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Biomedical subjects

E Clementi

Publications and source records attributed to E Clementi.

At least 19 recordsLinked to original sources
PubMed2006

The intradermal Leishmanin reaction induces antigen-specific maturation of canine dendritic cells with up-regulation of MHCII synthesis and expression.

Dendritic cells (DCs) are professional antigen-presenting cells that reside in many tissues, including the skin. This study showed that intradermal injection of leishmanin in Leishmania infantum-infected dogs induced the "up-regulation" of surface MHCII expression, associated with progressive ultrastrucutural changes characteristic of DC maturation, including the formation of multilaminar MHC class II-containing compartments and arrays of tubulo-vesicular structures. These changes were not observed in control dogs from L. infantum non-endemic areas. The results indicated that canine DCs were effector cells in delayed-type hypersensitivity, that the leishmanin reaction was specific for a cell-mediated reaction to L. infantum in infected dogs, and that canine DCs possessed ultrastructural organelles reminiscent of those in activated human DCs.

Animals↗
PubMed2002

Cyclic GMP-dependent inhibition of acid sphingomyelinase by nitric oxide: an early step in protection against apoptosis.

Activation of acid and neutral sphingomyelinases, and the ensuing generation of ceramide, contributes to the biological effects of tumour necrosis factor-alpha (TNF-alpha), one of which is apoptosis. While the mechanisms of activation of sphingomyelinases by the cytokine are being unravelled, less is known about regulation of their activity. Nitric oxide has previously been shown to exert a cyclic GMP-dependent inhibition of early apoptotic events triggered by TNF-alpha in the U937 monocytic cell line. We therefore investigated whether inhibition of sphingomyelinases by nitric oxide plays a role in regulating such early events. We found that activation of both acid and neutral sphingomyelinases, triggered in the first minutes after U937 cell stimulation with TNF-alpha, is regulated in an inhibitory fashion by nitric oxide, working through generation of cyclic GMP and activation of protein kinase G. Using a range of inhibitors selective for either sphingomyelinase we found that the acid sphingomyelinase contributes to activation of the initiator caspase-8 and early DNA fragmentation and that inhibition of the acid enzyme by nitric oxide accounts for cyclic GMP-dependent early protection from apoptosis. We also found that the protective effect by both cGMP and acid sphingomyelinase inhibitors progressively disappeared at later stages of the apoptotic process. Inhibition of sphingomyelinases represents a novel action of nitric oxide, which might be of physiological relevance in regulating initial phases of apoptosis as well as other biological actions of ceramide.

Apoptosis↗
PubMed2001

TNFalpha enhances the DNA single-strand breakage induced by the short-chain lipid hydroperoxide analogue tert-butylhydroperoxide via ceramide-dependent inhibition of complex III followed by enforced superoxide and hydrogen peroxide formation.

Treatment of U937 cells with nontoxic concentrations of TNFalpha increased the DNA strand scission induced by a short-chain lipid hydroperoxide analogue, tert-butylhydroperoxide. The following lines of evidence suggest that the enhancing effects of TNFalpha are mediated by inhibition of complex III and by the ensuing formation of superoxides and hydrogen peroxide: (a) the effects of TNFalpha were mimicked by the complex III inhibitor antimycin A; (b) the effects of TNFalpha, or antimycin A, were abolished by the complex I inhibitor rotenone, or by myxothiazol, an agent which inhibits the electron flow from the reduced coenzyme Q to cytochrome c(1) and therefore prevents ubisemiquinone formation; (c) the effects of TNFalpha, or antimycin A, were not observed in respiration-deficient cells; and (d) the effects of TNFalpha, or antimycin A, were sensitive to catalase. The TNFalpha-dependent inhibition of complex III appears to be mediated by ceramide. Three lines of evidence support this inference: (a) a synthetic cell-permeable ceramide analogue reproduced all the effects of TNFalpha, (b) TNFalpha promoted the formation of ceramide via a mechanism sensitive to inhibition of sphingomyelinases by tricyclodecan-9-yl-xanthogenate and imipramine, and (c) the TNFalpha-mediated enhancement of the tert-butylhydroperoxide-induced DNA-damaging response was prevented under conditions in which ceramide formation was inhibited.

Antimycin A↗
PubMed2001

Nitric oxide inhibits mitochondrial NADH:ubiquinone reductase activity through peroxynitrite formation.

This study was aimed at assessing the effects of long-term exposure to NO of respiratory activities in mitochondria from different tissues (with different ubiquinol contents), under conditions that either promote or prevent the formation of peroxynitrite. Mitochondria and submitochondrial particles isolated from rat heart, liver and brain were exposed either to a steady-state concentration or to a bolus addition of NO. NO induced the mitochondrial production of superoxide anions, hydrogen peroxide and peroxynitrite, the latter shown by nitration of mitochondrial proteins. Long-term incubation of mitochondrial membranes with NO resulted in a persistent inhibition of NADH:cytochrome c reductase activity, interpreted as inhibition of NADH:ubiquinone reductase (Complex I) activity, whereas succinate:cytochrome c reductase activity, including Complex II and Complex III electron transfer, remained unaffected. This selective effect of NO and derived species was partially prevented by superoxide dismutase and uric acid. In addition, peroxynitrite mimicked the effect of NO, including tyrosine nitration of some Complex I proteins. These results seem to indicate that the inhibition of NADH:ubiquinone reductase (Complex I) activity depends on the NO-induced generation of superoxide radical and peroxynitrite and that Complex I is selectively sensitive to peroxynitrite. Inhibition of Complex I activity by peroxynitrite may have critical implications for energy supply in tissues such as the brain, whose mitochondrial function depends largely on the channelling of reducing equivalents through Complex I.

Animals↗
PubMed2001

Human IgM anti-GM1 autoantibodies modulate intracellular calcium homeostasis in neuroblastoma cells.

Increased titers of IgM anti-GM1 antibodies are present in some patients with Lower Motor Neuron Disease (LMND) or Motor Neuropathy (MN), but their pathogenic role and the mechanism of action are unclear. Previous studies have shown that the B subunit of Cholera Toxin (CT), which binds and crosslinks ganglioside GM1, modulate intracellular calcium in murine neuroblastoma cells via the activation of L-type voltage-dependent calcium channels (VGCC). Therefore, using a fluorimetric approach, we have examined the hypothesis that the pentameric IgM anti-GM1 antibodies, could similarly alter calcium concentration in N18 neuroblastoma cells. Sera with human IgM anti-GM1 antibodies were obtained from 5 patients with LMND and 2 patients with MN. Human IgG anti-GM1, IgM anti-Myelin Associated Glycoprotein (MAG), IgM anti-sulfatide antibodies and lectin peanut agglutinin (PNA), that recognizes specifically the Gal(betal-3)GalNAc epitope, were used as control sera. Direct application of either human IgM anti-GM1 antibodies or the B subunit of CT to N18 neuroblastoma cells induced a sustained influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. Furthermore, the dihydropyridine L-type channel antagonists completely inhibited the manganese influx, suggesting that it is due to activation of an L-type VGCC. The magnitude of the influx was correlated with antibody titers. None of human IgG anti-GM1, IgM anti-MAG, IgM anti-sulfatide antibodies or PNA induce an ion influx, pointing to the selective participation of the pentameric IgM isotype of anti-GM1 in the modulation of L-type calcium channels opening. Given that L-type calcium channels are present on motor neurons, the modulation of L-type calcium channels by IgM GM1 antisera may have important implications in diseases such as LMND and MN.

Adult↗
PubMed2000

Activation of the endothelial nitric-oxide synthase by tumor necrosis factor-alpha. A novel feedback mechanism regulating cell death.

Cell death via apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plays an important role in many physiological and pathological conditions. The signal transduction pathway activated by this cytokine is known to be regulated by several intracellular messengers. In particular, in many systems nitric oxide (NO) has been shown to protect cells from TNF-alpha-induced apoptosis. However, whether NO can be generated by the cytokine to down-regulate its own apoptotic program has never been studied. We have addressed this question in HeLa Tet-off cell clones stably transfected with the endothelial NO synthase under a tetracycline-responsive promoter. Endothelial NO synthase, induced about 100-fold in these cells by removal of the antibiotic, retained the characteristics of the native enzyme of endothelial cells, both in terms of intracellular localization and functional activity. Expression of the endothelial NO synthase was sufficient to protect from TNF-alpha-induced apoptosis. This protection was mediated by the generation of NO. TNF-alpha itself stimulated endothelial NO synthase activity to generate NO through a pathway involving its lipid messenger, ceramide. Our results identify a novel mechanism of regulation of a signal transduction pathway activated by death receptors and suggest that NO may constitute a built-in mechanism by which TNF-alpha controls its own apoptotic program.

Adenosine Triphosphate↗
PubMed2000

Products of the phospholipase A(2) pathway mediate the dihydrorhodamine fluorescence response evoked by endogenous and exogenous peroxynitrite in PC12 cells.

A short-term exposure of PC12 cells to tert-butylhydroperoxide promotes a rapid oxidation of dihydrorhodamine sensitive to nitric oxide synthase inhibitors and peroxynitrite scavengers. This response was not directly caused by peroxynitrite, but rather appeared to be mediated by peroxynitrite-dependent activation of phospholipase A(2). The following lines of evidence support this inference: (i) the peroxynitrite-dependent dihydrorhodamine fluorescence response was blunted by low concentrations of two structurally unrelated phospholipase A(2) inhibitors; (ii) under similar conditions, the phospholipase A(2) inhibitors prevented release of arachidonic acid; (iii) low levels of arachidonic acid restored the dihydrorhodamine fluorescence response in nitric oxide synthase- as well as phospholipase A(2)-inhibited cells; (iv) the dihydrorhodamine fluorescence response induced by authentic peroxynitrite was also blunted by phospholipase A(2) inhibitors and restored upon addition of reagent arachidonic acid. We conclude that endogenous, or exogenous, peroxynitrite does not directly oxidize dihydrorhodamine in intact cells. Rather, peroxynitrite appears to act as a signalling molecule promoting release of arachidonic acid, which in turn leads to formation of species causing the dihydrorhodamine fluorescence response.

Animals↗
PubMed2000

Nitric oxide inhibits the tumor necrosis factor alpha -regulated endocytosis of human dendritic cells in a cyclic GMP-dependent way.

Tumor necrosis factor-alpha (TNFalpha)-induced maturation of dendritic cells (DC), with down-regulation of their endocytic ability, has been reported to be mediated by the accumulation of the lipid messenger ceramide. We have now studied the effects and mechanisms of action of NO on endocytosis, investigated with fluorescein isothiocyanate-labeled dextran using human monocyte-derived DC, both immature and after treatment with TNFalpha. Exposure of DC to NO, released by either bystander phagocytes or NO donors, reversed the inhibition of endocytosis induced by TNFalpha. The intracellular accumulation of ceramide induced by TNFalpha was also inhibited by NO. In addition, NO was found to exert an inhibitory effect downstream of the TNFalpha-triggered ceramide accumulation, because NO donors reversed the inhibition of endocytosis induced by the cell-permeant C(2)-ceramide. These effects of NO were mimicked by the membrane-permeant cyclic GMP analogue, 8-Br cyclic GMP, and prevented by inhibition of the soluble guanylyl cyclase. At variance with rodents, the inducible isoform of the NO synthase was expressed neither in immature human DC nor after cell treatment with TNFalpha, interferon-gamma, and lipopolysaccharide, suggesting that regulation of these cells depends on exogenous NO. NO, working through cyclic GMP, might therefore prolong the ability of human DC to internalize antigens at the site of inflammation and thus modulate the initial steps leading to antigen-specific immune responses.

Animals↗
PubMed2000

Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines.

Broad spectrum caspase inhibitors have been found to reduce neurodegeneration caused by cerebral ischemia. We studied whether blockade of group I caspases, mainly caspase-1, using the inhibitor Ac-YVAD.cmk reduced infarct volume and produced prolonged neuroprotection. Ac-YVAD.cmk (300 ng/rat) was injected intracerebroventricularly 10 min after permanent middle cerebral artery occlusion in the rat. Drug treatment induced a significant reduction of infarct volume not only 24 hr after ischemia (total damage, percentage of hemisphere volume: control, 41.1 +/- 2.3%; treated, 26.5 +/- 2.1%; p < 0.05) but also 6 d later (total damage: control, 30.6 +/- 2.2%; treated, 23.0 +/- 2.2%; p < 0.05). Ac-YVAD. cmk treatment resulted in a reduction not only of caspase-1 (control, 100 +/- 20.3%; treated, 3.4 +/- 10.4%; p < 0.01) but also of caspase-3 (control, 100 +/- 30.3%; treated, 13.2 +/- 9.5%; p < 0.05) activity at 24 hr and led to a parallel decrease of apoptosis as measured by nucleosome quantitation (control, 100 +/- 11.8%; treated, 47 +/- 5.9%; p < 0.05). Six days after treatment no differences in these parameters could be detected between control and treated animals. Likewise, brain levels of the proinflammatory cytokines IL-1beta and TNF-alpha were reduced at 24 hr (39.5 +/- 23.7 and 51.9 +/- 10.3% of control, respectively) but not at 6 d. Other cytokines, IL-10, MCP-1, MIP-2, and the gaseous mediator nitric oxide, were not modified by the treatment. These findings indicate that blockade of caspase-1-like activity induces a long-lasting neuroprotective effect that, in our experimental conditions, takes place in the early stages of damage progression. Finally, this effect is achieved by interfering with both apoptotic and inflammatory mechanisms.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Wild-type huntingtin protects from apoptosis upstream of caspase-3.

Expansion of a polyglutamine sequence in the N terminus of huntingtin is the gain-of-function event that causes Huntington's disease. This mutation affects primarily the medium-size spiny neurons of the striatum. Huntingtin is expressed in many neuronal and non-neuronal cell types, implying a more general function for the wild-type protein. Here we report that wild-type huntingtin acts by protecting CNS cells from a variety of apoptotic stimuli, including serum withdrawal, death receptors, and pro-apoptotic Bcl-2 homologs. This protection may take place at the level of caspase-9 activation. The full-length protein also modulates the toxicity of the poly-Q expansion. Cells expressing full-length mutant protein are susceptible to fewer death stimuli than cells expressing truncated mutant huntingtin.

Animals↗
PubMed2000

Nitric oxide inhibits tumor necrosis factor-alpha-induced apoptosis by reducing the generation of ceramide.

Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-alpha (TNF-alpha) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-alpha, however, ceramide potentiated, whereas NO inhibited, TNF-alpha-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-alpha-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.

Antigens, CD↗
PubMed2000

Continuous exposure to high concentrations of nitric oxide leads to persistent inhibition of oxygen consumption by J774 cells as well as extraction of oxygen by the extracellular medium.

Nitric oxide (NO) plays a key role in many physiological and pathophysiological events, including the control of cell respiration. Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of NO by cells. We have exposed the murine macrophage cell line J774 to high concentrations of NO, such as are generated in some pathological conditions, and determined their effect on oxygen consumption. We observed a persistent inhibition of respiration which was due to a redox-dependent, progressive inhibition of complex I activity. No other enzyme of the respiratory chain was inhibited in this way. At the same time, we detected a paradoxical removal of oxygen by the extracellular medium. This removal was due to a chemical interaction between dissolved oxygen and NO-related species released from cells exposed to NO. A similar removal of oxygen by the cell supernatant also occurred following activation of cells with cytokines and bacterial products. Thus, the amounts of NO generated during pathological conditions may contribute to tissue hypoxia both by inhibiting cell respiration and by promoting removal of oxygen from the extracellular medium.

Animals↗
PubMed2000

P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells.

ATP-induced Ca2+ transients were examined in individual PC12 cells of a well defined clone, before and after treatment with nerve growth factor (NGF) to induce a neurone-like phenotype. Using reverse transcriptase PCR these cells were found to express mRNA for several P2 receptors. In undifferentiated cells the ATP-induced Ca2+ response was entirely dependent on Ca2+ influx, could not be mimicked by UTP, alpha,beta-methylene ATP or dibenzoyl ATP or be blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). ATP had no significant effect on levels of cyclic AMP or inositol 1,4,5-trisphosphate (InsP3). These results suggest that in undifferentiated PC12 cells ATP mainly acts on a P2X receptor, possibly the P2X4 subtype. After treatment with NGF for 7 days the ATP response was increased and partially sensitive to PPADS. A component of the ATP-induced Ca2+ increase was due to mobilisation of intracellular Ca2+ stores and another to capacitative Ca2+ entry. UTP caused an increase in intracellular Ca2+, and InsP3 formation could be stimulated by ATP and UTP. ATP also caused a small increase in cyclic AMP, but this was abolished in the presence of indomethacin. Thus, after NGF treatment ATP acts partially via a P2Y receptor, possibly the P2Y2 subtype.

Adenosine Triphosphate↗
PubMed2000

Oxidative stress and S-nitrosylation of proteins in cells.

The effect of prolonged exposure to nitric oxide on enzymes involved in cell metabolism was investigated in T lymphocyte-derived Jurkat and L929 fibroblast human cell lines using a constant concentration of nitric oxide (1.5 microM) released by the nitric oxide donor DETA-NO (0.5 mM). Nitric oxide inhibited immediately the respiration of the cells acting reversibly at complex IV. With time, the inhibition became progressively persistent, i.e. not reversed by trapping of nitric oxide with oxyhaemoglobin, and was preceded by a decrease in the concentration of the intracellular reduced glutathione. This persistent effect of nitric oxide on respiration was due to inhibition of complex I activity which could be reversed by addition of reduced glutathione or by cold light, suggesting that it was due to S-nitrosylation of thiols necessary for the activity of the enzyme. The activity of other enzymes also known to be susceptible to inhibition by S-nitrosylation, i.e. glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase, was progressively decreased by exposure to nitric oxide with a similar time course to that observed for the inhibition of complex I. Furthermore, inhibition of these enzymes only occurred when the concentrations of reduced glutathione had previously fallen and could be prevented by increasing the intracellular concentrations of reduced glutathione. Our results suggest that S-nitrosylation of different enzymes by nitric oxide may occur only if the reducing potential of the cells is impaired.

Cell Line↗
PubMed2000

Endogenous and exogenous nitric oxide enhance the DNA strand scission induced by tert-butylhydroperoxide in PC12 cells via peroxynitrite-dependent and independent mechanisms, respectively.

A short-term exposure to tert-butylhydroperoxide (tB-OOH) promoted a concentration-dependent formation of DNA single-strand breaks in PC12 cells. These events were paralleled by an increase in the cytosolic concentration of Ca2+ that was in part cleared by the mitochondria. Unlike the extent of Ca2+ mobilization and/or mitochondrial Ca2+ clearance, the DNA strand scission evoked by the hydroperoxide was markedly reduced by the nitric oxide (NO) scavenger 2-phenyl-4,4,5,5-tetramethylimidazolin-1-oxyl-3-oxide (PTIO) or by the NO synthase inhibitor N-nitro-L-arginine methylester (L-NAME). Inhibitors of electron transport (rotenone and myxothiazol), ruthenium red (RR, a polycation which inhibits the calcium uniporter of mitochondria), or peroxynitrite scavengers (Trolox and L-methionine) were as effective as PTIO or L-NAME in inhibiting the DNA-damaging response mediated by tB-OOH. Rotenone, RR or peroxynitrite scavengers did not further reduce the residual DNA cleavage observed following treatment with tB-OOH in L-NAME-supplemented cells. Exogenous NO also increased the DNA damage caused by tB-OOH in L-NAME-supplemented cells and this response was blunted by RR or by inhibitors of electron transport but was insensitive to peroxynitrite scavengers. We conclude that both endogenous and exogenous NO enhance the DNA cleavage generated by tB-OOH in PC12 cells. However, only endogenous NO set the bases for an involvement of peroxynitrite in this DNA-damaging response.

Animals↗
PubMed1999

Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.

Animals↗
PubMed1999

The p75(NTR)-induced apoptotic program develops through a ceramide-caspase pathway negatively regulated by nitric oxide.

SK-N-BE neuroblastoma cell clones transfected with p75(NTR) and lacking Trk neurotrophin receptors, previously reported to undergo extensive spontaneous apoptosis and to be protected by nerve growth factor (NGF) (Bunone, G., Mariotti, A., Compagni, A., Morandi, E., and Della Valle, G. (1997) Oncogene 14, 1463-1470), are shown to exhibit (i) increased levels of the pro-apoptotic lipid metabolite ceramide and (ii) high activity of caspases, the proteases of the cell death cascade. In the p75(NTR)-expressing cells, these parameters were partially normalized by prolonged NGF treatment, which, in addition, decreased apoptosis, similar to caspase blockers. Conversely, exogenous ceramide increased caspase activity and apoptosis in both wild-type and p75(NTR)-expressing cells. A new p75(NTR)-expressing clone characterized by low spontaneous apoptosis exhibited high endogenous ceramide and low caspase levels. A marked difference between the apoptotic and resistant clones concerned the very low and high activities of nitric-oxide (NO) synthase, respectively. Protection from apoptosis by NO was confirmed by results with the NO donor S-nitrosoacetylpenicillamine and the NO-trapping agent hemoglobin. We conclude that the p75(NTR) receptor, while free of NGF, triggers a cascade leading to apoptosis; the cascade includes generation of ceramide and increased caspase activity; and the protective role of NO occurs at step(s) in between the latter events.

Apoptosis↗