Experimental study of the antigenicity of neural precursor cells of the central nervous system in C57BL/6 mice

Research studies focus on the autoimmunity, inflammation and demyelination in the nervous system aiming at developing innovative and more effective treatments. In multifactorial diseases, such as Multiple Sclerosis (MS), immunoregulatory treatments are administered, but treatments that can restore Central Nervous System (CNS) damage are quite limited. Neural Precursor Cells (NPCs) of CNS are under investigation as cell therapy, as they fulfill the characteristics of multipotency, self-renewal and differentiation. Studies of NPCs Transplantation in experimental models of MS, such as Experimental Autoimmune Encephalomyelitis (EAE), demonstrated clinical improvement of animals either by immunomodulation at the periphery or by remyelination through migration to the demyelinating areas. The existing immune disorder may lead to NPCs antigenic epitope recognition and to the production of autoantibodies or immune cells that target NPCs. Thus, the cross-talk between NPCs and the CNS immune mechanisms needs further investigation. The present dissertation focuses on the study of NPCs antigenicity and aims to answer whether their administration in normal mice may provoke immune responses, whether autoantibodies against NPCs niches are produced and whether unidentified NPCs antigenic epitopes are targeted. Initially, we identified the immunophenotype of undifferentiated NPCs isolated from newborn mice C57BL/6, for the expression of CD44, CD24 and CD133, verifying their properties of multipotency, self-renewal, migration and chemotaxis. NPCs were then subcutaneously inoculated to C57BL/6 mice and their effects were studied in comparison with mice injected with MOG, CFA and Νaive controls. The results demonstrated increased expression of genes related to antigen presentation, inflammation and chemotaxis, such as: CD80, CD86, CCL2, CCL5, CCL20, CXCL1, CXCL10, CXCL12, CXCR2, CXCR4 , IL17RA and TGF-β, in the brain. Additionally, immunohistochemical analysis of inflammatory markers: CD3, MAC-3 and B220, revealed induction of cellular and humoral immunity against NPCs niches, with a remarkable increase of macrophages in the subventricular zone (SVZ). Ten days after immunization, the presence of immune responses in the secondary lymphatic organs was verified by flow cytometry for the expression of CD11b, CD11c, B220, CD3, CD4, CD8, IFN-γ, IL-17, CD25 and FOXP3. Antisera from NPCs or MOG peptide immunized animals were analyzed and immunoreactivity against NPCs was identified. A dominant selective binding of antisera to SVZ of naive mice was observed, throughout the neonatal to adult stage. Antisera from NPCs inoculated animals targeted NPC antigenic epitopes, yielding specific bands on NPCs substrate. Proteomic and bioinformatic analysis of the aforementioned antigenic epitopes revealed over a thousand different set of proteins with at least two of them (protein CAR and CD166) having statistically significant sequence similarity with MOG protein. In vitro analysis of NPCs incubation with antisera showed that an impact on NPCs was exerted through activation of the apoptotic and/or autophagic pathways. In conclusion, the present thesis provided for the first time evidence of antigenic-immunogenic potential of NPCs, cells that are considered immune privileged and are proposed as a potential therapeutic tool for neurodegenerative disorders. The presence of a similar antigenic potential of human NPCs requires thorough investigation, contributing to better understanding of autoimmunity and the tight balance of CNS homeostasis.Ερευνητικές μελέτες των διαδικασιών αυτοανοσίας, φλεγμονής και απομυελίνωσης στο νευρικό σύστημα στοχεύουν στην ανάπτυξη καινοτόμων αποτελεσματικότερων θεραπειών. Σε πολυπαραγοντικές ασθένειες, όπως η Πολλαπλή Σκλήρυνση (ΠΣ), ανοσορυθμιστικές θεραπείες ήδη εφαρμόζονται, αλλά είναι περιορισμένες οι θεραπείες που δύνανται να αποκαταστήσουν τις βλάβες του Κεντρικού Νευρικού Συστήματος (ΚΝΣ) στους ασθενείς. Τα Νευρικά Προγονικά Κύτταρα (ΝΠΚ) του ΚΝΣ αποτελούν αντικείμενο μελέτης ως υποψήφια κυτταρική θεραπεία, καθώς πληρούν χαρακτηριστικά πολυδυναμίας, αυτοανανέωσης και ικανότητας διαφοροποίησης. Η μεταμόσχευσή τους σε πειραματικά μοντέλα προσομοίωσης της ΠΣ, όπως η Πειραματική Αυτοάνοση Εγκεφαλομυελίτιδα (ΠΑΕ), έδειξε κλινική βελτίωση των πειραματοζώων είτε μέσω δράσης στην περιφέρεια, είτε επιτελώντας επαναμυελίνωση μέσω μετανάστευσης στις παθολογικές εστίες. Η υπάρχουσα ανοσιακή διαταραχή ενδεχομένως οδηγεί σε αναγνώριση αντιγονικών επιτόπων των ΝΠΚ και σε παραγωγή ειδικών αντισωμάτων ή ανοσιακών κυττάρων έναντι των ΝΠΚ. Η γενικότερη συσχέτισή τους με το ΚΝΣ και με τους ανοσιακούς μηχανισμούς που ενεργοποιούνται χρήζουν περαιτέρω διερεύνησης. Η παρούσα διδακτορική διατριβή εστιάζει στη μελέτη της αντιγονικότητας των ΝΠΚ, δηλαδή εάν η χορήγησή τους στην περιφέρεια σε φυσιολογικούς μύες δύναται να προκαλέσει ανοσιακή αντίδραση εντός και εκτός του ΚΝΣ, εάν επάγεται παραγωγή αυτοαντισωμάτων έναντι θώκων νευρογένεσης του ΚΝΣ και εάν υπάρχουν άγνωστοι αντιγονικοί επίτοποι των ΝΠΚ που πιθανώς αποτελούν στόχο των αυτοαντισωμάτων. Αρχικά, διενεργήθη χαρακτηρισμός των απομονωμένων ΝΠΚ από εγκεφάλους νεογνών μυών C57BL/6, για έκφραση CD44, CD24 και CD133 και επαληθεύτηκαν οι ιδιότητες πολυδυναμίας, αυτοανανέωσης, μετανάστευσης και χημειοταξίας. Κατόπιν χορηγήθηκαν σε φυσιολογικούς μύες της φυλής C57BL/6 και μελετήθηκε η επίδραση τους, σε σύγκριση με μύες που έλαβαν MOG, CFA και φυσιολογικούς μάρτυρες. Τα αποτελέσματα κατέδειξαν αυξημένη έκφραση, κυρίως στον εγκέφαλο, γονιδίων που σχετίζονται με αντιγονοπαρουσίαση, φλεγμονή και χημειοταξία όπως: CD80, CD86, CCL2, CCL5, CCL20, CXCL1, CXCL10, CXCL12, CXCR2, CXCR4, IL17RA και ΤGF-β. Επιπροσθέτως, η ανοσοϊστοχημική μελέτη δεικτών φλεγμονής: CD3, MAC-3 και B220 φανέρωσε κινητοποίηση κυτταρικής και χυμικής ανοσίας έναντι περιοχών όπου εδράζουν ΝΠΚ, με αξιοσημείωτη την υπέρμετρη αύξηση μακροφάγων κατά τη χρόνια φάση στην υποκοιλιακή ζώνη (SVZ). Η ανάλυση των δευτερογενών λεμφικών οργάνων για έκφραση των δεικτών CD11b, CD11c, B220, CD3, CD4, CD8, IFN-γ, IL-17, CD25 και FOXP3, κατά την 10η ημέρα κατόπιν ανοσοποίησης, επαλήθευσε την κινητοποίηση της ανοσίας. Έπειτα, μελετήθηκαν αντιοροί που προέκυψαν από τα ανοσοποιημένα με ΝΠΚ ή MOG πειραματόζωα και διαπιστώθηκε ανοσοδραστικότητα έναντι ΝΠΚ με εκλεκτικότητα σύνδεσης σε κυτταρικούς πληθυσμούς της SVZ φυσιολογικών μυών τριών διαφορετικών ηλικιών. Οι αντιοροί από τα ανοσοποημένα με ΝΠΚ πειραματόζωα εμφάνισαν επαναληψιμότητα αναγνώρισης πρωτεϊνικών ζωνών σε υπόστρωμα ΝΠΚ, στοχεύοντας συγκεκριμένους αντιγονικούς επιτόπους. Η πρωτεομική και βιοπληροφορική επεξεργασία των παραπάνω πιθανών αντιγονικών επιτόπων, κατέδειξαν πάνω από χίλιες πρωτεΐνες, με τουλάχιστον δύο (πρωτεΐνη CAR και CD166) να εμφανίζουν στατιστικά σημαντικό ποσοστό ομολογίας με τη MOG. Οι αντιοροί μετά από in vitro μελέτη επίδρασης στα ΝΠΚ, ενεργοποίησαν μονοπάτια απόπτωσης ή/και αυτοφαγίας. Συμπερασματικά, η παρούσα ερευνητική προσέγγιση κατέδειξε για πρώτη φορά την πιθανή ύπαρξη αντιγονικού δυναμικού στα ΝΠΚ, κύτταρα τα οποία μέχρι σήμερα θεωρούνται μη ανοσογονικά και έχουν προταθεί ως κυτταρική θεραπεία σε νευροεκφυλιστικές παθήσεις. Η παρουσία αντίστοιχου αντιγονικού δυναμικού στα ανθρώπινα ΝΠΚ επιβάλλεται να διερευνηθεί για αποσαφήνιση του φαινομένου της αυτοανοσίας και της λεπτής ομοιοστατικής ισορροπίας στο ΚΝΣ

N-Acetylcysteine Administration Attenuates Sensorimotor Impairments Following Neonatal Hypoxic-Ischemic Brain Injury in Rats

Hypoxic ischemic (HI) brain injury that occurs during neonatal period has been correlated with severe neuronal damage, behavioral deficits and infant mortality. Previous evidence indicates that N-acetylcysteine (NAC), a compound with antioxidant action, exerts a potential neuroprotective effect in various neurological disorders including injury induced by brain ischemia. The aim of the present study was to investigate the role of NAC as a potential therapeutic agent in a rat model of neonatal HI brain injury and explore its long-term behavioral effects. To this end, NAC (50 mg/kg/dose, i.p.) was administered prior to and instantly after HI, in order to evaluate hippocampal and cerebral cortex damage as well as long-term functional outcome. Immunohistochemistry was used to detect inducible nitric oxide synthase (iNOS) expression. The results revealed that NAC significantly alleviated sensorimotor deficits and this effect was maintained up to adulthood. These improvements in functional outcome were associated with a significant decrease in the severity of brain damage. Moreover, NAC decreased the short-term expression of iNOS, a finding implying that iNOS activity may be suppressed and that through this action NAC may exert its therapeutic action against neonatal HI brain injury

The Role of Diet and Interventions on Multiple Sclerosis: A Review

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by inflammation and neurodegeneration. The most prominent clinical features include visual loss and sensorimotor symptoms and mainly affects those of young age. Some of the factors affecting its pathogenesis are genetic and/or environmental including viruses, smoking, obesity, and nutrition. Current research provides evidence that diet may influence MS onset, course, and quality of life of the patients. In this review, we address the role of nutrition on MS pathogenesis as well as dietary interventions that show promising beneficial results with respect to MS activity and progression. Investigation with large prospective clinical studies is required in order to thoroughly evaluate the role of diet in MS

Argininemia: Pathophysiology and Novel Methods for Evaluation of the Disease

Argininemia or arginase-1 deficiency constitutes a rare, genetic, metabolic disorder caused by mutations in arginase 1—the last enzyme of the urea cycle—that hydrolyses L-arginine to ornithine and urea. The disease is associated with progressive development of spasticity and other symptoms, including seizures, developmental delay, cognitive impairment, and hepatic pathology. The present review attempts to summarize the current knowledge on the pathophysiology of the disease and highlight novel methods for its evaluation. Different factors, such as the accumulation of arginine, ammonia, and guanidino compounds, act as neurotoxins and may account for the neurological sequelae observed in the disease. New markers, such as arginine/ornithine ratio along with metabolomics, machine learning algorithms, and genetic methods, can be useful in the early diagnosis of argininemia, while mobile phone apps can assist argininemic patients in adhering to the strict diet required. Neurophysiology, multi-modal imaging, and new modelling methods, such as induced pluripotent stem cells, hold promise for providing new insights into the pathophysiology of the disease. There are still many uncertainties regarding the underlying mechanisms of argininemia, but the use of novel modelling methods and new technology can lead to the decipherment of its pathophysiology, improvement of diagnostic accuracy, and better disease management

Additional file 1: of Humoral response in experimental autoimmune encephalomyelitis targets neural precursor cells in the central nervous system of naive rodents

Methods. Experimental groups, EAE induction and evaluation, Cell culture protocol, Preparation of samples for SDS-PAGE. (RTF 3.45 kb

Microbiome in Multiple Sclerosis: Where Are We, What We Know and Do Not Know

An increase of multiple sclerosis (MS) incidence has been reported during the last decade, and this may be connected to environmental factors. This review article aims to encapsulate the current advances targeting the study of the gut–brain axis, which mediates the communication between the central nervous system and the gut microbiome. Clinical data arising from many research studies, which have assessed the effects of administered disease-modifying treatments in MS patients to the gut microbiome, are also recapitulated

Stress hormones kinetics in ventricular fibrillation cardiac arrest and resuscitation: Translational and therapeutic implications

Background: Knowing the kinetics of endogenous stress hormones during cardiac arrest and cardiopulmonary resuscitation (CRP) will help to optimize personalized physiology-guided treatment. The aim of this study was to examine the dynamic changes in stress hormones in a swine model of ventricular fibrillation (VF) cardiac arrest. Methods: Ventricular fibrillation was induced in 10 healthy Landrace/Large White piglets, which were subsequently left untreated for 8 min. All animals were resuscitated according to the 2015 European Resuscitation Council guidelines. The concentration of adrenalin, noradrenalin, and cortisol was measured at baseline and at the 4th and 8th minute of VF-cardiac arrest, as well as at 30-min, 60-min, 24 h and 48 h post-ROSC. Results: By the end of the 4th min of VF, the animals of the ROSC group exhibited significantly higher adrenaline levels compared to those of the no-ROSC group (7264 pg/ml vs. 1648 pg/ml. p - 0.03). Noradrenaline was higher in the ROSC group at the 4th min of VI (3021 pg/ml vs. 1626 pg/ml, p = 0.02). Cortisol levels in the ROSC group were significantly lower by the end of the 8th min of VF[16.25 ng/ml vs. 92.82 ng/ml, p 0.031. With a cut-off point of 5970 pg/ml, adrenaline at the 4th min of VF exhibited 100% sensitivity and 80% specificity for predicting ROSC. Conclusion: Higher endogenous adrenaline and lower endogenous cortisol levels were associated with ROSC. (C) 2021 Elsevier Inc. All rights reserved