Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

CDNF and MANF in an experimental model of Parkinson's disease in rats

Parkinson s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra (SN). Current therapies of PD do not stop the progression of the disease and the efficacy of these treatments wanes over time. Neurotrophic factors are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. Neurotrophic factors are attractive candidates for neuroprotective or even neurorestorative treatment of PD. Thus, searching for and characterizing trophic factors are highly important approaches to degenerative diseases. CDNF (cerebral dopamine neurotrophic factor) and MANF (mesencephalic astrocyte-derived neurotrophic factor) are secreted proteins that constitute a novel, evolutionarily conserved neurotrophic factor family expressed in vertebrates and invertebrates. The present study investigated the neuroprotective and restorative effects of human CDNF and MANF in rats with unilateral partial lesion of dopamine neurons by 6-hydroxydopamine (6-OHDA) using both behavioral (amphetamine-induced rotation) and immunohistochemical analyses. We also investigated the distribution and transportation profiles of intrastriatally injected CDNF and MANF in rats. Intrastriatal CDNF and MANF protected nigrostriatal dopaminergic neurons when administered six hours before or four weeks after the neurotoxin 6-OHDA. More importantly, the function of the lesioned nigrostriatal dopaminergic system was partially restored even when the neurotrophic factors were administered four weeks after 6-OHDA. A 14-day continuous infusion of CDNF but not of MANF restored the function of the midbrain neural circuits controlling movement when initiated two weeks after unilateral injection of 6-OHDA. Continuous infusion of CDNF also protected dopaminergic TH-positive cell bodies from toxin-induced degeneration in the substantia nigra pars compacta (SNpc) and fibers in the striatum. When injected into the striatum, CDNF and GDNF had similar transportation profiles from the striatum to the SNpc; thus CDNF may act via the same nerve tracts as GDNF. Intrastriatal MANF was transported to cortical areas which may reflect a mechanism of neurorestorative action that is different from that of CDNF and GDNF. CDNF and MANF were also shown to distribute more readily than GDNF. In conclusion, CDNF and MANF are potential therapeutic proteins for the treatment of PD.

Putative neuroprotective compounds in in vitro models of dopaminergic neurodegeneration

Parkinson´s disease (PD) is a debilitating age-related neurological disorder that affects various motor skills and can lead to a loss of cognitive functions. The motor symptoms are the result of the progressive degeneration of dopaminergic neurons within the substantia nigra. The factors that influence the pathogenesis and the progression of the neurodegeneration remain mostly unclear. This study investigated the role of various programmed cell death (PCD) pathways, oxidative stress, and glial cells both in dopaminergic neurodegeneration and in the protective action of various drugs. To this end, we exposed dopaminergic neuroblastoma cells (SH-SY5Y cells) to 6-OHDA, which produces oxidative stress and activates various PCD modalities that result in neuronal degeneration. Additionally, to explore the role of glia, we prepared rat midbrain primary mixed-cell cultures containing both neurons and glial cell types such as microglia and astroglia and then exposed the cultures to either MPP plus or lipopolysaccharide. Our results revealed that 6-OHDA activated several PCD pathways including apoptosis, autophagic stress, lysosomal membrane permeabilization, and perhaps paraptosis in SH-SY5Y cells. Furthermore, we found that minocycline protected SH-SY5Y cells from 6-OHDA by inhibiting both apoptotic and non-apoptotic PCD modalities. We also observed an inconsistent neuroprotective effect of various dietary anti-oxidant compounds against 6-OHDA toxicity in vitro in SH-SY5Y cells. Specifically, quercetin and curcumin exerted neuroprotection only within a narrow concentration range and a limited time frame, whereas resveratrol and epigallocatechin 3-gallate provided no protection whatsoever. Lastly, we found that molecules such as amantadine may delay or even halt the neurodegeneration in primary cell cultures by inhibiting the release of neurotoxic factors from overactivated microglia and by enhancing the pro-survival actions of astroglia. Together these data suggest that the strategy of dampening oxidative species with anti-oxidants is less effective than preventing the production of toxic factors such as oxidative and pro-inflammatory molecules by pathologically activated microglia. This would subsequently prevent the activation of various PCD modalities that cause neuronal degeneration.

Role of GDNF and its Cross-Talk with Other Growth Factors in the Dopaminergic System

Parkinson´s Disease (PD) is a neurodegenerative movement disorder resulting from loss of dopaminergic (DA) neurons in substantia nigra (SN). Possible causative treatment strategies for PD include neurotrophic factors, which protect and in some cases restore the function of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors have been to date the most promising candidates for treatment of PD, demonstrating both neuroprotective and neurorestorative properties. We have investigated the role of GDNF in the rodent dopaminergic system and its possible crosstalk with other growth factors. We characterized the GDNF-induced gene expression changes by DNA microarray analysis in different neuronal systems, including in vitro cultured Neuro2A cells treated with GDNF, as well as midbrains from GDNF heterozygous (Hz) knockout mice. These microarray experiments, resulted in the identification of GDNF-induced genes, which were also confirmed by other methods. Further analysis of the dopaminergic system of GDNF Hz mice demonstrated about 40% reduction in GDNF levels, revealed increased intracellular dopamine concentrations and FosB/DeltaFosB expression in striatal areas. These animals did not show any significant changes in behavioural analysis of acute and repeated cocaine administration on locomotor activity, nor did they exhibit any changes in dopamine output following treatment with acute cocaine. We further analysed the significance of GDNF receptor RET signalling in dopaminergic system of MEN2B knock-in animals with constitutively active Ret. The MEN2B animals showed a robust increase in extracellular dopamine and its metabolite levels in striatum, increased tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels by immunohistochemical staining and Western blotting, as well as increased Th mRNA levels in SN. MEN2B mice had increased number of DA neurons in SN by about 25% and they also exhibited increased sensitivity to the stimulatory effects of cocaine. We also developed a semi-throughput in vitro micro-island assay for the quantification of neuronal survival and TH levels by computer-assisted methodology from limited amounts of tissue. This assay can be applied for the initial screening for dopaminotrophic molecules, as well as chemical drug library screening. It is applicable to any neuronal system for the screening of neurotrophic molecules. Since our microarray experiments revealed possible GDNF-VEGF-C crosstalk we further concentrated on studying the neurotrophic effects of VEGF-C. We showed that VEGF-C acts as a neurotrophic molecule for the DA neurons both in vitro and in vivo, however without additive effect when used together with GDNF. The neuroprotective effect for VEGF-C in vivo in rat 6-OHDA model of PD was demonstrated. The possible signalling mechanisms of VEGF-C in the nervous system were investigated - infusion of VEGF-C to rat brain induced ERK activation, however no direct activation of RET signalling in vitro was found. VEGF-C treatment of rat striatum lead to up-regulation of VEGFR-1-3, indicating that VEGF-C can regulate the expression level of its own receptor. VEGF-C dopaminotrophic activity in vivo was further supported by increased vascular tissue in the neuroprotection experiments.

Determinants of vascular cognitive impairment : White matter lesions, brain atrophy and their neuropsychological correlates

The concept of vascular cognitive impairment (VCI) covers a wide spectrum of cognitive dysfunctions related to cerebrovascular disease. Among the pathophysiological determinants of VCI are cerebral stroke, white matter lesions and brain atrophy, which are known to be important risk factors for dementia. However, the specific mechanisms behind the brain abnormalities and cognitive decline are still poorly understood. The present study investigated the neuropsychological correlates of particular magnetic resonance imaging (MRI) findings, namely, medial temporal lobe atrophy (MTA), white matter hyperintensities (WMH), general cortical atrophy and corpus callosum (CC) atrophy in subjects with cerebrovascular disease. Furthermore, the cognitive profile of subcortical ischaemic vascular disease (SIVD) was examined. This study was conducted as part of two large multidisciplinary study projects, the Helsinki Stroke Aging Memory (SAM) Study and the multinational Leukoaraiosis and Disability (LADIS) Study. The SAM cohort consisted of 486 patients, between 55 and 85 years old, with ischaemic stroke from the Helsinki University Hospital, Helsinki, Finland. The LADIS Study included a mixed sample of subjects (n=639) with age-related WMH, between 65 and 84 years old, gathered from 11 centres around Europe. Both studies included comprehensive clinical and neuropsychological assessments and detailed brain MRI. The relationships between the MRI findings and the neuropsychological test performance were analysed by controlling for relevant confounding factors such as age, education and other coexisting brain lesions. The results revealed that in elderly patients with ischaemic stroke, moderate to severe MTA was specifically related to impairment of memory and visuospatial functions, but mild MTA had no clinical relevance. Instead, WMH were primarily associated with executive deficits and mental slowing. These deficits mediated the relationship between WMH and other, secondary cognitive deficits. Cognitive decline was best predicted by the overall degree of WMH, whereas the independent contribution of regional WMH measures was low. Executive deficits were the most prominent cognitive characteristic in SIVD. Compared to other stroke patients, the patients with SIVD also presented more severe memory deficits, which were related to MTA. The cognitive decline in SIVD occurred independently of depressive symptoms and, relative to healthy control subjects, it was substantial in severity. In stroke patients, general cortical atrophy also turned out to be a strong predictor of cognitive decline in a wide range of cognitive domains. Moreover, in elderly subjects with WMH, overall CC atrophy was related to reduction in mental speed, while anterior CC atrophy was independently associated with frontal lobe-mediated executive functions and attention. The present study provides cross-sectional evidence for the involvement of WMH, MTA, general cortical atrophy and CC atrophy in VCI. The results suggest that there are multifaceted pathophysiological mechanisms behind VCI in the elderly, including both vascular ischaemic lesions and neurodegenerative changes. The different pathological changes are highly interrelated processes and together they may produce cumulative effects on cognitive decline.

Kuva tikkeinä kankaalla : Kokemuksia PE-design 6.0 -kirjontakuvion suunnitteluohjelman kahden automaattisen tekniikan käytettävyydestä

The aim of the thesis was to compare the correspondence of the outcome a computer assisted program appearance compared to the original image. The aspect of the study was directed to embroidery with household machines. The study was made from the usability point of view with Brother's PE-design 6.0 embroidery design programs two automatic techniques; multicoloured fragment design and multicoloured stitch surface design. The study's subject is very current because of the fast development of machine embroidery. The theory is based on history of household sewing machines, embroidery sewing machines, stitch types in household sewing machines, embroidery design programs as well as PE-design 6.0 embroidery design program's six automatic techniques. Additionally designing of embroidery designs were included: original image, digitizing, punching, applicable sewing threads as well as the connection between embroidery designs and materials used on embroidery. Correspondences of sewn appearances were examined with sewing experimental methods. 18 research samples of five original image were sewn with both techniques. Experiments were divided into four testing stages in design program. Every testing stage was followed by experimental sewing with Brother Super Galaxie 3100D embroidery machine. Experiments were reported into process files and forms made for the techniques. Research samples were analysed on images syntactic bases with sensory perception assessment. Original images and correspondence of the embroidery appearances were analysed with a form made of it. The form was divided into colour and shape assessment in five stage-similarity-scale. Based on this correspondence analysis it can be said that with both automatic techniques the best correspondence of colour and shape was achieved by changing the standard settings and using the makers own thread chart and edited original image. According to the testing made it is impossible to inform where the image editing possibilities of the images are sufficient or does the optimum correspondence need a separate program. When aiming at correspondence between appearances of two images the computer is unable to trace by itself the appearance of the original image. Processing a computer program assisted embroidery image human perception and personal decision making are unavoidable.

Lukeminen ja kirjallisuus modernissa opiskeluympäristössä : Tapaustutkimus kuuden peruskoulun 4.-6. luokan telemaattinen kirjallisuuspiiri Matilda -hankkeesta

Tutkimuksen tavoitteena oli kartoittaa ja kehittää modernia opiskeluympäristöä hyödyntävää kirjallisuuspiiritoimintaa ja sitä kautta edistää oppilaiden mahdollisuuksia osallistua elämyksellistä lukemista painottavaan kirjallisuuspiiriin sekä vahvistaa heidän kiinnostustaan tieto- ja viestintätekniikan käyttöön. Tutkimusaineisto kerättiin lukuvuonna 1997 1998 kyselyin ja haastatteluin sekä observoimalla lähiopetustilanteita ja virtuaaliryhmien sähköpostiviestintää. Lisäksi käytettiin valmiita dokumentteja ja asiakirjoja. Tutkimusjoukkona oli Matildaan osallistuneet 4.-6. luokan oppilaat kuudelta Espoon ala-asteelta sekä neljä tutoria, koordinaattori ja joitakin luokanopettajia Matilda-kouluista. Tutkimusongelmat suuntautuivat pääasiassa matildalaisten kokemuksiin opiskelusta modernissa opiskeluympäristössä, suhteesta tieto- ja viestintätekniikkaan sekä siihen, mitä uutta lukemiseen ja kirjallisuuteen liittyvää he kertoivat oppineensa ja kokeneensa. Koska tutkimuskohteen kaltaisia kokeiluita, joissa yhdistyy kirjallisuuspiiritoiminta ja moderni tieto- ja viestintätekniikka, ei ainakaan Suomessa ole aikaisemmin ollut, päädyttiin tutkimusmetodologian valinnassa kvalitatiiviseen otteeseen. Tutkimusote noudatti tapaustutkimuksen ja kehittämistutkimuksen periaatteita. Vaikka tutkimus oli menetelmältään kvalitatiivinen, aineistoa käsiteltiin myös kvantifioiden, mutta yleistyksiä tehtiin ainoastaan tapauksen suuntaan. Tutkimustulokset osoittivat, että opiskelu oli oppilaille positiivinen kokemus. He pitivät erityisesti etäopiskelun itsenäisestä työskentelystä ja lähiopetuspäivistä. Heidän suhtautumisensa tieto- ja viestintätekniikkaan muuttui positiivisemmaksi ja heidän taitonsa tieto- ja viestintätekniikan käyttäjinä kehittyivät. Oppilaat saivat positiivisia lukukokemuksia, heidän lukualueensa laajeni ja osa heistä kertoi lukutyylinsä muutoksista ja syvenemisestä. Oppilaat kokivat ongelmallisimmaksi hankkeen sekavan tutorointi-systeemin, kiireisen aikataulun sekä tietokoneisiin ja -verkkoon liittyvät ongelmat. kirjallisuuspiiri, kolmilukutaito, lukutaito, kirjallisuus, lukeminen, elämyksellinenlukeminen, kirjallisuudenopetus, moderni opiskeluympäristö, avoin ja joustava opiskeluympäristö, telematiikka, tieto- ja viestintätekniikka reading circle, teaching of literature, tri-literacy, literacy, reading, literature, aestheticreading, modern learning enviroment, open anf flexible learning enviroment, telematics, modern information technology

Arresting occlusal enamel caries lesions with pit and fissure sealants

The aim of this study was to evaluate the feasibility of pit and fissure sealants and the effectiveness of the two sealant methods applied in every-day practice in public dental health care in Finland. Two sealant methods were evaluated according to their effectiveness in preventing dentin caries and sealant retention. Application time with these sealant methods was compared. The survival rate of sealed first and second molars was followed for nine and 13 year periods, respectively. Caries risk evaluation and observed increased caries risk were the basis for considering sealant application. A questionnaire, sent to all public dental health centers in Finland, monitored the attitudes of the dental profession towards sealant application and explored the current policies used as well as changes noted in the sealant application protocol. DMFT (Decayed, Missing or Filled Teeth) index values collected from the health centers were evaluated. The difference in caries rate between two methods investigated was highly significant. When compared to the glass ionomer sealant method (GIC), the effectiveness of the resin-based method (RB) in preventing dentin caries was 74% and the rate difference 3%. The relative risk for RB-sealed surfaces vs. GIC-sealed surfaces of having detectable dentin caries was 0.3 (95% CI 0.12, 0.57). The retention rate of sealants was higher with RB than GIC (P<0.001). Application of RB sealant material was less time-consuming than application of GIC sealant. Occlusal dentin caries lesions were found in 4% and proximal caries in less than 2% of sealed teeth. The majority of respondents reported application of sealants on a systematic basis along with caries-risk evaluation. Those health centers sealing over suspected or detected enamel caries had lower average DMFT index values (1.0) when compared to DMFT values (1.2) of health centers applying sealants by alternative criteria. It is concluded that the RB sealant method is more effective than the GIC sealant method in preventing dentin caries. Sealant maintenance may increase the costs of a sealant program. Occlusal caries management may be improved if the applied sealant policies are changed towards an interceptive approach i.e. applying the sealants over detected or suspected enamel caries lesions instead of sealing sound teeth in a preventive manner.

The Role Of Fumarase (FH) In Tumorigenesis

In this study, a predisposing gene for a recently characterized cancer syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC), was identified and the role of the gene was investigated in other familial cancers and in nonsyndromic tumorigenesis. HLRCC is a dominantly inherited disorder predisposing predominantly to uterine and skin leiomyomas, and also to renal cell cancer and uterine leiomyosarcoma. The disease gene was recently localized in Finnish families to 1q42-q43 by a genome-wide linkage search. Independently in the UK, a clinically similar condition, multiple cutaneous and uterine leiomyomata (MCUL), was linked to the same chromosomal region, strongly suggesting that HLRCC and MCUL are actually a single syndrome. Linkage results were confirmed by detecting loss of heterozygosity (LOH) at the disease locus in most of the patients' tumors, suggesting that this predisposing gene acts as a tumor suppressor. Through detailed investigation by genotyping of microsatellite markers and haplotype construction in Finnish and UK HLRCC/MCUL families we were able to narrow the disease locus down to 1.6 Mb. Extensive mutation screening of known and predicted transcripts in the target region resulted in identification of the HLRCC predisposing gene, fumarase (fumarate hydratase, FH). FH is a key enzyme in energy metabolism, catalyzing fumarate to malate in the tricarboxylic acid cycle (TCAC) in mitochondria. Germline alterations in FH segregating with the disease were detected in 25 of 42 HLRCC/MCUL families including whole-gene deletions, truncating small deletions/insertions and nonsense mutations, as well as substitutions or deletions of highly conserved amino acids. Biallelic inactivation was detected in almost all studied tumors of HLRCC patients. Furthermore, FH enzyme activity was reduced in the patients' normal tissues and was completely or virtually absent from tumors. Based on these findings, we extensively demonstrated that mutations in FH underlie the HLRCC/MCUL syndrome. In our studies of other familial cancers, evidence for involvement of FH defects was not found in familial prostate and breast cancers. To investigate the role of FH in sporadic tumorigenesis, we analyzed 652 lesions, including a series of 353 nonsyndromic counterparts of tumor types associated with HLRCC. Mutations in nonsyndromic tumors were rare and appeared to be limited to tumor types observed in the hereditary form of the disease. Biallelic inactivation of FH was detected in a uterine leiomyosarcoma, a cutaneous leiomyoma, a soft-tissue sarcoma, and in two uterine leiomyomas. In the uterine leiomyosarcoma and the cutaneous lesion FH mutations originated from the germline whereas the soft-tissue sarcoma harbored purely somatic changes. In uterine leiomyomas somatic mutations were detected in the two out of five tumors with LOH at the FH locus. Our findings demonstrate that FH inactivation is also involved in nonhereditary tumor development, and further support the hypothesis that FH acts as a tumor suppressor. The role of FH in predisposition to malignancies, renal cell carcinoma and leiomyosarcoma is important in the diagnosis and prevention of cancer among HLRCC patients. This study is of general clinical interest, because prior to our findings, little was known about the molecular genetics of uterine leiomyomas, the most common tumors of women.

Human herpesvirus 6 in multiple sclerosis and encephalitis

Human herpesvirus 6 (HHV-6) was identified from patients with HIV and lymphoproliferative diseases in 1986. It is a β-herpesvirus and is divided into two subgroups, variants A and B. HHV-6 variant B is the cause of exanthema subitum, while variant A has not yet definitely proven to cause any disease. HHV-6, especially variant A, is a highly neurotropic virus and has been associated with many diseases of the central nervous system (CNS) such as encephalitis and multiple sclerosis (MS). The present studies were aimed to elucidate the role of HHV-6 and its two variants in neurological infections. Special attention was given to study the possible role of HHV-6 in the pathogenesis of MS. We studied the expression of HHV-6 antigens using immunohistochemistry in brain autopsy samples from patients with MS and controls. HHV-6 antigen was identified in 70% of MS specimens whereas 30% of control specimens expressed HHV-6 antigen. Serum and cerebrospinal fluid (CSF) samples were collected from patients with MS and patients with other neurological diseases (OND) from patients visiting Helsinki University Central Hospital Neurological Outpatient Clinic during the years 2003 and 2004. In addition, we studied 53 children with suspected encephalitis. We developed an immunofluorescence IgG-avidity assay for the detection of primary HHV-6A and HHV-6B infection. For HHV-6B antibodies, no differences were observed between patients with MS and OND. For HHV-6A both seroprevalence and mean titers were significantly higher in MS compared to OND. HHV-6A low-avidity IgG antibodies, suggestive of primary infection, were found in serum of two, three and one patient with definite MS, possible MS and OND, respectively. From pediatric patients with suspected encephalitis, six serum samples (11.3%) contained low-avidity antibodies, indicating a temporal association between HHV-6A infection and onset of encephalitis. Three out of 26 patients with CDMS and four out of 19 patients with CPMS had HHV-6 antibodies in their CSF compared to none of the patients with OND (p=0.06 and p=0.01, respectively). Two patients with CDMS and three patients with CPMS appeared to have specific intrathecal synthesis of HHV-6A antibodies. In addition, oligoclonal bands (OCB) were observed in the CSF of five out of nine MS patients tested, and in two the OCBs reacted specifically with HHV-6 antigen, which is a novel finding. These results indicate HHV-6 specific antibody production in the CNS and suggest that there is a subset of MS patients with an active or chronic HHV-6A infection in the CNS that might be involved in the pathogenesis of MS. Our studies suggest that HHV-6 is an important causative or associated virus in some neurological infections, such as encephalitis and it might contribute to the development of MS, at least in some cases. In conclusion, HHV-6 is a neurotropic virus that should be taken into consideration when studying acute and chronic CNS diseases of unknown origin.

Sindbis virus and Pogosta disease in Finland

Sindbis virus (SINV) (genus Alphavirus, family Togaviridae) is an enveloped virus with a genome of single-stranded, positive-polarity RNA of 11.7 kilobases. SINV is widespread in Eurasia, Africa, and Australia, but clinical infection only occurs in a few geographically restricted areas, mainly in Northern Europe. In Europe, antibodies to SINV were detected from patients with fever, rash, and arthritis for the first time in the early 1980s in Finland. It became evident that the causative agent of this syndrome, named Pogosta disease, was closely related to SINV. The disease is also found in Sweden (Ockelbo disease) and in Russia (Karelian fever). Since 1974, for unknown reason, the disease has occurred as large outbreaks every seven years in Finland. This study is to a large degree based on the material collected during the 2002 Pogosta disease outbreak in Finland. We first developed SINV IgM and IgG enzyme immunoassays (EIA), based on highly purified SINV, to be used in serodiagnostics. The EIAs correlated well with the hemagglutination inhibition (HI) test, and all individuals showed neutralizing antibodies. The sensitivities of the IgM and IgG EIAs were 97.6% and 100%, and specificities 95.2% and 97.6%, respectively. E1 and E2 envelope glycoproteins of SINV were shown to be recognized by IgM and IgG in the immunoblot early in infection. We isolated SINV from five patients with acute Pogosta disease; one virus strain was recovered from whole blood, and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, also representing the first human SINV isolates from Europe. Phylogenetic analysis indicated that the Finnish SINV strains clustered with the strains previously isolated from mosquitoes in Sweden and Russia, and seemed to have a common ancestor with South-African strains. Northern European SINV strains could be maintained locally in disease-endemic regions, but the phylogenetic analysis also suggests that redistribution of SINV tends to occur in a longitudinal direction, possibly with migratory birds. We searched for SINV antibodies in resident grouse (N=621), whose population crashes have previously coincided with human SINV outbreaks, and in migratory birds (N=836). SINV HI antibodies were found for the first time in birds during their spring migration to Northern Europe, from three individuals: red-backed shrike, robin, and song thrush. Of the grouse, 27.4% were seropositive in 2003, one year after a human outbreak, but only 1.4% of the grouse were seropositive in 2004. Thus, grouse might contribute to the human epidemiology of SINV. A total of 86 patients with verified SINV infection were recruited to the study in 2002. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in eight patients. IgM antibodies became detectable within the first eight days of illness, and IgG within 11 days. The acute phase of Pogosta disease was characterized by arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. Half of the patients reported in self-administered questionnaires joint symptoms to last > 12 months. Physical examination in 49 of these patients three years after infection revealed persistent joint manifestations. Arthritis (swelling and tenderness in physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness in palpation or in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, and additional 10.2% complained persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection three years earlier. A positive IgM antibody response persisted in 3/49 of the patients; both two patients with arthritis were in this group. Persistent symptoms of SINV infection might have considerable public health implications in areas with high seroprevalence. The age-standardized seroprevalence of SINV (1999-2003, N=2529) in the human population in Finland was 5.2%. The seroprevalence was high in North Karelia, Kainuu, and Central Ostrobothnia. The incidence was highest in North Karelia. Seroprevalence in men (6.0%) was significantly higher than in women (4.1%), however, the average annualized incidence in the non-epidemic years was higher in women than in men, possibly indicating that infected men are more frequently asymptomatic. The seroprevalence increased with age, reaching 15.4% in persons aged 60-69 years. The incidence was highest in persons aged 50-59 years.

CMV-, EBV-, and HHV-6-DNAemia after liver transplantation

Viral infections caused by herpesviruses are common complications after organ transplantation and they are associated with substantial morbidity and even mortality. Herpesviruses remain in a latent state in a host after primary infection and may reactivate later. CMV infection is the most important viral infection after liver transplantation. Less is known about the significance of human herpesvirus-6 (HHV-6). EBV is believed to play a major role in the development of post-transplant lymphoproliferative disorders (PTLD). The aim of this study was to investigate the CMV-, EBV- and HHV-6 DNAemia after liver transplantation by frequent monitoring of adult liver transplant patients. The presence of CMV, EBV and HHV-6 DNA were demonstrated by in situ hybridization assays and by real-time PCR methods from peripheral blood specimens. CMV and HHV-6 antigens were demonstrated by antigenemia assays and compared to the viral DNAemia. The response to antiviral therapy was also investigated. CMV-DNAemia appeared earlier than CMV pp65-antigenemia after liver transplantation. CMV infections were treated with ganciclovir. However, most of the treated patients demonstrated persistence of CMV-DNA for up to several months. Continuous CMV-DNA expression of peripheral blood leukocytes showed that the virus is not eliminated by ganciclovir and recurrences can be expected during several months after liver transplantation. HHV-6 DNAemia / antigenemia was common and occurred usually within the first three months after liver transplantation together with CMV. The HHV-6 DNA expression in peripheral blood mononuclear cells correlated well with HHV-6 antigenemia. Antiviral treatment significantly decreased the number of HHV-6 DNA positive cells, demonstrating the response to ganciclovir treatment. Clinically silent EBV reactivations with low viral loads were relatively common after liver transplantation. These EBV-DNAemias usually appeared within the first three months after liver transplantation together with betaherpesviruses (CMV, HHV-6, HHV-7). One patient developed high EBV viral loads and developed PTLD. These results indicate that frequent monitoring of EBV-DNA levels can be useful to detect liver transplant patients at risk of developing PTLD.

Obstetric and gynaecological aspects of HIV infection

The purpose of the present study was to examine the outcome of pregnancies among HIV-infected women in Helsinki, use of the levonorgestrel-releasing intrauterine system (LNG-IUS) among HIV-infected women and the prevalence and risk factors of cytological and histologically proven cervical lesions in this population. Between 1993 and 2003 a total of 45 HIV-infected women delivered 52 singleton infants. HIV infection was diagnosed during pregnancy in 40% of the mothers. Seventeen of the mothers received antiretroviral (ARV) medication prior to pregnancy and in 34 cases, the medication was started during pregnancy. A good virological response (i.e. HIV RNA load <1000/mL during the last trimester) to ARV medication was achieved in 36/40 (90%) of the patients in whom HI viral load measurements were performed. Of the infants, 92% were born at term, and their mean (±SD) birth weight was 3350±395 g. The Caesarean section rate was low, 25%. All newborns received ARV medication and none of the infants born to mothers with pre-delivery diagnosis of maternal HIV infection were infected. The safety and advantages of the LNG-IUS were studied prospectively (n=12) and retrospectively (n=6). The LNG-IUS was well tolerated and no cases of PID or pregnancy were noted. Menstrual bleeding was reduced significantly during use of the LNG-IUS; this was associated with a slight increase in haemoglobin levels. Serum oestradiol concentrations remained in the follicular range in all subjects. The key finding was that genital shedding of HIV RNA did not change after the insertion of the LNG-IUS. The mean annual prevalence of low-grade squamous intraepithelial lesions (SIL) was 15% and that of high-grade SIL was 5% among 108 systematically followed HIV-infected women during 1989 2003. A reduced CD4 lymphocyte count was associated with an increased prevalence of SIL, whereas duration of HIV infection, use of ARV medication and HI viral load were not. The cumulative risk of any type of SIL was 17% after one year and 48% after five years among patients with initially normal Pap smears. The risk of developing SIL was associated with young age and a high initial HI viral load. During the follow-up 51 subjects (n=153) displayed cervical intraepithelial neoplasia (CIN), (16% CIN1 and 18% CIN 2-3). Only one case of cancer of the uterine cervix was detected. Pap smears were reliable in screening for CIN. Both nulliparity (p<0.01) and bacterial vaginosis (p<0.04) emerged as significant risk factors of CIN. In conclusion, a combination of universal antenatal screening and multidisciplinary management allows individualized treatment and prevents vertical transmission of HIV. Use of the LNG-IUS is safe among HIV-infected women and cervicovaginal shedding of HIV RNA is not affected by use of the LNG-IUS. The risk of cervical pre-malignant lesions is high among HIV-infected women despite systematic follow-up.

Neuromagnetic studies on cortical somatosensory functions in infants and children : Normal development and effect of early brain lesions

Until recently, objective investigation of the functional development of the human brain in vivo was challenged by the lack of noninvasive research methods. Consequently, fairly little is known about cortical processing of sensory information even in healthy infants and children. Furthermore, mechanisms by which early brain insults affect brain development and function are poorly understood. In this thesis, we used magnetoencephalography (MEG) to investigate development of cortical somatosensory functions in healthy infants, very premature infants at risk for neurological disorders, and adolescents with hemiplegic cerebral palsy (CP). In newborns, stimulation of the hand activated both the contralateral primary (SIc) and secondary somatosensory cortices (SIIc). The activation patterns differed from those of adults, however. Some of the earliest SIc responses, constantly present in adults, were completely lacking in newborns and the effect of sleep stage on SIIc responses differed. These discrepancies between newborns and adults reflect the still developmental stage of the newborns’ somatosensory system. Its further maturation was demonstrated by a systematic transformation of the SIc response pattern with age. The main early adult­like components were present by age two. In very preterm infants, at term age, the SIc and SIIc were activated at similar latencies as in healthy fullterm newborns, but the SIc activity was weaker in the preterm group. The SIIc response was absent in four out of the six infants with brain lesions of the underlying hemisphere. Determining the prognostic value of this finding remains a subject for future studies, however. In the CP adolescents with pure subcortical lesions, contrasting their unilateral symptoms, the SIc responses of both hemispheres differed from those of controls: For example the distance between SIc representation areas for digits II and V was shorter bilaterally. In four of the five CP patients with cortico­subcortical brain lesions, no normal early SIc responses were evoked by stimulation of the palsied hand. The varying differences in neuronal functions, underlying the common clinical symptoms, call for investigation of more precisely designed rehabilitation strategies resting on knowledge about individual functional alterations in the sensorimotor networks.