Search

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to in vivo hemostasis. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of activated factor X (FXa). Heme was capable of inducing both TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Our results provide additional details on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

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.

Microparticles are sub-micron vesicles possessing protein and other materials derived from the plasma membrane of their parent cells, and literature suggests that they may have a role in the pathophysiology and downstream manifestations of sickle cell disease (SCD). The contributions of red blood cells microparticles (RMP) to the pathogenic mechanisms and clinical phenotypes of SCD are largely unknown. There is a controversy as to whether the proportions of intravascular hemolysis (approximately ≤ 30% of total hemolysis) would be enough to explain some complications seen in patients with SCD. We investigated RMP among 138 SCD patients and 39 HbAA individuals. Plasma RMPs were quantified by flow cytometry, plasma hemoglobin and heme by colorimetric assays, and haptoglobin and hemopexin by ELISA. The patients had higher RMP, plasma hemoglobin, and heme compared to the controls. On the contrary, haptoglobin and hemopexin were depleted in the patients. The RMP correlated positively with heme, lactate dehydrogenase, plasma hemoglobin, serum bilirubin, reticulocyte counts, and tricuspid regurgitant jet velocity of the patients. Contrarily, it correlated negatively with HbF, hemopexin, red blood cells counts, hemoglobin concentration, and haptoglobin. Although patients treated with hydroxyurea had lower RMP, this did not attain statistical significance. Patients with sickle leg ulcer and elevated tricuspid regurgitant jet velocity had higher levels of RMP. In conclusion, these data suggest that RMPs are associated with hemolysis and may have important roles in the pathophysiology and downstream complications of SCD.

Made available in DSpace on 2021-06-25T10:32:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-01 Fetal hemoglobin (HbF) induction constitutes a valuable and validated approach to treat the symptoms of sickle cell disease (SCD). Here, we synthesized pomalidomide–nitric oxide (NO) donor derivatives (3a–f) and evaluated their suitability as novel HbF inducers. All compounds demonstrated different capacities of releasing NO, ranging 0.3–30.3%. Compound 3d was the most effective HbF inducer for CD34+ cells, exhibiting an effect similar to that of hydroxyurea. We investigated the mode of action of compound 3d for HbF induction by studying the in vitro alterations in the levels of transcription factors (BCL11A, IKAROS, and LRF), inhibition of histone deacetylase enzymes (HDAC-1 and HDAC-2), and measurement of cGMP levels. Additionally, compound 3d exhibited a potent anti-inflammatory effect similar to that of pomalidomide by reducing the TNF-α levels in human mononuclear cells treated with lipopolysaccharides up to 58.6%. Chemical hydrolysis studies revealed that compound 3d was stable at pH 7.4 up to 24 h. These results suggest that compound 3d is a novel HbF inducer prototype with the potential to treat SCD symptoms. São Paulo State University (UNESP) School of Pharmaceutical Sciences Department of Molecular Medicine and Pediatrics Zucker School of Medicine at Hofstra/Northwell Laboratory of Developmental Erythropoiesis Les Nelkin Memorial Pediatric Oncology Laboratory The Feinstein Institutes for Medical Research Faculty of Medical Sciences State University of Campinas - UNICAMP São Paulo State University (UNESP) School of Pharmaceutical Sciences

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.

A trombocitemia essencial (TE) faz parte do grupo de síndromes mieloproliferativas e é caracterizada por um aumento na contagem de plaquetas. Uma mutação de ponto no gene JAK2 é comumente observada em paciente com TE, embora mutações nos genes CARL e MPL também estejam associadas ao surgimento da doença. As microvesículas (MVs) são pequenas estruturas liberadas por diversos tipos celulares mediante ativação e que são descritas como possíveis biomarcadores de gravidade clínica em diferentes doenças. Níveis elevados de MVs de plaquetas e de células endoteliais foram descritos na TE e as MVs de plaquetas foram associadas a risco trombótico na doença. Alguns trabalhos mostram que os níveis de MVs variam de acordo com a mutação existente nos genes JAK2, CARL e MPL, que estão relacionados a esse distúrbio, e que a terapia citorredutora com hidroxiureia pode modular essa contagem. Neste trabalho, investigamos os níveis de MVs de plaquetas e de plaquetas ativadas no plasma de pacientes com TE e sua associação com a mutação JAK2. Foram incluídos no estudo 16 pacientes com TE (11 com presença da mutação em JAK2 e 5 sem). Dentre os 16 pacientes estudados, 10 estavam em tratamento com hidroxiureia. O sangue foi coletado para o isolamento de MVs do plasma seguindo o protocolo descrito por nosso grupo (Olatunya et al., 2019). A quantificação das MVs foi realizada por citometria de fluxo. A população de MVs foi identificada pela marcação duplo-positiva de lactaderina+calceína e os números de MVs de plaquetas e plaquetas ativadas foram avaliados pela marcação com CD41 e CD41+CD62P, respectivamente. Os dados obtidos mostram que os pacientes com TE sem a mutação em JAK2 possuem níveis maiores de MVs de plaquetas ativadas em comparação ao grupo com a mutação (2,32 x 106 ± 182866 vs 1,68 x 106 ± 185198/mL, respectivamente p = 0,047). Não houve diferença nos níveis de MVs de plaquetas nos dois grupos estudados (3,00 x 106 ± 1,127 x 106 vs 5,948 x 106 ± 1,678 x 106/mL, respectivamente). Alguns trabalhos relacionaram a presença da mutação em JAK2 nos pacientes com TE a números elevados de MVs circulantes. Os nossos dados mostram que, na população estudada, os níveis de MVs de plaquetas ativadas parecem ser maiores nos pacientes que não possuem a mutação em JAK2. A possível relação da presença ou não da mutação com a modulação dos níveis de MVs na TE permanence desconhecida. Considerando a relevância dos estudos com MVs como possíveis biomarcadores de gravidade clínica, os dados obtidos podem fornecer novos insights para explorar a relação das mutações envolvidas nas síndromes mieloproliferativas e sua relação com os níveis de MVs liberados na circulação.

A anemia falciforme é uma doença hereditária recessiva causada por uma mutação em um único nucleotídeo no gene da beta globina. Esta mutação provoca uma alteração estrutural na qual o ácido glutâmico é substituído por uma valina acarretando na falcização das hemácias quando desoxigenadas. Alguns fármacos com ação hipometilante são capazes de induzir a expressão de gama globina através da inibição da metilação no promotor desse gene. Porém, pouco se sabe a respeito desta regulação e sobre os genes envolvidos neste processo. Visando compreender esta regulação, células CD34+, de indivíduos controle, foram tratadas com 1μM de Decitabina no 9°dia de cultura celular para indução da produção de Hemoglobina Fetal (HbF). Os níveis de expressão dos fatores de transcrição e modificadores da cromatina foram avaliados por meio da plataforma PCR Array (Qiagen™ Germany). Nesse ensaio, foram evidenciados 24 fatores de transcrição diferencialmente expressos, dentre eles, o HNF4α (Hepatocyte Nuclear Factor 4 Alpha) regulado positivamente em 26,1 vezes, HNF1α (Hepatocyte Nuclear Factor 1 Homeobox A) 20,0 vezes e o TCF7L2 (Transcription Factor 7 Like 2) 5,9 vezes, em células tratadas com Decitabina comparadas ao controle. Selecionamos o gene HNF4α como um candidato para silenciamento utilizando a ferramenta CRISPR/Cas9 em células HUDEP-2 (immortalized human erythroid progenitor) por ser o fator de transcriação com maior valor diferencial de expressão entre os listados. O HNF4α é descrito como um fator de transcrição que regula a expressão de vários genes hepáticos, podendo atuar no desenvolvimento do fígado, rim e intestinos e é expresso em tecido hematopoiético. As células HUDEP-2 foram tratadas com Decitabina 50 nM e coletadas após 72 horas. Os níveis de HbF foram mensurados por citometria de fluxo em triplicas biológicas. A porcentagem de células positivas para HbF em HUDEP-2, tratada com Decitabina, foi de 12,27 ± 0,7% n = 3, enquanto nas células controle 1,0 ± 0,06% n = 3, p < 0,0001. Estes resultados corroboram o aumento da expressão observado nas células CD34+. As células HUDEP-2 foram nucleofectedas com Cas9 high fidelity (104 pmol), complexo crRNA: tracrRNA (120 pmol) e 1uM de gRNA HNF4α usando o kit de células humanas CD34+ no dispositivo AMAXA Nucleofector 4D (Lonza). 48 horas após a nucleofecção, as células HUDEP-2 editadas foram submetidas à seleção de clones e expandidas por aproximadamente 28 dias. O DNA genômico dos clones foi sequenciado, e quatro clones -HNF4α-HUDEP-2 foram encontrados com indels no quarto éxon do gene HNF4α. Esses clones foram expandidos em cultura com um controle, e os níveis de HbF foram quantificados com anticorpo anti-HbF por citometria de fluxo. Os níveis de HbF destes quatro clones foram de 8,2 ± 2,4%, o nível do controle foi de 0,9 ± 0,02% p < 0,0258. Os clones editados, embora não homogeneamente, expressaram significativamente mais HbF do que o controle. Nossos resultados sugerem que o HNF4α pode desempenhar um papel importante na ativação do gene da gama globina em células HUDEP-2. Adicionalmente, o aumento na expressão dos genes TCF7L2 e HNF1α são sugestivos de um possível papel na regulação de HbF. Esses dados são importantes, pois indicam novos possíveis alvos para terapia gênica em hemoglobinopatias.

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.

Made available in DSpace on 2020-12-12T01:23:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Optical Society Universidade Estadual Paulista Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) National Institutes of Health Resveratrol (RVT) derivatives (10a-i) were designed, synthesized, and evaluated for their potential as gamma-globin inducers in treating Sickle Cell Disease (SCD) symptoms. All compounds were able to release NO at different levels ranging from 0 to 26.3%, while RVT did not demonstrate this effect. In vivo, the antinociceptive effect was characterized using an acetic acid-induced abdominal contortion model. All compounds exhibited different levels of protection, ranging from 5.9 to 37.3%; the compound 10a was the most potent among the series. At concentrations between 3.13 and 12.5 µM, the derivative 10a resulted in a reduction of 41.1–64.3% in the TNF-α levels in the supernatants of macrophages that were previously LPS-stimulated. This inhibitory effect was higher than that of RVT used as the control. In addition, the compound 10a and RVT induced double the production of the gamma-globin chains (γG + γA), compared to the vehicle, using CD34+ cells. Compound 10a also did not induce membrane perturbation and it was not mutagenic in the in vivo assay. Thus, compound 10a emerged as a new prototype of the gamma-globin-inducer group with additional analgesic and anti-inflammatory activities and proving to be a useful alternative to treat SCD symptoms. São Paulo State University (UNESP) School of Pharmaceutical Sciences University of Campinas (UNICAMP) Hematology and Hemotherapy Center Weill Cornell Medical College Department of Physiology and Biophysics São Paulo State University (UNESP) School of Pharmaceutical Sciences FAPESP: 2012/50359-2 FAPESP: 2014/00984-3 CNPq: 304731/2017-0 National Institutes of Health: GM21342

A new calorimetric technique is described which allows the measurement of metabolic heat rates in biological materials which are triggered by changes in the oxygen tension. It uses the high permeability for oxygen of a thin-walled Teflon flow channel of a chip calorimeter in order to adjust variable but defined oxygen concentrations in the samples. Using this technique, for the first time, the heat production rate of sickle cell erythrocytes (SS-RBCs) in sickled and non-sickled state could be compared. To measure heat rate changes in real time when sickled SS-RBCs return to the non-sickled state, the heat dissipation was measured during the re-oxygenation of the cell samples which had been treated before under anoxic conditions. We found higher heat production rate of the blood cells in non-sickled state, which is in agreement with the reported increased glucose uptake.

Made available in DSpace on 2018-12-11T16:50:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fetal hemoglobin (HbF) induction by hydroxyurea (HU) therapy is associated with decreased morbidity and mortality in sickle cell anemia (SCA) patients, but not all patients respond to or tolerate HU. This provides a rationale for developing novel HbF inducers to treat SCA. Thalidomide analogs have the ability to induce HbF production while inhibiting the release of tumor necrosis factor-alpha. Molecular hybridization of HU and thalidomide was used to synthesize 3- (1,3-dioxoisoindolin-2-yl) benzyl nitrate (compound 4C). In this study, we show that compound 4C increases HbF production in a transgenic SCA mouse model and reduces the production of pro-inflammatory cytokines by SCA mouse monocytes cultured ex vivo. Therefore, compound 4C is a novel drug designed to treat SCA with a unique combination of HbF-inducing and anti-inflammatory properties. Hematology and Hemotherapy Center, University of Campinas-UNICAMP, Campinas, Brazil Department of Clinical Pathology, School of Medicine, University of Campinas-UNICAMP, Campinas, Brazil São Paulo State University (UNESP), School of Pharmaceutical Science, Araraquara, Brazil Department of Anesthesiology and Perioperative Medicine, Augusta University, Augusta, GA, USA Department of Medicine, Medical College of Georgia, Augusta University, Augusta, GA, USA

Extracellular vesicles (EVs) are submicron structures released in blood circulation by different cell types which have been found to be increased in sickle cell disease (SCD) and are associated with clinical complications. The most abundant EVs in SCD patients derive from platelets, endothelial cells, and red blood cells (RBCs) and EVs have been explored as biomarkers of clinical severity. Crizanlizumab is a monoclonal antibody against P-selectin, an adhesion molecule expressed in activated platelets and endothelial cells. P-selectin facilitates the formation of heterocellular aggregates and is implicated in the pathophysiology of vaso-occlusive episodes (VOEs) in SCD. This study aimed to investigate the circulating levels of EVs in patients with SCD on standard of care or treated with crizanlizumab. We collected peripheral blood samples from 20 adults with SCD (Non treated group: 7 patients on hydroxyurea treatment and 7 without it. Treated group: 6 patients undergoing treatment with crizanlizumab in combination with hydroxyurea). Patients received the last dose of crizanlizumab at least a month prior to the study. EVs were identified by lactadherin+calcein stain and quantified by flow cytometry to determine the immunophenotype of their parent cell (platelet, endothelial cell, and RBC, with CD41+; CD146+/CD45-; CD235+, respectively). EV quantification was calculated in number per ml of blood as previously described by our group (Olatunya et al., 2019). We found that patients on crizanlizumab had lower total circulating EV counts than patients not receiving the drug (62.670.000,00 ± 15.600.000,00 vs 13.100.000,00 ± 3.513.000,00/mL, respectively, p=0,0076). The difference was statistically significant in platelet-derived EVs levels (5.397.000,00 ± 953.875,00 vs 2.413.000,00 ± 745.165,00/mL, p=0,0169), but not in endothelium-derived or RBC-derived EVs (345714 ± 101817 vs 220000 ± 64291, and 2.189.000,00 ± 1.648.000,00 vs 1.013.000,00 ± 572775, respectively). Crizanlizumab therapy has been shown to reduce the incidence of VOEs in SCD. EVs have been recognized as bio-effectors involved in VOEs, contributing to a hypercoagulable state, chronic inflammation, and endothelial damage. Our findings show an association between the use of crizanlizumab and lower EV levels, particularly of the platelet-derived type. While the anti-P-selectin activity of crizanlizumab could be expected to help remove platelets from circulation, clinical studies have not reported a reduction in platelet counts in patients treated with crizanlizumab. Therefore, we speculate that crizanlizumab may decrease the release of EV by activated platelets, reduce platelet activation, or contribute to EV removal from circulation. Our findings suggest that crizanlizumab therapy may modulate EV levels in the plasma of SCD patients and provide, for the first time, data to support exploring the use of extracellular vesicles as biomarkers to monitor the clinical response to this drug in patients. Further studies on EV expression of P-selectin and how crizanlizumab interacts with EVs and platelets may help clarify this particular effect of this drug. Disclosures Benites: Novartis: Honoraria. Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

Stress erythropoiesis (SE) is characterized by an increase in erythropoietic activity in the bone marrow and in extramedullary sites. The central macrophage present in the erythroblastic island (EBI) plays a key role in regulating SE through the expression of molecules mediating cell adhesion, iron metabolism, and those capable of identifying and engulfing damaged and senescent erythrocytes. Those receptors are also expressed in monocytes (MC), suggesting that their monocytic expression could be involved in erythropoiesis and erythrophagocytosis. CD14 +CD16 + intermediate MC (I-MC) express markers that are typically found in EBI macrophages and, together with CD14 +CD16 - classic MC (C-MC), are able to remove circulating iron. Polycythemia vera (PV) is characterized by autonomous overproduction of red blood cells (RBCs) with extramedullary hematopoiesis most often caused by an acquired JAK2 V617F mutation, resulting in a state of chronic SE. The depletion of macrophages from EBIs in animal model of PV reverses splenomegaly and erythrocytosis indicating that they are essential for the development of chronic SE. It is unknown if MC play the same role in humans or if they have different expressions of those key molecules, which could contribute to the severity of the disease. We aimed to investigate the role of MCs in SE present in PV by characterizing the expression of molecules relevant to RBC adhesion, anti-inflammation, erythrophagocytosis, and iron metabolism in MCs from PV patients and from healthy controls (HC). Peripheral blood MCs were isolated from HC (n=21) and PV patients (n=17) and phenotyped by flow cytometry (FC) for sialoadhesin (CD169), VCAM1 (CD106), the receptor for the hemoglobin-haptoglobin complex (CD163), mannose receptor (CD206), SIRPα (CD172), ferroportin (Fpn), and separated into subtypes according to expression of CD14 and CD16. We also evaluated MC erythrophagocytosis by determining positivity for intracellular glycophorin (CD235a) (nHC=13 and nPV=17). In PV, we observed significantly higher expression of CD169 and CD106, and lower expression of CD172 in C-MC (1,167±216.6 vs 1,834±241.9, p=0.009; 1,427±217.1 vs 2,849±182.3, p=0.0004; 104,707±9,546 vs 77,070±8,756, p=0.0428, respectively); higher CD169 and CD106 in I-MC (2,221±322.4 vs 3,150±321.8, p=0.0371; 2,186±201.7 vs 2,721±153.5, p=0.0238, respectively); and higher CD206, CD163, CD172, and CD106 in CD14 lowCD16 + non-classical MCs (NC-MC) (181.2±8.5 vs 268.6±13.4, p Our findings reveal an increase in the expression of markers relevant to the adhesion of erythrocytes to MCs in all MC subsets from PV patients along with more frequent erythrocyte phagocytosis in circulating cells. Further studies should yield better understanding of the role of MCs in SE and in the formation of EBIs, providing future targets for the treatment of chronic SE in PV patients. Disclosures Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.

Patients with homozygous beta thalassemia and the β⁰β⁰ genotype produce almost only fetal hemoglobin and a small proportion of hemoglobin A2. However, in the bone marrow of these patients there are descriptions of two different subpopulations of erythroblasts, one with high production of HbF (High HbF cells) and another with low production of HbF (Low HbF cells). The High F cells are probably those that survive through erythropoiesis and originate the viable red blood cells. The molecular mechanisms that are responsible for this high production of HbF are not clear. Therefore, the central aim of our work was to study, in cultures of CD34⁺ cells from β⁰β⁰ patients, differentiated to erythrocytes in vitro, the molecular characteristics of cells related to high production of HbF (High HbF), compared to those with low production of HbF (Low HbF). For this purpose, peripheral blood was collected from patients and CD34⁺ cells were identified, and further differentiated in vitro and then isolated by flow cytometry (FACS Aria). After separating the High HbF and Low HbF groups, the cells were morphologically evaluated by optical and confocal microscopy and image cytometry. Next RNA was extracted from the pool of isolated cells and the RNA sequencing assay (RNAseq) was performed, so that it was possible to analyze the transcripts that were differentially expressed between each of the groups. Finally, we validated the sequencing data by real-time PCR. The sorting was performed on the 10th day of cell culture, when the cells presented a double labeling profile for anti-transferrin (CD71) and anti-glycophorin (CD235) antibodies, the mean intensity and fluorescence (MIF) of the markers was 1058 and 1365 for CD71 in healthy control and β-thalassemia groups, respectively, and for CD235 was 430 in controls and 516 for patients. The cells were in an intermediate stage of maturation. After the isolation of sub-populations of High HbF and Low HbF cells, confocal microscopy indicated a clear difference in the intracellular HbF levels of the analyzed cells, confirming that the sorting by cytometry had occurred as desired. Imaging cytometry analysis revealed a MIF of 7.8 x 10⁵ for High HbF cells and 1.7 x 10⁵ for Low HbF cells, when we assessed the cell pools and when single cells were studied, these values ranged from 2.2 x 10⁵ for Low HbF cells to 1.9 x 10⁶ of mean intensity and fluorescence for High cells. After all data generated by RNAseq were filtered, we obtained a list of 16 enriched and differentially-expressed genes between High HbF and Low HbF cells. From these genes, it was possible to identify two that were apparently associated with the increased production of fetal hemoglobin, the genes ZBTB8B and LIN28B. ZBTB8B is a protein-coding gene with a zinc finger domain and may be involved in transcriptional regulation, it was included in our analyses since this gene is part of the same family as ZBTB7A, an important transcription factor that represses several genes involved in cell differentiation and proliferation and that has already been described as a regulator of HbF production. The LIN28B gene encodes an RNA-binding protein, which is described as a possible regulator of HbF levels during fetal development, by increasing the expression of gamma globin, culminating in the increase of intracellular levels of fetal hemoglobin through a direct action on the let-7 microRNA family and BCL11A gene.These results not only contribute to a better understanding of the mechanisms of gene regulation involved in the production of HbF, but indicate a potential new therapeutic approach to increase the production of HbF in hemoglobinopathies. Disclosures Costa: Novartis: Consultancy.

Pacientes com β-talassemia homozigótica e genótipo β0β0 produzem apenas hemoglobina fetal (HbF) e uma pequena proporção de hemoglobina A2. No entanto, na medula óssea desses pacientes foram descritas duas subpopulações de eritroblastos, uma com alta produção de HbF (High HbF) e outra com baixa produção de HbF (Low HbF). As células High HbF são provavelmente aquelas que sobrevivem à eritropoiese e dão origem aos glóbulos vermelhos viáveis. Entretanto, os mecanismos moleculares envolvidos nessa alta produção de HbF ainda não foram elucidados. Portanto, o objetivo do nosso projeto foi estudar, em cultura de células CD34+ de pacientes β0β0, diferenciadas para eritrócitos in vitro, as características moleculares das células relacionadas à alta produção de HbF (High HbF), frente aquelas com baixa produção de HbF (Low HbF). Para tanto, foi feita a coleta de sangue periférico dos pacientes e foram identificadas as células CD34+, essas foram diferenciadas in vitro e isoladas por citometria de fluxo (FACS Aria) de acordo com o conteúdo intracelular de HbF. Após a separação dos grupos High HbF e Low HbF, as células foram avaliadas morfologicamente por microscopia óptica, confocal e citometria de imagem. Em seguida, o RNA total foi extraído do pool de células isoladas e realizado o ensaio de sequenciamento de RNA (RNAseq), de forma que fosse possível analisar os transcritos que estavam diferencialmente expressos entre cada um dos grupos. Por fim, validamos os dados de sequenciamento por PCR em tempo real. O isolamento foi realizado no 10°dia de cultivo celular, quando as células apresentavam duplo perfil de marcação para anticorpos anti-transferrina (CD71) e anti-glicoforina (CD235), a intensidade média de fluorescência (MIF) dos marcadores foi de 1058 e 1365 para CD71 em grupos de controle saudável e β-talassemia, respectivamente, e para CD235 foi de 430 em controles e 516 em β0β0. As células apresentavam-se em estágio intermediário de maturação. Após o isolamento das subpopulações de células de High HbF e Low HbF, a microscopia confocal mostrou uma clara diferença nos níveis de HbF intracelular das células analisadas, confirmando que o isolamento por citometria havia ocorrido conforme o esperado. Ao analisarmos, por citometria de imagem, as subpopulações das células isoladas, obtivemos um MIF de 7,8 × 105 para células High HbF e 1,7 × 105 para células Low HbF. Quando fizemos as análises para single cell, esses valores variaram de 2,2 × 105 para as células Low HbF e 1,9 × 106 de MIF para as células High HbF. Após todos os dados gerados pelo RNAseq serem filtrados, obtivemos uma lista de 16 genes enriquecidos e diferencialmente expressos entre células High HbF e Low HbF. A partir desses genes, foi possível identificar dois que aparentemente estavam associados ao aumento da produção de hemoglobina fetal, os genes SMARCA1 e LIN28B. O gene LIN28B codifica uma proteína de ligação ao RNA, que é descrita como um possível regulador dos níveis de HbF durante o desenvolvimento fetal, por aumentar a expressão de gama globina. Já o gene SMARCA1 contribui para o complexo de remodelação da cromatina durante o processo de transcrição e pode estar envolvido com os níveis de expressão de HbF. Esses resultados não apenas contribuem para um melhor entendimento dos mecanismos de regulação gênica envolvidos na produção de HbF, mas indicam uma potencial nova abordagem terapêutica para aumentar a produção de HbF em hemoglobinopatias.

Sickle cell anemia (SCA) pathophysiology is characterized by the activation of sickle red blood cells, reticulocytes, leukocytes, platelets, and endothelial cells, and with the expression of several inflammatory molecules. Therefore, it is conceivable that variations in levels of proinflammatory cytokines may act as a signaling of differential clinical course in SCA. Here, we evaluated the clinical impact of proinflammatory cytokines interleukin 1-β (IL-1β), interleukin 6 (IL-6), and interleukin 8 (IL-8) in 79 patients with SCA, followed in a single reference center from northeastern Brazil. The main clinical/laboratory data were obtained from patient interview and medical records. The proinflammatory markers IL-1β, IL-6, and IL-8 were evaluated by using commercially available enzyme-linked immunosorbent assay kits. According to levels of the proinflammatory markers, we observed that patients who had a higher frequency of VOC per year (P = 0.0236), acute chest syndrome (P = 0.01), leg ulcers (P = 0.0001), osteonecrosis (P = 0.0006), stroke (P = 0.0486), and priapism (P = 0.0347) had higher IL-6 levels compared with patients without these clinical complications. Furthermore, increased levels of IL-8 were found in patients who presented leg ulcers (P = 0.0184). No significant difference was found for IL-1β levels (P > 0.05). In summary, the present study emphasizes the role of inflammation in SCA pathophysiology, reveals an association of IL-8 levels and leg ulcer occurrence, and indicates that IL-6 levels can be used as a useful predictor for poor outcomes in SCA.

The complex pathophysiology of sickle cell anemia (SCA) involves intravascular hemolytic processes and recurrent vaso-occlusion, driven by chronic vascular inflammation, which result in the disease's severe clinical complications, including recurrent painful vaso-occlusive episodes. Hydroxyurea, the only drug frequently used for SCA therapy, is a cytostatic agent, although it appears to exert nitric oxide/soluble guanylyl cyclase (sGC) modulating activity. As new drugs that can complement or replace the use of hydroxyurea are sought to further reduce vaso-occlusive episode frequency in SCA, we investigated the effects of the sGC agonists BAY 60-2770 (sGC activator) and BAY 41-2272 (sGC stimulator) in the presence or absence of hydroxyurea on SCA vaso-occlusive mechanisms and cell recruitment both ex vivo and in vivo. These agents significantly reduced stimulated human SCA neutrophil adhesive properties ex vivo in association with the inhibition of surface β2-integrin activation. A single administration of BAY 60-2770 or BAY 41-2272 decreased tumor necrosis factor cytokine-induced leukocyte recruitment in a mouse model of SCA vaso-occlusion. Importantly, the in vivo actions of both agonists were significantly potentiated by the coadministration of hydroxyurea. Erythroid cell fetal hemoglobin (HbF) elevation is also a major goal for SCA therapy. BAY 41-2272 but not BAY 60-2770 at the concentrations employed significantly induced γ-globin gene transcription in association with HbF production in cultured erythroleukemic cells. In conclusion, sGC agonist drugs could represent a promising approach as therapy for SCA, for use either as stand-alone treatments or in combination with hydroxyurea. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that stimulators and activators of sGC are potent inhibitors of the adhesion and recruitment of leukocytes from humans and in mice with sickle cell anemia (SCA) and may represent a promising approach for diminishing vaso-occlusive episode frequency in SCA. Hydroxyurea, a drug already frequently used for treating SCA, was found to potentiate the beneficial effects of sGC agonists in in vivo studies, implying that these classes of compounds could be used alone or in combination therapy.

Sickle cell anemia (SCA) pathophysiology is characterized by the activation of sickle red blood cells, reticulocytes, leukocytes, platelets, and endothelial cells, and with the expression of several inflammatory molecules. Therefore, it is conceivable that variations in levels of proinflammatory cytokines may act as a signaling of differential clinical course in SCA. Here, we evaluated the clinical impact of proinflammatory cytokines interleukin 1-β (IL-1β), interleukin 6 (IL-6), and interleukin 8 (IL-8) in 79 patients with SCA, followed in a single reference center from northeastern Brazil. The main clinical/laboratory data were obtained from patient interview and medical records. The proinflammatory markers IL-1β, IL-6, and IL-8 were evaluated by using commercially available enzyme-linked immunosorbent assay kits. According to levels of the proinflammatory markers, we observed that patients who had a higher frequency of VOC per year (P = 0.0236), acute chest syndrome (P = 0.01), leg ulcers (P = 0.0001), osteonecrosis (P = 0.0006), stroke (P = 0.0486), and priapism (P = 0.0347) had higher IL-6 levels compared with patients without these clinical complications. Furthermore, increased levels of IL-8 were found in patients who presented leg ulcers (P = 0.0184). No significant difference was found for IL-1β levels (P 0.05). In summary, the present study emphasizes the role of inflammation in SCA pathophysiology, reveals an association of IL-8 levels and leg ulcer occurrence, and indicates that IL-6 levels can be used as a useful predictor for poor outcomes in SCA.

Made available in DSpace on 2018-12-11T17:37:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-25 N-oxide derivatives 5(a-b), 8(a-b), and 11(a-c) were designed, synthesized and evaluated in vitro and in vivo as potential drugs that are able to ameliorate sickle cell disease (SCD) symptoms. All of the compounds demonstrated the capacity to releasing nitric oxide at different levels ranging from 0.8 to 30.1%, in vivo analgesic activity and ability to reduce TNF-α levels in the supernatants of monocyte cultures. The most active compound (8b) protected 50.1% against acetic acid-induced abdominal constrictions, while dipyrone, which was used as a control only protected 35%. Compounds 8a and 8b inhibited ADP-induced platelet aggregation by 84% and 76.1%, respectively. Both compounds increased γ-globin in K562 cells at 100 μM. The mechanisms involved in the γ-globin increase are related to the acetylation of histones H3 and H4 that is induced by these compounds. In vitro, the most promising compound (8b) was not cytotoxic, mutagenic and genotoxic. São Paulo State University (UNESP) School of Pharmaceutical Sciences Molecular and Clinical Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD 20892, United States Faculty of Medical Sciences State University of Campinas - UNICAMP São Paulo State University (UNESP) School of Dentistry Dipartimento di Scienza e Tecnologia del Farmaco Università degli Studi di Torino São Paulo State University (UNESP) School of Pharmaceutical Sciences São Paulo State University (UNESP) School of Dentistry

Made available in DSpace on 2019-10-06T16:08:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) β-thalassemia is a worldwide distributed monogenic red cell disorder, characterized by an absent or reduced beta globin chain synthesis. The unbalance of alpha-gamma chain and the presence of pathological free iron promote severe oxidative damage, playing crucial a role in erythrocyte hemolysis, exacerbating ineffective erythropoiesis and decreasing the lifespan of red blood cells (RBC). Catalase, glutathione peroxidase and peroxiredoxins act together to protect RBCs from hydrogen peroxide insult. Among them, peroxiredoxins stand out for their overall abundance and reactivity. In RBCs, Prdx2 is the third most abundant protein, although Prdxs 1 and 6 isoforms are also found in lower amounts. Despite the importance of these enzymes, Prdx1 and Prdx2 may have their peroxidase activity inactivated by hyperoxidation at high hydroperoxide concentrations, which also promotes the molecular chaperone activity of these proteins. Some studies have demonstrated the importance of Prdx1 and Prdx2 for the development and maintenance of erythrocytes in hemolytic anemia. Now, we performed a global analysis comparatively evaluating the expression profile of several antioxidant enzymes and their physiological reducing agents in patients with beta thalassemia intermedia (BTI) and healthy individuals. Furthermore, increased levels of ROS were observed not only in RBC, but also in neutrophils and mononuclear cells of BTI patients. The level of transcripts and the protein content of Prx1 were increased in reticulocyte and RBCs of BTI patients and the protein content was also found to be higher when compared to beta thalassemia major (BTM), suggesting that this peroxidase could cooperate with Prx2 in the removal of H 2 O 2 . Furthermore, Prdx2 production is highly increased in RBCs of BTM patients that present high amounts of hyperoxidized species. A significant increase in the content of Trx1, Srx1 and Sod1 in RBCs of BTI patients suggested protective roles for these enzymes in BTI patients. Finally, the upregulation of Nrf2 and Keap1 transcription factors found in BTI patients may be involved in the regulation of the antioxidant enzymes analyzed in this work. Universidade Federal de São Carlos (UFSCar) Departamento de Genética e Evolução Universidade de Campinas (UNICAMP) Departamento de Genética Evolução e Bioagentes Universidade Federal de Pernambuco (UFPE) Departamento de Genética Fundação de Hematologia e Hemoterapia do estado de Pernambuco (HEMOPE) Hemocentro da Universidade de Campinas (UNICAMP) Universidade Estadual Paulista (UNESP) Campus Litoral Paulista Universidade de São Paulo (USP) Departamento de Genética Biologia Evolutiva Universidade Estadual Paulista (UNESP) Campus Litoral Paulista FAPESP: 2011/50358-3

Sickle cell anemia is a recessive inherited disease caused by a single nucleotide polymorphism in the β-globin gene the resulting substitution of glutamic acid by valine causes red blood cell sickling when deoxygenated. Some hypomethylating agents are able to induce the expression of γ-globin by inhibiting DNA methylation at the gene promoter. However, it is not completely understood how this regulation occurs and which genes are involved in this process. To understand fetal hemoglobin (HbF) regulation, CD34+ cells were treated with 1µM of decitabine on the 9th day of culture to induce HbF production. The expression levels of transcription factors and chromatin modifiers were evaluated through the PCR Array platform (Qiagen™ Germany). The transcription factor, HNF4A (Hepatocyte Nuclear Factor 4 Alpha), was highly upregulated in cells treated with decitabine, compared to the control cells, and was chosen as a candidate for CRISPR/Cas9 knockout in HUDEP-2 cells (immortalized human erythroid progenitor cells). The HNF4A gene has been reported as a transcription factor, which regulates the expression of several hepatic genes, and is able to play a role in the development of the liver, kidney, and intestines. Moreover, HNF4A is expressed in the hematopoietic tissue. To the best of our knowledge, the association between HNF4A and gamma globin gene synthesis has not been previously described. HUDEP-2 cells were cultured and then treated with 50nM decitabine. After 72 hours, HbF levels were measured with anti-HbF antibody by flow cytometry in three biological replicates. The percentage of cells positive for HbF in decitabine-treated HUDEP-2 were 12.27 ± 0.7%, N=3, while in control cells the percentage was 1.0 ± 0.06%, N=3 (p Disclosures No relevant conflicts of interest to declare.

Dendritic cells (DCs) are the sentinels of the immune system able to recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs), promoting a bridge between the innate and adaptive immune systems. They form a heterogeneous group of cells with different development, phenotype and functions, and they are mainly classified in conventional DCs 1 (cDC1) and 2 (cDC2), plasmacytoid DCs (pDC) and inflammatory DCs. Changes in the development of DCs, in the ratio of the subsets or in the maturation and activation can impair immunity or tolerance, inducing susceptibility to infections, as well as the development of inflammatory and autoimmune diseases. Sickle cell disease (SCD), one of the most common hemoglobinophaties throughout the world, can be considered as a chronic inflammatory disease, with systemic release of TNF-α, IL-6, IL-1β and IL-8. Despite presenting more inflammation, SCD patients have dysfunction of lymphocytes T and B responses, and are more susceptible to infections. Although DCs are the main responsible for the activation and polarization of lymphocytes function and are able to produce the pro-inflammatory cytokines present in the serum of SCD patients, very little attention has been giving to these cells so far. In the present study, we characterized the subpopulations of circulating DCs in SCD patients. Our data of flow cytometry show that SCD patients (SS) have higher percentage (AA: 0.6±0.09, n=20; SS: 1.25±0.2 n=22; p Isolated circulating monocytes from patients are able to differentiate in vitro in DCs with the expression of the characteristic markers CD1c and CD209, however, unlike healthy individuals, a great percentage of these cells also express CD14 (AA: 5.5±2.2, n=16; SS: 17.6±6.1, n=14; p=0.02), indicating the presence of inflammatory DCs. These cells are more mature and activated in steady state as shown by CD83 (p=0.007), CD86 (p=0.0001) and HLA-DR (p Monocyte-derived DCs (mo-DCs) from patients are able to induce more proliferation of CD4+ and CD8+ T lymphocytes than mo-DCs from healthy individuals (AA: n=4; SS: n=4), and CD14+ DCs induce higher proliferation of T lymphocytes than CD14- DCs from the same patient (n=2 pairs of CD14- and CD14+ DCs). We have also seen that SCD patients have reduced percentage of regulatory T cells, as shown by Foxp3+ in the gate of CD3+CD4+ cells (AA: 4.0±0.26, n=17; SS: 3.1±0.3, n=17; p=0.01), and increased IL-17-producing CD4+ (AA: 1.1±0.08, n=18; SS: 1.7±0.18, n=17; p=0.05) and CD8+ (AA: 0.24±0.02; SS: 0.68±0.1, n=17; p=0.0001) T lymphocytes in the blood. Whether DCs are responsible for this skewing of T lymphocyte phenotype in SCD is still to be determined. Our data so far show that there are differences in circulating DC populations in SCD patients and they could explain the changes observed on T cell responses and the susceptibility to infections. The present data add new knowledge about the chronic inflammation in SCD, which is one of the major events in the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

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.

The endothelium plays an important role in sickle cell anemia (SCA) pathophysiology, interacting with red cells, leukocytes and platelets during the vaso-occlusive process and undergoing activation and dysfunction as a result of intravascular hemolysis and chronic inflammation. Blood outgrowth endothelial cells (BOECs) can be isolated from adult peripheral blood and have been used in diverse studies, since they have a high proliferative capacity and a stable phenotype during in vitro culture. This study aimed to establish BOEC cultures for use as an in vitro study model for endothelial function in sickle cell anemia. Once established, BOECs from steady-state SCA individuals (SCA BOECs) were characterized for their adhesive and inflammatory properties, in comparison to BOECs from healthy control individuals (CON BOECs). Cell adhesion assays demonstrated that control individual red cells adhered significantly more to SCA BOEC than to CON BOEC. Despite these increased adhesive properties, SCA BOECs did not demonstrate significant differences in their expression of major endothelial adhesion molecules, compared to CON BOECs. SCA BOECs were also found to be pro-inflammatory, producing a significantly higher quantity of the cytokine, IL-8, than CON BOECs. From the results obtained, we suggest that BOEC may be a good model for the in vitro study of SCA. Data indicate that endothelial cells of sickle cell anemia patients may have abnormal inflammatory and adhesive properties even outside of the chronic inflammatory and vaso-occlusive environment of patients.

Hereditary persistence of fetal hemoglobin deletion type-2 (HPFH-2) and Sicilian-δβ-thalassemia are conditions described as large deletions of the human β-like globin cluster, with absent β-globin chains and a compensatory variable increase in γ-globin. HPFH, in general, may be distinguished from DB-Thalassemia by higher fetal hemoglobin (HbF) levels, absence of anemia and hypochromic and microcytic erythrocytes. MicroRNAs (miRNAs) regulate a range of cellular processes including erythropoiesis and regulation of transcription factors such as the BCL11A and SOX6 genes, which are related to the regulation of γ-globin expression. In this report, a possible association among the overexpression of miRNAs and the expression of the γ-globin gene was analyzed in these two conditions. Forty-nine differentially expressed miRNAs were identified by microarrays in CD34+-derived erythroid cells of two subjects heterozygous for Sicilian-δβ-thalassemia, 2 for HPFH-2 and 3 for controls after 13 days of culture. Some of these miRNAs may participate in γ-globin gene regulation and red blood cell function. The BCL11A gene was found to be potentially targeted by 12 miRNAs that were up-regulated in HPFH-2 or in DB-Thal. A down-regulation of BCL11A gene expression in HPFH-2 was verified by quantitative polymerase chain reaction. These data suggest an important action for miRNA that may partially explain the phenotypic differences between HPFH-2 and Sicilian δβ-thalassemia and the increased expression of γ-globin in these conditions.

Sickle cell disease (SCD) is a complex disease that is characterized by the polymerization of deoxyhemoglobin S, altered red blood cell membrane biology, endothelial activation, hemolysis, a procoagulant state, acute and chronic inflammation, and vaso-occlusion. Among the physiological changes that occur during pregnancy, oxygen is consumed by fetal growth, and pregnant women with SCD are more frequently exposed to low oxygen levels. This might lead to red blood cells sickling, and, consequently, to vaso-occlusion. The mechanisms by which SCD affects placental physiology are largely unknown, and chronic inflammation might be involved in this process. This study aimed to evaluate the gene expression profile of inflammatory response mediators in the placentas of pregnant women with sickle cell cell anemia (HbSS) and hemoglobinopathy SC (HbSC). Our results show differences in a number of these genes. For the HbSS group, when compared to the control group, the following genes showed differential expression: IL1RAP (2.76-fold), BCL6 (4.49-fold), CXCL10 (−2.12-fold), CXCR1 (−3.66-fold), and C3 (−2.0-fold). On the other hand, the HbSC group presented differential expressions of the following genes, when compared to the control group: IL1RAP (4.33-fold), CXCL1 (3.05-fold), BCL6 (4.13-fold), CXCL10 (−3.32-fold), C3 (−2.0-fold), and TLR3 (2.38-fold). Taken together, these data strongly suggest a differential expression of several inflammatory genes in both SCD (HbSS and HbSC), indicating that the placenta might become an environment with hypoxia, and increased inflammation, which could lead to improper placental development.

Phthalimide derivatives containing furoxanyl subunits as nitric oxide (NO)-donors (3a-g) were designed, synthesized, and evaluated in vitro and in vivo for their potential uses in the oral treatment of sickle cell disease symptoms. All compounds (3a-g) demonstrated NO-donor properties at different levels. Moreover, compounds 3b and 3c demonstrated analgesic activity. Compound 3b was determined to be a promising drug candidate for the aforementioned uses, and it was further evaluated in K562 culture cells to determine its ability to increase levels of γ-globin expression. After 96 h at 5 μM, compound 3b was able to induce γ-globin expression by nearly three times. Mutagenic studies using micronucleus tests in peripheral blood cells of mice demonstrated that compound 3b reduces the mutagenic profile as compared with hydroxyurea. Compound 3b has emerged as a new leading drug candidate with multiple beneficial effects for the treatment of sickle cell disease symptoms and provides an alternative to hydroxyurea treatment.

Phosphatidylinositol phosphate kinases (PIPKs) are enzymes that participate in diverse intracellular signaling pathways. They are classified into 3 functionally distinct subfamilies - PIPKI (α, β, γ), PIPKII (α, β, γ), and PIPKIII - located in various subcellular compartments. Recently, the PIPKIIα and β-globin genes were found to be overexpressed in reticulocytes from 2 siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIa and the production of globins. The main aim of this study was to determine the expression profiles of PIPK genes in healthy individuals during in vitro erythropoiesis using quantitative real-time polymerase chain reaction and to compare these profiles with profiles of globin genes. Our results showed that expression of all PIPKs increases as the cells differentiate, coinciding with the expression profiles of globins. Analysis of the effects of globins on PIPK genes revealed that they varied significantly between the globins, the most noticeable being the effect of α-globin on PIPKIIα (P < 0.0001) and γ-globin on PIPKIIγ (P < 0.0001). The relationship between the expression of PIPKs and globin genes was statistically significant, particularly between PIPKIIα and α-globin (P = 0.0002) and PIPKIIγ and β-globin (P < 0.0001). Linear correlation analysis revealed a strong relationship between PIPKIIα and α-globin genes. This study is the first to establish the expression profiles of PIPK genes during in vitro erythropoiesis in healthy individuals and suggests a parallel between the expression of PIPK and globin genes, reinforcing the hypothesis that they may be related.

A novel series of thalidomide derivatives (4a-f) designed by molecular hybridization were synthesized and evaluated in vitro and in vivo for their potential use in the oral treatment of sickle cell disease symptoms. Compounds 4a-f demonstrated analgesic, anti-inflammatory, and NO-donor properties. Compounds 4c and 4d were considered promising candidate drugs and were further evaluated in transgenic sickle cell mice to determine their capacity to reduce the levels of the proinflammatory cytokine tumor necrosis factor α (TNFα). Unlike hydroxyurea, the compounds reduced the concentrations of TNFα to levels similar to those induced with the control dexamethasone (300 μmol/kg). These compounds are novel lead drug candidates with multiple beneficial actions in the treatment of sickle cell disease symptoms and offer an alternative to hydroxyurea treatment.

Sickle cell disease (SCD) is a serious condition, chronic and undoubtedly represents a public health problem worldwide. SCD is caused by a point mutation in codon 6 of the β globin gene resulting in the production of a structurally abnormal hemoglobin, hemoglobin S. Although the cause of the disease has been known for more than fifty years, therapeutic options are still quite limited. High levels of fetal hemoglobin (HbF) in the blood are associated with a better clinical outcome in SCD patients. In some individuals, the expression of γ-globin gene persists into adulthood in elevated levels, which is called hereditary persistence of fetal hemoglobin (HPFH). A single nucleotide mutation from C to G at position -195 of the HBG1 gene promoter, called non deletional HPFH Brazilian type (nd-HPFH-B), augments the levels of HbF in patients in 7%- 30%. Nd-HPFH-B has been described by our group, but the mechanism and how this single mutation rises HbF levels differently in red blood cells is still unknown. Genome editing using CRISPR/Cas9 in HUDEP-2 cell, an erythroid precursor line, has been developed through homologous direct repair from a small single DNA strand containing the guanine in -195 position at HBG1 gene promotor. All the other genes, including the second HBG1 allele were unaltered. This point mutation has been carried out by CRISPR/Cas9 high fidelity system, capable of performing a specific break in the DNA target sequence, that improves homologous recombination rate of the donor sequence containing the -195 C For the first time, we generated a HUDEP-2 cell line with the -195 C>T mutation in HBG1 promoter using CRISPR/Cas9 genome editing. The HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1uM ssODN -195, using CD34+ human cell kit and program E-001 in AMAXA Nucleofector 4D- device (Lonza). Seven days after nucleofection, the transformed cells were submitted to clonal selection for 25 days. The genomic DNA from 48 clones were submitted by Sanger Sequencing. The sequencing analysis showed highest Crispr/Cas9 efficiency in genomic DNA cut (77.08%; 37/48) and satisfactory ssODN -195 homologous recombination (10.4%; 5/48). Five nd-HPFH-B HUDEP-2 clones and three other clones without the mutation, but with indels after Cas9 DNA cut (controls), were expanded in culture and the HbF levels were measure with anti-HbF antibody by flow cytometry in two biological replicates. HbF levels in nd-HPFH-B HUDEP-2 clones were 6.02%±1.4, 8.25% ± 0.28, 10.18% ± 3.71, 11.95% ± 0.49, 26,3% ± 4,6 while in controls were 1.69% ± 0.26, 1.66% ± 0.26, 0.59% ± 0.06. Two nd-HPFH-B clones were differentiated into erythrocyte in vitro, and fetal hemoglobin levels persisted at high levels seen previously. In addition, α-globin, β-globin and γ-globin mRNA levels were evaluated in three nd-HPFH-B HUDEP-2 clones and two control clones. The mRNA HBG1/HBG1+HBB percentage in nd-HPFH-B were 96.16% ± 4.10 against 22.63% ± 9.64 in controls. The monoallelic single nucleotide mutation -195 C>G is capable to increase the fetal hemoglobin levels up to 30% in nd-HPFH-B HUDEP-2, and our results shows that this is a potential experimental in vitro model to be used in future studies. Disclosures No relevant conflicts of interest to declare.

Ceruloplasmin (CP) is a multicopper ferroxidase that oxidizes ferrous iron promoting the binding of ferric iron to transferrin. The secreted form of CP (sCP) produced mainly by the liver is essentially absent in patients with aceruloplasminemia, a rare type of hereditary iron overload with brain, liver and pancreatic siderosis. Alternative RNA splicing generates a form of CP that is anchored by glycosylphosphatidylinositol (GPI-CP) to the membrane of astrocytes and immune cells. GPI-CP has been reported to help stabilize ferroportin, the only known iron exporter in mammal cells, so we aimed to investigate whether ferroportin expression is abnormal in circulating blood cells in aceruloplasminemia and in paroxysmal nocturnal hemoglobinuria (PNH), a naturally-occurring human model of acquired deficiency of GPI-anchored proteins. Peripheral blood samples were collected from two patients with aceruloplasminemia with different mutations on the CP gene: CP c.2879-1 G>T (splice site mutation) and CP c.2756 T>C (missense mutation), both with undetectable levels of sCP (T were similar to those seen in the control. Nevertheless, monocytic expression of GPI-CP and ferroportin were significantly reduced in CP c.2756 T>C and PNH, when compared to the control. These data confirm previous observations that B lymphocytes and monocytes express GPI-CP and ferroportin, and concomitant reduction of both expressions in PNH and in CP c.2756 T>C support that GPI-CP fosters ferroportin stability on the cell membrane. We also show that, while germline mutations of the CP gene generally cause undetectable sCP, there is heterogeneity in GPI-CP expression, which may remain preserved, as observed in CP c.2879-1 G>T. Further studies are necessary to clarify why this splice site mutation would still allow GPI-anchoring, while the CP c.2756 T>C point mutation abrogates the ability to anchor GPI-CP. While the preservation of lymphocytic GPI-CP was not surprising in an essentially myeloid PNH clone, normal GPI-CP in B lymphocytes in aceruloplasminemia suggests there are lineage-specific differences in physiological expression of ceruloplasmin forms between B lymphocytes and monocytes, with possible implications to the importance of iron metabolism in immune responses. We also noticed that the CP c.2756 T>C patient with monocytic reduction ferroportin presented with slightly more intense anemia and microcytosis. This could result from lower expression of ferroportin in bone marrow macrophages, with impaired iron delivery to erythroblasts, in analogy to monocytes. Finally, acquired ferroportin deficiency in PNH monocytes implies that loss of GPI-anchored protein not only exposes these cells to lysis by complement, but also to intracellular iron retention, generation of reactive oxygen species and may be involved in the pathophysiology of PNH. In summary, our data show that heterogeneity in GPI-CP expression in B lymphocytes and monocytes results in differential expression of ferroportin in aceruloplasminemia and PNH, and future studies should aim at investigating the implications of dysregulated iron metabolism in immune cells. Disclosures Fertrin: Apopharma Inc.: Honoraria; Alexion Pharmaceuticals Brasil: Speakers Bureau.

Objectives: Erythroid differentiation is a dynamic process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. In this study, gene expression profiles during differentiation of human erythroid cells of a normal blood donor were evaluated using SAGE. Materials and methods: Global gene expression was evaluated in cells collected immediately before addition of erythropoietin (0 h) and 192 and 336 h after addition of this hormone. Real-time PCR was used to evaluate activation of differentially expressed genes. Results: The data indicate that global aspects of the transcriptome were similar during differentiation of the majority of the genes and that a relatively small set of genes is probably involved in modification of erythroid cells during differentiation. We have identified 93 differentially expressed genes during erythroid development, and expression of some of these was confirmed by qPCR. Various genes including EYA3, ERH, HES6, TIMELESS and TRIB3 were found to be homologous to those of Drosophila melanogaster and here are described for the first time during erythroid development. An important and unique carboxypeptidase inhibitor described in mammalians, LXN, was also identified. Conclusions: The results of this study amplify previously published data and may contribute to comprehension of erythroid differentiation and identification of new target genes involved in some erythroid concerning diseases.

We are reporting here the results of differential gene expression experiments comparing two siblings, a 21-yr-old male and a 19-yr-old female, with the same alpha-thalassemia genotype (-alpha(3.7)/(--SEA)) and quite different levels of Hb H in the peripheral blood (18.7 and 5%, respectively). By using mRNA differential-display reverse-transcription-PCR and suppression subtractive hybridization, two main transcripts were selected in both procedures and validated by qRT-PCR, one corresponding to the phosphatidylinositol phosphate 4-kinase type II-alpha (PIP4KIIA) gene and the other to the beta-globin gene, both over expressed in the patient with the higher percentage of Hb H. Type II PIP kinases produce phosphatidylinositol 4,5 biphosphate, a critical and pleiotropic regulatory molecule involved in diverse cellular activities, including gene expression. Our results suggest that PIP4KIIA may be one of the factors related to the regulation of the beta-globin gene expression and the different levels of Hb H in alpha-thalassemic patients.

Inflammation, cell adhesion to vascular endothelium, and endothelial injury contribute to sickle cell anemia (SCA) vaso-occlusion. Although alterations in inflammatory cytokines and biomarkers have been related, reports have been conflicting, and a conclusive role for these molecules in the disease remains to be established. Furthermore, the effect of hydroxyurea therapy (HU) on the release of inflammatory mediators is not understood. This study aimed to determine plasma levels and leukocyte gene expressions of inflammatory mediators in healthy controls, steady-state SCA patients, and SCA patients on HU therapy. TNF-α, IL-8, and PGE2 levels were significantly higher in the plasma of SCA individuals when compared with control individuals. HU therapy was associated with a significant reversal of augmented TNF-α and, interestingly, increased plasma anti-inflammatory IL-10. IFN-γ, IL-10, cyclooxygenase 2 (COX-2), and inducible NO synthase (iNOS) gene expressions were unaltered in SCA mononuclear cells (MC); however, gene expressions of TNF-α, IL-8, and the protective enzyme heme oxygenase-1 (HO-1) were significantly higher. HU therapy was not associated with significantly altered SCA MC inflammatory gene expression, although COX-2 mRNA expression was decreased. In SCA neutrophils, gene expressions of IL-8, IFN-γ, iNOS, and HO-1 were significantly higher than those of control subjects. Patients on HU demonstrated lower iNOS and higher IL-10 neutrophil gene expressions. Taken together, data suggest that alterations in the gene expressions and productions of a number of pro- and anti-inflammatory mediators are present in SCA and importantly, in those patients on HU therapy. Knowledge of these pathways may contribute to further the understanding of the pathophysiology of this disease.

Modulation of intracellular cyclic guanosine monophosphate (cGMP) may characterize a therapeutic target for sickle cell disease (SCD); cGMP-dependent signalling may be important for erythroid foetal haemoglobin induction and exert anti-inflammatory functions in leucocytes. As the inhibition of phosphodiesterases (PDEs), which regulate intracellular cGMP, can result in tissue-specific elevation of cGMP, we studied the gene expressions of cGMP-specific PDEs (-1A, -5A and -9A) in the reticulocytes and neutrophils of healthy controls, steady-state SCD patients and SCD patients on hydroxycarbamide therapy (SCDHC). PDE9A gene expression was found in numerous cell types; however, high expression was found in neutrophils, reticulocytes, CD34(+)-derived erythroid cells and K562 erythroleukaemic cells, indicating a high haematopoietic cell expression. PDE9A gene expression was, however, significantly higher in the reticulocytes and neutrophils of SCD individuals, compared to control cells; Western blotting confirmed the production of PDE9A protein in SCD neutrophils and K562 cells. Inhibition of PDE9A enzyme with the specific inhibitor, BAY73-6691, significantly increased production of the gamma-globin gene (HBG) in K562 cells and reversed the increased adhesive properties of SCD neutrophils. Since elevation of haematopoietic intracellular cGMP may be beneficial in SCD, the relatively limited tissue distribution of PDE9A suggests that it could represent a novel drug target worthy of further study.

Sickle cell disease (SCD) is a chronic inflammatory condition characterized by high leucocyte counts, altered cytokine levels and endothelial cell injury. As the removal of inflammatory cells by apoptosis is fundamental for the resolution of inflammation, we aimed to determine whether the leucocyte apoptotic process is altered in SCD. Neutrophils from SCD individuals showed an inhibition of spontaneous apoptosis when cultured in vitro, in the presence of autologous serum for 20 h. Intracellular cyclic adenosine monophosphate (cAMP) levels were approximately twofold increased in SCD neutrophils; possible cAMP-upregulating factors present in SCD serum include interleukin-8, granulocyte-macrophage colony-stimulating factor and prostaglandin. Accordingly, co-incubation of SCD neutrophils with KT5720, a cAMP-dependent protein kinase (PKA) inhibitor, abrogated increased SCD neutrophil survival. Caspase-3 activity was also significantly diminished in SCD neutrophils cultured for 16 h and this activity was restored when cells were co-incubated with KT5720. BIRC2 (encoding cellular inhibitor of apoptosis protein 1, cIAP(1)), MCL1 and BAX expression were unaltered in SCD neutrophils; however, BIRC3 (encoding the caspase inhibitor, cIAP(2)), was expressed at significantly higher levels. Thus, we report an inhibition of spontaneous SCD neutrophil apoptosis that appears to be mediated by upregulated cAMP-PKA signalling and decreased caspase activity. Increased neutrophil survival may have significant consequences in SCD; contributing to leucocytosis, tissue damage and exacerbation of the chronic inflammatory state.

Hydroxycarbamide (HC) (or hydroxyurea) has been reported to increase fetal haemoglobin levels and improve clinical symptoms in sickle cell anaemia (SCA) patients. However, the complete pathway by which HC acts remains unclear. To study the mechanisms involved in the action of HC, global gene expression profiles were obtained from the bone marrow cells of a SCA patient before and after HC treatment using serial analysis of gene expression. In the comparison of both profiles, 147 differentially expressed transcripts were identified. The functional classification of these transcripts revealed a group of gene categories associated with transcriptional and translational regulation, e.g. EGR-1, CENTB1, ARHGAP4 and RIN3, suggesting a possible role for these pathways in the improvement of clinical symptoms of SCA patients. The genes involved in these mechanisms may represent potential tools for the identification of new targets for SCA therapy.

Background Preeclampsia is one of the leading causes of maternal and neonatal morbidity and mortality in the world, but its appearance is still unpredictable and its pathophysiology has not been entirely elucidated. Genetic studies have associated single nucleotide polymorphisms in genes encoding nitric oxide synthase and matrix metalloproteases with preeclampsia, but the results are largely inconclusive across different populations. Objectives To investigate the association of single nucleotide polymorphisms (SNPs) in NOS3 (G894T, T-786C, and a variable number of tandem repetitions VNTR in intron 4), MMP2 (C-1306T), and MMP9 (C-1562T) genes with preeclampsia in patients from Southeastern Brazil. Methods This prospective case-control study enrolled 77 women with preeclampsia and 266 control pregnant women. Clinical data were collected to assess risk factors and the presence of severe complications, such as eclampsia and HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome. Results We found a significant association between the single nucleotide polymorphism NOS3 T-786C and preeclampsia, independently from age, height, weight, or the other SNPs studied, and no association was found with the other polymorphisms. Age and history of preeclampsia were also identified as risk factors. The presence of at least one polymorphic allele for NOS3 T-786C was also associated with the occurrence of eclampsia or HELLP syndrome among preeclamptic women. Conclusions Our data support that the NOS3 T-786C SNP is associated with preeclampsia and the severity of its complications.

Impairment of nitric oxide (NO)-mediated cavernosal relaxations in middle age contributes to erectile dysfunction. However, little information is available about the alterations of sympathetic neurotransmission and contraction in erectile tissue at middle age. This study aimed to evaluate the alterations of the contractile machinery associated with tyrosine hydroxylase (TH) in rat corpus cavernosum (RCC) at middle age, focusing on the role of superoxide anion. Male Wistar young (3.5-mo) and middle-aged (10-mo) rats were used. Electrical-field stimulation (EFS)- and phenylephrine-induced contractions were obtained in RCC strips. Levels of reactive-oxygen species (ROS) and TH mRNA expression, as well as protein expressions for α1/β1-subunits of soluble guanylyl cyclase (sGC), in RCC were evaluated. The neurogenic contractile responses elicited by EFS (4–32 Hz) were greater in RCC from the middle-aged group that was accompanied by elevated TH mRNA expression ( P < 0.01). Phenylephrine-induced contractions were also greater in the middle-aged group. A 62% increase in ROS generation in RCC from middle-aged rats was observed. The mRNA expression for the α1A-adrenoceptor remained unchanged among groups. Protein levels of α1/β1-sGC subunits were decreased in RCC from the middle-aged compared with young group. The NADPH oxidase inhibitor apocynin (85 mg·rat−1·day−1, 4 wk) fully restored the enhanced ROS production, TH mRNA expressions, and α1/β1-subunit sGC expression, indicating that excess of superoxide anion plays a major role in the sympathetic hyperactivity and hypercontractility in erectile tissue at middle age. Reduction of oxidative stress by dietary antioxidants may be an interesting approach to treat erectile dysfunction in aging population.

Aims Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin–angiotensin system (RAS) may be altered in an animal model of SCD. Main methods Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 ( AT1R and AT2R ) and the angiotensin-converting enzymes ( ACE1 and ACE2 ) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50–75 mg/kg/day, 4 weeks) treatment on these parameters were also determined. Key findings Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. Significance Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.

Sickle cell disease (SCD) is one of the most prevalent hematological diseases in the world. Despite the immense progress in molecular knowledge about SCD in last years few therapeutical sources are currently available. Nowadays the treatment is performed mainly with drugs such as hydroxyurea or other fetal hemoglobin inducers and chelating agents. This review summarizes current knowledge about the treatment and the advancements in drug design in order to discover more effective and safe drugs. Patient monitoring methods in SCD are also discussed.