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.

Genetic Variation and Clinical Manifestations in Inflammatory Bowel Disease

Crohn s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are characterised by chronic inflammation of the gastrointestinal tract. IBD prevalence in Finland is approximately 3-4 per 1000 inhabitants with a peak incidence in adolescence. The symptoms of IBD include diarrhoea, abdominal pain, fever, and weight loss. The precise aetiology of IBD is unknown but interplay of environmental risk factors and immunologic changes trigger the disease in a genetically susceptible individual. Twin and family studies have provided strong evidence for genetic factors in IBD susceptibility, and genetic factors may be more prominent in CD than UC. The first CD susceptibility gene was identified in 2001. Three common mutations R702W, G908R, and 1007fs of the CARD15/NOD2 gene are shown to associate independently with CD but the magnitude of association varies between different populations. The present study aimed at identifying mutations and genetic variations in IBD susceptibility and candidate genes. In addition, correlation to phenotype was also assessed. One of the main objectives of this study was to evaluate the role of CARD15 in a Finnish CD cohort. 271 CD patients were studied for the three common mutations and the results showed a lower mutation frequency than in other Caucasian populations. Only 16% of the patients carried one of the three mutations. Ileal location as well as stricturing and penetrating behaviour of the disease were associated with occurrence of the mutations. The whole protein coding region of CARD15 was screened for possible Finnish founder mutations. In addition to several sequence variants, five novel mutations (R38M, W355X, P727L, W907R, and R1019X) were identified in five patients. Functional consequences of these novel variants were studied in vitro, and these studies demonstrated a profound impairment of MDP response. Investigation of CARD15 mutation frequency in healthy people across three continents showed a large geographic fluctuation. No simple correlation between mutation frequency and disease incidence was seen in populations studied. The occurrence of double mutant carriers in healthy controls suggested that the penetrance of risk alleles is low. Other main objectives aimed at identifying other genetic variations that are involved in the susceptibility to IBD. We investigated the most plausible IBD candidate genes including TRAF6, SLC22A4, SLC22A5, DLG5, TLR4, TNFRSF1A, ABCB1/MDR1, IL23R, and ATG16L1. The marker for a chromosome 5 risk haplotype and the rare HLA-DRB1*0103 allele were also studied. The study cohort consisted of 699 IBD patients (240 CD and 459 UC), of which 23% had a first-degree relative with IBD. Of the several candidate genes studied, IL23R was associated with CD susceptibility, and TNFRSF1A as well as the HLA-DRB1*0103 allele with UC susceptibility. IL23R variants also showed association with the stricturing phenotype and longer disease duration in CD patients. In addition, TNFRSF1A variants were more common among familial UC and ileocolonic CD. In conclusion, the common CARD15 mutations were shown to account for 16% of CD cases in Finland. Novel CARD15 variants identified in the present study are most likely disease-causing mutations, as judged by the results of in vitro studies. The present study also confirms the IL23R association with CD susceptibility and, in addition, TNFRSF1A and HLA-DRB1*0103 allele association with UC of specific clinical phenotypes.

Regulating Food Consumption : Studies of change and variation in Europe

The role of people as buyers and eaters of food has changed significantly. From being protected by a paternalistic welfare state, people appear to be accorded more freedom and responsibility as individuals, where attention is redirected from the state towards market relations. Many have asserted that these changes are accompanied by fragmentation, individualisation, and privatisation, leading to individual uncertainty and lack of confidence. But empirical observations do not always confirm this, distrust is not necessarily growing and while responsibilities may change, the state still plays an active role. This dissertation explores changing relationships between states and markets, on the one hand, and ordinary people in their capacities as consumers and citizens, on the other. Do we see the emergence of new forms of regulation of food consumption? If so, what is the scope and what are the characteristics? Theories of regulation addressing questions about individualisation and self-governance are combined with a conceptualisation of consumption as processes of institutionalisation, involving daily routines, the division of labour between production and consumption, and the institutional field in which consumption is embedded. The analyses focus on the involvement of the state, food producers and scientific, first of all nutritional, expertise in regulating consumption, and on popular responses. Two periods come out as important, first when the ideas of “designing the good life” emerged, giving the state a very particular role in regulating food consumption, and, second, when this “designing” is replaced by ideas of choice and individual responsibility. One might say that “consumer choice” has become a mode of regulation. I use mainly historical studies from Norway to analyse the shifting role of the state in regulating food consumption, complemented with population surveys from six European countries to study how modernisation processes are associated with trust. The studies find that changing regulation is not only a question of societal or state vs individual responsibilities. Degrees of organisation and formalisation are important as well. While increasing organisation may represent discipline and abuses of power (including exploitation of consumer loyalty), organisation can also, to the consumer, provide higher predictability, systems to deal with malfeasance, and efficiency which may provide conditions for acting. The welfare state and the neo-liberal state have very different types of solutions. The welfare state solution is based on (national) egalitarianism, paternalism and discipline (of the market as well as households). Such solutions are still prominent in Norway. Individualisation and self-regulation may represent a regulatory response not only to a declining legitimacy of this kind of interventionism, but also increasing organisational complexity. This is reflected in large-scale re-regulation of markets as well as in relationships with households and consumers. Individualisation of responsibility is to the consumer not a matter of the number of choices that are presented on the shelves, but how choice as a form of consumer based involvement is institutionalised. It is recognition of people as “end-consumers”, as social actors, with systems of empowerment politically as well as via the provisioning system. ‘Consumer choice’ as a regulatory strategy includes not only communicative efforts to make people into “choosing consumers”, but also the provision of institutions which recognise consumer interests and agency. When this is lacking we find distrust as representing powerlessness. Individual responsibility-taking represents agency and is not always a matter of loyal support to shared goals, but involves protest and creativity. More informal (‘communitarian’) innovations may be an indication of that, where self-realisation is intimately combined with responsibility for social problems. But as solutions to counteract existing imbalances of power in the food market the impacts of such initiatives are probably more as part of consumer mobilisation and politicisation than as alternative provisioning.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

Variation i kvinnors namnfraser i officiella dokument i Helsingfors 1780-1930 : Socioonomastiska synvinklar på makt och identitet

This dissertation is an onomastic study of variation in women s name phrases in official documents in Finland during the period 1780−1930. The aim is to discuss from a socio-onomastic perspective both the changeover from patronymics to inherited family names and the use of surnames after marriage (i.e. whether women adopted their husbands family names or retained their maiden names), before new laws in this area entered into force in Finland in the early 20th century. In 1920, a law on family names that required fixed names put an end to the use of the patronymic as a person s only surname. After 1929, it was no longer possible for a married woman to retain her maiden name. Methodologically, to explain this development from a socio-onomastic perspective, I have based my study on a syntactic-semantic analysis of the actual name phrases. To be able to demonstrate the extensive material, I have elaborated a scheme to divide the 115 different types of name phrases into 13 main categories. The analysis of the material for Helsinki is based on frequency calculations of the different types of name phrases every thirtieth year, as well as on describing variation in the structure and semantic content of the name phrases, e.g. social variation in the use of titles and epithets. In addition to this, by applying a biographic-genealogical method, I have conducted two case studies of the usage of women s name phrases in the two chosen families. The study is based on parish registers from the period 1780−1929, estate inventory documents from the period 1780−1928, registration forms for liberty of trade from the period 1880−1908, family announcements on newspapers from the period 1829−1888, gravestones from the period 1796−1929 and diaries from the periods 1799−1801 and 1818−1820 providing a corpus of 5 950 name phrases. The syntactic-semantic analysis has revealed the overall picture of various ways of denoting women in official documents. In Helsinki, towards the end of the 19th century, the use of inherited family names seems to be almost fully developed in official contexts. At the late 19th century, a patronymic still appears as the only surname of some working-class women whereas in the early 20th century patronymics were only entered in the parish register as a kind of middle name. In the beginning of the 19th century, most married women were still registered under their maiden names, with a few exceptions among the bourgeoisie and upper class. The comparative analysis of name phrases in diaries, however, indicates that the use of the husband s family name by married women was a much earlier phenomenon in private contexts than in official documents. Keywords: socio-onomastics, syntactic-semantic analysis, name phrase, patronymic, maiden name, husband s family name

Associations of interleukin-1 (IL-1) gene variation and IL-1 receptor antagonist phenotypes with immunological responses, metabolic dysregulation and type 2 diabetes

The upstream proinflammatory interleukin-1 (IL-1) cytokines, together with a naturally occurring IL-1 receptor antagonist (IL-1Ra), play a significant role in several diseases and physiologic conditions. The IL-1 proteins affect glucose homeostasis at multiple levels contributing to vascular injuries and metabolic dysregulations that precede diabetes. An association between IL-1 gene variations and IL-1Ra levels has been suggested, and genetic studies have reported associations with metabolic dysregulation and altered inflammatory responses. The principal aims of this study were to: 1) examine the associations of IL-1 gene variation and IL-1Ra expression in the development and persistence of thyroid antibodies in subacute thyroiditis; 2) investigate the associations of common variants in the IL-1 gene family with plasma glucose and insulin concentrations, glucose homeostasis measures and prevalent diabetes in a representative population sample; 3) investigate genetic and non-genetic determinants of IL-1Ra phenotypes in a cross-sectional setting in three independent study populations; 4) investigate in a prospective setting (a) whether variants of the IL-1 gene family are predictors for clinically incident diabetes in two population-based observational cohort studies; and (b) whether the IL-1Ra levels predict the progression of metabolic syndrome to overt diabetes during the median follow-up of 10.8 and 7.1 years. Results from on patients with subacte thyroiditis showed that the systemic IL-1Ra levels are elevated during a specific proinflammatory response and they correlated with C-reactive protein (CRP) levels. Genetic variation in the IL-1 family seemed to have an association with the appearance of thyroid peroxidase antibodies and persisting local autoimmune responses during the follow-up. Analysis of patients suffering from diabetes and metabolic traits suggested that genetic IL-1 variation and IL-1Ra play a role in glucose homeostasis and in the development of type 2 diabetes. The coding IL-1 beta SNP rs1143634 was associated with traits related to insulin resistance in cross-sectional analyses. Two haplotype variants of the IL-1 beta gene were associated with prevalent diabetes or incident diabetes in a prospective setting and both of these haplotypes were tagged by rs1143634. Three variants of the IL-1Ra gene and one of the IL-1 beta gene were consistently identified as significant, independent determinants of the IL-1Ra phenotype in two or three populations. The proportion of the phenotypic variation explained by the genetic factors was modest however, while obesity and other metabolic traits explained a larger part. Body mass index was the strongest predictor of systemic IL-1Ra concentration overall. Furthermore, the age-adjusted IL-1Ra concentrations were elevated in individuals with metabolic syndrome or diabetes when compared to those free of metabolic dysregulation. In prospective analyses the systemic IL-1Ra levels were found as independent predictors for the development of diabetes in people with metabolic syndrome even after adjustment for multiple other factors, including plasma glucose and CRP levels. The predictive power of IL-1Ra was better than that of CRP. The prospective results also provided some evidence for a role of common IL-1 alpha promoter SNP rs1800587 in the development of type 2 diabetes among men and suggested that the role may be gender specific. Likewise, common variations in the IL-1 beta coding region may have a gender specific association with diabetes development. Further research on the potential benefits of IL-1Ra measurements in identifying individuals at high risk for diabetes, who then could be targeted for specific treatment interventions, is warranted. It has been reported in the recent literature that IL-1Ra secreted from adipose tissue has beneficial effects on glucose homeostasis. Furthermore, treatment with recombinant human IL-1Ra has been shown to have a substantial therapeutic potential. The genetic results from the prospective analyses performed in this study remain inconclusive, but together with the cross-sectional analyses they suggest gender-specific effects of the IL-1 variants on the risk of diabetes. Larger studies with more extensive genotyping and resequencing may help to pinpoint the exact variants responsible and to further elucidate the biological mechanisms for the observed associations. This would improve our understanding of the pathways linking inflammation and obesity with glucose and insulin metabolism.

Association between molecular variation in the phosphoglucose isomerase (Pgi) locus and migration propensity in the Glanville fritillary butterfly

Habitat fragmentation produces patches of suitable habitat surrounded by unfavourable matrix habitat. A species may persist in such a fragmented landscape in an equilibrium between the extinctions and recolonizations of local populations, thus forming a metapopulation. Migration between local populations is necessary for the long-term persistence of a metapopulation. The Glanville fritillary butterfly (Melitaea cinxia) forms a metapopulation in the Åland islands in Finland. There is migration between the populations, the extent of which is affected by several environmental factors and variation in the phenotype of individual butterflies. Different allelic forms of the glycolytic enzyme phosphoglucose isomerase (Pgi) has been identified as a possible genetic factor influencing flight performance and migration rate in this species. The frequency of a certain Pgi allele, Pgi-f, follows the same pattern in relation to population age and connectivity as migration propensity. Furthermore, variation in flight metabolic performance, which is likely to affect migration propensity, has been linked to genetic variation in Pgi or a closely linked locus. The aim of this study was to investigate the association between Pgi genotype and the migration propensity in the Glanville fritillary both at the individual and population levels using a statistical modelling approach. A mark-release-recapture (MRR) study was conducted in a habitat patch network of M. cinxia in Åland to collect data on the movements of individual butterflies. Larval samples from the study area were also collected for population level examinations. Each butterfly and larva was genotyped at the Pgi locus. The MRR data was parameterised with two mathematical models of migration: the Virtual Migration Model (VM) and the spatially explicit diffusion model. VM model predicted and observed numbers of emigrants from populations with high and low frequencies of Pgi-f were compared. Posterior predictive data sets were simulated based on the parameters of the diffusion model. Lack-of-fit of observed values to the model predicted values of several descriptors of movements were detected, and the effect of Pgi genotype on the deviations was assessed by randomizations including the genotype information. This study revealed a possible difference in the effect of Pgi genotype on migration propensity between the two sexes in the Glanville fritillary. The females with and males without the Pgi-f allele moved more between habitat patches, which is probably related to differences in the function of flight in the two sexes. Females may use their high flight capacity to migrate between habitat patches to find suitable oviposition sites, whereas males may use it to acquire mates by keeping a territory and fighting off other intruding males, possibly causing them to emigrate. The results were consistent across different movement descriptors and at the individual and population levels. The effect of Pgi is likely to be dependent on the structure of the landscape and the prevailing environmental conditions.

Seasonal variation in nitrification and nitrate-reduction pathways in coastal sediments in the Gulf of Finland, Baltic Sea

The Baltic Sea is one of the most eutrophic marine areas in the world. The role of nitrogen as a eutrophicating nutrient in the Baltic Sea has remained controversial, due to lack of understanding of nitrogen cycling in the area. We investigated the seasonal variation in sediment nitrification, denitrification, anaerobic ammonium oxidation (anammox), and dissimilatory nitrate reduction to ammonium (DNRA) at two coastal sites in the Gulf of Finland. In addition to the in situ rates, we assessed the potential for these processes in different seasons. The nitrification and nitrogen removal processes were maximal during the warm summer months, when the sediment organic content was highest. In colder seasons, the in situ rates of the nitrification and nitrate reduction processes decreased, but the potential for nitrification remained equal to or higher than that during the warm months. The denitrification and nitrification rates were usually higher in the accumulation basin, where the organic content of the sediment was higher, but the transportation area, despite lower denitrification rates and potential, typically had higher potential for nitrification than the accumulation basin. Anammox and DNRA were not significant nitrate sinks in any of the seasons sampled. The results also show that the denitrification rates in the coastal Gulf of Finland sediment have decreased, and that benthic denitrification might be a less important sink for fixed nitrogen than previously assumed.

Intraspecific variation in brain size and architecture : population divergence and phenotypic plasticity

Brain size and architecture exhibit great evolutionary and ontogenetic variation. Yet, studies on population variation (within a single species) in brain size and architecture, or in brain plasticity induced by ecologically relevant biotic factors have been largely overlooked. Here, I address the following questions: (i) do locally adapted populations differ in brain size and architecture, (ii) can the biotic environment induce brain plasticity, and (iii) do locally adapted populations differ in levels of brain plasticity? In the first two chapters I report large variation in both absolute and relative brain size, as well as in the relative sizes of brain parts, among divergent nine-spined stickleback (Pungitius pungitius) populations. Some traits show habitat-dependent divergence, implying natural selection being responsible for the observed patterns. Namely, marine sticklebacks have relatively larger bulbi olfactorii (chemosensory centre) and telencephala (involved in learning) than pond sticklebacks. Further, I demonstrate the importance of common garden studies in drawing firm evolutionary conclusions. In the following three chapters I show how the social environment and perceived predation risk shapes brain development. In common frog (Rana temporaria) tadpoles, I demonstrate that under the highest per capita predation risk, tadpoles develop smaller brains than in less risky situations, while high tadpole density results in enlarged tectum opticum (visual brain centre). Visual contact with conspecifics induces enlarged tecta optica in nine-spined sticklebacks, whereas when only olfactory cues from conspecifics are available, bulbus olfactorius become enlarged.Perceived predation risk results in smaller hypothalami (complex function) in sticklebacks. Further, group-living has a negative effect on relative brain size in the competition-adapted pond sticklebacks, but not in the predation-adapted marine sticklebacks. Perceived predation risk induces enlargement of bulbus olfactorius in pond sticklebacks, but not in marine sticklebacks who have larger bulbi olfactorii than pond fish regardless of predation. In sum, my studies demonstrate how applying a microevolutionary approach can help us to understand the enormous variation observed in the brains of wild animals a point-of-view which I high-light in the closing review chapter of my thesis.

Seasonal and short-term variation in denitrification and anammox at a coastal station on the Gulf of Finland, Baltic Sea

Benthic processes were measured at a coastal deposition area in the northern Baltic Sea, covering all seasons. The N-2 production rates, 90-400 mu mol N m(-2) d(-1), were highest in autumn-early winter and lowest in spring. Heterotrophic bacterial production peaked unexpectedly late in the year, indicating that in addition to the temperature, the availability of carbon compounds suitable for the heterotrophic bacteria also plays a major role in regulating the denitrification rate. Anaerobic ammonium oxidation (anammox) was measured in spring and autumn and contributed 10% and 15%, respectively, to the total N-2 production. The low percentage did, however, result in a significant error in the total N-2 production rate estimate, calculated using the isotope pairing technique. Anammox must be taken into account in the Gulf of Finland in future sediment nitrogen cycling research.