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  • Paavola, Jere (Helsingin yliopisto, 2014)
    Heart disease is the biggest killer world-wide, causing a quarter of all deaths. During the past two decades, it has also risen above infectious diseases as the leading cause of years of life lost. Heart failure, characterized by weak pump function of the heart, and disturbances in heart rhythm (arrhythmias) are common and interrelated mechanisms underlying cardiac mortality. Intracellular calcium ions are crucial to contraction and relaxation of the heart muscle, as well as to control of its rhythm. How calcium is handled and regulated is thus essential to normal cardiac function, and disturbances in these processes can have catastrophic consequences. Understanding the mechanisms of these disturbances is important for improving disease prevention, diagnosis, and management. The studies in this thesis focus on two conditions where cardiac calcium handling is impaired. Studies I - III examine the mechanisms of a genetic arrhythmia disease named catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by stress-induced ventricular tachycardia in a structurally normal heart. Study IV investigates cardiac function in a model of another genetic disease, autosomal dominant polycystic kidney disease (ADPKD). This systemic disease mainly affects the kidneys, and the mechanisms of the concomitant decline in cardiac function have thus far remained underinvestigated. We evaluated clinical data on cardiac function of CPVT patients, including 24h electrocardiograms, intracardiac monophasic action potential recordings, and exercise stress tests. We used cell models to study the underlying disease mechanisms in detail. During conditions of stress, CPVT cells showed increased spontaneous and irregular release of calcium from within the intracellular stores through the cardiac ryanodine receptors. These receptors, which function as intracellular calcium release channels, harbor the disease-causing mutation. The spontaneous release of calcium led to changes in the membrane potential of the cells, manifested as afterdepolarizations during the resting phase of the cardiac cycle. These afterdepolarizations were reproduced in the clinical monophasic action potential recordings of CPVT patients, and were shown to trigger arrhythmias in these patients. Changes in intracellular calcium alter the membrane potential, and these changes are reflected on the electrocardiogram. Thus, irregularities that might correspond to those observed in the cell model were then investigated in 24h electrocardiograms of CPVT patients. Increased irregularity of cardiac repolarization was found in the CPVT patients. Such irregularity was greater in the electrocardiograms of CPVT patients with a history of more severe arrhythmic events. Additionally, we found slowed depolarization in response to stress in CPVT cells and patients, suggesting reduced conduction velocity might contribute to an arrhythmic substrate in these patients. Cardiac function in ADPKD caused by mutations in polycystin-2, another intracellular calcium channel, was investigated in a zebrafish model lacking expression of the polycystin-2 protein. The zebrafish lacking polycystin-2 showed signs of heart failure, including reduced cardiac output, edema, and arrhythmias. Hearts, which were then examined in more detail ex vivo, showed impaired cycling of intracellular calcium, which is likely to underlie the cardiac dysfunction observed in vivo. The association of ADPKD with idiopathic dilated cardiomyopathy (IDCM) was examined using the Mayo ADPKD Mutation Database, which contains data on genotyped ADPKD patients. Examination of the ADPKD Database showed IDCM to be very common among ADPKD patients. IDCM was most prevalent in patients with mutations in polycystin-2, suggesting impaired calcium cycling as a potential pathomechanism. Studies I-III shed new light on mechanisms of arrhythmias in CPVT and related conditions, opening the way for future studies on arrhythmia risk and therapeutic evaluation. Furthermore, the results encourage pursuing the novel stem cell models for studying pathomechanisms and therapeutics. Study IV showed an association between ADPKD and IDCM. The zebrafish model suggested impaired calcium cycling as an underlying mechanism, highlighting the usefulness of zebrafish as a model in cardiac research.
  • Hernesniemi, Sari (Helsingin yliopisto, 2012)
    Chronic myeloid leukemia (CML) is a hematologic malignancy that originates from pluripotent hematopoietic stem cells. The genetic abnormality underlying this disease, as well as a subtype of acute lymphoblastic leukemia (ALL), is a translocation between chromosomes 9 and 22, which leads to the formation of the Philadelphia (Ph) chromosome and the oncogenic BCR-ABL1 fusion protein. Targeted inhibition of the BCR-ABL1 protein with imatinib and other specific tyrosine kinase inhibitors (TKIs) has revolutionized CML treatment and is currently regarded as the gold standard of targeted cancer therapy. In this doctoral thesis, different methodological approaches were used to characterize aberrant signaling activities in leukemic and normal cells with the ultimate aim of isolating novel prognostic markers for predicting TKI therapy outcome. First, the molecular mechanism of the disease was investigated in an ALL patient with a translocation between chromosomes 1 and 9. Cytogenetic characterization showed involvement of the ABL1 gene fused to an unknown gene. The RCSD1 gene was selected as a candidate and subsequent molecular dissection confirmed a RCSD1-ABL1 fusion. This novel fusion gene predicts sensitivity to TKI therapy in ALL patients. Second, several phosphoproteins related to immune cell function were analyzed both from the diagnostic phase CML patients and from patients under TKI therapy in order to reveal distinct signaling patterns. A single cell phosphoprotein method based on multiparameter flow cytometry was used to analyze phosphoprotein levels in different cell populations. The responsiveness of myeloid cells to ex vivo cytokine stimulation in diagnostic-phase patients was low, but was normalized in TKI-treated patients. In general, the blood leukocyte subsets responded normally to various cytokine stimulations, which indicated a non-immunosuppressive role for TKIs. This project also led to the development of a software assisted analysis program, which can conduct the whole range of flow cytometry data analysis, thereby diminishing the effort needed for manual cell population gating and interpretation. Lastly, biomarkers for therapy response were identified by analyzing aberrant signaling activities in leukemic cells collected at the time of diagnosis. The analysis was based on a phosphoproteomic array measuring phosphorylation of 46 different kinase targets. Several proteins had aberrant phosphorylation levels in patients with a poor therapy outcome − pSTAT5b being the most prominent. These biomarkers could be useful in guiding therapy selection at the time of diagnosis. In conclusion, the findings imply that TKI therapy responses can be linked to certain biological markers, which can possibly be utilized in clinical applications in the future.
  • Häkkinen, Margareeta (Helsingin yliopisto, 2015)
    Opioids are the most important drugs causing fatal poisonings. Determining whether an opioid death was poisoning may, however, be difficult even if involving appropriate toxicological laboratory investigation. Apart from heroin, little statistically significant data-analysis is available for interpretation of blood concentrations of opioids from various types of post-mortem cases. Tolerance, route of administration, and delay of death after drug administration all influence postmortem drug concentrations. In this thesis, quantitative blood concentration data extracted from the high-quality Finnish postmortem toxicology database was the investigative tool to overcome this problem. Opioid deaths typically involve drug abuse, and suspected drug-abuser deaths must, by Finnish law, undergo medico-legal examination. Medico-legal autopsy in these cases includes comprehensive drug screening and, based on its results, more specific drug quantification. This thesis combined concentration data stored in the postmortem toxicology database with information from death certificates issued by forensic pathologists to allow statistical comparisons between drug poisonings and other deaths, as well as between drug abusers and other users. Concentration data mainly involved drug concentrations in postmortem femoral blood, but drug concentrations in urine and parent drug/metabolite concentration ratios also allowed assessment of buprenorphine, codeine, and tramadol deaths. Opioid poisonings proved to be mainly unintentional polydrug poisonings, regularly involving benzodiazepines, gabapentinoids, and other psycholeptics. Buprenorphine and methadone blood concentrations in fatal poisonings remained within their therapeutic ranges, and these two opioids involved mostly abuse. Concentrations of the weak opioids tramadol and codeine were above their therapeutic ranges both in abuser cases and in fatal poisonings. Tramadol abuse was common but abuse of oxycodone, fentanyl, and codeine was rather low compared to their therapeutic use. Abuse of the gabapentinoids pregabalin and gabapentin was strongly associated with opioid abuse, and compared to gabapentin abuse, pregabalin abuse was proportionally more frequent. To prevent parenteral buprenorphine abuse, opioid maintenance treatment applied a combination product of buprenorphine-naloxone. This combination product is, however, abused as well, and monitoring its abuse is challenging. In this study, urine samples collected from living patients at different phases of opioid maintenance treatment supplemented the postmortem data. Based on the criteria established with these patients, combined with postmortem data and proper background information, a urine concentration limit was estimated for suspected parenteral abuse of the buprenorphine-naloxone product in postmortem cases. Deaths and fatal poisonings due to parenteral buprenorphine-naloxone abuse occurred frequently, and abuse of the combination product was proportionally even more fatal than was abuse of buprenorphine. The results of this study will assist in medico-legal cause-of-death investigations through providing quantitative reference concentrations for the interpretation of opioid-related deaths. Further, estimating the proportion attributable to prescription opioid abuse compared to that of other opioid use and creating abuser profiles for various opioids can promote public health through proper drug policy. In a clinical context, results may be helpful in evaluating possible drug abuse and compliance among prescription-drug users. Detecting abuse of these important yet addictive medications is vital in promoting welfare and drug safety.
  • Kolmonen, Marjo (Helsingin yliopisto, 2011)
    Human sport doping control analysis is a complex and challenging task for anti-doping laboratories. The List of Prohibited Substances and Methods, updated annually by World Anti-Doping Agency (WADA), consists of hundreds of chemically and pharmacologically different low and high molecular weight compounds. This poses a considerable challenge for laboratories to analyze for them all in a limited amount of time from a limited sample aliquot. The continuous expansion of the Prohibited List obliges laboratories to keep their analytical methods updated and to research new available methodologies. In this thesis, an accurate mass-based analysis employing liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS) was developed and validated to improve the power of doping control analysis. New analytical methods were developed utilizing the high mass accuracy and high information content obtained by TOFMS to generate comprehensive and generic screening procedures. The suitability of LC-TOFMS for comprehensive screening was demonstrated for the first time in the field with mass accuracies better than 1 mDa. Further attention was given to generic sample preparation, an essential part of screening analysis, to rationalize the whole work flow and minimize the need for several separate sample preparation methods. Utilizing both positive and negative ionization allowed the detection of almost 200 prohibited substances. Automatic data processing produced a Microsoft Excel based report highlighting the entries fulfilling the criteria of the reverse data base search (retention time (RT), mass accuracy, isotope match). The quantitative performance of LC-TOFMS was demonstrated with morphine, codeine and their intact glucuronide conjugates. After a straightforward sample preparation the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The hydrophilic interaction technique (HILIC) provided good chromatographic separation, which was critical for the morphine glucuronide isomers. A wide linear range (50-5000 ng/ml) with good precision (RSD<10%) and accuracy (±10%) was obtained, showing comparable or better performance to other methods used. In-source collision-induced dissociation (ISCID) allowed confirmation analysis with three diagnostic ions with a median mass accuracy of 1.08 mDa and repeatable ion ratios fulfilling WADA s identification criteria. The suitability of LC-TOFMS for screening of high molecular weight doping agents was demonstrated with plasma volume expanders (PVE), namely dextran and hydroxyethylstarch (HES). Specificity of the assay was improved, since interfering matrix compounds were removed by size exclusion chromatography (SEC). ISCID produced three characteristic ions with an excellent mean mass accuracy of 0.82 mDa at physiological concentration levels. In summary, by combining TOFMS with a proper sample preparation and chromatographic separation, the technique can be utilized extensively in doping control laboratories for comprehensive screening of chemically different low and high molecular weight compounds, for quantification of threshold substances and even for confirmation. LC-TOFMS rationalized the work flow in doping control laboratories by simplifying the screening scheme, expediting reporting and minimizing the analysis costs. Therefore LC-TOFMS can be exploited widely in doping control, and the need for several separate analysis techniques is reduced.
  • Virkki, Liisa (Helsingin yliopisto, 2014)
    Biological antirheumatic drugs are biotechnologically produced antibodies or fusion proteins that are used if rheumatic inflammation is not sufficiently ameliorated with conventional antirheumatic drugs. Approximately 10% of Finnish rheumatoid arthritis (RA) patients use biological drugs. Biological antirheumatic drugs target inflammatory mediators and cells. Tumor necrosis factor inhibitors (TNFi) have been used since the beginning of the 2000s, and they are still the primarily used type of biological antirheumatic drug. The efficacy and safety has been assessed in clinical drug trials usually of 6 to 12 months duration with strict inclusion and exclusion criteria. In practice these drugs are used for much longer and in very diverse patient populations. In this work, the effectiveness and adverse events (AEs) of TNFi treatment in a routine care setting were assessed using the national register of biological drugs. Biological drugs are expensive, and therefore also their cost-effectiveness and the outcomes of switching a TNFi to another one were assessed. The effectiveness of the TNFi infliximab (IFX) was similar to its efficacy in clinical trials. In about 2/3 of the patients clinically significant symptom relief was achieved within 3 months, and 42% achieved remission within the first year. The need of glucocorticoid and pain medication was reduced. In addition to methotrexate assessed in clinical trials, also other conventional antirheumatic drugs were suitable concomitant drugs. The most frequent AEs of TNFi treatment were infections, eczemas, and infusion reactions. AEs were reported in 17% of patients using biologicals, serious ones in 3.1%. No unexpected AEs were reported. 76% of RA patients benefited from IFX in terms of quality-adjusted life years (QALY); their functional capacity and subjective health improved. The median price of a QALY was 51884 . The best cost-effectiveness was achieved in patients with particularly high disease activity and significant response to the treatment. The IFX dose and methotrexate use also affected the cost-effectiveness. Switching a TNFi to another one was useful in cases where initial effectiveness was lost, and in cases of AEs such as infusion reactions, eczemas, or local injection site reactions, which did not require discontinuation of TNFi treatment altogether; in these cases the switch led to a similar or even better effect. The register research indicates that TNFi treatment is effective and relatively safe in RA refractory to other treatments. It was cost-effective in a significant portion of the patients; new drug alternatives (including biosimilars) and the contemporary treatment aim of remission may further improve the cost-effectiveness.
  • Kuusio, Hannamaria (Helsingin yliopisto, 2014)
    The shortage of general practitioners (GPs) threatens the effective functioning of public primary health care in many countries. Working as a GP has lost much of its attractiveness as a career option also among Finnish physicians during the past 15 years, and foreign-born physicians are being increasingly recruited to primary health care. The first aim of the present study was to examine the psychosocial work environment of physicians and how it is associated with their wellbeing and future career plans. GPs were compared to medical specialists and private physicians. The second aim of the study was to investigate the process of foreign-born physicians entering their profession in Finland and also to explore their job satisfaction, work-related stressors and future career interests. The study data was obtained from two surveys conducted in 2006 and 2010 among random samples of Finnish physicians (N=2,841, response rate 57%; and N=3780, response rate 56%, respectively). In 2010, a survey of all foreign-born physicians resident in Finland (N=1,292) was also conducted (553 respondents, response rate 43%). Qualitative theme interviews were conducted with foreign-born physicians in Finnish primary health care to explore their work history, career choices and plans, job satisfaction and health. The results showed that the work ability and self-rated health of Finnish GPs were lower than those of Finnish medical specialists and private physicians. Finnish GPs and medical specialists both reported more psychological distress than private physicians. Wellbeing differences were to some extent explained by higher job stressors among public-sector physicians. Furthermore, Finnish GPs expressed their intention to leave their job more often than other Finnish physicians. For foreign-born physicians, the extensive and challenging licensing process slowed down their career possibilities, particularly among physicians trained outside the EU/EEA. The job satisfaction of foreign-born public-sector physicians was lower than that of foreign-born private physicians, and they also reported higher work-related stressors. Foreign-born GPs more often expressed an intention to leave primary health care than foreign-born medical specialists, private physicians or Finnish GPs. The present study suggests that the retention of both Finnish and foreign-born GPs will remain a challenge due to the more often expressed intention among GPs to leave primary health care and higher stressors in comparison to private physicians. Investing in a more efficient and monicultural human resource policy in primary health care and giving GPs more influence in decisions concerning their work could attract more GPs to primary health care - both native and foreign.
  • Pekkarinen, Pirkka (Helsingin yliopisto, 2014)
    The human immune system consists of the innate and the adaptive immunity that together protect the body from pathogens. To complete this task, the immune system must be able to recognize and destroy the dangerous foreign structures but also not to react to host structures or innocuous foreign structures, such as proteins of food or the commensal microbes residing in the gut. The innate immunity includes the phagocytes, such as the macrophages and neutrophils, and multiple molecular defensive systems, most importantly the complement system. The innate immunity reacts quickly to pathogens but its functions remain unchanged with repeated encounters with the intruder. The adaptive immunity is slower in its response, but it is more specific and it has memory; upon repeated exposure to a given pathogen, the adaptive immunity is activated more rapidly. Immunological tolerance, the unresponsiveness to self antigens, is a feature of the adaptive immunity. The adaptive immunity includes T and B lymphocytes and the antibodies produced by the B lymphocytes. In spite of their interdependency, the innate and adaptive immune systems have often been studied separately. This thesis focuses on their interface by investigating the role of the innate complement system in the regulation of the adaptive immunity and of the T lymphocyte function in particular. We followed the immune response and the establishment of oral tolerance in a C3 deficient mouse model, where the function of the complement system is blocked. We also studied vaccination responses and mucosal immune homeostasis in C3 deficient human subjects. The mice were immunized with ovalbumin in Complete Freund s adjuvant. In order to induce oral tolerance, some of the mice were given ovalbumin to the gastrointestinal tract prior to the immunizations. The ensuing immune response was monitored by assessing the lymphocyte fractions by flow cytometry and by stimulating splenocytes with ovalbumin and monoclonal antibodies in vitro, and measuring the proliferative response with a radioactive thymidine incorporation assay. The expression of cytokines and transcription factors in isolated cells and tissue samples was analyzed with quantitative real-time PCR. Serum antibody levels were determined by ELISA. We isolated leukocytes from peripheral blood samples collected from the patients and healthy control subjects and analyzed the lymphocyte population with flow cytometry. Serum antibodies specific for intestinal commensal microbes and the vaccine antigens tetanus toxoid and diphtheria toxoid were measured with ELISA. Serum samples were also analyzed for the presence of a set of key cytokines. The results indicate that complement plays a crucial role in the regulation of the functional differentiation of the T helper lymphocytes central to the adaptive immunity. Immunization with ovalbumin produced a weaker T cell proliferative response in the C3 deficient mouse model compared to the wild-type controls. The response of the T lymphocytes was also qualitative different, since the development of a TH1 response was particularly impaired in the absence of a functional complement system, whereas the TH2 response showed no difference between the mouse strains. This was also reflected on the B lymphocyte response: The IgG2a and IgG3 response to the immunization was reduced in the C3 deficient mice but the IgE response was normal. In addition to the general attenuation of the adaptive immunity, the C3 deficiency resulted in a disturbance of the intestinal immune tolerance in both mice and men. The administration of a foreign protein into the gastrointestinal tract of the C3 deficient mice failed to prevent the systemic immune response to the subsequent immunization with the same protein, i.e. the establishment of oral tolerance failed. The C3 deficient human subjects had more mucosally homing activated T lymphocytes in the peripheral blood and higher levels of serum IgG specific for intestinal commensal microbes. A further sign of the deficient immune tolerance in the C3 deficient human system was the lack of IgG4 response to the vaccine antigens. IgG3 antibodies specific for vaccine antigens were present at higher concentrations in the patient sera and the levels of the inflammatory cytokines IL-12 and IL-21 were also elevated. In contrast to the mouse, the profile of serum cytokines and antibody subclasses in the C3 deficient human subjects pointed at a pronounced TH1 response. The work presented in this thesis defines the complement system as a versatile regulator of the adaptive immunity and helper T lymphocytes. The normal functional differentiation of the T lymphocytes requires signals from the complement system and the establishment of immune tolerance both in the mucosal and systemic immune systems is particularly dependent on complement. The results present novel information on the interplay of the innate and adaptive immune systems and will probably affect the treatment strategies for food allergies and inflammatory bowel diseases.
  • Kantola, Taru (Helsingin yliopisto, 2010)
    The Molecular Adsorbent Recirculating System (MARS) is an extracorporeal albumin dialysis device which is used in the treatment of liver failure patients. This treatment was first utilized in Finland in 2001, and since then, over 200 patients have been treated. The aim of this thesis was to evaluate the impact of the MARS treatment on patient outcome, the clinical and biochemical variables, as well as on the psychological and economic aspects of the treatment in Finland. This thesis encompasses 195 MARS-treated patients (including patients with acute liver failure (ALF), acute-on-chronic liver failure (AOCLF) and graft failure), and a historical control group of 46 ALF patients who did not undergo MARS. All patients received a similar standard medical therapy at the same intensive care unit. The baseline data (demographics, laboratory and clinical variables) and MARS treatment-related and health-related quality-of-life data were recorded before and after treatment. The direct medical costs were determined for a period of 3.5 years.Additionally, the outcome of patients (survival, native liver recovery and need for liver transplantation) and survival predicting factors were investigated. In the outcome analysis, for the MARS-treated ALF patients, their 6-month survival (75% vs. 61%, P=0.07) and their native liver recovery rate (49% vs. 17%, P<0.001) were higher, and their need for transplantations was lower (29% vs. 57%, P= 0.001) than for the historical controls. However, the etiological distribution of the ALF patients referred to our unit has changed considerably over the past decade and the percentage of patients with a more favorable prognosis has increased. The etiology of liver failure was the most important predictor of the outcome. Other survival predicting factors in ALF included hepatic encephalopathy, the coagulation factors and the liver enzyme levels prior to MARS treatment. In terms of prognosis, the MARS treatment of the cirrhotic AOCLF patient seems meaningful only when the patient is eligible for transplantation. The MARS treatment appears to halt the progression of encephalopathy and reduce the blood concentration of neuroactive amino acids, albumin-bound and water-soluble toxins. In general, the effects of the MARS treatment seem to stabilize the patients, thus allowing additional time either for the native liver to recover, or for the patients to endure the prolonged waiting for transplantation. Furthermore, for the ALF patients, the MARS treatment appeared to be less costly and more cost-efficient than the standard medical therapy alone. In conclusion, the MARS treatment appears to have a beneficial effect on the patient outcome in ALF and in those AOCLF patients who can be bridged to transplantation.
  • Reséndiz, Julio (Helsingin yliopisto, 2005)
  • Kankkunen, Päivi (Helsingin yliopisto, 2014)
    The human body is constantly exposed to a variety of microbes and their metabolites. In the early stage of an infection, the cells of innate immunity, including dendritic cells, macrophages, neutrophils and natural killer cells initiate a rapid immune response mediated via the different pattern recognition receptors (PRRs) expressed by these cells. PRRs, including membrane-bound Toll-like receptors, C-type lectin -like receptors (CLRs), and cytoplasmic NOD-like receptors (NLRs) recognize different conserved microbial structures such as microbial cell wall components. Moreover, particular NLRs are able to sense several heterogeneous danger signals such as ATP and microbial toxins of both endogenous and exogenous origins, respectively. Activation of PRRs with microbial structures and various danger signals leads to production of different cytokines such as pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 in immune cells. NLR containing multiprotein complexes called inflammasomes are considered as key mediators of inflammation. Inflammasome mediated signalling is needed for the processing of immature pro-IL-1β and pro-IL-18 into their biologically active IL-1β and IL-18 forms. Repeated fungal exposure and fungal infection is a significant cause of morbidity and mortality. Individuals with immunosuppression are especially prone to suffer fungal infections, mycoses. However, even individuals with normal immunity can experience fungal infection in certain conditions. Many individuals are at risk when exposed to fungi and their metabolites when living in water-damaged buildings. (1,3)-β-glucans and trichothecene mycotoxins are fungal metabolites and they are present on the materials and in indoor air of water-damaged buildings. The results suggest that a major fungal cell wall component, (1,3)-β-glucans, triggers secretion of IL-1β in human macrophages through CLR dectin-1, Syk tyrosine kinase and NLRP3 inflammasome dependent mechanism. Moreover, the results imply that the (1,3)-β-glucan induced inflammasome activation is dependent on the production of reactive oxygen species (ROS), efflux of K+, and on the function of lysosomal protease cathepsin B in human macrophages. In addition, the results reveal that fungal trichothecene mycotoxins activate NLRP3 inflammasome and resulted in IL-1β and IL-18 secretion in lipopolysaccharide-treated human macrophages. The trichothecene triggered inflammasome activation was dependent on the ROS production, K+ efflux and cathepsin B release. Moreover, trichothecene induced inflammasome activation was dependent on purinergic P2X7 receptor. In addition to Syk and Src tyrosine kinase mediated signalling participated on trichothecene triggered inflammasome induction. Collectively, both (1,3)-β-glucans and trichothecenes mycotoxins activate the NLRP3 inflammasome and result in inflammation in human macrophages.
  • Kajanne, Risto (Helsingin yliopisto, 2009)
    Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.
  • Olsson, Eija (Helsingin yliopisto, 2001)
  • Poukkanen, Meri (Helsingin yliopisto, 2015)
    Severe sepsis is the main cause of acute kidney injury (AKI) among critically ill patients. Septic AKI has been shown to associate with lower mean arterial pressure (MAP) levels. The initiation of renal replacement therapy (RRT) is mainly based on clinical judgment. The objectives of this study were to assess the incidence and 90-day mortality of patients with severe sepsis associated AKI treated in the intensive care units (ICUs), to evaluate the impact of MAP on development of AKI, to assess the differences in proportion of use of RRT in patients with septic shock, and to develop predictive model for one-year mortality among ICU-treated patients with AKI. All patients were from the prospective, observational FINNAKI study conducted in 17 Finnish ICUs over the five-month study period in 2011-2012. AKI was defined by Kidney Disease: Improving Global Outcomes (KDIGO) criteria. Of the 918 patients with severe sepsis, 488 had AKI (53%). Patients with septic AKI were older, had more comorbidities, and were more severely ill than patients without AKI. The 90-day mortality rate of the septic patients with AKI was higher (38%) compared to patients with severe sepsis without AKI (25%). The time-adjusted MAP during the first 24 hours in the ICU was lower in septic patients with development of AKI (74 mmHg) than in those septic patients without development of AKI (79 mmHg). The proportion of RRT-treated patients in patients with septic shock ranged from 3% to 35 % across Finnish ICUs. The variation in proportion of RRT among patients with septic shock between high- and low-RRT ICUs was explained by differences in case-mix and in severity of organ dysfunction. Indications for RRT or 90-day mortality did not differ between ICU groups. The one-year mortality among patients with AKI was 40%. Advanced age, number of co-morbidities, higher modified SAPS II score, mechanical ventilation and the lowest base excess value on the third day (D3), and the highest bilirubin value by D3 were predictors of one-year mortality. The severity of AKI, or the presence of severe sepsis, did not remain as predictors for one-year mortality. Conclusions: Over half of the patients with severe sepsis had AKI. Avoidance of time-adjusted MAP below 73mmHg may be beneficial for prevention of the progression of AKI in patients with severe sepsis. Despite 10-fold variation in proportion of RRT in patients with septic shock, the 90-day mortality of these patients was similar between high-RRT ICUs and low-RRT ICUs. The predictive model based on data by the third day in the ICU might be clinically useful in identifying patients with high risk for long-term mortality.
  • Usvasalo, Anu (Helsingin yliopisto, 2010)
    In recent reports, adolescents and young adults (AYA) with acute lymphoblastic leukemia (ALL) have had a better outcome with pediatric treatment than with adult protocols. ALL can be classified into biologic subgroups according to immunophenotype and cytogenetics, with different clinical characteristics and outcome. The proportions of the subgroups are different in children and adults. ALL subtypes in AYA patients are less well characterized. In this study, the treatment and outcome of ALL in AYA patients aged 10-25 years in Finland on pediatric and adult protocols was retrospectively analyzed. In total, 245 patients were included. The proportions of biologic subgroups in different age groups were determined. Patients with initially normal or failed karyotype were examined with oligonucleotide microarray-based comparative genomic hybridization (aCGH). Also deletions and instability of chromosome 9p were screened in ALL patients. In addition, patients with other hematologic malignancies were screened for 9p instability. aCGH data were also used to determine a gene set that classifies AYA patients at diagnosis according to their risk of relapse. Receiver operating characteristic analysis was used to assess the value of the set of genes as prognostic classifiers. The 5-year event-free survival of AYA patients treated with pediatric or adult protocols was 67% and 60% (p=0.30), respectively. White blood cell count larger than 100x109/l was associated with poor prognosis. Patients treated with pediatric protocols and assigned to an intermediate-risk group fared significantly better than those of the pediatric high-risk or adult treatment groups. Deletions of 9p were detected in 46% of AYA ALL patients. The chromosomal region 9p21.3 was always affected, and the CDKN2A gene was always deleted. In about 15% of AYA patients, the 9p21.3 deletion was smaller than 200 kb in size, and therefore, probably undetectable with conventional methods. Deletion of 9p was the most common aberration of AYA ALL patients with initially normal karyotype. Instability of 9p, defined as multiple separate areas of copy number loss or homozygous loss within a larger heterozygous area in 9p, was detected in 19% (n=27) of ALL patients. This abnormality was restricted to ALL; none of the patients with other hematologic malignancies had the aberration. The prognostic model identification procedure resulted in a model of four genes: BAK1, CDKN2B, GSTM1, and MT1F. The copy number profile combinations of these genes differentiated between AYA ALL patients at diagnosis depending on their risk of relapse. Deletions of CDKN2B and BAK1 in combination with amplification of GSTM1 and MT1F were associated with a higher probability of relapse. Unlike all previous studies, we found that the outcome of AYA patients with ALL treated using pediatric or adult therapeutic protocols was comparable. The success of adult ALL therapy emphasizes the benefit of referral of patients to academic centers and adherence to research protocols. 9p deletions and instability are common features of ALL and may act together with oncogene-activating translocations in leukemogenesis. New and more sensitive methods of molecular cytogenetics can reveal previously cryptic genetic aberrations with an important role in leukemic development and prognosis and that may be potential targets of therapy. aCGH also provides a viable approach for model design aiming at evaluation of risk of relapse in ALL.
  • Karinen, Heikki (Helsingin yliopisto, 2013)
    Acute mountain sickness (AMS) is a common problem while ascending at high altitude. AMS may progress rapidly with fatal results if the acclimatization process fails or symptoms are neglected and the ascent continues. It affects 25% of those ascending to altitudes of 1850 to 2750 m, 42% at altitudes of 3000 m, and even 84% of those attempting a tourist flight to Lhasa, Tibet (3860 m). The most common reason for altitude illness is a too rapid ascent. There is a need for a non-invasive, specific, and convenient field method for the detection of inadequate acclimatization and impending AMS. Arterial oxygen saturation (SpO2) measurement is useful in anticipating AMS, but it is susceptible to many disruptive factors in the field (for example temperature). Autonomic cardiac response to increasing altitude could be a low-cost non-invasive test to predict impending AMS, in addition to helping distinguish those who are at risk for AMS and those who are acclimatizing well. The purpose of the present study was to: 1. assess the prevalence of the symptoms and signs of acute mountain sickness among Finnish travelers climbing Mount Kilimanjaro, Tanzania; 2. investigate if post-exercise oxygen saturation (Ex-SpO2) at high altitudes predicts AMS better than arterial O2 saturation at rest (R-SpO2) or resting heart rate (HR) alone; 3. evaluate if heart rate variation (HRV) level or HRV changes have a relationship to AMS during ascent and provide new information on the deterioration of cardiac autonomic function as measured by HRV, not only at altitudes between 2400 and 5000 m, which are most frequent among climbers, but also at extreme altitudes above 5000 m in field conditions; 4. generate tools for AMS for non-medical persons to estimate the risk in field conditions; and 5. describe a typical AMS case and the difficulties to treat AMS at field conditions. The data was collected during several treks to Kilimanjaro and climbing expeditions to Denali, Shisha Pangma, Ulugh Muztagh, Island Peak, and Mount Everest 2001-2009. The incidence of AMS was very high (75%) for the trekkers at Mt Kilimanjaro. In climbing expeditions, subjects susceptible to AMS had lower SpO2 at rest and especially during exercise before the clinical manifestations of AMS so daily measurements of SpO2 were taken at these times to best predict the subsequent AMS at higher altitude if ascending continued. Subjects susceptible to AMS had lower root mean square successive differences and high-frequency power of HRV before the clinical manifestations of AMS than those who acclimatized well and did not get AMS 2-5 days later if ascending continued. The treatment of AMS in the field is difficult and time consuming, where possibilities for medical treatment and oxygen substitution might be limited. Prevention is the safest and the most efficient method of care. Realising the risk of mountain sickness, active inquiry about symptoms and correctly timed reaction to them, in other words interrupting the ascent or descending, helps to reduce, and even to prevent, the development of serious problems.
  • Kylänpää-Bäck, Marja-Leena (Helsingin yliopisto, 2001)
  • Lapatto-Reiniluoto, Outi (Helsingin yliopisto, 2001)
  • Åhlström, Annika (Helsingin yliopisto, 2006)
    Acute renal failure (ARF) is a clinical syndrome characterized by rapidly decreasing glomerular filtration rate, which results in disturbances in electrolyte- and acid-base homeostasis, derangement of extracellular fluid volume, and retention of nitrogenous waste products, and is often associated with decreased urine output. ARF affects about 5-25% of patients admitted to intensive care units (ICUs), and is linked to high mortality and morbidity rates. In this thesis outcome of critically ill patients with ARF and factors related to outcome were evaluated. A total of 1662 patients from two ICUs and one acute dialysis unit in Helsinki University Hospital were included. In study I the prevalence of ARF was calculated and classified according to two ARF-specific scoring methods, the RIFLE classification and the classification created by Bellomo et al. (2001). Study II evaluated monocyte human histocompatibility leukocyte antigen-DR (HLA-DR) expression and plasma levels of one proinflammatory (interleukin (IL) 6) and two anti-inflammatory (IL-8 and IL-10) cytokines in predicting survival of critically ill ARF patients. Study III investigated serum cystatin C as a marker of renal function in ARF and its power in predicting survival of critically ill ARF patients. Study IV evaluated the effect of intermittent hemodiafiltration (HDF) on myoglobin elimination from plasma in severe rhabdomyolysis. Study V assessed long-term survival and health-related quality of life (HRQoL) in ARF patients. Neither of the ARF-specific scoring methods presented good discriminative power regarding hospital mortality. The maximum RIFLE score for the first three days in the ICU was an independent predictor of hospital mortality. As a marker of renal dysfunction, serum cystatin C failed to show benefit compared with plasma creatinine in detecting ARF or predicting patient survival. Neither cystatin C nor plasma concentrations of IL-6, IL-8, and IL-10, nor monocyte HLA-DR expression were clinically useful in predicting mortality in ARF patients. HDF may be used to clear myoglobin from plasma in rhabdomyolysis, especially if the alkalization of diuresis does not succeed. The long-term survival of patients with ARF was found to be poor. The HRQoL of those who survive is lower than that of the age- and gender-matched general population.