Search

Thalassaemia is a diverse group of genetic disorders with a worldwide distribution affecting globin chain synthesis. The pathogenesis of thalassaemia lies in the unbalanced globin chain production, leading to ineffective erythropoiesis, increased haemolysis, and deranged iron homoeostasis. The clinical phenotype shows heterogeneity, ranging from close to normal without complications to severe requiring lifelong transfusion support. Conservative treatment with transfusion and iron chelation has transformed the natural history of thalassaemia major into a chronic disease with a prolonged life expectancy, albeit with co-morbidities and substantial disease burden. Curative therapy with allogeneic haematopoietic stem cell transplantation is advocated for suitable patients. The understanding of the pathogenesis of the disease is guiding therapeutic advances. Novel agents have shown efficacy in improving anaemia and transfusion burden, and initial results from gene therapy approaches are promising. Despite scientific developments, worldwide inequality in the access of health resources is a major concern, because most patients live in underserved areas.

Rationale and Objectives Molecular studies have shown the changes in bone marrow fat in relation to altered hematopoiesis. This study aims to investigate the changes in the bone marrow fat in patients affected by β-thalassemia by using chemical shift-encoded (CSE)–MRI. Materials and Methods Twenty-three subjects, comprising of six healthy (17-31 years old) and 17 β-thalassemia subjects (19-39 years old), were scanned using a multiecho fast low angle shot sequence (0.94 × 0.94 × 3.00 mm3) and a stimulated echo acquisition mode sequence using 3T MRI. Bone marrow proton density fat fraction (PDFF) was quantified in the left femur of each subject. Regression and Bland-Altman analysis were used to analyze agreement between CSE-MRI and 1H-MRS. PDFF distribution was analyzed using Hartigan's dip test and the computed Wasserstein distances. Jonckheere-Terpstra trend analysis was performed to evaluate the effect of disease severity on PDFF distribution. Results An excellent agreement was found between PDFF measured using CSE-MRI with 1H-MRS (R2 = 0.91; bias =-1.41%). Healthy subjects showed left-skewed or bimodal PDFF distribution while β-thalassemia subjects showed bimodal, normal or right-skewed distribution. Jonckheere-Terpstra test shows that PDFF distribution was increasingly different from the norm as disease severity increased (TJT = 166.0, z = 3.806, p Conclusion CSE-MRI is a promising tool to demonstrate spatial changes and variability in marrow fat distribution, resulting from ineffective erythropoiesis.

This review captures the anabolic and stimulatory effects observed with inhibition of the transforming growth factor β superfamily in muscle, blood, and bone. New medicinal substances that rectify activin, myostatin, and growth differentiation factor 11 signaling give hope to the many whose lives are affected by deterioration of these tissues. The review first covers the origin, structure, and common pathway of activins, myostatin, and growth differentiation factor 11 along with the pharmacodynamics of the new class of molecules designed to oppose the activin receptor signaling pathway. Current terminology surrounding this new class of molecules is inconsistent and does not infer functionality. Adopting inhibitors of the activin receptor signaling pathway (IASPs) as a generic term is proposed because it encapsulates the molecular mechanisms along the pathway trajectory. To conclude, a pragmatic classification of IASPs is presented that integrates functionality and side effects based on the data available from animals and humans. This provides researchers and clinicians with a tool to tailor IASPs therapy according to the need of projects or patients and with respect to side effects.

Well-controlled iron levels are indispensable for health. Iron deficiency is the most common cause of anemia, whereas iron overload, either hereditary or secondary due to disorders of ineffective erythropoiesis, causes widespread organ failure. Bone is particularly sensitive to fluctuations in systemic iron levels as both iron deficiency and overload are associated with low bone mineral density and fragility. Recent studies have shown that not only iron itself, but also iron-regulatory proteins that are mutated in hereditary hemochromatosis can control bone mass. This review will summarize the current knowledge on the effects of iron on bone homeostasis and bone cell activities, and on the role of proteins that regulate iron homeostasis, i.e. hemochromatosis proteins and proteins of the bone morphogenetic protein pathway, on bone remodeling. As disorders of iron homeostasis are closely linked to bone fragility, deeper insights into common regulatory mechanisms may provide new opportunities to concurrently treat disorders affecting iron homeostasis and bone.

Anemia is the most common clinical manifestation of myelodysplastic syndrome (MDS), and most patients become red blood cell transfusion dependent. Defective erythropoiesis includes impaired terminal erythroid maturation. There are limited options for treatments of anemia in lower-risk MDS after failure of erythroid-stimulating agents. Luspatercept is an activin receptor type IIB fusion ligand trap novel agent. Luspatercept showed promising activity for treating anemia in patients with MDS with ring sideroblast subtypes. This article reviews the mechanism of impaired erythropoiesis in MDS. It summarizes clinical data with luspatercept and foresees how to best use this treatment in practice.

The clinical manifestations of Sickle Cell Disease (SCD) include episodes of vascular occlusion, chronic hemolytic anemia and frequent infections. GDF-15, a multifactorial cytokine, is a member of the transforming growth factor- superfamily. Expression of the GDF-15 gene in cardiomyocytes, vascular smooth muscle cells, and endothelial cells is strongly upregulated in response to oxidative stress, inflammation and tissue injury, while high levels of serum GDF-15 associate with ineffective erythropoiesis and may reflect a certain type of bone marrow stress or erythroblast apoptosis. In this context we aimed to evaluate GDF-15 levels in 89 patients with HbS/βthal at steady phase and in 20 apparently healthy individuals, and correlate with clinical features of the disease and markers of hemolysis, iron burden, inflammation, coagulation and endothelial dysfunction. We found that: GDF-15 levels were elevated in patients with HbS/βthal compared to controls (1980.7 ± 159.8 vs 665.4 ± 50.9 pg/mL, p

Beta thalassemias are a significant global health problem. Globin chain imbalance leads to a complex physiologic cascade of hemolytic anemia, ineffective erythropoiesis, and iron overload. Management of the broad spectrum of phenotypes requires the careful use of red blood transfusions, supportive care, monitoring, and management of iron overload. In this article, the authors discuss recommendations for monitoring of individuals with thalassemia, as well as ongoing preclinical and clinical trials of therapies targeting different aspects of thalassemia pathophysiology.

Thalassemia is a common inherited monogenic disease. It is characterized by chronic hemolysis, ineffective erythropoiesis (IE) and iron overload. Despite advances in transfusion practices and chelation therapy, still many limitations in delivering these standard therapies exist. Challenges of currently available standard care and advances in understanding the underlying pathophysiological mechanisms in thalassemia stimulated research towards development of novel therapeutic targets. Agents reducing IE as Jak 2 inhibitors and Activin II receptor traps are promising and are currently in clinical trials. Other approaches targeting iron dysregulation as mini-hepcidins, exogenous transferrin and erythroferrone inhibitors are in preclinical studies. Gene therapy, a rapidly evolving field, has exhibited remarkable progress in recent years. Studies have focused on β or γ-globin addition, over expression of endogenous γ-globin-activating transcription factors, silencing of γ-globin repressors and genome editing of β-globin mutations or γ-globin repressors. In this article we provide an overview of emerging recent trends in treatment of thalassemia targeting IE, iron dysregulation and novel curative treatments as gene therapy and gene editing.

Erythropoiesis is the predominant consumer of iron in humans and other vertebrates. By decreasing the transcription of the gene encoding the iron-regulatory hormone hepcidin, erythropoietic activity stimulates iron absorption, as well as the release of iron from recycling macrophages and from stores in hepatocytes. The main erythroid regulator of hepcidin is erythroferrone (ERFE), synthesized and secreted by erythroblasts in the marrow and extramedullary sites. The production of ERFE is induced by erythropoietin (EPO) and is also proportional to the total number of responsive erythroblasts. ERFE acts on hepatocytes to suppress the production of hepcidin, through an as yet unknown mechanism that involves the bone morphogenetic protein pathway. By suppressing hepcidin, ERFE facilitates iron delivery during stress erythropoiesis but also contributes to iron overload in anemias with ineffective erythropoiesis. Although most of these mechanisms have been defined in mouse models, studies to date indicate that the pathophysiology of ERFE is similar in humans. ERFE antagonists and mimics may prove useful for the prevention and treatment of iron disorders.

The metabolic changes controlling the step-wise differentiation of human stem and progenitor cells (HSPC) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase (IDH1) plays a central role in this process. IDH1 downregulation augmented mitochondrial oxidation of αKG and inhibited reticulocyte generation. Furthermore, IDH1-knockdown resulted in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis –– oxidized ascorbate increased mitochondrial superoxide and significantly exacerbated the abnormal erythroblast phenotype of IDH1-downregulated progenitors whereas vitamin C, scavenging reactive oxygen species and reprogramming mitochondrial metabolism, rescued erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.

Beta-thalassaemia causes defective haemoglobin synthesis leading to ineffective erythropoiesis, chronic haemolytic anaemia, and subsequent clinical complications. Blood transfusion and iron chelation allow long-term disease control, and haematopoietic stem cell transplantation offers a potential cure for some patients. Nonetheless, there are still many challenges in the management of beta-thalassaemia. The main treatment option for most patients is supportive care; furthermore, the long-term efficacy and safety of current therapeutic strategies are limited and adherence is suboptimal. An increasing understanding of the underlying molecular and cellular disease mechanisms plus an awareness of limitations of current management strategies are driving research into novel therapeutic options. Here we provide an overview of the current pathophysiology, clinical manifestations, and global burden of beta-thalassaemia. We reflect on what has been achieved to date, describe the challenges associated with currently available therapy, and discuss how these issues might be addressed by novel therapeutic approaches in development.

The hallmarks of thalassemias are ineffective erythropoiesis and peripheral hemolysis leading to a cascade of events responsible for several clinical complications. This pathophysiologic mechanism can be partially controlled by blood transfusions or by correction of the severity of ineffective erythropoiesis. Thalassemias include a spectrum of phenotypes. Two main groups can be clinically distinguished: transfusion-dependent (TDT) and non-transfusion-dependent (NTDT) thalassemia. Both conditions are characterized by several clinical complications along life; some are shared, whereas some have higher prevalence in one group over the other. The authors present the most common clinical complications in TDT and NTDT and their management.

Stress erythropoiesis (SE) is characterized by an imbalance in erythroid proliferation and differentiation under increased demands of erythrocyte generation and tissue oxygenation. β-thalassemia represents a chronic state of SE, called ineffective erythropoiesis (IE), exhibiting an expansion of erythroid-progenitor pool and deposition of alpha chains on erythrocyte membranes, causing cell death and anemia. Concurrently, there is a decrease in hepcidin expression and a subsequent state of iron overload. There are substantial investigative efforts to target increased iron absorption under IE, by genetic and pharmacological agents. There are also avenues for targeting cell-contact and signaling within erythroblastic-islands under SE, for therapeutic benefits.

Congenital dyserythropoietic anaemias (CDAs) are a group of rare haematological disorders characterized by ineffective erythropoiesis and dyserythropoiesis and reduced numbers of red cells, often with an abnormal morphology. Pathogenic defects in CDAN1 , C15ORF41 , SEC23B , KIF23 , KLF1 and GATA1 genes have been identified in CDAs patients. In this study, we described 13 unrelated Chinese CDAs patients and identified 21 mutations, including 5 novel mutations in CDAN1 gene, and 5 novel mutations in SEC23B gene. Additionally, we predicted the molecular consequence of these missense mutations with Polymorphism Phenotyping v2 (Polyphen), Sorting Intolerant From Tolerant (SIFT), MutPred ( http://mutpred1.mutdb.org /) and Protein Variation Effect Analyzer (Provean, http://provean.jcvi.org/seq_submit.php ) and analyzed the conservation of the mutated amino acid among proteins from several mammalian species.

Objectives: Thalassemias are characterized by ineffective erythropoiesis and hemolysis due to imbalanced production and precipitation of globin chains. Adenosine triphosphate (ATP) levels are reduced in thalassemic red blood cells (RBCs), despite increased energy demands to maintain RBC cell integrity. Mitapivat (AG-348) is an oral activator of RBC pyruvate kinase (PKR), an enzyme regulating ATP production via glycolysis. In mice, mitapivat increased ATP synthesis and survival of thalassemic RBCs. Mitapivat has been shown to increase hemoglobin (Hb) in patients (pts) with PK deficiency and sickle cell disease. Results of the 24-week (wk) core period of a phase 2 open-label trial of mitapivat in adults with α- or β-non–transfusion-dependent thalassemia (NTDT; NCT03692052) are reported here. Material and methods: Key inclusion criteria: β-thalassemia ±α-globin gene mutations, Hb E β-thalassemia, or α-thalassemia (Hb H disease); Hb ≤10.0 g/dL; ≤5 RBC units transfused in prior 24 wks and none in prior 8 wks. Pts received mitapivat 50 mg orally twice daily (BID), then increased to 100 mg BID at Wk 6 visit, if tolerated. Primary endpoint was % pts who achieved a Hb response (increase ≥1.0 g/dL from baseline [BL] at any time between Wks 4–12, inclusive). Secondary endpoints included sustained Hb response (≥2 Hb increases of ≥1.0 g/dL vs BL during Wks 12–24 after achieving a primary Hb response), time to first ≥1.0 g/dL Hb increase, markers of hemolysis and erythropoiesis, and safety. Results: Of 20 pts (15 female) who received mitapivat, the mean age (SD) was 45.3 (11.8) yrs (range 29–67); mean (SD) BL Hb was 7.9 (1.4) g/dL (range 5.1–9.8). At Wk 6, all pts were escalated to 100 mg BID. Nineteen pts (95.0%) completed the core period, one discontinued due to adverse event (AE);17 pts continued to the extension period. Sixteen pts (80.0%, 90% CI 59.9, 92.9; p < 0.0001) achieved a Hb response, including 5/5 pts with α-thalassemia and 11/15 pts with β-thalassemia. Mean (SD) time to first Hb increase >1.0 g/dL was 4.5 (3.2) wks, and mean Hb (SD) increase from BL during Wks 12–24 was 1.3 (0.6) g/dL. A sustained response was achieved in 65% of pts, including 5/5 pts with α-thalassemia and 8/15 pts with β-thalassemia. Hb increases occurred across the spectrum of BL levels. Directional improvements in markers of erythropoiesis and hemolysis were also observed. The safety profile was consistent with previously published mitapivat studies. There was one serious grade 3 unrelated AE of renal impairment, leading to treatment discontinuation. There were three non-serious grade 3 AEs leading to dose reduction: 1 resulted in a permanent dose reduction to 50 mg BID, and 2 were successfully rechallenged at 100 mg BID. The most common non-serious AEs occurring in ≥25% of pts were initial insomnia (10/20), dizziness (6/20), and headache (5/20). Dose escalation to 100 mg BID was well tolerated and not associated with any increase in AEs. Twelve pts had DXA scans at BL and 6 months; no notable changes in bone mineral density were observed. Discussion: These results demonstrate that PKR activation with mitapivat was well tolerated and improved anemia, hemolysis, and ineffective erythropoiesis, and may represent a novel therapeutic approach for pts with α- or β-thalassemia. Conclusion: The study met its primary endpoint with 80% of pts achieving a Hb response.

Objectives Thalassemias are characterized by imbalanced globin-chain production resulting in excess α- or β-globin precipitation, hemolytic anemia, and ineffective erythropoiesis. Adenosine triphosphate (ATP) levels are reduced in thalassemic red blood cells (RBCs), despite increased energy demands. Mitapivat is an oral activator of RBC pyruvate kinase (PKR), a glycolytic enzyme that regulates ATP production. In a phase 2 study of patients (pts) with α- or β-non–transfusion-dependent thalassemia (NTDT), twice-daily (BID) dosing with mitapivat increased hemoglobin (Hb) levels by ≥1.0 g/dL in 80% of pts, supporting the broadening of mitapivat's development in thalassemia. The study designs of ENERGIZE (2021-000211-23) and ENERGIZE-T (2021-000212-34), two phase 3 trials to assess the efficacy and safety of mitapivat in adults with α- or β-NTDT or transfusion-dependent thalassemia (TDT), respectively, are reported here. Methods Both studies are phase 3, multicenter, randomized, double-blind, placebo-controlled trials. In ENERGIZE, approximately 171 eligible adults with NTDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 24 weeks (wks). Upon completion, eligible pts can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (β-thalassemia ±α-globin mutations, Hb E β-thalassemia, or α-thalassemia [Hb H disease]), Hb concentration ≤10.0 g/dL, and NTDT defined as ≤5 RBC units during the 24-wk period before randomization and no RBC transfusion ≤8 wks prior. The primary endpoint is an Hb response defined as a ≥1.0 g/dL increase in average Hb concentration from Wk 12 through 24 compared with baseline (BL). Secondary endpoints include pt-reported outcomes, changes in Hb, markers of hemolysis and erythropoiesis, and safety. In ENERGIZE-T, approximately 240 eligible adults with TDT will be randomized (2:1) to receive 100 mg mitapivat BID or placebo for 48 wks. Upon completion, eligible pts can transition to a 5-year, open-label extension. Key inclusion criteria: documented diagnosis of thalassemia (same genotypes as detailed for the ENERGIZE study), and TDT defined as 6–20 RBC units transfused and no transfusion-free period ≥6 wks during the 24 wks before randomization. The primary endpoint is a transfusion reduction response, defined as a ≥50% reduction in transfused RBC units with a reduction of ≥2 units of transfused RBCs in any consecutive 12-wk period through Wk 48 compared with BL. Secondary endpoints include additional measures of transfusion burden, changes in iron markers, and safety. Results Not yet available. Discussion ENERGIZE and ENERGIZE-T are the first pivotal studies to assess a potential treatment across a broad spectrum of pts with thalassemia (ie, pts with TDT and NTDT; α- and β-thalassemias). Conclusions ENERGIZE and ENERGIZE-T will evaluate the efficacy and safety of mitapivat, a novel, first-in-class oral activator of PKR. Both studies will start enrollment in 2021.

Myelodysplastic syndromes are characterised by ineffective erythropoiesis. Luspatercept (ACE-536) is a novel fusion protein that blocks transforming growth factor beta (TGF β) superfamily inhibitors of erythropoiesis, giving rise to a promising new investigative therapy. We aimed to assess the safety and efficacy of luspatercept in patients with anaemia due to lower-risk myelodysplastic syndromes.In this phase 2, multicentre, open-label, dose-finding study (PACE-MDS), with long-term extension, eligible patients were aged 18 years or older, had International Prognostic Scoring System-defined low or intermediate 1 risk myelodysplastic syndromes or non-proliferative chronic myelomonocytic leukaemia (white blood cell count13 000/μL), and had anaemia with or without red blood cell transfusion support. Enrolled patients were classified as having low transfusion burden, defined as requiring less than 4 red blood cell units in the 8 weeks before treatment (and baseline haemoglobin10 g/dL), or high transfusion burden, defined as requiring 4 or more red blood cell units in the 8 weeks before treatment. Patients received luspatercept subcutaneously once every 21 days at dose concentrations ranging from 0·125 mg/kg to 1·75 mg/kg bodyweight for five doses (over a maximum of 12 weeks). Patients in the expansion cohort were treated with 1·0 mg/kg luspatercept; dose titration up to 1·75 mg/kg was allowed, and patients could be treated with luspatercept for a maximum of 5 years. Patients in the base study were assessed for response and safety after 12 weeks in order to be considered for enrolment into the extension study. The primary endpoint was the proportion of patients achieving modified International Working Group-defined haematological improvement-erythroid (HI-E), defined as a haemoglobin concentration increase of 1·5 g/dL or higher from baseline for 14 days or longer in low transfusion burden patients, and a reduction in red blood cell transfusion of 4 or more red blood cell units or a 50% or higher reduction in red blood cell units over 8 weeks versus pre-treatment transfusion burden in high transfusion burden patients. Patient data were subcategorised by: luspatercept dose concentrations (0·125-0·5 mg/kg vs 0·75-1·75 mg/kg); pre-study transfusion burden (high transfusion burden vs low transfusion burden, defined as ≥4 vs4 red blood cell units per 8 weeks); pre-study serum erythropoietin concentration (200 IU/L, 200-500 IU/L, and500 IU/L); presence of 15% or more ring sideroblasts; and presence of SF3B1 mutations. Efficacy analyses were carried out on the efficacy evaluable and intention-to-treat populations. This trial is currently ongoing. This study is registered with ClinicalTrials.gov, numbers NCT01749514 and NCT02268383.Between Jan 21, 2013, and Feb 12, 2015, 58 patients with myelodysplastic syndromes were enrolled in the 12 week base study at nine treatment centres in Germany; 27 patients were enrolled in the dose-escalation cohorts (0·125-1·75 mg/kg) and 31 patients in the expansion cohort (1·0-1·75 mg/kg). 32 (63% [95% CI 48-76]) of 51 patients receiving higher dose luspatercept concentrations (0·75-1·75 mg/kg) achieved HI-E versus two (22% [95% CI 3-60]) of nine receiving lower dose concentrations (0·125-0·5 mg/kg). Three treatment-related grade 3 adverse events occurred in one patient each: myalgia (one [2%]), increased blast cell count (one [2%]), and general physical health deterioration (one [2%]). Two of these treatment-related grade 3 adverse events were reversible serious grade 3 adverse events: one patient (2%) had myalgia and one patient (2%) had general physical health deterioration.Luspatercept was well tolerated and effective for the treatment of anaemia in lower-risk myelodysplastic syndromes and so could therefore provide a novel therapeutic approach for the treatment of anaemia associated with lower-risk myelodysplastic syndromes; further studies are ongoing.Acceleron Pharma.

Context: Standard treatment of anemia in lower-risk myelodysplastic syndromes (LR-MDS) is based on erythropoietic-stimulating agents (ESAs), but some patients are primary refractory (PR) or lose response (secondary refractory, SR). High serum erythropoietin (sEPO) is the main negative predictor, and treatment of ESA-resistant anemia is challenging, so more predictors are needed. EPO promotes differentiation of committed erythroid cells (COMe) into erythroid precursors (EP), constituted by early (EEP) and late (LEP) precursors. Flow cytometry (FC) can characterize COMe and EP. RED score is an FC parameter that assesses dyserythropoiesis. Objective: We aimed to correlate COMe, EP, and RED score with response to ESAs, WHO category, and sEPO in 44 anemic LR-MDS patients. Design: We evaluated 10 negative controls, 17 PR patients, 17 SR patients, and 10 long responders (LR, response duration >24 months). Fifty-nine percent had sEPO 500 mU/mL(H-EPO); 23% had single lineage dysplasia (MDS-SLD), 34% multilineage dysplasia (MDS-MLD), 23% ring sideroblasts (MDS-RS), and 20% deletion of 5q (MDS-5q-). FC analysis was performed on bone marrow aspirates for all subgroups at diagnosis and within SR subgroup at loss of response to ESA (SRII). We used anti-CD34, CD36, CD105, CD117, CD33, CD71, and CD45 antibodies to identify COMe, total erythroid cells (TOTe), and EP. Coefficients of variance of CD71 and CD36 were used to calculate RED score. Results: TOTe and COMe were increased in LR-MDS compared to controls (p=0.013 and p=0.04, respectively), with the exception of MDS-5q- cases. In the PR subgroup, LEP were increased and COMe were reduced compared to LR. In the SRII group, COMe were lower than at diagnosis (p=0.039). RED score was higher in PR compared to responders (p=0.011). MDS-5q- cases showed reduced TOTe (p=0.047), while MDS-RS cases had higher LEP and RED score (p=0.008 and p=0.012, respectively). Finally, LEP were increased in the H-EPO group compared to L-EPO (p=0.023). Conclusions: These data suggest that COMe seem to be positively associated with ESA response, whereas higher LEP and RED score are negatively associated and also correlate with MDS-RS and H-EPO. Finally, MDS-5q- cases displayed fewer TOTe and COMe, suggesting premature apoptosis of erythroid precursors.

Rpl11 haploinsufficient mice develop a macrocytic anemia similar to patients with DBA. Here, we fully characterize this model from clinical and pathophysiological perspectives. Early erythroid precursors have increased heme content and high cytoplasmic reactive oxygen species, impairing erythroid differentiation at the colony-forming unit-erythroid (CFU-E)/proerythroblast stage and subsequently. Using single-cell analyses that link a cell's surface protein expression to its total transcriptome and unbiased analyses, we found GATA1, GATA1 target gene, and mitotic spindle pathway gene transcription were the pathways that decreased the most. Expression of ribosome protein and globin genes was amplified. These changes, as well as the other transcriptional changes that were identified, closely resemble findings in mice that lack the heme export protein FLVCR and, thus, suggest that heme excess and toxicity are the primary drivers of the macrocytic anemia. Consistent with this, treating Rpl11 haploinsufficient mice with corticosteroids increased the numbers of earliest erythroblasts but failed to overcome heme toxicity and improve the anemia. Rpl11 haploinsufficient mice uniquely upregulated mitochondrial genes, p53 and CDKN1A pathway genes, and DNA damage checkpoint genes, which should contribute further to erythroid marrow failure. Together our data establish Rpl11 haploinsufficient mice as an excellent model of DBA that can be used to study DBA pathogenesis and test novel therapies.

Hemoglobinopathies, thalassemia and sickle cell disease are among the most frequent monogenic diseases in the world. Transfusion has improved dramatically their prognosis, but provokes iron overload, which induces multiple organ damages. Iron overload is related to accumulation of iron released from hemolysis and transfused red cell, but also, in thalassemic patients, secondary to ineffective erythropoiesis, which increases intestinal iron absorption via decreased hepcidin production. Transfusion-related cardiac iron overload remains a main cause of death in thalassemia in well-resourced countries, and is responsible for severe hepatic damages in sickle cell disease. Regular monitoring by Magnetic Resonance Imaging (MRI) using myocardial T2* (ms) and Liver Iron Content (LIC) (mg of iron/g dry weight) are now standards of care in chronically transfused patients. Serum ferritin level measurements and record of the total number of transfused erythrocyte concentrates are also helpful tools. Three iron chelators are currently available, deferoxamine, which must be injected subcutaneously or intravenously, and two oral chelators, deferiprone and deferasirox. We will review the main characteristics of these drugs and their indications.

New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.

Background: We recently demonstrated that the reduced iron usage due to suppressed erythropoiesis is a major cause of the systemic iron overload in CBS knockout (CBS-/-) mice. However, the molecular mechanisms by which CBS deficiency suppresses erythropoiesis are unknown. Methods: Here, we therefore examined the changes in the ratio of granulocyte/erythroid cells, iron content, the expression of erythropoietin (EPO), iron-metabolism proteins including transferrin receptor 1 (TfR1), ferroportin 1 (Fpn1) and iron regulatory proteins (IRPs), and other molecules that affect iron-metabolism, including hypoxia inducible factor-2 subunit α (HIF-2α), nuclear factor erythroid 2-related factor-2 (Nrf2), and Kelch-like ECH-associated protein 1 (Keap1) in the blood, bone marrow, bone marrow-derived macrophages, liver or spleen of CBS-/- (homozygous), CBS+/- (heterozygous) and CBS+/+ (Wild Type) mice. Findings & Interpretation: Our findings demonstrated that CBS deficiency suppressed erythropoiesis by disrupting iron supply mainly via EPO/IRP/TfR1, Hepcidin/Fpn1, HIF-2α/EPO/Fpn1 and Keap1/Nrf2/Fpn1 pathways in macrophage or erythroid progenitors of rats. Funding: Supported by the National Natural Science Foundation of China (NSFC31571195), and the Competitive Earmarked Grants of the Hong Kong Research Grants Council (GRF14111815, GRF 14167817, GRF14107616). Declaration of Interest: The authors have no financial or competing interests to disclose. Ethical Approval: All animal care and experimental protocols were performed according to the Animal Management Rules of the Ministry of Health of China, and approved by the Animal Ethics Committees of Fudan University (NDFC31271132) and The Chinese University of Hong Kong (GRF14111815).

Growth differentiation factor-15 (GDF-15) is a member of TGF-β superfamily. Among hematopoietic cells, this factor is mainly produced by erythroid series and is recently considered a biomarker of ineffective erythropoiesis (IE). Whether IE induces enhanced GDF-15 expression or is prompted by it, has remained elusive. In this study we investigated how high levels of GDF-15 contribute to IE-associated erythroid dysplasia. We assessed mRNA levels of GDF-15 during erythroid maturation as well as in patients with IE using qRT-PCR. Later, the erythroid colony-forming capacity of GDF-15-treated hematopoietic stem cells (HSCs) was evaluated by CFC assay. Any effect of elevated levels of GDF-15 on erythroid maturation was ultimately examined by expression analysis of erythroid-associated transcription factors and flow cytometry analysis of CD235a expression. GDF-15 mRNA expression increased during erythroid differentiation and also in β-thalassemia and MDS patients which was directly correlated with erythropoiesis severity. Treating the cells with high GDF-15 concentration (50 ng/ml) resulted in an approximate 30% decline in the capacity of erythroid colony formation of HSCs and CD235a positive cells. Additionally, erythroid-specific transcription factors showed significant down-regulation in the early stages of erythroid differentiation. According to the expression level of GDF-15 and the role it plays in the erythroid system, high-levels of this factor could be an auto-modulatory mechanism to control the excessive production of erythroid cells.

Systemic iron overload (SIO) is a common challenge in patients with hematological diseases and develops as a result of ineffective erythropoiesis, multiple red blood cell (RBC) transfusions and disease-specific therapies. Iron homeostasis is tightly regulated as there is no physiological pathway to excrete iron from the body. Excess iron is, therefore, stored in tissues like liver, heart and bone marrow and can lead to progressive organ damage. The presence of free iron in the form of non-transferrin bound iron (NTBI) is especially detrimental. Reactive oxygen species can also cause stromal damage in the bone marrow and promote leukemic cell growth in vitro. In acute leukemias and myelodysplastic syndromes outcome is worse in patients with SIO compared to patients without. Especially in patients undergoing allogeneic HSCT presence of NTBI before or during transplant has been shown to negatively affect non-relapse mortality and overall survival. Although the mechanisms, of how these effects are mediated by SIO are not very well understood monitoring of iron status by serum markers and imaging techniques is, therefore, mandatory especially in these patients. Whether peri-interventional iron chelation may improve outcome of these patients is part of current clinical research.

Background: Iron overload, resulting from RBC transfusions or ineffective erythropoiesis, is associated with significant morbidity in MDS. Aims: To assess changes from baseline in serum ferritin (SF) and iron chelation therapy (ICT) use in the MEDALIST trial patients ( NCT02631070 ). Methods: Patients (IPSS-R-defined lower-risk MDS with RS; ≥18 years; unsatisfactory response to or ineligible for EPO-based therapy; required regular RBC transfusions) had luspatercept (1.0 mg/kg, titration up to 1.75 mg/kg) or placebo administered subcutaneously every 3 weeks. Results: Of 153 luspatercept-treated patients, 87 (56.9%) had low and 66 (43.1%) high baseline RBC transfusion burden (LTB and HTB; Conclusion: Luspatercept treatment resulted in SF decreases, particularly in HTB patients and those with high baseline SF who experienced significant RBC transfusion reduction. Overall, ICT use was reduced with luspatercept.

Aberrant cell cycle speed during hematopoiesis defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. We utilized a unique live-cell reporter of cell cycle speed using a Histone H2B-FT fusion protein containing the color-changing Fluorescent Timer (FT) protein, distinguishing faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence. In transgenic mice expressing this reporter, we characterized the stress erythropoietic response of spleen and bone marrow following phenylhydrazine-induced hemolytic anemia. Using flow cytometric sorting of erythroblast stages, we found that all stages divide more rapidly after the hemolytic insult, not only early progenitors as previously thought. We also observe that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. And the last nucleated cell stage, orthochromatophilic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate anemia. Mass spectrometry of sorted populations shows upregulation of cell cycle and metabolic pathway related genes. Using this system in in vitro human CD34+ primary cell culture, we assessed the erythropoietic response to known treatments of β-thalassemia and Diamond Blackfan Anemia, like Epo and dexamethasone. We found an increase in cell cycling rates with an increase in dexamethasone concentration but not that of Epo. These findings shed new light on the normal response to external stress, underscoring the possibility of precise quantification of cell cycle speed in animal models of anemia. This system can help in understanding stress hematopoiesis, allowing identification of cellular factors that facilitate response to external stress.

Patients with blood transfusion-dependent anemias develop transfusional iron overload (TIO), which may cause cardiosiderosis. In patients with an ineffective erythropoiesis, such as thalassemia major, common transfusion regimes aim at suppression of erythropoiesis and of enteral iron loading. Recent data suggest that maintaining residual, ineffective erythropoiesis may protect from cardiosiderosis. We investigated the common consequences of TIO, including cardiosiderosis, in a minipig model of iron overload with normal erythropoiesis. TIO was mimicked by long-term, weekly iron-dextran injections. Iron-dextran loading for around one year induced very high liver iron concentrations, but extrahepatic iron loading, and iron-induced toxicities were mild and did not include fibrosis. Iron deposits were primarily in reticuloendothelial cells, and parenchymal cardiac iron loading was mild. Compared to non-thalassemic patients with TIO, comparable cardiosiderosis in minipigs required about 4-fold greater body iron loads. It is suggested that this resistance against extrahepatic iron loading and toxicity in minipigs may at least in part be explained by a protective effect of the normal erythropoiesis, and additionally by a larger total iron storage capacity of RES than in patients with TIO. Parenteral iron-dextran loading of minipigs is a promising and feasible large-animal model of iron overload, that may mimic TIO in non-thalassemic patients.

INTRODUCTION: Erythropoiesis stimulating agents (ESA) are generally the first line of treatment of anaemia in lower risk myelodysplastic syndrome (MDS) patients. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including flow cytometry RED score, serum GDF-15, and hepcidin levels. METHODS: Inclusion criteria were: ESA naive, IPSS low or intermediate-1 MDS with Hemoglobin (Hb) level< 10g/dL. Patients received epoetin zeta 40 000 IU/week and erythroid response (HI-E, IWG 2006 criteria) was assessed at W12. sEPO level, iron parameters and hepcidin, flow cytometry (FC) Ogata and Red Score (RS), GDF-15, and molecular analysis by NGS were determined at baseline. RESULTS: 70 patients were included. Median age was 78 years. There were 22 RCMD, 19 RCUD, 14 RARS, 4 RAEB-1, 6 CMML, 2 del 5q-, 3 unclassifiable, R-IPSS was 13 very low, 47 low, 9 intermediate and high. Twenty patients were transfusion-dependent. HI-E was 48% and median duration of response was 26 months. At baseline, non-responders had significantly higher FC Red Score, lower hepcidin/ferritin ratio, lower IPSS. In multivariate analysis, only RS>4 at T0 (p=0.05) and hepcidin/ferritin 2000 pg/ml and hepcidin/ferritin

Heart disease is the leading cause of mortality and one of the main causes of morbidity in β-thalassemia. Growth differentiation factor-15 (GDF-15), a member of the transforming growth factor-β superfamily, is a marker of ineffective erythropoiesis in several anemias.To determine GDF-15 levels in children and adolescents with TI and the relation to hemolysis, iron overload and cardiovascular complications.GDF-15 was measured in 35 TI patients without symptoms for heart disease and correlated to echocardiographic parameters and carotid intima media thickness (CIMT).GDF-15 levels were significantly higher in TI patients compared with controls (p0.001). Transfusion dependent patients had higher GDF-15 than non-transfusion dependent patients. TI patients with splenectomy, pulmonary hypertension risk, and heart disease had higher GDF-15 levels than those without. GDF-15 was lower among hydroxyurea-treated patients. Multiple linear regression analysis revealed that transfusion index (p=0.012), serum ferritin (p0.001), tricuspid regurgitant jet velocity (p0.001), ejection fraction (p=0.01) and CIMT (p=0.007) were independently related to GDF-15. According to ROC curve analysis, the cutoff value of GDF-15 at 1500 pg/mL could differentiate patients with and without heart disease.GDF-15 would identify TI patients at increased risk of pulmonary and cardiovascular complications as well as subclinical atherosclerosis.

International audience; Alpha-hemoglobin stabilizing protein (AHSP), described as a chaperone of alpha-hemoglobin (α-Hb), is synthesized at a high concentration in the erythroid precursors. AHSP specifically recognizes the G and H helices of α-Hb and forms a stable complex with free α-Hb until its association with the partner β-subunits. Unlike the free β-Hb which are soluble and form homologous tetramers, freshly synthesized α-Hb chains are highly unstable molecular species which precipitate and generate reactive oxygen species within the erythrocyte precursors of the bone marrow leading to apoptosis and ineffective erythropoiesis. AHSP protects the free α-Hb chains in maintaining it in the soluble state. In this review, we report data from the literature and our laboratory concerning the key role of AHSP in the biosynthesis of Hb and its possible involvement in some disorders of the red blood cell as well as the hemoglobinopathies and we discuss its use as a prognostic tool in thalassemia syndromes.

High expression of growth differentiation factor-15 (GDF-15) contributes to pathological iron overload in thalassemia. Sickle cell syndromes are characterized by increased levels of erythropoiesis, although the primary defect involves the destruction of mature erythrocytes.To determine serum GDF-15 in 35 children and adolescents with sickle cell disease (SCD) compared to 35 healthy controls and assess its relation to markers of hemolysis, iron overload and vascular complications.GDF-15 was measured and correlated to genotype, frequency of sickling crises, hydroxyurea therapy and serum ferritin.GDF-15 levels were increased in SCD patients whether sickle cell anemia or sickle β° thalassemia compared with controls (p0.001) with no significant difference between patients' groups. GDF-15 was significantly higher in patients who had serum ferritin ≥2500 μg/L, previous cerebral stroke, and splenectomy. GDF-15 was not significantly related to frequency of sickling crises, pulmonary hypertension, or hydroxyurea therapy. On regression analysis, transfusion index, lactate dehydrogenase and serum ferritin were independently related to GDF-15.Increased GDF-15 in SCD reflects the importance of ineffective erythropoiesis in the pathophysiology and severity of anemia in SCD. GDF-15 levels are related to hemolysis and iron overload and may provide utility for identifying patients at increased risk of thrombotic events.

Sideroblastic anemias (SAs) may be acquired or congenital and share the features of disrupted utilization of iron in the erythroblast, ineffective erythropoiesis, and variable systemic iron overload. Congenital forms can have associated syndromic features or be nonsyndromic, and many of them have mutations in genes encoding proteins involved in heme biosynthesis, iron-sulfur cluster biogenesis, or mitochondrial protein synthesis. The mechanism(s) for the acquired clonal SA is undefined and is under intense study. Precise diagnosis of these disorders rests on careful clinical and laboratory evaluation, including molecular analysis. Supportive treatments usually provide for a favorable prognosis and often for normal survival.

In this article, the authors discuss new approaches to treating iron overload diseases using hepcidin mimetics or by modulating endogenous hepcidin expression. In particular, the authors discuss lipid nanoparticle encapsulated siRNA and antisense oligonucleotide-mediated inhibition of TMPRSS6, an upstream regulator of hepcidin, and treatment with transferrin or hepcidin mimetics, including the recently described minihepcidins. In each case, in animal models of β-thalassemia, not only do the interventions affect iron absorption but they also act as disease-modifying agents that ameliorate the ineffective erythropoiesis.

Background High prevalence and unresponsiveness to erythropoiesis-stimulating agents are 2 major limitations to the treatment of cancer-related anemia (CRA). They are often related to the dis-regulation of iron metabolism regulated by hepcidin, but the regulatory pathway of hepcidin in CRA is poorly understood. Enhanced GDF-15 levels contribute to the cancer progression and metastasis, and also have been found to suppress hepcidin expression in anemia characterized by ineffective erythropoiesis. The pathophysiological mechanisms and the relationship of GDF-15 and hepcidin in CRA remain to be elucidated. Methods The concentrations of hepcidin and GDF-15 as well as the hematological and the iron parameters were determined in sera from 131 patients with cancer and 40 healthy controls. Results Serum GDF-15 levels were increased significantly in patients with the severe CRA, compared with the mild or no CRA patients and the controls. Increasing GDF-15 levels corresponded to decreasing hepcidin concentrations. A trend toward a correlation between high levels of GDF-15 and poor prognosis of cancer was also found. Elevation of GDF-15 concentrations suppressed hepcidin expression at high concentrations. Conclusions Our findings suggest that tumor progression results in increased GDF-15 secretion, which may down-regulate hepcidin expression, resulting in iron overload in cancer patients; this phenomenon has also been found in some patients with sideropenic anemia due to chronic blood loss.

Background & Aim Blood supply shortage is emerging as an issue worldwide, partially due to an aging and shrinking population which diminishes the healthy blood donor pool available to national blood programmes. To secure a safe and consistent supply of blood, various strategies have been explored. With development of various molecular tools, ex vivo generation of RBCs is emerging as a promising technology to alleviate the blood supply shortage issue. Although much progress has been made in the production of enucleated RBCs from stem cells, further research is required to optimize the scalability of enucleated RBC production in a culture system, with emphasis on advanced studies into proliferation and maturation stimuli for erythropoetic progenitors. Meanwhile, the contrasting stories of erythroid hyperplasic anemia and aplastic anemia presents an interesting topic to investigate the underlying mechanisms behind erythroid cell proliferation and maturation. Beta thalassemia is caused by a defective Beta globin gene that destabilizes alpha-tetrameric formation in erythroid cells and causes premature cell death. The continued stimulation of ineffective erythropoiesis leads to erythroid hyperplasic anemia. Conversely, aplastic anemia is a condition characterized by massive erythroid cell depletion in the marrow. While both conditions present anemia clinically, the cascade of events leading up to anemia is different for each case, resulting in two dissimilar marrow microenvironments. Thus, we hypothesized that through studying the differential expressional patterns of progenitors from each condition, we can determine the important mechanistic pathways underlying erythropoiesis and erythroid cell maturation. Methods, Results & Conclusion In our study, we sought to discover the differential gene expression between Beta Thalassemia and Aplastic Anemia. To that end, we obtained the transcriptomic data of erythroid progenitor cells of Beta Thalassemia and Aplastic anemia, from publicly available databases. We performed exploratory gene expressional analysis to identify differentially expressed genes in each condition. Pathway analysis was then conducted on each set of differentially expressed genes to identify significantly perturbed signalling nodes. These genes and pathways were compared against known literature on erythroid cell proliferation and maturation, to identify novel genes and pathways in our analysis. Future work will test the application of these genes in ex vivo RBC production.

Despite fluctuations in dietary iron intake and intermittent losses through bleeding, the plasma iron concentrations in humans remain stable at 10–30μM. While most of the iron entering blood plasma comes from recycling, appropriate amount of iron is absorbed from the diet to compensate for losses and maintain nontoxic amounts in stores. Plasma iron concentration and iron distribution are similarly regulated in laboratory rodents. The hepatic peptide hepcidin was identified as the systemic iron-regulatory hormone. In the efferent arc, hepcidin regulates intestinal iron absorption, plasma iron concentrations, and tissue iron distribution by inducing degradation of its receptor, the cellular iron exporter ferroportin. Ferroportin exports iron into plasma from absorptive enterocytes, from macrophages that recycle the iron of senescent erythrocytes, and from hepatocytes that store iron. In the more complex and less well understood afferent arc, hepatic hepcidin synthesis is transcriptionally regulated by extracellular and intracellular iron concentrations through a molecular complex of bone morphogenetic protein receptors and their iron-specific ligands, modulators and iron sensors. Through as yet undefined pathways, hepcidin is also homeostatically regulated by the iron requirements of erythroid precursors for hemoglobin synthesis. In accordance with the role of hepcidin-mediated iron redistribution in host defense, hepcidin production is regulated by inflammation as well. Increased hepcidin concentrations in plasma are pathogenic in iron-restrictive anemias including anemias associated with inflammation, chronic kidney disease and some cancers. Hepcidin deficiency causes iron overload in hereditary hemochromatosis and ineffective erythropoiesis. Hepcidin, ferroportin and their regulators represent potential targets for the diagnosis and treatment of iron disorders and anemias. This article is part of a Special Issue entitled: Cell Biology of Metals.

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.

Anaemia is prevalent in patients with myelodysplastic syndromes (MDS), and most patients with MDS receive regular red blood cell transfusions, which can lead to iron overload. Some patients may already have iron overload before transfusions begin, as a result of ineffective erythropoiesis. Iron overload has been linked to hepatic, cardiac, and endocrine dysfunction, although its exact contribution to cardiac failure and other complications is difficult to determine due to the advanced age of MDS patients and the prevalence of comorbidities. In addition, in patients with lower-risk MDS, a high serum ferritin level has been associated with an increased risk of leukaemic evolution independent of other prognostic factors, possibly related in part to the accumulation of free iron radicals. Finally, iron overload has been associated with poorer outcome after allogeneic stem cell transplantation and possibly with increased risk of infection. Thus, iron overload may negatively influence survival in patients with MDS, especially those with lower-risk disease. Iron chelation therapy may be beneficial in these patients.

Aberrant promoter methylation may contribute to the hematopoietic disturbances in myelodysplastic syndromes (MDS). To explore a possible mechanism, we therefore analyzed expression of DNA methyltransferase (DNMT) subtypes kinetics and aberrant promoter methylation of key regulatory genes during MDS hematopoiesis. An in vitro model of MDS lineage-specific hematopoiesis was generated by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low-risk: RA/n=6, RARS/n=3; high-risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Promoter methylation analysis of key genes involved in the control of apoptosis (p73, survivin, DAPK), DNA-repair (hMLH1), differentiation (RARb, WT1) and cell cycle control (p14, p15, p16, CHK2) was performed by methylation specific PCR of bisulfite-treated genomic DNA. Expression of DNMT1, DNMT3a and DNMT3b was analyzed and correlated with gene promoter methylation for each lineage at different time points. DNMT expression (all isoforms) was increased during thrombopoiesis whereas elevated DNMT1 level were seen during erythropoiesis. Associations between aberrant promoter methylation and DNMT expression were found in high-risk MDS for all lineages and during erythropoiesis. Hypermethylation of p15, p16, p73, survivin, CHK2, RARb and DAPK were associated with elevated DNMT isoform expression. No general overexpression of DNMT subtype was detected during MDS hematopoiesis. However a negative association of DNMT3a and 3b expression with MDS disease risk (IPSS) could be observed. Our data indicate that all mammalian DNMT isoforms may be involved in the aberrantly methylated phenotype in MDS but seem also to be essential for the differentiation of normal hematopoietic stem cells. In particular elevated DNMT1 expression may in particular contribute to ineffective erythropoiesis in MDS.

Objective We aimed to create a molecular assay to monitor erythroid (red blood cell [RBC]) engraftment in any patient following allogeneic hematopoietic stem cell transplantation, independent of disease-specific mutations. Materials and Methods We identified 10 common single nucleotide polymorphisms (SNPs), expressed by genes encoding RBC antigens and structural proteins. These SNPs were polymerase chain reaction–amplified from total RNA extracted from peripheral blood, which contains nucleated erythroid progenitors. Mixing studies validated that each SNP can quantitatively measure donor/recipient DNA and RNA. Results We directly genotyped 23 patients who underwent hematopoietic stem cell transplantation and their human leukocyte antigen–matched donors and found a median of three informative SNPs (i.e., discordant between donor and recipient) per pair. By using the informative RBC SNPs to quantify donor-derived RBC transcripts, we compared rates of RBC engraftment in 13 patients with hemoglobinopathies vs donor mononuclear cell (white blood cell [WBC]) engraftment. Consistent with known ineffective erythropoiesis associated with hemoglobinopathies, we detected up to threefold greater RBC-specific compared to overall WBC engraftment in five of eight patients who were mixed chimeras by transplant day 30. The remaining three of eight who received ABH-incompatible grafts, demonstrated at least 0.5-fold lower RBC compared to WBC engraftment that was related to persistence of host-derived anti-isohemagglutinin antibodies. Conclusion This RNA-based assay can be used to monitor RBC-specific engraftment regardless of a patient's specific hemoglobin mutation or even diagnosis. We propose that panels of expressed SNPs informative for other cell lineages can be created to comprehensively assess the impact of novel stem cell–based therapies on lineage-specific engraftment.

Ineffective hematopoiesis, with premature death of marrow myeloid precursors, is a hallmark of myelodysplastic syndromes (MDS), with the apparent paradox of peripheral cytopenia associated with hypercellular bone marrow (BM). Excessive apoptosis appears relevant especially in low-risk MDS. Apoptosis, triggered by the BM microenvironment and/or intrinsic cellular defects, is regulated at different levels by numerous factors, such as oncogenes and their protein products, hematopoietic growth factors, immunologic factors, cell-cell or cellstromal interactions, critical adhesion receptors, and various cytokines. Deregulation of both the intrinsic and the extrinsic pathways have been reported in MDS cells. Many studies provide evidence that the activation of the Fas/Fas-ligand system might represent an important pathogenetic mechanism. Recently, it has been demonstrated that the erythroid apoptosis of low-risk MDS is initiated at a very early stage of stem cells and associated with mitochondrial dysfunction. There is a constitutive triggering to apoptosis via cytochrome C release from the mitochondrial intermembrane space, with subsequent activation of effector caspases and increased sensitivity to death ligands triggering the extrinsic apoptotic pathway. The role of the mitochondrial pathway might be relevant especially in refractory anemia with ring sideroblasts, where abnormalities in mitochondrial ferritin expression might directly influence iron homeostasis and contribute to alter the balance between cell growth and death. The pathogenesis of refractory anemia without ring sideroblasts seems to be more heterogeneous, with the involvement of alternative mechanisms, including T-cell–mediated BM failure. Elucidation of these pathogenetic mechanisms might lead to the development of novel therapeutic strategies.

Beta-thalassaemia is an inherited disease caused by defective synthesis of the beta-globin chain of haemoglobin, leading to an imbalance in globin chains. Excess alpha-globin chains precipitate in erythroid progenitor cells resulting in cell death, ineffective erythropoiesis and severe anaemia. Decreased alpha-globin synthesis leads to milder symptoms, exemplified by individuals who co-inherit alpha-thalassaemia and beta-thalassaemia. In this study, we set out to investigate whether co-inheritance of alpha- and beta-thalassaemia in mice leads to reduced anaemia. Heterozygous murine beta-globin knockout (KO) mice (beta+/-) which display severe anaemia were mated with heterozygous alpha-globin KO mice (alpha++/--). The resulting progeny were genotyped and classed as wild-type WT (alpha++/++;beta+/+), heterozygous alpha-KO (alpha++/--;beta+/+), heterozygous beta-KO (alpha++/++;beta+/-) or double heterozygous (DH) alpha-KO/beta-KO (alpha++/--;beta+/-). Mice were bled and full blood examinations (FBE) performed. FBE results for heterozygous beta-KO mice (beta+/-) showed marked reductions in haemoglobin and haematocrit levels and significant increases in red cell distribution widths and reticulocyte counts compared to WT mice. In contrast, FBE results for DH alpha-KO/beta-KO mice showed near normal red blood cell indices. These results indicate that reduction of alpha-globin expression leads to correction of the globin chain imbalance in beta-thalassaemic mice and therefore an improved phenotype. The analysis of DH alpha-KO/beta-KO mice leads to the following conclusions: (1) co-inheritance of alpha- and beta-thalassaemia in mice improves the thalassaemic phenotype, identical to the situation in humans; (2) the heterozygous murine beta-globin KO mouse model is a suitable in vivo model to test for therapeutic knockdown of alpha-globin.

Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of glycolysis causing chronic non-spherocytic haemolytic anaemia. The disease is transmitted as an autosomal recessive trait, clinical symptoms usually occurring in compound heterozygotes for two mutant alleles and in homozygotes. The severity of haemolysis is highly variable, ranging from very mild or fully compensated forms to life-threatening neonatal anaemia necessitating exchange transfusions. Erythrocyte PK is synthesised under the control of the PK-LR gene located on chromosome 1. One hundred eighty different mutations in PK-LR gene, mostly missense, have been so far reported associated to PK deficiency. First attempts to delineate the genotype-phenotype association were mainly based on the analysis of the enzyme's three-dimensional structure and the observation of the few homozygous patients. More recently, the comparison of the recombinant mutants of human red cell PK with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined. However, the clinical manifestations of red cell enzyme defects are not merely dependent on the molecular properties of the mutant protein but rather reflect the complex interactions of additional factors, including genetic background, concomitant functional polymorphisms of other enzymes, posttranslational or epigenetic modifications, ineffective erythropoiesis and differences in splenic function.

In myelodysplasia (MDS) the precise mechanism of ineffective erythropoiesis is not fully elucidated, but it is suggested that apoptosis may contribute to this process. We performed TdT-mediated dUTP-nick end labelling (TUNEL) staining of paraffin embedded bone marrow specimens to assess the amount of apoptotic cells in 21 MDS patients (7 RA, 3 RARS, 5 RAEB, 3 RAEB-T, 3 CMML) and five normal controls. In 10 MDS patients the TUNEL assay was performed in combination with immunostaining for Glycophorin-A (GpA) to determine apoptosis in the maturing erythroid compartment. To assess the proliferation of the bone marrow cells the expression of Ki-67 antigen was used as a marker. The mean apoptotic index (Al) in MDS patients was not increased (2.3 +/- 3.0% in MDS versus 4.8 +/- 1.2% in normal controls (P

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in alpha- and beta-globin hemizygous mice, lacking the two adult tandemly organized genes from either the alpha- or beta-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, beta-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for alpha-globin hemizygous mice. In contrast, however, beta-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of beta-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free alpha-chain than beta-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the beta-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in beta-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units approximately 100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units approximately 7-fold). This early compensatory mechanism was less pronounced in alpha-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for alpha- and beta-thalassemia.

The coinheritance of beta-thalassemia major with the genotype of Hb H disease is extremely rare, with few reported cases. We investigated the hematological, biochemical, biosynthetic, molecular and pathophysiological parameters to evaluate a rare male patient with this compound syndrome. The patient was studied at first diagnosis during hospitalization at 50 years of age and subsequently followed up for more than a year. Examinations included full hematological, biochemical, biosynthetic, molecular, pathophysiological and clinical parameters. Besides standard parameters, we additionally measured reticulocyte hemoglobin content (CHr), erythropoietin (Epo), soluble transferrin receptors (sTfR), oxygen pressure at 50% hemoglobin saturation (P50), 2,3-bisphosphoglycerate (2,3-BPG), total glutathione (GSHt), oxidized glutathione (GSSG), malonyldialdehyde (MDA), nontransferrin-bound iron (NTBI), vitamins A and E. The male patient was first hospitalized for a 2-day period at 50 years of age, following the finding of marked anemia (hematocrit 20%) during a blood test to investigate the cause of fatigue in the absence of weight-loss or other notable symptomatology. He had never been transfused, maintaining Hb 85-95 g/l. Definitive diagnosis was achieved through DNA studies, which showed coexistence of beta-thalassemia major (IVSI-6 T > C/IVSI-I G > A) with Hb H disease (-alpha(3.7)/-(Med)). Alpha/non-alpha globin chain biosynthesis was completely balanced. Parameters demonstrated a well-compensated anemia with ineffective erythropoiesis and oxidative stress, which was ameliorated following splenectomy. In conclusion, this case is a remarkable example that the coinheritance of severe forms of beta-thalassemia and alpha-thalassemia interact in a "synergistic" manner to almost complete balance the symptoms of classic thalassemia syndromes.

Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory process characterized by severe derangement of gas exchange in the advanced stages of disease. However, erythrocytosis is infrequent in IPF. The aim of this study was to investigate the potential relation between the blunted erythropoietic response and the chronic inflammation.Nine patients (6 men and 3 women) with IPF and profound hypoxemia (PO(2)65 mm Hg) and 34 sex- and age-matched healthy volunteers participated in the study.We evaluated the hematologic parameters, serum erythropoietin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 levels. We also studied the development of burst-forming unit-erythroid (BFU-E)-derived colonies in semisolid methylcellulose cultures in blood samples from all patients.Hemoglobin and serum erythropoietin levels were almost comparable between the two studied groups. On the contrary, serum TNF-alpha, IL-6, and IL-8 values were significantly higher in patients with IPF (p0.05, p0.01, and p0.001, respectively). IPF sera induced a significant growth inhibition of erythroid bursts arising from mononuclear cells of either patients or control subjects compared with heat-inactivated AB serum (p0.05 and p0.01, respectively). Moreover, there was an apparent increment in the number of BFU-E colonies when patients' mononuclear cells were cultured in comparison with those of healthy subjects (p0.05).Our findings suggest that in IPF there is an increased number of primitive erythroid progenitors, which fail to proliferate and differentiate in vivo, suggesting a kind of ineffective erythropoiesis. As a consequence, hemoglobin levels do not rise in proportion to the severity of hypoxemia. Cytokines released from alveolar macrophages seem to have not only local but also systemic effects, since the serum of these patients directly suppressed erythropoiesis; however, the suboptimal erythropoietic response to hypoxia cannot be entirely attributed to this suppression. It is possible that several other factors interfere, synergistically or additively.

Iron transport in the plasma is carried out by transferrin, which donates iron to cells through its interaction with a specific membrane receptor, the transferrin receptor (TfR). A soluble form of the TfR (sTfR) has been identified in animal and human serum. Soluble TfR is a truncated monomer of tissue receptor, lacking its first 100 amino acids, which circulates in the form of a complex of transferrin and its receptor. The erythroblasts rather than reticulocytes are the main source of serum sTfR. Serum sTfR levels average 5.0+/-1.0 mg/l in normal subjects but the various commercial assays give disparate values because of the lack of an international standard. The most important determinant of sTfR levels appears to be marrow erythropoietic activity which can cause variations up to 8 times below and up to 20 times above average normal values. Soluble TfR levels are decreased in situations characterized by diminished erythropoietic activity, and are increased when erythropoiesis is stimulated by hemolysis or ineffective erythropoiesis. Measurements of sTfR are very helpful to investigate the pathophysiology of anemia, quantitatively evaluating the absolute rate of erythropoiesis and the adequacy of marrow proliferative capacity for any given degree of anemia, and to monitor the erythropoietic response to various forms of therapy, in particular allowing to predict response early when changes in hemoglobin are not yet apparent. Iron status also influences sTfR levels, which are considerably elevated in iron deficiency anemia but remain normal in the anemia of inflammation, and thus may be of considerable help in the differential diagnosis of microcytic anemia. This is particularly useful to identify concomitant iron deficiency in a patient with inflammation because ferritin values are then generally normal. Elevated sTfR levels are also the characteristic feature of functional iron deficiency, a situation defined by tissue iron deficiency despite adequate iron stores. The sTfR/ferritin ratio can thus describe iron availability over a wide range of iron stores. With the exception of chronic lymphocytic leukemia (CLL) and high-grade non-Hodgkin's lymphoma and possibly hepatocellular carcinoma, sTfR levels are not increased in patients with malignancies. We conclude that soluble TfR represents a valuable quantitative assay of marrow erythropoietic activity as well as a marker of tissue iron deficiency.

Objectives 1) To study the mechanisms of anemia, erythroid hyperplasia, and red blood cell (RBC) clearance in murine models of sickle cell disease (Sickle) and β-thalassemia (Th1/Th1); 2) To determine the contribution of the phospholipid scramblase enzyme to phosphatidylserine (PS) exposure and RBC death in Sickle and Th1/Th1 mice. Methods We used a combination of flow-cytometric analysis and assays for phospholipid remodeling to determine the extent and sites of erythroid hyperplasia, PS exposure, and cell death. Results 1) Sickle RBCs have a much shorter half-life than Th1/Th1 RBCs (0.8 days vs 11 days). A significant proportion of Th1/Th1 peripheral reticulocytes mature into erythrocytes, however, ∼fivefold fewer Sickle reticulocytes mature. While erythroid hyperplasia exists in both Sickle and Th1/Th1 mice, Th1/Th1 produce fourfold more RBCs than necessary to maintain steady state, while Sickle produce no excess RBCs. 2) 61% of Sickle and 34% of Th1/Th1 RBCs are scramblase + as measured by internalization assays of the fluorescent phospholipid NBD-PC. The majority of NBD-PC + RBCs are also annexin-V + , supporting a mechanistic link between scramblase activity and PS exposure. A proportion of both reticulocytes and older RBCs in Sickle and Th1/Th1 mice have active scramblase, and the degree of scramblase activation in these strains correlates with the propensity for RBC death. Conclusions Sickle and Th1/Th1 mice are both anemic, with significant erythroid hyperplasia. Th1/Th1 mice display ineffective erythropoiesis while Sickle mice show rapid peripheral destruction of RBCs. PS exposure and phospholipid scramblase activity serve as markers of RBCs with altered phospholipid asymmetry and greater propensity for cell death.