Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosis and host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M.tuberculosis and, in particular, the proline-glutamic acid-polymorphicguanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

Mutational analysis of the insulin‑like growth factor 1 receptor tyrosine kinase domain in non-small cell lung cancer patients

The insulin‑like growth factor 1 receptor (IGF1R) pathway plays an important role in the pathogenesis of non‑small cell lung cancer (NSCLC) and also provides a mechanism of resistance to targeted therapies. IGF1R is therefore an ideal therapeutic target and several inhibitors have entered clinical trials. However, thus far the response to these inhibitors has been poor, highlighting the importance of predictive biomarkers to identify patient cohorts who will benefit from these targeted agents. It is well‑documented that mutations and/or deletions in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain predict sensitivity of NSCLC patients to EGFR TK inhibitors. Single‑nucleotide polymorphisms (SNPs) in the IGF pathway have been associated with disease, including breast and prostate cancer. The aim of the present study was to elucidate whether the IGF1R TK domain harbours SNPs, somatic mutations or deletions in NSCLC patients and correlates the mutation status to patient clinicopathological data and prognosis. Initially 100 NSCLC patients were screened for mutations/deletions in the IGF1R TK domain (exons 16‑21) by sequencing analysis. Following the identification of SNP rs2229765, a further 98 NSCLC patients and 866 healthy disease‑free control patients were genotyped using an SNP assay. The synonymous SNP (rs2229765) was the only aberrant base change identified in the IGF1R TK domain of 100 NSCLC patients initially analysed. SNP rs2229765 was detected in exon 16 and was found to have no significant association between IGF1R expression and survival. The GA genotype was identified in 53.5 and 49.4% of NSCLC patients and control individuals, respectively. No significant difference was found in the genotype (P=0.5487) or allele (P=0.9082) frequencies between the case and control group. The present findings indicate that in contrast to the EGFR TK domain, the IGF1R TK domain is not frequently mutated in NSCLC patients. The synonymous SNP (rs2229765) had no significant association between IGF1R expression and survival in the cohort of NSCLC patients.

PE_PGRS Antigens of Mycobacterium tuberculosis Induce Maturation and Activation of Human Dendritic Cells

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosismand host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid-polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappa B signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis. The Journal of Immunology, 2010, 184: 3495-3504.

Rotavirus nonstructural proteins: a structural perspective

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codesfor six structural proteins (VP1-6) and six non-structural proteins(NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus article. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of similar to 3.5 angstrom in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.

Identification of carbohydrate structures as receptors for localised adherent enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli strains of diffused adherent (DA) and localised adherent (LA) phenotypes were tested for their ability to bind to glycolipids. DA strains did not bind to the glycolipids tested, while LA strains bound to asialo GM1, asialo GM2, globoside and lacto-N-neotetraose in decreasing order of avidity. The minimum common sequence among the four glycolipids could be delineated as GalNac β 1–4 Gal as the binding epitope with GalNac β 1–3 Gal and GlcNac β 1–3 Gal serving as relatively weaker binders. The binding was not inhibited by a variety of free oligosaccharides or by the neoglycoproteins tested. Adhesion-negative mutants of an enteropathogenic LA strain showed a markedly reduced binding to asialo GM1 indicating that the recognition of GalNac β 1–4 Gal was correlated with the ability to adhere to HeLa cells. Thus recognition and binding to glycolipids could play an important role in colonisation through adherence to intestinal surfaces.

Genetic Mechanisms Underlying Autism Spectrum Disorders

Autism is a childhood-onset developmental disorder characterized by deficits in reciprocal social interaction, verbal and non-verbal communication, and dependence on routines and rituals. It belongs to a spectrum of disorders (autism spectrum disorders, ASDs) which share core symptoms but show considerable variation in severity. The whole spectrum affects 0.6-0.7% of children worldwide, inducing a substantial public health burden and causing suffering to the affected families. Despite having a very high heritability, ASDs have shown exceptional genetic heterogeneity, which has complicated the identification of risk variants and left the etiology largely unknown. However, recent studies suggest that rare, family-specific factors contribute significantly to the genetic basis of ASDs. In this study, we investigated the role of DISC1 (Disrupted-in-schizophrenia-1) in ASDs, and identified association with markers and haplotypes previously associated with psychiatric phenotypes. We identified four polymorphic micro-RNA target sites in the 3 UTR of DISC1, and showed that hsa-miR-559 regulates DISC1 expression in vitro in an allele-specific manner. We also analyzed an extended autism pedigree with genealogical roots in Central Finland reaching back to the 17th century. To take advantage of the beneficial characteristics of population isolates to gene mapping and reduced genetic heterogeneity observed in distantly related individuals, we performed a microsatellite-based genome-wide screen for linkage and linkage disequilibrium in this pedigree. We identified a putative autism susceptibility locus on chromosome 19p13.3 and obtained further support for previously reported loci at 1q23 and 15q11-q13. To follow-up these findings, we extended our study sample from the same sub-isolate and initiated a genome-wide analysis of homozygosity and allelic sharing using high-density SNP markers. We identified a small number of haplotypes shared by different subsets of the genealogically connected cases, along with convergent biological pathways from SNP and gene expression data, which highlighted axon guidance molecules in the pathogenesis of ASDs. In conclusion, the results obtained in this thesis show that multiple distinct genetic variants are responsible for the ASD phenotype even within single pedigrees from an isolated population. We suggest that targeted resequencing of the shared haplotypes, linkage regions, and other susceptibility loci is essential to identify the causal variants. We also report a possible micro-RNA mediated regulatory mechanism, which might partially explain the wide-range neurobiological effects of the DISC1 gene.

Physical activity and diabetic complications in patients with type 1 diabetes

Type 1 diabetes is associated with the risk for late diabetic complications which are divided into microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (cardiovascular disease, CVD) diseases. The risk for diabetic complication can be reduced by effective treatment, most importantly the glycaemic control. Glycaemia in type 1 diabetes is influenced by the interplay between insulin injections and lifestyle factors such as physical activity and diet. The effect of physical activity in patients with type 1 diabetes is not well known, however. The aim of this thesis was to investigate the physical activity and the physical fitness of patients with type 1 diabetes with special emphasis on glycaemic control and the diabetic complications. The patients included in the study were all part of the nationwide, multicenter Finnish Diabetic Nephropathy (FinnDiane) Study which aims to characterise genetic, clinical, and environmental factors that predispose to diabetic complications in patients with type 1 diabetes. In addition, subjects from the IDentification of EArly mechanisms in the pathogenesis of diabetic Late complications (IDEAL) Study were studied. Physical activity was assessed in the FinnDiane Study in 1945 patients by a validated questionnaire. Physical fitness was measured in the IDEAL Study by spiroergometry (cycle test with measurement of respiratory gases) in 86 young adults with type 1 diabetes and in 27 healthy controls. All patients underwent thorough clinical characterisation of their diabetic complication status. Four substudies were cross-sectional using baseline data and one study additionally used follow-up data. Physical activity, especially the intensity of activities, was reduced in patients affected by diabetic nephropathy, retinopathy, and CVD. Low physical activity was associated with poor glycaemic control, a finding most clear in women and evident also in patients with no signs of diabetic complications. Furthermore, low physical activity was associated with a higher HbA1c variability, which in turn was associated with the progression of renal disease and CVD during follow-up. A higher level of physical activity was also associated with better insulin sensitivity. The prevalence of the metabolic syndrome in type 1 diabetes was also lower the higher the physical activity. The aerobic physical fitness level of young adults with type 1 diabetes was reduced compared with healthy peers and in men an association between higher fitness level and lower HbA1c was observed. In patients with type 1 diabetes, a higher physical activity was associated with better glycaemic control and may thus be beneficial with respect to the prevention of diabetic complications.

Phospholipid cytochrome c interactions

The ability of the peripherally associated membrane protein cytochrome c (cyt c) to bind phospholipids in vitro was studied using fluorescence spectroscopy and large unilamellar liposomes. Previous work has shown that cyt c can bind phospholipids using two distinct mecha- nisms and sites, the A-site and the C-site. This binding is mediated by electrostatic or hydrophobic interactions, respectively. Here, we focus on the mechanism underlying these interactions. A chemically modified cyt c mutant Nle91 was used to study the ATP-binding site, which is located near the evolutionarily invariant Arg 91 on the protein surface. This site was also demonstrated to mediate phospholipid binding, possibly by functioning as a phospholipid binding site. Circular dichroism spectroscopy, time resolved fluorescence spectroscopy of zinc- porphyrin modified [Zn2+-heme] cyt c and liposome binding studies of the Nle91 mutant were used to demonstrate that ATP induces a conformational change in membrane- bound cyt c. The ATP-induced conformational changes were mediated by Arg 91 and were most pronounced in cyt c bound to phospholipids via the C-site. It has been previously reported that the hydrophobic interaction between phospho- lipids and cyt c (C-site) includes the binding of a phospholipid acyl chain inside the protein. In this mechanism, which is known as extended phospholipid anchorage, the sn-2 acyl chain of a membrane phospholipid protrudes out of the membrane surface and is able to bind in a hydrophobic cavity in cyt c. Direct evidence for this type of bind- ing mechanism was obtained by studying cyt c/lipid interaction using fluorescent [Zn2+- heme] cyt c and fluorescence quenching of brominated fatty acids and phospholipids. Under certain conditions, cyt c can form fibrillar protein-lipid aggregates with neg- atively charged phospholipids. These aggregates resemble amyloid fibrils, which are involved in the pathogenesis of many diseases. Congo red staining of these fibers con- firmed the presence of amyloid structures. A set of phospholipid-binding proteins was also found to form similar aggregates, suggesting that phospholipid-induced amyloid formation could be a general mechanism of amyloidogenesis.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Oral health and menopause

Hormone therapy (HT) is widely used to relieve climacteric symptoms in order to increase the well-being of the women. The benefits as well as side-effects of HT are well documented. The principal menopausal oral symptoms are dry mouth (DM) and sensation of painful mouth (PM) due to various causes. Profile studies have indicated that HT users are more health-conscious than non-users. The hypothesis of the present study was that there are differences in oral health between woman using HT and those not using HT. A questionnaire study of 3173 women of menopausal age (50-58 years old) was done to investigate the prevalence of self-assessed sensations of PM and DM. Of those women participating in the questionnaire study, a random sample of 400 (200 using, 200 not using HT) was examined clinically in a 2-year follow-up study. Oral status was recorded according to WHO methods using DMFT and CPITN indices. The saliva flows were measured, salivary total protein, albumin and immunoglobulin concentrations and selected periodontal micro-organisms were analysed, and panoramic tomography of the jaws was taken. The patients filled in a structured questionnaire on their systemic health, medication and health habits. According to our questionnaire study there was no significant difference in the occurrence of self- assessed PM or DM between the HT users and non-users. According to logistic regression analyses, climacteric complaints significantly correlated with the occurrence of PM (p=0.000) and DM (p=0.000) irrespective of the use of HT, indicating that PM and DM are associated with climacteric symptoms in general. There was no difference between the groups in DMFT index values at follow up. The number of filled teeth (FT) showed a significant (p<0.05) increase in the HT group at follow-up. Periodontitis was diagnosed in 79% of HT users at baseline and in 71% at the follow-up. The values for non-HT users were 80% vs. 76%, respectively (Ns.). The mean numbers of ≥ 6 mm deep periodontal pockets were 0.9 ± 1.7 at baseline vs. 1.1 ± 2.1 two years later in the HT group, and 1.0 ± 1.7 vs. 1.2 ± 1.9, respectively, in the non-HT group. In a large Finnish national health survey, the prevalence of peridontitis of women of this age group was lower, but the prevalence of severe periodontitis seemed to be higher than in our study. Salivary albumin, IgG and IgM concentrations decreased in the HT group during the 2-year follow up (p<0.05), possibly indicating an improvement in epithelial integrity. No difference was found in any other salivary parameters or in the prevalence of the periodontal bacteria between or within the groups. In conclusion, the present findings showed that 50 to 58 year old women living in Helsinki have fairly good oral and dental health. The occurrence of PM and DM seemed to be associated with climacteric symptoms in general, and the use of HT did not affect the oral symptoms studied.

Kirjallisuuskatsaus: Eturauhassyövän androgeeniresponsiiviset in vivo -mallit; Kokeellinen osa: Bioluminesenssiin perustuvan ortotooppisen eturauhassyöpämallin optimointi

Eturauhassyöpä on yksi yleisimmistä syövistä länsimaissa. Eturauhassyöpä on yleensä hitaasti kehittyvä tauti. Edetessään se voi kuitenkin muuntua aggressiivisemmaksi ja aiheuttaa metastaaseja, jotka ovat pääasiallisena syynä taudin kuolleisuuteen. Androgeenit ovat merkittäviä tekijöitä eturauhassyövän patogeneesissä ja eturauhassyöpäkudos on useimmiten riippuvainen androgeeneista. Tämän vuoksi hoidon tavoitteena on estää niiden eritys kirurgisella tai kemiallisella kastraatiolla ja/tai estää androgeenien vaikutus antiandrogeeneilla. Eturauhassyöpää sekä sen hoitoon tarkoitettuja uusia lääkehoitomahdollisuuksia tutkitaan kiivaasti. Eturauhassyövän tutkimiseen on kehitetty lukematon määrä erilaisia in vivo -malleja. Koska eturauhassyöpä on yleensä androgeeneille herkkä, kuvaavat androgeeniresponsiiviset eläinmallit ihmisen tautia parhaiten. Eturauhassyövän mallintamiseen in vivo voidaan käyttää eri eläinlajeja, mutta hiiri on ylivoimaisesti käytetyin mallieläin. Immuunipuutteisiin hiiriin voidaan aiheuttaa kasvaimia inokuloimalla ihmisen kasvainsoluja tai osia ihmisen kasvaimista. Ortotooppisesti eturauhaseen inokuloitavat kasvainmallit mallintavat eturauhassyövässä esiintyvää syöpäsolujen ja stroomasolujen välistä epänormaalia vuorovaikutusta. Muuntogeeniset hiirimallit ovat yhä yleisempiä eturauhassyövän tutkimuksessa. Muuntogeenisilla malleilla voidaan mallintaa taudin kehittymistä ja sen etenemistä kokonaisuudessaan parhaiten. Eturauhasessa olevaa kasvainta ja sen kasvua on vaikea seurata ilman prostataspesifisen antigeenin (PSA) pitoisuuden mittausta tai erityisiä kuvantamistekniikoita. Tällaisia menetelmiä, kuten optista kuvantamista, käytetään yhä enemmän hyödyksi erilaisissa eturauhassyövän in vivo -malleissa. Tutkielman kokeellisen osan tavoitteena oli optimoida bioluminesenssiin perustuva optinen kuvantamismenetelmä androgeeniresponsiivisessa LNCaP-luc2-solulinjassa ortotooppisessa eturauhassyöpämallissa. Bioluminesenssikuvantaminen perustuu kasvainsolujen ilmentämän lusiferaasin katalysoimaan reaktioon, jossa entsyymin substraatti, lusiferiini, hapettuu ja tuottaa näkyvää valoa. Lisäksi tavoitteena oli tutkia lääkehoitojen ja kastraation vasteita mallissa. Bioluminesenssiin perustuvalla kuvantamisella oli mahdollista seurata eturauhaskasvainten kasvua noninvasiivisesti, reaaliaikaisesti ja toistuvasti. Bioluminesenssikuvantamisen avulla kasvainten kvantitointi oli nopeampaa kuin ultraäänikuvantamisen avulla, ja kasvainten kasvua oli myös mahdollista seurata useammin kuin seerumin PSA-mittausten avulla. Bioluminesenssikuvantamisen todettiin korreloivan paremmin PSA-pitoisuuden kanssa kuin kasvaimen todelliseen kokoon lopetushetkellä. Seerumin PSA-pitoisuus korreloi kuitenkin bioluminesenssimittausta paremmin eturauhaskasvaimen kokoon tässä kokeessa. Kasvainten oletettua suurempaa kokoa voidaan pitää todennäköisimpänä syynä sille, ettei lääkehoitojen tai kastraation todettu vaikuttavan kasvainten kasvuun bioluminesenssikuvantamisella mitattuna. Bioluminesenssikuvantaminen ei sovellu suurille eikä nekroottisille kasvaimille, sillä kuvantamismenetelmä toimii vain elävillä soluilla. Bioluminesenssikuvantamisen hyödyntämisen kannalta oleellista tässä mallissa on myös lusiferiini-injektion onnistuminen. Jatkotutkimuksia tarvitaan edelleen mallin validoimiseksi mm. lääkehoitojen vasteiden osoittamiseksi.

Complement factor H dysfunction in atypical hemolytic uremic syndrome

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.

A systems perspective of host-pathogen interactions: predicting disease outcome in tuberculosis

The complex web of interactions between the host immune system and the pathogen determines the outcome of any infection. A computational model of this interaction network, which encodes complex interplay among host and bacterial components, forms a useful basis for improving the understanding of pathogenesis, in filling knowledge gaps and consequently to identify strategies to counter the disease. We have built an extensive model of the Mycobacterium tuberculosis host-pathogen interactome, consisting of 75 nodes corresponding to host and pathogen molecules, cells, cellular states or processes. Vaccination effects, clearance efficiencies due to drugs and growth rates have also been encoded in the model. The system is modelled as a Boolean network. Virtual deletion experiments, multiple parameter scans and analysis of the system's response to perturbations, indicate that disabling processes such as phagocytosis and phagolysosome fusion or cytokines such as TNF-alpha and IFN-gamma, greatly impaired bacterial clearance, while removing cytokines such as IL-10 alongside bacterial defence proteins such as SapM greatly favour clearance. Simulations indicate a high propensity of the pathogen to persist under different conditions.