Search Results (29)

Utilization of a blood pump to aid in circulating a patient’s blood, otherwise known as mechanical circulatory support, is an effective and often life-saving treatment for cardiac/pulmonary failure patients, yet adverse events remain a common complication often attributed to mechanical trauma inflicted on blood components. This work specifically focuses on erythrocyte-derived extracellular vesicles (ErEVs) as a marker of this mechanical trauma as they are elevated in patients with blood pumps and have been tied to adverse events. Despite this, ErEVs are typically neglected during device development which usually includes testing with animal blood, most commonly porcine and bovine. Flow cytometry was employed to monitor ErEVs generated during a 6 h perfusion of porcine or bovine red blood cells (RBCs) in a blood circulatory loop with the CentriMag blood pump. Successful measurement meant overcoming limitations in suitable stains for the RBCs and ErEVs of the two species. Between the two species, 12 different antibodies and dyes were evaluated, including multiple glycophorin A clones, the typical human erythrocyte antigen. Only CD46 and carboxyfluorescein succinimidyl ester (CFSE) were found to successfully and reliably label porcine and bovine RBCs, respectively. With these stains, statistically significant increases for both porcine and bovine ErEVs with perfusion time were observed. Bovine erythrocytes produced significantly more ErEVs than porcine, indicating they are more sensitive to mechanical trauma and could be useful in early-stage device development. The utility of CD46 and CFSE used for porcine and bovine ErEV detection was demonstrated for in vitro pump testing with implications for physiological and pathological research with these animals. Full article

Introduction: cigarette smoking is a major source of systemic oxidative stress and a well-established risk factor for cardiovascular disease. Heated tobacco products (HTPs) are increasingly promoted as reduced-risk alternatives, yet their cellular effects remain incompletely understood. Methods: this study compared the oxidative stress-mediated effects of conventional cigarette smoking and HTP use on human erythrocytes. Erythrocytes from healthy non-smokers, conventional smokers, and HTP users were analyzed using biochemical, functional, and cytological approaches to assess redox status, membrane and cytoskeletal organization, anion exchanger 1 (AE1) function, antioxidant response, and redox-sensitive signaling pathways. Results: conventional smokers exhibited higher intracellular reactive oxygen species (ROS) levels, thiol depletion, methemoglobin and hemichrome formation, whereas HTP users showed marked lipid peroxidation despite lower ROS availability. Both groups instead displayed altered expression and distribution of key membrane and cytoskeletal proteins, including glycophorin A, AE1, spectrin, ankyrin, and band 4.1, indicating impaired membrane–cytoskeleton interactions. Functional analyses revealed an accelerated AE1-mediated anion exchange in erythrocytes from conventional smokers, whereas cells from HTP users exhibited a reduced sulfate accumulation, indicating altered transport capacity. In both groups, G6PDH activity was significantly increased, and redox-sensitive signaling pathways involving ERK, AKT, and eNOS were activated, accompanied by sex-dependent alterations in estrogen receptor expression and distribution. Conclusions: collectively, these findings identify erythrocytes as sensitive biomarkers of tobacco-related systemic damage and indicate that smoking-induced erythrocyte dysfunction, including that associated with HTP use, may actively contribute to vascular impairment. This evidence challenges the assumption that heated tobacco products confer a substantially reduced cardiovascular risk compared with conventional cigarettes. Full article

Background/Objectives: Histological examination constitutes a fundamental methodology for establishing the vitality of a lesion. In cases where the corpse is preserved for an extended duration of time prior to the post-mortem evaluation, particularly if the body has undergone freezing and thawing cycles, post-mortem changes may obscure or alter evidence of traumatic injuries. Consequently, the reliability of hematoxylin and eosin (H&E) staining for the reliable detection of intralesional erythrocytes in suspected traumatic fatalities is potentially severely compromised. The primary objective of this study is to rigorously underscore the detrimental influence of freeze–thaw processes on histologic examination and to advocate the indispensable incorporation of immunohistochemical analysis, specifically employing anti-human glycophorin A antibodies, to ascertain the presence of red blood cells. Methods: Skin samples from 10 autopsy cases were subjected to serial freeze–thaw cycles and analyzed using anti-human Glycophorin A (GPA) immunohistochemistry staining to evaluate skin lesion vitality in freeze–thawed tissues compared to fresh controls. Results: Results indicated that while H&E reliability was limited to fresh tissue, anti-GPA staining remained stable across all freeze–thaw cycles. Conclusions: Forensic pathologists must remain acutely cognizant of the potential artifacts produced by freeze–thaw cycles. In these cases, anti-GPA staining proved to be a reliable asset for evaluating the vitality of a lesion. Full article

Mancozeb is a broad-spectrum fungicide used extensively in agriculture to protect crops against a wide range of plant diseases. Although its capacity to induce oxidative stress is well documented, the cytotoxic effects of mancozeb on red blood cells (RBCs) remain poorly characterized. The present study aimed to investigate the cytotoxic effects of mancozeb on isolated RBCs, with particular focus on oxidative stress-induced cellular and molecular alterations. Human RBCs were exposed to mancozeb (0.5–100 µM) for 24 h. No hemolytic activity was observed across the tested concentrations. However, 10 and 100 µM mancozeb induced a significant increase in intracellular reactive oxygen species (ROS), leading to lipid and protein oxidation and impaired Na⁺/K⁺-ATPase and anion exchanger 1 (AE1) function. These changes resulted in altered RBC morphology, reduced deformability, and increased methemoglobin levels. Alterations in glycophorin A distribution, anion exchanger 1 (AE1) clustering and phosphorylation, and α/β-spectrin and band 4.1 re-arrangement indicated disrupted membrane–cytoskeleton interactions. A release of extracellular vesicles (EVs) positive for glycophorin A and annexin-V was also observed, consistent with plasma membrane remodeling. Despite increased intracellular calcium, eryptosis remained minimal, possibly due to activation of protective estrogen receptor (ER)-mediated pathways involving ERK1/2 and AKT signaling. Activation of the cellular antioxidant system and the glutathione redox system (GSH/GSSG) occurred, with catalase (CAT) playing a predominant role, while superoxide dismutase (SOD) activity remained largely unchanged. These findings offer mechanistic insights regarding the potential health impact of oxidative stress induced by pesticide exposure. Full article

In forensic pathology, glycophorin A (GPA) presence in tissues has been studied through anti-GPA monoclonal antibodies with immunohistochemical techniques. The use of anti-GPA in distinguishing ante- from postmortem lesions, particularly in advanced stages of decomposition, is explored in this study. This systematic review assesses the current use of glycophorins, focusing on their application in forensic investigations to detect vital hemorrhagic infiltration. A total of 799 studies were identified, and after screening, 16 studies were included, encompassing case reports, experimental studies, and case-control analyses. The data extracted from these studies highlighted the diagnostic accuracy of GPA immunohistochemical staining in forensic cases. Of the 50 specimens analyzed, 48 were stained with anti-GPA serum and 2 with anti-glycophorin C (GPC) serum. The results showed that GPA staining was significantly more reliable in detecting vital hemorrhage compared to routine histology, which had a diagnostic accuracy of only 66%. Moreover, in an experimental study, GPA positivity was observed in 72.2% of bruises and vital tissues, even in highly decomposed specimens. The study further emphasized the robustness of GPA in distinguishing ante- from postmortem injuries, with particular relevance for cases involving significant decomposition. Overall, GPA’s high sensitivity in detecting vital hemorrhage, especially in decomposed bodies, underscores its potential as a reliable tool in forensic pathology. Despite some limitations due to the small number of studies, the evidence supports the inclusion of anti-GPA antibodies in routine forensic examinations, potentially enhancing the accuracy of wound vitality assessments in criminal investigations. Full article

Oxidative stress is widely recognized as a key mechanism in the development of hypertension. Under pathological conditions, such as in hypertension, oxidative stress leads to irreversible posttranslational modifications of proteins, which result in loss of protein function and cellular damage. We have previously documented physiological and morphological changes across various blood and bone marrow cell lineages, all of which exhibit elevated oxidative stress. While cytoskeletal changes in erythrocytes have been well characterized in hereditary diseases, this is the first study, to our knowledge, to investigate cytoskeletal reorganization in erythrocytes from hypertensive patients. To this end, we compared the expression patterns and subcellular distribution of key cytoskeletal proteins in erythrocytes from hypertensive individuals with those from normotensive subjects using Western blot, flow cytometry, and confocal microscopy. Our results revealed the presence of three erythrocyte subpopulations with differential expression of glycophorin A. The persistent oxidative environment in hypertensive patients causes dysregulation in the expression of glycophorin A, Band 3 protein, protein 4.1, and ankyrin, as well as the reorganization of spectrin. These alterations in protein expression and distribution suggest that oxidative stress in hypertensive individuals may induce structural modifications, ultimately impairing erythrocyte membrane elasticity and function. Full article

Background: The aim of the study was to investigate if feto-maternal transfusion was related to the size of the fetal-maternal interface, and, therefore, was larger in twin pregnancy in comparison with singleton pregnancy. Methods: Blood samples from women with singleton (n = 11), and monochorionic (n = 11) and dichorionic (n = 13) twin gestations were tested. Flow cytometry tests with hemoglobin F, glycophorin A, and hemoglobin F and carbonic anhydrase simultaneous staining were used to detect fetal red blood cells and maternal F cells. Results: In all cases, the volume of feto-maternal transfusion was estimated to be low. The highest rate of fetal red blood cells in the maternal circulation was observed in the blood of women with dichorionic twin gestations both before and after delivery. An increase in fetal red blood cells was observed after cesarean section in singletons and twins. The median rate of maternal F cells was 2.23% in singleton, 2.1% in monochorionic and 3.95% in dichorionic pregnancy. Conclusions: Feto-maternal transfusion during pregnancy may be related to the multiplicity and chorionicity of pregnancy. Full article

Previous omics research in patients with complex congenital heart disease and single-ventricle circulation (irrespective of the stage of palliative repair) revealed alterations in cardiac and systemic metabolism, inter alia abnormalities in energy metabolism, and inflammation, oxidative stress or endothelial dysfunction. We employed an affinity-proteomics approach focused on cell surface markers, cytokines, and chemokines in the serum of 20 adult Fontan patients with a good functioning systemic left ventricle, and we 20 matched controls to reveal any specific processes on a cellular level. Analysis of 349 proteins revealed 4 altered protein levels related to chronic inflammation, with elevated levels of syndecan-1 and glycophorin-A, as well as decreased levels of leukemia inhibitory factor and nerve growth factor-ß in Fontan patients compared to controls. All in all, this means that Fontan circulation carries specific physiological and metabolic instabilities, including chronic inflammation, oxidative stress imbalance, and consequently, possible damage to cell structure and alterations in translational pathways. A combination of proteomics-based biomarkers and the traditional biomarkers (uric acid, γGT, and cholesterol) performed best in classification (patient vs. control). A metabolism- and signaling-based approach may be helpful for a better understanding of Fontan (patho-)physiology. Syndecan-1, glycophorin-A, leukemia inhibitory factor, and nerve growth factor-ß, especially in combination with uric acid, γGT, and cholesterol, might be interesting candidate parameters to complement traditional diagnostic imaging tools and the determination of traditional biomarkers, yielding a better understanding of the development of comorbidities in Fontan patients, and they may play a future role in the identification of targets to mitigate inflammation and comorbidities in Fontan patients. Full article

Body fluid identification is fundamental in forensic science as it links a specific biological source to a genetic profile, thus providing critical clues for crime scene reconstruction. Blood is one of the most common body fluids found on the crime scene, and several strategies have been developed for its identification in recent decades. Usually, after a preliminary (or presumptive) test to determine the presence of blood (both human and non-human), a confirmatory test is needed to prove that the sample is human blood. Out of the confirmatory tests, immunochromatographic (IC) assays are the most commonly and widely used. This work gives a review of the use of commercial kits specifically developed to detect human hemoglobin or glycophorin A (a surface protein of human red cells) in forensics. Claimed sensitivity varies broadly (ranging from 0.06 to 75 nanoliters of fresh blood), but different values (as low as 0.002 nL) were found during validation procedures. Specificities are high, and the possibility of cross-reaction (with the risk of false-positive results) is so low that it can be considered negligible. False-negative results, however, can be found due to the so-called “hook effect” as well as to the target degradation/modification, which interferes with the Ag-Ab binding. In addition, the chemical compositions of the presumptive test, detergents, and washing can also promote false negative outcomes in peculiar situations. Although IC assays are rapid, inexpensive, specific, and easy to use even on the crime scene, their major limitation is represented by the destructive approach required by this kind of confirmatory test. Since the final goal of the forensic investigation is the genetic typing of a bloodstain, we will describe the strategies developed for IC assays of faint stains as well as the strategies adopted to ensure that exactly the same sample undergoes human blood identification and DNA typing. Full article

Consistent with well-established biochemical properties of coronaviruses, sialylated glycan attachments between SARS-CoV-2 spike protein (SP) and host cells are key to the virus’s pathology. SARS-CoV-2 SP attaches to and aggregates red blood cells (RBCs), as shown in many pre-clinical and clinical studies, causing pulmonary and extrapulmonary microthrombi and hypoxia in severe COVID-19 patients. SARS-CoV-2 SP attachments to the heavily sialylated surfaces of platelets (which, like RBCs, have no ACE2) and endothelial cells (having minimal ACE2) compound this vascular damage. Notably, experimentally induced RBC aggregation in vivo causes the same key morbidities as for severe COVID-19, including microvascular occlusion, blood clots, hypoxia and myocarditis. Key risk factors for COVID-19 morbidity, including older age, diabetes and obesity, are all characterized by markedly increased propensity to RBC clumping. For mammalian species, the degree of clinical susceptibility to COVID-19 correlates to RBC aggregability with p = 0.033. Notably, of the five human betacoronaviruses, the two common cold strains express an enzyme that releases glycan attachments, while the deadly SARS, SARS-CoV-2 and MERS do not, although viral loads for COVID-19 and the two common cold infections are similar. These biochemical insights also explain the previously puzzling clinical efficacy of certain generics against COVID-19 and may support the development of future therapeutic strategies for COVID-19 and long COVID patients. Full article

Healthcare-related homicidal cases are not novel within the medical–legal landscape, but investigations are often made difficult with the scarcity of material evidence related to the crime. For this reason, it is necessary to carefully analyze the clinical documentation and employ ancillary forensic resources such as radiology, histopathology, and toxicology. In the presented scenario, the observation of 14 deaths from abnormal bleeding in a First-Level Italian Hospital revealed the administration of massive doses of heparin by a nurse. On behalf of the Judicial Authority, a multidisciplinary medical team investigated the case through the following steps: a thorough review of the clinical documentation, exhumation of the bodies belonging to the deceased patients, performing PMCT and autopsy, and collecting tissue samples for histopathological, immunohistochemical, and toxicological investigations. All the analyzed cases have been characterized by the observation of fatal hemorrhagic episodes not explained with the clinical conditions of the patients, confirmed using autopsy observations and the histological demonstration of the vitality of the lesions. However, due to the limited availability of biological material for the toxicological analysis, the indirect evidence from hematological analyses in hospitalized patients was crucial in demonstrating heparin overdose and its link to the recorded deaths. The present scenario demonstrates the fundamental importance of a multidisciplinary approach to cases of judicial interest related to the healthcare context. Therefore, the illustrated methodologies can be interpreted as an operational framework for similar future cases. Full article

Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen. Full article

The key element in developing a successful malaria treatment is a good understanding of molecular mechanisms engaged in human host infection. It is assumed that oligosaccharides play a significant role in Plasmodium parasites binding to RBCs at different steps of host infection. The formation of a tight junction between EBL merozoite ligands and glycophorin receptors is the crucial interaction in ensuring merozoite entry into RBCs. It was proposed that sialic acid residues of O/N-linked glycans form clusters on a human glycophorins polypeptide chain, which facilitates the binding. Therefore, specific carbohydrate drugs have been suggested as possible malaria treatments. It was shown that the sugar moieties of N-acetylneuraminyl-N-acetate-lactosamine and 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which is its structural analog, can inhibit P. falciparum EBA-175-GPA interaction. Moreover, heparin-like molecules might be used as antimalarial drugs with some modifications to overcome their anticoagulant properties. Assuming that the principal interactions of Plasmodium merozoites and host cells are mediated by carbohydrates or glycan moieties, glycobiology-based approaches may lead to new malaria therapeutic targets. Full article

Anion exchanger-1 (AE1) is the main erythroid Cl⁻/HCO₃⁻ transporter that supports CO₂ transport. Glycophorin A (GPA), a component of the AE1 complexes, facilitates AE1 expression and anion transport, but Glycophorin B (GPB) does not. Here, we dissected the structural components of GPA/GPB involved in glycophorin-AE1 trafficking by comparing them with three GPB variants—GPBhead (lacking the transmembrane domain [TMD]), GPBtail (mainly the TMD), and GP.Mur (glycophorin B-A-B hybrid). GPB-derived GP.Mur bears an O-glycopeptide that encompasses the R18 epitope, which is present in GPA but not GPB. By flow cytometry, AE1 expression in the control erythrocytes increased with the GPA-R18 expression; GYP.Mur^+/+ erythrocytes bearing both GP.Mur and GPA expressed more R18 epitopes and more AE1 proteins. In contrast, heterologously expressed GPBtail and GPB were predominantly localized in the Golgi apparatus of HEK-293 cells, whereas GBhead was diffuse throughout the cytosol, suggesting that glycophorin transmembrane encoded an ER/Golgi retention signal. AE1 coexpression could reduce the ER/Golgi retention of GPB, but not of GPBtail or GPBhead. Thus, there are forward-trafficking and transmembrane-driven ER/Golgi retention signals encoded in the glycophorin sequences. How the balance between these opposite trafficking signals could affect glycophorin sorting into AE1 complexes and influence erythroid anion transport remains to be explored. Full article

The Sd^a histo-blood group antigen (GalNAcβ1-4(NeuAcα2-3)Galβ-R) is implicated in various infections and constitutes a potential biomarker for colon cancer. Sd(a−) individuals (2–4% of Europeans) may produce anti-Sd^a, which can lead to incompatible blood transfusions, especially if donors with the high-expressing Sd(a++)/Cad phenotype are involved. We previously reported the association of B4GALNT2 mutations with Sd(a−), which established the SID blood-group system. The present study provides causal proof underpinning this correlation. Sd(a−) HEK293 cells were transfected with different B4GALNT2 constructs and evaluated by immunostaining and glycoproteomics. The predominant SID^null candidate allele with rs7224888:T>C (p.Cys406Arg) abolished Sd^a synthesis, while this antigen was detectable as N- or O-glycans on glycoproteins following transfection of wildtype B4GALNT2. Surprisingly, two rare missense variants, rs148441237:A>G and rs61743617:C>T, found in a Sd(a−) compound heterozygote, gave results similar to wildtype. To elucidate on whether Sd(a++)/Cad also depends on B4GALNT2 alterations, this gene was sequenced in five individuals. No Cad-specific changes were identified, but a detailed erythroid Cad glycoprotein profile was obtained, especially for glycophorin-A (GLPA) O-glycosylation, equilibrative nucleoside transporter 1 (S29A1) O-glycosylation, and band 3 anion transport protein (B3AT) N-glycosylation. In conclusion, the p.Cys406Arg β4GalNAc-T2 variant causes Sd^a-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized. Full article