Epidemiology, treatment and outcome of Staphylococcus aureus bacteremia and endocarditis

Staphylococcus aureus is the second most common bloodstream isolate both in community- and hospital-acquired bacteremias. The clinical course of S. aureus bacteremia (SAB) is determined by its complications, particularly by the development of deep infections and thromboembolic events. Despite the progress of antimicrobial therapy, SAB is still associated with high mortality. However, injection drug users (IDUs) tend to have fewer complications and better prognosis than nonaddicts, especially in endocarditis. The present study was undertaken to investigate epidemiology, treatment and outcome of S. aureus bacteremia and endocarditis in Finland. In particular, differences in bacterial strains and their virulence factors, and host immune responses were compared between IDUs and nonaddicts. In Finland, 5045 SAB cases during 1995-2001 were included using the National Infectious Disease Register maintained by National Public Health Institute. The annual incidence of SAB increased, especially in elderly. While the increase in incidence may partly be explained by better reporting, it most likely reflects a growing population at risk, affected by such factors as age and/or severe comorbidity. Nosocomial infections accounted for 51% of cases, with no change in their proportion during the study period. The 28-day mortality was 17% and remained unchanged over time. A total of 381 patients with SAB were randomized to receive either standard antibiotic treatment or levofloxacin added to standard treatment. Levofloxacin combination therapy did not decrease the mortality, lower the incidence of deep infections, nor did it speed up the recovery during 3 months follow-up. However, patients with a deep infection appeared to benefit from combination therapy with rifampicin, as suggested also by experimental data. Deep infections were found in 84% of SAB patients within one week after randomization, and they appeared to be more common than previously reported. Endocarditis was observed in 74 of 430 patients (17%) with SAB, of whom 20 were IDUs and 54 nonaddicts. Right-sided involvement was diagnosed in 60% of addicts whereas 93% of nonaddicts had left-sided endocarditis. Unexpectedly, IDUs showed extracardiac deep infections, thromboembolic events and severe sepsis with the same frequency as nonaddicts. The prognosis of endocarditis was better among addicts due to their younger age and lack of underlying diseases in agreement with earlier reports. In total, all 44 IDUs with SAB were included and 20 of them had endocarditis. An equal number of nonaddicts with SAB were chosen as group matched controls. Serological tests were not helpful in identifying patients with a deep infection. No individual S. aureus strain dominated in endocarditis among addicts. Characterization of the virulence factors of bacterial strains did not reveal any significant differences in IDUs and nonaddicts.

Microsurgical Aneurysm Model in Rats and Mice: Development of Endovascular Treatment and Optimization of Magnetic Resonance Imaging

The rupture of a cerebral artery aneurysm causes a devastating subarachnoid hemorrhage (SAH), with a mortality of almost 50% during the first month. Each year, 8-11/100 000 people suffer from aneurysmal SAH in Western countries, but the number is twice as high in Finland and Japan. The disease is most common among those of working age, the mean age at rupture being 50-55 years. Unruptured cerebral aneurysms are found in 2-6% of the population, but knowledge about the true risk of rupture is limited. The vast majority of aneurysms should be considered rupture-prone, and treatment for these patients is warranted. Both unruptured and ruptured aneurysms can be treated by either microsurgical clipping or endovascular embolization. In a standard microsurgical procedure, the neck of the aneurysm is closed by a metal clip, sealing off the aneurysm from the circulation. Endovascular embolization is performed by packing the aneurysm from the inside of the vessel lumen with detachable platinum coils. Coiling is associated with slightly lower morbidity and mortality than microsurgery, but the long-term results of microsurgically treated aneurysms are better. Endovascular treatment methods are constantly being developed further in order to achieve better long-term results. New coils and novel embolic agents need to be tested in a variety of animal models before they can be used in humans. In this study, we developed an experimental rat aneurysm model and showed its suitability for testing endovascular devices. We optimized noninvasive MRI sequences at 4.7 Tesla for follow-up of coiled experimental aneurysms and for volumetric measurement of aneurysm neck remnants. We used this model to compare platinum coils with polyglycolic-polylactic acid (PGLA) -coated coils, and showed the benefits of the latter in this model. The experimental aneurysm model and the imaging methods also gave insight into the mechanisms involved in aneurysm formation, and the model can be used in the development of novel imaging techniques. This model is affordable, easily reproducible, reliable, and suitable for MRI follow-up. It is also suitable for endovascular treatment, and it evades spontaneous occlusion.

Combination antiretroviral therapy -associated lipodystrophy : insights into pathogenesis and treatment

Introduction: Combination antiretroviral therapy (cART) has decreased morbidity and mortality of individuals infected with human immunodeficiency virus type 1 (HIV-1). Its use, however, is associated with adverse effects which increase the patients risk of conditions such as diabetes and coronary heart disease. Perhaps the most stigmatizing side effect is lipodystrophy, i.e., the loss of subcutaneous adipose tissue (SAT) in the face, limbs and trunk while fat accumulates intra-abdominally and dorsocervically. The pathogenesis of cART-associated lipodystrophy is obscure. Nucleoside reverse transcriptase inhibitors (NRTI) have been implicated to cause lipoatrophy via mitochondrial toxicity. There is no known effective treatment for cART-associated lipodystrophy during unchanged antiretroviral regimen in humans, but in vitro data have shown uridine to abrogate NRTI-induced toxicity in adipocytes. Aims: To investigate whether i) cART or lipodystrophy associated with its use affect arterial stiffness; ii) lipoatrophic SAT is inflamed compared to non-lipoatrophic SAT; iii) abdominal SAT from patients with compared to those without cART-associated lipoatrophy differs with respect to mitochondrial DNA (mtDNA) content, adipose tissue inflammation and gene expression, and if NRTIs stavudine and zidovudine are associated with different degree of changes; iv) lipoatrophic abdominal SAT differs from preserved dorsocervical SAT with respect to mtDNA content, adipose tissue inflammation and gene expression in patients with cART-associated lipodystrophy and v) whether uridine can revert lipoatrophy and the associated metabolic disturbances in patients on stavudine or zidovudine based cART. Subjects and methods: 64 cART-treated patients with (n=45) and without lipodystrophy/-atrophy (n=19) were compared cross-sectionally. A marker of arterial stiffness, heart rate corrected augmentation index (AgIHR), was measured by pulse wave analysis. Body composition was measured by magnetic resonance imaging and dual-energy X-ray absorptiometry, and liver fat content by proton magnetic resonance spectroscopy. Gene expression and mtDNA content in SAT were assessed by real-time polymerase chain reaction and microarray. Adipose tissue composition and inflammation were assessed by histology and immunohistochemistry. Dorsocervical and abdominal SAT were studied. The efficacy and safety of uridine for the treatment of cART-associated lipoatrophy were evaluated in a randomized, double-blind, placebo-controlled 3-month trial in 20 lipoatrophic cART-treated patients. Results: Duration of antiretroviral treatment and cumulative exposure to NRTIs and protease inhibitors, but not the presence of cART-associated lipodystrophy, predicted AgIHR independent of age and blood pressure. Gene expression of inflammatory markers was increased in SAT of lipodystrophic as compared to non-lipodystrophic patients. Expression of genes involved in adipogenesis, triglyceride synthesis and glucose disposal was lower and of those involved in mitochondrial biogenesis, apoptosis and oxidative stress higher in SAT of patients with than without cART-associated lipoatrophy. Most changes were more pronounced in stavudine-treated than in zidovudine-treated individuals. Lipoatrophic SAT had lower mtDNA than SAT of non-lipoatrophic patients. Expression of inflammatory genes was lower in dorsocervical than in abdominal SAT. Neither depot had characteristics of brown adipose tissue. Despite being spared from lipoatrophy, dorsocervical SAT of lipodystrophic patients had lower mtDNA than the phenotypically similar corresponding depot of non-lipodystrophic patients. The greatest difference in gene expression between dorsocervical and abdominal SAT, irrespective of lipodystrophy status, was in expression of homeobox genes that regulate transcription and regionalization of organs during embryonal development. Uridine increased limb fat and its proportion of total fat, but had no effect on liver fat content and markers of insulin resistance. Conclusions: Long-term cART is associated with increased arterial stiffness and, thus, with higher cardiovascular risk. Lipoatrophic abdominal SAT is characterized by inflammation, apoptosis and mtDNA depletion. As mtDNA is depleted even in non-lipoatrophic dorsocervical SAT, lipoatrophy is unlikely to be caused directly by mtDNA depletion. Preserved dorsocervical SAT of patients with cART-associated lipodystrophy is less inflamed than their lipoatrophic abdominal SAT, and does not resemble brown adipose tissue. The greatest difference in gene expression between dorsocervical and abdominal SAT is in expression of transcriptional regulators, homeobox genes, which might explain the differential susceptibility of these adipose tissue depots to cART-induced toxicity. Uridine is able to increase peripheral SAT in lipoatrophic patients during unchanged cART.

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

Mechanisms of azole resistance and carcinogenic acetaldehyde production in chronic mucosal candidosis

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS1) is an autoimmune disease caused by a loss-of function mutation in the autoregulator gene (AIRE). Patients with APECED suffer from chronic mucocutaneous candidosis (CMC) of the oral cavity and oesophagus often since early childhood. The patients are mainly colonized with Candida albicans and decades of exposure to antifungal agents have lead to the development of clinical and microbiological resistance in the treatment of CMC in the APECED patient population in Finland. A high incidence of oral squamous cell carcinoma is associated with oral CMC lesions in the APECED patients over the age of 25. The overall aim of this study was firstly, to investigate the effect of long-term azole exposure on the metabolism of oral C. albicans isolates from APECED patients with CMC and secondly, to analyse the specific molecular mechanisms that are responsible for these changes. The aim of the first study was to examine C. albicans strains from APECED patients and the level of cross-resistance to miconazole, the recommended topical compound for the treatment of oral candidosis. A total of 16% of the strains had decreased susceptibility to miconazole and all of these isolates had decreased susceptibility to fluconazole. Miconazole MICs also correlated with MICs to voriconazole and posaconazole. A significant positive correlation between the years of miconazole exposure and the MICs to azole antifungal agents was also found. These included azoles the patients had not been exposed to. The aim of our second study was to determine if the APECED patients are continuously colonized with the same C. albicans strains despite extensive antifungal treatment and to gain a deeper insight into the genetic changes leading to azole resistance. The strains were typed using MLST and our results confirmed that all patients were persistently colonized with the same or a genetically related strain despite antifungal treatment between isolations. No epidemic strains were found. mRNA expression was analysed by Northern blotting, protein level by western blotting, and TAC1 and ERG11 genes were sequenced. The main molecular mechanisms resulting in azole resistance were gain-of-function mutations in TAC1 leading to over expression of CDR1 and CDR2, genes linked to azole resistance. Several strains had also developed point mutations in ERG11, another gene linked to azole resistance. In the third study we used gas chromatography to test whether the level of carcinogenic acetaldehyde produced by C. albicans strains isolated from APECED patients were different from the levels produced by strains isolated from healthy controls and oral carcinoma patients. Acetaldehyde is a carcinogenic product of alcohol fermentation and metabolism in microbes associated with cancers of the upper digestive tract. In yeast, acetaldehyde is a by-product of the pyruvate bypass that converts pyruvate into acetyl-CoA during fermentation. Our results showed that strains isolated from APECED patients produced mutagenic levels of acetaldehyde in the presence of glucose (100mM, 18g/l) and the levels produced were significantly higher than those from strains isolated from controls and oral carcinoma patients. All strains in the study, however, were found to produce mutagenic levels of acetaldehyde in the presence of ethanol (11mM). The glucose and ethanol levels used in this study are equivalent to those found in food and beverages and our results highlight the role of dietary sugars and ethanol on carcinogenesis. The aims of our fourth study were to research the effect of growth conditions in the levels of acetaldehyde produced by C. albicans and to gain deeper insight into the role of different genes in the pyruvate-bypass in the production of high acetaldehyde levels. Acetaldehyde production in the presence of glucose increased by 17-fold under moderately hypoxic conditions compared to the levels produced under normoxic conditions. Under moderately hypoxic conditions acetaldehyde levels did not correlate with the expression of ADH1 and ADH2, genes catalyzing the oxidation of ethanol to acetaldehyde, or PDC11, the gene catalyzing the oxidation of pyruvate to acetaldehyde but correlated with the expression of down-stream genes ALD6 and ACS1. Our results highlight a problem where indiscriminate use of azoles may influence azole susceptibility and lead to the development of cross-resistance. Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations that occur in strains may lead to the development of azole-resistant isolates and metabolic changes leading to increased production of carcinogenic acetaldehyde.

Treatment of illicit opioid and γ-hydroxybutyrate overdose by Helsinki emergency medical services

Aims: To examine the characteristics, incidence, treatment and outcome of presumed opioid, γ-hydroxybutyrate (GHB) and γ-butyrolactone (GBL) overdoses involving users of illicit drugs in Helsinki. GHB/GBL were included in this study, despite not being opioids, due to the relative ease with which they can cause potentially fatal respiratory depression. The incidence and time interval of recurrent opioid toxicity after prehospital administration of naloxone, an opioid antagonist, was studied in presumed heroin overdose patients. Naloxone has been reported to have many adverse effects and the effects of naloxone administered during an opioid overdose on the cardiovascular system and catecholamine levels in piglets were studied. Materials and methods: Patients included in these published retrospective studies were from the following time periods: Study I: 1995-2002, II: 1997-2000, III: 1995-2000, V: 2006-2007. Presumed opioid overdose patients were examined in studies I, II and III. GHB/GBL overdoses among injecting drug users was examined in study V. Recurrent opioid toxicity after prehospital naloxone administration in heroin overdose patients was examined in study III. The effects of naloxone (80 μg/kg i.v.) on the cardiovascular system and catecholamine levels administered during morphine overdose (8mg/kg i.v.) and under propofol anesthesia with spontaneous breathing were studied in eight piglets (IV). In this thesis, previously unpublished data on the incidence of opioid overdose between 2001-2007 and comparison of the characteristics of buprenorphine and heroin overdose patients encountered in 1995-2005 are also included. Results: Helsinki Emergency Medical Service (EMS) ambulances were dispatched annually to 34,153- 45,118 calls from 1995 to 2007. Of them, 7-8% were coded as intoxications or overdoses. During this time, 436 patients were treated by the EMS for presumed opioid overdose. The peak incidence of opioid overdoses was in the year 2000 (113 cases), after which they declined to 6-26 cases annually. The annual incidence of buprenorphine related overdoses increased from 4 (4% of opioid overdoses) in the year 2000 to 8 (30% of opioid overdoses) in 2007. The annual number of GHB related overdose patients treated by Helsinki EMS increased from 21 to 73 between 2004-2007. There appeared to be a peak in the incidence of both GHB/GBL and opioid related overdoses on Saturdays. Characteristics of opioid overdose patients The median age of opioid overdose patients was 28 years (22;33, 25- and 75-percentiles), and 84% were male. Buprenorphine overdose patients had more polydrug, such as alcohol and/or benzodiazepines, use in comparison with heroin overdose patients, 70% versus 33%, respectively. Severe respiratory depression was reported less often with buprenorphine overdoses compared to heroin overdoses, in 67.0% versus 85.4%, respectively. Outcome of heroin overdose patients with cardiac arrest Ninety four patients suffered cardiac arrest due to acute drug poisoning/overdose and were thus considered for resuscitation. Resuscitation was attempted in 72 cases. Cardiac arrest was caused by heroin overdose for 19 patients of which three (16%) were discharged alive. Other agents also induced cardiac arrest in 53 patients, of which six (11%) were discharged alive. The arrest was either EMS witnessed or occurring after the emergency call for all survivors of heroin induced cardiac arrest. Characteristics of GHB/GBL overdose patients The records of 100 GHB/GBL related overdose patients from 2006-2007 were retrieved. The median age of GHB/GBL overdose patients encountered on weekend nights was 24 years (22;27, 25- and 75-percentiles) and 49% were male. Polydrug use was reported in 62-80% of the cases. Thirty nine patients were encountered on Friday-Saturday or Saturday-Sunday night between 11 pm-6 am. The remaining sixty one patients were outside this time frame. There was a statistically significant difference between these two groups in history of chronic injecting drug use (33% vs. 59%, respectively, p=0.012). Recurrent heroin toxicity after prehospital naloxone administration Study III included 145 presumed heroin overdose patients. After prehospital care, 84 patients refused further care and were not transported to an Emergency Department (ED). Seventy one (85%) of them were administered naloxone by the EMS. During a 12-h follow up period, none of these patients developed severe recurrent opioid toxicity. The remaining 61 patients were transported to an ED. Prior to transportation, 52 (85%) patients were administered naloxone by the EMS. Fifteen of them were administered naloxone also in the ED and recurrent opioid toxicity was evident either on arrival at the ED or shortly thereafter. Prehospital naloxone was administered either intravenously, intramuscularly (i.m.) or subcutaneously (s.c.). There was a tendency for more frequent recurrent heroin toxicity among the patients with only intravenous administration of prehospital naloxone (13/36) compared with the patients with intramuscular or subcutaneous prehospital naloxone (2/16), p=0.106. The effects of naloxone on the cardiovascular system and catecholamine levels in piglets The administration of morphine to piglets resulted in an obvious respiratory depression, which was reversed by naloxone. Two severely hypoxemic piglets developed cardiac arrest after naloxone administration. In the other six animals, the administration of naloxone did not provoke arrhythmias, cardiac ischemia or visible evidence of pulmonary edema. There was a statistically significant (p=0.012) increase in norepinephrine levels after morphine administration and before naloxone administration: from 1.9 (1.3-2.3) ng/ml at baseline, to 31.7 (8.3-83.0) ng/ml (median, 25 and 75 percentiles parentheses) after morphine administration. After the administration of naloxone, the catecholamine levels continued to increase in only one of the animals. Conclusions: The incidence of buprenorphine related overdoses increased during the study period, but was still lower in comparison to those involving heroin. Injecting drug users have also started to use GHB/GBL. While recreational drug users use GHB/GBL during weekend nights, a GHB/GBL overdose patient encounter during weekdays has a more probable history of injecting drug use. Patients with cardiac arrest after heroin overdose have a poor prognosis. It appears to be safe to leave heroin overdose patients on scene after prehospital treatment with naloxone. Although no statistically significant difference was observed, it seems prudent to administer part of the total naloxone dose s.c. or i.m. to reduce the risk of recurrent respiratory depression. If transported to an ED, an observation period of one to two hours after the last naloxone dose seems adequate. The treating physician must be vigilant, however, due to the high prevalence of polydrug use and high morbidity after non fatal heroin overdose. Furthermore, care should be taken regarding possible chronic disorders and drug rehabilitation should be addressed. In the experimental animal study, two animals developed cardiac arrest after receiving naloxone while in hypoxemia and bradycardia. Further studies are required to assess the effect of naloxone during opioid-induced hypercapnia and hypoxemia in animals addicted to opioids.

Pharmacogenetic Variation at CYP2D6, CYP2C9, and CYP2C19: Population Genetic and Forensic Aspects

Pharmacogenetics deals with genetically determined variation in drug response. In this context, three phase I drug-metabolizing enzymes, CYP2D6, CYP2C9, and CYP2C19, have a central role, affecting the metabolism of about 20-30% of clinically used drugs. Since genes coding for these enzymes in human populations exhibit high genetic polymorphism, they are of major pharmacogenetic importance. The aims of this study were to develop new genotyping methods for CYP2D6, CYP2C9, and CYP2C19 that would cover the most important genetic variants altering the enzyme activity, and, for the first time, to describe the distribution of genetic variation at these loci on global and microgeographic scales. In addition, pharmacogenetics was applied to a postmortem forensic setting to elucidate the role of genetic variation in drug intoxications, focusing mainly on cases related to tricyclic antidepressants, which are commonly involved in fatal drug poisonings in Finland. Genetic variability data were obtained by genotyping new population samples by the methods developed based on PCR and multiplex single-nucleotide primer extension reaction, as well as by collecting data from the literature. Data consisted of 138, 129, and 146 population samples for CYP2D6, CYP2C9, and CYP2C19, respectively. In addition, over 200 postmortem forensic cases were examined with respect to drug and metabolite concentrations and genotypic variation at CYP2D6 and CYP2C19. The distribution of genetic variation within and among human populations was analyzed by descriptive statistics and variance analysis and by correlating the genetic and geographic distances using Mantel tests and spatial autocorrelation. The correlation between phenotypic and genotypic variation in drug metabolism observed in postmortem cases was also analyzed statistically. The genotyping methods developed proved to be informative, technically feasible, and cost-effective. Detailed molecular analysis of CYP2D6 genetic variation in a global survey of human populations revealed that the pattern of variation was similar to those of neutral genomic markers. Most of the CYP2D6 diversity was observed within populations, and the spatial pattern of variation was best described as clinal. On the other hand, genetic variants of CYP2D6, CYP2C9, and CYP2C19 associated with altered enzymatic activity could reach extremely high frequencies in certain geographic regions. Pharmacogenetic variation may also be significantly affected by population-specific demographic histories, as seen within the Finnish population. When pharmacogenetics was applied to a postmortem forensic setting, a correlation between amitriptyline metabolic ratios and genetic variation at CYP2D6 and CYP2C19 was observed in the sample material, even in the presence of confounding factors typical for these cases. In addition, a case of doxepin-related fatal poisoning was shown to be associated with a genetic defect at CYP2D6. Each of the genes studied showed a distinct variation pattern in human populations and high frequencies of altered activity variants, which may reflect the neutral evolution and/or selective pressures caused by dietary or environmental exposure. The results are relevant also from the clinical point of view since the genetic variation at CYP2D6, CYP2C9, and CYP2C19 already has a range of clinical applications, e.g. in cancer treatment and oral anticoagulation therapy. This study revealed that pharmacogenetics may also contribute valuable information to the medicolegal investigation of sudden, unexpected deaths.

Prevention of type 2 diabetes with lifestyle intervention - emphasis on dietary composition and identification of high-risk individuals

Type 2 diabetes is an increasing, serious, and costly public health problem. The increase in the prevalence of the disease can mainly be attributed to changing lifestyles leading to physical inactivity, overweight, and obesity. These lifestyle-related risk factors offer also a possibility for preventive interventions. Until recently, proper evidence regarding the prevention of type 2 diabetes has been virtually missing. To be cost-effective, intensive interventions to prevent type 2 diabetes should be directed to people at an increased risk of the disease. The aim of this series of studies was to investigate whether type 2 diabetes can be prevented by lifestyle intervention in high-risk individuals, and to develop a practical method to identify individuals who are at high risk of type 2 diabetes and would benefit from such an intervention. To study the effect of lifestyle intervention on diabetes risk, we recruited 522 volunteer, middle-aged (aged 40 - 64 at baseline), overweight (body mass index > 25 kg/m2) men (n = 172) and women (n = 350) with impaired glucose tolerance to the Diabetes Prevention Study (DPS). The participants were randomly allocated either to the intensive lifestyle intervention group or the control group. The control group received general dietary and exercise advice at baseline, and had annual physician's examination. The participants in the intervention group received, in addition, individualised dietary counselling by a nutritionist. They were also offered circuit-type resistance training sessions and were advised to increase overall physical activity. The intervention goals were to reduce body weight (5% or more reduction from baseline weight), limit dietary fat (< 30% of total energy consumed) and saturated fat (< 10% of total energy consumed), and to increase dietary fibre intake (15 g / 1000 kcal or more) and physical activity (≥ 30 minutes/day). Diabetes status was assessed annually by a repeated 75 g oral glucose tolerance testing. First analysis on end-points was completed after a mean follow-up of 3.2 years, and the intervention phase was terminated after a mean duration of 3.9 years. After that, the study participants continued to visit the study clinics for the annual examinations, for a mean of 3 years. The intervention group showed significantly greater improvement in each intervention goal. After 1 and 3 years, mean weight reductions were 4.5 and 3.5 kg in the intervention group and 1.0 kg and 0.9 kg in the control group. Cardiovascular risk factors improved more in the intervention group. After a mean follow-up of 3.2 years, the risk of diabetes was reduced by 58% in the intervention group compared with the control group. The reduction in the incidence of diabetes was directly associated with achieved lifestyle goals. Furthermore, those who consumed moderate-fat, high-fibre diet achieved the largest weight reduction and, even after adjustment for weight reduction, the lowest diabetes risk during the intervention period. After discontinuation of the counselling, the differences in lifestyle variables between the groups still remained favourable for the intervention group. During the post-intervention follow-up period of 3 years, the risk of diabetes was still 36% lower among the former intervention group participants, compared with the former control group participants. To develop a simple screening tool to identify individuals who are at high risk of type 2 diabetes, follow-up data of two population-based cohorts of 35-64 year old men and women was used. The National FINRISK Study 1987 cohort (model development data) included 4435 subjects, with 182 new drug-treated cases of diabetes identified during ten years, and the FINRISK Study 1992 cohort (model validation data) included 4615 subjects, with 67 new cases of drug-treated diabetes during five years, ascertained using the Social Insurance Institution's Drug register. Baseline age, body mass index, waist circumference, history of antihypertensive drug treatment and high blood glucose, physical activity and daily consumption of fruits, berries or vegetables were selected into the risk score as categorical variables. In the 1987 cohort the optimal cut-off point of the risk score identified 78% of those who got diabetes during the follow-up (= sensitivity of the test) and 77% of those who remained free of diabetes (= specificity of the test). In the 1992 cohort the risk score performed equally well. The final Finnish Diabetes Risk Score (FINDRISC) form includes, in addition to the predictors of the model, a question about family history of diabetes and the age category of over 64 years. When applied to the DPS population, the baseline FINDRISC value was associated with diabetes risk among the control group participants only, indicating that the intensive lifestyle intervention given to the intervention group participants abolished the diabetes risk associated with baseline risk factors. In conclusion, the intensive lifestyle intervention produced long-term beneficial changes in diet, physical activity, body weight, and cardiovascular risk factors, and reduced diabetes risk. Furthermore, the effects of the intervention were sustained after the intervention was discontinued. The FINDRISC proved to be a simple, fast, inexpensive, non-invasive, and reliable tool to identify individuals at high risk of type 2 diabetes. The use of FINDRISC to identify high-risk subjects, followed by lifestyle intervention, provides a feasible scheme in preventing type 2 diabetes, which could be implemented in the primary health care system.

Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Associations of gene polymorphisms and nutrition with calcium homeostasis and bone mineral density : Studies on skeletal nutrigenetics

Heredity explains a major part of the variation in calcium homeostasis and bone strength, and the susceptibility to osteoporosis is polygenetically regulated. Bone phenotype results from the interplay between lifestyle and genes, and several nutritional factors modulate bone health throughout life. Thus, nutrigenetics examining the genetic variation in nutrient intake and homeostatic control is an important research area in the etiology of osteoporosis. Despite continuing progress in the search for candidate genes for osteoporosis, the results thus far have been inconclusive. The main objective of this thesis was to investigate the associations of lactase, vitamin D receptor (VDR), calcium sensing receptor (CaSR) and parathyroid hormone (PTH) gene polymorphisms and lifestyle factors and their interactions with bone health in Finns at varying stages of the skeletal life span. Markers of calcium homeostasis and bone remodelling were measured from blood and urine samples. Bone strength was measured at peripheral and central bone sites. Lifestyle factors were assessed with questionnaires and interviews. Genetic lactase non-persistence (the C/C-13910 genotype) was associated with lower consumption of milk from childhood, predisposing females in particular to inadequate calcium intake. Consumption of low-lactose milk and milk products was shown to decrease the risk for inadequate calcium intake. In young adulthood, bone loss was more common in males than in females. Males with the lactase C/C-13910 genotype may be more susceptible to bone loss than males with the other lactase genotypes, although calcium intake predicts changes in bone mass more than the lactase genotype. The BsmI and FokI polymorphisms of the VDR gene were associated with bone mass in growing adolescents, but the associations weakened with age. In young adults, the A986S polymorphism of the calcium sensing receptor gene was associated with serum ionized calcium concentrations, and the BstBI polymorphism of the parathyroid gene was related to bone strength. The FokI polymorphism and sodium intake showed an interaction effect on urinary calcium excretion. A novel gene-gene interaction between the VDR FokI and PTH BstBI gene polymorphisms was found in the regulation of PTH secretion and urinary calcium excretion. Further research should be carried out with more number of Finns at varying stages of the skeletal life span and more detailed measurements of bone strength. Research should concern mechanisms by which genetic variants affect calcium homeostasis and bone strength, and the role of diet-gene and gene-gene interactions in the pathogenesis of osteoporosis.

F-18 Labelling Synthesis, Radioanalysis and Evaluation of A Dopamine Transporter and A Hypoxia Tracer

Positron emission tomography (PET) is an imaging technique in which radioactive positron-emitting tracers are used to study biochemical and physiological functions in humans and in animal experiments. The use of PET imaging has increased rapidly in recent years, as have special requirements in the fields of neurology and oncology for the development of syntheses for new, more specific and selective radiotracers. Synthesis development and automation are necessary when high amounts of radioactivity are needed for multiple PET studies. In addition, preclinical studies using experimental animal models are necessary for evaluating the suitability of new PET tracers for humans. For purification and analysing the labelled end-product, an effective radioanalytical method combined with an optimal radioactivity detection technique is of great importance. In this study, a fluorine-18 labelling synthesis method for two tracers was developed and optimized, and the usefulness of these tracers for possible prospective human studies was evaluated. N-(3-[18F]fluoropropyl)-2β-carbomethoxy-3β-(4-fluorophenyl)nortropane ([18F]β-CFT-FP) is a candidate PET tracer for the dopamine transporter (DAT), and 1H-1-(3-[18F]fluoro-2-hydroxypropyl)-2-nitroimidazole ([18F]FMISO) is a well-known hypoxia marker for hypoxic but viable cells in tumours. The methodological aim of this thesis was to evaluate the status of thin-layer chromatography (TLC) combined with proper radioactivity detection measurement systems as a radioanalytical method. Three different detection methods of radioactivity were compared: radioactivity scanning, film autoradiography, and digital photostimulated luminescence (PSL) autoradiography. The fluorine-18 labelling synthesis for [18F]β-CFT-FP was developed and carbon-11 labelled [11C]β-CFT-FP was used to study the specificity of β-CFT-FP for the DAT sites in human post-mortem brain slices. These in vitro studies showed that β-CFT-FP binds to the caudate-putamen, an area rich of DAT. The synthesis of fluorine-18 labelled [18F]FMISO was optimized, and the tracer was prepared using an automated system with good and reproducible yields. In preclinical studies, the action of the radiation sensitizer estramustine phosphate on the radiation treatment and uptake of [18F]FMISO was evaluated, with results of great importance for later human studies. The methodological part of this thesis showed that radioTLC is the method of choice when combined with an appropriate radioactivity detection technique. Digital PSL autoradiography proved to be the most appropriate when compared to the radioactivity scanning and film autoradiography methods. The very high sensitivity, good resolution, and wide dynamic range of digital PSL autoradiography are its advantages in detection of β-emitting radiolabelled substances.

Lake sediment research as a part of lake management - case studies and implications from southern Finland

In Finland one of the most important current issues in the environmental management is the quality of surface waters. The increasing social importance of lakes and water systems has generated wide-ranging interest in lake restoration and management, concerning especially lakes suffering from eutrophication, but also from other environmental impacts. Most of the factors deteriorating the water quality in Finnish lakes are connected to human activities. Especially since the 1940's, the intensified farming practices and conduction of sewage waters from scattered settlements, cottages and industry have affected the lakes, which simultaneously have developed in to recreational areas for a growing number of people. Therefore, this study was focused on small lakes, which are human impacted, located close to settlement areas and have a significant value for local population. The aim of this thesis was to obtain information from lake sediment records for on-going lake restoration activities and to prove that a well planned, properly focused lake sediment study is an essential part of the work related to evaluation, target consideration and restoration of Finnish lakes. Altogether 11 lakes were studied. The study of Lake Kaljasjärvi was related to the gradual eutrophication of the lake. In lakes Ormajärvi, Suolijärvi, Lehee, Pyhäjärvi and Iso-Roine the main focus was on sediment mapping, as well as on the long term changes of the sedimentation, which were compared to Lake Pääjärvi. In Lake Hormajärvi the role of different kind of sedimentation environments in the eutrophication development of the lake's two basins were compared. Lake Orijärvi has not been eutrophied, but the ore exploitation and related acid main drainage from the catchment area have influenced the lake drastically and the changes caused by metal load were investigated. The twin lakes Etujärvi and Takajärvi are slightly eutrophied, but also suffer problems associated with the erosion of the substantial peat accumulations covering the fringe areas of the lakes. These peat accumulations are related to Holocene water level changes, which were investigated. The methods used were chosen case-specifically for each lake. In general, acoustic soundings of the lakes, detailed description of the nature of the sediment and determinations of the physical properties of the sediment, such as water content, loss on ignition and magnetic susceptibility were used, as was grain size analysis. A wide set of chemical analyses was also used. Diatom and chrysophycean cyst analyses were applied, and the diatom inferred total phosphorus content was reconstructed. The results of these studies prove, that the ideal lake sediment study, as a part of a lake management project, should be two-phased. In the first phase, thoroughgoing mapping of sedimentation patterns should be carried out by soundings and adequate corings. The actual sampling, based on the preliminary results, must include at least one long core from the main sedimentation basin for the determining the natural background state of the lake. The recent, artificially impacted development of the lake can then be determined by short-core and surface sediment studies. The sampling must be focused on the basis of the sediment mapping again, and it should represent all different sedimentation environments and bottom dynamic zones, considering the inlets and outlets, as well as the effects of possible point loaders of the lake. In practice, the budget of the lake management projects of is usually limited and only the most essential work and analyses can be carried out. The set of chemical and biological analyses and dating methods must therefore been thoroughly considered and adapted to the specific management problem. The results show also, that information obtained from a properly performed sediment study enhances the planning of the restoration, makes possible to define the target of the remediation activities and improves the cost-efficiency of the project.

Environmental change and anthropogenic impact on lake sediments during the Holocene in the Finnish - Karelian inland area

This thesis discusses the prehistoric human disturbance during the Holocene by means of case studies using detailed high-resolution pollen analysis from lake sediment. The four lakes studied are situated between 61o 40' and 61o 50' latitudes in the Finnish Karelian inland area and vary between 2.4 and 28.8 ha in size. The existence of Early Metal Age population was one important question. Another study question concerned the development of grazing, and the relationship between slash-and-burn cultivation and permanent field cultivation. The results were presented as pollen percentages and pollen concentrations (grains cm 3). Accumulation values (grains cm 2 yr 1) were calculated for Lake Nautajärvi and Lake Orijärvi sediment, where the sediment accumulation rate was precisely determined. Sediment properties were determined using loss-on-ignition (LOI) and magnetic susceptibility (k). Dating methods used include both conventional and AMS 14C determinations, paleomagnetic dating and varve choronology. The isolation of Lake Kirjavalampi on the northern shore of Lake Ladoga took place ca. 1460 1300 BC. The long sediment cores from Finland, Lake Kirkkolampi and Lake Orijärvi in southeastern Finland and Lake Nautajärvi in south central Finland all extended back to the Early Holocene and were isolated from the Baltic basin ca. 9600 BC, 8600 BC and 7675 BC, respectively. In the long sediment cores, the expansion of Alnus was visible between 7200 - 6840 BC. The spread of Tilia was dated in Lake Kirkkolampi to 6600 BC, in Lake Orijärvi to 5000 BC and at Lake Nautajärvi to 4600 BC. Picea is present locally in Lake Kirkkolampi from 4340 BC, in Lake Orijärvi from 6520 BC and in Lake Nautajärvi from 3500 BC onwards. The first modifications in the pollen data, apparently connected to anthropogenic impacts, were dated to the beginning of the Early Metal Period, 1880 1600 BC. Anthropogenic activity became clear in all the study sites by the end of the Early Metal Period, between 500 BC AD 300. According to Secale pollen, slash-and-burn cultivation was practised around the eastern study lakes from AD 300 600 onwards, and at the study site in central Finland from AD 880 onwards. The overall human impact, however, remained low in the studied sites until the Late Iron Age. Increasing human activity, including an increase in fire frequency was detected from AD 800 900 onwards in the study sites in eastern Finland. In Lake Kirkkolampi, this included cultivation on permanent fields, but in Lake Orijärvi, permanent field cultivation became visible as late as AD 1220, even when the macrofossil data demonstrated the onset of cultivation on permanent fields as early as the 7th century AD. On the northern shore of Lake Ladoga, local activity became visible from ca. AD 1260 onwards and at Lake Nautajärvi, sediment the local occupation was traceable from 1420 AD onwards. The highest values of Secale pollen were recorded both in Lake Orijärvi and Lake Kirjavalampi between ca. AD 1700 1900, and could be associated with the most intensive period of slash-and-burn from AD 1750 to 1850 in eastern Finland.

Below-ground processes in meadow soil under elevated ozone and carbon dioxide : - Greenhouse gas fluxes, N cycling and microbial communities

This thesis focuses on how elevated CO2 and/or O3 affect the below-ground processes in semi-natural vegetation, with an emphasis on greenhouse gases, N cycling and microbial communities. Meadow mesocosms mimicking lowland hay meadows in Jokioinen, SW Finland, were enclosed in open-top chambers and exposed to ambient and elevated levels of O3 (40-50 ppb) and/or CO2 (+100 ppm) for three consecutive growing season, while chamberless plots were used as chamber controls. Chemical and microbiological analyses as well as laboratory incubations of the mesocosm soils under different treatments were used to study the effects of O3 and/or CO2. Artificially constructed mesocosms were also compared with natural meadows with regards to GHG fluxes and soil characteristics. In addition to research conducted at the ecosystem level (i.e. the mesocosm study), soil microbial communities were also examined in a pot experiment with monocultures of individual species. By comparing mesocosms with similar natural plant assemblage, it was possible to demonstrate that artificial mesocosms simulated natural habitats, even though some differences were found in the CH4 oxidation rate, soil mineral N, and total C and N concentrations in the soil. After three growing seasons of fumigations, the fluxes of N2O, CH4, and CO2 were decreased in the NF+O3 treatment, and the soil NH4+-N and mineral N concentrations were lower in the NF+O3 treatment than in the NF control treatment. The mesocosm soil microbial communities were affected negatively by the NF+O3 treatment, as the total, bacterial, actinobacterial, and fungal PLFA biomasses as well as the fungal:bacterial biomass ratio decreased under elevated O3. In the pot survey, O3 decreased the total, bacterial, actinobacterial, and mycorrhizal PLFA biomasses in the bulk soil and affected the microbial community structure in the rhizosphere of L. pratensis, whereas the bulk soil and rhizosphere of the other monoculture, A. capillaris, remained unaffected by O3. Elevated CO2 caused only minor and insignificant changes in the GHG fluxes, N cycling, and the microbial community structure. In the present study, the below-ground processes were modified after three years of moderate O3 enhancement. A tentative conclusion is that a decrease in N availability may have feedback effects on plant growth and competition and affect the N cycling of the whole meadow ecosystem. Ecosystem level changes occur slowly, and multiplication of the responses might be expected in the long run.