Hemoglobin Release Research Articles

Dysregulated Arginine Metabolism and Cardiopulmonary Dysfunction in Patients with Thalassemia

Pulmonary hypertension (PH) is common in thalassemia and contributes to mortality. Advancing age and a history of splenectomy are major risk factors in this population. The etiology of PH is multifactorial, involving a complex interaction of platelets, the coagulation system, erythrocytes, and endothelial cells along with inflammatory and vascular mediators. The long-term effect of splenectomy, red cell membrane pathology, coagulation abnormalities, low nitric oxide (NO) bioavailability, excess arginase activity, platelet activation, oxidative stress, iron overload, and chronic hemolysis play a role. The process of hemolysis disables the arginine-NO pathway through the simultaneous release of erythrocyte arginase and cell-free hemoglobin. Both NO and its obligate substrate arginine are rapidly consumed. The biological consequences of hemolysis on NO bioavailability ultimately translate into the clinical manifestations of PH. Guidelines for the management of PH in thalassemia have not yet been established; however, clinical trials are ongoing in an effort to guide future therapy.

Development of an in vitro system and model-based translational framework to assess haemolysis risk following intravenous abuse of medications containing polyethylene oxide

Multiple cases of potentially life-threatening thrombotic microangiopathy (TMA) have resulted from intravenous abuse of medications containing polyethylene oxide (PEO), most often Opana ER (oxymorphone hydrochloride extended release). No validated models are available to assess the risk of TMA with different formulations and extraction methods following intravenous abuse. We have developed an in vitro system that involves passing pooled blood containing the excipient of interest through a syringe needle and assessing haemolysis via haemoglobin release. Haemolysis is induced by high shear stress caused by the flow of blood containing PEO through a narrow-bore syringe needle, recapitulating the mechanism in small blood vessels. Using the in vitro system, we demonstrate that high-molecular-weight PEO (>1 MDa) induces haemolysis in a concentration-dependent manner under flowing but not static conditions. We use data from the in vitro system and published in vivo data to predict the time course of the haemolytic response in vivo via a pharmacometric model. The in vitro system is a novel method for investigating factors influencing PEO-induced haemolysis. In combination with our model-based translational framework, the in vitro system allows straightforward assessment of the haemolytic potential of PEO-containing medications, and may find application in gauging TMA risk following intravenous abuse.

Polyelectrolyte stiffness on gold nanorods mediates cell membrane damage.

Charge and surface chemistry of gold nanorods (AuNRs) are often considered the predictive factors for cell membrane damage. Unfortunately, extensive research on AuNR passivated with polyelectrolyte (PE) ligand shell (AuNR-PE) has hitherto left a vital knowledge gap between the mechanical stability of the ligand shell and the cytotoxicity of AuNR-PEs. Here, the agreement between unbiased coarse-grained molecular dynamics (CGMD) simulation and empirical outcomes on hemolysis of red blood cells by AuNR-PEs demonstrates for the first time, a direct impact of the mechanical stability of the PE shell passivating the AuNRs on the lipid membrane rupture. Such mechanical stability is ultimately modulated by the rigidity of the PE components. The CGMD simulation results also reveal the mechanism where the PE chain adsorbs near the surface of the lipid bilayer without penetrating the hydrophobic core of the bilayer, which allows the hydrophobic AuNR core to be in direct contact with the hydrophobic interior of the lipid bilayer, thereby perforating the lipid membrane to induce membrane damage.

Thiram-induced cytotoxicity and oxidative stress in human erythrocytes: an in vitro study

Tetramethylthiuram disulfide, commonly known as thiram, is an organosulfur compound which is used as a bactericide, fungicide and ectoparasiticide to prevent disease in seeds and crops. Being a fungicide there is a high probability of human occupational exposure to thiram and also via consumption of contaminated food. In this work, the cytotoxicity of thiram was studied under in vitro conditions using human erythrocytes as the cellular model. Erythrocytes were incubated with different concentrations of thiram (25–500 μM) for 4 h at 37 °C. Control cells (thiram untreated) were similarly incubated at 37 °C. Whole cells and hemolysates were analyzed for various biochemical parameters. Treatment of erythrocytes with thiram increased protein and lipid oxidation and hydrogen peroxide level in hemolysates but decreased glutathione and total sulfhydryl group content. This was accompanied by hemoglobin oxidation, heme degradation and release of free iron. Activities of all major antioxidant enzymes were inhibited. The antioxidant power of thiram treated erythrocytes was lowered resulting in decreased metal reducing and free radical quenching ability. These results suggest that thiram enhances the generation of reactive species that cause oxidative modification of cell components. This was confirmed by experiments that showed enhanced generation of reactive oxygen and nitrogen species in thiram treated erythrocytes. Activities of marker enzymes of glucose metabolism and erythrocyte membrane were also inhibited. All effects were seen in a thiram concentration-dependent manner. Electron microscopy further supported the damaging effect of thiram on erythrocytes. Thus thiram induces oxidative stress condition in human erythrocytes and causes oxidative modification of cell components.

Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021

Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by autochthonous Lactobacillus casei TA0021 strain. The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed. The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of >9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold. The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.

Thrombomodulin and Endothelial Dysfunction in Sickle Cell Anemia

Thrombomodulin (THBD) is a transmembrane protein that regulates endothelial function by 1) binding thrombin and inhibiting its interaction with fibrinogen, 2) augmenting protein C activation, and 3) down-regulating complement activation by inactivating C3a and C5a (PMID 29866818). Reduced THBD function has been implicated in several vasculopathies (PMID 29866818). Oxidative damage increases cleaved non-functional THBD in circulation and increased circulating non-functional THBD predicts the severity of organ damage in thrombotic microangiopathies (PMID 11190905). Vascular endothelial dysfunction is a hallmark feature of sickle cell disease (SCD) that leads to acute and chronic organ damage and may, in part, be mediated by hemolysis (PMID 28248201). Hemolysis leads to release of cell-free hemoglobin which may lead to vasculopathy through consumption of NO, activation of TLR4 pathways, and direct oxidative damage. We investigated whether decreased THBD activity may be implicated in the pathophysiology of SCD vasculopathy in endothelial cells, transgenic sickle mice, and in a prospective cohort of patients with sickle cell anemia. In vitro: We exposed endothelial cells (EA.hy926, ATTC® CRL-2922TM; Manassas, VA) to incremental doses of cell-free hemoglobin. With higher doses of cell-free hemoglobin we observed reduced surface endothelial cell THBD activity at 6 hours of incubation, assessed by cleavage of chromogenic substrate for activated protein C in the presence of thrombin (Figure 1A). In conjunction with the reduced endothelial THBD activity, there were increased concentrations of cleaved THBD in the supernatant by ELISA (R&D Systems, Minneapolis, MN) (Figure 1B). Transgenic sickle mice: At 6 months of age, transgenic sickle mice (Townes model, Jackson Laboratory; Bar Harbor, Maine) had higher plasma concentrations of cleaved non-functional THBD versus hemoglobin AA mice (Figure 2A). Furthermore, staining of the glomerular microvasculature demonstrated decreased endothelial-bound THBD (Figure 2B). Patients with sickle cell anemia: We evaluated whether plasma concentrations of cleaved non-functional THBD are predictive of acute multiorgan failure syndrome (PMID 8109600) in a cohort of 103 SCD patients recruited into a longitudinal kidney cohort study. Clinical and laboratory variables and plasma samples were obtained at the time of enrolment and the patients were monitored prospectively for acute multiorgan failure syndrome. Hemoglobinuria was defined by urine dipstick positive for blood with < 2 red blood cells/high power field. The median age of this cohort was 35 years (interquartile range, 28 - 44 years), 46% were female, 87% had hemoglobin SS genotype, and 47% were on hydroxyurea at the time of enrolment. In cross-sectional analysis, plasma THBD concentrations were greater in patients with hemoglobinuria, a marker of intravascular hemolysis-derived cell-free hemoglobin in circulation exceeding scavenging capability and filtering through the glomerulus, versus without hemoglobinuria (6.1 ± 0.6 µg/mL vs. 3.6 ± 0.4 µg/mL, P = 0.004) (Figure 3A). With a median follow up of 5.5 years (interquartile range, 1.4 - 5.9 years), 18 (17%) SCD patients had a multiorgan failure event. SCD patients with a multiorgan failure event were older (43 vs. 34 years, P = 0.01) but without significant differences in sex, SCD genotype, or hydroxyurea therapy. After adjusting for age, baseline THBD concentrations predicted a greater risk for multi-organ failure syndrome (log-transformed OR 4.0, 95% CI: 1.2 - 13.3; P = 0.01) (Figure 3B). In conclusion, circulating non-functional THBD, a protein that normally functions to reduce vasculopathy when bound to the endothelium, is increased after endothelial cell exposure to incremental doses of hemoglobin in vitro, in hemoglobin SS vs. AA mice, and in SCD patients with versus without hemoglobinuria. Furthermore, circulating THBD is a biomarker that predicted the risk for multi-organ failure syndrome on longitudinal follow up in SCD patients. Future studies investigating the role of THBD in SCD vasculopathy may help improve our understanding for the catastrophic multiorgan failure syndrome and therapies to augment endothelial cell THBD function may guide future intervention practices. Figure 1 Disclosures Gordeuk: Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding. Saraf:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

The Role of SOD2 in Maintaining Endothelial Cell Function in SCD

Sickle cell disease (SCD) is characterized by increased oxidative stress. Sources of oxidative stress include (1) intermittent vascular occlusion resulting in hypoxia-reoxygenation induced activation of prooxidant enzymes such as xanthine oxidase and NADPH oxidase, (2) increased fragility of red blood cell membranes causing hemolysis and release of free hemoglobin and heme which produce reactive oxygen species (ROS) through Fenton chemistry, and (3) respiratory chain leaked electrons reacting with oxygen to form superoxide. In SCD, the antioxidant defense system has been shown to be insufficient in its response to increased ROS production. In particular, peripheral blood expression of the mitochondrial targeted antioxidant superoxide dismutase 2 (SOD2) is decreased in SCD patients relative to non-sickle controls. We hypothesize that depletion of SOD2 may modify the progression of sickle pathology. In order to test our hypothesis we (Aim 1) genotyped 410 SCD patients from the Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (walk-PHaSST) study for a common polymorphism of SOD2 (rs4880), valine to alanine on the 16thamino acid (V16A), and investigated whether or not the polymorphism is associated with clinical indicators of endothelial dysfunction. (Aim 2) In primary human pulmonary arterial endothelial cells (hPAECs) we utilized an siRNA mediated knockdown of SOD2 (SOD2 KD) in order to examine its role in maintaining endothelial cell permeability. Permeability was measured by using electric-cell substrate impedance sensing (ECIS). In Aim 1, among the 410 SC anemia patients 129 (31%), 64 (16%) and 217 (53%) were homozygotes for the common valine allele (TT), homozygotes for the variant alanine allele (CC) and heterozygotes (TC), respectively. We examined association of each genotype with the following clinical parameters: history of pulmonary embolism, systolic blood pressure (SBP), pulse blood pressure, hemoglobin, mean corpuscular value, reticulocyte count, white blood cell count, platelet count, tricuspid regurgitant velocity (TRV), left mass index, right atrial area, left ventricle, right ventricular area at systolic, creatinine, six minute walk distance (6MWD). We found that homozygotes of alanine variant (CC) had higher systolic blood pressure (p=0.011), higher TRV (p=0.004), larger right ventricular area at systolic (p=0.023), as well as shorter 6MWD (p=0.006). All four of these clinical parameters are strong indicators of vasculopathy and endothelial damage. Based on our clinical findings, we extended our studies to isolated endothelial cells to define the role of SOD2 in endothelial cell permeability. In Aim 2, we used ECIS in order to measure resistance in SOD2 KD hPAECs. Decreased resistance as measured by ECIS has been shown to be an indicator of increased permeability. We found that SOD2 KD hPAECs had decreased baseline resistance as compared to hPAECs treated with a non-targeting siRNA sequence (siNT). This data supports that SOD2 plays a role in maintaining endothelial cell barrier function. We also, investigated whether free hemin would further accentuate endothelial barrier dysfunction in SOD2 KD cells. We serum starved siNT and siSOD2 KD hPAECs for four hours before treating with 2 mM hemin and measuring resistance. We found that after four hours of 2 mM hemin treatment, there was no further reduction of resistance in SOD2 KD hPAECs as compared to siNT hPAECs. In conclusion, we have found that in SCD patients SOD2 V16A is associated with clinically significant indicators of endothelial dysfunction and SOD2 is essential for the maintenance of endothelial cell barriers in hPAECs. Future directions will be aimed at further investigating the SOD2 V16A polymorphism, specifically we are interested in whether the polymorphism plays a role in barrier function. We will also investigate the pathways by which SOD2 depletion mediates endothelial cell barrier function. Taken together, our preliminary findings suggest that SOD2 functions as an essential mediator of endothelial function in SCD and thus can be used as a target for future SCD therapeutics. Disclosures Straub: Bayer Pharmaceuticals: Research Funding.

Structural Studies, Antimicrobial Activity and Protein Interaction of Photostable Terpyridine Silver(I) Complexes

Photostable mono‐ and binuclear silver(I) complexes derived from terpyridine ligands (4′‐(2‐pyridyl)‐2,2′:6′,2′′‐terpyridine (LPY) (1), 4′‐(4‐phenyl morpholine)‐2,2′:6′,2′′‐terpyridine (Lmorph) (2) and 1,4‐bis (2,2′:6′,2′′‐terpyridin‐4′yl) benzene (LDB) (3)) were synthesized and fully characterized in the solution and solid‐state including [1H–15N] heteronuclear multiple bond coherence (HMBC) NMR analysis and single‐crystal X‐ray analysis of five‐coordinates [Ag2(LPY)2(CH3CN)2](SO3CF3)2. Based on the NMR and ESI‐MS data, complex 1 exists as dimeric species, formed by head‐to‐tail interaction, while monomeric species of 3 predominantly exist in the same solvents. The protein binding affinity of 1–3 was investigated by using hen white egg lysozyme (HEWL). To explore where the metalation site of HEWL is, the interactions between the complexes and histidine were studied by NMR. The morpholine ligand and its Ag(I) complex (2) exhibited interesting antimicrobial activity. In comparison with AgOSO2CF3, compounds 1–3 displayed lower cytotoxicity to non‐malignant human embryonic kidney (HEK293) cells, and induced negligible haemoglobin release.

Hemolysis-associated hemoglobin cast nephropathy results from a range of clinicopathologic disorders

Intravascular hemolysis is relatively rare but can lead to acute kidney injury (AKI), from increased destruction of erythrocytes and release of free hemoglobin. Since hemolysis and hemoglobinuria are known causes of acute kidney injury we sought to define clinicopathologic findings and outcomes of patients with hemolysis-associated hemoglobin cast nephropathy through a retrospective analysis of 27 cases. The mean patient age was 47 years (range 19-79) and the female-to-male ratio was 1.3:1. All patients presented with AKI with a mean serum creatinine of 8.0 (range 2.9-17.0) mg/dL. Etiologies included autoimmune hemolytic anemia (30%), medication (26%), paroxysmal nocturnal hemoglobinuria (7%), procedural/mechanical causes (7%), transfusion of incompatible blood (4%), toxin ingestion (4%), disseminated intravascular coagulation (4%), and hemoglobinopathy (4%). All biopsies showed acute tubular injury and pigmented, proteinaceous casts characterized by positive hemoglobin immunohistochemistry. After a mean follow-up of nine months (range 0.5-26), the mean serum creatinine was 1.3 (range 0.6-3.3) mg/dL, with 78% of patients returning to normal kidney function. Thus, based on our clinicopathologic case series, hemolysis-associated hemoglobin cast nephropathy is an important entity for clinicians and pathologists to recognize as treatment hinges upon elimination of the pathogenic driver of intravascular hemolysis.

Extracellular Histones Induced Eryptotic Death in Human Erythrocytes.

Circulating or extracellular histones (EHs) in the bloodstream act as a damage-associated-molecular-pattern (DAMP) agent that plays a critical role in the pathogenesis of many diseases such as sepsis and sterile inflammation. To date, not much information is available to describe the mechanistic relationship between human erythrocytes and the cytotoxicity of EHs, the protein members from a highly conserved histone family across species. The present study explored this key question with a hypothesis that EHs induce eryptosis. Freshly isolated human red blood cells (RBCs) from healthy donors were treated with EHs or agents for positive controls in a physiological buffer for 3 or 24 h. After treatments, flow cytometry was employed to quantify surface phosphatidylserine (PS) exposure from annexin-V-RFP binding, cell shrinkage from flow cytometric forward scatter (FSC) analysis, Ca2+ rise by fluo-4, reactive oxygen species (ROS) production by H2DCFDA, and caspase-3 activation by FAM-DEVD-FMK measurement. Hemolysis and membarne permeabilization were estimated respectively from hemoglobin release into supernatant and calcein leakage from RBC ghosts. With positive controls for validation, EHs in the pathophsyiological range were found to accumulate annexin-V binding on cell surface, decrease FSC, upregulate ROS production, elevate Ca2+ influx and increase caspase-3 activity in a 3-h incubation. Of note, no RBC hemolysis and no calcein release from ghosts were obtained after EHs treatment for 24 h. Interestingly, external Ca2+ was not a prerequisite for the EHs-mediated ROS production and PS externalization. Also, the eryptotic hallmarks in the apoptotic RBCs were partially blocked by heparin and antibody (Ab) against Toll-like receptor 2 (TLR2). EHs act as a DAMP agent in the human RBCs that induces eryptosis. The cytotoxic effect is rapid as the hallmarks of eryptosis such as cell shrinkage, surface PS exposure, [Ca2+]i rise, ROS production and caspase-3 activation can be seen 3 h after treatment in a dose-dependent manner. The EHs' cytotoxic effects could be blocked by heparin and the Ab against TLR2.

Erythrocyte and plasma oxidative stress appears to be compensated in patients with sickle cell disease during a period of relative health, despite the presence of known oxidative agents

Sickle cell disease (SCD) is a monogenetic disease that results in the formation of hemoglobin S. Due to more rapid oxidation of hemoglobin S due to intracellular heme and adventitious iron in SCD, it has been thought that an inherent property of SCD red cells would be an imbalance in antioxidant defenses and oxidant production. Less deformable and fragile RBC in SCD results in intravascular hemolysis and release of free hemoglobin (PFHb) in the plasma, which might be expected to produce oxidative stress in the plasma. Thus, we aimed to characterize intracellular and vascular oxidative stress in whole blood and plasma samples from adult SCD patients and controls recruited into a large study of SCD at Children's Hospital of Los Angeles. We evaluated the cellular content of metHb and several components of the antioxidant system in RBC as well as oxidation of GSH and Prx-2 oxidation in RBC after challenge with hydroperoxides. Plasma markers included PFHb, low molecular weight protein bound heme (freed heme), hemopexin, isoprostanes, and protein carbonyls. While GSH was slightly lower in SCD RBC, protein carbonyls, NADH, NAD+ and total NADP+ + NADPH were not different. Furthermore, GSH or Prx-2 oxidation was not different after oxidative challenge in SCD vs. Control. Elevated freed heme and PFHb had a significant negative, non-linear association with hemopexin. There appeared to be a threshold effect for hemopexin (200 μg/ml), under which the freed heme rose acutely. Plasma F2-isoprostanes were not significantly elevated in SCD. Despite significant release of Hb and elevation of freed heme in SCD when hemopexin was apparently saturated, there was no clear indication of uncompensated vascular oxidative stress. This somewhat surprising result, suggests that oxidative stress is well compensated in RBCs and plasma during a period of relative health.

Pyridylbenzimidazole‐Based Gold(III) Complexes: Lysozyme Metalation, DNA Binding Studies, and Biological Activity

The lysozyme binding affinity of new Au(III) complexes, bearing pyridylbenzimidazole ligands, was investigated by ESI‐MS and UV/Vis. Metalation of lysozyme happened mainly by {Au}n+, {AuCl}0/n+ and {AuCl2}n+/–. The appendage sulfonate group of pyridylbenzimidazole ligand system played a role in determining the products of interaction of HEWL with Au(III) complexes. The hydrophilic sulfonate group inhibited the ligand cleavage via the participation in several coulombic and H‐bond interactions giving several AuLn+ containing adduct peaks (L = 1‐[(pyridin‐2‐yl) benzimidazole]‐propyl‐sulfonic acid). The stability of the complexes in presence of ascorbic acid was examined by UV/Vis and 13C NMR. To recognize if His15 side‐chain is the metalation site of HEWL, the interactions between the complexes and imidazole, as a simple model of histidine, were investigated by 1H and 13C NMR. The DNA binding studies of the complexes were reported. For this class of Au(III) complexes, it is preferred to decorate the pyridylbenzimidazole system with ethyl group rather than sulfonate and phthalimido group to have a complex with interesting antifungal activity against Candida albicans and Cryptococcus neoformans var. grubii. Au(III) complex, having sulfonate group, is noncytotoxic to non‐malignant cells (human embryonic kidney cells (HEK293)), shows negligible hemoglobin release and is safe to the normal cells if applicable.

Study of Desorbtion and Exemption of Terpeno-indole Alkaloids of Vinkristin and Vinblastin from Erythrocitary Cell Carriers

Introduction. Employees of the Department of Pharmaceutical Chemistry and Pharmaceutical Technology have obtained modified forms of terpino-indole alkaloids (ТIA) and vinbristine (VCR) and vinblastine (VBL) known in the treatment of cancer diseases by incorporating them into the erythrocyte carriers (EN) by the modified method of hypoosmotic lysis in the modified (aprotic solvent dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG 4000) and unmodified media.Aim. The aim of the work was to study the desorption and release of terpene-indole vincristine and vinblastine alkaloids from previously obtained cellular erythrocyte carriers.Materials and methods. In the experiment performed, the release characteristics of encapsulated preparations from erythrocytes were determined. TIA preparations were released from erythrocyte form in 6 hours: VCR – 54.5305%, VCR : PEG-4000 (1:5) – 53.3305%, VCR : PEG-4000 (1:10) – 40.1283%, VCR : PEG-4000 (1:20) – 39.9869%, VCR : DMSO (2 mg/ml) – 54.2354%, VBL – 68.0656%; VBL : PEG-4000 (1:10) – 63.8941%; VBL : PEG-4000 (1:20) – 60.7455%; VBL : PEG-400 (1:20) – 60.3529%; VBL : DMSO (2 mg/ml) – 64.5006%. The rate of release from an erythrocyte form encapsulated in a medium modified with DMSO is significantly higher than that encapsulated in an unmodified medium. The average rate of TIA release from erythrocyte forms encapsulated in PEG medium is approximately the same.Results and discussion. The results show that the release of hemoglobin from TIA encapsulated erythrocytes compared with non-encapsulated EN is small. The results show that, on average, 8.242 ± 0.3135% of hemoglobin is released from TIA encapsulated with EN, compared to control erythrocytes – 7.53% after 6 h of incubation at 37 °C. The release of hemoglobin from the TIA of encapsulated erythrocytes in modified media is less than that encapsulated in unmodified medium.Conclusion. Based on these results, it can be suggested that PEG can bind to the erythrocyte membrane and stabilize it.

Disruption of erythrocyte membrane asymmetry by triclosan is preceded by calcium dysregulation and p38 MAPK and RIP1 stimulation

Triclosan (TCS) is a broad-spectrum antimicrobial used in personal care products, household items, and medical devices. Owing to its apoptotic potential against tumor cells, TCS has been proposed for the treatment of malignancy. A major complication of chemotherapy is anemia, which may result from direct erythrocyte hemolysis or premature cell death known as eryptosis. Similar to nucleated cells, eryptotic cells lose membrane asymmetry and Ca2+ regulation, and undergo oxidative stress, shrinkage, and activation of a host of kinases. In this report, we sought to examine the hemolytic and eryptotic potential of TCS and dissect the underlying mechanistic scenarios involved there in. Hemolysis was spectrophotometrically evaluated by the degree of hemoglobin release into the medium. Flow cytometry was utilized to detect phosphatidylserine (PS) exposure by annexin-V binding, intracellular Ca2+ by Fluo-3/AM fluorescence, and oxidative stress by 2-,7-dichlorodihydrofluorescin diacetate (DCFH2-DA). Incubation of cells with 10–100 μM TCS for 1–4 h induced time- and dose-dependent hemolysis. Moreover, TCS significantly increased the percentage of eryptotic cells as evident by PS exposure (significantly enhanced annexin-V binding). Interestingly, TCS-induced eryptosis was preceded by elevated intracellular Ca2+ levels but was not associated with oxidative stress. Cotreatment of erythrocytes with 50 μM TCS and 50 μM SB203580 (p38 MAPK inhibitor), or 300 μM necrostatin-1 (receptor-interacting protein 1 (RIP1) inhibitor) significantly ameliorated TCS-induced PS externalization. We conclude that TCS is cytotoxic to erythrocytes by inducing hemolysis and stimulating premature death at least in part through Ca2+ mobilization, and p38 MAPK and RIP1 activation.

Getting Past the Spin: The CorWave LVAD, a Membrane Wave Pump Providing Physiologic Pulsatility without High Shear

Purpose A new LVAD is under development which employs a completely novel pumping technology. Magnetically driven oscillation of a polymer membrane creates a wave motion, gently pushing the blood through the pump. By changing the oscillation frequency and magnitude, pressure generation and blood flow rates can be dynamically adjusted over timescales as short as 30 msec. This approach offers a less damaging method for efficiently pumping blood while generating physiologic pulsatility. We evaluated pump actuation parameters and control algorithms in mock circulation loops and an acute animal study to evaluate and optimize pulse generation. Methods The LVAD was evaluated on a physiologic mock-loop to determine the optimal performance parameters. Pulsatility is created by switching rapidly between levels of pump output. A period of high output was generated following detection of systole to augment native cardiac output. Pump flow was reduced during diastole to avoid suck-down of the ventricle and limit back flow. The initial target was pump generated aortic dP/dt of >400 mmHg/sec under heart failure conditions with pump flow of 3-6 LPM. In parallel, hemolysis was measured using porcine blood. The pulsatility algorithm was evaluated with the pump implanted in an ovine model to validate the systole detection method. Results Evaluation on the mock circulatory loop achieved a high degree of pulsatility, defined as delta AoP=40mmHg, dP/dt=410mmHg/s, AoF=4.7 lpm, MAP=83mmHg vs. heart failure model of delta AoP=28mmHg, dP/dt=430mmHg/s, AoF=3.7lpm, MAP= 67mmHg. Generation of free hemoglobin at both high and low operating points are in a clinically acceptable range and well controlled transition between the two points does not significantly increase hemolysis. The acute animal study, the pulsatility algorithm correctly detected systole in >90% of beats. Conclusion Bench testing indicated that the pump provided augmented pulsatility to the failing heart, while achieving targeted levels of aortic flow and pressure without increased hemoglobin release. Augmented pulsatility offers a new mechanism to reduce bleeding and renal complications of VAD therapy with the intriguing possibly to increase cardiac recovery. Studies are underway to further evaluate the pulsatile operating mode algorithms and related hemoglobin release in an ovine heart failure model.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET)

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1–5 mM (0.02%–0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

Effects of Antibiotics on α-Toxin Levels during Staphylococcus aureus Culture: Implications for the Protection of Chondrocytes in a Model of Septic Arthritis.

Septic arthritis results from joint infection by Staphylococcus aureus, which produces potent α-toxin causing cell death, potentially leading to permanent cartilage damage. Treatment is by joint irrigation and antibiotics, although it is unclear if, following treatment with antibiotics which cause bacterial lysis, there is release of additional stored α-toxin. A rabbit erythrocyte hemolysis assay was optimised to assess biologically-active α-toxin from cultured S. aureus α-toxin strain DU5946. Hemoglobin release was measured spectrophotometrically following addition of a bacteriostatic antibiotic (linezolid) or a bacteriolytic antibiotic (penicillin). A bovine cartilage model of septic arthritis was used to test the protective effects of antibiotics against S. aureus infection. During S. aureus culture, α-toxin levels increased rapidly but the rate of rise was quickly (within 20 minutes) suppressed by linezolid (25 μg/mL). Penicillin also reduced the increase in α-toxin levels; however, the time course was relatively slow compared to linezolid even at high concentrations (50,000 U/mL). The efficacy of penicillin (250,000 U/mL) at reducing the rise in α-toxin was approximately 8% less than that of linezolid (P < 0.05) suggesting the presence of additional toxin. This could be due to a delayed action of penicillin, and/or release of a small pool of stored α-toxin from dying bacteria. In a bovine cartilage model, however, there was no difference between the protection of in situ chondrocytes against S. aureus by penicillin or linezolid (P > 0.05). The results suggested that equally effective protection of chondrocytes against S. aureus septic arthritis may be obtained by the bacteriostatic or bacteriolytic antibiotics tested.

The potential of biomimetic nanofibrous electrospun scaffold comprising dual component for bone tissue engineering

Oils play a putative choice for alleviating various symptoms associated with bone-related disorders. In this present study, polyurethane (PU) scaffold encompassing with Mahua oil (MO) and propolis (PP) were developed using the electrospinning technique. Morphological analysis showed the reduction in the diameter of the electrospun scaffold with blending of MO and MO/PP into the PU matrix. The strong interactions between PU, MO, and PP were evident through the infrared spectrum and thermal analysis. The wettability results showed the hydrophobic nature in electrospun PU/MO scaffold and hydrophilic behavior in electrospun PU/MO/PP scaffold. Mechanical testing indicated the enhancement in the strength of the PU due to the addition of MO and PP. Moreover, the fabricated scaffolds exhibited nontoxicity, low hemoglobin release and improved blood clotting time as evident in the coagulation studies. The cell proliferation studies showed the enhanced fibroblast cell adhesion in the developed nanocomposites than the pristine PU. Hence, the fabricated PU scaffolds blended with MO and PP having desirable properties can serve as a valuable candidate for bone tissue repair.

Histones Induce the Release of Extracellular Hemoglobin and Red Blood Cell-Derived Microvesicles with Procoagulant Activity

BackgroundHistones are nuclear proteins that can be released extracellularly following cell death or certain mechanisms of cell activation. Infusion of high doses of histones into mice causes lethality. Circulating levels of extracellular histones are elevated in several disease states associated with inflammation and thrombosis. The mechanisms by which extracellular histones induce morbidity and mortality are not fully understood. Histones may activate or be toxic to several cell types in vitro. Most of these activities have been shown to be dependent on histone signaling through toll-like receptors (TLRs). Red blood cells (RBC) do not express TLRs. However, previous studies have reported that histones promote RBC aggregation, increase shear-dependent RBC fragility, reduce deformability under flow and cause hemolytic anemia in mice. Histones also induce calcium influx and promote PS expression on RBC membranes thereby supporting the prothrombinase activity and shortening clotting times in plasma. Histones may insert into cell membranes (independently of TLRs), and form pore-like structures similar to ionophore. The role of this process in histone-induced RBC toxicity has not been fully investigated.ObjectiveWe assessed the ability of individual histones to promote the release of extracellular hemoglobin (Ex-Hb) and RBC-derived microvesicles (RBC-MVs), as well as RBC-MVs' ability to initiate or propagate thrombin generation (TG) in MV-free plasma.MethodsRBCs were isolated from citrated whole blood from healthy volunteers, and were re-suspended in buffer with or without 10uM A23187 (calcium ionophore), recombinant histones H2A, H2B, H3 or H4 for 1 hour at 37°C.To assess histone binding, RBCs were washed and dual-stained with fluorophore-conjugated anti-glycophorin A (GPA) and anti-panhistone antibodies. Stained RBCs were analyzed using imaging flow cytometry, and were considered positive for histone binding if the fluorescence intensity was > 95th percentile of RBCs processed in the absence of histones.RBCs were removed by centrifugation at 2,500g for 15 minutes x2. RBC-MVs present in the supernatant were pelleted by centrifugation at 20,000g for 30 min. Isolated RBC-MVs were then re-suspended in buffer to assess PS exposure using the microparticle activity assay (Hyphen Biomed). RBC-MVs were also re-suspended in re-calcified MV-free plasma supplemented with 4uM 41%PC/44%PE/15%PS lipid vesicles to assess their ability to initiate TG by monitoring the activity of thrombin on a fluorogenic substrate over time. To assess their ability to propagate TG, coagulation was initiated by 1pM tissue factor in MV-free plasma containing histone-induced RBC-MVs without addition of synthetic lipid vesicles. In this condition, phospholipids present on the surface of histone-induced RBC-MVs are the limiting factor for TG.The supernatant collected after pelleting RBC-MVs was used to measure Ex-Hb by absorbance at 540 nm.ResultsThe percentage of RBCs positive for histone binding was 21.6%, 2.3%, 76.9% and 98.1% for H2A, H2B, H3 and H4, respectively. Representative images of RBC staining for GPA and histone H4 is shown in Figure 1A.All histone types induced the release of Ex-Hb (Fig. 1B) and RBC-MVs that possess PS activity (Fig. 1C) in a dose-dependent manner, with the highest PS activity observed with H2A. RBC-MVs released by H2A or H2B clearly initiated TG in MV-free normal pool plasma (Fig.1D). They also significantly enhanced TF-initiated TG in the absence of additional lipid vesicles. The release of Ex-Hb and procoagulant RBC-MVs induced by histones were re-capitulated by ionophore to a greater degree, as expected (Fig.1B-E).ConclusionsAll 4 types of core histones bind to RBCs and induce the release of Ex-Hb. Our findings suggest that despite their lower binding to RBCs, histones H2A and H2B may have a greater ability to induce the release of procoagulant RBC-MVs that can initiate and propagate TG in plasma. The reason for the differential toxicity of histone types on RBCs and the discrepancy between the relative effect of histone-induced RBC-MVs on PS equivalents and TG are unknown. Histone-induced RBC toxicity appears to mimic calcium ionophore, supporting the idea that histones may insert and form functional pores in cell membranes. The contribution of Ex-Hb and procoagulant RBC-MVs to the morbidity and mortality of extracellular histones in vivo requires further studies. [Display omitted] DisclosuresMonroe:Novo Nordisk A/S: Honoraria, Research Funding.

The Ribonucleotide Reductase Inhibitor, Didox, Reduces the In Vivo Vascular Inflammation and Oxidative Stress Induced By Acute Hemolysis

Introduction: Several diseases and disorders, including hereditary and acquired hemolytic anemia, blood transfusion reactions, preeclampsia and some infections, incur intravascular hemolysis to varying degrees. The destruction of red blood cells and the release of hemoglobin (Hb) and heme into the circulation results in vascular inflammation, characterized by the recruitment of leukocytes to the vascular endothelium, reduced nitric oxide bioavailability and oxidative stress, all of which may contribute to complications seen in hemolytic diseases, such as pulmonary hypertension, cutaneous leg ulcers and priapism. Didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, has been shown to have anti-inflammatory and anti-oxidative effects. This molecule has potential as a cancer therapy and has demonstrated beneficial effects in numerous pathologies, diminishing vaso-occlusive processes in mice with sickle cell disease, and suppressing mast cell activation and degranulation, amongst other effects. Aims: The aim of this study was to evaluate the effects of didox on the vascular inflammatory effects of in vivo acute hemolysis. Methods: C57BL/6 mice received i.v. didox (10 mg/animal) or the same volume of saline vehicle immediately before receiving the hemolytic stimulus. In some mice, didox (10 mg) was given intraperitonally (i.p.) 30 min before hemolysis. Acute hemolysis (HEM) was induced by injecting mice (i.v.) with 100 µl sterile water; for non-hemolysis controls (CON), the same quantity of saline was administered i.v.. At 15 min post-hemolysis, blood was obtained by cardiac puncture for biochemical and flow cytometry analyses; other animals were submitted to cremaster muscle exposure followed by intravital microscopy. Results: Intravascular H2O administration successfully induced hemolysis within 15 min, as demonstrated by elevated plasma cell-free Hb and heme levels (plasma Hb: 0.20±0.05 and 0.66±0.16 mg/ml, P<0.01; total plasma heme: 26.3±4.9 and 87.1±18.4 µM, for CON and HEM, respectively, N=≥6, P<0.001). Interestingly, pre-administration of mice with didox slightly, but significantly, decreased plasma Hb and Heme (plasma Hb: 0.43±0.07 and 0.54±0.07 mg/ml, P<0.05; total plasma heme: 62.7.3±9.5 and 71.5±6.7 µM for i.v. and i.p. didox, respectively, N=6, P<0.05). Concomitantly, acute hemolysis significantly augmented leukocyte (WBC) recruitment and extravasation in the cremaster microcirculation (WBC adhesion: 3.07±0.28 and 13.41±1.02 WBC µm-1, P<0.001; WBC extravasation: 0.72±0.16 and 2.93±0.45 WBC (100x50 µm-2) for CON and HEM, respectively, N=≥5, P<0.001). Didox significantly abrogated this effect of hemolysis, decreasing WBC adhesion to 6.12±0.62 and 2.10±0.30 WBC µm-1 and WBC extravasation to 1.32±0.24 and 1.14±0.21 WBC (100x50 µm-2) (for i.v. and i.p. didox, respectively, N≥5, P<0.001). Flow cytometry demonstrated that hemolysis elevated both the generation of reactive oxygen species (ROS) in the granulocytes of mice and their expression of the Mac-1 integrin subunit, CD11b (ROS: 590±58 and 1140±187 MFI, P<0.01; CD11b: 3028±387 and 4534±416 MFI, for CON and HEM, respect. N=3-6, P<0.01). Importantly, the inhibition of vascular inflammation by didox was associated with a significant decrease in both ROS generation and CD11b expression by granulocytes (ROS: 591±53 and 699±120 MFI, P<0.01; CD11b 2399±126 and 2379±207 MFI, for i.v. and i.p. didox, respectively. N=3-6, P<0.01). Conclusion: Didox, when administrated both intravascularly and intraperitoneally in mice, can inhibit the significant elevations in leukocyte integrin expression and cellular recruitment in the microcirculation that are induced by acute hemolysis. This improvement in vascular inflammation is accompanied by the prevention of oxidative stress in the leukocytes. While didox was found to slightly reduce hemolysis in response to osmotic shock, an interesting observation in itself, it is probable that the beneficial effects of this molecule on vascular inflammation are mediated largely by its effect on oxidative stress parameters. As such, this molecule may provide a novel therapeutic approach to reduce the oxidative stress and vascular inflammation caused by hemolytic events. This includes potential clinical use to treat sickle cell disease and other disorders in which hemolysis is a contributing factor to the pathophysiology. DisclosuresElford:Molecules for Health, Inc.: Equity Ownership, Patents & Royalties: Didox.