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  • Carlsson, Emilia (Helsingin yliopisto, 2012)
    This thesis is based on the original observation of an allelic deletion of the recently described Neuron navigator 3 (NAV3) gene in patients with primary cutaneous T-cell lymphoma (CTCL) and CTCL-associated lung cancer. Thereafter mutations or copy number changes of NAV3 have been reported in melanoma, glioblastoma (GBM) and adrenal carcinoma. The aim of this study has been to shed light on the function and interactions of the NAV3 protein, as well as characterizing NAV3 copy number changes and their effect on patient survival in several forms of cancer. NAV3 is a novel cancer-associated gene at 12q21. The specific function of NAV3 is not known except that it carries actin-binding domains with ATPase activity, and is therefore likely to have an action on microtubules and cytoskeleton reorganization. The three Navigator genes, NAV1, NAV2 and NAV3 share homology among themselves and among different species. This suggests a central role for the encoded proteins in cell biology. In this study, NAV3 copy number changes have been studied by fluorescence in situ hybridisation (FISH) and array comparative genomic hybridisation (aCGH) in non-melanoma skin cancers, colorectal cancer and neural system tumours, and the relevant NAV3-regulated target genes have been identified. Furthermore, the expression levels of NAV3 and NAV3-regulated signalling molecules have been correlated to disease progression and patient outcome. In Basal cell carcinomas (BCC) and Squamous cell carcinomas (SCC) we found NAV3 copy number loss and corresponding absence of protein in 21% of the BCC and in 25% of the SCC tumours. In the nodular/superficial BCC subgroup, also low-level NAV3 amplification was found. NAV3 aberrations were independent of the known chromosome 6 amplification in BCC. Chromosome 12 polysomy, also independent of chromosome 6 polysomy, was found in 33% and 25% of the invasive type of BCC and in SCC, respectively. In colorectal carcinomas NAV3 deletion and chromosome 12 polysomy were detected in 30% and 70% of MSS carcinomas, and in 12.5% and 50% of MSI carcinomas, but also in 23% and 30% of adenomas, respectively. Low copy number amplification of NAV3 was found in 25% of MSS samples. 119 patient samples representing different central and peripheral nervous system tumours were studied for NAV3 copy number changes. Neuronally differentiated tumours (neuroblastomas and medulloblastomas) entailed more NAV3 aberrations than the glial tumours. In glial tumours, those with grade IV (gliobalstoma (GBM)) had significantly more NAV3 deletions than tumours with grades I, II or III. Log rank analysis also linked NAV3 deletion to a poor prognosis in gliomas. In contrast, glioma patients with NAV3 amplification showed better prognosis than those with normal NAV3 copy numbers. The FISH result was also supported by aCGH analysis, which showed results matching the FISH analysis for tumour samples with NAV3 amplification and deletion. To understand the in vivo functional consequences of NAV3 copy number changes, especially of NAV3 deletion, we silenced NAV3 in normal colon, GBM and primary keratinocyte cells, using a commercially available small inhibitory ribonucleic acid (siRNA) construct. Post transfection RNA samples from several time points were analyzed with Agilent 44K microarray. In GBM and colorectal cell lines we identified, among others, GnRHR and IL-23R as upregulated by NAV3 gene silencing. The upregulation of the selected genes were confirmed by quantitative PCR. In primary keratinocytes, NAV3 silencing led to consistent upregulation of 22 annotated genes, and several Wnt/HH pathway genes were slightly overexpressed too. Taken together the results of this thesis support the previously suggested role of NAV3 as a novel cancer associated gene, and we suggest that NAV3 affects the malignant potential of a given tumour through multiple pathways. This assumption is based on the fact that gene expression analysis of NAV3 silenced cells indicates that NAV3 affects genes with involvement in both inflammation and carcinogenesis.
  • den Hollander, Bjørnar (Helsingin yliopisto, 2015)
    In recent years there has been a large increase in the use of a new kind of amphetamine- type stimulants known as substituted cathinones. These compounds have a short history of human use, and little is known about their potential neurotoxicity. Two of the most popular substituted cathinones, 4-methylmethcathinone (4-MMC, mephedrone) and 3,4- methylenedioxymethcathinone (MDMC, methylone} are, aside from their β-ketone group, close structural analogues of potentially neurotoxic amphetamines such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy). This has led to concern about the potential neurotoxicity of these novel compounds, and warrants a closer investigation into their possible long-term neurotoxic effects. METHODS The long-term effects of METH and MDMA as well as 4-MMC and MDMC were assessed using a range of biochemical assays, including assessment of monoamine levels and their transporters. The effects on brain activity were investigated using manganese-enhanced magnetic resonance imaging. Furthermore, behavioral experiments assessing cognition and neuropsychiatric function were performed. Finally, in vitro experiments in a neuroblastoma cell line were performed to identify mechanisms responsible for the observed differences in toxicity between the amphetamines and cathinones. RESULTS Unlike METH and MDMA, which produced strong reductions in dopamine and serotonin levels or brain activation, 4-MMC produced few notable effects on monoamine levels and had only minor effects on brain activation, although MDMC produced a reduction in 5-HT levels similar to MDMA. No clear effects on behavioral tests of memory function were observed as both increases and decreases in test performance were seen following 4- MMC and MDMC. In vitro experiments revealed that cathinones differ from amphetamines in their redox properties, and 4-MMC produced different effects than METH on the mitochondrial electron transport chain. CONCLUSIONS The substituted cathinones 4-MMC and MDMC do not appear to be more neurotoxic than METH and MDMA. If anything, they show a more favorable safety profile. Therefore, these substances do not appear to present an imminent and severe threat to public health. From a harm reduction perspective, these compounds may be good alternatives toMETH and MDMA. However, future work is needed to assess with certainty the long- term effects of amphetamine-type stimulants in humans.
  • Heikkilä, Tero (Helsingin yliopisto, 2003)
  • Juutistenaho, Sari (Helsingin yliopisto, 2010)
    Cord blood is a well-established alternative to bone marrow and peripheral blood stem cell transplantation. To this day, over 400 000 unrelated donor cord blood units have been stored in cord blood banks worldwide. To enable successful cord blood transplantation, recent efforts have been focused on finding ways to increase the hematopoietic progenitor cell content of cord blood units. In this study, factors that may improve the selection and quality of cord blood collections for banking were identified. In 167 consecutive cord blood units collected from healthy full-term neonates and processed at a national cord blood bank, mean platelet volume (MPV) correlated with the numbers of cord blood unit hematopoietic progenitors (CD34+ cells and colony-forming units); this is a novel finding. Mean platelet volume can be thought to represent general hematopoietic activity, as newly formed platelets have been reported to be large. Stress during delivery is hypothesized to lead to the mobilization of hematopoietic progenitor cells through cytokine stimulation. Accordingly, low-normal umbilical arterial pH, thought to be associated with perinatal stress, correlated with high cord blood unit CD34+ cell and colony-forming unit numbers. The associations were closer in vaginal deliveries than in Cesarean sections. Vaginal delivery entails specific physiological changes, which may also affect the hematopoietic system. Thus, different factors may predict cord blood hematopoietic progenitor cell numbers in the two modes of delivery. Theoretical models were created to enable the use of platelet characteristics (mean platelet volume) and perinatal factors (umbilical arterial pH and placental weight) in the selection of cord blood collections with high hematopoietic progenitor cell counts. These observations could thus be implemented as a part of the evaluation of cord blood collections for banking. The quality of cord blood units has been the focus of several recent studies. However, hemostasis activation during cord blood collection is scarcely evaluated in cord blood banks. In this study, hemostasis activation was assessed with prothrombin activation fragment 1+2 (F1+2), a direct indicator of thrombin generation, and platelet factor 4 (PF4), indicating platelet activation. Altogether three sample series were collected during the set-up of the cord blood bank as well as after changes in personnel and collection equipment. The activation decreased from the first to the subsequent series, which were collected with the bank fully in operation and following international standards, and was at a level similar to that previously reported for healthy neonates. As hemostasis activation may have unwanted effects on cord blood cell contents, it should be minimized. The assessment of hemostasis activation could be implemented as a part of process control in cord blood banks. Culture assays provide information about the hematopoietic potential of the cord blood unit. In processed cord blood units prior to freezing, megakaryocytic colony growth was evaluated in semisolid cultures with a novel scoring system. Three investigators analyzed the colony assays, and the scores were highly concordant. With such scoring systems, the growth potential of various cord blood cell lineages can be assessed. In addition, erythroid cells were observed in liquid cultures of cryostored and thawed, unseparated cord blood units without exogenous erythropoietin. This was hypothesized to be due to the erythropoietic effect of thrombopoietin, endogenous erythropoietin production, and diverse cell-cell interactions in the culture. This observation underscores the complex interactions of cytokines and supporting cells in the heterogeneous cell population of the thawed cord blood unit.
  • Veijola, Lea (Helsingin yliopisto, 2007)
    Aims: Helicobacter pylori infection, although the prevalence is declining in Western world, is still responsible for several clinically important diseases. None of the diagnostic tests is perfect and in this study, the performance of three stool antigen tests was assessed. In areas of high H. pylori prevalence, the definition of patients with the greatest benefit from eradication therapy may be a problem; the role of duodenal gastric metaplasia in categorizing patients at risk for duodenal ulcer was evaluated in this respect. Whether persistent chronic inflammation and elevated H. pylori antibodies after successful eradication are associated with each other or with atrophic gastritis, a long term sequelae of H. pylori infection, were also studied. Patients and methods: The three stool antigen tests were assessed in pre- and post-eradication settings among 364 subjects in two studies as compared to the rapid urease test (RUT), histology, culture, the 13C-urea breath test (UBT) and enzyme immunoassay (EIA) based H. pylori serology. The association between duodenal gastric metaplasia with duodenal ulcer was evaluated in a retrospective study including 1054 patients gastroscopied due to clinical indications and 154 patients previously operated for duodenal ulcer. The extent of duodenal gastric metaplasia was assessed from histological specimens in different patient groups formed on the basis of gastroscopy findings and H. pylori infection. Chronic gastric inflammation (108 patients) and H. pylori antibodies and serum markers for atrophy (77 patients) were assessed in patients earlier treated for H. pylori. Results: Of the stool antigen tests studied, the monoclonal antibody-based EIA-test showed the highest sensitivity and specificity both in the pre-treatment setting (96.9% and 95.9%) and after therapy (96.9% and 97.8%). The polyclonal stool antigen test and the in-office test had at baseline a sensitivity of 91% and 94%, and a specificity of 96% and 89%, respectively and in a post-treatment setting, a sensitivity of 78% and 91%, and a specificity of 97%, respectively. Duodenal gastric metaplasia was strongly associated with H. pylori positive duodenal ulcer (odds ratio 42). Although common still five years after eradication, persistent chronic gastric inflammation (21%) and elevated H. pylori antibodies (33%) were neither associated with each other nor with atrophic gastritis. Conclusions: Current H. pylori infection can feasibly be diagnosed by a monoclonal antibody-based EIA test with the accuracy comparable to that of reference methods. The performance of the polyclonal test as compared to the monoclonal test was inferior especially in the post-treatment setting. The in-office test had a low specificity for primary diagnosis and hence positive test results should probably be confirmed with another test before eradication therapy is prescribed. The presence of widespread duodenal gastric metaplasia showed promising results in detecting patients who should be treated for H. pylori due to an increased risk of duodenal ulcer. If serology is used later on in patients with earlier successfully treated for H. pylori, it should be taken into account that H. pylori antibodies may persist elevated for years for unknown reason. However, this phenomenon was not found to be associated with persistent chronic inflammation or atrophic changes.
  • Kluger, Nicolas (Helsingin yliopisto, 2015)
    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, OMIM 240300) is a rare autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene located on chromosome 21 (21q22.3). AIRE deficiency causes a loss in central immune tolerance, leading to the failure to eliminate autoreactive T cells in the thymus and allowing their escape to the periphery. Because of a founder effect, APECED is particularly prevalent in Finland (1/25,000) but is observed worldwide with variable prevalence. APECED patients are susceptible to mucocutaneous candidiasis and multiple endocrine autoimmune diseases such as primary hypoparathyroidism, adrenal insufficiency, primary hypogonadism, type 1 diabetes, hypothyroidism, and hypophysitis. They may also develop additional nonendocrine autoimmune diseases, such as alopecia areata/totalis, vitiligo, gastro-intestinal (GI) diseases, keratitis or tubulointerstitial nephritis (TIN). In addition, the patients typically develop a variety of serum tissue-specific autoantibodies, which are predictive of the development of autoimmune disease and anticytokine antibodies such as those against type I interferons and Th17-related interleukin IL-17 and IL-22. The aim of this thesis was to study such manifestations of APECED that have not been well characterized before and also, to study health-related quality of life among Finnish APECED and Addison s disease/APS2 patients. We evaluated the clinical GI features and searched for novel markers of GI dysfunction in a Finnish cohort of 31 APECED patients. The main upper GI symptoms were dysphagia and retrosternal pain (45%) and the lower GI symptoms were constipation (48%), diarrhoea (45%) and malabsorption (16%). Previously, L-amino-acid decarboxylase (AADC) and tryptophan hydroxylase type 1 (TPH-1) antibodies have been demonstrated in APECED. AADC antibodies were found in 51% and TPH-1 antibodies in 39% of all patients. Also, a T cell response to AADC was detected in 43%. One third of the patients had autoimmune enteropathy (AIE)-related 75 kDa antigen (AIE-75, 33%) and villin (29%) autoantibodies, and antibodies against brush borders and Paneth cells (PCs) were detected in 29% and 20%, respectively. Mucosal intestinal IL-17 expression was decreased or negative in 77% of the intestinal samples. Duodenal chromogranin A and serotonin expression was absent or decreased in 50% and 66% of the patients, respectively. Of the clinical symptoms, constipation correlated with negative serotonin staining (p less than 0.05) and with AADC antibodies (p = 0.019). Importantly, we found a correlation between autoantibodies against AADC, which are critical for serotonin and DOPA synthesis, and constipation. Constipation was also associated with a lack of serotonin expression in the enteroendocrine cells (EECs). Paneth cells (PCs) were lacking in the duodenum in 20% of our intestinal samples, even though this was not associated with GI symptoms. In this Finnish APECED patient cohort, 17% (5/30) had moderate-to-severe renal failure, including 10% (3/30) with TIN requiring transplantation, haemodialysis or immunosuppressive treatment. However, the latter did not seem to be efficient in controlling disease progression. All 3 patients with TIN had circulating antibodies against the distal part of the nephron, as did 30% of all cohort cases. The pathogenic relevance of such circulating antibodies is still unclear. The immunological basis of hypoparathyroidism in APECED was explored by studying circulating calcium-sensing receptor (CaSR) and NALP5 antibodies. Although they were detected in 16 of 44 (36%) and 13 of 44 (30%) patients, respectively, we failed to find any clinically relevant statistical association. These APECED patients did not present circulating antibodies for other autoimmune diseases such as rheumatoid arthritis, celiac disease, bullous pemphigoid or pemphigus vulgaris. Some patients had antinuclear antibodies at a low-titre without clinical significance. Secondly, we evaluated the health-related quality of life among Finnish APECED and Addison s disease/APS2 patients and sought to determine which factors may predict a possible impairment. Using health-related quality of life (HRQoL) questionnaires for APECED (SF-36) and Addison s disease/APS2 patients (SF-36, 15D), we indeed observed impaired HRQoL. For the APECED patients, general health, emotional well-being and energy/vitality were the most diminished aspects of HRQoL. Among the patients with Addison s disease/APS2, compared to a large control population, physical or emotional role functioning, energy/vitality and general health were most affected. Discomfort and symptoms, vitality, and sexual activity were the most affected dimensions of the 15D scores. Affiliation with a patients association, female gender, the presence of non-APS2 inflammatory comorbidities, lower educational level and a longer disease duration were independent predictors of impaired HRQoL in these patients. Taken together, the results of this thesis show that APECED patients are genetically prone to develop autoantibodies to a multitude of tissue antigens but are still tolerant to some common autoantigens. The true clinical and biological relevance of these circulating autoantibodies has not yet been elucidated, and it is possible that they are only a reflection of T cell-mediated immunity. They may, however, have a cumulative effect and clinical disease may arise only in patients with a combination of circulating antibodies, as seen in diabetes type 1. This may explain why we failed to find any association between any single type of antibody and a given symptom. For the lower GI track manifestations, we hypothesise a cumulative effect of the autoimmunity directed against both the enteroendocrine cells and the Paneth cells, leading to a dysfunction in both the secretion of serotonin in the gut and the secretion of antimicrobiobial defensins. Such a disturbance would have an effect on the gut microbiota. The question of whether the neutralising antibodies against cytokines may have a paradoxical protective effect is open to debate. Lastly, despite having a high number of manifestations, patients with APECED seem to cope with their disease. Patients with Addison s disease have significantly impaired HRQoL compared to the general population.
  • Lilius, Tuomas (Helsingin yliopisto, 2014)
    Opioid analgesics are effective in relieving acute and chronic pain. However, adverse effects and the development of opioid dependence and tolerance may restrict the use of opioids and result in inadequate pain relief. The effects of four structurally and functionally different drugs already on the market, ibudilast, atipamezole, spironolactone, and ketamine, were studied in coadministration with morphine, the prototypical mu-opioid receptor agonist. Experiments were conducted using thermal and mechanical tests of nociception in male Sprague-Dawley rats. Morphine tolerance was produced during four days by subcutaneous or intrathecal delivery of morphine. Drug and metabolite concentrations were measured using high-pressure liquid chromatography-tandem mass spectrometry. The objective of the thesis study was to search for potential drugs to augment morphine antinociception and prevent opioid tolerance. Ibudilast, a phosphodiesterase and macrophage inhibitory factor inhibitor, had transient sedative effects, but it restored the antinociceptive effect of morphine in morphine-tolerant rats after single and repeated administration. It did not prevent the development of opioid tolerance. Atipamezole, an alpha-2-adrenoceptor antagonist used for the reversal of sedation in animals during anesthesia, was effective in augmenting intrathecal morphine antinociception in both opioid-naïve and opioid-tolerant animals. These effects were observed at doses lower than those required for the antagonism of alpha-2-adrenoceptors. In subcutaneous administration, low doses of atipamezole did not influence morphine antinociception. The mineralocorticoid receptor antagonist spironolactone dose-dependently enhanced morphine antinociception. This effect was mediated via the increased access of morphine to the central nervous system by the inhibition of the efflux protein P-glycoprotein. Spironolactone did not inhibit the metabolism of morphine to the pronociceptive metabolite morphine-3-glucuronide, and it did not prevent the development of opioid tolerance. The effects of ketamine in augmenting opioid analgesia in tolerance are thought to result from a beneficial pharmacodynamic interaction. When acute ketamine was administered to rats under chronic morphine treatment, the brain concentrations of morphine, ketamine and norketamine were increased compared with the situation where either morphine treatment or acute ketamine were administered alone. The results indicate a potentially beneficial pharmacokinetic interaction between the two drugs. The results of the thesis study demonstrate that ibudilast and atipamezole modulate nociception at systemic and spinal levels in preclinical models of pain, and they may prove advantageous as an adjuvant to opioid therapy. Spironolactone had a pharmacokinetic interaction with morphine, leading to increased morphine concentrations in the central nervous system. Ketamine, a drug used for the treatment of opioid tolerance in cancer patients, may undergo previously unrecognized beneficial pharmacokinetic interactions with morphine.
  • Gylfe, Alexandra (Helsingin yliopisto, 2014)
    Colorectal cancer (CRC) is the third most common cancer, and the second most common cause of cancer mortality. The aim of this thesis work was to gain novel insight into the molecular mechanisms behind CRC predisposition, as well as tumor progression and development. Microsatellite instability (MSI) arises due to a defective mismatch repair system and is characteristic for a subset of all CRCs. Here, we aimed to identify novel MSI target genes with potential oncogenic effects. We characterized all genes overexpressed in MSI CRCs and predicted to escape nonsense-mediated decay when mutated. The mitotic checkpoint kinase TTK was identified with protein-elongating mutations in 59% of MSI CRCs. TTK has an essential role in spindle assembly checkpoint (SAC) signaling, however, the mutated protein did not show SAC weakening. While no evidence of oncogenic mechanisms was observed, the high mutation frequency of TTK argues for biological significance. In another screening effort on MSI CRCs, we sought to identify novel oncogenes with activating hotspot mutations. The exomes of 25 tumor and respective healthy colon tissues were sequenced. A total of 15 candidate oncogenes with hotspot mutations were identified. Three genes, ZBTB2, PSRC1 and RANBP2, displayed hotspot mutations also in the validation set of 86 MSI CRCs. Interestingly, the CRC-associated mutant form of ZBTB2 increased cell proliferation. Additional work is needed to further clarify the role of the identified somatic mutations in CRC tumorigenesis. The candidate oncogenes identified in this thesis work might be used to develop personalized tumor profiling and therapy. Inherited susceptibility is estimated to be involved in approximately one-third of all CRCs. However, the great majority of inherited CRC susceptibility remains still molecularly unexplained. A recent systematic sequencing study on CRC reported a set of somatically mutated genes, termed candidate cancer (CAN) genes. We examined the mutational profiles of 15 top-ranked CAN genes for somatic mutations as well as for germline variants in 45 familial CRC cases. In our tumor set, six of the CAN genes were somatically mutated. In germline, three private missense variants were identified in CSMD3, EPHB6 and c10orf137. In another effort, we exome sequenced 96 independent cases with familial CRC. We identified 11 novel candidate CRC susceptibility genes with rare putative LoF variants. Seven loss-of-heterozygosity events, involving four genes, were observed in the data. In each occasion, the losses targeted the wild-type allele (P=0.0078), providing further support that true culprits are among the eleven genes. This study provides an interesting set of candidate predisposing genes, which might explain a subset of common familial CRC. The identified germline variants need to be validated in larger sample sets to provide firm evidence for disease predisposition. Additional work is also needed to characterize the detailed functional and clinical relevance of the identified candidate CRC predisposing genes. This information, then, can ultimately be translated into tools for cancer prevention and early diagnosis in individuals carrying true predisposition alleles.
  • Tigerstedt, Nina-Maria (Helsingin yliopisto, 2009)
    Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.
  • Broms, Ulla (Helsingin yliopisto, 2008)
    The worldwide health burden caused by the tobacco epidemic highlights the importance of study-ing determinants of smoking behaviour and key factors sustaining nicotine dependence. Despite vast-ranging preventive efforts, smoking remains one of the most deleterious health behaviours, and its genetic and environmental factors warrant continuous investigation. The heritability of smoking behaviour and nicotine dependence has been suggested to be relatively high. Earlier smoking behaviour, nicotine dependence, socio-economic position and demographic factors have all been shown to be associated with smoking cessation. This thesis aimed to examine various aspects of smoking behaviour and nicotine dependence from an epidemiological and genetic per-spective. Data for Studies I and IV were obtained from the Older Finnish Twin Cohort, a postal health sur-vey conducted in 1975, 1981 and 1990 on same-sexed pairs and in 1996-1997 on male-female adult pairs. The number of ever-smoking participants was 8941 in Study I and 3069 in Study IV. Data for Studies II and III came from the Family Study of Cigarette Smoking - Vulnerability to Nicotine Addiction. This study is linked to the Older Finnish Twin Cohort with new data collec-tion during 2001-2006 that focused on smoking twin pairs and their family members. The meas-ures included intensive telephone interviews, blood samples and additional postal questionnaires. The numbers of ever-smoking participants was 1370 in Study II and 529 in Study III. Study I examined whether a genetic component underlies smoking behaviour among Finnish adults. Genetic factors were important in the amount smoked and smoking cessation, with about half of the phenotypic differences explained by genetic variance. A novel finding was that genetic influences on amount smoked and smoking cessation were largely independent of genetic influ-ences on age at initiation. This result has implications for defining phenotypes in the search for genes underlying smoking behaviour. Furthermore, even if smoking initiation is postponed to a later age, potential vulnerability to subsequent nicotine dependence cannot be completely inhib-ited. Study II investigated the effect of genetic and environmental factors on nicotine dependence, as measured by the novel multidimensional Nicotine Dependence Syndrome Scale (NDSS). This scale was validated in the Finnish data. The NDSS correlated highly with other established nico-tine dependence scales (FTND and DSM-IV), suggesting that this new scale would be a feasible and valid measure for identifying nicotine-dependent smokers among the ever-smoking popula-tion. About one-third of the phenotypic variation in nicotine dependence in this sample was ex-plained by genetic influences. Study III aimed at identifying chromosomal regions harbouring genes that influence smoking be-haviour and nicotine dependence. Linkage analysis of family data revealed that for smoker and nicotine dependence phenotypes as well as for co-morbidity between nicotine dependence and alcohol use signals on specific chromosome regions (chromosomes 2q33, 5q12, 5q34 7q21, 7q31, 10q25, 11p15, 20p13) exist. Results further support the hypothesis that smoking behaviour phe-notypes have a genetic background. Study IV examined associations of smoking behaviour, socio-economic position and transition of marital status with smoking cessation. Indicators of socio-economic position were important pre-dictors of smoking cessation even when adjusted for previous smoking behaviour. Getting married was associated with an increased probability of cessation in men, a finding confirmed among dis-cordant twin pairs. Thus, having a partner appears to have a positive impact on smoking cessation. In conclusion, nicotine dependence and smoking behaviour demonstrate significant genetic liabil-ity, but also substantial environmental influences among Finnish adults. Smoking initiation should be prevented or at least postponed to a later age. Although genetic factors are important in nicotine dependence and smoking behaviour, societal actions still have a primary role in tobacco control and smoking prevalence. Future studies should examine the complex interactions between genetic and environmental factors in nicotine dependence.
  • Väisänen-Tommiska, Mervi (Helsingin yliopisto, 2006)
  • Heinonen-Guzejev, Marja (Helsingin yliopisto, 2009)
    Noise can be defined as unwanted sound. It may adversely affect the health and well-being of individuals. Noise sensitivity is a personality trait covering attitudes towards noise in general and a predictor of noise annoyance. Noise sensitive individuals are more affected by noise than less sensitive individuals. The determinants and characteristics related to noise sensitivity are rather poorly known. The risk of health effects caused by noise can be hypothesized to be higher for noise sensitive individuals compared to those who are not noise sensitive. A cardiovascular disease may be an example of outcomes. The general aim of the present study was to investigate the association of noise sensitivity with specific somatic and psychological factors, including the genetic component of noise sensitivity, and the association of noise sensitivity with mortality. The study was based on the Finnish Twin Cohort of same-sex twin pairs born before 1958. In 1988 a questionnaire was sent to twin pairs discordant for hypertension. 1495 individuals (688 men, 807 women) aged 31 88 years replied, including 573 twin pairs. 218 of the subjects lived in the Helsinki Metropolitan Area. Self-reported noise sensitivity, lifetime noise exposure and hypertension were obtained from the questionnaire study in 1988 and other somatic and psychological factors from the questionnaire study in 1981 for the same individuals. In addition, noise map information (1988 1992) from the Helsinki Metropolitan Area and mortality follow-up 1989 2003 were used. To evaluate the stability and validity of noise sensitivity, a new questionnaire was sent in 2002 to a sample of the subjects who had replied to the 1988 questionnaire. Of all subjects who had answered the question on noise sensitivity, 38 % were noise sensitive. Noise sensitivity was independent of noise exposure levels indicated in noise maps. Subjects with high noise sensitivity reported more transportation noise exposure than subjects with low noise sensitivity. Noise sensitive subjects reported transportation noise exposure outside the environmental noise map areas almost twice as often as non-sensitive subjects. Noise sensitivity was associated with hypertension, emphysema, use of psychotropic drugs, smoking, stress and hostility, even when lifetime noise exposure was adjusted for. Monozygotic twin pairs were more similar with regards noise sensitivity than dizygotic twin pairs, and quantitative genetic modelling indicated significant familiality. The best fitting genetic model provided an estimate of heritability of 36 %. Follow-up of subjects in the case-control study showed that cardiovascular mortality was significantly increased among noise sensitive women, but not among men. For coronary heart mortality the interaction of noise sensitivity and lifetime noise exposure was statistically significant in women. In conclusion, noise sensitivity has both somatic and psychological components. It does aggregate in families and probably has a genetic component. Noise sensitivity may be a risk factor for cardiovascular mortality in women.
  • Ilumets, Helen (Helsingin yliopisto, 2011)
    Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airway inflammation and largely irreversible airflow limitation. One major risk factor for COPD is cigarette smoking. Since the inflammatory process starts many years prior to the onset of clinical symptoms and still continues after smoking cessation, there is an urgent need to find simple non-invasive biomarkers that can be used in the early diagnosis of COPD and which could help in predicting the disease progression. The first aim of the present study was to evaluate the involvement of different oxidative/nitrosative stress markers, matrix metalloproteinases (MMPs) and their tissue inhibitor-1 (TIMP-1) in smokers and in COPD. Elevated numbers of inducible nitric oxide synthase (iNOS), nitrotyrosine, myeloperoxidase (MPO) and 4-hydroxy-2-nonenal (4-HNE) positive cells and increased levels of 8-isoprostane and lactoferrin were found in sputum of non-symptomatic smokers compared to non-smokers, and especially in subjects with stable mild to moderate COPD, and they correlated with the severity of airway obstruction. This suggests that an increased oxidant burden exists already in the airways of smokers with normal lung function values. However, none of these markers could differentiate healthy smokers from symptomatic smokers with normal lung function values i.e. those individuals who are at risk of developing COPD. In contrast what is known about asthma exhaled nitric oxide (FENO) was lower in smokers than in non-smokers, the reduced FENO value was significantly associated with neutrophilic inflammation and the elevated oxidant burden (positive cells for iNOS, nitrotyrosine and MPO). The levels of sputum MMP-8 and plasma MMP-12 appeared to differentiate subjects who have a risk for COPD development but these finding require further investigations. The levels of all studied MMPs correlated with the numbers of neutrophils, and MMP-8 and MMP-9 with markers of neutrophil activation (MPO, lactoferrin) suggesting that especially neutrophil derived oxidants may stimulate the tissue destructive MMPs already in lungs of smokers who are not yet experiencing any airflow limitation. When investigating the role of neutrophil proteases (neutrophil elastase, MMP-8, MMP-9) during COPD exacerbation and its recovery period, we found that levels of all these proteases were increased in sputum of patients with COPD exacerbation as compared to stable COPD and controls, and decreased during the one-month recovery period, giving evidence for a role of these enzymes in COPD exacerbations. In the last study, the effects of subject`s age and smoking habits were evaluated on the plasma levels of surfactant protein A (SP-A), SP-D, MMP-9 and TIMP-1. Long-term smoking increased the levels of all of these proteins. SP-A most clearly correlated with age, pack years and lung function decline (FEV1/FVC), and based on the receiver operating characteristic curve analysis, SP-A was the best marker for discriminating subjects with COPD from controls. In conclusion, these findings support the hypothesis that especially neutrophil derived oxidants may activate MMPs and induce an active remodeling process already in the lungs of smokers with normal lung function values. The marked increase of sputum levels of neutrophil proteases in smokers, stable COPD and/or during its exacerbations suggest that these enzymes play a role in the development and progression of COPD. Based on the comparison of various biomarkers, SP-A can be proposed to serve as sensitive biomarker in COPD development.
  • Louhelainen, Noora (Helsingin yliopisto, 2012)
    Chronic obstructive pulmonary disease (COPD) is characterized by airway obstruction that is mostly irreversible. The typical features of COPD are chronic inflammation in the small airways, with varying degrees of chronic bronchitis and/or emphysema. Long-term smoking is the major risk factor for COPD. Oxidative stress and protease/antiprotease imbalance play major roles in the pathogenesis of smoking related airway disease such as COPD. The diagnosis of COPD is based on spirometry. Diagnosis is however often delayed since during its early stages, clear symptoms are lacking, even though the lung damage is already extensive. The differential diagnosis of COPD from asthma is especially difficult in smokers. There is a clear need to develop reliable biomarkers that would detect early COPD, differentiate the sub-phenotypes of COPD as well as COPD from asthma and finally be useful in developing treatment strategies for different phenotypes and used in monitoring disease activity and progression. The first goal of this study was to compare different potential markers in various sample types that reflect oxidative stress in the lung, how they are related to each other, and to find the most specific markers for the early detection and monitoring of COPD and asthma and differentiation of these diseases as well as the disease from the condition of healthy smoker . Another goal was to examine the effects of smoking cessation on the best potential markers of oxidative and protease burden. Recent lung proteomic studies have indicated that SP-A could be potential marker for COPD; sputum proteomics was used to find other biomarkers for early COPD. FeNO levels were elevated in mild asthma and eosinophils were elevated in asthmatic adults and patients with COPD exacerbation when compared to healthy controls. No significant differences were detected in sputum 8-isoprostane levels between subjects with mild asthma and healthy controls, but in patients with COPD exacerbation, the 8-isoprostane levels were greatly increased when compared to healthy subjects. In the smoking cessation studies, smokers had elevated levels of all of the investigated markers at the baseline compared to non-smokers. Neutrophil counts increased after 3 months of quitting of smoking in the group of chronic bronchitis/COPD but at 6 months neutrophils had returned to the levels of non-smokers. 8-Isoprostane declined significantly but the levels of FeNO, nitrotyrosine and MMP-8 did not change significantly during 3 months after smoking cessation, although after 6 months, the levels of MMP-7, -8 and TIMP-1 decreased to the levels of non-smokers and only MMP-9 remained elevated at 6 months of smoking cessation. However, the symptoms declined significantly already after 3 months of smoking cessation. The plasma levels of SP-A, SP-D, MMP-9, TIMP-1 and the ratio of MMP-9/TIMP-1 were increased by long-term smoking and COPD, and SP-A correlated with the extent of airway limitation. In proteomic screening of sputum, PIGR was found to be elevated in smokers and in mild to moderate COPD, and this was confirmed in lung tissue and plasma specimens. To conclude, it seems that sputum 8-isoprostane is not sensitive enough in detecting oxidative stress in mild recent asthma while in COPD it may be useful. After smoking cessation, the symptoms decline significantly already after a few months but the oxidant and protease burdens seem to persist in the airways for months. SP-A and PIGR can be proposed as representing new promising markers in smoking related chronic airway inflammation and COPD.
  • Sipponen, Taina (Helsingin yliopisto, 2009)
    Background: The fecal neutrophil-derived proteins calprotectin and lactoferrin have proven useful surrogate markers of intestinal inflammation. The aim of this study was to compare fecal calprotectin and lactoferrin concentrations to clinically, endoscopically, and histologically assessed Crohn’s disease (CD) activity, and to explore the suitability of these proteins as surrogate markers of mucosal healing during anti-TNFα therapy. Furthermore, we studied changes in the number and expression of effector and regulatory T cells in bowel biopsy specimens during anti-TNFα therapy. Patients and methods: Adult CD patients referred for ileocolonoscopy (n=106 for 77 patients) for various reasons were recruited (Study I). Clinical disease activity was assessed with the Crohn’s disease activity index (CDAI) and endoscopic activity with both the Crohn’s disease index of severity (CDEIS) and the simple endoscopic score for Crohn’s disease (SES-CD). Stool samples for measurements of calprotectin and lactoferrin, and blood samples for CRP were collected. For Study II, biopsy specimens were obtained from the ileum and the colon for histologic activity scoring. In prospective Study III, after baseline ileocolonoscopy, 15 patients received induction with anti-TNFα blocking agents and endoscopic, histologic, and fecal-marker responses to therapy were evaluated at 12 weeks. For detecting changes in the number and expression of effector and regulatory T cells, biopsy specimens were taken from the most severely diseased lesions in the ileum and the colon (Study IV). Results: Endoscopic scores correlated significantly with fecal calprotectin and lactoferrin (p<0.001). Both fecal markers were significantly lower in patients with endoscopically inactive than with active disease (p<0.001). In detecting endoscopically active disease, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for calprotectin ≥200 μg/g were 70%, 92%, 94%, and 61%; for lactoferrin ≥10 μg/g they were 66%, 92%, 94%, and 59%. Accordingly, the sensitivity, specificity, PPV, and NPV for CRP >5 mg/l were 48%, 91%, 91%, and 48%. Fecal markers were significantly higher in active colonic (both p<0.001) or ileocolonic (calprotectin p=0.028, lactoferrin p=0.004) than in ileal disease. In ileocolonic or colonic disease, colon histology score correlated significantly with fecal calprotectin (r=0.563) and lactoferrin (r=0.543). In patients receiving anti-TNFα therapy, median fecal calprotectin decreased from 1173 μg/g (range 88-15326) to 130 μg/g (13-1419) and lactoferrin from 105.0 μg/g (4.2-1258.9) to 2.7 μg/g (0.0-228.5), both p=0.001. The relation of ileal IL-17+ cells to CD4+ cells decreased significantly during anti-TNF treatment (p=0.047). The relation of IL-17+ cells to Foxp3+ cells was higher in the patients’ baseline specimens than in their post-treatment specimens (p=0.038). Conclusions: For evaluation of CD activity, based on endoscopic findings, more sensitive surrogate markers than CDAI and CRP were fecal calprotectin and lactoferrin. Fecal calprotectin and lactoferrin were significantly higher in endoscopically active disease than in endoscopic remission. In both ileocolonic and colonic disease, fecal markers correlated closely with histologic disease activity. In CD, these neutrophil-derived proteins thus seem to be useful surrogate markers of endoscopic activity. During anti-TNFα therapy, fecal calprotectin and lactoferrin decreased significantly. The anti-TNFα treatment was also reflected in a decreased IL-17/Foxp3 cell ratio, which may indicate improved balance between effector and regulatory T cells with treatment.
  • Holmström, Miia (Helsingin yliopisto, 2006)
    Conventional invasive coronary angiography is the clinical gold standard for detecting of coronary artery stenoses. Noninvasive multidetector computed tomography (MDCT) in combination with retrospective ECG gating has recently been shown to permit visualization of the coronary artery lumen and detection of coronary artery stenoses. Single photon emission tomography (SPECT) perfusion imaging has been considered the reference method for evaluation of nonviable myocardium, but magnetic resonance imaging (MRI) can accurately depict structure, function, effusion, and myocardial viability, with an overall capacity unmatched by any other single imaging modality. Magnetocardiography (MCG) provides noninvasively information about myocardial excitation propagation and repolarization without the use of electrodes. This evolving technique may be considered the magnetic equivalent to electrocardiography. The aim of the present series of studies was to evaluate changes in the myocardium assessed with SPECT and MRI caused by coronary artery disease, examine the capability of multidetector computed tomography coronary angiography (MDCT-CA) to detect significant stenoses in the coronary arteries, and MCG to assess remote myocardial infarctions. Our study showed that in severe, progressing coronary artery disease laser treatment does not improve global left ventricular function or myocardial perfusion, but it does preserve systolic wall thickening in fixed defects (scar). It also prevents changes from ischemic myocardial regions to scar. The MCG repolarization variables are informative in remote myocardial infarction, and may perform as well as the conventional QRS criteria in detection of healed myocardial infarction. These STT abnormalities are more pronounced in patients with Q-wave infarction than in patients with non-Q-wave infarctions. MDCT-CA had a sensitivity of 82%, a specificity of 94%, a positive predictive value of 79%, and a negative predictive value of 95% for stenoses over 50% in the main coronary arteries as compared with conventional coronary angiography in patients with known coronary artery disease. Left ventricular wall dysfunction, perfusion defects, and infarctions were detected in 50-78% of sectors assigned to calcifications or stenoses, but also in sectors supplied by normally perfused coronary arteries. Our study showed a low sensitivity (sensitivity 63%) in detecting obstructive coronary artery disease assessed by MDCT in patients with severe aortic stenosis. Massive calcifications complicated correct assessment of the lumen of coronary arteries.
  • Ilonen, Ilkka (Helsingin yliopisto, 2011)
    Lung cancer accounts for more cancer-related deaths than any other cancer. In Finland, five-year survival ranges from 8% to 13%. The main risk factor for lung cancer is long-term cigarette smoking, but its carcinogenesis requires several other factors. The aim of the present study was to 1) evaluate post-operative quality of life, 2) compare clinical outcomes between minimally invasive and conventional open surgery, 3) evaluate the role of oxidative stress in the carcinogenesis of non-small lung cancer (NSCLC), and 4) to identify and characterise targeted agents for therapeutic and diagnostic use in surgery. For study I, pneumonectomy patients replied to 15D quality of life and baseline dyspnea questionnaires. Study III involved a prospective quality of life assessment using the 15D questionnaire after lobectomy or bi-lobectomy. Study IV was a retrospective comparison of clinical outcomes between 212 patients treated with open thoracotomy and 116 patients who underwent a minimally invasive technique. Study II measured parameters of oxidative metabolism (myeloperoxidase activity, glutathione content and NADPH oxidase activity) and DNA adducts. Study V employed the phage display method and identified a core motif for homing peptides. This method served in cell-binding, cell-localisation, and biodistribution studies. Following both pneumonectomy and lobectomy, NSCLC patients showed significantly decreased long-term quality of life. No significant correlation was noted between post-operative quality of life and pre-operative pulmonary function tests. Women suffered more from increased dyspnea after pneumonectomy which was absent after lobectomy or bi-lobectomy. Patients treated with video-assisted thoracoscopy showed significantly decreased morbidity and shorter periods of hospitalization than did open surgery patients. This improvement was achieved even though the VATS patients were older and suffered more comorbid conditions and poorer pulmonary function. No significant differences in survival were noted between these two groups. An increase in NADPH oxidase activity was noted in tumour samples of both adenocarcinoma and squamous cell carcinoma. This increase was independent from myeloperoxidase activity. Elevated glutathione content was noted in tumour tissue, especially in adenocarcinoma. After panning the clinical tumour samples with the phage display method, an amino acid sequence of ARRPKLD, the Thx, was chosen for further analysis. This method proved selective of tumour tissue in both in vitro and in vivo cell-binding assay, and biodistribution showed tumour accumulation. Because of the significantly reduced quality of life following pneumonectomy, other operative strategies should be implemented as an alternative (e.g. sleeve-lobectomy). To treat this disease, implementation of a minimally invasive surgical technique is safe, and the results showed decreased morbidity and a shorter period of hospitalisation than with thoracotomy. This technique may facilitate operative treatment of elderly patients with comorbid conditions who might otherwise be considered inoperable. Simultaneous exposure to oxidative stress and altered redox states indicates the important role of oxidative stress in the pathogenesis and malignant transformation of NSCLC. The studies showed with great specificity and with favourable biodistribution that Thx peptide is specific to NSCLC tumours. Thx thus shows promise in imaging, targeted therapy, and monitoring of treatment response.