Microglia and tumor (excluding TNF)

  1. Aoki K, Uchihara T, Sanjo N, Nakamura A, Ikeda K, Tsuchiya K, Wakayama Y (2003) Increased expression of neuronal apolipoprotein E in human brain with cerebral infarction. Stroke 34:875-880
    Abstract: BACKGROUND AND PURPOSE: Cellular origin of apolipoprotein E (ApoE) in the human brain and its roles in physiological and pathological conditions remain to be clarified. METHODS: Immunolocalization of ApoE was investigated in a series of autopsied human brains with or without infarction. ApoE expression was also estimated on immunoblot on protein extracts from autopsied brains and a cultured neuroblastoma cell line of human origin (GOTO) subjected to an oxidative stress induced by exposure to hydrogen peroxide (0.2 mmol/L). RESULTS: In addition to astrocytes and Microglia, neurons and degenerated axons in and around the ischemic foci contained ApoE-like immunoreactivity, which was more intense in recent ischemic foci. Immunoblot demonstrated an increase in expression of ApoE in brain extracts from ischemic lesion, and this increase was also pronounced in the cultured neuroblastoma cell line after the stress. CONCLUSIONS: Accumulation of ApoE in neurons in and around ischemic foci of the human brain is related to an increase in ApoE synthesis in neurons, as seen in cultured neuronal cells after oxidative stress. Intrinsic regenerative activity of neuron in reaction to external insults may be related to this increase in ApoE of neuronal origin

  2. Castilla EA, Prayson RA, Kanner AA, Rybicki LA, Tubbs RR, Vogelbaum MA, Barnett GH (2003) Cyclooxygenase-2 in oligodendroglial neoplasms. Cancer 98:1465-1472
    Abstract: BACKGROUND: Although increased expression of cyclooxygenase-2 (COX-2) has been described in association with a variety of neoplasms, including tumors of astrocytic derivation, limited data are available on COX-2 expression in oligodendrogliomas. METHODS: The current study retrospectively reviewed 53 oligodendrogliomas and 7 oligodendroglioma-predominant oligoastrocytomas (mixed gliomas) for COX-2 expression and MIB-1 proliferative index (by immunohistochemistry) and for chromosome 1p status (by fluorescence in situ hybridization). RESULTS: Patients included 35 males and 25 females, with a mean age of 41 years (range, 12-73 years) at the time of surgery. Forty-four tumor specimens were classified as World Health Organization (WHO) Grade II neoplasms and 16 as WHO Grade III tumors. MIB-1 labeling indices (marker of cell proliferation) ranged from 0 to 22.3 (mean 4.5). Twenty-eight tumor specimens demonstrated allelic loss on chromosome 1p. Positive staining was observed in 17 tumor specimens with COX-2 antibody. COX-2-positive tumor specimens were also evaluated with CD68 (macrophage/Microglial cell marker) by coimmunolabeling to confirm that the observed COX-2 immunostaining was not due to immunoreactive macrophages or Microglial cells. COX-2 expression, lack of allelic loss at chromosome 1p, and high proliferation indices were associated with decreased survival (P = 0.002, P = 0.009, and P = 0.015, respectively). No correlation with outcome was found with patient gender, age at diagnosis, or histologic grade. CONCLUSIONS: Chromosome 1p, COX-2 immunoreactivity, and MIB-1 labeling indices correlated with outcome and were associated with decreased survival. There was not a one-to-one correspondence between COX-2 immunoreactivity and lack of allelic loss at chromosome 1p. Tumors with expression of COX-2 by immunohistochemistry may, in theory, benefit from treatment with therapeutic agents that inhibit COX-2

  3. Das P, Estephan R, Banerjee P (2003) Apoptosis is associated with an inhibition of aminophospholipid translocase (APTL) in CNS-derived HN2-5 and HOG cells and phosphatidylserine is a recognition molecule in Microglial uptake of the apoptotic HN2-5 cells. Life Sci. 72:2617-2627
    Abstract: A balance of the activities of multiple enzymes maintains the typical asymmetry of plasma membrane lipids in healthy cells. Such enzyme activities are (a) the aminophopholipid translocase (APTL) (a lipid-selective P-type ATPase that catalyzes inward movement of aminophospholipids), (b) the scramblase (a calcium-dependent and ATP-independent enzyme that catalyzes both inward and outward movement of lipids), (c) the floppase (an ATP-dependent enzyme that catalyzes only outward movement of lipids). Activation or inhibition of any one of these enzymes would lead to a loss in this asymmetry. Apoptosis-associated externalization of phophatidylserine has been reported for many different cell-types, but the exact mechanism involved in this loss of membrane asymmetry has not been identified yet. In this report we demonstrate concurrence of APTL inhibition, caspase-3 activation and apoptosis in CNS-derived HN2-5 and HOG cells. Additionally, we provide data to demonstrate that the phagocytosis of apoptotic, CNS-derived HN2-5 cells by the Microglial cells requires recognition through phosphatidylserine (PS). Thus the enzyme aminopholipid translocase is inhibited during apoptosis of CNS-derived cells and this alone could account for the loss of plasma membrane lipid-asymmetry observed in these cells

  4. Deininger MH, Weinschenk T, Meyermann R, Schluesener HJ (2003) The allograft inflammatory factor-1 in Creutzfeldt-Jakob disease brains. Neuropathol.Appl.Neurobiol. 29:389-399
    Abstract: The allograft inflammatory factor-1 (AIF-1) is a 17-kDa IFN-gamma inducible Ca(2+)-binding EF-hand protein that is encoded within the HLA class III genomic region and is involved in immune dysfunction and smooth muscle cell activation. We used immunohistochemistry double labelling experiments to analyse the spatial distribution and cell-type-specific localization of AIF-1 in the brains of patients who died as a result of sporadic Creutzfeldt-Jakob disease (CJD) and neuropathologically unaltered controls. Significantly more AIF-1 immunoreactive macrophages/Microglial cells and, interestingly, neurones were observed in CJD patients compared to controls. Western blotting confirmed more prominent AIF-1 immunoreactive bands of approximately 50 kDa in four CJD patients compared to three controls. Chaotropic SDS-PAGE of the recombinant AIF-1 resulted in almost complete reduction of the 50 kDa band and mass spectrometry revealed only AIF-1-specific tryptic protein fragments suggesting that trimerized AIF-1 is the predominant form in vivo. Finally, we analysed mechanisms of neuronal AIF-1 induction. Following H2O2 challenge, a model of general cell stress, we observed the gradual induction of AIF-1 and, more interestingly, release to the supernatant of SKNSH neurones. Parallel reverse transcriptase polymerase chain reaction and sequencing was used to confirm AIF-1 mRNA expression

  5. Fiano V, Ghimenti C, Schiffer D (2003) Expression of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors in oligodendrogliomas in humans. Neurosci.Lett. 347:111-115
    Abstract: Cyclins are regulatory proteins of the cell cycle which bind and activate kinases. In gliomas, contrary to many malignancies, cyclin D1 is rarely amplified, but together with other cyclins, it increases with anaplasia. In a series of 23 surgical biopsies of grade II and III oligodendroglioma, cyclin D1, E, A, B1, CDK4-6, CDK2, Cdc2 and p27/Kip.1 have been studied by immunohistochemistry and Western blot. Cyclin D1 and A increased with anaplasia, showing a linear correlation with MIB.1 labeling index and an inverse correlation with p27/Kip.1 expression. Cyclin E and B1 and kinases were almost only expressed in grade III tumors. Normal oligodendrocytes and Microglia cells of the cortex and white matter showed a clear positivity for cyclin D1, but not for other cyclins or kinases

  6. Flannery T, Gibson D, Mirakhur M, McQuaid S, Greenan C, Trimble A, Walker B, McCormick D, Johnston PG (2003) The clinical significance of cathepsin S expression in human astrocytomas. Am.J.Pathol. 163:175-182
    Abstract: Early local invasion by astrocytoma cells results in tumor recurrence even after apparent total surgical resection, leading to the poor prognosis associated with malignant astrocytomas. Proteolytic enzymes have been implicated in facilitating tumor cell invasion and the current study was designed to characterize the expression of the cysteine proteinase cathepsin S (CatS) in astrocytomas and examine its potential role in invasion. Immunohistochemical analysis of biopsies demonstrated that CatS was expressed in astrocytoma cells but absent from normal astrocytes, oligodendrocytes, neurones and endothelial cells. Microglial cells and macrophages were also positive. Assays of specific activity in 59 astrocytoma biopsies confirmed CatS expression and in addition demonstrated that the highest levels of activity were expressed in grade IV tumors. CatS activity was also present in astrocytoma cells in vitro and the extracellular levels of activity were highest in cultures derived from grade IV tumors. In vitro invasion assays were carried out using the U251MG cell line and the invasion rate was reduced by up to 61% in the presence of the selective CatS inhibitor 4-Morpholineurea-Leu-HomoPhe-vinylsulphone. We conclude that CatS expression is up-regulated in astrocytoma cells and provide evidence for a potential role for CatS in invasion

  7. Giri RK, Selvaraj SK, Kalra VK (2003) Amyloid peptide-induced cytokine and chemokine expression in THP-1 monocytes is blocked by small inhibitory RNA duplexes for early growth response-1 messenger RNA. J.Immunol. 170:5281-5294
    Abstract: In Alzheimer's disease (AD) one finds increased deposition of A beta and also an increased presence of monocytes/macrophages in the vessel wall and activated Microglial cells in the brain. AD patients show increased levels of proinflammatory cytokines by activated Microglia. Here we used a human monocytic THP-1 cell line as a model for Microglia to delineate the cellular signaling mechanism involved in amyloid peptides (A beta(1-40) and A beta(1-42))-induced expression of inflammatory cytokines and chemokines. We observed that A beta peptides at physiological concentrations (125 nM) increased mRNA expression of cytokines (TNF-alpha, and IL-1 beta) and chemokines (monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1 beta (MIP-1 beta)). The cellular signaling involved activation of c-Raf, extracellular signal-regulated kinase-1 (ERK-1)/ERK-2, and c-Jun N-terminal kinase, but not p38 mitogen-activated protein kinase. This is further supported by the data showing that A beta causes phosphorylation of ERK-1/ERK-2, which, in turn, activates Elk-1. Furthermore, A beta mediated a time-dependent increase in DNA binding activity of early growth response-1 (Egr-1) and AP-1, but not of NF-kappa B and CREB. Moreover, A beta-induced Egr-1 DNA binding activity was reduced >60% in THP-1 cells transfected with small interfering RNA duplexes for Egr-1 mRNA. We show that A beta-induced expression of TNF-alpha, IL-1 beta, MCP-1, IL-8, and MIP-1 beta was abrogated in Egr-1 small inhibitory RNA-transfected cells. Our results indicate that A beta-induced expression of cytokines (TNF-alpha and IL-1 beta) and chemokines (MCP-1, IL-8, and MIP-1 beta) in THP-1 monocytes involves activation of ERK-1/ERK-2 and downstream activation of Egr-1. The inhibition of Egr-1 by Egr-1 small inhibitory RNA may represent a potential therapeutic target to ameliorate the inflammation and progression of AD

  8. Grant R, Kapoor V (2003) Inhibition of indoleamine 2,3-dioxygenase activity in IFN-gamma stimulated astroglioma cells decreases intracellular NAD levels. Biochem.Pharmacol. 66:1033-1036
    Abstract: Astroglia provide essential metabolic and neurotropic support to cells within the CNS and participate in the cellular immune response with Microglia/macrophages following activation by the pro-inflammatory cytokine IFN-gamma. Activation of glial cells results in local oxidative stress and induction of a number of proteins including the enzyme indoleamine 2,3-dioxygenase (IDO). As a rate-limiting enzyme, IDO regulates tryptophan catabolism via the kynurenine pathway producing a series of metabolic precursors (some of which are neurotoxic) before complete oxidation to the essential pyridine nucleotide NAD. Inhibition of this pathway may therefore prove therapeutic in neuroinflammatory disease by reducing production of cell toxins. However, kynurenine metabolism may also be cytoprotective through de novo synthesis of cellular NAD levels. We investigated the hypothesis that IDO activity is directly involved in maintenance of intracellular [NAD] in activated astroglial cells through control of de novo synthesis. Exposure to IFN-gamma increased IDO activity from 7+/-1 nmol to 129+/-11 nmol kynurenine/hr/mg protein. Inhibition of IDO activity with either 6-chloro-D-tryptophan (competitive inhibition), or 3-ethoxy beta-carboline (non-competitive inhibition) resulted in a dose-dependent decrease in IDO activity that correlated directly with decreasing [NAD] (R(2)=0.92 and 0.81, respectively). These results support the hypothesis that one important consequence of increasing IDO activity in astroglial cells during inflammation is to maintain NAD levels through de novo synthesis from tryptophan. Inhibition of kynurenine pathway metabolism under these conditions may significantly decrease cell viability and CNS functions unless alternate precursors for NAD synthesis are available

  9. Kang JQ, Chong ZZ, Maiese K (2003) Critical role for Akt1 in the modulation of apoptotic phosphatidylserine exposure and Microglial activation. Mol.Pharmacol. 64:557-569
    Abstract: Biological targets for neurodegenerative disease that focus on the intrinsic maintenance of cellular integrity and the extrinsic prevention of phagocytic cellular disposal offer the greatest promise for therapeutic intervention. Protein kinase B (Akt1), a serine-threonine kinase closely involved in cell growth and survival, offers a strong potential to address both intrinsic and extrinsic mechanisms of neuronal injury. We demonstrate that overexpression of a constitutively active form of Akt1 (myristoylated Akt1) in differentiated SH-SY5Y neuronal cells provides intrinsic cellular protection against apoptotic genomic DNA destruction and membrane phosphatidylserine (PS) exposure. Transfection of SH-SY5Y cells with a plasmid encoding a kinase-deficient dominant-negative Akt1 eliminates cytoprotection, suggesting that activation of Akt1 is necessary and sufficient to prevent apoptotic destruction. Apoptotic neuronal membrane PS exposure provides a unique pathway for Akt1 to offer extrinsic cellular protection and block Microglial activation, because independent cotreatment with an anti-PS receptor neutralizing antibody could also prevent Microglial proliferation. Akt1 maintains nuclear DNA integrity and membrane PS exposure through the specific inhibition of caspase 3-, 8-, and 9-like activities that were linked to mitochondrial membrane potential and cytochrome c release. Our work elucidates a novel capacity for Akt1 to maintain cellular integrity through a series of cysteine protease pathways and to uniquely regulate Microglial activation through the modulation of membrane PS residue externalization

  10. Kang JQ, Chong ZZ, Maiese K (2003) Akt1 protects against inflammatory Microglial activation through maintenance of membrane asymmetry and modulation of cysteine protease activity. J.Neurosci.Res. 74:37-51
    Abstract: In several cell systems, protein kinase B (Akt1) can promote cell growth and development, but the "antiapoptotic" pathways of this kinase that may offer protection against cellular inflammatory demise have not been defined. Given that early cellular membrane phosphatidylserine exposure is a critical component of apoptosis, we investigated the role of Akt1 during neuronal apoptotic injury. By employing differentiated SH-SY5Y neuronal cells that overexpress a constitutively active form of Akt1 (myristoylated Akt1), free radical-induced cell injury was assessed through trypan blue dye exclusion, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, and mitochondrial membrane potential. Membrane phosphatidylserine exposure was both necessary and sufficient for Microglial activation, insofar as cotreatment with an antiphosphatidylserine receptor-neutralizing antibody could prevent Microglial activity following neuronal loss of membrane asymmetry. Furthermore, expression of myristoylated Akt1 not only prevented cell injury through the prevention of membrane phosphatidylserine exposure and genomic DNA fragmentation but also inhibited Microglial activation and proliferation that required the inhibition of caspase 9-, caspase 3-, and caspase 1-like activities linked to cytochrome c release. Interestingly, Akt1 modulation of membrane phosphatidylserine exposure was primarily through caspase 1 activity. Removal of Akt1 activity abolished neuronal protection, suggesting that Akt1 functions as a critical pathway for the maintenance of cellular integrity and the prevention of phagocytic cellular removal during neurodegenerative insults

  11. Klegeris A, McGeer PL (2003) Toxicity of human monocytic THP-1 cells and Microglia toward SH-SY5Y neuroblastoma cells is reduced by inhibitors of 5-lipoxygenase and its activating protein FLAP. J.Leukoc.Biol. 73:369-378
    Abstract: To explore whether the proinflammatory products of the 5-lipoxygenase (5-LOX) pathway are involved in Microglia-mediated toxicity toward neuronal cells, we evaluated the effects of 5-LOX inhibitors using an in vitro assay system where human neuronal SH-SY5Y cells are exposed to toxic secretions from THP-1 monocytic cells or human Microglia. The specific 5-LOX inhibitors, REV 5901, zileuton, and 5-hydroxyeicosatetraenoic acid lactone; the nonselective LOX inhibitors, phenidone and dapsone; the dual 5-LOX/cyclooxygenase inhibitor, tepoxalin; and the selective inhibitor of the 5-LOX-activating protein (FLAP), MK-886, inhibited such toxicity. The toxicity was enhanced by the 5-LOX product leukotriene (LT)D(4) and reduced by the selective cysteinyl LT receptor (CysLT(1)) antagonist MK-571. The mRNAs for 5-LOX and FLAP were detected in THP-1 cells and human Microglia but not in SH-SY5Y cells. The data suggest that inhibition of proinflammatory LT production by 5-LOX inhibition could selectively reduce toxicity of Microglial cells and thus be beneficial in neuroinflammatory diseases

  12. Lambert JC, Luedecking-Zimmer E, Merrot S, Hayes A, Thaker U, Desai P, Houzet A, Hermant X, Cottel D, Pritchard A, Iwatsubo T, Pasquier F, Frigard B, Conneally PM, Chartier-Harlin MC, DeKosky ST, Lendon C, Mann D, Kamboh MI, Amouyel P (2003) Association of 3'-UTR polymorphisms of the oxidised LDL receptor 1 (OLR1) gene with Alzheimer's disease. J.Med.Genet. 40:424-430
    Abstract: Although possession of the epsilon 4 allele of the apolipoprotein E gene appears to be an important biological marker for Alzheimer's disease (AD) susceptibility, strong evidence indicates that at least one additional risk gene exists on chromosome 12. Here, we describe an association of the 3'-UTR +1073 C/T polymorphism of the OLR1 (oxidised LDL receptor 1) on chromosome 12 with AD in French sporadic (589 cases and 663 controls) and American familial (230 affected sibs and 143 unaffected sibs) populations. The age and sex adjusted odds ratio between the CC+CT genotypes versus the TT genotypes was 1.56 (p=0.001) in the French sample and 1.92 (p=0.02) in the American sample. Furthermore, we have discovered a new T/A polymorphism two bases upstream of the +1073 C/T polymorphism. This +1071 T/A polymorphism was not associated with the disease, although it may weakly modulate the impact of the +1073 C/T polymorphism. Using 3'-UTR sequence probes, we have observed specific DNA protein binding with nuclear proteins from lymphocyte, astrocytoma, and neuroblastoma cell lines, but not from the Microglia cell line. This binding was modified by both the +1071 T/A and +1073 C/T polymorphisms. In addition, a trend was observed between the presence or absence of the +1073 C allele and the level of astrocytic activation in the brain of AD cases. However, Abeta(40), Abeta(42), Abeta total, and Tau loads or the level of Microglial cell activation were not modulated by the 3'-UTR OLR1 polymorphisms. Finally, we assessed the impact of these polymorphisms on the level of OLR1 expression in lymphocytes from AD cases compared with controls. The OLR1 expression was significantly lower in AD cases bearing the CC and CT genotypes compared with controls with the same genotypes. In conclusion, our data suggest that genetic variation in the OLR1 gene may modify the risk of AD

  13. Lange-Dohna C, Zeitschel U, Gaunitz F, Perez-Polo JR, Bigl V, Rossner S (2003) Cloning and expression of the rat BACE1 promoter. J.Neurosci.Res. 73:73-80
    Abstract: The pathogenic processing of the amyloid precursor protein (APP) into beta-amyloid peptides, which give rise to beta-amyloid plaques in the brains of Alzheimer's disease patients, requires the enzymatic activity of the beta-site APP-cleaving enzyme 1 (BACE1). We report the cloning and sequence of a 1.5-kb DNA fragment upstream of the coding sequence of the rat BACE1 gene and the construction of a BACE1 promoter/luciferase reporter construct. The basal activity of this promoter construct was highest in neuronal cell lines such as BE(2)-C and PC12 and in the pancreatic cell line AR42J, somewhat lower in rat primary neurons, and astrocytic and Microglial cultures, very low in hepatocytes, and almost absent in fibroblasts and in the monocyte-macrophage cell line RAW264.7. The first 600 bp of this promoter are highly conserved among rat, mouse, and human, suggesting that this region contains regulatory elements that modulate BACE1 transcription. Indeed, this fragment contains several putative transcription factor binding sites such as MZF1, Sp1, four GATA-1 sites, and one YY1 site. Directed mutagenesis of GATA-1 elements led to altered luciferase expression, indicating that these sites are involved in the regulation of BACE1 transcription. Additionally, the analysis of promoter activities of deletion mutants suggests the presence of activators of BACE1 transcription between bases -514 to -753 and of suppressor elements between bases -754 and -1541. The BACE1 promoter sequence data and the constructs described here will be useful to identify factors that influence the expression of BACE1 in experimental paradigms in vitro

  14. Luder CG, Lang C, Giraldo-Velasquez M, Algner M, Gerdes J, Gross U (2003) Toxoplasma gondii inhibits MHC class II expression in neural antigen-presenting cells by down-regulating the class II transactivator CIITA. J.Neuroimmunol. 134:12-24
    Abstract: Major histocompatibility complex (MHC) class II expression by Microglia and astrocytes is critical for CD4+-mediated immune responses within the central nervous system. Here, we demonstrate that the obligate intracellular parasite, Toxoplasma gondii, down-regulates activation-induced MHC class II expression in human-derived glioblastoma cells as well as in primary astrocytes and Microglia from cortices of rat fetuses. Down-regulation of MHC class II proteins was predominantly observed in parasite-positive, but not parasite-negative, host cells of T. gondii-infected cell cultures. MHC class II transcript levels induced by IFN-gamma alone or in combination with TNF-alpha were also clearly diminished after parasitic infection. Furthermore, T. gondii dose-dependently down-regulated the transcript levels of the class II transactivator CIITA. These results suggest that T. gondii partially evade CD4+-mediated intracerebral immune responses, a mechanism which may contribute to long-term persistence of the parasite within the CNS

  15. Munch G, Gasic-Milenkovic J, Dukic-Stefanovic S, Kuhla B, Heinrich K, Riederer P, Huttunen HJ, Founds H, Sajithlal G (2003) Microglial activation induces cell death, inhibits neurite outgrowth and causes neurite retraction of differentiated neuroblastoma cells. Exp.Brain Res. 150:1-8
    Abstract: Activation of glial cells has been proposed to contribute to neuronal dysfunction and neuronal cell death in Alzheimer's disease. In this study, we attempt to determine some of the effects of secreted factors from activated murine N-11 Microglia on viability and morphology of neurons using the differentiated neuroblastoma cell line Neuro2a. Microglia were activated either by lipopolysaccharide (LPS), bacterial cell wall proteoglycans, or advanced glycation endproducts (AGEs), protein-bound sugar oxidation products. At high LPS or AGE concentrations, conditioned medium from Microglia caused neuronal cell death in a dose-dependent manner. At sublethal LPS or AGE concentrations, conditioned media inhibited retinoic acid-induced neurite outgrowth and stimulated retraction of already extended neurites. Among the many possible secreted factors, the contribution of NO or NO metabolites in the cytotoxicity of conditioned medium was investigated. Cell death and changes in neurite morphology were partly reduced when NO production was inhibited by nitric oxide synthase inhibitors. The results suggest that even in the absence of significant cell death, inflammatory processes, which are partly transmitted via NO metabolites, may affect intrinsic functions of neurons such as neurite extension that are essential components of neuronal morphology and thus may contribute to degenerative changes in Alzheimer's disease

  16. Phuong LK, Allen C, Peng KW, Giannini C, Greiner S, TenEyck CJ, Mishra PK, Macura SI, Russell SJ, Galanis EC (2003) Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme. Cancer Res. 63:2462-2469
    Abstract: Despite the most aggressive medical and surgical treatments, glioblastoma multiforme remains incurable with a median survival of <1 year. We investigated the antitumor potential of a novel viral agent, an attenuated strain of measles virus (MV), derived from the Edmonston vaccine lineage, genetically engineered to produce carcinoembryonic antigen (CEA). CEA production as the virus replicates can serve as a marker of viral gene expression. Infection of a variety of glioblastoma cell lines including U87, U118, and U251 at MOIs 0.1, 1, and 10 resulted in significant cytopathic effect consisting of excessive syncycial formation and massive cell death at 72-96 h from infection. terminal deoxynucleotidyltransferase-mediated nick end labeling assays demonstrated the mechanism of cell death to be predominantly apoptotic. The efficacy of this approach in vivo was examined in BALB/c nude mice by using both s.c. and intracranial orthotopic U87 tumor models. In the s.c. U87 model, mice with established xenografts were treated with a total dose of 8 x 10(7) plaque forming units of MV-CEA, administered i.v. Mice treated with UV light inactivated MV, and untreated mice with established U87 tumors were used as controls. There was statistically significant regression of s.c. tumors (P < 0.001) and prolongation of survival (P = 0.007) in MV-CEA treated animals compared with the two control groups. In the intracranial orthotopic U87 model, there was significant regression of intracranial U87 tumors treated with intratumoral administration of MV-CEA at a total dose of 1.8 x 10(6) plaque forming units as assessed by magnetic resonance image (P = 0.002), and statistically significant prolongation of survival as compared with mice that received UV-inactivated virus and untreated mice (P = 0.02). Histological examination of brains of MV-CEA-treated animals revealed complete regression of the tumor with the presence of a residual glial scar and reactive changes, mainly presence of hemosiderin-laden macrophages. In addition, CEA levels in the peripheral blood in both the s.c. and orthotopic models increased before tumor regression, indicating viral gene expression, and returned to normal when the tumors regressed. Ifnar(ko) CD46 Ge transgenic mice, susceptible to MV infection, were used to assess central nervous system toxicity of MV-CEA. Intracranial administration of MV-CEA into the caudate nucleus of Ifnar(ko) CD46 Ge did not result in clinical neurotoxicity. Pathologic examination demonstrated limited Microglial infiltration surrounding the injection site. In summary, MV-CEA has potent antitumor activity against gliomas in vitro, as well as in both s.c. and orthotopic U87 animal models. Monitoring CEA levels in the serum can serve as a low-risk method of detecting viral gene expression during treatment, and could allow dose optimization and individualization of treatment

  17. Platten M, Kretz A, Naumann U, Aulwurm S, Egashira K, Isenmann S, Weller M (2003) Monocyte chemoattractant protein-1 increases Microglial infiltration and aggressiveness of gliomas. Ann.Neurol. 54:388-392
    Abstract: Macrophages are thought to represent a first line of defense in anti-tumor immunity. Despite infiltration by Microglial cells, however, malignant gliomas are still highly aggressive tumors. We here identify monocyte chemoattractant protein-1 (MCP-1) as a critical chemoattractant for glioma-infiltrating Microglial cells. MCP-1-transfected rat CNS-1 gliomas were massively infiltrated by Microglial cells. Whereas MCP-1 did not promote the growth of CNS-1 cells in vitro, intracerebral CNS-1-transfected tumors grew more aggressively than control-transfected tumors. This provides the first functional evidence that MCP-1 recruits Microglial cells to gliomas and promotes their growth in vivo. Microglial cells may support rather than suppress glioma growth

  18. Sasaki A, Horikoshi Y, Yokoo H, Nakazato Y, Yamaguchi H (2003) Antiserum against human glucose transporter 5 is highly specific for Microglia among cells of the mononuclear phagocyte system. Neurosci.Lett. 338:17-20
    Abstract: Human monocytes and a variety of tissue macrophages, including Microglia, were studied immunohistochemically to determine the expression of a novel Microglial marker, human glucose transporter 5 (hGLUT5), in these cells. The hGLUT5 was not expressed in most peripheral macrophages in the normal state, but weakly expressed in some foamy macrophages in atherosclerotic lesions. There was no hGLUT5 reactivity in blood monocytes. In the lesions of brain infarcts, foamy macrophages (predominantly monocyte-derived cells) in the ischemic core were mostly negative for hGLUT5, while activated and phagocytic Microglia in the transitional zone were consistently positive. The present study indicated that unlike other Microglial markers, hGLUT5 is rarely present in peripheral macrophages, and that hGLUT5 immunohistochemistry is useful in distinguishing Microglia-derived macrophages from monocyte-derived macrophages in acute necrotic lesions

  19. Shimizu T, Sato K, Suzuki T, Tachibana K, Takeda K (2003) Induction of plasminogen activator inhibitor-2 is associated with suppression of invasive activity in TPA-mediated differentiation of human prostate cancer cells. Biochem.Biophys.Res.Commun. 309:267-271
    Abstract: We previously reported that 12-O-tetra-decanoylphorbol-13-acetate (TPA) induces Microglia-like differentiation and decreases malignancy in human prostate cancer TSU-Pr1 cells. To investigate the mechanism underlying differentiation and decrease of malignancy in TSU-Pr1 cells treated with TPA, we attempted to identify genes expressed differentially during the differentiation using differential display. We successfully detected plasminogen activator inhibitor type-2 (PAI-2) as one gene up-regulated by TPA treatment. The change in expression of PAI-2 by TPA was blocked by treatment with protein kinase C or mitogen-activated protein kinase inhibitors. We also found that secretion of PAI-2 protein was increased by TPA treatment. Moreover, we demonstrated that suppression of invasive activity of TSU-Pr1 cells by TPA treatment was blocked by co-treatment with anti-PAI-2 antibody. These results suggest that induction of PAI-2 is associated with suppression of invasive activity in TSU-Pr1 cells treated with TPA

  20. Ando H, Saio M, Ohe N, Tamakawa N, Yu H, Nakayama T, Yoshimura S, Kaku Y, Iwama T, Shinoda J, Sakai N, Takami T (2002) B7.1 immunogene therapy effectively activates CD(4+) tumor-infiltrating lymphocytes in the central nervous system in comparison with B7.2 gene therapy. Int.J.Oncol. 20:807-812
    Abstract: The B7 gene utilizing immunogene therapy is one of the most common methods against tumor growth. However, there is no known study that investigated the difference between B7.1 and B7.2 with regard to B7 gene therapy in the central nervous system (CNS). Therefore, to clarify the difference, we established B7.1 or B7.2 gene transduced tumor cells originating from the murine T cell lymphoma cell line EL4 (EL4-B7.1 or EL4-B7.2). First, we observed the survival time after intracranial inoculation of parent (IC-wt) or genetically modified tumor cells. All mice in control groups (IC-wt or IC-mock) were dead within 16 days. While there was significant survival elongation in the B7.2 modified group (IC-B7.2, p=0.0002), all mice in this group were dead of tumor growth within 22 days. On the other hand, 60% of mice inoculated with EL4-B7.1 (IC-B7.1) survived more than 120 days (p<0.0001). Second, to shed light on the anti-tumor immune response in situ, we tried to analyze CD(4+) tumor-infiltrating T lymphocytes (CD(4+) TIL). To purify and analyze CD(4+) TIL, we had to deplete F4/80(+) Microglia because of the CD4 expression. In terms of activation marker expression in CD(4+) TIL, a small population was activated (CD25, 9.8%; CD69, 15.8%) in the control group (IC-wt). In contrast, the activation marker positive CD4+ TIL percentage both in IC-B7.1 (CD25, 25.1%; CD69, 40.1%) and IC-B7.2 (CD25, 16.2%; CD69, 28.3%) appeared to reflect the survival curve in both groups. These findings strongly suggest that, in the CNS, B7.1 gene therapy could effectively introduce CD(4+) TIL activation compared with B7.2 gene therapy. This is the first study clearly describing the difference between B7.1 gene therapy and B7.2 gene therapy in the CNS in terms of the activation status of CD(4+) TIL in situ

  21. Badie B, Bartley B, Schartner J (2002) Differential expression of MHC class II and B7 costimulatory molecules by Microglia in rodent gliomas. J.Neuroimmunol. 133:39-45
    Abstract: To assess the immune function of Microglia and macrophages in brain tumors, the expression of MHC class II and B7 costimulatory molecules in three rodent glioma models was examined. Microglia and macrophages, which accounted for 5-12% of total cells, expressed B7.1 and MHC class II molecules in the C6 and 9L tumors, but not RG2 gliomas. Interestingly, the expression of B7.1 and MHC class II molecules by Microglia and macrophage was associated with an increase in the number of tumor-infiltrating lymphocytes in C6 and 9L tumors. B7.2 expression, which was present at low levels on Microglia and macrophages in normal brain, did not significantly change in tumors. Interestingly, the expression of all three surface antigens increased after Microglia were isolated from intracranial C6 tumors and cultured for a short period of time. We conclude that Microglia immune activity may be suppressed in gliomas and directly correlates to the immunogenecity of experimental brain tumors

  22. Bate C, Rutherford S, Gravenor M, Reid S, Williams A (2002) Cyclo-oxygenase inhibitors protect against prion-induced neurotoxicity in vitro. Neuroreport 13:1933-1938
    Abstract: The mechanisms of neuronal loss during the course of the prion diseases are not fully understood. In this study, neurones treated with certain non-steroidal anti-inflammatory drugs (NSAIDs) were protected against the otherwise toxic effects of a peptide derived from the prion protein, or extracts containing infectious prions (PrP ). These NSAIDs inhibit the cyclo-oxygenase (cox) enzymes that metabolise arachidonic acid to prostaglandins (PG). Conversely, drugs that inhibited the metabolism of arachidonic acid to leucotrienes enhanced neurotoxicity. Studies with selective inhibitors highlighted the importance of the cox-1 isoform in prion-induced neurotoxicity. The cox-1 inhibitors also inhibited neuronal PGE production and protected both neuroblastoma cells and primary cortical neurones against prions. They also reduced Microglia-mediated killing of prion-treated neurones

  23. Fukumitsu H, Takase-Yoden S, Furukawa S, Nemoto K, Ikeda T, Watanabe R (2002) Implantation of BDNF-producing packaging cells into brain. Cell Transplant. 11:459-464
    Abstract: In order to invent a screening system to check in vivo gene function and the efficiency of gene transfer mediated by a retroviral vector system, we established a novel packaging cell, PacC6/A8, that is transplantable to rat brains. The packaging cell is based on the gene of the neuropatogenic retrovirus, A8-V. For expression in the brain, a vector that expresses brain-derived neurotrophic factor (BDNF) tagged by c-Myc-His6 (LxA/bdmh) was constructed. After transfection of LxA/bdmh to PacC6/A8, a cloned cell line, PacC6/A8/bmh, was established. PacC6/A8/bmh cells stably produced pseudotyped retroviruses carrying LxA/bdmh. For a control, a retroviral vector that bears the gene that codes enhanced green fluorescent protein (EGFP) tagged by C-Mic-His6 was also created and used for the establishment of PacC6/A8/gfmh cells that produce pseudotyped retroviruses carrying LxA/gfmh. PacC6/A8/bmh and PacC6/A8/gfmh cells were injected to the brain of newborn rats. A tumor was formed in all the rats injected that did not exhibit any symptoms until 3-4 weeks after the injection. A histological study of the injected rats revealed that the transferred BDNF gene was expressed in the brain of rats injected with PacC6/A8/bmh cells, but not in rats with PacC6/A8/gfmh cells. Interestingly, many activated Microglia had migrated into the tumor induced by PacC6/A8/bmh cells, and expressed a high amount of BDNF

  24. Johnston JB, Silva C, Power C (2002) Envelope gene-mediated neurovirulence in feline immunodeficiency virus infection: induction of matrix metalloproteinases and neuronal injury. J.Virol. 76:2622-2633
    Abstract: The release of neurotoxins by activated brain macrophages or Microglia is one mechanism proposed to contribute to the development of neurological disease following infection by lentiviruses, including feline immunodeficiency virus (FIV). Since molecular diversity in the lentiviral envelope gene influences the expression of host molecules implicated in neuronal injury, the role of the envelope sequence in FIV neuropathogenesis was investigated by using the neurovirulent FIV strain V1CSF, the nonneurovirulent strain Petaluma, and a chimera (FIVCh) containing the V1CSF envelope gene in a Petaluma background. All three viruses replicated in primary feline macrophages with equal efficiency, but conditioned medium from V

  25. Klegeris A, McGeer PL (2002) Cyclooxygenase and 5-lipoxygenase inhibitors protect against mononuclear phagocyte neurotoxicity. Neurobiol.Aging 23:787-794
    Abstract: Neuroinflammation and oxidative stress are believed to be contributing factors to neurodegeneration in normal aging, as well as in age-related neurological disorders. Reactive Microglia are found in increased numbers in aging brain and are prominently associated with lesions in such age-related degenerative conditions as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). In vitro, stimulated Microglia or Microglial-like cells secrete neurotoxic materials and are generators of free radicals through their respiratory burst system. Agents that suppress Microglial activation are therefore candidates for neuroprotection. We have developed quantitative in vitro assays for measuring neurotoxicity of Microglia or other mononuclear phagocytes. Neuronal like SH-SY5Y cells are cultured in supernatants from activated cells of the human monocytic THP-1 line and their survival is followed. Respiratory burst is directly measured on the activated cells. We tested inhibitors of the cyclooxygenase (COX) or the 5-lipoxygenase (5-LOX) pathways as possible neuroprotective agents. The COX pathway generates inflammatory prostaglandins, while the 5-LOX pathway generates inflammatory leukotrienes. We found that inhibitors of both these pathways suppressed neurotoxicity in a dose-dependent fashion. They included the COX-1 inhibitor indomethacin; the COX-2 inhibitor NS-398; the mixed COX-1/COX-2 inhibitor ibuprofen; the nitric oxide (NO) derivatives of indomethacin, ibuprofen and flurbiprofen; the 5-LOX inhibitor REV 5901; and the 5-LOX activating protein (FLAP) inhibitor MK-886. The FLAP inhibitor also reduced respiratory burst activity in a more potent manner than indomethacin. Combinations of COX and 5-LOX inhibitors were more effective than single inhibitors. The data suggest that both COX inhibitors and 5-LOX inhibitors may be neuroprotective in vivo by suppressing toxic actions of Microglia/macrophages, and that combinations of the two might have greater therapeutic potential than single inhibitors of either class

  26. Linden DR, El Fakahany EE (2002) Microglial derived nitric oxide decreases serotonin content in rat basophilic leukemia (RBL-2H3) cells. Eur.J.Pharmacol. 436:53-56
    Abstract: Nitric oxide (NO) and serotonin (5-hydroxytryptamine; 5-HT) are important neuromodulators that are involved in a myriad of biochemical reactions. In this work, we describe a novel model co-culture system to study the interactions between NO and 5-HT. NO derived from cytokine stimulated Bv2 Microglial cells depleted 5-HT from RBL-2H3 cells. Reduction of 5-HT content by NO derived from the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was concentration-dependent, independent of intracellular Ca(2+) and inhibited by reduced glutathione (GSH). Collectively, these data indicate that this cell co-culture system is a viable model to study the mechanisms of interaction between nitrergic and serotonergic pathways

  27. Liu H, Ng CE, Tang BL (2002) Nogo-A expression in mouse central nervous system neurons. Neurosci.Lett. 328:257-260
    Abstract: The longest central nervous system (CNS) specific isoform of the major myelin-associated inhibitor of neurite growth, Nogo-A, has previously been shown to be expressed largely in oligodendrocytes. Using an antibody raised against a recombinant fusion protein comprising amino acids 223-399 of Nogo-A, we show in this report that Nogo-A is also expressed in the cell body of a distinct set of CNS neurons. The antibody detects a single protein band of 220 kDa in rat brain lysate and neuroblastoma cell lysates. Immunofluorescent analyses reveal that Nogo-A is found largely in the endoplasmic reticulum of neuroblastoma cell lines SH-SY5Y and NIE-115. In the mouse CNS, Nogo-A can be found in specific subsets of neuronal cell bodies in addition to oligodendrocytes, but not glial fibrillary associated protein positive astrocytes or MAC-1 positive Microglia. Our results provide a conclusive demonstration of the expression of Nogo-A in CNS neurons, which suggests that Nogo-A may have distinct endogenous roles in neurons other than its known ability to inhibit neurite growth

  28. Madigan MC, Penfold PL, King NJ, Billson FA, Conway RM (2002) Immunoglobulin superfamily expression in primary retinoblastoma and retinoblastoma cell lines. Oncol.Res. 13:103-111
    Abstract: Retinoblastoma (Rb) is the most common intraocular tumor of childhood. In this study we examined primary Rb specimens and Rb cell lines for the expression of immunoglobulin superfamily (IgSF) antigens: MHC class I and II (MHC-I and MHC-II), neural cell adhesion molecule (NCAM), intercellular adhesion molecule-1 (ICAM-1), and Thy-1, which play an important role in immune system and tumor cell interactions. MHC-I and-II, ICAM-1 (CD54), NCAM (CD56), and Thy-1 (CDw90) immunoreactivity was studied in eight primary Rb biopsy specimens using immunohistochemistry, three using immunoelectron microscopy, and six Rb cell lines using flow cytometry (FCM). Parenchymal and vascular-associated cells, phenotypically similar to retinal Microglia, strongly expressed MHC-II immunoreactivity and were distributed throughout primary Rb specimens. However, MHC-II expression on Rb cell lines was similar to nonspecific control levels. tumor cells in primary Rb specimens displayed high NCAM, moderate Thy-1, and low MHC-I and ICAM-1 immunolabeling. tumor vasculature expressed low to moderate MHC-I and ICAM-1 immunoreactivity and moderate Thy-1 immunoreactivity. NCAM was not detected on the vasculature of primary Rb specimens. Rb cell lines displayed variable expression of Thy-1, ICAM-1, and MHC-I. NCAM was highly expressed on five of six Rb cell lines. The high levels of constitutive NCAM immunoreactivity on Rb tumor cells confirm the neuroectodermal origins of this tumor. Additionally, the variable expression of Thy-1 may suggest separate neural lineages or differences in the maturational status ofsome Rb tumors. The presence of a population of infiltrating MHC-II-positive cells in primary Rb tumors has implications for immunomodulation of Rb growth

  29. Mentlein R, Held-Feindt J (2002) Pleiotrophin, an angiogenic and mitogenic growth factor, is expressed in human gliomas. J.Neurochem. 83:747-753
    Abstract: Pleiotrophin (PTN) is a mitogenic/angiogenic, 15.3 kDa heparin-binding peptide that is found in embryonic or early postnatal, but rarely in adult, tissues. Since developmentally regulated factors often re-appear in malignant cells, we examined PTN expression in human glioma cell lines, cell cultures derived from solid gliomas and glioma sections. PTN mRNA or protein was detected by reverse transcriptase-polymerase chain reaction, immunohistochemistry, western blot or enzyme-linked immunoassay in all WHO III and IV grade gliomas and cells analyzed in vitro or in situ. One WHO II grade glioma investigated was PTN negative. In vitro, PTN was synthesized in perinuclear regions of glioma cells, secreted into the cultivation medium, but its production varied considerably between glioma cells cultivated from different solid gliomas or glioma cell lines. In situ, PTN expression was restricted to distinct parts/cells of the tumour. PTN did not influence the proliferation of glioma cells themselves, but stimulated [3H]thymidine incorporation into DNA of Microglial cells. Furthermore, in Boyden chamber assays, PTN showed a strong chemotactic effect on murine BV-2 Microglial cells. PTN is supposed to be a paracrine growth/angiogenic factor that is produced by gliomas and contributes to their malignancy by targeting endothelial and Microglial cells

  30. Perini G, Della-Bianca V, Politi V, Della VG, Dal Pra I, Rossi F, Armato U (2002) Role of p75 neurotrophin receptor in the neurotoxicity by beta-amyloid peptides and synergistic effect of inflammatory cytokines. J.Exp.Med. 195:907-918
    Abstract: The neurodegenerative changes in Alzheimer's disease (AD) are elicited by the accumulation of beta-amyloid peptides (Abeta), which damage neurons either directly by interacting with components of the cell surface to trigger cell death signaling or indirectly by activating astrocytes and Microglia to produce inflammatory mediators. It has been recently proposed that the p75 neurotrophin receptor (p75(NTR)) is responsible for neuronal damage by interacting with Abeta. By using neuroblastoma cell clones lacking the expression of all neurotrophin receptors or engineered to express full-length or various truncated forms of p75(NTR), we could show that p75(NTR) is involved in the direct signaling of cell death by Abeta via the function of its death domain. This signaling leads to the activation of caspases-8 and -3, the production of reactive oxygen intermediates and the induction of an oxidative stress. We also found that the direct and indirect (inflammatory) mechanisms of neuronal damage by Abeta could act synergistically. In fact, TNF-alpha and IL-1beta, cytokines produced by Abeta-activated Microglia, could potentiate the neurotoxic action of Abeta mediated by p75(NTR) signaling. Together, our results indicate that neurons expressing p75(NTR), mostly if expressing also proinflammatory cytokine receptors, might be preferential targets of the cytotoxic action of Abeta in AD

  31. Repovic P, Benveniste EN (2002) Prostaglandin E2 is a novel inducer of oncostatin-M expression in macrophages and Microglia. J.Neurosci. 22:5334-5343
    Abstract: Oncostatin-M (OSM), a pluripotent cytokine of the interleukin-6 (IL-6) family, is produced in a number of inflammatory conditions. Known sources of OSM include monocytes-macrophages and T-cells. Here we present Microglia, the resident macrophages of the brain, as a source of OSM in the CNS. In this context, we describe a novel inducer of OSM, prostaglandin E(2) (PGE(2)). PGE(2) induces OSM expression in Microglia, monocytes, and macrophages of human and murine origin. PGE(2) induction of OSM is mimicked by cholera toxin, an activator of stimulatory G (G(s))-proteins; by forskolin, an activator of adenylate cyclase; and by the cAMP analog, dibutyryl-cAMP. PGE(2) induction of OSM gene expression is inhibited by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine, by the protein kinase A (PKA) inhibitor H-89, and by a dominant-negative PKA construct. These data indicate that PGE(2) signals via G(s)-protein-coupled receptor(s), adenylate cyclase, and PKA to induce OSM expression. Accordingly, other activators of cAMP signaling such as norepinephrine and PGE(1) induce OSM. The ability of PGE(2) to induce OSM expression was tested under more physiological conditions, using cocultures of astrocytes and monocytes. Treatment of the cocultures with IL-1beta or tumor necrosis factor-alpha (TNF-alpha) results in production of PGE(2) and OSM. PGE(2) produced in the cocultures is responsible for OSM induction, because pretreatment with indomethacin, an inhibitor of prostaglandin synthesis, as well as depletion of PGE(2), abrogate OSM expression induced by IL-1beta or TNF-alpha. These data suggest that in the CNS, OSM may be produced through collaboration of astrocytes and macrophages-Microglia

  32. Seo JH, Rah JC, Choi SH, Shin JK, Min K, Kim HS, Park CH, Kim S, Kim EM, Lee SH, Lee S, Suh SW, Suh YH (2002) Alpha-synuclein regulates neuronal survival via Bcl-2 family expression and PI3/Akt kinase pathway. FASEB J. 16:1826-1828
    Abstract: Alpha-synuclein (alpha-SN) is a ubiquitous protein that is especially abundant in the brain and has been postulated to play a central role in the pathogenesis of Parkinson's disease, Alzheimer's disease, and other neurodegenerative disorders. However, little is known about the neuronal functions of alpha-SN and the molecular and cellular mechanisms underlying neuronal loss. Here, we show that alpha-SN plays dual roles of neuroprotection and neurotoxicity depending on its concentration or level of expression. At nanomolar concentrations, a-SN protected neurons against serum deprivation, oxidative stress, and excitotoxicity through the PI3/Akt signaling pathway, and its protective effect was increased by Bcl-2 overexpression. Conversely, at both low micromolar and overexpressed levels in the cell, alpha-SN resulted in cytotoxicity. This might be related to decreased Bcl-xL expression and increased bax expression, which is subsequently followed by cytochrome c release and caspase activation and also by Microglia-mediated inflammatory responses via the NFkappaB and mitogen-activated protein kinase pathways

  33. Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, Moussignac RL (2002) Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J.Exp.Med. 196:1025-1037
    Abstract: Aspirin (ASA) is unique among current therapies because it acetylates cyclooxygenase (COX)-2 enabling the biosynthesis of R-containing precursors of endogenous antiinflammatory mediators. Here, we report that lipidomic analysis of exudates obtained in the resolution phase from mice treated with ASA and docosahexaenoic acid (DHA) (C22:6) produce a novel family of bioactive 17R-hydroxy-containing di- and tri-hydroxy-docosanoids termed resolvins. Murine brain treated with aspirin produced endogenous 17R-hydroxydocosahexaenoic acid as did human Microglial cells. Human COX-2 converted DHA to 13-hydroxy-DHA that switched with ASA to 17R-HDHA that also proved a major route in hypoxic endothelial cells. Human neutrophils transformed COX-2-ASA-derived 17R-hydroxy-DHA into two sets of novel di- and trihydroxy products; one initiated via oxygenation at carbon 7 and the other at carbon 4. These compounds inhibited (IC(50) approximately 50 pM) Microglial cell cytokine expression and in vivo dermal inflammation and peritonitis at ng doses, reducing 40-80% leukocytic exudates. These results indicate that exudates, vascular, leukocytes and neural cells treated with aspirin convert DHA to novel 17R-hydroxy series of docosanoids that are potent regulators. These biosynthetic pathways utilize omega-3 DHA and EPA during multicellular events in resolution to produce a family of protective compounds, i.e., resolvins, that enhance proresolution status

  34. Tan J, Town T, Crawford F, Mori T, DelleDonne A, Crescentini R, Obregon D, Flavell RA, Mullan MJ (2002) Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice. Nat.Neurosci. 5:1288-1293
    Abstract: We have shown that interaction of CD40 with CD40L enables Microglial activation in response to amyloid-beta peptide (Abeta), which is associated with Alzheimer's disease (AD)-like neuronal tau hyperphosphorylation in vivo. Here we report that transgenic mice overproducing Abeta, but deficient in CD40L, showed decreased astrocytosis and microgliosis associated with diminished Abeta levels and beta-amyloid plaque load. Furthermore, in the PSAPP transgenic mouse model of AD, a depleting antibody against CD40L caused marked attenuation of Abeta/beta-amyloid pathology, which was associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased circulating levels of Abeta. Conversely, in neuroblastoma cells overexpressing wild-type human APP, the CD40-CD40L interaction resulted in amyloidogenic APP processing. These findings suggest several possible mechanisms underlying mitigation of AD pathology in response to CD40L depletion, and validate the CD40-CD40L interaction as a target for therapeutic intervention in AD

  35. Tanimukai S, Hasegawa H, Nakai M, Yagi K, Hirai M, Saito N, Taniguchi T, Terashima A, Yasuda M, Kawamata T, Tanaka C (2002) Nanomolar amyloid beta protein activates a specific PKC isoform mediating phosphorylation of MARCKS in Neuro2A cells. Neuroreport 13:549-553
    Abstract: Myristoylated alanine-rich C kinase substrate (MARCKS), a protein associated with cell growth, neurosecretion and macrophage activation, is activated by protein kinase C (PKC) phosphorylation. We reported that amyloid beta protein (Abeta) activated MARCKS through a tyrosine kinase and PKC-delta in rat cultured Microglia. Here we report that Abeta signaling pathway through a specific PKC isoform is involved in the phosphorylation of MARCKS in Neuro2A cells. Selective PKC inhibitors but not tyrosine kinase inhibitors significantly inhibited the phosphorylation of MARCKS induced by Abeta. Abeta selectively activated PKC-alpha among the four PKC isoforms localized in Neuro2A cells. PKC-alpha activated by Abeta directly phosphorylated a recombinant MARCKS in vitro, Translocation of PKC-alpha from the cytoplasm to the membrane and accumulation of phospho-MARCKS in the cytoplasm were induced by Abeta. These results suggest involvement of a phosphoinositide signaling system through PKC-alpha in the phosphorylation of MARCKS in neurons, an event which may be associated with mechanisms underlying neurotrophic and neurotoxic effects of Abeta

  36. Traister RS, Lynch WP (2002) Reexamination of amphotropic murine leukemia virus neurovirulence: neural stem cell-mediated Microglial infection fails to induce acute neurodegeneration. Virology 293:262-272
    Abstract: The 4070A amphotropic murine leukemia virus (A-MuLV) has been variably reported to harbor neurovirulence determinants within its env gene. In this report we reexamined this issue by applying two approaches previously demonstrated to amplify murine leukemia virus neurovirulence. The first approach involved introducing the 4070A env gene into the background of Friend virus clone FB29 to enhance peripheral virus replication kinetics and central nervous system entry. The resulting chimeric virus, FrAmE, exhibited widespread vascular infection throughout the central nervous system (CNS); however, parenchymal infection was quite limited. Neither clinical neurological signs nor spongiform neurological changes accompanied FrAmE CNS infection. To overcome this CNS entry limitation, 4070A and FrAmE were delivered directly into the CNS via transplantation of infected C17.2 neural stem cells (NSCs). Significantly, NSC dissemination of either 4070A or FrAmE resulted in widespread, high-level amphotropic virus expression within the CNS parenchyma, including the infection of Microglia, the critical target required for inducing neurodegeneration. Despite the extensive CNS infection, no associated clinical neurological signs or acute neuropathological changes were observed. Interestingly, we observed the frequent appearance of circulating polytropic (MCF) virus in the serum of amphotropic virus-infected animals. However, neither peripheral inoculation of an amphotropic/MCF virus mixture nor transplantation of NSCs expressing both amphotropic and MCF viruses induced acute clinical neurological signs or spongiform neuropathology. Thus, the results generated in this study suggest that the 4070A env gene is not inherently neurovirulent. However, the frequent appearance of endogenous MCF viruses suggests the possibility that the interactions of amphotropic viruses with endogenous retroviral elements could contribute to the development of retrovirus-induced neurodegenerative disease

  37. Warntges S, Friedrich B, Henke G, Duranton C, Lang PA, Waldegger S, Meyermann R, Kuhl D, Speckmann EJ, Obermuller N, Witzgall R, Mack AF, Wagner HJ, Wagner A, Broer S, Lang F (2002) Cerebral localization and regulation of the cell volume-sensitive serum- and glucocorticoid-dependent kinase SGK1. Pflugers Arch. 443:617-624
    Abstract: The serum- and glucocorticoid-dependent kinase SGK1 is regulated by alterations of cell volume, whereby cell shrinkage increases and cell swelling decreases the transcription, expression and activity of SGK1. The kinase is expressed in all human tissues studied including the brain. The present study was performed to localize the sites of SGK1 transcription in the brain, to elucidate the influence of the hydration status on SGK1 transcription and to explore the functional significance of altered SGK1 expression. Northern blot analysis of human brain showed SGK1 to be expressed in all cerebral structures examined: amygdala, caudate nucleus, corpus callosum, hippocampus, substantia nigra, subthalamic nucleus and thalamus. In situ hybridization and immunohistochemistry in the rat revealed increased expression of SGK1 in neurons of the hippocampal area CA3 after dehydration, compared with similar slices from brains of euvolaemic rats. Additionally, several oligodendrocytes, a few Microglial cells, but no astrocytes, were positive for SGK1. The abundance of SGK1 mRNA in the temporal lobe, including hippocampus, was increased by dehydration and SGK1 transcription in neuroblastoma cells was stimulated by an increase of extracellular osmolarity. Co-expression studies in Xenopus laevis oocytes revealed that SGK1 markedly increased the activity of the neuronal K+ channel Kv1.3. As activation of K+ channels modifies excitation of neuronal cells, SGK1 may participate in the regulation of neuronal excitability

  38. Badie B, Schartner J, Prabakaran S, Paul J, Vorpahl J (2001) Expression of Fas ligand by Microglia: possible role in glioma immune evasion. J.Neuroimmunol. 120:19-24
    Abstract: The immune-privileged status of the central nervous system is thought to limit the application of immunotherapy for treatment of malignant brain tumors. Because the Fas pathway has been proposed to play a role in immune evasion, we examined the effect of tumor environment on the expression of Fas ligand (FasL) in a mouse glioma model. Immunoblotting revealed the expression of membrane-bound FasL to nearly double when murine G26 gliomas were propagated intracranially (IC) as compared to subcutaneously (SC). Further analysis by flow cytometry revealed Microglia, which were absent in the SC tumors, to account for half of the FasL expression in the IC tumors. Interestingly, when FasL activity was inhibited in IC tumors, the proportion of tumor-infiltrating leukocytes increased three-fold, reaching the same frequency as the SC tumors. These observations suggest that Microglia are a major source of FasL expression in brain tumors and possibly contribute to the local immunosuppressive milieu of malignant gliomas

  39. Bate C, Reid S, Williams A (2001) Killing of prion-damaged neurones by Microglia. Neuroreport 12:2589-2594
    Abstract: The loss of neurones that occurs in the transmissible spongiform encephalopathies, or prion diseases, can be reproduced in vitro by incubating neuronal cultures with either peptides derived from the prion protein or with partially purified prion preparations. In the present studies, the extent of neuronal loss on exposure to these prions or prion peptides was increased by the addition of Microglia, a process that was dependent upon the number of Microglia added, the concentration of prions/peptides present and the degree of fibrillarity of the prion peptides. Microglia also killed scrapie-infected neuroblastoma cells expressing infectious PrP(SC). Microglia secreted low amounts of interleukin (IL)-6 when incubated with peptides alone but up to 10 times as much IL-6 when incubated with peptide-treated neurones, suggesting that Microglia recognise peptide-induced changes in neurones

  40. Bu J, Akhtar N, Nishiyama A (2001) Transient expression of the NG2 proteoglycan by a subpopulation of activated macrophages in an excitotoxic hippocampal lesion. Glia 34:296-310
    Abstract: Cells that express the NG2 proteoglycan (NG2+ cells) constitute a large glial population in the normal mature rodent brain. They can differentiate into oligodendrocytes but are distinct from mature oligodendrocytes, astrocytes, Microglia, and neurons. Changes in NG2+ cells were examined in kainic acid-induced excitotoxic lesions of the hippocampus, and the relationship between NG2+ cells and reactive astrocytes and Microglia was investigated between 1 and 90 days after lesioning. Two types of reactive NG2+ cells with altered morphology and increased NG2 immunoreactivity were observed in the lesion. Early changes, consisting of an increase in NG2 immunoreactivity and the number of processes, were apparent 24 h after lesioning and persisted through 3 months. These cells were distinct from reactive astrocytes or activated Microglia/macrophages. A second type of reactive NG2+ cells appeared 2 weeks after injection, following an influx of macrophages. They had large, round cell bodies with short processes and expressed the Microglia/macrophage antigens OX42 and ED1. Single cells coexpressing NG2 and macrophage/Microglial antigens could be isolated from the lesion. The number of NG2+/OX42+ cells gradually declined and disappeared by 3 months after injection. They did not express glial fibrillary acidic protein or the alpha receptor for platelet-derived growth factor, indicating that they are distinct from astrocytes or oligodendrocyte progenitor cells. Cells that coexpressed NG2 and OX42 were never observed in hippocampal slice cultures treated with kainic acid, suggesting that NG2+/OX42+ cells are not derived from endogenous resident brain cells. These findings demonstrate that NG2 expression is transiently upregulated on activated macrophages/Microglia that appear during the chronic stage in an excitotoxic lesion in the adult CNS

  41. Eales-Reynolds LJ, Laver H, Mojtahedi H (2001) Evidence for the expression of the EGF receptor on human monocytic cells. Cytokine 16:169-172
    Abstract: Several malignancies over-express the epidermal growth factor receptor, ligation of which results in cellular differentiation and multiplication. Mononuclear phagocytes secrete this cytokine and its receptor has been detected on Microglial cells. This communication describes the expression (and its regulation) of epidermal growth factor receptor (EGFR) on U937 cells. We have shown that a few are EGFR-positive, with expression being up regulated by interleukin 6 (IL-6). Also, when cultured in the presence of serum with the monoclonal anti-EGFR, ICR62, U937s showed a reduced growth rate. By contrast, ICR9 caused a significant increase in cellular proliferation. Both antibodies induced cycle arrest in late G(1)/S phase. When the cells were cultured in the absence of serum, low antibody concentration (10 microg/ml) showed an early inhibitory effect on cell proliferation. By contrast, at high antibody concentrations (50 micro/ml), ICR62 significantly increased the proliferation of U937 cells. We suggest that these results provide indirect evidence for an autocrine action of EGF on U937 cells

  42. Elmlinger MW, Deininger MH, Schuett BS, Meyermann R, Duffner F, Grote EH, Ranke MB (2001) In vivo expression of insulin-like growth factor-binding protein-2 in human gliomas increases with the tumor grade. Endocrinology 142:1652-1658
    Abstract: Human central nervous system tumors and glioma cell lines highly express the insulin-like growth factor-binding protein (IGFBP)-2. As IGFBP-2 can affect tumor growth, we studied the relationship between IGFBP-2 expression and the malignancy of brain tumors in vivo. To do so, we investigated by immunohistochemistry the accumulation of IGFBP-1, -2, and -3 in 50 human gliomas classified by the WHO Malignancy Scale. Double labeling using anti-CD68 (monocytes/macrophages), antiglial fibrillary acidic protein, and anti-CD3 (T cells) antibodies was performed to further characterize the IGFBP-1, -2, and -3(+) cells. The expression of IGFBP messenger RNAs (mRNAs) was tested by RT-PCR in tumor samples from nine gliomas of different grades and in eight cell lines representing the cellular composition of human glioma. As controls, the accumulation of IGFBP-2 was investigated in normal brain and in the rat C6 glioblastoma model. IGFBP-1 and -3 accumulated in endothelial and macrophage/Microglial cells. IGFBP-2(+) macrophage/Microglial and glioma cells clustered in the immediate vicinity of focal necrosis of the human gliomas as well as of the rat C6 glioblastoma. The labeling score of IGFBP-1 accumulation in endothelial cells correlated negatively (P: = 0.0229), and that of IGFBP-2 accumulation in glioma cells correlated positively (P: < 0.0006) with the tumor grade of the gliomas. In addition, RT-PCR analysis confirmed mRNA expression of IGFBP-1, -2, and -3 by the gliomas and glial cells. Small amounts of IGFBP-1 and -3 mRNA, but high amounts of IGFBP-2 mRNA, were detectable in macrophage-like and glioma cell lines. The results suggest cell type-specific accumulation of IGFBP-1, -2, and -3 in human glial tumors of the brain. The increase in IGFBP-2 expression with this malignancy suggests a role of IGFBP-2 in the biology of human gliomas

  43. Fischer FR, Luo Y, Luo M, Santambrogio L, Dorf ME (2001) RANTES-induced chemokine cascade in dendritic cells. J.Immunol. 167:1637-1643
    Abstract: Dendritic cells (DC) are the most potent APCs and the principal activators of naive T cells. We now report that chemokines can serve as activating agents for immature DC. Murine bone marrow-derived DC respond to the CC chemokine RANTES (10-100 ng/ml) by production of proinflammatory mediators. RANTES induces rapid expression of transcripts for the CXC chemokines KC and macrophage inflammatory protein (MIP)-2, the CC chemokines MIP-1beta and MIP-1alpha, and the cytokines TNF-alpha and IL-6. Synthesis of KC, IL-6, and TNF-alpha proteins were also demonstrated. After 4 h, autoinduction of RANTES transcripts was observed. These responses are chemokine specific. Although DC demonstrated weak responses to eotaxin, DC failed to respond to other chemokines including KC, MIP-2, stromal-derived factor-1alpha, MIP-1beta, MIP-1alpha, monocyte chemoattractant protein-1, T cell activation gene 3, or thymus-derived chemotactic agent 4. In addition, RANTES treatment up-regulated expression of an orphan chemokine receptor termed Eo1. Chemokine induction was also observed after treatment of splenic DC and neonatal Microglia with RANTES, but not after treatment of thymocytes or splenocytes depleted of adherent cells. TNF-alpha-treated DC lose responsiveness to RANTES. DC from mice deficient for CCR1, CCR3, and CCR5 respond to RANTES, indicating that none of these receptors are exclusively used to initiate the chemokine cascade. RANTES-mediated chemokine amplification in DC may prolong inflammatory responses and shape the microenvironment, potentially enhancing acquired and innate immune responses

  44. Fleige G, Nolte C, Synowitz M, Seeberger F, Kettenmann H, Zimmer C (2001) Magnetic labeling of activated Microglia in experimental gliomas. Neoplasia. 3:489-499
    Abstract: Microglia, as intrinsic immunoeffector cells of the central nervous system (CNS), play a very sensitive, crucial role in the response to almost any brain pathology where they are activated to a phagocytic state. Based on the characteristic features of activated Microglia, we investigated whether these cells can be visualized with magnetic resonance imaging (MRI) using ultrasmall superparamagnetic iron oxides (USPIOs). The hypothesis of this study was that MR Microglia visualization could not only reveal the extent of the tumor, but also allow for assessing the status of immunologic defense. Using USPIOs in cell culture experiments and in a rat glioma model, we showed that Microglia can be labeled magnetically. Labeled Microglia are detected by confocal microscopy within and around tumors in a typical border-like pattern. Quantitative in vitro studies revealed that Microglia internalize amounts of USPIOs that are significantly higher than those incorporated by tumor cells and astrocytes. Labeled Microglia can be detected and quantified with MRI in cell phantoms, and the extent of the tumor can be seen in glioma-bearing rats in vivo. We conclude that magnetic labeling of Microglia provides a potential tool for MRI of gliomas, which reflects tumor morphology precisely. Furthermore, the results suggest that MRI may yield functional data on the immunologic reaction of the CNS

  45. Hentze H, Schwoebel F, Lund S, Keel M, Ertel W, Wendel A, Jaattela M, Leist M, Kehl M (2001) In vivo and in vitro evidence for extracellular caspase activity released from apoptotic cells. Biochem.Biophys.Res.Commun. 283:1111-1117
    Abstract: While caspases play an established role as intracellular executors of apoptosis, little is known about extracellular activities of this ubiquitously expressed family of proteases. We demonstrate here that recombinant caspase-3 retained enzymatic activity in various extracellular fluids. Experiments with cell lines, primary cells, and mice with fulminant CD95-triggered hepatitis showed that significant amounts of DEVD-aminofluoromethylcoumarine-cleaving activity, indicative of active effector caspases, were released into the medium/plasma during apoptosis. Furthermore, caspase activities were detected in liquor samples from human head trauma patients. These findings warrant closer investigation of DEVDase activity as a diagnostic marker, and of potential extracellular substrates for caspases

  46. Hodges JL, Ireland DD, Reiss CS (2001) The role of interleukin-18 in vesicular stomatitis virus infection of the CNS. Viral Immunol. 14:181-191
    Abstract: Intranasal application of vesicular stomatitis virus (VSV) results in the initial infection of the olfactory receptor neurons and a rapid progression of the virus through the mouse central nervous system (CNS). Interleukin-18 (IL-18) is an 18.3-kd cytokine that induces interferon gamma (IFN-gamma) production in mice. IL-18 is synthesized as an inactive precursor that is cleaved and activated by caspase-1/interleukin-1beta converting enzyme (ICE). IL-18 shares several biological properties with IL-12, including the ability to induce IFN-gamma production in T lymphocytes and natural killer (NK) cells. In the CNS, Microglia and astrocytes produce IL-18 and IL-12. We have previously shown that IL-12 promotes recovery from VSV encephalitis. This led us to examine the potential role of IL-18 in the pathogenesis of VSV encephalitis. We show that both IL-18 and caspase-1 mRNA are consistently present in the CNS of mice. The addition of exogenous IL-18 to cell cultures does not affect the production of VSV, and addition of exogenous IL-18 at the time of infection does not alter the morbidity or mortality of BALB/c mice. In vitro studies with neutralizing monoclonal antibody to IL-18 had no effect. From these results we conclude that in this system and under the experimental conditions used, unlike IL-12 and IFN-gamma, IL-18 does not play a significant role in the host response to VSV infection

  47. Hoozemans JJ, Rozemuller AJ, Janssen I, De Groot CJ, Veerhuis R, Eikelenboom P (2001) Cyclooxygenase expression in Microglia and neurons in Alzheimer's disease and control brain. Acta Neuropathol.(Berl) 101:2-8
    Abstract: Epidemiological studies suggest that non-steroidal anti-inflammatory drugs (NSAIDs) lower the risk of developing Alzheimer's disease (AD). Most NSAIDs act upon local inflammatory events by inhibiting the expression or activation of cylooxygenase (COX). In the present study the expression of COX-1 and COX-2 in AD and non-demented control temporal and frontal cortex was investigated using immunohistochemistry. COX-1 expression was detected in Microglial cells, while COX-2 expression was found in neuronal cells. In AD brains, COX-1-positive Microglial cells were primarily associated with amyloid beta plaques, while the number of COX-2-positive neurons was increased compared to that in control brains. No COX expression was detected in astrocytes. In vitro, primary human Microglial and astrocyte cultures, and human neuroblastoma cells (SK-N-SH) were found to secrete prostaglandin E2 (PGE2), especially when stimulated. PGE2 synthesis by astrocytes and SK-N-SH cells was stimulated by interleukin-1beta. Microglial cell PGE2 synthesis was stimulated by lipopolysaccharide only. Although astrocytes are used in studies in vitro to investigate the role of COX in AD, there are no indications that these cells express COX-1 or COX-2 in vivo. The different distribution patterns of COX-1 and COX-2 in AD could implicate that these enzymes are involved in different cellular processes in the pathogenesis of AD

  48. Ito A, Saito S, Masuko T, Oh-Eda M, Matsuura T, Satoh M, Nejad FM, Enomoto T, Orikasa S, Hakomori SI (2001) Monoclonal antibody (5F3) defining renal cell carcinoma-associated antigen disialosyl globopentaosylceramide (V3NeuAcIV6NeuAcGb5), and distribution pattern of the antigen in tumor and normal tissues. Glycoconj.J. 18:475-485
    Abstract: Renal cell carcinoma (RCC) has been characterized by high expression of three types of disialogangliosides: two based on lacto-series type 1 structure (disialosyl Lc(4), GalNAc disialosyl Lc(4)), the other based on globo-series structure (disialosyl globopentaosylceramide; disialosyl Gb5). The present study established a mAb, 5F3, directed to disialosyl Gb5. 5F3 was established after immunization with RCC cell line ACHN. The major disialoganglioside antigen isolated from ACHN cells, showing specific reactivity with 5F3, was characterized unequivocally as disialosyl Gb5 (V(3)NeuAcIV(6)NeuAcGb5) by identification of the core structure as globopentaosylceramide (Gb5) after enzymatic and acid hydrolysis, and by 2-dimensional (1)H-NMR spectroscopy. 5F3 does not react with monosialosyl Gb5 (V(3)NeuAcGb5), Gb5, or any lacto-series structures. 5F3 strongly stained 19 of 41 cases of primary RCC tissue. It reacted with proximal tubules (but not distal tubules) of kidney, Microglial cells of cerebrum and cerebellum, goblet cells of stomach and intestine, smooth muscle of various organs. It did not react with parenchymatous cells of various organs, except for kidney epithelia and prostate stroma. Immunostaining of RCC tissue by mAb 5F3, in combination with staining by other antibodies directed to globo-series and lacto-series structures, has prognostic significance in defining metastatic potential of RCC

  49. Keppler OT, Horstkorte R, Pawlita M, Schmidt C, Reutter W (2001) Biochemical engineering of the N-acyl side chain of sialic acid: biological implications. Glycobiology 11:11R-18R
    Abstract: N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, Microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications

  50. Le Y, Yazawa H, Gong W, Yu Z, Ferrans VJ, Murphy PM, Wang JM (2001) The neurotoxic prion peptide fragment PrP(106-126) is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1. J.Immunol. 166:1448-1451
    Abstract: Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP(106-126), was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP(106-126) is unknown. In this study, we show that PrP(106-126) is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP(106-126), FPRL1 underwent a rapid internalization and, furthermore, PrP(106-126) enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a "pattern recognition" receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases

  51. Platten M, Wick W, Weller M (2001) Malignant glioma biology: role for TGF-beta in growth, motility, angiogenesis, and immune escape. Microsc.Res.Tech. 52:401-410
    Abstract: Characteristics of human malignant glioma are excessive proliferation, infiltrative growth, angiogenesis and suppression of anti-tumor immune surveillance. Transforming growth factor-beta (TGF-beta), a versatile cytokine, is intimately involved in the regulation of these processes. Here, we discuss the interactions of TGF-beta with growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet derived growth factor (PDGF), metalloproteinases (MMP-2, MMP-9) and their inhibitor, plasmin activator inhibitor-1 (PAI-1), and immune cells, like natural killer cells, T-cells and Microglia. The differential effects of TGF-beta in glioma biology are outlined with emphasis on the induction of a survival advantage for glioma cells by enforced cell growth, migration, invasion, angiogenesis and immune paralysis. By virtue of its growth regulatory and immunomodulatory properties, TGF-beta promises to become a novel target for the experimental therapy of human malignant glioma

  52. Portis JL (2001) Genetic determinants of neurovirulence of murine oncornaviruses. Adv.Virus Res. 56:3-38

  53. Proescholdt MA, Merrill MJ, Ikejiri B, Walbridge S, Akbasak A, Jacobson S, Oldfield EH (2001) Site-specific immune response to implanted gliomas. J.Neurosurg. 95:1012-1019
    Abstract: OBJECT: Immunotherapy for glioblastoma has been uniformly ineffective. The immunological environment of the brain, with its low expression of major histocompatibility complex (MHC) molecules and limited access for inflammatory cells and humoral immune effectors due to the blood-brain barrier (BBB), may contribute to the failure of immunotherapy. The authors hypothesize that brain tumors are protected from immune surveillance by an intact BBB at early stages of development. To investigate the immunological characteristics of early tumor growth, the authors compared the host response to a glioma implanted into the brain and into subcutaneous tissue. METHODS: Samples of tumors growing in the brain or subcutaneously in rats were obtained for 7 consecutive days and were examined immunohistochemically for MHC Class I & II molecules, and for CD4 and CD8 lymphocyte markers. Additionally, B7-1 costimulatory molecule expression and lymphocyte-specific apoptosis were examined. CONCLUSIONS: On Days 3 and 4 after implantation, brain tumors displayed significantly lower MHC Class II expression and lymphocytic infiltration (p < 0.05). After Day 5, however, no differences were detected. The MHC Class II expressing cells within the brain tumors appeared to be infiltrating Microglia. Minimal B7-1 expression combined with lymphocyte-specific apoptosis were detected in both brain and subcutaneous tumors. Low MHC Class II expression and low lymphocytic infiltration at early time points indicate the importance of the immunologically privileged status of the brain during early tumor growth. These characteristics disappeared at later time points, possibly because the increasing perturbation of the BBB alters the specific immunological environment of the brain. The lack of B7-1 expression combined with lymphocyte apoptosis indicates clonal anergy of glioma-infiltrating lymphocytes regardless of implantation site

  54. Roth W, Isenmann S, Nakamura M, Platten M, Wick W, Kleihues P, Bahr M, Ohgaki H, Ashkenazi A, Weller M (2001) Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Res. 61:2759-2765
    Abstract: Decoy receptor 3 (DcR3) is a newly identified soluble protein that binds to CD95 ligand (CD95L) and inhibits its proapoptotic activity. Here we report that DcR3 is expressed by the majority of long-term and ex vivo malignant glioma cell lines as well as in human glioblastoma in vivo. Expression of DcR3 correlates with the grade of malignancy: 15 of 18 (83%) glioblastomas (WHO grade IV) but none of 11 diffuse astrocytomas (WHO grade II) exhibited DcR3 immunoreactivity. We also demonstrate that human malignant glioma cells engineered to release high amounts of DcR3 into the cell culture supernatant are protected from CD95L-induced apoptotic cell death. In contrast, DcR3 does not confer protection from the death ligand Apo2 ligand (TRAIL). Importantly, ectopic expression of DcR3 resulted in substantial differences in immune cell infiltration in the 9L rat gliosarcoma model. Thus, the infiltration of CD4+ and CD8+ T cells as well as Microglia/macrophages into glioma was substantially decreased in DcR3-producing tumors compared with control tumors. Chemotaxis assays revealed that DcR3 counteracts the chemotactic activity of CD95L against Microglial cells in vitro. These findings suggest that DcR3 may be involved in the progression and immune evasion of malignant gliomas

  55. Sugibayashi R, Shimizu T, Suzuki T, Yamamoto N, Hamada H, Takeda K (2001) Upregulation of p21(WAF1/CIP1) leads to morphologic changes and esterase activity in TPA-mediated differentiation of human prostate cancer cell line TSU-Pr1. Oncogene 20:1220-1228
    Abstract: We reported previously that human prostate cancer cell line TSU-Pr1 can differentiate into Microglia-like cells by 12-O-tetra-decanoylphorbol-13-acetate (TPA) treatment. In this study, we identified a signal transduction pathway involved in TPA-induced TSU-Pr1 cell differentiation and investigated the mechanism of growth arrest that accompanies this differentiation. TPA-induced differentiation and growth arrest of TSU-Pr1 cells were inhibited by treatment with Protein kinase C (PKC) inhibitor GF109203X and mitogen-activated protein (MAP) kinase inhibitor PD98059. Treatment of TSU-Pr1 cells with TPA for 15 min or longer resulted in translocation of PKCalpha, PKCgamma, and PKCepsilon from cytosolic to membrane fraction. Our results suggest that TPA-induced TSU-Pr1 cell differentiation is associated with activation of MAP kinase and PKCalpha, PKCgamma, and PKCepsilon. The mechanism of growth arrest in TSU-Pr1 cells that underwent TPA-induced differentiation were examined for factors in the signaling pathway downstream of MAP kinase that control the cell cycle. Upregulation of p21(WAF1/CIP1) cyclin-dependent kinase inhibitor protein was observed in a manner dependent on PKC or MAP kinase. Moreover, adenovirus-mediated overexpression of recombinant p21(WAF1/CIP1) in TSU-Pr1 cells result in growth arrest, morphological change to Microglia-like cells, and increased alpha-naphthyl acetate esterase activity, all of which are associated with cellular differentiation. Thus, our results indicate that p21(WAF1/CIP1) mediates TPA-induced growth arrest and differentiation of TSU-Pr1 cells

  56. Synowitz M, Ahmann P, Matyash M, Kuhn SA, Hofmann B, Zimmer C, Kirchhoff F, Kiwit JC, Kettenmann H (2001) GABA(A)-receptor expression in glioma cells is triggered by contact with neuronal cells. Eur.J.Neurosci. 14:1294-1302
    Abstract: The expression of functional GABA(A)-receptors in glioma cells correlates with low malignancy of tumours and cell lines from glioma lack these receptors. Here we show that contact with neurons induces the expression of functional GABA(A)-receptors. C6 and F98 glioma cell lines were labelled by recombinant expression of enhanced green fluorescent protein injected into rat brain and studied in acute slices after two to three weeks of tumour growth. The cells responded to GABA or the specific agonist, muscimol with a current typical for GABA(A)-receptors, as studied with the patch-clamp technique. To get insight into the mechanism of GABA(A) receptor induction, the C6 or F98 cells were co-cultured with neurons, astrocytes, oligodendrocytes and Microglia. Glioma cells expressed functional GABA(A) receptors within 24 h only in cultures where physical contact to neurons occurred. Activation of GABA(A)-receptors in the co-cultures attenuated glioma cell metabolism while blockade of the receptors increased metabolism. We conclude that with this form of interaction, neurons can influence tumour behaviour in the brain

  57. Carpentier AF, Xie J, Mokhtari K, Delattre JY (2000) Successful treatment of intracranial gliomas in rat by oligodeoxynucleotides containing CpG motifs. Clin.Cancer Res. 6:2469-2473
    Abstract: Phosphorothioate oligodeoxynucleotides with CpG motifs (CpG-ODNs) activate various immune cell subsets and induce production of numerous cytokines. To evaluate whether CpG-ODNs can induce rejection of established tumors, Lewis rats were inoculated intracerebrally with syngeneic CNS-1 glioma cells and subsequently injected with CpG-ODNs into the tumor bed. Although all of the control rats (n = 14) died within 23 days, 88% of the animals (n = 8) treated with a single CpG-ODN injection 5 days after tumor inoculation showed long-term survival (>90 days; P < 0.002). CpG-ODNs increased tumoral infiltration with macrophage/Microglial cells, CD8, and natural killer lymphocytes. CpG-ODN-cured animals were further protected against a second tumor challenge. CpG-ODNs had no effect on a s.c. CNS1 tumor in nude mice, which suggested that CpG-ODN is not directly cytotoxic and that immunostimulation is required for the antitumoral effect. These findings suggest that intratumoral injections of CpG-ODNs represent a new immunotherapeutic approach in human gliomas, which overcome the need for the selection and purification of a tumoral antigen

  58. Cohen J, Sugita Y, Chader GJ, Schwartz JP (2000) Recombinant forms of the neurotrophic factor pigment epithelium-derived factor activate cellular metabolism and inhibit proliferation of the RAW macrophage cell line. Neuroimmunomodulation. 7:51-58
    Abstract: Recombinant forms of the neurotrophic factor pigment epithelium-derived factor (PEDF) activate metabolism of RAW macrophage cells while simultaneously inhibiting their proliferation. The recombinant forms (rPEDF) acted with EC(50)s of 0.1-1 nM while full-length native bovine PEDF was inactive. Urea, which is the buffer used to extract recombinant PEDF, stimulated RAW cell proliferation, the first report of an effect of urea on non-kidney cells. PEDF acted within 12 h and its effects persisted up to 72 h with continuous exposure. Although rPEDF had no direct action on glioma cell lines, it increased the amount of a soluble factor released by RAW cells which was capable of blocking glioma cell division. Thus PEDF may function as a neuroimmune modulator, affecting both neural and immune system cells

  59. Dallasta LM, Wang G, Bodnar RJ, Draviam R, Wiley CA, Achim CL, Hamilton RL (2000) Differential expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in chronic murine retroviral encephalitis. Neuropathol.Appl.Neurobiol. 26:332-341
    Abstract: The cell adhesion molecules, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, are important mediators of immune interactions within the central nervous system (CNS). A wide variety of pro-inflammatory insults to the brain, including viral infection, result in upregulation of these molecules on brain endothelial cells, astrocytes, and Microglia. This study investigated the expression of ICAM-1 and VCAM-1 in chronic encephalitis induced by infection with a temperature sensitive (ts-1) strain of Moloney murine leukaemia virus (MoMuLV), an ecotropic murine retrovirus. During the late stages of disease, viral antigen was present in both endothelial cells and Microglia, but not astrocytes, in regions of spongiform change and gliosis. In these areas, ICAM-1 staining was detected on activated Microglia, but not on endothelial cells or astrocytes. In contrast, no cells showed increased VCAM-1 expression in the CNS. These findings demonstrate that there is cell-specific, differential expression of these adhesion molecules in ts-1 retroviral encephalitis. The lack of endothelial cell expression correlates with the characteristic lack of lymphocytic infiltrate in this chronic retroviral encephalitis and suggests that increased Microglial ICAM-1 expression may play a role in the pathogenesis of MoMuLV (ts-1)-mediated neurodegeneration

  60. Deininger MH, Seid K, Engel S, Meyermann R, Schluesener HJ (2000) Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/Microglial cells in rat and human gliomas. Acta Neuropathol.(Berl) 100:673-680
    Abstract: Allograft inflammatory factor-1 (AIF-1) is a Ca2+-binding peptide that constitutes a potential modulator of macrophage activation and function during the immune response of the brain. Peptides termed Microglia response factor-1 or ionized calcium-binding adaptor molecule- have been reported to be identical with AIF-1. We have investigated the expression of AIF-1 in the rat C6 glioblastoma and 9L gliosarcoma tumor models and additionally assessed AIF- expression in a diverse range of human astrocytomas by immunohistochemistry. AIF-1 was expressed by activated Microglial cells and a subset of infiltrating macrophages in areas of infiltrative tumor growth and in compact tumor areas in both rat and human gliomas. Double-labeling experiments in rats and humans characterized the nature and the functional status of AIF-1+ cells. AIF-1 expression was detected in cells expressing major histocompatibility complex class II molecules and in a subset of activated macrophages/Microglial cells. All MRP-8+ cells coexpressed AIF-1. In humans, there was a strong correlation of AIF-1-expressing activated macrophages/Microglial cells with tumor malignancy (P < 0.0001). These results suggest that AIF-1 defines a distinct subset of tumor-associated activated macrophages/ Microglial cells

  61. Deininger MH, Meyermann R, Trautmann K, Duffner F, Grote EH, Wickboldt J, Schluesener HJ (2000) Heme oxygenase (HO)-1 expressing macrophages/Microglial cells accumulate during oligodendroglioma progression. Brain Res. 882:1-8
    Abstract: Heme oxygenase (HO-1, HSP32) catalyzes the oxidation of heme to biliverdin and carbon monoxide, a putative neurotransmitter. In the brain, HO-1 expression has been associated with neuroprotection during oxidative stress and hypoxia. However, consecutive downstream mediation is involved in neoangiogenesis and consequent neoplastic outgrowth. We have analyzed HO-1 expression in 69 oligodendroglioma tissue samples, in rat intracranially transplanted C6 gliomas, and neuropathologically unaltered control brains by immunohistochemistry. Double labeling experiments confirmed the nature of HO-1 expressing cells. Reverse transcription-polymerase chain reaction was used to demonstrate HO-1 gene expression. HO-1 immunoreactivity was predominantly observed in macrophages/Microglial cells. The number of HO-1 expressing macrophages/Microglial cells was significantly lower in primary oligodendrogliomas than in their matched relapses (P<0.0001) and lower in primary anaplastic oligodendrogliomas than in their relapses (P=0.0006). Prominent accumulation of HO-1 expressing macrophages/Microglial cells was observed in perinecrotic areas of both experimental rat and human glioblastoma relapses. HO-1 expressing neurons, macrophages/Microglial cells and astrocytes were scattered in areas of infiltrative tumor growth. Surprisingly, HO-1 mRNA was detected in only one glioblastoma multiforme relapse. We conclude from these data that HO-1 expressing macrophages/Microglial cells accumulate during oligodendroglioma progression in areas of focal necrosis. However, overall biological function of this phenomenon remains to be determined

  62. Kingham PJ, Pocock JM (2000) Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release. J.Neurochem. 74:1452-1462
    Abstract: Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of Microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain Microglia or the murine N9 Microglial cell line with chromogranin A resulted in nitric oxide production, which triggered Microglial apoptosis. Exposure of Microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced Microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced Microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered Microglial apoptosis and may point to routes for the modulation of Microglial evoked neurotoxicity

  63. Prat E, Baron P, Meda L, Scarpini E, Galimberti D, Ardolino G, Catania A, Scarlato G (2000) The human astrocytoma cell line U373MG produces monocyte chemotactic protein (MCP)-1 upon stimulation with beta-amyloid protein. Neurosci.Lett. 283:177-180
    Abstract: Astrocytes associated with beta-amyloid (Abeta) accumulate in senile plaques of Alzheimer's disease (AD). To investigate the biological effects of Abeta/astrocyte interaction, we examined chemokine production by the human astrocytoma cell line U373MG stimulated with Abeta peptides. Northern blot analysis and specific immunoassays demonstrate that Abeta [1-42] and Abeta [25-35] induce mRNA expression and release of monocyte chemotactic protein (MCP)-1 but not of gamma-interferon inducible protein (IP)-10 by U373MG cells. The observation that Abeta induces astrocyte production of the potent Microglia chemoattractant MCP-1 contributes to understanding mechanism of damage exerted by Abeta in AD senile plaques

  64. Sandmair AM, Turunen M, Tyynela K, Loimas S, Vainio P, Vanninen R, Vapalahti M, Bjerkvig R, Janne J, Yla-Herttuala S (2000) Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions. Cancer Gene Ther. 7:413-421
    Abstract: Herpes simplex virus thymidine kinase (HSV-tk) gene transfer and ganciclovir (GCV) administration have been suggested for the treatment of malignant gliomas. To understand tissue responses and possible ways to improve the treatment effect, we studied tumor growth, tissue reactions, and survival time after HSV-tk/GCV treatment in a syngeneic BT4C rat glioma model by mixing various ratios of stably transfected HSV-tk-expressing BT4C-tk glioma cells with wild-type BT4C glioma cells (percentage of BT4C-tk cells: 0%, 1%, 10%, 30%, 50%, and 100%), followed by injection into BDIX rat brains (n = 79). With the exception of some animals with end-stage tumors, very little astroglia or Microglia reactivity was detected in the wild-type tumors as analyzed by immunocytochemistry using glial fibrillary acid protein (GFAP)-, vimentin-, human histocompatibility leukocyte antigen-DR-, OX-42-, and CD68-specific monoclonal antibodies. After 14 days of GCV treatment, tumors induced with > or = 10% BT4C-tk cells showed a significant reduction in tumor size (P < .05) and prolonged survival time (P < .01). Astrogliosis, as indicated by a strong GFAP and vimentin immunoreactivity, was seen in the tumor scar area. GFAP and vimentin reactivity was already present after the GCV treatment in tumors induced with 1% BT4C-tk cells. Much less human histocompatibility leukocyte antigen-DR-positive Microglia was seen in the treated animals, indicating low Microglia reactivity and immunoactivation against the tumor. However, GCV-treated tumors were positive for apoptosis, indicating that apoptosis is an important mechanism for cell death in the BT4C-tk glioma model. Our results suggest that > or = 10% transfection efficiency is required for a successful reduction in BT4C glioma tumor size with HSV-tk/GCV treatment in vivo. Tissue reactions after 14 days of GCV treatment are characterized by astrogliosis and apoptosis, whereas Microglia response and immunoactivation of the brain cells appear to play a minor role. Stimulation of the Microglia response by gene transfer or other means might improve the efficacy of the HSV-tk/GCV treatment in vivo

  65. Satoh J, Kuroda Y (2000) Amyloid precursor protein beta-secretase (BACE) mRNA expression in human neural cell lines following induction of neuronal differentiation and exposure to cytokines and growth factors. Neuropathology. 20:289-296
    Abstract: Recently, a novel amyloid precursor protein beta-secretase (designated BACE) was identified. Because activated Microglia and astrocytes play a role in amyloidogenesis in Alzheimer's disease, the constitutive and glial cytokine/growth factor-regulated expression of BACE was studied in human neural cell lines. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, BACE mRNA expression was identified in various human neural and non-neural cell lines. By northern blot analysis, the expression of BACE mRNA composed of five distinct transcripts (>8.0, 7.0, 6.0, 4.4 and 2.6 kb) was elevated markedly in NTera2 teratocarcinoma cells following retinoic acid-induced neuronal differentiation. But the levels of three major BACE mRNA species (7.0, 6.0 and 4.4 kb) were not significantly altered in NTera2-derived neurons, SK-N-SH neuroblastoma or U-373MG astrocytoma following exposure to tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, interferon-gamma, transforming growth factor-beta1, epidermal growth factor, basic fibroblast growth factor, brain-derived neurotrophic factor, dibutyryl cyclic adenosine monophosphate or phorbol 12-myristate 13-acetate. These results indicate that BACE mRNA is expressed constitutively in human neural cells and its expression is upregulated during neuronal differentiation, but it is unlikely to be regulated by activated glia-derived cytokines and growth factors

  66. Satoh J, Kuroda Y (2000) Beta-catenin expression in human neural cell lines following exposure to cytokines and growth factors. Neuropathology. 20:113-123
    Abstract: Beta-catenin acts as a key mediator of the Wnt/Wingless signaling pathway involved in cell proliferation, differentiation and survival. Recent studies have shown that an unstable interaction between beta-catenin and the mutant presenilin-1 induces neuronal apoptosis, and that beta-catenin levels are decreased in the brains of patients with Alzheimer's disease (AD). Since activated Microglia and astrocytes play a role in the process of neuronal degeneration in AD, the cytokine/growth factor-regulated expression of beta-catenin in human neural cell lines, including NTera2 teratocarcinoma-derived differentiated neurons (NTera2-N), IMR-32 neuroblastoma, SKN-SH neuroblastoma and U-373MG astrocytoma, was studied quantitatively following exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, transforming growth factor (TGF)-beta1, dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (cAMP) (dbcAMP) or phorbol 12-myristate 13-acetate (PMA). Beta-catenin mRNA expressed constitutively in all of these cell lines was unaffected by treatment with any factors examined. In contrast, beta-catenin protein levels were reduced markedly in NTera2-N cells by exposure to dbcAMP, EGF or bFGF, and in U-373MG cells by treatment with dbcAMP or PMA, but were unaffected in any cell lines by BDNF, TNF-alpha, IL-1beta, IL-6, IFN-gamma or TGF-beta1. These results indicate that beta-catenin is expressed constitutively in human neural cells and downregulated at a protein level by a set of growth factors in a cell type-specific manner

  67. Shieh JT, Martin J, Baltuch G, Malim MH, Gonzalez-Scarano F (2000) Determinants of syncytium formation in Microglia by human immunodeficiency virus type 1: role of the V1/V2 domains. J.Virol. 74:693-701
    Abstract: Microglia are the main reservoir for human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS), and multinucleated giant cells, the result of fusion of HIV-1-infected Microglia and brain macrophages, are the neuropathologic hallmark of HIV dementia. One potential explanation for the formation of syncytia is viral adaptation for these CD4(+) CNS cells. HIV-1(BORI-15), a virus adapted to growth in Microglia by sequential passage in vitro, mediates high levels of fusion and replicates more efficiently in Microglia and monocyte-derived-macrophages than its unpassaged parent (J. M. Strizki, A. V. Albright, H. Sheng, M. O'Connor, L. Perrin, and F. Gonzalez-Scarano, J. Virol. 70:7654-7662, 1996). Since the interaction between the viral envelope glycoprotein and CD4 and the chemokine receptor mediates fusion and plays a key role in tropism, we have analyzed the HIV-1(BORI-15) env as a fusogen and in recombinant and pseudotyped viruses. Its syncytium-forming phenotype is not the result of a switch in coreceptor use but rather of the HIV-1(BORI-15) envelope-mediated fusion of CD4(+)CCR5(+) cells with greater efficiency than that of its parental strain, either by itself or in the context of a recombinant virus. Genetic analysis indicated that the syncytium-forming phenotype was due to four discrete amino acid differences in V1/V2, with a single-amino-acid change between the parent and the adapted virus (E153G) responsible for the majority of the effect. Additionally, HIV-1(BORI-15) env-pseudotyped viruses were less sensitive to decreases in the levels of CD4 on transfected 293T cells, leading to the hypothesis that the differences in V1/V2 alter the interaction between this envelope and CD4 or CCR5, or both. In sum, the characterization of the envelope of HIV-1(BORI-15), a highly fusogenic glycoprotein with genetic determinants in V1/V2, may lead to a better understanding of the relationship between HIV replication and syncytium formation in the CNS and of the importance of this region of gp120 in the interaction with CD4 and CCR5

  68. Taniguchi Y, Ono K, Yoshida S, Tanaka R (2000) Antigen-presenting capability of glial cells under glioma-harboring conditions and the effect of glioma-derived factors on antigen presentation. J.Neuroimmunol. 111:177-185
    Abstract: The antigen-presenting capability of syngeneic rat glial cells was investigated under glioma-harboring conditions. Microglia induced a significant proliferation of glioma-primed splenocytes, but astrocytes did not. Furthermore, astrocytes suppressed the accessory cell function of Microglia. The presence of both indomethacin and anti-interleukin (IL)-10 neutralizing antibody during priming of Microglia enhanced splenocyte proliferation. The glioma culture supernatants down-regulated the interferon-gamma-induced expression of major histocompatibility complex class II molecules on Microglia. The down-regulation was blocked by indomethacin and anti-IL-10 antibody. The results suggest that Microglia but not astrocytes may function as antigen-presenting cells in glioma, and that glioma may suppress the antigen-presenting abilities of Microglia

  69. Thomas A, Gasque P, Vaudry D, Gonzalez B, Fontaine M (2000) Expression of a complete and functional complement system by human neuronal cells in vitro. Int.Immunol. 12:1015-1023
    Abstract: We demonstrate in vitro expression of complement components, i.e. C3, factor H (FH), factor B (FB), C4, C1-inhibitor (C1-inh), C1q, C5, C6, C7 and C9, by four human neuroblastoma cell lines IMR32, SKNSH, SH-SY5Y and KELLY. Activating proteins C4, C9 and C1q, and regulatory proteins FH and C1-inh were produced constitutively by the four cell lines. C3, C6 and FB were mainly produced by SKNSH and SH-SY5Y. Western blot experiments showed that secreted proteins were structurally similar to their serum counterparts. An additional polypeptide of 43 kDa with FH immunoreactivity was detected, which could correspond to the N-terminal truncated form found in plasma. Regulation of complement expression by inflammatory cytokines, lipopolysaccharide and dexamethasone was tested in vitro. These factors had no significant effects on activating synthesis of components C3, FB and C4, but expression of regulating components C1-inh and FH was strongly increased particularly by IFN-gamma and tumor necrosis factor-alpha. The rate of synthesis of complement components was dependent on the differentiation of neuroblastoma cells. This effect of differentiation was also observed on normal rat neurons. Rat cerebellar granule cells constitutively expressed mRNA for C4 and C1q, but expression of C3 mRNA was induced by differentiation. This study shows that neurons could be another local source of complement in the brain, besides astrocytes and Microglia. Human neuroblastoma cell lines can constitute an interesting model to analyze complement biosynthesis by human neurons. Local complement expression by neurons in vivo may be implicated in some physio-pathological processes

  70. Zhou X, Espey MG, Chen JX, Hofseth LJ, Miranda KM, Hussain SP, Wink DA, Harris CC (2000) Inhibitory effects of nitric oxide and nitrosative stress on dopamine-beta-hydroxylase. J.Biol.Chem. 275:21241-21246
    Abstract: Dopamine-beta-hydroxylase (DbetaH) is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the beta-carbon of dopamine to form norepinephrine. While norepinephrine causes vasoconstriction following reflex sympathetic stimulation, nitric oxide (NO) formation results in vasodilatation via a guanylyl cyclase-dependent mechanism. In this report, we investigated the relationship between NO and DbetaH enzymatic activity. In the initial in vitro experiments, the activity of purified DbetaH was inhibited by the NO donor, diethylamine/NO (DEA/NO), with an IC(50) of 1 mm. The inclusion of either azide or GSH partially restored DbetaH activity, suggesting the involvement of the reactive nitrogen oxide species, N(2)O(3). Treatment of human neuroblastoma cells (SK-N-MC) with diethylamine/NO decreased cellular DbetaH activity without affecting their growth rate and was augmented by the depletion of intracellular GSH. Co-culture of the SK-N-MC cells with interferon-gamma and lipopolysaccharide-activated macrophages, which release NO, also reduced the DbetaH activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N(2)O(3), can result in the inhibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation

  71. Arbour N, Ekande S, Cote G, Lachance C, Chagnon F, Tardieu M, Cashman NR, Talbot PJ (1999) Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E. J.Virol. 73:3326-3337
    Abstract: Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal Microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability

  72. Badie B, Schartner J, Klaver J, Vorpahl J (1999) In vitro modulation of Microglia motility by glioma cells is mediated by hepatocyte growth factor/scatter factor. Neurosurgery 44:1077-1082
    Abstract: OBJECTIVE: Considered as immune effector cells of the central nervous system, Microglia represent a major component of the inflammatory cells found in malignant gliomas. Although their role in brain tumor biology is unclear, accumulation of Microglia in malignant brain tumors may be mediated through active secretion of cytokines by glioma cells. Because hepatocyte growth factor/scatter factor (HGF/SF) has been shown to modulate glioma motility through an autocrine mechanism, and because Microglia have been reported to express the HGF/SF receptor Met, we hypothesized that Microglia recruitment by gliomas may also occur through the secretion of HGF/SF. METHODS: The effect of glioma cells in augmenting BV-2 murine Microglia motility was studied by using an in vitro Boyden chamber migration assay. To determine the chemokines involved in Microglia migration, neutralizing monoclonal antibodies against monocyte chemotactic protein-1 and HGF/SF were tested. Immunoblotting was used to check for the expression of HGF/SF by glioma cells, and the expression of Met by BV-2 cells was examined by flow cytometry. RESULTS: BV-2 migration was noted within 7 hours of incubation with both human (U251 MG and U373 MG) and murine (GL261) glioma cell lines. This migration corresponded to HGF/SF secretion by glioma cells and was completely inhibited by neutralizing monoclonal antibody against HGF/SF, but not monocyte chemotactic protein-1. Exposure of BV-2 cells to recombinant HGF/SF, but not monocyte chemotactic protein-1, resulted in their migration and down-regulation of Met in a dose-dependent fashion. CONCLUSION: HGF/SF, which plays a role in glioma motility and mitogenesis, may also act as a chemokine for Microglia and may be responsible for the Microglia infiltration in malignant gliomas. This active recruitment of Microglia may play an important role in glioma biology

  73. Chattopadhyay N, Ye CP, Yamaguchi T, Kerner R, Vassilev PM, Brown EM (1999) Extracellular calcium-sensing receptor induces cellular proliferation and activation of a nonselective cation channel in U373 human astrocytoma cells. Brain Res. 851:116-124
    Abstract: A receptor for extracellular calcium ions (Ca2+o), cloned from parathyroid gland, serves a critical function in Ca2+o homeostasis by regulating PTH release via "sensing" of its physiological agonist, Ca2+o. Its cloning from rat striatum revealed that the extracellular calcium-sensing receptor (CaR) could be involved in sensing ambient Ca2+o within the brain, where Ca2+ plays key roles in virtually all aspects of central nervous system (CNS) function. The CaR is expressed in neurons, oligodendrocytes, Microglia and the human astrocytoma cell line, U87 where its functions include control of cellular proliferation and modulation of ion channels, such as outward K+ channels and nonselective cation channels (NCC). In this report, we have shown that the CaR is expressed in U373 cells as assessed by RT-PCR using CaR-specific primers followed by sequencing of the amplified products, by Northern blot analysis using a CaR-specific probe as well as by Western analysis utilizing a specific polyclonal anti-CaR antiserum. Furthermore, agents known to activate the cloned CaR induce increases in cellular proliferation and the open probability of an NCC. Thus our study strongly suggests that elevated levels of Ca2+o, acting via the CaR, activate an NCC that could contribute to the associated CaR-induced stimulation of proliferation

  74. Czub M, Czub S, Gosztonyi G, Koutsilieri E, Sopper S, Muller JG, Gerlach M, Riederer P, ter M, V (1999) Effects of Selegiline in a retroviral rat model for neurodegenerative disease. J.Neurovirol. 5:458-464
    Abstract: Upon inoculation into neonatal rats, murine leukemia virus (MuLV) NT40 causes a non-inflammatory degeneration of the central nervous system. While Microglia cells appear to be the major target cells within the brain parenchyma for neurovirulent MuLV, degenerating neurons do not express retroviral gene products. In order to protect rats from neuronal damage we treated retrovirally infected rats once with monoamine oxidase (MAO) B inhibitor Selegiline which--under different conditions--exerts neuroprotective effects. Unexpectedly, when administered at 17 days post-infection (d.p.i.) a single intraperitoneal dose of Selegilin (1 mg/kg bodyweight) significantly shortened the incubation period for neurological disease. In contrast, Selegiline given in a lower dosage (0.05 mg/kg bodyweight) and/or at a different time point (13 d.p.i.) at the low (0.05 mg/kg bodyweight) and the high dose (1.0 mg/kg bodyweight) had no effect on the outcome of neurological disease. Animals treated with Selegiline (1.0 mg/kg bodyweight at 17 d.p.i.) contained higher amounts of viral loads in the CNS, higher numbers of brain cells expressing major histocompatibility complex class II molecules, and exhibited inhibition of MAO-B in comparison to untreated yet infected (control) animals. Supposedly, Selegiline activated the major target cell population of the CNS for MuLV-NT40, Microglia, with the consequence of enhanced susceptibility for retroviral infection and triggered endogenous mechanism(s) involved in the pathogenesis of retroviral neurodegeneration

  75. Davoust N, Jones J, Stahel PF, Ames RS, Barnum SR (1999) Receptor for the C3a anaphylatoxin is expressed by neurons and glial cells. Glia 26:201-211
    Abstract: Little is known about the expression of the receptor for complement anaphylatoxin C3a (C3aR) in the central nervous system (CNS). In this study, we provide the first evidence that neurons are the predominant cell type expressing C3aR in the normal CNS. By using in situ hybridization (ISH) and immunohistochemistry, we found that C3aR is constitutively expressed at high levels in cortical and hippocampal neurons as well as in Purkinje cells. Moreover, we showed that primary culture of human astrocytes and Microglia express the C3aR mRNA as assessed by RT-PCR. In situ hybridization performed on rat primary astrocytes confirmed the RT-PCR result demonstrating C3aR expression by astrocytes. In experimental allergic encephalitis (EAE), C3aR expression was elevated on Microglia, infiltrating monocyte-macrophage cells and a few astrocytes, whereas neuronal expression remained unchanged during the course of the disease. These data demonstrate that the C3aR is expressed primarily by neurons in the normal CNS and that its neuronal expression is not dramatically upregulated under inflammation. This is in contrast to the increased neuronal expression of the C5aR in several inflammatory CNS conditions. The high constitutive expression of the C3aR by neurons suggests this receptor may play an important role in normal physiological conditions in the CNS

  76. Deininger MH, Schluesener HJ (1999) Cyclooxygenases-1 and -2 are differentially localized to Microglia and endothelium in rat EAE and glioma. J.Neuroimmunol. 95:202-208
    Abstract: Cyclooxygenases (COX) mediate a wide variety of derangements observed during diseases of the brain. Their overexpression is involved in the mediation of inflammation, immunomodulation, blood flow, apoptosis and fever. Here, we have analyzed the localization of COX-1 and COX-2 in rat experimental autoimmune encephalomyelitis (EAE), C6 glioblastoma and 9L gliosarcoma by immunohistochemistry. In healthy brain, COX-1 was expressed in single macrophages/Microglial cells. Neurons and few endothelial cells expressed COX-2. In EAE, we observed an increase in COX-1+ macrophages/Microglial cells and COX-2+ endothelial cells that was closely linked to disease progression. Both COX-1+ macrophages/Microglial cells and COX-2+ endothelial cells were abundant in areas of cellular infiltration. In C6 and 9L tumors, high numbers of COX-1+ macrophages/Microglial cells and COX-2+ endothelial cells were found both in the tumor parenchyma and in areas of infiltrative tumor growth. Double labeling experiments confirmed expression of COX-2 in vWF+ (endothelial) cells and COX-1 in ED1+ (macophages), OX6+ (MHC class II) and in W3/13+ (lymphoblasts) cells. These data provide further evidence that expression of COX-1 in macrophages/Microglial cells and COX-2 in endothelial cells might represent important regulatory mechanisms in inflammatory processes associated with autoimmunity and neoplasia of the rat brain

  77. Dewey RA, Morrissey G, Cowsill CM, Stone D, Bolognani F, Dodd NJ, Southgate TD, Klatzmann D, Lassmann H, Castro MG, Lowenstein PR (1999) Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials. Nat.Med. 5:1256-1263
    Abstract: The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. We report here detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/Microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination. We detected strong widespread herpes simplex virus 1 thymidine kinase immunoreactivity and vector genomes throughout large areas of the brain. Thus, patient evaluation and the design of clinical trials in ongoing and future gene therapy for brain glioblastoma must address not only tumor-killing efficiency, but also long-term active brain inflammation, loss of myelin fibers and persistent transgene expression

  78. Engel S, Isenmann S, Stander M, Rieger J, Bahr M, Weller M (1999) Inhibition of experimental rat glioma growth by decorin gene transfer is associated with decreased Microglial infiltration. J.Neuroimmunol. 99:13-18
    Abstract: Decorin gene therapy for experimental malignant glioma is thought to involve antagonism of immunosuppression induced by glioma-derived transforming growth factor-beta (TGF-beta). TGF-beta is chemotactic for cells of the monocyte macrophage lineage but inhibits their functional activity in many in vitro paradigms. Here, we examined changes in the patterns of Microglial infiltration of rat C6 gliomas expressing a decorin transgene. We find that the number of OX42/ED-1-positive Microglial cells is reduced rather than enhanced in the presence of decorin. Decorin-expressing gliomas contain lower numbers of MHC class II antigen-expressing Microglial cells whereas the relative frequency of MHC I immunoreactivity among Microglial cells is increased. Interestingly, the reduction of TGF-beta levels in the tumors by decorin is associated with the de novo expression of inducible nitric oxide synthase (iNOS) in a minority of Microglial cells. These data suggest that Microglial cells do not participate in the regression of decorin-expressing rat C6 gliomas

  79. Flugel A, Labeur MS, Grasbon-Frodl EM, Kreutzberg GW, Graeber MB (1999) Microglia only weakly present glioma antigen to cytotoxic T cells. Int.J.Dev.Neurosci. 17:547-556
    Abstract: Microglia and brain macrophages represent a substantial fraction of the cells present in astrocytic gliomas. Yet, the functional role of Microglia in these tumors has remained enigmatic. We have compared rat Microglial cells and thymocytes with regard to their ability to present purified CNS proteins, MBP and S100beta, as well as C6 glioma cells to specific T lymphocytes. In addition, a new cytotoxicity assay based on fluorescence activated cell sorting of tumor cells carrying the green fluorescent protein was established. This assay was used to determine the influence of Microglial population density and activational state on C6 glioma cell survival in vitro. Microglia were consistently found to present MBP and S100beta less efficiently than thymocytes and appeared to be unable to present C6 glioma cells to cytotoxic T lymphocytes. In addition, high concentrations of Microglial cells attenuated the cytotoxic effects of these T cells on C6 glioma cells whereas thymocytes significantly supported their specific killing. It is suggested that defense functions of Microglial cells against C6 glioma are severely compromised and that the observed deficiency in antigen presentation may play an important role for astrocytoma growth in vivo

  80. Krohn K (1999) TGF-beta1-dependent differential expression of a rat homolog for latent TGF-beta binding protein in astrocytes and C6 glioma cells. Glia 25:332-342
    Abstract: Transforming growth factor-beta1 (TGF-beta1) is widely recognized for its multiple roles in development, cellular maintenance, and protection against injury. In the brain, TGF-beta1 upregulation in Microglia/macrophages is a predominant response to lesion and during pathology. However, the precise functions of TGF-beta1 in this context are still enigmatic. The present study investigates changes in astroglial gene expression as a major target of TGF-beta1 signaling in the brain. Differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to identify several gene fragments differentially regulated by TGF-beta1 in rat astrocytes and C6 glioma cells. Among the cDNAs regulated by TGF-beta1 in C6 cells two cDNAs showed homology to alpha-tropomyosin and glycerol-3-phosphate dehydrogenase, respectively. Cloning of a full length cDNA corresponding to a differentially regulated gene fragment revealed close homology to latent TGF-beta binding protein (LTBP)-2. Data using antisense LTBP-2 oligonucleotides to decrease LTBP-2 expression suggest that LTBP-2 functions to activate TGF-beta. Therefore, it is likely that upregulation of the rat LTBP-2 homolog mRNA in C6 cells and cortical astrocytes by TGF-1 might lead to self-activation and exaggeration of TGF-beta signaling. These data will extend our current understanding of TGF-beta1 functioning on lesion-related features of glial cells

  81. Mizuno T, Yoshihara Y, Kagamiyama H, Ohsawa K, Imai Y, Kohsaka S, Mori K (1999) Neuronal adhesion molecule telencephalin induces rapid cell spreading of Microglia. Brain Res. 849:58-66
    Abstract: Telencephalin (TLCN) is a neuronal surface glycoprotein whose expression is restricted to the telencephalon, the most rostral segment of the brain. TLCN binds to lymphocyte function-associated antigen-1 (LFA-1) integrin. In the central nervous system, LFA-1 is selectively and constitutively expressed by Microglia, suggesting that TLCN/LFA-1 binding may mediate cell-cell interactions between telencephalic neurons and Microglia. In the present study, we investigated the effects of recombinant TLCN protein on the morphology of Microglia. TLCN induced an intensive spreading of lamellipodia, causing a rapid change in Microglial morphology. In contrast, TLCN induced no significant change in morphology of neuroblastoma and fibroblasts. Furthermore, the TLCN-induced spreading of Microglia was accompanied by a clustering of LFA-1 on cell surface membrane. These results provide evidence that TLCN binding to the surface of Microglia transduces signals into Microglia that mediate or accelerate cell spreading and LFA-1 redistribution, implying that neuronal TLCN may control the state and/or function of Microglia in both physiological and pathological conditions

  82. Morelli L, Prat MI, Castano EM (1999) Differential accumulation of soluble amyloid beta peptides 1-40 and 1-42 in human monocytic and neuroblastoma cell lines. Implications for cerebral amyloidogenesis. Cell Tissue Res. 298:225-232
    Abstract: Alzheimer's disease (AD) is characterized by the massive deposition in the brain of the 40-42-residue amyloid beta protein (A(beta)). While A(beta)1-40 predominates in the vascular system, A(beta)1-42 is the major component of the senile plaques in the neuropil. The concentration of both A(beta) species required to form amyloid fibrils in vitro is micromolar, yet soluble A(betas) found in normal and AD brains are in the low nanomolar range. It has been recently proposed that the levels of A(beta) sufficient to trigger amyloidogenesis may be reached intracellularly. To study the internalization and intracellular accumulation of the major isoforms of A(beta), we used THP-1 and IMR-32 neuroblastoma cells as models of human monocytic and/or macrophagic and neuronal lineages, respectively. We tested whether these cells were able to internalize and accumulate 125I-A(beta)1-40 and 125I-A(beta)1-42 differentially when offered at nanomolar concentrations and free of large aggregates, conditions that mimic a prefibrillar stage of A(beta) in AD brain. Our results showed that THP-1 monocytic cells internalized at least 10 times more 125I-A(betas) than IMR-32 neuroblastoma cells, either isolated or in a coculture system. Moreover, 125I-A(beta)1-42 presented a higher adsorption, internalization, and accumulation of undigested peptide inside cells, as opposed to 125I-A(beta)1-40. These results support that A(beta)1-42, the major pathogenic form in AD, may reach supersaturation and generate competent nuclei for amyloid fibril formation intracellularly. In light of the recently reported strong neurotoxicity of soluble, nonfibrillar A(beta)1-42, we propose that intracellular amyloidogenesis in Microglia is a protective mechanism that may delay neurodegeneration at early stages of the disease

  83. Poulsen DJ, Favara C, Snyder EY, Portis J, Chesebro B (1999) Increased neurovirulence of polytropic mouse retroviruses delivered by inoculation of brain with infected neural stem cells. Virology 263:23-29
    Abstract: Following intraperitoneal (IP) inoculation of neonatal mice, the polytropic recombinant murine leukemia virus (MuLV), Fr98, induces a severe brain disease characterized by ataxia, seizures and death. In contrast, no apparent clinical neurological disease is seen after IP infection with Fr54, a polytropic MuLV differing from Fr98 in its envelope gene sequences. In the brain both Fr98 and Fr54 infect primarily capillary endothelial cells and Microglia. However, the level of Microglial infection by Fr98 is twofold higher than by Fr54, which might account for the difference in neurovirulence. In the present study, in order to test directly whether an increase in the number of Microglia infected by Fr54 would be sufficient to induce clinical disease, we attempted to increase the level of Fr54 in the brain by changing the route of infection. After intraventricular inoculation with Fr54-infected neural stem cells (clone C17.2), a well-established vehicle for delivery of viruses and genes to the brain, mice became ataxic and died 4 weeks postinfection. In these mice induction of brain disease was correlated with a higher level of viral antigen in the cerebrum and an increase in the number of infected Microglial cells in all brain regions examined compared with mice inoculated IP. In contrast, mice inoculated with neural stem cells infected with an ecotropic nonneurovirulent murine leukemia virus, FB29, developed no clinical disease in spite of evidence for widespread infection of Microglia in brain. Since the main differences between Fr54 and FB29 are in the SU (gp70) region of the envelope gene, this region is most likely to account for the differences in induction of CNS disease seen in the current experiments

  84. Sandmair AM, Loimas S, Poptani H, Vainio P, Vanninen R, Turunen M, Tyynela K, Vapalahti M, Yla-Herttuala S (1999) Low efficacy of gene therapy for rat BT4C malignant glioma using intra-tumoural transduction with thymidine kinase retrovirus packaging cell injections and ganciclovir treatment. Acta Neurochir.(Wien.) 141:867-872
    Abstract: BACKGROUND: The purpose of this study was to test the use of Herpes Simplex virus thymidine kinase (HSVtk) retrovirus packaging cell injections in the treatment of malignant brain tumours. METHODS: Therapeutic effect and tissue responses were examined in vivo in a syngeneic BT4C rat glioma model after HSVtk-producing PA317 packaging cell injections and intraperitoneal ganciclovir (GCV) medication. MRI was used to visualise the tumours before and after the treatment. Immunohistochemical stainings were performed to study astroglia and Microglia responses and apoptosis-mediated cell death. RESULTS: The results suggest that only a limited treatment effect can be achieved with HSVtk packaging cell injections with no prolonged survival rates. Histological examination showed a strong astroglia response but only a modest Microglia response after the treatment. HSVtk and GCV-induced cell death was at least partially mediated by apoptosis. It is concluded that HSVtk packaging cell injections and GCV treatment do not lead to eradication of malignant cells in a syngeneic BT4C rat glioma model. The lack of efficacy is most likely due to low gene transfer efficiency and limited life span of the injected packaging cell inside the tumours. CONCLUSIONS: Improvements in gene transfer efficiency, and stimulation of immunoresponse against tumour cells might lead to a more effective therapeutic response in vivo

  85. Satoh J, Kurohara K, Yukitake M, Kuroda Y (1999) The 14-3-3 protein detectable in the cerebrospinal fluid of patients with prion-unrelated neurological diseases is expressed constitutively in neurons and glial cells in culture. Eur.Neurol. 41:216-225
    Abstract: The 14-3-3 protein belongs to a family of 30-kD proteins originally identified by two-dimensional analysis of brain protein extracts. Recently, the detection of the 14-3-3 protein in the cerebrospinal fluid (CSF) is utilized as a highly reliable test for the premortem diagnosis of prion diseases such as Creutzfeldt-Jakob disease. For the initial step, to clarify the biological implication of the CSF 14-3-3 protein in these diseases, its expression was investigated in neural tissues and cultures and CSF samples from patients with a variety of neurological diseases by Western blot analysis and immunocytochemistry. The constitutive expression of the 14-3-3 protein was identified in all neural and nonneural tissues examined. It was expressed in all neurons, astrocytes, oligodendrocytes, and Microglia in culture with its location in both cytoplasmic and nuclear regions. The 14-3-3 protein was detected in the CSF of 8 out of 71 patients, including 1 Gerstmann-Straussler-Scheinker disease patient and 7 patients with prion-unrelated neurological diseases, such as meningoencephalitis of viral, bacterial, or tuberculous origin, multiple sclerosis, and mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. These results suggest that the 14-3-3 protein expressed constitutively at substantial levels in both neurons and glial cells might be released into the CSF as a disease-nonspecific consequence of the extensive brain damage and indicate that the analysis of the 14-3-3 protein in the CSF is not useful as a screening test for prion diseases

  86. van Den Pol AN, Mocarski E, Saederup N, Vieira J, Meier TJ (1999) Cytomegalovirus cell tropism, replication, and gene transfer in brain. J.Neurosci. 19:10948-10965
    Abstract: Cytomegalovirus (CMV) infects a majority of adult humans. During early development and in the immunocompromised adult, CMV causes neurological deficits. We used recombinant murine cytomegalovirus (mCMV) expressing either green fluorescent protein (GFP) or beta-galactosidase under control of human elongation factor 1 promoter or CMV immediate early-1 promoter as reporter genes for infected brain cells. In vivo and in vitro studies revealed that neurons and glial cells supported strong reporter gene expression after CMV exposure. Brain cultures selectively enriched in either glia or neurons supported viral replication, leading to process degeneration and cell death within 2 d of viral exposure. In addition, endothelial cells, tanycytes, radial glia, ependymal cells, Microglia, and cells from the meninges and choroid were infected. Although mCMV showed no absolute brain cell preference, relative cell preferences were detected. Radial glia cells play an important role in guiding migrating neurons; these were viral targets in the developing brain, suggesting that cortical problems including microgyria that are a consequence of CMV may be caused by compromised radial glia. Although CMV is a species-specific virus, recombinant mCMV entered and expressed reporter genes in both rat and human brain cells, suggesting that mCMV might serve as a vector for gene transfer into brain cells of non-murine species. GFP expression was sufficiently strong that long axons, dendrites, and their associated spines were readily detected in both living and fixed tissue, indicating that mCMV reporter gene constructs may be useful for labeling neurons and their pathways

  87. Cunningham TJ, Hodge L, Speicher D, Reim D, Tyler-Polsz C, Levitt P, Eagleson K, Kennedy S, Wang Y (1998) Identification of a survival-promoting peptide in medium conditioned by oxidatively stressed cell lines of nervous system origin. J.Neurosci. 18:7047-7060
    Abstract: A survival-promoting peptide has been purified from medium conditioned by Y79 human retinoblastoma cells and a mouse hippocampal cell line (HN 33.1) exposed to H2O2. A 30 residue synthetic peptide was made on the basis of N-terminal sequences obtained during purification, and it was found to exhibit gel mobility and staining properties similar to the purified molecules. The peptide maintains cells and their processes in vitro for the HN 33.1 cell line treated with H2O2, and in vivo for cortical neurons after lesions of the cerebral cortex. It has weak homology with a fragment of a putative bacterial antigen and, like that molecule, binds IgG. The peptide also contains a motif reminiscent of a critical sequence in the catalytic region of calcineurin-type phosphatases; surprisingly, like several members of this family, the peptide catalyzes the hydrolysis of para-nitrophenylphosphate in the presence of Mn2+. Application of the peptide to one side of bilateral cerebral cortex lesions centered on area 2 in rats results in an increase in IgG immunoreactivity in the vicinity of the lesions 7 d after surgery. Microglia immunopositive for IgG and ED-1 are, however, dramatically reduced around the lesions in the treated hemisphere. Furthermore, pyramidal neurons that would normally shrink, die, or disintegrate were maintained, as determined by MAP2 immunocytochemistry and Nissl staining. These survival effects were often found in both hemispheres. The results suggest that this peptide operates by diffusion to regulate the immune response and thereby rescue neurons that would usually degenerate after cortical lesions. The phosphatase activity of this molecule also suggests the potential for direct neuron survival-promoting effects

  88. Fiebich BL, Hull M, Lieb K, Schumann G, Berger M, Bauer J (1998) Potential link between interleukin-6 and arachidonic acid metabolism in Alzheimer's disease. J.Neural Transm.Suppl 54:268-278
    Abstract: Prostaglandins (PGs) and cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), have been implicated in the etiopathology of various inflammatory and degenerative disorders, including Alzheimer's disease (AD). Previously, we detected the presence of IL-6 in cortices of AD patients. On the other hand, non-steroidal antiinflammatory drugs (NSAIDs), potent inhibitors of prostaglandin synthesis, have been shown to be beneficial in the treatment of AD. Until now, it remained unclear whether and how these two observations were functionally connected. Here, we show that PGs are able to induce IL-6 synthesis in a human astrocytoma cell line. PGE1 and PGE2, but not PGD2 and PGF2 alpha, led to a rapid and transient induction of astrocytic IL-6 mRNA, followed by IL-6 protein synthesis. Furthermore, PGE2 potentiated IL-1 beta-induced IL-6 mRNA synthesis. These results suggest a possible link between the release of PGs from activated Microglia and the astrocytic synthesis of IL-6, which itself may affect neuronal cells, as hypothesized for Alzheimer's disease. Finally we demonstrate that Microglia are a strong source of PGE2 synthesis indicating that these cells may act as the origin of the pathogenic cascade

  89. Gasque P, Singhrao SK, Neal JW, Wang P, Sayah S, Fontaine M, Morgan BP (1998) The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis. J.Immunol. 160:3543-3554
    Abstract: The complement anaphylatoxins C5a and C3a are released at the inflammatory site, where they contribute to the recruitment and activation of leukocytes and the activation of resident cells. The distribution of the receptor for C5a (C5aR) has been well studied; however, the receptor for C3a (C3aR) has only recently been cloned, and its distribution is uncharacterized. Using a specific affinity-purified anti-C3aR peptide Ab and oligonucleotides for reverse transcriptase-PCR analysis, C3aR expression was characterized in vitro on myeloid and nonmyeloid cells and in vivo in the brain. C3aR was expressed by adult astrocytes, astrocyte cell lines, monocyte lines THP1 and U937, neutrophils, and monocytes, but not by K562 or Ramos. C3aR staining was confirmed by flow cytometry, confocal imaging, and electron microscopy analysis. A 65-kDa protein was immunoprecipitated by the anti-C3aR from astrocyte and monocyte cell lysates. Our results at the protein level were confirmed at the mRNA level. Using reverse transcriptase-PCR, Southern blot, and sequencing we found that C3aR mRNA was expressed by fetal astrocytes, astrocyte cell lines, and THP1, but not by K562 or Ramos. The astrocyte C3aR cDNA was identical with the reported C3aR cDNA. C3aR expression was not detected in normal brain sections. However, a strong C3aR staining was evident in areas of inflammation in multiple sclerosis and bacterial meningitis. In meningitis, C3aR was abundantly expressed by reactive astrocytes, Microglia, and infiltrating cells (macrophages and neutrophils). In multiple sclerosis, infiltrating lymphocytes did not express C3aR, but a strong staining was detected on smooth muscle cells (pericytes) surrounding blood vessels

  90. Itayasu T, Shimizu T, Iizumi T, Oshio S, Umeda T, Takeda K (1998) Phorbol ester induces differentiation of a human prostatic cancer cell line TSU-Pr1 into cells with characteristics of Microglia. Anticancer Res. 18:113-117
    Abstract: The effects of various reagents on the induction of differentiation of the human prostatic cancer cell line, TSU-Pr1, were examined. Among these agents, the phorbol ester, TPA, almost completely suppressed cell proliferation at the concentration of 10(-8) M, and induced remarkable morphologic changes yielding cells with the Microglial feature of an ameboid and/or ramified shape. More than 90% of the cells underwent the induction of morphologic changes by day 7 after treatment with 10(-8) M TPA. The expression of reliable Microglial markers, alpha-naphthyl acetate esterase activity and CD11b, was observed in the differentiated cells. The data presented here suggest that TPA induces differentiation of a human prostate cancer cell line into cells with the characteristics of Microglia

  91. Makman MH, Dobrenis K, Surratt CK (1998) Properties of mu 3 opiate alkaloid receptors in macrophages, astrocytes, and HL-60 human promyelocytic leukemia cells. Adv.Exp.Med.Biol. 437:137-148
    Abstract: An opiate alkaloid-selective receptor, designated mu(3), mediates inhibition by morphine of activation of human peripheral blood monocytes and granulocytes. The mu(3) receptor is present on several macrophage cell types including Microglia, on cultured astrocytes, and in brain and retina. Murine macrophage cell lines and human HL-60 leukemia cells contain high concentrations of these receptors. Binding of 3H-morphine to the receptor is displaced by morphine, etorphine, naloxone, diprenorphine and morphine 6-glucuronide, but not by morphine 3-glucuronide, fentanyl, benzomorphans, enkephalins, dynorphin, beta-endorphin, endomorphin-1, other opioid peptides or nociceptin (orphanin FQ). The mu(3) receptor appears to be much more sensitive to inactivation by reduced glutathione than are classical mu, delta and kappa receptors. Evidence is also presented for G protein-coupling of these receptors. These and other data raise the possibility that the mu(3) receptor is a member of a chemokine or of another related receptor family, rather than the opioid receptor family. The affinity for morphine of mu(3) receptors of granulocytic-differentiated HL-60 cells is markedly enhanced in the presence of levorphanol and certain benzomorphans. In contrast, receptors of monocytes, macrophage cell lines, Microglia, macrophage-differentiated HL-60 cells and astrocytes are not affected by levorphanol or benzomorphans. It is concluded that mu(3) receptors of granulocytic and promyelocytic cells differ from those of macrophage and astrocyte cell types, possibly due to differences in receptor subtype or to the presence of an additional component in the granulocytic and promyelocytic cells

  92. Shieh JT, Albright AV, Sharron M, Gartner S, Strizki J, Doms RW, Gonzalez-Scarano F (1998) Chemokine receptor utilization by human immunodeficiency virus type 1 isolates that replicate in Microglia. J.Virol. 72:4243-4249
    Abstract: The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in Microglia. To determine the role of specific chemokine coreceptors in infection of adult Microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured Microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones

  93. Stoica G, Barker R, Wu G, Lynn WS, Wong PK (1998) Quantitative variation of free amino acids in the central nervous system of MoMuLV-ts1-infected mice. In Vivo 12:395-401
    Abstract: A temperature-sensitive mutant of Moloney murine leukemia virus (MoMuLV-ts1) induces polioencephalomyelopathy and hind limb paralysis in highly susceptible FVB/N strains of neonatal mice. This disease is characterized by progressive motor neurons loss and severe gliosis within specific target areas of the central nervous system (CNS). The mechanism(s) of this neurodegeneration is unknown. In the neonatal infection of the CNS, the MoMuLV-ts1 virus was reported to replicate within the endothelial, ependymal, astrocytes and Microglial cells. Since no virus or viral products were recognized in the degenerating neurons, it is postulated that an indirect mechanism(s) caused the loss of neurons in the neonatally infected mice. This study was undertaken to investigate the possible pathogenic role of excitatory amino acids (EAAs) such as glutamate and other nonneurotransmitters amino acids (NAAs) in this animal model. The free amino acids concentration was analysed by a fluorometric HPLC method. The temporal measurements of the free amino acids concentration, glutamate, glutamine and arginine from the brain stem and spinal cord of MoMuLV-ts1-infected mice was significantly decreased when compared with the control non-infected mice. The concentration of EAAs during the course of this infection indicated a sharp decline in glutamate and its precursor, glutamine with early infection (10 days post infection-dpi). This deficiency persisted (20 and 30 dpi) in the spinal cord, where the neuronal loss was most severe, but not in the brain stem. A similar pattern occurs with the amino acid arginine. These observations suggest that an astrocyte-induced metabolic disturbance of glutamate and arginine in the CNS of developing mice, could be, in part responsible for the loss of motor neurons observed in this model

  94. Xia MQ, Berezovska O, Kim TW, Xia WM, Liao A, Tanzi RE, Selkoe D, Hyman BT (1998) Lack of specific association of presenilin 1 (PS-1) protein with plaques and tangles in Alzheimer's disease. J.Neurol.Sci. 158:15-23
    Abstract: Missense mutations in the presenilin-1 (PS-1) gene are causally related to the majority of familial early-onset Alzheimer's disease (FAD). PS-1 immunohistochemical expression in normal human brain and in brains with Alzheimer's disease (AD) has so far been controversial. Here, we report a study of PS-1 expression in brains, cell lines and peripheral blood mononuclear cells using a panel of well characterized PS-1-specific antibodies. These antibodies were characterized by immunofluorescent staining of PS-1 transfectants followed by flow cytometric analysis. In human brain, widespread neuronal staining was observed. PS-1 immunoreactivity was primarily confined to neuronal cell bodies and proximal dendrites. Weaker staining of Microglia was also detected, in accord with the finding of PS-1 immunoreactivity in monocytes. PS-1 expression is not particularly associated with neurons either containing or spared from neurofibrillary tangles, nor with senile plaques. The level of PS-1 expression does not differ between normal and AD brains. Immunoprecipitation from AD, FAD and control brains revealed only a 32 kDa N-terminal fragment and an 18-20 kDa C-terminal fragment. Little or no full length PS-1 was detected. The enriched presence of PS-1 in neurons implies an important role in neuronal function, however, the lack of apparent association of its expression with AD pathology signifies the need for a better understanding of its pathophysiological role

  95. Yang F, Sun X, Beech W, Teter B, Wu S, Sigel J, Vinters HV, Frautschy SA, Cole GM (1998) Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and Microglia in Alzheimer's disease. Am.J.Pathol. 152:379-389
    Abstract: During apoptosis, activation of a family of cysteine proteases related to interleukin-1beta-converting enzyme (ICE)-related proteases or "caspases" results in endoproteolytic cleavage of multiple substrates at specific aspartate residues. We have sought to develop new antibody probes for the neoepitopes in protein fragments produced by ICE-related proteolytic cleavage as specific markers of events tightly linked to apoptotic mechanisms. Here, we demonstrate that an antibody probe specific for the C terminus of a 32-kd actin fragment produced by ICE-like activity specifically labels apoptotic but not necrotic, differentiated human neuroblastoma cells in culture. Unlike probes for nonspecific DNA strand breaks confined to the nucleus or cell body, this method allows the detection of cytoskeletal fragments in cell processes as well as the perikaryon long before DNA fragmentation and cell death and therefore serves as a novel marker of apoptosis-related events in distal parts of cells such as axons and dendrites. To illustrate this new tool, we show that the antibody detects the processes and cell bodies of degenerating neurons and plaque-associated Microglia in Alzheimer's disease. In situ detection of caspase-cleaved actin provides a new means to evaluate the role of caspase activation in pathological and physiological processes

  96. Yeung MC, Geertsma F, Liu J, Lau AS (1998) Inhibition of HIV-1 gp120-induced apoptosis in neuroblastoma SK-N-SH cells by an antisense oligodeoxynucleotide against p53. AIDS 12:349-354
    Abstract: OBJECTIVES: This study examines the cytotoxicity potential and the mechanism of toxicity of the HIV-1 gp120 on human neuroblastoma cells. DESIGN: Previous data from our group have suggested that the HIV-1 envelope protein gp120 promotes the secretion of tumor necrosis factor-alpha and other factors by astrocytes and Microglial cells present in primary human brain cell cultures, thereby contributing to the injury of neurons in these cultures. This study investigates the cytotoxicity potential and the mechanism of toxicity of gp120 on human neuroblastoma cells. METHODS: SK-N-SH cells were treated with HIV-1 gp120, and was followed by in situ DNA fragmentation staining and small molecular weight DNA extraction studies to ascertain the induction of apoptosis by gp120 in these cells. To evaluate a potential role of the growth suppressor gene p53, gp120-treated SK-N-SH cells were subjected to reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses for the induction of p53. An antisense oligodeoxynucleotide against p53 was used to investigate the role of p53 in the gp120-induced apoptosis in these cells. RESULTS: Data from T7 DNA polymerase staining and small molecular weight DNA extraction studies demonstrated that gp120-induced DNA breakage in SK-N-SH cells with fragmentation patterns characteristic of apoptosis. RT-PCR and Western blot analyses revealed that the gp120-mediated induction of apoptosis was dependent on a gp120-induced and gp120-sustained upregulation of p53. The induction of p53 by gp120 was specific, since an antibody against gp120 prevented both the induction of p53 and subsequent apoptosis in SK-N-SH cells. The critical role of p53 was further illustrated by the effectiveness of a p53 antisense oligodeoxynucleotide to inhibit the gp120-induced apoptosis. As a control, the apoptosis-inducing potential of gp120 on SK-N-SH cells was not seen in the HIV-1 Gag proteins even when used at up to 5 nM. CONCLUSIONS: These results established that HIV-1 gp120 is potentially cytotoxic to human neuronal cells through the induction of p53, which may eventually lead to induction of apoptosis

  97. Bosman GJ, Renkawek K, Van Workum FP, Bartholomeus IG, De Grip WJ (1997) Involvement of neuronal anion exchange proteins in cell death in Alzheimer's disease. Gerontology 43:67-78
    Abstract: Anion exchange (AE) proteins are present in human neurons in the brain. Immunohistochemical data indicate that their apparent expression level increases with age, and especially with degeneration in Alzheimer's disease-affected brain areas. The increase in immunoreactivity is probably caused by changes in AE structure that lead to an increased accessibility of hitherto hidden epitopes. These epitopes correspond to regions in the membrane domain that are involved in generation of senescent cell-specific antigen from AE1 in aging erythrocytes. Elucidation of the molecular nature of these changes and the underlying mechanisms, will lead to insight in the processes that govern aging- and degeneration-associated perturbation of membrane integrity. AE-mediated chloride/bicarbonate exchange is a major component in the regulation of intracellular pH. The functional consequences of changes in AE structure may range from acidosis, disturbance of cytoskeleton integrity, and untimely or impaired recognition of cells by components of the immune system, such as Microglia. A molecular and physiological description of these changes will establish AE proteins as valuable tools in elucidating the processes of normal aging, and the disturbances in aging-related diseases such as Alzheimer's disease

  98. Du YS, Zhu H, Fu J, Yan SF, Roher A, Tourtellotte WW, Rajavashisth T, Chen X, Godman GC, Stern D, Schmidt AM (1997) Amyloid-beta peptide-receptor for advanced glycation endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: a proinflammatory pathway in Alzheimer disease. Proc.Natl.Acad.Sci.U.S.A 94:5296-5301
    Abstract: In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated Microglia. We demonstrate that binding of amyloid-beta peptide (Abeta) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Abeta, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor kappaB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Abeta deposits, and in cerebrospinal fluid from AD patients there was approximately 5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Abeta-stimulated neurons interacts with its cognate receptor, c-fms, on Microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of Microglia exposed to Abeta, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Abeta on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD

  99. Huettner C, Czub S, Kerkau S, Roggendorf W, Tonn JC (1997) Interleukin 10 is expressed in human gliomas in vivo and increases glioma cell proliferation and motility in vitro. Anticancer Res. 17:3217-3224
    Abstract: Interleukin 10 (IL-10) is a cytokine with a broad spectrum of immunosuppressive activity, but itoffs role in the oncogenesis of solid tumors is still unclear. In previous experiments we have shown that IL-10 specific mRNA is produced within glial tumors in vivo. The aim of the present study was to investigate the expression of the IL-10 protein in vivo and to identify the cells producing IL-10 within the tumor tissue. Expression levels significantly increased with malignancy of the gliomas. 87.5% of grade III and IV, but only 4% of grade II tumors expressed high levels of mRNA. Elevation of IL-10 serum levels was found in 11% of low grade and in 63.6% of high grade glioma patients. In situ hybridization analysis with combined immunohistochemistry revealed that: a) IL-10 is not produced by infiltrating B- or T- lymphocytes, b) both Microglia and astroglia contributed to IL-10 expression in malignant gliomas in vivo. These data suggested the functional role of IL-10 in glioma progression. Therefore, the effects of IL-10 on proliferation and migration of glioma cells were determined in vitro. Two human glioma cell lines were grown as monolayer as well as spheroids in the presence of different concentrations of IL-10. IL-10 increased cell proliferation significantly in both culture systems with a dose optimum of 25 ng/ml. Glioma cell motility was enhanced with 25 ng/ml as the optimal dose. Adding the IL-10 specific antibody reversed both effects. We conclude from our data that IL-10 is involved in the progression of glial tumors, especially in the enhancement of tumor cell proliferation and migration which promotes infiltration of the surrounding tissue

  100. Li Y, Kustova Y, Sei Y, Basile AS (1997) Regional changes in constitutive, but not inducible NOS expression in the brains of mice infected with the LP-BM5 leukemia virus. Brain Res. 752:107-116
    Abstract: Potential neurotoxins such as nitric oxide have been implicated in the pathogenesis of acquired immunodeficiency syndrome (AIDS) dementia complex. The LP-BM5 murine leukemia-infected mice, which develop immunological and cognitive deficits reminiscent of human HIV-1 infection, were employed to investigate the changes in brain constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) expression. Cerebellar and striatal cNOS enzymatic activity increased approximately 70% as early as 2 weeks after infection, declining to control levels by 12-16 weeks. In contrast, cNOS protein expression in the striatum and cerebellum was decreased 30% at 4 weeks, declining to 50% of control levels by 16 weeks post-infection. Staining intensity for cNOS, but not neuron number was reduced in the cerebral cortex, striatum, ventromedial hypothalamic nucleus and amygdala. Although iNOS protein expression was elevated in splenic monocytes, neither iNOS activity, mRNA nor protein was detected in the brains of mice 12 weeks after infection. These results indicate that neurons decrease cNOS protein expression to compensate for chronic cNOS activation, probably resulting from glutamatergic stimulation. The cNOS activation is contemporaneous with Microglial activation in LP-BM5-infected mice, and precedes the development of cognitive deficits. Moreover, the lack of iNOS induction in either infected macrophages or glial elements suggests that iNOS is not necessary for the development of these cognitive deficits

  101. Malhotra SK, Luong LT, Bhatnagar R, Shnitka TK (1997) Up-regulation of reactive astrogliosis in the rat glioma 9L cell line by combined mechanical and chemical injuries. Cytobios 89:115-134
    Abstract: The 9L rat glioma cells grown in culture, when subjected to a mechanical injury (scratch wound) and/or a chemical injury (CdCl2) manifest changes which are characteristic of an astrocyte reaction (astrogliosis) in the central nervous system. Such changes include cell hypertrophy and an increase in immunostaining for the astrocytic marker proteins, glial fibrillary acidic protein and J1-31 antigen. Mitochondria also increase in size and number, and the endoplasmic reticulum expands in area. These mechanical and chemical injuries are coordinated, and act synergistically to induce a considerably more intense astroglial reaction by 9L cells than can be elicited with either injurious agent alone, and this occurs without any interactions with Microglia, neurons or oligodendroglia. The phenomenon suggests that more than one transcriptional mechanism is involved in the activation of astrocytes, and that mechanical and CdCl2-induced injuries, respectively, probably affect different receptors and second- and third-messenger pathways. There are a number of questions concerning the molecular biology of reactive astrocytes which can be addressed through the use of the 9L rat glioma cell model. This model offers certain advantages over primary cultures of astrocytes, namely a low basal level of reactivity (because the cells are not subjected to mechanical injury prior to experimentation), an absence of contaminating Microglial cells, greater ease of reproducibility of results, lower costs and avoidance of the use of animals

  102. McDonald DR, Brunden KR, Landreth GE (1997) Amyloid fibrils activate tyrosine kinase-dependent signaling and superoxide production in Microglia. J.Neurosci. 17:2284-2294
    Abstract: Alzheimer's disease (AD) is a devastating neurological disorder characterized by loss of cognitive skills and progressive dementia. The pathological hallmark of AD is the presence of numerous senile plaques throughout the hippocampus and cerebral cortex associated with degenerating axons, neurofibrillary tangles, and gliosis. The core of the senile plaque primarily is composed of the 39-43 amino acid beta-amyloid peptide (Abeta), which forms fibrils of beta-pleated sheets. Although considerable genetic evidence implicates Abeta in the pathogenesis of AD, a direct causal link remains to be established. Senile plaques are foci of local inflammatory processes, as evidenced by the presence of numerous activated Microglia and acute phase proteins. Abeta has been shown to elicit inflammatory responses in Microglia; however, the intracellular events mediating these effects are largely unknown. We report that exposure of Microglia and THP1 monocytes to fibrillar Abeta led to time- and dose-dependent increases in protein tyrosine phosphorylation of a population of proteins similar to that elicited by classical immune stimuli such as immune complexes. The tyrosine kinases Lyn, Syk, and FAK were activated on exposure of Microglia and THP1 monocytes to Abeta, resulting in the tyrosine kinase-dependent generation of superoxide radicals. The present data support a role for oxidative damage in the pathogenesis of AD, provide an important mechanistic link between Abeta and the generation of reactive oxygen intermediates, and identify molecular targets for therapeutic intervention in AD

  103. Moffett JR, Els T, Espey MG, Walter SA, Streit WJ, Namboodiri MA (1997) Quinolinate immunoreactivity in experimental rat brain tumors is present in macrophages but not in astrocytes. Exp.Neurol. 144:287-301
    Abstract: Experimental tumors of the central nervous system were investigated with antibodies to quinolinate to assess the cellular distribution of this endogenous neurotoxin. In advanced F98 and RG-2 glioblastomas and E367 neuroblastomas in the striatum of rats, variable numbers of quinolinate immunoreactive cells were observed in and around the tumors, with the majority being present within tumors, rather than brain parenchyma. The stained cells were morphologically variable, including round, complex, rod-shaped, and sparsely dendritic cells. Neuroblastoma and glioma cells were unstained, as were neurons, astrocytes, oligodendrocytes, ependymal cells, endothelial cells, and cells of the choroid plexus and leptomeninges. Glial fibrillary acidic protein immunoreactivity was strongly elevated in astrocytes surrounding the tumors. Dual labeling immunohistochemistry with antibodies to quinolinate and glial fibrillary acidic protein demonstrated that astrocytes and the cells containing quinolinate immunoreactivity were morphologically disparate and preferentially distributed external and internal to the tumors, respectively, and no dual labeled cells were observed. Lectin histochemistry with Griffonia simplicifolia B4 isolectin and Lycopersicon esculentum lectin demonstrated numerous phagocytic macrophages and reactive Microglia in and around the tumors whose distribution was similar to that of quinolinate immunoreactive cells, albeit much more numerous. Dual labeling studies with antibodies to quinolinate and the lectins demonstrated partial codistribution of these markers, with most double-labeled cells having the morphology of phagocytes. The present findings suggest the possibility that quinolinate may serve a functional role in a select population of inflammatory cell infiltrates during the immune response to brain neoplasms

  104. Papavasiliou AK, Mehler MF, Mabie PC, Marmur R, Qingbin S, Keating RF, Kessler JA (1997) Paracrine regulation of colony-stimulating factor-1 in medulloblastoma: implications for pathogenesis and therapeutic interventions. Neurosurgery 41:916-923
    Abstract: OBJECTIVE: Colony-stimulating factor (CSF)-1, a chemotactic and mitogenic factor for macrophages and Microglia, is expressed in a variety of nervous system tumors and when present in nonneural malignancies, is associated with marked inflammatory infiltrates, dissemination, and poorer prognosis. This study investigated the paracrine effects of CSF-1 production by medulloblastoma cells on the macrophage/Microglial lineage. METHODS: A recurrent metastatic desmoplastic medulloblastoma was isolated from a 26-year-old man and propagated in tissue culture. Cellular phenotype and proliferation were assessed by immunocytochemical techniques; transcript expression for CSF-1, granulocyte macrophage-CSF, interleukin-3, and c-fms (the receptor for CSF-1) was examined with reverse transcriptase-polymerase chain reaction; and conditioned media and coculture paradigms were used to study cytokine effects on cellular proliferation. RESULTS: Serially passaged cells were uniformly immunoreactive for two lineage-independent neuroepithelial markers, nestin and vimentin. A subpopulation of cells with morphological characteristics of early differentiation stained for neurofilament 66 (7%) and microtubule-associated protein (5%) (markers of early neuronal precursors and postmitotic neurons, respectively) and for the Yp subunit of glutathione-S-transferase (3%) (a marker of early oligodendroglial progenitors). tumor cells expressed transcripts for CSF-1, but not for granulocyte macrophage-CSF, interleukin-3, or c-fms. Treatment of Microglia with serum-free medulloblastoma-conditioned media significantly increased proliferation (P < 0.001), suggesting the secretion of CSF-1. Coculture of medulloblastoma cells and Microglia significantly increased proliferation of both cell types (each condition, P < 0.01). CONCLUSION: These observations suggest that CSF-1 mediates important paracrine interactions between transformed cells and the immune system, resulting in increased growth rate and metastatic potential. Future therapeutic goals need to include immunotherapeutic protocols to modulate this interaction

  105. Qiu WQ, Ye Z, Kholodenko D, Seubert P, Selkoe DJ (1997) Degradation of amyloid beta-protein by a metalloprotease secreted by Microglia and other neural and non-neural cells. J.Biol.Chem. 272:6641-6646
    Abstract: Amyloid beta-protein (Abeta) is the major component of neuritic (amyloid) plaques in Alzheimer's disease, and its deposition is an early and constant event in the complex pathogenetic cascade of the disease. Although many studies have focused on the biosynthetic processing of the beta-amyloid precursor protein and on the production and polymerization of Abeta, understanding the degradation and clearance of Abeta has received very little attention. By incubating the conditioned medium of metabolically labeled Abeta-secreting cells with media of various cultured cell lines, we observed a time-dependent decrease in the amount of Abeta in the mixed media. The factor principally responsible for this decrease was a secreted metalloprotease released by both neural and non-neural cells. Among the cells examined, the Microglial cell line, BV-2, produced the most Abeta-degrading activity. The protease was completely blocked by the metalloprotease inhibitor, 1,10-phenanthroline, and partially inhibited by EDTA, whereas inhibitors of other protease classes produced little or no inhibition. Substrate analysis suggests that the enzyme was a non-matrix metalloprotease. The protease cleaved both Abeta1-40 and Abeta1-42 peptides secreted by beta-amyloid precursor protein-transfected cells but failed to degrade low molecular weight oligomers of Abeta that form in the culture medium. Lipopolysaccharide, a stimulator of macrophages/Microglia, activated BV-2 cells to increase their Abeta-degrading metalloprotease activity. We conclude that secreted Abeta1-40 and Abeta1-42 peptides are constitutively degraded by a metalloprotease released by Microglia and other neural cells, providing a potential mechanism for the clearance of Abeta in brain tissue

  106. Robertson SJ, Hasenkrug KJ, Chesebro B, Portis JL (1997) Neurologic disease induced by polytropic murine retroviruses: neurovirulence determined by efficiency of spread to Microglial cells. J.Virol. 71:5287-5294
    Abstract: Several murine leukemia viruses (MuLV) induce neurologic disease in susceptible mice. To identify features of central nervous system (CNS) infection that correlate with neurovirulence, we compared two neurovirulent MuLV, Fr98 and Fr98/SE, with a nonneurovirulent MuLV, Fr54. All three viruses utilize the polytropic receptor and are coisogenic, each containing a different envelope gene within a common genetic background. Both Fr98 and Fr98/SE induce a clinical neurologic disease characterized by hyperexcitability and ataxia yet differ in incubation period, 16 to 30 and 30 to 60 days, respectively. Fr54 infects the CNS but fails to induce clinical signs of neurologic disease. In this study, we compared the histopathology, regional virus distribution, and cell tropism in the brain, as well as the relative CNS viral burdens. All three viruses induced similar histopathologic effects, characterized by intense reactive astrogliosis and Microglial activation associated with minimal vacuolar degeneration. The infected target cells for each virus consisted primarily of endothelial and Microglial cells, with rare oligodendrocytes. Infection localized predominantly in white matter tracts of the cerebellum, internal capsule, and corpus callosum. The only feature that correlated with relative neurovirulence was viral burden as measured by both viral CA protein expression in cerebellar homogenates and quantification of infected cells. Interestingly, Fr54 (nonneurovirulent) and Fr98/SE (slow disease) had similar viral burdens at 3 weeks postinoculation, suggesting that they entered the brain with comparable efficiencies. However, spread of Fr98/SE within the brain thereafter exceeded that of Fr54, reaching levels of viral burden comparable to that seen for Fr98 (rapid disease) at 3 weeks. These results suggest that the determinants of neurovirulence in the envelope gene may influence the efficiency of virus spread within the brain and that a critical number of infected cells may be required for induction of clinical neurologic disease

  107. Rosales AA, Roque RS (1997) Microglia-derived cytotoxic factors. Part I: Inhibition of tumor cell growth in vitro. Brain Res. 748:195-204
    Abstract: The rarity of neoplasms in the adult mammalian retina has led us to hypothesize the presence or increased expression of 'tumor-inhibitory molecules' in the mature differentiated retina. We have begun to investigate the source(s) of these molecules, and the following study describes the inhibitory activity of a soluble Microglia-derived cytotoxic factor on the proliferation of C6 cells, a glial tumor cell line. C6 cells were treated for 24, 48, or 72 h with basal medium or basal medium conditioned by retina-derived Muller cells (MCCM) or Microglial cells (MGCM) and assayed for cell proliferation and/or cell death using various techniques involving fluorescent probes, lactate dehydrogenase release, or bromodeoxyuridine uptake. C6 cells increased in number from 24 to 72 h following incubation in basal medium or MCCM, but not in MGCM, where the cells rounded up and retracted their processes. The number of dead cells appeared to be the same in all groups at each time point. Similar findings were observed in the presence of 1-10% serum. About 25% of cells treated with basal medium for 72 h were positive for bromodeoxyuridine as compared with < 1% in MGCM-treated cultures. Our studies suggest that retina-derived Microglial cells secrete soluble product(s) that inhibit the growth of C6 cells in culture. These molecules may provide protection for the mature retina against the invasion of tumor cells and may prove useful in the treatment of cancer

  108. Saida K, Saida T, Kai K, Iwamura K (1997) Central nervous system lesions in rats infected with Friend murine leukemia virus-related PVC441: ultrastructural and immunohistochemical studies. Acta Neuropathol.(Berl) 93:369-378
    Abstract: Newborn F344 rats were injected intraperitoneally with PVC441 virus, a neuropathogenic variant of Friend murine leukemia virus, and developed paraparesis of hind limbs 35-40 days after infection. Immunohistochemical study using monoclonal anti-PVC441 antibody revealed that in the central nervous system endothelial cells but not neuronal or glial cells were infected with PVC441 virus. The major pathological changes were myelin vacuolation and oligodendrocyte degeneration in the white matter at the white-gray border zone. Anterior and lateral funiculi and intercalated myelin of anterior horns were dominantly affected in the spinal cord from the sacral to cervical level. The midbrain was also vacuolated. An ultrastructural study demonstrated that many viral particles were present outside the endothelial cells but only sparsely inside endothelial cells and pericytes. Endothelial cell membranes and tight junctions were also disrupted. Immunohistochemical studies with antibodies against major histo-compatibility complex class Ia, intercellular adhesion molecule-I, glial fibrillary acidic protein, neurofilament protein, CD3 and OX42 revealed the presence of abundant Microglia but not of lymphocytes or polymorphonuclear cells in the lesions. Axonal degeneration and astrogliosis were mild in degree. These pathological changes explain the observed spastic paraparesis in the rats, and represent a good model of spongiform diseases of the human central nervous system of retroviral origin, such as human T cell leukemia virus-associated myelopathy and AIDS

  109. Strizki JM, Turner JD, Collman RG, Hoxie J, Gonzalez-Scarano F (1997) A monoclonal antibody (12G5) directed against CXCR-4 inhibits infection with the dual-tropic human immunodeficiency virus type 1 isolate HIV-1(89.6) but not the T-tropic isolate HIV-1(HxB). J.Virol. 71:5678-5683
    Abstract: We used a monoclonal antibody (12G5) directed against an extracellular domain of CXCR-4 to investigate the role of this receptor in infection of immortalized lymphoid cell lines, peripheral blood mononuclear cells (PBMCs), and primary brain Microglia with a dual-tropic strain of human immunodeficiency virus (HIV-1(89.6)) and a T-tropic strain (HIV-1(IIIB)). Addition of antibody 12G5 to cells prior to and during infection with HIV-1(89.6) inhibited p24 production 100- to 10,000-fold in CEMx174 and 174-CD4 cells and about 10-fold in PBMC cultures but had no activity against infection of either monocyte-derived macrophages or brain Microglia. In contrast, 12G5 had little or no effect on infection of CEMx174 cells with HIV-1(IIIB) or HIV-1(HxB). To identify the region of the HIV-1(89.6) envelope that confers sensitivity to 12G5, we used chimeric molecular clones. Chimeras containing the V3 loop region of HIV-1(89.6) were inhibited by 12G5 to the same degree as wild-type HIV-1(89.6) whereas replication of those viruses containing the V3 loop of HIV-1(HxB) was not inhibited by the antibody. A similar pattern was seen in infections of a U87 glioblastoma line that coexpresses CD4 and CXCR-4. Antibody 12G5 was also able to block fusion between HeLa-CD4 cells and CEMx174 cells chronically infected with HIV-1(89.6) but had no effect on fusion mediated by cells chronically infected with HIV-1(IIIB). Taken together, these results suggest that different strains of HIV-1 may interact with different sites on CXCR-4 or may have different binding affinities for the coreceptor

  110. Sugawa M, Ikeda S, Kushima Y, Takashima Y, Cynshi O (1997) Oxidized low density lipoprotein caused CNS neuron cell death. Brain Res. 761:165-172
    Abstract: Death induced by oxidized low density lipoproteins (oxLDL) to embryonic CNS neuronal and neuroblastoma cells was investigated. Cell damage and viability were evaluated by LDH leakage and the MTT method, respectively. Dose- and time-dependent degeneration of neurons occurred after oxLDL (1-100 microg/ml) treatment but was absent after native low density lipoproteins (LDL). This degeneration was mediated, in part, by apoptosis because increased TUNEL and Hoechst dye-positive staining was observed. These effects occurred in the absence of Microglia. However, DNA degradation was not detected. The cytotoxicity was attenuated by pre-treatment with antioxidants. These results suggest that oxidation by oxLDL may be important in neurocytotoxicity in the brain

  111. Wilms H, Hartmann D, Sievers J (1997) Ramification of Microglia, monocytes and macrophages in vitro: influences of various epithelial and mesenchymal cells and their conditioned media. Cell Tissue Res. 287:447-458
    Abstract: Microglial cells are able to switch between an "active" amoeboid and a ramified "resting" morphology during development and after experiencing lesions. We have previously shown that in vitro Microglial morphology is controlled by their cellular environment, i. e. cells become ramified in astrocyte coculture but amoeboid on monolayers of fibroblasts. In the present study we have extended the analysis of the control of macrophage morphology by maintaining macrophages of different origins in coculture with different epithelial or mesenchymal cells and their conditioned media. Microglia, monocytes and spleen macrophages seeded onto monolayers of astrocytes, kidney epithelia or hepatoma cells developed the ramified morphology but remained amoeboid in fibroblast coculture. Ramification was also induced by media conditioned by these cells as well as by phorbolic esters, i.e. activators of protein kinase C. In double coculture assays, even small numbers of fibroblasts were able to override the "epithelial" influence. Likewise, Microglia remained amoeboid, when incubated on several constituents of the extracellular matrix. These results indicate that macrophage ramification is an active process initiated by diffusible factors secreted by various epithelial cells, possibly acting upon a protein-kinase-C-related receptor. We interprete the modification of macrophage morphology as a functional adaptation to the surrounding type of tissue that is enforced by its constituent cells. Thus, the specific morphologies of Microglia, hepatic von Kupffer's cells or peritubular kidney macrophages could be explained by similar epithelium-macrophage interaction

  112. Zachary JF, Baszler TV, French RA, Kelley KW (1997) Mouse Moloney leukemia virus infects Microglia but not neurons even though it induces motor neuron disease. Mol.Psychiatry 2:104-106
    Abstract: Motor neuron degeneration caused by ts1 MoMuLV occurs by an indirect mechanism and hypothetically appears associated with a two-cell or three-cell pathogenesis hypothesis. The first step in this hypothesis is associated with a small subset of resident Microglial cells that serve as the principal target cells for ts1 MoMuLV infection. The second step is likely linked to trophic events, probably mediated by cytokines, that lead to hypertrophy and activation of a substantial number of additional Microglial cells (autocrine effect) and adjacent astrocytes (paracrine effect). The third step in this hypothesis appears related to indirect neuronal degeneration mediated by cytotoxins produced by activated Microglial cells and astrocytes. In this last step, motor neurons located within these foci of activated Microglial cells and astrocytes are 'innocent bystander cells' and degenerate and die due to paracrine effects. The mechanism of motor neuron degeneration is poorly understood but is likely linked to a sequential cascade of trophic factors and cytokines resulting in a final common pathway for motor neuron death involving production of oxidative radicals, excitatory aminoacid neurotransmitter-like substances, prostaglandins, or nitric oxide

  113. Nam M, Johnston P, Lal B, Indurti R, Wilson MA, Laterra J (1996) Endothelial cell-based cytokine gene delivery inhibits 9L glioma growth in vivo. Brain Res. 731:161-170
    Abstract: Malignant brain neoplasms present great therapeutic challenges due to their extremely aggressive behavior and relative isolation by the blood-brain and blood-tumor barriers. Endothelial cells may be versatile platforms for delivering genes to solid tumors by virtue of their location at blood-tissue interfaces and their proliferation in response to endothelial mitogens produced by tumors. Immortalized rat brain endothelial cells that express the E. coli lacZ reporter gene and the gene for murine interleukin-2 (RBEZ-IL2) were co-inoculated with 9L glioma cells to Fisher rats to examine the effects of endothelial cell-based cytokine delivery on glioma growth in vivo. 9L glioma growth was not affected by the implantation of control RBEZ cells. The growth of subcutaneous and intracranial 9L gliomas was significantly inhibited by RBEZ-IL2 cells (P < 0.005 and P < 0.01, respectively) when compared to control transfected RBEZ cells. Rats receiving intracranial 9L glioma cells with RBEZ-IL2 cells showed increased survival (P < 0.001). Histologic and immunohistologic analysis showed enhanced activation of Microglia/macrophages and CD8-positive T lymphocytes and/or natural killer cells within brain at sites of 9L inoculation with RBEZ-IL2 cells. This report establishes that immortalized endothelial cells can be used for cytokine gene delivery and to activate anti-tumor host responses to experimental gliomas within the central nervous system

  114. Personett DA, Chouinard M, Sugaya K, McKinney M (1996) Simplified RT/PCR quantitation of gene transcripts in cultured neuroblastoma (SN49) and Microglial (BV-2) cells using capillary electrophoresis and laser-induced fluorescence. J.Neurosci.Methods 65:77-91
    Abstract: We developed a simplified protocol for sensitive quantitation of mRNA using polymerase chain reaction (PCR) amplification of cDNA made by reverse transcriptase (RT), as resolved with capillary electrophoresis (CE) and detected with laser-induced fluorescence (LIF). The conditions required for adequate accuracy of the simplified version of the RT/PCR quantitation, in which a single concentration of external standard and amplification to within or near the plateau phase are used, were established for assay of mRNAs expressed at high, moderate, and low abundance. The mRNAs for the cytosolic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH) and the growth-associated protein GAP-43 in cultured SN49 neuroblastoma cells were used as target genes for high and moderate levels of expression, respectively. Using cultured mouse Microglial cells (BV-2), we demonstrated the utility of this RT/PCR/CE/LIF protocol to quantitate a low-abundance mRNA, encoding a form of nitric oxide synthase (i-NOS) induced by treatment with endotoxin. The appearance of i-NOS mRNA after endotoxin treatment of BV-2 cells was confirmed by Northern blot analysis and in situ hybridization histochemistry, and functional enzyme activity was followed by release of nitric oxide (as nitrite) into the medium. The many advantages of the 'single-point' RT/PCR/CE/LIF protocol for quantitating mRNAs of interest include: simplified protocol, elimination of the use of radiotracers, high sensitivity and precision, and semi-automation of the quantitation phase of analysis

  115. Petanceska S, Canoll P, Devi LA (1996) Expression of rat cathepsin S in phagocytic cells. J.Biol.Chem. 271:4403-4409
    Abstract: Cysteine lysosomal proteases are essential for turnover of intracellular and extracellular proteins. These enzymes are strongly implicated in normal and pathological processes involving tissue remodeling. Among the cysteine proteases, cathepsin S seems to be best suited for such a process since it retains most of its enzymatic activity at neutral pH. In situ hybridization analyses of the adult rat brain, spleen, and lung reveal that cathepsin S mRNA is preferentially expressed in cells of mononuclear-phagocytic origin. After entorhinal cortex lesion of adult rat brain (a paradigm for neuronal degeneration and reactive synaptogenesis), cathepsin S mRNA is dramatically increased in activated Microglia in the deafferented dentate gyrus and in macrophages at the wound site, suggesting a role in lesion-induced tissue remodeling. This possibility is further supported by the finding that cathepsin S degrades a number of extracellular matrix molecules at neutral pH and by the finding that inflammatory mediators stimulate its secretion from the Microglia and macrophages. These data suggest that cathepsin S is an important player in degenerative disorders associated with the cells of the mononuclear phagocytic system

  116. Shrikant P, Benos DJ, Tang LP, Benveniste EN (1996) HIV glycoprotein 120 enhances intercellular adhesion molecule-1 gene expression in glial cells. Involvement of Janus kinase/signal transducer and activator of transcription and protein kinase C signaling pathways. J.Immunol. 156:1307-1314
    Abstract: It is well established that the two major glial cells in the central nervous system (CNS), astrocytes and Microglia, are key participants in mediating the neurologic dysfunction associated with HIV infection of the CNS. In this study, we investigated the ability of the major envelope glycoprotein of HIV, glycoprotein 120 (gp120), to regulate intercellular adhesion molecule-1 (ICAM-1) expression in glial cells, because ICAM-1 is important in mediating immune responsiveness in the CNS, facilitating entry of HIV-infected cells into the CNS, and promoting syncytia formation. Our results indicate that gp120 enhances ICAM-1 gene expression in primary rat astrocytes, primary human astrocytes, a human astroglioma cell line CRT, and primary rat Microglia. The signal transduction events involved in gp120-mediated enhancement of ICAM-1 appear to involve activation of both protein kinase C and tyrosine kinase, because inhibitors of protein kinase C and tyrosine kinase abrogate gp120-mediated ICAM-1 expression in both astrocytes and Microglia. Moreover, gp120 induces tyrosine phosphorylation of signal transducer and activator of transcription (STAT-1 alpha) as well as the Janus kinase (JAK2) in glial cells. We also demonstrate that gp120-mediated ICAM-1 expression has functional significance, as it enhances the ability of monocytic cells to bind to gp120-stimulated human astrocytes in an ICAM-1/beta 2 integrin-dependent fashion. These results provide new insights into how gp120 can influence the involvement of glial cells in the pathogenesis of AIDS dementia complex

  117. Tanaka M, Sato A, Okada Y, Makino M, Tabira T (1996) Evidence that brain capillary endothelial cells can be infected with murine leukaemia retrovirus, LP-BM5. Neurodegeneration. 5:287-291
    Abstract: Some mice infected with murine leukaemia retrovirus, LP-BM5 including ecotropic, mink cell focus-inducing murine leukaemia virus, and a replication-defective genome, have been reported to show weakness, ataxia, or selective deficits in spatial learning after developing an immunodeficiency syndrome similar to human AIDS. In the central nervous system, astrocytes and Microglial cells have been shown to be infected by this virus. We present here findings that the ecotropic virus and defective genome can infect murine brain capillary endothelial cells, and infected endothelial cells show an impaired function as target cells against myelin basic protein (MBP) specific T cell clone

  118. Czub M, Czub S, Rappold M, Mazgareanu S, Schwender S, Demuth M, Hein A, Dorries R (1995) Murine leukemia virus-induced neurodegeneration of rats: enhancement of neuropathogenicity correlates with enhanced viral tropism for macrophages, Microglia, and brain vascular cells. Virology 214:239-244
    Abstract: A highly neuropathogenic retrovirus, NT40, was generated by serially passaging an infectious molecular clone of Friend murine leukemia virus, FB29, through F344 Fisher rats. NT40 induced severe neurological signs such as reflex abnormalities and ataxia within 4-6 weeks following neonatal inoculation. FB29 led to only very mild neurological dysfunctions with longer incubation periods. Pathological alterations were characterized by mild (FB29) to extensive (NT40) noninflammatory spongiform degeneration, mainly of brain-stem areas. Infectious center assays revealed that viral titers in brain tissues of NT40-infected rats were 100-fold higher than those of FB29-infected animals. Employing immunohistochemistry, in situ hybridization, and flow cytometry, NT40 was found to infect many endothelial cells of brain blood vessels and Microglia, whereas FB29 infected only Microglia and those to a lower extent. However, when isolated from adult diseased rats, Microglial cells turned out in both cases to be nonproductively infected with either FB29 or NT40. Of peripheral organs, we found enhanced levels of NT40 in peritoneal macrophages but not in spleen, thymus, or serum when compared to FB29. Altogether these data suggest that an expanded cellular tropism within the CNS and elevated viral titers in macrophages and Microglia correlated with enhancement of neuropathogenicity

  119. Dobrenis K, Makman MH, Stefano GB (1995) Occurrence of the opiate alkaloid-selective mu3 receptor in mammalian Microglia, astrocytes and Kupffer cells. Brain Res. 686:239-248
    Abstract: Evidence is presented for occurrence of opiate alkaloid-selective, opioid-peptide-insensitive receptor binding sites, labeled with [3H]morphine, in primary cultures of cat Microglia and cat astrocytes, as well as on highly purified preparations of rat Kupffer cells. These receptors have been designated mu3 on the basis of their close similarity to receptors first found to be present on human peripheral blood monocytes. Exposure of the Microglia to morphine and etorphine caused marked quantifiable changes in cellular morphology, including assumption of a more rounded shape and retraction of cytoplasmic processes; in contrast, several opioid peptides were without effect on morphology. The effects of morphine on Microglial morphology were blocked by the opiate antagonist naloxone. These effects of drugs on morphology were as predicted for action via the mu3 receptor. Opiate alkaloid binding sites previously detected on the rat C6 glioma cell line were also characterized here as of the mu3 receptor subtype. It is proposed that mu3 receptors have broad distribution in different macrophage cell types of bone marrow lineage, including Microglia and Kupffer cells. Furthermore, these receptors are not restricted to cells of bone marrow lineage, since they are also present on astrocytes

  120. Dyck JR, Fliegel L (1995) Specific activation of the Na+/H+ exchanger gene during neuronal differentiation of embryonal carcinoma cells. J.Biol.Chem. 270:10420-10427
    Abstract: We examined the regulation of the Na+/H+ exchanger gene during differentiation of the P19 mouse embryonal carcinoma cells. Treatment of P19 cells with retinoic acid induces the development of neurons, astroglia, and Microglia cells. Upon retinoic acid-induced differentiation of P19 cells, there was an early and rapid 10-fold increase in NHE1 transcription. A proximal cis-acting AP-2 site of the NHE1 promoter was sufficient for stimulation of transcription of the gene by differentiation. Bandshift experiments demonstrated that in retinoic acid-treated cells there was an elevated level of AP-2 transcription factor binding to the AP-2 consensus site of the Na+/H+ exchanger gene. In the differentiation defective mutant RAC65, the effect of differentiation on Na+/H+ exchanger gene expression was reduced by 60%. Examination of Na+/H+ exchanger activity showed that retinoic acid-treated P19 cells recovered from an acid load at a rate approximately three times greater than untreated cells. The increases in gene expression and protein activity preceded major changes in cell morphology, suggesting that the initiation of differentiation is linked to NHE1 gene expression. Our findings show for the first time that the NHE1 gene is activated early in cell differentiation and that this activation may play an important role in the process of neuronal cell differentiation

  121. Gocht A, Lohler J, Scheidel P, Stegner HE, Saeger W (1995) Gliomatosis peritonei combined with mature ovarian teratoma: immunohistochemical observations. Pathol.Res.Pract. 191:1029-1035
    Abstract: Gliomatosis peritonei (GP) is the metastatic implantation of glial cells within the peritoneal cavity of patients with ovarian teratomas. The case of a young woman is presented, who initially developed a mature teratoma in the left ovary that was surgically removed. Nine years later a mature teratoma in the right ovary was excised, upon which GP was found in the greater omentum. To identify the cellular composition of the ovarian teratoma and of the omental implants, immunostainings were performed using antibodies against glial and neuronal antigens as well as against determinants of hematopoietic cells. In the teratoma the neuroectodermal part was strongly HNK-1-positive and contained

  122. Janabi N, Peudenier S, Heron B, Ng KH, Tardieu M (1995) Establishment of human Microglial cell lines after transfection of primary cultures of embryonic Microglial cells with the SV40 large T antigen. Neurosci.Lett. 195:105-108
    Abstract: Four continuous cell lines of human Microglial cells were obtained by transfection of enriched cultures of human embryonic brain-derived macrophages with a plasmid encoding for the large T antigen of SV40. The transformed cells had the macrophagic characteristics of adherence and intra-cytoplasmic non-specific esterase activity. They could phagocytize zymosan particles but the phagocytic activity remained low. They expressed several macrophagic antigens but not the monocytic markers CD14, CD4, CD68/Ki-M6 and CD11c. The cells could be activated to express class II major histocompatibility complex antigens after interferon-gamma activation. Finally, interleukin-6 was produced spontaneously by the cells and this production was further increased after interleukin-1 alpha stimulation

  123. Puy L, MacLusky NJ, Becker L, Karsan N, Trachtenberg J, Brown TJ (1995) Immunocytochemical detection of androgen receptor in human temporal cortex characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues. J.Steroid Biochem.Mol.Biol. 55:197-209
    Abstract: Immunocytochemical and biochemical studies have demonstrated the presence of androgen receptor protein in various regions of the rodent and non-human primate cortex. Localization of androgen receptor in the human brain has, however, not been studied as extensively, because of difficulties in obtaining suitable tissue samples. In the present study, we have localized androgen receptors in both frozen and paraffin-embedded temporal cortex from epileptic patients undergoing resection. Polyclonal antibodies were raised against fusion proteins containing fragments of the human androgen receptor protein. The antibodies were affinity-purified against the corresponding fusion protein. Immunoprecipitation and Western blotting using extracts from human cell lines demonstrated the specificity of the antibodies for the human androgen receptor and lack of cross-reactivity with other steroid hormone receptors. Immunocytochemistry was performed on frozen and paraffin sections of human temporal cortex and in paraffin-embedded benign hyperplastic prostates (BPH), as well as prostate and breast carcinomas, by the streptavidin-biotin-peroxidase method. Antigen-retrieval was performed in paraffin-embedded sections using microwave irradiation. Specific nuclear and cytoplasmic immunoreactivity for androgen receptor was detected in neurons, astrocytes, oligodendrocytes, and Microglia cells of the temporal cortex. In contrast, only nuclear staining was observed in BPH, prostate and breast carcinomas. Immunoprecipitation of human temporal cortex lysate and subsequent Western blot analysis demonstrated the expression of a 98 kDa immunoreactive protein, slightly smaller than the reported molecular weight of the wild-type androgen receptor. These results provide further evidence for the expression of androgen receptor in the human temporal cortex. The use of these immunocytochemical techniques should enable the retrospective determination of possible changes in androgen receptor expression in a variety of archival paraffin-embedded tissues, including samples of the human central nervous system

  124. Viola JJ, Ram Z, Walbridge S, Oshiro EM, Trapnell B, Tao-Cheng JH, Oldfield EH (1995) Adenovirally mediated gene transfer into experimental solid brain tumors and leptomeningeal cancer cells. J.Neurosurg. 82:70-76
    Abstract: Among the appealing features of adenoviruses as vectors for transfer of genes into the central nervous system (CNS) are that they are not neurotoxic, they can accommodate the insertion of several large genes, they are not associated with the hazards of insertional mutagenesis, and they can be concentrated to a high-titer preparation. The authors evaluated the feasibility of using adenovirally mediated gene transfer into cultured human glioma cells and in rat models of solid brain tumors and meningeal cancer. Replication-deficient adenoviral vector particles carrying a nuclear-localizing lacZ gene were injected into established 9L cerebral gliomas in Fischer rats. In addition, the adenoviral vector was injected into the subarachnoid space, either simultaneously with intrathecal tumor inoculation or after establishing leptomeningeal cancer. The brains and spinal cords were removed at various intervals for histochemical evaluation for beta-galactosidase activity using X-Gal staining. Additional rats received a stereotactic intracerebral injection of the vector into normal brain. No clinical abnormalities were observed in the injected rats. Injection of the adenoviral vector into normal brain resulted in diffuse transduction of astrocytes, Microglia, neurons, and endothelial cells at the injection site. Injection of a high-concentration vector preparation into cerebral gliomas resulted in effective tumor transduction. Intrathecal injection of the vector in rats with meningeal cancer resulted in transduction of the infiltrating tumor in the subarachnoid space when injections were given simultaneously with, or 7 days after, tumor inoculation. Transduction rates of both solid and leptomeningeal tumors correlated with the number of injected particles. These results suggest that adenoviral vectors can efficiently transduce solid brain tumors and that the vectors survive in the cerebrospinal fluid for a sufficient period of time to allow leptomeningeal tumor transduction. Adenoviral vector should be evaluated for its potential use in therapeutic gene transfer approaches in malignancies of the CNS

  125. Frei K, Malipiero U, Piani D, Fontana A (1994) Microglia and tumor rejection. Neuropathol.Appl.Neurobiol. 20:206-208

  126. Kida S, Ellison DW, Steart PV, Iannotti F, Weller RO (1994) Perivascular edema fluid pathway in astrocytic tumors. Acta Neurochir.Suppl (Wien.) 60:384-386
    Abstract: Perivascular spaces are anatomical routes for the bulk flow drainage of fluid from the gray matter to the subarachnoid space in normal rat brain. Perivascular cells are the resident scavengers in perivascular spaces. Following focal brain damage, perivascular cells upregulate MHC Class II antigens associated with uptake of edema fluid. Similar cells can be defined in damaged human brain. In the present investigation, the distribution of MHC Class II upregulated perivascular cells was measured in 30 astrocytic tumors and adjacent edematous tissues by immunocytochemistry using the following antibodies: HLA-DR (MHC Class II), PGM1 and MAC387 (macrophages). Perivascular cells were PGM1+/MA

  127. Kurpad SN, Wikstrand CJ, Bigner DD (1994) Immunobiology of malignant astrocytomas. Semin.Oncol. 21:149-161

  128. Otero GC, Merrill JE (1994) Cytokine receptors on glial cells. Glia 11:117-128
    Abstract: Given what evidence there is for the molecular and functional nature of cytokines and their cognate binding proteins in the immune system and the emerging similarities or even identities for these ligands and receptors in the nervous system, two general models may be relevant. The first emerging pattern is that receptors for related but distinct trophic factors in the CNS are in many instances multichain complexes with one or more shared components. The shared components of the receptor complex may be either signal- or nonsignal-transducing chains. A second emerging motif is that related ligands and related receptors fall into gene families. Undoubtedly, these models will facilitate the cloning of novel members of these families whose function is quite specific to the nervous system and in particular to glial cells. This article will review the function of the receptors for cytokines and families of differentiation/survival/growth factors as they operate on astrocytes, Microglia, and oligodendrocytes in development, health, and disease

  129. Vasilakos JP, Carroll RT, Emmerling MR, Doyle PD, Davis RE, Kim KS, Shivers BD (1994) Interleukin-1 beta dissociates beta-amyloid precursor protein and beta-amyloid peptide secretion. FEBS Lett. 354:289-292
    Abstract: A heightened production of interleukin 1 beta (IL-1 beta) has been reported in Microglial-associated amyloid deposits in Alzheimer's disease (AD) brains. These plaques are composed predominantly of beta/A4 peptide derived from beta-amyloid precursor protein (beta APP). We demonstrate that short-term (1 h) IL-1 beta-treatment increases beta APPs secretion and concomitantly decreases cell-associated beta APP in human H4 neuroglioma cells. Long-term (5 h) IL-1 beta treatment did not alter secreted or cell-associated beta APP content. In contrast, the secretion of beta/A4-containing epitope was not affected by short-term IL-1 beta stimulation; however, long-term IL-1 beta treatment decreased the amount of beta/A4-containing epitope secreted from the cells. These results show that IL-1 beta modifies the processing and secretion of beta APP to exacerbate perhaps the neuropathology of AD

  130. Cupp C, Taylor JP, Khalili K, Amini S (1993) Evidence for stimulation of the transforming growth factor beta 1 promoter by HIV-1 Tat in cells derived from CNS. Oncogene 8:2231-2236
    Abstract: Infection by human immunodeficiency virus type 1 (HIV-1), the etiologic agent of the acquired immunodeficiency syndrome (AIDS), is often complicated with a high incidence of neurologic disorders. It is believed that HIV-1, in addition to infecting both macroglial and Microglial cells, may influence the expression of several strategic genes of uninfected neighboring or latently infected brain cells. It is suspected that the viral-encoded transregulatory protein, Tat, facilitates cross-communications between these cells. In support of this concept, earlier studies demonstrated that Tat is released from the infected cells, and has the capacity to be taken up by the uninfected cells and exert its biological activity on the responsive gene. Recent studies in several laboratories suggest the involvement of Tat in altering the expression of a limited number of cellular regulatory factors which, in turn, may mediate the altered physiology of the cells. In this communication, we demonstrate the ability of the HIV-1 Tat protein to increase expression of transforming growth factor beta 1 (TGF-beta 1), a cytokine with potent immunosuppressive activity, in human astrocytic glial cells. Implications of the Tat-mediated induction of TGF-beta 1 expression and cytokine involvement in the regulation of immune response and central nervous system (CNS) pathology are discussed

  131. Kuchelmeister K, Bergmann M, Gullotta F (1993) Cellular changes in the cerebellar granular layer in AIDS-associated PML. Neuropathol.Appl.Neurobiol. 19:398-401
    Abstract: Six cases of AIDS-associated progressive multifocal leukoencephalopathy (PML) exhibited peculiar cellular changes in the cerebellar granular layer. These cells without discernible cytoplasm showed hypochromatic nuclei about twice as large as those of normal granule cells. They were restricted exclusively to the granular layer and always surrounded PML foci. An astrocytic, leukocytic or macrophage/Microglial nature was largely excluded by immunocytochemistry. Human immunodeficiency virus (HIV) antigen p 24 could not be found in these cells and there was no unequivocal detection of JC virus (JCV) DNA and no ultrastructural evidence of papovavirus particles in them. They possibly represent altered cerebellar granule cells abortively or latently infected with JCV

  132. Nagra RM, Wong PK, Wiley CA (1993) Expression of major histocompatibility complex antigens and serum neutralizing antibody in murine retroviral encephalitis. J.Neuropathol.Exp.Neurol. 52:163-173
    Abstract: Murine leukemia virus infection serves as a model for noninflammatory degeneration of the central nervous system (CNS). During the course of infection with either of the molecularly cloned viruses pNE-8 or ts-1, we observed that ts-1 spread twice as rapidly as pNE-8, and ascended higher in the neuraxis. Endothelial cells were infected first, followed by oligodendrocytes and neurons, while astrocytes containing glial fibrillary acidic protein were not infected. Additionally, ts-1 also infected macrophages/Microglia. Major histocompatibility complex (MHC) class I beta 2-microglobulin expression was minimal in pNE-8 infected mice, while it was elevated in endothelial cells of early ts-1 lesions, and in macrophages/Microglia during later stages. Occasional infected cells expressed beta 2-microglobulin while rare endothelial and parenchymal cells expressed MHC class II in both viral infections. Limited intra-CNS MHC expression may be one of the mechanisms of viral persistence and will present a barrier to developing immunotherapy for CNS retroviral infections. The few mice that escaped lethal infection had higher serum titers of neutralizing antibodies and showed no neuropathologic changes or detectable virus in the CNS. Higher titers of neutralizing antibodies may protect the CNS from infection

  133. Constam DB, Philipp J, Malipiero UV, ten Dijke P, Schachner M, Fontana A (1992) Differential expression of transforming growth factor-beta 1, -beta 2, and -beta 3 by glioblastoma cells, astrocytes, and Microglia. J.Immunol. 148:1404-1410
    Abstract: The type beta transforming growth factors (TGF) are potent regulators of the growth and functions of lymphocytes and macrophages. Recently the human glioblastoma cell line 308 was shown to produce TGF-beta 2. The relevance of this finding was evaluated further by comparing human glioblastoma cells with their nontransformed animal counterpart, astrocytes, with regard to the production of the three TGF-beta isoforms observed so far in mammals. In this report astrocytes are demonstrated to secrete also TGF-beta 2 and to express TGF-beta 1, -beta 2, and -beta 3 mRNA in vitro. In contrast, cultured murine brain macrophages release TGF-beta 1 and are positive for TGF-beta 1 mRNA only. Glia cell-derived TGF-beta 1 and -beta 2 are detected in latent form whereas both latent and active TGF-beta are identified in the supernatant of three human glioblastoma cell lines tested. These cell lines, however, show heterogeneity in regard to the isoform of TGF-beta expressed but share with astrocytes the inability to release TGF-beta 3. Provided production and activation of latent TGF-beta occur in vivo, astrocytes and Microglia may then be expected to exert regulatory influences on immune mediated diseases of the central nervous system

  134. Frei K, Piani D, Malipiero UV, Van Meir E, de Tribolet N, Fontana A (1992) Granulocyte-macrophage colony-stimulating factor (GM-CSF) production by glioblastoma cells. Despite the presence of inducing signals GM-CSF is not expressed in vivo. J.Immunol. 148:3140-3146
    Abstract: One of the morphologic hallmarks of human gliomas are inflammatory infiltrates with accumulation of macrophages in the tumor site. The signals leading to the macrophage response are only at the beginning of being understood. Novel chemotactic factors that have recently been characterized as secretory products of glioblastoma cells may attract mononuclear cells from the blood. Within the tumor tissue blood-derived monocytes and macrophages of the brain tissue, the Microglial cells, may increase in cell numbers due to tumor-derived growth factors. Both astrocytoma cell lines and cultured astrocytes have been shown recently to produce granulocyte-macrophage (GM)-CSF. We show that in vitro not only astrocytoma but also glioblastoma cell lines secrete GM-CSF when stimulated with TNF-alpha or IL-1. However, there is no evidence for GM-CSF production by glioblastoma cells in vivo: fresh tumor samples lack the mRNA for GM-CSF and the protein is not detectable in the tumor cyst fluids or the cerebrospinal fluids of glioblastoma patients. This contrasts IL-1 and IL-6 that are detectable in the tumor cyst fluids and IL-6 also in the cerebrospinal fluids of the patients. Unlike GM-CSF, transforming growth factor-beta 2 mRNA is expressed in ex vivo tested glioblastoma tissues. Absence of GM-CSF in vivo may be explained by the presence of tumor-derived inhibitory factors, such as transforming growth factor-beta 2 and PGE which suppress GM-CSF production by glioblastoma cells in vitro. The accumulation of macrophages at the tumor site may be due to local elaboration of chemoattractants and/or not yet defined growth factors rather than due to GM-CSF production

  135. Simmons ML, Murphy S (1992) Induction of nitric oxide synthase in glial cells. J.Neurochem. 59:897-905
    Abstract: Primary astrocyte cultures, C6 glioma cells, and N18 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL-6 fibroblasts. Treatment of astrocyte cultures for 16-18 h with lipopolysaccharide (LPS) induced NOS-like activity that was L-arginine and NADPH dependent, Ca2+ independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced Microglial cell contamination from 7 to 1%, with a loss of 44% of NOS-like activity. C6 cells treated with LPS also showed Ca(2+)-independent and L-arginine-dependent NOS-like activity. N18 cells demonstrated constitutive Ca(2+)-dependent NOS-like activity that was not enhanced by LPS induction. These data indicate that NOS-like activity can be induced in Microglia, astrocytes, and a related glioma cell line as it can in numerous other cell types, but not in neuron-like N18 cells

  136. Achim CL, Schrier RD, Wiley CA (1991) Immunopathogenesis of HIV encephalitis. Brain Pathol. 1:177-184
    Abstract: HIV infection leads to severe immunosuppression and in a sub-population of patients, encephalitis. Whether systemic immunosuppression is required for CNS infection is still unclear. However, latent infection of monocytes/macrophages is an important mechanism by which HIV escapes immune surveillance and enters the CNS. Unlike other viral encephalitides, HIV predominantly infects macrophages/Microglia and not neurons and glia. These cells produce retroviral proteins and cytokines which may be neurotoxic. Despite significant MHC expression within the CNS, there is a limited infiltration of immune cells, possibly due to a defect in systemic immunity. Anti-retroviral therapy by decreasing viral replication and reversing immunosuppression, may arrest nervous system damage

  137. Jung HW, Berens ME, Krouwer HG, Rosenblum ML (1991) A three-dimensional micro-organ culture system optimized for in vitro growth of human malignant brain tumors. Neurosurgery 29:390-398
    Abstract: A brain tumor is composed not only of tumor cells, but also of normal glial, mesenchymal, endothelial, and Microglial cells, as well as lymphocytes and macrophages. Therefore, homogeneous cultures of tumor cells, currently used for chemosensitivity testing, do not accurately model in situ tumors. We have developed an in vitro growth assay for brain tumors that includes normal host cells and is potentially useful for studies of chemotherapy and biological response modifiers. Human glioblastoma xenografts (U251-MG) were resected from mice, minced, and explanted into agarose-coated culture wells. After 5 to 7 days, microtumors emerged as expanding spheroids, which grew most efficiently in minimum essential medium supplemented with 20% fetal calf serum, 90% of which was replaced on alternate days. The growth rate and bromodeoxyuridine labeling index were similar in the microtumors and the xenografts, and light microscopy revealed highly cellular, pleomorphic tumors with high mitotic activity in both. Immunohistochemical studies also demonstrated the persistence of macrophages in both xenografts and microtumors. Microtumors treated for 2 hours with 75 mumol/L 1,3-bis-(2-chloroethyl)-1-nitrosourea showed a growth delay of 1.5 days; no effects were observed after treatment with lower doses. This in vitro system for brain tumor culture may provide a useful technique for the study of new therapies as an alternative to in vivo xenograft studies using immunodeficient animals

  138. Sutter A, Hekmat A, Luckenbach GA (1991) Antibody-mediated tumor cytotoxicity of Microglia. Pathobiology 59:254-258
    Abstract: The status of Microglial cells as potent effector cells in antibody-mediated tumor cytotoxicity (ADCC) could be established. Microglia (greater than or equal to 99.9% pure) derived from brain cortices of newborn mice were shown to lyse human tumor cell lines expressing different levels of epidermal growth factor (EGF) receptors in the presence of MAb 425, a monoclonal murine anti-primate EGF receptor antibody. MAb 425 mediates Microglial ADCC (MiADCC) at concentrations as low as 10(-11) M. Antibody ligands binding unilaterally to either EGF receptors on target cells or Fc receptors on Microglia have little effect on MiADCC. At 10(-10) M MAb 425, a 10(3)-fold excess of MAb 425 F(ab')2 fragments or irrelevant antibodies of identical isotype did not block MAb-425-induced MiADCC. Formation of effector-target cell contacts seems to be critical for MiADCC and MiADCC could not be inhibited by anti-tumor necrosis factor-alpha antibodies. In addition to its stimulatory effect on MiADCC, MAb 425 bound to EGF receptors exerted a microgliotrophic effect. Factor(s) derived from astrocytes enhance MiADCC

  139. Dozic S, Suvakovic V, Cvetkovic D, Jevtovic D, Skender M (1990) Neoplastic angioendotheliomatosis (NAE) of the CNS in a patient with AIDS subacute encephalitis, diffuse leukoencephalopathy and meningo-cerebral cryptococcosis. Clin.Neuropathol. 9:284-289
    Abstract: A 12-year-old, hemophilic boy died with acquired immune deficiency syndrome (AIDS) after a clinical course characterized by progressive psycho-organic syndrome and opportunistic infections. Postmortem neuropathological examination revealed a cerebral form of neoplastic angioendotheliomatosis (NAE), leukoencephalopathy, giant cell encephalitis and meningo-cerebral cryptococcosis. The most unusual finding was the presence of proliferated neoplastic cells within lumina of some blood vessels throughout the central nervous system (CNS). These cells displayed cytologic features of malignancy and stained positively for common leukocyte antigen. Coronal sections showed diffuse cerebral and cerebellar leukoencephalopathy with most pronounced loss of myelin and axons in deep white matter, while the subcortical arcuate fibers and the corpus callosum were partially spared. In these areas numerous small foci of severe myelin loss were present. Microglial nodules and distinctive multinucleated giant cells (MGC) were numerous. Intracytoplasmic and intranuclear acidophilic inclusions were found in a few multinuclear and mononuclear cells. Close contact between mononuclear and multinuclear cells suggesting their fusion was also observed. As far as we know this is the first case of NAE encountered in AIDS, one of the rare primary cerebral forms and the youngest reported case of NAE up to now. This case could be considered as one proof more that NAE is a special form of malignant lymphoma

  140. Vazeux R, Cumont M, Girard PM, Nassif X, Trotot P, Marche C, Matthiessen L, Vedrenne C, Mikol J, Henin D, . (1990) Severe encephalitis resulting from coinfections with HIV and JC virus. Neurology 40:944-948
    Abstract: We observed 3 cases of progressive multifocal leukoencephalopathy (PML) among frozen CNS samples obtained at autopsy from 102 adult AIDS patients. In 2 patients, PML was associated with severe HIV encephalitis. In those 2 cases, the areas of extensive JC-induced demyelination were massively infiltrated by HIV infected macrophages/Microglial cells with evidence for localized increase of HIV encephalitis in PML lesions. Using immunohistochemistry and in situ hybridization, we demonstrated that each virus infects, in a latent or productive fashion, different CNS cell populations. Therefore, the extension of HIV encephalitis could not be related to an intracellular transactivation of 1 virus by the other. However, the results are consistent with dissemination of viral infection by the recruitment of HIV-infected macrophages to damaged areas of the brain. This phenomenon might be generalized to other pathogens that are frequently associated with HIV CNS infection. Early detection and treatment of opportunistic CNS lesions could be important to prevent extension of HIV encephalitis