Future Perspectives in HLA Typing Technologies

A wide range of malignant and nonmalignant diseases require hematopoietic stem cell transplantation (HSCT) as last resort therapeutic approach. Graft versus host disease (GVHD), which is one of the major causes of transplant-related mortality, is minimized whenever increased matching of human leukocyte antigens (HLAs) between donor and recipient is present. Suitable donor selection is determined with the utilization of HLA typing. HLAs are highly polymorphic glycoproteins encoded by a region of genes known as the major histocompatibility complex (MHC). Their biological function is to present antigenic peptides to T lymphocytes. However, they also play important role in HSCT acceptance/rejection. During the previous years, various techniques have been acquired in order to better characterize the HLA profile of transplant donors and recipients. This effort is particularly challenging due to MHC size, but most importantly due to high sequence variability in specific regions of the respective genetic loci, between individuals. Initially, HLA typing was performed using serological typing, hybridization techniques, and restriction fragment length polymorphism (RFLP) approaches. Later on, polymerase chain reaction (PCR) based techniques and direct sequencing (dideoxy-based Sanger sequencing) capillary electrophoretic analyses arose. Nowadays, 2nd and 3rd generation sequencing (NGS) technologies show great potential in effectively identifying these polymorphic regions

Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

BACKGROUND: We have showed that secretory Apolipoprotein J/Clusterin (sCLU) is down-regulated in senescent, stressed or diseased red blood cells (RBCs). It was hypothesized that sCLU loss relates to RBCs vesiculation, a mechanism that removes erythrocyte membrane patches containing defective or potentially harmful components. METHODOLOGY/PRINCIPAL FINDINGS: To investigate this issue we employed a combination of biochemical and microscopical approaches in freshly prepared RBCs or RBCs stored under standard blood bank conditions, an in vitro model system of cellular aging. We found that sCLU is effectively exocytosed in vivo during membrane vesiculation of freshly prepared RBCs. In support, the RBCs' sCLU content was progressively reduced during RBCs ex vivo maturation and senescence under cold storage due to its selective exocytosis in membrane vesicles. A range of typical vesicular components, also involved in RBCs senescence, like Band 3, CD59, hemoglobin and carbonylated membrane proteins were found to physically interact with sCLU. CONCLUSIONS/SIGNIFICANCE: The maturation of RBCs is associated with a progressive loss of sCLU. We propose that sCLU is functionally involved in the disposal of oxidized/defected material through RBCs vesiculation. This process most probably takes place through sCLU interaction with RBCs membrane proteins that are implicit vesicular components. Therefore, sCLU represents a pro-survival factor acting for the postponement of the untimely clearance of RBCs

A swift risk analysis for COVID-19 testing facilities using rapid tests

Introduction. COVID-19 is an infectious disease of International Concern, due to the wide-spread geographic impact and high transmissibility, causing severe illnesses. Many testing facilities were set-up for monitoring the spread of the SARS-CoV-2 virus, at the early days of the coronavirus pandemic. From Biosafety aspect this study investigates a reliable risk assessment method to identify and mitigate the risks of COVID-19 testing facilities using Rapid diagnostic tests (POCT), in order to protect the staff, the people who got tested, the community and the environment. Material and methods. Many techniques have been used so far for performing a risk assessment. In the present study, SWIFT analysis suitable for biosafety facilities and for risks of different magnitude, was used for identifying threats and hazards and to calculate the risks for COVID-19 testing facilities. Results. Our analysis showed several initial and potential risks, which could lead to unwanted exposure or release of the SARS-CoV-2, and/or unwanted infection of staff and patients. With minor adjustments of the testing facility, by creating standard operating procedures and awareness of the potential risks, most of the identified risks could be mitigated. Conclusions. Our study demonstrated that when setting up a COVID-19 testing facility, a proper risk assessment should be part of the process, in order to ensure the safety of staff, patients, and the environment. Additionally, we proposed a number of multiple mitigation measures and recommendations, with the goal to reduce the risks during the rapid testing diagnostic procedure.Introducere. COVID-19 este o boală infecțioasă cu un impact geografic larg răspândit și transmisibilitate ridicată, care poate provoacăboli grave. Încă de la debutul pandemiei de COVID-19 au fost înființate multe stații de testare pentru monitorizarea răspândirii virusu-lui SARS-CoV-2. Din punct de vedere al biosecurității acest studiu investighează o metodă de evaluare a riscurilor în vederea identificării și atenuării riscurilor stațiilor de testare COVID-19, care utilizează teste de diagnosticare rapidă (POCT) pentru a proteja personalul, pacienții, comunitatea și mediul. Material și metode. În prezentul studiu au fost aplicate diferite tehnici pentru realizarea unei evaluări a riscurilor. A fost utilizată analiza SWIFT pentru instalațiile de biosecuritate și pentru riscuri de diferită amploare pentru identificarea amenințărilor și pericolelor, și pentru a calcula riscurile pentru stațiile de testare COVID-19. Rezultate. Analiza noastră a identificat mai multe riscuri inițiale și potențiale, care ar putea duce la expunerea sau eliberarea nedorită a SARS-CoV-2 și/sau la infectarea nedorită a personalului și a pacienților. Cu ajustări minore ale stațiilor de testare, prin crearea de proceduri standard de operare și conștientizarea riscurilor potențiale, majoritatea riscurilor identificate ar putea fi atenuate. Concluzii. Prezentul studiu a demonstrat că atunci când se înființează o unitate de testare COVID-19, o evaluare adecvată a riscurilor ar trebui să facă parte din proces pentru a asigura siguranța personalului, a pacienților și a mediului. În plus, am propus o serie de măsuri și recomandări multiple de atenuare cu scopul de a reduce riscurile în timpul procedurii de diagnosticare a testării rapide

Reply to Ghirardello et al Letter to the Editor

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The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation

Oxidative phenomena are considered to lie at the root of the accelerated senescence observed in red blood cells (RBCs) stored under standard blood bank conditions. It was recently shown that the addition of uric (UA) and/or ascorbic acid (AA) to the preservative medium beneficially impacts the storability features of RBCs related to the handling of pro-oxidant triggers. This study constitutes the next step, aiming to examine the links between hemolysis, redox, and metabolic parameters in control and supplemented RBC units of different storage times. For this purpose, a paired correlation analysis of physiological and metabolism parameters was performed between early, middle, and late storage in each subgroup. Strong and repeated correlations were observed throughout storage in most hemolysis parameters, as well as in reactive oxygen species (ROS) and lipid peroxidation, suggesting that these features constitute donor-signatures, unaffected by the diverse storage solutions. Moreover, during storage, a general “dialogue” was observed between parameters of the same category (e.g., cell fragilities and hemolysis or lipid peroxidation and ROS), highlighting their interdependence. In all groups, extracellular antioxidant capacity, proteasomal activity, and glutathione precursors of preceding time points anticorrelated with oxidative stress lesions of upcoming ones. In the case of supplemented units, factors responsible for glutathione synthesis varied proportionally to the levels of glutathione itself. The current findings support that UA and AA addition reroutes the metabolism to induce glutathione production, and additionally provide mechanistic insight and footing to examine novel storage optimization strategies

Cellular study on transfused packed red blood cells

The aim of this PhD thesis was the cellular study of pRBCs intended to transfusion that have been stored in CPDA units and the further correlation of the results with relevant clinical studies. The results of the study were as follow: 1. The oxidation of the RBC is a part of storage lesion. 2. The hemoglobin is increasingly bound to the membrane and the cytoskeleton during storage. 3. Some of the main integral proteins of the RBC membrane and the lipid raft-associated leave the membrane, but other cytoplasmic ones become membrane-associated. 4. There is evidence of FAS oligomerization. 5. At the same time, proteolytic fragments representing active caspase-8 and -3 were evident. 6. RBCs lose their physiological shape during storage. From the membrane spikes of echino- and shero-echinocytes, microvesicles are released. 7. The microvesicles released in the units of pRBCs had diameter of 50-210nm. The microvesicles that were isolated from units stored for short periods (1-2 days) were smaller than those released after 35 days of storage (137±37nm, statistically important difference). The latter were characterized by tails, membrane gaps and formation of protein aggregates. All the microvesicles collected from "fresh" units and the majority of those collected on the last day of storage contained hemoglobin, so they were RBC-derived. 8. The degree of vesiculation was increased during storage. 9. The vesicles do not contain the skeletal proteins but have actin, band-3 and GpA. 10. By Western blot analysis the vesicles do not contain aquaporin, but contain the lipid raft associated proteins and proteins related to the membrane pathway of classical apoptosis. The vesicles contain proteins related to phagocytosis signaling events. 11. The proteins of the microvesicles were heavily carbonylated in relation to the RBC membrane of origin. In conclusion, the protein oxidation is a probably important part of the storage lesion. During storage, various mechanisms of cell signaling are activated and are mainly related to cell aging, cell clearance and, probably, programmed cell death. The stored RBCs-derived vesicles bear the same signaling components with their parent cells and, as a result, they probably are also post-transfusionally removed by the same mechanisms. In addition, the role of vesiculation seems to be at least of dual importance: a) one negative parameter is associated with the resulted permanent membrane loss, but b) another positive parameter is probably associated with the specific protein composition of the collected vesicles: since they contain harmful, oxidized and high components signaling potential their release during storage may prevent the premature removal or death of otherwise functional RBCs. Recent clinical studies, supporting the practice of using more "fresh" pRBCs for transfusion purposes, especially in critically affected patients, corroborated the results of the present study.Σκοπός της παρούσας διατριβής ήταν η κυτταροβιολογική μελέτη των συμπυκνωμένων ερυθροκυττάρων (ΣΕ) που προορίζονται για μετάγγιση προκειμένου να βρεθούν οι άριστες συνθήκες συντήρησης ex vivo που εξασφαλίζουν καλή βιωσιμότητα και λειτουργικότητα. Η μεθοδολογία περιλάμβανε απομόνωση και ανάλυση πρωτεϊνών των ΣΕ σε διάφορα χρονικά σημεία της αποθήκευσης με τεχνικές ηλεκτροφόρησης, ανοσοαποτυπώματος κατά Western και μικροσκοπίας (ηλεκτρονικής, φωτονικής και φθορισμού). Τα αποτελέσματα έδειξαν πως: 1. Η οξείδωση της μεμβράνης, του κυτταροσκελετού και του κυτταροπλάσματος είναι μέρος της αποθηκευτικής βλάβης των ΣΕ. 2. Η αιμοσφαιρίνη μετατοπίζεται από το κυτταρόπλασμα στη μεμβράνη αλλά και στον υπομεμβρανικό κυτταροσκελετό. 3. Οι κύριες πρωτεΐνες του ερυθροκυττάρου καθώς και εκείνες που συσχετίζονται με τις λιπιδικές σχεδίες εγκαταλείπουν τη μεμβράνη και άλλες φαίνεται να μετατοπίζονται από το κυτταρόπλασμα στη μεμβράνη. 4. Υπάρχουν ενδείξεις ενεργοποίησης του μεμβρανικού μονοπατιού της απόπτωσης. 5. Παρατηρήθηκαν πρωτεολυτικά θραύσματα των κασπασών-8 και -3 που αντιπροσωπεύουν τις ενεργοποιημένες μορφές τους. 6. Τα ΣΕ, καθώς αυξάνει ο χρόνος της αποθήκευσης μετασχηματίζονται αρχικά σε ακανθοκύτταρα και στη συνέχεια σε σφαιρο-ακανθοκύτταρα. 7. Τα μικροκυστίδια που απομονώθηκαν από τους ασκούς των ΣΕ είχαν διάμετρο από 50-210nm ενώ τα μικροκυστίδια που απομονώθηκαν από ασκούς αποθηκευμένους για μία ημέρα ήταν μικρότερα σε μέγεθος (137±37 nm) από αυτά που απομονώθηκαν στο τέλος της αποθηκευτικής περιόδου (35η ημέρα, 137±37nm). 8. Η κυστιδιοποίηση αυξάνει με την πάροδο του χρόνου αποθήκευσης. 9. Τα κυστίδια δεν περιέχουν σκελετικά συστατικά ενώ φέρουν ακτίνη, ζώνη-3 και γλυκοφορίνη-Α. 10. Πειράματα ανοσοεντόπισης έδειξαν την απουσία υδατοπορίνης στα κυστίδια, την παρουσία πρωτεϊνών που σχετίζονται με τις λιπιδικές σχεδίες, πρωτεΐνες που συμμετέχουν στο μεμβρανικό μονοπάτι της απόπτωσης και πρωτεΐνες που συμμετέχουν αρνητικά ή θετικά σε πορείες κυτταρικής εκκαθάρισης. 11. Οι κυστιδιακές πρωτεΐνες βρέθηκαν σε μεγάλο βαθμό καρβονυλιωμένες. Συμπερασματικά, τα αποτελέσματα αυτής της μελέτης περιέγραψαν τη σταδιακή αναδιαμόρφωση της ερυθροκυτταρικής μεμβράνης που λαμβάνει χώρα κατά την αποθήκευση. Η πρωτεϊνική οξείδωση φαίνεται να συνιστά σημαντικό καθοριστή της αποθηκευτικής βλάβης των ΣΕ. Κατά την αποθήκευση ενεργοποιούνται διάφοροι μηχανισμοί κυτταρικής σηματοδότησης, που σχετίζονται με τη γήρανση, την εκκαθάριση και ίσως τον προγραμματισμένο κυτταρικό θάνατο των ερυθρών. Τα κυστίδια που παράγονται στον ασκό φέρουν τα ίδια σηματοδοτικά συστατικά με τα κύτταρα προέλευσης τους. Επιπρόσθετα, ο ρόλος της κυστιδιοποίησης κατά την αποθήκευση φαίνεται να είναι διττός: αφενός μεν είναι αρνητικός καθώς προκαλεί μη-αντιστρεπτή μείωση της κυτταρικής επιφάνειας, από την άλλη όμως εξασφαλίζει στο ερυθροκύτταρο την απομάκρυνση οξειδωμένων και επιζήμιων συστατικών τα οποία θα προκαλούσαν τον πρώιμο θάνατο των ερυθροκυττάρων στους ασκούς της μετάγγισης. Τα αποτελέσματα της μελέτης αυτής επιβεβαιώθηκαν πρόσφατα από κλινικές μελέτες που υποστηρίζουν τη χρήση φρέσκων ασκών για μετάγγιση ειδικά σε ασθενείς που είναι ανοσοκατεσταλμένοι ή βρίσκονται σε μονάδες εντατικής θεραπείας

In Sickness and in Health: Erythrocyte Responses to Stress and Aging

Mature red blood cells (RBC) are the most abundant host cell in our body [...

Blood Cell-Derived Microvesicles in Hematological Diseases and beyond

Microvesicles or ectosomes represent a major type of extracellular vesicles that are formed by outward budding of the plasma membrane. Typically, they are bigger than exosomes but smaller than apoptotic vesicles, although they may overlap with both in size and content. Their release by cells is a means to dispose redundant, damaged, or dangerous material; to repair membrane lesions; and, primarily, to mediate intercellular communication. By participating in these vital activities, microvesicles may impact a wide array of cell processes and, consequently, changes in their concentration or components have been associated with several pathologies. Of note, microvesicles released by leukocytes, red blood cells, and platelets, which constitute the vast majority of plasma microvesicles, change under a plethora of diseases affecting not only the hematological, but also the nervous, cardiovascular, and urinary systems, among others. In fact, there is evidence that microvesicles released by blood cells are significant contributors towards pathophysiological states, having inflammatory and/or coagulation and/or immunomodulatory arms, by either promoting or inhibiting the relative disease phenotypes. Consequently, even though microvesicles are typically considered to have adverse links with disease prognosis, progression, or outcomes, not infrequently, they exert protective roles in the affected cells. Based on these functional relations, microvesicles might represent promising disease biomarkers with diagnostic, monitoring, and therapeutic applications, equally to the more thoroughly studied exosomes. In the current review, we provide a summary of the features of microvesicles released by blood cells and their potential implication in hematological and non-hematological diseases

Apolipoprotein J/Clusterin is a novel structural component of human erythrocytes and a biomarker of cellular stress and senescence.

BACKGROUND: Secretory Apolipoprotein J/Clusterin (sCLU) is a ubiquitously expressed chaperone that has been functionally implicated in several pathological conditions of increased oxidative injury, including aging. Nevertheless, the biological role of sCLU in red blood cells (RBCs) remained largely unknown. In the current study we identified sCLU as a component of human RBCs and we undertook a detailed analysis of its cellular topology. Moreover, we studied the erythrocytic membrane sCLU content during organismal aging, in conditions of increased organismal stress and accelerated RBCs senescence, as well as during physiological in vivo cellular senescence. METHODOLOGY/PRINCIPAL FINDINGS: By using a combination of molecular, biochemical and high resolution microscopical methods we found that sCLU is a novel structural component of RBCs extra- and intracellular plasma membrane and cytosol. We observed that the RBCs membrane-associated sCLU decreases during organismal aging or exposure to acute stress (e.g. smoking), in patients with congenital hemolytic anemia, as well as during RBCs in vivo senescence. In all cases, sCLU reduction paralleled the expression of typical cellular senescence, redox imbalance and erythrophagocytosis markers which are also indicative of the senescence- and oxidative stress-mediated RBCs membrane vesiculation. CONCLUSIONS/SIGNIFICANCE: We propose that sCLU at the mature RBCs is not a silent remnant of the erythroid precursors, but an active component being functionally implicated in the signalling mechanisms of cellular senescence and oxidative stress-responses in both healthy and diseased organism. The reduced sCLU protein levels in the RBCs membrane following cell exposure to various endogenous or exogenous stressors closely correlates to the levels of cellular senescence and redox imbalance markers, suggesting the usefulness of sCLU as a sensitive biomarker of senescence and cellular stress