Applications of Treatment Effects Models and Semiparametric Estimation

This thesis is composed of an introductory chapter and four applications each of them constituting an own chapter. The common element underlying each of the chapters is the econometric methodology. The applications rely mostly on the leading econometric techniques related to estimation of causal effects. The first chapter introduces the econometric techniques that are employed in the remaining chapters. Chapter 2 studies the effects of shocking news on student performance. It exploits the fact that the school shooting in Kauhajoki in 2008 coincided with the matriculation examination period of that fall. It shows that the performance of men declined due to the news of the school shooting. For women the similar pattern remains unobserved. Chapter 3 studies the effects of minimum wage on employment by employing the original Card and Krueger (1994; CK) and Neumark and Wascher (2000; NW) data together with the changes-in-changes (CIC) estimator. As the main result it shows that the employment effect of an increase in the minimum wage is positive for small fast-food restaurants and negative for big fast-food restaurants. Therefore, it shows that the controversial positive employment effect reported by CK is overturned for big fast-food restaurants and that the NW data are shown, in contrast to their original results, to provide support for the positive employment effect. Chapter 4 employs the state-specific U.S. data (collected by Cohen and Einav [2003; CE]) on traffic fatalities to re-evaluate the effects of seat belt laws on the traffic fatalities by using the CIC estimator. It confirms the CE results that on the average an implementation of a mandatory seat belt law results in an increase in the seat belt usage rate and a decrease in the total fatality rate. In contrast to CE, it also finds evidence on compensating-behavior theory, which is observed especially in the states by the border of the U.S. Chapter 5 studies the life cycle consumption in Finland, with the special interest laid on the baby boomers and the older households. It shows that the baby boomers smooth their consumption over the life cycle more than other generations. It also shows that the old households smoothed their life cycle consumption more as a result of the recession in the 1990s, compared to young households.

PERFECT Stroke : PERFormance, Effectiveness, and Costs of treatment episodes in Stroke

Stroke is a major cause of death and disability, incurs significant costs to healthcare systems, and inflicts severe burden to the whole society. Stroke care in Finland has been described in several population-based studies between 1967 and 1998, but not since. In the PERFECT Stroke study presented here, a system for monitoring the Performance, Effectiveness, and Costs of Treatment episodes in Stroke was developed in Finland. Existing nationwide administrative registries were linked at individual patient level with personal identification numbers to depict whole episodes of care, from acute stroke, through rehabilitation, until the patients went home, were admitted to permanent institutional care, or died. For comparisons in time and between providers, patient case-mix was adjusted for. The PERFECT Stroke database includes 104 899 first-ever stroke patients over the years 1999 to 2008, of whom 79% had ischemic stroke (IS), 14% intracerebral hemorrhage (ICH), and 7% subarachnoid hemorrhage (SAH). A 18% decrease in the age and sex adjusted incidence of stroke was observed over the study period, 1.8% improvement annually. All-cause 1-year case-fatality rate improved from 28.6% to 24.6%, or 0.5% annually. The expected median lifetime after stroke increased by 2 years for IS patients, to 7 years and 7 months, and by 1 year for ICH patients, to 4 years 5 months. No change could be seen in median SAH patient survival, >10 years. Stroke prevalence was 82 000, 1.5% of total population of Finland, in 2008. Modern stroke center care was shown to be associated with a decrease in both death and risk of institutional care of stroke patients. Number needed to treat to prevent these poor outcomes at one year from stroke was 32 (95% confidence intervals 26 to 42). Despite improvements over the study period, more than a third of Finnish stroke patients did not have access to stroke center care. The mean first-year healthcare cost of a stroke patient was ~20 000 , and among survivors ~10 000 annually thereafter. Only part of this cost was incurred by stroke, as the same patients cost ~5000 over the year prior to stroke. Total lifetime costs after first-ever stroke were ~85 000 . A total of 1.1 Billion , 7% of all healthcare expenditure, is used in the treatment of stroke patients annually. Despite a rapidly aging population, the number of new stroke patients is decreasing, and the patients are more likely to survive. This is explained in part by stroke center care, which is effective, and should be made available for all stroke patients. It is possible, in a suitable setting with high-quality administrative registries and a common identifier, to avoid the huge workload and associated costs of setting up a conventional stroke registry, and still acquire a fairly comprehensive dataset on stroke care and outcome.

Optimal Use of Treatment Modalities and Treatment Results of Osteosarcoma and Soft Tissue Sarcomas

Soft tissue sarcomas are malignant tumours of mesenchymal origin. Because of infiltrative growth pattern, simple enucleation of the tumour causes a high rate of local recurrence. Instead, these tumours should be resected with a rim of normal tissue around the tumour. Data on the adequate margin width are scarce. At Helsinki University Central Hospital (HUCH) a multidisciplinary treatment group started in 1987. Surgical resection with a wide margin (2.5 cm) is the primary aim. In case of narrower margin radiation therapy is necessary. The role of adjuvant chemotherapy remains unclear. Our aims were to study local control by the surgical margin and to develop a new prognostic tool to aid decision-making on which patients should receive adjuvant chemotherapy. Patients with soft tissue sarcoma of the extremity or the trunk wall referred to HUCH during 1987-2002 form material in Studies I and II. External validation material comes from the Lund university sarcoma registry. The smallest surgical margin of at least 2.5 centimetres yielded local control of 89 per cent at five years. Amputation rate was 9 per cent. The proposed prognostic model with necrosis, vascular invasion, size on a continuous scale, depth, location and grade worked well both in Helsinki material and in the validation material, and it also showed good calibration. Based on the present study, we recommend the smallest surgical margin of 2-3 centimetres in soft tissue sarcoma irrespective of grade. Improvement in local control was present but modest in margins wider than 1 centimetre. In cases where gaining a wider margin would lead to a considerable loss of function, smaller margin is to be considered combined to radiation therapy. Patients treated with inadequate margins should be offered radiation therapy irrespective of tumour grade. Our new prognostic model to estimate 10-year survival probability in patients with soft tissue sarcoma of the extremities or trunk wall showed good dicscrimination and calibration. For time being the prognostic model is available for scientific use and further validations. In the future, the model may aid in clinical decision-making. For operable osteosarcoma, neoadjuvant multidrug chemotherapy followed by delayed surgery and multidrug adjuvant chemotherapy is the treatment of choice. Overall survival rates at five years are approximately 75 per cent in modern trials with classical osteosarcoma. All patients diagnosed and reported to the Finnish Cancer Registry with osteosarcoma in Finland during 1971-2005 form the material in Studies III and IV. Limb-salvage rate increased from 23 per cent to 78 per cent during 1971-2005. The 10-year sarcoma-specific survival for the whole study population improved from 32 per cent to 62 per cent. It was 75 per cent for patients with a local high-grade osteosarcoma of the extremity diagnosed during 1991-2005. This study outlines the improved prognosis of osteosarcoma patients in Finland with modern chemotherapy. The 10-year survival rates are good also in an international scale. Nonetheless, their limb-salvage rate remains inferior to those seen for highly selected patient series. Overall, the centralisation of osteosarcoma treatment would most likely improve both survival and limb-salvage rates even further.

Outcome of singleton pregnancy after assisted reproductive treatment

Singleton pregnancies achieved by means of assisted reproductive treatment (ART) are associated with increased obstetric and neonatal risks in comparison with spontaneously conceived singleton pregnancies. The impact of infertility- and treatment-related factors on these risks is not properly understood. In addition, the psychological effects of infertility and its treatment on the experience of pregnancy have scarcely been studied. Thus, the aim of the present study was to evaluate the importance of infertility- and treatment-related factors on prediction of pregnancy outcome, obstetric and neonatal risks, fear-of-childbirth and pregnancy-related anxiety. The subjects consisted of infertile women who achieved a singleton pregnancy by means of in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). The control groups comprised spontaneously conceiving women with singleton gestations. Early pregnancy outcome was assessed by means of assay of serum human chorionic gonadoptrophin (hCG) in single samples. Other outcome data were collected from patient records, national Health Registers and via prospective questionnaire surveys. Viable pregnancies were associated with significantly higher serum hCG levels 12 days after embryo transfer than non-viable pregnancies. Among singleton pregnancies, aetiological subgroup, treatment type or the number of transferred embryos did not impair the predictive value of single hCG assessment. According to the register-based data, age-, parity- and socioeconomic status- adjusted risks of gestational hypertension, preterm contractions and placenta praevia were more frequent in the ART pregnancies than in the control pregnancies. Significantly higher rates of induction of delivery and Caesarean section occurred in the ART group than in the control group. The risks of preterm birth and low birth weight (LBW) were increased after ART pregnancy. Duration or aetiology of infertility, treatment type (fresh or frozen IVF or ICSI) or rank of treatment did not contribute to the risks of preterm birth or LBW. In addition, the risks of preterm birth and LBW remained elevated in spite of of the number of transferred embryos. Although mean duration of pregnancy was shorter and mean birth weight lower in the ART pregnancies than in the control pregnancies, these differences were hardly of clinical significance. Fear-of-childbirth and pregnancy-related anxiety were equally common to women conceiving by means of ART, or spontaneously. Partnership of five to ten years appeared to be protective as regards severe fear-of-childbirth, whereas long preceding infertility (≥ seven years) had the opposite effect. In conclusion, an early hCG assessment maintained its good predictive value regardless of infertility- or patient-related factors. Further, we did not recognise any infertility- or patient-related factors that would expose infertile women to increased obstetric or neonatal risks. However, a long period of infertility was associated with severe fear-of-childbirth.

Barrett's Esophagus and Associated Adenocarcinoma : studies on pathogenesis, clinical staging and cost-utility of cancer treatment

Esophageal and gastroesophageal junction (GEJ) adenocarcinoma is rapidly increasing disease with a pathophysiology connected to oxidative stress. Exact pre-treatment clinical staging is essential for optimal care of this lethal malignancy. The cost-effectiviness of treatment is increasingly important. We measured oxidative metabolism in the distal and proximal esophagus by myeloperoxidase activity (MPA), glutathione content (GSH), and superoxide dismutase (SOD) in 20 patients operated on with Nissen fundoplication and 9 controls during a 4-year follow-up. Further, we assessed the oxidative damage of DNA by 8-hydroxydeoxyguanosine (8-OHdG) in esophageal samples of subjects (13 Barrett s metaplasia, 6 Barrett s esophagus with high-grade dysplasia, 18 adenocarcinoma of the distal esophagus/GEJ, and 14 normal controls). We estimated the accuracy (42 patients) and preoperative prognostic value (55 patients) of PET compared with computed tomography (CT) and endoscopic ultrasound (EUS) in patients with adenocarcinoma of the esophagus/GEJ. Finally, we clarified the specialty-related costs and the utility of either radical (30 patients) or palliative (23 patients) treatment of esophageal/GEJ carcinoma by the 15 D health-related quality-of-life (HRQoL) questionnaire and the survival rate. The cost-utility of radical treatment of esophageal/GEJ carcinoma was investigated using a decision tree analysis model comparing radical, palliative, and hypothetical new treatment. We found elevated oxidative stress ( measured by MPA) and decreased antioxidant defense (measured by GSH) after antireflux surgery. This indicates that antireflux surgery is not a perfect solution for oxidative stress of the esophageal mucosa. Elevated oxidative stress in turn may partly explain why adenocarcinoma of the distal esophagus is found even after successful fundoplication. In GERD patients, proximal esophageal mucosal anti-oxidative defense seems to be defective before and even years after successful antireflux surgery. In addition, antireflux surgery apparently does not change the level of oxidative stress in the proximal esophagus, suggesting that defective mucosal anti-oxidative capacity plays a role in development of oxidative damage to the esophageal mucosa in GERD. In the malignant transformation of Barrett s esophagus an important component appears to be oxidative stress. DNA damage may be mediated by 8-OHdG, which we found to be increased in Barrett s epithelium and in high-grade dysplasia as well as in adenocarcinoma of the esophagus/GEJ compared with controls. The entire esophagus of Barrett s patients suffers from increased oxidative stress ( measured by 8-OhdG). PET is a useful tool in the staging and prognostication of adenocarcinoma of the esophagus/GEJ detecting organ metastases better than CT, although its accuracy in staging of paratumoral and distant lymph nodes is limited. Radical surgery for esophageal/GEJ carcinoma provides the greatest benefit in terms of survival, and its cost-utility appears to be the best of currently available treatments.

Mid-urethral slings for treatment of stress urinary incontinence : long- and mid-term follow-up

Urinary incontinence is a common problem, affecting one third of the women at least at some time during their lives. The prevalence of urinary incontinence increases with advancing age, and the everyday impact of incontinence on women and on health services is enormous. Urinary incontinence is usually divided into three different subtypes, of which stress urinary incontinence (SUI) is the most common. Surgical treatment is often needed to cure SUI, and modern mid-urethral sling procedures give the possibility to cure this condition with a low risk of adverse events, a problem often associated with the so-called traditional incontinence operations. Life expectancy among women in Western countries has grown beyond 80 years of age. Long-term efficacy of treatment options for urinary incontinence therefore becomes an important issue in a world with limited eco-nomic resources. The purpose of the present study was to prospectively evaluate the long-term efficacy and safety of the first minimally invasive mid-urethral tape procedure, the Tension-free Vaginal Tape (TVT) procedure. The long-term (5-year) follow-up results of the TVT procedure as a repeat operation af-ter an unsuccessful mid-urethral tape operation were studied and the reasons for failure of the first operation were analyzed. Another purpose was to compare the original TVT procedure with a newer modification, the Tension-free Vaginal Tape Obturator (TVT-O) procedure within a multi-centre, randomized context in order to find out possible differences between these procedures re-garding efficacy and complications and the effects on symptoms of urgency. The first study of the present thesis is a prospective, Nordic, three-centre follow-up study of 90 women suffering from SUI, who were treated by means of the TVT procedure. The mean follow-up time was more than eleven years, and the study is the first to be published in connection with more than ten years of follow-up. The second study is a retrospective analysis of 26 women who were treated with a repeat TVT procedure after an unsuccessful primary mid-urethral tape procedure. The third and fourth studies concern 273 women in seven centres in Finland who were ran-domly assigned to the TVT and TVT-O procedures, the 3-year follow-up results of which are pre-sented in this thesis. After eleven years of follow-up, 90% of the women had a negative cough stress test result and a negative 24-h pad test result. The subjective cure rate measured as the women s global impression of cure was 77%, the rate of improvement 20%, and only 3% thought that the treatment had failed. No late-onset adverse effects were found. The repeat TVT procedure was successful in 75% of the cases when women who were cured and women who were significantly improved were included. The reasons for failure of the first operation could be separated into four different groups: tape material-related, operation technique-related, concomitant illness-related and a group with no identifiable reason. There were no intra-operative complications during the repeat operation. In the randomized trial comparing the TVT with the TVT-O procedure a cough stress test results were negative in 94.6% and 89.5% of the women in the two groups, respectively, after a 3-year follow-up period. There were no statistical differences in the cure rate or the rate of complications be-tween the two procedures. Symptoms of urgency were analyzed more closely and the main finding was that the prevalence of urgency symptoms decreased significantly after both mid-urethral sling procedures. The TVT operation was found to be an effective and safe procedure even after eleven years of follow-up. Long-term follow-up after a repeat TVT procedure revealed that the TVT procedure can well be considered after an unsuccessful mid-urethra tape procedure, because 75% of the patients showed significantly improvement of their incontinence. The TVT and TVT-O procedures showed no statistically significant differences in efficacy and rate of complications after three years of follow-up. In most cases these procedures alleviate preoperative symptoms of urgency and the risk of developing de novo urgency is low.

Neurochemical regulation of auditory information processing studied with EEG/MEG: application to schizophrenia

Cognitive impairments of attention, memory and executive functions are a fundamental feature of the pathophysiology of schizophrenia. The neurophysiological and neurochemical changes in the auditory cortex are shown to underlie cognitive impairmentsin schizophrenia patients. Functional state of the neural substrate of auditory information processing could be objectively and non-invasively probed with auditory event-related potentials (ERPs) and event- related fields (ERFs). In the current work, we explored the neurochemical effect on the neural origins of auditory information processing in relation to schizophrenia. By means of ERPs/ERFs we aimed to determine how neural substrates of auditory information processing are modulated by antipsychotic medication in schizophrenia spectrum patients (Studies I, II) and by neuropharmacological challenges in healthy human subjects (Studies III, IV). First, with auditory ERPs we investigated the effects of olanzapine (Study I) and risperidone (Study II) in a group of patients with schizophrenia spectrum disorders. After 2 and 4 weeks of treatment, olanzapine has no significant effects on mismatch negativity(MMN) and P300, which, as it has been suggested, respectively reflect preattentive and attention-dependent information processing. After 2 weeks of treatment, risperidone has no significant effect on P300, however risperidone reduces P200 amplitude. This latter effect of risperidone on neural resources responsible for P200 generation could be partly explained through the action of dopamine. Subsequently, we used simultaneous EEG/MEG to investigate the effects of memantine (Study III) and methylphenidate (Study IV) in healthy subjects. We found that memantine modulates MMN response without changing other ERP components. This could be interpreted as being due to the possible influence of memantine through the NMDA receptors on auditory change- detection mechanism, with processing of auditory stimuli remaining otherwise unchanged. Further, we found that methylphenidate does not modulate the MMN response. This finding could indicate no association between catecholaminergic activities and electrophysiological measures of preattentive auditory discrimination processes reflected in the MMN. However, methylphenidate decreases the P200 amplitudes. This could be interpreted as a modulation of auditory information processing reflected in P200 by dopaminergic and noradrenergic systems. Taken together, our set of studies indicates a complex pattern of neurochemical influences produced by the antipsychotic drugs in the neural substrate of auditory information processing in patients with schizophrenia spectrum disorders and by the pharmacological challenges in healthy subjects studied with ERPs and ERFs.

Routine Measurement of Health-Related Quality of Life in Assessing Cost-Effectiveness in Secondary Health Care

Aims: The aims of this study were 1) to identify and describe health economic studies that have used quality-adjusted life years (QALYs) based on actual measurements of patients' health-related quality of life (HRQoL); 2) to test the feasibility of routine collection of health-related quality of life (HRQoL) data as an indicator of effectiveness of secondary health care; and 3) to establish and compare the cost-utility of three large-volume surgical procedures in a real-world setting in the Helsinki University Central Hospital, a large referral hospital providing secondary and tertiary health-care services for a population of approximately 1.4 million. Patients and methods: So as to identify studies that have used QALYs as an outcome measure, a systematic search of the literature was performed using the Medline, Embase, CINAHL, SCI and Cochrane Library electronic databases. Initial screening of the identified articles involved two reviewers independently reading the abstracts; the full-text articles were also evaluated independently by two reviewers, with a third reviewer used in cases where the two reviewers could not agree a consensus on which articles should be included. The feasibility of routinely evaluating the cost-effectiveness of secondary health care was tested by setting up a system for collecting HRQoL data on approximately 4 900 patients' HRQoL before and after operative treatments performed in the hospital. The HRQoL data used as an indicator of treatment effectiveness was combined with diagnostic and financial indicators routinely collected in the hospital. To compare the cost-effectiveness of three surgical interventions, 712 patients admitted for routine operative treatment completed the 15D HRQoL questionnaire before and also 3-12 months after the operation. QALYs were calculated using the obtained utility data and expected remaining life years of the patients. Direct hospital costs were obtained from the clinical patient administration database of the hospital and a cost-utility analysis was performed from the perspective of the provider of secondary health care services. Main results: The systematic review (Study I) showed that although QALYs gained are considered an important measure of the effectiveness of health care, the number of studies in which QALYs are based on actual measurements of patients' HRQoL is still fairly limited. Of the reviewed full-text articles, only 70 reported QALYs based on actual before after measurements using a valid HRQoL instrument. Collection of simple cost-effectiveness data in secondary health care is feasible and could easily be expanded and performed on a routine basis (Study II). It allows meaningful comparisons between various treatments and provides a means for allocating limited health care resources. The cost per QALY gained was 2 770 for cervical operations and 1 740 for lumbar operations. In cases where surgery was delayed the cost per QALY was doubled (Study III). The cost per QALY ranges between subgroups in cataract surgery (Study IV). The cost per QALY gained was 5 130 for patients having both eyes operated on and 8 210 for patients with only one eye operated on during the 6-month follow-up. In patients whose first eye had been operated on previous to the study period, the mean HRQoL deteriorated after surgery, thus precluding the establishment of the cost per QALY. In arthroplasty patients (Study V) the mean cost per QALY gained in a one-year period was 6 710 for primary hip replacement, 52 270 for revision hip replacement, and 14 000 for primary knee replacement. Conclusions: Although the importance of cost-utility analyses has during recent years been stressed, there are only a limited number of studies in which the evaluation is based on patients own assessment of the treatment effectiveness. Most of the cost-effectiveness and cost-utility analyses are based on modeling that employs expert opinion regarding the outcome of treatment, not on patient-derived assessments. Routine collection of effectiveness information from patients entering treatment in secondary health care turned out to be easy enough and did not, for instance, require additional personnel on the wards in which the study was executed. The mean patient response rate was more than 70 %, suggesting that patients were happy to participate and appreciated the fact that the hospital showed an interest in their well-being even after the actual treatment episode had ended. Spinal surgery leads to a statistically significant and clinically important improvement in HRQoL. The cost per QALY gained was reasonable, at less than half of that observed for instance for hip replacement surgery. However, prolonged waiting for an operation approximately doubled the cost per QALY gained from the surgical intervention. The mean utility gain following routine cataract surgery in a real world setting was relatively small and confined mostly to patients who had had both eyes operated on. The cost of cataract surgery per QALY gained was higher than previously reported and was associated with considerable degree of uncertainty. Hip and knee replacement both improve HRQoL. The cost per QALY gained from knee replacement is two-fold compared to hip replacement. Cost-utility results from the three studied specialties showed that there is great variation in the cost-utility of surgical interventions performed in a real-world setting even when only common, widely accepted interventions are considered. However, the cost per QALY of all the studied interventions, except for revision hip arthroplasty, was well below 50 000, this figure being sometimes cited in the literature as a threshold level for the cost-effectiveness of an intervention. Based on the present study it may be concluded that routine evaluation of the cost-utility of secondary health care is feasible and produces information essential for a rational and balanced allocation of scarce health care resources.

Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.

Hormone therapy in elderly women; a comparative study with alendronate of the effects on bone, cardiovascular risk factors, periodontal conditions and quality of life

Thirty percent of 70-year-old women have osteoporosis; after age of 80 its prevalence is up to 70%. Postmenopausal women with osteoporosis seem to be at an increased risk for cardiovascular events, and deterioration of oral health, as shown by attachment loss of teeth, which is proportional to the severity of osteoporosis. Osteoporosis can be treated with many different medication, e.g. estrogen and alendronate. We randomized 90 elderly osteoporotic women (65-80 years of age) to receive hormone therapy (HT)(2mg E2+NETA), 10mg alendronate, and their combination for two years and compared their effects on bone mineral density (BMD) and turnover, two surrogate markers of the risk of cardiovascular diseases, C-reactive protein (CRP) and E-selectin, as well as oral health. The effect of HT on health-related quality of life (HRQoL) was studied in the population-based cohort of 1663 postmenopausal women (mean age 68 yr) (585 estrogen users and 1078 non-users). BMD was measured with dual-energy X-ray absorptiometry (DXA) at 0, 12 and 24 months. Urinary N-telopeptide (NTX) of type I collagen, a marker of bone resorption, and serum aminoterminal propeptide of human type I procollagen (PINP), a marker of bone formation, were measured every six months of treatment. Serum CRP and E-selectin, were measured at 0, 6, and 12 months. Dental, and periodontal conditions, and gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 levels were studied to evaluate the oral health status and for the mouth symptoms a structured questionnaire was used. The HRQoL was measured with 15D questionnaire. Lumbar spine BMD increased similarly in all treatment groups (6.8-8.4% and 9.1-11.2%). Only HT increased femoral neck BMD at both 12 (4.9%) and 24 months (5.8%), at the latter time point the HT group differed significantly from the other groups. HT reduced bone marker levels of NTX and PINP significantly less than other two groups.Oral HT significantly increased serum CRP level by 76.5% at 6 and by 47.1% (NS) at 12 months, and decreased serum E-selectin level by 24.3% and 30.0%. Alendronate had no effect on these surrogate markers. Alendronate caused a decrease in the resting salivary flow rate and tended to increase GCF MMP-8 levels. Otherwise, there was no effect on the parameters of oral health. HT improved the HRQoL of elderly women significantly on the dimensions of usual activities, vitality and sexual activity, but the overall improvement in HRQoL was neither statistically significant nor clinically important. In conclusion, bisphosphonates might be the first option to start the treatment of postmenopausal osteoporosis in the old age.

Venous thromboembolism during pregnancy and the impact of thrombophilia in pregnancy complications

Venous thromboembolism (VTE) is the greatest single cause of maternal mortality in pregnant women in developed countries. Pregnancy is a hypercoagulable state and brings about an enhanced risk of deep venous thrombosis (DVT) in otherwise healthy women. Traditionally, unfractionated heparin (UFH) has been used for treatment of DVT during pregnancy. We showed in our observational study that low molecular weight heparin (LMWH) is as effective and safe as UFH in the treatment of DVT during pregnancy. Although DVT during pregnancy is often massive, increasing the risk of developing long-term consequences, namely post-thrombotic syndrome (PTS), only 11% of all patients had confirmed PTS 3 4 years after DVT. In our studies the prevalence of PTS was not dependent on treatment (UFH vs LMWH). Low molecular weight heparin is more easily administered, few laboratory controls are required and the hospital stay is shorter, factors that lower the costs of treatment. Cervical insufficiency is defined as repeated very preterm delivery during the second or early third trimester. Infection is a well-known risk factor of preterm delivery. We found overpresentation of thrombophilic mutations (FV Leiden, prothrombin G20210A)among 42 patients with cervical insufficiency compared with controls (OR 6.7, CI 2.7 18.4). Thus, thrombophilia might be a risk factor of cervical insufficiency possibly explained by interaction of coagulation and inflammation processes. The presence of antiphospholipid (aPL) antibodies increases the risk for recurrent miscarriage (RM). Annexins are proteins which all bind to anionic phospholipids (PLs) preventing clotting on vascular phospholipid surfaces. Plasma concentrations of circulating annexin IV and V were investigated in 77 pregnancies at the beginning of pregnancy among women with a history of RM, and in connection to their aPL antibody status. Control group consisted unselected pregnant patients (n=25) without history of adverse pregnancy outcome. Plasma levels of annexin V were significantly higher at the beginning (≤5th week) of pregnancy in women with aPL antibodies compared with those without aPL antibodies (P=0.03). Levels of circulating annexin V were also higher at the 6th (P= 0.01) and 8th week of pregnancy in subjects with aPL antibodies (P=0.01). Results support the hypothesis that aPL could displace annexin from anionic phospholipid surfaces of syncytiotrophoblasts (STBs) and may exert procoagulant activities on the surfaces of STBs Recurrent miscarriage (RM) has been suggested to be caused by mutations in genes coding for various coagulation factors resulting in thrombophilia. In the last study of my thesis were investigated the prevalence of thrombomodulin (TM) and endothelial protein C receptor polymorphism EPCR among 40 couples and six women suffering RM. This study showed that mutations in the TM or EPCR genes are not a major cause of RM in Finnish patients.

Hemodynamics and outcome of septic shock

Septic shock is a common killer in intensive care units (ICU). The most crucial issue concerning the outcome is the early and aggressive start of treatment aimed at normalization of hemodynamics and the early start of antibiotics during the very first hours. The optimal targets of hemodynamic treatment, or impact of hemodynamic treatment on survival after first resuscitation period are less known. The objective of this study was to evaluate different aspects of the hemodynamic pattern in septic shock with special attention to prediction of outcome. In particular components of early treatment and monitoring in the ICU were assessed. A total of 401 patients, 218 with septic shock and 192 with severe sepsis or septic shock were included in the study. The patients were treated in 24 Finnish ICUs during 1999-2005. 295 of the patients were included in the Finnish national epidemiologic Finnsepsis study. We found that the most important hemodynamic variables concerning the outcome were the mean arterial pressures (MAP) and lactate during the first six hours in ICU and the MAP and mixed venous oxygen saturation (SvO2) under 70% during first 48 hours. The MAP levels under 65 mmHg and SvO2 below 70% were the best predictive thresholds. Also the high central venous pressure (CVP) correlated to adverse outcome. We assessed the correlation and agreement of SvO2 and mean central venous oxygen saturation (ScvO2) in septic shock during first day in ICU. The mean SvO2 was below ScvO2 during early sepsis. Bias of difference was 4.2% (95% limits of agreement 8.1% to 16.5%) by Bland-Altman analysis. The difference between saturation values correlated significantly to cardiac index and oxygen delivery. Thus, the ScvO2 can not be used as a substitute of SvO2 in hemodynamic monitoring in ICU. Several biomarkers have been investigated for their ability to help in diagnosis or outcome prediction in sepsis. We assessed the predictive value of N-terminal pro brain natriuretic peptide (NT-proBNP) on mortality in severe sepsis or septic shock. The NT-proBNP levels were significantly higher in hospital nonsurvivors. The NT-proBNP 72 hrs after inclusion was independent predictor of hospital mortality. The acute cardiac load contributed to NTproBNP values at admission, but renal failure was the main confounding factor later. The accuracy of NT-proBNP, however, was not sufficient for clinical decision-making concerning the outcome prediction. The delays in start of treatment are associated to poorer prognosis in sepsis. We assessed how the early treatment guidelines were adopted, and what was the impact of early treatment on mortality in septic shock in Finland. We found that the early treatment was not optimal in Finnish hospitals and this reflected to mortality. A delayed initiation of antimicrobial agents was especially associated with unfavorable outcome.

Individual variation in in vitro efficacy of antiplatelet medication - aspirin and clopidogrel

Antiplatelet medication is known to decrease adverse effects in patients with atherothrombotic disease. However, despite ongoing antiplatelet medication considerable number of patients suffer from atherothrombotic events. The aims of the study were 1) to evaluate the individual variability in platelet functions and compare the usability of different methods in detecting it, 2) to assess variability in efficacy of antiplatelet medication with aspirin (acetylsalicylic acid) or the combination of aspirin and clopidogrel and 3) to investigate the main genetic and clinical variables as well as potential underlying mechanisms of variability in efficacy of antiplatelet medication. In comparisons of different platelet function tests in 19 healthy individuals PFA-100® correlated with traditional methods of measuring platelet function and was thus considered appropriate for testing individual variability in platelet activity. Efficacy of ongoing 100mg aspirin daily was studied in 101 patients with coronary artery disease (CAD). Aspirin response was measured with arachidonic acid (AA)-induced platelet aggregation, which reflects cyclo-oxygenase (COX)-1 dependent thromboxane (Tx) A2 formation, and PFA-100®, which evaluates platelet activation under high shear stress in the presence of collagen and epinephrine. Five percent of patients failed to show inhibition of AA-aggregation and 21% of patients had normal PFA-100® results despite aspirin and were thus considered non-responders to aspirin. Interestingly, the two methods of assessing aspirin efficacy, platelet aggregation and PFA-100®, detected different populations as being aspirin non-responders. It could be postulated that PFA-100® actually measures enhanced platelet function, which is not directly associated with TxA2 inhibition exerted by aspirin. Clopidogrel efficacy was assessed in 50 patients who received a 300mg loading dose of clopidogrel 2.5 h prior to percutaneous coronary intervention (PCI) and in 51 patients who were given a loading dose of 300mg combined with a five day treatment of 75mg clopidogrel daily mimicking ongoing treatment. Clopidogrel response was assessed with ADP-induced aggregations, due to its mechanism of action as an inhibitor of ADP-induced activation. When patients received only a loading dose of clopidogrel prior to PCI, 40% did not gain measurable inhibition of their ADP-induced platelet activity (inhibition of 10% or less). Prolongation of treatment so that all patients had reached a plateau of inhibition exerted by clopidogrel, decreased the incidence of non-responders to 20%. Polymorphisms of COX-1 and GP VI, as well as diabetes and female gender, were associated with decreased in vitro aspirin efficacy. Diabetes also impaired the in vitro efficacy of short-term clopidogrel. Decreased response to clopidogrel was associated with limited inhibition by ARMX, an antagonist of P2Y12-receptor, suggesting the reason for clopidogrel resistance to be receptor-dependent. Conclusions: Considerable numbers of CAD patients were non-responders either to aspirin, clopidogrel or both. In the future, platelet function tests may be helpful to individually select effective and safe antiplatelet medication for these patients.

The role of mast cells in ischemic and hemorrhagic brain injury

Stroke, ischemic or hemorrhagic, belongs among the foremost causes of death and disability worldwide. Massive brain swelling is the leading cause of death in large hemispheric strokes and is only modestly alleviated by available treatment. Thrombolysis with tissue plasminogen activator (TPA) is the only approved therapy in acute ischemic stroke, but fear of TPA-mediated hemorrhage is often a reason for withholding this otherwise beneficial treatment. In addition, recanalization of the occluded artery (spontaneously or with thrombolysis) may cause reperfusion injury by promoting brain edema, hemorrhage, and inflammatory cell infiltration. A dominant event underlying these phenomena seems to be disruption of the blood-brain barrier (BBB). In contrast to ischemic stroke, no widely approved clinical therapy exists for intracerebral hemorrhage (ICH), which is associated with poor outcome mainly due to the mass effect of enlarging hematoma and associated brain swelling. Mast cells (MCs) are perivascularly located resident inflammatory cells which contain potent vasoactive, proteolytic, and fibrinolytic substances in their cytoplasmic granules. Experiments from our laboratory showed MC density and their state of granulation to be altered early following focal transient cerebral ischemia, and degranulating MCs were associated with perivascular edema and hemorrhage. (I) Pharmacological MC stabilization led to significantly reduced ischemic brain swelling (40%) and BBB leakage (50%), whereas pharmacological MC degranulation raised these by 90% and 50%, respectively. Pharmacological MC stabilization also revealed a 40% reduction in neutrophil infiltration. Moreover, genetic MC deficiency was associated with an almost 60% reduction in brain swelling, 50% reduction in BBB leakage, and 50% less neutrophil infiltration, compared with controls. (II) TPA induced MC degranulation in vitro. In vivo experiments with post-ischemic TPA administration demonstrated 70- to 100-fold increases in hemorrhage formation (HF) compared with controls HF. HF was significantly reduced by pharmacological MC stabilization at 3 (95%), 6 (75%), and 24 hours (95%) of follow-up. Genetic MC deficiency again supported the role of MCs, leading to 90% reduction in HF at 6 and 24 hours. Pharmacological MC stabilization and genetic MC deficiency were also associated with significant reduction in brain swelling and in neutrophil infiltration. Importantly, these effects translated into a significantly better neurological outcome and lower mortality after 24 hours. (III) Finally, in ICH experiments, pharmacological MC stabilization resulted in significantly less brain swelling, diminished growth in hematoma volume, better neurological scores, and decreased mortality. Pharmacological MC degranulation produced the opposite effects. Genetic MC deficiency revealed a beneficial effect similar to that found with pharmacological MC stabilization. In sum, the role of MCs in these clinically relevant scenarios is supported by a series of experiments performed both in vitro and in vivo. That not only genetic MC deficiency but also drugs targeting MCs could modulate these parameters (translated into better outcome and decreased mortality), suggests a potential therapeutic approach in a number of highly prevalent cerebral insults in which extensive tissue injury is followed by dangerous brain swelling and inflammatory cell infiltration. Furthermore, these experiments could hint at a novel therapy to improve the safety of thrombolytics, and a potential cellular target for those seeking novel forms of treatment for ICH.