Lysis Of Erythrocytes Research Articles

INTERACTION OF DEXTRIN CAPPED CADMIUM SULFIDE QUANTUM DOTS WITH BLOOD COMPONENTS: COAGULATION AND HEMOLYSIS STUDIES

Evaluate at interaction of dextrin capped cadmium sulfide quantum dots with blood components. The intravenous administration of quantum dots (QDs) has been widely reported as a promising alternative for delivery of drugs to specific cells. Therefore, previous studies have pointed out the importance of studying of, at least, factors like coagulation effect; and their hemolytic character when nanoparticles are intended to be administered for intravenous injection. The hemolytic activity and platelets aggregation of the nanoparticles was tested on isolated rat erythrocytes and platelets. Atomic force microscopy (AFM) facilitates the real‐time studies of blood cells and the morphological, structural, optical properties of the CdS‐dex QDs. In this work a methodology for erythrocytes and platelets study by platelet aggregation, hemolysis and AFM was developed. The results show that CdS‐Dex/QDs caused platelet aggregation at concentration‐dependent manner, this effect being greater at 1000 ug/mL, where the increase was up to 57% (p<0.05). The AFM measurements establish a more direct correlation between the surface topography of platelets with the surface nanostructure and reveal that modified the morphology of the platelets from the concentration of 0.01 ug/mL. The CdS‐Dex/QDs do not cause lysis of erythrocytes significantly, although they modified their morphology, suggesting that they can cause eriptosis. The CdS‐Dex/QDs, caused effects in oval and biconcave morphology of the erythrocytes, making it round and of edges not defined, semi‐flat and with cracks in its surface and lacking central depression in a concentration‐dependent manner and observed in its surface some artifacts that may correspond to agglomerated NPs. We found that CdS‐dex/QDs caused aggregation of rats platelets in vitro; however; there were both quantitative and qualitative differences depends to various concentrations.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prediction of Hemolytic Toxicity for Saponins by Machine-Learning Methods.

Saponins are a type of compounds bearing a hydrophobic steroid/triterpenoid moiety and hydrophilic carbohydrate branches. The majority of the saponins demonstrate a broad range of prominent pharmacological activities. Nevertheless, many saponins also possess harmful hemolytic toxicity, which can cause the lysis of erythrocytes and thereby hamper their applications in medicine. As such, the organic synthesis of diverse saponins with versatile therapeutic effects and without hemolytic toxicity has gained considerable interests among medicinal/organic chemists. To date, the non-hemolytic saponins of interests have usually been designed by the traditional trial-and-error method or discovered by serendipity. It would be more efficient to develop an in silico method to rationally design promising saponins without hemolytic toxicity prior to the laborious organic synthesis, despite the fact that there is, so far, no computational model to predict the hemolytic toxicity of saponins. To this end, we manually curate 331 hemolytic and 121 non-hemolytic saponins from the literature for the first time and build the first machine-learning-based hemolytic toxicity classification model for the saponins, which provides encouraging performance with 95% confidence intervals for accuracy (0.906 ± 0.009), precision (0.904 ± 0.012), specificity (0.711 ± 0.039), sensitivity (0.978 ± 0.010), F1-score (0.939 ± 0.006), and Matthews correlation coefficient (0.756 ± 0.025) on the test set by averaging over 19 different random data-partitioning schemes. Moreover, we have developed a free program called "e-Hemolytic-Saponin" for the automatic prediction and design of hemolytic/non-hemolytic saponins. To the best of our knowledge, we herein compile the first comprehensive saponin dataset focused on hemolytic toxicity, build the first informative model of hemolytic toxicity for the saponins, and implement the first convenient software that will enable organic/medicinal chemists to automatically predict and design the saponins of interests.

Nano-erythrocyte membrane-chaperoned 5-fluorouracil liposomes as biomimetic delivery platforms to target hepatocellular carcinoma cell lines

Nano-erythrocyte coating has been developed as an interesting biomimetic platform to provide hybrid nano-carriers with innate functions to target liver cancer. This goal was achieved by coating nano-erythrocyte membranes (NEMs) onto 5-fluorouracil (5-FU)-loaded liposomes (LPs) to produce NEM-5-FU-LPs. This framework is used to promote the escape of 5-FU-LPs from degradation during systemic circulation. NEMs were obtained by hypotonic lysis of erythrocytes to produce ghost erythrocytes (GEs) followed by extrusion through polycarbonate membranes. Chimeric NEM-5-FU-LPs were fabricated via the fusion of NEMs and artificial LPs. The resultant chaperoned LPs were characterized based on particle size, morphology, entrapment efficiency (EE %), stability, protein content and phosphatidylserine exposure and their in vitro release profiles and cytotoxic efficacy were also determined. The present results revealed that 5-FU-LPs, NEM-5-FU and NEM-5-FU-LPs exhibited nanosize, spherical shapes and unimodal size distributions <0.3. In addition, the vesicles presented a zeta potential with EE% of 24.6–30.7% and an appropriate stability for 3 weeks. NEM-5-FU-LPs retained the erythrocyte membrane proteins as confirmed by PAGE and displayed a sustained release profile up to 48 h when compared to NEM-5-FU and the 5-FU solution. Moreover, hybrid NEM-5-FU-LPs induced a late cytotoxic effect after 48 h compared to the other formulations. Thus, mantling of 5-FU-LPs by NEMs could enhance vesicle controllability and their targetability to liver cancer cells.

Transportan 10 improves the pharmacokinetics and pharmacodynamics of vancomycin

In the presented study, transportan 10 (TP10), an amphipathic cell penetrating peptide (CPP) with high translocation activity, was conjugated with vancomycin (Van), which is known for poor access to the intracellular bacteria and the brain. The antibacterial activity of the conjugates was tested on selected clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus sp. It turned out that all of them had superior antimicrobial activity in comparison to that of free Van, which became visible particularly against clinical MRSA strains. Furthermore, one of the conjugates was tested against MRSA - infected human cells. With respect to them, this compound showed high bactericidal activity. Next, the same conjugate was screened for its capacity to cross the blood brain barrier (BBB). Therefore, qualitative and quantitative analyses of the conjugate’s presence in the mouse brain slices were carried out after its iv administration. They indicated the conjugate’s presence in the brain in amount >200 times bigger than that of Van. The conjugates were safe with respect to erythrocyte toxicity (erythrocyte lysis assay). Van in the form of a conjugate with TP10 acquires superior pharmacodynamic and pharmacokinetic.

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S.aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S.aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.

The Mycoplasma pneumoniae HapE alters the cytokine profile and growth of human bronchial epithelial cells.

Mycoplasma pneumoniae is one of the most common pathogenic causes of community-acquired pneumonia. Hydrogen sulfide, alanine, and pyruvate producing enzyme (HapE) is a recently discovered M. pneumoniae virulence factor that can produce H2S to promote erythrocyte lysis. However, other cytotoxic effects of HapE have not been explored. The present study examined the effects of this enzyme on normal human bronchial epithelial (NHBE) cells, in an attempt to identify additional mechanisms of M. pneumoniae pathogenesis. Recombinant HapE was purified for use in downstream assays. MTT and colony formation assays were conducted to determine the effects of HapE on cell viability and growth, while flow cytometry was used to examine changes in cell proliferation and cell cycle function. ELISA was performed to examine changes in the cytokine profile of HapE-treated cells. HapE treatment arrested NHBE cells in S phase and inhibited cell proliferation in a concentration-dependent manner. The anti-inflammatory factors interleukin (IL)-4 and IL-6 were significantly enhanced following HapE treatment. Increased secretion of pro-inflammatory factors was not observed. The effects of HapE on the respiratory epithelium may have an impact on the efficiency of host immune surveillance and pathogen elimination, and contribute to the pathogenesis of M. pneumoniae.

Effects of trace mineral amount and source on aspects of oxidative metabolism and responses to intramammary lipopolysaccharide challenge in midlactation dairy cows

Trace minerals have important roles in immune function and oxidative metabolism; however, little is known about the relationships between supplementation level and source with outcomes in dairy cattle. Multiparous Holstein cows (n=48) beginning at 60 to 140 days in milk were utilized to determine the effects of trace mineral amount and source on aspects of oxidative metabolism and responses to intramammary lipopolysaccharide (LPS) challenge. Cows were fed a basal diet meeting National Research Council (NRC) requirements except for no added zinc (Zn), copper (Cu) or manganese (Mn). After a 4-week preliminary period, cows were assigned to one of four topdress treatments in a randomized complete block design with a 2×2 factorial arrangement of treatments: (1) NRC inorganic (NRC levels using inorganic (sulfate-based) trace mineral supplements only); (2) NRC organic (NRC levels using organic trace mineral supplements (metals chelated to 2-hydroxy-4-(methythio)-butanoic acid); (3) commercial inorganic (approximately 2×NRC levels using inorganic trace mineral supplements only; and (4) commercial organic (commercial levels using organic trace mineral supplements only). Cows were fed the respective mineral treatments for 6 weeks. Treatment effects were level, source and their interaction. Activities of super oxide dismutase and glutathione peroxidase in erythrocyte lysate and concentrations of thiobarbituric acid reactive substances (TBARS) and total antioxidant capacity (TAC) in plasma were measured as indices of oxidative metabolism. Effects of treatment on those indices were not significant when evaluated across the entire experimental period. Plasma immunoglobulin G level was higher in cows supplemented with organic trace minerals over the entire treatment period; responses assessed as differences of before and after Escherichia coli J5 bacterin vaccination at the end of week 2 of treatment period were not significant. Cows were administered an intramammary LPS challenge during week 5; during week 6 cows fed commercial levels of Zn, Cu and Mn tended to have higher plasma TAC and cows fed organic sources had decreased plasma TBARS. After the LPS challenge, the extent and pattern of response of plasma cortisol concentrations and clinical indices (rectal temperature and heart rate) were not affected by trace mineral level and source. Productive performance including dry matter intake and milk yield and composition were not affected by treatment. Overall, results suggest that the varying level and source of dietary trace minerals do not have significant short-term effects on oxidative metabolism indices and clinical responses to intramammary LPS challenge in midlactation cows.

Hemolysis, tocopherol, and lipid oxidation in erythrocytes and muscle tissue in chickens, ducks, and turkeys

Muscle from turkeys is more sensitive to lipid oxidation during post mortem storage compared with that of chicken and duck which may involve increased lysis of turkey erythrocytes that releases hemoglobin oxidant. Three separate experiments were conducted to study characteristics of chicken, duck, and turkey erythrocytes in which dietary tocopherols were standardized. In Experiment I, tocopherol, fatty acid composition, and lipid oxidation capacity were measured in erythrocytes from chickens, ducks, and turkeys. Tocopherol content was greater in chicken erythrocytes compared with that of duck and turkey (P < 0.05). Oleic and linoleic acid content was higher in chicken erythrocytes compared with that of turkey (P < 0.05). Lipid oxidation capacity of erythrocytes in washed turkey muscle (WTM) at pH 5.8 ranked chicken > duck > turkey (P < 0.05). In Experiment II, hemolysis was measured in erythrocytes from turkeys and chickens. Detergent-induced hemolysis (pH 7.4) was on average 12-fold greater for turkey erythrocytes compared with that of chicken (P < 0.05). In Experiment III, the ability of lysed and non-lysed erythrocytes to promote lipid oxidation was examined. Lysed erythrocytes promoted lipid oxidation in WTM more effectively than intact erythrocytes (P < 0.05). Reasons that turkey erythrocytes were more labile to detergent-induced hemolysis whereas chicken erythrocytes more effectively promoted lipid oxidation in the WTM model system are discussed. These studies describe variation in chemical and physical properties of erythrocytes from chickens, ducks, and turkeys that can influence progression of lipid oxidation in poultry muscle.

Synergistic cytotoxic effect of sodium dichloroacetate combined with chemotherapeutic drugs on B16F10 murine melanoma cell line.

The incidence of skin cancer has increased dramatically in recent decades, particularly melanoma. In this study, the cytotoxic effects of sodium dichloroacetate (DCA) in combination with chemotherapeutic drugs such as doxorubicin (DOX) and cisplatin (CIS) were evaluated. In vitro, B16F10, macrophages, and murine erythrocytes were treated with DCA (ranged from 3.66 × 104 to 3.66 × 105 μmol/L), DOX (1.38 × 10-4 to 1.38 × 10-3 μmol/L) and CIS (0.16 to 1.28 μmol/L) alone or in combination and were incubated for 72 h. Cell viability and hemolysis were determined by the MTT method and released hemoglobin, respectively. The results obtained on B16F10 cells indicate that the treatment with DCA alone showed a half-maximal inhibitory concentration (IC50) of 1.49 × 105 μmol/L, DOX caused an IC50 of 1.12 × 10-4 μmol/L and CIS-induced an IC50 of 1.14 μmol/L. Combinations of treatments with IC50 of DCA+CIS or DCA+DOX resulted in significantly decreased cell viability by 60 and 95%, respectively. Finally, the treatments alone or in combination did not cause lysis of murine erythrocytes and did not affect the cell viability of macrophages. Our results suggest that DCA enhances cytotoxicity induced by CIS or DOX on B16F10 cells without affecting erythrocytes and macrophages integrity.

Induction and evaluation of inflammatory markers by zinc oxide nanoparticles: A comparative study using in vitro and in vivo approaches.

The incidence of skin cancer has increased dramatically in recent decades, particularly melanoma. In this study, the cytotoxic effects of sodium dichloroacetate (DCA) in combination with chemotherapeutic drugs such as doxorubicin (DOX) and cisplatin (CIS) were evaluated. In vitro, B16F10, macrophages, and murine erythrocytes were treated with DCA (ranged from 3.66 × 104 to 3.66 × 105 μmol/L), DOX (1.38 × 10-4 to 1.38 × 10-3 μmol/L) and CIS (0.16 to 1.28 μmol/L) alone or in combination and were incubated for 72 h. Cell viability and hemolysis were determined by the MTT method and released hemoglobin, respectively. The results obtained on B16F10 cells indicate that the treatment with DCA alone showed a half-maximal inhibitory concentration (IC50) of 1.49 × 105 μmol/L, DOX caused an IC50 of 1.12 × 10-4 μmol/L and CIS-induced an IC50 of 1.14 μmol/L. Combinations of treatments with IC50 of DCA+CIS or DCA+DOX resulted in significantly decreased cell viability by 60 and 95%, respectively. Finally, the treatments alone or in combination did not cause lysis of murine erythrocytes and did not affect the cell viability of macrophages. Our results suggest that DCA enhances cytotoxicity induced by CIS or DOX on B16F10 cells without affecting erythrocytes and macrophages integrity.

The oxidative stress index in human population with arterial hypertension and diabetes mellitus

Context: A clinical study was conducted in a cohort of human subjects from 60 to 75 years of age in order to assess the correlation between disease diagnosis and oxidative stress (OS) damage in diabetes mellitus (DM) and arterial hypertension (AHT). Aims: To provide a sound basis to design a clinical protocol for antioxidant adjuvant therapy for DM and AHT. Methods: The values of the OS index were determined from the experimental values of biomarkers in erythrocyte lysates obtained from human blood samples including total antioxidant levels, specific biomarkers of oxidative damage, and antioxidant enzyme activities. Three study groups were formed: Group I: DM patients (110 subjects), group II: AHT patients (112 subjects); and Control group: Healthy volunteers (123 subjects). The data of all groups were analyzed using SPSS 9.0 software. A nonparametric Friedman test was used to compare several related subgroups, and changes within the groups and subgroups were tested using the Wilcoxon paired test. A Mann-Whitney U test was used to estimate significant differences (p&lt;0.05) between the subgroups. Results: Severe OS was observed in subgroup IIc (AHT III), moderate OS was observed in subgroups Ib (DM II) and IIb (AHT II), mild OS was observed in subgroup Ia (DM I), and no OS was observed in subgroup IIa (AHT I). Conclusions: The results support the possible design of clinical trial protocols for adjuvant antioxidant therapy in order to increase the efficacy of standard therapies for AHT II and III and for DM II. Antioxidant therapies for DM I and AHT I are not recommended due to the presence of only mild OS or no OS, respectively.

Isolation of Human Regulatory T Lymphocytes by Fluorescence-Activated Cell Sorting.

Regulatory T cells (Tregs) are a population of lymphocytes that exerts suppressive effects upon the immune system. In human peripheral blood, the major population of T lymphocytes with suppressive capacity are defined by expression of the T cell co-receptor CD4 and the interleukin-2 receptor α-chain (CD25), combined with minimal expression of the interleukin-7 receptor α subunit (CD127). We begin by outlining the method for isolating peripheral blood mononuclear cells (PBMCs) from human blood by centrifugation of whole blood overlayed on a hydrophilic polysaccharide, with an additional erythrocyte lysis step. The protocol that follows utilizes Fluorescence-Activated Cell Sorting (FACS) for the isolation of this CD4+CD25+CD127lo population of regulatory T cells, with high yield and purity, from immunostained PBMCs. Prior to FACS isolation, this protocol exploits magnetic immunoselection for pre-enrichment of CD25+ PBMC, which reduces the duration of the subsequent FACS isolation.

ВПЛИВ ФЕНІЛГІДРАЗИНУ НА ГІПЕРТОНІЧНИЙ СТРЕС ЕРИТРОЦИТІВ ССАВЦІВ І АНТИГЕМОЛІТИЧНУ АКТИВНІСТЬ АМФІФІЛЬНИХ СПОЛУК

In the research work the initial level of erythrocyte damage of the investigated mammals has been shown to be significantly differed under hypertonic conditions. The level of human erythrocyte lysis in 4.0 mol/l NaCl at 37 °C made 90 %, for bovine cells it was 80 % and 60 % for equine ones. During the medium temperature decrease down to 0 °C the resistance of human, bovine and equine erythrocytes did not rise significantly. It has been demonstrated that sensitivity of mammalian erythrocytes to hypertonic stress at 37 °C after treatment with phenylhydrazine depended on the species of cells. Herewith the sensitivity of modified bovine and human erythrocytes was reduced and the equine ones increased. Sensitivity of rabbit erythrocytes after modification did not change statistically and significantly. Under hypertonic stress at temperature of 0 °C the rise in the damage rate of modified erythrocytes of all the investigated mammal species was observed. Trifluoperazine and dodecyl-b,D-maltoside has been established to manifest a high protective effect under hypertonic stress of erythrocytes for all the studied mammal species, excluding the rabbit erythrocytes. Trifluoperazine has a somewhat higher anithemolytic activity (about 80–90 %) if compared with dodecyl-b,D-maltoside (70 %). When decreasing in experiment the temperature down to 0 °C the antihemolytic activity of trifluoperazine and dodecyl-b,D-maltoside was strongly decreased. It has been shown that under hypertonic stress antihemolytic activity of amphiphilic compounds after modification of mammalian erythrocytes with phenylhydrazine was strongly decreased.

Скрининг-тест для определения функциональной активности классического пути системы комплемента

Цель настоящей работы - разработка доступного теста для скрининга функциональной активности системы комплемента по классическому пути. Материалы и методы. В работе исследовали сыворотки крови 90 относительно здоровых лиц с избыточной массой тела (ИМТ &gt;25) без метаболического синдрома. Иммуноферментный анализ функциональной активности системы комплемента по классическому пути проводили с использованием коммерческого набора. Реакцию лизиса эритроцитов барана, сенсибилизированных антителами (ЕА), оценивали турбидиметрически по снижению оптической плотности суспензии при длине волны 620 нм, степень лизиса определяли по калибровочному графику. Результаты. Разработан простой скрининг-тест для определения функциональной активности классического пути системы комплемента. Корреляционный анализ показал высокую степень сходимости результатов (r = 0,496), полученных разными методами. Заключение. Скрининг-тест для определения функциональной активности классического пути системы комплемента является быстрым, информативным и доступным для рутинных исследований. The aim of this work was to develop an accessible test for screening the functional activity of the complement system classic pathway. Materials and methods. Blood serum from 90 relatively healthy, overweight (BMI&gt;25) individuals without the metabolic syndrome was studied. Enzyme immunoassay of the complement system functional activity following the classic pathway was performed using a commercial kit. The lysis of antibody-sensitized sheep erythrocytes was evaluated turbidimetrically by a decrease in suspension optical density at a wavelength of 620 nm; the lysis intensity was determined by the calibration curve. Results. A simple screening test was developed to determine the functional activity of the complement classic pathway. A high degree of correlation (r = 0.496) was observed between results obtained by different methods. This screening test for evaluating the functional activity of classic complement system pathway is fast, informative, and available for routine research.

Modulation of Human Complement System by Antimicrobial Peptide Arenicin-1 from Arenicola marina.

Antimicrobial peptides from marine invertebrates are known not only to act like cytotoxic agents, but they also can display some additional activities in mammalian organisms. In particular, these peptides can modulate the complement system as was described for tachyplesin, a peptide from the horseshoe crab. In this work, we investigated the influence on complement activation of the antimicrobial peptide arenicin-1 from the marine polychaete Arenicola marina. To study effects of arenicin on complement activation in human blood serum, we used hemolytic assays of two types, with antibody sensitized sheep erythrocytes and rabbit erythrocytes. Complement activation was also assessed, by the level of C3a production that was measured by ELISA. We found that the effect of arenicin depends on its concentration. At relatively low concentrations the peptide stimulates complement activation and lysis of target erythrocytes, whereas at higher concentrations arenicin acts as a complement inhibitor. A hypothetical mechanism of peptide action is proposed, suggesting its interaction with two complement proteins, C1q and C3. The results lead to the possibility of the development of new approaches for therapy of diseases connected with complement dysregulation, using peptide regulators derived from natural antimicrobial peptides of invertebrates.

Hemostatic Effect of the Combination of Factor VIII Concentrate with Tranexamic Acid (TXA) in the Prophylactic Setting in Severe Hemophilia a

IntroductionHemophilia is an X linked disorder characterized by an increased tendency to spontaneous bleeding resulting from profound compromise of the hemostatic response with delayed development of a clot and the formation of clots that are vulnerable to fibrinolysis. Optimization of clot stability by adding TXA to low concentrations of factor concentrates may be a way to improve prophylaxis in patients with severe hemophilia (sH) without substantially increasing the cost or the burden of more frequent infusions.AimsWe proposed to study whether hemostasis is improved with the addition of TXA to low FVIII plasma concentrations (equivalent to trough levels in prophylactic setting). We specifically studied in vitro the clot stability in patients with sHA; and in vivo the hemostatic response to trauma-induced joint bleed and tail clip in FVIII knock-out (KO) mice.MethodsAfter obtaining informed consent, blood samples of 12 adults with sHA were spiked in vitro to achieve final concentrations (FC) of 0-3-10 and 30 IU/dL of FVIII, then studied with and without TXA at FC 0.1mg/ml. Whole blood (WB) was assessed with ROTEM performed after addition of tPA in 6 patients. Thrombin generation (TG) was measured in plasma (PL) of 6 other patients. Clots obtained from TG were examined under electron microscopy (EM) to evaluate the quality of fibrin clot structure. Diameters of randomly selected 100 fibrin fibers were measured in each clot.In animal studies, 13 FVIII KO mice were given FVIII to a plasma concentration of 3 IU/dL to mimic trough FVIII levels in prophylactic setting; 7 were also infused with TXA (FC 0.1mg/ml). 10 minutes after infusion, joint bleeding was induced by knee puncture using a standardized protocol. 2 days post trauma, MRI was done and the joint bleed (surface of hyper signal into the joint) was compared between the groups with and w/o TXA.10 other FVIII KO mice were infused with 3 IU/dL rFVIII, of which 5 were also infused with TXA (FC 0.1mg/ml). 10 minutes after infusion, the extremity of the tail was cut at 2mm, and then immersed in pre-heated water-filled tube at 37°C. Blood was collected during 10 minutes. After lysis of erythrocytes, the amount of hemoglobin in collected blood was measured using spectrophotometry; means of optical density (OD) were compared between the 2 groups.ResultsA dose dependent improvement of TG was observed after adding FVIII alone (p=0.024). As expected, the addition of TXA had no effect on TG capcity. Fibrin fiber diameters were significantly decreased with TXA+FVIII compared to FVIII alone (results shown in fig1), suggesting a stronger fibrin network, as thin fibrin fibers are characteristic of a robust fibrin mesh. Surprisingly, ROTEM was fully normalized after addition of TXA only, with no exogenous FVIII.In FVIII KO mice, the addition of TXA to 3 IU/dL rFVIII significantly decreased joint bleeding compared to 3 IU/dL rFVIII alone (p=0.022) (fig2).With the tail clip experiment, the addition of TXA to 3 IU/dLFVIII showed a trend toward improved bleeding when compared to 3 IU/dLFVIII alone; with means of OD±SEM 0,31±0,19 and 0,94±0,45 respectively, but did not reach statistical significance (p=0,15) (fig3).ConclusionOur in vitro and in vivo results suggest a potential benefit of TXA when used in combination with FVIII in prophylactic setting. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiR-181b regulates ER stress induced neuron death through targeting Heat Shock Protein A5 following intracerebral haemorrhage

Endoplasmic reticulum (ER) stress acts as a protein folding and contributes to neuronal damage and neurological deterioration following intracerebral hemorrhage (ICH). Heat Shock Protein A5 (HSPA5) serves as an essential regulator of the endoplasmic reticulum (ER) stress response. However, the specific mechanism has not been will identified. Primary cortical neurons from C57BL/6 mice were subjected to erythrocyte lysates. Cell viability, microRNA and HSPA5 levels, and ER stress was detected. The interaction between microRNA and the target HSPA5 was identified by dual luciferase reporter gene assay. In addition, inflammatory cytokines, brain edema, and neurological functions in ICH mice were also assessed. Erythrocyte lysates induced ER stress and neuron damage, downregulated miR-181b and upregulated HSPA5 levels. MiR-181b suppressed HSPA5 expression by directly binding its 3′-untranslated region. Correspondingly, our data demonstrated that overexpression of miR-181b attenuated erythrocyte lysates induced neuronal necrosis and apoptosis. In vivo, downregulated miR-181b increased the HSPA5 level, along with significant elevations of pro-inflammatory cytokines, brain edema, and neurological injury following ICH. HSPA5 pathway plays an important role in ER stress induced brain damage following ICH. In addition, miR-181b has neuroprotective effects that alleviates neurological injury and represents a promising therapeutic strategy in ICH.

Endometriosis Is a Cause of Infertility. Does Reactive Oxygen Damage to Gametes and Embryos Play a Key Role in the Pathogenesis of Infertility Caused by Endometriosis?

Approximately, 10–15% of women of reproductive age are affected by endometriosis, which often leads to infertility. Endometriosis often has an inherited component, and several causative predisposing factors are hypothesized to underlie the pathogenesis of endometriosis. One working hypothesis is the theory of retrograde menstruation. According to the theory of retrograde menstruation, components of refluxed blood, including apoptotic endometrial tissue, desquamated menstrual cells, lysed erythrocytes, and released iron, induce inflammation in the peritoneal cavity. This in turn activates macrophage release of reactive oxygen species (ROS), leading to oxidative stress via the respiratory burst. Refluxed blood promotes the Fenton reaction, terminating in the production of hydroxyl radical, the most potently destructive ROS. In this article, we review the papers that demonstrate decreased quantity and quality of oocytes and embryos retrieved from IVF/ICSI patients with endometriosis. We discuss literature data demonstrating that ROS are generated in endometriotic tissues that have physical proximity to gametes and embryos, and demonstrating adverse impacts on oocyte, sperm and embryo microtubule apparatus, chromosomes, and DNA. Data that addresses the notions that endometriosis causes oocyte and fetal aneuploidy and that these events are mediated by ROS species are also discussed. Literature data are also discussed that employ use of anti-oxidant molecules to evaluate the importance of ROS-mediated oxidative damage in the pathogenesis of endometriosis. Studies are discussed that have employed anti-oxidants compounds as therapeutics to improve oocyte and embryo quality in infertile subjects, and improve fertility in patients with endometriosis.

Efficient immunoaffinity chromatography of lymphocytes directly from whole blood

We show that defined lymphocytes can be rapidly purified by immunoaffinity chromatography starting directly from whole blood. The method relies on low-affinity Fab-fragments attached to a column-matrix combined with the reversible Strep-tag technology. Compared to established cell enrichment protocols, the Strep-tag affinity chromatography of cells is independent of erythrocyte lysis or centrifugation steps, allowing for simple cell-enrichment with good yields, high purities, and excellent functionality of purified cells.

A Balanced Proinflammatory and Regulatory Cytokine Signature in Young African Children Is Associated With Lower Risk of Clinical Malaria.

The effect of timing of exposure to first Plasmodium falciparum infections during early childhood on the induction of innate and adaptive cytokine responses and their contribution to the development of clinical malaria immunity is not well established. As part of a double-blind, randomized, placebo-controlled trial in Mozambique using monthly chemoprophylaxis with sulfadoxine-pyrimethamine plus artesunate to selectively control timing of malaria exposure during infancy, peripheral blood mononuclear cells collected from participants at age 2.5, 5.5, 10.5, 15, and 24 months were stimulated ex vivo with parasite schizont and erythrocyte lysates. Cytokine messenger RNA expressed in cell pellets and proteins secreted in supernatants were quantified by reverse-transcription quantitative polymerase chain reaction and multiplex flow cytometry, respectively. Children were followed up for clinical malaria from birth until 4 years of age. Higher proinflammatory (interleukin [IL] 1, IL-6, tumor necrosis factor) and regulatory (IL-10) cytokine concentrations during the second year of life were associated with reduced incidence of clinical malaria up to 4 years of age, adjusting by chemoprophylaxis and prior malaria exposure. Significantly lower concentrations of antigen-specific T-helper 1 (IL-2, IL-12, interferon-γ) and T-helper 2 (IL-4, IL-5) cytokines by 2 years of age were measured in children undergoing chemoprophylaxis compared to children receiving placebo (P < .03). Selective chemoprophylaxis altering early natural exposure to malaria blood stage antigens during infancy had a significant effect on T-helper lymphocyte cytokine production >1 year later. Importantly, a balanced proinflammatory and anti-inflammatory cytokine signature, probably by innate cells, around age 2 years was associated with protective clinical immunity during childhood. NCT00231452.