Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 4 uppsatser.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

Τόσο τα βλαστικά όσο και τα προγονικά κύτταρα συντονίζουν τον πολλαπλασιασμό και τη διαφοροποίηση για να δώσουν γένεση στον κατάλληλο αριθμό λειτουργικά εξειδικευμένων κυττάρων κατά την οργανογένεση. Διαφορετικές πειραματικές προσεγγίσεις ανέδειξαν τον ρόλο της πρωτεΐνης Geminin στη διατήρηση των προγονικών κυττάρων, τη συμμετοχή της στις αποφάσεις καθορισμού της αναπτυξιακής τους τύχης αλλά και την συμβολή της στην οργανογένεση. Αν και ο ακριβής μηχανισμός δεν είναι ξεκάθαρος, η Geminin φαίνεται να επηρεάζει την κατεύθυνση του πολλαπλασιασμού έναντι της διαφοροποίησης. Για να διαλευκανθεί ο ρόλος της Geminin in vivo στην κυτταρική διαίρεση και διαφοροποίηση των προγονικών κυττάρων, δημιουργήθηκαν ποντίκια που στερούνται την έκφραση της Geminin ειδικά στα κύτταρα της λεμφοειδούς σειράς μέσω Cre ανασυνδυασμού. Προηγούμενες μελέτες έδειξαν ότι η απουσία της Geminin έχει ελάχιστες επιπτώσεις στην ανάπτυξη και διαφοροποίηση των θυμοκυττάρων ενώ τα ώριμα περιφερειακά Τ κύτταρα που δεν εκφράζουν Geminin εμφανίζουν πολλές επιπτώσεις στον πολλαπλασιασμό μετά από in vitro ενεργοποίηση. Τα φυσικά φονικά κύτταρα (Natural Killer Cells) αναπτύσσονται στο μυελό των οστών από κοινά αρχέγονα κύτταρα και καθορίζονται από την ικανότητά τους να σκοτώνουν καρκινικούς στόχους χωρίς προηγούμενη ενεργοποίηση. Τα κύτταρα αυτά παίζουν σημαντικό ρόλο στις μη ειδικές αποκρίσεις και πέρα από την ισχυρή κυτταροτοξική δράση έναντι ευαίσθητων κυττάρων-στόχων χαρακτηρίζονται από την ικανότητα να απελευθερώνουν διάφορες κυτταροκίνες αποκρινόμενα άμεσα σε ενδοκυτταρικές μολύνσεις. Επιπρόσθετα, αποτελούν έναν κρίσιμο κυτταρικό πληθυσμό για την προστασία των ιικών μολύνσεων, καθώς αναγνωρίζουν με ειδικό τρόπο ιικά μολυσμένα κύτταρα και τα σκοτώνουν απευθείας. Για τους λόγους αυτούς παρουσιάζει ενδιαφέρον ο έλεγχος των τυχόν επιπτώσεων της απουσίας της Geminin στα ΝΚ κύτταρα. Με βασικό πειραματικό εργαλείο την κυτταρομετρία ροής, μελετήσαμε την αναπτυξιακή πορεία των φυσικών φονικών κυττάρων απουσία της Geminin. Σε κυτταρικά εναιωρήματα από σπλήνα και μυελό των οστών από Fl/koCD2Cre και Fl/WT πειραματόζωα, NK1.1+TCRβ- ΝΚ κύτταρα αναλύονται με κυτταρομετρία ροής για τον έλεγχο της έκφρασης μορίων-δεικτών των ώριμων ΝΚ κυττάρων. / The interplay of proliferation and differentiation is essential for normal development and organogenesis. Stem cells as wells as progenitor cells coordinate these cellular decisions to give rise to the appropriate number of differentiated cells with specific function. Geminin was identified through two parallel lines of investigation as both a regulator of DNA replication and a regulator of differentiation of neural cells. In order to address the in vivo role of Geminin in regulating progenitor cell self-renewal and differentiation decisions in different cellular systems, have been generated mice that allow the conditional inactivation of the mouse Geminin gene in cells giving rise to the lymphoid lineage. Natural Killer Cells are lymphocytes of the innate immunity with effector function such as perforin-dependent cytotoxicity and interferon-γ secretion. Several lines of evidence implicate them in the early control of virus infection, in tumor immunoserveillance and in the regulation of immune responses. Mature NK cells express a wide variety of cell surface receptors that enable them to recognize targets expressing low surface amounts of major histocompatibility complex class I molecules or high surface amounts of molecules induced by stress or microbial molecules. NK cells generation occurs in the bone marrow from hematopoietic stem cells. In this study, we shed light to the in vivo role of Geminin in the development and differentiation of Natural Killer Cells. Cell suspensions from bone marrow and spleen from Fl/koCD2Cre mice that lack Geminin expression in the lymphoid lineage and Fl/WT mice that serves as controls were analyzed with flow cytometry. NK1.1+TCRβ- ΝΚ cells were analysed for surface receptors, markers of mature NK cells.

In this thesis, the monocrotaline (MCT)-induced rat model of pulmonary arterial hypertension (PAH) was used to investigate the role of innate immunity in the pathogenesis of PAH and the mode of action of experimental therapies. The first section of this thesis is an investigation of the therapeutic mechanism of human, early and late-outgrowth endothelial progenitor cells (EPCs) in the MCT-induced, nude rat model of PAH. While late-outgrowth EPCs provided no therapeutic benefit in this model, early EPCs (E-EPCs) prevented the elevation of right ventricular systolic pressure (RVSP, P<0.001) and right ventricular (RV) hypertrophy (P<0.01). Ablation of natural killer (NK) and natural killer T cells with anti-asialo GM-1 antiserum (ASGM-1) enhanced human cell retention in the lung but abrogated the therapeutic capacity of E-EPCs. In vitro studies demonstrated that E-EPCs are similar to monocyte-derived regulatory dendritic cells (DCs) and possess the capacity to stimulate both autologous and rat NK cells in co-culture. Imatinib mesylate has been reported to reverse established PAH both clinically and in the MCT model. Imatinib can also induce NK activation through inhibition of c-kit signaling in DCs, suggesting that imatinib and the DC-like E-EPCs may prevent PAH through a similar, NK-mediated mechanism. In the second section of this thesis, imatinib prevented MCT-induced increases in RVSP (P<0.001) and RV hypertrophy (P<0.01) in immunocompetent Fisher 344 rats, but not in nude rats or Fisher rats following ablation of NK cells and T lymphocytes with ASGM-1. These data suggest that the stimulation of NK activity by imatinib is insufficient to prevent disease in the absence of T lymphocytes. Hyaluronan (HA) fragments are a potent inflammatory stimulus, capable of inducing macrophage activation and DC maturation. In the third section of this thesis, HA synthesis and degradation were investigated in the MCT model of PAH. While the early stages of disease were characterized by enhanced hyaluronidase-1 activity and a loss of high molecular weight (HMW) HA, severe disease was associated with HMW HA synthesis and HA accumulation in the lungs. The early degradation of HMW HA may drive inflammation and stimulate pathological vascular remodeling in PAH.

In this thesis, the monocrotaline (MCT)-induced rat model of pulmonary arterial hypertension (PAH) was used to investigate the role of innate immunity in the pathogenesis of PAH and the mode of action of experimental therapies. The first section of this thesis is an investigation of the therapeutic mechanism of human, early and late-outgrowth endothelial progenitor cells (EPCs) in the MCT-induced, nude rat model of PAH. While late-outgrowth EPCs provided no therapeutic benefit in this model, early EPCs (E-EPCs) prevented the elevation of right ventricular systolic pressure (RVSP, P<0.001) and right ventricular (RV) hypertrophy (P<0.01). Ablation of natural killer (NK) and natural killer T cells with anti-asialo GM-1 antiserum (ASGM-1) enhanced human cell retention in the lung but abrogated the therapeutic capacity of E-EPCs. In vitro studies demonstrated that E-EPCs are similar to monocyte-derived regulatory dendritic cells (DCs) and possess the capacity to stimulate both autologous and rat NK cells in co-culture. Imatinib mesylate has been reported to reverse established PAH both clinically and in the MCT model. Imatinib can also induce NK activation through inhibition of c-kit signaling in DCs, suggesting that imatinib and the DC-like E-EPCs may prevent PAH through a similar, NK-mediated mechanism. In the second section of this thesis, imatinib prevented MCT-induced increases in RVSP (P<0.001) and RV hypertrophy (P<0.01) in immunocompetent Fisher 344 rats, but not in nude rats or Fisher rats following ablation of NK cells and T lymphocytes with ASGM-1. These data suggest that the stimulation of NK activity by imatinib is insufficient to prevent disease in the absence of T lymphocytes. Hyaluronan (HA) fragments are a potent inflammatory stimulus, capable of inducing macrophage activation and DC maturation. In the third section of this thesis, HA synthesis and degradation were investigated in the MCT model of PAH. While the early stages of disease were characterized by enhanced hyaluronidase-1 activity and a loss of high molecular weight (HMW) HA, severe disease was associated with HMW HA synthesis and HA accumulation in the lungs. The early degradation of HMW HA may drive inflammation and stimulate pathological vascular remodeling in PAH.

No description available.

Natural killer (NK) cells are important in limiting tumor dissemination. The NK activity in C57B1/6 mice bearing Lewis lung carcinoma (LLC) was monitored during tumor development. During the initial period of tumor growth, NK activity was enhanced. As tumor growth progressed, NK activity became suppressed. Depletion of macrophages from the spleen cells of tumor-bearing mice restored the NK cytotoxic response. Plasma prostaglandin E2 (PGE2) concentrations were measured by a radioimmunoassay and found to become elevated during the course of tumor growth. To determine whether the suppressed NK activity might have been a result of the elevated levels of PGE2, mice were treated with a prostaglandin synthesis inhibitor, indomethacin. Indomethacin treatment prevented the rise in plasma PGE2 concentrations and the suppression in NK activity. These results support the hypothesis that the suppression of NK activity in tumor bearers is mediated by PGE2 which might be produced by the host's suppressor macro-phages.

Surgery precipitates a hypercoagulable state and has been shown to increase the development of cancer metastases in animal models, however mechanism(s) responsible for this are largely unknown. We hypothesize that the prometastatic effect of surgery may be secondary to postoperative hypercoagulable state. Surgical stress was induced in mice by partial hepatectomy or nephrectomy, preceded by intravenous injection of CT26-LacZ or B16F10-LacZ cells to establish pulmonary metastases with or without perioperative anticoagulation and their lung tumor cell emboli (TCE) were quantified. Fibrinogen and platelets were fluorescently labeled prior to surgical stress to evaluate TCE-associated fibrin and platelet clots. Surgery significantly increased metastases while anticoagulation with five different agents attenuated this effect. Fibrin and platelet clots were associated with TCE significantly more frequently in surgically stressed mice. Surgery promotes the formation of fibrin and platelet clots around TCE and this appears to be the mechanism for the increase in metastases seen following surgery.

Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / Herpes simplex virus type I (HSV-I) produces acute muco-cutaneous infections, followed by spread to sensory nerve ganglia, and establishment of latency. In the peripheral nervous system, primary sensory neurons, which are found in dorsal root ganglia of the of the spinal nerves, are the target for HSV and they may undergo either productive or latent intection. Productive infection of sensory neurons generates the potential for lethal spread of virus through the nervous system but in immunocom petent hosts, viral replication is terminated by limely development of an adaptive immune response. The infection of dorsal root ganglia that follows cutaneous inoculation of the flanks of mice with HSV provides a well-characterized model of peripheral nervous system infection. The mechanisms responsible for clearance of HSV are complex. At mucosal and cutaneous sites, local innate immune mechanisms act to interrupt the initial spread of virus to the nervous system, while adaptive immunity is important in limiting replication in the ganglia and extension of the virus to adjacent dennatomes. Thus actions of both the innate and the adaptive immune systems are vital in defence against replicating HSV-1, while it is thought that latent infection in the ganglia is contained by the surveillance of the adaptive immune system. Natural killer T (NKT) cells are a conserved subpopulation of lymphocytes that recognize glycolipid antigens presented by the invariant MHC class I-like molecule CD1d. Upon activation through their semi-invariant T cell receptor, these cells rapidly release large amounts of immuno-modulating Th1 and Th2 cytokines. NKT cells have, therefore, been implicated in immune responses controlling various diseases, including infection, cancer, and autoimmunity, as well as having an involvement in allo-graft survival. Consideration of the important contributions of innate and adaptive immunity to clearance of HSV prompted this investigation of the role of CD1d and of CD1d-restricted NKT cells in the pathogenesis of HSV infection. The first part of this thesis (Chapter 3 and 4) describes investigations into the role of NKT cells in immunity to HSV-1, using a zosteriform model of infection and two gene knockout strains of C57BL/6 mice. CD1d GKO and Ja18 GKO mice, which are deficient in NKT cells, are compromised in controlling HSV-1 as evidenced by mortality, virus loads in skin and dorsal root ganglia, presence and size of skin lesions, persistence of HSV antigen, neuronal damage and extent of latency. Comparisons between wild type (NKT cell replete), Ja18 GKO (deficient in invariant Va14⁺ NKT cells) and CD1d GKO (deficient in all CD1d-dependant NKT cells) mice allowed assessment of CD1d-dependant NKT cell subsets in defence against the virus at various stages of infection. It was concluded that both subsets play important roles in controlling the virus and in preventing lethal neuro-invasive disease, that both are vital adjuncts to the adaptive immune response and that without them, low doses of neuropathogenic HSV-1 can establish quickly and cause fatal infections. The NKT-cell population appears to be quite dynamic in its response to a range of pathogens and other disease processes. The study described in Chapter 5 presents evidence suggesting that the response of NKT cells during HSV infection is no less dynamic. In the axillary lymph nodes, observations on numbers of cells expressing NK1.1 antigen and the invariant TCR suggest that NKT cells are activated in the regional lymph nodes draining the infection site. Observations on lymphocytes prepared from liver and spleen also suggested activation of NKT cells, indicating that NKT cells at these sites are also activated during the course of acute HSV infection. The role of NKT cells in the control of HSV infection was further examined by adoptive transfer studies, to investigate whether the defect in handling of HSV-1 by Ja18 GKO mice could be complemented by the adoptive transfer of lymphocytes from wt mice (Chapter 6). Finally, the relevance of activated NKT cells in the anti-HSV response was examined by observing the effects of a-GalactosylCeramide therapy on the severity of HSV-1 infection (Chapter 6). Activation of NKT cells by this compound delayed the onset of HSV disease, decreased prevalence and severity of zosteriform lesions and reduced viral titres in skin and ganglia. The beneficial effects of a-GalactosylCeramide on the outcome and severity of HSV infection in the skin were dose-dependent. Collectively, the studies described in this thesis provide insights into how NKT cells, normally a rare population of cells, has the ability to regulate the protective immune response to HSV-1. As more understanding is gained about how NKT cells become activated during HSV-1 infection, and how they mediate their antiviral effects, other ways may be developed to modulate and activate this interesting subset to the benefit of infected individuals. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1277283 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007

Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / Herpes simplex virus type I (HSV-I) produces acute muco-cutaneous infections, followed by spread to sensory nerve ganglia, and establishment of latency. In the peripheral nervous system, primary sensory neurons, which are found in dorsal root ganglia of the of the spinal nerves, are the target for HSV and they may undergo either productive or latent intection. Productive infection of sensory neurons generates the potential for lethal spread of virus through the nervous system but in immunocom petent hosts, viral replication is terminated by limely development of an adaptive immune response. The infection of dorsal root ganglia that follows cutaneous inoculation of the flanks of mice with HSV provides a well-characterized model of peripheral nervous system infection. The mechanisms responsible for clearance of HSV are complex. At mucosal and cutaneous sites, local innate immune mechanisms act to interrupt the initial spread of virus to the nervous system, while adaptive immunity is important in limiting replication in the ganglia and extension of the virus to adjacent dennatomes. Thus actions of both the innate and the adaptive immune systems are vital in defence against replicating HSV-1, while it is thought that latent infection in the ganglia is contained by the surveillance of the adaptive immune system. Natural killer T (NKT) cells are a conserved subpopulation of lymphocytes that recognize glycolipid antigens presented by the invariant MHC class I-like molecule CD1d. Upon activation through their semi-invariant T cell receptor, these cells rapidly release large amounts of immuno-modulating Th1 and Th2 cytokines. NKT cells have, therefore, been implicated in immune responses controlling various diseases, including infection, cancer, and autoimmunity, as well as having an involvement in allo-graft survival. Consideration of the important contributions of innate and adaptive immunity to clearance of HSV prompted this investigation of the role of CD1d and of CD1d-restricted NKT cells in the pathogenesis of HSV infection. The first part of this thesis (Chapter 3 and 4) describes investigations into the role of NKT cells in immunity to HSV-1, using a zosteriform model of infection and two gene knockout strains of C57BL/6 mice. CD1d GKO and Ja18 GKO mice, which are deficient in NKT cells, are compromised in controlling HSV-1 as evidenced by mortality, virus loads in skin and dorsal root ganglia, presence and size of skin lesions, persistence of HSV antigen, neuronal damage and extent of latency. Comparisons between wild type (NKT cell replete), Ja18 GKO (deficient in invariant Va14⁺ NKT cells) and CD1d GKO (deficient in all CD1d-dependant NKT cells) mice allowed assessment of CD1d-dependant NKT cell subsets in defence against the virus at various stages of infection. It was concluded that both subsets play important roles in controlling the virus and in preventing lethal neuro-invasive disease, that both are vital adjuncts to the adaptive immune response and that without them, low doses of neuropathogenic HSV-1 can establish quickly and cause fatal infections. The NKT-cell population appears to be quite dynamic in its response to a range of pathogens and other disease processes. The study described in Chapter 5 presents evidence suggesting that the response of NKT cells during HSV infection is no less dynamic. In the axillary lymph nodes, observations on numbers of cells expressing NK1.1 antigen and the invariant TCR suggest that NKT cells are activated in the regional lymph nodes draining the infection site. Observations on lymphocytes prepared from liver and spleen also suggested activation of NKT cells, indicating that NKT cells at these sites are also activated during the course of acute HSV infection. The role of NKT cells in the control of HSV infection was further examined by adoptive transfer studies, to investigate whether the defect in handling of HSV-1 by Ja18 GKO mice could be complemented by the adoptive transfer of lymphocytes from wt mice (Chapter 6). Finally, the relevance of activated NKT cells in the anti-HSV response was examined by observing the effects of a-GalactosylCeramide therapy on the severity of HSV-1 infection (Chapter 6). Activation of NKT cells by this compound delayed the onset of HSV disease, decreased prevalence and severity of zosteriform lesions and reduced viral titres in skin and ganglia. The beneficial effects of a-GalactosylCeramide on the outcome and severity of HSV infection in the skin were dose-dependent. Collectively, the studies described in this thesis provide insights into how NKT cells, normally a rare population of cells, has the ability to regulate the protective immune response to HSV-1. As more understanding is gained about how NKT cells become activated during HSV-1 infection, and how they mediate their antiviral effects, other ways may be developed to modulate and activate this interesting subset to the benefit of infected individuals. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1277283 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2007