Corticosteroids limit microglial activation occurring during acute stress.

Our previous studies demonstrated that exposure of animals to acute stress immediately induced morphological microglial activation in the brain. Here we investigated the effects of adrenal corticoids on microglial activation following acute stress. We compared microglial activation in vivo in adrenalectomized (ADX), Sham-operated (SHM), and adrenalectomy plus corticosterone (CORT) administered rats exposed to a 2-h period of acute water restraint stress. We found that: (1) acute stress induced microglial activation in SHM rats; (2) acute stress robustly enhanced microglial activation in ADX rats; (3) CORT treatment significantly reduced the effects of adrenalectomy. Thus, while acute stress has the ability to activate microglia, the magnitude of activation is negatively regulated by CORT. Glucocorticoids may serve as an important endogenous suppressive signal limiting neuroinflammation that might otherwise occur during stress.

Immunological responses of astroglia in the rat brain under acute stress: interleukin 1 beta co-localized in astroglia.

In previous studies, we demonstrated that acute stress induces microglial activation, without inducing any inflammatory responses; however, the effect of acute stress on astroglia, another glial cell subtype in the brain, remains to be elucidated. We determined the effect of acute stress on astroglia, particularly in terms of morphological changes and inflammatory properties. In contrast to microglia, the morphology of astroglia was not altered following a 2-h period of acute stress. Interestingly, the number of astroglia immunoreactive to interleukin 1 beta (IL-1ß) significantly increased in several brain regions including the hippocampus, hypothalamus, amygdala, and periaqueductal gray following the acute stress. Confocal microscopy revealed that IL-1ß is exclusively co-localized in astroglia, and not in neurons or microglia. The present study demonstrates that exposing rats to acute stress increases IL-1ß immunoreactivity in astroglia in specific regions of the brain, and the mechanism of astroglial response to acute stress clearly differs from that of microglial response. Thus, astroglia may play important roles in neuroimmunomodulation through IL-1ß during times of acute stress.

Balloon occlusion test of the internal carotid artery: correlation with stump pressure and 99mTc-HMPAO SPECT.

PURPOSE: To evaluate the correlation of stump pressure during balloon occlusion test and relative cerebral blood flow (relative CBF) as measured by 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO) single-photon emission computed tomography (SPECT) after test occlusion. MATERIAL AND METHODS: Balloon occlusion test of the internal carotid artery (ICA) was performed in 25 patients. The count ratio of occluded hemisphere to non-occluded hemisphere was calculated on 99mTc-HMPAO SPECT. The ratio of mean stump pressure to mean arterial pressure during carotid occlusion during the balloon occlusion test was compared with the count ratio of 99mTc-HMPAO SPECT. RESULTS: Two patients failed to tolerate even brief carotid occlusion. The other 23 patients showed no ischemic deficit during occlusion of the ICA. In 13 of these 23 patients, the ratios of mean stump pressure to mean arterial pressure were more than 50%, and the count ratios on SPECT were more than 85%. In 10 of 23 patients, the ratios of mean stump pressure to mean arterial pressure were less than 50%, and the count ratios on SPECT were variable. CONCLUSION: Maintenance of a mean stump pressure of 50% or more of the mean systemic pressure during test occlusion indicates adequate cerebral blood flow during carotid occlusion.

TNF-alpha mediates the development of anaemia in a murine Trypanosoma brucei rhodesiense infection, but not the anaemia associated with a murine Trypanosoma congolense infection.

Development of anaemia in inflammatory diseases is cytokine-mediated. Specifically, the levels of tumour necrosis factor-alpha (TNF-alpha), produced by activated macrophages, are correlated with severity of disease and anaemia in infections and chronic disease. In African trypanosomiasis, anaemia develops very early in infection around the time when parasites become detectable in the blood. Since the anaemia persists after the first waves of parasitaemia when low numbers of trypanosomes are circulating in the blood, it is generally assumed that anaemia is not directly induced by a parasite factor, but might be cytokine-mediated, as in other cases of anaemia accompanying inflammation. To clarify the role of TNF-alpha in the development of anaemia, blood parameters of wild type (TNF-alpha+/+), TNF-alpha-null (TNF-alpha-/-) and TNF-alpha-hemizygous (TNF-alpha-/+) trypanotolerant mice were compared during infections with the cattle parasite Trypanosoma congolense. No differences in PCV, erythrocyte numbers or haemoglobin were observed between TNF-alpha-deficient and wild type mice, suggesting that the decrease in erythrocytes was not mediated by TNF-alpha. Erythropoetin (EPO) levels increased during infection and no significant differences in EPO levels were observed between the three mouse strains. In contrast, during an infection with the human pathogen Trypanosoma brucei rhodesiense, the number of red blood cells in TNF-alpha-deficient mice remained significantly higher than in the wild type mice. These data suggest that more than one mechanism promotes the development of anaemia associated with trypanosomiasis.

Wnt-1 promotes neuronal differentiation and inhibits gliogenesis in P19 cells.

Wnt-1, the vertebrate counterpart of the Drosophila wingless gene, plays an important role in the early morphogenesis of neural tissues. In this report, we have shown that overexpression of Wnt-1 can direct embryonic carcinoma P19 cells to differentiate into neuron-like cells in the absence of retinoic acid. Immunocytochemistry showed that these cells expressed neuronal markers, such as the neurofilament (NF) and microtubule-associated protein 2 (MAP2), but failed to express the glial cell marker, glial fibrillary acidic protein (GFAP). RT-PCR revealed that two basic helix-loop-helix (bHLH) genes, Mash-1 and Ngn-1, were up-regulated during the differentiation stage of Wnt-1-overexpressing P19 cells. These results suggest that the Wnt-1 gene promotes neuronal differentiation and inhibits gliogenesis during the neural differentiation of P19 cells, and that neural bHLH genes might be involved in this process.

A role of N-cadherin in neuronal differentiation of embryonic carcinoma P19 cells.

N-cadherin is one of the important molecules for cell to cell interaction in the development of the central nervous system (CNS). In this report, we have shown that N-cadherin mRNA and protein were increased rapidly in retinoic acid (RA)-induced neuronal differentiation of embryonic carcinoma P19 cells. To explore possible roles for N-cadherin during this process, N-cadherin-overexpressing P19 cell lines were established. These transfected cells could differentiate into neurofilament-expressing neurons in the absence of RA. RT-PCR revealed that the expression patterns of development-related genes, such as Oct-3/4, nestin, Notch-1, and Mash-1 were similar between the transfected P19 cells and the RA-induced wild-type P19 cells during their neuronal differentiation. On the contrary, the Wnt-1 gene was up-regulated in the N-cadherin-overexpressing P19 cells, but could not be detected in the wild-type P19 cells. These results suggest N-cadherin may play a role in neuronal differentiation of P19 cells, possibly through the Wnt-1 signaling pathway.

Virological properties and nucleotide sequences of Cas-E-type endogenous ecotropic murine leukemia viruses in South Asian wild mice, Mus musculus castaneus.

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of Mus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4(r) gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4(r) gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4(r) gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the Fv-4(r) gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic env gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.

Isolation and characterisation of fetal bovine brain cells in primary culture.

Primary cultures and cryopreservation procedure of bovine brain cells were established as in vitro experimental systems to study the responses of bovine brain cells to neuropathogenic agents. Brain cells were dissociated by papain from the cerebellum of a bovine fetus at 90 to 120 days old, and were cultured in different media. In a medium containing 1 per cent fetal bovine serum (FBS), neuronal cells were maintained and they formed clusters on glial and fibroblastic cell sheets. In a medium containing 10 per cent FBS, the proportion of neurones decreased, and fibroblastic and microglial cells dominated. In a serum-free medium containing epidermal growth factor, the highest neuronal proportion was obtained. Optimal cryopreservation condition for the brain tissues was investigated by changing the concentrations of DMSO and FBS. Brain cells could be cultured from cryopreserved tissue with only slightly reduced growth profiles and varying cell proportions in comparison to those prepared from fresh tissue.

Preferential infection of neuronal and astroglia cells by Akabane virus in primary cultures of fetal bovine brain.

Akabane virus is a member of the genus Bunyavirus; it is pathogenic for ruminants and transmitted by arthropod vectors. Infection of adult cattle and sheep causes a transient viremia without obvious clinical signs, while infection of pregnant animals often causes fetal abnormalities including hydranencephaly, poliomyelitis and arthrogryposis. Infectious virus or viral antigens is present in the brain, spinal cord and skeletal muscle of infected fetuses. To understand the interaction between Akabane virus and bovine brain cells, we investigated the viral tropism using primary cultures of fetal bovine brain. The cultured neuronal cells, astroglia cells and microglia cells were distinguished by cell type specific antisera. Akabane virus was found to infect neuronal cells and astroglia cells, which led to degenerative death. No microglia cells were found infected. In some brain cultures, we observed different sensitivities of the cells to two Akabane virus strains: an attenuated strain infected and spread more readily than wild type virus. This difference was not observed in a hamster fibroblast cell line. Both viral and host determinants might be involved in the different susceptibility of brain cells to Akabane virus infection.

The inhibitory effect of interferon gamma and tumor necrosis factor alpha on intracellular multiplication of Neospora caninum in primary bovine brain cells.

Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-gamma) and tumor necrosis factors alpha (TNF-alpha) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-gamma inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-alpha, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.

Properties of the naturally occurring soluble surface glycoprotein of ecotropic murine leukemia virus: binding specificity and possible conformational change after binding to receptor.

Ecotropic murine leukemia virus (MuLV) infection is initiated by the interaction between the surface glycoprotein (SU) of the virus and its cell-surface receptor mCAT-1. We investigated the SU-receptor interaction by using a naturally occurring soluble SU which was encoded by the envelope (env) gene of a defective endogenous MuLV, Fv-4(r). Binding of the SU to mCAT-1-positive mouse cells was completed by 1 min at 37 degrees C. The SU could not bind to mouse cells that were persistently infected by ecotropic MuLVs (but not amphotropic or dualtropic MuLVs) or transfected with wild-type ecotropic env genes or a mutant env gene which can express only precursor Env protein that is restricted to retention in the endoplasmic reticulum. These cells were also resistant to superinfection by ecotropic MuLVs. Thus, superinfection resistance correlated with the lack of SU-binding capacity. After binding to the cells, the SU appeared to undergo some conformational changes within 1 min in a temperature-dependent manner. This was suggested by the different properties of two monoclonal antibodies (MAbs) reactive with the same C-terminal half of the Fv-4(r) SU domain, including a proline-rich motif which was shown to be important for conformation of the SU and interaction between the SU and the transmembrane protein. One MAb reacting with the soluble SU bound to cells was dissociated by a temperature shift from 4 to 37 degrees C. Such dissociation was not observed in cells synthesizing the SU or when another MAb was used, indicating that the dissociation was not due to a temperature-dependent release of the MAb but to possible conformational changes in the SU.

Evidence for a mouse brain-specific variant of alpha-tubulin.

While studying the neural precursor cell intermediate filament protein known as nestin in the developing mouse brain, we observed a strong cross-reaction of our nestin antibody with a 50 kDa protein that appeared on embryonic day 10 and continued to accumulate until postnatal day 1. Here we report evidence that this protein is a brain-specific variant form of alpha-tubulin and discuss its implications.

Morphogenic activity of fibroblast growth factor-2 on primary neural precursor cells in three-dimensional culture.

Mouse neural precursor cells (NPC) were dissociated from fetal heads at the 10th day of gestation. When clumps of NPC were cultured in collagen gel, they grew and reorganized neural tube-like structures in medium containing fetal calf serum at 10% and supplemented with insulin, transferrin, cholera toxin and selenite. However, dissociated NPC died when they were cultured in collagen gel at low density in the same medium. Addition of fibroblast growth factor-2 (FGF-2) to this culture stimulated growth of NPC and formation of neural tube-like structures. The requirement for FGF-2 disappeared in high seeding density culture: they grew and formed neural tube-like structures without FGF-2. The structures formed in collagen gel were immunohistochemically positive against anti-FGF-2 antibody. The results show that the three-dimensional culture system provides a useful tool to study the roles of FGF-2 in morphogenesis of the central nervous system.

Efficiency of neural differentiation of mouse P19 embryonal carcinoma cells is dependent on the seeding density.

Serum-free culture conditions for retinoic acid-induced neural differentiation of mouse P19 embryonal carcinoma cells were determined for future ex vivo retroviral gene transfer and brain transplantation studies. Neural differentiation of P19 cells was dependent on the seeding densities, and both neurons and astroglia differentiated efficiently at high seeding densities (2 x 10(4) and 5 x 10(4) cells/cm2) but not at low seeding density (1 x 10(4) cells/cm2). In addition, P19 cells cultured at 5 x 10(4) cells/cm2 showed neural differentiated whether or not they were infected with Friend leukemia virus FrC6-V, which inhibited neural differentiation at 2 x 10(4) cells/cm2. These results indicate that FrC6-V-infected P19 embryonal carcinoma cells should be seeded at high density to achieve efficient neural differentiation in vitro for ex vivo gene transfer with a FrC6-V-derived retroviral vector system.

Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterisation.

The morphological and biochemical characterisation of adult chicken hepatocytes in a serum-free culture are described. When cultured in positively charged plastic dishes, chicken hepatocytes formed a monolayer cell sheet. The monolayer morphology of these chicken hepatocytes was quite distinct from the spheroid shape of rat hepatocytes cultured under similar conditions. Electron microscopy showed that the cytoplasmic organelles of chicken hepatocytes were well preserved in vitro. Two-dimensional gel electrophoresis showed that the chicken hepatocytes secreted liver-specific proteins. Several enzymes of glucose-6-phosphatase, cytochrome P-450 or glutathione S-transferase, involved in metabolic and biotransformation pathways in the liver, were retained in the chicken hepatocytes in a serum free condition. These findings suggest that the primary culture of adult chicken hepatocytes with a serum-free culture system could be useful to study the hepatic metabolic pathway in the chicken and its response to various chemicals.

Isolation of a germline-transmissible embryonic stem (ES) cell line from C3H/He mice.

We have isolated three embryonic stem (ES) cell lines from C3H/He mice using mouse STO cells as a feeder layer. One ES cell line (H-1) was male, and two (H-2 and H-3) were female, as determined by polymerase chain reaction, in situ hybridization, and karyotype analyses. All were immunocytochemically reactive with a C3H strain-specific antibody. Injection of cells from the female ES H-3 line into C57BL/6 blastocysts yielded four chimeras with slight coat color chimerism. All chimeras were male, and as expected, no germline-transmission was observed. By contrast, when male ES H-1 cells were injected into the perivitelline space of 8-cell C57BL/6 embryos, one male mouse with overt coat color chimerism was recovered, and it produced ES H-1-derived offspring exclusively. This germline-transmissible C3H/He cell line represents a novel addition to those ES lines currently employed for gene manipulation studies of development.

A possible role of the nestin protein in the developing central nervous system in rat embryos.

Rat embryos at the head-hold stage (Slc:SD strain; 9.5 days of gestation) were cultured for 48 h in rat serum with the anti-nestin peptide antiserum. The antiserum identified a single band in Western blots of the tissue extracts from rat embryos and stained the cells from the neural tube, migrating neural crest, and somites immunohistochemically. The antiserum-treated embryos appeared to develop normally for the most part. However, histological observation disclosed that the ventral portion of the neural tube was deformed. The cells in the deformed portion did not show the elongated shape but were round. These round cells tended to crowd near the ventricular surface, and a gap was observed between the original pial surface and cells arranged in the most pialward region. The penetration of the anti-nestin peptide antibody into the embryos from the culture medium was confirmed by visualization of the penetrated antibody using biotinylated anti-rabbit IgG antibody raised in goats and Texas red-conjugated streptavidin. These results indicate that the nestin protein plays an important role in the organization or the maintenance of neuroepithelial cells of the elongated shape spanning the neural tube from the luminar to the pial side.

Purification and activity of recombinant chicken stem cell factor produced by using a baculovirus vector.

Stem cell factor is a pleiotropic cytokine that plays an essential role in the development of hematopoietic cells, germ cells, and melanocytes. To obtain recombinant soluble chicken stem cell factor (chSCF), a baculovirus containing the cDNA encoding chSCF polypeptide from amino acids -25 to 170 was constructed. The chSCF produced in insect cells infected with the virus was purified by ion exchange column chromatography. The ability of the purified protein to induce the outgrowth of neurites from chicken dorsal root ganglia cultured in vitro was demonstrated.

[Basic and clinical studies on serum cytokeratin 19 fragment assay using Centocor CYFRA 21-1 kit in patients with lung cancer].

We evaluated the newly developed tumor marker assay kit, "Centocor CYFRA 21-1", an immunoradiometric assay (IRMA) kit for determining the serum cytokeratin 19 fragment using the sera of healthy subjects, patients with benign lung diseases and patients with lung cancer. The assay procedure is simple and based on the one-step IRMA system. There were no problems in reproducibility, dilution test and recovery test. The minimum detectable dose was 0.3 ng/ml. The antigen measured by this kit was immunologically cross-reactive with tissue polypeptide antigen (TPA) and CYFRA 21-1 concentration was closely correlated with TPA concentration in the patient's serum (r = 0.86, p < 0.01). The cut-off value of serum CYFRA 21-1 based on the assay results of this kit was calculated to be 1.6 ng/ml from the receiver operating characteristic curve. Three of 47 healthy subjects (6.4%) and 9 of 30 patients with benign lung diseases (30.0%) showed a concentration over the cut-off value. By contrast, serum CYFRA 21-1 concentration was elevated in 31 of 50 patients with lung cancer (62.0%), 11 of 13 squamous cell carcinoma patients (84.6%), 8 of 12 small cell carcinoma patients (66.7%), 4 of 7 large cell carcinoma patients (57.1%) and 8 of 18 adenocarcinoma patients (44.4%). In addition, the positive rate of serum CYFRA 21-1 in patients with lung cancer gradually increased with staging of the disease: 50.0% in stage I, 50.0% in stage II, 61.9% in stage III, and 76.9% in stage IV. Thus, our results suggested that the Centocor CYFRA 21-1 kit is a useful assay system for serum cytokeratin 19 fragment as a tumor marker in patients with lung cancer.

[Evaluation of BEKI TPS radioassay kit as a one-step immunoradiometric assay utilizes monoclonal and polyclonal antibodies specific for serum tissue polypeptide antigen].

We evaluated the usefulness of the BEKI TPS radioassay kit as a one-step immunoradiometric assay (IRMA) utilizes monoclonal and polyclonal antibodies specific for serum tissue polypeptide antigen (TPA). This IRMA was found to be highly sensitive to serum TPA; minimum detectable concentration of TPA was 20 U/L. There were no problems in the intraassay and interassay reproducibility and recovery test. However, dilution test of the patient's serum with higher TPA concentrations showed no linear relationship between TPA concentrations and diluted serum samples. The antigen measured by this IRMA was immunologically similar to TPA, and the TPS concentration was closely correlated (r = 0.835, p < 0.01) with the TPA concentration in 101 patient's serum. Four out of 77 healthy subjects (5.2%) and 24 out of 74 patients with benign diseases (32.4%) showed a serum concentration over cut-off value of 71 U/L. The serum TPS concentration was elevated in 80 of 232 patients with malignant diseases (34.5%) including 21 of 26 with hepatocellular carcinoma (80.8%) and 9 of 15 with cholangiocarcinoma (60.0%). In addition, the serum TPA level during the clinical course of patients with malignant diseases was a very useful indicator for the effect of treatment. Thus, our findings suggested that BEKI TPS IRMA kit is a useful assay system for serum TPA as a tumor marker that can be performed by a simple assay operation within about 2 hours.