Cellular Mechanisms of Sleep Homeostasis : Studies on Brain Energy Metabolism and Aging

Sleep is governed by a homeostatic process in which the duration and quality of previous wake regulate the subsequent sleep. Active wakefulness is characterized with high frequency cortical oscillations and depends on stimulating influence of the arousal systems, such as the cholinergic basal forebrain (BF), while cessation of the activity in the arousal systems is required for slow wave sleep (SWS) to occur. The site-specific accumulation of adenosine (a by-product of ATP breakdown) in the BF during prolonged waking /sleep deprivation (SD) is known to induce sleep, thus coupling energy demand to sleep promotion. The adenosine release in the BF is accompanied with increases in extracellular lactate and nitric oxide (NO) levels. This thesis was aimed at further understanding the cellular processes by which the BF is involved in sleep-wake regulation and how these processes are affected by aging. The BF function was studied simultaneously at three levels of organization: 1) locally at a cellular level by measuring energy metabolites 2) globally at a cortical level (the out-put area of the BF) by measuring EEG oscillations and 3) at a behavioral level by studying changes in vigilance states. Study I showed that wake-promoting BF activation, particularly with glutamate receptor agonist N-methyl-D-aspatate (NMDA), increased extracellular adenosine and lactate levels and led to a homeostatic increase in the subsequent sleep. Blocking NMDA activation during SD reduced the high frequency (HF) EEG theta (7-9 Hz) power and attenuated the subsequent sleep. In aging, activation of the BF during SD or experimentally with NMDA (studies III, IV), did not induce lactate or adenosine release and the increases in the HF EEG theta power during SD and SWS during the subsequent sleep were attenuated as compared to the young. These findings implicate that increased or continuous BF activity is important for active wake maintenance during SD as well as for the generation of homeostatic sleep pressure, and that in aging these mechanisms are impaired. Study II found that induction of the inducible NO synthase (iNOS) during SD is accompanied with activation of the AMP-activated protein kinase (AMPK) in the BF. Because decreased cellular energy charge is the most common cause for AMPK activation, this finding implicates that the BF is selectively sensitive to the metabolic demands of SD as increases were not found in the cortex. In aging (study III), iNOS expression and extracellular levels of NO and adenosine were not significantly increased during SD in the BF. Furthermore, infusion of NO donor into the BF did not lead to sleep promotion as it did in the young. These findings indicated that the NO (and adenosine) mediated sleep induction is impaired in aging and that it could at least partly be due to the reduced sensitivity of the BF to sleep-inducing factors. Taken together, these findings show that reduced sleep promotion by the BF contributes to the attenuated homeostatic sleep response in aging.

Antizyme Inhibitor 2 : A Novel Regulator of Cellular Polyamine Homeostasis

Polyamines are organic polycations that participate in various physiological functions, including cell proliferation, differentiation and apoptosis. Cellular polyamines originate from endogenous biosynthesis and exogenous sources. Their subcellular pool is under strict control, achieved by regulating their uptake and metabolism. Polyamine-induced proteins called antizymes (AZ) act as key regulators of intracellular polyamine concentration. They regulate both the transport of polyamines and the activity and degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. AZs themselves are negatively regulated by antizyme inhibitor (AZIN). AZIN functions as a positive regulator of cellular polyamine homeostasis, which by binding to AZs reactivates ODC and induces the uptake of polyamines. In various pathological conditions, including cancer, polyamine levels are misregulated. Polyamine homeostasis has therefore become an attractive target for therapeutic interventions and it is thus crucial to characterize the molecular basis underlying the homeostatic regulation. A novel human AZIN-resembling protein was previously identified in our group. The purpose of this study was to elucidate the function and distribution of this protein, termed as an antizyme inhibitor 2 (AZIN2). According to my results, AZIN2 functions as a novel regulator of polyamine homeostasis. It shows no enzymatic activity, but instead it binds AZs and negates their activity, which subsequently leads to reactivation of ODC and inhibition of its degradation. Expression of AZIN2 is restricted to terminally differentiated cells, such as mast cells (MC) and neurosecretory cells. In these actively secreting cell types, AZIN2 localizes to subcellular vesicles or granules where its function is important for the vesicle-mediated secretion. In MCs, AZIN2 localizes to the serotonin-containing subset of MC granules, and its expression is coupled to MC activation. The functional role of polyamines as potential mediators of MC activity was also investigated, and it was observed that the secretion of serotonin is selectively dependent on activation of ODC. In neurosecretory cells, AZIN2-positive vesicles localize mainly to the trans-Golgi network (TGN). Depletion of AZIN2 or cellular polyamines causes selective fragmentation of the TGN and retards secretion of proteins. Since addition of exogenous polyamines reverses these effects, the data indicate that AZIN2 and its downstream effectors, polyamines, are functionally implicated in the regulation of secretory vesicle transport. My studies therefore reveal a novel function for polyamines as modulators of both constitutive and regulated secretion. Based on the results, I propose that the role of AZIN2 is to act as a local in situ activator of polyamine biosynthesis.

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

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

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Cholesterol Disturbances in Inherited Neurodegenerative Diseases : Studies on Npc1, Ppt1 and Ctsd deficient mice

The central nervous system (CNS) is the most cholesterol-rich organ in the body. Cholesterol is essential to CNS functions such as synaptogenesis and formation of myelin. Significant differences exist in cholesterol metabolism between the CNS and the peripheral organs. However, the regulation of cholesterol metabolism in the CNS is poorly understood compared to our knowledge of the regulation of cholesterol homeostasis in organs reached by cholesterol-carrying lipoprotein particles in the circulation. Defects in CNS cholesterol homeostasis have been linked to a variety of neurodegenerative diseases, including common diseases with complex pathogenetic mechanisms such as Alzheimer s disease. In spite of intense effort, the mechanisms which link disturbed cholesterol homeostasis to these diseases remain elusive. We used three inherited recessive neurodegenerative disorders as models in the studies included in this thesis: Niemann-Pick type C (NPC), infantile neuronal ceroid lipofuscinosis and cathepsin D deficiency. Of these three, NPC has previously been linked to disturbed intracellular cholesterol metabolism. Elucidating the mechanisms with which disturbances of cholesterol homeostasis link to neurodegeneration in recessive inherited disorders with known genetic lesions should shed light on how cholesterol is handled in the healthy CNS and help to understand how these and more complex diseases develop. In the first study we analyzed the synthesis of sterols and the assembly and secretion of lipoprotein particles in Npc1 deficient primary astrocytes. We found that both wild type and Npc1 deficient astrocytes retain significant amounts of desmosterol and other cholesterol precursor sterols as membrane constituents. No difference was observed in the synthesis of sterols and the secretion of newly synthesized sterols between Npc1 wild type, heterozygote or knockout astrocytes. We found that the incorporation of newly synthesized sterols into secreted lipoprotein particles was not inhibited by Npc1 mutation, and the lipoprotein particles were similar to those excreted by wild type astrocytes in shape and size. The bulk of cholesterol was found to be secreted independently of secreted NPC2. These observations demonstrate the ability of Npc1 deficient astrocytes to handle de novo sterols, and highlight the unique sterol composition in the developing brain. Infantile neuronal ceroid lipofuscinosis is caused by the deficiency of a functional Ppt1 enzyme in the cells. In the second study, global gene expression studies of approximately 14000 mouse genes showed significant changes in the expression of 135 genes in Ppt1 deficient neurons compared to wild type. Several genes encoding for enzymes of the mevalonate pathway of cholesterol biosynthesis showed increased expression. As predicted by the expression data, sterol biosynthesis was found to be upregulated in the knockout neurons. These data link Ppt1 deficiency to disturbed cholesterol metabolism in CNS neurons. In the third study we investigated the effect of cathepsin D deficiency on the structure of myelin and lipid homeostasis in the brain. Our proteomics data, immunohistochemistry and western blotting data showed altered levels of the myelin protein components myelin basic protein, proteolipid protein and 2 , 3 -cyclic nucleotide 3 phosphodiesterase in the brains of cathepsin D deficient mice. Electron microscopy revealed altered myelin structure in cathepsin D deficient brains. Additionally, plasmalogen-derived alkenyl chains and 20- and 24-carbon saturated and monounsaturated fatty acids typical for glycosphingolipids were found to be significantly reduced, but polyunsaturated species were significantly increased in the knockout brains, pointing to a decrease in white matter. The levels of ApoE and ABCA1 proteins linked to cholesterol efflux in the CNS were found to be altered in the brains of cathepsin D deficient mice, along with an accumulation of cholesteryl esters and a decrease in triglycerols. Together these data demonstrate altered myelin architecture in cathepsin D deficient mice and link cathepsin D deficiency to aberrant cholesterol metabolism and trafficking. Basic research into rare monogenic diseases sheds light on the underlying biological processes which are perturbed in these conditions and contributes to our understanding of the physiological function of healthy cells. Eventually, understanding gained from the study of disease models may contribute towards establishing treatment for these disorders and further our understanding of the pathogenesis of other, more complex and common diseases.

Transcriptional regulation by the orphan nuclear receptor ERRgamma

The nuclear receptor (NR) superfamily is comprised of receptors for small lipopfilic ligands such as steroid hormones, thyroid hormone, retinoids, and vitamin D. NRs are ligand-inducible transcription factors capable of both activating and repressing their target gene expression. They control a wide range of biological functions connected to growth, development, and homeostasis. In addition to the ligand-regulated receptors, the family includes a large group of receptors whose physiological ligands are unknown. These receptors are referred to as orphan NRs. Estrogen-related receptor gamma (ERRgamma) belongs to the ERR subfamily of orphan NRs together with the related ERRalpha and ERRbeta. ERRs share amino acid sequence homology with the classical estrogen receptors (ERs) but they are unable to bind natural estrogenic ligands. ERRgamma is expressed in several embryonic and adult tissues but its biological role is still largely unknown. ERRgamma activates reporter gene expression in transfected cells independently of added hormones implying that ERRgamma harbors constitutive activity. However, the intrinsic activity of ERRgamma can be inhibited by synthetic compounds such as the selective estrogen receptor modulator 4-hydroxytamoxifen (4-OHT). Ligands of NRs can act as agonists that activate transcription, as antagonists that prevent activation of transcription, or as inverse agonists that antagonize the constitutive transcriptional activity of receptor. Most of the synthetic ERRgamma ligands act as inverse agonists but recently, a synthetic ERRgamma agonist GSK4716 was identified. This demonstrates that it is possible to design and identify agonists for ERRgamma. Prior to this thesis work, the structural and functional characteristics of ERRgamma were largely unknown. The aim of this study was to define the functional requirements for ERRgamma-mediated transcriptional regulation and to examine the cross-talk between ERRgamma and other NRs. Due to the fact that natural physiological ligands of ERRgamma are unknown, another aim of this study was to seek new natural compounds that may affect transcriptional activity of ERRgamma. Plant-derived phytoestrogens have previously been shown to act as ligands for ERs and ERRalpha, and therefore the effects of these compounds were also studied on ERRgamma-mediated transcriptional regulation. This work demonstrated that ERRgamma-mediated transcriptional regulation was dependent on DNA-binding, dimerization and activation function-2. Heterodimerization with ERRalpha inhibited the transcriptional activity of ERRgamma. In addition to 4-OHT, another anti-estrogen, 4-hydroxytoremifene (4-OHtor), was identified as an inverse agonist of ERRgamma. Interestingly, ERRgamma activated transcription in the presence of 4-OHT and 4-OHtor on activator protein-1 binding sites. ERRgamma was found to interact with another orphan NR Nurr1 by repressing the ability of Nurr1 to activate transcription of the osteopontin gene. Transcriptional activity of ERRgamma was shown to be stimulated by the phytoestrogen equol. Structural model analysis and mutational experiments indicated that equol was able to bind to the ligand binding domain of ERRgamma. The growth inhibitory effect of ERRgamma on prostate cancer cells was found to be enhanced by equol. In summary, this study demonstrates that despite the absence of an endogenous physiological ligand, the activity of ERRgamma can be modulated in other ways such as dimerization with related receptors or by cross-talk with other transcription factors as well as by binding some synthetic or natural compounds.

Function and regulation of Cdk7

Understanding the process of cell division is crucial for modern cancer medicine due to the central role of uncontrolled cell division in this disease. Cancer involves unrestrained proliferation as a result of cells loosing normal control and being driven through the cell cycle, where they normally would be non-dividing or quiescent. Progression through the cell cycle is thought to be dependent on the sequential activation of cyclin-dependent kinases (Cdks). The full activation of Cdks requires the phosphorylation of a conserved residue (threonine-160 on human Cdk2) on the T-loop of the kinase domain. In metazoan species, a trimeric complex consisting of Cdk7, cyclin H and Mat1 has been suggested to be the T-loop kinase of several Cdks. In addition, Cdk7 have also been implicated in the regulation of transcription. Cdk7, cyclin H, and Mat1 can be found as subunits of general transcription factor TFIIH. Cdk7, in this context, phosphorylates the Carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (RNA pol II), specifically on serine-5 residues of the CTD repeat. The regulation of Cdk7 in these and other functions is not well known and the unambiguous characterization of the in vivo role of Cdk7 in both T-loop activation and CTD serine-5 phosphorylation has proved challenging. In this study, the fission yeast Cdk7-cyclin H homologous complex, Mcs6-Mcs2, is identified as the in vivo T-loop kinase of Cdk1(Cdc2). It also identifies multiple levels of regulation of Mcs6 kinase activity, i.e. association with Pmh1, a novel fission yeast protein that is the apparent homolog of metazoan Mat1, and T-loop phosphorylation of Mcs6, mediated by Csk1, a monomeric T-loop kinase with similarity to Cak1 of budding yeast. In addition, Skp1, a component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase is identified by its interactions with Mcs2 and Pmh1. The Skp1 association with Mcs2 and Pmh1 is however SCF independent and does not involve proteolytic degradation but may reflect a novel mechanism to modulate the activity or complex assembly of Mcs6. In addition to Cdk7, also Cdk8 has been shown to have CTD serine-5 kinase activity in vitro. Cdk8 is not essential in yeast but has been shown to function as a transcriptional regulator. The function of Cdk8 is unknown in flies and mammals. This prompted the investigation of murine Cdk8 and its potential role as a redundant CTD serine-5 kinase. We find that Cdk8 is required for development prior to implantation, at a time that is co-incident with a burst of Cdk8 expression during normal development. The results does not support a role of Cdk8 as a serine-5 CTD kinase in vivo but rather shows an unexpected requirement for Cdk8, early in mammalian development. The results presented in this thesis extends our current knowledge of the regulation of the cell cycle by characterizing the function of two distinct cell cycle regulating T-loop kinases, including the unambiguous identification of Mcs6, the fission yeast Cdk7 homolog, as the T-loop kinase of Cdk1. The results also indicate that the function of Mcs6 is conserved from fission yeast to human Cdk7 and suggests novel mechanisms by which the distinct functions of Cdk7 and Mcs6 could be regulated. These findings are important for our understanding of how progression of the cell cycle and proper transcription is controlled, during normal development and tissue homeostasis but also under condition where cells have escaped these control mechanisms e.g. cancer.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

The scientific basis and development of a matrix metalloproteinase (MMP) -8 specific chair-side test for monitoring of periodontal health and disease from gingival crevicular fluid

Matrix metalloproteinase (MMP) -8, collagenase-2, is a key mediator of irreversible tissue destruction in chronic periodontitis and detectable in gingival crevicular fluid (GCF). MMP-8 mostly originates from neutrophil leukocytes, the first line of defence cells which exist abundantly in GCF, especially in inflammation. MMP-8 is capable of degrading almost all extra-cellular matrix and basement membrane components and is especially efficient against type I collagen. Thus the expression of MMP-8 in GCF could be valuable in monitoring the activity of periodontitis and possibly offers a diagnostic means to predict progression of periodontitis. In this study the value of MMP-8 detection from GCF in monitoring of periodontal health and disease was evaluated with special reference to its ability to differentiate periodontal health and different disease states of the periodontium and to recognise the progression of periodontitis, i.e. active sites. For chair-side detection of MMP-8 from the GCF or peri-implant sulcus fluid (PISF) samples, a dip-stick test based on immunochromatography involving two monoclonal antibodies was developed. The immunoassay for the detection of MMP-8 from GCF was found to be more suitable for monitoring of periodontitis than detection of GCF elastase concentration or activity. Periodontally healthy subjects and individuals suffering of gingivitis or of periodontitis could be differentiated by means of GCF MMP-8 levels and dipstick testing when the positive threshold value of the MMP-8 chair-side test was set at 1000 µg/l. MMP-8 dipstick test results from periodontally healthy and from subjects with gingivitis were mainly negative while periodontitis patients sites with deep pockets ( 5 mm) and which were bleeding on probing were most often test positive. Periodontitis patients GCF MMP-8 levels decreased with hygiene phase periodontal treatment (scaling and root planing, SRP) and even reduced during the three month maintenance phase. A decrease in GCF MMP-8 levels could be monitored with the MMP-8 test. Agreement between the test stick and the quantitative assay was very good (κ = 0.81) and the test provided a baseline sensitivity of 0.83 and specificity of 0.96. During the 12-month longitudinal maintenance phase, periodontitis patients progressing sites (sites with an increase in attachment loss ≥ 2 mm during the maintenance phase) had elevated GCF MMP-8 levels compared with stable sites. General mean MMP-8 concentrations in smokers (S) sites were lower than in non-smokers (NS) sites but in progressing S and NS sites concentrations were at an equal level. Sites with exceptionally and repeatedly elevated MMP-8 concentrations during the maintenance phase were clustered in smoking patients with poor response to SRP (refractory patients). These sites especially were identified by the MMP-8 test. Subgingival plaque samples from periodontitis patients deep periodontal pockets were examined by polymerase chain reaction (PCR) to find out if periodontal lesions may serve as a niche for Chlamydia pneumoniae. Findings were compared with the clinical periodontal parameters and GCF MMP-8 levels to determine the correlation with periodontal status. Traces of C. pneumoniae were identified from one periodontitis patient s pooled subgingival plaque sample by means of PCR. After periodontal treatment (SRP) the sample was negative for C. pneumoniae. Clinical parameters or biomarkers (MMP-8) of the patient with the positive C. pneumoniae finding did not differ from other study patients. In this study it was concluded that MMP-8 concentrations in GCF of sites from periodontally healthy individuals, subjects with gingivitis or with periodontitis are at different levels. The cut-off value of the developed MMP-8 test is at an optimal level to differentiate between these conditions and can possibly be utilised in identification of individuals at the risk of the transition of gingivitis to periodontitis. In periodontitis patients, repeatedly elevated GCF MMP-8 concentrations may indicate sites at risk of progression of periodontitis as well as patients with poor response to conventional periodontal treatment (SRP). This can be monitored by MMP-8 testing. Despite the lower mean GCF MMP-8 concentrations in smokers, a fraction of smokers sites expressed very high MMP-8 concentrations together with enhanced periodontal activity and could be identified with MMP-8 specific chair-side test. Deep periodontal lesions may be niches for non-periodontopathogenic micro-organisms with systemic effects like C. pneumoniae and possibly play a role in the transmission from one subject to another.

Oral Immune Defense against Chronic Hyperplastic Candidosis

Candida yeast species are widespread opportunistic microbes, which are usually innocent opportunists unless the systemic or local defense system of the host becomes compromised. When they adhere on a fertile substrate such as moist and warm, protein-rich human mucosal membrane or biomaterial surface, they become activated and start to grow pseudo and real hyphae. Their growth is intricately guided by their ability to detect surface defects (providing secure hiding , thigmotropism) and nutrients (source of energy, chemotropism). The hypothesis of this work was that body mobilizes both non-specific and specific host defense against invading candidal cells and that these interactions involve resident epithelial cells, rapidly responding non-specific protector neutrophils and mast cells as well as the antigen presenting and responding den-dritic cell lymphocyte plasma cell system. It is supposed that Candida albicans, as a result of dar-winistic pressure, has developed or is utilizing strategies to evade these host defense reactions by e.g. adhering to biomaterial surfaces and biofilms. The aim of the study was to assess the host defense by taking such key molecules of the anti-candidal defense into focus, which are also more or less characteristic for the main cellular players in candida-host cell interactions. As a model for candidal-host interaction, sections of chronic hyperplastic candidosis were used and compared with sections of non-infected leukoplakia and healthy tissue. In this thesis work, neutrophil-derived anti-candidal α-defensin was found in the epithelium, not only diffusely all over in the epithelium, but as a strong α-defensin-rich superficial front probably able to slow down or prevent penetration of candida into the epithelium. Neutrophil represents the main host defence cell in the epithelium, to which it can rapidly transmigrate from the circulation and where it forms organized multicellular units known as microabscesses (study I). Neutrophil chemotactic inter-leukin-8 (IL-8) and its receptor (IL-8R) were studied and were surprisingly also found in the candidal cells, probably helping the candida to keep away from IL-8- and neutrophil-rich danger zones (study IV). Both leukocytes and resident epithelial cells contained TLR2, TLR4 and TLR6 receptors able to recognize candidal structures via utilization of receptors similar to the Toll of the banana fly. It seems that candida can avoid host defence via stimulation of the candida permissive TLR2 instead of the can-dida injurious TLR4 (study V). TLR also provides the danger signal to the immune system without which it will not be activated to specifically respond against candidal antigens. Indeed, diseased sites contained receptor activator of nuclear factor kappa B ligand (RANKL; II study), which is important for the antigen capturing, processing and presenting dendritic cells and for the T lymphocyte activation (study III). Chronic hyperplastic candidosis provides a disease model that is very useful to study local and sys-temic host factors, which under normal circumstances restrain C. albicans to a harmless commensal state, but failure of which in e.g. HIV infection, cancer and aging may lead to chronic infection.

Complement system and alcoholic liver disease

Alcoholic liver disease (ALD) is a well recognized and growing health problem worldwide. ALD advances from fatty liver to inflammation, necrosis, fibrosis and cirrhosis. There is accumulating evidence that the innate immune system is involved in alcoholic liver injury. Within the innate and acquired immune systems, the complement system participates in inflammatory reactions and in the elimination of invading foreign, as well as endogenous apoptotic or injured cells. The present study aimed at evaluating the role of the complement system in the development of alcoholic liver injury. First, in order to study the effects of chronic ethanol intake on the complement system, the deposition of complement components in liver and the expression of liver genes associated with complement in animals with alcohol-induced liver injury were examined. It was demonstrated that chronic alcohol exposure leads to hepatic deposition of the complement components C1, C3, C8 and C9 in the livers of rats. Liver gene expression analysis showed that ethanol up-regulated the expression of transcripts for complement factors B, C1qA, C2, C3 and clusterin. In contrast, ethanol down-regulated the expression of the complement regulators factor H, C4bp and factor D and the terminal complement components C6, C8α and C9. Secondly, the role of the terminal complement pathway in the development of ALD was evaluated by using rats genetically deficient in the complement component C6 (C6-/-). It was found that chronic ethanol feeding induced more liver pathology (steatosis and inflammatory changes) in C6-/- rats than in wild type rats. The hepatic triacylglyceride content and plasma alanine aminotransferase activity increased in C6-/- rats, supporting the histopathological findings and elevation of the plasma pro-/anti-inflammatory TNF-/IL-10 ratio was also more marked in C6-/- rats. Third, the role of the alternative pathway in the development of alcoholic liver steatosis was characterized by using C3-/- mice. In C3-/- mice ethanol feeding tended to reduce steatosis and had no further effect on liver triacylglyceride, liver/body weight ratio nor on liver malondialdehyde level and serum alanine aminotransferase activity. In C3-/- mice alcohol-induced liver steatosis was reduced also after an acute alcohol challenge. In both wild type and C3-/- mice ethanol markedly reduced serum cholesterol and ApoA-I levels, phospholipid transfer protein activity and hepatic mRNA levels of fatty acid binding proteins and fatty acid -oxidation enzymes. In contrast, exclusively in C3-/- mice, ethanol treatment increased serum and liver adiponectin levels but down-regulated the expression of transcripts of lipogenic enzymes, adiponectin receptor 2 and adipose differentiation-related protein and up-regulated phospholipase D1. In conclusion, this study has demonstrated that the complement system is involved in the development of alcohol-induced liver injury. Chronic alcohol exposure causes local complement activation and induction of mRNA expression of classical and alternative pathway components in the liver. In contrast expression of the terminal pathway components and soluble regulators were decreased. A deficient terminal complement pathway predisposes to alcoholic liver damage and promotes a pro-inflammatory cytokine response. Complement component C3 contributes to the development of alcohol-induced fatty liver and its consequences by affecting regulatory and specific transcription factors of lipid homeostasis.

VEGF-C/VEGFR-3 and PDGF-B/PDGFR-b pathways in embryonic lymphangiogenesis

The circulatory system comprises the blood vascular system and the lymphatic vascular system. These two systems function in parallel. Blood vessels form a closed system that delivers oxygen and nutrients to the tissues and removes waste products from the tissues, while lymphatic vessels are blind-ended tubes that collect extravasated fluid and cells from the tissues and return them back to blood circulation. Development of blood and lymphatic vascular systems occurs in series. Blood vessels are formed via vasculogenesis and angiogenesis whereas lymphatic vessels develop via lymphangiogenesis, after the blood vascular system is already functional. Members of the vascular endothelial growth factor (VEGF) family are regulators of both angiogenesis and lymphangiogenesis, while members of the platelet-derived growth factor (PDGF) family are major mitogens for pericytes and smooth muscle cells and regulate formation of blood vessels. Vascular endothelial growth factor C (VEGF-C) is the major lymphatic growth factor and signaling through its receptor vascular endothelial growth factor receptor 3 (VEGFR-3) is sufficient for lymphangiogenesis in adults. We studied the role of VEGF-C in embryonic lymphangiogenesis and showed that VEGF-C is absolutely required for the formation of lymph sacs from embryonic veins. VEGFR-3 is also required for normal development of the blood vascular system during embryogenesis, as Vegfr3 knockout mice die at mid-gestation due to failure in remodeling of the blood vessels. We showed that sufficient VEGFR-3 signaling in the embryo proper is required for embryonic angiogenesis and in a dosage-sensitive manner for embryonic lymphangiogenesis. Importantly, mice deficient in both VEGFR-3 ligands, Vegfc and Vegfd, developed a normal blood vasculature, suggesting VEGF-C- and VEGF-D- independent functions for VEGFR-3 in the early embryo. Platelet-derived growth factor B (PDGF-B) signals via PDGFR-b and regulates formation of blood vessels by recruiting pericytes and smooth muscle cells around nascent endothelial tubes. We showed that PDGF-B fails to induce lymphangiogenesis when overexpressed in adult mouse skin using adenoviral vectors. However, mouse embryos lacking Pdgfb showed abnormal lymphatic vessels, suggesting that PDGF-B plays a role in lymphatic vessel maturation and separation from blood vessels during embryogenesis. Lymphatic vessels play a key role in immune surveillance, fat absorption and maintenance of fluid homeostasis in the body. However, lymphatic vessels are also involved in various diseases, such as lymphedema and tumor metastasis. These studies elucidate the basic mechanisms of embryonic lymphangiogenesis and add to the knowledge of lymphedema and tumor metastasis treatments by giving novel insights into how lymphatic vessel growth could be induced (in lymphedema) or inhibited (in tumor metastasis).

Studies on immune activation in rheumatoid arthritis and reactive arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, progressive joint destruction, and disability. Reactive arthritis (ReA) is a sterile joint inflammation following a distant mucosal infection. The clinical course of these diseases is variable and cannot be predicted with reasonable accuracy by clinical and laboratory markers. The predictive value of circulating soluble interleukin-2 receptor (sIL-2R), a marker of lymphocyte activation, measured by Immulite® automated immunoassay analyzer, was evaluated in two cohorts of RA patients. In 175 patients with active early RA randomized to treatment with either on disease-modifying antirheumatic drug (DMARD) or a combination of 3 DMARDs and prednisolone, low baseline sIL-2R level predicted remission after 6 months in patients treated with a single DMARD. In 24 patients with active RA refractory to DMARDs, low baseline sIL-2R level predicted rapid clinical response to treatment with infliximab, an anti-tumour necrosis factor antibody. Furthermore, in a cohort of 26 patients with acute ReA, high baseline sIL-2R level predicted remission after 6 months. Levels of circulating soluble E-selectin (sE-selectin), a marker of endothelial activation, were measured annually by enzyme-linked immunosorbent assay (ELISA) in a cohort of 85 patients with early RA. During a five-year follow-up, sE-selectin levels were associated with activity and outcome of RA. The levels of neutrophil and monocyte CD11b/CD18 expression measured by flow cytometry, and circulating levels of sE-selectin measured by ELISA, and procalcitonin by immunoluminometric assay, were compared in 28 patients with acute ReA and 16 patients with early RA. The levels of the markers were comparable in ReA, RA, and healthy control subjects. In conlusion, sIL-2R may provide a new predictive marker in early RA treated with a single DMARD and refractory RA treated with infliximab. In addition, sIL-2R level predicts remission in acute ReA.

Stanniocalcin-1 in cell stress and differentiation

Stanniocalcin-1 (STC-1) is a 56 kD homodimeric protein which was originally identified in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. STC-1 was considered unique to fish until the cloning of cDNA for human STC-1 in 1995 and mouse Stc-1 in 1996. STC-1 is conserved through evolution with human and salmon STC-1 sharing 60% identity and 80% similarity. The surprisingly high homology between mammalian and fish STC-1 and the protective actions of STC-1 in terminally differentiated neurons, originally reported by my colleagues, prompted me to further study the role of STC-1 in cell stress and differentiation. One purpose was to determine whether there is an inter-relationship between terminally differentiated cells and STC-1 expression. The study revealed an accumulation of STC-1 in mature megakaryocytes and adipocytes, i.e. postmitotic cells with limited or lost proliferative capacity. Still proliferating uninduced cells were negative for STC-1 mRNA and protein, whereas differentiating cells accumulated STC-1 in their cytoplasm. Interestingly, in liposarcomas the grade inversely correlated with STC-1 expression. Another aim was to study how STC-1 gene expression is regulated. Given that IL-6 is a cytokine with neuroprotective actions, by unknown mechanisms, we examined whether IL-6 regulates STC-1 gene expression. Treatment of human neural Paju cells with IL-6 induced a dose-dependent upregulation of STC-1 mRNA levels. This induction of STC-1 expression by IL-6 occurred mainly through the MAPK signaling pathway. Furthermore, I studied the role of IL-6-mediated STC-1 expression as a mechanism of cytoprotection conferred by hypoxic preconditioning (HOPC) in brain and heart. My findings show that Stc-1 was upregulated in brain after hypoxia treatment. In the brain of IL-6 deficient mice, however, no upregulation of Stc-1 expression was evident. After induced brain injury the STC-1 response in brains of IL-6 transgenic mice, with IL-6 overexpression in astroglial cells, was stronger than in brains of WT mice. These results indicate that IL-6-mediated expression of STC-1 is one molecular mechanism of HOPC-induced tolerance to brain ischemia. The protection conferred by HOPC in heart occurs during a bimodal time course comprising early and delayed preconditioning. Interestingly, my results showed that the expression of Stc-1 in heart was upregulated in a biphasic manner during HOPC. IL-6 deficient mice did not, however, show a similar biphasic manner of Stc-1 upregulation as did WT mice. Instead, only an early upregulation of Stc-1 expression was evident. The results suggest that the upregulation of Stc-1 during the delayed preconditioning is IL-6-dependent. The upregulated expression of Stc-1 during the early preconditioning, however, is only partly IL-6-dependent and possibly also directly mediated by HIF-1. These findings suggest that STC-1 is a pro-survival protein for terminally differentiated cells and that STC-1 expression may in fact be regulated by stress. In addition, I show that STC-1 gene upregulation, mediated in part by IL-6, is a new mechanism of protection conferred by HOPC in brain and heart. Because of its importance for fundamental biological processes, such as differentiation and cytoprotection, STC-1 may have therapeutic implications for management of stroke, neurodegenerative diseases, cancer, and obesity.