Cultures Of Human Glial Cells Research Articles

Localization of Viral Antigens Improves Understanding of Congenital Rubella Syndrome Pathophysiology

Rubella virus (RV) is responsible for benign postnatal and deadly antenatal infections. Its involvement in developmental defects in children following congenital transmission was first made by Sir Norman McAlister Gregg in 1941. Indeed, the maternal primary infection results in an RV viremia and placental transfer. If it occurs before 12 weeks of gestation, during organogenesis, it can result in severe fetal damage causing miscarriage or malformations primarily affecting the cardiovascular system, eyes, ears and the central nervous system; it is fatal in nearly one third of cases during the first year of life (Tondury and Smith, 1966). This severe RV infection acquired in utero early in pregnancy is named as Congenital Rubella Syndrome or CRS. In 2014, 141 CRS cases were reported from 114 countries mostly from South-East Asia (n = 86/141), although reporting is probably inconsistent according to the authors themselves (Grant et al., 2015). This number had been estimated as closer to 100,000 cases each year in developing countries alone (Cutts and Vynnycky, 1999). To better understand the pathogenesis of CRS in the absence of an animal model, the first histopathological explorations were conducted in some of the 20,000 fetuses infected during the 1964–1965 epidemics in the US when about 12.5 M Rubella cases were reported (National Communicable Disease Center, 1969). Although these studies identified histological lesions in the heart, blood vessels, crystalline lens, ears, brain, teeth and liver consistent with clinical features and sequelae of CRS, they did not solved the mechanisms of virus teratogenicity (Tondury and Smith, 1966, Esterly and Oppenheimer, 1969). In particular, until recently, the exact nature of the cell types infected with RV remained unknown.

MicroRNA-146a: a key regulator of astrocyte-mediated inflammatory response.

Increasing evidence supports the involvement of microRNAs (miRNA) in the regulation of inflammation in human neurological disorders. In the present study we investigated the role of miR-146a, a key regulator of the innate immune response, in the modulation of astrocyte-mediated inflammation. Using Taqman PCR and in situ hybridization, we studied the expression of miR-146a in epilepsy-associated glioneuronal lesions which are characterized by prominent activation of the innate immune response. In addition, cultured human astrocytes were used to study the regulation of miR-146a expression in response to proinflammatory cytokines. qPCR and western blot were used to evaluate the effects of overexpression or knockdown of miR-146a on IL-1β signaling. Downstream signaling in the IL-1β pathway, as well as the expression of IL-6 and COX-2 were evaluated by western blot and ELISA. Release several cytokines was evaluated using a human magnetic multiplex cytokine assay on a Luminex® 100™/200™ platform. Increased expression of miR-146a was observed in glioneuronal lesions by Taqman PCR. MiR-146a expression in human glial cell cultures was strongly induced by IL-1β and blocked by IL-1β receptor antagonist. Modulation of miR-146a expression by transfection of astrocytes with anti-miR146a or mimic, regulated the mRNA expression levels of downstream targets of miR-146a (IRAK-1, IRAK-2 and TRAF-6) and the expression of IRAK-1 protein. In addition, the expression of IL-6 and COX-2 upon IL-1β stimulation was suppressed by increased levels of miR-146a and increased by the reduction of miR-146a. Modulation of miR-146a expression affected also the release of several cytokines such as IL-6 and TNF-α. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation and suggests that this miR may represent a novel target for therapeutic strategies.

Autoregulation of co‐chaperone BAG3 gene transcription

The Bcl-2-associated athanogene, BAG, protein family through their BAG domain associates with the heat shock protein 70 (HSP-70) and modulates its chaperone activity. One member of this family, BAG3, appears to play an important role in protein homeostasis, as its expression promotes cell survival. Expression of BAG3 is enhanced by a variety of stress-inducing agents. Here we describe a role for BAG3 to modulate transcription of its own promoter through a positive feedback loop involving its 5'-UTR sequence. Activation of the BAG3 promoter is mediated by the BAG domain and is independent of BAG3 association with the UTR sequence. Autoactivation of the BAG3 gene is observed in several cultures of human glial cells including gliomas, but not in several other non-glial cell lines such as He La and others. Results from cell fractionation and immunocytochemistry showed BAG3 in the cytoplasm as well as the nuclei of glial cells. These observations suggest that BAG3 gene expression is controlled by its own product and that this may be critical for the biological activity of BAG3 in some cell types.

Expression of CXCL8, CXCR1, and CXCR2 in Neurons and Glial Cells of the Human and Rabbit Retina

Several eye diseases are accompanied by inflammatory processes. The authors examined the expression of the proinflammatory chemokine CXCL8 and the corresponding receptors in healthy human retinas, in cellular membranes from patients with proliferative vitreoretinopathy (PVR) or human glial cell cultures and in an animal model of PVR in rabbit eyes. The authors used immunohistochemical methods, Western blotting, RT-PCR, and real time RT-PCR to characterize the expression of CXCL8, CXCR1, and CXCR2 in human and rabbit retinas. Functionality of the receptors in cultured glial cells was tested by Ca(2+) imaging. Immunohistochemical examinations of normal human and rabbit retinas revealed a distinct expression of CXCR1 and CXCR2 in several neuronal cell types. CXCL8 mRNA was demonstrated only by RT-PCR in normal retinas, and receptor expression was confirmed by Western blotting and RT-PCR. The presence of CXCR1 and CXCR2, but not CXCL8, was detected by immunostaining in glial fibrillary acidic protein-positive glial cells of cellular PVR membranes. Immunoreactivity for CXCL8, CXCR1, and CXCR2 was observed in virtually all cultured glial cells and in the human Müller cell line MIO-M1. Müller cells responded to the application of CXCL8 with increased cytosolic Ca(2+) concentrations. In PVR rabbit retinas, CXCR1 expression is increased in Müller cells, and CXCL8 and CXCR2 are strongly expressed in microglial cells. Expression of CXCL8 and CXCL8 receptors in glial cells of human PVR membranes and rabbit PVR retinas suggests an involvement in glial reactivity. Furthermore, the prominent expression of CXCR1 and CXCR2 in neurons of the healthy human and rabbit retina suggests additional physiological functions.

Effects of human retinal glial cells under hypoxic conditions on circulating endothelial progenitor cells

The specific signaling events by which circulating endothelial progenitor cells (EPCs) responding to alterations of the retinal vasculature induced by hypoxia are not yet completely understood. Our goal was to generate a suitable experimental model for the identification of such factors as well as the effects of these factors on the EPCs. Primary cultures of human retinal glial cells were incubated under hypoxic or normoxic conditions. Effects of hypoxia were confirmed by analysis of HIF-lalpha and VEGF expression. Circulating EPCs (CD34 +, CD133+ double-positive cells) were isolated by FACS Vantage from the human periphery blood. EPCs were treated with conditioned media from retinal glial cells incubated under either hypoxic or normoxic conditions. Effects of conditioned media on the proliferation and differentiation of EPCs were evaluated. Incubation of human retinal glial cells at low oxygen concentration for 24 hours lead to HIF-lalpha expression and a significant increase of VEGF released to the media[ (319. 16 +/-34. 12) pg/ml versus (220. 28+/-24. 33) pg/ml] as compared to the control cultures incubated at normoxic conditions (P <0. 01). Treatment of EPCs with conditioned medium from retinal glial cells incubated at low oxygen concentration resulted in an increase of proliferative activity (day 3: 0. 83+/-0. 10 versus 0. 61+/-0. 22,P <0. 05; day 7: 1. 53+/-0. 21 versus 0. 88+/-0. 25, P <0. 05) and presence of differentiated endothelial cells as compared to cells cultured with conditioned media from normoxic cultures. Conditioned media from retinal glial cells contain factors capable of activating the proliferation and differentiation of circulating EPCs. Analysis of the effects of conditioned medium from neuronal glial cells incubated under hypoxic conditions on circulating human EPCs might be useful in providing an effective experimental method for the identification of the specific signaling molecules involved in the activation of circulating EPCs for the regeneration of retinal blood vessels.

Selective inhibition of human glial inducible nitric oxide synthase by interferon‐β: Implications for multiple sclerosis

Nitric oxide generated from the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of multiple sclerosis. Because significant species- and cell-specific differences exist in the expression of iNOS, we used primary human glial cell cultures to screen for an inhibitor of iNOS expression. Remarkably, among numerous soluble factors tested, interferon-beta (IFN-beta) alone showed a selective and potent inhibition of interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma)-induced iNOS expression in astrocytes. Inhibition of iNOS may provide a mechanism by which IFN-beta can ameliorate inflammation and cytotoxicity in the central nervous system of patients with multiple sclerosis.

Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization.

We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3-9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte-specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP-positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1-3%) were present in the human adult astrocyte cultures, using a panel of macrophage-specific markers. However, the monoclonal antibodies (mAbs KP1, EBM1, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/ microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non-lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay).

A Study on Tumor Necrosis Factor, Tumor Necrosis Factor Receptors, and Nitric Oxide in Human Fetal Glial Cultures

This chapter presents the main novel findings concerning human fetal astrocyte tumor necrosis factor receptors (TNF-Rs) and nitric oxide (NO). The focus is on interleukin-1β (IL-1β) and Interferon-γ (IFN-γ) treatment that leads to increased soluble TNF-R concentrations in astrocyte supernatants without a detectable change in surface TNF-Rs, the inhibition of cytokine-induced soluble TNF-Rs by pentoxifylline (PTX); but it does not affect surface TNF-R, the association of elevated soluble TNF-R levels with increased TNF and NO production. It is concluded that during CNS inflammation, such as MS, NO production is accompanied by changes in TNF-Rs, which may either limit or prolong the effects that NO has on surrounding cells or invading pathogens. Indeed, NO concentrations may be regulated, in part, by modulating TNF-R shedding. Recent findings suggest that TNF effects on a target cell are mediated, in part, by the presence of soluble extracellular TNF receptor (TNF-R) fragments cleaved from intact surface receptors by proteases. These soluble TNF-Rs can bind to TNF and inhibit or prolong its activity, and are additional pathways by which TNF can be modulated. Thus, the maintenance of NO levels within a nonpathological range may depend not only on limiting inducible nitric oxide synthase (iNOS), but also on regulating cytokine pathways associated with iNOS activation. One of the aims of the chapter is to discuss the evidence of the production and regulation of TNF, TNF-Rs, and NO production in human glial cell cultures. These studies are based on the hypothesis that TNF, IL-1, and NO mediate demyelination in multiple sclerosis (MS) plaques and neuronal damage in acquired immunodeficiency syndrome (AIDS) dementia, both of which may contribute to the clinical manifestations of these conditions.

The identification of four protein kinase C isoforms in human glioblastoma cell lines: PKC alpha, gamma, epsilon, and zeta

Levels of protein kinase C (PKC) isoforms in eight human glioblastoma cell lines and two normal human glial cell cultures were determined. Earlier studies identified PKC-alpha and PKC-gamma in these cell lines but PKC-beta was not present. In this study, PKC-epsilon and PKC-zeta are demonstrated immunologically in these cell lines and also in two normal human glial cell cultures. Protein kinase C-delta was not present. When levels of the four isoforms in the tumor cells were compared to levels in the normal cells, no increase was observed in PKC-alpha or PKC-gamma, but PKC-epsilon was elevated three to 30 times in six of the eight tumors, and PKC-zeta was elevated approximately two times in all of the tumors. Incubation of cell line A172 with phorbol ester for 6 hours resulted in a 48-fold maximum increase in the nuclear PKC-epsilon and a sevenfold increase in the plasma membrane fraction with no change in the cytoplasmic fraction. A similar incubation for 4 hours produced a 0.5- to onefold increase of PKC-zeta in cytoplasmic, nuclear, and plasma membrane fractions. Other researchers have shown that overexpression of PKC-epsilon in fibroblasts results in tumorigenesis, and that blocking PKC-zeta function inhibits deoxyribonucleic acid synthesis. These data suggest that alteration in the expression of PKC-epsilon and PKC-zeta could be a factor in the conversion of normal glial cells to glioblastomas.

Detection of human immunodeficiency virus DNA in cultured human glial cells by means of the polymerase chain reaction

This report describes the use of the polymerase chain reaction (PCR) for the detection of viral genomic sequences in latently infected cells. Infection with human immunodeficiency virus in cultures of human glial cells was demonstrated, using nucleic acid amplification followed by dot blot hybridization. It was not possible to detect any viral antigen production in the cultures, and attempts to recover virus by highly sensitive coculture techniques were unsuccessful, indicating that the infection was latent. The PCR technique provides a simple approach to the study of viral infection in cases where viral replication is absent, or where genomic copies are present at such low numbers that they are otherwise undetectable.

The waste-product theory of aging: Transformation to unlimited growth in cell cultures

A differential equation governing intracellular waste content is solved numerically to determine the circumstances under which the growth of an in vitro cell population is limited. Parameter values derived from data on human glial cell cultures are employed. It is assumed that a) waste accumulation depresses the rate of cellular reproduction and b) intracellular waste is diluted by cell division, but is not otherwise eliminated. Population size depends upon two parameters: the rate of waste production and the rate of cell division in the absence of waste. If the rate of waste production is sufficient, the population size approaches an asymptote as in phase III growth in vitro. If a lower rate of waste production allows the cells to outmultiply the waste, growth is unlimited as in a tranformed cell population. The asymptotic population size and the threshold for unlimited growth are remarkably sensitive to small changes in the values of the two rate parameters unless the ratio of their values is constant. This suggests that there may be a cellular mechanism that relates the waste production and cell division rates.

Antigen expression by glial cells grown in culture

Cell type-specific markers are currently available for identifying major cell types in neural cell cultures. The markers considered specific for glial cells are numerous, but only galactocerebroside and myelin basic protein for oligodendrocytes, and glial fibrillary acidic protein for astrocytes are widely adopted by investigators for that purpose. Other surface and intracellular markers which are specific for oligodendrocytes or astrocytes in vitro are briefly described. The possibility of using different classes of gangliosides as cell type-specific markers in neural cell culture is discussed. The presence of "transitional" or "bipotential" glial cells that express both oligodendrocytic and astrocytic phenotypes in human glial cell cultures and the regulatory effect of cyclic AMP derivatives on these cells are reported. In addition, the presence of Ia antigens on the surface of a selected population of cultured human oligodendrocytes and astrocytes is described.

Identification and characterization of non-dividing cell populations in phase II cultures of human glial cells

It has been reported that cultures of normal cells in phase II contain a non-multiplying cell population, the size of which increases with passage number. In phase II cultures of normal human glial cells we have found two subpopulations of non-proliferating cells, one of which has a characteristic morphology, and differs from the actively dividing cells in a number of respects: (1) they are larger although of various sizes and are well spread over a very large substratum area: (2) they contain a great number of granules showing acid phosphatase activity, being heavy metal positive and displaying the characteristic natural fluorescence of lipofuscin pigment; (3) they frequently contained a central somewhat irregular nucleus with various numbers of darkly straining nucleolar-like structures. Cytophotometric nuclear DNA measurements of the described “large” cell population show a decreased proportion of diploid cells as compared to their smaller sister cells. Moreover, with increasing passage number, the DNA values for large cells shift towards higher ploidy levels resulting in a scattered aneuploid pattern in the oldest passage. This “large” cell subpopulation consists of between 2% and 3% of all passages and become greatly decreased following subcultivation. The other subpopulation of non-dividing cells is generally morphologically similar to the dividers, increases in size with passage number and is the more important in the phase III phenomenon.

A quantitative analysis of the aging of human glial cells in culture.

The kinetics of aging of normal human diploid brain cells in culture have been determined using the miniclone technique in which cells are cloned in the presence of a large number of other cells. The miniclone technique records the behaviour of every viable cell in the sample, not merely those cells capable of forming visible clones. This technique permits the direct measurement of the reproductive potential of individual cells growing in bulk culture and of the dispersion of the sizes of colonies generated by dividing cells. The fraction of cells that are able to divide declines smoothly and continuously from the beginning of in vitro cultures of human glial cells. There is a broad distribution of colony sizes; even at the earliest passages there are significant numbers of small colonies. With increasing age of the culture there is a shift in the distribution, so that fewer large colonies and more small colonies occur. The distribution of intermitotic times is almost identical in young and middle-aged cultures. Our data seem to exclude quite positively any description in terms of a catastrophe or any abrupt change in the population. On the contrary, the decline in reproductive potential may be described adequately either as a linear change with time, or as predicted by the mortality theory of Shall and Stein (1979), in which the single constant, gamma, describes the change in reproductive potential over the entire lifetime.

Electron probe X-ray microanalysis of residual bodies in aged cultured human glial cells.

Secondary lysosomes of the residual body type are frequent in nondividing cells from phase III cultures of human glial cells. These organelles have previously been shown to be analogous to lipofuscin granules of postmitotic cells in vivo. Most recent studies favor the assumption that residual bodies mainly result from incomplete degradation within the lysosomal vacuome of endogenous cellular components such as mitochondria and endoplasmic reticulum. Since iron occurs in several metalloenzymes produced by such organelles, it should then be possible to demonstrate accumulated iron within residual bodies. X-ray dispersive analysis of sectioned biological material is often hampered by diffusion and dissolution during preparation, as well as by too low a concentration of the elements. In this study we cultured glial cells on Formvar-coated gold grids and studied them unsectioned, after brief glutaraldehyde fixation and freeze-drying, in a transmission electron microscope at 100 kV in TEM and STEM mode. It was then possible to demonstrate iron in residual bodies of aged cells, presumably because the type of preparation utilized does not permit much dissolution.

Ammonium ion interferes with the lysosomal degradation of glycosaminoglycans in cultures of human glial cells

Summary Supplementation of human normal glial cells with NH 4 Cl led to marked effects on the metabolism of sulphated glycosaminoglycans (GAG). Sulphated GAG accumulated intracellularly in the presence of NH 4 Cl. Further, the degradation of sulphated GAG was partially inhibited. These phenomena were accompanied by an accumulation of lysosomes within the glial cells. The characteristics of NH 4 Cl-treated cells were thus similar to those displayed by cultured cells from patients with mucopolysaccharidosis.

Ammonium ion interferes with the lysosomal degradation of glycosaminoglycans in cultures of human glial cells

Ammonium ion interferes with the lysosomal degradation of glycosaminoglycans in cultures of human glial cells

Somatomedin A and B: demonstration of two different somatomedinlike components in human plasma.

This chapter discusses the presence of two different somatomedinlike components in human plasma. Assay methods were developed for the estimation of sulfate uptake in monolayer cultures of human glial cells, fibroblasts, or chick chondrocytes. An enhanced uptake of 35 SO 4 , obeying a linear dose–response relationship, was demonstrated after stimulation of the cultures with appropriate amounts of human serum. The 35 S-labeled chondroitin sulfate recovered from stimulated chondrocyte cultures had the same degree of sulfation, the same ratio of chondroitin 4-sulfate to chondroitin 6-sulfate and the same macromolecular behavior on gel chromatography as that isolated from nonstimulated cultures. A preparation of somatomedin (SM) from human plasma stimulated both sulfate uptake in chick cartilage and thymidine uptake in human glial cells or fibroblasts. Gel chromatography on Sephadex G-75, high voltage paper electrophoresis at pH 2, and isoelectric focusing showed that the cartilage factor was not identical with the glial or fibroblast factor, but differed both in regard to charge properties and molecular size. The tentative designations SM-A and SM-B were suggested for the cartilage and glial-fibroblast-stimulating factors, respectively.

The response of cultured human normal glial cells to growth factors.

This chapter discusses the response of cultured human normal glial cells to growth factors. Normal surface-attached animal cells in culture divide at the maximal rate only as long as free space is available. When the whole vessel surface is covered by cells, proliferation rate decreases, and a stationary phase appears at the terminal cell density. In this study, an assay system for growth factors was developed. Putative growth factors are added to serum-free human glial cell cultures, and the incorporation of tritiated thymidine is determined. The finding of a glial cell-stimulating polypeptide somatomedin B is reported. Fibroblast growth factor (FGF) was found to be a potent glial cell growth promoter. Human osteosarcoma cells in culture, release a growth factor active in the glial cell assay. The factor is nondialyzable, trypsin sensitive, and fairly heat stable. The chapter discusses its possible role in the deficient growth control of the osteosarcoma cells. The major part of the growth-promoting activity of human serum was found to be platelet dependent. Serum prepared from platelet-poor plasma contained less activity than serum from platelet-rich plasma.