Red Cell Production Research Articles

A Chinese family carrying novel mutations in SEC23B and HFE2, the genes responsible for congenital dyserythropoietic anaemia II (CDA II) and primary iron overload, respectively

A Chinese family carrying novel mutations in <scp><i>SEC23B</i></scp> and <scp><i>HFE2</i></scp>, the genes responsible for congenital dyserythropoietic anaemia <scp>II</scp> (<scp>CDA II</scp>) and primary iron overload, respectively

Cell therapy: Promise fulfilled?

Cellular immunotherapy has been widely accepted as a new powerful modality of cancer treatment. The last 2 decades have seen impressive results in its application against haemato-oncologic malignancies, melanomas and prostate carcinoma. Cellular immunotherapy has since found applicability beyond cancer into autoimmunity and continues to expand in its clinical applicability. The discovery that stem cells have the ability to differentiate into more mature cell types, like neurones and myocardium, has focused research on using exogenous cells to repair damaged tissues. This led to numerous clinical trials using stem cells in myocardial infarction, cardiomyopathy and spinal cord damage. Results have ranged from modest to significant clinical outcomes with continuing debate on the exact process of regeneration achieved. The intertwining between cell therapy and transfusion medicine now includes research on progenitor cells for the production of mature red cells. It is also clear that cell therapy has enabled an improved understanding of the pathogenesis and clinical course of many diseases, while perhaps its role in regenerative medicine is most enticing. However, the critical role of manufacturing in terms of cost, complexity, reproducibility, and regulatory matters remains a central issue in the consideration of whether cell therapy has met all of its promise.

Expression of fetal hemoglobin in adult humans exposed to high altitude hypoxia

In humans, acute erythroid expansion can lead to maturation of red blood cell (RBC) precursors containing fetal hemoglobin (F red cells). This can occur in patients after recovery from bone marrow transplantation, or in individuals affected by sickle cell or thalassemic syndromes. An accelerated erythroid lineage expansion is also a hallmark of the adaptive response to high altitude hypoxia. To explore the possible effect of this environment on F red cell production, we analyzed RBCs from five subjects during and after 17 days spent at high altitude and investigated the expression of fetal hemoglobin by different methodological approaches. By flow cytometry, we found a moderate increase of circulating F red cells during and after the hypoxia exposure, with respect to control cells analyzed before a stay at high altitude. The increased expression of γ-globin (as the specific subunit contained in F hemoglobin together with α-globin) was further confirmed by immunoblotting of young RBC hemolysates and quantitative RT-PCR of transcripts purified from a reticulocyte-enriched RBC fraction. Thus, in healthy adults the exposure to high altitude hypoxia induces maturation of F red cells at a level higher than under normal condition. The effect appears reduced after return to normoxia.

Clinical and hematologic manifestations in patients with Diamond Blackfan anemia in Korea

BackgroundDiamond Blackfan anemia (DBA), characterized by impaired red cell production, is a rare condition that is usually symptomatic in early infancy. The purpose of this study was to assess nationwide experiences of DBA encountered over a period of 20 years.MethodsThe medical records of 56 patients diagnosed with DBA were retrospectively reviewed from November 1984 to July 2010. Fifteen institutions, including 13 university hospitals, participated in this study.ResultsThe male-to-female ratio of patients with DBA was 1.67:1. The median age of diagnosis was 4 months, and 74.1% were diagnosed before 1 year of age. From 2000 to 2009, annual incidence was 6.6 cases per million. Excluding growth retardation, 38.2% showed congenital defects: thumb deformities, ptosis, coarctation of aorta, ventricular septal defect, strabismus, etc. The mean hemoglobin concentration was 5.1±1.9 g/dL, mean corpuscular volume was 93.4±11.6 fL, and mean number of reticulocytes was 19,700/mm3. The mean cellularity of bone marrow was 75%, with myeloid:erythroid ratio of 20.4:1. After remission, 48.9% of patients did not need further steroids. Five patients with DBA who received hematopoietic transplantation have survived. Cancer developed in 2 cases (3.6%).ConclusionThe incidence of DBA is similar to data already published, but our study had a male predilection. Although all patients responded to initial treatment with steroids, about half needed further steroids after remission. It is necessary to collect further data, including information regarding management pathways, from nationwide DBA registries, along with data on molecular analyses.

From Hematopoietic Stem Cell to Erythroblast: Regulation of Red Cell Production At Multiple Levels

Abstract SCI-1 Hematopoietic stem cells (HSC) undergo self-renewal and also generate all types of blood cells, including red cells, myeloid cells, and all immune system cells. Formation of red cells from HSC involves multiple cellular stages, including early erythroid-specific progenitors (burst-forming unit-erythroid [BFU-E]) that respond to several growth factors and an erythropoietin (Epo)-responsive progenitor, and the colony-forming unit-erythroid (CFU-E) that in 3 days generates ∼30 reticulocytes. In adult animals most CFU-E cells undergo apoptosis. A short-term need for red cells is met by increased Epo production by the kidney, which rescues increasing numbers of CFU-Es from apoptosis and increases red cell production over a few days. Chronic stress, such as in certain types of anemias and inflammatory diseases, leads to a marked increase in numbers of HSC and many types of progenitor cells. I will focus most of my talk on stress-triggered BFU-E proliferation and formation of CFU-Es, since our very recent work showed how glucocorticoids – an important class of stress hormones – as well as HIF-1α stimulate red cell production. Acting synergistically these allow ∼300 times more CFU-Es and erythroblasts to be formed from each BFU-E. More specifically, these molecules stimulate limited self-renewal of BFU-Es during cell division, thus maintaining progenitor immaturity and allowing over time increased numbers of CFU-E cells and thus mature red cells to be formed. This explains why certain corticosteroids are useful in treating some non-Epo responsive anemias, and I will present a molecular dissection of glucocorticoid and HIF-1α action on BFU-E progenitors that has led to the identification of genes critical for self-renewal and that suggest other possible avenues for treatment of chronic anemias. Disclosures: No relevant conflicts of interest to declare.

The Role of the Distal Erythropoietin Receptor in Iron Deficiency Anemia

Abstract 1312Anemia affects the quality of life and the life expectancy of millions of people in the U.S. Many patients are either intolerant or unresponsive to available treatments, so alternative strategies are needed. Red blood cell production requires the action of erythropoietin (Epo) on red blood cell precursors in the bone marrow. Iron restriction results in loss of Epo-responsiveness and anemia, despite increased serum Epo levels. Iron infusion restores Epo-responsiveness suggesting that iron dominantly regulates Epo-receptor (EpoR) signaling. Understanding how iron restriction regulates EpoR signaling pathways has major clinical significance. Agonists could offer an iron-free approach that enhances the response to Epo in anemia due to iron deficiency or chronic diseases. In addition, antagonists could be used to treat polycythemia vera or other myeloproliferative disorders. We have discovered that the aconitases, multifunctional iron-sulfur cluster proteins that convert citrate into isocitrate are key in connecting iron to Epo-signaling in early erythroid progenitors (GC Bullock, et. al. Blood 2010;116:97). We also discovered that isocitrate, the downstream product of aconitase, can enhance the effectiveness of Epo during iron deficiency in vitro and in vivo in mice with IDA. These observations suggest that isocitrate or derivatives of isocitrate that synergize with erythropoiesis stimulating agents (ESAs) have important therapeutic application in the treatment of anemia. Deletion of EpoR in mice is incompatible with life, however mice and humans that express truncated EpoR show increased production of red blood cells. These observations suggest that the distal cytoplasmic domain of the EpoR inhibits production of red cells and may play a critical role in iron deficiency anemia. EpoR mutant mice lacking the distal half of the cytoplasmic domain of the EpoR (EpoR-H mice) and mice with the same EpoR truncation mutation plus an additional mutation of tyrosine 343 (EpoR-HM mice) show near normal levels of steady state erythropoiesis. To determine the role of the distal domain in erythroid suppression during iron deficiency, EpoR-H, EpoR-HM and EpoR-wildtype mice were fed a low iron diet and compared by weekly CBCs and flow cytometry. EpoR-H mutant mice continue to efficiently produce red blood cells during iron deficiency. And this occurs despite a decrease in hemoglobin. EpoR-HM mice produce fewer rbcs than EpoR-H mice, however rbc production by EpoR-HM mice resists the suppressive effects of iron restriction. Similar experiments also suggest that the distal EpoR is necessary for the isocitrate-mediated enhancement of Epo-driven erythropoiesis. In addition to aconitase/isocitrate and the distal EpoR other candidate key signaling components of this Epo-dependent, iron-responsive pathway have been identified in our recent preliminary experiments. These components include specific protein kinase C (PKC) isozymes, AKT1 and ERK1/2. These findings support a new model of iron sensing by aconitase/isocitrate that alters EpoR signaling to decrease red blood cell production and conserve iron when supplies are low. This model fits better than older “heme-deficiency” models because disorders in heme synthesis block red cell differentiation at a later stage. This model also has potential to explain changes seen in other tissues during chronic iron deficiency. Nutritional iron restriction may have unmasked a new role for the distal EpoR in red cell development and implicated new iron-responsive Epo signaling pathways that can be used to develop new therapeutic agonists and antagonists of Epo. Disclosures:No relevant conflicts of interest to declare.

Transfusion Complications in Thalassemia: A Report From the Centers for Disease Control and Prevention (CDC)

Abstract 340Transfusions are the primary therapy for thalassemia (thal) but have significant cumulative risks. The Thalassemia Clinical Research Network recently highlighted the increased rate of alloimmunization in this population1. In 2004, the CDC established a national blood safety monitoring program for thal. The goals of the program were to monitor blood safety and strategies for the management of complications. This report focuses on immune and non-immune transfusion reactions. Methods:The CDC Thalassemia Blood Safety Network is a consortium of thal centers that follow patients longitudinally to determine the transfusion-related complications. Serial demographic, physical, and laboratory data are collected along with blood samples analyzed at the CDC. Data collected includes ethnicity, genotype, phenotype, transfusion data, immunizations, type of red cell product, degree of antigen matching, and chelation therapy.Chronically transfused (CT) patients were defined as requiring at least 8 transfusions annually. Intermittently transfused (IT) patients received less than 8 transfusions annually. Statistical analyses included continuous variables, logistic regression, and multivariant analysis. Results:There were 407 patients including 327 CT patients and 80 IT patients. The genotype of chronically transfused patients included: TM (81%), TI (6%), E-Beta-Thal (6%), and Hemoglobin H/CS (4%). The average age of CT patients was 22.3 years ± 13 and 21.4 years ± 17 in IT patients. The average units CT patients received was 149 ± 103.Hemosiderosis occurred in both patient groups and correlated with transfusion history. The average ferritin (m/L) in each group, respectively, was 2261 and 689. 78% of the total population has received chronic chelation therapy. Multiple organ dysfunction was common in the population, including cardiac disease (13%), gonadal failure (17%), growth hormone deficiency (8%), hypothyroidism (8%), hypoparathyroidism (1.2%), diabetes (10%), and thrombotic events (5.4%). 45% of the population was splenectomized.Transfusion reactions occurred in 48% of patients, including allergic (45%), multiple etiologies (32%), febrile (17%), and hemolytic (5%). Transfusion-associated infection included 58 cases of Hepatitis-C and 4 cases of HIV. Two Hepatitis-C cases occurred after specific blood testing was initiated. Recent cases of bacteremia, malaria, and Babesiosis have been documented. 21% of CT patients and 13% of IT patients developed an alloantibody. The most common antigen specificity included E (25%), Kell (13%), C (9%), Jka (5%), Kpa (4%), HLA (4%), V (3%), c (3%), D (2.5%), WA-1 (2.5%), and S (2.5%). 45% of alloimmunized patients had multiple antibodies. Race, transfusion burden, age and splenectomy predicted alloimmunization. Alloimmunization was found in 31% of splenectomized patients vs. 10% of non-splenectomized patients (p <.0001). The alloimmunization prevalence rose from 10% in young children compared to 54% in older patients. The average units transfused in patients with alloantibodies was 215 ± 104 vs. 126 ± 92 in patients without alloantibodies (p <.0001).Autoantibodies occurred in 6.5% of the population and increased with age and splenectomy. Alloimmunization increased the risk of autoantibodies: 26% of patients with an alloantibody had an autoantibody whereas only 2% of patients without alloantibodies had an autoantibody (p<.0001). Overall, 81% of patients with an autoantibody had an alloantibody.Local institutional policies were the major determinant in the type of units administered. In the last year, 32% of the units were washed and 80% radiated. 40% of patients received standard ABO matching, 47% limited phenotypic matching, and 12% full extended matching. Conclusion:Transfusion-related morbidity is a major problem in thal. Hemosiderosis immunologic and non-immunologic reactions are common. Transfusion-related infections have decreased but new, emerging pathogens have been noted. Disclosures:No relevant conflicts of interest to declare.

Hepcidin Production in Ineffective Erythropoiesis and Chronic Hemolysis: Insights on the Crosstalk Between Erythropoiesis and Iron Metabolism

Abstract 346 Background and Aims:Ineffective erythropoiesis in thalassemias and congenital dyserythropoietic anemia has been associated with hepcidin deficiency and spontaneous iron overload, potentially mediated by an abnormally high production of GDF-15, a cytokine overexpressed by maturing erythroblasts (Tanno et al. Nat Med 2007;13:1096–101; Tamary et al., Blood 2008; 112:5241–5244). In this study, we focused on analyzing hepcidin levels in different disorders affecting red cell production. Patients and Methods:Twenty patients with transfusion independent β-thalassemia intermedia (TI) were enrolled as a model of hemoglobinopathy with ineffective erythropoiesis, and were compared to 53 patients with sickle cell anemia (SCA), in which erythropoiesis is effective. Seventeen healthy volunteers and 13 patients with other anemic states with chronic hemolysis (hereditary spherocytosis, autoimmune hemolytic anemia, pyrimidine 5’-nucleotidase deficiency) were recruited for comparison. Fourteen patients with megaloblastic anemia due to cobalamin deficiency were studied before, during and after treatment as an alternative model of ineffective erythropoiesis. We have determined hepcidin levels as previously described (Ganz et al. Blood 2008;112:4292–4297), as well as serum ferritin, transferrin saturation (TS) and GDF-15 in all groups. Levels of C reactive protein (CRP) and IL-6, an inducer of hepcidin production, were also determined in controls, SCA and TI. Results and Discussion:IL-6 levels in SCA patients were increased [mean± standard error of mean (1.69±0.22pg/ml, p=0.047)] when compared to TI (1.33±0.21pg/ml) and controls (0.77±0.18pg/ml), and although there was a positive correlation between IL-6 and hepcidin levels (rS=0.25, p=0.039) this did not correspond to an absolute increase in hepcidin in this group. Indeed, circulating hepcidin levels were lower both in TI (56.24±12.14ng/ml) and SCA patients (52.53±10.97ng/ml) than in controls and other hemolytic anemias (80.23±14.3ng/ml and 120.7±12.2ng/ml, respectively, p=0.013), and did not correlate with the elevated levels of GDF-15 found in the 3 groups of patients (p>0.05). Ferritin levels correlated with both hepcidin and GDF-15 levels (rS=0.38, p=0.0005, and rS=0.63, p<0.0001, respectively). CRP correlated only weakly with IL-6 (rS=0.26, p=0.03) and was not significantly different among the groups studied. Ferritin and TS were considerably higher in TI patients than in the remaining groups (p=0.003). Ineffective erythropoiesis in cobalamin deficiency was neither associated with spontaneous ferritin or TS elevation, nor with decreased hepcidin in spite of high GDF-15 levels. Hepcidin levels at the time of diagnosis were higher than those in SCA (p<0.01) and decreased during vitamin B12 supplementation (108.8±23.36ng/ml vs. 59.37±16.23ng/ml, p=0.045). Hepcidin/ferritin ratios were significantly lower in SCA (28.91±3.08) and TI (13.76±3.98) than in controls and cobalamin deficiency (320.1±109.2 and 279.9±60.2, respectively, p<0.0001, graph represents minimum/maximum values and quartiles), better representing relative hepcidin deficiency in hemoglobin disorders than isolated hepcidin levels. Conclusions:Although both ineffective erythropoiesis and chronic hemolysis were associated with high levels of GDF-15 and low hepcidin in hemoglobinopathies as a result of erythroid compartment expansion, a similar pattern was not confirmed in other hemolytic anemias or megaloblastic anemia. In the latter case, ineffective erythropoiesis did not suppress hepcidin, and regulation of hepcidin production during recovery seemed intact and occurred independently from hepcidin-inhibitory GDF-15 production. Despite elevated IL-6 levels in SCA, chronic inflammation appeared insufficient to overcome the influence of the erythropoietic drive to increase overall hepcidin levels in these patients, but might explain why hepcidin/ferritin ratios in SCA are higher than in TI patients, and normal hepcidin/ferritin ratios corresponded to normal iron status in cobalamin deficiency patients. Our data suggest that hepcidin/ferritin ratio may be a more accurate measurement to detect patients prone to systemic iron overload secondary to increased erythropoietic activity, and corroborate the concept that chronic inflammation may protect SCA from overt iron overload to some extent. [Display omitted] Disclosures:Olbina:Intrinsic LifeSciences LLC: Employment, Equity Ownership; Hepcidin C-ELISA: Co-developer. Westerman:Intrinsic LifeSciences LLC: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Hepcidin C-ELISA: Patents & Royalties; Centocor-Ortho R&D, Inc.: Honoraria.

A Robust Ex Vivo culture System for Production of Mature Human Reticulocytes From a Single Donor

Abstract▪2096▪This icon denotes a clinically relevant abstractWe have formulated a robust culture system simulating human erythropoiesis, producing mature reticulocytes from adult peripheral blood CD34+ progenitor cells in 20 days. Expansion of cell numbers over the course of the culture exceeded 104-fold and enucleation efficiency was up to 90% without the use of feeder layers. Mature reticulocytes and erythrocytes were isolated from culture by passage through a standard leucocyte filter. Scanning and transmission electron microscopy of the reticulocytes revealed morphological features indistinguishable from those of mature reticulocytes isolated from the peripheral blood of normal donors. Post-filtration cells expressed adult haemoglobin and had a capacity to bind and release oxygen comparable to that of adult peripheral blood erythrocytes. Examination of blood group active- and other cell surface antigens revealed a normal glycosylation profile and normal cell surface protein expression. Most notably, the cells did not express cryptantigens T and Tn. LORCA analysis indicated that cultured cells had slightly reduced membrane stability and deformability compared to peripheral blood erythrocytes. Haematology parameters demonstrated a typical mean cell volume of 131fL and mean cell haemoglobin of 37.7pg. This, scalable approach has allowed us to culture cells from a single apheresis cone to produce 1010 reticulocytes, which represents a packed cell volume of 1ml. These results clearly demonstrate the feasibility of producing cultured red cell products suitable for transfusion therapy ex vivo from adult peripheral blood stem cells. Disclosures:No relevant conflicts of interest to declare.

Correction of Murine Sickle Cell Disease Through Interference with Fetal Hemoglobin Silencing

Abstract 351The switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin is critical to the pathogenesis of the major hemoglobin disorders, sickle cell disease (SCD) and β-thalassemias. Increased HbF expression ameliorates the clinical severity of these conditions, forming the basis for the goal of elucidating mechanisms of globin switching and developing target-based therapies to enhance HbF production. The rationale for this therapeutic approach rests on the hypothesis that manipulation of a single target would lead to sufficient HbF reactivation for clinical benefit. Despite recent advances in understanding the complex processes of developmental globin switching and HbF silencing, this hypothesis is untested. The transcription factor BCL11A is a critical mediator of HbF switching and silencing. Previously we showed that BCL11A controls globin switching in mice and silencing of HbF expressed from human β-globin locus transgenes in mouse fetal liver, and contributes to HbF silencing in primary human erythroid cells. Furthermore, we demonstrated that BCL11A is required in vivo for HbF silencing in adult animals, yet dispensable for red cell production. These findings illustrate favorable features of BCL11A as a potential target for reactivation of HbF in the β-hemoglobinopathies.Here, we test whether impairment of BCL11A is sufficient to ameliorate disease symptoms in a relevant preclinical model of a major β-hemoglobin disorder, the humanized SCD mouse model (“Berkeley” SCD model, Paszty et al. Science 278:876–878, 1997). These mice express exclusively human sickle hemoglobin and faithfully recapitulate the principal hematologic and histopathologic phenotypes of patients with SCD. We introduced BCL11A-null alleles into the “Berkeley” SCD model (SCD Bcl11afl/fl EpoR-Cre+, hereafter called SCD/Bcl11a−/− mice). Loss of BCL11A in SCD animals restored the hematologic deficits to normal values. Compared with sickle animals, SCD/Bcl11a−/− mice have marked increases in RBC counts (6.4 ± 0.5 to 9.8 ± 0.4 × 1012/L; non-sickle control 10.1 ± 0.2 × 1012/L ) and hemoglobin level (7.8 ± 0.6 to 13.6 ± 0.7 g/L; non-sickle control 13.1 ± 0.3 g/L), accompanied by a significant reduction in reticulocyte counts (38.2% ± 3.9% to 7.0% ± 0.3%; non-sickle control 3.1% ± 0.6%). Consistent with correction of hematologic parameters, sickled erythrocytes were absent in the peripheral blood of SCD/Bcl11a−/− mice and red cell lifespan was substantially normalized. In addition, tissue damage in multiple organs was prevented in SCD/Bcl11a−/− mice. Urine osmolality of SCD/Bcl11a−/− mice was restored (from 1037 ± 82 mOsm in SCD mice to 2133 ± 333 mOsm in SCD/Bcl11a−/− mice), indicating improved renal function.To assess the extent to which these phenotypic findings were related to HbF induction, we determined the level of γ-globin expression and cellular distribution of HbF in SCD/Bcl11a−/− mice. Compared with SCD mice, expression of γ-globin genes was markedly elevated in adult SCD/Bcl11a−/− mice (1.3% to 28.3% of total β-like human globins; P = 0.00017). Peripheral blood of control and SCD mice contained few F cells (3.6% and 7.2%, respectively). In contrast, peripheral blood of SCD/Bcl11a−/− mice displayed strong pancellular staining of HbF, and F cells contributed to 85.1% of total RBCs (P = 3.9 × 10−5). The level of HbF expression achieved in the absence of BCL11A is well beyond that needed to impede HbS polymerization.In conclusion, we demonstrate that inactivation of a single component involved in HbF regulation, the transcription factor BCL11A, provides phenotypic correction of SCD in a mouse model. Our findings provide a crucial “proof of principle” for targeted reactivation of HbF as a realistic therapeutic goal. Disclosures:No relevant conflicts of interest to declare.

Clinical and Hematologic Manifestations in Patients with Diamond-Blackfan Anemia

Abstract 4378 BackgroundDiamond-Blackfan anemia (DBA), characterized by impaired red cell production, is a rare condition that usually occurs symptomatic in early infancy. The purpose of this study was to assess the nationwide experiences of DBA that were encountered over a period of 20 years as a part of further molecular analyses. MethodsMedical records of 56 patients diagnosed to have DBA were reviewed retrospectively from Nov. 1984 to July 31, 2010. Fifteen clinics including 13 university hospitals have been participated. ResultsMale to female ratio was 1.67:1 (males 35 cases, females 21). The median age of diagnosis was 4 months (range birth to 52 months of age), with the diagnosis of 74.1% before 1 year of age. From 2000 to 2009, 33 cases with DBA were registered, which showed the incidence of 6.6 cases per million. Excluding growth retardation, 21 cases (38.2%) showed congenital defects: thumb deformities (8 cases), ptosis (5), coarctation of aorta (5), ventricular septal defect (4), strabismus (3), cleft palate (2), mitral valve prolapsed (2). The mean hemoglobin (Hb) concentration was 5.1±1.9 g/dL, and median, 5.2 g/dL. The mean MCV was 93.4±11.6 fL, mean HbF 7.7±11.3%, mean reticulocytes 19,700/mm3 and mean adenosine deaminase 21.3. The mean cellularity of bone marrow was 75%, with the ME ratio of 20.4:1. Among 47 patients who had been treated with steroids, 23 patients (48.9%) did not need further steroids after remission. The mean duration of steroid use was 7 years and 2 months (maximum 22 years). Among 28 cases who received steroid, 9 patients had been controlled with the oral steroid of 5 mg or less per day. Eight patients with DBA that received hematopoietic transplantation have survived. Cancers developed in 2 cases (3.6%). ConclusionThe incidence of DBA is similar with the data already published, but male predilection in our study. Although all patients responded to initial treatment with steroids, about a half needed further steroids after remission. It is necessary to collect the further data including management pathways from nationwide DBA registries, with the molecular analyses. Disclosures:No relevant conflicts of interest to declare.

Burn Injury Dampens Erythroid Cell Production Through Reprioritizing Bone Marrow Hematopoietic Response

Anemia in burn patients is due to surgical blood loss and anemia of critical illness. Because the commitment paradigm of common bone marrow progenitors dictates the production of erythroid, myeloid, and lymphoid cells, we hypothesized that skewed bone marrow lineage commitment decreases red cell production and causes anemia after a burn injury. After anesthesia, B(6)D(2)F(1) mice received a 15% total body surface area dorsal scald burn. The sham group did not receive scald burn. Femoral bone marrow was harvested on 2, 5, 7, 14, and 21 postburn days (PBD). Total bone marrow cells were labeled with specific antibodies to erythroid (CD71/Ter119), myeloid (CD11b), and lymphoid (CD19) lineages and analyzed by flow cytometry. To test whether erythropoietin (EPO) could increase red blood cell production, EPO was administered to sham and burn animals and their reticulocyte response was measured on PBD 2 and PBD 7. Burn injury reduced the erythroid cells of the bone marrow from 35% in sham to 17% by PBD 5 and remained at similar level until PBD 21. Myeloid cells, however, increased from 42% in sham to 60% on PBD 5 and 77% on PBD 21. Burn injury reduced reticulocyte counts on PBD 2 and PBD 7 indicating that the erythroid compartment is severely depleted. This depleted compartment, however, responded to EPO but was not sufficient to change red cell production. Burn injury skews the bone marrow hematopoietic commitment away from erythroid and toward myeloid cells. Shrinkage of the erythroid compartment contributes to resistance to EPO and the anemia of critical illness.

Rhepo for Reduction of Perinatal Arterial Stroke: A Feasibility and Safety Study

Background: Perinatal Arterial Stroke (PAS) occurs in 1 per 2600 newborns. No causative therapy is available. Experimental studies in a neonatal stroke model suggest neuroprotective effect of recombinant erythropoietine (rhEPO). We therefore performed a feasibilty and safety study of rhEPO in neonates with PAS. Patients: 17 neonates with MRI-proven PAS were included, and treated with 3 subsequent doses of rhEPO of 1000 IU/kg: immediately after MRI-diagnosis of PAS and at 24 and 48 hrs after the first dose. MRI/MRA was repeated one week and 3 months after the diagnosis of PAS. During the first weeks hemodynamic, coagulation and hematologic parameters (red cell, white cell and platelet counts), liver and renal function, and Le/Ri regional cerebral saturation (rScO2) with NIRS were monitored. Results: HR, blood pressure and coagulation were always in the normal range, as were liver and renal functions. The table summarizes red, white and platelet counts (median and ranges). Hct's were always in the normal range. Slightly higher rScO2-values were detected at the infarction side.(day1-3: 0.69[0.56-0.95] vs 0.63[0.54-0.85]). Table Conclusion: Treatment with rhEPO (1000 IU/kg daily for 3 d) in neonates with PAS had no effect on red and white cell production or on platelets or coagulation. Cerebral oxygenation was higher on the infarction side. rhEPO therapy seems to be safe, but its effectivity remains to be confirmed.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

Negative Autoregulation by Fas Stabilizes Adult Erythropoiesis and Accelerates Its Stress Response

Erythropoiesis maintains a stable hematocrit and tissue oxygenation in the basal state, while mounting a stress response that accelerates red cell production in anemia, blood loss or high altitude. Thus, tissue hypoxia increases secretion of the hormone erythropoietin (Epo), stimulating an increase in erythroid progenitors and erythropoietic rate. Several cell divisions must elapse, however, before Epo-responsive progenitors mature into red cells. This inherent delay is expected to reduce the stability of erythropoiesis and to slow its response to stress. Here we identify a mechanism that helps to offset these effects. We recently showed that splenic early erythroblasts, ‘EryA’, negatively regulate their own survival by co-expressing the death receptor Fas, and its ligand, FasL. Here we studied mice mutant for either Fas or FasL, bred onto an immune-deficient background, in order to avoid an autoimmune syndrome associated with Fas deficiency. Mutant mice had a higher hematocrit, lower serum Epo, and an increased number of splenic erythroid progenitors, suggesting that Fas negatively regulates erythropoiesis at the level of the whole animal. In addition, Fas-mediated autoregulation stabilizes the size of the splenic early erythroblast pool, since mutant mice had a significantly more variable EryA pool than matched control mice. Unexpectedly, in spite of the loss of a negative regulator, the expansion of EryA and ProE progenitors in response to high Epo in vivo, as well as the increase in erythropoietic rate in mice injected with Epo or placed in a hypoxic environment, lagged significantly in the mutant mice. This suggests that Fas-mediated autoregulation accelerates the erythropoietic response to stress. Therefore, Fas-mediated negative autoregulation within splenic erythropoietic tissue optimizes key dynamic features in the operation of the erythropoietic network as a whole, helping to maintain erythroid homeostasis in the basal state, while accelerating the stress response.

Severe acquired anaemia in Africa: new concepts

Severe anaemia is common in Africa. It has a high mortality and particularly affects young children and pregnant women. Recent research provides new insights into the mechanisms and causes of severe acquired anaemia and overturns accepted dogma. Deficiencies of vitamin B12 and vitamin A, but not of iron or folic acid, are associated with severe anaemia. Bacterial infections and, in very young children, hookworm infections are also common in severe anaemia. Irrespective of the aetiology, the mechanism causing severe anaemia is often red cell production failure. Severe anaemia in Africa is therefore a complex multi-factorial syndrome, which, even in an individual patient, is unlikely to be amenable to a single intervention. Policies and practices concerning anaemia diagnosis, treatment and prevention need to be substantially revised if we are to make a significant impact on the huge burden of severe anaemia in Africa.

Determination of Some Ethnomedicinally Important Constituents of Aegle marmelos Fruit During Different Stages of Ripening

Aim Aegle marmelos is a medicinal herb belonging to the Rutacae family. The fruit of A. marmelos at each stage of ripening is used as ethnomedicine to cure various diseases. The aim of the present study was to determine the components contributing to the medicinal value of the A. marmelos fruit at different stages of ripening. Methods High-performance liquid chromatography (HPLC) was used to determine polyphenol, vitamins, organic acids and sugars in A. marmelos fruit at different stages of ripening. Results Tannin, a polyphenol responsible for astringent and antimicrobial properties of A. marmelos fruit was found to increase during ripening. Riboflavin, a vital medicinal component was detected in traceable amount only in full-ripe A. marmelos fruit. Riboflavin contributes towards body growth, reproduction and red cell production. The content of ascorbic acid (vitamin C) which is useful in preventing scurvy decreased significantly as fruit ripens. Conclusions The difference in the medicinal effect of A. marmelos fruit at each stage of ripening might be due to the presence of different amount of polyphenol, vitamins and organic acids.

EBV‐associated hemophagocytic syndrome

General aspect of the marrow: macrophages with prominent phagocytosis of red cells, platelets, and erythroblasts. The marrow is overall hypocellular (MGG, original magnification ×400). High-power view of two characteristic hemophagocytic macrophages. (A) A macrophage with ingested red cells, platelets, erythroblasts, neutrophil, and assorted debris. (B) A macrophage which has engulfed a large number of erythroblasts (MGG, original magnification ×1,000). Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH) is a rare syndrome characterized by fever, pancytopenia, organomegaly, multiorgan dysfunction, and hemophagocytosis by a large number of macrophages in bone marrow and other tissues [1]. Central to the pathophysiology is the impaired activity of NK and cytotoxic T lymphocytes resulting in uncontrolled immune activation, hypercytokinemia, and proliferation of macrophages. The diagnosis of HLH relies upon well-established criteria [2]. The syndrome has been described in familial conditions or as a secondary phenomenon to infections, malignancies, and collagen vascular diseases. HLH in the context of infection is best described in association with Epstein-Barr virus (EBV). EBV-associated HLH can occur either in primary infection or as a consequence of viral reactivation. EBV can also be the trigger in both familial and secondary cases. Although uncommon, hemophagocytic syndrome is a serious complication of EBV infection and can be fatal in many cases. Early recognition and prompt initiation of treatment are of great importance to reduce the high morbidity and mortality associated with this condition. The history and representative images from a case of EBV-associated hemophagocytic syndrome are described. The patient's consent has been obtained. A 25-year-old woman was admitted with fever, pancytopenia, and hepatic dysfunction. Five weeks previously, she was diagnosed with infectious mononucleosis on the basis of pharyngitis, fever, and positive EBV antibodies. In the weeks after diagnosis, she continued to complain of intermittent fevers and sweating. Past medical and family history were unremarkable. Physical examination revealed temperature 38°C, icteric sclera, and hepatosplenomegaly. Laboratory values included hemoglobin 8.1 g/dL, leucocytes 2,100 μL−1, neutrophils 720 μL−1, platelets 40,000 μL−1, bilirubin 7.4 mg/dL, ALT 135 U/L, LDH 1,080 U/L, triglycerides 459 mg/dL, and ferritin 6,070 ng/mL. Peripheral blood film morphology was unremarkable. The direct and indirect antiglobulin test and screening for cold autoagglutinins gave negative results. The reticulocyte automated count and absolute value were 1.11% and 31,900 μL−1, respectively, indicating impaired production of red cells. Bone marrow aspiration showed numerous macrophages with striking phagocytosis of blood cells and marrow precursors (Images 1 and 2). EBV serologies indicated primary infection, and the EBV-PCR result was 18,000 copies/mL. Genetic testing for familial HLH was negative. The patient was treated with methylprednisolone and intravenous immunoglobulins and eventually recovered completely.

Tumor necrosis factor alpha-mediated inhibition of erythropoiesis involves GATA-1/GATA-2 balance impairment and PU.1 over-expression

Many physiological perturbations can cause anemia. In cancer patients, activation of the immune system leads to the production of proinflammatory cytokines including tumor necrosis factor alpha (TNFα), that have been shown to inhibit red-cell production via poorly understood mechanisms. Treatment of anemia by human recombinant erythropoietin (EPO) is strongly suspected to induce tumor growth. This study focuses on the mechanisms involved in TNFα-mediated inhibition of erythropoiesis. CD34 + hematopoietic stem/progenitor cells (HSPCs) were isolated from human cord blood. Erythropoiesis was achieved in vitro by stimulating cells with EPO. We show that TNFα clearly affected erythroid development, as assessed by May-Grünwald/Giemsa staining, flow cytometry analysis and fluorescent microscopy. The amount of hemoglobin-producing cells as well as the expression of GATA-1 target erythro-specific genes (EPO receptor, glycophorin A and globins) was found decreased after TNFα treatment of HSPC. In correlation, TNFα induced the expression of the transcription factors GATA-2 and PU.1, described as inhibitors of erythropoiesis. In this regard, TNFα promoted the formation of the GATA-1/PU.1 complex that has been reported to block the transcriptional activity of GATA-1. Our results clearly demonstrate that TNFα prevents EPO-mediated erythropoiesis of HSPC as an early event, by directly affecting erythroid cell development.

Optimization of in vitro erythropoiesis from CD34 + cord blood cells using design of experiments (DOE)

In vitro culture studies of hematopoietic cells (HCs) have traditionally been limited to dose-response, empirical methods which can be uninformative, misleading and suboptimal. Herein, we use design of experiments methodology to quantitatively define and optimize in vitro erythropoiesis from human cord blood CD34 + cells to enucleation. DOE was used to theoretically define the effects of 7 cytokines known to affect red cell maturation in humans. Of these, stem cell factor (SCF) and erythropoietin (EPO) were found to be the most significant factors in red cell production ( p < 0.01). DOE defined process characteristics and interactions of SCF and EPO on total cell expansion and maturation of cord blood HCs towards the erythroid lineage and delineated optimal in vitro concentrations of each cytokine (75 ng/mL and 4.5 IU/mL, respectively). The DOE-optimized cytokine cocktail achieved an average of 26,460-fold total cell expansion and accelerated erythroid maturation yielding over 90% glycophorin-A-expressing cells by day 7. Maturation of red cells was confirmed by morphology, immunophenotype, clonogenic capacity, gene and protein expression of adult and neonatal hemoglobins (α, β, and γ) and by progressive expression of GATA-1 with maturation. The use of DOE was effective and efficient for the analysis, characterization and optimization of in vitro erythropoiesis to the stage of enucleation and provides a systematic platform for the use of other growth factors in vitro for the study of hematopoiesis and expansion of cell products.