RESUMEN

We have recently found that D(-)lentiginosine, a synthetic iminosugar exerting glucosidase inhibitory activity, but not its natural enantiomer lentiginosine, is endowed with an unexpected, pro-apoptotic activity. Here, we investigated mechanisms involved in apoptosis induced by D(-)lentiginosine in MOLT-3, HT-29 and SH-SY5Y tumour cell lines. The results showed that D(-)lentiginosine increased caspase 9 expression at 18 h in all the cell lines from 1.5-3.1 folds. Cytochrome c in the cytoplasm was found to be increased from 2.3-2.6 folds in treated cells with respect to control cells. These effects were accompanied by a remarkable collapse of the mitochondrial membrane potential and by the downregulation of anti-apoptotic genes, as well as the upregulation of pro-apoptotic genes of the Bcl-2 family. U937Bcl-2 transfectants, highly expressing Bcl-2, were reluctant to undergo apoptosis even following treatment with 500 µM D(-)lentiginosine, whereas apoptosis by D(-)lentiginosine was induced also in U937 cells, naturally deficient in P53. Thus, our study establishes that the enantiomer of a natural iminosugar is endowed with a possible anti-tumorigenic effect that might be ascribed not only to their capacity to inhibit glycosidases but also to other unknown mechanisms. These data encourage further investigation on similar compounds to make them an interesting platform for the generation of new anticancer drugs.

Asunto(s)

Alcaloides/farmacología , Apoptosis/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Células HT29 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Estereoisomerismo , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

In this study, we investigated molecular mechanisms underlying low susceptibility to apoptosis induced by the nucleoside analog azidothymidine (AZT) and the role of nuclear factor-κB (NF-κB) activation in these phenomena. A preliminary screening in different cell lines indicated U937 monocytic cell line as suitable to this purpose. Treatment of U937 cells even with suprapharmacological concentrations of AZT induced only moderate levels of apoptosis. Surprisingly, SuperArray analysis showed that AZT induced the transcriptional activity of both pro- and anti-apoptotic genes. Interestingly, moreover, several genes upregulated by AZT were NF-κB related. In fact, AZT, after an initial inhibition of NF-κB activation with respect to control, induced a transient, but consistent, increase in NF-κB-binding activity. Inhibition of NF-κB activation in U937 cells, stably transfected with a dominant-negative IκBα or by pharmacological treatment, sensitized them to apoptosis induced by AZT and impaired the upregulation of anti-apoptotic genes in response to AZT treatment, with respect to control cells. These results indicate that NF-κB activation by AZT has a role in protecting target cells from apoptotic cell death, improving our understanding of the toxicology and the therapeutic usage of this drug.

Asunto(s)

Antimetabolitos/farmacología , Apoptosis , FN-kappa B/metabolismo , Zidovudina/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , FN-kappa B/antagonistas & inhibidores , Análisis de Secuencia por Matrices de Oligonucleótidos , Células U937

RESUMEN

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

Asunto(s)

Transformación Celular Viral , Retrovirus Endógenos/fisiología , Melanoma/virología , Activación Viral/fisiología , Células CACO-2 , Proliferación Celular , Transformación Celular Viral/genética , Células Cultivadas , Células Clonales/virología , Progresión de la Enfermedad , Retrovirus Endógenos/genética , Humanos , Células Jurkat , Células K562 , Melanocitos/patología , Melanocitos/ultraestructura , Melanocitos/virología , Melanoma/etiología , Melanoma/genética , Melanoma/patología , Modelos Biológicos , ARN Viral/aislamiento & purificación , Virión/crecimiento & desarrollo , Activación Viral/genética

Asunto(s)

Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Animales , Compuestos de Bifenilo/metabolismo , Compuestos de Bifenilo/farmacología , Humanos , Mitocondrias/metabolismo , Nitrofenoles/metabolismo , Nitrofenoles/farmacología , Piperazinas/metabolismo , Piperazinas/farmacología , Proteínas Quinasas/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo

RESUMEN

BACKGROUND AND OBJECTIVE: Cord blood obtained at delivery can be used for hematopoietic precursor cells (HPC) transplantation. The major limit for its success is represented by the low cellular yield of the stem cell population. The objective of this study was to determine the role played by apoptosis in the numerical control of CD34+ cell counts. DESIGN AND METHODS: Umbilical cord blood samples were collected from 15 women at the time of the delivery and cord blood units processed. Cells, collected following 24h and 48h of incubation, were analysed by flow cytometry using the gating strategy. RESULTS: Remarkable levels of apoptosis were detected in the stem cell population and a significant difference between apoptosis mean values at 24h and 48h within CD34+ cells were found. The difference between the percentage of apoptosis in CD34+ cells and that in the remaining population was significant both at 24h and at 48h. CONCLUSIONS: CD34+ cells have a higher likelihood to undergo apoptosis in comparison to the remaining ones present in umbilical cord blood. This process of cellular death plays a major role in the control of CD34+ cell counts in placental blood and influence, for this reason, the possibility of success of a cord blood transplantation.

Asunto(s)

Antígenos CD34/sangre , Apoptosis/fisiología , Sangre Fetal/fisiología , Células Madre Hematopoyéticas/fisiología , Adulto , Anexina A5/metabolismo , Separación Celular , Células Cultivadas , Femenino , Sangre Fetal/citología , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Humanos , Leucocitos Mononucleares/citología , Recuento de Linfocitos , Fosfatidilserinas/análisis , Fosfatidilserinas/metabolismo , Embarazo , Factores de Tiempo

RESUMEN

Antiretrovirals have progressively entered clinical practice since the discovery of the association between the acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) infection. Among the classes of drugs which have shown efficacy against HIV, nucleoside-based reverse transcriptase inhibitors (NRTIs) have been extensively investigated in both their basic and therapeutic aspects. The basic mechanism of the effects of NRTIs relies on interaction with both viral and host cell functions. This implies that NRTIs could act not only by inhibiting viral genome replication, but also by interfering with some basic mechanism regulating cell growth and death. According to these characteristics, NRTIs might share several similarities with antineoplastic agents, including side effects. In this article, we have briefly reviewed the pharmacological activities of NRTIs in viral replication, neoplastic growth and cellular functions. These aspects might be involved both in the beneficial and adverse effects of NRTIs.

Asunto(s)

Nucleósidos/farmacología , Nucleósidos/uso terapéutico , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Farmacorresistencia Viral/fisiología , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nucleósidos/química , Inhibidores de la Transcriptasa Inversa/química

RESUMEN

In the present transectional study, Fas ligand (Fas-L) levels, either in membrane or in soluble form, in cells from multiple sclerosis (MS) patients were investigated. Expression of Fas was evaluated after PHA stimulation of peripheral blood mononuclear cells from MS patients with relapsing-remitting or secondary-progressive disease, and in healthy donors. There was statistically significant decreased expression (p = 0.001), as well as release of Fas-L, (p = 0.045) in lymphocytes from MS patients, in comparison with healthy donors. Moreover, levels of Fas-L production were inversely correlated with the EDSS scores of patients in an highly significant way. Impairment of Fas-L release in stimulated PBMC from MS patients might influence the ability to eliminate autoreactive clones in vivo.

Asunto(s)

Linfocitos/metabolismo , Glicoproteínas de Membrana/biosíntesis , Esclerosis Múltiple Crónica Progresiva/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Adulto , Anciano , Proteína Ligando Fas , Femenino , Humanos , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Crónica Progresiva/metabolismo , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Fitohemaglutininas/farmacología

RESUMEN

The present study investigated the effect of 3'-azido 3'deoxythymidine (AZT) treatment on in vitro infection of human cord blood mononuclear cells (CBMCs) exposed to HTLV-1 by cocultivation with the MT-2 cell line. Cultures of CBMCs were grown in IL-2 and were either left untreated or were treated with concentrations of AZT ranging from 0.0078 to 32 microM. HTLV-1-infected cultures were monitored at different times of culture by evaluating proliferation activity, cell growth and the presence and expression of HTLV-1 genes. Results showed that untreated cultures infected with HTLV-1 were able to grow for several weeks, while those treated with AZT at 0.03 microM or higher concentrations were limited in their growth capacity. Moreover, the addition of AZT at the moment of infection significantly inhibited cell proliferation in a dose-dependent fashion. In the presence of AZT, detection of proviral DNA and, more remarkably, viral RNA expression were clearly reduced. In addition, treatment with AZT resulted in a noticeable decrease in Tax protein expression. Using treatment with relatively low doses of AZT, effective in exerting an antiviral action, cytotoxicity on CBMCs was not observed, whereas higher doses induced apoptosis in uninfected CBMCs. These data show that CBMCs are protected by AZT against HTLV-1 transmission even at low, non-toxic doses.

Asunto(s)

Virus Linfotrópico T Tipo 1 Humano/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/virología , Zidovudina/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Técnicas de Cocultivo , ADN Viral/análisis , Relación Dosis-Respuesta a Droga , Sangre Fetal , Productos del Gen tax/biosíntesis , Virus Linfotrópico T Tipo 1 Humano/aislamiento & purificación , Virus Linfotrópico T Tipo 1 Humano/fisiología , Humanos , Provirus/aislamiento & purificación , ARN Viral/análisis , Replicación Viral/efectos de los fármacos

Asunto(s)

Apoptosis , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , ARN Viral/sangre , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Estudios de Cohortes , Estudios de Seguimiento , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/genética , Humanos , Estudios Longitudinales , Carga Viral

RESUMEN

Nuclear phosphoprotein HMGA1a, high mobility group A1a, (previously HMGI) has been investigated during apoptosis. A change in the degree of phosphorylation of HMGA1a has been observed during apoptosis induced in four leukemic cell lines (HL60, K562, NB4, and U937) by drugs (etoposide, camptothecin) or herpes simplex virus type-1. Both hyper-phosphorylation and de-phosphorylation of HMGA1a have been ascertained by liquid chromatography-mass spectrometry. Hyper-phosphorylation (at least five phosphate groups/HMGA1a molecule) occurs at the early apoptotic stages and is probably related to HMGA1a displacement from DNA and chromatin release from the nuclear scaffold. De-phosphorylation (one phosphate or no phosphate groups/HMGA1a molecule) accompanies the later formation of highly condensed chromatin in the apoptotic bodies. We report for the first time a direct link between the degree of phosphorylation of HMGA1a protein and apoptosis according to a process that involves the entire amount of HMGA1a present in the cells and, consequently, whole chromatin. At the same time we report that variously phosphorylated forms of HMGA1a protein are also mono-methylated.

Asunto(s)

Apoptosis , Proteínas del Grupo de Alta Movilidad/metabolismo , Leucemia/metabolismo , Leucemia/patología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteína HMGA1a , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Datos de Secuencia Molecular , Fosforilación , Factores de Transcripción/genética , Células Tumorales Cultivadas

RESUMEN

Increasing evidence suggests that regulation of apoptosis in infected cells is associated with several viral infections. The gammaherpesvirus bovine herpesvirus 4 (BHV-4) has been shown to harbor genes with antiapoptotic potentialities. However, here we have demonstrated that productive infection of adherent, permissive cell lines by BHV-4 resulted in a cytopathic effect characterized by induction of apoptosis. This phenomenon was confirmed using different techniques to detect apoptosis and using different virus strains and cell targets. Apoptosis induced by BHV-4 was inhibited by (1) treatment with doses of heparin, which completely inhibited virus attachment and infectivity; (2) UV treatment, which completely abrogated infectivity; and (3) treatment with a dose of phosphonoacetic acid, which blocked virus replication. Virus-induced apoptosis was associated with a down-regulation of Bcl-2 expression and was reduced by Z-VAD-FMK, but not by Z-DEVD-FMK (caspase-3-specific) caspase inhibitors. Inhibition of apoptosis by Z-VAD-FMK treatment during infection did not modify virus yield. Therefore, despite the presence of antiapoptotic genes in its genoma, BHV-4 could complete its cycle of productive infection while inducing apoptosis of infected cells. This finding might have implications for the pathobiology of BHV-4 and other gammaherpesviruses in vivo.

Asunto(s)

Apoptosis , Gammaherpesvirinae/fisiología , Replicación Viral/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Bovinos , Enfermedades de los Bovinos/virología , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Femenino , Gammaherpesvirinae/efectos de los fármacos , Genes bcl-2/efectos de los fármacos , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Mastitis Bovina/virología , Oligopéptidos/farmacología , Ácido Fosfonoacético/farmacología , Trastornos Puerperales/veterinaria , Trastornos Puerperales/virología , Replicación Viral/efectos de los fármacos

RESUMEN

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces < 10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death. Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.

Asunto(s)

Apoptosis/fisiología , Astrocitos/patología , Comunicación Celular/fisiología , VIH , Macrófagos/virología , Receptor fas/fisiología , Fármacos Anti-VIH/farmacología , Apoptosis/efectos de los fármacos , Células Cultivadas , Proteína Ligando Fas , Productos del Gen tat/fisiología , Proteína gp120 de Envoltorio del VIH/fisiología , Homeostasis , Humanos , Macrófagos/patología , Masculino , Glicoproteínas de Membrana/fisiología , Persona de Mediana Edad , Necrosis , Zidovudina/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

To test whether Helicobacter pylori may contribute to the inflammatory response following myocardial infarction, the levels of IgG antibodies to Helicobacter pylori and some parameters of leukocyte activity were measured in 63 patients and 61 comparable controls. Helicobacter pylori-positive patients showed a significantly higher expression of the adhesion molecule LFA-1 on neutrophils than Helicobacter pylori-negative patients (433+/-29.0 vs. 398.8+/-38.9 mean fluorescence channels; P<0.0001), whereas no significant difference for any parameters tested was found in control subjects. These data suggest a role of Helicobacter pylori in inducing a leukocyte response following myocardial infarction.

Asunto(s)

Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Leucocitos/inmunología , Infarto del Miocardio/inmunología , Anciano , Anticuerpos Antibacterianos/sangre , Estudios de Casos y Controles , Femenino , Infecciones por Helicobacter/microbiología , Humanos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Masculino , Persona de Mediana Edad , Infarto del Miocardio/complicaciones , Infarto del Miocardio/microbiología

RESUMEN

OBJECTIVE: The aim of this study was to investigate susceptibility to spontaneous or anti-Fas-induced apoptosis in peripheral blood mononuclear cells (PBMC) from HIV-positive patients before and during highly active anti-retroviral therapy (HAART). DESIGN: A longitudinal study was performed on 12 evaluable patients on HAART. This cohort was analysed prior to and at week 2, 4, 8, 16 and 24 after beginning HAART. Variations in CD4 and CD8 cells, viral load, susceptibility to spontaneous or anti-Fas-induced apoptosis in the presence of IL-2, IL-4 or IL-12 were studied. Expression of Fas and Bcl-2 were also assessed. METHODS: Levels of HIV RNA were determined by a quantitative reverse transcription-PCR assay. Apoptosis was evaluated by staining isolated nuclei with propidium iodide followed by multiparameter flow cytometry analysis. RESULTS: Spontaneous apoptosis of PBMC was promptly inhibited after the start of treatment. Similarly, anti-Fas-induced apoptosis diminished greatly during treatment. Expression of Fas decreased significantly, while that of Bcl-2 remained statistically unchanged during the first 24 weeks of therapy. Levels of apoptosis correlated inversely to CD4 cell counts and directly to viral load in a highly significant way. Expression of Fas was directly correlated to apoptosis. Interleukin (IL)-2, but not IL-4 or IL-12, protected PBMC of HIV-positive individuals from spontaneous or anti-Fas-induced apoptosis before and during HAART. CONCLUSION: These results suggest that regulation of apoptosis and of Fas expression are involved in immunoreconstitution during HAART.

Asunto(s)

Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Apoptosis , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Linfocitos/fisiología , Adulto , Anciano , Recuento de Linfocito CD4 , Células Cultivadas , Femenino , Humanos , Interleucinas/farmacología , Estudios Longitudinales , Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiología , Carga Viral , Receptor fas/inmunología , Receptor fas/metabolismo

Asunto(s)

Anticuerpos Antibacterianos/sangre , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/inmunología , Técnicas para Inmunoenzimas , Pruebas de Fijación de Látex , Adulto , Anciano , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Humanos , Persona de Mediana Edad , Juego de Reactivos para Diagnóstico

RESUMEN

In this study we examined the effect of the synthetic peptide thymosin-alpha1 (T(alpha)1) on MHC class I expression in FRTL-5 cells. Treatment with T(alpha)1 increased expression of MHC class I surface molecules and mRNA, which reached its peak (153 +/- 8 % of the control value) after 12 h. Chloramphenicol acetyltransferase (CAT) analysis, following transfection with a plasmid containing the regulatory sequence of MHC class I (or its deletion derivatives) with the CAT reporter gene, and electrophoretic mobility shift assay experiments demonstrated that the action of T(alpha)1 was at the transcriptional level, and its mechanism of action is likely due to increased binding between the complex p50/fra-2 and the enhancer A sequence of the 5' flanking region of a swine class I gene (PD1). An increase in the expression of MHC class I surface molecules was also observed by flow cytometry in murine and human tumor cell lines and in primary cultures of human macrophages. This study shows for the first time an effect of Talpha1 on the regulation of gene expression at the molecular level, and may further contribute to explaining the results obtained using Talpha1 in the control of infectious diseases and tumor growth.

Asunto(s)

Genes MHC Clase I/efectos de los fármacos , Timosina/análogos & derivados , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Antígeno 2 Relacionado con Fos , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Complejo Mayor de Histocompatibilidad , Ratones , Mutación , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Porcinos , Timalfasina , Timosina/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transfección , Células Tumorales Cultivadas

RESUMEN

BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.

Asunto(s)

Apoptosis , Núcleo Celular/patología , Citometría de Flujo/métodos , Linfocitos/patología , Animales , Núcleo Celular/metabolismo , Electroforesis , Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Necrosis , Fosfatidilserinas/metabolismo

RESUMEN

We investigated the sensitivity to cell death of peripheral blood mononuclear cells (PBMCs) from patients with multiple sclerosis (MS). PBMCs from MS patients, following PHA stimulation, were less sensitive to cell death than those from healthy donors (mean +/- s.e.m., 22.5 +/- 1.9 in MS patients vs 36.5 +/- 2.8 in healthy controls; p = 0.0003). However, when Fas-agonist antibody was added, the increase in respect to apoptosis induced by mitogen alone was even higher in MS patients than in controls. In addition, PHA-activated PBMCs from MS patients showed higher surface expression of Fas than controls, while Bcl-2 expression was decreased. This finding raised the question of whether an impaired generation of apoptotic signals may be contributing to the immune component of MS.

Asunto(s)

Apoptosis/fisiología , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Mitógenos/farmacología , Esclerosis Múltiple/fisiopatología , Adulto , Anticuerpos/farmacología , Complejo CD3/metabolismo , Membrana Celular/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/inmunología , Esclerosis Múltiple/sangre , Esclerosis Múltiple/patología , Fitohemaglutininas/farmacología , Valores de Referencia , Receptor fas/inmunología , Receptor fas/metabolismo

RESUMEN

Human CD4+ Th1 and Th2 clones were infected with human T-lymphotropic virus type I (HTLV-I) and followed up for a 12 month period in culture. PCR analysis showed that proviral DNA and viral mRNA were present in both Th1 and Th2 infected clones, throughout the entire culture period. Thus, HTLV-I exhibited neither preferential tropism nor exerted differential immortalizing activity in Th1 versus Th2 cells. All the infected clones immediately lost their antigen dependency for growth and continuously proliferated in IL-2-conditioned medium without need for additional stimulation. Infected Th1 and Th2 clones equally showed high expression of CD25, HLA-DR, CD44, CD30 and CD45RO. Infection with HTLV-I altered the cytokine profile in Th1 and Th2 clones. Both types of clones produced IL-6 and TNF-alpha. Th1 infected clones retained their ability to secrete IFN-gamma, but lost IL-2 gene expression. Th2 infected clones lost IL-4 gene expression, retained the ability to produce small amounts of IL-5 and acquired IFN-gamma expression.

Asunto(s)

Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Células TH1/virología , Células Th2/virología , Antígenos CD/análisis , Citocinas/biosíntesis , Humanos , Activación de Linfocitos

RESUMEN

It has been previously reported that following severe brain damage, a deficit of cellular immunity could be detected in the early phase after the occurence of the lesion. We report here the results of a cross-sectional study on long term effects of severe brain damage on immunological and neuro-endocrine changes in patients who recovered from prolonged coma caused by head injury. Results obtained from post-comatose (PC) patients were compared with those obtained from two control groups made up of spinal-cord injury (SCI) patients and healthy subjects, respectively. The following parameters were studied: lymphomonocyte subsets; interleukin 2 (IL-2) production; natural killer (NK) activity and serum levels of adrenocorticotrophic hormone (ACTH), cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, tri-iodothyronine (T3) and thyroxine (T4). With respect to healthy controls the PC1 subgroup, i.e. patients examined 3-6 months after injury, showed a statistically significant decrease in IL-2 production, NK activity and CD25+ lymphocytes. Similar immunological disturbances were observed in SCI but not in the PC2 subgroup, i.e. patients examined later than 6 months after injury. The same sub-group of PC1 patients showed high serum levels of cortisol and PRL. These results could be related to the immunological status and may be interpreted as a transient but prolonged condition of chronic stress or "chronic alarm reaction". Copyright Rapid Science Ltd