Επιδράσεις μεταλλαγών της αιμοσφαιρίνης στην πρωτεόσταση ερυθροκυττάρων κατά τη γήρανση

Τα ερυθροκύτταρα κατά την αποθήκευση τους υφίστανται μία σειρά αλλοιωτικών αλλαγών που αφορούν βιοχημικά και μορφολογικά τους χαρακτηριστικά και αναφέρονται με τον όρο αποθηκευτική βλάβη. Η διατήρηση της λειτουργικότητας του κυττάρου και η διασφάλιση της ακεραιότητας της μεμβράνης του προϋποθέτει την ενεργοποίηση μίας σειράς προστατευτικών μηχανισμών, που έγκεινται στην εξουδετέρωση των ROS, την επιδιόρθωση ή καταστροφή αλλοιωμένων μορίων (π.χ. οξειδωμένες πρωτεΐνες) ή τέλος την «θυσία» αυτών, μέσω κυστιδιοποίησης. Το 20S πρωτεάσωμα, ένα κεντρικό πρωτεολυτικό σύμπλοκο εξοπλισμένο με ενεργότητες κασπάσης, θρυψίνης και χυμοθρυψίνης, το οποίο έχει βρεθεί σε περίπου 20πλάσια αφθονία από το 26S πρωτεάσωμα στα ερυθροκύτταρα, αποτελεί μέρος των μηχανισμών καταστροφής πρωτεϊνών που έχουν υποστεί βλάβη σε αυτά. Στόχος της παρούσας διπλωματικής εργασίας είναι η διερεύνηση της λειτουργίας και της τοπολογίας του πρωτεασώματος στα ερυθροκύτταρα δύο ομάδων αιμοδοτών, η πρώτη αποτελούμενη από 9 ετερόζυγους φορείς β-θαλασσαιμίας και η δεύτερη από 10 αιμοδότες μάρτυρες. Μελετήθηκαν τόσο δείγματα φρέσκου αίματος όσο και αποθηκευμένων ερυθροκυττάρων σε CPD-SAGM σε εβδομαδιαία βάση μέχρι το τέλος της αποθήκευσης. Σε αυτά μετρήθηκαν τα επίπεδα και τριών πρωτεασωμικών ενεργοτήτων σε δείγματα κυτοσολίων και μεμβρανών και τα επίπεδα των ενδογενών και επαγόμενων επιπέδων ROS με φθορισμομετρία. Επιπλέον, εκτελέσθηκε ανοσοαποτύπωση κατά Western για την εύρεση πρωτεϊνών σχετιζόμενων με στρες στις μεμβράνες των ερυθροκυττάρων των αιμοδοτών και μέτρηση των επιπέδων των καρβονυλιωμένων πρωτεϊνών στη μεμβράνη με τη μέθοδο Oxyblot. Τέλος, πραγματοποιήθηκε εστιασμένη στο πρωτεάσωμα ανάλυση και σύγκριση δικτύων συσχετίσεων μεμβρανικών και κυστιδιακών παραμέτρων των δύο ομάδων στο τέλος της αποθήκευσης. Κατόπιν ανάλυσης των δεδομένων, παρατηρήθηκε στο σύνολο των αιμοδοτών μία χωροεξαρτώμενη αλλαγή στα επίπεδα των πρωτεασωμικών ενεργοτήτων. Συγκεκριμένα, σημειώθηκε πτώση αυτών στα δείγματα των κυτοσολίων και αύξηση στα δείγματα των μεμβρανών, που μαρτυρά μία μετατόπιση του συμπλόκου προς τη μεμβράνη κατά τη διάρκεια της αποθήκευσης. Ακόμη, η ομάδα των ετερόζυγων παρουσίασε υψηλότερες, στατιστικά σημαντικές τιμές ενεργοτήτων χυμοθρυψίνης και κασπάσης στη μεμβράνη στο δεύτερο μισό της αποθήκευσης έναντι των μαρτύρων. Παράλληλα, οι ετερόζυγοι εμφάνισαν υψηλές τιμές συσχέτισης των ενεργοτήτων με τα επαγόμενα από οξειδωτικούς παράγοντες επίπεδα ROS στο μέσο και το τέλος της αποθήκευσης. Οι ενδείξεις για αυξημένη και αποτελεσματική λειτουργία του πρωτεασώματος στην ομάδα αυτή ενισχύονται περαιτέρω από το γεγονός ότι τα επίπεδα καρβονυλίωσης πρωτεϊνών στις μεμβράνες των ετερόζυγων ήταν σημαντικά χαμηλότερα από αυτά των μαρτύρων. Τα παραπάνω αποτελέσματα υποστηρίζουν την πρόταση πως τα ερυθροκύτταρα των ετερόζυγων φορέων β-θαλασσαιμίας είναι πιο αποτελεσματικά στην αντιμετώπιση της αποθηκευτικής βλάβης χάρη στην ήπια οξειδωτική πίεση που δέχονται in vivo. Τέλος, το κομμάτι της εργασίας που αφορούσε τη σύγκριση δικτύων και το πώς κυρίως πρωτεασωμικές παράμετροι της μεμβράνης επηρεάζουν το φαινότυπο των κυστιδίων ανέδειξε διαφορές στο πλήθος και το είδος των διασυνδέσεων. Οι ετερόζυγοι παρουσίασαν αφενός περισσότερες συσχετίσεις και αφετέρου διαφοροποιημένο πρότυπο πυρήνωσης αυτών. Στην ομάδα των μαρτύρων οι περισσότερες διασυνδέσεις αφορούσαν σκελετικά και σχετικά με G-πρωτεΐνες χαρακτηριστικά, ενώ στους ετερόζυγους αφορούσαν διαφορετικές παραμέτρους G-πρωτεϊνών και σκελετικών στοιχείων, αντιοξειδωτικούς μηχανισμούς αλλά και ορισμένες βασικές αιματολογικές παραμέτρους. Το γεγονός αυτό μαρτυρά πως το πρωτεάσωμα στους 73 δεύτερους κατέχει έναν κεντρικότερο ρόλο όσον αφορά στη διαδικασία διαλογής πρωτεϊνών κατά την κυστιδιοποίηση.During storage in the cold, the erythrocytes undergo a series of defects and alterations pertinent to their biochemical and morphological characteristics that are collectively referred to as “storage lesion”. The safe-keeping of RBC functionality and membrane homeostasis require the activation of protective mechanisms that neutralize ROS, repair or destroy affected molecules (i.e. oxidized proteins) or "sacrifice" these molecules via vesiculation. The 20S proteasome, a supermolecular proteolytic complex, which is equipped with caspase-like, chymothrypsin-like and thrypsin-like activities and has been reported in a 20fold abundance compared to the 26S proteasome in erythrocytes, plays an important role in the disposal of oxidized and misfolded proteins. The aim of this Bachelor Thesis is the study of the proteasome's function and topology in the erythrocytes of two groups of blood donors, one that consisted of heterozygotes for β-thalassaemia (n=9) and one of controls (n=10). Samples used for assays included both fresh blood and CPD-SAGM units of RBCs, tested on a weekly basis. Proteasomal activities in cytosol and membrane fractions, along with intrinsic and induced ROS levels were measured by fluorescence assays, while immunoblotting and Oxyblot analyses were performed in order to assess differences in the levels of specific stress-related proteins in the RBC membranes. Lastly, a network analysis focused on proteasome components was performed for membrane and vesicle parameters in both groups at the end of the storage period. After analyzing the collected measurements, an overall fraction-dependent change in all three proteasomal activities was observed; namely a decrease in cytosol samples and an increase in membrane samples that suggest a transfer to the latter during storage. Moreover, the erythrocytes of heterozygotes exhibited significantly higher levels of caspase-like and chymothrypsin-like activities in middle and late storage compared to controls. They also exhibited high values of the R2 correlation coefficient between proteasomal activities and ROS levels, induced or otherwise, during the second half of storage, contrary to controls. The proteasome's higher efficiency in this group was further corroborated by the fact that the Protein Carbonylation Index's values in the heterozygotes' membranes during the same period were significantly lower than those of controls. These findings support the hypothesis that erythrocytes from heterozygotes for β-thalassaemia cope better with oxidative stress during storage, thanks to the low, sustained oxidative pressure they experience in vivo. Finally, the part of this thesis that concerns the networks of membrane and vesicle parameters revealed that heterozygotes exhibit more correlations and a different pattern of hub-formation compared to controls. In the control network, most correlations were noted between proteasomal subunits and skeletal or G-protein-related parameters, while, in the heterozygotes' network, most correlations were observed between the proteasome and a number of anti-oxidant and different skeletal and G-protein-related components, along with some hematological parameters. These differences suggest that the role of the proteasome in the process of protein sorting for vesiculation might be of higher importance for this group

Red Blood Cell Proteasome in Beta-Thalassemia Trait: Topology of Activity and Networking in Blood Bank Conditions

Proteasomes are multi-catalytic complexes with important roles in protein control. Their activity in stored red blood cells (RBCs) is affected by both storage time and the donor’s characteristics. However, apart from their abundancy in the membrane proteome, not much is known about their topology, activity, and networking during the storage of RBCs from beta-thalassemia trait donors (beta Thal(+)). For this purpose, RBC units from fourteen beta Thal(+) donors were fractionated and studied for proteasome activity distribution and interactome through fluorometric and correlation analyses against units of sex- and aged-matched controls. In all the samples examined, we observed a time-dependent translocation and/or activation of the proteasome in the membrane and a tight connection of activity with the oxidative burden of cells. Proteasomes were more active in the beta Thal(+) membranes and supernatants, while the early storage networking of 20S core particles and activities showed a higher degree of connectivity with chaperones, calpains, and peroxiredoxins, which were nonetheless present in all interactomes. Moreover, the beta Thal(+) interactomes were specially enriched in kinases, metabolic enzymes, and proteins differentially expressed in beta Thal(+) membrane, including arginase-1, piezo-1, and phospholipid scramblase. Overall, it seems that beta Thal(+) erythrocytes maintain a considerable “proteo-vigilance” during storage, which is closely connected to their distinct antioxidant dynamics and membrane protein profile

Corpuscular Fragility and Metabolic Aspects of Freshly Drawn Beta-Thalassemia Minor RBCs Impact Their Physiology and Performance Post Transfusion: A Triangular Correlation Analysis In Vitro and In Vivo

The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor–recipient matching optimization. We hereby propose a “triangular” strategy for studying the links comprising the transfusion chain—donor, blood product, recipient—as exemplified in two cohorts of control and beta-thalassemia minor (βThal+) donors (n = 18 each). It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules (e.g., phosphocreatine) and purine metabolism factors (IMP, hypoxanthine) were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed “triangularity model” can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes

The Post-Storage Performance of RBCs from Beta-Thalassemia Trait Donors Is Related to Their Storability Profile

Blood donors with beta-thalassemia traits (βThal+) have proven to be good “storers”, since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored βThal+ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted βThal+ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that βThal+ RBCs present a marginal trend for higher recovery, regardless of the recipient’s immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored βThal+ RBCs. Overall, it seems that the intrinsic physiology of βThal+ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials

Epidemiology and outcomes of hospital-acquired bloodstream infections in intensive care unit patients: the EUROBACT-2 international cohort study

Purpose In the critically ill, hospital-acquired bloodstream infections (HA-BSI) are associated with significant mortality. Granular data are required for optimizing management, and developing guidelines and clinical trials. Methods We carried out a prospective international cohort study of adult patients (≥ 18 years of age) with HA-BSI treated in intensive care units (ICUs) between June 2019 and February 2021. Results 2600 patients from 333 ICUs in 52 countries were included. 78% HA-BSI were ICU-acquired. Median Sequential Organ Failure Assessment (SOFA) score was 8 [IQR 5; 11] at HA-BSI diagnosis. Most frequent sources of infection included pneumonia (26.7%) and intravascular catheters (26.4%). Most frequent pathogens were Gram-negative bacteria (59.0%), predominantly Klebsiella spp. (27.9%), Acinetobacter spp. (20.3%), Escherichia coli (15.8%), and Pseudomonas spp. (14.3%). Carbapenem resistance was present in 37.8%, 84.6%, 7.4%, and 33.2%, respectively. Difficult-to-treat resistance (DTR) was present in 23.5% and pan-drug resistance in 1.5%. Antimicrobial therapy was deemed adequate within 24 h for 51.5%. Antimicrobial resistance was associated with longer delays to adequate antimicrobial therapy. Source control was needed in 52.5% but not achieved in 18.2%. Mortality was 37.1%, and only 16.1% had been discharged alive from hospital by day-28. Conclusions HA-BSI was frequently caused by Gram-negative, carbapenem-resistant and DTR pathogens. Antimicrobial resistance led to delays in adequate antimicrobial therapy. Mortality was high, and at day-28 only a minority of the patients were discharged alive from the hospital. Prevention of antimicrobial resistance and focusing on adequate antimicrobial therapy and source control are important to optimize patient management and outcomes

The role of centre and country factors on process and outcome indicators in critically ill patients with hospital-acquired bloodstream infections

Purpose: The primary objective of this study was to evaluate the associations between centre/country-based factors and two important process and outcome indicators in patients with hospital-acquired bloodstream infections (HABSI). Methods: We used data on HABSI from the prospective EUROBACT-2 study to evaluate the associations between centre/country factors on a process or an outcome indicator: adequacy of antimicrobial therapy within the first 24 h or 28-day mortality, respectively. Mixed logistical models with clustering by centre identified factors associated with both indicators. Results: Two thousand two hundred nine patients from two hundred one intensive care units (ICUs) were included in forty-seven countries. Overall, 51% (n = 1128) of patients received an adequate antimicrobial therapy and the 28-day mortality was 38% (n = 839). The availability of therapeutic drug monitoring (TDM) for aminoglycosides everyday [odds ratio (OR) 1.48, 95% confidence interval (CI) 1.03-2.14] or within a few hours (OR 1.79, 95% CI 1.34-2.38), surveillance cultures for multidrug-resistant organism carriage performed weekly (OR 1.45, 95% CI 1.09-1.93), and increasing Human Development Index (HDI) values were associated with adequate antimicrobial therapy. The presence of intermediate care beds (OR 0.63, 95% CI 0.47-0.84), TDM for aminoglycoside available everyday (OR 0.66, 95% CI 0.44-1.00) or within a few hours (OR 0.51, 95% CI 0.37-0.70), 24/7 consultation of clinical pharmacists (OR 0.67, 95% CI 0.47-0.95), percentage of vancomycin-resistant enterococci (VRE) between 10% and 25% in the ICU (OR 1.67, 95% CI 1.00-2.80), and decreasing HDI values were associated with 28-day mortality. Conclusion: Centre/country factors should be targeted for future interventions to improve management strategies and outcome of HABSI in ICU patients

Presentation, management, and outcomes of older compared to younger adults with hospital-acquired bloodstream infections in the intensive care unit: a multicenter cohort study

Purpose: Older adults admitted to the intensive care unit (ICU) usually have fair baseline functional capacity, yet their age and frailty may compromise their management. We compared the characteristics and management of older (≥ 75 years) versus younger adults hospitalized in ICU with hospital-acquired bloodstream infection (HA-BSI). Methods: Nested cohort study within the EUROBACT-2 database, a multinational prospective cohort study including adults (≥ 18 years) hospitalized in the ICU during 2019-2021. We compared older versus younger adults in terms of infection characteristics (clinical signs and symptoms, source, and microbiological data), management (imaging, source control, antimicrobial therapy), and outcomes (28-day mortality and hospital discharge). Results: Among 2111 individuals hospitalized in 219 ICUs with HA-BSI, 563 (27%) were ≥ 75 years old. Compared to younger patients, these individuals had higher comorbidity score and lower functional capacity; presented more often with a pulmonary, urinary, or unknown HA-BSI source; and had lower heart rate, blood pressure and temperature at presentation. Pathogens and resistance rates were similar in both groups. Differences in management included mainly lower rates of effective source control achievement among aged individuals. Older adults also had significantly higher day-28 mortality (50% versus 34%, p < 0.001), and lower rates of discharge from hospital (12% versus 20%, p < 0.001) by this time. Conclusions: Older adults with HA-BSI hospitalized in ICU have different baseline characteristics and source of infection compared to younger patients. Management of older adults differs mainly by lower probability to achieve source control. This should be targeted to improve outcomes among older ICU patients