RESUMEN
Immunophenotyping of bone marrow (BM) precursors has been used as an ancillary diagnostic tool in myelodysplastic syndromes (MDS), but there is no general agreement about which variables are the most relevant for prognosis. We developed a parsimonious prognostic model based on BM cell populations well-defined by phenotype. We analyzed 95 consecutive patients with primary MDS diagnosed at our Institution between 2005 and 2012 where BM immunophenotyping had been performed at diagnosis. Median follow-up: 42 months (4-199). Median age: 67 years (33-79). According to IPSS-R, 71 cases were low or intermediate risk. Flow variables significant in the univariate Cox analysis: "%monocytes/TNCs", "% CD16+ monocytes/TNCs", "total alterations in monocytes", "% myeloid CD34+ cells", "number of abnormal expressions in myeloblasts" and "% of B-cell progenitors". In the multivariate model remained independent: "% myeloid CD34+ cells", B-cell progenitors" and "% CD16+ monocytes/TNCs". These variables were categorized by the extreme quartile risk ratio strategy in order to build the score: % myeloid CD34+ cells" (≥ 2.0% = 1 point), B-cell progenitors" (< 0.05% 1 point) and "CD16+ monocytes/TNCs" (≥ 1.0% 1 point). This score could separate patients with a different survival. There was a weak correlation between the score and IPSS-R. Both had independent prognostic values and so, the flow score adds value for the prognostic evaluation in MDS.
Asunto(s)
Células de la Médula Ósea/inmunología , Médula Ósea/patología , Modelos Estadísticos , Síndromes Mielodisplásicos/mortalidad , Adulto , Anciano , Antígenos CD34/metabolismo , Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Estudios de Casos y Controles , Separación Celular , Estudios de Factibilidad , Femenino , Citometría de Flujo , Estudios de Seguimiento , Proteínas Ligadas a GPI/metabolismo , Humanos , Inmunofenotipificación , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/inmunología , Síndromes Mielodisplásicos/patología , Pronóstico , Receptores de IgG/metabolismo , Medición de Riesgo/métodosRESUMEN
The complex pathophysiology of sickle cell anemia (SCA) involves intravascular hemolytic processes and recurrent vaso-occlusion, driven by chronic vascular inflammation, which result in the disease's severe clinical complications, including recurrent painful vaso-occlusive episodes. Hydroxyurea, the only drug frequently used for SCA therapy, is a cytostatic agent, although it appears to exert nitric oxide/soluble guanylyl cyclase (sGC) modulating activity. As new drugs that can complement or replace the use of hydroxyurea are sought to further reduce vaso-occlusive episode frequency in SCA, we investigated the effects of the sGC agonists BAY 60-2770 (sGC activator) and BAY 41-2272 (sGC stimulator) in the presence or absence of hydroxyurea on SCA vaso-occlusive mechanisms and cell recruitment both ex vivo and in vivo. These agents significantly reduced stimulated human SCA neutrophil adhesive properties ex vivo in association with the inhibition of surface ß2-integrin activation. A single administration of BAY 60-2770 or BAY 41-2272 decreased tumor necrosis factor cytokine-induced leukocyte recruitment in a mouse model of SCA vaso-occlusion. Importantly, the in vivo actions of both agonists were significantly potentiated by the coadministration of hydroxyurea. Erythroid cell fetal hemoglobin (HbF) elevation is also a major goal for SCA therapy. BAY 41-2272 but not BAY 60-2770 at the concentrations employed significantly induced γ-globin gene transcription in association with HbF production in cultured erythroleukemic cells. In conclusion, sGC agonist drugs could represent a promising approach as therapy for SCA, for use either as stand-alone treatments or in combination with hydroxyurea. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that stimulators and activators of sGC are potent inhibitors of the adhesion and recruitment of leukocytes from humans and in mice with sickle cell anemia (SCA) and may represent a promising approach for diminishing vaso-occlusive episode frequency in SCA. Hydroxyurea, a drug already frequently used for treating SCA, was found to potentiate the beneficial effects of sGC agonists in in vivo studies, implying that these classes of compounds could be used alone or in combination therapy.
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Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/metabolismo , Hidroxiurea/farmacocinética , Guanilil Ciclasa Soluble/metabolismo , Animales , Benzoatos/farmacología , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Eritroides/efectos de los fármacos , Células Eritroides/metabolismo , Hemoglobina Fetal/metabolismo , Humanos , Hidrocarburos Fluorados/farmacología , Células K562 , Masculino , Ratones , Ratones Endogámicos C57BL , Pirazoles/farmacología , Piridinas/farmacología , Enfermedades Vasculares/tratamiento farmacológico , Enfermedades Vasculares/metabolismo , Vasodilatadores/farmacologíaRESUMEN
Hereditary anemias are a group of heterogeneous disorders including hemolytic anemias and hyporegenerative anemias, as congenital dyserythropoietic anemia (CDA). Causative mutations occur in a wide range of genes leading to deficiencies in red cell production, structure, or function. The genetic screening of the main genes is important for timely diagnosis, since routine laboratory tests fail in a percentage of the cases, appropriate treatment decisions, and genetic counseling purposes. A conventional gene-by-gene sequencing approach is expensive and highly time-consuming, due to the genetic complexity of these diseases. To overcome this problem, we customized a targeted sequencing panel covering 35 genes previously associated to red cell disorders. We analyzed 36 patients, and potentially pathogenic variants were identified in 26 cases (72%). Twenty variants were novel. Remarkably, mutations in the SPTB gene (ß-spectrin) were found in 34.6% of the patients with hereditary spherocytosis (HS), suggesting that SPTB is a major HS gene in the Southeast of Brazil. We also identified two cases with dominant HS presenting null mutations in trans with α-LELY in SPTA1 gene. This is the first comprehensive genetic analysis for hereditary anemias in the Brazilian population, contributing to a better understanding of the genetic basis and phenotypic consequences of these rare conditions in our population.
Asunto(s)
Anemia Diseritropoyética Congénita/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Espectrina/genética , Esferocitosis Hereditaria/genética , Brasil , Femenino , Humanos , MasculinoRESUMEN
Umbilical cord blood contains undifferentiated mesenchymal stem cells (MSCs) with chondrogenic potential that may be used for the repair of joint damage. The role of growth factors during the process of chondrogenesis is still not entirely understood. The objective of this study was to evaluate the formation of chondrocytes, cartilaginous matrix and type II collagen from human umbilical cord blood stem cells exposed to two different growth factors, BMP-6 and BMP-2, while being cultured as a micromass or a monolayer. Umbilical cord blood was obtained from full-term deliveries, and then, mononuclear cells were separated and cultured for expansion. Afterward, these cells were evaluated by flow cytometry using antibodies specific for MSCs and induced to chondrogenic differentiation in micromass and monolayer cultures supplemented with BMP-2 and BMP-6. Cellular phenotype was evaluated after 7, 14 and 21 days by RT-PCR and Western blot analysis to identify the type II collagen and aggrecan. The expanded cells displayed surface antigens characteristic of mesenchymal progenitor cells and were negative for hematopoietic differentiation antigens. Type II collagen and aggrecan mRNAs were expressed from day 14 in cells stimulated with BMP-2 or BMP-6. Type II collagen was demonstrated by Western blotting in both groups, and the greatest expression was observed 21 days after the cells were stimulated with BMP-2 cultured in micromass. BMP-2 in micromass culture was more efficient to induce the chondrogenesis.
Asunto(s)
Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 6/farmacología , Condrocitos/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Agrecanos/genética , Agrecanos/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Condrogénesis/fisiología , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Sangre Fetal/citología , Expresión Génica/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/metabolismoRESUMEN
OBJECTIVES: Erythroid differentiation is a dynamic process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. In this study, gene expression profiles during differentiation of human erythroid cells of a normal blood donor were evaluated using SAGE. MATERIALS AND METHODS: Global gene expression was evaluated in cells collected immediately before addition of erythropoietin (0 h) and 192 and 336 h after addition of this hormone. Real-time PCR was used to evaluate activation of differentially expressed genes. RESULTS: The data indicate that global aspects of the transcriptome were similar during differentiation of the majority of the genes and that a relatively small set of genes is probably involved in modification of erythroid cells during differentiation. We have identified 93 differentially expressed genes during erythroid development, and expression of some of these was confirmed by qPCR. Various genes including EYA3, ERH, HES6, TIMELESS and TRIB3 were found to be homologous to those of Drosophila melanogaster and here are described for the first time during erythroid development. An important and unique carboxypeptidase inhibitor described in mammalians, LXN, was also identified. CONCLUSIONS: The results of this study amplify previously published data and may contribute to comprehension of erythroid differentiation and identification of new target genes involved in some erythroid concerning diseases.
Asunto(s)
Diferenciación Celular/genética , Células Eritroides/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Antígenos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Células Eritroides/citología , Eritropoyetina/farmacología , Genoma/genética , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Fosfatasas/genética , ARN Mensajero/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: To identify clinical and genetic risk factors for moderate hyperbilirubinemia during the first week of life. STUDY DESIGN: Using univariate and multivariate multiple regression analyses, the RR for clinical factors, the African variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency (G202A/A376G), and (TA)(n) UGT1A1 polymorphisms were established in a cohort of 608 Brazilian newborn infants. Hyperbilirubinemia was monitored until 134.5 ± 49.8 h of life (IQR, 111.0 to 156.7). The dependent variable was total bilirubinemia (TB) ≥12.9 mg per 100 ml estimated by transcutaneous or plasma bilirubin measurements. RESULT: The African variant of G6PD deficiency and (TA)(7)/(TA)(7) and (TA)(7)/(TA)(8) polymorphisms present in 6.1 and 12.0% of newborns, respectively, were not risk factors for moderate hyperbilirubinemia. Coexpression of G6DP deficiency and UGT1A1 polymorphisms occurred in 0.49% of the subjects. Independent clinical predictors for TB≥ 12.9 mg per 100 ml were gestational age <38 weeks and reference curve percentiles >P40th. CONCLUSION: In this study, G6PD deficiency and UGT1A1 gene promoter polymorphisms were not risk factors for moderate hyperbilirubinemia. Genetic factors may vary considerably in importance among different populations.
Asunto(s)
Comparación Transcultural , Hiperbilirrubinemia Neonatal/diagnóstico , Hiperbilirrubinemia Neonatal/genética , Brasil , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Tamización de Portadores Genéticos , Genotipo , Deficiencia de Glucosafosfato Deshidrogenasa/diagnóstico , Deficiencia de Glucosafosfato Deshidrogenasa/genética , Glucuronosiltransferasa/genética , Humanos , Recién Nacido , Kernicterus/diagnóstico , Kernicterus/genética , Masculino , Tamizaje Neonatal , Polimorfismo Genético/genética , Estudios Prospectivos , Factores de RiesgoRESUMEN
OBJECTIVE: There are few studies regarding vitamin B12 deficiency in developing countries. In Brazil, a late diagnosis of vitamin B12 deficiency progressing to severe neurological damage is common. Thus, the aim of the present study was to verify the frequency of vitamin B12 deficiency in two Brazilian populations (elderly and adult participants) and to compare different methods of vitamin B12 deficiency detection. DESIGN: Five hundred participants were recruited from health centres from south-east Brazil and were separated into two groups: 60 years old or more and 30-59 years old. Vitamin B12 and folate concentrations were measured using electrochemiluminescence immunoassay (ECI) and RIA. Methylmalonic acid (MMA) was measured by LC coupled to tandem MS. Full blood counts were acquired using standard methods. RESULTS: All participants had normal blood count results and mean cell volume less than 99 fl; none of them presented folate deficiency according to the results, which were all greater than 3 ng/ml. Cobalamin levels less than 200 pmol/l were identified by one of the two or by both methods in 7.2 % of the participants aged 60 years or more and 6.4 % of the participants aged 30-59 years. MMA levels were higher in older subjects (P = 0.007) compared with younger subjects. A greater correlation of MMA v. RIA was observed than of MMA v. ECI (P = 0.0017 v. P = 0.014). MMA quantification estimated that cobalamin deficiency was present in more than 11 % of the subjects for both studied groups. CONCLUSIONS: The study shows that vitamin B12 deficiency is frequent in Brazilian adults and suggests that RIA is more sensitive than ECl for measuring cobalamin levels.
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Ácido Metilmalónico/sangre , Deficiencia de Vitamina B 12/epidemiología , Vitamina B 12/sangre , Adulto , Factores de Edad , Anciano , Brasil/epidemiología , Humanos , Inmunoensayo/métodos , Persona de Mediana Edad , Prevalencia , Deficiencia de Vitamina B 12/sangre , Deficiencia de Vitamina B 12/diagnósticoRESUMEN
The JAK2 V617F mutation, present in the majority of polycythemia vera (PV) patients, causes constitutive activation of JAK2 and seems to be responsible for the PV phenotype. However, the transcriptional changes triggered by the mutation have not yet been totally characterized. In this study, we performed a large-scale gene expression study using serial analysis of gene expression in bone marrow cells of a newly diagnosed PV patient harboring the JAK2 V617F mutation and in normal bone marrow cells of healthy donors. JUNB was one of the genes upregulated in PV, and we confirmed, by quantitative real-time PCR, an overexpression of JUNB in hematopoietic cells of other JAK2 V617F PV patients. Using Ba/F3-EPOR cell lines and primary human erythroblast cultures, we found that JUNB was transcriptionally induced after erythropoietin addition and that JAK2 V617F constitutively induced JunB protein expression. Furthermore, JUNB knockdown reduced not only the growth of Ba/F3 cells by inducing apoptosis, but also the clonogenic and proliferative potential of human erythroid progenitors. These results establish a role for JunB in normal erythropoiesis and indicate that JunB may play a major role in the development of JAK2 V617F myeloproliferative disorders.
Asunto(s)
Proliferación Celular , Eritrocitos/patología , Janus Quinasa 2/genética , Mutación Missense , Trastornos Mieloproliferativos/etiología , Proteínas Proto-Oncogénicas c-jun/genética , Médula Ósea/patología , Linaje de la Célula , Eritropoyesis , Humanos , Policitemia Vera/genética , Proteínas Proto-Oncogénicas c-jun/fisiología , Células Tumorales CultivadasRESUMEN
Inflammation, cell adhesion to vascular endothelium, and endothelial injury contribute to sickle cell anemia (SCA) vaso-occlusion. Although alterations in inflammatory cytokines and biomarkers have been related, reports have been conflicting, and a conclusive role for these molecules in the disease remains to be established. Furthermore, the effect of hydroxyurea therapy (HU) on the release of inflammatory mediators is not understood. This study aimed to determine plasma levels and leukocyte gene expressions of inflammatory mediators in healthy controls, steady-state SCA patients, and SCA patients on HU therapy. TNF-alpha, IL-8, and PGE(2) levels were significantly higher in the plasma of SCA individuals when compared with control individuals. HU therapy was associated with a significant reversal of augmented TNF-alpha and, interestingly, increased plasma anti-inflammatory IL-10. IFN-gamma, IL-10, cyclooxygenase 2 (COX-2), and inducible NO synthase (iNOS) gene expressions were unaltered in SCA mononuclear cells (MC); however, gene expressions of TNF-alpha, IL-8, and the protective enzyme heme oxygenase-1 (HO-1) were significantly higher. HU therapy was not associated with significantly altered SCA MC inflammatory gene expression, although COX-2 mRNA expression was decreased. In SCA neutrophils, gene expressions of IL-8, IFN-gamma, iNOS, and HO-1 were significantly higher than those of control subjects. Patients on HU demonstrated lower iNOS and higher IL-10 neutrophil gene expressions. Taken together, data suggest that alterations in the gene expressions and productions of a number of pro- and anti-inflammatory mediators are present in SCA and importantly, in those patients on HU therapy. Knowledge of these pathways may contribute to further the understanding of the pathophysiology of this disease.
Asunto(s)
Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/tratamiento farmacológico , Citocinas/sangre , Hidroxiurea/uso terapéutico , Mediadores de Inflamación/sangre , Leucocitos/metabolismo , Adulto , Anemia de Células Falciformes/genética , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidroxiurea/farmacología , Leucocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Apoptotic protease activating factor 1 (APAF-1) has a critical role in the regulation of apoptosis. In the present study, the mRNA expression analysis of different APAF-1 transcripts (APAF-1S, APAF-1LC, APAF-1LN, and APAF-1XL) was analyzed in bone marrow samples from 37 patients with acute myeloid leukemia (newly diagnosed, with no previous treatment). APAF-1XL and APAF-1LN transcripts (with and without an extra WD-40 repeat region, respectively) were detected in all samples, although the major form expressed was APAF-1XL in 65% of the samples (group 1), while 35% of the samples expressed primarily APAF-1LN (group 2). Only 46% of the patients presented complete remission in response to remission induction therapy (represented by less than 5% marrow blasts and hematological recovery), all but 2 cases being from group 1, 21.6% did not attain complete remission (only 1 case from group 1), and 32.4% of the patients died early. Lower expression of APAF-1XL (APAF-1XL/APAF-1LN ratio <1.2) was associated with a poor response to therapy (P = 0.0005, Fisher exact test). Both groups showed similar characteristics regarding white blood cell counts, cytogenetic data or presence of gene rearrangements associated with good prognosis as AML1-ETO, CBFB-MYH11 and PML/RARA. Since it has been shown that only the isoforms with the extra WD-40 repeat region activate procaspase-9, we suggest that low procaspase-9 activation may also be involved in the deregulation of apoptosis and chemotherapy resistance in acute myeloid leukemia.
Asunto(s)
Factor Apoptótico 1 Activador de Proteasas/genética , Leucemia Mieloide Aguda/genética , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Células de la Médula Ósea/química , Estudios de Casos y Controles , ADN Complementario/genética , Densitometría , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción , Transcripción Genética/genética , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
Apoptotic protease activating factor 1 (APAF-1) has a critical role in the regulation of apoptosis. In the present study, the mRNA expression analysis of different APAF-1 transcripts (APAF-1S, APAF-1LC, APAF-1LN, and APAF-1XL) was analyzed in bone marrow samples from 37 patients with acute myeloid leukemia (newly diagnosed, with no previous treatment). APAF-1XL and APAF-1LN transcripts (with and without an extra WD-40 repeat region, respectively) were detected in all samples, although the major form expressed was APAF-1XL in 65 percent of the samples (group 1), while 35 percent of the samples expressed primarily APAF-1LN (group 2). Only 46 percent of the patients presented complete remission in response to remission induction therapy (represented by less than 5 percent marrow blasts and hematological recovery), all but 2 cases being from group 1, 21.6 percent did not attain complete remission (only 1 case from group 1), and 32.4 percent of the patients died early. Lower expression of APAF-1XL (APAF-1XL/APAF-1LN ratio <1.2) was associated with a poor response to therapy (P = 0.0005, Fisher exact test). Both groups showed similar characteristics regarding white blood cell counts, cytogenetic data or presence of gene rearrangements associated with good prognosis as AML1-ETO, CBFB-MYH11 and PML/RARA. Since it has been shown that only the isoforms with the extra WD-40 repeat region activate procaspase-9, we suggest that low procaspase-9 activation may also be involved in the deregulation of apoptosis and chemotherapy resistance in acute myeloid leukemia.
Asunto(s)
Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Factor Apoptótico 1 Activador de Proteasas/genética , Leucemia Mieloide Aguda/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Células de la Médula Ósea/química , Estudios de Casos y Controles , Densitometría , ADN Complementario/genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Mensajero/genética , Factores de Transcripción , Insuficiencia del Tratamiento , Transcripción Genética/genética , Biomarcadores de Tumor/genética , Adulto JovenRESUMEN
1. The major effect associated with hydroxyurea (HU) treatment of sickle cell anaemia (SCA) patients is an increase in fetal haemoglobin (HbF) synthesis, which inhibits the polymerization of haemoglobin S. 2. Hydroxyurea improves clinical symptoms by reducing the frequency of pain and vaso-occlusive crises, acute chest syndrome, transfusion requirements and hospitalization. 3. The molecular mechanisms responsible for HU-mediated induction of fetal globin transcription are not completely understood. Therefore, the aim of the present study was to identify differentially expressed genes participating in these mechanisms. 4. We established two suppression subtractive hybridization (SSH) libraries from reticulocytes obtained from SCA patients either not on or on HU treatment. The gene expression of some of the genes identified was subsequently evaluated by real-time polymerase chain reaction (PCR). 5. Genes identified with altered expression included SUDS3, FZD5 and PHC3, which may be associated with the regulation of globin expression. 6. This is the first demonstration of an association between HU treatment and the expression of genes identified in erythroid cells.
Asunto(s)
Perfilación de la Expresión Génica , Hidroxiurea/farmacología , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Rasgo Drepanocítico/metabolismo , Adulto , Femenino , Regulación de la Expresión Génica , Biblioteca de Genes , Humanos , MasculinoRESUMEN
OBJECTIVE: The aim of this investigation was to study the effect of vitamin E treatment in oxidative stress of red and white cells of beta-thalassaemia intermedia patients. METHODS: Nine patients undergoing occasional transfusions (5 females/4 males), median age 39 years (range 15-74), were recruited for oral daily administration of 400 IU vitamin E for 3 months. Twenty-seven milliliters of peripheral blood was obtained before and after 3 months of treatment, and 3 months after treatment completion. In the case of transfused patients (n = 4), blood was obtained at least 30 days after transfusion. Reactive oxygen species (ROS) was measured by flow cytometry; red blood cell (RBC) reduced glutathione (GSH) was measured by dinitrothiocyanobenzene reduction, serum malondialdehyde was measured in terms of thiobarbituric acid-reactive substances (TBARS), and alpha-haemoglobin-stabilizing protein (AHSP) mRNA expression was measured by real-time polymerase chain reaction of reticulocyte RNA extracts. RESULTS: beta-Thalassaemia patients presented basal levels of RBC ROS, GSH and serum TBARS statistically different compared with healthy controls. However, after vitamin E administration, patients presented a significant reduction in erythrocyte RBC ROS and serum TBARS levels. In parallel, red cell GSH was significantly increased after treatment. Peripheral mononuclear cells and T lymphocytes also demonstrated a reduction in ROS production. Therefore, after treatment, no significant differences were detected comparing patients and normal controls. Three months after treatment completion, all measurements showed a tendency of returning to basal values. A significant reduction in reticulocyte number was observed after vitamin E treatment. Vitamin E treatment did not modify levels of haemoglobin or AHSP mRNA expression. CONCLUSION: Although vitamin E is not capable of reducing anaemia in these patients, it could be useful for reducing oxidative damage in other target organs of beta-thalassaemic patients. Finally, this is the first study to analyse the effects of vitamin E on ROS production in red and white blood cells and AHSP mRNA expression.
Asunto(s)
Antioxidantes/uso terapéutico , Vitamina E/uso terapéutico , Talasemia beta/tratamiento farmacológico , Talasemia beta/fisiopatología , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: Human umbilical cord blood (UCB) is an important source of haematopoietic stem cells; however, the behaviour of progenitor cells obtained from premature and full-term neonates is still a controversy subject. Thus, the aim of this study was to evaluate cell cycle parameters and the proliferative capacity of UCB progenitor cells from premature and full-term neonates. MATERIAL AND METHODS: Clonogenic assays were performed with methylcellulose, medium supplemented with recombinant stimulating growth factors and the colonies were scored on the seventh day and the 14th day of culture. A cell cycle study was carried out by DNA analysis using flow cytometry and 30 000 events were acquired; p107 and p130 expressions were analysed by Western blotting. RESULTS: Cultures obtained from UCB of premature neonates showed an early growth of colony-forming unit (CFU)-burst forming unit erythroid/CFU-granulocyte, erythrocyte, macrophage and megakaryocyte (BFU-E/GEMM), and CFU-granulocyte, macrophage (GM) by the seventh day of culture (P < 0.001). Therefore, the number and morphological characteristics of these colonies were comparable with those obtained from full-term neonates, on the 14th day of culture. At the 14th day, a large amount of CFU-GM was detected in the premature group (P < 0.0032). The premature culture on the 14th day showed fibroblasts and was comparable to those of full-term neonates on the 21st day in terms of number and morphology of the colonies. DNA analysis showed that the number of cells in S-phase was also higher in premature samples when compared to full-term neonates, P < 0.0021 (0 h = 12.8 vs. 2.5%; 16 h = 10.5 vs. 5.9%; 20 h = 13.5 vs. 10.3%; 24 h = 13.8 vs. 9.1%; 48 h = 14.0 vs. 5.4%; 72 h = 20.5 vs. 8.9%; 96 h = 13.8 vs. 7.7%). The Western blotting results demonstrated that p107 and p130 cell cycle protein expressions were higher in premature cells than in full-term cells. CONCLUSION: These results suggest that the higher capacity of proliferation and early differentiation of premature UCB might not be related only to the amount of stem/progenitor cells but also to a different timing of cell cycle entry.
Asunto(s)
Ciclo Celular , Proliferación Celular , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Recien Nacido Prematuro/sangre , Diferenciación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , ADN/análisis , Citometría de Flujo , Humanos , Recién NacidoRESUMEN
BACKGROUND AND PURPOSE: Reticulated platelet (RP) count provides an estimate of thrombopoiesis. The objective was to evaluate RP in patients in different stages of sickle cell disease (SCD) and to determine the relationship between interleukin-6 (IL-6), interleukin-3 (IL-3) and thrombopoietin (TPO) and RP count and degree of activation. METHODS: Eighty-nine adult patients with SCD were studied: 38 were in the steady state, 27 in hemolytic crisis (HC) and 24 in vaso-occlusive crisis (VOC). RPs and activated platelets were analyzed by flow cytometry. Soluble P-selectin, IL-6, IL-3 and thrombopoietin (TPO) levels were measured by ELISA tests. RESULTS: The patients in VOC had a higher absolute number of RPs and CD62P+ platelets than did the control group or patients in the steady state. A significant correlation was observed between the absolute number of CD62P+ platelets and RPs in patients in the steady state, HC and VOC. In the steady-state group of patients, the level of soluble P-selectin was found to be dependent on the RP values. IL-3 and TPO serum levels were higher in patients in the steady state, HC and VOC than in the control group. IL-6 serum levels were higher in HC and VOC patients than in the control group and higher in patients in the steady state than in the VOC group. CONCLUSION: Our results suggest that PRs contribute to the vaso-occlusive process in sickle cell disease. Increased interleukin serum levels probably indicate that inflammatory process is involved in the vascular-occlusive phenomenon. However, it appears that these inflammatory mediators do not have an effect on thrombopoiesis in sickle-cell-disease patients.
Asunto(s)
Anemia de Células Falciformes/sangre , Plaquetas/metabolismo , Adulto , Anemia de Células Falciformes/fisiopatología , Estudios de Casos y Controles , Femenino , Humanos , Interleucina-3/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana Edad , Selectina-P/sangre , Recuento de Plaquetas , Índice de Severidad de la Enfermedad , Trombopoyetina/sangreRESUMEN
Cytokines are associated with inflammatory responses including febrile non-hemolytic transfusion reactions (FNHTR). Moreover, there are some polymorphisms of these cytokine genes associated with different levels of gene expression. The aim of the present study was to investigate the association of inflammatory cytokine gene polymorphisms with the occurrence of FNHTR in multitransfused patients. We studied two groups of transfused patients: one presenting FNHTR before 20 transfusions of red blood cells concentrates and the other which never presented FNHTR even after 20 transfusions. The gene polymorphisms studied were IL1B-511C/T and +3953C/T, IL1RN (intron 2, variable number tandem repeat), IL6-174G/C, IL10-1082G/A and -819C/T, TNF-308G/A and LTA+253G/A using polymerase chain reaction and restriction digestion or sequencing methods. An association of IL1RN*2.2 genotype with the occurrence of precocious FNHTR (P < 0.025) was detected. This allele and this genotype have been related with higher serum levels of interleukin (IL)-1beta in vivo and higher promoter activity. No other association was demonstrated. The association of gene polymorphisms related with the increase of inflammatory cytokine gene expression may be a relevant factor in FNHTR and requires confirmation.
Asunto(s)
Citocinas/genética , Fiebre/etiología , Polimorfismo Genético , Reacción a la Transfusión , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Genotipo , Hemólisis , Humanos , Inflamación , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Persona de Mediana Edad , Sialoglicoproteínas/genéticaRESUMEN
Sickle cell disease (SCD) is recognized as a chronic inflammatory condition. Cytokines are released in response to stress or pathological situations, and influence hematopoiesis. The aim of this study was to evaluate interleukin-3 (IL-3), interferon-gamma (IFN-gamma), and neopterin (NP) levels in steady-state patients with sickle cell anemia (SS) (n = 35) and SC hemoglobinopathy (n = 15) in order to verify the possible action of those cytokines and NP on iron metabolism and hematopoiesis. Serum IL-3 concentration was higher in SS and SC groups than in controls, whereas IFN-gamma levels did not differ among groups. SS patients presenting hemoglobin fetal (HbF) >or=8.5% had significantly higher IL-3 levels than those with HbF <8.5% (P = 0.0338). No correlation was observed among inflammatory and iron metabolism parameters. Significant correlations were observed between IL-3 and Hb levels (r = 0.4633, P = 0.0457), and IL-3 and HbF levels (r = 0.6011, P = 0.0065). A negative correlation was observed between IL-3 and reticulocyte counting (r = -0.4632, P = 0.0457) only in the SS group. NP levels were significantly higher in the SS and SC groups than in controls, but did not differ between SS and SC. No correlation was observed between NP and iron metabolism parameters. These data suggest that IL-3 stimulates hematopoiesis, and that SS patients, even in steady state, have macrophage/monocyte activation (represented by high levels of NP) that probably contributes to their chronic inflammatory condition.
Asunto(s)
Enfermedad de la Hemoglobina SC/sangre , Interleucina-3/sangre , Neopterin/sangre , Adolescente , Adulto , Eritropoyesis , Pruebas Hematológicas , Hematopoyesis/fisiología , Enfermedad de la Hemoglobina SC/patología , Hemoglobinas/análisis , Humanos , Interferón gamma/sangre , Persona de Mediana EdadRESUMEN
Patients with chronic renal insufficiency (CRI) have reduced hemoglobin levels, mostly as a result of decreased kidney production of erythropoietin, but the relation between renal insufficiency and the magnitude of hemoglobin reduction has not been well defined. Hereditary hemochromatosis is an inherited disorder of iron metabolism. The importance of the association of hemochromatosis with treatment for anemia among patients with CRI has not been well described. We analyzed the frequency of the C282Y and H63D mutations in the HFE gene in 201 Brazilian individuals with CRI undergoing hemodialysis. The analysis of the effects of HFE mutations on iron metabolism and anemia with biochemical parameters was possible in 118 patients of this study (hemoglobin, hematocrit, ferritin levels, transferrin saturation, and serum iron). A C282Y heterozygous mutation was found in 7/201 (3.4%) and H63D homozygous and heterozygous mutation were found in 2/201 (1.0%) and 46/201 (22.9%), respectively. The allelic frequencies of the HFE mutations (0.017 for C282Y mutation and 0.124 for H63D mutation) did not differ between patients with CRI and healthy controls. Regarding the biochemical parameters, no differences were observed between HFE heterozygous and mutation-negative patients, although ferritin levels were not higher among patients with the H63D mutation (P = 0.08). From what we observed in our study, C282Y/H63D HFE gene mutations are not related to degrees of anemia or iron stores in CRI patients receiving intravenous iron supplementation (P > 0.10). Nevertheless, the present data suggest that the H63D mutation may have an important function as a modulating factor of iron overload in these patients.
Asunto(s)
Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Fallo Renal Crónico/genética , Proteínas de la Membrana/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil , Estudios de Casos y Controles , Femenino , Genotipo , Hemocromatosis/sangre , Proteína de la Hemocromatosis , Humanos , Fallo Renal Crónico/sangre , Fallo Renal Crónico/terapia , Masculino , Persona de Mediana Edad , Mutación/genética , Diálisis RenalRESUMEN
Patients with chronic renal insufficiency (CRI) have reduced hemoglobin levels, mostly as a result of decreased kidney production of erythropoietin, but the relation between renal insufficiency and the magnitude of hemoglobin reduction has not been well defined. Hereditary hemochromatosis is an inherited disorder of iron metabolism. The importance of the association of hemochromatosis with treatment for anemia among patients with CRI has not been well described. We analyzed the frequency of the C282Y and H63D mutations in the HFE gene in 201 Brazilian individuals with CRI undergoing hemodialysis. The analysis of the effects of HFE mutations on iron metabolism and anemia with biochemical parameters was possible in 118 patients of this study (hemoglobin, hematocrit, ferritin levels, transferrin saturation, and serum iron). A C282Y heterozygous mutation was found in 7/201 (3.4 percent) and H63D homozygous and heterozygous mutation were found in 2/201 (1.0 percent) and 46/201 (22.9 percent), respectively. The allelic frequencies of the HFE mutations (0.017 for C282Y mutation and 0.124 for H63D mutation) did not differ between patients with CRI and healthy controls. Regarding the biochemical parameters, no differences were observed between HFE heterozygous and mutation-negative patients, although ferritin levels were not higher among patients with the H63D mutation (P = 0.08). From what we observed in our study, C282Y/H63D HFE gene mutations are not related to degrees of anemia or iron stores in CRI patients receiving intravenous iron supplementation (P > 0.10). Nevertheless, the present data suggest that the H63D mutation may have an important function as a modulating factor of iron overload in these patients.