Staphylococcus epidermidis Strategies to Avoid Killing by Human Neutrophils

Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs) contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs), including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS). These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus

Crowding by Invisible Flankers

BACKGROUND: Human object recognition degrades sharply as the target object moves from central vision into peripheral vision. In particular, one's ability to recognize a peripheral target is severely impaired by the presence of flanking objects, a phenomenon known as visual crowding. Recent studies on how visual awareness of flanker existence influences crowding had shown mixed results. More importantly, it is not known whether conscious awareness of the existence of both the target and flankers are necessary for crowding to occur. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that crowding persists even when people are completely unaware of the flankers, which are rendered invisible through the continuous flash suppression technique. Contrast threshold for identifying the orientation of a grating pattern was elevated in the flanked condition, even when the subjects reported that they were unaware of the perceptually suppressed flankers. Moreover, we find that orientation-specific adaptation is attenuated by flankers even when both the target and flankers are invisible. CONCLUSIONS: These findings complement the suggested correlation between crowding and visual awareness. What's more, our results demonstrate that conscious awareness and attention are not prerequisite for crowding

Human Placental-Specific Epipolymorphism and its Association with Adverse Pregnancy Outcomes

Interindividual variation in DNA-methylation level is widespread in the human genome, despite its critical role in regulating gene expression. The nature of this variation, including its tissue-specific nature, and the role it may play in human phenotypic variation and disease is still poorly characterized. The placenta plays a critical role in regulating fetal growth and development in ways that have lifelong effects on health. To identify genes with a high degree of interindividual DNA methylation variation in the human placenta, we surveyed the human genome using the Illumina GoldenGate Methylation Cancer panel targeting 1505 CpG sites of 807 genes. While many sites show a continuous pattern of methylation levels, WNT2, TUSC3 and EPHB4 were identified to have a polymorphic “on-or-off” pattern of DNA methylation variation at their promoter region which was confirmed by pyrosequencing. Methylation of these genes can be found in 7%–25% of over 100 placentas tested. The methylation state at the promoter of these genes is concordant with mRNA allelic expression. In three informative cases TUSC3 was observed to be methylated on the maternal allele, and it is thus possible this represents a polymorphically imprinted gene. Furthermore, TUSC3 promoter methylation showed evidence for association with preeclampsia. A biological significance of these methylation allelic polymorphisms (MAPs) to human placental diversity is further implied by their placental specificity and absence in mouse. An extended study of blood suggests that MAPs may also be found in other tissues, implicating their utility for tissue-specific association with complex disorders. The identification of such “epipolymorphism” in other tissues and their use in association studies, should improve our understanding of interindividual phenotypic variability and complex disease susceptibility

Oxygen Tension Modulates Neurite Outgrowth in PC12 Cells Through A Mechanism Involving HIF and VEGF

Cell-based approaches are a promising therapeutic strategy for treating injuries to the nervous system, but the optimal means to promote neurite extension and direct cellular behavior are unclear. Previous studies have examined the behavior of neural-like cells in ambient air (21% oxygen tension), yet these conditions are not representative of the physiological oxygen microenvironment of neural tissues. We hypothesized that neuronal differentiation of a model neural cell line (PC12) could be controlled by modulating local oxygen tension. Compared to ambient conditions, PC12 cells cultured in reduced oxygen exhibited significant increases in neurite extension and total neurite length, with 4% oxygen yielding the highest levels of both indicators. We confirmed neurite extension was mediated through oxygen-responsive mechanisms using small molecules that promote or inhibit HIF-1α stabilization. The hypoxic target gene Vegf was implicated as a neurotrophic factor, as neurite formation at 21% oxygen was mimicked with exogenous VEGF, and a VEGF-neutralizing antibody attenuated neurite formation under reduced oxygen conditions. These findings demonstrate that behavior of neural-like cells is driven by the oxygen microenvironment via VEGF function, and suggest promising approaches for future applications in neural repair

Technical Variability Is Greater than Biological Variability in a Microarray Experiment but Both Are Outweighed by Changes Induced by Stimulation

INTRODUCTION: A central issue in the design of microarray-based analysis of global gene expression is that variability resulting from experimental processes may obscure changes resulting from the effect being investigated. This study quantified the variability in gene expression at each level of a typical in vitro stimulation experiment using human peripheral blood mononuclear cells (PBMC). The primary objective was to determine the magnitude of biological and technical variability relative to the effect being investigated, namely gene expression changes resulting from stimulation with lipopolysaccharide (LPS). METHODS AND RESULTS: Human PBMC were stimulated in vitro with LPS, with replication at 5 levels: 5 subjects each on 2 separate days with technical replication of LPS stimulation, amplification and hybridisation. RNA from samples stimulated with LPS and unstimulated samples were hybridised against common reference RNA on oligonucleotide microarrays. There was a closer correlation in gene expression between replicate hybridisations (0.86-0.93) than between different subjects (0.66-0.78). Deconstruction of the variability at each level of the experimental process showed that technical variability (standard deviation (SD) 0.16) was greater than biological variability (SD 0.06), although both were low (SD<0.1 for all individual components). There was variability in gene expression both at baseline and after stimulation with LPS and proportion of cell subsets in PBMC was likely partly responsible for this. However, gene expression changes after stimulation with LPS were much greater than the variability from any source, either individually or combined. CONCLUSIONS: Variability in gene expression was very low and likely to improve further as technical advances are made. The finding that stimulation with LPS has a markedly greater effect on gene expression than the degree of variability provides confidence that microarray-based studies can be used to detect changes in gene expression of biological interest in infectious diseases

Discretization Provides a Conceptually Simple Tool to Build Expression Networks

Biomarker identification, using network methods, depends on finding regular co-expression patterns; the overall connectivity is of greater importance than any single relationship. A second requirement is a simple algorithm for ranking patients on how relevant a gene-set is. For both of these requirements discretized data helps to first identify gene cliques, and then to stratify patients

In vivo reversal of the anticoagulant effect of rivaroxaban with four-factor prothrombin complex concentrate

Four-factor prothrombin complex concentrate (PCC) 50 iu/kg is able to swiftly restore haemostatic parameters in healthy subjects on rivaroxaban. We hypothesized that lower dosages of PCC may be sufficient to restore normal haemostasis. In this double-blind, crossover, placebo-controlled study, we compared the effects of PCC 37.5 iu/kg, PCC 25 iu/kg, and placebo on thrombin generation (endogenous thrombin potential, ETP) and prothrombin time in six healthy subjects receiving twice-daily rivaroxaban 15 mg for 2.5 days. Fifteen min after infusion of PCC 37.5 iu/kg, ETP increased from 47 ± 16% to 64 ± 22% (P = 0.03; pre-rivaroxaban ETP: 92 ± 14%) and remained higher than after placebo over 24 h (P = 0.001). PCC 25 iu/kg did not modify ETP within 15 min (53 ± 11% to 59 ± 12%; P = 0.14) and was not different from placebo over 24 h (P = 0.31). ETP reached pre-rivaroxaban levels within 6 h after PCC 37.5 iu/kg infusion and within 12-24 h after PCC 25 iu/kg infusion. Both dosages restored rivaroxaban-induced prothrombin time prolongation after 15 min (P < 0.001). Placebo did not have an effect on coagulation parameters. 37.5 iu/kg of PCC leads to partial restoration of thrombin generation, whereas 25 iu/kg does not. PCC 37.5 iu/kg may be insufficient for immediate full reversal of peak therapeutic rivaroxaban level

Pharmacokinetics of dabigatran etexilate and rivaroxaban in patients with short bowel syndrome requiring parenteral nutrition: The PDER PAN study

Background and aims: Patients on parenteral nutrition for short bowel syndrome (SBS) have a high risk of thrombotic complications and are often treated with parenteral anticoagulation. Direct oral anticoagulants are absorbed proximally in the digestive tract and may represent alternative regimens in selected SBS patients. In our pilot study, we provided pharmacokinetics parameters of dabigatran etexilate and rivaroxaban in this setting and compared peak (Cmax), trough (Ctrough) concentrations, and areas-under-the-concentration-time-curve (AUC(0) (-) (t)) to reference values retrieved from phase I-III studies. Methods: We enrolled 6 adults with a remaining small bowel length <= 200 cm, normal renal/hepatic function, and intact stomach. In our crossover study, patients were exposed to twice-daily dabigatran etexilate 150 mg and once-daily rivaroxaban 20 mg. Results: After 5 days of dabigatran dosing, Ctrough and Cmax geometric means were 39 mu g/L (90% CI: 23-66) and 88 mu g/L (90% CI: 56-137), respectively; AUC(0-12) h was 958 mu g*h/L (90% CI: 635-1445). After 5 days of rivaroxaban dosing, Ctrough and Cmax geometric means were 9 mu g/L (90% CI: 1-71) and 167 mu g/L (90% CI: 102-276), respectively; AUC(0-24) h was 1720 mu g*h/L (90% CI: 899-3300). Absorption was negligible in one patient with ultra-short (similar to 15 cm) bowel. For dabigatran, Cmax ratio was 0.57 (SD 0.33) and Ctrough ratio was 0.35 (SD 0.44). For rivaroxaban, the mean observed-to-reference ratios AUC(0-24) h and Cmax ratios were 0.73 (SD 0.32) and 0.76 (SD 0.34), respectively. Conclusions: While in SBS patients there is some absorption of the oral anticoagulants dabigatran etexilate and rivaroxaban, it appears to be lower than reference values. Plasma drug levels showed significant inter-individual variabilit