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  • Kaislasuo, Janina (Helsingin yliopisto, 2015)
    The proportion of nulligravid and nulliparous women is increasing as women delay childbirth in developed countries. Simultaneously, contraceptive failure, unintended pregnancies and abortions, especially in women below the common childbearing age, are a global problem. By promoting intrauterine devices (IUDs) and subdermal implants, referred to as long-acting reversible contraceptives (LARCs), among these women, contraceptive failure caused by non-compliance of the user can be minimized, in addition to providing easy and efficient long-term contraception. However, the risk of difficulties at IUD insertion in nulligravid/nulliparous women, as well as small uterine size, have both been considered as barriers limiting the use of intrauterine contraception (IUC) in these women. The present studies were designed to study the barriers to IUC in nulligravid and nulliparous women. To compare both types of IUC available, we used the levonorgestrel-releasing intrauterine system (LNG-IUS) and the copper-releasing NovaT (TCu380Ag), with identical frames measuring 32 x 32 mm. To exclude any effect of prior pregnancy on the uterine cavity or the cervix, only nulligravid women were included. Difficulties at insertion, menstrual diaries kept after insertion (months 1 3) and at the end of the study (months 10 12) as well as adverse events were compared against uterine cavity measurements and pre-insertion menstrual characteristics reported by the women. In addition, as uterine perforation is mainly seen as a complication related to insertion, we retrospectively analysed women treated for this rare complication between 1996 and 2009 in our hospital district area. We gave 165 nulligravid women requesting their first IUD a free choice between the two IUDs after contraceptive counselling. The majority, 113 women (68.5%), chose the LNG-IUS and 52 women (31.5%) chose the copper IUD. Insertion was easy in 89% of the women. The women were satisfied, with only 17/135 women (12.6%) available for follow-up discontinuing because of adverse events. The reported numbers of days of bleeding and pain were similar to that in earlier reports on parous women. Severe pain at insertion was reported by 56.5% of the women and severe dysmenorrhoea the only factor predicting severe pain (OR 7.9, 95% CI 2.5 24.9, p less than 0.001). Dysmenorrhoea was also related to more pain during the first months with both devices. Baseline spontaneous bleeding predicted bleeding with the LNG-IUS, but not with the copper IUD. Among women using the LNG-IUS, scanty menstrual bleeding (OR 8.2, 95% CI 1.4 48.2, p=0.02) and smoking (OR 8.2, 95% CI 1.8 38.6, p=0.007) predicted amenorrhoea at one year. Uterine measurements, particularly fundal cavity width, were small in comparison to the devices in a majority of the women. The odds of a difficult or failed insertion increased with shorter uterine length and a steeper flexion angle, but the great majority of insertions, even in small and more flexed uteri, were uneventful. Cervical tightness was the main reason for problems in cases of difficult insertion. No uterine threshold measurements predicting difficulties were found. Small uterine measurements were associated with both less bleeding and less pain among LNG-IUS users. Women with the widest fundal widths reported significantly more pain at the end of the one-year follow-up period compared with those with smaller widths. Uterine size did not affect bleeding in connection with the copper IUD, but there was a slight tendency towards more pain during long-term use among women with smaller uterine cavity measurements, although size groups were small with this device. Uterine size did not predict adverse events. We found 75 cases of surgically treated uterine perforation during the 15 year long study period. The incidence of perforation was low, 0.4/1000 insertions, and similar with both types of IUC. Postpartum insertion, earlier presented as the main risk factor of uterine perforation, was also common in this population (64%). The majority of cases, 71%, presented with complaints of abnormal bleeding or pain, but 29% were asymptomatic and diagnosed in connection with missing threads or pregnancy. Pregnancy was more common with a misplaced copper IUD, 33% vs. 7% with a misplaced LNG-IUS (p=0.009). We found no severe complications or intra-abdominal adhesions caused by the misplaced devices. Adhesions were local and more common in copper IUD users (58% vs. 20%, p=0.002). In conclusion, nulligravid women are satisfied users of modern IUC, with continuation rates and bleeding and pain profiles similar to those in parous women. Small uterine cavity measurements are not a barrier to IUC and pre-insertion ultrasonographic evaluation of uterine cavity size is unnecessary. As dysmenorrhoea predicts both severe insertion pain and pain during the first months of IUD use, analgesia and counselling for these women should be highlighted. Although rare, the risk of uterine perforation is increased during the postpartum period, probably reflecting uterine involution as the main reason for this complication. Neither symptoms nor surgical findings are severe in connection with current devices.
  • Virtanen, Anni (Helsingin yliopisto, 2015)
    High coverage amongst those at risk and a high attendance rate are essential in achieving a good impact in a cervical cancer screening programme. In Finland, attendance in the programme is approximately 70% with a slight decreasing trend. There is wide variation in the current invitation practice between municipalities. The introduction of human papillomavirus (HPV) testing in cervical cancer screening has brought about a new possible means of improving attendance rates, as HPV-testing can be performed on self-collected samples. This offers the opportunity to supply sampling devices directly to the homes of the women (self-sampling). The aim of this study was to investigate the effects and feasibility of using self-taken samples for HPV-testing to conduct cervical cancer screening of non-attendees to the Finnish cervical screening programme. The effect on attendance to the screening programme, on overall screening test coverage (including also testing outside the screening programme), on the yield of precancerous lesions detected by screening and on the costs of a screening programme were assessed, as were women s views on this new screening modality. The effects of self-sampling were first studied as a first reminder (i.e. among non-attendees after the primary invitation) in a randomized setting in comparison to a reminder letter, and then in a non-randomized setting as a second reminder after two invitation letters. As a first reminder to non-attendees after the primary invitation, a self-sampling test resulted in somewhat higher attendance than a reminder letter. The difference was small, and in terms of resulting costs (price per extra screened woman and price per detected CIN2+ lesion), a reminder letter with a pre-assigned appointment time is a more feasible choice than a self-sampling test. However, self-sampling can be used to increase screening attendance as a second reminder after two invitation letters. Overall attendance rates increased by 4-8%, and the combined effect of reminder letters and self-sampling showed a 12-23% increase. The yield of detected CIN2+ lesions increased by 25-33% with two reminders. As opportunistic screening is very common in Finland, the increase in overall test coverage remained smaller than the increase in the uptake to the programme. Based on a questionnaire study conducted alongside self-sampling, self-sampling at home helps to overcome both practical and emotional barriers to traditional screening. Women who took part in screening by self-sampling reported mainly positive experiences, but negative experiences were more common among women with a mother tongue other than Finnish or Swedish. The invitation protocol preceding the self-sampling option must be carefully arranged to achieve optimal attendance. A total attendance of well over 80% is achievable in the national programme if personal invitations and reminder letters to non-attendees are sent, scheduled appointments are used in both letters and self-sampling tests are sent to those women who still do not attend.
  • Parviainen, Suvi (Helsingin yliopisto, 2015)
    It has been estimated that up to half of people living in industrial societies will get cancer. Almost all cancer deaths are caused by metastatic disease and only few metastatic solid tumors can be cured with available therapies. Therefore, novel therapeutic modalities are needed. Genetically engineered oncolytic viruses such as adenoviruses and vaccinia viruses are a promising therapeutic approach given their capacity to specifically replicate in and kill tumor cells as well as to reach distant metastasis. Oncolytic viruses have demonstrated good safety, tolerability and promising signs of anti-tumor efficacy in several clinical trials, but the efficacy of the therapy needs improvement to reach its full clinical potential. Adenoviruses are one of the most studied vectors in oncolytic virotherapy. Two major obstacles limiting the efficacy have been insufficient transduction of the tumor cells and the recognition of the virus by the immune system, which rapidly clears the virus from systemic circulation, hampering the overall efficacy. This study shows that novel capsid modifications can increase the transduction efficacy and oncolytic potency of the virus, and that embedding oncolytic virus in a silica implant might help to circumvent the problem of clearance by prompting only modest immune responses against the virus. Recently, arming oncolytic viruses with immunomodulatory molecules has been a major focus in virotherapy. Immunotherapy means inducing the patient´s immune system to recognize and attack the tumor and has been recently linked with oncolytic virotherapy, as the replication of the virus in the tumor can be immunogenic per se and also release cancer epitopes available for antigen-presenting cells. Host immune responses have been shown to be a key player in oncolytic virotherapy as the balance between anti-viral and anti-tumoral immunity determines the efficacy of the therapy. Oncolytic vaccinia virus possesses unique immunogenicity and mechanisms of action that are distinct from other treatment modalities, and its self-perpetuating nature provides an ideal platform for therapeutic transgenic insertion. Therefore we engineered novel oncolytic vaccinia viruses with granulocyte-macrophage colony-stimulating factor (GMCSF) or CD40-ligand and studied their ability to increase the therapeutical outcome and anti-tumor immune responses in preclinical animal models. In the final part of the thesis the life cycle of oncolytic vaccinia virus was studied in more detail as we were able to show that the life cycle was compromised in a feline squamous cell carcinoma cell line. This finding might be important for a deeper understanding of the vaccinia virus-host cell interactions.
  • Tohmola, Niina (Helsingin yliopisto, 2015)
    Metabolites are low molecular weight compounds participating in different functions of cellular systems. Metabolites can be used as diagnostic biomarkers for numerous diseases. Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a powerful tool in quantification of metabolites from various sample matrices. Good sensitivity and specificity are the main benefits of the technique. Mass spectrometry is commonly used in industry, drug research and clinical diagnostics. Extensive validation of newly developed analytical methods will construct the basis to a reliable assay, and it is significant especially when analysing e.g. patient samples. The aim of this study was to develop quantitative assays for metabolites from biological samples for biomedical research and clinical diagnostics. We designed and constructed an on-line high performance liquid chromatography (HPLC) equipment and validated an assay for direct quantification of extracellular metabolites from cell cultivation. Automated sampling for LC-MS/MS analysis of intracellular metabolites was connected to the on-line system. The on-line analysis improves the methodology and shortens the time of analysis. Furthermore, a frequent sampling data can provide valuable information about physiological indications in various cell cultivations. On-line HPLC is suitable for various biotechnological applications because of its ability to monitor and collect data during cell cultivation. We developed and validated LC-MS/MS assays for neuroendocrine tumor (NET) biomarkers 5-hydroxyindole acetic acid (5-HIAA) and vanillylmandelic acid (VMA) from human serum. Generally, urinary HPLC assays are used for the determination of NET markers. HPLC assays have certain limitations and 24-h urine collection is laborious. Our LC-MS/MS assays are specific, fast and well suited for diagnostics of NETs. Furthermore, guidelines for urine collection advise to refrain from serotonin-containing foods for three days before sample collection. We showed that such a diet restriction before serum 5-HIAA assay is not necessary. Instead, one day serotonin-free diet before sampling is sufficient because the half-life of 5-HIAA in circulation was found to be 1.3 hours. All assays developed during this study were sensitive and had a wide linear range. Our serum 5-HIAA LC-MS/MS assay is routinely used for the analysis of NET patient samples at the Helsinki University Central Hospital Laboratory, HUSLAB. Serum VMA LC-MS/MS assay will be in routine use in the HUSLAB in near future. Furthermore, On-line HPLC Ltd, (Helsinki, Finland) has commercialized the on-line HPLC equipment developed in this study.
  • Rantanen, Ville (Helsingin yliopisto, 2015)
    Images provide invaluable information to Biomedicine. Especially, microscopy as an information source has been providing knowledge for research and clinical diagnostics. We have moved away from simply looking at the images to quantifiable computerized image analysis. Over the last decades, image analysis developers have prepared algorithms and software to address various scientific enquiries using images. These software are often created for a single purpose. Naturally, not even the most generic software can include all the algorithms ever created. From an image analysis developer point of view, the choice of software creates limitations. It limits the developer to the algorithms included and to the language it was developed in. Even if the software is modular and extendable, a specific language is required and the earlier algorithm implementations would have to be ported. This thesis presents an integration platform for image analysis: Anima. It is capable of using existing software and including them in analysis workflows. Since image analysis is very case specific, custom processing commands are frequently needed. Anima comes with a large number of data and image analysis components developed directly for the platform, as well as components that send custom commands to the integrated software. All of the components can be executed in a single analysis pipeline. Anima itself is built on top of Anduril, another software, inheriting its software architecture. Anduril gives Anima the power of parallel processing and rerun prevention mechanism, speeding up the development cycle of new algorithms. The usability of Anima for method development is shown by implementing new segmentation algorithms and visualization tools. The tools and methods are all suited to large data sets. To display the modularity, the tools are published as separate programs that are then integrated in Anima. The usefulness of the platform is shown by applying it in different biomedical research settings. The settings include different organisms: human, rat and nematode; different sample material: brain tissue, lymphatic nodes and serum; and different medical interests: cerebral ischemia, cancer and allergy. Anima is a versatile open-source image analysis platform, that encourages the use of best practices of programming habits. It makes the development of analysis workflows and individual algorithms more efficient.
  • Häkkinen, Margareeta (Helsingin yliopisto, 2015)
    Opioids are the most important drugs causing fatal poisonings. Determining whether an opioid death was poisoning may, however, be difficult even if involving appropriate toxicological laboratory investigation. Apart from heroin, little statistically significant data-analysis is available for interpretation of blood concentrations of opioids from various types of post-mortem cases. Tolerance, route of administration, and delay of death after drug administration all influence postmortem drug concentrations. In this thesis, quantitative blood concentration data extracted from the high-quality Finnish postmortem toxicology database was the investigative tool to overcome this problem. Opioid deaths typically involve drug abuse, and suspected drug-abuser deaths must, by Finnish law, undergo medico-legal examination. Medico-legal autopsy in these cases includes comprehensive drug screening and, based on its results, more specific drug quantification. This thesis combined concentration data stored in the postmortem toxicology database with information from death certificates issued by forensic pathologists to allow statistical comparisons between drug poisonings and other deaths, as well as between drug abusers and other users. Concentration data mainly involved drug concentrations in postmortem femoral blood, but drug concentrations in urine and parent drug/metabolite concentration ratios also allowed assessment of buprenorphine, codeine, and tramadol deaths. Opioid poisonings proved to be mainly unintentional polydrug poisonings, regularly involving benzodiazepines, gabapentinoids, and other psycholeptics. Buprenorphine and methadone blood concentrations in fatal poisonings remained within their therapeutic ranges, and these two opioids involved mostly abuse. Concentrations of the weak opioids tramadol and codeine were above their therapeutic ranges both in abuser cases and in fatal poisonings. Tramadol abuse was common but abuse of oxycodone, fentanyl, and codeine was rather low compared to their therapeutic use. Abuse of the gabapentinoids pregabalin and gabapentin was strongly associated with opioid abuse, and compared to gabapentin abuse, pregabalin abuse was proportionally more frequent. To prevent parenteral buprenorphine abuse, opioid maintenance treatment applied a combination product of buprenorphine-naloxone. This combination product is, however, abused as well, and monitoring its abuse is challenging. In this study, urine samples collected from living patients at different phases of opioid maintenance treatment supplemented the postmortem data. Based on the criteria established with these patients, combined with postmortem data and proper background information, a urine concentration limit was estimated for suspected parenteral abuse of the buprenorphine-naloxone product in postmortem cases. Deaths and fatal poisonings due to parenteral buprenorphine-naloxone abuse occurred frequently, and abuse of the combination product was proportionally even more fatal than was abuse of buprenorphine. The results of this study will assist in medico-legal cause-of-death investigations through providing quantitative reference concentrations for the interpretation of opioid-related deaths. Further, estimating the proportion attributable to prescription opioid abuse compared to that of other opioid use and creating abuser profiles for various opioids can promote public health through proper drug policy. In a clinical context, results may be helpful in evaluating possible drug abuse and compliance among prescription-drug users. Detecting abuse of these important yet addictive medications is vital in promoting welfare and drug safety.
  • Nieminen, Tarja (Helsingin yliopisto, 2015)
    Social capital has been widely discussed in research. An increasing amount of literature has linked social capital to various health outcomes and well-being. However, both health and social capital are complex phenomena, and there is still inconsistency in the research findings. The general aim of this study was to examine the associations between social capital, health behaviour and health among adult Finnish population. The conceptualization and operationalization of social capital varies according to discipline and level. In this study, social capital is measured at the individual level assuming that an individual s investment in group activity reflects social capital seen as a resource related to social networks and group membership. Individual benefits are accessed through social connections in varied groups and society. Thus the resources do not reside within the individual but rather in the structure of person s social networks. Social capital was measured on three dimensions in this study: 1) social support, 2) social networks and participation and 3) trust and reciprocity. The association between these dimensions and health were examined. Health was investigated as self-rated health, psychological well-being and mortality. This study utilised the data of the Health 2000 Survey conducted in 2000−2001. Of people aged 30 and over, 89% participated in the home interview and 80% in the general health examination. The study material presents the whole population unusually well. The National Institute for Health and Welfare (THL; formerly the National Public Health Institute, KTL) had the overall responsibility for the project. In addition, the project organization involved a wide range of research and funding agencies. This survey contains a rich armoury of questions about health and illnesses, health behaviour, capacity for work, functional capacity and use of health services. Furthermore, it includes a broad selection of questions used in measuring social capital. The results found an accumulation of social capital and general welfare for the same groups: the highest levels of social capital were found among the young, well-educated and married people. However, all socio-demographic subgroups seem to benefit from social capital. Regardless of all socio-demographic characteristics, high levels of social capital were associated with good health, associations which varied among different health-related behaviours, but social participation had a strong statistical association with all components of health and all health behaviours. Regardless of chronic diseases people with high levels of social capital felt healthier than those with low levels. The positive association between social capital and survival was statistically significant among men and suggestive among women. These findings indicate that social capital contributes to health. Health inequalities between population sub-groups are still substantial. Health could be promoted and health inequalities reduced by developing tools for increasing social participation especially in those groups lacking social capital−and who often also suffer from several health problems.
  • Lehto, Hanna (Helsingin yliopisto, 2015)
    Objective: Aneurysms of the vertebral artery (VA) and its branch posterior inferior cerebellar artery (PICA) are rare, comprising only about 1 to 3% of all intracranial aneurysms. The series published thus far on these lesions are small. We aim to describe the special anatomical and morphological features of these aneurysms compared to aneurysms in other locations, and to describe the variety of symptoms they cause. We describe their treatment and analyze the outcome. Additionally, we describe their anatomy imaged with computed tomography angiography. Patients and methods: We reviewed retrospectively 9 709 consecutive patients with intracranial aneurysms treated in the Department of Neurosurgery at Helsinki University Central Hospital, Finland, between 1934 and 2011. The study population included 268 patients with 284 VA or PICA aneurysms or both. Follow-up data came from the Population Registry Centre (dates of death), Statistics Finland (causes of death), from written questionnaires to patients still alive, medical records of the Department of Neurosurgery, and for those deceased, medical records from all public health services. Results: Among all the aneurysm patients, 5.1% had an aneurysm in the VA or PICA. Most aneurysms, 51%, were located at the VA PICA junction. The proportion of fusiform aneurysms was 28%. Compared to patients with ruptured aneurysms at other locations, patients with a ruptured VA or PICA aneurysm were older and had a higher Fisher grade. Ruptured distal PICA aneurysms also re-bled more regularly. Compared to other ruptured aneurysms, ruptured VA and PICA aneurysms were smaller and more often fusiform. At least one VA or PICA aneurysm was treated in 209 (78%) patients. The most common technique for aneurysm occlusion was clipping, used in 107 aneurysms. Total occlusion of the aneurysm was achieved among saccular aneurysms in 90%, and among fusiform aneurysms in 61%. Within one year of aneurysm diagnosis, 26% of the patients were dead. Among those who survived a minimum one year and in whom the VA or PICA aneurysm received active treatment; those returning to an independent or their previous stage of life amounted to 92%. Conclusion: In treatment of VA and PICA aneurysms, their special anatomical and morphological features are challenge. Despite this, and often severe hemorrhage, most patients surviving the initial stage make a good recovery.
  • Cousminer, Diana (Helsingin yliopisto, 2015)
    Puberty is a highly variable developmental stage marked by the development of secondary sex characteristics and the attainment of reproductive maturity. Variation during childhood developmental phases correlates with altered disease risk in adulthood; variation in pubertal growth and timing, in particular, correlates with adult risk for type 2 diabetes, obesity, adverse cardiovascular heath, and hormone-dependent cancers. While normal variation in age at menarche (AAM) has recently been investigated in large-scale genome-wide association (GWA) studies, the genetic regulation of male puberty remains less understood. Moreover, extreme variation in pubertal timing is a common cause for referral to pediatric specialists, while the underlying genetic factors are largely unknown. This work aimed to identify both common and rare genetic variants influencing pubertal growth and timing in both sexes. Utilizing Finnish population-based samples with frequent height measurements across puberty, we ran GWA of growth during late puberty and uncovered an association for variants near LIN28B, the most robust menarche-associated locus. Investigation of the longitudinal effects of two partly-correlated markers, one tagging a pubertal timing effect and one tagging an effect on adult stature, revealed distinct sex-specific association patterns with height growth from birth until adulthood. Thus, the LIN28B locus tags an important developmental regulator of both growth and maturational development. We then expanded to include European-wide samples within the Early Growth Genetics (EGG) Consortium. Genetic mapping of three pubertal growth traits revealed 9 novel pubertal growth variants in addition to LIN28B, 5 of which also associated with pubertal timing, and one which associated with childhood and adult body mass index (BMI). Longitudinal investigation of these variants showed diverse patterns of association with height growth, some of which contradicted epidemiological correlations between rapid prepubertal growth, advanced puberty, and reduced final adult stature. Given the complex relationships between these traits, tracking individual unique effects across multiple periods of growth may help uncover the pathways linking childhood development with adult health outcomes. Also within the EGG Consortium, GWA meta-analysis of Tanner genital and breast staging data uncovered the first robust male puberty locus on chromosome 16 near MKL2, a locus which also associates with advanced menarche, decreased pubertal growth, and shorter adult stature. Furthermore, part of the genetic architecture underlying the onset of puberty is shared between males and females, evidenced by the high correlation between menarche-advancing alleles and earlier male genital development. However, while BMI-increasing alleles strongly correlate with advanced breast development in girls, our data shows that these variants play a more complex role in male puberty. Finally, we performed targeted sequencing of the pericentromeric region of chromosome 2 previously robustly linked with constitutional delay of growth and puberty (CDGP), an extreme delay in normal pubertal timing, in multiply affected Finnish families. Analysis of shared low-frequency variation in genes and regulatory regions of the best functional candidate genes revealed 6 protein-altering variants in a single gene, DNAH6, in 10 of the families. However, follow-up sequencing in an additional 135 Finnish CDGP cases failed to provide evidence for enrichment of DNAH6 mutations compared to a large, unique set of SISu Finnish population controls. DNAH6 is potentially an appropriate candidate gene that may be involved in the regulation of steroid hormone biosynthesis by the cytoskeleton. This study highlights the difficulties of detecting susceptibility variants under a linkage signal for complex traits. Taken together, these results advance our understanding of the genetics of pubertal timing and development in both sexes. However, more work is needed to understand how each genetic locus functions in the biology of puberty and childhood growth, and further study of the genetic loci highlighted in this work may help pinpoint the mechanisms that link the timing of this important developmental stage with adult health and risk for common diseases. Keywords: puberty, development, growth, genome-wide association studies (GWAS), targeted sequencing, constitutional delay of growth and puberty (CDGP)
  • Honkasalo, Mikko (Helsingin yliopisto, 2015)
    Diabetes is a common chronic disease with growing prevalence in Finland like worldwide. It shortens the life expectancy and the quality of life. Despite the development in medication and devices there has been only modest improvement in the outcome, especially among type 1 diabetic patients. The aim of this study was to compare the outcome of overall diabetes care in municipalities with different primary health care models of organising the follow-up of type 1 diabetes and type 2 diabetes with special treatment problems. The study also aimed at estimating the feasibility of various indicators of the standard of diabetes care. The outcome, use and costs of health services connected with diabetes and its complications were compared in two suburban communities, Kouvola and Nurmijärvi. In Kouvola the follow-up of all patients had been based on family doctors already over 15 years whereas in Nurmijärvi the follow-up of T1D patients and the complicated T2D patients had been centralized to 1-2 doctors for the same time. The diabetic populations of these municipalities resembled each other. In the centralized system T1D became cheaper for the municipality. Differences in the quality parameters were minor. Both these results were obviously due to more consultations of the specialist level in Kouvola. However, T1D patients were significantly more satisfied with the centralized follow-up model. In conclusion, the centralized follow-up of the most demanding diabetes in PHC is cost-effective and results in high patient satisfaction. The centralized model is better in the follow-up of T1D but in T2D there were no differences between these two models.
  • Koskela-Niska, Virpi (Helsingin yliopisto, 2015)
    Both ovarian and tubal cancers are rare. Therefore only a few studies explored such postmenopausal hormone therapies (HT) as used in Finland, as a risk factor for these cancers. I compared the incidence of ovarian and tubal cancer in HT users with the incidences of these cancers in the background population in two nationwide cohort studies. To exclude a number of potential confounders I conducted two case-control studies to further elucidate HT use as a risk factor for these cancers. The cohort studies included all women aged 50 or older who had used HT-regimens for 6 months or longer in 1994-2008. These women were identified from the Finnish Medical Reimbursement Register. The ovarian cancer cohort study included 224,015 women using estradiol-progestin therapy (EPT), whereas in the primary fallopian tube carcinoma (PFTC) cohort study included altogether 365,601 women using EPT (n=247,781) or estradiol-only therapy (ET)(n=117,820). These women were followed from the first HT purchase to the diagnosis of ovarian or tubal cancer, death, emigration or to the end of the study period through the national Finnish Cancer Registry. Relative risks of these cancers in HT users were estimated by comparing the incidence of ovarian or tubal cancer in HT users to the age-matched comparable background population and calculating standardized incidence ratios (SIR) and their 95% confidence intervals (CI). In the case-control studies, all 50-year-old or older women with incident ovarian cancer (n=3,958) or PFTC (n= 360) during 1995-2007 were identified from the Cancer Registry. The Population Register provided control women, 3 per each case of ovarian cancer and 10 per each case of PFTC, and parity data. The controls had to be alive and without ovarian/primary fallopian tube cancer and they were matched for age (+/- one month) and place or residence. The controls with an oophorectomy (n=506) or a salpingectomy (n=158) were excluded leaving 11,325 and 3,442 controls, respectively. Odds ratios (OR) with 95% CIs for various HT regimens were estimated by using conditional logistic regression adjusted for parity, ages at deliveries, hysterectomy and sterilization. The use of any type of EPT for less than five years did not modify the overall risk of ovarian cancer. Instead, the use of sequential EPT for five years or longer was associated with an elevation in the overall risk of ovarian cancer (SIR 1.21, 95% CI 1.06-1.37). The risk rises were most marked for serous (1.56; 1.33-1.80) and mixed cancer (1.54; 1.22-1.91) whereas the risk for mucinous cancer was decreased (0.47; 0.22-0.86). The risk increase connected to EPT use did not depend on the progestin type, mode or route of administration of EPT. The use of ET for five years or longer was linked with an increase in the risk for serous ovarian cancer (OR 1.45, 95% CI 1.20-1.75) while the risk of mucinous cancer was decreased (0.35; 0.19-0.67). As a whole the use of ET was accompanied with borderline rise in overall ovarian cancer risk (1.15; 0.99-1.32). In the users of sequential EPT, the risk rise was similar for endometroid (1.88; 1.24-2.86) and serous (1.32; 1.01-1.71) cancers. The use of continuous EPT, tibolone or E+LNG-IUS did not modify the risk of ovarian cancer. The use of ET was not a risk factor for PFTC. However, the use of sequential EPT for five years or longer was accompanied with a risk elevation (SIR 2.15, 95% CI 1.66-2.72), which was further increased if the use of sequential EPT continued for 10 years or longer (3.36; 2.02-5.24). In contrast, the use of continuous EPT did not modify the risk for PFTC. The uses of E+LNG-IUS for five years or longer was associated with increased risks of PFTC (ORs 2.84, 95% CI 1.10-7.38 and 3.37; 2.23-5.08, respectively), but this finding was based on low number of cases and controls. Postmenopausal hormone therapy use modifies the risks of both ovarian and fallopian tube cancers. The risk of serous and endometroid ovarian cancer rises but the risk of mucinous cancer decreases. The use of sequential EPT regimen is characterized with the highest risk elevations. In absolute terms, in 10,000 women using sequential EPT for five years or longer, there would be four extra cases of ovarian cancer and two extra cases of PFTC after follow-up of five years. These data should be incorporated into modern guidelines for the optimal use of HT.
  • Lithovius, Raija (Helsingin yliopisto, 2015)
    As health care resources are limited, it is crucial to identify potential subgroups of patients with different resource needs and the major cost drivers in order to improve understanding of the cost structure of diabetes care. About one-third of the patients with type 1 diabetes develop diabetic nephropathy (DN), which is the most burdensome diabetic complication and the leading cause of end-stage renal disease (ESRD), requiring dialysis or kidney transplantation. Pharmaceutical interventions play an important role not only in controlling glycaemia, but also in preventing and treating related co-morbidities. The aim was to evaluate the use and costs of prescription medication in a large nationwide cohort of patients with type 1 diabetes, stratified by severity stages of diabetic kidney disease, and to study to what extent target values of the American Diabetes Association (ADA) guidelines were met in normal clinical settings and how achievement of the most relevant targets predicts the prognosis of the patients. All sub-studies are part of the ongoing, multicentre Finnish Diabetic Nephropathy Study (FinnDiane). Studies I (N=3,717), II (N=1,905), III (N=330) and V (N=3,151) have longitudinal and Study IV (N=3,678) a cross-sectional design. To obtain information on purchases of prescription medications, co-morbidities and mortality, the FinnDiane data were linked with several national registers. In patients with ESRD, the 11-year cumulative costs increased fourfold or even 15-fold, when diabetes medications were excluded compared to those without severe complications. The cost of diabetes medications remained quite stable irrespective of the presence of complications and duration of diabetes. The costs were significantly higher in patients with macroalbuminuria than in those with earlier stages of DN, and the gap continued to increase until the end of follow-up. A large gap exists between evidence-based guidelines and clinical practice since only a minority of all patients reached the targets for HbA1C, blood pressure (BP) and LDL cholesterol proposed by the ADA. Failure to reach the targets was associated with increased risk of cardiovascular disease and mortality. One of the novel findings was the high prevalence of treatment-resistant hypertension (RH) which increased in parallel with the worsening of DN in patients with type 1 diabetes. Progression to a more severe stage of DN has a substantial impact on prescription medication costs, highlighting the importance of early intervention to prevent or delay the onset of diabetic kidney disease. The treatment targets of HbA1C, BP and LDL cholesterol have not been achieved. Successful implementation of these targets would be a key for the optimal prevention of CVD and mortality.
  • Laurila, Pirkka-Pekka (Helsingin yliopisto, 2015)
    Cardiovascular disease is the leading cause of death worldwide, and is characterized by disturbances in lipid metabolism. High blood LDL cholesterol is the most important risk factors for atherosclerosis, a pathological state in which the circulating lipid molecules accumulate in the blood vessel wall. Blood levels of HDL cholesterol are inversely associated with cardiovascular risk, and low HDL-cholesterol is a significant risk factor for cardiovascular disease. In this thesis, new genes predisposing to low HDL-cholesterol level were searched using genome-wide association analysis in Finnish individuals with extremely low or high HDL-cholesterol. Several new genes predisposing to low HDL-cholesterol were discovered, of which many were known to be associated with immune system and inflammatory reaction of the body, but their role in cholesterol metabolism has not previously been characterized. Some of the subjects appeared to be genetically more prone to inflammation than others, especially in the blood and adipose tissue. The more inflammation inducing genetic variants one had, the stronger was the inflammatory state of the body, especially in blood and adipose tissue, and the lower the HDL-cholesterol level. The inflammation may block the transport of cholesterol from vessel walls to circulation leading to lower HDL-cholesterol levels in the circulation. This thesis also examined the composition of HDL particles by determining the concentration of all know lipid species in HDL particles. The quality of HDL particles appeared to vary considerably. In individuals, whose HDL-cholesterol levels were low, the quality of HDL particles was also impaired; they contained smaller amounts of plasmalogens which are fat molecules known to be antioxidant and thus protective to arteries. In individuals with high HDL-cholesterol levels in the circulation, the lipid composition of the HDL particle was more beneficial regarding heart disease risk. This thesis further demonstrates that not only the quantity but also the quality of HDL particles is genetically regulated. The genetic variants regulating the level of HDL-cholesterol also govern its quality, highlighted by inflammation-increasing genetic variants also impairing HDL quality. This thesis work provides novel insights about the molecular background of HDL cholesterol and validates the strong link between inflammation and low HDL-cholesterol levels. The studies show how genetically induced inflammation reduces blood HDL-cholesterol levels and impairs HDL particle quality, potentially predisposing to cardiovascular disease.
  • Soininen, Antti (Helsingin yliopisto, 2015)
    Hydrogen free diamond-like carbon (DLC) coatings have been the subject of investigation all around the world for the last 30 years. One of the major problems in producing of thick high-quality DLC coatings has been the inadequate adhesion of the deposited film to the substrate. This obstacle is finally overcome by depositing an intermediate adhesion layer produced with high energy (>2 keV) carbon plasma before application of a high-quality coating produced with a low energy unit. To the best of our knowledge, this can be achieved (with reasonable yield for industrial purposes) only with the filtered pulsed arc discharge (FPAD) method developed by our group. In this thesis a new combination of in situ surface oxide reduction method with FPAD is presented. Novel anti-soiling DLC polymer hybrid coatings (DLC-p-h) can be deposited using a slightly modified FPAD system. Both the coatings and their deposition method are recent innovations invented in our research group. This method makes possible to combine diamond and polymer properties in the resulting coatings. Two novel hybrid coatings have been developed: DLC polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC polydimethylsiloxane hybrid (DLC-PDMS-h). These hybrid coatings are highly hydrophobic and oleophobic (dirt repelling). Therefore these novel coatings could probably be used as antifouling and wear resistant coatings to which pathogenic bacteria would adhere less than to conventional biomaterials in biomedical applications. These DLC-p-h coatings are not produced anywhere else in the world. In this thesis bacterial adhesion to DLC was studied under dynamic conditions. Our experiments demonstrated that the bacterial adhesion to DLC was similar to the adhesion to AISI 316L surgical steel commonly used in medical applications. This suggested that DLC coating can be used on implants made from AISI 316L or other materials without increasing the risk of implant-related infections. Adhesion of bacteria and human cells (hMSC, hOB, Saos-2) to our novel DLC-p-h coatings was also studied. Bacterial adhesion tests showed a potential application of DLC-PTFE-h coating as a less biofouling surface than DLC, titanium and oxidized silicon surfaces. Cell adhesion studies showed less adhesion on DLC-PDMS-h surfaces than on DLC or titanium surfaces and some of the cells even underwent programmed cell death caused by lack or loss of adhesion. Osteogenic differentiation study on DLC-PDMS-h surface showed impaired or delayed osteogenesis. Cytocompatibility and cytotoxicity tests proved that DLC-PTFE-h and DLC-PDMS-h coatings are biocompatible. In summary, these studies suggest that DLC-PTFE-h coatings could be used in medical applications where bone integration would be preferred while DLC-PDMS-h coating in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth.
  • Merkkiniemi, Katja (Helsingin yliopisto, 2015)
    The presence of certain cancer-related genetic and epigenetic alterations in the tumor affect patients´ response to specific cancer therapies. The accurate screening of these predictive biomarkers in molecular diagnostics is important since it enables the tailoring of an optimal treatment based on molecular characteristics of the tumor. Depending on the type of gene alteration, a wide variety of methods could be applied in biomarker testing. Among the novel methods is next-generation sequencing (NGS) technology, enabling simultaneous detection of multiple alterations. The aim of this thesis was to analyze predictive or potentially predictive genetic and epigenetic alterations of diffuse gliomas and non-small cell lung cancer (NSCLC), and to evaluate the feasibility of pyrosequencing and targeted NGS in the detection of these alterations in formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens. In Study I, we assessed the genetic and epigenetic profile of diffuse gliomas by applying methylation-specific pyrosequencing to detect MGMT promoter hypermethylation, array comparative genomic hybridization to detect chromosomal copy number alterations, and immunohistochemistry (IHC) to detect IDH1 mutation status. MGMT hypermethylation, IDH1 mutations, and losses of chromosome arms 1p and 19q were typical changes in oligodendroglial tumors (grades II-III), whereas losses of 9p and 10q were frequently seen in glioblastomas (grade IV). Furthermore, we detected significant associations of 1) MGMT hypermethylation with IDH1 mutations and loss of 19q, 2) unmethylated MGMT with losses of 9p and 10q and gain of 7p, 3) IDH1 mutations with MGMT hypermethylation, 1p loss, and combined loss of 1p/19q, and 4) non-mutated IDH1 with losses of 10q. Pyrosequencing proved to be a feasible method for determination of MGMT methylation status in FFPE sample material. In Studies II and III, we compared targeted NGS with fluorescence in situ hybridization, IHC, and real-time reverse-transcription PCR in the detection of ALK fusion (Study II), and with real-time PCR in the detection of EGFR, KRAS, and BRAF mutations (Study III). All analyses were successfully performed on all FFPE samples. A good concordance was observed between the results obtained by different methods, and targeted NGS also proved to be advantageous in the identification of novel and rare variants with a potential predictive value. In Study IV, we determined the frequency of ALK fusion in 469 Finnish NSCLC patients, and the association of ALK fusion with clinicopathological characteristics and with the presence of mutations in 22 other driver genes. We detected ALK fusion at a frequency of 2.3%, suggesting that it is a relatively rare alteration in Finnish NSCLC patients. The presence of ALK fusion was significantly linked to younger age and never-/ex-light smoking history. Although most of the ALK-positive tumors had adenocarcinoma histology, also ALK-positive large cell carcinomas were detected. Characterization of ALK-positive cases by targeted NGS showed a coexistence of ALK fusion with mutations in MET, TP53, CTNNB1, and PIK3CA, but the value of these co-occurrences requires further examination. In conclusion, our studies indicate that certain genetic and epigenetic alterations occur together, and the simultaneous screening of multiple alterations may thus allow one to obtain a more comprehensive picture of the molecular background of the tumor, which could facilitate prediction of tumor behavior, prognosis, and treatment response. Our results show the feasibility of pyrosequencing and targeted NGS in FFPE tumor tissue material and also the advantages of targeted NGS over other commonly used methods in the detection of gene rearrangements and mutations, particularly the ability to simultaneously identify multiple alterations.
  • 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.
  • Alestalo, Paula Kaarina (Helsingin yliopisto, 2015)
    ABSTRACT The general objective of this thesis was to study management and leadership in the Public Dental Service (PDS) in Finland during the major Dental Care Reform in 2003–2011. The specific aims were to study 1) how dentists became leaders in the PDS and 2) what characterised their leadership, 3) distribution of leadership positions between women and men, 4) chief dentists’ position in the municipal hierarchy, as seen from their own superiors’ and subordinates’ points of view and 5) chief dentists’ attitudes to the Dental Care Reform and the changes it caused in the work environment. Four questionnaire surveys based on four data sets were carried out in 2003. The target groups were the chief dentists of the municipal PDS units (health centres) (n=265), the chief physicians (n=233), the line managers (superiors) of the chief dentists, PDS dentists who were subordinates to the chief dentists (n=365), and the chairpersons of Municipal Boards of Social Affairs and Health (MBSH) (n=233). In 2011, the target group was the chief dentists alone (n=161). Factor analysis, linear regression analysis, parametric and non-parametric tests were used in the analysis of the materials. The results showed that only fewer than a fifth (17%) of chief dentists were full-time leaders in 2011 and they worked in the largest health centres. The rest also provided patient care, to varying degrees. In 2003 and 2011, nearly two thirds (62%) of the chief dentists identified themselves as leaders instead of seeing themselves only as dentists among other dentists in the PDS, though fewer than a third (31%) of them had applied for their posts. More precisely, just 21% of female and 43% of male chief dentists (p<0.001) had applied for the chief dentist posts they held. Chief dentists felt they were better people-oriented leaders than goal-oriented managers (p<0.001). Nevertheless, 49% p<0.001) of their job satisfaction was explained by the fact that they were motivated to lead, their position as superiors to their subordinates was good, they had enough (3-4 on a scale 1-4) decision making power, they were good goal-oriented managers (good in sum variables ≥3 on a scale 1-4 from very bad to very good), and had received enough (3 on a scale 1-3) leadership education. In addition, if they had a good position as subordinates to their superiors, chief physicians and MBSH chairpersons (p<0.001), and when they had applied for their posts, they felt contented (p<0.001). In 2003, their subordinates, the PDS dentists, appreciated their people-oriented female superiors highest. Chief dentists themselves as subordinates felt they received little feedback and support from their superiors (chief physicians), and their own subordinates, the PDS dentists, felt the same (p<0.001). Chief dentists’ superiors (chief physicians and MBSH chairpersons) evaluated the chief dentists as good managers and leaders, whereas the chief dentists’ subordinates evaluated their superiors in less positive terms. In 2003, more than half (59%) of the chief dentists were very positive (4 on a scale 1-4) towards the recently introduced reforms of the Health Insurance Act (HIA) promoting adults’ treatment in the private sector by increasing reimbursement for dental care. Fewer than half (43%) were very positive towards the reform of the Primary Health Care Act (PHCA) improving adults’ access to care in the public sector (p<0.001). In 2011, only 20% totally agreed (4 on a scale 1-4) that the implementation of the Dental Care Reform had succeeded. The respondents’ general opinion was that the timetable for the Reform was too tight and not enough resources were allocated. In general, the chief dentists considered their position as isolated and rather weak in the municipal hierarchy. Effective and rewarding leadership in the PDS needs more attention in the future.
  • Hämäläinen, Harri (Helsingin yliopisto, 2015)
    ABSTRACT The purpose of this study was to explore bone mineral content (BMC) and bone mineral density (BMD) development and related factors in patients with rheumatoid arthritis (RA) between 15 and 20 years from disease onset (I), in premenopausal women with RA (II), and in young adults with juvenile idiopathic arthritis (JIA) (III), and to ascertain osteoporosis (OP) drug use in patients with early RA (IV). BMD of the lumbar spine and the femoral neck were measured by dual-energy X-ray absorptiometry in patients with RA in two longitudinal studies and in young adults with JIA in a cross-sectional study. In assessing BMD at 15 years from disease onset in an inception cohort of RF-positive RA patients, it was found that eighteen out of 59 (31%) patients had OP. However, the decreases in central bone mineral in this patient group were of low degree and after the subsequent five years no essential change in central BMD was found. None of the explanatory variables: sex, age, ESR, HAQ, Larsen score, and cumulative prednisolone dose between 15-20 years from disease onset, proved to be a significant predictor of BMD change at the lumbar spine and femoral neck from 15- to 20-year check-ups (I). In assessment of BMC and BMD development in premenopausal, regularly cycling RA patients with and without GCs and in controls, it was found that RA patients with GCs had lower BMD values than those without GCs at commencement of follow-up. Furthermore, the mean BMD decreased significantly in both lumbar spine (P 0.002) and femoral neck (P smaller than 0.001) only in the RA patients with GCs during the 2-year follow-up. However, there was no statistically significant difference between the three groups in change in BMC or projectional area in the lumbar spine or femoral neck. Comparing results on bone mineral density change between the three groups it is relevant to report changes both in bone mineral content and in projectional area to clarify the basics of the bone mineral density change. BMD is expressed as BMC per projectional area. Only weight was found to be a significant predictor of BMD change (II). Assessment of BMC and BMD development in young adults with JIA and controls assumed to have reached their peak bone mass, showed that three (2.6%) out of 116 patients with JIA had OP. The male and female JIA patients had lower weight- and height-adjusted BMD values in the femoral neck than the controls. Dividing the patients into two groups, those with active and those with inactive JIA, both groups had lower BMC values in the femoral neck than the controls (P smaller than 0.001). Comparing BMC values in the femoral neck in both men and women with JIA a difference was found only among men (P 0.006). Among men, use of GCs and weight were significantly associated with BMC in the femoral neck. Among women, use of GCs, weight and also height were associated statistically significantly with BMC in the femoral neck, and among women GC use and height were also associated with BMC in the lumbar spine (III). A total of 14 878 incident cases of RA were studied when evaluating the implementation of pharmaceutical OP drug use. Out of this total, 1351 (9%) patients with RA had purchased OP drugs, mainly bisphosphonates, during the first year after commencement of antirheumatic treatment. Of GC users, 14% of women and 6% of men were prescribed OP drugs. In addition, 8% of females and 3% of males not taking GCs received OP medication. Women were more prone to use OP medication. Of the RA patients who took GCs, 38% of women and 24% of men received concomitant calcium and vitamin D preparations by prescription during the same year, whereas the corresponding percentages for patients without GCs were 21% and 13%. (IV). Study results suggest that bone loss takes place in earlier disease course in RA and JIA and bone loss is in the long-term disease course in RA of low degree. Further studies are needed to elucidate bone loss and OP development in early rheumatoid arthritis and to better focus the timing and means of OP prevention.
  • Ho Huu, Tho (Helsingin yliopisto, 2015)
    The aim of this study was to utilize molecular biology knowledge to develop novel amplification-based technologies, which enable enrichment of challenging nucleic acid targets to a level sufficient for detection, including those with extremely long, GC-rich and/or repetitive sequences and low-abundance single nucleotide RNA variants in the excess of alternative variants. We have introduced multiple heat pulses in the extension step of a PCR cycling protocol to generate a novel amplification technology (HPE-PCR), which temporarily destabilize secondary structures, in order to enhance DNA polymerase extension over GC-rich sequences. Different GC rich target sequences in the human genome, extremely long Fragile X GGC repeats and Myotonic Dystrophy type 1 CTG repeats have been used as models to develop and validate this novel technology. In order to detect a low-abundance RNA variant, we devised a novel technology using a competitive Extendable Blocking Probe (ExBP) in the reverse transcription reaction for allele-specific priming with superior selectivity. In ExBP-reverse transcription, the mismatch priming site on the alternative variant is blocked by a perfectly matched ExBP. This initiates formation of a stable cDNA-RNA hybrid that completely blocks false cross-priming by the target specific primer. In experimental models, the ExBP-based reverse transcription assay allowed for detection of multiple mutation types on different genes in at least 1000-fold excess of wildtype RNA and detection was linear over a 4 log dynamic range. This technique not only reveals the presence or absence of rare mutations with an exceptionally high selectivity, but also provides a convenient tool for accurate determination of RNA variants in different settings, such as quantification of allele-specific expression. In conclusion, we have established HPE-PCR and ExBP-RT techniques to enable enrichment of different nucleic acid templates that are currently challenging to detect by PCR, such as long, GC rich and/or repetitive sequences as well as low-abundance point mutations in a vast excess of wildtype alleles. These techniques expand the capacities of current PCR technology; provide versatile and convenient research tools and open many new possibilities for its applications in molecular diagnostics.
  • Liikanen, Ilkka (Helsingin yliopisto, 2015)
    Cancer remains a major cause of death and novel treatment modalities are needed. Oncolytic immunotherapy is a safe and promising approach, where cancer-selective viruses kill only cancer cells and mount an immune response against the tumor. We aimed to improve oncolytic adenoviral immunotherapy by combining it with chemotherapy and radiotherapy, and by identifying resistance mechanisms and biomarkers. We first showed that combining radiotherapy with adenoviral vector proteins E4orf3 and E4orf6, but not E1B55K, enhanced DNA damage accumulation and cancer cell killing, and inhibited prostate tumor growth in mice. This intrinsic ability of adenoviruses to radiosensitize cells could be harnessed against cancer cells by selective targeting, thus increasing efficacy while reducing the harmful side-effects of radiotherapy. In study two, we established two ovarian cancer mouse models, where tumors relapse despite the presence of functional oncolytic adenovirus, with tumor stroma maintaining the virus resistance. We identified upregulated interferon signaling in the resistant tumors by microarray, while pathway analyses suggested potential therapeutic targets, and myxovirus resistance protein A (MxA) was found a protein level indicator correlating with resistance to virus. Our results provide a putative biomarker and targets, which can help in detecting and overcoming resistance against oncolytic adenovirus. Antitumor T-cell activation appears to require autophagy and immunogenic cell death (ICD). In a translational study, we demonstrated preclinically that oncolytic adenovirus together with low-dose temozolomide and cyclophosphamide increased ICD and autophagy, resulting in tumor growth inhibition. Combination therapy was found safe in 41 treatments given to patients with refractory solid tumors in the context of an advanced therapy access program (ATAP). Increase of an ICD marker protein high-mobility group box 1 (HMGB1) and antitumor T-cell activity suggested activation of immune responses. Disease stabilization or better was observed in 67% of evaluable treatments, and as an estimated effect on survival, combination-treated patients trended for increased overall survival over non-randomized control patients. Biomarkers are urgently needed for identification of cancer patients likely to benefit from immunotherapy. Because HMGB1 protein is emerging a key player in immunomodulation, we addressed the biomarker value of HMGB1 serum level in an ATAP cohort of 202 cancer patients treated with oncolytic adenoviruses: Patients with low-baseline HMGB1 showed significantly improved overall survival and disease control rate in multivariate analyses as compared to high-baseline patients. Both patient groups showed good safety. HMGB1-low patients seemed to benefit from immunogenic virus constructs and antitumor T-cell activity. Thus, we have identified HMGB1 as a novel prognostic and predictive biomarker for oncolytic immunotherapy, which may distinguish between immunologically inert and responsive cancer patients. This thesis provides rationale for combining oncolytic adenoviruses with radiotherapy, low-dose temozolomide and cyclophosphamide. We report safety, possible signs of efficacy, and immunological effects in altogether 238 patient treatments, and introduce promising biomarkers for oncolytic immunotherapy. Our results can help in designing clinical trials and developing oncolytic adenovirus treatments.