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Ischemic stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. The comparative analysis of the gene expression profile of ECFCs from IS patients versus controls seems to indicate that there is a persistent angiogenic process even after a long time this complication has occurred. Thus, this is an original study which may lead to new insights into the molecular basis of SCA stroke and contribute to a better understanding of the role of endothelial cells in stroke recovery.
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Anemia Falciforme , Acidente Vascular Cerebral , Humanos , Células Endoteliais/metabolismo , Transcriptoma , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/complicações , Anemia Falciforme/complicações , Isquemia , Perfilação da Expressão Gênica , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Família de Proteínas EGF/genética , Família de Proteínas EGF/metabolismoRESUMO
BACKGROUND: (TA) n repeat sequence (rs8175347) of UGT1A1 gene promoter polymorphism is associated with serum bilirubin levels and gallstones among different sickle cell anaemia (SCA) populations. There are no data on UGT1A1 polymorphisms and their impact on Nigerian SCA patients. In this study, we determined the distribution of the UGT1A1 (TA) n genotypes among a group of young Nigerian SCA patients and healthy controls. In addition, the influence of UGT1A1 (TA) n genotypes on the laboratory and clinical events among the patients was determined. METHODS: The distribution of the UGT1A1 (TA) n genotypes among 101 young Nigerian SCA patients and 64 normal appropriate controls were determined and studied. The UGT1A1 (TA) n genotypes were further classified into subgroups and used to differentiate the clinical events and laboratory parameters of the patients. RESULTS: Four (TA) n alleles:(TA)5, 6, 7, and 8 were found. These were associated with 10 genotypes: TA5/5, 5/6, 5/7, 5/8, 6/6, 6/7, 6/8, 7/7, 7/8, 8/8. The normal (wild-type)-(TA) 6/6), low- (TA) 7/7, 7/8, 8/8), intermediate- (TA) 5/7, 5/8, 6/7, 6/8), and high-activity (TA) 5/5, 5/6,) genotypes were found in 24.8, 24.8, 41.5, and 8.9% patients and 20.3, 15.6, 61, and 3.1% controls respectively. The general genotype distribution of the patients and control group were not significantly different. There were significant differences in serum bilirubin and lactate dehydrogenase (LDH) of the patients when differentiated by the UGT1A1 (TA) n genotypes (p<0.05). Asymptomatic gallstones were found in 5.9% of patients and were significantly of the low-activity genotypes sub-group 5 (20%) vs 1(1.3%) p = 0.0033. Although, bilirubin and fetal hemoglobin (HbF) of patients with gallstones were significantly different from those without gallstone, only the serum bilirubin was associated with UGT1A1 (TA) n genotypes on multivariate analysis (p < 0.0001). CONCLUSION: This study highlights the contribution of UGT1A1 polymorphisms, a non-globin genetic factor, to the laboratory and clinical manifestations of young Nigerian SCA patients for the first time. It also shows that children with co-inheritance of low UGT1A1 (TA) n affinity genotypes may be at risk of gallstone, hence the need to follow them up.
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Anemia Falciforme/genética , Glucuronosiltransferase/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Adolescente , Anemia Falciforme/complicações , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Cálculos Biliares/complicações , Cálculos Biliares/genética , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Nigéria , Adulto JovemRESUMO
Aceruloplasminemia is a rare form of brain iron overload of autosomal recessive inheritance that results from mutations in the CP gene, encoding the iron oxidase ceruloplasmin. Homozygous aceruloplasminemia causes progressive neurodegenerative disease, anemia, and diabetes, and is usually diagnosed late in life upon investigation of anemia, high ferritin, or movement disorders, but its heterozygous state is less characterized and believed to be silent. Here we report two heterozygotes for new mutations causing aceruloplasminemia from whom peripheral blood samples were collected for complete blood counts, iron studies, and genotyping by automated sequencing. We then performed a systematic review of preview reports of heterozygotes with data on genotype and clinical findings. Heterozygosity for aceruloplasminemia invariably causes reduced ceruloplasmin levels, and similarly to previews reports in the literature, our cases did not present with anemia. Mild hyperferritinemia was found only in two reports. Nevertheless, 5 out of 11 variants have been associated with significant neurological symptoms despite the presence of one wild-type alelle. This review contributes to better genetic counseling of heterozygotes for CP gene variants and supports that measuring ceruloplasmin levels may be useful when investigating patients with movement disorders or rare cases of unexplained high ferritin.
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Ceruloplasmina/deficiência , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Adulto , Ceruloplasmina/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação/genética , Adulto JovemRESUMO
Early reduction of BCR-ABL1 transcript levels has been associated with improved outcome in chronic myeloid leukemia (CML) treatment. We evaluated 54 chronic-phase CML patients treated with imatinib who switched therapy to dasatinib (n = 33) or nilotinib (n = 21). BCR-ABL1 transcript levels were measured in peripheral blood using real-time quantitative PCR (RQ-PCR) every 3 months from the start of second-line treatment. Patients with BCR-ABL transcript levels >10% at 3 months and >1% at 6 months had significantly inferior progression-free (PFS) and event-free survival (EFS) than patients with RQ-PCR <10% at 3 months and <1% at 6 months (66 vs. 100%, p = 0.01, and 33 vs. 73%, p = 0.02, respectively). Patients with RQ-PCR <10% at 3 months and >1% at 6 months also had inferior PFS and EFS than patients with RQ-PCR <10% at 3 months and <1% at 6 months (48 vs. 100%, p = 0.002, and 25 vs. 73%, p < 0.0001, respectively). Two measurements of BCR-ABL levels were better than a single one to stratify chronic-phase CML patients as failure after second-line therapy.
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Proteínas de Fusão bcr-abl/sangue , Mesilato de Imatinib/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , RNA Mensageiro/sangue , RNA Neoplásico/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Falha de TratamentoRESUMO
Pyrimidine-5'-nucleotidase type I (P5'NI) deficiency is an autosomal recessive condition that causes nonspherocytic hemolytic anemia, characterized by marked basophilic stippling and pyrimidine nucleotide accumulation in erythrocytes. We herein present two African descendant patients, father and daughter, with P5'N deficiency, both born from first cousins. Investigation of the promoter polymorphism of the uridine diphospho glucuronosyl transferase 1A (UGT1A) gene revealed that the father was homozygous for the allele (TA7) and the daughter heterozygous (TA6/TA7). P5'NI gene (NT5C3) gene sequencing revealed a further change in homozygosity at amino acid position 56 (p.R56G), located in a highly conserved region. Both patients developed gallstones; however the father, who had undergone surgery for the removal of stones, had extremely severe intrahepatic cholestasis and, liver biopsy revealed fibrosis and siderosis grade III, leading us to believe that the homozygosity of the UGT1A polymorphism was responsible for the more severe clinical features in the father. Moreover, our results show how the clinical expression of hemolytic anemia is influenced by epistatic factors and we describe a new mutation in the P5'N gene associated with enzyme deficiency, iron overload, and severe gallstone formation. To our knowledge, this is the first description of P5'N deficiency in South Americans.
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5'-Nucleotidase/deficiência , Anemia Hemolítica Congênita/genética , Colestase/genética , Doença de Gilbert/genética , Glicoproteínas/genética , Sobrecarga de Ferro/genética , Cirrose Hepática/genética , 5'-Nucleotidase/genética , Adulto , Alelos , Anemia Hemolítica Congênita/complicações , Anemia Hemolítica Congênita/enzimologia , Anemia Hemolítica Congênita/patologia , Criança , Colestase/complicações , Colestase/enzimologia , Colestase/patologia , Consanguinidade , Epistasia Genética , Feminino , Doença de Gilbert/complicações , Doença de Gilbert/enzimologia , Doença de Gilbert/patologia , Heterozigoto , Homozigoto , Humanos , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/enzimologia , Sobrecarga de Ferro/patologia , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática/complicações , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Masculino , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Hydroxyurea (HU), or hydroxycarbamide, is used for the treatment of some myeloproliferative and neoplastic diseases, and is currently the only drug approved by the FDA for use in sickle cell disease (SCD). Despite the relative success of HU therapy for SCD, a genetic disorder of the hemoglobin ß chain that results in red-cell sickling, hemolysis, vascular inflammation and recurrent vasoocclusion, the exact mechanisms by which HU actuates remain unclear. We hypothesized that HU may modulate endothelial angiogenic processes, with important consequences for vascular inflammation. The effects of HU (50-200 µM; 17-24 h) on endothelial cell functions associated with key steps of angiogenesis were evaluated using human umbilical vein endothelial cell (HUVEC) cultures. Expression profiles of the HIF1A gene and the miRNAs 221 and 222, involved in endothelial function, were also determined in HUVECs following HU administration and the direct in vivo antiangiogenic effects of HU were assessed using a mouse Matrigel-plug neovascularization assay. Following incubation with HU, HUVECs exhibited high cell viability, but displayed a significant 75% inhibition in the rate of capillary-like-structure formation, and significant decreases in proliferative and invasive capacities. Furthermore, HU significantly decreased HIF1A expression, and induced the expression of miRNA 221, while downregulating miRNA 222. In vivo, HU reduced vascular endothelial growth factor (VEGF)-induced vascular development in Matrigel implants over 7 days. Findings indicate that HU is able to inhibit vessel assembly, a crucial angiogenic process, both in vitro and in vivo, and suggest that some of HU's therapeutic effects may occur through novel vascular mechanisms.
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Anemia Falciforme/tratamento farmacológico , Inibidores da Angiogênese/química , Hidroxiureia/química , Animais , Movimento Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Marcação In Situ das Extremidades Cortadas , Inflamação/tratamento farmacológico , Úlcera da Perna/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Transtornos Mieloproliferativos/tratamento farmacológico , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Growth differentiation factor 15 (GDF-15) is a bone marrow-derived cytokine whose ability to suppress iron regulator hepcidin in vitro and increased concentrations found in patients with ineffective erythropoiesis (IE)suggest that hepcidin deficiency mediated by GDF-15 may be the pathophysiological explanation for nontransfusional iron overload. We aimed to compare GDF-15 production in anemic states with different types of erythropoietic dysfunction. Complete blood counts, biochemical markers of iron status, plasma hepcidin, GDF-15, and known hepcidin regulators [interleukin-6 and erythropoietin (EPO)] were measured in 87 patients with red cell disorders comprising IE and hemolytic states: thalassemia, sickle cell anemia, and cobalamin deficiency. Healthy volunteers were also evaluated for comparison. Neither overall increased EPO,nor variable GDF-15 concentrations correlated with circulating hepcidin concentrations (P = 0.265 and P = 0.872). Relative hepcidin deficiency was found in disorders presenting with concurrent elevation of GDF-15 and soluble transferrin receptor (sTfR), a biomarker of erythropoiesis, and sTfR had the strongest correlation with hepcidin (r(s) = 0.584, P < 0.0001). Our data show that high concentrations of GDF-15 in vivo are not necessarily associated with pathological hepcidin reduction, and hepcidin deficiency was only found when associated with sTfR overproduction. sTfR elevation may be a necessary common denominator of erythropoiesis-driven mechanisms to favor iron absorption in anemic states and appears a suitable target for investigative approaches to iron disorders.
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Eritrócitos/metabolismo , Fator 15 de Diferenciação de Crescimento/sangue , Doenças Hematológicas/sangue , Hepcidinas/sangue , Receptores da Transferrina/sangue , Transferrina/metabolismo , Anemia Ferropriva/sangue , Estudos de Casos e Controles , Eritropoese , Feminino , Humanos , Ferro/sangue , Ferro/metabolismo , Deficiências de Ferro , Sobrecarga de Ferro/sangue , MasculinoRESUMO
BACKGROUND: Iron overload (IO) is a complex condition in which clinical, behavioral and genetic factors contribute to the phenotype. In multiethnic and non-Caucasian populations, mutations in HFE gene alone cannot explain IO in most of the cases, and additional genetic and environmental factors must be investigated. Bone Morphogenetic Proteins (BMPs) play a central role in iron homeostasis by modulating HAMP transcription through the signaling pathway that includes SMAD and HJV. In this study, we aimed to explore the clinical relevance of BMP6 mutations in a cohort of Brazilian patients with IO. METHODS: 41 patients with IO were evaluated. Blood samples were collected to analyze BMP6 mutations through New Sequence Generations (NGS). Frequency of variants and mutations were analyzed and correlated with clinical and environmental characteristics. RESULTS: We identified BMP6 mutations in three patients with IO. The p.Arg257His mutation was identified in two patients and the p.Leu71Val mutation was identified in one patient. Two of these patients had additional risk factors for IO (HFE mutations and diabetes mellitus). CONCLUSION: BMP6 mutations, when combined to other genetic and clinical risk factors, may contribute to IO. Functional studies and THE evaluation of large cohorts are necessary to fully address BMP6 role in IO.
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HbSC disease, a less severe form of sickle cell disease, affects the retina more frequently and patients have higher rates of proliferative retinopathy that can progress to vision loss. This study aimed to identify differences in the expression of endothelial cell-derived molecules associated with the pathophysiology of proliferative sickle cell retinopathy (PSCR). RNAseq was used to compare the gene expression profile of circulating endothelial colony-forming cells from patients with SC hemoglobinopathy and proliferative retinopathy (n = 5), versus SC patients without retinopathy (n = 3). Real-time polymerase chain reaction (qRT-PCR) was used to validate the RNAseq results. A total of 134 differentially expressed genes (DEGs) were found. DEGs were mainly associated with vasodilatation, type I interferon signaling, innate immunity and angiogenesis. Among the DEGs identified, we highlight the most up-regulated genes ROBO1 (log2FoldChange = 4.32, FDR = 1.35E-11) and SLC38A5 (log2FoldChange = 3.36 FDR = 1.59E-07). ROBO1, an axon-guided receptor, promotes endothelial cell migration and contributes to the development of retinal angiogenesis and pathological ocular neovascularization. Endothelial SLC38A5, an amino acid (AA) transporter, regulates developmental and pathological retinal angiogenesis by controlling the uptake of AA nutrient, which may serve as metabolic fuel for the proliferation of endothelial cells (ECs) and consequent promotion of angiogenesis. Our data provide an important step towards elucidating the molecular pathophysiology of PSCR that may explain the differences in ocular manifestations between individuals with hemoglobinopathies and afford insights for new alternative strategies to inhibit pathological angiogenesis.
Assuntos
Proteínas do Tecido Nervoso , Receptores Imunológicos , Neovascularização Retiniana , Proteínas Roundabout , Adulto , Feminino , Humanos , Masculino , Angiogênese , Células Endoteliais/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Receptores Imunológicos/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologiaRESUMO
Increased γ-globin production and consequent fetal hemoglobin (Hb F, α2γ2) formation is an important modulator of the clinical and hematological features of hemolytic anemias, such as sickle cell disease and ß-thalassemia (ß-thal). Hb F genes are genetically regulated, but despite numerous studies, the molecular basis of hemoglobin (Hb) switching is not completely understood. Hereditary persistence of fetal Hb (HPFH) is a consequence of impaired switching in adult life, which results in the continued expression of the γ-globin gene. This study was undertaken to identify genes that could be involved in Hb switching and/or maintenance of elevated Hb F levels. Two libraries were constructed using reticulocytes from normal donors and from Brazilian HPFH subjects. Results suggest that the maintenance of Hb F levels could be associated with some gene/protein expression modifications, such as low expression of KLF1, a transcription factor known to contribute to the regulation and modulation of Hb switching, decreased expression of MIER1, known for the recruitment of chromatin remodeling enzymes, and decreased expression of HOOK3. These data suggest new genes that may play a role in globin gene regulation, γ-globin gene expression and augmentation of Hb F levels, and may represent newly-defined cellular pathways for the control of Hb switching in erythroid cells.
Assuntos
Hemoglobina Fetal/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , gama-Globinas/genética , Hemoglobina Fetal/metabolismo , Biblioteca Gênica , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reticulócitos/metabolismo , Ativação Transcricional , gama-Globinas/metabolismoRESUMO
Among sickle cell anemia (SCA) complications, proliferative sickle cell retinopathy (PSCR) is one of the most important, being responsible for visual impairment in 10-20% of affected eyes. The aim of this study was to identify differentially expressed genes (DEGs) present in pathways that may be implicated in the pathophysiology of PSCR from the transcriptome profile analysis of endothelial progenitor cells. RNA-Seq was used to compare gene expression profile of circulating endothelial colony-forming cells (ECFCs) from HbSS patients with and without PSCR. Furthermore, functional enrichment analysis and protein-protein interaction (PPI) networks were performed to gain further insights into biological functions. The differential expression analysis identified 501 DEGs, when comparing the groups with and without PSCR. Furthermore, functional enrichment analysis showed associations of the DEGs in 200 biological processes. Among these, regulation of mitogen-activated protein (MAP) kinase activity, positive regulation of phosphatidylinositol 3-kinase (PI3K), and positive regulation of Signal Transducer and Activator of Transcription (STAT) receptor signaling pathway were observed. These pathways are associated with angiogenesis, cell migration, adhesion, differentiation, and proliferation, important processes involved in PSCR pathophysiology. Moreover, our results showed an over-expression of VEGFC (vascular endothelial growth factor-C) and FLT1 (Fms-Related Receptor Tyrosine Kinase 1) genes, when comparing HbSS patients with and without PSCR. These results may indicate a possible association between VEGFC and FLT1 receptor, which may activate signaling pathways such as PI3K/AKT and MAPK/ERK and contribute to the mechanisms implicated in neovascularization. Thus, our findings contain preliminary results that may guide future studies in the field, since the molecular mechanisms of PSCR are still poorly understood.
Assuntos
Células Progenitoras Endoteliais , Doenças Retinianas , Humanos , Células Progenitoras Endoteliais/metabolismo , Transcriptoma/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Perfilação da Expressão GênicaRESUMO
Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.
Assuntos
Doença de Chagas/parasitologia , Trato Gastrointestinal/parasitologia , Coração/parasitologia , Trypanosoma cruzi/isolamento & purificação , Doença de Chagas/patologia , DNA de Protozoário/análise , Humanos , Reação em Cadeia da Polimerase/métodosRESUMO
STAT3 is a cytokine-signaling transcription factor critical for gene regulation. Gain-of-function (GOF) mutations in STAT3 are associated with lymphoproliferation, autoimmune cytopenias, increased susceptibility to infection, early-onset solid-organ autoimmunity, short stature, and eczema. We studied the JAK/STAT signaling pathway gene expression and the cytokine profile in two families carrying STAT3-GOF variants to shed light on the STAT3-GOF-associated variable expressivity, including the identification of disease markers. Considering 92 target genes, KIT and IL2RA were downregulated only in patients with high clinical penetrance, while CXCL8 was markedly downregulated for all of them. Unlike previous studies, SOCS3-a STAT3 inhibitor-was not upregulated in patients. In addition, low levels of IL-2 and a reduced numbers of Tregs cells were strikingly prevalent in patients. This study shows a disruptive role of STAT3-GOF variants in the regulatory axis activities CXCL8/STAT3, KIT/STAT3, IL2/CD25/Treg, which, by slightly different mechanisms, underlie the broad clinical spectrum seen in the studied patients. In addition, we suggest the investigation of CXCL8 as a biomarker for identifying STAT3-GOF mutation.
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Mutação com Ganho de Função , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-2/metabolismo , Interleucina-8/metabolismo , Fator de Transcrição STAT3/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Adulto , Pré-Escolar , Hibridização Genômica Comparativa , Citocinas/sangue , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Imunofenotipagem , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
INTRODUCTION: Mutations affecting genes involved in oxidative and signaling pathways may be associated with kidney disease in sickle cell anemia. We determined the allele and genotype frequencies of some polymorphisms in the promoter regions of the Heme Oxygenase-1 (HMOX1) [rs2071746 (A>T) and (GT)n repeats, short (S) and long (L) alleles] and Bone Morphogenetic Protein Receptor type-1B (BMPR1B) [rs17022863 (A>G), rs4331783 (A>G) and rs1470409 (A>G)] genes in 75 adult patients with sickle cell anemia and 160 healthy controls and investigated whether these polymorphisms may influence the estimated glomerular filtration rate for the patients. METHODS: The single nucleotide polymorphisms were genotyped using the TaqMan assays, the HMOX1(GT)n repeats were determined by polymerase chain reaction fragment size analysis and the estimated glomerular filtration rate was calculated by the Modification of Diet in Renal Disease formula. RESULTS: Regarding the HMOX1rs2071746, the estimated glomerular filtration rate median was significantly higher in TT patients (p=0.019), including when TT was compared with AT+AA (p=0.009); for the (GT)n repeats, the estimated glomerular filtration rate medians of SS, SL and LL significantly differed (p=0.009), being the LL estimated glomerular filtration rate median significantly higher, when compared with the LS+SS (p=0.005). These results suggest that both the homozygotes, TT for rs2071746 and LL for (GT)n repeats, lead to a higher risk of developing renal complications. Concerning the BMPR1B, the frequencies of GG for rs17022863 and AA for rs4331783 were significantly higher in patients than in controls (p=0.002 and p=0.008, respectively), however no association with estimated glomerular filtration rate was found. CONCLUSION: These results contribute to a better understanding of the genetic factors related to the development of nephropathy in sickle cell anemia patients.
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Sickle cell disease (SCD), one of the most common hemoglobinopathies worldwide, is characterized by a chronic inflammatory component, with systemic release of inflammatory cytokines, due to hemolysis and vaso-occlusive processes. Patients with SCD demonstrate dysfunctional T and B lymphocyte responses, and they are more susceptible to infection. Although dendritic cells (DCs) are the main component responsible for activating and polarizing lymphocytic function, and are able to produce pro-inflammatory cytokines found in the serum of patients with SCD, minimal studies have thus far been devoted to these cells. In the present study, we identified the subpopulations of circulating DCs in patients with SCD, and found that the bloodstream of the patients showed higher numbers and percentages of DCs than that of healthy individuals. Among all the main DCs subsets, inflammatory DCs (CD14+ DCs) were responsible for this rise and correlated with higher reticulocyte count. The patients had more activated monocyte-derived DCs (mo-DCs), which produced MCP-1, IL-6, and IL-8 in culture. We found that a CD14+ mo-DC subset present in culture from some of the patients was the more activated subset and was mainly responsible for cytokine production, and this subset was also responsible for IL-17 production in co-culture with T lymphocytes. Finally, we suggest an involvement of heme oxygenase in the upregulation of CD14 in mo-DCs from the patients, indicating a potential mechanism for inducing inflammatory DC differentiation from circulating monocytes in the patients, which correlated with inflammatory cytokine production, T lymphocyte response skewing, and reticulocyte count.
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Anemia Falciforme/imunologia , Células Dendríticas/imunologia , Diferenciação Celular/imunologia , Humanos , Inflamação/imunologia , Ativação Linfocitária/imunologia , Células Th17/imunologiaRESUMO
PURPOSE: To determine the various haptoglobin genotypes and their influence on the clinico-laboratory manifestations among young Nigerian sickle cell anemia (SCA) patients. PATIENTS AND METHODS: A total of 101 SCA patients and 64 controls were studied. SCA was diagnosed by polymerase chain reaction (PCR). Haptoglobin genotype was determined by PCR followed by agarose gel electrophoresis. The patients' laboratory and clinical parameters were differentiated by haptoglobin genotypes. RESULTS: The Hp1 and Hp2 alleles frequencies were 0.62 and 0.38 in the patients and 0.73 and 0.27 in the controls, respectively, and these did not differ significantly (p>0.05). The haptoglobin genotype distribution among the patients and controls were Hp1-1, 43 (42.6%); Hp2-1, 40 (39.6%); Hp2-2, 18 (17.8%) and Hp1-1, 35 (54.7%); Hp2-1, 24 (37.5%); Hp2-2, 5 (7.8%), respectively, with no difference between the two groups (P>0.05). No significant difference was found in the clinical events and laboratory parameters of the patients when partitioned according to the various haptoglobin genotypes (P> 0.05). CONCLUSION: This study found that haptoglobin gene polymorphism does not have a significant influence on the clinico-laboratory manifestations among SCA patients.
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OBJECTIVE: alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression. MATERIALS AND METHODS: Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains. RESULTS: RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. CONCLUSION: These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.
Assuntos
Antígenos CD34/efeitos dos fármacos , Proteínas Sanguíneas/efeitos dos fármacos , Eritropoetina/farmacologia , Globinas/efeitos dos fármacos , Hemina/farmacologia , Hemoglobinas/biossíntese , Chaperonas Moleculares/efeitos dos fármacos , Antígenos CD34/biossíntese , Apoptose/efeitos dos fármacos , Proteínas Sanguíneas/biossíntese , Proteínas Sanguíneas/genética , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Clonagem Molecular , Células Precursoras Eritroides/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Globinas/metabolismo , Humanos , Células K562 , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Interferência de RNA , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Norovirus is a highly prevalent pathogen that can infect a wide range of host species. Thus far, there have only been two reports of norovirus infection in rats. Diagnostic assays for the detection of norovirus are well established, but a specific molecular assay for the diagnosis of norovirus infection in laboratory rats has not yet been reported. In this study, we describe the development of a sensitive, semi-nested RT-PCR assay for detection of norovirus in fecal samples from Rattus norvegicus, reared in animal facilities under different sanitary barrier conditions. Additionally, we describe the first report of the presence of norovirus in rat colonies from Brazilian animal facilities.
Assuntos
Infecções por Caliciviridae/diagnóstico , Fezes/virologia , Norovirus/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Doenças dos Roedores/diagnóstico , Animais , Brasil , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
ABSTRACT Introduction Mutations affecting genes involved in oxidative and signaling pathways may be associated with kidney disease in sickle cell anemia. We determined the allele and genotype frequencies of some polymorphisms in the promoter regions of the Heme Oxygenase-1 (HMOX1) [rs2071746 (A > T) and (GT)n repeats, short (S) and long (L) alleles] and Bone Morphogenetic Protein Receptor type-1B (BMPR1B) [rs17022863 (A > G), rs4331783 (A > G) and rs1470409 (A > G)] genes in 75 adult patients with sickle cell anemia and 160 healthy controls and investigated whether these polymorphisms may influence the estimated glomerular filtration rate for the patients. Methods The single nucleotide polymorphisms were genotyped using the TaqMan assays, the HMOX1(GT)n repeats were determined by polymerase chain reaction fragment size analysis and the estimated glomerular filtration rate was calculated by the Modification of Diet in Renal Disease formula. Results Regarding the HMOX1rs2071746, the estimated glomerular filtration rate median was significantly higher in TT patients (p = 0.019), including when TT was compared with AT + AA (p = 0.009); for the (GT)n repeats, the estimated glomerular filtration rate medians of SS, SL and LL significantly differed (p = 0.009), being the LL estimated glomerular filtration rate median significantly higher, when compared with the LS + SS (p = 0.005). These results suggest that both the homozygotes, TT for rs2071746 and LL for (GT)n repeats, lead to a higher risk of developing renal complications. Concerning the BMPR1B, the frequencies of GG for rs17022863 and AA for rs4331783 were significantly higher in patients than in controls (p = 0.002 and p = 0.008, respectively), however no association with estimated glomerular filtration rate was found. Conclusion These results contribute to a better understanding of the genetic factors related to the development of nephropathy in sickle cell anemia patients.