Medulloblastoma cell line secretes platelet-derived growth factor

Medulloblastoma is the most common malignant childhood brain tumor in which aggressive growth produces recurrence in approximately 50% of appropriately treated cases and metastases along the neuraxis in 30%. To date, no studies exist concerning the production of autocrine growth factors by this brain tumor type. Malignant brain tumors in adults often produce platelet-derived growth factor (PDGF). A medulloblastoma cell line, TE-671, has been used for many years in pediatric neuro-oncologic studies. We assayed this medulloblastoma cell line for the production of PDGF. PDGF is produced by medulloblastoma cells grown in monolayer tissue culture and stimulates PDGF-sensitive 3T3 fibroblasts to incorporate tritiated thymidine in a dose-dependent fashion. This biologic activity is blocked by PDGF antibodies in a dose-dependent relationship. We postulate that PDGF produced by medulloblastoma cells plays a role in the growth of this tumor by stimulating mitogenic activity.     

Intracerebral transplantation of the A7 immortalized astrocytic cell line

The A7 cell line is an astrocyte-like cell immortalized by SV40 large T antigen, using retroviral-mediated gene transfer. These cells were transplanted into rat brains, and the graft-host interaction was investigated immunohistochemically. The A7 cells survived focally 2, 6 and 8 weeks after transplantation and retained the immunocytochemical properties observed in vitro. No immunological response was observed. GAP-43 and N-cadherin immunoreactivities were not expressed by A7 cells, but were seen in the matrix within the area of the graft and in the surrounding brain tissue. This indicates that A7 cells may stimulate expression of GAP-43 and N-cadherin immunoreactivity by host tissue. Expression of Thy 1.1 was not observed within the graft site after 2 weeks of survival, but 6 and 8 weeks after transplantation Thy 1.1 was observed within the graft area, indicating the possible co-existence of grafted cells and host tissue. Although indirect, these observations suggest that the A7 cells induce changes in host brain, including possible growth or regeneration of host tissue into the graft area.     

An immortalized cell line expresses properties of activated microglial cells.

Murine cultured microglial cells were immortalized after infection with a v-raf/v-myc recombinant retrovirus. This immortalized cell line (BV-2) shares properties with body macrophages with respect to the antigen profile, their phagocytic capacity and antimicrobial activity. BV-2 cells are not constitutively able to kill tumor cells in vitro, but acquire antitumor activity following an increase in [Ca++]i. BV-2 cells, like microglial cells, are however, distinct from peripheral macrophages by their expression of inwardly rectifying K+ channels in concert with a lack in outwardly rectifying K+ channels and the formation of spineous processes. The BV-2 cell line thus represents a suitable model for in vitro studies of activated microglial cells.     

Immunocytochemical and functional characterization of an immortalized type 1 astrocytic cell line

The DI TNC₁ cell line has been derived from cultures of rat brain astrocytes targeted oncogenesis. Cultured astrocytes are known to promote neurite outgrowth via the production of adhesion molecules found either on the cell surface or in the extracellular matrix. We sought to investigate whether DI TNC₁ cells retained the ability to produce such neurite-inducing molecules, and promote neurite growth. We found by immunofluorescence that DI TNC₁ cells expressed laminin, N-CAM and 1A1. The latter is a cell adhesion molecule that is expressed exclusively on astrocytes of the type 1 lineage. In vitro neurite outgrowth assays were also used to assess the functional properties of these cells. Monolayers of DI TNC₁ cells were almost as effective a substrate as monolayers of astrocytes purified from the neonatal rat brain in their ability to support neurite outgrowth. In addition, PC12 cells grown on extracellular matrix derived from either DI TNC₁ cells or neonatal astrocytes displayed significantly more neurite growth than cells plated on plastic. This effect was partially inhibited by preincubation of the extracellular matrix with anti-laminin antibodies. Taken together, these results suggest that the immortalized DI TNC₁ cells show many similarities to neonatal astrocytes. Given the heterogeneity of cultured astrocytes, this homogeneous cell line may prove to be particularly useful for future investigations on interactions between glia and neurons.     

Effects of dopaminergic cell degeneration on electrophysiological characteristics and GAD65/GAD67 expression in the substantia nigra: different action on GABA cell subpopulations.

The motor disturbances occurring in Parkinson's disease have been partially attributed to a hyperactivity of gamma-aminobutyric acid (GABA)-ergic nigral cells largely in the substantia nigra pars reticulata (SNr) secondary to the degeneration of dopaminergic nigrostriatal neurons. However, some aspects of this response remain unclear. In this work, different electrophysiological and neurochemical parameters were studied in GABAergic cells of the SN after unilateral nigrostriatal dopaminergic lesion using 6-hydroxydopamine injection in rats. Our data showed that 1) the SN under normal conditions contains different subsets of GABAergic cells according to their firing pattern and glutamic acid decarboxylase mRNA levels, and 2) the response of these GABAergic cell subgroups was different after the ipsi- and contralateral dopaminergic cell degeneration. These findings indicate a complex regulation of nigral GABAergic activity after nigrostriatal dopaminergic degeneration that probably involves local mechanisms, the nigro-striato-nigral loop, as well as interhemispheric mechanisms whose anatomical basis remains unstudied.     

Up-regulation of GAD65 and GAD67 in remaining hippocampal GABA neurons in a model of temporal lobe epilepsy.

In the pilocarpine model of chronic limbic seizures, subpopulations of glutamic acid decarboxylase (GAD)-containing neurons within the hilus of the dentate gyrus and stratum oriens of the CA1 hippocampal region are vulnerable to seizure-induced damage. However, many gamma-aminobutyric acid (GABA) neurons remain in these and other regions of the hippocampal formation. To determine whether long-term changes occur in the main metabolic pathway responsible for GABA synthesis in remaining GABA neurons, the levels of mRNA and protein labeling for the two forms of GAD (GAD65 and GAD67) were studied in pilocarpine-treated animals that had developed spontaneous seizures. Qualitative and semiquantitative analyses of nonradioactive in situ hybridization experiments demonstrated marked increases in the relative amounts of GAD65 and GAD67 mRNAs in remaining hippocampal GABA neurons. In addition, immunohistochemical studies demonstrated parallel increases in the intensity of terminal labeling for both GAD65 and GAD67 isoforms throughout the hippocampal formation. These increases were most striking for GAD65, the isoform of GAD that is particularly abundant in axon terminals. These findings demonstrate that, in a neuronal network that is capable of generating seizures, both GAD65 and GAD67 are up-regulated at the gene and protein levels in the remaining GABA neurons of the hippocampal formation. This study provides further evidence for the complexity of changes in the GABA system in this model of temporal lobe epilepsy.     

Cerebral GABA release and GAD activity in protein- and tryptophan-restricted rats during development

To evaluate the effects on the GABAergic system, Wistar rats were raised on a chronically protein- and tryptophan-restricted diet with 8% protein, based on either Purina chow or corn. There was a significant decrease in both body and cerebral weight in the restricted animals compared with the control group fed with a 23% protein diet. In animals fed mainly corn, glutamic acid decarboxylase (GAD) activity increased significantly at the ages studied (14, 30, and 60 days) in the cerebral cortex and hippocampus. In the same way, gamma-aminobutyric acid (GABA) release decreased significantly in early life in both brain regions, then increased in 30-60-day-old animals corn-fed predominantly in the cerebral cortex. The reduction in GABA release may be attributable to a decrease in GABAergic cell density, which could induce an over-activation of 5-hydroxytryptamine (5-HTergic) receptors, leading in turn to the observed enhancement of GAD activity. Taken together, these results may represent a plastic response by GABAergic neurons to (5-HTergic under-stimulation in mainly corn-fed animals.     

Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity 3H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light 3H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib. High-affinity GABA uptake was demonstrated in the termination zone of the projection from the anterior olfactory nucleus to the anterior piriform cortex. Cell bodies of origin of this projection displayed heavy retrograde labeling with 3H-GABA. Matching neuropil and cellular labeling was demonstrated with the GABA-BSA antiserum but not with the GAD antiserum, thus suggesting that GABA is normally present in these cells but is taken up from the neuropil rather than synthesized. No comparable high-affinity GABA uptake was demonstrated in the association fiber systems that originate in the piriform cortex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bovine anterior pituitary progenitor cell line expresses interleukin (IL)-18 and IL-18 receptor

In the anterior pituitary gland, inflammatory mediators regulate cell function through an immuno-endocrine pathway. Recent studies have shown that undifferentiated stem cells act as immunomodulators. These studies prompted us to establish a progenitor cell line from the bovine anterior pituitary gland and to detail its function. First, we localised interleukin (IL)-18 by immunohistochemistry to the marginal cell layer of Rathke's pouch that is assumed to embody a stem/progenitor cell compartment of the postnatal pituitary gland. A cloned anterior pituitary-derived cell line from the bovine anterior pituitary gland was established from single cell clone by the limiting dilution method and was designated as bovine anterior pituitary-derived cell line (BAPC)-1. BAPC-1 cells constantly expressed mRNAs for IL-18 and IL-18 receptor, and grew steadily and rapidly in the medium containing epidermal growth factor and basic fibroblast growth factor. The cell line also expressed the mRNAs for the stem/progenitor cell- related factors such as Nanog, Oct-4, Ptch1, Nestin, Notch1, Hes1, Lrp and Fzd4, and the mRNAs for embryonic pituitary-related factors, such as Lhx3, PitX1 and Pit-1. The nuclei of BAPC-1 were immunostained positively for Pit-1, Hes1 and beta-catenin antibodies. Furthermore, BAPC-1 cells expressed mRNAs for cytokine such as IL-1alpha, IL-6, IL-7, IL-12 and IL-15. Stimulation of BAPC-1 cells with IL-18 increased expression of mRNAs for IL-1alpha, IL-6, IL-1beta and IL-8. At day 6 in culture, BAPC-1 cells also express growth hormone mRNA. These results strongly suggest that BAPC-1 is a stem/progenitor cell line and modulates the immuno-endocrine function of the anterior pituitary cells through its cytokine production.     

实验性癫痫与γ-氨基丁酸和谷氨酸脱羧酶的关系

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Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content

The M213-20 and M213-1L cell lines were immortalized from rat striatum using the tsA58 allele of the SV40 large T antigen, contain the GAD enzyme, and produce GABA (Giordano et al., 1994, Exp. Neurol. 124:395-400). Cell lines that produce large amounts of GABA may be useful for transplantation into the brain in conditions such as Huntington's disease or epilepsy, where localized application of GABA may be of therapeutic value. We have explored the potential use of the pREP10 plasmid vector, which replicates episomally, to increase GAD expression and GABA production in M213-20 and M213-1L cells. Human GAD(67) cDNA was transfected into M213-20 and M213-1L, and subclones were isolated with hygromycin selection. Immunochemical studies showed increased GAD(67) expression compared to the parent M213-20 and M213-1L cell lines. Staining for the EBNA antigen and Southern blots demonstrated that the pREP10 plasmid was stably maintained in the cells for at least 12-15 months in culture. Several clones were isolated in which GABA concentrations were increased by as much as 4-fold (M213-1L) or 44-fold (M213-20) compared to the parent cell lines or 12-fold (M213-1L) and 94-fold (M213-20) greater than rat striatal tissue (1.678 +/- 0.4 micromol/g prot). The ability of these cells to continue to produce large amounts of GABA while being maintained in culture for extended periods suggests that similar methods might be used with human cell lines to produce cells that can be transplanted into the brain to deliver GABA for therapeutic purposes.     

gamma-Aminobutyric acid (GABA) enhances glutamate cytotoxicity in a cerebellar cell line

A temperature-sensitive rat cerebellar cell line SC9 has been used to study the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in glutamate cytotoxicity. GABA increases glutamate toxicity in a dose-dependent fashion, but NMDA and kainic acid were not toxic in the presence or absence of GABA. The specificity of this cytotoxicity was further indicated by the NMDA-selective antagonist 2-amino-7-phosphonoheptanoic acid (APV), which does not block glutamate effect. These observations, as well as binding experiments with 3H-glutamate, suggest that glutamate cytotoxicity in these cells depends on quisqualate-selective uptake sites of the amino acid. The study may open therefore a novel pathway for understanding the cytotoxic effect of excitatory amino acids in brain structures that are enriched with GABA and glutamate uptake sites.     

Non-correspondence of [3H]GABA uptake and GAD localization in goldfish amacrine cells

The effects induced by penicillin (PEN) upon the synaptic responses of CA1 hippocampal pyramidal cells (HPCs) were studied in the 'in vitro' slice. Low concentrations of PEN (0.17-0.34 mM) evoked an increase in amplitude and duration of the orthodromic excitatory post-synaptic potential induced by stratum radiatum, while stimuli which were subthreshold in control conditions became effective in eliciting action potentials. These changes were not paralleled by any decrease of the recurrent inhibitory post-synaptic potential due to inhibitory interneurons located at or near the soma. However, the latter decreased and then disappeared as PEN concentrations were brought to levels higher than 0.68 mM. Since low concentrations of PEN increase CA1 HPCs responsiveness without decreasing somatic inhibition, it is concluded that this action is probably due to a reduced dendritic inhibitory mechanism.