Approaches for improving the safety and efficacy of adenoviral gene therapy

Cancer is a devastating disease with poor prognosis and no curative treatment, when widely metastatic. Conventional therapies, such as chemotherapy and radiotherapy, have efficacy but are not curative and systemic toxicity can be considerable. Almost all cancers are caused due to changes in the genetic material of the transformed cells. Cancer gene therapy has emerged as a new treatment option, and past decades brought new insights in developing new therapeutic drugs for curing cancer. Oncolytic viruses constitute a novel therapeutic approach given their capacity to replicate in and kill specifically tumor cells as well as reaching tumor distant metastasis. Adenoviral gene therapy has been suggested to cause liver toxicity. This study shows that new developed adenoviruses, in particular Ad5/19p-HIT, can be redirected towards kidney while adenovirus uptake by liver is minimal. Moreover, low liver transduction resulted in a favorable tumor to liver ratio of virus load. Further, we established a new immunocompetent animal model Syrian hamsters. Wild type adenovirus 5 was found to replicate in Hap-T1 hamster tumors and normal tissues. There are no antiviral drugs available to inhibit adenovirus replication. In our study, chlorpromazine and cidofovir efficiently abrogated virus replication in vitro and showed significant reduction in vivo in tumors and liver. Once safety concerns were addressed together with the new given antiviral treatment options, we further improved oncolytic adenoviruses for better tumor penetration, local amplification and host system modulation. Further, we created Ad5/3-9HIF-Δ24-VEGFR-1-Ig, oncolytic adenovirus for improved infectivity and antiangiogenic effect for treatment of renal cancer. This virus exhibited increased anti-tumor effect and specific replication in kidney cancer cells. The key player for good efficacy of oncolytic virotherapy is the host immune response. Thus, we engineered a triple targeted adenovirus Ad5/3-hTERT-E1A-hCD40L, which would lead to tumor elimination due to tumor-specific oncolysis and apoptosis together with an anti-tumor immune response prompted by the immunomodulatory molecule. In conclusion, the results presented in this thesis constitute advances in our understanding of oncolytic virotherapy by successful tumor targeting, antiviral treatment options as a safety switch in case of replication associated side-effects, and modulation of the host immune system towards tumor elimination.  

Growth factor induction in intervertebral disc tissue: Observations in basic mechanisms of disc degeneration and rearrangement

The intervertebral disc is composed of concentrically arranged components: annulus fibrosus, the transition zone, and central nucleus pulposus. The major disc cell type differs in various parts of the intervertebral disc. In annulus fibrosus a spindle shaped fibroblast-like cell mainly dominates, whereas in central nucleus pulposus the more rounded chondrocyte-like disc cell is the major cell type. At birth the intervertebral disc is well vascularized, but during childhood and adolescence blood vessels become smaller and less numerous. The adult intervertebral disc is avascular and is nourished via the cartilage endplates. On the other hand, degenerated and prolapsed intervertebral discs are again vascularized, and show many changes compared to normal discs, including: nerve ingrowth, change in collagen turnover, and change in water content. Furthermore, the prolapsed intervertebral disc tissue has a tendency to decrease in size over time. Growth factors are polypeptides which regulate cell growth, extracellular matrix protease activity, and vascularization. Oncoproteins c-Fos and c-Jun heterodimerize, forming the AP-1 transcription factor which is expressed in activated cells. In this thesis the differences of growth factor expression in normal intervertebral disc, the degenerated intervertebral disc and herniated intervertebral disc were analyzed. Growth factors of particular interest were basic fibroblast growth factor (bFGF or FGF-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGFβ). Cell activation was visualized by the expression of the AP-1 transcription promoters c-Fos and c-Jun. The expression was shown with either mono- or polyclonal antibodies by indirect avidin-biotin-peroxidase immunohistochemical staining method. The normal control material was collected from a tissue bank of five organ donors. The degenerated disc material was from twelve patients operated on for painful degenerative disc disease, and herniated disc tissue material was obtained from 115 patients operated on for sciatica. Normal control discs showed only TGFβ immunopositivity. All other factors studied were immunonegative in the control material. Prolapsed disc material was immunopositive for all factors studied, and this positivity was located either in the disc cells or in blood vessels. Furthermore, neovascularization was noted. Disc cell immunoreaction was shown in chondrocyte-like disc cells or in fibroblast-like disc cells, the former being expressed especially in conglomerates (clusters of disc cells). TGFβ receptor induction was prominent in prolapsed intervertebral disc tissue. In degenerated disc material, the expression of growth factors was analyzed in greater detail in various parts of the disc: nucleus pulposus, anterior annulus fibrosus and posterior annulus fibrosus. PDGF did not show any immunoreactivity, whereas all other studied growth factors were localized either in chondrocyte-like disc cells, often forming clusters, in fibroblast-like disc cells, or in small capillaries. Many of the studied degenerated discs showed tears in the posterior region of annulus fibrosus, but expression of immunopositive growth factors was detected throughout the entire disc. Furthermore, there was a difference in immunopositive cell types for different growth factors. The main conclusion of the thesis, supported by all substudies, is the occurrence of growth factors in disc cells. They may be actively participating in a network regulating disc cell growth, proliferation, extracellular matrix turnover, and neovascularization. Chondrocyte-like disc cells, in particular, expressed growth factors and oncoproteins, highlighting the importance of this cell type in the basic pathophysiologic events involved in disc degeneration and disc rearrangement. The thesis proposes a hypothesis for cellular remodelling in intervertebral disc tissue. In summary, the model presents an activation pattern of different growth factors at different intervertebral disc stages, mechanisms leading to neovascularization of the intervertebral disc in pathological conditions, and alteration of disc cell shape, especially in annulus fibrosus. Chondrocyte-like disc cells become more numerous, and these cells are capable of forming clusters, which appear to be regionally active within the disc. The alteration of the phenotype of disc cells expressing growth factors from fibroblast-like disc cells to chondrocyte-like cells in annulus fibrosus, and the numerous expression of growth factor expressing disc cells in nucleus pulposus, may be a key element both during pathological degeneration of the intervertebral disc, and during the healing process after trauma.

Affected sibling pairs with juvenile idiopathic arthritis : An immunogenetic study of the disease in multicase families

Genetic susceptibility to juvenile idiopathic arthritis (JIA) was studied in the genetically homogeneous Finnish population by collecting families with two or three patients affected by this disease from cases seen in the Rheumatism Foundation Hospital. The number of families ranged in different studies from 37 to 45 and the total number of patients with JIA, from among whom these cases were derived, was 2 000 to 2 300. Characteristics of the disease in affected siblings in Finland were compared with a population-based series and with a sibling series from the United States. A thorough clinical and ophthalmological examination was made of all affected patients belonging to sibpair series. Information on the occurrence of chronic rheumatic diseases in parents was collected by questionnaire and diagnoses were confirmed from hospital records. All patients, their parents and most of the healthy sibs were typed for human leukocyte antigen (HLA) alleles in loci A, C, B, DR and DQ. The HLA allele distribution of the cases was compared with corresponding data from Finnish bone marrow donors. The genetic component in JIA was found to be more significant than previously believed. A concordance rate of 25% for a disease with a population prevalence of 1 per 1000 implied a relative risk of 250 for a monozygotic (MZ) twin. An estimate for the sibling risk of an affected individual was about 15- to 20-fold. The disease was basically similar in familial and sporadic cases; the mean age at disease onset was however lower in familial cases, (4.8 years vs 7.4 years). Three sibpairs (3.4 expected) were concordant for the presence of asymptomatic uveitis. Uveitis would thus not appear to have any genetic component of its own, separate from the genetic basis of JIA. Four of the parents had JIA (0.2 cases expected), four had a type of rheumatoid factor-negative arthritis similar to that seen in juvenile patients but commencing in adulthood, and one had spondyloarthropathy (SPA). These findings provide additional support for the conception of a genetic predisposition to JIA and suggest the existence of a new disease entity, JIA of adult onset. Both the linkage analysis of the affected sibpairs and the association analysis of nuclear families provided overwhelming evidence of a major contribution of HLA to the genetic susceptibility to JIA. The association analysis in the Finnish population confirmed that the most significant associations prevailed for DRB1*0801, DQB1*0402, as expected from previous observations, and indicated the independent role of Cw*0401.

Molecular basis for the development of diagnostic methods and specific treatment modalities for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown aetiology and poor prognosis. IPF is characterized by alveolar epithelial damage that leads tissue remodelling and ultimately to the loss of normal lung architecture and function. Treatment has been focused on anti-inflammatory therapies, but due to their poor efficacy new therapeutic modalities are being sought. There is a need for early diagnosis and also for differential diagnostic markers for IPF and other interstitial lung diseases. The study utilized patient material obtained from bronchoalveolar lavage (BAL), diagnostic biopsies or lung transplantation. Human pulmonary fibroblast cell cultures were propagated and asbestos-induced pulmonary fibrosis in mice was used as an experimental animal model of IPF. The possible markers for IPF were scanned by immunohistochemistry, RT-PCR, ELISA and western blot. Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in tissue remodelling. Microarray studies have introduced potential markers that could serve as additional tools for the assessment of IPF and one of the most promising was MMP 7. MMP-7 protein levels were measured in the BAL fluid of patients with idiopathic interstitial lung diseases or idiopathic cough. MMP-7 was however similarly elevated in the BAL fluid of all these disorders and thus cannot be used as a differential diagnostic marker for IPF. Activation of transforming growth factor (TGF)-ß is considered to be a key element in the progression of IPF. Bone morphogenetic proteins (BMP) are negative regulators of intracellular TGF-ß signalling and BMP-4 signalling is in turn negatively regulated by gremlin. Gremlin was found to be highly upregulated in the IPF lungs and IPF fibroblasts. Gremlin was detected in the thickened IPF parenchyma and endothelium of small capillaries, whereas in non-specific interstitial pneumonia it localized predominantly in the alveolar epithelium. Parenchymal gremlin immunoreactivity might indicate IPF-type interstitial pneumonia. Gremlin mRNA levels were higher in patients with end-stage fibrosis suggesting that gremlin might be a marker for more advanced disease. Characterization of the fibroblastic foci in the IPF lungs showed that immunoreactivity to platelet-derived growth factor (PDGF) receptor-α and PDGF receptor-β was elevated in IPF parenchyma, but the fibroblastic foci showed only minor immunoreactivity to the PDGF receptors or the antioxidant peroxiredoxin II. Ki67 positive cells were also observed predominantly outside the fibroblastic foci, suggesting that the fibroblastic foci may not be composed of actively proliferating cells. When inhibition of profibrotic PDGF-signalling by imatinib mesylate was assessed, imatinib mesylate reduced asbestos-induced pulmonary fibrosis in mice as well as human pulmonary fibroblast migration in vitro but it had no effect on the lung inflammation.

Cyclosporine A in the treatment of Interstitial Cystitis

Painful bladder syndrome/interstitial cystitis (PBS/IC) is a chronic urinary bladder disorder of unknown etiology characterized by symptoms of bladder pain and urinary frequency. PBS/IC is a chronic disease in which drug therapy has not led to significant success over the course of time. If the symptoms of PBS/IC are refractory to standard treatments, a possible cure might demand surgical intervention involving cystectomy. The eventual autoimmune etiology in mind, immunosuppressive drug therapy with cyclosporine A (CyA) was started to patients with refractory PBS/IC. CyA is a potent anti-inflammatory drug, a calcineurin inhibitor which inhibits T lymphocyte IL-2 produc-tion. T cells are present in abundance in inflammation of the bladder in PBS/IC. On the basis of a pilot, short-term study with CyA on PBS/IC, use of CyA was continued empirically over the long term. We conducted a prospective, randomized, six-month study in 64 patients comparing the effect of CyA with the FDA approved treatment, pentosan polysulfate sodium (PPS). We measured the drug effect on patient s symptoms, the potassium sensitivity test, and on urinary biomarkers. We further tested the impact of CyA, PPS, DMSO and BCG therapy on a health-related quality of life questionnaire and evaluated the response rate to treatment with these therapies. Long-term use of CyA was safe and effective in PBS/IC patients. The good clinical effect matured individually during the years in which CyA was continued. Cessation of medication led to the reappearance of symptoms, and restarting CyA to renewed alleviation, so that CyA was administered as continuous medication. The response rate to CyA increased during the study period, comprising 75% of CyA patients at six months. 19% of patients responded to PPS therapy. Adverse effects were more common in the CyA group, underlining the importance of monitoring the drug safety and appropriate titration of the dose. The potassium sensitivity test is positive in the majority of PBS/IC patients. Successful therapy of PBS/IC can alter nerve sensitivity to external potassium. This effect was seen more often after CyA therapy. Successful treatment of PBS/IC with CyA resulted to decreasing urinary levels of EGF. IL-6 levels in urine were higher among older patient with a longer history of PBS/IC. In these patients, reduced levels of urinary IL-6 were measured after CyA therapy. Patients who experience the best treatment response have improved quality of life according to the post-treatment health-related quality of life (HRQOL) questionnaire. CyA had more impact on the ma-jority of the aspects of QoL than PPS. Despite DMSO therapy being more successful than BCG in the count of responders, DMSO and BCG had equal effects on the HRQOL questionnaire.

Vascular dilatory function and cardiovascular risk factors in women with a history of pre-eclampsia

Women with a history of pre-eclampsia have an increased risk of cardiovascular disease in later life. The mechanisms which mediate this heightened risk are poorly understood; it was long believed that pre-eclampsia was a separate disease without any connection to other pathologies. The present study was undertaken to investigate the cardiovascular risk milieu, vascular dilatory function and cardiovascular risk factors, in women with pre-eclampsia, 5 6 years after index pregnancy. The aim was to understand better the cardiovascular risks associated with pre-eclampsia and add tools to the evaluation of cardiovascular risk in women. --- The study involved 30 women with previous severe pre-eclampsia and 21 controls. The 2-day study protocol included venous occlusion plethysmography and pulse wave analysis for assessment of vascular dilatory function and central pulse wave reflection, respectively, office and ambulatory blood pressure measurements, assessment of insulin sensitivity, using a minimal model technique, and tests regarding renal function, lipid metabolism, sympathetic activity and inflammation. Vasodilatory function was impaired in women with a history of pre-eclampsia; this was seen in both endothelium-dependent and endothelium-independent vasodilatation. Proteinuria during pre-eclampsia did not predict changes in vasodilatation, and renal function was similar in the two groups. Insulin sensitivity was related to vasodilatation and features of metabolic syndrome, but only in the patient group, despite similar insulin sensitivity in the control group. Arterial pressure was higher in the patient group than in the controls and correlated with endothelin-1 levels in the patient group, whilst the overall difference between the groups was diminished in 24 hour arterial pressure measurements. Additionally, women with previous pre-eclampsia were characterized by increased sympathetic activity. Impaired vasodilatory function at the vascular smooth muscle level seems to characterize clinically healthy women with a history of pre-eclampsia. These vascular changes and the features of metabolic syndrome may be related to the increased risk of cardiovascular disease. Furthermore, increased blood pressure in combination with enhanced sympathetic activity may be additive as regards this risk. These women should be informed about their potential cardiovascular risk profile and the possibilities to minimize it via their own actions. Medical cardiovascular risk assessment in women should include obstetric history.

Effects of SLCO1B1 polymorphism on the pharmacokinetics of the oral antidiabetic drugs repaglinide, nateglinide, rosiglitazone, and pioglitazone

Organic anion-transporting polypeptide 1B1 (OATP1B1), encoded by the SLCO1B1 gene, is an influx transporter expressed on the sinusoidal membrane of human hepatocytes. The common c.521T>C (p.Val174Ala) single-nucleotide polymorphism (SNP) of the SLCO1B1 gene has been associated with reduced OATP1B1 transport activity in vitro and increased plasma concentrations of several of its substrate drugs in vivo in humans. Another common SNP of the SLCO1B1 gene, c.388A>G (p.Asn130Asp), defining the SLCO1B1*1B (c.388G-c.521T) haplotype, has been associated with increased OATP1B1 transport activity in vitro. The aim of this thesis was to investigate the role of SLCO1B1 polymorphism in the pharmacokinetics of the oral antidiabetic drugs repaglinide, nateglinide, rosiglitazone, and pioglitazone. Furthermore, the effect of the SLCO1B1 c.521T>C SNP on the extent of interaction between gemfibrozil and repaglinide as well as the role of the SLCO1B1 c.521T>C SNP in the potential interaction between atorvastatin and repaglinide were evaluated. Five crossover studies with 2-4 phases were carried out, with 20-32 healthy volunteers in each study. The effects of the SLCO1B1 c.521T>C SNP on single doses of repaglinide, nateglinide, rosiglitazone, and pioglitazone were investigated in Studies I and V. In Study II, the effects of the c.521T>C SNP on repaglinide pharmacokinetics were investigated in a dose-escalation study, with repaglinide doses ranging from 0.25 to 2 mg. The effects of the SLCO1B1*1B/*1B genotype on repaglinide and nateglinide pharmacokinetics were investigated in Study III. In Study IV, the interactions of gemfibrozil and atorvastatin with repaglinide were evaluated in relation to the c.521T>C SNP. Plasma samples were collected for drug concentration determinations. The pharmacodynamics of repaglinide and nateglinide was assessed by measuring blood glucose concentrations. The mean area under the plasma repaglinide concentration-time curve (AUC) was ~70% larger in SLCO1B1 c.521CC participants than in c.521TT participants (P ≤ 0.001), but no differences existed in the pharmacokinetics of nateglinide, rosiglitazone, and pioglitazone between the two genotype groups. In the dose-escalation study, the AUC of repaglinide was 60-110% (P ≤ 0.001) larger in c.521CC participants than in c.521TT participants after different repaglinide doses. Moreover, the AUC of repaglinide increased linearly with repaglinide dose in both genotype groups (r > 0.88, P 0.001). The AUC of repaglinide was ~30% lower in SLCO1B1*1B/*1B participants than in SLCO1B1*1A/*1A (c.388AA-c.521TT) participants (P = 0.007), but no differences existed in the AUC of nateglinide between the two genotype groups. In the drug-drug interaction study, the mean increase in the repaglinide AUC by gemfibrozil was ~50% (P = 0.002) larger in c.521CC participants than in c.521TT participants, but the relative (7-8-fold) increases in the repaglinide AUC did not differ significantly between the genotype groups. In c.521TT participants, atorvastatin increased repaglinide peak plasma concentration and AUC by ~40% (P = 0.001) and ~20% (P = 0.033), respectively. In each study, after repaglinide administration, there was a tendency towards lower blood glucose concentrations in c.521CC participants than in c.521TT participants. In conclusion, the SLCO1B1 c.521CC genotype is associated with increased and the SLCO1B1*1B/*1B genotype with decreased plasma concentrations of repaglinide, consistent with reduced and enhanced hepatic uptake, respectively. Inhibition of OATP1B1 plays a limited role in the interaction between gemfibrozil and repaglinide. Atorvastatin slightly raises plasma repaglinide concentrations, probably by inhibiting OATP1B1. The findings on the effect of SLCO1B1 polymorphism on the pharmacokinetics of the drugs studied suggest that in vivo in humans OATP1B1 significantly contributes to the hepatic uptake of repaglinide, but not to that of nateglinide, rosiglitazone, or pioglitazone. SLCO1B1 polymorphism may be associated with clinically significant differences in blood glucose-lowering response to repaglinide, but probably has no effect on the response to nateglinide, rosiglitazone, or pioglitazone.

Pharmacogenetics of SLCO1B1: Population genetics and effect on statins

Kohonneiden kolesterolipitoisuuksien alentamisessa käytettävien statiinien hyödyt sydän- ja verisuonisairauksien estossa on vahvasti osoitettu ja niiden käyttö on niin Suomessa kuin muuallakin maailmassa kasvanut voimakkaasti – Suomessa statiininkäyttäjiä on noin 600 000. Statiinilääkitys on pitkäaikaisessakin käytössä melko hyvin siedetty, mutta yleisimpinä haittavaikutuksina voi ilmetä lihasheikkoutta, -kipua ja -kramppeja, jotka voivat edetä jopa henkeä uhkaavaksi lihasvaurioksi. Lihashaittariski suurenee suhteessa statiiniannokseen ja plasman statiinipitoisuuksiin. Statiinien plasmapitoisuuksissa, tehossa ja haittavaikutusten ilmenemisessä on suuria potilaskohtaisia eroja. SLCO1B1-geenin koodaama OATP1B1-kuljetusproteiini kuljettaa monia elimistön omia aineita ja lääkeaineita verenkierrosta solukalvon läpi maksasoluun, mm. statiineja, joiden kolesterolia alentava vaikutus ja poistuminen elimistöstä tapahtuvat pääosin maksassa. Erään SLCO1B1-geenin nukleotidimuutoksen (c.521T>C) tiedetään heikentävän OATP1B1:n kuljetustehoa. Tässä väitöskirjatyössä selvitettiin SLCO1B1-geenin perinnöllistä muuntelua suomalaisilla ja eri väestöissä maailmanlaajuisesti. Lisäksi selvitettiin SLCO1B1:n muunnosten vaikutusta eri statiinien pitoisuuksiin (farmakokinetiikka) ja vaikutuksiin (farmakodynamiikka) sekä kolesteroliaineenvaihduntaan. Näihin tutkimuksiin valittiin SLCO1B1-genotyypin perusteella terveitä vapaaehtoisia koehenkilöitä, joille annettiin eri päivinä kerta-annos kutakin tutkittavaa statiinia: fluvastatiinia, pravastatiinia, simvastatiinia, rosuvastatiinia ja atorvastatiinia. Verinäytteistä määritettiin plasman statiinien ja niiden aineenvaihduntatuotteiden sekä kolesterolin ja sen muodostumista ja imeytymistä kuvaavien merkkiaineiden pitoisuuksia. Toiminnallisesti merkittävien SLCO1B1-geenimuunnosten esiintyvyydessä todettiin suuria eroja eri väestöjen välillä. Suomalaisilla SLCO1B1 c.521TC-genotyypin (geenimuunnos toisessa vastinkromosomissa) esiintyvyys oli noin 32 % ja SLCO1B1 c.521CC-genotyypin (geenimuunnos molemmissa vastinkromosomeissa) esiintyvyys noin 4 %. Globaalisti geenimuunnosten esiintyvyys korreloi maapallon leveyspiirien kanssa siten, että matalaan transportteriaktiivisuuteen johtavat muunnokset olivat yleisimpiä pohjoisessa ja korkeaan aktiivisuuteen johtavat päiväntasaajan lähellä asuvilla väestöillä. SLCO1B1-genotyypillä oli merkittävä vaikutus statiinien plasmapitoisuksiin lukuun ottamatta fluvastatiinia. Simvastatiinihapon plasmapitoisuudet olivat keskimäärin 220 %, atorvastatiinin 140 %, pravastatiinin 90 % ja rosuvastatiinin 70 % suuremmat c.521CC-genotyypin omaavilla koehenkilöillä verrattuna normaalin c.521TT-genotyypin omaaviin. Genotyypillä ei ollut merkittävää vaikutusta minkään statiinin tehoon tässä kerta-annostutkimuksessa, mutta geenimuunnoksen kantajilla perustason kolesterolisynteesinopeus oli suurempi. Tulokset osoittavat, että SLCO1B1 c.521T>C geenimuunnos on varsin yleinen suomalaisilla ja muilla ei-afrikkalaisilla väestöillä. Tämä geenimuunnos voi altistaa erityisesti simvastatiinin, mutta myös atorvastatiinin, pravastatiinin ja rosuvastatiinin, aiheuttamille lihashaitoille suurentamalla niiden plasmapitoisuuksia. SLCO1B1:n geenimuunnoksen testaamista voidaan tulevaisuudessa käyttää apuna valittaessa sopivaa statiinilääkitystä ja -annosta potilaalle, ja näin parantaa sekä statiinihoidon turvallisuutta että tehoa.

Language learning in infancy

Although immensely complex, speech is also a very efficient means of communication between humans. Understanding how we acquire the skills necessary for perceiving and producing speech remains an intriguing goal for research. However, while learning is likely to begin as soon as we start hearing speech, the tools for studying the language acquisition strategies in the earliest stages of development remain scarce. One prospective strategy is statistical learning. In order to investigate its role in language development, we designed a new research method. The method was tested in adults using magnetoencephalography (MEG) as a measure of cortical activity. Neonatal brain activity was measured with electroencephalography (EEG). Additionally, we developed a method for assessing the integration of seen and heard syllables in the developing brain as well as a method for assessing the role of visual speech when learning phoneme categories. The MEG study showed that adults learn statistical properties of speech during passive listening of syllables. The amplitude of the N400m component of the event-related magnetic fields (ERFs) reflected the location of syllables within pseudowords. The amplitude was also enhanced for syllables in a statistically unexpected position. The results suggest a role for the N400m component in statistical learning studies in adults. Using the same research design with sleeping newborn infants, the auditory event-related potentials (ERPs) measured with EEG reflected the location of syllables within pseudowords. The results were successfully replicated in another group of infants. The results show that even newborn infants have a powerful mechanism for automatic extraction of statistical characteristics from speech. We also found that 5-month-old infants integrate some auditory and visual syllables into a fused percept, whereas other syllable combinations are not fully integrated. Auditory syllables were paired with visual syllables possessing a different phonetic identity, and the ERPs for these artificial syllable combinations were compared with the ERPs for normal syllables. For congruent auditory-visual syllable combinations, the ERPs did not differ from those for normal syllables. However, for incongruent auditory-visual syllable combinations, we observed a mismatch response in the ERPs. The results show an early ability to perceive speech cross-modally. Finally, we exposed two groups of 6-month-old infants to artificially created auditory syllables located between two stereotypical English syllables in the formant space. The auditory syllables followed, equally for both groups, a unimodal statistical distribution, suggestive of a single phoneme category. The visual syllables combined with the auditory syllables, however, were different for the two groups, one group receiving visual stimuli suggestive of two separate phoneme categories, the other receiving visual stimuli suggestive of only one phoneme category. After a short exposure, we observed different learning outcomes for the two groups of infants. The results thus show that visual speech can influence learning of phoneme categories. Altogether, the results demonstrate that complex language learning skills exist from birth. They also suggest a role for the visual component of speech in the learning of phoneme categories.

Childhood otitis media - risk factors and surgical management

Background: Otitis media (OM) is one of the most common childhood diseases. Approximately every third child suffers from recurrent acute otitis media (RAOM), and 5% of all children have persistent middle ear effusion for months during their childhood. Despite numerous studies on the prevention and treatment of OM during the past decades, its management remains challenging and controversial. In this study, the effect of adenoidectomy on the risk for OM, the potential risk factors influencing the development of OM and the frequency of asthma among otitis-prone children were investigated. Subjects and methods: One prospective randomized trial and two retrospective studies were conducted. In the prospective trial, 217 children with RAOM or chronic otitis media with effusion (COME) were randomized to have tympanostomy with or without adenoidectomy. The age of the children at recruitment was between 1 and 4 years. RAOM was defined as having at least 3 episodes of AOM during the last 6 months or at least 5 episodes of AOM during the last 12 months. COME was defined as having persistent middle ear effusion for 2-3 months. The children were followed up for one year. In the first retrospective study, the frequency of childhood infections and allergy was evaluated by a questionnaire among 819 individuals. In the second retrospective study, data of asthma diagnosis were analysed from hospital discharge records of 1616 children who underwent adenoidectomy or had probing of the nasolacrimal duct. Results: In the prospective randomized study, adenoidectomy had no beneficial effect on the prevention of subsequent episodes of AOM. Parental smoking was found to be a significant risk factor for OM even after the insertion of tympanostomy tubes. The frequencies of exposure to tobacco smoke and day-care attendance at the time of randomization were similar among children with RAOM and COME. However, the frequencies of allergy to animal dust and pollen and parental asthma were lower among children with COME than those with RAOM. The questionnaire survey and the hospital discharge data revealed that children who had frequent episodes of OM had an increased risk for asthma. Conclusions: The first surgical intervention to treat an otitis-prone child younger than 4 years should not include adenoidectomy. Interventions to stop parental smoking could significantly reduce the risk for childhood RAOM. Whether an otitis-prone child develops COME or RAOM, seems to be influenced by genetic predisposition more strongly than by environmental risk factors. Children who suffer from repeated upper respiratory tract infections, like OM, may be at increased risk for developing asthma.

Transition to parenthood after assisted reproductive treatment: Follow-up study of singleton pregnancies

Infertility treatments are relatively easily available in most Western countries today, but the psychological consequences of these high-tech treatments have scarcely been addressed. The purpose of this controlled longitudinal study was to explore the early environment of the infant born by assisted reproductive treatment (ART). We focused on the parents mental well-being, marital relations and experience of parenting. In addition to this, we assessed parent child interaction and parents mental representations of their child after long-standing infertility and several unsuccessful ART attempts. The subjects were infertile couples who achieved a singleton pregnancy by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The control group comprised of spontaneously conceiving couples with singleton pregnancies. ART women showed fewer depressive symptoms than controls during pregnancy and after delivery, but the difference vanished by the end of the child s first year. ART men consistently had lower levels of anxiety symptoms, sleeping difficulties, and social dysfunction than control men. Control women experienced a decrease in dyadic consensus during the child s first year, which did not happen among ART women. After the child was born, ART men reported a higher level of sexual affection compared with control men. Psychic symptoms and stressful life events were differently related to marital relations in ART and control groups. The parenting experiences of ART mothers were in general at a higher level, compared with controls, and they changed in a positive direction during the child s first year. Fathering experiences were at the same level in both groups, and they changed positively in both groups by the end of the child s first year. The parenting experiences of ART mothers and fathers were more resilient to certain child-related stressors than those of control group. Both mothers and fathers with long-term infertility showed more sensitive behaviour with their child in toddler-age than in infancy. Correspondingly, children s cooperation increased. Mothers often mentioned a fear of miscarriage and difficulty in creating representations of the child during pregnancy. Descriptions of the infants were mainly rich, vivid and loaded with positive features. In conclusion, ART parents in general seem to adapt well to the transition to parenthood. Former infertility and ART do not seem to constitute a risk for parents mental health, marital relations or experience of parenting. Even longstanding infertility with several unsuccessful treatment attempts did not create a risk as regards parenting behaviour or parents mental representations of their child. In this group, however, women were found to have fear for losing the child and difficulty in creating representations of the child during pregnancy, which in some cases may indicate need for psychosocial support. Even though our results are encouraging, infertility and infertility treatments are generally considered as a stressful experience. It is a challenge for health authorities to recognize those couples who need professional help to overcome the distressing experiences of infertility and ART.

Pathophysiology of Congenital Nephrotic Syndrome of the Finnish type

Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal recessive disease which is highly enriched in the Finnish population. It is caused by mutations in the NPHS1 gene encoding for nephrin, which is a major component of the glomerular filtration barrier in the kidney. Patients with NPHS1 have heavy proteinuria and nephrotic syndrome (NS) from birth and develop renal fibrosis in early childhood. Renal transplantation (TX) is the only curative treatment for NPHS1. These patients form the largest group of pediatric kidney transplant children in our country. The NPHS1 kidneys are removed in infancy and they serve as an excellent human material for studies of the pathophysiology of proteinuric kidney diseases. Sustained proteinuria is a major factor leading to end-stage renal failure and understanding this process is crucial for nephrology. In this study we investigated the glomerular and tubulointerstitial changes that occur in the NPHS1 kidneys during infancy as well as the expression of nephrin in non-renal tissues. We also studied the pathology and management of recurrent proteinuria in kidney grafts transplanted to NPHS1 children. Severe renal lesions evolved in patients with NPHS1 during the first months of life. Glomerular sclerosis developed through progressive mesangial sclerosis, and capillary obliteration was an early consequence of this process. Shrinkage of the glomerular tuft was common, whereas occlusion of tubular opening or protrusion of the glomerular tuft into subepithelial space or through the Bowman's capsule were not detected. Few inflammatory cells were detected in the mesangial area. The glomerular epithelial cells (podocytes) showed severe ultrastructural changes and hypertrophy. Podocyte proliferation and apoptosis were rare, but moderate amounts of podocytes were detached and ended up in the urine. The results showed that endocapillary lesions not extracapillary lesions, as generally believed were important for the sclerotic process in the NPHS1 glomeruli. In the tubulointerstitium, severe lesions developed in NPHS1 kidneys during infancy. Despite heavy proteinuria, tubular epithelial cells (TECs) did not show transition into myofibroblasts. The most abundant chemokines in NPHS1 tissue were neutrophil activating protein-2 (NAP-2), macrophage inhibiting factor (MIF), and monocyte chemoattractant protein-1 (MCP-1). Interstitial inflammation and fibrosis were first detected in the paraglomerular areas and the most abundant inflammatory cells were monocytes/macrophages. Arteries and arterioles showed intimal hypertrophy, but the pericapillary microvasculature remained quite normal. However, excessive oxidative stress was evident in NPHS1 kidneys. The results indicated that TECs were relatively resistant to the heavy tubular protein load. Nephrin was at first thought to be podocyte specific, but some studies especially in experimental animals have suggested that nephrin might also be expressed in non-renal tissues such as pancreas and central nervous system. The knowledge of nephrin biology is important for the evaluation of nephrin related diseases. In our study, no significant amounts of nephrin protein or mRNA were detected in non-renal tissues of man and pig as studied by immunohistochemistry and in situ hybridization. The phenotype analysis of NPHS1 children, who totally lack nephrin, revealed no marked impairment in the neurological, testicular, or pancreatic function speaking against the idea that nephrin would play an important functional role outside the kidney. The NPHS1 kidneys do not express nephrin and antibodies against this major glomerular filter protein have been observed in NPHS1 children after renal TX most likely as an immune reaction against a novel antigen. These antibodies have been associated with the development of recurrent NS in the kidney graft of NPHS1 patients. In our study, a third of the NPHS1 patients homozygous for Fin-Major mutation developed recurrent NS in the transplanted graft. Re-transplantations were performed to patients who lost their graft due to recurrent NS and heavy proteinuria immediately developed in all cases. While 73% of the patients had detectable serum anti-nephrin antibodies, the kidney biopsy findings were minimal. Introduction of plasma exchange (PE) to the treatment of recurrent nephroses increased the remission rate from 54% to 89%. If remission was achieved, recurrent NS did not significantly deteriorate the long term graft function. In conclusion, the results show that the lack of nephrin in podocyte slit diaphragm in NPHS1 kidneys induces progressive mesangial expansion and glomerular capillary obliteration and inflicts interstitial fibrosis, inflammation, and oxidative stress with surprisingly little involvement of the TECs in this process. Nephrin appears to have no clinical significance outside the kidney. Development of antibodies against nephrin seems to be a major cause of recurrent NS in kidney grafts of NPHS1 patients and combined use of PE and cyclophosphamide markedly improved remission rates.

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.

Development of a new flexor tendon repair technique performed with bioabsorbable poly-L/D-lactide (PLDLA) suture

Sormen koukistajajännevamman korjauksen jälkeisen aktiivisen mobilisaation on todettu johtavan parempaan toiminnalliseen lopputulokseen kuin nykyisin yleisesti käytetyn dynaamisen mobilisaation. Aktiivisen mobilisaation ongelma on jännekorjauksen pettämisriskin lisääntyminen nykyisten ommeltekniikoiden riittämättömän vahvuuden vuoksi. Jännekorjauksen lujuutta on parannettu kehittämällä monisäieommeltekniikoita, joissa jänteeseen tehdään useita rinnakkaisia ydinompeleita. Niiden kliinistä käyttöä rajoittaa kuitenkin monimutkainen ja aikaa vievä tekninen suoritus. Käden koukistajajännekorjauksessa käytetään yleisesti sulamattomia ommelmateriaaleja. Nykyiset käytössä olevat biohajoavat langat heikkenevät liian nopeasti jänteen paranemiseen nähden. Biohajoavan laktidistereokopolymeeri (PLDLA) 96/4 – langan vetolujuuden puoliintumisajan sekä kudosominaisuuksien on aiemmin todettu soveltuvan koukistajajännekorjaukseen. Tutkimuksen tavoitteena oli kehittää välittömän aktiivisen mobilisaation kestävä ja toteutukseltaan yksinkertainen käden koukistajajännekorjausmenetelmä biohajoavaa PLDLA 96/4 –materiaalia käyttäen. Tutkimuksessa analysoitiin viiden eri yleisesti käytetyn koukistajajänneompeleen biomekaanisia ominaisuuksia staattisessa vetolujuustestauksessa ydinompeleen rakenteellisten ominaisuuksien – 1) säikeiden (lankojen) lukumäärän, 2) langan paksuuden ja 3) ompeleen konfiguraation – vaikutuksen selvittämiseksi jännekorjauksen pettämiseen ja vahvuuteen. Jännekorjausten näkyvän avautumisen todettiin alkavan perifeerisen ompeleen pettäessä voima-venymäkäyrän myötöpisteessä. Ydinompeleen lankojen lukumäärän lisääminen paransi ompeleen pitokykyä jänteessä ja suurensi korjauksen myötövoimaa. Sen sijaan paksumman (vahvemman) langan käyttäminen tai ompeleen konfiguraatio eivät vaikuttaneet myötövoimaan. Tulosten perusteella tutkittiin mahdollisuuksia lisätä ompeleen pitokykyä jänteestä yksinkertaisella monisäieompeleella, jossa ydinommel tehtiin kolmen säikeen polyesterilangalla tai nauhamaisen rakenteen omaavalla kolmen säikeen polyesterilangalla. Nauhamainen rakenne lisäsi merkitsevästi ompeleen pitokykyä jänteessä parantaen myötövoimaa sekä maksimivoimaa. Korjauksen vahvuus ylitti aktiivisen mobilisaation jännekorjaukseen kohdistaman kuormitustason. PLDLA 96/4 –langan soveltuvuutta koukistajajännekorjaukseen selvitettiin tutkimalla langan biomekaanisia ominaisuuksia ja solmujen pito-ominaisuuksia staattisessa vetolujuustestauksessa verrattuna yleisimmin jännekorjauksessa käytettävään punottuun polyesterilankaan (Ticron®). PLDLA –langan todettiin soveltuvan hyvin koukistajajännekorjaukseen, sillä se on polyesterilankaa venymättömämpi ja solmujen pitävyys on parempi. Viimeisessä vaiheessa tutkittiin PLDLA 96/4 –langasta valmistetulla kolmisäikeisellä, nauhamaisella jännekorjausvälineellä tehdyn jännekorjauksen kestävyyttä staattisessa vetolujuustestauksessa sekä syklisessä kuormituksessa, joka simuloi staattista testausta paremmin mobilisaation toistuvaa kuormitusta. PLDLA-korjauksen vahvuus ylitti sekä staattisessa että syklisessä kuormituksessa aktiivisen mobilisaation edellyttämän vahvuuden. Nauhamaista litteää ommelmateriaalia ei aiemmin ole tutkittu tai käytetty käden koukistajajännekorjauksessa. Tässä tutkimuksessa ommelmateriaalin nauhamainen rakenne paransi merkitsevästi jännekorjauksen vahvuutta, minkä arvioidaan johtuvan lisääntyneestä kontaktipinnasta jänteen ja ommelmateriaalin välillä estäen ompeleen läpileikkautumista jänteessä. Tutkimuksessa biohajoavasta PLDLA –materiaalista valmistetulla rakenteeltaan nauhamaisella kolmisäikeisellä langalla tehdyn jännekorjauksen vahvuus saavutti aktiivisen mobilisaation edellyttämän tason. Lisäksi uusi menetelmä on helppokäyttöinen ja sillä vältetään perinteisten monisäieompeleiden tekniseen suoritukseen liittyvät ongelmat.

Mitochondrial helicase Twinkle in mtDNA copy number regulation and a late-onset disease model

Mitochondrial diseases are caused by disturbances of the energy metabolism. The disorders range from severe childhood neurological diseases to muscle diseases of adults. Recently, mitochondrial dysfunction has also been found in Parkinson s disease, diabetes, certain types of cancer and premature aging. Mitochondria are the power plants of the cell but they also participate in the regulation of cell growth, signaling and cell death. Mitochondria have their own genetic material, mtDNA, which contains the genetic instructions for cellular respiration. Single cell may host thousands of mitochondria and several mtDNA molecules may reside inside single mitochondrion. All proteins needed for mtDNA maintenance are, however, encoded by the nuclear genome, and therefore, mutations of the corresponding genes can also cause mitochondrial disease. We have here studied the function of mitochondrial helicase Twinkle. Our research group has previously identified nuclear Twinkle gene mutations underlying an inherited adult-onset disorder, progressive external ophthalmoplegia (PEO). Characteristic for the PEO disease is the accumulation of multiple mtDNA deletions in tissues such as the muscle and brain. In this study, we have shown that Twinkle helicase is essential for mtDNA maintenance and that it is capable of regulating mtDNA copy number. Our results support the role of Twinkle as the mtDNA replication helicase. No cure is available for mitochondrial disease. Good disease models are needed for studies of the cause of disease and its progression and for treatment trials. Such disease model, which replicates the key features of the PEO disease, has been generated in this study. The model allows for careful inspection of how Twinkle mutations lead to mtDNA deletions and further causes the PEO disease. This model will be utilized in a range of studies addressing the delay of the disease onset and progression and in subsequent treatment trials. In conclusion, in this thesis fundamental knowledge of the function of the mitochondrial helicase Twinkle was gained. In addition, the first model for adult-onset mitochondrial disease was generated.