α-Synuclein pathology in very elderly Finns : A population-based clinico-neuropathologic and molecular genetic study of Dementia with Lewy bodies

Populations in developed countries are ageing fast. The elderly have the greatest incidence of de-mentia, and thus the increase in the number of demented individuals, increases the immediate costs for the governments concerning healthcare and hospital treatment. Attention is being paid to disorders behind cognitive impairment with behavioural and psychological symptoms, which are enormous contributors to the hospital care required for the elderly. The highest dreams are in prevention; however, before discovering the tools for preventing dementia, the pathogenesis behind dementia disorders needs to be understood. Dementia with Lewy bodies (DLB), a relatively recently discovered dementia disorder compared to Alzheimer’s disease (AD), is estimated to account for up to one third of primary degenerative dementia, thus being the second most common cause of dementia in the elderly. Nevertheless, the impact of neuropathological and genetic findings on the clinical syndrome of DLB is not fully established. In this present series of studies, the frequency of neuropathological findings of DLB and its relation to the clinical findings was evaluated in a cohort of subjects with primary degenerative dementia and in a population-based prospective cohort study of individuals aged 85 years or older. α-synuclein (αS) immunoreactive pathology classifiable according to the DLB consensus criteria was found in one fourth of the primary degenerative dementia subjects. In the population-based study, the corresponding figure was one third of the population, 38% of the demented and one fifth of the non-demented very elderly Finns. However, in spite of the frequent discovery of αS pathology, its association with the clinical symptoms was quite poor. Indeed, the common clinical features of DLB, hypokinesia and visual hallucinations, associated better with the severe neurofibrillary AD-type pathology than with the extensive (diffuse neocortical) αS pathology when both types of pathology were taken into account. The severity of the neurofibrillary AD-type pathology (Braak stage) associated with the extent of αS pathology in the brain. In addition, the genetic study showed an interaction between tau and αS; common variation in the αS gene (SNCA) associated significantly with the severity of the neurofibrillary AD-type pathology and nominally significantly with the extensive αS pathology. Further, the relevance and temporal course of the substantia nigra (SN) degeneration and of the spinal cord αS pathology were studied in relation to αS pathology in the brain. The linear association between the extent of αS pathology in the brain and the neuron loss in SN suggests that in DLB the degeneration of SN proceeds as the αS pathology extends from SN to the neocortex instead of early destruction of SN seen in Parkinson’s disease (PD). Furthermore, the extent of αS pathology in the brain associated with the severity of αS pathology in the thoracic and sacral autonomic nuclei of the spinal cord. The thoracic αS pathology was more common and more severe compared to sacral cord, suggesting that the progress of αS pathology proceeds downwards from the brainstem towards the sacral spinal cord.

Target Cell Topography and Cytoskeletal Reorganization in Natural Killer Cell Activity

The immune system has to recognize and destroy abnormal or infected cells to maintain homeostasis. Natural killer (NK) cells directly recognize and kill transformed or virus-infected cells without prior sensitization. We have studied both virus-infected and tumor cells in order to identify the target structures involved in triggering NK activity. Mouse/human cell hybrids containing various human chromosomes were used as targets. The human chromosome responsible for activating NK cell killing was identified to chromosome number 6. The results suggest that activated NK cells recognize ligands that are encoded on human chromosome 6. We showed that the ligand on the target cell side was intercellular adhesion molecule 2 (ICAM-2). There was no difference in the level of expression of ICAM-2, however, but a drastic difference was seen in the distribution of the molecule: ICAM-2 was evenly distributed on the surface of the NK-resistant cells, but almost totally redistributed to the tip of uropods, bud-like extensions, which were absent from the parental cells. Interestingly, the gene coding for cytoskeletal linker protein ezrin has been localized to human chromosome 6, and there was a colocalization of ezrin and ICAM-2 in the uropods. Furthermore, the transfected human ezrin into NK cell-resistant cells induced uropod formation, ICAM-2 and ezrin redistribution to newly formed uropods, and sensitized target cells to NK cell killing. These data reveal a novel form of NK cell recognition: target structures are already present on normal cells; they become detectable only after abnormal redistribution into hot spots on the target cell membrane. NK cells are central players in the defence against virus infections. They inhibit the spread of infection, allowing time for specific immune responses to develop. The virus-proteins that directly activate human NK cell killing are largely unknown. We studied the sensitivity of virus-specific early proteins of Semliki Forest virus (SFV) to NK killing. The viral non-structural proteins (nsP1-4) translated early in the virus cycle were transfected in NK-resistant cells. Viral early gene nsP1 alone efficiently sensitized target cells to NK activity, and the tight membrane association of nsP1 seems to be critical in the triggering of NK killing. NsP1 protein colocalized with (redistributed) ezrin in filopodia-like structures to which the NK cells were bound. The results suggest that also in viral infections NK cells react to rapid changes in membrane topography. Based on the results of this thesis, a new model of target cell recognition of NK cells can be suggested: reorganization of the cytoskeleton induces alterations in cell surface topography, and this new pattern of surface molecules is recognized as "altered-self".

Prognostic significance of cyclooxygenase-2 and associated molecules in gastric cancer

Background and aims: Low stage and curative surgery are established factors for improved survival in gastric cancer. However, not all low-stage patients have a good prognosis. Cyclooxygenase-2 (COX-2) is known to associate with reduced survival in several cancers, and has been shown to play an important role in gastric carcinogenesis. Since new and better prognostic markers are needed for gastric cancer, we studied the prognostic significance of COX-2 and of markers that associate with COX-2 expression. We also studied markers reflecting proliferation and apoptosis, and evaluated their association with COX-2. Our purpose was to construct an accurate prognostic model by combining tissue markers and clinicopathogical factors. Materials and methods: Of 342 consecutive patients who underwent surgery for gastric cancer at Meilahti Hospital, Helsinki University Central Hospital, 337 were included in this study. Low stages I to II were represented by 141 (42%) patients, and high stages III to IV by 196 (58%). Curative surgery was performed on 176 (52%) patients. Survival data were obtained from the national registers. Slides from archive tissue blocks were prepared for immunohistochemistry by use of COX-2, human antigen R (HuR), cyclin A, matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), and Ki-67 antibodies. Immunostainings were scored by microscopy, and scores were entered into a database. Associations of tumor markers with clinicopathological factors were calculated, as well as associations with p53, p21, and results of flow cytometry from earlier studies. Survival analysis was performed by the Kaplan-Meier method, and Cox multivariate models were reconstructed. Cell culture experiments were performed to explore the effect of small interfering (si)RNA of HuR on COX-2 expression in a TMK-1 gastric cancer cell line. Results: Overall 5-year survival was 35.1%. Study I showed that COX-2 was an independent prognostic factor, and that the prognostic impact of COX-2 was more pronounced in low-stage patients. Cytoplasmic HuR expression also associated with reduced survival in gastric cancer patients in a non-independent manner. Cell culture experiments showed that HuR can regulate COX-2 expression in TMK-1 cells in vitro, with an association also between COX-2 and HuR tissue expression in a clinical material. In Study II, cyclin A was an independent prognostic factor and was associated with HuR expression in the gastric cancer material. The results of Study III showed that epithelial MMP-2 associated with survival in univariate, but not in multivariate analysis. However, MMP-9 showed no prognostic value. MMP-2 expression was associated with COX-2 expression. In Study IV, the prognostic power of COX-2 was compared with that of all tested markers associated with survival in Studies I to III, as well as with p21, p53, and flow cytometry results. COX-2 and p53 were independent prognostic factors, and COX-2 expression was associated with that of p53 and Ki-67 and also with aneuploidy. Conclusions: COX-2 is an independent prognostic factor in gastric cancer, and its prognostic power emerges especially in low stage cancer. COX-2 is regulated by HuR, and is associated with factors reflecting invasion, proliferation, and apoptosis. In an extended multivariate model, COX-2 retained its position as an independent prognosticator. COX-2 can be considered a promising new prognostic marker in gastric cancer.

Toxoplasma gondii - alkueläimen esiintyvyys suomalaisissa lampaissa

Lisensiaatin tutkielma koostuu kirjallisuuskatsauksesta, jossa käsitellään Toxoplasma gondii – alkueläintä ja sen vaikutusta lampaisiin ja ihmisiin sekä kokeellisesta osasta, jossa tutkittiin T. gondii – alkueläin vasta-aineiden esiintyvyyttä lampailla Suomessa. T. gondii on laajalle levinnyt zoonoottinen alkueläin, jonka pääisäntänä toimivat kissaeläimet ja väli-isäntinä lähes kaikki tasalämpöiset eläimet. T. gondii voi aiheuttaa vakavia seurauksia mm. lampaalle sekä ihmiselle. Toksoplasmoosi aiheuttaa lampaalle ohimenevän kuumeen sekä mahdollisesti abortointeja ja sikiökuolemia, mikäli tartunta on saatu tiineyden aikana. T. gondii – alkueläimen aiheuttamat infektiot ovat hyvin yleisiä ihmisillä, mutta kliininen tauti rajoittuu suurilta osin riskiryhmiin. Raskauden aikana saatu toksoplasmoosi voi aiheuttaa sikiövaurioita myös ihmisellä. T. gondii - alkueläimellä infektoitunut lampaanliha on eräs mahdollinen lähde ihmisten tartunnoille. Euroopan elintarviketurvallisuusviranomainen, EFSA, suosittelee, että loista tulisi alkaa monitoroida lampailla, kun sopivat serologiset menetelmät ovat saatavilla. T. gondii - alkueläimen esiintyvyydestä lampailla Suomessa ei ole aiempaa tietoa, mutta sen oletettiin olevan samankaltainen kuin muissa Pohjoismaissa. Ruotsissa alkueläintä löytyy 19 % lampaista ja Norjassa 16 %. T. gondii – alkueläimen esiintyvyys määritettiin tutkimalla 1940 lammasseerumia kaupallisella suoralla agglutinaatiotestillä (Toxo-Screen DA). Näytteet ovat kerätty ympäri Suomea 97 tilalta ja jokaiselta tilalta tutkittiin 20 näytettä. Käytetty testi havaitsee IgG- luokan T. gondii vasta-aineet seeruminäytteestä agglutinaation avulla. T. gondii – vasta-aineita löytyi maan laajuisesti 477 lampaalta 1940 tutkitusta eli seroprevalenssi on 24,6 %. Tuloksen 95 %:n luottamusväli on 22,7% – 26,5%. Matalin esiintyvyys alkueläimellä oli Lapin läänissä ja korkein Ahvenanmaalla. Seroprevalenssitulos on oletettua suuruusluokkaa. Tutkituista tiloista 76 %:lla löytyi ainakin yksi lammas, jolta havaittiin vasta-aineita loista vastaan. Suhteellisesti eniten tiloja, jossa oli ainakin yksi seropositiivinen lammas, oli Itä-Suomen läänissä ja vähiten Lapin läänissä. Tutkimuksessa tutkittiin vain yli vuoden ikäisiä lampaita, joten karitsojen T. gondii - vasta-aineiden esiintyvyydestä ei saatu tietoa. Se on yleensä aikuisia lampaita matalampi. Tutkimuksen tulokset osoittavat, että T. gondii – alkueläintartuntoja esiintyy Suomessa lampailla ja että lampaat altistuvat T. gondii – alkueläimelle varsinkin eteläisimmissä osissa Suomea. Suomalainen lampaan liha on potentiaalinen tartunnan lähde ihmisten T. gondii – infektioille, mikäli lihaa ei käsitellä tavoilla, jotka tuhoavat loisen, esimerkiksi riittävällä kuumennuksella tai pakastamalla.

Progression of diffuse gliomas - from the first diagnosis to recurrence

Gliomas are the most frequent primary brain tumours. The cardinal features of gliomas are infiltrative growth pattern and progression from low-grade tumours to a more malignant phenotype. These features of gliomas generally prevent their complete surgical excision and cause their inherent tendency to recur after initial treatment and lead to poor long-term prognosis. Increasing knowledge about the molecular biology of gliomas has produced new markers that supplement histopathological diagnostics. Molecular markers are also used to evaluate the prognosis and predict therapeutic response. The purpose of this thesis is to study molecular events involved in the malignant progression of gliomas. Gliomas are highly vascularised tumours. Contrast enhancement in magnetic resonance imaging (MRI) reflects a disrupted blood-brain barrier and is often seen in malignant gliomas. In this thesis, 62 astrocytomas, oligodendrogliomas and oligoastrocytomas were studied by MRI and immunohistochemistry. Contrast enhancement in preoperative MRI was associated with angiogenesis, tumour cell proliferation and histological grade of gliomas. Activation of oncogenes by gene amplification is a common genetic aberration in gliomas. EGFR amplification on chromosome 7p12 occurs in 30-40% of glioblastomas. PDGFRA, KIT and VEGFR2 are receptor tyrosine kinase genes located on chromosome 4q12. Amplification of these genes was studied using in situ hybridisation in the primary and recurrent astrocytomas, oligodendrogliomas and oligoastrocytomas of 87 patients. PDGFRA, KIT or VEGFR2 amplification was found in 22% of primary tumours and 36% of recurrent tumours including low-grade and malignant gliomas. The most frequent aberration was KIT amplification, which occurred in 10% of primary tumours and in 27% of recurrent tumours. The expression of ezrin, cyclooxygenase 2 (COX-2) and HuR was studied immunohistochemically in a series of primary and recurrent gliomas of 113 patients. Ezrin is a cell membrane-cytoskeleton linking-protein involved in the migration of glioma cells. The COX-2 enzyme is implicated in the carcinogenesis of epithelial neoplasms and is overexpressed in gliomas. HuR is an RNA-stabilising protein, which regulates the expression of several proteins including COX-2. Ezrin, COX-2 and HuR were associated with histological grade and the overall survival of glioma patients. However, in multivariate analysis they were not independent prognostic factors. In conclusion, these results suggest that contrast enhancement in MRI can be used as a surrogate marker for the proliferative and angiogenic potential of gliomas. Aberrations of PDGFRA, KIT and VEGFR2 genes, as well as the dysregulated expression of ezrin, COX-2 and HuR proteins, are linked to the progression of gliomas.

Temperature-Sensitive Src-Transformed Mdck Cells nn 3d Cultures as a Model of Malignant Transformation of Epithelial Cells

The equilibrium between cell proliferation, differentiation, and apoptosis is crucial for maintaining homeostasis in epithelial tissues. In order for the epithelium to function properly, individual cells must gain normal structural and functional polarity. The junctional proteins have an important role both in binding the cells together and in taking part in cell signaling. Cadherins form adherens junctions. Cadherins initiate the polarization process by first recognizing and binding the neighboring cells together, and then guiding the formation of tight junctions. Tight junctions form a barrier in dividing the plasma membranes to apical and basolateral membrane domains. In glandular tissues, single layered and polarized epithelium is folded into tubes or spheres, in which the basal side of the epithelial layer faces the outer basal membrane, and the apical side the lumen. In carcinogenesis, the differentiated architecture of an epithelial layer is disrupted. Filling of the luminal space is a hallmark of early epithelial tumors in tubular and glandular structures. In order for the transformed tumor cells to populate the lumen, enhanced proliferation as well as inhibition of apoptosis is required. Most advances in cancer biology have been achieved by using two-dimensional (2D) cell culture models, in which the cells are cultured on flat surfaces as monolayers. However, the 2D cultures are limited in their capacity to recapitulate the structural and functional features of tubular structures and to represent cell growth and differentiation in vivo. The development of three-dimensional (3D) cell culture methods enables the cells to grow and to be studied in a more natural environment. Despite the wide use of 2D cell culture models and the development of novel 3D culture methods, it is not clear how the change of the dimensionality of culture conditions alters the polarization and transformation process and the molecular mechanisms behind them. Src is a well-known oncogene. It is found in focal and adherens junctions of cultured cells. Active src disrupts cell-cell junctions and interferes with cell-matrix binding. It promotes cell motility and survival. Src transformation in 2D disrupts adherens junctions and the fibroblastic phenotype of the cells. In 3D, the adherens junctions are weakened, and in glandular structures, the lumen is filled with nonpolarized vital cells. Madin-Darby canine kidney (MDCK) cells are an epithelial cell type commonly used as a model for cell polarization. Its-src-transformed variants are useful model systems for analyzing the changes in cell morphology, and they play a role in src-induced malignant transformation. This study investigates src-transformed cells in 3D cell cultures as a model for malignant transformation. The following questions were posed. Firstly: What is the role of the composition and stiffness of the extracellular matrix (ECM) on the polarization and transformation of ts v-src MDCK cells in 3D cell cultures? Secondly: How do the culture conditions affect gene expression? What is the effect of v-src transformation in 2D and in 3D cell models? How does the shift from 2D to 3D affect cell polarity and gene expression? Thirdly: What is the role of survivin and its regulator phosphatase and tensin homolog protein (PTEN) in cell polarization and transformation, and in determining cell fate? How does their expression correlate with impaired mitochondrial function in transformed cells? In order to answer the above questions, novel methods of culturing and monitoring cells had to be created: novel 3D methods of culturing epithelial cells were engineered, enabling real time monitoring of a polarization and transformation process, and functional testing of 3D cell cultures. Novel 3D cell culture models and imaging techniques were created for the study. Attention was focused especially on confocal microscopy and live-cell imaging. Src-transformation disturbed the polarization of the epithelium by disrupting cell adhesion, and sensitized the cells to their environment. With active src, the morphology of the cell cluster depended on the composition and stiffness of the matrix. Gene expression studies revealed a broader impact of src transformation than mere continuous activity of src-kinase. In 2D cultures, src transformation altered the expression of immunological, actin cytoskeleton and extracellular matrix (ECM). In 3D, the genes regulating cell division, inhibition of apoptosis, cell metabolism, mitochondrial function, actin cytoskeleton and mechano-sensing proteins were altered. Surprisingly, changing the culture conditions from 2D to 3D affected also gene expression considerably. The microarray hit survivin, an inhibitor of apoptosis, played a crucial role in the survival and proliferation of src-transformed cells.

Trichinella spiralis ja Trichinella nativa -loisten erittyminen ulosteisiin rotalla kokeellisen infektion jälkeen

Trichinella-suvun loiset ovat maailmanlaajuisesti levinneitä sukkulamatoja, jotka ovat infektiivisiä useille eläinlajeille ja tarttuvat myös ihmiseen. Loiset aiheuttavat ongelmia muun muassa lihateollisuudessa, haittaavat tuotantoeläinten terveyttä ja ovat elintarviketurvallisuusriski. Eri Trichinella-lajien infektiivisyys eri isäntäeläinlajeissa vaihtelee. Esimerkiksi Trichinella spiralis aiheuttaa rotassa voimakkaamman infektion kuin Trichinella nativa, mutta syytä loislajien erilaiseen infektiviteettiin samassa isäntäeläinlajissa ei tiedetä. Trichinella-loisten elämänkiertoon kuuluu sekä enteraali- eli suolistovaihe että parenteraalivaihe eli suoliston ulkopuolella tapahtuva vaihe. Vielä on epävarmaa, missä vaiheessa elämänkiertoa loislajien selviytyminen rotassa eroaa toisistaan. Tutkielmani kokeellisen osuuden tarkoituksena oli selvittää rotan ulosteita tutkimalla, kiinnittyykö toinen tutkituista Trichinella-lajeista (T. spiralis tai T. nativa) paremmin suolen seinämään ja tuleeko toinen nopeammin ulos suolesta. Mikäli rotan heikosti infektoivat T. nativa -loiset tulevat T. spiralis -loisia nopeammin ulosteen mukana ulos suolistosta, voidaan olettaa suolistovaiheen immuunipuolustuksen olevan ainakin osatekijä rotan kyvyssä puolustautua T. nativa –infektioita vastaan. Työ suoritettiin infektoimalla kuusi rottaa T. spiralis -loisella ja kuusi rottaa T. nativa -loisella. Lisäksi tutkimuksessa oli mukana kolme kontrollirottaa, joita ei infektoitu. Rottien ulosteet kerättiin seitsemän viikon ajalta, ja näytteet tutkittiin FLOTAC-menetelmällä. Ulosteista etsittiin Trichinella-loisten aikuis- ja toukkamuotoja. Ulostenäytteistä ei löytynyt yhtään loista. Kokeen jälkeen rotat lopetettiin ja niiden suolet tutkittiin, mutta suolistakaan ei löytynyt loisia. Lopetettujen eläinten lihasnäytteitä tutkimalla eläinten todettiin infektoituneen kyseessä olleelle loislajille tyypillisellä voimakkuudella. Kontrollirotista ei löydetty loisia. Koska rottien ulosteista tai suolista ei löytynyt loisia huolimatta onnistuneista infektoinneista, voidaan todeta käytetyn menetelmän olleen kokeeseen sopimaton. Mikäli loisia olisi löytynyt ulosteista, olisi ollut tarpeellista verrata eri lajeilla infektoitujen ryhmien tuloksia. Tieto siitä, tapahtuuko rotan suolistossa jotain, mikä heikentää toisen Trichinella-lajin infektiivisyyttä, olisi ollut merkittävä. Saadut tulokset olisivat olleet hyödyksi pohdittaessa parempia keinoja Trichinella-tartuntojen ennaltaehkäisyyn ja infektioiden hoitoon.

Role of CIP2A in carcinogenesis

Worldwide and notably in the developed countries, cancer is an increasing cause of morbidity and mortality, being the second most common cause of death after ischemic heart disease. Now and in the future new cancer cases need to be diagnosed earlier. Prognostic factors may be helpful in recognizing and handling those patients who need more aggressive therapy, and it is also desirable to predict treatment response accurately. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein predominantly expressed in malignant tissues and inhibiting protein phosphatase 2A (PP2A) activity; it is a promising target for cancer therapy. The aim of this thesis was to evaluate the prognostic role of CIP2A in solid cancers, and for this purpose to explore expression of CIP2A, and investigating regulation of CIP2A in order to gain insight into signalling pathways leading to alteration in prognosis. Patients diagnosed with gastric, serous ovarian, tongue, or colorectal cancer at Helsinki University Central Hospital were included. Tumour tissue microarrays assembled from specimens from these patients were prepared and stained immunohistochemically for CIP2A protein expression. Associations with clinicopathologic parameters and other biomarkers were explored, and survival analyses were done according to the Kaplan-Meier method. Study of the role of CIP2A in intracellular signalling in vitro involved gastric, ovarian, and tongue cancer cell lines. We found CIP2A to be highly expressed in gastric, ovarian, tongue, and colorectal cancer specimens. CIP2A was associated with clinicopathologic parameters characterizing an aggressive disease, namely advanced stage, high grade, p53 immunopositivity, and high proliferation index. CIP2A led to recognition of gastric, ovarian, and tongue cancer patients with poor prognosis, however, with a cancer type-specific cut-off level for prognostic significance. In tongue cancer, it served as an independent prognostic marker. In contrast, in colorectal cancer, CIP2A provided no prognostic value. In cancer cell lines, CIP2A was highly expressed at both protein and mRNA levels, and promoted cell proliferation and anchorage-independent growth. In gastric cancer, we demonstrated with a MYCER construct in mouse embryo fibroblasts that activation of MYC led to increased CIP2A mRNA expression, and hence we suggested that a positive feedback mechanism between CIP2A and MYC may potentiate and prolong the oncogenic activity of these proteins. We demonstrated in ovarian cancer an association between CIP2A and EGFR protein overexpression and EGFR gene amplification. In ovarian and tongue cancer cells we showed that depletion of EGFR downregulates CIP2A expression. In conclusion, high CIP2A expression occurred frequently among patients with aggressive disease. CIP2A may serve as a prognostic marker in gastric, ovarian, and tongue cancer and thus may help in tailoring therapy for cancer patients. The positive feedback mechanism between CIP2A and MYC, as well as the positive regulation of CIP2A by EGFR, are a few signalling pathways regulating and regulated by CIP2A. These and other mechanisms need to be studied further, however. CIP2A is a potential target for therapy, and its potential role as predictive marker and as a tumour marker in serum requires exploration.