Biomedical subjects

A Klegeris

Publications and source records attributed to A Klegeris.

At least 19 recordsLinked to original sources

PubMed2001

Inflammatory cytokine levels are influenced by interactions between THP-1 monocytic, U-373 MG astrocytic, and SH-SY5Y neuronal cell lines of human origin.

We measured the secretion of interleukin (IL)1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) from human monocytic (THP-1), astrocytic (U-373 MG) and neuronal (SH-SY5Y) cell lines alone and in co-culture, with and without stimulation by a combination of lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) or amyloid beta peptide 1-40 (Abeta). LPS+IFN-gamma stimulation increased IL-1beta secretion 16-fold from THP-1 cells. It increased IL-6 secretion 23-fold from THP-1 cells and 2.5-fold from U-373 MG cells. It increased TNF-alpha secretion 3.4-fold from THP-1 cells, but did not influence its secretion from U-373 MG cells. It did not affect the secretion of any of the cytokines from SH-SY5Y cells. Abeta stimulation increased IL-6 secretion 2.3-fold from U-373 MG cells but did not influence secretion of IL-1beta or TNF-alpha. Abeta stimulation also failed to influence secretion of any of the cytokines from THP-1 or SH-SY5Y cells. When THP-1 and U-373 MG cells were cocultured, IL-1beta and IL-6 secretion, but not TNF-alpha secretion, were significantly reduced from the levels obtained in independent cultures, suggesting that a mutual suppressive action may occur between microglia and astrocytes.

Amyloid beta-Peptides↗

PubMed2001

Induction of complement C9 messenger RNAs in human neuronal cells by inflammatory stimuli: relevance to neurodegenerative disorders.

Neurons express proteins of the classical complement pathway, including C9. Both the mRNA and protein levels for C9 are sharply upregulated in brain areas affected by Alzheimer's disease (AD). Since little is known about the signals that are responsible for this upregulation, we evaluated in human SH-SY5Y neuroblastoma cells the factors which stimulate C9 production. Interferon-gamma, phorbol myristate acetate and interleukin-6 all stimulated C9 mRNA expression but the inflammatory cytokines tumor necrosis factor-alpha, interleukin-1 beta, as well as the anaphylatoxin C5a and the bacterial lipopolysaccharide, were ineffective. Immunohistochemical analysis of postmortem human brains for C9 protein demonstrated its presence in many cortical pyramidal neurons in AD, Down's syndrome, the parkinsonism dementia complex of Guam and pallido-ponto-nigral degeneration, as well as in thalamic neurons of progressive supranuclear palsy and ballooned neurons of Pick's disease. Since C9 is required for the membrane attack complex of complement to become functional, interfering with signaling pathways that stimulate its production could offer new therapeutic strategies for treating various neurodegenerative disorders.

Aged↗

PubMed2000

Expression of complement messenger RNAs by human endothelial cells.

This study evaluated complement mRNA expression in human brain microvessel endothelial cells (HBMEC), human umbilical vein endothelial cells (HUVEC), and cells of the human derived ECV304 line. Cerebral endothelial cells and HUVEC expressed detectable levels of complement gene mRNAs for the C1q B-chain, C1r, C1s, C2, C3, C4, C5, C7, C8 gamma-subunit and C9. In addition to C6 mRNA, C1q and C9 were not detected in ECV304 cells. These results indicate that endothelial cells may be a source of complement proteins in brain and other organs of the body.

Cell Line↗

PubMed2000

Inhibitory action of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) on some mononuclear phagocyte functions.

Peripheral benzodiazepine receptors (PBRs) are widely distributed throughout the body, but their functions are unknown. They are found on mononuclear phagocytes, and they are up-regulated in a number of neurological and other disease states. We explored the functional consequences of PBR ligand binding to mononuclear-derived cells using the high-affinity ligands 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) and 4'-chlorodiazepam (7-chloro-5-(4'-chlorophenyl)-1, 3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; Ro 5-4864). The functions were the following: respiratory burst; secretion of glutamate, interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha); toxicity of culture supernatants towards SH-SY5Y human neuroblastoma cells; and expression of the inflammatory surface markers HLA-DR and Fcgamma RII (CDw32). PK 11195 inhibited the respiratory burst response, reduced release of glutamate and IL-1beta, and suppressed secretion of products cytotoxic to neuronal cells. Selectivity was suggested by the failure of PK 11195 to influence TNF-alpha secretion or expression of HLA-DR and CDw32. Powerful ligands of PBRs, such as PK 11195, may be useful inhibitors of selective macrophage functions, retarding both local and systemic inflammation. Since PK 11195 readily enters the brain, it may be beneficial in treating central as well as peripheral inflammatory diseases.

Animals↗

PubMed2000

Interaction of various intracellular signaling mechanisms involved in mononuclear phagocyte toxicity toward neuronal cells.

Microglia become activated in a wide range of neurodegenerative disorders, including Alzheimer's disease. Such activation may lead to autodestruction of neurons. It is demonstrated here that activation of both human microglia and monocytic THP-1 cells by a combination of lipopolysaccharide and interferon-gamma results in secretion of neurotoxins that kill human neuronal SH-SY5Y cells. This neurotoxicity can be partially blocked by inhibitors of cytosolic phospholipase A2, cGMP-selective phosphodiesterases, or protein kinase C. When combinations of these inhibitors, or combinations of an inhibitor plus nordihydroguaiaretic acid, or the nonsteroidal anti-inflammatory drug diclofenac were tried, additive reductions in neurotoxicity were observed. It is concluded that the stimulants activated multiple intracellular pathways, and that combination therapies inhibiting these pathways might be beneficial for treating neurodegenerative disorders.

Cell Death↗

PubMed2000

R-(-)-Deprenyl inhibits monocytic THP-1 cell neurotoxicity independently of monoamine oxidase inhibition.

R-(-)-Deprenyl (deprenyl, selegiline), a monoamine oxidase B (MAO-B) inhibitor, delays progression of Parkinson's disease. This action could be mediated by inhibition of MAO-B but there may also be unrelated mechanisms. Direct neuroprotective and antiapoptotic actions of deprenyl have previously been observed in vitro. Here we describe an antineurotoxic action of deprenyl which is independent of direct neuronal effects. We employed a previously described assay in which human neuroblastoma SH-SY5Y cells are exposed to cell-free supernatants of stimulated human monocytic THP-1 cells. Deprenyl reduced the secretion of neurotoxic products by such stimulated cells in a concentration-dependent manner, while the MAO inhibitors iproniazid, isocarboxazid, nialamide, tranylcypromine, phenelzine, and clorgyline were without effect. No antineurotoxic action was observed when deprenyl was added directly to SH-SY5Y cells. Messenger RNAs for MAO-A and MAO-B were not detected in THP-1 cells by reverse transcriptase-polymerase chain reaction analysis of total RNA extracts. Such mRNAs were easily detected in extracts of SH-SY5Y cells under comparable conditions. MAO enzymatic activity was also undetectable in THP-1 cell lysates, while it was readily observed in SH-SY5Y cells. It was concluded that the effect of deprenyl on THP-1 cells was not mediated by MAO and that deprenyl itself was not protecting neurons. These data suggest that deprenyl may have utility in neurodegenerative diseases due to its antineurotoxic actions.

Alzheimer Disease↗

PubMed1999

Toxicity of human THP-1 monocytic cells towards neuron-like cells is reduced by non-steroidal anti-inflammatory drugs (NSAIDs).

There is mounting evidence that inflammatory processes, including activation of microglia, are upregulated in Alzheimer's disease. The importance of this phenomenon is indicated by multiple epidemiological studies showing that patients taking non-steroidal anti-inflammatory drugs (NSAIDs) have a substantially reduced prevalence of Alzheimer's disease. The pharmacological actions of anti-inflammatory drugs in brain are still uncertain. As a step towards identifying key pharmacological targets, we developed a neurotoxicity assay based on the property of supernatant media from stimulated human monocytic THP-1 cells to cause human neuroblastoma cell death. Similar neurotoxicity was observed when postmortem human microglia were substituted for THP-1 cells, establishing the validity of the assay for simulating neurotoxicity in human brain. A combination of lipopolysaccharide and interferon-gamma was used to activate the THP-1 cells. NSAIDs were effective in inhibiting neurotoxicity by this assay, while steroidal anti-inflammatories and propentofylline had no effect. The neuroprotective potency of NSAIDs appeared to be unrelated to their selective ability to inhibit cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2). It is suggested that inhibition of monocyte cytotoxicity might be responsible for the apparent beneficial effects of NSAIDs in Alzheimer's disease.

Analysis of Variance↗

PubMed1997

beta-amyloid protein enhances macrophage production of oxygen free radicals and glutamate.

Cells of the monocyte phagocytic system can generate superoxide and glutamate anions, both of which are neurotoxic at high levels. We used rat peritoneal macrophages as a model system to test the effects of various stimulants on the production of these molecules. Glutamate production by such cells was enhanced, in a concentration-dependent manner, by treatment with serum-opsonized zymosan (OZ), lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and beta-amyloid peptide Abeta (1-40); but not by treatment with the reverse Abeta (40-1) or the Abeta (25-35) subfragment. Superoxide anion production by the cells was stimulated by OZ, PMA, Abeta (1-40), and Abeta (25-35). Moreover, Abeta and its subfragment, when used as priming agents, also enhanced the stimulatory effect of PMA. However, they did not act as priming agents for OZ, suggesting a competition for receptors or intracellular signaling pathways linked to those receptors. Inflammatory mediators, including Abeta, could place glutamate-sensitive neurons at risk by enhancing glutamate and oxygen free radical production by monocyte-derived cells. Such mechanisms could contribute to the pathogenesis of neurodegenerative disorders, including Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed1997

Interaction of Alzheimer beta-amyloid peptide with the human monocytic cell line THP-1 results in a protein kinase C-dependent secretion of tumor necrosis factor-alpha.

Immunological mechanisms, including stimulation of brain microglia and elevation of various inflammatory cytokines, have been implicated in the pathogenesis of Alzheimer's disease, where accumulation of beta-amyloid peptide (A beta) is one of its main pathological features. In this study we investigated the interaction of human monocyte-like cells with synthetic beta-amyloid peptide A beta (1-40) and its subfragment A beta (25-35). THP-1 cells (a transformed human monocyte cell line) were used with or without prior differentiation by phorbol myristate acetate (PMA), and cell activation was assessed by the secretion of tumor necrosis factor-alpha (TNF-alpha). First, it was shown that THP-1 cells could be induced to secrete significant amounts of TNF-alpha by interleukin-1, lipopolysaccharide, interferon-gamma (IFN-gamma) and PMA alone or in combination with each other. Next it was shown that A beta (1-40) could also induce secretion of TNF-alpha by THP-1 cells, but the effect was diminished when this peptide was applied in combination with IFN-gamma. The A beta subfragment A beta (25-35) was ineffective in inducing TNF-alpha production. The cellular action of A beta (1-40) appears to involve protein kinase C since pretreatment of THP-1 cells by PMA or the protein kinase C inhibitor H-7 diminished the cellular response to A beta (1-40). Identification of the pathway by which extracellular A beta activates the intracellular PKC-dependent secretion of TNF-alpha may help in developing new therapeutic strategies for Alzheimer's disease.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Regulation of glutamate in cultures of human monocytic THP-1 and astrocytoma U-373 MG cells.

Glutamate, an excitatory neurotransmitter, is neurotoxic at high concentrations. Neuroglial cells, including astrocytes and microglia, play an important role in regulating its extracellular levels. Cultured human monocytic THP-1 cells increased their glutamate secretion following 18 and 68 h exposure to the inflammatory mediators zymosan, phorbol myristate acetate (PMA), lipopolysaccharide, interferon-gamma, tumor-necrosis factor-alpha and interleukin-1beta. Cultured astrocytoma U-373 MG cells increased their glutamate secretion following similar exposure to zymosan and PMA. DL-Alpha-aminopimelic acid, an inhibitor of the glutamate secretion system, reduced extracellular glutamate in both cell culture systems, while the high-affinity glutamate uptake inhibitors D-Aspartic acid, DL-threo-beta-hydroxyaspartic acid and L-trans-pyrrolidine-2,4-dicarboxylic acid increased extracellular glutamate in U-373 MG, but not THP-1 cell cultures. In co-cultures of THP-1 and U-373 MG cells, extracellular glutamate levels were increased significantly by the Alzheimer beta-amyloid peptide (1-40) and were decreased significantly by the anti-inflammatory drug dexamethasone. These data indicate that inflammatory stimuli may increase extracellular glutamate while antiinflammatory drugs decrease it.

Amyloid beta-Peptides↗

PubMed1996

Acetylcholinesterase-induced respiratory burst in macrophages: evidence for the involvement of the macrophage mannose-fucose receptor.

It has long been suggested that acetylcholinesterase is capable of functioning in a non-cholinergic manner. However, very little is known about the molecular structures which mediate the interaction between this protein and the cellular membrane. Previously it was demonstrated that acetylcholinesterase interacted in a carbohydrate-specific manner with peritoneal macrophages and induced the 'respiratory burst' [1]. This study aimed to establish whether a carbohydrate-binding site exists on the acetylcholinesterase molecule itself, or alternatively, whether the macrophage carbohydrate-binding receptor is involved. No carbohydrate binding properties intrinsic to acetylcholinesterase were detected using affinity chromatography with immobilised monosaccharides, erythrocyte agglutination and gel-diffusion techniques. The interaction between acetylcholinesterase and several monosaccharide columns observed in this study appeared to be due to ionic interactions. Moreover, it was shown that a specific inhibitor of the enzymatic activity of AChE, BW284C51, could inhibit the peritoneal cell response not only to acetylcholinesterase, but also to several other stimuli, thus exhibiting a non-specific effect on macrophages. However, the inhibitory effects of specific ligands of the macrophage mannose-fucose receptor and the inability of non-glycosylated acetylcholinesterase to interact with macrophages suggested that the effect of acetylcholinesterase on peritoneal cells is most probably mediated by the macrophage mannose-fucose receptor. The role of the mannose-fucose receptor in triggering the respiratory burst response was supported by the fact that several ligands of these receptors were capable of inducing the functional response of macrophages.

Acetylcholinesterase↗

PubMed1995

A possible interaction between acetylcholinesterase and dopamine molecules during autoxidation of the amine.

Acetylcholinesterase has an action in the central nervous system, independent of hydrolysis of acetylcholine. This study explored the possible interaction between the two molecules: the effects of acetylcholinesterase on the autoxidation of the catecholamine were tested, and, in turn, modification of the catalytic activity of the enzyme by products of dopamine oxidation were studied. Acetylcholinesterase selectively inhibited the speed of quinone production from dopamine as well as accumulation of hydrogen peroxide, whilst the rate of generation of superoxide was increased. Analysis of absorption spectra revealed the formation of a new product, which appeared after mixing acetylcholinesterase and dopamine in neutral pH. In all cases, butyrylcholinesterase was ineffective. Incubation of acetylcholinesterase in the presence of dopamine resulted in a significant decrease in the catalytic activity of the enzyme. The effects of application of preparations modifying autoxidation of dopamine (SOD, catalase, peroxidase) suggested that inactivation of the enzyme occurred as a result of the direct interaction of a quinone and/or semiquinone oxidation product with enzyme, as opposed to any effects of reactive oxygen species. Because acetylcholinesterase and dopamine are co-released from the neurons degenerating in Parkinson's disease, a direct chemical interaction between these two molecules could have significance both for the normal functioning of the substantia nigra and for related pathological states.

Acetylcholinesterase↗

PubMed1995

Autoxidation of dopamine: a comparison of luminescent and spectrophotometric detection in basic solutions.

Oxidation products of catecholamines, particularly dopamine, could play an important role in the physiology and pathology of the nervous system. This study has therefore characterised autoxidation of dopamine monitored in a variety of systems. Lucigenin-dependent chemiluminescence and reduction of cytochrome c were exploited to register generation of the byproduct superoxide anion, whereas the quinone product was detected by a direct spectrophotometric measurement. Accumulation of hydrogen peroxide was followed as an increase in luminol-dependent chemiluminescence. In all cases, basic solutions were used to initiate the oxidation of dopamine. The results obtained could be interpreted as specific reactions at the particular stages of the autoxidation process: the luminol-dependent chemiluminescence system detected accumulation of hydrogen peroxide during dopamine oxidation, whereas the lucigenin-dependent chemiluminescence indicated generation of superoxide anion. Furthermore, cytochrome c reduction, observed during dopamine oxidation, was probably attributed to a direct interaction with dopamine semiquinone. In addition, the effects of superoxide dismutase, catalase, and peroxidase were examined in each of the systems: Each enzyme exhibited a different effect in each system used. The possible reaction mechanisms leading to different action of enzymes affecting reactive oxygen species are discussed. The methods described here of monitoring dopamine autoxidation could thus be used in parallel to detect the effects of different preparations on various stages of the dopamine autoxidation process.

Acridines↗

PubMed1994

Inhibition of respiratory burst in macrophages by complement receptor blockade.

Respiratory burst activity was induced in rat peritoneal macrophages by opsonized zymosan. Inhibitors were tested by administering them before or after the inducing agent: OX-42, an anti-rat macrophage complement receptor type 3 antibody, was active at an estimated concentration of 2.1 nM, and was more than 100-fold more potent when administered before, rather than after, opsonized zymosan. Indomethacin and dapsone, two agents with antiinflammatory activity, were also more effective before opsonized zymosan, but only in the 10(-3) to 10(-4) molar range. Inhibitors of eicosanoid synthesis, as well as the antiinflammatory prostaglandin E2, also reduced the respiratory burst.

Animals↗

PubMed1994

Activation of macrophages by Alzheimer beta amyloid peptide.

Microglia (brain resident macrophages) have been found to be closely associated with beta amyloid containing plaques in brain tissue affected by Alzheimer disease (AD). To investigate whether beta amyloid peptide (beta AP) may activate microglia, the effects of synthetic beta AP (amino acids 1-40) and a subfragment (amino acids 25-35) on rat peritoneal macrophages were assessed using four different assays for activation. These peptides were compared with substance P, which has previously been shown to activate macrophages. Both beta amyloid peptides activated macrophages, as assessed by increased respiratory burst-associated oxygen consumption, by luminol-dependent chemiluminescence, and by aggregation. In addition, beta amyloid peptide (1-40) caused a significant increase in macrophage nitric oxide production, while subfragment (25-35) did not. Substance P caused significant activation as assessed by oxygen consumption and chemiluminescence, but not by aggregation or nitric oxide induction.

Amino Acid Oxidoreductases↗

PubMed1994

The subthalamo-nigral pathway regulates movement and concomitant acetylcholinesterase release from the substantia nigra.

Within the substantia nigra acetylcholinesterase is released independently of cholinergic transmission: this release could be related to some aspects of motor control. To investigate this possibility, acetylcholinesterase release was continuously monitored in relation to specific movements evoked by central electrical stimulation. Increased intensities of stimulation of the subthalamic nucleus in awake guinea-pigs produced a behavioural response, ranging from a decrease in spontaneous movement, to chewing, to both chewing and circling movements. Enhancement of acetylcholinesterase release occurred only when large scale movements (circling as well as chewing) were evoked by subthalamic stimulation: however, a similar protocol of stimulation during ketamine-induced anaesthesia did not produce any comparable movements nor any concomitant change in the release of acetylcholinesterase. Perfusion of the glutamate agonist N-methyl-D-aspartate (NMDA) into the substantia nigra also induced an increase in release of acetylcholinesterase from the substantia nigra of conscious animals, whereas (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) did not significantly enhance acetylcholinesterase levels. It is concluded that AChE release in the substantia nigra can occur as a result of activation of glutamatergic subthalamic afferents, and that this activation may also be associated with changes in movement.

Acetylcholinesterase↗

PubMed1994

Acetylcholinesterase activation of peritoneal macrophages is independent of catalytic activity.

1. In diverse tissues, acetylcholinesterase appears to play a critical role in the functional state of cells completely dependent of cholinergic transmission. However, very little is known about the mechanisms and actual molecular structures mediating the fundamental interactions between this protein and the cellular membrane. 2. In this study, peritoneal macrophages were used as a model system to study the possible interaction between acetylcholinesterase, acting in a non-cholinergic capacity, and the cellular membrane. 3. When acetylcholinesterase was incubated with macrophages harvested from rat peritoneum, the rate of oxygen consumption was increased in a concentration-dependent manner that was independent of mitochondrial block with sodium cyanide. Furthermore, heat inactivation of enzymatic activity or application of BW 284C51 at a concentration which totally blocks catalytic activity did not eliminate the effect. 4. In contrast, incubation with bovine serum albumin or butyrylcholinesterase actually retarded oxygen consumption. 5. The effect of acetylcholinesterase depended on the presence of divalent cations and was inhibited by mannan and D-mannose, but not D-galactose. It is concluded that acetylcholinesterase can induce a "respiratory burst" in macrophages independent of its conventional catalytic site but involving either the mannose receptor of the monocyte-derived macrophage or a possible sugar binding site on acetylcholinesterase itself.

Acetylcholinesterase↗

PubMed1994

Rat brain microglia and peritoneal macrophages show similar responses to respiratory burst stimulants.

Respiratory burst activity was compared between cultured newborn rat microglia and directly harvested adult rat peritoneal macrophages using a Clarke oxygen electrode system. Both types of cells showed stimulated oxygen consumption almost immediately after the administration of opsonized zymosan, phorbol myristate acetate, concanavalin A, or tuftsin. The absolute values of stimulated oxygen consumption after administration of these agents ranged from 0.11 to 0.99 nmol per min per million cells, with some variation in relative response of microglia compared with peritoneal macrophages. After lysis of cells with deoxycholate, or disruption by sonication, oxygen consumption was restored by NADPH for stimulated microglia but not stimulated astrocytes. The potential for stimulated microglia to generate oxygen free radicals may have implications in several degenerative neurological diseases where activated microglia are found in association with the lesions.

Animals↗