Young Erythrocytes Research Articles

Differential Responses of Young and Old Erythrocytes Stored with Vitamin C and Vitamin E in Additive Solution-7.

Oxidative stress (OS) causes biochemical and morphological alterations in erythrocytes. The primary factors contributing to OS are aging and storage. Antioxidants significantly alleviate OS. Therefore, this study aimed to investigate the response of young and old erythrocytes to vitamin C and vitamin E during storage. Erythrocytes were separated into young and old by the Percoll method. Each erythrocyte subpopulation was categorized into the i) Control (additive solution-7 [AS-7]) and ii) vitamin C and vitamin E in AS-7 (VC+VE) groups and stored for 21 days at 4°C. OS, antioxidant, and aging markers were analyzed on days 1, 14, and 21. The activity of antioxidant enzymes was similar throughout storage in young cells. However, superoxide dismutase activity elevated in old cells (Control and VC+VE) on days 1 and 21. Catalase (CAT) activity increased on days 14 and 21, whereas glutathione peroxidase (GPX) increased on days 1 and 14 in old Controls. However, in old VC+VE, CAT increased on day 21 and GPX increased on day 1. Advanced oxidation protein products, superoxides, glutathione, and uric acid increased in old cells throughout storage. Malondialdehyde decreased in old VC+VE compared with old Control on days 14 and 21. Sialic acids and glutamate oxaloacetate transaminase activity were higher in young cells compared to old cells. Young cells exhibited lower oxidative changes throughout storage. Vitamin C and vitamin E were effective in maintaining the redox balance in old cells. These findings emphasize the need for specific approaches for different subpopulations during erythrocyte banking.

Diagnostic Utility of Reticulocyte Hemoglobin Equivalent Parameter in the Evaluation of Microcytic Hypochromic Anemia – Our Experience from Northeast India

Background: Reticulocytes are red blood cell precursors with an average lifespan of 1–2 days. Reticulocyte hemoglobin equivalent (Ret-He) is an early marker of iron deficiency (ID) erythropoiesis and reflects real-time information regarding the synthesis of young erythrocytes in the bone marrow. The objective of this study is to determine the diagnostic utility of Ret-He in patients having microcytic hypochromic anemia in comparison with serum ferritin and iron studies. Objectives: The objective of this study is to determine the diagnostic utility of the Ret-He parameter in patients having microcytic hypochromic anemia in comparison with the serum ferritin, iron studies and its role in routine hematological screening for nutritional deficiency anemia like ID. Design and Settings: The design involves observational study. Materials and Methods: A hospital-based observational study was carried out in a referral hospital. Hematological parameters were processed using Sysmex XN1000 (Sysmex Corporation, Kobe, Japan) analyzer and were compared with Serum iron studies using biochemistry analyzer (VITROS® 250 Chemistry System) from 201 participants who presented with microcytic hypochromic anemia. Main Outcome and Measures: Relationship of Ret-He parameter and its diagnostic utility for screening of iron-deficient states. Sample Size: The sample size was 201. Results: When serum ferritin is taken as “the gold standard” to detect ID, with Ret-He cutoff of 27.15 pg/cell, we found a statistically significant positive correlation between Ret-He and serum ferritin (P < 0.001). We found a sensitivity of 57.37% and specificity of 75.95% with area under the curve of 0.681, positive predictive value of 100%, negative predictive value of 3.8%, and accuracy of 62.19% for Ret-He in detecting ID anemia (IDA). Also found a statistically significant negative correlation between Ret-He and total iron binding capacity (P < 0.001). There was no statistical correlation between Ret-He and serum iron levels in our study. Conclusions: The present study suggests Ret-He is one of the better and more reliable hematological parameters indicating ID and, when used along with biochemical parameters like serum ferritin, can give valuable inputs in a better screening and diagnosis of IDA; hence proper treatment is possible. Limitations: The multicentric study is required to standardize Ret-He reference values, and follow-up to therapy of the subjects was not done. Additional hemoglobin variant analysis data would have been favorable to the study.

Функциональные морфоденситометрические параметры хроматина ядра и цитоплазмы эритробластов и эритроцитов птиц в постэмбриональном онтогенезе

Abstract. The purpose is to characterize the development of the synthetic activity of polychromatophilic erythroblasts and erythrocytes, in particular the status of the hemoglobin-synthesized function of avian erythroid cells in early postembryonic ontogenesis. Methods. The experimental part of the study was carried out in the conditions poultry farm of “Chebarkul’skaya ptitsa” (Chebarkul district of the Chelyabinsk region, Russia). The whole blood of Hubbard ISA F15 broiler chickens of four age groups (n = 40) was studied: group I – 1-day-old chicks; II – 7-day-old chickens; III – 23-day-old broilers; IV – 42-day-old chickens. The scientific novelty. Functional morpho-densitometric parameters of chromatin of the nucleus and cytoplasm of erythroblasts and red blood cells of birds in postembryonic ontogenesis. Cytophysiological and epigenetic parameters of hemoglobin synthesis by erythroblasts and young erythrocytes are important in elucidating the regulation of the functions of blood cells in normal, adaptation and disorders. Criteria for the synthetic activity of erythroblasts and maturing erythrocytes of animals and humans are optical density as an indicator of the concentration of euchromatin and the metabolic dynamics of the cytoplasm, as well as area as an indicator of the distribution of euchromatin in the stroma of the nucleus and the size’s of the cytoplasm. Results. According to the results of calculation of spectral, morphometric and opticometric values of nuclear chromatin, cytoplasm and their indexed ratios for polychromatophilic erythroblasts and erythrocytes, hemoglobin synthesis in early postembryonic ontogenesis is characterized on a model organism of broiler chickens. According to the results of determining the geometric (n = 30) and optical (n = 300) parameters of euchromatin, heterochromatin of the nucleus and cytoplasm, their ratios and indices were calculated for polychromatophilic erythroblasts, polychromatophilic erythrocytes and mature erythrocytes in peripheral blood smears (n = 158) stained by Pappenheim. Cytochemical reactions reflecting the physiological and biochemical interrelations of erythroblasts and erythrocytes are the basis of a complex morpho-densitometric test of the activity level of hemoglobin synthesis by erythroid cells in early postnatal ontogenesis.

Erythrocyte PIG-A mutant frequencies in cancer patients receiving cisplatin.

Cisplatin is a primary chemotherapy choice for various solid tumors. DNA damage caused by cisplatin results in apoptosis of tumor cells. Cisplatin-induced DNA damage, however, may also result in mutations in normal cells and the initiation of secondary malignancies. In the current study, we have used the erythrocyte PIG-A assay to evaluate mutagenesis in non-tumor hematopoietic tissue of cancer patients receiving cisplatin chemotherapy. Twenty-one head and neck cancer patients undergoing treatment with cisplatin were monitored for the presence of PIG-A mutant total erythrocytes and the young erythrocytes, reticulocytes (RETs), in peripheral blood for up to five and a half months from the initiation of the anti-neoplastic chemotherapy. PIG-A mutant frequency (MF) in RETs increased at least two-fold in 15 patients at some point of the monitoring, while the frequency of total mutant RBCs increased at least two-fold in 6 patients. A general trend for an increase in the frequency of mutant RETs and total mutant RBCs was observed in 19 and 18 patients, respectively. Only in one patient did both RET and total RBC PIG-A MFs did not increase at any time-point over the monitoring period. Cisplatin chemotherapy induces moderate increases in the frequency of PIG-A mutant erythrocytes in head and neck cancer patients. Mutagenicity measured with the flow cytometric PIG-A assay may serve as a tool for predicting adverse outcomes of genotoxic antineoplastic therapy.

ВЕТСАНЭКСПЕРТИЗА И МОРФОЛОГИЯ КРОВИ МОЛОДИ ВОБЛЫ ПРИ ПАРАЗИТАРНОЙ ИНВАЗИИ

A study of the morphological parameters of the peripheral blood of juvenile volva during parasitic invasions by trematodes A. muehlingi and P. cuticula was carried out to study the degree of negative impact of these parasites on early juvenile vobla. The analysis confirmed that parasitic invasion influenced changes in hematological parameters. A natural increase in the number of neutrophils and eozonophils in infested fish was shown. Reliably high rates of young erythrocytes were revealed, twice as high as in fish free from parasites. The identified deviations in the composition of the white blood of juvenile roach were due to the protective mechanism and immunological reactions of the body to infection by parasites. The presence of degenerative forms of erythrocytes in blood smears of both infected and uninfected early juvenile vobla may indicate that these changes are caused not only by the parasitic load, but also by negative changes in abiotic environmental factors.

Red Blood Cell Distribution Width, Erythrocyte Indices, and Elongation Index at Baseline in a Group of Trained Subjects.

Regular exercise elicits adaptive changes in several organs and physiological processes, including erythrocyte properties. In a group of 79 subjects (62 men and 17 women; mean age 31.37 ± 10.19 years) who trained several times a week as they practiced amateur sports, we evaluated the elongation index, markers of erythrocyte deformability, red blood cell distribution width (RDW), indicators of erythrocyte anisocytosis, hematocrit, hemoglobin, and the main erythrocyte indices (MCV, MCH, MCHC) in basal conditions. In comparison with a group of healthy, but not training, volunteers, the values of the elongation index, and not the RDW, are increased, and this datum is accompanied by an increase in MCV and MCHC, likely related to an increased presence of circulating young erythrocytes in training subjects. We also divided the same group according to the median of the VO2max, observing that the subgroup above the median shows both an increase in the elongation index values and a decrease in MCH and MCHC. In trained subjects, there is no correlation between the values of the elongation index and the RDW, while the interrelations among the elongation index, RDW, and main erythrocyte indices appear to be of particular interest and of a certain complexity.

Selective elimination of younger erythrocytes in blood circulation and associated molecular changes in benzo (a) pyrene induced mouse model of lung cancer.

Anemia is a common feature in cancer patients. The present research was conducted to explore the mechanisms of induction of anemia in a mouse model of lung cancer. The lung cancer was induced by treating orally with BaP (50 mg/kg body weight, twice a week for four weeks). The erythrocyte kinetics were studied using a double in vivo biotinylation (DIB) technique. ROS production and apoptosis analysis were done by staining with the CMH2DCFDA stain and anti-mouse Annexin V antibody, followed by flow cytometry. The expression of antioxidant, apoptotic, anti-apoptotic and inflammatory genes was analyzed by quantitative PCR (RT-qPCR). BaP-induced tumour reduced body weight and induced persistent haemolytic anaemia. The kinetics data suggest that, though reticulocyte production was enhanced, the proportion of young erythrocytes did not increase in the same proportion. The young aged erythrocytes were selectively eliminated from blood circulation, but intermediate and old aged erythrocytes persisted for a longer duration. The tumour progression leads to a significant increase in ROS production and apoptosis in the erythrocytes. The molecular data suggests that the expression levels of antioxidants (SOD1, catalase, and GPX1) and erythropoietin (Epo) were significantly increased. The anti-inflammatory genes Interleukin-6 (IL-6), Interleukin-10 (IL-10) were significantly decreased.Apoptotic genes Bax, and caspase 3 were significantly decreased while Bcl 2 was significantly increased in the blood of tumour-bearing mice. The overall data suggest that erythrocyte turnover is severely modulated with the progression of tumor. The apoptosis, ROS levels, antioxidant, anti-apoptotic, and Epo gene expressions were increased, but proapoptotic and anti-inflammatory gene expression were suppressed.

#3627 PHOSPHATIDYLSERINE EXPOSURE AND SHORTENED LIFE SPAN OF ERYTHROCYTES IN RENAL ANEMIA

Abstract Background and Aims The lifespan of erythrocytes in patients with renal anemia is shortened (∼70 days) compared to that of healthy erythrocytes (120 days), but the pathological mechanisms and causes are unclear. Senescent erythrocytes express phosphatidylserine (PS) on their outer membrane, which is a signal for being removed by macrophages. The PS localization in the cellular membrane is mainly regulated by two transporters: flippases and scramblases. Flippases, type Ⅳ P-type ATPases (P4-ATPases), maintain PS localization in the inner leaflet by transporting PS from the outer leaflet of the cell membrane. When scramblases are activated, they facilitate equal distribution of phospholipids between the inner and outer lipid bilayers, resulting in the exposure of PS on the outer leaflet. Recently, we have revealed that reduced flippase activity contributes to PS exposure in healthy senescent erythrocytes [1]. In this study, we examined the percentage of PS-exposed cells and the transporter activity in erythrocytes from renal anemia to elucidate the mechanism of the shortened lifespan. Method This study was approved by the Ethics Committee of Tokyo Women's Medical University (#3835, #4822R2). Erythrocytes from the renal anemic patients undergoing maintenance hemodialysis were separated with the centrifugation using Percoll-density gradient according to our previous report. Resultant heavy and light fractions were used as senescent and young erythrocytes, respectively. We analyzed the percentage of PS-exposed cells (n = 12), flippase/scramblase activities (n = 7), and the factors involved in those enzymatic activities such as intracellular K+ (n = 10) and ATP concentration (n = 9). We also measured protein 4.1a/b and HbA1c as an index of erythrocytes’ age. Results The cells from renal anemia were separated to heavy (senescent) and light (young) fractions, which was similar to the distributions previously performed on healthy erythrocytes. The PS-exposed erythrocytes in the heavy fraction were slightly more than those of the light fraction (p < 0.01, Wilcoxon test), and the flippase activity of the heavy fraction decreased more than that of the light fraction (p < 0.01, paired t-test). Intracellular K+ concentration decreased in the heavy fraction more than in the light fraction similar to the healthy senescent erythrocytes, but there was no statistical difference in the intracellular ATP concentration between the heavy and light fractions. The measurement of protein 4.1a/b and HbA1c in these fractions is still in progress. Conclusion Similar to healthy erythrocytes, reduced flippase activity contributes to PS exposure in senescent erythrocytes of renal anemia, maybe due to the decreased intracellular K+ concentration. This may occur in an earlier stage of senescence than the healthy ones, which may explain the mechanism of the shortened life span.

HEMOPHAGOCYTOSIS IN THE RED BONE MARROW OF PATIENTS WITH COVID-19 WITH SARS-COV-2 INFECTION

Red bone marrow samples investigation in deceased COVID-19 patients enabled to identify the phenomena of secondary hemophagocytosis. Analysis of the data showed that phagocytic reactions during infection of patients with SARS-CoV-2 are manifested both in relation to erythrocytes and leukocytes. The data obtained make it possible to expand the strategy of therapeutic measures, taking into account the new data on the mechanisms of the pathogenesis of COVID-19 in severe viral infection based on morphological findings and additional information on the involvement of young erythrocytes and lymphocytes in the structure of the red bone marrow in the cascade of pathological reactions. The results obtained confirm a wide range of aggressive damaging effects of SARS-CoV-2 in the development of multiple organ failure against the background of COVID-19 and the involvement of the red bone marrow in the pathological process. The authors supplemented information about the mechanisms of hypoxia in COVID-19, which is not only a consequence of damage to the respiratory epithelium, but also the result of damage to erythrocyte differons both at the level of red bone marrow and in peripheral blood. This fact must be taken into account in the development of a treatment strategy and in the creation of new drugs for the treatment of infected patients with various strains of SARS-CoV-2.

Осмотическая резистентность эритроцитов у девушек с гиподинамией в зависимости от полиморфизма I/D гена АСЕ

The purpose of this paper was to study erythrocyte osmotic resistance (EOR) and its relationship with the quantitative and morphofunctional parameters of red blood cells in young women with limited physical activity depending on the I/D polymorphism of the ACE gene. Materials and methods. The study involved 200 healthy women aged 18–22 years leading a sedentary lifestyle. Their blood samples were taken for genotyping and to determine the haemoglobin (HGB) level and red blood cell count (RBC), their mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) as well as haematocrit (HCT). EOR was studied by measuring the optical density of haemoglobin solutions after erythrocyte destruction in a series of hypotonic NaCl solutions with the concentration decreasing from 0.8 % to 0.1 %. Based on the results of osmotic haemolysis, erythrograms were produced and the NaCl concentrations were determined, at which 10, 50 and 90 % of the red blood cells were destroyed. Results. Carriers of the DD genotype were characterized by the lowest resistance to haemolysis in all three erythrocyte populations. According to the analysis of factor structures, in girls with the DD genotype only relationships between the total and individual parameters of erythrocytes were established. In carriers of the I allele (ID and II genotypes) the dominant factor combined the total red blood cell parameters (RBC, HGB, HCT) and the EOR of old, mature, and young erythrocytes. Thus, carriers of the I allele (ID and II genotypes) of the ACE gene are characterized by a higher resistance of mature and old cell populations than those of the DD genotype, while EOR variations in the former are interconnected with changes in the quantitative parameters of red blood cells. At the same time, an increase in EOR is combined with an increase in erythrocyte functional activity. DD genotype carriers showed no correlations between EOR and the quantitative and morphofunctional parameters of red blood cells.

Variation in Glucose-6-Phosphate Dehydrogenase activity following acute malaria.

Primaquine and tafenoquine are the only licensed drugs with activity against Plasmodium vivax hypnozoites but cause haemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Malaria also causes haemolysis, leading to the replacement of older erythrocytes with low G6PD activity by reticulocytes and young erythrocytes with higher activity. Aim of this study was to assess the impact of acute malaria on G6PD activity. Selected patients with uncomplicated malaria were recruited in Bangladesh (n = 87), Indonesia (n = 75), and Ethiopia (n = 173); G6PD activity was measured at the initial presentation with malaria and a median of 176 days later (range 140 to 998) in the absence of malaria. Among selected participants (deficient participants preferentially enrolled in Bangladesh but not at other sites) G6PD activity fell between malaria and follow up by 79.1% (95%CI: 40.4 to 117.8) in 6 participants classified as deficient (<30% activity), 43.7% (95%CI: 34.2 to 53.1) in 39 individuals with intermediate activity (30% to <70%), and by 4.5% (95%CI: 1.4 to 7.6) in 290 G6PD normal (≥70%) participants. In Bangladesh and Indonesia G6PD activity was significantly higher during acute malaria than when the same individuals were retested during follow up (40.9% (95%CI: 33.4-48.1) and 7.4% (95%CI: 0.2 to 14.6) respectively), whereas in Ethiopia G6PD activity was 3.6% (95%CI: -1.0 to -6.1) lower during acute malaria. The change in G6PD activity was apparent in patients presenting with either P. vivax or P. falciparum infection. Overall, 66.7% (4/6) severely deficient participants and 87.2% (34/39) with intermediate deficiency had normal activities when presenting with malaria. These findings suggest that G6PD activity rises significantly and at clinically relevant levels during acute malaria. Prospective case-control studies are warranted to confirm the degree to which the predicted population attributable risks of drug induced haemolysis is lower than would be predicted from cross sectional surveys.

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.

Micronuclei and Nuclear Buds Induced by Cyclophosphamide in Crocodylus moreletii as Useful Biomarkers in Aquatic Environments.

Simple SummaryCrocodiles are territorial reptiles that are exposed to both aquatic and terrestrial environments. Analyzing their state of health and the environment in which they live is essential to detect changes that could affect them by exposure to agents that damage their genetic material, putting their health or other species that share said habitat, including humans, at risk. There are several ways to determine exposure to harmful agents, a very simple and direct one is to analyze the nuclei of blood cells under a microscope, a test that can also be carried out directly in their habitat and that gives rapid results on the effects of agents on those at that moment they are exposed. This study demonstrates the possibility of analyzing the blood of Crocodylus moreletii to quickly analyze its exposure to toxic agents in a sample of its blood by evaluating two abnormal structures in its cells under the microscope, demonstrating that evidence of damage can be observed only by analyzing a drop of their blood.Micronuclei (MN) are used to assess genotoxic exposure, whereas nuclear buds (NBs) have been linked to genotoxic events. Crocodylus moreletii was studied to identify MN and NBs. Three groups were formed: Group 1 (water) and groups 2 and 3 (7 or 10 mg/kg of cyclophosphamide). A drop of blood was obtained daily from the claw tip at 0 to 120 h. Spontaneous micronucleated erythrocytes (MNEs) and erythrocytes with nuclear buds (NBEs) were counted. The frequencies of micronucleated young erythrocytes (MNYEs) and NB young erythrocytes (NBYEs) were evaluated, including the ratio of young erythrocytes (YE)/1000 total erythrocytes. No significant differences were observed in the YE proportion on sampling days; group 1 did not show differences for any parameter, whereas group 2 showed significant differences in MNEs and NBEs, and group 3 showed differences in NBEs and NBYEs. Some mitotic activity in circulation was observed in YEs. In conclusion, NBEs could be a more sensitive biomarker to genotoxic damage than MNEs. The identification of these biomarkers leads us to propose Crocodylus moreletii as a possible environment bioindicator because these parameters could be useful to analyze the in vivo health status of these reptiles and for biomonitoring genotoxic pollutants in their habitats.

Aging-Related Changes in Erythropoietic Activity and Iron Metabolism in a Mouse Model of Congenital Erythrocytosis with Human Gain-of-Function Erythropoietin Receptor

We previously created and characterized a mouse model of congenital erythrocytosis with low erythropoietin (EPO) levels from a gain-of-function mutation of the human erythropoietin receptor gene (mtHEPOR) (Divoky et al. PNAS. 2001; 98:986; Divoky et al. JMM Berl. 2016; 94:597). These mice develop fetal erythrocytosis, followed by transient amelioration of erythrocytosis in perinatal life, and reappearance at 3-6 weeks of age. Similarly, erythrocytosis is observed in heterozygous mtHEPOR patients postnatally but not at birth. We previously reported dynamic changes of the erythron with iron homeostasis during ontogenesis in these mice (Kralova et al. Blood 2017; 130: 170). We observed that while perinatal mtHEPOR mice exhibit relative iron deficiency, aged mice had iron overload. Here, we evaluated developmentally-determined factors associated with hyperactivation of EPOR signaling which could cause a transition from iron deficiency (neonates) to hyperferremia and increased iron deposition (aged mice). To assess the consequences of different levels of EPOR-JAK2-STAT5 signaling, we studied hetero- and homozygous mtHEPOR mice that differ in their severity of erythrocytosis.We found that prenatally and perinatally, mtHEPOR hetero- and homozygous mice have increased erythroferrone (Erfe) transcripts and reduced hepcidin, consistent with the known inverse correlation between Erfe and hepcidin and in accordance with increased numbers of immature erythroid progenitors in the fetal hepatic circulation. At birth, previously normal Epo expression decreased and remained low in adulthood. Iron deficiency, observed in mtHEPOR hetero- and homozygotes at postnatal day 7, was likely related to increased iron consumption by augmented erythropoiesis at this stage.Postnatally, hepcidin levels increased in mutant mice, accompanied by low Erfe induction and iron accumulation in the liver and spleen as reflected by the upregulation of hepatic Bmp6 expression in mature adult (aged ~6.5 months) and old (~16 months) mtHEPOR homozygotes. We hypothesized that this could be a consequence of diminished iron consumption due to a progressive decline of erythropoiesis in mtHEPOR mice, possibly mediated by premature aging of erythroid progenitors with cell-autonomously increased proliferative history and/or increased inflammation.Indeed, young mutant erythrocytes had decreased erythrocyte survival and expression of a senescent marker CD47, an inhibitor of erythrocytes' phagocytosis. Additionally, a progressive decline in the percentage of Ter119-positive bone marrow cells and immature erythroblasts was observed in mtHEPOR hetero- and homozygotes with aging. Clonogenic assays of old mice revealed suppression of early (BFU-E) and late (CFU-E) erythroid progenitors and myeloid bias of hematopoiesis, paralleled by the up-regulation of PU.1 expression, elevation of platelet counts, and an increase in megakaryocytes chiefly in the bone marrow of mtHEPOR homozygotes. Serum levels of inflammatory cytokines did not indicate systemic inflammation; however, induced transcripts of IL-6, Inf-γ, Tgf-β, and Tnf-α, mainly in mtHEPOR homozygotes showed local bone marrow inflammatory stress. These data indicate progressive attenuation of erythroid drive in mtHEPOR homozygotes, and less so in mtHEPOR heterozygotes, paralleled by a decline in hematocrit levels with aging. In response to attenuated erythropoietic activity, iron consumption was reduced in mtHEPOR mice, leading to iron accumulation in the liver and spleen accompanied by markedly increased hepcidin synthesis.Our data suggest that even in the absence of systemic inflammation, albeit with possible paracrine inflammatory signals, known to affect bone marrow remodeling and hematopoietic aging, life-lasting prolonged activation of EPOR-JAK2-STAT5 signaling promoted exhaustion of erythroid progenitors and resulted in an age-related decline of accelerated erythropoiesis in this mouse model of congenital erythrocytosis with human gain-of-function EPOR.Grant support: Czech grant agencies projects GA17-05988S, NV19-07-00412 and LTAUSA17142, Palacky University project IGA_LF_2021_004. DisclosuresNo relevant conflicts of interest to declare.

Erythrocytes, a New Contributor to Age-Associated Loss of Blood-Brain Barrier Integrity.

Blood exchanges between young and old partners demonstrate old blood has a detrimental effect on brain health of young animals. Previous studies primarily investigate soluble blood factors, such as transforming growth factor‐beta, on the brain and the blood–brain barrier (BBB). However, the role of blood cellular components, particularly erythrocytes, has not been defined. Erythrocyte morphology and rigidity change as mammals age, altering their transport within the capillary bed. This impacts downstream biological events, such as the release of reactive oxygen species and hemoglobin, potentially compromising the BBB. Here, a micro electrical BBB (µE‐BBB), with cocultured endothelial and astrocytic cells, and a built‐in trans‐endothelial electrical resistance (TEER) system is described to monitor the effect of capillary shear stress on erythrocytes derived from young and old mice and people and the subsequent effects of these cells on BBB integrity. This is monitored by the passage of fluorescein isothiocyanate‐dextran and real‐time profiling of TEER across the BBB after old and young erythrocyte exposure. Compared to young erythrocytes, old erythrocytes induce an increased permeability by 42% and diminished TEER by 2.9% of the µE‐BBB. These results suggest that changes in circulating erythrocytes are a biomarker of aging in the context of BBB integrity.

Rodent Malaria Erythrocyte Preference Assessment by an Ex Vivo Tropism Assay.

Circulating red blood cells consist of young erythrocytes (early and late reticulocytes) and mature erythrocytes (normocytes). The human malaria parasites, Plasmodium falciparum and P. vivax, have a preference to invade reticulocytes during blood-stage infection. Rodent malaria parasites that also prefer reticulocytes could be useful tools to study human malaria reticulocyte invasion. However, previous tropism studies of rodent malaria are inconsistent from one another, making it difficult to compare cell preference of different parasite species and strains. In vivo measurements of cell tropism are also subjected to many confounding factors. Here we developed an ex vivo tropism assay for rodent malaria with highly purified fractions of murine reticulocytes and normocytes. We measured invasion into the different erythrocyte populations using flow cytometry and evaluated the tropism index of the parasite strains. We found that P. berghei ANKA displayed the strongest reticulocyte preference, followed by P. yoelii 17X1.1, whereas P. chabaudi AS and P. vinckei S67 showed mixed tropism. These preferences are intrinsic and were maintained at different reticulocyte and normocyte availabilities. Our study shed light on the true erythrocyte preference of the parasites and paves the way for future investigations on the receptor-ligand interactions mediating erythrocyte tropism.

Hematological parameters and protein metabolism in the blood of pregnant rats under the effect of vanadium citrate

Dose-dependent changes in protein metabolism in the blood and hematological parameters of pregnant rats under the effect of vanadium citrate are presented in the article. The animals were divided into five groups: group I – non-pregnant females, II – pregnant females consuming pure water without additives, III, IV, V – females which during the mating and pregnancy period received the solution of vanadium citrate at concentrations of 0.03, 0.125 and 0.50 μg V/mL water. The research findings show that in pregnant animals of group II, the level of urea and alkaline phosphatase activity increased, meanwhile aspartate aminotransferase activity decreased, as compared to the non-pregnant females of group І. The levels of total protein and albumin decreased; however, the content of β-globulins increased in the pregnant animals of group II, as compared with that in group I. Also, in the rats of group II, there was a decrease in hemolysis time, total content of erythrocytes and hemoglobin, the content of old and mature erythrocytes, while the content of young erythrocytes increased, as compared to group I. The platelet content and thrombocrit in rats of group II increased in comparison with group I. The content of leukocytes and lymphocytes in pregnant animals of group II decreased, while the content of granulocytes increased, in contrast to non-pregnant rats. Under the effect of vanadium citrate at concentrations of 0.03–0.50 μg V/mL, there was a significant increase in the maximum number of prohemolized erythrocytes, the time of maximum hemolysis was delayed by 0.4–0.6 min, as compared with the pregnant rats of group II. This did not affect the time of total hemolysis in rats of groups III and V, as compared with the pregnant animals in group II. Under the effect of vanadium citrate, an increase in the content of young erythrocytes was observed, as compared with group II. The hemoglobin content decreased at the concentration of 0.125 μg V/mL, while at the concentration of 0.50 μg V/mL it increased, as compared to the pregnant animals of group II. Also, under the effect of vanadium citrate there was a decrease in the mean hemoglobin concentration in the erythrocyte. In pregnant animals fed with vanadium citrate solutions, the platelet content and thrombocrit, the relative width of platelet distribution by volume decreased, as compared with the pregnant rats of group II. The content of leukocytes, lymphocytes and granulocytes under the effect of vanadium citrate increased, as compared with the pregnant animals in group II. Under the effect of vanadium citrate at the concentration of 0.03 μg V/mL, the level of albumin, creatinine and aspartate aminotransferase activity increased in blood plasma in comparison with group II. Meanwhile, at the concentration of 0.125 μg V/mL, the relative content of γ-globulins and aspartate aminotransferase activity increased, alkaline phosphatase activity and urea level decreased in comparison with group II. However at the concentration of 0.50 μg V/mL, the relative α- and γ-globulins content and aspartate aminotransferase activity increased, at the same time, the relative β-globulins content and urea level decreased in comparison with group II. Therefore, vanadium citrate normalizes the indicators of protein metabolism during pregnancy, thus it can be considered as a potential dietary drug for the pregnant.

Influence of AS-7 on the storage lesion in young and old circulating erythrocytes

Blood and its components are stored to meet the demands of blood transfusion. Erythrocytes undergo progressive modifications during storage known as storage lesions. Storage solutions were developed to improve shelf life and extend red cell viability. Therefore, the objective of this study is to analyze the effects of AS-7 on young and old erythrocytes during storage. Blood was collected from the blood bank at Kempegowda Institute of Medical Sciences (KIMS) hospital, Bengaluru. Erythrocytes were isolated from whole blood and separated based on its age using Percoll density gradient. The young and old erythrocytes were stored in AS-7 for 35 days and every 5th day, oxidative stress markers – Hemoglobin (Hb), Oxidative Hemolysis, Mechanical Fragility, Sialic Acid, Superoxides, Glucose, Lactate Dehydrogenase (LDH), Glutathione, antioxidant capacity (TACCUPRAC), Plasma Membrane Redox System (PMRS), antioxidant enzymes, lipid peroxidation, and protein oxidation products were assessed. Hb, glucose, TACCUPRAC, and superoxide dismutase reduced, while oxidative hemolysis, mechanical fragility, protein oxidation, and lipid peroxidation products increased in young and old cells over storage. LDH, PMRS, catalase, advanced oxidation protein products, and conjugate dienes were significant in old cells from day 5 itself, whereas in young cells towards the end of storage (from day 25). Oxidative insult was higher in old cells compared to young cells. AS-7 was beneficial to young erythrocytes during storage and thus laying the foundation for the possibilities of utilizing young cells as models for storage studies.

Effect of Rat Exposure to Moderate Pressure Oxygen Atmosphere on Resistance of Erythrocytes against Hemolysis.

Exposure of male Wistar rats to oxygen atmosphere at moderate pressure (1.10-1.15 atm) for 4 h resulted in significant, transient, and reversible decrease in hemolytic resistance of peripheral blood erythrocytes. In 2 days after oxygen exposure, the bone marrow released young erythrocytes with enhanced hemolytic resistance as a compensation for oxygen injury. These findings suggest that normobaric oxygen load can be viewed as a stress factor of moderate intensity.

Changes in Mean Corpuscular Volume and RBC Distribution Width Predict Erythrocyte Engraftment Following ABO-Incompatible Hematopoietic Stem Cell Transplantation.

The purpose of this study was to identify laboratory parameters representing erythrocyte engraftment to be used as an indicator to change the recipient to donor ABO group and Rh type following an ABO-incompatible hematopoietic stem cell transplant (HSCT). Studies have shown that ABO incompatibility does not have an effect on outcome of HSCT; however, the serologic consequences of these ABO-incompatible transplants can make it difficult to decide when to begin support with donor ABO/Rh-type blood products. This study explored the use of RBC distribution width (RDW), mean corpuscular volume, and hemoglobin as regularly tested laboratory parameters that could be used as surrogate markers for RBC engraftment in 65 patients who received ABO/Rh-incompatible HSCT. The appearance of engrafted donor RBCs correlated with a peak in RDW (P = .002). In addition, our findings suggest that serologic changes in ABO/Rh appear to correspond with a peak in RDW (P = .002). High values of RDW likely result from a substantial proportion of large, young erythrocytes from recent engraftment with smaller, older pretransplant erythrocytes from the recipient. Our findings suggest that peak RDW may be an indicator of erythrocyte engraftment, following an ABO/Rh-incompatible HSCT.