Mature Red Cells Research Articles

Re-challenging patients who developed pure red cell aplasia with epoetin: can it be done?

Most, if not all, nephrologists are aware of the explosion of epoetin-associated cases of pure red cell aplasia, particularly in Europe, from 1998 onwards [1–4]. The dramatic increase in reported cases was largely associated with a particular brand of epoetin alfa (Eprex; Erypo) [5], but a few cases have also been reported with epoetin beta [3]. Epoetin-associated pure red cell aplasia is characterized by a sudden loss of response to the drug, with the haemoglobin falling to 5 or 6 g/dl, and requiring regular blood transfusions to maintain a more respectable haemoglobin. The white cell and platelet counts are characteristically normal, but there is usually a profound decrease in circulating reticulocytes (to <10 10/l). Bone marrow examination usually shows a severe reduction in erythroblasts (to <5%) and/or evidence of red cell maturation arrest. Granulopoiesis and megakaryopoiesis is usually preserved. The condition is caused by the development of circulating anti-erythropoietin antibodies, which are usually initially detected by immunoassay, and subsequently confirmed to be neutralizing in a bioassay [1,6]. These antibodies crossreact with all commercially available erythropoietic products, as well as endogenous erythropoietin, and hence it is recommended that patients with this condition should not be switched to an alternative agent [3]. The condition is rare, and follow-up of most cases is too short to determine its natural course, but it is generally believed that immunosuppressive therapy may promote recovery [4]. We now report on three patients who developed epoetin-associated pure red cell aplasia, who were treated with immunosuppressive therapy, and who have now been re-challenged with another epoetin.

Hypoxia‐induced erythropoietin expression in human neuroblastoma requires a methylation free HIF‐1 binding site

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.

CDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP.

Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

Degradation of AP2 during reticulocyte maturation enhances binding of hsc70 and Alix to a common site on TFR for sorting into exosomes.

Reticulocytes release small membrane vesicles termed exosomes during their maturation in erythrocytes. The transferrin receptor (TfR) is completely lost from the red cell surface by its segregation in the secreted vesicles where it interacts with the heat shock cognate 70 kDa protein (hsc70). We have now determined a region of the TfR that can potentially interact with hsc70. The peptide P1 (YTRFSLARQV) from the TfR cytosolic domain: (i). binds to hsc70 (ii). with an increased affinity in oxidative conditions, (iii). competes for binding of an unfolded protein to hsc70, and (iv). inhibits the interaction of hsc70 with a recombinant protein corresponding to the cytosolic domain of the receptor. This peptide encompasses the internalization motif (YTRF) of the receptor, and accordingly an affinity column made with the immobilized peptide retains hsc70 and also the AP2 adaptor complex. On the other hand, we show that AP2 is degraded by the proteasome system during reticulocyte maturation and that the presence of the proteasome inhibitor during in vitro red cell maturation inhibits AP2 degradation and specifically decreases TfR secretion via exosomes. Finally, coimmunoprecipitation of Alix with the exosomal TfR, and binding of P1 peptide to the Alix homolog PalA suggest that Alix also interacts with the YTRF motif and contributes to exosomal TfR sorting.

Erythropoietic changes in rats after 2.45 GHz nonthermal irradiation

The purpose of this study was to observe the erythropoietic changes in rats subchronically exposed to radiofrequency microwave (RF/MW) irradiation at nonthermal level. Adult male Wistar rats (N=40) were exposed to 2.45 GHz continuous RF/MW fields for 2 hours daily, 7 days a week, at 5 – 10 mW/cm2. Exposed animals were divided into four subgroups (n=10 animals in each subgroup) in order to be irradiated for 2, 8, 15 and 30 days. Animals were sacrified on the final irradiation day of each treated subgroup. Unexposed rats were used as control (N=24). Six animals were included into the each control subgroup. Bone marrow smears were examined to determine absolute counts of anuclear cells and erythropoietic precursor cells. The absolute erythrocyte count, haemoglobin and haematocrit values were observed in the peripheral blood by an automatic cell counter. The bone marrow cytogenetic analysis was accomplished by micronucleus (MN) tests. In the exposed animals erythrocyte count, haemoglobin and haematocrit were increased in peripheral blood on irradiation days 8 and 15. Concurrently, anuclear cells and erythropoietic precursor cells were significantly decreased (p<0.05) in the bone marrow on day 15, but micronucleated cells' frequency was increased. In the applied experimental condition, RF/MW radiation might cause disturbance in red cell maturation and proliferation, and induce micronucleus formation in erythropoietic cells.

PTP-MEG2 is activated in polycythemia vera erythroid progenitor cells and is required for growth and expansion of erythroid cells

AbstractPolycythemia vera (PV) is a human clonal hematologic disorder. Previously we demonstrated that erythroid colony-forming cells (ECFCs) from PV patients contained a hyperactive membrane-associated tyrosine phosphatase. We now show that this phosphatase corresponded to protein tyrosine phosphatase (PTP)-MEG2, an intracellular enzyme with a putative lipid-binding domain. The increased activity of PTP-MEG2 in PV cells is due to its elevated distribution in the membrane fraction. With the development of ECFCs to mature red cells, the protein level of PTP-MEG2 decreased gradually, but membrane-associated PTP-MEG2 was sustained for a longer period of time in PV cells, which correlated with an enhanced colony-forming capability of the cells. Importantly, expression of dominant-negative mutant forms of PTP-MEG2 suppressed in vitro growth and expansion of both normal and PV ECFCs. The data indicate that PTP-MEG2 has an important role in the development of erythroid cells. (Blood. 2003;102:4354-4360)

Cooperative signaling between cytokine receptors and the glucocorticoid receptor in the expansion of erythroid progenitors: molecular analysis by expression profiling

AbstractErythroid progenitors undergo renewal (proliferation without apparent differentiation) in response to erythropoietin (Epo), stem cell factor (SCF), and glucocorticoids (dexamethasone) (Dex). SCF and Dex cooperate with Epo to promote proliferation and inhibit differentiation of erythroid progenitors, while Epo alone is required to protect erythroid cells from apoptosis during terminal red cell maturation. To examine the mechanism of the synergistic interactions of Epo, SCF, and Dex, we analyzed gene expression patterns using DNA chip–based large-scale comparative gene profiling using microarrays enriched in hematopoietic transcripts or containing randomly selected genes. Differentially regulated genes were validated by real-time reverse transcription–polymerase chain reaction (RT-PCR). The results reveal cooperative regulation of gene expression by glucocorticoids and Epo/SCF on a number of genes, such as CIS, BTG1, VDUP1, CXCR4, GILZ, and RIKEN29300106B05. While Epo and SCF never showed opposite effects on gene expression, Dex either enhanced or attenuated the effect of Epo and/or SCF. Several glucocorticoid receptor (GR)–target genes were regulated by Dex only in the presence of Epo and/or SCF, suggesting that the GR functions in the context of a larger transactivation complex to regulate these genes. The data also suggest that modulation of cytokine-induced signals by the GR is an important mechanism in erythroid progenitor renewal.

The role of the high-density lipoprotein receptor SR-BI in the lipid metabolism of endocrine and other tissues.

Because cholesterol is a precursor for the synthesis of steroid hormones, steroidogenic tissues have evolved multiple pathways to ensure adequate supplies of cholesterol. These include synthesis, storage as cholesteryl esters, and import from lipoproteins. In addition to endocytosis via members of the low-density lipoprotein receptor superfamily, steroidogenic cells acquire cholesterol from lipoproteins by selective lipid uptake. This pathway, which does not involve lysosomal degradation of the lipoprotein, is mediated by the scavenger receptor class B type I (SR-BI). SR-BI is highly expressed in steroidogenic cells, where its expression is regulated by various trophic hormones, as well as in the liver. Studies of genetically manipulated strains of mice have established that SR-BI plays a key role in regulating lipoprotein metabolism and cholesterol transport to steroidogenic tissues and to the liver for biliary secretion. In addition, analysis of SR-BI-deficient mice has shown that SR-BI expression is important for alpha-tocopherol and nitric oxide metabolism, as well as normal red blood cell maturation and female fertility. These mouse models have also revealed that SR-BI can protect against atherosclerosis. If SR-BI plays similar physiological and pathophysiological roles in humans, it may be an attractive target for therapeutic intervention in cardiovascular and reproductive diseases.

Perspectives transfusionnelles du contrôle ex vivo de l’hématopoïèse

Our increasing knowledge of the mechanisms of blood regulation allows us to envisage the production of cells in quantities sufficient enough to have transfusional interest. We present the state of the art on our capacity to amplify hematopoietic stem cells (HSC) and to force their differentiation into the erythroid lineage. We describe the difficulties met ex vivo for simultaneously reaching a very high level of expansion to have meaningful transfusional interests and obtaining a complete terminal maturation up to enucleation. We describe here a procedure for massively amplifying HSC from human cord blood to produce erythroblastic precursors able to differentiate in vivo into mature red cells after infusion. Although we do not suggest to replace classical blood transfusion, we consider that this approach may become complementary.

Tired blood. Part 1.

"Tired blood" is a common term used to describe anemia because of the association between fatigue, often the first sign of anemia, and the idea that it has "something to do with blood." Today, millions of people have diagnosed anemia, and millions more with chronic diseases are at risk for developing anemia. Anemia is not a specific disease; rather, it is an indication that something has either disturbed the maturation of essential red blood cells or interfered with the oxygen carry capacity of red cells. In this two-part series about tired blood, the etiology and pathophysiology of selected forms of anemia are discussed and pharmacotherapy designed to address the condition of anemia is identified. Part 1 addresses anemia related to red blood cell development, and Part 2 explores the physiology of hemoglobin-related anemia and inherited anemia.

Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency

We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the common PKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine -248 and 2 single nucleotide substitutions, nucleotide (nt) -324T>A and nt -83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream -324T>A and -248delT mutations were nonfunctional polymorphisms. In contrast, the -83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt -87 and nt -83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.

Two Distinct Domains of Protein 4.1 Critical for Assembly of Functional Nuclei in Vitro

Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly.

Protein and lipid trafficking induced in erythrocytes infected by malaria parasites.

The human malaria parasite Plasmodium falciparum develops in a parasitophorous vacuolar membrane (PVM) within the mature red cell and extensively modifies structural and antigenic properties of this host cell. Recent studies shed significant new, mechanistic perspective on the underlying processes. There is finally, definitive evidence that despite the absence of endocytosis, transmembrane proteins in the host red cell membrane are imported in to the PVM. These are not major erythrocyte proteins but components that reside in detergent resistant membrane (DRM) rafts in red cell membrane and are detected in rafts in the PVM. Disruption of either erythrocyte or vacuolar rafts is detrimental to infection suggesting that raft proteins and lipids are essential for the parasitization of the red cell. On secretory export of parasite proteins: an ER secretory signal (SS) sequence is required for protein secretion to the PV. Proteins carrying an additional plastid targeting sequence (PTS) are also detected in the PV but subsequently delivered to the plastid organelle within the parasite, suggesting that the PTS may have a second function as an endocytic sorting signal. A distinct but yet undefined peptidic motif underlies protein transport across the PVM to the red cell (although all of the published data does not yet fit this model). Further multiple exported proteins transit through secretory 'cleft' structures, suggesting that clefts may be sorting compartments assembled by the parasite in the red cell.

The exosome pathway in K562 cells is regulated by Rab11.

During maturation, reticulocytes lose some membrane proteins that are not required on the mature red cell surface. The proteins are released into the extracellular medium associated with vesicles that are formed by budding of the endosomal membrane into the lumen of the compartment; this process results in the formation of multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles, termed exosomes. K562 cells release exosomes with similar characteristics to reticulocyte exosomes, in particular the transferrin receptor (TfR) is found associated with the vesicles. Interestingly, this cell line has been shown to possess high amounts of Rab11 compared with other Rab proteins. To assess the regulation of transferrin receptor release via exosome secretion by Rab11 in this cell type, K562 cells were stably transfected with GFP-Rab11wt or the GTP- and GDP-locked mutants. The distribution of the proteins was assessed by fluorescence microscopy. Transferrin recycling and the number of TfRs present on the surface of the transfected cells were reduced by overexpression of either Rab11wt or the mutants. The amount of released exosomes was analyzed by measuring different molecular markers present on these vesicles either biochemically or by western blot. Overexpression of the dominant-negative mutant Rab11S25N inhibited exosome release, whereas the secretion of exosomes was slightly stimulated in cells transfected with Rab11wt. Taken together, the results demonstrate that in K562 cells Rab11 modulates the exosome pathway although the exact step involved is still not known.

Prevalence of Hepatitis C Virus Antibody in Polytransfused β-Thalassemia Major Patients

Prevalence of Hepatitis C Virus Antibody in Polytransfused β-Thalassemia Major Patients

MONITORING DIETARY COMPLIANCE IN COELIAC DISEASE USING RED CELL DISTRIBUTION WIDTH

SUMMARYRed cell distribution width (RDW) as measured on modern blood analysers is a sensitive marker of nutritional deficiency affecting red blood cell production and maturation. Such deficiencies are common in coeliac disease and it may therefore be possible indirectly to assess response to a gluten‐free diet by monitoring the RDW. We measured RDW, iron, B12, folate and endomysial antibodies (EMA) in 46 newly diagnosed coeliac patients at diagnosis and after a gluten‐free diet. RDW was elevated in 76% of patients at diagnosis. There was a significant decrease in RDW (17.3% vs 13.8%) after 12 months of diet, corresponding to sero‐conversion of EMA and normalisation of serum iron, B12 and folate levels. We conclude that monitoring RDW is a simple indirect means of assessing response to a gluten‐free diet in coeliac disease.

Failure of red blood cell maturation in mice with defects in the high-density lipoprotein receptor SR-BI

Mammalian erythrocytes undergo a unique maturation process in which they discard their nuclei and organelles and assume a flexible biconcave shape. We found that altered plasma lipoprotein metabolism can profoundly influence these events. Abnormal erythrocyte morphology was observed in hypercholesterolemic mice lacking the high-density lipoprotein receptor SR-BI. This was exacerbated by feeding mice a high-cholesterol diet or, more dramatically, by inactivating the apolipoprotein E gene. Erythrocytes fromSR-BI−/−/apolipoprotein E−/−mice and SR-BI−/−mice that were fed cholesterol had markedly increased membrane cholesterol. Their morphology appeared immature, with macrocytosis, irregular shape, and large autophagolysosomes. Autophagolysosomes fromSR-BI−/−/apolipoprotein E−/−erythrocytes were expelled when the erythrocytes were transfused into wild-type animals or incubated in vitro with normolipidemic serum or the cholesterol-sequestering agent methyl cyclodextrin. We propose that autophagocytosis and phagolysosome expulsion are essential steps in erythroid maturation and that expulsion is inhibited in the presence of markedly increased cellular cholesterol.

Failure of red blood cell maturation in mice with defects in the high-density lipoprotein receptor SR-BI.

Mammalian erythrocytes undergo a unique maturation process in which they discard their nuclei and organelles and assume a flexible biconcave shape. We found that altered plasma lipoprotein metabolism can profoundly influence these events. Abnormal erythrocyte morphology was observed in hypercholesterolemic mice lacking the high-density lipoprotein receptor SR-BI. This was exacerbated by feeding mice a high-cholesterol diet or, more dramatically, by inactivating the apolipoprotein E gene. Erythrocytes from SR-BI(-/-)/apolipoprotein E(-/-) mice and SR-BI(-/-) mice that were fed cholesterol had markedly increased membrane cholesterol. Their morphology appeared immature, with macrocytosis, irregular shape, and large autophagolysosomes. Autophagolysosomes from SR-BI(-/-)/apolipoprotein E(-/-) erythrocytes were expelled when the erythrocytes were transfused into wild-type animals or incubated in vitro with normolipidemic serum or the cholesterol-sequestering agent methyl cyclodextrin. We propose that autophagocytosis and phagolysosome expulsion are essential steps in erythroid maturation and that expulsion is inhibited in the presence of markedly increased cellular cholesterol.

Review : Reticulocyte maturation: mitoptosis and exosome release

During the differentiation of erythroid cells, a vast program of maturation takes place, leading to decay or elimination of organelles, including the nucleus, mitochondria, ribosomes, lysosomes, endoplasmic reticulum and Golgi apparatus. During the last step of red cell maturation, remaining organelles, primarily mitochondria and ribosomes but also vestiges of others are finally cleared from the cell. This cleaning session also affects specific proteins that are partially or entirely removed from the cell surface. The interplay of the various events and their causal relationships are approached here.

Temporal differences in membrane loss lead to distinct reticulocyte features in hereditary spherocytosis and in immune hemolytic anemia

AbstractSpherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.