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  • Hekim, Can (Helsingin yliopisto, 2012)
    Prostate cancer is the most common cancer in males and a major cause of cancer death in industrial- ized countries. It is generally a very slowly growing cancer and potentially curable at early stages by radical prostatectomy or radiotherapy. However, radical therapy is associated with side effects. Therefore, there is need for novel treatments for advanced prostate cancer, and for curing or slowing down the growth of the tumors. Proteases play important roles in the progression of prostate and other cancers. Prostate produces high levels of two kallikreins, human kallikrein 2 (hK2, kallikrein-related peptidase 2, KLK2) and prostate specific antigen (PSA, KLK3). These proteases are secreted into seminal fluid and mediate liquefaction of the seminal clot that forms after ejaculation. Furthermore, enzymatically active PSA has been shown to reduce angiogenesis in vitro and in vivo, which may contribute to the slow growth of prostate cancer. PSA expression is lower in malignant than in normal prostatic epithelium. It is further reduced in poorly differentiated tumors, in which the expression of hK2 is increased. hK2 may mediate tumor growth and invasion by participating in proteolytic cascades degrading extracelullar matrix and thereby promoting tumor spread. Both PSA and hK2 degrade insulin-like growth factor- binding protein-3 (IGFBP-3) in vitro. IGFBP-3 is an important regulator of cell proliferation and survival via IGF-system, and independently. hK2 is more potent than PSA in degrading IGFBP-3. Because its expression is increased in prostate cancer, degradation of IGFBP-3 by hK2 locally in the prostate may promote prostate cancer growth. By using phage display technology we developed biologically active peptides, which specifically inhibit the enzymatic activity of hK2. The peptides were characterized and the motifs required for their inhibitory activity were determined. These may be used to target hK2 for treatment and their binding property be utilized in tumor imaging. However, the peptides were degraded in plasma within minutes and thus, have limited use in vivo. By head-to-tail cyclization we were able to improve plasma stability of the original linear hK2-inhibiting peptide, while the activity towards hK2 was retained. When secreted from the prostate, most of PSA is free and enzymatically active. In circulation major portion of PSA occurs in complexes with protease inhibitors and, thus, is inactive. To justify the use of mouse models for evaluation of the function of PSA and for studies in therapeutic modalities based on modulation of PSA activity it is important to know whether PSA complexation is similar in mouse and man. We characterized the circulating forms of PSA in mouse, by directly adding to mouse serum or using subcutaneous PSA-producing human prostate cancer cell xenograft tumor models. When added to mouse serum, over 70% of PSA forms complexes within 30 min. The complexes contained α2-macroglobulin and serpins of the α1-antitrypsin (AAT) family. Thus, in mouse serum, PSA forms complexes similar to those in man, but the major immunoreactive complex contains AAT rather than α1-antichymotrypsin (ACT). In mice bearing LNCaP xenograft tumors 70% of immunore- active PSA occurs in complex with AAT. Hence, the metabolism of PSA produced by xenograft tumor models in mice is similar to that of human prostate tumors with respect to the fate of released PSA and would allow the evaluation of treatment modalities based on PSA activity. We studied the degradation of IGFBP-3 by hK2 and identified the cleavage sites by mass spectrometry. Furthermore, we showed that hK2-inhibiting peptides inhibit hK2 activity towards natural protein substrates, including IGFBP-3. As degradation of IGFBP-3 leads to release of IGF-I, which may stimulate cancer growth, these peptides may be useful for treatment of prostate cancer and other diseases associated with increased hK2 activity. The peptides identified in this study have potential therapeutic value for treatment of prostate cancer. They can also be used as lead molecules for the development of peptide analogues suitable for imaging and treatment of prostate cancer.
  • Putkonen, Hanna (Helsingin yliopisto, 2003)
  • Eviö, Sirpa (Helsingin yliopisto, 2006)
    Thirty percent of 70-year-old women have osteoporosis; after age of 80 its prevalence is up to 70%. Postmenopausal women with osteoporosis seem to be at an increased risk for cardiovascular events, and deterioration of oral health, as shown by attachment loss of teeth, which is proportional to the severity of osteoporosis. Osteoporosis can be treated with many different medication, e.g. estrogen and alendronate. We randomized 90 elderly osteoporotic women (65-80 years of age) to receive hormone therapy (HT)(2mg E2+NETA), 10mg alendronate, and their combination for two years and compared their effects on bone mineral density (BMD) and turnover, two surrogate markers of the risk of cardiovascular diseases, C-reactive protein (CRP) and E-selectin, as well as oral health. The effect of HT on health-related quality of life (HRQoL) was studied in the population-based cohort of 1663 postmenopausal women (mean age 68 yr) (585 estrogen users and 1078 non-users). BMD was measured with dual-energy X-ray absorptiometry (DXA) at 0, 12 and 24 months. Urinary N-telopeptide (NTX) of type I collagen, a marker of bone resorption, and serum aminoterminal propeptide of human type I procollagen (PINP), a marker of bone formation, were measured every six months of treatment. Serum CRP and E-selectin, were measured at 0, 6, and 12 months. Dental, and periodontal conditions, and gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 levels were studied to evaluate the oral health status and for the mouth symptoms a structured questionnaire was used. The HRQoL was measured with 15D questionnaire. Lumbar spine BMD increased similarly in all treatment groups (6.8-8.4% and 9.1-11.2%). Only HT increased femoral neck BMD at both 12 (4.9%) and 24 months (5.8%), at the latter time point the HT group differed significantly from the other groups. HT reduced bone marker levels of NTX and PINP significantly less than other two groups.Oral HT significantly increased serum CRP level by 76.5% at 6 and by 47.1% (NS) at 12 months, and decreased serum E-selectin level by 24.3% and 30.0%. Alendronate had no effect on these surrogate markers. Alendronate caused a decrease in the resting salivary flow rate and tended to increase GCF MMP-8 levels. Otherwise, there was no effect on the parameters of oral health. HT improved the HRQoL of elderly women significantly on the dimensions of usual activities, vitality and sexual activity, but the overall improvement in HRQoL was neither statistically significant nor clinically important. In conclusion, bisphosphonates might be the first option to start the treatment of postmenopausal osteoporosis in the old age.
  • Salmela, Marja (Helsingin yliopisto, 2010)
    There is only little information available on the 4-6-year-old child s hospital-related fears, and on the coping with such fears, as expressed by the children themselves. However, previous data collected from parents and hospital personnel indicate that hospitalization is an anxiety-producing experience for young children. The purpose of this study was to describe the experience of hospital-related fears and the experience of coping with hospital-related fears of 4-6-year-old children. The aim of this study was to form a descriptive model of the subjective experience of hospital-related fears and coping strategies of 4-6-year old children. The data were collected by interviewing 4-6-year-old children from a hospital and kindergarten settings in Finland from 2004 to 2006. Ninety children were interviewed in order to describe the hospital-related fear and the experience of fear, and 89 to describe their coping with the fear and the experience of coping. The children were chosen through purposive sampling. The data were gathered by semi-structured interview, supported by pictures. The data about hospital-related fears and on strategies for coping with hospital-related fears were reviewed by qualitative and quantitative methods. The experience of hospital-related fears and coping with these fears were analyzed using Colaizzi s Method of Phenomenological Analysis. The results revealed that more than 90 % of the children said they were afraid of at least one thing in hospital. Most of the fears could be categorized as nursing interventions, fears of being a patient, and fears caused by the developmental stage of the child. Children interviewed in the hospital expressed substantially more fears than children interviewed in kindergarten. Children s meanings of hospital-related fears were placed into four main clusters: 1) insecurity, 2) injury, 3) helplessness, 4) and rejection. The results also showed that children have plenty of coping strategies, to deal with their fears, especially such strategies in which the children themselves play an active role. Most often mentioned coping strategies were 1) the presence of parents and other family members, 2) the help of the personnel, 3) positive images and humour, 4) play, and 5) the child s own safety toy. The children interviewed in the hospital mentioned statistically significantly more often play, positive imagination and humour as their coping strategy than children interviewed in kindergarten. The meaning of coping with hospital fears consisted of six clusters: pleasure, security, care, understanding the meaning of the situation participating, and protecting oneself. Being admitted to a hospital is an event which may increase the fears of a 4-6-year-old child. Children who have personal experience of being admitted to a hospital describe more fears than healthy children in kindergarten. For young children, hospital-related fear can be such a distressing experience that it reflects on their feelings of security and their behaviour. Children can sometimes find it difficult to admit their fear. Children need the help of adults to express their hospital-related fears, the objects of the fears, and to cope with the fears. Personnel should be aware of children s fears and support them in the use of coping strategies. In addition to the experiences of security and care, pre-school-aged children need active coping strategies that they can use themselves, regardless of the presence of the parents or nurses. Most of all, children need the possibility to play and experience pleasure. Children can also be taught coping strategies which give them an active, positive role.
  • Vironen, Jaana (Helsingin yliopisto, 2005)
  • Härmä, Maiju (Helsingin yliopisto, 2007)
    Rejection and infections are the two most common complications after liver transplantation. Human herpesvirus-6 (HHV-6) belongs to the betaherpesviruses, together with its close relatives cytomegalovirus (CMV) and human herpesvirus-7 (HHV-7). The impact of CMV in liver transplantation is well characterized, but the roles of the other two betaherpesviruses have been acknowledged only recently. Although, HHV-6 reactivation after transplantation is usually asymptomatic, the virus may infect the liver transplant, cause an intragraft lymphocyte dominated inflammatory reaction and graft dysfunction. HHV-6 is also suggested to be associated with liver allograft rejection but the mechanisms are unclear. The aim of this study was to investigate the intragraft immunological processes associated with HHV-6, the involvement of HHV-6 in acute liver failure (ALF) and the hepatic HHV-6 infection of the same patients after transplantation. In addition, the occurrence of HHV-6 and HHV-7 was investigated in liver transplant patients with symptomatic CMV infection. HHV-6 infection of the liver graft was associated with portal lymphocyte infiltration and with a significant increase of adhesion molecules (ICAM-1 and VCAM-1) and the number of cells expressing their ligand molecules (LFA-1, VLA-4) and class II antigens. HHV-6 infection was associated with significant immunological changes, but the immune response was limited to lymphocyte infiltration and the adhesion molecule level. However, one third of these patients developed chronic rejection during the follow-up. Of the patients with ALF of unknown origin, most patients demonstrated HHV-6 antigens in the liver, whereas the opposite was seen in ALF patients with a known disease. After transplantation, HHV-6 recurrence was found in the liver transplant in half of these patients with pre-transplant HHV-6 infection of the liver, whereas no post-transplant HHV-6 infection of the liver was seen in patients without pre-transplant HHV-6. Our studies further demonstrated that both HHV-6 and HHV-7 antigenemia often appeared in association with CMV disease in liver transplant patients. The time-related occurrence of the viruses differed, as HHV-6 appeared early after transplantation and regularly preceded CMV whereas HHV-7 often appeared concurrently with CMV. In conclusion, these results indicate that all three betaherpesviruses are common after liver transplantation, often associated with each other. The immunological events caused by HHV-6 in the liver transplant may be involved in, or trigger mechanisms of allograft rejection. In addition, HHV-6 could be one of the causes of ALF, and pre-transplant HHV-6 infection in ALF patients is a risk factor for post-transplant HHV-6 infection of the graft. These results strongly support the clinical significance of HHV-6 in liver transplantation. Even though the reactivation is usually asymptomatic, in some individuals HHV-6 infection may lead to severe manifestations, such as liver failure or in transplant patients, graft dysfunction and rejection.
  • Virtanen, Oskari (Helsingin yliopisto, 2009)
    Human herpesvirus 6 (HHV-6) was identified from patients with HIV and lymphoproliferative diseases in 1986. It is a β-herpesvirus and is divided into two subgroups, variants A and B. HHV-6 variant B is the cause of exanthema subitum, while variant A has not yet definitely proven to cause any disease. HHV-6, especially variant A, is a highly neurotropic virus and has been associated with many diseases of the central nervous system (CNS) such as encephalitis and multiple sclerosis (MS). The present studies were aimed to elucidate the role of HHV-6 and its two variants in neurological infections. Special attention was given to study the possible role of HHV-6 in the pathogenesis of MS. We studied the expression of HHV-6 antigens using immunohistochemistry in brain autopsy samples from patients with MS and controls. HHV-6 antigen was identified in 70% of MS specimens whereas 30% of control specimens expressed HHV-6 antigen. Serum and cerebrospinal fluid (CSF) samples were collected from patients with MS and patients with other neurological diseases (OND) from patients visiting Helsinki University Central Hospital Neurological Outpatient Clinic during the years 2003 and 2004. In addition, we studied 53 children with suspected encephalitis. We developed an immunofluorescence IgG-avidity assay for the detection of primary HHV-6A and HHV-6B infection. For HHV-6B antibodies, no differences were observed between patients with MS and OND. For HHV-6A both seroprevalence and mean titers were significantly higher in MS compared to OND. HHV-6A low-avidity IgG antibodies, suggestive of primary infection, were found in serum of two, three and one patient with definite MS, possible MS and OND, respectively. From pediatric patients with suspected encephalitis, six serum samples (11.3%) contained low-avidity antibodies, indicating a temporal association between HHV-6A infection and onset of encephalitis. Three out of 26 patients with CDMS and four out of 19 patients with CPMS had HHV-6 antibodies in their CSF compared to none of the patients with OND (p=0.06 and p=0.01, respectively). Two patients with CDMS and three patients with CPMS appeared to have specific intrathecal synthesis of HHV-6A antibodies. In addition, oligoclonal bands (OCB) were observed in the CSF of five out of nine MS patients tested, and in two the OCBs reacted specifically with HHV-6 antigen, which is a novel finding. These results indicate HHV-6 specific antibody production in the CNS and suggest that there is a subset of MS patients with an active or chronic HHV-6A infection in the CNS that might be involved in the pathogenesis of MS. Our studies suggest that HHV-6 is an important causative or associated virus in some neurological infections, such as encephalitis and it might contribute to the development of MS, at least in some cases. In conclusion, HHV-6 is a neurotropic virus that should be taken into consideration when studying acute and chronic CNS diseases of unknown origin.
  • Nicklen, Jenna (Helsingin yliopisto, 2016)
    Background: Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) whereby the host immune system attacks against the myelin. MS affects predominantly young adults and leads to neurological disability. Although it has worldwide penetrance, MS has different incidence rates in different parts of the world. The incidence may also vary among different parts of the country, as seen in Finland. The final diagnosis of MS may often be delayed due to the heterogeneity and relapsing nature of the disease. Occurring symptoms depend on the location of the inflammation in the central nervous system. The most common form of the disease is relapsing-remitting multiple sclerosis (RRMS), in which patients typically recover from all of their symptoms. Infections are often seen before the disease progresses or relapses. Therefore, infectious agents, especially viruses, have been under suspicion for triggering an autoimmune reaction that leads to demyelination. Human herpesviruses are considered to be possible triggers for MS pathogenesis. Objective: Most patients who have been diagnosed with MS have oligoclonal bands (OCBs) of immunoglobulins in their cerebrospinal fluid (CSF). The OCBs are intrathecally produced antibodies mainly consisting of IgG, IgA, and IgM class antibodies. Clinically, the most important antibody group in OCB analysis is the IgG class, which was the main focus in this study. The objective was to study if the OCBs of patients contain antibodies against highly neurotropic and common childhood viruses: human herpesvirus-6 (HHV-6) and varicella-zoster virus (VZV). Another objective was to study if the patients, who have virus-reactive OCBs in their CSF, have some distinguishable features. The main aim was to study the possible role of these viruses in the pathogenesis of MS, focusing on the presence of antibodies during the early stages of the disease. Methods: OCB-positive CSF-serum sample pairs were systematically collected over the course of one year. A retrospective re-detection of OCBs by isoelectric focusing (IEF) was made, and HHV-6A, HHV-6B and VZV-reactive OCBs were localized with affinity driven immunoblotting. HHV-6 IgG antibodies were analyzed from the serum with immunofluorescence assay (IFA). The binding capacity of the IgG antibodies was analyzed and the infections (primary vs. past infection) were classified with avidity testing. During the clinical evaluation, the medical records of the patients were analyzed without knowing the results of the IFA or virus-reactive OCBs. This study has been ethically approved. Results: We had 18 immunocompetent adult patients with serologically primary HHV-6A or HHV-6B infections. None of them had any typical signs of a virus infection (e.g. fever or rash). Of those 18 patients 11 were diagnosed with MS with a primary infection during the early stage of the disease. Of 79 patients, 26 had HHV-6A-, HHV-6B-, or VZV reactive OCBs in their CSF. Of those 26 patients 62% were diagnosed with MS and had these virus-reactive OCBs during the early stage of MS. Patients who had any studied virus-reactive OCBs in their CSF seemed to differ from those without, including: more OCBs (p=0.001-0.003), lower protein concentration (p=0.012), and higher IgG index (p=0.007-0.014) in the CSF. They were also younger (p=0.047). Conclusion:Virus-reactive OCBs are possibly associated with MS disease pathogenesis. HHV-6 and VZV may have some association with MS disease. The pathogenesis of multiple sclerosis may have distinguishable subgroups, including pathogenesis triggered by infections with different viruses.
  • Chen, Ren Wei (Helsingin yliopisto, 2004)
  • Riipinen, Anita (Helsingin yliopisto, 2011)
    Human parvovirus B19 (B19V) is known to cause anemia, hydrops fetalis, and fetal death especially during the first half of pregnancy. Women who are in occupational contact with young children are at increased risk of B19V infection. The role of the recently discovered human parvovirus, human bocavirus (HBoV), in reproduction is unknown. The aim of this research project was to establish a scientific basis for assessing the work safety of pregnant women and for issuing special maternity leave regulations during B19V epidemics in Finland. The impact of HBoV infection on the pregnant woman and her fetus was also defined. B19V DNA was found in 0.8% of the miscarriages and in 2.4% of the intrauterine fetal death (IUFD; fetal death after completed 22 gestational weeks). All control fetuses (from induced abortions) were B19V-DNA negative. The findings on hydropic B19V DNA-positive IUFDs with evidence of acute or recent maternal B19V infection are in line with those of previous Swedish studies. However, the high prevalence of B19V-related nonhydropic IUFDs noted in the Swedish studies was mostly without evidence of maternal B19V infection and was not found during the third trimester. HBoV was not associated with miscarriages or IUFDs. Almost all of the studied pregnant women were HboV-IgG positive, and thus most probably immune to HBoV. All preterm births, perinatal deaths, smallness for gestational age (SGA) and congenital anomaly were recorded among the infants of child-care employees in a nationwide register-based cohort study over a period of 14 years. Little or no differences in the results were found between the infants of the child-care employees and those of the comparison group. The annual B19V seroconversion rate was over two-fold among the child-care employees, compared to the women in the comparison group. The seropositivity of the child-care employees increased with age, and years from qualification/joining the trade union. In general, the child-care employees are not at increased risk for adverse pregnancy outcome. However, at the population level, the risk of rare events, such as adverse pregnancy outcomes attributed to infections, could not be determined. According to previous studies, seronegative women had a 5 10% excess risk of losing the fetus during the first half of their pregnancy, but thereafter the risk was very low. Therefore, an over two-fold increased risk of B19V infection among child-care employees is considerable, and should be taken into account in the assessment of the occupational safety of pregnant women, especially during the first half of their pregnancy.
  • Mikkola, Milla (Helsingin yliopisto, 2013)
    Human pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells) are defined by two important characteristics: unlimited self-renewal capacity and ability to switch on various differentiation pathways. These unique properties make them valuable tools for basic research and for the development of regenerative therapies. Since the discovery of stem cells, diverse culture conditions have been developed. Pluripotent stem cells are cultured either in the presence of feeder cells or with extracellular matrix components that together with growing colonies create a niche supporting the growth of undifferentiated cells. Current standard in vitro cell culture techniques are based on the use of xenogeneic reagents. However, this is not compatible with the clinical applications of human pluripotent stem cells. The aim of this study was to develop optimal culture conditions for pluripotent stem cells without the use of xenogeneic reagents, based on the analysis of their cell surface glycan expression. Initially, postnatal human feeder cells, foreskin fibroblasts, were shown to support the derivation of new embryonic stem cell lines and continued undifferentiated growth of these cells. However, the growth rate of stem cells was significantly lower on human feeder cells than on mouse embryonic fibroblasts. This feature restricts the use of human feeder cells for large-scale cell production of stem cells. Next, a number of human embryonic stem cell lines were derived and characterized in detail. The results show that despite of similar basic characteristics in the undifferentiated state, the differentiation capacity of stem cell lines varies. This highlights the need to identify markers that reliably predict cell lineage propensity of the stem cells lines. To identify such predictive markers we conducted a global analysis of cell surface glycans expressed on pluripotent human stem cells. The results show that embryonic stem cells have a unique glycan fingerprint that differs from their differentiated derivatives. This suggests that information of stem cell surface glycans can be used to design markers that define specific stages of differentiation. In addition, this information can be used to develop defined reagents for stem cell culture. Based on the analysis of stem cell surface glycans, specific glycan-binding lectins were studied for their capacity to support human pluripotent stem cells. This lead to the discovery of a lectin, Erythrina Cristagalli (ECA) as a potent simple defined matrix for human pluripotent stem cells. This simple defined matrix, combined with a defined culture medium and the use of a Rho-kinase inhibitor at the time of cell propagation, allowed more efficient production of high-quality human pluripotent stem cells than Matrigel®, the current standard acellular matrix used for stem cell culture. Taken together, these studies advance the development of technologies needed for the efficient generation of fully undifferentiated human pluripotent stem cells in defined conditions.
  • Kakkola, Laura (Helsingin yliopisto, 2008)
    Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.
  • Itkonen, Outi (Helsingin yliopisto, 2008)
    Human pancreatic juice contains two major trypsinogen isoenzymes called trypsinogen-1 and -2, or cationic and anionic trypsinogen, respectively. Trypsinogen isoenzymes are also expressed in various normal and malignant tissues. We aimed at developing monoclonal antibodies (MAbs) and time-resolved immunofluorometric methods recognizing human trypsinogen-1 and -2, respectively. Using these MAbs and methods we purified, characterized and quantitated trypsinogen isoenzymes in serum samples, ovarian cyst fluids and conditioned cell culture media. In sera from healthy subjects and patients with extrapancreatic disease the concentration of trypsinogen-1 is higher than that of trypsinogen-2. However, in acute pancreatitis we found that the concentration of serum trypsinogen-2 is 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentration is only 15-fold. This suggested that trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis. In human ovarian cyst fluids tumor-associated trypsinogen-2 (TAT-2) is the predominant isoenzyme. Most notably, in mucinous cyst fluids the levels of TAT-2 were higher in borderline and malignant than in benign cases. The increased levels in association with malignancy suggested that TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. Serum samples from patients who had undergone pancreatoduodenectomy contained trypsinogen-2. Trypsinogen-1 was detected in only one of nine samples. These results suggested that the expression of trypsinogen is not restricted to the pancreas. Determination of the isoenzyme pattern by ion exchange chromatography revealed isoelectric variants of trypsinogen isoenzymes in serum samples. Intact trypsinogen isoenzymes and tryptic and chymotryptic trypsinogen peptides were purified and characterized by mass spectrometry, Western blot analysis and N-terminal sequencing. The results showed that pancreatic trypsinogen-1 and -2 are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin, thus Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic trypsinogens.
  • Keränen, Mikko (Helsingin yliopisto, 2014)
    Cardiac transplantation is one of the most effective therapies for end-stage heart disease. Short-term survival is good, whereas long-term survival remains relatively poor due to chronic rejection and immunosuppression related morbidities. Early injury of the allografts during brain death donation, organ preservation, ischemia-reperfusion, and alloimmune response may have devastating effects on long-term survival of cardiac allograft recipients. The common denominator for all of these is ischemia. Transcription factor hypoxia-inducible factor-1 (HIF-1) is the master regulator of gene transcription during low oxygen (O2) tension. HIF-1 regulates most of the major adaptive functions that promote survival during cellular stress, particularly during hypoxia. HIF-1 also couples with nuclear factor-κb (NF-κB), thus connecting the two innate stress responses: hypoxic response and innate immunity. The activity of HIF-1 is dictated by the stability of the O2-sensitive subunit HIF-1α. The role of HIF-1 in cardiac transplantation is virtually unknown. Our results show that the HIF-1 pathway is activated during allograft ischemia, ischemia-reperfusion injury, and acute and chronic rejection in the rat. Pharmacological augmentation of the HIF pathway in the donor enhanced ischemia-reperfusion injury, whereas in the recipient it had anti-inflammatory effects and prolonged survival in the rat. Transgenic augmentation of the HIF-pathway in the recipient myelomonocytic cells prevented the development of acute rejection and prolonged survival in mice. Cardiomyocyte-targeted gene transfer of HIF-1α reduced cardiomyocyte apoptosis and the development of cardiac allograft vasculopathy in the rat. The results of this experimental study indicate that HIF-1 has a pivotal role in cardiac allografts. Accumulation of HIF-1α reflects injury of the allograft and could be used as a marker of ischemic injury that requires medical attention. Augmentation of the HIF pathway cell-specifically appears to diminish cardiac allograft rejection, whereas wide-ranging non-targeted HIF stabilization in the transplant may have a detrimental outcome.
  • Heliövaara-Peippo, Satu (Helsingin yliopisto, 2012)
    Menorrhagia significantly impairs the quality of life of many women and causes major health care costs. Hysterectomy has been the standard treatment for menorrhagia. Levonorgestrel-releasing intrauterine system (LNG-IUS) has been advocated as an alternative to surgery. The VuoKKo trial set out to compare the LNG-IUS and hysterectomy in the treatment of menorrhagia, and it consisted of 236 women, aged 35 49 years, referred for menorrhagia to the five university hospitals in Finland between 1994 and 1997. Of these women, 117 were randomized to treatment with hysterectomy and 119 to treatment with LNG-IUS. The follow-up visits took place at 6 and 12 months after treatment and again 5 and 10 years after randomization. The objective of this study was to compare the cost-effectiveness and effects on health-related quality of life (HRQoL), pain, lower urinary tract symptoms (LUTS) and cardiovascular disease (CVD) risk factors of these two treatment modalities during a 10-year follow-up. The continuation rate at 10 years was 94%. Of the 119 women assigned to LNG-IUS, 55 (46%) subsequently underwent hysterectomy over the 10-year period. HRQoL and psychosocial well-being improved during the first 5 years, but diminished between 5 and 10 years, presumably due to ageing. No significant differences emerged between the groups, but the overall costs per participant were lower in the LNG-IUS group ($3423) than in the hysterectomy group ($4937). Both hysterectomy and LNG-IUS decreased pain substantially. The occurrence of pain decreased most during the first 6 months and monthly frequent pain decreased most of all. Lower abdominal pain score (occurrence and intensity) decreased in both groups, but back pain score decreased only in the LNG-IUS group. Women treated by hysterectomy used more medication for urinary incontinence than women treated by LNG-IUS (12% vs. 1%, p=0.006). Urinary tract infections (34% vs. 14%, p=0.002), feeling of incomplete emptying (19% vs. 9%, p=0.04) and stress urinary incontinence (48% vs. 34%, p=0.04) were more common in women with hysterectomy than in LNG-IUS users. The CVD risk profile showed that women treated by hysterectomy had higher levels of inflammatory cytocines, TNF-α at 5 years (median 108.59 vs. 49.02 pg/ml) and hsCRP at 10 years (median 1.55 vs. 0.78 mg/ml) than women treated by LNG-IUS. Women treated by hysterectomy used more often estrogen therapy than women treated by LNG-IUS (56% vs. 27%, p PIENEMPI 0.001) 10 years after the treatment. LNG-IUS is a safe and cost-effective alternative to hysterectomy in the treatment of menorrhagia. The overall costs were approximately 31% lower in the LNG-IUS group than in the hysterectomy group. Both treatments have a favourable effect on HRQoL, but hysterectomy seems to predispose women more to CVD risk factors and LUTS over the long run.
  • Brummer, Tea (Helsingin yliopisto, 2012)
    In the 1990 s and up until 2002, annual numbers of hysterectomies for benign disease in Finland exceeded 10 000; only cataract surgeries and cesarean sections were more commonly performed on women. Hysterectomy is traditionally performed through laparotomy, meaning abdominal hysterectomy (AH), which currently is still the most common surgical approach for hysterectomy worldwide. In Finland, as well, a national cohort of hysterectomy for benign disease in 1996 showed AH as being the most common method, with 58%, but unlike in other nations, laparoscopic hysterectomy (LH) was fairly common, and performed for as many as 24%. Current guidelines state, that vaginal hysterectomy (VH) should be performed in preference to AH when possible. When VH is not possible, LH may avoid the need for AH; but LH is associated with an increased risk for urinary tract injuries. The aim of this study was to evaluate in a national setting the current trends of hysterectomy for benign disease, focusing on the incidence and risk factors for various complications. In addition, analysis concerns the coverage and effect of thrombosis and antibiotic prophylaxis. Prior to the national prospective FINHYST study in 2006, major complications of LH in 2000-2005 were analysed from the registers of the Patient Insurance Centre in Finland and compared to those of the previous decade. This register also served in verification of major complications involved in FINHYST, in which all 46 public hospitals where hysterectomies were performed collaborated, in addition to 7 private clinics. Detailed data were collected on intraoperative and on early- and late-onset postoperative complications. Risk factors for complications were analysed by logistic regression adjusted for confounders. In this thesis, also complications of FINHYST in 2006 were compared to the prospective national hysterectomy study in 1996, as a 10-year follow-up Since 2002, annual numbers of hysterectomies for benign disease in Finland gradually declined, and in 2007-2010 remained stable at an average 5 750 per year. By method, VH has been the most common approach in Finland since 2002, and LH surpassed AH in 2005. Urinary tract injuries, particularly ureter injuries with LH, were reduced to 0.3% in 2000-2005, representing a nearly three-fold reduction since the 1990 s. The FINHYST study in 2006, which covered 79.4% of national hysterectomies for benign disease, comprised 1 255 AHs (24%), 1 679 LHs (32%), and 2 345 VHs (44%), with respective overall complications occurring in 19.2%, 15.4%, and 11.7%, and major complications (organ injuries, reoperations or venous thromboembolism) in 4.0%, 4.3%, and 2.6%. No deaths occurred. Most bladder and bowel injuries were detected and repaired intraoperatively (88% and 83%). Risk factors for complications overall were adhesiolysis (OR 2.48), uterine size of 500 g or more (OR 1.44), and concomitant surgery (OR 1.28). Major complication risk factors were adhesiolysis (OR 2.41), more specifically bladder injuries associated with caesarean section (OR 4.01) and uterine size of 500 g or more (OR 2.88). Postoperative infections were associated with those overweight (OR 1.61), obese (OR 1.67), or extremely obese (OR 1.82), compared to those of normal weight. Pharmaceutical thrombosis prophylaxis (TP) was given to 64.8%. TP was associated with postoperative haemorrhage or haematoma in VH performed for prolapse (OR 4.82), and in AH (OR 2.87). Age of 55 or over reduced the risk for complications overall (OR 0.61), and for infections (OR 0.66); operative haemorrhage decreased with age. Antibiotic prophylaxis was given to 97.5%: cefuroxime alone to 38.5%, metronidazole alone to 9.9%, and metronidazole in combination with cefuroxime to 43.0%. Overall, 54% received metronidazole, which had no significant independent risk-reducing effect, nor any interactive effect when combined with cefuroxime, for total infections in any type of hysterectomy. Cefuroxime, however, had a risk-reducing effect (OR 0.29); also for all hysterectomy methods separately. No method was independently associated with complications, except for infections, for which AH, compared to LH, led to a risk for febrile events, wound infections, and urinary tract infections. Compared to AH, in both the hysterectomy methods (LH and VH) in which the vault is closed vaginally, the risk for pelvic infection was 5-fold. In conclusion, while numbers of hysterectomies in Finland have declined, the minimally invasive methods have overtaken AH. Such a trend promoting faster recovery from surgery has reduced complications, particularly infections. The follow-up also showed a reduction in ureter injuries in LH, and bowel injuries in VH. During the time that VH became the most common method for hysterectomy, its complications nearly halved.
  • Tian, Li (Helsingin yliopisto, 2001)
  • Eriksson, Johanna (Helsingin yliopisto, 2014)
    Cutaneous melanoma is one of the most aggressive malignancies, typified by a high metastatic tendency and refractoriness to treatments. Identification of the molecular mechanisms behind the development and progression of melanomas could provide new insights into treating this challenging disease. The aim of this study was to identify and characterize genes and proteins associated with melanoma development and progression by comparing genome-wide gene expression profiles from different stages of melanoma, and to evaluate their potential as diagnostic and prognostic markers and therapeutic targets. A further aim was to develop sensitive RT-PCR and immunohistochemical assays for the detection of melanoma micrometastases. We found the outgrowth of melanoma metastases to be associated with the activation of stromal fibroblasts, increased TGFβ/Smad2 signaling, and an up-regulation of TGFβ-target genes encoding extracellular matrix proteins fibronectin, collagen-I, periostin, and versican. These proteins were found to form around tumor cells fibrillar networks, which, suggested by our functional studies, promote the growth and migration of tumor cells as well as stromal fibroblasts and endothelial cells. In addition, periostin and cellular fibronectin were found to be specifically up-regulated in newly-formed tumor blood vessels. We further found a common up-regulation of collagen triple helix repeat containing 1 (CTHRC1) and cysteine cathepsin B and L1 proteases during melanoma progression. Our analyses suggest that CTHRC1 is, together with fibronectin and integrin β3, part of the pro-invasive and pro-angiogenic transcriptional program induced by the NFATC2 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2) transcription factor. Cathepsins B and L1 (and TGFβ signaling), in turn, were found to play essential roles in the co-invasive process of melanoma cells and activated fibroblasts. Further, our results suggest that high expression of CTHRC1, fibronectin, and cathepsin B in primary melanomas may serve as prognostic factors predicting poor survival. Combined, these results suggest that TGFβ receptors, fibronectin, periostin, and CTHRC1, as well as cathepsins B and L1 are attractive therapeutic targets against advanced melanomas. In addition, we identified the best gene expression markers for the detection of melanoma lymph node metastases. Of the identified genes, melan-A (MLANA), tyrosinase (TYR), melanoma inhibitory activity (MIA), preferentially expressed antigen in melanoma (PRAME), and osteopontin (SPP1) were tested as potential melanoma micrometastasis markers by RT-PCR and immunohistochemistry (IHC). Graded MLANA- and TYR-RT-PCR analyses were found to detected clinically significant metastases better than IHC, suggesting that quantifiable RT-PCR analyses should be used to confirm and complement histological and immunohistochemical examinations. Further, the melanoma-specific genes, PRAME and SPP1, may be used to differentiate melanoma cells from benign nevus cells occasionally residing in lymph nodes.
  • Salmikangas, Paula (Helsingin yliopisto, 2001)