Erythroblastic islands foster granulopoiesis in parallel to terminal erythropoiesis.

The erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry, in vitro island reconstitution, and single-cell RNA sequencing of adult mouse bone marrow (BM) EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with BM EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis and erythropoiesis.

Etavopivat, a Pyruvate Kinase Activator in Red Blood Cells, for the Treatment of Sickle Cell Disease.

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability.

Safety, Pharmacokinetics, and Pharmacodynamics of Etavopivat (FT-4202), an Allosteric Activator of Pyruvate Kinase-R, in Healthy Adults: A Randomized, Placebo-Controlled, Double-Blind, First-in-Human Phase 1 Trial.

Etavopivat (FT-4202) is an orally administered, small-molecule allosteric activator of erythrocyte pyruvate kinase-R (PKR) in clinical development for the treatment of sickle cell disease and other hemoglobin disorders. This randomized, placebo-controlled, double-blind, first-in-human combination single-ascending dose and multiple-ascending dose phase 1 trial (NCT03815695) evaluated the safety and pharmacokinetics/pharmacodynamics of etavopivat in 90 healthy adult subjects. In 4 single-ascending dose cohorts, 8 participants were randomized 3:1 to a single oral dose of either etavopivat (n = 6) or placebo (n = 2).

Automated Oxygen Gradient Ektacytometry: A Novel Biomarker in Sickle Cell Anemia.

Sickle cell anemia (SCA) is a hereditary hemoglobinopathy with a variable phenotype. There is no single biomarker that adequately predicts disease severity and can be used to monitor treatment response in patients in clinical trials and clinical care. The use of clinical outcomes, such as vaso-occlusive crises (VOC), requires long and expensive studies, sometimes with inconclusive results.

Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation.

RhoA GTPase has been shown in vitro in cell lines and in vivo in nonmammalian organisms to regulate cell division, particularly during cytokinesis and abscission, when 2 daughter cells partition through coordinated actomyosin and microtubule machineries. To investigate the role of this GTPase in the rapidly proliferating mammalian erythroid lineage, we developed a mouse model with erythroid-specific deletion of RhoA. This model was proved embryonic lethal as a result of severe anemia by embryonic day 16.

Gene targeting RhoA reveals its essential role in coordinating mitochondrial function and thymocyte development.

Thymocyte development is regulated by complex signaling pathways. How these signaling cascades are coordinated remains elusive. RhoA of the Rho family small GTPases plays an important role in actin cytoskeleton organization, cell adhesion, migration, proliferation, and survival.

Identification of a murine erythroblast subpopulation enriched in enucleating events by multi-spectral imaging flow cytometry.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.

Erythrocyte NADPH oxidase activity modulated by Rac GTPases, PKC, and plasma cytokines contributes to oxidative stress in sickle cell disease.

Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease (SCD). One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle red blood cells (RBC). Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane.

Signaling and cytoskeletal requirements in erythroblast enucleation.

To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3.

Effect of epinephrine and insulin resistance on human monocytes obtained from lean and obese healthy participants: a pilot study.

We assessed the effect of epinephrine on human monocytes. Monocytes were isolated from 16 healthy obese and 10 lean healthy subjects. Insulin sensitivity was assessed by euglycemic hyperinsulinemic clamp.

The ambiguous role of the Na+-H+ exchanger isoform 1 (NHE1) in leptin-induced oxidative stress in human monocytes.

Leptin, a 16-kDa cytokine produced mainly by the adipose tissue, is known to increase energy expenditure while at the same time lowering food intake by acting directly on the hypothalamus. ObRb, the leptin receptor mostly involved in intracellular signaling, is expressed in a wide range of tissues, thus allowing leptin to affect a much broader diversity of biological processes. High concentrations of leptin are encountered in patients with hyperleptinemia, a condition which very often accompanies obesity and which is a direct result of leptin resistance.

Signaling components involved in leptin-induced amplification of the atherosclerosis-related properties of human monocytes.

Background/aims: Leptin, a 16-kDa cytokine that is released mainly by the adipose tissue, is known to affect a wide assortment of processes, ranging from energy homeostasis to angiogenesis and the immune response. In the present study, the effect of leptin on atherosclerosis-related properties of human monocytes was investigated. methods: Monocytes were isolated from whole blood obtained from healthy donors who had normal body mass index values.

Inhibition of the Na+-H+ exchanger isoform-1 and the extracellular signal-regulated kinase induces apoptosis: a time course of events.

Aims: The present study attempts to shed light on the role and the relative position of the Na(+)/H(+) exchanger isoform 1 (NHE1) and the extracellular signal-regulated kinase (ERK) in HEp-2 cell signaling pathways concerning a diverse range of cellular functions such as regulation of intracellular pH (pHi), DNA synthesis, production of reactive oxygen species (ROS) and apoptosis.

Methods: Pharmacological inhibition with cariporide (highly specific inhibitor of NHE1) and PD98059 (specific inhibitor of the upstream activator of ERK) was implemented. Fluorescence spectrometry, atomic absorption spectrometry and ELISA methods were used in order to obtain the results.