Luspatercept stimulates erythropoiesis, increases iron utilization, and redistributes body iron in transfusion-dependent thalassemia.

Luspatercept, a ligand-trapping fusion protein, binds select TGF-ß superfamily ligands implicated in thalassemic erythropoiesis, promoting late-stage erythroid maturation. Luspatercept reduced transfusion burden in the BELIEVE trial (NCT02604433) of 336 adults with transfusion-dependent thalassemia (TDT). Analysis of biomarkers in BELIEVE offers novel physiological and clinical insights into benefits offered by luspatercept. Transfusion iron loading rates decreased 20% by 1.4 g (~7 blood units; median iron loading rate difference: -0.05 ± 0.07 mg Fe/kg/day, p< .0001) and serum ferritin (s-ferritin) decreased 19.2% by 269.3 ± 963.7 µg/L (p < .0001), indicating reduced macrophage iron. However, liver iron content (LIC) did not decrease but showed statistically nonsignificant increases from 5.3 to 6.7 mg/g dw. Erythropoietin, growth differentiation factor 15, soluble transferrin receptor 1 (sTfR1), and reticulocytes rose by 93%, 59%, 66%, and 112%, respectively; accordingly, erythroferrone increased by 51% and hepcidin decreased by 53% (all p < .0001). Decreased transfusion with luspatercept in patients with TDT was associated with increased erythropoietic markers and decreasing hepcidin. Furthermore, s-ferritin reduction associated with increased erythroid iron incorporation (marked by sTfR1) allowed increased erythrocyte marrow output, consequently reducing transfusion needs and enhancing rerouting of hemolysis (heme) iron and non-transferrin-bound iron to the liver. LIC increased in patients with intact spleens, consistent with iron redistribution given the hepcidin reduction. Thus, erythropoietic and hepcidin changes with luspatercept in TDT lower transfusion dependency and may redistribute iron from macrophages to hepatocytes, necessitating the use of concomitant chelator cover for effective iron management.

The clinical relevance of detectable plasma iron species in iron overload states and subsequent to intravenous iron-carbohydrate administration.

Many disorders of iron homeostasis (e.g., iron overload) are associated with the dynamic kinetic profiles of multiple non-transferrin bound iron (NTBI) species, chronic exposure to which is associated with deleterious end-organ effects. Here we discuss the chemical nature of NTBI species, challenges with measuring NTBI in plasma, and the clinical relevance of NTBI exposure based on source (iron overload disorder vs. intravenous iron-carbohydrate complex administration). NTBI is not a single entity but consists of multiple, often poorly characterized species, some of which are kinetically non-exchangeable while others are relatively exchangeable. Prolonged presence of plasma NTBI is associated with excessive tissue iron accumulation in susceptible tissues, with consequences, such as endocrinopathy and heart failure. In contrast, intravenous iron-carbohydrate nanomedicines administration leads only to transient NTBI appearance and lacks evidence for association with adverse clinical outcomes. Assays to measure plasma NTBI are typically technically complex and remain chiefly a research tool. There have been two general approaches to estimating NTBI: capture assays and redox-activity assays. Early assays could not avoid capturing some iron from transferrin, thus overestimating NTBI. By contrast, some later assays may have promoted the donation of NTBI species to transferrin during the assay procedure, potentially underestimating NTBI levels. The levels of transferrin saturation at which NTBI species have been detectable have varied between different methodologies and between patient populations studied.

Outcome predictors for maternal red blood cell alloimmunisation with anti-K and anti-D managed with intrauterine blood transfusion.

Red blood cell (RBC) alloimmunisation with anti-D and anti-K comprise the majority of cases of fetal haemolytic disease requiring intrauterine red cell transfusion (IUT). Few studies have investigated which haematological parameters can predict adverse fetal or neonatal outcomes. The aim of the present study was to identify predictors of adverse outcome, including preterm birth, intrauterine fetal demise (IUFD), neonatal death (NND) and/or neonatal transfusion. We reviewed the records of all pregnancies alloimmunised with anti-K and anti-D, requiring IUT over 27 years at a quaternary fetal centre. We reviewed data for 128 pregnancies in 116 women undergoing 425 IUTs. The median gestational age (GA) at first IUT was significantly earlier for anti-K than for anti-D (24·3 vs. 28·7 weeks, P = 0·004). Women with anti-K required more IUTs than women with anti-D (3·84 vs. 3·12 mean IUTs, P = 0·036) and the fetal haemoglobin (Hb) at first IUT was significantly lower (51.0 vs. 70.5 g/l, P = 0·001). The mean estimated daily decrease in Hb did not differ between the two groups. A greater number of IUTs and a slower daily decrease in Hb (g/l/day) between first and second IUTs were predictive of a longer period in utero. Earlier GA at first IUT and a shorter interval from the first IUT until delivery predicted IUFD/NND. Earlier GA and lower Hb at first IUT significantly predicted need for phototherapy and/or blood product use in the neonate. In the anti-K group, a greater number of IUTs was required in women with a higher titre. Furthermore, the higher the titre, the earlier the GA at which an IUT was required in both groups. The rate of fall in fetal Hb between IUTs decreased, as the number of transfusions increased. Our present study identified pregnancies at considerable risk of an unfavourable outcome with anti-D and anti-K RBC alloimmunisation. Identifying such patients can guide pregnancy management, facilitates patient counselling, and can optimise resource use. Prospective studies can also incorporate these characteristics, in addition to laboratory markers, to further identify and improve the outcomes of these pregnancies.

Intravenous iron preparations transiently generate non-transferrin-bound iron from two proposed pathways.

Intravenous iron-carbohydrate complex preparations (IVIPs) are non-interchangeable pro-drugs: their pharmacokinetics (PK) varies determined by semi-crystalline iron core and carbohydrate shell structures, influences pharmacodynamics (PD) and thus efficacy and safety. Examining PK/PD relationships of 3 IVIPs we identify a two-pathway model of transient NTBI generation following single dose administration. 28 hypoferremic non-anemic patients randomized to 200mg iron as ferric carboxymaltose (Fe-carboxymaltose), iron sucrose (Fe-sucrose), iron isomaltoside 1000 (Fe-isomaltoside-1000), n=8/arm, or placebo, n=4, on a 2-week PK/PD study, had samples analysed for total serum iron, IVIP-iron, transferrin-bound iron (TBI) by HPLC-ICP-MS, transferrin saturation (TSAT), serum ferritin (s-Ferritin) by standard methods, non-TBI (NTBI) and hepcidin as published before. IVIP-dependent increases in these parameters returned to baseline in 48-150h, except for s-Ferritin and TSAT. NTBI was low with Fe-isomaltoside-1000 (0.13µM at 8h), rapidly increased with Fe-sucrose (0.8µM at 2h, 1.25µM at 4h), and delayed for Fe-carboxymaltose (0.57µM at 24h). NTBI AUCs were 7-fold greater for Fe-carboxymaltose and Fe-sucrose than for Fe-isomaltoside-1000. Hepcidin peak time varied, but not AUC or mean levels. s-Ferritin levels and AUC were highest for Fe-carboxymaltose and greater than placebo for all IVIPs. We propose 2 mechanisms for the observed NTBI kinetics: rapid and delayed NTBI appearance consistent with direct (circulating IVIP-to-plasma) and indirect (IVIP-to-macrophage-to-plasma) iron release based on IVIP plasma half-life and s-Ferritin dynamics. IVIPs generate different, broadly stability- and PK-dependent, NTBI and s-Ferritin signatures, which may influence iron bioavailability, efficacy and safety. Longer-term studies should link NTBI exposure to subsequent safety and efficacy parameters and potential clinical consequences.

Atherosclerosis is aggravated by iron overload and ameliorated by dietary and pharmacological iron restriction.

AIMS: Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE-/- FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). METHODS AND RESULTS: Compared with normo-ferremic ApoE-/- mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE-/- FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE-/- FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. CONCLUSION: Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.

Practical guide to quantification of hepatic iron with MRI.

Our intention is to demystify the MR quantification of hepatic iron (i.e., the liver iron concentration) and give you a step-by-step approach by answering the most pertinent questions. The following article should be more of a manual or guide for every radiologist than a classic review article, which just summarizes the literature. Furthermore, we provide important background information for professional communication with clinicians. The information regarding the physical background is reduced to a minimum. After reading this article, you should be able to perform adequate MR measurements of the LIC with 1.5-T or 3.0-T scanners. KEY POINTS: • MRI is widely accepted as the primary approach to non-invasively determine liver iron concentration (LIC). • This article is a guide for every radiologist to perform adequate MR measurements of the LIC. • When using R2* relaxometry, some points have to be considered to obtain correct measurements-all explained in this article.

Eltrombopag: a powerful chelator of cellular or extracellular iron(III) alone or combined with a second chelator.

Eltrombopag (ELT) is a thrombopoietin receptor agonist reported to decrease labile iron in leukemia cells. Here we examine the previously undescribed iron(III)-coordinating and cellular iron-mobilizing properties of ELT. We find a high binding constant for iron(III) (log ß2=35). Clinically achievable concentrations (1 µM) progressively mobilized cellular iron from hepatocyte, cardiomyocyte, and pancreatic cell lines, rapidly decreasing intracellular reactive oxygen species (ROS) and also restoring insulin secretion in pancreatic cells. Decrements in cellular ferritin paralleled total cellular iron removal, particularly in hepatocytes. Iron mobilization from cardiomyocytes exceeded that obtained with deferiprone, desferrioxamine, or deferasirox at similar iron-binding equivalents. When combined with these chelators, ELT enhanced cellular iron mobilization more than additive (synergistic) with deferasirox. Iron-binding speciation plots are consistent with ELT donating iron to deferasirox at clinically relevant concentrations. ELT scavenges iron citrate species faster than deferasirox, but rapidly donates the chelated iron to deferasirox, consistent with a shuttling mechanism. Shuttling is also suggested by enhanced cellular iron mobilization by ELT when combined with the otherwise ineffective extracellular hydroxypyridinone chelator, CP40. We conclude that ELT is a powerful iron chelator that decreases cellular iron and further enhances iron mobilization when combined with clinically available chelators.

Sotatercept, a novel transforming growth factor ß ligand trap, improves anemia in ß-thalassemia: a phase II, open-label, dose-finding study.

ß-thalassemia, a hereditary blood disorder caused by defective synthesis of hemoglobin ß globin chains, leads to ineffective erythropoiesis and chronic anemia that may require blood transfusions. Sotatercept (ACE-011) acts as a ligand trap to inhibit negative regulators of late-stage erythropoiesis in the transforming growth factor ß superfamily, correcting ineffective erythropoiesis. In this phase II, open-label, dose-finding study, 16 patients with transfusion-dependent ß -thalassemia and 30 patients with non-transfusion-dependent ß-thalassemia were enrolled at seven centers in four countries between November 2012 and November 2014. Patients were treated with sotatercept at doses of 0.1, 0.3, 0.5, 0.75, or 1.0 mg/kg to determine a safe and effective dose. Doses were administered by subcutaneous injection every 3 weeks. Patients were treated for ≤22 months. Response was assessed as a ≥20% reduction in transfusion burden sustained for 24 weeks in transfusion-dependent ß-thalassemia patients, and an increase in hemoglobin level of ≥1.0 g/dL sustained for 12 weeks in non-transfusion-dependent ß-thalassemia patients. Sotatercept was well tolerated. After a median treatment duration of 14.4 months (range 0.6-35.9), no severe life-threatening adverse events were observed. Thirteen percent of patients reported serious but manageable adverse events. The active dose of sotatercept was ≥0.3 mg/kg for patients with non-transfusion-dependent ß-thalassemia and ≥0.5 mg/kg for those with transfusion-dependent ß-thalassemia. Of 30 non-transfusion-dependent ß-thalassemia patients treated with ≥0.1 mg/kg sotatercept, 18 (60%) achieved a mean hemoglobin increase ≥1.0 g/dL, and 11 (37%) an increase ≥1.5 g/dL, sustained for ≥12 weeks. Four (100%) transfusion-dependent ß-thalassemia patients treated with 1.0 mg/kg sotatercept achieved a transfusion-burden reduction of ≥20%. Sotatercept was effective and well tolerated in patients with ß-thalassemia. Most patients with non-transfusion-dependent ß-thalassemia treated with higher doses achieved sustained increases in hemoglobin level. Transfusion-dependent ß-thalassemia patients treated with higher doses of sotatercept achieved notable reductions in transfusion requirements. This trial was registered at ClinicalTrials.gov with the number NCT01571635.

Immunophenotypic analysis of cell cycle status in acute myeloid leukaemia: relationship to cytogenetics, genotype and clinical outcome.

Cell cycle status may play an important role in directing patient therapy. We therefore determined the cell cycle status of leukaemic cells by immunophenotypic analysis of bone marrow trephine biopsies from 181 patients with acute myeloid leukaemia (AML) and correlated the results with biological features and clinical outcome. There was considerable heterogeneity between patients. The presenting white cell count significantly correlated with the proportion of non-quiescent cells (P < 0·0001), of cycling cells beyond G1 (P < 0·0001) and the speed of cycling (P < 0·0001). Profiles in acute promyelocytic leukaemia (APL) differed from non-APL and were consistent with more differentiated cells with reduced proliferative potential, but no significant differences were observed between non-APL cytogenetic risk groups. NPM1 mutations but not FLT3 internal tandem duplication (FLT3ITD ) were significantly associated with a higher proportion of cells beyond G1 (P = 0·002) and faster speed of cycling (P = 0·003). Resistance to standard cytosine arabinoside and daunorubicin induction chemotherapy was significantly related to a slower speed of cycling (P = 0·0002), as was a higher relapse rate (P = 0·05), but not with the proportion of non-quiescent cells or actively cycling cells. These results show a link between the cycling speed of AML cells and the response to chemotherapy, and help to identify a group with a very poor prognosis.

Residual erythropoiesis protects against myocardial hemosiderosis in transfusion-dependent thalassemia by lowering labile plasma iron via transient generation of apotransferrin.

Cardiosiderosis is a leading cause of mortality in transfusion-dependent thalassemias. Plasma non-transferrin-bound iron and its redox-active component, labile plasma iron, are key sources of iron loading in cardiosiderosis. Risk factors were identified in 73 patients with or without cardiosiderosis. Soluble transferrin receptor-1 levels were significantly lower in patients with cardiosiderosis (odds ratio 21). This risk increased when transfusion-iron loading rates exceeded the erythroid transferrin uptake rate (derived from soluble transferrin receptor-1) by >0.21 mg/kg/day (odds ratio 48). Labile plasma iron was >3-fold higher when this uptake rate threshold was exceeded, but non-transferrin-bound iron and transferrin saturation were comparable. The risk of cardiosiderosis was decreased in patients with low liver iron, ferritin and labile plasma iron, or high bilirubin, reticulocyte counts or hepcidin. We hypothesized that high erythroid transferrin uptake rate decreases cardiosiderosis through increased erythroid re-generation of apotransferrin. To test this, iron uptake and intracellular reactive oxygen species were examined in HL-1 cardiomyocytes under conditions modeling transferrin effects on non-transferrin-bound iron speciation with ferric citrate. Intracellular iron and reactive oxygen species increased with ferric citrate concentrations especially when iron-to-citrate ratios exceeded 1:100, i.e. conditions favoring kinetically labile monoferric rather than oligomer species. Excess iron-binding equivalents of apotransferrin inhibited iron uptake and decreased both intracellular reactive oxygen species and labile plasma iron under conditions favoring monoferric species. In conclusion, high transferrin iron utilization, relative to the transfusion-iron load rate, decreases the risk of cardiosiderosis. A putative mechanism is the transient re-generation of apotransferrin by an active erythron, rapidly binding labile plasma iron-detectable ferric monocitrate species.

Synergistic intracellular iron chelation combinations: mechanisms and conditions for optimizing iron mobilization.

Iron chelators are increasingly combined clinically but the optimal conditions for cellular iron mobilization and mechanisms of interaction are unclear. Speciation plots for iron(III) binding of paired combinations of the licensed iron chelators desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) suggest conditions under which chelators can combine as 'shuttle' and 'sink' molecules but this approach does not consider their relative access and interaction with cellular iron pools. To address this issue, a sensitive ferrozine-based detection system for intracellular iron removal from the human hepatocyte cell line (HuH-7) was developed. Antagonism, synergism or additivity with paired chelator combinations was distinguished using mathematical isobologram analysis over clinically relevant chelator concentrations. All combinations showed synergistic iron mobilization at 8 h with clinically achievable concentrations of sink and shuttle chelators. Greatest synergism was achieved by combining DFP with DFX, where about 60% of mobilized iron was attributable to synergistic interaction. These findings predict that the DFX dose required for a half-maximum effect can be reduced by 3·8-fold when only 1 µmol/l DFP is added. Mechanisms for the synergy are suggested by consideration of the iron-chelate speciation plots together with the size, charge and lipid solubilities for each chelator. Hydroxypyridinones with low lipid solubilities but otherwise similar properties to DFP were used to interrogate the mechanistic interactions of chelator pairs. These studies confirm that synergistic cellular iron mobilization requires one chelator to have the physicochemical properties to enter cells, chelate intracellular iron and subsequently donate iron to a second 'sink' chelator.

Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients.

In transfusional iron overload, extra-hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non-transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond-Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin-iron utilization (DBA).

Biopsy-based calibration of T2* magnetic resonance for estimation of liver iron concentration and comparison with R2 Ferriscan.

BACKGROUND: There is a need to standardise non-invasive measurements of liver iron concentrations (LIC) so clear inferences can be drawn about body iron levels that are associated with hepatic and extra-hepatic complications of iron overload. Since the first demonstration of an inverse relationship between biopsy LIC and liver magnetic resonance (MR) using a proof-of-concept T2* sequence, MR technology has advanced dramatically with a shorter minimum echo-time, closer inter-echo spacing and constant repetition time. These important advances allow more accurate calculation of liver T2* especially in patients with high LIC. METHODS: Here, we used an optimised liver T2* sequence calibrated against 50 liver biopsy samples on 25 patients with transfusional haemosiderosis using ordinary least squares linear regression, and assessed the method reproducibility in 96 scans over an LIC range up to 42 mg/g dry weight (dw) using Bland-Altman plots. Using mixed model linear regression we compared the new T2*-LIC with R2-LIC (Ferriscan) on 92 scans in 54 patients with transfusional haemosiderosis and examined method agreement using Bland-Altman approach. RESULTS: Strong linear correlation between ln(T2*) and ln(LIC) led to the calibration equation LIC = 31.94(T2*)-1.014. This yielded LIC values approximately 2.2 times higher than the proof-of-concept T2* method. Comparing this new T2*-LIC with the R2-LIC (Ferriscan) technique in 92 scans, we observed a close relationship between the two methods for values up to 10 mg/g dw, however the method agreement was poor. CONCLUSIONS: New calibration of T2* against liver biopsy estimates LIC in a reproducible way, correcting the proof-of-concept calibration by 2.2 times. Due to poor agreement, both methods should be used separately to diagnose or rule out liver iron overload in patients with increased ferritin.

Oral ferroportin inhibitor vamifeport for improving iron homeostasis and erythropoiesis in ß-thalassemia: current evidence and future clinical development.

INTRODUCTION: In ß-thalassemia, imbalanced globin synthesis causes reduced red blood cell survival and ineffective erythropoiesis. Suppressed hepcidin levels increase ferroportin-mediated iron transport in enterocytes, causing increased iron absorption and potentially iron overload. Low hepcidin also stimulates ferroportin-mediated iron release from macrophages, increasing transferrin saturation (TSAT), potentially forming non-transferrin-bound iron, which can be toxic. Modulating the hepcidin-ferroportin axis is an attractive strategy to improve ineffective erythropoiesis and limit the potential tissue damage resulting from iron overload. There are no oral ß-thalassemia treatments that consistently ameliorate anemia and prevent iron overload. AREAS COVERED: The preclinical and clinical development of vamifeport (VIT-2763), a novel ferroportin inhibitor, was reviewed. PubMed, EMBASE and ClinicalTrials.gov were searched using the search term 'VIT-2763'. EXPERT OPINION: Vamifeport is the first oral ferroportin inhibitor in clinical development. In healthy volunteers, vamifeport had comparable safety to placebo, was well tolerated and rapidly decreased iron levels and reduced TSAT, consistent with observations in preclinical models. Data from ongoing/planned Phase II studies are critical to define its potential in ß-thalassemia and other conditions associated with iron overabsorption and/or ineffective erythropoiesis. If vamifeport potentially increases hemoglobin and reduces iron-related parameters, it could be a suitable treatment for non-transfusion-dependent and transfusion-dependent ß-thalassemia.

Right ventricular volumes and function in thalassemia major patients in the absence of myocardial iron overload.

AIM: We aimed to define reference ranges for right ventricular (RV) volumes, ejection fraction (EF) in thalassemia major patients (TM) without myocardial iron overload. METHODS AND RESULTS: RV volumes, EF and mass were measured in 80 TM patients who had no myocardial iron overload (myocardial T2* > 20 ms by cardiovascular magnetic resonance). All patients were receiving deferoxamine chelation and none had evidence of pulmonary hypertension or other cardiovascular comorbidity. Forty age and sex matched healthy non-anemic volunteers acted as controls. The mean RV EF was higher in TM patients than controls (males 66.2 +/- 4.1% vs 61.6 +/- 6%, p = 0.0009; females 66.3 +/- 5.1% vs 62.6 +/- 6.4%, p = 0.017), which yielded a raised lower threshold of normality for RV EF in TM patients (males 58.0% vs 50.0% and females 56.4% vs 50.1%). RV end-diastolic volume index was higher in male TM patients (mean 98.1 +/- 17.3 mL vs 88.4 +/- 11.2 mL/m2, p = 0.027), with a higher upper limit (132 vs 110 mL/m2) but this difference was of borderline significance for females (mean 86.5 +/- 13.6 mL vs 80.3 +/- 12.8 mL/m2, p = 0.09, with upper limit of 113 vs 105 mL/m2). The cardiac index was raised in TM patients (males 4.8 +/- 1.0 L/min vs 3.4 +/- 0.7 L/min, p < 0.0001; females 4.5 +/- 0.8 L/min vs 3.2 +/- 0.8 L/min, p < 0.0001). No differences in RV mass index were identified. CONCLUSION: The normal ranges for functional RV parameters in TM patients with no evidence of myocardial iron overload differ from healthy non-anemic controls. The new reference RV ranges are important for determining the functional effects of myocardial iron overload in TM patients.

Extracts of Thai Perilla frutescens nutlets attenuate tumour necrosis factor-α-activated generation of microparticles, ICAM-1 and IL-6 in human endothelial cells.

Elevation of endothelial microparticles (EMPs) play an important role in the progression of inflammation-related vascular diseases such as cardiovascular diseases (CVDs). Thai perilla (Perilla frutescens) nutlets are rich in phenolic compounds and flavonoids that exert potent antioxidant and anti-inflammatory effects. We found that the ethyl acetate (EA) and ethanol (Eth) extracts of Thai perilla nutlets contain phenolic compounds such as luteolin, apigenin, chryseoriol and their glycosides, which exhibit antioxidant activity. The goal of the present study was to investigate the effects of the extracts on endothelial activation and EMPs generation in tumour necrosis factor-α (TNF-α)-induced EA.hy926 cells. We found that TNF-α (10 ng/ml) activated EA.hy926 cells and subsequently generated EMPs. Pre-treatment with the extracts significantly attenuated endothelial activation by decreasing the expression of the intracellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Only the Eth extract showed protective effects against overproduction of interleukin-6 (IL-6) in the activated cells. Furthermore, the extracts significantly reduced TNF-α-enhanced EMPs generation in a dose-dependent manner. In conclusion, Thai perilla nutlet extracts, especially the Eth extract, may have potential to protect endothelium against vascular inflammation through the inhibition of endothelial activation and the generation of endothelial microparticles (EMPs).

Interaction of Transfusion and Iron Chelation in Thalassemias.

The relationship between blood transfusion intensity, chelatable iron pools, and extrahepatic iron distribution is described in thalassemia. Risk factors for cardiosiderosis are discussed with particular reference to the balance of transfusional iron loading rate and transferrin-iron utilization rate as marked by plasma levels of soluble transferrin receptors. Low transfusion regimens increase residual erythropoiesis allowing for apotransferrin-dependent clearance of non-transferrin-bound iron species otherwise destined for myocardium. The impact of transfusion rates on chelation dosing required for iron balance is also shown.

Clinical and methodological factors affecting non-transferrin-bound iron values using a novel fluorescent bead assay.

Nontransferrin-bound iron (NTBI) is a heterogeneously speciated plasma iron, typically detectable when transferrin saturation (TfSat) exceeds 75%. Here, we examine factors affecting NTBI levels by a recently discovered direct chelator-based (CP851) fluorescent bead-linked flow-cytometric assay (bead-NTBI), compared with the established indirect nitrilotriacetate (NTA) assay in 122 iron-overloaded patients, including 64 on recent iron chelation therapy and 13 healthy volunteers. Both methods correlated (r = 0.57, P < 0.0001) but with low agreement, attributable to 2 major factors: (1) the NTA method, unlike the bead method, is highly dependent on TfSat, with NTBI under-estimation at low TfSat and over-estimation once Tf is saturated, (2) the bead method detects <3-fold higher values than the NTA assay in patients on recent deferiprone-containing chelation due to greater detection of chelate complexes but lower values for patients on deferasirox. The optimal timing of sample collection relative to chelation dosing requires further study. Patients with splenectomy, high-storage iron, and increased erythropoiesis had greater discrepancy between assays, consistent with differential access by both methods to the NTBI pools associated with these clinical variables. The bead-NTBI assay has advantages over the NTA assay, being less dependent on TfSat, hence of less tendency for false-negative or false-positive values at low and high TfSat, respectively.