RESUMO
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.
Assuntos
Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Hidroxiureia/farmacocinética , Guanilil Ciclase Solúvel/metabolismo , Animais , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Hemoglobina Fetal/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Células K562 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Piridinas/farmacologia , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/metabolismo , Vasodilatadores/farmacologiaRESUMO
The impaired bioavailability of endogenous nitric oxide (NO) in sickle cell anemia (SCA) may be influenced by polymorphisms in the endothelial nitric oxide synthase gene (eNOS). We compared allelic/genotypic frequencies of the eNOS polymorphisms T-786C, VNTR4a/b and G894T between 89 adult SCA patients and 100 healthy controls, and investigated the relationship between these SNPs and markers of hemolysis [lactate dehydrogenase (LDH), indirect bilirubin (IB) and reticulocyte counts], inflammation [interleukins IL-1ß, IL-6, IL-8, Tumor Necrosis Factor (TNF-α) and C-reactive protein (CRP)] and endothelial dysfunction (ED) [soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), soluble L-selectin (sL-selectin), von Willebrand Factor (vWF) antigen and D-dimers] in the patients. The frequencies of the mutant -786C allele and -786C/C genotype were significantly higher in patients (p = 0.02 and p = 0.04, respectively) but not significantly correlated with the markers. For VNTR4a/b and G894T, the allelic/genotypic frequencies did not statistically differ between patient and control groups. Patients carrying the 4a allele and those with the 894G/G genotype showed a significant decrease in IB (p = 0.02 and p = 0.04, respectively), and only patients with the 4a allele exhibited reduced IL-1ß (p = 0.01). The correlation profiles between markers of inflammation and ED significantly differed between patients carrying the mutant alleles and those with wild-type genotypes. This appears to be the first report on the relationship between eNOS gene polymorphisms and markers of hemolysis, inflammation and ED in Brazilian SCA patients. Our results indicate that the SNPs analyzed may influence the phenotypic variability of these patients.
Assuntos
Anemia Falciforme/enzimologia , Anemia Falciforme/genética , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Hemólise , Molécula 1 de Adesão Intercelular/sangue , Óxido Nítrico Sintase Tipo III/genética , Polimorfismo de Nucleotídeo Único , Molécula 1 de Adesão de Célula Vascular/sangue , Fator de von Willebrand/análise , Adulto , Alelos , Anemia Falciforme/sangue , Anemia Falciforme/epidemiologia , Bilirrubina/sangue , Biomarcadores/sangue , Brasil/epidemiologia , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Frequência do Gene , Haplótipos , Humanos , Inflamação/sangue , L-Lactato Desidrogenase/sangue , Masculino , Contagem de Reticulócitos , Adulto JovemRESUMO
Sickle cell retinopathy (SCR) develops in up to 30% of sickle cell disease patients (SCD) during the second decade of life. Treatment for this affection remains palliative, so studies on its pathophysiology may contribute to the future development of novel therapies. SCR is more frequently observed in hemoglobin SC disease and derives from vaso-occlusion in the microvasculature of the retina leading to neovascularization and, eventually, to blindness. Circulating inflammatory cytokines, angiogenic factors, and their interaction may contribute to the pathophysiology of this complication. Angiopoietin (Ang)-1, Ang-2, soluble vascular cell adhesion molecule-1, intercellular adhesion molecule (ICAM)-1, E-selectin, P-selectin, IL1-ß, TNF-α, pigment epithelium derived factor (PEDF) and vascular endothelial growth factor plasmatic levels were determined in 37 SCD patients with retinopathy, 34 without retinopathy, and healthy controls. We observed that sICAM-1 is significantly decreased, whereas PEDF is elevated in HbSC patients with retinopathy (P=0.012 and P=0.031, respectively). Ang-1, Ang-2 and IL1-ß levels were elevated in SCD patients (P=0.001, P<0.001 and P=0.001, respectively), compared to controls, and HbSS patients presented higher levels of Ang-2 compared to HbSC (P<0.001). Our study supports the possible influence of sICAM-1 and PEDF on the pathophysiology of retinal neovascularization in SCD patients.
Assuntos
Anemia Falciforme/sangue , Proteínas do Olho/sangue , Molécula 1 de Adesão Intercelular/sangue , Fatores de Crescimento Neural/sangue , Neovascularização Retiniana/sangue , Serpinas/sangue , Adulto , Anemia Falciforme/complicações , Angiopoietina-1/sangue , Angiopoietina-2/sangue , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-1beta/sangue , Masculino , Pessoa de Meia-Idade , Neovascularização Retiniana/etiologia , Fator de Necrose Tumoral alfa/sangueRESUMO
O objetivo deste trabalho foi avaliar a variação do perfil proteico e do cálcio solúvel na coagulação do leite pelo etanol nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Amostras de leite de 61 animais foram avaliadas quanto à estabilidade ao etanol nas concentrações de 66 a 92 por cento (v/v) nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Três amostras, após 24 horas de armazenamento a 4ºC, foram ultracentrifugadas em quadruplicata (40.000 x g) a 4ºC e a 20ºC, respectivamente, por 60 minutos. Em seguida, o sobrenadante foi retirado e submetido à análise do cálcio solúvel pela técnica via úmida (digestão nitroperclórica) e leitura em espectrofotômetro de absorção atômica. O perfil proteico foi analisado pela técnica de eletroforese capilar empregando kit específico para determinação proteica. Os resultados mostraram uma correlação positiva entre o aumento da temperatura das amostras e a estabilidade do leite frente às diferentes concentrações de etanol. A porcentagem de cálcio solúvel no sobrenadante após ultracentrifugação foi maior nas amostras tratadas a 4ºC (P<0,05). As amostras ultracentrifugadas na temperatura de 4ºC apresentaram quantidades superiores de β-caseína no sobrenadante em comparação com as amostras tratadas a 20ºC. O abaixamento da temperatura favoreceu a migração da β-caseína e do cálcio coloidal para a fase solúvel do leite, o que possivelmente favoreceu o aumento da instabilidade das amostras no teste do etanol. Os resultados sugerem que a temperatura ideal para a realização de teste de estabilidade do leite frente ao etanol deveria ser de 21ºC...
The aim of this study was to evaluate the variation in protein profile and soluble calcium in milk coagulation by ethanol at 4ºC, 10ºC, 15ºC and 20ºC. Milk samples from 61 dairy cows were evaluated for stability of ethanol concentrations from 66 to 92 percent (v/v) at temperatures of 4°C, 10°C, 15°C and 20°C. Three samples were ultracentrifuged (40,000 x g) after 24 hours of storage at 4°C and 20°C, respectively, for 60 minutes. Their supernatants were removed and subjected to analyses of soluble calcium through nitro-perchloric digestion and atomic absorption spectrophotometry. The protein profiles were determined by capillary electrophoresis using a specific kit for protein determination. The results showed a positive correlation between the increase in temperature of the samples and the stability of milk against various concentrations of ethanol. The percentage of soluble calcium in the supernatant after centrifugation was higher in samples treated at 4°C (P<0.05). The samples ultracentrifuged at 4°C showed higher amounts of β-casein in the supernatant compared with samples stored at 20°C. The lowering of the temperature favored the migration of β-casein and colloidal calcium to the soluble phase of milk, which may also have favored the instability of milk in the ethanol test. According to the results, the milk sample temperature for the ethanol stability test should be 21ºC...
Assuntos
Animais , Cálcio/química , Etanol/efeitos adversos , Proteínas do Leite/química , Ultracentrifugação , Leite/metabolismo , Temperatura de TransiçãoRESUMO
The purpose of this study was to test the precision and accuracy of three-dimensional (3D) linear measurements for Le Fort I osteotomy, obtained from multi-slice computed tomography (MSCT) and cone beam computed tomography (CBCT) scans. The study population consisted of 11 dried skulls submitted to 64-row MSCT and CBCT scans. Three-dimensional reconstructed images (3D-CT) were generated, and linear measurements (n=11) based on anatomical structures and landmarks of interest for Le Fort I osteotomy were performed independently by two oral and maxillofacial radiologists, twice each, using Vitrea software; this allows true 3D measurement on 3D-CT images. The results demonstrated no statistically significant differences between the inter-examiner and intra-examiner analyses, and physical and true 3D linear measurements using MSCT and CBCT images. Regarding examiner accuracy, no statistically significant differences were found for the comparisons among the physical and the MSCT and the CBCT linear measurements by either examiner. For examiners 1 and 2, the analysis intra-examiner correlation coefficient ranged from 0.87 to 0.96 and 0.82 to 0.98, respectively, using MSCT, and from 0.84 to 0.98 and 0.80 to 0.98, respectively, using CBCT, indicating almost perfect agreement for all analyses performed. 3D linear measurements obtained from MSCT and CBCT images were considered precise and accurate for Le Fort I osteotomy and thus accurate and helpful for Le Fort I osteotomy planning.
Assuntos
Tomografia Computadorizada de Feixe Cônico , Imageamento Tridimensional , Osteotomia de Le Fort , Crânio/diagnóstico por imagem , Adulto , Cadáver , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Interpretação de Imagem Radiográfica Assistida por Computador , Tomografia Computadorizada por Raios XRESUMO
BCR-ABL-negative myeloproliferative neoplasms (MPNs) are most frequently characterized by the JAK2V617F gain-of-function mutation, but several studies showed that JAK2V617F may not be the initiating event in MPN development, and recent publications indicate that additional alterations such as chromatin modification and microRNA (miRNA) deregulation may have an important role in MPN pathogenesis. Here we report that 61 miRNAs were significantly deregulated in CD34+ cells from MPN patients compared with controls (P<0.01). Global miRNA analysis also revealed that polycythemia vera (JAKV617F) and essential thrombocythemia (JAK2 wild type) patients have significantly different miRNA expression profiles from each other. Among the deregulated miRNAs, expression of miR-134, -214 and -433 was not affected by changes in JAK2 activity, suggesting that additional signaling pathways are responsible for the deregulation of these miRNAs in MPN. Despite its upregulation in MPN CD34+ and during normal erythropoiesis, both overexpression and knockdown studies suggest that miR-433 negatively regulates CD34+ proliferation and differentiation ex vivo. Its novel target GBP2 is downregulated during normal erythropoiesis and regulates proliferation and erythroid differentiation in TF-1 cells, indicating that miR-433 negatively regulates hematopoietic cell proliferation and erythropoiesis by directly targeting GBP2.
Assuntos
Biomarcadores Tumorais/genética , Diferenciação Celular , Proliferação de Células , Células Eritroides/citologia , MicroRNAs/genética , Transtornos Mieloproliferativos/genética , Antígenos CD34/metabolismo , Células Cultivadas , Células Eritroides/metabolismo , Eritropoese/fisiologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Humanos , Janus Quinase 2/genética , Luciferases/metabolismo , Mutação/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
MDM2/p53 pathway plays an important role in the control of apoptotic and proliferation mechanisms, and alterations in this pathway have been described in myelodysplastic syndromes (MDS). We investigated the frequency of MDM2 SNP309, TP53 Arg72Pro polymorphisms in de novo MDS and the association of these polymorphisms with clinical characteristics. Our results showed that the frequencies of genotypes for MDM2 SNP309 and TP53 Arg72Pro did not differ between MDS and healthy controls and that these polymorphisms were not associated with clinical and laboratory parameters, disease progression and overall survival, suggesting that MDM2 and TP53 polymorphisms are not involved in risk for MDS, or in the clinical and laboratory characteristics of the disease.
Assuntos
Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , DNA de Neoplasias/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
Neutrophils participate in the initiation and progression of rheumatoid arthritis (RA) although the exact mechanisms responsible for neutrophil accumulation in rheumatoid joints are not understood. This study compared the adhesive and chemotactic functions of neutrophils from RA patients in activity (DAS28 > 3.2) and not in activity (DAS28 < 2.6) and observed the effects of different treatment approaches on these functions. Neutrophils were isolated from healthy controls (CON), and patients with active or inactive RA in use of therapy not specific for RA (NSAIDs), in use of DMARDs and in use of anti-TNF-α therapy. Adhesive and chemotactic properties were evaluated using in vitro assays; adhesion molecule expression was assessed by flow cytometry and real-time PCR and circulating chemokines were determined by ELISA. No significant alterations in the adhesive and chemotactic properties of neutrophils from active RA were observed when compared to CON neutrophils, independently of treatment regimen. In contrast, neutrophils from RA patients in disease remission presented reduced adhesive properties and a lower spontaneous chemotactic capacity, in association with decreased adhesion molecule expression, although profiles of alterations differed for those patients on DMARDs and those on anti-TNF-α therapy. Circulating levels of the major neutrophilic chemokines, IL-8 and epithelial neutrophil activating peptide-78, were also significantly decreased in those patients demonstrating a clinical response. Remission of RA appears to be associated with ameliorations in aspects important for neutrophil adhesion and chemotaxis; whether these alterations contribute to decrease neutrophil migration to the synovial fluid, with consequent improvements in the clinical manifestations of RA, remains to be determined.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Quimiocinas/sangue , Quimiotaxia de Leucócito/efeitos dos fármacos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Artrite Reumatoide/sangue , Artrite Reumatoide/diagnóstico , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Infliximab , Interleucina-8/sangue , Interleucina-8/farmacologia , Selectina L/sangue , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Indução de Remissão , Adulto JovemRESUMO
Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.
Assuntos
Adolescente , Adulto , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Adulto Jovem , Sondas de DNA/genética , Reação em Cadeia da Polimerase Multiplex , Mutação/genética , alfa-Globinas/genética , Talassemia alfa/genética , Brasil , Genótipo , Linhagem , Fenótipo , Sensibilidade e Especificidade , Talassemia alfa/diagnósticoRESUMO
The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.
Assuntos
Animais , Masculino , Camundongos , Anemia/terapia , Caspase 9/genética , Dimerização , Eritropoetina , Expressão Gênica/genética , Terapia Genética/métodos , Tacrolimo/análogos & derivados , Caspase 9/administração & dosagem , Eritropoetina , Vetores Genéticos/genética , Hematócrito , Injeções Intramusculares , Lentivirus/genética , Plasmídeos/uso terapêutico , Tacrolimo/uso terapêuticoRESUMO
BACKGROUND AND OBJECTIVES: The human neutrophil antigen-2 (HNA-2) is expressed on a subpopulation of neutrophils as most subjects present a negative plus a positive HNA-2 population of neutrophils. The number of neutrophils expressing HNA-2 is variable and may increase in pregnancy, infections, myeloproliferative disorders and after G-CSF. This study investigated the presence of polymorphisms in the gene encoding HNA-2 (CD177) in individuals presenting different patterns of antigen expression and determined the association of single nucleotide polymorphisms (SNPs) with the heterogeneous HNA-2 expression. MATERIALS AND METHODS: Flow cytometry was employed to analyse the HNA-2 expression on neutrophils from 135 healthy subjects using two monoclonal antibodies (TAG4, 7D8). Sequencing reactions were performed on subjects whose antigen expression was low (< or = 50%), high (> or = 80%) or atypical (a nonreactive population plus two distinct positive cell populations). RESULTS: Five SNPs were detected, two of them (A793C, G1084A) were related to a low expression of HNA-2 (P = 0.031 and P = 0.004). Atypical antigen expression was observed in 5.9% (8/135) of the individuals, three nonpregnant women and five men. In these cases, the cDNA sequences revealed three SNPs (A134T, G156A and G1333A) strongly related to this atypical HNA-2 expression (P = 0.004, P = 0.006 and P < 0.0001, respectively). CONCLUSIONS: Our data show that polymorphisms in the CD177 are associated with variations in the HNA-2 expression and may be the cause of atypical expressions.
Assuntos
Isoantígenos/genética , Glicoproteínas de Membrana/genética , Neutrófilos/imunologia , Receptores de Superfície Celular/genética , Adulto , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI , Humanos , Isoantígenos/biossíntese , Isoantígenos/sangue , Isoantígenos/imunologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/imunologia , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/sangue , Receptores de Superfície Celular/imunologia , Adulto JovemRESUMO
The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.
Assuntos
Humanos , Regulação da Expressão Gênica/genética , Globinas/genética , Haplótipos/genética , Mutação/genética , Polimorfismo Genético/genética , Elementos Reguladores de Transcrição/genética , Luciferases/genéticaRESUMO
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.
Assuntos
Proliferação de Células , Eritrócitos/patologia , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/etiologia , Proteínas Proto-Oncogênicas c-jun/genética , Medula Óssea/patologia , Linhagem da Célula , Eritropoese , Humanos , Policitemia Vera/genética , Proteínas Proto-Oncogênicas c-jun/fisiologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Fator Apoptótico 1 Ativador de Proteases/genética , Leucemia Mieloide Aguda/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Células da Medula Óssea/química , Estudos de Casos e Controles , DNA Complementar/genética , Densitometria , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição , Transcrição Gênica/genética , Falha de Tratamento , Adulto JovemRESUMO
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.
Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Fator Apoptótico 1 Ativador de Proteases/genética , Leucemia Mieloide Aguda/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células da Medula Óssea/química , Estudos de Casos e Controles , Densitometria , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/tratamento farmacológico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/genética , Fatores de Transcrição , Falha de Tratamento , Transcrição Gênica/genética , Biomarcadores Tumorais/genética , Adulto JovemRESUMO
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.
Assuntos
Ciclo Celular , Proliferação de Células , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Recém-Nascido Prematuro/sangue , Diferenciação Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , DNA/análise , Citometria de Fluxo , Humanos , Recém-NascidoRESUMO
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.
Assuntos
Anemia Falciforme/sangue , Plaquetas/metabolismo , Adulto , Anemia Falciforme/fisiopatologia , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-3/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Contagem de Plaquetas , Índice de Gravidade de Doença , Trombopoetina/sangueRESUMO
INTRODUCTION: Chronic graft-vs-host disease (cGVHD) is a major cause of morbidity in long-term survivors of allogeneic hematopoietic progenitor cell transplantation. Herpesviruses are involved in the occurrence and progression of various oral diseases. AIM: The aim of this study was to investigate the role of human herpesvirus 6 (HHV6) in patients with oral manifestations of cGVHD. MATERIALS AND METHODS: Peripheral blood and oral fluids (whole saliva, gingival crevicular fluid and parotid gland saliva) from 19 cGVHD patients, and 28 blood donors were examined for HHV6. Oral tissue samples were collected from 12 cGVHD patients and 12 healthy individuals. Nested polymerase chain reaction was employed to identify the HHV6. RESULTS AND CONCLUSION: The virus was detected in whole saliva in 13 cGVHD patients (68%) and in 19 blood donors (67%). HHV6 was not identified in any of the gingival crevicular fluid and parotid gland saliva samples in cGVHD patients. In the control group 14.3% of both, four gingival crevicular fluid and four parotid gland saliva samples were positive. Two oral tissue samples of cGVHD patients were positive for HHV6. These results indicate that patients with oral manifestations of cGVHD and healthy individuals present high and similar incidence of HHV6 in blood and oral fluids. These data do not support the importance of HHV6 in oral lesions of cGVHD.
Assuntos
Doença Enxerto-Hospedeiro/virologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Herpesvirus Humano 6/patogenicidade , Doenças da Boca/virologia , Adulto , Estudos de Casos e Controles , DNA Viral/análise , Feminino , Líquido do Sulco Gengival/virologia , Doença Enxerto-Hospedeiro/etiologia , Humanos , Líquen Plano Bucal/etiologia , Líquen Plano Bucal/virologia , Masculino , Pessoa de Meia-Idade , Doenças da Boca/etiologia , Mucosa Bucal/virologia , Úlceras Orais/etiologia , Úlceras Orais/virologia , Saliva/virologia , Glândulas Salivares Menores/virologiaRESUMO
Hereditary hemorrhagic telangiectasia (HHT) or Osler-Rendu-Weber disease is a systemic fibrovascular dysplasia with an autosomal dominant inheritance pattern. Mutations in two genes, endoglin and ALK-1, are known to cause HHT, both of which mediate signaling by transforming growth factor beta ligands in vascular endothelial cells. Ten patients were analyzed. Diagnosis of HHT was carried out by means of family history, recurrent bleeding, and the presence of multiple telangiectases lesions. Conformation-sensitive gel electrophoresis analyses with consistent abnormal migration patterns were cloned and sequenced using the MegaBace 1000 DNA automated analyzer. Three novel mutations were identified in the coding sequence of the ALK-1 gene in five patients and their families, which demonstrated clinical manifestations of HHT type 2. These mutations included a G insertion and a T deletion of single base pairs in exons 3 and 7, as well as missense mutations in exons 7 and 8 of the ALK-1 gene. These data indicate that loss-of-function mutations in a single allele of the ALK1 locus are sufficient to contribute to defects in maintaining endothelial integrity. We suggest the high rate of mutation detection and the small size of the ALK-1 gene make genomic sequencing a viable diagnostic test for HHT2.
Assuntos
Receptores de Activinas Tipo II/genética , Mutação/genética , Telangiectasia Hemorrágica Hereditária/enzimologia , Telangiectasia Hemorrágica Hereditária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Análise Mutacional de DNA , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
Hemoglobin (Hb) Indianapolis [beta112 (G14) Cys-->Arg] is a rare and slightly unstable beta-globin variant. All carriers described to date were clinically normal with only mild reticulocytosis. We report here a case of a Brazilian patient in whom hemolytic anemia and acute renal failure were probably caused by the presence of this variant.