratory rat indicates that the white-footed mouse may be more resistant to the toxic effects of chemicals than the laboratory rat and may further indicate the lesser sensitivity of wildlife species to chemical insult. Chronic NOAEL values for the test species based on the laboratory studies indicate that, by the oral route of exposure, TNB and RDX are not highly toxic to mammalian species. However, as seen with TNB, values are less conservative when chronic studies are available or when studies were conducted with wildlife species. Insufficient data were located to calculate NOAELs for avian species. In the absence of criteria or guidelines for terrestrial plants, invertebrates, and soil heterotrophic processes, LOECs were used as screening benchmarks for effect levels in the environment. In most cases, too few data were available to derive a screening benchmark or to have a high degree of confidence in the benchmarks that were derived. (ABSTRACT TRUNCATED)

Investigation of the role of delayed-type-hypersensitivity responses to myelin in the pathogenesis of Theiler's virus-induced demyelinating disease.

The contribution of autoimmune responses to the pathogenesis of Theiler's virus-induced demyelinating disease was investigated. Delayed-type hypersensitivity responses to myelin were examined in both symptomatic and asymptomatic mice at different times post-infection, in order to determine whether autoreactivity correlates with the development of demyelination. The results indicate that although autoimmune responses probably do not play a major role in the initiation of demyelination at early times post-infection, autoreactivity to myelin antigens dose eventually develop in symptomatic animals, perhaps through the mechanism of epitope spreading. Autoimmunity to myelin components is therefore an additional factor that may contribute to lesion progression in chronically diseased animals.

Cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells.

In a murine model of rickettsial disease in which, as in human rickettsioses, endothelial cells are the major target of infection, depletion of IFN-gamma or TNF-alpha converts a sublethal infection into a uniformly fatal disease with overwhelming rickettsial growth and decreased nitric oxide (NO) synthesis. The kinetics of NO production and rickettsial survival and growth were examined on Days 1, 2, and 3 after inoculation of endothelial cells with Rickettsia conorii under four different experimental conditions: (a) no cytokine treatment, (b) treatment with IFN-gamma and TNF-alpha, (c) treatment with cytokines and NG monomethyl-L-arginine, a competitive inhibitor of NO synthesis, and (d) treatment with sodium nitroprusside, a source of NO. Endothelial cells were examined for the presence of inducible nitric oxide synthase mRNA by specific reverse transcriptase-PCR after stimulation with IFN-gamma and TNF-alpha. Cytokine-stimulated and unstimulated rickettsiae-infected endothelial cells were examined by electron microscopy to observe the cellular and rickettsial events. Transformed and diploid mouse endothelial cells stimulated by the combination of recombinant murine IFN-gamma and TNF-alpha killed intracellular Rickettsia conorii by a mechanism that required the synthesis of NO. The antirickettsial effect and NO synthesis were inhibited by treatment of endothelial cells with NG monomethyl-L-arginine. Addition of nitroprusside, which released NO, also exerted a strong antirickettsial effect in the absence of IFN-gamma and TNF-alpha. Endothelial inducible nitric oxide synthase mRNA was detected 4 hours after cytokine stimulation, increased substantially at 8 hours, and decreased to low levels by 72 hours. Ultrastructural evaluation revealed that endothelial cells effected rickettsial killing in association with autophagy. Double membranes of endothelial cell granular endoplasmic reticulum surrounded rickettsiae, which were also observed being destroyed within phagolysosomes. This study demonstrated for the first time that endothelial cells are capable of killing rickettsiae. When stimulated by the combination of IFN-gamma and TNF-alpha, mouse endothelial cells kill Rickettsia conorii by an NO-dependent mechanism. Within the endothelium, NO exerts a rickettsicidal effect.

Characteristics of cloned cerebrovascular endothelial cells following infection with Theiler's virus. I. Acute infection.

The present study describes the replication of Theiler's virus in cloned cerebrovascular endothelial cells (CVE) isolated from strains of mice that are either susceptible or resistant to Theiler's virus-induced demyelination (TVID). CVE isolated from all strains of mice were equally permissive to Theiler's virus infection. Interferon-gamma and tumor necrosis factor-alpha were found to inhibit the replication of Theiler's virus in CVE. A correlation between susceptibility to demyelination and the ability of Theiler's virus to induce MHC Class I on CVE was demonstrated.