Researching glutamate – induced cytotoxicity in different cell lines: a comparative/collective analysis/study

Although glutamate is one of the most important excitatory neurotransmitters of the central nervous system, its excessive extracellular concentration leads to uncontrolled continuous depolarization of neurons, a toxic process called, excitotoxicity. In excitotoxicity glutamate triggers the rise of intracellular Ca2+ influx, followed by up regulation of nNOS, dysfunction of mitochondria, ROS production, ER stress and release of lysosomal enzymes. Excessive calcium concentration is the key mediator of glutamate toxicity through over activation of ionotropic and metabotropic receptors. In addition, glutamate accumulation can also inhibit cystine uptake by reversing the action of the cystine/glutamate antiporter. Reversal of the antiporter action reinforces the aforementioned events by depleting neurons of cysteine and eventually glutathione’s reducing potential. Various cell lines have been employed in the pursuit to understand the mechanism(s) by which excitotoxicity affects the cells leading them ultimately to their demise. In some cell lines glutamate toxicity is exerted mainly through over activation of NMDA, AMPA or Kainate receptors whereas in other cell lines lacking such receptors, the toxicity is due to glutamate induced oxidative stress. However in the greatest majority of the cell lines ionotropic-glutamate receptors are present, co-existing to cystine/glutamate antiporters and metabotropic glutamate receptors, supporting the assumption that excitotoxicity effect in these cells is accumulative. Different cell lines differ in their responses when exposed to glutamate. In this review article the responses of PC12, SH-SY5Y, HT-22, NT-2, OLCs, C6, primary rat cortical neurons, RGC-5 and SCN2.2 cell systems are systematically collected and analyzed

Influence of conception and delivery mode on stress response marker Oct4B1 and imprinted gene expression related to embryo development: A cohort study

Background: Recent scientific data support that the mode of conception and delivery may influence epigenetic regulation and therefore embryo development. Octamer-binding transcription factor 4-B1 (OCT4B1), a novel variant of OCT4 with yet unknown biological function, is suggested to have a potential role in mediating cellular stress response. Furthermore, Insulinlike Growth Factor 2 (IGF2), Mesoderm-specific Transcript (MEST) and paternally expressed gene 10 (PEG10) are genes known as imprinted and are regulated via means of epigenetic regulation. The influence of delivery mode and conception on epigenetic regulation is an active research field. Objective: Our aim was to correlate the expression level of Oct4B1 and the expression and methylation level of IGF2, MEST, and PEG10 imprinted genes with the mode of delivery and conception in the umbilical cord blood of newborns. Materials and Methods: Samples of umbilical cord blood from infants born after vaginal delivery, caesarean section (CS) with the infant in cephalic position and CS due to breech position were examined. Furthermore, the investigation included infants conceived through means of assisted reproductive technology. Results: No statistically significant differences were found in mRNA expression levels between different modes of conception and delivery (p = 0.96). Oct4B1, IGF2, MEST, and PEG10 expression levels do not seem to be significantly affected by different modes of conception and delivery. Conclusion: These results indicate that the expression and methylation patterns of Oct4B1, IGF2, MEST and PEG10 in umbilical cord blood are not affected by the conception and delivery mode. Key words: Conception, Fertilization in vitro, Genomic imprinting, Fetal blood

Design of a laboratory bioreactor for engineering articular cartilage based on 3D printed nasal septum-like scaffolds

«Η εκφύλιση των χόνδρων είναι μια σοβαρή πάθηση που επηρεάζει μεγάλο μέρος του πληθυσμού σε όλο το ηλικιακό φάσμα. Επί του παρόντος χρησιμοποιούνται διάφορες τεχνικές αποκατάστασης για μικρής έκτασης βλάβες όπως η αρθροπλαστική απόξεσης και ο υποχονδρικός τρυπανισμός, οι οποίες δεν μπορούν να επιδιορθώσουν βλάβες μεγαλύτερης έκτασης. Η Αναγεννητική Ιατρική προωθεί την Μηχανική Ιστών στο προσκήνιο των σύγχρονων μηχανικών τεχνικών, συνδυάζοντας καινοτόμα βιοσυμβατά υλικά, νέες μεθόδους μηχανικής ιστών όπως η τεχνολογία 3D εκτύπωσης και βιοδιαδικασίες που αποσκοπούν στην δημιουργία ποιοτικών μοσχευμάτων για εκτεταμένες βλάβες των χόνδρων. Κατάλληλο κυτταρικό περιβάλλον για δημιουργία ιστών μπορεί να επιτευχθεί με την ανάπτυξη αυτών των μοσχευμάτων σε βιοαντιδραστήρες. O κάθε βιοαντιδραστήρας χρησιμοποιεί διαφορετικές αρχές καλλιέργειας, και ορισμένοι από αυτούς όπως οι μικτού τύπου και οι βιοαντιδραστήρες διαπότισης επιστρατεύουν την άσκηση μηχανικών δυνάμεων επί του ικριώματος ώστε να επιτευχθεί μεγάλη κυτταρική πυκνότητα και ενισχυμένες μηχανικές ιδιότητες που οδηγούν στην δημιουργία καλύτερης ποιότητας χόνδρου. Αυτές οι ιδιαιτερότητες των βιοαντιδραστήρων μπορούν να αποτελέσουν εφαλτήριο κατασκευής εργαστηριακών βιοαντιδραστήρων, για την καλλιέργεια 3D εκτυπωμένων ρινικών διαφραγμάτων ως ένα λειτουργικό παράδειγμα υαλώδους χόνδρου».Cartilage degeneration is a severe disease affecting a significant part of the population at all ages. Various treatment modalities are currently used for small-sized cartilage defects, such as abrasion arthroplasty and subchondral drilling, but fail to repair larger-scale damages. Regenerative Medicine pushes Tissue Engineering (TE) to the forefront of modern engineering techniques combining novel biocompatible materials, new tissue engineering methods, like 3D printing technology and bioprocesses trying to create quality transplants for large cartilage defects. The appropriate cell environment for engineered tissues can be achieved through growth of the tissue-engineered constructs into bioreactors. Each bioreactor uses different principles for culturing processes, and some of them mostly mixed and perfusion bioreactors, use different kind of mechanical forces on the scaffold to achieve high cell densities, enhanced mechanical properties leading to better quality of engineered cartilage. These advantageous particularities can be used to create a laboratory bioreactor design, for culturing 3D printed nasal septum cartilage as a working example of hyaline cartilage

Translational research for nasal septum cartilage regeneration with chondrocytes derived from differentiated human adipose mesenchymal stem cells

Η εργασία αφορά στη μεταφραστική έρευνα ιστοτεχνολογίας και συγκεκριμένα στη δημιουργία ανθρώπινου ρινικού διαφράγματος με τη χρήση ηλεκτρονικά υποβοηθούμενου σχεδιασμού και τρισδιάστατης εκτύπωσης τρισδιάστατου (3D) πορώδους ικριώματος χιτοζάνης/ζελατίνης (CAD/CAM). Το ικρίωμα θα χρησιμοποιηθεί για να αποικιστεί από χρονδροκύτταρα που προκύπτουν από διαφοροποιημένα μεσεγχυματικά κύτταρα ανθρώπου προερχόμενα από λιπώδη ιστό (Adipose Tissue Mesenchymal Stem Cells-AD- MSCs). Η όλη διαδικασία επιτυγχάνεται με τη χρήση βιοαντιδραστήρα.Τα μεσεγχυματικά κύτταρα είναι πολυδύναμα βλαστοκύτταρα που μπορούν να απομονωθούν από το μυελό των οστώνκαι το λιπώδη ιστό. Τα κύτταρα αυτά έχουν τη δυνατότητα να διαφοροποιούνται, υπό εργαστηριακές συνθήκες, σε οστεοκύτταρα, χονδροκύτταρα, και λιποκύτταρα. Στην παρούσα μελέτη ανθρώπινα μεσεγχυματικά κύτταρα απομονώθηκαν από λιπώδη ιστό και καλλιεργήθηκαν in vitro. Η έκφραση των αντιγόνων επιφανείας CD90, CD73, σε συνδυασμό με την απουσία του μάρτυρα CD45 επιβεβαιώνουν την επιτυχή απομόνωση μεσεγχυματικών βλαστικών κυττάρων, με χρήση κυτταρομετρίας ροής. Έπειτα από 21 ημέρες από την επαγωγή στοχευόμενης διαφοροποίησης τα βλαστοκύτταρα διαφοροποιήθηκαν σε χονδροκύτταρα και χαρακτηρίστηκαν ιστολογικά με χρώση κυανού της τολουιδίνης και μοριακά με RTPCR για δείκτες διαφοροποίησης όπως η αγκρεκάνη. Με τη χρήση του τρισδιάστατου εκτυπωτή δημιουργήθηκε υπό κλίμακα ικρίωμα ρινικού χόνδρου από PLA. Η διαδικασία θα ολοκληρωθεί με την εκτύπωση του υπό διερεύνηση υλικού χιτοζάνης/ζελατίνης σε 3D ικρίωμα και αφού εμποτιστεί με χονδροκύτταρα θα μεταφερθεί στον βιοαντιδραστήρα.Mesenchymal stem cells (MSCs) are multipotent cells isolated from various tissues, mainly from the bone marrow and adipose tissue. Their ability to differentiate into osteoblasts, chondrocytes or adipocytes renders them a promising clinical tool for injury repair and tissue regeneration. In the current study, MSCs were isolated from human adipose tissue (hAD-MSCs) and were triggered to differentiate into chondrocytes in vitro. Expression of mesenchymal stem cell markers, such as CD90 and CD73, in combination with the absence of hematopoietic markers, such as CD45, proves via flow cytometry the successful isolation of MSCs. Histologic staining with Toluidine blue and real time PCR analysis for the expression of the chondrogenic marker aggrecan (ACAN) verified the successful chondrogenic differentiation of AD-MSCs. Using Poly Lactic-Acid as scaffolding material, a three-dimensional scaffold with customized architecture, controlled porosity and interconnected porous structure was fabricated using 3D printing. The produced scaffold represents the morphology of the nasal septum cartilage. We aspire, to see this scaffold with the differentiated chondrocytes and culture the complex under the appropriate micoenvironmental conditions of a bioreactor system in order to regenerate a potential cartilage transplant. This in vitro study expands the potentials of human AD-MSCs to be used in clinic for alleviation of cartilage defects and tissue engineering in Greece and worldwide

Study of the molecular response of neural cells after exposure to hypoxic conditions

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral haemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16 hour oxygen glucose deprivation protocol (OGD) in a PC12 cell line model. Furthermore it investigates the effect of cathepsin D inhibition using the inhibitor Pepstatin A on the expression of the proteins implicated in UPR. Expression of glucose regulated protein 78 (GRP78) and glucose regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose regulated protein 75 (GRP75), heat shock cognate 70 (HSC70) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. Addition of pepstatin does not modify significantly the trends observed in the OGD samples. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. Pepstatin reveals that the autophagic phenomena elicited by OGD are not completed by the 16 hour mark. The above observations warrant further investigation to elucidate whether autophagy acts as a prosurvival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model hypoxia modulates UPR and induces autophagy.Ως υποξία ορίζεται η έλλειψη επαρκούς οξυγόνωσης του ιστού η οποία επιφέρει μεγάλο στρες στα κύτταρα. Η υποξία διαδραματίζει σημαντικό ρόλο σε πλειάδα παθολογικών καταστάσεων όπως στο αγγειακό εγκεφαλικό επεισόδιο, στη τραυματική εγκεφαλική κάκωση, στην εγκεφαλική αιμορραγία, στην περιγεννητική ασφυξία και μπορεί να οδηγήσει σε κυτταρικό θάνατο διαμέσου εξαντλήσεως των ενεργειακών αποθεμάτων του κυττάρου και αύξησης της παραγωγής ελευθέρων ριζών. Η παρούσα μελέτη ερευνά τα αποτελέσματα της υποξίας στην απόκριση μη σωστά αναδιπλωμένων πρωτεϊνών (Unfolded Protein Response, UPR) στην κυτταρική σειρά PC12 χρησιμοποιώντας ένα πρωτόκολλο αποστέρησης οξυγόνου-γλυκόζης διάρκειας 16 ωρών. Επιπρόσθετα ερευνά το αποτέλεσμα της αναστολής των λυσοσωμικών Καθεψινών D, χρησιμοποιώντας τον αναστολέα Pepstatin A, στην έκφραση πρωτεϊνών που εμπλέκονται στην UPR. Η έκφραση των πρωτεϊνών GRP78 και GRP94, βασικών συντελεστών της UPR, μελετήθηκε μαζί με την έκφραση των πρωτεϊνών GRP75, HSC70 και GAPDH υπό το πρίσμα των μηχανισμών κυτταρικού θανάτου. Τα κύτταρα που εκτέθηκαν στο πρωτόκολλο παρουσίασαν αύξηση της έκφρασης της GRP78 και της GRP94 με ταυτόχρονη μείωση της έκφρασης της GRP75. Η προσθήκη Pepstatin A δεν προκάλεσε σημαντική τροποποίηση αυτών των τάσεων. Τα παραπάνω ευρήματα συνοδεύονται με ανίχνευση αυτοφαγίας στα δείγματα που εκτέθηκαν σε υποξικές συνθήκες ενώ δεν ανιχνεύτηκε σημαντική διαφορά στα επίπεδα απόπτωσης του κυτταρικού πληθυσμού. Η προσθήκη Pepstatin A αποκαλύπτει πως τα αυτοφαγικά φαινόμενα που λαμβάνουν χώρα δεν έχουν ολοκληρωθεί ως τις 16 ώρες που κρατάει το πρωτόκολλο. Οι παραπάνω παρατηρήσεις αξίζουν περαιτέρω διερεύνηση έτσι ώστε να διελευκανθεί αν η αυτοφαγία δρα ως κυτταροπροστατευτικός μηχανισμός ο οποίος κατά τη διάρκεια βαριάς και εκτενούς υποξίας επιφέρει τον κυτταρικό θάνατο

Ceramides during Pregnancy and Obstetrical Adverse Outcomes

Ceramides are a group of sphingolipids located in the external plasma membrane layer and act as messengers in cellular pathways such as inflammatory processes and apoptosis. Plasma ceramides are biomarkers of cardiovascular disease, type 2 diabetes mellitus, Alzheimer’s disease, various autoimmune conditions and cancer. During pregnancy, ceramides play an important role as stress mediators, especially during implantation, delivery and lactation. Based on the current literature, plasma ceramides could be potential biomarkers of obstetrical adverse outcomes, although their role in metabolic pathways under such conditions remains unclear. This review aims to present current studies that examine the role of ceramides during pregnancy and obstetrical adverse outcomes, such as pre-eclampsia, gestational diabetes mellitus and other complications

mRNA in the Context of Protein Replacement Therapy

Protein replacement therapy is an umbrella term used for medical treatments that aim to substitute or replenish specific protein deficiencies that result either from the protein being absent or non-functional due to mutations in affected patients. Traditionally, such an approach requires a well characterized but arduous and expensive protein production procedure that employs in vitro expression and translation of the pharmaceutical protein in host cells, followed by extensive purification steps. In the wake of the SARS-CoV-2 pandemic, mRNA-based pharmaceuticals were recruited to achieve rapid in vivo production of antigens, proving that the in vivo translation of exogenously administered mRNA is nowadays a viable therapeutic option. In addition, the urgency of the situation and worldwide demand for mRNA-based medicine has led to an evolution in relevant technologies, such as in vitro transcription and nanolipid carriers. In this review, we present preclinical and clinical applications of mRNA as a tool for protein replacement therapy, alongside with information pertaining to the manufacture of modified mRNA through in vitro transcription, carriers employed for its intracellular delivery and critical quality attributes pertaining to the finished product

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

In the quest for a formidable weapon against the SARS-CoV-2 pandemic, mRNA therapeutics have stolen the spotlight. mRNA vaccines are a prime example of the benefits of mRNA approaches towards a broad array of clinical entities and druggable targets. Amongst these benefits is the rapid cycle “from design to production” of an mRNA product compared to their peptide counterparts, the mutability of the production line should another target be chosen, the side-stepping of safety issues posed by DNA therapeutics being permanently integrated into the transfected cell’s genome and the controlled precision over the translated peptides. Furthermore, mRNA applications are versatile: apart from vaccines it can be used as a replacement therapy, even to create chimeric antigen receptor T-cells or reprogram somatic cells. Still, the sudden global demand for mRNA has highlighted the shortcomings in its industrial production as well as its formulation, efficacy and applicability. Continuous, smart mRNA manufacturing 4.0 technologies have been recently proposed to address such challenges. In this work, we examine the lab and upscaled production of mRNA therapeutics, the mRNA modifications proposed that increase its efficacy and lower its immunogenicity, the vectors available for delivery and the stability considerations concerning long-term storage

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells

Background/Aims: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. Methods: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. Results: Administration of glutamate in PC12 cells in doses as low as 10 μM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. Conclusion: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion