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  • 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.
  • Kuivalainen, Anna-Maria (Helsingin yliopisto, 2015)
    Background: Bone marrow aspiration and/or biopsy (BMAB) is a procedure used to diagnose and follow up various haematological diseases. It is usually performed at either the sternum or the iliac crest. The procedure often causes pain despite local infiltration anaesthesia. The objective of this study was to evaluate different means of pain relief during BMAB in adult patients. Special attention was paid to pre-procedural anxiety and its effect on pain. The commonly used local anaesthetic lidocaine was compared with articaine, an anaesthetic known for its ability to penetrate bone tissue. The effect of warming and buffering the lidocaine solution, measures expected to improve the anaesthetic action, was examined. Also investigated were sublingual fentanyl and inhaled 50% nitrous oxide (N2O) in oxygen (O2) as means of analgesia and sedation during BMAB. Patients: The patient population comprised 646 adult outpatients from the Department of Haematology, Helsinki University Central Hospital, Finland. Patients were randomized to treatment groups in trials comparing one intervention with another or with placebo. The studies were all patient-blinded. One study was observational and investigated the association between pain and pre-procedural anxiety. Patient recruitment was performed between 2007 and 2014. Main results: Pre-procedural anxiety intensified pain during BMAB in all trials. Median NRS (Numeral Rating Scale, 0 = no pain, 10 = worst pain imaginable) during infiltration was 3.0 (range 0 10, interquartile range (IQR) 3.0), puncture 2.0 (range 0 10, IQR 3.0), aspiration 4.0 (range 0 10, IQR 4.0), biopsy 4.0 (range 0 10, IQR 4.0) and immediately after BMAB 0 (range 0 9.0, IQR 1.0). Scores of 8 10 comprised 8.1%, 4.7%, 13.9%, and 12.4% of the scores for infiltration, puncture, aspiration and biopsy, respectively. Possible supplemental analgesia or sedation given on patient request in addition to local anaesthesia and study intervention did not lower pain scores during BMAB. Articaine was not found to be superior to lidocaine as a local anaesthetic. Warming and buffering the lidocaine solution diminished pain during infiltration, but did not lower the pain scores during other phases of BMAB. Sublingual fentanyl (200 µg or 100 µg) did not provide significant pain relief relative to placebo when administered 6 64 minutes before BMAB. Dizziness was a frequent side-effect. Inhalation of 50% N2O in O2 was no more effective than inhalation of 50% O2. No significant differences in adverse effects emerged between patients receiving N2O/O2 and those receiving 50% O2. Interestingly, 86% of N2O patients and 83% of placebo patients would choose the same analgesia method during their next BMAB. Conclusions: Many patients undergoing BMAB suffer intense pain during the procedure. Pre-procedural anxiety was strongly associated with pain during the various phases of BMAB. The pain from local anaesthetic infiltration with articaine and lidocaine was similar. Buffering and warming the local anaesthetic solution clearly reduced the infiltration pain. However, neither these measures nor the use of sublingual fentanyl or inhalation of N2O had an impact on the pain caused by aspiration and biopsy.
  • Snäll, Johanna (Helsingin yliopisto, 2015)
    Background and purpose Short-term glucocorticoids (GCs) are frequently used in association with oral and maxillofacial surgery to prevent postoperative pain, edema, and nausea. However, the influence on tissue repair and the anti-inflammatory and immunosuppressive features of GCs may have an adverse impact on healing of the surgical site. The main aim of this study was to determine the occurrence of disturbance in surgical wound healing (DSWH) and pulp necrosis (PN) after surgical treatment of facial fractures and the influence of perioperative administration of GCs on these complications. Patients This study comprised four populations of patients (Studies I-IV) treated for facial fractures. For Study I, the medical records of 280 consecutive patients who had undergone open reduction of different types of facial fractures or reconstruction of orbital wall fracture were assembled retrospectively. Prospective Studies II-IV consisted of patients with mandibular fractures (n=41) (Study II) and patients with a simple zygomatic complex (ZC) fractures (n=64) (Study III). The fourth population (n=24) (Study IV) was extracted from the population of patients with mandibular fractures recruited for Study II. Methods In the retrospective study (Study I), the outcome variable was DSWH, which was established when any kind of aberrant wound healing and/or sign of infection in the surgical site occurred. The primary predictor variable was the perioperative use of GC. Patients recruited for Studies II and III were randomly assigned to one of two groups. Patients in the study group received dexamethasone (DXE) (Oradexon®), whereas patients in the control group received no GC. The main outcome variables were DSWH (Studies II-III) and PN of teeth in the area of mandibular fracture (Study IV). The primary predictor variable was the perioperative use of DXE. Results In patients with ZC fractures (Study III), DSWH was significantly associated with perioperative use of DXE as well as with intraoral surgical approach. In patients operated on for different types of facial fractures (Study I), DSWH was associated significantly with intraoral surgical approach. DSWH occurred more frequently in patients receiving GCs, however, without statistical significance. In patients undergoing intraoral surgery for mandibular fractures (Study II), DSWH occurred more frequently in the DXE group. Also PN occurred more frequently in the DXE group (Study IV). The delay of DSWH was notably longer in the DXE groups (Study I-III). Particularly PN (Study IV) was observed much later in the DXE group. Conclusions Perioperative DXE cannot be recommended in association with surgery of ZC fractures. Moreover, GCs should be used with caution in association with surgery of other facial fractures as well, particularly when the intraoral approach is used.
  • Tolonen, Nina (Helsingin yliopisto, 2015)
    Background: Cardiovascular disease is the most common cause of death in patients with type 1 diabetes, and the premature mortality rates are especially high in patients with diabetic nephropathy. Diabetic retinopathy is the leading cause of vision loss among the working-age population in industrialized countries. Early identification and aggressive treatment of risk factors are crucial to reduce the incidence of diabetic complications. Aims: To examine the relationships between lipid profiles and diabetic nephropathy, diabetic retinopathy, and incident coronary artery disease (CAD) events in a large nationwide cohort of patients with type 1 diabetes. Subjects and methods: These studies are part of the ongoing Finnish Diabetic Nephropathy Study (FinnDiane). Studies I (N=2927) and III (N=1465) have a cross-sectional design. At follow-up, renal status was verified by a review of all available medical files (Study II, N=2304), and data on CAD events were retrieved from the Finnish Hospital Discharge Register and the Causes of Death Register (Study IV, N=3520). Results: The recommended lipid concentrations of current treatment guidelines were poorly met, especially regarding the target for LDL cholesterol. Triglycerides and apolipoprotein (Apo) B were independent predictors of progression to micro- and macroalbuminuria, and total cholesterol was an independent predictor of progression to end-stage renal disease. HDL and HDL2 cholesterol were independently associated with proliferative diabetic retinopathy (PDR). In patients with PDR, the correlations between albumin excretion rate (AER) and lipid variables were strong. However, in patients without retinopathy no significant correlations were observed. In multivariate models, ApoB, triglycerides, non-HDL cholesterol, ApoB/ApoA-I ratio, and triglyceride/HDL cholesterol ratio were the strongest lipid predictors of an incident CAD event. Conclusions: Lipid abnormalities were associated with an increased risk of all three diabetic complications studied, i.e. diabetic nephropathy, retinopathy, and incident CAD events. Triglycerides and ApoB were independently associated with AER and estimated glomerular filtration rate (eGFR) and predicted the progression to micro- and macroalbuminuria as well as incident CAD events. Far lower concentrations of triglycerides than the currently recommended cut-off level (less than 1.7 mmol/l) increased the risk of progression of renal disease. Total and LDL cholesterol were poor predictors of an incident CAD event in patients with normal AER, in patients with HbA1c below the median (8.3%, 67mmol/l) of the cohort, and in women, in whom the ratios of atherogenic and anti-atherogenic lipoproteins and lipids performed better. Current treatment recommendations may need to be revised to reflect residual CAD risk in patients with type 1 diabetes.
  • Tainio, Juuso (Helsingin yliopisto, 2015)
    Renal transplantation (RTx) has become an established treatment modality for end-stage renal disease in children. Along with the improvements in pre- and post-transplant care, the patient and graft outcomes have improved significantly during the past two decades. This attracts more attention to avoiding secondary complications and long-term side effects of the post-RTx immunosuppressive medication. Several risk factors cast a shadow over patients normal physical and mental development, but detailed reports on long-term outcome after pediatric RTx are scarce. This thesis was designed to investigate pubertal development and subsequent male fertility and semen quality with special emphasis on the effects of immunosuppressive medication on reproductive function. The study also aimed to analyze the prevalence of metabolic syndrome and its components in pediatric RTx patients and the association of these parameters with the long-term graft function. The onset of pubertal development occurred at the mean age of 12.7 years in boys with 22% considered delayed. In girls, however, no delayed development occurred, with the age at onset of puberty and menarche averaging 10.7 years and 12.5 years, respectively. Pubertal growth continued relatively long resulting in average final height of -1.7 height standard deviation score in boys and -1.2 in girls. The reproductive hormone levels were normal in a great majority of the patients. In young adult males who had received RTx in childhood, the free testosterone levels were lower and luteinizing hormone levels were higher in comparison with age-matched healthy controls. The RTx patients had also smaller testicular volumes and total sperm counts than the controls. Only a quarter of the RTx men who provided a semen sample had normospermia. Patients with a history of cyclophosphamide therapy had the worst outcome. Metabolic data were collected at several time points during a 13-year follow-up post-RTx. Hypertriglyceridemia associated with a worse kidney graft function at 1.5 and 5 years post-RTx, and it predicted the subsequent rate of kidney function decrease after 1.5 years post-RTx. Beyond the first postoperative year, other metabolic risk factors associated modestly with the long-term kidney graft function in pediatric RTx patients. The ambulatory BP monitoring data were retrospectively analyzed 5 to 10 years post-transplantation. The BP profiles were similar between renal, heart, and liver transplant groups. Hypertension was common especially at nighttime and the nocturnal BP dipping was often blunted. The use of antihypertensive medication did not notably change the ambulatory BP profile in RTx recipients. The BP variables correlated poorly with the metabolic parameters or kidney graft function.
  • Mäkinen, Netta (Helsingin yliopisto, 2015)
    Uterine leiomyomas, or fibroids, are benign tumors arising from the smooth muscle lining of the uterus, the myometrium. Although they represent one of the most common neoplasms in women with an estimated prevalence of 20-40% during the reproductive years, the molecular mechanisms underlying their tumorigenesis have remained relatively unknown. The aim of this thesis was to elucidate the molecular genetic characteristics of uterine leiomyomas using next-generation sequencing technology. Exome sequencing of 18 uterine leiomyomas and the respective normal myometrium from 17 Finnish (Caucasian) patients led to the identification of recurrent somatic mutations in mediator complex subunit 12 (MED12) gene. This, and further Sanger sequencing of 207 additional leiomyomas revealed that a remarkable 70% (159/225) of the tumors harbor MED12 mutations. MED12 is a component of the Mediator complex, which participates in the regulation of global as well as gene-specific transcription. All the observed mutations resided in exon 2 or the intron 1-exon 2 junction, an evolutionarily conserved region of the gene, suggesting that malfunction of the region contributes to tumorigenesis. This was the first time MED12 mutations have been implicated in human tumorigenesis. To validate the finding and determine the frequency of MED12 exon 2 mutations in another ethnic group, a series of 28 uterine leiomyomas from 18 South African patients underwent Sanger sequencing for the mutations. Altogether 50% (14/28) of the tumors displayed a mutation, indicating that MED12 mutations occur frequently also in uterine leiomyomas of South African women. Overall, the result confirms the role of these mutations in the growth and development of leiomyomas regardless of ethnicity. Original identification of MED12 exon 2 mutations took place in a series of histopathologically conventional uterine leiomyomas which account for approximately 90% of the tumors. To assess the frequency of MED12 mutations in rarer clinical leiomyoma subtypes, 103 histopathological uterine leiomyoma variants, as well as 34 uterine leiomyomas from 14 patients with hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome, a disease caused by heterozygous germline mutations in fumarate hydratase (FH) gene, entered the study. Both the histopathological leiomyoma variants (17%; 18/103) and leiomyomas from HLRCC-patients (9%; 3/34) harbored MED12 mutations significantly less frequently than conventional leiomyomas (P less than 0.001), proposing that MED12 mutation positivity is a key characteristic of conventional leiomyomas. Of note, none of the MED12 mutation-positive tumors from HLRCC-patients displayed biallelic FH inactivation, a well-known attribute of tumors in HLRCC, suggesting that MED12 mutations and biallelic FH inactivation may be mutually exclusive. Exome sequencing of 27 uterine leiomyomas (12 MED12 mutation-negative and 15 MED12 mutation-positive) and their corresponding normal myometrium revealed no additional recurrent somatic mutations in either MED12 mutation-negative or -positive tumors. The result emphasizes the significance of MED12 mutations for the tumorigenesis of uterine leiomyomas. Alterations undetectable by exome sequencing, such as structural rearrangements, intronic variants, and epigenetic events, probably have an impact to the development of MED12 mutation-negative lesions. The discovery of MED12 mutations is a giant step forward in understanding the pathogenesis of uterine leiomyomas, hopefully leading to improved diagnosis, personalized medical treatments, and prognosis in the future.
  • Haario, Peppi (Helsingin yliopisto, 2015)
    Insomnia symptoms and short sleep duration constitute a notable public-health problem. They impair wellbeing and are risk factors for several diseases, both of which may affect societal functioning. The aim of this study was to examine whether insomnia symptoms are associated with subsequent unhealthy behaviours and weight gain, and whether unhealthy behaviours and weight are associated with subsequent insomnia symptoms. Additionally, the aim was to examine whether sleep duration is associated with weight gain. The data were derived from the Helsinki Health Study cohort baseline and follow-up mail surveys. The baseline data were collected in 2000-2002 among employees of the City of Helsinki aged 40-60 (n=8960, response rate 67%). The follow-up data were collected in 2007 (n=7332, response rate 83%). Insomnia symptoms were measured on frequency about having trouble falling asleep, waking up at night, having trouble staying asleep and waking up tired after one s usual amount of sleep, all during the previous four weeks. The respondents were also asked about their average sleep duration week days. Unhealthy behaviours included heavy and binge drinking, smoking, physical inactivity and unhealthy food habits. Weight was assessed using body mass index (BMI), kilograms and weight gain. Analyses of variance, logistic regression and multinomial regression were used in the statistical analyses. Poor sleep, more specifically insomnia symptoms and short sleep duration, were associated with weight gain, and BMI showed an association with changes in insomnia symptoms. These associations were evident mainly among women. Insomnia symptoms showed a bidirectional association with heavy drinking, and were also associated with subsequent physical inactivity. Additionally, binge drinking showed an association with subsequent insomnia symptoms. This study confirms associations among poor sleep and weight and mainly weak and inconsistent associations among insomnia symptoms and unhealthy behaviours.
  • Vanhanen, Jenni (Helsingin yliopisto, 2015)
    Neuronal histamine and its H3 receptor (H3R) regulate several physiological functions and are involved in the pathophysiology of various central nervous system disorders such as Parkinson s disease, Alzheimer s disease, Tourette syndrome and narcolepsy. Studies conducted in experimental animals have also suggested a role for histamine and especially H3R in the effects of drugs of abuse. In this thesis, the main aim was to study how histamine and H3R regulate alcohol-related behaviors. Furthermore, our goal was to investigate the underlying mechanisms in the observed behaviors. By using both wild type mice in different background strains and genetically modified mice, we studied whether histamine and H3R regulate the behavioral responses to alcohol. Three different H3R antagonists (ciproxifan, JNJ-10181457 and JNJ-39220675) were used and it was found that both pharmacological antagonism and genetic knockout of H3R (H3R KO) lead to diminished alcohol consumption and reward. By using histamine deficient histidine decarboxylase knockout (HDC KO) mice, we found that the lack of histamine does not alter alcohol consumption or reward but histamine is indeed required for the H3R-mediated alcohol reward inhibition. We also found that JNJ-39220675 inhibited the acute stimulation of amphetamine, but failed to inhibit the rewarding properties of amphetamine. This indicates that although H3R antagonists inhibit alcohol reward, they may not possess the same ability on psychostimulants, such as amphetamine. The findings obtained from the behavioral experiments led us to hypothesize that H3R interacts with the dopaminergic system. This was further studied on a molecular level using both radioactive in situ hybridization and semi-quantitative Western blotting. We found that compared with control mice, H3R KO mice displayed lower levels of dopamine D1 receptor messenger RNA in the striatum, which is an area important in the regulation of e.g. reward. In addition, we found that activation of dopamine D1 and D2 receptors resulted in abnormal striatal cell signaling in the absence of H3Rs. Taken together, these findings demonstrate that H3R is an important regulator of alcohol-related behaviors. The mechanism by which H3R regulates these phenomena might involve the interaction between the striatal H3R and dopamine receptors. In addition, these results provide preclinical evidence that H3R antagonists may serve as a novel approach to treat alcohol dependence.
  • Poukkanen, Meri (Helsingin yliopisto, 2015)
    Severe sepsis is the main cause of acute kidney injury (AKI) among critically ill patients. Septic AKI has been shown to associate with lower mean arterial pressure (MAP) levels. The initiation of renal replacement therapy (RRT) is mainly based on clinical judgment. The objectives of this study were to assess the incidence and 90-day mortality of patients with severe sepsis associated AKI treated in the intensive care units (ICUs), to evaluate the impact of MAP on development of AKI, to assess the differences in proportion of use of RRT in patients with septic shock, and to develop predictive model for one-year mortality among ICU-treated patients with AKI. All patients were from the prospective, observational FINNAKI study conducted in 17 Finnish ICUs over the five-month study period in 2011-2012. AKI was defined by Kidney Disease: Improving Global Outcomes (KDIGO) criteria. Of the 918 patients with severe sepsis, 488 had AKI (53%). Patients with septic AKI were older, had more comorbidities, and were more severely ill than patients without AKI. The 90-day mortality rate of the septic patients with AKI was higher (38%) compared to patients with severe sepsis without AKI (25%). The time-adjusted MAP during the first 24 hours in the ICU was lower in septic patients with development of AKI (74 mmHg) than in those septic patients without development of AKI (79 mmHg). The proportion of RRT-treated patients in patients with septic shock ranged from 3% to 35 % across Finnish ICUs. The variation in proportion of RRT among patients with septic shock between high- and low-RRT ICUs was explained by differences in case-mix and in severity of organ dysfunction. Indications for RRT or 90-day mortality did not differ between ICU groups. The one-year mortality among patients with AKI was 40%. Advanced age, number of co-morbidities, higher modified SAPS II score, mechanical ventilation and the lowest base excess value on the third day (D3), and the highest bilirubin value by D3 were predictors of one-year mortality. The severity of AKI, or the presence of severe sepsis, did not remain as predictors for one-year mortality. Conclusions: Over half of the patients with severe sepsis had AKI. Avoidance of time-adjusted MAP below 73mmHg may be beneficial for prevention of the progression of AKI in patients with severe sepsis. Despite 10-fold variation in proportion of RRT in patients with septic shock, the 90-day mortality of these patients was similar between high-RRT ICUs and low-RRT ICUs. The predictive model based on data by the third day in the ICU might be clinically useful in identifying patients with high risk for long-term mortality.
  • den Hollander, Bjørnar (Helsingin yliopisto, 2015)
    In recent years there has been a large increase in the use of a new kind of amphetamine- type stimulants known as substituted cathinones. These compounds have a short history of human use, and little is known about their potential neurotoxicity. Two of the most popular substituted cathinones, 4-methylmethcathinone (4-MMC, mephedrone) and 3,4- methylenedioxymethcathinone (MDMC, methylone} are, aside from their β-ketone group, close structural analogues of potentially neurotoxic amphetamines such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy). This has led to concern about the potential neurotoxicity of these novel compounds, and warrants a closer investigation into their possible long-term neurotoxic effects. METHODS The long-term effects of METH and MDMA as well as 4-MMC and MDMC were assessed using a range of biochemical assays, including assessment of monoamine levels and their transporters. The effects on brain activity were investigated using manganese-enhanced magnetic resonance imaging. Furthermore, behavioral experiments assessing cognition and neuropsychiatric function were performed. Finally, in vitro experiments in a neuroblastoma cell line were performed to identify mechanisms responsible for the observed differences in toxicity between the amphetamines and cathinones. RESULTS Unlike METH and MDMA, which produced strong reductions in dopamine and serotonin levels or brain activation, 4-MMC produced few notable effects on monoamine levels and had only minor effects on brain activation, although MDMC produced a reduction in 5-HT levels similar to MDMA. No clear effects on behavioral tests of memory function were observed as both increases and decreases in test performance were seen following 4- MMC and MDMC. In vitro experiments revealed that cathinones differ from amphetamines in their redox properties, and 4-MMC produced different effects than METH on the mitochondrial electron transport chain. CONCLUSIONS The substituted cathinones 4-MMC and MDMC do not appear to be more neurotoxic than METH and MDMA. If anything, they show a more favorable safety profile. Therefore, these substances do not appear to present an imminent and severe threat to public health. From a harm reduction perspective, these compounds may be good alternatives toMETH and MDMA. However, future work is needed to assess with certainty the long- term effects of amphetamine-type stimulants in humans.
  • Kalliola, Satu (Helsingin yliopisto, 2015)
    Background: The diagnosis of asthma in young children is based mostly on symptoms. The need for objective methods for diagnosing asthma in this age group is therefore obvious. Lung function in preschool children can be assessed with impulse oscillometry (IOS), which involves no voluntary breathing manoeuvers. Because most children with asthma have normal baseline lung function, the use of bronchoprovocative tests may improve diagnostics. Fractional concentration of nitric oxide (FeNO) is suggested to be a good measure for airway inflammation, and the method is also available for preschoolers. Aims: To examine new methods for evaluating airway hyperresresponsiveness and inflammation in young children. Further aims were to study the effect of parental smoking on lung function and airway inflammation in wheezy children and whether children with severe exercise induced bronchoconstriction exhibit small airways dysfunction. Methods: A total of 272 children (3 to 8 years old), 231 with obstructive syndromes and 41 healthy controls, were examined. Children with various clinical characteristics were recruited: troublesome lung symptoms, a history of bronchopulmonary dysplasia (BPD), early wheezing symptoms and multiple-trigger wheezing. Airway hyperresponsiveness was evaluated with exercise test, methacholine and mannitol challenge tests using IOS. FeNO measurements with two different analyzers were examined. Parental reports and children s urinary cotinine measurements served to monitor exposure to environmental tobacco smoke. Results: Exercise test with IOS succesfully identified children with probable asthma, and the methacholine challenge test was able to differentiate children with probable asthma, BPD and early wheezing from the controls. The mannitol challenge test, however, was unable to distinguish between the study groups. Furthermore, children with severe exercise-induced bronchoconstriction (EIB) exhibited small airways dysfunction. A portable FeNO analyzer proved to be more difficult than a stationary device to use in young children. In addition, its poorer accuracy in low FeNO levels diminishes its feasibility in this age group. However, a portable analyzer differentiated children with asthma from the controls. Children with smoking mothers had poorer lung function and higher FeNO than children with non-smoking mothers. Urinary cotinine concentrations closely reflected reported smoking in the family. A father s smoking had no effect on children s FeNO or lung function. Conclusions: The exercise test with IOS succesfully identified children with probable asthma. The methacholine challenge test aids in evaluating airway hyperresponsiveness in young children, although its cut-off value for this age group requires re-evaluation. A portable FeNO analyzer can also serve as a screening tool in young children, because it differentiates asthmatics from the controls with reasonable accuracy. Children with severe exercise induced bronchoconstriction exhibited small airways dysfunction, which suggests that peripheral airways are involved even in young asthmatic children. Maternal smoking clearly deteriorates lung function and increases bronchial inflammation in young children with wheeze. This objective finding with cotinine measurements emphasizes current knowledge; young children should not be exposed to environmental tobacco smoke.
  • Maksimovic, Milica (Helsingin yliopisto, 2015)
    Bipolar disorder, schizophrenia and schizoaffective disorder are extremely debilitating illnesses that encompass affective and/or psychotic symptoms. Not only is there common symptomatology and genetic susceptibility, but the pharmacotherapy approaches are also similar. Nonetheless, molecular mechanisms underpinning these diseases are not yet fully understood. The theory that there is a dopaminergic dysfunction cannot account for all of the symptoms. Nor can the compounds that act on dopaminergic mechanisms successfully alleviate the symptoms. There is evidence to suggest that there are imbalances in other neurotransmitter systems, particularly the main excitatory pathway - the glutamatergic system. Glutamatergic transmission is essential for development,learning and memory and many other physiological functions of the brain. Glutamatergic receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type mediate the majority of the fast synaptic neurotransmission. Here, Gria1−/− mice, lacking the GluA1 subunit of AMPA receptors, with concurrent schizophrenia-like and affective symptoms were used. The predictive validity was addressed using the standard and novel glutamate-modulating pharmacotherapeutics. The hyperactivity, the most robust feature of Gria1−/− mice and a hallmark of psychotic disorders, was attenuated by drug-treatments. Importantly, chronic treatments with lithium, valproate, topiramate, lamotrigine and perampanel were effective, evidence of their pharmacological efficacy after the acute, often sedative, treatment phase. In addition, excessive novelty-induced activation of the dorsal hippocampus of Gria1−/− mice as measured by c-Fos expression was blunted by the drug-treatments of which all are known to reduce the activity of the glutamatergic transmission. Other behaviours relevant to the schizoaffective symptomatology such as disinhibited risk-taking, less despair-like behaviour and highly exploratory phenotype as well as social deficits were partially responsive to treatment with mood-stabilisers. Moreover, Gria1−/− mice exhibited a slightly higher preference for sucrose and made more impulsive choices towards sucrose. The Gria1−/− mice may represent a suitable model for the screening of the preclinical efficacy of novel drugs on the hyperactive behaviour linked to manic episode of bipolar disorder, schizophrenia and schizoaffective disorder.
  • Tamminen, Jenni (Helsingin yliopisto, 2014)
    The aim of this thesis was to extend the understanding of molecular mechanisms of asbestos exposure related diseases asbestosis (lung fibrosis), lung cancer and mesothelioma. Epithelial-to-mesenchymal transition (EMT) is implicated in fibrosis and in cancer, both asbestos induced diseases. The first of the three studies investigated whether asbestos fibers induced EMT. In the next two studies, the focus was on mesothelioma. In these two studies, the aim was to recognize novel potential therapeutic target molecules and pathways involved in chemoresistance and invasion. We found that exposure to asbestos led to loss of epithelial characteristics when lung epithelial cells retained the type II airway epithelial cell phenotype. This loss of epithelial characteristics was found not to depend on TGF-β or Smad signaling cascades. The MAPK/ERK pathway was found to mediate epithelial plasticity in response to asbestos exposure. A screen for new gremlin-1 interacting proteins discovered fibrillin-2. This finding was validated in mesothelioma tumor samples in which gremlin-1 and fibrillin-2 co-localized in the tumors. Primary mesothelioma cells were harvested from pleural effusion samples of mesothelioma patients. These cells recapitulated primary tumor characteristics and overexpressed gremlin-1, fibrillin-2 and transcription factor slug. Silencing of gremlin-1 downregulated slug, reverted the EMT-phenotype and sensitized the cells to the cytotoxic effect of chemotherapeutic drugs. The concomitant overexpression of gremlin-1 and fibrillin-2 enables gremlin targeting to fibrillin-2 containing fibers in mesothelioma extracellular matrix, where it supports the chemoresistant EMT phenotype through transcription factor slug. Mesothelioma tumors as well as primary mesothelioma cells and mesothelioma cell lines were found to overexpress activin-A and activin-B. Canonical Smad3 response to activin stimulation was attenuated in mesothelioma cells. Attenuation of the Smad3 response associated with migratory and invasive phenotype. Mesothelioma cells switch from canonical Smad3 signaling to non-canonical MAPK/ERK pathway, and this promotes migration and invasion. Invasion, migration and ERK activation was impaired by sequestering extracellular activins by soluble activin-receptor. Likewise, inhibiting MAPK/ERK upstream kinase impaired migration and invasion The results presented in this thesis support the concept that pathological EMT is a central mechanism in the development and progression of asbestos induced fibrotic and malignant diseases. They also suggest gremlin-1 and activin-A as new potential therapeutic targets in mesothelioma.
  • Kaseva, Nina (Helsingin yliopisto, 2014)
    Advancements in neonatal care are resulting in increasing numbers of adult survivors after preterm birth at very low birth weight (VLBW, ≤ 1500 g). VLBW is associated with risk factors of non-communicable diseases, e.g. cardiovascular disease, osteoporosis and diabetes. We investigated mechanisms underlying the effects of preterm birth on later health in VLBW adults, with a focus on 1) physical activity, 2) nutrition and 3) stress response. The participants come from a follow-up cohort study, the Helsinki Study of Very Low Birth Weight Adults. Different subgroups from the original birth cohort (born in 1978-1985) have as young adults participated in the studies of this thesis. The controls, born at term, were group-matched for birth hospital, age and sex. We evaluated physical activity by self-report and objective measurement. The participants completed a physical activity questionnaire and underwent wrist-worn accelerometer measurement. To assess dietary intake, the participants completed a 3-day food record. For evaluation of stress response, the participants underwent the Trier Social Stress Test (TSST). In conjunction with TSST, we measured heart rate, salivary cortisol, plasma ACTH, cortisol, glucose, insulin, adrenalin and noradrenalin. 1) Based on self-report, healthy VLBW adults undertake approximately 50% less conditioning leisure-time physical activity, with lower yearly frequency, total time, total volume and energy expenditure than controls. We were unable to confirm our finding of lower physical activity with wrist-worn accelerometer measurement. 2) VLBW adults had lower consumption of vegetables, fruits, berries and milk products. This was combined with lower intake of calcium and vitamin D. 3) VLBW adults showed a lower hypothalamic-pituitary-adrenal axis (HPAA) response to stress than controls. This was accompanied by a lower insulin response. No evidence of a higher sympathetic-adrenal-medullary (SAM) system stress response was found. Furthermore, we observed a lower noradrenalin response to stress in VLBW women. This study showed that VLBW adults undertake less conditioning leisure-time physical activities and have unhealthier diets, both factors that negatively affect future health in this high-risk population. They may in part underlie the increased risk for chronic non-communicable diseases in VLBW individuals. Further, a lower HPAA response to stress was found in VLBW adults than in controls. For SAM stress response, the results were similar in VLBW and control groups, with lower noradrenalin response to stress in VLBW women only. These findings reinforce the supposition that stress response is programmed early in life. In sum, this study increased understanding of possible mechanisms linking preterm birth and adult risk of disease.
  • 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.
  • Raj, Rahul (Helsingin yliopisto, 2014)
    Background: Prognostic models are important tools for heterogeneity adjustment in traumatic brain injury (TBI). Prognoses after TBI have been particularly challenging to predict, with limited availability of robust prognostic models. TBI patients are by definition trauma patients, and often treated in the intensive care unit (ICU). Several prognostic models for ICU and trauma patients have been developed, although their applicability in patients with TBI is uncertain. Recently, however, some new prognostic models specifically designed for patients with TBI were introduced. Still, the optimal type of prognostic model in TBI remains unknown. Aim: To investigate the applicability of different types of prognostic models in patients with TBI and to develop novel models with enhanced performance to previous models, focusing on long- term outcome prediction. Methods: Four patient databases of patients with TBI treated in the ICU were used to validate three TBI specific models, two computerized tomography (CT) scoring systems, one trauma scoring system, and three intensive care scoring systems. Models were validated by assessing their discrimination using area under the curve (AUC), calibration, and explanatory variation. Logistic regression was used for model customization and development. Models were internally validated using a resample bootstrap technique or a split-sample technique. Primary outcome was six-month mortality and unfavorable neurological outcome by the Glasgow Outcome Scale. 30-day in-hospital mortality was used for the trauma scoring system. Results: Study populations ranged from 342 to 9,915 patients. The TBI models showed the best performance with AUCs between 0.80 and 0.85, followed by the intensive care scoring systems and the CT scores with AUCs between 0.68 to 0.80 and 0.63 to 0.70, respectively. Most models showed poor calibration, although good calibration was achieved following customization. The trauma scoring system exhibited modest to good discrimination (AUC 0.76-0.89) for short-term mortality prediction, but poor calibration. Several new prognostic models, with statistically significant superior performance to previous models were created, among them a combined TBI-ICU model ( IMPACT-APACHE ) and a novel CT scoring system ( The Helsinki CT score ). Using a TBI specific model, based on admission characteristics, up to 40 % of the patient s final long-term outcome could be predicted. Conclusion: The TBI models showed superior predictive performance to the intensive care and trauma scoring systems, showing that TBI patients are a highly specific population in the trauma and ICU setting. Thus, the use of a TBI specific model is advocated in the setting of TBI. The newly proposed models were found to be significant improvements over previous models, but require external validation to show generalizability.
  • Pietiläinen, Olli (Helsingin yliopisto, 2014)
    Severe mental disorders including schizophrenia often segregate within the same families. Twin and family studies suggest that this co-occurrence is largely genetic, which implies that the different mental disorders have a shared genetic background. Some symptomatic features, such as cognitive impairment also manifest to a variable degree in the majority of severe mental disorders. Cognitive impairment occurs already before the onset of the disease and healthy family members of patients perform worse in cognitive tests than do the general population, which suggests that the cognitive impairment is indicative of genetic loading of the disease. Furthermore, the cognitive impairment persists throughout the disease and is associated with poorer outcome. This led us to hypothesize that the genetic architecture of schizophrenia is more similar to developmental disorders than had been considered earlier. Specifically, we hypothesized that rare high impact genetic variants play a role in the genetic risk for schizophrenia. Rare recurrent large-scale structural variation has long known to cause developmental syndromes, such as Prader-Willi syndrome or Velocardiofacial syndrome. In this study we investigated the role of large-scale chromosomal copy number variants in the genetic background of schizophrenia and other traits hypothesized to reflect abnormal neuronal development. In this study four chromosomal deletions on 1q21, 15q11.2, 15q13.3 and 22q11.2 were identified to be associated with schizophrenia. Three of the deletions occurred recurrently, whereas the deletion on 22q11.22 was a founder mutation enriched especially in the North-East region of Finland. On a population level, carriers of large deletions were found to have more intellectual disability or sub-normality (IQ<85) than non-carriers. Also milder learning difficulties as measured by repeated grades in school were more common among carriers of large deletions. The four deletions specifically identified as associating with schizophrenia are linked to variable phenotypes with the strongest effect manifesting in intellectual disabilities. The regional enrichment of the deletion on 22q11.22 also enabled the assessment of recessive effects related to the deletion. Four individuals, all presenting with a neurodevelopmental phenotype and/or schizophrenia, were identified as homozygous for the deletion. This deletion overlaps one gene encoding for topoisomerase 3 beta (TOP3β) that forms a protein complex with FMRP, the fragile X mental retardation protein, via tudor domain containing 3 (TDRD3) protein. The results of this study imply that rare high risk variants are present in a sub set of schizophrenia patients and that these variants are shared with developmental disorders. The study also demonstrates that special populations such as population isolates can provide useful study designs in identifying rare genetic risk variants, especially with recessive effects for complex traits.
  • Rahkonen, Petri (Helsingin yliopisto, 2014)
    Despite advancements in neonatal intensive care and increased survival of infants born extremely preterm, many of them still develop with motor, sensory, cognitive, and behavioral impairments. Predicting adverse neurodevelopmental outcome as early as possible is a challenge in neonatology. Structural neuroimaging methods partly fail to detect milder brain abnormalities that may interfere with later developmental outcome, and neurological assessment is more unreliable in the neonatal period than in childhood. Thus, additional methods are needed for earlier and more accurate recognition of extremely preterm infants with adverse neurodevelopmental outcome. The first purpose of this study was to assess the value of measuring higher cortical function by neurophysiological methods in predicting outcome of infants born extremely preterm. Second, we aimed to examine possible difficulties in behavioral somatosensory processing and in mother-child interaction and their associations with developmental outcome. The lack of somatosensory evoked magnetic fields from the secondary somatosensory cortex in magnetoencephalography at term equivalent age, reflecting abnormal higher cortical functioning during somatosensory processing, was associated with worse neuromotor outcome of extremely preterm infants at two years of corrected age, not foreseen with structural neuroimaging methods. Further, we showed that responses from the secondary somatosensory cortex can also be detected by measuring somatosensory evoked potentials during electroencephalography. The quality of mother-infant interaction in mother- extremely preterm child dyads did not differ from that in mother-term child dyads. However, among children born extremely preterm worse child adjustment and lower quality of maternal and dyadic behavior were associated with lower neurocognitive outcomes. Half of the children born extremely preterm presented atypical behavioral sensory processing at two years of corrected age. Sensation seeking was common in extremely preterm children with neonatal neuroanatomical lesions. In conclusion, the functional neurophysiological methods magnetoencephalography and measurement of somatosensory evoked potentials during electroencephalography hold promise as valuable additional tools in predicting outcome of children born extremely preterm. The quality of mother-infant interaction may play a significant role in optimizing cognitive outcome after extremely preterm birth. Atypical behavioral sensory processing in children born extremely preterm is common, but the pathogenesis and developmental significance of this phenomenon call for more research in the future.