RESUMO
Qinghaosu, known as artemisinin (ARS), has been for over two millennia, one of the most common herbs prescribed in traditional Chinese medicine (TCM). ARS was developed as an antimalarial drug and currently belongs to the established standard treatments of malaria as a combination therapy worldwide. In addition to the antimalarial bioactivity of ARS, anticancer activities have been shown both in vitro and in vivo. Like other natural products, ARS acts in a multi-specific manner also against hematological malignancies. The chemical structure of ARS is a sesquiterpene lactone, which contains an endoperoxide bridge essential for activity. The main mechanism of action of ARS and its derivatives (artesunate, dihydroartemisinin, artemether) toward leukemia, multiple myeloma, and lymphoma cells comprises oxidative stress response, inhibition of proliferation, induction of various types of cell death as apoptosis, autophagy, ferroptosis, inhibition of angiogenesis, and signal transducers, as NF-κB, MYC, amongst others. Therefore, new pharmaceutically active compounds, dimers, trimers, and hybrid molecules, could enhance the existing therapeutic alternatives in combating hematologic malignancies. Owing to the high potency and good tolerance without side effects of ARS-type drugs, combination therapies with standard chemotherapies could be applied in the future after further clinical trials in hematological malignancies.
Assuntos
Antineoplásicos/farmacologia , Artemisininas/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Animais , Antimaláricos/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Artemisininas/efeitos adversos , Artemisininas/química , Neoplasias Hematológicas/patologia , HumanosRESUMO
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.
Assuntos
Células da Medula Óssea/imunologia , Medula Óssea/patologia , Modelos Estatísticos , Síndromes Mielodisplásicas/mortalidade , Adulto , Idoso , Antígenos CD34/metabolismo , Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Separação Celular , Estudos de Viabilidade , Feminino , Citometria de Fluxo , Seguimentos , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/patologia , Prognóstico , Receptores de IgG/metabolismo , Medição de Risco/métodosRESUMO
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.
Assuntos
Anemia Diseritropoética Congênita/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Espectrina/genética , Esferocitose Hereditária/genética , Brasil , Feminino , Humanos , MasculinoRESUMO
Human milk is an important source of microorganisms for infant gut colonisation. Although the maternal antibiotic prophylaxis is an important strategy to prevent maternal/neonatal sepsis, it has to be investigated how it may affect the human milk microbiota, especially the genus Bifidobacterium, which has been associated to health benefits. Here, we investigated the impact of the maternal antibiotic prophylaxis on the human milk Bifidobacterium spp. and total bacteria counts, in the first week (short-term) and first month (medium-term) after delivery. Human milk samples were collected from 55 healthy lactating women recruited from the University Hospital of the University of São Paulo at days 7±3 and 30±4 after vaginal delivery. Twenty one volunteers had received maternal antibiotic prophylaxis (MAP group) and 34 had not received MAP (no-MAP group) during or after labour. Total DNA was isolated from milk samples, and the bacterial counts were estimated by quantitative PCR (qPCR). We found lower levels of Bifidobacterium in the MAP group in the first week after delivery (median = 2.1 vs 2.4 log of equivalent cells/ml of human milk, for MAP and no-MAP groups, respectively; P=0.01), although there were no statistical differences in total bacteria count. However, no differences were found in Bifidobacterium counts between the groups at day 30±4 (median = 2.5 vs 2.2 log of equivalent cells/ml of human milk, for MAP and no-MAP groups, respectively; P=0.50). Our results suggest that MAP has a significant impact on Bifidobacterium counts in human milk, reducing this population in the first week after delivery. However, throughout the first month after delivery, the Bifidobacterium counts tend to recover, reaching similar counts to those found in no-MAP group at day 30±4 after delivery.
Assuntos
Antibacterianos/administração & dosagem , Antibioticoprofilaxia/métodos , Carga Bacteriana , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/isolamento & purificação , Leite Humano/microbiologia , Período Pós-Parto , Adolescente , Adulto , Antibacterianos/efeitos adversos , Antibioticoprofilaxia/efeitos adversos , Brasil , Feminino , Voluntários Saudáveis , Hospitais Universitários , Humanos , Recém-Nascido , Masculino , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
The aim of this study was to evaluate the assimilation of the prebiotics fructooligosaccharides (FOS), galactooligosaccharides (GOS), and Konjac glucomannan oligosaccharides (KGMO) by three human (H1, H2 and H3) and pig (P1, P2 and P3) faecal microbiotas in the presence of the potentially probiotic strain Lactobacillus amylovorus DSM 16698, using an in vitro batch fermentation model. Total bacteria and L. amylovorus populations were quantified using qPCR and biochemical features (pH, production of short chain fatty acids (SCFA), lactate, ammonia, and carbohydrate assimilation) were determined. L. amylovorus did not have a competitive advantage under in vitro conditions, reflected by its reduced relative abundance during fermentation despite the carbohydrate sources added. Pig microbiota sustained more stable probiotic counts. Intermittently produced lactate was possibly assimilated by the microbiota and converted to other SCFA as the carbohydrates were assimilated, with H3 probably having a methanogenic metabolism with high lactate and acetate consumption except in the presence of FOS, which assimilation resulted in the highest total SCFA for this volunteer. Addition of FOS also resulted in lower pH and ammonia, which might have been used as nitrogen source by pig microbiota. KGMO needed longer fermentation periods to be completely assimilated by both human and porcine faecal microbiotas. Overall, our results reinforce the notion that care must be taken when generalising the effects claimed for a given probiotic or potentially probiotic strain, including the combination with different prebiotic substrates, since they may vary considerably among individuals, which is important when studying potentially pro- and prebiotic combinations for application as functional foods and feed ingredients.
Assuntos
Carboidratos da Dieta/metabolismo , Microbioma Gastrointestinal , Lactobacillus acidophilus/metabolismo , Prebióticos , Animais , Cromatografia por Troca Iônica , Ácidos Graxos Voláteis/metabolismo , Fezes/microbiologia , Fermentação , Frutose/metabolismo , Galactose/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mananas/metabolismo , Oligossacarídeos/metabolismo , SuínosRESUMO
BACKGROUND AND OBJECTIVES: Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96 h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation. MATERIALS AND METHODS: Twenty UCB units were analyzed at 24 and 96 h after collection, frozen for 6 months, thawed and re-evaluated. MNCs were analyzed by flow cytometry, viability by 7-AAD and clonogenic assays (CFU) were performed. RESULTS: After 96 h of storage, no substantial loss of MNC was found (median 7.320 × 10(6 ) × 6.05 × 10(6) ). Percentage and viability CD34(+) cells, B-cell precursors and mesenchymal stem cells were not affected. However, mature B and T lymphocytes as well as granulocytes had a substantial loss. CFU growth was observed in all samples. Prefreezing storage of 96 h was associated with a relative loss of colony formation (median 12%). Postthaw, this loss had a median of 49% (24 h samples) to 56% (96 h samples). CONCLUSION: The delay of 96 h before UCB processing is possible, without a prohibitive impairment of CD34(+) loss in number and functionality.
Assuntos
Antígenos CD34/metabolismo , Criopreservação/métodos , Sangue Fetal/citologia , Células-Tronco/citologia , Antígenos CD34/genética , Sobrevivência Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Células-Tronco/metabolismo , TemperaturaRESUMO
The present study evaluated the plasma fatty acid levels and the vascular prostaglandin (PG) release in a rabbit model of early hypercholesterolemia with endothelial dysfunction. Rabbits were fed either a control diet (CD) or a diet containing 1 % cholesterol (HD) for 5-6 weeks. The level of fatty acids was measured in plasma. The levels of PG and nitric oxide (NO) released from the aorta were also determined. Vascular morphology of the aorta was characterized by intima and media thickness measurements. The rabbits fed with HD had higher levels of arachidonic acid (ARA) and lower levels of oleic acid. The linoleic acid level was unchanged. PGI(2) and NO were diminished and PGF(2α) levels, the PGI(2)/TXA(2) ratio and the intima/media ratio were increased in rabbits fed with HD. In conclusion, feeding HD for a short period increased ARA plasma levels and unbalanced release of vasodilator/vasoconstrictor PG redirected the pathway to vasoconstrictor metabolite release. These lipid metabolism alterations in addition to the reduced NO levels and the moderate changes in the vascular morphology contributed to the endothelial dysfunction in this animal model. Therefore, the present findings support the importance of early correction or prevention of high cholesterol levels to disrupt the endothelial dysfunction process that leads to cardiovascular disease.
Assuntos
Endotélio Vascular/fisiopatologia , Ácidos Graxos Ômega-6/sangue , Ácidos Graxos/sangue , Homeostase , Hipercolesterolemia/sangue , Hipercolesterolemia/metabolismo , Prostaglandinas/metabolismo , Animais , Colesterol/administração & dosagem , Colesterol/efeitos adversos , Ácidos Graxos/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Masculino , Prostaglandinas/sangue , CoelhosRESUMO
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.
Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 6/farmacologia , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Agrecanas/genética , Agrecanas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/metabolismo , Condrogênese/fisiologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Sangue Fetal/citologia , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/metabolismoRESUMO
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
AIMS: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. METHODS AND RESULTS: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell-free supernatants containing bacteriocins, added to 3-h-old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto-aggregation was strain-specific, and values ranged from 7·2% for ET35 to 12·1% for ET05. Various degrees of co-aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco-2 cells was within the range reported for Lactobacillus rhamnosus GG, a well-known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61·964·6%), Lact. fermentum (78·9%), Lact. delbrueckii (43·7%) and Ped. acidilactici (51·3%), which are higher than the one recorded for Lact. rhamnosus GG (53·3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain-dependent manner. CONCLUSIONS: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco-2 cells was within the range reported for Lact. rhamnosus GG, a well-known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics.
Assuntos
Antibacterianos/biossíntese , Bacteriocinas/biossíntese , Probióticos , Salmão/microbiologia , Animais , Antibacterianos/farmacologia , Aderência Bacteriana , Bacteriocinas/farmacologia , Células CACO-2 , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/metabolismo , Enterococcus faecium/fisiologia , Humanos , Lactobacillus/efeitos dos fármacos , Lactobacillus/metabolismo , Lactobacillus/fisiologia , Listeria monocytogenes/efeitos dos fármacos , Pediococcus/efeitos dos fármacos , Pediococcus/metabolismo , Pediococcus/fisiologia , Alimentos MarinhosRESUMO
Multiparametric flow cytometry is a useful co-criterion for diagnostic confirmation of MDS in patients with peripheral cytopenias and a normal karyotype. We examined the impact on patients' survival of several phenotypic aberrancies detected by a small 4-color panel of monoclonal antibodies (MoAbs). Diagnosis of the patients (54) was made by WHO criteria using peripheral blood counts, bone marrow (BM) morphology and karyotype. Flow cytometry was performed at diagnosis, and features obtained were compared to normal BM (24). We could detect 16 alterations: 4 in granulocytic precursors, 4 in monocytes, 6 in CD34+ cells, beside changes in plasmacytoid dendritic cells and basophil precursors. The total number of changes in RAEB was higher (median 8) than in cases with of abnormalities) were independent risk factors for a shorter survival. Our panel was sufficient to confirm the diagnosis of MDS and permitted to detect independent prognostic features.
Assuntos
Síndromes Mielodisplásicas/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/análise , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/imunologia , Fatores de RiscoRESUMO
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
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.
Assuntos
Diferenciação Celular/genética , Células Eritroides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Antígenos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Células Eritroides/citologia , Eritropoetina/farmacologia , Genoma/genética , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genéticaRESUMO
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 microg/50 g ptet-mEpoD and 0.5 microg/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 microg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% 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
Anemia/terapia , Caspase 9/genética , Dimerização , Eritropoetina/administração & dosagem , Expressão Gênica/genética , Terapia Genética/métodos , Tacrolimo/análogos & derivados , Animais , Caspase 9/administração & dosagem , Eritropoetina/genética , Vetores Genéticos/genética , Hematócrito , Injeções Intramusculares , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/uso terapêutico , Proteínas Recombinantes , Tacrolimo/uso terapêuticoRESUMO
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.
Assuntos
Comparação Transcultural , Hiperbilirrubinemia Neonatal/diagnóstico , Hiperbilirrubinemia Neonatal/genética , Brasil , Estudos de Coortes , Feminino , Seguimentos , Triagem de Portadores Genéticos , Genótipo , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Deficiência de Glucosefosfato Desidrogenase/genética , Glucuronosiltransferase/genética , Humanos , Recém-Nascido , Kernicterus/diagnóstico , Kernicterus/genética , Masculino , Triagem Neonatal , Polimorfismo Genético/genética , Estudos Prospectivos , Fatores de RiscoRESUMO
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.
Assuntos
Ácido Metilmalônico/sangue , Deficiência de Vitamina B 12/epidemiologia , Vitamina B 12/sangue , Adulto , Fatores Etários , Idoso , Brasil/epidemiologia , Humanos , Imunoensaio/métodos , Pessoa de Meia-Idade , Prevalência , Deficiência de Vitamina B 12/sangue , Deficiência de Vitamina B 12/diagnósticoRESUMO
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.
Assuntos
Anemia Falciforme/sangue , Anemia Falciforme/tratamento farmacológico , Citocinas/sangue , Hidroxiureia/uso terapêutico , Mediadores da Inflamação/sangue , Leucócitos/metabolismo , Adulto , Anemia Falciforme/genética , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Leucócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
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.