Changes in the microenvironment have been implicated in the transition of pre-invasive ductal carcinoma in-situ (DCIS) to invasive breast cancer. Normal myoepithelial cells have a tumour suppressor phenotype but they are altered in DCIS and ultimately lost with transition to invasive cancer. A consistent change in DCIS is up-regulation of the integrin αvβ6 in myoepithelial cells. Preliminary observations identified a correlation between myopeithelial αvβ6 and an increased peri-ductal inflammatory infiltrate. The hypothesis of this study is that the altered myoepithelial phenotype influences the peri-ductal inflammatory environment, which in turn mediates a pro-apoptotic effect on myoepithelial cells contributing to their loss. To investigate this, the inflammatory infiltrate was characterised in a series of DCIS tissue in relation to αvβ6 status. This demonstrated significantly higher levels of CD4+ve and FOXP3+ve T cells around αvβ6+ve DCIS ducts compared to αvβ6-ve ducts (P=<0.01), suggesting an increase in Treg cells. In-vivo, Matrigel plugs containing injected into the flanks of female C57/Blk6 normal mice generated influx of higher levels of CD4+ve cells (p=0.005) and FOXP3+ T cells (p=0.007) in the presence of αvβ6+ve myoepithelial cells compared to αvβ6-ve cells, supporting the findings in human tissue samples. Since Treg cells produce TRAIL that can induce apoptosis, we investigated the influence of αvβ6 on myoepithelial cells on the levels of TRAIL in T cells and the hypothesis that αvβ6-positive myoepithelial ells may be more susceptible to TRAIL-induced apoptosis, leading to loss of the myoepithelial barrier. Firstly, levels of TRAIL in Jurkat and primary T cell populations co-cultured with β4 (ii) or β6 myoepithelial cells were measured. This demonstrated a higher level of TRAIL in primary T cells co-cultured β6 myoepithelial cells compared to those co-cultured with β4 myoepithelial cells. β6+ve and β6-ve myoepithelial cells were exposed to TRAIL, and this demonstrated that TRAIL enhanced apoptosis, measured by cleaved PARP, in β6+ve cells. Furthermore, these cells showed loss of the anti-apoptotic protein Galectin-7, and knockdown of Galectin-7 in normal β6-ve myoepithelial cells rendered them more susceptible to TRAIL-induced apoptosis. In DCIS tissues, an inverse relationship between αvβ6 and Galectin-7 in myoepithelial cells was demonstrated, and Cytokine Array analysis showed that αvβ6+ve myoepithelial cells express higher levels of IL-16, which has a role in Treg cell recruitment. Taken together these results suggest that expression of αvβ6 by myoepithelial cells in DCIS generates a tumour-promoter peri-ductal inflammatory infiltrate through altered cytokine release, is associated with reduced galectin-7 expression and enhances myoepithelial cell apoptosis in response to TRAIL. This provides a potential mechanism by which myoepithelial cells may be lost during evolution of DCIS and so contribute to progression to invasive disease.

En France, les sites ostréicoles de la méditerranée sont confrontés régulièrement à de fortes mortalités de juvéniles de Crassostrea gigas mais également à des efflorescences récurrentes du dinoflagellé producteur de toxines paralysantes (PSTs), Alexandrium catenella. Parmi les pathogènes associés de manière récurrente à ces mortalités, on retrouve des souches de Vibrio appartenant au clade Splendidus. Nous nous intéressons ici aux interactions entre A. catenella et l’huître C. gigas confrontée à des vibrios pathogènes. Dans une première partie, nous avons montré qu'en conditions expérimentales contrôlées, A. catenella augmentait la sensibilité de l’huître C. gigas au pathogène Vibrio tasmaniensis LGP32. In situ, nous avons également constaté la coïncidence entre la mortalité des huîtres en 2014 et la présence des PSTs dans leurs tissus. Dans une seconde partie, nous avons étudié les interactions entre les PSTs produites par A. catenella et les cellules immunitaires de l’huître, les hémocytes. Un résultat important de cette thèse a été de montrer que la saxitoxine, une des toxines produites par A. catenella, se lie à des structures granulaires présentes dans le cytoplasme des hémocytes de C. gigas et induit une mort hémocytaire caspases-dépendante. Cette mort est indépendante de la production d’espèces réactives de l’oxygène. Nous avons également démontré que la toxine majoritaire des cellules d’A. catenella, la gonyautoxine 5, est la plus toxique sur les hémocytes de C. gigas. Parmi les populations hémocytaires touchées, les hyalinocytes sont très sensibles à ce stress toxique. Les hémocytes étant les cellules immunocompétentes de l’huître qui jouent le rôle central dans la défense contre les infections, nous supposons que leur mort cellulaire induite par les PSTs affecte négativement la défense des mollusques bivalves et explique l'augmentation de la susceptibilité des huîtres à l'infection par Vibrio tasmaniensis LGP32 quand elles sont exposées à A. catenella. / In France, oyster sites in the Mediterranean Sea are regularly confronted to high mortalities of Crassostrea gigas juveniles and to recurrent blooms of the dinoflagellate producer of Paralytic Shellfish Toxins (PSTs), Alexandrium catenella. Among the pathogens associated to these mortalities, we found Vibrio strains belonging to Splendidus clade. We here focus on the interactions between A. catenella and the oyster C. gigas challenged with pathogenic vibrios. In the first part of this work, we have shown that, in vivo, A. catenella increased the susceptibility of the oyster C. gigas to the pathogen Vibrio tasmaniensis LGP32. In situ, we also established the coincidence between oyster mortality in 2014 and the presence of PSTs in their tissues. In the second part of this work, we studied the interactions between the PSTs produced by A. catenella and oyster immune cells, the hemocytes. An important result of this thesis was that saxitoxin, a toxin produced by A. catenella, binds to granular structures in the cytoplasm of C. gigas hemocytes and induces their caspase-dependent cell death. This death is independent of the production of reactive oxygen species. We also demonstrated that the major toxin of A. catenella cells, the gonyautoxin 5, is the most toxic on C. gigas hemocytes. Among affected hemocyte populations, the hyalinocytes are very sensitive to this toxic stress. As hemocytes are oyster immunocompetent cells and therefore play the central role in the defense against infections, we can presume that their cell death induced by the PSTs negatively affects the defense of bivalve mollusks and explains the increased susceptibility of oysters to the infection by Vibrio tasmaniensis LGP32 when exposed to A. catenella.

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Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Muscle injuries are associated with changes in skeletal muscle as well as the immune system. All studies investigating possible treatment modalities have found both positive and negative effects on muscle recovery. Since no universally accepted treatment modality exists, this thesis aims to determine whether a plant-derived antioxidant, proanthocyanidolic oligomer (PCO), might prove beneficial as treatment for sports injuries in order for athletes to return to the sports field quicker. The difference in recovery of muscle following both chronic (supplementation started 14 days prior to injury and continued thereafter) and acute supplementation (supplementation started two hours after injury) were also investigated. Both chronic and acute PCO supplementation in a rat hindlimb contusion injury model resulted in earlier muscle recovery, verified by an earlier satellite cell response compared to the placebo group. This effect was most prominent already at the four hour time point following injury, compared to day seven and three after chronic and acute placebo treatment respectively. PCO supplementation also resulted in quicker foetal myosin heavy chain (MHCf) expression compared to placebo treatment. Chronic supplementation specifically resulted in a blunted circulatory pro-inflammatory cytokine response, whilst allowing for a significant increase in IL-10, an anti-inflammatory cytokine, on day three (in the PCO group only). At tissue level, the response of the muscle pro-inflammatory cytokines, TNF- and IL- 6, coincided with the satellite cell response. Macrophage infiltration into the injured muscle also followed a similar pattern to that seen for the pro-inflammatory cytokines. Macrophages invaded the injured area quicker when supplemented with PCO chronically, however, macrophage infiltration could not explain the cytokine response seen with acute supplementation. Both chronic and acute supplementation with PCO was responsible for a severely blunted neutrophil response, a novel finding of this particular antioxidant. The main findings of the in vivo rodent study were that PCO was able to blunt the neutrophil response, whilst allowing for earlier macrophage infiltration. To establish possible mechanisms by which PCO might exert these beneficial effects, further analysis included determining macrophage phenotypes and neutrophil numbers in circulation. An in vitro neutrophil migration assay was also employed to further elucidate PCO’s ability to blunt neutrophil infiltration into the injured area. For this study, conditioned plasma were harvested from experimental animals and added together with neutrophils from control rats and granulocyte colony stimulating factor (G-CSF) to the insert of the migration chamber. A chemotactic factor, N-formyl methionine-leucine-phenylalanine (fMLP), was added to the bottom well and neutrophils were allowed to migrate for two hours. Results from this study indicated that neutrophil migration was attenuated in vitro in the presence of conditioned plasma from PCO supplemented rats only. The studies in this thesis on the effect of PCO on parameters of muscle and the immune system led to the following main conclusions: a) PCO supplementation resulted in earlier muscle recovery as a result of earlier satellite cell activation and MHCf synthesis; b) PCO favours an anti-inflammatory cytokine reaction, whilst blunting the pro-inflammatory cytokine response; and c) PCO blunted the neutrophil response whilst facilitating earlier macrophage infiltration into the injured area. The specific mechanism of action of PCO to blunt the neutrophil response specifically, possibly includes the ability to suppress adhesion molecule expression on the neutrophils themselves. However, this warrants further investigation. / AFRIKAANSE OPSOMMING: Spier beserings word geassosiëer met veranderinge in skeletspier sowel as die immuunstelsel. Meeste studies wat moontlike behandelingsopsies ondersoek, het beide positiewe en negatiewe spierherstel gerapporteer. Omrede daar geen universele behandelingsmoontlikheid bestaan nie, is die doel van hierdie tesis om die effek van ‘n plantgebaseerde anti-oksidant, pro-antosianiedoliese oligomeer (PSO), as ‘n voordelige behandelingstrategie vir sportbeserings te toets. Die verskil in spierherstel na beide kroniese (supplementering wat 14 dae voor besering begin is, en volgehou is daarna) en akute supplementering (supplementering het twee uur na besering begin), is ook ondersoek. Beide kroniese en akute PSO supplementering, in ‘n rot agterbeen-kneusbeseringmodel, het gelei tot vroeë spierherstel. Die bevindinge is geverifiëer deur ‘n vroeë satelietselrespons in vergelyking met die plasebo groep. Hierdie effek was reeds opvallend vier uur na besering, in vergelyking met die dag sewe en dag drie tydpunt tydens kroniese en acute plasebo behandeling onderskeidelik. In vergelyking met die kontrole groep, het PSO supplementering ook gelei to vininger uitdrukking van miosienswaarketting (MHCf). Kroniese supplementering het spesifiek gelei to ‘n onderdrukte sirkulatoriese pro-inflammatoriese sitokien response, terwyl ‘n betekenisvolle toename in IL-10 op dag drie (in die PSO groep alleenlik) waargeneem is. Op weefselvlak, het die pro-inflammatoriese sitokiene, IL-6 en TNF- , dieselfde patron gevolg as die van satelietselle. Makrofaaginfiltrasie binne die beseerde spier het ook ‘n soorgelyke patroon gevolg. Makrofage het die beseerde area vinniger geïnfiltreer in die kronies PSO-gesupplementeerde groep, maar kon nie die sitokienrespons, wat waargeneem is met akute supplementasie, verklaar nie. Beide kroniese en akute PSO supplementering was verantwoordelik vir ‘n onderdrukte neutrofiel respons, wat ‘n nuwe bevinding is vir hierdie spesifieke anti-oksidant. Die hoof bevindinge in die in vivo rotstudies, is dat PSO instaat is om die neutrofielrespons te onderdruk, en sodoende vroeë makrofaaginfiltrasie teweeg te bring. Om meganismes waarby PSO hierdie voordelige effekte veroorsaak te ondersoek, is verdere analises gedoen om makrofaagfenotipe en neutrofielgetalle in die sirkulasie te bepaal. ‘n In vitro neutrofielmigrasie studie is ook aangewend om PSO se vermoë om neutrofielinfiltrasie in die beseerde area te onderdruk, te ondersoek. Neutrofiele van kontrole rotte, tesame met gekondisioneerde plasma van eksperimentele diere en granulosiet-kolonie stimulerende faktor (G-KSF), is toegelaat om vir twee ure in die teenwoordigheid van ‘n chemotaktiese faktor, N-formiel metionien-leusien-fenielalanien (fMLP) te migreer. Resultate van hierdie studie het aangetoon dat neutrofielmigrasie, in vitro, alleenlik onderdruk word in die teenwoordigheid van gekondisioneerde plasma van PSO-gesupplementeerde rotte. Die studies in hierdie tesis oor die effek van PSO op parameters van spier en die immuunsisteem, het tot die volgende hoofgevolgtrekkings gelei: a) PSO supplementering het vroeë spierherstel, as gevolge van vroeë satelietselaktivering en MHCf sintese, teweeg gebring; b) PSO verkies ‘n anti-inflammatoriese sitokien reaksie, terwyl dit die proinflammatoriese sitokienrespons onderdruk; en c) PSO onderdruk die neutrofielrespons, terwyl vroeë makrofaaginfiltrasie in die beseerde area gefasiliteer word. Die spesifieke meganisme van aksie van PSO, om die neutrofielrespons te onderdruk, kan moontlik die vermoë van neutrofiele om adhesie molekule uit te druk, insluit. Hierdie aanname moet egter verder ondersoek word.

Les IgIV, une préparation thérapeutique d'IgG normales, sont utilisées dans le traitement de diverses maladies auto-immunes et inflammatoires. Les mécanismes par lesquels les IgIV exercent une activité anti-inflammatoire ne sont pas complètement compris. Elles interagissent avec de nombreux composants du système immunitaire et modulent leurs fonctions. Des études récentes ont rapporté que l'hème oxygénase-1 (HO-1) joue un rôle important dans la régulation de la réponse inflammatoire dans un certain nombre de pathologies. Plusieurs agents thérapeutiques exercent des effets anti-inflammatoires grâce à l'induction de l'HO-1. Etant donné le rôle commun anti-inflammatoire de l'HO-1 et des IgIV, j'ai étudié l'implication de l'HO-1 dans les mécanismes d'action des IgIV. J'ai montré que les effets des IgIV ne sont pas associés à l'induction de l'HO-1, que ce soit dans des cellules de l'immunité innée comme les monocytes, cellules dendritiques ou macrophages, ou dans les reins et foie de souris avec une encéphalomyélite auto-immune expérimentale traitées par les IgIV. Des données récentes dans des modèles expérimentaux suggèrent que les IgIV induisent la sécrétion d’IL-4 des basophiles en augmentant l’IL-33 des cellules innées SIGN-R1+. J’ai rapporté que les IgIV induisent directement l’activation de basophiles pré-stimulés avec l’IL-3 alors que contrairement au modèle murin, l’IL-33 n’est pas indispensable. L’activation des basophiles par les IgIV est associée à l’expression augmentée de CD69 et la sécrétion d’IL-4, d’IL-6 et d’IL-8. Ces fonctions sont médiées par les fragments F(ab’)2 qui se lient à des IgE membranaires et activent la voie Syk. / Intravenous immunoglobulin (IVIG), a therapeutic normal immunoglobulin G preparation, is used in the therapy of various autoimmune and inflammatory conditions. The mechanisms by which IVIG exerts anti-inflammatory effects are not completely understood. It interacts with numerous components of the immune system including dendritic cells, macrophages, T and B cells and modulates their functions. Recent studies have reported that heme oxygenase-1 (HO-1) pathway plays an important role in the regulation of inflammatory response in several pathologies. Several therapeutic agents exert anti-inflammatory effects via induction of HO-1. Therefore, in view of common anti-inflammatory role exerted by both HO-1 and IVIG, I investigated if mechanisms of IVIG implicate HO-1. I show that anti-inflammatory effects of IVIG were not associated with an induction of HO-1 either in innate cells such as monocytes, dendritic cells and macrophages or in the kidneys or liver of experimental autoimmune encephalomyelitis. Recent data in experimental models suggest that IVIG induces IL-4 in basophils by enhancing IL-33 in SIGN-R1+ innate cells. I reported that IVIG directly induces activation of IL-3-primed basophils while unlike mice IL-33 was dispensable. The activation of basophils by IVIG was associated with enhanced expression of CD69 and secretion of IL-4, IL-6 and IL-8. These functions of IVIG are mediated via F(ab’)2 fragments that bind to basophil surface IgE and activate Syk pathway.

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 2002</p> / digitalisering@umu

Moving towards label-free technologies is essential for many clinical and research applications. Raman spectroscopy is a powerful tool in the field of biomedicine for label-free cell characterisation and disease diagnosis, owing to its high chemical specificity. However, Raman scattering is a relatively weak process and can require long acquisition times, thus hampering its integration to clinical technologies. Multimodal analysis is currently pushing the boundaries in biomedicine, obtaining more information than would be possible using a single mode and overcoming any limitations specific to a single technique. Digital holographic microscopy (DHM) is a rapid and label-free quantitative phase imaging modality, providing complementary information to Raman spectroscopy, and is thus an ideal candidate for combination in a multimodal system. Firstly, this thesis explores the use of wavelength modulated Raman spectroscopy (WMRS), for the classification of immune cell subsets. Following this a multimodal approach, combining Raman spectroscopy and DHM, is demonstrated, where each technique is considered individually and in combination. The complementary modalities provide a wealth of information (both chemical and morphological) for cell characterisation, which is a step towards achieving a label-free technology for the identification of human immune cells. The suitability of WMRS to discriminate between closely related neuronal cell types is also explored. Furthermore optical spectroscopic techniques are useful for the analysis of food and beverages. The use of Raman and fluorescence spectroscopy to successfully discriminate between various whisky and extra-virgin olive oil brands is demonstrated, which may aid the detection of counterfeit or adulterated samples. The use of a compact Raman device is utilised, demonstrating the potential for in-field analysis. Finally, monodisperse and highly spherical nanoparticles are synthesised. A short study demonstrates the potential for these nanoparticles to benefit the techniques of surface enhanced Raman spectroscopy and optical trapping, by way of minimising variability.

Bacillus anthracis, agent de la maladie du charbon, est aussi un agent majeur de la menace biologique. Sa virulence est liée à deux principaux facteurs : une capsule et deux toxines, la toxine oedémateuse (ET = EF + PA) et la toxine létale (LT = LF + PA). EF est une adénylate cyclase, calcium et calmoduline dépendante, produisant une élévation de la concentration en AMPc intracellulaire tandis que LF est une métalloprotéase à zinc clivant la majorité des Mitogen Activated Protein Kinase Kinases. Les toxines jouent un rôle central dans la pathogénie de la maladie du charbon et dans la dérégulation des fonctions des cellules du système immunitaire. Le cytosquelette d'actine participe activement aux fonctions de phagocytose et de migration des macrophages et des cellules dendritiques.Cependant, peu d'études analysent l'implication du cytosquelette d'actine des cellules immunitaires dans la physiopathologie des toxines. ET induit une rétraction temps-dépendant des cellules dendritiques et des macrophages normalisés sur des micropatterns de fibronectine, s'accompagnant d'une dépolymérisation de l'actine et d'une perte des points d'ancrage des cellules dendritiques. Précocement, ET active la cofiline par l'activation de la voie de signalisation AMPc – PKA – Protéines phosphatases. Malgré ces altérations du cytosquelette d'actine, ET n'induit pas de modification des capacités phagocytaires des cellules dendritiques, à l'exception d'une dérégulation de la maturation des phagosomes. ET conduit également à une augmentation de la migration des cellules dendritiques in vitro par activation et expression de CCR7 et CXCR4 à la surface des cellules dendritiques.A l'inverse, LT conduit à un étalement temps-dépendant des cellules dendritiques normalisées, accompagné d'une dérégulation de la dynamique de l'actine provoquant des regroupements anormaux d'actine filament. LT active les myosines phosphatases via la voie RhoA-ROCK pour déphosphoryler les myosines II. A la différence de ET, LT inhibe les capacités phagocytaires des cellules dendritiques mais ne conduit pas à une modification de la migration des cellules dendritiques in vitro. / Bacillus anthracis, the agent of anthrax, is also a major agent of biological warfare threat. Its virulence is caused by two main factors : the capsule and two toxins, edema toxin (ET = PA + EF) and lethal toxin (LT = LF + PA). EF is a calcium and calmodulin-dependent adenylate cyclase, producing a rise in intracellular cAMP concentration, while LF is a zinc metalloprotease cleaving the majority of Mitogen Activated Protein Kinase Kinases. The toxins play a central role in the pathogenesis of the disease and the deregulation of the functions of immune cells. The actin cytoskeleton is actively participating in the phagocytosis and the migration of macrophages and dendritic cells.However, few studies analyze the involvement of the actin cytoskeleton of immune cells in the pathogenesis of toxins. ET induces a time-dependent retraction of dendritic cells and macrophages on fibronectin micropatterns, accompanied by actin depolymerization and a loss of the anchor points of dendritic cells. ET early activates cofilin by activating the cAMP - PKA - Protein phosphatases signaling pathway. Despite these alterations of the actin cytoskeleton, ET does not induce any change in the phagocytic capacity of dendritic cells, except for a deregulation of the phagosomes maturation. ET also leads to an increase in the migration of dendritic cells in vitro by activation and expression of CCR7 and CXCR4 on the surface of dendritic cells.In contrast, LT results in a time-dependent spreading of micropatterned dendritic cells, accompanied by a dysregulation of actin dynamics causing abnormal combinations of actin filament. LT activates myosin phosphatase via the RhoA-ROCK pathway to dephosphorylate myosin II. Unlike ET, LT inhibits the dendritic cells phagocytosis but does not lead to a change in dendritic cells migration in vitro.