Toxicity of dioxin to developing teeth and salivary glands : An experimental study

Dioxins are ubiquitous environmental poisons having unequivocal adverse health effects on various species. The majority of their effects are thought to be mediated by the aryl hydrocarbon receptor (AhR). Developing human teeth may be sensitive to dioxins and the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to rodent teeth. Mechanisms of TCDD toxicity can be studied only experimentally. The aim of the present thesis work was to delineate morphological end points of developmental toxicity of TCDD in rat and mouse teeth and salivary glands in vivo and in vitro and to characterize their cellular and molecular background. Mouse embryonic teeth and submandibular gland explants were grown in organ culture without/with TCDD at various concentrations, examined stereomicroscopically and processed for histological examination. The effects of TCDD on cellular mechanisms essential for organogenesis were investigated. The expression of various genes eliciting the response to TCDD exposure or involved in tooth and salivary gland development was studied at the mRNA and/or protein levels by in situ hybridization and immunohistochemistry. Association of the dental effects of TCDD with the resistance of a rat strain to TCDD acute lethality was analyzed in two lactationally exposed rat strains. The effect of TCDD on rat molar tooth mineralization was studied in tissue sections. TCDD dose- and developmental stage-dependently interfered with tooth formation. TCDD prevented early mouse molar tooth morphogenesis and altered cuspal morphology by enhancing programmend cell death, or apoptosis, in dental epithelial cells programmed to undergo apotosis. Cell proliferation was not affected. TCDD impaired mineralization of rat molar dental matrices, possibly by specifically reducing the expression of the mineralization-related dentin sialophosphoprotein gene shown in cultured mouse teeth. The impaired mineralization of rat teeth was accompanied by decreased expression of AhR and the TCDD-inducible xenobiotic-metabolozing enzyme P4501 A1 (CYP1A1), suggesting mediation of the TCDD effect by the AhR pathway. The severe interference by TCDD with rat incisor formation was independent of the genotypic variation of AhR determining the resistance of a rat strain to TCDD acute lethality. The impairment by TCDD of mouse submandibular gland branching morphogenesis was associated with CYP1A1 induction and involved blockage of EGF receptor signalling. In conclusion, TCDD exposure is likely to have activated the AhR pathway in target organs with the consequent activation of other signalling pathways involving developmentally regulated genes. The resultant phenotype is organ specific and modified by epithelial-mesenchymal interactions and dependent on dose as well as the stage of organogenesis at the time of TCDD exposure. Teeth appear to be responsive to TCDD exposure throughout their development.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

Periodontitis and peri-implantitis biomarkers in human oral fluids and the null-allele mouse model

Tissue destruction associated with the periodontal disease progression is caused by a cascade of host and microbial factors and proteolytic enzymes. Aberrant laminin-332 (Ln-332), human beta defensin (hBD), and matrix metalloproteinase (MMP) functions have been found in oral inflammatory diseases. The null-allele mouse model appears as the next step in oral disease research. The MMP-8 knock-out mouse model allowed us to clarify the involvement of MMP-8 in vivo in oral and related inflammatory diseases where MMP-8 is suggested to play a key role in tissue destruction. The cleaved Ln-332 γ2-chain species has been implicated in the apical migration of sulcular epithelial cells during the formation of periodontal pockets. We demonstrated that increased Ln-332 fragment levels in gingival crevicular fluid (GCF) are strongly associated with the severity of inflammation in periodontitis. Porphyromonas gingivalis trypsin-like proteinase can cleave an intact Ln-332 γ2-chain into smaller fragments and eventually promote the formation of periodontal pockets. hBDs are components of an innate mucosal defense against pathogenic microbes. Our results suggest that P. gingivalis trypsin-like proteinase can degrade hBD and thus reduce the innate immune response. Elevated levels and the increased activity of MMPs have been detected in several pathological tissue-destructive conditions where MMPs are shown to cleave extracellular matrix (ECM) and basement membrane (BM) molecules and to facilitate tissue destruction. Elevated levels of MMP-8 have been reported in many inflammatory diseases. In periodontitis, MMP-8 levels in gingival crevicular fluid (GCF) and in peri-implant sulcular fluid (PISF) are elevated at sites of active inflammation, and the increased levels of MMP-8 are mainly responsible for collagenase activity, which leads to tissue destruction. MMP-25, expressed by neutrophils, is involved in inflammatory diseases and in ECM turnover. MMP-26 can degrade ECM components and serve as an activator of other MMP enzymes. We further confirmed that increased levels and activation of MMP-8, -25, and -26 in GCF, PISF, and inflamed gingival tissue are associated with the severity of periodontal/peri-implant inflammation. We evaluated the role of MMP-8 in P. gingivalis-induced periodontitis by comparing MMP-8 knock-out (MMP8-/-) and wild-type mice. Surprisingly, MMP-8 significantly attenuated P. gingivalis-induced site-specific alveolar bone loss. We also evaluated systemic changes in serum immunoglobulin and lipoprotein profiles among these mouse groups. P. gingivalis infection increased HDL/VLDL particle size in the MMP-8-/- mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total LPS and IgG antibody levels were enhanced in both mice groups. P. gingivalis-induced periodontitis, especially in MMP-8-/- mice, is associated with severe alveolar bone loss and with systemic inflammatory and lipoprotein changes that are likely to be involved in early atherosclerosis.

Studies on Matrix Metalloproteinase (MMP-2, -9 and -14) and β2 Integrin Targeting as Potential Anticancer Therapeutics

Proteolytic enzymes, such as matrix metalloproteinases (MMP), are associated to the progression of several cancers. They degrade extracellular components, which helps tumors to expand and cancer cells to escape from the primary site. Of all MMPs, gelatinases (MMP-2 and -9) and membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), in particular, are often associated to more aggressive types of head and neck carcinomas as well as to a poorer outcome in patient survival. Although therapies during the last decades have advanced, the mortality of the disease is still rather high and adjuvant therapies are searched for continuously. MMP-9 and MT1-MMP are also involved in neo-angiogenesis, which is necessary for tumor expansion. For this reason, we have identified synthetic peptides-targeting gelatinases and MT1-MMP, and have also evaluated their anticancer effects in vitro and in vivo. Antigelatinolytic peptides effectively inhibited tongue-carcinoma cell invasion and reduced the growth of xenografted tumors. In tumor samples of mice that were treated with antigelatinolytic peptides, the micro-vessel density was significantly reduced. We also identified a novel MT1-MMP targeting peptide and demonstrated that it exerted anticancer effects against several malignant cell lines in vitro. The effects of MT1-MMP inhibition on tongue-squamous cell carcinomas were evaluated by using xenograft tumors, which it effectively inhibited. Tranexamic acid was also demonstrated to inhibit tongue-squamous cell carcinoma invasion, most probably due to its ability to prevent the plasmin-mediated activation of proMMP-9. Leukocyte β2 integrins are another interesting option when evaluating targets for the therapeutic intervention of inflammatory conditions or malignancies of hematopoietic origin, since β2 integrins are expressed mainly by leukocytes. We identified a novel technique for screening small-molecule libraries against β2 integrins, and by using this technique we identified a novel αMβ2 integrin-binding chemical (IMB-10). IMB-10 significantly enhances leukocyte adhesion and inhibits their motility. We also demonstrated that IMB-10 can be used to inhibit inflammation and lymphoma growth in vivo. Interestingly, IMB-10 also reduced leukocyte tumor infiltration and inhibited tumor invasion.

Cone beam computed tomography in oral radiology

In dentistry, basic imaging techniques such as intraoral and panoramic radiography are in most cases the only imaging techniques required for the detection of pathology. Conventional intraoral radiographs provide images with sufficient information for most dental radiographic needs. Panoramic radiography produces a single image of both jaws, giving an excellent overview of oral hard tissues. Regardless of the technique, plain radiography has only a limited capability in the evaluation of three-dimensional (3D) relationships. Technological advances in radiological imaging have moved from two-dimensional (2D) projection radiography towards digital, 3D and interactive imaging applications. This has been achieved first by the use of conventional computed tomography (CT) and more recently by cone beam CT (CBCT). CBCT is a radiographic imaging method that allows accurate 3D imaging of hard tissues. CBCT has been used for dental and maxillofacial imaging for more than ten years and its availability and use are increasing continuously. However, at present, only best practice guidelines are available for its use, and the need for evidence-based guidelines on the use of CBCT in dentistry is widely recognized. We evaluated (i) retrospectively the use of CBCT in a dental practice, (ii) the accuracy and reproducibility of pre-implant linear measurements in CBCT and multislice CT (MSCT) in a cadaver study, (iii) prospectively the clinical reliability of CBCT as a preoperative imaging method for complicated impacted lower third molars, and (iv) the tissue and effective radiation doses and image quality of dental CBCT scanners in comparison with MSCT scanners in a phantom study. Using CBCT, subjective identification of anatomy and pathology relevant in dental practice can be readily achieved, but dental restorations may cause disturbing artefacts. CBCT examination offered additional radiographic information when compared with intraoral and panoramic radiographs. In terms of the accuracy and reliability of linear measurements in the posterior mandible, CBCT is comparable to MSCT. CBCT is a reliable means of determining the location of the inferior alveolar canal and its relationship to the roots of the lower third molar. CBCT scanners provided adequate image quality for dental and maxillofacial imaging while delivering considerably smaller effective doses to the patient than MSCT. The observed variations in patient dose and image quality emphasize the importance of optimizing the imaging parameters in both CBCT and MSCT.

The function of Bmps and Runx2 in normal tooth development and in the pathogenesis of cleidocranial dysplasia

The development of many embryonic organs is regulated by reciprocal and sequential epithelial-mesenchymal interactions. These interactions are mediated by conserved signaling pathways that are reiteratively used. Cleidocranial dysplasia (CCD) is a congenital syndrome where both bone and tooth development is affected. The syndrome is characterized by short stature, abnormal clavicles, general bone dysplasia, and supernumerary teeth. CCD is caused by mutations in RUNX2, a transcription factor that is a key regulator of osteoblast differentiation and bone formation. The first aim of this study was to analyse the expression of a family of key signal molecules, Bone morphogenetic protein (Bmp) at different stages of tooth development. Bmps have a variety of functions and they were originally discovered as signals inducing ectopic bone formation. We performed a comparative in situ hybridisation analysis of the mRNA expression of Bmp2-7 from initiation of tooth development to differentiation of dental hard tissues. The expression patterns indicated that the Bmps signal between the epithelial and mesenchymal tissues during initiation and morphogenesis of tooth development, as well as during the differentiation of odontoblasts and ameloblasts. Furthermore, they are also part of the signalling networks whereby the enamel knot regulates the patterning of tooth cusps. The second aim was to study the role of Runx2 during tooth development and thereby to gain better understanding of the pathogenesis of the tooth phenotype in CCD. We analysed the tooth phenotype of Runx2 knockout mice and examined the patterns and regulation of Runx2 gene expression.. The teeth of wild-type and Runx2 mutant mice were compared by several methods including in situ hybridisation, tissue culture, bead implantation experiments, and epithelial-mesenchymal recombination studies. Phenotypic analysis of Runx2 -/- mutant tooth development showed that teeth failed to advance beyond the bud stage. Runx2 expression was restricted to dental mesenchyme between the bud and early bell stages of tooth development and it was regulated by epithelial signals, in particular Fgfs. We searched for downstream targets of Runx2 by comparative in situ hybridisation analysis. The expression of Fgf3 was downregulated in the mesenchyme of Runx2 -/- teeth. Shh expression was absent from the enamel knot in the lower molars of Runx2 -/- and reduced in the upper molars. In conclusion, these studies showed that Runx2 regulates key epithelial-mesenchymal interactions that control advancing tooth morphogenesis and histodifferentiation of the epithelial enamel organ. In addition, in the upper molars of Runx2 mutants extra buddings occured at the palatal side of the tooth bud. We suggest that Runx2 acts as an inhibitor of successional tooth formation by preventing advancing development of the buds. Accordingly, we propose that RUNX2 haploinsuffiency in humans causes incomplete inhibition of successional tooth formation and as a result supernumerary teeth.

Oral Health among Young Adults and the Middle-aged in Iran

The aim of the present study was to assess oral health and treatment needs among adult Iranians according to socio-demographic status, smoking, and oral hygiene, and to investigate the relationships between these determinants and oral health. Data for 4448 young adult (aged 18) and 8301 middle-aged (aged 35 to 44) Iranians were collected in 2002 as part of a national survey using the World Health Organization (WHO) criteria for sampling and clinical diagnoses, across 28 provinces by 33 calibrated examiners. Gender, age, place of residence, and level of education served as socio-demographic information, smoking as behavioural and modified plaque index (PI) as the biological risk indicator for oral hygiene. Number of teeth, decayed teeth (DT), filled teeth (FT), decayed, missing, filled teeth (DMFT), community periodontal index (CPI), and prosthodontic rehabilitation served as outcome variables of oral health. Mean number of DMFT was 4.3 (Standard deviation (SD) = 3.7) in young adults and 11.0 (SD = 6.4) among middle-aged individuals. Among young adults the D-component (DT = 70%), and among middle-aged individuals the M-component (60%) dominated in the DMFT index. Among young adults, visible plaque was found in nearly all subjects. Maximum (max) PI was associated with higher mean number of DT, and higher periodontal treatment needs. A healthy periodontium was a rare condition, with 8% of young adults and 1% of middle-aged individuals having a max CPI = 0. The majority of the CPI findings among young adults consisted of calculus (48%) and deepened periodontal pockets (21%). Respective values for middle-aged individuals were 40% and 53%. Having a deep pocket (max CPI = 4) was more likely among young adults with a low level of education (Odds ratio (OR) = 2.7, 95% Confidence interval (CI) = 1.9–4.0) than it was among well-educated individuals. Among middle-aged individuals, having calculus or a periodontal pocket was more likely in men (OR = 1.8, 95% CI = 1.6–2.0) and in illiterate subjects (OR = 6.3, 95% CI = 5.1–7.8) than it was for their counterparts. Among young adults, having 28 teeth was more (p < 0.05) prevalent among men (72% vs. 68% for women), urban residents (71% vs. 67% for rural residents), and those with a high level of education (73% vs. 60% for those with a low level). Among middle-aged individuals, having a functional dentition was associated with younger age (OR = 2.0, 95% CI = 1.7−2.5) and higher level of education (OR = 1.8, 95% CI = 1.6−2.1). Of middle-aged individuals, 2% of 35- to 39-year-olds and 5% of those aged 40 to 44 were edentulous. Among the dentate subjects (n = 7,925), prosthodontic rehabilitation was more prevalent (p < 0.001) among women, urban residents, and those with a high level of education than it was among their counterparts. Among those having 1 to 19 teeth, a removable denture was the most common type of prosthodontic rehabilitation. Middle-aged individuals lacking a functional dentition were more likely (OR = 6.0, 95% CI = 4.8−7.6) to have prosthodontic rehabilitation than were those having a functional dentition. In total, 81% of all reported being non-smokers, and 32% of men and 5% of women were current smokers. Heavy smokers were the most likely to have deepened periodontal pockets (max CPI ≥ 3, OR = 2.9, 95% CI = 1.8−4.7) and to have less than 20 teeth (OR = 2.3, 95% CI = 1.5−3.6). The findings indicate impaired oral health status in adult Iranians, particularly those of low socio-economic status and educational level. The high prevalence of dental plaque and calculus and considerable unmet treatment needs call for a preventive population strategy with special emphasis on the improvement of oral self-care and smoking cessation to tackle the underlying risk factors for oral diseases in the Iranian adult population.

Oral Actinomyces Species in Health and Disease: Identification, Occurence and Importance of Early Colonization

The genus Actinomyces consists of a heterogeneous group of gram-positive, mainly facultatively anaerobic or microaerobic rods showing various degrees of branching. In the oral cavity, streptococci and Actinomyces form a fundamental component of the indigenous microbiota, being among initial colonizers in polymicrobial biofilms. The significance of the genus Actinomyces is based on the capability of species to adhere to surfaces such as on teeth and to co-aggregate with other bacteria. Identification of Actinomyces species has mainly been based on only a few biochemical characteristics, such as pigmentation and catalase production, or on the use of a single commercial kit. The limited identification of oral Actinomyces isolates to species level has hampered knowledge of their role both in health and disease. In recent years, Actinomyces and related organisms have attracted the attention of clinical microbiologists because of a growing awareness of their presence in clinical specimens and their association with disease. This series of studies aimed to amplify the identification methods for Actinomyces species. With the newly developed identification scheme, the age-related occurrence of Actinomyces in healthy mouths of infants and their distribution in failed dental implants was investigated. Adhesion of Actinomyces species to titanium surfaces processed in various ways was studied in vitro. The results of phenotypic identification methods indicated a relatively low applicability of commercially available test kits for reliable identification within the genus Actinomyces. However, in the study of conventional phenotypic methods, it was possible to develop an identification scheme that resulted in accurate differentiation of Actinomyces and closely related species, using various different test methods. Genotypic methods based on 16S rRNA sequence analysis of Actinomyces proved to be a useful method for genus level identification and further clarified the species level identification with phenotypic methods. The results of the study of infants showed that the isolation frequency of salivary Actinomyces species increased according to age: thirty-one percent of the infants at 2 months but 97% at 2 years of age were positive for Actinomyces. A. odontolyticus was the most prominent Actinomyces colonizer during the study period followed in frequency by A. naeslundii and A. viscosus. In the study of explanted dental implants, Actinomyces was the most prevalent bacterial genus, colonizing 94% of the fixtures. Also in the implants A. odontolyticus was revealed as the most common Actinomyces species. It was present in 84% of Actinomyces -positive fixtures followed in frequency by A. naeslundii, A. viscosus and A. israelii. In an in vitro study of titanium surfaces, different Actinomyces species showed variation regarding their adhesion to titanium. Surface roughness as well as albumin coating of titanium had significant effects on adhesion. The use of improved phenotypic and molecular diagnostic methods increased the accuracy of the identification of the Actinomyces to species level. This facilitated an investigation of their occurrence and distribution in oral specimens in both health and disease.

Preadolescents and Their Mothers as Oral Health-Promoting Actors : Non-biologic Determinants of Oral Health among Turkish and Finnish Preadolescents

Is oral health becoming a part of the global health culture? Oral health seems to turn out to be part of the global health culture, according to the findings of a thesis-research, Institute of Dentistry, University of Helsinki. The thesis is entitled as “Preadolescents and Their Mothers as Oral Health-Promoting Actors: Non-biologic Determinants of Oral Health among Turkish and Finnish Preadolescents.” The research was supervised by Prof.Murtomaa and led by Dr.A.Basak Cinar. It was conducted as a cross-sectional study of 611 Turkish and 223 Finnish school preadolescents in Istanbul and Helsinki, from the fourth, fifth, and sixth grades, aged 10 to 12, based on self-administered and pre-tested health behavior questionnaires for them and their mothers as well as the youth’s oral health records. Clinically assessed dental status (DMFT) and self-reported oral health of Turkish preadolescents was significantly poorer than the Finns`. A similar association occurred for well-being measures (height and weight, self-esteem), but not for school performance. Turkish preadolescents were more dentally anxious and reported lower mean values of toothbrushing self-efficacy and dietary self-efficacy than did Finns. The Turks less frequently reported recommended oral health behaviors (twice daily or more toothbrushing, sweet consumption on 2 days or less/week, decreased between-meal sweet consumption) than did the Finns. Turkish mothers reported less frequently dental health as being above average and recommended oral health behaviors as well as regular dental visits. Their mean values for dental anxiety was higher and self-efficacy on implementation of twice-daily toothbrushing were lower than those of the Finnish. Despite these differences between the Turks and Finns, the associations found in common for all preadolescents, regardless of cultural differences and different oral health care systems, assessed for the first time in a holistic framework, were as follows: There seems to be interrelation between oral health and general-well being (body height-weight measures, school performance, and self-esteem) among preadolescents: • The body height was an explanatory factor for dental health, underlining the possible common life-course factors for dental health and general well-being. • Better school performance, high levels of self-esteem and self-efficacy were interrelated and they contributed to good oral health. • Good school performance was a common predictor for twice-daily toothbrushing. Self-efficacy and maternal modelling have significant role for maintenance and improvement of both oral- and general health- related behaviors. In addition, there is need for integration of self-efficacy based approaches to promote better oral health. • All preadolescents with high levels of self-efficacy were more likely to report more frequent twice-daily toothbrushing and less frequent sweet consumption. • All preadolescents were likely to imitate toothbrushing and sweet consumption behaviors of their mothers. • High levels of self-efficacy contributed to low dental anxiety in various patterns in both groups. As a conclusion: • Many health-detrimental behaviors arise from the school age years and are unlikely to change later. Schools have powerful influences on children’s development and well-being. Therefore, oral health promotion in schools should be integrated into general health promotion, school curricula, and other activities. • Health promotion messages should be reinforced in schools, enabling children and their families to develop lifelong sustainable positive health-related skills (self-esteem, self-efficacy) and behaviors. • Placing more emphasis on behavioral sciences, preventive approaches, and community-based education during undergraduate studies should encourage social responsibility and health-promoting roles among dentists. Attempts to increase general well-being and to reduce oral health inequalities among preadolescents will remain unsuccessful if the individual factors, as well as maternal and societal influences, are not considered by psycho-social holistic approaches.

Inflammation-induced atherogenesis, liver alterations, and cardiovascular outcome

Cardiovascular diseases, which presently are considered inflammatory diseases, affect millions of people worldwide. Chronic infections may contribute to the systemic inflammation suggested to increase the risk for cardiovascular diseases. Such chronic infections are periodontitis and Chlamydia pneumoniae infection. They are highly prevalent as approximately 10% of adult population and 30% of people over 50 years old are affected by severe periodontitis and 70-80% of elderly people are seropositive for C. pneumoniae. Our general aim was to investigate the role of infection and inflammation in atherosclerosis both in animal and human studies. We aimed to determine how the two pathogens alter the atherosclerosis-associated parameters, and how they affect the liver inflammation and lipid composition. Furthermore, we evaluated the association between matrix metalloproteinase-8 (MMP-8), a proteinase playing a major role in inflammation, and the future cardiovascular diseases (CVD) events in a population-based cohort. For the animal experiments, we used atherosclerosis-susceptible apolipoprotein E deficient (apoE-/-) mice. They were kept in germ free conditions and fed with a normal chow diet. The bacteria were administered either intravenously (A. actinomycetemcomitans) or intranasally (C. pneumoniae). Several factors were determined from serum as well as from aortic and hepatic tissues. We also determined how cholesterol efflux, a major event in the removal of excess cholesterol from the tissues, and endothelial function were affected by these pathogens. In the human study, serum MMP-8 and its tissue inhibitor (TIMP-1) concentrations were measured and their associations during the follow-up time of 10 years with CVD events were determined. An infection with A. actinomycetemcomitans increased concentrations of inflammatory mediators, MMP production, and cholesterol deposit in macrophages, decreased lipoprotein particle size, and induced liver inflammation. C. pneumoniae infection also elicited an inflammatory response and endothelial dysfunction, as well as induced liver inflammation, microvesicular appearance and altered fatty acid profile. In the population-based cohort, men with increased serum MMP-8 concentration together with subclinical atherosclerosis (carotid artery intima media thickness > 1mm) had a three-fold increased risk for CVD death during the follow-up. The results show that infections with A. actinomycetemcomitans and C. pneumoniae induce proatherogenic changes, as well as affect the liver. These data therefore support the concept that common infections have systemic effects and could be considered as cardiovascular risk factors. Furthermore, our data indicate that, as an independent predictor of fatal CVD event, serum MMP-8 could have a clinical significance in diagnosing cardiovascular diseases.

Drug Analysis without Primary Reference Standards : Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material

Drug Analysis without Primary Reference Standards: Application of LC-TOFMS and LC-CLND to Biofluids and Seized Material Primary reference standards for new drugs, metabolites, designer drugs or rare substances may not be obtainable within a reasonable period of time or their availability may also be hindered by extensive administrative requirements. Standards are usually costly and may have a limited shelf life. Finally, many compounds are not available commercially and sometimes not at all. A new approach within forensic and clinical drug analysis involves substance identification based on accurate mass measurement by liquid chromatography coupled with time-of-flight mass spectrometry (LC-TOFMS) and quantification by LC coupled with chemiluminescence nitrogen detection (LC-CLND) possessing equimolar response to nitrogen. Formula-based identification relies on the fact that the accurate mass of an ion from a chemical compound corresponds to the elemental composition of that compound. Single-calibrant nitrogen based quantification is feasible with a nitrogen-specific detector since approximately 90% of drugs contain nitrogen. A method was developed for toxicological drug screening in 1 ml urine samples by LC-TOFMS. A large target database of exact monoisotopic masses was constructed, representing the elemental formulae of reference drugs and their metabolites. Identification was based on matching the sample component s measured parameters with those in the database, including accurate mass and retention time, if available. In addition, an algorithm for isotopic pattern match (SigmaFit) was applied. Differences in ion abundance in urine extracts did not affect the mass accuracy or the SigmaFit values. For routine screening practice, a mass tolerance of 10 ppm and a SigmaFit tolerance of 0.03 were established. Seized street drug samples were analysed instantly by LC-TOFMS and LC-CLND, using a dilute and shoot approach. In the quantitative analysis of amphetamine, heroin and cocaine findings, the mean relative difference between the results of LC-CLND and the reference methods was only 11%. In blood specimens, liquid-liquid extraction recoveries for basic lipophilic drugs were first established and the validity of the generic extraction recovery-corrected single-calibrant LC-CLND was then verified with proficiency test samples. The mean accuracy was 24% and 17% for plasma and whole blood samples, respectively, all results falling within the confidence range of the reference concentrations. Further, metabolic ratios for the opioid drug tramadol were determined in a pharmacogenetic study setting. Extraction recovery estimation, based on model compounds with similar physicochemical characteristics, produced clinically feasible results without reference standards.

Early-onset depressive disorders,related mental health disorders and substance use-A prospective : longitudinal study of Finnish twins born 1983-1987

Early-onset psychiatric illnesses effects scatter to academic achievements as well as functioning in familial and social environments. From a public health point of view, depressive disorders are the most significant mental health disorders that begin in adolescence. Using prospective and longitudinal design, this study aimed to increase the understanding of early-onset depressive disorders, related mental health disorders and developing substance use in a large population-derived sample of adolescent Finnish twins. The participants of this study, FinnTwin12, an ongoing longitudinal population-based study, came from Finnish families with twins born in 1983-87 (exhaustive of five birth cohorts, identified from Finland s Central Population Register). With follow-up ongoing at age 20-24, this thesis assessed adolescent mental health in the first three waves, starting from baseline age 11-12 to follow-ups at age 14 and 17½. Some 5600 twins participated in questionnaire assessments of a wide range of health related behaviors. Mental health was further assessed among an intensively studied subsample of 1852 adolescents, who completed also professionally administered interviews at age 14, which provided data for full DSM-IV/III-R (Diagnostic and Statistical Manual for Mental Health disorders, 4th and 3rd editions) diagnoses. The participation rates of the study were 87-92%. The results of the study suggest, that the diagnostic criteria for major depressive disorder (MDD) may not capture youth with clinically significant early-onset depressive conditions outside clinical settings. Milder cases of depression, namely adolescents fulfilling the diagnostic criteria for minor depressive disorder, a qualitatively similar condition to MDD with fewer symptoms are also associated with marked suicidal thoughts, plans and attempts, recurrences and a high degree of comorbidity. Prospectively and longitudinally, early-onset depressive disorders were of substantial importance in the context of other mental health disorders and substance use behaviors: These data from a large population-derived sample established a substantial overlap between early-onset depressive disorders and attention deficit hyperactivity disorder in adolescent females, both of them significantly predictive for development of substance use among girls. Only in females baseline DSM-IV ADHD symptoms were strong predictors of alcohol abuse and dependence and illicit drug use at age 14 and frequent alcohol use and illicit drug use at age 17.½ when conduct disorder and previous substance use were controlled for. Early-onset depressive disorders were also prospectively and longitudinally associated to daily smoking behavior, smokeless tobacco use, frequent alcohol use and illicit drug use and eating disorders. Analysis of discordant twins suggested that these predictive associations were independent of familial confounds, such as family income, structure and parental models. In sum, early-onset depressive disorders predict subsequent involvement of substance use and psychiatric morbidity. A heightened risk for substance use is substantial also among those depressed below categorical diagnosis of MDD. Whether early recognition and interventions among these young people hold potential for substance use prevention further in their lives has potential public health significance and calls for more research. Data from this population-derived sample with balanced representation of boys and girls, suggested that boys and girls with ADHD behaviors may differ from each other in their vulnerability to substance use and depressive disorders: the data suggest more adverse substance use outcome for girls that was not attenuated by conduct disorder or previous substance use. Further, the prospective associations of early-onset depressive disorders and future elevated levels of addictive substance use is not explained by familial factors supporting future substance use, which could have important implications for substance use prevention.

Evidence-based perspective on CP rehabilitation : Reviews on physiotherapy, physiotherapy-related motor-based interventions and orthotic devices

This thesis utilises an evidence-based approach to critically evaluate and summarize effectiveness research on physiotherapy, physiotherapy-related motor-based interventions and orthotic devices in children and adolescents with cerebral palsy (CP). It aims to assess the methodological challenges of the systematic reviews and trials, to evaluate the effectiveness of interventions in current use, and to make suggestions for future trials Methods: Systematic reviews were searched from computerized bibliographic databases up to August 2007 for physiotherapy and physiotherapy-related interventions, and up to May 2003 for orthotic devices. Two reviewers independently identified, selected, and assessed the quality of the reviews using the Overview Quality Assessment Questionnaire complemented with decision rules. From a sample of 14 randomized controlled trials (RCT) published between January 1990 and June 2003 we analysed the methods of sampling, recruitment, and comparability of groups; defined the components of a complex intervention; identified outcome measures based on the International Classification of Functioning, Disability and Health (ICF); analysed the clinical interpretation of score changes; and analysed trial reporting using a modified 33-item CONSORT (Consolidated Standards of Reporting Trials) checklist. The effectiveness of physiotherapy and physiotherapy-related interventions in children with diagnosed CP was evaluated in a systematic review of randomised controlled trials that were searched from computerized databases from January 1990 up to February 2007. Two reviewers independently assessed the methodological quality, extracted the data, classified the outcomes using the ICF, and considered the level of evidence according to van Tulder et al. (2003). Results: We identified 21 reviews on physiotherapy and physiotherapy-related interventions and five on orthotic devices. These reviews summarized 23 or 5 randomised controlled trials and 104 or 27 observational studies, respectively. Only six reviews were of high quality. These found some evidence supporting strength training, constraint-induced movement therapy or hippotherapy, and insufficient evidence on comprehensive interventions. Based on the original studies included in the reviews on orthotic devices we found some short-term effects of lower limb casting on passive range of movement, and of ankle-foot orthoses on equinus walk. Long term effects of lower limb orthoses have not been studied. Evidence of upper limb casting or orthoses is conflicting. In the sample of 14 RCTs, most trials used simple randomisation, complemented with matching or stratification, but only three specified the concealed allocation. Numerous studies provided sufficient details on the components of a complex intervention, but the overlap of outcome measures across studies was poor and the clinical interpretation of observed score changes was mostly missing. Almost half (48%) of the applicable CONSORT-based items (range 28 32) were reported adequately. Most reporting inadequacies were in outcome measures, sample size determination, details of the sequence generation, allocation concealment and implementation of the randomization, success of assessor blinding, recruitment and follow-up dates, intention-to-treat analysis, precision of the effect size, co-interventions, and adverse events. The systematic review identified 22 trials on eight intervention categories. Four trials were of high quality. Moderate evidence of effectiveness was established for upper extremity treatments on attained goals, active supination and developmental status, and of constraint-induced therapy on the amount and quality of hand use and new emerging behaviours. Moderate evidence of ineffectiveness was found for strength training's effect on walking speed and stride length. Conflicting evidence was found for strength training's effect on gross motor function. For the other intervention categories the evidence was limited due to the low methodological quality and the statistically insignificant results of the studies. Conclusions: The high-quality reviews provide both supportive and insufficient evidence on some physiotherapy interventions. The poor quality of most reviews calls for caution, although most reviews drew no conclusions on effectiveness due to the poor quality of the primary studies. A considerable number of RCTs of good to fair methodological and reporting quality indicate that informative and well-reported RCTs on complex interventions in children and adolescents with CP are feasible. Nevertheless, methodological improvement is needed in certain areas of the trial design and performance, and the trial authors are encouraged to follow the CONSORT criteria. Based on RCTs we established moderate evidence for some effectiveness of upper extremity training. Due to limitations in methodological quality and variations in population, interventions and outcomes, mostly limited evidence on the effectiveness of most physiotherapy interventions is available to guide clinical practice. Well-designed trials are needed, especially for focused physiotherapy interventions.

Predictors of disability retirement: From early intentions to retirement

The strong tendency of elderly employees to retire early and the simultaneous aging of the population have been major topics of policy and scientific debate. A key concern has been the financing of future pension schemes and possible labour shortage, especially in social and health services within the public sector. The aging of the population is inevitable, but efforts can be made to prevent or postpone early exit from the labour force, e.g., by identifying and intervening in the factors that contribute to the process of early retirement due to disability. The associations of intentions to retire early, poor mental health and different psychosocial factors with the process of disability retirement are still poorly understood. The purpose of this study was to investigate the associations of intentions to retire early, poor mental health, work and family related psychosocial factors and experiences of earlier life stages with the process of disability retirement. The data were derived from the Helsinki Health Study (HHS, N=8960) and the Health and Social Support Study (HeSSup, N=25 901). The Helsinki Health Study is an ongoing employee cohort study among middle-aged women and men. The Health and Social Support Study is an ongoing longitudinal study of a working-age sample representative of the Finnish population. The analyses were restricted to respondents 40 years of age or older. Age and gender adjusted prevalence and incidence rates were calculated. Associations were studied by using logistic, multinomial and Cox regression. Strong intentions to retire early were common among employees. Poor mental health, unfavourable working conditions and work-to-family conflicts were clearly associated with increased intentions to retire early. Strong intentions to retire early predicted disability retirement. Risk of disability retirement increased in a dose-response manner with increasing number of childhood adversities. Poor mental and somatic health, life dissatisfaction, heavy alcohol consumption, current smoking, obesity and low socioeconomic status were also predictors of disability retirement. The impact of poor mental health and adverse experiences from earlier life stages, work and family related psychosocial factors, e.g., work-family interface, the subjective experience of well-being and health related risk behaviours on the process of disability retirement should be recognised. Preventive measures against disability retirement should be launched before subjective experience of ill health, work disability and strong intentions to retire early emerge.

Routine Measurement of Health-Related Quality of Life in Assessing Cost-Effectiveness in Secondary Health Care

Aims: The aims of this study were 1) to identify and describe health economic studies that have used quality-adjusted life years (QALYs) based on actual measurements of patients' health-related quality of life (HRQoL); 2) to test the feasibility of routine collection of health-related quality of life (HRQoL) data as an indicator of effectiveness of secondary health care; and 3) to establish and compare the cost-utility of three large-volume surgical procedures in a real-world setting in the Helsinki University Central Hospital, a large referral hospital providing secondary and tertiary health-care services for a population of approximately 1.4 million. Patients and methods: So as to identify studies that have used QALYs as an outcome measure, a systematic search of the literature was performed using the Medline, Embase, CINAHL, SCI and Cochrane Library electronic databases. Initial screening of the identified articles involved two reviewers independently reading the abstracts; the full-text articles were also evaluated independently by two reviewers, with a third reviewer used in cases where the two reviewers could not agree a consensus on which articles should be included. The feasibility of routinely evaluating the cost-effectiveness of secondary health care was tested by setting up a system for collecting HRQoL data on approximately 4 900 patients' HRQoL before and after operative treatments performed in the hospital. The HRQoL data used as an indicator of treatment effectiveness was combined with diagnostic and financial indicators routinely collected in the hospital. To compare the cost-effectiveness of three surgical interventions, 712 patients admitted for routine operative treatment completed the 15D HRQoL questionnaire before and also 3-12 months after the operation. QALYs were calculated using the obtained utility data and expected remaining life years of the patients. Direct hospital costs were obtained from the clinical patient administration database of the hospital and a cost-utility analysis was performed from the perspective of the provider of secondary health care services. Main results: The systematic review (Study I) showed that although QALYs gained are considered an important measure of the effectiveness of health care, the number of studies in which QALYs are based on actual measurements of patients' HRQoL is still fairly limited. Of the reviewed full-text articles, only 70 reported QALYs based on actual before after measurements using a valid HRQoL instrument. Collection of simple cost-effectiveness data in secondary health care is feasible and could easily be expanded and performed on a routine basis (Study II). It allows meaningful comparisons between various treatments and provides a means for allocating limited health care resources. The cost per QALY gained was 2 770 for cervical operations and 1 740 for lumbar operations. In cases where surgery was delayed the cost per QALY was doubled (Study III). The cost per QALY ranges between subgroups in cataract surgery (Study IV). The cost per QALY gained was 5 130 for patients having both eyes operated on and 8 210 for patients with only one eye operated on during the 6-month follow-up. In patients whose first eye had been operated on previous to the study period, the mean HRQoL deteriorated after surgery, thus precluding the establishment of the cost per QALY. In arthroplasty patients (Study V) the mean cost per QALY gained in a one-year period was 6 710 for primary hip replacement, 52 270 for revision hip replacement, and 14 000 for primary knee replacement. Conclusions: Although the importance of cost-utility analyses has during recent years been stressed, there are only a limited number of studies in which the evaluation is based on patients own assessment of the treatment effectiveness. Most of the cost-effectiveness and cost-utility analyses are based on modeling that employs expert opinion regarding the outcome of treatment, not on patient-derived assessments. Routine collection of effectiveness information from patients entering treatment in secondary health care turned out to be easy enough and did not, for instance, require additional personnel on the wards in which the study was executed. The mean patient response rate was more than 70 %, suggesting that patients were happy to participate and appreciated the fact that the hospital showed an interest in their well-being even after the actual treatment episode had ended. Spinal surgery leads to a statistically significant and clinically important improvement in HRQoL. The cost per QALY gained was reasonable, at less than half of that observed for instance for hip replacement surgery. However, prolonged waiting for an operation approximately doubled the cost per QALY gained from the surgical intervention. The mean utility gain following routine cataract surgery in a real world setting was relatively small and confined mostly to patients who had had both eyes operated on. The cost of cataract surgery per QALY gained was higher than previously reported and was associated with considerable degree of uncertainty. Hip and knee replacement both improve HRQoL. The cost per QALY gained from knee replacement is two-fold compared to hip replacement. Cost-utility results from the three studied specialties showed that there is great variation in the cost-utility of surgical interventions performed in a real-world setting even when only common, widely accepted interventions are considered. However, the cost per QALY of all the studied interventions, except for revision hip arthroplasty, was well below 50 000, this figure being sometimes cited in the literature as a threshold level for the cost-effectiveness of an intervention. Based on the present study it may be concluded that routine evaluation of the cost-utility of secondary health care is feasible and produces information essential for a rational and balanced allocation of scarce health care resources.