The effects of selective estrogen receptor modulators on the death of breast cancer cells and osteoblastic cells

Selektiivisten estrogeenireseptorin muuntelijoiden (serm) vaikutus rintasyöpäsolujen ja luun solujen kuolemaan Selektiiviset estrogeenireseptorin muuntelijat (SERMit) ovat ryhmä kemialliselta rakenteeltaan erilaisia yhdisteitä jotka sitoutuvat solunsisäisiin estrogeenireseptoreihin toimien joko estrogeenin kaltaisina yhdisteinä tai estrogeenin vastavaikuttajina. Tamoksifeeni on SERM –yhdiste, jota on jo pitkään käytetty estrogeenireseptoreita (ER) ilmentävän rintasyövän lääkehoidossa. Tamoksifeeni sekä estää rintasyöpäsolujen jakaantumista että toisaalta aikaansaa niiden apoptoosin eli ohjelmoidun solukuoleman muuntelemalla ER-välitteisesti kohdesolun geenien ilmentymistä. Viimeaikaiset tutkimustulokset ovat kuitenkin osoittaneet tamoksifeenilla olevan myös nopeampia, nongenomisia vaikutusmekanismeja. Tässä väitöskirjatyössä tutkimme niitä nopeita vaikutusmekanismeja joiden avulla tamoksifeeni vaikuttaa rintasyöpäsolujen elinkykyyn. Osoitamme että tamoksifeeni farmakologisina pitoisuuksina aikaansaa nopean mitokondriaalisen solukuolemaan johtavan signallointireitin aktivoitumisen rintasyöpäsoluissa. Tämän lisäksi tutkimme myös tamoksifeenin aiheuttamaan mitokondriovaurioon johtavia tekijöitä. Tutkimustuloksemme osoittavat että ER-positiivisissa rintasyöpäsoluissa tamoksifeeni indusoi pitkäkestoisen ERK-kinaasiaktivaation, joka voidaan estää 17-beta-estradiolilla. Tamoksifeenin aikaansaama nopea solukuolema on pääosin ER:sta riippumaton tapahtuma, mutta siihen voidaan vaikuttaa myös ER-välitteisin mekanismein. Sen sijaan epidermaalisen kasvutekijäreseptorin (EGFR) voitiin osoittaa osallistuvan tamoksifeenin nopeiden vaikutusten välittämiseen. Tämän lisäksi vertailimme myös estradiolin ja eri SERM-yhdisteiden kykyä suojata apoptoosilta käyttämällä osteoblastiperäisiä soluja. Pytyäksemme vertailemaan ER-isotyyppien roolia eri yhdisteiden suojavaikutuksissa, transfektoimme U2OS osteosarkoomasolulinjan ilmentämään pysyvästi joko ERalfaa tai ERbetaa. Tulostemme mukaan sekä estradioli että uusi SERM-yhdiste ospemifeeni suojaavat osteoblastin kaltaisia soluja etoposidi-indusoidulta apoptoosilta. Sekä ERalfa että ERbeta pystyivät välittämään suojavaikutusta, joskin vaikutukset erosivat toisistaan. Lisäksi havaitsimme edellä mainitun suojavaikutuksen olevan yhteydessä muutoksiin solujen sytokiiniekspressiossa. Tietoa SERM-yhdisteiden anti-ja proapoptoottisten vaikutusmekanismeista eri kohdekudoksissa voidaan mahdollisesti hyödyntää kehiteltäessä uusia kudosspesifisiä SERM-yhdisteitä.

Virulence properties of Aggregatibacter actinomycetemcomitans biofilm and characterisation of its putative cytokine exploitation

Biofilms are surface-attached multispecies microbial communities that are embedded by their self-produced extracellular polymeric substances. This lifestyle enhances the survival of the bacteria and plays a major role in many chronic bacterial infections. For instance, periodontitis is initiated by multispecies biofilms. The phases of active periodontal tissue destruction and notably increased levels of proinflammatory mediators, such as the key inflammatory mediator interleukin (IL)-1beta, are typical of the disease. The opportunistic periodontal pathogen Aggregatibacter actinomycetemcomitans is usually abundant at sites of aggressive periodontitis. Despite potent host immune system responses to subgingival invaders, A. actinomycetemcomitans is able to resist clearance attempts. Moreover, some strains of A. actinomycetemcomitans can generate genetic diversity through natural transformation, which may improve the species’ adjustment tothe subgingival environment in the long term. Some biofilm forming species are known to bind and sense human cytokines. As a response to cytokines, bacteria may increase biofilm formation and alter their expression of virulence genes. Specific outer membrane receptors for interferon-γ or IL-1β have been characterised in two Gram-negative pathogens. Because little is known about periodontal pathogens’ ability to sense cytokines, we used A. actinomycetemcomitans as a model organism to investigate how the species responds to IL-1beta. The main aims of this thesis were to explore cytokine binding on single-species A. actinomycetemcomitans biofilms and to determine the effects of cytokines on the biofilm formation and metabolic activity of the species. Additionally, the cytokine’s putative internalisation and interaction with A. actinomycetemcomitans proteins were studied. The possible impact of biofilm IL-1beta sequestering on the proliferation and apoptosis of gingival keratinocyte cells was evaluated in an organotypic mucosa co-culture model. Finally, the role of the extramembranous domain of the outer membrane protein HofQ (emHofQ) in DNA binding linked to DNA uptake in A. actinomycetemcomitans was examined. Our main finding revealed that viable A. actinomycetemcomitans biofilms can bind and take up the IL-1β produced by gingival cells. At the sites of pathogen-host interaction, the proliferation and apoptosis of gingival keratinocytes decreased slightly. Notably, the exposure of biofilms to IL-1beta caused their metabolic activity to drop, which may be linked to the observed interaction of IL-1beta with the conserved intracellular proteins DNA binding protein HU and the trimeric form of ATP synthase subunit beta. A Pasteurellaceaespecific lipoprotein, which had no previously determined function, was characterized as an IL-1beta interacting membrane protein that was expressed in the biofilm cultures of all tested A. actinomycetemcomitans strains. The use of a subcellular localisation tool combined with experimental analyses suggested that the identified lipoprotein, bacterial interleukin receptor I (BilRI), may be associated with the outer membrane with a portion of the protein oriented towards the external milieu. The results of the emHofQ study indicated that emHofQ has both the structural and functional capability to bind DNA. This result implies that emHofQ plays a role in DNA assimilation. The results from the current study also demonstrate that the Gram-negative oral species appears to sense the central proinflammatory mediator IL-1beta.

Immunomodulation of allergic immune response during specific immunotherapy

Atopic, IgE-mediated allergies are one of the major public health problems in Finland and other Western countries. These diseases are characterized by type 2 T helper (Th2) cell predominated immune responses (interleukin-4 (IL-4), IL-5) against ubiquitous environmental allergens. Despite of adequate pharmacological treatment, more than 20% of the patients with allergic rhinitis develop asthma. Allergen specific immunotherapy (SIT) is the only treatment currently available to affect to the natural course of allergic diseases. This treatment involves repeated administration of allergens to the patients either via sublingual route (sublingual immunotherapy, SLIT) or by subcutaneous injections (subcutaneous immunotherapy, SCIT). Successful treatment with SCIT or SLIT has been shown to provide long-term remission in symptoms, and prevent disease progression to asthma, but the immunological mechanisms behind these beneficial effects are not yet completely understood. Increased knowledge of such mechanisms could not only help to improve SIT efficacy, but also provide tools to monitor the development of clinical response to SIT in individual patients, and possibly also, predict the ultimate therapeutic outcome. The aim of this work was to clarify the immunological mechanisms associated with SIT by investigating the specific allergen-induced immune responses in peripheral blood mononuclear cells (PBMC) of allergic rhinitis patients during the course of SLIT and SCIT. The results of this work demonstrate that both therapies induced increases in the protective, Th2-balancing Th1 type immune responses in PBMC, e.g. by up-regulating signaling lymphocytic activation molecule (SLAM) and interferon gamma (IFN-γ) expression, and augmented tolerogenic T regulatory (Treg) cell type responses against the specific allergens, e.g. by increasing IL-10 or Forkhead box P3 (FOXP3) expression. The induction of allergen-specific Th1 and Treg type responses during SLIT were dependent on the treatment dose, favoring high allergen dose SLIT. During SCIT, the early decrease in Th2 type cytokine production - in particular of IL-4 mRNA and IL-4/IFN-γ expression ratio - was associated with the development of good therapeutic outcome. Conversely, increases in both Th2 (IL-5) and Th1 (IFN-γ, SLAM) type responses and IL-10 mRNA production were seen in the patients with less effective outcome. In addition, increase in Th17 type cytokine (IL-17) mRNA production was found in the PBMC of patients with less effective outcome during both SLIT and SCIT. These data strengthen the current hypothesis that immunomodulation of allergen-specific immune responses from the prevailing Th2-biased responses towards a more Th1 type, and induction of tolerogenic Treg cells producing IL-10 represent the two key mechanisms behind the beneficial effects of SIT. The data also give novel insight into the mechanisms why SIT may fail to be effective in some patients by demonstrating a positive correlation between the proinflammatory IL-17 responses, Th2 type IL-5 production and clinical symptoms. Taken together, these data indicate that the analysis of Th1, Th2, Treg ja Th17-associated immune markers such as IL-10, SLAM, IL-4, IL-5 and IL-17 could provide tools to monitor the development of clinical response to SIT, and thereby, predict the ultimate clinical outcome already in the early course of the treatment.