FT-4202, a selective pyruvate kinase R activator for sickle cell disease.

Anemia in patients with sickle cell disease (SCD) increases 2,3-diphosphoglycerate (2,3-DPG), decreasing hemoglobin-oxygen (HbO) affinity to improve oxygen offloading and promote hemoglobin polymerization (sickling) of red blood cells (RBCs). We report the discovery of FT-4202, an investigational, selective pyruvate kinase type-R (PKR) activator with a multimodal mechanism of action and potential to increase ATP and decrease 2,3-DPG, resulting in increased HbO affinity, decreased Hb polymerization, and improved RBC health. FT-4202 was identified via structure-enabled lead optimization medicinal chemistry using X-ray crystallography, molecular modeling, and thermal shift assays.

The RoxyScan is a novel measurement of red blood cell deformability under oxidative and shear stress.

Exposure to both oxidative and shear stress, a condition that the red blood cell (RBC) continuously experiences in the circulation in vivo can be mimicked in a Couette type viscometer and monitored by ektacytometry. RBCs maintain their deformation and orientation under shear stress and oxidative stress until a threshold is reached at which these conditions appear to overwhelm the elaborate and complex pathways that maintain a proper redox environment in the cell. Oxidative stress under shear alters the ability of the cell to deform, changes cell morphology, its orientation in the shear stress field, and appears to alter intracellular and membrane characteristics.

Effect of voxelotor on cardiopulmonary testing in youths with sickle cell anemia in a pilot study.

Background: Individuals with sickle cell anemia (SCA) exhibit decreased exercise capacity. Anemia limits oxygen-carrying capacity and affects cardiopulmonary fitness. The drug voxelotor raises hemoglobin in SCA.

Assessment of total and unbound cell-free heme in plasma of patients with sickle cell disease.

Intravascular hemolysis results in the release of cell-free hemoglobin and heme in plasma. In sickle cell disease, the fragility of the sickle red blood cell leads to chronic hemolysis, which can contribute to oxidative damage and activation of inflammatory pathways. The scavenger proteins haptoglobin and hemopexin provide pathways to remove hemoglobin and heme, respectively, from the circulation.

Blood draw site and analytic device influence hemoglobin measurements.

Anemia is a continuing global public health concern and a priority for international action. The prevalence of anemia is estimated from the hemoglobin (Hb) levels within target populations, yet the procedures for measuring Hb are not standardized and different approaches may result in discrepancies. Several analytical variables have been proposed to influence Hb measurements, but it is difficult to understand the impact on specific variables from large population or field studies.

Implications for the metabolic fate of oral glutamine supplementation within plasma and erythrocytes of patients with sickle cell disease: A pharmacokinetics study.

Objectives: L-Glutamine is FDA-approved for sickle cell disease (SCD), yet the mechanism(s)-of-action are poorly understood. We performed a pharmacokinetics (pK) study to determine the metabolic fate of glutamine supplementation on plasma and erythrocyte amino acids in patients with SCD.

Design: A pK study was performed where patients with SCD fasting for > 8 h received oral L-glutamine (10 g).

Vincristine-induced anemia in hereditary spherocytosis.

Unlabelled: Vincristine, a vinca alkaloid, is widely used in many hematologic disorders and pediatric cancers, and acts by binding to the tubulin protein, to inhibit effective cell division. Vincristine-induced anemia has been observed, but its mechanism is not well understood. We describe a case involving serious vincristine-induced anemia in a patient with congenital spherocytosis and provide the explanation to the underlying mechanism.

Image-Based Flow Cytometry and Angle-Resolved Light Scattering to Define the Sickling Process.

Red blood cells (RBCs) from sickle cell patients exposed to a low oxygen tension reveal highly heterogeneous cell morphologies due to the polymerization of sickle hemoglobin (HbS). We show that angle-resolved light scattering approach with the use of image-based flow cytometry provides reliable quantitative data to define the change in morphology of large populations of RBCs from sickle cell patients when the cells are exposed for different times to low oxygen. We characterize the RBC morphological profile by means of a set of morphological and physical parameters, which includes cell shape, size, and orientation.

Featured Article: Depletion of HDL high density lipoprotein and altered functionality of HDL in blood from sickle cell patients.

In sickle cell disease (SCD), alterations of cholesterol metabolism is in part related to abnormal levels and activity of plasma proteins such as lecithin cholesterol acyltransferase (LCAT), and apolipoprotein A-I (ApoA-I). In addition, the size distribution of ApoA-I high density lipoproteins (HDL) differs from normal blood. The ratio of the amount of HDL particle relative to the smaller higher density pre-β HDL (HDL) particle was shifted toward HDL.

Simvastatin reduces vaso-occlusive pain in sickle cell anaemia: a pilot efficacy trial.

Sickle cell anaemia (SCA) is a progressive vascular disease characterized by episodic vaso-occlusive pain. Despite the broad impact of inflammation on acute and chronic clinical manifestations of SCA, no directed anti-inflammatory therapies currently exist. Statins are cholesterol-lowering agents shown to confer protection from vascular injury by suppressing inflammation.

Targeting tumor hypoxia with the epigenetic anticancer agent, RRx-001: a superagonist of nitric oxide generation.

This study reveals a novel interaction between deoxyhemoglobin, nitrite and the non-toxic compound, RRx-001, to generate supraphysiologic levels of nitric oxide (NO) in blood. We characterize the nitrite reductase activity of deoxyhemoglobin, which in the presence of bound RRx-001 reduces nitrite at a much faster rate, leading to markedly increased NO generation. These data expand on the paradigm that hemoglobin generates NO via nitrite reduction during hypoxia and ischemia when nitric oxide synthase (NOS) function is limited.

Featured Article: Alterations of lecithin cholesterol acyltransferase activity and apolipoprotein A-I functionality in human sickle blood.

In sickle cell disease (SCD) cholesterol metabolism appears dysfunctional as evidenced by abnormal plasma cholesterol content in a subpopulation of SCD patients. Specific activity of the high density lipoprotein (HDL)-bound lecithin cholesterol acyltransferase (LCAT) enzyme, which catalyzes esterification of cholesterol, and generates lysoPC (LPC) was significantly lower in sickle plasma compared to normal. Inhibitory amounts of LPC were present in sickle plasma, and the red blood cell (RBC) lysophosphatidylcholine acyltransferase (LPCAT), essential for the removal of LPC, displayed a broad range of activity.

A look inside the mechanistic black box: Are red blood cells the critical effectors of RRx-001 cytotoxicity?

The therapeutic potential of epi-immunotherapeutic anticancer agent RRx-001 in cancer has been validated with preclinical and clinical studies, since RRx-001 has successfully completed a phase 1 trial and multiple single-agent and combination phase 2 trials with preliminary evidence of promising activity are underway. Previous experimental work has implicated diverse anticancer mechanisms such as oxidative stress, ATP and NADPH depletion, anti-angiogenesis and epigenetic modulation in the overall antitumor effect of RRx-001. The hypothesis of this study was that the RRx-001 red blood cells are the essential and de facto intermediaries responsible for the reprograming of tumor behavior via transfer of their intracellular and membrane contents.

Dysregulated arginine metabolism and cardiopulmonary dysfunction in patients with thalassaemia.

Pulmonary hypertension (PH) commonly develops in thalassaemia syndromes, but is poorly characterized. The goal of this study was to provide a comprehensive description of the cardiopulmonary and biological profile of patients with thalassaemia at risk for PH. A case-control study of thalassaemia patients at high versus low PH-risk was performed.

Microparticles as biomarkers of osteonecrosis of the hip in sickle cell disease.

Osteonecrosis of the femoral head (ONFH) is a common complication of sickle cell disease (SCD). To examine the association between microparticles and ONFH in SCD, we compared plasma microparticle levels in 20 patients with and without ONFH. Microparticles were quantified using nanoparticle tracking analysis and found to be 2·3-fold higher in patients with ONFH compared to patients without ONFH, and 2·5-fold higher than in healthy controls.

The capacity of red blood cells to reduce nitrite determines nitric oxide generation under hypoxic conditions.

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation.

Elevated tricuspid regurgitant jet velocity in subgroups of thalassemia patients: insight into pathophysiology and the effect of splenectomy.

A high tricuspid regurgitant jet velocity (TRV) signifies a risk for or established pulmonary hypertension (PH), which is a serious complication in thalassemia patients. The underlying pathophysiology in thalassemia subgroups and potential biomarkers for early detection and monitoring are not well defined, in particular as they relate to spleen removal. To better understand some of these unresolved aspects, we examined 76 thalassemia patients (35 non-transfused), 25 splenectomized non-thalassemia patients (15 with hereditary spherocytosis), and 12 healthy controls.

RBC deformability and amino acid concentrations after hypo-osmotic challenge may reflect chronic cell hydration status in healthy young men.

Biomarkers of chronic cell hydration status are needed to determine whether chronic hyperosmotic stress increases chronic disease risk in population-representative samples. In vitro, cells adapt to chronic hyperosmotic stress by upregulating protein breakdown to counter the osmotic gradient with higher intracellular amino acid concentrations. If cells are subsequently exposed to hypo-osmotic conditions, the adaptation results in excess cell swelling and/or efflux of free amino acids.

Sildenafil therapy in thalassemia patients with Doppler-defined risk of pulmonary hypertension.

Pulmonary hypertension is a common but often overlooked complication associated with thalassemia syndromes. There are limited data on the safety and efficacy of selective pulmonary vasodilators in this at-risk population. We, therefore, designed a 12-week, open-label, phase 1/2, pilot-scale, proof-of-principle trial of sildenafil therapy in 10 patients with β-thalassemia and at increased risk of pulmonary hypertension based on an elevated tricuspid regurgitant jet velocity >2.

Compromised mitochondrial fatty acid synthesis in transgenic mice results in defective protein lipoylation and energy disequilibrium.

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan.

A pilot study of the short-term use of simvastatin in sickle cell disease: effects on markers of vascular dysfunction.

Sickle cell disease (SCD) is characterized by progressive vascular injury and its pathophysiology is strikingly similar to that of atherosclerosis. Statins decrease inflammation and improve endothelial function in cardiovascular disease, but their effect in SCD is not known. In this pilot study, we examined the safety and effect of short-term simvastatin on biomarkers of vascular dysfunction in SCD.

Secretory phospholipase A2: a marker of infection in febrile children presenting to a pediatric ED.

Background: Fever is a common presenting complaint to the emergency department (ED), and the evaluation of the febrile child remains a challenging task.

Objective: The aim of this study was to examine the relationship between secretory phospholipase A2 (sPLA2) and infection in febrile children.

Methods: A prospective convenience sample of children presenting with fever to an urban pediatric ED were studied.

Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of tropomodulin 3 in red blood cells and disruption of the membrane skeleton.

The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton.

Human term placenta as a source of hematopoietic cells.

The main barrier to a broader clinical application of umbilical cord blood (UCB) transplantation is its limiting cellular content. Thus, the discovery of hematopoietic progenitor cells in murine placental tissue led us investigate whether the human placenta contains hematopoietic cells, sites of hematopoiesis, and to develop a procedure of processing and storing placental hematopoietic cells for transplantation. Here we show that the human placenta contains large numbers of CD34-expressing hematopoietic cells, with the potential to provide a cellular yield several-fold greater than that of a typical UCB harvest.

Hydroxycarbamide-induced changes in E/beta thalassemia red blood cells.

In thalassemia, fetal hemoglobin (HbF) augmentation with hydroxycarbamide (also known as hydroxyurea) is not always successful. The expected parallel effects on red cell (RBC) membrane deformability, cell hydration, and membrane phospholipid organization, all important for extending RBC life span and increasing Hb, have been infrequently examined. We analyzed these characteristics in 15 nontransfused E/beta(0) thalassemia patients treated with HU (mean 10.