Artery-to-vein anastomoses in unequally divided placentas and their association with birthweight discordance.

INTRODUCTION: This study investigated the impact of the shared intertwin circulation in unequally divided monochorionic (MC) placentas on fetal growth. METHODS: This retrospective analysis included color-dyed, unequally shared placentas from two tertiary centers. Exclusions included twin-twin transfusion syndrome, twin anemia polycythemia sequence, and lethal anomalies. Measurement of the external diameters and areas of the artery-to-artery (AA), artery-to-vein (AV), and vein-to-vein (VV) anastomoses was performed. The ratio of the shared circulation (AV ratio) was determined by comparing the areas of the summed venous components of shared AV anastomoses to those in the individual AV anastomoses of the smaller placental part. The birth weight ratio/placental ratio (BWR/PR), total AV size areas and net AV transfusion were calculated. Univariable and multivariable linear regressions were performed to assess the relationship between BWR/PR, the AV ratio, the areas of the different anastomoses and cord insertion discordance. RESULTS: Among 352 placentas, 97 % (340) had intertwin AV anastomoses, and 50 % (176) were from pregnancies with selective growth restriction. The AV ratio, AA, VV, total AV areas, and cord insertion discordance negatively correlated with BWR/PR. Multivariable linear regression confirmed the independent negative association between BWR/PR and the AV ratio, suggesting that a larger shared circulation benefits the twin with the smaller placental part. Type III sFGR placentas exhibited the highest AV ratio, resulting in the lowest BWR/PR. DISCUSSION: A larger shared circulation mitigates the impact of an unequally divided placenta on fetal growth. This effect surpasses the influence of AA and VV diameters and is most prominent in Type III sFGR placentas.

Non-canonical Wnt signalling regulates scarring in biliary disease via the planar cell polarity receptors.

The number of patients diagnosed with chronic bile duct disease is increasing and in most cases these diseases result in chronic ductular scarring, necessitating liver transplantation. The formation of ductular scaring affects liver function; however, scar-generating portal fibroblasts also provide important instructive signals to promote the proliferation and differentiation of biliary epithelial cells. Therefore, understanding whether we can reduce scar formation while maintaining a pro-regenerative microenvironment will be essential in developing treatments for biliary disease. Here, we describe how regenerating biliary epithelial cells express Wnt-Planar Cell Polarity signalling components following bile duct injury and promote the formation of ductular scars by upregulating pro-fibrogenic cytokines and positively regulating collagen-deposition. Inhibiting the production of Wnt-ligands reduces the amount of scar formed around the bile duct, without reducing the development of the pro-regenerative microenvironment required for ductular regeneration, demonstrating that scarring and regeneration can be uncoupled in adult biliary disease and regeneration.

Pericardial complications and postcardiac injury syndrome after cardiovascular implantable electronic device placement : A meta-analysis and systematic review.

BACKGROUND: Postcardiac injury syndrome (PCIS) is an emerging condition including pericarditis with or without pericardial effusion after an injury to cardiac tissue. Data are lacking on its incidence and clinical predictors after cardiovascular implantable electronic device (CIED) placement. We therefore performed this meta-analysis to determine the incidence of PCIS. METHODS: Medline, Embase, and Cochrane CENTRAL databases were searched according to PRISMA guidelines from February 2007 to February 2017 for studies evaluating pericardial complications subsequent to CIED implantation. Primary outcome was the total number of cases of pericarditis, pericardial effusion, and cardiac tamponade documented. RESULTS: Of 2931 references, 22 articles (enrolling 188,944 patients) were included. Pooled estimates from random-effects analysis showed an overall incidence of 5.82 per 1000 patients (95% confidence interval [CI], 4.33-8.17) at 30 days, and 1.60 per 1000 (95% CI: 0.13-3.07) at 1 year. Advanced age and prior coronary artery bypass graft (CABG) surgery were associated with increased rates of pericardial complications. CONCLUSION: Our analysis revealed that CIED implantations are associated with a low incidence (0.6%) of pericardial complications at 30 days. Patients with advanced age and prior CABG are high-risk patients for pericardial complications.

Non-invasive genotyping with a massively parallel sequencing panel for the detection of SNPs in HPA-axis genes.

We designed a genotyping panel for the investigation of the genetic underpinnings of inter-individual differences in aggression and the physiological stress response. The panel builds on single nucleotide polymorphisms (SNPs) in genes involved in the three subsystems of the hypothalamic-pituitary-adrenal (HPA)-axis: the catecholamine, serotonin and corticoid metabolism. To promote the pipeline for use with wild animal populations, we used non-invasively collected faecal samples from a wild population of Assamese macaques (Macaca assamensis). We targeted loci of 46 previously reported SNPs in 21 candidate genes coding for elements of the HPA-axis and amplified and sequenced them using next-generation Illumina sequencing technology. We compared multiple bioinformatics pipelines for variant calling and variant effect prediction. Based on this strategy and the application of different quality thresholds, we identified up to 159 SNPs with different types of predicted functional effects among our natural study population. This study provides a massively parallel sequencing panel that will facilitate integrating large-scale SNP data into behavioural and physiological studies. Such a multi-faceted approach will promote understanding of flexibility and constraints of animal behaviour and hormone physiology.

COMT Val158 Met moderates the link between rank and aggression in a non-human primate.

The COMT Val158 Met polymorphism is one of the most widely studied genetic polymorphisms in humans implicated in aggression and the moderation of stressful life event effects. We screened a wild primate population for polymorphisms at the COMT Val158 Met site and phenotyped them for aggression to test whether the human polymorphism exists and is associated with variation in aggressive behavior. Subjects were all adults from 4 study groups (37 males, 40 females) of Assamese macaques (Macaca assamensis) in their natural habitat (Phu Khieo Wildlife Sanctuary, Thailand). We collected focal animal behavioral data (27 males, 36 females, 5964 focal hours) and fecal samples for non-invasive DNA analysis. We identified the human COMT Val158 Met polymorphism (14 Met/Met, 41 Val/Met and 22 Val/Val). Preliminary results suggest that COMT genotype and dominance rank interact to influence aggression rates. Aggression rates increased with rank in Val/Val, but decreased in Met/Met and Val/Met individuals, with no significant main effect of COMT genotype on aggression. Further support for the interaction effect comes from time series analyses revealing that when changing from lower to higher rank position Val/Val individuals decreased, whereas Met/Met individuals increased their aggression rate. Contradicting the interpretation of earlier studies, we show that the widely studied Val158 Met polymorphism in COMT is not unique to humans and yields similar behavioral phenotypes in a non-human primate. This study represents an important step towards understanding individual variation in aggression in a wild primate population and may inform human behavioral geneticists about the evolutionary roots of inter-individual variation in aggression.

Comparison of commercially available target enrichment methods for next-generation sequencing.

Isolating high-priority segments of genomes greatly enhances the efficiency of next-generation sequencing (NGS) by allowing researchers to focus on their regions of interest. For the 2010-11 DNA Sequencing Research Group (DSRG) study, we compared outcomes from two leading companies, Agilent Technologies (Santa Clara, CA, USA) and Roche NimbleGen (Madison, WI, USA), which offer custom-targeted genomic enrichment methods. Both companies were provided with the same genomic sample and challenged to capture identical genomic locations for DNA NGS. The target region totaled 3.5 Mb and included 31 individual genes and a 2-Mb contiguous interval. Each company was asked to design its best assay, perform the capture in replicates, and return the captured material to the DSRG-participating laboratories. Sequencing was performed in two different laboratories on Genome Analyzer IIx systems (Illumina, San Diego, CA, USA). Sequencing data were analyzed for sensitivity, specificity, and coverage of the desired regions. The success of the enrichment was highly dependent on the design of the capture probes. Overall, coverage variability was higher for the Agilent samples. As variant discovery is the ultimate goal for a typical targeted sequencing project, we compared samples for their ability to sequence single-nucleotide polymorphisms (SNPs) as a test of the ability to capture both chromosomes from the sample. In the targeted regions, we detected 2546 SNPs with the NimbleGen samples and 2071 with Agilent's. When limited to the regions that both companies included as baits, the number of SNPs was ∼1000 for each, with Agilent and NimbleGen finding a small number of unique SNPs not found by the other.

Verapamil is associated with an increased risk of cancer in the elderly: the Rotterdam study.

The association between the use of calcium channel blockers (CCB) and cancer has received ample attention, but is still controversial. In this study, we have tested the hypothesis that the observed association between CCB and cancer in earlier studies could be explained by residual confounding or by misclassification of exposure because of the use of cross-sectional data on drug use. Data from the Rotterdam Study, a prospective population-based cohort study in the municipal area Ommoord, were used. The study population consisted of a cohort of 3204 participants aged 71 years or older who were followed from a baseline interview in the period 1991-1993 for the occurrence of incident cancer. Data on drug use were gathered at baseline and through the seven community pharmacies which served the Ommoord region during the study period between 1 January 1991 and 1 January 1999. Incident cancer events were gathered from a nationwide registry of hospitalisation data and from a specialised cancer centre in the Rotterdam region. We performed three analyses. First, we followed the method, and adjusted for the same risk factors, as in the earlier studies. In the second analysis, we included all risk factors that were univariately associated with cancer in the Rotterdam Study. In the third analysis, we included exposure to CCBs as time-varying co-variates, while adjusting for potential confounders. The relative risk (RR) of cancer associated with CCB was 1.4 (95% Confidence Interval (CI): 0.9-2.0) in the first analysis and lowered to 1.2 (95% CI: 0.8-1.8) upon adjustment for the different co-variates in the second. In both analyses, however, verapamil was significantly associated with cancer with RRs of 2.1 (95% CI: 1.1-4.0) and 2.0 (1.01-3.9), respectively, whereas no associations were found with the other CCB in this study, i.e. diltiazem and nifedipine. A significantly increased risk of cancer was found for intermediate daily doses of verapamil and diltiazem. Intake of other antihypertensives such as beta-blocking agents, diuretics and ACE-inhibitors was not associated with cancer. In the third analysis with exposure to CCB as time-varying co-variates, the risk increase was non-significant for use of 2 years or less, 1.0 (95% CI: 0.7-1.5), and for use for a cumulative period of more than 2 years, 1.3 (95% CI: 0.8-2.0). However, in all models the hazard ratio was statistically significantly increased for verapamil, but not for diltiazem and nifedipine. On the basis of these analyses, we found no increase in cancer in users of diltiazem and nifedipine, nor in users of other antihypertensives. In line with earlier studies, however, we found an increased risk of cancer in users of verapamil. At variance with the conclusions from several other studies, we think that it is too early to conclude that CCB are not associated with cancer.

Cell surface presentation of recombinant (poly-) peptides including functional T-cell epitopes by the AIDA autotransporter system.

For the efficient surface presentation and release of virulence factors especially pathogenic Gram-negative bacteria have developed several distinct secretion mechanisms. An increasing number of pathogens in various species employs a mechanism denoted the 'autotransporter' pathway. This pathway is characterised by an outer membrane translocator module representing the C-terminal domain of the transported protein itself. An intriguing potential application of such systems involves the transport and surface expression of recombinant proteins or peptides, like e.g. the presentation of antigens for the generation of live oral vectors as vaccine carriers. Here we report on the incorporation of heterologous (poly-) peptides in permissive sites of the translocator module of the adhesin-involved-in-diffuse-adherence (AIDA) autotransporter system. We demonstrate the presentation of the B subunit of the heat labile enterotoxin of Escherichia coli (LTB) as well as of functional T-cell epitopes of Yersinia enterocolitica heat-shock protein 60 (Y-hsp60) on the surface of E. coli.

DNA vaccination using coexpression of cytokine genes with a bacterial gene encoding a 60-kDa heat shock protein.

Coexpression of cytokine genes together with antigen-encoding genes in DNA vaccination vectors can increase humoral and cellular immune responses and may steer them in a Th1 or Th2 direction. In this study, the modulatory effect of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma coexpressed with the 60-kDa heat shock protein (Hsp60) of Yersinia enterocolitica O:8 (Y-Hsp60) was studied. DNA vaccination with gamma-hsp60 evoked specific humoral and cellular immune responses as well as reduction of the splenic bacterial load upon challenge with Y. enterocolitica in a mouse infection model. Coexpression of IL-2 or IFN-gamma enhanced Y. enterocolitica-specific total IgG (P < 0.05) and IgG2a antibody responses. Coexpression of IFN-gamma also improved the proliferative T cell responses upon stimulation with Y-Hsp60. A reduction of the splenic bacterial load as compared with the plasmid encoding Y-Hsp60 only was found for the IFN-gamma coexpressing vector. Thus, coexpression of cytokine genes such as IFN-gamma in DNA vaccination vectors might improve immunity and help to overcome the side effects of standard adjuvants.

Rational live oral carrier vaccine design by mutating virulence-associated genes of Yersinia enterocolitica.

Three different Yersinia enterocolitica serotype O8 strains harboring mutations in virulence-associated genes coding for Yersinia adhesin A (YadA), Mn-cofactored superoxide dismutase (SodA), and high-molecular-weight protein 1 were analyzed for their ability to colonize and persist in tissues after orogastric immunization of C57BL/6 mice. We demonstrated that all three Yersinia mutant strains were markedly impaired in their ability to disseminate into the spleens and livers of immunized mice but were able to colonize the Peyer's patches for at least 12 days, resulting in the induction of significant antibody titers against Yersinia outer proteins (Yops) and in the priming of Yersinia antigen-specific CD4+ Th1 cells isolated from spleens. The high level of attenuation did not diminish the immunogenic properties of the mutant strains. In fact, mice immunized with a single oral dose of any of the mutant strains were protected against a lethal oral-challenge infection with wild-type Y. enterocolitica. Moreover, adoptive transfer of Yersinia-specific antibodies from sera of mice immunized with the mutant WAP-314 sodA revealed that this protection could be mediated by Yersinia-specific immunoglobulins.

DNA immunization confers systemic, but not mucosal, protection against enteroinvasive bacteria.

Naked plasmid DNA (pRc/Y-hsp60) with a cytomegalovirus promoter and a sequence encoding Yersinia enterocolitica 60-kDa heat shock protein (Y-HSP60) was used for vaccination. After intramuscular injection of pRc/Y-hsp60, Y-hsp60 mRNA could be detected by reverse transcription-PCR in muscle, liver and spleen. A single immunization with pRc/Y-hsp60 induced significant Y-HSP60-specific T cell responses after 1 week. IFN-gamma production by spleen cells upon stimulation with Y-HSP60 was strictly dependent on the presence of CD4+ T cells, indicating the generation of a Th1 response upon DNA immunization. DNA immunization in addition induced strong Y-HSP60-specific IgG2a, weak IgG1, but not IgA antibodies. Immunization of BALB/c and C57BL/6 mice with pRc/Y-hsp60 conferred protection against disseminated Y. enterocolitica infection in spleen, but not at the site of mucosal entry, the Peyer's patches. Furthermore, pRc/Y-hsp60 vaccination did not induce cross-protection against related pathogens. Vaccination of beta2-microglobulin- and H2-I-Abeta-deficient mice was not protective, suggesting that both CD4+ and CD8+ T cells are required for protective immunity induced by DNA vaccination.

In vivo tracking and protective properties of Yersinia-specific intestinal T cells.

After invasion via M cells enteropathogenic Yersinia enterocolitica subsequently establish an infection at three different sites: (i) Peyer's patches (PP), (ii) mesenteric lymph nodes (MLN), and after systemic dissemination in (iii) spleen, liver and lung. In order to characterize protective properties of intestinal T cells at the different sites of Y. enterocolitica infection, PP and MLN T cells were isolated from Y. enterocolitica-infected C57B1/6 mice and Yersinia-specific T cell lines were generated. These T cells exhibited the phenotype of CD4 Th1 cells. The adoptive transfer of Yersinia-specific Th1 cells from PP and MLN conferred protection against a lethal orogastric inoculum with Y. enterocolitica as revealed by survival post-infection. However, determination of bacterial counts in infected organs revealed that the transfer of PP T cells conferred protection in spleen but not in MLN and PP, whereas the transfer of T cells from MLN reduced bacterial counts in both spleen and MLN but not in PP. To elucidate the different protection pattern we wanted to track the transferred cells in vivo. For this purpose the cells were labelled with the stable green fluorescent cell linker PKH2-GL prior to the adoptive transfer. In vivo tracking of these cells revealed that the distribution pattern of transferred T cells in spleen, MLN and PP correlated closely with the protection pattern observed after Yersinia infection. Thus, most cells were recovered from the spleen, while only few cells were recovered from MLN and PP. In keeping with these results a rapid and significant increase in interferon-gamma (IFN-gamma) production in the spleen of mice after adoptive transfer of T cell lines was observed. Taken together, the present results demonstrate that intestinal CD4 Th1 cells from PP and MLN may be involved in the defence against Y. enterocolitica at different sites of the infection, and that PKH2-GL labelling is a suitable tool to characterize T cell functions in vivo.

Shifts in the TH1/TH2 balance during human pregnancy correlate with apoptotic changes.

An important prerequisite for a successful pregnancy is that the maternal immune system does not reject the fetus. Down-regulation of the T helper 1 (TH1) associated cellular immune response could therefore be essential. With flow cytometric techniques, we show on a single cell level that both CD4+ and CD8+ T cells from peripheral blood produce less TH1 cytokines (i.e. IFN-gamma and IL-2) and more TH2 cytokines (i.e. IL-4) during normal human pregnancy and shortly after delivery than during non-pregnancy. The TH1/TH2 cytokine ratio in T cells of women during pregnancy and after delivery was significantly decreased. In contrast the TH1/TH2 ratio was elevated to near normal in women with recurrent spontaneous abortions, indicating a marked shift towards TH1 immunity. Fas antigen (CD95) on T cells was significantly elevated during pregnancy and in the post-delivery phase whereas the intracellular expression of anti-apoptotic protein Bcl-2 remained unchanged. Nevertheless Fas-mediated apoptosis in T cells was markedly reduced during normal human pregnancy. We hypothesize that TH1 cells undergo predominantly Fas-mediated apoptosis during pregnancy as has been shown in some TH2-prone diseases (e.g. SLE, HIV) where an elevated Fas expression on peripheral T cells is observed. This could explain the exacerbated occurrence of TH2-associated diseases in pregnancy.

Ambiguous role of interleukin-12 in Yersinia enterocolitica infection in susceptible and resistant mouse strains.

Endogenous interleukin-12 (IL-12) mediates protection against Yersinia enterocolitica in C57BL/6 mice by triggering gamma interferon (IFN-gamma) production in NK and CD4+ T cells. Administration of exogenous IL-12 confers protection against yersiniae in Yersinia-susceptible BALB/c mice but exacerbates yersiniosis in resistant C57BL/6 mice. Therefore, we wanted to dissect the different mechanisms exerted by IL-12 during Yersinia infections by using different models of Yersinia-resistant and -susceptible mice, including resistant C57BL/6 mice, susceptible BALB/c mice, intermediate-susceptible wild-type 129/Sv mice, 129/Sv IFN-gamma-receptor-deficient (IFN-gamma R-/-) mice and C57BL/6 tumor necrosis factor (TNF) receptor p55 chain-deficient (TNFR p55-/-) mice. IFN-gamma R-/- mice turned out to be highly susceptible to infection by Y. enterocolitica compared with IFN-gamma R+/+ mice. Administration of IL-12 was protective in IFN-gamma R+/+ mice but not in IFN-gamma R-/- mice, suggesting that IFN-gamma R-induced mechanisms are essential for IL-12-induced resistance against yersiniae. BALB/c mice could be rendered Yersinia resistant by administration of anti-CD4 antibodies or by administration of IL-12. In contrast, C57BL/6 mice could be rendered more resistant by administration of transforming growth factor beta (TGF-beta). Furthermore, IL-12-triggered toxic effects in C57BL/6 mice were abrogated by coadministration of TGF-beta. While administration of IL-12 alone increased TNF-alpha levels, administration of TGF-beta or TGF-beta plus IL-12 decreased both TNF-alpha and IFN-gamma levels in Yersinia-infected C57BL/6 mice. Moreover, IL-12 did not induce toxicity in Yersinia-infected TNFR p55-/- mice, suggesting that TNF-alpha accounts for IL-12-induced toxicity. Taken together, IL-12 may induce different effector mechanisms in BALB/c and C57BL/6 mice resulting either in protection or exacerbation. These results are important for understanding the critical balance of proinflammatory and regulatory cytokines in bacterial infections which is decisive for beneficial effects of cytokine therapy.

The perception of bodily sensations, with special reference to hypochondriasis.

Bodily sensations are relevant to problems such as hypochondriasis, but the issue of whether people are accurate in their perception remains unclear. The accuracy of perception of bodily sensations was analysed in 20 male and 20 female volunteers using two methods: a heart beat tracking procedure and the within-S correlational approach described by Steptoe and Vögele (1992, Behaviour Research and Therapy, 30, 597-607). The correlational approach involved monitoring of heart rate, skin conductance level and total respiratory resistance during relaxation and task periods, and computing correlations between appropriate physiological parameters and ratings of heart rate, sweaty hands and difficulty with breathing. In general, subjective ratings of bodily sensations were tied more closely with feelings of distress than with objective physiological state. Error scores on the heart beat tracking procedure showed no association with hypochondriacal concerns or with vigilant and avoidant coping styles measured with the Mainz Coping Inventory. Individuals varied considerably in accuracy as assessed with the correlational approach. However, there was a significant negative association between hypochondriacal concerns and accuracy of perception of sweat gland activity. The results are discussed in relation to measures of somatic perception and the experience of bodily sensations.

Immunity against Yersinia enterocolitica by vaccination with Yersinia HSP60 immunostimulating complexes or Yersinia HSP60 plus interleukin-12.

Microbial heat shock proteins (HSP) are dominant antigens for the host immune response. Because of the high sequence homology between mammalian and microbial HSP, their value as component of a subunit vaccine has been the subject of controversy. Previous work from this laboratory, however, demonstrated for the first time that the adoptive transfer of HSP60-reactive CD4+ alphabeta T-cell clones confers protection against bacterial infection in mice but does not induce autoimmunity. In the present study, we have therefore evaluated the potential role of Yersinia HSP60 (Y-HSP60) as a vaccine in the Yersinia enterocolitica mouse infection model. For this purpose, immunostimulating complexes (ISCOM) which included Y-HSP60 were constructed. Parenteral administration of this vaccine induced high Y-HSP60-specific serum antibody responses as well as T-cell responses. This reaction was parallelled by immunity against a lethal challenge with Y. enterocolitica. In contrast, mucosal application of Y-HSP60-ISCOM failed to induce systemic Y-HSP60-specific T-cell responses and thus failed to induce immunity against yersiniae. Likewise, vaccination with purified recombinant Y-HSP60 induced antibody responses but only weak T-cell responses. Therefore, this vaccination protocol was not protective. However, when interleukin-12 was used as an adjuvant, purified Y-HSP60 induced significant Y-HSP60-specific T-cell responses and thus induced protection against subsequent challenge with yersiniae. These studies suggest that (i) microbial HSP might be promising candidates for the design of subunit vaccines and (ii) interleukin-12 is an efficient alternative adjuvant to ISCOM particles for induction of protective CD4 Th1-cell-dependent immune responses against bacterial pathogens.