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  • Väisänen-Tommiska, Mervi (Helsingin yliopisto, 2006)
  • Heinonen-Guzejev, Marja (Helsingin yliopisto, 2009)
    Noise can be defined as unwanted sound. It may adversely affect the health and well-being of individuals. Noise sensitivity is a personality trait covering attitudes towards noise in general and a predictor of noise annoyance. Noise sensitive individuals are more affected by noise than less sensitive individuals. The determinants and characteristics related to noise sensitivity are rather poorly known. The risk of health effects caused by noise can be hypothesized to be higher for noise sensitive individuals compared to those who are not noise sensitive. A cardiovascular disease may be an example of outcomes. The general aim of the present study was to investigate the association of noise sensitivity with specific somatic and psychological factors, including the genetic component of noise sensitivity, and the association of noise sensitivity with mortality. The study was based on the Finnish Twin Cohort of same-sex twin pairs born before 1958. In 1988 a questionnaire was sent to twin pairs discordant for hypertension. 1495 individuals (688 men, 807 women) aged 31 88 years replied, including 573 twin pairs. 218 of the subjects lived in the Helsinki Metropolitan Area. Self-reported noise sensitivity, lifetime noise exposure and hypertension were obtained from the questionnaire study in 1988 and other somatic and psychological factors from the questionnaire study in 1981 for the same individuals. In addition, noise map information (1988 1992) from the Helsinki Metropolitan Area and mortality follow-up 1989 2003 were used. To evaluate the stability and validity of noise sensitivity, a new questionnaire was sent in 2002 to a sample of the subjects who had replied to the 1988 questionnaire. Of all subjects who had answered the question on noise sensitivity, 38 % were noise sensitive. Noise sensitivity was independent of noise exposure levels indicated in noise maps. Subjects with high noise sensitivity reported more transportation noise exposure than subjects with low noise sensitivity. Noise sensitive subjects reported transportation noise exposure outside the environmental noise map areas almost twice as often as non-sensitive subjects. Noise sensitivity was associated with hypertension, emphysema, use of psychotropic drugs, smoking, stress and hostility, even when lifetime noise exposure was adjusted for. Monozygotic twin pairs were more similar with regards noise sensitivity than dizygotic twin pairs, and quantitative genetic modelling indicated significant familiality. The best fitting genetic model provided an estimate of heritability of 36 %. Follow-up of subjects in the case-control study showed that cardiovascular mortality was significantly increased among noise sensitive women, but not among men. For coronary heart mortality the interaction of noise sensitivity and lifetime noise exposure was statistically significant in women. In conclusion, noise sensitivity has both somatic and psychological components. It does aggregate in families and probably has a genetic component. Noise sensitivity may be a risk factor for cardiovascular mortality in women.
  • Ilumets, Helen (Helsingin yliopisto, 2011)
    Chronic obstructive pulmonary disease (COPD) is a slowly progressive disease characterized by airway inflammation and largely irreversible airflow limitation. One major risk factor for COPD is cigarette smoking. Since the inflammatory process starts many years prior to the onset of clinical symptoms and still continues after smoking cessation, there is an urgent need to find simple non-invasive biomarkers that can be used in the early diagnosis of COPD and which could help in predicting the disease progression. The first aim of the present study was to evaluate the involvement of different oxidative/nitrosative stress markers, matrix metalloproteinases (MMPs) and their tissue inhibitor-1 (TIMP-1) in smokers and in COPD. Elevated numbers of inducible nitric oxide synthase (iNOS), nitrotyrosine, myeloperoxidase (MPO) and 4-hydroxy-2-nonenal (4-HNE) positive cells and increased levels of 8-isoprostane and lactoferrin were found in sputum of non-symptomatic smokers compared to non-smokers, and especially in subjects with stable mild to moderate COPD, and they correlated with the severity of airway obstruction. This suggests that an increased oxidant burden exists already in the airways of smokers with normal lung function values. However, none of these markers could differentiate healthy smokers from symptomatic smokers with normal lung function values i.e. those individuals who are at risk of developing COPD. In contrast what is known about asthma exhaled nitric oxide (FENO) was lower in smokers than in non-smokers, the reduced FENO value was significantly associated with neutrophilic inflammation and the elevated oxidant burden (positive cells for iNOS, nitrotyrosine and MPO). The levels of sputum MMP-8 and plasma MMP-12 appeared to differentiate subjects who have a risk for COPD development but these finding require further investigations. The levels of all studied MMPs correlated with the numbers of neutrophils, and MMP-8 and MMP-9 with markers of neutrophil activation (MPO, lactoferrin) suggesting that especially neutrophil derived oxidants may stimulate the tissue destructive MMPs already in lungs of smokers who are not yet experiencing any airflow limitation. When investigating the role of neutrophil proteases (neutrophil elastase, MMP-8, MMP-9) during COPD exacerbation and its recovery period, we found that levels of all these proteases were increased in sputum of patients with COPD exacerbation as compared to stable COPD and controls, and decreased during the one-month recovery period, giving evidence for a role of these enzymes in COPD exacerbations. In the last study, the effects of subject`s age and smoking habits were evaluated on the plasma levels of surfactant protein A (SP-A), SP-D, MMP-9 and TIMP-1. Long-term smoking increased the levels of all of these proteins. SP-A most clearly correlated with age, pack years and lung function decline (FEV1/FVC), and based on the receiver operating characteristic curve analysis, SP-A was the best marker for discriminating subjects with COPD from controls. In conclusion, these findings support the hypothesis that especially neutrophil derived oxidants may activate MMPs and induce an active remodeling process already in the lungs of smokers with normal lung function values. The marked increase of sputum levels of neutrophil proteases in smokers, stable COPD and/or during its exacerbations suggest that these enzymes play a role in the development and progression of COPD. Based on the comparison of various biomarkers, SP-A can be proposed to serve as sensitive biomarker in COPD development.
  • Louhelainen, Noora (Helsingin yliopisto, 2012)
    Chronic obstructive pulmonary disease (COPD) is characterized by airway obstruction that is mostly irreversible. The typical features of COPD are chronic inflammation in the small airways, with varying degrees of chronic bronchitis and/or emphysema. Long-term smoking is the major risk factor for COPD. Oxidative stress and protease/antiprotease imbalance play major roles in the pathogenesis of smoking related airway disease such as COPD. The diagnosis of COPD is based on spirometry. Diagnosis is however often delayed since during its early stages, clear symptoms are lacking, even though the lung damage is already extensive. The differential diagnosis of COPD from asthma is especially difficult in smokers. There is a clear need to develop reliable biomarkers that would detect early COPD, differentiate the sub-phenotypes of COPD as well as COPD from asthma and finally be useful in developing treatment strategies for different phenotypes and used in monitoring disease activity and progression. The first goal of this study was to compare different potential markers in various sample types that reflect oxidative stress in the lung, how they are related to each other, and to find the most specific markers for the early detection and monitoring of COPD and asthma and differentiation of these diseases as well as the disease from the condition of healthy smoker . Another goal was to examine the effects of smoking cessation on the best potential markers of oxidative and protease burden. Recent lung proteomic studies have indicated that SP-A could be potential marker for COPD; sputum proteomics was used to find other biomarkers for early COPD. FeNO levels were elevated in mild asthma and eosinophils were elevated in asthmatic adults and patients with COPD exacerbation when compared to healthy controls. No significant differences were detected in sputum 8-isoprostane levels between subjects with mild asthma and healthy controls, but in patients with COPD exacerbation, the 8-isoprostane levels were greatly increased when compared to healthy subjects. In the smoking cessation studies, smokers had elevated levels of all of the investigated markers at the baseline compared to non-smokers. Neutrophil counts increased after 3 months of quitting of smoking in the group of chronic bronchitis/COPD but at 6 months neutrophils had returned to the levels of non-smokers. 8-Isoprostane declined significantly but the levels of FeNO, nitrotyrosine and MMP-8 did not change significantly during 3 months after smoking cessation, although after 6 months, the levels of MMP-7, -8 and TIMP-1 decreased to the levels of non-smokers and only MMP-9 remained elevated at 6 months of smoking cessation. However, the symptoms declined significantly already after 3 months of smoking cessation. The plasma levels of SP-A, SP-D, MMP-9, TIMP-1 and the ratio of MMP-9/TIMP-1 were increased by long-term smoking and COPD, and SP-A correlated with the extent of airway limitation. In proteomic screening of sputum, PIGR was found to be elevated in smokers and in mild to moderate COPD, and this was confirmed in lung tissue and plasma specimens. To conclude, it seems that sputum 8-isoprostane is not sensitive enough in detecting oxidative stress in mild recent asthma while in COPD it may be useful. After smoking cessation, the symptoms decline significantly already after a few months but the oxidant and protease burdens seem to persist in the airways for months. SP-A and PIGR can be proposed as representing new promising markers in smoking related chronic airway inflammation and COPD.
  • Sipponen, Taina (Helsingin yliopisto, 2009)
    Background: The fecal neutrophil-derived proteins calprotectin and lactoferrin have proven useful surrogate markers of intestinal inflammation. The aim of this study was to compare fecal calprotectin and lactoferrin concentrations to clinically, endoscopically, and histologically assessed Crohn’s disease (CD) activity, and to explore the suitability of these proteins as surrogate markers of mucosal healing during anti-TNFα therapy. Furthermore, we studied changes in the number and expression of effector and regulatory T cells in bowel biopsy specimens during anti-TNFα therapy. Patients and methods: Adult CD patients referred for ileocolonoscopy (n=106 for 77 patients) for various reasons were recruited (Study I). Clinical disease activity was assessed with the Crohn’s disease activity index (CDAI) and endoscopic activity with both the Crohn’s disease index of severity (CDEIS) and the simple endoscopic score for Crohn’s disease (SES-CD). Stool samples for measurements of calprotectin and lactoferrin, and blood samples for CRP were collected. For Study II, biopsy specimens were obtained from the ileum and the colon for histologic activity scoring. In prospective Study III, after baseline ileocolonoscopy, 15 patients received induction with anti-TNFα blocking agents and endoscopic, histologic, and fecal-marker responses to therapy were evaluated at 12 weeks. For detecting changes in the number and expression of effector and regulatory T cells, biopsy specimens were taken from the most severely diseased lesions in the ileum and the colon (Study IV). Results: Endoscopic scores correlated significantly with fecal calprotectin and lactoferrin (p<0.001). Both fecal markers were significantly lower in patients with endoscopically inactive than with active disease (p<0.001). In detecting endoscopically active disease, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for calprotectin ≥200 μg/g were 70%, 92%, 94%, and 61%; for lactoferrin ≥10 μg/g they were 66%, 92%, 94%, and 59%. Accordingly, the sensitivity, specificity, PPV, and NPV for CRP >5 mg/l were 48%, 91%, 91%, and 48%. Fecal markers were significantly higher in active colonic (both p<0.001) or ileocolonic (calprotectin p=0.028, lactoferrin p=0.004) than in ileal disease. In ileocolonic or colonic disease, colon histology score correlated significantly with fecal calprotectin (r=0.563) and lactoferrin (r=0.543). In patients receiving anti-TNFα therapy, median fecal calprotectin decreased from 1173 μg/g (range 88-15326) to 130 μg/g (13-1419) and lactoferrin from 105.0 μg/g (4.2-1258.9) to 2.7 μg/g (0.0-228.5), both p=0.001. The relation of ileal IL-17+ cells to CD4+ cells decreased significantly during anti-TNF treatment (p=0.047). The relation of IL-17+ cells to Foxp3+ cells was higher in the patients’ baseline specimens than in their post-treatment specimens (p=0.038). Conclusions: For evaluation of CD activity, based on endoscopic findings, more sensitive surrogate markers than CDAI and CRP were fecal calprotectin and lactoferrin. Fecal calprotectin and lactoferrin were significantly higher in endoscopically active disease than in endoscopic remission. In both ileocolonic and colonic disease, fecal markers correlated closely with histologic disease activity. In CD, these neutrophil-derived proteins thus seem to be useful surrogate markers of endoscopic activity. During anti-TNFα therapy, fecal calprotectin and lactoferrin decreased significantly. The anti-TNFα treatment was also reflected in a decreased IL-17/Foxp3 cell ratio, which may indicate improved balance between effector and regulatory T cells with treatment.
  • Holmström, Miia (Helsingin yliopisto, 2006)
    Conventional invasive coronary angiography is the clinical gold standard for detecting of coronary artery stenoses. Noninvasive multidetector computed tomography (MDCT) in combination with retrospective ECG gating has recently been shown to permit visualization of the coronary artery lumen and detection of coronary artery stenoses. Single photon emission tomography (SPECT) perfusion imaging has been considered the reference method for evaluation of nonviable myocardium, but magnetic resonance imaging (MRI) can accurately depict structure, function, effusion, and myocardial viability, with an overall capacity unmatched by any other single imaging modality. Magnetocardiography (MCG) provides noninvasively information about myocardial excitation propagation and repolarization without the use of electrodes. This evolving technique may be considered the magnetic equivalent to electrocardiography. The aim of the present series of studies was to evaluate changes in the myocardium assessed with SPECT and MRI caused by coronary artery disease, examine the capability of multidetector computed tomography coronary angiography (MDCT-CA) to detect significant stenoses in the coronary arteries, and MCG to assess remote myocardial infarctions. Our study showed that in severe, progressing coronary artery disease laser treatment does not improve global left ventricular function or myocardial perfusion, but it does preserve systolic wall thickening in fixed defects (scar). It also prevents changes from ischemic myocardial regions to scar. The MCG repolarization variables are informative in remote myocardial infarction, and may perform as well as the conventional QRS criteria in detection of healed myocardial infarction. These STT abnormalities are more pronounced in patients with Q-wave infarction than in patients with non-Q-wave infarctions. MDCT-CA had a sensitivity of 82%, a specificity of 94%, a positive predictive value of 79%, and a negative predictive value of 95% for stenoses over 50% in the main coronary arteries as compared with conventional coronary angiography in patients with known coronary artery disease. Left ventricular wall dysfunction, perfusion defects, and infarctions were detected in 50-78% of sectors assigned to calcifications or stenoses, but also in sectors supplied by normally perfused coronary arteries. Our study showed a low sensitivity (sensitivity 63%) in detecting obstructive coronary artery disease assessed by MDCT in patients with severe aortic stenosis. Massive calcifications complicated correct assessment of the lumen of coronary arteries.
  • Ilonen, Ilkka (Helsingin yliopisto, 2011)
    Lung cancer accounts for more cancer-related deaths than any other cancer. In Finland, five-year survival ranges from 8% to 13%. The main risk factor for lung cancer is long-term cigarette smoking, but its carcinogenesis requires several other factors. The aim of the present study was to 1) evaluate post-operative quality of life, 2) compare clinical outcomes between minimally invasive and conventional open surgery, 3) evaluate the role of oxidative stress in the carcinogenesis of non-small lung cancer (NSCLC), and 4) to identify and characterise targeted agents for therapeutic and diagnostic use in surgery. For study I, pneumonectomy patients replied to 15D quality of life and baseline dyspnea questionnaires. Study III involved a prospective quality of life assessment using the 15D questionnaire after lobectomy or bi-lobectomy. Study IV was a retrospective comparison of clinical outcomes between 212 patients treated with open thoracotomy and 116 patients who underwent a minimally invasive technique. Study II measured parameters of oxidative metabolism (myeloperoxidase activity, glutathione content and NADPH oxidase activity) and DNA adducts. Study V employed the phage display method and identified a core motif for homing peptides. This method served in cell-binding, cell-localisation, and biodistribution studies. Following both pneumonectomy and lobectomy, NSCLC patients showed significantly decreased long-term quality of life. No significant correlation was noted between post-operative quality of life and pre-operative pulmonary function tests. Women suffered more from increased dyspnea after pneumonectomy which was absent after lobectomy or bi-lobectomy. Patients treated with video-assisted thoracoscopy showed significantly decreased morbidity and shorter periods of hospitalization than did open surgery patients. This improvement was achieved even though the VATS patients were older and suffered more comorbid conditions and poorer pulmonary function. No significant differences in survival were noted between these two groups. An increase in NADPH oxidase activity was noted in tumour samples of both adenocarcinoma and squamous cell carcinoma. This increase was independent from myeloperoxidase activity. Elevated glutathione content was noted in tumour tissue, especially in adenocarcinoma. After panning the clinical tumour samples with the phage display method, an amino acid sequence of ARRPKLD, the Thx, was chosen for further analysis. This method proved selective of tumour tissue in both in vitro and in vivo cell-binding assay, and biodistribution showed tumour accumulation. Because of the significantly reduced quality of life following pneumonectomy, other operative strategies should be implemented as an alternative (e.g. sleeve-lobectomy). To treat this disease, implementation of a minimally invasive surgical technique is safe, and the results showed decreased morbidity and a shorter period of hospitalisation than with thoracotomy. This technique may facilitate operative treatment of elderly patients with comorbid conditions who might otherwise be considered inoperable. Simultaneous exposure to oxidative stress and altered redox states indicates the important role of oxidative stress in the pathogenesis and malignant transformation of NSCLC. The studies showed with great specificity and with favourable biodistribution that Thx peptide is specific to NSCLC tumours. Thx thus shows promise in imaging, targeted therapy, and monitoring of treatment response.
  • Ståhl, Minna (Helsingin yliopisto, 2014)
    Neck pain is a common health problem globally. In addition to individual suffering, it causes a huge burden on societies. Some evidence suggests that neck pain in adolescence predicts frequent neck symptoms also in adulthood. A steady increase in prevalence of frequent and persistent neck pain among Finnish adolescents was observed in the 1990s, which might predict an increasing amount of adults with chronic neck pain in the near future. Thus, better understanding neck pain in children and adolescents might provide further clues to the origins of adult neck pain, and help to plan and target needed prevention programs against chronic neck pain in the whole population. The aim of this study was to fulfil the gaps of knowledge regarding neck pain in the early years of life by investigating the occurrence, co-occurrence, intensity, consequences, natural course, risk and prognostic factors, heritability, and secular trends of neck pain alone versus concomitant neck and low back pain in preadolescent to adolescent populations. Three different types of data were used: 1) A longitudinal data of 9 to 12-year-old Finnish schoolchildren (N=1268), with follow-ups at baseline, and 1 and 4 years later, 2) cross-sectional data from 11-year-old Finnish twins (FinnTwin 12 baseline study, N=1797 pairs), and data from 8 cross-sectional surveys between 1991 and 2011 among 12 to 18-year-old Finns (Adolescent Health and Lifestyle Surveys, N= 52 331). In the longitudinal study, of those without any musculoskeletal pain at baseline 21% reported neck pain at the 1-year follow-up, and 43% at the 4-year follow-up. Thus, the occurrence of neck pain increased with age. Neck pain had occurred more to girls (57%) than boys (29%) only at the 4-year follow-up. Thus, gender difference in prevalence of neck pain appeared first in midadolescence. Neck pain occurred more often together with other musculoskeletal pain(s)(most often with leg or other axial pain) than as a single pain (14% vs. 8% at the 1-year follow-up; 34% vs. 10% at the 4-year follow-up). The intensity of pain increased linearly (p < 0.001) with the frequency of pain, thus, it seems that the frequency of neck pain pain reflects the intensity quite well. About a quarter of those that had experienced neck pain at the 4-year follow-up, had used painkillers, and reported some disability. However, severe disability was rare (4%). In majority (71%), the frequency of neck symptoms fluctuated between no, monthly and at least weekly pain during the follow- up. Thus, the natural course of neck pain from preadolescence to adolescence was mainly fluctuating. However, 5% reported frequent neck pain (symptoms at least once a week during the last 3 months) at baseline, and at both follow-ups indicating a chronic type problem already at adolescence. Other musculoskeletal pains (only in girls) and/or psychosomatic symptoms at baseline predicted new onset of frequent neck pain during the 4-year follow-up. The same factors were found to predict a persistent course of symptoms from preadolescence to adolescence. Additionally, girls with frequent pain at baseline had 5.9 (compared 4.6 for boys) times more likely frequent symptoms also during the follow-up compared to those with no neck pain at baseline. Neither level of physical activity, nor joint hypermobility in preadolescence, was found to be a risk factor for neck pain in adolescence, nor had predicting value for the future course of neck pain. The results from the twin study showed substantial heritability (68%) for neck pain. In addition to genetic influence, unique environmental factors (e.g. personal hobbies) seem to play an important role in liability towards neck pain, while common environmental factors (e.g. home) were found to be less important. Repeated cross-sectional studies from 1991 to 2011 showed that neck pain has become increasingly common among Finnish adolescents. In 2011, nearly half of the Finnish 16 to 18-year-old girls, and fifth of the boys reported that they have had neck pain at least weekly during the last 6 months. A steady increase was observed in the prevalence of concomitant neck and low back pain over the last two decades (prevalence increased 2 to 3-fold), while the prevalence of neck pain alone increased only in the 1990s. The results of this thesis strengthen the evidence that chronic neck pain might originate from the early years of life. Genetic and psychological factors seem to be important in the aetiology of adolescent neck pain. The possibility that common genetic factors influence both neck pain and symptoms of psychological distress requires investigation. In light of the current knowledge, school age children with neck pain and multiple other medically unexplained symptoms, and perhaps also chronic neck pain sufferers in the family, could be a good target group for future preventive interventions against chronic neck pain. However, before interventions, it should be explored whether single neck pain and neck pain co-occurring with other musculoskeletal pains are two distinct, partly overlapping, or consecutive phenomena with, at least partly, underlying aetiologies.
  • Sahlstedt, Leila (Helsingin yliopisto, 2015)
    Hematopoietic stem cell transplantation (HSCT) is an intensive treatment often complicated by organ injuries. Non-transferrin-bound iron (NTBI), as an inducer of free oxygen radicals, is a potential factor in the pathogenesis of these complications. We studied the appearance and timing of NTBI in transplant patients and the possibility to prevent the occurrence of NTBI by binding it with apotransferrin administration. We showed that NTBI appears regularly during the peritransplantation period in HSCT, both in allogeneic and autologous transplantation. We could demonstrate, for the first time, the exact timing and duration of the changes in transferrin saturation and the appearance and the length of the presence of NTBI. We could also show and confirm the 80% saturation of transferrin to be a reliable threshold for NTBI. In allogeneic HSCT fully saturated transferrin and the appearance of NTBI was observed by day -4 before HSCT (day 0). NTBI was detectable on average for 14 days. In autologous HSCT a similar steep rise was found in the transferrin saturation. The mean time for the first to the last NTBI-positive samples was 6.1 days, the intensity of the conditioning seemed to have an effect on the appearance of NTBI. The disappearance or marked reduction of the NTBI positive samples coincided with the recovery of the bone marrow function. Our studies indicate that the presence of NTBI is largely due to reduced utilization of iron by erythropoiesis and the reduction of serum transferrin levels caused by the transplantation process. As NTBI might contribute to the complications of HSCT, we assessed the possibility to bind NTBI during the peritransplant period with apotransferrin infusions. With a single apotransferrin dose given on day +3 NTBI disappeared from the sera of all the six patients, but reappeared 12-24 hours later. Twenty patients were given repeated doses of apotransferrin at three dose levels. The daily loading doses were given at the start of conditioning, followed by maintenance doses every other day given until day +7 to day +13. Five of the eight patients given the highest dose were NTBI negative through the study period. With lower doses there was a nonsignificant trend towards fewer days with demonstrable NTBI. Our in vitro study demonstrating the toxic effect of NTBI on hematopoietic progenitors and the beneficial effect of binding NTBI with apotransferrin on colony growth supports the possibility that binding of NTBI with exogenous apotransferrin may turn out to be clinically useful. Our studies showed that the appearance of NTBI could be prevented or reduced by apotransferrin. However, the clinical utility of apotransferrin administration and the effects on transplantation outcomes remain to be investigated in further studies.
  • Juhila, Juuso (Helsingin yliopisto, 2007)
    Nephrin is a transmembrane protein belonging to the immunoglobulin superfamily and is expressed primarily in the podocytes, which are highly differentiated epithelial cells needed for primary urine formation in the kidney. Mutations leading to nephrin loss abrogate podocyte morphology, and result in massive protein loss into urine and consequent early death in humans carrying specific mutations in this gene. The disease phenotype is closely replicated in respective mouse models. The purpose of this thesis was to generate novel inducible mouse-lines, which allow targeted gene deletion in a time and tissue-specific manner. A proof of principle model for succesful gene therapy for this disease was generated, which allowed podocyte specific transgene replacement to rescue gene deficient mice from perinatal lethality. Furthermore, the phenotypic consequences of nephrin restoration in the kidney and nephrin deficiency in the testis, brain and pancreas in rescued mice were investigated. A novel podocyte-specific construct was achieved by using standard cloning techniques to provide an inducible tool for in vitro and in vivo gene targeting. Using modified constructs and microinjection procedures two novel transgenic mouse-lines were generated. First, a mouse-line with doxycycline inducible expression of Cre recombinase that allows podocyte-specific gene deletion was generated. Second, a mouse-line with doxycycline inducible expression of rat nephrin, which allows podocyte-specific nephrin over-expression was made. Furthermore, it was possible to rescue nephrin deficient mice from perinatal lethality by cross-breeding them with a mouse-line with inducible rat nephrin expression that restored the missing endogenous nephrin only in the kidney after doxycycline treatment. The rescued mice were smaller, infertile, showed genital malformations and developed distinct histological abnormalities in the kidney with an altered molecular composition of the podocytes. Histological changes were also found in the testis, cerebellum and pancreas. The expression of another molecule with limited tissue expression, densin, was localized to the plasma membranes of Sertoli cells in the testis by immunofluorescence staining. Densin may be an essential adherens junction protein between Sertoli cells and developing germ cells and these junctions share similar protein assembly with kidney podocytes. This single, binary conditional construct serves as a cost- and time-efficient tool to increase the understanding of podocyte-specific key proteins in health and disease. The results verified a tightly controlled inducible podocyte-specific transgene expression in vitro and in vivo as expected. These novel mouse-lines with doxycycline inducible Cre recombinase and with rat nephrin expression will be useful for conditional gene targeting of essential podocyte proteins and to study in detail their functions in the adult mice. This is important for future diagnostic and pharmacologic development platforms.
  • Grönholm, Mikaela (Helsingin yliopisto, 2005)
  • Ola, Roxana (Helsingin yliopisto, 2012)
    Glial-cell-line-derived neurotrophic factor (GDNF) signaling through the receptor tyrosine kinase Ret and its co-receptor Gfrα1 is indispensable for the ureteric bud (UB) outgrowth from Wolffian ducts during kidney differentiation. To search for novel genes regulated by GDNF in Wolffian ducts and early UB epithelium, we performed a genome wide analysis of Wolffian ducts exposed by GDNF in vitro, and identified 69 up-regulated genes. Plakophilin2 gene encoding for the armadillo protein- Pkp2 and Visinin like 1 gene, encoding for a neuronal calcium sensor protein- VSNL1 were among the highly up-regulated genes and their expression was missing in the Gdnf- null kidneys. Analysis of the Pkp2- null allele kidneys revealed a defect in ureteric branching. The Pkp2-/- kidney explants did not undergo UB formation and branching in vitro, but responded to GDNF by producing supernumerary buds, suggesting that Pkp2 is not essential for UB formation from the Wolffian duct. VSNL1 characterized the tip domain of the UB epithelium regardless of the inducer. In the UB tips, VSNL1 displayed a unique mosaic expression pattern that demonstrated the heterogeneity of cells in the tip. VSNL1 expression was mutually exclusive with β-catenin activity as shown in Bat-gal Wnt-reporter mice. VSNL1 was downregulated in both β-catenin stabilized and β-catenin deficient kidneys. Moreover, VSNL1 transduction compromised β-catenin stability in cultured cells. The results suggest an antagonistic effect between VSNL1 and β-catenin. The spatial and temporal expression of VSNL1 and its mRNA was further addressed during mouse embryogenesis. Vsnl1 was specifically confined to the cardiac anlagen. All myocardial cells that contributed to the remodeling of the venous pole of the heart were positive for Vsnl1. An expansion of the VSNL1 expression domain to the ventricles was seen soon after the first week of postnatal life, and this pattern was maintained to adulthood. The function of PlexinB2 during renal development was finally addressed by analyzing the kidney phenotype in Plxnb2- null mice. These embryos displayed hypoplastic kidneys, and occasional unilateral double kidneys and ureters. The defect in branching was intrinsic to the ureteric epithelium. By co-immunoprecipitation experiments, PlexinB2 was shown to interact with Ret suggesting that PlexinB2, activated by Sema4C ligand, may modulate ureteric branching by a direct regulation of Ret activity.
  • Sillanpää, Heidi (Helsingin yliopisto, 2014)
    Lyme borreliosis (LB) is a bacterial infection spread by the bite of Ixodes sp. ticks. Borrelia burgdorferi sensu lato group of spirochaetes is causing the infection. Less than 10 species are known to be pathogenic to humans. Distribution of B. burgdorferi sensu lato species varies geographically and LB is endemic in Europe, Asia and North America. The typical initial manifestation of LB is a ring-like rash, erythema migrans (EM). EM appears around the tick bite site after a couple of days or weeks. Diagnosis of LB is based on EM and the infection is treated with antibiotics. In some cases, the tick bite or EM may go unnoticed. Manifestations of late-stage LB are Lyme arthritis, cardiac symptoms, neuroborreliosis (LNB) manifesting as meningoradiculitis or meningitis, or chronic atrophic skin changes (acrodermatitis chronica atrophicans). These late-stage manifestations may appear within weeks, months, or in rare occasions after years. Frequently, laboratory tests are needed to confirm the clinical diagnosis. Laboratory testing is mainly based on the detection of Borrelia specific antibodies, usually with ELISA method or immunoblotting. There are no markers of active LB and antibody responses may persist for years after the treatment of the infection. The aim of this study was to identify new immunological markers for the laboratory diagnostics of LB. A large amount of serum and cerebrospinal fluid (CSF) samples were collected from patients with different manifestations of LB from Europe and the United States. Five peptide variants of IR6 (= invariable region 6 of the VlsE protein) as antigens were compared to commercial C6 Immunoassay in the serodiagnosis of early and disseminated stages of LNB. Recombinant decorin binding proteins A (DbpA) and B (DbpB) (three variants of each) as antigens were evaluated in the serodiagnosis of LNB. CXCL13 Immunoassay was used to measure chemokine CXCL13 levels in children with early LNB. As a conclusion, the use of a combination of different IR6 peptide variants in one assay may improve the laboratory diagnostics of LB by increasing the sensitivity of the test. DbpA and DbpB may serve as supplementary antigens in diagnostic assays. Appearance of CXCL13 in the CSF seems to be an early immunological marker of inflammation in CNS. Measurement of CXCL13 concentrations in the CSF may improve the diagnostics of LNB.
  • Moza, Monica (Helsingfors universitet, 2008)
    In skeletal and cardiac muscle cells, the contractile unit sarcomere consists of actin filaments that have constant length, a strict spatial distribution and are aligned with myosin filaments. Actin filaments from adjacent sarcomeres are cross-linked by actin-associated proteins which form the Z-disk. Sarcomeres provide the muscle cell its shape and they are the structural and functional basis of the muscle contraction. In response to intra- and extracellular stimuli, the actin cytoskeleton is continuously reorganizing in non-muscle or smooth muscle cells in order to determine the cell morphology, the cytokinesis, coordinated cell movements and the intracellular transport of organelles. Myotilin, palladin and myopalladin form a small family of proteins containing immunoglobulin-like (Igl) domains. They are associated with the actin cytoskeleton, and all members of the family are suggested to have roles as a scaffold and as regulators of actin organization. Myotilin and myopalladin are expressed as a single isoform only in the striated muscle. Myotilin expression level is higher in the skeletal muscle than in the heart, while myopalladin expression in higher in the heart than in the skeletal muscle. Their expression levels might be directly correlated with their function. On the contrary, the palladin gene gives rise to several palladin isoforms expressed as a result of alternative splicing events. Palladin is expressed not only in the striated muscle but also in other tissue types, such as the nervous tissue, and different isoforms apparently play cell-specific roles. The three members of the family are localized at the Z-disk in the striated muscle and here they have a common binding partner, the bona-fide Z-disk protein, alpha-actinin. Point mutations in myotilin gene causes muscle disorders of variable phenotype: limb-girdle muscular dystrophy A (LGMD1A), myofibrillar myopathy (MFM) and spheroid body myopathy (SBM). On the other hand, palladin gene mutation has been reported to be associated with familial pancreatic cancer and increased risk for myocardial infarction. In this study we found that, as shown for other poly-L-proline-containing proteins, palladin directly binds a key molecule involved in actin dynamics, profilin. Profilin has a dual function: it is a promoter of actin assembly and it can also act as an actin-sequestring protein resulting in actin depolymerisation. Palladin was shown to interact with profilin via its second polyproline region and, further more, we demonstrated that the interaction is highly dynamic and of low affinity. In cells, palladin colocalized with profilin in actin-rich bundles near cell edges. These results indicate that 90-92 kDa palladin associates with profilin in regions in which novel actin filaments form and that palladin may be involved in the coordination of actin filament formation. Alpha-actinin, an important actin cross-linking molecule, connects the cytoskeleton to transmembrane proteins and serves as a scaffold for signalling molecules. We found that palladin binds to and colocalize with alpha-actinin. The interaction is mediated via a highly conserved region in both myotilin and palladin which is considered as a new binding domain for alpha-actinin. Pain, stiffness and tenderness in the muscle, or delayed onset muscle soreness (DOMS) appears in untrained persons who perform eccentric exercise. All aspects of DOMS are not yet fully understood, however it is considered that new sarcomeres are formed. This process includes modifications in the Z-disk architecture and composition, such as temporary loss of -actinin, titin and nebulin and polymerization of G-actin to form foci of increased F-actin labelling in skeletal muscle fibers after the exercise to finally result in supranumerary sarcomeres. Our studies showed that myotilin follows the widened distribution patterns observed for F-actin, suggesting that myotilin acts as a cross-linker and an F-actin stabilizer. In these structures, myotilin is probably replacing alpha-actinin and, similarly with alpha-actinin, might be an anchoring site for signalling or scaffolding molecules that are recruited to drive the formation of the new sarcomeres. We propose myotilin as a guardian of the Z-disk as apparently myotilin contributes to actin organization in both mature and forming Z-disks. To further study the functions of myotilin we generated knockout mice. While loss of all palladin isoforms leads to embryonical lethality in mice, we showed that myotilin knockout mouse survives and has no obvious morphological or functional alteration in striated muscle or other organs. Myotilin absence is apparently compensated by other proteins, possibly by members of myotilin/palladin/myopalladin subfamily. This finding is in concordance with the finding that the transgenic expression of a mutant myotilin leads to morphological alterations observed in myotilinopathies, i.e. mutated myotilin appears to have a toxic effect on the structure of the skeletal muscle, whereas myopathy due to myotilin deficiency has not been reported. Further, we have generated and investigated a mouse that lacks myotilin and expresses low levels of the 200 kDa palladin. While the 200 kDa palladin hypomorph mice shows ultrastructural modifications of the heart architecture, we showed that combined absence of myotilin and low levels of 200 kDa palladin led to morphological and functional alterations in skeletal muscle. This work showed that 200 kDa palladin has an important role in the maintenance of normal architecture of the cardiomyocytes and that myotilin deficiency apparently exacerbated the cardiomyocyte defects. This indicates that in skeletal muscle myotilin and 200 kDa palladin may have a similar function, while in the heart their functions might be different.
  • Bister, Ville (Helsingin yliopisto, 2007)
    Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.
  • Kallio, Suvi (Helsingin yliopisto, 2009)
    Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Both environmental factors and several predisposing genes are required to generate MS. Despite intensive research these risk factors are still largely unknown, the pathogenesis of MS demyelination is poorly understood, and no curative treatment exists. Both prevalence and familial occurrence of MS are exceptionally high in a Finnish population subisolate, Southern Ostrobothnia, presumably due to enrichment of predisposing genetic variants within this region. Previous linkage scan on MS pedigrees from Southern Ostrobothnia detected three main MS loci on chromosomes 5p, 6p (HLA) and 17q. Linkage studies in other populations have also provided independent evidence for the location of MS susceptibility genes in these regions. Further, these loci are syntenic to the experimental autoimmune encephalomyelitis (EAE) susceptibility loci of rodents. In this thesis work an effort was made to localize MS predisposing alleles of the linked loci outside the HLA region by studying familial MS cases from the Southern Ostrobothnia isolate. Analysis of the 5p locus revealed one region, flanking the complement component 7 (C7) gene. The identified relatively rare haplotype seems to have a fairly large effect on genetic susceptibility of MS (frequency MS 12%, controls 4%; p=0.000003, OR=2.73). Evidence for association with alleles of the region and MS was seen also in more heterogeneous populations. Convincingly, plasma C7 protein levels and complement activity correlated with the risk haplotype identified. The finding stimulated us to study other complement cascade genes in MS. No evidence for association could be observed with the complement component coding genes outside 5p. A scan of the 17q locus provided evidence for association with variants of the protein kinase C alpha (PRKCA) gene (p=0.0001). Modest evidence for association with PRKCA was observed also in Canadian MS families. Finally we used a candidate gene based approach to identify potential MS loci. Mutations of DAP12 and TREM2 cause a recessively inherited CNS white matter disease PLOSL. Interestingly, DAP12 and TREM2 are located in MS regions on 6p and 19q, and we tested them as potential candidate genes in the Finnish MS sample. No evidence for association with MS was observed. This thesis provides an example of how extended families from special populations can be utilized in fine-mapping of the linked loci. A first relatively rare MS variant was identified utilizing the strength of a Finnish population subisolate. This variant seems to have an effect on activity of the complement system, which has previously been suggested to have an important role in the pathogenesis of MS.
  • Lundán, Tuija (University of Helsinki, 2008)
    Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.
  • Peltonen, Lauri (Helsingfors universitet, 2008)
    Polylactic- and polyglycolic acid-based degradable biomaterials are in wide clinical use especially in orthopedic and craniomaxillofacial surgery, and in general they have proved to be safe and well-tolerated implant materials. Thus far, they have not been used in reconstructive human middle ear surgery. Common problems related to implant surgery are microbial biofilm formation on medical implants in vivo and subsequent infection. A postoperative implant infection often leads to re-operation and implant removal. The present thesis clarifies the surface properties of polylactic-glycolic acid (PLGA) in relation to bacterial adherence—the first step in biofilm formation—and the biocompatibility of the material in the middle ear. Furthermore, a new application has been studied for imaging of the middle ear with a lower radiation dose than with the present computed tomography (CT) of the ear. This method, cone-beam computed tomography (CBCT), has been used particularly in dental and maxillofacial imaging, where it allows accurate, 3D imaging of a defined area. The first two studies of this thesis tested in vitro adherence of two common pathogens of a chronically or postoperatively infected ear, Staphylococcus aureus and Pseudomonas aeruginosa, onto the surfaces of PLGA and of two common implant materials used in ear surgery: silicone and titanium. In addition, the effect of albumin coating on adherence to these materials was tested and compared. Next, applicability of the PLGA material to reconstructive middle ear surgery was explored by study of its biocompatibility in the middle ear in an animal model in which chinchillas were operated on, followed-up, and killed at 6 months postoperatively. Biocompatibilities of the implanted materials were evaluated according to clinical and histological findings. Suitability of the CBCT for imaging of the ear was tested by imaging human cadaver temporal bones and comparing scaled accuracies of the images created by CBCT with those constructed with traditional multislice helical CT. There proved to be no more bacterial adherence in vitro to the PLGA materials tested than to silicone and titanium. Albumin coating significantly reduced bacterial adherence to every material tested. The biocompatibility of PLGA in the middle ear of the chinchilla was very good. In histological samples, only a mild inflammatory reaction was evident around the degrading PLGA plates. Clinically, no infections or extrusion of the implanted material occurred. Limited CBCT was found to be at least as accurate as conventional multislice helical CT in showing the clinically and surgically important landmarks of the middle and inner ear. The effective radiation dose was essentially lower with the CBCT than with the helical CT used in this study. Visualization of the middle ear structures and implants of operated temporal bones was clear and of diagnostic value. Based on the results of the thesis, it can be concluded that PLGA is a safe and well-tolerated implant material in the middle ear area, and there is no greater in vitro adherence of S. aureus and P. aeruginosa to PLGA surfaces than to titanium and silicone. In addition, coating of these materials with albumin means that adherence of the bacteria to their surfaces can be substantially reduced, suggesting that this method may be useful in implant surgery as a preventive maneuver against postoperative infections. Furthermore, CBCT seems to be a promising new method for imaging the structures of the temporal bone. The diagnostic accuracy of CBCT corresponds to that of helical CT, while the radiation dose to patient per investigation remains lower with CBCT. Focused imaging with the lesser radiation exposure expands the possibilities for imaging the ear, especially when follow-up and limited uni- or bilateral examination is needed.
  • Moore, Henna (2013)
    The nucleolus is a subnuclear structure governing ribosome biogenesis. In the past few years there has been increasing evidence that in addition to function as a ribosome factory the nucleolus bears several other cellular functions, including regulation of tumor suppressor and oncogene activities, and sensing cellular and proteotoxic stress. Alterations in nucleolar structures and protein localization are indicative of changes in nucleolar function. When exposed to certain types of cellular stress, the nucleolar structure is disrupted and the localization of several nucleolar proteins is reorganized. This process, nucleolar stress, leads to the stabilization of the tumor suppressor protein p53. In this thesis work, we used quantitative proteomics of isolated nucleoli and dynamic imaging of live cells to explore the changes in localization of nucleolar proteins in response to various types of cellular stress, including ultraviolet (UV) and ionizing radiation (IR). Furthermore, we used fluorescent photobleaching and biochemical analyses of nucleoli to study nucleolar and nucleolus-associated proteins in cells exposed to UV radiation and proteasomal stress. We found that nucleolar substructures and the nucleolar proteome underwent extensive reorganization in response to UV damage whereas the responses to IR were more limited. These results were supported by concurrent repression of RNA polymerase I (RNA Pol I) transcription that we observed in UV but not in IR treated cells. However, proteomic analysis imminently after the IR insult revealed that IR caused a rapid and transient response in certain DNA repair proteins and RNA Pol I components. Although these responses were not extensive, they were significant and likely relevant in the early cellular response to IR. We also identified a novel nucleolar stress body, which we named nucleolus-associated RNA-protein aggregate (NoA). It formed by the inhibition of proteasomal activity and contained conjugated ubiquitin, polyadenylated RNA and numerous nuclear proteasomal target proteins, including cellular regulators, key players in oncogenesis and stress response proteins. The formation of NoA was dependent on ubiquitin availability as it was rescued by promoting ubiquitin recycling. Interestingly, the inhibition of chromosome region maintenance 1 protein homolog (CRM1)-dependent nuclear export further promoted the formation of NoA. These results suggest that the nucleolar activities are linked with the proteasomal function, and surveillance of RNA and nuclear proteasomal target proteins. Finally, we found that proteasomal activity was required for the nucleolar responses of nucleophosmin (NPM) and other granular component (GC) proteins in cells exposed to UV radiation. These results support the role of the nucleolus as a sensitive stress responder and suggest a role for the proteasome in nucleolar activities.