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Autophagy Jul 2012The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal...
The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal rearrangements and changes to cellular architecture. Autophagy is implicated in the sequential removal of erythroid organelles during erythropoiesis, although how this is regulated during late stages of erythroid differentiation, and the potential contribution of autophagy during reticulocyte maturation, remain unclear. Using an optimized ex vivo differentiation system for human erythropoiesis, we have observed that maturing reticulocytes are characterized by the presence of one or few large vacuolar compartments. These label strongly for glycophorin A (GYPA/GPA) which is internalized from the plasma membrane; however, they also contain organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting that they are endocytic/autophagic hybrid structures. Interestingly, we observed the release of these vacuoles by exocytosis in maturing reticulocytes, and speculate that autophagy is needed to concentrate the final remnants of the reticulocyte endomembrane system in autophagosome/endosome hybrid compartments that are primed to undergo exocytosis.
Topics: Exocytosis; Glycophorins; Humans; Membrane Fusion; Phagosomes; Reticulocytes; Transport Vesicles
PubMed: 22659916
DOI: 10.4161/auto.20648 -
American Journal of Hematology Dec 2021Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection,... (Randomized Controlled Trial)
Randomized Controlled Trial
Athletes abuse recombinant human erythropoietin (rhEPO) and erythropoiesis stimulating agents to increase hemoglobin mass and improve performance. To evade detection, athletes have developed sophisticated blood doping regimens, which often include rhEPO micro-dosing. Detection of these methods requires biomarkers with increased sensitivity and a sample matrix that is more amenable to frequent testing in the field. We have developed a method to measure two immature reticulocyte proteins, CD71 and ferrochelatase (FECH), and one total erythrocyte protein, Band 3, in dried blood spots (DBS). This method was tested in response to rhEPO administration after low doses, 40 IU/kg, micro-doses, 900 IU, or saline injection in 20 healthy subjects. During administration of low-dose rhEPO, the mean CD71/Band 3 and FECH/Band 3 ratio increased by 412 ± 197% and 250 ± 44%, respectively. The mean response for the current biomarker, RET%, increased by 195 ± 35%. During administration of rhEPO micro-doses, CD71/Band 3 increased to 127 ± 25% on day 35 and 139 ± 36% on day 39, while no increase was observed in RET%. After rhEPO administration, during the washout phase, mean values decreased to a minimum of 64 ± 4% and 64 ± 11% for CD71/Band 3 and RET%, respectively. However, CD71/Band 3 remained below 75% of baseline for at least 4 weeks after rhEPO injection, while RET% returned to baseline levels. The results demonstrate that immature reticulocyte proteins have a larger response to rhEPO administration than the current biomarker, RET%, and can be monitored in the DBS matrix.
Topics: Adolescent; Adult; Cell Tracking; Dried Blood Spot Testing; Erythropoietin; Humans; Male; Middle Aged; Placebo Effect; Recombinant Proteins; Reticulocytes; Substance Abuse Detection; Young Adult
PubMed: 34626008
DOI: 10.1002/ajh.26368 -
PloS One 2013The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous...
BACKGROUND
The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these 'sub-vital' stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.
DESIGN AND METHODS
Here we use highly purified populations of reticulocytes isolated from cord blood, sorted by flow cytometry into four sequential subpopulations based on transferrin receptor (CD71) expression: CD71high, CD71medium, CD71low and CD71negative. Each of these subgroups was phenotyped in terms of their, morphology, membrane antigens, biomechanical properties and metabolomic profile.
RESULTS
Superficially CD71high and CD71medium reticulocytes share a similar gross morphology (large and multilobular) when compared to the smaller, smooth and increasingly concave reticulocytes as seen in the in the CD71low and CD71negativesamples. However, between each of the four sample sets we observe significant decreases in shear modulus, cytoadhesive capacity, erythroid receptor expression (CD44, CD55, CD147, CD235R, and CD242) and metabolite concentrations. Interestingly increasing amounts of boric acid was found in the mature reticulocytes.
CONCLUSIONS
Reticulocyte maturation is a dynamic and continuous process, confounding efforts to rigidly classify them. Certainly this study does not offer an alternative classification strategy; instead we used a nondestructive sampling method to examine key phenotypic changes of in reticulocytes. Our study emphasizes a need to focus greater attention on reticulocyte biology.
Topics: Antigens, CD; Fetal Blood; Flow Cytometry; Humans; Reticulocytes
PubMed: 24116088
DOI: 10.1371/journal.pone.0076062 -
Journal of Applied Physiology... Sep 2005Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine...
Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulation, even under severe erythropoietic stress. The goals of this study were to determine whether reticulocytes could be detected and characterized in horses that are anemic or have been treated with EPO using a modern hematology analyzer. Anemia was induced in six horses by removal of 30 ml of blood/kg of body wt over 24 h. After 28 days, the horses were treated twice with 55 U/kg of EPO (Eprex), and after 65 days they were treated thrice with 73 U/kg of EPO. Blood samples were analyzed with the ADVIA120 instrument every 3-5 days and bone marrow samples 7 days after anemia and EPO treatments. Analysis of blood reticulocyte parameters by ANOVA in a randomized complete block design determined that anemia and EPO induced significant (P < or = 0.05) increases in red cell distribution width and reticulocyte mean cell volume. Parameters changed only after EPO treatment were cellular hemoglobin concentration mean, mean cell volume, reticulocyte concentration, proportion of macrocytic reticulocytes, and reticulocyte cellular hemoglobin. These findings indicate that horses under erythropoietic stress and after EPO treatment release reticulocytes with unique characteristics into circulation.
Topics: Anemia; Animals; Dose-Response Relationship, Drug; Erythropoietin; Hematopoiesis; Horse Diseases; Horses; Recombinant Proteins; Reticulocyte Count; Reticulocytes
PubMed: 16103516
DOI: 10.1152/japplphysiol.00438.2005 -
Journal of Clinical Laboratory Analysis 2003Hemoglobin F (HbF) is an effective inhibitor of HbS polymerization. Hydroxyurea (HU) is used to increase HbF synthesis and improve the clinical course of sickle cell...
Hemoglobin F (HbF) is an effective inhibitor of HbS polymerization. Hydroxyurea (HU) is used to increase HbF synthesis and improve the clinical course of sickle cell disease (SCD) patients. We studied a series of laboratory parameters concerning HbF production and reticulocyte response, and compared data between two groups: 1) 13 SCD patients treated with HU, and 2) 33 untreated SCD patients. Higher values of Hb concentration, mean cell volume (MCV), mean cell hemoglobin (MCH), mean reticulocyte volume (MRV), HbF concentration, percentage of F-cells, and amount of HbF/F-cells were observed in the treated group of patients. There was no correlation between Hb and HbF elevations. The reticulocyte count, immature reticulocyte count, mean fluorescence index (MFI), and neutrophil count were significantly lower in treated patients. Taken together, these findings suggest that a decreased hemolytic process occurred in patients undergoing HU treatment. There was a significant correlation between MCV and HbF, between MRV and HbF, and between MRV and F-cell in patients taking HU. These data indicate that macroreticulocytes correspond to F-reticulocytes, and that an increase in MRV in SCD patients using HU may be an indirect signal of F-cell production. The concentration of HbF/F-cells was higher in patients treated with HU, but this increase apparently was independent of F-cell production. Reticulocyte (RTC) parameters, as assessed by hematological analyzers, may be useful for following erythropoietic changes in patients receiving HU, and can indirectly indicate HbF and F-cell production induced by HU therapy.
Topics: Adult; Anemia, Sickle Cell; Antisickling Agents; Fetal Hemoglobin; Humans; Hydroxyurea; Neutrophils; Reference Values; Reticulocyte Count; Reticulocytes
PubMed: 12640630
DOI: 10.1002/jcla.10070 -
Haematologica Mar 2001Transferrin receptor (TfR) expression in erythroid cells is regulated by a number of factors, including iron status and erythropoietin (Epo) stimulation. However, the...
BACKGROUND AND OBJECTIVES
Transferrin receptor (TfR) expression in erythroid cells is regulated by a number of factors, including iron status and erythropoietin (Epo) stimulation. However, the impact of these factors on reticulocyte TfR expression in vivo has never been studied. A soluble form of TfR (sTfR) is present in serum in proportion to the mass of cellular TfR. Although sTfR shedding by reticulocytes and erythroblasts has been demonstrated in vitro, the contribution of reticulocyte TfR to serum sTfR has never been evaluated in vivo.
DESIGN AND METHODS
We measured directly the total number of reticulocyte TfR in normal rats of different age and iron status, as well as in animals experiencing various conditions and treatments aimed at altering erythropoietic activity and iron status, including rHuEpo therapy, hemolytic anemia, phlebotomies, hypertransfusions, thiamphenicol-induced red cell aplasia or inflammation. In addition, we examined the impact of repeated hypertransfusions with normal, reticulocyte-poor and reticulocyte-rich blood on serum sTfR levels.
RESULTS
The number of TfR molecules per reticulocyte was around 50,000 in young rats but was around 100,000 in older animals. These values remained constant in most conditions and in particular were not influenced by iron supplementation or iron overload. However, functional iron deficiency as well as rHuEpo therapy resulted in increased reticulocyte TfR expression. In addition, TfR numbers in reticulocytes were elevated in the early phase of recovery after acute hemolysis or red cell aplasia but normalized soon after. Hypertransfusion experiments clearly demonstrated that reticulocytes can contribute substantially to sTfR levels in vivo.
INTERPRETATION AND CONCLUSIONS
TfR numbers are regulated in vivo by the same factors as in vitro, in particular iron deficiency and erythropoietin stimulation. Circulating reticulocytes contribute significantly to serum sTfR levels.
Topics: Animals; Iron Overload; Male; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, Transferrin; Reticulocytes; Solubility
PubMed: 11255270
DOI: No ID Found -
Proceedings of the National Academy of... Jul 1974Protein synthesis in reticulocyte lysates ceases abruptly in the absence of added hemin or in the presence of double-stranded RNA. A similar effect of double-stranded...
The effects of hemin and double-stranded RNA on alpha and beta globin synthesis in reticulocyte and Krebs II ascites cell-free systems and the relationship of these effects to an initiation factor preparation.
Protein synthesis in reticulocyte lysates ceases abruptly in the absence of added hemin or in the presence of double-stranded RNA. A similar effect of double-stranded RNA is observed in Krebs II ascites cell-free systems translating exogenous globin mRNA. The shut-off of protein synthesis is due to inhibition of initiation and can be prevented or reversed by addition of the initiation factor preparation M(3). Preparations of M(1), M(2), and dissociation factor are ineffective under these conditions. The effects of added hemin, M(3), and globin mRNA on the synthesis of alpha and beta globin chains have been studied in the reticulocyte and ascites cell extracts. When the concentration of M(3) is rate limiting, the synthesis of beta chains exceeds that of alpha chains. When the concentration of mRNA is rate limiting, synthesis of alpha and beta chains is more nearly equal.
Topics: Animals; Carcinoma, Krebs 2; Cell-Free System; Globins; Heme; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Protein Biosynthesis; RNA; RNA, Messenger; Rabbits; Reticulocytes; Tritium
PubMed: 4528029
DOI: 10.1073/pnas.71.7.2863 -
Environmental and Molecular Mutagenesis Mar 2021The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and... (Review)
Review
The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig-a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described.
Topics: Animals; Biological Assay; Flow Cytometry; Male; Mutagenicity Tests; Mutagens; Mutation; Rats; Reticulocytes; Rodentia
PubMed: 33608913
DOI: 10.1002/em.22427 -
Haematologica Dec 2018The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss...
The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes are currently poorly understood and, at present, reticulocytes derived through culture fail to undergo the final transition to erythrocytes. Here, we used high-throughput proteomic methods to highlight differences between erythrocytes, cultured reticulocytes and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) whose abundance and phosphorylation status differs between reticulocytes and erythrocytes and localized it in the proximity of autophagosomal vesicles. An circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with non-muscle myosin II inhibition, we demonstrate the involvement of non-muscle myosin IIA in reticulocyte remodeling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation.
Topics: Cell Differentiation; Cells, Cultured; Cytoplasmic Vesicles; Cytoskeletal Proteins; Erythrocytes; Erythropoiesis; Humans; Molecular Motor Proteins; Myosin Heavy Chains; Myosin Type II; Phosphorylation; Proteomics; Reticulocytes
PubMed: 30076174
DOI: 10.3324/haematol.2018.199083 -
Annals of Laboratory Medicine Jul 2018
Comparative Study
Topics: Calibration; Humans; Reticulocyte Count; Reticulocytes
PubMed: 29611390
DOI: 10.3343/alm.2018.38.4.375