Human embryonic stem cell-derived oligodendrocyte progenitor cells express the serotonin receptor and are susceptible to JC virus infection.

We studied the susceptibility of human embryonic stem cell-derived oligodendrocyte progenitor cells to infection with JC virus, the causative agent of progressive multifocal leukoencephalopathy (PML). A human embryonic stem cell line, H7, was used to derive an enriched population of cells expressing the oligodendrocyte progenitor cell-specific marker NG2. These cells expressed the 5HT2a receptor (5HT2aR) for JC virus and were highly susceptible to infection. Infection was reduced by treatment with anti-5HT2aR antibodies and by the 5HT2aR antagonists ritanserin and ketanserin. This is the first demonstration that human embryonic stem cell-derived oligodendrocyte progenitor cells are susceptible to JC virus infection and indicates that cells poised to replenish mature oligodendrocytes in PML lesions may also be a target of viral infection.

Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes.

Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.

Lack of CCR2 results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus.

In the present study, we evaluated the role of CCR2 in a model of viral-induced neurologic disease. An orchestrated expression of chemokines, including the CCR2 ligands monocyte chemoattractant protein-1/CCL2 and monocyte chemoattractant protein-3/CCL7, occurs within the CNS following infection with mouse hepatitis virus (MHV). Infection of mice lacking CCR2 (CCR2(-/-)) with MHV resulted in increased mortality and enhanced viral recovery from the brain that correlated with reduced (p < or = 0.04) T cell and macrophage/microglial (determined by F4/80 Ag expression, p < or = 0.004) infiltration into the CNS. Moreover, MHV-infected CCR2(-/-) mice displayed a significant decrease in Th1-associated factors IFN-gamma (p < or = 0.001) and RANTES/CCL5 (p < or = 0.002) within the CNS as compared with CCR2(+/+) mice. Further, peripheral CD4(+) and CD8(+) T cells from immunized CCR2(-/-) mice displayed a marked reduction in IFN-gamma production in response to viral Ag and did not migrate into the CNS of MHV-infected recombination-activating gene (RAG)1(-/-) mice following adoptive transfer. In addition, macrophage/microglial infiltration into the CNS of RAG1(-/-) mice receiving CCR2(-/-) splenocytes was reduced (p < or = 0.05), which correlated with a reduction in the severity of demyelination (p < or = 0.001) as compared with RAG1(-/-) mice receiving splenocytes from CCR2(+/+) mice. Collectively, these results indicate an important role for CCR2 in host defense and disease by regulating leukocyte activation and trafficking.

Reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor CCR5 following infection with a neurotropic coronavirus.

Studies were performed to investigate the contributions of the CC chemokine receptor CCR5 in host defense and disease development following intracranial infection with mouse hepatitis virus (MHV). T cell recruitment was impaired in MHV-infected CCR5(-/-) mice at day 7 postinfection (pi), which correlated with increased (P < or = 0.03) titers within the brain. However, by day 12 pi, T cell infiltration into the CNS of infected CCR5(-/-) and CCR5(+/+) mice was similar and both strains exhibited comparable viral titers, indicating that CCR5 expression is not essential for host defense. Following MHV infection of CCR5(+/+) mice, greater than 50% of cells expressing CCR5 antigen were activated macrophage/microglia (determined by F4/80 antigen expression). In addition, infected CCR5(-/-) mice exhibited reduced (P < or = 0.02) macrophage (CD45(high)F4/80(+)) infiltration, which correlated with a significant reduction (P < or = 0.001) in the severity of demyelination compared to CCR5(+/+) mice. These data indicate that CCR5 contributes to MHV-induced demyelination by allowing macrophages to traffic into the CNS.

Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis.

Intracerebral infection of mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a chronic demyelinating disease with clinical and histological similarities with the human demyelinating disease multiple sclerosis (MS). Following MHV infection, chemokines including CXC chemokine ligand (CXCL)10 (IFN inducible protein 10 kDa), CXCL9 (monokine induced by IFN-gamma), and CC chemokine ligand 5 (RANTES) are expressed during both acute and chronic stages of disease suggesting a role for these molecules in disease exacerbation. Previous studies have shown that during the acute phase of infection, T lymphocytes are recruited into the CNS by the chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice with established demyelination were treated with antisera against these two chemokines, and disease severity was assessed. Treatment with anti-CXCL10 reduced CD4+ T lymphocyte and macrophage invasion, diminished expression of IFN-gamma and CC chemokine ligand 5, inhibited progression of demyelination, and increased remyelination. Anti-CXCL10 treatment also resulted in an impediment of clinical disease progression that was characterized by a dramatic improvement in neurological function. Treatment with antisera against CXCL9 was without effect, demonstrating a critical role for CXCL10 in inflammatory demyelination in this model. These findings document a novel therapeutic strategy using Ab-mediated neutralization of a key chemokine as a possible treatment for chronic human inflammatory demyelinating diseases such as MS.

Expression of Mig (monokine induced by interferon-gamma) is important in T lymphocyte recruitment and host defense following viral infection of the central nervous system.

Induction of a Th1 immune response against viral infection of the CNS is important in contributing to viral clearance. The present studies demonstrate a role for the T cell chemoattractant chemokine Mig (monokine induced by IFN-gamma) in contributing to a Th1 response against mouse hepatitis virus infection of the CNS. Analysis of the kinetics of Mig expression revealed mRNA transcripts present at days 7 and 12 postinfection (p.i.) but not early (day 2) or late (day 35) in the infection. To determine functional significance, mouse hepatitis virus-infected mice were treated with anti-Mig antisera, and the severity of disease was evaluated. Such treatment resulted in a marked increase in mortality that correlated with a >3 log increase in viral burden within the brains as compared with control mice treated with normal rabbit serum. Anti-Mig-treated mice displayed a significant decrease (p < 0.005) in CD4(+) and CD8(+) T cell recruitment into the CNS as compared with normal rabbit serum-treated mice. In addition, anti-Mig treatment resulted in a significant decrease (p < 0.05) in levels of IFN-gamma and IFN-beta that coincided with increased (p < 0.02) expression of the anti-inflammatory Th2 cytokine IL-10 within the CNS. Collectively, these data indicate that Mig is important in contributing to host defense by promoting a protective Th1 response against viral infection of the CNS.

Regional hypomyelination and dysplasia in transgenic mice with astrocyte-directed expression of interferon-gamma.

Interferon-gamma (IFN-gamma), traditionally associated with a variety of physiological and pathological processes of the immune system, manifests an array of biological effects on cells of the nervous system. Clinical and in vitro studies support a key role for IFN-gamma in the pathogenesis of immune-mediated demyelinating disorders such as multiple sclerosis (MS). To investigate the role of this cytokine within the central nervous system (CNS), transgenic mice were derived in which IFN-gamma transgene expression was selectively targeted to astrocytes, a potentially important cellular source of this cytokine. Here we report that astrocyte-directed expression of IFN-gamma results in regional hypomyelination and selective disruption of brain histogenesis, which included severe cerebellar and hippocampal dysplasia. Transgenic mice were markedly ataxic and the majority died prior to reaching sexual maturity. This study demonstrates that astrocyte-directed expression of IFN-gamma profoundly affects the differentiation and morphogenesis of the brain and provides additional evidence that this cytokine has deleterious consequences on myelin-producing cells, independent of the cellular source.

Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope.

Hybrids of tobacco mosaic virus (TMV) were constructed with the use of fusion to the coat protein peptides of 10 or 15 amino acids, containing the 5B19 epitope from the spike protein of murine hepatitis virus (MHV) and giving rise to TMV-5B19 and TMV-5B19L, respectively. The TMV hybrids were propagated in tobacco plants, and the virus particles were purified. Immunogold labeling, with the use of the monoclonal MAb5B19 antibody, showed specific decoration of hybrid TMV particles, confirming the expression and display of the MHV epitope on the surface of the TMV. Mice were immunized with purified hybrid viruses after several regimens of immunization. Mice that received TMV-5B19L intranasally developed serum IgG and IgA specific for the 5B19 epitope and for the TMV coat protein. Hybrid TMV-5B19, administered by subcutaneous injections, elicited high titers of serum IgG that was specific for the 5B19 epitope and for coat protein, but IgA that was specific against 5B19 was not observed. Mice that were immunized with hybrid virus by subcutaneous or intranasal routes of administration survived challenge with a lethal dose (10 x LD50) of MHV strain JHM, whereas mice administered wild-type TMV died 10 d post challenge. Furthermore, there was a positive correlation between the dose of administered immunogen and protection against MHV infection. These studies show that TMV can be an effective vaccine delivery vehicle for parenteral and mucosal immunization and for protection from challenge with viral infection.

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus.

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.

Expression of inflammatory cytokines and inducible nitric oxide synthase in brains of SIV-infected rhesus monkeys: applications to HIV-induced central nervous system disease.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) can lead to severe impairments in cognition, behavior, and motor skills. The mechanism(s) by which HIV-1 induces CNS disease are not well understood. Recent evidence suggests that expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) may contribute to HIV-1-induced neurologic disease. We sought to determine if these factors were present in the CNS of rhesus monkeys with simian immunodeficiency virus (SIV)-induced CNS disease. MATERIALS AND METHODS: Total NO production in cerebral spinal fluid (CSF) from infected monkeys was determined by measuring nitrite (NO2-) and nitrate (NO3-) (stable NO degradation products) utilizing Greiss reagents. In situ hybridization revealed iNOS, interferon-gamma (IFNgamma), and interleukin 1 beta (IL-1 beta) mRNA in the brains of SIV-infected monkeys. Microglia were isolated from animals infected with SIV. Following stimulation with LPS, induction of iNOS mRNA in isolated microglia was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: Serial CSF samples from an SIV-infected monkey reveal increased levels of NO2-/NO3-. In situ hybridization demonstrated iNOS, IFN gamma, and IL-1 beta mRNAs in post-mortem brain tissue of SIV-infected monkeys. Furthermore, stimulated microglia from an SIV-infected monkey could produce iNOS mRNA. CONCLUSIONS: The presence of iNOS in the brain and NO2-/NO3- in the CSF indicates that NO is produced in the CNS of SIV-infected monkeys. The data suggest that iNOS and NO may be contributing to SIV-induced CNS disease.

Antihistoplasma effect of activated mouse splenic macrophages involves production of reactive nitrogen intermediates.

The mechanism by which recombinant murine gamma interferon (rMuIFN-gamma) and bacterial lipopolysaccharide (LPS) activate mouse resident splenic macrophages to inhibit the intracellular growth of the fungus Histoplasma capsulatum was examined. Growth inhibition depended on L-arginine metabolism. The growth inhibitory state normally induced by rMuIFN-gamma and LPS in resident splenic macrophages did not occur when the macrophages were cultured in the presence of NG-monomethyl-L-arginine, a competitive inhibitor of L-arginine metabolism. Resident splenic macrophages treated with rMuIFN-gamma and LPS produced nitrite (NO2-), an end product of L-arginine metabolism. When macrophages were cultured in the presence of NG-monomethyl-L-arginine together with rMuIFN-gamma and LPS, only baseline levels of NO2- were detected. Spleen cells from H. capsulatum-infected mice produced high levels of NO2- in culture. The production of NO2- correlated with in vitro inhibition of the intracellular growth of H. capsulatum. Anti-tumor necrosis factor alpha antibody did not block NO2- production by the immigrant splenic macrophages and did not abolish the antihistoplasma activity.

Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity.

The antihistoplasma activity of recombinant murine gamma interferon (rMuIFN-gamma)-treated macrophages of the RAW 264.7 cell line depends on the generation of nitric oxide (NO.) from L-arginine. Macrophages of the P388D1 cell line treated with rMuIFN-gamma do not produce NO. or inhibit the intracellular growth of Histoplasma capsulatum. NO. is generated by the inducible enzyme nitric oxide synthase (iNOS) formed by stimulated macrophages. Northern (RNA) blot analysis of RAW 264.7 cells revealed the expression of iNOS mRNA after exposure to rMuIFN-gamma. In contrast, rMuIFN-gamma-treated P388D1 cells did not produce detectable levels of iNOS. These data suggest that the failure of P388D1 cells to generate NO. and to restrict the intracellular growth of H. capsulatum is due to a lack of expression of iNOS following treatment with rMuIFN-gamma.

Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide.

Rat ameboid microglia are able to lyse rat oligodendrocytes in vitro. The lysis is inhibited by transforming growth factor-beta, antagonists of nitric oxide (NO) production, as well as antibodies to TNF-alpha, intercellular adhesion molecule-1 (ICAM-1), and leukocyte functional Ag-1. Ameboid microglial cells spontaneously produce detectable levels of the NO metabolite nitrite (NO2-). Stimuli such as PMA, LPS, and/or IFN-gamma induce micromolar concentrations of NO2- within 24 h. TNF-alpha increases IFN gamma but not LPS-induced NO2- production. Incubation with target oligodendrocytes also increases NO2- production in a contact-dependent manner. NO2- production is inhibited by NO synthase antagonists, transforming growth factor-beta, and anti TNF-alpha. Neither antileukocyte functional Ag-1 nor anti-ICAM-1 inhibit NO2- production by microglia in the presence or absence of oligodendrocytes. Indeed, anti-ICAM-1 treatment increases NO2- production. There is a correlation between ameboid microglial cell killing of oligodendrocytes and NO2- production suggesting NO may be a mechanism of death of the oligodendrocyte and possibly play a role in lesion formation in multiple sclerosis.

Gamma interferon cooperates with lipopolysaccharide to activate mouse splenic macrophages to an antihistoplasma state.

Inhibition of the intracellular growth of Histoplasma capsulatum by murine resident red pulp splenic macrophages was examined. Splenic macrophages, unlike resident peritoneal macrophages, required a prolonged preincubation (18 h) with recombinant murine gamma interferon (rMuIFN-gamma) for activation. To be fully activated, the splenic macrophages required incubation with rMuIFN-gamma in combination with 0.1 microgram of lipopolysaccharide (LPS) per ml. Splenic macrophages stimulated with rMuIFN-gamma, LPS, or rMuIFN-gamma and LPS produced tumor necrosis factor alpha (TNF-alpha), but recombinant murine TNF-alpha (rMuTNF-alpha) did not activate macrophages when used alone or as a second signal with rMuIFN-gamma. Anti-TNF-alpha antibody did not block IFN-gamma-LPS activation of splenic macrophages to any significant extent. One hundred micromolar ferrous sulfate antagonized IFN-gamma-LPS activation of splenic macrophages, indicating that iron was involved in the fungistatic activity of cytokine-stimulated phagocytes. Our results indicate that (i) splenic macrophages differ significantly from peritoneal macrophages in their requirements for activation and (ii) the mechanism by which splenic macrophages exert their antifungal effects involves iron.

Iron limitation and the gamma interferon-mediated antihistoplasma state of murine macrophages.

The zoopathogenic fungus Histoplasma capsulatum requires iron for growth. Intracellular growth of the fungus within mouse peritoneal macrophages is inhibited by recombinant murine gamma interferon (IFN-gamma). Such treatment of mouse peritoneal macrophages induces a marked downshift in transferrin receptors. We tested whether the antihistoplasma effect of IFN-gamma-treated macrophages is the result of iron deprivation. Treatment of mouse peritoneal macrophages with the intracellular iron chelator deferoxamine inhibits the intracellular growth of H. capsulatum. Exposure of macrophages to holotransferrin antagonizes the effect of both recombinant murine IFN-gamma and deferoxamine treatments. These results suggest that iron restriction may be one of the bases for the IFN-gamma-induced antihistoplasma effect of mouse macrophages.

Electron-capture gas chromatography as a sensitive method for measuring subnanogram amounts of cholesterol in saliva and urine.

This is a sensitive method, suitable for measuring subanogram amounts of cholesterol. Cholesterol and the internal standard, epicoprostanol (5-beta-cholestan-3-alpha-ol), are derivatized with pentafluorobenzoyl chloride and detected by electron-capture gas chromatography. The pentafluorobenzoyl esters of cholesterol and the internal standard are easily formed and possess excellent chromatographic and electron-capturing properties. The lower limit of detection of the method, approximately 100 pg injected, is about 500-fold as sensitive as chromatographic methods involving flame-ionization detection. Within-day and between-day coefficients of variation were 4.2% and 8.2%, respectively, for determinations of a urinary cholesterol concentration of 570 micrograms/L (1.47 mumol/L). Such sensitivity permits analysis for cholesterol in (e.g.) physiological fluids, tissue samples, and cell cultures that contain very low concentrations of cholesterol.