RESUMO
Kawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19(+) B lymphocytes and CD19(+) /CD86(+) activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-α production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19(+) B cells, the number of IgA-, IgM- and IgG-SC and the production of IL-6 and TNF-α. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-α inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD.
Assuntos
Células Produtoras de Anticorpos/metabolismo , Células Produtoras de Anticorpos/patologia , Imunoglobulina A/metabolismo , Síndrome de Linfonodos Mucocutâneos/imunologia , Receptor Toll-Like 9/agonistas , Células Produtoras de Anticorpos/efeitos dos fármacos , Antígenos CD19/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Pré-Escolar , Feminino , Humanos , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Imunoglobulinas Intravenosas/uso terapêutico , Lactente , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Células Matadoras Naturais/patologia , Contagem de Linfócitos , Masculino , Síndrome de Linfonodos Mucocutâneos/terapia , Oligodesoxirribonucleotídeos/farmacologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Although apoptosis is considered one of the major mechanisms of CD4(+) T cell depletion in HIV-infected patients, the virus-infected cells somehow appear to be protected from apoptosis, which generally occurs in bystander cells. Vpr is an auxiliary HIV-1 protein, which, unlike the other regulatory gene products, is present at high copy number in virus particles. We established stable transfectants of CD4+ T Jurkat cells constitutively expressing low levels of vpr. These clones exhibited cell cycle characteristics similar to those of control-transfected cells. Treatment of control clones with apoptotic stimuli (i.e., cycloheximide/tumor necrosis factor alpha (TNF-alpha), anti-Fas antibody, or serum starvation) resulted in a massive cell death by apoptosis. In contrast, all the vpr-expressing clones showed an impressive protection from apoptosis independently of the inducer. Notably, vpr antisense phosphorothioate oligodeoxynucleotides render vpr-expressing cells as susceptible to apoptosis induced by cycloheximide and TNF-alpha as the control clones. Moreover, the constitutive expression of HIV-1 vpr resulted in the upregulation of bcl-2, an oncogene endowed with antiapoptotic activities, and in the downmodulation of bax, a proapoptotic factor of the bcl-2 family. Altogether, these results suggest that low levels of the endogenous vpr protein can interfere with the physiological turnover of T lymphocytes at early stages of virus infection, thus facilitating HIV persistence and, subsequently, viral spread. This might explain why apoptosis mostly occurs in bystander uninfected cells in AIDS patients.
Assuntos
Síndrome da Imunodeficiência Adquirida/etiologia , Apoptose/genética , Linfócitos T CD4-Positivos/metabolismo , Produtos do Gene vpr/metabolismo , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular , Cicloeximida/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Humanos , Immunoblotting , Células Jurkat/citologia , Células Jurkat/virologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/análise , Transfecção/genética , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Virais/análise , Proteínas Virais/metabolismo , Proteína X Associada a bcl-2 , Receptor fas/imunologia , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Subcellular organelles such as mitochondria, endoplasmic reticulum (ER) and the Golgi complex are involved in the progression of the cell death programme. We report here that soon after ligation of Fas (CD95/Apo1) in type II cells, elements of the Golgi complex intermix with mitochondria. This mixing follows centrifugal dispersal of secretory membranes and reflects a global alteration of membrane traffic. Activation of apical caspases is instrumental for promoting the dispersal of secretory organelles, since caspase inhibition blocks the outward movement of Golgi-related endomembranes and reduces their mixing with mitochondria. Caspase inhibition also blocks the FasL-induced secretion of intracellular proteases from lysosomal compartments, outlining a novel aspect of death receptor signalling via apical caspases. Thus, our work unveils that Fas ligand-mediated apoptosis induces scrambling of mitochondrial and secretory organelles via a global alteration of membrane traffic that is modulated by apical caspases.
Assuntos
Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Receptores de Morte Celular/metabolismo , Receptor fas/metabolismo , Apoptose , Caspases/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proteína Ligante Fas/metabolismo , Células HeLa , Humanos , Células Jurkat , Ligantes , Lisossomos/enzimologia , Organelas/metabolismo , Peptídeo Hidrolases/metabolismo , Transdução de SinaisRESUMO
Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.
Assuntos
Apoptose , Toxinas Bacterianas/farmacologia , Citotoxinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Proteínas de Escherichia coli , Pinocitose/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Compartimento Celular , Células Cultivadas , Citotoxinas/genética , Citotoxinas/metabolismo , Endossomos , Ativação Enzimática , Células Epiteliais/fisiologia , Humanos , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/fisiologia , Células Tumorais Cultivadas , Células U937 , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Cytoskeletal components play a major role in the human immunodeficiency virus-1 (HIV-1) infection. A wide variety of molecules belonging to the microfilament system, including actin filaments and actin binding proteins, as well as microtubules have a key role in regulating both cell life and death. Cell shape maintenance, cell polarity and cell movements as well as cytoplasmic trafficking of molecules determining cell fate, including apoptosis, are in fact instructed by the cytoskeleton components. HIV infection and viral particle production seem to be controlled by cytoskeleton as well. Furthermore, HIV-associated apoptosis failure can also be regulated by the actin network function. In fact, HIV protein gp120 is able to induce cytoskeleton-driven polarization, thus sensitizing T cells to CD95/Fas-mediated apoptosis. The microfilament system seems thus to be a sort of cytoplasmic supervisor of the viral particle, the host cell and the bystander cell's very fate.
Assuntos
Citoesqueleto de Actina/virologia , Apoptose , Produtos do Gene nef/fisiologia , Produtos do Gene tat/fisiologia , Produtos do Gene vpr/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1/fisiologia , Linfócitos T/virologia , Proteínas Virais/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Produtos do Gene nef/farmacologia , Produtos do Gene tat/farmacologia , Produtos do Gene vpr/farmacologia , Proteína gp120 do Envelope de HIV/farmacologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/ultraestrutura , Proteínas Virais/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.
Assuntos
Apoptose/fisiologia , Gangliosídeo G(M3)/metabolismo , Microdomínios da Membrana/metabolismo , Mitocôndrias/metabolismo , Linfócitos T/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Linfócitos T/citologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Proteína X Associada a bcl-2/metabolismo , beta-Ciclodextrinas/farmacologia , Receptor fas/metabolismoRESUMO
The cytotoxic effect of the combination of N-methylformamide (NMF) with 5-fluorouracil (5-FU) on cell survival of the human colon cancer line HT29 was assessed. The differentiating activity of NMF was evidenced by morphological maturation and conversion of cell culture characteristics to those consistent with a more benign phenotype. In combination experiments, the noncytotoxic concentration of 1% NMF was chosen and doses of 5-FU ranging from 5 to 25 micrograms/ml were employed. Two main schedules were tested either on exponentially or stationarily growing cells: (a) 1% NMF for 72 h followed by 12-h exposure to 5-FU; (b) 5-FU for 12 h followed by 72-h exposure to 1% NMF. The results obtained demonstrated that the 5-FU----NMF sequence determined a powerful reduction in the surviving fraction of HT29 cells, while the reverse sequence did not increase the killing effect of 5-FU given alone. Immunocytochemical and scanning electron microscopy studies seemed to confirm that the association in which the differentiating agent followed the 5-FU treatment strongly impaired cellular integrity and function and that cytoskeletal elements, particularly microfilaments, and surface structures could play an essential role in the mechanisms of cytotoxicity. Furthermore, the results of this work indicate that drug sequence is a critical factor for the optimal combination of 5-FU and NMF.
Assuntos
Adenocarcinoma/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/patologia , Fluoruracila/administração & dosagem , Formamidas/administração & dosagem , Adenocarcinoma/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Citoesqueleto/efeitos dos fármacos , Formamidas/farmacologia , Humanos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Type I Interferon (IFN) and all-trans retinoic acid (RA) inhibit cell proliferation of squamous carcinoma cell lines (SCC). Examinations of growth-affected cell populations show that SCC lines ME-180 and SiHa treated with IFN-beta undergo a specific slower progression through the S phase that seems to trigger cellular death. In combination treatment RA potentiates IFN-beta effect in SCC ME-180 but not in SiHa cell line, partially resistant to RA antiproliferative action. RA added as single agent affects cell proliferation differently by inducing a slight G1 accumulation. The IFN-beta-induced S phase lengthening parallels the increased expression of PML, a nuclear phosphoprotein specifically up-regulated at transcriptional level by IFN, whose overexpression induces cell growth inhibition and tumor suppression. We report that PML up-regulation may account for the alteration of cell cycle progression induced by IFN-beta in SCC by infecting cells with PML-PINCO recombinant retrovirus carrying the PML-3 cDNA under the control of the 5' LTR. In fact PML overexpression reproduces the IFN-beta-induced S phase lengthening. These findings provide important insight into the mechanism of tumor suppressing function of PML and could allow PML to be included in the pathways responsible for IFN-induced cell growth suppression.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Interferon Tipo I/farmacologia , Proteínas de Neoplasias/biossíntese , Proteínas Nucleares , Fase S/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Neoplasias do Colo do Útero/patologia , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores do Crescimento/administração & dosagem , Inibidores do Crescimento/farmacologia , Humanos , Interferon Tipo I/administração & dosagem , Proteínas de Neoplasias/genética , Proteína da Leucemia Promielocítica , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Recombinantes , Fatores de Transcrição/genética , Tretinoína/administração & dosagem , Tretinoína/farmacologia , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/metabolismoRESUMO
The possible cytotoxic effects of fusinite, a new charcoal-like electron paramagnetic resonance (EPR) oxygen probe, were evaluated in three cell types with very different characteristics and growth features: K562 (an erythroleukemic cell line which grows in suspension), A431 (an epidermal carcinoma cell line which grows in monolayer) and primary cultures of murine fibroblasts (which also grow in adhesion culture) utilizing morphological and functional studies as well as growth analyses. Scanning and transmission electron microscopy as well as fluorescence microscopy were used for the morphological analyses while conductometric relaxation studies in the radiowave frequency range, membrane resistance measurements and adenine nucleotide levels were utilized for the more subtle functional evaluation of cell parameters. The results show that the presence of fusinite particles, even after long internalization times, does not induce any cytotoxic effects in the cells studied. Thus, from these results, it can be deduced that fusinite is non-toxic as well as highly stable, inert and very sensitive to oxygen, and can be used with great success for cell studies where determination of oxygen concentration is important.
Assuntos
Carbono/toxicidade , Oxigênio/análise , Marcadores de Spin , Nucleotídeos de Adenina/análise , Nucleotídeos de Adenina/metabolismo , Divisão Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Células Cultivadas , Condutividade Elétrica , Espectroscopia de Ressonância de Spin Eletrônica , Microscopia Eletrônica , Microscopia de Fluorescência , Sondas Moleculares/toxicidade , FagocitoseRESUMO
The feasibility of using EPR and the paramagnetic derivative of coal 'fusinite' to measure intracellular oxygen concentration in cultured cells in which this substance was internalized in the cytoplasm was examined. First, the possible cytotoxic effects of fusinite on cultured cells were ruled out by both morphological as well as by growth characteristics analyses. After construction of a calibration curve in which the EPR spectral linewidth of this substance was measured in response to known oxygen concentrations, the efficacy of using fusinite in the determination of intracellular oxygen concentration in cells was also tested by flowing different known oxygen gas mixtures outside cultured cells. The results indicate that fusinite is able of measuring the variations in cytoplasmic oxygen concentration that exist in response to the different gas mixtures. In addition, as an example of a possible use of fusinite, data are also presented demonstrating a decrease in cytoplasmic oxygen concentration during respiration in cells with a limited supply of oxygen. In fact, as the oxygen is consumed by the cells, the linewidth of fusinite narrows giving an intracellular oxygen concentration corresponding to zero. From the results obtained, fusinite appears to represent a new extremely precise biophysical cellular oxygen probe which may prove useful in the understanding of the complex interrelationships between oxygen and normal cell physiology and/or pathology.
Assuntos
Carbono , Carvão Mineral , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Sondas Moleculares , Oxigênio/análise , Divisão Celular , Microscopia Eletrônica , Consumo de OxigênioRESUMO
We have previously shown that CD4+ T Jurkat cells constitutively expressing low levels of the human immunodeficiency virus 1 (HIV-1) vpr protein were less susceptible to undergo apoptosis than control cells.1 In this study we have investigated the role of vpr in affecting mechanisms of importance in the control of apoptosis. Vpr-expressing clones consistently aggregated in clusters with time in culture, whereas mock-transfected cells grew as dispersed cultures. The analysis of adhesion molecules involved in cell-to-cell as well as in cell-substrate interactions showed a higher expression of cadherin and integrins alpha5 and alpha6 in vpr-transfected clones with respect to mock-transfected cells. This up-modulation was specifically blocked by cell exposure to antisense oligonucleotides targeted at the vpr. In addition, F-actin microfilament cytoskeletal organization, known to be involved in cell-cell interaction pathways and in the modulation of cell surface molecule expression, was significantly improved in vpr-expressing clones, in which filament polymerization was increased. We thus envisage that vpr viral protein can maintain cell survival via a specific activity on cytoskeleton-dependent cell adhesion pathways, i.e. by inducing anoikis-resistance. These particular effects of vpr might enhance the homing, spreading and survival of the infected lymphocytes, thus contributing to virus persistence in the course of acute HIV-1 infection.
Assuntos
Citoesqueleto de Actina/fisiologia , Apoptose , Adesão Celular/fisiologia , Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Divisão Celular , Citoplasma/metabolismo , Citoesqueleto , Expressão Gênica , Produtos do Gene vpr/genética , Homeostase , Humanos , Integrina alfa5 , Integrina alfa6 , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiologia , Células Jurkat , Mitocôndrias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transfecção , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Cell-cell and cell-matrix interactions play a pivotal role in numerous cell functions including cell survival and death. In this work, we report evidence that the Rho-dependent cell spreading activated by a protein toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), is capable of hindering apoptosis in HEp-2 cells. In addition to the promotion of cell spreading, CNF1 protects cells from the experimentally-induced rounding up and detachment and improves the ability of cells to adhere to each other and to the extracellular matrix by modulating the expression of proteins related to cell adhesion. In particular, the expression of integrins such as alpha 5, alpha 6 and alpha v, as well as of some heterotypic and homotypic adhesion-related proteins such as the Focal Adhesion Kinase, E-cadherin, alpha and beta catenins were significantly increased in cells exposed to CNF1. Our results suggest, however, that the promotion of Rho-dependent cell spreading is the key mechanism in protecting cells against apoptosis rather than cell adhesion per se. A toxin inducing cell spreading without activating Rho, such as Cytochalasin B, was in fact ineffective in favouring cell survival. These data are of relevance (i) for the understanding of the role of the actin-dependent and especially Rho-dependent cellular activities involved in apoptosis regulation and (ii) in providing some clues to understanding the mechanisms by which bacteria, by controlling cell fate, might exert their pathogenic activity.
Assuntos
Apoptose/fisiologia , Toxinas Bacterianas/farmacologia , Adesão Celular/fisiologia , Citotoxinas/farmacologia , Proteínas de Escherichia coli , Proteínas de Ligação ao GTP/fisiologia , Actinas/metabolismo , Antígenos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Adesão Celular/efeitos dos fármacos , Adesão Celular/efeitos da radiação , Linhagem Celular , Citocalasina B/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/efeitos da radiação , Escherichia coli/patogenicidade , Humanos , Microscopia Eletrônica de Varredura , Raios Ultravioleta , Proteínas rho de Ligação ao GTPRESUMO
CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.
Assuntos
Apoptose/fisiologia , Proteína gp120 do Envelope de HIV/toxicidade , Fosfoproteínas/metabolismo , Linfócitos T/metabolismo , Receptor fas/metabolismo , Apoptose/efeitos dos fármacos , Antígenos CD4/metabolismo , Proteínas do Citoesqueleto , Humanos , Interleucina-2/metabolismo , Ativação Linfocitária/fisiologia , FosforilaçãoRESUMO
The aim of this study was to analyze (i) phenotype, (ii) in vitro spontaneous and induced apoptosis, (iii) glutathione (GSH) intracellular content and (iv) inhibitors of apoptosis of potential therapeutical use in peripheral blood mononuclear cells (PBMC) from HIV+ long term non progressors (LTNP), in comparison with progressors (HIV+P) and seronegative controls (HIV-). Three groups of subjects were studied: 15 HIV+P (patients losing >150 CD4+/year), 9 LTNP (subjects infected by HIV for at least 7 years without clinical and immunological signs of progression, with a mean of 898 CD4+/microL) and 18 HIV-. All subjects were living in a large community for former drug addicts, and were matched for age and sex. We used flow cytometry for analyzing PBMC phenotype and apoptosis; high performance liquid chromatography for measuring intracellular GSH content. PBMC phenotype of LTNP shared characteristics with those of both HIV- and HIV+P. Indeed, LTNP showed a normal number CD4+ cells (an inclusion criteria), but significantly increased numbers of CD8+ lymphocytes, activated T cells, CD19+, CD5+ B lymphocytes and CD57+ cells, as well as a decrease in CD19+, CD5- B lymphocytes and CD16+ cells. In LTNP, spontaneous apoptosis was similar to that of HIV- and significantly lower than that of HIV+P. Adding interleukin-2 (IL-2) or nicotinamide (NAM) significantly decreased spontaneous apoptosis in LTNP and HIV+P. Pokeweed mitogen-induced apoptosis was also similar in LTNP and HIV-, but significantly lower than that of HIV+P. In HIV+P, but also in LTNP, spontaneous apoptosis was inversely correlated to the absolute number and percentage of CD4+ cells and directly correlated to the number and percentage of activated T cells present in peripheral blood. GSH intracellular content was greatly decreased in PBMC from HIV+P and slightly, but significantly, reduced in LTNP. Adding 2-deoxy-D-ribose, an agent provoking apoptosis through GSH depletion, to quiescent PBMC resulted in similar levels of massive cell death in the three groups. This phenomenon was equally prevented in the three groups by N-acetyl-cysteine but not by IL-2. A complex immunological situation seems to occur in LTNP. Indeed, PBMC from LTNP are characterized by a normal in vitro tendency to undergo apoptosis despite the presence of a strong activation of their immune system, unexpectedly similar to that of HIV+P. Our data suggest that NAM and IL-2 are possible candidates for reducing spontaneous apoptosis in HIV infection.
RESUMO
Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, which acts as a free-radical scavenger, was investigated as a protective agent against ultraviolet-light-induced damage in human keratinocytes. First, we demonstrate that carboxyfullerene is not cytotoxic for these cells. In addition, this compound significantly reduces the ultraviolet-B-induced inhibition of keratinocyte proliferation and protects keratinocytes from apoptosis caused by ultraviolet B irradiation in a time- and dose-dependent fashion. Furthermore, the percentage of cells with depolarized mitochondria is significantly lower in ultraviolet-B-irradiated keratinocytes pretreated with carboxyfullerene than in cells provided with diluent alone. Carboxyfullerene also protects human keratinocytes from apoptosis induced by exposure to deoxy-D-ribose, a sugar that causes cell death through a pathway involving oxidative stress. On the other hand, ultraviolet B downregulates bcl-2 levels in human keratinocytes, and carboxyfullerene fails to prevent this effect. These results suggest that carboxy- fullerene protects human keratinocytes from ultraviolet B damage possibly via a mechanism interfering with the generation of reactive oxygen species from depolarized mitochondria without the involvement of bcl-2.
Assuntos
Carbono/farmacologia , Ácidos Carboxílicos/farmacologia , Fulerenos , Queratinócitos/citologia , Protetores contra Radiação/farmacologia , Raios Ultravioleta , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Desoxirribose/farmacologia , Citometria de Fluxo , Humanos , Membranas Intracelulares/fisiologia , Potenciais da Membrana , Mitocôndrias , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Apoptosis or programmed cell death (PCD) is a type of death occurring in various physiological processes. Several data suggest that: (1) apoptosis may play a critical role in AIDS pathogenesis; (2) an increase of endocellular free radical levels can be associated with activation of previously latent HIV virus. Tumor necrosis factor (TNF), a cytokine capable of inducing oxygen free radicals and apoptosis, appears also to be involved in HIV activation. The present findings, which elucidate a relationship between the percentage of apoptotic cells, reduced glutathione (GSH) depletion and an increase of p24 antigenemia, suggest that pretreatment with N-acetylcysteine (NAC) is capable of decreasing the above-mentioned phenomena in HIV-infected U937 cells.
Assuntos
Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Bisbenzimidazol , Morte Celular/efeitos dos fármacos , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Glutationa/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/crescimento & desenvolvimento , Humanos , Leucócitos Mononucleares/microbiologia , Microscopia de Fluorescência , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Toxins A and B from the anaerobic bacterium Clostridium difficile are the causative agents of the antibiotic-associated pseudomembraneous colitis. At the subcellular level, they inhibit the Rho family GTPases, thus causing alterations of the actin cytoskeleton. The cytoskeletal integrity is also controlled by the redox state of cells. Therefore, we have evaluated whether an oxidative imbalance could be involved in the toxin-induced cytopathic effects. Our results indicate that both toxins induce oxidative stress with a significant depletion of protein SH-groups. These responses and the cytoskeleton-dependent cell retraction and rounding are significantly counteracted by N-acetylcysteine but not by alpha-tocopherol. Our study provides the first evidence that the thiol supplier N-acetylcysteine impairs the cellular intoxication by acting on the cytoskeleton integrity. This also suggests a possible beneficial role for this drug during therapeutic intervention.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Proteínas de Bactérias , Toxinas Bacterianas/toxicidade , Enterotoxinas/toxicidade , Células Epiteliais/efeitos dos fármacos , Glutationa/metabolismo , Cromanos/farmacologia , Citoesqueleto/efeitos dos fármacos , Células Epiteliais/patologia , Humanos , Oxirredução , Vitamina E/farmacologiaRESUMO
It has been hypothesized that programmed cell death (PCD), an active cell suicide process occurring in place of necrosis, can be associated with the pathogenesis of acquired immunodeficiency syndrome (AIDS). The entry of human immunodeficiency virus (HIV) into competent cells is mediated by the CD4 molecule present on the surface of certain lymphocyte subpopulations as well as on some cultured cell lines, e.g. U937 myelomonocytic cells. The present paper focuses on some specific aspects of PCD induced by the cytokine tumor necrosis factor (TNF). The results obtained indicate that the exposure of U937 cells to cycloheximide facilitates TNF-mediated PCD via a short term cell death program and modifies the expression of CD4 surface molecules. This change in surface antigen expression, manifested by internalization of the CD4 molecule, occurs in cells in which apoptosis has been triggered, but not in cells undergoing necrosis. These results indicate that the progression of cell death could be associated with specific alterations of certain surface molecules and could have a role in the entry of HIV into cells.
Assuntos
Apoptose , Antígenos CD4/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Membrana Celular/imunologia , Cicloeximida/farmacologia , Regulação para Baixo , HIV/fisiologia , Humanos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Galectins are a family of proteins involved in several cell processes, including their survival and death. Galectin-3 has in particular been described as an anti-apoptotic molecule entangled with a number of subcellular activities including anoikis resistance. In this work we partially address the mechanisms underlying this activity pointing at two key factors in injury progression: the alteration of mitochondrial membrane potential and the formation of reactive oxygen species. Overexpression of galectin-3 appears in fact to exert a protective effect towards both these events. On the basis of these data, we propose a reappraisal of the role of galectin-3 as a regulator of mitochondrial homeostasis.
Assuntos
Antígenos de Diferenciação/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Morte Celular/fisiologia , Feminino , Galectina 3 , Homeostase , Humanos , Membranas Intracelulares/fisiologia , Potenciais da Membrana , Microscopia Eletrônica de Varredura , Mitocôndrias/ultraestrutura , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Vitamina K/farmacologiaRESUMO
The intracellular molecular oxygen concentration in control and menadione-treated K562 (an erythroleukemic cell line that grows in suspension) and A431 (an epidermal carcinoma that grows in monolayer) cells was measured directly by using the new electron paramagnetic resonance (EPR) probe fusinite. Because the oxidizing agent menadione is known to damage mitochondria and the cytoplasmic membrane in other cell systems, before conducting measurements of oxygen concentration in K562 and A431 cells, it was necessary to establish injury in these systems as well. Consequently, morphological and flow cytometric analyses were conducted after menadione treatment. The data presented here show that the two cell lines are heavily damaged by menadione. Once this menadione-induced injury was demonstrated, measurements of oxygen concentration were carried out in both K562 and A431 cells. Treatment with this quinone induces a sharp increase in intracytoplasmic molecular oxygen in both cell lines (from about 1% to about 10 and 15% in K562 and A431 cells, respectively). In addition, to gain a more complete understanding of the effects of menadione on cells, the extracellular molecular oxygen concentration and the oxygen consumption rate were also measured in control and menadione-treated K562 cells. These measurements demonstrate that menadione treatment results in an increase in the extracellular oxygen concentration (from about 5% in controls to 15% in treated cells) as well as a decrease in the oxygen consumption rate (from about 10 ng O/min/10(6) cells in controls to 3 ng O/min/10(6) cells after menadione exposure). The importance of the new EPR probe fusinite in monitoring directly cellular functions in which oxygen is involved and the effects of menadione on cellular oxygen balance are discussed.