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. We hypothesized that voxelotor improves exercise capacity in youths with SCA. METHODS: In a single-center, open-label, single-arm, longitudinal interventional pilot study (NCT04581356), SCA patients aged 12 and older, stably maintained on hydroxyurea, were treated with 1500 mg voxelotor daily, and performed cardiopulmonary exercise testing before (CPET#1) and after voxelotor (CPET#2). A modified Bruce Protocol was performed on a motorized treadmill, and breath-by-breath gas exchange data were collected. Peak oxygen consumption (peak VO2 ), anaerobic threshold, O2 pulse, VE/VCO2 slope, and time exercised were compared for each participant. The primary endpoint was change in peak VO2 . Hematologic parameters were measured before each CPET. Patient Global Impression of Change (PGIC) and Clinician Global Impression of Change (CGIC) surveys were collected. RESULTS: Ten hemoglobin SS patients aged 12-24 completed the study. All demonstrated expected hemoglobin rise, with average +1.6 g/dL (p = .003) and P50 left shift of average -11 mmHg (p < .0001) with decreased oxygen off-loading at low pO2 . The change in % predicted peak VO2 from CPET#1 to CPET#2 ranged from -12.8% to +11.3%, with significant improvement of more than 5% in one subject, more than 5% decrease in five subjects, and insignificant change of less than 5% in four subjects. All 10 CGIC and seven of 10 PGIC responses were positive. CONCLUSION: In a plot study of 10 youths with SCA, voxelotor treatment did not improve peak VO2 in 9 out of 10 patients.

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. These parameters define the cell as discocyte, sickle, elongated, as well as irregularly or abnormal RBC shaped cells, including echinocytes, holly-leaf, and granular structures. In contrast to microscopy, quick assessment of large numbers of cells provides statistically relevant information of the dynamic process of RBC sickling in time. The use of this approach facilitates the understanding of the processes that define the propensity of sickle blood samples to change their shape, and the ensuing vaso-occlusive events in the circulation of the patients. Moreover, it assists in the evaluation of treatments that include the use of anti-sickling agents, gene therapy-based hemoglobin modifications, as well as other approaches to improve the quality of life of sickle cell patients. © 2019 International Society for Advancement of Cytometry.

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. We previously documented a reduction in soluble biomarkers of inflammation in patients with sickle cell disease treated with simvastatin. To determine the potential clinical efficacy of simvastatin, we treated 19 SCA patients with single daily dose simvastatin for 3 months and assessed changes from baseline in the frequency and intensity of diary-reported pain and levels of circulating nitric oxide metabolites (NOx), high sensitivity C-reactive protein (hs-CRP), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), ICAM-3, E-selectin, and vascular endothelial growth factor (VEGF). Treatment with simvastatin resulted in a significant reduction in the frequency of pain (P = 0·0003), oral analgesic use (P = 0·003) and circulating hs-CRP (P = 0·003), soluble (s)E-selectin (P = 0·01), sICAM-1 (P = 0·02), sICAM-3 (P = 0·02) and sVEGF (P = 0·01). Simvastatin had no effect on pain intensity or levels of NOx, sP-selectin and sVCAM-1. The observed reductions in pain rate and markers of inflammation were greatest in subjects receiving hydroxycarbamide (HC), suggesting a synergistic effect of simvastatin. These results provide preliminary clinical data to support a larger trial of simvastatin in SCA.

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. These results suggest that microparticles may be a clinically useful biomarker of ONFH in SCD. Further investigations are needed to determine the functional relevance of microparticles in the pathogenesis of ONFH in SCD.

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. A single cross-sectional measurement for variables reflecting cardiopulmonary status and biological pathophysiology were obtained, including Doppler-echocardiography, 6-min-walk-test, Borg Dyspnoea Score, New York Heart Association functional class, cardiac magnetic resonance imaging (MRI), chest-computerized tomography, pulmonary function testing and laboratory analyses targeting mechanisms of coagulation, inflammation, haemolysis, adhesion and the arginine-nitric oxide pathway. Twenty-seven thalassaemia patients were evaluated, 14 with an elevated tricuspid-regurgitant-jet-velocity (TRV) ≥ 2·5 m/s. Patients with increased TRV had a higher frequency of splenectomy, and significantly larger right atrial size, left atrial volume and left septal-wall thickness on echocardiography and/or MRI, with elevated biomarkers of abnormal coagulation, lactate dehydrogenase (LDH) levels and arginase concentration, and lower arginine-bioavailability compared to low-risk patients. Arginase concentration correlated significantly to several echocardiography/MRI parameters of cardiovascular function in addition to global-arginine-bioavailability and biomarkers of haemolytic rate, including LDH, haemoglobin and bilirubin. Thalassaemia patients with a TRV ≥ 2·5 m/s have additional echocardiography and cardiac-MRI parameters suggestive of right and left-sided cardiac dysfunction. In addition, low arginine bioavailability may contribute to cardiopulmonary dysfunction in ß-thalassaemia.

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. An elevated TRV (>2.5 m/s) was found in 25/76 (33 %) of the patients, confined to non-transfused or those with a late start of transfusions, including patients with hemoglobin H-constant spring, a finding not previously described. These non or late-transfused patients (76 % splenectomized) had significantly increased platelet activation (sCD40L), high platelet count, endothelial activation (endothelin-1), and hemolysis (LDH, plasma-free Hb), while hypercoagulable and inflammatory markers were not significantly increased. The same markers were increased in the seven patients with confirmed PH on cardiac catheterization, suggesting their possible role for screening patients at risk for PH. A combination of hemolysis and absence of spleen is necessary for developing a high TRV, as neither chronic hemolysis in the non-splenectomized thalassemia patients nor splenectomy without hemolysis, in the non-thalassemia patients, resulted in an increase in TRV.

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. The application of the RoxyScan technology allows the comparison of oxidant effects and the role of antioxidant systems. This provides the opportunity to study the ability of RBC to deal with oxidative stress in various conditions, including RBC disorders such as sickle cell disease (SCD).

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 ß-thalassemia and at increased risk of pulmonary hypertension based on an elevated tricuspid regurgitant jet velocity >2.5 m/s on Doppler-echocardiography. Variables compared at baseline and after 12 weeks of sildenafil treatment included Doppler-echocardiographic parameters, 6-minute walked distance, Borg Dyspnea Score, New York Heart Association functional class, pulmonary function, and laboratory parameters. Treatment with sildenafil resulted in a significant decrease in tricuspid regurgitant jet velocity by 13.3% (3.0±0.7 versus 2.6±0.5 m/s, P=0.04), improved left ventricular end systolic/diastolic volume, and a trend towards a improved New York Heart Association functional class. No significant change in 6-minute walked distance was noted. Sildenafil was well tolerated, although minor expected adverse events were commonly reported. The total dose of sildenafil (mg) was strongly correlated with percent change in nitric oxide metabolite concentration in the plasma (ρ=0.80, P=0.01). There were also significant increases in plasma and erythrocyte arginine concentrations. Our study suggests that sildenafil is safe and may improve pulmonary hemodynamics in patients at risk of pulmonary hypertension; however, it was not demonstrated to improve the distance walked in 6 minutes. Clinical trials are needed to identify the best treatment strategy for pulmonary hypertension in patients with ß-thalassemia. (clinicaltrials.gov identifier: NCT00872170).

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. Heme also intercalates in membranes of blood cells and endothelial cells in the vasculature and associates with other plasma components such as albumin and lipoproteins. Hemopexin has a much higher affinity and can strip heme from the other pools and detoxify plasma from cell-free circulatory heme. However, due to chronic hemolysis, hemopexin is depleted in individuals with sickle cell disease. Thus, cell-free unbound heme is expected to accumulate in plasma. We developed a methodology for the accurate quantification of the fraction of heme, which is pathologically relevant in sickle cell disease, that does not appear to be sequestered to a plasma compartment. Our data show significant variation in the concentration of unbound heme, and rather unexpectedly, the size of the unbound fraction does not correlate to the degree of hemolysis, as measured by the concentration of bound heme. Very high heme concentrations (>150 µM) were obtained in some plasma with unbound concentrations that were several fold lower than in plasma with much lower hemolysis (<50 µM). These findings underscore the long-term effects of chronic hemolysis on the blood components and of the disruption of the essential equilibrium between release of hemoproteins/heme in the circulation and adaptative response of the scavenging/removal mechanisms. Understanding the clinical implications of this loss of response may provide insights into diagnostic and therapeutic targets in patients with sickle cell disease.

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. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.

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. Therefore, we designed a highly controlled protocol that minimized most technical parameters to specifically investigate the impact of blood draw site and analytic device on Hb measurements. A diverse cohort of sixty healthy adults each provided a sequential capillary and venous blood sample that were measured for Hb using an automated hematology analyzer (ADVIA-2120) and two point-of-care devices (HemoCue 201+ and HemoCue 301). Comparing blood draw sites, the mean Hb content was 0.32-0.47 g/dL (2-4%) higher in capillary compared to venous blood from the same donors. Comparing different Hb measuring instruments, the mean Hb content was 0.19-0.46 g/dL (1-4%) higher measured with HemoCue devices compared to ADVIA-2120 in both capillary and venous blood from the same donors. The maximum variance in measurement was also higher with HemoCue devices using blood from venous (5-6% CV) and capillary (21-25% CV) sites compared to ADVIA-2120 (0.6-2% CV). Other variables including blood collection tube manufacturer did not affect mean Hb content. These results demonstrate that even when most technical variables are minimized, the blood draw site and the analytical device can have a small but statistically significant effect on the mean and dispersion of Hb measurements. Even in this study, the few participants identified as mildly anemic using venous blood measured by ADVIA-2120 would not have been classified as anemic using their capillary blood samples or point-of-care analyzers. Thus, caution is warranted when comparing Hb values between studies having differences in blood draw site and Hb measuring device. Future anemia testing should maintain consistency in these analytical variables.

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). Blood was analyzed at baseline, 30/60/90 min/2/3/4/8 hrs. A standardized diet was administered to all participants at 3 established time-points (after 2/5/7hrs). A subset of patients also had pK studies performed without glutamine supplementation to follow normal diurnal fluctuations in amino acids. SETTING: Comprehensive SCD Center in Oakland, California RESULTS: Five patients with SCD were included, three of whom performed pK studies both with and without glutamine supplementation. Average age was 50.6 ± 5.6 years, 60% were female, 40% SS, 60% SC. Plasma glutamine levels increased significantly after oral glutamine supplementation, compared to minimal fluctuations with diet. Plasma glutamine concentration peaked within 30-min of ingestion (p = 0.01) before decreasing to a plateau by 2-h that remained higher than baseline by 8 h. Oral glutamine also increased plasma arginine concentration, which peaked by 4-h (p = 0.03) and remained elevated through 8-h. Erythrocyte glutamine levels began to increase by 8-h, while erythrocyte arginine concentration peaked at 4-h. CONCLUSIONS: Oral glutamine supplementation acutely improved glutamine and arginine bioavailability in both plasma and erythrocytes. This is the first study to demonstrate that glutamine therapy increases arginine bioavailability and may provide insight into shared mechanisms-of-action between these conditionally-essential amino acids.

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. We treated 26 SCD patients with simvastatin, 20 or 40 mg/d, for 21 d. Plasma nitric oxide metabolites (NOx), C-reactive protein (CRP), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), tissue factor (TF) and vascular endothelial growth factor (VEGF) were analyzed and responses to simvastatin were compared between the two treatment groups. Simvastatin increased NOx levels by 23% in the low-dose (P =0·01) and 106% in the moderate-dose (P =0·01) groups, and by 52% overall (P=0·0008). CRP decreased similarly in both dose groups and by 68% overall (P =0·02). Levels of IL-6 decreased by 50% (P=0·04) and 71% (P<0·05) in the low- and moderate-dose groups, respectively. Simvastatin had no effect on VEGF, VCAM1 or TF. Simvastatin was well-tolerated and safe. Our preliminary findings showing a dose-related effect of simvastatin on levels of NOx, CRP and IL-6 suggest a potential therapeutic role for simvastatin 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. Blood and urine cultures, a complete blood count, and serum concentrations of sPLA2 were obtained, and patients were compared based on their final diagnosis of either a viral or bacterial infection. RESULTS: In the 76 patients enrolled, 60 were diagnosed with a viral infection, 14 with a bacterial infection, 1 with Kawasaki disease, and 1 with acute lymphoblastic leukemia. The difference in the serum concentration of sPLA2 in patients with viral infections (22 ± 34 ng/mL) versus those with bacterial infections (190 ± 179 ng/mL) was statistically significant (P < .0001). Receiver operator characteristic curve analysis revealed that sPLA2 was more accurate at predicting bacterial infection (area under the curve = 0.89) than the total white blood cell count (area under the curve = 0.71) and that a value of more than 20 ng/mL had a sensitivity of 93%, specificity of 67%, positive predictive value of 39%, and negative predictive value of 97%. CONCLUSION: Secretory phospholipase A2 differs significantly in children with viral versus bacterial infection and seems to be a reliable screening test for bacterial infection in febrile children.

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. Cells from fresh or cryopreserved placental tissue generated erythroid and myeloid colonies in culture, and also produced lymphoid cells after transplantation in immunodeficient mice. These results suggest that human placenta could become an important new source of hematopoietic cells for allogeneic transplantation.

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 10.2 months). Membrane deformability and cell hydration mildly improved in association with increased HbF levels approaching statistical significance (r = 0.51, P = 0.06). All measures improved considerably in splenctomized patients. These findings underscore the disappointing results of hydroxyurea treatment in clinical trials and the importance of examining the effect on RBC characteristics for the development and understanding of HbF-enhancing agents.

Interaction of an annexin V homodimer (Diannexin) with phosphatidylserine on cell surfaces and consequent antithrombotic activity.

Annexin V (AV), a protein with anticoagulant activity, exerts antithrombotic activity by binding to phosphatidylserine (PS), inhibiting activation of serine proteases important in blood coagulation. The potential use of this protein as an anticoagulant is limited as it rapidly passes from the blood into the kidneys due to its relatively small size (36 kDa). We used recombinant DNA technology to produce a homodimer of human AV (DAV, 73 kDa), which exceeds the renal filtration threshold, and has a 6.5-hour half-life in the rat circulation. Human red blood cells with externalized PS were used to show that DAV had a higher affinity for PS-exposing cells than AV. DAV labeling sensitively identifies PS-exposing cells, was found to be a potent inhibitor of the activity of the prothombinase complexes and inhibits the ability of secretory phospholipaseA(2) to hydrolyze phospholipids of PS-exposing cells, reducing the formation of mediators of blood coagulation and reperfusion injury. DAV exerts dose-dependent antithrombotic activity in rat veins. This combination of activities suggests that DAV is a valuable probe to measure PS exposure and may be efficacious as a novel drug in a wide range of clinical situations.

Featured Article: Depletion of HDL3 high density lipoprotein and altered functionality of HDL2 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 HDL2 particle relative to the smaller higher density pre-ß HDL (HDL3) particle was shifted toward HDL2. This lipoprotein imbalance is exacerbated during acute vaso-occlusive episodes (VOE) as the relative levels of HDL3 decrease. HDL3 deficiency in SCD plasma was found to relate to a slower ApoA-I exchange rate, which suggests an impaired ABCA1-mediated cholesterol efflux in SCD. HDL2 isolated from SCD plasma displayed an antioxidant capacity normally associated with HDL3, providing evidence for a change in function of HDL2 in SCD as compared to HDL2 in normal plasma. Although SCD plasma is depleted in HDL3, this altered capacity of HDL2 could account for the lack of difference in pro-inflammatory HDL levels in SCD as compared to normal. Exposure of human umbilical vein endothelial cells to HDL2 isolated from SCD plasma resulted in higher mRNA levels of the acute phase protein long pentraxin 3 (PTX3) as compared to incubation with HDL2 from control plasma. Addition of the heme-scavenger hemopexin protein prevented increased expression of PTX3 in sickle HDL2-treated cells. These findings suggest that ApoA-I lipoprotein composition and functions are altered in SCD plasma, and that whole blood transfusion may be considered as a blood replacement therapy in SCD. Impact statement Our study adds to the growing evidence that the dysfunctional red blood cell (RBC) in sickle cell disease (SCD) affects the plasma environment, which contributes significantly in the vasculopathy that defines the disease. Remodeling of anti-inflammatory high density lipoprotein (HDL) to pro-inflammatory entities can occur during the acute phase response. SCD plasma is depleted of the pre-ß particle (HDL3), which is essential for stimulation of reverse cholesterol from macrophages, and the function of the larger HDL2 particle is altered. These dysfunctions are exacerbated during vaso-occlusive episodes. Interaction of lipoproteins with endothelium increases formation of inflammatory mediators, a process counteracted by the heme-scavenger hemopexin. This links hemolysis to lipoprotein-mediated inflammation in SCD, and hemopexin treatment could be considered. The use of RBC concentrates in transfusion therapy of SCD patients underestimates the importance of the dysfunctional plasma compartment, and transfusion of whole blood or plasma may be warranted.

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. The functionality of sickle HDL appeared to be altered as evidenced by a decreased HDL-Apolipoprotein A-I exchange in sickle plasma as compared to control. Increased levels of oxidized proteins including ApoA-I were detected in sickle plasma. In vitro incubation of sickle plasma with washed erythrocytes affected the ApoA-I-exchange supporting the view that the RBC blood compartment can affect cholesterol metabolism in plasma. HDL functionality appeared to decrease during acute vaso-occlusive episodes in sickle patients and was associated with an increase of secretory PLA2, a marker for increased inflammation. Simvastatin treatment to improve the anti-inflammatory function of HDL did not ameliorate HDL-ApoA-I exchange in sickle patients. Thus, the cumulative effect of an inflammatory and highly oxidative environment in sickle blood contributes to a decrease in cholesterol esterification and HDL function, related to hypocholesterolemia in SCD.

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. To test this hypothesis, and thereby resolve the "black box" incompleteness in the continuity of the mechanism, the fate of red blood cells incubated with RRx-001 was explored in vitro and in vivo both in healthy animals and in tumor-bearing mice. The collective results establish that RRx-001-derivatized red blood cells are the critical "missing links" to explain the specificity and anticancer activity of RRx-001, including its immunomodulatory effects on tumor-associated macrophages. These experimental results delineate a novel erythrocyte-based mechanism without precedent in the annals of oncology and open the door to rational combination strategies with RRx-001 both in cancer therapy and beyond, particularly in disease states that affect red blood cell and vascular function such as malaria, leishmaniasis, sickle-cell disease and hemorrhagic shock.