Potential Of Red Blood Cells Research Articles

Red blood cells as potential materials for microRNA biomarker study: overcoming heparin-related challenges.

microRNAs (miRNAs) have been intensively studied as valuable biomarkers in cardiometabolic disease. Typically, miRNAs are detected in plasma or serum but the use of samples collected in heparinized tubes is problematic for miRNA studies utilizing qPCR. Heparin and its derivatives interfere with qPCR-based analysis, leading to a substantial reduction or even complete loss of detectable miRNA levels. Given that red blood cells (RBCs) express abundant miRNAs, whose expression is altered in cardiometabolic disease, RBCs could serve as an attractive alternative in biomarker studies. Here, we aim to explore the stability of miRNAs in RBCs collected from whole blood with different anticoagulants and thereby the potential of RBCs as alternative materials for miRNA biomarker studies. miRNA profiling was performed in human RBCs via RNA sequencing, followed by qPCR validation of selected miRNAs in RBCs and plasma in both heparinized and EDTA tubes. RNA sequencing revealed abundant miRNA presence in RBCs isolated from blood collected in EDTA tubes. miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were detected at comparable levels in RBCs isolated from both heparinized and EDTA tubes, but not in plasma from heparinized tubes. Of note, miR-210 levels were consistently lower in RBCs from individuals with type 2 diabetes compared to healthy controls, regardless of anticoagulant type, supporting their potential as biomarker materials. In conclusion, RBCs offer a promising alternative for miRNA biomarker studies, overcoming heparin-related challenges.

Cytoplasmic anion/cation imbalances applied across the membrane capacitance may form a significant component of the resting membrane potential of red blood cells

Electrical aspects of cell function manifest in many ways. The most widely studied is the cell membrane potential, Vm, but others include the conductance and capacitance of the membrane, the conductance of the enclosed cytoplasm, as well as the charge at the cell surface (an electrical double layer) producing an extracellular electrical potential, the ζ-potential. Empirical relationships have been identified between many of these, but not the mechanisms that link them all. Here we examine relationships between Vm and the electrical conductivities of both the cytoplasm and extracellular media, using data from a suspensions of red blood cells. We have identified linear relationships between extracellular medium conductivity, cytoplasm conductivity and Vm. This is in contrast to the standard model of a resting membrane potential which describes a logarithmic relationship between Vm and the concentration of permeable ions in the extracellular medium. The model here suggests that Vm is partially electrostatic in origin, arising from a charge imbalance at an inner electrical double-layer, acting across the membrane and double-layer capacitances to produce a voltage. This model describes an origin for coupling between Vm and ζ, by which cells can alter their electrostatic relationship with their environment, with implications for modulation of membrane ion transport, adhesion of proteins such as antibodies and wider cell–cell interactions.

Impact of Polyethylene Glycol Functionalization of Graphene Oxide on Anticoagulation and Haemolytic Properties of Human Blood.

Graphene oxide (GO) is one of the most explored nanomaterials in recent years. It has numerous biomedical applications as a nanomaterial including drug and gene delivery, contrast imaging, cancer treatment, etc. Since most of these applications need intravenous administration of graphene oxide and derivatives, the evaluation of their haemocompatibility is an essential preliminary step for any of the developed GO applications. Plentiful data show that functionalization of graphene oxide nanoparticles with polyethylene glycol (PEG) increases biocompatibility, thus allowing PEGylated GO to elicit less dramatic blood cell responses than their pristine counterparts. Therefore, in this work, we PEGylated graphene oxide nanoparticles and evaluated the effects of their PEGylation on the structure and function of human blood components, especially on the morphology and the haemolytic potential of red blood cells (RBCs). Further, we studied the effect of PEGylation on some blood coagulation factors, including plasma fibrinogen as well as on the activated partial thromboplastin (aPTT), prothrombin time (PT) and platelet aggregation. Our findings provide important information on the mechanisms through which PEGylation increases GO compatibility with human blood cells. These data are crucial for the molecular design and biomedical applications of PEGylated graphene oxide nanomaterials in the future.

The Change of Electrical Properties of Blood Corpuscles under in vitro Mechanical Stress

Changes in the electrical properties of blood corpuscles that are experiencing mechanical stress modeled in vitro have been studied. An increase in the concentration of ATP molecules in the intercellular space in response to the mechanical effect of moving plasma layers both in the blood of healthy people and patients with acute lymphoblastic leukemia is demonstrated. The surface potential of red blood cells and platelets becomes more positive both in the blood of healthy people and in leukemia patients. In contrast, the negative charge of lymphocytes in healthy people decreases in response to mechanical stress but increases in the blood of patients with acute lymphoblastic leukemia.

Коррекция состояния организма низкоинтенсивным лазерным излучением при моделировании адреналовой токсемии у крыс

Срыв адаптационных возможностей организма может стать патогенетической основой различных заболеваний. Длительный выброс адреналина в кровь вызывает кататоксические эффекты, что делает актуальным поиск эффективных средств и методов коррекции гиперадреналинемии. Цель работы - изучение влияния низкоинтенсивного лазерного излучения (НИЛИ) на электрофоретическую подвижность эритроцитов, процессы липопероксидации и структуру надпочечников при адреналовой токсемии у крыс. Методика. Адреналовую токсемию моделировали внутрибрюшинным введением адреналина-гидрохлорида (0,1 мг/кг). Животным 1-й группы вводили только адреналин, животных 2-й - через 30 мин после введения адреналина облучали НИЛИ, крысы 3-й группы получали только воздействие НИЛИ, контролем служили интактные животные (4-я группа). Воздействие НИЛИ проводилось на затылочную область в течение 10 мин. В качестве источника излучения применяли аппарат лазерный терапевтический «Успех» (ГП «Восход»), основанный на низкоинтенсивном импульсном излучении (длина волны - 0,89±0,05 мкм, частота импульсов 415 Гц, плотность средней мощности излучения - 193 мкВт/см2, площадь облучаемой поверхности 20 см2). Забор крови производили через 1 ч, 24 ч и 1 нед после начала эксперимента. Определяли уровень малонового диальдегида по реакции с тиабарбитуровой кислотой; электрофоретическую подвижность эритроцитов изучали методом микроэлектрофореза. Структуру надпочечников изучали морфологическими методами. Результаты. Показано, что НИЛИ на фоне адреналина (2-я группа) через 1 нед нивелировало снижение электрофоретической подвижности эритроцитов, восстанавливало уровень малонового диальдегида до значений интактной группы. Морфологическая картина надпочечников в этой группе не отличалась от таковой у интактных животных, тогда как при действии адреналина (1-я группа) не происходило восстановления исследуемых показателей до физиологической нормы. Заключение. Полученные в ходе эксперимента данные свидетельствуют о корригирующем действии низкоинтенсивного лазерного излучения при адреналовой токсемии у животных. Механизм действия низкоинтенсивного лазерного излучения, вероятно, носит системный характер и связан с воздействием на стресс-реализующие системы организма. Maladaptation results in the third stage of stress, exhaustion, and, thus, may underlie many diseases. Prolonged release of epinephrine into blood exerts catatoxic effects; therefore, searching for effective methods for correction of hyperadrenalinemia is relevant. Presently, low-intensity laser therapy (LILT) is commonly used in medicine. However, the introduction of LILT to clinical practice is largely empirical. The aim of this study was to analyze the effect of LILT on electrokinetic potential of red blood cells (RBC), lipid peroxidation (LPO), and the morphology of adrenal glands in adrenal toxemia in rats. Methods. Adrenal toxemia was modeled by intraperitoneal administration of epinephrine hydrochloride (0.1 mg/kg) to rats. Animals of group 1 received epinephrine alone; animals of group 2 received LILT 30 min prior to epinephrine administration; rats of group 3 received LILT alone; and intact rats of group 4 were used as control. Blood was withdrawn at one hour, 24 h, and one week of the experiment onset. The RBC zeta potential, which characterizes the morpho-functional state of membranes, was determined by measuring the effect of low-intensity laser therapy on RBC electrophoretic mobility using microelectrophoresis. LPO as an indicator of cell membrane injury was determined by concentration of malonic dialdehyde (MDA). The structure of adrenal glands was studied histologically. Results. LILT combined with epinephrine (group 2) alleviated the decrease in RBC electrophoretic mobility and restored the MDA level to values of the intact group at one week. Morphological structure of adrenal glands in group 2 did not differ from that in control whereas epinephrine (group 1) did not restore the study parameters to physiologically normal values. Conclusion. The study showed that LILT exerted a correcting effect in experimental adrenal toxemia. The mechanism of this effect is likely systemic and related with the effect on stress systems of the body.

Characterization of ferucarbotran-loaded RBCs as long circulating magnetic contrast agents.

The biomedical application of contrast agents based on superparamagnetic iron oxide nanoparticles is still limited because of their short intravascular half-life. The potential of red blood cells (RBCs) loaded with new ferucarbotran nanoparticles as magnetic contrast agents with longer blood retention time has been investigated. Ferucarbotran was loaded into RBCs by a procedure of hypotonic dialysis and isotonic resealing. Ferucarbotran amounts encapsulated in RBCs were determined by NMR. The survival of ferucarbotran-loaded RBCs and bulk ferucarbotran was evaluated in the mouse bloodstream. Blood retention time of these RBC constructs is longer (∼14 days) than the bulk ferucarbotran (∼1 h) with a slower Fe clearance from liver and spleen. Ferucarbotran-loaded RBCs could be used as potential contrasting agents for diagnostic applications in MRI/magnetic particle imaging.

Transmembrane Potential of Red Blood Cells Under Low Ionic Strength Conditions

Background/Aims: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached. Methods: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC₄(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method. Results: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min. Conclusions: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media.

Human erythrocytes as nanoparticle carriers for magnetic particle imaging

The potential of red blood cells (RBCs) loaded with iron oxide nanoparticles as a tracer material for magnetic particle imaging (MPI) has been investigated. MPI is an emerging, quantitative medical imaging modality which holds promise in terms of sensitivity in combination with spatial and temporal resolution. Steady-state and dynamic magnetization measurements, supported by semi-empirical modeling, were employed to analyze the MPI signal generation using RBCs as novel biomimetic constructs. Since the superparamagnetic iron oxide (SPIO) bulk material that is used in this study contains nanoparticles with different sizes, it is suggested that during the RBC loading procedure, a preferential entrapment of nanoparticles with hydrodynamic diameter ⩽60 nm occurs by size-selection through the erythrocyte membrane pores. This affects the MPI signal of an erythrocyte-based tracer, compared to bulk. The reduced signal is counterbalanced by a higher in vivo stability of the SPIO-loaded RBCs constructs for MPI applications.

Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study

Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

Effects of sperminated polymers on the pulmonary absorption of insulin

Sperminated pullulans (SP) and gelatin (SG) having different numbers of amino groups were prepared as sperminated polymers for absorption enhancement and the absorption-enhancing effects on the pulmonary absorption of insulin in rats and the permeation of FITC-dextran (MW 4400, FD4) through Calu-3 cell monolayers were evaluated. In both in vivo and in vitro experimental systems, SP and SG acted as enhancers and their enhancing effects correlated with the amino group content. The zeta potential of red blood cells treated with SP and SG was positive and the degree was also correlated with the amino group content. SP and SG interact directly with the luminal surface of mucus membranes via an ion-ion interaction and then may induce signals that open tight junctions resulting in intercellular permeation of water soluble drugs.

Membrane surface charge and morphological and mechanical properties of young and old erythrocytes

Abstract The membrane surface charge and the morphological and mechanical properties of living erythrocytes in normal physiological conditions were studied as a function of cell age. The young and old cells were separated from fresh rabbit blood by using Percoll density gradients. A novel multi-dimensional microscope was employed to perform real-time, non-invasive in situ measurements on the membrane bending elastic modulus and the cell shape and size. A phase-analysis micro-electrophoresis laser scattering technique was used to measure the surface charge density. The results show that the membrane Zeta potential of red blood cells is reduced when they become old, thus leading to an increase of their membrane bending elastic modulus and a marked change of their size and shape.

Antidiabetic effects of an oral administration of Lactobacillus casei in a non-insulin-dependent diabetes mellitus (NIDDM) model using KK-Ay mice.

The antidiabetic effects of Lactobacillus casei (LC) on a non-insulin-dependent diabetes mellitus (NIDDM) model, KK-Ay mice, were investigated. The oral administration of LC to male 4-week-old KK-Ay mice, or raising the mice on a 0.05% LC-containing diet significantly decreased the plasma glucose at 8 to 10 weeks of age compared with the control group. The body weights of the LC-treated groups were lower than those of the control group, although the food intake was nearly the same in all groups. Phenotypic analysis of spleen cell surface markers revealed that the increase in CD4+ T cells at 12 weeks was significantly inhibited by the oral treatment with LC. Cytokine production, especially that of interferon-gamma and interleukin 2, was also inhibited in the oral LC-treated group. The plasma insulin levels of the LC-treated groups were also lower than those of the control group, and the insulin binding potential of red blood cells in the LC-treated mice was augmented more than that in the control group. Taken together, these findings led us to conclude that the oral administration of LC in the NIDDM model mice, KK-Ay, was involved in the decrease in the plasma glucose level and modified the host immune responses.

P-112 - Antidiabetic effects of an oral administration of Lactobacillus casei in a non-insulin-dependent diabetes mellitus (NIDDM) model using KK-Ay mice

The antidiabetic effects of Lactobacillus casei (LC) on a non-insulin-dependent diabetes mellitus (NIDDM) model, KK-Ay mice, were investigated. The oral administration of LC to male 4-week-old KK- Ay mice, or raising the mice on a 0.05% LC-containing diet significantly decreased the plasma glucose at 8 to 10 weeks of age compared with the control group. The body weights of the LC-treated groups were lower than those of the control group, although the food intake was nearly the same in all groups. Phenotypic analysis of spleen cell surface markers revealed that the increase in CD4+ T cells at 12 weeks was significantly inhibited by the oral treatment with LC. Cytokine production, especially that of interferon-γ and interleukin 2, was also inhibited in the oral LC-treated group. The plasma insulin levels of the LC-treated groups were also lower than those of the control group, and the insulin binding potential of red blood cells in the LC-treated mice was augmented more than that in the control group. Taken together, these findings led us to conclude that the oral administration of LC in the NIDDM model mice, KK-Ay, was involved in the decrease in the plasma glucose level and modified the host immune responses.

Decreased amino acid uptake by erythrocytes during postoperative sepsis

We describe a patient with postoperative sepsis associated with reduced ability to transport amino acids into erythrocytes. Administration of amino acid infusion showed no effect on plasma or red blood cell concentrations of the amino acids during the sepsis period. However, when the site of sepsis was removed, patient recovery was associated with marked increases of all amino acids, particularly in the red blood cells. The case illustrates the potential of red blood cells as a marker of amino acid utilization and demonstrates the association of sepsis with effects on cellular uptake.

Potential difference and the distribution of ions across the human red blood cell membrane; a study of the mechanism by which the fluorescent cation, diS-C3-(5) reports membrane potential.

1. The mechanism by which the fluorescent, cationic dye diS-C3-(5) responds to the membrane potential of red blood cells has been investigated. 2. The dye in aqueous solution absorbs most strongly at 650 nm. Addition of white, haemoglobin-free membranes red shifts the absorption maximum ca. 20 nm, while addition of membrane-free cell lysate results in the appearance of a new dye absorption peak at 590 nm. Thus the dye binds both to cell membranes and to cell contents. The component of the cytoplasm which binds the dye is non-dialysable, presumably haemoglobin. 3. Dye added to a suspension of intact cells shows a strong absorption at 590 nm indicating that the dye has bound to the cell contents and that the membrane is permeable to the dye. 4. The amount of dye which partitions into (and on to) the cells can be determined, as reported by Sims, Waggoner, Wang & Hoffman (1974), from the fluorescence of the dye remaining in the supernatant after the cells are centrifuged to the bottom of the suspension. In most conditions the proportion of the cell associated dye which is either free inside the cell or bound to the outside face of the membrane is negligible compared to the proportion bound to the cell contents. 5. On the assumption that the dye is not actively transported, the ratio of the equilibrium dye activities inside and outside the cell, ai/ao, is determined by the membrane potential according to the Nernst relation. Driving the membrane potenial negative then increases the cell associated dye by increasing the activity ratio and hence ai and the amount of dye bound to cell contents. 6. At the known Donnan equilibrium potential the internal dye activity can be calculated from the external activity. An empirical relation between cell associated dye and internal activity has been determined by measuring the dye partition between cells and medium at different external activities. 7. Using this empirial relation, and providing that any changes in cell composition do not affect the dye binding, the internal activity at any potential can be calculated from the measured amount of cell associated dye. The external activity can be estimated fluorimetrically. The membrane potential is then calculated from the activity ratio. 8. The membrane potenial of cells has been altered by adding valinomycin in the presence of different K gradients. Under the conditions used, the 'constant field' permeability for K-Val is 15-20 times that of Cl. 9. Dye binding to haemoglobin is influenced by pH and thus dye partitioning into cells changes with intracellular pH. Increasing intracellular pH increases the amount of dye partitioned, while decreasing pH decreases this amount. 10. When large potentials are produced with valinomycin there is no change in intracellular pH. This result indicates that in red blood cells intracellular pH is determined by the external pH and the Cl concentration ratio and not by the membrane potentials. 11. DiS-C3-(5) can be used to estimate potentials across resealed ghost membranes...

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10 3–10 4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of 131I-labeled albumin and repeated dielectric breakdown.

Agglutination and the electrical surface potential of red blood cells.

THE reduction of the electrical surface potential of erythrocytes has been considered for a long time as a possible cause of cell agglutination.