Irradiated Erythrocytes Research Articles

Clinical features of methods for collecting hematopoietic stem cells in patients with malignant neoplasms weighing up to 15 kg and early age

Relevance. Autologous hematopoietic stem cell transplantation (auto-HSCT) is an integral part of the treatment of patients with high-risk malignancies. However, carrying out the mobilization and collection of hematopoietic stem cells (HSC) for patients weighing up to 15 kg is a complex task that requires a special approach and multidisciplinary interaction.The purpose of the study is to present the experience of collecting HSC in young children weighing up to 15 kg at the Research Institute of Pediatric Oncology and Hematology of the Federal State Budgetary Institution N.N. Blokhin National Medical Research Centre of Oncology, Ministry of Health of Russia.Materials and methods. The study included 30 patients weighing up to 15 kg who received treatment from January 2020 to May 2021 at the Research Institute of Pediatric Oncology and Hematology of the Federal State Budgetary Institution N.N. Blokhin National Medical Research Centre of Oncology, Ministry of Health of Russia. Median age was 30.6 (12–48) months, median body weight was 12.2 (7.8–15) kg. Apheresis in children weighing up to 15 kg was performed in the resuscitation and intensive care unit with the condition of pre-filling the cell separator circuit with a donor irradiated erythrocyte suspension to prevent hypovolemic complications. Thirty two apheresis was performed on a cell separator of the “Spectra Optia” type. To mobilize HSC, preparations of granulocyte colony-stimulating factor with the main active ingredient filgrastim were used.Results. Twenty eight HSC apheresis procedures were successful on the first attempt, 2 patients underwent repeated apheresis (a total of 32 procedures were performed). The median number of CD34+ cells obtained was 13.9 × 106/kg (0.04–92.0 × 106/kg), and the median apheresis duration was 251 (160–415) min. In 2 children during the procedure, an immediate organization of repeated venous access was required.Conclusions. Performing HSC apheresis in children weighing up to 15 kg and young children is a safe and effective technique with the participation of a multidisciplinary team. The apheresis algorithm proposed by us makes it possible to carry out a high-quality collection of CD34+ cells, sufficient for auto-HSCT.

Evolutionary conservation of a regulative pathway of erythropoiesis in Poikilothermic vertebrates

Apoptosis, programmed cell death, plays a central role in haematopoiesis. Mature erythrocytes of non-mammalian vertebrates maintain a permanent nucleus; these cells can undergo apoptosis (eryptosis), as do other somatic cells of a given non-mammalian vertebrate. In this study, we have investigated the expression and subcellular distribution of Bcl-2, Bcl-XL and Bax proteins in the maturation phases and after X-ray irradiation of nucleated erythrocytes of Torpedo marmorata and Caretta caretta and the effect of X-ray irradiation on nucleated circulating erythrocytes of Torpedo marmorata. The cellular distribution of proteins was detected in erythrocytes by using immunocytochemistry at light microscopy and immunoelectron microscopy. The electrophoretic separation and immunoblotting of pro- and anti-apoptotic proteins of immature and mature erythroid cells was performed too, after X-ray irradiation of torpedoes. The results of the immunocytochemical analyses show an increase, in the expression level of Bax in mature as compared to young erythrocytes and a corresponding decrease of Bcl-2 and Bcl-XL. This maturation pattern of Bax, Bcl-2 and Bcl-XL was abrogated in X-ray irradiated torpedo erythrocytes. On the basis of these observations, Bax, Bcl-2 and Bcl-XL seems to play a role in the erythropoiesis of Torpedo marmorata Risso and in Caretta caretta. In conclusion, the same apoptotic proteins of somatic cells appear to be conserved in circulating nucleated erythrocytes thus suggesting to play a role in the maturation of these cells.

Photothermal biological effects of monomeric erythrocyte using optical tweezers

The changes of mechanical properties and biological activities of monomeric erythrocytes are studied using optical tweezers micromanipulation technology. Firstly, the mechanical properties of irradiated erythrocyte membranes are obtained. Weaker power laser irradiation can delay the decay of the mechanical properties of erythrocytes and promote the biological activity of erythrocytes, while higher power laser irradiation damages erythrocytes. The stronger the laser irradiation is, the more obvious and rapid the damage will be. The temperature of the cell surface will be changed by regulating the laser power and irradiation time, so the biological functions of erythrocyte can be controlled. Secondly, the finite element simulation of the temperature change on the cell surface under the condition of laser irradiation is carried out using simulation software, and the precise temperature of the cell surface irradiated cumulatively by a laser with different powers is obtained. Finally, the processes of abscission, unfolding, and denaturation of hemoglobins in erythrocytes at different temperatures due to the photothermal effect are analyzed using the model. The mechanism of laser irradiation on the elasticity of erythrocyte membranes is also obtained.

Fullerenol C60(OH)36 protects human erythrocyte membrane against high-energy electrons

Fullerenols (polyhydroxylated fullerene C60) are nanomaterial with potentially broad applicability in biomedical sciences with high antioxidant ability, thus, we investigated the radioprotecting potential of fullerenol C60(OH)36 on human erythrocytes irradiated by high-energy electrons of 6 MeV. The results demonstrate that C60(OH)36 at concentration of 150 μg/mL protects the erythrocytes against the radiation-induced hemolysis (comparing to non-protected cells, we observed 30% and 39% protection for 0.65 and 1.3 kGy irradiation doses, respectively). The protecting effect was confirmed by 32% decreased release of potassium cations comparing to the cells irradiated without C60(OH)36. Measurements of the amount of lactate dehydrogenase (LDH) released from the irradiated erythrocytes showed that the size of the pores formed by irradiation was not sufficient to release LDH across the erythrocyte membranes. We also report a significant decrease of the affinity of acetylcholinesterase (AChE) for the substrate in the presence of fullerenol, indicating the relatively strong adsorption of C60(OH)36 to components of plasma membrane. Changes in membrane fluidity detected by fluorescence spectroscopy and conformational changes in membrane proteins detected by spin labeling suggest the dose-dependent formation of disulfide groups as an effect of oxidation and this process was inhibited by C60(OH)36. We suppose that scavenging the ROS as well as adsorption of fullerenol to membrane proteins and steric protection of -SH groups against oxidation are responsible for the observed effects.

Сравнение лабораторных показателей патогенредуциро-ванных и рентгеноблученных эритроцитных взвесей

Сравнение лабораторных показателей патогенредуциро-ванных и рентгеноблученных эритроцитных взвесей

The influence of very small doses of alpha radiation on the stability of erythrocytes.

Our aim was to study the influence of low doses (0.2-4 μGy) of α radiation on the stability of human erythrocytes isolated from healthy and diabetic erythrocytes. Absorption spectroscopy was used to measure the level of red blood cell (RBC) hemolysis, along with Mössbauer spectroscopy, which is a highly specific method suited to monitoring various hemoglobin forms. States of hemoglobin are sensitive to a homeostatic imbalance in red blood cells. Changes in the membrane skeleton organization of irradiated erythrocytes isolated from healthy donors were studied using atomic force microscopy (AFM). Hemolysis, in healthy red blood cells, showed characteristic discontinuities, depending on the α particle flux and the exposure time to the low doses applied. This phenomenon was not observed in severe diabetic cases, which could be a result of modified protein-lipid-sugar complexes and the attenuation/absence of some antioxidative enzymatic processes in their RBC membranes. Similar effects were also observed for red blood cells treated with low doses of neutron and γ-radiation. AFM measurements demonstrated a reorganization of the RBC membrane skeleton network depending on the time of RBC exposure to α radiation. This suggests that the changes in the activity of the acute defense processes against free radicals which are activated within the erythrocyte membrane irradiated with α-particles could additionally be up- or down regulated by modifications to the membrane-skeleton network. However, even the highest dose of α radiation applied in these studies did not cause any significant changes in the ability of hemoglobin to transport oxygen. Microsc. Res. Tech. 80:131-143, 2017. © 2016 Wiley Periodicals, Inc.

Effects of Very Low Dose Fast Neutrons on Cell Membrane And Secondary Protein Structure in Rat Erythrocytes.

The effects of ionizing radiation on biological cells have been reported in several literatures. Most of them were mainly concerned with doses greater than 0.01 Gy and were also concerned with gamma rays. On the other hand, the studies on very low dose fast neutrons (VLDFN) are rare. In this study, we have investigated the effects of VLDFN on cell membrane and protein secondary structure of rat erythrocytes. Twelve female Wistar rats were irradiated with neutrons of total dose 0.009 Gy (241Am-Be, 0.2 mGy/h) and twelve others were used as control. Blood samples were taken at the 0, 4th, 8th, and 12th days postirradiation. Fourier transform infrared (FTIR) spectra of rat erythrocytes were recorded. Second derivative and curve fitting were used to analysis FTIR spectra. Hierarchical cluster analysis (HCA) was used to classify group spectra. The second derivative and curve fitting of FTIR spectra revealed that the most significant alterations in the cell membrane and protein secondary structure upon neutron irradiation were detected after 4 days postirradiation. The increase in membrane polarity, phospholipids chain length, packing, and unsaturation were noticed from the corresponding measured FTIR area ratios. This may be due to the membrane lipid peroxidation. The observed band shift in the CH2 stretching bands toward the lower frequencies may be associated with the decrease in membrane fluidity. The curve fitting of the amide I revealed an increase in the percentage area of α-helix opposing a decrease in the β-structure protein secondary structure, which may be attributed to protein denaturation. The results provide detailed insights into the VLDFN effects on erythrocytes. VLDFN can cause an oxidative stress to the irradiated erythrocytes, which appears clearly after 4 days postirradiation.

Could Na,K-ATPase play a role in potassium leakage from irradiated erythrocytes?

BackgroundIn developing countries, the access to red blood cell (RBC) irradiators is restricted. Thus, it is a common practice in blood banks to stock irradiated RBC units until they expire. The aim of this work is to elucidate the involvement of Na,K-ATPase in potassium leakage from prophylactically irradiated RBCs. MethodsWhole blood was collected from healthy donors, and blood concentrates were irradiated with 25Gy of γ-radiation within 24h of collection. At days 3, 5, 7, 9, 11, 14 and 28 post-irradiation, fractions were removed and centrifuged and Na,K-ATPase activity from ghost membranes was determined. ResultsThe inhibition of Na,K-ATPase activity in RBCs reached 12.6% by day 7 of storage and up to 50% by day 14 of storage. The addition of vitamin C prevented the irradiation-induced loss of Na,K-ATPase activity. The irradiation of RBCs provoked an increase in potassium plasma levels and a decrease in sodium plasma levels. The incubation of RBCs with ouabain did not change the sodium or potassium levels in the plasma, and the addition of vitamin C only partially prevented a decrease in sodium levels caused by irradiation. ConclusionBecause the inhibition of Na,K-ATPase by ouabain did not cause potassium accumulation in the plasma, we conclude that the irradiation-induced inhibition of the pump is not a key factor driving this effect.

Oxidative stress-induced posttranslational modifications of human hemoglobin in erythrocytes

Posttranslational modifications (PTMs) have been reported in hemoglobin (Hb) treated with ROS/RNS in cell-free experiments. However, little is known about oxidative PTMs of Hb occurring within the erythrocytes. The aim of this study is to characterize the patterns of Hb PTMs in erythrocytes under oxidative stress. Using mass spectrometry, we investigated specifically methionine/tryptophan oxidation, tyrosine nitration, and the modification via 4-hydroxynonenal (HNE), a product of lipid-peroxidation, on Hb. We demonstrated that the treatment with H2O2/nitrite induced higher levels of Hb oxidation/nitration in purified Hb preparations than in unpurified hemolysates and erythrocytes, indicating that ROS/RNS are primarily removed by antioxidative mechanisms. We further studied Hb from erythrocytes exposed to γ-irradiation. An irradiation of 30–100Gy triggered a remarkable increase of intracellular ROS. However, 30Gy did not induce apparent changes in Hb oxidation/nitration and hemolysis, while Hb oxidation/nitration and hemolysis were significantly enhanced by 100Gy, suggesting that Hb oxidation/nitration are the consequence of overwhelmed antioxidative mechanisms after oxidative attack and reflect the severity of the oxidative damage of erythrocytes. Although irradiation was known to induce lipid-peroxidation, we could not detect HNE-Hb adducts in irradiated erythrocytes. Analyzing PTM patterns suggests Hb nitration as a more suitable indicator of the oxidative damage of erythrocytes.

Micro-Raman spectroscopy study of the effect of Mid-Ultraviolet radiation on erythrocyte membrane

Mid-Ultraviolet (UVB) has a significant influence on human health. In this study, human erythrocytes were exposed to UVB to investigate the effects of UVB radiation on erythrocytes membrane. And Micro-Raman spectroscopy was employed to detect the damage. Principal component analysis (PCA) was used to classify the control erythrocytes and the irradiated erythrocytes. Results showed that the erythrocytes membrane was damaged by Mid-Ultraviolet (UVB) radiation. The intensity of the Raman peaks at 1126cm−1 and 1082cm−1 were used to calculate the Longitudinal Order-Parameters in Chains (Strans) which can present the liquidity and ionic permeability of erythrocyte membrane. After UVB radiation for 30min, both the liquidity and ionic permeability decreased. At the same time, the intensity of the peaks at 1302cm−1 (α-helix), 1254cm−1 (random coil), 1452cm−1 and 1430cm−1 (CH2/CH3 stretch) have also changed which indicated the membrane protein also been damaged by UVB. In the whole process of radiation, the more UVB radiation dose the more damage on the erythrocyte membrane.

Spectroscopic analysis of irradiated erythrocytes

The aim of the present work is to study the effect of gamma radiation on the lipid part of the erythrocyte membrane, and to test the efficiency of lipoic acid as a radioprotector. This effect was evaluated using electron paramagnetic resonance (EPR), and Fourier transform infrared (FT-IR) spectroscopy. The results showed an increase in the number of spin density by 14%, 22% and 65% after exposure to 25, 50 and 100 Gy respectively; whereas there was a decline in the obtained density after incubation with lipoic acid by a factor of approximately 32%. The FT-IR spectra of the irradiated erythrocytes samples showed a marked decrease in the intensity of all characteristic peaks, which increased as the irradiation dose increased. The second-derivative of these spectra, allow the conformationally sensitive membrane acyl chain methylene stretching modes to be separated from the protein (mostly hemoglobin) vibrations that dominate the spectra of intact cells. The 2850 cm −1 band showed changes in the band shape and position after exposure to 50 and 100 Gy. Therefore it can be concluded that the band at 2850 cm −1 only is useful in monitoring the radiation effect of the lipids cell membrane intact cells.

Study of the Influence of Pulse Infrared Radiation on Optical and Hemolysis Resistant Properties of Erythrocytes

Resistance to hemolysis of erythrocytes in lysing isotonic water solution of ammonium chloride becomes apparent in the dynamics of the change of their optical properties. Optical properties of erythrocytes were registered on light scattering from single cells with a scanning flow cytometer. It was shown experimentally during hemolysis the dynamics of the change of the integral intensity of light scattering of erythrocytes is different for irradiated and non-irradiated human blood. Qualitatively, the effect is similar for the both sources of pulse radiation: at the beginning of hemolysis the mean integral intensity of irradiated erythrocytes is increasing more than that is of non-irradiated ones. Quantitatively, at the same mean power density the interrupted irradiation of CO2 laser caused greater effect than continuous one, but less than pulsed terahertz irradiation, i.e. the effect is greater for shorter pulse. On the base of the obtained results one can suggest that the observed effect on pulse infrared radiation of low mean power caused by unsteady heating of erythrocytes.

Poloxamine 1107 sealing of radiopermeabilized erythrocyte membranes

Purpose : Lipid peroxidation-mediated permeabilization of cell membranes following intense ionizing irradiation is well documented. This form of membrane radiopermeabilization leads to rapid exhaustion of cellular high-energy compounds, resulting in the acute onset of cellular necrosis. Strategies to reverse the process of necrosis and preserve cell viability require membrane sealing. This report documents the relative efficacy of Poloxamine 1107, a non-ionic surfactant, compared with other polymers, in sealing radiopermeabilized cell membranes. Materials and methods : Isolated erythrocytes were exposedto 600Gy 60Co irradiation at a dose rate of 1.3Gy/s. Different polymer compounds were added 10min later to the irradiated cell suspensions. At 2h later the haemoglobin content in the supernatants was determined spectrophotometrically. Results : Compared with the non-treated irradiated control, Poloxamine 1107 significantly reduced the leakage of haemoglobin from irradiated erythrocytes. Poloxamer 188 and dextran at equal concentrations had no significant reverse effect on the irradiation-mediated increased membrane permeability. The amount of haemoglobin released from irradiated erythrocytes was inversely related to the Poloxamine 1107 concentration. Conclusions : This study demonstrates the capability of Poloxamine 1107 to seal radiopermeabilized cell membranes. Thus, surfactants such as Poloxamine 1107 might be useful as a therapeutic agent in the treatment of high-dose radiation injuries since cellular necrosis due to metabolic exhaustion following radiopermeabilization of their membranes might be prevented.

Effect of microwaves, high energy electrons and gamma irradiation on the osmotic resistance of human erythrocyte membranes

The effects of 5 MeV electrons and of gamma irradiation at 0°C as well as the effects of 2.45 GHz microwaves at −196°C on the osmotic resistance of human erythrocyte membranes are presented. Both electron and gamma radiation in the range 0–400 Gy induced no haemolysis, indicating that the membrane modifications due to radiation interaction do not reach a critical point to cause swelling of the cells and consequent lysis. The microwave irradiated erythrocytes showed a haemolysis degree between 6–16% in the irradiation time range of 0–5 min. The osmotic stress experiments performed after irradiation showed that the gamma irradiated erythrocytes exhibited an almost similar behaviour at all irradiation doses whereas the electron irradiated samples showed a much larger increase in the haemlysis degree, and in the case of a certain electron dose (100 Gy), the haemolysis was found much smaller than for the control sample. A similar behaviour of the erythrocytes was found in the case of microwave irradiation of the cryoprotected erythrocytes at −196°C: the sample irradiated for 6 min developed a haemolysis degree much smaller than for the control (70% from the nonirradiated sample haemolysis). Our experimental data suggest that electron radiation and gamma radiation have different impacts on the erythrocyte haemolysis in the dose range used. Much more experimental data is necessary in order to clarify the similarity between the results obtained in the case of electron irradiation at 0°C and microwave irradiation at −196°C, in the two different sets of irradiation conditions (liquid samples at 0°C and frozen samples at −196°C).

Membrane solubilization in erythrocytes as a measure of radiation exposure to fast neutrons

Membrane solubilization and osmotic fragility of rat erythrocytes irradiated in vivo with fast neutron fluences ranging from to using a source were measured instantaneously using a light scattering technique. The solubilization of erythrocyte membrane by a non-ionic detergent, octylglucoside (OG), was found to exhibit a two stage transition from vesicular form to mixed micellar form in the range of detergent concentrations 1.5-7.8 mM. The coexistence phase, vesicular/mixed micellar, was shifted towards higher detergent concentrations with increase in the neutron fluence, indicating increasing membrane resistance to the detergent and hence change in the natural membrane permeation properties. The technique shows an adequate sensitivity in detecting membrane damage in erythrocytes and has potential as a biophysical marker of radiation exposure. The osmotic fragility of irradiated erythrocytes shows a decreasing trend with increasing irradiation fluence measured directly and two weeks post-irradiation. Blood films photographed two weeks post-irradiation show developed elliptocytosis and crenated cell anaemia.

Kinetics of electroinduced pores as a probe of membrane modification produced by ionizing radiation

In this work, we have attempted to demonstrate that the study of erythrocyte electroporation can be used to characterize modifications induced at the membrane level by the exposure of living cells to ionizing radiation. Wistar rats were contaminated by chronic ingestion of tritiated water (HTO) in a range of absorbed doses between 0 and 49 cGy. Erythrocyte suspensions were subjected to single electrical pulses and, by modelling the time evolution of light absorption at 610 nm, kinetic parameters describing the electroporated suspension were calculated. A significant increase in the apparent haemolysis rate was observed for the irradiated erythrocytes when subjected to long, high-intensity electrical pulses. In milder electropulsation conditions, when the reversible electroporation process was favoured, the irradiated erythrocytes showed a significant increase in both the radius pore resealing rate and the maximum area of the electroporated surface (for a dose of 49 cGy) compared with the control. The results indicate that the modifications of the parameters due to the effects of irradiation on the erythrocytes reflect an increase in the membrane fluidity without disturbing the association between the cytoskeleton and membrane proteins.

Radioprotection of sheep erythrocytes by polyvinylpyrrolidone

The protective effect of polyvinylpyrrolidone of mol. wt 10,000 and 40,000, on sheep erythrocytes irradiated up to 13 kGy, and stored for one month at 4°C was studied. Addition of these compounds at a level of 10–15% reduced hemolysis from 90% in irradiated erythrocyte solution to approximately 10%. The high efficiency of this protective agent is discussed.

Effect of thiol reactive reagents and ionizing radiation on the permeability of erythrocyte membrane for non-electrolyte spin labels.

The paper presents some results on the effect of PCMB and NEM on the transport of non-electrolyte spin labels: TEMPO and TEMPOL across non-irradiated and irradiated porcine erythrocyte. Irradiated erythrocytes exhibited increased inhibitory effect of thiol reactive compounds in the TEMPO and TEMPOL transport compared to non-irradiated erythrocytes.

Modification of allograft survival in rats by blood transfusion to the donor.

In the BN/Ro-to-WAG/Ro rat donor-host combination, third-party blood transfusions given to the donor shortly before transplantation can markedly influence heart allograft survival if the immune response of the recipient has been modulated. The WAG recipients were either transfused with BN donor blood, which leads to a permanent graft survival, or postoperatively treated with a single i.m. injection of 15 mg/kg cyclosporine, which leads to a moderate prolongation of graft survival. In the transfused recipients, blood transfusions to the donor had a detrimental influence on graft survival. In the cyclosporine-treated recipients, blood transfusions to the donor had a beneficial effect. There was no significant difference in graft survival after a single transfusion or multiple transfusions to the donor. Recipient-type blood transfusion to the donor did not influence graft survival, nor did irradiated third-party blood or third-party erythrocytes. However, third-party leukocyte suspensions had a significant influence on heart allograft survival. It is concluded that third-party viable leukocytes are responsible for the induction of the donor transfusion phenomenon. It is also postulated that the third-party leukocytes interact with the dendritic cell population of the graft, leading to a reduction in its immunogenicity.

Therapy in adenosine deaminase and purine nucleoside phosphorylase deficient patients

The discovery of the causal association of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency with some forms of primary immunodeficiency disease had led to new approaches to therapy, such as enzyme replacement. In ADA deficiency, bone marrow transplantation remains the primary method of choice. If no suitable bone marrow donor is available, enzyme replacement with irradiated erythrocyte transfusions should be considered. The latter therapy may be sustained by treatment with thymic factors. In ADA deficiency, bone marrow transplantation and, in about 50% of the cases, also enzyme replacement, may result in clinical and neurological improvement with concurrent (partial) restoration of immune function and (partial) disappearance of the metabolic abnormalities present before treatment. In PNP deficiency, enzyme replacement has been evaluated carefully in only two patients. The results disclose profound changes in the purine excretion patterns after each transfusion, and a slow but partial restoration of in vitro T cell function. Treatment of ADA and PNP deficiency with continued enzyme replacement by erythrocyte transfusions has certain risks which hopefully can be overcome in the near future by loading the patient's own blood cells with the missing enzyme.