Hemolysis Of Human Red Blood Cells Research Articles

Characterization of Nanoprecipitated PET Nanoplastics by 1H NMR and Impact of Residual Ionic Surfactant on Viability of Human Primary Mononuclear Cells and Hemolysis of Erythrocytes.

Manufactured nanoplastic particles (NPs) are indispensable for in vitro and in vivo testing and a health risk assessment of this emerging environmental contaminant is needed. The high surface area and inherent hydrophobicity of plastic materials makes the production of NPs devoid of any contaminants very challenging. In this study, we produced nanoprecipitated polyethylene terephthalate (PET) NPs (300 nm hydrodynamic diameter) with an overall yield of 0.76%. The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. For a wide range of surfactant/NP ratios (17:100 to 1.2:100), the measured zeta potential changed from -42.10 to -34.93 mV, but with an NP concentration up to 100 μg/mL, no clear differences were observed in the cellular assays performed in protein-rich media on primary human cells. The remaining impurities contributed to the outcome of the biological assays applied in protein-free buffers, such as human red blood cell hemolysis. The presence of SDS increased the NP-induced hemolysis by 1.5% in protein-rich buffer and by 7.5% in protein-free buffer. As the size, shape, zeta potential, and contaminants of NPs may all be relevant parameters for the biological effects of NPs, the relative quantification of impurities exemplified in our work by the application of 1H NMR for PET NPs and the ionic surfactant SDS could be a valuable auxiliary method in the quality control of manufactured NPs.

Antioxidant and Neuroprotective Effects of the First Tryptophyllin Found in Snake Venom (Bothrops moojeni)

In this study, we report the isolation, characterization, and synthesis of the peptide BmT-2 belonging to the tryptophyllins family, isolated from the venom of the snake Bothrops moojeni. This is the first time a tryptophyllin is identified in snake venom. We tested whether BmT-2 had cytotoxic effects and antioxidant activity in a set of experiments that included both in vitro and cell-based assays. BmT-2 presented a radical scavenging activity toward ABTS• and AAPH-derived radicals. BmT-2 protected fluorescein, DNA molecules, and human red blood cells (RBCs) from free radicals generated by the thermal decomposition of AAPH. The novel tryptophyllin was not toxic in cell viability tests, where it (up to 0.4 mg/mL) did not cause hemolysis of human RBCs and did not cause significant loss of cell viability, showing a CC50 > 1.5 mM for cytotoxic effects against SK-N-BE(2) neuroblastoma cells. BmT-2 prevented the arsenite-induced upregulation of Nrf2 in Neuro-2a neuroblasts and the phorbol myristate acetate-induced overgeneration of reactive oxygen species and reactive nitrogen species in SK-N-BE(2) neuroblastoma cells. Electronic structure calculations and full atomistic reactive molecular dynamics simulations revealed the relevant contribution of aromatic residues in BmT-2 to its antioxidant properties. Our study presents a novel peptide classified into the family of the tryptophyllins, which has been reported exclusively in amphibians. Despite the promising results on its antioxidant activity and low cytotoxicity, the mechanisms of action of BmT-2 still need to be further elucidated.

Abstract 607: Humanized mice model for efficacy and toxicity evaluation of drugs targeting CD47-SIRPα-PD1-PD-L1 axes

Abstract CD47 is widely expressed on the surface of cells. CD47 interacts with inhibitory receptor signaling protein alpha (SIRPα) to mediate a series of activities such as apoptosis, proliferation, and immune response. Tumor cells can escape the immune surveillance of macrophages through the CD47-SIRPa signaling pathway. Blocking CD47-SIRPα interactions has been shown to promote the destruction of cancer cells by phagocytes. Furthermore, there is growing evidence that targeting of the CD47-SIRPα axis may also promote antigen-presenting cell function. These identify the CD47-SIRPα axis as a promising innate immune checkpoint in cancer. We have generated humanized SIRPα and double humanized CD47/SIRPα mice model for drug efficacy and toxicity evaluation. The humanized mice with human extracellular domain and mouse cytoplasmic fragment of target protein showed similar immune cell population compared with wild type mice. The anti-CD47 antibody, Magrolimab (5F9) analog, displayed anti-tumor effect when administrated to humanized SIRPα mice bearing MC38-hCD47 (MC38 cells engineered to express human CD47) tumor cells. The tumor regression was also observed when treating humanized CD47/SIRPα mice bearing MC38-hCD47 with tested anti-CD47 antibody. Dual checkpoint blockade of CD47 and PD-L1 or PD1 have been reported as a synergistic therapy that activates both innate and adaptive immune response against tumors.We also generated humanized PD1/SIRPα and PD1/PDL1/SIRPα/CD47 mice model for efficacy and toxicity evaluation of drugs targeting for CD47/SIRPα-PD1/PD-L1 axes. Treatment with anti-CD47, anti-SIRPα and anti-PD-L1 alone showed tumor regression efficacy, and combination of anti-PD-L1 and anti-SIRPα or anti-CD47 showed improved anti-tumor effect compared with alone treatment. Those data suggested the humanized syngeneic mice model are useful for the efficacy evaluation of drugs targeting of axes.However, the administration of anti-CD47 blocking antibodies may cause haematotoxicity, including anaaemia and thrombocytopenia, because of the ubiquitous expression of CD47.In vitro Erythrocyte binding activity analysis showed that red blood cells(RBC) collected from humanized CD47/SIRPα mice could bind with Magrolimab analog, while another anti-CD47 antibody has low affinity with RBC. Furthermore, treatment with Magrolimab analog in CD47/SIRPα humanized mice caused haemagglutination and Hemolysis of human RBCs, which were not observed when treating with tested anti-CD47 antibody. Thus, the humanized CD47 and double humanized CD47/SIRPα mice are also suitable for CD47 antibody toxicity evaluation. In conclusion, humanized CD47/SIRPα and quadruple humanized PD1/PDL1/SIRPα/CD47 mice model provide a validated preclinical mouse model to interrogate new therapeutic agents targeting SIRPα-PD1 axes in immune oncology therapy. Citation Format: Juan Liang, Cunxiang Ju, Song Li, Weiwei Yu, Hongyan Sun, Santi Suryani Chen, Jing Zhao, Xiang Gao, Mark Moore. Humanized mice model for efficacy and toxicity evaluation of drugs targeting CD47-SIRPα-PD1-PD-L1 axes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 607.

In vitro antidiabetic, anti-inflammatory and antioxidant potential of the ethanol extract of Uromastyx hardwickii skin

Purpose: To evaluate the in vitro antidiabetic, anti-inflammatory and antioxidant potential of the ethanol extract of Uromastyx hardwickii Skin (UHSEE).
 Methods: The in vitro effects of UHSEE at various concentrations (10 - 250 µg/mL) on the activities of ߙ-amylase, ߙ-glucosidase and glucose uptake by yeast cells were used to evaluate its antidiabetic potential. Nitric oxide (NO), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide inhibitory assay were employed to determine its antioxidant effects, while the anti-inflammatory effects were evaluated using human red blood cell (HRBC) membrane stabilization assay.
 Results: UHSEE inhibited ߙ-amylase and ߙ-glucosidase enzymes but increased glucose uptake by yeast cells in a concentration-dependent manner (p < 0.05). It also inhibited NO, DPPH, hydrogen peroxide and HRBC hemolysis in a concentration-dependent manner (p < 0.05).
 Conclusion: Uromastyx hardwickii skin exhibits promising good antidiabetic, antioxidant and antiinflammatory properties in vitro. However, its true potentials in this regard needs to be evaluted in vivo.

Antagonistic effect of the Fengzhecao extract against human red blood cell hemolysis induced by wasp venom

To explore the antagonistic effect of Fengzhecao extract against human red blood cell (RBC) hemolysis induced by wasp venom. Water extract method was used to extract dried Fengzhecao and vacuum-dried to obtain Fengzhecao extract. It was diluted into 1 g/L for next use. Wasp venom was collected from the wasp workers. A, B, O, AB type healthy blood donors' suspended RBC solution was obtain to make washed RBC solutions and adjust the RBCs count (4.0-80.0)×109/L (the number of RBC counted on the hemocytometer is 1-20 cells/small checker). According to treatment factors, they were divided into the normal saline controlled group (NS group; 200 μL RBC solution+20 μL normal saline), Fengzhecao extract group (FZC group; 200 μL RBC solution+10 μL Fengzhecao extract+10 μL normal saline), wasp venom group (FD group; 200 μL RBC solution+10 μL wasp venom+10 μL normal saline), and Fengzhecao extract+wasp venom group (FCD group; 200 μL RBC solution+10 μL Fengzhecao extract+10 μL wasp venom), with 10 blood samples per group of every blood type. The solutions were put into the glass test tube respectively, and then into 37 centigrade water bath thermostat. After 10 minutes, the blood cell counting plate was directly observed under the microscope and the RBCs was counted. Differences in RBC count was compared between the same treatment factors of different blood types and between different treatment factor groups of the same blood type. There was no statistically significant difference in RBC count between blood types under the same treatment factors. The RBC count (×109/L) of the type A, B, O, AB in the NS group were 5.567±1.368, 5.146±1.690, 4.577±0.774, 5.197±1.587 (F = 0.852, P = 0.475), the FZC group were 5.751±1.489, 5.268±1.418, 4.727±1.174, 5.298±1.229 (F = 0.987, P = 0.410), the FD group were 0.546±0.450, 0.804±0.428, 0.679±0.283, 0.846±0.453 (F = 1.089, P = 0.366), and the FCD group were 5.532±1.330, 5.051±1.596, 4.589±0.879, 5.140±1.492 (F = 0.820, P = 0.492), respectively. Comparison of RBC count between groups with different treatment factors of the same blood type was done. There was no significant difference between the FZC group and the NS group, indicating that the extract of Fengzhecao extract had no effect on hemolysis of RBC; in the FD group, it was significantly lower than the NS group (all P < 0.05), indicating that wasp venom had a significant hemolytic effect on RBC; but there was no statistically significant difference in RBC count between the FCD group and the NS group, indicating that the Fengzhecao extract antagonizes the hemolytic effect of wasp venom without affecting the RBC count; however, the RBC count in the FCD group was significantly higher than that in the FD group (all P < 0.05), further indicating that the Fengzhecao extract antagonizes the hemolytic effect of wasp venom. Wasp venom has a significant hemolytic effect which can be effectively antagonized by Fengzhecao extract and has nothing to do with the human ABO blood type.

Biocompatibility, cytotoxicity and antimicrobial effects of gentamicin-loaded CaCO3 as a drug delivery to osteomyelitis

Nowadays, the drug delivery systems are considered as a strategy to decrease the side effects and increase the therapeutic effect of antibiotics in osteomyelitis. In the present study, biocompatibility, cytotoxicity, antimicrobial, and biofilm inhibitory effects of gentamicin-loaded CaCO3 against Pseudomonas aeruginosa were determined. The antimicrobial and biofilm effects of gentamicin-loaded CaCO3 were studied against P. aeruginosa by minimum inhibitory (MIC) and minimum biofilm inhibitory concentrations (BIC), respectively. Biocompatibility of gentamicin-loaded CaCO3 was examined by the effect on the hemolysis rate of human red blood cells (HRBCs) and erythrocyte sedimentation rate (ESR). The SDS-PAGE assay was performed to show the protein-gentamicin-loaded CaCO3 interaction by human blood plasma. Cytotoxicity of the gentamicin-loaded CaCO3 was determined by the MTT assay (hBM-MSC cell viability). In the current study, the gentamicin-loaded CaCO3 has shown antibacterial and antibiofilm effects against all isolates at different concentrations higher than MICs and BICs of gentamicin. Gentamicin-loaded CaCO3 has no significant effect on HRBCs hemolysis and ESR. A low interaction of gentamicin-loaded CaCO3 was observed with apolipoprotein A1 with a molecular weight of 28 kDa than albumin in HBP (66.5 kDa). The viability of hBM-MSC cells treated with serial concentrations of gentamicin-loaded CaCO3 was 95–100%. According to these results, gentamicin-loaded CaCO3 display the favorable biocompatibility characterization, cytotoxicity; and antibacterial and biofilm activities and can be a promising option for chronic and untreatable osteomyelitis.

Anti-inflammatory, anti-diarrheal, thrombolytic and cytotoxic activities of an ornamental medicinal plant: Persicaria orientalis

Persicaria orientalis, an ornamental medicinal plant, has been used in traditional medicine for the treatment of various diseases. Although the plant is reported to have some important pharmacological effects, many medicinal values remain unidentified. Our objective was to evaluate the anti-inflammatory, anti-diarrheal, thrombolytic, and cytotoxic properties of the methanol extract of P. orientalis leaves (Po-MeOH). Anti-inflammatory activity was measured by the inhibition of hypotonicity-induced human red blood cell hemolysis and albumin denaturation technique in vitro of Po-MeOH. Diarrheal episodes were examined in mice with castor oil-induced diarrhea. The clot lysis and brine shrimp lethality bioassay in vitro were used to evaluate the thrombolytic and cytotoxic activities of the plant extract, respectively. Using in vitro anti-inflammatory models, the results demonstrated that Po-MeOH at the five different dose ranges from 31.25 to 500 μg/mL significantly (p<0.05) protected (0.98%-50.71%) the erythrocyte membrane against lysis induced by hypotonic medium solution and protein denaturation (38.27%-79.22%) of bovine albumin, respectively. The extract exhibited a significant reduction of severity (75.17%) of castor oil-induced diarrhea in mice at the highest dose of 400 mg/kg compared to loperamide (82.06%) at 5 mg/kg. Po-MeOH also showed 33.14% clot lytic activity in the thrombolytic test and cytotoxicity with LC50 value 58.91 μg/mL in the brine shrimp bioassay. These findings suggest that Po-MeOH has significant anti-inflammatory and anti-diarrheal effects along with moderate thrombolytic and lower cytotoxic properties that may warrant the further exploration.

Hemolytic activity is mediated by the endogenous lectin in the mosquito hemolymph serum

Although cytolysis of invading organisms is an innate form of immunity used by invertebrates, so far the underlying mechanism remains less explored. The pupal hemolymph of the mosquito Armigeres subalbatus induces an activity that causes hemolysis of human red blood cells (HRBC). This hemolytic activity was inhibited by sialic acid (N-acetylneuraminic acid) and serine protease inhibitors. We purified the sialic acid-specific lectin(s) from the pupal hemolymph using formaldehyde-fixed HRBC and determined the sequence of the amino-terminal 19 amino acid residues. A polyclonal antibody produced against this N-terminal peptide clearly inhibited the hemolytic activity of the hemolymph in vitro, thus suggesting that the hemolysis of HRBC is caused by the lectin present in the mosquito hemolymph. We suggest that mosquitoes possess a cytolysis system.

The Use of ELISA to Monitor Amplified Hemolysis by the Combined Action of Osmotic Stress and Radiation: Potential Applications

A new assay has been developed to study the osmotic fragility of red blood cells (RBCs) and the involvement of oxygen-derived free radicals and other oxidant species in causing human red blood cell hemolysis. The amount of hemoglobin released into the supernatant, which is a measure of human red blood cell hemolysis, is monitored using an ELISA reader. This ELISA-based osmotic fragility test compared well with the established osmotic fragility test, with the added advantage of significantly reduced time and the requirement of only 60 mul of blood. This small amount of blood was collected fresh by finger puncture and was immediately diluted 50 times with PBS, thus eliminating the use of anticoagulants and the subsequent washings. Since exposure of RBCs to 400 Gy gamma radiation caused less than 5% hemolysis 24 h after irradiation, the RBC hemolysis induced by gamma radiation was amplified by irradiating the cell in hypotonic saline. The method was validated by examining the protective effect of Trolox, an analog of vitamin E and reduced glutathione (GSH), a well-known radioprotector, against human RBC hemolysis caused by the combined action of radiation and osmotic stress. Trolox, a known membrane stabilizer and an antioxidant, and GSH offered significant protection. This new method, which is simple and requires significantly less time and fewer RBCs, may offer the ability to study the effects of antioxidants and membrane stabilizers on human red blood cell hemolysis induced by radiation and oxidative stress and assess the osmotic fragility of erythrocytes.

The Two PM2.5 (Fine) and PM2.5–10 (Coarse) Fractions: Evidence of Different Biological Activity

Recent studies have shown that an increased concentration of environmental particulate matter (PM10) is related to many respiratory diseases. One major issue is whether the toxicity of the particles resides in some particular fraction as defined by chemical composition and size. The overall purpose of this study was to compare the in vitro toxicity of coarse (PM2.5–10) and fine (PM2.5) particulate matter, collected in an urban area of Rome, in relation to their physicochemical composition as assessed by analytic electron microscopy and atomic absorption spectroscopy. In particular, our aim was to evaluate the importance of particle physicochemical components in the induced toxicity. The in vitro toxicity assays used included human red blood cell hemolysis, cell viability, and nitric oxide (NO) release in the RAW 264.7 macrophage cell line. The hemolytic potential has been widely used as an in vitro toxicity screen and as a useful indicator of oxidative damage to biomembranes. We found that human erythrocytes underwent dose-dependent hemolysis when they were incubated with varying concentrations of fine and coarse particles. The hemolytic potential was greater for the fine particles than for the coarse particles in equal mass concentration. However, when data were expressed in terms of PM surface per volume unit of suspension, the two fractions did not show any significant hemolytic differences. This result suggested that the oxidative stress induced by PM on the cell membranes could be due mainly to the interaction between the particle surfaces and the cell membranes. RAW 264.7 macrophage cells challenged with particles showed decreased viability and an increased release of NO, a key inflammatory mediator, and both effects were not dose dependent in the tested concentration range. The fine particles were the most effective and the differences beween the two size fractions in inducing these biological effects remained unchanged when the basis of comparison was changed from weight to surface measures. It seemed therefore that these differences relied on the different physicochemical nature of the particles. The main chemical difference between the two fractions resided in a greater abundance of C-rich particles with S traces in the fine fraction. Therefore, we cautiously suggest a role for these particles in the induction of toxicity.

Hemolysis of human red blood cells by riboflavin-Cu(II) system

The photodynamic action of riboflavin is generally considered to involve the generation of reactive oxygen species, whose production is enhanced when Cu(II) is present in the reaction. In the present study we report that photoactivated riboflavin causes K+ loss from fresh human red blood cells (RBC) in a time dependent manner. Addition of Cu(II) further enhances the K+ loss and also leads to significant hemolysis. Riboflavin in a 2:1 stoichiometry with Cu(II) leads to maximum K+ loss and up to 45% hemolysis. Bathocuproine, a specific Cu(I)-sequestering agent, when present in the reaction, inhibits the hemolysis completely. Free radical scavengers like superoxide dismutase, potassium iodide and mannitol inhibited the hemolysis up to 55% or more. However, thiourea was the most effective scavenger showing 90% inhibition. These results suggest that K+ leakage and hemolysis of human RBC are basically free radical mediated reactions.

Haemolytic activity of Helicobacter pylori against human and animal erythrocytes

Clinical isolates and reference strains of H. pylori (n = 28) were investigated for lytic activity against red blood cells (RBC) of man, sheep, horse, rabbit, hamster and guinea pig. The test medium (HTA) consisted of agar base supplemented with newborn calf serum and washed RBC. In contrast to previous reports, haemolysis of human RBC was detected. Blood group A RBC supported the haemolytic growth of all strains studied. On HTA with blood group 0 RBC, one strain failed to grow and five strains grew nonhaemolytically. The diversity of growth and haemolysis on HTA containing the human and several animal RBC species allowed a differentiation of the strains into eight groups. Using a liquid RBC assay for quantitative measurement of haemolysis, extracts of broth-grown cells of strain ATCC 43504 showed an average haemolysis rate of 75% against sheep RBC and of 83% against human A RBC. Cell-free culture supernatants, however, showed an average haemolysis rate of 57% against sheep RBC and no activity against human A RBC. The data indicate that H. pylori expresses an intracellular haemolytic factor and a cell surface-associated or secreted haemolytic factor which have different RBC specificities. The demonstration of the sensitivity of human RBC supports the hypothesis that the haemolytic property of H. pylori is involved in the pathogenesis of human infection.

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes.

The interaction of Actinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.

Role of ascorbic acid in lead acetate induced lipid peroxidation and hemolysis in human RBC

Lipid peroxidation and hemolysis in humand red blood cells (RBCs) increased on their exposure to increasing concentrations of lead acetate (0.01–1.0mM). However pretreatment of RBCs with ascorbic acid (2mM and 4mM) significantly reduced the effect of lead acetate on lipid peroxidation and hemolysis in human RBCs.

Thermal instability of red blood cell membrane bilayers: temperature dependence of hemolysis.

Rates of human red blood cell hemolysis were measured as a function of temperature. Three distinct temperature intervals for hemolysis were noted: a) At temperatures equal to or less than 37 degrees C no hemolysis was observed for the duration of the incubation (30 hr). b) For temperatures exceeding 45 degrees C hemolysis rates are rapid and are accompanied by gross changes in cellular morphology. The activation energy for hemolysis is 80 kcal/mole; this value is characteristic of protein denaturation and enzyme inactivation suggesting that these processes contribute to hemolysis at these high temperatures. c) Between 38 and 45 degrees C the energy of activation is 29 kcal/mole, indicating that a fundamentally different process than protein inactivation is responsible for hemolysis at these relatively low temperatures. A mechanism based on the concept of the critical bilayer assembly temperature of cell membranes (N.L. Gershfeld, Biophys. J. 50:457-461, 1986) accounts for hemolysis at these relatively mild temperatures: The unilamellar state of the membrane is stable at 37 degrees C, but is transformed to a multibilayer when the temperature is raised; hemolysis results because formation of the multibilayer requires exposing lipid-free areas of the erythrocyte surface. An analysis of the activation energy for hemolysis is presented that is consistent with the proposed unilamellar-multibilayer transformation.

The haemolysis of human red blood-cells by freezing and thawing

The destructive processes which occur when red blood-cells are frozen and thawed have been observed. The results of these observations suggest that the principal damaging effect associated with freezing is the concentration of the electrolytes present within and without the cell. The destructive action of exposure to concentrated salt solutions is not instantaneous and the survival of rapidly frozen and thawed cells is attributed to this factor. The nature of the damage caused by exposure to strong salt solutions is complex. At moderate concentrations the blood-cell becomes sensitive to thermal and mechanical shock; at high concentrations the cell structure is dispersed by an apparently lyotropic effect.

A skin reaction in carcinoma from the subcutaneous injection of human red blood cells

Numerous investigators have shown that if the blood serum of a patient suffering from carcinoma be mixed with normal human red blood cells hemolysis occurs. The reaction takes place in from 50 per cent. to 80 per cent. of patients with malignant disease. It occurs with considerable frequency in tuberculosis, and more rarely in other diseases.