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Background: Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Methods: Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Results: Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Conclusion: Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

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La anemia por deficiencia de hierro es un problema prevalente a nivel global que aparece en niños, adolescentes y mujeres en edad fértil, son los más afectados. La hemoglobina reticulocitaria es un nuevo biomarcador prometedor para el diagnóstico temprano. Objetivo. Evaluar la hemoglobina reticulocitaria para el diagnóstico precoz de anemia por deficiencia de hierro. Metodología. Se realizó una revisión sistemática en bases de datos biomédicas como PubMed, Scielo, Researchgate, Base, Cochrane Library y DOAJ; se incluyeron 24 estudios observacionales (2018-2023) sobre el uso de la hemoglobina reticulocitaria en el diagnóstico de anemia por deficiencia de hierro; se extrajeron datos sobre las características de los estudios, los valores de sensibilidad y especificidad de este biomarcador. Resultados. La hemoglobina reticulocitaria presentó una sensibilidad agrupada de 90% y una especificidad de 89,5% en los estudios analizados. También mostró una diferencia de medias significativa de -2,88 (IC 95%: -3,36 a -2,40) entre grupos con y sin anemia por deficiencia de hierro. Se encontró una heterogeneidad sustancial entre los resultados de los diferentes estudios (I2=95%; p<0,00001). Conclusión. La hemoglobina reticulocitaria demostró elevada sensibilidad y especificidad, así como una diferencia significativa entre grupos con y sin la condición, lo que evidencia su utilidad como prueba para la detección temprana de la anemia por deficiencia de hierro.

Iron deficiency anemia is a prevalent global health problem that appears in children, adolescents and women of childbearing age, who are the most affected. Reticulocyte hemoglobin is a promising new biomarker for early diagnosis. Objective. To evaluate reticulocyte hemoglobin for the early diagnosis of iron deficiency anemia. Methodology. A systematic review was conducted searching biomedical databases including PubMed, Scielo, Researchgate, Base, Cochrane Library and DOAJ; 24 observational studies (2018-2023) were included on the use of reticulocyte hemoglobin in the diagnosis of iron deficiency anemia; data were extracted on the characteristics of the studies and the sensitivity and specificity values of this biomarker. Results. Reticulocyte hemoglobin showed a pooled sensitivity of 90% and a specificity of 89.5% in the studies analyzed. It also showed a significant mean difference of -2.88 (95% CI: -3.36 to -2.40) between groups with and without iron deficiency anemia. Substantial heterogeneity was found among the results of the different studies (I2=95%; p<0.00001). Conclusion. Reticulocyte hemoglobin demonstrated high sensitivity and specificity, as well as a significant difference between groups with and without the condition, which shows its usefulness as a test for the early detection of iron deficiency anemia.

A anemia por deficiência de ferro é um problema de saúde global prevalente que aparece em crianças, adolescentes e mulheres em idade fértil, sendo os mais afetados. A hemoglobina reticulocitária é um novo biomarcador promissor para o diagnóstico precoce. Objetivo. Avaliar a hemoglobina reticulocitária para o diagnóstico precoce da anemia por deficiência de ferro. Metodologia. Foi realizada uma revisão sistemática com busca em bases de dados biomédicas incluindo PubMed, Scielo, Researchgate, Base Cochrane Library e DOAJ; foram incluídos 24 estudos observacionais (2018-2023) sobre o uso da hemoglobina reticulocitária no diagnóstico de anemia por deficiência de ferro; foram extraídos dados sobre as características dos estudos e os valores de sensibilidade e especificidade deste biomarcador. Resultados. A hemoglobina reticulocitária apresentou sensibilidade agrupada de 90% e especificidade de 89,5% nos estudos analisados. Também mostrou uma diferença média significativa de -2,88 (IC 95%: -3,36 a -2,40) entre grupos com e sem anemia por deficiência de ferro. Encontrou-se heterogeneidade substancial entre os resultados dos diferentes estudos (I2=95%; p<0,00001). Conclusão. A hemoglobina reticulocitária demonstrou elevada sensibilidade e especificidade, bem como uma diferença significativa entre grupos com e sem a condição, o que evidencia a sua utilidade como teste para a detecção precoce da anemia por deficiência de ferro.

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OBJECTIVE: Compare erythropoiesis-related factors between different stages of canine chronic kidney disease (CKD). ANIMALS: 8 healthy adult dogs (controls), and 24 dogs with CKD, equally divided into 3 groups based on International Renal Interest Society-CKD Guidelines (stage 2, 3, and 4) were recruited between December 2012 and December 2014. METHODS: The following were assessed in all dogs and then compared between groups: bone marrow cytology, CBC, reticulocyte count, urinalysis, serum biochemistry, blood pressure, occult gastrointestinal bleeding, and serum concentrations of parathyroid hormone (PTH), erythropoietin, interleukin-1ß, interleukin-3, tumor necrosis factor-α (TNFα), and interferon-γ. RESULTS: Erythropoiesis inducing and suppressing factors and the results of the bone marrow cytology of dogs in stage 2 CKD did not differ from the control group. The presence of reticulocytosis in CKD stage 2 suggests that blood loss or erythrocyte destruction might be contributing to developing anemia. Anemia in dogs with progressive CKD was associated with increasing PTH and TNFα and with elevation of the ratio of myeloid to erythroid precursor cells caused by hypoplasia of the erythroid series. The latter was represented mainly by a decrease in the population of polychromatophilic rubricytes and metarubricytes. CLINICAL RELEVANCE: Increased PTH and TNFα seem to contribute to the reduced percentage of polychromatophilic rubricytes and erythroid population, thereby aggravating the anemia of dogs with advanced CKD. Gastrointestinal blood loss contributes to anemia in all canine CKD stages.

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BACKGROUND: Sickle cell disease (SCD) is a highly prevalent genetic disease caused by a point mutation in the HBB gene, which can lead to chronic hemolytic anemia and vaso-occlusive events. Patient-derived induced pluripotent stem cells (iPSCs) hold promise for the development of novel predictive methods for screening drugs with anti-sickling activity. In this study, we evaluated and compared the efficiency of 2D and 3D erythroid differentiation protocols using a healthy control and SCD-iPSCs. METHODS: iPSCs were subjected to hematopoietic progenitor cell (HSPC) induction, erythroid progenitor cell induction, and terminal erythroid maturation. Differentiation efficiency was confirmed by flow cytometry analysis, colony-forming unit (CFU) assay, morphological analyses, and qPCR-based gene expression analyses of HBB and HBG2. RESULTS: Both 2D and 3D differentiation protocols led to the induction of CD34+/CD43+ HSPCs. The 3D protocol showed good efficiency (>50%) and high productivity (45-fold) for HSPC induction and increased the frequency of BFU-E, CFU-E, CFU-GM, and CFU-GEMM colonies. We also produced CD71+/CD235a+ cells (>65%) with a 630-fold cell expansion relative to that at the beginning of the 3D protocol. After erythroid maturation, we observed 95% CD235a+/DRAQ5- enucleated cells, orthochromatic erythroblasts, and increased expression of fetal HBG2 compared to adult HBB. CONCLUSION: A robust 3D protocol for erythroid differentiation was identified using SCD-iPSCs and comparative analyses; however, the maturation step remains challenging and requires further development.

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ABSTRACT The development of red blood cells (RBCs), or erythropoiesis, occurs in specialized niches in the bone marrow, called erythroblastic islands, composed of a central macrophage surrounded by erythroblasts at different stages of differentiation. Upon anemia or hypoxemia, erythropoiesis extends to extramedullary sites, mainly spleen and liver, a process known as stress erythropoiesis, leading to the expansion of erythroid progenitors, iron recruitment and increased production of reticulocytes and mature RBCs. Macrophages are key cells in both homeostatic and stress erythropoiesis, providing conditions for erythroid cells to survive, proliferate and differentiate. During RBCs aging and injury, macrophages play a fundamental role again, performing the clearance of these cells and recycling iron for new erythroblasts in development. Thus, macrophages are crucial components of the RBCs turnover and in this review, we aimed to cover the main known mechanisms involved in the process of birth and death of RBCs, highlighting the importance of macrophage functions in the whole RBC lifecycle.

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The development of red blood cells (RBCs), or erythropoiesis, occurs in specialized niches in the bone marrow, called erythroblastic islands, composed of a central macrophage surrounded by erythroblasts at different stages of differentiation. Upon anemia or hypoxemia, erythropoiesis extends to extramedullary sites, mainly spleen and liver, a process known as stress erythropoiesis, leading to the expansion of erythroid progenitors, iron recruitment and increased production of reticulocytes and mature RBCs. Macrophages are key cells in both homeostatic and stress erythropoiesis, providing conditions for erythroid cells to survive, proliferate and differentiate. During RBCs aging and injury, macrophages play a fundamental role again, performing the clearance of these cells and recycling iron for new erythroblasts in development. Thus, macrophages are crucial components of the RBCs turnover and in this review, we aimed to cover the main known mechanisms involved in the process of birth and death of RBCs, highlighting the importance of macrophage functions in the whole RBC lifecycle.

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Introduction: To a considerable extent, the magnitude of blood volume (BV) and hemoglobin mass (Hbmass) contribute to the maximum O2-uptake (VO2max), especially in endurance-trained athletes. However, the development of Hbmass and BV and their relationships with VO2max during childhood are unknown. The aim of the present cross-sectional study was to investigate Hbmass and BV and their relationships with VO2max in children and adolescents. In addition, the possible influence of endurance training and chronic hypoxia was evaluated. Methods: A total of 475 differently trained children and adolescents (girls n = 217, boys n = 258; untrained n = 171, endurance trained n = 304) living at two different altitudes (∼1,000 m, n = 204, ∼2,600 m, n = 271) and 9-18 years old participated in the study. The stage of puberty was determined according to Tanner; Hbmass and BV were determined by CO rebreathing; and VO2max was determined by cycle ergometry and for runners on the treadmill. Results: Before puberty, there was no association between training status and Hbmass or BV. During and after puberty, we found 7-10% higher values in the trained groups. Living at a moderate altitude had a uniformly positive effect of ∼7% on Hbmass in all groups and no effect on BV. The VO2max before, during and after puberty was strongly associated with training (pre/early puberty: boys +27%, girls +26%; mid puberty: +42% and +45%; late puberty: +43% and +47%) but not with altitude. The associated effects of training in the pre/early pubertal groups were independent of Hbmass and BV, while in the mid- and late pubertal groups, 25% of the training effect could be attributed to the elevated Hbmass. Conclusions: The associated effects of training on Hbmass and BV, resulting in increased VO2max, can only be observed after the onset of puberty.

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BACKGROUND: The presence of Plasmodium vivax malaria parasites in the human bone marrow (BM) is still controversial. However, recent data from a clinical case and experimental infections in splenectomized nonhuman primates unequivocally demonstrated the presence of parasites in this tissue. METHODS: In the current study, we analyzed BM aspirates of 7 patients during the acute attack and 42 days after drug treatment. RNA extracted from CD71+ cell suspensions was used for sequencing and transcriptomic analysis. RESULTS: We demonstrated the presence of parasites in all patients during acute infections. To provide further insights, we purified CD71+ BM cells and demonstrated dyserythropoiesis and inefficient erythropoiesis in all patients. In addition, RNA sequencing from 3 patients showed that genes related to erythroid maturation were down-regulated during acute infections, whereas immune response genes were up-regulated. CONCLUSIONS: This study thus shows that during P. vivax infections, parasites are always present in the BM and that such infections induced dyserythropoiesis and ineffective erythropoiesis. Moreover, infections induce transcriptional changes associated with such altered erythropoietic response, thus highlighting the importance of this hidden niche during natural infections.

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BACKGROUND: Anemia is one of the most frequent complications in patients with chronic kidney disease (CKD). Despite being multifactorial, the relative or absolute deficiency of erythropoietin production is the leading cause. Recent studies have shown that uremic toxins produced by the gut microbiota also may play a role in the genesis of anemia in these patients. OBJECTIVE: To evaluate the possible association between uremic toxins plasma levels and anemia in patients with CKD on hemodialysis (HD). METHODS: This cross-sectional study evaluated one hundred fifty-four patients (53.2% men, 51.2 ± 11.2 years, hemoglobin (Hb) levels of 11.2 ± 1.6 g/dL). Biochemical variables such as urea, creatinine, hemoglobin, hematocrit, were measured according to standard methods and uremic toxins such as indoxyl sulfate (IS), indole-3-acetic acid (IAA), p-cresyl sulfate (p-CS) plasma levels were measured by reverse-phase high-performance liquid chromatography (RP-HPLC). RESULTS: The levels of uremic toxins such as IS, IAA, p-CS were increased in all patients. However, no correlation was found between uremic toxins plasma levels and anemia parameters. Only patients with Hb < 11 g/dL presented a negative correlation between hematocrit and IAA plasma levels. CONCLUSION: There is no strong evidence that uremic toxins produced by the gut microbiota may be associated with anemia in patients with CKD on HD.

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La hemoglobina reticulocitaria es un nuevo parámetro dentro de los autoanalizadores hematológicos de cuarta generación, siendo indispensable en el diagnóstico y manejo de eritropoyesis deficiente en hierro, especialmente la deficiencia funcional de hierro, el secuestro de hierro y la deficiencia absoluta de hierro. Además, este parámetro demuestra ser más preciso que las pruebas bioquímicas como el hierro sérico, la ferritina y la saturación de transferrina, en la detección precoz de eritropoyesis deficiente en hierro. El objetivo de la investigación fue describir la utilidad clínica de la hemoglobina reticulocitaria (CHr) en el diagnóstico temprano de eritropoyesis por deficiencia de hierro absoluto en mujeres adolescentes. El tipo de investigación fue descriptivo, analítico, el diseño de campo transversal. La muestra voluntaria, no aleatoria estuvo constituida por 62 mujeres adolescentes con edades comprendidas entre los 14 y 19 años. Como resultado se encontró que el 97% de la muestra tiene disminución de la CHr, indicando eritropoyesis deficiente en hierro, mientras que un 3% de las adolescentes presentan valores normales para la CHr, se realizó la relación diagnostica entre pruebas de laboratorio tales como CHr y el Hierro sérico. También se reportó que el 93% de la muestra presenta déficit de hierro sin anemia, y un 7% tiene anemia ferropénica, el rango de edad con mayor predominio de anemia ferropénica fue entre los 14 y 16 años. Se concluye que la CHr es de utilidad clínica y una nueva herramienta de diagnóstico temprano de eritropoyesis por deficiencia de hierro.

Reticulocyte hemoglobin is a new parameter within the fourth generation hematological autoanalyzers, being indispensable in the diagnosis and management of iron deficient erythropoiesis, especially functional iron deficiency, iron sequestration and absolute iron deficiency. Moreover, this parameter proves to be more accurate than biochemical tests such as serum iron, ferritin and transferrin saturation in the early detection of iron deficient erythropoiesis. The aim of the research was to describe the clinical utility of reticulocyte hemoglobin (CHr) in the early diagnosis of absolute iron deficiency erythropoiesis in adolescent females. The type of research was descriptive, analytical, cross-sectional field design. The voluntary, non-random sample consisted of 62 adolescent females aged between 14 and 19 years. As a result, it was found that 97% of the sample had decreased CHr, indicating iron deficient erythropoiesis, while 3% of the adolescents had normal values for CHr. The diagnostic relationship between laboratory tests such as CHr and serum iron was performed. It was also reported that 93% of the sample presented iron deficiency without anemia, and 7% had iron deficiency anemia; the age range with the highest prevalence of iron deficiency anemia was between 14 and 16 years of age. It is concluded that HRH is clinically useful and a new tool for early diagnosis of erythropoiesis due to iron deficiency.

A hemoglobina reticulócita é um novo parâmetro dentro da quarta geração de auto-analisadores hematológicos, sendo indispensável no diagnóstico e manejo da eritropoiese com deficiência de ferro, especialmente deficiência funcional de ferro, seqüestro de ferro e deficiência absoluta de ferro. Além disso, este parâmetro prova ser mais preciso do que testes bioquímicos como ferro sérico, ferritina e saturação da transferrina na detecção precoce de eritropoiese com deficiência de ferro. O objetivo da pesquisa foi descrever a utilidade clínica da hemoglobina reticulocitária (RCHr) no diagnóstico precoce da eritropoiese absoluta de deficiência de ferro em mulheres adolescentes. O tipo de pesquisa foi descritivo, analítico, de corte transversal do campo. A amostra voluntária e não aleatória consistiu de 62 fêmeas adolescentes com idades entre 14 e 19 anos. Como resultado, descobriu-se que 97% da amostra tinha uma diminuição na HRH, indicando uma eritropoiese com deficiência de ferro, enquanto 3% das adolescentes tinham valores normais para HRH. Também foi relatado que 93% da amostra tinha deficiência de ferro sem anemia, e 7% tinha anemia por deficiência de ferro; a faixa etária com maior prevalência de anemia por deficiência de ferro era entre 14 e 16 anos. Conclui-se que o RHH é clinicamente útil e uma nova ferramenta para o diagnóstico precoce da eritropoiese devido à deficiência de ferro.

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