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  • Palojärvi, Anniina (Helsingin yliopisto, 2014)
    A complex interaction prevails between coagulation and inflammation. The coagulation system is adapted to thrombotic challenges during and after birth, leading to specific features in the coagulation system of the newborn. Inflammation and intra-alveolar fibrin formation characterize respiratory distress syndrome (RDS). Tissue factor (TF) is a link between inflammation and coagulation pathways. In the very low birth weight (VLBW) infant, birth and intensive care present major immunological challenges, and the developmental immaturity of the immune system adds to the risks of intensive care. A protective response to a strong inflammatory stimulus downregulates the antigen-presenting molecules (Human leukocyte antigen (HLA)-DR) on monocytes. If severe, however, the response may lead to immunodepression or immunoparalysis, which in adults is associated with increased morbidity and death. Antenatal betamethasone treatment for mothers at risk for premature delivery effectively reduces neonatal morbidity and mortality. This thesis evaluates the bidirectional interaction between inflammation and coagulation and combines the findings in VLBW infants with clinical morbidity. Patients were 56 VLBW infants, with a gestational age (GA) of less than 32 weeks and a birth weight of less than 1500 g. A control population comprised 25 healthy infants with a GA of more than 34 weeks. VLBW infants showed low monocyte HLA-DR expression. On day 3, 45% of infants presented with immunodepression. VLBW infants showed high plasma TF in circulation, but which did not lead to coagulation. In VLBW infants, tissue factor pathway inhibitor (TFPI) was the main anticoagulant in the first days; on day 3, however, the TF surge exceeded the TFPI capacity, leaving the VLBW infant with a pool of plasma TF. In VLBW infants, antenatal betamethasone associated with immunodepression and suppressed both proinflammatory IL-6 and anti-inflammatory IL-10. VLBW infants are postnatally in a state of immunodepression, which associated with RDS, low GA, and subsequent infections. Immunodepression may represent a link between low GA and risk for infections. TF is expressed in the lungs of a VLBW infant and leaks into circulation. TFPI could not control the TF surge on day 3. Plasma TF does not lead to thrombin formation, thereby making it available for inflammatory pathways (e.g. protease-activated receptors (PARs)). VLBW infants showed an inflammatory profile similar to adult sepsis or acute respiratory distress syndrome, with additional VLBW-specific features: a lower net anti-inflammatory cytokine effect without TF-induced activation of coagulation. Antenatal betamethasone (BM) was associated in a time-dependent manner to immunodepression, those infants with BM less than 24 hours before birth showing the deepest nadir in HLA-DR expression.
  • Pietola, Laura (Helsingin yliopisto, 2012)
    Hearing loss is the most common sensory deficit in humans. Genetic defects play a major part among patients with sensorineural hearing loss. Usher syndrome (USH) is an autosomal recessive disorder defined by bilateral sensorineural hearing loss and a visual impairment phenotypically similar to retinitis pigmentosa (RP). USH is divided into three main clinical types (USH1, USH2 and USH3), based on the severity and progression of the hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of RP. The most common form of Usher syndrome in Finland is USH3, which comprises 40% of all USH cases. USH3 is caused by mutations in the clarin 1 (CLRN1) gene. To date, treatment methods for sensorineural hearing loss are limited to rehabilitation with traditional hearing aids or cochlear implantation. An ideal cure would be targeted, long-term or permanent, and should cause as little damage as possible to the inner ear structure. Our studies focused on cochlear gene therapy and Usher syndrome type 3 (USH3). Finnish USH3 patients answered three questionnaires which evaluated their quality of life after cochlear implantation. We also collected data of these patients audiological tests and speech discrimination tests from their patient records. We developed an in vitro model suitable for cochlear gene transfer studies from a detached mouse round window membrane (RWM) and also tested the suitability of adeno-associated virus vectors and lentivirus vectors for cochlear gene therapy applications in cell lines and in the mouse cochlea. Cochlear implantation is beneficial for USH3 patients and improves their quality of life. The detached mouse RWM model is suitable for inner ear gene transfer studies in vitro. Manipulation of the RWM with AgNO3, trichloracetic acid or histamine-glycerol did not increase the permeability of the membrane. Lentivirus vectors are safe and can be used in gene transfer into the perilymph. Silencing of p53 protein may decrease apoptosis in the kanamycin-damaged mouse cochlea. AAV-delivered clarin-1 ribozyme may induce apoptosis in cochlear hair cells and cells of the stria vascularis. Apoptosis could explain the progressive nature of USH3.
  • Bidel, Siamak (Helsingin yliopisto, 2008)
    Type 2 diabetes is one of the diseases that largely determined by lifestyle factors. Coffee is one of the most consumed beverages in the world and recently released data suggest the effects of coffee consumption on type 2 diabetes. The objective of the present study was to evaluate the effects of habitual coffee consumption on various aspects of type 2 diabetes and its most common complications. This study is part of the national FINRISK studies. Baseline surveys were carried out between 1972 and 1997. The surveys covered two eastern regions in 1972 and 1977, but were expanded to include a third region in southwestern Finland in 1982, 1987, 1992, and 1997. The Helsinki capital area was included in the survey in 1992 and 1997 and the Oulu province, in northern Finland, in 1997. Each survey was drawn from an independent random sample of the national register of subjects aged 25-64. In 1997, an additional sample of subjects aged 65-74 was conducted. The blood pressure, weight, and height of subjects were measured. By using self-administered questionnaires data were collected on medical history, socioeconomic factors, physical activity, smoking habits, and alcohol, coffee, and tea consumption. Higher coffee consumption was associated with higher body mass index, occupational physical activity and cigarette smoking, and lower blood pressure, education level, leisure time physical activity, tea consumption and alcohol use. Age, body mass index, systolic blood pressure and current smoking were positively associated with the risk of type 2 diabetes, however, education, and occupational, commuting and leisure time physical activity were inversely associated. The significant inverse association between coffee consumption and the risk of type 2 diabetes was found in both sexes but the association was stronger in women. Coffee consumption was significantly and inversely associated with fasting glucose, 2-hour plasma glucose, fasting insulin, impaired fasting glucose, impaired glucose regulation, and hyperinsulinemia among both men and women and with isolated impaired glucose tolerance among women. Serum gamma-glutamyltransferase modified the association between coffee consumption and incident diabetes. Among subjects with high serum -glutamyltransferase (>75th percentile), coffee consumption showed an inverse association for women, as well as men and women combined. An inverse association also occurred between coffee consumption and the risk of total, cardiovascular disease, and coronary heart disease mortality among patients with type 2 diabetes. The results of this study showed that habitual coffee consumption may be associated with a reduced risk of type 2 diabetes. Coffee consumption may have some effects on several markers of glycemia, and may lower the incident of type 2 diabetes in high normal serum -glutamyltransferase levels. Total, cardiovascular disease, and coronary heart disease mortality rate among subjects with type 2 diabetes may also be reduced by coffee consumption.
  • Rapeli, Pekka (National Institute for Health and Welfare (THL), Finland, 2014)
    Opioid substitution treatment with buprenorphine or methadone is the most effective treatment for opioid-dependence. In this study attention, working memory, and episodic memory functioning in opioid-substituted patients was examined cross-sectionally and longitudinally. Methadone patients performed worse in many attention-related reaction time tasks in relation to controls or buprenorphine patients. In each drug group, drug treatment variables predicted 10% of the attention performance. In most working memory tests both drug groups performed worse than controls. A group by time interaction in one working memory test in buprenorphine patients may indicate improvement of function. Use of benzodiazepine medication predicted impaired working memory performance. In verbal episodic memory tests treatment with more than one other psychoactive drug (than opioid or benzodiazepine) and frequent substance abuse in the past month predicted 20% of performance. Almost normal cognitive performance in stable opioid substitution treatment supports the idea of efficient compensation of the opioid abuse history related neural burden. The results are relevant for patients and prescribers when choosing treatment options or rehabilitation goals.
  • Kivitie-Kallio, Satu (Helsingin yliopisto, 2000)
  • Sevastianova, Ksenia (Helsingin yliopisto, 2011)
    Introduction: Combination antiretroviral therapy (cART) has decreased morbidity and mortality of individuals infected with human immunodeficiency virus type 1 (HIV-1). Its use, however, is associated with adverse effects which increase the patients risk of conditions such as diabetes and coronary heart disease. Perhaps the most stigmatizing side effect is lipodystrophy, i.e., the loss of subcutaneous adipose tissue (SAT) in the face, limbs and trunk while fat accumulates intra-abdominally and dorsocervically. The pathogenesis of cART-associated lipodystrophy is obscure. Nucleoside reverse transcriptase inhibitors (NRTI) have been implicated to cause lipoatrophy via mitochondrial toxicity. There is no known effective treatment for cART-associated lipodystrophy during unchanged antiretroviral regimen in humans, but in vitro data have shown uridine to abrogate NRTI-induced toxicity in adipocytes. Aims: To investigate whether i) cART or lipodystrophy associated with its use affect arterial stiffness; ii) lipoatrophic SAT is inflamed compared to non-lipoatrophic SAT; iii) abdominal SAT from patients with compared to those without cART-associated lipoatrophy differs with respect to mitochondrial DNA (mtDNA) content, adipose tissue inflammation and gene expression, and if NRTIs stavudine and zidovudine are associated with different degree of changes; iv) lipoatrophic abdominal SAT differs from preserved dorsocervical SAT with respect to mtDNA content, adipose tissue inflammation and gene expression in patients with cART-associated lipodystrophy and v) whether uridine can revert lipoatrophy and the associated metabolic disturbances in patients on stavudine or zidovudine based cART. Subjects and methods: 64 cART-treated patients with (n=45) and without lipodystrophy/-atrophy (n=19) were compared cross-sectionally. A marker of arterial stiffness, heart rate corrected augmentation index (AgIHR), was measured by pulse wave analysis. Body composition was measured by magnetic resonance imaging and dual-energy X-ray absorptiometry, and liver fat content by proton magnetic resonance spectroscopy. Gene expression and mtDNA content in SAT were assessed by real-time polymerase chain reaction and microarray. Adipose tissue composition and inflammation were assessed by histology and immunohistochemistry. Dorsocervical and abdominal SAT were studied. The efficacy and safety of uridine for the treatment of cART-associated lipoatrophy were evaluated in a randomized, double-blind, placebo-controlled 3-month trial in 20 lipoatrophic cART-treated patients. Results: Duration of antiretroviral treatment and cumulative exposure to NRTIs and protease inhibitors, but not the presence of cART-associated lipodystrophy, predicted AgIHR independent of age and blood pressure. Gene expression of inflammatory markers was increased in SAT of lipodystrophic as compared to non-lipodystrophic patients. Expression of genes involved in adipogenesis, triglyceride synthesis and glucose disposal was lower and of those involved in mitochondrial biogenesis, apoptosis and oxidative stress higher in SAT of patients with than without cART-associated lipoatrophy. Most changes were more pronounced in stavudine-treated than in zidovudine-treated individuals. Lipoatrophic SAT had lower mtDNA than SAT of non-lipoatrophic patients. Expression of inflammatory genes was lower in dorsocervical than in abdominal SAT. Neither depot had characteristics of brown adipose tissue. Despite being spared from lipoatrophy, dorsocervical SAT of lipodystrophic patients had lower mtDNA than the phenotypically similar corresponding depot of non-lipodystrophic patients. The greatest difference in gene expression between dorsocervical and abdominal SAT, irrespective of lipodystrophy status, was in expression of homeobox genes that regulate transcription and regionalization of organs during embryonal development. Uridine increased limb fat and its proportion of total fat, but had no effect on liver fat content and markers of insulin resistance. Conclusions: Long-term cART is associated with increased arterial stiffness and, thus, with higher cardiovascular risk. Lipoatrophic abdominal SAT is characterized by inflammation, apoptosis and mtDNA depletion. As mtDNA is depleted even in non-lipoatrophic dorsocervical SAT, lipoatrophy is unlikely to be caused directly by mtDNA depletion. Preserved dorsocervical SAT of patients with cART-associated lipodystrophy is less inflamed than their lipoatrophic abdominal SAT, and does not resemble brown adipose tissue. The greatest difference in gene expression between dorsocervical and abdominal SAT is in expression of transcriptional regulators, homeobox genes, which might explain the differential susceptibility of these adipose tissue depots to cART-induced toxicity. Uridine is able to increase peripheral SAT in lipoatrophic patients during unchanged cART.
  • Liikanen, Ilkka (Helsingin yliopisto, 2015)
    Cancer remains a major cause of death and novel treatment modalities are needed. Oncolytic immunotherapy is a safe and promising approach, where cancer-selective viruses kill only cancer cells and mount an immune response against the tumor. We aimed to improve oncolytic adenoviral immunotherapy by combining it with chemotherapy and radiotherapy, and by identifying resistance mechanisms and biomarkers. We first showed that combining radiotherapy with adenoviral vector proteins E4orf3 and E4orf6, but not E1B55K, enhanced DNA damage accumulation and cancer cell killing, and inhibited prostate tumor growth in mice. This intrinsic ability of adenoviruses to radiosensitize cells could be harnessed against cancer cells by selective targeting, thus increasing efficacy while reducing the harmful side-effects of radiotherapy. In study two, we established two ovarian cancer mouse models, where tumors relapse despite the presence of functional oncolytic adenovirus, with tumor stroma maintaining the virus resistance. We identified upregulated interferon signaling in the resistant tumors by microarray, while pathway analyses suggested potential therapeutic targets, and myxovirus resistance protein A (MxA) was found a protein level indicator correlating with resistance to virus. Our results provide a putative biomarker and targets, which can help in detecting and overcoming resistance against oncolytic adenovirus. Antitumor T-cell activation appears to require autophagy and immunogenic cell death (ICD). In a translational study, we demonstrated preclinically that oncolytic adenovirus together with low-dose temozolomide and cyclophosphamide increased ICD and autophagy, resulting in tumor growth inhibition. Combination therapy was found safe in 41 treatments given to patients with refractory solid tumors in the context of an advanced therapy access program (ATAP). Increase of an ICD marker protein high-mobility group box 1 (HMGB1) and antitumor T-cell activity suggested activation of immune responses. Disease stabilization or better was observed in 67% of evaluable treatments, and as an estimated effect on survival, combination-treated patients trended for increased overall survival over non-randomized control patients. Biomarkers are urgently needed for identification of cancer patients likely to benefit from immunotherapy. Because HMGB1 protein is emerging a key player in immunomodulation, we addressed the biomarker value of HMGB1 serum level in an ATAP cohort of 202 cancer patients treated with oncolytic adenoviruses: Patients with low-baseline HMGB1 showed significantly improved overall survival and disease control rate in multivariate analyses as compared to high-baseline patients. Both patient groups showed good safety. HMGB1-low patients seemed to benefit from immunogenic virus constructs and antitumor T-cell activity. Thus, we have identified HMGB1 as a novel prognostic and predictive biomarker for oncolytic immunotherapy, which may distinguish between immunologically inert and responsive cancer patients. This thesis provides rationale for combining oncolytic adenoviruses with radiotherapy, low-dose temozolomide and cyclophosphamide. We report safety, possible signs of efficacy, and immunological effects in altogether 238 patient treatments, and introduce promising biomarkers for oncolytic immunotherapy. Our results can help in designing clinical trials and developing oncolytic adenovirus treatments.
  • Hannila-Handelberg, Tuula (Helsingin yliopisto, 2009)
    Most of the diseases affecting public health, like hypertension, are multifactorial by etiology. Hypertension is influenced by genetic, life style and environmental factors. Estimation of the influence of genes to the risk of essential hypertension varies from 30 to 50%. It is plausible that in most of the cases susceptibility to hypertension is determined by the action of more than one gene. Although the exact molecular mechanism underlying essential hypertension remains obscure, several monogenic forms of hypertension have been identified. Since common genetic variations may predict, not only to susceptibility to hypertension, but also response to antihypertensive drug therapy, pharmacogenetic approaches may provide useful markers in finding relations between candidate genes and phenotypes of hypertension. The aim of this study was to identify genetic mutations and polymorphisms contributing to human hypertension, and examine their relationships to intermediate phenotypes of hypertension, such as blood pressure (BP) responses to antihypertensive drugs or biochemical laboratory values. Two groups of patients were investigated in the present study. The first group was collected from the database of patients investigated in the Hypertension Outpatient Ward, Helsinki University Central Hospital, and consisted of 399 subjects considered to have essential hypertension. Frequncies of the mutant or variant alleles were compared with those in two reference groups, healthy blood donors (n = 301) and normotensive males (n = 175). The second group of subjects with hypertension was collected prospectively. The study subjects (n=313) underwent a protocol lasting eight months, including four one-month drug treatment periods with antihypertensive medications (thiazide diuretic, β-blocker, calcium channel antagonist, and an angiotensin II receptor antagonist). BP responses and laboratory values were related to polymorphims of several candidate genes of the renin-angiotensin system (RAS). In addition, two patients with typical features of Liddle’s syndrome were screened for mutations in kidney epithelial sodium channel (ENaC) subunits. Two novel mutations causing Liddle’s syndrome were identified. The first mutation identified located in the beta-subunit of ENaC and the second mutation found located in the gamma-subunit, constituting the first identified Liddle mutation locating in the extracellular domain. This mutation showed 2-fold increase in channel activity in vitro. Three gene variants, of which two are novel, were identified in ENaC subunits. The prevalence of the variants was three times higher in hypertensive patients (9%) than in reference groups (3%). The variant carriers had increased daily urinary potassium excretion rate in relation to their renin levels compared with controls suggesting increased ENaC activity, although in vitro they did not show increased channel activity. Of the common polymorphisms of the RAS studied, angiotensin II receptor type I (AGTR1) 1166 A/C polymorphism was associated with modest changes in RAS activity. Thus, patients homozygous for the C allele tended to have increased aldosterone and decreased renin levels. In vitro functional studies using transfected HEK293 cells provided additional evidence that the AGTR1 1166 C allele may be associated with increased expression of the AGTR1. Common polymorphisms of the alpha-adducin and the RAS genes did not significantly predict BP responses to one-month monotherapies with hydroclorothiazide, bisoprolol, amlodipin, or losartan. In conclusion, two novel mutations of ENaC subunits causing Liddle’s syndrome were identified. In addition, three common ENaC polymorphisms were shown to be associated with occurrence of essential hypertension, but their exact functional and clinical consequences remain to be explored. The AGTR1 1166 C allele may modify the endocrine phenotype of hypertensive patients, when present in homozygous form. Certain widely studied polymorphisms of the ACE, angiotensinogen, AGTR1 and alpha-adducin genes did not significantly affect responses to a thiazide, β-blocker, calcium channel antagonist, and angiotensin II receptor antagonist.
  • Korvenoja, Antti (Helsingin yliopisto, 2007)
    MEG directly measures the neuronal events and has greater temporal resolution than fMRI, which has limited temporal resolution mainly due to the larger timescale of the hemodynamic response. On the other hand fMRI has advantages in spatial resolution, while the localization results with MEG can be ambiguous due to the non-uniqueness of the electromagnetic inverse problem. Thus, these methods could provide complementary information and could be used to create both spatially and temporally accurate models of brain function. We investigated the degree of overlap, revealed by the two imaging methods, in areas involved in sensory or motor processing in healthy subjects and neurosurgical patients. Furthermore, we used the spatial information from fMRI to construct a spatiotemporal model of the MEG data in order to investigate the sensorimotor system and to create a spatiotemporal model of its function. We compared the localization results from the MEG and fMRI with invasive electrophysiological cortical mapping. We used a recently introduced method, contextual clustering, for hypothesis testing of fMRI data and assessed the the effect of neighbourhood information use on the reproducibility of fMRI results. Using MEG, we identified the ipsilateral primary sensorimotor cortex (SMI) as a novel source area contributing to the somatosensory evoked fields (SEF) to median nerve stimulation. Using combined MEG and fMRI measurements we found that two separate areas in the lateral fissure may be the generators for the SEF responses from the secondary somatosensory cortex region. The two imaging methods indicated activation in corresponding locations. By using complementary information from MEG and fMRI we established a spatiotemporal model of somatosensory cortical processing. This spatiotemporal model of cerebral activity was in good agreement with results from several studies using invasive electrophysiological measurements and with anatomical studies in monkey and man concerning the connections between somatosensory areas. In neurosurgical patients, the MEG dipole model turned out to be more reliable than fMRI in the identification of the central sulcus. This was due to prominent activation in non-primary areas in fMRI, which in some cases led to erroneous or ambiguous localization of the central sulcus.
  • Rautemaa, Riina (Helsingin yliopisto, 2001)
  • Alitalo, Antti (Helsingin yliopisto, 2004)
  • Seppänen, Mikko (Helsingin yliopisto, 2007)
    The study assessed whether plasma concentrations of complement factors C3, C4, or immunoglobulins, serum classical pathway hemolytyic activity, or polymorphisms in the class I and II HLA genes, isotypes and gene numbers of C4, or allotypes of IgG1 and IgG3 heavy chain genes were associated with severe frequently recurring or chronic mucosal infections. According to strict clinical criteria, 188 consecutive voluntary patients without a known immunodeficiency and 198 control subjects were recruited. Frequencies of low levels in IgG1, IgG2, IgG3 and IgG4 were for the first time tested from adult general population and patients with acute rhinosinusitis. Frequently recurring intraoral herpes simplex type 1 infections, a rare form of the disease, was associated with homozygosity in HLA -A*, -B*, -C*, and -DR* genes. Frequently recurrent genital HSV-2 infections were associated with low levels of IgG1 and IgG3, present in 54% of the recruited patients. This association was partly allotype-dependent. The G3mg,G1ma/ax haplotype, together with low IgG3, was more common in patients than in control subjects who lacked antibodies against herpes simplex viruses. This is the first found immunogenetic deficiency in otherwise healthy adults that predisposes to highly frequent mucosal herpes recurrences. According to previous studies, HSV effectively evades the allotype G1ma/ax of IgG1, whereas G3mg is associated with low IgG3. Certain HLA genes were more common in patients than in control subjects. Having more than one C4A or C4B gene was associated with neuralgias caused by the virus. Low levels of IgA, IgG1, IgG2, IgG3, and IgG4 were common in the general adult population, but even more frequent in patients with chronic sinusitis. Only low IgG1 was more common chronic than in acute rhinosinusitis. Clinically, nasal polyposis and bronchial asthma were associated with complicated disease forms. The best differentiating immunologic parameters were C4A deficiency and the combination of low plasma IgG4 together with low IgG1 or IgG2, performing almost equally. The lack of C4A, IgA, and IgG4, all known to possess anti-inflammatory activity, together with a concurrently impaired immunity caused by low subclass levels, may predispose to chronic disease forms. In severe chronic adult periodontitis, any C4A or C4B deficiency combined was associated with the disease. The new quantitative analysis of C4 genes and the conventional C4 allotyping method complemented each other. Lowered levels of plasma C3 or C4 or both, and serum CH50 were found in herpes and periodontitis patients. In rhinosinusitis, there was a linear trend with the highest levels found in the order: acute > chronic rhinosinusitis > general population > blood donors with no self-reported history of rhinosinusitis. Complement is involved in the defense against the tested mucosal infections. Seemingly immunocompetent patients with chronic or recurrent mucosal infections frequently have subtle weaknesses in different arms of immunity. Their susceptibility to chronic disease forms may be caused by these. Host s subtly impaired immunity often coincides with effective immune evasion from the same arms of immunity by the disease-causing pathogens. The interpretation of low subclass levels, if no additional predisposing immunologic factors are tested, is difficult and of limited value in early diagnosis and treatment.
  • Bhattacharjee, Arnab (Helsingin yliopisto, 2014)
    Complement is comprised of a cascade of proteins that recognizes and attacks the invading microbes and thus is the first line of defense for the human body against invading pathogens. It is initiated via different activation pathways that lead to C3b deposition on the target and sequentially to the formation of lytic membrane attack complexes (MAC). One of the complement activation pathways the alternative pathway (AP) can be activated on any surface, self or non-self, and is therefore tightly regulated. Complement Factor H (CFH) is the most important complement down-regulator and it mediates target discrimination between self and non-self cells. Several point mutations in CFH and/or autoantibodies (AA) against it are found to be directly associated with atypical haemolytic uremic syndrome (aHUS), a severe and often fatal disease triggered by the impaired regulation of AP on self surfaces leading to complement attack. ----- CFH is composed of 20 homologous complement control protein domains (CCP). The N-terminal domains 1 to 4 (CFH1-4) mediate inactivation of C3b on the self-surfaces and the C-terminal domains 19 and 20 (CFH19-20) are critical for the ability of CFH to discriminate between self and non-self structures. Self-surfaces are rich in anionic sialic acids (SA) and glycosaminoglycans (GAGs) that are not present on pathogenic microbes. CFH19-20 contains binding sites for both the C3d part of C3b and self-surface polyanions that enhance avidity of CFH to C3b on self surfaces and thus enhance C3b inactivation. CFH mutations that have been found in aHUS patients are mostly located in CFH19-20. ----- The previously solved X-ray crystal structure of CFH19-20 illuminated the location of aHUS related mutations. The aims of this thesis work were to study the functionality of CFH on a molecular level by studying the molecular structure of CFH C-terminus and its mutants along with structures of CFH in complex with its different interacting partners, as well as the structure of the domains of CFH-related protein-1 highly homologous to CFH19-20. The structures solved and the their relevance are described in the four articles attached to this thesis. ------ In the first article, stability of the CFH C-terminus fold by aHUS mutation(s) was studied by analyzing the binding of the CFH19-20 mutant proteins to C3d/C3b using radioligand assays and affinity chromatography. The X-ray crystal structures of CFH19-20 with two different point mutations (of residues indicated to be involved in binding C3d/C3b) were solved. It was shown that these mutations did not result in the disruption of the basic fold of CFH19-20, but maintained the same fold with a prominent difference in the surface charge distribution in the zone of the residues. The results clearly indicated that the aHUS mutations on CFH do not disrupt the basic structural fold, but induce anomaly in the charge distribution of the molecule, explaining the effects of the mutations to its C3b/heparin binding abilities suggested to be critical for the pathogenesis of the disease. In the second article, we revealed the rationale of the molecular mechanism of CFH19-20 mediated self nonself discrimination and showed why point mutations in CFH19-20 lead to aHUS. The CFH19-20 :C3d structure reported in this article revealed two independent binding interfaces between CFH19-20 and C3d, namely the CFH19 site and the CFH20 site . The results of deeper analysis of this structure also showed that the simultaneous binding of the CFH19-20 via the CFH19 site to C3b and via CFH20 site to C3d was possible. In the third article, the X-ray crystal structure of CFH19-20 was solved in complex with the outer surface protein E (OspE) from Borrelia burgdorferi in order to understand the molecular mechanism of sequestering CFH by microbes which results in complement evasion. The nucleomagnetic resonance (NMR) structure of the OspE protein from Borrelia burgdorferi reported in this paper was required for solving the structure of OspE in complex with CFH19-20. Chemical shift perturbations studies using NMR also confirmed the physiological viability of the complex structure. This complex structure was the first structure of CFH19-20 in complex with any microbial protein and thus answered the puzzle of the molecular mimicry used by microbes involving CFH C-terminus in order to evade complement. ----- In the fourth article the structure of the CFHR1 domains 4 and 5 (CFHR14-5) was solved and used to explain why AA bind to a common epitope on CFH domain 20, which is highly homologous to domain 5 of CFHR1. The CFHR14-5 structure revealed an important structural bigamy of CFH and its related proteins that can be used to understand why CFHR-1 deficiency and formation of AA against CFH (CFH- AA) lead to autoimmune aHUS (AI-aHUS). We extensively studied CFH-AA from more than a dozen of patients and their binding behavior to CFH and CFHR1. The results suggest a novel hypothesis on the pathogenesis of AI-aHUS. In conclusion, the results have revealed the molecular mechanisms beneath different functionalities of the C-terminus of CFH. It is not only the most important molecule to facilitate the target discrimination by the AP, but is also a prominent tool used by the microbes in order to evade complement attack. Furthermore, the CFH C- terminus also houses the AA binding epitope and thus also plays a role in the pathogenesis of AI-aHUS. We were also quite surprised to find out that the molecular structure of CFH C-terminus is extremely stable, and hardly undergoes any changes in its conformation in the presence of other ligands. Cumulatively, the structures of the CFH in presence of its different partners of interactions contributed greatly to the knowledge pool of the understanding of the molecular mechanisms associated with CFH in complement activation and regulation in health and disease.
  • Lehtinen, Markus (Helsingin yliopisto, 2011)
    Alternative pathway (AP) of complement can be activated on any surface, self or non-self. In atypical hemolytic uremic syndrome (aHUS) the AP regulation on self surfaces is insufficient and leads to complement attack against self-cells resulting usually in end-stage renal disease. Factor H (FH) is one of the key regulators of AP activation on the self surfaces. The domains 19 and 20 (FH19-20) are critical for the ability of FH to discriminate between C3b-opsonized self and non-self surfaces and are a hot-spot for mutations that have been described from aHUS patients. FH19-20 contains binding sites for both the C3d part of C3b and self surface polyanions that are needed for efficient C3b inactivation. To study the dysfunction of FH19-20, crystallographic structures of FH19-20 and FH19-20 in complex with C3d (FH19-20:C3d) were solved and aHUS-associated and structurally interesting point mutations were induced to FH19-20. Functional defects caused by these mutations were studied by analyzing binding of the FH19-20 mutant proteins to C3d, C3b, heparin, and mouse glomerular endothelial cells (mGEnCs). The results revealed two independent binding interfaces between FH19-20 and C3d - the FH19 site and the FH20 site. Superimposition of the FH19-20:C3d complex on the previously published C3b and FH1-4:C3b structures showed that the FH20 site on C3d is partially occluded, but the FH19 site is fully available. Furthermore, binding of FH19-20 via the FH19 site to C3b did not block binding of the functionally important FH1-4 domains and kept the FH20 site free to bind heparin or an additional C3d. Binding assays were used to show that FH20 domain can bind to heparin while FH19-20 is bound to C3b via the FH19 site, and that both the FH19 site and FH20 are necessary for recognition of non-activator surfaces. Simultaneous binding of FH19 site to C3b and FH20 to anionic self structures are the key interactions in self-surface recognition by FH and thereby enhanced avidity of FH explains how AP discriminates between self and non-self. The aHUS-associated mutations on FH19-20 were found to disrupt binding of the FH19 or FH20 site to C3d/C3b, or to disrupt binding of FH20 to heparin or mGEnC. Any of these dysfunctions leads to loss of FH avidity to C3b bearing self surfaces explaining the molecular pathogenesis of the aHUS-cases where mutations are found within FH19-20.
  • Cheng, Zhu-Zhu (Helsingin yliopisto, 2005)
  • Hakulinen, Juha (Helsingin yliopisto, 2003)
  • Junnikkala, Sami (Helsingin yliopisto, 2002)