Aggregation Of Human Erythrocytes Research Articles

Anti-Inflammatory Effects of the 35kDa Hyaluronic Acid Fragment (B-HA/HA35).

Hyaluronic acid (HA) and HA fragments interact with a variety of human body receptors and are involved in the regulation of various physiological functions and leukocyte trafficking in the body. Accordingly, the development of an injectable HA fragment with good tissue permeability, the identification of its indications, and molecular mechanisms are of great significance for its clinical application. The previous studies showed that the clinical effects of injectable 35kDa B-HA result from B-HA binding to multiple receptors in different cells, tissues, and organs. This study lays the foundation for further studies on the comprehensive clinical effects of injectable B-HA. We elaborated on the production process, bioactivity assay, efficacy analyses, and safety evaluation of an injectable novel HA fragment with an average molecular weight of 35 kDa (35 kDa B-HA), produced by recombinant human hyaluronidase PH20 digestion. The results showed that 35 kDa B-HA induced human erythrocyte aggregation (rouleaux formation) and accelerated erythrocyte sedimentation rates through the CD44 receptor. B-HA application and injection treatment significantly promoted the removal of mononuclear cells from the site of inflammation and into the lymphatic circulation. At a low concentration, 35 kDa B-HA inhibited production of reactive oxygen species and tumor necrosis factor by neutrophils; at a higher concentration, 35 kDa B-HA promoted the migration of monocytes. Furthermore, 35 kDa B-HA significantly inhibited the migration of neutrophils with or without lipopolysaccharide treatment, suggesting that in local tissues, higher concentrations of 35 kDa B-HA have antiinflammatory effects. After 99mTc radiolabeled 35 kDa B-HA was intravenously injected into mice, it quickly entered into the spleen, liver, lungs, kidneys and other organs through the blood circulation. This study demonstrated that the HA fragment B-HA has good tissue permeability and antiinflammatory effects, laying a theoretical foundation for further clinical studies.

Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76

ABSTRACT Background Some strains of Streptococcus mitis exhibit β-hemolysis due to the β-hemolytic activity of cholesterol-dependent cytolysin (CDC). Recently, a gene encoding an atypical lectinolysin-related CDC was found in S. mitis strain Nm-76. However, the product of this gene remains uncharacterized. We aimed to characterize this atypical CDC and its molecular functions and contribution to the pathogenicity of S. mitis strain Nm-76. Methods Phylogenetic analysis of the CDC gene was conducted based on the web-deposited information. The molecular characteristics of CDC were investigated using a gene-deletion mutant strain and recombinant proteins expressed in Escherichia coli. Results The gene encoding CDC found in Nm-76 and its homolog are distributed among many S. mitis strains. This CDC is phylogenetically different from other previously characterized CDCs, such as S. mitis-derived human platelet aggregation factor (Sm-hPAF)/lectinolysin and mitilysin. Because this CDC possesses an additional N-terminal domain, including a discoidin motif, it was termed discoidinolysin (DLY). In addition to the preferential lysis of human cells, DLY displayed N-terminal domain-dependent facilitation of human erythrocyte aggregation and intercellular associations between human cells. Conclusion DLY functions as a hemolysin/cytolysin and erythrocyte aggregation/intercellular association molecule. This dual-function DLY could be an additional virulence factor in S. mitis.

A Study of the Mechanism of the FeCl3-Induced Aggregation of Human Erythrocytes

Abstract—Aggregation of intact and ATP-depleted human erythrocytes, which was induced by FeCl3 at a final concentration of 200 μM, was studied. The shape of erythrocytes and the stability of their cytoskeleton, the effect of shear flow on erythrocyte deformation, and the content of anionic sites in cell membranes were investigated. It is shown that the ATP depletion of erythrocytes led to a change in their shape: discocytes were transformed into echinocytes. This was accompanied by a decrease in the membrane cytoskeleton stability, disturbance in the deformation of erythrocytes, and reduction in the content of anionic sites in the membrane of ATP-depleted erythrocytes. Aggregation of such erythrocytes significantly decreased. A possible mechanism of FeCl3-induced aggregation of human erythrocytes is discussed.

РОЛЬ НАРУШЕНИЙ РЕОЛОГИЧЕСКИХ СВОЙСТВ ЭРИТРОЦИТОВ В ПАТОГЕНЕЗЕ ВОСПАЛИТЕЛЬНЫХ ЗАБОЛЕВАНИЙ КИШЕЧНИКА У ДЕТЕЙ

Цель исследования: изучение роли нарушений реологических свойств эритроцитов в патогенезе воспалительных заболеваний кишечника у детей. Материалы и методы. Обследовано 37 детей с воспалительными заболеваниями кишечника (ВЗК): 17 детей с болезнью Крона (БК) и 20 — с язвенным колитом (ЯК). Исследовали деформируемость эритроцитов, спонтанную агрегацию тромбоцитов в искусственном сдвиговом потоке, динамическую вязкость крови, морфологию агрегатов эритроцитов, состояние цитоскелета эритроцитов методом термоиндукции, состояние гемостаза на тромбоэластографе, среднемолекулярные пептиды, сорбционную емкость эритроцитов, связывающую способность альбумина. Результаты. Выявлено, что ВЗК сопровождаются значительным ухудшением гемореологических свойств крови, что является важным фактором патогенеза этих заболеваний и лежит в основе нарушений микроциркуляции при ВЗК. Установлено, что при БК нарушения большинства исследованных показателей не только существеннее, чем при ЯК, но и стабильнее — они сохраняются даже после проведенного лечения. Также как и реологические нарушения, эндогенная интоксикация сохраняется при БК и после проведенного лечения. Заключение. Нарушения реологических свойств крови являются важным фактором, обуславливающим ишемические повреждения кишечника. Это дает основание рекомендовать использование при ВЗК адъювантных методов борьбы с гипоксией и нарушениями микроциркуляции. Aim: to study the role of disturbances in erythrocytes rheological properties in pathogenesis of inflammatory bowel diseases (IBD) in children. Materials and methods. The study included 37 patients with IBD: 17 with Crohn’s disease (CD) and 20 with ulcerative colitis (UC). Erythrocyte deformability, spontaneous platelet aggregation, dynamical blood viscosity, morphology of erythrocyte aggregates, the state of erythrocyte cytoskeleton by thermoinduction method, hemostasis state by thromboelastograph, medium molecular peptides, erythrocyte sorption capacity, binding ability of albumin were researched. Results. We revealed that IBD accompanied by a significant degradation of blood hemorheological properties. It is an important factor in the pathogenesis of these diseases and is at the core of microcirculation disorders at IBD. It was found that at CD disorders the majority of studied parameters were not only more significant than in UC, but they also remained much more stable even after the treatment. At CD endogenous intoxication as well as rheological disorders persisted after treatment. Conclusion. Disorders of blood rheological properties are an important factor causing ischemic damage of the intestine. This gives reason for recommending the use of adjuvant methods to reduce hypoxia and microcirculatory disturbances at IBD.

Preparation of blue-color-emitting graphene quantum dots and their in vitro and in vivo toxicity evaluation

We synthesized and characterized blue-color-emitting graphene quantum dots (BGQDs) which is better than that of other GQDs synthesized from organic molecules. We investigated the biocompatibility of BGQDs with cells and erythrocytes. Evaluation of epithelial carcinoma cancer cells indicated that most cells successfully took up BGQDs and toxicity testing showed no effect on cell viability. We also examined alterations in deformability and aggregation of human erythrocytes exposed to BGQDs at different concentrations and incubation times. These results suggest that BGQDs are biocompatible and do not cause cell membrane damage, and thus, are promising nanomaterials for various medical diagnoses and treatments.

Effect of vinca alkaloids on human erythrocyte aggregation

Effect of vinca alkaloids on human erythrocyte aggregation

The effect of centrifugation at various g force levels on rheological properties of rat, dog, pig and human red blood cells.

Laboratory investigations often require centrifugation of blood samples for various erythrocyte tests. Although there is a lack of data about the effect of centrifugation at various g force levels on erythrocyte rheological properties. We aimed to investigate the effect of a 10-minute centrifugation at 500, 1000 or 1500 g at 15°C of rat, dog, pig and human venous (K3-EDTA, 1.5 mg/ml) blood samples. Hematological parameters, erythrocyte deformability, cell membrane stability, osmotic gradient ektacytometry (osmoscan) and erythrocyte aggregation were determined. Hematological and erythrocyte deformability parameters showed interspecies differences, centrifugation caused no significant alterations. Cell membrane stability for human erythrocytes centrifuged at higher g level showed less decrease in deformability. Osmoscan O min parameter showed slight elevation in dog centrifuged aliquots. Erythrocyte aggregation parameters changed unexpectedly. Rat and dog erythrocyte aggregation indices significantly dropped in centrifuged aliquots. Pig erythrocyte aggregation indices increased significantly after centrifugation. Human erythrocyte aggregation was the most stable one among the investigated species. The used centrifugation protocols caused the largest alterations in erythrocyte aggregation in a controversial way among the investigated species. On the other hand, erythrocyte deformability parameters were stable, cell membrane stability and osmoscan data show minor shifts.

Lysophosphatidic acid and human erythrocyte aggregation

The effects of lysophosphatidic acid on the morphology and aggregation of human erythrocytes has been studied. Morphology of erythrocytes and their aggregates were studied by light microscopy. It has been shown that lysophosphatidic acid changes the shape of red blood cells: diskocyte become echinocytes. Aggregation of red blood cells (rouleaux) was significantly reduced in autoplasma. At the same time there is a strong aggregation of echinocytes. This was accompanied by the formation of microvesicles. Adding normal plasma to echinocytes restores shape and aggregation of red blood cells consisting of "rouleaux". A possible mechanism of action of lysophosphatidic acid on erythrocytes is discussed.

Lipid Metabolism as a Therapeutic Target

Targeting lipid metabolism (biosynthesis and catabolism) associated with human diseases and pathogens with therapeutics has gained much momentum in recent years. This has come about in part due to the wide availability of sequenced genomes, the advancement in analytical techniques such as mass spectrometry and deep sequencing, and the increased understanding of signaling molecules. Collectively, this has advanced the knowledge of lipid metabolism for translational purposes, that is, diagnosis or treatment, and the papers and reviews in this special issue highlight some of these aspects. One review in this issue by S. Young et al. outlines the major pathways in eukaryotic sphingolipid metabolism and catabolism and discusses these in relation to the possibilities of their therapeutic intervention against cancers, Alzheimer's disease, inherited diseases, and numerous important human pathogens. Such a pathogenic disease is leishmaniases, which is the subject of the paper by H. Ali et al., who investigate the reliance of old and new world Leishmania species on host sphingolipids and how an infection may or may not influence host sphingolipid biosynthesis. Another review in this issue addresses the link between serum triglyceride levels caused by a dysregulation of lipoprotein lipase and the risk of development of various cancers, atherosclerosis, chylomicronemia, obesity, and type 2 diabetes. S. Takasu et al. conclude that as lipoprotein lipase plays important roles in many of these conditions and as such it is appropriate to treat it as a general target for chemopreventive and chemotherapeutic agents. This approach is nicely highlighted by the paper by R. Noriega-Cisneros et al., who investigate the effect of chronic administration of ethanolic extract of Eryngium carlinae in serum of streptozotocin-induced diabetic rats. They clearly show reduced levels of creatinine, uric acid, total cholesterol and triglycerides, thus as a general approach this could be used to reduce hyperlipidemia related to cardiovascular risk in diabetes mellitus. In a related area, Saldanha et al. look at the behavior of human erythrocyte aggregation in presence of autologous lipoprotein, finding that human blood aliquots enriched with their own LDL-C and HDL-C showed higher levels of erythrocyte aggregation compared to controls. N. Nikolic et al. show in their paper that overexpression of peroxisome proliferator-activated receptor γ coactivator-1α increases oxidative capacity of human skeletal muscle cells by improving lipid metabolism, thus increasing expression of genes involved in regulation of mitochondrial function and biogenesis and decreasing expression of the fast fiber-type gene marker MHCIIa. They conclude that obesity and obesity-related diseases could be therapeutically targeted by increasing expression of peroxisome proliferator-activated receptor γ coactivator-1α. While M. Cahova et al., in their study on liver lysosomal lipase activity, conclude that overproduction of diacylglycerol may represent the causal link between high-fat die-induced hepatic triacylglycerol accumulation and hepatic insulin resistance via PKCe activation. On a personal note, I would like to highlight that through my own research on protozoan parasites and that with various collaborators studying a wide variety of human pathogens and model systems for human diseases, mitochondrial dysfunction as a result of not maintaining its lipid homeostasis seems to be a leading cause for many of downstream affects. Thus, I would suggest a better understanding of mitochondrial lipid metabolism (both biosynthetic and catabolic) will aid the development of effective novel therapeutics and diagnostics in the future. Terry K. Smith Todd B. Reynolds Paul W. Denny

Behaviour of human erythrocyte aggregation in presence of autologous lipoproteins.

The aim of this work was to evaluate in vitro the effect of autologous plasma lipoprotein subfractions on erythrocyte tendency to aggregate. Aliquots of human blood samples were enriched or not (control) with their own HDL-C, LDL-C, or VLDL-C fractions obtained from the same batch by density gradient ultracentrifugation. Plasma osmolality and erythrocyte aggregation index (EAI) were determined. Blood aliquots enriched with LDL-C and HDL-C showed significant higher EAI than untreated aliquots, whereas enrichment with VLDL-C does not induce significant EAI changes. For the same range of lipoprotein concentrations expressed as percentage of osmolality variation, the EAI variation was positive and higher in presence of HDL-C than upon enrichment with LDL-C (P < 0.01). Particle size, up to LDL diameter values, seems to reinforce erythrocyte tendency to aggregate at the same plasma osmolality (particle number) range of values.

Cationic Derivatives of Dextran and Hydroxypropylcellulose as Novel Potential Heparin Antagonists

Cationic derivatives of dextran (Dex) and hydroxypropylcellulose (HPC) were studied as potential alternatives of protamine sulfate (PS) used in the reversal of anticoagulant activity of heparin. The modification was performed by the attachment of cationic groups to the Dex main chain or by grafting short side chains of a polycation onto HPC. The cationic derivatives of these polysaccharides were found to bind heparin with the efficiency increasing with growing degree of cationic modification. The degree of cationic modification and consequently the ζ potential of the polymers do not have to be high to achieve effective heparin binding. The size of the complexes of cationic Dex with unfractionated heparin (UFH) is a few micrometers. For complexes of cationic HPC and UFH the size is much below 1 μm, both below and above the lower critical solution temperature of HPC. None of the cationic polysaccharides studied caused hemolysis. The concentrations of the polymers inducing the aggregation of human erythrocytes in vitro were determined.

Hen lysozyme amyloid fibrils induce aggregation of erythrocytes and lipid vesicles

We have studied the interaction of hen egg white lysozyme (HEWL) amyloid fibrils with human erythrocytes and lipid vesicles. The fibrils caused extensive aggregation of human erythrocytes and lipid vesicles without any significant lysis. The membrane activity of HEWL fibrils suggests that the interaction of lysozyme fibrils with cellular membranes could be a contributing factor under disease conditions.

Incoherent light transport in an anisotropic random medium: A probe of human erythrocyte aggregation and deformation

This paper investigates the flow induced disaggregation, deformation and orientation of several modified human red blood cells suspended in concentrated, physiological like conditions (volume fraction in erythrocytes of 0.4). The aim is to determine simultaneously, and under flow, the aggregate sizes as well as the deformation and orientation of the cells. The measurement method uses steady, incoherent, unpolarized light transport while the sample is sheared in a flow cell controlled by a rheometer. Several blood samples were prepared to alter the erythrocyte's aggregating, deformability and shape properties. The measurements using these samples show a clear relationship between the intrinsic properties of the cells and the evolution of aggregate sizes, average cell orientation and anisotropy as a function of the applied shear, which may lead to clinical applications. In other words, the careful analysis of the incoherent light transport in concentrated media provides quantitative insight into their microscopic details. In particular, the topological properties (average anisotropy and orientation) and size of the suspended objects can be determined.

Extra- and Intracellular Mechanisms of Changes in Erythrocyte Aggregation

Reversible aggregation is a form of erythrocyte behavior. The presence of α- and β-adrenergic receptors on erythrocyte membranes suggests that aggregation can be influenced by agonists of these receptors. Human erythrocyte aggregation (EA) was studied in the presence of α- and β-agonists of adrenergic receptors (in the concentration range from 10−6 to 10−8 M). The α2-agonist clonidine stimulated EA most strongly (by 163%, P < 0.01). The cell reaction decreased from the α-agonist clonidine to the β-agonist metaproterenol. The phosphodiesterase inhibitor papaverine and the penetrating cAMP analogue dibutyryl cAMP (dB-cAMP) increased the intracellular cAMP and decreased EA by 46–50% (P < 0.05). Clonidine-induced stimulation of EA sharply decreased upon erythrocyte incubation in the presence of clonidine + dB-cAMP and became even lower than in the control. Thus, α-agonists of adrenergic receptors markedly stimulated EA. The adenylate cyclase-cAMP system is likely involved as an intracellular signaling pathway.

Comparison and simulation of different levels of erythrocyte aggregation with pig, horse, sheep, calf, and normal human blood

Erythrocyte aggregation levels in pig, horse, sheep, and calf blood samples were investigated and compared to that of normal human blood. The aggregation kinetics and adhesive forces between red cells, and an index of structure of the aggregates were determined with an erythroaggregameter (Regulest, France) at constant hematocrit (0.40 1/1) and temperature (37 °C). The adhesive forces and the index of structure in pig blood were close to those of normal human blood. The results for horse blood showed a very high level of aggregation kinetics and adhesive forces between red cells. For sheep and calf blood, little erythrocyte aggregation was found. To simulate different levels of red cell hyperaggregation in humans, a volume of horse plasma was replaced by isotonic NaCl in different proportions (5 to 40% V/V). The kinetics of rouleaux formation and especially the adhesive forces between erythrocytes were systematically decreased, while the index of structure was raised with increasing concentrations of isotonic NaCl. By replacing the porcine plasma with isotonic NaCl, normal and hypoaggregating levels of human red cells were simulated. The aggregation kinetics and the adhesive forces were reduced and the index of structure was raised when the concentration of isotonic NaCl was increased. In summary, large differences in the aggregation parameters were found between mammals. This study also showed that different human erythrocyte aggregation levels can be simulated by diluting the concentration of plasma proteins in equine and porcine bloods.

The amino-terminal peptide of HIV-1 glycoprotein 41 fuses human erythrocytes

The ability of synthetic peptides based on the amino-terminus of HIV-1 glycoprotein 41 000 (gp41) to fuse human erythrocytes was investigated. Previous site-directed mutagenesis studies have shown an important role for the N-terminal gp41 domain in HIV-fusion, in which replacement of hydrophobic amino acids with polar residues inhibits viral infection and syncytia formation. Here, a synthetic peptide (FP; 23 amino acid residues 519–541) corresponding to the N-terminus of HIV-1 gp41, and also a FP analog (FP526L/R) with Arg replacing Leu-526, were prepared with solid phase techniques. The lipid mixing and leakage of resealed ghosts triggered by these peptides were examined with fluorescence quenching techniques. Peptide-induced aggregation of human erythrocytes was studied using Coulter counter sizing and scanning electron microscopy (SEM). Using resealed erythrocyte ghosts at physiologic pH, FP induces rapid lipid mixing between red cell membranes at doses previously shown to hemolyze intact cells. FP also causes leakage from resealed ghosts, and promotes the formation of multicelled aggregates with whole erythrocytes. Contrarily, similar FP526L/R concentrations did not induce red cell lysis, lipid mixing, leakage or aggregation. Since the fusogenic potency of FP and FP526L/R parallels earlier gp41 mutagenesis studies showing that substitution of Arg for Leu-526 blocks fusion activity, these data suggest that the N-terminal gp41 domain in intact HIV participates in fusion.

Effects of ultrasound on agglutination and aggregation of human erythrocytes in vitro

A new experimental approach has shown that human erythrocytes of different blood groups were induced to form more agglutinates at a sound pressure of 70–240 kPa in vitro than the control erythrocytes. Similar effects were observed for alcian blue and dextran stimulated aggregation and for spontaneous aggregation. The increase of agglutination or aggregation was reversible. Heating and acoustic cavitation were shown not to be responsible for this effect. Bulk fluid movement produced by ultrasound irradiation appeared to cause the described phenomenon. Possible underlying mechanisms connecting the acoustic streaming and agglutination or aggregation behaviour of the cells are proposed.

Effect of pH on the velocity of erythrocyte aggregation.

The effect of pH on the velocity of aggregation of human erythrocytes was quantitatively examined with a rheoscope combined with a video-camera, an image analyzer and a computer, in relation to the morphological changes of erythrocytes and their aggregates. (i) With increasing pH of the medium, the velocity of erythrocyte aggregation increased. (ii) The rouleaux formed at high pH were longer in shape and more stable against the increase of shear rate than those formed at low pH. (iii) With increasing pH, the diameter of erythrocyte increased, the (maximum) thickness decreased, and the cell volume decreased. The pH dependency of erythrocyte aggregation may be mainly due to the morphological change of erythrocytes, and partly due to the changes of erythrocyte deformability and of interaction with macromolecules.

Effect of temperature on the velocity of erythrocyte aggregation

The velocity of the aggregation of human erythrocytes was examined in the range of 5–43°C with a rheoscope combined with a video camera, an image analyzer and a computer. (1) With increasing temperature, the velocity of erythrocyte aggregation induced by fibrinogen, immunoglobulin G and artificial macromolecules (dextran of 70 kDa and poly(glutamic acid) of 50 kDa) increased. However, the relationship between the velocity of erythrocyte aggregation and the temperature was different among these macromolecules. (2) In 70% autologous plasma, the velocity of erythrocyte aggregation was minimum at 15–18°C, and increased at both higher and lower temperatures. (3) The shape of erythrocyte aggregates in 12 μmol/l fibrinogen (containing 770 μmol/l albumin) and in 70% autologous plasma was dependent on temperature: three-dimensional below 15–18°C and one-dimensional (mainly rouleaux) above 15–18°C. However, the shape of aggregates in 27 μmol/l immunoglobulin G (containing 770 μmol/l albumin) was three-dimensional in all temperature ranges. (4) The temperature dependency of erythrocyte aggregation was discussed in terms of the changes of medium viscosity, of erythrocyte properties and of bridging macromolecules.

NMR investigation of hydroxyethyl starch‐induced aggregation of human erythrocytes

Aggregation of human erythrocytes at physiological hematocrit by hydroxyethyl starch (HES) was investigated with a 1H nuclear magnetic resonance method which measures the erythrocyte mean water exchange time, tau a. A relationship that takes into account the effect of the aggregate as the water-exchanging unit was used to transform tau a values into the number of cells per aggregate, n. The procedure permits the determination of the time course of aggregation as well as its initial and equilibrium status. The values of n at equilibrium range from about 2 to 4 cells over the concentration of 1 to 5% HES in isotonic saline. The aggregation of erythrocytes in the presence of HES was found to follow first-order kinetics. The feasibility of this technique for application in the study of diseased states is indicated.