Prolonged activation of cytomegalovirus early gene e1-promoter exclusively in neurons during infection of the developing cerebrum.

The brain is the major target of congenital cytomegalovirus (CMV) infection. It is possible that neuron disorder in the developing brain is a critical factor in the development of neuropsychiatric diseases in later life. Previous studies using mouse model of murine CMV (MCMV) infection demonstrated that the viral early antigen (E1 as a product of e1 gene) persists in the postnatal neurons of the hippocampus (HP) and cerebral cortex (CX) after the disappearance of lytic infection from non-neuronal cells in the periventricular (PV) region. Furthermore, neuron-specific activation of the MCMV-e1-promoter (e1-pro) was found in the cerebrum of transgenic mice carrying the e1-pro-lacZ reporter construct. In this study, in order to elucidate the mechanisms of e1-pro activation in cerebral neurons during actual MCMV infection, we have generated the recombinant MCMV (rMCMV) carrying long e1-pro1373- or short e1-pro448-EGFP reporter constructs. The length of the former, 1373 nucleotides (nt), is similar to that of transgenic mice. rMCMVs and wild type MCMV did not significantly differed in terms of viral replication or E1 expression. rMCMV-infected mouse embryonic fibroblasts showed lytic infection and activation of both promoters, while virus-infected cerebral neurons in primary neuronal cultures demonstrated the non-lytic and persistent infection as well as the activation of e1-pro-1373, but not -448. In the rMCMV-infected postnatal cerebrum, lytic infection and the activation of both promoters were found in non-neuronal cells of the PV region until postnatal 8 days (P8), but these disappeared at P12, while the activation of e1-pro-1373, but not -448 appeared in HP and CX neurons at P8 and were prolonged exclusively in these neurons at P12, with preservation of the neuronal morphology. Therefore, e1-pro-448 is sufficient to activate E1 expression in non-neuronal cells, however, the upstream sequence from nt -449 to -1373 in e1-pro-1373 is supposed to work as an enhancer necessary for the neuron-specific activation of e1-pro, particularly around the second postnatal week. This unique activation of e1-pro in developing cerebral neurons may be an important factor in the neurodevelopmental disorders induced by congenital CMV infection.

Different exercises can modulate the differentiation/maturation of neural stem/progenitor cells after photochemically induced focal cerebral infarction.

INTRODUCTION: Exercise therapies during rehabilitation significantly promote recovery from various deficits after cerebral infarction, which is mediated by neuronal plasticity with distinct inputs. Although adult neurogenesis can also be modulated by neuronal activity before synaptogenesis, how distinct exercises contribute to the neurological reorganization of the injured cerebral cortex remains unclear. In the present study, we aimed to elucidate the effects of different exercise therapies on motor recovery and neuronal reorganization after photochemically induced focal cerebral infarction. METHODS: Here, we examined the effects of three different exercises-(a) forced lower-intensity and (b) higher-intensity treadmill exercises, and (c) voluntary exercise with wheel running-on motor recovery and adult neurogenesis in a rat model of focal cerebral infarction. Photochemically induced thrombosis (PIT) was used to generate focal infarction in rats that was mostly confined to their motor cortices. RESULTS: Beam walking tests showed that recovery after PIT-induced cortical infarction differed in acute and chronic stages and was influenced by the type of exercise. Furthermore, forced low-intensity training had more positive effects on functional recovery than other exercises or control. To evaluate the production of newly generated cells including de novo neurogenesis, we performed lineage analysis with BrdU labeling and immunofluorescence experiments. Lower-intensity treadmill exercise increased the number of BrdU/NeuN colabeled cells, but not total BrdU-retaining or BrdU/Sox2-colabeled cells, in the peri-infarct region of the ipsilateral cortex. In contrast, high-intensity treadmill or voluntary exercises had the opposite effects. CONCLUSIONS: These results suggest that neuronal maturation can be differently modulated by distinct exercises and that low-intensity treadmill exercise could result in more potent generation of mature neurons. This also suggests the possibility that the generation of neural stem/progenitor cells and differentiation might be modulated by rehabilitation-mediated neural plasticity.

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 µl of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-µl syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 µl of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Cytomegalovirus initiates infection selectively from high-level ß1 integrin-expressing cells in the brain.

Cytomegalovirus (CMV) is a prevalent pathogen in intrauterine infections that causes congenital anomalies such as CMV encephalitis, which is characterized by the focal areas of reactive gliosis, reactive mononuclear cells, microglial nodules, and ventriculoencephalitis. To elucidate the mechanisms of CMV susceptibility in the developing brain, cell tropism and the infectious dynamics of CMV infection were investigated. We evaluated intraventricular and intravascular infections from the perspective of the distribution of CMV and its receptor (ß1 integrin) in the earliest phase of infection. Murine CMV (MCMV) immediate early 1-positive cells were colocalized mainly with meninges and choroid plexus (after intraventricular infection) or with endothelial cells and pericytes (after intravascular infection). Using green fluorescent protein-expressing recombinant MCMV particles and fluorescent microbeads (100 to 300 nm), we revealed that CMV particle size is the primary factor determining the initial CMV distribution. ß1 Integrin inhibition using a shRNA and functional blocking antibody significantly reduced MCMV infection. IHC analysis, flow cytometric, and brain slice analyses strongly support that high-level ß1 integrin-expressing cells (eg, endothelial cells, pericytes, meninges, choroid plexus, and neural stem progenitor cells) are the first targets of MCMV. Therefore, our data demonstrate that the initial distributions of MCMV particles and ß1 integrin determine the distinct pattern of infection in the brain in the acute phase.

Aberrant fetal macrophage/microglial reactions to cytomegalovirus infection.

OBJECTIVE: Congenital cytomegalovirus (CMV) infection is the leading viral cause of neurodevelopmental disorders in humans, with the most severe and permanent sequelae being those affecting the cerebrum. As the fetal immune reactions to congenital CMV infection in the brain and their effects on cerebral development remain elusive, our aim was to investigate primitive innate immunity to CMV infection and its effects on cerebral corticogenesis in a mouse model for congenital CMV infection using a precise intraplacental inoculation method. METHODS: At 13.5 embryonic days (E13.5), pregnant C57BL/6 mice were intraplacentally infected with murine CMV (MCMV). Placentas and fetal organs were collected at 1, 3, and 5 days postinfection and analyzed. RESULTS: MCMV antigens were found frequently in perivascular macrophages, and subsequently in neural stem/progenitor cells (NSPCs). With increased expression of inducible nitric oxide synthase and proinflammatory cytokines, activated macrophages infiltrated into the infectious foci. In addition to the infected area, the numbers of both meningeal macrophages and parenchymal microglia increased even in the uninfected areas of MCMV-infected brain due to recruitment of their precursors from other sites. A bromodeoxyuridine (BrdU) incorporation experiment demonstrated that MCMV infection globally disrupted the self-renewal of NSPCs. Furthermore, BrdU-labeled neurons, particularly Brn2(+) neurons of upper layers II/III in the cortical plate, decreased in number significantly in the MCMV-infected E18.5 cerebrum. INTERPRETATION: Brain macrophages are crucial for innate immunity during MCMV infection in the fetal brain, while their aberrant recruitment and activation may adversely impact on the stemness of NSPCs, resulting in neurodevelopmental disorders.

Effects of intrapulmonary viral tropism and cytokine expression on the histological patterns of cytomegalovirus pneumonia.

Pulmonary cytomegalovirus (CMV) infection causes fatal CMV pneumonia (CMVp) in immunocompromised patients; however, the mechanisms underlying CMV-infection-induced pulmonary lesion development remain largely unknown. We examined the relationship between CMVp patterns and intrapulmonary viral tropism, including expression of inflammatory cytokines and related molecules. Double immunohistochemistry of CMV antigen and cellular markers showed that epithelial tropism was associated with a diffuse alveolar damage (DAD) pattern (CMVp-DAD) while stromal tropism was associated with a predominantly interstitial inflammation/fibrosis (IIF) (CMVp-IIF) or a combination of DAD and IIF (CMVp-complex). Transforming growth factor (TGF)-ß1 expression was relevant to CMV-induced tissue injury, and its expression was higher in CMVp-complex and CMVp-IIF than in CMVp-DAD. Expression of integrin ß6 (ITGB6), an adhesion molecule and important activator of TGF-ß1 in interstitial pneumonia, was lost in CMV-infected pneumocytes, especially CMVp-DAD, whereas CMV-negative pneumocytes in CMVp-complex and CMVp-IIF showed overexpression. Diffuse interleukin (IL)-8 up-regulation and strong expression were present in both CMV-infected pneumocytes and stromal cells only in CMVp-IIF cases with marked interstitial neutrophilic infiltration. On the basis of viral tropism and the expression of TGF-ß1, ITGB6, and IL-8, we conclude that CMV-infected pulmonary cells play an important role in the development of diverse CMVp patterns.

Mouse embryonic stem cells inhibit murine cytomegalovirus infection through a multi-step process.

In humans, cytomegalovirus (CMV) is the most significant infectious cause of intrauterine infections that cause congenital anomalies of the central nervous system. Currently, it is not known how this process is affected by the timing of infection and the susceptibility of early-gestational-period cells. Embryonic stem (ES) cells are more resistant to CMV than most other cell types, although the mechanism responsible for this resistance is not well understood. Using a plaque assay and evaluation of immediate-early 1 mRNA and protein expression, we found that mouse ES cells were resistant to murine CMV (MCMV) at the point of transcription. In ES cells infected with MCMV, treatment with forskolin and trichostatin A did not confer full permissiveness to MCMV. In ES cultures infected with elongation factor-1α (EF-1α) promoter-green fluorescent protein (GFP) recombinant MCMV at a multiplicity of infection of 10, less than 5% of cells were GFP-positive, despite the fact that ES cells have relatively high EF-1α promoter activity. Quantitative PCR analysis of the MCMV genome showed that ES cells allow approximately 20-fold less MCMV DNA to enter the nucleus than mouse embryonic fibroblasts (MEFs) do, and that this inhibition occurs in a multi-step manner. In situ hybridization revealed that ES cell nuclei have significantly less MCMV DNA than MEF nuclei. This appears to be facilitated by the fact that ES cells express less heparan sulfate, ß1 integrin, and vimentin, and have fewer nuclear pores, than MEF. This may reduce the ability of MCMV to attach to and enter through the cellular membrane, translocate to the nucleus, and cross the nuclear membrane in pluripotent stem cells (ES/induced pluripotent stem cells). The results presented here provide perspective on the relationship between CMV susceptibility and cell differentiation.

Aggressive progression of breast cancer with microscopic pulmonary emboli possessing a stem cell-like phenotype independent of its origin.

Microscopic pulmonary tumor embolism is difficult to diagnose. Herein is presented the case of a patient who suffered from acute dyspnea and breast cancer on the right side. Two weeks after the breast cancer diagnosis the patient began to experience dyspnea. After 2 weeks of dyspnea, the patient died without an accurate diagnosis of dyspnea. Autopsy indicated massive microscopic pulmonary emboli of the breast cancer. Immunohistochemistry showed that most of the cancer cells in the primary site were negative for estrogen receptors, progesterone receptors Her2/neu oncogene (triple negative), and stem cell-like markers (OCT3/4, NANOG2, CD44, CD24, aldehyde dehydrogenase 1 (ALDH1)). The breast cancer cells in the lung (the metastasized site), however, were triple negative, but were enriched in stem cell-like markers (OCT3/4(+), NANOG2(+), CD44(+)/CD24(-/low), ALDH1(+)). This is a significant case report indicating that vascular emboli themselves contain the essential molecular signature of 'stemness' independent of the origin.

Effects of cytomegalovirus infection on embryogenesis and brain development.

Congenital cytomegalovirus (CMV) infection is a significant cause of brain disorders, such as microcephaly, mental retardation, hearing loss and visual disorders in humans. The type and severity of brain disorder may be dependent on the stage of embryonic development when the congenital infection occurs. Developmental disorders may be associated with the type of embryonic cells to which CMV is susceptible and the effects of the infection on the cellular functions of these cells. Early murine embryos, including embryonic stem (ES) cells, are not susceptible to CMV infection. A part of the embryonic cells acquire susceptibility during early development. Mesenchymal cells are the targets of infection at midgestation, affecting organogenesis of the brain, eyes and oral-facial regions. In contrast to ES cells, neural stem progenitor cells (NSPC) from fetal brains are susceptible to murine CMV (MCMV) infection. The viral infection inhibits proliferation and differentiation of the NSPC to neuronal and glial cells in addition to induction of neuronal cell loss. These cellular events may cause brain malformations, such as microcephaly and polymicrogyria. Furthermore, MCMV persists in neuronal cells in developing brains, presumably resulting in neuronal dysfunction.

Characterization of the guinea pig cytomegalovirus genome locus that encodes homologs of human cytomegalovirus major immediate-early genes, UL128, and UL130.

We reported previously that the guinea pig cytomegalovirus (CMV) stock purchased from the American Type Culture Collection contained two types of strains, one containing and the other lacking a 1.6 kb locus, and that the 1.6 kb locus was required for efficient viral growth in animals but not in cell culture. In this study, we characterized the genetic contents of the locus, and found that i) the 1.6 kb locus encodes homologs of human CMV UL128 and UL130, GP129 and GP131, respectively, ii) these genes are expressed with late gene kinetics, iii) GP131 protein (pGP131) localized to cell surface only in the presence of glycoproteins H and L, and iv) pGP131 is a virion component. Therefore, it is plausible that pGP131 forms a complex with glycoproteins H and L and becomes a virion component as does UL130 protein (pUL130). Since pUL130 is one of the glycoproteins essential for infection of endothelial and epithelial cells in human and primates, functional and immunological analyses of this GPCMV homolog of pUL130 may help to illuminate the in vivo role of pUL130.

Identification of a 1.6 kb genome locus of guinea pig cytomegalovirus required for efficient viral growth in animals but not in cell culture.

Guinea pig cytomegalovirus (GPCMV) provides a useful model for studies of congenital CMV infection. During characterization of the GPCMV genome sequence, we identified two types of strains in a virus stock purchased from ATCC. One of them, GPCMV/del, lacks a 1.6 kb locus that positionally corresponds to murine CMV (MCMV) M129-M133. Growth of GPCMV/del in cell culture was marginally better than that of the other strain, GPCMV/full, which harbors the 1.6 kb locus. However, in animals infected intraperitoneally with virus stocks containing both strains, GPCMV/full disseminated more efficiently than GPCMV/del, including 200-fold greater viral load in salivary glands. Viral DNA, transcripts of the immediate-early 2 gene homolog, and viral antigens were more abundant in animals infected with GPCMV/full than in those infected with GPCMV/del. Although the observed phenomena have some similarity with the growth properties of MCMV strains defective in mck-1/mck-2(M129/131) and those defective in sgg(M132), no M129-M132 homologs were found in the 1.6 kb locus. Since one of the ORFs in the locus has a weak sequence similarity with HCMV UL130, which relates to cell tropism, further studies will be required to learn the mechanism for efficient GPCMV growth in animal.

Induction of cytomegalovirus-infected labyrinthitis in newborn mice by lipopolysaccharide: a model for hearing loss in congenital CMV infection.

Congenital cytomegalovirus (CMV) infection is the most common infectious cause of sensorineural hearing loss in children. Here, we established an experimental model of hearing loss after systemic infection with murine CMV (MCMV) in newborn mice. Although almost no viral infection was observed in the inner ears and brains by intraperitoneal (i.p.) infection with MCMV in newborn mice, infection in these regions was induced in combination with intracerebral (i.c.) injection of bacterial lipopolysaccharide (LPS). The susceptibility of the inner ears was higher than that of the brains in terms of viral titer per unit weight. In the labyrinths, the viral infection was associated with the mesenchymal vessels and accompanied by inflammatory cells induced by LPS, causing hematogenous targets of infection in the labyrinths. Viral infection also spread in the perilymph regions such as the scala tympani and scala vestibuli, probably from infected brains via meningogenic and cochlear nerve routes. Viral infection was not observed in the scala media in the endolymph, including the Corti organ. However, viral infection was observed in the spiral limbus, including the stria vascularis. These results suggest that hearing loss caused by labyrinthitis after congenital CMV infection may be enhanced by inflammation caused by systemic bacterial infection in the neonatal period.

Roles of neural stem progenitor cells in cytomegalovirus infection of the brain in mouse models.

Cytomegalovirus (CMV) is the most significant infectious cause of brain disorders in humans. Although the brain is the principal target organ for CMV infection in infants with congenital infection and in immunocompromised patients, little has been known about cellular events in pathogenesis of the brain disorders. Mouse models have been developed by the authors for studying the cell tropism, infectious dynamics of CMV infection and the effects of CMV infection on proliferation, regeneration and differentiation of neural cells. It has been shown, using brain slice cultures and neurospheres, that neural stem progenitor (NSP) cells are the most susceptible to CMV infection in developing brains. The NSP cells are also susceptible to CMV infection in adult and aged brains. The susceptibility can be enhanced by stimulation of neurogenesis. It was shown that latent murine CMV infection occurs in NSP cells by demonstrating the reactivation in brain slice culture or neurospheres. It is hypothesized that CMV brain disorder such as microcephaly is caused by disturbance of cellular events in the ventricular regions, including proliferation and differentiation of the neural stem cells, whereas neurons are also targets in persistent CMV infection, presumably resulting in functional disorders such as mental retardation.

Congenital anaplastic astrocytoma differentiated into pilocytic astrocytoma: an autopsy case.

We report an autopsy case of congenital astrocytoma and its histopathological changes during 5 years of the patient's development from birth to death. At birth, a right exophthalmic tumor was observed, and MRI revealed that the tumor occupied the right orbital space and had also affected the suprasellar diencephalic structures. The right orbital tumor, which was enucleated at 2 months of age, was a highly cellular tumor with moderate pleomorphism resembling anaplastic astrocytoma. On the other hand, at autopsy, a brain tumor was found in the right diencephalic region with features of pilocytic astrocytoma, accompanied by leptomeningeal dissemination. A biopsy specimen, which was obtained from the chiasmatic part of the tumor at 4 months of age, showed an intermediate appearance between the orbital tumor and the brain tumor obtained at autopsy. Immunohistochemical examination confirmed that all three phases of the tumors showed an astrocytic lineage, and the Ki-67 labeling index decreased rapidly after 2 months of age. We believe that this congenital anaplastic astrocytoma differentiated into a pilocytic astrocytoma during the 5 years of the patient's development. The transformation of the congenital astrocytoma from anaplastic to pilocytic forms can be attributed to the nature of the tumor, namely postmitotic neoplastic cells are characterized by their ability to undergo self-differentiation, along with the organotropism of the developing brain.

Enhancement of susceptibility of adult mouse brain to cytomegalovirus infection by infusion of epidermal growth factor.

Neural precursor cells, including neural stem and progenitor cells, in the subventricular zone (SVZ) are the main targets for cytomegalovirus (CMV) infection in developing brains. The neural precursor cells in the SVZ of the adult brain have been reported to respond by proliferating after infusion with epidermal growth factor (EGF). Here we report the susceptibility of the precursor cells in the adult mouse brain to murine CMV (MCMV) infection. Adult mouse brains from 10-, 25-, and 70-week-old (W) mice were infused with either phosphate-buffered saline or EGF into the brain for 3 days, and then intracerebrally infected with MCMV for 5 days. The susceptibility of the adult brains to MCMV was significantly increased by infusion of EGF in terms of viral titers and viral antigen-positive cells. The susceptibility of the young adult brain from 10-week-old mice to MCMV was higher than that of the adult brains from 25-week-old or 70-week-old mice. Both the ependymal and the SVZ cells were susceptible to MCMV infection. The number of virus-infected cells in the SVZ was significantly increased by infusion of EGF, whereas the number of infected ependymal cells was not significantly increased. Among the virus-infected cells in the SVZ, 73% were positive for nestin, 87% were positive for Musashi, 86% were positive for GFAP, and 96% were positive for PCNA. These results indicate that the susceptibility of the adult brain to MCMV is correlated with the proliferative ability of the neural precursor cells in the SVZ of the adult brain.

Cyclosporine inhibits mouse cytomegalovirus infection via a cyclophilin-dependent pathway specifically in neural stem/progenitor cells.

The potential of neural stem and progenitor cell (NSPC) transplantation in neurodegenerative disease raises a concern about immunosuppressive agents and opportunistic neurotropic pathogens that may interfere with engraftment. Cytomegalovirus (CMV) is an important opportunistic pathogen infecting the central nervous system, where it may remain latent for life, following transplacental transmission. Cyclosporine (Cs), an immunosuppressive drug used in organ transplantation, where its use is associated with CMV reactivation, suppressed murine CMV (MCMV) infection in cultured NSPCs but not in fibroblasts. This activity of Cs appears to be mediated via cyclophilin (CyP) rather than via calcineurin. First, the calcineurin-specific inhibitor FK506 failed to suppress replication. Second, the CyP-specific inhibitor NIM811 strongly suppressed replication in NSPC. NSPCs maintained in the presence of NIM811 retained viral genomes for several weeks without detectable viral gene expression or obvious deleterious effects. The withdrawal of NIM811 reactivated viral replication, suggesting that the inhibitory mechanism was reversible. Finally, inhibition of endogenous CyP A (CyPA) by small interfering RNA also inhibited replication in NSPCs. These results show that MCMV replication depends upon cellular CyPA pathways in NSPCs (in a specific cell type-dependent fashion), that CyPA plays an important role in viral infection in this cell type, and that inhibition of viral replication via CyP leads to persistence of the viral genome without cell damage. Further, the calcineurin-signaling pathway conferring immunosuppression in T cells does not influence viral replication in a detectable fashion.

Pathogenesis of cytomegalovirus-associated labyrinthitis in a guinea pig model.

Cytomegalovirus infects fetuses through the placenta, resulting in various congenital disorders in newborns, including hearing loss. We developed a monoclonal antibody to guinea pig cytomegalovirus (GPCMV) that was available for immunohistochemistry, and investigated the expression of the GPCMV antigen in animal models of direct and congenital infections. Injection of GPCMV, directly to the inner ear, increased the sound pressure level and resulted in labyrinthitis with severe inflammation. Immunohistochemistry detected GPCMV-infected cells mainly in the scala tympani, scala vestibule and spinal ganglion, but rarely in the cochlear duct. Injection of GPCMV to 5-week pregnant guinea pigs resulted in severe labyrinthitis in fetuses. Immunohistochemistry detected GPCMV-infected cells in the perilymph area and spinal ganglion, but not in the endolymph area, including hair cells. These data suggest that the virus spreads via the perilymph and neural routes in the inner ear of both models of direct and congenital infections.