Structural basis of cytoskeletal regulation by twinfilin

The actin cytoskeleton is required, in all eukaryotic organisms, for several key cellular functions such as cell motility, cytokinesis, and endocytosis. In cells, actin exists either in a monomeric state (G-actin) or in a filamentous form (F-actin). F-actin is the functional form, which can assemble into various structures and produce direct pushing forces that are required for different motile processes. The assembly of actin monomers into complicated three-dimensional structures is tightly regulated by a large number of actin regulating proteins. One central actin regulating protein is twinfilin. Twinfilin consists of two actin depolymerizing-factor homology (ADF-H) domains, which are capable of binding actin, and is conserved from yeast to mammals. Previously it has been shown that twinfilin binds to and sequesters G-actin, and interacts with the heterodimeric capping protein. More recently it has been found that twinfilin also binds to the fast growing actin filament ends and prevents their growth. However, the cellular role of twinfilin and the molecular mechanisms of these interactions have remained unclear. In this study we characterized the molecular mechanisms behind the functions of twinfilin. We demonstrated that twinfilin forms a high-affinity complex with ADP-bound actin monomers (ADP-G-actin). Both ADF-H domains are capable of binding G-actin, but the C-terminal domain contains the high-affinity binding site. Our biochemical analyses identified twinfilin s C-terminal tail region as the interaction site for capping protein. Contrary to G-actin binding, both ADF-H domains of twinfilin are required for the actin filament barbed end capping activity. The C-terminal domain is structurally homologous to ADF/cofilin and binds to filament sides in a similar manner, providing the main affinity for F-actin during barbed end capping. The structure of the N-terminal domain is more distant from ADF/cofilin, and thus it can only associate with G-actin or the terminal actin monomer at the filament barbed end, where it regulates twinfilin s affinity for barbed ends. These data suggest that the mechanism of barbed end capping is similar for twinfilin and gelsolin family proteins. Taken together, these studies revealed how twinfilin interacts with G-actin, filament barbed ends, and capping protein, and also provide a model for how these activities evolved through a duplication of an ancient ADF/cofilin-like domain.

Genetic aspects of outcome in kidney transplantation: cytokine and thrombosis associated candidate genes and gene expression biomarkers

Kidney transplantation (Tx) is the treatment of choice for end stage renal disease. Immunosuppressive medications are given to prevent an immunological rejection of the transplant. However, immunosuppressive drugs increase e.g. the risk of infection, cancer or nephrotoxicity. A major genetic contributors to immunological acceptance of the graft are human leukocyte antigen (HLA) genes. Also other non-HLA gene polymorphisms may predict the future risk of complications before Tx, possibly enabling individualised immunotherapy. Graft function after Tx is monitored using non-specific clinical symptoms and laboratory markers. The definitive diagnosis of graft rejection however relies on a biopsy of the graft. In the acute rejection (AR) diagnostics there is a need for an alternative to biopsy that would be an easily repeatable and simple method for regular use. Frequent surveillance of acute or subclinical rejection (SCR) may improve long-term function. In this thesis, associations between cytokine and thrombosis associated candidate genes and the outcome of kidney Tx were studied. Cytotoxic and co-stimulatory T lymphocyte molecule gene expression biomarkers for the diagnosis of the AR and the SCR were also investigated. We found that polymorphisms in the cytokine genes tumor necrosis factor and interleukin 10 (IL10) of the recipients were associated with AR. In addition, certain IL10 gene polymorphisms of the donors were associated with the incidence of cytomegalovirus infection and occurrence of later infection in a subpopulation of recipients. Further, polymorphisms in genes related to the risk of thrombosis and those of certain cytokines were not associated with the occurrence of thrombosis, infarction, AR or graft survival. In the study of biomarkers for AR, whole blood samples were prospectively collected from adult kidney Tx patients. With real-time quantitative PCR (RT-QPCR) gene expression quantities of CD154 and ICOS differentiated the patients with AR from those without, but not from the patients with other causes of graft dysfunction. Biomarkers for SCR were studied in paediatric kidney Tx patients. We used RT-QPCR to quantify the gene expression of immunological candidate genes in a low-density array format. In addition, we used RT-QPCR to validate the results of the microarray analysis. No gene marker differentiated patients with SCR from those without SCR. This research demonstrates the lack of robust markers among polymorphisms or biomarkers in investigated genes that could be included in routine analysis in a clinical laboratory. In genetic studies, kidney Tx can be regarded as a complex trait, i.e. several environmental and genetic factors may determine its outcome. A number of currently unknown genetic factors probably influence the results of Tx.

Clinical aspects of exhaled nitric oxide in adults : Associations with atopy, bronchial hyperresponsiveness, smoking and chronic obstruction

Measurement of fractional exhaled nitric oxide (FENO) has proven useful in assessment of patients with respiratory symptoms, especially in predicting steroid response. The objective of these studies was to clarify issues relevant for the clinical use of FENO. The influence of allergic sensitization per se on FENO in healthy asymptomatic subjects was studied, the association between airway inflammation and bronchial hyperresponsiveness (BHR) in steroid-naive subjects with symptoms suggesting asthma was examined, as well as the possible difference in this association between atopic and nonatopic subjects. Influence of smoking on FENO was compared between atopic and nonatopic steroid-naive asthmatics and healthy subjects. The short-term repeatability of FENO in COPD patients was examined in order to assess whether the degree of chronic obstruction influences the repeatability. For these purposes, we studied a random sample of 248 citizens of Helsinki, 227 army conscripts with current symptoms suggesting asthma, 19 COPD patients, and 39 healthy subjects. FENO measurement, spirometry and bronchodilatation test, structured interview. skin prick tests, and histamine and exercise challenges were performed. Among healthy subjects with no signs of airway diseases, median FENO was similar in skin prick test-positive and –negative subjects, and the upper normal limit of FENO was 30 ppb. In atopic and nonatopic subjects with symptoms suggesting asthma, FENO associated with severity of exercise- or histamine-induced BHR only in atopic patients. FENO in smokers with steroid-naive asthma was significantly higher than in healthy smokers and nonsmokers. Among atopic asthmatics, FENO was significantly lower in smokers than in nonsmokers, whereas no difference appeared among nonatopic asthmatics. The 24-h repeatability of FENO was equally good in COPD patients as in healthy subjects. These findings indicate that allergic sensitization per se does not influence FENO, supporting the view that elevated FENO indicates NO-producing airway inflammation, and that same reference range can be applied to both skin prick test-positive and -negative subjects. The significant correlation between FENO and degree of BHR only in atopic steroid-naive subjects with current asthmatic symptoms supports the view that pathogenesis of BHR in atopic asthma is strongly involved in NO-producing airway inflammation, whereas in development of BHR in nonatopic asthma other mechanisms may dominate. Attenuation of FENO only in atopic but not in nonatopic smokers with steroid-naive asthma may result from differences in mechanisms of FENO formation as well as in sensitivity of these mechanisms to smoking in atopic and nonatopic asthma. The results suggest, however, that in young adult smokers, FENO measurement may prove useful in assessment of airway inflammation. The short-term repeatability of FENO in COPD patients with moderate to very severe disease and in healthy subjects was equally good.

Obstetric and gynaecological aspects of HIV infection

The purpose of the present study was to examine the outcome of pregnancies among HIV-infected women in Helsinki, use of the levonorgestrel-releasing intrauterine system (LNG-IUS) among HIV-infected women and the prevalence and risk factors of cytological and histologically proven cervical lesions in this population. Between 1993 and 2003 a total of 45 HIV-infected women delivered 52 singleton infants. HIV infection was diagnosed during pregnancy in 40% of the mothers. Seventeen of the mothers received antiretroviral (ARV) medication prior to pregnancy and in 34 cases, the medication was started during pregnancy. A good virological response (i.e. HIV RNA load <1000/mL during the last trimester) to ARV medication was achieved in 36/40 (90%) of the patients in whom HI viral load measurements were performed. Of the infants, 92% were born at term, and their mean (±SD) birth weight was 3350±395 g. The Caesarean section rate was low, 25%. All newborns received ARV medication and none of the infants born to mothers with pre-delivery diagnosis of maternal HIV infection were infected. The safety and advantages of the LNG-IUS were studied prospectively (n=12) and retrospectively (n=6). The LNG-IUS was well tolerated and no cases of PID or pregnancy were noted. Menstrual bleeding was reduced significantly during use of the LNG-IUS; this was associated with a slight increase in haemoglobin levels. Serum oestradiol concentrations remained in the follicular range in all subjects. The key finding was that genital shedding of HIV RNA did not change after the insertion of the LNG-IUS. The mean annual prevalence of low-grade squamous intraepithelial lesions (SIL) was 15% and that of high-grade SIL was 5% among 108 systematically followed HIV-infected women during 1989 2003. A reduced CD4 lymphocyte count was associated with an increased prevalence of SIL, whereas duration of HIV infection, use of ARV medication and HI viral load were not. The cumulative risk of any type of SIL was 17% after one year and 48% after five years among patients with initially normal Pap smears. The risk of developing SIL was associated with young age and a high initial HI viral load. During the follow-up 51 subjects (n=153) displayed cervical intraepithelial neoplasia (CIN), (16% CIN1 and 18% CIN 2-3). Only one case of cancer of the uterine cervix was detected. Pap smears were reliable in screening for CIN. Both nulliparity (p<0.01) and bacterial vaginosis (p<0.04) emerged as significant risk factors of CIN. In conclusion, a combination of universal antenatal screening and multidisciplinary management allows individualized treatment and prevents vertical transmission of HIV. Use of the LNG-IUS is safe among HIV-infected women and cervicovaginal shedding of HIV RNA is not affected by use of the LNG-IUS. The risk of cervical pre-malignant lesions is high among HIV-infected women despite systematic follow-up.

Essential Thrombocythaemia : Diagnosis, Prognostic Aspects, and the Outcome of Finnish patients

Essential thrombocythaemia (ET) is a myeloproliferative disease (MPD) characterized by thrombocytosis, i.e. a constant elevation of platelet count. Thrombocytosis may appear in MPDs (ET, polycythaemia vera, chronic myeloid leukaemia, myelofibrosis) and as a reactive phenomenon. The differential diagnosis of thrombocytosis is important, because the clinical course, need of therapy, and prognosis are different in patients with MPDs and in those with reactive thrombocytosis. ET patients may remain asymptomatic for years, but serious thrombohaemorrhagic and pregnancy-related complications may occur. The complications are difficult to predict. The aims of the present study were to evaluate the diagnostic findings, clinical course, and prognostic factors of ET. The present retrospective study consists of 170 ET patients. Two thirds had a platelet count < 1000 x 109/l. The diagnosis was supported by an increased number of megakaryocytes with an abnormal morphology in a bone marrow aspirate, aggregation defects in platelet function studies, and the presence of spontaneous erythroid and/or megakaryocytic colony formation in in vitro cultures of haematopoietic progenitors. About 70 % of the patients had spontaneous colony formation, while about 30 % had a normal growth pattern. Only a fifth of the patients remained asymptomatic. Half had a major thrombohaemorrhagic complication. The proportion of the patients suffering from thrombosis was as high as 45 %. About a fifth had major bleedings. Half of the patients had microvascular symptoms. Age over 60 years increased the risk of major bleedings, but the occurrence of thrombotic complications was similar in all age groups. Male gender, smoking in female patients, the presence of any spontaneous colony formation, and the presence of spontaneous megakaryocytic colony formation in younger patients were identified as risk factors for thrombosis. Pregnant ET patients had an increased risk of complications. Forty-five per cent of the pregnancies were complicated and 38 % of them ended in stillbirth. Treatment with acetylsalicylic acid alone or in combination with platelet lowering drugs improved the outcome of the pregnancy. The present findings about risk factors in ET as well as treatment outcome in the pregnancies of ET patients should be taken into account when planning treatment strategies for Finnish patients.

Pathobiological Aspects of Nonreheumatic Aortic Valve Stenosis

Aortic valve stenosis (AS) is an active disease process akin to atherosclerosis, with chronic inflammation, lipid accumulation, extracellular matrix remodeling, fibrosis, and extensive calcification of the valves being characteristic features of the disease. The detailed mechanisms and pathogenesis of AS are still incompletely understood, however, and pharmacological treatments targeted toward components of the disease are not currently available. In this thesis project, my coworkers and I studied stenotic aortic valves obtained from 86 patients undergoing valve replacement for clinically significant AS. Non-stenotic control valves (n=17) were obtained from patients undergoing cardiac transplantation or from organ donors without cardiac disease. We identified a novel inflammatory factor, namely mast cell, in stenotic aortic valves and present evidence showing that this multipotent inflammatory cell may participate in the pathogenesis of AS. Using immunohistochemistry and double immunofluorescence stainings, we found that a considerable number of mast cells accumulate in stenotic valves and, in contrast to normal valves, the mast cells in diseased valves were in an activated state. Moreover, valvular mast cells contained two effective proteases, chymase and cathepsin G, which may participate in adverse remodeling of the valves either by inducing fibrosis (chymase and cathepsin G) or by degrading elastin fibers in the valves (cathepsin G). As chymase and cathepsin G are both capable of generating the profibrotic peptide angiotensin II, we also studied the expression and activity of angiotensin-converting enzyme (ACE) in the valves. Using RT-PCR, imunohistochemistry, and autoradiography, we observed a significant increase in the expression and activity of ACE in stenotic valves. Besides mast cell-derived cathepsin G, aortic valves contained other elastolytic cathepsins (S, K, and V). Using immunohistochemistry, RT-PCR, and fluorometric microassay, we showed that the expression and activity of these cathepsins were augmented in stenotic valves. Furthermore, in stenotic but not in normal valves, we observed a distinctive pattern of elastin fiber degradation and disorganization. Importantly, this characteristic elastin degradation observed in diseased valves could be mimicked by adding exogenous cathepsins to control valves, which initially contained intact elastin fibers. In stenotic leaflets, the collagen/elastin ratio was increased and correlated positively with smoking, a potent AS-accelerating factor. Indeed, cigarette smoke could also directly activate cultured mast cells and fibroblasts. Next, we analyzed the expression and activity of neutral endopeptidase (NEP), which parallels the actions of ACE in degrading bradykinin (BK) and thus inactivates antifibrotic mechanisms in tissues. Real-time RT-PCR and autoradiography revealed NEP expression and activity to be enhanced in stenotic valves compared to controls. Furthermore, both BK receptors (1 and 2) were present in aortic valves and upregulated in stenotic leaflets. Isolated valve myofibroblasts expressed NEP and BK receptors, and their upregulation occurred in response to inflammation. Finally, we observed that the complement system, a source of several proinflammatory mediators and also a potential activator of valvular mast cells, was activated in stenotic valves. Moreover, receptors for the complement-derived effectors C3a and C5a were expressed in aortic valves and in cultured aortic valve myofibroblasts, in which their expression was induced by inflammation as well as by cigarette smoke. In conclusion, our findings revealed several novel mechanisms of inflammation (mast cells and mast cell-derived mediators, complement activation), fibrosis (ACE, chymase, cathepsin G, NEP), and elastin fiber degradation (cathepsins) in stenotic aortic valves and highlighted these effectors as possible pathogenic contributors to AS. These results support the notion of AS as an active process with inflammation and extracellular matrix remodeling as its key features and identify possible new targets for medical therapy in AS.

Atrial remodeling in atrial fibrillation : Epidemiological, clinical and magnetocardiographic aspects

Atrial fibrillation is the most common arrhythmia requiring treatment. This Thesis investigated atrial fibrillation (AF) with a specific emphasis on atrial remodeling which was analysed from epidemiological, clinical and magnetocardiographic (MCG) perspectives. In the first study we evaluated in real-life clinical practice a population-based cohort of AF patients referred for their first elective cardioversion (CV). 183 consecutive patients were included of whom in 153 (84%) sinus rhythm (SR) was restored. Only 39 (25%) of those maintained SR for one year. Shorter duration of AF and the use of sotalol were the only characteristics associated with better restoration and maintenance of SR. During the one-year follow-up 40% of the patients ended up in permanent AF. Female gender and older age were associated with the acceptance of permanent AF. The LIFE-trial was a prospective, randomised, double-blinded study that evaluated losartan and atenolol in patients with hypertension and left ventricular hypertrophy (LVH). Of the 8,851 patients with SR at baseline and without a history of AF 371 patients developed new-onset AF during the study. Patients with new-onset AF had an increased risk of cardiac events, stroke, and increased rate of hospitalisation for heart failure. Younger age, female gender, lower systolic blood pressure, lesser LVH in ECG and randomisation to losartan therapy were independently associated with lower frequency of new-onset AF. The impact of AF on morbidity and mortality was evaluated in a post-hoc analysis of the OPTIMAAL trial that compared losartan with captopril in patients with acute myocardial infarction (AMI) and evidence of LV dysfunction. Of the 5,477 randomised patients 655 had AF at baseline, and 345 patients developed new AF during the follow-up period, median 3.0 years. Older patients and patients with signs of more serious heart disease had and developed AF more often. Patients with AF at baseline had an increased risk of mortality (hazard ratio (HR) of 1.32) and stroke (HR 1.77). New-onset AF was associated with increased mortality (HR 1.82) and stroke (HR of 2.29). In the fourth study we assessed the reproducibility of our MCG method. This method was used in the fifth study where 26 patients with persistent AF had immediately after the CV longer P-wave duration and higher energy of the last portion of atrial signal (RMS40) in MCG, increased P-wave dispersion in SAECG and decreased pump function of the atria as well as enlarged atrial diameter in echocardiography compared to age- and disease-matched controls. After one month in SR, P-wave duration in MCG still remained longer and left atrial (LA) diameter greater compared to the controls, while the other measurements had returned to the same level as in the control group. In conclusion is not a rare condition in either general population or patients with hypertension or AMI, and it is associated with increased risk of morbidity and mortality. Therefore, atrial remodeling that increases the likelihood of AF and also seems to be relatively stable has to be identified and prevented. MCG was found to be an encouraging new method to study electrical atrial remodeling and reverse remodeling. RAAS-suppressing medications appear to be the most promising method to prevent atrial remodeling and AF.