Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

Genetic Basis of Pituitary Adenoma Predisposition

Much of the global cancer research is focused on the most prevalent tumors; yet, less common tumor types warrant investigation, since A rare disorder is not necessarily an unimportant one . The present work discusses a rare tumor type, the benign adenomas of the pituitary gland, and presents the advances which, during the course of this thesis work, contributed to the elucidation of a fraction of their genetic background. Pituitary adenomas are benign neoplasms of the anterior pituitary lobe, accounting for approximately 15% of all intracranial tumors. Pituitary adenoma cells hypersecrete the hormones normally produced by the anterior pituitary tissue, such as growth hormone (GH) and prolactin (PRL). Despite their non-metastasizing nature, these adenomas can cause significant morbidity and have to be adequately treated; otherwise, they can compromise the patient s quality of life, due to conditions provoked by hormonal hypersecretion, such as acromegaly in the case of GH-secreting adenomas, or due to compressive effects to surrounding tissues. The vast majority of pituitary adenomas arise sporadically, whereas a small subset occur as component of familial endocrine-related tumor syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 is caused by germline mutations in the MEN1 tumor suppressor gene (11q13), whereas the majority of CNC cases carry germline mutations in the PRKAR1A gene (17q24). Pituitary adenomas are also encountered in familial settings outside the context of MEN1 and CNC, but unlike in the latter syndromes, their genetic background until recently remained elusive. Evidence in previous literature supported the notion that a tumor suppressor gene on 11q13, residing very close to but still distinct from MEN1, causes genetic susceptibility to pituitary tumors. The aim of the study was to identify the genetic cause of a low penetrance form of Pituitary Adenoma Predisposition (PAP) in families from Northern Finland. The present work describes the methodological approach that led to the identification of aryl hydrocarbon receptor interacting protein (AIP) as the gene causing PAP. Combining chip-based technologies (SNP and gene expression arrays) with traditional gene mapping methods and genealogy data, we showed that germline AIP mutations cause PAP in familial and sporadic settings. PAP patients were diagnosed with mostly adenomas of the GH/PRL-secreting cell lineage. In Finland, two AIP mutations accounted for 16% of all patients diagnosed with GH-secreting adenomas, and for 40% of patients being younger than 35 years of age at diagnosis. AIP is suggested to act as a tumor suppressor gene, a notion supported by the nature of the identified mutations (most are truncating) and the biallelic inactivation of AIP in the tumors studied. AIP has been best characterized as a cytoplasmic interaction partner of aryl hydrocarbon receptor (AHR), also known as dioxin receptor, but it has other partners as well. The mechanisms that underlie AIP-mediated pituitary tumorigenesis are to date largely unknown and warrant further investigation. Because AIP was identified in the genetically homogeneous Finnish population, it was relevant to examine its contribution to PAP in other, more heterogeneous, populations. Analysis of pituitary adenoma patient series of various ethnic origins and differing clinical settings revealed germline AIP mutations in all cohorts studied, albeit with low frequencies (range 0.8-7.4%). Overall, PAP patients were typically diagnosed at a young age (range 8-41 years), mainly with GH-secreting adenomas, without strong family history of endocrine disease. Because many PAP patients did not display family history of pituitary adenomas, detection of the condition appeared challenging. AIP immunohistochemistry was tested as a molecular pre-screening tool on mutation-positive versus mutation-negative tumors, and proved to be a potentially useful predictor of PAP. Mutation screening of a large cohort of colorectal, breast, and prostate tumors did not reveal somatic AIP mutations. These tumors, apart from being the most prevalent among men and women worldwide, have been associated with acromegaly, particularly colorectal neoplasia. In this material, AIP did not appear to contribute to the pathogenesis of these common tumor types and other genes seem likely to play a role in such tumorigenesis. Finally, the contribution of AIP in pediatric onset pituitary adenomas was examined in a unique population-based cohort of sporadic pituitary adenoma patients from Italy. Germline AIP mutations may account for a subset of pediatric onset GH-secreting adenomas (in this study one of seven GH-secreting adenoma cases or 14.3%), and appear to be enriched among young (≤25 years old) patients. In summary, this work reveals a novel tumor susceptibility gene, namely AIP, which causes genetic predisposition to pituitary adenomas, in particular GH-secreting adenomas. Moreover, it provides molecular tools for identification of individuals predisposed for PAP. Further elaborate studies addressing the functional role of AIP in normal and tumor cells will hopefully expand our knowledge on endocrine neoplasia and reveal novel cellular mechanisms of pituitary tumorigenesis, including potential drug targets.

Genotyping for genetic association studies: Methods and applications

In this thesis, two separate single nucleotide polymorphism (SNP) genotyping techniques were set up at the Finnish Genome Center, pooled genotyping was evaluated as a screening method for large-scale association studies, and finally, the former approaches were used to identify genetic factors predisposing to two distinct complex diseases by utilizing large epidemiological cohorts and also taking environmental factors into account. The first genotyping platform was based on traditional but improved restriction-fragment-length-polymorphism (RFLP) utilizing 384-microtiter well plates, multiplexing, small reaction volumes (5 µl), and automated genotype calling. We participated in the development of the second genotyping method, based on single nucleotide primer extension (SNuPeTM by Amersham Biosciences), by carrying out the alpha- and beta tests for the chemistry and the allele-calling software. Both techniques proved to be accurate, reliable, and suitable for projects with thousands of samples and tens of markers. Pooled genotyping (genotyping of pooled instead of individual DNA samples) was evaluated with Sequenom s MassArray MALDI-TOF, in addition to SNuPeTM and PCR-RFLP techniques. We used MassArray mainly as a point of comparison, because it is known to be well suited for pooled genotyping. All three methods were shown to be accurate, the standard deviations between measurements being 0.017 for the MassArray, 0.022 for the PCR-RFLP, and 0.026 for the SNuPeTM. The largest source of error in the process of pooled genotyping was shown to be the volumetric error, i.e., the preparation of pools. We also demonstrated that it would have been possible to narrow down the genetic locus underlying congenital chloride diarrhea (CLD), an autosomal recessive disorder, by using the pooling technique instead of genotyping individual samples. Although the approach seems to be well suited for traditional case-control studies, it is difficult to apply if any kind of stratification based on environmental factors is needed. Therefore we chose to continue with individual genotyping in the following association studies. Samples in the two separate large epidemiological cohorts were genotyped with the PCR-RFLP and SNuPeTM techniques. The first of these association studies concerned various pregnancy complications among 100,000 consecutive pregnancies in Finland, of which we genotyped 2292 patients and controls, in addition to a population sample of 644 blood donors, with 7 polymorphisms in the potentially thrombotic genes. In this thesis, the analysis of a sub-study of pregnancy-related venous thromboses was included. We showed that the impact of factor V Leiden polymorphism on pregnancy-related venous thrombosis, but not the other tested polymorphisms, was fairly large (odds ratio 11.6; 95% CI 3.6-33.6), and increased multiplicatively when combined with other risk factors such as obesity or advanced age. Owing to our study design, we were also able to estimate the risks at the population level. The second epidemiological cohort was the Helsinki Birth Cohort of men and women who were born during 1924-1933 in Helsinki. The aim was to identify genetic factors that might modify the well known link between small birth size and adult metabolic diseases, such as type 2 diabetes and impaired glucose tolerance. Among ~500 individuals with detailed birth measurements and current metabolic profile, we found that an insertion/deletion polymorphism of the angiotensin converting enzyme (ACE) gene was associated with the duration of gestation, and weight and length at birth. Interestingly, the ACE insertion allele was also associated with higher indices of insulin secretion (p=0.0004) in adult life, but only among individuals who were born small (those among the lowest third of birth weight). Likewise, low birth weight was associated with higher indices of insulin secretion (p=0.003), but only among carriers of the ACE insertion allele. The association with birth measurements was also found with a common haplotype of the glucocorticoid receptor (GR) gene. Furthermore, the association between short length at birth and adult impaired glucose tolerance was confined to carriers of this haplotype (p=0.007). These associations exemplify the interaction between environmental factors and genotype, which, possibly due to altered gene expression, predisposes to complex metabolic diseases. Indeed, we showed that the common GR gene haplotype associated with reduced mRNA expression in thymus of three individuals (p=0.0002).

Oncolytic adenoviruses for treatment of ovarian cancer

Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.

New molecular strategies for prevention of fibroproliferative vascular disease : An experimental approach to restenosis

Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.

Vascular growth factors and progenitor cells in cardiac allograft arteriosclerosis

Heart transplantation is the only therapeutic modality for many end-stage heart diseases but poor long-term survival remains a challenging problem. This is mainly due to the development of cardiac allograft arteriosclerosis (TxCAD) that is an accelerated form of coronary artery disease. Both traditional cardiovascular and transplantation-related risk factors for TxCAD have been identified but options for therapy are limited. TxCAD involves dysfunction of cardiac allograft vascular cells. Activated endothelial cells (EC) regulate allograft inflammation and secrete smooth muscle cell (SMC) growth factors. In turn, SMC and their progenitors invade the intima of the injured vessels and occlude the affected coronary arteries. Different vascular growth factors have to be delicately regulated in normal vascular development. In the present study, experimental heterotopic transplantation models were used to study the role of angiogenic and pro-inflammatory vascular endothelial growth factor (VEGF), EC growth factor angiopoietin (Ang), and SMC mitogen platelet-derived growth factor (PDGF) in the development of TxCAD. Pharmacological and gene transfer approaches were used to target these growth factors and to assess their therapeutic potential. This study shows that alloimmune response in heart transplants upregulates VEGF expression, and induces allograft angiogenesis that involves donor-derived primitive EC. Intracoronary adenoviral VEGF gene transfer increased macrophage infiltration, intimal angiogenesis and TxCAD. VEGF inhibition with PTK787 decreased allograft inflammation and TxCAD, and simultaneous PDGF inhibition with imatinib further decreased TxCAD. Specific inhibition of two VEGF-receptors (VEGFR) decreased allograft inflammation and TxCAD, and VEGFR-2 inhibition normalized the density of primitive and mature capillaries in the allografts. Adenovirus-mediated transient Ang1 expression in the allograft had anti-inflammatory and anti-arteriosclerotic effects. Adeno-associated virus (AAV)-mediated prolonged Ang1 or Ang2 expression had similar anti-inflammatory effects. However, AAV-Ang1 activated allograft SMC whereas AAV-Ang2 had no effects on SMC activation and decreased the development of TxCAD. These studies indicate an interplay of inflammation, angiogenesis and arteriosclerosis in cardiac allografts, and show that vascular growth factors are important regulators in the process. Also, VEGF inhibition, PDGF inhibition and angiopoietin therapy with clinically-relevant pharmacological agents or novel gene therapy approaches may counteract vascular dysfunction in cardiac allografts, and have beneficial effects on the survival of heart transplant patients in the future.

Diet and the development of atherosclerosis: a whole-diet approach from childhood to adulthood

Cardiovascular diseases (CVDs) are the leading cause of mortality in the world. Studies of the impact of single nutrients on the risk for CVD have often provided inconclusive results, and recent research in nutritional epidemiology with a more holistic whole-diet approach has proven fruitful. Moreover, dietary habits in childhood and adolescence may play a role in later health and disease, either independently or by tracking into adulthood. The main aims of this study were to find childhood and adulthood determinants of adulthood diet, to identify dietary patterns present among the study population and to study the associations between long-term food choices and cardiovascular health in young Finnish adults. The study is a part of the multidisciplinary Cardiovascular Risk in Young Finns study, which is an ongoing, prospective cohort study with a 21-year follow-up. At baseline in 1980, the subjects were children and adolescents aged 3 to 18 years (n included in this study = 1768), and young adults aged 24 to 39 years at the latest follow-up study in 2001 (n = 1037). Food consumption and nutrient intakes were assessed with repeated 48-hour dietary recalls. Other determinations have included comprehensive risk factor assessments using blood tests, physical measurements and questionnaires. In the latest follow-up, ultrasound examinations were performed to study early atherosclerotic vascular changes. The average intakes showed substantial changes since 1980. Intakes of fat and saturated fat had decreased, whereas the consumption of fruits and vegetables had increased. Intake of fat and consumption of vegetables in childhood and physical activity in adulthood were important health behavioural determinants of adult diet. Additionally, a principal component analysis was conducted to identify major dietary patterns at each study point. A similar set of two major patterns was recognised throughout the study. The traditional dietary pattern positively correlated with the consumption of traditional Finnish foods, such as rye, potatoes, milk, butter, sausages and coffee, and negatively correlated with fruit, berries and dairy products other than milk. This type of diet was independently associated with several risk factors of CVD, such as total and low-density lipoprotein cholesterol, apolipoprotein B and C-reactive protein concentrations among both genders, as well as with systolic blood pressure and insulin levels among women. The traditional pattern was also independently associated with intima media thickness (IMT), a subclinical predictor of CVD, in men but not in women. The health-conscious pattern, predominant among female subjects, non-smokers and urbanites, was characterised by more health-conscious food choices such as vegetables, legumes and nuts, tea, rye, fish, cheese and other dairy products, as well as by the consumption of alcoholic beverages. This pattern was inversely, but less strongly, associated with cardiovascular risk factors. Tracking of the dietary pattern scores was observed, particularly among subjects who were adolescents at baseline. Moreover, a long-term high intake of protein concurrent with a low intake of fat was positively associated with IMT. These findings suggest that food behaviour and food choices are to some extent established as early as in childhood or adolescence and may significantly track into adulthood. Long-term adherence to traditional food choices seems to increase the risk for developing CVD, especially among men. Those with intentional or unintentional low fat diets, but with high intake of protein may also be at increased risk for CVD. The findings offer practical, food-based information on the relationship between diet and CVD and encourage further use of the whole-diet approach in epidemiological research. The results support earlier findings that long-term food choices play a role in the development of CVD. The apparent influence of childhood habits is important to bear in mind when planning educational strategies for the primary prevention of CVD. Further studies on food choices over the entire lifespan are needed.

Identification of Isoflavonoid Metabolites in Humans

Epidemiological studies have associated high soy intake with a lowered risk for certain hormone-dependent diseases, such as breast and prostate cancers, osteoporosis, and cardiovascular disease. Soy is a rich source of isoflavones, diphenolic plant compounds that have been shown to possess several biological activities. Soy is not part of the traditional Western diet, but many dietary supplements are commercially available in order to provide the proposed beneficial health effects of isoflavones without changing the original diet. These supplements are usually manufactured from extracts of soy or red clover, which is another important source of isoflavones. However, until recently, detailed studies of the metabolism of these compounds in humans have been lacking. The aim of this study was to identify urinary metabolites of isoflavones originating from soy or red clover using gas chromatography - mass spectrometry (GC-MS). To examine metabolism, soy and red clover supplementation studies with human volunteers were carried out. In addition, the metabolism of isoflavones was investigated in vitro by identification of metabolites formed during a 24-h fermentation of pure isoflavones with a human fecal inoculum. Qualitative methods for identification and analysis of isoflavone metabolites in urine and fecal fermentation samples by GC-MS were developed. Moreover, a detailed investigation of fragmentation of isoflavonoids in electron ionization mass spectrometry (EIMS) was carried out by means of synthetic reference compounds and deuterated trimethylsilyl derivatives. After isoflavone supplementation, 18 new metabolites of isoflavones were identified in human urine samples. The most abundant urinary metabolites of soy isoflavones daidzein, genistein, and glycitein were found to be the reduced metabolites, i.e. analogous isoflavanones, a-methyldeoxybenzoins, and isoflavans. Metabolites having additional hydroxyl and/or methoxy substituents, or their reduced analogs, were also identified. The main metabolites of red clover isoflavones formononetin and biochanin A were identified as daidzein and genistein. In addition, reduced and hydroxylated metabolites of formononetin and biochanin A were identified; however, they occurred at much lower levels in urine samples than daidzein or genistein or their reduced metabolites. The results of this study show that the metabolism of isoflavones is diverse. More studies are needed to determine whether the new isoflavonoid metabolites identified here have biological activities that contribute to the proposed beneficial effects of isoflavones on human health. Another task is to develop validated quantitative methods to determine the actual levels of isoflavones and their metabolites in biological matrices in order to assess the role of isoflavones in prevention of chronic diseases.

Traditional Village Governance (Desa Adat) and Inter-ethnic Conflict in Bali : A Study of Conflict between Balinese and Migrants: Cases from Municipality of Denpasar

Regional autonomy in Indonesia was initially introduced as a means of pacifying regional disappointment at the central government. Not only did the Regional Autonomy Law of 1999 give the Balinese a chance to express grievance regarding the centralist policies of the Jakarta government but also provided an opportunity to return to the regional, exclusive, traditional village governance (desa adat). As a result, the problems faced by the island, particularly ethnic conflicts, are increasingly handled by the mechanism of this traditional type of governance. Traditional village governance with regard to ethnic conflicts (occurring) between Balinese and migrants has never been systematically analyzed. Existing analyses emphasized only the social context, but do not explain either the cause of conflicts and the ensuing problems entails or the virtues of traditional village governance mechanisms for mediating in the conflict. While some accounts provide snapshots, they lack both theoretical and conflict study perspective. The primary aim of this dissertation is to explore the expression and the causes of conflict between the Balinese and migrants and to advance the potential of traditional village governance as a means of conflict resolution with particular reference to the municipality of Denpasar. One conclusion of the study is that the conflict between the Balinese and migrants has been expressed on the level of situation/contradiction, attitudes, and behavior. Yet the driving forces behind the conflict itself consist of the following factors: absence of cooperation; incompatible position and perception; inability to communicate effectively; and problem of inequality and injustice, which comes to the surface as a social, cultural, and economic problem. This complex of factors fuels collective fear for the future of both groups. The study concludes that traditional village governance mechanisms as a means of conflict resolution have not yet been able to provide an enduring resolution for the conflict. Analysis shows that the practice of traditional village governance is unable to provide satisfactory mechanisms for the conflict as prescribed by conflict resolution theory. Traditional village governance, which is derived from the exclusive Hindu-Balinese culture, is accepted as more legitimate among the Balinese than the official governance policies. However, it is not generally accepted by most of the Muslim migrants. In addition, traditional village governance lacks access to economic instruments, which weakens its capacity to tackle the economic roots of the conflict. Thus the traditional mechanisms of migrant ordinance , as practiced by the traditional village governance have not yet been successful in penetrating all aspects of the conflict. Finally, one of the main challenges for traditional village governance s legal development is the creation of a regional legal system capable of accommodating rapid changes in line with the national and international legal practices. The framing of the new laws should be responsive to the aspirations of a changing society. It should not only protect the various Balinese communities interests, but also that of other ethnic groups, especially those of the minority. In other words, the main challenge to traditional village governance is its ability to develop flexibility and inclusiveness.

Novel surgical and imaging methods of the middle ear and temporal bone

Välikorvaleikkauksiin usein liittyvän välikorvan ja kuuloluuketjun kirurgisen rekonstruktion tavoitteena on luoda olosuhteet, jotka mahdollistavat hyvän kuulon sekä välikorvan säilymisen tulehduksettomana ja ilmapitoisena. Välikorvan rekonstruktiossa on käytetty implanttimateriaaleina perinteisesti potilaan omia kudoksia sekä tarvittaessa erilaisia hajoamattomia biomateriaaleja, mm. titaania ja silikonia. Ongelmana biomateriaalien käytössä voi olla bakteerien adherenssi eli tarttuminen vieraan materiaalin pintaan, mikä saattaa johtaa biofilmin muodostumiseen. Tämä voi aiheuttaa kroonisen, huonosti antibiootteihin reagoivan infektion kudoksessa, mikä usein käytännössä johtaa uusintaleikkaukseen ja implantin poistoon. Maitohappo- ja glykolihappopohjaiset biologisesti hajoavat polymeerit ovat olleet kliinisessä käytössä jo vuosikymmeniä. Niitä on käytetty erityisesti tukimateriaaleina mm. ortopediassa sekä kasvo- ja leukakirurgiassa. Niitä ei ole toistaiseksi käytetty välikorvakirurgiassa. Korvan kuvantamiseen käytetään ensisijaisesti tietokonetomografiaa (TT). TT-tutkimuksen ongelmana on potilaan altistuminen suhteellisen korkealle sädeannokselle, joka kasvaa kumulatiivisesti, jos kuvaus joudutaan toistamaan. Väitöskirjatyö selvittää uuden, aiemmin kliinisessä työssä rutiinisti lähinnä hampaiston ja kasvojen alueen kuvantamiseen käytetyn rajoitetun kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen. Väitöskirjan kahdessa ensimmäisessä osatyössä tutkittiin ja verrattiin kahden kroonisia ja postoperatiivisia korvainfektioita aiheuttavan bakteerin, Staphylococcus aureuksen ja Pseudomonas aeruginosan, in vitro adherenssia titaanin, silikonin ja kahden eri biohajoavan polymeerin (PLGA) pintaan. Lisäksi tutkittiin materiaalien albumiinipinnoituksen vaikutusta adherenssiin. Kolmannessa osatyössä tutkittiin eläinmallissa PLGA:n biokompatibiliteettia eli kudosyhteensopivuutta kokeellisessa välikorvakirurgiassa. Chinchillojen välikorviin istutettiin PLGA-materiaalia, eläimiä seurattiin, ja ne lopetettiin 6 kk:n kuluttua operaatiosta. Biokompatibiliteetin arviointi perustui kliinisiin havaintoihin sekä kudosnäytteisiin. Neljännessä osatyössä tutkittiin kartiokeila-TT:n soveltuvuutta korvan alueen kuvantamiseen vertaamalla sen tarkkuutta perinteisen spiraali-TT:n tarkkuuteen. Molemmilla laitteilla kuvattiin ohimo- eli temporaaliluita korvan alueen kliinisesti ja kirurgisesti tärkeiden rakenteiden kuvantumisen tarkkuuden arvioimiseksi. Viidennessä osatyössä arvioitiin myös operoitujen temporaaliluiden kuvantumista kartiokeila-TT:ssa. Bakteeritutkimuksissa PLGA-materiaalin pintaan tarttui keskimäärin korkeintaan saman verran tai vähemmän bakteereita kuin silikonin tai titaanin. Albumiinipinnoitus vähensi bakteeriadherenssia merkitsevästi kaikilla materiaaleilla. Eläinkokeiden perusteella PLGA todettiin hyvin siedetyksi välikorvassa. Korvakäytävissä tai välikorvissa ei todettu infektioita, tärykalvon perforaatioita tai materiaalin esiin työntymistä. Kudosnäytteissä näkyi lievää tulehdusreaktiota ja fibroosia implantin ympärillä. Temporaaliluutöissä rajoitettu kartiokeila-TT todettiin vähintään yhtä tarkaksi menetelmäksi kuin spiraali-TT välikorvan ja sisäkorvan rakenteiden kuvantamisessa, ja sen aiheuttama kertasäderasitus todettiin spiraali-TT:n vastaavaa huomattavasti vähäisemmäksi. Kartiokeila-TT soveltui hyvin välikorvaimplanttien ja postoperatiivisen korvan kuvantamiseen. Tulokset osoittavat, että PLGA on välikorvakirurgiaan soveltuva, turvallinen ja kudosyhteensopiva biomateriaali. Biomateriaalien pinnoittaminen albumiinilla vähentää merkittävästi bakteeriadherenssia niihin, mikä puoltaa pinnoituksen soveltamista implanttikirurgiassa. Kartiokeila-TT soveltuu korvan alueen kuvantamiseen. Sen tarkkuus kliinisesti tärkeiden rakenteiden osoittamisessa on vähintään yhtä hyvä ja sen potilaalle aiheuttama sädeannos pienempi kuin nykyisen korva-spiraali-TT:n. Tämä tekee menetelmästä spiraali-TT:aa potilasturvallisemman vaihtoehdon erityisesti, jos potilaan tilanne vaatii seurantaa ja useampia kuvauksia, ja jos halutaan kuvata rajoitettuja alueita uni- tai bilateraalisesti.

Determinants of outcome in critically ill patients

Assessment of the outcome of critical illness is complex. Severity scoring systems and organ dysfunction scores are traditional tools in mortality and morbidity prediction in intensive care. Their ability to explain risk of death is impressive for large cohorts of patients, but insufficient for an individual patient. Although events before intensive care unit (ICU) admission are prognostically important, the prediction models utilize data collected at and just after ICU admission. In addition, several biomarkers have been evaluated to predict mortality, but none has proven entirely useful in clinical practice. Therefore, new prognostic markers of critical illness are vital when evaluating the intensive care outcome. The aim of this dissertation was to investigate new measures and biological markers of critical illness and to evaluate their predictive value and association with mortality and disease severity. The impact of delay in emergency department (ED) on intensive care outcome, measured as hospital mortality and health-related quality of life (HRQoL) at 6 months, was assessed in 1537 consecutive patients admitted to medical ICU. Two new biological markers were investigated in two separate patient populations: in 231 ICU patients and 255 patients with severe sepsis or septic shock. Cell-free plasma DNA is a surrogate marker of apoptosis. Its association with disease severity and mortality rate was evaluated in ICU patients. Next, the predictive value of plasma DNA regarding mortality and its association with the degree of organ dysfunction and disease severity was evaluated in severe sepsis or septic shock. Heme oxygenase-1 (HO-1) is a potential regulator of apoptosis. Finally, HO-1 plasma concentrations and HO-1 gene polymorphisms and their association with outcome were evaluated in ICU patients. The length of ED stay was not associated with outcome of intensive care. The hospital mortality rate was significantly lower in patients admitted to the medical ICU from the ED than from the non-ED, and the HRQoL in the critically ill at 6 months was significantly lower than in the age- and sex-matched general population. In the ICU patient population, the maximum plasma DNA concentration measured during the first 96 hours in intensive care correlated significantly with disease severity and degree of organ failure and was independently associated with hospital mortality. In patients with severe sepsis or septic shock, the cell-free plasma DNA concentrations were significantly higher in ICU and hospital nonsurvivors than in survivors and showed a moderate discriminative power regarding ICU mortality. Plasma DNA was an independent predictor for ICU mortality, but not for hospital mortality. The degree of organ dysfunction correlated independently with plasma DNA concentration in severe sepsis and plasma HO-1 concentration in ICU patients. The HO-1 -413T/GT(L)/+99C haplotype was associated with HO-1 plasma levels and frequency of multiple organ dysfunction. Plasma DNA and HO-1 concentrations may support the assessment of outcome or organ failure development in critically ill patients, although their value is limited and requires further evaluation.

Kohti leikkaussalin tuloksellista toiminnanohjausta

Background: The aging population is placing increasing demands on surgical services, simultaneously with a decreasing supply of professional labor and a worsening economic situation. Under growing financial constraints, successful operating room management will be one of the key issues in the struggle for technical efficiency. This study focused on several issues affecting operating room efficiency. Materials and methods: The current formal operating room management in Finland and the use of performance metrics and information systems used to support this management were explored using a postal survey. We also studied the feasibility of a wireless patient tracking system as a tool for managing the process. The reliability of the system as well as the accuracy and precision of its automatically recorded time stamps were analyzed. The benefits of a separate anesthesia induction room in a prospective setting were compared with the traditional way of working, where anesthesia is induced in the operating room. Using computer simulation, several models of parallel processing for the operating room were compared with the traditional model with respect to cost-efficiency. Moreover, international differences in operating room times for two common procedures, laparoscopic cholecystectomy and open lung lobectomy, were investigated. Results: The managerial structure of Finnish operating units was not clearly defined. Operating room management information systems were found to be out-of-date, offering little support to online evaluation of the care process. Only about half of the information systems provided information in real time. Operating room performance was most often measured by the number of procedures in a time unit, operating room utilization, and turnover time. The wireless patient tracking system was found to be feasible for hospital use. Automatic documentation of the system facilitated patient flow management by increasing process transparency via more available and accurate data, while lessening work for staff. Any parallel work flow model was more cost-efficient than the traditional way of performing anesthesia induction in the operating room. Mean operating times for two common procedures differed by 50% among eight hospitals in different countries. Conclusions: The structure of daily operative management of an operating room warrants redefinition. Performance measures as well as information systems require updating. Parallel work flows are more cost-efficient than the traditional induction-in-room model.

Ischemic Stroke in Young Adults

Stroke is the second leading cause of death and the leading cause of disability worldwide. Of all strokes, up to 80% to 85% are ischemic, and of these, less than 10% occur in young individuals. Stroke in young adults—most often defined as stroke occurring under the age of 45 or 50—can be particularly devastating due to long expected life-span ahead and marked socio-economic consequences. Current basic knowledge on ischemic stroke in this age group originates mostly from rather small and imprecise patient series. Regarding emergency treatment, systematic data on use of intravenous thrombolysis are absent. For this Thesis project, we collected detailed clinical and radiological data on all consecutive patients aged 15 to 49 with first-ever ischemic stroke between 1994 and 2007 treated at the Helsinki University Central Hospital. The aims of the study were to define demographic characteristics, risk factors, imaging features, etiology, and long-term mortality and its predictors in this patient population. We additionally sought to investigate, whether intravenous thrombolysis is safe and beneficial for the treatment of acute ischemic stroke in the young. Of our 1008 patients, most were males (ratio 1.7:1), who clearly outnumbered females after the age of 44, but females were preponderant among those aged <30. Occurrence increased exponentially. The most frequent risk factors were dyslipidemia (60%), smoking (44%), and hypertension (39%). Risk factors accumulated in males and along aging. Cardioembolism (20%) and cervicocerebral artery dissection (15%) were the most frequent etiologic subgroups, followed by small-vessel disease (14%), and large-artery atherosclerosis (8%). A total of 33% had undetermined etiology. Left hemisphere strokes were more common in general. Posterior circulation infarcts were more common among those aged <45. Multiple brain infarcts were present in 23% of our patients, 13% had silent infarcts, and 5% had leukoaraiosis. Of those with silent brain infarcts, majority (54%) had only a single lesion, and most of the silent strokes were located in basal ganglia (39%) and subcortical regions (21%). In a logistic regression analysis, type 1 diabetes mellitus in particular predicted the presence of both silent brain infarcts (odds ratio 5.78, 95% confidence interval 2.37-14.10) and leukoaraiosis (9.75; 3.39-28.04). We identified 48 young patients with hemispheric ischemic stroke treated with intravenous tissue plasminogen activator, alteplase. For comparisons, we searched 96 untreated control patients matched by age, gender, and admission stroke severity, as well as 96 alteplase-treated older controls aged 50 to 79 matched by gender and stroke severity. Alteplase-treated young patients recovered more often completely (27% versus 10%, P=0.010) or had only mild residual symptoms (40% versus 22%, P=0.025) compared to age-matched controls. None of the alteplase-treated young patients had symptomatic intracerebral hemorrhage or died within 3-month follow-up. Overall long-term mortality was low in our patient population. Cumulative mortality risks were 2.7% (95% confidence interval 1.5-3.9%) at 1 month, 4.7% (3.1-6.3%) at 1 year, and 10.7% (9.9-11.5%) at 5 years. Among the 30-day survivors who died during the 5-year follow-up, more than half died due to vascular causes. Malignancy, heart failure, heavy drinking, preceding infection, type 1 diabetes, increasing age, and large-artery atherosclerosis causing the index stroke independently predicted 5-year mortality when adjusted for age, gender, relevant risk factors, stroke severity, and etiologic subtype. In sum, young adults with ischemic stroke have distinct demographic patterns and they frequently harbor traditional vascular risk factors. Etiology in the young is extremely diverse, but in as many as one-third the exact cause remains unknown. Silent brain infarcts and leukoaraiosis are not uncommon brain imaging findings in these patients and should not be overlooked due to their potential prognostic relevance. Outcomes in young adults with hemispheric ischemic stroke can safely be improved with intravenous thrombolysis. Furthermore, despite their overall low risk of death after ischemic stroke, several easily recognizable factors—of which most are modifiable—predict higher mortality in the long term in young adults.