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1.
Br J Haematol ; 2019 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-31884679

RESUMO

Severe bacterial infection is a major complication causing morbidity and mortality in ß-thalassaemia/HbE patients. Innate immunity constitutes the first line of defence against bacterial infection. This study aimed to comprehensively investigate the innate immune phenotype and function related to factors predisposing to infection in non-transfusion-dependent (NTD) ß°-thalassaemia/HbE patients. Twenty-six patients and 17 healthy subjects were recruited to determine complement activity (C3, C4, mannose-binding lectin and CH50) and surface receptor expression including markers of phagocytosis (CD11b, CD16 and C3bR), inflammation (C5aR) and migration (CD11b, CXCR1 and CXCR2) on neutrophils and monocytes. In addition, phagocytosis and oxidative burst activity of neutrophils and monocytes against Escherichia coli and neutrophil migration were examined. Decreased C3 and surface expression of CD11b and C3bR on neutrophils were found in patients. However, phagocytosis of neutrophils in patients was still in the normal range. Interestingly, patients displayed a significant reduction of surface expression of CXCR2 [1705 ± 217 mean fluorescent intensity (MFI)] on neutrophils, leading to impaired neutrophil migration (9·2 ± 7·7%) when compared to neutrophils from healthy subjects (2261 ± 627 MFI and 27·8 ± 9% respectively). Moreover, surface expression of CXCR2 on neutrophils was associated with splenectomy status, serum ferritin and haemoglobin levels. Therefore, impaired neutrophil migration could contribute to the increased susceptibility to infection seen in NTD ß°-thalassaemia/HbE patients.

2.
PLoS One ; 14(10): e0223996, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31661492

RESUMO

α0-Thalassemia is an inherited hematological disorder caused by the deletion of α-globin genes. The Southeast Asian deletion (--SEA) is the most common type of α0-thalassemia observed in Southeast Asian countries. Regarding WHO health policy, an effective α0-thalassemia screening strategy is needed to control new severe α-thalassemia cases. In this study, a monoclonal antibody panel was used to develop immunochromatographic (IC) strip tests for detecting the Hb Bart's and ζ-globin chain. Among 195 samples, all α0-thalassemia traits (78 α0-thalassemia (--SEA) and 4 α0-thalassemia (--THAI)) had low MCV or MCH values. The sensitivity, specificity, PPV and NPV of the IC strip tests for ζ-globin and Hb Bart's for screening α0-thalassemia (--SEA) within the low MCV or MCH samples were 100%, 65.2%, 90.7%, 100% and 96.2%, 47.8%, 86.6%, 78.6%, respectively. All 4 α0-thalassemia (--THAI) traits were negative for ζ-globin chains but positive for Hb Bart's using the IC strip tests. These results led to a α0-thalassemia screening being proposed in which blood samples are first evaluated by MCV, MCH and Hb typing. Samples with high MCV and MCH values are excluded for the presence of the α0-thalassemia gene. Samples with low MCV or MCH values are assayed using the developed IC strip tests, where only samples testing positive are further assayed for α0-thalassemia by PCR. Patients with Hb H, EA Bart's or EF Bart's diseases do not need to use this IC strip assay. Thus, in this study, a simple and cost effective α0-thalassemia point of care test was developed.

3.
Nitric Oxide ; 93: 6-14, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31513867

RESUMO

Inhaled sodium nitrite has been reported to decrease pulmonary artery pressure in hemoglobin E/ß-thalassemia (HbE/ß-thal) patients with pulmonary hypertension. This study investigated the pharmacokinetics and pharmacodynamics of inhaled nebulized sodium nitrite in 10 healthy subjects and 8 HbE/ß-thal patients with high estimated pulmonary artery pressure. Nitrite pharmacokinetics, fraction exhaled nitric oxide (FENO), estimated right ventricular systolic pressure (eRVSP) measured by echocardiography, and platelet activation were determined. Nebulized sodium nitrite at doses used in this study (37.5 and 75 mg for healthy subjects and 15 mg for HbE/ß-thal patients) was well tolerated and did not cause changes in methemoglobin levels and systemic blood pressure. Absorption of inhaled nitrite was rapid with the absolute bioavailability of 18%. In whole blood, nitrite exhibited the dose-independent pharmacokinetics with clearance (CL) of 1.5 l/h/kg, volume of distribution (Vd) of 1.3 l/kg and half-life (t1/2) of 0.6 h. CL and Vd of nitrite was higher in red blood cells (RBC) than whole blood and plasma. HbE/ß-thal patients had lower nitrite CL and longer t1/2 in RBC than healthy subjects. FENO increased immediately after inhalation. Following nitrite inhalation, eRVSP remained unchanged but platelet activation was suppressed as evidenced by inhibition of adenosine diphosphate (ADP)-induced P-selectin expression and increase in phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets. There were no changes in markers of oxidative and nitrosative stress after inhalation. Our results support further development of inhaled nebulized sodium nitrite for treatment of pulmonary hypertension in ß-thalassemia.

4.
Biol Proced Online ; 21: 15, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31388336

RESUMO

Background: Couples who carry α-thalassemia-1 deletion are at 25% risk of having a fetus with hemoglobin Bart's hydrops fetalis. Southeast Asian deletion (--(SEA)) is the most common type of α-thalassemia 1 among Southeast Asian populations. Thus, identification of the (--(SEA)) α-thalassemia 1 carrier is necessary for controlling severe α-thalassemia in Southeast Asian countries. Results: Using our generated anti ζ-globin chain monoclonal antibodies (mAbs) clones PL2 and PL3, a simple immunostick test for detecting ζ-globin chain presence in whole blood lysates was developed. The procedure of the developed immunostick test was as follows. The immunostick paddles were coated with 50 µg/mL of mAb PL2 as capture mAb, or other control antibodies. The coated immunostick was dipped into cocktail containing tested hemolysate at dilution of 1:500, 0.25 µg/mL biotin-labeled mAb PL3 and horseradish peroxidase-conjugated streptavidin at dilution of 1:1000. The immunostick was then dipped in precipitating substrate and the presence of ζ-globin chain in the tested sample was observed by the naked eye. Upon validation of the developed immunostick test with various types of thalassemia and normal subjects, 100% sensitivity and 82% specificity for detection of the (--(SEA)) α-thalassemia-1 carriers were achieved. The mAb pre-coated immunostick can be stored at room temperature for at least 20 weeks. Conclusion: In this study, a novel simple immunostick test for the screening of (--(SEA)) α-thalassemia 1 carriers was presented. The developed immunostick test, within a single test, contains both positive and negative internal procedural controls.

5.
Ann Hematol ; 98(9): 2045-2052, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31243572

RESUMO

Thalassemia has a high prevalence in Thailand. Oxidative damage to erythroid cells is known to be one of the major etiologies in thalassemia pathophysiology. Oxidative stress status of thalassemia is potentiated by the heme, nonheme iron, and free iron resulting from imbalanced globin synthesis. In addition, levels of antioxidant proteins are reduced in α-thalassemia and ß-thalassemia erythrocytes. However, the primary molecular mechanism for this phenotype remains unknown. Our study showed a high expression of miR-144 in ß- and α-thalassemia. An increased miR-144 expression leads to decreased expression of nuclear factor erythroid 2-related factor 2 (NRF2) target, especially in α-thalassemia. In α-thalassemia, miR-144 and NRF2 target are associated with glutathione level and anemia severity. To study the effect of miR-144 expression, the gain-loss of miR-144 expression was performed by miR inhibitor and mimic transfection in the erythroblastic cell line. This study reveals that miR-144 expression was upregulated, whereas NRF2 expression and glutathione levels were decreased in comparison with the untreated condition after miR mimic transfection, while the reduction of miR-144 expression contributed to the increased NRF2 expression and glutathione level compared with the untreated condition after miR inhibitor transfection. Moreover, miR-144 overexpression leads to significantly increased sensitivity to oxidative stress at indicated concentrations of hydrogen peroxide (H2O2) and rescued by miR-144 inhibitor. Taken together, our findings suggest that dysregulation of miR-144 may play a role in the reduced ability of erythrocyte to deal with oxidative stress and increased RBC hemolysis susceptibility especially in thalassemia.


Assuntos
Eritrócitos/metabolismo , MicroRNAs/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Estresse Oxidativo , Regulação para Cima , Talassemia alfa/metabolismo , Talassemia beta/metabolismo , Eritrócitos/patologia , Feminino , Glutationa/biossíntese , Glutationa/genética , Hemólise , Humanos , Peróxido de Hidrogênio/metabolismo , Células K562 , Masculino , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Talassemia alfa/genética , Talassemia alfa/patologia , Talassemia beta/genética , Talassemia beta/patologia
6.
J Clin Pathol ; 72(8): 520-524, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31010830

RESUMO

AIMS: Iron overload is a major factor contributing to the overall pathology of thalassaemia, which is primarily mediated by ineffective erythropoiesis and shorter mature red blood cell (RBC) survival. Iron accumulation in RBCs generates reactive oxygen species (ROS) that cause cellular damage such as lipid peroxidation and RBC membrane deformation. Abnormal RBCs in patients with thalassaemia are commonly known as microcytic hypochromic anaemia with poikilocytosis. However, iron and ROS accumulation in RBCs as related to RBC morphological changes in patients with thalassaemia has not been reported. METHODS: Twenty-one patients with thalassaemia, including HbH, HbH with Hb Constant Spring and ß-thalassaemia/HbE (splenectomy and non-splenectomy) genotypes, and five normal subjects were recruited. RBC morphology was analysed by light and scanning electron microscopy. Systemic and RBC iron status and oxidative stress were examined. RESULTS: Decreased normocytes were observed in the samples of patients with thalassaemia, with RBC morphological abnormality being related to the type of disease (α-thalassaemia or ß-thalassaemia) and splenic status. Target cells and crenated cells were mainly found in splenectomised patients with ß-thalassaemia/HbE, while target cells and teardrop cells were found in non-splenectomised patients. Patients with thalassaemia had high levels of serum ferritin, red cell ferritin and ROS in RBCs compared with normal subjects (p<0.05). Negative correlations between the amount of normocytes and serum ferritin (rs=-0.518, p=0.011), red cell ferritin (rs=-0.467, p=0.025) or ROS in RBCs (rs=-0.672, p<0.001) were observed. CONCLUSIONS: Iron overload and its consequent intracellular oxidative stress in RBCs were associated with reduce normocytes in patients with thalassaemia.


Assuntos
Eritrócitos Anormais/ultraestrutura , Sobrecarga de Ferro/patologia , Ferro/sangue , Estresse Oxidativo , Talassemia/patologia , Adolescente , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Eritrócitos Anormais/metabolismo , Feminino , Ferritinas/sangue , Humanos , Sobrecarga de Ferro/sangue , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/sangue , Talassemia/sangue , Adulto Jovem
7.
Sci Rep ; 9(1): 6059, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988349

RESUMO

A hypercoagulable state leading to a high risk of a thrombotic event is one of the most common complications observed in ß-thalassemia/HbE disease, particularly in patients who have undergone a splenectomy. However, the hypercoagulable state, as well as the molecular mechanism of this aspect of the pathogenesis of ß-thalassemia/HbE, remains poorly understood. To address this issue, fifteen non-splenectomized ß-thalassemia/HbE patients, 8 splenectomized ß-thalassemia/HbE patients and 20 healthy volunteers were recruited to this study. Platelet activation and hypercoagulable parameters including levels of CD62P and prothrombin fragment 1 + 2 were analyzed by flow cytometry and ELISA, respectively. A proteomic analysis was conducted to compare the platelet proteome between patients and normal subjects, and the results were validated by western blot analysis. The ß-thalassemia/HbE patients showed significantly higher levels of CD62P and prothrombin fragment 1 + 2 than normal subjects. The levels of platelet activation and hypercoagulation found in patients were strongly associated with splenectomy status. The platelet proteome analysis revealed 19 differential spots which were identified to be 19 platelet proteins, which included 10 cytoskeleton proteins, thrombin generation related proteins, and antioxidant enzymes. Our findings highlight markers of coagulation activation and molecular pathways known to be associated with the pathogenesis of platelet activation, the hypercoagulable state, and consequently with the thrombosis observed in ß-thalassemia/HbE patients.

8.
PLoS One ; 13(9): e0203955, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30235277

RESUMO

Nitric oxide (NO) can be generated from nitrite by reductase activity of deoxygenated hemoglobin (deoxyHb) apparently to facilitate tissue perfusion under hypoxic condition. Although hemoglobin E (HbE) solutions have been shown to exhibit decreased rate of nitrite reduction to NO, this observation has never been reported in erythrocytes from subjects with hemoglobin E/ß-thalassemia (HbE/ß-thal). In this study, we investigated the nitrite reductase activity of deoxyHb dialysates from 58 non-splenectomized and 23 splenectomized HbE/ß-thal subjects compared to 47 age- and sex-matched normal subjects, and examined its correlation with platelet activity. Iron-nitrosyl-hemoglobin (HbNO) was measured by tri-iodide reductive chemiluminescence as a marker of NO generation. HbNO produced from the reaction of nitrite with deoxyHb dialysate from both non-splenectomized and splenectomized HbE/ß-thal subjects was lower than that of normal (AA) hemoglobin subjects. P-selectin expression, a marker of platelet activation, at baseline and in reactivity to stimulation by adenosine diphosphate (ADP), were higher in HbE/ß-thal subjects than normal subjects. HbNO formation from the reactions of nitrite and deoxyHb inversely correlated with baseline platelet P-selectin expression, HbE levels, and tricuspid regurgitant velocity (TRV). Nitrite plus deoxygenated erythrocytes from HbE/ß-thal subjects had a lower ability to inhibit ADP-induced P-selectin expression on platelets than erythrocytes from normal subjects. We conclude that deoxyHb in erythrocytes from HbE/ß-thal subjects has a decreased ability to reduce nitrite to NO, which is correlated with increased platelet activity in these individuals.


Assuntos
Hemoglobina E/metabolismo , Hemoglobinas/metabolismo , Nitrito Redutases/metabolismo , Ativação Plaquetária/fisiologia , Talassemia beta/metabolismo , Adulto , Plaquetas/metabolismo , Feminino , Humanos , Masculino , Selectina-P/metabolismo
9.
Sci Rep ; 8(1): 13033, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158562

RESUMO

Thromboembolic complication occurs frequently in ß-thalassaemia/HbE patients, particularly in splenectomised patients. Endothelial cells play an important role in thrombosis. There is strong evidence of endothelial cell activation and dysfunction in ß-thalassaemia. Microparticles (MPs) are associated with thrombosis and endothelial cell dysfunction in many diseases including ß-thalassaemia. However, the effect of thalassaemic-MPs on endothelial cells mediating thrombus formation has not been elucidated. In this study, the effects of circulating MPs from ß-thalassaemia/HbE patients on endothelial cell functions were investigated. The results showed that MPs directly induce tissue factor, interleukin (IL)-6, IL-8, intracellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin expression in human umbilical vein endothelial cells (HUVECs). Notably, the levels of these endothelial cell activation markers were significantly increased in HUVECs treated with MPs obtained from splenectomised ß-thalassaemia/HbE patients when compared to MPs from non-splenectomised patients or normal subjects. The increased endothelial cell activation ultimately lead to increased monocyte-endothelial cell adhesion. THP-1 and HUVECs adhesion induced by MPs from normal subjects, non-splenectomised and splenectomised patients increased to 2.0 ± 0.4, 2.3 ± 0.4 and 3.8 ± 0.4 fold, respectively when compared to untreated cells. This finding suggests that MPs play an important role on thrombosis and vascular dysfunction in ß-thalassaemia/HbE disease, especially in splenectomised cases.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Hemoglobinopatias/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Tromboembolia/patologia , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Citocinas/metabolismo , Hemoglobinopatias/complicações , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células THP-1/fisiologia , Tromboplastina/metabolismo
10.
Can J Physiol Pharmacol ; 96(9): 879-885, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29806986

RESUMO

Iron chelation can improve endothelial function. However, effect on endothelial function of deferiprone has not been reported. We hypothesized deferiprone could promote nitric oxide (NO) production in endothelial cells. We studied effects of deferiprone on blood nitrite and blood pressure after single oral dose (25 mg/kg) in healthy subjects and hemoglobin E/ß-thalassemia patients. Further, effects of deferiprone on NO production and endothelial NO synthase (eNOS) phosphorylation in primary human pulmonary artery endothelial cells (HPAEC) were investigated in vitro. Blood nitrite levels were higher in patients with deferiprone therapy than those without deferiprone (P = 0.023, n = 16 each). Deferiprone increased nitrite in plasma and whole blood of healthy subjects (P = 0.002 and 0.044) and thalassemia patients (P = 0.003 and 0.046) at time 180 min (n = 20 each). Asymptomatic reduction in diastolic blood pressure (P = 0.005) and increase in heart rate (P = 0.009) were observed in healthy subjects, but not in thalassemia patients. In HPAEC, deferiprone increased cellular nitrite and phospho-eNOS (Ser1177) (P = 0.012 and 0.035, n = 6) without alteration in total eNOS protein and mRNA. We conclude that deferiprone can induce NO production by enhancing eNOS phosphorylation in endothelial cells.


Assuntos
Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Piridonas/farmacologia , Adulto , Pressão Sanguínea/efeitos dos fármacos , Deferiprona , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Talassemia/metabolismo , Talassemia/patologia , Talassemia/fisiopatologia
11.
Nitric Oxide ; 76: 174-178, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28964835

RESUMO

Pulmonary hypertension is a life-threatening complication in ß-thalassemia. Inhaled sodium nitrite has vasodilatory effect on pulmonary vasculature. However, its effect on pulmonary artery pressure (PAP) in ß-thalassemia subjects with pulmonary hypertension has never been reported. In this study, we investigated the change in PAP during inhalation of sodium nitrite in 5 ß-thalassemia patients. We demonstrated that sodium nitrite administered by nebulization rapidly decreased PAP as measured by echocardiography and right heart catheterization. The effect of nitrite was short as PAP returned to baseline at end of inhalation. Our findings support acute pulmonary vasodilation effect of nitrite in ß-thalassemia with pulmonary hypertension.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/tratamento farmacológico , Artéria Pulmonar/efeitos dos fármacos , Nitrito de Sódio/administração & dosagem , Nitrito de Sódio/farmacologia , Talassemia beta/complicações , Administração por Inalação , Adulto , Relação Dose-Resposta a Droga , Ecocardiografia , Feminino , Coração/efeitos dos fármacos , Humanos , Masculino , Nitrito de Sódio/sangue
12.
Stem Cell Res ; 20: 80-83, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28395745

RESUMO

The thalassemias are a group of genetic disorders characterized by a deficiency in the synthesis of globin chains. In this study the MUi009-A human induced pluripotent stem cell line was successfully generated from peripheral blood CD34+ haematopoietic progenitors of a 32year old male who had coinherited a homozygous ß°-thalassemia mutation at codon 41/42 (-TCTT) and a heterozygous α-thalassemia 4.2 deletion. The MUi009-A cell line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into the three germ layers. The cell line may provide a tool for drug testing and gene therapy studies.


Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Talassemia alfa/patologia , Adulto , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Deleção de Genes , Genótipo , Heterozigoto , Humanos , Cariótipo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Microscopia de Fluorescência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Talassemia alfa/genética , Talassemia alfa/metabolismo
13.
Stem Cell Res ; 20: 84-87, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28395746

RESUMO

Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C) is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.


Assuntos
Reprogramação Celular , Hemoglobinas Anormais/genética , Células-Tronco Pluripotentes Induzidas/citologia , Antígenos CD34/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Análise Mutacional de DNA , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariótipo , Microscopia de Fluorescência , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Talassemia alfa/genética , Talassemia alfa/metabolismo , Talassemia alfa/patologia
14.
Biosens Bioelectron ; 82: 140-5, 2016 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-27084987

RESUMO

We selected and modified DNA aptamers specifically bound glycated human serum albumin (GHSA), which is an intermediate marker for diabetes mellitus. Our aptamer truncation study indicated that the hairpin-loop structure with 23 nucleotides length containing triple G-C hairpins and 15-nucleotide loop, plays an important role in GHSA binding. Fluorescent quenching graphene oxide (GO) and Cy5-labeled G8 aptamer were used in this study to develop simple and sensitive graphene based aptasensor for GHSA detection. The limit of detection (LOD) of our aptasensor was 50 µg/mL, which was lower than other existing methods. In addition, with the nuclease resistance system, our GHSA detection platform could also be used in clinical samples. Importantly, our approach could significantly reveal the higher levels of GHSA concentrations in diabetes than normal serums. These indicate that our aptasensor has a potential for diagnosis and monitoring of diabetes mellitus.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Diabetes Mellitus/diagnóstico , Grafite/química , Albumina Sérica/análise , Sequência de Bases , Carbocianinas/química , Diabetes Mellitus/sangue , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Óxidos/química
15.
J Gene Med ; 11(11): 1012-9, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19653252

RESUMO

BACKGROUND: Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. METHODS: We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. RESULTS: Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. CONCLUSIONS: The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.


Assuntos
Dependovirus/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Vetores Genéticos/genética , Mutação , Recombinação Genética , Células Cultivadas , Dependovirus/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Marcação de Genes/métodos , Vetores Genéticos/metabolismo , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Modelos Genéticos , Transdução Genética
16.
Int J Hematol ; 78(5): 421-8, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14704034

RESUMO

Interferon (IFN)-gamma is a survival factor for mature erythroid progenitor cells. To elucidate related survival mechanisms, we compared the role of phosphatidylinositol 3-kinase (PI3-kinase) in the survival signals of IFN-gamma and erythropoietin (EPO). Human erythroid colony-forming cells (ECFCs) purified from peripheral blood were used, and Ly294002 was used as a PI3-kinase inhibitor. Treating ECFCs with a high concentration of Ly294002 (50 micromol/L) in the presence of EPO and/or IFN-gamma reduced cell viability by inducing apoptosis. However, treating cells with a lower concentration of Ly294002 (10 micromol/L) did not affect the antiapoptotic function of IFN-gamma and abolished the antiapoptotic effect of EPO. Adding IFN-gamma or EPO induced Bcl-x expression in ECFCs, as determined by Western blotting, and expression was suppressed in the presence of Ly294002. We also examined the phosphorylation of the protein kinase Akt, the downstream target of PI3-kinase. EPO stimulation significantly increased the level of Akt phosphorylation, but IFN-gamma did not. These results suggest that IFN-gamma plays a role in preventing the apoptosis of erythroid progenitor cells by affecting Bcl-x expression, thereby reducing the disruption of the mitochondrial transmembrane potential via PI3-kinase pathways that are related to but distinct from the EPO pathway.


Assuntos
Apoptose/efeitos dos fármacos , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoetina/farmacologia , Interferon gama/farmacologia , Proteínas Serina-Treonina Quinases , Transdução de Sinais/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Células Precursoras Eritroides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Células-Tronco/farmacologia , Proteína bcl-X
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