Red Cell Production Research Articles

Treatment in Vitro with a Combination of Bcl-Xl Inhibitor-ABT-737 and a JAK2 Inhibitor Selectively Eliminates JAK2V617F MPN Progenitor Cells.

Abstract 752The Ph- myeloproliferative neoplasm (MPN) are associated with excessive production of red cells, platelets and granulocytes which largely determines their clinical manifestations. A mutation in the JAK2 tyrosine kinase (JAK2V617F) was identified in the majority of patients with MPNs. The JAK2V617F mutation has been shown to play a pivotal role in the pathogenesis of MPNs. We have reported that erlotinib (Tarceva), a kinase inhibitor which inhibits the epidermal growth factor induced kinase activity, is also a potent inhibitor of JAK2V617F activity. It has been shown that erythroblasts from patients with polycythemia vera (PV) express elevated levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL. In addition, we have recently documented that megakaryocytes derived from patients with primary myelofibrosis (PMF) undergo a delayed pattern of apoptosis in vitro which might be attributed to the over-expression of Bcl-xL. We hypothesize that a combination of a JAK2V617F inhibitor and a Bcl-xL inhibitor might be capable of selectively eliminating MPN cells while sparing normal cells, and therefore, providing an optimal treatment strategy for Ph− MPNs.We then evaluated the combinations of a JAK2V617 inhibitor (erlotinib or INCB018424) and a Bcl-xL inhibitor (ABT-737) for their ability to selectively eliminate MPN (JAK2V617F positive) cells while sparing normal cells in vitro using a variety of screening systems. We first tested the ability of each of the JAK2 inhibitors alone or in combination with ABT-737 to induce death of HEL cells, which harbors JAK2V617F. Among these treatments, ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1 nM) were shown to have similar capability of inducing HEL cell apoptosis (50-70%) which was significantly greater than that by each of the single agents (<5%). However, the combination of ABT-737 (0.25 uM) plus erlotinib (1.0 uM) was the least potent of inducing normal CD34+ cells to undergo apoptosis (∼5%) as compared to ABT-737 plus INCB018424 (∼20%). We next examined the effects of each of the JAK2 inhibitors alone or in combination with ABT-737 on CD34+ cells isolated from patients with PV or PMF. PV or PMF CD34+ cells were incubated in the absence or presence of agent(s) for 4 days; the percentage of apoptotic cells was then determined using Annexin V/PI staining by flow cytometry. A fraction of both untreated and treated CD34+ cells were assayed for hematopoietic colonies in the presence of SCF, IL-6, IL-3, G-CSF and EPO; individual colonies were then randomly plucked for JAK2V617F genotyping using nested allele-specific PCR. Similar to HEL cells, the percentage of MPD CD34+ cells undergoing apoptosis were similar when cells were treated with ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1.0 nM) (∼40%), but were significantly higher than that of MPN CD34+ cells treated with each of the agents alone (∼5%). Interestingly, when the percentages of JAK2V617F positive colonies within each of the treated and untreated cell populations were analyzed, MPN CD34+ cells treated with ABT-737 plus each of the JAK2 inhibitors contained a significantly lower percentage of JAK2V617F positive colonies than those untreated or treated with each of the single agents. These data indicate that the combination of ABT-737 plus a JAK2 inhibitor specifically targets on the JAK2V617F positive progenitor cells. Collectively, we identified an agent combination of ABT-737 (0.25 uM) plus a JAK2 inhibitor that is capable of selectively eliminating JAK2V617F positive MPN progenitor cells while sparing normal progenitor cells in vitro. Disclosures:No relevant conflicts of interest to declare.

Use of Jak2 Inhibitors to Limit Ineffective Erythropoiesis and Iron Absorption in Mice Affected by β-Thalassemia and Other Disorders of Red Cell Production.

Abstract 2020Poster Board I-1042β-thalassemia intermedia (TI) and major (TM) are characterized by Ineffective Erythropoiesis (IE). We hypothesized that the kinase Jak2 plays a major role in IE and splenomegaly. To test this hypothesis we administered a Jak2 inhibitor (TG101209) to mice affected by TI, showing that this treatment was associated with a marked decrease in IE, and a moderate decrease in hemoglobin (Hb) levels (∼1 g/dL). This last observation indicates that the use of a Jak2 inhibitor might exacerbate anemia in thalassemia. However, we hypothesized that using standard transfusion to treat TM mice would also be adequate to prevent any further anemia caused by Jak2 inhibition while still allowing for decreased splenomegaly. Therefore, we analyzed the erythropoiesis and iron metabolism in TM animals treated with a Jak2 inhibitor and transfused. Use of TG101209 in TM mice not only reduced the spleen size dramatically (0.42±0.15 g and 0.19±0.10 g respectively in transfused+placebo (N=4) vs transfused+TG101209 (N=8), P= 0.007), but also allowed the mice to maintain higher Hb levels (respectively 7.3±1.1 g/dl vs 9.3±1.2 g/dl, P=0.019). This was likely due to reduced spleen size and limited red cell sequestration. Contrary to TM mice treated with transfusion+placebo, no foci of extra-medullary hematopoiesis were detectable in the parenchema of mice treated with TG101209. Hamp1 expression inversely correlated with the spleen weight, possibly indicating that suppression of IE (due both to blood transfusion and TG101209 administration) had a positive effect on Hamp1 expression. Hb levels also directly correlated with Hamp1 expression in the same animals. In this case, however, only transfusion played a role in increasing Hamp1 expression, although TG101209 undoubtedly had a positive effect by reducing the spleen size and thereby indirectly increasing the Hb levels.The suppression of erythropoiesis by blood transfusion limits the extent of our interpretations as it may mask the effect of the Jak2 inhibitor. Therefore we hypothesized that the administration of a tailored and reduced dose of the drug could be effective in reducing the splenomegaly in non-transfused TI mice, without affecting the Hb levels. We also hypothesized that the suppression of erythropoiesis would also lead to increased Hamp1 expression in the presence of iron overload. Compared to mice treated with placebo (N=5), analysis of TI mice treated with a tailored dose of 100mg/kg/day of the drug (N=11) showed a significant decrease in spleen size (0.18±0.05 g and 0.27±0.05 g, P=0.006 for drug treated mice and placebo treated mice respectively). Of note no significant difference of Hb levels was detectable between the 2 groups. In the drug treated mice we observed a significant decrease of the immature erythroid cell population (P=0.012) and amelioration of the architecture of the spleen, with the reappearance of white pulp foci and a significant restoration of the splenic lymphocitic populations. Drug treated mice showed increased levels of Hamp1 mRNA that inversely correlated with the spleen weight, suggesting a direct feedback between erythropoietic rate and expression of Hamp1.To determine if the use of Jak2 inhibitors could be beneficial in a mouse model mimicking a human form of hereditary ellyptocytosis, we treated mice KO for the 4.1R protein isoforms with TG101209, in presence or absence of blood transfusions. These mice exhibit moderate splenomegaly and anemia and the drug treatment was effective in reducing the spleen weight and the associated IE. We also plan to analyze Sickle Cell mice that we are treating with a Jak2 inhibitor. In conclusion our data show that the administration of Jak2 inhibitors is efficient in decreasing the spleen size and ameliorating the pathologic iron metabolism in thalassemia, both in the presence or absence of blood transfusions. Moreover we show that Jak2 inhibitors could transform the therapeutic approach for other forms of anemias. Disclosures:No relevant conflicts of interest to declare.

Macrophage Depletion Leads to Modification of Thalassemic Phenotype.

Abstract 2023Poster Board I-1045Macrophages represent an important link between erythropoiesis and iron metabolism. They support maturation and differentiation of erythroblasts and clear senescent red cells from circulation, thus recycling iron within the hematopoietic system. However, little is known about the role of macrophages in conditions of ineffective erythropoiesis like beta-thalassemia intermedia (β-TI) or sickle cell anemia (SCD). Our goal was to study the role of macrophages under such conditions, where there is a large expansion of the erythron, the lifespan of the red cells is decreased, and both iron absorption and organ iron content are increased.Clodronate liposomes have been shown to efficiently eliminate macrophages from spleen, liver and bone marrow when administered by intravenous injection. Therefore, we utilized this technique to eliminate macrophages from the spleen, liver and bone marrow of β-TI and SCD mice. Three different treatment schedules were used; a) acute high dose; b) medium term split dose; c) chronic low dose. Animals were analyzed for erythropoietic and pathological parameters.All treatments effectively depleted splenic and bone marrow macrophages as shown by flow citometric and immunohistochemical analyses. At doses that led to only a small decrease of hemoglobin (Hb) levels in normal mice, clodronate treatment induced high level of mortality in β-TI and SCD mice (70% and 75%, respectively). In contrast, administration of clodronate to mice affected by beta-thalassemia major, mice that do not make any adult or fetal Hb/mature red cells, caused no mortality suggesting that circulating abnormal red cells might be responsible for the mortality observed in β-TI and SCD mice. Pathological analysis of β-TI mice revealed the formation of thrombi in the lungs and heart to be the likely cause of death. Analysis of SCD mice is in progress. β-TI and SCD have been described as hypercoagulable states with a high risk of thrombosis. Damaged red cell membranes and ROS have been implicated as factors that increase the risk of thrombosis in β-TI and SCD patients. We hypothesize that clodronate treatment enhances this process. To test this hypothesis, we administered an anti-coagulant (heparin) and an anti-oxidant (NAC) to β-TI mice. Both these treatments resulted in increased survival from 31% to 65% compared to clodronate alone. Analysis of SCD animals is in progress. β-TI mice that survived clodronate treatment exhibited a milder thalassemic phenotype, characterized by increased Hb (∼1.5g/dL), HCT (∼7%) and RBC count (∼2e6 cells/ul), partially associated with a decreased RBC clearance. Ineffective erythropoiesis and splenomegaly were also ameliorated (∼40% decrease in spleen size). Animals depleted of macrophages also showed a marked decrease in serum iron parameters, but no difference in their organ iron concentrations. Analysis of hepcidin expression is in progress. To further corroborate these observations, β-TI mice are being crossed with mice expressing the human diphtheria toxin (CD11b-DTR) which allows for conditional depletion of macrophages.Our data suggests that macrophages play a major role in conditions of abnormal red cell production and ineffective erythropoiesis. In the former case, they have an important protective function related to the high risk of thrombosis associated with β-TI and SCD. In the latter, macrophages may act as negative modulators of erythropoietic development. The amelioration of splenomegaly and ineffective erythropoiesis observed suggests that erythroid cells might proliferate less and/or differentiate more in the absence of macrophages. One hypothesis is that macrophages sense erythroid maturation and/or differentiation and act as modulators of these processes to ensure the release of high quality red cells into the circulation. Second, macrophages, under conditions of iron overload, might produce inflammatory cytokines that further impair erythropoiesis in thalassemia. Analysis of RNA extracted from macrophages and of inflammatory cytokines in β-TI mice before and after clodronate treatment is in progress. Disclosures:No relevant conflicts of interest to declare.

Identification of high oxygen affinity hemoglobin variants in the investigation of patients with erythrocytosis

Erythrocytosis is a disorder of red cell production that can arise from several different causes. Familial cases have been classified into four groups (ECYT1-4) by Online Mendelian Inheritance in Man (OMIM) as listed at the website: http://www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM. Erythrocytosis associated with mutations in the erythropoietin receptor belong to group ECYT1 and arise from a primary cause that is intrinsic to the red cell.1 The other three categories of erythrocytosis, ECYT2-4, are secondary and are characterized by dysregulated erythropoietin (Epo) production resulting from defects in the oxygen sensing pathway.1 OMIM classification does not consider other secondary causes of erythrocytosis that are related to abnormal oxygen delivery such as hemoglobin (Hb) variants or 2,3-biphosphoglycerate mutase (BPG) deficiencies. Approximately 100 hemoglobin variants with a high affinity for oxygen have been described that cause a decrease in the supply of oxygen to tissues.2 These Hb variants are inherited in an autosomal dominant manner with an associated family history.2 Both α and β globin genes can be affected2 and the serum Epo would be inappropriately normal for the associated raised hematocrit. During the differential diagnosis of erythrocytosis, a high oxygen affinity Hb can be excluded by studying the oxygen binding properties of freshly drawn blood.2–4 Blood gas analyzers available in all hospitals will calculate P50 values. High affinity Hbs cause a left shift in the oxygen dissociation curve and thus lower the P50 value. Not all high affinity Hb variants are detected by routine Hb electrophoresis and isoelectric focusing in polyacry-lamide and hence a number are misdiagnosed. Over the last decade we have developed a data base of erythrocytosis patients referred from centers throughout the UK and Ireland.5 Before patients can be included in the registry all secondary causes of erythrocytosis should be excluded. During an audit of the data base we decided it may be useful to confirm the absence of high oxygen affinity Hb variants. Patients with a strong family history and serum Epo within the reference range or above were selected for β globin gene sequencing. Those patients negative for a β chain variant were further screened for an alpha globin gene variant. Four different high oxygen affinity Hb variants, Olympia (β20Va→Met),1,6,7 Pierre-Benite (β90Glu→Asp),6,8 Santa Clara (β97His→Asn)6,9 and Heathrow (β103Phe→Leu),6,10 were detected in 6 families from a data base of 205 individuals giving a prevalence of 3%. All are electrophoretically silent and hence difficult to detect by routine laboratory tests.6 Variants can be confirmed by high performance liquid chromatography or by mass spectrometry and identified by PCR-direct sequencing. 6 The hematological indices of the patients are listed in Table 1. In all cases the hematocrit and the hemoglobin level was raised, except for UPN224 which was found to be iron deficient. White blood cell and platelet counts were in the normal range. The serum Epo level was either elevated or inappropriately normal for the elevated Hb. Thrombotic events have been reported for only Patients 214 and 225. Table 1. Hematologic indices of erythrocytosis patients with hemoglobin variants. The hemoglobin tetramer is composed of 2 chains of α and 2 chains of β globin arranged in two identical halves, containing one chain of each globin type. As Hb transfers from the low oxygen affinity state (known as T or tense) to the high affinity state (R or relaxed) there is an accompanying movement within the α1β2 subunit.1 Many of the amino acid changes associated with a high affinity phenotype map to the α1β2 interface or the C-terminal of the β chain.1 These mutations prevent the transition of the Hb molecule to the T state and hence are unable to release the bound oxygen molecules. The Santa Clara variant cannot achieve the T state9 while Pierre-Benite is thought to stabilize Hb in the T quaternary structure8,11 and Hb Olympia12 affects the surface of the protein causing it to self-associate. Other mutations cause structural alteration of the hemepocket. The presence of several aromatic amino acids around the hememoiety is crucial for the correct orientation of the hemegroup. Replacement of Phe103 with Leu (Hb Heathrow) causes the affinity of the hemegroup for oxygen to be increased.1 During the differential diagnosis of erythrocytosis the first step would be the calculation of P50 using the routine blood gas analyzer. In those patients with a low value for P50 sequencing of the globin gene to establish the actual variant would be performed. In the absence of P50 values, as with patients in this study, it is important to eliminate the possibility of Hb variants by sequencing t he globin genes, which confirmed 6 positive cases on the registry.

Differential gene expression in umbilical cord blood and maternal peripheral blood

Umbilical cord blood (UCB) has become a useful alternative source of hematopoietic stem cells for clinical and research applications. UCB represents neonatal blood and differs from adult blood in many aspects, displaying different cell composition and various features of cellular immaturity. To understand molecular basis of phenotypic differences between neonatal and adult blood, we studied variations in transcriptome of UCB and maternal peripheral blood (PB). Using Illumina microarrays, we determined gene expression profiles of UCB and PB samples obtained from 30 mothers giving birth to living baby. Out of 20,589 tested genes, 424 genes were down-regulated and 417 genes were up-regulated in UCB compared with PB. Reduced expression of many immunity-related pathways (e.g. TLR pathway, Jak-STAT pathway, cytokine-cytokine receptor interaction) in neonatal blood cells may contribute to the poor response to antigens, increasing susceptibility to infections at the time of disappearance of protective maternal antibodies. On the other hand, overexpression of erythropoiesis-related genes (glycophorins, fetal hemoglobins, enzymes catalysing heme synthesis and erythrocyte differentiation) in UCB probably enforces red cell production in newborns. Our study demonstrates that neonatal and maternal bloods show specific gene expression profiles, likely reflecting differences in phenotypes of immunologically immature and fully evolved hematopoietic cells.

High-dose epoetin alfa as induction treatment for severe anemia in multiple myeloma patients

Anemia is a common finding in patients with multiple myeloma (MM): it is present in two-thirds of patients (pts) at diagnosis and reflects the course of the disease, since it worsens during resistant or progressive disease and ameliorates when the disease is controlled by treatment [1, 2]. Generally, anemia during myeloma is normochromic and normocytic, characterized by shortened erythrocyte survival with failure of bone marrow to compensate red cell production. The multifactor basis of bone marrow impairment includes: (a) impaired availability of storage iron, (b) inadequate erythropoietin response to the level of anemia, and (c) overproduction of cytokines capable of direct erythropoiesis inhibition. These cytokines (including tumor necrosis factor, interleukin-1 and interleukin-6) may also decrease reutilization of iron stores from reticuloendothelial cells and may interfere with kidney erythropoietin production [3]. Recombinant human erythropoietin (rHuEPO) has been extensively used throughout the course of the disease to increase hemoglobin (Hb) concentration, reduce transfusion requirement and improve the quality of life. Treatment of anemia in lymphoproliferative disorders with erythropoiesis-stimulating agents (ESA) has been shown to be safe, efficacious and effective. Response rate (RR) varied from 31 to 78%, according to the criteria utilized for response [4]. In MM, RR was influenced by the disease duration, ratio between observed and predicted serum erythropoietin concentration and Epo dose. RR at 4 weeks (wk) represents the most powerful predictor of a durable response [5]. Recently, ASCO/ASH guidelines for the use of epoetin and darbepoetin have been revised: FDA-approved starting dose of epoetin is 150 IU/kg 3 times/week or 40,000 IU/week subcutaneously; FDAapproved starting dose of darbepoetin is 2.25 lg/kg/week or 500 lg every 3 week subcutaneously. Alternative starting doses or dosing schedules have shown no consistent differences in transfusion and Hb response, although they may be considered to improve cost/effectiveness. Dose escalation should follow those approved by FDA; no convincing evidences indicate that other schedules may be associated with better results. The threshold for initiating ESA therapy should be a concentration of Hb B 10 g/dL [6]. The aim of this study is to evaluate the safety and efficacy of a regimen based on rHuEPO alfa 40000 IU twice/week as starting dose with weekly IV iron supplementation in MM pts with severe anemia. Between January 2003 and March 2004, 20 pts (10 M/ 10 F) with MM were enrolled in this open-label trial. The starting dose consisted of 40000 IU subcutaneously twice/ week for 5 doses (up to 8 doses if no major response was obtained at week 3), maintained by rHuEPO at the conventional schedule in the responder patients. All pts received iron supplementation (oral or IV). The main pretreatment characteristics were the following: median age 67.5 years (58–81); 9 pts were in first line treatment, 6 pts in second line and 5 received more than 3 lines of T. Caravita A. Siniscalchi P. Niscola S. Amadori P. de Fabritiis Department of Hematology, University ‘‘Tor Vergata’’, St. Eugenio Hospital, Rome, Italy

Safe use of recombinant activated factor VIIa for recalcitrant postoperative haemorrhage in cardiac surgery

The aim of this case series is to review the effect of recombinant activated factor VIIa (rFVIIa) on refractory haemorrhage, despite aggressive treatment with conventional blood products and medications at our institution. All patients undergoing cardiac surgery who received rFVIIa as rescue therapy for persistent uncontrollable haemorrhage were studied. We examined coagulation immediately before and after rFVIIa was given; international normalized ratio (INR), activated partial thromboplastin (APTT) fibrinogen and platelet levels, in addition to the use of red cell and non-red cell blood products, morbidity and mortality. Thirty patients (0.6%) received 31 doses of rFVIIa for bleeding refractory to conventional treatment. Twenty received rFVIIa in theatre after primary surgery, three after re-exploration and eight in the intensive care unit (ICU). Hospital mortality was 6.5% (2/30) and there were no documented thromboembolic phenomena. There was significant reduction in red blood cell and product transfusion before and after rFVIIa administration (P<0.001). There was significant correction in coagulation parameters after rFVIIa. Recombinant FVIIa appears to be safe, and is effective in reducing red blood cell and product transfusion requirements and may impact on early and late outcomes in this small complex subgroup of patients.

Time course of anemia during six months follow up following intensive care discharge and factors associated with impaired recovery of erythropoiesis*

Anemia is a common complication of critical illness, but its duration after intensive care discharge and possible contributory factors have not been studied. Our aim was to follow patients discharged anemic from the intensive care unit (ICU) for up to 6 months and determine the duration of and possible reasons for persisting anemia. Prospective observational cohort study of intensive care (ICU) survivors with moderate-severe anemia at the time of ICU discharge. Erythropoietic and inflammatory markers were measured at regular intervals over six months to assess red cell production and factors limiting recovery from anemia. An 18-bed medico-surgical ICU in a Scottish university teaching hospital. Patients who required >24 hrs of ventilatory support and were discharged from intensive care with hemoglobin < 100 g/L were studied prospectively over 6 months. 30 patients were recruited; 19 completed 6 months follow-up, 6 died during the study period, and 5 completed part of the follow up. Patients with ongoing renal failure or chronic hematologic disorders were excluded. 47% (9 of 19) of patients completing 6 months follow up recovered from their anemia. The median time to recovery was 11 wks (1, 3 quartiles: 9, 26 wks). 10 patients (53%) were still anemic 6 months after ICU discharge. No patients developed iron, vitamin B12 or folate deficiency. An inappropriately low erythropoietin response to anemia was observed in virtually all patients and did not distinguish nonrecovering patients. Patients with delayed recovery or persisting anemia during the 13 wks following ICU discharge had higher levels of circulating inflammatory markers (IL-6 and C-reactive protein) and did not exhibit reticulocytosis during the weeks following discharge. Anemia persists in many patients following critical illness and is associated with ongoing inflammation, inappropriate erythropoietin response and poor marrow red cell production.

CNTO 530 functions as a potent EPO mimetic via unique sustained effects on bone marrow proerythroblast pools

AbstractAnemia as associated with numerous clinical conditions can be debilitating, but frequently can be treated via administration of epoetin-alfa, darbepoietin-alfa, or methoxy-PEG epoetin-beta. Despite the complexity of EPO-EPO receptor interactions, the development of interesting EPO mimetic peptides (EMPs) also has been possible. CNTO 530 is one such novel MIMETIBODY Fc-domain dimeric EMP fusion protein. In a mouse model, single-dose CNTO 530 (unlike epoetin-alfa or darbepoietin-alfa) bolstered red cell production for up to 1 month. In 5-fluorouracil and carboplatin-paclitaxel models, CNTO 530 also protected against anemia with unique efficiency. These actions were not fully accounted for by half-life estimates, and CNTO 530 signaling events therefore were studied. Within primary bone marrow erythroblasts, kinetics of STAT5, ERK, and AKT activation were similar for CNTO 530 and epoetin-alfa. p70S6K activation by CNTO 530, however, was selectively sustained. In vivo, CNTO 530 uniquely stimulated the enhanced formation of PODXLhighCD71high (pro)erythroblasts at frequencies multifold above epoetin-alfa or darbepoietin-alfa. CNTO 530 moreover supported the sustained expansion of a bone marrow–resident KitnegCD71highTer119neg progenitor pool. Based on these distinct erythropoietic and EPOR signaling properties, CNTO 530 holds excellent promise as a new EPO mimetic.

Regulation of LMO2 mRNA and protein expression in erythroid differentiation

new erythrocytes daily to replace the 1% of old cells removed from the circulation. Red cell production increases several-fold after blood loss or hemolysis but does not overshoot because it is so tightly regulated. This regulation involves three basic processes in erythroid progenitor and precursor cell populations: proliferation, differentiation, and survival. Although they act in concert from the earliest cell committed to erythropoiesis, the burst-forming unit-erythroid (BFU-E), through the last cell division of erythroblasts, each of these three processes can be regulated independently of another. After blood loss or hemolysis, stem cell factor (SCF/Kit-ligand) and glucocorticoids increase proliferation of cells in the BFU-E to colonyforming unit-erythroid (CFU-E) stages. 1 However, SCF and glucocorticoids have little effect on survival or differentiation of these erythroid progenitor cells. Erythopoietin (EPO), the major physiologic regulator of erythropoiesis, is regulated by hypoxia at the level of its transcription. 2 EPO promotes the survival of erythroid cells in the CFU-E through basophilic erythroblast stages without affecting their proliferation or differentiation. Contact with macrophages in erythroblastic islands regulates proliferation of these EPO-dependent cells without affecting their survival or differentiation. 3

Current and Upcoming Erythropoiesis-Stimulating Agents, Iron Products, and Other Novel Anemia Medications

Treatment for anemia has come a long way in the last 20 years since the first recombinant human erythropoietins were licensed for the management of anemia in chronic kidney disease. The first-generation epoetins were succeeded by the development and production of a longer-acting erythropoietin (EPO) analog, darbepoetin alpha, which allowed less frequent dosing, usually once weekly or once every 2 weeks. More recently, another EPO-related molecule has been manufactured called Continuous Erythropoietin Receptor Activator with an even longer half-life, and although for patent reasons this is not available in the United States, it is licensed and is already being used in Europe. Other molecules are in development or are becoming licensed in Europe, including biosimilar epoetin products/follow-on biologics, and elsewhere in the world there are cheaper-production "copy" epoetins. Indeed, it is estimated that up to 80 such products may be sold in countries with less stringent regulatory control of pharmaceutical products. Two different biosimilar epoetins have already been licensed in Europe, one under 2 different brand names and one under 3 different brand names, and others may follow. Hematide is a synthetic peptide-based EPO receptor agonist that, interestingly, has no structural homology with EPO, and yet is still able to activate the EPO receptor and stimulate erythropoiesis. This agent is currently in phase III clinical trials. Research continues for orally active antianemic therapies, and several strategies are being investigated, although none is imminently available. Two new intravenous iron preparations have recently been developed, one in the United States (Ferumoxytol; AMAG Pharmaceuticals, Inc., Cambridge, MA) and one recently licensed in Europe (ferric carboxymaltose [Ferinject; Vifor Pharma, Zurich, Switzerland]). In conclusion, the development of effective therapies for the treatment of anemia has been a highly active field, both scientifically and economically, over the last two decades.

Drug-induced immune hemolytic anemia

Drug-induced immune hemolytic anemia (DIIHA) is rare, and a specialized laboratory is often required to provide the optimal serological tests to confirm the diagnosis. The most common drugs associated with DIIHA and the hypotheses for the mechanisms thought to be involved have changed during the last few decades. The drugs most frequently associated with DIIHA at this time are cefotetan, ceftriaxone, and piperacillin. DIIHA is attributed most commonly to drug-dependent antibodies that can only be detected in the presence of drug (eg, cephalosporin antibodies). DIIHA can also be associated with drug-independent antibodies; such antibodies do not need drug to be present to obtain in vitro reactions (eg, fludarabine). In these latter cases, the drug affects the immune system, causing production of red cell (RBC) autoantibodies; the clinical and laboratory findings are identical to autoimmune hemolytic anemia (AIHA), other than the remission associated with discontinuing the drug. Some of the mechanisms involved in DIIHA are controversial. The most acceptable one involves drugs, like penicillin, that covalently bind to proteins (eg, RBC membrane proteins); RBCs become coated with drug in vivo and, a drug antibody (usually IgG) attaches to the drug-coated RBCs that are subsequently cleared by macrophages. The most controversial is the so-called immune complex mechanism, which has been revised to suggest that most drugs are capable of binding to RBC membrane proteins, but not covalently like penicillins. The combined membrane plus drug can create an immunogen; the antibodies formed can be IgM or IgG and often activate complement, leading to acute intravascular lysis and sometimes renal failure; fatalities are more common in this group. It is still unknown why and how some drugs induce RBC autoantibodies, sometimes causing AIHA.

Blood Transfusion: Quality and Safety Issues in Thalassemia, Basic Requirements and New Trends

Guidelines for minimizing risks from alloimmunization, other transfusion reactions, and infection risks are presented based on the Thalassemia International Federation (TIF) guidelines. Future developments including pretreatment of the red cell product that may reduce infection risks are discussed. The rationale for guidelines about the transfusion regime that optimizes the balance between over- and undertransfusing patients is discussed. The optimal approach is still debated and may vary with the patient population in question. Because of changes in the preparation of red cell products in recent years, there is variability in iron content of a ‘unit’ of blood. It is recommended that each center involved in the treatment of thalassemia knows the average hematocrit and volume of the blood product that they are using, as this impacts on response to chelation therapy.

An update on iron physiology

Iron is an essential micronutrient, as it is required for adequate erythropoietic function, oxidative metabolism and cellular immune responses. Although the absorption of dietary iron (1-2 mg/d) is regulated tightly, it is just balanced with losses. Therefore, internal turnover of iron is essential to meet the requirements for erythropoiesis (20-30 mg/d). Increased iron requirements, limited external supply, and increased blood loss may lead to iron deficiency (ID) and iron-deficiency anemia. Hepcidin, which is made primarily in hepatocytes in response to liver iron levels, inflammation, hypoxia and anemia, is the main iron regulatory hormone. Once secreted into the circulation, hepcidin binds ferroportin on enterocytes and macrophages, which triggers its internalization and lysosomal degradation. Thus, in chronic inflammation, the excess of hepcidin decreases iron absorption and prevents iron recycling, which results in hypoferremia and iron-restricted erythropoiesis, despite normal iron stores (functional ID), and anemia of chronic disease (ACD), which can evolve to ACD plus true ID (ACD + ID). In contrast, low hepcidin expression may lead to iron overload, and vice versa. Laboratory tests provide evidence of iron depletion in the body, or reflect iron-deficient red cell production. The appropriate combination of these laboratory tests help to establish a correct diagnosis of ID status and anemia.

Low red cell production may protect against severe anemia during a malaria infection—Insights from modeling

The malaria parasite causes lysis of red blood cells, resulting in anemia, a major cause of mortality and morbidity. Intuitively, one would expect the production of red blood cells to increase in order to compensate for this loss. However, it has been observed that this response is weaker than would be expected. Furthermore, iron supplementation for iron deficient children in malaria endemic regions can paradoxically adversely affect the clinical outcome of malaria infection. A possible explanation may lie in the preference that some malaria parasites show for infecting immature red blood cells (reticulocytes). In the presence of a parasite preference for immature red cells, a rise in red cell production can ‘fuel the fire’ of infection by increasing the availability of the parasite's preferred target cell. We present a mathematical model of red blood cell production and infection in order to explore this hypothesis. We assess the effect of varying the reticulocyte replacement rate and preference of the parasite for reticulocytes on four key outcome measures assessing anemia and parasitemia. For a given level of parasite preference for reticulocytes we uncover an optimal erythropoietic response which minimizes disease severity. Increasing red blood cell production much above this optimum confers no benefit to the patient, and in fact can increase the degree of anemia and parasitemia. These conclusions are consistent with epidemiological studies demonstrating that both iron deficiency and anemia are protective against severe malaria, whilst iron supplementation in malaria endemic regions is with an increased number of malaria related adverse effects. Thus, suppression of red blood cell production, rather than being an unfortunate side effect of inflammation, may be a host protective effect against severe malarial anemia.

Invited Commentary

The perioperative use of red cell or coagulation products in a critical care setting has been debated in the adult literature. The literature in pediatrics is comparatively scant but certainly merits attention. The study by Szekely and colleagues [1] concluded that the total amount of blood transfusion was independently associated with an increased risk for infection in pediatric patients undergoing congenital heart disease operations with the use of cardiopulmonary bypass. Platelet transfusion was associated with a lower incidence of nonvascular pulmonary complications.

Exogenous iron increases hemoglobin in β–thalassemic mice

Beta-thalassemia results from beta-globin gene mutations that lead to ineffective erythropoiesis, shortened red cell survival, and anemia. Patients with beta-thalassemia develop iron overload, despite which, hepcidin levels are low. This suggests that hepcidin regulation in beta-thalassemia is more sensitive to factors unrelated to iron state. Our preliminary data demonstrates that Hbb(th1/th1) mice, a model of beta-thalassemia intermedia, have lower bone marrow iron levels while levels in the liver and spleen are increased; the later account for the increased systemic iron burden in beta-thalassemia intermedia. We hypothesized that exogenous iron would improve anemia in beta-thalassemia intermedia despite systemic iron overload and further suppress hepcidin secondary to progressive expansion of erythroid precursors. We investigate parameters involved in red cell production, precursor apoptosis, parenchymal iron distribution, and hepcidin expression in iron treated Hbb(th1/th1) mice. Exogenous iron results in an expansion of erythroid precursors in the liver and spleen, leading to an increase in the number of red cells, reticulocytes, and hemoglobin production. A decrease in hepcidin expression is also observed. These findings demonstrate for the first time that iron results in expansion of extramedullary erythropoiesis, which improves anemia and suggests that expansion of extramedullary erythropoiesis itself results in hepcidin suppression in beta-thalassemia intermedia.

DYRK3 Dual-specificity Kinase Attenuates Erythropoiesis during Anemia

During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.

Stage-Restricted Non-Metabolic Function for Aconitase Enzymes in Erythropoietin Programming of Erythroid Growth and Differentiation in Vivo

Erythropoietin (EPO) is the main cytokine responsible for red blood cell production in the human marrow. Signal transduction through the EPO receptor consists of multiple limbs that promote the proliferation, differentiation, and survival of erythroid progenitors. Regulatory loops that modulate EPO signaling are not fully understood, but could prove critical in providing new therapeutic approaches to EPO-refractory anemias. One such poorly understood loop is the regulation of EPO-driven erythropoiesis by iron. Previously presented work has established that iron deprivation acts in a lineage specific manner to block erythroid development and inhibit the activity of aconitase, a Krebs cycle enzyme that interconverts citrate and isocitrate. In the current studies, the causal relationship between aconitase inhibition and erythroid blockade was characterized in normal donor-derived mobilized human primary hematopoietic CD34+ cells and in adult wild type C57BL/6 mice. We hypothesized that critical thresholds of aconitase activity are required for specific facets of EPO signaling during discrete phases of erythroid development. CD34+ cells in erythroid-promoting culture conditions (EPO and iron) were strongly growth inhibited in a dose-dependent manner when treated with fluoroacetate, a reversible aconitase inhibitor. Interestingly, there was no increase in overall cell death in the growth impaired cultures, providing the first evidence that growth and survival signals in primary erythroid precursors can be dissociated. Aconitase blockade also inhibited EPO-dependent erythroid maturation, with decreased glycophorin A upregulation, diminished CD34 downregulation, and a sharp decrease in globin chain protein levels after 5 days of culture. Biochemical analysis of known EPO targets showed no alterations in the phosphorylation status of STAT5, Akt and PKCalpha, effectively ruling out a role for the corresponding pathways in the impaired growth and differentiation. Moreover, intracellular ATP levels were unaffected by aconitase inhibition, and alterations in AMPK activation, an intracellular sensor of the ATP:AMP ratio, could not be detected. These results argue against energy starvation as the cause of the observed developmental defects. Cell cycle analysis using propidium iodide showed no evidence for phase-specific arrest, excluding standard checkpoint mechanisms. Delayed addition or washout of fluoroacetate at various time points during cultures identified the existence of aconitase-dependent and subsequent aconitase-independent phases of human erythroid development. To extend these studies to in vivo erythropoiesis, C57BL/6 mice underwent fluoroacetate (n=10) or saline (n=10) treatment with continuous infusion pumps at a drug dose of 4 mg/kg/day. In vivo blockade of aconitase over a two-week period resulted in an anemia characterized by a significant decrease in mean hemoglobin (11.6 vs. 14.7 g/dL, P<0.001), hematocrit (37.3 vs. 44%, P<0.001) and red cell number (7.49 vs. 9.32 × 1012 cells/liter, P<0.001). No thrombocytopenia or neutropenia was noted. Reticulocytes were diminished (6.6 vs. 7.9%, P<0.001), and mean serum EPO levels were 3 fold higher in the treated animals (533.2 vs. 172.3 pg/ml, P<0.001). Flow cytometric analysis of the marrow erythroid compartment consistently showed accumulation of cells at a Ter119-bright C71-intermediate stage (n=3 for each group). In summary, our data establish a new function for aconitase in EPO-driven erythropoiesis that is distinct from its metabolic role in cellular energy homeostasis. Sustained aconitase activity is required for the proliferation and maturation of erythroid precursors, but does not impact survival or specific cell cycle phases. Furthermore, this requirement for aconitase activity is stage specific and restricted to an early phase of EPO-dependent erythroid development. Taken together, these results suggest the existence of a new regulatory loop in which levels of aconitase activity modulate EPO-mediated growth and maturation. This novel checkpoint could provide new targets in the treatment of many disorders of red cell production.

In Vitro and in Vivo Abrogation of An Erythroid Iron Restriction Developmental Checkpoint by Isocitrate through a Novel Erythropoietin- Coupled Signaling Mechanism

Erythropoietin (Epo) signaling drives normal erythropoiesis by promoting the survival, proliferation and maturation of committed erythroid progenitor cells. Epo acts at an early stage in erythroid development, prior to the initiation of hemoglobin synthesis. Under conditions of iron restriction, erythroid progenitors become refractory to Epo, resulting in hypoplastic anemia. The resulting iron restriction checkpoint protects iron stores from depletion by preventing Epo-driven erythroid expansion and inappropriate iron utilization for hemoglobin synthesis. In addition to diminished body stores, defects in iron uptake or intracellular trafficking can also activate this checkpoint and contribute to Epo-refractory anemias, e.g. sideroblastic anemias. Our previous work using primary human hematopoietic cultures had implicated aconitase enzymes, which interconvert citrate and isocitrate, as critical regulators of the erythroid iron-restriction checkpoint. In those studies, supplying cells with isocitrate had completely abrogated the block in erythroid development caused by iron restriction. In the current studies, we examine the mechanism for isocitrate rescue of erythropoiesis in iron deprived human progenitors and determine the in vivo effects of isocitrate administration in mice with iron deficiency anemia. Initial experiments addressed whether the activity of isocitrate was due to its catabolism to yield ATP and succinyl CoA, a precursor of heme. Several independent findings argued against such a metabolic mechanism. Firstly, erythroid progenitors showed no changes in cellular [ATP] with iron deprivation −/+ isocitrate. Secondly, a bioactive analog of alpha-ketoglutarate, TaKG, failed to rescue erythropoiesis under iron deprivation. Thirdly, isocitrate promoted erythroid differentiation in progenitors with blockade in mitochondrial biogenesis, induced by chloramphenicol. In these last studies, isocitrate reversed chloramphenicol inhibition of glycophorin A and globin expression; exogenous hemin by contrast reversed only the inhibition of globin expression. The combination of isocitrate and hemin, however, showed strong synergy in the rescue of growth and globin expression in cholaramphenicol treated progenitors. Subsequent experiments tested the hypothesis that isocitrate functions as a second messenger in erythroid development. Accordingly, iron deprived erythroid progenitors exposed to a range of Epo levels (0.05–20 U/ml) underwent isocitrate treatment. Remarkably, isocitrate showed no rescue of iron deprived erythroid cultures with 0.05 U/ml Epo, a dose that still promotes erythroid differentiation in high iron cultures. Partial rescue was obtained with 0.2 U/ml Epo, and complete rescue with > 4.5 U/ml. Previous studies have suggested that Epo-mediated calcium signaling shows a strong dosage dependency, requiring relatively high doses of Epo to activate calcium influx. In human CD34+ progenitors exposed to 4.5 U/ml Epo for either 5 hours or 3 days, iron deprivation induced a drop in steady state intracellular calcium levels. Inclusion of isocitrate in the medium restored the intracellular calcium to the levels seen in the iron-replete cultures. Finally, the in vivo activity of isocitrate was assessed in a murine model of iron deficiency anemia. Strikingly, intraperitoneal injections of isocitrate (200 mg/kg/day for 5 days) significantly augmented the red cell counts in C57BL/6 weanlings subjected to a low iron diet: mean RBC of 9.3 ± 0.35 × 10e12 cells/liter for the isocitrate group compared to an average RBC of 7.4 ± 0.51 × 10e12 cells/liter for the saline control group (P=0.012, N = 6 for each group). Interestingly, this increase accompanied a parallel decrease in red cell mean corpuscular hemoglobin concentration. Taken together, our results suggest a role for isocitrate as a second messenger coupled to Epo signaling and potentially involved in intracellular calcium regulation. In vivo administration abrogates the iron restriction checkpoint on red cell production, as in ex vivo studies, but cannot drive hemoglobin synthesis in the face of limited iron stores. The ability of isocitrate to collaborate with hemin in overriding erythroid mitochondrial defects offers novel therapeutic avenues for sideroblastic anemias.