PubMed Health⌕ Search

Biomedical subjects

L F Eng

Publications and source records attributed to L F Eng.

At least 19 recordsLinked to original sources
PubMed1996

Astrocyte-astrocytoma cell line interactions in culture.

Astrocytomas are the most common brain tumors arising in the CNS and account for 65% of all primary brain tumors. Astrocytes have been shown to have the highest predisposition to malignant transformation compared to any other CNS cell type. The majority of astrocytomas are histologically malignant neoplasm. Previous studies have shown that resident astrocytes are the first cell type to react to tumors and surround them. However, the role of these astrocytes in tumor formation and progression has not been determined. In the present study, we have co-cultured astrocytes with a permanent cell line S635c15 (derived from anaplastic astrocytoma) in order to understand the cellular interactions between astrocytes and astrocytoma cells. Our studies demonstrate that astrocytes in contact with the tumor cells become reactive and fibrous with an increase in glial fibrillary acidic protein (GFAP) immunoreactivity as early as 4 days in culture. By 8 days, astrocytes formed glial boundaries around the tumor cells which grew as round colonies. The astrocytic processes surrounding the tumor cells were also intensely GFAP positive. Since the behavior of these cells observed in culture is very similar to their interaction seen in vivo, this co-culture system may serve as an in vitro model for astrocyte and astrocytoma cell line interaction and aid in our understanding of the molecular and cellular mechanisms during early stages of tumor formation and cell interactions.

Animals↗
PubMed1996

Inflammation in EAE: role of chemokine/cytokine expression by resident and infiltrating cells.

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) which has many clinical and pathological features in common with multiple sclerosis (MS). Comparison of the histopathology of EAE and MS reveals a close similarity suggesting that these two diseases share common pathogenetic mechanisms. Immunologic processes are widely accepted to contribute to the initiation and continuation of the diseases and recent studies have indicated that microglia, astrocytes and the infiltrating immune cells have separate roles in the pathogenesis of the MS lesion. The role of cytokines as important regulatory elements in these immune processes has been well established in EAE and the presence of cytokines in cells at the edge of MS lesions has also been observed. However, the role of chemokines in the initial inflammatory process as well as in the unique demyelinating event associated with MS and EAE has only recently been examined. A few studies have detected the transient presence of selected chemokines at the earliest sign of leukocyte infiltration of CNS tissue and have suggested astrocytes as their cellular source. Based on these studies, chemokines have been postulated as a promising target for future therapy of CNS inflammation. This review summarized the events that occur during the inflammatory process in EAE and discusses the roles of cytokine and chemokine expression by the resident and infiltrating cells participating in the process.

Animals↗
PubMed1995

Macrophage inflammatory protein 1-alpha mRNA expression in an immortalized microglial cell line and cortical astrocyte cultures.

Macrophage inflammatory protein 1 (MIP-1) is a recently characterized inflammatory and chemokinetic cytokine. Proinflammatory stimuli have been shown to induce expression of MIP-1 by macrophages. We hypothesized that microglia and astrocytes express MIP-1 alpha because of their many immunologic similarities to macrophages. MIP-1 alpha mRNA was examined with quantitative reverse transcription and polymerase chain reaction in an immortalized mouse microglial cell line (BV-2) and in mouse cortical astrocyte cultures. We found that in both the BV-2 microglial cell line and in astrocyte cultures, MIP-1 alpha mRNA was strongly induced by lipopolysaccharide and the phorbol ester PMA. MIP-1 alpha mRNA was reduced by dBcAMP, interferon-gamma, and PGE1. Dexamethasone decreased MIP-1 alpha mRNA levels in astrocyte cultures, but not in BV-2 microglial cells. Interleukin-1 beta, tumor necrosis factor alpha, and MIP-1 alpha had no effect on MIP-1 alpha mRNA expression. These findings demonstrate that MIP-1 alpha mRNA is expressed by cultured glial cells and is regulated by proinflammatory and anti-inflammatory stimuli. MIP-1 alpha may be expressed by microglia and astrocytes in vivo, and may help modulate cerebral inflammation.

Animals↗
PubMed1995

Astrogliosis in culture. IV. Effects of basic fibroblast growth factor.

Previous studies have shown that the mechanical wounding of 3-week-old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti-bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti-bFGF antibody (100 micrograms/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc-AMP) reduced injury-associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti-bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty-four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti-bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc-AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis.

Animals↗
PubMed1995

Leukemia inhibitory factor mRNA is expressed in cortical astrocyte cultures but not in an immortalized microglial cell line.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine synthesized by a variety of cell types. In the nervous system LIF affects neuronal differentiation, and may be important during cerebral infection and inflammation. To clarify the cellular source of LIF in the brain, we examined the expression of LIF mRNA by primary cortical astrocyte cultures and an immortalized microglial cell line. The microglial cell line did not express LIF mRNA in response to pro-inflammatory agents such as lipopolysaccharide (LPS) that induced expression of other cytokine mRNAs. In contrast, primary astrocyte cultures grown in serum-containing medium expressed LIF mRNA constitutively, and this expression was regulated by pro-inflammatory and anti-inflammatory stimuli. Agents which activate the cAMP and protein kinase C second messenger systems also increased LIF mRNA in astrocyte cultures. These results suggest that astrocytes, but not microglia, may be an important source of LIF during cerebral inflammation and infection.

Animals↗
PubMed1995

Chondroitin sulfate proteoglycan staining in astrocyte-Schwann cell co-cultures.

Transplantation of Schwann cells (SCs) in the central nervous system (CNS) for remyelination in pathological situations has been considered a promising approach. However, numerous studies have indicated that astrocytes have a restrictive effect on SC migration within the CNS. We have previously established an in vitro model which demonstrates the restrictive effect of astrocytes on SCs (Ghirnikar and Eng, Glia 4:367-377, 1994). Using this culture model, in the present study, we have characterized the molecular basis underlying astrocyte-SC interaction and demonstrated chondroitin sulfate proteoglycan (CSP) staining in the co-cultures. Following 1-2 weeks of incubation, CSP staining was specifically associated with SCs co-cultured with astrocytes. Staining with antibodies specific for the different chondroitin sulfate isomers revealed the presence of both, chondroitin-4- and 6-sulfates in SCs. In contrast, SCs when cultured alone, or in the presence of astrocytes conditioned medium did not show CSP staining. These data suggest that CSP staining is associated with SCs following co-culture with astrocytes and mediated by cell to cell contact. We hypothesize that the CSP, alone or in combination with other molecules expressed by astrocytes and/or SCs, may be involved in the restrictive effects of astrocytes on SCs. Identification of molecules involved in the unfavorable interaction between astrocytes and SCs will have an important bearing on efforts to remyelinate demyelinated axons by SC transplantation within the damaged CNS.

Animals↗
PubMed1995

Gene expression in astrocytes during and after ischemia.

Involvement of the IEGs in brain injury and ischemia is under intensive investigation (Gubits et al., 1993). There are several families of the IEGs. They include the fos, jun, and zinc finger genes that encode transcription factors. Products of the fos family (c-fos, fra-1, fra-2, and fos B) bind to members of the jun family (c-jun, jun B, jun D) via leucine zippers, and this dimer then binds to the AP-1 site (consensus sequence -TGACTCA-) in the promoter of target genes, which in turn regulate the expression of late response genes that produce long-term changes in cells. For example, c-fos may regulate the long-term expression of preproenkephalin, nerve growth factor, dynorphin, vasoactive intestinal polypeptide, tyrosine hydroxylase and other genes with AP-1 sites in their promoters (Curran and Morgan, 1987; Sheng and Greenberg, 1990). It is likely that the c-fos gene up-regulation observed in ischemic astrocytes leads to the changes observed in the expressions of hsp and cytoskeleton protein genes in this experimental model. This is supported by the findings of Sarid (1991) and Pennypacker et al. (1994) who have shown that AP-1 DNA binding activity in hippocampus recognized an AP-1 sequence from the promoter region of the GFAP which is a potential target gene. van de Klundert et al. (1992) also suggested the involvement of AP-1 in transcriptional regulation of vimentin. IEGs can be induced within minutes by extracellular stimuli including transmitters, peptides, and growth factors. In this study, we have shown that c-fos induction by ischemia was rapid and transient.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Tumor necrosis factor-alpha and basic fibroblast growth factor decrease glial fibrillary acidic protein and its encoding mRNA in astrocyte cultures and glioblastoma cells.

Tumor necrosis factor-alpha is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor-alpha. Treatment with tumor necrosis factor-alpha for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor-alpha treatment increased the expression of the cytokine interleukin-6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor-alpha, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor-alpha induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor-alpha. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor-alpha treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.

Animals↗
PubMed1994

Astrogliosis in culture: III. Effect of recombinant retrovirus expressing antisense glial fibrillary acidic protein RNA.

Injury to the central nervous system (CNS) either from trauma or due to demyelinating/degenerating diseases results in a typical response of astrocytes, termed astrogliosis. This reaction is characterized by astrocyte proliferation, extensive hypertrophy of nuclei, cell body, and cytoplasmic processes and an increase in immunodetectable glial fibrillary acidic protein (GFAP). GFAP accumulation may cause a physical barrier preventing the reestablishment of a functional environment. Our studies have aimed at modulating astrogliosis by inhibiting or delaying GFAP synthesis in damaged and reactive astrocytes. The present study investigates the use of a recombinant retrovirus expressing antisense GFAP RNA in controlling the response of mechanically injured astrocytes. A 650 bp fragment from the coding region of mouse GFAP cDNA was cloned in the antisense orientation under the control of long terminal repeat (LTR) promoter of Moloney murine leukemia virus. Increase in GFAP as detected by immunocytochemical staining in injured astrocytes was inhibited by treatment with retrovirus expressing antisense GFAP RNA. Also, astrocytes at the site of injury in these scratched cultures did not show cell body hypertrophy compared to control cultures. These observations demonstrate that the increase in GFAP at the site of injury can be inhibited using retroviral treatment and indicate the potential of retrovirus-mediated gene transfer in modulating scar formation in the CNS in vivo. These studies also shed light on the role of GFAP in maintaining the morphology of astrocytes.

Animals↗
PubMed1994

Astrocyte-Schwann cell interactions in culture.

After injury, either as a result of trauma or degenerating/demyelinating diseases, axons of the central nervous system (CNS) normally fail to regenerate. Transplantation of glial cells, particularly Schwann cells, into areas of injury or demyelination has been considered a promising approach to promote recovery. However, the extent of Schwann cell interaction with CNS axons is greatly influenced by the presence of astrocytes which redefine the CNS-PNS (peripheral nervous system) boundary in a lesioned CNS, thereby preventing invasion of Schwann cells. The molecular basis for this restrictive effect of astrocytes on Schwann cells is not known. In the present study, we have cocultured astrocytes and Schwann cells to develop an in vitro model to characterize this interaction. Astrocytes in contact with Schwann cells appeared hypertrophied and showed increased staining for glial fibrillary acidic protein (GFAP). In cocultures maintained for 2-3 weeks, segregation of the two cell types was observed, Schwann cells appeared in groups, and each group was surrounded and separated from one another by astrocytic processes. Since the behavior of these two cell types observed in culture is very similar to their interaction seen in vivo, this coculture model may be useful in further studying the relationship between astrocytes and Schwann cells.

Animals↗
PubMed1994

GFAP and astrogliosis.

One of the most remarkable characteristics of astrocytes is their vigorous response to diverse neurologic insults, a feature that is well conserved across a variety of different species. The astroglial response occurs rapidly and can be detected within one hour of a focal mechanical trauma (Mucke et al., 1991). Prominent reactive astrogliosis is seen; in AIDS dementia; a variety of other viral infections; prion associated spongiform encephalopathies; inflammatory demyelinating diseases; acute traumatic brain injury; neurodegenerative diseases such as Alzheimer's disease. The prominence of astroglial reactions in various diseases, the rapidity of the astroglial response and the evolutionary conservation of reactive astrogliosis indicate that reactive astrocytes fulfill important functions of the central nervous system (CNS). Yet, the exact role reactive astrocytes play in the injured CNS has so far remained elusive. This chapter summaries the various experimental models and diseases that exhibit astrogliosis and increase in glial fibrillary acidic protein (GFAP). Recent in vitro studies to inhibit GFAP synthesis are also presented.

Animals↗
PubMed1994

Development of a monoclonal antibody specific for the COOH-terminal of beta-amyloid 1-42 and its immunohistochemical reactivity in Alzheimer's disease and related disorders.

The beta-amyloid peptide (beta AP) has been characterized by protein sequencing techniques as a 39-43 amino acid protein with heterogeneous COOH-termini. Controversy exists regarding the predominant form of beta AP in neuritic plaques (NP) and cerebral vasculature of Alzheimer's disease (AD) brain. A monoclonal antibody was developed that selectively recognizes the free COOH-terminal of beta AP 1-42 but not beta AP species with shorter or longer COOH-termini. Brain sections from AD and related disorders were examined using this antibody. In AD samples, the antibody stained diffuse amyloid and NP cores, many intraneuronal and extraneuronal neurofibrillary tangles (NFT), but not cerebrovascular amyloid. Pick and Lewy bodies lacked immunoreactivity. These findings suggest that beta AP 1-42 is present in early and mature amyloid deposits and NFT, but that species of beta AP other than 1-42 comprise human vascular deposits.

Adult↗
PubMed1993

Reverse transcription and polymerase chain reaction technique for quantification of mRNA in primary astrocyte cultures.

The reverse transcription and polymerase chain reaction technique (RT-PCR) was assessed for the quantification of changes in mRNA levels from primary astrocyte cultures. The effects of dibutyryl cyclic AMP (dBcAMP) on glial fibrillary acidic protein (GFAP) mRNA and the effects of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS) on interleukin-6 (IL-6) mRNA were examined. Two quantitative PCR methods were used: one involved carrying out the reaction in the exponential phase and the other involved the coamplification of a competitive target sequence. Increased GFAP mRNA in response to chronic dBcAMP treatment and increased IL-6 mRNA in response to TNF-alpha/IL-1 beta were readily detected. Both RT-PCR techniques were found to be suitable for the detection of large as well as smaller (twofold) changes in mRNA levels. The advantages and limitations of RT-PCR for mRNA quantification are discussed.

Animals↗
PubMed1993

Astrogliosis in culture: I. The model and the effect of antisense oligonucleotides on glial fibrillary acidic protein synthesis.

Astrogliosis is a predictable response of astrocytes to various types of injury caused by physical, chemical, and pathological trauma. It is characterized by hyperplasia, hypertrophy, and an increase in immunodetectable glial fibrillary acidic protein (GFAP). As GFAP accumulation is one of the prominent features of astrogliosis, inhibition or delay in GFAP synthesis in damaged and reactive astrocytes might affect astrogliosis and delay scar formation. The aim of this study is to investigate the possibility of utilizing antisense oligonucleotides in controlling the response of astrocytes after mechanically induced injury. We scratched primary astrocyte cultures prepared from newborn rat cerebral cortex with a plastic pipette tip as an injury model and studied the astrogliotic responses in culture. Injured astrocytes became hyperplastic, hypertrophic, and had an increased GFAP content. These observations demonstrate that injured astrocytes in culture are capable of becoming reactive and exhibit gliotic behaviors in culture without neurons. The increase in GFAP content in injured astrocytes could be inhibited by incubating the scratched culture with commercially available liposome complexed with 3' or 5' antisense oligonucleotides (20 nt) in the coding region of mouse GFAP. The scratch model provides a simple system to examine in more detail the mechanisms involved in triggering glial reactivity and many of the cellular dynamics associated with scar formation. Antisense oligonucleotide treatment could inhibit the GFAP synthesis in injured astrocytes, hence it may be applicable in modifying scar formation in CNS injury in vivo.

Animals↗
PubMed1993

Immunohistochemical localization of basic fibroblast growth factor in cultured rat astrocytes and oligodendrocytes.

The distribution of basic fibroblast growth factor in cultured astrocytes and oligodendrocytes was examined using immunocytochemistry. The results demonstrate a localization of basic fibroblast growth factor immunoreactivity predominantly in astrocyte nuclei at all stages of differentiation. Cytoplasmic and process staining was best detected during early stages of differentiation, under normal growth conditions or as a result of treatment with dibutyryl cyclic adenosine monophosphate. Astrocytes at all stages of differentiation bound antibody-complexed bFGF, suggesting the presence of cell-associated low affinity binding sites for the growth factor. Our studies also show the presence of immunoreactivity for basic fibroblast growth factor in process-bearing oligodendrocytes. These results suggest a role for endogenous basic fibroblast growth factor in astrocyte and oligodendrocyte growth and function.

Animals↗