Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Human parvovirus infections during pregnancy : Special reference to the child-care employees

Human parvovirus B19 (B19V) is known to cause anemia, hydrops fetalis, and fetal death especially during the first half of pregnancy. Women who are in occupational contact with young children are at increased risk of B19V infection. The role of the recently discovered human parvovirus, human bocavirus (HBoV), in reproduction is unknown. The aim of this research project was to establish a scientific basis for assessing the work safety of pregnant women and for issuing special maternity leave regulations during B19V epidemics in Finland. The impact of HBoV infection on the pregnant woman and her fetus was also defined. B19V DNA was found in 0.8% of the miscarriages and in 2.4% of the intrauterine fetal death (IUFD; fetal death after completed 22 gestational weeks). All control fetuses (from induced abortions) were B19V-DNA negative. The findings on hydropic B19V DNA-positive IUFDs with evidence of acute or recent maternal B19V infection are in line with those of previous Swedish studies. However, the high prevalence of B19V-related nonhydropic IUFDs noted in the Swedish studies was mostly without evidence of maternal B19V infection and was not found during the third trimester. HBoV was not associated with miscarriages or IUFDs. Almost all of the studied pregnant women were HboV-IgG positive, and thus most probably immune to HBoV. All preterm births, perinatal deaths, smallness for gestational age (SGA) and congenital anomaly were recorded among the infants of child-care employees in a nationwide register-based cohort study over a period of 14 years. Little or no differences in the results were found between the infants of the child-care employees and those of the comparison group. The annual B19V seroconversion rate was over two-fold among the child-care employees, compared to the women in the comparison group. The seropositivity of the child-care employees increased with age, and years from qualification/joining the trade union. In general, the child-care employees are not at increased risk for adverse pregnancy outcome. However, at the population level, the risk of rare events, such as adverse pregnancy outcomes attributed to infections, could not be determined. According to previous studies, seronegative women had a 5 10% excess risk of losing the fetus during the first half of their pregnancy, but thereafter the risk was very low. Therefore, an over two-fold increased risk of B19V infection among child-care employees is considerable, and should be taken into account in the assessment of the occupational safety of pregnant women, especially during the first half of their pregnancy.

Stereochemical and steric control of enzymatic glucuronidation : A rational approach for the design of novel Inhibitors for the human UDP-glucuronosyltransferase 2B7

The UDP-glucuronosyltransferases (UGTs) are enzymes of the phase II metabolic system. These enzymes catalyze the transfer of α-D-glucuronic acid from UDP-glucuronic acid to aglycones bearing nucleophilic groups affording exclusively their corresponding β-D-glucuronides to render lipophilic endobiotics and xenobiotics more water soluble. This detoxification pathway aids in the urinary and biliary excretion of lipophilic compounds thus preventing their accumulation to harmful levels. The aim of this study was to investigate the effect of stereochemical and steric features of substrates on the glucuronidation catalyzed by UGTs 2B7 and 2B17. Furthermore, this study relates to the design and synthesis of novel, selective inhibitors that display high affinity for the key enzyme involved in drug glucuronidation, UGT2B7. The starting point for the development of inhibitors was to assess the influence of the stereochemistry of substrates on the UGT-catalyzed glucuronidation reaction. A set of 28 enantiomerically pure alcohols was subjected to glucuronidation assays employing the human UGT isoforms 2B7 and 2B17. Both UGT enzymes displayed high stereoselectivity, favoring the glucuronidation of the (R)-enantiomers over their respective mirror-image compounds. The spatial arrangement of the hydroxy group of the substrate determined the rate of the UGT-catalyzed reaction. However, the affinity of the enantiomeric substrates to the enzymes was not significantly influenced by the spatial orientation of the nucleophilic hydroxy group. Based on these results, a rational approach for the design of inhibitors was developed by addressing the stereochemical features of substrate molecules. Further studies showed that the rate of the enzymatic glucuronidation of substrates was also highly dependent on the steric demand in vicinity of the nucleophilic hydroxy group. These findings provided a rational approach to turn high-affinity substrates into true UGT inhibitors by addressing stereochemical and steric features of substrate molecules. The tricyclic sesquiterpenols longifolol and isolongifolol were identified as high-affinity substrates which displayed high selectivity for the UGT isoform 2B7. These compounds served therefore as lead structures for the design of potent and selective inhibitors for UGT2B7. Selective and potent inhibitors were prepared by synthetically modifying the lead compounds longifolol and isolongifolol taking stereochemical and steric features into account. The best inhibitor of UGT2B7, β-phenyllongifolol, displayed an inhibition constant of 0.91 nM.

Environmental change and human history in the Jabal Harun area, Jordan

The research is related to the Finnish Jabal Harun Project (FJHP), which is part of the research unit directed by Professor Jaakko Frösén. The project consists of two interrelated parts: the excavation of a Byzantine monastery/pilgrimage centre on Jabal Harun, and a multiperiod archaeological survey of the surrounding landscape. It is generally held that the Near Eastern landscape has been modified by millennia of human habitation and activity. Past climatic changes and human activities could be expected to have significantly changed also the landscape of the Jabal Harun area. Therefore it was considered that a study of erosion in the Jabal Harun area could shed light on the environmental and human history of the area. It was hoped that it would be possible to connect the results of the sedimentological studies either to wider climatic changes in the Near East, or to archaeologically observable periods of human activity and land use. As evidence of some archaeological periods is completely missing from the Jabal Harun area, it was also of interest whether catastrophic erosion or unfavourable environmental change, caused either by natural forces or by human agency, could explain the gaps in the archaeological record. Changes in climate and/or land-use were expected to be reflected in the sedimentary record. The field research, carried out as part of the FJHP survey fieldwork, included the mapping of wadi terraces and cleaning of sediment profiles which were recorded and sampled for laboratory analyses of facies and lithology. To obtain a chronology for the sedimentation and erosion phases also OSL (optically stimulated luminescence) dating samples were collected. The results were compared to the record of the Near Eastern palaeoclimate, and to data from geoarchaeological studies in central and southern Jordan. The picture of the environmental development was then compared to the human history in the area, based on archaeological evidence from the FJHP survey and the published archaeological research in the Petra region, and the question of the relationship between human activity and environmental change was critically discussed. Using the palaeoclimatic data and the results from geoarchaeological studies it was possible to outline the environmental development in the Jabal Harun area from the Pleistocene to the present.It is appears that there was a phase of accumulation of sediment before the Middle Palaeolithic period, possibly related to tectonic movement. This phase was later followed by erosion, tentatively suggested to have taken place during the Upper Palaeolithic. A period of wadi aggradation probably occurred during the Late Glacial and continued until the end of the Pleistocene, followed by significant channel degradation, attributed to increased rainfall during the Early Holocene. It seems that during the later Holocene channel incision has been dominant in the Jabal Harûn area although there have been also small-scale channel aggradation phases, two of which were OSL-dated to around 4000-3000 BP and 2400-2000 BP. As there is no evidence of tectonic movements in the Jabal Harun area after the early Pleistocene, it is suggested that climate change and human activity have been the major causes of environmental change in the area. At a brief glance it seems that many of the changes in the settlement and land use in the Jabal Harun area can be explained by climatic and environmental conditions. However, the responses of human societies to environmental change are dependent on many factors. Therefore an evaluation of the significance of environmental, cultural, socio-economic and political factors is needed to decide whether certain phenomena are environmentally induced. Comparison with the wider Petra region is also needed to judge whether the phenomena are characteristic of the Jabal Harun area only, or can they be connected to social, political and economic development over a wider area.

Studies of working memory in interrupted human-computer interaction

Distraction in the workplace is increasingly more common in the information age. Several tasks and sources of information compete for a worker's limited cognitive capacities in human-computer interaction (HCI). In some situations even very brief interruptions can have detrimental effects on memory. Nevertheless, in other situations where persons are continuously interrupted, virtually no interruption costs emerge. This dissertation attempts to reveal the mental conditions and causalities differentiating the two outcomes. The explanation, building on the theory of long-term working memory (LTWM; Ericsson and Kintsch, 1995), focuses on the active, skillful aspects of human cognition that enable the storage of task information beyond the temporary and unstable storage provided by short-term working memory (STWM). Its key postulate is called a retrieval structure an abstract, hierarchical knowledge representation built into long-term memory that can be utilized to encode, update, and retrieve products of cognitive processes carried out during skilled task performance. If certain criteria of practice and task processing are met, LTWM allows for the storage of large representations for long time periods, yet these representations can be accessed with the accuracy, reliability, and speed typical of STWM. The main thesis of the dissertation is that the ability to endure interruptions depends on the efficiency in which LTWM can be recruited for maintaing information. An observational study and a field experiment provide ecological evidence for this thesis. Mobile users were found to be able to carry out heavy interleaving and sequencing of tasks while interacting, and they exhibited several intricate time-sharing strategies to orchestrate interruptions in a way sensitive to both external and internal demands. Interruptions are inevitable, because they arise as natural consequences of the top-down and bottom-up control of multitasking. In this process the function of LTWM is to keep some representations ready for reactivation and others in a more passive state to prevent interference. The psychological reality of the main thesis received confirmatory evidence in a series of laboratory experiments. They indicate that after encoding into LTWM, task representations are safeguarded from interruptions, regardless of their intensity, complexity, or pacing. However, when LTWM cannot be deployed, the problems posed by interference in long-term memory and the limited capacity of the STWM surface. A major contribution of the dissertation is the analysis of when users must resort to poorer maintenance strategies, like temporal cues and STWM-based rehearsal. First, one experiment showed that task orientations can be associated with radically different patterns of retrieval cue encodings. Thus the nature of the processing of the interface determines which features will be available as retrieval cues and which must be maintained by other means. In another study it was demonstrated that if the speed of encoding into LTWM, a skill-dependent parameter, is slower than the processing speed allowed for by the task, interruption costs emerge. Contrary to the predictions of competing theories, these costs turned out to involve intrusions in addition to omissions. Finally, it was learned that in rapid visually oriented interaction, perceptual-procedural expectations guide task resumption, and neither STWM nor LTWM are utilized due to the fact that access is too slow. These findings imply a change in thinking about the design of interfaces. Several novel principles of design are presented, basing on the idea of supporting the deployment of LTWM in the main task.

Social perception and cognition : processing of gestures, postures and facial expressions in the human brain

Humans are a social species with the internal capability to process social information from other humans. To understand others behavior and to react accordingly, it is necessary to infer their internal states, emotions and aims, which are conveyed by subtle nonverbal bodily cues such as postures, gestures, and facial expressions. This thesis investigates the brain functions underlying the processing of such social information. Studies I and II of this thesis explore the neural basis of perceiving pain from another person s facial expressions by means of functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG). In Study I, observing another s facial expression of pain activated the affective pain system (previously associated with self-experienced pain) in accordance with the intensity of the observed expression. The strength of the response in anterior insula was also linked to the observer s empathic abilities. The cortical processing of facial pain expressions advanced from the visual to temporal-lobe areas at similar latencies (around 300 500 ms) to those previously shown for emotional expressions such as fear or disgust. Study III shows that perceiving a yawning face is associated with middle and posterior STS activity, and the contagiousness of a yawn correlates negatively with amygdalar activity. Study IV explored the brain correlates of interpreting social interaction between two members of the same species, in this case human and canine. Observing interaction engaged brain activity in very similar manner for both species. Moreover, the body and object sensitive brain areas of dog experts differentiated interaction from noninteraction in both humans and dogs whereas in the control subjects, similar differentiation occurred only for humans. Finally, Study V shows the engagement of the brain area associated with biological motion when exposed to the sounds produced by a single human being walking. However, more complex pattern of activation, with the walking sounds of several persons, suggests that as the social situation becomes more complex so does the brain response. Taken together, these studies demonstrate the roles of distinct cortical and subcortical brain regions in the perception and sharing of others internal states via facial and bodily gestures, and the connection of brain responses to behavioral attributes.

Human brain mechanisms of auditory and audiovisual selective attention

Selective attention refers to the process in which certain information is actively selected for conscious processing, while other information is ignored. The aim of the present studies was to investigate the human brain mechanisms of auditory and audiovisual selective attention with functional magnetic resonance imaging (fMRI), electroencephalography (EEG) and magnetoencephalography (MEG). The main focus was on attention-related processing in the auditory cortex. It was found that selective attention to sounds strongly enhances auditory cortex activity associated with processing the sounds. In addition, the amplitude of this attention-related modulation was shown to increase with the presentation rate of attended sounds. Attention to the pitch of sounds and to their location appeared to enhance activity in overlapping auditory-cortex regions. However, attention to location produced stronger activity than attention to pitch in the temporo-parietal junction and frontal cortical regions. In addition, a study on bimodal attentional selection found stronger audiovisual than auditory or visual attention-related modulations in the auditory cortex. These results were discussed in light of Näätänen s attentional-trace theory and other research concerning the brain mechanisms of selective attention.

Dynamics of contour, object and face processing in the human visual cortex

The neural basis of visual perception can be understood only when the sequence of cortical activity underlying successful recognition is known. The early steps in this processing chain, from retina to the primary visual cortex, are highly local, and the perception of more complex shapes requires integration of the local information. In Study I of this thesis, the progression from local to global visual analysis was assessed by recording cortical magnetoencephalographic (MEG) responses to arrays of elements that either did or did not form global contours. The results demonstrated two spatially and temporally distinct stages of processing: The first, emerging 70 ms after stimulus onset around the calcarine sulcus, was sensitive to local features only, whereas the second, starting at 130 ms across the occipital and posterior parietal cortices, reflected the global configuration. To explore the links between cortical activity and visual recognition, Studies II III presented subjects with recognition tasks of varying levels of difficulty. The occipito-temporal responses from 150 ms onwards were closely linked to recognition performance, in contrast to the 100-ms mid-occipital responses. The averaged responses increased gradually as a function of recognition performance, and further analysis (Study III) showed the single response strengths to be graded as well. Study IV addressed the attention dependence of the different processing stages: Occipito-temporal responses peaking around 150 ms depended on the content of the visual field (faces vs. houses), whereas the later and more sustained activity was strongly modulated by the observers attention. Hemodynamic responses paralleled the pattern of the more sustained electrophysiological responses. Study V assessed the temporal processing capacity of the human object recognition system. Above sufficient luminance, contrast and size of the object, the processing speed was not limited by such low-level factors. Taken together, these studies demonstrate several distinct stages in the cortical activation sequence underlying the object recognition chain, reflecting the level of feature integration, difficulty of recognition, and direction of attention.

Human brain networks of auditory attention and working memory

This thesis examines brain networks involved in auditory attention and auditory working memory using measures of task performance, brain activity, and neuroanatomical connectivity. Auditory orienting and maintenance of attention were compared with visual orienting and maintenance of attention, and top-down controlled attention was compared to bottom-up triggered attention in audition. Moreover, the effects of cognitive load on performance and brain activity were studied using an auditory working memory task. Corbetta and Shulman s (2002) model of visual attention suggests that what is known as the dorsal attention system (intraparietal sulcus/superior parietal lobule, IPS/SPL and frontal eye field, FEF) is involved in the control of top-down controlled attention, whereas what is known as the ventral attention system (temporo-parietal junction, TPJ and areas of the inferior/middle frontal gyrus, IFG/MFG) is involved in bottom-up triggered attention. The present results show that top-down controlled auditory attention also activates IPS/SPL and FEF. Furthermore, in audition, TPJ and IFG/MFG were activated not only by bottom-up triggered attention, but also by top-down controlled attention. In addition, the posterior cerebellum and thalamus were activated by top-down controlled attention shifts and the ventromedial prefrontal cortex (VMPFC) was activated by to-be-ignored, but attention-catching salient changes in auditory input streams. VMPFC may be involved in the evaluation of environmental events causing the bottom-up triggered engagement of attention. Auditory working memory activated a brain network that largely overlapped with the one activated by top-down controlled attention. The present results also provide further evidence of the role of the cerebellum in cognitive processing: During auditory working memory tasks, both activity in the posterior cerebellum (the crus I/II) and reaction speed increased when the cognitive load increased. Based on the present results and earlier theories on the role of the cerebellum in cognitive processing, the function of the posterior cerebellum in cognitive tasks may be related to the optimization of response speed.

Feature integration in human vision

The earliest stages of human cortical visual processing can be conceived as extraction of local stimulus features. However, more complex visual functions, such as object recognition, require integration of multiple features. Recently, neural processes underlying feature integration in the visual system have been under intensive study. A specialized mid-level stage preceding the object recognition stage has been proposed to account for the processing of contours, surfaces and shapes as well as configuration. This thesis consists of four experimental, psychophysical studies on human visual feature integration. In two studies, classification image a recently developed psychophysical reverse correlation method was used. In this method visual noise is added to near-threshold stimuli. By investigating the relationship between random features in the noise and observer s perceptual decision in each trial, it is possible to estimate what features of the stimuli are critical for the task. The method allows visualizing the critical features that are used in a psychophysical task directly as a spatial correlation map, yielding an effective "behavioral receptive field". Visual context is known to modulate the perception of stimulus features. Some of these interactions are quite complex, and it is not known whether they reflect early or late stages of perceptual processing. The first study investigated the mechanisms of collinear facilitation, where nearby collinear Gabor flankers increase the detectability of a central Gabor. The behavioral receptive field of the mechanism mediating the detection of the central Gabor stimulus was measured by the classification image method. The results show that collinear flankers increase the extent of the behavioral receptive field for the central Gabor, in the direction of the flankers. The increased sensitivity at the ends of the receptive field suggests a low-level explanation for the facilitation. The second study investigated how visual features are integrated into percepts of surface brightness. A novel variant of the classification image method with brightness matching task was used. Many theories assume that perceived brightness is based on the analysis of luminance border features. Here, for the first time this assumption was directly tested. The classification images show that the perceived brightness of both an illusory Craik-O Brien-Cornsweet stimulus and a real uniform step stimulus depends solely on the border. Moreover, the spatial tuning of the features remains almost constant when the stimulus size is changed, suggesting that brightness perception is based on the output of a single spatial frequency channel. The third and fourth studies investigated global form integration in random-dot Glass patterns. In these patterns, a global form can be immediately perceived, if even a small proportion of random dots are paired to dipoles according to a geometrical rule. In the third study the discrimination of orientation structure in highly coherent concentric and Cartesian (straight) Glass patterns was measured. The results showed that the global form was more efficiently discriminated in concentric patterns. The fourth study investigated how form detectability depends on the global regularity of the Glass pattern. The local structure was either Cartesian or curved. It was shown that randomizing the local orientation deteriorated the performance only with the curved pattern. The results give support for the idea that curved and Cartesian patterns are processed in at least partially separate neural systems.