Browsing by department "Institute of Biomedicine"

Respiratory distress syndrome is a major cause of neonatal morbidity and mortality in premature infants caused by immaturity of the lungs. In recent years lamellar body count (LBC) has replaced phospholipid tests as the primary fetal lung maturity assessment method. Our aim was to develop new reference values for LBC and compare the performance of LBC and phospholipids. We also wanted to estimate the impact of new references on the costs and accuracy of the fetal lung maturity determination. Data was collected on all mothers who had LBC testing at Women's Hospital (Helsinki University Central Hospital) during 2009-2011 (n=299) and their fetuses. As in other recent studies, LBC was found to perform slightly better than phospholipids. Specificity and sensitivity values for LBC were calculated with ROC analysis and the results led to the recommendation of a single cutoff value for LBC and the discontinuation of phospholipid testing in non-diabetic mothers.

Basilar artery occlusion (BAO) is one of the most severe neurological diseases associated with high mortality and morbidity. According to previous studies, aetiology, risk factors and outcome of BAO differ from other stroke subtypes, and little information exists on the characteristics of young adult BAO patients. This study is a retrospective observational case-control study of a large registry of consecutive young adult stroke patients (aged 15 to 49 years) treated in the Helsinki University Central Hospital between 1994 and 2007, first describing in detail the risk factors, aetiology, symptom severity and subsequent mortality and disability of young adult BAO-patients, and comparing them with other young adult stroke patients. We found that smoking is significantly less common in BAO patients than in non-BAO-patients (25% vs 45.2%, respectively), large artery atherosclerosis and vertebral or basilar artery dissection are significantly more common in BAO-patients than in non-BAO-patients (25% vs 7.8% and 32.2% vs 7.8%) whereas cardioembolisms, small vessel disease, and internal carotid artery dissection were significantly less common in BAO-patients. Stroke symptoms were significantly more difficult in BAO patients vs non-BAO patients (NIHSS medians 9 [range 0-35] vs 3 [range 0-30]) and follow-up disability defined by the modified Rankin Scale was significantly higher in BAO -patients (good outcome of mRS 0-2 in 53.6% of BAO patients vs 82.3% in non-BAO-patients at 3 months) along with mortality (BAO vs non-BAO-patients 28.6% vs 14.7%). Further development in diagnostics, treatments and rehabilitation is needed to manage BAO in young adult patients.

Introduction: Patients with rheumatoid arthritis (RA) have disturbances in the hypothalamic-pituitary-adrenal (HPA) axis. These are reflected in altered circadian rhythm of circulating serum cortisol, melatonin and IL-6 levels and in chronic fatigue. We hypothesized that the molecular machinery responsible for the circadian timekeeping is perturbed in RA. The aim of this study was to investigate the expression of circadian clock in RA. Methods: Gene expression of thirteen clock genes was analyzed in the synovial membrane of RA and control osteoarthritis (OA) patients. BMAL1 protein was detected using immunohistochemistry. Cell autonomous clock oscillation was started in RA and OA synovial fibroblasts using serum shock. The effect of pro-inflammatory stimulus on clock gene expression in synovial fibroblasts was studied using IL-6 and TNF-a. Results: Gene expression analysis disclosed disconcerted circadian timekeeping and immunohistochemistry revealed strong cytoplasmic localization of BMAL1 in RA patients. Perturbed circadian timekeeping is at least in part inflammation independent and cell autonomous, because RA synovial fibroblasts display altered circadian expression of several clock components, and perturbed circadian production of IL-6 and IL-1b after clock resetting. However, inflammatory stimulus disturbs the rhythm in cultured fibroblasts. Throughout the experiments ARNTL2 and NPAS2 appeared to be the most affected clock genes in human immune-inflammatory conditions. Conclusion: We conclude that the molecular machinery controlling the circadian rhythm is disturbed in RA patients.

In our research we studied if the overexpression of VEGF-A would save the impaired tumor growth in PROX1 silenced colorectal cancer xenografts. We conducted both small hairpin PROX1 and VEGF-A gene transfers to SW1222 colorectal cancer cells using lentiviral vectors. SW1222 cell lines were then injected to Nod scid gamma mice and grown for 14 days for analysis. We also established 3D cocultures of genetically modified SW1222 cell line together with lymphatic endothelial cells or blood endothelial cells to analyze lymphangiogenesis and angiogenesis in vitro. We found out that overexpression of VEGF-A rescues the growth of PROX1 silenced tumors. Large necrotic areas in the central of the PROX1 silenced tumors remain even though VEGF-A overexpression induces greater vascularity in these xenografts. In 3D cocultures silencing of PROX1 did not affect in vitro lymphangiogenesis or angiogenesis.

Tutkimuksessa tutkittiin NR4A-ryhmän tumareseptoreiden määrän muutosta maksakudoksessa energiarajoituksen ja resveratrolin vaikutuksista. Tutkimus tehtiin hiirillä ja laboratoriomenetelmänä oli Western Blot. NR4A-tumareseptoriryhmään kuuluvat Nur77, Nurr1 ja NOR-1. Ne säätelevät useissa kudoksissa useita eri solunsisäisiä tapahtumia. Maksassa ne säätelevät eri metabolisia prosesseja, etenkin glukoneogeneesiä ja lipogeneesiä. Lipogeneesillä on merkitystä rasvamaksan syntyyn ja glukoneogeneesillä taas on merkitystä veren sokeripitoisuudelle ja lisääntynyt glukoneogeneesi on merkittävä tekijä Tyypin 2 Diabetes Mellitus-taudissa esiintyvän hyperglykemian kannalta. Saamiemme tulosten mukaan Nur77:n määrä nousee selvästi energiarajoituksen vaikutuksesta runsasrasvaista ravintoa syöneeseen kontrolliryhmään verrattuna. NOR-1:n määrä nousee selvästi suurella (4 g/kg) rehupitoisuudella resveratrolia. Nurr1:n tuloksissa ei löytynyt statistisesti merkittäviä eroja ryhmien välillä. Tulkintani mukaan resveratroli on potentiaalinen maksan rasvoittumisen estäjä ja glukoneogeneesiä lisäävä aine stimuloimalla NR4A-ryhmän tumareseptoreita maksassa. Täten resveratroli ei ainakaan hepatosyyttien NR4A-reitin kannalta selitä sitä tosiasiaa että resveratroli vähentää diabeettista hyperglykemiaa.

Recently, our research group together with the Hematology Research Unit Helsinki, found that 31 of 77 patients (40%) with T-cell large granular lymphocytic leukemia have somatic point mutations in the Src Homology 2 (SH2) domain of the STAT3 gene. LGL leukemia is a rare and indolent disease characterized by the clonal expansion of large granular lymphocytes of unknown etiology. The aim of this master's thesis study was to elucidate whether the identified Y640F and D661V mutations can cause hyperactivity of the STAT3 protein and excessive proliferation of T-cells. STAT3 is a transcription factor that is known to have a key role in cell proliferation and apoptosis, and which is activated by the phosphorylation of receptor-associated kinases. The phosphorylation of tyrosine residue 705 in STAT3 induces dimerization and localization of the STAT3 dimer to the nucleus. In several cancers STAT3 protein has been reported to be constitutively active. Furthermore, previously published data strongly support our hypothesis that the mutations identified in LGL patients might cause STAT3 to be hyperactive resulting in inhibition of apoptotic pathways in cytotoxic T cells. In order to evaluate the function of the novel mutations, expression constructs of STAT3 containing the D661V and Y640F mutations were generated. In addition, lentiviral vectors were produced to establish a T-cell line (Jurkat) with stable expression of mutant STAT3. After the successful generation of STAT3 constructs and cell line models, several functional assays were performed. Transcriptional activity of STAT3 was measured by luciferase reporter assay and immunocytochemistry was used to determine whether the mutations promote nuclear localization of STAT3. STAT3 phosphorylation was examined by immunoblotting. In addition, quantitative RT-PCR was used to detect differential expression of five STAT3 target genes from patient samples. The results, particularly the luciferase reporter assay, indicated a significant difference between the mutant and wild type STAT3 providing strong evidence that the STAT3 mutants are transcriptionally more active. The localization assay, imaged by fluorescence microscopy, showed more STAT3 D661V and Y640F protein present in the nucleus when compared to wild type STAT3. However, the proliferation rate of mutant STAT3 expressing Jurkat cells was not increased. In addition, STAT3 target gene expression levels of two patient samples did not show large differences when compared to healthy LGL cells. As a result of these findings, it can be strongly hypothesized that aberrant STAT3 signaling underlies the cause of T-cell LGL leukemia. Understanding the molecular basis of LGL leukemia is important in order to develop diagnostic and therapeutic strategies for patients suffering from the disease. Since constitutively active STAT3 is common amongst many cancers and autoimmune disorders, activating mutations could possibly be found in these diseases. More careful sequencing studies of STAT3 upstream molecules are warranted as well, and will be performed in the future from leukemic LGL samples.

Unverricht-Lundborg disease (EPM1/ULD, OMIM 254800) is an autosomal recessive inherited severe type of epilepsy with myoclonus and progressive neurological degeneration. The incidence of EPM1 in Finland is 1:20.000 births per year, and there are about 200 diagnosed cases. The age of disease onset is between 6 and 16 years. The symptoms start with epileptic seizures, stimulus sensitive myoclonus, and generalized tonic-clonic seizures, and progress within a few years to ataxia, incoordination, and dysarthria. Fourteen EPM1-associated loss-of-function mutations in the gene cystatin B (CSTB) have been described. CSTB is a ubiquitously expressed intracellular cysteine proteinase inhibitor, counteracting i.e. cathepsins B, L, and K. The expression levels of CSTB are higher in cerebellar Purkinje cells and in Bergmann glia of the adult central nervous system. There is a Cstb-deficient mouse model for EPM1, which shows progressive death of neurons and widespread gliosis. It has been earlier shown that Cstb knockdown sensitizes cerebellar granule neurons to cathepsin B mediated oxidative stress, resulting in cell death. This master's thesis is based on a previously done gene expression profiling of primary microglia of Cstb-/- mice, which revealed a downregulation of type I and type II interferon-regulated genes on the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) –signaling pathway. The two aims of this study were to create an in vitro disease model for EPM1 in the human cervical adeno-carcinoma cell line HeLa, and in the murine leukemic cell line RAW264.7 by siRNA mediated RNA inhibition, and to study the effects of Cstb knockdown in selected interferon regulated genes of the JAK/STAT signaling pathway. Cystatin B was successfully knocked down in both cell lines HeLa and RAW264.7, and the obtained kinetics of Cstb knockdown in the cell line RAW264.7 provided with valuable information for the sec-ond part of the study. In the cell line HeLa, downregulation of CSTB did not change the expression lev-els of the genes of the JAK/STAT signaling pathway. In the cell line RAW264.7, CSTB knockdown on protein level was followed by a downregulation of the genes Stat1, Stat2, and Irf9. These results were in concordance with the results that had been obtained from the previously performed gene expression profiling of Cstb-/- microglia.

Epithelial tissue is characterized by close cell-cell and cell-extracellular matrix (ECM) contacts as well as by apico-basal polarization. Integrity of these two features is important for functionality of epithelium. Additionally, proteins regulating polarity and cell junctions have been linked to cell cycle and apoptosis control. Consequently, defects in many of the polarity proteins have been linked to oncogenic events and loss of polarity is a hallmark of advanced cancers but whether it is causal to tumorigenesis is yet unknown. However, large body of knowledge on apico-basal polarity regulation and its connection on homeostasis control is derived from studies in Drosophila. This is mainly due to fact that efficient high throughput organotypic three dimensional (3D) culture methods enabling apico-basal polarization have not been available until the last decade. Large screens for epithelial polarity regulators have not been carried out in mammalian cells. Moreover, as cancer is the leading cause of death in developed countries and most of the cancers originate from epithelial tissues, knowledge of polarity regulation can be medically relevant. Oncogene MYC is overexpressed or amplified in variety of human cancers. The tumorigenic function of MYC is mainly due to its ability to drive cell cycle. We have previously shown that intact epithelial architecture is protective from cell cycle deregulating activities of MYC in 3D MCF10A mammary epithelial cell model and in vivo. This resistance can be overcome by inactivating LKB1 which is the human homologue of the polarity protein PAR4 implying a tumour suppressive role for epithelial architecture in mammalian cells. To identify regulators of epithelial architecture in mammalian cells, we have established lentiviral shRNA library (human epithelial architecture library, hEAL) encompassing 219 constructs targeting 77 genes associated with polarity regulation in Drosophila. We have previously screened the shRNA constructs for downregulation and quantified their effects on acinar morphology in the MCF10A 3D model. In this Master's Thesis I have validated the downregulation and phenotypes observed in a subset of the shRNAs during primary screening of the constructs of the library. Additionally, the possible co-operation with downregulation of the polarity regulators and conditional activation of MYC was determined. Most dramatically, downregulation of Wnt pathway gene DVL3 was shown to cause formation of enlarged multiacinar structures, which have increased proliferation. Additionally, downregulation of another Wnt pathway gene, GSK3β, resulted in acini with increased size and filled lumens. Thus these results propose a role for these genes in epithelial architecture regulation and tumour suppression in the used model even though apico-basal polarization of the acini was intact and no synergy with MYC was observed. Interestingly, no role in epithelial architecture regulation for Hippo pathway related genes FAT4 and MOBKL1A was found. Importantly, this study was able to validate primary screen showing relevance of the pipeline. Lastly, the study characterized the synthetic lethality phenotype found in the primary screen caused by downregulation of GTPase RHOA and chronic MYC activation. The shRHOA acini exhibited perturbed α6-integrin localization. When combined with MYC activation, the percentage of apoptotic acini was significantly increased. Importantly, the results suggest the observed synthetic lethality to be specific for the 3D context and to be associated with MEK/ERK and ROCK pathways. Taken together, in this study I have validated the role of novel epithelial architecture regulators and candidate tumour suppressors in MCF10A cells which may have medical relevance by helping to characterize tumorigenic processes. Furthermore, I characterized a novel 3D specific RHOA-MYC synthetic lethal interaction, which may prove to have therapeutic significance in MYC-driven cancers in future.

Apotti on Helsingin ja Uudenmaan sairaanhoitopiirin sekä alueen kuntien potilastietojärjestelmähanke. Helsingin kaupungin terveyslautakunta kokoontui ensimmäisen kerran ottamaan kantaa Apottiin 11.9.2012. Ennen kokousta sosiaalisessa mediassa oli virinnyt vilkas keskustelu, jossa lähinnä kritisoitiin hanketta. (Hanketta kritisoitiin muun muassa sen korkeasta kustannusarviosta ja siitä, että hankkeessa oltaisiin ajautumassa niin sanottuun toimittajaloukkuun.) Keskustelun keskiössä oli 9.7.2012 perustettu Facebook-ryhmä Terveydenhuollon tietojärjestelmät korjattava. Keskusteluun osallistuivat suoraan ja välillisesti myös perinteisempi media ja potilastietojärjestelmähankkeen vastuuhenkilöt. Keskustelun tärkeimmät pitkät puheenvuorot ja lyhyemmät kommentit ovat hajallaan ja sekavassa järjestyksessä eri internetsivustoilla. Tämän tutkielman ensisijaisena tavoitteena on etsiä nämä keskustelun tärkeimmät puheenvuorot ja esitellä ne kronologisessa järjestyksessä (luvut 2-7 ja 13-29). Tutkielman toissijaisena tavoitteena on pohtia joitakin keskustelussa esiinnousseita teemoja (luvut 8-12 ja 30).

γ-Aminobutyric acid type A (GABAA) receptors are ligand gated ion channels mediating most inhibitory signals in the brain. GABAA receptor consists of five subunits, and there are 16 different subunits known. This forms base to pharmacological diversity of GABAA receptors. In this study autoradiography on subunit gene knock out mouse models was used to visualize the meaning of different subunits in forming GABAA receptors exhibiting special kinds of pharmacology, ie. GABA-insensitive [35S]TBPS-binding and high-affinity [3H]muscimol binding. In receptors exhibiting GABA-insensitive TBPS-binding GABA fails to cause full dissociation of TBPS from the ionophore, which means that the receptors are not fully activated by GABA. This study revealed, that contrary to what was previously believed, the forebrain GABA-insensitive [35S]TBPS binding is most likely caused by GABAA receptors consisting of α1 and β subunits or α1, β and δ subunits. High-affinity muscimol binding in the α1 KO mice was found to be unaltered, whereas α4 KO, δ KO and α4 + δ KO mice exhibited diminished high-affinity [3H]muscimol binding. Contrary to what was previously considered, this study points out that α1β GABAA receptors are mediating GIS-binding in the forebrain. Changes in high-affinity [3H]muscimol binding has also been found to correlate with changes of muscimols effects in vivo in knock out mouse models.