RESUMEN
Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Eritrocitos/fisiología , Mitocondrias/fisiología , Animales , Calcio/metabolismo , Calcio/farmacología , Caspasa 3 , Caspasas/metabolismo , Caspasas/farmacología , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Eritrocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Leupeptinas/metabolismo , Leupeptinas/farmacología , Activación de Macrófagos/inmunología , Ratones , Modelos Biológicos , Oligopéptidos/metabolismo , Oligopéptidos/farmacologíaRESUMEN
In vivo phagocytosis of senescent red blood cells (RBCs) by macrophages occurs 120 days after their release into the circulation. It depends on two sequential signals that trigger phagocytosis: (1) desialylation of membrane glycoconjugates with the exposure of the penultimate beta-galactosyl residues and (2) exposure of phosphatidylserine in the membrane outer leaflet. Leukodepleted and nonleukodepleted RBCs were compared using flow cytometric procedures to determine whether the in vitro deterioration of RBCs during storage might be attributable to an identical mechanism of desialylation induced by leukocyte neuraminidases, resulting in exposure of beta-galactosyl and subsequently phosphatidylserine residues - signals of senescent RBCs. Without prior leukodepletion, stored RBCs showed an increased population of senescent RBCs (using light scatter measurements), extensive desialylation with the exposure of beta-galactosyl residues (using specific fluorescein isothiocyanate [FITC]-lectins), significant exposure of phosphatidylserine in the outer leaflet of the RBC membrane (using FITC-annexin V), and extensive in vitro phagocytosis (using PKH-26-labeled RBCs). There were minimal changes observed with the leukodepleted RBCs. These results lead to the conclusion that leukocyte enzymes, including neuraminidases, are definitive contributers to the desialylation of RBCs during storage and to the exposure of phosphatidylserine residues. These deleterious effects resulting from highly active leukocyte enzymes are preventable by prior leukodepletion of the stored RBCs. Previously developed flow cytometric procedures to detect in vivo "RBC senescence" have been applied and proved to be reliable criteria to monitor the viability of stored RBCs.
Asunto(s)
Conservación de la Sangre/métodos , Envejecimiento Eritrocítico , Eritrocitos/citología , Adulto , Animales , Supervivencia Celular , Citometría de Flujo , Humanos , Leucocitos/enzimología , Ratones , Persona de Mediana Edad , Neuraminidasa/química , Fagocitosis , Fosfatidilserinas/análisis , Manejo de EspecímenesRESUMEN
This study prospectively analysed the relationships between immunophenotypic and cytogenetic features of blast cells in 432 acute non-lymphoblastic leukemias (ANLL) at presentation. An abnormal karyotype was detected in 232 cases (54%). These abnormalities were related to immunophenotypic markers as detected using a consensual panel of monoclonal antibodies allowing lineage assignment and investigation of myeloid marker expression on blast cells. In univariate analysis, CD9, CD10, CD15, CD34 and TdT expression appeared significantly associated with chromosomal anomalies. Multivariate analysis identified CD34 and CD9 expression as independently predictive of the presence of at least one cytogenetic abnormality (P < 10(-4) and P < 0.03, respectively). Significant associations between immunophenotypic and karyotypic features were observed both within individual FAB subgroups and independently from morphological criteria. Specific features were seen in five ANLL entities: M0 or M1/B lineage antigen positivity/t(9;22) or del(11)(q23); M2/CD13-/t(8;21); M4/CD13+, CD34+, CD36+/inv(16); M4 or M5/lack of B lineage antigen/del(11)(q23) or t(9;11). More practically, and although the relationships demonstrated only represent a fraction of homogeneous immunophenotypic subgroups, identification of such immunophenotypic features should prompt careful karyotypic examination, eventually using molecular biology analysis on non-growing cells.
Asunto(s)
Aberraciones Cromosómicas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide/genética , Leucemia Mieloide/inmunología , Antígenos CD/análisis , Bandeo Cromosómico , Inversión Cromosómica , Mapeo Cromosómico , Femenino , Eliminación de Gen , Reordenamiento Génico , Humanos , Inmunofenotipificación/métodos , Cariotipificación , Leucemia Mieloide/clasificación , Leucemia Mieloide/patología , Leucemia Mieloide Aguda/clasificación , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/inmunología , Síndromes Mielodisplásicos/patología , Estudios Prospectivos , Eliminación de Secuencia , Translocación GenéticaRESUMEN
We analyzed P glycoprotein (PGP) expression and its correlation with hematological parameters and outcome in 50 cases of newly diagnosed adult acute lymphoblastic leukemia (ALL). PGP expression was evaluated by flow cytometry using MRK16 monoclonal antibody (MoAb) and/or immunocytochemistry on marrow slides, using JSB1 MoAb. Thirty-two of the 50 patients (64%) were PGP positive by at least one of the two methods, which gave concordant results in 15 of the 18 cases in which they were both used. No correlation between PGP expression and clinical and hematological parameters including WBC counts, immunophenotype and karyotype was seen, although there was a trend for more frequent CD34 expression in PGP-positive cases. All patients were treated with intensive chemotherapy. We found no difference in complete remission (CR) rate, actuarial disease-free survival and survival in PGP-positive and PGP-negative cases. Our findings suggest that the clinical significance of PGP expression is less clear in ALL than in AML. Wider use of functional techniques of evaluation of mdr1 gene expression, which assess the 'pumping' activity of PGP, and their correlation with quantitative analysis of mdr1 mRNA and protein, would probably improve knowledge of the role of PGP in ALL. Analysis of other mechanisms of drug resistance, especially multidrug resistance-associated protein (MRP) expression, would also be useful.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Adulto , Biomarcadores , Femenino , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Pronóstico , Análisis de SupervivenciaRESUMEN
The use of flow cytometry as a common tool has opened the field of quantimetric immunophenotyping. This could help refine the definition of leukemic proliferations, and perhaps provide new prognostic criteria. We investigated whether this approach could be applied to multicentric immunophenotyping. The reproducibility of quantitative measurements on different types of flow cytometers was tested using a single batch of calibrating and "blind" fluorescent beads. Less than 7% CV was obtained between 20 different laboratories. We then explored the variations noted with control or normal lymphocytes, using various CD4 monoclonal antibodies, which were found to be lower than 10%. We finally applied this strategy to acute lymphoblastic leukemias (ALL), performing quantitative measurements of CD10 using one single aliquoted batch of FITC-CD10 in comparison with locally used reagents. PreB-ALL could indeed be further stratified depending on the density of CD10 expression. Use of the same monoclonal antibody is however recommended, since PreB-ALL also seem to display epitopic variations of the CD10 molecule.
Asunto(s)
Neprilisina/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Antígenos CD4/análisis , Citometría de Flujo , Humanos , Reproducibilidad de los ResultadosRESUMEN
We performed cytogenetic and molecular analysis of the BCR-ABL rearrangement by polymerase chain reaction (PCR) in 39 consecutive cases of adult acute lymphoblastic leukemia (ALL). Eleven patients had a Philadelphia (Ph) chromosome. Thirteen patients had a BCR-ABL rearrangement, involving minor breakpoint cluster region (m-bcr, situated in intron 1 of the BCR gene) in 11 cases, and major breakpoint cluster region (M-bcr, 'specific' of chronic myeloid leukemia) in the remaining two cases. All of the 12 BCR-ABL cases studied immunologically were of early B, CALLA-positive immunophenotype. The 13 BCR-ABL positive cases included the 11 Ph-positive cases, and two patients with normal karyotype at diagnosis. In the two Ph-negative BCR-positive cases, seven (patient 1) and 18 (patient 2) mitoses had been examined at diagnosis. In patient 1, Ph negativity at diagnosis could certainly be explained by the small number of mitoses analyzed, as a Ph chromosome was found at relapse. This was less probable in patient 2, who raised the issue of whether authentic Ph-negative BCR-ABL-positive ALL exists (as in the chronic myeloid leukemia model) or not. Whatever the explanation, our results suggest that molecular detection of BCR-ABL should be more widely used in B-lineage ALL.
Asunto(s)
Proteínas de Fusión bcr-abl/genética , Genes abl , Leucemia Linfoide/genética , Adulto , Secuencia de Bases , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Cromosoma Filadelfia , Reacción en Cadena de la Polimerasa , Translocación GenéticaRESUMEN
We performed immunocytochemical detection of myeloperoxidase (MPO), using monoclonal antibody MPO-7, in 15 consecutive cases of adult acute leukemia (AL) unclassified by conventional cytological and cytochemical criteria and 7 AML-M1 with less than 10% of cytochemically MPO-positive blasts. In AL with negative MPO cytochemistry the anti-MPO reaction was positive in 5 of the 15 patients with 3, 3, 7, 11 and 45% positive blasts respectively. In AML-M1, immunocytochemistry was positive in a larger percentage of blasts than cytochemistry in 2 cases. Immunological detection of myeloid surface markers was positive in all 15 cases of unclassified AL (including the 10 AL with negative anti-MPO reaction). Eleven of the 22 patients from this study had mixed lymphoid-myeloid phenotype. Discrepancy between immunological MPO detection and light cytochemistry was more frequent in patients with mixed immunophenotype than in patients without lymphoid markers. No relationship between MPO-antigen positivity and clinical or biological features was seen. These findings confirm immunological detection of MPO as useful for the diagnosis of poorly differentiated AL. The high incidence of inactive MPO detectable only by immunocytochemistry in mixed lineage AL needs to be confirmed.
Asunto(s)
Leucemia/enzimología , Peroxidasa/análisis , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/análisis , Médula Ósea/enzimología , Médula Ósea/inmunología , Médula Ósea/patología , Femenino , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Humanos , Inmunohistoquímica , Inmunofenotipificación , Leucemia/sangre , Leucemia/patología , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Peroxidasa/sangreRESUMEN
To contribution the help of flow cytometry in the diagnosis of mesothelioma in pleural effusions, we studied the aneuploid frequency in paraffin-embedded pleural mesotheliomas. The low aneuploidy rate (31%) explains the moderate yield of mesothelioma diagnosis in pleural effusions. Because of the possibility of false aneuploidies, flow cytometry cannot replace of a cytologic or histopathologic evidence.
Asunto(s)
Citometría de Flujo/métodos , Mesotelioma/diagnóstico , Neoplasias Pleurales/diagnóstico , ADN de Neoplasias/análisis , Humanos , Mesotelioma/genética , Neoplasias Pleurales/genéticaRESUMEN
The level of sCD23 produced in the course of human immunodeficiency virus (HIV) infection was measured in patients grouped according to the Centers for Disease Control by using an immunoradiometric assay. Soluble CD23 was evaluated in supernatants of peripheral blood mononuclear cell (PBMC) (10(6) cells/ml) stimulated by phytohemagglutinin (PHA). Compared with healthy controls (m +/- S.D. = 1.0 +/- 0.34 U/ml, n = 7), higher values were observed in some of the patients of group II (asymptomatic) (m +/- S.D. = 2 +/- 1.33, n = 9) and some of the patients of group IV (AIDS) (m +/- S.D. = 1.3 +/- 1.40, n = 8). Those results prompted us to compare the plasma levels of sCD23 in group II and group IV HIV-infected patients and in healthy individuals. Soluble CD23 plasma levels in healthy patients (n = 42) ranged from 0 to 1.5 U/ml (m +/- S.D. = 0.9 +/- 0.33), in group II patients (n = 17) from 0 to 3 U/ml (m +/- S.D. = 0.92 +/- 0.83) and in group IV patients (n = 73) from 0 to 2.9 U/ml (m +/- S.D. = 1.15 +/- 0.71). The differences between the patients and the healthy individuals were not statistically significant but individual sCD23 values higher than 2 U/ml were obtained in 6% of the group II patients and 16.7% of the group IV patients. Increased values of sCD23 were obtained in plasma from patients with secondary infectious diseases (groups IV-Cl and IV-C2) and from patients without secondary infectious diseases (group II, group IV-A and group IV-B).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Receptores de IgE/análisis , Síndrome de Inmunodeficiencia Adquirida/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Ensayo Inmunorradiométrico , Recuento de Leucocitos , Activación de Linfocitos/inmunología , Fitohemaglutininas , Receptores de IgE/biosíntesis , Solubilidad , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Variations in cytokine production in patients with human immunodeficiency virus (HIV) infection could be involved in the physiopathology and in the progression of the disease. Therefore we studied the level of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha) produced in patients with HIV infection at stage II (asymptomatic seropositives) and stage IV (AIDS) of the CDC classification, by using an enzyme amplified sensitivity immunoassay. We measured the level of GM-CSF and TNF alpha in supernatant of phytohemagglutinin-activated peripheral blood mononuclear cells from patients and healthy individuals. In one out of 10 stage II patients and 4 out of 14 stage IV patients, we obtained higher levels of GM-CSF than the mean + 2 S.D. of controls, but in 3 stage IV patients with very low CD4+ T lymphocyte counts (< 50/mm-3) compared to other patients, the GM-CSF values were very low. High levels of TNF alpha were detected in 3 out of 10 stage II and 6 out of 11 stage IV patients. The high values of TNF alpha were associated with high values of GM-CSF in stage II and in most of AIDS patients except those with very low CD4+ T cell counts, who produced low levels of GM-CSF. Plasma levels of cytokines were evaluated in 10 stage II, 22 stage IV patients and 20 controls. Increased levels of GM-CSF (more than 9 pg/ml) were observed in the plasma from 8 out of 10 stage II patients and 17 out of 22 stage IV patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Infecciones por VIH/inmunología , VIH-1 , Factor de Necrosis Tumoral alfa/análisis , Linfocitos T CD4-Positivos , Células Cultivadas , Citometría de Flujo , Humanos , Recuento de Leucocitos , Leucocitos Mononucleares/inmunología , Fitohemaglutininas/farmacologíaRESUMEN
We report two cases of secondary acute lymphoblastic leukemia (ALL) with t (4;11) (q21;q23) translocation occurring after chemotherapy and radiotherapy for a prior cancer. Seven previously published cases of secondary ALL with t (4;11) (q21;q23) are also reviewed. Most patients had received a combination of topoisomerase II inhibitors (anthracyclines, mitoxantrone, or the epipodophillotoxin derivatives VP16 or VM26) and cyclophosphamide, which have also been implicated in the pathogenesis of secondary acute myeloid leukemia (AML) with 11q23 rearrangements. These observations give further support to the existence of a subgroup of secondary acute leukemias with cytogenetic findings "specific" for de novo ALL and AML, especially those with translocations involving the 11q23 region.
Asunto(s)
Cromosomas Humanos Par 11 , Cromosomas Humanos Par 4 , Neoplasias Primarias Secundarias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Antígenos de Diferenciación/análisis , Femenino , Humanos , Persona de Mediana Edad , Neoplasias Primarias Secundarias/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunologíaRESUMEN
Mutations of exons 5 to 8 of the p53 gene were looked for in 39 cases of B-cell chronic lymphocytic leukemia (CLL) using polymerase chain reaction single-strand conformation polymorphism analysis and DNA sequencing. All patients also had cytogenetic analysis. A point mutation, leading to an amino acid change in the p53 protein was found in four cases, involving exon 7 (one case) or exon 8 (three cases). Mutations seemed to predominate in advanced clinical stages (Binet's stage C). All four patients with 17p monosomy had a mutation whereas no mutation was found in the 35 patients with cytogenetically normal 17p. These findings suggest that p53 mutations are relatively rare in B-cell CLL, and largely predominate or may even be restricted to patients with 17p monosomy (who constitute about 5% of all B-cell CLL patients in large published series). In those patients, the mutations may play a role in leukemogenesis through loss of tumor suppressive activity of normal p53 genes.