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1.
Exp Hematol ; 82: 8-23, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32007479

RESUMO

Establishing an in vitro "red blood cell matrix" that would allow uninterrupted access to a stable, homogeneous reticulocyte population would facilitate the establishment of continuous, long-term in vitro Plasmodium vivax blood stage cultures. In this study, we have explored the suitability of the erythroleukemia K562 cell line as a continuous source of such reticulocytes and have investigated regulatory factors behind the terminal differentiation (and enucleation, in particular) of this cell line that can be used to drive the reticulocyte production process. The Duffy blood group antigen receptor (Fy), essential for P. vivax invasion, was stably introduced into K562 cells by lentiviral gene transfer. miRNA-26a-5p and miRNA-30a-5p were downregulated to promote erythroid differentiation and enucleation, resulting in a tenfold increase in the production of reticulocytes after stimulation with an induction cocktail compared with controls. Our results suggest an interplay in the mechanisms of action of miRNA-26a-5p and miRNA-30a-5p, which makes it necessary to downregulate both miRNAs to achieve a stable enucleation rate and Fy receptor expression. In the context of establishing P. vivax-permissive, stable, and reproducible reticulocytes, a higher enucleation rate may be desirable, which may be achieved by the targeting of further regulatory mechanisms in Fy-K562 cells; promoting the shift in hemoglobin production from fetal to adult may also be necessary. Despite the fact that K562 erythroleukemia cell lines are of neoplastic origin, this cell line offers a versatile model system to research the regulatory mechanisms underlying erythropoiesis.


Assuntos
Leucemia Eritroblástica Aguda , Plasmodium vivax/crescimento & desenvolvimento , Reticulócitos , Diferenciação Celular , Sistema do Grupo Sanguíneo Duffy/biossíntese , Sistema do Grupo Sanguíneo Duffy/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Células K562 , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/parasitologia , Leucemia Eritroblástica Aguda/patologia , MicroRNAs/biossíntese , MicroRNAs/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Reticulócitos/metabolismo , Reticulócitos/parasitologia , Reticulócitos/patologia
2.
Int J Hematol ; 111(4): 579-584, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31820325

RESUMO

Pregnancy-associated primary red cell aplasia (pPRCA) is a rare disorder that may occur at various time points during pregnancy. Unlike pregnancy-associated aplastic anemia, pPRCA is usually reversible, and no maternal deaths attributable to pPRCA have been reported. Herein, we report a woman diagnosed with pPRCA in two consecutive pregnancies. Corticosteroids were found to be ineffective, and she required a large number of red blood cell transfusions during both pregnancies. Despite severe anemia developing in both pregnancies, two healthy babies were vaginally born and spontaneous remission of pPRCA was seen after delivery. Interestingly, in both events of pPRCA described here, a transient rise of reticulocytes was observed precedent to the authentic recovery phase of reticulocytes and remission of pPRCA, which is a novel finding that has not been reported. The significance of this phenomenon has yet to be elucidated. Along with this case report, we review all 15 cases with 21 events of pPRCA in the literature, including the present case.


Assuntos
Complicações Hematológicas na Gravidez , Aplasia Pura de Série Vermelha , Adulto , Anemia Aplástica/etiologia , Anemia Aplástica/terapia , Transfusão de Eritrócitos , Feminino , Humanos , Gravidez , Complicações Hematológicas na Gravidez/terapia , Resultado da Gravidez , Aplasia Pura de Série Vermelha/congênito , Aplasia Pura de Série Vermelha/terapia , Indução de Remissão , Reticulócitos/patologia , Índice de Gravidade de Doença
4.
Br J Haematol ; 187(3): 386-395, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31273765

RESUMO

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or ß-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2  = 0·15; slope = 9·09) and, less significantly, in HE (R2  = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2  ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2  = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.


Assuntos
Membrana Eritrocítica/enzimologia , Eritrócitos Anormais/enzimologia , Piruvato Quinase/metabolismo , Esferocitose Hereditária/enzimologia , Adolescente , Adulto , Idoso , Anemia Hemolítica Congênita não Esferocítica/enzimologia , Anemia Hemolítica Congênita não Esferocítica/patologia , Anemia Falciforme/enzimologia , Anemia Falciforme/patologia , Criança , Pré-Escolar , Membrana Eritrocítica/patologia , Eritrócitos Anormais/patologia , Feminino , Doença da Hemoglobina C/enzimologia , Doença da Hemoglobina C/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/enzimologia , Erros Inatos do Metabolismo dos Piruvatos/patologia , Reticulócitos/enzimologia , Reticulócitos/patologia , Esferocitose Hereditária/patologia , Talassemia beta/enzimologia , Talassemia beta/patologia
5.
J Clin Lab Anal ; 33(4): e22844, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30945356

RESUMO

BACKGROUND: There is currently no single index for the diagnostic screening of hereditary spherocytosis (HS). However, hematology analyzers are widely used in hospital laboratories because of their highly automated performance and quality control procedure, and detection of some blood cell parameters may be useful for the early screening of HS. METHODS: We investigated the values of blood cell parameters for the screening and differential diagnosis of HS. We performed a descriptive study of 482 samples (67 cases of HS, 59 cases of G6PD deficiency, 57 cases of AIHA, 199 cases of thalassemia, and 100 cases of healthy controls) that were run on Beckman Coulter LH780 Hematology Analyzer. RESULTS: HS was characterized by increased MCHC, decreased MRV, MSCV-MCV < 0, and increased Ret with no concomitant increase in IRF. The areas under the ROC curves were MSCV-MCV (0.97; 95% CI 0.95-1.0) > MRV (0.94; 95% CI 0.91-0.97) > MCHC (0.92; 95% CI 0.88-0.97) > Ret/IRF (0.77; 95% CI 0.7-0.84). MSCV-MCV ≤ 0.6 fl was valuable parameter for the diagnostic screening of HS, with a sensitivity of 95.5% and specificity of 94.9%. CONCLUSION: These indices have high reference values for differentiating HS from thalassemia, AIHA, and G6PD deficiency.


Assuntos
Índices de Eritrócitos , Esferocitose Hereditária/sangue , Adolescente , Adulto , Anemia Hemolítica Autoimune/sangue , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Curva ROC , Reticulócitos/patologia , Sensibilidade e Especificidade , Esferocitose Hereditária/diagnóstico , Talassemia/sangue
6.
Leukemia ; 33(1): 217-229, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30050123

RESUMO

Reticulocytes shed CD71 from the cell membrane and eliminate mitochondria during terminal maturation, but it is unknown whether these two events are coordinated. We demonstrate that timely removal of CD71 is coupled with mitochondrial clearance, which can be disrupted by null mutation of immediate early response gene X-1 (IEX-1), leading to generation of aberrant CD71-positive and mitochondria-negative (CD71+Mito-) reticulocytes. CD71+Mito- reticulocytes were also present in a subset of patients with myelodysplastic syndromes (MDS) in direct proportion to reduced mitochondrial membrane potential (∆ψm). Mitochondrial abnormality caused by either IEX-1 deficiency or agents that dissipate ∆ψm could trigger premature clearance of mitochondria in reticulocytes. Premature clearance of mitochondria or addition of anti-oxidants lowered intracellular reactive oxygen species (ROS) that in turn hindered CD71 shedding and reticulocyte maturation. In contrast, introduction of ROS accelerated CD71 shedding via release of exosomes that contained a high proportion of Fe3+ over Fe2+, suggesting dual functions of CD71 shedding both in removal of toxic Fe3+ from reticulocytes and in limiting importation of Fe3+ into the cells. These observations emphasize the coordination of mitochondrial and CD71 clearance in erythroid terminal maturation and offer new insights into a role for mitochondrial degeneration in the pathogenesis of some MDS-associated anemia.


Assuntos
Antígenos CD/metabolismo , Eritropoese , Proteínas Imediatamente Precoces/fisiologia , Mitocôndrias/patologia , Síndromes Mielodisplásicas/patologia , Receptores da Transferrina/metabolismo , Reticulócitos/patologia , Animais , Autofagia , Estudos de Casos e Controles , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Síndromes Mielodisplásicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reticulócitos/metabolismo
7.
Transfusion ; 59(1): 385-395, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30520533

RESUMO

BACKGROUND: Alloanti-M was once regarded as not clinically significant, with a few exceptions in extremely rare cases. However, an increasing number of cases of severe hemolytic disease of the fetus and newborn (HDFN), resulting in fetal hydrops and recurrent abortion caused by alloanti-M, have been reported mainly in the Asian population. STUDY DESIGN AND METHODS: Three pregnant Chinese women with a history of abnormal pregnancy with hydrops fetalis were encountered. During this pregnancy, a series of clinical examinations and an alloantibody identification against RBCs and platelets were conducted. Intrauterine transfusion and postnatal transfusion were then performed in the fetuses. In addition, the HDFN cases caused by alloanti-M reported in different ethnic groups as well as their clinical and serologic features are also summarized. RESULTS: Three pregnant women were identified with an M-N+ phenotype and IgM mixed with IgG alloanti-M in serum. Their fetuses were found by ultrasound examination and cord blood testing to have severe anemia. Additionally, an M+N+ phenotype and IgG alloanti-M were detected in the cord blood of the three fetuses with titers ranging from 1:1 to 1:128. Moreover, low reticulocyte counts and negative direct antiglobulin tests were also shown in two of the fetuses. After receiving intrauterine transfusions and postnatal transfusions several times, these three fetuses eventually survived and then healthfully developed in the follow-up tracking. CONCLUSION: Alloanti-M immunization can cause severe HDFN with hyporegenerative anemia, often seen in the Asian population, and suppression of erythropoiesis might account for it.


Assuntos
Eritroblastose Fetal/patologia , Anemia/patologia , Transfusão de Sangue Intrauterina , Eritropoese/fisiologia , Feminino , Feto , Humanos , Recém-Nascido , Masculino , Gravidez , Reticulócitos/patologia
8.
Biomedica ; 38(3): 379-387, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30335243

RESUMO

Introduction: The quantification of chromosomal instability is an important parameter to assess genotoxicity and radiosensitivity. Most conventional techniques require cell cultures or laborious microscopic analyses of chromosomes or nuclei. However, a flow cytometry that selects the reticulocytes has been developed as an alternative for in vivo studies, which expedites the analytical procedures and increases up to 20 times the number of target cells to be analyzed. Objectives: To standardize the flow cytometry parameters for selecting and quantifying the micronucleated reticulocytesCD71+ (MN-RET) from freshly drawn peripheral blood and to quantify the frequency of this abnormal cell subpopulation as a measure of cytogenetic instability in populations of healthy volunteers (n =25), and patients (n=25), recently diagnosed with high-grade gliomas before the onset of treatment. Materials and methods: Blood cells were methanol-fixed and labeled with anti-CD-71-PE for reticulocytes, antiCD-61-FITC for platelet exclusion, and propidium iodide for DNA detection in reticulocytes. The MN-RETCD71+ cell fraction was selected and quantified with an automatic flow cytometer. Results: The standardization of cytometry parameters was described in detail, emphasizing the selection and quantification of the MN-RETCD71+ cellular fraction. The micronuclei basal level was established in healthy controls. In patients, a 5.2-fold increase before the onset of treatment was observed (p <0.05). Conclusion: The data showed the usefulness of flow cytometry coupled with anti-CD-71-PE and anti-CD-61-FITC labeling in circulating reticulocytes as an efficient and high resolution method to quantify chromosome instability in vivo. Finally, possible reasons for the higher average of micronuclei in RETCD71+ cells from untreated high-grade glioma patients were discussed.


Assuntos
Instabilidade Cromossômica , Citometria de Fluxo/métodos , Glioblastoma/genética , Micronúcleos com Defeito Cromossômico , Reticulócitos/patologia , Separação Celular/métodos , Feminino , Glioblastoma/sangue , Glioblastoma/patologia , Humanos , Masculino , Gradação de Tumores , Fatores de Risco , Manejo de Espécimes/métodos
9.
Int J Lab Hematol ; 40(6): 697-703, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30184319

RESUMO

INTRODUCTION: Development of additional parameters for complete blood count has emerged in recent hematology analyzers, leading to many publications. However, few studies have been conducted on advanced RBC parameters and hemolytic anemias. We investigated the interest of Sysmex unique parameters, MicroR and HypoHe, as well as the immature fraction of reticulocytes (IRF) in combination with complete blood and reticulocyte count, for screening hereditary spherocytosis (HS) and pyruvate kinase deficiency. METHODS: We analyzed 182 samples using Sysmex XE-5000 analyzers from a cohort of red cell disorder patients from the Rouen University Hospital. These included 47 HS, 17 pyruvate kinase deficiencies, sickle cell diseases and trait, ß-thalassemia minor, iron deficiencies, and 489 samples from a routine group. RESULTS: Combining five parameters (hemoglobin level, reticulocyte count, IRF, MicroR, and %HypoHe), we developed a specific screening tool for HS allowing a sensitivity of 100% and a specificity of 92.1% and a specific screening tool for pyruvate kinase deficiencies allowing a sensitivity of 100% and a specificity of 96.5%. These parameters were also found accurate in infants and in HS without anemia. CONCLUSION: We propose a costless, easy-to-use, and efficient approach to detect HS and pyruvate kinase deficiencies using Sysmex analyzers. These screening tools may help diagnosis of these disorders, help prevent complications, and result in a better management of these patients.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/sangue , Eritrócitos Anormais/metabolismo , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/sangue , Reticulócitos/metabolismo , Esferocitose Hereditária/sangue , Anemia Hemolítica Congênita não Esferocítica/patologia , Contagem de Células Sanguíneas/instrumentação , Contagem de Células Sanguíneas/métodos , Eritrócitos Anormais/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Piruvato Quinase/sangue , Erros Inatos do Metabolismo dos Piruvatos/patologia , Reticulócitos/patologia , Esferocitose Hereditária/patologia
10.
Hematology ; 23(10): 844-848, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29993346

RESUMO

BACKGROUND: Patients with thalassaemia who received regular transfusions had increased iron accumulation, leading to iron overload, which was associated with oxidative stress. Mitochondrial ferritin, encoded by the FTMT gene is an iron-storage protein in the mitochondria. The aim of this work was to investigate the expression levels of FTMT in the reticulocytes of patients with alpha-thalassaemia who were regularly transfused and rarely transfused compared with healthy controls and to evaluate the relationships of the levels of FTMT mRNA with malondialdehyde (MDA) and ferritin in these patients. METHODS: The levels of FTMT mRNA in the reticulocytes of patients (30 regularly transfused and 30 rarely transfused) and 30 healthy individuals were assessed by quantitative reverse transcription-polymerase chain reaction. The levels of ferritin and MDA were analysed by ELISA and by a thiobarbituric acid reactive substance assay, respectively. RESULTS: The levels of FTMT mRNA, ferritin and MDA in both groups of patients were significantly increased compared with those in the healthy controls. In addition, the levels of FTMT mRNA, ferritin and MDA in the regularly transfused patients were significantly higher than those in the rarely transfused patients. Furthermore, the relative expression levels of FTMT in patients correlated with those of MDA and ferritin. CONCLUSION: These results suggest that the elevation of expression levels of FTMT in the reticulocytes of patients with alpha-thalassaemia may be associated with iron loading and oxidative stress.


Assuntos
Ferritinas/biossíntese , Regulação da Expressão Gênica , Proteínas Mitocondriais/biossíntese , RNA Mensageiro/biossíntese , Reticulócitos/metabolismo , Talassemia alfa/sangue , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Reticulócitos/patologia , Talassemia alfa/patologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-29875078

RESUMO

The mutagenic potencies of 1,3-propane sultone (PS), N-propyl-N-nitrosourea (PNU), and mitomycin C (MMC) were investigated in three independent laboratories in Korea using the Pig-a assay in vivo. Sprague-Dawley rats were treated with vehicle or test substance on three consecutive days. Blood samples were collected for measuring Pig-a mutant phenotypes (CD59-deficient erythrocytes, RBCCD59-; CD59-deficient reticulocytes, RETCD59-) on days -1, 15, and 29 after the first treatment. In some studies, blood was collected for determining DNA damage (comet assay) on day 3 and measuring micronucleated reticulocytes (MN-RET) on day 4. Treatment with the alkylating agents PS and PNU induced dose-dependent increases in the frequency of RBCCD59- on days 15 and 29, and caused maximum elevations in the frequency of RETCD59- on day 15. Inter-laboratory comparison of the day 29 Pig-a assay data confirmed the mutagenic potencies of PS and PNU, and showed good agreement among the test sites. Treatment with the DNA cross-linker MMC induced increases in the frequencies of RBCCD59- and RETCD59- on days 15 and 29 (all three laboratories). MN-RETs increased significantly in animals treated with PS, PNU, or MMC, but biologically significant increases in DNA damage were observed only with PS and PNU, and not with MMC. The results of this study indicate that the Pig-a assay is a sensitive, reproducible method for evaluating the in vivo mutagenicity of various test substances, in particular, DNA cross-linkers and alkylating agents. Our limited data on integrating the Pig-a assay with the comet and micronucleus assays indicate that a short-term treatment protocol evaluating these three endpoints in a single set of animals may be a robust strategy for evaluating in vivo genotoxicity.


Assuntos
Laboratórios/normas , Proteínas de Membrana/genética , Mitomicina/toxicidade , Mutação , Compostos de Nitrosoureia/toxicidade , Reticulócitos/patologia , Tiofenos/toxicidade , Alquilantes/toxicidade , Animais , Reagentes para Ligações Cruzadas/toxicidade , Dano ao DNA , Masculino , Proteínas de Membrana/sangue , Testes de Mutagenicidade , Ratos , Ratos Sprague-Dawley , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo
12.
Mutat Res ; 820: 8-18, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28676266

RESUMO

The use of computed tomography (CT scans) has increased dramatically in recent decades, raising questions about the long-term safety of CT-emitted x-rays especially in infants who are more sensitive to radiation-induced effects. Cancer risk estimates for CT scans typically are extrapolated from models; therefore, new approaches measuring actual DNA damage are needed for improved estimations. Hence, changes in a dosimeter of DNA double-strand breaks, micronucleated reticulocytes (MN-RETs) measured by flow cytometry, were investigated in mice and infants exposed to CT scans. In male C57BL/6N mice (6-8 weeks-of-age), there was a dose-related increase in MN-RETs in blood samples collected 48h after CT scans delivering targeted exposures of 1-130 cGy x-rays (n=5-10/group, r=0.994, p=0.01), with significant increases occurring at exposure levels as low as 0.83 cGy x-rays compared to control mice (p=0.002). In paired blood specimens from infants with no history of a prior CT scan, there was no difference in MN-RET frequencies found 2h before (mean, 0.10±0.07%) versus 48h after (mean, 0.11±0.05%) a scheduled CT scan/cardiac catheterization. However, in infants having prior CT scan(s), MN-RET frequencies measured at 48h after a scheduled CT scan (mean=0.22±0.12%) were significantly higher than paired baseline values (mean, 0.17±0.07%; p=0.032). Increases in baseline (r=0.722, p<0.001) and 48-h post exposure (r=0.682, p<0.001) levels of MN-RETs in infants with a history of prior CT scans were significantly correlated with the number of previous CT scans. These preliminary findings suggest that prior CT scans increase the cellular responses to subsequent CT exposures. Thus, further investigation is needed to characterize the potential cancer risk from single versus repeated CT scans or cardiac catheterizations in infants.


Assuntos
Cateterismo Cardíaco/efeitos adversos , Quebras de DNA de Cadeia Dupla , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Reticulócitos/patologia , Tomografia Computadorizada por Raios X/efeitos adversos , Animais , Relação Dose-Resposta à Radiação , Feminino , Citometria de Fluxo , Instabilidade Genômica , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos Endogâmicos C57BL , Micronúcleos com Defeito Cromossômico/estatística & dados numéricos , Projetos Piloto , Gravidez , Estudos Prospectivos , Doses de Radiação , Irradiação Corporal Total
13.
Cell Death Dis ; 8(7): e2914, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28682312

RESUMO

Anaemia is a major global health problem arising from diverse causes and for which improved therapeutic strategies are needed. Erythroid cells can undergo apoptotic cell death and loss of pro-survival BCL-XL is known to trigger apoptosis during late-stage erythroid development. However, the mechanism by which loss or pharmacological blockade of BCL-XL leads to erythroid cell apoptosis remains unclear. Here we sought to identify the precise stage of erythropoiesis that depends on BCL-XL. We also tested whether deficiency of BIM or PUMA, the two main pro-apoptotic antagonists of BCL-XL, could prevent reticulocyte death and anaemia caused by BCL-XL loss. Using an in vivo mouse model of tamoxifen-inducible Bclx gene deletion and in vitro assays with a BCL-XL-selective inhibitor, we interrogated each stage of erythrocyte differentiation for BCL-XL dependency. This revealed that reticulocytes, but not orthochromatic erythroblasts, require BCL-XL for their survival. Surprisingly, concurrent loss of BIM or PUMA had no significant impact on the development of anemia following acute BCL-XL deletion in vivo. However, analysis of mixed bone marrow chimaeric mice revealed that loss of PUMA, but not loss of BIM, partially alleviated impaired erythropoiesis caused by BCL-XL deficiency. Insight into how the network of pro-survival and pro-apoptotic proteins works will assist the development of strategies to mitigate the effects of abnormal cell death during erythropoiesis and prevent anaemia in patients treated with BCL-XL-specific BH3-mimetic drugs.


Assuntos
Anemia/genética , Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Proteína 11 Semelhante a Bcl-2/genética , Regulação da Expressão Gênica , Proteínas Supressoras de Tumor/genética , Proteína bcl-X/genética , Anemia/metabolismo , Anemia/patologia , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteína 11 Semelhante a Bcl-2/deficiência , Benzotiazóis/farmacologia , Sobrevivência Celular , Modelos Animais de Doenças , Eritroblastos/metabolismo , Eritroblastos/patologia , Eritropoese/genética , Deleção de Genes , Humanos , Isoquinolinas/farmacologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Domínios Proteicos , Reticulócitos/metabolismo , Reticulócitos/patologia , Transdução de Sinais , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/deficiência , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/deficiência
14.
Radiat Res ; 188(4.2): 495-504, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28741984

RESUMO

Computed tomography (CT) scans are a routine diagnostic imaging technique that utilize low-energy X rays with an average absorbed dose of approximately 10 mGy per clinical whole-body CT scan. The growing use of CT scans in the clinic has raised concern of increased carcinogenic risk in patients exposed to ionizing radiation from diagnostic procedures. The goal of this study was to better understand cancer risk associated with low-dose exposures from CT scans. Historically, low-dose exposure preceding a larger challenge dose increases tumor latency, but does little to impact tumor frequency in Trp53+/- mice. To assess the effects of CT scans specifically on tumor progression, whole-body CT scans (10 mGy/scan, 75 kVp) were started at four weeks after 4 Gy irradiation, to allow for completion of tumor initiation. The mice were exposed to weekly CT scans for ten consecutive weeks. In this study, we show that CT scans modify cellular end points commonly associated with carcinogenesis in cancer-prone Trp53+/- heterozygous mice. At five days after completion of CT scan treatment, the multiple CT scans did not cause detectable differences in bone marrow genomic instability, as measured by the formation of micronucleated reticulocytes and H2AX phosphorylation in lymphoid-type cells, and significantly lowered constitutive and radiation induced levels of apoptosis. The overall lifespan of 4 Gy exposed cancer-initiated mice treated with multiple CT scans was increased by approximately 8% compared to mice exposed to 4 Gy alone (P < 0.017). Increased latency periods for lymphoma and sarcoma (P < 0.040) progression contributed to the overall increase in lifespan. However, repeated CT scans did not affect carcinoma latency. To our knowledge, this is the first reported study to show that repeated CT scans, when administered after tumor initiation, can improve cancer morbidity by delaying the progression of specific types of radiation-induced cancers in Trp53+/- mice.


Assuntos
Neoplasias Induzidas por Radiação/etiologia , Neoplasias Induzidas por Radiação/patologia , Tomografia Computadorizada por Raios X/efeitos adversos , Animais , Apoptose/efeitos da radiação , Carcinogênese/efeitos da radiação , Progressão da Doença , Suscetibilidade a Doenças , Relação Dose-Resposta à Radiação , Feminino , Histonas/metabolismo , Masculino , Camundongos , Estadiamento de Neoplasias , Neoplasias Induzidas por Radiação/metabolismo , Reticulócitos/patologia , Reticulócitos/efeitos da radiação , Medição de Risco , Análise de Sobrevida , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
15.
Eur J Haematol ; 98(6): 584-589, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28295642

RESUMO

OBJECTIVE: Here, we present a 7-year-old patient suffering from severe haemolytic anaemia. The most common cause of chronic hereditary non-spherocytic haemolytic anaemia is red blood cell pyruvate kinase (PK-R) deficiency. Because red blood cells rely solely on glycolysis to generate ATP, PK-R deficiency can severely impact energy supply and cause reduction in red blood cell lifespan. We determined the underlying cause of the anaemia and investigated how erythroid precursors in the patient survive. METHODS: PK activity assays, Western blot and Sanger sequencing were employed to determine the underlying cause of the anaemia. Patient erythroblasts were cultured and reticulocytes were isolated to determine PK-R and PKM2 contribution to glycolytic activity during erythrocyte development. RESULTS: We found a novel homozygous mutation (c.583G>A) in the PK-R coding gene (PKLR). Although this mutation did not influence PKLR mRNA production, no PK-R protein could be detected in the red blood cells nor in its precursors. In spite of the absence of PK-R, the reticulocytes of the patient exhibited 20% PK activity compared with control. Western blotting revealed that patient erythroid precursors, like controls, express residual PKM2. CONCLUSIONS: We conclude that PKM2 rescues glycolysis in PK-R-deficient erythroid precursors.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Proteínas de Transporte/genética , Eritroblastos/enzimologia , Proteínas de Membrana/genética , Piruvato Quinase/deficiência , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/genética , Reticulócitos/enzimologia , Hormônios Tireóideos/genética , Anemia Hemolítica Congênita não Esferocítica/enzimologia , Anemia Hemolítica Congênita não Esferocítica/patologia , Sequência de Bases , Diferenciação Celular , Criança , Consanguinidade , Eritroblastos/patologia , Expressão Gênica , Glicólise/genética , Homozigoto , Humanos , Masculino , Proteínas de Membrana/deficiência , Mutação , Células Mieloides/citologia , Células Mieloides/enzimologia , Cultura Primária de Células , Erros Inatos do Metabolismo dos Piruvatos/enzimologia , Erros Inatos do Metabolismo dos Piruvatos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reticulócitos/patologia , Hormônios Tireóideos/deficiência
16.
Ann Hematol ; 96(5): 733-738, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28247057

RESUMO

New automated hematology analyzers have led to the availability of novel hematological parameters, including the immature platelet fraction (IPF) and the immature reticulocyte fraction (IRF), both of potential interest in patients with myeloproliferative neoplasms (MPNs). We performed a prospective analysis of 217 patients with MPN, including 32 (15%) with essential thrombocythemia (ET), 43 (20%) with polycythemia vera (PV), and 142 (65%) with myelofibrosis (MF); the IPF and IRF were measured by the Sysmex XN analyzer. As compared to patients with ET, both a higher IPF and IRF were observed among patients with PV and MF. Factors associated with high IPF among patients with PV/ET were male sex, thrombocytopenia, and diagnosis of PV; among patients with MF, they were elevated peripheral blasts, low platelet count, JAK2 V617F mutation, and previous therapy. Factors associated with high IRF among patients with PV/ET were low hemoglobin, high reticulocyte count, and PV diagnosis; among patients with MF, they were peripheral blasts and elevated reticulocytes. The IPF and IRF represent novel parameters in patients with MPN with potential relevant clinical implications. Comparison with healthy subjects and those with secondary polycythemia is needed to confirm our preliminary findings.


Assuntos
Contagem de Células Sanguíneas , Plaquetas/patologia , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/diagnóstico , Reticulócitos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Transformação Celular Neoplásica , Diagnóstico Diferencial , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade , Transtornos Mieloproliferativos/mortalidade , Fenótipo , Policitemia Vera/sangue , Policitemia Vera/diagnóstico , Mielofibrose Primária/sangue , Mielofibrose Primária/diagnóstico , Estudos Prospectivos , Fatores de Risco , Trombocitemia Essencial/sangue , Trombocitemia Essencial/diagnóstico , Adulto Jovem
17.
Arch Toxicol ; 91(6): 2443-2453, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28197649

RESUMO

Temozolomide (TMZ), a monofunctional alkylating agent, was selected as a model compound to determine its quantitative genotoxic dose-response relationship in different tissues (blood, liver, and jejunum) and endpoints [Pig-a-, comet-, and micronucleus assay (MNT)] in male rats. TMZ was administered p.o. over 5 consecutive days (day 1-5), followed by a treatment-free period of 50 days (day 6-56) and a final administration prior to necropsy (day 57-59). TMZ showed a dose-dependent increase in DNA damage in all interrogated endpoints. A statistically significant increase in Pig-a mutant phenotypes was observed on day 44 starting at 7.5 mg/kg/day for mutant reticulocytes (for RETCD59-) and at 3.75 mg/kg/day for mutant red blood cells (RBCCD59-), respectively. In addition, a statistically significant increase in cytogenetic damage, as measured by micronucleated reticulocytes, was observed starting at 3.75 mg/kg/day on day 3 and 1.5 mg/kg/day on day 59. DNA strand breaks, as detected by the comet assay, showed a dose-dependent and statistically significant increase in liver, blood, and jejunum starting at doses of 3.75, 3.75, and 7.5 mg/kg/day, respectively. The dose-response relationships of the Pig-a, MNT, and comet data were analyzed for possible points of departure (PoD) using the benchmark-dose (BMD) software PROAST with different critical effect sizes (CES) (BMD0.1, BMD0.5, BMD1, and BMD1SD). Overall, PoD values show a high concordance between different tissues and endpoints, underlining the suitability of this experimental design to explore quantitative dose-response relationships in a variety of different tissues and endpoints, while minimizing animal use.


Assuntos
Dano ao DNA , Dacarbazina/análogos & derivados , Eritrócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Mutagênicos/toxicidade , Animais , Ensaio Cometa , Dacarbazina/toxicidade , Relação Dose-Resposta a Droga , Eritrócitos/patologia , Jejuno/efeitos dos fármacos , Jejuno/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Testes para Micronúcleos , Ratos Wistar , Reticulócitos/efeitos dos fármacos , Reticulócitos/patologia , Temozolomida
18.
Eur J Pharmacol ; 796: 69-75, 2017 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-27993641

RESUMO

Janus family kinases (JAKs) are essential molecules for cytokine responses and attractive targets for the treatment of transplant rejection and autoimmune diseases. Several JAK inhibitors have shown demonstrable effects on acute rejection in experimental cardiac transplant models. However, little is known about the potential benefits of JAK inhibitors on chronic rejection outcomes such as vasculopathy and fibrosis. Here, we examined the pharmacological profile of a novel JAK inhibitor, AS2553627, and explored its therapeutic potential in chronic rejection as well as acute rejection in a rat cardiac transplant model. AS2553627 potently inhibited JAK kinases but showed no inhibition of other kinases, including TCR-associated molecules. The compound also suppressed proliferation of IL-2 stimulated human and rat T cells. In a rat cardiac transplant model, oral administration of AS2553627 alone or co-administration with a sub-therapeutic dose of tacrolimus effectively prolonged cardiac allograft survival, suggesting the efficacy in treating acute rejection. To evaluate the effect on chronic rejection, recipient rats were administered a therapeutic dose of tacrolimus for 90 days. In combination with tacrolimus, AS2553627 significantly reduced cardiac allograft vasculopathy and fibrosis that tacrolimus alone did not inhibit. AS2553627 at the effective dose in rat transplantation models did not significantly reduce reticulocyte counts in peripheral whole blood after in vivo erythropoietin administration, indicating a low risk for anemia. These results suggest that AS2553627 may be a therapeutic candidate for the prevention of not only acute but also chronic rejection in cardiac transplantation.


Assuntos
Aloenxertos , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/efeitos adversos , Janus Quinases/antagonistas & inibidores , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Aloenxertos/patologia , Animais , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Interações Medicamentosas , Rejeição de Enxerto/sangue , Rejeição de Enxerto/patologia , Humanos , Reticulócitos/efeitos dos fármacos , Reticulócitos/patologia , Tacrolimo/farmacologia , Fatores de Tempo
19.
Vet Clin Pathol ; 45(4): 594-597, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27755775

RESUMO

A 9-year-old, female Maltese dog was referred to the Veterinary School of Toulouse with a 2-day history of anorexia and weakness. On clinical examination, the dog had hyperthermia (39.7°C), abdominal discomfort, and polypnea. Significant laboratory findings included pigmenturia, hyperbilirubinemia, hypercreatininemia, hyperfibrinogenemia, abnormal Snap canine pancreas-specific lipase, and pancytopenia with a nonregenerative anemia. A peripheral blood smear revealed numerous intraerythrocytic large Babesia but no polychromasia. There was a discrepancy between the absolute automated reticulocyte count (Sysmex reticulocyte count: 60 × 109 /L; RI 19.4-150.1 × 109 /L) and the manual reticulocyte count (3.6 × 109 /L) as well as the absence of polychromasia. The optical red blood cell scattergram showed an abnormal isolated reticulocyte cluster at the location of low-fluorescence ratio cells. These findings were interpreted as erythrocytes parasitized by large Babesia. The discrepancy between the Sysmex reticulocyte count and the manual reticulocyte count has been reported previously in people with falciparum malaria and numerous intra-erythrocytic Plasmodium falciparum organisms. This spurious reticulocyte profile and reticulocyte count were observed with the Sysmex XT-2000iV and the ProCyte using the same fluorescent dye polymethine but not with the LaserCyte using new methylene blue which does not stain Babesia organisms on a blood smear performed for manual reticulocyte counting.


Assuntos
Babesia/isolamento & purificação , Babesiose/sangue , Doenças do Cão/sangue , Reticulócitos/patologia , Animais , Babesiose/parasitologia , Babesiose/patologia , Contagem de Células Sanguíneas/instrumentação , Contagem de Células Sanguíneas/veterinária , Doenças do Cão/parasitologia , Doenças do Cão/patologia , Cães , Eritrócitos/parasitologia , Eritrócitos/patologia , Feminino , Contagem de Reticulócitos/instrumentação , Contagem de Reticulócitos/veterinária , Reticulócitos/parasitologia
20.
Br J Haematol ; 174(6): 970-82, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27291690

RESUMO

Very Late Antigen-4 (VLA-4, α4ß1-integrin, ITGA4) orchestrates cell-cell and cell-endothelium adhesion. Given the proposed role of VLA-4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA-4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA-4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA-4, while VLA-4 staining of non-SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA-4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM-1) was blocked by natalizumab in a dose-dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF-α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 µg/ml). This indicates that VLA-4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM-1 and that the VLA-4 adhesion to VCAM-1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA-4 blockade may be beneficial in sickle cell disease.


Assuntos
Anemia Falciforme/sangue , Adesão Celular/efeitos dos fármacos , Integrina alfa4beta1/antagonistas & inibidores , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Natalizumab/farmacologia , Reticulócitos/efeitos dos fármacos , Reticulócitos/patologia , Adolescente , Adulto , Anemia Falciforme/diagnóstico , Anemia Falciforme/tratamento farmacológico , Biomarcadores , Membrana Celular/metabolismo , Criança , Pré-Escolar , Simulação por Computador , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Hemodinâmica , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Lactente , Masculino , Pessoa de Meia-Idade , Natalizumab/química , Natalizumab/metabolismo , Ligação Proteica , Multimerização Proteica , Reticulócitos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adulto Jovem
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