Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.

Uterine cervix nitric oxide and human papillomavirus infection in women

Most women acquire genital high risk human papillomavirus (HPV) infection during their lifetime, but seldom the infection persists and leads to cervical cancer. However, currently it is not possible to identify the women who will develop HPV mediated cervical cancer and this often results to large scale follow-up and overtreatment of the likely spontaneously regressing infection. Thus, it is important to obtain more information on the course of HPV and find markers that could help to identify HPV infected women in risk for progression of cervical lesions and ultimately cancer. Nitric oxide is a free radical gas that takes part both in immune responses and carcinogenesis. Nitric oxide is produced also by cervical cells and therefore, it is possible that cervical nitric oxide could affect also HPV infection. In the present study, including 801 women from the University of Helsinki between years of 2006 and 2011, association between HPV and cervical nitric oxide was evaluated. The levels of nitric oxide were measured as its metabolites nitrate and nitirite (NOx) by spectrophotometry and the expression of nitric oxide producing enzymes endothelial and inducible synthases (eNOS, iNOS) by Western blotting. Women infected with HPV had two-times higher cervical fluid NOx levels compared with non-infected ones. The expression levels of both eNOS and iNOS were higher in HPV-infected women compared with non-infected. Another sexually transmitted disease Chlamydia trachomatis that is an independent risk factor for cervical cancer was also accompanied with elevated NOx levels, whereas vaginal infections, bacterial vaginosis and candida, did not have any effect on NOx levels. The meaning of the elevated HPV related cervical nitric oxide was evaluated in a 12 months follow-up study. It was revealed that high baseline cervical fluid NOx levels favored HPV persistence with OR 4.1. However, low sensitivity (33%) and high false negative rate (67%) restrict the clinical use of the current NOx test. This study indicated that nitric oxide favors HPV persistence and thus it seems to be one of the cofactor associated with a risk of carcinogenesis.

Prognostic markers in head and neck carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Well-known risk factors include tobacco smoking and alcohol consumption. Overall survival has improved, but is still low especially in developing countries. One reason for this is the often advanced stage of the disease at the time of diagnosis, but also lack of reliable prognostic tools to enable individualized patient treatment to improve outcome. To date, the TNM classification still serves as the best disease evaluation criterion, although it does not take into account the molecular basis of the tumor. The need for surrogate molecular markers for more accurate disease prediction has increased research interests in this field. We investigated the prevalence, physical status, and viral load of human papillomavirus (HPV) in HNSCC to determine the impact of HPV on head and neck carcinogenesis. The prevalence and genotyping of HPV were assessed with an SPF10 PCR microtiter plate-based hybridization assay (DEIA), followed by a line probe-based genotyping assay. More than half of the patients had HPV DNA in their tumor specimens. Oncogenic HPV-16 was the most common type, and coinfections with other oncogenic and benign associated types also existed. HPV-16 viral load was unevenly distributed among different tumor sites; the tonsils harbored significantly greater amounts of virus than other sites. Episomal location of HPV-16 was associated with large tumors, and both integrated and mixed forms of viral DNA were detected. In this series, we could not show that the presence of HPV DNA correlated with survival. In addition, we investigated the prevalence and genotype of HPV in laryngeal carcinoma patients in a prospective Nordic multicenter study based on fresh-frozen laryngeal tumor samples to determine whether the tumors were HPV-associated. These patients were also examined and interviewed at diagnosis for known risk factors, such as tobacco smoking and alcohol consumption, and for several other habituations to elucidate their effects on patient survival. HPV analysis was performed with the same protocols as in the first study. Only 4% of the specimens harbored HPV DNA. Heavy drinking was associated with poor survival. Heavy drinking patients were also younger than nonheavy drinkers and had a more advanced stage of disease at diagnosis. Heavy drinkers had worse oral hygiene than nonheavy drinkers; however, poor oral hygiene did not have prognostic significance. History of chronic laryngitis, gastroesophageal reflux disease, and orogenital sex contacts were rare in this series. To clarify why vocal cord carcinomas seldom metastasize, we determined tumor lymph vessel (LVD) and blood vessel (BVD) densities in HNSCC patients. We used a novel lymphatic vessel endothelial marker (LYVE-1 antibody) to locate the lymphatic vessels in HNSCC samples and CD31 to detect the blood microvessels. We found carcinomas of the vocal cords to harbor less lymphatic and blood microvessels than carcinomas arising from sites other than vocal cords. The lymphatic and blood microvessel densities did not correlate with tumor size. High BVD was strongly correlated with high LVD. Neither BVD nor LVD showed any association with survival in our series. The immune system plays an important role in tumorigenesis, as neoplastic cells have to escape the cytotoxic lymphocytes in order to survive. Several candidate HLA class II alleles have been reported to be prognostic in cervical carcinomas, an epithelial malignancy resembling HNSCC. These alleles may have an impact on head and neck carcinomas as well. We determined HLA-DRB1* and -DQB1* alleles in HNSCC patients. Healthy organ donors served as controls. The Inno-LiPA reverse dot-blot kit was used to identify alleles in patient samples. No single haplotype was found to be predictive of either the risk for head and neck cancer, or the clinical course of the disease. However, alleles observed to be prognostic in cervical carcinomas showed a similar tendency in our series. DRB1*03 was associated with node-negative disease at diagnosis. DRB1*08 and DRB1*13 were associated with early-stage disease; DRB1*04 had a lower risk for tumor relapse; and DQB1*03 and DQB1*0502 were more frequent in controls than in patients. However, these associations reached only borderline significance in our HNSCC patients.

The role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-α (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene’s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.

Structural and Functional Studies on Trimeric Autotransporters

Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.

Synthesis of neoglycoconjugates and oligosaccharides with potential anti-Helicobacter pylori activity

The significance of carbohydrate-protein interactions in many biological phenomena is now widely acknowledged and carbohydrate based pharmaceuticals are under intensive development. The interactions between monomeric carbohydrate ligands and their receptors are usually of low affinity. To overcome this limitation natural carbohydrate ligands are often organized as multivalent structures. Therefore, artificial carbohydrate pharmaceuticals should be constructed on the same concept, as multivalent carbohydrates or glycoclusters. Infections of specific host tissues by bacteria, viruses, and fungi are among the unfavorable disease processes for which suitably designed carbohydrate inhibitors represent worthy targets. The bacterium Helicobacter pylori colonizes more than half of all people worldwide, causing gastritis, gastric ulcer, and conferring a greater risk of stomach cancer. The present medication therapy for H. pylori includes the use of antibiotics, which is associated with increasing incidence of bacterial resistance to traditional antibiotics. Therefore, the need for an alternative treatment method is urgent. In this study, four novel synthesis procedures of multivalent glycoconjugates were created. Three different scaffolds representing linear (chondroitin oligomer), cyclic (γ-cyclodextrin), and globular (dendrimer) molecules were used. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc all representing analogues of the tissue binding epitopes for H. pylori. The first synthetic method included the reductive amination of scaffold molecules modified to express primary amine groups, and in the case of dendrimer direct amination to scaffold molecule presenting 64 primary amine groups. The second method described a direct procedure for amidation of glycosylamine modified oligosaccharides to scaffold molecules presenting carboxyl groups. The final two methods that were created both included an oxime-linkage on linkers of different length. All the new synthetic procedures synthesized had the advantage of using unmodified reducing sugars as starting material making it easy to synthesize glycoconjugates of different specificity. In addition, the binding activity of an array of neoglycolipids to H. pylori was studied. Consequently, two new neolacto-based structures, Glcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer and GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer, with binding activity toward H. pylori were discovered. Interestingly, N-methyl and N-ethyl amide modification of the GlcAβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-Cer glucuronic acid residue resulted in more effective H. pylori binding epitopes than the parent molecule.

Randomised Evaluation of New Technologies within the Population‐Based Cervical Cancer Screening Programme in Finland: Cross‐Sectional Performance and Validity

A randomised and population-based screening design with new technologies has been applied to the organised cervical cancer screening programme in Finland. In this experiment the women invited to routine five-yearly screening are individually randomised to be screened with automation-assisted cytology, human papillomavirus (HPV) test or conventional cytology. By using the randomised design, the ultimate aim is to assess and compare the long-term outcomes of the different screening regimens. The primary aim of the current study was to evaluate, based on the material collected during the implementation phase of the Finnish randomised screening experiment, the cross-sectional performance and validity of automation-assisted cytology (Papnet system) and primary HPV DNA testing (Hybrid Capture II assay for 13 oncogenic HPV types) within service screening, in comparison to conventional cytology. The parameters of interest were test positivity rate, histological detection rate, relative sensitivity, relative specificity and positive predictive value. Also, the effect of variation in performance by screening laboratory on age-adjusted cervical cancer incidence was assessed. Based on the cross-sectional results, almost no differences were observed in the performance of conventional and automation-assisted screening. Instead, primary HPV screening found 58% (95% confidence interval 19-109%) more cervical lesions than conventional screening. However, this was mainly due to overrepresentation of mild- and moderate-grade lesions and, thus, is likely to result in overtreatment since a great deal of these lesions would never progress to invasive cancer. Primary screening with an HPV DNA test alone caused substantial loss in specificity in comparison to cytological screening. With the use of cytology triage test, the specificity of HPV screening improved close to the level of conventional cytology. The specificity of primary HPV screening was also increased by increasing the test positivity cutoff from the level recommended for clinical use, but the increase was more modest than the one gained with the use of cytology triage. The performance of the cervical cancer screening programme varied widely between the screening laboratories, but the variation in overall programme effectiveness between respective populations was more marginal from the very beginning of the organised screening activity. Thus, conclusive interpretations on the quality or success of screening should not be based on performance parameters only. In the evaluation of cervical cancer screening the outcome should be selected as closely as possible to the true measure of programme effectiveness, which is the number of invasive cervical cancers and subsequent deaths prevented in the target population. The evaluation of benefits and adverse effects of each new suggested screening technology should be performed before the technology becomes an accepted routine in the existing screening programme. At best, the evaluation is performed randomised, within the population and screening programme in question, which makes the results directly applicable to routine use.

Primary Fallopian tube carcinoma:occurrence, risk and prognostic factors

The aim of the study was to clarify the occurrence, and etiological and prognostic factors of primary fallopian tube carcinoma (PFTC). We studied the sociodemographic determinants of the incidence of PFTC in Finland and the role of chlamydial infections and human papillomavirus infections as risk factors for PFTC. Serum tumor markers were studied as prognostic factors for PFTC. We also evaluated selected reproductive factors (parity, sterilization and hysterectomy) as risk or protective factors of PFTC. The risks of second primary cancers after PFTC were also studied. The age-adjusted incidence of PFTC in Finland increased to 5.4 / 1,000,000 in 1993 97. The incidence rate was higher in the cities, but the relative rise was higher in rural areas. Women in the two highest social classes showed a 1.8 fold incidence compared with those in the lowest. Women in agriculture and those not working outside the home showed only half the PFTC incidence of those in higher socioeconomic occupations. Pretreatment serum concentrations of hCGβ, CA125 and TATI were evaluated as prognostic markers for PFTC. Elevated hCGβ values (above the 75th percentile, 3.5 pmol/L; OR 2.49, 95% CI 1.22 5.09), stage and histology were strong independent prognostic factors for PFTC. The effects of parity, sterilization and hysterectomy on the risk of PFTC were studied in a case control-study with 573 PFTC cases from the Finnish Cancer Registry. In multivariate analysis parity was the only significant protective factor as regards PFTC, with increasing protection associated with increasing number of deliveries. In univariate analysis sterilization gave borderline protection against PFTC and the protective effect increased with time since the operation. In multivariate analysis the protection did not reach statistical significance. Chlamydial and human papillomavirus (HPV) infections were studied in two separate seroepidemiological case-control studies with 78 PFTC patients. The incidence of women with positive HPV or chlamydial serology was the same in PFTC patients and in the control group and was not found to be a risk factor for PFTC. Finally, the possible risk of a second primary cancer after diagnosis and treatment of PFTC in a cohort of 2084 cases from 13 cancer registries followed for second primary cancers within the period 1943 2000 was studied. In PFTC patients, second primary cancers were 36% more common than expected (SIR 1.36, 95% CI 1.13 1.63). In conclusion, the incidence of PFTC has increased in Finland, especially in higher social classes and among those in certain occupations. Elevated serum hCGβ reflect a worsened prognosis. Parity is a clear protective factor, as is previous sterilization. After PFTC there is a risk of second primary cancers, especially colorectal, breast, lung and bladder cancers and non-lymphoid leukemia. The excess of colorectal and breast cancers after PFTC may indicate common effects of earlier treatments, or they could reflect common effects of lifestyle or genetic, immunological or environmental background.

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.

Examination of Genetic Components Affecting Human Obesity-Related Quantitative Traits

Obesity increases the risk for several conditions, including type 2 diabetes mellitus, cardiovascular disease, hypertension, osteoarthirits and certain types of cancer. Twin- and family studies have shown that there is a major genetic component in the determination of body mass. In recent years several technological and scientific advance have been made in obesity research. For instance, novel replicated loci have been revealed by a number of genome wide association studies. This thesis aimed to investigate the association of genetic factors and obesity-related quantitative traits. The first study investigated the role of the lactase gene in anthropometric traits. We genetically defined lactose persistence by genotyping 31 720 individuals of European descent. We found that lactase persistence was significantly correlated with weight and body mass index but not with height. In the second study we performed the largest whole genome linkage scan for body mass index to date. The sample consisted of 4401 twin families and 10 535 individuals from six European countries. We found supporting evidence for two loci (3q29 and 7q36). We observed that the heritability estimate increased substantially when additional family members were removed from the analyses, which suggests reduced environmental variance in the twin sample. In the third study we assessed metabonomic, transcriptomic and genomic variation in a Finnish population cohort of 518 individuals. We formed gene expression networks to portray pathways and showed that a set of highly correlated genes of an inflammatory pathway associated with 80 serum metabolites (of 134 quantified measures). Strong association was found, for example, with several lipoprotein subclasses. We inferred causality by using genetic variation as anchors. The expression of the network genes was found to be dependent on the circulatory metabolite concentrations.

Distribution and adhesion-promoting activity of alpha4 and alpha5 chain laminins in human endothelia and carcinomas

Basement membranes are specialized sheets of extracellular matrix found in contact with epithelia, endothelia, and certain isolated cells. They support tissue architecture and regulate cell behaviour. Laminins are among the main constituents of basement membranes. Due to differences between laminin isoforms, laminins confer structural and functional diversity to basement membranes. The first aim of this study was to gain insights into the potential functions of the then least characterized laminins, alpha4 chain laminins, by evaluating their distribution in human tissues. We thus created a monoclonal antibody specific for laminin alpha4 chain. By immunohistochemistry, alpha4 chain laminins were primarily localized to basement membranes of blood vessel endothelia, skeletal, heart, and smooth muscle cells, nerves, and adipocytes. In addition, alpha4 chain laminins were found in the region of certain epithelial basement membranes in the epidermis, salivary gland, pancreas, esophagus, stomach, intestine, and kidney. Because of the consistent presence of alpha4 chain laminins in endothelial basement membranes of blood vessels, we evaluated the potential roles of endothelial laminins in blood vessels, lymphatic vessels, and carcinomas. Human endothelial cells produced alpha4 and alpha5 chain laminins. In quantitative and morphological adhesion assays, human endothelial cells barely adhered to alpha4 chain-containing laminin-411. The weak interaction of endothelial cells with laminin-411 appeared to be mediated by alpha6beta1 integrin. The alpha5 chain-containing laminin-511 promoted endothelial cell adhesion better than laminin-411, but it did not promote the formation of cell-extracellular matrix adhesion complexes. The adhesion of endothelial cells to laminin-511 appeared to be mediated by Lutheran glycoprotein together with beta1 and alphavbeta3 integrins. The results suggest that these laminins may induce a migratory phenotype in endothelial cells. In lymphatic capillaries, endothelial basement membranes showed immunoreactivity for laminin alpha4, beta1, beta2, and gamma1 chains, type IV and XVIII collagens, and nidogen-1. Considering the assumed inability of alpha4 chain laminins to polymerize and to promote basement membrane assembly, the findings may in part explain the incomplete basement membrane formation in these vessels. Lymphatic capillaries of ovarian carcinomas showed immunoreactivity also for laminin alpha5 chain and its receptor Lutheran glycoprotein, emphasizing a difference between normal and ovarian carcinoma lymphatic capillaries. In renal cell carcinomas, immunoreactivity for laminin alpha4 chain was found in stroma and basement membranes of blood vessels. In most tumours, immunoreactivity for laminin alpha4 chain was also observed in the basement membrane region of tumour cell islets. Renal carcinoma cells produced alpha4 chain laminins. Laminin-411 did not promote adhesion of renal carcinoma cells, but inhibited their adhesion to fibronectin. Renal carcinoma cells migrated more on laminin-411 than on fibronectin. The results suggest that alpha4 chain laminins have a counteradhesive function, and may thus have a role in detachment and invasion of renal carcinoma cells.

Characterization of cytokines, matrix metalloproteinases and toll-like receptors in human periodontal tissue destruction

Periodontal Disease affects the supporting structures of the teeth and is initiated by a microbial biofilm called dental plaque. Severity ranges from superficial inflammation of the gingiva (gingivitis) to extensive destruction of connective tissue and bone leading to tooth loss (periodontitis). In periodontitis the destruction of tissue is caused by a cascade of microbial and host factors together with proteolytic enzymes. Matrix metalloproteinases (MMPs) are known to be central mediators of the pathologic destruction in periodontitis. Initially plaque bacteria provide pathogen-associated molecular patterns (PAMPs) which are sensed by Toll-like receptors (TLRs), and initiate intracellular signaling cascades leading to host inflammation. Our aim was to characterize TNF-α (tumor necrosis factor-alpha) and its type I and II receptors in periodontal tissues, as well as, the effects of TNF-α, IL-1β (interleukin-1beta) and IL-17 on the production and/or activation of MMP-3, MMP-8 and MMP-9. Furthermore we mapped the TLRs in periodontal tissues and assessed how some of the PAMPs binding to the key TLRs found in periodontal tissues affect production of TNF-α and IL-1β by gingival epithelial cells with or without combination of IL-17. TNF-α and its receptors were detected in pericoronitis. Furthermore, increased expression of interleukin-1β and vascular cell adhesion molecule-1 was found as a biological indicator of TNF-α ligand-receptor interaction. MMP-3, -8, and 9 were investigated in periodontitis affected human gingival crevicular fluid and gingival fibroblasts produced pro-MMP-3. Following that, the effect of IL-17 was studied on MMP and pro-inflammatory cytokine production. IL-17 was increased in periodontitis and up-regulated IL-1β, TNF-α, MMP-1 and MMP-3. We continued by demonstrating TLRs in gingival tissues, in which significant differences between patients with periodontitis and healthy controls were found. Finally, enzyme-linked immunosorbent assays were performed to show that the gingival cells response to inflammatory responses in a TLR-dependent manner. Briefly, this thesis demonstrates that TLRs are present in periodontal tissues and present differences in periodontitis compared to healthy controls. The cells of gingival tissues respond to inflammatory process in a TLR-dependent manner by producing pro-inflammatory cytokines. During the destruction of periodontal tissues, the release (IL-1β and TNF-α) and co-operation with other pro-inflammatory cytokines (IL-17), which in turn increase the inflammation and thus be more harmful to the host with the increased presence of MMPs (MMP-1, MMP-3, MMP-8, MMP-9) in diseased over healthy sites.

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.