Long-Term Antibody Response to SARS-CoV-2 in Children.

Almost 2 years into the pandemic and with vaccination of children significantly lagging behind adults, long-term pediatric humoral immune responses to SARS-CoV-2 are understudied. The C19.CHILD Hamburg (COVID-19 Child Health Investigation of Latent Disease) Study is a prospective cohort study designed to identify and follow up children and their household contacts infected in the early 2020 first wave of SARS-CoV-2. We screened 6113 children < 18 years by nasopharyngeal swab-PCR in a low-incidence setting after general lockdown, from May 11 to June 30, 2020. A total of 4657 participants underwent antibody testing. Positive tests were followed up by repeated PCR and serological testing of all household contacts over 6 months. In total, the study identified 67 seropositive children (1.44%); the median time after infection at first presentation was 83 days post-symptom onset (PSO). Follow-up of household contacts showed less than 100% seroprevalence in most families, with higher seroprevalence in families with adult index cases compared to pediatric index cases (OR 1.79, P = 0.047). Most importantly, children showed sustained seroconversion up to 9 months PSO, and serum antibody concentrations persistently surpassed adult levels (ratio serum IgG spike children vs. adults 90 days PSO 1.75, P < 0.001; 180 days 1.38, P = 0.01; 270 days 1.54, P = 0.001). In a low-incidence setting, SARS-CoV-2 infection and humoral immune response present distinct patterns in children including higher antibody levels, and lower seroprevalence in families with pediatric index cases. Children show long-term SARS-CoV-2 antibody responses. These findings are relevant to novel variants with increased disease burden in children, as well as for the planning of age-appropriate vaccination strategies.

Effect of ultraviolet photofunctionalization of dental titanium implants on osseointegration.

OBJECTIVE: The aim of the current study was to evaluate the effect of ultraviolet (UV) photofunctionalization of dental titanium implants with exposure to the oral cavity on osseointegration in an animal model. METHODS: Forty-eight titanium implants (Camlog® Conelog® 4.3 mmx9.0 mm) were placed epicrestally into the edentulous jaws of three minipigs and implant stability was assessed by measuring the implant stability quotient (ISQ). Prior to implantation half of the implants were photofunctionalized with intense UV-light. After three months, the implants were exposed and ISQ was measured again. After six months of implant exposure, the minipigs were sacrificed and the harvested specimens were analyzed using histomorphometric, light, and fluorescence microscopy. MAIN RESULTS: Forty-two of 48 implants osseointegrated. The overall mean bone-implant contact area (BIC) was (64±22)%. No significant differences were found in BIC or ISQ value (multivariate analysis of variance (MANOVA), P>0.05) between implants with and without exposure to UV photofunctionalization. CONCLUSIONS: No significant effects were observed on osseointegration of dental titanium implants nine months after exposure of UV photofunctionalization.

Replication of Merkel cell polyomavirus induces reorganization of promyelocytic leukemia nuclear bodies.

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC), a rare but aggressive skin cancer. The virus is highly prevalent: 60-80 % of adults are seropositive; however, cells permissive for MCPyV infection are unknown. Consequently, very little information about the MCPyV life cycle is available. Until recently, MCPyV replication could only be studied using a semi-permissive in vitro replication system (Neumann et al., 2011; Feng et al., 2011, Schowalter et al., 2011). MCPyV replication most likely depends on subnuclear structures such as promyelocytic leukemia protein nuclear bodies (PML-NBs), which are known to play regulatory roles in the infection of many DNA viruses. Here, we investigated PML-NB components as candidate host factors to control MCPyV DNA replication. We showed that PML-NBs change in number and size in cells actively replicating MCPyV proviral DNA. We observed a significant increase in PML-NBs in cells positive for MCPyV viral DNA replication. Interestingly, a significant amount of cells actively replicating MCPyV did not show any Sp100 expression. While PML and Daxx had no effect on MCPyV DNA replication, MCPyV replication was increased in cells depleted for Sp100, strongly suggesting that Sp100 is a negative regulator of MCPyV DNA replication.

In Vitro Replication Assay for Merkel Cell Polyomavirus (MCPyV).

Merkel cell polyomavirus (MCPyV) genomes are clonally integrated in tumor cells of ∼95% of all Merkel cell carcinoma (MCC) cases. The virus is highly prevalent; however, where the virus persists and which cell types are permissive for MCPyV replication is still unknown. As a consequence, very little information is available about the life cycle and no fully permissive in vitro replication system has been established. Recently, semi-permissive replication systems based on wild-type MCPyV genomes recovered from the skin of healthy donors or synthetic MCPyV genomes constructed from consensus sequences have been established. The transfection of this intramolecular re-circularized MCPyV DNA into some human cell lines recapitulates efficient DNA replication of the viral genome, viral gene expression as well as moderate levels of virus particle formation. However, serial transmission of infectious virus is still restricted in these cells.

A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence.

Merkel cell polyomavirus (MCPyV) is considered the etiological agent of Merkel cell carcinoma and persists asymptomatically in the majority of its healthy hosts. Largely due to the lack of appropriate model systems, the mechanisms of viral replication and MCPyV persistence remain poorly understood. Using a semi-permissive replication system, we here report a comprehensive analysis of the role of the MCPyV-encoded microRNA (miRNA) mcv-miR-M1 during short and long-term replication of authentic MCPyV episomes. We demonstrate that cells harboring intact episomes express high levels of the viral miRNA, and that expression of mcv-miR-M1 limits DNA replication. Furthermore, we present RACE, RNA-seq and ChIP-seq studies which allow insight in the viral transcription program and mechanisms of miRNA expression. While our data suggest that mcv-miR-M1 can be expressed from canonical late strand transcripts, we also present evidence for the existence of an independent miRNA promoter that is embedded within early strand coding sequences. We also report that MCPyV genomes can establish episomal persistence in a small number of cells for several months, a time period during which viral DNA as well as LT-Ag and viral miRNA expression can be detected via western blotting, FISH, qPCR and southern blot analyses. Strikingly, despite enhanced replication in short term DNA replication assays, a mutant unable to express the viral miRNA was severely limited in its ability to establish long-term persistence. Our data suggest that MCPyV may have evolved strategies to enter a non- or low level vegetative stage of infection which could aid the virus in establishing and maintaining a lifelong persistence.

MTSS1 is a metastasis driver in a subset of human melanomas.

In cancers with a highly altered genome, distinct genetic alterations drive subsets rather than the majority of individual tumours. Here we use a sequential search across human tumour samples for transcript outlier data points with associated gene copy number variations that correlate with patient's survival to identify genes with pro-invasive functionality. Employing loss and gain of function approaches in vitro and in vivo, we show that one such gene, MTSS1, promotes the ability of melanocytic cells to metastasize and engages actin dynamics via Rho-GTPases and cofilin in this process. Indeed, high MTSS1 expression defines a subgroup of primary melanomas with unfavourable prognosis. These data underscore the biological, clinical and potential therapeutic implications of molecular subsets within genetically complex cancers.

High-affinity Rb binding, p53 inhibition, subcellular localization, and transformation by wild-type or tumor-derived shortened Merkel cell polyomavirus large T antigens.

UNLABELLED: Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE: MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential.

An attempt at a molecular prediction of metastasis in patients with primary cutaneous melanoma.

BACKGROUND: Current prognostic clinical and morphological parameters are insufficient to accurately predict metastasis in individual melanoma patients. Several studies have described gene expression signatures to predict survival or metastasis of primary melanoma patients, however the reproducibility among these studies is disappointingly low. METHODOLOGY/PRINCIPAL FINDINGS: We followed extended REMARK/Gould Rothberg criteria to identify gene sets predictive for metastasis in patients with primary cutaneous melanoma. For class comparison, gene expression data from 116 patients with clinical stage I/II (no metastasis) and 72 with III/IV primary melanoma (with metastasis) at time of first diagnosis were used. Significance analysis of microarrays identified the top 50 differentially expressed genes. In an independent data set from a second cohort of 28 primary melanoma patients, these genes were analyzed by multivariate Cox regression analysis and leave-one-out cross validation for association with development of metastatic disease. In a multivariate Cox regression analysis, expression of the genes Ena/vasodilator-stimulated phosphoprotein-like (EVL) and CD24 antigen gave the best predictive value (p = 0.001; p = 0.017, respectively). A multivariate Cox proportional hazards model revealed these genes as a potential independent predictor, which may possibly add (both p = 0.01) to the predictive value of the most important morphological indicator, Breslow depth. CONCLUSION/SIGNIFICANCE: Combination of molecular with morphological information may potentially enable an improved prediction of metastasis in primary melanoma patients. A strength of the gene expression set is the small number of genes, which should allow easy reevaluation in independent data sets and adequately designed clinical trials.

Replication, gene expression and particle production by a consensus Merkel Cell Polyomavirus (MCPyV) genome.

Merkel Cell Polyomavirus (MCPyV) genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag). These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle.

Methods from the theory of random heterogeneous media for quantifying myocardial morphology in normal and dilated hearts.

In the present study, descriptors from the theory of random heterogeneous media were used to characterize the morphology of the myocardial interstitial space in histological sections from hearts of healthy subjects and of patients with idiopathic dilated cardiomyopathy (DCM). Histological sections from resected DCM hearts (n = 9) were compared with donor hearts showing no signs of cardiac disease (n = 6). From control to DCM, the area fraction phi(1) of the interstitial space increased from 0.13 +/- 0.05 to 0.27 +/- 0.08, the chord-length z from 1.67 +/- 0.61 to 5.56 +/- 1.78 microm, the pore-size delta from 0.72 +/- 0.13 to 1.73 +/- 0.40 microm, the distance r (min) of the first local minimum in the two-point correlation function from 10.99 +/- 1.09 to 18.57 +/- 4.36 mum, whereas specific interface length s and decay-rate gamma of the lineal-path function decreased from 0.20 +/- 0.07 to 0.16 +/- 0.04 microm(-1) and from 0.39 +/- 0.09 to 0.16 +/- 0.05 microm(-1), respectively. All descriptors (except for s) were significantly different (p < 0.05) between control and DCM, reflecting an increasingly heterogeneous morphology in DCM hearts. Our results suggest that (1) descriptors originally developed to characterize the morphology of random heterogeneous media are well suited for histomorphometry of DCM, and (2) among the descriptors studied, either pore-size delta or chord-length z qualify best to discriminate between control and DCM hearts.

Assessment of regional differences in tariquidar-induced P-glycoprotein modulation at the human blood-brain barrier.

We attempted to assess regional differences in cerebral P-glycoprotein (P-gp) function by performing paired positron emission tomography (PET) scans with the P-gp substrate (R)-[(11)C]verapamil in five healthy subjects before and after i.v. infusion of tariquidar (2 mg/kg). Comparison of tariquidar-induced changes in distribution volumes (DVs) in 42 brain regions of interest (ROIs) failed to detect significant differences among brain ROIs. Statistical parametric mapping analysis of parametric DV images visualized symmetrical bilateral clusters with moderately higher DV increases in response to tariquidar administration in cerebellum, parahippocampal gyrus, olfactory gyrus, and middle temporal lobe and cortex, which might reflect moderately decreased P-gp function and expression.

Age dependency of cerebral P-gp function measured with (R)-[11C]verapamil and PET.

PURPOSE: The aim of this study was to assess the influence of age on the functional activity of the multidrug efflux transporter P-glycoprotein (P-gp) at the human blood-brain barrier. METHODS: Seven young (mean age: 27 +/- 4 years) and six elderly (mean age: 69 +/- 9 years) healthy volunteers underwent dynamic (R)-[(11)C]verapamil (VPM) positron emission tomography (PET) scans and arterial blood sampling. Parametric distribution volume (DV) images were generated using Logan linearisation, and age groups were compared with statistical parametric mapping (SPM). Brain regions that SPM analysis had shown to be most affected by age were analysed by a region of interest (ROI)-based approach using a maximum probability brain atlas, before and after partial volume correction (PVC). RESULTS: SPM analysis revealed significant clusters of DV increases in cerebellum, temporal and frontal lobe of elderly compared to younger subjects. In the ROI-based analysis, elderly subjects showed significant DV increases in amygdala (+30%), insula (+26%) and cerebellum (+25%) before PVC, and in insula (+33%) after PVC. CONCLUSIONS: Increased VPM DV values in the brains of elderly subjects suggest a decrease in cerebral P-gp function with increasing age.

[Different attitudes towards hypertension and urinary tract incontinence in elderly individuals participating in a health promotion project]. / Unterschiedliches Gesundheitsverhalten bezüglich Bluthochdruck und Harninkontinenz bei gesundheitsbewusst lebenden älteren Menschen.

BACKGROUND: The aim of the study was to evaluate health associated attitudes of elderly individuals taking part in a community based health promotion programme. It was of interest to focus on mobility, hypertension and urinary incontinence, problems frequently observed in geriatrics. METHODS: Participants in the health promotion programme were asked to answer a standardized questionnaire about their individual risk factors and habits concerning some of the main risk factors in the elderly: social isolation, immobility, hypertension, urinary incontinence. Mobility and risk assessment for falls was assessed in a standardized way using the Chair-rising- and Timed-upand-go tests. RESULTS: The percentage of people with a healthy life style was significantly higher than in a comparable population group. Gait disturbance, mobility limitation and risk of falls were not present. 38.7 % suffered from urinary incontinence. Hypertension was, if present, diagnosed and adequately treated. The participants knew their prescribed drugs and seemed to have an excellent compliance. DISCUSSION: The participants in the health promotion programme showed an exceptionally high level of health awareness. Nevertheless, the incidence for reported urinary incontinence was significantly higher than in known epidemiological studies. The calm, reassuring atmosphere in which the questionnaire was completed and the large amount of time spent with the participants could explain this finding. The difference in dealing with hypertension and urinary incontinence is obvious. Further health promotion and disease prevention programmes for the elderly should be focused on the complex process of ageing and not only on specific single topics.

Optimized arterial trees supplying hollow organs.

Computer models of arterial trees can be generated from optimization principles using the algorithm of constrained constructive optimization (CCO). Up to now this algorithm could handle only tissue areas of convex shape, without concavities. CCO is now generalized to cope also with non-convex organ shapes, possibly featuring external as well as internal concavities. This allows the modeling of a much larger class of interesting real arterial systems. The concept of a generalized domain-potential was developed to represent arbitrary non-convex shapes mathematically and incorporate them as boundary conditions to optimization. Domain-potentials may be derived from analytical representations as well as from finite element triangulations obtained from organ images. To demonstrate the feasibility of the concept, the optimized growth of an arterial tree model is confined to some part of an elliptical shell, representing the free wall of the left ventricle of the heart.

Error estimation of geometrical data obtained by histomorphometry of oblique vessel sections: a computer model study.

The errors of radius and wall thickness of a single vessel due to oblique sectioning in histomorphometry are expressed as a function of the circular shape factor (CSF) of the section's lumen, assuming cylindrical geometry and the absence of tissue deformation. Using computer model trees generated by constrained constructive optimization, mean errors are estimated for an ensemble of vessel segments. A geometrical exclusion criterion for segments cut too obliquely is defined on the basis of a CSF-cutoff value. It is shown that CSF-values ranging from 0.95 to 0.9 are reasonable choices for a cutoff and lead to mean errors of the same order of magnitude (9.6% [9.3%] to 15.4% [14.8%] for the radius [wall thickness]) as errors due to histological tissue processing.

The spatial pattern of coronary capillaries in patients with dilated, ischemic, or inflammatory cardiomyopathy.

INTRODUCTION: The goal of the present study was to compare the pattern of coronary capillaries in healthy participants and in patients with end-stage heart failure due to idiopathic dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), or inflammatory cardiomyopathy (InfCM). METHODS: Capillary patterns were studied in histological sections from resected hearts from patients with DCM (n=5), ICM (n=5), or InfCM (n=5) and compared with donor hearts showing no signs of cardiac disease (n=3). Patterns were characterized by the distribution of Voronoi polygon areas, A, associated with the centers of capillary profiles, nearest-neighbor distances, d, intercapillary distances (ICD), as well as by means of the pair correlation function g(r). The coefficient of variation of A, CV, was used to characterize capillary patterns as regular, random, or clustered. RESULTS: CV increased from 30.5% (control) to 33.8% (DCM), 36.6% (ICM), and to 40.3% (InfCM). d was minimal in the control group (16.5+/-4.3 microm) and increased to 18.1+/-5.2 microm in the DCM group and to 20.9+/-6.8 and 20.6+/-6.6 microm in the ICM and InfCM groups, respectively. ICD increased from 25.6+/-7.9 (control) to 28.5+/-9.2 (DCM), 34.4+/-12.2 (ICM), and 33.6+/-12.0 microm (InfCM). In all groups, g(r) was markedly different from random points; in the control and DCM group, g(r) showed a weak but distinct first maximum, characteristic for short-range order in a point pattern. CONCLUSIONS: Our data suggest that, for the patients studied, (1) the pattern of coronary capillaries is not purely random, (2) the regularity of the pattern diminishes from the control to DCM, ICM, and InfCM, and (3) ICD increases, whereas capillary density (CD) decreases, from control to DCM, ICM, and InfCM.

Fractal properties of perfusion heterogeneity in optimized arterial trees: a model study.

Regional blood flows in the heart muscle are remarkably heterogeneous. It is very likely that the most important factor for this heterogeneity is the metabolic need of the tissue rather than flow dispersion by the branching network of the coronary vasculature. To model the contribution of tissue needs to the observed flow heterogeneities we use arterial trees generated on the computer by constrained constructive optimization. This method allows to prescribe terminal flows as independent boundary conditions, rather than obtaining these flows by the dispersive effects of the tree structure. We study two specific cases: equal terminal flows (model 1) and terminal flows set proportional to the volumes of Voronoi polyhedra used as a model for blood supply regions of terminal segments (model 2). Model 1 predicts, depending on the number Nterm of end-points, fractal dimensions D of perfusion heterogeneities in the range 1.20 to 1.40 and positively correlated nearest-neighbor regional flows, in good agreement with experimental data of the normal heart. Although model 2 yields reasonable terminal flows well approximated by a lognormal distribution, it fails to predict D and nearest-neighbor correlation coefficients r1 of regional flows under normal physiologic conditions: model 2 gives D = 1.69 +/- 0.02 and r1 = -0.18 +/- 0.03 (n = 5), independent of Nterm and consistent with experimental data observed under coronary stenosis and under the reduction of coronary perfusion pressure. In conclusion, flow heterogeneity can be modeled by terminal positions compatible with an existing tree structure without resorting to the flow-dispersive effects of a specific branching tree model to assign terminal flows.

Voronoi polyhedra analysis of optimized arterial tree models.

Topological and metric properties of Voronoi polyhedra (VP) generated by the distal end points of terminal segments in arterial tree models grown by the method of constrained constructive optimization (CCO) are analyzed with the aim to characterize the spatial distribution of their supply sites relative to randomly distributed points as a reference model. The distributions of the number Nf of Voronoi cell faces, cell volume V, surface area S, area A of individual cell faces, and asphericity parameter alpha of the CCO models are all significantly different from the ones of random points, whereas the distributions of V, S, and alpha are also significantly different among CCO models optimized for minimum intravascular volume and minimum segment length (p < 0.0001). The distributions of Nf, V, and S of the CCO models are reasonably well approximated by two-parameter gamma distributions. We study scaling of intravascular blood volume and arterial cross-sectional area with the volume of supplied tissue, the latter being represented by the VP of the respective terminal segments. We observe scaling exponents from 1.20 +/- 0.007 to 1.08 +/- 0.005 for intravascular blood volume and 0.77 +/- 0.01 for arterial cross-sectional area. Setting terminal flows proportional to the associated VP volumes during tree construction yields a relative dispersion of terminal flows of 37% and a coefficient of skewness of 1.12.

Heterogeneous perfusion is a consequence of uniform shear stress in optimized arterial tree models.

Using optimized computer models of arterial trees we demonstrate that flow heterogeneity is a necessary consequence of a uniform shear stress distribution. Model trees are generated and optimized under different modes of boundary conditions. In one mode flow is delivered to the tissue as homogeneously as possible. Although this primary goal can be achieved, resulting shear stresses between blood and the vessel walls show very large spread. In a second mode, models are optimized under the condition of uniform shear stress in all segments which in turn renders flow distribution heterogeneous. Both homogeneous perfusion and uniform shear stress are desirable goals in real arterial trees but each of these goals can only be approached at the expense of the other. While the present paper refers only to optimized models, we assume that this dual relation between the heterogeneities in flow and shear stress may represent a more general principle of vascular systems.