Inhibition of major integrin αVβ3 reduces Staphylococcus aureus attachment to sheared human endothelial cells.

BACKGROUND: Vascular endothelial dysfunction with associated oedema and organ failure is one of the hallmarks of sepsis. While a large number of microorganisms can cause sepsis, Staphylococcus aureus is one of the primary etiological agents. Currently there are no approved specific treatments for sepsis and therefore the initial management bundle focuses on cardiorespiratory resuscitation and mitigation against the immediate threat of uncontrolled infection. The continuous emergence of antibiotic resistant strains of bacteria urges the development of new therapeutic approaches for this disease. OBJECTIVE: The objective of this study was to identify the molecular mechanisms leading to endothelial dysfunction as a result of Staphylococcus aureus binding. METHODS: Stahpylococcus aureus Newman and clumping factor A-deficient binding to endothelium were measured in vitro and in the mesenteric circulation of C57Bl/6 mice. The effect of the αVβ3 blocker, cilengitide, on bacterial binding, endothelial VE-cadherin expression, apoptosis, proliferation and permeability were assessed. RESULTS: Here we show that the major Staphylococcus aureus cell wall protein clumping factor A binds to endothelial cell integrin αVβ3 in the presence of fibrinogen. This interaction results in disturbances in barrier function mediated by VE-cadherin in endothelial cell monolayers and ultimately cell death by apoptosis. Using a low concentration of cilengitide, ClfA binding to αVβ3 was significantly inhibited both in vitro and in vivo. Moreover, preventing Staphylococcus aureus from attaching to αVβ3 resulted in a significant reduction in endothelial dysfunction following infection. CONCLUSION: Inhibition of Staphylococcus aureus ClfA binding to endothelial cell αVβ3 using cilengitide prevents endothelial dysfunction. This article is protected by copyright. All rights reserved

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

miR330-3p Dysregulation Plays a Critical Role in Disrupting the Endothelial Barrier During Staphylococcus aureus Infection

Sepsis is a life-threatening condition defined as an extreme inflammatory host response to infection. One of the most commonly isolated causative microorganisms from sepsis patients is the Gram-positive bacteria Staphylococcus aureus (S. aureus). Upon entry to the bloodstream, S. aureus locates and adheres to the inner lining of the blood vessels, the endothelium, and triggers the typical characteristics of sepsis such as a sustained and dysregulated immune response, hyper-coagulation and endothelial dysfunction. These manifestations are thought to result from early dysregulation of the host endothelium in response to infection, of which one of the primary characteristics is loss of endothelial barrier integrity. Loss of barrier integrity is attributed to a loss of critical junction proteins such as VE-cadherin and ZO-1, located at cell-to-cell junction points. VE-cadherin is a central regulator of endothelial junctions and plays a pivotal role in mediating the permeability of the endothelial monolayer. Currently, recommended management strategies for sepsis include an aggressive empiric administration of antibiotics and fluid resuscitation. Antibiotic treatment however has been inadequate and indeed, eradication of bacteria from the host is not sufficient to reverse adverse effects of the infection. This is in part due to the gap in understanding as to how infection of the endothelium triggers early endothelial dysfunction and as a result, many clinical trials and treatments have been ineffective and disappointing. Investigating these early pathogenic events in sepsis will be critical for preventing progression of the infection, as breakdown of the endothelial barrier provides a gateway for bacteria to access metastatic sites and major organs throughout the body. If left untreated, bacterial dissemination can lead to organ infection, organ failure and death. miRNAs are signalling molecules responsible for regulating approximately 60% of all genes within the human genome. They are implemented in nearly all human biological processes including inflammation, immune responses and endothelial function. Unsurprisingly, their dysregulation has been associated with a variety of clinically important human diseases such as cancers and immune diseases. They however, have not been widely studied in the pathogenesis of sepsis. The overall goal of the work presented in this thesis was to investigate early signals triggered by S. aureus infection of the human endothelium that lead to the endothelial dysfunction associated with sepsis. We examine the impact of S. aureus infection upon the intrinsic expression of endothelial miRNAs, and focus on identifying the role of miRNAs in maintaining the integrity of the endothelial barrier through regulating the expression of important junction proteins. In this study, we uncover the vast interference implemented by S. aureus into the expression of intrinsic endothelial miRNA. Specifically, we demonstrate that S. aureus infection causes dysregulated and enhanced expression of intrinsic miR330-3p. We identify a previously unknown role for miR330-3p as an important regulator of critical junction protein VE-cadherin within the endothelium. In addition, we demonstrate that the increase of miR330-3p implemented by S. aureus leads to the vascular leak observed during sepsis as the enhanced expression of miR330-3p leads to a harmful loss of junction protein VE-cadherin. Our work suggests that S. aureus manipulates host signalling mechanisms by triggering abnormal expression of miRNA in the endothelium, in order to enhance the infection. By increasing expression of miR330-3p, and thereby hindering expression of VE-cadherin, S. aureus ensures that the endothelium provides a passageway to secondary sites and organs throughout the body. Further work is required to investigate the expression of miR330-3p in response to S. aureus infection in vivo.</p

Coordinated Molecular Cross-Talk between Staphylococcus aureus, Endothelial Cells and Platelets in Bloodstream Infection

Staphylococcus aureus is an opportunistic pathogen often carried asymptomatically on the human body. Upon entry to the otherwise sterile environment of the cardiovascular system, S. aureus can lead to serious complications resulting in organ failure and death. The success of S. aureus as a pathogen in the bloodstream is due to its ability to express a wide array of cell wall proteins on its surface that recognise host receptors, extracellular matrix proteins and plasma proteins. Endothelial cells and platelets are important cells in the cardiovascular system and are a major target of bloodstream infection. Endothelial cells form the inner lining of a blood vessel and provide an antithrombotic barrier between the vessel wall and blood. Platelets on the other hand travel throughout the cardiovascular system and respond by aggregating around the site of injury and initiating clot formation. Activation of either of these cells leads to functional dysregulation in the cardiovascular system. In this review, we will illustrate how S. aureus establish intimate interactions with both endothelial cells and platelets leading to cardiovascular dysregulation

Reactive Nitrogen Species-Induced Cell Death Requires Fas-Dependent Activation of c-Jun N-Terminal Kinase

Nitrogen dioxide is a highly toxic reactive nitrogen species (RNS) recently discovered as an inflammatory oxidant with great potential to damage tissues. We demonstrate here that cell death by RNS was caused by c-Jun N-terminal kinase (JNK). Activation of JNK by RNS was density dependent and caused mitochondrial depolarization and nuclear condensation. JNK activation by RNS was abolished in cells lacking functional Fas or following expression of a truncated version of Fas lacking the intracellular death domain. In contrast, RNS induced JNK potently in cells expressing a truncated version of tumor necrosis factor receptor 1 or cells lacking tumor necrosis factor receptor 1 (TNF-R1), illustrating a dependence of Fas but not TNF-R1 in RNS-induced signaling to JNK. Furthermore, Fas was oxidized, redistributed, and colocalized with Fas-associated death domain (FADD) in RNS-exposed cells, illustrating that RNS directly targeted Fas. JNK activation and cell death by RNS occurred in a Fas ligand- and caspase-independent manner. While the activation of JNK by RNS or FasL required FADD, the cysteine-rich domain 1 containing preligand assembly domain required for FasL signaling was not involved in JNK activation by RNS. These findings illustrate that RNS cause cell death in a Fas- and JNK-dependent manner and that this occurs through a pathway distinct from FasL. Thus, avenues aimed at preventing the interaction of RNS with Fas may attenuate tissue damage characteristic of chronic inflammatory diseases that are accompanied by high levels of RNS