Rapid functionalisation and detection of viruses via a novel Ca2+-mediated virus-DNA interaction

Current virus detection methods often take significant time or can be limited in sensitivity and specificity. The increasing frequency and magnitude of viral outbreaks in recent decades has resulted in an urgent need for diagnostic methods that are facile, sensitive, rapid and inexpensive. Here, we describe and characterise a novel, calcium-mediated interaction of the surface of enveloped viruses with DNA, that can be used for the functionalisation of intact virus particles via chemical groups attached to the DNA. Using DNA modified with fluorophores, we have demonstrated the rapid and sensitive labelling and detection of influenza and other viruses using single-particle tracking and particle-size determination. With this method, we have detected clinical isolates of influenza in just one minute, significantly faster than existing rapid diagnostic tests. This powerful technique is easily extendable to a wide range of other enveloped pathogenic viruses and holds significant promise as a future diagnostic tool

Study of role of caspase 8 during maturation, differentiation and function of T cells using in vitro and transgenic mouse systems

Within the framework of this PhD thesis, mechanisms that are involved in the activation, differentiation and function of T lymphocytes have been studied. Specifically, the role of components of the pro-inflammatory tumor necrosis factor (TNF) signaling pathway (TNF receptor I and caspase 8) in regulating incoming TCR-mediated signals to shape effector T cell responses in vitro and in vivo have been investigated. The TNF/TNFRI signaling pathway, in which caspase 8 is involved, is a major activator of NF-κB which is essential for T cell activation. NF-κB deficiency results in attenuation of immune responses and diminished IL-2 production by T cells. TNF- and TNF receptor I (TNFRI)-deficient mice are resistant to initiation and show delayed resolution of disease in paradigms of autoimmune disease such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). Available knowledge shows that TNF/TNFRI signalling plays a significant role in advancing both the initiation and the resolution of autoimmune diseases. However, the contribution of TNF/TNFRI signaling to T-cell activation and effector responses has not yet been determined. Recent findings show that caspase 8, an apical caspase in apoptosis signaling, also plays an essential role in T cell activation. Following TCR engagement, caspase 8 nucleates a complex with BCL10 and MALT1, which then binds TRAF6 and relocates to T cell lipid rafts. This results in NF-κB activation which is essential for the development of proper immune responses and differential induction of gene expression in T cells. In humans, individuals that carry mutations in the caspase 8 gene show defective T cell, B cell and NK cell activation, and develop an immunodeficiency syndrome. Since a body of mechanistic data now exists to show that NF-κB mediates the effect of caspase 8 in T cells, this study is focused on the investigation of TNFRI/NF-κB signaling pathway in TCR mediated T cell activation, both in vivo and in vitro using CD3+-enriched primary T cells and mice deficient in TNFRI. We show that following TCR engagement, TNFRI KO T cells showed significantly delayed proliferation, cell division, upregulation of interleukin 2 (IL-2) and IL-2 receptor a chain (CD25) mRNA and cell-surface expression of CD25 compared to WT cells. Interestingly, TNFRI KO mice developed defective primary T-cell responses. Effector T cell maturation as measured by cytokine production following TCR stimulation, was similarly delayed in TNFRI KO cells with cytokine production being initially delayed but later increased compared to WT control cells. Further, it seems that TNFRI-mediated cytokine regulation might involve a T-cell autonomous effect. Our results show a novel role for TNFRI as a positive T-cell costimulatory molecule that is important for timely T-cell activation, effector cytokine production and the development of primary immune responses in mice.Η διατριβή αυτή επικεντρώνεται στη μελέτη των μηχανισμών που διέπουν την ενεργοποίηση, τη διαφοροποίηση και τη λειτουργία των Τ λεμφοκυττάρων κατά τη διάρκεια των ανοσολογικών αποκρίσεων. Πιο συγκεκριμένα, μελετάται η συμβολή και η εμπλοκή του μεταγωγού μονοπατιού σήματος του υποδοχέα 1 του TNF (TNFRI) και της κασπάσης 8 στο μεταγωγό μονοπάτι σήματος του υποδοχέα των Τ λεμφοκυττάρων (T cell receptor - TCR). Το μεταγωγό μονοπάτι σήματος του TNF/TNFRI, στο οποίο συμμετέχει και η κασπάση 8, είναι ένα από τα κύρια μονοπάτια τα οποία οδηγούν στην ενεργοποίηση του μεταγραφικού παράγοντα NF-κB. Ο NF-κΒ είναι απαραίτητος για την ενεργοποίηση των Τ λεμφοκυττάρων, καθώς έχει δειχθεί ότι η απενεργοποίησή του οδηγεί σε μειωμένες ανοσολογικές αποκκρίσεις και σημαντικά μειωμένη παραγωγή της ιντερλευκίνης 2. Ποντίκια με απενεργοποιημένο το γονίδιο του TNF ή του TNFRI εμφανίζουν καθυστερημένη έναρξη εκδήλωσης συμπτωμάτων αυτοάνοσων ασθενειών (όπως είναι η πειραματική αυτοάνοση εγκεφαλομυελίτιδα - ΕΑΕ - και η επαγόμενη από το κολλαγόνο αρθρίτιδα - CIA) όπως επίσης και χειρότερη κλινική εικόνα κατά τη χρόνια φάση των ασθενειών σε σύγκριση με τα φυσιολογικού τύπου ποντίκια. Αυτό δείχνει ότι το μεταγωγό μονοπάτι σήματος του TNF/TNFRI είναι θεμελιώδους σημασίας για την έναρξη αλλά και πορεία των νόσων αυτών. Ωστόσο, η συνεισφορά του μονοπατιού αυτού στην ενεργοποίηση και τις ανοσολογικές αποκρίσεις των δραστικών Τ λεμφοκυττάρων δεν έχει καθοριστεί πλήρως. Πρόσφατες μελέτες έχουν δείξει ότι η κασπάση 8, μια εναρκτήριος κασπάση των αποπτωτικών μονοπατιών, παίζει πολύ σημαντικό ρόλο στην ενεργοποίηση των Τ λεμφοκυττάρων. Κατόπιν ενεργοποίησης του Τ υποδοχέα η κασπάση 8 δημιουργεί σύμπλοκο με τα μόρια BCL10 και MALT1 συνεισφέροντας, με τη βοήθεια του μορίου TRAF6, στη στρατολόγησή τους στις λιπιδικές σχεδίες των Τ λεμφοκυττάρων. Αυτό οδηγεί στην ενεργοποίηση του μεταγραφικού παράγοντα NF-κB που είναι απαραίτητος για τις σωστές ανοσολογικές αποκρίσεις και τη διαφοροποιημένη γονιδιακή έκφραση των Τ λεμφοκυττάρων. Έχει βρεθεί ότι μεταλλάξεις του γονιδίου της κασπάσης 8 σε ανθρώπους προκαλούν μειωμένη ενεργοποίηση των Τ και Β λεμφοκυττάρων όπως και των φυσικών κυττάρων φονιάδων, και ως εκ τούτου οδηγούν σε ανοσολογική ανεπάρκεια. .................

Mesenchymal Stem Cell Protection of Neurons against Glutamate Excitotoxicity Involves Reduction of NMDA-Triggered Calcium Responses and Surface GluR1, and Is Partly Mediated by TNF

Mesenchymal stem cells (MSC) provide therapeutic effects in experimental CNS disease models and show promise as cell-based therapies for humans, but their modes of action are not well understood. We previously show that MSC protect rodent neurons against glutamate excitotoxicity in vitro, and in vivo in an epilepsy model. Neuroprotection is associated with reduced NMDA glutamate receptor (NMDAR) subunit expression and neuronal glutamate-induced calcium (Ca2+) responses, and increased expression of stem cell-associated genes. Here, to investigate whether MSC-secreted factors modulate neuronal AMPA glutamate receptors (AMPAR) and gene expression, we performed longitudinal studies of enriched mouse cortical neurons treated with MSC conditioned medium (CM). MSC CM did not alter total levels of GluR1 AMPAR subunit in neurons, but its distribution, reducing cell surface levels compared to non-treated neurons. Proportions of NeuN-positive neurons, and of GFAP- and NG2-positive glia, were equal in untreated and MSC CM-treated cultures over time suggesting that neurons, rather than differentially-expanded glia, account for the immature gene profile previously reported in MSC CM-treated cultures. Lastly, MSC CM contained measurable amounts of tumor necrosis factor (TNF) bioactivity and pre-treatment of MSC CM with the TNF inhibitor etanercept reduced its ability to protect neurons. Together these results indicate that MSC-mediated neuroprotection against glutamate excitotoxicity involves reduced NMDAR and GluR1-containing AMPAR function, and TNF-mediated neuroprotection

Factors Influencing Quality of Life in Breast Cancer Patients Six Months after the Completion of Chemotherapy

Purpose: To assess breast cancer patients’ quality of life six months after the completion of adjuvant chemotherapy, and to investigate factors affecting this. Methods: The study was conducted in one large hospital located in a major Greek city. A convenience sample of 61 breast cancer outpatients was recruited. A questionnaire, including the SF-36 scale and questions regarding demographic and clinical information, was used to collect data. Results: The mean age of the patients was 51.52 ± 12.10. The effect of age on the physical role was significant (p = 0.003). Τhe effect of menopausal status on physical role was also found to be significant (p = 0.003); this might be explained by age. Regarding the treatment type, patients who received hormone therapy in addition to surgery and chemotherapy reported a significantly higher quality of life in terms of bodily pain (p = 0.04) and vitality (p = 0.04) than patients who underwent only surgery and chemotherapy. Conclusions: Quality of life is affected by factors such as age, menopausal status, and previous therapy. Health care professionals should be more aware of the factors that influence the quality of life domains (physical role, bodily pain, vitality) within this group of cancer patients in order to meet their needs following acute treatment

IKKβ deletion from CNS macrophages increases neuronal excitability and accelerates the onset of EAE, while from peripheral macrophages reduces disease severity

Abstract Background Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. Methods We used constitutive MφIKKβΚΟ mice, in which depletion of IKKβ, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚβKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. Results Global depletion of IKKβ from myeloid cells in MφIKKβΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKβ only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKβ, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKβ resulted in enhanced late long-term potentiation in EAE. Conclusions IKKβ-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKβ deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKβ on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS

Prevalence, Wellbeing, and Symptoms of Dysmenorrhea among University Nursing Students in Greece

Dysmenorrhea (pain during menstruation) is one of the most common medical conditions among women of reproductive age. Dysmenorrhea has been studied around the world but not yet in Greece. The aim of the present study was to investigate the prevalence, characteristics, and impact of dysmenorrhea on the wellbeing (exercising, and social and academic functioning) among nursing students in Greece. A cross-sectional study of 637 nursing students was conducted by administering a questionnaire at a university in Athens. The prevalence of dysmenorrhea was 89.2% and the rate of severe intensity was 52.5%. Factors that were associated with severe dysmenorrhea were family history (p = 0.02), early menarche (p = 0.05) and menstruation duration (p = 0.05). Women with moderate and severe pain reported using pain relievers (non-steroidal anti-inflammatory drugs (NSAIDs), paracetamol etc., p < 0.0005). Finally, activities affected by severe pain were class attendance (p = 0.01), personal studying (p < 0.0005), exercising (p < 0.0005), and socializing (p < 0.0005). Exam attendance (p = 0.27) and clinical placement attendance (p = 0.48) were not affected by severe dysmenorrhea. Dysmenorrhea has a high prevalence among nursing students and seems to affect important aspects of wellbeing and academic performance when the pain intensity is severe. The present findings lay the foundation for further investigation of dysmenorrhea both in the Greek population and cross-culturally