Hemolysis Of Cells Research Articles

Biocompatibility testing of composite biomaterial designed for a new petal valve construction for pulsatile ventricular assist device

This paper presents the results of biocompatibility testing performed on several biomaterial variants for manufacturing a newly designed petal valve intended for use in a pulsatile ventricular assist device or blood pump. Both physical vapor deposition (PVD) and plasma-enhanced chemical vapor deposition (PECVD) were used to coat titanium-based substrates with hydrogenated tetrahedral amorphous carbon (ta-C:H) or amorphous hydrogenated carbon (a-C:H and a-C:H, N). Experiments were carried out using whole human blood under arterial shear stress conditions in a cone-plate analyzer (ap. 1800 1/s). In most cases, tested coatings showed good or very good haemocompatibility. Type a-C:H, N coating proved to be superior in terms of activation, risk of aggregation, and the effects of generating microparticles of apoptotic origin, and also demonstrated excellent mechanical properties. Therefore, a-C:H, N coatings were selected for further in vivo studies. In vivo animal studies were carried out according to the ISO 10993 standard. Intradermal reactivity was assessed in three rabbits and sub-acute toxicity and local effects after implantation were examined in 12 rabbits. Based on postmortem examination, no organ failure or wound tissue damage occurred during the required period of observation. In summary, our investigations demonstrated high biocompatibility of the biomaterials in relation to thrombogenicity, toxicity, and wound healing. Prototypes of the petal valves were manufactured and mounted on the pulsatile ventricular assist device. Hydrodynamic features and impact on red blood cells (hemolysis) as well as coagulation (systemic thrombogenicity) were assessed in whole blood.

MDS-126: Categorized Hematologic Response to Pegcetacoplan in Patients with Paroxysmal Nocturnal Hemoglobinuria: Post Hoc Analysis of Data from Phase 1b (PADDOCK) and Phase 2a (PALOMINO) Trials

Context: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematologic disease characterized by complement-mediated red blood cell hemolysis. Despite treatment with C5-inhibitors to control intravascular hemolysis (IVH), anemia persists in ~70% of patients due to persistent IVH and mostly C3-mediated extravascular hemolysis. Objective: This post hoc analysis categorized the hematologic response to pegcetacoplan (C3-inhibitor recently approved by the FDA) in the PADDOCK (phase 1b, two-cohort [NCT02588833]; n=20) and PALOMINO (phase 2a, single-cohort [NCT03593200]; n=4) open-label trials, which assessed safety/efficacy of pegcetacoplan in complement inhibitor-naive patients. Design: Hematologic response to treatment was categorized at Days 113 and 337 for the combined trials (n=24), per criteria in Risitano AM, et al. Front Immunol. 2019;10:1157: complete- no transfusions required, stable hemoglobin (Hb; normal range), no evidence of hemolysis (lactate dehydrogenase [LDH] ≤1.5×upper limit of normal [ULN] U/L, absolute reticulocyte count [ARC] ≤150,000/µL); major- no transfusion, normal Hb, but with evidence of hemolysis (LDH >1.5×ULN U/L and/or ARC >150,000/µL); good- no transfusions, but with chronic mild anemia or evidence of hemolysis; partial-chronic moderate anemia and/or occasional transfusions ( 6 units/6 months). Patients with missing data at either timepoint for ≥1 parameter were not evaluated. Manual categorization was independently conducted by two blinded observers. Results: Most patients achieved at least a good hematologic response at Days 113 (75.0%; 18/24) and 337 (62.5%; 15/24). Categorized hematologic responses were as follows: Day 113— good-to-complete 75.0%, partial 4.2%, minor 8.3%, no response 4.2%; Day 337— good-to-complete 62.5%, partial 20.8%, minor 4.2%, no response 0.0%. Of patients with evaluable data, 19.0% (4/21) achieved ≥1 category of improvement in hematologic response at Day 337 versus Day 113. In PADDOCK, 2 patients at Day 113 and 3 patients at Day 337 were unevaluable due to missing data. Conclusions: In the PADDOCK and PALOMINO trials, a substantial proportion of patients achieved complete, major, or good hematological responses to pegcetacoplan at Day 113, which was sustained to Day 337, suggesting that pegcetacoplan treatment in complement inhibitor-naive patients can lead to clinical improvements in PNH symptoms.

In Vitro and In Silico Toxicological Properties of Natural Antioxidant Therapeutic Agent Azimatetracantha. LAM.

Plant-derived antioxidants are a large group of natural products with the capacity to reduce radical-scavenging. Due to their potent therapeutic and preventive actions, these compounds receive a lot of attention from scientists, particularly pharmacologists. The pharmacological activities of the Azima tetracantha Lam. (AT) plant, belonging to the Salvadoraceae family, reported here justifies its traditional use in treating several diseases or disorders. This study aims to look at the propensity of certain plant compounds found in natural AT plant extracts that might play a critical role as a secondary metabolite in cervical cancer treatment. There is a shortage of information on the plant’s phytochemical and biological characteristics. Methanol (MeOH) solvent extracts of the dried AT plant were screened phytochemically. Its aqueous extract was tested for antioxidant, antiseptic, anti-inflammatory, and anticancerous properties. Absorption Distribution Metabolism and Excretion (ADME/T), Docking, and HPLC were also performed. In clinical treatment, the plant shown no adverse effects. The antioxidant activity was evaluated and showed the highest concentration at 150 µg/mL (63.50%). MeOH leaf extract of AT exhibited the highest and best inhibitory activity against Staphylococcus aureus (15.3 mm/1000) and displayed a high antiseptic potential. At a 200 µg/mL concentration, MeOH leaves-extract inhibited red blood cells (RBC) hemolysis by 66.56 ± 0.40, compared with 62.33 ± 0.40 from the standard. Albumin’s ability to suppress protein denaturation ranged from 16.75 ± 0.65 to 62.35 ± 0.20 inhibitions in this test, providing even more support for its favorable anti-inflammatory properties. The ADME/T studies were considered for a potential cancer drug molecule, and one of our compounds from MeOH extract fills the ADME and toxicity parameters. The forms of compound 4 showed a strong hydrogen-bonding interaction with the vital amino acids (ASN923, THR410, LEU840TRY927, PHE921, and GLY922). A total of 90% of cell inhibition was observed when HeLa cell lines were treated with 300 µg/mL of compound 4 (7-acetyl-3a1-methyl- 4,14-dioxo-1,2,3a,3a1,4,5,5a,6,8a,9b,10,11,11a-tetradecahydro-2,5a epoxy5,6a (methanooxymethano)phenaleno[1′,9′:5,6,7]indeno[1,7a-b]oxiren-2-yl acetate). The polyphenol compounds demonstrated significant advances in anticancer drug properties, and it could lead to activation of cancer cell apoptosis.

SERUM LACTATE DEHYDROGENASE AS A BIOCHEMIAL MARKER FOR MATERNAL AND PERINATAL OUTCOME IN PRE-ECLAMPSIA

Background: Pre-eclampsia affects about 5-10% of all pregnancies and is a major cause of maternal, fetal and neonatal morbidity and mortality, particularly in developing countries. FOGSI and other studies show the incidence of pre eclampsia in India ranges between 11-13%. Lactate Dehydrogenase (LDH) is mainly an intracellular enzyme. LDH is present in many body tissues, especially heart, liver, kidney, skeletal muscle, brain, blood cells and lungs. Its level is increased in the scenario of increased cell injury, hemolysis and cell death. Cellular enzymes in the extracellular space although of no further metabolic function in this space, are still of benet because they serve as indicators suggestive of disturbance of cellular integrity induced by pathological conditions and is used to detect cell damage or cell death.This can be further used as help in making decision, regarding the management strategies to improve the maternal and foetal outcome. Objectives: To compare serum LDH levels in the normal pregnant women and in women with preeclampsia and eclampsia in ante-partum period and to correlate the severity of the disease, maternal and perinatal outcome with Lactic Dehydrogenase (LDH) levels in serum in patients of pre-eclampsia and eclampsia. Material and Methods: A prospective comparative study was conducted in the department of Obstetrics and Gynaecology, Ispat General Hospital, Rourkela. Out of 150 women studied, 50 were normal pregnant women, 32 were of mild preeclampsia, 35 were of severe preeclampsia and 33 of eclampsia. The statistical analysis was done by Chi-square test, Fischer Exact test, analysis of variance and student ''t'' test (two tailed and independent). Results: LDH levels were signicantly elevated in women with preeclampsia and eclampsia (p value <0.001). LDH levels had signicant direct correlation with increasing blood pressure (p value <0.001) as well as poor maternal and perinatal outcome. Conclusion: High serum LDH levels correlate well with the severity of the disease and poor maternal and fetal outcomes in patients of preeclampsia and eclampsia and can be considered as a supportive biochemical and prognostic marker from early third trimester.

Blood compatibility evaluations of CaCO3 particles

CaCO3 particles, due to their unique properties such as biodegradation, pH-sensitivity, and porous surface, have been widely used as carrier materials for delivering drugs, genes, vaccines, and other bioactive molecules. In these applications, CaCO3 particles are often administered intravenously. In this sense, the interaction between CaCO3 particles and blood components plays a key role in their delivery efficacy and biosafety, though the hemocompatibility of CaCO3 particles has not been evaluated until now. Deficiency in the biosafety information has delayed the clinical use of CaCO3 particles in delivery systems. In this work, we investigated the biosafety of CaCO3 particles, focusing on their in vitro and in vivo effects on key blood components (red blood cells, platelets, etc) and coagulation functions. We found in vitro that high concentrations of CaCO3 particles can cause the aggregation and hemolysis of red blood cells, with platelet activation and coagulation prolongation. In vivo, we found that intravenously injected CaCO3 particles at 50 mg kg−1 significantly disturbed the red blood cells, and platelet-related blood routine indexes, but did not induce visible abnormalities in the tissue structures of the key organs. Overall, these effects may be due to the enormous adsorption capability of the porous surface of CaCO3 particles. 0.1 mg ml−1 of the CaCO3 particles exhibit excellent compatibility for their practical applications. These results would be expected to greatly promote the in vivo applications and clinical use of CaCO3 particles in biomedicine.

Interaction of 4\u2032-methylflavonoids with biological membranes, liposomes, and human albumin

The aim of the study was to compare the impact of three synthesized chemical compounds from a group of methylated flavonoids, i.e. 2′-hydroxy-4-methylchalcone (3), 4′-methylflavanone (4), and 4′-methylflavone (5), on a red blood cell membranes (RBCMs), phosphatidylcholine model membranes (PC), and human serum albumin (HSA) in order to investigate their structure–activity relationships. In the first stage of the study, it was proved that all of the compounds tested do not cause hemolysis of red blood cells and, therefore, do not have a toxic effect. In biophysical studies, it was shown that flavonoids have an impact on the hydrophilic and hydrophobic regions of membranes (both RBCMs and PC) causing an increase in packing order of lipid heads and a decrease in fluidity, respectively. Whereas, on the one hand, the magnitude of these changes depends on the type of the compound tested, on the other hand, it also depends on the type of membrane. 4′-Methylflavanone and 4′-methylflavone are located mainly in the hydrophilic part of lipid membranes, while 2′-hydroxy-4-methylchalcone has a greater impact on the hydrophobic area. A fluorescence quenching study proved that compounds (3), (4) and (5) bind with HSA in a process of static quenching. The binding process is spontaneous whereas hydrogen bonding interactions and van der Waals forces play a major role in the interaction between the compounds and HSA.

Calcium-oxidative stress signaling axis and casein kinase 1α mediate eryptosis and hemolysis elicited by novel p53 agonist inauhzin

Inauhzin (INZ) is a novel p53 agonist with antitumor activity. Anemia is a common side effect of chemotherapy and may arise from red blood cell (RBC) hemolysis or eryptosis. In this study, we investigate the mechanisms of INZ toxicity in human RBCs. RBCs were isolated from healthy donors and treated with antitumor concentrations of INZ (5–500 μM) for 24 h at 37 °C. Hemoglobin was photometrically measured, and cells were stained with Annexin-V-FITC for phosphatidylserine (PS), Fluo4/AM for calcium, and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) for oxidative stress. INZ caused significant dose-responsive, calcium-dependent hemolysis starting at 40 μM. Furthermore, INZ significantly increased Annexin-positive cells and Fluo4 and DCF fluorescence. The cytotoxicity of INZ was also significantly mitigated in presence of D4476. INZ possesses hemolytic and eryptotic potential characterized by cell membrane scrambling, intracellular calcium overload, cell shrinkage, and oxidative stress secondary to calcium influx from the extracellular space.

A new insight into the cellular mechanisms of envenomation: Elucidating the role of extracellular vesicles in Loxoscelism

Envenomation by the Loxosceles genus spiders is a recurring health issue worldwide and specially in the Americas. The physiopathology of the envenomation is tightly associated to the venom’s rich toxin composition, able to produce a local dermonecrotic lesion that can evolve systemically and if worsened, might result in multiple organ failure and lethality. The cellular and molecular mechanisms involved with the physiopathology of Loxoscelism are not completely understood, however, the venom’s Phospholipases D (PLDs) are known to trigger membrane injury in various cell types. Here, we report for the first time the Loxosceles venom’s ability to stimulate the production of extracellular vesicles (EVs) in various human cell lineages. Components of the Loxosceles venom were also detectable in the cargo of these vesicles, suggesting that they may be implicated in the process of extracellular venom release. EVs from venom treated cells exhibited phospholipase D activity and were able to induce in vitro hemolysis in human red blood cells and alter the HEK cell membranes’ permeability. Nonetheless, the PLD activity was inhibited when an anti-venom PLDs monoclonal antibody was co-administered with the whole venom. In summary, our findings shed new light on the mechanisms underlying cellular events in the context of loxoscelism and suggest a crucial role of EVs in the process of envenomation.

Thermal responsive poly-N-isopropylacrylamide/galactomannan copolymer nanoparticles as a potential amphotericin delivery carrier

Thermo responsive copolymers were synthesized by radical polymerization of the N-isopropylacrylamide (NIPAm) onto galactomannan (GM) chain. All copolymers showed thermal responsiveness and a critical aggregation concentration (CAC) at 25 °C greater than at 50 °C. Below the lower critical solution temperature (LCST), copolymer nanoparticles presented sizes between 16 and 50 nm, whereas above LCST, they aggregate, presenting sizes in the range 145.8–194.0 nm, depending on NIPAm/GM molar ratio. Amphotericin-B (AmB) was encapsulated in the copolymer nanoparticles with encapsulation efficiency of 41.4 ± 2.76% and 69.0 ± 2.92% respectively for CP1 and CP2. In vitro AmB delivery from the CP1 nanoparticles was found to exhibit controlled release profile. Hemolysis assay was carried out using erythrocytes cells and samples of AmB loaded copolymer nanoparticles (CP1-AmB), unloaded copolymer nanoparticles (CP1) and commercial AmB were evaluated. Unloaded copolymer nanoparticles show hemocompatibility while the percentage of hemolysis of erythrocytes cells for CP1-AmB sample was 8.7 times lower than that observed for commercial AmB. CP1-AmB showed similar minimum inhibitory concentration (MIC) as commercial AmB, in four Candida albicans strains. As CP1-AmB nanoparticle show better hemocompatibility than commercial AmB, this thermo responsive copolymer shows potential as AmB nanocarrier device to be used in fungal infection.

Hybrid Bionanocomposite Containing Magnesium Hydroxide Nanoparticles Embedded in a Carboxymethyl Cellulose Hydrogel Plus Silk Fibroin as a Scaffold for Wound Dressing Applications.

Based on the promising biomedical developments in wound healing strategies, herein, a new nanobiocomposite scaffold was designed and presented by incorporation of carboxymethyl cellulose hydrogels prepared using epichlorohydrin as a cross-linking agent (CMC hydrogel), a natural silk fibroin (SF) protein, and magnesium hydroxide nanoparticles (Mg(OH)2 NPs). Biological evaluation of the CMC hydrogel/SF/Mg(OH)2 nanobiocomposite scaffold was conducted via in vitro cell viability assays and in vivo assays, red blood cell hemolysis, and antibiofilm assays. Considering the cell viability percentage of Hu02 cells (84.5%) in the presence of the prepared nanobiocomposite after 7 days, it was indicated that this new nanoscaffold was biocompatible. The signs of excellent hemocompatibility and the high antibacterial activity were observed due to the low-point hemolytic effect (8.3%) and high-level potential in constraining the P. aeruginosa biofilm formation with a low OD value (0.13). Moreover, in vivo wound healing assay results indicated that the wound healing method was faster in mice treated with the prepared nanobiocomposite scaffold (82.29%) than the control group (75.63%) in 12 days. Apart from the structural characterization of the CMC hydrogel/SF/Mg(OH)2 nanobiocomposite through FTIR, EDX, FESEM, and TG analyses, compressive mechanical tests, contact angle, porosity, and swelling ratio studies indicated that the combination of the CMC hydrogel structure with SF protein and Mg(OH)2 NPs could significantly impact Young's modulus (from 11.34 to 10.14 MPa), tensile strength (from 299.35 to 250.78 MPa), elongation at break (12.52 to 12.84%), hydrophilicity, and water uptake capacity (92.5%).

HO-1 and Heme: G-Quadruplex Interaction Choreograph DNA Damage Responses and Cancer Growth.

Many anti-cancer therapeutics lead to the release of danger associated pattern molecules (DAMPs) as the result of killing large numbers of both normal and transformed cells as well as lysis of red blood cells (RBC) (hemolysis). Labile heme originating from hemolysis acts as a DAMP while its breakdown products exert varying immunomodulatory effects. Labile heme is scavenged by hemopexin (Hx) and processed by heme oxygenase-1 (HO-1, Hmox1), resulting in its removal and the generation of biliverdin/bilirubin, carbon monoxide (CO) and iron. We recently demonstrated that labile heme accumulates in cancer cell nuclei in the tumor parenchyma of Hx knockout mice and contributes to the malignant phenotype of prostate cancer (PCa) cells and increased metastases. Additionally, this work identified Hx as a tumor suppressor gene. Direct interaction of heme with DNA G-quadruplexes (G4) leads to altered gene expression in cancer cells that regulate transcription, recombination and replication. Here, we provide new data supporting the nuclear role of HO-1 and heme in modulating DNA damage response, G4 stability and cancer growth. Finally, we discuss an alternative role of labile heme as a nuclear danger signal (NDS) that regulates gene expression and nuclear HO-1 regulated DNA damage responses stimulated by its interaction with G4.

Silicane Derivative Increases Doxorubicin Efficacy in an Ovarian Carcinoma Mouse Model: Fighting Drug Resistance.

The development of cancer resistance continues to represent a bottleneck of cancer therapy. It is one of the leading factors preventing drugs to exhibit their full therapeutic potential. Consequently, it reduces the efficacy of anticancer therapy and causes the survival rate of therapy-resistant patients to be far from satisfactory. Here, an emerging strategy for overcoming drug resistance is proposed employing a novel two-dimensional (2D) nanomaterial polysiloxane (PSX). We have reported on the synthesis of PSX nanosheets (PSX NSs) and proved that they have favorable properties for biomedical applications. PSX NSs evinced unprecedented cytocompatibility up to the concentration of 300 μg/mL, while inducing very low level of red blood cell hemolysis and were found to be highly effective for anticancer drug binding. PSX NSs enhanced the efficacy of the anticancer drug doxorubicin (DOX) by around 27.8-43.4% on average and, interestingly, were found to be especially effective in the therapy of drug-resistant tumors, improving the effectiveness of up to 52%. Fluorescence microscopy revealed improved retention of DOX within the drug-resistant cells when bound on PSX NSs. DOX bound on the surface of PSX NSs, i.e., PSX@DOX, improved, in general, the DOX cytotoxicity in vitro. More importantly, PSX@DOX reduced the growth of DOX-resistant tumors in vivo with 3.5 times better average efficiency than the free drug. Altogether, this paper represents an introduction of a new 2D nanomaterial derived from silicane and pioneers its biomedical application. As advances in the field of material synthesis are rapidly progressing, novel 2D nanomaterials with improved properties are being synthesized and await thorough exploration. Our findings further provide a better understanding of the mechanisms involved in the cancer resistance and can promote the development of a precise cancer therapy.

UJI AKTIVITAS ANTIINFLAMASI INFUSA BUNGA PUKUL EMPAT SECARA IN VITRO

The stew of Mirabilis jalapa L. flower has been used traditionally as an anti-inflammatory. Phytochemical screening of Mirabilis jalapa L. flower showed flavonoid content which is known to have anti-inflammatory activity. This study aims to determine the anti-inflammatory activity of Mirabilis jalapa L. flower infusion using the red blood cell membrane stabilization method by inducing hypotonicity and heat. The test group consisted of Mirabilis jalapa L. flower infusion with a concentration of 2.5%; 5%; and 10% w / v, positive control (diclofenac sodium 0.01% w / v and aspirin 0.01% w / v), and negative control (distilled water). The anti-inflammatory activity of Mirabilis jalapa L. flower infusion was determined by the percentage of membrane stabilization and inhibition of red blood cell hemolysis. Data from the test results were statistically analyzed both parametric and nonparametric with SPSS. Mirabilis jalapa L. flower infusion contains flavonoids, tannins and terpenoids. The test results showed that the 10% concentration of Mirabilis jalapa L. flower infusion had the largest percentage of red blood cell hemolysis protection on hypotonicity induction, that is 99.50% (p>0.05) and the largest percentage of hemolysis inhibition on heat induction, that is 50.27% ( p≤0.05). From these results, it can be concluded that the Mirabilis jalapa L. flower infusion has potential as an anti-inflammatory by stabilizing the red blood cell membrane.

In Vitro Combination Studies of ACH-4471 with Eculizumab to Assess a Potential “Switch” Treatment Approach for Paroxysmal Nocturnal Hemoglobinuria

Introduction: ACH-4471 is an oral complement factor D (fD) inhibitor that has demonstrated clinical efficacy in an ongoing phase 2 clinical study for naïve patients suffering from paroxysmal nocturnal hemoglobinuria (PNH). PNH is a rare clonal blood disorder in which erythrocytes lacking the GPI-anchored complement regulators CD55 and CD59 are susceptible to destruction by the terminal complement complex (also known as membrane attack complex, MAC) following the normal low-level activation of the complement alternative pathway (AP) in the fluid phase. The therapeutic agent eculizumab, a monoclonal antibody directed against the terminal complement component C5, blocks MAC assembly on erythrocyte membranes and so prevents most intravascular hemolytic destruction. Eculizumab treatment however leads to increased deposition of complement C3 fragments on PNH membranes, which can result in extravascular phagocytic elimination of opsonized erythrocytes (A. M. Risitano et al, Blood, 2009, 113:4094) and incomplete inhibition of intravascular hemolysis (M. J. Harder et al, Blood, 2017, 129:970), and consequently to the continued anemia and transfusion dependence observed in a significant subset of patients. As an AP inhibitor, in contrast to eculizumab, ACH-4471 prevents both MAC assembly and C3 fragment deposition on PNH cell surfaces and therefore is expected to address the medical needs of these suboptimal responders to eculizumab. To assess this notion directly and also to support a future switch treatment approach, a further clinical study has been planned to evaluate ACH-4471 co-administration in PNH patients who respond suboptimally to eculizumab. In the present study, we conducted in vitro combination experiments to explore the potential pharmacological consequences when ACH-4471 is added on to eculizumab or another anti-C5 monoclonal antibody, using functional AP assays that include the hemolysis of erythrocytes from a PNH patient.Methods: Concentration series of ACH-4471 were assessed in pairwise combinations with eculizumab and a second monoclonal antibody inhibitor of complement C5. Functional assays for human serum AP activity included a single-plate ELISA-based functional assay (Wieslab) and the hemolysis of red blood cells from a PNH patient under AP-restricted conditions but at neutral pH. Analyses of interactions were performed using a three-dimensional surface graphing method and, where applicable, a median-effect plot method.Results: We assessed pairwise interactions using two assays for AP function. In the AP Wieslab assay, interactions between ACH-4471 and an anti-C5 monoclonal antibody ranged from additive to moderately synergistic. Next, to better approximate the physiological setting, we assessed inhibitor interactions against the AP-mediated hemolysis of red blood cells obtained from a PNH patient. ACH-4471 in this assay showed strongly synergistic interactions when tested with eculizumab. In light of a recent report on incomplete inhibition by eculizumab following strong complement activation, we propose a possible mechanism for synergy between ACH-4471 and eculizumab wherein ACH-4471 reduces the density of deposited C3b clusters, to which C5 binding is thereby more readily inhibited by eculizumab.Conclusions: ACH-4471 showed strong synergy with eculizumab. These results suggest the potential clinical utility of ACH-4471 as a PNH therapeutic agent that may be used in suboptimal eculizumab responders in support of a “switch” treatment strategy. DisclosuresPatel:Achillion: Employment. Thanassi:Achillion: Employment. Yang:Achillion: Employment. Brodsky:Alexion Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Grant Funding. Huang:Achillion: Employment. Podos:Achillion: Employment.

GWAS of Osmotic Hemolysis in 12,352 Healthy Blood Donors Identifies Red Cell Genetic Variants Associated with Steady State Hemolysis in Patients with Sickle Cell Disease

Background: The evolutionary pressure of endemic malaria has produced highly variable genes encoding erythrocyte proteins and enzymes, many of which modulate cellular structure and function. We hypothesized that a high throughput screen of red blood cell susceptibility to osmotic hemolysis, coupled with a GWAS study, would identify rare and common genetic variants associated with cellular structure and function during red blood cell storage and in hemolytic diseases like sickle cell and malaria.Methods: The REDS III RED Blood Cell Omics (RBC-Omics) study enrolled 13,403 successful blood donors at 4 major hubs. Two labs were used for red blood cell phenotyping of leukoreduced RBC unit-derived samples stored for ~42 days under blood bank conditions. Red blood cell osmotic fragility was assayed in 12,352 donors, including 1,483 African American, 1,477 Asian and 960 Hispanic donors. DNA was isolated and genotyped using a custom Affymetrix Axiom TM-Array, which contained approximately 875,000 SNPs enriched for blood and transfusion related polymorphisms.Results: Genome wide association analysis in the complete RBC-omics data set revealed 18 genes/loci to be associated with osmotic hemolysis at a genome wide significance threshold of P<5x10-8. The genes included candidates with clear internal validity such as HBB (specifically rs344 the HbS variant) and promotor regions of HBA2 (thalassemic variants), as well as SNPs in logical candidates, such as Ankyrin, Spectrin, and Aquaporin 1, mutations in which are responsible for spherocytosis and red cell membrane water transport defects, among others. To test the potential relevance of these finding to human disease, we evaluated the top 100 regions in the RBC-Omics studies in the 852 pediatric and adult patients with sickle cell disease enrolled in the PUSH and Walk-PHASST screening studies with genome SNP data, and analyzed the association of the SNPs with the intensity of steady state hemolysis, as measured by a principle component analysis of reticulocyte count, bilirubin, lactate dehydrogenase and aspartate amino transferase. After multiple testing correction, numerous genes were associated with the intensity of hemolysis in both studies, including SNPs in red cell metabolism enzymes genes (HK1, TKTL1 MTOR signaling), ion channels (PIEZO1), as well as regulatory gene (ZNF573) and less obvious genes (FLN, SBF2, GAB3, and TMEM116).Conclusion: An analysis of the genetic variability underlying normal human red cell susceptibility to osmotic stress hemolysis identified new candidate genes that are also associated with the severity of red blood cell hemolysis during the disease stress of human SCD. We hypothesize that many of these candidate SNPs may modulate the severity of red cell hemolysis in other diseases like malaria and potential outcomes after routine red blood cell storage and transfusion. These studies highlight the discovery of a number of new metabolic and ion/water transport variants that are associated with the severity of steady state hemolysis and under disease stress. DisclosuresMast:Novo Nordisk: Research Funding. Gordeuk:Emmaus Life Sciences: Consultancy.

Neutrophil Extracellular Traps Promote Lung Arteriole Occlusion in Sickle Cell Disease

Introduction:Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder, which leads to red blood cell sickling and hemolysis. Acute systemic painful vaso-occlusive crisis (VOC) is the predominant pathophysiology requiring emergency medical care by SCD patients. 10-20% of SCD patients hospitalized with VOC tend to develop acute chest syndrome (ACS), a type of lung injury within next few days, suggesting a role for pulmonary vaso-occlusion in ACS. The cellular, molecular and biophysical mechanism of pulmonary vaso-occlusion has just started to unfold and a profound understanding is needed for the development of rescue therapies to halt the progression of VOC to ACS. Our recent findings using quantitative fluorescence intravital lung microscopy (qFILM) revealed that lung vaso-occlusion is enabled by the entrapment of embolic neutrophil-platelet aggregates in the pulmonary arterioles of transgenic humanized SCD mice. Neutrophil extracellular traps (NETs) are web-like structures of decondensed nuclear DNA decorated with citrunilated-histones and neutrophil granule proteins. NETs are released by activated neutrophils under inflammatory conditions and are known to possess pro-inflammatory and pro-thrombotic properties. Recently, hemolysis driven release of cell free hemoglobin or hemin has been shown to promote NETs formation in the pulmonary microcirculation of SCD mice. Nevertheless, how these NETs can serve to facilitate pulmonary vaso-occlusion and promote ACS is still unclear.Materials and Methods:Townes knock-in humanized SS (hα/hα:βS/βS) and AS (hα/hα:βA/βS) mice were used as SCD and control mice, respectively. SS and AS mice were intravascularly (IV) challenged with Oxy-hemoglobin (Oxy-Hb) and hemin to model VOC. Fluorescently-conjugated anti-mouse CD49b and Ly6G mAbs, Sytox Orange, and FITC dextran were administered IV to visualize platelets, neutrophils, extracellular DNA and lung vasculature in the mouse, respectively. Lung microcirculation was visualized in real time in vivo using qFILM and analyzed as described elsewhere. Also, SCD or control human blood with or without treatment with Oxy-Hb or hemin was perfused through in vitro microfluidic microchannels presenting a combination of P-selectin, ICAM-1 and IL-8, and neutrophil-platelet interactions as well as NET formation was visualized using Quantitative Microfluidic Fluorescence Microscopy (qMFM). Fluorescent anti-human mAbs against CD49b, CD16, citrullinated histones (H3-Cit) and neutrophil elastase were added to human blood to visualize platelets, neutrophils, histones, and neutrophil elastase, respectively.Results and Discussion:Our initial findings using qFILMreveal that non-lethal dose of 20 µmole/kg IV hemin or 10 µmole/kg IV Oxy-Hb led to entrapment of neutrophil-platelet embolic aggregates in the lung arteriolar bottlenecks of SCD but not control mice. These aggregates resulted loss of blood perfusion in the lung, followed by shedding of NETs from neutrophils into the blood circulation.Conclusion: Erythroid DAMPs, hemin and Oxy-Hb promote occlusion of lung arterioles by neutrophil-platelet aggregates in SCD mice. These aggregates promote loss of blood flow and release of NETs from neutrophils in the lung arterioles. DisclosuresKato:Bayer: Research Funding; Global Blood Therapeutics: Consultancy; Novartis: Consultancy; MAST Therapeutics: Research Funding.

Access to Care for Medicaid and Commercially-Insured United States Patients with Sickle Cell Disease

Access to Care for Medicaid and Commercially-Insured United States Patients with Sickle Cell Disease

Effects of Oral N -Acetylcysteine on Oxidative Stress in Patients with Sickle Cell Disease

Oxidative stress (OS) has been demonstrated to play a central role in the pathophysiology of sickle cell disease (SCD). The antioxidant N -acetylcysteine (NAC) is an important precursor of cysteine, which is the rate limiting substrate for glutathione (GSH) synthesis, and can thereby exert intracellular antioxidant effects. Yet, because of its thiol group, NAC can also act extracellularly as a reducing agent to break protein disulfide bonds, or to scavenge reactive oxidants. Pilot studies with NAC in SCD have demonstrated various beneficial effects, reducing markers of red blood cell membrane damage, hemolysis and OS. The major plasma modulator of OS is the amino acid cysteine (Cys), with the ratio of its reduced form to the oxidized form reflecting the plasma redox state. Under oxidative stress, relatively low levels of free thiol Cys and high levels of protein bound Cys are observed (Fu et al. Blood 2015; 126:1044). The aim of this study was to assess the effects of oral NAC on the thiol redox balance in SCD patients. Moreover, we explored correlations of the thiol redox balance at baseline with other clinical and pathophysiological markers of SCD.Plasma was analyzed from 2 completed prospective studies that evaluated the effect of oral NAC in SCD: 1) an open label, uncontrolled, pilot study consisting of 6 weeks of NAC 1,200 -2,400mg daily, and 6 weeks follow-up in patients with HbSS or HbSβ⁰-disease (N=10, Nur et al. Ann Hematol. 2012; 91:1097-105); and 2) a randomized, placebo-controlled trial (RCT) of oral NAC 1,200mg daily for 6 months in HbSS, HbSC, HbSβ⁰ or HbSβ⁺ patients (N=96, Sins et al. Br J Haematol. 2017 ) . Using liquid chromatography-tandem mass-spectrometry, we assessed the following markers of the thiol redox balance: total NAC, total and free (reduced) Cys, and protein bound (oxidized) Cys.In both studies, oral NAC significantly increased levels of total NAC and free Cys, and decreased levels of protein bound Cys, as compared to baseline (figure 1). In the pilot study, total Cys was decreased as compared to baseline, however, no significant difference was observed in the RCT. In addition, when comparing the mean change from baseline of these parameters between the NAC arm and the placebo arm of the RCT, similar results were found. Yet, differences in mean change of protein bound and total Cys did not reach statistical significance in this analysis (results not shown).Multivariate linear regression analysis of baseline thiol redox values in the RCT was performed, adjusting for genotype and use of HU. In this analysis, higher levels of free Cys (less OS) were independently associated with a higher MCV (standardized β [sβ] 0.36, P<.001), TAT (sβ 0.19, P=0.040) and levels of Nε-(carboxymethyl)lysine (CML, advanced glycation end product; sβ 0.27, P=.005), lower sP-selectin levels (sβ -0.33, P=.001) and a lower number of hospital admissions in the 3 years prior to randomization (sβ -0.21, P=.027). Higher levels of protein bound Cys (more OS) were independently associated with VWF:ag levels (sβ 0.24, P=.016) and the HbSS/HbSβ0 genotype (sβ -0.25, P=.020). Lastly, higher levels of total Cys (more OS) were independently associated with older age (sβ 0.21, P=.027) and the HbSS/HbSβ0 genotype (sβ -0.21, P=.042), and higher levels of VWF:ag (sβ 0.28, P=.004), CML (sβ 0.17, P=.046) and prothrombin activation fragment F1-2 (sβ 0.18, P=.038).In line with previous intravenous studies, oral treatment with NAC significantly improved the thiol plasma redox balance in patients with SCD. These effects appeared to be most apparent in the uncontrolled pilot study, possibly due to poor adherence and a relatively low dosage in the RCT. Nevertheless, these findings emphasize the role of NAC as an extracellular reducing agent of protein disulfide bonds, beyond acting as a substrate for glutathione synthesis or as a direct scavenger of reactive oxygen species. Moreover, a higher extracellular oxidant burden at baseline was independently associated with endothelial and coagulation activation, and with the number of hospital admissions. Thereby, we provide indirect evidence that improvement of the thiol redox balance may be an important therapeutic target in SCD, improving other pathophysiological processes of the disease, and potentially even reducing the number of hospital admissions. Hence, the use of NAC and other thiol antioxidants in SCD deserves further investigation. [Display omitted] DisclosuresRijneveld:Jazz pharmaceuticals: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Delannoy:Innate Pharma: Honoraria.

Human Urine Alters Methicillin-Resistant Staphylococcus aureus Virulence and Transcriptome.

Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of hospital-associated urinary tract infections (UTI), especially in catheterized individuals. Despite being rare, MRSA UTI are prone to potentially life-threatening exacerbations such as bacteremia that can be refractory to routine antibiotic therapy. To delineate the molecular mechanisms governing MRSA urinary pathogenesis, we exposed three S. aureus clinical isolates, including two MRSA strains, to human urine for 2 h and analyzed virulence characteristics and changes in gene expression. The in vitro virulence assays showed that human urine rapidly alters adherence to human bladder epithelial cells and fibronectin, hemolysis of sheep red blood cells (RBCs), and surface hydrophobicity in a staphylococcal strain-specific manner. In addition, transcriptome sequencing (RNA-Seq) analysis of uropathogenic strain MRSA-1369 revealed that 2-h-long exposure to human urine alters MRSA transcriptome by modifying expression of genes encoding enzymes catalyzing metabolic pathways, virulence factors, and transcriptional regulators. In summary, our results provide important insights into how human urine specifically and rapidly alters MRSA physiology and facilitates MRSA survival in the nutrient-limiting and hostile urinary microenvironment. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is an uncommon cause of urinary tract infections (UTI) in the general population. However, it is important to understand MRSA pathophysiology in the urinary tract because isolation of MRSA in urine samples often precedes potentially life-threatening MRSA bacteremia. In this report, we describe how exposure to human urine alters MRSA global gene expression and virulence. We hypothesize that these alterations may aid MRSA in acclimating to the nutrient-limiting, immunologically hostile conditions within the urinary tract leading to MRSA UTI.

Hemolysis estimation in turbulent flow for the FDA critical path initiative centrifugal blood pump.

Hemolysis in medical devices and implants has been a primary concern over the past fifty years. Turbulent flow in particular can cause cell trauma and hemolysis in such devices. In this work, the effects of turbulence on red blood cell (RBC) damage are examined by simulating the flow field through a centrifugal blood pump that has been identified as a case study through the critical path initiative of the US Food and Drug Administration (FDA). In this study, a new model was employed to predict hemolysis in the turbulent flow environment in the pump selected for the FDA critical path initiative. The operating conditions for a centrifugal blood pump were specified by the FDA for rotational speeds of 2500 and 3500rpm. The model is based on the analysis of the smaller eddies within the turbulent flow field, since it is assumed that turbulent flow eddies with sizes comparable to the dimensions of the RBCs lead to cell trauma. The Kolmogorov length scale of the velocity field is used to identify such small eddies. Using model parameters obtained in prior work through comparisons to capillary and jet flow, it is found that hemolysis for the 2500-rpm pump was predicted well, while hemolysis for the 3500-rpm pump was overpredicted. Results indicate refinement of the model and empirical constants with better experimental data is needed.