Effects of Metabolic Risk Factors and Type 1 Diabetes on Brain Glucose and Brain Metabolites

The metabolic syndrome and type 1 diabetes are associated with brain alterations such as cognitive decline brain infarctions, atrophy, and white matter lesions. Despite the importance of these alterations, their pathomechanism is still poorly understood. This study was conducted to investigate brain glucose and metabolites in healthy individuals with an increased cardiovascular risk and in patients with type 1 diabetes in order to discover more information on the nature of the known brain alterations. We studied 43 20- to 45-year-old men. Study I compared two groups of non-diabetic men, one with an accumulation of cardiovascular risk factors and another without. Studies II to IV compared men with type 1 diabetes (duration of diabetes 6.7 ± 5.2 years, no microvascular complications) with non-diabetic men. Brain glucose, N-acetylaspartate (NAA), total creatine (tCr), choline, and myo-inositol (mI) were quantified with proton magnetic resonance spectroscopy in three cerebral regions: frontal cortex, frontal white matter, thalamus, and in cerebellar white matter. Data collection was performed for all participants during fasting glycemia and in a subgroup (Studies III and IV), also during a hyperglycemic clamp that increased plasma glucose concentration by 12 mmol/l. In non-diabetic men, the brain glucose concentration correlated linearly with plasma glucose concentration. The cardiovascular risk group (Study I) had a 13% higher plasma glucose concentration than the control group, but no difference in thalamic glucose content. The risk group thus had lower thalamic glucose content than expected. They also had 17% increased tCr (marker of oxidative metabolism). In the control group, tCr correlated with thalamic glucose content, but in the risk group, tCr correlated instead with fasting plasma glucose and 2-h plasma glucose concentration in the oral glucose tolerance test. Risk factors of the metabolic syndrome, most importantly insulin resistance, may thus influence brain metabolism. During fasting glycemia (Study II), regional variation in the cerebral glucose levels appeared in the non-diabetic subjects but not in those with diabetes. In diabetic patients, excess glucose had accumulated predominantly in the white matter where the metabolite alterations were also the most pronounced. Compared to the controls values, the white matter NAA (marker of neuronal metabolism) was 6% lower and mI (glia cell marker) 20% higher. Hyperglycemia is therefore a potent risk factor for diabetic brain disease and the metabolic brain alterations may appear even before any peripheral microvascular complications are detectable. During acute hyperglycemia (Study III), the increase in cerebral glucose content in the patients with type 1 diabetes was, dependent on brain region, between 1.1 and 2.0 mmol/l. An every-day hyperglycemic episode in a diabetic patient may therefore as much as double brain glucose concentration. While chronic hyperglycemia had led to accumulation of glucose in the white matter, acute hyperglycemia burdened predominantly the gray matter. Acute hyperglycemia also revealed that chronic fluctuation in blood glucose may be associated with alterations in glucose uptake or in metabolism in the thalamus. The cerebellar white matter appeared very differently from the cerebral (Study IV). In the non-diabetic men it contained twice as much glucose as the cerebrum. Diabetes had altered neither its glucose content nor the brain metabolites. The cerebellum seems therefore more resistant to the effects of hyperglycemia than is the cerebrum.

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.

At the Limits of Presentation : Coming-into-Presence and its Aesthetic Relevance in Jean-Luc Nancy's Philosophy

This study investigates the significance of art in Jean-Luc Nancy s philosophy. I argue that the notion of art contributes to some of Nancy s central ontological ideas. Therefore, I consider art s importance in its own right whether art does have ontological significance, and if so, how one should describe this with respect to the theme of presentation. According to my central argument, with his thinking on art Nancy attempts to give one viewpoint to what is called the metaphysics of presence and to its deconstruction. On which grounds, as I propose, may one say that art is not reducible to philosophy? The thesis is divided into two main parts. The first part, Presentation as a Philosophical Theme, is a historical genesis of the central concepts associated with the birth of presentation in Nancy s philosophy. I examine this from the viewpoint of the differentiation between the ontological notions of presentation and representation by concentrating on the influence of Martin Heidegger and Jacques Derrida, as well as of Hegel and Kant. I give an overview of the way in which being or sense for Nancy is to be described as a coming-into-presence or presentation . Therefore, being takes place in its singular plurality. I argue that Nancy redevelops Heidegger s account of being in two principal ways: first, in rethinking the ontico-ontological difference, and secondly, by striving to radicalize the Heideggerian concept of Mitsein, being-with . I equally wish to show the importance of Derrida s notion of différance and its inherence in Nancy s questioning of being that rests on the unfoundedness of existence. The second part, From Ontology to Art, draws on the importance of art and the aesthetic. If, in Nancy, the question of art touches upon its own limit as the limit of nothingness, how is art able to open its own strangeness and our exposure to this strangeness? My aim is to investigate how Nancy s thinking on art finds its place within the conceptual realm of its inherent difference and interval. My central concern is the thought of originary ungroundedness and the plurality of art and of the arts. As for the question of the difference between art and philosophy, I wish to show that what differentiates art from thought is the fact that art exposes what is obvious but not apparent, if apparent is understood in the sense of givenness. As for art s ability to deconstruct Nancy s ontological notions, I suggest that in question in art is its original heterogeneity and diversity. Art is a matter of differing art occurs singularly, as a local difference. With this in mind, I point out that in reflecting on art in terms of spacing and interval, as a thinker of difference Nancy comes closer to Derrida and his idea of différance than to the structure of Heidegger s ontological difference.

Encounters with the Virtual : The Experience of Art In Gilles Deleuze's Philosophy

My thesis concerns the notion of existence as an encounter, as developed in the philosophy of Gilles Deleuze (1925 1995). What this denotes is a critical stance towards a major current in Western philosophical tradition which Deleuze nominates as representational thinking. Such thinking strives to provide a stable ground for identities by appealing to transcendent structures behind the apparent reality and explaining the manifest diversity of the given by such notions as essence, idea, God, or totality of the world. In contrast to this, Deleuze states that abstractions such as these do not explain anything, but rather that they need to be explained. Yet, Deleuze does not appeal merely to the given. He sees that one must posit a genetic element that accounts for experience, and this element must not be naïvely traced from the empirical. Deleuze nominates his philosophy as transcendental empiricism and he seeks to bring together the approaches of both empiricism and transcendental philosophy. In chapter one I look into the motivations of Deleuze s transcendental empiricism and analyse it as an encounter between Deleuze s readings of David Hume and Immanuel Kant. This encounter regards, first of all, the question of subjectivity and results in a conception of identity as non-essential process. A pre-given concept of identity does not explain the nature of things, but the concept itself must be explained. From this point of view, the process of individualisation must become the central concern. In chapter two I discuss Deleuze s concept of the affect as the basis of identity and his affiliation with the theories of Gilbert Simondon and Jakob von Uexküll. From this basis develops a morphogenetic theory of individuation-as-process. In analysing such a process of individuation, the modal category of the virtual becomes of great value, being an open, indeterminate charge of potentiality. As the virtual concerns becoming or the continuous process of actualisation, then time, rather than space, will be the privileged field of consideration. Chapter three is devoted to the discussion of the temporal aspect of the virtual and difference-without-identity. The essentially temporal process of subjectification results in a conception of the subject as composition: an assemblage of heterogeneous elements. Therefore art and aesthetic experience is valued by Deleuze because they disclose the construct-like nature of subjectivity in the sensations they produce. Through the domain of the aesthetic the subject is immersed in the network of affectivity that is the material diversity of the world. Chapter four addresses a phenomenon displaying this diversified indentity: the simulacrum an identity that is not grounded in an essence. Developed on the basis of the simulacrum, a theory of identity as assemblage emerges in chapter five. As the problematic of simulacra concerns perhaps foremost the artistic presentation, I shall look into the identity of a work of art as assemblage. To take an example of a concrete artistic practice and to remain within the problematic of the simulacrum, I shall finally address the question of reproduction particularly in the case recorded music and its identity regarding the work of art. In conclusion, I propose that by overturning its initial representational schema, phonographic music addresses its own medium and turns it into an inscription of difference, exposing the listener to an encounter with the virtual.

Childhood and adolescent antecedents of work stress and early atherosclerosis

Work stress is after musculoskeletal disorders the second most common work-related health problem in the European Union, affecting 28% of EU employees. Furthermore, a 50% excessive cardiovascular disease risk among employees with work stress is reported. High job demands combined with low job control according to the Job Demands-Job Control model, or high effort combined with low rewards according to Effort-Reward Imbalance model, are likely to produce work stress in the majority of employees. Atherosclerotic wall thickening is a validated marker of an increased risk of cardiovascular disease. This study examined the role of childhood and adolescent factors as antecedents of work stress and early atherosclerosis, and in the relationship between them. The Cardiovascular Risk in Young Finns Study, (the CRYF project) started in 1980 when the participants were at the age of three to 18 years. Follow-ups have been conducted every three years until 1992, after that in 1997 and 2001, and the latest is ongoing in 2008. The participants parents reported their socioeconomic position in 1980 and 1983, and their life satisfaction in 1983. Biological risk factors were measured in 1980 and 2001. Type A behaviour was reported in 1986, 1989 and 2001. In the 2001 follow-up when the participants were aged 24 to 39, work stress was assessed from responses to questionnaires on job demands-job control and effort-reward imbalance, and education. Ultrasound measurement of the carotid intima-media thickness (CIMT) was used to assess atherosclerosis. There were 755, 746, 1014 and 494 participants in studies I-IV, respectively. The results showed that low parental socioeconomic position and parental life dissatisfaction during childhood and adolescence predicted higher levels of job strain 18 years later, and that education mediated the relationship between parental socioeconomic position and job strain. Childhood and adolescent family factors were not related to the effort-reward imbalance. Parental life satisfaction was associated with high rewards at work among the men, and high parental socioeconomic position was associated with high reward among the women. Among the men, the eagerness-energy component of Type A behaviour across different developmental periods predicted increased CIMT. Among the women, hard-driving component of Type A behaviour predicted decreased CIMT. Low leadership characteristic in adolescence and early adulthood was associated with both high job strain and increased CIMT, and attenuated the relationship between job strain and CIMT to non-significance in men. The current findings add to the literature on the relationship between job strain and health literature in adopting a developmental perspective. The results imply that work stress does not completely originate from work. There are childhood and adolescent environmental and dispositional effects on work stress and CIMT several years later, and these partly seem to operate through educational attainment.

Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

The Role of Genes on Chromosome Locus 4q12 in Human Central Nervous System Tumors

Human central nervous system (CNS) tumors are a heterogeneous group of tumors occurring in brain, brainstem and spinal cord. Malignant gliomas (astrocytic and oligodendroglial tumors), which arise from the neuroepithelial cells are the most common CNS neoplasms in human. Malignant gliomas are highly aggressive and invasive tumors, and have a very poor prognosis. The development and progression of gliomas involve a stepwise accumulation of genetic alterations that generally affect either signal transduction pathways activated by receptor tyrosine kinases (RTKs), or cell cycle arrest pathways. Constitutive activation or deregulated signaling by RTKs is caused by gene amplification, overexpression or mutations. The aberrant RTK signaling results in turn in the activation of several downstream pathways, which ultimately lead to malignant transformation and tumor proliferation. Many genetic abnormalities implicated in nervous system tumors involve the genes located at the chromosomal region 4q12. This locus harbors the receptor tyrosine kinases KIT, PDGFRA and VEGFR2, and other genes (REST, LNX1) with neural function. Gene amplification and protein expression of KIT, PDGFRA, and VEGFR2 was studied using clinical tumor material. REST and LNX1, as well as NUMBL, the interaction partner of LNX1, were studied for their gene mutations and amplifications. In our studies, amplification of LNX1 was associated with KIT and PDGFRA amplification in glioblastomas, and coamplification of KIT, PDGFRA and VEGFR2 was detected in medulloblastomas and CNS primitive neuroectodermal tumors. PDGFRA amplification was also correlated with poor overall survival. Coamplification of KIT, PDGFRA and VEGFR2 was observed in a subset of human astrocytic and oligodendroglial tumors. We suggest that genes at 4q12 could be a part of a larger amplified region, which is deregulated in gliomas, and could be used as a prognostic marker of tumorigenic process. The signaling pathways activated due to gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment. This study also includes the characterization of KIT overexpressing astrocytes, analyzed by various in vitro functional assays. Our results show that overexpression of KIT in mouse astrocytes promotes cell proliferation and anchorage-independent growth, as well as phenotypic changes in the cells. Furthermore, the increased proliferation is partly inhibited by imatinib, a small molecule inhibitor of KIT. These results suggest that KIT may play a role in astrocyte growth regulation, and might have an oncogenic role in brain tumorigenesis. Elucidation of the altered signaling pathways due to specific gene amplifications, activating gene mutations, and overexpressed proteins may be useful as therapeutic targets for glioma treatment.

Markers of systemic inflammation in diagnostics and in prediction of outcome of community-acquired infection

Sepsis is associated with a systemic inflammatory response. It is characterised by an early proinflammatory response and followed by a state of immunosuppression. In order to improve the outcome of patients with infection and sepsis, novel therapies that influence the systemic inflammatory response are being developed and utilised. Thus, an accurate and early diagnosis of infection and evaluation of immune state are crucial. In this thesis, various markers of systemic inflammation were studied with respect to enhancing the diagnostics of infection and of predicting outcome in patients with suspected community-acquired infection. A total of 1092 acutely ill patients admitted to a university hospital medical emergency department were evaluated, and 531 patients with a suspicion of community-acquired infection were included for the analysis. Markers of systemic inflammation were determined from a blood sample obtained simultaneously with a blood culture sample on admission to hospital. Levels of phagocyte CD11b/CD18 and CD14 expression were measured by whole blood flow cytometry. Concentrations of soluble CD14, interleukin (IL)-8, and soluble IL-2 receptor α (sIL-2Rα) were determined by ELISA, those of sIL-2R, IL-6, and IL-8 by a chemiluminescent immunoassay, that of procalcitonin by immunoluminometric assay, and that of C-reactive protein by immunoturbidimetric assay. Clinical data were collected retrospectively from the medical records. No marker of systemic inflammation, neither CRP, PCT, IL-6, IL-8, nor sIL-2R predicted bacteraemia better than did the clinical signs of infection, i.e., the presence of infectious focus or fever or both. IL-6 and PCT had the highest positive likelihood ratios to identify patients with hidden community-acquired infection. However, the use of a single marker failed to detect all patients with infection. A combination of markers including a fast-responding reactant (CD11b expression), a later-peaking reactant (CRP), and a reactant originating from inflamed tissues (IL-8) detected all patients with infection. The majority of patients (86.5%) with possible but not verified infection showed levels exceeding at least one cut-off limit of combination, supporting the view that infection was the cause of their acute illness. The 28-day mortality of patients with community-acquired infection was low (3.4%). On admission to hospital, the low expression of cell-associated lipopolysaccharide receptor CD14 (mCD14) was predictive for 28-day mortality. In the patients with severe forms of community-acquired infection, namely pneumonia and sepsis, high levels of soluble CD14 alone did not predict mortality, but a high sCD14 level measured simultaneously with a low mCD14 raised the possibility of poor prognosis. In conclusion, to further enhance the diagnostics of hidden community-acquired infection, a combination of inflammatory markers is useful; 28-day mortality is associated with low levels of mCD14 expression at an early phase of the disease.

Regulation of strawberry growth and development

Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.

Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

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

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

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.

Novel matrix metalloproteinases in intestinal inflammation and in cancer of the gastrointestinal tract

Matrix metalloproteinases (MMPs) comprise a family of 23 zinc-dependent human endopeptidases that can degrade virtually all components of the extracellular matrix (ECM). They are classified into eight subgroups according to their structure and into six subgroups based on their substrate-specificity. MMPs have been implicated in inflammation, tissue destruction, cell migration, arthritis, vascular remodeling, angiogenesis, and tumor growth and invasion. MMPs are inhibited by their natural inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Different MMPs function in the same tasks depending on the tissue or cancer subtype. I investigated the role of recently discovered MMPs, especially MMPs-19 and -26, in intestinal inflammation, in intestinal and cutaneous wound healing, and in intestinal cancer. Several MMPs and TIMPs were studied to determine their exact location at tissue level and to obtain information on possible functions of MMPs in such tissues and diseases as the healthy intestine, inflammatory bowel disease (IBD), neonatal necrotizing enterocolitis (NEC), pyoderma gangrenosum (PG), and colorectal as well as pancreatic cancers. In latent celiac disease (CD), I attempted to identify markers to predict later onset of CD in children and adolescents. The main methods used were immunohistochemistry, in situ hybridization, and Taqman RT-PCR. My results show that MMP-26 is important for re-epithelialization in intestinal and cutaneous wound healing. In colon and pancreatic cancers, MMP-26 seems to be a marker of invasive potential, although it is not itself expressed at the invasive front. MMP-21 is upregulated in pancreatic cancer and may be associated with tumor differentiation. MMPs-19 and -28 are associated with normal tissue turnover in the intestine, but they disappear in tumor progression as if they were protective markers . MMP-12 is an essential protease in intestinal inflammation and tissue destruction, as seen here in NEC and in previous CD studies. In patients with type 1 diabetes (T1D), MMPs-1, -3, and -12 were upregulated in the intestinal mucosa. Furthermore, MMP-7 was strongly elevated in NEC. In a model of aberrant wound repair, PG, MMPs-8, -9, and 10 and TNFα may promote ECM destruction, while absence of MMP-1 and MMP-26 from keratinocytes retards re-epithelialization. Based on my results, I suggest MMP-26 to be considered a putative marker for poor prognosis in pancreatic and colon cancer. However, since it functions differently in various tissues and tumor subtypes, this use cannot be generalized. Furthermore, MMP-26 is a beneficial marker for wound healing if expressed by migrating epithelial cells. MMP-12 expression in latent CD patients warrants research in a larger patient population to confirm its role as a specific marker for CD in pathologically indistinct cases. MMP-7 should be considered one of the most crucial proteases in NEC-associated tissue destruction; hence, specific inhibitors of this MMP are worth investigating. In PG, TNFα inhibitors are potential therapeutic agents, as shown already in clinical trials. In conclusion, studies of several MMPs in specific diseases and in healthy tissues are needed to elucidate their roles at the tissue level. MMPs and TIMPs are not exclusively destructive or reparative in tissues. They seem to function differently in different tissues. To identify selective MMP inhibitors, we must thoroughly understand the MMP profile (degradome) and their functions in various organs not to interfere with normal reparative functions during wound repair or beneficial host-response effects during cancer initiation and growth.

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.

Determinants of Coronary and Carotid Atherosclerosis in Finnish Patients with Clinically Suspected Coronary Artery Disease. A Quantitative Angiography and Ultrasound Study

Background. Cardiovascular disease (CVD) remains the most serious threat to life and health in industrialized countries. Atherosclerosis is the main underlying pathology associated with CVD, in particular coronary artery disease (CAD), ischaemic stroke, and peripheral arterial disease. Risk factors play an important role in initiating and accelerating the complex process of atherosclerosis. Most studies of risk factors have focused on the presence or absence of clinically defined CVD. Less is known about the determinants of the severity and extent of atherosclerosis in symptomatic patients. Aims. To clarify the association between coronary and carotid artery atherosclerosis, and to study the determinants associated with these abnormalities with special regard to novel cardiovascular risk factors. Subjects and methods. Quantitative coronary angiography (QCA) and B-mode ultrasound were used to assess coronary and carotid artery atherosclerosis in 108 patients with clinically suspected CAD referred for elective coronary angiography. To evaluate anatomic severity and extent of CAD, several QCA parameters were incorporated into indexes. These measurements reflected CAD severity, extent, and overall atheroma burden and were calculated for the entire coronary tree and separately for different coronary segments (i.e., left main, proximal, mid, and distal segments). Maximum and mean intima-media thickness (IMT) values of carotid arteries were measured and expressed as mean aggregate values. Furthermore, the study design included extensive fasting blood samples, oral glucose tolerance test, and an oral fat-load test to be performed in each participant. Results. Maximum and mean IMT values were significantly correlated with CAD severity, extent, and atheroma burden. There was heterogeneity in associations between IMT and CAD indexes according to anatomical location of CAD. Maximum and mean IMT values, respectively, were correlated with QCA indexes for mid and distal segments but not with the proximal segments of coronary vessels. The values of paraoxonase-1 (PON1) activity and concentration, respectively, were lower in subjects with significant CAD and there was a significant relationship between PON1 activity and concentration and coronary atherosclerosis assessed by QCA. PON1 activity was a significant determinant of severity of CAD independently of HDL cholesterol. Neither PON1 activity nor concentration was associated with carotid IMT. The concentration of triglycerides (TGs), triglyceride-rich lipoproteins (TRLs), oxidized LDL (oxLDL), and the cholesterol content of remnant lipoprotein particle (RLP-C) were significantly increased at 6 hours after intake of an oral fatty meal as compared with fasting values. The mean peak size of LDL remained unchanged 6 hours after the test meal. The correlations between total TGs, TRLs, and RLP-C in fasting and postprandial state were highly significant. RLP-C correlated with oxLDL both in fasting and in fed state and inversely with LDL size. In multivariate analysis oxLDL was a determinant of severity and extent of CAD. Neither total TGs, TRLs, oxLDL, nor LDL size were linked to carotid atherosclerosis. Insulin resistance (IR) was associated with an increased severity and extent of coronary atherosclerosis and seemed to be a stronger predictor of coronary atherosclerosis in the distal parts of the coronary tree than in the proximal and mid parts. In the multivariate analysis IR was a significant predictor of the severity of CAD. IR did not correlate with carotid IMT. Maximum and mean carotid IMT were higher in patients with the apoE4 phenotype compared with subjects with the apoE3 phenotype. Likewise, patients with the apoE4 phenotype had a more severe and extensive CAD than individuals with the apoE3 phenotype. Conclusions. 1) There is an association between carotid IMT and the severity and extent of CAD. Carotid IMT seems to be a weaker predictor of coronary atherosclerosis in the proximal parts of the coronary tree than in the mid and distal parts. 2) PON1 activity has an important role in the pathogenesis of coronary atherosclerosis. More importantly, the study illustrates how the protective role of HDL could be modulated by its components such that equivalent serum concentrations of HDL cholesterol may not equate with an equivalent, potential protective capacity. 3) RLP-C in the fasting state is a good marker of postprandial TRLs. Circulating oxLDL increases in CAD patients postprandially. The highly significant positive correlation between postprandial TRLs and postprandial oxLDL suggests that the postprandial state creates oxidative stress. Our findings emphasize the fundamental role of LDL oxidation in the development of atherosclerosis even after inclusion of conventional CAD risk factors. 4) Disturbances in glucose metabolism are crucial in the pathogenesis of coronary atherosclerosis. In fact, subjects with IR are comparable with diabetic subjects in terms of severity and extent of CAD. 5) ApoE polymorphism is involved in the susceptibility to both carotid and coronary atherosclerosis.