The role of Wilms’ Tumor gene product (WT1)in CD95-mediated apoptosis in T-cell leukaemias

Abstract Apoptosis, also known as programmed cell death, is a very critical process for the maintenance of tissue homeostasis in multicellular organisms. In our immune system, which is comprised of different cell types with different but interconnected functions, apoptosis plays a crucial role in preserving homeostasis during development, throughout adult life and upon termination of immune responses. Any defects in apoptosis can potentially lead to autoimmunity or cancer. CD95 (Fas/APO-1) belongs to the family of the so-called death receptors, and upon binding with its ligand, the CD95L, can instruct CD95-bearing cells to undergo apoptosis. The CD95/CD95L system is crucial for homeostasis of the immune system, and deregulation of CD95-mediated apoptosis results in several diseases including autoimmune syndromes, leukaemias or AIDS. CD95-mediated apoptosis is used by the T-lymphocytes of the immune system at several stages during their development and after the termination of an immune response, so that the quality and quantity of activated T cells are controlled. In these cases, the T cells being activated through their TCR die via CD95-mediated apoptosis upon restimulation, a process collectively known as activation-induced cell death (AICD). One characteristic of cancer is its resistance to apoptosis. Leukaemias are no exception. One of the ways a cancerous T-lymphocyte evades death is by deregulating its CD95/CD95L system by e.g., downregulating the CD95L. NF-AT and Egr proteins (1-3) are amongst the different transcription factors that take part in the regulation of the CD95L. Upon TCR stimulation different signalling pathways lead to the activation of NF-AT and the transcriptional activation of Egr factors. These then bind on the CD95L promoter and activate it. Wilms’ tumor gene 1 (WT1) encodes a four-zinc-finger protein. Alternative splicing at the pre-mRNA level yields several transcript variants, of which the best characterized are the so called WT1-KTS and WT1+KTS isoforms. The WT1-KTS isoform, which lacks three amino acids between zinc-fingers three and four, is suggested to be a transcription factor that belongs to the Egr family and has target sequences similar to Egr1. WT1 is expressed during the early stages of haematopoiesis and has been implicated in leukaemogenesis. Its role in leukaemias is controversial as it is either found to be mutated or overexpressed, and overexpression is linked with more immature leukaemias. It is therefore uncertain whether WT1 is a tumor suppressor or tumor promoter. It was threfore of interest to analyze the expression of WT1 in T cell leukaemias and understand its role in CD95-mediated apoptosis

Επιμόρφωση και Επαγγελματική Ανάπτυξη των Εκπαιδευτικών

Τα τελευταία χρόνια παρατηρείται η ανάγκη δημιουργίας ενός εκπαιδευτικού συστήματος που θα είναι ευέλικτο και ικανό να προσαρμοστεί στον μεταβαλλόμενο κόσμο, ο οποίος αναζητά διαρκώς την καινοτομία. Καθίσταται σαφές ότι το νέο κοινωνικό πλαίσιο της υψηλής νεωτερικότητας και της παγκοσμιοποίησης – το οποίο είναι από μόνο του σύνθετο, συμπιεσμένο και αβέβαιο – επηρεάζει και απασχολεί την κοινωνία σε πολλούς τομείς. Ειδικότερα, στον τομέα της εκπαίδευσης έχει απασχολήσει πολλούς ερευνητές, καθώς προσπαθούν να ερμηνεύσουν τις αλλαγές αλλά και τις επιδράσεις που έχει επιφέρει η νέα πραγματικότητα σε αυτόν. Με βάση τη νέα πραγματικότητα, η ανάγκη της επιμόρφωσης και της επαγγελματικής ανάπτυξης των εκπαιδευτικών, της διεύρυνσης των δεξιοτήτων, καθώς και της συνεχούς πιστοποίησης των νέων ικανοτήτων τους, θεωρείται δεδομένη. Επομένως, ενόσω ο εκπαιδευτικός καλείται να αναπροσαρμοστεί και να ανταποκριθεί σε νέες συνθήκες στο πλαίσιο των ευρύτερων αλλαγών στο χώρο της εκπαίδευσης, είναι αναγκαία η συνεχής επαγγελματική του ανάπτυξη, αφού μέσω αυτής διαμορφώνει την επαγγελματική του ταυτότητα αποκτώντας γνώσεις, δεξιότητες και προσωπικές πεποιθήσεις, οι οποίες τον καθιστούν ικανό να εκπληρώσει τον επαγγελματικό του ρόλο και να διαμορφώσει ώριμους, σκεπτόμενους μαθητές με τις απαιτούμενες ικανότητες και δεξιότητες, οι οποίοι θα εξελιχθούν μετέπειτα σε υπεύθυνους, ενεργούς πολίτες. Σκοπός της παρούσας μελέτης είναι να καταστεί σαφής η σπουδαιότητα της επιμόρφωσης και της επαγγελματικής ανάπτυξης των εκπαιδευτικών και η διερεύνηση της αλληλεπίδρασης των δύο αυτών εννοιών στους Νηπιαγωγούς. Για την ολοκλήρωση της εργασίας επιλέχθηκε η ποσοτική έρευνα με τη χρήση ερωτηματολογίου και απευθύνθηκε σε εκπαιδευτικούς Πρωτοβάθμιας Εκπαίδευσης και πιο συγκεκριμένα σε Νηπιαγωγούς. Η έρευνα επικεντρώθηκε στην ανάδειξη της αναγκαιότητας συμμετοχής των εκπαιδευτικών σε επιμορφωτικά προγράμματα, στα κίνητρα που τους οδηγούν να συμμετέχουν σε ανάλογα προγράμματα και στα οφέλη που μπορούν να αποκομίσουν από τη συμμετοχή τους σε αυτά.In recent years, there has been a need to create an education system that is flexible and capable of adapting to the changing world, which is constantly looking for innovation. It is becoming clear that the new social context of high modernity and globalization – which is in itself complex, compressed and uncertain – affects and concerns society in many areas. In particular, in the field of education it has engaged many researchers, as they try to interpret the changes and the effects that the new reality has brought to it. Based on the new reality, the need for the training and professional development of teachers, the expansion of skills, as well as the continuous certification of their new competences, is taken for granted. Therefore, while every teacher is called upon to adapt and respond to new conditions in the context of the wider changes in the field of education, her/his continuous professional development is necessary, since through it, s/he shapes her/his professional identity by acquiring knowledge, skills and personal beliefs, which make her/him able to fulfill the professional role and form mature and thinking students with the required abilities and skills, who will later develop into responsible, active citizens. The purpose of this study is to clarify the importance of teacher training and professional development and to investigate the interaction of these two concepts in kindergarten teachers. For the completion of the project, the quantitative survey was selected by using a questionnaire and it was addressed to primary education teachers and more specifically to kindergarten teachers. The research focused on highlighting the need for teachers to participate in training programs, the motivations that lead them to participate in such programs and the benefits they can derive from participating in them

Glioma Associated Stem Cells (GASCs) Isolation and Culture.

Glioma Associated Stem Cells (GASCs) represent a population of nontumorigenic multipotent stem cells hosted in the microenvironment of human gliomas. In vitro, these cells are able, through the release of exosomes, to increase the biological aggressiveness of glioma-initiating cells. The clinical importance of this finding is supported by the strong prognostic value associated with the GASCs surface immunophenotype thus suggesting that this patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor strom

Topoisomerase II beta mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs

Background: Glioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation. Methods: In this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage. Results: We found several differentially expressed genes and we identified topoisomerase II beta (Top2 beta) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2 beta was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2 beta expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2 beta increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2 beta depletion in NCH421k cells. Conclusion: We suggest that Top2 beta may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics

Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy.

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression

Systemic T cells immunosuppression of glioma stem cell-derived exosomes is mediated by monocytic myeloid-derived suppressor cells

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression

Topoisomerase II\u3b2 mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs

The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy

Nanothermodynamics mediates drug delivery

© Springer International Publishing Switzerland 2015. The efficiency of penetration of nanodrugs through cell membranes imposes further complexity due to nanothermodynamic and entropic potentials at interfaces. Action of nanodrugs is effective after cell membrane penetration. Contrary to diffusion of water diluted common molecular drugs, nanosize imposes an increasing transport complexity at boundaries and interfaces (e.g., cell membrane). Indeed, tiny dimensional systems brought the concept of “nanothermodynamic potential,” which is proportional to the number of nanoentities in a macroscopic system, from either the presence of surface and edge effects at the boundaries of nanoentities or the restriction of the translational and rotational degrees of freedom of molecules within them. The core element of nanothermodynamic theory is based on the assumption that the contribution of a nanosize ensemble to the free energy of a macroscopic system has its origin at the excess interaction energy between the nanostructured entities. As the size of a system is increasing, the contribution of the nanothermodynamic potential to the free energy of the system becomes negligible. Furthermore, concentration gradients at boundaries, morphological distribution of nanoentities, and restriction of the translational motion from trapping sites are the source of strong entropic potentials at the interfaces. It is evident therefore that nanothermodynamic and entropic potentials either prevent or allow enhanced concentration very close to interfaces and thus strongly modulate nanoparticle penetration within the intracellular region. In this work, it is shown that nano-sized polynuclear iron (III)-hydroxide in sucrose nanoparticles have a nonuniform concentration around the cell membrane of macrophages in vivo, compared to uniform concentration at hydrophobic prototype surfaces. The difference is attributed to the presence of entropic and nanothermodynamic potentials at interfaces

Selective aggregation of PAMAM dendrimer nanocarriers and PAMAM/ZnPc nanodrugs on human atheromatous carotid tissues: a photodynamic therapy for atherosclerosis

© 2015, Spyropoulos-Antonakakis et al.; licensee Springer. Photodynamic therapy (PDT) involves the action of photons on photosensitive molecules, where atomic oxygen or OH− molecular species are locally released on pathogenic human cells, which are mainly carcinogenic, thus causing cell necrosis. The efficacy of PDT depends on the local nanothermodynamic conditions near the cell/nanodrug system that control both the level of intracellular translocation of nanoparticles in the pathogenic cell and their agglomeration on the cell membrane. Dendrimers are considered one of the most effective and promising drug carriers because of their relatively low toxicity and negligible activation of complementary reactions. Polyamidoamine (PAMAM) dendrite delivery of PDT agents has been investigated in the last few years for tumour selectivity, retention, pharmacokinetics and water solubility. Nevertheless, their use as drug carriers of photosensitizing molecules in PDT for cardiovascular disease, targeting the selective necrosis of macrophage cells responsible for atheromatous plaque growth, has never been investigated. Furthermore, the level of aggregation, translocation and nanodrug delivery efficacy of PAMAM dendrimers or PAMAM/zinc phthalocyanine (ZnPc) conjugates on human atheromatous tissue and endothelial cells is still unknown. In this work, the aggregation of PAMAM zero generation dendrimers (G0) acting as drug delivery carriers, as well as conjugated G0 PAMAM dendrimers with a ZnPc photosensitizer, to symptomatic and asymptomatic human carotid tissues was investigated by using atomic force microscopy (AFM). For the evaluation of the texture characteristics of the AFM images, statistical surface morphological and fractal analytical methodologies and Minkowski functionals were used. All statistical quantities showed that the deposition of nanodrug carriers on healthy tissue has an inverse impact when comparing to the deposition on atheromatous tissue with different aggregation features between G0 and G0/ZnPc nanoparticles and with considerably larger G0/ZnPc aggregations on the atheromatous plaque. The results highlight the importance of using PAMAM dendrimer carriers as a novel and promising PDT platform for atherosclerosis therapies