Treatment with NS3623, a novel Cl-conductance blocker, ameliorates erythrocyte dehydration in transgenic SAD mice: a possible new therapeutic approach for sickle cell disease

The dehydration of sickle red blood cells (RBCs) through the Ca-activated K channel depends on the parallel movement of Cl ions. To study whether Cl-conductance block might prevent dehydration of sickle RBCs, a novel Cl-conductance inhibitor (NS3623) was characterized in vitro using RBCs from healthy donors and sickle cell patients and in vivo using normal mice and a transgenic mouse model of sickle cell disease (SAD mice). In vitro, NS3623 reversibly blocked human RBC Cl-conductance (gCl) with an IC50 value of 210 nmol/L and a maximal block of 95%. In vivo, NS3623 inhibited RBC gCl after oral administration to normal mice (ED50 = 25 mg/kg). Although gCl, at a single dose of 100 mg/kg, was still 70% inhibited 5 hours after dosing, the inhibition disappeared after 24 hours. Repeated administration of 100 mg/kg twice a day for 10 days caused no adverse effects; therefore, this regimen was chosen as the highest dosing for the SAD mice. SAD mice were treated for 3 weeks with 2 daily administrations of 10, 35, and 100 mg/kg NS3623, respectively. The hematocrit increased, and the mean corpuscular hemoglobin concentration decreased in all groups with a concomitant increase in the intracellular cation content. A loss of the densest red cell population was observed in conjunction with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes, with some echinocytes present at the highest dosage. These data indicate feasibility for the potential use of Cl-conductance blockers to treat human sickle cell disease

Bitopertin, a selective oral GLYT1 inhibitor, improves anemia in a mouse model of \u3b2-thalassemia

Anemia of \u3b2-thalassemia is caused by ineffective erythropoiesis and reduced red cell survival. Several lines of evidence indicate that iron/heme restriction is a potential therapeutic strategy for the disease. Glycine is a key initial substrate for heme and globin synthesis. We provide evidence that bitopertin, a glycine transport inhibitor administered orally, improves anemia, reduces hemolysis, diminishes ineffective erythropoiesis, and increases red cell survival in a mouse model of \u3b2-thalassemia (Hbbth3/+ mice). Bitopertin ameliorates erythroid oxidant damage, as indicated by a reduction in membrane-associated free \u3b1-globin chain aggregates, in reactive oxygen species cellular content, in membrane-bound hemichromes, and in heme-regulated inhibitor activation and eIF2\u3b1 phosphorylation. The improvement of \u3b2-thalassemic ineffective erythropoiesis is associated with diminished mTOR activation and Rab5, Lamp1, and p62 accumulation, indicating an improved autophagy. Bitopertin also upregulates liver hepcidin and diminishes liver iron overload. The hematologic improvements achieved by bitopertin are blunted by the concomitant administration of the iron chelator deferiprone, suggesting that an excessive restriction of iron availability might negate the beneficial effects of bitopertin. These data provide important and clinically relevant insights into glycine restriction and reduced heme synthesis strategies for the treatment of \u3b2-thalassemia

Missense mutations in the ABVB6 transporter cause dominat familial pseudohyperkaliemia

Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by increased serum [K1] inwhole blood stored at or below room temperature, without additional hematological abnormalities. Functionalgene mapping and sequencing analysis of the candidate genes within the 2q35–q36 critical intervalidentified—in 20 affected individuals among three multigenerational FP families—two novel heterozygousmissense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutionsaltered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that, inerythrocyte membranes, bears the Langereis blood group antigen system. The ABCB6 R375Q mutation didnot alter the levels of mRNA or protein, or protein localization in mature erythrocytes or erythroid precursorcells, but it is predicted to modestly alter protein structure. ABCB6 mRNA and protein levels increase duringin vitro erythroid differentiation of CD341 erythroid precursors and the erythroleukemia cell lines HELand K562. These data suggest that the two missense mutations in residue 375 of the ABCB6 polypeptidefound in affected individuals of families with chromosome 2-linked FP could contribute to the red cell K1leak characteristic of this condition

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): study protocol for a two-factorial randomized controlled multi-arm trial with cluster sampling

Background: To achieve higher effectiveness in population-based SARS-CoV-2 surveillance and to reliably predict the course of an outbreak, screening, and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic. Methods: This trial implements a two-factorial, randomized, controlled, multi-arm, prospective, interventional, single-blinded design with cluster sampling and four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy based on individuals' self-collection of saliva samples which are then sent to and analyzed by a laboratory. The targeted sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (2500 participants per arm). Strategies differ with respect to tested population groups (individuals vs. all household members) and testing approach (without vs. with pre-screening survey). The trial is complemented by an economic evaluation and qualitative assessment of user experiences. Primary outcomes include costs per completely screened person, costs per positive case, positive detection rate, and precision of positive detection rate. Discussion: Systems for active surveillance of the general population will gain more importance in the context of pandemics and related disease prevention efforts. The pandemic parameters derived from such active surveillance with routine population monitoring therefore not only enable a prospective assessment of the short-term course of a pandemic, but also a more targeted and thus more effective use of local and short-term countermeasures. Trial registration: ClinicalTrials.gov DRKS00023271. Registered November 30, 2020, with the German Clinical Trials Register (Deutsches Register Klinischer Studien

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in M-thalassemic mice

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From disgusting and complicated to simple and brilliant: Implementation perspectives and lessons learned from users and rejectors of mail-in SARS-CoV-2 gargle tests

BackgroundDespite the important role of testing as a measure against the COVID-19 pandemic, user perspectives on SARS-CoV-2 tests remain scarce, inhibiting an improvement of testing approaches. As the world enters the third year of the pandemic, more nuanced perspectives of testing, and opportunities to expand testing in a feasible and affordable manner merit consideration.MethodsConducted amid the second pandemic wave (late 2020–early 2021) during and after a multi-arm trial evaluating SARS-CoV-2 surveillance strategies in the federal state Baden-Württemberg, Germany, this qualitative sub-study aimed to gain a deeper understanding of how test users and test rejectors perceived mail-in SARS-CoV-2 gargle tests. We conducted 67 semi-structured in-depth interviews (mean duration: 60 min) via telephone or video call. Interviews were audio-recorded, transcribed verbatim and analyzed inductively using thematic analysis. The Consolidated Framework for Implementation Research guided the findings' presentation.ResultsRespondents generally described gargle sampling as simple and comfortable. However, individual perceptions of the testing method and its feasibility varied widely from disgusting and complicated to simple and brilliant. Self-sampling was appreciated for lowering infection risks during testing, but also considered more complex. Gargle-sampling increased participants' self-efficacy to sample correctly. Communication (first contact, quantity and content of information, reminders, support system) and trust (in the study, its institutional affiliation and test method) decisively influenced the intervention's acceptability.ConclusionUser-driven insights on how to streamline testing include: consider communication, first impressions of tests and information as key for successful mail-in testing; pay attention to the role of mutual trust between those taking and administering tests; implement gargle self-sampling as a pleasant alternative to swab testing; offer multiple test methods to increase test up-take

Epeleuton, a novel synthetic ω-3 fatty acid, reduces hypoxia/ reperfusion stress in a mouse model of sickle cell disease

Inflammatory vasculopathy is critical in sickle cell disease (SCD)-associated organ damage. An imbalance between pro-inflammatory and pro-resolving mechanisms in response to different triggers such as hypoxia/reoxygenation or infections has been proposed to contribute to the progression of SCD. Administration of specialized pro-resolving lipid mediators may provide an effective therapeutic strategy to target inflammatory vasculopathy and to modulate inflammatory response. Epeleuton (15 hydroxy eicosapentaenoic acid ethyl ester) is a novel, orally administered, second-generation ω-3 fatty acid with a favorable clinical safety profile. In this study we show that epeleuton re-programs the lipidomic pattern of target organs for SCD towards a pro-resolving pattern. This protects against systemic and local inflammatory responses and improves red cell features, resulting in reduced hemolysis and sickling compared with that in vehicle-treated SCD mice. In addition, epeleuton prevents hypoxia/reoxygenation-induced activation of nuclear factor-κB with downregulation of the NLRP3 inflammasome in lung, kidney, and liver. This was associated with downregulation of markers of vascular activation in epeleuton-treated SCD mice when compared to vehicle-treated animals. Collectively our data support the potential therapeutic utility of epeleuton and provide the rationale for the design of clinical trials to evaluate the efficacy of epeleuton in patients with SCD

Resveratrol Induces Erythroid Maturation by Activating FOXO3 and Improves in Vivo Erythropoiesis in Normal and Beta -Thalassemic Mice

Resveratrol is a polyphenolic stilbene with anti-oxidant, anti-inflammatory and anti-tumoral bioactivities . High concentrations of resveratrol (50 \u3bcM) have been reported to induce HbF synthesis in an in vitro model of normal and beta-thalassemic erythropoiesis (Fibach E. Int J Mol Med 2012; Rodrigue CM. BJH 2001) and to improve erythropoiesis in a mouse model for Fanconi Anemia (Zhang Q. Blood 2010). Beta thalassemia (b-thal) is characterized by ineffective erythropoiesis and increased cellular oxidative stress. We studied the effects of resveratrol (5 \ub5M) on erythropoiesis in vitro from peripheral CD34+ cells of healthy and b-thal subjects. Erythroid maturation was evaluated at 7, 9, 11 and 14 days of culture by cytofluorimetric analysis using the CD71-GPA-CD36 strategy that allows to separate CFU-E, Pro-E, Int-E and Late-Erythroblasts (Merryweather-Clarke AT. Blood 2011). Resveratrol reduced cell growth in both cell types, with a reduction of CFU-E, increased Int-E at day 7 and 9, and increased Int-E and Late-E at 11 and 14 days. The early maturation of erythroid progenitors was confirmed by morphological analysis of the cells. We sorted CFU-E cells (at 7 days) from resveratrol treated and untreated cells and analyzed the cell cycle, cyclinD1 and p21 expression. In both cell types resveratrol induced increased frequency of S-G2/M cells compared to untreated cells with increased p21 levels, suggesting decreased cycling of CFU-E with increased maturation of erythroblasts. No changes of gamma chain mRNA levels were present in cells treated with resveratrol (5 \ub5M). Since FOXO3 is a key regulator of erythroid redox required for normal erythroid maturation (Marinkovic D. JCI 2007), FOXO3 expression and activity was assessed in sorted CFU (7day) and Int-E (11 day) with and without resveratrol. FOXO3a mRNA levels were increased in resveratrol treated cells in both sorted cell populations. We used nuclear localization as a surrogate assay for FOXO3a activity and found resveratrol increased the overall expression of FOXO3 protein in the nucleus without impacting significantly the nuclear/cytoplasmic ratio. Interestingly, resveratrol did not appear to modify FOXO1 expression or subcellular localization. These results suggest that resveratrol enhances specifically expression of FOXO3 in human erythroblasts. Dietary resveratrol supplementation (2.4 mg/Kg) was studied in wild-type and Hbb3th+/- mice (2 months of age) for 6 months. In resveratrol Hbb3th+/- treated mice increased Hb levels (8.3\ub10.6 vs 10.3\ub10.5 g/dL, n=12; P<0.05) and decreased reticulocyte count (33.9\ub10.8 vs 23.7\ub1 8.2 %, n=12; P<0.05) were observed. Significant increased MCV (34.6\ub10.6 vs 41.6\ub1 5.4 fL, n=12; P<0.05) and MCH ( 9.7\ub1 0.6 vs 12.8 \ub1 2.1 pg, n=12; P<0.05) were also noted. Flow cytometric evidence of decreased ineffective erythropoiesis and reduced spleen/ body weight ratio were also observed. These data indicate that resveratrol affects erythroid maturation both in vitro and in vivo, and that these effects have possible therapeutic relevance for the treatment of thalassemias

Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD341 cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells