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  • Hahtola, Sonja (Helsingin yliopisto, 2008)
    Cutaneous T-cell lymphomas (CTCL) represent a group of non-Hodgkin lymphomas showing a growing incidence especially in the Western world. The mechanisms leading to the disease are largely unknown, diagnosis is difficult and therefore often delayed, and no curative therapy exists. CTCL presents with skin symptoms although the malignant cells are not derived of human skin but of human immune system instead. The malignant cells are mature T helper memory cells, and preferentially express cytokines characteristic to T-helper 2 (Th2) type immune response. Chromosomal instability is a typical feature of CTCL. Some secondary cancers occur in CTCL patients more often than in general population, the most common of which are lung cancers and non-Hodgkin lymphomas. The aim of the study was to identify genes relevant to CTCL pathogenesis to clarify the poorly understood pathomechanisms behind the disease group. The two most common subgroups of CTCL, mycosis fungoides (MF) and Sezary syndrome (SS), as well as the difficult to diagnose subcutaneous panniculitis like T-cell lymphoma (SPTL), were studied. To reveal the molecular pathogenesis underlying CTCL-associated lung cancer, CTCL-associated lung cancer samples were analysed and compared to primary / reference lung cancer samples. Identification of potential novel diagnostic markers as well as target molecules for therapy was a special focus of the study. To achieve this, patient derived material was studied with molecular cytogenetic techniques, microarrays and gene expression analyses. This study identified the first specific, recurrent gene-level aberration in CTCL, namely the deletion / translocation of neuron navigator 3 (NAV3) gene, characteristic to many different CTCL subgroups, namely MF, SS, and SPTL. NAV3 is a putative haploinsufficient tumor suppressor influencing the differentiation of T-helper cells. The pathways affected by its aberrant function, are currently being studied. Novel insights to CTCL pathogenesis were achieved through the observation that several genes specific for Th1 type immune response (e.g. T-bet, RANTES, and NKG7) were downregulated in CTCL. For the first time, CTCL-associated lung cancers were observed to show chromosomal aberrations differing from primary lung cancers. This finding warrants further prospective studies to identify the common underlying factors between CTCL and CTCL-associated lung cancer. Demonstration of NAV3 deletion by FISH provides a novel diagnostic tool, and overexpression of certain membrane antigens (e.g. MS4A4A, LIR9 and CD52) will provide the basis for developing novel antibody-based therapeutic means.
  • Aavikko, Mervi (Helsingin yliopisto, 2014)
    Identification of tumor susceptibility genes has contributed significantly to our understanding on molecular basis of cancer. Over one hundred high risk tumor susceptibility genes have been identified during past few decades, but some remain still to be characterized, including those that do not cause other clinically recognizable syndromic features, and present with incomplete penetrance. The aim of this study was to assess the familial aggregation of cancers in Finland and to identify novel tumor predisposition families and genes in families with Kaposi sarcoma (KS), nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), and intracranial meningioma. First, to assess the familial aggregation of cancers in Finland, we conducted a computerized clustering of 878 593 patients in FCR, based on family name at birth, municipality of birth, and tumor type. Additional clustering, based on family name at birth and tumor type, was also conducted. We calculated a cluster score to assess the frequency of familial aggregation of different tumor types. The top scoring types included those with well-established genetic background, but also those with unknown genetic etiology such as KS. We performed genealogy analysis from the clustered KS cases, and identified a family with five affected individuals and several smaller KS families. Furthermore, we showed that KS incidence is unevenly distributed in Finland, with more cases in the western and northeastern than other parts of the country. Second, we examined the genetic susceptibility in the KS family with five affected cases. We mapped the shared genomic regions and performed whole genome and exome sequencing. We identified 14 candidate variants, among them a missense variant (c.1337C>T, p.Thr446Ile) in the DNA binding domain of STAT4. We showed that healthy variant carriers had decreased IFN-γ production in the activated T-cells. We studied STAT4 in a large number of familial and sporadic KS cases, but detected no additional mutation carriers. Our results suggest STAT4 as a KS predisposing gene. However, further genetic and functional validation is needed to claim causality. Third, a family of four cousins with a rare subtype of Hodgkin lymphoma, namely NLPHL, was studied for genetic predisposition. Linkage analysis and exome sequencing revealed a frameshift mutation (c.2437-2438_delAG) in NPAT segregating with NLPHL in the family. We sequenced NPAT from a large number of HL patients, and detected an in-frame deletion (c.2171-2173delCTT, p.Ser725del) that was more prevalent in cases than in population matched controls (odds ratio 4.11, P=0.018), supporting the role of NPAT as a candidate predisposition gene. Fourth, we studied a family with five affected siblings with intracranial meningiomas, four of whom had multiple meningiomas. We identified a c.367C>T (p.Arg123Cys) mutation in SUFU that segregated with the meningiomas in the family. The tumors from the affected individuals showed loss of heterozygosity of the wild-type allele at the site of the mutation. We showed that the mutation led to lowered SUFU activity and dysregulated Hedgehog signaling. Our genetic and functional analyses indicate that germline SUFU mutations predispose to meningiomas, particularly multiple meningiomas.
  • Vaahtomeri, Kari (Helsingin yliopisto, 2011)
    Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.
  • Kyttälä, Mira (Helsingin yliopisto, 2006)
    Meckel syndrome (MKS, MIM 249000) is an autosomal recessive developmental disorder causing death in utero or shortly after birth. The hallmarks of the disease are cystic kidney dysplasia and fibrotic changes of the liver, occipital encephalocele with or without hydrocephalus and polydactyly. Other anomalies frequently seen in the patients are incomplete development of the male genitalia, club feet and cleft lip or palate. The clinical picture has been well characterized in the literature while the molecular pathology underlying the disease has remained unclear until now. In this study we identified the first MKS gene by utilizing the disease haplotypes in Finnish MKS families linked to the MKS1 locus on chromosome 17q23 (MKS1) locus. Subsequently, the genetic heterogeneity of MKS was established in the Finnish families. Mutations in at least four different genes can cause MKS. These genes have been mapped to the chromosomes 17q23 (MKS1), 11q13 (MKS2), 8q22 (MKS3) and 9q33 (MKS4). Two of these genes have been identified so far: The MKS1 gene (this work) and the MKS3 gene. The identified MKS1 gene was initially a novel human gene which is conserved among species. We found three different MKS mutations, one of them being the Finnish founder mutation. The information available from MKS1 orthologs in other species convinced us that the MKS1 gene is required for normal ciliogenesis. Defects of the cilial system in other human diseases and model organisms actually cause phenotypic features similar to those seen in MKS patients. The MKS3 (TMEM67) gene encodes a transmembrane protein and the gene maps to the syntenic Wpk locus in the rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. The available information from these two genes suggest that MKS1 would encode a structural component of the centriole required for normal ciliary functions, and MKS3 would be a transmembrane component most likely required for normal ciliary sensory signaling. The MKS4 locus was localized to chromosme 9q32-33 in this study by using an inbred Finnish family with two affected and two healthy children. This fourth locus contains TRIM32 gene, which is associated to another well characterized human ciliopathy, Bardet Biedl syndrome (BBS). Future studies should identify the MKS4 gene on chromosome 9q and confirm if there are more than two genes causing MKS Finnish families. The research on critical signaling pathways in organogenesis have shown that both Wnt and Hedgehog pathways are dependent on functional cilia. The MKS gene products will serve as excellent model molecules for more detailed studies of the functional role of cilia in organogenesis in more detail.
  • Siintola, Eija (Helsingin yliopisto, 2008)
    The neuronal ceroid lipofuscinoses (NCLs) are a group of mostly autosomal recessively inherited neurodegenerative disorders. The aim of this thesis was to characterize the molecular genetic bases of these, previously genetically undetermined, NCL forms. Congenital NCL is the most aggressive form of NCLs. Previously, a mutation in the cathepsin D (CTSD) gene was shown to cause congenital NCL in sheep. Based on the close resemblance of the phenotypes between congenital NCLs in sheep and human, CTSD was considered as a potential candidate gene in humans as well. When screened for mutations by sequencing, a homozygous nucleotide duplication creating a premature stop codon was identified in CTSD in one family with congenital NCL. While in vitro the overexpressed truncated mutant protein was stable although inactive, the absence of CTSD staining in brain tissue samples of patients indicated degradation of the mutant CTSD in vivo. A lack of CTSD staining was detected also in another, unrelated family with congenital NCL. These results imply that CTSD deficiency underlies congenital NCL. While initially Turkish vLINCL was considered a distinct genetic entity (CLN7), mutations in the CLN8 gene were later reported to account for the disease in a subset of Turkish patients with vLINCL. To further dissect the genetic basis of the disease, all known NCL genes were screened for homozygosity by haplotype analysis of microsatellite markers and/or sequenced in 13 mainly consanguineous, Turkish vLINCL families. Two novel, family-specific homozygous mutations were identified in the CLN6 gene. In the remaining families, all known NCL loci were excluded. To identify novel gene(s) underlying vLINCL, a genomewide single nucleotide polymorphism scan, homozygosity mapping, and positional candidate gene sequencing were performed in ten of these families. On chromosome 4q28.1-q28.2, a novel major facilitator superfamily domain containing 8 (MFSD8) gene with six family-specific homozygous mutations in vLINCL patients was identified. MFSD8 transcript was shown to be ubiquitously expressed with a complex pattern of alternative splicing. Our results suggest that MFSD8 is a novel lysosomal integral membrane protein which, as a member of the major facilitator superfamily, is predicted to function as a transporter. Identification of MFSD8 emphasizes the genetic heterogeneity of Turkish vLINCL. In families where no MFSD8 mutations were detected, additional NCL-causing genes remain to be identified. The identification of CTSD and MFSD8 increases the number of known human NCL-causing genes to eight, and is an important step towards the complete understanding of the genetic spectrum underlying NCLs. In addition, it is a starting point for dissecting the molecular mechanisms behind the associated NCLs and contributes to the challenging task of understanding the molecular pathology underlying the group of NCL disorders.
  • Hodgson, Ulla (Helsingin yliopisto, 2006)
  • Rauta, Virpi (Helsingin yliopisto, 2006)
    IgA nephropathy (IgAN) is the most common primary glomerulonephritis. In one third of the patients the disease progresses, and they eventually need renal replacement therapy. IgAN is in most cases a slowly progressing disease, and the prediction of progression has been difficult, and the results of studies have been conflicting. Henoch-Schönlein nephritis (HSN) is rare in adults, and prediction of the outcome is even more difficult than in IgAN. This study was conducted to evaluate the clinical and histopathological features and predictors of the outcome of IgAN and HSN diagnosed in one centre (313 IgAN patients and 38 HSN patients), and especially in patients with normal renal function at the time of renal biopsy. The study also aimed to evaluate whether there is a difference in the progression rates in four countries (259 patients from Finland, 112 from UK, 121 from Australia and 274 from Canada), and if so, can this be explained by differences in renal biopsy policy. The third aim was to measure urinary excretions of cytokines interleukin 1ß (IL-1ß) and interleukin 1 receptor antagonist (IL-1ra) in patients with IgAN and HSN and the correlations of excretion of these substances with histopathological damage and clinical factors. A large proportion of the patients diagnosed in Helsinki as having IgAN had normal renal function (161/313 patients). Four factors, (hypertension, higher amounts of urinary erythrocytes, severe arteriolosclerosis and a higher glomerular score) which independently predicted progression (logistic regression analysis), were identified in mild disease. There was geographic variability in renal survival in patients with IgAN. When age, levels of renal function, proteinuria and blood pressure were taken into account, it showed that the variability related mostly to lead-time bias and renal biopsy indications. Amount of proteinuria more than 0.4g/24h was the only factor that was significantly related to the progression of HSN. the Hypertension and the level of renal function were found to be factors predicting outcome in patients with normal renal function at the time of diagnosis. In IgAN patients, IL-1ra excretion into urine was found to be decreased as compared with HSN patients and healthy controls. Patients with a high IL-1ra/IL-1ß ratio had milder histopathological changes in renal biopsy than patients with a low/normal IL-1ra/IL-1ß ratio. It was also found that the excretion of IL-1ß and especially IL-1ra were significantly higher in women. In conclusion, it was shown that factors associated with outcome can reliably be identified even in mild cases of IgAN. Predicting outcome in adult HSN, however, remains difficult.
  • Kotiranta-Ainamo, Anna (Helsingin yliopisto, 2006)
    The purpose of this work was to elucidate the ontogeny of interleukin-10 (IL-10) secretion from newborn mononuclear cells (MCs), and to examine its relation to the secretion of interferon-g (IFN-g) and immunoglobulins (Igs). The initial hypothesis was that the decreased immunoglobulin (Ig) synthesis of newborn babies was the result of immature cytokine synthesis regulation, which would lead to excessive IL-10 production, leading in turn to suppressed IFN-g secretion. Altogether 57 full-term newborns and 34 adult volunteers were enrolled. Additionally, surface marker compositions of 29 premature babies were included. Enzyme-linked immunoassays were used to determine the amount of secreted IL-10, IFN-g, and Igs, and the surface marker composition of MC were analyzed with a FACScan flow cytometer. The three most important findings were: 1. Cord blood MC, including CD5+ B cells, are able to secrete IL-10. However, when compared with adults, the secretion of IL-10 was decreased. This indicates that reasons other than excessive IL-10 secretion are responsible of reduced IFN-g secretion in newborns. 2. As illustrated by the IL-10 and IFN-g secretion pattern, newborn cytokine profile was skewed towards the Th2 type. However, approximately 25% of newborns had an adult like cytokine profile with both good IL10 and IFN-g secretion, demonstrating that fullterm newborns are not an immunologically homogenous group at the time of birth. 3. There were significant differences in the surface marker composition of MCs between individual neonates. While gestational age correlated with the proportion of some MC types, it is evident that there are many other maternal and fetal factors that influence the maturity and nature of lymphocyte subpopulations in individual neonates. In conclusion, the reduced ability of neonates to secrete Ig and IFN-g is not a consequence of high IL-10 secretion. However, individual newborns differ significantly in their ability to secrete cytokines as well as Igs.
  • Sinkkonen, Saku (Helsingin yliopisto, 2004)
  • Mustonen, Antti (Helsingin yliopisto, 2009)
    Acute knee injury is a common event throughout life, and it is usually the result of a traffic accident, simple fall, or twisting injury. Over 90% of patients with acute knee injury undergo radiography. An overlooked fracture or delayed diagnosis can lead to poor patient outcome. The major aim of this thesis was retrospectively to study imaging of knee injury with a special focus on tibial plateau fractures in patients referred to a level-one trauma center. Multi-detector computed tomography (MDCT) findings of acute knee trauma were studied and compared to radiography, as well as whether non-contrast MDCT can detect cruciate ligaments with reasonable accuracy. The prevalence, type, and location of meniscal injuries in magnetic resonance imaging (MRI) were evaluated, particularly in order to assess the prevalence of unstable meniscal tears in acute knee trauma with tibial plateau fractures. The possibility to analyze with conventional MRI the signal appearance of menisci repaired with bioabsorbable arrows was also studied. The postoperative use of MDCT was studied in surgically treated tibial plateau fractures: to establish the frequency and indications of MDCT and to assess the common findings and their clinical impact in a level-one trauma hospital. This thesis focused on MDCT and MRI of knee injuries, and radiographs were analyzed when applica-ble. Radiography constitutes the basis for imaging acute knee injury, but MDCT can yield information beyond the capabilities of radiography. Especially in severely injured patients , sufficient radiographs are often difficult to obtain, and in those patients, radiography is unreliable to rule out fractures. MDCT detected intact cruciate ligaments with good specificity, accuracy, and negative predictive value, but the assessment of torn ligaments was unreliable. A total of 36% (14/39) patients with tibial plateau fracture had an unstable meniscal tear in MRI. When a meniscal tear is properly detected preoperatively, treatment can be combined with primary fracture fixation, thus avoiding another operation. The number of meniscal contusions was high. Awareness of the imaging features of this meniscal abnormality can help radiologists increase specificity by avoiding false-positive findings in meniscal tears. Postoperative menisci treated with bioabsorbable arrows showed no difference, among different signal intensities in MRI, among menisci between patients with operated or intact ACL. The highest incidence of menisci with an increased signal intensity extending to the meniscal surface was in patients whose surgery was within the previous 18 months. The results may indicate that a rather long time is necessary for menisci to heal completely after arrow repair. Whether the menisci with an increased signal intensity extending to the meniscal surface represent improper healing or re-tear, or whether this is just the earlier healing feature in the natural process remains unclear, and further prospective studies are needed to clarify this. Postoperative use of MDCT in tibial plateau fractures was rather infrequent even in this large trauma center, but when performed, it revealed clinically significant information, thus benefitting patients in regard to treatment.
  • Meretoja, Tuomo (Helsingin yliopisto, 2007)
    Breast cancer is the most common form of potentially fatal cancer in women in the Western world. Better understanding of the breast cancer disease process together with developments in treatments have led to improved survival and reduced risk of recurrence, significantly influencing the acceptance of breast reconstructions as part of breast cancer treatment. Skin-sparing mastectomy followed by immediate breast reconstruction has proved superior to other forms of breast reconstruction in terms of aesthetic outcome. However, due to the relatively recent introduction of skin-sparing mastectomy concerns on the surgical and oncological safety of the operation persist. The aim of the present study is to evaluate the surgical and oncological safety of skin-sparing mastectomy and immediate breast reconstruction in a consecutive patient series with ensuing follow-up. Subsequent aims of the study are to examine possibilities of reducing surgical complications of the operation and to assess the feasibility of sentinel node biopsy together with immediate breast reconstruction. The study population comprises a consecutive series of patients having undergone skin-sparing mastectomy followed by immediate breast reconstruction at the Helsinki University Central Hospital between 1992 and 2006. In Study I, the hospital records of 207 patients, operated between 1992 and 2001, were analyzed for surgical complications and recurrences of breast cancer during follow-up. In Study II, 60 consecutive patients were randomized into either conventional diathermy or radiofrequency coagulation groups to examine possibilities of reducing skin-flap complications. Study III consists of 62 consecutive breast cancer patients evaluated for the feasibility of sentinel node biopsy simultaneously with immediate breast reconstruction. In Study IV, hospital records were analyzed to examine local recurrence of breast cancer in a consecutive series of 146 patients with Stage I or II disease. Post-operative complications in Study I included native skin-flap necrosis (10.1%), hematoma (10.1%), anastomose thrombosis (5.3%), infection (3.4%), hernia (2.6%) and loss of one microvascular flap (0.7%). The Stage I and II patients in Study IV had a local recurrence rate of 2.7%, an isolated regional lymph node recurrence rate of 2.1% and a systemic recurrence rate of 2.7%, during a mean follow-up time of 51 months. The Stage III patients in study I had a locoregional recurrence rate of 31.3% during follow-up. Radiofrequency coagulation in Study II did not decrease skin-flap complications when compared with conventional diathermy. An increased skin-flap complication rate in Study II was associated with smoking and the type of skin incision used. In Study III, eleven patients had tumor positive sentinel nodes, nine of which were detected intraoperatively. Skin-sparing mastectomy followed by immediate breast reconstruction is a safe procedure both surgically and oncologically, especially for early stage breast cancer. Tennis racket type incision is associated with an increased skin-flap complication rate. Sentinel node biopsy with intraoperative assessment of sentinel node metastases is feasible in patients undergoing immediate breast reconstruction.
  • Alfakry, Hatem (2014)
    ABSTRACT An inflammation-associated mechanism of atherogenesis is the current well-accepted hypothesis. The general hypothesis of this series of studies was that periodontitis, as a chronic infectious disease that induces immune-inflammatory responses, detrimentally affects cardiovascular diseases (CVDs). Inflammation is considered a key issue in the pathogenesis of periodontitis and plays an important role in all stages. The aim of this thesis was to determine the possible diagnostic involvement and roles of neutrophilic biomarkers and their regulators (matrix metalloproteinase (MMP) -7, -8, -9, tissue inhibitor of metalloproteinase (TIMP) -1, MMP-8/TIMP-1, myeloperoxidase (MPO), polymorphonuclear neutrophil (PMN) elastase, C-reactive protein (CRP), and heat shock protein antibodies (HSP60) in serum of patients with periodontitis and CVDs, and to investigate whether local periodontal therapy associated with or without antibiotic or anti-MMP medication could reduce these measurable indices of inflammatory biomarkers. Overall, we wished to provide information on potential survival benefits and biomarker diagnostics to patients at risk of recurrent CVD. This thesis consists of four original studies. - In Study I, serum and salivary samples were analysed to determine whether serum antibody levels to hHSP60 are associated with serum antibody levels and salivary carriage of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as with the dental status, in 141 acute coronary syndrome (ACS) patients. A strong positive correlation between IgG-class antibodies against HSP60 and A. actinomycetemcomitans was found. Mean serum IgG antibody levels to HSP60 were significantly higher in A. actinomycetemcomitans IgG- and IgA-seropositive than -seronegative patients. Cross-reactivity in the systemic IgGclass antibody response to HSP60 and A. actinomycetemcomitans was detected in ACS patients. - In Study II, serum samples were collected from 141 ACS patients to investigate the association between serum neutrophilic markers and risk of recurrent ACS. Recurrent ACS events were registered during a one-year follow-up. Results showed that high serum MPO concentrations and MMP-8⁄TIMP-1 ratio may be considered predictive factors for recurrent ACS events, especially in non-periodontitis patients or in those not receiving antimicrobial medication, while elevated TIMP-1 concentration may be a protective factor. - In Study III, during each visit in a 10-month follow-up, serum samples were collected from 31 non-smoking males who had had coronary bypass surgery. Subjects were randomly assigned to receive placebo or 100 mg of doxycycline daily for 4 months. At the end of treatment, serum levels of MMP-8, MMP-7 and MMP-8/TIMP-1 were and remained lower in the doxycycline group relative to the placebo group. Doxycycline, a MMP-inhibitor, might prevent or reduce the risk of secondary myocardial infarctions by providing a systemic antiproteolyticand inflammatory shield. - In Study IV, 120 subjects (aged 48±7 years) suffering from severe generalized periodontitis were randomized to receive either control periodontal therapy (CPT 59 patients) or intensive periodontal therapy (IPT 61 patients). Blood samples were obtained from the patients at baseline and at 1, 7, 30, 60 and 180 days. Acute impairment in endothelial function and increased neutrophil activity were observed one day following IPT relative to CPT (increased CRP, IL-6, endotoxin activity, reactive oxygen species (ROS), lipid oxidation, MMP-8, MPO, MMP-8/TIMP-1, and reduction in TIMP-1 levels), while at 6 months following IPT relative to CPT, improvements in endothelial function were associated with a reduction in ROS production (determination of reactive oxygen metabolites (D-ROM), p<0.01), increased antioxidant potential (BAP, p<0.05) and reduced neutrophilic enzyme activity ratios (MMP-8/TIMP-1, p<0.05). Changes in circulating markers of oxidative stress, endotoxin and neutrophil activity reflected the acute and chronic influences of periodontal treatment on endothelial function. In conclusion, cross-reactivity between host cells and bacterial pathogens might contribute to CVD initiation and progression. Immune and proteolytic processes as part of a systemic persisting low-grade inflammation can provide a link between periodontal disease and CVD. Prevention and treatment of chronic inflammatory conditions, such as chronic periodontitis, might have a beneficial effect and reduce the systemic biomarkers. Suppression of systemic inflammation and improvement of endothelial functions may also lead to reduced CVDassociated proteolytic destructive events and oxidative stress.
  • Oksjoki, Riina (Helsingin yliopisto, 2006)
    Atherosclerosis is an inflammatory disease characterized by accumulation of lipids in the inner layer of the arterial wall. During atherogenesis, various structures that are recognized as non-self by the immune system, such as modified lipoproteins, are deposited in the arterial wall. Accordingly, atherosclerotic lesions and blood of humans and animals with atherosclerotic lesions show signs of activation of both innate and adaptive immune responses. Although immune attack is initially a self-protective reaction, which is meant to destroy or remove harmful agents, a chronic inflammatory state in the arterial wall accelerates atherosclerosis. Indeed, various modulations of the immune system of atherosclerosis-prone animals have provided us with convincing evidence that immunological mechanisms play an important role in the pathogenesis of atherosclerosis. This thesis focuses on the role of complement system, a player of the innate immunity, in atherosclerosis. Complement activation via any of the three different pathways (classical, alternative, lectin) proceeds as a self-amplifying cascade, which leads to the generation of opsonins, anaphylatoxins C3a and C5a, and terminal membrane-attack complex (MAC, C5b-9), all of which regulate the inflammatory response and act in concert to destroy their target structures. To prevent uncontrolled complement activation or its attack against normal host cells, complement needs to be under strict control by regulatory proteins. The complement system has been shown to be activated in atherosclerotic lesions, modified lipoproteins and immune complexes containing oxLDL, for instance, being its activators. First, we investigated the presence and role of complement regulators in human atherosclerotic lesions. We found that inhibitors of the classical and alternative pathways, C4b-binding protein and factor H, respectively, were present in atherosclerotic lesions, where they localized in the superficial proteoglycan-rich layer. In addition, both inhibitors were found to bind to arterial proteoglycans in vitro. Immunohistochemical stainings revealed that, in the superficial layer of the intima, complement activation had been limited to the C3 level, whereas in the deeper intimal layers, complement activation had proceeded to the terminal C5b-9 level. We were also able to show that arterial proteoglycans inhibit complement activation in vitro. These findings suggested to us that the proteoglycan-rich layer of the arterial intima contains matrix-bound complement inhibitors and forms a protective zone, in which complement activation is restricted to the C3 level. Thus, complement activation is regulated in atherosclerotic lesions, and the extracellular matrix is involved in this process. Next, we studied whether the receptors for the two complement derived effectors, anaphylatoxins C3a and C5a, are expressed in human coronary atherosclerotic lesions. Our results of immunohistochemistry and RT-PCR analysis showed that, in contrast to normal intima, C3aR and C5aR were highly expressed in atherosclerotic lesions. In atherosclerotic plaques, the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR, and a small fraction of them also expressed C3aR, mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. These results suggested that intimal cells can respond to and become activated by complement-derived anaphylatoxins. Finally, we wanted to learn, whether oxLDL-IgG immune complexes, activators of the classical complement pathway, could have direct cellular effects in atherogenesis. Thus, we tested whether oxLDL-IgG immune complexes affect the survival of human monocytes, the precursors of macrophages, which are the most abundant inflammatory cell type in atherosclerotic lesions. We found that OxLDL-IgG immune complexes, in addition to transforming monocytes into foam cells, promoted their survival by decreasing their spontaneous apoptosis. This effect was mediated by cross-linking Fc receptors with ensuing activation of Akt-dependent survival signaling. Our finding revealed a novel mechanism by which oxLDL-IgG immune complexes can directly affect the accumulation of monocyte-macrophages in human atherosclerotic lesions and thus play a role in atherogenesis.
  • Kumar, Arun (Helsingin yliopisto, 2014)
    Many unknown species of human DNA viruses have recently (2005-2013) been discovered by using modern molecular and bioinformatic tools. The clinical and pathogenic roles of these viruses are presently known only fragmentarily; however they were found in symptomatic patients, and some have been shown to cause severe infectious illness, or cancer. Some of these emerging DNA viruses are examined in this thesis: Human Bocavirus 1 (HBoV1), Merkel cell polyomavirus (MCV or MCPyV) and Trichodysplasia spinulosa-associated polyomavirus (TSV or TSPyV). Viruses like these are of fundamental importance in the genesis of not only of acute but also of chronic or late-onset illness. The immunobiology and pathogenesis of these new viruses along with the already known DNA virus (parvovirus B19 or B19) can be found by the immunological and molecular methods. For years it was thought that parvovirus B19, was the sole human-pathogen among its family members. In 2005 a new pathogenic species, HBoV1 (previously denoted HBoV), was discovered by random-PCR from a nasopharyngeal aspirate. The existing data strongly suggest that HBoV1 causes a respiratory illness in young children. The aim of our study was to increase our knowledge on HBoV1-specific Th-cell immunity by examining T-cell proliferation and cytokine responses in asymptomatic adults. HBoV1-specific response was compared to those elicited by B19. B19-specific Th-cell immunity appears to be more divergent (in terms of cytokine response patterns) than the HBoV1-specific one. The present study also suggests that interleukin-13 (IL-13) response induced by HBoV1 may contribute to the airway pathology like asthma or bronchiolitis. A novel concept of CD4+ T-cells with cytolytic potential (CD4+ CTL) is emerging. Very recently, CD4+ CTL have been implicated in the control of persistent viral infections, e.g., Epistein-Barr virus (EBV), hepatitis C virus (HCV) and HIV-1. While human parvovirus B19 can establish persistence, yet no data exist on the presence of B19-specific CD4+ CTLs. Detection of vigorous B19-specific granzyme B (GrB) and perforin responses in seropositive individuals points to a role of CD4+ CTL also in B19 immunity. Such cells could function within immune regulation and in the triggering of autoimmune phenomena such as Systemic Lupus Erythematosus (SLE) or rheumatoid arthritis (RA). The newly discovered MCV resides in approximately 80% of Merkel cell carcinomas (MCC). The integration of MCV genome in-to the genome of host cell has been suggested to be the primary reason for this rare and aggressive skin cancer. Here we studied the T-cell immunity against this carcinogenic virus. We found that interferon-γ (IFN-γ) is the dominant cytokine among MCV-seropositive individuals and suggest that IFN-γ plays an important role in surveillance against MCV-induced disease. Our studies also suggested a role for IL-13 and IL-10 in anti-tumor immunity and immune regulation, respectively. TSV, while exhibiting high seroprevalence in general population, has been detected in trichodysplasia spinulosa (TS) skin lesions, suggesting an etiological role in this disease. In order to characterize Th-cell immunity against TSV, and to permit its comparisons with MCV-specific Th-cell immunity, we studied TSV and MCV-specific proliferation and cytokine responses in healthy volunteers and in one MCC patient. While an association between humoral and cellular responses was detectable with MCV, it was found to be weaker than the humoral and cellular responses detectable with TSV. Despite the significant homology in amino acid sequences of VP1, Th-cell crossreactivity was not evident between these viruses. As CD8+ T-cells specific for MCV LT-Ag oncoprotein clearly provide an important defence mechanism against MCC, the MCV VP1-specific Th-cells may also be important in preventing the oncogenic process, by suppressing MCV replication with antiviral cytokines such as IFN-γ. Parvoviruses (HBoV1 and B19) and polyomaviruses (MCV and TSV) induce effector CD4+ T-cell responses that are best known for their ability to protect against viral infections. Besides helper functions, CD4+ T-cell contribute to viral control and elimination by CD4-mediated cytotoxic effector functions. Thus, understanding of the CD4+ T-cell immunity is of key importance in the development of vaccines and therapeutic agents against life threatening infectious pathogens.
  • Suvilehto, Jari (Helsingin yliopisto, 2009)
    In the first part of this thesis the association of different forms of sinonasal diseases and plasma concentrations of C3, C4, immunoglobulins, immunoglobulin G subclasses, C4A and C4B gene numbers were studied in 287 adult patients and 150 sex-matched adult controls. Patients were well characterized and stratified into groups using strict clinical criteria and females and males were also studied as separate groups. Severe primary antibody antibody deficiencies were rare in patients coming to sinonasal operations. Female patients had more recurrent sinusitis and other mucosal infections and males had more nasal polyposis. Upregulation of complement activity was seen in acute rhinosinusitis patients (high levels of plasma C3, C4, and complement classical pathway activity CH50) and male patients coming to sinonasal operations (high levels of plasma C3 and C4). In females, total and partial C4B deficiencies and lower levels of IgG1 and IgG3 were associated with rhinosinusitis leading to sinonasal operations. C4A deficiencies were found to predispose to severe chronic rhinosinusitis in females and males. In female patients with chronic or recurrent rhinosinusitis with nasal polyposis C4B deficiencies seem to predispose to the disease, but in males with a similar disease C4B deficiencies seem to be protective. This suggests a different pathophysiology between sexes in this form of sinonasal disease. In the second part of this thesis work 213 children coming to elective tonsillectomy were studied and compared with 155 randomly selected school children. An association with recurrent upper respiratory tract infections and hypersensitivity disorders was seen especially in children under 7 years of age. However, this association was not seen in levels of specific IgE to respiratory allergens in the same age group. Both symptomatic respiratory allergy and specific IgE to respiratory allergens became more common in boys than girls over 7 years of age. We were able to show that although both rhinoviruses and bacterial pathogens were found in the tonsils, no association between their presence and clinical forms of tonsillar disease was seen. The ability of GAS to bind complement regulators FH and C4BP did not differ between strains causing tonsillar diseases or septicemia, suggesting that other virulence mechanisms of the bacteria are more important.
  • Marschan, Emma (Helsingin yliopisto, 2007)
    Epidemiological and experimental studies suggest that changes in gut microbial balance are associated with increases in the prevalence of allergic diseases. Probiotics are proposed to provide beneficial immunoregulatory signals which aid in oral tolerance achievement and alleviation of symptoms of allergic diseases. The present study evaluates both the immunological mechanisms of probiotics in infants with allergic diseases and their preventive aspect among infants prone to allergy. Furthermore, the purpose of the study was to characterise the immunological features of cord blood mononuclear cells (CBMCs) in infants at high genetic risk for allergy. GATA-3 expression (p = 0.03), interleukin (IL) -2(p = 0.026), and IL-5 (p = 0.013) secretion of stimulated CBMCs were higher in IgE-sensitized infants at age 2 than in non-allergic, non-sensitized infants. Lactobacillus GG (LGG) treatment increased secretion of IFN-γ by PBMCs in vitro in infants with cow s milk allergy (CMA) (p = 0.006) and in infants with IgE-associated eczema (p = 0.017), when compared to levels in the placebo group. A probiotic mixture, increased secretion of IL-4 by PBMCs in vitro in infants with CMA (p = 0.028), when compared with placebo-group levels. The LGG treatment induced higher plasma C-reactive protein (CRP) (p = 0.021) and IL-6 (p = 0.036) levels in infants with IgE-associated eczema than in the placebo group. The probiotic mixture induced higher plasma IL-10 levels in infants with eczema (p = 0.016). In the prevention study of allergic dis-eases, the infants receiving the probiotic mixture had higher plasma levels of CRP (p = 0.008), total IgA (p = 0.016), total IgE (p = 0.047), and IL-10 (p = 0.002) than did infants in the placebo group. Increased CRP level at age 6 months was associated with a decreased risk for eczema at age 2 not only in the infants who received probiotics but also in the placebo group (p = 0.034). In conclusion, the priming of the GATA-3 and IL-5 pathway can occur in utero, and a primary feature of T-cells predisposing to IgE-sensitization seems to directly favour Th2 deviation. LGG treatment induced increased plasma levels of CRP and IL-6 in infants with IgE-associated eczema, suggesting an activation of innate immu-nity. The probiotic mixture, when given to allergy-prone infants, induced inflammation, detected as increased plasma CRP levels, which at age 6 months was associated with decreased risk for eczema at age 2.The probiotic-induced response in allergy prone infants was characterized by their higher plasma IL-10, total IgE, and CRP levels, without induction of an allergen-specific IgE response. In this respect, the probiotics in infancy appear to induce protective immune profiles that are characteristic for chronic low-grade inflammation, a response resembling that of helminth-like infections.
  • Österlund, Pamela (Helsingin yliopisto, 2003)
  • Nissinen, Riikka (Helsingin yliopisto, 2003)