The animal model of feline immunodeficiency virus (FIV) infection of cats was used to dissect the pathogenic role of microglia within the first 6 months of infection. Applying real-time PCR, microglia-associated FIV replication was first detectable at 14 days past inoculation (dpi) and remained at elevated levels throughout the whole observation period. In contrast, FIV RNA levels within paired serum samples declined again after an initial peak between 14 dpi and 28 dpi. Concomitant with the onset of viral reproduction, microglia transiently upregulated expression of MHC class I and class II molecules. Virus-induced microglial activation was followed by a mild infiltration of peripheral leukocytes into the CNS parenchyma. The presented data suggest that microglia is infected by FIV very early after peripheral entry of the virus. Virus replicating microglia withstands eradication by brain-infiltrating leukocytes resulting in formation of a brain-resident virus reservoir, which probably cannot be cleared by peripheral chemotherapy.

It is widely accepted that human immunodeficiency virus (HIV) invades the central nervous system (CNS) shortly after peripheral infection to establish a persistent infection of tissue-resident microglial cells. To what extent this early CNS infection is of pathogenic relevance is a matter of discussion. It is conceivable, however, that infected microglia releases virus variants of enhanced neurotropism and/or neurovirulence compared to peripheral isolates. Moreover, microglial variants may exhibit high resistance to antiviral therapeutics that poorly penetrate into brain tissue. The molecular basis of these biological properties is suspected to be associated with specific sequences in the viral env gene, particularly within the V3 loop. Therefore, we analyzed in the animal model of feline immunodeficiency virus (FIV) infection of cats lentiviral V3 sequences in highly purified microglial cells and blood from acutely infected animals. Compared to the inoculated virus, nucleotide sequence alterations in serum samples were rarely detectable, if at all. In contrast, up to 19 nucleotide exchanges could be identified within FIV V3 from microglia, resulting in a mutation frequency of up to 14.5% with respect to the deduced amino acid sequence. These findings suggest selection of specific virus variants by brain-resident target cells that might have implications for antiretroviral drug design.

Mouse models of systemic and gastrointestinal infection with the yeast Candida albicans were used to investigate the ability of a commercial mannan antigen enzyme immunoassay and a commercial (1-->3) beta-D-glucan limulus assay to detect systemic infection and to differentiate between colonization and infection. Both assays were positive in all i.v. infected mice and negative in all uninfected control mice. In gastrointestinal infection both tests were positive whenever organ cultures were positive. In colonized mice with no detectable dissemination, there were mostly negative results with the glucan assay whereas the mannan assay was positive or intermediate in all colonized mice. Therefore, in the mouse model used, glucan detection appeared to be superior for differentiation between colonization and dissemination.

Expression of CD1 by microglia, macrophages and T cells was investigated ex vivo. In the healthy central nervous system (CNS), resident microglia, macrophages and T cells express levels of CD1 significantly lower than that expressed by splenic macrophages and T cells. During experimental autoimmune encephalomyelitis (EAE), CD1 expression by microglia and the number of CD1+ microglia increase. Macrophages and T cells strongly upregulate CD1 expression in the CNS, but not in the spleen. Whereas the function of CD1 expressed by T cells remains unclear, the expression by microglia and macrophages provides the CNS with a (glyco)lipidic-presenting molecule in an inflammatory and demyelinating environment.

ABSTRACT Intravenous infection of cats with feline immunodeficiency virus was used as a model system to study activation of virus replication in brain-resident microglial cells in vitro. Virus release by ramified microglial cells isolated from subclinically infected animals was detectable in cell-free tissue culture supernatant only by reverse transcription and nested PCR of gag-specific RNA sequences and not by virion-associated reverse transcriptase activity. In contrast, cocultivation of in vivo-infected microglial cells with mitogen-activated peripheral blood mononuclear cells (PBMC) regularly allows detection of high virus yields in cell-free tissue culture fluid. Besides uptake and multiplication of microglia-derived virus in PBMC, release of virus from microglia is stimulated by cell contact with PBMC. The data suggest that T lymphocytes patrolling the central nervous system could reactivate the semilatent state of lentiviruses in microglial cells in the course of clinically silent central nervous system infection.

Infection of microglial cells by the human immunodeficiency virus (HIV) is supposed to play an important role in the pathogenesis of AIDS-related central nervous system (CNS) complications. So far, however, experimental data about interactions between HIV and ramified microglia from the adult CNS were only occasionally reported, making it difficult to understand the exact nature of pathogenic events contributing to HIV-encephalopathy. Therefore, we used the animal model of feline immunodeficiency virus (FIV) infection of domestic cats to establish an experimental system which is suitable for studying the relationships between an immunodeficiency virus and the mature ramified microglia of the central nervous system. By means of density gradient centrifugation approximately 95% pure microglial cells could be isolated from adult feline brain that were characterized by their CD45(low) phenotype. Resident microglia extracted from the CNS of experimentally infected cats harbored FIV-specific DNA and cocultivation with mitogen-activated, but uninfected peripheral blood mononuclear cells (PBMC) resulted in recovery of high-titered infectious virus. Double labeling of brain cell monocultures explanted from persistently infected animals for both microglia and FIV markers disclosed less than 1% of viral antigen expressing microglial cells. This suggests that during the subclinical phase of the infection only a small number of brain-resident macrophages are productively infected. However, interaction of FIV-infected microglia and inflammatory lymphocytes may promote viral replication, thus supporting viral spread in brain tissue.

We compared the contribution of T cell memory to the clearance of the fungus Candida albicans from the liver, kidneys and brain of Balb/c mice in a model of secondary systemic infection. In secondary infection, the fungi were more rapidly eliminated from the liver and kidneys than during primary infection. This was most pronounced in the liver where the fungi were eliminated at day 14 of infection. In contrast, in the brain, cultivable yeasts were still detectable 35 days after infection. Although both CD4(+) and CD8(+) cells could be detected in the brain with immunohistology, these cells appeared later in infection and in lower numbers than in the liver, and there were no significant differences in the numbers of T cells detected in the brain between primary and secondary infection. In contrast to the liver and the kidneys where an effect of T cells on the fungal load could be demonstrated, depletion of neither CD4(+) nor CD8(+) nor Thy-1.2(+) cells resulted in a significant increase of the amount of fungi in the brain above levels measured in secondarily infected mice treated with irrelevant antibodies. We conclude that the contribution of CD4(+) and CD8(+) cells to the clearance of C. albicans in secondary infection is organ-dependent and that T cell memory is inefficient in the brain.

Clinical course, recovery of infectious virus from brain tissue and histopathology of the central nervous system were examined in g-irradiated Lewis rats reconstituted by naive lymphocytes before infection with coronavirus MHV-4 (strain JHM). Up to 9 days past infection, no differences were seen between immunologically competent and immuno-deficient animals in terms of onset and progression of neurological disease. However, in the latter animals neurological symptoms were dominated by signs of encephalitis instead of paralytic disease as usually seen in immunocompetent animals. Nevertheless, despite high titers of infectious virus in the CNS of immunodeficient animals only mild histopathological changes were noticeable. In contrast, infectious virus in the CNS of immunologically competent animals was below the detection limit of the assay. Paralytic disease and tissue destruction were T lymphocyte mediated because g-irradiated rats that were reconstituted by CD4 + or CD8 + T lymphocyte enriched cells in the absence of B lymphocytes revealed an earlier onset of clinical symptoms and a more rapid deterioration of their clinical state compared to fully competent animals. Whereas in CD4 + T cell reconsituted animals infectious virus was moderately reduced and tissue destruction as well as inflammatory changes in the CNS were focal, in CD8 + T cell reconstituted animals vacuolizing white matter inflammation was diffuse without reduction of infectious virus in brain tissue. From the presented data we conclude that in the acute stage of JHMV-induced encephalomyelitis of Lewis rats: (i) tissue destruction and paralytic clinical symptomatology are mainly T cell-mediated; (ii) CD4 + T lymphocytes can directly contribute to reduction of viral load in the brain and (iii) only coordinated action of both, the T and the B cell compartment enables animals to survive the infection and recover from disease.

Clinical course, recovery of infectious virus from brain tissue and histopathology of the central nervous system were examined in gamma-irradiated Lewis rats reconstituted by naive lymphocytes before infection with coronavirus MHV-4 (strain JHM). Up to 9 days past infection, no differences were seen between immunologically competent and immuno-deficient animals in terms of onset and progression of neurological disease. However, in the latter animals neurological symptoms were dominated by signs of encephalitis instead of paralytic disease as usually seen in immunocompetent animals. Nevertheless, despite high titers of infectious virus in the CNS of immunodeficient animals only mild histopathological changes were noticeable. In contrast, infectious virus in the CNS of immunologically competent animals was below the detection limit of the assay. Paralytic disease and tissue destruction were T lymphocyte mediated because gamma-irradiated rats that were reconstituted by CD4+ or CD8+ T lymphocyte enriched cells in the absence of B lymphocytes revealed an earlier onset of clinical symptoms and a more rapid deterioration of their clinical state compared to fully competent animals. Whereas in CD4+ T cell reconstituted animals infectious virus was moderately reduced and tissue destruction as well as inflammatory changes in the CNS were focal, in CD8+ T cell reconstituted animals vacuolizing white matter inflammation was diffuse without reduction of infectious virus in brain tissue. From the presented data we conclude that in the acute stage of JHMV-induced encephalomyelitis of Lewis rats: (i) tissue destruction and paralytic clinical symptomatology are mainly T cell-mediated; (ii) CD4+ T lymphocytes can directly contribute to reduction of viral load in the brain and (iii) only coordinated action of both, the T and the B cell compartment enables animals to survive the infection and recover from disease.

Borna disease virus (BDV) is a neurotropic agent with capacity to cause encephalomyelitis in a wide range of animal species, including horses and cats. Recent studies also point to a link between BDV and human neuropsychiatric disorders. The pathogenesis of Borna disease (BD) has been proposed to be immune-mediated, mainly through the effects of cytotoxic T cells. We used flow cytometric analysis in order to characterize the peripheral and intracerebral T cell immune response in cats naturally infected with BDV. Our results show the presence of two different CD8+ cell populations (CD8+low and CD8+high) in the blood, spleen and brain of these cats. In the brain, CD8+low cells predominated over CD8+high cells. Since CD8+low cells have been suggested to represent a non-MHC-restricted T cell population, the recruitment of such cells to the brains of BDV-infected cats could possibly be of importance for the clearance of virus from neurones.

In a rat model, we have investigated the effects of adoptively transferred virus-specific immune cells on an established retroviral infection of various organs. The experimental design required inoculation of neonatal Fisher rats with a molecular clone of Friend murine leukemia virus (F-MuLV; FB29) which resulted in virus-specific immunotolerance, while infection of adult rats lead to a virus-specific humoral and cellular immune response. Adoptive transfer of virus-specific immune cells from immunized to immunotolerant (i.e., neonatally inoculated) rats was performed at around 15 days postpartum, a time when retroviral titers had already reached high levels in serum, spleen, thymus, and central nervous system (CNS). Seven days post-transfer (dpt), virus titers began to decline by 3-5 logs first in sera and at around 11-15 dpt, in spleens and thymi. Approximately 19 days post-transfer viral titers increased again. In the CNS, viral titers appeared not to change after adoptive transfer, although we observed an influx of activated T-cells and natural killer cells (NK-cells), but not of B-cells, into the CNS as well as an upregulation of major histocompatibility complex class I and II molecules between 8 and 21 dpt on both microglia and other brain cells. From these data we conclude that MuLV-infected cells of lymphoid organs can be eliminated by an antiviral immune response. In the CNS, however, most virus-infected cells escaped an immunological attack in spite of the presence of T- and NK-cells and may thus function as a reservoir for MuLVs.

Oral infection of athymic nude and immunocompetent Lewis rats with Toxoplasma gondii induced a chronic nonlethal encephalitis. The histopathological pattern of Toxoplasma encephalitis was significantly different in both groups of animals and there were substantially larger numbers of Toxoplasma cysts in the brains of athymic rats. Combined immunohistochemical and flow cytometric analyses of intracerebral leukocytes identified alpha beta TCR+ CD4+ and CD8+ T cells; macrophages, and natural killer cells as inflammatory cell populations in immunocompetent rats, whereas in athymic rats natural killer cells, macrophages, and gamma delta TCR+ CD8+ CD3+ T cells contributed to the intracerebral inflammatory infiltrates. These findings not only point to a major participation of alpha beta TCR+ T cells to the intracerebral immune response, but also indicate that they are not essential to prevent the development of a lethal Toxoplasma encephalitis. In addition, microglia were strongly activated in both strains with simultaneous up-regulation of major histocompatibility complex class I and II antigens and CD4. Activation of microglia was most prominent in athymic rats, demonstrating that immunodeficiency does not preclude an up-regulation of these molecules including the human immunodeficiency virus receptor CD4 on microglial cells.

A highly neuropathogenic retrovirus, NT40, was generated by serially passaging an infectious molecular clone of Friend murine leukemia virus, FB29, through F344 Fisher rats. NT40 induced severe neurological signs such as reflex abnormalities and ataxia within 4-6 weeks following neonatal inoculation. FB29 led to only very mild neurological dysfunctions with longer incubation periods. Pathological alterations were characterized by mild (FB29) to extensive (NT40) noninflammatory spongiform degeneration, mainly of brain-stem areas. Infectious center assays revealed that viral titers in brain tissues of NT40-infected rats were 100-fold higher than those of FB29-infected animals. Employing immunohistochemistry, in situ hybridization, and flow cytometry, NT40 was found to infect many endothelial cells of brain blood vessels and microglia, whereas FB29 infected only microglia and those to a lower extent. However, when isolated from adult diseased rats, microglial cells turned out in both cases to be nonproductively infected with either FB29 or NT40. Of peripheral organs, we found enhanced levels of NT40 in peritoneal macrophages but not in spleen, thymus, or serum when compared to FB29. Altogether these data suggest that an expanded cellular tropism within the CNS and elevated viral titers in macrophages and microglia correlated with enhancement of neuropathogenicity.

Intracerebral infection of Lewis (LEW) inbred rats with the neurotropic strain of the murine coronavirus JHM (JHMV) frequently results in a monophasic paralytic disease. In contrast, infection of Brown Norway (BN) inbred rats does not lead to clinical disease. Previous findings indicated that in both rat strains brain-infiltrating leukocytes consisted mainly of CD8+ T lymphocytes. Here, we phenotypically as well as functionally characterised this T cell subset after isolation from the central nervous system (CNS). Using JHMV-infected target cells, MHC class I restricted, cytotoxic T lymphocytes were demonstrated to be present in the leukocyte fraction from the CNS of both, susceptible LEW and disease-resistant BN rats. However, compared to infected, but healthy BN rats, diseased LEW rats generated an enhanced cytotoxic immune response which became most prominent at the maximum of neurological disease. Recently published observations from our laboratory demonstrated a strong virus-specific antibody response in the CNS of BN rats. In LEW rats, however, the response was delayed and of low magnitude. This suggests, that consequences of cytotoxic T lymphocyte action in JHMV-infected CNS tissue largely depend on the efficacy of an accompanying virus-specific humoral immune response.

The CD4+ T lymphocyte response in the central nervous system (CNS) and cervical lymph nodes (CLNs) of rats with different susceptibility to coronavirus-induced encephalitis was investigated. The majority of CD4+ T lymphocytes entering the virus-infected CNS in the course of the infection are primed cells that neither proliferate ex vivo nor can be stimulated to proliferation by viral antigens or mitogen in vitro. In contrast, T lymphocytes taken from CLNs of the same animals revealed a strong proliferative response. Restimulation of CLN lymphocytes by viral antigens disclosed a striking difference between the disease-resistant rat strain Brown Norway (BN) and the susceptible Lewis (LEW) strain. Whereas BN lymphocytes responded as early as 5 days post infection, it took more than 11 days until a comparable proliferation was detectable in LEW lymphocytes. From these data we postulate that the majority of T lymphocytes entering the virus-infected brain after sensitisation and expansion in cervical lymph nodes is unresponsive to further proliferation signals and that the kinetics and magnitude of T lymphocyte stimulation in CLNs play an important role in the clinical course of the infection.

Neural-specific T cells are held to play a pivotal role in the Guillain-Barr6 syndrome, and experimental allergic neuritis (EAN).Here, the effects of intraneural accumulation of nonneural-specific T cell on bloodnerve barrier permeability and peripheral nerve function were assessed.Rat ovalbumin (OA)-specific T cells were activated in vitro and on day 0 intravenously transferred to female adult Lewis rats.Rats were then given intraneural injections of OA or casein into left and fight tibial nerves respectively.On days 3 and 4, selected rats also received intravenous purified immunoglobulin from rabbits with myelin-induced EAN, Rapid accumulation of ct/(~ T cells and ED1 + macrophages and marked increases in blood-nerve barrier permeability in OA but not casein injected nerves followed transfer of 2x10 6 T cells.5x10 6 T ceils induced decreases in proximal/distal CMAP amplitude ratios but also severe reductions in distal CMAP amplitudes and Wallerian degeneration in OA nerves.Demyelination was occasionally observed in nerves proximal to sites of OA injection.5x10 5 T cells also induced decreases in amplitude ratio but with only minor axonal degeneration and reductions in distal amplitudes.Conduction block and demyelination were considerably augmented in animals also receiving anti-myelin antibody.lntraneural accumulation of nonneural-specific T ceils can orchestrate demyelination, axonal degeneration, or both.P06.

The repertoire of tools for therapeutic intervention in demyelinating disorders of the CNS, such as multiple sclerosis or postinfectious encephalomyelitis, is still very limited.This deficit is mainly because the pathogene- sis of these serious complications is far from being completely understood.In this context, animal models of virus-induced demyelinating encephalomyelitis are gaining in importance as a promising platform for the development of therapeutic strategies.Among these model systems intracerebral infection of rodents by the murine coronavirus JHM (JHMV) is par- ticularly interesting.'We review our current data on the role of the humoral immune sys- tem response in the clinical course of JHMV infections in rat inbred strains.

Intracerebral infection of rodents with the murine coronavirus JHM is a well established animal model to study the pathology of virus-induced primary demyelination of the central nervous system (CNS). Although it was assumed by Weinerl that cytopathogenic effects of the virus play the dominant role in this axonal loss of myelin sheaths, a growing body of evidence suggests now a significant contribution of the virus-specific immune response to the histopathological changes in the central nervous system as well as to the clinical course of the infection. In this context, in mice action of CD8+ T-lymphocytes appears to be a two-edged sword. On the one hand, they are necessary to clear JHM virus from infected brain tissue,2,3 on the other hand, in vivo depletion of this lymphoid subset reduces drastically the appearance of white matter destruction4,5 and adoptive transfer of either viral-specific6 or naive syngeneic CD8+ splenocytes7 in immunosuppressed animals fully reconstitutes neurological disease. This suggests that cytotoxic T-lymphocytes may cause demyelination by killing of virus-infected oligodendrocytes and thereby contribute to the clinical symptomatology of the infection.