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Summary Increasing evidence suggests that free haem and iron exert vasculo‐toxic and pro‐inflammatory effects by activating endothelial and immune cells. In the present retrospective study, we compared serum samples from transfusion‐dependent patients with β‐thalassaemia major and intermedia, hereditary spherocytosis and sickle cell disease (SCD). Haemolysis, transfusions and ineffective erythropoiesis contribute to haem and iron overload in haemolytic patients. In all cohorts we observed increased systemic haem and iron levels associated with scavenger depletion and toxic ‘free’ species formation. Endothelial dysfunction, oxidative stress and inflammation markers were significantly increased compared to healthy donors. In multivariable logistic regression analysis, oxidative stress markers remained significantly associated with both haem‐ and iron‐related parameters, while soluble vascular cell adhesion molecule 1 (sVCAM‐1), soluble endothelial selectin (sE‐selectin) and tumour necrosis factor α (TNFα) showed the strongest association with haem‐related parameters and soluble intercellular adhesion molecule 1 (sICAM‐1), sVCAM‐1, interleukin 6 (IL‐6) and vascular endothelial growth factor (VEGF) with iron‐related parameters. While hereditary spherocytosis was associated with the highest IL‐6 and TNFα levels, β‐thalassaemia major showed limited inflammation compared to SCD. The sVCAM1 increase was significantly lower in patients with SCD receiving exchange compared to simple transfusions. The present results support the involvement of free haem/iron species in the pathogenesis of vascular dysfunction and sterile inflammation in haemolytic diseases, irrespective of the underlying haemolytic mechanism, and highlight the potential therapeutic benefit of iron/haem scavenging therapies in these conditions.

Ravulizumab, a novel long‐acting complement component 5 (C5) inhibitor administered every 8 weeks (q8w), was non‐inferior to eculizumab for all efficacy outcomes in two randomised, open‐label, phase 3 trials in C5 inhibitor‐naïve (Study 301) and eculizumab‐experienced (Study 302) adult patients with paroxysmal nocturnal haemoglobinuria (PNH). This pre‐specified analysis characterised ravulizumab pharmacokinetics (PK), pharmacodynamics (PD; free C5 levels), and PD differences between medications (Study 301, n = 246; Study 302, n = 195). Ravulizumab PK parameters were determined using non‐compartmental analysis. Serum free C5 was quantified with a Gyros‐based fluorescence assay (ravulizumab) and an electrochemiluminescence ligand‐binding assay (eculizumab). Ravulizumab PK parameters were numerically comparable in both studies; the median time to maximum concentrations ranged from 2·3 to 2·8 and 2·3 to 2·6 h in studies 301 and 302, respectively. Ravulizumab steady‐state serum concentrations were achieved immediately after the first dose and sustained throughout treatment. For ravulizumab, the mean (SD) post hoc terminal elimination half‐life was 49·7 (8·9) days. Serum free C5 concentrations

Summary Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off‐target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.

A previous dose‐finding study has suggested that romiplostim is effective in patients with refractory aplastic anaemia (AA) and 10 µg/kg once weekly was recommended as a starting dose. In this Phase II/III, multicentre, open‐label study, romiplostim was administered subcutaneously at a fixed dose of 10 µg/kg once weekly for 4 weeks (weeks 1–4) followed by weekly doses (5, 10, 15 and 20 µg/kg) titrated by platelet response for up to 52 weeks (weeks 5–52). A total of 31 patients with AA who were refractory to immunosuppressive therapy (IST) and thrombocytopenia (platelet count of ≤30 × 109/l) were enrolled. The primary efficacy endpoint of the proportion of patients achieving any haematological (platelet, neutrophil and erythrocyte) response at week 27 was 84% [95% confidence interval (CI) 66–95%]. Trilineage response was 39% (95% CI 22–58%) at week 53. The most common treatment‐related adverse events (AEs) were headache and muscle spasms (each 13%). All AEs were mild or moderate except for three patients with Grade 3 hepatic AEs; no AEs necessitated romiplostim discontinuation. Two patients developed cytogenetic abnormalities, of whom one returned to normal karyotype at last follow‐up. High‐dose romiplostim is effective and well tolerated in the treatment of patients with AA refractory to IST.

Summary Ticagrelor is an antiplatelet agent for adults with coronary artery disease. The inhibition of platelet activation may decrease the frequency of vaso‐occlusion crisis (VOC) in sickle cell disease (SCD). The HESTIA2 study (NCT02482298) randomised 87 adults with SCD (aged 18–30 years) 1:1:1 to twice‐daily ticagrelor 10, 45 mg or placebo for 12 weeks. Numerical decreases from baseline in mean proportion of days with patient‐reported pain (primary endpoint) were seen in all three groups, as well as in pain intensity and analgesic use, with no significant differences between placebo and ticagrelor treatment groups. Plasma ticagrelor concentrations and platelet inhibition increased with dose. Adverse events were distributed evenly across groups and two non‐major bleeding events occurred per group. Ticagrelor was well tolerated with a low bleeding risk, but no effect on diary‐reported pain was detected. Potential effects on frequency of VOCs will need to be evaluated in a larger and longer study.

Summary For decades, folic acid has routinely been given to prevent or treat anaemia in children, pregnant women and people with sickle cell disease. However, there is no conclusive evidence that folate deficiency anaemia constitutes a public health problem in any of these groups. Industrial flour fortification is recommended and implemented in many countries to combat neural tube defects. Dietary folates or folic acid can antagonise the action of antifolate drugs that play a critical role in the prevention and treatment of malaria. Randomised trials have shown that folic acid supplementation increases the rate of treatment failures with sulfadoxine‐pyrimethamine. The efficacy of antifolate drugs against Plasmodium is maximized in the absence of exogenous folic acid, suggesting that there is no safe minimum dose of ingested folic acid. We here review the safety and benefits of interventions to increase folate status in malaria‐endemic countries. We conclude that formal cost‐benefit analyses are required.

Immune aplastic anaemia (AA) is caused by cytotoxic T lymphocytes (CTLs) that destroy haematopoietic stem and progenitor cells. Enhanced type 1 T helper (Th1) responses and reduced regulatory T cells (Tregs) are involved in the immune pathophysiology. CD24hiCD38hi regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL‐10). We investigated circulating B‐cell subpopulations, including CD24hiCD38hi Bregs, as well as total B cells, CD4+ T cells, CD8+ T cells and natural killer cells in 104 untreated patients with severe and very severe AA, aged ≥18 years. All patients were treated with standard immunosuppressive therapy (IST) plus eltrombopag. CD24hiCD38hi Bregs were markedly reduced in patients with AA compared to healthy individuals, especially in very severe AA, but residual Bregs remained functional, capable of producing IL‐10; total B‐cell counts and the other B‐cell subpopulations were similar to those of healthy individuals. CD24hiCD38hi Bregs did not correlate with responses to IST, and they recovered to levels present in healthy individuals after therapy. Mature naïve B‐cell counts were unexpectedly associated with IST response. Markedly reduced CD24hiCD38hi Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA.

Summary Non‐transfusion‐dependent thalassaemias (NTDT) encompass a spectrum of anaemias rarely requiring blood transfusions. Increased iron absorption, driven by hepcidin suppression secondary to erythron expansion, initially causes intrahepatic iron overload. We examined iron metabolism biomarkers in 166 NTDT patients with β thalassaemia intermedia (n = 95), haemoglobin (Hb) E/β thalassaemia (n = 49) and Hb H syndromes (n = 22). Liver iron concentration (LIC), serum ferritin (SF), transferrin saturation (TfSat) and non‐transferrin‐bound iron (NTBI) were elevated and correlated across diagnostic subgroups. NTBI correlated with soluble transferrin receptor (sTfR), labile plasma iron (LPI) and nucleated red blood cells (NRBCs), with elevations generally confined to previously transfused patients. Splenectomised patients had higher NTBI, TfSat, NRBCs and SF relative to LIC, than non‐splenectomised patients. LPI elevations were confined to patients with saturated transferrin. Erythron expansion biomarkers (sTfR, growth differentiation factor‐15, NRBCs) correlated with each other and with iron overload biomarkers, particularly in Hb H patients. Plasma hepcidin was similar across subgroups, increased with >20 prior transfusions, and correlated inversely with TfSat, NTBI, LPI and NRBCs. Hepcidin/SF ratios were low, consistent with hepcidin suppression relative to iron overload. Increased NTBI and, by implication, risk of extra‐hepatic iron distribution are more likely in previously transfused, splenectomised and iron‐overloaded NTDT patients with TfSat >70%.

Eculizumab is the first and only medication approved for paroxysmal nocturnal haemoglobinuria (PNH) and atypical haemolytic uraemic syndrome (aHUS) treatment. However, eculizumab safety based on long-term pharmacovigilance is unknown. This analysis summarises safety data collected from spontaneous and solicited sources from 16 March 2007 through 1 October 2016. Cumulative exposure to eculizumab was 28518 patient-years (PY) (PNH, 21016 PY; aHUS, 7502 PY). Seventy-six cases of meningococcal infection were reported (025/100 PY), including eight fatal PNH cases (003/100 PY). Susceptibility to meningococcal infections remained the key risk in patients receiving eculizumab. The meningococcal infection rate decreased over time; related mortality remained steady. The most commonly reported serious nonmeningococcal infections were pneumonia (118%); bacteraemia, sepsis and septic shock (111%); urinary tract infection (41%); staphylococcal infection (26%); and viral infection (25%). There were 434 reported cases of eculizumab exposure in pregnant women; of 260 cases with known outcomes, 70% resulted in live births. Reporting rates for solid tumours (approximate to 06/100 PY) and haematological malignancies (approximate to 074/100 PY) remained stable over time. No new safety signals affecting the eculizumab benefit-risk profile were identified. Continued awareness and implementation of risk mitigation protocols are essential to minimise risk of meningococcal and other Neisseria infections in patients receiving eculizumab.

Summary Quantitative sensory testing (QST) is used in a variety of pain disorders to characterize pain and predict prognosis and response to specific therapies. In this study, we aimed to confirm results in the literature documenting altered QST thresholds in sickle cell disease (SCD) and assess the test–retest reliability of results over time. Fifty‐seven SCD and 60 control subjects aged 8–20 years underwent heat and cold detection and pain threshold testing using a Medoc TSAII. Participants were tested at baseline and 3 months; SCD subjects were additionally tested at 6 months. An important facet of our study was the development and use of a novel QST modelling approach, allowing us to model all data together across modalities. We have not demonstrated significant differences in thermal thresholds between subjects with SCD and controls. Thermal thresholds were consistent over a 3‐ to 6‐month period. Subjects on whom hydroxycarbamide (HC) was initiated shortly before or after baseline testing (new HC users) exhibited progressive decreases in thermal sensitivity from baseline to 6 months, suggesting that thermal testing may be sensitive to effective therapy to prevent vasoocclusive pain. These findings inform the use of QST as an endpoint in the evaluation of preventative pain therapies.

Summary Phosphatidylserine (PS) exposure increases as red cells age, and is an important signal for the removal of senescent cells from the circulation. PS exposure is elevated in red cells from sickle cell anaemia (SCA) patients and is thought to enhance haemolysis and vaso‐occlusion. Although precise conditions leading to its externalisation are unclear, high intracellular Ca2+ has been implicated. Red cells from SCA patients are also exposed to an increased oxidative challenge, and we postulated that this stimulates PS exposure, through increased Ca2+ levels. We tested four different ways of generating oxidative stress: hypoxanthine and xanthine oxidase, phenazine methosulphate, nitrite and tert‐butyl hydroperoxide, together with thiol modification with N‐ethylmaleimide (NEM), dithiothreitol and hypochlorous acid (HOCl), in red cells permeabilised to Ca2+ using bromo‐A23187. Unexpectedly, our findings showed that the four oxidants significantly reduced Ca2+‐induced PS exposure (by 40–60%) with no appreciable effect on Ca2+ affinity. By contrast, NEM markedly increased PS exposure (by about 400%) and slightly but significantly increased the affinity for Ca2+. Dithiothreitol modestly reduced PS exposure (by 25%) and HOCl had no effect. These findings emphasise the importance of thiol modification for PS exposure in sickle cells but suggest that increased oxidant stress alone is not important.

Summary The World Health Organization recommends universal iron supplementation of 30–60 mg/day in pregnancy but coverage is low in most countries. Its efficacy is uncertain, however, and there has been a vigorous debate in the last decade about its safety, particularly in areas with a high burden of malaria and other infectious diseases. We reviewed the evidence on the safety and efficacy of antenatal iron supplementation in low‐income countries. We found no evidence that daily supplementation at a dose of 60 mg leads to increased maternal Plasmodium infection risk. On the other hand, recent meta‐analyses found that antenatal iron supplementation provides benefits for maternal health (severe anaemia at postpartum, blood transfusion). For neonates, there was a reduced prematurity risk, and only a small or no effect on birth weight. A recent trial showed, however, that benefits of antenatal iron supplementation on maternal and neonatal health vary by maternal iron status, with substantial benefits in iron‐deficient women. The benefits of universal iron supplementation are likely to vary with the prevalence of iron deficiency. As a consequence, the balance between benefits and risks is probably more favourable in low‐income countries than in high‐income countries despite the higher exposure to infectious pathogens.

Summary Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next‐generation‐sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical‐grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically‐reliable diagnostic test and minimize false‐negative results we developed an open‐source tool (CoverMi) to accurately determine base‐coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33‐gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS‐based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.

To date, there is a lack of long-term safety and efficacy data for iron chelation therapy in transfusion-dependent patients with sickle cell disease (SCD). To evaluate the long-term safety and efficacy of deferasirox (a once-daily oral iron chelator), patients with SCD completing a 1-year, Phase II, randomized, deferoxamine (DFO)-controlled study entered a 4-year extension, continuing to receive deferasirox, or switching from DFO to deferasirox. Average actual deferasirox dose was 19·4 ± 6·3 mg/kg per d. Of 185 patients who received at least one deferasirox dose, 33·5% completed the 5-year study. The most common reasons for discontinuation were withdrawal of consent (23·8%), lost to follow-up (9·2%) and adverse events (AEs) (7·6%). Investigator-assessed drug-related AEs were predominantly gastrointestinal [including nausea (14·6%), diarrhoea (10·8%)], mild-to-moderate and transient in nature. Creatinine clearance remained within the normal range throughout the study. Despite conservative initial dosing, serum ferritin levels in patients with ≥ 4 years deferasirox exposure significantly decreased by -591 μg/l (95% confidence intervals, -1411, -280 μg/l; P = 0·027; n = 67). Long-term deferasirox treatment for up to 5 years had a clinically acceptable safety profile, including maintenance of normal renal function, in patients with SCD. Iron burden was substantially reduced with appropriate dosing in patients treated for at least 4 years.

Several lines of evidence suggest that sickle cell disease (SCD) is associated with a chronic inflammatory state. In this study of 70 children with SCD at steady state evaluated by a broad panel of biomarkers representing previously examined mechanisms of pathogenicity in SCD, high sensitivity C-reactive protein (hs-CRP), a marker of low-grade, systemic inflammation, emerged as the most significant laboratory correlate of hospitalizations for pain or vaso-occlusive (VOC) events. While markers of increased haemolytic status, endothelial activation and coagulation activation all correlated positively with VOC events by univariate analysis, baseline hs-CRP levels provided the most significant contribution to the association in multiple regression models (22%), and, hs-CRP, along with age, provided the best fit in negative binomial models. These data highlight the clinical relevance of the role of inflammation in paediatric VOC, providing both a rationale for future therapeutic strategies targeting inflammation in microvessel occlusive complications of SCD, and the potential clinical use of hs-CRP as a biomarker in childhood SCD.

High cerebral blood flow velocity (CBFv) and low haemoglobin oxygen saturation (SpO(2)) predict neurological complications in sickle cell anaemia (SCA) but any association is unclear. In a cross-sectional study of 105 Kenyan children, mean CBFv was 120 +/- 34.9 cm/s; 3 had conditional CBFv (170-199 cm/s) but none had abnormal CBFv (>200 cm/s). After adjustment for age and haematocrit, CBFv > or =150 cm/s was predicted by SpO(2) < or = 95% and history of fever. Four years later, 10 children were lost to follow-up, none had suffered neurological events and 11/95 (12%) had died, predicted by history of fever but not low SpO(2). Natural history of SCA in Africa may be different from North America and Europe.

Reactivation of fetal haemoglobin (HbF) expression in adult life could provide an effective therapeutic approach for patients with haemoglobinopathies and thalassaemias. Among HbF induction agents, hydroxycarbamide (HC) has been successfully used to treat sickle cell disease (SCD) and selected thalassaemia syndromes (Rodgers et al, 1990; Zeng et al, 1995; Fucharoen et al, 1996). Hydroxycarbamide is believed to exert its beneficial effects in SCD via a variety of mechanisms, including augmented HbF synthesis by erythroid regeneration or NO-related increases in soluble guanylate cyclase activity and cyclic guanidine monophosphate (cGMP) that stimulate HBG expression (Cokic et al, 2003). Additional mechanisms may also include myelosuppression with a reduction in circulating neutrophils, increased erythrocyte water content, modified erythrocyte endothelial cell interactions and altered vascular tone by increasing NO bioavailability (Okpala, 2005). Many patients treated with HC increase HbF levels from baseline, with wide variability in the magnitude of this response. The underlying reasons for this spectrum of HC responsiveness are poorly understood (Steinberg et al, 1997; Bakanay et al, 2005). Presently, a major research priority is the identification of genetic variants underlying both human diseases and differences in pharmacological responsiveness to drugs (Collins et al, 2003). In SCD, this paradigm is being applied to elucidate the pathogenesis of vaso-occlusive and vascular complications and to develop individualized SCD therapies (Steinberg, 2005). Genetic variants in drug-metabolizing enzymes, transporters or targets could potentially influence the efficacy or toxicity of drugs used in SCD. The HC-induced small guanosine triphosphate (GTP)-binding protein, secretion-associated and RAS-related (SAR) protein has recently been shown to play a pivotal role in HBG induction by causing cell apoptosis and G1/S-phase arrest by reduction of phosphatidylinositol 3 (PI3) kinase and extracellular protein-related kinase (ERK) phosphorylation with increased p21 and GATA-2 expression (Tang et al, 2005). SAR1A belongs to the small GTPase superfamily and encodes a GTP-binding protein SAR1a. This protein plays a key role in initializing transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The localization of SAR1A in the endoplasmic reticulum and its association with HBG expression demonstrated in our recent study suggest that SAR1A also plays a special role in haemoglobin regulation (Tang et al, 2005). However, the precise pathway(s) through which SAR1A regulates HBG remain unknown. SAR1A may increase the transport of membrane-bound transcription factor precursors from the ER to the Golgi. The proteolytic cleavage of the precursor proteins in the Golgi activate cytosolic fragments that enter the nucleus and regulate erythroid-specific transcription factors, such as GATA, eventually modulating HBG expression. Moreover, previous studies have illuminated a pivotal role of the p38 mitogen-activated protein kinase (MAPK) pathway during GTP-mediated erythroid differentiation of K562 cells with the accumulation of HBG mRNA (Osti et al, 1997; Moosavi et al, 2007). Activators of the soluble guanylate cyclase (sGC) and protein kinase G (PKG) pathways have been implicated in the regulation of HBG expression in both erythroleukemic cells and in primary erythroblasts (Ikuta et al, 2001). Expression of HBG and the sGC alpha subunit are correlated, indicating that GTP-binding proteins may participate in HBG induction. Preliminary data from our study indicated that HC inducibility is transcriptionally regulated and localized to elements in the SAR1A promoter. Accordingly, we hypothesized that DNA sequence variation within the SAR1A promoter might explain differences in individual responses to HC therapy. To test this hypothesis, we identified the single nucleotide polymorphism (SNPs) in the SAR1A promoter by DNA sequencing and examined these variants in an association study of sickle cell anaemia patients treated with HC.

Bacteraemia is a leading cause of morbidity in sickle cell anaemia (SCA), but information from studies in Africa is limited. We evaluated 890 admissions from 648 SCA patients at a tertiary hospital in Tanzania. Bacteraemia was present in 43 admissions (4·8%); isolates included Staphylococcus aureus (12/43; 28%), non-Typhi Salmonella (9/43; 21%), Streptococcus pneumoniae (3/43; 7%) and Salmonella Typhi (2/43; 5%). Compared to SCA patients without bacteraemia, SCA patients with bacteraemia had significantly lower haemoglobin [71 g/l vs. 62 g/l, odds ratio 0·72 (95% confidence interval 0·56-0·91), P < 0·01]. Further exploration is needed of the relationship between anaemia and bacterial infections in SCA in Africa.

Severe forms of alpha and beta thalassaemia have been estimated to affect approximately 68000 births annually. Individuals who carry thalassaemic genes are protected against death from malaria infection; the global distribution of thalassaemic genes thus matches the historical distribution of malaria. Screening programmes are a vital tool to counter the thalassaemias by: (i) identifying individual carriers and allowing them to make informed reproductive choices, and (ii) generating population level gene-frequency estimates, to help ensure the optimal allocation of public health resources. For both of these functions it is vital that the screen performed is suitably sensitive. One popular first-stage screening option for beta thalassaemia in low-income countries is the One Tube Osmotic Fragility Test (OTOFT). Here we introduce a population genetic framework within which to quantify the likely sensitivity and specificity of the OTOFT in different epidemiological contexts. We demonstrate that the co-occurrence of alpha thalassaemia, and other malaria related erythrocyte poly-morphisms such as Southeast Asian Ovalocytosis and glucose-6-phosphate dehydrogenase deficiency, could reduce the sensitivity of OTOFTs for beta thalassaemia to below 70%. Our results highlight a potential hazard of the widespread application of OTOFTs and emphasize the fact that the public health impact of any single genetic adaptation to malaria cannot be considered in isolation.

Summary ATOH8 has previously been shown to be an iron-regulated transcription factor, however its role in iron metabolism is not known. ATOH8 expression in HEK293 cells resulted in increased endogenous HAMP mRNA levels as well as HAMP promoter activity. Mutation of the E-box or SMAD response elements within the HAMP promoter significantly reduced the effects of ATOH8, indicating that ATOH8 activates HAMP transcription directly as well as through bone morphogenic protein (BMP) signalling. In support of the former, Chromatin immunoprecipitation assays provided evidence that ATOH8 binds to E-box regions within the HAMP promoter while the latter was supported by the finding that ATOH8 expression in HEK293 cells led to increased phosphorylated SMAD1,5,8 levels. Liver Atoh8 levels were reduced in mice under conditions associated with increased erythropoietic activity such as hypoxia, haemolytic anaemia, hypotransferrinaemia and erythropoietin treatment and increased by inhibitors of erythropoiesis. Hepatic Atoh8mRNA levels increased in mice treated with holo transferrin, suggesting that Atoh8 responds to changes in plasma iron. ATOH8 is therefore a novel transcriptional regulator of HAMP, which is responsive to changes in plasma iron and erythroid activity and could explain how changes in erythroid activity lead to regulation of HAMP.

Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium, CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine.

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -β (TGF-β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-β family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis.

Summary Transcranial Doppler ultrasonography measures cerebral blood flow velocity (CBFv) of basal intracranial vessels and is used clinically to detect stroke risk in children with sickle cell anaemia (SCA). Co‐inheritance in SCA of alpha‐thalassaemia and glucose‐6‐phosphate dehydrogenase (G6PD) polymorphisms is reported to associate with high CBFv and/or risk of stroke. The effect of a common functional polymorphism of haptoglobin (HP) is unknown. We investigated the effect of co‐inheritance of these polymorphisms on CBFv in 601 stroke‐free Tanzanian SCA patients aged

As a leading cause of childhood mortality worldwide, selection pressure by Plasmodium falciparum continues to shape the human genome. Severe disturbances within the microcirculation result from the adhesion of infected erythrocytes to host receptors on monocytes, platelets, and endothelium. In this prospective study, we compared expression of all major host cytoadhesion receptors among Ugandan children presenting with uncomplicated malaria (n = 1078) versus children with severe malaria (n = 855), including cerebral malaria (n = 174), severe anaemia (n = 522), and lactic acidosis (n = 154). We report a significant survival advantage attributed to blood group O and increased monocyte expression of CD36 and ICAM1 (CD54). The high case fatality rate syndromes of cerebral malaria and lactic acidosis were associated with high platelet CD36 expression and thrombocytopenia, and severe malaria anaemia was characterized by low ICAM1 expression. In a logistic regression model of disease severity, odds ratios for the mitigating effects of blood group O, CD36, and ICAM1 phenotypes were greater than that of sickle haemoglobin. Host genetic adaptations to Plasmodium falciparum suggest new potential malaria treatment strategies.

Severe anaemia is a life-threatening complication of falciparum malaria associated with loss of predominantly non-parasitized red blood cells (npRBCs). This poorly elucidated process might be influenced by (i) rosettes, i.e. npRBCs cytoadherent to haemozoin-containing parasitized RBCs (pRBCs) and (ii) generation in pRBCs of 4-hydroxynonenal (4-HNE) through haemozoin-catalysed lipid peroxidation. We explored whether close proximity in rosettes may facilitate 4-HNE transfer to npRBCs, which is likely to enhance their phagocytosis and contribute to malaria anaemia. Fluorescence microscopy and flow cytometry data indicated 4-HNE transfer to npRBCs in rosettes. Rosettes were formed by 64·8 ± 1·8% varO-expressing pRBCs, and 8·7 ± 1·1% npRBCs were positive for 4-HNE-protein-conjugates, while low-rosetting parasites generated only 2·4 ± 1·1% 4-HNE-conjugate-positive npRBCs. 4-HNE transfer decreased after blocking rosetting by monoclonal antibodies. A positive linear relationship between rosette frequency and 4-HNE-conjugates in npRBCs was found in 40 malaria patients, a first indication for a role of rosetting in npRBCs modifications in vivo. Children with severe malaria anaemia had significantly higher percentages of 4-HNE-conjugate-positive npRBCs compared to children with uncomplicated malaria. In conclusion, 4-HNE transfer from pRBCs to npRBCs in rosettes is suggested to play a role in the phagocytic removal of large numbers of npRBCs, the hallmark of severe malaria anaemia.

The highest approved dose of deferasirox is currently 30 mg/kg per d in many countries; however, some patients require escalation above 30 mg/kg per d to achieve their therapeutic goals. This retrospective analysis investigated the efficacy (based on change in serum ferritin levels) and safety of deferasirox >30 mg/kg per d in adult and paediatric patients with transfusion-dependent anaemias, including β-thalassaemia, sickle cell disease and the myelodysplastic syndromes. In total, 264 patients pooled from four clinical trials received doses of >30 mg/kg per d; median exposure to deferasirox >30 mg/kg per d was 36 weeks. In the overall population there was a statistically significant median decrease in serum ferritin of 440 μg/l (P< 0·0001) from pre-dose-escalation to the time-of-analysis; significant decreases were also observed in adult and paediatric patients, as well as β-thalassaemia patients. The adverse event profile in patients who received deferasirox doses of >30 mg/kg per d was consistent with previously published data. There was no worsening of renal or liver function following dose escalation. Deferasirox >30 mg/kg per d effectively reduced iron burden to levels lower than those achieved prior to dose escalation in patients with transfusion-dependent anaemias. This has important implications for patients who are heavily transfused and may require higher doses to reduce body iron burden.

Deferasirox is a once-daily, oral iron chelator developed for treating transfusional iron overload. Preclinical studies indicated that the kidney was a potential target organ of toxicity. As patients with sickle cell disease often have abnormal baseline renal function, the primary objective of this randomised, open-label, phase II trial was to evaluate the safety and tolerability of deferasirox in comparison with deferoxamine in this population. Assessment of efficacy, as measured by change in liver iron concentration (LIC) using biosusceptometry, was a secondary objective. A total of 195 adult and paediatric patients received deferasirox (n = 132) or deferoxamine (n = 63). Adverse events most commonly associated with deferasirox were mild, including transient nausea, vomiting, diarrhoea, abdominal pain and skin rash. Abnormal laboratory studies with deferasirox were occasionally associated with mild non-progressive increases in serum creatinine and reversible elevations in liver function tests. Discontinuation rates from deferasirox (11·4%) and deferoxamine (11·1%) were similar. Over 1 year, similar dose-dependent LIC reductions were observed with deferasirox and deferoxamine. Once-daily oral deferasirox has acceptable tolerability and appears to have similar efficacy to deferoxamine in reducing iron burden in transfused patients with sickle cell disease.