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  • 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.
  • 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)
  • Mätzke, Sorjo (Helsingin yliopisto, 2004)
  • Hernandez Rios, Marcela (Helsingin yliopisto, 2012)
    Chronic periodontitis results from a complex aetiology, including the formation of a subgingival biofilm and the elicitation of the host s immune and inflammatory response. The hallmark of chronic periodontitis is alveolar bone loss and soft periodontal tissue destruction. Evidence supports that periodontitis progresses in dynamic states of exacerbation and remission or quiescence. The major clinical approach to identify disease progression is the tolerance method, based on sequential probing. Collagen degradation is one of the key events in periodontal destructive lesions. Matrix metalloproteinase (MMP)-8 and MMP-13 are the primary collagenolytic MMPs that are associated with the severity of periodontal inflammation and disease, either by a direct breakdown of the collagenised matrix or by the processing of non-matrix bioactive substrates. Despite the numerous host mediators that have been proposed as potential biomarkers for chronic periodontitis, they reflect inflammation rather than the loss of periodontal attachment. The aim of the present study was to determine the key molecular MMP-8 and -13 interactions in gingival crevicular fluid (GCF) and gingival tissue from progressive periodontitis lesions and MMP-8 null allele mouse model. In study (I), GCF and gingival biopsies from active and inactive sites of chronic periodontitis patients, which were determined clinically by the tolerance method, and healthy GCF were analysed for MMP-13 and tissue inhibitor of matrix metalloproteinases (TIMP)-1. Chronic periodontitis was characterised by increased MMP-13 levels and the active sites showed a tendency of decreased TIMP-1 levels associated with increments of MMP-13 and total protein concentration compared to inactive sites. In study (II), we investigated whether MMP-13 activity was associated with TIMP-1, bone collagen breakdown through ICTP levels, as well as the activation rate of MMP-9 in destructive lesions. The active sites demonstrated increased GCF ICTP levels as well as lowered TIMP-1 detection along with elevated MMP-13 activity. MMP-9 activation rate was enhanced by MMP-13 in diseased gingival tissue. In study (III), we analysed the potential association between the levels, molecular forms, isoenzyme distribution and degree of activation of MMP-8, MMP-14, MPO and the inhibitor TIMP-1 in GCF from periodontitis progressive patients at baseline and after periodontal therapy. A positive correlation was found for MPO/MMP-8 and their levels associated with progression episodes and treatment response. Because MMP-8 is activated by hypochlorous acid in vitro, our results suggested an interaction between the MPO oxidative pathway and MMP-8 activation in GCF. Finally, in study (IV), on the basis of the previous finding that MMP-8-deficient mice showed impaired neutrophil responses and severe alveolar bone loss, we aimed to characterise the detection patterns of LIX/CXCL5, SDF-1/CXCL12 and RANKL in P. gingivalis-induced experimental periodontitis and in the MMP-8-/- murine model. The detection of neutrophil-chemoattractant LIX/CXCL5 was restricted to the oral-periodontal interface and its levels were reduced in infected MMP-8 null mice vs. wild type mice, whereas the detection of SDF-1/CXCL12 and RANKL in periodontal tissues increased in experimentally-induced periodontitis, irrespectively from the genotype. Accordingly, MMP-8 might regulate LIX/CXCL5 levels by undetermined mechanisms, and SDF-1/CXCL12 and RANKL might promote the development and/or progression of periodontitis.
  • Anttila, Timo Verneri (Helsingin yliopisto, 2010)
    Migraine is the common cause of chronic episodic headache, affecting 12%-15% of the Caucasian population (41 million Europeans and some half a million Finns), and causes considerable loss of quality of life to its sufferers, as well as being linked to increased risk for a wide range of conditions, from depression to stroke. Migraine is the 19th most severe disease in terms of disability-adjusted life years, and 9th among women. It is characterized by attacks of headache accompanied by sensitivity to external stimuli lasting 4-72 hours, and in a third of cases by neurological aura symptoms, such as loss of vision, speech or muscle function. The underlying pathophysiology, including what triggers migraine attacks and why they occur in the first place, is largely unknown. The aim of this study was to identify genetic factors associated with the hereditary susceptibility to migraine, in order to gain a better understanding of migraine mechanisms. In this thesis, we report the results of genetic linkage and association analyses on a Finnish migraine patient collection as well as migraineurs from Australia, Denmark, Germany, Iceland and the Netherlands. Altogether we studied genetic information of nearly 7,000 migraine patients and over 50,000 population-matched controls. We also developed a new migraine analysis method called the trait component analysis, which is based on individual patient responses instead of the clinical diagnosis. Using this method, we detected a number of new genetic loci for migraine, including on chromosome 17p13 (HLOD 4.65) and 10q22-q23 (female-specific HLOD 7.68) with significant evidence of linkage, along with five other loci (2p12, 8q12, 4q28-q31, 18q12-q22, and Xp22) detected with suggestive evidence of linkage. The 10q22-q23 locus was the first genetic finding in migraine to show linkage to the same locus and markers in multiple populations, with consistent detection in six different scans. Traditionally, ion channels have been thought to play a role in migraine susceptibility, but we were able to exclude any significant role for common variants in a candidate gene study of 155 ion transport genes. This was followed up by the first genome-wide association study in migraine, conducted on 2,748 migraine patients and 10,747 matched controls followed by a replication in 3,209 patients and 40,062 controls. In this study, we found interesting results with genome-wide significance, providing targets for future genetic and functional studies. Overall, we found several promising genetic loci for migraine providing a promising base for future studies in migraine.