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.

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.

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.

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.

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.

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.

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.

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.

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.

A case of clear cell adenocarcinoma of the müllerian duct in persistent müllerian duct syndrome: the first reported case.

We report a case of a 67-year-old man with clear cell adenocarcinoma of the remnant uterus in persistent Müllerian duct syndrome. He had a normal penis, urethra, and scrotum, and there was also a vagina and uterus. He died in a traffic accident, and clear cell adenocarcinoma was discovered incidentally at autopsy. Clear cell adenocarcinoma of the remnant uterus metastasized to the retroperitoneal lymph nodes and bilateral lungs. Persistent Müllerian duct syndrome is characterized by the persistence of Müllerian derivatives in otherwise normally virilized males. A variety of germ cell tumors of the testis have been reported in association with persistent Müllerian duct syndrome. However, no malignant change of the persistent Müllerian duct structures has been reported. This represents the first reported case of malignant change of the persistent Müllerian duct structures in persistent Müllerian duct syndrome.

Primary prostatic endodermal sinus tumor (yolk sac tumor) combined with a small focal seminoma.

We report on a primary endodermal sinus tumor (EST) (yolk sac tumor) combined with a focal seminoma of the prostate occurring in a 24-year-old man. The prostate was widely infiltrated with neoplasms that penetrated the capsule and invaded into the bladder wall and urethra. Most areas of the tumor were composed of papillary and glandular epithelium in the fibrous or myxoid stroma. Schiller-Duval bodies and periodic acid-Schiff-positive hyaline bodies were focally present. In addition to yolk sac tumor, solid nests of seminoma were found in some areas. Immunohistochemistry using specific antibodies for alpha-fetoprotein and cytokeratin showed positive reaction on the EST portion, and placental alkaline phosphatase revealed positive staining in the seminoma portion and a part of EST. Tumor cells exhibited negative staining for prostate-specific antigen, prostatic acid phosphatase, carcinoembryonic antigen, vimentin, chromogranin A, and human chorionic gonadotropin. Despite radical surgery and ordinary cisplatin-based chemotherapy, the patient died 8 months after operation. At autopsy, only EST elements had metastasized to the lungs, liver, and brain, and no tumors were found in either testis. To our knowledge, this is the first reported case of a primary EST combined with a focal seminoma in the prostate.

Brain slice culture for analysis of developmental brain disorders with special reference to congenital cytomegalovirus infection.

Cytomegalovirus (CMV) is the most significant infectious cause of congenital abnormalities of the central nervous system (CNS) with variation from the fatal cytomegalic inclusion disease to functional brain disorder. The phenotype and degree of the brain disorder depends on infection time during the developing stage, virulence, route of infection and the viral susceptibility of the cells. The pathogenesis of the CMV infection to the CNS seems to be strongly related to neural migration, neural death, cellular compositions and the immune system of the brain. To understand the complex mechanism of this disorder, we used organotypic brain slice cultures. In the brain slice culture system, migration of CMV-infected neuronal cells was observed, which reflects infectious dynamics in vivo. Neural progenitor cells or glial immature cells in the subventricular zone and marginal area are most susceptible to murine cytomegalovirus (MCMV) infection in this system. The susceptibility declined as the number of immature glial cells decreased with age. The immature glial cells proliferated in brain slice cultures during prolonged incubation, and the susceptibility to MCMV infection also increased in association with the proliferation of these cells. The brain slice from an immunocompromised mouse (Beige-SCID mouse) unexpectedly showed lower susceptibility than that of an immunocompetent mouse during any prolonged incubation. These results suggest that the number of immature glial cells might determine the susceptibility of CMV infection to the brain, independent of the immune system. We reviewed recent findings of CMV infection to the brain from the perspective of brain slice cultures and the possibility that this system could be a useful method to investigate mechanisms of congenital anomaly of the brain.

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.

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.

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.