Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

Phylogenetics, taxon delimitation and phylogenetic diversity

Throughout the history of Linnean taxonomy, species have been described with varying degrees of justification. Many descriptions have been based on only a few ambiguous morphological characters. Moreover, species have been considered natural, well-defined units whereas higher taxa have been treated as disparate, non-existent creations. In the present thesis a few such cases were studied in detail. Often the species-level descriptions were based on only a few specimens and the variation previously thought to be interspecific was found to be intraspecific. In some cases morphological characters were sufficient to resolve the evolutionary relationships between the taxa, but generally more resolution was gained by the addition of molecular evidence. However, both morphological and molecular data were found to be deceptive in some cases. The DNA sequences of morphologically similar specimens were found to differ distinctly in some cases, whereas in other closely related species the morphology of specimens with identical DNA sequences differed substantially. This study counsels caution when evolutionary relationships are being studied utilizing only one source of evidence or a very limited number of characters (e.g. barcoding). Moreover, it emphasizes the importance of high quality data as well as the utilization of proper methods when making scientific inferences. Properly conducted analyses produce robust results that can be utilized in numerous interesting ways. The present thesis considered two such extensions of systematics. A novel hypothesis on the origin of bioluminescence in Elateriformia beetles is presented, tying it to the development of the clicking mechanism in the ancestors of these animals. An entirely different type of extension of systematics is the proposed high value of the white sand forests in maintaining the diversity of beetles in the Peruvian Amazon. White sand forests are under growing pressure from human activities that lead to deforestation. They were found to harbor an extremely diverse beetle fauna and many taxa were specialists living only in this unique habitat. In comparison to the predominant clay soil forests, considerably more elateroid beetles belonging to all studied taxonomic levels (species, genus, tribus, and subfamily) were collected in white sand forests. This evolutionary diversity is hypothesized to be due to a combination of factors: (1) the forest structure, which favors the fungus-plant interactions important for the elateroid beetles, (2) the old age of the forest type favoring survival of many evolutionary lineages and (3) the widespread distribution and fragmentation of the forests in the Miocene, favoring speciation.

CMV-, EBV-, and HHV-6-DNAemia after liver transplantation

Viral infections caused by herpesviruses are common complications after organ transplantation and they are associated with substantial morbidity and even mortality. Herpesviruses remain in a latent state in a host after primary infection and may reactivate later. CMV infection is the most important viral infection after liver transplantation. Less is known about the significance of human herpesvirus-6 (HHV-6). EBV is believed to play a major role in the development of post-transplant lymphoproliferative disorders (PTLD). The aim of this study was to investigate the CMV-, EBV- and HHV-6 DNAemia after liver transplantation by frequent monitoring of adult liver transplant patients. The presence of CMV, EBV and HHV-6 DNA were demonstrated by in situ hybridization assays and by real-time PCR methods from peripheral blood specimens. CMV and HHV-6 antigens were demonstrated by antigenemia assays and compared to the viral DNAemia. The response to antiviral therapy was also investigated. CMV-DNAemia appeared earlier than CMV pp65-antigenemia after liver transplantation. CMV infections were treated with ganciclovir. However, most of the treated patients demonstrated persistence of CMV-DNA for up to several months. Continuous CMV-DNA expression of peripheral blood leukocytes showed that the virus is not eliminated by ganciclovir and recurrences can be expected during several months after liver transplantation. HHV-6 DNAemia / antigenemia was common and occurred usually within the first three months after liver transplantation together with CMV. The HHV-6 DNA expression in peripheral blood mononuclear cells correlated well with HHV-6 antigenemia. Antiviral treatment significantly decreased the number of HHV-6 DNA positive cells, demonstrating the response to ganciclovir treatment. Clinically silent EBV reactivations with low viral loads were relatively common after liver transplantation. These EBV-DNAemias usually appeared within the first three months after liver transplantation together with betaherpesviruses (CMV, HHV-6, HHV-7). One patient developed high EBV viral loads and developed PTLD. These results indicate that frequent monitoring of EBV-DNA levels can be useful to detect liver transplant patients at risk of developing PTLD.

Tumour markers as prognostic factors of survival of gastric cancer patients

The incidence of gastric cancer in the last decades has declined rapidly in the industrialised countries. Worldwide, however, gastric cancer is still the second most common cause of cancer death. Although surgery is currently the most effective treatment, the rapid progress in adjuvant chemotherapy and radiation therapy requires a re-evaluation of prognosis assessment. The TNM staging system of the UICC is ubiquitously used; it groups patients by decreasing survival times from stage I to stage IV based on the spread of disease, i.e. depth of tumour penetration (T), extent of spread to lymph nodes (N), and the presence or absence of distant (M) metastases. This is by far the most consistent prognostic classification system today. However, even within the stage groups there are patients that follow a varying course of disease. Our knowledge of the molecular differences between tumours of the same stage and morphology has been accumulating over the years and methods for a more accurate assessment of the phenotype of neoplasias are of value when evaluating the prognosis of individual patients with gastric cancer. In this study, the immunohistochemical expression of tumour markers involved in different phases in tumourigenesis was examined. The aim was to find new markers which could provide prognostic information in addition to what is provided by the TNM variables. A total of 337 specimens from the primary tumour of patients who underwent surgery for gastric cancer were collected and the immunohistochemical expression of seven different biomarkers was analysed. DNA ploidy and S-phase fraction (SPF) was assessed by flow cytometry. Finally, all biomarkers and clinicopathological prognostic factors were combined and evaluated by a multivariate Cox regression model to elucidate which specific factors provide independent prognostic information. By univariate survival analysis the following variables were significant prognostic factors: epithelial and stromal syndecan-1 expression, stromal tenascin-C expression, expression of tumour-associated trypsin inhibitor (TATI) in cancer cells, nuclear p53 expression, nuclear p21 expression, DNA ploidy, and SPF. By multivariate survival analysis adjusted for all available clinicopathological and biomolecular variables, p53 expression, p21 expression, and DNA ploidy emerged as independent prognostic biomarkers, together with penetration depth of the tumour, presence of nodal metastases, surgical cure of the cancer, and age of the patient at the time of diagnosis.

Airway Inflammation and Bronchial Hyperresponsiveness in Elite Cross-Country Skiers and in Patients with Newly Diagnosed Asthma: A Bronchial Biopsy Study

The objective of these studies was to evaluate possible airway inflammation and remodeling at the bronchial level in cross-country skiers without a prior diagnosis of asthma, and relate the findings to patients with mild chronic asthma and patients with newly diagnosed asthma. We also studied the association of airway inflammatory changes and bronchial hyperresponsivess (BHR), and treatment effects in cross-country skiers and in patients with newly diagnosed asthma. Bronchial biopsies were obtained from the subjects by flexible bronchoscopy, and the inflammatory cells (eosinophils, mast cells, T-lymphocytes, macrophages, and neutrophils) were identified by immunohistochemistry. Tenascin (Tn) immunoreactivity in the bronchial basement membrane (BM) was identified by immunofluorescence staining. Lung function was measured with spirometry, and BHR was assessed by methacholine (skiers) or histamine (asthmatics) challenges. Skiers with BHR and asthma-like symptoms were recruited to a drug-intervention study. Skiers were given treatment (22 weeks) with placebo or budesonide (400 µg bid). Patients with newly diagnosed asthma were given treatment for 16 weeks with placebo, salmeterol (SLM) (50 µg bid), fluticasone propionate (FP) (250 µg bid), or disodium cromoglicate (DSCG) (5 mg qid). Bronchial biopsies were obtained at baseline and at the end of the treatment period. In the skiers a distinct airway inflammation was evident. In their bronchial biopsy specimens, T-lymphocyte, macrophage, and eosinophil counts were, respectively greater by 43-fold (P<0.001), 26-fold (P<0.001, and 2-fold (P<0.001) in skiers, and by 70-fold (p>0.001), 63-fold (P<0.001), and 8-fold (P<0.001) in asthmatic subjects than in controls. In skiers, neutrophil counts were more than 2-fold greater than in asthmatic subjects (P<0.05). Tn expression was higher in skiers than in controls and lower in skiers than in mild asthmatics. No significant changes were seen between skiers with or without BHR in the inflammatory cell counts or Tn expression. Treatment with inhaled budesonide did not attenuate asthma-like symptoms, the inflammatory cell infiltration, or BM Tn expression in the skiers. In newly diagnosed asthmatic patients, SLM, FP, and DSCG reduced asthma symptoms, and need for rescue medication (P<0.04). BHR was reduced by doubling doses 2.78, 5.22, and 1.35 respectively (all P<0.05). SLM and placebo had no effect on cell counts or Tn expression. FP and DSCG reduced eosinophil counts in the bronchial biopsy specimens (P<0.02 and <0.048, respectively). No significant change in tenascin expression appeared in any treatment group. Regarding to atopy, no significant differences existed in the inflammatory cell counts in the bronchial mucosa of subjects with newly diagnosed asthma or in elite cross country skiers. Tn expression in the BM was significantly higher in atopic asthma than in those with nonatopic asthma. Airway inflammation occurred in elite cross-country skiers with and without respiratory symptoms or BHR. Their inflammatory cell pattern differed from that in asthma. Infiltration with eosinophils, macrophages, and mast cells was milder, but lymphocyte counts did not differ from counts in asthmatic airways. Neutrophilic infiltration was more extensive in skiers than in asthmatics. Remodeling took place in the skiers’ airways, as reflected by increased expression of BM tenascin These inflammatory changes and Tn expression may be caused by prolonged exposure of the lower airways to inadequately humidified cold air. In skiers inflammatory changes and remodeling were not reversed with anti-inflammatory treatment. In contrast, in patients with newly diagnosed asthma, anti-inflammatory treatment did attenuate eosinophilic inflammation in the bronchial mucosa. In skiers, anti-inflammatory treatment did not attenuate BHR as it did in asthmatic patients. The BHR in skiers was attenuated spontaneously during placebo treatment, with no difference from budesonide treatment. Lower training intensity during the treatment period may explain this spontaneous decrease in BHR. The origin of BHR probably differs in skiers and in asthmatics. No significant association between BHR and inflammatory cell counts or between BHR and Tn expression was evident in cross-country skiers or asthmatic subjects. Airway remodeling differed between atopic and nonatopic asthma. As opposed to nonatopic asthma, Tn expression was higher in atopic asthma and is related to inflammatory cell densities.

Conjunctival Melanoma in Finland 1967-2000: a population-based study

Eighty-five new cases of conjunctival melanoma (CM) were diagnosed in Finland between 1967 and 2000. The annual crude incidence of CM was 0.51 per million inhabitants. The average age-adjusted incidence of 0.54 doubled during the study period, analogous to the increase in the incidence of cutaneous malignant melanoma during this period, suggesting a possible role for ultraviolet radiation in its pathogenesis. Nonlimbal tumors were more likely than limbal ones to recur and they were associated with decreased survival. Increasing tumor thickness and recurrence of the primary tumor were other clinical factors related to death from CM. The histopathologic specimens of 85 patients with CM melanoma were studied for cell type, mitotic count, tumor-infiltrating lymphocytes and macrophages, mean vascular density, extravascular matrix loops and networks, and mean diameter of the ten largest nucleoli (MLN). The absence of epithelioid cells, increasing mitotic count and small MLN were associated with shorter time to recurrence according to the Cox univariate regression. None of the histopathologic variables was associated with mortality from CM. Four (5%) patients had a CM limited to the cornea without evidence of a tumor other than primary acquired melanosis of the conjunctiva. Because there are no melanocytes in the cornea, the origin of these melanomas most likely is the limbal conjunctiva. All four corneally displaced CM were limited to the epithelium, and none of the patients developed metastases. An anatomic sub-classification based on my patients and world literature was developed for corneally displaced CM. In 20 patients the metastatic pattern could be determined. Ten patients had initial systemic metastases detected, nine had initial regional metastases, and in one case the two types were detected simultaneously. The patients most likely to develop either type of initial metastases were those with nonlimbal conjunctival melanoma, those with a primary tumor more than 2 mm thick, and those with a recurrent conjunctival melanoma. Approximately two thirds of the patients had limbal CM, a location associated with good prognosis. One third, however, had a primary CM originating outside the limbus. In these patients the chance of developing local recurrences as well as systemic metastases was significantly higher than in patients with limbal CM. Each recurrence accompanies an increased risk of developing metastases, and recurrences contribute to death along with increasing tumor thickness and nonlimbal tumor location. In my data, an equal number of patients with initial locoregional and systemic metastasis existed. Patients with limbal primary tumors less than 2 mm in thickness rarely experienced metastases, unless the tumor recurred. Consequently, the patients most likely to benefit from sentinel lymph node biopsy are those who have nonlimbal tumors, CM that are over 2 mm thick, or recurrent CM. The histopathology of CM differs from that of uveal melanoma. Microvascular factors did not prove to be of prognostic importance, possibly due to the fact that CM at least as often disseminates first to the regional lymph nodes, unlike uveal melanoma that almost always disseminates hematogenously.

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.

Long-term results of operative treatment for Hirschsprung's disease and internal anal sphincter achalasia

The aim of the study was to evaluate long-term results of operative treatment for Hirschsprung's disease(HD) and internal anal sphincter achalasia. Fecal continence and quality of life were evaluated by a questionnaire in 100 adult patients who had undergone surgery for HD, during 1950-75. Fecal continence was evaluated using a numerical scoring described by Holschneider. Fifty-four of the 100 patients underwent clinical examination, rigid sigmoidoscopy and manometric evaluation. In anorectal manometry basal resting pressure(BRP)and maximal squeeze pressure(MSP) were measured and voluntary sphincter force(VSF) was calculated by subtracting the BRP from MSP. The results of operative treatment for adult HD were compared with the results of the patients operated in childhood. In adult HD the symptoms are such mild that the patients attain adolescence or even adulthood. The patients with HD and cartilage-hair-hypoplasia were specifically evaluated. The outcome of the patients with internal anal sphincter achalasia operated on by myectomy was evaluated by a questionnaire and continence was evaluated using a numerical scoring described by Holschneider. Of the 100 patients operated on for HD 38 patients had completely normal bowel habits. A normal or good continence score was found in 91 our of 100 patients. Nine patients had fair continence. One of the patients with fair continence had Down's syndrome and two were mentally retarded for other reasons. Only one patient suffered from constipation. In anorectal manometry the difference in BRP between patients with normal and good continence was statistically significant, whereas the difference between good and fair continence groups was not statistically significant. The differences on MSP and VSF between patient groups with different continence outcome were not statistically significant. The differences between patient groups and normal controls were statistically significant in BRP and MSP. In VSF there was not statistically significant difference between the patients and the normal controls. The VSF reflects the working power of the muscles including external sphincter, levator ani and gluteal muscles. The patients operated at adult age had as good continence as patients operated in childhood. The patients with HD and cartilage-hair-hypoplasia had much more morbidity and mortality than non-cartilage-hair-hypoplasia HD patients. The mortality was as high as 38%. In patients with internal anal sphincter achalasia the constipation was cured or alleviated by myectomy whereas a significant number suffered from soiling-related social problems.

Probiotics and prebiotics in the primary prevention of allergic diseases

The rapid increase in allergic diseases in developed, high-income countries during recent decades is attributed to several changes in the environment such as urbanization and improved hygiene. This relative lack of microbial stimulation is connected to a delay in maturation of the infantile immune system and seems to predispose especially genetically prone infants to allergic diseases. Probiotics, which are live ingestible health-promoting microbes, may compensate for the lack of microbial stimulation of the developing gut immune system and may thus be beneficial in prevention of allergies. Prebiotics, which are indigestible nutrients by us, promote the growth and activity of a number of bacterial strains considered beneficial for the gut. In a large cohort of 1 223 infants at hereditary risk for allergies we studied in a double-blind placebo-controlled manner whether probiotics administered in early life prevent allergic diseases from developing. We also evaluated their safety and their effects on common childhood infections, vaccine antibody responses, and intestinal immune markers. Pregnant mothers used a mixture of four probiotic bacteria or a placebo, from their 36th week of gestation. Their infants received the same probiotics plus prebiotic galacto-oligosaccharides for 6 months. The 2-year follow-up consisted of clinical examinations and allergy tests, fecal and blood sampling, and regular questionnaires. Among the 925 infants participating in the 2-year follow-up the cumulative incidence of any allergic disease (food allergy, eczema, asthma, rhinitis) was comparable in the probiotic (32%) and the placebo (35%) group. However, eczema, which was the most common manifestation (88%) of all allergic diseases, occurred less frequently in the probiotic (26%) than in the placebo group (32%). The preventive effect was more pronounced against atopic (IgE-associated) eczema which, of all atopic diseases, accounted for 92%. The relative risk reduction of eczema was 26% and of atopic eczema 34%. To prevent one case of eczema, the number of mother-infant pairs needed to treat was 16. Probiotic treatment was safe without any undesirable outcome for neonatal morbidity, feeding-related behavior, serious adverse events, growth, or for vaccine-induced antibody responses. Fewer infants in the probiotic than in the placebo group received antibiotics during their first 6 months of life and thereafter to age 2 years suffered from fewer respiratory tract infections. As a novel finding, we discovered that high fecal immunoglobulin A (IgA) concentrations at age 6 months associated with reduced risk for atopic (IgE-associated) diseases by age 2 years. In conclusion, although feeding probiotics to high-risk newborn infants showed no preventive effect on the cumulative incidence of any allergic diseases by age 2, they apparently prevented eczema. This probiotic effect was more pronounced among IgE-sensitized infants. The treatment was safe and seemed to stimulate maturation of the immune system as indicated by increased resistance to respiratory infections and improved vaccine antibody responses.

Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

Ischemia-reperfusion injury in human liver transplantation : Mechanisms and effects on graft function

Liver transplantation is an established therapy for both acute and chronic liver failure. Despite excellent long-term outcome, graft dysfunction remains a problem affecting up to 15-30% of the recipients. The etiology of dysfunction is multifactorial, with ischemia-reperfusion injury regarded as one of the most important contributors. This thesis focuses on the inflammatory response during graft procurement and reperfusion in liver transplantation in adults. Activation of protein C was examined as a potential endogenous anti-inflammatory mechanism. The effects of inflammatory responses on graft function and outcome were investigated. Seventy adult patients undergoing liver transplantation in Helsinki University Central Hospital, and 50 multiorgan donors, were studied. Blood samples from the portal and the hepatic veins were drawn before graft procurement and at several time points during graft reperfusion to assess changes within the liver. Liver biopsies were taken before graft preservation and after reperfusion. Neutrophil and monocyte CD11b and L-selectin expression were analysed by flow cytometry. Plasma TNF-α, IL-6, IL-8, sICAM-1, and HMGB1 were determined by ELISA and Western-blotting. HMGB1 immunohistochemistry was performed on liver tissue specimens. Plasma protein C and activated protein C were determined by an enzyme-capture assay. Hepatic IL-8 release during graft procurement was associated with subsequent graft dysfunction, biliary in particular, in the recipient. Biliary marker levels increased only 5 7 days after transplantation. Thus, donor inflammatory response appears to influence recipient liver function with relatively long-lasting effects. Hepatic phagocyte activation and sequestration, with concomitant HMGB1 release, occurred during reperfusion. Neither phagocyte activation nor plasma cytokines correlated with postoperative graft function. Thus, activation of the inflammatory responses within the liver during reperfusion may be of minor clinical significance. However, HMGB1 was released from hepatocytes and were also correlated with postoperative transaminase levels. Accordingly, HMGB1 appears to be a marker of hepatocellular injury.

Sireenit, floksit ja iirikset- Puu-Käpylän koristekasvit

Puu-Käpylä (“Wooden Käpylä”), a neighbourhood of Helsinki, is the earliest example of the Garden City Movement in Finland. The suburb of valuable wooden architecture was built between 1920 and 1925, with the aim to provide a healthy housing area for working-class families with many children. The houses were erected by a co-operative (Käpylän kansanasunnot, “People?s Dwellings”) and they are protected by the city plan since 1960?s. However, the historical value of the sheltered courtyards has not been investigated. The aim of this study was to survey the garden flora of Puu-Käpylä and to evaluate the authenticity of the courtyard gardens. The survey covered the area of one residential quarter (1.2 ha) with twelve 2-storey semi-detached timber houses arranged around a common yard, which was originally appointed for the tenants? vegetable gardens. The houses are still rented, and each flat is allowed a small lot of the courtyard for cultivation. A complete list was made of all perennial, ornamental plant taxa present in the quarter. Spring bulbs were missed due to the timing of the survey. Generally, the plants were recorded on species level, with the exception of common lilacs, shrub roses, irises and peonies that were thoroughly studied for cultivar identification. It was assumed that plants initially grown in the courtyard could be distinguished by studying Finnish garden magazines, books and nursery catalogues published in the 1920?s and by comparing the present vegetation to surviving documents from the quarter. The total number of ornamental plant taxa identified was 172, of which 17 were trees, 47 shrubs, 7 climbers and 101 herbaceous perennials. The results indicated that a major part of the shrubs, climbers and perennials presumably originated from the 1970?s or later, whereas ca. 70 % of the tree specimens were deemed as original. The survey disclosed a heritage variety of common lilac, resembling cultivar „Prince Notger?, a specific peony taxon, Paeonia humilis Retz., cultivated in Nordic countries since long ago, and a few historic iris varieties. Well-preserved design elements included front gardens on one side of the quarter, a maple alley on another side as well as trees at the garden gates. Old garden books and magazines did not shed much light on the Finnish garden flora commonly used in the period when Puu-Käpylä was built. However, they gave a valuable picture of contemporary planting design. Nursery catalogues offered insight into the assortment of ornamental plants traded in the 1920?s. Conclusions on the authenticity of the current flora were mainly drawn on the basis of old photographs and a vegetation survey map drawn in the 1970?s. This study revealed a need for standardization of syrvey methods applied when investigating garden floras. Uniform survey techniques would make the results comparable and enable a future compilation of data from e.g. historic gardens.

Real-Time PCR : A Molecular Approach to Investigate the Role of Intestinal Microbiota in the Pathophysiology of Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is a common multifactorial functional intestinal disorder, the pathogenesis of which is not completely understood. Increasing scientific evidence suggests that microbes are involved in the onset and maintenance of IBS symptoms. The microbiota of the human gastrointestinal (GI) tract constitutes a massive and complex ecosystem consisting mainly of obligate anaerobic microorganisms making the use of culture-based methods demanding and prone to misinterpretation. To overcome these drawbacks, an extensive panel of species- and group-specific assays for an accurate quantification of bacteria from fecal samples with real-time PCR was developed, optimized, and validated. As a result, the target bacteria were detectable at a minimum concentration range of approximately 10 000 bacterial genomes per gram of fecal sample, which corresponds to the sensitivity to detect 0.000001% subpopulations of the total fecal microbiota. The real-time PCR panel covering both commensal and pathogenic microorganisms was assessed to compare the intestinal microbiota of patients suffering from IBS with a healthy control group devoid of GI symptoms. Both the IBS and control groups showed considerable individual variation in gut microbiota composition. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients, whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. In the screening of intestinal pathogens, 17% of IBS samples tested positive for Staphylococcus aureus, whereas no positive cases were discovered among healthy controls. Furthermore, the methodology was applied to monitor the effects of a multispecies probiotic supplementation on GI microbiota of IBS sufferers. In the placebo-controlled double-blind probiotic intervention trial of IBS patients, each supplemented probiotic strain was detected in fecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group. The combination of assays developed and applied in this thesis has an overall coverage of 300-400 known bacterial species, along with the number of yet unknown phylotypes. Hence, it provides good means for studying the intestinal microbiota, irrespective of the intestinal condition and health status. In particular, it allows screening and identification of microbes putatively associated with IBS. The alterations in the gut microbiota discovered here support the hypothesis that microbes are likely to contribute to the pathophysiology of IBS. The central question is whether the microbiota changes described represent the cause for, rather than the effect of, disturbed gut physiology. Therefore, more studies are needed to determine the role and importance of individual microbial species or groups in IBS. In addition, it is essential that the microbial alterations observed in this study will be confirmed using a larger set of IBS samples of different subtypes, preferably from various geographical locations.

Identification of molecules relevant for the invasiveness of fibrosarcomas and melanomas

Cancer is becoming the leading cause of deaths in the world. As 90% of all deaths from cancer are caused by metastasis, discovery of the mechanisms behind cancer cell invasion and metastasis is of utmost importance. Only new effective therapies targeting cancer progression can reduce cancer mortality rates. The aim of this study was to identify molecules that are relevant for tumor cell invasion and spreading in fibrosarcomas and melanomas, and to analyze their potential for cancer biomarkers or therapeutic targets. First, the gene expression changes of normal cells and transformed cells showing high invasiveness, S-adenosylmethionine decarboxylase (AdoMetDC)-transfected murine fibroblasts and human melanoma cells, were studied by microarray analyses. The function of the identified candidate molecules were then studied in detail in these cell lines. Finally, the physiological relevance of the identified changes was studied by immunohistochemical analyses of human sarcoma and melanoma specimens or by a mouse xenograft model. In fibrosarcoma cells, the most remarkable change detected was a dramatic up-regulation of the actin-sequestering molecule thymosin beta 4 (TB4), which was shown to be important for the transformed phenotype of the AdoMetDC-transfected cells (Amdc-s and -as). A sponge toxin latrunculin A, inhibiting the binding of TB4 to actin, was found to selectively inhibit the migration and invasion of these cells. Further, Amdc-s-induced mouse tumors and human high-grade sarcomas were found to show intense TB4 immunostaining. In addition to TB4, integrin subunits alfa 6 and beta 7 (ItgA6 and ItgB7) were found to be up-regulated in Amdc-s and -as cells. ItgA6 was shown to dimerize mainly with ItgB1 in Amdc-s. Inhibition of ItgA6 or ItgB1 function with neutralizing antibodies fully blocked the invasiveness of Amdc-s cells, and importantly also human HT-1080 fibrosarcoma cells, in three-dimensional (3D)-Matrigel mimicking tumor extracellular matrix (ECM). By immunohistochemical analyses, strong staining for ITGA6 was detected in human high-grade fibrosarcomas and other sarcomas, especially at the invasion fronts of the tumors. In the studied melanoma cell lines, the expression levels of the adhesion-related ECM proteins tenascin-C (TN-C), fibronectin (FN), and transforming growth factor beta-induced (TGFBI) were found to be highly up-regulated. By immunohistochemistry, intense TN-C and FN staining was detected in invasive and metastatic melanoma tumors, showing co-localization (together with procollagen-I) in tubular meshworks and channels around the invading melanoma cells. In vitro, TN-C and FN were further found to directly stimulate the migration of melanoma cells in 3D-collagen-I matrix. The third candidate protein, TGFBI, was found to be an anti-adhesive molecule for melanoma cells, and knockdown of its expression in metastatic melanoma cells (TGFBI-KD cells) led to dramatically impaired tumor growth in immunocompromized mice. Interestingly, the control tumors showed intense TGFBI immunostaining in the invasion fronts, showing partial co-localization with the fibrillar FN staining, whereas the small TGFBI-KD cell-induced tumors displayed amorphous, non-fibrillar FN staining. These data suggest an important role for TGFBI in FN fibrillogenesis and melanoma progression. In conclusion, we have identified several invasion-related molecules, which show potential for cancer diagnostic or prognostic markers, or therapeutic targets. Based on our previous and present fibrosarcoma studies, we propose the possibility of using ITGA6 antagonists (affecting tumor cell adhesion) in combination with TB4 inhibitors (affecting tumor cell migration) and cathepsin L inhibitors (affecting the degradation of basement membrane and ECM proteins) for the treatment of fibrosarcomas and other tumors overexpressing these molecules. With melanoma cells, in turn, we point to the importance of three secreted ECM proteins, TN-C, FN, and TGFBI, in melanoma progression. Of these, especially the potential of TN-C as a prognostic melanoma biomarker and TGFBI as a promising therapeutic target molecule are clearly worth additional studies.

mRNA-binding protein HuR in breast carcinogenesis

Breast cancer is the most common cancer among women. Although its prognosis has improved nowadays, methods to predict the progression of the disease or to treat it are not comprehensive. This thesis work was initiated to elucidate in breast carcinogenesis the role of HuR, a ubiquitously expressed mRNA-binding protein that regulates gene expression posttranscriptionally. HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm, and this nucleocytoplasmic translocation is important for its function as a RNA-stabilizing and translational regulator. HuR has been associated with diverse cellular processes, for example carcinogenesis. The specific aims of my thesis work were to study the prognostic value of HuR in breast cancer and to clarify the mechanisms by which HuR contributes to breast carcinogenesis. My ultimate goal is, by better understanding the role of HuR in breast carcinogenesis, to aid in the discovery of novel targets for cancer therapies. HuR expression and localization was studied in paraffin-embedded preinvasive (atypical ductal hyperplasia, ADH, and ductal carcinoma in situ, DCIS) specimens as well in sporadic and familial breast cancer specimens. Our results show that cytoplasmic HuR expression was already elevated in ADH and remained elevated in DCIS as well as in cancer specimens. Clinicopathological analysis showed that cytoplasmic HuR expression associated with the more aggressive form of the disease in DCIS, and in cancer specimens it proved an independent marker for poor prognosis. Importantly, cytoplasmic HuR expression was significantly associated with poor outcome in the subgroups of small (2 cm) and axillary lymph node-negative breast cancers. HuR proved to be the first mRNA stability protein the expression of which is associated in breast cancer with poor outcome. To explore the mechanisms of HuR in breast carcinogenesis, lentiviral constructs were developed to inhibit and to overexpress the HuR expression in a breast epithelial cell line (184B5Me). Our results suggest that HuR mediates breast carcinogenesis by participating in processes important in cell transformation, in programmed cell death, and in cell invasion. Global gene expression analysis shows that HuR regulates genes participating in diverse cellular processes, and affects several pathways important in cancer development. In addition, we identified two novel target transcripts (connective tissue growth factor, CTGF, and Ras oncogene family member 31, RAB31) for HuR. In conclusion, because cytoplasmic HuR expression in breast cancer can predict the outcome of the disease it could serve in clinics as a prognostic marker. HuR accumulates in the cytoplasm even at its non-invasive stage (ADH and DCIS) of the carcinogenic process and supports functions essential in cell alteration. These data suggest that HuR contributes to carcinogenesis of the breast epithelium.