RESUMEN
PURPOSE, OBJECTIVE: The aim of our study was to investigate the quality of life of patients undergoing cleft lip or cleft lip and palate as well as the perception of quality of life of patients perceived by their parents slot. PATIENTS AND METHODS: Fifty-one adolescents and young adults (29 boys and 22 girls) who underwent cleft lip (FL 22) or lip and palate (CLP 29) and their parents have received psychological and surgical joint consultation. The minimum age for inclusion was 10 years (mean age 15.5 years). A quality of life questionnaire (VSP-A) "children" was given between 10 and 11 years and a questionnaire "adolescents" beyond. Parents have them answered a questionnaire of perceived quality of life of their children. The results were analyzed and compared to a control population (Statistics Student test). Perceived parents lived in relation to their children was obtained through the study of linear regression curves. RESULTS: The response rate to the questionnaires was 66.7% for parents, 85.7% for children and 63.6% for teenagers. The quality of life of the patients was assessed by the patients to 65.1/100 on average. The index of overall quality of life was superimposed on the control population (p=0.66). Perceived quality of life of patients by their parents was fairly close to the quality of life described by patients (66.5). On the areas of family, education, recreation, quality index was proportionately less than for other areas. For each of these areas, parents overestimated the quality of life of their child. Compared to the control group the fields of education, leisure, vitality quality index were significantly lower in the/FL population P.
Asunto(s)
Labio Leporino/psicología , Fisura del Paladar/psicología , Familia/psicología , Calidad de Vida , Adolescente , Labio Leporino/cirugía , Fisura del Paladar/cirugía , Femenino , Francia , Humanos , Masculino , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the expression of the recombinant oncoprotein Bcr-Abl. In this investigation, we studied an imatinib-resistant cell line (K562-r) generated from the K562 cell line in which none of the previously described mechanisms of resistance had been detected. A threefold increase in the expression of the heat-shock protein 70 (Hsp70) was detected in these cells. This increase was not associated to heat-shock transcription factor-1 (HSF-1) overexpression or activation. RNA silencing of Hsp70 decreased dramatically its expression (90%), and was accompanied by a 34% reduction in cell viability. Overexpression of Hsp70 in the imatinib-sensitive K562 line induced resistance to imatinib as detected by a large reduction in cell death in the presence of 1 muM of imatinib. Hsp70 level was also increased in blast cells of CML patients resistant to imatinib, whereas the level remained low in responding patients. Taken together, the results demonstrate that overexpression of Hsp70 can lead to both in vitro and in vivo resistance to imatinib in CML cells. Moreover, the overexpression of Hsp70 detected in imatinib-resistant CML patients supports this mechanism and identifies potentially a marker and a therapeutic target of CML evolution.
Asunto(s)
Resistencia a Antineoplásicos/genética , Proteínas HSP70 de Choque Térmico/biosíntesis , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Regulación hacia Arriba , Biomarcadores de Tumor , Línea Celular Tumoral , Proteínas de Fusión bcr-abl/genética , Regulación Neoplásica de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismoRESUMEN
Anti-leukemia activity of human macrophages involves the generation of nitric oxide (NO) derivatives. However, leukemic transformation may involve mechanisms that rescue cells from NO-mediated apoptosis. In the present work, we analyzed the effects of exogenous NO on the proliferation of BCR-ABL(+) chronic myelogenous leukemia (CML) cells. As normal leukocytes, the proliferation of leukemia cells was inhibited by SNAP (S-nitroso-N-acetyl-penicillamine), GEA (Oxatriazolium amino-chloride), and SIN-1 (Morpholino-sydnonimine), whereas SNP (sodium nitroprusside) had no effect on leukemia cell growth. SIN-1 induced higher anti-proliferation activity in BCR-ABL(+) cells, compared to normal hemopoietic cells. Inhibition of leukemia cell proliferation correlated with increased apoptosis and DEVDase activity. The simultaneous addition of exogenous iron reversed NO-mediated inhibition of cell growth, caspase activation and apoptosis in all BCR-ABL(+) cells tested. The quantification of intracellular iron levels in leukemia cells indicated that NO induced an early, dose-dependent decrease in ferric iron levels. Accordingly, elevation of intracellular iron protected leukemia cells from NO-mediated apoptosis. Together, the present work reveals the presence of an iron-dependant mechanism for leukemia cell rescue from NO-induced growth inhibition and apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Hierro/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Óxido Nítrico/farmacología , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas/efectos de los fármacos , Ferricianuros/farmacología , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Donantes de Óxido Nítrico/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina/farmacología , Transducción de Señal/fisiologíaRESUMEN
F2The host defense against tumor cells is in part based upon the production of nitric oxide (NO) by activated macrophages. However, carcinogenesis may involve mechanisms that protect tumor cells from NO-mediated apoptosis. In the present study, we have assessed the effects of exogenous NO on the proliferation and survival of human liver (AKN-1), lung (A549), skin (HaCat), and pancreatic (Capan-2) tumor cell lines, compared with normal skin-derived epithelial cell cultures. Except to the HaCat cell line, all of the other human epithelioid cells were sensitive to the antiproliferation effect of S-nitroso-N-acetyl-penicillamine or Deta NONOate, whereas tumor cells had low if any response to sodium nitroprusside. Growth inhibition with exogenous NO correlated with increased apoptosis, but was not mediated by cyclic GMP, peroxynitrite generation, or poly(ADP-ribose) polymerase modulation, all of which involved in NO-mediated growth inhibition of normal skin-derived epithelial cell cultures. The simultaneous addition of iron-containing compounds protected tumor cells from NO-mediated growth inhibition and apoptosis. Intracellular iron quantification indicated that, as deferoxamine, exogenous NO significantly decreased intracellular ferric iron levels in tumor cells. Together, the current study reveals that intracellular iron elevation rescues tumor cells from NO-mediated iron depletion and subsequent growth inhibition and apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Hierro/fisiología , Neoplasias/patología , Óxido Nítrico/farmacología , Apoptosis/fisiología , Ciclo Celular/efectos de los fármacos , Ferricianuros/farmacología , Humanos , Hierro/metabolismo , Neoplasias/metabolismo , Donantes de Óxido Nítrico/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina , Transducción de Señal/fisiología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
CD40 ligand (CD40L)/CD40 interactions play a central role in T-cell-dependent B-cell activation as previously shown by in vitro studies, the phenotype of CD40L knockout mice and the defective expression of CD40L in patients who have X-linked immunodeficiency with hyper-IgM. The distribution of CD40 in cells other than of myeloid and lymphoid lineages has suggested additional functions for this receptor/ligand couple. Here we show that CD40L stimulates myelopoiesis with a noticeable effect on megakaryocytopoiesis in cocultures of hematopoietic progenitor cells and bone marrow stromal cells. These results suggest a mechanism by which T-cell or platelet-associated or soluble CD40L may regulate myelopoiesis. (Blood. 2000;95:3758-3764)
Asunto(s)
Endotelio Vascular/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Leucopoyesis/fisiología , Glicoproteínas de Membrana/farmacología , Proteínas de la Membrana/biosíntesis , Trombopoyetina/biosíntesis , Animales , Células de la Médula Ósea/citología , Ligando de CD40 , Células COS , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Endotelio Vascular/citología , Femenino , Sangre Fetal/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Recién Nacido , Leucopoyesis/efectos de los fármacos , Ratones , Ratones Noqueados , Embarazo , Venas UmbilicalesRESUMEN
BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.
Asunto(s)
Ciclina B/metabolismo , Linfocitos T/metabolismo , Western Blotting , Butiratos/farmacología , División Celular , Colchicina/farmacología , Ciclina B1 , ADN/análisis , Replicación del ADN , Citometría de Flujo , Humanos , Interfase , Células Jurkat/metabolismo , Activación de Linfocitos , Mitosis , Fitohemaglutininas/farmacología , Fase S , Timidina/farmacologíaRESUMEN
We investigated transfection rates of CD34+ haematopoietic progenitor cells (HPC) or haematopoietic cell lines (TF-1, KG1a and K562) using the LacZ gene as a reporter and cationic liposomes. The transfection efficiency of CD34+ haematopoietic progenitor cells (HPC) or TF-1, KG1a and K562 grown in suspension is very low (average percentage of 0.013 for HPC and 0.03 for cell lines). Adhesion of HPC or cell lines to plates by immunological or physical methods significantly enhances transfection efficiency; however, the percentage of transfected cells still remained low. We found that adhesion of TF-1, KG1a and K562 HC to MS-5 stroma cells or NIH-3T3 fibroblast cells increased transfection efficiency. Under these conditions transfection is achieved in 11.2-25% (mean 18.30%) for the cell lines and 13.6% (range 8.2-24.2%) for CD34+ HPC. These results indicate that liposome-mediated transfection of HC is significantly increased when cells are grown in adherence to stroma or fibroblast monolayers.
Asunto(s)
Adhesión Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Células del Estroma/citología , Transfección/métodos , beta-Galactosidasa/genética , Células 3T3 , Animales , Resinas de Intercambio de Catión , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Portadores de Fármacos , Fibroblastos/citología , Fibroblastos/fisiología , Genes Reporteros , Humanos , Células K562 , Cinética , Lípidos , Liposomas , Ratones , Células del Estroma/fisiologíaRESUMEN
Flt3-ligand (FL) is a cytokine that is of paramount importance in the proliferation of primitive hematopoietic progenitors. In this study, we show that endothelial cells (EC) produce large amounts of soluble FL and express a membrane-bound form of the molecule. Bone marrow microvascular EC also produce FL, suggesting that EC are an important source of FL in the bone marrow. High concentrations of FL in EC supernatants contrast with its undetectable levels in long-term bone marrow cultures. A single mRNA for FL is detected, suggesting that soluble FL derives from the membrane-bound species by proteolytic release. FL mRNA is stable with a half-life of about 3 h. II-1alpha increases FL mRNA levels and membrane and soluble FL expression. Glucocorticoids, known inhibitors for many hematopoietic growth factors do not down-regulate the expression of FL. On the contrary, GC increase the expression of both species of FL. The neutralization of FL in cocultures EC/ hematopoietic progenitors results in an acceleration of the maturation of the progenitors. IFN-alpha, MIP-1 alpha and TGF-beta stimulate production of membrane-bound and soluble FL. This stimulation is essential to explain their modulatory effect on the generation of clonogenic cells in cocultures EC/hematopoietic progenitors. Leukemia (2000) 14, 153-162.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas de la Membrana/biosíntesis , Secuencia de Bases , Células de la Médula Ósea/citología , Diferenciación Celular , Células Cultivadas , Clonación Molecular , Técnicas de Cocultivo , Citocinas/farmacología , Cartilla de ADN , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Proteínas de la Membrana/genética , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have analysed the potential of an endothelialized hydroxyapatite matrix (HAP), a synthetic bone substitute, as a cellularized support for the expansion of haemopoietic progenitor cells. Scanning electron microscopy (SEM) showed that the endothelial cells (EC) tended to form a monolayer fitting closely to the matrix, and that the progenitors adhered to the EC layer. Endothelialized HAP supported the proliferation and differentiation of the progenitors with the addition of the exogenous cytokines IL-1, and IL-3. The expanded cell population essentially belonged to the myeloid and monocytic lineages, with a smaller percentage of the megakaryocyte lineage. In comparative experiments CD34+ progenitors were expanded on endothelialized tissue culture flasks, and a significant higher viability of the expanded cells was found with the endothelialized HAP. A high percentage (approx. 40%) of mature granulocytes was generated in accordance with the presence of differentiating cytokines such as G-CSF and GM-CSF at high concentrations in the coculture medium. Other cytokines that could be detected were IL-6, M-CSF, SCF, flt3-ligand. More than 50% of the expanded cell population was able to phagocytose bacteria and to generate an oxydative burst. These data indicate that cellularized HAP may be a useful matrix for stromal cell-based expansion systems.
Asunto(s)
Durapatita , Células Madre Hematopoyéticas/citología , Diferenciación Celular , División Celular , Citocinas/metabolismo , Endotelio/citología , Citometría de Flujo , Células Madre Hematopoyéticas/metabolismo , Humanos , Fenotipo , Cordón Umbilical/citologíaRESUMEN
Experiments by flow cytometry (FCM) after nuclei isolation have never been done to investigate cyclins. We have conducted different experiments by FCM using whole cells and isolated nuclei to study the immunolocalization and kinetic patterns of cyclin B1 and cyclin E in various leukemic cell lines. During asynchronous growth, all whole cells had a scheduled, cell cycle phase-restricted expression of cyclin B1. By using a washless immunostaining of unfixed nuclei, cyclin B1 was detected in all cell cycle phases, including G1, although to a lesser extent than in G2/M, suggesting that in whole cells the cyclin B1 epitope is masked and accessible only in isolated nuclei. When the cells were synchronized at the G1/S boundary by thymidine or in the G1 phase by sodium n-butyrate, an identical accumulation of cyclin B1 was observed. As for cyclin E, its expression was higher with thymidine treatment than with sodium n-butyrate, particularly in nuclei. The elevated cyclin B1 level in the cells arrested at the G1/S boundary may reflect the increased half-life of this protein stabilized as the result of cyclin E overexpression. However, our FCM data also support the notion that accumulation of human cyclin B1 in leukemic cell lines begins during the G1 phase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G1 phase of the cell cycle confirms this finding. It is possible, therefore, that tumor transformation or leukemic phenotype may invariably be associated with altered cyclin B1 expression.
Asunto(s)
Ciclo Celular , Ciclina B/biosíntesis , Ciclina E/biosíntesis , Leucemia/metabolismo , Leucemia/patología , Western Blotting , Ciclo Celular/efectos de los fármacos , División Celular , Separación Celular , Ciclina B/metabolismo , Ciclina B1 , Ciclina E/metabolismo , Citometría de Flujo , Fase G1 , Humanos , Células Jurkat , Células K562 , Cinética , Células Tumorales Cultivadas , Células U937RESUMEN
We investigated the ability of endothelial cells (EC) to support hematopoiesis in contact and non-contact cocultures with isolated CD34+ or CD34/CD38low cells. In the absence of exogenous cytokines, umbilical vein EC (HUVEC) efficiently support proliferation of hematopoietic cells and generation of colony-forming cells (CFC). Cytokines (IL-6, LIF, G-CSF, GM-CSF, M-CSF, but not IL-1, IL-3, IL-7) were detected in HUVEC coculture supernatants. Neutralization of these cytokines profoundly inhibited the ability of EC supernatants to support the differentiation of hematopoietic progenitors and led to an accumulation of immature cells. Contact cocultures were significantly more efficient than non-contact cocultures. The expanded cell population essentially belonged to the myeloid and monocytic lineages. Contact cocultures generated cells expressing the CD61 or CD41 antigens. Interleukin-1alpha (IL-1alpha) augmented EC capacity to support hematopoiesis, this property resulting from the upregulation of cytokine expression. Glucocorticoids (GC) reduced this capacity by downregulating the biosynthesis of cytokines by EC and not by a direct effect on the progenitor cells. EC from the bone marrow microvasculature (BMEC) support the proliferation and the differentiation of hematopoietic progenitors. Synergistic increase in progenitor cells expansion and generation of CFC occurred when EC cocultures were added with exogenous cytokines. Supernatants of IL-1alpha-stimulated EC potentiated the effects of an association of IL-1, IL-3, IL-6, LIF, SCF, Flt3-ligand, TPO, G-CSF, GM-CSF, M-CSF and IL-11 on the proliferation of hematopoietic progenitors suggesting that EC may produce other soluble growth factors potentiating the action of the above set of cytokines.
Asunto(s)
Endotelio Vascular/fisiología , Glucocorticoides/farmacología , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Interleucina-1/farmacología , Antígenos CD34/metabolismo , Médula Ósea/irrigación sanguínea , Diferenciación Celular , División Celular , Células Cultivadas , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados , Citocinas/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Sangre Fetal , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Microcirculación , Fenotipo , Venas UmbilicalesRESUMEN
The CD34 antigen is present at all differentiation stages of hematopoietic cells, from immature progenitor cells to committed precursor cells. In vivo, transplantation of CD34+ cells is sufficient to allow hematopoietic recovery after myeloablative chemotherapy, but a neutropenic period of 9-12 days still exists, even when hematopoietic growth factors are given posttransplantation. After ex vivo expansion cultures in the presence of cytokines, CD34+ cells can generate mature precursor cells in a stroma-free liquid culture system. This could lead to a shortening of the aplasia duration, but the persistence of primitive progenitor cells in the expanded CD34+ compartment remains to be demonstrated. In this study, CD34+ cells were isolated from eight peripheral blood (PB) and eight cord blood (CB) samples using either Isolex 50 (n = 6), Ceprate LC CD34 kit (n = 6), or Microcellector T-25 Stem Cell kit (n = 4). We have evaluated the functional potential of CD34+ cells after 7 days of ex vivo expansion culture in the presence of 500 UI/ml of interleukin-1 (IL-1), 10 ng/ml of IL-3, and 10 ng/ml of stem cell factor (SCF). The expansions of nucleated cells, granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive committed precursors, IL-1 + IL-3 + SCF + erythropoietin (EPO)-responsive multilineage progenitors, and 5-fluorouracil (5-FU)-resistant quiescent progenitor were 8-fold, 59-fold, 4.4-fold, and 2.2-fold, respectively. There was no significant difference in the amplification/expansion parameters between cultures initiated with CD34+ cells from PBSC or CB. Our data confirm that cytokine-mediated ex vivo expansion of blood CD34+ cells can produce large numbers of committed precursors and does not significantly affect the compartment containing more immature progenitors. Cytokine-mediated expansion could be of great interest in autologous transplantation to decrease the duration of marrow aplasia.
Asunto(s)
Antígenos CD34/análisis , Células Madre Hematopoyéticas/citología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , División Celular/efectos de los fármacos , Células Cultivadas , Resistencia a Medicamentos , Sangre Fetal/citología , Sangre Fetal/efectos de los fármacos , Fluorouracilo/farmacología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Humanos , LeucaféresisRESUMEN
Autologous haematopoietic progenitor cell (HPC) transplantation is increasingly used to restore haematopoiesis after high-dose chemotherapy treatments. The present study was designed to analyse the ability of hydroxyapatite (HAP) seeded with endothelial cells (EC) to support the proliferation and differentiation of CD34+ HPC in static culture conditions. HAP is endothelializable as assessed by scanning electron microscopy and time-course DNA synthesis analysis using tritiated thymidine incorporated in EC isolated from human umbilical vein cord. Short-term coculture experiments in which CD34+ cells isolated from human cord blood were seeded on endothelialized HAP, were performed. Results show that endothelialized HAP is permissive to CD34+ cell expansion with a maximum expansion obtained between days 7 and 14 of coculture in the presence of IL-1 and IL-3 when compared with other experiments omitting either EC or interleukins. From morphological analyses, the expanded cell population mainly belonged to the myelocytic lineage with 33% mature cells (polymorphonuclear neutrophils and monocytes) at day 14 of coculture. The immature HPC could remain trapped within HAP while giving rise to a more mature progeny that exit from HAP microenvironment.
RESUMEN
Recruitment of quiescent, clonogenic blasts from patients with acute myeloid leukemia (AML) by hematopoietic growth factors (HGFs) may improve the cytotoxic effects of cell-cycle-specific drugs like cytosine-arabinoside (Ara-C). Using the culture methods described by Nara and McCulloch and making a distinction between self-renewing and post-deterministic mitoses, we analyzed the effects of stem cell factor (SCF), a growth factor acting on early hematopoietic progenitor and stem cells. First, we demonstrated that SCF, used in combination with other HGFs included in fetal calf serum (FCS) and/or in 5637 cell line supernatant (5637-CM), stimulated both colony formation and self-renewal of blast progenitors from 10 patients, unlike SCF alone. We tested the effects of SCF on the recruitment of cells in the S-phase by using a bromodeoxyuridine/DNA (BrdUrd/DNA) staining method in flow cytometry (FCM). We showed that SCF stimulated proliferation of AML cells significantly in 9/18 patients with AML. Second, we tested the influence of SCF on the sensitivity to Ara-C of self-renewing leukemic cells from 18 patients with AML. We showed that SCF was efficient in increasing the toxicity of Ara-C on the self-renewing blast progenitors, especially with high concentrations of Ara-C. However, a large patient-to-patient heterogeneity was found and the activity of SCF was not correlated with its effect on the cell cycle. These data indicate that SCF can enhance sensitivity to Ara-C of some leukemic cells with self-renewing capacity.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Citarabina/farmacología , Leucemia Mieloide/patología , Células Madre Neoplásicas/efectos de los fármacos , Factor de Células Madre/farmacología , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bovinos , Ciclo Celular/efectos de los fármacos , Medios de Cultivo/farmacología , Medio de Cultivo Libre de Suero/farmacología , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Femenino , Sangre Fetal/química , Citometría de Flujo , Factores de Crecimiento de Célula Hematopoyética/aislamiento & purificación , Factores de Crecimiento de Célula Hematopoyética/farmacología , Humanos , Leucemia Mieloide/metabolismo , Masculino , Metilcelulosa , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Proteínas Recombinantes/farmacología , Suspensiones , Ensayo de Tumor de Célula MadreRESUMEN
We evaluated the expansion capacity of untreated and 5-fluorouracil (5-FU)-resistant peripheral blood stem cells (PBSC) after 7-day incubation with interleukin-1 (IL-1) plus IL-3 plus stem cell factor (SCF) or with medium alone. We found a significant increase in the proportion of CD34+ cells in the PBSC fraction resistant to 25 micrograms/mL 5-FU after 7-day incubation with IL-1 plus IL-3 plus SCF as compared with the untreated fraction (p = 0.011). We also showed that 5-FU-resistant PBSC have a greater capacity for expansion of IL-1/IL-3/SCF-responsive immature progenitors (p = 0.05), amplification of IL-3 plus GM-CSF responsive progenitors (p = 0.01), and production of committed single growth factor-responsive (granulocyte-macrophage colony-stimulating factor [GM-CSF]) precursors (p = 0.01) than the untreated PBSC. The expansion of all types of progenitors and CD34+ cells was only observed after 7-day incubation with IL-1 plus IL-3 plus SCF. These results suggest that PBSC contain a primitive stem cell population with an enhanced expansion capacity that is identified by 5-FU resistance. As these cells can be expanded in vitro, they may then be suitable for a number of clinical applications.
Asunto(s)
Fluorouracilo/farmacología , Factores de Crecimiento de Célula Hematopoyética/administración & dosificación , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-1/administración & dosificación , Interleucina-3/administración & dosificación , Antígenos CD/análisis , Antígenos CD34 , Células Sanguíneas/efectos de los fármacos , Ciclo Celular , Separación Celular/métodos , Citaféresis , Resistencia a Medicamentos , Humanos , Factor de Células MadreRESUMEN
CD34 positive (CD34+) cells contain all hematopoietic progenitors from stem cells to committed precursors. Therefore the transplantation of purified bone marrow or blood CD34+ cells is sufficient for hematopoietic recovery after a myeloablative radiochemotherapy. Using different techniques, CD34+ progenitors can be induced to undergo terminal differentiation in a stroma-free liquid culture system in the presence of cytokines. In the present study, we have evaluated the functional potential of CD34+ blood progenitors after ex-vivo expansion cultures. CD34+ cells were isolated from 16 samples (PBSC n = 8 and Cord Blood (CB) n = 8) using either ISOLEX 50 (n=6), CEPRATE LC CD34 kit (n = 6) or MICROCELLECTOR T-25 Stem Cell kit (n = 4). CD34+ cells were cultured for seven days in the presence of 500 UI/ML of IL-1, 10 ng/ml of IL-3 and 10 ng/ml of SCF. We obtained an 8-fold expansion of nucleated cells. We observed a 59-fold expansion of GM-CSF responsive committed precursors, a 4.4-fold expansion of IL-1+IL-3+SCF+Epo responsive multilineage progenitors and a 2.2-fold expansion of the 5-FU resistant quiescent progenitors. We did not observe any significant difference in the amplification/expansion parameters between cultures initiated with CD34+ cells from PBSC or CB. Our data show that cytokine mediated ex-vivo expansion of blood CD34+ cells can produce a large number of committed precursors without affecting the compartment of the most immature progenitors. These results suggest that cytokine-mediated amplification technology could be of great interest in the autologous transplantation setting.
Asunto(s)
Antígenos CD4/sangre , Citocinas/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Resistencia a Medicamentos , Fluorouracilo/farmacología , Humanos , Proteínas Recombinantes/farmacologíaRESUMEN
To further characterize the primitive stem cell subpopulation present in chemotherapy mobilized peripheral blood stem cells (PBSC), we evaluated the functional characteristics of 5-FU-resistant PBSC and normal bone marrow (BM) cells after 7 days incubation with IL-1 + IL-3+SCF. The resulting 5-FU-resistant cells were evaluated for (1) the production of GM-CSF-responsive clonogenic elements (CE), (2) the production of IL-3+GM-CSF-responsive CE, and (3) their self-renewal capacity (production of IL-1+IL-3+SCF-responsive CE). We also evaluated the percentage of CD34+ cells, the percentage of cells in S phase of the cell cycle, and the number of nucleated cells before and after cytokine-mediated expansion. We demonstrated an overall loss in nucleated cells after cytokine-mediated expansion in all cell fractions. We demonstrated a significantly greater increase in the percentage of CD34+ cells in the 5-FU-resistant PBSC fraction as compared to 5-FU-resistant BM cells (p = 0.012). We also showed that 5-FU-resistant PBSC have a greater capacity for self-renewal, amplification of IL-3+GM-CSF-responsive progenitors, and the production of committed GM-CSF-responsive progenitors as compared with BM cells, but this did not reach statistical significant. These results suggest that PBSC contain a truly primitive stem cell with an enhanced self-renewal and differentiative capacity that is recruited by 5-FU resistance and IL-1+IL-3+CSF-mediated expansion.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Médula Ósea/efectos de los fármacos , Citocinas/farmacología , Fluorouracilo/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Antígenos CD/sangre , Antígenos CD34 , Núcleo Celular/fisiología , Resistencia a Medicamentos/fisiología , Humanos , Fase SRESUMEN
OBJECTIVE: To investigate the link between alcohol consumption and glycoregulation. PATIENTS AND METHODS: Cross-sectional study during the annual occupational health check-up at the work site, concerning 7402 workers, excluding known diabetic persons and pregnant women. Alcohol consumption was recorded as the number of glasses of alcoholic beverage per day as stated by the subjects. A proxy for the glycoregulation was the level of capillary blood glucose measured with a reflectance meter (Reflolux) at the time of the visit. The association of alcohol consumption with impaired glycoregulation was assessed by multivariate analysis including potential confounders. RESULTS: Mean capillary blood glucose increased with increasing alcohol consumption, in both men and women. This association remained significant (p < 0.001) after adjustment for risk factors (multiple linear regression). Among 366 subjects with a fasting glycaemia or an oral glucose tolerance test, 26 were diabetic. Alcohol consumption was found independently associated by logistic regression with diabetes mellitus. CONCLUSION: Alcohol consumption might be a target for primary and secondary prevention of impaired glycoregulation and diabetes mellitus.