Young Red Blood Cells Research Articles

Large-scale production of Plasmodium falciparum gametocytes for malaria drug discovery

The tightly controlled induction of Plasmodium falciparum gametocytes in large-scale culture is a fundamental requirement for malaria drug discovery applications including, but not limited to, high-throughput screening. This protocol uses magnetic separation for isolation of hemozoin-containing parasites in order to (i) increase parasitemia, (ii) decrease hematocrit and (iii) introduce higher levels of young red blood cells in a culture simultaneously within 2-4 h. These parameters, along with red blood cell lysis products that are generated through schizont rupture, are highly relevant for enabling optimum induction of gametocytogenesis in vitro. No other previously published protocols have applied this particular approach for parasite isolation and maximization of fresh red blood cells before inducing gametocytogenesis, which is essential for obtaining highly synchronous gametocyte classical stages on a large scale. In summary, 500-1,000 million stage IV gametocytes can be obtained within 16 d from an initial 10 ml of asexual blood-stage culture.

Haemoglobin mass alterations in healthy humans following four-day head-down tilt bed rest.

What is the central question of this study? Is haemoglobin mass (Hbmass) decreased following 4days of head-down tilt bed rest (HDTBR), and does increased red blood cell (RBC) destruction mediate this adaptation? What is the main finding and its importance? Haemoglobin mass was increased immediately following HDTBR, before decreasing below baseline 5days after return to normal living conditions. The transient increase in Hbmass might be the result of decreased RBC destruction, but it is also possible that spleen contraction after HDTBR contributed to this adaptation. Our data suggest that the decreased Hbmass 5days following HDTBR resulted from decreased RBC production, not increased RBC destruction. Rapid decreases in haemoglobin mass (Hbmass) have been reported in healthy humans following spaceflight and descent from high altitude. It has been proposed that a selective increase in the destruction of young red blood cells (RBCs) mediates these decreases, but conclusive evidence demonstrating neocytolysis in humans is lacking. Based on the proposed triggers and time course of adaptation during spaceflight, we hypothesized that Hbmass would be reduced after 4 days of -6deg head-down tilt bed rest (HDTBR) and that this would be associated with evidence for increased RBC destruction. We assessed Hbmass in seven healthy, recreationally active men before (PRE), 5h after (POST) and 5 days after (POST5) 4days of HDTBR. The concentration of erythropoietin decreased from 7.1±1.8 mIU ml(-1) at PRE to 5.2±2.8 mIU ml(-1) at POST (mean±SD; P=0.028). Contrary to our hypothesis, Hbmass was increased from 817±135g at PRE to 849±141g at POST (P=0.014) before decreasing below PRE to 789±139g at POST5 (P=0.027). From PRE to POST, the concentration of haptoglobin increased from 0.54±0.32 to 0.68±0.28gl(-1) (P=0.013) and the concentration of bilirubin decreased from 0.50±0.24 to 0.32±0.11mg dl(-1) (P=0.054), suggesting that decreased RBC destruction might have contributed to the increased Hbmass. However, it is possible that spleen contraction following HDTBR also played a role in the increase in Hbmass at POST, but as the transient increase in Hbmass was unexpected, we did not collect data that would provide direct evidence for or against spleen contraction. From PRE to POST5, the concentration of soluble transferrin receptor decreased from 20.7±3.9 to 17.1±3.3nmoll(-1) (P=0.018) but the concentrations of ferritin, haptoglobin and bilirubin were not significantly altered, suggesting that the decrease in Hbmass was mediated by decreased RBC production rather than increased RBC destruction. Peak oxygen uptake decreased by 0.31±0.16l min(-1) from PRE to POST (P=2×10(-4) ) but was not significantly altered at POST5 compared with PRE. Overall, these findings indicate that 4days of HDTBR does not increase RBC destruction and that re-examination of the time course and mechanisms of Hbmass alterations following short-term spaceflight and simulated microgravity is warranted.

Absence of Polycythemia in Obstructive Sleep Apnea (OSA) Is Caused By Neocytolysis

The number of red blood cells (RBC) is tightly controlled to maintain tissue oxygen delivery. During hypoxia, RBC mass is increased by erythropoietin (EPO), which is regulated by hypoxia-inducible transcription factors (HIFs). Neocytolysis is a pathophysiological mechanism that overcorrects the high RBC mass generated during chronic hypoxemia by preferential destruction of young RBCs after normoxia is restored. We recently showed that neocytolysis is caused by excessive mitochondrial-derived reactive oxygen species (ROS) in reticulocytes mediated by downregulation of HIF-regulated Bnip3L (Bnip3L regulates mitophagy) and a decrease in catalase in hypoxia-generated erythrons. Catalase changes are due to hypoxia-induced miR-21 that downregulates catalase. We showed that removal of ROS in plasma by PEG-catalase attenuated neocytolysis, indicating that excessive ROS in plasma from reticulocytes diffuses into RBCs, resulting in preferential destruction of hypoxia-born RBCs with low catalase (Song J, et al. J Mol. Med., 2015 93:857-66). Neocytolysis is well described in astronauts and mountaineers, and here we report this phenomenon in obstructive sleep apnea (OSA).OSA results in chronic intermittent hypoxia (CIH), which is associated with increased incidence of cardiovascular disease, insulin resistance, and cancer. However, the mechanisms of CIH-induced morbidities are still unknown. Despite CIH, polycythemia is rarely present in OSA; our analysis of 190 consecutive OSA patients at the University of Utah showed that only 4% of OSA patients had polycythemia that corrects after therapy with continuous positive airway pressure therapy (CPAP). Several studies reported that CIH also increases ROS. We hypothesize that neocytolysis might explain why most patients with OSA do not have polycythemia, and that increased ROS in blood cells during OSA may account for oxidant related injury seen with OSA associated diseases.We analyzed rate of erythropoiesis by EPO levels, HIFs (by HIF-regulated genes transcripts), catalase and miR-21, mitochondrial mass and Bnip3L transcripts, and ROS in blood cells in 30 OSA patients without polycythemia and without chronic cardiopulmonary disease before and after 3 months of CPAP. Patients ranged in OSA severity according to an apnea-hypopnea index from mild to severe. An average of 99.0 minutes was spent with oxygen saturations of 89% or lower, with a 4% oxygen desaturation index of 20.5/hr. The average lowest oxygen saturation value was 78.0%. EPO was higher before CPAP (p=0.0054), indicating that uncorrected OSA augments erythropoiesis in spite of normal hematocrit. We then reasoned that CIH of OSA might cause overcorrection of increased erythropoiesis by neocytolysis. We first tested HIFs changes during CIH indirectly by measuring selected HIF-regulated gene transcripts (GLUT1, HK1, PDK1, NOX2, SOD2, TFRC, and EGLN1) in blood cells. We found that CIH-mediated downregulation of HIF-regulated gene transcripts is consistent with intermittent rapid abrogation of apnea cycles. Further, blunted HIFs levels were differently regulated in reticulocytes, platelets and granulocytes. ROS were found to be increased during OSA in B-cells, T-cells, monocytes, granulocytes, and mature RBCs, but not in platelets. The elevated ROS corrected after CPAP treatment. These changes of ROS in blood cells coexisted with changes in mitochondrial mass and BNIP3L transcript levels; CPAP treatment decreased mitochondrial mass in reticulocytes and granulocytes but not in lymphocytes or monocytes. Catalase levels in granulocytes, platelets and reticulocytes were lower in uncorrected OSA and corrected after CPAP treatment; reverse correlation with miR-21 was found.These results indicate that neocytolysis occurs in uncorrected OSA resulting in increased mitochondrial mass in reticulocytes (and presumably also in erythroid progenitors) and granulocytes and that excessive ROS generated by increased mitochondrial mass preferentially destroy RBCs with low catalase,resulting in an absence of polycythemia in untreated OSA patients, supporting the central role of HIFs in OSA pathophysiology. Furthermore, increased ROS production may be causally related to other pathologies in OSA such as increased sympathetic tone due to stimulation of carotid bodies by ROS. DisclosuresNo relevant conflicts of interest to declare.

Polycythemia of Mice with Human Gain-of-Function EPOR (mtHEPOR) Is Transiently Corrected in Perinatal Life in Association with Low Epo and Increased Erythrocyte Phosphatidylserine Exposure

Gain-of-function mutations of erythropoietin receptor (EPOR) resulting from truncation of the cytoplasmic domain of EPOR are associated with primary familial and congenital polycythemia (PFCP). We generated and previously reported a PFCP mouse model where the murine EpoR gene (mEpoR) was replaced with wild-type human EPOR (wtHEPOR) or mutant human EPOR (mtHEPOR) genes. We showed that mtHEPOR mice become polycythemic at 3-6 weeks of age, but not at birth, similar to the polycythemia phenotype of affected humans, while the wtHEPOR mice were anemic (Divoky et al, Proc Natl Acad Sci U S A 2001;98:986). In addition, we previously reported (Yoon et al, ASH Annual Meeting Abstracts 2005 106: Abstract 567) that mtHEPOR fetuses are polycythemic at the mid- and late-gestation stages (embryonic days, E12.4-18.5), while wtHEPOR fetuses are anemic, similar to what we observed in adult mice. The mtHEPOR fetal liver cells (FLCs) revealed augmented and sustained activation of Stat5, while wtHEPOR FLCs had significantly reduced Stat5 activation. We also previously presented (Yoon and Prchal, ASH Annual Meeting Abstracts 2007 110: Abstract 3664) that the fetal polycythemia of mtHEPOR mice is associated with a delayed switch from primitive to definitive erythropoiesis, and that polycythemia in mtHEPOR mice is transiently corrected in perinatal life and reappears at 3 weeks after birth. Here, we hypothesized that the change of oxygen tension at delivery (from the uterus to ambient atmosphere) and differences in Epo levels may account for the inexplicable perinatal absence of polycythemia in mtHEPOR mice.Epo levels in adult mice were substantially elevated in wtHEPOR (1271±240 pg/mL) and significantly reduced in mtHEPOR (98±51 pg/mL) when compared to mEpoR mice (265±62 pg/mL). We then proposed that destruction of young red blood cells (RBCs), termed neocytolysis (a mechanism that overcorrects increased RBC mass generated during chronic hypoxemia after restoration of normoxic conditions) contributes to the anemia of newborns (Song et al, J Mol Med 2015;93:857). When adult mice were challenged by rapid conversion from 10 days of hypoxia (12% of O2) to normoxia, the mtHEPOR mice had greater changes of hematocrit (drop of 16.9%) than other genotypes (drop of 14.8% for mEpoR and13.8% for wtHEPOR). Similarly, the most pronounced decrease of hematocrit after birth, at perinatal day 7, (PN7) was detected in mtHEPOR mice (drop of 56%) followed by mEpoR (drop of 48%) and only a relatively moderate decrease of hematocrit in wtHEPOR neonates (drop of 24%). In parallel, PN7 mtHEPOR newborns had the lowest levels of Epo (310±102 pg/mL), while PN7 wtHEPOR neonates had Epo levels markedly increased (2757±912 pg/mL); the Epo levels of control mEpoR at PN7 littermates were 494±102 pg/mL. This suggests that Epo protects from neocytolysis. These changes corresponded to exposure of phosphatidylserine; a stimulus for RBC removal from circulation. There was a gradual increase of phosphatidylserine-positive RBCs in mtHEPOR neonates and, to a lower extent, also in mEpoR between PN0 and PN7 with a maximum positivity at PN7 (mean fluorescence: 6.5±2.8 and 6.2±1.7 for mtHEPOR and mEpoR, respectively). This correlated with a maximal decrease of hematocrit and lowest Epo levels at PN7 in mtHEPOR. The proportion of phosphatidylserine-positive RBCs then declined with age (mean fluorescence of 0.6 in adult mtHEPOR mice). In contrast, wtHEPOR mice had the lowest numbers of phosphatidylserine-positive RBCs, which were comparable at all analyzed time-points (mean fluorescence at PN0=0.7±0.1; PN7=1.3±0.3; in adults=0.6±0.04), consistent with the lowest reduction in perinatal hematocrit and very high Epo levels in these mice.In conclusion, our study describes that transient correction of polycythemia of the mtHEPOR PFCP mouse model in the perinatal period is associated with low Epo levels and increased erythrocyte phosphatidylserine exposure. Increased phosphatidylserine exposure on the membrane of mtHEPOR RBCs is consistent with accelerated destruction of these cells by macrophages, thus contributing to the decrease of hematocrit in mtHEPOR neonatal mice.Authorship: JS and BK: equal credit as first authorsAcknowledgment: VD and MH were supported by the Czech Science Foundation (project P301/12/1503) and by the Ministry of Health Czech Republic (project NT13587). DisclosuresNo relevant conflicts of interest to declare.

Restoring the youth of aged red blood cells and extending their lifespan in circulation by remodelling membrane sialic acid.

Membrane sialic acid (SA) plays an important role in the survival of red blood cells (RBCs), the age-related reduction in SA content negatively impacts both the structure and function of these cells. We have therefore suggested that remodelling the SA in the membrane of aged cells would help recover cellular functions characteristic of young RBCs. We developed an effective method for the re-sialylation of aged RBCs by which the cells were incubated with SA in the presence of cytidine triphosphate (CTP) and α-2,3-sialytransferase. We found that RBCs could be re-sialylated if they had available SA-binding groups and after the re-sialylation, aged RBCs could restore their membrane SA to the level in young RBCs. Once the membrane SA was restored, the aged RBCs showed recovery of their biophysical and biochemical properties to similar levels as in young RBCs. Their life span in circulation was also extended to twofold. Our findings indicate that remodelling membrane SA not only helps restore the youth of aged RBCs, but also helps recover injured RBCs.

Rheological properties of young and aged erythrocytes in chronic venous disease patients with varicose veins

We hypothesize that heterogeneity in RBC rheology can be attributed not only to the aging process but also to various disease states. The present study aims at investigating the rheological properties of young and old RBC in chronic venous disease (CVD) patients. RBC were separated on the basis of their density. RBC rheology was assessed with Laser Optical Rotational Cell Analyzer. For medium and high shear stress elongation index (EI) of young RBC from CVD patients was significantly higher than of young cells from controls. The same was observed for the old cells. Threshold shear rate (THR) of young RBC from CVD patients was significantly higher than of young cells from controls indicating a decreased tendency towards the formation of aggregates and of their stability in CVD patients. The same situation was observed for the old cells. To sum up, the present study demonstrated that heterogeneity in RBC rheology is both affected by age and CVD. Additionally, CVD-induced changes in RBC rheology occur to different degrees among the subpopulations of young and old RBC. CVD patients subpopulations of RBC had higher deformability and increased tendency towards aggregates formation and of their stability when compared to appropriate subpopulations of controls.

HIF-mediated increased ROS from reduced mitophagy and decreased catalase causes neocytolysis.

During prolonged hypoxia, hypoxia-inducible factors (HIFs) mediate an increase in erythropoiesis, leading to an increased red blood cell (RBC) mass and polycythemia. Upon return to normoxia, the increased RBC mass is abruptly overcorrected by the preferential destruction of hypoxia-formed young RBCs, a phenomenon termed neocytolysis. The molecular and biochemical mechanisms involved in neocytolysis are unknown. We developed a murine model of neocytolysis by exposing mice to 12 % oxygen for 10 days followed by return to normoxia. Upon return to normoxia, there was excessive accumulation of reactive oxygen species (ROS) in RBCs from an increased reticulocyte mitochondrial mass correlating with decreased Bnip3L transcripts (Bnip3L mediates reticulocyte mitophagy) and reduced catalase activity. During hypoxia, upregulated miR-21 resulted in low catalase activity in young RBCs. Furthermore, neocytolysis was attenuated by antioxidants and plasma catalase and blunted in mice that had constitutively high expression of HIFs. Among human neonates studied, we report data supporting the existence of neocytolysis during the first week of life. Together, these experiments indicate that the major mechanisms causing neocytolysis involve (1) production of young RBCs with low catalase during hypoxia and (2) lysis of the young RBCs after return to normoxia, mediated by ROS from an increased mitochondrial mass. We report a mouse model of neocytolysis. Neocytolysis is caused by excessive ROS formation mediated by HIF. ROS is generated from increased mitochondria in reticulocytes. Hypoxia-generated RBCs have low catalase and are preferentially destroyed. Reduced catalase is regulated by increased microRNA-21.

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells.

AimTo investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.MethodRBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.ResultsThe proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine1177 phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N5-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation.ConclusionThis study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC.

N‐Methyl D‐Aspartate (NMDA) Receptors in Human Red Blood Cells in Health and Disease

In search for the molecular identity of the Ca2+ uptake pathways in red blood cells (RBCs) we have recently discovered glutamate/homocysteic acid-sensitive Ca2+ transporter which appeared to be the erythroid NMDA receptor. Using ex-vivo erythropoiesis system and RBCs of healthy human subjects and sickle cell disease patients we were able to characterize subunit composition, function and regulation of these receptors in erythroid precursor cells and in the circulating RBCs. The number of the NMDA receptors per cell changed from ~300 000 in proeythroblasts to ~30 in young RBCs decreasing further to ~4 in senescent cells of healthy humans. Activation of these receptors with glutamate and glycine resulted in transient increase in the intracellular calcium which could be blocked by the NMDA receptor antagonists. Activation of the NMDA receptors in RBC triggered transient shrinkage due to the activation of Gardos channels, stimulation of NO synthase and induction of oxidative stress. Calcium uptake was furthermore inducing transient decrease in hemoglobin oxygen affinity. Abnormally high numbers of NMDA receptor copies found in RBCs of patients with sickle cell disease were most likely caused by the disturbed clearance of the receptors from reticulocytes. In RBCs of patients receptor-mediated calcium uptake was high during asymptomatic periods and further up-regulated during vaso-occlusive haemolytic crises. Hyper-activation of the NMDA receptors caused oxidation, dehydration and promoted polymerisation of deoxygenated haemoglobin S. Sickle cell transformation was largely prevented by treatment of RBCs with the NMDA receptor antagonist memantine.

CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite

Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47(hi))-expressing young RBC. Importantly, C57BL/6 CD47(-/-) mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47(-/-) mice was associated with a higher percentage of splenic F4/80(+) cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47(hi) young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC.

Neocytolysis Is Mediated by down Regulation of Catalase By Mir-21 Resulting in defective Clearance of reticulocyte mitochondrial ROS

Oxygen homeostasis is tightly controlled by the number of red blood cells (RBCs). Hypoxia increases RBCs by enhanced erythropoiesis mediated by hypoxia-inducible factors (HIFs). Upon return to normoxia, the increase in RBCs is overcorrected by preferential destruction of young RBCs, a process termed neocytolysis. Neocytolysis was first described in astronauts and later in people descending from high altitude. The molecular mechanism of neocytolysis is obscure. We hypothesized that neocytolysis occurs because of rapid transient changes of HIF levels, resulting in increased reactive oxygen species (ROS) from mitochondria in reticulocytes and defective antioxidant protection of young RBCs generated in hypoxia.We developed a neocytolysis model by exposing mice to 12% oxygen (equivalent to 4500m altitude) for 10 days. This model recapitulates the RBC changes observed in humans exposed to hypoxia for ~30 days. Upon return to normoxia, ROS were markedly increased in reticulocytes and mature RBCs, but not in neutrophils, B- or T-cells, or monocytes. Reduction of ROS by antioxidant (N-acetyl-L cysteine) treatment attenuated hemolysis and decreased hematocrit.To test whether HIFs (transcription factors regulating hypoxic response) contribute to neocytolysis, we repeated these experiments using Chuvash mice (which bear a VHLR200Wmutation, resulting in constitutively high HIF). These mice also showed attenuated hemolysis and decreased hematocrit; in addition, their reticulocyte half-life was higher (36.6 vs.17.8 hours in wild type). Similar findings were also observed with treatment of mice with dimethyloxallyl glycine (DMOG), an inhibitor of prolyl hydroxylase (another negative regulator of HIF). These experiments indicate the essential role of HIF pathways in neocytolysis.Mitochondria are a major source of ROS in cells. During terminal differentiation of RBCs, mitochondria are removed from reticulocytes by mitophagy. After hypoxia treatment, mitochondria mass increased in reticulocytes concomitant with the reduction of HIF-regulated Bnip3L (a mediator of mitophagy) transcripts. These increased ROS were of mitochondrial origin, as detected by Mito-Sox staining.To pursue the mechanism behind the preferential destruction of young RBCs, we investigated antioxidant enzymes. After hypoxia treatment, catalase decreased by 30%, but not glutathione peroxidase, superoxide dismutase or NADH oxidase. The decreased catalase in RBCs produced during hypoxia was unexpected, as it was shown previously that catalase is regulated by HIF2 (as we also show, to a lower degree by HIF1 in our Hif1a-/-embryo) suggesting alternate negative regulator(s) of catalase in hypoxia. Several hypoxia-regulated microRNAs (miRs) are reported to control oxidative stress; we found that miR-451, miR-205 and miR-21 were expressed in erythroid progenitors and reticulocytes and induced after 10 day-hypoxia exposure. To verify whether these miRs regulate catalase expression, we overexpressed and downregulated these miRs in K562 and HEL erythroid cell lines, and found that only miR-21 regulated catalase. Further, we found increased miR-21 after 10 day-hypoxia exposure, with a concomitant decrease of catalase transcripts and activity resulting in impaired ROS scavenging.We conclude that neocytolysis is mediated by excessive generation of ROS from increased mitochondrial mass due to reduced Bnip3L in reticulocytes upon return to normoxia. The reticulocyte ROS then interact with hypoxia-produced young RBCs having miR-21-downregulated catalase, resulting in their preferential destruction. We show that increased mitochondrial ROS and miR-21-downregulated catalase provide the molecular basis of neocytolysis. DisclosuresNo relevant conflicts of interest to declare.

Production of mature red blood cell by using peripheral blood mononuclear cells

Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.

Вплив продукту декарбоксилювання L-аргініну на морфофункціональні показники еритрону за умов експериментального цукрового діабету у щурів

This paper reports the results of a study of the impact of the agmatine treatment on erythrocyte resistance to acid hemolytic, reticulocyte count and reticulocyte production index, erythrocyte surface architectonics in streptozotocin-induced diabetic rats. Our results indicate that treatment of diabetic rats with agmatine causes a suppression of erythropoiesis and increases the resistance of erythrocytes against HCl-induced hemolysis. It was shown a 10% increase in the number of young red blood cells and 35% reduction in the number of structurally transformed erythrocytes that are capable of restoring normal biconcave disc shape under physiological condition. Our data demonstrate an improvement of morphofunctional state of red blood cells in diabetes and reflect the positive effect of agmatine treatment on erythrone due to the glucose-lowering action.

The quantification of reticulocyte maturation and neocytolysis in normal and erythropoietin stimulated rats.

A technique has recently been proposed for obtaining the reticulocyte (RET) age distribution from the flow cytometric reticulocyte count. It allows for a quantitative characterization of reticulocyte dynamics. In this work this technique was applied to characterize the blood, bone marrow and spleen reticulocytes in homeostatic and erythropoietically stimulated rats in order to determine the reticulocyte maturation times in the bone marrow and blood; and to confirm the presence of ineffective erythropoiesis (neocytolysis). The latter was done by comparing the reticulocyte removal rate from blood with bilirubin formation after erythropoiesis stimulation. A single subcutaneous dose (4050 IU/kg) of recombinant human erythropoietin (rHuEPO) was administered to rats, then their reticulocytes were stained with thiazole orange and the distribution of the fluorescent signal measured using flow cytometry. The obtained signal distribution of the reticulocytes was transformed to the age distribution and a set of basic parameters reflecting reticulocyte dynamics was determined. Bilirubin concentrations were measured to directly assess the presence of reticulocyte irreversible removal. The bilirubin formation was found to be considerably modulated by rHuEPO and corresponded well to the determined reticulocyte removal rate. The initial increase and subsequent decrease of the reticulocyte maturation time in blood was quantitated and directly linked with RET mobilization from the bone marrow. A substantial number (60%) of reticulocytes is sequestrated during homeostasis in rats. This number increases and then decreases after rHuEPO administration, as also reflected by bilirubin formation. Flow cytometry seems to be an excellent method for studying RET dynamics and the presence of young RBC neocytolysis.

Iron supplementation and iron deficiency anemia impact malaria pathogenesis (LB472)

Iron deficiency is the most widespread nutritional deficiencies in the developing world and iron is commonly administered compounds through mass distribution campaigns. However, recent evidence suggests that iron deficiency may protect against malaria and that iron supplementation may increase susceptibility to malaria, complicating recommendations for universal supplementation in malaria‐endemic regions. It has been previously hypothesized that iron deficiency inhibited malaria growth through iron deprivation, as is the case with other pathogens. However, our work has generated a novel hypothesis for the effect of iron deficiency and iron supplementation on parasite growth ‐ namely that changes in RBC physiology and RBC age population structure drive resistance of RBCs from IDA donors and increased susceptibility of RBCs from iron supplemented IDA donors to malaria Using an in vitro system with RBCs from donors with well‐defined physiological iron states, we have investigated the cellular mechanisms associated with malaria protection by iron‐deficiency anemia (IDA). We described multiple IDA impacts on parasites, including attenuated invasion of IDA red blood cells (RBCs) and reduced production of parasite progeny. We demonstrate that as iron deficient individuals increase their rate of erythropoiesis in response to iron supplementation, their RBCs becomes more susceptible to infection. Additionally, we show that the replacement of iron deficient RBCs with young normocytic iron‐replete RBCs increases susceptibility to P. falciparum infection. Our results provide a cautionary message to well‐intentioned public health workers who follow the current World Health Organization recommendation to routinely provide iron supplements to iron‐deficient children.Grant Funding Source: National Institute of Child Health and Human Development under award number U01HD061235

Neocytolysis: none, one or many? A reappraisal and future perspectives

Neocytolysis is the hypothesis formulated to explain experimental evidence of selective lysis of young red blood cells (RBCs) (neocytes) associated with decreased plasma levels of erythropoietin (EPO). In humans, it appears to take place whenever a fast RBC mass reduction is required, i.e., in astronauts during the first days of spaceflight under weightlessness, where a fast reduction in plasma volume and increase in haematocrit occur. EPO plasma levels then decline and a decrease in RBC mass takes place, apparently because of the selective lysis of the youngest, recently generated RBCs (neocytes). The same process seems to occur in people descending to sea level after acclimatization at high altitude. After descent, the polycythaemia developed at high altitude must be abrogated, and a rapid reduction in the number of circulating RBCs is obtained by a decrease in EPO synthesis and the lysis of what seem to be young RBCs. In vivo, neocytolysis seems to be abolished by EPO administration. More recent research has ascribed to neocytolysis the RBC destruction that occurs under such disparate pathophysiologic conditions as nephropathy, severe obstructive pulmonary disease, blood doping, and even malaria anaemia. According to the theory, EPO's central role would be not only to stimulate the production of new RBCs in conditions of anaemia, as maintained by the orthodox view, but also that of a cytoprotective factor for circulating young RBCs. Why neocytes are specifically destroyed and how is this related to decreased EPO levels has not yet been elucidated. Changes in membrane molecules of young RBCs isolated from astronauts or mountain climbers upon return to normal conditions seem to indicate a higher susceptibility of neocytes to ingestion by macrophages. By limiting the context to space missions and high altitude expeditions, this review will address unresolved and critical issues that in our opinion have not been sufficiently highlighted in previous works.

A Newly Modified Hemoglobin H Inclusion Test as a Secondary Screening for α⁰-Thalassemia in Southeast Asian Populations

Screening for α⁰-thalassemia is usually associated with a high false-positive rate, leading to an unnecessary PCR workload for accurate diagnosis. We have developed a modified Hb H inclusion test for use as a secondary screening. This test was performed on young red blood cell enriched fractions using dextran sedimentation. The study was performed in 100 subjects positive on initial screening. Confirmatory tests included Hb analysis and a multiplex PCR assay to identify α⁰-thalassemia deletions. A modified Hb H inclusion test was positive in 31 cases, 30 of whom were α⁰-thalassemia carriers (97%). The remaining case (3.0%) was homozygous for α⁺-thalassemia. The remaining 69 cases with a negative Hb H inclusion test included normal subjects, α⁺-thalassemia carriers and β-thalassemia carriers. Two of them (2/69, 3.0%) were found to be double heterozygotes for β⁰-thalassemia and α⁰-thalassemia. The overall sensitivity and specificity of the modified Hb H inclusion test for screening of α⁰-thalassemia were 94.0 and 99.0%, respectively. Therefore, we recommend the use of this test in combination with Hb analysis to exclude cases with αβ-thalassemia. This should lead to a significant reduction in the number of cases referred for PCR analysis of α⁰-thalassemia by about 50.0%.

Impact of glucose‐6‐phosphate dehydrogenase deficiency on sickle cell anaemia expression in infancy and early childhood: a prospective study

In patients with sickle cell anaemia (SCA), concomitant glucose-6-phosphate dehydrogenase (G6PD) deficiency is usually described as having no effect and only occasionally as increasing severity. We analysed sequential clinical and biological data for the first 42months of life in SCA patients diagnosed by neonatal screening, including 27 G6PD-deficient patients, who were matched on sex, age and parents' geographic origin to 81 randomly selected patients with normal G6PD activity. In the G6PD-deficient group, steady-state haemoglobin was lower (-6·2g/l, 95% confidence interval (CI), [-10·1;-2·3]) and reticulocyte count higher (247×10(9) /l, 95%CI, [97; 397]). The acute anaemic event rate was 3 times higher in the G6PD-deficient group (P<10(-3) ). A higher proportion of G6PD-deficient patients required blood transfusion (20/27 [74%] vs. 37/81 [46%], P<10(-3) ), for acute anaemic events, and also vaso-occlusive and infectious events. No significant between-group differences were found regarding the rates of vaso-occlusive, infectious, or cerebrovascular events. G6PD deficiency in babies with SCA worsens anaemia and increases blood transfusion requirements in the first years of life. These effects decrease after 2years of age, presumably as the decline in fetal haemoglobin levels leads to increased sickle cell haemolysis and younger red blood cells with higher G6PD activity.

Longer storage of red blood cells is associated with increased in vitro erythrophagocytosis

Refrigerated storage of red blood cells (RBCs) induces numerous changes that may target the cells for erythrophagocytosis following transfusion. The influence of storage upon the phagocytosis of unseparated and fractionated young and old stored RBCs was investigated using two in vitro quantitative phagocytosis assays. Leucocyte-depleted RBC units were sampled at day 1 or 42 of storage. Young and old RBCs were fractionated at day 1 by density centrifugation and stored in paediatric packs for up to 42 days. RBCs were labelled with the fluorescent dye PKH26 and incubated with the human monocytic cell line THP-1. Erythrophagocytosis was quantified by flow cytometry and plate fluorometric assays. A higher proportion of THP-1 cells phagocytosed RBCs stored for 42 days compared with 1 day (41% and 24% respectively; P<0·0001). This was associated with an increased mean number of RBCs phagocytosed per THP-1 cell (5·2±0·6 and 3·3±0·2 respectively; P<0·002). Erythrophagocytosis of fractionated young and old RBCs increased with longer storage duration up to 28 days (P<0·05). However, no significant differences were observed between erythrophagocytosis of young and old RBCs. The susceptibility of stored RBCs to erythrophagocytosis significantly increased with longer storage time of the RBC units. Storage duration of RBCs had a greater influence on in vitro erythrophagocytosis than the chronological age of the RBCs at donation.

P31: The effects of red blood cell age on nitric oxide and nitrite-dependent signaling: Implications for transfusion related toxicity

RBC are critical hubs for regulated NO-homeostasis, affecting both inhibition (NO and nitrite oxidation) and stimulatory (nitrite-reduction to NO) pathways. How RBC affect this balance, and whether this changes in disease remains unclear. Importantly, biophysical and biochemical properties of the RBC are key parameters, and which change as a function of RBC age in vivo and storage time ex vivo. Transfusion with older RBC is associated with increased morbidity and mortality in critically ill patients with dysfunction in NO-bioavailability underlying, in part, increased inflammation (e.g., in the lung leading to acute lung injury) and circulatory dysfunction. We hypothesized that changes in RBC morphology/ biochemical composition that occur during aging/ storage results in a shift in the balance presented above towards a greater inhibition of NO-signaling both by accelerated rates of NO-and nitrite oxidation. Comparisons of NO dioxygenation and nitrite oxidation kinetics were made between fresh and stored RBC as well as RBC of different ages. Functional assessment of changes in NO/nitrite reaction kinetics were evaluated by testing effects of RBC on NO-or nitrite-dependent vasodilation of aortic rings ex vivo and in vivo using a murine model of storage lesion in the context of trauma-hemorrhage and acute lung injury. NO-scavenging kinetics by intact oxygenated RBC were ∼40-fold higher with RBC stored for 42 days compared to freshly isolated RBC; an effect that translated to a greater inhibition of NO-dependent vasodilation. Similarly, RBC isolated from freshly drawn blood and separated as a function of endogenous age, showed that older RBC scavenged NO ∼2-fold faster compared to younger RBC, which also translated to a greater degree of inhibition of NO-dependent vasodilation. Both stored and older RBC exhibited increased rates of NO 2 - oxidation, which was not however associated with any deficit in NO 2 - -dependent vasodilation. Transfusion of fresh or stored RBC into mice after trauma-hemorrhage led to significant acute lung injury in the latter. Collectively, these data suggest that endogenous NO-signaling will be modulated by the relative distribution of young vs. older RBC. Also the storage of RBC under current blood banking conditions results in cells with higher NO-scavenging activities that are predicted to complement increased hemolysis in contributing to pro-inflammatory stresses associated with transfusion. In addition, we posit that increased oxidation of nitrite, which would decrease an endogenous substrate for NO-formation during hypoxia, also contributes to storage lesion pathology and raises the concept of nitrite-based therapy to replete NO-signaling during trauma-hemorrhage. Supported by NIH T32 HL007918.