Heme oxygenase-1 in cardiovascular diseases

Myocardial infarction (MI) and heart failure are major causes of morbidity and mortality worldwide. Treatment of MI involves early restoration of blood flow to limit infarct size and preserve cardiac function. MI leads to left ventricular remodeling, which may eventually progress to heart failure, despite the established pharmacological treatment of the disease. To improve outcome of MI, new strategies for protecting the myocardium against ischemic injury and enhancing the recovery and repair of the infarcted heart are needed. Heme oxygenase-1 (HO-1) is a stress-responsive and cytoprotective enzyme catalyzing the degradation of heme into the biologically active reaction products biliverdin/bilirubin, carbon monoxide (CO) and free iron. HO-1 plays a key role in maintaining cellular homeostasis by its antiapoptotic, anti-inflammatory, antioxidative and proangiogenic properties. The present study aimed, first, at evaluating the role of HO-1 as a cardioprotective and prohealing enzyme in experimental rat models and at investigating the potential mechanisms mediating the beneficial effects of HO-1 in the heart. The second aim was to evaluate the role of HO-1 in 231 critically ill intensive care unit (ICU) patients by investigating the association of HO-1 polymorphisms and HO-1 plasma concentrations with illness severity, organ dysfunction and mortality throughout the study population and in the subgroup of cardiac patients. We observed in an experimental rat MI model, that HO-1 expression was induced in the infarcted rat hearts, especially in the infarct and infarct border areas. In addition, pre-emptive HO-1 induction and CO donor pretreatment promoted recovery and repair of the infarcted hearts by differential mechanisms. CO promoted vasculogenesis and formation of new cardiomyocytes by activating c-kit+ stem/progenitor cells via hypoxia-inducible factor 1 alpha, stromal cell-derived factor 1 alpha (SDF-1a) and vascular endothelial growth factor B, whereas HO-1 promoted angiogenesis possibly via SDF-1a. Furthermore, HO-1 protected the heart in the early phase of infarct healing by increasing survival and proliferation of cardiomyocytes. The antiapoptotic effect of HO-1 persisted in the late phases of infarct healing. HO-1 also modulated the production of extracellular matrix components and reduced perivascular fibrosis. Some of these beneficial effects of HO-1 were mediated by CO, e.g. the antiapoptotic effect. However, CO may also have adverse effects on the heart, since it increased the expression of extracellular matrix components. In isolated perfused rat hearts, HO-1 induction improved the recovery of postischemic cardiac function and abrogated reperfusion-induced ventricular fibrillation, possibly in part via connexin 43. We found that HO-1 plasma levels were increased in all critically ill patients, including cardiac patients, and were associated with the degree of organ dysfunction and disease severity. HO-1 plasma concentrations were also higher in ICU and hospital nonsurvivors than in survivors, and the maximum HO-1 concentration was an independent predictor of hospital mortality. Patients with the HO-1 -413T/GT(L)/+99C haplotype had lower HO-1 plasma concentrations and lower incidence of multiple organ dysfunction. However, HO-1 polymorphisms were not associated with ICU or hospital mortality. The present study shows that HO-1 is induced in response to stress in both experimental animal models and severely ill patients. HO-1 played an important role in the recovery and repair of infarcted rat hearts. HO-1 induction and CO donor pretreatment enhanced cardiac regeneration after MI, and HO-1 may protect against pathological left ventricular remodeling. Furthermore, HO-1 induction potentially may protect against I/R injury and cardiac dysfunction in isolated rat hearts. In critically ill ICU patients, HO-1 plasma levels correlate with the degree of organ dysfunction, disease severity, and mortality, suggesting that HO-1 may be useful as a marker of disease severity and in the assessment of outcome of critically ill patients.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.

Physical activity and diabetic complications in patients with type 1 diabetes

Type 1 diabetes is associated with the risk for late diabetic complications which are divided into microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (cardiovascular disease, CVD) diseases. The risk for diabetic complication can be reduced by effective treatment, most importantly the glycaemic control. Glycaemia in type 1 diabetes is influenced by the interplay between insulin injections and lifestyle factors such as physical activity and diet. The effect of physical activity in patients with type 1 diabetes is not well known, however. The aim of this thesis was to investigate the physical activity and the physical fitness of patients with type 1 diabetes with special emphasis on glycaemic control and the diabetic complications. The patients included in the study were all part of the nationwide, multicenter Finnish Diabetic Nephropathy (FinnDiane) Study which aims to characterise genetic, clinical, and environmental factors that predispose to diabetic complications in patients with type 1 diabetes. In addition, subjects from the IDentification of EArly mechanisms in the pathogenesis of diabetic Late complications (IDEAL) Study were studied. Physical activity was assessed in the FinnDiane Study in 1945 patients by a validated questionnaire. Physical fitness was measured in the IDEAL Study by spiroergometry (cycle test with measurement of respiratory gases) in 86 young adults with type 1 diabetes and in 27 healthy controls. All patients underwent thorough clinical characterisation of their diabetic complication status. Four substudies were cross-sectional using baseline data and one study additionally used follow-up data. Physical activity, especially the intensity of activities, was reduced in patients affected by diabetic nephropathy, retinopathy, and CVD. Low physical activity was associated with poor glycaemic control, a finding most clear in women and evident also in patients with no signs of diabetic complications. Furthermore, low physical activity was associated with a higher HbA1c variability, which in turn was associated with the progression of renal disease and CVD during follow-up. A higher level of physical activity was also associated with better insulin sensitivity. The prevalence of the metabolic syndrome in type 1 diabetes was also lower the higher the physical activity. The aerobic physical fitness level of young adults with type 1 diabetes was reduced compared with healthy peers and in men an association between higher fitness level and lower HbA1c was observed. In patients with type 1 diabetes, a higher physical activity was associated with better glycaemic control and may thus be beneficial with respect to the prevention of diabetic complications.

Diabetesassocierade autoantikroppar hos barn med nydiagnostiserad typ 1 diabetes i relation till demografiska och kliniska faktorer

Syftet med studien var att undersöka sambanden mellan de diabetesassocierade autoantikropparna ICA, IAA, GADA, IA-2A och klinisk manifestation, HLA-genotyp, släktanamnes samt demografiska faktorer såsom ålder och kön hos finländska barn under 15 år med nydiagnostiserad typ 1 diabetes. Analyserna baserades på ett utdrag ur det finländska pediatriska diabetesregistret (2257 barn). Antikroppsfrekvenserna fastställdes utgående från halterna i serum. Alla barn HLA-genotypades och indelades i DR3- och DR4-positiva. Småbarnen (?5 år) hade ofta 3-4 positiva antikroppar. Äldre barn hade färre autoantikroppar men en allvarligare metabolisk dekompensering vid diagnostillfället. Diabetisk ketoacidos var vanligare hos flickor. I gruppen med endast en positiv autoantikropp var IA-2A-barnen oftare acidotiska, i övrigt påverkade inte antikroppsprofilen den kliniska bilden. Högriskgenotypen DR4/non-DR3 var associerad med IA-2A, som verkar fungera som en markör för betacelldestruktion. Det omfattande patientmaterialet gav stöd åt tidigare rapporter om samband mellan autoantikroppar och ålder, kön samt genotyp. Den allvarligare metaboliska dekompenseringen hos äldre barn tyder på att de inte diagnostiseras lika snabbt som småbarn.

Lapsuusiän kantasolusiirrot HUS:ssa vuosina 1993-2006

Tutkielma kuvaa lapsuusiän kantasolusiirtojen merkittävimmät tulokset ja komplikaatiot keskeisissä tautiryhmissä modernin hoidon aikakaudella HUS:ssä. Aineistona on HUS:n Lasten ja nuorten sairaalan veri- ja syöpätautien sekä kantasolusiirtoyksikön potilaista kerätty, vuonna 1993 perustetun ProLapsi-rekisterin sisältämä kliininen kantasolusiirtoaineisto vuosilta 1993-2006. Aineisto sisältää runsaat 90% Suomessa tehdyistä lasten allogeenisista kantasolusiirroista (n=233) sekä kaikki HUS:in autologiset siirrot (n=117) ko. aikajaksolla. Tutkielma on toteutettu kvantitatiivisia tutkimusmenetelmiä käyttäen. Suurin allogeenisen kantasolusiirron saaneiden potilaiden diagnoosiryhmä oli akuutti lymfoblastileukemia, ja suurin autologisen kantasolusiirron saaneiden ryhmä neuroblastoomapotilaat. Allogeenisista kantasolusiirroista 38% tehtiin HLA-identtiseltä sukulaisluovuttajalta ja 53% rekisteriluovuttajan soluilla. Kumulatiivinen kokonaisselviytyminen oli merkitsevästi parempaa sukulaisluovuttajan soluilla tehdyissä siirroissa kuin rekisteriluovuttajan (p=0,003). Allogeenisen kantasolusiirron saaneista potilaista 71% sai jonkin asteisen akuutin ja 42% kroonisen käänteishyljinnän (GVHD). Sekä akuutin että kroonisen GVHD:n vaikeus puolestaan korreloi kuolleisuuteen. Allogeenisen kantasolusiirron pitkäaikaisvaikutusta arvioitiin seurantatietojen perusteella. 58%:lla elämänlaatu arvioitiin normaaliksi, 35%:lla hieman rajoittuneeksi ja 7%:lla heikoksi.

Infliximabi-infuusioiden akuuttien haittavaikutusten esto asetyylisalisyylihapolla lapsipotilaiden IBD- ja reumasairauksien hoidossa

IBD- ja reumasairaudet ovat elämänlaatua merkittävästi heikentäviä, kroonisia tulehduksellisia sairauksia, joiden keskivaikeiden ja vakavien muotojen hoidossa suoneen annettava biologinen TNF-alfa -tulehdustekijän vasta-aine infliksimabi (Remicade®) on vankassa asemassa. Infliksimabin hyvän tehon kääntöpuolena esiintyy yleisesti haittoja, joista infuusion aikana tai pian sen jälkeen ilmenevät allergistyyppiset reaktiot ovat hoitoa hankaloittava ja jopa vaarallinen alaluokka. Infuusioreaktioiden estoon ei nykyisellään ole todistettavasti tehokkaita keinoja. Parasetamoli ja setiritsiini osoittautuivat tässä käyttötarkoituksessa tehottomiksi. HUS:n Lasten ja nuorten sairaalassa aloitettiin 11.3.2009 hoitokokeilu asetosalisyylihapolla (ASA, Disperin®), annosteltuna painonmukaisesti per os, päämääränä selvittää prospektiivisesti ASA:n käyttömahdollisuudet infuusioreaktioiden ehkäisyssä. Tämän tutkielman aineisto kerättiin esilääkekokeilun alun ja 24.6.2010 välisellä ajalla (yhteensä 67 viikkoa) infliksimabi-infuusiossa Lasten ja nuorten sairaalan osasto 2:lla käyneiden IBD- ja reumapotilaiden asiakirjoista. Vain 1 (0,2 %) ASA:n kanssa annetuista infuusioista johti infuusioreaktioon kun aiemmin parasetamolin ja setiritsiinin kanssa todettiin 11 (2,9 %) reaktiota. GraphPad Prism 5 -ohjelmistolla tehdyn tilastoanalyysin perusteella tulokset osoittavat ASA:n olevan erittäin lupaava infuusioreaktioiden estolääke.

MECHANISMS AND EFFECTS OF MITOCHONDRIAL DNA INSTABILITY AND COPY NUMBER MANIPULATION

Defects in mitochondrial DNA (mtDNA) maintenance cause a range of human diseases, including autosomal dominant progressive external ophthalmoplegia (adPEO). This study aimed to clarify the molecular background of adPEO. We discovered that deoxynucleoside triphosphate (dNTP) metabolism plays a crucial in mtDNA maintenance and were thus prompted to search for therapeutic strategies based on the modulation of cellular dNTP pools or mtDNA copy number. Human mtDNA is a 16.6 kb circular molecule present in hundreds to thousands of copies per cell. mtDNA is compacted into nucleoprotein clusters called nucleoids. mtDNA maintenance diseases result from defects in nuclear encoded proteins that maintain the mtDNA. These syndromes typically afflict highly differentiated, post-mitotic tissues such as muscle and nerve, but virtually any organ can be affected. adPEO is a disease where mtDNA molecules with large-scale deletions accumulate in patients tissues, particularly in skeletal muscle. Mutations in five nuclear genes, encoding the proteins ANT1, Twinkle, POLG, POLG2 and OPA1, have previously been shown to cause adPEO. Here, we studied a large North American pedigree with adPEO, and identified a novel heterozygous mutation in the gene RRM2B, which encodes the p53R2 subunit of the enzyme ribonucleotide reductase (RNR). RNR is the rate-limiting enzyme in dNTP biosynthesis, and is required both for nuclear and mitochondrial DNA replication. The mutation results in the expression of a truncated form of p53R2, which is likely to compete with the wild-type allele. A change in enzyme function leads to defective mtDNA replication due to altered dNTP pools. Therefore, RRM2B is a novel adPEO disease gene. The importance of adequate dNTP pools and RNR function for mtDNA maintenance has been established in many organisms. In yeast, induction of RNR has previously been shown to increase mtDNA copy number, and to rescue the phenotype caused by mutations in the yeast mtDNA polymerase. To further study the role of RNR in mammalian mtDNA maintenance, we used mice that broadly overexpress the RNR subunits Rrm1, Rrm2 or p53R2. Active RNR is a heterotetramer consisting of two large subunits (Rrm1) and two small subunits (either Rrm2 or p53R2). We also created bitransgenic mice that overexpress Rrm1 together with either Rrm2 or p53R2. In contrast to the previous findings in yeast, bitransgenic RNR overexpression led to mtDNA depletion in mouse skeletal muscle, without mtDNA deletions or point mutations. The mtDNA depletion was associated with imbalanced dNTP pools. Furthermore, the mRNA expression levels of Rrm1 and p53R2 were found to correlate with mtDNA copy number in two independent mouse models, suggesting nuclear-mitochondrial cross talk with regard to mtDNA copy number. We conclude that tight regulation of RNR is needed to prevent harmful alterations in the dNTP pool balance, which can lead to disordered mtDNA maintenance. Increasing the copy number of wild-type mtDNA has been suggested as a strategy for treating PEO and other mitochondrial diseases. Only two proteins are known to cause a robust increase in mtDNA copy number when overexpressed in mice; the mitochondrial transcription factor A (TFAM), and the mitochondrial replicative helicase Twinkle. We studied the mechanisms by which Twinkle and TFAM elevate mtDNA levels, and showed that Twinkle specifically implements mtDNA synthesis. Furthermore, both Twinkle and TFAM were found to increase mtDNA content per nucleoid. Increased mtDNA content in mouse tissues correlated with an age-related accumulation of mtDNA deletions, depletion of mitochondrial transcripts, and progressive respiratory dysfunction. Simultaneous overexpression of Twinkle and TFAM led to a further increase in the mtDNA content of nucleoids, and aggravated the respiratory deficiency. These results suggested that high mtDNA levels have detrimental long-term effects in mice. These data have to be considered when developing and evaluating treatment strategies for elevating mtDNA copy number.

Identification of genetic susceptibility loci for migraine

Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.

Very Low Birth Weight Infants as Young Adults : Focus on aspects of cognition, behavior and sleep

Major advances in the treatment of preterm infants have occurred during the last three decades. Survival rates have increased, and the first generations of preterm infants born at very low birth weight (VLBW; less than 1500 g) who profited from modern neonatal intensive care are now in young adulthood. The literature shows that VLBW children achieve on average lower scores on cognitive tests, even after exclusion of individuals with obvious neurosensory deficits. Evidence also exists for an increased risk in VLBW children for various neuropsychiatric disorders such as attention-deficit hyperactivity disorder (ADHD) and related behavioral symptoms. Up till now, studies extending into adulthood are sparse, and it remains to be seen whether these problems persist into adulthood. The aim of this thesis was to study ADHD-related symptoms and cognitive and executive functioning in young adults born at VLBW. In addition, we aimed to study sleep disturbances, known to adversely affect both cognition and attention. We hypothesized that preterm birth at VLBW interferes with early brain development in a way that alters the neuropsychological phenotype; this may manifest itself as ADHD symptoms and impaired cognitive abilities in young adulthood. In this cohort study from a geographically defined region, we studied 166 VLBW adults and 172 term-born controls born from 1978 through 1985. At ages 18 to 27 years, the study participants took part in a clinic study during which their physical and psychological health was assessed in detail. Three years later, 213 of these individuals participated in a follow-up. The current study is part of a larger research project (The Helsinki Study of Very Low Birth Weight Adults), and the measurements of interest for this particular study include the following: 1) The Adult Problem Questionnaire (APQ), a self-rating scale of ADHD-related symptoms in adults; 2) A computerized cognitive test battery designed for population studies (CogState®) which measures core cognitive abilities such as reaction time, working memory, and visual learning; 3) Sleep assessment by actigraphy, the Basic Nordic Sleep Questionnaire, and the Morningness-Eveningness Questionnaire. Actigraphs are wrist-worn accelerometers that separate sleep from wakefulness by registering body movements. Contrary to expectations, VLBW adults as a group reported no more ADHD-related behavioral symptoms than did controls. Further subdivision of the VLBW group into SGA (small for gestational age) and AGA (appropriate for gestational age) subgroups, however, revealed more symptoms on ADHD subscales pertaining to executive dysfunction and emotional instability among those born SGA. Thus, it seems that intrauterine growth retardation (for which SGA served as a proxy) is a more essential predictor for self-perceived ADHD symptoms in adulthood than is VLBW birth as such. In line with observations from other cohorts, the VLBW adults reported less risk-taking behavior in terms of substance use (alcohol, smoking, and recreational drugs), a finding reassuring for the VLBW individuals and their families. On the cognitive test, VLBW adults free from neurosensory deficits had longer reaction times than did term-born peers on all tasks included in the test battery, and lower accuracy on the learning task, with no discernible effect of SGA status over and above the effect of VLBW. Altogether, on a group level, even high-functioning VLBW adults show subtle deficits in psychomotor processing speed, visual working memory, and learning abilities. The sleep studies provided no evidence for differences in sleep quality or duration between the two groups. The VLBW adults were, however, at more than two-fold higher risk for sleep-disordered breathing (in terms of chronic snoring). Given the link between sleep-disordered breathing and health sequelae, these results suggest that VLBW individuals may benefit from an increased awareness among clinicians of this potential problem area. An unexpected finding from the sleep studies was the suggestion of an advanced sleep phase: The VLBW adults went to bed earlier according to the actigraphy registrations and also reported earlier wake-up times on the questionnaire. In further study of this issue in conjunction with the follow-up three years later, the VLBW group reported higher levels of morningness propensity, further corroborating the preliminary findings of an advanced sleep phase. Although the clinical implications are not entirely clear, the issue may be worth further study, since circadian rhythms are closely related to health and well-being. In sum, we believe that increased understanding of long-term outcomes after VLBW, and identification of areas and subgroups that are particularly vulnerable, will allow earlier recognition of potential problems and ultimately lead to improved prevention strategies.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Mental Disorders among Burn Patients

ABSTRACT Mental disorders and suicide attempts among acute burn patients were investigated in a collaborative research project between National Institute for Health and Welfare and Departments of Psychiatry and Plastic surgery in University of Helsinki in Finland. This project was realized in two parts. The first cohort of burn patients consisted of all burn patients admitted to the Helsinki Burn Centre during 1989 97. In this retrospective cohort, 5.7% (N=46) of the total of 811 burn patients had attempted suicide. The burn severity of suicide attempters was markedly higher than in the other burn patients. Suicide attempters were more often unemployed or on disability pension and had psychiatric history before the injury. The second sample was a prospective cohort of all acute consecutive burn patients admitted to the Helsinki Burn Centre during 18 months in 2006- 2007. All subjects (N=107) of the cohort were interviewed with the Structured Clinical Interview for DSM-IV for Axis I and II mental disorders (SCID-I and SCID-II) at baseline and then 86 % of all (N=92) with SCID-I at the end of six-month follow-up. Most (61%) patients had at least one lifetime mental disorder before burn; 47 % substance-related, 10% psychotic and 23% personality disorders. The overall prevalence of Axis I mental disorders increased significantly from the month prior to burn to acute care but decreased significantly from acute care to six months. However, more than one half (55%) of the cohort suffered from some mental disorder during follow-up. Less than one half of the burn patients with estimated need for psychiatric care received psychiatric care. Burn severity independently and strongly predicted risk for mental disorders during follow-up and pre-burn psychiatric history, severe burns and estimated need for psychiatric care significantly predicted psychiatric care received. The proportion of patients with self-inflicted burns is not high but mental disorders are common among burn patients. Mental disorders may predispose to burns. After burn injury, more than half of the patients suffer from mental disorders and a strong relationship exists between burn severity and some post-burn mental disorders. A minority of the patients with unequivocal need for psychiatric care actually receive it. Psychiatric consultations and care follow mainly the course of acute burn treatment.

Prognostic significance of cyclooxygenase-2 and associated molecules in gastric cancer

Background and aims: Low stage and curative surgery are established factors for improved survival in gastric cancer. However, not all low-stage patients have a good prognosis. Cyclooxygenase-2 (COX-2) is known to associate with reduced survival in several cancers, and has been shown to play an important role in gastric carcinogenesis. Since new and better prognostic markers are needed for gastric cancer, we studied the prognostic significance of COX-2 and of markers that associate with COX-2 expression. We also studied markers reflecting proliferation and apoptosis, and evaluated their association with COX-2. Our purpose was to construct an accurate prognostic model by combining tissue markers and clinicopathogical factors. Materials and methods: Of 342 consecutive patients who underwent surgery for gastric cancer at Meilahti Hospital, Helsinki University Central Hospital, 337 were included in this study. Low stages I to II were represented by 141 (42%) patients, and high stages III to IV by 196 (58%). Curative surgery was performed on 176 (52%) patients. Survival data were obtained from the national registers. Slides from archive tissue blocks were prepared for immunohistochemistry by use of COX-2, human antigen R (HuR), cyclin A, matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), and Ki-67 antibodies. Immunostainings were scored by microscopy, and scores were entered into a database. Associations of tumor markers with clinicopathological factors were calculated, as well as associations with p53, p21, and results of flow cytometry from earlier studies. Survival analysis was performed by the Kaplan-Meier method, and Cox multivariate models were reconstructed. Cell culture experiments were performed to explore the effect of small interfering (si)RNA of HuR on COX-2 expression in a TMK-1 gastric cancer cell line. Results: Overall 5-year survival was 35.1%. Study I showed that COX-2 was an independent prognostic factor, and that the prognostic impact of COX-2 was more pronounced in low-stage patients. Cytoplasmic HuR expression also associated with reduced survival in gastric cancer patients in a non-independent manner. Cell culture experiments showed that HuR can regulate COX-2 expression in TMK-1 cells in vitro, with an association also between COX-2 and HuR tissue expression in a clinical material. In Study II, cyclin A was an independent prognostic factor and was associated with HuR expression in the gastric cancer material. The results of Study III showed that epithelial MMP-2 associated with survival in univariate, but not in multivariate analysis. However, MMP-9 showed no prognostic value. MMP-2 expression was associated with COX-2 expression. In Study IV, the prognostic power of COX-2 was compared with that of all tested markers associated with survival in Studies I to III, as well as with p21, p53, and flow cytometry results. COX-2 and p53 were independent prognostic factors, and COX-2 expression was associated with that of p53 and Ki-67 and also with aneuploidy. Conclusions: COX-2 is an independent prognostic factor in gastric cancer, and its prognostic power emerges especially in low stage cancer. COX-2 is regulated by HuR, and is associated with factors reflecting invasion, proliferation, and apoptosis. In an extended multivariate model, COX-2 retained its position as an independent prognosticator. COX-2 can be considered a promising new prognostic marker in gastric cancer.

Cystatin C, a measure of renal function, as prognostic risk marker in acute heart failure : Studies on the cardiorenal syndrome

Acute heart failure (AHF) is a complex syndrome associated with exceptionally high mortality. Still, characteristics and prognostic factors of contemporary AHF patients have been inadequately studied. Kidney function has emerged as a very powerful prognostic risk factor in cardiovascular disease. This is believed to be the consequence of an interaction between the heart and kidneys, also termed the cardiorenal syndrome, the mechanisms of which are not fully understood. Renal insufficiency is common in heart failure and of particular interest for predicting outcome in AHF. Cystatin C (CysC) is a marker of glomerular filtration rate with properties making it a prospective alternative to the currently used measure creatinine for assessment of renal function. The aim of this thesis is to characterize a representative cohort of patients hospitalized for AHF and to identify risk factors for poor outcome in AHF. In particular, the role of CysC as a marker of renal function is evaluated, including examination of the value of CysC as a predictor of mortality in AHF. The FINN-AKVA (Finnish Acute Heart Failure) study is a national prospective multicenter study conducted to investigate the clinical presentation, aetiology and treatment of, as well as concomitant diseases and outcome in, AHF. Patients hospitalized for AHF were enrolled in the FINN-AKVA study, and mortality was followed for 12 months. The mean age of patients with AHF is 75 years and they frequently have both cardiovascular and non-cardiovascular co-morbidities. The mortality after hospitalization for AHF is high, rising to 27% by 12 months. The present study shows that renal dysfunction is very common in AHF. CysC detects impaired renal function in forty percent of patients. Renal function, measured by CysC, is one of the strongest predictors of mortality independently of other prognostic risk markers, such as age, gender, co-morbidities and systolic blood pressure on admission. Moreover, in patients with normal creatinine values, elevated CysC is associated with a marked increase in mortality. Acute kidney injury, defined as an increase in CysC within 48 hours of hospital admission, occurs in a significant proportion of patients and is associated with increased short- and mid-term mortality. The results suggest that CysC can be used for risk stratification in AHF. Markers of inflammation are elevated both in heart failure and in chronic kidney disease, and inflammation is one of the mechanisms thought to mediate heart-kidney interactions in the cardiorenal syndrome. Inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) correlate very differently to markers of cardiac stress and renal function. In particular, TNF-α showed a robust correlation to CysC, but was not associated with levels of NT-proBNP, a marker of hemodynamic cardiac stress. Compared to CysC, the inflammatory markers were not strongly related to mortality in AHF. In conclusion, patients with AHF are elderly with multiple co-morbidities, and renal dysfunction is very common. CysC demonstrates good diagnostic properties both in identifying impaired renal function and acute kidney injury in patients with AHF. CysC, as a measure of renal function, is also a powerful prognostic marker in AHF. CysC shows promise as a marker for assessment of kidney function and risk stratification in patients hospitalized for AHF.

Prognostic importance of the tumour microenvironment in follicular lymphoma patients treated with immunochemotherapy

Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma. It is an indolent and clinically heterogeneous disease, which is generally considered incurable. Currently, immunochemotherapy has significantly improved the outcome of FL patients. This is based on the combination of rituximab, a monoclonal anti-CD20 antibody, with chemotherapy, and is used at present as a standard first-line therapy in FL. Thus far, however, patients have been selected for treatment based on clinical risk factors and indices that were developed before the rituximab era. Therefore, there is a growing need to understand the molecular mechanisms underlying the disease, which would not only provide information to predict survival in the rituximab era, but also enable the design of more targeted therapeutic strategies. In this study, our aim was to identify genes predicting the outcome in FL patients treated with immunochemotherapy. Thus, we performed a cDNA microarray with 24 FL patients. When gene expression differences from diagnostic tumour samples were related to the clinical outcome, we identified novel genes with a prognostic impact on survival. The expression of selected genes was further characterized with quantitative PCR and immunohistochemistry (IHC). Interestingly, the prognostic influence of these genes was often associated with their expression in non-malignant cells instead of tumour cells. Based on the observed gene expression patterns, we analyzed the abundance and prognostic value of non-malignant immune cells in 95-98 FL patients treated with immunochemotherapy. We observed that a high content of tumour-associated macrophages was a marker of a favourable prognosis. In contrast, the accumulation of mast cells correlated with a poor outcome and was further associated with tumour vascularity. Increased microvessel density also correlated with an inferior outcome. In addition, we used the same microarray data with a systems biology approach to identify signalling pathways or groups of genes capable of separating patients with favourable or adverse outcomes. Among the transcripts, there were many genes associated with signal transducers and activators of the transcription (STAT5a) pathway. When IHC was used as validation, STAT5a expression was mostly observed in T-cells and follicular dendritic cells, and expression was found to predict a favourable outcome. In cell cultures, rituximab was observed to induce the expression of STAT5a-associated interleukins in human lymphoma cell lines, which might provide a possible link for the cross-talk between rituximab-induced FL cells and their microenvironment. In conclusion, we have demonstrated that the microenvironment has a prognostic role in FL patients treated with immunochemotherapy. The results also address the importance of re-evaluating the prognostic markers in the rituximab era of lymphoma therapies.

Consequences of Vertebral Fractures

Vertebral fractures occur due to forces applied to spinal structures. When the bone tissue is weakened, vertebral fractures can result from a minor trauma. Adult vertebral fractures are commonly considered to be an indication for osteoporosis. In children osteoporosis is a rare condition, and pediatric vertebral fractures are usually clearly trauma-related. The aims of this dissertation are to produce knowledge of the epidemiology of osteoporotic vertebral fractures and to analyse their association with total and cause-specific mortality, to find indicators with which to identify individuals who are at great risk of subsequent fractures, to study the incidence of pediatric vertebral fractures and need for their operative treatment and hospital care. The Mobile-Clinic and Mini-Finland Health surveys of the adult population were used as materials in this research. Record linkages to the Finnish Hospital Discharge Register and the Official Cause of Death register were used to study mortality and hospitalization in the same population group. These registers were also used to evaluate epidemiology, mortality, hospitalization and the need for operative management of pediatric vertebral fracture patients. The main findings and conclusions of the present dissertation are: 1. The presence of a thoracic vertebral fracture in adults is a significant predictor of cancer and respiratory mortality. In women, but not in men, vertebral fractures strongly predict mortality due to injuries. Most of these deaths in the study group were hip fracture related. 2. Severe thoracic vertebral fracture in adults was a strong predictor of a subsequent hip fracture, whereas mild or moderate fractures and the number of compressed vertebrae were much weaker predictors, 3. Pediatric spinal fractures were rare: The incidence was 66 per one million children per year. In younger children cervical spine was most often affected, whereas in older children fractures of the thoracic and lumbar spine were more common. Maturation of spinal structures seems to play a major role in the typical injury patterns in children. Thirty per cent of pediatric spinal fractures required surgical treatment. The current study focuses on consequences of vertebral fractures in general, without evaluating further the causation of the studied phenomena. Further studies are needed to clarify the mechanisms of association between vertebral fractures and specific causes of mortality. A severe vertebral fracture appears to indicate a substantial risk of a subsequent hip fracture. If such a fracture is identified from a chest radiograph, urgent clinical evaluation, treatment of osteoporosis and protective measures against falls are recommended.