Plasmodium falciparum Glutamic Acid-Rich Protein-Independent Polyclonal Antibodies Inhibit Malaria Parasite Growth in Human Erythrocytes.

Plasmodium falciparum glutamic acid-rich protein (PfGARP) is a recently characterized cell surface antigen encoded by Plasmodium falciparum, the causative agent of severe human malaria pathophysiology. Previously, we reported that the human erythrocyte band 3 (SLC4A1) serves as a host receptor for PfGARP. Antibodies against PfGARP did not affect parasite invasion and growth. We surmised that PfGARP may play a role in the rosetting and adhesion of malaria. Another study reported that antibodies targeting PfGARP exhibit potent inhibition of parasite growth. This inhibition occurred without the presence of any immune or complement components, suggesting the activation of an inherent density-dependent regulatory system. Here, we used polyclonal antibodies against PfGARP and a monoclonal antibody mAb7899 to demonstrate that anti-PfGARP polyclonal antibodies, but not mAb7899, exerted potent inhibition of parasite growth in infected erythrocytes independent of PfGARP. These findings suggest that an unknown malaria protein(s) is the target of growth arrest by polyclonal antibodies raised against PfGARP.

Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

How Clot Composition Influences Fibrinolysis in the Acute Phase of Stroke: A Proteomic Study of Cerebral Thrombi.

BACKGROUND: The dramatic clinical improvement offered by mechanical thrombectomy raised questions about the relevance of prior intravenous thrombolysis in large-vessel occlusion strokes. Hence, studying intravenous thrombolysis susceptibility and its dependence on thrombus composition is crucial. We used an observational proteomic study of whole thrombi retrieved by mechanical thrombectomy to identify factors associated with fibrin content and fibrinolytic activity (FA). METHODS: In 104 stroke patients, the thrombi proteome was established by mass spectrometry coupled to liquid chromatography. FA was estimated in clots both outside (FAout) by measuring D-dimer levels at the blood-thrombus interface and inside (FAin) by evaluating the ratio of fibrinogen α to its plasmin-cleaved forms using proteomics coupled with protein electrophoresis. The factors associated with fibrin content, FAin, and FAout were determined by intravenous thrombolysis-adjusted linear regression. RESULTS: FAout (P<0.0001) and FAin (P=0.0147) were driven by recombinant tissue-type plasminogen activator (r-tPA) administration (47/104) and thrombus composition. Indeed, FAout was greater with fibrin-rich than erythrocyte-rich thrombi, presumably because of more (r)tPA substrates. Thus, FAout was increased with cardioembolic thrombi (72/104), which are rich in fibrin (P=0.0300). Opposite results were found inside the thrombus, suggesting that (r)tPA penetrability was hampered by the density of the fibrinous cap. Moreover, blood cells had a strong impact on thrombus structure and susceptibility to (r)tPA. Indeed, fibrin content was negatively associated with erythrocyte-specific proteins in the thrombus, admission hematocrit (P=0.0139), and hemoglobin level (P=0.0080), which underlines the key role of erythrocytes in thrombus composition. Also, an increased number of neutrophils impaired FAout (P=0.0225), which suggests that their aggregation around the thrombus prevented the (r)tPA attack. Only FAout was significantly associated with reduced thrombus weight (P=0.0310), increased recanalization rate (P=0.0150), good clinical outcome (P=0.0480), and reduced mortality (P=0.0080). CONCLUSIONS: Proteomics can offer new insights into the close relationship between thrombus composition and susceptibility to fibrinolysis, paving the way for new adjuvant therapies.

Renal Clearable Nanodots-Engineered Erythrocytes with Enhanced Circulation and Tumor Accumulation for Photothermal Therapy of Hepatocellular Carcinoma.

Living cell-mediated nanodelivery system is considered a promising candidate for targeted antitumor therapy; however, their use is restricted by the adverse interactions between carrier cells and nanocargos. Herein, a novel erythrocyte-based nanodelivery system is developed by assembling renal-clearable copper sulfide (CuS) nanodots on the outer membranes of erythrocytes via a lipid fusion approach, and demonstrate that it is an efficient photothermal platform against hepatocellular carcinoma. After intravenous injection of the nanodelivery system, CuS nanodots assembled on erythrocytes can be released from the system, accumulate in tumors in response to the high shear stress of bloodstream, and show excellent photothermal antitumor effect under the near infrared laser irradiation. Therefore, the erythrocyte-mediated nanodelivery system holds many advantages including prolonged blood circulation duration and enhanced tumor accumulation. Significantly, the elimination half-life of the nanodelivery system is 74.75 ± 8.77 h, which is much longer than that of nanodots (33.56 ± 2.36 h). Moreover, the other two kinds of nanodots can be well assembled onto erythrocytes to produce other erythrocyte-based hitchhiking platforms. Together, the findings promote not only the development of novel erythrocyte-based nanodelivery systems as potential platforms for tumor treatment but also their further clinical translation toward personalized healthcare.

Chemical Resolution of an Epitope Recognized by Blood Group Antibodies Capable of Binding Both A and B Red Blood Cells.

The high specificity of human antibodies to blood group A and B antigens is impressive, especially when considering the structural difference between these antigens (tetrasaccharides) is a NHAc versus a OH-group on the terminal monosaccharide residue. It is well established that in addition to anti-A and anti-B there is a third antibody, anti-A,B capable of recognizing both A and B antigens. To analyze this AB specificity, we synthesized a tetrasaccharide, where the NHAc of the A antigen was replaced with NH2. This NH2-group was used to attach the glycan to an affinity resin, creating an AB-epitope (ABep) adsorbent where the critical site for recognition by A and B antibodies was not accessible, while the rest of the (conformationally compact) tetrasaccharide remained accessible. Anti-ABep antibodies were isolated from blood group O donors and found to have expected A,B-specificity against immobilized and red cell bound synthetic antigens, including ABep, and were able to agglutinate both A and B red cells. The amount of these anti-ABep (anti-A,B) antibodies found in the blood of group O donors was comparable to levels of anti-A and anti-B found in group B and A individuals. Using STD-NMR the location for the AB-epitope on the tetrasaccharide was found.

Investigation of the cytotoxicity, genotoxicity and antioxidant prospects of JM-20 on human blood cells: A multi-target compound with potential therapeutic applications.

JM-20 is a 1,5-benzodiazepine compound fused to a dihydropyridine fraction with different pharmacological properties. However, its potential toxic effects on blood cells have not yet been reported. Thus, the present study aimed to investigate, for the first time, the possible cytotoxicity of JM-20 through cell viability, cell cycle, morphology changes, reactive species (RS) to DCFH-DA, and lipid peroxidation in human leukocytes, its hemolytic effect on human erythrocytes, and its potential DNA genotoxicity using plasmid DNA in vitro. Furthermore, the compound's ability to reduce the DPPH radical was also measured. Human blood was obtained from healthy volunteers (30 ± 10 years old), and the leukocytes or erythrocytes were immediately isolated and treated with different concentrations of JM-20. A cytoprotective effect was exhibited by 10 µM JM-20 against 1 mM tert-butyl hydroperoxide (t-but-OOH) in the leukocytes. However, the highest tested concentrations of the compound (20 and 50 µM) changed the morphology and caused a significant decrease in the cell viability of leukocytes (p < 0.05, in comparison with Control). All tested concentrations of JM-20 also resulted in a significant increase in intracellular RS as measured by DCFH-DA in these cells (p < 0.05, in comparison with Control). On the other hand, the results point out a potent antioxidant effect of JM-20, which was similar to the classical antioxidant α-tocopherol. The IC50 value of JM-20 against the lipid peroxidation induced by (FeII) was 1.051 µM ± 0.21, while the IC50 value of α-tocopherol in this parameter was 1.065 µM ± 0.34. Additionally, 50 and 100 µM JM-20 reduced the DPPH radical in a statistically similar way to the 100 µM α-tocopherol (p < 0.05, in comparison with the control). No significant hemolysis in erythrocytes, no cell cycle changes in leukocytes, and no genotoxic effects in plasmid DNA were induced by JM-20 at any tested concentration. The in silico pharmacokinetic and toxicological properties of JM-20, derivatives, and nifedipine were also studied. Here, our findings demonstrate that JM-20 and its putative metabolites exhibit similar characteristics to nifedipine, and the in vitro and in silico data support the low toxicity of JM-20 to mammals.

IL-1ß Impacts Vascular Integrity and Lymphatic Function in the Embryonic Omentum.

BACKGROUND: The chromatin-remodeling enzyme BRG1 (brahma-related gene 1) regulates gene expression in a variety of rapidly differentiating cells during embryonic development. However, the critical genes that BRG1 regulates during lymphatic vascular development are unknown. METHODS: We used genetic and imaging techniques to define the role of BRG1 in murine embryonic lymphatic development, although this approach inadvertently expanded our study to multiple interacting cell types. RESULTS: We found that omental macrophages fine-tune an unexpected developmental process by which erythrocytes escaping from naturally discontinuous omental blood vessels are collected by nearby lymphatic vessels. Our data indicate that circulating fibrin(ogen) leaking from gaps in omental blood vessels can trigger inflammasome-mediated IL-1ß (interleukin-1ß) production and secretion from nearby macrophages. IL-1ß destabilizes adherens junctions in omental blood and lymphatic vessels, contributing to both extravasation of erythrocytes and their uptake by lymphatics. BRG1 regulates IL-1ß production in omental macrophages by transcriptionally suppressing the inflammasome trigger RIPK3 (receptor interacting protein kinase 3). CONCLUSIONS: Genetic deletion of Brg1 in embryonic macrophages leads to excessive IL-1ß production, erythrocyte leakage from blood vessels, and blood-filled lymphatics in the developing omentum. Altogether, these results highlight a novel context for epigenetically regulated crosstalk between macrophages, blood vessels, and lymphatics.

Erythrocytes membrane fluidity changes induced by adenylyl cyclase cascade activation: study using fluorescence recovery after photobleaching.

In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade.

Isolation and functional identification of immune cells in hemolymph of blood clams Tegillarca granosa.

Blood clam Tegillarca granosa is a type of economically cultivated bivalve mollusk with red blood, and it primarily relies on hemocytes in its hemolymph for immune defense. However, there are currently no reports on the isolation and identification of immune cells in T. granosa, which hinders our understanding of their immune defense. In this study, we employed single-cell transcriptome sequencing (scRNA-seq) to visualize the molecular profile of hemocytes in T. granosa. Based on differential expression of immune genes and hemoglobin genes, hemocytes can be molecularly classified into immune cells and erythrocytes. In addition, we separated immune cells using density gradient centrifugation and demonstrated their stronger phagocytic capacity compared to erythrocytes, as well as higher levels of ROS and NO. In summary, our experiments involved the isolation and functional identification of immune cells in hemolymph of T. granosa. This study will provide valuable insights into the innate immune system of red-blood mollusks and further deepen the immunological research of mollusks.

Hyaluronic acid impacts hematological endpoints and spleen histological features in African catfish (Clarias gariepinus).

Since its identification in the vitreous humour of the eye and laboratory biosynthesis, hyaluronic acid (HA) has been a vital component in several pharmaceutical, nutritional, medicinal, and cosmetic uses. However, little is known about its potential toxicological impacts on aquatic inhabitants. Herein, we investigated the hematological response of Clarias gariepinus to nominal doses of HA. To achieve this objective, 72 adult fish were randomly and evenly distributed into four groups: control, low-dose (0.5 mg/l HA), medium-dose (10 mg/l HA), and high-dose (100 mg/l HA) groups for two weeks each during both the exposure and recovery periods. The findings confirmed presence of anemia, neutrophilia, leucopoenia, lymphopenia, and eosinophilia at the end of exposure to HA. In addition, poikilocytosis and a variety of cytomorphological disturbances were observed. Dose-dependent histological alterations in spleen morphology were observed in the exposed groups. After HA removal from the aquarium for 2 weeks, the groups exposed to the two highest doses still exhibited a notable decline in red blood cell count, hemoglobin concentration, mean corpuscular hemoglobin concentration, and an increase in mean corpuscular volume. Additionally, there was a significant rise in neutrophils, eosinophils, cell alterations, and nuclear abnormalities percentages, along with a decrease in monocytes, coupled with a dose-dependent decrease in lymphocytes. Furthermore, only the highest dose of HA in the recovered groups continued to cause a significant increase in white blood cells. White blood cells remained lower, and the proportion of apoptotic RBCs remained higher in the high-dose group. The persistence of most of the haematological and histological disorders even after recovery period indicates a failure of physiological compensatory mechanisms to overcome the HA-associated problems or insufficient duration of recovery. Thus, these findings encourage the inclusion of this new hazardous agent in the biomonitoring program and provide a specific pattern of hematological profile in HA-challenged fish. Further experiments are highly warranted to explore other toxicological hazards of HA using dose/time window protocols.

Is post-hypertonic lysis of human red blood cells caused by excessive cell volume regulation?

Human red blood cells (RBC) exposed to hypertonic media are subject to post-hypertonic lysis - an injury that only develops during resuspension to an isotonic medium. The nature of post-hypertonic lysis was previously hypothesized to be osmotic when cation leaks were observed, and salt loading was suggested as a cause of the cell swelling upon resuspension in an isotonic medium. However, it was problematic to account for the salt loading since the plasma membrane of human RBCs was considered impermeable to cations. In this study, the hypertonicity-related behavior of human RBCs is revisited within the framework of modern cell physiology, considering current knowledge on membrane ion transport mechanisms - an account still missing. It is recognized here that the hypertonic behavior of human RBCs is consistent with the acute regulatory volume increase (RVI) response - a healthy physiological reaction initiated by cells to regulate their volume by salt accumulation. It is shown by reviewing the published studies that human RBCs can increase cation conductance considerably by activating cell volume-regulated ion transport pathways inactive under normal isotonic conditions and thus facilitate salt loading. A simplified physiological model accounting for transmembrane ion fluxes and membrane voltage predicts the isotonic cell swelling associated with increased cation conductance, eventually reaching hemolytic volume. The proposed involvement of cell volume regulation mechanisms shows the potential to explain the complex nature of the osmotic response of human RBCs and other cells. Cryobiological implications, including mechanisms of cryoprotection, are discussed.

Iron supplementation in the diets of hybrid catfish (Ictalurus punctatus × I. furcatus) juveniles affected haematocrit levels and potentially decreased disease resistance to Edwardsiella ictaluri.

To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.

Study of the frequencies of erythrocyte abnormalities as in situ biomarkers of genotoxic risk of chemicals in special fish stock in water supply reservoirs.

In three water-supply reservoirs in the catchment area of the Odra River (Czech Republic), a special fish stock was monitored for control of health to estimate the mutagenic effect of chemicals. The results contribute to obtaining initial information about the morphology of erythrocyte abnormalities classified in 21 categories in 16 fish species in reservoirs with abundant salmonids (the Morávka Reservoir) or with the prevalence of cyprinids (the Kruzberk and Sance Reservoirs), not directly exposed to the adverse environmental effects such as industrial, urban, agricultural and intensive farming activities. The different intensities and prevalence of nuclear abnormalities (NA) and cytoplasmic abnormalities (CA) in fish from the same reservoir habitat show that to be able to obtain an objective view of the genotoxic risk of chemicals, it is necessary to respect the different requirements of the fish for the exploitation of the food available in the biotope and to subject all representatives of piscivorous, omnivorous and benthophagous fishes in the reservoir to cytogenetic analysis. The occurrence of certain categories of erythrocyte abnormalities in diseased fish draws attention to the need to know the state of health of the fish and to employ this knowledge to exclude parasitological, viral and other infectious agents. These results are the first report of the frequencies of erythrocyte abnormalities in native fish. They should serve to check which of the categories examined could be of use in assessing the genotoxic risk in other stagnant and running aquatic ecosystems affected by anthropogenic activities.

Environmental cadmium exposure perturbs systemic iron homeostasis via hemolysis and inflammation, leading to hepatic ferroptosis in common carp (Cyprinus carpio L.).

Cadmium (Cd) pollution is considered a pressing challenge to eco-environment and public health worldwide. Although it has been well-documented that Cd exhibits various adverse effects on aquatic animals, it is still largely unknown whether and how Cd at environmentally relevant concentrations affects iron metabolism. Here, we studied the effects of environmental Cd exposure (5 and 50 µg/L) on iron homeostasis and possible mechanisms in common carp. The data revealed that Cd elevated serum iron, transferrin saturation and iron deposition in livers and spleens, leading to the disruption of systemic iron homeostasis. Mechanistic investigations substantiated that Cd drove hemolysis by compromising the osmotic fragility and inducing defective morphology of erythrocytes. Cd concurrently exacerbated hepatic inflammatory responses, resulting in the activation of IL6-Stat3 signaling and subsequent hepcidin transcription. Notably, Cd elicited ferroptosis through increased iron burden and oxidative stress in livers. Taken together, our findings provide evidence and mechanistic insight that environmental Cd exposure could undermine iron homeostasis via erythrotoxicity and hepatotoxicity. Further investigation and ecological risk assessment of Cd and other pollutants on metabolism-related effects is warranted, especially under the realistic exposure scenarios.

Atomic force microscopy reveals involvement of the cell envelope in biomechanical properties of sickle erythrocytes.

BACKGROUND: Intracellular hemoglobin polymerization has been supposed to be the major determinant for the elevated rigidity/stiffness of sickle erythrocytes from sickle cell anemia (SCA) patients. However, the contribution of the cell envelope remains unclear. RESULTS: In this study, using atomic force microscopy (AFM), we compared the normal and sickled erythrocyte surfaces for stiffness and topography. AFM detected that sickle cells had a rougher surface and were stiffer than normal erythrocytes and that sickle cell ghosts had a rougher surface (for both outer and inner surfaces) and were thicker than normal ghosts, the latter implying a higher membrane-associated hemoglobin content/layer in the sickle cell envelope. Compared to healthy subjects, the SCA patients had lower plasma lipoprotein levels. AFM further revealed that a mild concentration of methyl-ß-cyclodextrin (MßCD, a putative cholesterol-depleting reagent) could induce an increase in roughness of erythrocytes/ghosts and a decrease in thickness of ghosts for both normal and sickle cells, implying that MßCD can alter the cell envelope from outside (cholesterol in the plasma membrane) to inside (membrane-associated hemoglobin). More importantly, MßCD also caused a more significant decrease in stiffness of sickle cells than that of normal erythrocytes. CONCLUSIONS: The data reveal that besides the cytosolic hemoglobin fibers, the cell envelope containing the membrane-associated hemoglobin also is involved in the biomechanical properties (e.g., stiffness and shape maintenance) of sickle erythrocytes.

Hypoxia alters the upper thermal limits and blood physiology in zebrafish, Danio rerio.

Hypoxic aquatic environments occur more frequently as a result of climate change, thereby exerting challenges on the physiological and metabolic functions of aquatic animals. In this study, a model fish, zebrafish (Danio rerio) was used to observe the climate-induced hypoxic effect on the upper thermal limit (critical thermal maximum; CTmax), hemoglobin, and blood glucose levels, and abnormalities of erythrocytes at cellular and nuclear level. The value of CTmax decreased significantly under hypoxia (39.10 ± 0.96 °C) compared to normoxia (43.70 ± 0.91 °C). At CTmax, hemoglobin levels were much lower (9.33 ± 0.60 g/dL) and blood glucose levels were significantly higher (194.20 ± 11.33 mg/L) under hypoxia than they were under normoxia and at the beginning of the experiment. Increased frequencies of abnormalities in the erythrocytes at both cellular (fusion, twin, elongated, spindle and tear drop shaped) and nuclear (micronucleus, karyopyknosis, binuclei, nuclear degeneration and notched nuclei) levels were also found under hypoxia compared to normoxia. These results suggest that hypoxic conditions significantly alter the temperature tolerance and subsequent physiology in zebrafish. Our findings will aid in the development of effective management techniques for aquatic environments with minimum oxygen availability.

Diascopy and histopathological evaluation of nonblanching erythematous dermatoses in dogs.

BACKGROUND: Diascopy is a point-of-care diagnostic test used to differentiate skin erythema due to vascular dilation from haemorrhage. In the veterinary literature, only a handful of diseases have been described to be associated with a negative (nonblanching) diascopy result, and histological investigation of haemorrhage has been inconsistent. OBJECTIVES: Retrospective study to undertake a histopathological investigation of canine, nonblanching erythematous dermatoses for the presence or absence of haemorrhage and vascular changes. MATERIALS AND METHODS: Skin biopsies from dogs presented with moderate-to-severe nonblanching erythema were evaluated histologically. Additionally, clinical data about each patient were analysed. RESULTS: Twenty cases were identified with nonblanching erythema. Diagnoses included vasculopathy (n = 6), canine eosinophilic dermatitis (n = 3), cutaneous epitheliotropic T-cell lymphoma (n = 2), and one case each of sterile granuloma and pyogranuloma syndrome, German shepherd dog pyoderma, multiple mast cell tumours, haemangiosarcoma, exfoliative cutaneous lupus erythematosus, canine leishmaniosis with sebaceous adenitis, sebaceous adenitis with concurrent dermatophytosis, calcinosis cutis and canine atopic dermatitis with insect-bite reaction. One or more vascular changes were present in all 20 cases and included perivascular oedema, endothelial swelling and neutrophilic infiltration of vessel walls. Haemorrhage was identified in 17 of 20 cases (85%). Three cases without dermal haemorrhage were calcinosis cutis, sebaceous adenitis with dermatophytosis and canine atopic dermatitis with insect-bite reaction. CONCLUSIONS AND CLINICAL RELEVANCE: Negative diascopy was associated with haemorrhage and vascular pathological findings in the majority of cases, yet not all. Haemorrhage was identified histologically in all diseases previously reported as nonblanching as well as in a few additional diseases.

The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells.

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Chloride Gradient Is Involved in Ammonium Influx in Human Erythrocytes.

The ammonia/ammonium (NH3/NH4+, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH4+ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl- and HCO3-). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH4+ buffer, RBCs concentration-dependently swelled and lysed when [NH4+] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH4+-induced cell swelling and hemolysis, and the restoration of [Cl-] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH4+ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs.

Studies on the Thermal Decomposition Course of Nitrogen-Rich Heterocyclic Esters as Potential Drug Candidates and Evaluation of Their Thermal Stability and Properties.

Drug candidates must undergo thermal evaluation as early as possible in the preclinical phase of drug development because undesirable changes in their structure and physicochemical properties may result in decreased pharmacological activity or enhanced toxicity. Hence, the detailed evaluation of nitrogen-rich heterocyclic esters as potential drug candidates, i.e., imidazolidinoannelated triazinylformic acid ethyl esters 1-3 (where R1 = 4-CH3 or 4-OCH3 or 4-Cl, and R2 = -COOC2H5) and imidazolidinoannelated triazinylacetic acid methyl esters 4-6 (where R1 = 4-CH3 or 4-OCH3 or 4-Cl, and R2 = -CH2COOCH3)-in terms of their melting points, melting enthalpy values, thermal stabilities, pyrolysis, and oxidative decomposition course-has been carried out, using the simultaneous thermal analysis methods (TG/DTG/DSC) coupled with spectroscopic techniques (FTIR and QMS). It was found that the melting process (documented as one sharp peak related to the solid-liquid phase transition) of the investigated esters proceeded without their thermal decomposition. It was confirmed that the melting points of the tested compounds increased in relation to R1 and R2 as follows: 2 (R1 = 4-OCH3; R2 = -COOC2H5) < 6 (R1 = 4-Cl; R2 = -CH2COOCH3) < 5 (R1 = 4-OCH3; R2 = -CH2COOCH3) < 3 (R1 = 4-Cl; R2 = -COOC2H5) < 1 (R1 = 4-CH3; R2 = -COOC2H5) < 4 (R1 = 4-CH3; R2 = -CH2COOCH3). All polynitrogenated heterocyclic esters proved to be thermally stable up to 250 °C in inert and oxidising conditions, although 1-3 were characterised by higher thermal stability compared to 4-6. The results confirmed that both the pyrolysis and the oxidative decomposition of heterocyclic ethyl formates/methyl acetates with para-substitutions at the phenyl moiety proceed according to the radical mechanism. In inert conditions, the pyrolysis process of the studied molecules occurred with the homolytic breaking of the C-C, C-N, and C-O bonds. This led to the emission of alcohol (ethanol in the case of 1-3 or methanol in the case of 4-6), NH3, HCN, HNCO, aldehydes, CO2, CH4, HCl, aromatics, and H2O. In turn, in the presence of air, cleavage of the C-C, C-N, and C-O bonds connected with some oxidation and combustion processes took place. This led to the emission of the corresponding alcohol depending on the analysed class of heterocyclic esters, NH3, HCN, HNCO, aldehydes, N2, NO/NO2, CO, CO2, HCl, aromatics, and H2O. Additionally, after some biological tests, it was proven that all nitrogen-rich heterocyclic esters-as potential drug candidates-are safe for erythrocytes, and some of them are able to protect red blood cells from oxidative stress-induced damage.