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  • Keski-Säntti, Petra (Helsingin yliopisto, 2011)
    The occurrence of occupational chronic solvent encephalopathy (CSE) seems to decrease, but still every year reveals new cases. To prevent CSE and early retirement of solvent-exposed workers, actions should focus on early CSE detection and diagnosis. Identifying the work tasks and solvent exposure associated with high risk for CSE is crucial. Clinical and exposure data of all the 128 cases diagnosed with CSE as an occupational disease in Finland during 1995-2007 was collected from the patient records at the Finnish Institute of Occupational Health (FIOH) in Helsinki. The data on the number of exposed workers in Finland were gathered from the Finnish Job-exposure Matrix (FINJEM) and the number of employed from the national workforce survey. We analyzed the work tasks and solvent exposure of CSE patients and the findings in brain magnetic resonance imaging (MRI), quantitative electroencephalography (QEEG), and event-related potentials (ERP). The annual number of new cases diminished from 18 to 3, and the incidence of CSE decreased from 8.6 to 1.2 / million employed per year. The highest incidence of CSE was in workers with their main exposure to aromatic hydrocarbons; during 1995-2006 the incidence decreased from 1.2 to 0.3 / 1 000 exposed workers per year. The work tasks with the highest incidence of CSE were floor layers and lacquerers, wooden surface finishers, and industrial, metal, or car painters. Among 71 CSE patients, brain MRI revealed atrophy or white matter hyperintensities or both in 38% of the cases. Atrophy which was associated with duration of exposure was most frequently located in the cerebellum and in the frontal or parietal brain areas. QEEG in a group of 47 patients revealed increased power of the theta band in the frontal brain area. In a group of 86 patients, the P300 amplitude of auditory ERP was decreased, but at individual level, all the amplitude values were classified as normal. In 11 CSE patients and 13 age-matched controls, ERP elicited by a multimodal paradigm including an auditory, a visual detection, and a recognition memory task under single and dual-task conditions corroborated the decrease of auditory P300 amplitude in CSE patients in single-task condition. In dual-task conditions, the auditory P300 component was, more often in patients than in controls, unrecognizable. Due to the paucity and non-specificity of the findings, brain MRI serves mainly for differential diagnostics in CSE. QEEG and auditory P300 are insensitive at individual level and not useful in the clinical diagnostics of CSE. A multimodal ERP paradigm may, however, provide a more sensitive method to diagnose slight cognitive disturbances such as CSE.
  • Ojajärvi, Anneli (Helsingin yliopisto, 2006)
    Objective and background. Tobacco smoking, pancreatitis and diabetes mellitus are the only known causes of pancreatic cancer, leaving ample room for yet unidentified determinants. This is an empirical study on a Finnish data on occupational exposures and pancreatic cancer risk, and a non-Bayesian and a hierarchical Bayesian meta-analysis of data on occupational factors and pancreatic cancer. Methods. The case-control study analyzed 595 incident cases of pancreatic cancer and 1,622 controls of stomach, colon, and rectum cancer, diagnosed 1984-1987 and known to be dead by 1990 in Finland. The next-of-kin responded to a mail questionnaire on job and medical histories and lifestyles. Meta-analysis of occupational risk factors of pancreatic cancer started off with 1,903 identified studies. The analyses were based on different subsets of that database. Five epidemiologists examined the reports and extracted the pertinent data using a standardized extraction form that covered 20 study descriptors and the relevant relative risk estimates. Random effects meta-analyses were applied for 23 chemical agents. In addition, hierarchical Bayesian models for meta-analysis were applied to the occupational data of 27 job titles using job exposure matrix as a link matrix and estimating the relative risks of pancreatic cancer associated with nine occupational agents. Results. In the case-control study, logistic regressions revealed excess risks of pancreatic cancer associated with occupational exposures to ionizing radiation, nonchlorinated solvents, and pesticides. Chlorinated hydrocarbon solvents and related compounds, used mainly in metal degreasing and dry cleaning, are emerging as likely risk factors of pancreatic cancer in the non-Bayesian and the hierarchical Bayesian meta-analysis. Consistent excess risk was found for insecticides, and a high excess for nickel and nickel compounds in the random effects meta-analysis but not in the hierarchical Bayesian meta-analysis. Conclusions. In this study occupational exposure to chlorinated hydrocarbon solvents and related compounds and insecticides increase risk of pancreatic cancer. Hierarchical Bayesian meta-analysis is applicable when studies addressing the agent(s) under study are lacking or very few, but several studies address job titles with potential exposure to these agents. A job-exposure matrix or a formal expert assessment system is necessary in this situation.
  • Airaksinen, Liisa (Helsingin yliopisto, 2010)
    Occupational rhinitis is mainly caused by work environment and not by stimuli encountered outside the workplace. It differs from rhinitis that is worsened by, but not mainly caused by, workplace exposures. Occupational rhinitis can develop in response to allergens, inhaled irritants, or corrosive gases. The thesis evaluated the use of challenge tests in occupational rhinitis diagnostics, studied the long-term health-related quality of life among allergic occupational rhinitis patients, and the allergens of wheat grain among occupational respiratory allergy patients. The diagnosed occupational rhinitis was mainly allergic rhinitis, which was caused by occupational agents, most commonly flours and animal allergens. The non-IgE-mediated rhinitis reactions were less frequent and caused more often asthma than rhinitis. Both nasal challenges and inhalation challenges were found to be safe tests. The inhalation challenge tests had considerably resource-intensive methodology. However, the evaluation of nasal symptoms and signs together with bronchial reactions saved time and expense compared with the organization of multiple individual challenges. The scoring criteria used matched well with the weighted amount of discharge ≥ 0.2 g and in most cases gave comparable results. The challenge tests are valuable tools when there is uncertainty whether the patient's exposure should be reduced or discontinued. It was found that continuing exposure decreases health-related quality of life among patients with allergic occupational rhinitis despite of rhinitis medications, still approximately ten years after the diagnosis. Health-related quality of life among occupational rhinitis patients without any longer occupational exposure was mainly similar than that of the healthy controls. This highlights the importance of the reduction and cessation of occupational exposure. To achieve this, 17% of occupational rhinitis patients had been re-educated. Alpha-amylase inhibitors, lipid transfer protein 2G, thaumatin -like protein, and peroxidase I were found to be relevant allergens in Finnish patients with occupational respiratory wheat allergy. Of these allergens, thaumatin-like protein and lipid transfer protein 2G were found as new allergens associated with baker's rhinitis and asthma. The knowledge of the new clinically relevant proteins can be used in the future in the development of better standardized diagnostic preparations.
  • Pöntynen, Nora (Helsingin yliopisto, 2008)
    Autoimmune diseases affect 5 % of the population and come in many forms, such as diabetes, rheumatoid arthritis and MS. However, how and why autoimmune diseases arise are not yet fully resolved. In this thesis, the onset of autoimmunity was investigated using both patient samples and a mouse model of autoimmunity. Autoimmune diseases are usually complex, due to a number of different causative genes and environmental factors. However, a few monogenic autoimmune diseases have been described, which are caused by mutations in only one gene per disease. One of such disease is called APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) and is enriched in the Finnish population. The causative gene behind APECED is named AIRE from AutoImmune REgulator. How malfunction of just one gene product can cause the multitude of disease components found in APECED is not yet resolved. This thesis sought out to find out more about the functions of AIRE, in order to reveal why APECED and other autoimmune diseases arise and what goes wrong? Usually, immune cells are taught to distinguish between self and non-self during their development. That way, immune cells can fight off bacteria and microbes while leaving the tissues and organs of the host organism itself unharmed. In APECED, the development of immune cells called αβ T cells is incomplete. The cells are not able to fully distinguish between self and non-self. This leads to autodestruction of self tissues and autoimmune disease. One of the achievements of this thesis was the finding that the development of another set of T cells called γδ T cells is not affected by AIRE in mice or in men. Instead, we found that another type of immune cell important in tolerance, called the dendritic cell is defective in APECED patients and is not able to respond to microbial stimulus in a normal fashion. Finally, we studied Aire-deficient mice and found that autoantibodies expressed in the mice were not targeted against the same molecules as those found in APECED patients. This indicates differences in the autoimmune pathology in mice and men. More work is still required before we understand the mechanisms of tolerance and autoimmunity well enough to be able to cure APECED, let alone the more complex autoimmune diseases. Yet altogether, the findings of this thesis work bring us one step closer to finding out why and how APECED and common autoimmune diseases arise.
  • Bramante, Simona (Helsingin yliopisto, 2015)
    Despite the numerous advances in cancer therapy over the past 50 years, cancer still remains the major cause of mortality worldwide, and thus new and more efficient treatments are needed. Oncolytic viruses have shown promising results in preclinical and clinical studies for the treatment of solid tumors, but their efficacy often remains low. A multitude of viral strains, such as adenovirus, have been engineered to become tumor-selective and to stimulate the immune system against the tumors. One example of oncolytic virus is Ad5/3-D24-GMCSF, a tumor-selective 5/3 chimeric oncolytic adenovirus armed with the immunostimulatory granulocyte-macrophage colony-stimulating factor (GM-CSF). In this thesis, we studied the utility of Ad5/3-D24-GMCSF in the treatment of sarcoma, melanoma and breast cancer. The virus showed strong oncolytic potential in vitro and antitumor efficacy in immunodeficient animal models. Furthermore, replication-linked GM-CSF production stimulated the differentiation of human monocytes into macrophages, important for induction of antitumor immune responses. In immunocompetent Syrian hamsters with soft-tissue sarcoma (STS) tumors, Ad5/3-D24-GMCSF reached non-injected tumors through vascular circulation, suggesting its utility for the treatment of metastatic disease. We showed that combining Ad5/3-D24-GMCSF with chemotherapeutics that possess immunogenic properties (doxorubicin and ifosfamide) and that selectively reduce circulating regulatory T-cells (cyclophosphamide), enhanced adenoviral replication and induced immunogenic cell death, setting the stage for clinical testing of combination regimens. Finally we reported safety and possible signs of efficacy in 40 patients with advanced sarcoma, melanoma and breast cancer, who were treated in the context of an Advanced Therapy Access Program (ATAP). Treatments were overall well-tolerated, and objective signs of treatment benefits were also observed. Therefore, our results confirm previous good data regarding Ad5/3-D24-GMCSF as a promising agent for treatment of cancer. Furthermore, our data may prove useful for clinical development of oncolytic adenoviruses combined with low-dose chemotherapy for the treatment of advanced sarcoma, melanoma and breast cancer, and may help to design optimal clinical trials.
  • Bauerschmitz, Gerd Johannes (Helsingin yliopisto, 2007)
    Gene therapy is a promising novel approach for treating cancers resistant to or escaping currently available modalities. Treatment approaches are based on taking advantage of molecular differences between normal and tumor cells. Various strategies are currently in clinical development with adenoviruses as the most popular vehicle. Recent developments include improving targeting strategies for gene delivery to tumor cells with tumor specific promoters or infectivity enhancement. A rapidly developing field is as well replication competent agents, which allow improved tumor penetration and local amplification of the anti-tumor effect. Adenoviral cancer gene therapy approaches lack cross-resistance with other treatment options and therefore synergistic effects are possible. This study focused on development of adenoviral vectors suitable for treatment of various gynecologic cancer types, describing the development of the field from non-replicating adenoviral vectors to multiple-modified conditional replicating viruses. Transcriptional targeting of gynecologic cancer cells by the use of the promoter of vascular endothelial growth factor receptor type 1 (flt-1) was evaluated. Flt-1 is not expressed in the liver and thus an ideal promoter for transcriptional targeting of adenoviruses. Our studies implied that the flt-1 promoter is active in teratocarcinomas.and therefore a good candidate for development of oncolytic adenoviruses for treatment of this often problematic disease with then poor outcome. A tropism modified conditionally replicating adenovirus (CRAd), Ad5-Δ24RGD, was studied in gynecologic cancers. Ad5-Δ24RGD is an adenovirus selectively replication competent in cells defective in the p16/Rb pathway, including many or most tumor cells. The fiber of Ad5-Δ24RGD contains an integrin binding arginine-glycine-aspartic acid motif (RGD-4C), allowing coxackie-adenovirus receptor independent infection of cancer cells. This approach is attractive because expression levels of CAR are highly variable and often low on primary gynecological cancer cells. Oncolysis could be shown for a wide variety of ovarian and cervical cancer cell lines as well as primary ovarian cancer cell spheroids, a novel system developed for in vitro analysis of CRAds on primary tumor substrates. Biodistribution was evaluated and preclinical safety data was obtained by demonstrating lack of replication in human peripheral blood mononuclear cells. The efficicacy of Ad5-Δ24RGD was shown in different orthotopic murine models including a highly aggressive intraperitoneal model of disseminated ovarian cancer cells, where Ad5-Δ24RGD resulted in complete eradication of intraperitoneal disease in half of the mice. To further improve the selectivity and specificity of CRAds, triple-targeted oncolytic adenoviruses were cloned, featuring the cyclo-oxygenase-2 (cox-2) promoter, E1A transcomplementation and serotype chimerism. Those viruses were evaluated on ovarian cancer cells for specificity and oncolytic potency with regard to two different cox2 versions and three different variants of E1A (wild type, delta24 and delta2delta24). Ad5/3cox2Ld24 emerged as the best combination due to enhanced selectivity without potency lost in vitro or in an aggressive intraperitoneal orthotopic ovarian tumor model. In summary, the preclinical therapeutic efficacy of the CRAds tested in this study, taken together with promising biodistribution and safety data, suggest that these CRAds are interesting candidates for translation into clinical trials for gynecologic cancer.
  • Raki, Mari (Helsingin yliopisto, 2009)
    Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.
  • Rajecki, Maria (Helsingin yliopisto, 2011)
    Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.
  • Parry, Mikko (Helsingin yliopisto, 2010)
    Cells are packed with membrane structures, defining the inside and outside, and the different subcellular compartments. These membranes consisting mainly of phospholipids have a variety of functions in addition to providing a permeability barrier for various compounds. These functions involve cellular signaling, where lipids can act as second messengers, or direct regulation of membrane associating proteins. The first part of this study focuses on relating some of the physicochemical properties of membrane lipids to the association of drug compounds to membranes. A fluorescence based method is described allowing for determination of the membrane association of drugs. This method was subsequently applied to a novel drug, siramesine, previously shown to have anti-cancer activity. Siramesine was found to associate with anionic lipids. Especially interesting is its strong affinity for a second messenger lipid phosphatidic acid. This is the first example of a small molecule drug compound specifically interacting with a cellular lipid. Phosphatidic acid in cells is required for the activation of many signaling pathways mediating growth and proliferation. This provides an intriguing possibility for a simple molecular mechanism of the observed anti-cancer activity of siramesine. In the second part the thermal behavior and self assembly of charged and uncharged membrane assemblies was studied. Strong inter-lamellar co-operativity was observed for multilamellar DPPC vesicles using fluorescence techniques together with calorimetry. The commonly used membrane models, large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) were found to possess different biophysical properties as interlamellar interactions of MLVs drive segregation of a pyrene labeled lipid analogue into clusters. The effect of a counter-ion lattice on the self assembly of a cationic gemini surfactant was studied. The presence of NaCl strongly influenced the thermal phase behavior of M-1 vesicles, causing formation of giant vesicles upon exceeding a phase transition temperature, followed by a subsequent transition into a more homogenous dispersion. Understanding the underlying biophysical aspects of cellular membranes is of fundamental importance as the complex picture of the structure and function of cells is evolving. Many of the cellular reactions take place on membranes and membranes are known to regulate the activity of many peripheral and intergral membrane associating proteins. From the point of view of drug design and gene technology, membranes can provide an interesting target for future development of drugs, but also a vehicle sensitive for environmental changes allowing for encapsulating drugs and targeting them to the desired site of action.
  • Gaal, Emilia (Helsingin yliopisto, 2012)
    Cerebrovascular diseases continue to challenge us by robbing lives and leaving many disabled still in their prime working age. Some cerebrovascular diseases are more acute in nature, and some erode the quality of life over a long period of time. A life-threatening form of acute cerebrovascular disease is brought on by the rupture of an intracranial aneurysm (IA). Most IAs are berry-shaped pouches at the forking site of cerebral arteries. Since according to autopsy results, 2-5% of the population harbours IA, it is a common disease. Most IA go unnoticed during one s lifetime, however, often the first symptom they give is their deadly rupture. Likely, both environmental factors and a compound genetic susceptibility, contribute to the risk of IA, making it a complex disease. The aim of studies I-III was to test whether in humans common genetic variants contribute to the susceptibility to IA (I,II), and to seek genetic evidence for their pathomechanism (III). In multinational genome-wide association studies (I,II) we identified 5 loci with strong statistical evidence of association with IA, and a further 14 loci with suggestive evidence. Further, we found that suggestive IA risk locus at 5q26 is strongly associated with high systolic blood pressure in over 210 000 individuals of European descent, highlighting the connection between hypertension and IA (III). To gain further insight into cerebral vasculopathies and to facilitate the development of novel therapies, in study (IV) we turned our attention to vascular growth factor induced angiogenesis in a model organism. We tested by viral gene transfer the known vascular growth factors in the murine central nervous system and characterised extensively the angiogenesis upon treatment. The aim of the study was to identify the best candidate vascular growth factor(s) for therapeutic brain angiogenesis. We identified placenta growth factor as the most safe and efficient candidate for therapeutic revascularisation of the central nervous system. We envision a placenta growth factor enhanced multiple bur hole indirect extracranial-intracranial bypass as a novel therapeutic approach in the brain, possibly aiding the treatment of diseases such as chronic cerebral hypoperfusion, complex IAs and stroke.
  • Kankuri, Esko (Helsingin yliopisto, 2002)
  • Hiekkalinna, Tero (Helsingin yliopisto, 2012)
    In this thesis, we developed software for automated genome-wide linkage and linkage disequilibrium analysis based on common gene mapping methods for qualitative and quantitative phenotypes. We further developed likelihood-based software for joint linkage and/or linkage disequilibrium (LD) analysis in general pedigrees based on a novel variation of the classical lod score approach, the so-called pseudomarker method, and evaluated its statistical properties as compared with the existing family-based association methods. This was done using real-life migraine and schizophrenia pedigree structures from Finland. In addition, we compared various study designs for association analysis and investigated statistical properties of the likelihood ratio test for conditional analysis of LD given linkage. First, we automated the laborious process of running a variety of genome-wide linkage and linkage disequilibrium analysis software packages, including ANALYZE, MERLIN, GENEHUNTER, and SOLAR. With this software tool, data file format conversion, and running of the analyses are completely automated. This tool has been applied to many large genome-wide mapping studies. Second, we developed user-friendly PSEUDOMARKER software, which performs likelihood-based linkage and/or linkage disequilibrium analysis in general pedigrees. This software allows for joint analysis of heterogeneous relationship structures, such as singletons (i.e. cases and controls), triads, sibships, and large multigenerational pedigrees. The performance of this software was evaluated in comparison to the existing repertoire of other family-based association methods. Third, we performed an extensive simulation study to investigate the statistical properties (i.e. type-I error and power) of PSEUDOMARKER and other commonly used family-based association methods. Our results demonstrate that many widely-used methods are not valid for testing LD in the presence of linkage, and likelihood-based methods which can properly account for missing data and individual relationships in pedigrees, such as PSEUDOMARKER, outperform the other approaches over a wide variety of etiological models. We also demonstrated that association mapping in families is far more powerful than in population-based samples. Fourth, we investigated the statistical properties of the likelihood ratio test for association conditional on linkage when inaccurate parametric models were used. Our results showed that while under most situations they perform appropriately despite the parametric model being improperly specified, under certain conditions, when there is complete linkage between disease and marker loci, overly-deterministic dominant analysis models can lead to false inferences of LD in the presence of linkage when the true etiological model is recessive in character. In this study, we have developed powerful and easy to use tools for analysis of linkage and LD in general pedigrees and unrelated individuals jointly, and have demonstrated the superiority of such methods in the general case. Our results provide important information for the human genetics community about optimal ways to collect and analyze data.
  • Kivisaari, Reetta (Helsingin yliopisto, 2008)
    Background: Opiod dependence is a chronic severe brain disorder associated with enormous health and social problems. The relapse back to opioid abuse is very high especially in early abstinence, but neuropsychological and neurophysiological deficits during opioid abuse or soon after cessation of opioids are scarcely investigated. Also the structural brain changes and their correlations with the length of opioid abuse or abuse onset age are not known. In this study the cognitive functions, neural basis of cognitive dysfunction, and brain structural changes was studied in opioid-dependent patients and in age and sex matched healthy controls. Materials and methods: All subjects participating in the study, 23 opioid dependents of whom, 15 were also benzodiazepine and five cannabis co-dependent and 18 healthy age and sex matched controls went through Structured Clinical Interviews (SCID) to obtain DSM-IV axis I and II diagnosis and to exclude psychiatric illness not related to opioid dependence or personality disorders. Simultaneous magnetoencephalography (MEG) and electroencephalography (EEG) measurements were done on 21 opioid-dependent individuals on the day of hospitalization for withdrawal therapy. The neural basis of auditory processing was studied and pre-attentive attention and sensory memory were investigated. During the withdrawal 15 opioid-dependent patients participated in neuropsychological tests, measuring fluid intelligence, attention and working memory, verbal and visual memory, and executive functions. Fifteen healthy subjects served as controls for the MEG-EEG measurements and neuropsychological assessment. The brain magnetic resonance imaging (MRI) was obtained from 17 patients after approximately two weeks abstinence, and from 17 controls. The areas of different brain structures and the absolute and relative volumes of cerebrum, cerebral white and gray matter, and cerebrospinal fluid (CSF) spaces were measured and the Sylvian fissure ratio (SFR) and bifrontal ratio were calculated. Also correlation between the cerebral measures and neuropsychological performance was done. Results: MEG-EEG measurements showed that compared to controls the opioid-dependent patients had delayed mismatch negativity (MMN) response to novel sounds in the EEG and P3am on the contralateral hemisphere to the stimulated ear in MEG. The equivalent current dipole (ECD) of N1m response was stronger in patients with benzodiazepine co-dependence than those without benzodiazepine co-dependence or controls. In early abstinence the opioid dependents performed poorer than the controls in tests measuring attention and working memory, executive function and fluid intelligence. Test results of the Culture Fair Intelligence Test (CFIT), testing fluid intelligence, and Paced Auditory Serial Addition Test (PASAT), measuring attention and working memory correlated positively with the days of abstinence. MRI measurements showed that the relative volume of CSF was significantly larger in opioid dependents, which could also be seen in visual analysis. Also Sylvian fissures, expressed by SFR were wider in patients, which correlated negatively with the age of opioid abuse onset. In controls the relative gray matter volume had a positive correlation with composite cognitive performance, but this correlation was not found in opioid dependents in early abstinence. Conclusions: Opioid dependents had wide Sylvian fissures and CSF spaces indicating frontotemporal atrophy. Dilatation of Sylvian fissures correlated with the abuse onset age. During early withdrawal cognitive performance of opioid dependents was impaired. While intoxicated the pre-attentive attention to novel stimulus was delayed and benzodiazepine co-dependence impaired sound detection. All these changes point to disturbances on frontotemporal areas.
  • Sampo, Mika (Helsingin yliopisto, 2011)
    Soft tissue sarcomas are malignant tumours of mesenchymal origin. Because of infiltrative growth pattern, simple enucleation of the tumour causes a high rate of local recurrence. Instead, these tumours should be resected with a rim of normal tissue around the tumour. Data on the adequate margin width are scarce. At Helsinki University Central Hospital (HUCH) a multidisciplinary treatment group started in 1987. Surgical resection with a wide margin (2.5 cm) is the primary aim. In case of narrower margin radiation therapy is necessary. The role of adjuvant chemotherapy remains unclear. Our aims were to study local control by the surgical margin and to develop a new prognostic tool to aid decision-making on which patients should receive adjuvant chemotherapy. Patients with soft tissue sarcoma of the extremity or the trunk wall referred to HUCH during 1987-2002 form material in Studies I and II. External validation material comes from the Lund university sarcoma registry. The smallest surgical margin of at least 2.5 centimetres yielded local control of 89 per cent at five years. Amputation rate was 9 per cent. The proposed prognostic model with necrosis, vascular invasion, size on a continuous scale, depth, location and grade worked well both in Helsinki material and in the validation material, and it also showed good calibration. Based on the present study, we recommend the smallest surgical margin of 2-3 centimetres in soft tissue sarcoma irrespective of grade. Improvement in local control was present but modest in margins wider than 1 centimetre. In cases where gaining a wider margin would lead to a considerable loss of function, smaller margin is to be considered combined to radiation therapy. Patients treated with inadequate margins should be offered radiation therapy irrespective of tumour grade. Our new prognostic model to estimate 10-year survival probability in patients with soft tissue sarcoma of the extremities or trunk wall showed good dicscrimination and calibration. For time being the prognostic model is available for scientific use and further validations. In the future, the model may aid in clinical decision-making. For operable osteosarcoma, neoadjuvant multidrug chemotherapy followed by delayed surgery and multidrug adjuvant chemotherapy is the treatment of choice. Overall survival rates at five years are approximately 75 per cent in modern trials with classical osteosarcoma. All patients diagnosed and reported to the Finnish Cancer Registry with osteosarcoma in Finland during 1971-2005 form the material in Studies III and IV. Limb-salvage rate increased from 23 per cent to 78 per cent during 1971-2005. The 10-year sarcoma-specific survival for the whole study population improved from 32 per cent to 62 per cent. It was 75 per cent for patients with a local high-grade osteosarcoma of the extremity diagnosed during 1991-2005. This study outlines the improved prognosis of osteosarcoma patients in Finland with modern chemotherapy. The 10-year survival rates are good also in an international scale. Nonetheless, their limb-salvage rate remains inferior to those seen for highly selected patient series. Overall, the centralisation of osteosarcoma treatment would most likely improve both survival and limb-salvage rates even further.
  • Sarkonen, Nanna (Helsingin yliopisto, 2007)
    The genus Actinomyces consists of a heterogeneous group of gram-positive, mainly facultatively anaerobic or microaerobic rods showing various degrees of branching. In the oral cavity, streptococci and Actinomyces form a fundamental component of the indigenous microbiota, being among initial colonizers in polymicrobial biofilms. The significance of the genus Actinomyces is based on the capability of species to adhere to surfaces such as on teeth and to co-aggregate with other bacteria. Identification of Actinomyces species has mainly been based on only a few biochemical characteristics, such as pigmentation and catalase production, or on the use of a single commercial kit. The limited identification of oral Actinomyces isolates to species level has hampered knowledge of their role both in health and disease. In recent years, Actinomyces and related organisms have attracted the attention of clinical microbiologists because of a growing awareness of their presence in clinical specimens and their association with disease. This series of studies aimed to amplify the identification methods for Actinomyces species. With the newly developed identification scheme, the age-related occurrence of Actinomyces in healthy mouths of infants and their distribution in failed dental implants was investigated. Adhesion of Actinomyces species to titanium surfaces processed in various ways was studied in vitro. The results of phenotypic identification methods indicated a relatively low applicability of commercially available test kits for reliable identification within the genus Actinomyces. However, in the study of conventional phenotypic methods, it was possible to develop an identification scheme that resulted in accurate differentiation of Actinomyces and closely related species, using various different test methods. Genotypic methods based on 16S rRNA sequence analysis of Actinomyces proved to be a useful method for genus level identification and further clarified the species level identification with phenotypic methods. The results of the study of infants showed that the isolation frequency of salivary Actinomyces species increased according to age: thirty-one percent of the infants at 2 months but 97% at 2 years of age were positive for Actinomyces. A. odontolyticus was the most prominent Actinomyces colonizer during the study period followed in frequency by A. naeslundii and A. viscosus. In the study of explanted dental implants, Actinomyces was the most prevalent bacterial genus, colonizing 94% of the fixtures. Also in the implants A. odontolyticus was revealed as the most common Actinomyces species. It was present in 84% of Actinomyces -positive fixtures followed in frequency by A. naeslundii, A. viscosus and A. israelii. In an in vitro study of titanium surfaces, different Actinomyces species showed variation regarding their adhesion to titanium. Surface roughness as well as albumin coating of titanium had significant effects on adhesion. The use of improved phenotypic and molecular diagnostic methods increased the accuracy of the identification of the Actinomyces to species level. This facilitated an investigation of their occurrence and distribution in oral specimens in both health and disease.
  • Ahmed Haji Omar, Abdirisak (Helsingin yliopisto, 2015)
    Oral (OSCC) and cutaneous (CSCC) squamous cell carcinomas are epithelial neoplasms, which are both derived from keratinocyte cells. However, the etiology and prognosis of OSCC and CSCC are different. The main etiological factors behind OSCC are tobacco smoke and alcohol consumption, and for CSCC it is UV-radiation. OSCC has poorer prognosis than CSCC. In order to be invasive, cancer cells have to pass various barriers. They have to disrupt cell-to-cell junctions, penetrate basement membranes, and invade connective tissue. The pattern of invasion of tumors varies strikingly. Some invade in large border fronts while others invade in single cell manner. Expression of the transmembrane proteins, E-cadherin and syndecan-1, in cell membrane are lost during tumor invasion, and therefore loosening cell adhesion. Matrix metalloproteinases (MMP) are tissue proteinases, which have a proteolytic role in various physiological events and during tumor progression MMPs are capable of degrading extracellular matrix (ECM) proteins but also have an immunomodulatory role. Toll-like receptors (TLRs) are part of the innate immunity and can recognize exogenous pathogen associated molecular patterns or endogenous damage associated molecular patterns. Cancer cells may use TLRs to induce tumor-promoting inflammation. The aim of the study was to examine possible cellular and molecular differences between OSCC and CSCC explaining their different behaviors as cancers despite having cellular similarities. The study included 36 OSCC and 27 CSCCs from patients with early stage histological risk assessment (HRA) model and histological risk assessment score (HRS). We performed immunohistochemical staining for E-cadherin, Snail (Snail1), Syndecans (1 and 2), MMPs (7, 8 and 9) and TLRs (4 and 5). In vitro, with oral and cutaneous cell lines the effect of TLR-5 ligand flagellin on proliferation, migration and invasion was studied. OSCC patients had worse disease-specific survival than CSCC patients and this correlated with the invasion depths of the OSCC tumors. OSCC had a more severe histological pattern of invasion than CSCC. E-cadherin and Syndecan-1 expression decreased in the invasive front of OSCCs and CSCCs. Syndecan-1 expression in the tumor stroma was higher in OSCC than in CSCC in tumors with invasion depth over 4 mm. MMP-7 was mainly expressed in the invasive front of OSCC and CSCC and was stronger in OSCC than in CSCC. MMP-8 and MMP-9 were mainly expressed in the peritumoral inflammatory cells. TLR-5 expression was stronger in OSCC than in CSCC. In vitro, TLR-5 ligand flagellin increased proliferation, migration and invasion of less aggressive oral and cutaneous cell lines, but failed to do so with the most aggressive oral cancer cell line. In conclusion, the OSCC patients of this study had poorer disease specific survival than CSCC patients. Increased stromal syndecan-1 expression in OSCC, MMP-7 expression in the invasive front of the tumor, and MMP-9 expression in inflammatory cells could partly explain the differences in survival between OSCC and CSCC.
  • Saarela, Riitta (Helsingin yliopisto, 2014)
    ABSTRACT This study formed part of a developmental project in the Helsinki Metropolitan Area of Finland during 2003 2011 that aimed to develop nutritional care in long-term care facilities. The aim of this study was to assess tooth brushing/denture cleaning habits, dentition, chewing problems, and swallowing difficulties and their associations with nutritional status and eating habits. Furthermore, the aim was to explore the prognostic value of dentition, chewing problems and swallowing difficulties in relation to mortality. In addition, the adequacy of the dietary intake of energy, protein, and other nutrients was examined. Of all the residents in assisted living facilities (N = 2188) in the cities of Helsinki and Espoo, 67% (1475) participated in this study in 2007. Trained registered nurses familiar with the residents assessed each participant and collected demographic data, the medical history, information on the functional and cognitive status, tooth brushing/dentition cleaning habits, dentition, oral symptoms, eating habits and diets. The nutritional status was assessed with the Mini Nutritional Assessment (MNA). In addition, 343 volunteer residents provided one-day food diaries. Their energy, protein, and nutrient intakes were calculated from these detailed food diaries and compared with the recommendations of the Finnish National Nutrition Council as a measure of dietary adequacy. Information on three-year mortality was retrieved from central registers on 6 July 2010. The mean age of the residents was 83 years and 79% of them were women. The educational level was low; 56% of the residents had a primary school level of education or less. In activities of daily living, most residents (84%) required at least prompting or assistance in dressing, hygiene and the keeping of personal effects, or required considerable help with personal care, often involving incontinence. Over half of the residents (55%) had cognitive impairment. Edentulousness was common; more than half of the residents (52%) had lost all their teeth, while 7% (n = 94) were totally edentulous without prosthesis. Of the residents, 17% did not clean or had not cleaned their teeth/dentures daily. According to the MNA, 13% were malnourished, 65% were at risk of malnutrition and 22% were well nourished. Edentulousness without prostheses and infrequent tooth brushing were associated with malnutrition, oral symptoms and infrequent use of oral health care services. Residents with chewing problems (n = 287) were older, had more comorbidities and were more likely to be malnourished according to the MNA, to be dependent in activities of daily living (ADL) and to have poorer subjective health than those without chewing problems. In logistic regression analysis including age, sex, MNA class and the Charlson´s Comorbidity Index as covariates, chewing problems still independently predicted mortality (OR = 1.46, 95% CI = 1.10 1.93). Of the residents, 12% (n = 173) had swallowing difficulties and they were more likely to be malnourished that those residents without swallowing difficulties. Swallowing difficulties also had an independent predictive value for mortality (OR = 1.49, 95% Cl = 1.04-2.12). Large proportions of volunteer participants in the subsample that provided one-day food diaries received less than the recommended amounts of energy, protein or micronutrients. The dietary intake of protein was significantly lower among edentulous subjects without dentures than those with natural teeth. In the adjusted (age, gender and Charlson s comorbidity index) logistic regression model, being in Group 1 (edentulous participants without dentures) and Group 2 (edentulous participants with some removable dentures in one or both jaws) predicted a poorer protein intake (less than 60 g/day; OR 2.4, 95% CI 1.0 5.7, p = 0.042 and OR 1.6, 95% CI 1.0 2.6, p = 0.045, respectively) compared with the reference Group 3 (dentulous participants all or some natural teeth and with or without removable dentures in one or both jaws; OR = 1). Oral problems were common among frail older residents in assisted living facilities and they were associated with nutritional problems. These findings suggest the need for co-operation between nursing staff and oral care personnel.
  • Helenius, L Miia J (Helsingin yliopisto, 2005)