Lymphatic vascular maturation : Roles of FOXC2, NFATc1 and liprin ß1

The blood vascular system is a closed circulatory system, responsible for delivering oxygen and nutrients to the tissues. In contrast, the lymphatic vascular system is a blind-ended transport system that collects the extravasated tissue fluid from the capillary beds, and transports it back to the blood circulation. Failure in collecting or transporting the lymph, due to defects in the lymphatic vasculature, leads to accumulation of extra fluid in the tissues, and consequently to tissue swelling lymphedema. The two vascular systems function in concert. They are structurally related, but their development is regulated by separate, however overlapping, molecular mechanisms. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, processes largely mediated by members of the vascular endothelial growth factor (VEGF) family and their tyrosine kinase receptors. The lymphatic vessels are formed after the cardiovascular system is already functional. This process, called lymphangiogenesis, is controlled by distinct members of the VEGF family, together with the transcription factors Prox1 and Sox18. After the primary formation of the vessels, the vasculature needs to mature and remodel into a functional network of hierarchically organized vessels: the blood vasculature into arteries, capillaries and veins; and the lymphatic vasculature into lymphatic capillaries, responsible for the uptake of the extravasated fluid from the tissues, and collecting vessels, responsible for the transport of the lymph back to the blood circulation. A major event in the maturation of the lymphatic vasculature is the formation of collecting lymphatic vessels. These vessels are characterized by the presence of intraluminal valves, preventing backflow of the lymph, and a sparse coverage of smooth muscle cells, which help in pumping the lymph forward. In our study, we have characterized the molecular and morphological events leading to formation of collecting lymphatic vessels. We found that this process is regulated cooperatively by the transcription factors Foxc2 and NFATc1. Mice lacking either Foxc2 or active NFATc1 fail to remodel the primary lymphatic plexus into functional lymphatic capillaries and collecting vessels. The resulting vessels lack valves, display abnormal expression of lymphatic molecules, and are hyperplastic. Moreover, the lymphatic capillaries show aberrant sprouting, and are abnormally covered with smooth muscle cells. In humans, mutations in FOXC2 lead to Lymphedema-Distichiasis (LD), a disabling disease characterized by swelling of the limbs due to insufficient lymphatic function. Our results from Foxc2 mutant mice and LD patients indicate that the underlying cause for lymphatic failure in LD is agenesis of collecting lymphatic valves and aberrant recruitment of periendothelial cells and basal lamina components to lymphatic capillaries. Furthermore, we show that liprin β1, a poorly characterized member of the liprin family of cytoplasmic proteins, is highly expressed in lymphatic endothelial cells in vivo, and is required for lymphatic vessel integrity. These data highlight the important role of FOXC2, NFATc1 and liprin β1 in the regulation of lymphatic development, specifically in the maturation and formation of the collecting lymphatic vessels. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results also suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

Structure-Based Design of DevR Inhibitor Active against Nonreplicating Mycobacterium tuberculosis

Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organ ` isms. Our findings suggest that compound 10 ``locks'' DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.

Absence of parallelism between polyamine and nucleic acid contents during induced growth of cucumber cotyledons

The cytokinins (benzyladenine or benzyladenosine) decreased spermidine and spermine contents despite increasing putrescine content, when administered to isolated cotyledons of Cucumis sativus L. var. Guntur in organ culture. KCl decreased putrescine contents, although marginally increasing polyamine contents. The cytokinins and/or KCl augmented nucleic acid biosynthesis and accumulation, resulting in enhanced growth and differentiation of the isolated cotyledons. These observations show that polyamine accumulation and growth are not always coupled.

A promoter-level mammalian expression atlas

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.

Functional and cellular analysis of autoimmune regulator (AIRE) protein

Autoimmune diseases are a major health problem. Usually autoimmune disorders are multifactorial and their pathogenesis involves a combination of predisposing variations in the genome and other factors such as environmental triggers. APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) is a rare, recessively inherited, autoimmune disease caused by mutations in a single gene. Patients with APECED suffer from several organ-specific autoimmune disorders, often affecting the endocrine glands. The defective gene, AIRE, codes for a transcriptional regulator. The AIRE (autoimmune regulator) protein controls the expression of hundreds of genes, representing a substantial subset of tissue-specific antigens which are presented to developing T cells in the thymus and has proven to be a key molecule in the establishment of immunological tolerance. However, the molecular mechanisms by which AIRE mediates its functions are still largely obscure. The aim of this thesis has been to elucidate the functions of AIRE by studying the molecular interactions it is involved in by utilizing different cultured cell models. A potential molecular mechanism for exceptional, dominant, inheritance of APECED in one family, carrying a glycine 228 to tryptophan (G228W) mutation, was described in this thesis. It was shown that the AIRE polypeptide with G228W mutation has a dominant negative effect by binding the wild type AIRE and inhibiting its transactivation capacity in vitro. The data also emphasizes the importance of homomultimerization of AIRE in vivo. Furthermore, two novel protein families interacting with AIRE were identified. The importin alpha molecules regulate the nuclear import of AIRE by binding to the nuclear localization signal of AIRE, delineated as a classical monopartite signal sequence. The interaction of AIRE with PIAS E3 SUMO ligases, indicates a link to the sumoylation pathway, which plays an important role in the regulation of nuclear architecture. It was shown that AIRE is not a target for SUMO modification but enhances the localization of SUMO1 and PIAS1 proteins to nuclear bodies. Additional support for the suggestion that AIRE would preferably up-regulate genes with tissue-specific expression pattern and down-regulate housekeeping genes was obtained from transactivation studies performed with two models: human insulin and cystatin B promoters. Furthermore, AIRE and PIAS activate the insulin promoter concurrently in a transactivation assay, indicating that their interaction is biologically relevant. Identification of novel interaction partners for AIRE provides us information about the molecular pathways involved in the establishment of immunological tolerance and deepens our understanding of the role played by AIRE not only in APECED but possibly also in several other autoimmune diseases.

The role of MADS and TCP transcription factors in Gerbera hybrida flower development

Angiosperms represent a huge diversity in floral structures. Thus, they provide an attractive target for comparative developmental genetics studies. Research on flower development has focused on few main model plants, and studies on these species have revealed the importance of transcription factors, such as MADS-box and TCP genes, for regulating the floral form. The MADS-box genes determine floral organ identities, whereas the TCP genes are known to regulate flower shape and the number of floral organs. In this study, I have concentrated on these two gene families and their role in regulating flower development in Gerbera hybrida, a species belonging to the large sunflower family (Asteraceae). The Gerbera inflorescence is comprised of hundreds of tightly clustered flowers that differ in their size, shape and function according to their position in the inflorescence. The presence of distinct flower types tells Gerbera apart from the common model species that bear only single kinds of flowers in their inflorescences. The marginally located ray flowers have large bilaterally symmetrical petals and non-functional stamens. The centrally located disc flowers are smaller, have less pronounced bilateral symmetry and carry functional stamens. Early stages of flower development were studied in Gerbera to understand the differentiation of flower types better. After morphological analysis, we compared gene expression between ray and disc flowers to reveal transcriptional differences in flower types. Interestingly, MADS-box genes showed differential expression, suggesting that they might take part in defining flower types by forming flower-type-specific regulatory complexes. Functional analysis of a CYCLOIDEA-like TCP gene GhCYC2 provided evidence that TCP transcription factors are involved in flower type differentiation in Gerbera. The expression of GhCYC2 is ray-flower-specific at early stages of development and activated only later in disc flowers. Overexpression of GhCYC2 in transgenic Gerbera-lines causes disc flowers to obtain ray-flower-like characters, such as elongated petals and disrupted stamen development. The expression pattern and transgenic phenotypes further suggest that GhCYC2 may shape ray flowers by promoting organ fusion. Cooperation of GhCYC2 with other Gerbera CYC-like TCP genes is most likely needed for proper flower type specification, and by this means for shaping the elaborate inflorescence structure. Gerbera flower development was also approached by characterizing B class MADS-box genes, which in the main model plants are known regulators of petal and stamen identity. The four Gerbera B class genes were phylogenetically grouped into three clades; GGLO1 into the PI/GLO clade, GDEF2 and GDEF3 into the euAP3 clade and GDEF1 into the TM6 clade. Putative orthologs for GDEF2 and GDEF3 were identified in other Asteraceae species, which suggests that they appeared through an Asteraceae-specific duplication. Functional analyses indicated that GGLO1 and GDEF2 perform conventional B-function as they determine petal and stamen identities. Our studies on GDEF1 represent the first functional analysis of a TM6-like gene outside the Solanaceae lineage and provide further evidence for the role of TM6 clade members in specifying stamen development. Overall, the Gerbera B class genes showed both commonalities and diversifications with the conventional B-function described in the main model plants.

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.

Blended learning in medical imaging pre-clinical skills training – does it work?

Undergraduate Medical Imaging (MI)students at QUT attend their first clinical placement towards the end of semester two. Students undertake two (pre)clinical skills development units – one theory and one practical. Students gain good contextual and theoretical knowledge during these units via a blended learning model with multiple learning methods employed. Students attend theory lectures, practical sessions, tutorial sessions in both a simulated and virtual environment and also attend pre-clinical scenario based tutorial sessions. The aim of this project is to evaluate the use of blended learning in the context of 1st year Medical Imaging Radiographic Technique and its effectiveness in preparing students for their first clinical experience. It is hoped that the multiple teaching methods employed within the pre-clinical training unit at QUT builds students clinical skills prior to the real situation. A quantitative approach will be taken, evaluating via pre and post clinical placement surveys. This data will be correlated with data gained in the previous year on the effectiveness of this training approach prior to clinical placement. In 2014 59 students were surveyed prior to their clinical placement demonstrated positive benefits of using a variety of learning tools to enhance their learning. 98.31%(n=58)of students agreed or strongly agreed that the theory lectures were a useful tool to enhance their learning. This was followed closely by 97% (n=57) of the students realising the value of performing role-play simulation prior to clinical placement. Tutorial engagement was considered useful for 93.22% (n=55) whilst 88.14% (n=52) reasoned that the x-raying of phantoms in the simulated radiographic laboratory was beneficial. Self-directed learning yielded 86.44% (n=51). The virtual reality simulation software was valuable for 72.41% (n=42) of the students. Of the 4 students that disagreed or strongly disagreed with the usefulness of any tool they strongly agreed to the usefulness of a minimum of one other learning tool. The impact of the blended learning model to meet diverse student needs continues to be positive with students engaging in most offerings. Students largely prefer pre -clinical scenario based practical and tutorial sessions where 'real-world’ situations are discussed.

Determinants of outcome in critically ill patients

Assessment of the outcome of critical illness is complex. Severity scoring systems and organ dysfunction scores are traditional tools in mortality and morbidity prediction in intensive care. Their ability to explain risk of death is impressive for large cohorts of patients, but insufficient for an individual patient. Although events before intensive care unit (ICU) admission are prognostically important, the prediction models utilize data collected at and just after ICU admission. In addition, several biomarkers have been evaluated to predict mortality, but none has proven entirely useful in clinical practice. Therefore, new prognostic markers of critical illness are vital when evaluating the intensive care outcome. The aim of this dissertation was to investigate new measures and biological markers of critical illness and to evaluate their predictive value and association with mortality and disease severity. The impact of delay in emergency department (ED) on intensive care outcome, measured as hospital mortality and health-related quality of life (HRQoL) at 6 months, was assessed in 1537 consecutive patients admitted to medical ICU. Two new biological markers were investigated in two separate patient populations: in 231 ICU patients and 255 patients with severe sepsis or septic shock. Cell-free plasma DNA is a surrogate marker of apoptosis. Its association with disease severity and mortality rate was evaluated in ICU patients. Next, the predictive value of plasma DNA regarding mortality and its association with the degree of organ dysfunction and disease severity was evaluated in severe sepsis or septic shock. Heme oxygenase-1 (HO-1) is a potential regulator of apoptosis. Finally, HO-1 plasma concentrations and HO-1 gene polymorphisms and their association with outcome were evaluated in ICU patients. The length of ED stay was not associated with outcome of intensive care. The hospital mortality rate was significantly lower in patients admitted to the medical ICU from the ED than from the non-ED, and the HRQoL in the critically ill at 6 months was significantly lower than in the age- and sex-matched general population. In the ICU patient population, the maximum plasma DNA concentration measured during the first 96 hours in intensive care correlated significantly with disease severity and degree of organ failure and was independently associated with hospital mortality. In patients with severe sepsis or septic shock, the cell-free plasma DNA concentrations were significantly higher in ICU and hospital nonsurvivors than in survivors and showed a moderate discriminative power regarding ICU mortality. Plasma DNA was an independent predictor for ICU mortality, but not for hospital mortality. The degree of organ dysfunction correlated independently with plasma DNA concentration in severe sepsis and plasma HO-1 concentration in ICU patients. The HO-1 -413T/GT(L)/+99C haplotype was associated with HO-1 plasma levels and frequency of multiple organ dysfunction. Plasma DNA and HO-1 concentrations may support the assessment of outcome or organ failure development in critically ill patients, although their value is limited and requires further evaluation.

Outcomes of ruptured abdominal aortic aneurysm

Ruptured abdominal aortic aneurysm (RAAA) is a life-threatening event, and without operative treatment the patient will die. The overall mortality can be as high as 80-90%; thus repair of RAAA should be attempted whenever feasible. The quality of life (QoL) has become an increasingly important outcome measure in vascular surgery. Aim of the study was to evaluate outcomes of RAAA and to find out predictors of mortality. In Helsinki and Uusimaa district 626 patients were identified to have RAAA in 1996-2004. Altogether 352 of them were admitted to Helsinki University Central Hospital (HUCH). Based on Finnvasc Registry, 836 RAAA patients underwent repair of RAAA in 1991-1999. The 30-day operative mortality, hospital and population-based mortality were assessed, and the effect of regional centralisation and improving in-hospital quality on the outcome of RAAA. QoL was evaluated by a RAND-36 questionnaire of survivors of RAAA. Quality-adjusted life years (QALYs), which measure length and QoL, were calculated using the EQ-5D index and estimation of life expectancy. The predictors of outcome after RAAA were assessed at admission and 48 hours after repair of RAAA. The 30-day operative mortality rate was 38% in HUCH and 44% nationwide, whereas the hospital mortality was 45% in HUCH. Population-based mortality was 69% in 1996-2004 and 56% in 2003-2004. After organisational changes were undertaken, the mortality decreased significantly at all levels. Among the survivors, the QoL was almost equal when compared with norms of age- and sex-matched controls; only physical functioning was slightly impaired. Successful repair of RAAA gave a mean of 4.1 (0-30.9) QALYs for all RAAA patients, although non-survivors were included. The preoperative Glasgow Aneurysm Score was an independent predictor of 30-day operative mortality after RAAA, and it also predicted the outcome at 48- hours for initial survivors of repair of RAAA. A high Glasgow Aneurysm Score and high age were associated with low numbers of QALYs to be achieved. Organ dysfunction measured by the Sequential Organ Failure Assessment (SOFA) score at 48 hours after repair of RAAA was the strongest predictor of death. In conclusion surgery of RAAA is a life-saving and cost-effective procedure. The centralisation of vascular emergencies improved the outcome of RAAA patients. The survivors had a good QoL after RAAA. Predictive models can be used on individual level only to provide supplementary information for clinical decision-making due to their moderate discriminatory value. These results support an active operation policy, as there is no reliable measure to predict the outcome after RAAA.

Cytomegalovirus in the Process of Chronic Allograft Nephropathy

Cytomegalovirus (CMV) is a major cause of morbidity, costs and even mortality in organ transplant recipients. CMV may also enhance the development of chronic allograft nephropathy (CAN), which is the most important cause of graft loss after kidney transplantation. The evidence for the role of CMV in chronic allograft nephropathy is somewhat limited, and controversial results have also been reported. The aim of this study was to investigate the role of CMV in the pathogenesis of CAN. Material for the purpose of this study was available from altogether 70 kidney transplant recipients who received a kidney transplant between the years 1992-2000. CMV infection was diagnosed with pp65 antigenemia test or by viral culture from blood, urine, or both. CMV proteins were demonstrated in the kidney allograft biopsies by immunohistochemisrty and CMV-DNA by in situ hybridization. Cytokines, adhesion molecules, and growth factors were demonstrated from allograft biopsies by immunohistochemistry, and from urinary samples by ELISA-methods. CMV proteins were detectable in the 6-month protocol biopsies from 18/41 recipients with evidence of CMV infection. In the histopathological analysis of the 6-month protocol biopsies, presence of CMV in the allograft together with a previous history of acute rejection episodes was associated with increased arteriosclerotic changes in small arterioles. In urinary samples collected during CMV infection, excretion of TGF-β was significantly increased. In recipients with increased urinary excretion of TGF-β, increased interstitial fibrosis was recorded in the 6- month protocol biopsies. In biopsies taken after an active CMV infection, CMV persisted in the kidney allograft in 17/48 recipients, as CMV DNA or antigens were detected in the biopsies more than 2 months after the last positive finding in blood or urine. This persistence was associated with increased expression of TGF-β, PDGF, and ICAM-1 and with increased vascular changes in the allografts. Graft survival and graft function one and two years after transplantation were reduced in recipients with persistent intragraft CMV. Persistent intragraft CMV infection was also a risk factor for reduced graft survival in Cox regression analysis, and an independent risk factor for poor graft function one and two years after transplantation in logistic regression analysis. In conclusion, these results show that persistent intragraft CMV infection is detrimental to kidney allografts, causing increased expression of growth factors and increased vascular changes, leading to reduced graft function and survival. Effective prevention, diagnosis and treatment of CMV infections may a major factor in improving the long term survival of kidney allograft.

Studies of Immune Regulation in Type 1 Diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. In T1D insulin producing pancreatic β cells are destroyed. The disease process begins years before the clinical diagnosis of T1D. During the pathogenesis of T1D, pancreatic islets are infiltrated by cells of the immune system and T-lymphocytes are considered to be the main mediators of the β-cell destruction. In children with an active β-cell destruction process, autoantibodies against β-cell antigens appear in the blood. Individuals at increased risk of developing T1D can often be identified by detecting serum autoantibodies against β-cell antigens. Immunological aberrancies associated with T1D are related to defects in the polarization of T cells and in the function of regulatory mechanisms. T1D has been considered as an organ-specific autoimmune disease mediated by uncontrolled Th1-responses. In human T1D, the evidence for the role of over-expression of cytokines promoting cytotoxicity is controversial. For the past 15 years, regulatory T cells (Tregs) have been recognized as having a key role in the initiation and maintenance of tolerance, limiting harmful autoantigen-specific inflammation processes. It is possible that, if regulatory mechanisms fail to be initiated, the subtle inflammation targeting β cells lead to insulitis and eventually to overt T1D in some individuals. In the present thesis, we studied the induction of Tregs during the generation of T-cell responses in T1D. The results suggest that the generation of regulatory mechanisms and effector mechanisms upon T-cell activation is aberrant in children with T1D. In our studies, an in vitro cytotoxic environment inhibited the induction of genes associated with regulatory functions upon T-cell activation. We also found T1D patients to have an impaired cytotoxic response against coxsackievirus B4. Ineffective virus clearance may increase the apoptosis of β cells, and thus the risk of β-cell specific autoimmunity, due to the increased presentation of β-cell-derived peptides by APCs to T cells in pancreatic lymph nodes. Recently, a novel T helper cell subset called Th17 has been discovered. Animal models have associated Th17 cells and especially co-producers of IL-17 and IFN-γ with the pathogenesis of T1D. We aimed to characterize the role of Th17 immunity in human T1D. We demonstrated IL-17 activation to be a major alteration in T1D patients in comparison to healthy children. Moreover, alterations related to the FOXP3-mediated regulatory mechanisms were associated with the IL-17 up-regulation seen in T1D patients. These findings may have therapeutic implications for the treatment and prevention of T1D.

Surveillance of Surgical Site Infections following Major Hip and Knee Surgery in Finland

Background and aims. Since 1999, hospitals in the Finnish Hospital Infection Program (SIRO) have reported data on surgical site infections (SSI) following major hip and knee surgery. The purpose of this study was to obtain detailed information to support prevention efforts by analyzing SIRO data on SSIs, to evaluate possible factors affecting the surveillance results, and to assess the disease burden of postoperative prosthetic joint infections in Finland. Methods. Procedures under surveillance included total hip (THA) and total knee arthroplasties (TKA), and the open reduction and internal fixation (ORIF) of femur fractures. Hospitals prospectively collected data using common definitions and written protocol, and also performed postdischarge surveillance. In the validation study, a blinded retrospective chart review was performed and infection control nurses were interviewed. Patient charts of deep incisional and organ/space SSIs were reviewed, and data from three sources (SIRO, the Finnish Arthroplasty Register, and the Finnish Patient Insurance Centre) were linked for capture-recapture analyses. Results. During 1999-2002, the overall SSI rate was 3.3% after 11,812 orthopedic procedures (median length of stay, eight days). Of all SSIs, 56% were detected after discharge. The majority of deep incisional and organ/space SSIs (65/108, 60%) were detected on readmission. Positive and negative predictive values, sensitivity, and specificity for SIRO surveillance were 94% (95% CI, 89-99%), 99% (99-100%), 75% (56-93%), and 100% (97-100%), respectively. Of the 9,831 total joint replacements performed during 2001-2004, 7.2% (THA 5.2% and TKA 9.9%) of the implants were inserted in a simultaneous bilateral operation. Patients who underwent bilateral operations were younger, healthier, and more often males than those who underwent unilateral procedures. The rates of deep SSIs or mortality did not differ between bi- and uni-lateral THAs or TKAs. Four deep SSIs were reported following bilateral operations (antimicrobial prophylaxis administered 48-218 minutes before incision). In the three registers, altogether 129 prosthetic joint infections were identified after 13,482 THA and TKA during 1999-2004. After correction with the positive predictive value of SIRO (91%), a log-linear model provided an estimated overall prosthetic joint infection rate of 1.6% after THA and 1.3% after TKA. The sensitivity of the SIRO surveillance ranged from 36% to 57%. According to the estimation, nearly 200 prosthetic joint infections could occur in Finland each year (the average from 1999 to 2004) after THA and TKA. Conclusions. Postdischarge surveillance had a major impact on SSI rates after major hip and knee surgery. A minority of deep incisional and organ/space SSIs would be missed, however, if postdischarge surveillance by questionnaire was not performed. According to the validation study, most SSIs reported to SIRO were true infections. Some SSIs were missed, revealing some weakness in case finding. Variation in diagnostic practices may also affect SSI rates. No differences were found in deep SSI rates or mortality between bi- and unilateral THA and TKA. However, patient materials between these two groups differed. Bilateral operations require specific attention paid to their antimicrobial prophylaxis as well as to data management in the surveillance database. The true disease burden of prosthetic joint infections may be heavier than the rates from national nosocomial surveillance systems usually suggest.

Childhood-onset mitochondrial diseases : with emphasis on molecular diagnostics of neurological disorders

Childhood-onset mitochondrial diseases comprise a heterogeneous group of disorders, which may manifest with almost any symptom and affect any tissue or organ. Due to challenging diagnostics, most children still lack a specific aetiological diagnosis. The aim of this thesis was to find molecular causes for childhood-onset mitochondrial disorders in Finland. We identified the underlying cause for 25 children, and found three new diseases, which had not been diagnosed in Finland before. These diseases caused severe progressive infantile-onset encephalomyopathies, and were due to defects in mitochondrial DNA (mtDNA) maintenance. Furthermore, the thesis provides the molecular background of Finnish patients with ‘leukoencephalopathy with brain stem and spinal cord involvement and elevated brain lactate’ (LBSL). A new phenotype was identified to be due to mutations in Twinkle, resembling ‘infantile onset spinocerebellar ataxia’ (IOSCA). These mutations caused mtDNA depletion in the liver, thus confirming the essential role of Twinkle in mtDNA maintenance, and expanding the molecular background of mtDNA depletion syndromes. The major aetiology for infantile mitochondrial myopathy in Finland was discovered to be due to mutations in thymidine kinase 2 (TK2). A novel mutation with Finnish ancestry was identified, and a genotype-phenotype correlation with mutation-specific distribution of tissue involvement was found, thus proving that deficient TK2 may cause multi-tissue depletion and impair neuronal function. This work established the molecular diagnosis and advanced the knowledge of phenotypes among paediatric patients with polymerase gamma (POLG) mutations. The patients showed severe early-onset encephalopathy with intractable epilepsy. POLG mutations are not a prevalent cause of children’s ataxias, although ataxia is a major presenting symptom among adults. Our findings indicate that POLG mutations should be investigated even if typical MRI, histochemical or biochemical abnormalities are lacking. LBSL patients showed considerable variation in phenotype despite identical mutations. A common, most likely European, ancestry, and a relative high carrier frequency of these mutations in Finland were discovered; suggesting that LBSL may be a quite common leukoencephalopathy in other populations as well. The results suggest that MRI findings are so unique that the diagnosis of LBSL is possible to make without genetic studies. This thesis work has resulted in identification of new mitochondrial disorders in Finland, enhancing the understanding of the clinical variability and the importance of tissue-specificity of these disorders. In addition to providing specific diagnosis to the patients, these findings give light to the underlying pathogenetic mechanisms of childhood-onset mitochondrial disorders.

The incidence and outcome of severe sepsis in Finland : The Finnsepsis Study

Severe sepsis is associated with common occurrence, high costs of care and significant mortality. The incidence of severe sepsis has been reported to vary between 0.5/1000 and 3/1000 in different studies. The worldwide Severe Sepsis Campaign, guidelines and treatment protocols aim at decreasing severe sepsis associated high morbidity and mortality. Various mediators of inflammation, such as high mobility group box-1 protein (HMGB1) and vascular endothelial growth factor (VEGF), have been tested for severity of illness and outcome in severe sepsis. Long-term survival with quality of life (QOL) assessment is important outcome after severe sepsis. The objective of this study was to evaluate the incidence, severity of organ dysfunction and outcome of severe sepsis in intensive care treated patients in Finland (study I)). HMGB1 and VEGF were studied in predicting severity of illness, development and type of organ dysfunction and hospital mortality (studies II and III). The long-term outcome and quality of life were assessed and quality-adjusted life years and cost per one QALY were estimated (study IV). A total of 470 patients with severe sepsis were included in the Finnsepsis Study. Patients were treated in 24 Finnish intensive care units in a 4-month period from 1 November 2004 to 28 February 2005. The incidence of severe sepsis was 0.38 /1,000 in the adult population (95% confidence interval 0.34-0.41). Septic shock (77%), severe oxygenation impairment (71.4%) and acute renal failure (23.2%) were the most common organ failures. The ICU, hospital, one-year and two-year mortalities were 15.5%, 28.3%, 40.9% and 44.9% respectively. HMGB1 and VEGF were elevated in patients with severe sepsis. VEGF concentrations were lower in non-survivors than in survivors, but HMGB1 levels did not differ between patients. Neither HMGB1 nor VEGF were predictive of hospital mortality. The QOL was measured median 17 months after severe sepsis and QOL was lower than in reference population. The mean QALY was 15.2 years for a surviving patient and the cost for one QALY was 2,139 . The study showed that the incidence of severe sepsis is lower in Finland than in other countries. The short-term outcome is comparable with that in other countries, but long-term outcome is poor. HMGB1 and VEGF are not useful in predicting mortality in severe sepsis. The mean QALY for a surviving patient is 15.2 and as the cost for one QALY is reasonably low, the intensive care is cost-effective in patients with severe sepsis.