Red Blood Cell Homeostasis Research Articles

Combating hypoxemia in COVID-19 patients with a natural oxygen carrier, HEMO2Life® (M101)

BackgroundInfection with SARS-CoV-2 is responsible for the COVID-19 crisis affecting the whole world. This virus can provoke acute respiratory distress syndrome (ARDS) leading to overcrowed the intensive care unit (ICU). Over the last months, worldwide experience demonstrated that the ARDS in COVID-19 patients are in many ways “atypical”. The mortality rate in ventilated patients is high despite the application of the gold standard treatment (protective ventilation, curare, prone position, inhaled NO). Several studies suggested that the SARS-CoV-2 could interact negatively on red blood cell homeostasis. Furthermore, SarsCov2 creates Reactive Oxygen Species (ROS), which are toxic and generate endothelial dysfunction. Hypothesis/objective(s)We hypothesis that HEMO2Life® administrated intravenously is safe and could help symptomatically the patient condition. It would increase arterial oxygen content despite lung failure and allow better tissue oxygenation control. The use of HEMO2Life® is also interesting due to its anti-oxidative effect preventing cytokine storm induced by the SARS-CoV-2. Evaluation of the hypothesis: Hemarina is based on the properties of the hemoglobin of the Arenicola marina sea-worm (HEMO2Life®). This extracellular hemoglobin has an oxygen capacity 40 times greater than the hemoglobin of vertebrates. Furthermore, the size of this molecule is 250 times smaller than a human red blood cell, allowing it to diffuse in all areas of the microcirculation, without diffusing outside the vascular sector. It possesses an antioxidative property du a Superoxide Dismutase Activity. This technology has been the subject of numerous publications and HEMO2Life® was found to be well-tolerated and did not induce toxicity. It was administered intravenously to hamsters and rats, and showed no acute effect on heart rate and blood pressure and did not cause microvascular vasoconstriction. In preclinical in vivo models (mice, rats, and dogs), HEMO2Life® has enabled better tissue oxygenation, especially in the brain. This molecule has already been used in humans in organ preservation solutions and the patients showed no abnormal clinical signs. Consequences of the hypothesisThe expected benefits of HEMO2Life® for COVID-19 patients are improved survival, avoidance of tracheal intubation, shorter oxygen supplementation, and the possibility of treating a larger number of patients as molecular respirator without to use an invasive machine.

An Adaptive, Randomized, Placebo-Controlled, Double-Blind, Multi-Center Study of Oral FT-4202, a Pyruvate Kinase Activator in Patients with Sickle Cell Disease (PRAISE)

An Adaptive, Randomized, Placebo-Controlled, Double-Blind, Multi-Center Study of Oral FT-4202, a Pyruvate Kinase Activator in Patients with Sickle Cell Disease (PRAISE)

The Relationship Between Aggregation and Deformability of Red Blood Cells in Health and Disease.

The molecular organization of the membrane of the red blood cell controls cell morphology and function and is thereby a main determinant of red blood cell homeostasis in the circulation. The role of membrane organization is prominently reflected in red blood cell deformation and aggregation. However, there is little knowledge on whether they are controlled by the same membrane property and if so, to what extent. To address the potential interdependence of these two parameters, we measured deformation and aggregation in a variety of physiological as well as pathological conditions. As a first step, we correlated a number of deformability and aggregation parameters in red blood cells from healthy donors, which we obtained in the course of our studies on red blood cell homeostasis in health and disease. This analysis yielded some statistically significant correlations. Also, we found that most of these correlations were absent in misshapen red blood cells that have an inborn defect in the interaction between the membrane and the cytoskeleton. The observations suggest that deformability and aggregation share at least one common, membrane-related molecular mechanism. Together with data obtained after treatment with various agents known to affect membrane organization in vitro, our findings suggest that a phosphorylation-controlled interaction between the cytoskeleton and the integral membrane protein band 3 is part of the membrane-centered mechanism that plays a role in deformability as well as aggregation.

Germline heterozygous mutations in Nxf1 perturb RNA metabolism and trigger thrombocytopenia and lymphopenia in mice

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.

Macrophage Hypercellularity Accompanies Erythroid Hyperplasia in Sickle Cell Mice and during Recovery from Blood Loss in Wild Type Mice

Red blood cell (RBC) homeostasis tightly balances production of new erythroid cells and clearance of damaged or senescent RBCs. Macrophages play a central role in this process in the specialized niches termed erythroblastic island. Macrophages expressing CD169 (Sialoadhesin or Siglec-1), F4/80, CD11b, VCAM-1, ER-HR3 and Ly-6G play important roles in physiological and pathological erythropoiesis. Sickle cell disease (SCD) is characterized by chronic stress erythropoiesis as a compensatory mechanism for anemia. Recent publications have detected macrophage hypercellularity correlated with erythroid hyperplasia in genetic models of erythrocytosis and monocyte-derived macrophage proliferation in the spleen. Because macrophages are integral part of the erythropoietic niche, our objective was to assess the macrophage compartment in transgenic sickle cell mice (SS) at steady state compared to AA controls. Furthermore, we used acute blood loss in C57BL/6J wild type (WT) to confirm if macrophage hypercellularity accompanies erythroid hyperplasia in a mouse model without erythrocytosis or sickling. Using flow cytometry, macrophages were identified with F4/80, CD45, CD11b and CD169, while erythroid progenitor cells were identified with Ter119 and CD71 in the BM and spleen of SS mice and age-matched control (AA) mice. Each subset of macrophages is 3.8 to 6.8-fold higher in SS mice bone marrow compared to AA at steady state (p≤0.05, Fig 1a). Findings were similar for other CD169 subsets in the spleens of SS mice. This macrophage hypercellularity was accompanied by expected erythroid hyperplasia, with 12-fold higher immature erythroid progenitor cells (CD71hiTer119hi) in SS mice than AA controls (p≤0.001). To confirm the increase in macrophage numbers that accompanied erythroid hyperplasia in SS mice as a response to erythroid stress, we induce stress with acute blood loss in WT mice by phlebotomy (once). Interestingly, we found macrophage hypercellularity accompanies erythroid hyperplasia in WT mice during recovery from acute blood loss. WT mice showed about 47-61% increases in CD169 macrophages subsets in the marrow and 47-83% increase in the spleen during recovery from acute blood loss (5-7 days) compared to untreated mice (Fig. 1b). This macrophage hypercellularity was accompanied with 88% increase in immature erythroid progenitor cell. Similarly, we investigated if there was age dependence in macrophage hypercellularity that accompanies erythroid hyperplasia in SS mice. In SS mice, there was no significant differences in macrophage numbers in the BM juvenile mice (5 weeks old) compared to young adults (14 weeks old), presumably because the higher demand for erythroid production to compensate for anemia had already started in the juvenile animals. However, there was a significantly higher macrophage hypercellularity in the spleen at 14 weeks compared to juvenile animals (p≤0.001). There was also a reduction in hematocrit (p≤0.05) and hemoglobin (p≤0.01) from 5 to 14 weeks in SS mice, although this does not correlate to either macrophage hypercellularity or erythroid hyperplasia. We also quantified mRNA expression of heme oxygenase 1 (Hmox1), a critical regulator of erythroid stress response, in the BM of untreated SS mice, and untreated and stressed WT mice. As expected, BM cells from SS mice at steady state expressed significantly higher Hmox1 expression than in WT mice (p≤0.001). Acute blood loss induced-stress raised the Hmox1 expression in WT mice to comparable levels to that of SS mice at steady state. Our investigation is the first to our knowledge to detect BM macrophage hypercellularity is associated with erythroid hyperplasia in response to acute blood loss and in congenital hemolytic anemia. Our data of macrophage hypercellularity in SCD opens up further research opportunity to elucidate the role of these cells in multiorgan complication in SCD. Figure 1: Macrophage hypercellularity accompanies erythroid hyperplasia in sickle cell disease and during recovery from acute blood loss. (a) BM macrophage quantification (per femur) in untreated sickle cell mice compared to AA controls (n = 5). Student's Ttest * SS mice compared to AA mice. (b) Acute blood loss induced macrophage hypercellularity in the BM of WT mice (n = 4 - 5). Student's Ttest * Untreated compared to stressed WT mice. Figure 1 Disclosures Ofori-Acquah: Shire Human Genetic Therapies Inc: Other: Financial Relationship. Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding.

Safety, toxicity and immunogenicity of a malaria vaccine based on the circumsporozoite protein (FMP013) with the adjuvant army liposome formulation containing QS21 (ALFQ)

Antibodies to Circumsporozoite protein (CSP) confer protection against controlled human malaria infection (CHMI) caused by the parasite Plasmodium falciparum. Although CSP is highly immunogenic, it does not induce long lasting protection and efforts to improve CSP-specific immunological memory and duration of protection are underway. We have previously reported that the clinical grade CSP vaccine FMP013 was immunogenic and protective against malaria challenge in mice when combined with the Army Liposomal Formulation adjuvant containing immune modulators 3D-PHAD™ and QS21 (ALFQ). To move forward with clinical evaluation, we now report the safety, toxicity and immunogenicity of clinical grade FMP013 and ALFQ in Rhesus macaques. Three groups of Rhesus (n = 6) received half or full human dose of FMP013 + ALFQ on a 0-1-2 month schedule, which showed mild local site reactions with no hematologic derangements in red blood cell homeostasis, liver function or kidney function. Immunization induced a transient systemic inflammatory response, including elevated white blood cell counts, mild fever, and a few incidences of elevated creatine kinase, receding to normal range by day 7 post vaccination. Optimal immunogenicity in Rhesus was observed using a 1 mL ALFQ + 20 µg FMP013 dose. Doubling the FMP013 antigen dose to 40 µg had no effect while halving the ALFQ adjuvant dose to 0.5 mL lowered immunogenicity. Similar to data generated in mice, FMP013 + ALFQ induced serum antibodies that reacted to all regions of the CSP molecule and a Th1-biased cytokine response in Rhesus. Rhesus antibody response to FMP013 + ALFQ was found to be non-inferior to historical benchmarks including that of RTS,S + AS01 in humans. A four-dose GLP toxicity study in rabbits confirmed no local site reactions and transient systemic inflammation associated with ALFQ adjuvant administration. These safety and immunogenicity data support the clinical progression and testing of FMP013 + ALFQ in a CHMI trial in the near future.

Red Blood Cell Homeostasis and Altered Vesicle Formation in Patients With Paroxysmal Nocturnal Hemoglobinuria.

A subset of the red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) lacks GPI-anchored proteins. Some of these proteins, such as CD59, inhibit complement activation and protect against complement-mediated lysis. This pathology thus provides the possibility to explore the involvement of complement in red blood cell homeostasis and the role of GPI-anchored proteins in the generation of microvesicles (MVs) in vivo. Detailed analysis of morphology, volume, and density of red blood cells with various CD59 expression levels from patients with PNH did not provide indications for a major aberration of the red blood cell aging process in patients with PNH. However, our data indicate that the absence of GPI-anchored membrane proteins affects the composition of red blood cell-derived microvesicles, as well as the composition and concentration of platelet-derived vesicles. These data open the way toward a better understanding on the pathophysiological mechanism of PNH and thereby to the development of new treatment strategies.

The role of the HO-1/Heme axis on macrophage cytokine production

Abstract Transfusion Related Immunomodulation (TRIM) is a phenomenon that was first described over 30 years ago when it was reported that patients receiving blood transfusions prior to renal transplants had improved graft survival rates. The prevailing hypothesis for these observations at the time was that donor leukocytes were responsible for the effects; however, transfusion of leukoreduced and irradiated blood products still resulted in TRIM, suggesting that another component of the blood products, such as red blood cells (RBCs), might mediate the immunosuppressive effects. RBCs have been shown to affect the activation of immune cells and immune responses. For example, monocytes co-cultured with RBCs and stimulated with LPS produce lower levels of TNF-a than those that are not co-cultured. This suggests that RBCs can shape the immune response that develops during antigen presentation. Splenic red pulp macrophages are critical for clearance of both RBCs and heme, and these processes are essential for maintaining RBC homeostasis and preventing heme-mediated toxicity. Inside macrophages, heme is degraded by the enzyme Heme Oxygenase-1 (HO-1) and HO-1 activity leads to increased IL-10 and decreased TNF-a secretion. We hypothesized that erythrophagocytosis by macrophages during RBC transfusion may result in the activation of a heme-mediated HO-1 dependent signaling pathway that causes immunosuppressive effects. We used the robust TLR stimulation models of LPS and CpG challenge, and tested whether RBC transfusion or heme signaling could modulate the resulting macrophage cytokine response. We found that CORM-2 (a synthetic CO releasing molecule) downregulates the macrophage pro-inflammatory cytokine profile.

Red Blood Cell Homeostasis: Mechanisms and Effects of Microvesicle Generation in Health and Disease.

Red blood cells (RBCs) generate microvesicles to remove damaged cell constituents such as oxidized hemoglobin and damaged membrane constituents, and thereby prolong their lifespan. Damage to hemoglobin, in combination with altered phosphorylation of membrane proteins such as band 3, lead to a weakening of the binding between the lipid bilayer and the cytoskeleton, and thereby to membrane budding and microparticle shedding. Microvesicle generation is disturbed in patients with RBC-centered diseases, such as sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, spherocytosis or malaria. A disturbance of the membrane-cytoskeleton interaction is likely to be the main underlying mechanism, as is supported by data obtained from RBCs stored in blood bank conditions. A detailed proteomic, lipidomic and immunogenic comparison of microvesicles derived from different sources is essential in the identification of the processes that trigger vesicle generation. The contribution of RBC-derived microvesicles to inflammation, thrombosis and autoimmune reactions emphasizes the need for a better understanding of the mechanisms and consequences of microvesicle generation.

Splenomegaly: Pathophysiological bases and therapeutic options.

The spleen is the largest immune organ in the human body and is also essential for red blood cell homeostasis and iron recycling. An average human spleen is approximately 10 centimetres in length and weighs 150g. Pathological conditions can result in the spleen weighing in excess of 2000g and extending over 30 centrimetres in length. This remarkable property of the spleen to expand is termed splenomegaly. Splenomegaly can occur as a physiological response to stress or as a chronic process that is often detrimental to the wellbeing of the individual. Here, we will discuss the normal function and physiology of the spleen, the pathophysiological bases of splenomegaly and the commonly available therapeutic options. Additionally we will address experimental systems to determine the regulatory mechanisms underlying splenomegaly.

Role of Macrophages in Erythropoiesis -Review

Terminal erythroid differentiation and its regulation occurs in the bone marrow specialized structure termed erythroblastic islands. The role of macrophages in erythropoiesis is closely related with the integrity of erythroblastic island, which is reliant on adhesive interactions between erythroid and macrophage cells. In regulating red blood cell homeostasis of adult, macrophages have 2 major roles, retaining erythroblast in bone marrow, controlling the final maturation of erythroblasts into reticulocytes and clearing old erythrocytes. Recent research demonstrated that body can affect erythropoiesis by regulating the function of macrophages in physiological or pathological conditions. This paper review focuses on the new advances of this aspect in recent years, including adhesion factor and regulatory factor between macrophage and red blood cells, role of macrophage in development of red blood cells, effect of macrophage on erythropoiesis in different conditiones and so on.

Acetylcholinesterase provides new insights into red blood cell ageing in vivo and in vitro.

During its 120 days sojourn in the circulation, the red blood cell (RBC) remodels its membrane. Acetylcholinesterase (AChE) is a glycosylphosphatidylinositol (GPI)-linked enzyme that may serve as a marker for membrane processes occurring this ageing-associated remodelling process. Expression and enzymatic activity of AChE were determined on RBCs of various ages, as obtained by separation based on volume and density (ageing in vivo), and on RBCs of various times of storage in blood bank conditions (ageing in vitro), as well as on RBC-derived vesicles. During ageing in vivo, the enzymatic activity of AChE decreases, but not the AChE protein concentration. In contrast, neither AChE activity nor concentration show a consistent, significant decrease during ageing in vitro. CD59, another GPI-linked protein that protects against complement-induced removal, also remains constant during storage. The cellular content of the integral membrane protein glycophorin A, however, decreases with storage time in the more dense RBC fractions. The latter are enriched in echinocytes and other misshapen cells during storage. Our findings suggest that, during RBC ageing, GPI-linked proteins and integral membrane proteins are differentially sorted. Also, the vesicles that are generated in vitro show a fast and extensive loss of AChE activity, but not of AChE expression. Thus, AChE characteristics may constitute sensitive biomarkers of RBC ageing in vivo, and a source of information on the structural and functional changes that GPI-linked proteins undergo during ageing in vivo and in vitro. This information may help to understand RBC homeostasis and the effects of transfusion, especially in immunologically compromised patients.

Pathophysiological aspects of red blood cells in end-stage renal disease patients resistant to recombinant human erythropoietin therapy.

Modified, bioreactive red blood cells (RBCs) and RBC-derived microvesicles (MVs) likely contribute to the hematological and cardiovascular complications in end-stage renal disease (ESRD). This study assesses the physiological profile of RBCs in patients with ESRD receiving standard or high doses of recombinant human erythropoietin (rhEPO). Blood samples from twenty-eight patients under sustained hemodialysis, responsive, or not to standard rhEPO administration were examined for RBC morphology, fragility, hemolysis, redox status, removal signaling, membrane protein composition, and microvesiculation before and after dialysis. Acute effects of uremic plasma on RBC features were examined in vitro through reconstitution experiments. Overall, the ESRD RBCs were characterized by pathological levels of shape distortions, surface removal signaling, and membrane exovesiculation, but reduced fragility compared to healthy RBCs. Irreversible transformation of RBCs was found to be a function of baseline Hb concentration. The more toxic uremic context in non-responsive patients compared to rhEPO responders was blunted in part by the antioxidant, antihemolytic, and anti-apoptotic effects of high rhEPO doses, and probably, of serum uric acid. A selective lower expression of RBC membrane in complement regulators (CD59, clusterin) and of CD47 "marker-of-self" was detected in non-responders and responders, respectively. Evidence for different short-term dialysis effects and probably for a different erythrocyte vesiculation mechanism in rhEPO responsive compared to non-responsive patients was also revealed. Deregulation of RBC homeostasis might involve diverse molecular pathways driving erythrocyte signaling and removal in rhEPO non-responders compared to responsive patients.

How do red blood cells know when to die?

Human red blood cells (RBCs) are normally phagocytized by macrophages of splenic and hepatic sinusoids at 120 days of age. The destruction of RBCs is ultimately controlled by antagonist effects of phosphatidylserine (PS) and CD47 on the phagocytic activity of macrophages. In this work, we introduce a conceptual model that explains RBC lifespan as a consequence of the dynamics of these molecules. Specifically, we suggest that PS and CD47 define a molecular algorithm that sets the timing of RBC phagocytosis. We show that significant changes in RBC lifespan described in the literature can be explained as alternative outcomes of this algorithm when it is executed in different conditions of oxygen availability. The theoretical model introduced here provides a unified framework to understand a variety of empirical observations regarding RBC biology. It also highlights the role of RBC lifespan as a key element of RBC homeostasis.

Erythropoietin Regulation by Angiotensin II.

The renin-angiotensin system (RAS) is a key regulator of blood pressure and blood volume homeostasis. The RAS is primarily comprised of the precursor protein angiotensinogen and the two proteases, renin and angiotensin-converting enzyme (ACE). Angiotensin I (Ang I) is derived from angiotensinogen by renin, but appears to have no biological activity. In contrast, angiotensin II (Ang II) that has a variety of biological functions in the cells is converted from Ang I through removal of two-C-terminal residues by ACE. The physiological effects of Ang II are due to Ang II signaling through specific receptor binding, resulting in muscle contraction leading to increased blood pressure and volume. To modulate RAS, three classes of drugs have been developed: (1) renin inhibitors to prevent angiotensinogen conversion to Ang I, (2) ACE inhibitors, to prevent Ang I processing to Ang II and (3) angiotensin receptor blockers, to inhibit Ang II signaling through its receptor. Studies using the RAS inhibitors and Ang II demonstrated that RAS signaling mediates actions of Ang II in the regulation of proliferation and differentiation of specific hematopoietic cell types, especially in the red blood cell lineage. Accumulating evidence indicates that RAS regulates EPO, an essential mediator of red cell production, for human anemia and erythropoiesis in vivo and in vitro. The regulation of EPO expression by Ang II may be responsible for maintaining red blood cell homeostasis. This review highlights the biological roles of RAS for blood cell and EPO homeostasis through Ang II signaling. The molecular mechanism for Ang II-induced EPO production of the cell or tissue type-specific expression is discussed.

The Proteome of the Red Blood Cell: An Auspicious Source of New Insights into Membrane-Centered Regulation of Homeostasis.

During the past decade, the hand-in-hand development of biotechnology and bioinformatics has enabled a view of the function of the red blood cell that surpasses the supply of oxygen and removal of carbon dioxide. Comparative proteomic inventories have yielded new clues to the processes that regulate membrane–cytoskeleton interactions in health and disease, and to the ways by which red blood cells communicate with their environment. In addition, proteomic data have revealed the possibility that many, hitherto unsuspected, metabolic processes are active in the red blood cell cytoplasm. Recent metabolomic studies have confirmed and expanded this notion. Taken together, the presently available data point towards the red blood cell membrane as the hub at which all regulatory processes come together. Thus, alterations in the association of regulatory proteins with the cell membrane may be a sine qua non for the functional relevance of any postulated molecular mechanism. From this perspective, comparative proteomics centered on the red blood cell membrane constitute a powerful tool for the identification and elucidation of the physiologically and pathologically relevant pathways that regulate red blood cell homeostasis. Additionally, this perspective provides a focus for the interpretation of metabolomic studies, especially in the development of biomarkers in the blood.

Red Blood Cell Homeostasis: Pharmacological Interventions to Explore Biochemical, Morphological and Mechanical Properties.

During their passage through the circulation, red blood cells (RBCs) encounter severe physiological conditions consisting of mechanical stress, oxidative damage and fast changes in ionic and osmotic conditions. In order to survive for 120 days, RBCs adapt to their surroundings by subtle regulation of membrane organization and metabolism. RBC homeostasis depends on interactions between the integral membrane protein band 3 with other membrane and cytoskeletal proteins, and with key enzymes of various metabolic pathways. These interactions are regulated by the binding of deoxyhemoglobin to band 3, and by a signaling network revolving around Lyn kinase and Src family kinase-mediated phosphorylation of band 3. Here we show that manipulation of the interaction between the lipid bilayer and the cytoskeleton, using various pharmacological agents that interfere with protein-protein interactions and membrane lipid organization, has various effects on: (1) morphology, as shown by high resolution microscopy and quantitative image analysis; (2) organization of membrane proteins, as indicated by immunofluorescence confocal microscopy and quantitative as well as qualitative analysis of vesicle generation; (3) membrane lipid organization, as indicated by flow cytometric analysis of phosphatidylserine exposure; (4) deformability, as assessed in capillary-mimicking circumstances using a microfluidics system; (5) deformability as determined using a spleen-mimicking device; (6) metabolic activity as indicated by metabolomics. Our data show that there is a complex relationship between red cell morphology, membrane organization and deformability. Also, our data show that red blood cells have a relatively high resistance to disturbance of membrane organization in vitro, which may reflect their capacity to withstand mechanical, oxidative and osmotic stress in vivo.

Developmental plasticity of red blood cell homeostasis

Most human physiologic set points like body temperature are tightly regulated and show little variation between healthy individuals. Red blood cell (RBC) characteristics such as hematocrit and mean cell volume are stable within individuals but can vary by 20% from one healthy person to the next. The mechanisms for the majority of this inter-individual variation are unknown and do not appear to involve common genetic variation. Here, we show that environmental conditions present during development, namely in utero iron availability, can exert long-term influence on a set point related to the RBC life cycle. In a controlled study of rhesus monkeys and a retrospective study of humans, we use a mathematical model of in vivo RBC population dynamics to show that in utero iron deficiency is associated with a lowered threshold for RBC clearance and turnover. This in utero effect is plastic, persisting at least 2 years after birth and after the cessation of iron deficiency. Our study reports a rare instance of developmental plasticity in the human hematologic system and also shows how mathematical modeling can be used to identify cellular mechanisms involved in the adaptive control of homeostatic set points.

A novel method for measuring the ATP-related compounds in human erythrocytes.

The ATP-related compounds in whole blood or red blood cells have been used to evaluate the energy status of erythrocytes and the degradation level of the phosphorylated compounds under various conditions, such as chronic renal failure, drug monitoring, cancer, exposure to environmental toxics, and organ preservation. The complete interpretation of the energetic homeostasis of erythrocytes is only performed using the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid value. For the first time, we report a liquid chromatographic method using a diode array detector that measures all of these compounds in a small human whole blood sample (125 μL) within an acceptable time of 20 min. The stability was evaluated for all of the compounds and ranged from 96.3 to 105.1% versus the day zero values. The measurement had an adequate sensitivity for the ATP-related compounds (detection limits from 0.001 to 0.097 μmol/L and quantification limits from 0.004 to 0.294 μmol/L). This method is particularly useful for measuring inosine monophosphate, inosine, hypoxanthine, and uric acid. Moreover, this assay had acceptable linearity (r > 0.990), precision (coefficients of variation ranged from 0.1 to 2.0%), specificity (similar retention times and spectra in all samples) and recoveries (ranged from 89.2 to 104.9%). The newly developed method is invaluable for assessing the energetic homeostasis of red blood cells under diverse conditions, such as in vitro experiments and clinical settings.

Hypoxia-inducible factor-1α(Pro-582-Ser) polymorphism prevents iron deprivation in healthy blood donors.

Frequent blood loss induces progressive depletion of iron stores, leading to iron deficiency and, ultimately, to overt iron-deficient anaemia. The erythropoietin-mediated bone marrow response to anaemia is under the control of hypoxia-inducible factors (HIF), the master regulators of oxygen and iron homeostasis. Since the HIF-1α(Pro-582-Ser) variant is associated with elevated trans-activation capacity of hypoxia responsive elements of target genes, we investigated whether the HIF-1α(Pro-582-Ser) polymorphism might influence the response to repeated blood withdrawals. Using polymerase chain reaction analysis and DNA sequencing, we retrospectively investigated the presence of HIF-1α(Pro-582-Ser) in a series of 163 blood donors. Haematological findings, serum ferritin levels and frequency of donations were compared according to the mutational status of the HIF-1α gene. We found that male carriers of the HIF-1α(Pro-582-Ser) polymorphism had higher haemoglobin and ferritin levels than individuals homozygous for the wild-type allele. Moreover, the HIF-1α(Pro-582-Ser) polymorphism protected regular blood donors from developing iron deficiency and anaemia and predicted uninterrupted donation activity. These findings show for the first time that the HIF-1α(Pro-582-Ser) polymorphism significantly affects red blood cell and iron homeostasis after blood loss, conferring to male carriers a resistance to anaemia. Regarding the female gender, large series of individuals should be investigated to establish whether there is an effect of the HIF-1α(Pro-582-Ser) polymorphism in this population. Although these data need to be confirmed in prospective studies, they could have important implications in blood donor selection and donation procedures.